AU2005200246A1 - Neisseria genomic sequences and methods of their use - Google Patents

Description

AUSTRALIA

Patents Act 1990 CHIRON CORPORATION, THE INSTITUTE FOR GENOMIC RESEARCH COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Neisseria genomic sequences and methods of their use The following statement is a full description of this invention including the best method of performing it known to us: r NEISSERIA GENOMIC SEQUENCES AND METHODS OF THEIR USE This is a divisional application from Australian Patent Application No.

32492/00, the entire contents of which are herein incorporated by reference.

All documents cited herein are incorporated by reference in their entirety. In particular, the contents of international patent application WO 00/66791 are fully incorporated herein.

This invention relates to methods of obtaining antigens and immunogens, the antigens and immunogens so obtained, and nucleic acids from the bacterial species: Neisseria meningitidis. In particular, it relates to genomic sequences from the bacterium; more particularly its serogroup.

BACKGROUND

Neisseria meningitidis is a non-motile, gram negative diplococcus human pathogen. It colonizes the pharynx, causing meningitis and, occasionally, septicaemia in the absence of meningitis. It is closely related to N. gonorrhoea, although one feature that clearly differentiates meningococcus from gonococcus is the presence of a polysaccharide capsule that is present in all pathogenic meningococci.

N. meningitidis causes both endemic and epidemic disease. In the United States the attack rate is 0.6-1 per 100,000 persons per year, and it can be much greater during outbreaks. (see Lieberman et al. (1996) Safety and Immunogenicity of a Serogroups A/C Neisseria meningitidis Oligosaccharide-Protein Conjugate Vaccine in Young Children. JAMA 275 1499-1503; Schuchat et al (1997) Bacterial Meningitis in the United States in 1995. N Engl J Med 337 970-976). In developing countries, endemic disease rates are much higher and during epidemics incidence rates can reach 500 cases per 100,000 persons per year. Mortality is extremely high, at 10-20% in the United States, and much higher in developing countries. Following the introduction of the conjugate vaccine against Haemophilus influenzae, N. meningitidis is the major cause of bacterial meningitis at all ages in the United States (Schuchat et al (1997) supra).

Based on the organism's capsular polysaccharide, 12 serogroups of N.

meningitidis have been identified. Group A is the pathogen most often implicated in epidemic disease in sub-Saharan Africa. Serogroups B and C are responsible for the vast majority of cases in the United States and in most developed countries: Serogroups W135 and Y are responsible for the rest of the cases in the United States and developed countries. The meningococcal vaccine currently in use is a tetravalent polysaccharide vaccine composed of serogroups A, C, Y and W135. Although efficacious in adolescents and adults, it induces a poor immune response and short duration of protection, and cannot be used in infants Morbidity and Mortality weekly report, Vol. 46, No. RR-5 (1997)). This is because polysaccharides are Tcell independent antigens that induce a weak immune response that cannot be boosted by repeated immunization. Following the success of the vaccination against H. influenzae, conjugate vaccines against serogroups A and C have been developed and are at the final stage of clinical testing (Zollinger WD "New and Improved Vaccines Against Meningococcal Disease". In: New Generation Vaccines, supra, pp. 469-488; Lieberman et al (1996) supra; Costantino et al (1992) Development and phase I clinical testing of a conjugate vaccine against meningococcus A (menA) and C (menC) (Vaccine 10:691-698)).

Meningococcus B (MenB) remains a problem, however. This serotype currently is responsible for approximately 50% of total meningitis in the United States, Europe, and South America. The polysaccharide approach cannot be used because the MenB capsular polysaccharide is a polymer of a(2-8)-linked N-acetyl neuraminic acid that is also present in mammalian tissue. This results in tolerance to the antigen; indeed, if an immune response were elicited, it would be anti-self, and therefore undesirable. In order to avoid induction of autoimmunity and to induce a protective immune response, the capsular polysaccharide has, for instance, been chemically modified substituting the N-acetyl groups with N-propionyl groups, leaving the specific antigenicity unaltered (Romero Outschoorn (1994) Current status of Meningococcal group B vaccine candidates: capsular or non-capsular? Clin Microbiol Rev 7(4):559-575).

Alternative approaches to MenB vaccines have used complex mixtures of outer membrane proteins (OMPs), containing either the OMPs alone, or OMPs enriched in porins, or deleted of the class 4 OMPs that are believed to induce antibodies that block bactericidal activity. This approach produces vaccines that are not well characterized. They are able to protect against the homologous strain, but are not effective at large where there are many antigenic variants of the outer membrane proteins. To overcome the antigenic variability, multivalent vaccines containing up to nine different porins have been constructed

I

Poolman JT (1992) Development of a meningococcal vaccine. Infect. Agents Dis.

4:13-28). Additional proteins to be used in outer membrane vaccines have been the opa and opc proteins, but none of these approaches have been able to overcome the antigenic variability Ala'Aldeen Borriello (1996) The meningococcal transferrin-binding proteins 1 and 2 are both surface exposed and generate bactericidal antibodies capable of killing homologous and heterologous strains. Vaccine 14(1):49- 53).

A certain amount of sequence data is available for meningococcal and gonococcal genes and proteins EP-A-0467714, W096/29412), but this is by no means complete. The provision of further sequences could provide an opportunity to identify secreted or surface-exposed proteins that are presumed targets for the immune system and which are not antigenically variable or at least are more antigenically conserved than other and more variable regions. Thus, those antigenic sequences that are more highly conserved are preferred sequences. Those sequences specific to Neisseria meningitidis or Neisseria gonorrhoeae that are more highly conserved are further preferred sequences. For instance, some of the identified proteins could be components of vaccines of efficacious vaccines against meingococcus B, some could be components of vaccines against all meningococcal serotypes, and others could be components of vaccines against, all pathogenic N'eisseriae. The identification of sequences from the bacterium will also facilitate the production of biological probes, particularly organism-specific probes.

It is thus a desire of the invention to provide Neisserial DNA sequences which encode proteins predicted and/or shown to be antigenic or immunogenic, can be used as probes or amplification primers, and can be analysed by bioinformatics.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

3A BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 illustrates the products of protein expression and purification of the predicted ORF 919 as cloned and expressed in E. coli.

Fig. 2 illustrates the products of protein expression and purification of the predicted ORF 279 as cloned and expressed in E. coli.

Fig. 3 illustrates the products of protein expression and purification of the predicted ORF 576-1 as cloned and expressed in E. coli.

Fig. 4 illustrates the products of protein expression and purification of the predicted ORF 519-1 as cloned and expressed in E. coli.

Fig. 5 illustrates the products of protein expression and purification of the predicted ORF 121-1 as cloned and expressed in E. coli.

Fig. 6 illustrates the products of protein expression and purification of the predicted ORF 128-1 as cloned and expressed in E. coli.

Fig. 7 illustrates the products of protein expression and purification of the predicted ORF 206 as cloned and expressed in E. coli.

Fig. 8 illustrates the products of protein expression and purification of the predicted ORF 287 as cloned and expressed in E. coli.

Fig. 9 illustrates the products of protein expression and purification of the predicted ORF 406 as cloned and expressed in E. coli.

Fig. 10 illustrates the hydrophilicity plot, antigenic index and AMPHI regions of the products of protein expression the predicted ORF 919 as cloned and expressed in E. coli.

Fig. 11 illustrates the hydrophilicity plot, antigenic index and AMPHI regions of the products of protein expression the predicted ORF 279 as cloned and expressed in E. coli.

Fig. 12 illustrates the hydrophilicity plot, antigenic index and AMPHI regions of the products of protein expression the predicted ORF 576-1 as cloned and expressed in E. coli.

Fig. 13 illustrates the hydrophilicity plot, antigenic index and AMPHI regions of the products of protein expression the predicted ORF 519-1 as cloned and expressed in E. coli.

Fig. 14 illustrates the hydrophilicity plot, antigenic index and AMPHI regions of the products of protein expression the predicted ORF 121-1 as cloned and expressed in E. coli.

Fig. 15 illustrates the hydrophilicity plot, antigenic index and AMPHI regions of the products of protein expression the predicted ORF 128-1 as cloned and expressed in E. coli.

Fig. 16 illustrates the hydrophilicity plot, antigenic index and AMPHI regions of the products of protein expression the predicted ORF 206 as cloned and expressed in E. coli.

Fig. 17 illustrates the hydrophilicity plot, antigenic index and AMPHI regions of the products of protein expression the predicted ORF 287 as cloned and expressed in E. coli.

Fig. 18 illustrates the hydrophilicity plot, antigenic index and AMPHI regions of the products of protein expression the predicted ORF 406 as cloned and expressed in E. coli.

THE INVENTION The first complete sequence of the genome ofN. meningitidis was disclosed as 961 partial contiguous nucleotide sequences, shown as SEQ ID NOs:1-961 of co-owned PCT/US99/23573 (the '573 application), filed 8 October 1999 (to be published April 2000).

A single sequence full length genome ofN. meningitidis was also disclosed as SEQ ID NO.

1068 of the '573 application. The invention is based on a full length genome of N. meningitidis which appears as SEQ ID NO. 1 in the present application as Appendix A hereto. The 961 sequences of the '573 application represent substantially the whole genome of serotype B ofN. meningitidis There is partial overlap between some of the 961 contiguous sequences ("contigs") shown in the 961 sequences, which overlap was used to construct the single full length sequence shown in SEQ ID NO. 1 in Appendix A hereto, using the TIGR Assembler Sutton et al., TIGR Assembler: A New Tool for Assembling Large Shotgun Sequencing Projects, Genome Science and Technology, 1:9-19 (1995)].

Some of the nucleotides in the contigs had been previously released. (See ftp: 1lftp.tigr.org/pub/data/n_meningitidis on the world-wide web or The coordinates of the 2508 released sequences in the present contigs are presented in Appendix A of the '573 application. These data include the contig number (or as presented in the first column; the name of the sequence as found on WWW is in the second column; with the coordinates of the contigs in the third and fourth columns, respectively. The sequences of certain MenB ORFs presented in Appendix B of the '573 application feature in International Patent Application filed by Chiron SpA on October 9, 1998 (PCT/IB98/01665) and January 14, 1999 (PCT/IB99/00103) respectively. Appendix B hereto provides a listing of 2158 open reading frames contained within the full length sequence found in SEQ ID NO. 1 in Appendix A hereto. The information set forth in Appendix B hereto includes the "NMB" name of the sequence, the putative translation product, and the beginning and ending nucleotide positions within SEQ ID NO. 1 which comprise the open reading frames. These open reading frames are referred to herein as the "NMB open reading frames".

In a first aspect, the invention provides nucleic acid including the N. meningitidis nucleotide sequence shown in SEQ ID NO. 1 in Appendix A hereto. It also provides nucleic acid comprising sequences having sequence identity to the nucleotide sequence disclosed herein. Depending on the particular sequence, the degree of sequence identity is preferably greater than 50% 60%, 70%, 80%, 90%, 95%, 99% or more). These sequences include, for instance, mutants and allelic variants. The degree of sequence identity cited herein is determined across the length of the sequence determined by the Smith-Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular) using an affme gap search with the following parameters: gap open penalty 12, gap extension penalty 1.

The invention also provides nucleic acid including a fragment of one or more of the nucleotide sequences set out herein, including the NMB open reading frames shown in Appendix B hereto. The fragment should comprise at least n consecutive nucleotides from the sequences and, depending on the particular sequence, n is 10 or more 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40,45, 50, 60, 75, 100 or more). Preferably, the fragment is unique to the genome ofN. meningitidis, that is to say it is not present in the genome of another organism. More preferably, the fragment is unique to the genome of strain B ofN. meningitidis. The invention also provides nucleic acid that hybridizes to those provided herein. Conditions for hybridizing are disclosed herein.

The invention also provides nucleic acid including sequences complementary to those described above for antisense, for probes, or for amplification primers).

Nucleic acid according to the invention can, of course, be prepared in many ways by chemical synthesis, from DNA libraries, from the organism itself, etc.) and can take various forms single-stranded, double-stranded, vectors, probes, primers, etc.). The term "nucleic acid" includes DNA and RNA, and also their analogs, such as those containing modified backbones, and also peptide nucleic acid (PNA) etc.

It will be appreciated that, as SEQ ID NOs: 1-961 of the '573 application represent the substantially complete genome of the organism, with partial overlap, references to SEQ ID NOs: 1-961 of the '573 application include within their scope references to the complete genomic sequence, that is, SEQ ID NO. 1 hereof. For example, where two SEQ ID NOs overlap, the invention encompasses the single sequence which is formed by assembling the two overlapping sequences, which full sequence will be found in SEQ ID NO. 1 hereof.

Thus, for instance, a nucleotide sequence which bridges two SEQ ID NOs but is not present in its entirety in either SEQ ID NO is still within the scope of the invention. Such a sequence will be present in its entirety in the single full length sequence of SEQ ID NO. 1 of the present application.

The invention also provides vectors including nucleotide sequences of the invention expression vectors, sequencing vectors, cloning vectors, etc.) and host cells transformed with such vectors.

According to a further aspect, the invention provides a protein including an amino acid sequence encoded within a N. meningitidis nucleotide sequence set out herein. It also provides proteins comprising sequences having sequence identity to those proteins.

Depending on the particular sequence, the degree of sequence identity is preferably greater than 50% 60%, 70%, 80%, 90%, 95%, 99% or more). Sequence identity is determined as above disclosed. These homologous proteins include mutants and allelic variants, encoded within the N. meningitidis nucleotide sequence set out herein.

The invention further provides proteins including fragments of an amino acid sequence encoded within a N. meningitidis nucleotide sequence set out in the sequence listing. The fragments should comprise at least n consecutive amino acids from the sequences and, depending on the particular sequence, n is 7 or more 8, 10, 12, 14, 16, 18, 20 or more). Preferably the fragments comprise an epitope from the sequence.

The proteins of the invention can, of course, be prepared by various means recombinant expression, purification from cell culture, chemical synthesis, etc.) and in various forms native, fusions etc.). They are preferably prepared in substantially isolated form substantially free from other N. meningitidis host cell proteins).

Various tests can be used to assess the in vivo immunogenicity of the proteins of the invention. For example, the proteins can be expressed recombinantly or chemically synthesized and used to screen patient sera by immunoblot. A positive reaction between the protein and patient serum indicates that the patient has previously mounted an immune response to the protein in question; the protein is an immunogen. This method can also be used to identify immunodominant proteins.

The invention also provides nucleic acid encoding a protein of the invention.

In a further aspect, the invention provides a computer, a computer memory, a computer storage medium floppy disk, fixed disk, CD-ROM, etc.), and/or a computer database containing the nucleotide sequence of nucleic acid according to the invention.

Preferably, it contains one or more of the N. meningitidis nucleotide sequences set out herein.

-8- This may be used in the analysis of the N. meningitidis nucleotide sequences set out herein. For instance, it may be used in a search to identify open reading frames (ORFs) or coding sequences within the sequences.

In a further aspect, the invention provides a method for identifying an amino acid sequence, comprising the step of searching for putative open reading frames or proteincoding sequences within a N. meningitidis nucleotide sequence set out herein. Similarly, the invention provides the use of a N. meningitidis nucleotide sequence set out herein in a search for putative open reading frames or protein-coding sequences.

Open-reading frame or protein-coding sequence analysis is generally performed on a computer using standard bioinformatic techniques. Typical algorithms or program used in the analysis include ORFFINDER (NCBI), GENMARK [Borodovsky Mclninch (1993) Computers Chem 17:122-133], and GLIMMER [Salzberg et al. (1998) NuclAcids Res 26:544-548].

A search for an open reading frame or protein-coding sequence may comprise the steps of searching a N. meningitidis nucleotide sequence set out herein for an initiation codon and searching the upstream sequence for an in-frame termination codon. The intervening codons represent a putative protein-coding sequence. Typically, all six possible reading frames of a sequence will be searched.

An amino acid sequence identified in this way can be expressed using any suitable system to give a protein. This protein can be used to raise antibodies which recognize epitopes within the identified amino acid sequence. These antibodies can be used to screen N. meningitidis to detect the presence of a protein comprising the identified amino acid sequence.

Furthermore, once an ORF or protein-coding sequence is identified, the sequence can be compared with sequence databases. Sequence analysis tools can be found at NCBI (http://www.ncbi.nlm.nih.gov) the algorithms BLAST, BLAST2, BLASTn, BLASTp, tBLASTn, BLASTx, tBLASTx [see also Altschul et al. (1997) Gapped BLAST and PSI- BLAST: new generation of protein database search programs. Nucleic Acids Research 25:2289-3402]. Suitable databases for comparison include the nonredundant GenBank, EMBL, DDBJ and PDB sequences, and the nonredundant GenBank CDS translations, PDB, SwissProt, Spupdate and PIR sequences. This comparison may give an indication of the function of a protein.

Hydrophobic domains in an amino acid sequence can be predicted using algorithms such as those based on the statistical studies ofEsposti et al. [Critical evaluation of the hydropathy of membrane proteins (1990) Eur JBiochem 190:207-219]. Hydrophobic domains represent potential transmembrane regions or hydrophobic leader sequences, which suggest that the proteins may be secreted or be surface-located. These properties are typically representative of good immunogens.

Similarly, transmembrane domains or leader sequences can be predicted using the PSORT algorithm (http://www.psort.nibb.ac.jp), and functional domains can be predicted using the MOTIFS program (GCG Wisconsin PROSITE).

The invention also provides nucleic acid including an open reading frame or proteincoding sequence present in a N. meningitidis nucleotide sequence set out herein.

Furthermore, the invention provides a protein including the amino acid sequence encoded by this open reading frame or protein-coding sequence.

According to a further aspect, the invention provides antibodies which bind to these proteins. These may be polyclonal or monoclonal and may be produced by any suitable means known to those skilled in the art.

The antibodies of the invention can be used in a variety of ways, for confirmation that a protein is expressed, or to confirm where a protein is expressed. Labeled antibody fluorescent labeling for FACS) can be incubated with intact bacteria and the presence of label on the bacterial surface confirms the location of the protein, for instance.

According to a further aspect, the invention provides compositions including protein, antibody, and/or nucleic acid according to the invention. These compositions may be suitable as vaccines, as immunogenic compositions, or as diagnostic reagents.

The invention also provides nucleic acid, protein, or antibody according to the invention for use as medicaments as vaccines) or as diagnostic reagents. It also provides the use of nucleic acid, protein, or antibody according to the invention in the manufacture of a medicament for treating or preventing infection due to Neisserial bacteria (ii) a diagnostic reagent for detecting the presence of Neisserial bacteria or of antibodies raised against Neisserial bacteria. Said Neisserial bacteria may be any species or strain (such as N. gonorrhoeae) but are preferably N. meningitidis, especially strain A, strain B or strain C.

In still yet another aspect, the present invention provides for compositions including proteins, nucleic acid molecules, or antibodies, More preferable aspects of the present invention are drawn to immunogenic compositions of proteins. Further preferable aspects of the present invention contemplate pharmaceutical immunogenic compositions of proteins or vaccines and the use thereof in the manufacture of a medicament for the treatment or prevention of infection due to Neisserial bacteria, preferably infection of MenB.

The invention also provides a method of treating a patient, comprising administering to the patient a therapeutically effective amount of nucleic' acid, protein, and/or antibody according to the invention.

According to further aspects, the invention provides various processes.

A process for producing proteins of the invention is provided, comprising the step of culturing a host cell according to the invention under conditions which induce protein expression. A process which may further include chemical synthesis of proteins and/or chemical synthesis (at least in part) ofnucleotides.

A process for detecting polynucleotides of the invention is provided, comprising the steps of: contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and detecting said duplexes.

A process for detecting proteins of the invention is provided, comprising the steps of: contacting an antibody according to the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and detecting said complexes.

Another aspect of the present invention provides for a process for detecting antibodies that selectably bind to antigens or polypeptides or proteins specific to any species or strain of Neisserial bacteria and preferably to strains ofN. gonorrhoeae but more preferably to strains ofN. meningitidis, especially strain A, strain B or strain C, more preferably MenB, where the process comprises the steps of: contacting antigen or polypeptide or protein according to the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and detecting said complexes.

-11- Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

Methodology Summary of standard procedures and techniques.

General This invention provides Neisseria meningitidis MenB nucleotide sequences, amino acid sequences encoded therein. With these disclosed sequences, nucleic acid probe assays and expression cassettes and vectors can be produced. The proteins can also be chemically synthesized. The expression vectors can be transformed into host cells to produce proteins.

The purified or isolated polypeptides can be used to produce antibodies to detect MenB proteins. Also, the host cells or extracts can be utilized for biological assays to isolate agonists or antagonists. In addition, with these sequences one can search to identify open reading frames and identify amino acid sequences. The proteins may also be used in immunogenic compositions and as vaccine components.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes Iand ii (D.N Glover ed. 1985); Oligonucleotide Synthesis Gait ed, 1984); Nucleic Acid Hybridization Hames S.J. Higgins eds. 1984); Transcription and Translation Hames S.J. Higgins eds. 1984); Animal Cell Culture Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 155; Gene Transfer Vectorsfor Mammalian Cells Miller and M.P. Calos eds. 1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, and Handbook of Experimental Immunology, Volumes I-IV Weir and C.C.

Blackwell eds 1986).

Standard abbreviations for nucleotides and amino acids are used in this specification.

-12- All publications, patents, and patent applications cited herein are incorporated in full by reference.

Expression systems The Neisseria MenB nucleotide sequences can.be expressed in a variety of different expression systems; for example those used with mammalian cells, plant cells, baculoviruses, bacteria, and yeast.

i. Mammalian Systems Mammalian expression systems are known in the art. A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream transcription of a coding sequence structural gene) into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element, usually located within 100 to 200 bp upstream of the TATA box.

An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation (Sambrook et al. (1989) "Expression of Cloned Genes in Mammalian Cells." In Molecular Cloning: A Laboratory Manual, 2nd ed.).

Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter. In addition, sequences derived from non-viral genes, such as the murine metallothionein gene, also provide useful promoter sequences. Expression may be either constitutive or regulated (inducible). Depending on the promoter selected, many promotes may be inducible using known substrates, such as the use of the mouse mammary tumor virus (MMTV) promoter with the glucocorticoid responsive element (GRE) that is induced by glucocorticoid in hormone-responsive transformed cells (see for example, U.S. Patent 5,783,681).

-13- The presence of an enhancer element (enhancer), combined with the promoter elements described above, will usually increase expression levels. An enhancer is a regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to homologous or heterologous promoters, with synthesis beginning at the normal RNA start site. Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter (Maniatis et al. (1987) Science 236:1237; Alberts et al. (1989) Molecular Biology of the Cell, 2nd Enhancer elements derived from viruses may be particularly useful, because they usually have a broader host range. Examples include the SV40 early gene enhancer (Dijkema et al (1985) EMBO J. 4:761) and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus (Gorman et al. (1982b) Proc. Natl. Acad. Sci. 79:6777) and from human cytomegalovirus (Boshart et al. (1985) Cell 41:521). Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion (Sassone- Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis et al. (1987) Science 236:1237).

A DNA molecule may be expressed intracellularly in mammalian cells. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.

Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cells. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The adenovirus tripartite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.

Usually, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3' terminus of the -14mature mRNA is formed by site-specific post-transcriptional cleavage and polyadenylation (Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988) "Termination and 3' end processing of eukaryotic RNA. In Transcription and splicing (ed. B.D. Hames and D.M.

Glover); Proudfoot (1989) Trends Biochem. Sci. 14:105). These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA.

Examples of transcription terminator/polyadenylation signals include those derived from (Sambrook et al (1989) "Expression of cloned genes in cultured mammalian cells." In Molecular Cloning: A Laboratory Manual).

Usually, the above-described components, comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs.

Enhancers, introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired. Expression constructs are often maintained in a replicon, such as an extrachromosomal element plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria. Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate. For example, plasmids containing the replication systems ofpapovaviruses, such as SV40 (Gluzman (1981) Cell 23:175) or polyomavirus, replicate to extremely high copy number in the presence of the appropriate viral T antigen. Additional examples of mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus. Additionally, the replicon may have two replication systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a prokaryotic host for cloning and amplification. Examples of such mammalian-bacteria shuttle vectors include pMT2 (Kaufman et al. (1989) Mol. Cell. Biol. 9:946) and pHEBO (Shimizu et al. (1986) Mol.

Cell. Biol. 6:1074).

The transformation procedure used depends upon the host to be transformed.

Methods for introduction ofheterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells Hep G2), and a number of other cell lines.

ii. Plant Cellular Expression Systems There are many plant cell culture and whole plant genetic expression systems known in the art. Exemplary plant cellular genetic expression systems include those described in patents, such as: U.S. 5,693,506; US 5,659,122; and US 5,608,143. Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytochemistry 30:3861- 3863 (1991). Descriptions of plant protein signal peptides may be found in addition to the references described above in Vaulcombe et al., Mol. Gen. Genet. 209:33-40 (1987); Chandler et al., Plant Molecular Biology 3:407-418 (1984); Rogers, J. Biol. Chem. 260:3731- 3738 (1985); Rothstein et al., Gene 55:353-356 (1987); Whittier et al., Nucleic Acids Research 15:2515-2535 (1987); Wirsel et al., Molecular Microbiology 3:3-14 (1989); Yu et al., Gene 122:247-253 (1992). A description of the regulation of plant gene expression by the phytohormone, gibberellic acid and secreted enzymes induced by gibberellic acid can be found in R.L. Jones and J. MacMillin, Gibberellins: in: Advanced Plant Physiology,.

Malcolm B. Wilkins, ed., 1984 Pitman Publishing Limited, London, pp. 21-52. References that describe other metabolically-regulated genes: Sheen, Plant Cell, 2:1027-1038(1990); Maas et al., EMBO J. 9:3447-3452 (1990); Benkel and Hickey, Proc. Natl. Acad. Sci.

84:1337-1339 (1987) Typically, using techniques known in the art, a desired polynucleotide sequence is inserted into an expression cassette comprising genetic regulatory elements designed for operation in plants. The expression cassette is inserted into a desired expression vector with companion sequences upstream and downstream from the expression cassette suitable for expression in a plant host. The companion sequences will be ofplasmid or viral origin and provide necessary characteristics to the vector to permit the vectors to move DNA from an original cloning host, such as bacteria, to the desired plant host. The basic bacterial/plant vector construct will preferably provide a broad host range prokaryote replication origin; a prokaryote selectable marker; and, for Agrobacterium transformations, T DNA sequences for Agrobacterium-mediated transfer to plant chromosomes. Where the heterologous gene is not -16readily amenable to detection, the construct will preferably also have a selectable marker gene suitable for determining if a plant cell has been transformed. A general review of suitable markers, for example for the members of the grass family, is found in Wilmink and Dons, 1993, Plant Mol. Biol. Reptr, 11(2):165-185.

Sequences suitable for permitting integration of the heterologous sequence into the plant genome are also recommended. These might include transposon sequences and the like for homologous recombination as well as Ti sequences which permit random insertion of a heterologous expression cassette into a plant genome. Suitable prokaryote selectable markers include resistance toward antibiotics such as ampicillin or tetracycline. Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.

The nucleic acid molecules of the subject invention may be included into an expression cassette for expression of the protein(s) of interest. Usually, there will be only one expression cassette, although two or more are feasible. The recombinant expression cassette will contain in addition to the heterologous protein encoding sequence the following elements, a promoter region, plant 5' untranslated sequences, initiation codon depending upon whether or not the structural gene comes equipped with one, and a transcription and translation termination sequence. Unique restriction enzyme sites at the 5' and 3' ends of the cassette allow for easy insertion into a pre-existing vector.

A heterologous coding sequence may be for any protein relating to the present invention. The sequence encoding the protein of interest will encode a signal peptide which allows processing and translocation of the protein, as appropriate, and will usually lack any sequence which might result in the binding of the desired protein of the invention to a membrane. Since, for the most part, the transcriptional initiation region will be for a gene which is expressed and translocated during germination, by employing the signal peptide which provides for translocation, one may also provide for translocation of the protein of interest. In this way, the protein(s) of interest will be translocated from the cells in which they are expressed and may be efficiently harvested. Typically secretion in seeds are across the aleurone or scutellar epithelium layer into the endosperm of the seed. While it is not required that the protein be secreted from the cells in which the protein is produced, this facilitates the isolation and purification of the recombinant protein.

-17- Since the ultimate expression of the desired gene product will be in a eucaryotic cell it is desirable to determine whether any portion of the cloned gene contains sequences which will be processed out as introns by the host's splicosome machinery. If so, site-directed mutagenesis of the "intron" region may be conducted to prevent losing a portion of the genetic message as a false intron code, Reed and Maniatis, Cell 41:95-105, 1985.

The vector can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA. Crossway, Mol. Gen. Genet, 202:179-185, 1985. The genetic material may also be transferred into the plant cell by using polyethylene glycol, Krens, et al., Nature, 296, 72-74, 1982. Another method of introduction of nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, Klein, et al., Nature, 327, 70-73, 1987 and Knudsen and Muller, 1991, Planta, 185:330-336 teaching particle bombardment of barley endosperm to create transgenic barley. Yet another method of introduction would be fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies, Fraley, et al., Proc. Natl. Acad. Sci. USA, 79, 1859-1863, 1982.

The vector may also be introduced into the plant cells by electroporation. (Fromm et al., Proc. Natl Acad. Sci. USA 82:5824, 1985). In this technique, plant protoplasts are electroporated in the presence ofplasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.

All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred gene. It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables. Some suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, -18- Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura.

Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced from the protoplast suspension.

These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable.

In some plant cell culture systems, the desired protein of the invention may be excreted or alternatively, the protein may be extracted from the whole plant. Where the desired protein of the invention is secreted into the medium, it may be collected.

Alternatively, the embryos and embryoless-half seeds or other plant tissue may be mechanically disrupted to release any secreted protein between cells and tissues. The mixture may be suspended in a buffer solution to retrieve soluble proteins. Conventional protein isolation and purification methods will be then used to purify the recombinant protein.

Parameters of time, temperature pH, oxygen, and volumes will be adjusted through routine methods to optimize expression and recovery of heterologous protein.

iii. Baculovirus Systems The polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector. Vector construction employs techniques which are known in the art. Generally, the components of the expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the -19homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.

After inserting the DNA sequence encoding the protein into the transfer vector, the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome are allowed to recombine. The packaged recombinant virus is expressed and recombinant plaques are identified and purified. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA ("MaxBac" kit). These techniques are generally known to those skilled in the art and fully described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) (hereinafter "Summers and Smith").

Prior to inserting the DNA sequence encoding the protein into the baculovirus genome, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are usually assembled into an intermediate transplacement construct (transfer vector). This construct may contain a single gene and operably linked regulatory elements; multiple genes, each with its owned set of operably linked regulatory elements; or multiple genes, regulated by the same set of regulatory elements. Intermediate transplacement constructs are often maintained in a replicon, such as an extrachromosomal element plasmids) capable of stable maintenance in a host, such as a bacterium. The replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification.

Currently, the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc373. Many other vectors, known to those of skill in the art, have also been designed. These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamHI cloning site 32 basepairs downstream from the ATT; see Luckow and Summers, Virology (1989) 17:31.

The plasmid usually also contains the polyhedrin polyadenylation signal (Miller et al.

(1988) Ann. Rev. Microbiol., 42:177) and a prokaryotic ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. A baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream to transcription of a coding sequence structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A baculovirus transfer vector may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene. Expression may be either regulated or constitutive.

Structural genes, abundantly transcribed at late times in a viral infection cycle, provide particularly useful promoter sequences. Examples include sequences derived from the gene encoding the viral polyhedron protein, Friesen et al., (1986) "The Regulation of Baculovirus Gene Expression," in: The Molecular Biology ofBaculoviruses (ed. Walter Doerfler); EPO Publ. Nos. 127 839 and 155 476; and the gene encoding the plO protein, Vlak et al., (1988), J. Gen. Virol. 69:765.

DNA encoding suitable signal sequences can be derived from gpnes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al.

(1988) Gene, 73:409). Alternatively, since the signals for mammalian cell posttranslational modifications (such as signal peptide cleavage, proteolytic cleavage, and phosphorylation) appear to be recognized by insect cells, and the signals required for secretion and nuclear accumulation also appear to be conserved between the invertebrate cells and vertebrate cells, leaders of non-insect origin, such as those derived from genes encoding human (alpha) ainterferon, Maeda et al., (1985), Nature 315:592; human gastrin-releasing peptide, Lebacq- Verheyden et al., (1988), Molec. Cell. Biol. 8:3129; human IL-2, Smith et al., (1985) Proc.

Nat'lAcad. Sci. USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and human glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also be used to provide for secretion in insects.

A recombinant polypeptide or polyprotein may be expressed intracellularly or, if it is expressed with the proper regulatory sequences, it can be secreted. Good intracellular expression of nonfused foreign proteins usually requires heterologous genes that ideally have a short leader sequence containing suitable translation initiation signals preceding an ATG start signal. If desired, methionine at the N-terminus may be cleaved from the mature protein by in vitro incubation with cyanogen bromide.

Alternatively, recombinant polyproteins or proteins which are not naturally secreted can be secreted from the insect cell by creating chimeric DNA molecules that encode a fusion -21protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in insects. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the translocation of the protein into the endoplasmic reticulum.

After insertion of the DNA sequence and/or the gene encoding the expression product precursor of the protein, an insect cell host is co-transformed with the heterologous DNA of the transfer vector and the genomic DNA of wild type baculovirus usually by cotransfection. The promoter and transcription termination sequence of the construct will usually comprise a 2-5kb section of the baculovirus genome. Methods for introducing heterologous DNA into the desired site in the baculovirus virus are known in the art. (See Summers and Smith supra; Ju et al. (1987); Smith et al., Mol. Cell. Biol. (1983) 3:2156; and Luckow and Summers (1989)). For example, the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene. Miller et al., (1989), Bioessays 4:91. The DNA sequence, when cloned in place of the polyhedrin gene in the expression vector, is flanked both 5' and 3' by polyhedrin-specific sequences and is positioned downstream of the polyhedrin promoter.

The newly formed baculovirus expression vector is subsequently packaged into an infectious recombinant baculovirus. Homologous recombination occurs at low frequency (between about 1% and about thus, the majority of the virus produced after cotransfection is still wild-type virus. Therefore, a method is necessary to identify recombinant viruses. An advantage of the expression system is a visual screen allowing recombinant viruses to be distinguished. The polyhedrin protein, which is produced by the native virus, is produced-at very high levels in the nuclei of infected cells at late times after viral infection. Accumulated polyhedrin protein forms occlusion bodies that also contain embedded particles. These occlusion bodies, up to 15 pm in size, are highly refractile, giving them a bright shiny appearance that is readily visualized under the light microscope. Cells infected with recombinant viruses lack occlusion bodies. To distinguish recombinant virus from wild-type virus, the transfection supernatant is plaqued onto a monolayer of insect cells by techniques known to those skilled in the art. Namely, the plaques are screened under the light microscope for the presence (indicative of wild-type virus) or absence (indicative of 22 recombinant virus) of occlusion bodies. Current Protocols in Microbiology Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990); Summers and Smith, supra; Miller et al. (1989).

Recombinant baculovirus expression vectors have been developed for infection into several insect cells. For example, recombinant baculoviruses have been developed for, inter alia: Aedes aegypti Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni (PCT Pub. No. WO 89/046699; Carbonell et al., (1985) J. Virol. 56:153; Wright (1986) Nature 321:718; Smith et al., (1983) Mol. Cell. Biol.

3:2156; and see generally, Fraser, et al. (1989) In Vitro Cell. Dev. Biol. 25:225).

Cells and cell culture media are commercially available for both direct and fusion expression of heterologous polypeptides in a baculovirus/expression system; cell culture technology is generally known to those skilled in the art. See, Summers and Smith supra.

The modified insect cells may then be grown in an appropriate nutrient medium, which allows for stable maintenance of the plasmid(s) present in the modified insect host.

Where the expression product gene is under inducible control, the host may be grown to high density, and expression induced. Alternatively, where expression is constitutive, the product will be continuously expressed into the medium and the nutrient medium must be continuously circulated, while removing the product of interest and augmenting depleted nutrients. The product may be purified by such techniques as chromatography, HPLC, affinity chromatography, ion exchange chromatography, etc.; electrophoresis; density gradient centrifugation; solvent extraction, or the like. As appropriate, the product may be further purified, as required, so as to remove substantially any insect proteins which are also secreted in the medium or result from lysis of insect cells, so as to provide a product which is at least substantially free of host debris, proteins, lipids and polysaccharides.

In order to obtain protein expression, recombinant host cells derived from the transformants are incubated under conditions which allow expression of the recombinant protein encoding sequence. These conditions will vary, dependent upon the host cell selected.

However, the conditions are readily ascertainable to those of ordinary skill in the art, based upon what is known in the art.

23 iv. Bacterial Systems Bacterial expression techniques are known in the art. A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream transcription of a coding sequence structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression may occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal to the RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli coli) (Raibaud et al. (1984) Annu. Rev. Genet. 18:173). Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) (Chang et al. (1977) Nature 198:1056), and maltose.

Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) (Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al. (1981) Nucl.

Acids Res. 9:731; U.S. Patent 4,738,921; EPO Publ. Nos. 036 776 and 121 775). The betalactamase (bla) promoter system (Weissmann (1981) "The cloning of interferon and other mistakes." In Interferon 3 (ed. I. Gresser)), bacteriophage lambda PL (Shimatake et al. (1981) Nature 292:128) and T5 Patent 4,689,406) promoter systems also provide useful promoter sequences.

In addition, synthetic promoters which do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter Patent 4,551,433). For -24example, the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor (Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21). Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of nonbacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system (Studier et al.

(1986) J. Mol. Biol. 189:113; Tabor et al. (1985) Proc Natl. Acad. Sci. 82:1074). In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO Publ. No. 267 851).

In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes. In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon (Shine et al. (1975) Nature 254:34). The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3' end of E. coli 16S rRNA (Steitz et al. (1979) "Genetic signals and nucleotide sequences in messenger RNA." In Biological Regulation and Development: Gene Expression (ed. R.F.

Goldberger)). To express eukaryotic genes and prokaryotic genes with weak ribosomebinding site, it is often necessary to optimize the distance between the SD sequence and the ATG of the eukaryotic gene (Sambrook et al. (1989) "Expression of cloned genes in Escherichia coli." In Molecular Cloning: A Laboratory Manual).

A DNA molecule may be expressed intracellularly. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo or in vitro incubation with a bacterial methionine Nterminal peptidase (EPO Publ. No. 219 237).

Fusion proteins provide an alternative to direct expression. Usually, a DNA sequence encoding the N-terminal portion of an endogenous bacterial protein, or other stable protein, is fused to the 5' end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the bacteriophage lambda cell gene can be linked at the 5' terminus of a foreign gene and expressed in bacteria. The resulting fusion protein preferably retains a site for a processing enzyme (factor Xa) to cleave the bacteriophage protein from the foreign gene (Nagai et al. (1984) Nature 309:810). Fusion proteins can also be made with sequences from the lacZ (Jia et al. (1987) Gene 60:197), trpE (Allen et al. (1987) J. Biotechnol. 5:93; Makoffet al. (1989) J. Gen. Microbiol. 135:11), and Chey (EPO Publ. No. 324 647) genes. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein. Through this method, native foreign protein can be isolated (Miller et al. (1989) Bio/Technology 7:698).

Alternatively, foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria Patent 4,336,336).

The signal sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). Preferably there are processing sites, which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene.

DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) (Masui et al.

(1983), in: Experimental Manipulation of Gene Expression; Ghrayeb et al. (1984) EMBOJ.

3:2437) and the E. coli alkaline phosphatase signal sequence (phoA) (Oka et al. (1985) Proc.

Natl. Acad. Sci. 82:7212). As an additional example, the signal sequence of the alphaamylase gene from various Bacillus strains can be used to secrete heterologous proteins from B. subtilis (Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EPO Publ. No. 244 042).

Usually, transcription termination sequences recognized by bacteria are regulatory regions located 3' to the translation stop codon, and thus together with the promoter flank the -26coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E. coli as well as other biosynthetic genes.

Usually, the above described components, comprising a promoter, signal sequence (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element plasmids) capable of stable maintenance in a host, such as bacteria. The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and amplification. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 20 plasmids. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.

Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome. For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (EPO Publ. No. 127 328). Integrating vectors may also be comprised of bacteriophage or transposon sequences.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed.

Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline (Davies etal. (1978) Annu. Rev. Microbiol. 32:469). Selectable -27markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.

Alternatively, some of the above described components can be put together in transformation vectors. Transformation vectors are usually comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either extra-chromosomal replicons or integrating vectors, have been developed for transformation into many bacteria. For example, expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis (Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EPO Publ. Nos. 036 259 and 063 953; PCT Publ. No. WO 84/04541), Escherichia coli (Shimatake et al. (1981) Nature 292:128; Amann et al. (1985) Gene 40:183; Studier et al. (1986) J. Mol. Biol. 189:113; EPO Publ. Nos. 036 776, 136 829 and 136 907), Streptococcus cremoris (Powell et al. (1988) Appl. Environ. Microbiol. 54:655); Streptococcus lividans (Powell et al. (1988) Appl.

Environ. Microbiol. 54:655), Streptomyces lividans Patent 4,745,056).

Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaCl 2 or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary with the bacterial species to be transformed. (See use of Bacillus: Masson et al. (1989) FEMS Microbiol. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA -79:5582; EPO Publ. Nos. 036 259 and 063 953; PCT Publ. No. WO 84/04541; use of Campylobacter: Miller etal. (1988) Proc. Natl.

Acad. Sci. 85:856; and Wang et al. (1990) J. Bacteriol. 172:949; use of Escherichia coli: Cohen et al. (1973) Proc. Natl. Acad. Sci. 69:2110; Dower et al. (1988) Nucleic Acids Res.

16:6127; Kushner (1978) "An improved method for transformation of Escherichia coli with ColEl-derived plasmids. In Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering (eds. H.W. Boyer and S. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159; Taketo (1988) Biochim. Biophys. Acta 949:318; use of Lactobacillus: Chassy et al. (1987) FEMS Microbiol. Lett. 44:173; use of Pseudomonas: Fiedler et al.

(1988) Anal. Biochem 170:38; use of Staphylococcus: Augustin et al. (1990) FEMS Microbiol. Lett. 66:203; use of Streptococcus: Barany et al. (1980) J. Bacteriol. 144:698; -28- Harlander (1987) "Transformation of Streptococcus lactis by electroporation, in: Streptococcal Genetics (ed. J. Ferretti and R. Curtiss III); Perry et al. (1981) Infect. Immun.

32:1295; Powell et al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc.

4th Evr. Cong. Biotechnology 1:412.

v. Yeast Expression Yeast expression systems are also known to one of ordinary skill in the art. A yeast promoter is any DNA sequence capable of binding yeast RNA polymerase and initiating the downstream transcription of a coding sequence structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the end of the coding sequence. This transcription initiation region usually includes an RNA polymerase bindingsite (the "TATA Box") and a transcription initiation site. A yeast promoter may also have a second domain called an upstream activator sequence (UAS), which, if present, is usually distal to the structural gene. The UAS permits regulated (inducible) expression. Constitutive expression occurs in the absence of a UAS. Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.

Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences.

Examples include alcohol dehydrogenase (ADH) (EPO Publ. No. 284 044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO Publ. No. 329 203). The yeast PH05 gene, encoding acid phosphatase, also provides useful promoter sequences (Myanohara et al. (1983) Proc. Natl.

Acad. Sci. USA 80:1).

In addition, synthetic promoters which do not occur in nature also function as yeast promoters. For example, UAS sequences of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region Patent Nos. 4,876,197 and 4,880,734). Other examples of hybrid promoters include promoters which consist of the regulatory sequences of -29either the ADH2, GAL4, GALO0, OR PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EPO Publ. No. 164 556).

Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription. Examples of such promoters include, inter alia, (Cohen et al. (1980) Proc. Natl. Acad. Sci. USA 77:1078; Henikoffet al. (1981) Nature 283:835; Hollenberg et al. (1981) Curr. Topics Microbiol.

Immunol. 96:119; Hollenberg et al. (1979) "The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae," in: Plasmids ofMedical, Environmental and Commercial Importance (eds. K.N. Timmis and A. Puhler); Mercerau- Puigalon et al. (1980) Gene 11:163; Panthier et al. (1980) Curr. Genet. 2:109;).

A DNA molecule may be expressed intracellularly in yeast. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the Nterminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.

Fusion proteins provide an alternative for yeast expression systems, as well as in mammalian, plant, baculovirus, and bacterial expression systems. Usually, a DNA sequence encoding the N-terminal portion of an endogenous yeast protein, or other stable protein, is fused to the 5' end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the yeast or human superoxide dismutase (SOD) gene, can be linked at the 5' terminus of a foreign gene and expressed in yeast. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. See EPO Publ. No. 196056. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein. Through this method, therefore, native foreign protein can be isolated WO88/024066).

Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.

DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (EPO Publ. No. 012 873; JPO Publ. No.

62:096,086) and the A-factor gene Patent 4,588,684). Alternatively, leaders of nonyeast origin, such as an interferon leader, exist that also provide for secretion in yeast (EPO Publ, No. 060 057).

A preferred class of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a "pre" signal sequence, and a "pro" region. The types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (usually about to about 50 amino acid residues) Patent Nos. 4,546,083 and 4,870,008; EPO Publ.

No. 324 274). Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast alpha factor. (See PCT Publ. No. WO 89/02463.) Usually, transcription termination sequences recognized by yeast are regulatory regions located 3' to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of anmRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes.

Usually, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element plasmids) capable of stable maintenance in a host, such as yeast or bacteria. The replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host for cloning and amplification. Examples of such yeast-bacteria shuttle vectors include YEp24 (Botstein et al.

(1979) Gene 8:17-24), pCl/1 (Brake et al. (1984) Proc. Natl. Acad. Sci USA 81:4642-4646), and YRpl7 (Stinchcomb et al. (1982) J Mol. Biol. 158:157). In addition, a replicon may be -31either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20. Enter a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host. See Brake et al., supra.

Alternatively, the expression constructs can be integrated into the yeast genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct. Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome (Orr- Weaver et al. (1983) Methods in Enzymol. 101:228-245). An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for inclusion in the vector. See Orr-Weaver et al., supra. One or more expression construct may integrate, possibly affecting levels of recombinant protein produced (Rine et al. (1983) Proc.

Natl. Acad. Sci. USA 80:6750). The chromosomal sequences included in the vector can occur either as a single segment in the vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration of only the expression construct.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed.

Selectable markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TRP1, and ALG7, and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively. In addition, a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal. For example, the presence of CUP1 allows yeast to grow in the presence of copper ions (Butt et al. (1987) Microbiol, Rev. 51:35 1).

Alternatively, some of the above described components can be put together into transformation vectors. Transformation vectors are usually comprised of a selectable marker -32that is either maintained in a replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts. For example, expression vectors and methods of introducing exogenous DNA into yeast hosts have been developed for, inter alia, the following yeasts: Candida albicans (Kurtz, et al. (1986) Mol.

Cell. Biol. 6:142); Candida maltosa (Kunze, etal. (1985)J. Basic Microbiol. 25:141); Hansenula polymorpha (Gleeson, et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302); Kluyveromycesfragilis (Das, et al. (1984) J. Bacteriol.

158:1165); Kluyveromyces lactis (De Louvencourt et al. (1983) J. Bacteriol. 154:737; Van den Berg etal. (1990) Bio/Technology 8:135); Pichia guillerimondii (Kunze et al. (1985) J.

Basic Microbiol. 25:141); Pichia pastoris (Cregg, et al. (1985) Mol. Cell. Biol. 5:3376; U.S.

Patent Nos. 4,837,148 and 4,929,555); Saccharomyces cerevisiae (Hinnen et al. (1978) Proc.

Natl. Acad. Sci. USA 75:1929; Ito etal. (1983) J. Bacteriol. 153:163); Schizosaccharomyces pombe (Beach and Nurse (1981) Nature 300:706); and Yarrowia lipolytica (Davidow, et al.

(1985) Curr. Genet. 10:380471 Gaillardin, et al. (1985) Curr. Genet. 10:49).

Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and usually include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformation procedures usually vary with the yeast species to be transformed. See [Kurtz et al. (1986) Mol. Cell. Biol. 6:142; Kunze et al. (1985) J. Basic Microbiol. 25:141; Candida]; [Gleeson et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302; Hansenula]; [Das et al. (1984) J.

Bacteriol. 158:1165; De Louvencourt et al. (1983) J Bacteriol. 154:1165; Van den Berg et al. (1990) Bio/Technology 8:135; Kluyveromyces]; [Cregg et al. (1985) Mol. Cell. Biol.

5:3376; Kunze et.al. (1985) J. Basic Microbiol. 25:141; U.S. PatentNos. 4,837,148 and 4,929,555; Pichia]; [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75;1929; Ito et al.

(1983) J. Bacteriol. 153:163 Saccharomyces]; [Beach and Nurse (1981) Nature 300:706; Schizosaccharomyces]; [Davidow et al. (1985) Curr. Genet. 10:39; Gaillardin et al. (1985) Curr. Genet. 10:49; Yarrowia].

-33- Definitions A composition containing X is "substantially free of" Y when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95% or even O 99% by weight.

The term "heterologous" refers to two biological components that are not found together in nature. The components may be host cells, genes, or regulatory regions, such as promoters. Although the heterologous components are not found together in nature, they can function together, as when a promoter heterologous to a gene is operably linked to the gene.

C' Another example is where a Neisserial sequence is heterologous to a mouse host cell.

An "origin of replication" is a polynucleotide sequence that initiates and regulates replication of polynucleotides, such as an expression vector. The origin of replication behaves as an autonomous unit of polynucleotide replication within a cell, capable of replication under its own control. An origin of replication may be needed for a vector to replicate in a particular host cell. With certain origins of replication, an expression vector can be reproduced at a high copy number in the presence of the appropriate proteins within the cell.

Examples of origins are the autonomously replicating sequences, which are effective in yeast; and the viral T-antigen, effective in COS-7 cells.

A "mutant" sequence is defined as a DNA, RNA or amino acid sequence differing from but having homology with the native or disclosed sequence. Depending on the particular sequence, the degree of homology between the native or disclosed sequence and the mutant sequence is preferably greater than 50% 60%, 70%, 80%, 90%, 95%, 99% or more) which is calculated as described above. As used herein, an "allelic variant" of a nucleic acid molecule, or region, for which nucleic acid sequence is provided herein is a nucleic acid molecule, or region, that occurs at essentially the same locus in the genome of another or second isolate, and that, due to natural variation caused by, for example, mutation or recombination, has a similar but not identical nucleic acid sequence. A coding region allelic variant typically encodes a protein having similar activity to that of the protein encoded by the gene to which it is being compared. An allelic variant can also comprise an alteration in the 5' or 3' untranslated regions of the gene, such as in regulatory control regions.

(see, for example, U.S. Patent 5,753,235).

-34- Antibodies As used herein, the term "antibody" refers to a polypeptide or group of polypeptides composed of at least one antibody combining site. An "antibody combining site" is the three-dimensional binding space with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows a binding of the antibody with the antigen. "Antibody" includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanized antibodies, altered antibodies, univalent antibodies, Fab proteins, and single domain antibodies.

Antibodies against the proteins of the invention are useful for affinity chromatography, immunoassays, and distinguishing/identifying Neisseria MenB proteins.

Antibodies elicited against the proteins of the present invention bind to antigenic polypeptides or proteins or protein fragments that are present and specifically associated with strains of Neisseria meningitidis MenB. In some instances, these antigens may be associated with specific strains, such as those antigens specific for the MenB strains. The antibodies of the invention may be immobilized to a matrix and utilized in an immunoassay or on an affinity chromatography column, to enable the detection and/or separation of polypeptides, proteins or protein fragments or cells comprising such polypeptides, proteins or protein fragments. Alternatively, such polypeptides, proteins or protein fragments may be immobilized so as to detect antibodies bindably specific thereto.

Antibodies to the proteins of the invention, both polyclonal and monoclonal, may be prepared by conventional methods. In general, the protein is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat. Rabbits and goats are preferred for the preparation of polyclonal sera due to the volume of serum obtainable, and the availability of labeled anti-rabbit and anti-goat antibodies. Immunization is generally performed by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). A dose of 50-200 jig/injection is typically sufficient. Immunization is generally boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant. One may alternatively generate antibodies by in vitro immunization using methods known in the art, which for the purposes of this invention is considered equivalent to in vivo immunization. Polyclonal antisera is obtained by bleeding the immunized animal into a glass or plastic container, incubating the blood at 25 0

C

for one hour, followed by incubating at 4 0 C for 2-18 hours. The serum is recovered by centrifugation 1,000g for 10 minutes). About 20-50 ml per bleed may be obtained from rabbits.

Monoclonal antibodies are prepared using the standard method ofKohler Milstein (Nature (1975) 256:495-96), or a modification thereof. Typically, a mouse or rat is immunized as described above. However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen. B-cells that express membrane-bound immunoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium hypoxanthine, aminopterin, thymidine medium, The resulting hybridomas are plated by limiting dilution, and are assayed for the production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens). The selected MAb-secreting hybridomas are then cultured either in vitro in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).

If desired, the antibodies (whether polyclonal or monoclonal) may be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms (particularly 32 P and 1251), electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3',5,5'-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer. "Specific binding partner" refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art.

It should be understood that the above description is not meant to categorize the various -36labels into distinct classes, as the same label may serve in several different modes. For example, 1.25I may serve as a radioactive label or as an electron-dense reagent. HRP may serve as enzyme or as antigen for a MAb. Further, one may combine various labels for desired effect. For example, MAbs and avidin also require labels in the practice of this invention: thus, one might label a MAb with biotin, and detect its presence with avidin labeled with 125, or with an anti-biotin MAb labeled with HRP. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.

Antigens, immunogens, polypeptides, proteins or protein fragments of the present invention elicit formation of specific binding partner antibodies. These antigens, immunogens, polypeptides, proteins or protein fragments of the present invention comprise immunogenic compositions of the present invention. Such immunogenic compositions may further comprise or include adjuvants, carriers, or other compositions that promote or enhance or stabilize the antigens, polypeptides, proteins or protein firagments of the present invention. Such adjuvants and carriers will be readily apparent to those of ordinary skill in the art.

Pharmaceutical Compositions Pharmaceutical compositions can include either polypeptides, antibodies, or nucleic acid of the invention. The pharmaceutical compositions will comprise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the claimed invention.

The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature, when given to a patient that is febrile. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in -37advance. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgment of the clinician.

For purposes of the present invention, an effective dose will be from about 0.01 mg/ kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.

1.

t A pharmaceutical composition can also contain a pharmaceutically acceptable carrier.

SThe term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents.

The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier.

Delivery Methods Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated.

-38- Direct delivery of the compositions will generally be accomplished by injection, S either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and IN transdermal and transcutaneous applications, needles, and gene guns or hyposprays. Dosage S treatment may be a single dose schedule or a multiple dose schedule.

I Vaccines Vaccines according to the invention may either be prophylactic to prevent C1 infection) or therapeutic to treat disease after infection).

Such vaccines comprise immunizing antigen(s) or immunogen(s), immunogenic polypeptide, protein(s) or protein fragments, or nucleic acids ribonucleic acid or deoxyribonucleic acid), usually in combination with "pharmaceutically acceptable carriers," which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents ("adjuvants"). Furthermore, the immunogen or antigen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.

Preferred adjuvants to enhance effectiveness of the composition include, but are not lirhited to: aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example MF59 (PCT Publ. No. WO 90/14837), containing Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, MA), SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr- MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a -39larger particle size emulsion, and Ribi T adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting ofmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL CWS (Detox T m saponin adjuvants, such as StimulonTM (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); cytokines, such as interleukins IL-1, IL-2, IL-4, IL-5, IL-6, 11-7, IL-12, etc.), interferons gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; detoxified mutants of a bacterial ADP-ribosylating toxin such as a cholera toxin a pertussis toxin or an E. coli heat-labile toxin (LT), particularly LT-K63, LT-R72, CT-S109, PT-K9/G129; see, WO 93/13302 and WO 92/19265; and other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Alum and MF59 are preferred.

As mentioned above, muramyl peptides include, but are not limited to, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-Disoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(l'-2'dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

The vaccine compositions comprising immunogenic compositions which may include the antigen, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Alternatively, vaccine compositions comprising immunogenic compositions may comprise an antigen, polypeptide, protein, protein fragment or nucleic acid in a pharmaceutically acceptable carrier.

More specifically, vaccines comprising immunogenic compositions comprise an immunologically effective amount of the immunogenic polypeptides, as well as any other of the above-mentioned components, as needed. By "immunologically effective amount", it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

Typically, the vaccine compositions or immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.

The immunogenic compositions are conventionally administered parenterally, by injection, either subcutaneously or intramuscularly. Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal and transcutaneous applications. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents.

As an alternative to protein-based vaccines, DNA vaccination may be employed Robinson Torres (1997) Seminars in Immunology 9:271-283; Donnelly et al. (1997) Annu Rev Immunol 15:617-648).

Gene Delivery Vehicles Gene therapy vehicles for delivery of constructs, including a coding sequence of a therapeutic of the invention, to be delivered to the mammal for expression in the mammal, can be administered either locally or systemically. These constructs can utilize viral or non-viral vector approaches in in vivo or ex vivo modality. Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters.

Expression of the coding sequence in vivo can be either constitutive or regulated.

The invention includes gene delivery vehicles capable of expressing the contemplated nucleic acid sequences. The gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vector. The viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, -41paramyxovirus, parvovirus, picomavirus, poxvirus, or togavirus viral vector. See generally, Jolly (1994) Cancer Gene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly (1995) Human Gene Therapy 6:185-193; and Kaplitt (1994) Nature Genetics 6:148-153.

Retroviral vectors are well known in the art, including B, C and D type retroviruses, xenotropic retroviruses (for example, NZB-X1, NZB-X2 and NZB9-1 (see O'Neill (1985) J.

Virol. 53:160) polytropic retroviruses MCF and MCF-MLV (see Kelly (1983) J. Virol.

45:291), spumaviruses and lentiviruses. See RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.

Portions of the retroviral gene therapy vector may be derived from different retroviruses. For example, retrovector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.

These recombinant retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see US patent 5,591,624). Retrovirus vectors can be constructed for site-specific integration into host cell DNA by incorporation of a chimeric integrase enzyme into the retroviral particle (see W096/37626). It is preferable that the recombinant viral vector is a replication defective recombinant virus.

Packaging cell lines suitable for use with the above-described retrovirus vectors are well known in the art, are readily prepared (see W095/30763 and W092/05266), and can be used to create producer cell lines (also termed vector cell lines or "VCLs") for the production of recombinant vector particles. Preferably, the packaging cell lines are made from human parent cells HT1080 cells) or mink parent cell lines, which eliminates inactivation in human serum.

Preferred retroviruses for the construction of retroviral gene therapy vectors include Avian Leukosis Virus, Bovine Leukemia, Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma Virus. Particularly preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe (1976) J Virol 19:19-25), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey Sarcoma Virus and -42- Rauscher (ATCC No. VR-998) and Moloney Murine Leukemia Virus (ATCC No. VR-190).

Such retroviruses may be obtained from depositories or collections such as the American Type Culture Collection ("ATCC") in Rockville, Maryland or isolated from known sources using commonly available techniques.

Exemplary known retroviral gene therapy vectors employable in this invention include those described in patent applications GB2200651, EP0415731, EP0345242, EP0334301, W089/02468; W089/05349, W089/09271, W090/02806, W090/07936, W094/03622, W093/25698, W093/25234, W093/11230, W093/10218, W091/02805, W091/02825, W095/07994, US 5,219,740, US 4,405,712, US 4,861,719, US 4,980,289, US 4,777,127, US 5,591,624. See also Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967; Ram (1993) Cancer Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res 33:493-503; Baba (1993) JNeurosurg 79:729-735; Mann (1983) Cell 33:153; Cane (1984) Proc Natl Acad Sci 81:6349; and Miller (1990) Human Gene Therapy 1.

Human adenoviral gene therapy vectors are also known in the art and employable in this invention. See, for example, Berkner (1988) Biotechniques 6:616 and Rosenfeld (1991) Science 252:431, and W093/07283, W093/06223, and W093/07282. Exemplary known adenoviral gene therapy vectors employable in this invention include those described in the above referenced documents and in W094/12649, W093/03769, W093/19191, W094/28938, W095/11984, W095/00655, W095/27071, W095/29993, W095/34671, W096/05320, W094/08026, W094/11506, W093/06223, W094/24299, W095/14102, W095/24297, W095/02697, W094/28152, W094/24299, W095/09241, W095/25807, W095/05835, W094/18922 and W095/09654. Alternatively, administration of DNA linked to killed adenovirus as described in Curiel (1992) Hum. Gene Ther. 3:147-154 may be employed. The gene delivery vehicles of the invention also include adenovirus associated virus (AAV) vectors. Leading and preferred examples of such vectors for use in this invention are the AAV-2 based vectors disclosed in Srivastava, W093/09239. Most preferred AAV vectors comprise the two AAV inverted terminal repeats in which the native D-sequences are modified by substitution ofnucleotides, such that at least 5 native nucleotides and up to 18 native nucleotides, preferably at least 10 native nucleotides up to 18 native nucleotides, most preferably 10 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides. The native -43- D-sequences of the AAV inverted terminal repeats are sequences of 20 consecutive nucleotides in each AAV inverted terminal repeat there is one sequence at each end) which are not involved in HP formation. The non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position.

Other employable exemplary AAV vectors are pWP-19, pWN-1, both of which are disclosed in Nahreini (1993) Gene 124:257-262. Another example of such an AAV vector is psub201 (see Samulski (1987) J. Virol. 61:3096). Another exemplary AAV vector is the Double-D ITR vector. Construction of the Double-D ITR vector is disclosed in US Patent 5,478,745.

Still other vectors are those disclosed in Carter US Patent 4,797,368 and Muzyczka US Patent 5,139,941, Chartejee US Patent 5,474,935, and Kotin W094/288157. Yet a further example of an AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhancer and albumin promoter and directs expression predominantly in the liver. Its structure and construction are disclosed in Su (1996) Human Gene Therapy 7:463-470.

Additional AAV gene therapy vectors are described in US 5,354,678, US 5,173,414, US 5,139,941, and US 5,252,479.

The gene therapy vectors comprising sequences of the invention also include herpes vectors. Leading and preferred examples are herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in US 5,288,641 and EP0176170 (Roizman). Additional exemplary herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in W095/04139 (Wistar Institute), pHSVlac described in Geller (1988) Science 241:1667-1669 and in W090/09441 and W092/07945, HSV Us3::pgC-lacZ described in Fink (1992) Human Gene Therapy 3:11-19 and HSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), and those deposited with the ATCC as accession numbers ATCC VR-977 and ATCC VR-260.

Also contemplated are alpha virus gene therapy vectors that can be employed in this invention. Preferred alpha virus vectors are Sindbis viruses vectors. Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described in US patents 5,091,309, 5,217,879, and W092/10578. More particularly, those alpha virus vectors described in U.S. Serial No. 08/405,627, filed March 15, 1995,WO94/21792, W092/10578, -44- W095/07994, US 5,091,309 and US 5,217,879 are employable. Such alpha viruses may be obtained from depositories or collections such as the ATCC in Rockville, Maryland or isolated from known sources using commonly available techniques. Preferably, alphavirus vectors with reduced cytotoxicity are used (see USSN 08/679640).

DNA vector systems such as eukarytic layered expression systems are also useful for expressing the nucleic acids of the invention. SeeWO95/07994 for a detailed description of eukaryotic layered expression systems. Preferably, the eukaryotic layered expression systems of the invention are derived from alphavirus vectors and most preferably from Sindbis viral vectors.

Other viral vectors suitable for use in the present invention include those derived from poliovirus, for example ATCC VR-58 and those described in Evans, Nature 339 (1989) 385 and Sabin (1973) J. Biol. Standardization 1:115; rhinovirus, for example ATCC VR-1110 and those described in Arnold (1990) J Cell Biochem L401; pox viruses such as canary pox virus or vaccinia virus, for example ATCC VR-111 and ATCC VR-2010 and those described in Fisher-Hoch (1989) Proc Natl Acad Sci 86:317; Flexner (1989) Ann NYAcad Sci 569:86, Flexner (1990) Vaccine 8:17; in US 4,603,112 and US 4,769,330 and WO89/01973; virus, for example ATCC VR-305 and those described in Mulligan (1979) Nature 277:108 and Madzak (1992) JGen Virol 73:1533; influenza virus, for example ATCC VR-797 and recombinant influenza viruses made employing reverse genetics techniques as described in US 5,166,057 and in Enami (1990) Proc Natl Acad Sci 87:3802-3805; Enami Palese (1991) J Virol 65:2711-2713 and Luytjes (1989) Cell 59:110, (see also McMichael (1983) NEJMed 309:13, and Yap (1978) Nature 273:238 and Nature (1979) 277:108); human immunodeficiency virus as described in EP-0386882 and in Buchschacher (1992) J. Virol.

66:2731; measles virus, for example ATCC VR-67 and VR-1247 and those described in EP- 0440219; Aura virus, for example ATCC VR-368; Bebaru virus, for example ATCC VR-600 and ATCC VR-1240; Cabassou virus, for example ATCC VR-922; Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; Fort Morgan Virus, for example ATCC VR-924; Getah virus, for example ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example ATCC VR-927; Mayaro virus, for example ATCC VR-66; Mucambo virus, for example ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example ATCC VR-371; Pixuna virus, for example ATCC VR-372 and ATCC VR-1245; Tonate virus, for example ATCC VR-925; Triniti virus, for example ATCC VR-469; Una virus, for example ATCC E VR-374; Whataroa virus, for example ATCC VR-926; Y-62-33 virus, for example ATCC VR-375; O'Nyong virus, Eastern encephalitis virus, for example ATCC VR-65 and ATCC VR-1242; Western encephalitis virus, for example ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus, for example ATCC VR-740 and those Sdescribed in Hamre (1966) Proc Soc Exp Biol Med 121:190.

Delivery of the compositions of this invention into cells is not limited to the above mentioned viral vectors. Other delivery methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example see US Serial No. 08/366,787, filed December 30, 1994 and Curiel (1992) Hum Gene Ther 3:147-154 ligand linked DNA, for example see Wu (1989) JBiol Chem 264:16985-16987, eucaryotic cell delivery vehicles cells, for example see US Serial No.08/240,030, filed May 9, 1994, and US Serial No. 08/404,796, deposition of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as described in US Patent 5,149,655, ionizing radiation as described in US5,206,152 and in W092/11033, nucleic charge neutralization or fusion with cell membranes. Additional approaches are described in Philip (1994) Mol Cell Biol 14:2411-2418 and in Woffendin (1994) Proc Natl Acad Sci 91:1581-1585.

Particle mediated gene transfer may be employed, for example see US Serial No.

60/023,867. Briefly, the sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in Wu Wu (1987) J. Biol. Chem. 262:4429-4432, insulin as described in Hucked (1990) Biochem Pharmacol 40:253-263, galactose as described in Plank (1992) Bioconjugate Chem 3:533-539, lactose or transferrin.

Naked DNA may also be employed to transform a host cell. Exemplary naked DNA introduction methods are described in WO 90/11092 and US 5,580,859. Uptake efficiency may be improved using biodegradable latex beads. DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads. The method may be improved -46further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.

Liposomes that can act as gene delivery vehicles are described in U.S. 5,422,120, W095/13796, W094/23697, WO91/14445 and EP-524,968. As described in USSN.

60/023,867, on non-viral delivery, the nucleic acid sequences encoding a polypeptide can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin. Other delivery systems include the use of liposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously-active promoters. Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al (1994) Proc. Natl. Acad. Sci. USA 91(24):11581-11585. Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials. Other conventional methods for gene delivery that can be used for delivery of the. coding sequence include, for example, use of hand-held gene transfer particle gun, as described in U.S. 5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. 5,206,152 and WO92/11033 Exemplary liposome and polycationic gene delivery vehicles are those described in US 5,422,120 and 4,762,915; inWO 95/13796; W094/23697; and W091/14445; in EP- 0524968; and in Stryer, Biochemistry, pages 236-240 (1975) W.H. Freeman, San Francisco; Szoka (1980) Biochem Biophys Acta 600:1; Bayer (1979) Biochem Biophys Acta 550:464; Rivnay (1987) Meth Enzymol 149:119; Wang (1987) Proc Natl Acad Sci 84:7851; Plant (1989) Anal Biochem 176:420.

A polynucleotide composition can comprise a therapeutically effective amount of a gene therapy vehicle, as the term is defined above. For purposes of the present invention, an effective dose will be from about 0.01 mg/ kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.

-47- Delivery Methods Once formulated, the polynucleotide compositions of the invention can be administered directly to the subject; delivered ex vivo, to cells derived from the subject; or in vitro for expression of recombinant proteins. The subjects to be treated can be mammals or birds. Also, human subjects can be treated.

Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, transdermally or transcutaneously, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a tumor or lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications, needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule. See W098/20734.

Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in W093/14778. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoetic, lymph cells, macrophages, dendritic cells, or tumor cells, Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by the following procedures, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.

Polynucleotide and Polypeptide pharmaceutical compositions In addition to the pharmaceutically acceptable carriers and salts described above, the following additional agents can be used with polynucleotide and/or polypeptide compositions.

A. Polypeptides One example are polypeptides which include, without limitation: asialoorosomucoid (ASOR); transferrin; asialoglycoproteins; antibodies; antibody fragments; ferritin; interleukins; interferons, granulocyte, macrophage colony stimulating factor (GM-CSF), -48granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), stem cell factor and erythropoietin. Viral antigens, such as envelope proteins, can also be used. Also, proteins from other invasive organisms, such as the 17 amino acid peptide from the circumsporozoite protein ofplasmodium falciparum known as RII.

0 t B. Hormones, Vitamins, Etc.

SOther groups that can be included in a pharmaceutical composition include, for example: hormones, steroids, androgens, estrogens, thyroid hormone, or vitamins, folic acid.

"1 C. Polyalkylenes, Polysaccharides, etc.

Also, polyalkylene glycol can be included in a pharmaceutical compositions with the desired polynucleotides and/or polypeptides. In a preferred embodiment, the polyalkylene glycol is polyethlylene glycol. In addition, mono-, di-, or polysaccarides can be included. In a preferred embodiment of this aspect, the polysaccharide is dextran or DEAE-dextran. Also, chitosan and poly(lactide-co-glycolide) may be included in a pharmaceutical composition.

D. Lipids, and Liposomes The desired polynucleotide or polypeptide can also be encapsulated in lipids or packaged in liposomes prior to delivery to the subject or to cells derived therefrom.

Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid or polypeptide. The ratio of condensed polynucleotide to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight (1991) Biochim. Biophys. Acta. 1097:1-17; Straubinger (1983) Meth. Enzymol. 101:512-527.

Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. Cationic liposomes have been shown to mediate intracellular delivery ofplasmid DNA (Felgner (1987) Proc. Natl.

Acad. Sci. USA 84:7413-7416); mRNA (Malone (1989) Proc. Natl. Acad. Sci. USA 86:6077-6081); and purified transcription factors (Debs (1990) J. Biol. Chem.

265:10189-10192), in functional form.

-49- Cationic liposomes are readily available. For example, N(1 2 3 -dioleyloxy)propyl)-NN,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, NY. (See, also, Feigner supra). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, Szoka (1978) Proc.

Natl. Acad. Sci. USA 75:4194-4198; W090/11092 for a description of the synthesis of DOTAP (1, 2 -bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials.

Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art. See Straubinger (1983) Meth.

Immunol. 101:512-527; Szoka (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198; Papahadjopoulos (1975) Biochim. Biophys. Acta 394:483; Wilson (1979) Cell 17:77); Deamer Bangham (1976) Biochim. Biophys. Acta 443:629; Ostro (1977) Biochem.

Biophys. Res. Commun. 76:836; Fraley (1979) Proc. Natl. Acad. Sci. USA 76:3348); Enoch Strittmatter (1979) Proc. Natl. Acad. Sci. USA 76:145; Fraley (1980) J. Biol. Chem. (1980) 255:10431; Szoka Papahadjopoulos (1978) Proc. Natl. Acad. Sci. USA 75:145; and Schaefer-Ridder (1982) Science 215:166.

E. Lipoproteins In addition, lipoproteins can be included with the polynucleotide or polypeptide to be delivered. Examples of lipoproteins to be utilized include: chylomicrons, HDL, IDL, LDL, and VLDL. Mutants, fragments, or fusions of these proteins can also be used. Also, modifications of naturally occurring lipoproteins can be used, such as acetylated LDL. These lipoproteins can target the delivery ofpolynucleotides to cells expressing lipoprotein receptors. Preferably, if lipoproteins are including with the polynucleotide to be delivered, no other targeting ligand is included in the composition.

Naturally occurring lipoproteins comprise a lipid and a protein portion. The protein portion are known as apoproteins. At the present, apoproteins A, B, C, D, and E have been isolated and identified. At least two of these contain several proteins, designated by Roman numerals, AI, AII, AIV; CI, CII, CIII.

A lipoprotein can comprise more than one apoprotein. For example, naturally occurring chylomicrons comprises of A, B, C, and E; over time these lipoproteins lose A and acquire C and E apoproteins. VLDL comprises A, B, C, and E apoproteins, LDL comprises apoprotein B; and HDL comprises apoproteins A, C, and E.

The amino acid sequences of these apoproteins are known and are described in, for example, Breslow (1985) Annu Rev. Biochem 54:699; Law (1986) Adv. Exp Med. Biol.

151:162; Chen (1986) JBiol Chem 261:12918; Kane (1980) Proc NatlAcad Sci USA 77:2465; and Utermann (1984) Hum Genet 65:232.

Lipoproteins contain a variety of lipids including, triglycerides, cholesterol (free and esters), and phopholipids. The composition of the lipids varies in naturally occurring lipoproteins. For example, chylomicrons comprise mainly triglycerides. A more detailed description of the lipid content of naturally occurring lipoproteins can be found, for example, in Meth. Enzymol. 128 (1986). The composition of the lipids are chosen to aid in conformation of the apoprotein for receptor binding activity. The composition of lipids can also be chosen to facilitate hydrophobic interaction and association with the polynucleotide binding molecule.

Naturally occurring lipoproteins can be isolated from serum by ultracentrifugation, for instance. Such methods are described in Meth. Enzymol. (supra); Pitas (1980) J. Biochem.

255:5454-5460 and Mahey (1979) J Clin. Invest 64:743-750.

Lipoproteins can also be produced by in vitro or recombinant methods by expression of the apoprotein genes in a desired host cell. See, for example, Atkinson (1986) Annu Rev Biophys Chem 15:403 and Radding (1958) Biochim Biophys Acta 30: 443.

Lipoproteins can also be purchased from commercial suppliers, such as Biomedical Techniologies, Inc., Stoughton, Massachusetts, USA.

-51 Further description of lipoproteins can be found in Zuckermann et al., PCT. Appln.

No. US97/14465.

F. Polycationic Agents Polycationic agents can be included, with or without lipoprotein, in a composition with the desired polynucleotide and/or polypeptide to be delivered.

Polycationic agents, typically, exhibit a net positive charge at physiological relevant pH and are capable of neutralizing the electrical charge of nucleic acids to facilitate delivery to a desired location. These agents have both in vitro, ex vivo, and in vivo applications.

Polycationic agents can be used to deliver nucleic acids to a living subject either intramuscularly, subcutaneously, etc.

The following are examples of useful polypeptides as polycationic agents: polylysine, polyarginine, polyornithine, and protamine. Other examples of useful polypeptides include histones, protamines; human serum albumin, DNA binding proteins, non-histone chromosomal proteins, coat proteins from DNA viruses, such as QX174, transcriptional factors also contain domains that bind DNA and therefore may be useful as nucleic aid condensing agents. Briefly, transcriptional factors such as C/CEBP, c-jun, c-fos, AP-1, AP-2, AP-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP, and TFIID contain basic domains that bind DNA sequences.

Organic polycationic agents include: spermine, spermidine, and purtrescine.

The dimensions and of the physical properties of a polycationic agent can be extrapolated from the list above, to construct other polypeptide polycationic agents or to produce synthetic polycationic agents.

G. Synthetic Polycationic Agents Synthetic polycationic agents which are useful in pharmaceutical compositions include, for example, DEAE-dextran, polybrene. Lipofectin T M and lipofectAMINE T M are monomers that form polycationic complexes when combined with polynucleotides or polypeptides.

-52- Immunodiagnostic Assays Neisseria MenB antigens, or antigenic fragments thereof, of the invention can be used in immunoassays to detect antibody levels (or, conversely, anti-Neisseria MenB antibodies can be used to detect antigen levels). Immunoassays based on well defined, recombinant antigens can be developed to replace invasive diagnostics methods. Antibodies to Neisseria MenB proteins or fragments thereof within biological samples, including for example, blood or serum samples, can be detected. Design of the immunoassays is subject to a great deal of variation, and a variety of these are known in the art. Protocols for the immunoassay may be based, for example, upon competition, or direct reaction, or sandwich type assays. Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.

Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the compositions of the invention, in suitable containers, along with the remaining reagents and materials (for example, suitable buffers, salt solutions, etc.) required for the conduct of the assay, as well as suitable set of assay instructions.

Nucleic Acid Hybridization "Hybridization" refers to the association of two nucleic acid sequences to one another by hydrogen bonding. Typically, one sequence will be fixed to a solid support and the other will be free in solution. Then, the two sequences will be placed in contact with one another under conditions that favor hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase sequence to the solid support (Denhardt's reagent or BLOTTO); concentration of the sequences; use of compounds to increase the rate of association of sequences (dextran sulfate or polyethylene glycol); and the -53stringency of the washing conditions following hybridization. See Sambrook et al. (supra) Volume 2, chapter 9, pages 9.47 to 9.57.

"Stringency" refers to conditions in a hybridization reaction that favor association of very similar sequences over sequences that differ. For example, the combination of temperature and salt concentration should be chosen that is approximately 120 to 200°C below the calculated Tm of the hybrid under study. The temperature and salt conditions can often be determined empirically in preliminary experiments in which samples of genomic DNA immobilized on filters are hybridized to the sequence of interest and then washed under conditions of different stringencies. See Sambrook et al. at page 9.50.

Variables to consider when performing, for example, a Southern blot are the complexity of the DNA being blotted and the homology between the probe and the sequencesbeing detected. The total amount of the fragment(s) to be studied can vary a magnitude of 10, from 0.1 to 1 jg for a plasmid or phage digest to 10' 9 to 10 8 g for a single copy gene in a highly complex eukaryotic genome. For lower complexity polynucleotides, substantially shorter blotting, hybridization, and exposure times, a smaller amount of starting polynucleotides, and lower specific activity of probes can be used. For example, a single-copy yeast gene can be detected with an exposure time of only 1 hour starting with 1 gg of yeast DNA, blotting for two hours, and hybridizing for 4-8 hours with a probe of 108 cpm/pg. For a single-copy mammalian gene a conservative approach would start with 10 ig of DNA, blot overnight, and hybridize overnight in the presence of 10% dextran sulfate using a probe of greater than 108 cpm/pg, resulting in an exposure time of-24 hours.

Several factors can affect the melting temperature (Tm) of a DNA-DNA hybrid between the probe and the fragment of interest, and consequently, the appropriate conditions for hybridization and washing. In many cases the probe is not 100% homologous to the fragment. Other commonly encountered variables include the length and total G+C content of the hybridizing sequences and the ionic strength and formamide content of the hybridization buffer. The effects of all of these factors can be approximated by a single equation: Tm= 81 16.6(logloCi) 0.6(%formamide) 600/n where Ci is the salt concentration (monovalent ions) and n is the length of the hybrid in base pairs (slightly modified from Meinkoth Wahl (1984) Anal. Biochem. 138:267-284).

-54- In designing a hybridization experiment, some factors affecting nucleic acid hybridization can be conveniently altered. The-temperature of the hybridization and washes and the salt concentration during the washes are the simplest to adjust. As the temperature of the hybridization increases stringency), it becomes less likely for hybridization to occur between strands that are nonhomologous, and as a result, background decreases. If the radiolabeled probe is not completely homologous with the immobilized fragment (as is frequently the case in gene family and interspecies hybridization experiments), the hybridization temperature must be reduced, and background will increase. The temperature of the washes affects the intensity of the hybridizing band and the degree of background in a similar manner. The stringency of the washes is also increased with decreasing salt concentrations.

In general, convenient hybridization temperatures in the presence of 50% formamide are 42 0 C for a probe with is 95% to 100% homologous to the target fragment, 37C for to 95% homology, and 32°C for 85% to 90% homology. For lower homologies, formamide content should be lowered and temperature adjusted accordingly, using the equation above. If the homology between the probe and the target fragment are not known, the simplest approach is to start with both hybridization and wash conditions which are nonstringent. If non-specific bands or high background are observed after autoradiography, the filter can be washed at high stringency and reexposed. If the time required for exposure makes this approach impractical, several hybridization and/or washing stringencies should be tested in parallel.

Nucleic Acid Probe Assays Methods such as PCR, branched DNA probe assays, or blotting techniques utilizing nucleic acid probes according to the invention can determine the presence of cDNA or mRNA. A probe is said to "hybridize" with a sequence of the invention if it can form a duplex or double stranded complex, which is stable enough to be detected.

The nucleic acid probes will hybridize to the Neisserial nucleotide sequences of the invention (including both sense and antisense strands). Though many different nucleotide sequences will encode the amino acid sequence, the native Neisserial sequence is preferred because it is the actual sequence present in cells. mRNA represents a coding sequence and so a probe should be complementary to the coding sequence; single-stranded cDNA is complementary to mRNA, and so a cDNA probe should be complementary to the non-coding sequence.

The probe sequence need not be identical to the Neisserial sequence (or its complement) some variation in the sequence and length can lead to increased assay sensitivity if the nucleic acid probe can form a duplex with target nucleotides, which can be detected. Also, the nucleic acid probe can include additional nucleotides to stabilize the formed duplex. Additional Neisserial sequence may also be helpful as a label to detect the formed duplex. For example, a non-complementary nucleotide sequence may be attached to the 5' end of the probe, with the remainder of the probe sequence being complementary to a Neisserial sequence. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the a Neisserial sequence in order to hybridize therewith and thereby form a duplex which can be detected.

The exact length and sequence of the probe will depend on the hybridization conditions, such as temperature, salt condition and the like. For example, for diagnostic applications, depending on the complexity of the analyte sequence, the nucleic acid probe typically contains at least 10-20 nucleotides, preferably 15-25, and more preferably at least nucleotides, although it may be shorter than this. Short primers generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.

Probes may be produced by synthetic procedures, such as the triester method of Matteucci et al. Am. Chem. Soc. (1981) 103:3185), or according to Urdea et al. (Proc.

Natl. Acad. Sci. USA (1983) 80: 7461), or using commercially available automated oligonucleotide synthesizers.

The chemical nature of the probe can be selected according to preference. For certain applications, DNA or RNA are appropriate. For other applications, modifications may be incorporated backbone modifications, such as phosphorothioates or methylphosphonates, can be used to increase in vivo half-life, alter RNA affinity, increase nuclease resistance etc. see Agrawal Iyer (1995) Curr Opin Biotechnol 6:12-19; Agrawal (1996) TIBTECH 14:376-387); analogues such as peptide nucleic acids may also be 56 used see Corey (1997) TIBTECH 15:224-229; Buchardt et al. (1993) TIBTECH 11:384- 386).

One example of a nucleotide hybridization assay is described by Urdea et al. in international patent application W092/02526 (see also U.S. Patent 5,124,246).

Alternatively, the polymerase chain reaction (PCR) is another well-known means for detecting small amounts of target nucleic acids. The assay is described in: Mullis et al. (Meth.

Enzymol. (1987) 155: 335-350); US patent 4,683,195; and US patent 4,683,202. Two "primer" nucleotides hybridize with the target nucleic acids and are used to prime the reaction. The primers can comprise sequence that does not hybridize to the sequence of the amplification target (or its complement) to aid with duplex stability or, for example, to incorporate a convenient restriction site. Typically, such sequence will flank the desired Neisserial sequence.

A thermostable polymerase creates copies of target nucleic acids from the primers using the original target nucleic acids as a template. After a threshold amount of target nucleic acids are generated by the polymerase, they can be detected by more traditional methods, such as Southern blots. When using the Southern blot method, the labeled probe will hybridize to the Neisserial sequence (or its complement).

Also, mRNA or cDNA can be detected by traditional blotting techniques described in Sambrook et al (supra). mRNA, or cDNA generated from mRNA using a polymerase enzyme, can be purified and separated using gel electrophoresis. The nucleic acids on the gel are then blotted onto a solid support, such as nitrocellulose. The solid support is exposed to a labeled probe and then washed to remove any unhybridized probe. Next, the duplexes containing the labeled probe are detected. Typically, the probe is labeled with a radioactive moiety.

EXAMPLES

The invention is based on the 961 nucleotide sequences from the genome of N. meningitidis set out in Appendix C, SEQ ID NOs:1-961 of the '573 application, which together represent substantially the complete genome of serotype B of N. meningitidis, as well as the full length genome sequence shown in Appendix D, SEQ ID NO 1068 of the '573 -57application, and the full length genome sequence shown in Appendix A hereto, SEQ ID NO.

1.

It will be self-evident to the skilled person how this sequence information can be utilized according to the invention, as above described.

The standard techniques and procedures which may be employed in order to perform the invention to utilize the disclosed sequences to predict polypeptides useful for vaccination or diagnostic purposes) were summarized above. This summary is not a limitation on the invention but, rather, gives examples that may be used, but are not required.

These sequences are derived from contigs shown in Appendix C (SEQ ID NOs 1-961) and from the full length genome sequence shown in Appendix D (SEQ ID NO 1068), which were prepared during the sequencing of the genome of N meningitidis (strain The full length sequence was assembled using the TIGR Assembler as described by G.S. Sutton et al., TIGR Assembler: A New Tool for Assembling Large Shotgun Sequencing Projects, Genome Science and Technology, 1:9-19 (1995) [see also R. D. Fleischmann, et al., Science 269, 496- 512.(1995); C. M. Fraser, et al., Science 270, 397-403 (1995); C. J. Bult, et al., Science 273, 1058-73 (1996); C. M. Fraser, et. al, Nature 390, 580-586 (1997); Tomb, et. al., Nature 388, 539-547 (1997); H. P. Klenk, et al., Nature 390, 364-70 (1997); C. M. Fraser, et al., Science 281, 375-88 (1998); M. J. Gardner, et al., Science 282, 1126-1132 (1998); K. E.

Nelson, et al., Nature 399, 323-9 (1999)]. Then, using the above-described methods, putative translation products of the sequences were determined. Computer analysis of the translation products were determined based on database comparisons. Corresponding gene and protein sequences, if any, were identified in Neisseria meningitidis (Strain A) and Neisseria gonorrhoeae. Then the proteins were expressed, purified, and characterized to assess their antigenicity and immunogenicity.

In particular, the following methods were used to express, purify, and biochemically characterize the proteins of the invention.

Chromosomal DNA Preparation N. meningitidis strain 2996 was grown to exponential phase in 100 ml of GC medium, harvested by centrifugation, and resuspended in 5 ml buffer (20% Sucrose, 50 mM Tris-HC1, mM EDTA, adjusted to pH After 10 minutes incubation on ice, the bacteria were -58lysed by adding 10 ml lysis solution (50 mM NaCI, 1% Na-Sarkosyl, 50 pg/ml Proteinase K), and the suspension was incubated at 37 0 C for 2 hours. Two phenol extractions (equilibrated to pH 8) and one ChCl 3 /isoamylalcohol (24:1) extraction were performed. DNA was precipitated by addition of 0.3M sodium acetate and 2 volumes ethanol, and was collected by centrifugation. The pellet was washed once with 70% ethanol and redissolved in 4 ml buffer mM Tris-HC1, 1mM EDTA, pH The DNA concentration was measured by reading the OD at 260 nm.

Oligonucleotide design Synthetic oligonucleotide primers were designed on the basis of the coding sequence of each ORF, using the meningococcus B sequence when available, or the gonococcus/meningococcus A sequence, adapted to the codon preference usage of meningococcus. Any predicted signal peptides were omitted, by deducing the amplification primer sequence immediately downstream from the predicted leader sequence.

For most ORFs, the 5' primers included two restriction enzyme recognition sites (BamHI-NdeI, BamHI-Nhel, or EcoRI-NheI, depending on the gene's restriction pattern); the 3' primers included a Xhol restriction site. This procedure was established in order to direct the cloning of each amplification product (corresponding to each ORF) into two different expression systems: pGEX-KG (using either BamHI-Xhol or EcoRI-Xhol), and pET2 lb+ (using either NdeI-XhoI or NheI-Xhol).

primer tail: CGCGGATCCCATATG (BamHI-NdeI) CGCGGATCCGCTAGC (BamHI-Nhel) CCGGAATTCTAGCTAGC (EcoRI-NheI) 3'-end primer tail: CCCGCTCGAG (XhoI) For some ORFs, two different amplifications were performed to clone each ORF in the two expression systems. Two different 5' primers were used for each ORF; the same 3' XhoI primer was used as before: primer tail: GGAATTCCATATGGCCATGG (NdeI) primer tail: CGGGATCC (BamHI) 59 Other ORFs were cloned in the pTRC expression vector and expressed as an amino-terminus His-tag fusion. The predicted signal peptide may be included in the final product. NheI-BamHI restriction sites were incorporated using primers: primer tail: GATCAGCTAGCCATATG (NheI) 3'-end primer tail: CGGGATCC (BamHI) As well as containing the restriction enzyme recognition sequences, the primers included nucleotides which hybridizeed to the sequence to be amplified. The number of hybridizing nucleotides depended on the melting temperature of the whole primer, and was determined for each primer using the formulae: Tm =4 2 (tail excluded) Tm= 64.9 0.41 GC) 600/N (whole primer) The average melting temperature of the selected oligos were 65-70 0 C for the whole oligo and 50-55 0 C for the hybridising region alone.

Oligos were synthesized by a Perkin Elmer 394 DNA/RNA Synthesizer, eluted from the columns in 2 ml NH4-OH, and deprotected by 5 hours incubation at 56 The oligos were precipitated by addition of 0.3M Na-Acetate and 2 volumes ethanol. The samples were then centrifuged and the pellets resuspended in either 100pl or lml of water. OD260 was determined using a Perkin Elmer Lambda Bio spectophotometer and the concentration was determined and adjusted to 2-10 pmol/pl.

Table 1 shows the forward and reverse primers used for each amplification. In certain cases, it might be noted that the sequence of the primer does not exactly match the sequence in the ORF. When initial amplifications are performed, the complete 5' and/or 3' sequence may not be known for some meningococcal ORFs, although the corresponding sequences may have been identified in gonoccus. For amplification, the gonococcal sequences could thus be used as the basis for primer design, altered to take account ofcodon preference. In particular, the following codons may be changed: ATA->ATT; TCG--TCT; CAG->CAA; AAG-AAA; GAG-GAA; CGA and CGG-CGC; GGG-GGC.

Amplification The standard PCR protocol was as follows: 50-200 ng of genomic DNA were used as a template in the presence of 20-40 LM of each oligo, 400-800 pM dNTPs solution, lx PCR buffer (including 1.5 mM MgCl 2 2.5 units TaqIDNA polymerase (using Perkin-Elmer AmpliTaQ, GIBCO Platinum, Pwo DNA polymerase, or Tahara Shuzo Taq polymerase).

In some cases, PCR was optimsed by the addition of 10tl DMSO or 50 gl 2M betaine.

After a hot start (adding the polymerase during a preliminary 3 minute incubation of the whole mix at 95 0 each sample underwent a double-step amplification: the first 5 cycles were performed using as the hybridization temperature the one of the oligos excluding the restriction enzymes tail, followed by 30 cycles performed according to the hybridization temperature of the whole length oligos. The cycles were followed by a final 10 minute extension step at 72 0

C.

The standard cycles were as follows: Denaturation Hybridisation Elongation First 5 cycles 30 seconds 30 seconds 30-60 seconds 50-55 0 C 72 0

C

Last 30 cycles 30 seconds 30seconds 30-60 seconds 9500 65-70 0 C 72°C The elongation time varied according to the length of the ORF to be amplified.

The amplifications were performed using either a 9600 or a 2400 Perkin Elmer GeneAmp PCR System. To check the results, 1/10 of the amplification volume was loaded onto a 1-1.5% agarose gel and the size of each amplified fragment compared with a DNA molecular weight marker.

The amplified DNA was either loaded directly on a 1% agarose gel or first precipitated with ethanol and resuspended in a suitable volume to be loaded on a 1% agarose gel. The DNA fragment corresponding to the right size band was then eluted and purified from gel, using the Qiagen Gel Extraction Kit, following the instructions of the manufacturer.

The final volume of the DNA fragment was 30 1 l or 50l of either water or 10mM Tris, pH Digestion of PCR fragments The purified DNA corresponding to the amplified fragment was split into 2 aliquots and double-digested with: -61- NdeI/XhoI or NheIlXhoI for cloning into pET-21b+ and further expression of the S protein as a C-terminus His-tag fusion SBamHI/XhoI or EcoRI/XhoI for cloning into pGEX-KG and further expression of the protein as a GST N-terminus fusion.

fO For ORF 76, NheI/BamHI for cloning into pTRC-HisA vector and further expression of the protein as N-terminus His-tag fusion.

Each purified DNA fragment was incubated (37 0 C for 3 hours to overnight) with units of each restriction enzyme (New England Biolabs in a either 30 or 40 tl final volume in the presence of the appropriate buffer. The digestion product was then purified using the

C

1 QIAquick PCR purification kit, following the manufacturer's instructions, and eluted in a final volume of 30 (or 50) pl of either water or 10mM Tris-HCl, pH 8.5. The final DNA concentration was determined by 1% agarose gel electrophoresis in the presence of titrated molecular weight marker.

Digestion of the cloning vectors (pET22B, pGEX-KG and pTRC-His A) .pg plasmid was double-digested with 50 units of each restriction enzyme in 200 pl reaction volume in the presence of appropriate buffer by overnight incubation at 37 0 C. After loading the whole digestion on a 1% agarose gel, the band corresponding to the digested vector was purified from the gel using the Qiagen QIAquick Gel Extraction Kit and the DNA was eluted in 50 d of 10 mM Tris-HC1, pH 8.5. The DNA concentration was evaluated by measuring OD 260 of the sample, and adjusted to 50 pg/pl. 1 pll of plasmid was used for each cloning procedure.

Cloning The fragments corresponding to each ORF, previously digested and purified, were ligated in both pET22b and pGEX-KG. In a final volume of 20 pl, a molar ratio of 3:1 fragment/vector was ligated using 0.5 pl of NEB T4 DNA ligase (400 units/il), in the presence of the buffer supplied by the manufacturer. The reaction was incubated at room temperature for 3 hours. In some experiments, ligation was performed using the Boheringer "Rapid Ligation Kit", following the manufacturer's instructions.

-62- In order to introduce the recombinant plasmid in a suitable strain, 100 pl E. coli competent cells were incubated with the ligase reaction solution for 40 minutes on ice, then at 37°C for 3 minutes, then, after adding 800 l LB broth, again at 37 0 C for 20 minutes. The cells were then centrifuged at maximum speed in an Eppendorfmicrofuge and resuspended in approximately 200 l1 of the supematant. The suspension was then plated on LB ampicillin (100 mg/ml).

The screening of the recombinant clones was performed by growing randomly-chosen colonies overnight at 37 OC in either 2 ml (pGEX or pTC clones) or (pET clones) LB broth 100 pg/ml ampicillin. The cells were then pelletted and the DNA extracted using the Qiagen QIAprep Spin Miniprep Kit, following the manufacturer's instructions, to a final volume of 30 p1. 5 pl of each individual miniprep (approximately Ig) were digested with either NdeIUXhoI or BamHIXhoI and the whole digestion loaded onto a 1agarose gel (depending on the expected insert size), in parallel with the molecular weight marker (1Kb DNA Ladder, GIBCO). The screening of the positive clones was made on the base of the correct insert size.

Cloning Certain ORFs may be cloned into the pGEX-HIS vector using EcoRI-PstI, EcoRI-Sall, or Sall-PstI cloning sites. After cloning, the recombinant plasmids may be introduced in the E.coli host W3110.

Expression Each ORF cloned into the expression vector may then be transformed into the strain suitable for expression of the recombinant protein product. 1 pl of each construct was used to transform 30 pl of E.coli BL21 (pGEX vector), E.coli TOP 10 (pTRC vector) or E.coli BL21- DE3 (pET vector), as described above. In the case of the pGEX-His vector, the same E.coli strain (W3110) was used for initial cloning and expression. Single recombinant colonies were inoculated into 2ml LB+Amp (100 jg/ml), incubated at 37 0 C overnight, then diluted 1:30 in 20 ml of LB+Amp (100 pg/ml) in 100 ml flasks, making sure that the OD 600 ranged between 0.1 and 0.15. The flasks were incubated at 30 C into gyratory water bath shakers until OD indicated exponential growth suitable for induction of expression (0.4-0.8 OD for -63pET and pTRC vectors; 0.8-1 OD for pGEX and pGEX-His vectors). For the pET, pTRC and pGEX-His vectors, the protein expression was induced by addiction of 1mM IPTG, whereas in the case ofpGEX system the final concentration of IPTG was 0.2 mM. After 3 hours incubation at 30 0 C, the final concentration of the sample was checked by OD. In order to check expression, 1ml of each sample was removed, centrifuged in a microfuge, the pellet resuspended in PBS, and analysed by 12% SDS-PAGE with Coomassie Blue staining. The whole sample was centrifuged at 6000g and the pellet resuspended in PBS for further use.

GST-fusion proteins large-scale purification.

A single colony was grown overnight at 37 0 C on LB+Amp agar plate. The bacteria were inoculated into 20 ml of LB+Amp liquid colture in a water bath shaker and grown overnight. Bacteria were diluted 1:30 into 600 ml of fresh medium and allowed to grow at the optimal temperature (20-37 0 C) to OD 550 0.8-1. Protein expression was induced with 0.2mM IPTG followed by three hours incubation. The culture was centrifuged at 8000 rpm at 4°C. The supernatant was discarded and the bacterial pellet was resuspended in 7.5 ml cold PBS. The cells were disrupted by sonication on ice for 30 sec at 40W using a Branson sonifier B-15, frozen and thawed two times and centrifuged again. The supernatant was collected and mixed with 150p.l Glutatione-Sepharose 4B resin (Pharmacia) (previously washed with PBS) and incubated at room temperature for 30 minutes. The sample was centrifuged at 700g for 5 minutes at 4C. The resin was washed twice with 10 ml cold PBS for 10 minutes, resuspended in lml cold PBS, and loaded on a disposable column. The resin was washed twice with 2ml cold PBS until the flow-through reached OD2so of 0.02-0.06.

The GST-fusion protein was eluted by addition of 7 0 0 pl cold Glutathione elution buffer reduced glutathione, 50mM Tris-HC1) and fractions collected until the OD 28 0 was 0.1.

211pl of each fraction were loaded on a 12% SDS gel using either Biorad SDS-PAGE Molecular weight standard broad range (Ml) (200, 116.25, 97.4, 66.2, 45, 31, 21.5, 14.4, kDa) or Amersham Rainbow Marker (220, 66, 46, 30, 21.5, 14.3 kDa) as standards. As the MW of GST is 26kDa, this value must be added to the MW of each GST-fusion protein.

-64- His-fusion soluble proteins large-scale purification.

A single colony was grown overnight at 37 0 C on a LB Amp agar plate. The bacteria were inoculated into 20ml of LB+Amp liquid culture and incubated overnight in a water bath shaker. Bacteria were diluted 1:30 into 600ml fresh medium and allowed to grow at the optimal temperature (20-37 0 C) to OD 5 so 0.6-0.8. Protein expression was induced by addition of 1 mM IPTG and the culture further incubated for three hours. The culture was centrifuged at 8000 rpm at 4 0 C, the supernatant was discarded and the bacterial pellet was resuspended in 7.5ml cold 10mM imidazole buffer (300 mM NaC1, 50 mM phosphate buffer, mM imidazole, pH The cells were disrupted by sonication on ice for 30 sec at using a Branson sonifier B-15, frozen and thawed two times and centrifuged again. The supernatant was collected and mixed with 1501l Ni2+-resin (Pharmacia) (previously washed with 10mM imidazole buffer) and incubated at room temperature with gentle agitation for minutes. The sample was centrifuged at 700g for 5 minutes at 4 0 C. The resin was washed twice with 10 ml cold 10mM imidazole buffer for 10 minutes, resuspended in lml cold imidazole buffer and loaded on a disposable column. The resin was washed at 4 C with 2ml cold 10mM imidazole buffer until the flow-through reached the O.D 280 of 0.02- 0.06. The resin was washed with 2ml cold 20mM imidazole buffer (300 mM NaCI, 50 mM phosphate buffer, 20 mM imidazole, pH 8) until the flow-through reached the O.D 2 80 of 0.02- 0.06. The His-fusion protein was eluted by addition of 700 l cold 250mM imidazole buffer (300 mM NaCI, 50 mM phosphate buffer, 250 mM imidazole, pH 8) and fractions collected until the O.D 2 8 0 was 0.1. 21 ll of each fraction were loaded on a 12% SDS gel.

His-fusion insoluble proteins large-scale purification.

A single colony was grown overnight at 37 OC on a LB Amp agar plate. The bacteria were inoculated into 20 ml of LB+Amp liquid culture in a water bath shaker and grown overnight. Bacteria were diluted 1:30 into 600ml fresh medium and let to grow at the optimal temperature (37 0 C) to O.D 550 0.6-0.8. Protein expression was induced by addition of 1 mM IPTG and the culture further incubated for three hours. The culture was centrifuged at 8000rpm at 4 0 C. The supernatant was discarded and the bacterial pellet was resuspended in 7.5 ml buffer B (urea 8M, 10mM Tris-HCl, 100mM phosphate buffer, pH The cells were disrupted by sonication on ice for 30 sec at 40W using a Branson sonifier B-15, frozen and thawed twice and centrifuged again. The supernatant was stored at -20 0 C, while the pellets were resuspended in 2 ml guanidine buffer (6M guanidine hydrochloride, 100mM phosphate buffer, 10 mM Tris-HCl, pH 7.5) and treated in a homogenizer for 10 cycles. The product was centrifuged at 13000 rpm for 40 minutes. The supernatant was mixed with 1501l Ni 2 +-resin (Pharmacia) (previously washed with buffer B) and incubated at room temperature with gentle agitation for 30 minutes. The sample was centrifuged at 700 g for minutes at 4 0 C. The resin was washed twice with 10 ml buffer B for 10 minutes, resuspended in Iml buffer B, and loaded on a disposable column. The resin was washed at room temperature with 2ml buffer B until the flow-through reached the OD 28 0 of 0.02-0.06.

The resin was washed with 2ml buffer C (urea 8M, 10mM Tris-HC1, 100mM phosphate buffer, pH 6.3) until the flow-through reached the O.D 2 80 of 0.02-0.06. The His-fusion protein was eluted by addition of 7001l elution buffer (urea 8M, 10mM Tris-HCl, 100mM phosphate buffer, pH 4.5) and fractions collected until the OD 280 was 0.1. 211l of each fraction were loaded on a 12% SDS gel.

His-fusion proteins renaturation glycerol was added to the denatured proteins. The proteins were then diluted to using dialysis buffer I (10% glycerol, 0.5M arginine, 50mM phosphate buffer, reduced glutathione, 0.5mM oxidised glutathione, 2M urea, pH 8.8) and dialysed against the same buffer at 4 0 C for 12-14 hours. The protein was further dialysed against dialysis buffer II (10% glycerol, 0.5M arginine, 50mM phosphate buffer, 5mM reduced glutathione, oxidised glutathione, pH 8.8) for 12-14 hours at 4 0 C. Protein concentration was evaluated using the formula: Protein (mg/ml) (1.55 x OD280) (0.76 x OD 260 Mice immunisations of each purified protein were used to immunise mice intraperitoneally. In the case of some ORFs, Balb-C mice were immunised with AI(OH) 3 as adjuvant on days 1, 21 and 42, and immune response was monitored in samples taken on day 56. For other ORFs, CD1 mice could be immunised using the same protocol. For other ORFs, CD1 mice could be immunised using Freund's adjuvant, and the same immunisation protocol was used, except that the immune response was measured on day 42, rather than 56. Similarly, for still other -66- ORFs, CD1 mice could be immunised with Freund's adjuvant, but the immune response was measured on day 49.

C ELISA assay (sera analysis) The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated t overnight at 37 0 C. Bacterial colonies were collected from the agar plates using a sterile O dracon swab and inoculated into 7ml of Mueller-Hinton Broth (Difco) containing 0.25% C'i Glucose. Bacterial growth was monitored every 30 minutes by following OD620. The 0 bacteria were let to grow until the OD reached the value of 0.3-0.4. The culture was cN centrifuged for 10 minutes at 10000 rpm. The supernatant was discarded and bacteria were washed once with PBS, resuspended in PBS containing 0.025% formaldehyde, and incubated for 2 hours at room temperature and then overnight at 4 0 C with stirring. 100l bacterial cells were added to each well of a 96 well Greiner plate and incubated overnight at 4°C. The wells were then washed three times with PBT washing buffer Tween-20 in PBS). 200 pl of saturation buffer Polyvinylpyrrolidone 10 in water) was added to each well and the plates incubated for 2 hours at 37 0 C. Wells were washed three times with PBT. 200 p1 of diluted sera (Dilution buffer: 1% BSA, 0.1% Tween-20, 0.1% NaN 3 in PBS) were added to each well and the plates incubated for 90 minutes at 37 0 C. Wells were washed three times with PBT. 100 p1 of HRP-conjugated rabbit anti-mouse (Dako) serum diluted 1:2000 in dilution buffer were added to each well and the plates were incubated for 90 minutes at 37°C.

Wells were washed three times with PBT buffer. 100 pl of substrate buffer for HRP (25 ml of citrate buffer pH5, 10 mg of O-phenildiamine and 10 pl of H 2 0) were added to each well and the plates were left at room temperature for 20 minutes. 100 pl H 2

SO

4 was added to each well and OD 490 was followed. The ELISA was considered positive when OD490 was times the respective pre-immune sera.

FACScan bacteria Binding Assay procedure.

The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37 0 C. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into 4 tubes containing 8ml each Mueller-Hinton Broth (Difco) containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by following -67-

OD

62 0 The bacteria were let to grow until the OD reached the value of 0.35-0.5. The culture was centrifuged for 10 minutes at 4000 rpm. The supernatant was discarded and the pellet was resuspended in blocking buffer BSA, 0.4% NaN 3 and centrifuged for 5 minutes at 4000 rpm. Cells were resuspended in blocking buffer to reach OD 620 of 0.07. 100ul bacterial cells were added to each well of a Costar 96 well plate. 100pl of diluted (1:200) sera (in blocking buffer) were added to each well and plates incubated for 2 hours at 4 0 C. Cells were centrifuged for 5 minutes at 4000 rpm, the supematant aspirated and cells washed by addition of 200ld/well of blocking buffer in each well. 100l of R-Phicoerytrin conjugated F(ab) 2 goat anti-mouse, diluted 1:100, was added to each well and plates incubated for 1 hour at 4°C. Cells were spun down by centrifugation at 4000rpm for 5 minutes and washed by addition of 200dl/well of blocking buffer. The supernatant was aspirated and cells resuspended in 200gl/well of PBS, 0.25% formaldehyde. Samples were transferred to FACScan tubes and read. The condition for FACScan setting were: FL1 on, FL2 and FL3 off; FSC-H Treshold:92; FSC PMT Voltage: E 02; SSC PMT: 474; Amp. Gains 7.1; FL-2 PMT: 539. Compensation values: 0.

OMV preparations Bacteria were grown overnight on 5 GC plates, harvested with a loop and resuspended in 10 ml 20mM Tris-HC1. Heat inactivation was performed at 56 0 C for 30 minutes and the bacteria disrupted by sonication for 10' on ice 50% duty cycle, 50% output). Unbroken cells were removed by centrifugation at 5000g for 10 minutes and the total cell envelope fraction recovered by centrifugation at 50000g at 4 0 C for 75 minutes. To extract cytoplasmic membrane proteins from the crude outer membranes, the whole fraction was resuspended in 2% sarkosyl (Sigma) and incubated at room temperature for 20 minutes. The suspension was centrifuged at 10000g for 10 minutes to remove aggregates, and the supernatant further ultracentrifuged at 50000g for 75 minutes to pellet the outer membranes. The outer membranes were resuspended in 10mM Tris-HC1, pH8 and the protein concentration measured by the Bio-Rad Protein assay, using BSA as a standard.

-68- Whole Extracts preparation SBacteria were grown overnight on a GC plate, harvested with a loop and resuspended in iml of20mM Tris-HCl. Heat inactivation was performed at 56 0 C for 30' minutes.

Western blotting SPurified proteins (500ng/lane), outer membrane vesicles (5 p.g) and total cell extracts O (25pig) derived from MenB strain 2996 were loaded on 15% SDS-PAGE and transferred to a nitrocellulose membrane. The transfer was performed for 2 hours at 150mA at 4 0 C, in O transferring buffer (0.3 Tris base, 1.44 glycine, 20% methanol). The membrane was C1 saturated by overnight incubation at 4°C in saturation buffer (10% skimmed milk, 0.1% Triton X100 in PBS). The membrane was washed twice with washing buffer skimmed milk, 0.1% Triton X100 in PBS) and incubated for 2 hours at 37 0 C with 1:200 mice sera diluted in washing buffer. The membrane was washed twice and incubated for 90 minutes with a 1:2000 dilution of horseradish peroxidase labeled anti-mouse Ig. The membrane was washed twice with 0.1% Triton X100 in PBS and developed with the Opti-4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water.

Bactericidal assay MC58 strain was grown overnight at 37 0 C on chocolate agar plates. 5-7 colonies were collected and used to inoculate 7ml Mueller-Hinton broth. The suspension was incubated at 37 0 C on a nutator and let to grow until OD 620 was in between 0.5-0.8. The culture was aliquoted into sterile 1.5ml Eppendorf tubes and centrifuged for 20 minutes at maximum speed in a microfuge. The pellet was washed once in Gey's buffer (Gibco) and resuspended in the same buffer to an OD 620 of 0.5, diluted 1:20000 in Gey's buffer and stored at 25 0

C.

501pl of Gey's buffer/l% BSA was added to each well of a 96-well tissue culture plate. 25Vl of diluted (1:100) mice sera (dilution buffer: Gey's buffer/0.2% BSA) were added to each well and the plate incubated at 4 0 C. 25gl of the previously described bacterial suspension were added to each well. 251l of either heat-inactivated (56 0 C waterbath for minutes) or normal baby rabbit complement were added to each well. Immediately after the addition of the baby rabbit complement, 22pl of each sample/well were plated on Mueller- -69- Hinton agar plates (time The 96-well plate was incubated for 1 hour at 37 0 C with rotation and then 221 of each sample/well were plated on Mueller-Hinton agar plates (time After overnight incubation the colonies corresponding to time 0 and time lh were counted.

The following DNA and amino acid sequences are identified by titles of the following form: m, or a] or pep], where means a sequence from N. gonorrhoeae, "m" means a sequence from N. meningitidis B, and means a sequence from N. meningitidis A; means the number of the sequence; "seq" means a DNA sequence, and "pep" means an amino acid sequence. For example, "g001.seq" refers to an N. gonorrohoeae DNA sequence, number 1. The presence of the suffix or to these sequences indicates an additional sequence found for the same ORF. Further, open reading frames are identified as ORF where means the number of the ORF, corresponding to the number of the sequence which encodes the ORF, and the ORF designations may be suffixed with or indicating that the ORF corresponds to a N. gonorrhoeae sequence or a N. meningitidis A sequence, respectively. Computer analysis was performed for the comparisons that follow between and peptide sequences; and therein the "pep" suffix is implied where not expressly stated.

EXAMPLE 1 The following ORFs were predicted from the contig sequences and/or the full length sequences using the methods herein described.

Localization of the ORFs ORF: contig: 279 gnm4.seq The following partial DNA sequence was identified in N. meningitidis <SEQ ID 2>: m279.seq 1 ATAACGCGGA TTTGCGGCTG CTTGATTTCA ACGGTTTTCA GGGCTTCGGC 51 AAGTTTGTCG GCGGCGGGTT TCATCAGGCT GCAATGGGAA GGTACGGACA 101 CGGGCAGCGG CAGGGCGCGT TTGGCACCGG CTTCTTTGGC GGCAGCCATG 151 GCGCGTCCGA CGGCGGCGGC GTTGCCTGCA ATCACGATTT GTCCGGGTGA 201 GTTGAAGTTG ACGGCTTCGA CCACTTCGCT TTGGGCGGCT TCGGCACAAA 251 TGGCTTTAAC CTGCTCATCT TCCAAGCCGA GAATCGCCGC CATTGCGCCC 301 ACGCCTTGCG GTACGGCGGA CTGCATCAGT TCGGCGCGCA GGCGCACGAG 351 TTTGACCGCG TCGGCAAAAT TCAATGCGCC GGCGGCAACG AGTGCGGTGT 401 ATTCGCCGAG GCTGTGTCCG GCAACGGCGG CAGGCGTTTT GCCGCCCGCT 451 TCTAAATAG This corresponds to the amino acid sequence <SEQ ID 3; ORF 279>: ni279.pep 1 ITRICGCLIS TVFRASASLS AAGFIRLQWE GTDTGSQRAR LAPASLAAAJ4 51 ARPTAAALPA ITICPGELKL TASTTSLWAA SAQMALTCSS SKPRIAAIAP 101 TPCGTADCIS SARRRTSLTA SAKFNAPAAT SAVYSPRLCP ATAAGVPPA 151 SK* The following partial DNA sequence was identified in Ngonorrhoeae <SEQ ID 4>: g279. seq 51 101 251 301 351 401 atgacgcgga aagtttgtcg cc99cagcgg gtgcgtccga 9ttgaagttg tctgcctg~ac acgccttgcg tttgacggca attcgccgag ttt9g9Cgtg gcggCgggtt cagggcgcgt cggcggcgsc acggcttcga ctgttcatct 9tacggcgga tcggcaaaat gctgtgtcc9 cttgatttca acggttttga gtgtttcggc tcatcaggct ttggctccg9 gttgcctgca ccacttcgcc tccaaaccca ctgratcagt ccaatgcttc 9caacggcg9 gcaat99gaa 99aac99ata Cttctttggc ggcag.ccatg atcacgactt gtccgggcga ctgtgcggat tcggcacaaa aAatggccgc cattgcgcct tcggcgcgca ggcggacgag.

9gcggcgaca agcgc9gtgt caggcgtttt gccgcccact 451 tccaaatag This corresponds to the amino acid sequinc<E D5 OF29n> g279-pep ne<E )5.OF29g> 1 MTRICQCLIS TVLSVSASLS AAGFIIRLQWE GTDTGSGRiAR'LAPASLAAAm 51 VRPTAAALPA ITTCPGELO. TASTTSPCAD SAQICLTCSS, sxPJ4AAIAp 101 TPCGTADCIS SAP.RRTSLTA SAKSNASAAT SAVYSPRLCP ATAPAGVPPT 151 SK* ORF 279 shows 89.5% identity over a 152 aa overlap with a predicted ORF (ORF 279.ng) from N. gonorrhoeae: 2030 40 so m2 79..pep ITIrCITFAALAGILWGDGGALpSAAApAAp 9279 MTRCGCLISTVLSVSASLSAAFIRLWETTGSGRAASLAAJVPpTAJALPA 20 30 .40 so 80 90 100 110 120 M279 ,pep IT GLLATSWMOATSSPL

ATC;ACSAMST

g279 XTTcpG.ELIUTASTTSPCADSAQICLTCSSSKPMGAAIAPTPCG;T2CISSARRRTSLTA so -90 100 110 120 130 140 150 M279 .pep SAKFNAATSAVYSPRLCPATAAGVLPPASOC 9279 SAKSNASAATSAVYSPRLCPATAAGVLPPTSKoc 130 140 The following partial DNA sequence was identified in N. -meningitidis <SEQ ID 6>: a279. seq I ATGACNCNGA TTTGCGGCTG CTTGATTTCA ACGGTTTNNA GGGCTTCGGC 51 GAGTTTGTCG GCGGCGGGTT TCATGAGGCT GCAATGGGAA GGTACNGACA 101 CNGGCAGCGG CAGGGCGCGT TTGGCGCCGG CTTCTTTGGC GGCAAGCATA 151 GCGCQCTCGA CGGCGGCGGC ATTGCCTGCA ATCACGACTT GTCCGGGCGA 201 GTTGAAGTTG ACGGCTTCAA CCACTTCATC CTGTGCGGAT TCGGCGCAAA .251 TTTGTTTTAC CTGTTCATCT TCCAAGCCGA GAATCGCCGC CATTGCGCCC 301 ACGCCTTGCG GTACGGCGGA- CTGCATCAGT TCGGCGCGCA NGCGCACGAG 351 TTTGACCGCG TCGGCAAAAT CCAATGCGCC GGCGGCAACN AGTGCGGTGT RECTIFIED SHEET (RULE 91) ISAIEP -71- 401 ATTtGCCGAN GCTGTGTCCG GCAACGGCGG CAGGCGtTTT GCCGCCCGCT 451 TCCGAATAG This corresponds to the amino acid sequence <SEQ I .D 7; ORF 279.a>: a279. pep 1 MTXICGCLIS TVXRASASLS AAGFMRLQWE GTDTGSGRAR LAPASLAASI 51 ARSTAAALPA ITTCPGELKL TASTTSSCAD SAQICFTCSS SKPRIAAIAP 101 TPCGTADCIS SARXRTSLTA SAKSNAPAAT SAVYSPXLCP ATAAGVLPPA 151 SE* m279/a279, ORFs 279 and 279.a showed a 88.2% identity in 152 aa, overlap 20 30 40 50 m27 9. pep ITR ICGCLISTVFRASASLSAAGFIRLQWEGTDtGSGLAPASLAMAPTA1ALPA a27 9 MTXICGCLISTVXRASASLSAAGFRLQWGTDTGSGLAPASLASIRSTAAPA 20 30 40 50 80 .90 100 110 120 m27 9. pep ITICPGELILTASTTSLWAASAQMALTCSSSKPRIAAIAPTPCGTADCISSARRRTSLTA a27 9 ITTCPGELKLTASTTSSCADSAQICFTCSSSKPRIAAIAPTPCGTADCISSARXRTSLTA 80 90 100. 110 120 130 140 150 m27 9. pep SAKFNAPAATSAVYSPRLCPATAGVLPPASKo( a27 9 SAKSNAPAATSAVYSPXLCPATAAGVLPPASEX 130 140 150 519 and 519-1 gnm7.seq The following partial DNA sequence was identified in N. meningitidis <SEQ ID 8>: m519.seq(pril 1 .TCCGTTATCG GGCGTATGGA GTTGGACAAA ACGTTTGAAG AACGCGACGA 51 AA'rCAACAGT ACTGTTGTTG CGGCTTTGGA CGAGGCGGCC GGGg CTrgGG 101 GTGTGAAGGT TTTGCGT'rAT GAGATTAAAG ACTTGGTTCC GCCGCAAGAA 151 ATCCTTCGCT CAATGCAGGC GCAAATTACT GCCGAACGCG AAAAAkCGCGC 201 CCGTATCGCC GAATCCGAAG GTCGTAAAAT CGAACAAATC AACCTTGCCA 25.1. GTGGTCAGCG CGAAGCCGAA ATCCAACAAT CCGAAGGCGA GGCTCAGGCT 301 GCGGTCAATGCGTCAAATGC CGAGAAAATC GCCCGCATCA ACCGCGCCAA 351 AGGTGAAGCG GAATCCTTGC GCCTTGTTGC CGAAGCCAAT GCCGAAGCCA 401 TCCGTCAAAT TGCCGCCGCC CTTCAAACCC AAGGCGGTGC GGATGCGGTC 451 AATCTGAAGA TTGCGGAACA ATACGTCGCT GCGTTCAACA ATCTTGCCAA 501 AGAAAGCAAT ACGCTGATTA TGCCCGCCAA TGTTGCCGAC ATCGGCAGCC 551 TGATTTCTGC CGGTATGAAA ATTATCGACA GCAGCAAAAC CGCCAAaTAA This corresponds to the amino acid sequence <SEQ ID 9; ORF 519>: m519.pep (partial) 1 .SVIGR4ELD)K TFEERDEINS TVVAALDEAA GAWGVKVLRY EIKDLVPPQE 51 ILRSMQAQIT AEREKR.ARIA ESEGRKIEQI NL.ASGQREAE IQQSEGEAQA 101 AVNASNAEKI ARINRAXGEA ESLRLVAEAN AE-AIROIAAA LQOQGGADAV 151 NLKIAEQYVA AFNNLAKESN TLIMPANVAD IGSLISAGMK The following partial DNA sequence was identified inN gonorrhoeae <SEQ ID g519. seq 1 atggaatttt tcattatctt gttggcagcc gtcgccgttt tcggcttcaa 51 atcctttgtc gtcatccccc agcaggaagt ccacgttgtc gaaaggctcg 72 101 ggcgtttcca tcgcgccctg acggccggtt tgaatatttt gattccctt 151 atcgacCgcg tcgcctaccg ccattcgctg aaagaaatcc ctttagacgt 201 acccagccag gtctgcatca cgcgcgataa tacgcaattg actgttgacg 251 gcatcatcta tttccaagta accgatccca aactcgcctc atacggttcg 301 agcaactaca ttatggcaat tacccagctt gcccaaacga cgctgcgttc 351 cgttatcggg cgtatggagt tggacaaaac gtttgaagaa cgcgacgaaa 401 tcaacagtac cgtcgtctcc gccctcgatg aagccgccgg ggcttggggt 451 gtgaaagtcc tccgttacga aatcaaggat ttggttccgc .cgcaagaaat 501 ccttcgcgca atgcaggcac aaattaccgc cgaacgcgaa aaacgcgccc 551 gtattgccga atccgaaggc cgtaaaatcg aacaaatcaa ccttgccagt 601 ggtcagcgtg aagccgaaat ccaacaatcc gaaggcgagg ctcaggctgc 651 ggtcaatgcg tccaatgccg agaaaatcgc ccgcatcaac cgcgccaaag 701 gcgaagcgga atccctgcgc cttgttgccg aagccaatgc cgaagccaac 751 cgtcaaAttg ccgccgccct tcaaacccaa agcggggcgg atgcggtcaa 801 tctgAagatt gcgggacaat acgttaccgc gttcaaaaat cttgccaaag 851 aagacaatac gcggattaag cccgccaagg ttgccgaaat cgggaaccct 901 aattttcggc ggcatgaaaa attttcgCCa gaagcaaaaa cggccaaata 951 a This corresponds to the amino acid sequence <SEQ ID 11; ORE 5 19.ng>: 9519 .PeP 1 MEFFIILLAA VAVFGFKSFV VIPQQEVHvV ERLGRFHRAL TAGLNILIPF 51 IDRVAYRHSL KEIPLDVPSQ vC-ITRDNTQL TVDGIIYPQV TDPKaLASYGS 101 SNYIMAITQL AQTTLRSVIG RMELDKTFEE RDEINSTVVS ALDEAAGAWG 151 VKVLRYEIKD LVPPQEIIRA MQAQITAERE KRARIAESEG RKIEQINLAS 261 GOREAEIQQS EGEAQAAVNA SNAEKIARIN RAKcGEAESLR LVAEANAEA1N 251 RQIAAALQTQ SGADAVNLKI AGQYVTAFKN LAXEDNTRIK PAKVAEIGNP 301 NFRRHEKFSP EAKTAK* ORE 519 shows 87.5% identity over a 200 aa overlap with a predicted ORE (ORE 5 19.ng) from N. gonorrlioeae.

tn519/g519 20 m519.pep

SVIGRMELDKTFEERDEINSTVALDA

100 110 120 130 140 50 60 70 80 mS19 .pep GAGKLYIDVPELSOQTEERRASGKEIIAGRA 150 160 170 180 190 200 100 110 120 130 140 150 19. pep IQSGAAVANEIRNAGAELLAAAARIALTGAA 210 220 230 240 250 260 160 170 180 190 200 m519 .pep NLIEYAFNAENLMANAIS-SGKISKA 9 NLKIAGYVTAFNLAKEDNTRIKPAK

AEINFRRHEKSPEAKTAK

270 280 290 300 310 The following partial DNA sequence was identified in N. meningitidis <SEQ ID 12>: a519. seq 73

ATGGAATTTT

ATCCTTTGTT

GGCGTTTCCA

ATCGACCGCG

ACCCAGCCAG

GTATCATCTA

AGCAACTACA

CGTTATCGGG

TCAACAGCAC

GTGAAGGTTT

CCTTCGCTCA

GTATCGCCGA

TCATTATCTT

GTCATCCCAC

TCGCGCCCTG

TCGCCTACCG

GTCTGCATCA

TTTCCAAGTA

TTATGGCGAT

CGTATGGAAT

CGTCGTCTCC

TGCGTTATGA

ATGCAGGCC

ATCCGAAGGT

GCTGGCAGCC GTCGTTGTTT AGCAGGAAGT CCACGTTGTC ACGGCCGGTT TGAATATTTT CCATTCGCTG AAAGAAATCC CGCGCGACAA TACGCAGCTG ACCGACCCCA AACTCGCCTC GGTCAGCGCG AAGCCGAAAT GGTCAATGCG TCAAATGCCG GTGAAGCGGA ATCCTTGCGC CGTCAAATTG CCGCCGCCCT

TCTGAAGATTGCGGAACAAT

AAAGCAATAC GCTGATTATG ATTTCTGCCG GTATGAAAAT

TACCCAGCTT

TGGACAAAAC

GCCCTCGATG

GATTAAAGAC

AAATTACTGC

CGTAAAATCG

CCAACAATCC

AGAAAATCGC

CTTGTTGCCG

TCAAACCCAA

ACGTCGCCGC

CCCGCCAATG

TATCGACAGC

GCCCAAACGA

GTTTGAAGAA

AAGCCGCCGG

TTGGTTCCGC

TGAACGCGAA

AACAAATCAA

GAAGGCGAGG

CCGCATCAAC

AAGCCAATGC

GGCGGTGCGG

GTTCAACAAT

TTGCCGACAT

AGCAAAACCG

TCGGCTTCAA

GAAAGGCTCG

GATTCCCTTT

CTTTAGACGT

ACTGTTGACG

ATACGGTTCG

CGCTGCGTTC

CGCGACGAAA

AGCTTGGGGT

CGCAAGAAAT

*AAACGCGCCC

CCTTGCCAGT

CTCAGGCTGC

CGCGCCAAAG

CGAAGCCATC

ATGCGGTCAA

CTTGCCAAAG

CGGCAGCCTG

CCAAATAA

This corresponds to the amino acid sequence <SEQ 13; ORF 519.a>: a519.pep 1 MEFFIILLAA VVVFGFKSFV VIPQQEVHVV ERLGRFHRAL TAGLNILIPF 51 IDRVAYRHSL KEIPLDVPSQ VCITRDNTQL TVDGIIYFQV TDPKLASYGS 101 SNYIMAITOL AQTTLRSVIG RIMELDKTFEE RDEINSTVVS ALDMAGAWG 151 VKVLRYEIKD LVPPQEILRS MQAQITAERE KRARIAESEG RKIEQINLAS 201 GQRE.AEIQQS EGEAQAAVNA SNAEKIARIN RAKGEAESLR LVAEANAEAI 251* RQIAAALQTQ GGADAVNLKI AEQYVAAFNN LAKESNTLIM PANVADIGSL 301 ISAGMKIIDS SKTAK* M519/aS19 OR~s 519 and 519.a. showed a 99.5% identity in 199 aa overlap .m519.peP a51 9 10 20

SVIGRMELDKTFEERDEINSTVAALDEAA

YFQVTDPKLASYGSSNYIMAITQLAQTTLRSVIGRMELDKTFEERDEINSTVSALDEAA

100 110 120 130 140 50 60 70 80 m519 .pep GAGKLYIbVPELSQQTARKAIEERIQNAGRA 150 160 170 180 190 200 100 110 120 130 140 150 m51 9. pep IQQSEGEAQAAVNASNAEKIAI(iRIGEAESLRLVAJMAEAIRQIAALTGGADAV a51 9 IQSGAAVANEIRNAGAELLAAAARIALTGAA 210 220 230 240 250 260 160 170 180 190 200 m51 9. pep NLKIAEQYVAAFNNLAKESNTLIMPANVADIGSLISAGKIIDSSKTAJX a51 9 NLKIAEQYVAAFNNLAKESNTLIMPAN4VADIGSLISAGMKIIDSSKTAKX 270 280 290 300 310 Further work revealed the following DNA sequence identified in N. meningitidis <SEQ ID 14>: m519-1.-seq 74

ATGGAATTTT

ATCCTTTGTT

GGCGTTTCCA

ATCGACCGCG

ACCCAGCCAG

GCATCATCTA

AGCAACTACA

CGTTATCGGG

TCAACAGTAC

GTGAAGGTTT

CCTTCGCTCA

GTATCGCCGA

GGTCAGCGCG

GGTCAATGCG

GTGAAGCGGA

CGTCAAATTG

TCTGAAGATT

AAAGCAATAC

ATTTCTGCCG

TCATTATCTT

GTCATCCCAC

TCGCGCCCTG

TCGCCTACCG

GTCTGCATCA

TTTCCAAGTA

TTATGGCGAT

CGTATGGAGT

TGTTGTTGCG

TGCGTTATGA

ATGCAGGCGC

ATCCGAAGGT

AAGCCGAAAT

TCAAATGCCG

ATCCTTGCGC

CCGCCGCCCT

GCGGAACAAT

GCTGATTATG

GTATGAAAAT

GTTGGTAGCC

AACAGGAAGT

ACGGcCGGTT

CCATTCGCTG

CGCGCGACAA

ACCGACCCCA

TACCCAGCTT

TGGACAAAAC

GCTTTGGACG

GATTAAAGAC

AAATTACTGC

CGTAAAATCG

CCAACAATCC

AGAAAATCGC

CTTGTTGCCG

TCAAACCCAA

ACGTCGCTGC

CCCGCCAATG

GTCGCCGTTT

CCACGTTGTC

TGAATATTTT

AAAGAAATCC

TACGCAGCTG

AACTCGCCTC

GCCCAAACGA

GTTTGAAGAA

AGGCGGCCGG

TTGGTTCCGC

CGAACGCGAA

AACAAATCAA

GAAGGCGAGG

CCGCATCAAC

AAGCCAATGC

GGCGGTGCGG

GTTCAACAAT

TTGCCGACAT

TCGGTTTCAA

GAAAGGCTGG

GATTCCCTTT

CTTTAGACGT

ACTGTTGACG

ATACGGTTCG

CGCTGCGTTC

CGCGACGAAA

GGCTTGGGGT

CGCAAGAAAT

AAACGCGCCC

CCTTGCCAGT

CTCAGGCTGC

CGCGCCAAAG

CGAAGCCATC

ATGCGGTCAA

CTTGCCAAAG

CGGCAGCCTG

TA1U(GACAGiC AGCAAAACCG CCAAATAA .This corresponds to the amino acid sequence <SEQ ID) 15; ORF 5 19-1>: M519-1.

1 MEFFIILLVA VAVFGFKSFV.VIPOQEVHVV

ERLGRFHRAL

.1 IDRVAYRHSL KEIPLDVPSQ VCITRDNTQL TVDGIIYFQV '1 SNYIMAITQL AQTTLRSVIG RMELDKTFEE RDEINSTVVA .1 VKVLRYEIKD LVPPQEILRS MQAQITAERE KRARIAESEG 1 GQREAEIQQS EGEAQAAVNA SNAEKIARIN RAI(GEAESLR '1 RQIAAALQTQ GGADAVNLKI AEQYVAAFNN LAI(ESNTLIM '1 ISAGMKIIDS SKTAK* TAGLN ILlPF

TDPKLASYGS

ALDEAAGAWG

RKIEQINLAS

LVAEANAEAI

PANVADIGSL

The following DNA sequence was identified in N. gonorrhoeae <SEQ II) 16>: g519-1. seq 1 51 1 101 C 151 201 7 251. G 301 351 C 401 T1 *451 G 501 C 551

G

601 G~ 651 G 701 G 751 C 801 TI 851 901 kTGGAATTTT

~TCCTTTGTC

~GCGTTTCCA

TCGACCGCG

~CCCAGCCAG

~CATCATCTA

LGCAACTACA

.GTTATCGGG

CAACAGTAC

.TGAAAGTCC

~CTTCGCGCA

;TATTGCCGA

;GTCAGCGTG

;GTCAATGCG

;CGAAGcCGA

:GTCAAATTG

CTGAAGATT

LAAGCAATAC

LT.TTCTGCCG

TCATTATCTT

GTCATCcCCCC

TCGCGCCCTG

TCGCCTACCG

GTCTGCATCA

TTTCCAAGTA

TTATGGCAAT

CGTATGGAGT

CGTCGTCTCC

TCCGTTACGA

ATGCAGGCAC

ATCCGAAGGC

AAGCCGAAAT

TCCAATGCCG

ATCCCTGCGC

CCGCCGCCCT

GCGGAACAAT

GCTGATTATG

GCATGAAAAT

GTTGGCAGCC

AGCAGGAAGT

ACGGCCGGTT

CCATTCGCTG

CGCGCGATAA

ACCGATCCCA

TACCCAGCTT

TGGACAAAAC

GCCCTCGATG

AATCAAGGAT

AAATTACCC

CGTAAAATCG

CCAACAATCC

AGAAAATCGC

CTTGTTGCCG

TCAAACCCAA

ACGTAGCCGC

CCCGCCAATG

TATCGACAGC

GTCGCCGTTT

CCACGTTGTC

TGAATATTTT

AAAGAAATCC

TACCCAATTG

AACTCGCCTC

GCCCAAACGA

GTTTGAAGAA

AAGCCGCCGG

TTGGTTCCGC

CGAACGCGAA

AACAAATCAA

GAAGGCGAGG

CCGCATCAAC

AAGCCAATGC

GGCGGGGCGG

GTTCAACAAT

TTGCCGACAT

AGCAAAACCG

TCGGCTTCAA

GAAAGGCTCG

GATTCCCTTT,

CTTTAGACGT

ACTGTTGACG

ATACGGTTCG

CGCTGCGTTC

CGCGACGAAA

GGCTTGGGGT

CGCAAGAAAT

AAACGCGCCC

CCTTGCCACT

CTCAGGCTGC

CGCGCCAAAG

CGAAGCCATC

ATGCGGTCAA

CTTGCCAAAG

CGGCAGCCTG

CCAAATAA

This corresponds to the amino acid sequence <SEQ ID 17; ORF 519-1.ng>: g519-1. pep 1 MEFFIILLAA VAVFGFKSFV 1 IDRVAYRHSL KEIPLDVPSQ 1 SNYIMAITQL AQTTLRSVIG 1 VKVLRYEIKD LVPPQEILRA 1 GQREAEIQQs EGEAQAAVNA 1 RQIAAALQTQ GGADAVl4LKI 1 ISAGMKIIDS SKTAI(*

VIPQQEVHVV

VCITRDNTQL

RMELDKTFEE

MQAQITAERE

SNAEKIARIN

AEQYVAAFNN

ERLGRFHRAL

TVDGIIYFQV

RDEINSTVVS

KRARIAESEG

RAI<GEAESLR

LAKESNTLIM

TAGLNILIPF

TDPKLASYGS

ALDEAAGAWG

RKIEQINLAS

LVAEANAEAI

PANVADIGSL

75 m519-i/g519-1 ORE's 51.9-1 arnd 519-1.ng showed a 99.0% identity in 315 aa overlap g519-1 .pep m519-1 g519-1 .pep m51i9-1 g519-1 .pep m51 9-i g519-1 .pep M519-1 g519-1 .pep 19-1 g519-1 .pep m51 9-1 20 1 30 40 50 MEFFI ILLAAVAVFGFKSFVVIPQQEVHVVERLGRFHRALTAGLNILIPFIDRVAYRHSL MEFFI ILLVAVAVFGFKSFVVI PQQEVHVVERLGRFHRALTAGLNILI

PFIDRVAYRHSL

20 30 40 50 80 90 100 110 120 KEIPLDVPSQVCITRDNTQLTVDGI

IYFQVTDPKLASYGSSNYIMITQJQTTLRSVIG

KEI PLDVPSQVCITRDNTOLTVDGI

IYFQVTDPI(LASYGSSNYIMAITQLAQTTLRSVIG

80 90 100 110 120 130 140 150 160 170 180

RMELDKTFEERDEINSTVVSALDEAAGAWGVKVLRYEIKDLVPPQEILRAM.QAQITAERE

RMELDKTFEERDEINSTVVAALDEAAGAWGVKVLRYEIKDLVPPQEILRSMQAQITAERE

130 140 150 160 170 180 190 200 210 220 230 240 190 200 210 220 230 240 250 260 270 280 290 300

LVAEANAIRQIAAALQTQGGADAVNLKIQYVNNAKSNTLIMPAVADIGSL

250 260 270 280 290 300 310

ISAGMKIIDSSKTAKX

ISAGMKII DSSKTAKX 310 The following DNA sequence was identified in N. meningitidis <SEQ ID) 18>: a519-1. seq 1 ATGGAATTTT TCATTATCTT 51 ATCCTTTGTT GTCATCCCAC 101 GGCGTTTCCA TCGCGCCCTG 151 ATCGACCGCG TCGCCTACCG 201 ACCCAGCCAG GTCTGCATCA 251 GTATCATCTA TTTCCAAGTA 301 AGCAACTACA TTATGGCGAT 351 CGTTATCGGG CGTATGGAAT 401 TCAACAGCAC CGTCGTCTCC 451 GTGAAGGTTT

TGCGTTATGA

501 CCTTCGCTCA ATGCAGGCGC 551' GTATCGCCGA ATCCGAAGGT 601 GGTCAGCGCG AAGCCGAAAT 651 *GGTCAATGCG TCAAATGCCG 701 GTGAAGCGGA ATCCTTGCGC 751 CGTCAAATTG CCGCCGCCCr 801 TCTGAAGATT GCGGAACAAT 851 AAAGCAATAC GCTGATTATG 901 ATTTCTGCCG GTATGAAAAT

GCTGGCAGCC

AGCAGGAAGT

ACGGCCGGTT

CCATTCGC.TG,

CGCGCGACAA

ACCGACCCCA

TACCCAGCTT

TGGACAAAAC

GCCCTCGATG

GATTAAAGAC

AAATTACTGC

CGTAAAATCG

CCAACAATCC

AGAAAATCGC

CTTGTTGCCG

TCAAACCCAA

ACGTCGCCGC

CCCGCCAATG

TATCGACAGC

GTCGTTGTTT TCGGCTTCAA CCACGTTGTC GAAAGGCTCG TGAATATTTT GATTCCCTTT AAAGAAATCC CTTTAGACGT TACGCAGCTG ACTGTTGACG AACTCGCCTC ATACGGTTCG GCCCAAACGA CGCTGCGTTC GTTTGAAGAA CGCGACGAAA AAGCCGCCGG AGCTTGGGGT TTGGTTCCGC CGCAAGAAAT TGAACGCGAA AAACGCGCCC AACAAATCAA CCTTGCCAGT GAAGGCGAGG CTCAGGCTGC CCGCATCAAC CGCGCCAAAG AAGCCAATGC CGAAGCCATC GGCGGTGCGG ATGCGGTCAA GTTCAACAAT CTTGCCAAAC TTGCCGACAT CGGCAGCCTG AGCAAAACCG CCAAATAA 76 This corresponds to the amino acid sequence <SEQ ID 19; _ORF 5 19-LIa>: a519-1.pep.

1 MEFE'IILLAA VVVFGFKSEV VIPQQEVHVV ERLGRFHRAL TAGLNILIPF 51 IDRVAYRHSL KEIPLDVPSQ VCITRDNTQL TVDGIIYFQV TDPKLASYGS 101 SNYIMAITQL AQTTLRSVIG RNELDKTFEE RDEINSTVVS ALDEAAGAWG 151 vkVLRYEIKD LVPPQEILRS MQAQITAERE KRARIAESEG RKIEQINLAS 201 GQREAEIQQS EGEAQAAVNA SNAEKIARIN RAKGEAESLR LVAEANAEAI 251 RQIAAALQTQ GGADAVNLKI AEQYVAAFNN LAKESNTLIM.PANVADIGSL 301 ISAGMKIIDS SKTAK* m519-1/a519-1 ORFs 519-1 and 519-lI.a showed a 99. 0% identity in 315 aa overlap 20 30 40 50 a519-1 .pep MEFFIILLAAVVVFGFKSFVVIPQQEVHVVERLGRFHPALTAGLNILIPFIDRVAYRHSL xn519-1 MEFILAAFFSVIQEHVELRHATGNLPIRARS 20 30 40 50 80 90 100 110 120 a519-1 .pep KEIPLDVPSQVCITRDNTQLTVDGIIYFQVTDPKASYGSSNYIMAITQLQTTLRSVIG m51 9-1 KEIPLDVPSQVCITRDNTQLTVD

IIYFQVTDPKLASYGSSNYIMITOJAQTTLRSVIG

80 90 100 110 120 a519-1. .pep M519-1 a519-1 .pep mu519-1 a519-1. pep 19-1 a519-1 .pep M519-1 130 140 150 160 170 180

RMELDKTFEERDEINSTVSALDEAAGAWGVKVLRYEIKDLVPPQEILRSMQAQITAERE

RMELDKTFEERDEINSTVVAALDEAAGAWGVKVLRYEIKDLVPPQEILRSMQAQITER

130 140 150 160 170 180 190 200 210 220 230 240 190 200 210 220 230 240.

250 260 270 280 290 300 LVAANAEAIRQIAAALQTQGGADAVNLKI

QYAANLKSNTLIMPVADIGSL

250 260 270 .280 290 300 310

ISAGMKIIDSSKTAKX

ISAGMKIIDSSKTAKX

310 576 and 576-1 gnm22.seq The following partial DNA sequence was identified in N. meningitidis <SEQ ID) m576.seq.. (partial) 1 ATGCAGCAGG CAAGCTATGC GATGGGCGTG GACATOGGAC GCTCCCTGAA 51 GCAAATGAAG GAACAGGGCG CGGAAATCGA TTTGAAAGTC TTTACCGAAG 101 CCATGCAGGC AGTGTATGAC GGCAAAGAAA TCAAAATGAC CGAAGAGCAG 151 GCTCAGGAAG TCATGATGAA ATTCCTTCAG GAACAACAGG CTAAAGCCGT 201 AGAAAAACAC AAGGCGGACG CGAAGGCCAA TAAAGAAAAA GGCGAAGCCT 77 TTCTGAAAGA AAATGCCGCC AAAGACGGCG TGAAGACCAC TGCTTCCGGC CTGCAATACA AAATCACCAA ACAGGGCGAA GGCAAACAGC CGACCAAAGA CGACATCGTT ACCGTGGAAT ACGAAGGCCG CCTGATTGAC GGTACGGTAT TCGACAGCAG CAAAGCCAAC GGCGGCCCGG TCACCTTCcc TTTGAGCCAA GTGATTCCGG GTTGGACCGA AGgCGTACAG CTTCTGAAAG AAGGCGGCGA AGCCACGTTC TACATCCCGT CCAACCTTGC CTACCGCGAA CAGGGTGCGG GCGACAAAAT CGGTCCGAAC GCCACTTTGG TATTTGATGT GAAACTGGTC AAAATCGGCG CACCCGAAAA CGCGCCCGCC AAGCAGCCGG CTCAAGTCGA CATCAAAAAA GTAAATTAA This correspond Is to the amino acid sequence <SEQ ID 2 1; ORF 5 76>: m576.pep.. (partial) 1 .MQQASYAMGV DIGRSLKQMK EOGAEIDLKV FTEAkdQAVYD GKEIKMTEEO 51 AQEVMMKFLQ EQQAKAVEKH KADAKANKEK GEAFLKENAA KDGVKTTASG 101 LQYKITKQGE GKQPTKDDIV TVEYEGRLID GTVFDSSKAN GGPVTFPLSQ 151 VIPGWTEGVQ LLKEGGEATF YIPSNLAYRE QGAGDKIGPN ATLVFDVKLV 201 KIGAPENAPA KQPAQVDIKK VN* The following partial DNA sequence was identified in N. gonorrhoeae <SEQ ID 22>: g576.seq..(partial) *..atgggcgtgg ggaaatcgat gcaaagaaat ttcctgcagg gaaggccaac aagacggcgt cagggtgaag cgaaggccgc gcggcccggc ggcgtacggc caaccttgcc ccactttggt gcgcccgcca acatcggacg ttgaaagtct caaaatgacc agcagcaggc aaagaaaaag gaagaccact gcaaacagcc ctgattgacg caccttccct ttctgaaaga taccgcgaac atttgacgtg agcagccgga ctccctgaaa ttaccgatgc gaagagcagg taaagccgta gcgaagcctt gcttccggtc gacaaaagac gtaccgtatt ttgagccaag aggcggcgaa agggtgcggg aaactggtca tcaagtcgac caaatgaagg catgcaggca cccaggaagt gaaaaacaca cctgaaggaa tgcagtacaa gacatcgtta cgacagcagc tgattccggg gccacgttct cgaaaaaatc aaatcggcgc atcaaaaaag aacagggcgc gtgtatgacg gatgatgaaa aggcggatgc aatgccgccg aatcaccaaa ccgtggaata aaagccaacg ttggaccgaa acatcccgtc ggiccgaacg acccgaaaac taaattaa This corresponds to the amino acid sequence <SEQ ID 23; ORF 576.ng>: g576.pep.. (partial) 1 .MGVDIGRSLK QMKEQGAEID LKVFTDANQA VYDGKEIKMrT EEQAQEVMMK 51 FLQEQQAKAV EKHKADAKAN KEKGEAFLKE NAAEDGVKTT ASGLQYKITK 101 QGEGKQPTKD DIVTVEYEGR LIDGTVFDSS KANGGPATFP LSQVIPGWTE 151 GVRLLKEGGE ATFYIPSNLA YREQGAGEKI GPNATLVFDV KLVKIGAPEN 201 APAKQPDQVD IKKVl4* Computer analysis of this amino acid sequence gave the following results: Homology with a predicted ORE from N. gonorrhoeae m576/g576 97.2% identity in 215 aa overlap 20 30 40 50 m57 6.pep MQQASYAGVDIGRSLKQKEQGAIDLKVEOAVYDKEIMTEEQAQEVMKFLQ g57 6

MGVDIGRSLKMKEQGAEIDLKVFTDQAVYDGKEITEEQAQEMKF-Q

20 30 40 80 90 100 110 120 m57 6. pep EQQAKAVEKHKADAKNKEKGEAFLKENAKDGVTTASGLQYKITKQGEGKQPTKDDIV g57 6 EQQAKAVEKHKADAKANKEKGEAFLKENADGVKTTASGLQYKITKQGEGKQPTKDDIV 70 80 90 100 110 78 130 140 150 160 170 m576.pep TVEYEGRLIDGTVFDSSKGGVVTFPLSQVIPGWTEGVQLLKEGGEATYIPSNAYRE g5,76 TEYEGRLIDGTVFDSSKANGGPATFPLSQVIPGWTEGVRLLEGGEATFYIPSNAYR 120 130 140 150 160 170 1.90 200 210 220 m57 6. pep QGAGD1<IGPNATLVFDVKLVKIGAPENAPAQPAQVIIKKNX g57 6 OGAGEKIGPNATLVFDVKLVKIGAPENPA<QPDQVDIKKVNX 180 190 200 210 The following partial DNA sequence was identified in N. meningitidis <SEQ ID 24>: a576. seq 1 ATGAACACCA. TTTTCAAAAT CAGCGCACTG ACCCTTTCCG CCGCTTTGGC 51 ACTTTCCGCC TGCGGCAAAA AAGAAkGCCGC CCCCGCATCT GCATCCGAIAC 101 CTGCCGCCGC TTCTTCCGCG CAGGGCGACA CCTCTTCGAT CGGCAGCACG 11ATGCAGCAGG CAAGCTATGC GATGGGCGTG GACATCGGAC GCTCCCTGAA 201 GCAAATGAAG GAACAGGGCG CGGAAATCGA TTTGAAAGTC TTTACCGAAG 251 CCATGCAGGC AGTGTATGAC GGCAAAGAAA TCAAAATGAC CGAAGAGCAG 301 GCTCAGGAAG TCATGATGAA ATTCCTTCAG GAACAACAGG CTAAAGCCGT 351 AGAAAAACAC AAGGCGGACG CGAAGGCCAA TAAAGAAAA1A GGCGAAGCCT 401 TTCTGAAAGA AAATGCCGCC AAAGACGGCG TGAAGACCAC TGCTTCCGGC 451 CTGCAATACA AAATCACCAA ACAGGGCGAA GGCAAACAGC CGACCAAAGA 501 CGACATCGTT ACCGTGGAAT ACGAAGGCCG CCTGATTGAC GGTACGGTAT 551 TCGACAGCAG CAAAGCCAAC GGCGGCCCGG TCACCTTCCC TTTGAGCCAA 601 GTGATTCTGG GTTGGACCGA AGGCGTACAG CTTCTGAAAG AAGGCGGCGA 651 AGCCACGTTC TACATCCCGT CCAACCTTGC CTACCGCGAA CAGGGTGCGG 701 GCGACAAAAT CGGCCCGAAC GCCACTTTGG TATTTGATGT GAAACTGGTC 751 AAAATCGGCG CACCCGAAAA CGCGCCCGCC AAGCAGCCGG CTCAAGTCGA 801 CATCAAAAAA GTAAATTAA This corresponds to the amino, acid sequence <SEQ ID 25; ORF 576.a>: a57 6. pep 1 MNTIFKISAL TLSAALALSA CGKKEAAPAS ASEPAAASSA QGDTSSIGSTr 51 MQQASYAMGV DIGRSLKQMK EQGAEIDLKV FI'EAMQAVYD GKEIKMTEEQ 101 AQEVMMKFLQ.EQQAKAVEKH KADAKANKEK GEAFLKENAA KDGVK'rTASG 151 LQYKITKQGE GKQPTKDDIV TVEYEGRLID GTVFDSSKAN GGPVTFPLSQ 201 VILGWTEGVQ LLKEGGEATF YIPSNLAYRE QGAGDKIGPN ATLVFDVKLV 251 KIGAPENAPA KQPAQVDIKK VNm576/a576 ORFs 576 and 576.a showed a 99.5% identity in 222 aa overlap 7 6. pep a576 10 20

MQQASYAMGVDIGRSLKQEGEIDLKV

CGKKEAAPASASEPAAASSAQGDTSSISTMQQASYMGVDIGRSLKMKEQGEIDLKV

40 50 60 70 50 60 70 80 m57 6. pep FT~QVDKIMEQQVMFQEQKVKKDKNEGALEA a57 6 FTMAYGEKTEAEMKLEQAAEHA~<NEGALEA 100 110 120 .130 140 100 110 120 130 140 150 m57 6. pep KGKTSLYIKGGQTDITJYGLDTFSIGPTPS a57 6 KDGVKTTASGLQYKITKQGEGKPTKDDIVTEYEGRLIDGTVFDSSKANGGPVTFPLSQ 150 160 170 180 190 200 79 .160 170 180 190 200 210 m57 6. pep VIPGWTEGVQLLKEGGEATYIPSNLYREQGAGDKIGPNATLVFDVKLVKIGAPENAPA a57 6 VILGWTEGVQLLKEGGEATFYIPSNAYRQGAGDKIGPNATLVFDVKLVIGAPENAPA 210 220 230 240 250 260 220 m576.pep KQPAQVDIKKVNX a576 KQPAQVDIKKVNX 270 Further work revealed the following DNA sequence identified in N meningitidis <SEQ ID 26>: m57 6-1. seq*

ATGAACACCA

ACTTTCCGCC

CTGCCGCCGC

ATGCAGCAGG

GCAAATGAAG

CCATGCAGGC

GCTCAGGAAG

AGAAAAACAC

TTCTGAAAGA

CTGCAATACA

CGACATCGTT

TCGACAGCAG

GTGATTCCGG

AGCCACGTTC

GCGACAAAAT

AAAATCGGCG.

TTTTCAAAAT

TGCGGCAAAA

TTCTTCCGCG

CAAGCTATGC

GAACAGGGCG

AGTGTATGAC

TCATGATGAA

AAGGCGGACG

CAGCGCACTG

AAGAAGCCGC

CAGGGCGACA

GATGGGCGTG

CGGAAATCGA

GGCAAAGAAA

ATTCCTTCAG

CGAAGGCCAA

ACCCTTTCCG

CCCCGCATCT

CCTCTTCGAT

GACATCGGAC

TTTGAAAGTC

TCAAAATGAC

GAACAACAGG

TAAAGAAAAA

CCGCTTTGGC

GCATCCGAAC

CGGCAGCACG

GCTCCCTGAA

TTTACCGAAG

CGAAGAGCAG

CTAAAGCCGT

AAATGCCGCC AAAGACGGCG TGAAGACCAC TGCTTCCGGC AAATCACCAA ACAGGGCGAA GGCAAACAGC CGACCAAAGA.

ACCGTGGAAT ACGAAGGCCG CCTGATTGAC GGTACGGTAT CAAAGCCAAC GGCGG CCCGG TCACCTTCCC TTTGAGCCAA GTTGGACCGA AGGCGTACAG CTTCTGAAAG AAGGCGGCGA TACATCCCGT CCAACCTTGC CTACCGCGAA CAGGGTGCGG CGGTCCGAAC GCCACTTTGG TATTTGATGT GAAACTGGTC CACCCGAAAA CGCGCCCGCC AAGCAGCCGG CTCAAGTCGA 8 01 CATCAAAAAA GTA-AATTAA This corresponds to the amino acid sequence <SEQ ID 27; ORF 576-1>: m57 6-1. pep 1, MNTIFKISAL TLSAALALSA U1 MQQASYAMGV DIGRSLKQMK )l AQEVMM~KFLQ EQQAKAVEKH U LQYKITKQGE GKQPTKDDIV )l vIPGWTEGVQ LLKEGGEATF i1 KIGAPENAPA KQPAQVDIKK

CGKKEAAPAS

EQGAEI DLKV

KADAKANKEI<

TVEYEGRLID

YIPSNLAYRE

jN* ASEPAAASSA QGDTSSIGST FTEAMQAVYD GKEIKMTEEQ GEAFLKENAA KDGVKTTASG GTVFDSSKAN GGPVTFPLSQ QGAGDKIGPN ATLVFDVKLV The following DNA sequence was identified in N gonorrhoeae <SEQ ID1 28>: g576- seq

ATGAACACCA

ACTTTCCGCC

CTGCCGCCGC

ATGCACCAGG

ACAAATGAAG

CCATGCAGGC

GCCCAGGAAG

AGAAAAACAC

TCCTGAAGGA

CTGCAGTACA

CGACATCGTT

TCGACAGCAG

GTGATTCCGG

.AGCCACGTTC

GCGAAAAAAT

AAAATCGGCG

TTTTCAAAAT

TGCGGCAAAA

TTCTGCCGCG

CAAGCTATGC

GAACAGGGCG

AGTGTATGAC

TGATGATGAA

AAGGCGGATG

AAATGCCGCC

AAATCACCAA

ACCGTGGAAT

CAAAGCCAAC

GTTGGACCGA

TACATCCCGT

CGGTCCGAAC

CACCCGAAAA

CAGCGCACTG

AAGAAGCCGC

CAGGGCGACA

AATGGGCGTG

CGGAAATCGA

GGCAAAGAAA

ATTCCTGCAG

CGAAGGCCAA

AAAGACGGCG

ACAGGGTGAA

ACGAAGGCCG

GGCGGCCcGG

AGGCGTACGG

CCAACCTTGC

ACCCTTTCCG

CCCCGCATCT

CCTCTTCAAT

GACATCGGAC

TTTGAAAGTC

TCAAAATGAC

GAGCAGCAGG

CAAAGAAAAA

TGAAGACCAC

GGCAAACAGC

CCTGATTGAC

CCACCTTCCC

CTTCTGAAAG

CTACCGCGAA

CCGCTTTGGC

GCATCCGAAC

CGGCAGCACG

GCTCCCTGAA

TTTACCGATG

CGAAGAGCAG

CTAAAGCCGT

GGCGAAGCCT

TGCTTCCGGT

CGACAAAAGA

GGTACCGTAT

TTTGAGCCAA

AAGGCGGCGA

GCCACTTTGG TATTTGACGT GAAACTGGTC CGCGCCCGCC AAGCAGCCGG ATCAAGTCGA 801 CATCAAAAAA GTAAATTAA This corresponds to the amino acid sequence <SEQ ID 29; ORF 576-1.ng>: g576-1 .pep 1 MNTIFKISAL TLSAALALSA.CGKKEAAPAS ASEPAAASAA QGDTSSIG.' 51 MQQASYANGV DIGRSLKQM.K EQGAEIDLKV FTDAMQAVYD GKEIKMTEE 101 AQEVMMKFLQ EQQAKAVEKH KADAKANKEK GEAFLKENAA KDGVKTTAS 151 LQYKITKQGE GKQPTKDDIV TVEYEGRLID GTVFDSSKAN GGPATFPLS 201 VIPGWTEGVR LLKEGGEATF YIPSN'LAYRE QGAGEKIGPN ATLVFDVKI 251 KIGAPENAPA KQPDQVDIKK VN*

T

Q

G

Q

V

g576-1/m576-1 ORE's 576-1 and 576-1.ng showed a 97.8% identity in 272 aa overlap g57 6-1. pep m516-1 g576-1 .pep m576-1 g57 6-1. pep mu57 6-1 g57 6-1. pep m57 6-1 g576-1. .pep m57 6-1 20 30 40 50 MNTIFKISALTLSAALALSACGKKEIAPASASEP1XASSAQGDTSSIGSTMQQASYAMGV 20 30 40 50 80 90 100 110 120 DIGRSLKQMKEQGAEI DLKVFTDAMQAVYDGKEIKIMTEEQAQEVKFLQEQ0AKVEKH 80 90 100 110 120 130 140 150. 160 170 180

KADAKANKEKGEAFLKENAAKDGVITTASGLQYKITKQGEGKQPTKDDIVTVEYEGRLID

KADAKANKEKGEAFLKENAAJ(OGVKTTASGLQYKITKQGEGKQPTKDDIVTVEYEGRLID

130 140 150 160 170 180 190 200 210 220 230 240 GTVFDSSKANGGPATFPLSQVI

PGWTEGVRLLKEGGEATFYIPSNLAYREQGAGEKIGPN

GTVFDSSKANGGPVTFPLSQVI

PGWTEGVQLLKEGGEATFYIPSNLAYREQGAGDKIGPN

190 200 210 220 230 240 250 260 270

ATLVFDVKLVKIGAPENAPA<QPDQVDIKKVNX

ATLVFDVKLVKIGAPENAPKQPAQVDIKKVNX

250 260 270 The following DNA sequence was identified in N meningitidis <SEQ ID a57 6-1. seq

ATGAACACCA

ACTTTCCGCC

CTGCCGCCGC

ATGCAGCAGG

GCAAATGAAG

CCATGCAGGC

GCTCAGGAAG

AGAAAAACAC

TTCTGAAAGA

CTGCAATACA

CGACATCGTT

TCGACAGCAG

GTGATTCTGG

AGCCACGTTC

GCGACAAAAT

TTTTCAAAAT CAGCGCACTG TGCGGCAAAA AAGAAGCCGC TTCTTCCGCG CAGGGCGACA CAAGCTATGC GATGGGCGTG GAACAGGGCG CGGAAATCGA AGTGTATGAC GGCAAAGAAA TCATGATGAA ATTCCTTCAG AAGGCGGACG CGAAGGCCAA AAATGCCGCC AAAGACGGCG AAATCACCAA ACAGGGCGAA ACCGTGGAAT ACGAAGGCCG CAAAGCCAAC GGCGGCCCGG GTTGGACCGA AGGCGTACAG TACATCCCGT CCAACCTTGC CGGCCCGAAC GCCACTTTGG

ACCCTTTCCG

CCCCGCATCT

CCTCTTCGAT

GACATCGGAC

TTTGAAAGTC

TCAAAATGAC

GAACAACAGG

TAAAGAAAAA

TGAAGACCAC

GGCAAACAGC

CCTGATTGAC

TCACCTTCCC

CTTCTGAAAG

CTACCGCGAA

TATTTGATGT

CCGCTTTGGC

GCATCCGAAC

CGGCAGCACG

GCTCCCTGAA

TTTACCGAAG

CGAAGAGCAG

CTAAAGCCGT

GGCGAAGCCT

TGCTTCCGGC

CGACCAAAGA

GGTACGGTAT

TTTGAGCCAA

AAGGCGGCGA

CAGGGTGCGG

GAAACTGGTC

-81 751 AAAATCGGCG CACCCGAAAA CGCGCCCGCC AAGCAGCCGG CTCAAGTCGA 801 CATCAAAAAA GTAAATTAA This corresponds to the amino acid sequence <SEQ ID 31; ORF 576-1.a>: a576-l.pep 1 MNTIFKISAL.TLSAALALSA CGKKEAAPAS ASEPAAASSA QGDTSSI 51 MQQASYAI'GV DIGRSLKQMK EQGAEIDLKV FTEAMQAVYD GKEIKMTJ 101 AQEVMMKFLQ EQQAKAVEKH KADAKI NKEK GEAFLKENAA KDGVKTT2 151 LQYKITKQGE GKQPTKDDIV TVEYEGRLID GTVFDSSKAN GGPVTFP] 201 VILGWTEGVQ LLKEGGEATF YIPSNLAYRE QGAGDKIGPN ATLVFDVI 251 KIGAPENAPA KOPA0VDIKK VNl*

GST

D.SG

KLV

a576-1/m576-1 ORFs 576-1 and 576-l.a 99.6% identity in 272 aa overlap a576-1 .pep m57 6-1 a576-l .pep m57 6-1 a57 6-1. pep.

m576-1 a57 6-1. pep.

m576-1 20 30 40 s0 MNT IFKI SALTLSAALALSACGKKEAAPASASEPAAASSAQGDTSS

IGSTMQQASYAMGV

MNTI FKI SALTLSAALALSACGKKEAAPASASEPAAASSAQGDTSS

IGSTMQQASYAMGV

20 30 40 50 80 90 '100 110 120 DIGRSLKQMKEQGAEI DLKVFTEAMQAVYDGKEIKTEEQAQEVMKFLQEQQKVEY1I DIGRSLKQMKEQGAE IDLKVFTEAMQAVYDGKEIKM~TEEQAQEVMMJKFLQEQQAKAJVEKH 80 90 100 110 120 130 140 150 160 170 180

KADAKANKEKGEAFLKENAAKDGVKTTASGLQYKITKQGEGKQPTKDDIVTVEYEGRLID

KADAKANKEKGEAFLKENAAXDGVKTTASGLQYKITKQGEGKQPTKDDIVTVEYEGRLI

D

130 140 150 160 170 180 190 200 210 220 230 240 GTVFDSSKANGGPVTFPLSQVILGWTEGVQLLKEGGEATFYI

PSNLAYREQGAGDKIGPN

GTVFDSSKAN'GGPVTFPLSQVI PGWTEGVQLLKEGGEATFYI PSNLAYREQGAGDKIGPN 190 200 210 220 230 240 250 260 270

ATLVFDVKLVKIGAPENAPAKQPAQVDIK(VNX

ATLVFDVKLVKIGAPENAPAKQPAQVDIKKVNX

250 260 270 gnm43 .seq a576-1 .pep m57 6-1 919 and 919-2 The following partial DNA sequence was identified in N. meningitidis <SEQ ID 32>: M919.seq

ATGAAAAAAT

CCTCGCCGCC

CATCCGTCAT

GGAACGACGG

GTCCCTGCCC

TCCGCCTCGG

TGCGCCCAAG

TTTTGAACGC

ACCTATTCCG CGCCGCCCTG TGCCAAAGCA AGAGCATCCA CAACGGCCCG GACCOOCCOG TCGGCGGCGG CGGGGCCGTC CACTGGGCGG CGCAGGATTT CTGCGCCAAT TTGAAAAACC CCTTTCAAAC CCCCGTCCAT TATTTCACGC CGTGGCAGGT

TACGGCATCG

AACCTTTCCG

TCGGCATCCC

TATACCGTTG

CGCC.AAAAGC

GCCAAGGCTG

TCCTTTCAGG

TGCAGGCAAC

CCGCCGCCAT

CAACCCGACA

CGACCCCGCC

TACCGCACCT

CTGCAATCCT

GCAGGATGTG

CAAAACAGTT

GGAAGCCTTG

82 401 CCGGTACGGT 451 CGGACGGCAC 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301

CTCCGTCCCC

TCAGGCAGAC

CATACCGCCG

CAAAGGCAGG

A.AATCAACGG

GAAGACCCTG

GAAAACCCCG

AACATCCyTA

AAACTCGGAC

TCCGCAACGC

TCCGCGAGCT

ACGCCGC'rGA

CTTGGGTGCG

CCCTCAACCG

GCGGTGCGCG

TGCCGGCAAA

GTATGAAGCC

TACCGGCTAT TACGAACCGG TGCTGAAGGG CGACGACAGG AAGCCCGCTT CCCGATTTAC GGTATTCCCG ACGATTTTAT CTGCCTGCCG GTTTGCGGAG CGGAAAAGCC CTTGTCCGCA GGGAAAAAAC AGCGGCACAA TCGACAATAC CGGCGGCACA ACCTCTCCcG ATTCCCCATC TTTGAAGGAA GCCGCTTCCT CGGCGCGCTT GACGGCAAAG TCGAACTTTT TTTTATGCAC

TCCGGCAAAT

CGTTTCCATC

AAACCTCCAT

CTCGCCGAAG

TGCCGGAAGC

TGGGGGAATA

CCCTTATTTG

CCTGATTATG

TGGATTATTT

CAGAAAACCA

CGAATACCGc

ACATCCGCAT

GGACGCTATA

GCAGGGCATT

TTTTGGGTCA

AGCAATGACG

TGCCGGCGCA

TCGCCACCGC

GCGCAGGATA

TTGGGGATAC

CGGGATATGT

CCGTAA

ACCGCGCGCA CAACAGCAAT CCCCTACCAC ACGCGCAACC CCCCGATACT CGGTTACGCC ATCCAAGGCT CGGGCCGTCT CGGCTATGCC GACAAAAACG TGGCGGATAA GGGCTACCTC AAGTCTTATA TGCGGCAAAA AAACCCCAGC TATATCTTTT GCCCTGTCGG CGCACTGGGC GTCGACCGGC ACTACATTAC CCATCCGGTT ACCCGCAAAG CCGGCAGCGC GATTAAAGGC GGCGACGAAG CCGGCGAACT CTGGCAGCTC CTACCCAACG This corresponds to the amino acid sequence <SEQ ID) 33; ORF 919>: M919.pep MKKYLFRAAL YGIAAAILAA GTTVGGGGAV YTVVPHLSLP CAQAFQTpVH SFOAKQFFER RTAQARFPIY GIPDDFISVP HTADLSRFPI TARTTAIKGR EDPVELFFMH IQGSGRLKTP KLGQTSMQGI KSYMRQNPQR TPLMGEYAGA VDRHYITLGA AVRVDYFWGY GDEAGELAGK CQSKSIOTFP QPDTSVI14GP DRPVGIPDPA HWAAQDFAKS LOS FRLGCAN LKNRQGWQDV YFTPWQVAGN GSLAGTVTGY YEPVLKGDDR LPAGLRSGKA LVRIRQTGKNl SGTIDNTGGT FEGSRFLPYH TRI4QINGGAL DGKAPILGYA SGKYIRIGYA DIQ4EHPYVSI GRYMADKGYL ELAEVLGQNPS YIFFRELAGS SNDGPVGALG PLFVATAHPV TRKALNRLIM AQDTGSAI KG QKTTGYVWQL LPNGMKPEYR P*' The following partial DNA sequence was identified in N. meningitidis <SEQ IID 34>: m919-2. seq 51 51 151 201 251 301 351 401 451 501 .551 601 651 701 751 801 851 901 951 1001 1051 1101 1151

ATGAAAAAAT

CC:TCGCCGCC

CATCCGTCAT

GGAACGACGG

GTCCCTGCCC

TCCGCCTCGG

TGCGCCCAAG

TTTTGAACGC

CCGGTACGGT

CGGACGGCAC

CTCCGTCCCC

TCAGGCAGAC

CATACCGCCG

.CAAAGGCAGG

AAATCAACGG

GAAGACCCTG

GAAAACCCCG

AACATCCCTA

AAACTCGGAC

TCCGCAACGC

TCCGCGAGCT

ACGCCGCTGA

CTTGGGTGCG

CCCTCAACCG

ACCTATTCCG CGCCGCCCTG TGCCAAAGCA AGAGCATCCA

CAACGGCCCG

TCGGCGGCGG

CACTGGGCGG

CTGCGCCAAT

CCTTTCAAAC

TATTTCACGC

TACCGGCTAT.

AAGCCCGCTT

CTGCCTGCCG

GGGAAAAAAC

ACCTCTCCCG

TTTGAAGGAA

CGGCGCGCTT

TCGAACTTTT

TCCGGCAAAT

CGTTTCCATC

AAACCTCCAT

CTCGCCGAAG

TGCCGGAAGC

TGGGGGAATA

CCCTTATTTG

CCTGATTATG

GACCGGCCGG

CGGGGCCGTC

CGCAGGATTT

TTGAAAAACC

CCCCGTCCAT

CGTGGCAGGT

TACGAACCGG

CCCGATTTAC

GTTTGCGGAG

AGCGGCACAA

ATTCCCCATC

GCCGCTTCCT

GACGGCAAAG

TTTTATGCAC

ACATCCGCAT

GGACGCTATA

GCAGGGCATT

TTTTGGGTCA

AGCAATGACG

TGCCGGCGCA

TCGCCACCGC

GCGCAGGATA

TACQGCATCG

AACCTTTCCG

TCGGCATCCC

TATACCGTTG

CGCCAAAAGC

GCCAAGGCTG

TCCTTTCAGG

TGCAGGCAAC

TGCTGAAGGG

GGTATTCCCG

CGGAAAAGCC

TCGACAATAC

ACCGCGCGCA

CCCCTACCAC

CCCCGATACT

ATCCAAGGCT

CGGCTATGCC

TGGCGGATAA

AAGTCTTATA

AAACCCCAGC

GCCCTGTCGG

GTCGACCGC

CCATCCGGTT

CCGGCAGCGC

CCGCCGCCAT

CAACCCGACA

CGACCCCGCC

TACCGCACCT

CTGCAATCCT

GCAGGATGTG

CAAAACAGTT

GGAAGCCTTG

CGACGACAGG

ACGATTTTAT

CTTGTCCGCA

CGGCGGCACA

CAACAGCAAT

ACGCGCAACC

CGGTTACGCC

CGGGCCGTCT

GACAAAAACG

GGGCTACCTC

TGCGGCAAAA

TATATCTTTT

CGCACTGGGC

ACTACATTAC

ACCCGCAAAG

GATTAAAGGC

83 1201 GCGGTGCGCG 1251 TGCCGGCAAA 1301. GTATGAAGCC TGGATTATTT TTGGGGATAC GGCGACGAAG CCGGCGAACT CAGAAAACCA CGGGATATGT CTGGCAGCTC CTACCCAACG CGAATACCGC CCGTAA This corresponds to the amino acid sequence <SEQ ID 35; ORF 919-2>: m919-2 .pep MKPCYLFRAAL YGIAAAILAA GTTVGGGGAV YTVVPHLSLP CAQAFQTPVH SFQAKQFFER RTAQARFPIY GIPDDFISVP HTADLSRFPI TARTTAIKGR EDPVELFFMH IQGSGRLKTP KLGQTSMQGI KSYMRQNPQR TPLM"GEYAGA VDRHYITLGA

CQSKSIQTFP

HWAAQDFAKS

YFI'PWQVAGN

LPAGLRSGKA

FEGSRFLPYH

SGKYIRIGYA

LAEVLGQN PS

PLFVATAHPV

QPDTSVINGP DRPVGIPDPA LQSFRLGCAN LKNRQGWODV GSLAGTVTGY YEPVLKGDDR LVRIRQTGKN SGTI DNTGGT TP.NQINGGAL DGKAPILGYA DKNEHPYVSI GRYMADKGYL YIFFRELAGS SNDGPVGALG TRKALNRLIM AQDTGSAIKG LPNGMKPEYR P* 401 AVRVDYFWGY GDEAGELAGK QKTTGYVWQL The following partial DNA sequence was identified in N.gonorrhoeae <SEQ ID 36>: g919.Seq 1 51 1L01 151 201 251 361 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 *1101 1151 1201 1251 13.01

ATGAAAAAAC

CctcgCCGCC

CATCCGTCAT

GGAACGACGG

GTCCATGCCC

TCCGCCTCGG

TGCGCCCAAG

TTTTGAACGC

Caggtacggt

CGGACGGAAC

CTCCG'rCCCG TCAGGCAGac

CATACCGCCG

caaaGGCAGG AAAtcaacGG GAagaccCcG GAAAACCCcg AACAtCCgT a AAGctcgggc

TCCGCAACGC

TCCGCGAGCT

ACGCCACTGA

CTTGGGCGCG

CCCTCAACCG

GCGGTGCGCG

TGCCGGCAAA

GCATGAAGCC

ACCTGCTCCG CTCCGCCCTG TACG~catCG TGCCAAAgca

CAACGGCCCG

TTGCCGGCGG

CACTGGGCGG

CTGCGCCAAT

CCTTTCAAAC

TATTTCACGC

TACCGGCTAT

GGGCCCGCTT

CTGCCTGCCG

9GgcGAAAAAC

ACCTCTCCCG

TTTGAaggAA CGGCgcgcTT tcgaacttTT tccg9Caaat tgtttccatc agAdCTCGAT

CTCGCCGAAG

TGCCGGAAGC

TGGGGCGAATA

CCCTTATTTG

CCTGATTATG

TGGATTATTT

CAGAAAACCA

CGAATACCGC

gGAGCATCCA AACCTTTCCG GACCGGCCGG CCGGCATCCC CCGCCgccAT

CAACCCGACA

CGACCCCGCC

CGGGGCCGTC

CGCaggATIT

TTGAAAAACC

CCCCGTGCAT

cgtGGCaggt

TACGAACCGG

CCCGATTTAC

GTTTGCGGGG

AGCGGCACGA

ATTCCCCATC

GCCGCTTCCT

GACGGCAAag

TTTCATGCAC

acatCCGCAt ggACGctaTA GCAGGgcatc

TTTTGGGTCA

GGCAATGAGG

CGCCOGCGCA

TCGCCACCGC

GCGCAGGATA

TTGGGGTTAC

CGGGATACGT

CCGTGA

TATACCGTTG TGCCGCACCT TGCCAAAAGC CTGCAATCCT GCCAAGGCTG GCAGGATGTG TCCTTTCAG3G CAAAGcGgTT tgcaggcaAC GGAAGCCTTG TGCTGAAGGG CGACGGCAGG GGTATTCCCG ACGATTTTAT CGGAAAAAAC CTTGTCCGCA TCGACAATGC CGGCGGCACG ACCGCGCGCA CAACGGcaat CCCTTACCAC ACGCGCAACC cccCCATCCT CggttacgcC AtccaaggCT CGGGCCGCCT cggaTacgcc gacAAAAACG TGGCGGACAA AGGCTACCTC aaagcCTATA TGCGGCAAAA AAACCC.CAGC TATATCTTTT GCCCCGTCGG CGCACTGGGC ATCGACCGGC ACTACATTAC CCATCCGGTT ACCCGCAAAG CAGGCAGCGC GATCAAAGGC GGCGACGAAG CCGGCGAACT CTGGCAGCTC CTGCCCAACG This corresponds to the amino acid sequence <SEQ ID1 37; ORF 919.ng>: g919 .pep MKKH-LLRSAL YGIAAAILAA GTTVAGGGAV YTVVPHLSMP CAQAFQTPVH- SFQAXRFFER RTERARFPIY GIPDDFISVP MTADLSRFPI TARTTAIKGR EDPVELFFMH IQGSGRLKTP KLGQTSMQGI KAYMRQNPQR TPLMGEYAGA IDRHYITLGA AVRVDYFWGY GDEAGELAGK

CQSRSIOTFP

HWAAQDFAKS

YFTPWQVAGN

LPAGLRGGKN

FEGSRFLPYH

SGKYIRIGYA

LAEVLGQNPS

PLFVATAHPV

OKTTGYVWQL

QPDTSVINGP

LQS FRLGCAN

GSLAGTVTGY

LVRIRQTGQN

TRNQINGGAL

DKNEHPYVSI

YIFFRELAGS

TRXALNRLIM

LPNGMKPEYR

DRPAGI PDPA LIG4ROGWQDV

YEPVLKGDGR

SGTIDNAGGT

DGKAPILGYA

GRYMAflKGYL

GNEGPVGALG,

AQDTGSAIKG

84 ORE 919 shows 9 5.9 identity over a 441 aa, overlap with a predicted ORF (ORE 919.ng) from N. gonorrhoeae: M919/g919 20 30 40 s0 m919 .pep MKKYLFRAALYGIAAAILAACOSKSIQTFPQPDTSVINGPDRPVGIPDPAGT..JGOGAV g91 9 MKKHLLRSALYGIAAAILAACQSRSIQTFPQPDTSVINGPDRPAGI

PDPAGTTVAGGG-AV

20 30 40 50 80 90 100 110 120 M91 9. pep YTVHSPW~DASQFLCNLNQWDCQFTVSQKFE 80 90 100 110 120 130 140 150 160 170 180 m919 .pep YFTPWQVAGNGSLAGTVTGYYEPVLKGDDRTAQRFPIYGIPDD)FISVPLPAGLRSGKA 9919 YFTPWQVAGNGSLAGTVTGYYEPKDGRTEARFPIYGIPDDFISVPLPAGLRGKN 130 140 iso 160 170 180 190 200 20 220 230 240 m919 .pep LVIOGNGXNWHALRPTRTIGFGRLYMQNCk g9 19 LVIQGNGINGTTDSFIATJIGFGRLYTNIGA 190 200 210 .220 230 240 250 260 270 280 290 300 m9 19 .pep DGKAPILGYAEDPVELFFHIQGSGR1KTPSGKYIRIGYADKNEHPYVSIGRY?.DKGYL 250 260 270 280 290 300 310 320 330 340 350 360 m919 .1pep KLQSQISMQPRAVGNSYFRLGSDPGLTLGYG g9 19 KLOSQIAMQPRAVGNSYFRLGGEPGLTLGYG 310 320 330 340 350 360 3.70 380 390 400 410 420 m9 19. pep VDHILALVTHVRANLMQTSIGVVYWYDAEM 370 380 390 .400 410 420 430 440 m919 .pep QKTTGYVWQLLPNGMKPEYRPX 9919 QKTTGYVWQLLPNGMKPEYRPX 430 440 The following partial DNA sequence was identified in Nmeningitidis <SEQ 11) 38>: .a919. seq 85 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301

ATGAAAAAAT

CCTCGCCGCC

CATCCGTCAT

GGAACGACGG

GTCCCTGCCC

TCCGCCTCGG

TGCGCCCAAG

TTTTGAACGC

.CCGGTACGGT

CGGACGGCAC

CTCCGTCCCC

TCAGGCAGAC

CATACCGCCG

CAAAGGCAGG

AAATCAACGG

GAAGACCCCG

GAAAACCCCG

AACATCCCTA

AAGCTCGGGC

CCCGCAACGC

TCCGAGAGCT

ACGCCGCTGA

CTTGGGCGCG

CCCTCAACCG

GCGGTGCGCG

TGCCGGCAAA

GTATGAAGCC

ACCTATTCCG

TGCCAAAGCA

CAACGGCCCG

TCGGCGGCGG

CACTGGGCGG

CTGCGCCAAT

CCTTTCAAAC

TATTTCACGC

TACCGGCTAT

AAGCCCGCTT

CTGCCTGCCG

GGGAAAAAAC

ACCTCTCCCA

TTTGAAGGAA

CGGCGCGCTT

TCGAACTTTT

TCCGGCAAAT

CGTTTCCATC

AGACCTCGAT

CTCGCCGAAG

TACCGGAAGC

TGGGCGAGTA

CCCTTATTTG

CCTGATTATG

TGGATTATTT

CAGAAAACCA

CGAATACCGC

CGCCGCCCTG

AGAGCATCCA

GACCGGCCGG

CGGGGCCGTT

CGCAGGATTT

TTGAAAAACC

CCCCGTCCAT

CGTGGCAGGT

TACGAGCCGG

CCCGATTTAC

GTTTGCGGAG

AGCGGCACAA

ATTCCCCATC

GCCGCTTCCT

GACGGCAAAG

TTTTATGCAC

ACATCCGCAT

GGACGCTATA

GCAGGGCATC

TTTTGGGGCA

AGCAATGACG

CGCCGGCGCA

TCGCCACCGC

GCGCAGGATA

TTGGGGATAC

CGGGATATGT

CCGTAA

TGCGGCATCG

AACCTTTCCG

TCGGCATCCC

TATACCGTTG

CGCCAAAAGC

GCCAAGGCTG

TCCGTTCAGG

TGCAGGCAAC

TGCTGAAGGG

GGTATTCCCG

CGGAAAAGCC

TCGACAATAC

ACTGCGCGCA

CCCCTACCAC

CCCCGATACT

ATCCAAGGCT

CGGCTATGCC

TGGCGGACAA

AAAGCCTATA

AAACCCCAGC

GCCCTGTCGG

GTCGACCGGC

CCATCCGGTT

CCGGCAGCGC

GGCGACGAAG

CTGGCAGCTT

CCGCCGCCAT

CAACCCGACA

CGACCCCGCC

TGCCGCACCT

CTGCAATCCT

GCAGGATGTG

CAAAACAGTT

GGAAGCCTTG

CGACGACAGG

ACGATTTTAT

CTTGTCCGCA

CGGCGGCACA

CAACGGCAAT

ACGCGCAACC

CGGTTACGCC

CGGGCCGTCT

GACAAAAACG

AGGCTACCTC

TGCAGCAAAA

TATATCTTTT

CGCACTGGGC

ACTACATTAC

ACCCGCAAAG

GATTAAAGGC

CCGGCGAACT

CTGCCCAACG

This corresponds to the amnino acid sequence <SEQ ID) 39; OR" 919.a>: a919 .pep 1 MKKYLFRAAL CGIAAAILAA CQSKSIQTFP QPDTSVINGP DRPVGI 51 GTTVGGGGAV YTVVPHLSLP HWAAQDFAKS.LQSFRLGCAN LKNRQC 101 CAQAFQTPVH SVQAXQFFER YFTPWQVAGN GSLAGTVTGY YEPVIJ 151. RTAQARFPIY GIPDDFISVP LPAGLRSGKA LVRIRQTGKN SGTIDb 201 HTADLSQFPI TARTTAIKGR.FEGSRFLPYH TRNQINGGAL DGKAP) 251 EDPVELFFMH IQGSGRLKTP SGKYIRIGYA DKNEHPYVSI GRYMAI 301 KLGQTSMQGI KAYMQQNPQR LAEVLGQNPS YIFFRELTGS SNDGPN 351 TPLM'GEYAGA VDRHYITLGA PLFVATAHPV TRKALNRLIM AQDTGI 401 AVRVDYFWGY GDEAGELAGK QKTTGYVWQL LPNGMKPEYR P*

PDPA

;WQDV

(GDDR

JTGGT

ELGYA

)KGYL

TGALG

AIKG

m919/a9l9 ORFs 919 and 919.a showed a 98.6% identity in 441 aa overlap 20 30 40 50 m919 .pep MKIYLFRAALYGIAAAILAACQSKSIQTFPQPDTSVINGPDRPVGIPDPAGTTVGGGGAV *a91 9 MKKYLFRAALCGIAAAILAACQSKSIQTFPQPDTSVINGPDRPVGIPDPAGTTVGGGGAV 20. 30 40 50 80 90 100 110 120 m9 19. pep YTVVPHLSLPHWAAQDFAKSLQSFRLGCAN.LKNROGWQDvcAQAFQTPVHSFQAJKQFFER 80 90 *100 110 120 130 140 150 160 170 180 m919 .pep YFTPWQVAGNGSLATVTGYYEPVLKGDDRRTAQARFPIYGIPDDFISVPLPAGLRSGKA a919 YFTPWQVAGNGSLAGTVTGYEPVLKGDDRRTAQARFPIYGI

PDDFISVPLPAGLRSGKA

130 140 150 160 170 180 190 .200 210 220 230 240 m919 .pep LVRIRQTGKNSGTIDNTGGTHTADLSRFPITARTTAIKGRFEGSRFLPYHTRNQINGGAL 86 a91 9 LVRIRQTGKNSGTIDNTGGTHTADLSQFPITARTTAIKGRFEGSRFLPYHTRNQINGGAL 190 200 210 220 230 240 250 260 270 280 290 300 m919 .pep DGKAPILGYAEDPVELFFMHIQGSGRLKTPSGKYIRIGYADKNEHPYVSIGRYMI3DKGYL a91 9 DGKAPILGYAEDPVELFFMHIQGSGRLKTPSGKYIRIGYADNEHPYVSIGRYADKGYL 250 260 270 280 290 300 310 320 330 340 350 360 m91 9. pep KLGQTSMQGIKSYMRQNPQRLAEVLGQNPSYIFFRELAGSSNDGPVGALGTPLMGEYAGA a919, KLGQTSMQGIKAYMQQNPQRLAEVLGQNPSYIFFRELTGSSNDGPVGALGTPLMGEYAGA 310 320 330 340 350 360 370 30 390 400 410 420 m919 .pep VDRHYITLGAPLFVATAHPVTRKLNRLIMAQDTGSAIKGAVRVDYFWGYGDEAGELAJGK a91 9 VDRHYITLGAPLFVATAHPVTRKALNRLIMAQDTGSAIKGAVRVDYFWGYGDEAGELAGK 370 380 390 400 410 420 430 440 m91 9. pep QKTTGYVWQLLPNGMKPEYRPX a919 QKTTGYVWQLLPN~GMKPEYRPX 430 440 121 and 121-1 The following partial DNA sequence was identified in N. meningitidis <SEQ 11)40>: m121. seq ATGGAAACAC AGCTTTACAT GGCGGATGCC GTACTGATAC AAGGGCACGC CTTTACCCCC GATTTGCAGG ACACAGGCGC GCAAGAACTC AGCCGCCTAT 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101

GTCAAAACCT

ACCGTCCGAC

GCCGCTGCTG

xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxCAGC

CATATTGCCG

AACGCCACCC

GAAACCTACC

TTCCCGTTTT

CAGATGCCCG

TTAATGGCGG

CACCGCCGAC

CGTGGTTGGC

GCAACCGGCG

A-

CGCACCGTCC

ACGCGCCGGA

GCGxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx

TTCCTTACGA

CAACTGCTCG

TAAAAGCACG

TTGACGGCGG

ACCGCGCAAA

TCAAATGTAC

CGGCATCATG

GGATGGACGG

TACCCCGGCA

AGACGAACTG

ATGCGCAAAC

GACATTACCG

ACACGGTTAC

xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx

CAAAAACGGT

ACAGGCTGCT

GGGCiGCGAAC

CGAAAACCGA

CCGTTTGCGA

ATTTGCGACG

TCGGGAACCA GCATGGACGG CGGCAAATGG CTGGGCGCGG GGTTACGCCG CCAATTGCTG CACCGCAGCA GGATTTTGTC CGCCGCCGAA CTGCTGTGCA CCCTCGGCTG CCACGGGCAA AGCATACAGC TTGCCGATTT xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx GCAAAGTCGG CACAAGGCAA CGCCCACCCG TATTTCGCAC TGTTTGCCAT AAATTGGCTC TACGACGTAT TGCGGACGCT CGCCGTCTCA CACGCAGCGG GCGGCATCCG CAATCCTGTT ACACGCGTTT CCCTGCACAG GGTGGAAGCC GCCGnATTTG TTCCCGGTAG TCCGCACAAA ATTTGGCAGA ATGTTTCGGC CTGAACCTCG ATCCGCAATG GGCGTGTTGG ATTAATCGCA CATCCAAACC GTGTATTCTG AnCGCGGGAT ATTATTATTG This corresponds to the amino acid sequence <SEQ ID 41; OR" 121>: m3.2l.pep 1 METOLYIGIN SGTSMDGADA VLIRMDGGKW LGAEGHAFTP YPGRLRRQLL 51 DLODTGADEL HRSRILSQEL SRLYAQTAAE LLCSQNLAPS DITALGCHiGQ 87

TVRHAPEHGY

xxxxxxxxxx xxQLPYDKNG

ETYLDGGENR

LMADLAECFG

ATGASKPCIL

SIQLADLPLL

xxxxxxxxxx

AKSAQGNILP

YDVLRTLSRF

TRVSLHSTAD

XAGYYY*

Axxxxxxxxx

XXXXXXXXXX

QLLDRLLARP

TAQTVCDAVS

LNLDPQWVEA

xxxxxxxxxx xxxxxxxxxx

YFAQRHPKST

HAAADARQMY

AXFAWLAACW

xxxxxxxxxx xxxxxxxxxx

GRELFAINWL

ICDGGIRNPV

INRI PGSPHK The following partial DNA sequence was identified in N. gonorrhoeae <SEQ ID 42>: g12 1. seq 1 51 101 151 201 251 301 351 401 451 501 551 601.

651 701 751 801 851 901 951 1001 1051 ATGGAAACAC AGCTTTACAT CGGCATTATG TCGGGAACCA GGCGGATGCC GTGCTGGTAC GGATGGACGG CGGCAAATGG AAGGGCACGC CTTTACCCCC TACCCTGACC GGTTGCGCCG GATTTGCAGG ACACAGGOAC AGACGAACTG CACCGCAGCA GCAAGAACTC AGCCGCCTGT ACGCGCAAAC CGCCGCCGAA GTCAAAACCT CGCTCCGTGC GACATTACCG CCCTCGGCTG ACCGTCCGAC ACGCGCCGGA ACACGGTtac AGCATACAGC GCCGCTrGCTG GCGGAACT~a cgcggatttT TACCGTCggc GCCGCGACCT TGCTGCCGCC GGacaAGGTG CGCCGCTCGT CACGAAGCCC TGTTCCGCGA TGACAGGGAA ACACGCGTGG CGGCGGGATT GCCAACATCA GCGTACTCCC CCCCGGCGCA GCTTCGACAC AGGGCCGGGC AATATGCTGA TGGAcgcgtg cacTGGcagc TGCCTTACGA CAAAAacggt gcAAAGgcgg cAtatTGCcg cAACTGCTCG gcaggctGCT CGCCcaccCG AACCCcaccc aaAAAGCACG GGgcGCGaac TgtttgcccT gaaacctAcc ttgacggcgg cgqaaaaccga tacgacgtat ttcccgattc accgcgcaaA ccgTttggga cgccgtctca CAGATGCCCG TCAAATGTAC ATTTGCGGCG GCGGCATCCG TTAATGGCGG ATTTGGCAGA ATGTTTCGGC ACACGCGTTT CACCGCCGAA CTGAACCTCG ATCCTCAATG GGTGGAGGCG cgtggttggC GGCGTGTTGG ATTAACCGCA TTCCCGGTAG GCGACCGGCG CATCCAAACC GTGTATTCTG GGCGCGGGAT

GTATGGACGG

CTGGGCGCGG

CAAATTGCTG

GGAiTGTTGTC

CTGCTGTGCA

CCACGGGCAA

TTGCCGATTT

gacttcCGCA

CCCCGCCTTT

TACTGAACAT

CCCGCCTTCG

gacgcaggca cacAAGGCAA

TATTTCTCAC

AAattggctc tgcggacgct

CACGCAGCGG

CAATCCTGTT

CCCTGCACAG

gccgCATTtg

TCCGCACAAA

ATTATTATTG

1101 A This corresponds to the amino acid sequence <SEQ ID 43; ORF 121.ng>: g12, -pep

METQLYIGIM

DLQDTGTDEL

TVRHAPEHGY

HEALFRDDRE

HWQLPYDKNG

ETYLDGGENR

iLMADLAECFG

ATGASKPCIL

SGTSMDGADA

HRSRMLSQEL

SIQLADLPLL

TRVVLNIGGI

AKAAQGN IL P

YDVLRTLSRF

TRVSLHSTAE

GAGYYY*

VLVRMDGGKW

SRLYAQTAAE

AELTRIFTVG.

ANISVLPPGA

QLLGRLLAHP

TAQTVWDAVS

LNLDPQWVEA

LGAEGHAFrP

LLCSONLAPC

DFRSRDLAAG

PAFGFDTGPG

YFSQPHPKST

HAAADARQMY

AAFAWLAACW

YPDRLRRKLL

DITALGCHGQ

GQGAPLVPAF

NMLMDAWTQA

GRELFALNWL

ICGGGIRNPV

INRIPGSPHK

ORF 12 1 shows 73.5% identity over a 366 aa. overlap with a predicted ORF (ORF12I.ng) from N. gonorrhoeae: ml2l/gl21 20 30 40 50 m121 .pep METQLYIGIMSGTSMDGADAVLIRMDGGKWLGEGJIJFPYPGRLRRQLLLDTGADEL g12 1 METQLYIGIMSGTSMDGADAVLVRMDGGKWLGAGHAFTPYPDRLRJ(LLDLQDTGTDEL 20 30 40 50 80 90 100 110 120 m121 .pep HRSRILSQELSRLYAQTAAELLCSQNLAPSDITALGCHGQTVRHAPEHGYSIQLADLPLL g12 1 HRSRMLSQELSRLYAQTAA2LLCSNLAPCDITALGCHGQTVRHAJPEHGYSIQLADLPLL 80 90 100 .110 120 130 140 150 160 170 180 88 ml 21.pep AXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX g12 1 AELTRIFTVGDFRSRDLAAGGGAPLVPAFHELFRDDRETRVLNIGGIANISVLPPGA 130 140 150 160 170 180 190 200 210 220 230 240 m121 .pep XXXXXXXXXXXXXXXXXXXXQLPYDKGASAQGNILPQLLDRLLAPYFAQRHPKST g 121 PAFGFDTGPGNML14DAWTQAHWQLPYDKNGAKAQGNILPQLLGRLL.11PYFSQPHPKST 190 200 210 220 230 240 250 20 270 280 290 300 ml2 1.pep GRELFAINWLETYLDGGENRYDVLRTLSRFTAQTVCDAVSAADAQMYICDGGIRNPV g12 1 GRELFALNWLETYLDGGENRYDVLRTLSRFAQTVWDAVSHAJ~AADARQMYICGGGIRNPV 250 260 270 280 290 300 310 320 330 340 350 360 m 121 .pep LMDACGRSHTDNDQVAAFWACIRPSHAGSPI g121 LMADLAECFGTRVSLHSTAELNLDPQWVEAFAWLAAJCWINRIPGSPHKAYTGASKPCIL 310 320 330 340 350 360 m121.pep XAGYYYX g121 GAGYYYX The follow ing partial DNA sequence was identified in N. meningitidis <SEQ ID 44>: a12l. seq 51 101 151 201 251 301 *351 401 451 501 *551 601 651 701 751 801 851 901 951 1001 1051 1101

ATGGAAACAC

GGCGGATGCC

AAGGGCACGC

GATTTGCAGG

GCAAGAACTC

GTCAAAACCT

ACCGTCAGAC

GCCGCTGCTG

GCCGCGACCT

CACGAAGCCC

CGGCGGGATT

6CTTCGACAC

CACTGGCAGC

CATATTGCCG

AACCCCACCC

GAAACCTACC

TTCCCGATTC.

CAGATGCCCG

TTAATGGCGG

CACCGCCGAA

CATGGATGGC

GCAACCGGCG

A

AGCTTTACAT

GTACTGA:TAC

CTTTACCCCC

ACACAGGCGC

AGCCGCCTGT

CGCGCCGTCC

ACGCGCCGGA

GCGGAACGGA

TGCGGCCGGC

TGTTCCGCGA

GCCAACATCA

AGGACCGGGC

TTCCTTACGA

CAACTGCTCG

TAAAAGCACG

TTGACGGCGG

ACCGCGCAAA

TCAAATGTAC

ATTTGGCAGA

CTGAACCTCG

GGCGTGTTGG

CATCCAAACC

CGGCATCATG

GGATGGACGG

TACCCCGGCA

GGACGAACTG

ACGCGCAAAC

GACATTACCG

ACACAGTTAC

CTCAGATTTT

GGACAAGGCG

CGACAGGGAA

GCGTACTCCC

AATATGCTGA

CAAAAACGGT

ACAGGCTGCT

GGGCGCGAAC

CGAAAACCGA

CCGTTTTCGA

ATTTGCGGCG

ATGTTTCGGC

ATCCGCAATG

GTCAACCGCA

GTGTATTCTG

TCGGGAACCA GCATGGACGG CGGCAAATGG CTGGGCGCGG GGTTACGCCG CAAATTGCTG CACCGCAGCA GGATGTTGTC CGCCGCCGAA CTGCTGTGCA CCCTCGGCTG CCACGGGCAA AGCGTACAGC TTGCCGATTT TACCGTCGGC GACTTCCGCA CGCCGCTCGT CCCCGCCTTT ACACGCGCGG TACTGAACAT CCCCGACGCA CCCGCCTTCG TGGACGCGTG GATGCAGGCA GCAAAGGCGG CACAAGGCAA CGCCCACCCG TATTTCGCAC TGTTTGCCCT AAATTGGCTC TACGACGTAT TGCGGACGCT CGCCGTCTCA CACGCAGCGG GCGGCATCCG CAATCCTGTT ACACGCGTTT CCCTGCACAG GGTAGAAGCC GCCGCGTTCG TTCCCGGTAG TCCGCACAAA GGCGCGGGAT ATTATTATTG This corresponds to the amino acid sequence <SEQ ID 45; ORF 121.a>: al2l. pep 1 METQLYIGIM SGTSMDGADA VLIRMDGGKW LGAEGHAFTP YPGRIJ 51 DLQDTGADEL HRSRMLSQEL SRLYAQTAAE LLCSQNLAPS DITALC 101 TVRHAPEHSY SVQLADL PLL AERTQIFTVG DFRSRDLAAG GQGAPI 151 HEALFRDDRE TRAVLNIGGI AISVLPPDA PAFGFDTGPG NMLMW 201 HWQLPYDKNG AKAAQGNILP QLLDRLLAHP YFAQPHPKST GRELF; 251 ETYLDGGENR YDVLRTLSRF' TAQTVFDAVS HAAADARQMY ICGGGI 301 LMADLAECFG TRVSLHSTAE LNLDPOW'JEA AFAWMAAC JOT

~KLL

CHGQ

WPAF

~WMQA

~LNWL

iuqPV

;SPHK

89 351 ATGASKPCIL GAGYYY* m12I/a121 ORFs 121 and 121.a 74.0% identity in 366 aa overlap 20 n0 40 50 m121 .pep METQLYIGIMSGTSMDGADAVLIRDGGKWLGAEGHAFPYPGRLRRQLLDLQDTGADEL a121 MEQYGMGSDAALRDGWLAGATYGLRLDQTAE 20 30 40 50 80 90 .100 110 120 m12 1. pep HRSRILSQELSRLYAOTAAELLCSQNLAPSDITLGCHGQTVRJAPEHGYSIQLADLPLL a12 1 HRSRMLSQELSRLYAQTAAELLCSQNLAPSDITALGCHGQTVRHAPEHSYSVQJADLPLL 80 90 100 110 120 130 140 150 160 170 180 ml 21.pep AXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX a121 AERTQIFTVGDFRSRDLAGGQGAPLVPAFHEALFDDRETJ

VLNIGGIANISVLPPDA

130 140 150 160 170 180 190 200 210 220 230 240 m12l1..pep XXXXXXXXXXXXXXXXXXXXXQLPYDKNGSAQGNILPLLRLIJHPYFAQRHPKST a12 1 PAFGFDTGPGNMLMDAWMQAJWQLPYDKNGAAGNILPQLLDRLAPYFAQPHPKST 190 200 210 220 230 240 250 260 270 280 290 300 m12 pep GRELFAINWLETYLDGGENRYDVLRTLRFTAQTVCDAVSADAQMYICDGGIRNPV a12 1 GRELFALNWLETYLDGGENRYDVLRTLSRFAQTVFDAVSAADARQMYICGGGIRNPV 250 260 270 280 290 300 310 320 330 340 350 360 m121 .pep LMADLAECFGTRVSLHSTADLNLDPQWVEAXFAWLAACWINRIPGSPHKTGASKPCIL a12 1 LMADLAECFGTRVSLHSTAELNLDPQWVEAAAWMACWVNRIPGSPHKTGASKPCIL 310 320 330 340 350 360 nl2l.pep XAGYYYX HIM11 a121 GAGYYYX Further work revealed the DNA sequence identified in N. meningitidis <SEQ ID 46>: m121-1 .seq ATGGAAACAC AGCTTTACAT CGGCATCATG GGCGGATGCC GTACTGATAC GGATGGACGG AAGGGCACGC CTTTACCCCC TACCCCGGCA GATTTGCAGG ACACAGGCGC AGACGAACTG GCAAGAACTC AGCCGCCTAT ATGCGCAAAC

GTCAAAACCT

ACCGTCCGAC

GCCGCTGCTG

GCCGCGACCT

CACGAAGCCC

CGGCGGGATT

GCTTCGACAC

CACTGGCAGC

CATATTGCCG

AACCCCACCC

GAAACCTAC

CGCACCGTCC GACATTACCG ACGCGCCGGA ACACGGTTAC GCGGAACGGA CGCGGATTTT TGCGGCCGGC GGACAAGGCG TGTTCCGCGA CAACAGGGAA GCCAACATCA GCGTACTCCC AGGGCCGGGC AATATGCTGA TTCCTTACGA CAAAAACGGT CAACTGCTCG ACAGGCTGCT TCGGGAACCA GCATGGACGG CGGCAAATGG CTGGGCGCGG GGTTACGCCG CCAATTGCTG CACCGCAGCA GGATTTTGTC CGCCGCCGAA CTGCTGTGCA CCCTCGGCTG CCACGGGCAA AGCATACAGC TTGCCGATTT TACCGTCGGC CACTTCCGCA CGCCACTCGT CCCCGCCTTT ACACGCGCGG TACTGAACAT CCCCGACGCA CCCGCCTTCG TGGACGCGTG GACGCAGGCA GCAAAGGCGG CACAAGGCAA CGCCCACCCG TATTTCGCAC TAAAAGCACG GGGCGCGAAC TGTTTGCCCT AAATTGGCTC TTGACGGCGG CGAAAACCGA TACGACGTAT TGCGGACGCT b TTCCCGTTTT ACCGCGCAAA CCGTTTGCGA CGCCGTCTCA CACGCAGCGG 851 CAGATGCCCG TCAAATGTAC ATTTGCGGCG GCGGCATCCG CAATCCTGTT 901 TTAATGGCGG ATTTGGCAGA ATGTTTCGGC ACACGCGTTT CCCTGCACAG 951 CACCGCCGAC CTGAACCTCG ATCCGCAATG GGTGGAAGCC GCCGNATTTG 1001 CGTGGTTGGC GGCGTGTTGG ATTAATCGCA TTCCCGGTAG TCCGCACAAA 1051 GCAACCGGCG CATCCAAACC GTGTATTCTG ANCGCGGGAT ATTATTATTG 1101 A This corresponds to the amino acid sequence <ZSEQ ID 47; OR" 12 1-1>: m121-1 .pep

METQLYIGIM

DLQDTGADEL

TVRHAPEHGY

HEALFRDNRE

HWQLPYDKNG

ETYLDGGENR

LMADLAECFG

ATGASKPCIL

SGTSr4DGADA HRSRILSQ9L

SIQLADLPLL

TRAVLNIGGI

AKAAQGNILP

YDVLRTLSRF

TRVSLHSTAD

XAGYYY*

VLIRMDGGKW

SRLYAQTAAE

AEkTRIEFTVG

ANISVLPPDA

QLLDRLLAHP

TAQTVCDAVS

LNL6PQWVFA

LGP

1 EGHAFrP

LLCSQNLAPS

DFRSRDLAAG

PAFGFDTGPG

YFAQPHPKST

HAAADPIRQMY

AXFAWLAACW

YPGRLRRQLL

DITALGCHGQ

GQGAPLVPAF

NMLMDAWTQA

GRELFALNWL

ICGGGIRNPV

INRIPGSPHI(

m121-1/gl2l overlap m121-1 .pep g121 m121-1 .pep gl21 m121-1 .pep gi 21 m121-l.pep m121-4 .pep g121 m121-1 .pep g121 m121-1 .pep gl2l OR~s 121-1 and 121-1.ng showed a 95.6% identity in 366 aa 2030 *40 s0 METQLYIGIMSGTSMDGADAVLIR4DGGKWLGAEGHAFTPYPGRLPRQLLDLODTGADEL METQLYIGIMSGTSMDGADAVLVRDGGKWLGEGApIPYPDRLRRKLLDLQDTGTDEL 20 30 40 s0 .80 90 100 110 120

HRSRILSQELSRLYAQTAAELLCSQNLAPSDITALGCHGQTVRHAJPEHGYSIQLADLPLL

HRSRMLSQELSRLYAQtAELLCSQNLAPCDITAGCHGQTVRJ4JPEHGYSIQLADLPLL 80 90 100 110 120 130 140 150 160 170 180

AERTRIFTVGDFRSRDLAAGGQGAPLVPAFHEALFRDNRETPRAVLNIGGIANISVLPPDA

AELTRIFTVGDFRSRDLAAGGQGAPLVPAFHEALFRDDRETRVLNIGGIANISVLPPGA

130 140 150 .160 170 180 190 200 210 220 230 240

PAFGFDTGPGNMLMDAWTQAHWQLPYDKNGACAAQGNILPQLLDRLJAJHPYFAQPHPKST

PAFGFDTGPGNMLMDAWTOAJWLPYD4GAKAAQGNILPQLLGRLLAJ4PYFSQPHPKST 190 .200 210 220 230 240 250 .260 270 .280 290 300

GRELFALNWLETYLDGGENRYDVLRTLSRFAQTVWDAVSAADAQMYICGGGIRN~PV

250 260 270 280 290 300 310 320 330 340. 350 360 LMDACGRSHTDNLPWEAFWACIRPGS

PHKATGASKPCIL

LMADLAECFGTRVSLHSTAELNLDPQWVEAAAFAWLAACWINRI PGS PHKATGASI{PCI L .310 320 330 340 350 360

XAGYYYX

I1I1II

GAGYYYX

191 The following partial DNA sequence was identified in N. meningitidis <SEQ ED 48>: a121-1. seq

ATGGAAACAC

GGCGGATGCC

AAGGGCACGC

GATTTGCAGG

GCAAGAACTC

GTCAAAACCT

ACCGTCAGAC

GCCGCTGCTG

GCCGCGACCT

CACGAAGCCC

CGGCGGGATT

GCTTCGACAC

CACTGGCAGC

CATATTGCCG

AACCCCACCC

AGCTTTACAT

GTACTGATAC

CTTTACCCCC

ACACAGGCGC

AGCCGCCTGT

CGCGCCGTCC

ACGCGCCGGA

GCGGAACGGA

TGCGGCCGGC

TGTTCCGCGA

GCCAACATCA

AGGACCGGGC

TTCCTTACGA

CAACTGCTCG

TAAAAGCACG

CGGCATCATG TCGGGAACCA GCATGGACGG GGATGGACGG CGGCAAATGG CTGGGCGCGG TACCCCGGCA GGTTACGCCG CAAATTGCTG GGACGAACTG CACCGCAGCA GGATGTTGTc ACGCGCAAAC CGCCGCCGAA CTGCTGTGCA GACATTACCG CCCTCGGCTG CCACGGGCAA ACACAGTTAC AGCGTACAGC TTGCCGATTT CTCAGATTTT TACCGTCGGC GACTTCCGCA GGACAAGGCG CGCCGCTCGT CCCCGCCTTT CGACAGGGAA ACACGCGCGG TACTGAACAT GCGTACTCCC CCCCGACGCA CCCGCCTTCG AATATGCTGA TGGACGCGTG GATGCAGGCA CAAAAACGGT GCAAAGGCGG CACAAGGCAA ACAGGCTGCT CGCCCACCCG TATTTCGCAC GGGCGCGAAc TGTTTGCCCT AAATTGGCTC CGAAAACCGA TACGACGTAT TGCGGACGCT CCGTTTTCGA CGCCGTCTCA CACGCAGCGG ATTVT CGGCG GCGGCATCCG CAATCCTGTT ATGTTTCGGC ACACGCGTTT CCCTGCACAG ATCCGCAATG GGTAGAAGCC

GCCGCGTTCG

GTCAACCGCATTCCCGGTAG

TCCGCACAAA

GTGTATTCTG GGCGCGGGAT ATTATTATTG 751 801 851 901 951 1001 1051 1101 GAAACCTACC TTGACGGCGG TTCCCGATTC ACCGCGCAAA CAGATGCCCG TCAAATGTAC TTAATGGCGG ATTTGGCAGA CACCGCCGAA CTGAACCTCG CATGGATGGC GGCGTGTTGG GCAACCGGCG CATCCAAACC

A

This corresponds to the amino acid sequence <SEQ ID 49; ORE 121-l.a>: a121-1.pep 1 METQLYIGIM SGTSMDGADA VLIRMDGGKW LGAflGHArrP YPGRLRR 51 DLQDTGADEL HPRSR1'LSQEL SRLYAQTAAE LLCSQNLAPS DITALGC 101 TVRHAPEHSY SVQLADLPLL AERTQIETVG DFRSROLAAG GQGAPLV -11HEALFRDD.E TPAVLNIGGI ANISVLPPDA PAFGFDTGPG NMLMDAW 201 HWQLPYDKNG AKAAQGNILP QLLDRLLAHP YFAQPHPKST GRELFALI 251 ETYLDGGENR YDVLRTLSRF TAOTVFDAVS HAAADARQMY ICGGGIRI 301 LMADLAECFG TRVSLHSTAE LNLDPQWVEA AAFAWMAACW VNRIPGS 351 ATGASKPCIL GAGYYY*

KLL

HiGo

PAF

TWL

PHK

m121-1/al21-1 ORFs 121-1 and 121-1.a. showed a 96.4% identity in 366 aa overlap m121-1 .pep a121-l m121-1 .pep a121-l m121-1 .pep a121-1 m121-1 .pep a12l-1 20 30 40 s0

METQLYIGIMSGTSMDGADAVLIRMDGGI(XLGAEJJATPYPGRLRRKLLDLQDTGADEL

20 30 40 50 80 90 100 110 .120

HRSRILSQELSRLYAQT.AALLCSQNLAPSDITAGCHGQTVJJAPEHGYSIQLADLPLL

80 90 100 110 120 130 140 150 160 170 180 AERTRI ~VDRRLAGGALPFELRNERALIGAIVPD AERTQI TGFSDAGQGPVAHAFDDERVNGINSVLPPDA 130 140 150 160 170 180 190 200 210 220 230 240 190 200 210 220 230 240 92 ml21-1 pep al21-l m12l-l. pep al2l 250 260 270 280 290 300

GRELFALNWLETYLDGGENRYDVLRTLSRFTAQTVCDAVSHAAADARQMYICGGGIRNPV

GRELFALNWLETYLDGGENRYDVLRTLSRFTAQTVFDAVSHAAADAROMYICGGGIRN

PV

250 260 270 280 290 300 310 320 330 340 350 360 LMDACGRSHTDNLPWEA

WACIRPGSPHKATGASKPCIL

LMADLAECFGTRVSLHSTAELNLDPQWVEAAWHJWMACWJNRIPGSPHKATGASKPCIL

310 320 330 340 350 360 ml21-l. pep a121 128 a nd 128-1

XAGYYYX

HIM11

GAGYYYX

The following partial DNA sequence was identified in N. meningitidis <SEQ ID) m128. seq 1 51 101 151 201 251 *.301.

351 .1 51 101 151 201 251 301 351 401 451 501 551 601 *651 701 751 851 951 1001 (partial) ATGACTrGACA ACGCACTGCT AATCAAAACC GAAGACATCA CGCGCGAACA AATCGCCGCC AACACTGTCG AACCCCTGAC GGGCGTGGTG TCGCACCTCA CCGTCTATAA CGAACTGATG GGACAAGACA TCGAGCTGTA CGAATTCGAC ACCCTCTCCC TACGCCAGCG AAAAACTGCG wGTCAAAAAA TAyTTCCCyG AAMTCAAAAA ACTMTACGGC TGGCACAAAG

ACGTGCGCTA

AGGCGGCGTT TATATGGATT CGTGGATGAA CGACTACAAA CAAyTGCCCA CCGCCTACCT CAGGGAAGCC CGCyTGAGCC CCGGACACGG GCTGCACCAC TCCGGCATCA ACGGCGTACA TATGGAAAAT TTCGTTTGGO ACGAAGAAAC CGcgcTTCCC GCCGCCAAAA ACTTCCAAsG CGCCCTCTTT GATATGATGA AAAACTGGCA ACAGGTTTTA CAGCCGCCCG AATACAAc CG

CCATTTGGGC

AACCCGCCCT

ATCAAAGCCC

CGGCATCACC

ACTGCGTCGC

CCCGAAATCA

CAACCGCTTC

CCGCACAAAA

CGAAGCCAAA

TCGGCAAwGT

ATCGGATTTA

TTkTGAATTG

TGTACGCACG

GGCCGCCGCC

CGTCTGCAAC

ACGACGAAAT

.CTGCTTACCC

ATGGGACGCG

AATACAATGT

yTGCCGAAAG

CGGCATGTTC

TTTACAGCGA

GACAGCGTGC

CTTCGCCTTG

GAAGAACCCC GTTTTGATCA GCAAACCGCC ATCGCCGAAG AAACGCACAC CGGCTGGGCA GAACGCGTCG GCAGGATTTG CGACACGCCC GAACTGCGCG CCGTCTTCTT CACCGAAATC AAAACCATCA AAAATTCCCC AACCAAACTC AACCAC TACGCGTTCA GCGAAACCGA ATTAAACGGA CTGTTCGCCC CCGAAAAAAC yGTCCCCGTC CAACAAAACG GCGAAMCCAT CGAAGGCAAA CGCGGCGGCG GTTTTTCAGA CGGCACGCTG TTCGCCCCAC CCGTCQGCGO CCTCATCCTC TTCCACGAAA AAGTGGACGA ACTGGGCGTA GTCGAACTGC CCAGCCAGTT CTTGGCACAA MTGTCAGCCC AACTCTTiSGA CAAAWTGCTC yTSGTCCGGC AAWTGGAGTT AGACGACGAA GOCCGTCTGA GCAAAAAAGT CGCCGTCATC AGCTTCGGCC ACATCTTCGC CGCGTGGGCG GAAGTATTGA GCGACGATGT CGCCGCCACA GTCGGGGnAT CGCGCAGCGG CGAACCGAGC ATAGACGCAC

TCTGA

AGGCGGCTAT TCCGCAGCTn ATTACAGCTA GCGCGGACGC ATACGCCGCC TTTGAAGAAA GGCAAACGCT TTTGGCAGGA AATCCTCGCC nGCAGAATCC TTCAAAGCCT TCCGCGGCCG TCTTGCGCCA CAGCGGTTTC GACAACGCGG This corresponds to the amino, acid sequence <SEQ ID 5 1; ORE 128>: m128.pep (partial) 1 MTDNALLHLG EEPRFDOIKT EDIKPALQTA IAEAREQIAA IKAQTHTGWA 51 NTVEPLTGIT ERVGRIWGVV SHLNCVADTP ELRAVYNELM PEITVPFTEI 101 GQDIELYNRF KTIKNSPEFD TLSPAOKTKL NH 1 YASEKIREAK YAFSETXVKK YFPVGXVLNG LFAQXKKLYG IGFTEKTVPV 93

WHKDVRYXEL

QLPTAYLVCN

SGINGVXWDA

AAKNFQXGMF

OPPEYNRFAL

G KRFWQEILA

QQNGEXIGGV

FAPPVGGREA

VELPSQFMEN

XVRQXEFALF

SFGHI FAGGY

VGXSRSGAES

YMDLYAREGK RGGAWMNDYK GRRRFSDGTL RLSHDEILIL FHETGHOLHH LLTQMDELGV FVWEYNVLAQ XSAHEETGVP LPKELXDOCL DMMIYSEDDE GRLKNWQQVL DSVRKKVAVI SAAXYSYAWA EVLSADAYAA FEESDDVAAT FKAFRGREPS IDALLDRHSGF DNAV* The following partial DNA sequence was identified in N. gonorrhoeae <SEQ ID) 52>: g128. seq 1. atgattgaca aCgCActgct ccacttgggc gaagaacCCC GTTTTaatca 51 aatccaaacc gaagACAtca AACCCGCCGT CCAAACCGCC ATCGCCGAAG 101 CGCGCGGACA AATCGCCGCC GTCAAAGCGC AAACGCACAC CGGCTGGGCG 151 AACACCGTCG AGCGTCTGAC CGGCATCACC GAACGCGTCG GCAGGATTTG 201 GGGCGTCGTG TCCCATCTCA ACTCCGTiCGT CGACACGCCC GAACTGCGCG 251 CCGTCTATAA CGAACTGATG CCTGAAATCA CCGTCTTCTT CACCGAAATC 301 GGACAAGACA TCGAACTGTA CAACCGCTTC AAAACCATCA AAAATTCCCC 351 CGAATTTGCA ACGCTTTCCC CCGCACAAAA AACCAAGCTC GATCACGACC 401 TGCGCGATTT CGTATTGAGC GGCG.CGGAAC TGCCGCCCGA ACGGCAGGCA 451 GAACTGGCAA AACTGCAAAC CGAAGGCGCG CAACTTTCCG CCAAATTCTC 501 CCAAAACGTC CTAGACGCGA CCGACGCGTT CGGCATTTAC T.TTGACGATG 1 CCGCACCGC'r TGCCGGCATT CCCGAAGACG CGCTCGCCAT GTTTGCCGCC 601 GCCGCGCAAA GCGAAGGCAA AACAGGTTAC AAAATCGGCT TGCAGATTCC 6.51 GCACTACCTT GCCGTTA TCC AATACGCCGG CAACCGCGAA CTGCGCGAAC 701 AAATCTACCG CGCCTACOTT ACCCGTGCCA GCGAACTTTC AAACGACGGC 751 AAATTCGACA ACACCGCCAA CATCGACCGC ACGCTCGAAA ACGCATTGAA *801 AACCGccaaa cTGCTCGGCT TTAAAAATTA CGCCGAATTG TCGCTGGCAA 851 CCAAAATGGC GGACACGCCC GAACAGGTTT TAAACTTCCT GCACGACCTC 901 GCCCGCCGCG CCAAACCCTA CGCCGAAAAA GACCTCGCCG AAGTCAAAGC *951 CTTCGCCCGC GAACACCTCG GTCTCGCCGA CCCGCAGCCG TGGGACTTGA 1001 GCTACGCCGG CGAAAAACTG CGCGAAGCCA AATACGCATT CAGCGAAACC 1051 GAAGTCAAAA AATACTTCCC CGTCGGCAAA GTTCTGGCAG GCCTGTTCGC 1101 CCAAATCAAA AAACTCTACG GCATCGGATT CGCCGAAAAA ACCGTTCCCG 1151 TCTGGCACAA AGACGTGCGC TATrrTGAAT TGCAACAAAA CGGCAAAACC 1201 ATCGGCGGCG TTTATATGGA TITGTACGCA CGCGAAGGCA AACGCGGCGG 1251 CGCGTGGATG AACGACtaca AAGGCCGCCG CCGCTTTGCC GACGgcacGC 1301 TGCAACTGCC CACCGCCTAC CTCGTCTGCA ACTTCGCCCC GCCCGTCGGC 1351 GGCAAAGAAG CGCGTTTAAG CCACGACGAA ATCCTCACCC TCTTCCACGA 1401 AacCGGCCAC GGACTGCACC ACCTGCTTAC CCAAGTGGAC GAACTGGGCG 1451 TGTCCGGCAT CAAcggcgtA GAATGGGACG CGGTCGAACT GCCCAGCCAG -1501 TTTATGGAAA ACTTCGTTTG GGAATACAAT GTATTGGC-AC AAATGTCCGC 1551 CCACGAAGAA AccgGCGAGC CCCTGCCGAA AGAACTCTTC GACAAAATGC 1601 TcgcCGCCAA AAACTTCCAG CGCGGTATGT TCCTCGTCCG GCAAATGGAG 1651 TTCGCCCTCT TCGATATGAT GATTTACAGT GAAAGCGACG AATGCCGTCT 1701 GAAAAACtGG CAGCAGGTTT TAGACAGCGT GCGCAAAGAA GTcGCCGTCA 1751 TCCAACCGCC CGAATACAAC CGCTTCGCCA ACAGCTTCGG CCacatctTC 1801 GCcggcGGCT ATTCCGCAGG CTATTACAGC TACGCATGGG CCGAAGTCCt 1851 cAGCACCGAT GCCTACGCCG CCTTGAAGA AAGcGACGac gtcGCCGCCA 1901 CAGGCAAACG CTTCTGGCAA GAAAtccttg ccgtcggcgg ctCCCGCAGC 1951 gc§GCGGAAT CCTTCAAAGC CTTCCGCGGA CGCGAACCGA GCATAGACGC 2001 ACTGCTGCGC CAaagcgqT TCGACAACGC gGCttaA This corresponds to the amino acid sequence <SEQ ID 53; ORE 128.ng>: 9128 .pep

MIDNALLHLG

NTVERLTGIT

GQDIELYNR.F

ELAKLQTEGA

AAQSEGKTGY

EEPRFNQIQT

ERVGRIWGVV

ICTI KNSPEFA

QLSAXFSQNV

KIGLQIPHYL

EDIKPAVQTA

SHLNSVVDTP

TLSPAOKTKaJ

LDATDAFGIY

AVIQYAGNRE

IAEARGOIAA

ELRAVYNELM

DHDLRDFVLS

FDDAAPLAGI

LREQIYRAYV

VKAQTHTGWA

PEITVFFTEI

GAELPPERQA

PEDALAMFAA

TRASELSNDG

94 251 KFDNTANIDR TLENALKTAK LLGFKNYAEL SLATIG{ADTP EQVLNFLHDL 301 ARRAKPYAEK DLAEVKAFAR EHLGLADPQP WDLSYAGEKL REAICYAFSET 351 EVKKYFPVGK VLAGLFAQ1K KLYGIGFAEK TVPVWHKDVR YFELQQNGKT 401 IGGVYMDLYA REGKRGGAWM NDYKGRRRFA DGTLQLPTAY LVCNFAPPVG 451 GKEARLSHDE ILTLFHETGH GLHHLLTQVD ELGVSGINGV EWDAVELPSQ 501 FMENFVWEYN VLAOMSAHEE TGEPLPKELF DKa4LAAKNFQ RGMFLVRQME 551 FALFDMMIYS ESDECRLKNW QQVLADSVRKE VAVIQPPEYN RFASFGHIF 601 AGGYSAGYYS YAWAEVLSTD AYAAFEESDD VAATGKRFWQ EILAVGGSRS 651 AAESFKAFRG REPSIDALLR QSGFDNJAA* ORE 128 shows 91.7% identity over a 475 aa overlap with a predicted ORF (ORE 128.ng) from N. gonorrhoeae: m128/g128 20 30 40 50 9128. pep MINLHGERNITDKAQAIERQAV~TTWNVRTI m12 8 MTNLHGERDITDKAQAIEROAIATTWNVPTI 20 30 40 50 80 90 100 110 120 g12 8-.pep ERGIGVHNVDPLAYEMPIVFEGDtYRKIUPF m12 8 ERGIGVHNVDPLAYEMPIVFEGDEYRKINPF 80 90 100 110 120 130 140 150 160 170 180 9128. pep TL.AKKDDRFLGEPEQEALTGQSKSNLADFI m128 TLSPAQKTKLNH 130 340 350 360 9128 .pep

YAGKREAYAFSETEVKYFPVGVLAG

m128

YASEKLREAKYAFSETXVKKYFPVGXVLNG

20 370 380 390 400 410 420 g12 8. pep LFAOIKKYI ETPWKDRPLQGTGVMDYRGRGWNY m12 8 LFQKLGGTKVVHDRXLQNEIGYDYRGRGWNY 50 60 70 80 430 440 450 460 470 480 9128. pep GRRAGLLTYVNAPVGERLHELLHTHLHLOIEG m12 8 GRRRFSDGTLOLPTAYLVCNFAPPVGGREARIJSHDE ILl LFHETGI{GLHHLLTQVDELGV 100 110 120 130 140 150 490 500 510 520 530 540 9128 .pep SGINGVEWDAVELPSQFMENFVWEYNVLAQMSAHEETGEPLPKELFDI LAAKqFQRGMF HIM11 IIIIIIIIIIIIIIIIIIIIIII 1111111 111111.11 1111111 111 m12 8 SGINGVXWDAVELPSQFENFVWEYLAQXSAHEETGVPLPKLXDCZLAAKNpQXGMF 160 170 180 190 200 210 560 570 580 5960 590 600 95 *g128 .pep LVROMEFALFMMIYSESDECRLKWQOVLDSVREVAVIPPEYRFASFGHIFAGIJY m12 8 XVRQXEFALFDMMIYSEDDEGRLWQQVLSVRVAVIQPPEYRFSFGHIFAGGY 220 230 240 250 260 270 610 620 630 640 650 660 9128.pep SAGYYSYAWAEVLSTDAYAAFEESDDVATGKRIFWQEILAVGGSRSAASFKAYRGREPS m12 8 SAAXYSYAWAEVLSADAYAAFEESDDVAATGKRFWQEILAVGXSRSGAESFKAFRGREPS 280 290 300 310 320 330 670 679 9128 .pep IDALLRQSGFDNAAX mn128 IDALLRHSGFDNAVX 340 The following partial DNA sequence was identified in N. meningitidis <SEQ ID 54>: a128.seq 51 101 151 201 251 301 351 401 451 .501 551 601 651 701 751 801 851 901 951 100 1051 110*1 1151 1201 1251 1301 1351 1401 1451 1501 *1551 1601 1651 *1701 1751 1801 1851 1901 1951 2001

ATGACTGACA

AATCAAAACC

CGCGCGAACA

AACACTGTCG

GGGCGTGGTG

CCGCCTACAA

GGACAAGACA

CGAGTTCGAC

TGCGCGATTT

GAATTGGCAA

CCAAAACGTC

CCGCACCGCT

GCCGCGCAAA

GCACTACCTC

AAATCTACCG

AAATTCGACA

AACCGCCAAA

CCAAAATGGC

GCCCGCCGCG

CTTCGCCCGC

GCTACGCCGG

GAAGTCAAAA

CCAAATCAAA

TCTGGCACAA

ATAGGCGGCG

CGCGTGGATG

TGCAACTGCC

GGCAAAGAAG

AACCGGACAC

TATCCGGCAT

TTTATGGAAA

CCACGAAGAA

TCGCCGCCAA

TTCGCCCTCT

GAAAAACTGG

TCCGACCGCC

GCAGGCGGCT

GAGCGCGGAC

CAGGCAAACG

GCGGCAGAAT

ACTCTTGCGC

ACGCACTGCT CCATTTGGGC GAAGACATCA AACCCGCCCT

AATCGCCGCC

AACCCCTGAC

TCGCACCTCA

TGAATTAATG

TCGAGCTGTA

ACCCTCTCCC

CGTCCTCAGC

AACTGCAAAC

CTAGACGCGA

TGCCGGCATT

GCGAAGGCAA

GCCGTCATCC

CGCCTACGTT

ACACCGCCAA

CTGCTCGGCT

GGACACCCCC

CCAAACCCTA

GAAAGCCTCG

CGAAAAACTG

AATACTTCCC

AAACTCTACG

AGACGTGCGC

ATCAAAGCCC

CGGCATCACC

ACTCCGTCAC

CCCGAAATTA

CAACCGCTTC

ACGCGCAAAA

GGCGCGGAAC

CGAAGGCGCG

CCGACGCGTT

CCCGAAGACG

AACAGGCTAC

AATACGCCGA

ACCCGCGCCA

CATCGACCGC

TCAAAAACTA

GAACAAGTTT

CGCCGAAAAA

GCCTCGCCGA

CGCGAAGCCA

CGTCGGCAAA

GCATCGGATT

TATTTTGAAT

GAAGAACCCC

GCAAACCGCC

AAACGCACAC

GAACGCGTCG

CGACACGCCC

CCGTCTTCTT

AAAACCATCA

AACCAAACTC

TGCCGCCCGA

CAACTTTCCG

CGGCATTTAC

CGCTCGCCAT

AAAATCGGTT

CAACCGCAAA

GCGAGCTTTC

ACGCTCGAAA

CGCCGAATTG

TAAACTTCCT

GACCTCGCCG

TTTGCAACCG

AATACGCATT

GTATTAAACG

TACCGAAAAA

TGCAACAAAA

CGCGAAGGCA

CCGTTTTTCA

ACTTCACCCC

ATCCTCACCC

CCAAGTCGAC

CAGTCGAACT

GTCTTGGCGC

AGAACTCTTC

TCCTCGTCCG

GAAGACGACG

GCGCAAAGAA

ACAGCTTCGG

TACGCGTGGG

AAGCGACGAT

CCGTCGGCGG

CGCGAACCGA

GGCTTGA

GTTTTGATCA

ATTGCCGAAG

CGGCTGGGCA

GCAGGATTTG

GAACTGCGCG

CACCGAAATC

AAAACTCCCC

AACCACGATC

ACAGCAGGCA

CCAAATTCTC

TTTGACGATG

GTTTGCCGCT

TGCAGATTCC

CTGCGCGAAC

AGACGACGGC

ACGCCCTGCA

TCGCTGGCAA

GCACGACCTC

AAGTCAAAGC

TGGGACTTGG

CAGCGAAACC

GACTGTTCGC

ACCGTCCCCG

CGGCGAAACC

AACGCGGCGG

GACGGCACGC

GCCCGTCGGC

TCTTCCACGA

GAACTGGGCG

GCCCAGTCAG

AAATGTCCGC

GACAAAATGC

CCAAATGGAG

AAGGCCGTCT

GTCGCCGTCG

CCACATCTTC

CGGAAGTATT

GTCGCCGCCA

ATCGCGCAGC

GCATAGACGC

TTTATATGGA TTTGTACG CA AACGACTACA AAGGCCGCCG CACCGCCTAC CTCGTCTGCA CCCGCTTGAG CCATGACGAA GGCCTGCACC ACCTGCTTAC CAACGGCGTA GAATGGGACG ATTTCGTTTG GGAATACAAT ACCGGCGTTC CCCTGCCGAA AAACTTCCAA CGCGGAATGT TTGATATGAT GATTTACAGC CAACAGGTTT TAGACAGCGT CGAATACAAC CGCTTCGCCA ATTCCGCAGG CTATTACAGC GCATACGCCG CCTTTGAAGA CTTTTGGCAG GAAATCCTCC CCTTCAAAGC CTTCCGCGGA CACAGCGGCT TCGACAACGC 96 This corresponds to the amino, acid sequence <SEQ ID 55; ORF 128.a>: a128.pep 1 MTDNALLHLG EEPRFDQIKT EDIKPALQTA IAEAREQIAA IKAQTHTGWA 51 NTVEPLTGIT ERVGRIWGVV SHLNSVTDTP ELRAAYNELM PEITVFFTEI 101 GQDIELYNRF IKTIKNSPEFD TLSHAQI(TKL NHDLRDFVLS GAELPPEQQA 151 ELAKLQTEGA QLSAKFSQNV LDATDAFGIY FDDAAPLAGI PEDALAMFAA 201 AAQSEGKTGY KIGLOIPHYL AVIQYADNRK LREQIYRAYV TRASELSDDG 251 KFDNTANIDR TLENALQTAK LLGFKNYAEL SLATKMADTP EQVLNFLHDL 301 ARRAKPYAEK DLAEVKAFAR ESLGLADLQP WDLdYAGEKL REAKYAFSET 351 EVKKYFPVGK VLNGLFAQIK KLYGIGFTEK TVPVWH -KDVR YFELQQNGET 401 IGGVYMDLYA REGKRGGAWM NDYKGRRRFS DGTLQLPTAY LVCNFTPPVG 451 GKLARLSHDE ILTLFHETGH GLH-HLLTQVD ELGVSGINGV EWDAVELPSQ 501 FMENE'VWEYN VLAQMSAHEE TGVPLPKELF DKMLAAKNFQ RGMFLVRQME 551 FALFDMMIYS EDDEGRLKNW QQVLDSVRKE VAVVRPPEYN RFANSFGHIF .601 AGGYSAGYYS YAWAEVLSAD AYAAFEESDD VAATGKRFWQ EILAVGGSRS 651 AAESFKAFRG REPSIDALLR HSGFDNAA* m128/a128 ORFs 128 and 128.a showed a 66.0% identity in 677 aa overlap 20 30 40 50 m128 .pep MTNLHGERDITDKAQAIERQAIATTWNVPTI a128 MTNLHGERDITDKAQAIERQAIATTWNVPTI 20 30 40 50 80 90 100 110 120 zn128 .pep ERVGRIWGVVSHLNCVADTPELRAVYNELMPEITVFFTEIGQDIELYNRFTIQNSPEFD a12 8 ERVGRIWGVVSHLNSVTDTPELRAAYNELMPEITVFFTEIGQDIELYNRFKTI(NSPE.D s0 90 100 110 120 11301 m128.pep TLSPAQI(TKLNH a12 8 TLSHAQKTKLNHDLRDFVLSGAELPPEQQAELAI(LQTEGAQLSAKFSONVLDATDAFGIY 130 140 150 160 170 180 m128..pep a128 FDDAAPLAGIPEDALAMFAAAAQSEGKTGYKIGLQIPHYLAVIQYADNRLREQIYpJAYV 190 200 210 220 230 240 m128 .pep a128 T1RASELSDDGKFDNTANIDRTLENALQTAI(LLGFKNYAELSLATKM4ADTPEOVLNFLHDL 250 260 270 280 290 300 140 150 m128

YASEKLREAKYAFSETXVKKYFPVGX

a 128 ARRKPYAEKDLAEVKAFARESLGLADLQPWDLGYAGEKLREAKYAFSETEVK(YFPVGK 310 320 330 340 350 360 160 170 180 190 200 210 m128 .pep VLNGLFAOXKKLYGIGFTEKTVPWHKDVRYXELQQNGEXIGGVYMDLYRGKRGGAWM a128 VLNGLEAQIKKLYGIGFTEKTVPVWiHKDVRYFELQQNGETIGGVYMDLYARGKRGGAWM 370 380 390 400 410 420 220 230 240 250 260 270 m128 .pep 'NDYKGRRRFSDGTLQLPTAYLVCNFAPPVGGREARLSHDEILILFHETGHGLHHLLTQVD 97 al 28 NDYKGRRRFSDGTLQLPTAYLVCRFTPPVGGKEARLSHDEILTLFHETGHGLHHLLTQVD 430 440 450 460 470 480 280 290 300 310 320 330 m12 8. pep ELVGNVWAEPQMNVENVAXAETVLKLDXAKF a12 8 ELVGNVWAEPQMNVENVAMAETVLKLDMAKF 490 500 510 .520 530 540 340, 350 360 370 380 390 m128 .pep XGFVQEAF MYEDGLNQVDVKVVQPYRASGI a 128 11 11 550 560 570 .580 590 600 400 410 420 430 440 450 m128.pep AGYAXSAAVSDYAESDVAGRWELVXRGEFAR a 128 AGYAYSAAVSDYAESDVAGRWELVGRAEFAR 610 620 630 640 650 660 460 470 m128 .pep REPSIDALLRHSGFDNAVX a128 REPSIDALLRHSGFDNAAX 670 Further work revealed the DNA sequence identified in N. meningifidis <SE 56>: M128-1. se4 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401

ATGACTGACA.

AATCAAAACC

CGCGCGAACA

AACACTGTCG

GGGCGTGGTG

CCGTCTATAA

GGACAAGACA

CGAATTCGAC

TGCGCGATTT

GAACTGGCAA

CCAAAACGTC

CCGCACCGCT

GCCGCGCAAA

ACGCACTGCT

GAAGACATCA

AATCGCCGCC

AACCCCTGAC

TCGCACCTCA

CGAACTGATG

TCGAGCTGTA

ACCCTCTCCC

CGTCCTCAGC

AACTGCAAAC

CTAGACGCGA

TGCCGGCATT

GCGAAAGCAA

CCATTTGGGC

AACCCGCCCT

ATCAAAGCCC

CGGCATCACC

ACTCCGTCGC

CCCGAAATCA

CAACCGCTTC

CCGCACAAAA

GGCGCGGAAC

CGAAGGCGCG

CCGACGCGTT

CCCGAAGACG

AACAGGCTAC

GAAGAACCCC GTTTTGATCA GCAAACCGCC ATCGCCGAAG AAACGCACAC CCGCTGGGCA GAACGCGTCG GCAGGATTTG CGACACGCCC GAACTGCGCG CCGTCTTCTT CACCGAAATC AAAACCATCA AAAATTCCCC AACCAAACTC AACCACGATC TGCCCCCGA ACAGCAGGCA CAACTTTCCG CCAAATTCTC CGGCATTTAC TTTGACGATG CGCTCGCCAT GTTTGCCGCC AAAATCGGCT TGCAGATTCC ACACTACCTC GCCGTCATCC AATACGCCGA CAACCGCGAA CTGCGCGAAC AAATCTACCG CGCCTACGTT ACCCGCGCCA GCGAACTTTC AGACGACGGC AAATTCGACA ACACCGCCAA CATCGACCGC ACGCTCGCAA ACGCCCTGCA AACCGCCAAA CTGCTCGGCT TCAAAAACTA CGCCGAATTG TCGCTGGCAA *CCAAAATGGC GGACACGCCC GAACAAGTTT TAAACTTCCT GCACGACCTC GCCCGCCGCG CCAAACCCTA CGCCGAAAAA GACCTCGCCG AAGTCAAAGC CTTCGCCCGC GAAAGCCTGA ACCTCGCCGA TTTGCAACCG TGGGACTTGG GCTACGCCAG CGAAAAACTG CGCGAAGCCA AATACGCGTT CAGCGAAACC GAAGTCAAAA AATACTTCCC CGTCGGCAAA GTATTAAACG GACTGTTCGC CCAAATCAAA AAACTCTACG GCATCGGATT TACCGAAAAA ACCGTCCCCG TCTGGCACAA AGACGTGCGC TATTTTGAAT TGCAACAAAA CGGCGAAACC ATAGGCGGCG TTTATATGGA TTTGTACGCA CGCGAAGGCA AACGCGGCGG CGCGTGGATG AACGACTACA AAGGCCGCCG CCGTTTTTCA GACGGCACGC TGCAACTGCC CACCGCCTAC CTdGTCTGCA ACTTCGCCCC ACCCGTCGGC GGCAGGGAAG CCCGCCTGAG CCACGACGAA ATCCTCATCC TCTTCCACGA AACCGGACAC. GGGCTGCACC ACCTGCTTAC CCAAGTGGAC GAACTGGGCG 98 1451 1501 1551 1601 1651 1701 1751 1801 .1851 1901 1951 2001 TATCCGGCAT CAACGGCGTA GAATGGGACG TTTATGGAAA ATTTCGTTTG CCACGAAGAA ACCGGCGTTC TCGCCGCCAA AAACTTCCAA

TTCGCCCTCT

GAAAAACTGG

TCCAGCCGCC

6CAGGCGGCT

GAGCGCGGAC

CAGGCAAACG

GCGGCAGAAT

ACTCTTGCGC.

TTGATATGAT

CAACAGGTTT

CGAATACAAC

ATTCCGCAG

GCATACGCCG

CTTTTGGCAG

CCTTCAAAGC

CACAGCGGTT

GGAATACAAT

CCCTGCCGAA

CGCGGCATGT

GATTTACAGC

TAGACAGCGT

CGCTTCGCCT

CTATTACAGC

CCTTTGAAGA

GAAATCCTCG

CTTCCGCGGC

TCGACAACGC

CGGTCGAACT

GTCTTGGCAC

AGAACTCTTC

TCCTCGTCCG

GAAGACGACG

GCGCAAAAAA

TGAGCTTCGG

TACGCGTGGG

AAGCGACGAT

CCGTCGGCGG

CGCGAACCGA

GGTCTGA

GCCCAGCCAG

AAATGTCAGC

GACAAAATGC

GCAAATGGAG

AAGGCCGTCT

GTCGCCGTCA

CCACATCTTC

CGGAAGTATT

GTCGCCGCCA

ATCGCGCAGC

GCATAGACGC

This corresponds to the amino acid sequence <SEQ ID 57; ORE 128-1>: m128-1 .pep.

1 MTDNALLHLG EEPRFDQIKT EDIKPA-LQT. IAEAREQIAA IKAQTH 51 NTVEPLTGIT ERVGRIWGVV SHLNSVADTP ELRAVYNELM PEITVF 101 GQDIELYNRF KTIKNSPEFD TLSPAQKTKL NHDLRDFVLS GAELPP 151 ELAKLQTEGA QLSAKFSQNV LDATDAFGIY FDDAAPLAGI PEDALP.

201 AAQSESKTGY KIGLQIPHYL AVIQYADNRE LREQIYRAYV TRASEI 251 KFDNTANIDR TLANALQTAK LLGFKNYAEL SLATKMADTP EQVLNP 301 ARRAKPYAEK DLAEVKAFAR ESLNLADLQP WDLGYASEKL REAKYA 351 EVKKYF'PVGK VLNGLFAQIK KLYGIGFTEK TVPVWHKDVR YFELQQ 401 IGGVYMDLYA REGKRGGAWM NDYKGRRRFS DGTLQLPTAY LVCNFA 451 GREARLSHDE ILILFHETGH GLHHLLTQVD ELGVSGINGV EWDAVE 501 FMENFVWEYN VLAQMSAHEE TGVPLPKELF DK1MLAAKNFQ RGk4FLV 551 FALFDMNIYS EDDEGRLKNw QQVLDSVRKK VAVIQPPEYN RFALSE 601 AGGYSAGYYS YAWAEVLSAD AYAAFEESDD VAATGKRFWQ EILAVG 651 AAESFKAFR G REPSIDALLR HSGFDNAV* tTGWA

TTEI

~EQQA

MFAA

*SDDG

THDL

LFSET

~NGET

LPPVG

:LPSQ

'RQME

'GHIF

;GSRS

The following partial DNA sequence was identified in N gonorrhoeae <SEQ ID 58>: g128-1.seq (partial) 1 ATGATTGACA ACGCACTGCT CCACTTGGGC GAAGAACCCC GTTTTAATCA 51 AATCAAAACC GAAGACATCA AACCCGCCGT CCAAACCGCC ATCGCCGAAG 101 CGCGCGGACA AATCGCCGCC GTCAAAGCGC AAACGCACAC CGGCTGGGCG 151 *AACACCGTCG AGCGTCTGAC CGGCATCACC GAACGCGTCG GCAGGATTTG 201 GGGCGTCGTG TCCCATCTCA ACTCCGTCGT CGACACGCCC GAACTGCGCG 251 CCGTCTATAA CGAACTGATG CCTGAAATCA CCGTCTTCTT CACCGAAATC 301 GGACAAGACA TCGAACTGTA CAACCGCTTC AAAACCATCA AAAATTCCCC 351 CGAATTTGCA ACGCTTTCCC CCGCACAAAA AACCAAGCTC GATCACGACC 401 TGCGCGATTT CGTATTGAGC GGCGCGGAAC TGCCGCCCGA ACGGCAGGCA 451 GAACTGGCAA AACTGCAAAC CGAAGGCGCG CAACTTTCCG CCAAATTCTC 501 CCAAAACGTC CTAGACGCGA CCGACGCGTT CGGCATTTAC TTTGACGATG 551 CCGCACCGCT TGCCGGCATT CCCGAAGACG CGCTCGCCAT GTTTGCCGCC 601 *GCCGCGCAAA GCGAAGGCAA AACAGGTTAC AAAATCGGCT TGCAGATTCC 651 GCACTACCTT GCCGTTATCC AATACGCCGG CAACCGCGAA CTGCGCGAAC 701 AAATCTACCG CGCCTACGTT ACCCGTGCCA 6CGAACTTTC AAACGACGGC 751 AAATTCGACA ACACCGCCAA CATCGACCGC ACGCTCGAAA ACGCATTGAA 801 AACCGCCAAA CTGCTCGGCT TTAAAAATTA CGCCGAATTG TCGCTGGCAA 851 CCAAAATGGC GGACACGCCC GAACAGGTTT TAAACTTCCT GCACGACCTC 901 GCCCGCCGCG CCAAACCCTA CGCCGAAAAA GACCTCGCCG AAGTCAAAGC 951 CTTCGCCCGC GAACACCTCG*GTCTCGCCGA CCCGCAGCCG TGGGACTTGA 1001 GCTACGCCGG CGAAAAACTG CGCGAAGCCA AATACGCATT CAGCGAAACC 1051 GAAGTCAAAA AATACTTCCC CGTCGGCAAA GTTCTGGCAG GCCTGTTCGC 1101 CCAAATCAAA AAACTCTACG GCATCGGATT CGCCGAAAAA ACCGTTCCCG 1151 TCTGGCACAA AGACGTGCGC TATTTTGAAT TGCAACAAAA CGGCAAAACC 1201 ATCGGCGGCG TTTATATGGA TTTGTACGCA CGCGAAGGCA AACGCGGCGG 1251 CGCGTGGATG AACGACTACA AAGGCCGCCG CCGCTTTGCC GACGGCACGC 1301 TGCAACTGCC CACCGCCTAC CTCGTCTGCA ACTTCGCCCC GcccGTCGGC 1351 GGCAAAGAAG CGCGTTTAAG CCACGACGAA ATCCTCACCC TCTTCCACGA 1401 AACCGGCCAC GGACTGCACC ACCTGCTTAC CCAAGTGGAC GAACTGGGCG 1451 TGTCCGGCAT CAACGGCGTA AAA 99 This corresponds to the amino acid sequence <SEQ ID) 59; ORE 128-1.ng>: g128-1.pep (partial) 1 MIDNALLHLG EEPRFNQIKT 51 NTVERLTGIT ERVGRIWGVV .101 GQDIELYNRF KTIKNSPEFA 151 ELAKLQTEGA QLSAKFSQNV 201 AAQSEGKTGY KIGLQIPHYL 251 KFDNTANIDR TLENALKTAK 301 ARRAKPYAEK DLAEVKAFAR 351 tVKKYFPVGK VLAGLFAQIK 401 IGGVYMDLYA REGKRGGAWM 451 GKEARLSHDE ILTLFHETGH EDIKPAVQTA IAEARGQIAA VKAQTHTGWA SHLNSVVDTP ELRAVYNELM PEITVFFTEI TLSPAQKTKL DHDLRDFVLS GAELPPERQA LDATDAFGIY FDDAAPLAGI PEDALAMFAA AVIQYAGNRE LREQIYRAYV TRASELSNDG LLGFKNYAEL .SLATKMADTP EQVLNFLHDL EHLGLADPQP WDLSYAGEKL REAKYAFSET KLYGIGFAEK TVPVWHKDVR YFELQQNGKT NDYKGRRRFA DGTLQLPTAY LVCNFAPPVG GLHHLLTQVD ELGVSGINGV K m128-1/gI2B-1 OR~s 128-1 and 12.8-1.ng showed a 94.5% identity in 491 aa overlap gi 28-1. pep m12 8-1 g128-1 .pep m128-1 gl28-1. pep m1 28-1 g128- pep m128-1 g128-1 .pep m128-1 g128-1. pep *m128-i g128-1. pep m128-1 g128-1 .pep 20 30 40 50 20 30 40 50 80 *90 100 110 120 ERVGRIWGVVSHLNSVTPELAVYNELMPEITVFTE IGQDIELYNRFKTIgrqSPEFA 80 90 100 110 120 130 140 150 160 170 180

TLSPAQKTKLDHDLRDFVLSGAELPPERQELLQTEGAQLSAKFSQNVLDATDAFGIY

1111111111:111111111111111:111111111111111111111111111111

IY

130 140 150 160 170 180 190 200 210 220 230 240 FDDAAPLAGIPEDALAMFAAAAQSEGKTGYKIGLQI

PHYLAVIQYAGNRELREQIYRAYV

FDDAAPLAGI PEDALAMFAAAAQSESKTGYKIGLQI

PHYLAVIQYADNRELREQIYRAYV

190 200 210 220 .230 240 250 260 270 280 .290 300

TRASELSNDGKFDNTNIDRTLENALKTAKLLGFNYAELSLATKMADTPEQVLNFLHDL

250 260 270 280 290 300 310 320 330 340 350 360 310 320 330 .340 350 360 370 .380 390 400 410 420 VLAGLFAQIKKLYGIGFAEKTVPVW~iDVRYFLQQNGKTIGGVYMDLYAEGKRGGAWM

VLNGLFAQIKKLYGIGTEKTVPVWHKDVRYELQQNGETIGGVYMDLYRGKRGGAWM

370 380 390 400 410 420 430 440 450 460 470 480 NDYKGRRRFADGTLQLPTAYLVCNFAPPVGGKEARLSHDEILTLFHETGH4GLHHLLTQVD -100m1 28-1 NDYKGRRRFSDGTLQLPTAYLVCNFAPPVGGREARLSHDETLILFHETGHGLHHLLTQVD 430 440 450 460 470 480 490

ELGVSGINGVK

490. 500 510 1520 530 540 g128-1. pep.

m1 28-1 The following DNA sequence was identified in Nk meningitidis <SEQ 11D a128-1. seq 1 ATGACTGACA ACGC.ACTGCT CCATTTGGGC GAAGAACCCC GTTTTGATCA 51 AATCAAAACC GAAGACATCA AACCCGCCCT GCAAACCGCC ATTGCCGAAG 101 CGCGCGAAOA AATCGCCGCC ATCAAAGCCC AAACGCACAC CGGCTGGGCA *151 AACACTGTCG AACCCCTGAC CGGCATCACC GAACGCGTCG GCAGGATTTG 201 GGGCGTGGTG TCGCACOTCA ACTCCGTCAC CGACACGCCC GAACTGCGCG 251 CCGCCTACAA TGAATTAATG CCCGAMTTA CCGTCTTCTT CACCGAAATC 3 01 GGACAAGACA TCGAGCTGTA CAACCGCTTC AAAACCATCA WAACTCCCC 351 CGAGTTCGAC ACCCTCTCCC ACGCGCAAAA AACCAAACTC AACCACGATC 401 TGCGCGATTT CGTCCTCAGC GGCGCGGAAC TGCCGCCCGA ACAGCAGGCA 451 GAATTGGCAA AACTGCAAC CGAAGGCGCG CAACTTTCCG CCAAATTCTC 501 CCAAAACGTC CTAGACGCGA CCGACGCGTT CGGCATTTAC TTTGACGATG 551 CCGCACCGCT TGCCGGCATT CCCGAAGACG CGCTCGCCAT GTTTGCCGCT .601 GCCGCGCAAA GCGAAGGCAA AACAGGCTAC AP.AATCGGTT TGCAGATTCC 651 GCACTACCTC GCCGTCATCC AATACGCCGA CAACCGCAAA CTGCGCGAAC 701 AAATCTACCG CGCCTACGTT ACCCGCGCCA GCGAGCT'rTC AGACGACGGC 751 AAATTCGACA ACACCGCCAA CATCGACCGC ACGCTCGAAA ACGCCCTGCA 801 AACCGCCAAA CTGCTCGGCT TCAAAAACTA CGCCGAATTG TCGCTGGCAA 851 CCAAAATGGC GGACACCCCC GAACAAGTTT TAAACTTCCT GCACGACCTC 901 'GCCCGCCGCG CCAAACCCTA CGCCGAAAAA GACCTCGCCG AAGTCAAAGC 951 CTTCGCCCGC GAAAGCCTCG GCCTCGCCGA TTTGCAACCG TGGGACTTGG 1001 GCTACGCCGG CGAAAAACTG CGCGAAGCCA AATACGCATT CAGCGAAACC 1051. GAAGTCAAAA AATACTTCCC CGTCGGCAAA GTATTAAACG GACTGTTCGC 1101 CCAAATCAAA AAACTCTACG GCATCGGATT .TACCGAAAAA ACCGTCCCCG 1151 .TCTGGCACAA AGACGTGCGC TATTTTGAAT TGCAACAAAA CGGCGAAAC 1201 ATAGGCGGCG TTTATATGGA TTTGTACGCA CGCGAAGGCA AACGCGGCGG 1251 CGCGTGGATG AACGACTACA AAGGCCGCCG CCGTTTTTCA GACGGCACGC 1301 TGCAACTGCC CACCGCCTAC CTCGTCTGCA ACTTCACCCC GCCCGTCGGC 1351 GGCAAAGAAG CCdGCTTGAG CCATGACGAA ATCCTCACCC TCTTCCACGA 1401 AACCGGACAC GGCCTGCACc ACCTGCTTAC CCAAGTCGAC GAACTGGGCG 1451 TATCCGGCAT CAACGGCGTA GATGGC CArGTCGCYTln~ GrOiCr,' 1501 1551 1601 1651 1701 .1751 1801 1851 1901 1951 2001 TTTATGGAAA ATTTCGTTTG CCACGAAGAA ACCGGCGTTC TCGCCGCCAA AAACTTCCAA TTCGCCCTCT TTGATAT.GAT G AAAAACTGG CAACAGGTTT TCCGACCGCC CGAATACAAC GCAGGCGGCT ATTCCGCAGG GAGCGCGGAC GCATACGCCG CAGGCAAACG

CTTTTGGCAG

GCGGCAGAAT CCTTCAAAGC ACTCTTGCGC CACAGCGGCT

GGAATACAAT

CCCTGCCGAA

CGCGGAATGT

GATTTACAGC

TAGACAGCGT

CGCTTCGCCA

CTATTACAGC

CCTTTGAAGA

GAAATCCTCG

CTTCCGCGGA

TCGACAACC

GTCTTGGCGc AAATGTCCGC AGAACTCTTC GACAAAATGC TCCTCGTCCG CCAAATGGAG GAAGACGACG AAGGCCGTCT GCGCAAAGAA GTCGCCGTCG ACAGCTTCGG CCACATCTTC TACGCGTGGG CGGAAGTATT AAGCGACGAT GTCGCCGCCA CCGTCGGCGG ATCGCGCAGC CGCGAACCGA GCATAGACGC

GGCTTGA

This corresponds to the amino acid sequence <SEQ ID 61; ORF 128-1.a>: a128-1. pep 1 MTDNALLHLG EEPRFDQfl(T EDIKPALQTA IAEAREQIAA IKAQTHTGWA 51 *NTVEPLTGIT ERVGRIWGVV SHLNSVTDTP ELRAAYNELM PEITVFFTEI 101 GQDIELYNRF KTIKNSPEFD TLSHAQKTKL NHDLRDFVLS GAELPPEQQA 151 ELAKLQTEGA QLSAKFSQNV LDATDAFGIY FDDAAPLAGI PEDALAHFAA 201 AAQSEGKTGY KIGLQIPHYL AVIQYADNRX LREQIYRAYV TRASELSDDG 251 KFDNTANIDR TLENALOTAK LLGFKNYAEL SLATKMADTP EQVLNFLHDL 101

APRAKPYAEK

EVKKYFPVGK

IGGVYMDLYA

GKEAP.LSHDE

FMENFVWEYN

FALFDMMIYS

AGGYSAGYYS

AAESFKAFRG

DLAEVKAFAR ESLGLADLOP VLNGLFAQIK KLYGIGErEK REGPCRGGAWM NDYKGRRRFS ILTLFHETGH GLHHLLTQVD VLAQMSAJIEE TGVPLPKELF EDDEGRLKNW QQVLDSVRKE YAWAEVLSAD AYAAFEESDD REPSIDALLR HSGFDNAA*

WDLGYAGEKL

TVPVWHKDVR

DGTLQLPTAY

ELGVSGINGV

DlQ4LAAKNFQ

VAVVRPPEYN

VAATGKRFWQ

REAKYAFSET

YFELQQNGET

LVCNFI'PPVG

EWDAVELPSQ

RGMFLVRQME

RFAINSFGHI F

EILAVGGSRS

m128-1/a128-1 ORFs 128-1 and 128-l.a showed a 97.8% identity in 677 aa overlap a128-1. pep m128-1 a128-1. pep, m128-1 a128-1. pep ml128-1 a128 pep ml128-1 a128-1. pep m12 8-1 a128-1 .pep, m128- 1 a128-1. pep m128 -1 a128-1. pep mr128 -1 a128-1 .pep 20 30 40 50 20 30 40 50 80 90 100 110 120

ERVGRIWGVVSHLNSVTDTPELRYNELPEIFTIGQDIELYNRFTI

4 S PEFD ERGIGVHNSATERVNEMETFTIGQDIELYNRFKTIKNS

PEFD

80 90 100 110 120 130 140 150 160 170 180

TLSHAQKTKLNHDLRDFVLSGALPPEQALA<LTEGALSAJ<FSQNVLDATDAFGIY

TLSPAQKTKLNHDLRDFVLSGAELPPEQQAELALQTEGALSAKJFSQNVLDATDAFGIY

130 140 150 160 170. 180 190 200 210 220 230 240 FDDAAPLAGI PEDALAMFAAAAQSEGKTGYKIGLQI PHYLAVIQYADNRKLREQIYp.AYV FDDAAPLAGI PEDALAMFAAAAQSESKTGYKIGLQIPHYLAVIQYADNRELREQIYRAYV 190 200 210 220 230 240 250 260 270 280 290 300 TRASELSDDGKFDNTANI DRTLENALQTAKLLGFKNYAELSLATMADTPEQVLNFLHDL 250 260 270 280 290 300 310 320 330 340 350 360

ARRKPYAEKDLAEVKAF'ARESLGLADLQPWDLGYAGELREAYAFSETEVKKJYFPVGK

ARiAKP11EK111EVKA IIES11:1DL11WD1GYASEK1REAKYAFSETiiiiYFPVGK 310 320 330 340 350 360 370 380 390 400 .410 420 370 380 390 400 410 420 430 440 .450 460 470 480

NDYKGRRRFSDGTLLPTAYLVCNFTPPVGGKALSHDEILTLFHETGHGLHHLLTQVD

NDYKGRRRFSDGTLQLPTAYLVCNFAPPVGGREAJRLSHDEILILFHETGHGLHHLLTQVD

430 440 450 460 470 480 490 500 510 520 530 540

ELVGNVWAEPOMNVENVAMAETVLKLDMAKF

-102m128-1 a128-1.pep m12 8-1 .a128-1 .pep m.2 8-1 a128-1 .pep m12 8-1 490 500 510 520 530 540 550 560 570 580 590 600 550 560 570 .580 590 600 610 620 630 640 650 660 610 620 630 640 650 660 670 679 REPSI DALLRHSGFDNAAX 11111111IIIII:

REPSIDALLRHSGFDNAVX

670 The following partial DNA sequence was identified in N. meningitidis <SEQ ID 62>: m206.seq

ATGTTTCCCC

CGCCTCATGC.

AGACAGTCCG

CAAGGCTCGC

CTACAAATGG

TGATTCAATT

GCCCGCGACA

GGCCGGCGAC

ACGTCGGACT

CCGACAAAAC

GGCACGACCT

GCAAATCCAA

*AGGAACTCAT

GGCGGCAGCA

CGTTTACAAr

TGGCGGCGGC

CTCGTATTCT

CTACATCGGC

CCTTTTCCTC

CCGGCAAACA

GCCGTCCGCA

GCTCCACAGC

GCACCGCAAC

AACGCCCTCA

AAGCCGsAAA TC AACACCGG

AACGGCGAAT

TGTCTCAGCG

CCGCCAACCG

TCAGCCACAT

CTCGGACTCA

CGGCTTCGAT

ACGTCAAGCT

ATCCCCGACA

CGGCGCACAC

TCATCCATGC

CACTGCTCCT

AAACCCAAAC

CGACCGCACA

TCGGCACGCC

TGCAGCGGCA

GCCGCGCACC

GCCGCyTCAA

CGCTACTCAC

451 GGCAAAACCA TCAAAACCGA AAAACTCTCC ACACCGTTTr ACGCCAAAAA 501 CTACCTCGGC GCACATACTT TTTTTACAGA ATGA This corresponds to the amino acid sequence <SEQ ]D 63; ORF 206>: m206 .pep..

1 MFPPDKTLFL CLSALLLASC GTTSGKHRQP KPKQTVRQIQ AVRISUIDRT 51 QGSQELMLHs LGLIGTPYKw GGSSTATGFD CSGMIQFVYK NALNVKLPRT.

101 ARDMAAASRK IPDSRXKAGD LVFFNTGGIUJ RYSHVGLYIG NGEFIHAPSS 15,1 GKTIKTEKLS TPFYAKIUYLG AHTFFTE* The following partial DNA sequence was identified in N. gonorrhoeae <SEQ ID 64>: g206.seq atgttttccc cgcctcatgc agacagtccg caaggctcgc ctacaaatgg tgattcaatt gcccgcgaca ggccggcgac acgtcggact ggcaaaacca ctaccttgga ccgacaaaac ggcacgacct gcaaatccaa aggaactcat ggcggcagca g9tttacaaa tggcggcggc atcgtattct ctacatcggc cCttttcctc ccggcaaaca gccgtccgca gCtCcacagc gcaccgcaac aacgccctca aagccgcaaa tcaacaccgg aacggcgaat tgtctcggcg ccgccaaccg tcagccacat ct cggactca cggct tcgac acgtcaagct atccccgaca Cggcgcacac tcatccatgc acaccgtttt atga caCtgctcct aaacccaaac cggccgoaca tcggcacgcc tgcagcggca gccgcgcacc gccgcctcaa cgctactcac ccccggcagc acgccaaaaa tCaaaaccga'aaaactctcc gcgcatacgt tttttacaga 103 This corresponds to the amino acid sequence <SEQ ID 65; ORF 206.ng>: 1 MFSPDKTLFL CLGALLLASC GTTSGKHRQP KPKQTVRQIQ AVRISHIGRT 51 QGSQELMLHS LGLIGTPYKW GGSSTATGFD CSGMIQLVYK NALNVKLPRT 101 ARDMAAASRK IPDSRLKAGD IVFFNTGGAH RYSHVGLYIG NGEFIHAPGS 151 GKTIKTEKLS TPEFYAIGNYLG AHTFFTE* ORF 206 shows 96.0% identity over a 177 aa overlap with a predicted ORF (ORF 206.ng) from N gonorrhoeae: m2 06/g2 06 2030 40 50 m206 .pep MFPPDKTLFLCLSALLLASCGTTSKHQPKPKQTVRJQIQAVRISHIDRTQGSQELMLHS g2 06 MFPKLLLALACTSKRPKKTRIARSIROSEMH 20 .30 40 50 80 90 100 110 120 m206 .pep LGITYWGSAGDSMQVKANKPTR)AARIDRKG g 206 LGITYWGSAGDSMQVKNLVLRADAARIDRKG 80 90 100 110 120 130 140 150 160 170 m2 06.pep LVFFNTGGAHRYSHVIGYEIHAPGFII4SSrEKSTPFYYLGHTFFEX g2 06 IVFFNTGGAHRYSHVGLYIGNGEFIHAPGSGTI KTEKL STPFYAKI4YLGAHTFFTE 130 .140 150 160 170 The following partial DNA sequence was identified in N meningitidis <SEQ ID 66>: a2 06.seq 1 ATGTTTCCCC CCGACAAAAC CCTTTTCCTC TGTCTCAGCG CACTGCTCCT 51 CGCCTCATGC GGCACGACCT CCGGCAAACA CCGCCAACCG AAACCCAAAC 101 AGACAGTCCG GCAAATCCAA GCCGTCCGCA TCAGCCACAT CGACCGCACA *151 CAAGGCTCGC AGGAACTCAT GCTCCACAGC CTCGGACTCA TCGGCACGCC 201 CTACAAATGG GGCGGCAGCA GCACCGCAAC CGGCTtCGAT TGCAGCGGCA 251 TGATTCAATT CGTTTACAAA AACGCCCTCA ACGTCAAGCT GCCGCGCACC 301 GCCCGCGACA TGGCGGCGGC AAGCCGCAAA ATCCCCGACA GCCGCCTTAA 351 GGCCGGCGAC CTCGTATTCT TCAAOACCGG CGGCGCACAC CGCTACTCAC 401 ACGTCGGACT CTATATCGGC AACGGCGAAT TCATCCATGC CCCCAGCAGC 451 GGCAAAACCA TCAAAACCGA AAAACTCTCC ACACCGTTTT ACGCCAAAAA 501 CTACCTCGGC GCACATACTT TCTTTACAGA ATGA This corresponds to the amino acid sequence <SEQ ID 67; ORF 206.a>: a206.pep 1 MFPPDKTLFL CLSALLLASC GTTSGKHRQP KPKQTVRQIQ AVRISHIDRT 51 QGSQELMLHS LGLIGTPYKW GGSSTATGFD CSGMIQFVY( NALNVKLPRT 101 ARDMAAASR( IPDSRLKAGD LVFFNTGGAH RYSHVGLYIG NGEFIHAPSS 151 GKTIKTEKLS TPFYAKNYLG AHTFFTE* ni206/a206 ORFs 206 and 206.a showed a 99.4% identity in 177 aa overlap 20 30 40 50 r2 06. .pep MFPPDKTLFLCLSALLLASCGTTSGKHRQPKPKQTVRQIQAVRISHIDRTQGSQELMLHS a206 MFPPDKTLFLCLSALLLASCGTTSGKHRQPKPKQTVRQIQAVRISHIDRTQGSQELMLHS .20 30 40 50 -104- 80 90 100 110 120 m206 .pep LGITYWGSAGDSMQVKNLVLRADAARIDRKG a206 LGLIGTPYKWGGSSTATGFDCSGMIQFVYKNALNVKLPRTRDMAAASRKPDSRLKAGD 80 90 100 110 120 130 140 150 160 170 m2 06. pep LVFFNTGGAHRYSHVGLYIGNGEFIAPSSGKTIKTEKLSTPFYKNYLGHTFFTEX 6 LVFTGHYHGYGGFRPSGTKELTFANLATFE 130 140 150 160 170 287, The folIlowing partial DNA sequence was identified in N meningitidis <SEQ ID) 68>: m287.seq ATGTTTAAAC GCAGCGTAAT CGCAATGGCT CTGCGGGGGC GGCGGTGGCG GATCGCCCGA TGTCAAAACC TGCCGCCCCT GTTGTTTCTG GAAGATGCGC CACAGGCAGG TTCTCAAGGA AGGCAGTCAA GATATGGCGG CGGTTTCGGA GTGCGGTAAC AGCGGATAAT CCCAAAAATG GATATGCCGC AAAATGCCGC CGGTACAGAT CCCGGATCCG AATATGCTTG CCGGAAATAT CCGGGGAATC GTCTCAGCCG GCAAACCAAC GACGGAATGC AGGGGGACGA TCCGTCGGCA TACGGCTGCC CAAGGTGCAA ATCAAGCCGG CTTCAGATCC CATCCCCGCG TCAAACCCTG AATTTTGGAA GGGTTGATTT GGCTAATGGc GCAAAATATA ACGTTGACCC ACTGTAAAGG

TGTATTTTTG

TGTCAAGTCG

AAAAAGAGAC

CAGGGCGCGC

AGAAAATACA

AAGACGAGGT

AGTTCGACAC

CCCTTTCAGC

GCGGACACGC

AGAGGCAAAG

CATCCGCACA

GGCAATGGCG

GGCACAAAAT

701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451

ATTTCTTGGA

GATGCAGACA

TGTCGGTTTG

TTATCTTTTA

GCACGGTCGA

TCAGGCGGAT

ATTCCGGCAA

GGGGCGGAAA

ACCGGCAAAA

TACTGCATTT

TTTGCCGCAA

CAGCGGCGAT.

ATGGAAACGG

TCCGGAAAGT

CTATCGCCCG

AAAAAGAGCA

TGAAGAAGTA

AAATAAGTAA

GTTGCCGATA

TAAACCTAAA

*GGCGGTCGCT

ACGCTGATTG

TATCTTCGCG

AATTGCCCGG

GGCGAAATGC

CCATACGGAA

AAGTCGATTT

GATTTGCATA

CTTTAAGGGG

TTTACGGCCC

ACAGATGCGG

GGAT*TGA

CAGCTAAAAT

TTACAAGAAA

GTGTGCAGAT

CCCACTTCAT

TCCGGCCGAG

TCGATGGGGA

CCCGAAGGGA

CGGATCGTAT

TTGCGGGCGC

AACGGCCGTC

CGGCAGCAAA

TGGGTACGCA

ACTTGGACGG

GGCCGGCGAG

AAAAGGGCGG

GGAAAATCAA GCAACGGATG CGGATATGGC AAATGCGGCG GGCGGGCAAA ATGCCGGCAA AAACAATCAA GCCGCCGGTT CACCTGCGAA TGGCGGTAGC GTTTTGATTG ACGGGCCGTC CGATTCTTGT AGTGGCAATA CAGAATTTGA AAAATTAAGT GATGGGAAGA ATGATAAATT GAAGGGAATC AATCAATATA TTGCGCGATT TAGGCGTTCT ATGCCGCTGA TTCCCGTCAA AGCGGTCAGC CTGACGGGGC ATTACCGGTA TCTGACTTAC GCCCTTCGTG TTCAAGGCGA GGCCGTGTAC AACGGCGAAG CGTACCCGAC CAGGGGCAGG TCTGTGGACG GCATTATCGA AAAATTCAAA GCCGCCATCG AAAATGGCAG CGGGGATGTT GAAGTGGCGG GAAAATACAG ATTCGGCGTG TTTGCCGGCA This corresponds to the amino acid sequence <SEQ ID 69; ORE 287>: m287.pep MFKRSVIAMA CIFALSACGG GGGGSPDVKS EDAPQAGSQG QGAPSAQGSQ

DMAAVSEENT

DMPQNAAGTD SSTPNHTPDP NMLAGNMENQ DGMQGDDPSA GGQNAGNTAA QGA1NQAGNNQ NFGRVDLANG VLIDGPSQNI TLTHCKGDSC DADKISNYK( DGKNDKFVGL VADSVQMKGI ARSRRSLPAE MPLIPVNQAD TLIVDGEAVS

ADTLSKPAAP

GNGGAVTADN

ATDAGESSQP

AAGSSDPIPA

SGNNFLDEEV

NQYI IFYKPK

LTGHSGNIFA

VVSEKETEAK

PKNEDEVAQN

AN'QPDMANAA

SNPAPANGGS

QLKSEFEKLS

PTS FAR FRRS

PEGNYRYLTY

105 351 GAEKLPGGSY 401 FAAXVDFGSK 451 SGKFYGPAGE ALRVQGE PAK

SVDGIIDSGD

EVAGKYSYRP

GEMLAGAAVY

DLHk4GTQXFK

TDAEKGGFGV

NGEVLHFHTE

AAIDGNGFcG

FAGKKEQD*

NGRPYPTRGR

1WTENGSGDV The following partial DNA sequence was identified in N. gonorrhoeae <SEQ CK1 g287.seq 1 51 101 151 201 251 301 351 .401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 atgtttaaac ctgtgggggc cgtcaaaacc ctgccgaaag cgatacgcag tt tcggcaga aaaaatgaag atccgcaaat cccccgcgtC acgaacgtgg gt tgacccac aagaagcacc attaagcgat tgctgacagg cggacaaacc .gagattccgc ggaagcggtc ggaattaccg tatgccctcc cacggccgtg gtccgtaccc aaatctgtgg gcaaaaattc cggaaaatgg gaggaagtgg cggattcggc gcagtgtgat ggcggtggcg ggCCgCCdCC aaaagaaaga gacgcaaccg aaatacaggc acgcgggggc caaacaggga aaaCcctgcc gcaattctgt tgtaaaggcg gtcaaaatca ataaaaaaga gtaaaaaagg acctactcgt tgattcccgi agcCtgacgg gtatctgact gtgqtgcaagg tacaacggcg gtccggaggc acggcattat aaagccgcca cggcggggat cgggaaaata gtgtttgccg tgcaatggct gatcgcccga gttgttgctg tgaggaggca ccggagaagg aatggcggtg gcaaaatgat acaaccaacc cctgcgaatg tgtgattgac attcttgtaa gaatttgaaa cgagcaacgg atggaactad tctgcacggt caatcaggcc ggcattccgg tacggggcgg cgaaccggca aagtgctgca aggtttgccg cgacagcggc tcgatggaaa gtttccggaa cagctatcgc gcaaaaaaga tgtatttttc tgtcaagtcg Aaaatgccgg.

gcgggcggtg cagccaagat cggcaacaac atgccgcaaa cgccggttct gcggtagcga ggaccgtcgc tggtgataat aattaagtga gagaattttg caaatatatc cgaggaggtc gatadgctga caatatcttc aaaaattgcc aaaggcgaaa tttccatatg caaaagtcga gatgatttgc cggctttaag ggttttacgg ccgacagatg tcgggattga CCCtttcagc gcggacacgc ggaaggggtg cgccgcaagc atggcggcag ggacaacccc atgccgccga tcagattccg ttttggaagg aaaatataac ttattggatg tgaagaaaaa tcggtttggt atcttctata gcttccggcc ttgtggatgg gcgcccgaag cggcggatcg tgcttgttgg gaaaacggcc tttcggcagc atatgggtac gggacttgga cccggccggC ctgaaaaggg This corresponds to the amnino, acid sequence <SEQ ID) 71; ORE 287.ng>: g287.pep 1 MFKRSVIAMA CIFPLSACGG GGGGSPDVKS ADTPSKPAAP VVAENAGEGV 51 LPKEKI<DEEA AGGAPOADTQ DATAGEGSQD MAAVSAENTG NGGAATTDNP 101 KNEDAGAQND MPQNAAESAN QTGNNQPAGS SDSAPASNPA PANGGSDFGR 151 TNVGNSVVID GPSQNITLTH CKGDSCNGDN LLDEEAPSKS EFEKLSDEEK 201 IKRYKKDEQR ENFVGLVADR VKKDGTNKYI IFYTDKPPTR SARSRRSLPA 251 EIPLIPVNQA DTLIVDGEAV SLTGHSGNIF APEGNYRYLT YGAEKLPGGS 301 YALRVQGEPA KGEMLVGTAV YNGEVLHFHM ENGRPYPSGG RFAAKVD'GS 351 KSVbGIIDSG DDLHMGTQKF KAAIDGNGFK GTWTENGGGD VSGRFYGPAG 401 EEVAGKYSYR PTDAEKGGFG VFAGKKDRD* m287/g287 OR~s 287 and 287.ng showed a 70.1% identity in 499 aa overlap 20 30 40 49 m287. pep MFKRSVIAMACIFALSACGGGGGGSPDVKSADTLSKPAAPVVSE

KETEA

g287 MFKRSVIAMACIFPLSACGGGGGSPDVKSADTPSKPAPVANAGEGVLPKEKKDEEA 20 30 40 50 60 70 so 90 100 109 m287 .pep KEDAPQAGSQGQGAPSAQGSQDMJAAVSEENTGNGGAVTADNPKJNEDEVANDMPQNAAGT g2 87 AGAQDQ-AAESDAVANTNGATNKEAANMQA- 80 90 100 110 -106- 110 120 130 140 150 160 169 m287 .pep DSTNTDNLGMNrTaESPNPMNAGQDPAGNGT g2 87 170 180 190 200 210 220 229 m287 .pep AQAQGNAGSPPSPPNGSFRDAGLDPOILHKD g28 7 -ESANQTGNNQPAGSSDSAPASNPAPANGGSDFGRTNVGNSVVIDGPSQNITLTHCKGDS 120 130 140 10160 170 230 240 250 260 270 280 289 in287 .pep CSNFDEQKEELDDINKKGNKVLASQKIQIFK g287 CNGDNLLDEEAPSKSEFEKLSDEEKIKRYKkDEQPNVGLVADRVKDGTKYIIFYTD 180 190 200 210 220 230 290 300 310 320- 330 340 349 mr287 .pep KPTSFARFRRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPENYRYLT 240 250 260 270 280 290 350 360 370 380 390 400 409 n2 87. pep YGELGSARQEAGMAAVYGVHHEGPPRRAKDG g2 87 YG AEKLPGGSYALRVQGEPGEMLVGTAGEVLHFJENGRPYPSGGRFKDFGS 300 310 320 330 340 350 410 420 430 440 450 460 469 m2 87. pep KSVDGIIDSGDDLHMGTQKFKAAIDGNGFKGTWTENGSGDVSGKF.YGPAGEEVAGKYSYR g2 87 KSVDGIIDSGDDLHGTQKFKAIDGNGF(GTWTENGGGDVSGRFGPAGEEVAGKYSYR 360 370 380 390 400 410 470 480 489 m287 .pep PTDAEKGGFGVFAGKKEQDX g287 PTDAEKGGFGVFAGKKDRDX 420 430 The following partial DNA sequence was. identified in N. meningitidis <SEQ ID 72>: a287. seq I ATGTTTAAAC GCAGTGTGAT TGCAATGGCT TGTATTGTTG CCCTTTCAGC 51 CTGTGGGGGC GGCGGTGGCG GATCGCCCGA TGTTAAGTCG GCGGACACGC 101 TGTCAAAACC TGCCGCCCCT GTTGTTACTG AAGATGTCGG.GGAAGAGGTG 151 CTGCCGAAAG AAAAGAAAGA TGAGGAGGCG GTGAGTGGTG CGCCGCAAGC 201 CGATACGCAG GACGCAACCG CCGGAAAAGG CGGTCAAGAT ATGGCGGCAG 251 TTTCGGCAGA AAATACAGGC AATGGCGGTG CGGCAAOAAC GGATAATCCC 301 GAAAATAAAG ACGAGGGACC GCAAAATGAT ATGCCGCAAA ATGCCGCCGA 351 TACAGATAGT TCGACACCGA ATCACACCCC TGCACCGAAT ATGCCAACCA 401 GAGATATGGG AAACCAAGCA CCGGATGCCG GGGAATCGGC ACAACCGGCA 451 AACCAACCGG ATATGGCAAA TGCGGCGGAC GGAATGCAGG GGGACGATCC 501 GTCGGCAGGG GAAAATGCCG GCAATACGGC AGATCAAGCT GCAAATCAAG 551 CTGAAAACAA TCAAGTCGGC GGCTCTCAAA ATCCTGCCTC TTCAACCAAT 601 CCTAACGCCA CGAATGGCGG CAGCGATTTT GGAAGGATAA ATGTAGCTAA 651 TGGCATCAAG CTTGACAGCG GTTCGGAAAA TGTAACGTTG ACACATTGTA 701 AAGACAAAGT ATGCGATAGA GATTTCTTAG ATGAAGAAGC ACCACCAAAA 751 TCAGAATTTG AAAAATTAAG TGATGAAGAA AAAATTAATA AATATAAAAA 801 AGACGAGCAA CGAGAGAATT TTGTCGGTTT GGTf1TGCTGAC AGGGTAGAAA -107- 851 901 951 1001 1051 1101 1151 120 1251 1301 1351 1401 1451

AGAATGGAAC

TCTTCATCTG

GGCCGAGATG

ATGGGGAAGC

GAAGGGAATT

ATCGTATGCC

CGGGCACGGC

GGCCGTCCGT

CAGCAAATCT

GTACGCAAAA

TGGACGGAAA

CGGCGAAGAA

AGGGCGGATT

TAACAAATAT

CGCGATTCAG

CCGCTGATTC

GGTCAGCCTG

ACCGGTATCT

CTCAGTGTGC

CGTGTACAAC

CCCCGTCCGG

GTGGACGGCA

ATTCAAAGCC

ATGGCGGCGG

GTGGCGGGA.

CGGCGTGTTT

GTCATCATTT

GCGTTCTGCA

CCGTCAATCA

ACGGGGCATT

GACTTACGGG

AAGGCGAACC

GGCGAAGTGC

AGGCAGGTTT

TTATCGACAG

GTTATCGATG

GGATGTTTcC

AATACAGCTA

GCCGGCAAAA

ATAAAGACAA

CGGTCGAGGC

GGCGGATACG

CCGGCAATAT

GCGGAAAAAT

GGCAAAAGGC

TGCATTTCCA

GCCGCAAAAG

CGGCGATGAT

GAAACGGCTT

GGAAGGTTTT

TCGCCCGACA

AAGAGCAGGA

GTCCGCTTCA

GGTCGCTTCC

CTGATTGTCG

CTTCGCGCCC

TGTCCGGCGG

GAAATGCTTG

TATGGAAAAC

TCGATTTCGG

TTGCATATGG

TAAGGGGACT

ACGGCCCGGC

GATGCGGAAA

TTGA

This corresponds to the amino acid sequence <SEQ ID 73; ORF 287.a>: a287.pep MFKRSVIANA CIVALSACGG LPKEKKDEEA VSGAPQADTQ ENKDEGPQND MPQNAADTDs NQPDMANAAD GMQGDDPSAG PNATNGGSDF GRINVANGIK SEFEKLSDEE KINKYKKDEQ SSSARFRRSA RSRRSLPAEM EGNYRYLTYG AEKLSGGSYA GRPSPSGGRF AAKVDFGSKS

GGGGSPDVKS

DATAGKGGQD

STPNHTPAPN

ENAGNTADQA

LDSGSENVTL

RENEVGLVAD

PLI PVNQADT LSVQGE PAKG VDGI IDSGDD ADTLSKPAAP VVTEDVGEEV MAAVSAENTG NGGAATTDNP MPTRDMGNQA PDAGESAQPA AN'QAENNQVG GSQNPASSTN THCKDKVCDR DFLDEEAPPK RVEKNGTNKY VIIYKDKSAS LIVDGEAVSL TGHSGNI FAP EMLAGTAVYN GEVLHFHMEN LHMGTQkFKA VIDGNGFKGT DAEKGGFGVF AGKKEOD* WTENGGGDVS GRFYGPAGEE VAGKYSYRPT m287/a287 ORFs 287 and 287.a showed a 77.2% identity in 501 aa overlap 20 30 40 49 m2 87. pep MFKRSVIAMACIFALSACGGGGGGS PDVKSADTLSKPA.APWVSE------------

KETEA

a2 87 MFKRSVIAMACIVALSACGGGGGGSPDVKSADTLSKPAPVTEDVGEEVLPKEKKDEEA 20 30 40 50 m287.pep a2 87 60 70 80 90 100 109 80 90 100 110 110 120 130 140 150 160 169 ru287 .pep DSSTPNHTPDPNMLAGNMENQATDAESSQPNQPDMANAADGM0GDDPSAGGQNAGNTA a2 87 DSSTPNHTPAPNMPTRDMGNQAPDAGESAQPNQPDANAADGMQGDDPSAG-ENAGNTA 120 130 140 150 160 170 170 180 190 200 210 220 229 m287.pep AQGANQAGNQAGSSDPIPASNPAPAGGSNFGRVDLAGVLIDGPSQNITLTHCKGDS a287 DQAANQANQVGGSQNPASSTNPNATNGGSbFGRINVAGIKLDSGSENVTLTHCKDKV 180 190 200 210 220 230 230 240 250 260 270 280 289 m287 .pep CSGNNFLDEEVQLKSEEKLSDADKISNYKDGKNDKEVGLVADSVQMKGINQYIIFYKP a287 CDRFDEPKEELDEIKKKERNVLARENTKVIK 240 250 260 270 280 290 290 300 310 320. 330 340 m287 .pep KP--TSFARFRRSARSRRSLPAEMPLIPIJNQADTLIVDGEAVSLTGHSGNIFAPEGNYRY -108a287 KSASSSSARFRRSARSRRSLPAESMPLIPVNQADTLIVDGEAVSLTGHSGNI

FAPEGNYRY

300 310 320 330 340 350 350 360 370 380 390 400 m28 7. pep LTYGAEKLPGGSYALRVQGEPAKGEAGAVYNGEVLHFHTENGRPYPTRGRFAKDF a287 LTYGAEKLSGGSYALSVQGEPKGEMLAGTANGEVLHFMENGRPSPSGGRFAIOVF 360 370 380 390 400 410 410 420 430 440 450 460 m287. pep GSKSVDGI IDSGDDLHNMGTQKFKAAIDGNGFKGTWTENGSGDVSGKFYGPAGEEVAGKYS a287 GSKSVDGIIDSGDDLHMGTQKFKAVIDGNGFKGTWTENGGGDVSGRFYGPAGEEVAGKYS 420 430 440 450 460 470 470 480 489 *m287 .pep YRPTDAEKGGFGVFAGKKEQDX a287 YRPTDAEKGGFGVFAGKKEQDX 480 490 406 The following partial DNA sequence was identified in N. meningitidis <SEQ ID 74>: m406 .seq

ATGCAAGCAC

CGCCTGCGGG

TTGCGGTCGA

GACATGGATT

CACTATGGGC

TTGATGCACT

GATTACACCT

TTTGACAGGT.

CTCGCACCCA

ATTGGCGGGA

CGACACTGCC

GCATAGACGT

ATCGACGTAT

TGCCGAAACA

GGCTGCTGAT

ACACTGACAG

ACAAGAACTT

TACAGGCATT

GACCAAGGTT

GATTCGTGGC

ATCCACGTTA

TTAACCACTT

ATCAGACGGT

TGGGATTA

TTTCTTTCCC

TGTTTCTCCT

TCGGAACGAT

CTGAAAGCCC

ACCTATTCTT

GTATTCCATC

GTGGCCGCTT

ACACGGACG.A

CAGGCAGTTT

GAATACATAA

CGAAACCACC

CTTTATCTAC

AGCGGAAGTA

TTTTCAGTTT TTATTTTATC GCATGGCGGA GGTAAACGCT CTGCCAGAGC TGCCGTTAAA AAAGTTGCAT TGTACATTGC GACAGGGGGT CGCTACTCCA ACAGCCCTGC CGTCCGTACC GCTGAAACAA CATCAGGCGG ACTTAATGCC CCTGCACTCT AAAGCAGTCT GGGCTTAAAT TCGAAATGAA.ACCTTGACGA

CTAACCCGCG

ACTTGGTACA GACCGTATTT.TTCCTGCGCG GCCAATGCCG ATACAGATOT GTITTATTAAC ACGCAACAGA ACCGAAATGC ACCTATACAA AAACAAAACT GGAA TATTTC GCAGTAGACA ATCAAACCAA AAACCAATGC GTTTGAAGCT ATTGTGGATG GGGCCGTATA AAGTAAGCAA GATTAATGGT CGATTTCTCC GATATCCGAC 701 GAACCAATAA AAAATTGCTC 751 GCCTATAAAG AAAATTACGC 801 AGGAAT TAAA CCGACGGAAG.

*851 CATACGGCAA TCATACGGGT AACTCCGCCC CATCCGTAGA GGCTGATAAC 901 AGTCATGAGG GGTATGGATA CAGCGATGAA GTAGTGCGAC AACATAGACA 951 AGGACAACCT TGA This corresponds to the amino acid sequence <SEQ ID 75; ORF 406>: m406 .pep 1 MOARLLIPIL FSVFILSACG TLTGIPSHGG GKRFAVEQEL VAASARAAVK 51 DMDLQALHGR KVALYIATMG DQGSGSLTGG RYSIDALIRG EYINSPAVRT 101 DYTYPRYETT AETTSGGLTG LTTSLSTLNA PALSRTQSDG SGSKSSLGLN 151 IGGMGDYRNE TLTTNPRDTA FLSHLVQTVF FLRGIDVVSP ANADTDVFIN 201 IDVFGTIRNR TEMHLYNAET LKAOTKLEYF AVDRTNKKLL IKPKTNAFBA 251 AYKENYALWM GPYKVSKGIK PTEGLI4VDFS DIRPYGNMTG NSAPSVEADN 301 SHEGYGYSDE VVRQHRQGQP -109- The following partial DNA sequence was identified in N gonorrhoeae <SEQ ID) 76>: g406.seq

I

51 101 151 201 251 301 351 401 451 501 551 601 651 7 01 751 801 851 901 951

ATGCGGGCAC

CGCCTGCGGG

TCGCGGTCGA

GACATGGATT

AACTATGGGC

TTGATGCACT

GATTACACCT

TTTGACGGGT

CGCGCACCCA

ATTGGCGGGA

CGACACTGCC

GCATAGACGT

ATCGACGTAT

TGCCGAAACA

GGCTGCTGAT

ACACTGACAG

ACAAGAACTT

TACAGGCATT

GACCAAGGTT

GATTCGCGGC

ATCCGCGTTA

TTAACCACTT

ATCAGACGGT

TGGGGGATTA

TTTCTTTCCC

TGTTTCTCCT

ACCTATTCTT

GTATTCCATC

GTGGCCGCTT

ACACGGACGA

CAGGCAGTTT

GAATACATAA

CGAAACCAC

CTTTATCTAC

AGCGGAAGTA

TCGAAATGAA

ACTTGGTGCA

GCCAATGCCG

TTTTCAGTTT TTATT2'TATC GCATGGCGGA GGCAAACGCT CTGCCAGAGc TGCCGTTAAA

AAAGTTGCAT.TGTACATTGC

GACAGGGGGT CGCTACTCCA ACAGCCCTGC CGTCCGCACC GCTGAAACAA CATCAGGCGG .ACTTAATGCC CCTGCACTCT GGAGCAGTCT GGGCTrTAAAT ACCTTGACGA CCAACCCGCG GACCGTATTT TTCCTGCGCG ATACAGATGT GTTTATTAAC ACCGAAATGC ACCTATACAA GGAATATTTC GCAGTAGACA AAACCAATGC GTTTGAAGCT GGGCCGTATA AAGTAAGCAA CGATTTCTCC GATATCCAAC CATCCGTAGA GGCTGATAAC GCAGTGCGAC AACATAGACA TCGGAACGAT ACGCAACAGA CTGAAAGCCC AAACAAAACT GAACCAATAA AAAATTGCTC ATCAAACCCA GCCTATAAAG AAAATTACGC ATTGTGGATG AGGAATCAAA CCGACGGAAG GATTGATGGT CATACGGCAA TCATACGGGT AACTCCGCCC AGTCATGAGG GGTATGGATA CAGCGATGAA AGGGCAACCT TGA This corresponds to the amino acid sequence <SEQ ID 77; ORF 406.ng>: g406.pep 1 MRARLLIPIL FSVFILSACG TLTGIPSHGG GKRFAVEQEL VAASARAAVK 51 DMDLQALHGR KVALYIATMG DOGSGSLTGG RYSIDALIRG EYINSPAVRT 101 DYTYPRYETT AETTSGGLTG LTTSLSTLjNA PALSRTQSDG SGSRSSLGLN 151 IGGMGDYRNE TLTTNPRDTA FLSHLVQTVF FLRGIDVVSP ANADTDVFIN 201 IDVFGTIRNR TEMHLYNAET LKAOTKLEYF AVDRTNKKJJL IKPKTNAFEA 251 AYKENYALWM GPYKVSKGIK PTEGLMVDFS DIQPYGNHTG NSAPSVEADN 301 SHEGYGYSDE AVRQHRQGQP* OR F.406.ng shows 98.8% identity over a 320 aa overlap with a predicted ORE (0RF406.a) .from N gonorrhoeae: g406/m4O6 20 30 40 50 g406 .pep MRRLPLSFLAGLGPHGKFVOLASRAKMrALG m4 06 MOARLLIPILFSVFILSACGTLTGI PSHGGGRFAV QELVvAAVKDMDrLGR 20 30 40 s0 80 90 100 110 120 9406 .pep KVLYIATMGDOGSGSLTGGRYIDALIRGEYINSPAVRTDYTYPRYETTAETTSGGLTG m4 06 KVLYIATMGDQGSGSLTGGRYSIDALIRGEYINSPARTDYTYPRYETTAETTSGGLTG 80 90, 100 110 120 130 140 150 160 170 180 g406. pep LTTSLSTMAPALSRTQSDGSGSRSSLGLNIGG4GDYRNETLTTNPRDTAFLSHLVQTJF 130 140 150 160 170 180 190 90 200 210. 2202324 230 240 110- 9406.pep FLGDVPNDDFNDFTRRTMLNELATLYADTKL m4 06 FLGDVPNDDFNDFTRRTMLNELATLYADTKL 190 200 210 220 230 240 250 260 270 280 290 300 94 06.pep IKKNFAYEYLMPKSGKTG4]FDOYNTNASED m4 06 IKPKTNAFEAAYKENYALWMGPYKVSKGITEGFSDIRPYGHTGNSAPSEADN 250 260 .270 280 290 300 310 320 g406.pep SHEGYGYSDEAVRQHiRQGOPX m4 06 SHEGYGYSDEVVRQHRQGQPX 310 320 The following partial DNA sequence was identified in N. meningitidis <SEQ ID 78>: a406. seq 51 101 151 201 251 301 *351 .401 451 501 551 601 *651 701 751 .801 851 901 951

ATGCAAGCAC

CGCCTGCGGG

TCGCGGTCGA

GACATGGATT

AACTATGGGC

TTGATGCACT

GATTACACCT

TTTGACAGGT

CGCGCACCCA

ATTGGCGGGA

CGACACTGCC

GCATAGACGT

ATCGACGTAT

TGCCGAAACA

GAACCAATAA

GCCTATAAAG

AGGAATTAAA

CATACGGCAA

AGTCATGAGG

AGGGCAACCT

GGCTGCTGAT

ACACTGACAG

ACAAGAACTT

TACAGGCATT

GACCAAGGTT

GATTCGTGGC

ATCCACGTTA

TTAACCACTT

ATCAGACGGT

TGGGGGATTA

TTTCTTTCCC

TGTTTCTCCT

TCGGAACGAT

CTGAAAGCCC

AAAATTGCTC

AAAATTACGC

CCGACAGAAG

TCATATGGGT

GGTATGGATA

TGA

ACCTATTCTT

GTATTCCATC

GTGGCCGCTT

ACACGGACGA

CAGGCAGTTT

GAATACATAA

CGAAACCACC

CTTTATCTAC

AGCGGAAGTA

TCGAAATGAA

ACTTGGTACA

GCCAATGCCG

ACGC AACAGA

AAACAAAACT

ATCAAACCAA

ATTGTGGATG

GATTAATGGT

AACTCTGCCC

CAGCGATGAA

TTTTCAGTTT

GCATGGCG.GA

CTGCCAGAGC

AAAGTTGCAT

GACAGGGGGT

ACAGCCCTGC

GCTGAAACAA

ACTTAATGCC

AAAGCAGTCT

ACCTTGACGA

GACCGTATTT

ATACGGATGT

ACCGAAATGC

GGAATATTTC.

AAACCAATGC

GGACCGTATA

CGATTTCTCC

CATCCGTAGA

GCAGTGCGAC

TTATTTTATC

GGTAAACGCT

TGCCGTTAAA

TGTACATTGC

CGCTACTCCA

CGTCCGTACC

CATCAGGCGG

CCTGCACTCT

GGGCTTAAAT

CTAACCCGCG

TTCCTGCGCG

GTTTATTAAC

ACCTATACAA

GCAGTAGACA

GTTTGAAGCT

)AAGTAAGCAA

GATATCCAAC

GGCTGATAAC

GACATAGACA

This corresponds to the amino acid sequence <SEQ ID) 79; ORF 406.a>: a406.pep 1 MQARLLIPIL SVFILSACG TLTGIPSHGG GKRFAVEQEL VAASA 51 DMDLQALHGR KVALYIATMG DQGSGSLTGG RYSIDALIRG EYINSI 101 DYTYPRYETT AETTSGGLTG LTTSLSTLNA PALSRTQSDG SGSKS1 151 IGGMGDYRNE TLTTNPRDTA FLSHLVQTVF FLRGIDVVSP AI4ADTI 201. IDVFGTIRNR TEMHLYNAET LKAQTKLEYF AVDRTNKKLL IK*PKTI 251 AYKENYALWM GPYKVSKGIK PTEGLMVDFS DIOPYGNHMG NSAPS~ 301 SHEGYGYSDE AVRRHRQGQP

.AAVK

?AVRT

~LGLN

)VFfl4

AFEA

TEADN

m406/a406 ORFs 406 and 406.a showed a 98.8% identity in 320 aa overlap 20 30 40 50 m4 06..pep MQRLPLSFLAGLGPHGGRAEEVAAAVDDOLG a4 06 MQARLLIPILFSVFILSACGTLTGIPSHGGGKRFA

ELVSRVKDMDLQAHGR

20 30 40 50 80 90 100 110 120 m4 06. pep KVALYIATMGDQGSGSLTGGRYSIDALIRGEYINSPAVRTDYTYPRYETTAETTSGGLTG -1illa4 06 KVALYIATMGDQGSGSLTGGRYSIDALIRGEYINSPAVRTDYTYPRYETTAETTSGGLTG 170 80 90 100 110 120 130 .140 150 160 170 180 m4 06. pep LTTSLSTLNAPALSRTQSDGSGSKSSLGLNIGGMGDYRNETLTTNPRDTAFLSHLVQTVF a4 06 LTSSLAASTSGGKSGNIGGYNTTNRTFSLQV 130 140 150 160 170 180 190 200 210 220 230 240 mn406 .pep FLRGIDVVSPANADTDVFINIDVFGTIRNRTEMHLYNETLKAQTKLEYFAVDRTNKKLL a406 FLRGIDVSPANADTDVFINIDVFGTIRNRTEMHLYETLKQTKLEYFAVDRTNKKLL 190 200 210 220 230 240 250 260 270 280 290 300 m4 06. pep IKPKTNAFEAAYKENYALWMGPYVSKGIKPTEGLVDFSDIRPYGNHTGNSAPSVEADN a4 06 IKKNFAYEYLMPYVKIPELVFDOYNMNASED 250 260 270 .280 290 300 310 320 m4 06. pep SHEGYGYSDEVVRQHRQGQPX a4 06 SHEGYGYSDEAVIRRHRQGQPX 310 320 The following partial DNA sequence was identified in N. meningitidis <SEQ ID) m726.aeq 1 51 .101 151 201 251 301 351 401 451 501 551 601

ATGACCATCT

CCCCGAAGGC

CAGGACAGGC

GTTTTAACCC

ATGGAAAATC

CCGCCTTGGC

CTCTTGGCGG

AAAAGAAGCC

TGGCGCAAAT

AAAGTTATCG

CGGAAAGCGT

CCGGATTGGA

GGCTGA

ATTTCAAAAA

GCGGTTGCCG

GCAGGGCGGG

CGCCGCGCCC

AGCAAAGCCG

ATTCCGCCTC

GCTATCCCCA

CTCGCGCGGC

CGCCGCCGCA

AAAAATCCGC

CAGCAGCTCG

CGGCTTTTAC

TCCGCGCCGA

CAGATTGCCG

GTCCGATTAC

CCGCCGCCGC

GCGGAAAAGG

AGTGGAAATC

AGGCGGACAA

AGGGGCGTGG

CCGCCTGGCT

AAGACAAATT

GACGACACAT

AGAATACGCC

CAGATTCCGA

CACGAATGGG

CCGTTTCGCC

CGGACGAACT

GACAGCTTTT

.CAACGCCCCG

AATTGGACGT

GTTGCCGCCG

GAACACCATC

AAGAATGGAC

TGGGCGGCAT

GCCCTTTTGG

CGGCCGCCCC

ACGGCAAAAA

AAACAAAAAA

CAAAAACAGC

ACAGGCAGGA

ACCCCGATGC

TTTGATTGAA

GCGCGATTAT

GAAACCGCGC

GCTAAACATC

CGCGCTGGAA AAGGAAATCG This corresponds to the amino acid sequence <SEQ 11) 81; ORF 726>: m726.pep

I

51 151 201

MTIYFKNGFY

VLTPPRPSDY

LLAGYPQVEI

KVIEKSARLA

DDTLGGI PEG AVAVRAEEYA HEWDGKKWKI SKAAAAARFA DSFYRQEKEA LARQADNNAP VAAGAIIGKR QQLEDKLNTI.

ALLAGQAOGG QIAADSDGRP KQKTAI.AFRL AEKADELKNS TPMLAQIAAA RGVELDVLIE ETAPGLDALE KEIEEWTLNI The following partial DNA sequence was identified in.N. ineningitidis <SEQ ID 82>: m907-2 .seq 1 ATGAGAAAAC CGACCGATAC CCTACCCGTT AATCTGCAAC GCCGCCGCCT 51 GTTGTGTGCC GCCGGTGCGT TGTTGCTCAG TCCTCTGGCG CACGCCGGCG 101 CGCAACGTGA GGAAACGCTT GCCGACGATG TGGCTTCCGT GATGAGGAGT 112

TCTGTCGGCA

GGGCGAGCGT

CCGAGGAGGA

AGCCGGGCCG

AAGCGCGTTC

TGCAGGTTAT

CTGTTCGACA

TTACCGGAAT

ACGGCAGCTT

CGCAACCGCT

GCGTCAATCC

TGGTTGTCTG

GGAGCGGCGC

GTTTGGATAC

CGCCAGTATG

GCCGTTTTGG

TCCGCACCAA

CTTGAAAAAG

GGGCAGCAAT

GGCAGTGGCG

GCCGAGGCTG

CCATGTCGGC

AGGCTGCTGG

GCAGATTGTG

CA ATCAGCGG

AAAAACTACA

CCTGCGTTAC

GCAACATCGT

AAATATCCGA

TTGA

.GTGTTTGACA

A.CGTTTGGCA

TCAATATCCA

TTGGGGCTGA

TGTCGGCGCG

TCGGCAAACC

GGCTGTACCA

CCGCGCGCTT

ACGCCGTTTT

ATCCGAAAGA

AGGTTCGTCC

GTACGAAAGC

TTGAGGTGGA

CGCGGCCTGA

GGCGCACAAC

TCCTGCGCCA

GCCCGCTTTA

GGGCGCGTGG

This corresponds. to the amino acid sequence <S EQ ED 83; ORE 907-2>: m907-2 .peaj 1* 51 101 151 201

MRKPTDTLPV

SVGSVNPPRL

SRAGLDTQIV

LFDIRTNLRY

RNRWQWR-

NLQRP.RLLCA AGALLLSPLA HAGAQREETL ADDVASVMRS VFDNPKEGER WLSAMSARLA RFVPEEEERR RLLVNIQYES LGLIEVESAF RQYAISGVGA RGLMQVMPFWKNYIGKPAHN GCTILRHYRN LEKGNIVRAL ARFNGSLGSN KYPNAVLGAW The following partial DNA sequence was identified in N. meningitidis <SEQ ID 84>: rn953.seq 51 1.01 151 201 251 301 351 401 451 501 551 ATGAAAAAAA TCATCTTCGC CTCCGCCGCC ACCTACA1AAG CCATCGACCA TTTCAACACC ACCGGTTCCG TCGAGTTCGA CACCATCCCC ATTGCCAACC ACCTGA1AATC AGCCGACATC TTTGTTTCCA CCAAATTCAA CGGCAACCTG ACCATGCACG AAAAATTCAA CTGCTACCAA GGCGACTTCA GCACCACCAT CGTTAACGTT GGTATGACCA CAGCCAAACA ATAA CGCACTCGCA GCCGCCGCCA TGGACGAATA TCACGCCAAC AGCACCAXCG TCGGCGGTTT CCAAGCAAAA CGCGACGGTA TGCAAAGCGG TTCGCAACAC TTCGATGCCG CCCAATATCC CTTCAACGGC AAAAAACTGG GCAAAACCGC CCCCGTCAAA AGCCCGATGG AGAAAACCGA CGACCGCACC AAATGGGGCA AAAGCGTCCG CATCGACATC

TCAGTACTGC

GCCCGTTTCG

TTACGGTCTG

AAATCGACAT

TTTACCGACC

GGACATCCGC

TTTCCGTTGA

CTCAAAGCCG

AGTTTGTGGC

TGGACTACCT

CAAATCGAGG

This corresponds to the amino acid sequence <SEQ ID 85; ORE 953>: m953.pap 1 MKKIIFAALA AAAISTASAA TYKVDEYHAN ARFAIDHENT STNVGGFYGL 51 TGSVEFDQAK RDGKIDITIP IANLQSGSQH FTDHLKSADI FDAAQYPDIR 101 FVSTKFNFNG KKLVSVDGNL TMHGKTAPVK LKAEKFNCYQ SPMEKTEVCG 151 GDFSTTIDRT KWGMDYLVNV GMTKSVRIDI QIEAAKQ* The follo Wing partial DNA sequence was identified in N. meningitidis <SEQ ID 86>: orf1-1. so( 51 101 151 201 251 301 351 401 451 501

ATGAAAACAA

AACCGGCCGC

TCGGCATTCT

TACCAATACT

GGCGAAAGAT,

CAATGACAAA

GTGGCGGCAT

CGGCTATAAC

ATCGTTTTAC

AAAGGCCATC

TGTCACAGAT

CCGACAAACG

ATCCGCTTCT

TCCCCAAGCC

ATCGCGACTT

ATTGAGGTTT

AGCCCCGATG

TGGTGGGCGA

AACGTTGATT

TTATAAAATT

CTTATGGCGG

GCAGAACCTG

GACAACCGAA

CGCCTGCTTA

TGGGCGGGAC

TGCCGAAAAT

ACAACAAAAA

ATTGATTTTT

TCAATATATT

TTGGTGCGGA

GTGAAACGGA

CGATTATCAT

TTGAAATGAC

ACACACCGCA AAGCCCCGAA CTTAGCCATA TGCCTGTCGT ACACTTATTT CGGCATCAAC AAAGGCAAGT TTGCAGTCGG AGGGGAGTTG GTCGGCAAAT CTGTGGTGTC GCGTAACGGC GTGAGCGTGG CACATAACGG AGGAAGAAAT CCCGATCAAC ATAATTATAA AGCAGGGACT ATGCCGCGTT .TGCATAAATT CAGTTATATG GATGGGCGGA 113 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651 2701 2751 2801 2851 2901 2951 3001 3051 3101 3151 3201 3251 3301 3351 3401 3451 3501 3551 3601 3651 AATATATCGA TCAAAATAAT AGGCAATATT GGCGATCTGA ATATCATATT GCAAGTGCGT CACAAAkATGG ATCAGGTGGT AAACATAGCC CATATGGTTT TGGCTCACCA ATGTTTATCT ATGGGGTATT GCAAACGGGC CAGCTGGTTC GTAAAGATTG CCATTCAGTA TTCTACGAAC ACGATAATAA TGGCACAGGA CTGCCTAATA GATTAAAAAC TACCCTGACC GTGTTCGTAT TGGGGCAGGC TGAAGATGAG CCCAATAACC GCGAAAGTTC ATTCTTGGCT CGTTGGTGGC AATACCTTTG GGCACAGTCA ACTTAGGTAG TGAAAAAATT TTTACCAACA GGAGGCTCAT TTGGCGACAG ATGATGCCCA AAAGCAAAAG TGGTTAATTA AACCCCTATA TAGGAAAAAG CAATGGCTTC GTTCTATGAT GAAATCTTTG CTGGAGATAC CACGTCAAAA TGGGAAATAC TCTTTTAACG

ATCICGAGACA

GTTATCGACC

GGAAAAGGCG

ATTATATTTC

GGCAAGGCGC

GTAAACGGCG

GCACGTTCAA

GTACAGTCAT

TTTAGTGAAA

CGATAATCAG

GTTTGGATTT

GATGAAGGGG

TACCATTACA

TGGATAGCAA

ACGACCAAAA

GCAAGAGAAC.

CAGACTGAAT

AATTGATACT

CAAGGAGATT

GGGCGTTCAT

TGGCAAACGA

GCCAAAGGGG

TTTGGATCAG

TCGGCTTGGT

TTCAACCCCG

AAACGGGCAT

CGATGATTGT

GGCAATAAAG

AAAAGAAATT

CGAACGGGCG

AAAATCAATG

ACGAACCGTT

CTGTTTATCA

AATGGAGAAA

TACCAGCAAC

TTACGGTCTC

ATCAGTGAAG

CCGCCTGTCC

AAAACCAAGG

CAGGCAGACG

CAGCGGCAGG

ACAAACTCTA

TCGCTTTCGT

CAACCACAAT

ATATTGCTAC

GCCTACAACG

GCTCAACCTT

CCAAACATGA ACACAATTCT CAATTGTTTA ATGTTTCTTT TGCTGCAGGT GGTGTCAACA ATATTTCCTT TATTGACGAA ATCAATCAAG GTGCTGGAGG GCCTGAAAAT AACGAAACTT ACAGTACCGT TACTTGGAAA AAAATCGGCA AAGGCACGCT CTCGATCAGC GTGGGCGACG ATAAAGGCAA AAAACAAGCC GGTACGGTGC. AACTGAATGC TTTCGGCTTT CGCGGCGGAC TCCACCGTAT TCAAAATACC CAAGACAAAG AATCCACCGT AACCGGCAAT AACAACAGCT GTTGGTTTGG CGAGAAAGAT GTTTACCAGC CCGCCGCAGA AG~ACCGCACC CTGCTGCTTT CCGGCGGAAC AAATTTAAAC GGCAACATCA CGCAAACAAA CGGCAAACTrG TTTTTCAGCG GCAGACCAAC ACCGCACGCC TACAATCATT TAAACGACCA TTGGTCGCAA AAAGAGGGCA TTCCTCGCGG GGAAATCGTG TGGGACAACG ACTGGATCAA CCGCACATTT AAAGCGGAAA ACTTCCAAAT TAAAGGCGGA CAGGCGGTGG TTTCCCGCAA TGTTGCCAAA GTGAAAGGCG ATTGGCATTT GAGCAATCAC GCCCAAGCAG TTTTTGGTGT CCCACCGCATCAAAGCCACA CAATCTGTAC ACGTTCGGAC TGGACGGGTC TGACAAATTG TGTCGAAAAA ACCATTACCG ACGATAAAGT GATTGCTTCA TTGACTAAGA CCGACATCAG CGGCAATGTC GATCTTGCCG ATCACGCTCA TTTAAATCTC ACAGGGCTTG CCACACTCAA CGGCAATCTT AGTGCAAATG GCGATACACG TTATACAGTC AGCCACAACG CCACCCAAAA CGGCAACCTT AGCCTCGTGG GCAATGCCCA AGCAACATTT AATCAAGCCA CATTAAACGG CAACACATCG GCTTCGGGCA ATGCTTCATT TAATCTAAGC GACCACGCCG TACAAAACGG CAGTCTGACG CTTTCCGGCA ACGCTAAGGC AAACGTAAGC CATTCCGCAC TCAACGGTAA TGTCTCCCTA GCCGATAAGG CAGTATTCCA TTTTGAAAGC AGCCGCTTTA CCGGACAAAT. CAGCGGCGGC AAGGATACGG CATTACACTT AAAAGACAGC GAATGGACGC TGCCGTCAGG CACGGAATTA GG CAATTTAA ACCTTGACAA CGCCACCATT ACACTCAATT CCGCCTATCG CCACGATGCG GCAGGGGCGC AAACCGGCAG TGCGACAGAT GCGCCGCGCC GCCGTTCGCG CCGTTCG CGC CGTTCCCTAT TATCCGTTAC ACCGCCAACT TCGGTAGAAT CCCGTTTCAA CACGCTGACG GTAAACGGCA AATTGAACGG TCAGGGAACA TTCCGCTTTA TGTCGGAACT CTTCGGCTAC CGCAGCGACA AATTGAAGCT GGCGGAAAGT TCCGAAGGCA CTTACACCTT GGCGGTCAAC AATACCGGCA ACGAACCTGC AAGCCTCGAA CAATTGACGG TAGTGGAAGG AAAAGACAAC AAACCGCTGT CCGAAAACCT TAATTTCACC CTGCAAAACG AACACGTCGA TGCCGGCGCG TGGCGTTACC AACTCATCCG CAAAGACGGC GAGTTCCGCC TGCATAATCC GGTCAAAGAA CAAGAGCTTT CCGACAAACT CGGCAAGGCA GAAGCCAAAA AACAGGCGGA AAAAGACAAC GCGCAAAGCC TTGACGCGCT GATTGCGGCC GGGCGCGATG CCGTCGAAAA GACAGAAAGC GTTGCCGAAC CGGCCCGGCA GGCAGGCGGG GAAAATGTCG GCATTATGCA GGCGGAGGAA GAGAAAAAAC GGGTGCAGGC GGATAAAGAC ACCGCCTTGG CGAAACAGCG CGAAGCGGAA ACCCGGCCGG CTACCACCGC CTTCCCCCGC GCCCGCCGCG CCCGCCGGGA TTTGCCGCAA CTGCAACCCC AACCGCAGCC CCAACCGCAG CGCGACCTGA TCAGCCGTTA TGCCAATAGC GGTrTTGAGTG AATTTTCCGC CACGCTCAAC AGCGTTTTCG CCGTACAGGA CGAATTAGAC CGCGTATTTG CCGAAGACCG CCGCAACGCC GTTTGGACAA GCGGCATCCG GGACACCAAA CACTACCGTT CGCAAGATTT CCGCGCCTAC CGCCAACAAA 114- 3701 3751 380 3851 3901 3951 4001 4051 4101.

4151 4201 .4251 4301 4351

CCGACCTGCG

GGCATCCTGT

CGGCAACTCG

TCGACAGGTT

AGCCTTTCAG

CGGCATTCAG

CGCACATCGG

GAAAACGTCA

GGGCATTAAG

CGCCTTATTT

ACACGCGTCA

TGCGGAATGG

ACGCTGCCGC

ATCAAATTAG

CCAAATCGGT ATGC TTTCGCACAA CCGG GCACGGCTTG CCCA CTACATCGGC ATCA ACGGCATCGG AGGC GCACGATACC GCGC CGCAACGCGC TATT' ATATCGCCAC CCCCC GCAGATTATT CATT GAGCCTGTCC TATA ATACCGCCGT ATTG( GGCGTAAACG CCGA CGCCAAAGGC CCGC GCTACCGCTG GTAA PkGAAAA ACCTCGGCAG PCCGAA AACACCTTCG

CGGCGC

GCGCGG

DAAATC

CGGTTT

TCGTCC

GGCCTT

CAAACC

CCGATG

GCTCAG

kATCAA.

%.ACTGG

CGTTTTCGGG

GCGCGGGTTT

CGCCGCCGCG

CGGCGGATTC

AAAAAGCGGA

GCATTCAACC

GGCGCAACAC

CCGCTTCGGG

GATTTCGGCA

AGGTTTCACG

AAGCGCAACA

CGGGCGCGTC

ACGACGGCAT

CAATACGGCA

TAGCAGCGGC

TGCTGCATTA

GGCATCGAAC

TTACCGCTAC

GCTACCGCGC

ATTTCCATCA

CAAAGTCCGA

AAACCCGCAG

CTGTCCCTCC

CAGCGCGGGC

This corresponds to the amino acid sequence <SEQ ID 87; ORF orfl-1I>: 1 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 .1101 1151 1201 1251 1301 1401 1451

MKTTDKRTTE

YQYYRDFAEN

VAALVGDQYI

KGHPYGGDYH

RQYWRSDEDE

KHSPYGFLPT

QLVRKDWFYD

LPNRLKTRTV

GKGELILTSN

VNGVANDRLS

FSEIGLVSGR

DEGAMIVNHN

TTKTNGRLNL

YNHLNDHWSQ

VKGDWHLSNH

LTKTDISGNV

SLVGNAQATF

HSALNGNVSL

GNLNLDNATI

SVESRFNTLT

NTGNEPASLE

EFRLHNPVKE

*VAEPARQAGG

ARRARRDLPQ

*RVFAEDRRNA

GI LFSHNRTE.

SLSDGIGGKI

ENVNIATPGL

TRVNTAVLAQ

IKLGYRW*

THRKAPKTGR IRFSPAYLAI CLSFGILPQA WAGHTYFGIN KGKFAVGAKD IEVYNKKGEL VGKSMTKAPM IDFSVVSRNG VSVAHNGGYN NVDFGAEGRN P DQHRFTYKI VKRNNYKAGT MPRLHKrVTD. AEPVEMTSYM DGPXYIDQNN YPDRVRIGAG PNNRESSYHI ASAYSWLVGG NTFAQNGSGG. GTVNLGSEKI GGSFGDSGSP MFIYDAQKQK WLINGVLQTG NPYIGKSNGF EIFAGDTHSV FYEPRQNGKY SFNDDNNGTG KINAKHEHNS QLFNVSLSET AREPVYHAAG GVNSYRPRLN NGENISFIDE INOGAGGLYF QGDFrVSPEN NETWQGAGVH ISEDSTVTWK KIGKGTLHVQ AKGENQGSIS VGDGTVILDQ QADDKGKKQA GTVQLNADNQ FNPDKLYFGF RGGRLDLNGH SLSFHRIQNT QDKESTVTIT GNKDIATTGN NNSLDSKKEI AYNGWFGEKD VYQPAAEDRT LLLSGGTNLN GNITQTNGKL FFSGRPTPHA KEGIPRGEIV WDNDWINRTF KAENFQIKGG QAVVSRNVA( AQAVFGVAPH QSHTICTRSD WTGLTNCVEK TITDDKVIAS DLADIIAHLNL TGLATLNGNL SAGDTRYTV SHNATQNGNL NQATLNGNTS ASGNASFNLS DHAVQNGSLT LSGNAKANVS .ADKAVFHFES SRFFGQISGG KDTALHLKDS EWTLPSGTEL TLNSAYRHDA AGAQ'rGSATD APRRRSRRSR RSLLSVTPPT VNGKLNGQGT FRFMSELFGY RSDKLKLAES SEGTYTLAVN QLTVVEGKDN KPLSENLNFT LQNEHVDAGA WRYQLIRKDG QELSDKLGkA EAKKQAEKDN AQSLDALIAA GRDAVEKTES ENVGIMQAEE EKKRVQADKD TALAKQREAE TRPATTAFPR LQPQPQPQPQ RDLISRYANS GLSEFSATLN SVFAVQDELD VWTSGIRDTK HYRSQDFRAY RQQTDLRQIG MQKNLGSGRV NTFDDGIGNS ARLAHGAVFG QYGIDRFYIG ISAGAGFSSG RRRVLHYGIQ ARYRAGFGGF GIEPHIGATR YFVQKADYRY AFNRYRAGIK ADYSFKPAQH ISITPYLSLS YTDAASGKVR DFGKTRSAEW GVNAEIKGFI' LSLHAAAAKG PQLEAQHSAG The following partial DNA sequence was identified in N. meningitidis <SEQ ID 88>: orf46-2 s 51 101 151 201 251 301 351

TTGGGCATTT

CCTGCCGATG

GGCAGGTTCT

TTCGGCAGCA

AAAAATACAA

TTAAAGGAAA

GTCCATTCCC

CGGTAGTCCC

CCCGCAAAAT

CATGCACACG

CGACCGTCAG

GGGGGGAACT

AGCCATCAGT

TATCGGCTAC

CCTTCGACAA

GTTGACGGAT

ATCCCTTATT

CCTCAGATTT

CATTTCGAAC

TGCCGAGCGC

TGGGCAACCT

ATTGTCCGCT

CCATGCCTCA

TTAGCCTTTA

CTGTCCATAC

GGCAAACGAT

CCGACGGGAA

AGCGGCCATA

GATGATTCAA

MTCCGATcA

CATTCCGATT

CCGCATCCAT

TGGCAGTGTG

TCTTTTATCC

ATACCACCTA

TCGGATTGGG

CAGGCGGCCA

CGGGC.ACGAA

CTGATGAAGC

TGGGACGGAT

115- 401 451 501 551 601 651 701 751 1 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651

ACGAACACCA

CCCGCTCCCA

T.GCCCAAAAT

GGCTTGCCGA

GGCGACGGAT

GGGCAATGCC

TCATCGGCGC

ATAAGCGAAG

CACCGAAAAC

TCAAAGACTA

AATGCCGCAC

CCCCATCAAA

TCACGGCACA

AAAGGGAAAT

ATACCCGTCC

GTTACGGCAA

AAAAATGTCA

TGACGGTAAA

AGCTCGATAT

GTGTTTGATG

GACAACTCGT

ATATAAACAG

AAACTAAAAT

TACCGATAGC

AAGAGAATGG

AAAGCATATA

TCCCGCCGAc GGCTATGACG GGCCACAGGG CGGCGGCTAT AAGGCGCGAG GGATATATAC AGCTACGACA TAAAAGGCGT ATCCGCCTCA ACCTGACCGA CAACCGCAGC ACCGGACAAC CCGTTTCCAC AATGCCGGTA GTATGCTGAC GCAAGGAGTA TCAAACGCGC CACCOGATAC AGCCCCGAGC TGGACAGATC GCCGAAGCCT.TCAACGGCAC TGCAGATATC GTTAAAAACA GGCAGGAGAA ATTGTCGGCG CAGGCGATGC CGTGCAGGGC GCTCAAACAT TGCTGTCATG CACGGCTTGG GTCTGCTTTC AAGATGGCGC GCATCAACGA TTTGGCAGAT ATGGCGCAAC TGCCGCAGCA GCCATCCGCG ATTGGGCAGT CCAAAACCCC AAGGCATAGA AGCCGTCAGC AATATCTTTA TGGCAGCCAT GGGATTGGAG CTGTTCGGGG AAAATACGGC TTGGGCGGCA TCCTATCAAG CGGTCGCAGA. TGGGCGCGAT CGCATTGCCG CCGCCGTCAG CGACAATTTT GCCGATGCGG CATACGCCAA CCTTACCATT CCCGAAATAT CCGTTCAAAC TTGGAGCAGC AGAAAACATC ACCTCCTCAA CCGTGCCGCC GTCAAACGGC AACTGGCAGA CCAACGCCAC CCGAAGACAG GCGTACCGTT GGGTTTCCGA ATTTTGAGAA GCACGTGAAA TATGATACGA TCAAGAATTA TCGGGGGGCGGTATACCTAA

GGCTAAGCCT

CGAAACCGAG ATGGGAGGTT GATAGGAAGC TTAATAAATT GAGCAGGTGG AGAAAAATGT TCAGGAAATA AGGAACGGTA TAACTTTAGC CAACATGCTC AACTAGAGAG GGAAATTAAT CTGCCGATGA AATTAATTTT GCAGATGGAA TGGGAAAATT ATGAATGACA AGGCTTTTAG TAGGCTTGTG AAATCAGTTA CTTCACAAAT CCAGTTGTGG AGTACGTTGA AATAAATGGA TCGTAAGAGG AAATAATRGG GTTTTTGCTG CAGAATACCT CATGAATTAA AATTTAAAAA AGTTGACTTT CCTGTTCCTA GAAAAATCCT ACTGATGTCT TGAATGAATC AGGTAATGTT GTTATAGGAG TAAATAA 1701 TGGCAGGATA 1751 ATACTAGTTG 1801 AAGAGACCTC This corresponds to the amino acid sequence <SEQ ID 89; ORE orf46-2>: orf46-2.pep 1 LGISRKISLI 51 FGSRGELAER 101 VHSPFDNHAS 151 PAPKGARDIY 201 GDGFKRATRY 251 ISEGSNIAVM 301 NAAQGIEAVS 351 KGKSAVSDNF 401 KNVKLADQ.H 451 VFDAKPRWEV 501 KLKSADEINF 551 KAYIVRGNNR 601 KRPRYRSK* LSILAVCLPM HAHASDLAND SFIRQVLDRQ HFEPDGKYHL SGHIGLGKIQ SHQLGNLMIQ QAAIKGNIGY IVRFSDHGHE HSDSDEAGSP VDGFSLYRIH WDGYEHHPAD GYDGPQGGGY SYDIKGVAQN IRLNLTDNRS TGQRLADRFH NAGSMLTQGV SPELDRSGNA AEAFNGTADI VKNIIGAAGE IVGAGDAVQG HGLGLLSTEN KMARINDLAD MAQLKDYAAA AIRDWAVQNP NIFMAAI PIK GIGAVRGKYG LGGITAHPIK RSQMGAIALP ADAAYAKYPS PYHSRNIRSN LEQRYGKENI TSSTVPPSNG PKTGVPFDGK GFPNFEKHVI( YDTKLDIQEL SGGGIPKAKP DRKLNKLTTR EQVEKNVQEI RNGNINSNFS QHAQLEREIN ADGMGKFTDS t4NDKAFSRLV KSVKENGFI'W.PVVEYVE

ING

VFAAEYLGRI HELKFKKVDF PVPNTSWKNp TDVLNESGNV Using the above-described procedures, the following oligonucleotide primers were employed in the polymerase chain reaction (PCR) assay in order to clone the OR~s as indicated: Oligonucleotides used for PCR Table 1 116 ORF I Primer]I Sequence fRestriction sites .51M I 519 576 919 121 128 206 287 406 Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

I

CGCGGATCCCATATG-TTGCCTGCAATCACGATT

<SEQ) ID 90> CCCGCTCGAG-1TAGAAGCGGGCGGCMA

<SEQ

ID. 91 CGCGGATCCCATATG-TTCAAATCC1TTTGTCGTCA <SEQ ID 92> CCCGCTCGAG-TGGCGG1-ITrGCTGC <SEQ ID 93>

CGCGGATCCCATATG-GCCGCCCCCGCATCT

<SEQ ID 94> CCCGCTCGAG-ATAC1TflTGATGTCGAC <SEQ ID 95>

CGCGGATCCCATATG-TGCCAAAGCAAGAGCATC

<SEQ ID 96> CCCGCTCGAG-CGGGCGGTATT1CGGG <SEQ ID 97> CGCGGATCCCATATG-GAAACACAGCMrACAT <SEQ ID 98> CCCGCTCGAG-ATAATMATATCCCGCGCCC

<SEQ

ID 99> CGCGGATCCCATATG-ACTGACMACGCACT

<SEQ

ID 100> CCCGCTCGAG-GACCGCGTVGTCGAAA <SEQ ID 101>

CGCGGATCCCATATG-AAACACCGCCAACCGA

<SEQ ID 102>

CCCGCTCGAG-TTCTGTAAAAAAAGTATGTGC

<SEQ ID 103> CCGGAATTCTAGCTAGC-C1--CAGCCTGCGGG <SEQ ID 104> CCCGCTCGAG-ATCCTGCTCTTI-GCC <SEQ ID 105>

CGCGGATCCCATATG-TGCGGGACACTGACAG

<SEQ ID 106> CCCGCTCGAG-AGGTTGTCC1TGTCTATG

<SEQ

ID 107> BamHI-Ndel Xhol BamHI-Ndel XhoI BamHI-Ndel XhoI BamHI-Ndel Xhol BamHI-NdeI Xhol BamHl-Ndel Xhol BamHI-Ndbt Xhol EcoRl-NheI Xhol BamHI-Ndel Xhol EXAMPLE 2 Expression of ORF 919 The primer described in Table 1 for ORF 919 was used to locate and clone ORF 919.

The predicted gene 919 was cloned in pET vector and expressed in E. coli. The product of -117protein expression and purification was analyzed by SDS-PAGE. In panel A) is shown the analysis of919-His fusion protein purification. Mice were immunized with the purified 919- His and sera were used for Western blot (panel FACS analysis (panel bactericidal assay (panel and ELISA assay (panel Symbols: Ml, molecular weight marker; PP, purified protein, TP, N. meningitidis total protein extract; OMV, N. meningitidis outer membrane vesicle preparation. Arrows indicate the position of the main recombinant protein product and the N. meningitidis immunoreactive band These experiments confirm that 919 is a surface-exposed protein and that it is a useful immunogen. The hydrophilicity plots, antigenic index, and amphipatic regions of ORF 919 are provided in Figure 10. The AMPHI program is used to predict putative T-cell epitopes (Gao et al 1989, J. Immunol 143:3007; Roberts et al. 1996, AIDS Res Human Retroviruses 12:593; Quakyi et al. 1992, Scand Jlmmunol Suppl 11:9). The nucleic acid sequence of ORF 919 and the amino acid sequence encoded thereby is provided in Example 1.

EXAMPLE 3 Expression of ORF 279 The primer described in Table 1 for ORF 279 was used to locate and clone ORF 279.

The predicted gene 279 was cloned in pGex vector and expressed in E. coli. The product of protein expression and purification was analyzed by SDS-PAGE. In panel A) is shown the analysis of 279-GST purification. Mice were immunized with the purified 279-GST and sera were used for Western blot analysis (panel FACS analysis (panel bactericidal assay (panel and ELISA assay (panel Symbols: Ml, molecular weight marker; TP, N.

meningitidis total protein extract; OMV, N. meningitidis outer membrane vescicle preparation. Arrows indicate the position of the main recombinant protein product and the N. meningitidis immunoreactive band These experiments confirm that 279 is a surface-exposed protein and that it is a useful immunogen. The hydrophilicity plots, antigenic index, and amphipatic regions of ORF 279 are provided in Figure 11. The AMPHI program is used to predict putative T-cell epitopes (Gao et al 1989, J. Immunol 143:3007; Roberts et al. 1996, AIDS Res Human Retroviruses 12:593; Quakyi et al. 1992, ScandJ Immunol Suppl 11:9). The nucleic acid sequence of ORF 279 and the amino acid sequence encoded thereby is provided in Example 1.

-118- EXAMPLE 4 Expression of ORF 576 The primer described in Table 1 for ORF 576 was used to locate and clone ORF 576.

The predicted gene 576 was cloned in pGex vector and expressed in E. coli. The product of protein purification was analyzed by SDS-PAGE. In panel A) is shown the analysis of 576- GST fusion protein purification. Mice were immunized with the purified 576-GST and sera were used for Western blot (panel FACS analysis (panel bactericidal assay (panel D), and ELISA assay (panel Symbols: M1, molecular weight marker; TP, N. meningitidis total protein extract; OMV, N meningitidis outer membrane vescicle preparation. Arrows indicate the position of the main recombinant protein product and the N. meningitidis immunoreactive band These experiments confirm that ORF 576 is a surface-exposed protein and that it is a useful immunogen. The hydrophilicity plots, antigenic index, and amphipatic regions of ORF 576 are provided in Figure 12. The AMPHI program is used to predict putative T-cell epitopes (Gao et al 1989, J. Immunol 143:3007; Roberts et al. 1996, AIDS Res Human Retroviruses 12:593; Quakyi et al. 1992, Scand Jmmunol Suppl 11:9).

The nucleic acid sequence of ORF 576 and the amino acid sequence encoded thereby is provided in Example 1.

EXAMPLE Expression of ORF 519 The primer described in Table 1 for ORF 519 was used to locate and clone ORF 519.

The predicted gene 519 was cloned in pET vector and expressed in E. coli. The product of protein purification was analyzed by SDS-PAGE. In panel A) is shown the analysis of 519- His fusion protein purification. Mice were immunized with the purified 519-His and sera were used for Western blot (panel FACS analysis (panel bactericidal assay (panel D), and ELISA assay (panel Symbols: M1, molecular weight marker; TP, N. meningitidis total protein extract; OMV, N. meningitidis outer membrane vesicle preparation. Arrows indicate the position of the main recombinant protein product and the N. meningitidis immunoreactive band These experiments confirm that 519 is a surface-exposed protein -119and that it is a useful immunogen. The hydrophilicity plots, antigenic index, and amphipatic regions of ORF 519 are provided in Figure 13. The AMPHI program is used to predict putative T-cell epitopes (Gao et al 1989, J. Immunol 143:3007; Roberts et al. 1996, AIDS Res Human Retroviruses 12:593; Quakyi et al. 1992, ScandJImmunol Suppl 11:9). The nucleic acid sequence of ORF 519 and the amino acid sequence encoded thereby is provided in Example 1.

EXAMPLE 6 Expression of ORF 121 The primer described in Table 1 for ORF 121 was used to locate and clone ORF 121.

The predicted gene 121 was cloned in pET vector and expressed in E. coli. The product of protein purification was analyzed by SDS-PAGE. In panel A) is shown the analysis of 121- His fusion protein purification. Mice were immunized with the purified 121-His and sera were used for Western blot analysis (panel FACS analysis (panel bactericidal assay (panel and ELISA assay (panel Results show that 121 is a surface-exposed protein.

Symbols: Ml, molecular weight marker; TP, N. meningitidis total protein extract; OMV, N.

meningitidis outer membrane vescicle preparation. Arrows indicate the position of the main recombinant protein product and the N. meningitidis immunoreactive band These experiments confirm that 121 is a surface-exposed protein and that it is a useful immunogen.

The hydrophilicity plots, antigenic index, and amphipatic regions of ORF 121 are provided in Figure 14. The AMPHI program is used to predict putative T-cell epitopes (Gao et al 1989, J.

Immunol 143:3007; Roberts et al. 1996, AIDS Res Human Retroviruses 12:593; Quakyi et al.

1992, Scand Jlmmunol Suppl 11:9). The nucleic acid sequence of ORF 121 and the amino acid sequence encoded thereby is provided in Example 1.

EXAMPLE 7 Expression of ORF 128 The primer described in Table 1 for ORF 128 was used to locate and clone ORF 128.

The predicted gene 128 was cloned in pET vector and expressed in E. coli. The product of protein purification was analyzed by SDS-PAGE. In panel A) is shown the analysis of 128- His purification. Mice were immunized with the purified 128-His and sera were used for -120- Western blot analysis (panel FACS analysis (panel bactericidal assay (panel D) and ELISA assay (panel Results show that 128 is a surface-exposed protein. Symbols: Ml, molecular weight marker; TP, N. meningitidis total protein extract; OMV, N. meningitidis outer membrane vesicle preparation. Arrows indicate the position of the main recombinant protein product and the N. meningitidis immunoreactive band These experiments confirm that 128 is a surface-exposed protein and that it is a useful immunogen. The hydrophilicity plots, antigenic index, and amphipatic regions of ORF 128 are provided in Figure 15. The AMPHI program is used to predict putative T-cell epitopes (Gao et al 1989, J.

Immunol 143:3007; Roberts et al. 1996, AIDS Res Human Retroviruses 12:593; Quakyi et al.

1992, ScandJlmmunol Suppl 11:9). The nucleic acid sequence of ORF 128 and the amino acid sequence encoded thereby is provided in Example 1.

EXAMPLE 8 Expression of ORF 206 The primer described in Table 1 for ORF 206 was used to locate and clone ORF 206.

The predicted gene 206 was cloned in pET vector and expressed in E. coli. The product of protein purification was analyzed by SDS-PAGE. In panel A) is shown the analysis of 206- His purification. Mice were immunized with the purified 206-His and sera were used for Western blot analysis (panel It is worthnoting that the immunoreactive band in protein extracts from meningococcus is 38 kDa instead of 17 kDa (panel To gain information on the nature of this antibody staining we expressed ORF 206 in E. coli without the His-tag and including the predicted leader peptide. Western blot analysis on total protein extracts from E.

coli expressing this native form of the 206 protein showed a recative band at a position of 38 kDa, as observed in meningococcus. We conclude that the 38 kDa band in panel B) is specific and that anti-206 antibodies, likely recognize a multimeric protein complex. In panel C is shown the FACS analysis, in panel D the bactericidal assay, and in panel E) the ELISA assay. Results show that 206 is a surface-exposed protein. Symbols: Ml, molecular weight marker; TP, N. meningitidis total protein extract; OMV, N. meningitidis outer membrane vesicle preparation. Arrows indicate the position of the main recombinant protein product (A) and the N. meningitidis immunoreactive band These experiments confirm that 206 is a surface-exposed protein and that it is a useful immunogen. The hydrophilicity plots, -121 antigenic index, and amphipatic regions of ORF 519 are provided in Figure 16. The AMPHI program is used to predict putative T-cell epitopes (Gao et al 1989, J. Immunol 143:3007; Roberts et al. 1996, AIDS Res Human Retroviruses 12:593; Quakyi et al. 1992, ScandJ Immunol Suppl 11:9). The nucleic acid sequence of ORF 206 and the amino acid sequence encoded thereby is provided in Example 1.

EXAMPLE 9 Expression of ORF 287 The primer described in Table 1 for ORF 287 was used to locate and clone ORF 287.

The predicted gene 287 was cloned in pGex vector and expressed in E. coli. The product of protein purification was analyzed by SDS-PAGE. In panel A) is shown the analysis of 287- GST fusion protein purification. Mice were immunized with the purified 287-GST and sera were used for FACS analysis (panel bactericidal assay (panel and ELISA assay (panel Results show that 287 is a surface-exposed protein. Symbols: Ml, molecular weight marker. Arrow indicates the position of the main recombinant protein product These experiments confirm that 287 is a surface-exposed protein and that it is a useful immunogen.

The hydrophilicity plots, antigenic index, and amphipatic regions of ORF 287 are provided in Figure 17. The AMPHI program is used to predict putative T-cell epitopes (Gao et al 1989, J.

Immunol 143:3007; Roberts et al. 1996, AIDS Res Human Retroviruses 12:593; Quakyi et al.

1992, Scand Jmmunol Suppl 11:9). The nucleic acid sequence of ORF 287 and the amino acid sequence encoded thereby is provided in Example 1.

EXAMPLE Expression of ORF 406 The primer described in Table 1 for ORF 406 was used to locate and clone ORF 406.

The predicted gene 406 was cloned in pET vector and expressed in E. coli. The product of protein purification was analyzed by SDS-PAGE. In panel A) is shown the analysis of 406- His fusion protein purification. Mice were immunized with the purified 406-His and sera were used for Western blot analysis (panel FACS analysis (panel bactericidal assay (panel and ELISA assay (panel Results show that 406 is a surface-exposed protein.

Symbols: Ml, molecular weight marker; TP, N. meningitidis total protein extract; OMV, N.

-122meningitidis outer membrane vescicle preparation. Arrows indicate the position of the main recombinant protein product and the N. meningitidis immunoreactive band These experiments confirm that 406 is a surface-exposed protein and that it is a useful immunogen.

The hydrophilicity plots, antigenic index, and amphipatic regions of ORF 406 are provided in Figure 18. The AMPHI program is used to predict putative T-cell epitopes (Gao et al 1989, J.

Immunol 143:3007; Roberts et al. 1996, AIDS Res Human Retroviruses 12:593; Quakyi et al.

1992, Scand Jmmunol Suppl 11:9). The nucleic acid sequence of ORF 406 and the amino acid sequence encoded thereby is provided in Example 1.

The foregoing examples are intended to illustrate but not to limit the invention.

~I

Appendix B Appendix B NMB Open Reading Frames NMB0001 acetyltransferase, putative 491 3 NMB0002 hypothetical protein 890 498 NMB0003 glutamyl-tRNA synthetase 2305 914 NMB0004 EpiH/GdmH-related protein 3154 2513 NMB0005 arsenate reductase 3504 3154 sC NMB0006 thioredoxin-related protein 3628 4304 t NMB0007 cell division ATP-binding protein FtsE 4304 4951 CK1 NMB0008 cell division protein FtsX, putative 4951 5865 0 NMB0009 BolA/YrbA family protein 5959 6204 0 NMB0010 phosphoglycerate kinase 7485 6277 CK1 NMB0011 UDP-N-acetylglucosamine 1-carboxyvinyltransferase 8819 7569 fl NMB0012 conserved hypothetical .protein 103.10 9342 0 NMB0013 conserved hypothetical protein 10792 10346 NMB0014 3-deoxy-D-manno-octulosonic-acid transferase 12104 10836 CK1 NMB0015 6-phosphogluconate dehydrogenase, decarboxylating 13615 12170 NMB0016 hypothetical protein 13911 14144 NMB0017 UDP-3-O-3-hydroxymyristoyl N-acetylglucosamine deacetylase 16137 15217 NMB0018 pilin PilE 17734 17225 NMB0019 pilS cassette 18932 18513 NMB0020 pilS cassette 19646 19263 NMB0021 pilS cassette 20297 19914 NMB0022 pilS cassette 21157 20894 NMB0023 pilS cassette 21882 21466 NMB0024 pilS cassette 22474 22061 NMB0025 large pilS cassette 23489 22821 NMB0026 pilS cassette 23868 23594 NMB0027 FKBP-type peptidyl-prolyl cis-trans isomerase 24226 23900 NMB0028 hypothetical protein 24522 24307 NMB0029 glycerate dehydrogenase 24644 25594 NMB0030 methionyl-tRNA synthetase 27729 25675 NMB0031 glucosamine--fructose-6-phosphate aminotransferase (isomerizing) 29683 27848 NMB0032 hypothetical protein 29959 30483 NMB0033 membrane-bound lytic murein transglycosylase A, putative 32229 30907 NMB0034 conserved hypothetical protein 32440 33276 NMB0035 conserved hypothetical protein 33276 34439 NMB0036 conserved hypothetical protein 34706 35968 NMB0037 phnA protein 36372 36046 NMB0038 UDP-N-acetylglucosamine pyrophosphorylase 37817 36450 NMB0039 hypothetical protein 38144 37875 NMB0040 hydrolase, putative 38850 38140 NMB0041 ABC transporter, periplasmic solute-binding protein 38909 39907 NMB0042 conserved hypothetical protein 40004 40849 NMB0043 conserved hypothetical protein 40878 41360 NMB0044 peptide methionine sulfoxide reductase 43033 41468 NMB0045 signal recognition particle protein 43179 44441 NMB0046 hypothetical protein 44451 44672 NMB0047 conserved hypothetical protein 45072 45353 NMB0048 conserved hypothetical protein FRAMESHIFT 47969 48109 NMB0049 pilC2 protein FRAMESHIFT 48116 51279 NMB0050 conserved hypothetical protein 55173 53026 NMB0051 twitching motility protein 56685 55462 NMB0052 twitching motility protein PilT 57891 56851 NMB0053 conserved hypothetical protein 58011 58694 NMB0054 hypothetical protein 58697 59101 NMB0055 pyrroline-5-carboxylate reductase 59153 59941 Appendix B NMB0056 DnaK suppressor protein 60091 60504 NMB0057 hypothetical protein 66347 66700 NMB0058 hypothetical protein 66731 66885 NMB0059 dnaJ protein 66972 68090 NMB0060 conserved hypothetical protein 68289 70304 NMB0061 dTDP-6-deoxy-L-lyxo-4-hexulose reductase FRAMESHIFT 70923 69924 NMB0062 glucose-l-phosphate thymidylyltransferase 71828 70965 NMB0063 dTDP-D-glucose 4,6-dehydratase 72958 71894 NMB0064 UDP-glucose 4-epimerase 74093 73077 NMB0065 hypothetical protein 74476 75399 NMB0066 rRNA adenine N-6-methyltransferase 75687 76418 NMB0067 polysialic acid capsule biosynthesis protein SiaD, truncation 77283 76609 NMB0068 polysialic acid capsule biosynthesis protein SiaC 78416 77370 NMB0069 polysialic acid capsule biosynthesis protein SiaB 79103 78420 NMB0070 polysialic acid capsule biosynthesis protein synX 80240 79110 NMB0071 capsule polysaccharide export outer membrane protein CtrA 80375 81547 NMB0072 capsule polysaccharide export inner-membrane protein CtrB 81565 82725 NMB0073 capsule polysaccharide export inner-membrane protein CtrC 82728 83522 NMB0074 capsule polysaccharide export ATP-binding protein CtrD 83522 84169 NMB0075 transcriptional accessory protein Tex, putative 84236 86506 NMB0076 methyltransferase HphIm(C), FRAMESHIFT 86540 87539 NMB0077 site-specific DNA methylase, truncation 87529 87876 NMB0078 UDP-glucose 4-epimerase, truncation 87922 88575 NMB0079 dTDP-D-glucose 4,6-dehydratase 88694 89758 NMB0080 glucose-l-phosphate thymidylyltransferase 89824 90687 NMB0081 dTDP-4-keto-6-deoxy-D-glucose-3,6-epimerase 90729 91280 NMB0082 capsule polysaccharide modification protein LipA 91308 93419 NMB0083 capsule polysaccharide modification protein LipB 93559 94815 NMB0084 conserved hypothetical protein FRAMESHIFT 95185 96587 NMB0085 sodium/glutamate symporter 96808 98019 NMB0086 hypothetical protein 98121 99134 NMB0087 hypothetical protein 99148 99342 NMB0088 outer membrane protein PI, putative 101170 99773 NMB0089 pyruvate kinase II 102957 101488 NMB0090 IS1016 family transposase, putative FRAMESHIFT 103217 103857 NMB0091 hypothetical protein 104399 104632 NMB0092 hypothetical protein 104629 104853 NMB0093 hypothetical protein 104856 104939 NMB0094 hypothetical protein 105228 105413 NMB0095 hypothetical protein 105423 105572 NMB0096 hypothetical protein 105676 105843 NMB0097 secretion protein, putative POINT MUTATION 105860 107344 NMB0098 ABC transporter, ATP-binding protein FRAMESHIFT 107313 109396 NMB0099 hypothetical protein 109624 109484 NMB0100 hypothetical protein 109770 109627 NMB0101 IS1016 family transposase, putative FRAMESHIFT 109850 110489 NMB0102 hypothetical protein 110608 111123 NMB0103 bacteriocin resistance protein, putative 111896 111405 NMB0104 hypothetical protein 113073 112402 NMB0105 PhnO-related protein 114197 113358 NMB0106 aspartate carbamoyltransferase, catalytic subunit 114436 115353 NMB0107 aspartate carbamoyltransferase, regulatory subunit 115366 115821 NMB0108 hypothetical protein 115889 116551 NMB0109 conserved hypothetical protein 117948 116620 NMB0110 polypeptide deformylase 118018 118518 NMB0111 methionyl-tRNA formyltransferase 118608 119531 NMB0112 16S RNA methyltransferase 119613 120869 NMB0113 hypothetical protein 120892 121431 NMB0114 nitrogen regulation protein NtrY, putative 121434 123551 NMB0115 nitrogen assimilation regulatory protein NtrX 123547 124821 Appendix B -3- NMB0116 DNA processing chain A 124915 126105 NMB0117 smg protein, putative 126134 126592 NMB0118 DNA topoisomerase I 126667 128970 NMB0119 hypothetical protein 129741 129049 NMB0120 hypothetical protein 130312 129764 NMB0121 conserved hypothetical protein 130431 130805 NMB0122 conserved hypothetical protein 130897 131463 NMB0123 ferredoxin, 4Fe-4S bacterial type 131589 131837 NMB0124 translation elongation factor Tu 132257 133438 NMB0125 preprotein translocase subunit SecE 133638 133913 NMB0126 transcription antitermination protein NusG 133918 134451 NMB0127 50S ribosomal protein LII 134555 134986 NMB0128 50S ribosomal protein Li 134989 135681 NMB0129 hypothetical protein 135753 135893 NMB0130 50S ribosomal protein L10 135914 136411 NMB0131 50S ribosomal protein L7/LI2 136472 136840 NMB0132 DNA-directed RNA polymerase, beta subunit FRAMESHIFT 137027 141208 NMB0133 DNA-directed RNA polymerase, beta' subunit 141368 145540 NMB0134 hypothetical protein 145835 146089 NMB0135 conserved hypothetical protein 146089 146235 NMB0136 30S ribosomal protein S12 146417 146785 NMB0137 30S ribosomal protein S7 146906 147373 NMB0138 elongation factor G (EF-G) 147395 149497 NMB0139 translation elongation factor Tu 149586 150767 NMB0140 30S ribosomal protein S10 150788 151096 NMB0141 transposase, truncation 151241 151603 NMB0142 50S ribosomal protein L3 151777 152418 NMB0143 50S ribosomal protein L4 152421 153038 NMB0144 50S ribosomal protein L23 153038 153349 NMB0145 50S ribosomal protein L2 153358 154188 NMB0146 30S ribosomal protein S19 154198 154473 NMB0147 50S ribosomal protein L22 154485 154811 NMB0148 30S ribosomal protein S3 154824 155513 NMB0149 50S ribosomal protein L16 155500 155913 NMB0150 50S ribosomal protein L29 155916 156104 NMB0151 30S ribosomal protein S17 156107 156367 NMB0152 50S ribosomal protein L14 156592 156957 NMB0153 50S ribosomal protein L24 156972 157292 NMB0154 50S ribosomal protein L5 157305 157841 NMB0155 30S ribosomal protein S14 157847 158149 NMB0156 30S ribosomal protein S8 158168 158557 NMB0157 50S ribosomal protein L6 158574 159104 NMB0158 50S ribosomal protein L18 159121 159471 NMB0159 30s ribosomal protein,S5 159493 160008 NMB0160 50S ribosomal protein L30 160004 160186 NMB0161 50S ribosomal protein LI5 160191 160622 NMB0162 preprotein translocase SecY subunit 160637 161944 NMB0163 translation initiation factor IF-i 161952 162167 NMB0164 50S ribosomal protein L36 162191 162301 NMB0165 30S ribosomal protein S13 162370 162729 NMB0166 30S ribosomal protein 51 162752 163144 NMB0167 30S ribosomal protein S4 163167 163784 NMB0168 DNA-directed RNA polymerase, alpha subunit 163813 164796 NMB0169 50S ribosomal protein L17 164823 165188 NMB0170 septum site-determining protein MinC 165338 166048 NMB0171 septum site-determining protein MinD 166079 166891 NMB0172 cell division topological specificity factor 166898 167158 NMB0173 transcriptional regulator, LysR family 167165 168082 NMB0174 valyl-tRNA synthetase 171252 168418 NMB0175 conserved hypothetical protein 172158 171352 NMB0176 D-amino acid dehydrogenase, small subunit 173595 172342 NMB0177 sodium/alanine symporter, putative 175065 173677 NMB0178 acyl-(acyl-carrier-protein)--UDP-N-acetylglucosamine 0acyltransferase 176198 175425 Appendix B -4- NMB0179 (3R)-hydroxymyristoyl-(acyl carrier protein) dehydratase 176734 176288 NMB0180 UDP-3-O-(3-hydroxymyristoyl)-glucosamine N-acyltransferase 177814 176771 NMB0181 outer membrane protein OmpH, putative 178347 177850 NMB0182 outer membrane protein Omp85 180806 178416 NMB0183 conserved hypothetical protein 182203 180866 NMB0184 1-deoxy-D-xylulose 5-phosphate reductoisomerase 183422 182241 NMB0185 phosphatidate cytidylyltransferase 184275 183481 NMB0186 undecaprenyl pyrophosphate synthetase 185024 184281 NMB0187 ribosome recycling factor 185637 185083 NMB0188 conserved hypothetical protein 186944 185820 NMB0189 hypothetical protein 187355 187774 NMB0190 glucose inhibited division protein B 187935 188555 NMB0191 ParA family protein 188657 189427 NMB0192 ribonuclease HII 191274 190693 NMB0193 glucose inhibited division protein A 193238 191346 NMB0194 amino acid symporter, putative 194991 193567 NMB0195 pyridoxal phosphate biosynthetic protein PdxA 195133 196137 NMB0196 ribonuclease E 200197 197441 NMB0197 hypothetical protein 200321 200605 NMB0198 ribosomal large subunit pseudouridine synthase C 200690 201679 NMB0199 lipid-A-disaccharide synthase 201730 202899 NMB0200 hypothetical protein 203501 203115 NMB0201 hypothetical protein 203724 204131 NMB0202 hypothetical protein 204152 204322 NMB0203 dihydrodipicolinate reductase 205207 204401 NMB0204 lipoprotein, putative 205594 205220 NMB0205 ferric uptake regulation protein 205813 206244 NMB0206 leucyl/phenylalanyl-tRNA--protein transferase 206317 207039 NMB0207 glyceraldehyde 3-phosphate dehydrogenase 208326 207298 NMB0208 ferredoxin, 4Fe-4S bacterial type 209364 208528 NMB0209 glutathione-regulated potassium-efflux system protein 209513 211486 NMB0210 site-specific DNA methylase, truncation 212082 212401 NMB0211 L-serine dehydratase 214093 212711 NMB0212 DNA gyrase subunit B 216580 214193 NMB0213 hypothetical protein 216736 217719 NMB0214 oligopeptidase A 217810 219843 NMB0215 conserved hypothetical protein 221035 220472 NMB0216 catalase 222945 221434 NMB0217 RNA polymerase sigma-54 factor RpoN, putative 223293 224141 NMB0218 glycosyltransferase 226194 225067 NMB0219 3-oxoacyl-(acyl-carrier-protein) synthase II 227746 226502 NMB0220 acyl carrier protein 228138 227905 NMB0221 dihydroorotate dehydrogenase 228370 229374 NMB0222 hypothetical protein 229540 230010 NMB0223 hypothetical protein 230140 230355 NMB0224 glutamate-ammonia-ligase adenylyltransferase 230556 233243 NMB0225 transposase, IS30 family FRAMESHIFT 234513 233551 NMB0226 conserved hypothetical protein 235470 234781 NMB0227 conserved hypothetical protein 236771 235581 NMB0228 conserved hypothetical protein 237637 236903 NMB0229 conserved hypothetical protein FRAMESHIFT 238552 237662 NMB0230 conserved hypothetical protein 239196 238552 NMB0231 hypothetical protein 239356 239255 N NMB0232 DNA helicase II 239380 241584 NMB0233 hypothetical protein 241663 241761 NMB0234 hypothetical protein 242111 242647 NMB0235 hypothetical protein 243052 242894 NMB0236 hypothetical protein 243168 243063 NMB0237 hypothetical protein 243535 243179 NMB0238 IS1016 family transposase, degenerate 243588 243849 NMB0239 hypothetical protein 244051 244668 Appendix B NMB0240 NMB0241 NMB0242 NMB0243 NMB0244 NMB0245 NMB0246 NMB0247 NMB0248 NMB0249 NMB0250 NMB0251 NMB0252 NMB0253 NMB0254 NMB0255 NMB0256 NMB0257 NMB0258 NMB0259 NMB0260 NMB0261 NMB0262 NMB0263 NMB0264 NMB0265 NMB0266 NMB0267 NMB0268 NMB0269 NMB0270 NMB0271 NMB0272 NMB0273 NMB0274 NMB0275 NMB0276 NMB0277 NMB0278 NMB0279 NMB0280 NMB0281 NMB0282 NMB0283 NMB0284 NMB0285 NMB0286 NMB0287 NMB0288 NMB0289 NMB0290 NMB0291 NMB0292 NMB0293 NMB0294 NMB0295 NMB0296 NMB0297 NMB0298 NMB0299 NMB0300 NMB0301 NMB0302 NMB0303 hypothetical protein 244694 246142 NADH dehydrogenase I, A subunit 246607 246960 NADH dehydrogenase I, B subunit 246954 247433 NADH dehydrogenase I, C subunit 247449 248039 NADH dehydrogenase I, D subunit 248032 249285 NADH dehydrogenase I, E subunit 249288 249758 NADH dehydrogenase I, F subunit 250151 251449 hypothetical protein 251452 251886 conserved hypothetical protein 252175 252411 NADH dehydrogenase I, G subunit 252726 254984 NADH dehydrogenase I, H subunit 254990 256063 NADH dehydrogenase I, I subunit 256147 256623 hypothetical protein 256657 257361 NADH dehydrogenase I, J subunit 257400 258068 NADH dehydrogenase I, K subunit 258068 258370 cell filamentation protein Fic-related protein 258407 258979 hypothetical protein 259106 259444 NADH dehydrogenase I, L subunit 259496 261517 NADH dehydrogenase I, M subunit 261616 263109 NADH dehydrogenase I, N subunit 263122 264561 hypothetical protein 264612 264995 geranyltranstransferase 265863 265087 exodeoxyribonuclease, small subunit 266188 265967 conserved hypothetical protein 267358 266438 ABC transporter, ATP-binding protein 269219 267366 Holliday junction DNA helicase RuvA 269966 269385 conserved hypothetical protein 270374 270051 conserved hypothetical protein 271155 270439 RNA methyltransferase, TrmH family 271749 271288 competence protein 272539 271817 bioH protein, putative 272538 273284 hypothetical protein 273284 274069 hypothetical protein 274527 274820 hypothetical protein 274861 275283 ATP-dependent DNA helicase RecQ 277728 275431 indole-3-glycerol phosphate synthase 278575 277796 conserved hypothetical protein 279582 278629 virulence factor MviN 281255 279717 thiol:disulfide interchange protein DsbA 281470 282165 conserved hypothetical protein 283229 282228 organic solvent tolerance protein, putative 283431 285704 peptidyl-prolyl cis-trans isomerase 285809 286852 ribonuclease II-related protein 290243 288366 conserved hypothetical protein 290552 291181 adenylosuccinate lyase 291256 292623 O-antigen acetylase FRAMESHIFT 292707 294573 conserved hypothetical protein 295481 294870 probable ATP-dependent helicase DinG 297668 295521 hypothetical protein 297740 297967 deoxyribodipyrimidine photolyase, FRAMESHIFT 299363 298066 transcriptional regulator, putative 300264 299356 conserved hypothetical protein 300372 300767 conserved hypothetical protein 300819 301421 TonB-dependent receptor, putative 301610 303718 thiol:disulfide interchange protein DsbA 303836 304528 signal recognition particle protein 306232 304865 CcsA-related protein 306452 307255 hypothetical protein 307272 307367 hypothetical protein 307401 307583 comEA-related protein 313097 313540 hypothetical protein 313603 313904 Hypothetical protein 313958 314161 IS1016C2 transposase, degenerate 314284 314933 transposase, degenerate 315024 315307 Appendix B -6- NMB0304 class 5 outer membrane protein, degenerate 315549 315295 NMB0305 hypothetical protein 315891 315736 NMB0306 hypothetical protein 316061 316252 NMB0307 phospho-2-dehydro-3-deoxyheptonate aldolase, phe-sensitive 316403 317455 NMB0308 dihydrofolate reductase 317526 318011 NMB0309 conserved hypothetical protein 318840 318367 NMB0310 conserved hypothetical protein 319280 318855 NMB0311 hypothetical protein 319392 319634 NMB0312 virulence-associated protein VapA FRAMESHIFT 321089 323177 NMB0313 conserved hypothetical protein 323422 324885 NMB0314 hypothetical protein 326057 325092 NMB0315 conserved hypothetical protein 326135 327424 NMB0316 conserved hypothetical protein 328616 327933 NMB0317 conserved hypothetical protein 329164 328694 NMB0318 fatty acid efflux system protein 329606 330757 NMB0319 fatty acid efflux system protein 330784 332307 NMB0320 hypothetical protein 332373 332519 NMB0321 50S ribosomal protein L28 332560 332790 NMB0322 50S ribosomal protein L33 332825 332977 NMB0323 UbiH family protein 334353 333172 NMB0324 50S ribosomal protein L27 334964 334695 NMB0325 50S ribosomal protein L21 335297 334992 NMB0326 octaprenyl-diphosphate synthase 335521 336492 NMB0327 conserved hypothetical protein FRAMESHIFT 336500 336944 NMB0328 hypothetical protein 336993 337165 NMB0329 type IV pilus assembly protein 337388 339061 NMB0330 conserved hypothetical protein 339358 339152 NMB0331 kinase, putative 339983 339354 NMB0332 type IV prepilin peptidase 340845 339988 NMB0333 pilus assembly protein PilG 342151 340922 NMB0334 glucose-6-phosphate isomerase 342508 344148 NMB0335 2,3,4,5-tetrahydropyridine-2-carboxylate N-succinyltransferase 344361 345179 NMB0336 enoyl-(acyl-carrier-protein) reductase 345337 346119 NMB0337 branched-chain amino acid aminotransferase, putative 347364 346369 NMB0338 hypothetical protein 347506 347985 NMB0339 conserved hypothetical protein 347999 349165 NMB0340 lactoylglutathione lyase FRAMESHIFT 349193 349605 NMB0341 tspA protein 352407 349783 NMB0342 intracellular septation protein A 352613 353140 NMB0343 conserved hypothetical protein 353158 353433 NMB0344 BolA/YrbA family protein 353436 353711 NMB0345 cell-binding factor, putative 353763 354626 NMB0346 hypothetical protein 354700 355455 NMB0347 conserved hypothetical protein 355531 356019 NMB0348 conserved hypothetical protein 356053 357060 NMB0349 glutamyl-tRNA synthetase-related protein 358020 357136 NMB0350 hypothetical protein 358760 358311 NMB0351 transaldolase 359966 358914 NMB0352 sugar isomerase, KpsF/GutQ family 360063 361034 NMB0353 conserved hypothetical protein 361255 361788 NMB0354 hypothetical protein 361788 362366 NMB0355 conserved hypothetical protein 362350 362877 NMB0356 ABC transporter, ATP-binding protein 362924 363685 NMB0357 monofunctional biosynthetic peptidoglycan transglycosylase 364858 364160 NMB0358 shikimate 5-dehydrogenase 365670 364864 NMB0359 glutamate--ammonia ligase 365970 367385 NMB0360 AmpG-related protein 367544 368824 NMB0361 conserved hypothetical protein 368824 369096 NMB0362 hypothetical protein 369205 369282 NMB0363 hypothetical protein 369610 369744 NMB0364 FrpC operon protein 370088 370858 Appendix B -7- NMB0365 iron-regulated protein FrpC, truncation 370878 371150 NMB0366 hypothetical protein 372373 371243 NMB0367 hypothetical protein 372823 372440 NMB0368 hypothetical protein 373350 372895 NMB0369 hypothetical protein 373720 373334 NMB0370 hypothetical protein 374229 373855 NMB0371 hypothetical protein 374658 374254 NMB0372 hypothetical protein 375341 374667 NMB0373 hypothetical protein 375915 375559 NMB0374 MafB-related protein 377321 375921 NMB0375 mafA protein 378266 377328 NMB0376 hypothetical protein 378379 378266 NMB0377 conserved hypothetical protein 379516 378389 NMB0378 phosphate permease, putative 379807 381378 NMB0379 oxygen-independent coproporphyrinogen III oxidase 383155 381737 NMB0380 trans.criptional regulator, Crp/Fnr family 383360 384091 NMB0381 cys regulon transcriptional activator 385157 384210 NMB0382 outer membrane protein class 4 385521 386246 NMB0383 hypothetical protein 386270 386494 NMB0384 hypothetical protein 386773 387066 NMB0385 thiamin-monophosphate kinase 387100 388053 NMB0386 phosphatidylglycerophosphatase A 388049 388531 NMB0387 ABC transporter, ATP-binding protein 390270 388597 NMB0388 sugar transporter, putative 390657 392009 NMB03B9 aldose 1-epimerase 392016 393023 NMB0390 maltose phosphorylase 393260 395515 NMB0391 beta-phosphoglucomutase 395531 396193 NMB0392 1-aspartate oxidase 397882 396377 NMB0393 multidrug resistance protein 398266 397934 NMB0394 quinolinate synthetase A 399530 398421 NMB0395 conserved hypothetical protein 399732 400667 NMB0396 nicotinate-nucleotide pyrophosphorylase 400888 401766 NMB0397 hypothetical protein 401797 402081 NMB0398 transcriptional regulator, ArsR family 402176 402454 NMB0399 exodeoxyribonuclease III 402517 403284 NMB0400 transposase, truncated 404230 404799 NMB0401 proline dehydrogenase 409441 405839 NMB0402 sodium/proline symporter 411216 409693 NMB0403 hypothetical protein 411644 411555 NMB0404 conserved hypothetical protein 411699 412016 NMB0405 competence protein ComM 412033 413526 NMB0406 conserved hypothetical protein 413629 414495 NMB0407 thiol:disulfide interchange protein DsbA 414501 415142 NMB0408 bacitracin resistance protein 415178 415996 NMB0409 conserved hypothetical protein 417783 416575 NMB0410 conserved hypothetical protein 418062 418514 NMBO411 conserved hypothetical protein 418514 419497 NMB0412 cell division protein FtsL-related protein 419491 419757 NMB0413 penicillin-binding protein 2 419821 421563 NMB0414 UDP-N-acetylmuramoylalanyl-D-glutamate--2,6-diaminopimelate ligase 421591 423066 NMB0415 conserved hypothetical protein FRAMESHIFT 423092 424736 NMB0416UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-diaminopimelate--Dalanyl-D- alanyl ligase 424864 426228 NMB0417 hypothetical protein 426234 426407 NMB0418 phospho-N-acetylmuramoyl-pentapeptide-transferase 426657 427736 NMB0419 conserved hypothetical protein 427865 428458 NMB0420 UDP-N-acetylmuramoylalanine--D-glutamate ligase 428545 429879 NMB0421 cell division protein FtsW 430062 431330 NMB0422 UDP-N-acetylglucosamine--N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase 431337 432401 NMB0423 UDP-N-acetylmuramate--alanine ligase 432559 433965 NMB0424 D-alanine--D-alanine ligase 434081 434992 Appendix B -8- NMB0425 cell division protein FtsQ 435006 435710 NMB0426 cell division protein FtsA 435799 437040 NMB0427 cell division protein FtsZ 437162 438337 NMB0428 conserved hypothetical protein 438479 439786 NMB0429 hypothetical protein 440162 440263 NMB0430 carboxyphosphonoenolpyruvate phosphonomutase, putative 440412 441287 NMB0431 methylcitrate synthase/citrate synthase 2 441376 442527 NMB0432 conserved hypothetical protein 442683 443468 NMB0433 aconitate hydratase 1 443549 446152 NMB0434 conserved hypothetical protein 446958 448124 NMB0435 acetate kinase 448541 449737 NMB0436 conserved hypothetical protein 450078 450716 NMB0437 conserved hypothetical protein 451289 450849 NMB0438 hypothetical protein 451463 451828 NMB0439 conserved hypothetical protein 451876 453027 NMB0440 prephenate dehydrogenase, putative 453959 453090 NMB0441 nitrilase 454044 454853 NMB0442 opacity protein FRAMESHIFT 455681 454888 NMB0443 transposase, IS30 family 456456 457418 NMB0444 conserved hypothetical protein 457979 458830 NMB0445 bicyclomycin resistance protein, putative 459352 460581 NMB0446 chorismate mutase/prephenate dehydratase 460662 461747 NMB0447 DNA repair protein RecO 461787 462575 NMB0448 pyridoxal phosphate biosynthetic protein PdxJ 462602 463327 NMB0449 hypothetical protein 463482 463703 NMB0450 hypothetical protein 463968 464411 NMB0451 hypothetical protein 464424 465188 NMB0452 holo-(acyl-carrier protein) synthase 465391 465765 NMB0453 mutT protein 465850 466656 NMB0454 hypothetical protein 466652 467071 NMB0455 conserved hypothetical protein 467123 468262 NMB0456 N-acetylmuramoyl-L-alanine amidase 469573 468326 NMB0457 conserved hypothetical protein 470031 469573 NMB0458 glutamate racemase 470233 471042 NMB0459 conserved hypothetical protein 473202 472096 NMB0460 transferrin-binding protein 2 475573 477708 NMB0461 transferrin-binding protein 1 477798 480542 NMB0462 spermidine/putrescine ABC transporter, periplasmic spermidine/putrescine-binding protein 483195 481819 NMB0463 30S ribosomal protein S20 483261 483521 NMB0464 phospholipase Al, putative 483685 484830 NMB0465 conserved hypothetical protein 484976 485674 NMB0466 aspartyl-tRNA synthetase 485735 487540 NMB0467 hypothetical protein 487694 487975 NMB0468 biosynthetic arginine decarboxylase 488145 490034 NMB0469 agmatinase 490136 491056 NMB0470 C4-dicarboxylate transporter 491257 492720 NMB0471 conserved hypothetical protein 494006 492933 NMB0472 8-amino-7-oxononanoate synthase 494229 495368 NMB0473 conserved hypothetical protein 495381 496025 NMB0474 biotin synthesis protein BioC, putative 496016 496795 NMB0475 hypothetical protein 497063 498451 NMB0476 hypothetical protein 498457 499551 NMB0477 conserved hypothetical protein 499566 500099 NMB0478 hypothetical protein 500104 500745 NMB0479 conserved hypothetical protein 500771 501127 NMB0480 TspB-related protein 502193 501801 NMB0481 hypothetical protein 502509 502180 NMB0482 hypothetical protein 502900 502625 NMB0483 Hypothetical protein 503191 502910 NMB0484 hypothetical protein 503396 503202 NMB0485 hypothetical protein 503691 503404 NMB0486 conserved hypothetical protein FRAMESHIFT 505078 503739 Appendix B NMB0487 hypothetical protein 505244 505152 NMB0488 hypothetical protein 505800 505309 NMB0489 hypothetical protein 506682 505804 NMB0490 PspA-related protein 507809 506910 NMB0491 hypothetical protein 508744 508304 NMB0492 hypothetical protein 509383 509063 NMB0493 hemagglutinin/hemolysin-related protein 517494 509386 NMB0494 DNA helicase, truncation 518107 517625 NMB0495 replication protein 519187 518207 NMB0496 hemolysin activator-related protein 519134 520810 NMB0497 hemagglutinin/hemolysin-related protein 520922 526826 NMB0498 hypothetical protein 526836 527342 NMB0499 hypothetical protein 527471 529090 NMB0500 hypothetical protein 529102 529476 NMB0501 hypothetical protein 529757 530128 NMB0502 hypothetical protein 530166 532115 NMB0503 hypothetical protein 532134 532562 NMB0504 hypothetical protein 532780 532992 NMB0506 hypothetical protein 533691 535208 NMB0507 hypothetical protein 535208 535693 NMB0508 hypothetical protein 535883 536152 NMB0509 hypothetical protein 536335 537114 NMB0510 hypothetical protein 537136 537396 NMB0511 hypothetical protein 537506 539425 NMB0512 hypothetical protein 539437 539856 NMB0513 hypothetical protein 539896 540294 NMB0514 hypothetical protein 540420 540656 NMB0515 hypothetical protein 540656 541036 NMB0516 hypothetical protein 541042 541974 NMB0517 hypothetical protein 542172 542020 NMB0518 hypothetical protein 542486 542734 NMB0519 hypothetical protein 542725 542925 NMB0520 hypothetical protein 542931 543107 NMB0521 hypothetical protein 543492 543947 NMB0522 transposase, truncated 543958 544080 NMB0523 ABC transporter, ATP-binding protein, truncation 544162 544441 NMB0524 ribonuclease BN, putative 545691 544474 NMB0525 aluminum resistance protein, putative 546236 546892 NMB0526 hypothetical protein 546923 547438 NMB0527 6-pyruvoyl tetrahydrobiopterin synthase, putative 547448 547867 NMB0528 conserved hypothetical protein 548139 548507 NMB0529 conserved hypothetical protein 548507 549142 NMB0530 glycosyl hydrolase, family 3 550869 549787 NMB0531 conserved hypothetical protein 552446 550929 NMB0532 protease DO 554147 552651 NMB0533 endonuclease III 554914 554288 NMB0534 conserved hypothetical protein 555373 554963 NMB0535 glucose/galactose transporter 555906 557183 NMB0536 Na+/H+ antiporter 557477 558853 NMB0537 conserved hypothetical protein 559809 558988 NMB0538 conserved hypothetical protein 560326 559820 NMB0539 porphobilinogen deaminase 560445 561377 NMB0540 aspartate aminotransferase 562977 561787 NMB0541 hypothetical protein 563556 563062 NMB0542 hypothetical protein 563672 563872 NMB0543 L-lactate permease, putative 565630 564047 NMB0544 conserved hypothetical protein 566621 565902 NMB0545 conserved hypothetical protein 566870 570352 NMB0546 alcohol dehydrogenase, propanol-preferring 571566 570523 NMB0547 type IV pilin protein 572238 571852 NMB0548 AcrA/AcrE family protein 572464 573639 NMB0549 ABC transporter, ATP-binding protein 573708 575639 NMB0550 thiol:disulfide interchange protein DsbC 576837 576058 NMB0551 primosomal protein n' 576975 579161 Appendix B NMB0552 hypothetical protein 580284 579214 NMB0553 transposase, putative, POINT MUTATION 581288 580335 NMB0554 dnaK protein 584451 582526 NMB0555 hypothetical protein 584931 584662 NMB0556 repressor protein, putative 585119 585802 NMB0557 conserved hypothetical protein 585937 586272 NMB0558 hypothetical protein 586435 586896 NMB0559 ubiquinone biosynthesis protein AarF 586934 588442 NMB0560 serine acetyltransferase 589620 588805 NMB0561 grpE protein 589804 590379 NMB0562 conserved hypothetical protein 590874 590662 MB0563 thiamine biosynthesis lipoprotein ApbE 591955 590903 NMB0564 Na(+)-translocating NADH-quinone reductase, subunit F 593325 592111 NMB0565 Na(+)-translocating NADH-quinone reductase, subunit E 593932 593342 NMB0566 Na(+)-translocating NADH-quinone reductase, subunit D 594562 593939 NMB0567 Na(+)-translocating NADH-quinone reductase, subunit C 595338 594565 NMB0568 Na(+)-translocating NADH-quinone reductase, subunit B 596563 595334 NMB0569 Na(+)-translocating NADH-quinone reductase, subunit A 597909 596569 NMB0570 hypothetical protein 599680 598262 NMB0571 conserved hypothetical protein 600400 600044 NMB0572 hypothetical protein 601002 600400 NMB0573 transcriptional regulator, AsnC family 601612 601052 NMB0574 glycine cleavage system T protein 602042 603139 NMB0575 glycine cleavage system H protein 603304 603687 NMB0576 glutamyl-tRNA reductase 603842 605086 NMB0577 NosR-related protein 605365 605934 NMB0578 copper ABC transporter, periplasmic copper-binding protein 605991 607022 NMB0579 copper ABC transporter, ATP-binding protein 607083 607700 NMB0580 protein disulfide isomerase NosL, putative 607842 608333 NMB0581 electron transfer flavoprotein-ubiquinone oxidoreductase 610085 608427 NMB0582 NMB0583 NMB0584 NMB0585 NMB0586 NMB0587 NMB0588 NMB0589 NMB0590 NMB0591 NMB0592 NMB0593 NMB0594 NMB0595 NMB0596 NMB0597 NMB0598 NMB0599 NMB0600 NMB0601 NMB0602 NMB0603 NMB0604 NMB0605 NMB0606 NMB0607 bacteriocin resistance protein, putative 610757 610218 IS1016C2 transposase 612651 611986 FrpC operon protein 613242 614054 iron-regulated protein FrpA, putative 614074 617979 adhesin, putative 619176 618265 membrane protein 620128 619256 ABC transporter, ATP-binding protein 620907 620155 50s ribosomal protein L19 621563 621201 tRNA (guanine-Nl)-methyltransferase FRAMESHIFT 622329 621582 16S rRNA processing protein RimM 622838 622332 30S ribosomal protein S16 623099 622857 conserved hypothetical protein 625570 623147 sensor histidine kinase 627094 625691 DNA-binding response regulator 627785 627111 hypothetical protein 629789 627978 hypothetical protein 630132 629782 Maf/YceF/YhdE family protein 630749 630144 conserved hypothetical protein 631572 630805 hypothetical protein 632272 631589 conserved hypothetical protein 632479 632279 hitA protein 632849 632529 phosphoribosyl-ATP cyclohydrolase 633244 632924 alcohol dehydrogenase, zinc-containing 634449 633388 histone deacetylase family protein 636107 635001 conserved hypothetical protein 636235 636498 protein-export membrane protein SecD 636710 638563 Appendix B -11- NMB0608 protein-export membrane protein SecF 638570 639502 NMB0609 30s ribosomal protein S15 639728 639994 NMB0610 spermidine/putrescine ABC transporter, ATP-binding protein 640243 641499 NMB0611 spermidine/putrescine ABC transporter, permease protein 641518 642480 NMB0612 spermidine/putrescine ABC transporter, permease protein 642483 643367 NMB0613 hypothetical protein 643392 643496 NMB0614 oxidoreductase, putative 643496 644788 NMB0615 ammonium transporter AmtB, putative 646340 645039 NMB0616 IS1016 family transposase, degenerate 647272 646871 NMB0617 transcription termination factor Rho 648837 647581 NMB0618 phosphoenolpyruvate synthase 651441 649060 NMB0619 conserved hypothetical protein 651853 652671 NMB0620 phosphoglycolate phosphatase 653575 652916 NMB0621 conserved hypothetical protein 654440 653616 NMB0622 outer membrane lipoprotein carrier protein 654867 655487 NMB0623 spermidine/putrescine ABC transporter, periplasmic spermidine/putrescine-binding protein 655763 656899 NMB0624 galactosyltransferase-related protein FRAMESHIFT 657035 658253 NMB0625 conserved hypothetical protein 658297 658824 NMB0626 peptide chain release factor 3 660797 659205 NMB0627 phosphoribosyl-AMP cyclohydrolase 661299 660907 NMB0628 hisF protein 662097 661333 NMB0629 phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase 662847 662113 NMB0630 amidotransferase HisH 663518 662883 NMB0631 phosphate acetyltransferase Pta, putative 665151 663652 NMB0632 iron(III) ABC transporter, ATP-binding protein 666394 665339 NMB0633 iron(III) ABC transporter, permease protein 667932 666418 NMB0634 iron(III) ABC transporter, periplasmic binding protein 668995 668003 NMB0635 transposase, IS30 family 670247 669285 NMB0636 hypothetical protein 670794 670414 NMB0637 argininosuccinate lyase 672228 670855 NMB0638 UTP--glucose-l-phosphate uridylyltransferase 673116 672250 NMB0639 conserved hypothetical protein 673743 673147 NMB0640 hypothetical protein 673969 673739 NMB0641 inorganic pyrophosphatase 674610 674080 NMB0642 dATP pyrophosphohydrolase 675169 674714 NMB0643 MafB-related protein 675614 677437 NMB0644 hypothetical protein 677443 677904 NMB0645 ribonuclease FRAMESHIFT 677948 678275 NMB0646 ribonuclease inhibitor barstar 678290 678574 NMB0647 hypothetical protein 679091 680326 NMB0648 hypothetical protein 680357 680776 NMB0649 hypothetical protein 680970 681191 NMB0650 hypothetical protein 681167 681583 NMB0651 hypothetical protein 681687 682073 NMB0652 mafA protein 682199 683137 NMB0653 MafB-related protein 683144 684409 NMB0654 hypothetical protein 684415 684729 NMB0655 hypothetical protein 684867 685571 NMB0656 hypothetical protein 685600 685926 NMB0657 hypothetical protein 686024 686224 NMB0658 Hypothetical protein 686055 686312 NMB0659 hypothetical protein 686346 686744 NMB0660 hypothetical protein 686929 687315 NMB0661 bis(5'-nucleosyl)-tetraphosphatase, symmetrical/Trk system potassium uptake protein TrkG FRAMESHIFT 689659 687362 NMB0662 ribonuclease, putative 690126 689740 NMB0663 outer membrane protein NsgA 690786 690265 NMB0664 hypothetical protein 691151 690960 Appendix B -12- NMB0665 oxygen-independent coprophorphyrinogen III oxidase family protein 692546 691374 NMB0666 DNA ligase 695128 692606 NMB0667 hypothetical protein 696562 695279 NMB0668 ampD protein 697352 696783 NMB0669 conserved hypothetical protein 697436 698428 NMB0670 thymidylate kinase 698491 699108 NMB0671 malate oxidoreductase (NAD) 699333 700610 NMB0672 tetraacyldisaccharide 4'-kinase 701160 702191 NMB0673 hypothetical protein 702394 702978 NMB0674 conserved hypothetical protein 703050 703229 NMB0675 3-deoxy-D-manno-octulosonate cytidylyltransferase 703229 703987 NMB0676 hypothetical protein 704013 704411 NMB0677 hypothetical protein 704610 704723 NMB0678 tryptophan synthase, alpha subunit 705306 706088 NMB0679 acetyl-CoA carboxylase, carboxyl transferase beta subunit 706129 706998 NMB0680 cryptic protein 707672 707064 NMB0681 conserved hypothetical protein 707781 708002 NMB0682 dihydroorotase 708368 709399 NMB0683 N utilization substance protein B 710195 709773 NMB0684 riboflavin synthase, beta subunit 710749 710276 NMB0685 hypothetical protein 711120 710800 NMB0686 ribonuclease III 711287 712003 NMB0687 GTP-binding protein Era 712003 712974 NMB0688 N-(5'-phosphoribosyl)anthranilate isomerase 715446 714823 NMB0689 transcription elongation factor GreB 715996 715508 NMB0690 amidophosphoribosyltransferase 717640 716099 NMB0691 colicin V production protein, putative 718450 717956 NMB0692 tpc protein 719441 718446 NMB0693 folylpolyglutamate synthase/dihydrofolate synthase 720728 719457 NMB0694 folI protein 721205 720762 NMB0695 hypothetical protein 721569 721213 NMB0696 amino acid ABC transporter, ATP-binding protein FRAMESHIFT 722369 NMB0697 NMB0698 NMB0699 NMB0700 NMB0701 NMB0702 NMB0703 NMB0704 NMB0705 NMB0706 NMB0707 NMB0708 NMB0709 NMB0710 NMB0711 NMB0712 NMB0713 NMB0714 NMB0715 NMB0716 NMB0717 NMB0718 NMB0719 NMB0720 NMB0721 NMB0722 NMB0723 NMB0724 NMB0725 721645 dimethyladenosine transferase 723321 722545 hypothetical protein 723518 724204 tryptophan synthase, beta subunit 724290 725489 IgA-specific serine endopeptidase 731118 725674 hypothetical protein 731531 731280 competence protein ComA 732529 734601 competence lipoprotein ComL 735635 734835 ribosomal large subunit pseudouridine synthase D 735634 736755 transporter 737858 736914 conserved hypothetical protein 738418 739194 rare lipoprotein B, putative 739249 739725 DNA polymerase III, delta subunit 739730 740725 Hypothetical protein 740849 741265 Hypothetical protein 741293 741856 conserved hypothetical protein FRAMESHIFT 742826 741946 RNA polymerase sigma-32 factor 744182 743313 apolipoprotein N-acyltransferase, putative 746012 744441 conserved hypothetical protein FRAMESHIFT 746771 746019 Hypothetical protein 746967 747284 Hypothetical protein 747440 747727 cytochrome, putative 748209 747796 ferrochelatase 749572 748493 queuine tRNA-ribosyltransferase 750697 749585 threonyl-tRNA synthetase 751005 752915 translation initiation factor 3 752990 753454 50S ribosomal protein L35 753604 753798 50S ribosomal protein L20 753814 754170 phenylalanyl-tRNA synthetase, alpha chain 754519 755508 modification methylase HgaI-1 755694 756749 Appendix B -13- NMB0726 type II restriction enzyme HgaI 756755 758221 NMB0727 N-6 adenine-specific DNA methylase 758221 758868 NMB0728 phenylalanyl-tRNA synthetase, beta chain 758896 761256 NMB0729 integration host factor, alpha subunit 761333 761632 NMB0730 hypothetical protein 762257 762739 NMB0731 hypothetical protein 763002 763226 NMB0732 adenosylmethionine-8-amino-7-oxononanoate aminotransferase 763559 764857 NMB0733 dethiobiotin synthase 764857 765501 NMB0734 hypothetical protein 765519 765992 NMB0735 4-hydroxybenzoate octaprenyltransferase 766025 766912 NMB0736 PTS system, nitrogen regulatory IIA protein 767100 767546 NMB0737 HPr kinase/phosphatase, putative 767551 768510 NMB0738 conserved hypothetical protein 768494 769345 NMB0739 conserved hypothetical protein 769429 770943 NMB0740 DNA repair protein RecN 771255 772925 NMB0741 conserved hypothetical protein 775384 773948 NMB0742 conserved hypothetical protein 775684 776040 NMB0743 ubiquinone/menaquinone biosynthesis methlytransferase UbiE 776097 776831 NMB0744 hypothetical protein 777054 777530 NMB0745 2-amino-4-hydroxy-6-hydroxymethyldihydropteridinepyrophosphokinase 778153 777662 NMB0746 conserved hypothetical protein 778537 778166 NMB0747 conserved hypothetical protein 779157 778594 NMB0748 host factor-I 779535 779245 NMB0749 penicillin-binding protein 4 780602 779667 NMB0750 bacterioferritin comigratory protein 780923 781360 NMB0751 integrase/recombinase XerD 781415 782287 NMB0752 bacterioferritin-associated ferredoxin, putative 782462 782659 NMB0753 conserved hypothetical protein 782828 783058 NMB0754 hypothetical protein 783066 783173 NMB0755 hypothetical protein 783194 783334 NMB0756 dTDP-L-rhamnose synthase, putative 784398 783481 NMB0757 phosphoribosylaminoimidazole-succinocarboxamide synthase 784598 785458 NMB0758 polyribonucleotide nucleotidyltransferase 785695 787815 NMB0759 conserved hypothetical protein 788619 787894 NMB0760 diaminopimelate epimerase 789006 789854 NMB0761 hypothetical protein 789940 790164 NMB0762 hypothetical protein 790198 790653 NMB0763 cysteine synthase 790653 791582 NMB0764 conserved hypothetical protein 792048 792950 NMB0765 signal peptidase I 794128 793112 NMB0766 GTP-binding protein LepA 796064 794274 NMB0767 5-methylthioadenosine nucleosidase/S-adenosylhomocysteine nucleosidase 796909 796211 NMB0768 twitching motility protein PilT 797095 798204 NMB0769 DNA polymerase III, delta prime subunit, putative 798241 799215 NMB0770 type IV pilus assembly protein PilZ, putative 799222 799569 NMB0771 conserved hypothetical protein 799577 800353 NMB0772 conserved hypothetical protein 800382 800594 NMB0773 conserved hypothetical protein 800698 801006 NMB0774 uracil phosphoribosyltransferase 801115 801738 NMB0775 hypothetical protein 801764 802081 NMB0776 conserved hypothetical protein 802335 802751 NMB0777 uroporphyrinogen-III synthase HemD, putative 802796 803533 NMB0778 uroporphyrin-III C-methyltransferase HemX, putative 803611 804882 NMB0779 hypothetical protein 804882 806102 NMB0780 hypothetical protein 806138 806575 NMB0781 uroporphyrinogen decarboxylase 806732 807793 NMB0782 DNA repair protein RadA 807982 809358 NMB0783 conserved hypothetical protein 810116 809640 NMB0784 phage shock protein E precursor, putative 810717 810361 Appendix B -14- NMB0785 exodeoxyribonuclease V 135 KD polypeptide 814370 810759 NMB0786 conserved hypothetical protein 815358 814453 NMB0787 amino acid ABC transporter, periplasmic amino acid-binding protein 815643 816467 NMB0788 amino acid ABC transporter, permease protein 816514 817173 NMB0789 amino acid ABC transporter, ATP-binding protein 817186 817938 NMB0790 phosphoglucomutase 819343 817964 NMB0791 peptidyl-prolyl cis-trans isomerase 820019 819513 NMB0792 transporter, NadC family 821553 820141 NMB0793 hypothetical protein 821759 821553 NMB0794 hypothetical protein 822146 821787 NMB0795 peptidyl-tRNA hydrolase 822988 822413 NMB0796 conserved hypothetical protein 823319 823044 NMB0797 conserved hypothetical protein 823749 823315 NMB0798 cell division protein FtsH 825932 823968 NMB0799 cell division protein FtsJ 826616 825999 NMB0800 conserved hypothetical protein 826726 827007 NMB0801 delta-aminolevulinic acid dehydratase 827193 828191 NMB0802 cystathionine gamma-synthase 829414 828260 NMB0803 conserved hypothetical protein 829606 830376 NMB0804 NAD(P)H nitroreductase, putative 830489 831151 NMB0805 transposase, IS30 family 831295 832257 NMB0806 conserved hypothetical protein 833050 832295 NMB0807 conserved hypothetical protein 833965 833078 NMB0808 hypothetical protein 834551 833988 NMB0809 conserved hypothetical protein 835399 834605 NMB0810 transcriptional regulator, TetR family 836104 835457 NMB0811 UDP-N-acetylpyruvoylglucosamine reductase 837156 836119 NMB0812 conserved hypothetical protein 838579 837203 NMB0813 hypothetical protein 838634 838819 NMB0814 histidyl-tRNA synthetase 838914 840062 NMB0815 adenylosuccinate synthetase 840163 841464 NMB0816 hypothetical protein 841592 841903 NMB0817 hypothetical protein 841932 842312 NMB0818 hypothetical protein 842329 842736 NMB0819 hypothetical protein 842856 843245 NMB0820 hypothetical protein 843456 843845 NMB0821 hypothetical protein 843962 844519 NMB0822 heat shock protein HtpX 845866 844826 NMB0823 adenylate kinase 845878 846522 NMB0824 orotidine 5'-phosphate decarboxylase 847051 847788 NMB0825 ADP-heptose synthase, putative 847846 848814 NMB0826 C-5 cytosine-specific DNA methylase 848854 850086 NMB0827 type II restriction enzyme-related protein FRAMESHIFT 850091 851119 NMB0828 ADP-L-glycero-D-mannoheptose-6-epimerase 851251 852252 NMB0829 type I restriction enzyme EcoR124II M protein 852329 853870 NMB0830 conserved hypothetical protein 853870 854877 NMB0831 type I restriction enzyme S protein, degenerate 855046 856216 NMB0832 anticodon nuclease 856277 857416 NMB0833 type I restriction enzyme-related protein 857416 857799 NMB0834 transposase, IS30 family 858756 857794 NMB0835 type I restriction enzyme EcoR124II R protein, putative 858832 861594 NMB0836 ATP-dependent Clp protease, ATP-binding subunit ClpA 863945 861639 .NMB0837 conserved hypothetical protein 864249 863950 NMB0838 cold-shock domain family protein 864492 864692 NMB0839 pmbA protein 866323 864995 NMB0840 conserved hypothetical protein 866446 866979 NMB0841 hypothetical protein 867029 867742 NMB0842 single-stranded-DNA-specific exonuclease RecJ 867814 869511 NMB0843 polyA polymerase 869811 871169 NMB0844 hypothetical protein 871345 871665 NMB0845 PhoH-related protein 872732 871782 Appendix B NMB0846 LPS biosynthesis protein-related protein 873905 872874 NMB0847 hypothetical protein 874235 874065 NMB0848 hypothetical protein 874369 875070 NMB0849 deoxycytidine triphosphate deaminase, putative 875703 875140 NMB0850 hypothetical protein 876185 875772 NMB0851 recombination associated protein RdgC 877146 876250 NMB0852 essential GTPase 878634 877180 NMB0853 conserved hypothetical protein 879413 878787 NMB0854 histidyl-tRNA synthetase 880709 879417 NMB0855 bacteriocin resistance protein, putative 881459 880806 NMB0856 hypothetical protein 882208 881744 NMB0857 hypothetical protein 882441 882268 NMB0858 hypothetical protein 882645 882448 NMB0859 hypothetical protein 883025 882651 NMB0860 hypothetical protein 883340 883086 NMB0861 hypothetical protein 883975 883433 NMB0862 hypothetical protein 884091 883975 NMB0863 hypothetical protein 884410 884141 NMB0864 hypothetical protein 884966 884679 NMB0865 hypothetical protein 885445 884975 NMB0866 hypothetical protein 886357 885491 NMB0867 YabO/YceC/SfhB family protein 886521 887441 NMB0868 conserved hypothetical protein 888163 887525 NMB0869 hypothetical protein 889009 888221 NMB0870 3-methyl-2-oxobutanoate hydroxymethyltransferase 889502 890290 NMB0871 pantoate--beta-alanine ligase 890416 891249 NMB0872 conserved hypothetical protein 891416 893257 NMB0873 outer membrane lipoprotein LolB, putative 893400 893978 NMB0874 conserved hypothetical protein 893991 894833 NMB0875 ribose-phosphate pyrophosphokinase 895258 896238 NMB0876 50S ribosomal protein L25 896308 896877 NMB0877 penicillin-binding protein 898174 897008 NMB0878 threonine dehydratase 898322 899845 NMB0879 sulfate ABC transporter, ATP-binding protein 900978 899908 NMB0880 sulfate ABC transporter,' permease protein 901835 900978 NMB0881 sulfate ABC transporter, permease protein 902923 902090 NMB0882 hypothetical protein 903214 903543 NMB0883 conserved hypothetical protein 903878 904384 NMB0884 superoxide dismutase 905491 904907 NMB0885 replicative DNA helicase 905655 907058 NMB0886 fimbrial protein FimT 907370 908035 NMB0887 type IV pilus assembly protein PilV, putatve 908056 908667 NMB0888 hypothetical protein 908667 909605 NMB0889 hypothetical protein 909587 910177 NMB0890 type IV pilin-related protein 910170 910655 NMB0891 hypothetical protein 911708 911944 NMB0892 AzlC-related protein 912795 912376 NMB0893 deoxyuridine 5'-triphosphate nucleotidohydrolase 912995 913444 NMB0894 aminotransferase, class I 913525 914709 NMB0895 conserved hypothetical protein 914975 915751 NMB0896 integrase, FRAMESHIFT 916283 917352 NMB0897 hypothetical protein 917468 917845 NMB0898 hypothetical protein 917894 918079 NMB0899 hypothetical protein 918396 918749 NMB0900 hypothetical protein 919621 920535 NMB0901 D-lactate dehydrogenase-related protein 920880 920599 NMB0902 hypothetical protein 921133 920945 NMB0903 hypothetical protein 921429 921139 NMB0904 hypothetical protein 921686 921429 NMB0905 hypothetical protein 921936 921724 NMB0906 hypothetical protein 922860 922009 NMB0907 hypothetical protein 923244 922888 NMB0908 hypothetical protein 923512 923315 NMB0909 hypothetical protein 924280 923759 Appendix B -16- NMB0910 transcriptional regulator 925000 924287 NMB0911 transposase, IS30 family 926382 925420 NMB0912 hypothetical protein 926526 927149 NMB0913 pemK protein 927552 927208 NMB0914 pemI protein 927790 927557 NMB0915 hypothetical protein 928640 928152 NMB0916 hypothetical protein 928799 928662 NMB0917 death-on-curing protein 929446 929081 NMB0918 hypothetical protein 929574 929446 NMB0919 IS1106 transposase, putative 930929 929973 NMB0920 isocitrate dehydrogenase 934317 932095 NMB0921 hypothetical protein 934522 934325 NMB0922 alpha-2,3-sialyltransferase 934750 935862 NMB0923 cytochrome c 936488 936033 NMB0924 oxidoreductase, short-chain dehydrogenase/reductase family 936607 937425 NMB0925 acyl CoA thioester hydrolase family protein 937925 937482 NMB0926 opacity protein 940336 939513 NMB0927 proline iminopeptidase 941840 942769 NMB0928 hypothetical protein 944025 942832 NMB0929 dihydrodipicolinate synthase 944909 944037 NMB0930 xanthine/uracil permease family protein 945369 946757 NMB0931 RNA methyltransferase, TrmH family 947574 946825 NMB0932 conserved hypothetical protein 948129 947644 NMB0933 cytidine and deoxycytidylate deaminase family protein 948580 948137 NMB0934 DNA transformation protein tfoX-related protein 948853 948625 NMB0935 tRNA delta(2)-isopentenylpyrophosphate transferase 949798 948860 NMB0936 hypothetical protein 951481 950180 NMB0937 elongation factor P (EF-P) 951788 952345 NMB0938 hypothetical protein 953235 952402 NMB0939 conserved hypothetical protein 953933 953355 NMB0940 homoserine O-acetyltransferase 955069 953933 NMB0941 50S ribosomal protein L36 955756 955634 NMB0942 50S ribosomal protein L31, putative 956031 955759 NMB0943 5,10-methylenetetrahydrofolate reductase 956231 957106 NMB0944 methyltransferase 957247 959520 NMB0945 hypothetical protein 959535 959696 NMB0946 peroxiredoxin 2 family protein/glutaredoxin 959802 960536 NMB0947 lipoamide dehydrogenase, putative 960788 962188 NMB0948 succinate dehydrogenase, cytochrome b556 subunit 962470 962844 NMB0949 succinate dehydrogenase, hydrophobic membrane anchor protein 962841 963179 NMB0950 succinate dehydrogenase, flavoprotein subunit 963185 964945 NMB0951 succinate dehydrogenase, iron-sulfur protein 965068 965772 NMB0952 conserved hypothetical protein 965779 966024 NMB0953 hypothetical protein 966024 966104 NMB0954 citrate synthase 966139 967419 NMB0955 2-oxoglutarate dehydrogenase, El component 967627 970452 NMB0956 2-oxoglutarate dehydrogenase, E2 component, dihydrolipoamide succinyltransferase 970555 971733 NMB0957 2-oxoglutarate dehydrogenase, E3 component, lipoamide dehydrogenase 972101 973531 NMB0958 hypothetical protein 973659 973943 NMB0959 succinyl-CoA synthetase, beta subunit 974045 975208 NMB0960 succinyl-CoA synthetase, alpha subunit 975222 976109 NMB0961 funZ protein 978267 976675 NMB0962 excinuclease ABC, subunit A 981150 978304 NMB0963 phosphatidylserine decarboxylase precursor-related protein 981305 982099 NMB0964 TonB-dependent receptor 985503 983230 NMB0965 hypothetical protein 985832 985564 Appendix B -17- NMB0966 para-aminobenzoate synthase glutamine amidotransferase component II 985925 986512 NMB0967 anthranilate phosphoribosyltransferase 986579 987634 NMB0968 hypothetical protein 987644 987729 NMB0969 hypothetical protein 988030 987792 NMB0970 conserved hypothetical protein, FRAMESHIFT 988106 989527 NMB0971 hypothetical protein 989493 989780 NMB0972 hypothetical protein 989788 989982 NMB0973 hypothetical protein 989993 990274 NMB0974 hypothetical protein 990284 990559 NMB0975 hypothetical protein 990690 991004 NMB0976 TspB-related protein 990991 991383 NMB0977 modulator of drug activity B, putative 991676 992146 NMB0978 NAD(P) transhydrogenase, beta subunit 993742 992360 NMB0979 hypothetical protein 994205 993825 NMB0980 NAD(P) transhydrogenase, alpha subunit 995750 994212 NMB0981 phosphoserine phosphatase 996040 996870 NMB0982 chloride channel protein-related protein 997018 998157 NMB0983 phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase 998324 999901 NMB0984 transposase, putative, degenerate 1000517 1001457 NMB0985 E16-related protein 1001522 1002016 NMB0986 hypothetical protein 1001997 1002425 NMB0987 N-acetylmuramoyl-L-alanine amidase, putative 1002736 1003278 NMB0988 hypothetical protein 1003278 1003478 NMB0989 hypothetical protein 1003484 1003645 NMB0990 hypothetical protein 1003859 1004260 NMB0991 IS1106 transposase 1005417 1004308 NMB0992 adhesin 1007326 1005554 NMB0993 rubredoxin 1009428 1009261 NMB0994 acyl-CoA dehydrogenase family protein 1011202 1010114 NMB0995 macrophage infectivity potentiator-related protein 1012020 1011340 NMB0996 hypothetical protein 1012411 1012043 NMB0997 D-lactate dehydrogenase 1014397 1012709 NMB0998 oxidoreductase, putative 1014921 1018751 NMB0999 NifR3/SMM1 family protein 1018935 1019933 NMB1000 IS1106 transposase, putative FRAMESHIFT 1020537 1021551 NMB1001 integrase protein, degenerate 1023183 1022614 NMB1002 hypothetical protein 1024370 1023498 NMB1003 hypothetical protein 1024711 1024418 NMB1004 hypothetical protein 1024962 1024720 NMB1005 hypothetical protein 1025179 1024958 NMB1006 hypothetical protein 1025360 1025184 NMB1007 transcriptional regulator 1025451 1025819 NMB1008 hypothetical protein 1025824 1026444 NMB1009 conserved hypothetical protein 1026440 1026631 NMB1010 hypothetical protein 1026658 1027218 NMB1011 hypothetical protein 1027252 1028196 NMB1012 hypothetical protein 1028284 1028784 NMB1013 hypothetical protein 1028801 1028971 NMB1014 conserved hypothetical protein 1029045 1029635 NMB1015 IS150 transposase, putative FRAMESHIFT 1029653 1030443 NMB1016 conserved hypothetical protein 1031794 1031192 NMB1017 sulfate ABC transporter, periplasmic sulfate-binding protein 1033574 1032522 NMB1018 conserved hypothetical protein 1034162 1033683 NMB1019 phosphoribosylaminoimidazole carboxylase, ATPase subunit 1035345 1034212 NMB1020 hypothetical protein 1035887 1035345 NMB1021 anthranilate synthase component I 1037359 1035887 NMB1022 transposase, IS30 family 1038444 1037482 NMB1023 conserved hypothetical protein 1039543 1038587 NMB1024 conserved hypothetical protein 1040502 1039639 NMB1025 conserved hypothetical protein 1040896 1040537 Appendix B -18- NMB1026 conserved hypothetical protein 1040971 1041447 NMB1027 dnaJ protein, truncation 1041473 1042192 NMB1028 conserved hypothetical protein 1042197 1043069 NMB1029 aspartate ammonia-lyase 1044541 1043147 NMB1030 conserved hypothetical protein 1045565 1045005 NMB1031 3-isopropylmalate dehydrogenase 1046798 1045731 NMB1032 type II restriction enzyme NlaIV 1047563 1046835 NMB1033 modification methylase NlaIV 1048850 1047582 NMB1034 3-isopropylmalate dehydratase, small subunit 1049666 1049028 NMB1035 hypothetical protein 1049982 1049731 NMB1036 3-isopropylmalate dehydratase, large subunit 1051488 1050082 NMB1037 glutamate--cysteine ligase 1051748 1053094 NMB1038 DNA repair protein RadC 1053220 1053894 NMB1039 conserved hypothetical protein 1053970 1054692 NMB1040 hypothetical protein 1054848 1056125 NMB1041 GTP-binding protein 1056133 1057308 NMB1042 cation transport ATPase, E1-E2 family 1057308 1059776 NMB1043 hypothetical protein 1059940 1060142 NMB1044 ferredoxin--NADP reductase 1061316 1060543 NMB1045 hypothetical protein 1062298 1061507 NMB1046 threonine synthase 1063753 1062347 NMB1047 hypothetical protein 1064197 1063829 NMB1048 hypothetical protein 1065918 1064452 NMB1049 transcriptional regulator, putative 1066174 1067085 NMB1050 transposase, IS30 family 1068512 1067550 NMB1051 ABC transporter, ATP-binding protein 1070544 1068637 NMB1052 dedA protein 1071207 1070566 NMB1053 class 5 outer membrane protein 1072189 1071374 NMB1054 IS1106 transposase, degenerate 1073920 1072988 NMB1055 serine hydroxymethyltransferase 1075474 1074227 NMB1056 hypothetical protein 1075753 1075544 NMB1057 gamma-glutamyltranspeptidase 1077776 1075959 NMB1058 conserved hypothetical protein FRAMESHIFT 1078161 1077902 NMB1059 conserved hypothetical protein 1078505 1078720 NMB1060 fructose-1,6-bisphosphatase 1079840 1078869 NMB1061 conserved hypothetical protein 1080931 1080089 NMB1062 conserved hypothetical protein 1081610 1081011 NMB1063 dihydroneopterin aldolase 1081666 1082019 NMB1064 conserved hypothetical protein 1082056 1082589 NMB1065 crcB protein 1083465 1083109 NMB1066 hypothetical protein 1084174 1083497 NMB1067 cell division protein FtsK 1084339 1087380 NMB1068 gamma-glutamyl phosphate reductase 1088870 1087611 NMB1069 glutamate 5-kinase 1089992 1088886 NMB1070 2-isopropylmalate synthase 1090477 1092027 NMB1071 conserved hypothetical protein 1092125 1092784 NMB1072 prolipoprotein diacylglyceryl transferase 1093721 1092873 NMB1073 conserved hypothetical protein 1094922 1093795 NMB1074 acetylglutamate kinase 1095092 1095985 NMB1075 conserved hypothetical protein 1098302 1097637 NMB1076 DnaA-related protein 1098967 1098302 NMB1077 ABC transporter, ATP-binding protein, truncation 1099623 1099075 NMB1078 transcriptional regulator, UmuD/LexA family 1100312 1099875 NMB1079 hypothetical protein 1100580 1100425 NMB1080 ner protein FRAMESHIFT 1100802 1101061 NMB1081 bacteriophage transposase 1101126 1103108 NMB1082 hypothetical protein 1103120 1103317 NMB1083 bacteriophage DNA transposition protein B, putative 1103481 1104650 NMB1084 hypothetical protein 1104655 1105173 NMB1085 N-acetylmuramoyl-L-alanine amidase, putative 1105319 1105861 NMB1086 hypothetical protein 1106234 1106467 NMB1087 hypothetical protein 1106758 1107060 NMB1088 conserved hypothetical protein 1107278 1107111 Appendix B -19- NMB1089 hypothetical protein 1107506 1107841 NMB1090 hypothetical protein 1107856 1108119 NMB1091 hypothetical protein 1108119 1108313 NMB1092 hypothetical protein 1108319 1108822 NMB1093 hypothetical protein 1109412 1108825 NMB1094 hypothetical protein 1109497 1111044 NMB1095 conserved hypothetical protein 1111047 1112612 NMB1096 conserved hypothetical protein 1112602 1113894 NMB1097 cryptic Mu-phage G protein, putative 1114007 1114419 NMB1098 I protein, putative 1114653 1115711 NMB1099 transposase, IS30 family 1116767 1115805 NMB1100 hypothetical protein 1116795 1117274 NMB1101 conserved hypothetical protein 1117277 1117696 NMB1102 hypothetical protein 1117746 1118336 NMB1103 hypothetical protein 1118336 1118530 NMB1104 phage sheath protein 1118536 1119942 NMB1105 hypothetical protein 1120010 1120384 NMB1106 hypothetical protein 1120391 1120753 NMB1107 hypothetical protein 1121610 1121011 NMB1108 hypothetical protein 1121780 1123933 NMB1109 phage virion protein, putative 1123936 1125264 NMB1110 tail protein, 43 kDa 1125257 1126399 NMB1111 baseplate assembly protein V, putative 1126399 1127064 NMBI112 conserved hypothetical protein 1127168 1127512 NMB1113 conserved hypothetical protein FRAMESHIFT 1127529 1128580 NMB1114 conserved hypothetical protein 1128580 1129137 NMB1115 tail fibre protein, putative 1129151 1131121 NMBI116 hypothetical protein 1131560 1132084 NMB1117 hypothetical protein 1132350 1132204 NMB1118 conserved hypothetical protein 1132762 1132478 NMB1119 conserved hypothetical protein 1132842 1133444 NMB1120 hypothetical protein 1133426 1133719 NMB1121 conserved hypothetical protein 1133719 1133925 NMB1122 ABC transporter, ATP-binding protein FRAMESHIFT 1135181 1134041 NMB1198 conserved hypothetical protein 1199352 1198465 NMB1161 hypothetical protein 1167620 1167426 NMB1162 hypothetical protein 1168307 1167663 NMB1163 hypothetical protein 1168675 1168307 NMB1164 hypothetical protein 1169353 1168685 NMB1165 oxidoreductase, short chain dehydrogenase/reductase family 1170237 1169521 NMB1128 conserved hypothetical protein 1139597 1138287 NMB1167 hypothetical protein 1171869 1171666 NMB1168 phytoene synthase, putative 1172903 1172034 NMB1131 chaperone protein HscA 1142897 1141038 NMB1132 hypothetical protein 1143630 1142977 NMB1171 conserved hypothetical protein ankyrin-related protein 1176464 1175706 NMB1172 ferredoxin, 2Fe-2S type 1176860 1176522 NMBI173 hypothetical protein 1177278 1177138 NMB1136 hypothetical protein 1146017 1145337 NMB1175 conserved hypothetical protein 1178247 1178053 NMB1176 conserved hypothetical protein 1178719 1178321 NMB1139 acetyl-CoA carboxylase, carboxyl transferase alpha subunit 1147851 1146895 NMB140 mesJ protein FRAMESHIFT 1149229 1147948 NMB1179 RNA methyltransferase, TrmH family 1182124 1181516 NMB1180 hypothetical protein 1182411 1182178 NMB1181 hypothetical protein 1182945 1182583 NMB1182 hypothetical protein 1183262 1182960 NMB1145 UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-mesodiaminopimelate ligase 1152664 1151291 NMB1146 biotin synthetase 1153923 1152874 NMB1185 hypothetical protein 1186675 1186043 Appendix B NMB1148 hypothetical protein 1154845 1154693 NMB1187 hypothetical protein 1187052 1186912 NMB1150 dihydroxy-acid dehydratase 1157144 1155288 NMB1189 sulfite reductase hemoprotein, beta-component 1191122 1189356 NMB1190 sulfite reductase (NADPH) flavoprotein, alpha component 1192963 1191152 NMB1153 sulfate adenylyltransferase, subunit 1 1162210 1160927, plasmid protein NMB1192 sulfate adenylyltransferase, subunit 2 1195208 1194288 NMB1155 phosphoadenosine phosphosulfate reductase 1163950 1163213 NMB1194 siroheme synthase 1197448 1196000 NMB1195 hypothetical protein 1197732 1197577 NMB1158 nickel-dependent hydrogenase, b-type cytochrome subunit 1166365 1165712 NMB1197 conserved hypothetical protein 1199352 1198465 NMB1199 GTP-binding protein TypA 1201433 1199625 NMB1200 ribonuclease II family protein 1202272 1204644 NMB1201 IMP dehydrogenase 1206449 1204989 NMB1202 hypothetical protein 1207237 1206779 NMB1203 protein-PII uridylyltransferase 1209886 1207331 NMB1204 transcriptional regulator 1210255 1209938 NMB1205 hypothetical protein 1210426 1210283 NMB1206 bacterioferritin B 1211053 1210583 NMB1207 bacterioferritin A 1211545 1211084 NMB1208 hypothetical protein 1211610 1211810 NMB1209 hypothetical protein 1211900 1212100 NMB1210 toxin-activating protein, putative 1212121 1212585 NMB1211 hypothetical protein 1212984 1212745 NMB1212 hypothetical protein 1213319 1212984 NMB1213 hypothetical protein 1213678 1213319 NMB1214 hemagglutinin/hemolysin-related protein 1220496 1213678 NMB1215 hypothetical protein 1220814 1220659 NMB1216 lipoic acid synthetase 1221989 1221009 NMB1217 lipoate-protein ligase B 1222554 1221985 NMB1218 conserved hypothetical protein 1222882 1222610 NMB1219 transporter, putative 1223067 1224134 NMB1220 stomatin/Mec-2 family protein 1225281 1224337 NMB1221 hypothetical protein 1225703 1225299 NMB1222 uracil-DNA glycosylase 1225784 1226440 NMB1223 site-specific DNA methylase, degenerate 1226520 1229028 NMB1224 hypothetical protein 1229552 1229154 NMB1225 hypothetical protein 1230112 1229600 NMB1226 ABC transporter, ATP-binding protein 1232500 1230581 NMB1227 conserved hypothetical protein 1232972 1232580 NMB1228 homoserine dehydrogenase 1233145 1234449 NMB1229 hypothetical protein 1234445 1234876 NMB1230 DNA-binding protein HU-beta 1235207 1234941 NMB1231 ATP-dependent protease La 1237851 1235392 NMB1232 conserved hypothetical protein 1238285 1239202 NMB1233 exodeoxyribonuclease V, alpha subunit 1240978 1239236 NMB1234 ABC transporter, ATP-binding protein 1241741 1241049 NMB1235 conserved hypothetical protein 1242981 1241737 NMB1236 hypothetical protein 1243186 1243461 NMB1237 recombination protein RecR 1244140 1243523 NMB1238 peptidyl-prolyl cis-trans isomerase-related protein 1245742 1244207 NMB1239 conserved hypothetical protein 1246176 1245805 NMB1240 ABC transporter, ATP-binding protein 1246326 1247951 NMB1241 tRNA nucleotidyltransferase 1248026 1249276 NMB1242 hypothetical protein 1249502 1249807 NMB1243 Holliday junction DNA helicase RuvB 1249892 1250920 NMB1244 ribulose-phosphate 3-epimerase 1251674 1250949 NMB1245 hypothetical protein 1252367 1252035 NMB1246 conserved hypothetical protein 1253294 1252434 Appendix B -21- NMB1247 riboflavin synthase, alpha subunit 1254006 1253305 NMB1248 molybdopterin-guanine dinucleotide biosynthesis protein A FRAMESHIFT 1254659 1254085 NMB1249 nitrate/nitrite sensory protein NarX, putative 1254901 1256670 NMB1250 transcriptional regulator, LuxR family 1256670 1257323 NMB1251 transposase, IS30 family 1258731 1257769 NMB1252 phosphoribosylformylglycinamidine cyclo-ligase 1259914 1258883 NMB1253 hypothetical protein 1260672 1261346 NMB1254 GTP cyclohydrolase II 1261342 1261932 NMB1255 glycosyl transferase, degenerate 1262256 1263263 NMB1256 GTP cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase 1263728 1264816 NMB1257 site-specific DNA methylase, degenerate 1265357 1265130 NMB1258 conserved hypothetical protein 1267046 1265739 NMB1259 transposase, IS30 family 1267584 1268546 NMB1260 type III restriction-modification system EcoPI enzyme, subunit res 1271565 1268629 NMB1261 type III restriction-modification system EcoPI enzyme, subunit mod POINT MUTATION FRAMESHIFT 1273661 1271581 NMB1262 peptidyl-prolyl cis-trans isomerase 1274334 1273780 NMB1263 CobW-related protein 1275316 1274402 NMB1264 conserved hypothetical protein 1275771 1275502 NMB1265 conserved hypothetical protein 1276061 1275771 NMB1266 zinc uptake regulation protein, putative 1276582 1276109 NMB1267 low molecular weight protein tyrosine-phosphatase 1277108 1276656 NMB1268 conserved hypothetical protein 1278348 1277236 NMB1269 hypothetical protein 1279559 1278465 NMB1270 conserved hypothetical protein 1281272 1279644 NMB1271 mercury transport periplasmic protein, putative 1281584 1281375 NMB1272 hypothetical protein 1281765 1281625 NMB1273 alginate O-acetylation protein AlgI, putative 1282215 1283648 NMB1274 hypothetical protein 1283662 1284642 NMB1275 hypothetical protein 1284642 1286083 NMB1276 long-chain-fatty-acid--CoA ligase 1286122 1287672 NMB1277 transporter, BCCT family 1289792 1287768 NMB1278 site-specific recombinase 1290081 1292084 NMB1279 membrane-bound lytic murein transglycosylase B, putative 1293319 1292213 NMB1280 very long chain acyl-CoA dehydrogenase-related protein 1294948 1293524 NMB1281 transcription-repair coupling factor 1295133 1299269 NMB1282 aspartate 1-decarboxylase 1299421 1299801 NMB1283 2-dehydro-3-deoxyphosphooctonate aldolase 1299826 1300665 NMB1284 hypothetical protein 1300683 1301120 NMB1285 enolase 1301171 1302454 NMB1286 conserved hypothetical protein 1302471 1302746 NMB1287 ferredoxin, putative 1303080 1302793 NMB1288 ribonucleoside-diphosphate reductase, beta subunit 1304479 1303328 NMB1289 type II restriction enzyme, putative 1305706 1304522 NMB1290 C-5 cytosine-specific DNA-methylase 1306712 1305702 NMB1291 ribonucleoside-diphosphate reductase, alpha subunit 1309049 1306773 NMB1292 hypothetical protein 1309394 1309209 NMB1293 hypothetical protein 1309563 1309886 NMB1294 l-acyl-sn-glycerol-3-phosphate acyltransferase 1310967 1310203 NMB1295 formamidopyrimidine-DNA glycosylase 1311882 1311058 NMB1296 hypothetical protein 1312599 1311937 NMB1297 membrane-bound lytic murein transglycosylase D 1312778 1314751 NMB1298 ribosomal small subunit pseudouridine synthase A 1314822 1315511 NMB1299 sodium- and chloride-dependent transporter, degenerate 1316091 1317454 NMB1300 cytidylate kinase 1317701 1318354 NMB1301 30S ribosomal protein Sl 1318513 1320195 NMB1302 integration host factor, beta subunit 1320209 1320520 Appendix B -22- NMB1303 transcriptional regulator, MerR family 1321281 1320877 NMB1304 alcohol dehydrogenase, class III 1321402 1322535 NMB1305 esterase, putative 1322547 1323371 NMB1306 conserved hypothetical protein 1323765 1324913 NMB1307 nucleoside diphosphate kinase 1324975 1325397 NMB1308 conserved hypothetical protein 1325543 1326634 NMB1309 fimbrial biogenesis and twitching motility protein, putative, 1326640 1327398 NMB1310 gcpE protein 1327417 1328679 NMB1311 hypothetical protein 1328970 1328737 NMB1312 ATP-dependent Clp protease, proteolytic subunit 1329655 1329128 NMB1313 trigger factor 1331148 1329838 NMB1314 cell division protein FtsK 1333791 1331356 NMB1315 uracil permease 1334014 1335222 NMB1316 hypothetical protein 1335289 1335726 NMB1317 hypothetical protein 1335865 1336266 NMB1318 CDP-diacylglycerol--serine 0-phosphatidyltransferse 1336343 1337086 NMB1319 conserved hypothetical protein 1337090 1337860 NMB1320 50S ribosomal protein L9 1338540 1338091 NMB1321 30S ribosomal protein S18 1338787 1338560 NMB1322 primosomal replication protein n, putative 1339096 1338797 NMB1323 30S ribosomal protein S6 1339465 1339100 NMB1324 thioredoxin reductase 1340571 1339624 NMB1325 cation transport ATPase, E1-E2 family 1340710 1342869 NMB1326 excinuclease ABC, subunit C 1342969 1344819 NMB1327 conserved hypothetical protein 1345045 1346445 NMB1328 conserved hypothetical protein 1346570 1347283 NMB1329 hypothetical protein 1347649 1347840 NMB1330 hypothetical protein 1348276 1347917 NMB1331 excinuclease ABC, subunit B 1350416 1348392 NMB1332 carboxy-terminal peptidase 1352229 1350748 NMB1333 conserved hypothetical protein 1354146 1352359 NMB1334 hypothetical protein 1354238 1354471 NMB1335 creA protein 1354474 1355031 NMB1336 conserved hypothetical protein 1355036 1355581 NMB1337 conserved hypothetical protein 1355577 1356029 NMB1338 isomerase, putative 1356698 1356045 NMB1339 prolyl-tRNA synthetase_ 1358473 1356764 NMB1340 hypothetical protein 1358924 1359151 NMB1341 pyruvate dehydrogenase, El component 1359167 1361827 NMB1342 pyruvate dehydrogenase, E2 component, dihydrolipoamide acetyltransferase FRAMESHIFT 1361979 1363583 NMB1343 hypothetical protein 1363680 1364114 NMB1344 pyruvate dehydrogenase, E3 component, lipoamide dehydrogenase 1364135 1365916 NMB1345 hypothetical protein 1367830 1366283 NMB1346 TonB-dependent receptor, putative FRAMESHIFT 1369731 1367957 NMB1347 extragenic suppressor protein SuhB 1370786 1370004 NMB1348 RNA methylase, putative 1371030 1371842 NMB1349 hypothetical protein 1371906 1372760 NMB1350 hypothetical protein 1372967 1373305 NMB1351 fmu and fmv protein, putative 1373656 1374909 NMB1352 hypothetical protein 1375272 1375703 NMB1353 aldehyde dehydrogenase family protein 1377097 1375757 NMB1354 conserved hypothetical protein 1377755 1377105 NMB1355 glutamyl-tRNA (Gln) amidotransferase subunit C, putative 1377906 1378193 NMB1356 Glu-tRNA(Gln) amidotransferase, subunit A 1378259 1379701 NMB1357 conserved hypothetical protein 1379701 1380630 NMB1358 Glu-tRNA(Gln) amidotransferase, subunit B 1380676 1382103 NMB1359 CDP-6-deoxy-delta-3,4-glucoseen reductase, putative 1382318 1383325 NMB1360 pyridoxamine 5-phosphate oxidase 1384090 1383461 Appendix B NMB1361 conserved hypothetical protein 1384312 1385361 NMB1362 oxalate/formate antiporter, putative 1386974 1385436 NMB1363 exodeoxyribonuclease, large subunit 1388622 1387270 NMB1364 NH(3)-dependent NAD+ synthetase NadE, putative 1388819 1389637 NMB1365 conserved hypothetical protein 1390183 1389713 NMB1366 thioredoxin 1390481 1390810 NMB1367 conserved hypothetical protein 1391930 1390869 NMB1368 ATP-dependent RNA helicase, putative 1392141 1393526 NMB1369 hypothetical protein 1394572 1394021 NMB1370 hypothetical protein 1395217 1394860 NMB1371 acetylornithine aminotransferase 1395561 1396754 NMB1372 ATP-dependent Clp protease, ATP-binding subunit ClpX 1398104 1396863 NMB1373 ribosome-binding factor A 1398295 1398663 NMB1374 tRNA pseudouridine synthase B 1398699 1399619 NMB1375 modification methylase, putative FRAMESHIFT 1399839 1401945 NMB1376 conserved hypothetical protein POINT MUTATION 1401938 1404712 NMB1377 L-lactate dehydrogenase 1406036 1404867 NMB1378 conserved hypothetical protein 1406327 1406770 NMB1379 nifS protein 1406802 1408013 NMB1380 nifU protein 1408280 1408663 NMB1381 HesB/YadR/YfhF family protein 1408693 1409070 NMB1382 conserved hypothetical protein 1409254 1409036 NMB1383 chaperone protein HscB 1409336 1409833 NMB1384 DNA gyrase subunit A 1409934 1412681 NMB1385 IS1016 family transposase, degenerate 1412841 1413241 NMB1386 transposase, putative FRAMESHIFT 1413303 1413955 NMB1387 hypothetical protein 1414840 1414292 NMB1388 glucose-6-phosphate isomerase 1416500 1414857 NMB1389 RpiR/YebK/YfhH family protein 1417469 1416624 NMB1390 glucokinase 1418505 1417522 NMB1391 oxidoreductase, Sol/DevB family 1419181 1418489 NMB1392 glucose-6-phosphate 1-dehydrogenase 1420906 1419464 NMB1393 phosphogluconate dehydratase 1421474 1423306 NMB1394 4-hydroxy-2-oxoglutarate aldolase/2-deydro-3-deoxyphosphogluconate aldolase 1423490 1424125 NMB1395 alcohol dehydrogenase, zinc-containing 1425427 1424390 NMB1396 A/G-specific adenine glycosylase 1425581 1426627 NMB1397 hypothetical protein 1426793 1426972 NMB1398 Cu-Zn-superoxide dismutase 1427047 1427604 NMB1399 IS1106 transposase 1429146 1428175 NMB1400 ABC transporter family protein 1431631 1429406 NMB1401 IS1016C2 transposase 1432983 1432447 NMB1402 hypothetical protein 1433320 1433751 NMB1403 FrpA/C-related protein 1433795 1433983 NMB1404 hypothetical protein 1434021 1434746 NMB1405 FrpA/C-related protein 1434763 1435962 NMB1406 hypothetical protein 1436396 1436755 NMB1407 FrpA-related protein, degenerate 1436755 1437881 NMB1408 hypothetical protein 1437960 1438451 NMB1409 FrpA/C-related protein 1438582 1439007 NMB1410 hypothetical protein 1439247 1439783 NMB1411 IS1016C2 transposase 1440610 1439960 NMB1412 FrpC operon protein 1441216 1442022 NMB1413 IS1016 family transposase, putative FRAMESHIFT 1442715 1442132 NMB1414 FrpC operon protein 1442798 1443568 NMB1415 iron-regulated protein FrpC 1443588 1449074 NMB1416 aminopeptidase N 1452022 1449422 NMB1417 conserved hypothetical protein 1452947 1452156 NMB1418 HtrB/MsbB family protein 1454563 1453697 NMB1419 crossover junction endodeoxyribonuclease RuvC 1455150 1454617 NMB1420 factor-for-inversion stimulation protein Fis, putative 1455392 1455156 NMB1421 nifR3 protein 1456432 1455425 Appendix B NMB1422 ATP-dependent RNA helicase, putative 1456798 1458168 NMB1423 conserved hypothetical protein 1458746 1459870 NMB1424 hypothetical protein 1459903 1460928 NMB1425 lysyl-tRNA synthetase, heat inducible 1462560 1461052 NMB1426 hypothetical protein 1463968 1462718 NMB1427 hypothetical protein 1464208 1464032 NMB1428 aminopeptidase, putative 1464426 1466219 NMB1429 outer membrane protein PorA 1468209 1467034 NMB1430 transcription elongation factor GreA 1470964 1470491 NMB1431 hypothetical protein 1471298 1471050 NMB1432 3-phosphoshikimate 1-carboxyvinyltransferase 1471360 1472658 NMB1433 conserved hypothetical protein FRAMESHIFT 1473237 1472707 NMB1434 cardiolipin synthetase family protein 1474971 1473448 NMB1435 drug resistance translocase family protein 1476489 1475086 NMB1436 conserved hypothetical'protein 1476774 1477550 NMB1437 conserved hypothetical protein 1477550 1478248 NMB1438 conserved hypothetical protein 1478248 1479699 NMB1439 phosphoribosylaminoimidazole carboxylase, catalytic subunit 1480370 1479888 NMB1440 hypothetical protein 1481131 1480421 NMB1441 O-methyltransferase, putative 1481799 1481134 NMB1442 mismatch repair protein MutL 1482139 1484112 NMB1443 DNA polymerase III, subunits gamma and tau 1484210 1486321 NMB1444 conserved hypothetical protein 1486404 1486736 NMB1445 recA protein 1489556 1488513 NMB1446 3-dehydroquinate dehydratase 1489810 1490571 NMB1447 ATP-dependent DNA helicase Rep 1490594 1492606 NMB1448 DNA-damage-inducible protein P 1493734 1492781 NMB1449 TonB-dependent receptor POINT MUTATION 1496967 1493881 NMB1450 ferredoxin--NADP reductase 1497241 1498017 NMB1451 DNA polymerase III, epsilon subunit 1499643 1498234 NMB1452 conserved hypothetical protein 1500459 1501595 NMB1453 hypothetical protein 1502335 1501847 NMB1454 ferredoxin, 4Fe-4S bacterial type 1503891 1502398 NMB1455 hypothetical protein 1504075 1503959 NMB1456 hypothetical protein 1504347 1504153 NMB1457 transketolase 1504419 1506395 NMB1458 fumarate hydratase, class II 1506547 1507932 NMB1459 conserved hypothetical protein 1508923 1508003 NMB1460 single-strand binding protein 1509972 1509451 NMB1461 drug resistance translocase family protein 1511361 1509979 NMB1462 transglycosylase, putative 1512092 1511472 NMB1463 IS1106 transposase, degenerate 1512998 1512596 NMB1464 conserved hypothetical protein 1513541 1513053 NMB1465 opacity protein FRAMESHIFT 1515309 1514483 NMB1466 conserved hypothetical protein 1515639 1516367 NMB1467 exopolyphosphatase 1516487 1517992 NMB1468 hypothetical protein 1518527 1518207 NMB1469 hypothetical protein 1518607 1518527 NMB1470 hypothetical protein 1519392 1518850 NMB1471 tryptophanyl-tRNA synthetase 1520471 1519464 NMB1472 clpB protein 1520732 1523308 NMB1473 aminotransferase, class I 1524612 1523401 NMB1474 4-oxalocrotonate tautomerase, putative 1524910 1524704 NMB1475 conserved hypothetical protein 1525255 1526058 NMB1476 glutamate dehydrogenase, NAD-specific 1527384 1526122 NMB1477 hypothetical protein 1527562 1527396 NMB1478 phosphoglycolate phosphatase FRAMESHIFT 1527786 1528489 NMB1479 regulatory protein RecX 1528560 1529018 NMB1480 hypothetical protein 1529095 1529253 NMB1481 hypothetical protein 1529262 1529393 NMB1482 acyl CoA thioester hydrolase family protein 1529409 1529888 NMB1483 lipoprotein NlpD, putative 1531499 1530255 NMB1484 stationary-phase survival protein SurE 1532501 1531758 Appendix B NMB1485 conserved hypothetical protein 1534074 1532521 NMB1486 hypothetical protein 1534263 1534126 NMB1487 fimbrial assembly protein 1535230 1534445 NMB1488 succinate-semialdehyde dehydrogenase (NADP+) 1536772 1535342 NMB1489 hypothetical protein 1537259 1537750 NMB1490 hypothetical protein 1538345 1537917 NMB1491 hypothetical protein 1538785 1538699 NMB1492 hypothetical protein 1538860 1538795 NMB1493 carbon starvation protein A 1538892 1540970 NMB1494 conserved hypothetical protein 1540963-1541154 NMB1495 hypothetical protein 1541371 1541562 NMB1496 conserved hypothetical protein 1541673 1542230 NMB1497 TonB-dependent receptor 1543234 1545996 NMB1498 aspartokinase, alpha and beta subunits 1549220 1548006 NMB1499 ribonuclease PH 1550148 1549423 NMB1500 conserved hypothetical protein 1550694 1550233 NMB1501 HesA/MoeB/ThiF family protein 1550911 1551684 NMB1502 hypothetical protein 1551825 1552349 NMB1503 hypothetical protein 1552608 1552814 NMB1504 conserved hypothetical protein 1552706 1553557 NMB1505 nicotinate phosphoribosyltransferase 1553601 1554806 NMB1506 arginyl-tRNA synthetase 1554901 1556616 NMB1507 hypothetical protein 1556714 1557070 NMB1508 hypothetical protein 1557130 1558584 NMB1509 amino acid ABC transporter, permease protein 1560344 1559601 NMB1510 thermonuclease family protein 1561224 1560526 NMB1511 ribose 5-phosphate isomerase A 1561934 1561266 NMB1512 YgbB/YacN family protein 1562493 1562014 NMB1513 conserved hypothetical protein 1563214 1562528 NMB1514 DNA polymerase III, epsilon subunit 1563945 1563214 NMB1515 transporter, putative 1565411 1564104 NMB1516 fixS protein 1565589 1565404 NMB1517 hypothetical protein 1565885 1565589 NMB1518 acetate kinase 1566236 1567429 NMB1519 thiol:disulfide interchange protein DsbD 1569752 1567950 NMB1520 hypothetical protein 1570337 1569819 NMB1521 phytoene synthase-related protein 1571249 1570425 NMB1522 FKBP-type peptidyl-prolyl cis-trans isomerase SlyD 1571803 1571324 NMB1523 hypothetical protein 1572276 1572569 NMB1524 oxidoreductase, putative 1572682 1574046 NMB1525 VirG-related protein FRAMESHIFT 1576262 1574233 NMB1526 small major protein B 1577081 1576638 NMB1527 ADP-heptose--LPS heptosyltransferase II 1578146 1577139 NMB1528 methylated-DNA--protein-cysteine methyltransferase, putative 1579353 1578547 NMB1529 conserved hypothetical protein FRAMESHIFT 1579597 1580409 NMB1530 succinyl-diaminopimelate desuccinylase 1582228 1581086 NMB1531 conserved hypothetical protein 1582961 1582344 NMB1532 conserved hypothetical protein 1583504 1582998 NMB1533 H.8 outer membrane protein 1584150 1583602 NMB1534 hypothetical protein 1584287 1584150 NMB1535 hypothetical protein 1584404 1584874 NMB1536 preprotein translocase SecA subunit 1584984 1587731 NMB1537 DNA primase 1587879 1589648 NMB1538 RNA polymerase sigma factor RpoD 1589838 1591763 NMB1539 IS1106 transposase 1591913 1592917 NMB1540 lactoferrin-binding protein A 1597271 1594443 NMB1541 lactoferrin-binding protein B 1599481 1597271 NMB1542 hypothetical protein 1600504 1600722 NMB1543 conserved hypothetical protein 1600871 1602082 NMB1544 hypothetical protein 1602097 1602405 NMB1545 hypothetical protein 1602412 1602609 NMB1546 hypothetical protein 1602795 1603076 NMB1547 hypothetical protein 1603107 1603406 Appendix B -26- NMB1548 tspB protein, putative 1603741 1605384 NMB1549 hypothetical protein 1606176 1606325 NMB1550 conserved hypothetical protein 1606332 1606613 NMB1551 conserved hypothetical protein 1606617 1607717 NMB1552 pilin gene inverting protein PivNM-1A 1608019 1608972 NMB1553 transposase, truncation 1612022 1611708 NMB1554 CTP synthase 1613884 1612253 NMB1555 long-chain-fatty-acid--CoA ligase 1615666 1613999 NMB1556 tRNA (5-methylaminomethyl-2-thiouridylate) -methyltransferase 1616840 1615740 NMB1557 conserved hypothetical protein 1617439 1616969 NMB1558 diacylglycerol kinase 1618115 1617735 NMB1559 glutathione synthetase 1619386 1618430 NMB1560 glutaminyl-tRNA synthetase 1621164 1619479 NMB1561 transcriptional regulator, DeoR family 1622049 1621279 NMB1562 conserved hypothetical protein 1622994 1622095 NMB1563 transcriptional regulator, GntR family 1623859 1623146 NMB1564 conserved hypothetical protein 1624850 1624431 NMB1565 hypothetical protein 1625639 1624971 NMB1566 phosphoribosylglycinamide formyltransferase 1626281 1625658 NMB1567 macrophage infectivity potentiator 1627206 1626391 NMB1568 DNA polymerase holoenzyme chi subunit, putative 1627905 1627468 NMB1569 aminopeptidase A/I, FRAMESHIFT 1629499 1627971 NMB1570 conserved hypothetical protein 1629544 1630656 NMB1571 conserved hypothetical protein 1630656 1631723 NMB1572 aconitate hydratase 2 1631936 1634518 NMB1573 ornithine carbamoyltransferase, catabolic 1634663 1635655 NMB1574 ketol-acid reductoisomerase 1636895 1635885 NMB1575 conserved hypothetical protein 1637268 1636978 NMB1576 acetolactate synthase III, small subunit 1637826 1637338 NMB1577 acetolactate synthase III, large subunit 1639564 1637840 NMB1578 conserved hypothetical protein 1640685 1641335 NMB1579 ATP phosphoribosyltransferase 1641417 1642067 NMB1580 hypothetical protein 1642174 1643070 NMB1581 histidinol dehydrogenase 1643070 1644356 NMB1582 histidinol-phosphate aminotransferase 1644405 1645499 NMB1583 imidazoleglycerol-phosphate dehydratase 1645499 1646413.

NMB1584 3-hydroxyacid dehydrogenase 1646511 1647377 NMB1585 transcriptional regulator, MarR family 1647658 1648086 NMB1586 hypothetical protein 1648100 1648963 NMB1587 protease, putative 1650120 1649020 NMB1588 CDP-diacylglycerol--glycerol-3-phosphate 3-phosphatidyltransferase 1651479 1650919 NMB1589 hypothetical protein 1652036 1651797 NMB1590 conserved hypothetical protein 1652675 1652343 NMB1591 transcriptional regulator MtrA 1652804 1653706 NMB1592 hypothetical protein 1653729 1654313 NMB1593 conserved hypothetical protein 1654445 1655305 NMB1594 spermidine/putrescine ABC transporter, periplasmic spermidine/putrescine-binding protein 1656479 1655352 NMB1595 alanyl-tRNA synthetase 1656684 1659305 NMB1596 hypothetical protein 1659348 1659551 NMB1597 hypothetical protein 1659569 1659997 NMB1598 hypothetical protein 1660094 1660282 NMB1599 hypothetical protein 1660300 1660584 NMB1600 hypothetical protein 1660624 1660878 NMB1601 IS1106 transposase 1661075 1662079 NMB1602 transposase, putative 1663112 1661997 NMB1603 tellurite resistance protein, putative 1663289 1664230 NMB1604 phosphoglycerate mutase 1664989 1664309 NMB1605 topoisomerase IV subunit A 1665137 1667437 NMB1606 sensor histidine kinase 1667460 1669033 NMB1607 sigma-54 dependent response regulator 1669029 1669493 NMB1608 conserved hypothetical protein 1669600 1670349 Appendix B -27- NMB1609 trans-sulfuration enzyme family protein 1672860 1671694 NMB1610 hypothetical protein 1673766 1673008 NMB1611 hypothetical protein 1673866 1674114 NMB1612 amino acid ABC transporter, periplasmic amino acid-binding protein 1674169 1674972 NMB1613 fumarate hydratase, class I 1675282 1676802 NMB1614 Trk system potassium uptake protein TrkA 1676903 1678312 NMB1615 hypothetical protein 1678758 1679018 NMB1616 phosphomethylpyrimidine kinase 1679755 1680558 NMB1617 tellurite resistance protein, putative 1681480 1680614 NMB1618 ribonuclease HI 1681594 1682028 NMB1619 conserved hypothetical protein 1682889 1683290 NMB1620 conserved hypothetical protein 1683333 1684514 NMB1621 glutathione peroxidase 1685113 1684583 NMB1622 nitric oxide reductase 1687547 1685295 NMB1623 major anaerobically induced outer membrane protein 1687918 1689087 NMB1624 conserved hypothetical protein 1689215 1689967 NMB1625 pilin gene inverting protein PivNM-lB 1691651 1690698 NMB1626 conserved hypothetical protein 1693053 1691953 NMB1627 conserved hypothetical protein 1693338 1693057 NMB1628 tspB protein, putative 1695347 1693797 NMB1629 Hypothetical protein 1695690 1695328 NMB1630 hypothetical protein 1696057 1695758 NMB1631 hypothetical protein 1696449 1696088 NMB1632 hypothetical protein 1696752 1696555 NMB1633 hypothetical protein 1697067 1696759 NMB1634 conserved hypothetical protein 1698296 1697091 NMB1635 hypothetical protein 1698662 1698444 NMB1636 opacity protein FRAMESHIFT 1700231 1701047 NMB1637 conserved hypothetical protein 1701808 1701254 NMB1638 YhbX/YhjW/YijP/YjdB family protein 1703518 1701887 NMB1639 hypothetical protein 1703921 1703595 NMB1640 phosphoserine aminotransferase 1705027 1703924 NMB1641 conserved hypothetical protein 1705374 1705820 NMB1642 N utilization substance protein A 1705851 1707350 NMB1643 translation initiation factor IF-2 1707365 1710250 NMB1644 hypothetical protein 1711755 1710418 NMB1645 hypothetical protein 1713169 1711832 NMB1646 hemolysin, putative 1713312 1713935 NMB1647 amino acid symporter, putative 1715420 1714005 NMB1648 conserved hypothetical protein 1715747 1716472 NMB1649 disulfide bond formation protein B 1717022 1716537 NMB1650 leucine-responsive regulatory protein 1718177 1717716 NMB1651 alanine racemase 1718502 1719557 NMB1652 conserved hypothetical protein 1720979 1719627 NMB1653 conserved hypothetical protein 1721266 1720997 NMB1654 conserved hypothetical protein 1722129 1721395 NMB1655 adenine specific methylase, putative 1723321 1722413 NMB1656 hypothetical protein 1723454 1724044 NMB1657 comE operon protein 1-related protein 1725327 1724713 NMB1658 DNA/pantothenate metabolism flavoprotein 1731065 1732246 NMB1659 guanosine-3',5'-bis(diphosphate) 3'-pyrophosphohydrolase 1734472 1732319 NMB1660 DNA-directed RNA polymerase, omega subunit 1734770 1734567 NMB1661 guanylate kinase 1735446 1734832 NMB1662 adenine phosphoribosyltransferase 1735607 1736170 NMB1663 conserved hypothetical protein 1737007 1736222 NMB1664 protease, putative 1737332 1738684 NMB1665 conserved hypothetical protein 1739253 1738870 NMB1666 hypothetical protein 1739498 1739253 NMB1667 hypothetical protein 1740061 1739858 NMB1668 hemoglobin receptor 1742596 1740224 NMB1669 iron-starvation protein PigA 1743420 1742794 NMB1670 PqiA family protein 1743706 1745214 Appendix B NMB1671 pqiB protein 1745210 1746868 NMB1672 conserved hypothetical protein 1746871 1747386 NMB1673 DNA-3-methyladenine glycosylase I, putative 1747393 1747941 NMB1674 GDSL lipase family protein 1747934 1748572 NMB1675 hypothetical protein 1748797 1749102 NMB1676 glycine dehydrogenase (decarboxylating) 1749136 1751984 NMB1677 cytochrome c5 1753288 1752452 NMB1678 aromatic-amino-acid aminotransferase 1754906 1753716 NMB1679 tRNA (uracil-5-)-methyltransferase 1756015 1754930 NMB1680 chorismate synthase 1756162 1757259 NMB1681 hypothetical protein 1757354 1757776 NMB1682 topoisomerase IV subunit B 1759838 1757856 NMB1683 MutT/nudix family protein 1760429 1759908 NMB1684 seryl-tRNA synthetase 1760595 1761887 NMB1685 D-lactate dehydrogenase 1762966 1761971 NMB1686 peptide chain release factor 1 1764167 1763094 NMB1687 conserved hypothetical protein 1765042 1764275 NMB1688 L-asparaginase I 1766051 1765053 NMB1689 dedA protein, putative 1767007 1766327 NMB1690 phosphoglucomutase/phosphomannomutase family protein 1768532 1767201 NMB1691 dihydropteroate synthase 1769519 1768665 NMB1692 chorismate mutase-related protein 1770552 1769662 NMB1693 hypothetical protein 1770643 1772754 NMB1694 conserved hypothetical protein 1774305 1772824 NMB1695 hypothetical protein 1774424 1775401 NMB1696 acyl carrier protein 1775800 1775558 NMB1697 acyl carrier protein, putative 1776072 1775815 NMB1698 acyltransferase, putative 1776827 1776072 NMB1699 hypothetical protein 1777185 1776823 NMB1700 hypothetical protein 1777345 1777707 NMB1701 hypothetical protein 1777763 1778260 NMB1702 3-oxoacyl-(acyl-carrier-protein) reductase 1778291 1779016 NMB1703 3-oxoacyl-(acyl-carrier-protein) synthase II 1779013 1780260 NMB1704 beta-1,4-glucosyltransferase 1780467 1781222 NMB1705 alpha-1,2-N-acetylglucosamine transferase 1781226 1782287 NMB1706 hypothetical protein 1782329 1782496 NMB1707 sodium- and chloride-dependent transporter 1782677 1784011 NMB1708 NosX-related protein 1784846 1784189 NMB1709 thymidylate synthase 1785648 1784857 NMB1710 glutamate dehydrogenase, NADP-specific 1786032 1787363 NMB1711 transcriptional regulator, GntR family 1788280 1787504 NMB1712 L-lactate permease-related protein 1788711 1789007 NMB1713 transposase, IS30 family 1790361 1789399 NMB1714 multidrug efflux pump channel protein 1791874 1790474 NMB1715 multiple transferable resistance system protein MtrD 1795132 1791932 NMB1716 membrane fusion protein 1796382 1795147 NMB1717 trancscriptional regulator MtrR 1796785 1797414 NMB1718 hypothetical protein 1797953 1797699 NMB1719 efflux pump component MtrF 1798240 1799805 NMB1720 exodeoxyribonuclease V 125 kD polypeptide 1803085 1799879 NMB1721 conserved hypothetical protein 1804596 1803190 NMB1722 cytochrome C555 FRAMESHIFT 1804923 1804801 NMB1723 cytochrome c oxidase, subunit III 1806129 1805035 NMB1724 cytochrome-c oxidase, subunit II 1806939 1806331 NMB1725 cytochrome c oxidase, subunit I 1808411 1806969 NMB1726 conserved hypothetical protein 1808726 1810471 NMB1727 conserved hypothetical protein 1810539 1810964 NMB1728 biopolymer transport protein ExbD 1812088 1811657 NMB1729 biopolymer transport protein ExbB 1812753 1812094 NMB1730 TonB protein 1813661 1812822 NMB1731 conserved hypothetical protein 1813916 1814551 NMB1732 transporter, putative 1815806 1815009 Appendix B -29- NMB1733 hypothetical protein 1816445 1815945 NMB1734 glutaredoxin 1817423 1816785 NMB1735 GTP pyrophosphokinase 1817566 1819776 NMB1736 transposase, putative FRAMESHIFT 1820048 1820856 NMB1737 secretion protein, putative 1822426 1821026 NMB1738 secretion protein, putative 1823922 1822498 NMB1739 hypothetical protein 1824158 1824508 NMB1740 hypothetical protein 1824635 1825042 NMB1741 conserved hypothetical protein FRAMESHIFT 1825116 1826455 NMB1742 hypothetical protein 1826503 1826790 NMB1743 hypothetical protein 1826798 1826992 NMB1744 hypothetical protein 1827003 1827284 NMB1745 hypothetical protein 1827294 1827569 NMB1746 hypothetical protein 1827700 1827987 NMB1747 tspB protein, putative 1828031 1829533 NMB1748 conserved hypothetical protein 1829537 1829824 NMB1749 conserved hypothetical protein 1829837 1830919 NMB1750 pilin gene inverting protein PivNM-2 1831548 1832495 NMB1751 transposase, degenerate 1833264 1832887 NMB1752 conserved hypothetical protein FRAMESHIFT 1833772 1833299 NMB1753 VapD-related protein 1834647 1835081 NMB1754 cryptic plasmid protein A-related protein 1835182 1835084 NMB1755 hypothetical protein 1835328 1835669 NMB1756 hypothetical protein 1835980 1836171 NMB1757 hypothetical protein 1836529 1836756 NMB1758 hypothetical protein 1837008 1837217 NMB1759 conserved hypothetical protein 1837403 1838764 NMB1760 conserved hypothetical protein 1839128 1839631 NMB1761 conserved hypothetical protein 1839797 1841047 NMB1762 hemolysin activation protein HecB, putative 1843162 184137*8 NMB1763 toxin-activating protein, putative 1843675 1843220 NMB1764 hypothetical protein 1844155 1843844 NMB1765 hypothetical protein 1844466 1844170 NMB1766 hypothetical protein 1845460 1844450 NMB1767 hypothetical protein 1845945 1845532 NMB1768 hemagglutinin/hemolysin-related protein 1853493 1845952 •NMB1769 IS1016 family transposase, putative truncation 1853631 1853822 NMB1770 transposase, IS30 family 1854072 1855034 NMB1771 hypothetical protein 1855539 1855108 NMB1772 hypothetical protein 1857374 1855539 NMB1773 hypothetical protein 1857783 1857412 NMB1774 hypothetical protein 1858438 1858064 NMB1775 hypothetical protein 1860252 1858450 NMB1776 hypothetical protein 1860353 1860252 NMB1777 hypothetical protein 1861364 1861122 NMB1778 hypothetical protein 1861489 1861388 NMB1779 hemagglutinin/hemolysin-related protein 1867499 1861515 NMB1780 hemolysin activation protein HecB, putative 1869350 1867611 NMB1781 hypothetical protein 1869919 1869752 NMB1782 hypothetical protein 1870236 1869937 NMB1783 secretion protein, putative FRAMESHIFT 1871826 1870605 NMB1784 hypothetical protein 1872240 1871890 NMB1785 hypothetical protein 1872472 1872236 NMB1786 hypothetical protein 1873623 1872472 NMB1787 N-acetyl-gamma-glutamyl-phosphate reductase 1874156 1875196 NMB1788 ATP-dependent DNA helicase RecG 1878304 1876265 NMB1789 protein-export protein SecB 1878833 1878393 NMB1790 glutaredoxin 3 1879111 1878857 NMB1791 cytoplasmic axial filament protein FRAMESHIFT 1879236 1880813 NMB1792 sensor histidine kinase 1881795 1880854 NMB1793 response regulator, putative FRAMESHIFT 1882272 1881854 NMB1794 citrate transporter 1883808 1882498 NMB1795 hypothetical protein 1884071 1883916 NMB1796 conserved hypothetical protein 1884950 1884381 Appendix B NMB1797 penicillin-binding protein 3 1885109 1886515 NMB1798 IS1016 family transposase, putative FRAMESHIFT 1887236 1886597 NMB1799 S-adenosylmethionine synthetase 1888654 1887488 NMB1800 hypothetical protein 1888703 1888903 NMB1801 HtrB/MsbB family protein 1889000 1889893 NMB1802 0-sialoglycoprotein endopeptidase 1891004 1889943 NMB1803 cytochrome c-type biogenesis protein, putative 1892308 1891124 NMB1804 cytochrome c-type biogenesis protein, putative 1894316 1892304 NMB1805 cytochrome c4 1895153 1894533 NMB1806 conserved hypothetical protein 1895353 1895985 NMB1807 penicillin-binding protein 1 1898505 1896112 NMB1808 pilM protein 1898657 1899769 NMB1809 pilN protein FRAMESHIFT 1899775 1900371 NMB1810 pilO protein 1900375 1901019 NMB1811 pilP protein 1901040 1901582 NMB1812 pilQ protein FRAMESHIFT 1901604 1903908 NMB1813 shikimate kinase 1904813 1905322 NMB1814 3-dehydroquinate synthase 1905405 1906481 NMB1815 conserved hypothetical protein 1907451 1908290 NMB1816 conserved hypothetical protein 1908323 1908784 NMB1817 riboflavin-specific deaminase 1908819 1909925 NMB1818 lipopolysaccharide biosynthesis protein, putative 1910123 1911541 NMB1819 hypothetical protein 1911541 1911693 NMB1820 pilin glycosylation protein PglB 1911712 1912950 NMB1821 pilin glycosylation protein PglC 1913086 1914258 NMB1822 pilin glycosylation protein PglD 1914309 1916216 NMB1823 valine--pyruvate aminotransferase 1916275 1917564 NMB1824 conserved hypothetical protein 1918455 1917622 NMB1825 hypothetical protein 1919103 1918903 NMB1826 conserved hypothetical protein 1919452 1919084 NMB1827 DNA polymerase III, alpha subunit 1919852 1923283 NMB1828 conserved hypothetical protein 1924652 1923723 NMB1829 TonB-dependent receptor 1926848 1924725 NMB1830 phosphoglycolate phosphatase, putative 1926996 1927652 NMB1831 lytB protein 1928711 1927746 NMB1832 lipoprotein signal peptidase 1929267 1928743 NMB1833 isoleucyl-tRNA synthetase 1933332 1930546 NMB1834 riboflavin kinase/FMN adenylyltransferase 1934394 1933477 NMB1835 tyrosyl-tRNA synthetase 1936217 1934925 NMB1836 lipopolysaccharide biosynthesis protein WbpC, putative 1938151 1936283 NMB1837 hypothetical protein 1938466 1938215 NMB1838 GTP-binding protein, putative 1939615 1938527 NMB1839 formate--tetrahydrofolate ligase 1941406 1939733 NMB1840 conserved hypothetical protein 1941581 1942009 NMB1841 mannose-1-phosphate guanyltransferase-related protein 1942741 1942049 NMB1842 4-hydroxyphenylacetate 3-hydroxylase, small subunit, putative 1943257 1942760 NMB1843 transcriptional regulator, MarR family 1943812 1943375 NMB1844 hypothetical protein 1943938 1943819 NMB1845 thioredoxin 1944662 1944156 NMB1846 Mrp/NBP35 family protein 1945032 1946108 NMB1847 pilCl protein FRAMESHIFT 1947287 1950374 NMB1848 hypothetical protein 1952279 1951938 NMB1849 carbamoyl-phosphate synthase, small subunit 1952589 1953719 NMB1850 hypothetical protein 1954091 1954363 NMB1851 hypothetical protein 1954440 1954697 NMB1852 conserved hypothetical protein 1954697 1955083 NMB1853 hypothetical protein 1955422 1955691 NMB1854 hypothetical protein 1955768 1956406 NMB1855 carbamoyl-phosphate synthase, large subunit 1956438 1959650 NMB1856 transcriptional regulator, LysR family 1960777 1959881 NMB1857 modulator of drug activity B 1961016 1961591 Appendix B -31- NMB1858 hypothetical protein 1961977 1961594 NMB1859 S-adenosylmethionine:tRNA ribosyltransferase-isomerase 1963108 1962071 NMB1860 acetyl-CoA carboxylase, biotin carboxyl carrier protein 1963464 1963916 NMB1861 acetyl-CoA carboxylase, biotin carboxylase 1964031 1965389 NMB1862 ribosomal protein L11 methyltransferase 1965653 1966537 NMB1863 oligoribonuclease 1966558 1967118 NMB1864 glutamate-l-semialdehyde 2,1-aminomutase 1968808 1967528 *NMB1865 hypothetical protein 1968821 1969036 NMB1866 conserved hypothetical protein 1969593 1970918 NMB1867 l-deoxyxylulose-5-phosphate synthase 1972919 1971009 NMB1868 integrase/recombinase XerC 1973909 1973007 NMB1869 fructose-bisphosphate aldolase 1974093 1975154 NMB1870 hypothetical protein 1975177 1976136 NMB1871 conserved hypothetical protein 1976286 1976960 NMB1872 ribosomal-protein-alanine acetyltransferase, putative 1976960 1977397 NMB1873 DNA polymerase, bacteriophage-type, putative 1977394 1978128 NMB1874 orotate phosphoribosyltransferase 1978193 1978831 NMB1875 hypothetical protein 1978908 1979339 NMB1876 N-acetylglutamate synthase 1979339 1980646 NMB1877 prolyl oligopeptidase family protein 1980850 1982862 NMB1878 transcriptional regulator, AraC family 1983567 1982983 NMB1879 hypothetical protein 1983936 1983628 NMB1880 ABC transporter, periplasmic solute-binding protein, putative 1984172 1985134 NMB1881 conserved hypothetical protein 1985694 1986014 NMB1882 TonB-dependent receptor 1986131 1988305 NMB1883 hypothetical protein 1988727 1988440 NMB1884 conserved hypothetical protein 1989047 1988727 NMB1885 protein-L-isoaspartate O-methyltransferase 1989783 1989130 NMB1886 conserved hypothetical protein 1990389 1989889 NMB1887 triosephosphate isomerase 1990568 1991338 NMB1888 protein-export membrane protein SecG 1991348 1991695 NMB1889 hypothetical protein 1992486 1992575 NMB1890 conserved hypothetical protein 1992709 1993074 NMB1891 helix-turn-helix family protein 1993074 1993382 NMB1892 hypothetical protein 1993495 1993704 NMB1893 conserved hypothetical protein FRAMESHIFT 1994615 1993771 NMB1894 leucyl-tRNA synthetase, truncation 1994851 1994723 NMB1895 DNA adenine methylase, truncation 1994987 1994847 NMB1896 type II restriction enzyme DpnI 1995774 1994974 NMB1897 leucyl-tRNA synthetase 1998538 1995911 NMB1898 lipoprotein 1998808 1999320 NMB1899 hypothetical protein 1999330 1999770 NMB1900 polyphosphate kinase 1999849 2001996 NMB1901 IS1016C2 transposase, degenerate 2002232 2002770 NMB1902 DNA polymerase III, beta subunit 2004113 2003013 NMB1903 chromosomal replication initiator protein DnaA 2005904 2004351 NMB1904 ribosomal protein L34 2006196 2006327 NMB1905 ribonuclease P protein component 2006333 2006695 NMB1906 conserved hypothetical protein 2006763 2006981 NMB1907 60 kd inner-membrane protein 2007156 2008790 NMB1908 conserved hypothetical protein 2009599 2008877 NMB1909 Maf/YceF/YhdE family protein 2010236 2009649 NMB1910 conserved hypothetical protein 2010384 2010884 NMB1911 50S ribosomal protein L32 2010921 2011097 NMB1912 conserved hypothetical protein 2011275 2011799 NMB1913 fatty acid/phospholipid synthesis protein 2011891 2012943 NMB1914 hypothetical protein 2013082 2013330 NMB1915 hypothetical protein 2013360 2013746 NMB1916 3-oxoacyl-(acyl-carrier-protein) synthase III 2013931 2014890 NMB1917 conserved hypothetical protein 2014940 2015344 Appendix B -32- NMB1918 malonyl CoA-acyl carrier protein transacylase 2015441 2016364 NMB1919 ABC transporter, ATP-binding protein 2016505 2018367 NMB1920 GMP synthase 2018470 2020032 NMB1921 3-oxoacyl-(acyl-carrier-protein) reductase 2020097 2020840 NMB1922 IS1106 transposase, degenerate 2021273 2021118 NMB1923 conserved hypothetical protein 2021377 2021757 NMB1924 inositol monophosphatase family protein 2022673 2021981 NMB1925 conserved hypothetical protein 2022876 2023598 NMB1926 lacto-N-neotetraose biosynthesis glycosyl transferase LgtE 2025680 2024841 NMB1927 lacto-N-neotetraose biosynthesis glycosyl transferase-related protein 2025817 2025725 NMB1928 lacto-N-neotetraose biosynthesis glycosyl transferase LgtB 2026656 2025832 NMB1929 lacto-N-neotetraose biosynthesis glycosyl transferase LgtA 2027747 2026701 NMB1930 glycyl-tRNA synthetase, beta chain 2029827 2027767 NMB1931 hypothetical protein 2030256 2029912 NMB1932 glycyl-tRNA synthetase, alpha chain 2031238 2030336 NMB1933 ATP synthase Fl, epsilon subunit 2032065 2031646 NMB1934 ATP synthase Fl, beta subunit 2033473 2032079 NMB1935 ATP synthase Fl, gamma subunit 2034386 2033514 NMB1936 ATP synthase Fl, alpha subunit 2035958 2034414 NMB1937 ATP synthase Fl, delta subunit 2036502 2035972 NMB1938 ATP synthase FO, B subunit 2036977 2036510 NMB1939 ATP synthase FO, C subunit 2037284 2037051 NMB1940 ATP synthase FO, A subunit 2038207 2037344 NMB1941 hypothetical protein 2038550 2038200 NMB1942 hypothetical protein 2038997 2038707 NMB1943 hypothetical protein 2039340 2039170 NMB1944 ParB family protein 2040252 2039395 NMB1945 3-octaprenyl-4-hydroxybenzoate carboxy-lyase 2040407 2040976 NMB1946 outer membrane lipoprotein 2041904 2041044 NMB1947 ABC transporter, permease protein 2042749 2042066 NMB1948 ABC transporter, ATP-binding protein 2043488 2042754 NMB1949 soluble lytic murein transglycosylase, putative 2044018 2045865 NMB1950 30S ribosomal protein S21 2046157 2046366 NMB1951 conserved hypothetical protein 2046405 2046944 NMB1952 stringent starvation protein B 2047538 2047149 NMB1953 stringent starvation protein A 2048215 2047613 NMB1954 hypothetical protein 2050146 2048488 NMB1955 cadmium resistance protein 2050933 2050310 NMB1956 50S ribosomal protein L31 2051451 2051239 NMB1957 acetyltransferase-related protein FRAMESHIFT 2051688 2052197 NMB1958 thioredoxin, putative 2052770 2052273 NMB1959 conserved hypothetical protein 2053150 2052770 NMB1960 hypothetical protein 2053632 2053153 NMB1961 VacJ-related protein 2054464 2053640 NMB1962 hypothetical protein 2054739 2054464 NMB1963 conserved hypothetical protein 2055380 2054793 NMB1964 conserved hypothetical protein 2055911 2055420 NMB1965 conserved hypothetical protein 2056738 2055965 NMB1966 ABC transporter, ATP-binding protein 2057586 2056789 NMB1967 transcriptional regulator, AraC family 2057759 2058673 NMB1968 aldehyde dehydrogenase A 2058936 2060375 NMB1969 serotype-l-specific antigen, putative 2061412 2064657 NMB1970 para-aminobenzoate synthetase component I/4-amino-4deoxychorismate lyase, putative 2065692 2067470 NMB1971 conserved hypothetical protein 2069049 2067535 NMB1972 chaperonin, 60 kDa 2071379 2069748 NMB1973 chaperonin, 10 kDa 2071762 2071475 NMB1974 IS1016C2 transposase, degenerate 2071990 2072639 NMB1975 sodium- and chloride-dependent transporter 2072855 2074387 NMB1976 diaminopimelate decarboxylase 2075759 2074518 Appendix B NMB1977 hypothetical protein 2075940 2075773 NMB1978 cyaY protein 2076011 2076331 NMB1979 conserved hypothetical protein 2076361 2077374 NMB1980 conserved hypothetical protein 2077403 2077819 NMB1981 conserved hypothetical protein 2077844 2078347 NMB1982 DNA polymerase I 2078496 2081309 NMB1983 hypothetical protein 2082658 2083326 NMB1984 IS1106 transposase FRAMESHIFT 2083391 2084499 NMB1985 adhesion and penetration protein 2089191 2084821 NMB1986 hypothetical protein 2089756 2089328 NMB1987 thiophene and furan oxidation protein ThdF 2090041 2091384 NMB1988 iron-regulated outer membrane protein FrpB 2092611 2094752 NMB1989 iron(III) ABC transporter, periplasmic binding protein 2095472 2096434 NMB1990 iron(III) ABC transporter, permease protein 2096601 2097566 NMB1991 iron(III) ABC transporter, permease protein 2097559 2098530 NMB1992 hypothetical protein 2098577 2099200 NMB1993 iron(III) ABC transporter, ATP-binding protein 2099286 2100041 NMB1994 adhesin/invasin, putative 2100342 2101433 NMB1995 nitrogen regulatory protein P-II, FRAMESHIFT 2101839 2101423 NMB1996 phosphoribosylformylglycinamidine synthase 2101990 2105949 NMB1997 hydroxyacylglutathione hydrolase 2106047 2106802 NMB1998 serine-type peptidase 2107119 2111411 NMB1999 magnesium transporter 2111646 2113097 NMB2000 conserved hypothetical protein 2114094 2113189 NMB2001 conserved hypothetical protein 2114339 2115091 NMB2002 hypothetical protein 2115113 2115328 NMB2003 conserved hypothetical protein 2115476 2115820 NMB2004 conserved hypothetical protein 2115820 2116509 NMB2005 glutamate N-acetyltransferase/amino-acid acetyltransferase 2116579 2117796 NMB2006 chloride channel protein-related protein 2117859 2119265 NMB2007 ATP-dependent RNA helicase HrpA, truncation 2119458 2120846 NMB2008 ABC transporter, ATP-binding protein-related protein 2120993 2122633 NMB2009 ATP-dependent RNA helicase HrpA, degenerate 2122680 2122859 NMB2010 YhbX/YhjW/YijP/YjdB family protein 2123074 2124648 NMB2011 ATP-dependent RNA helicase HrpA, truncation 2124717 2128133 NMB2012 transcriptional regulator, HTH 3 family 2129260 2128172 NMB2013 hypothetical protein 2129920 2129279 NMB2014 hypothetical protein 2130249 2130004 NMB2015 hypothetical protein 2130614 2130880 NMB2016 type IV pilin-related protein 2131493 2131047 NMB2017 ComEA-related protein 2132027 2131584 NMB2018 conserved hypothetical protein 2138411 2137752 NMB2019 lipopolysaccharide core biosynthesis protein KdtB 2138949 2138440 NMB2020 conserved hypothetical protein 2139756 2139076 NMB2021 conserved hypothetical protein 2140179 2139916 NMB2022 conserved hypothetical protein 2140722 2140255 NMB2023 conserved hypothetical protein 2141162 2140779 NMB2024 conserved hypothetical protein 2141826 2141224 NMB2025 conserved hypothetical protein 2142422 2141826 NMB2026 ABC transporter, permease protein 2144046 2142454 NMB2027 gluconate permease 2144385 2145767 NMB2028 thermoresistant gluconokinase 2145790 2146305 NMB2029 homoserine kinase FRAMESHIFT 2147564 2146650 NMB2030 3-demethylubiquinone-9 3-methyltransferase 2148329 2147604 NMB2031 tryptophan transporter 2148481 2149719 NMB2032 lipopolysaccharide glycosyl transferase, FRAMESHIFT 2149872 2150922 NMB2033 histidinol-phosphatase, putative 2151173 2151733 NMB2034 1-acyl-sn-glycerol-3-phosphate acyltransferase, putative 2151765 2152505 NMB2035 conserved hypothetical protein 2152505 2153194 Appendix B -34- NMB2036 tRNA pseudouridine synthase A 2154495 2155390 NMB2037 hypothetical protein 2155415 2155651 NMB2038 PemK-related protein 2155642 2155962 NMB2039 major outer membrane protein PIB 2157487 2158479 NMB2040 thiamine biosynthesis protein ThiC 2161479 2159581 NMB2041 thiamin pyrophosphokinase-related protein 2162093 2162965 NMB2042 spermidine/putrescine ABC transporter, ATP-binding protein 2162977 2163912 NMB2043 IS1106 transposase, putative POINT MUTATION 2165702 2164734 NMB2044 phosphoenolpyruvate-protein phosphotransferase 2168278 2166506 NMB2045 phosphocarrier protein HPr 2168547 2168281 NMB2046 PTS system, IIAB component 2169074 2168619 NMB2047 hypoxanthine-guanine phosphoribosyltransferase, putative 2169697 2169137 NMB2048 DNA ligase 2170590 2169769 NMB2049 glyoxalase II family protein 2170682 2171311 NMB2050 conserved hypothetical protein 2173305 2171524 NMB2051 ubiquinol--cytochrome c reductase, cytochrome cl 2174444 2173647 NMB2052 ubiquinol--cytochrome c reductase, cytochrome b 2175793 2174447 NMB2053 ubiquinol--cytochrome c reductase, iron-sulfur subunit 2176393 2175815 NMB2054 conserved hypothetical protein 2177265 2176519 NMB2055 transcriptional regulator, LysR family 2177396 2178322 NMB2056 30S ribosomal protein S9 2178972 2178583 NMB2057 50S ribosomal protein L13 2179413 2178985 NMB2058 conserved hypothetical protein 2180081 2179779 NMB2059 hypothetical protein 2180421 2180095 NMB2060 glycerol-3-phosphate dehydrogenase (NAD+) 2181465 2180479 NMB2061 phosphoenolpyruvate carboxylase 2184290 2181591 NMB2062 thiF protein 2184460 2185227 NMB2063 slyX protein, putative 2186018 2185797 NMB2064 conserved hypothetical protein 2187407 2186022 NMB2065 hemK protein FRAMESHIFT 2188764 2187496 NMB2066 tldD protein 2190271 2188832 NMB2067 conserved hypothetical protein 2190661 2191881 NMB2068 D-amino acid oxidase flavoprotein, putative 2191881 2192978 NMB2069 thiamin-phosphate pyrophosphorylase 2193003 2193617 NMB2070 hypothetical protein 2194042 2194233 NMB2071 thiG protein 2194450 2195235 NMB2072 hypothetical protein 2195352 2195492 NMB2073 hypothetical protein 2195580 2195780 NMB2074 hypothetical protein 2196867 2196004 NMB2075 BirA protein/Bvg accessory factor 2198657 2196882 NMB2076 aut protein 2199160 2198657 NMB2077 methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase FRAMESHIFT 2199800 2200650 NMB2078 conserved hypothetical protein 2201296 2200718 NMB2079 aspartate-semialdehyde dehydrogenase 2201472 2202584 NMB2080 hypothetical protein 2203345 2202818 NMB2081 hypothetical protein 2203700 2203359 NMB2082 exodeoxyribonuclease 2204466 2203690 NMB2083 cysteinyl-tRNA synthetase 2205970 2204552 NMB2084 hypothetical protein 2206648 2205985 NMB2085 hypothetical protein 2207707 2206661 NMB2086 GTP-binding protein 2208944 2207793 NMB2087 hypothetical protein 2209792 2209433 NMB2088 conserved hypothetical protein 2210766 2209894 NMB2089 conserved hypothetical protein 2210812 2211156 NMB2090 phosphoheptose isomerase 2211164 2211754 NMB2091 hemolysin, putative 2211821 2212426 NMB2092 hypothetical protein 2212437 2213066 NMB2093 methionine aminopeptidase 2213109 2213885 NMB2094 hypothetical protein 2214043 2214339 NMB2095 adhesin complex protein, putative 2214580 2214951 Appendix B NMB2096 malate:quinone oxidoreductase 2216608 2215145 NMB2097 hypothetical protein 2216749 2216663 NMB2098 conserved hypothetical protein 2217735 2217148 NMB2099 conserved hypothetical protein 2218377 2217799 NMB2100 hypothetical protein 2218455 2218685 NMB2101 30S ribosomal protein S2 2218861 2219586 NMB2102 elongation factor TS (EF-TS) 2219718 2220569 NMB2103 uridylate kinase 2220789 2221505 NMB2104 mafA protein FRAMESHIFT 2221692 2222652 NMB2105 mafB protein 2222695 2224143 NMB2106 hypothetical protein 2224143 2224496 NMB2107 MafB-related protein 2224527 2225288 NMB2108 hypothetical protein 2225301 2225504 NMB2109 hypothetical protein 2225639 2225887 NMB2110 hypothetical protein 2225887 2226255 NMB2111 MafB-related protein 2226268 2227110 NMB2112 hypothetical protein 2227306 2227572 NMB2113 hypothetical protein 2227598 2227897 NMB2114 MafB-related protein 2227948 2228583 NMB2115 hypothetical protein 2228589 2228930 NMB2116 hypothetical protein 2228971 2229312 NMB2117 MafB-related protein, degenerate 2229645 2230340 NMB2118 hypothetical protein 2230340 2230654 NMB2119 MafB-related protein 2230709 2231464 NMB2120 hypothetical protein 2231471 2231869 NMB2121 hypothetical protein 2232031 2232372 NMB2122 MafB-related protein 2232409 2232510 NMB2123 hypothetical protein 2232518 2232871 NMB2124 hypothetical protein 2232922 2233047 NMB2125 hypothetical protein 2233047 2233418 NMB2126 IS1016 family transposase, putative FRAMESHIFT 2234296 2233462 NMB2127 protease, putative 2235364 2234381 NMB2128 CinA-related protein 2236204 2235407 NMB2129 argininosuccinate synthase 2236517 2237857 NMB2130 hypothetical protein 2237908 2238147 NMB2131 hypothetical protein 2238143 2238355 NMB2132 transferrin-binding protein-related protein 2239900 2238437 NMB2133 sodium/dicarboxylate symporter family protein 2241384 2240158 NMB2134 conserved hypothetical protein 2241857 2243761 NMB2135 conserved hypothetical protein 2243771 2247985 NMB2136 peptide transporter 2249471 2250925 NMB2137 hypothetical protein 2251451 2251660 NMB2138 peptide chain release factor 2 2252924 2251824 NMB2139 conserved hypothetical protein 2253920 2253030 NMB2140 conserved hypothetical protein 2254265 2254711 NMB2141 hypothetical protein 2254787 2255092 NMB2142 conserved hypothetical protein 2255187 2256050 NMB2143 conserved hypothetical protein 2256043 2256786 NMB2144 sigma factor, putative 2256811 2257395 NMB2145 hypothetical protein 2257404 2257580 NMB2146 hypothetical protein 2257703 2257810 NMB2147 hypothetical protein 2257842 2258261 NMB2148 transposase, IS30 family 2258738 2259700 NMB2149 hypothetical protein 2260052 2259795 NMB2150 conserved hypothetical protein 2261006 2260440 NMB2151 phosphoribosylamine--glycine ligase 2262344 2261076 NMB2152 hypothetical protein 2262502 2262816 NMB2153 conserved hypothetical protein 2263482 2262874 NMB2154 electron transfer flavoprotein, alpha subunit 2264480 2263548 NMB2155 electron transfer flavoprotein, beta subunit 2265240 2264494 NMB2156 heptosyltransferase I 2266435 2265470 NMB2157 pyrazinamidase/nicotinamidase PncA, putative 2267107 2266475 NMB2158 conserved hypothetical protein 2267221 2267898 NMB2159 glyceraldehyde 3-phosphate.dehydrogenase 2269163 2268162 Appendix B -36- NMB2160 DNA mismatch repair protein MutS 2269607 2272198 NMB0505 hypothetical protein 533467 533186 NMB1123 hypothetical protein 1135584 1135390 NMB1124 hypothetical protein 1136271 1135627 NMB1125 hypothetical protein 1136639 1136271 NMB1126 hypothetical protein 1137317 1136649 NMB1127 oxidoreductase, short chain dehydrogenase/reductase family 1138201 1137485 NMB1129 hypothetical protein 1139833 1139630 NMB1130 phytoene synthase, putative 1140867 1139998 NMB1133 conserved hypothetical protein ankyrin-related protein 1144428 1143670 NMB1134 ferredoxin, 2Fe-2S type 1144824 1144486 NMB1135 hypothetical protein 1145242 1145102 NMB1137 conserved hypothetical protein 1146211 1146017 NMB1138 conserved hypothetical protein 1146683 1146285 NMB1141 RNA methyltransferase, TrmH family 1150088 1149480 NMB1142 hypothetical protein 1150375 1150142 NMB1143 hypothetical protein 1150909 1150547 NMB1144 hypothetical protein 1151226 1150924, lipoprotein NMB1147 hypothetical protein 1154639 1154007, homology to plasmid proteins Y4SH RISHN and PXO2 BACAN NMB1149 hypothetical protein 1155016 11.54876 NMB1151 sulfite reductase hemoprotein, beta-component 1159086 1157320 NMB1152 sulfite reductase (NADPH) flavoprotein, alpha component 1160927 1159116 NMB1154 sulfate adenylyltransferase, subunit 2 1163172 1162252 NMB1156 siroheme synthase 1165412 1163964 NMB1157 hypothetical protein 1165696 1165541 NMB1159 conserved hypothetical protein 1167316 1166429, inner membrane NMB1160 conserved hypothetical protein 1167316 1166429 NMB1166 conserved hypothetical protein 1171633 1170323 NMB1169 chaperone protein HscA 1174933 1173074 NMB1170 hypothetical protein 1175666 1175013 NMB1174 hypothetical protein 1178053 1177373 NMB1177 acetyl-CoA carboxylase, carboxyl transferase alpha subunit 1179887 1178931 NMB1178 mesJ protein FRAMESHIFT 1181265 1179984 NMB1183 UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-mesodiaminopimelate ligase 1184700 1183327 NMB1184 biotin synthetase 1185959 1184910 NMB1186 hypothetical protein 1186881 1186729 NMB1188 dihydroxy-acid dehydratase 1189180 1187324 NMB1191 sulfate adenylyltransferase, subunit 1 1194246 1192963 NMB1193 phosphoadenosine phosphosulfate reductase 1195986 1195249 NMB1196 nickel-dependent hydrogenase, b-type cytochrome subunit 1198401 1197748

Claims (20)

1. A method for identifying an amino acid sequence, comprising the step of searching for putative open reading frames or protein-coding sequences within one or more ofN. meningitidis nucleotide sequences selected from the group consisting of SEQ ID NO 1 and the NMB open reading frames.

2. A method according to claim 1, comprising the steps of searching a N. meningitidis nucleotide sequence for an initiation codon and searching the upstream sequence for an in-frame termination codon.

3. A method for producing a protein, comprising the step of expressing a protein comprising an amino acid sequence identified according to any one of claims 1-2.

4. A method for identifying a protein in N. mengitidis, comprising the steps of producing a protein according to claim 3, producing an antibody which binds to the protein, and determining whether the antibody recognises a protein produced by N. menigitidis. Nucleic acid comprising an open reading frame or protein-coding sequence identified by a method according to any one of claims 1-2.

6. A protein obtained by the method of claim 3.

7. Nucleic acid comprising one or more of the N. meningitidis nucleotide sequences selected from the group consisting of SEQ ID NO 1 and the NMB open reading frames.

8. Nucleic acid comprising a nucleotide sequence having greater than sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO 1 and the NMB open reading frames. -124-

9. Nucleic acid comprising a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NO 1 and the NMB open reading frames. Nucleic acid according to claim 9, wherein the fragment is unique to the genome ofN. meningitidis.

11. Nucleic acid complementary to the nucleic acid of any one of claims 7-10. In

12. A protein comprising an amino acid sequence encoded within one or more of C' the N. meningitidis nucleotide sequences selected from the group consisting of SEQ ID NO 1 and the NMB open reading frames.

13. A protein comprising an amino acid sequences having greater than sequence identity to an amino acid sequence encoded within one or more of the N. meningitidis nucleotide sequences selected from the group consisting of SEQ ID NO 1 and the NMB open reading frames.

14. A protein comprising a fragment of an amino acid sequence encoded within one or more of the N. meningitidis nucleotide sequences selected from the group consisting of SEQ ID NO 1 and the NMB open reading frames. Nucleic acid encoding a protein according to any one of claims 6-8.

16. A computer, a computer memory, a computer storage medium or a computer database containing the nucleotide sequence of a nucleic acid according to any one of claims 7-11.

17. A computer, a computer memory, a computer storage medium or a computer database containing one or more of the N. meningitidis nucleotide sequences selected from the group consisting of SEQ ID NO 1 and the NMB open reading frames. r -125-

18. A polyclonal or monoclonal antibody which binds to a protein according to any one of claims 12-14 or 6.

19. A nucleic acid probe comprising nucleic acid according to any one of claims 7-10, or An amplification primer comprising nucleic acid according to any one of claims 5, 7-10, or

21. A composition comprising nucleic acid according to any one of claims 7-10, or 15; protein according to any one of claims 12-14; and/or an antibody according to claim 18.

22. The use of a composition according to claim 21 as a medicament or as a diagnostic reagent.

23. The use of a composition according to claim 21 in the manufacture of a medicament for treating or preventing infection due to Neisserial bacteria and/or a diagnostic reagent for detecting the presence of Neisserial bacteria or of antibodies raised against Neisserial bacteria.

24. A method of treating a patient, comprising administering to the patient a therapeutically effective amount of a composition according to claim 21.