Anna Puglia

Eight structurally related halogenated aliphatic hydrocarbons mono-, di- and trichloroacetaldehyde (the last in the anhydrous and hydrate form), moni-, di- and trichloroethanol and allyl chloride, were tested for their ability to induce gene mutations in prokaryotic and eukaryotic microorganisms. The genetic systems employed were the Salmonella reversion test with strain TA1535 and TA100, with and without metabolic activation, a forward and a back-mutation system in S. coelicolor and two forward mutation systems in A. nidulans. Each compound was tested with the spot and plate incorporation assay techniques. Mono-, di- and trichloroacetaldehyde were mutagenic in all the microorganisms employed; all the halogenated ethanols were positive in A. nidulans, while in S. typhimurium and in S. coelicolor the only active forms were respectively the mono- and dichloroderivatives. Allyl chloride was active in S. typhimurium and S. coelicolor and negative in A. nidulans. The technical approach a...

A spontaneous chromosomalmutation(plc A(-)) in Streptomyces coelicolor A3(2) made the inborn plasmid SCP1 susceptible to curing by UV irradiation. Lack of the SCP1 plasmid (in SCP1(-) strains) prevented the occurrence of the co-mutation process after MNNG treatment, although it left the susceptibility to the lethal effect of the mutagen virtually unaffected. SCP1(-) strains were, however, ultrasensitive to the lethal effect of UV. Curing a plc A(-) strain of its SCPI plasmid made it refractory to co-mutation by MNNG and sensitive to the lethal effect of UV; reinfected by the plasmid, the strain resumed both the co-mutation proficiency and the UV-resistance. The occurrence on the SCP1 plasmid of a gene comparable to the uvrE gene of E. coli (Nevers and Spatz 1975) was assumed.

The actinomycete Nonomuraea sp. ATCC 39727 produces the glycopeptide A40926, the precursor of dalbavancin. Biosynthesis of A40926 is encoded by the dbv gene cluster which contains 37 protein coding sequences that participate in antibiotic biosynthesis, regulation, immunity, and export. In addition to the positive regulatory protein Dbv4, the A40926 biosynthetic gene cluster encodes two additional putative regulators, Dbv3 and Dbv6. Independent mutations in these genes, combined with bioassays and LC-MS analyses, demonstrated that Dbv3 and Dbv4 are both required for antibiotic production, while inactivation of dbv6 had no effect. In addition, over-expression of dbv3 led to higher levels of A40926 production. Transcriptional and quantitative RT-PCR analyses showed that Dbv4 is essential for the transcription of two operons, dbv14-dbv8 and dbv30-dbv35, while Dbv3 positively controls the expression of four monocistronic transcription units (dbv4, dbv29, dbv36, dbv37) and of six operons (dbv2-dbv1, dbv14-dbv8, dbv17-dbv15, dbv21-dbv20, dbv24-dbv28, dbv30-dbv35). We propose a complex and coordinated model of regulation in which Dbv3 directly or indirectly activates transcription of dbv4 and controls biosynthesis of 4-hydroxyphenylglycine and the heptapeptide backbone, A40926 export and some tailoring reactions (mannosylation and hexose oxidation), while Dbv4 regulates directly biosynthesis of 3,5-dihydroxyphenylglycine and other tailoring reactions including the four cross-links, halogenation, glycosylation and acylation. This manuscript expands knowledge of the regulatory mechanisms used to control the biosynthesis of the glycopeptide antibiotic A40926 in the actinomycete Nonomuraea sp. ATCC 39727. A40926 is the precursor of dalbavancin, approved for treatment of Gram positive bacterial skin infections. Therefore, the understanding of the regulation of its biosynthesis is also of industrial importance. So far, the regulatory mechanisms used to control other two similar glycopeptides (balhimycin and teicoplanin) have been elucidated and, beyond a common step, different clusters seem to have devised different strategies to control glycopeptide production. Thus, our work provides one more example of the pitfalls of deducing regulatory roles from bioinformatic analyses only, even when analyzing gene clusters directing the synthesis of structurally related compounds.

We have further characterized the genomic region of Streptomyces coelicolor A3(2) that contains genes involved in the biosynthesis of histidine. A 2,357-base pair fragment contained in plasmid pSCH3328 that complemented hisD mutations has been sequenced. Computer analysis revealed an open reading frame that encodes a protein with significant homology to the Escherichia coli, Salmonella typhimurium and Mycobacterium smegmatis hisD product, Saccharomyces cerevisiae HIS4C, and Neurospora crassa his3 gene products. Two other contiguous open reading frames oriented divergently with respect to hisD did not show significant similarity with any of the his genes or to other sequences included in the gene bank. S1 nuclease mapping and primer extension experiments indicate that the transcription initiation site of the his-specific mRNA coincides with the GUG translation initiation codon of the hisD cistron.

The 'bald'(bld) mutants of filamentous bacterium Streptomyces coelicolor A3(2) are characterized by a lack of aerial myceliumand spores. A 'bald' mutant was isolated exhibiting unusual features. It forms slightly sculptured colonies producing a red-orange mycelial pigment, large amounts of agarase and methylenomycin A; it is also highly resistant to U.V. killing. The bld mutation (bld F1) never reverted to bld+ phenotype and was localized very closed to met A.

... production in pleiotropic bld mutants of Streptomyces coelicolor A3(2) ROSA PASSANTINO,ANNA-MARIA PUGLIA' and KEITH CHATER** ... However, a bald colony producing large amounts of blue pigment (presumed to be actinorhodin) was isolated. ...

ABSTRACT The process leading to gene recombination can be interrupted in the filamentous bacteria Streptomyces coelicolor by growing mixed cultures on cellophane disks lying on complete medium. The mycelium is harvested, broken, diluted and the broken hyphae plated at different time intervals. By this means some markers can be excluded from heteroclones or from recombinant progeny in early samples. The recombinant pattern clearly changes with time, with an increase of markers contributed to the recombinant progeny. In crosses between male (NF) and female (UF) strains, the maleness is the first donor trait to appear in the cells of the recipient parent. The fertility factor does not produce a transfer origin on the donor chromosomes; the donor contribution may extend on either side or on both sides of the factor which appears to be compulsory for zygote formation. The longer the time of contact between parental cells, the longer the segment of the donor chromosome contributing to the recombinant progeny. When spores are formed they contain almost exclusively recombinant nuclei derived from segregation processes.

ABSTRACT Initial Fertility (IF) strains of Streptomyces coelicolor are able to convert recipient strains (UF) to the IF condition by contact, without concomitant transfer of chromosomal markers. The conversion is prevented by the presence of acridine orange in the medium of the mixed culture. Acridine orange is also moderately effective in inducing the formation of UF variants from IF-treated strains. No effect of the drug is observed on UF variant formation from Normal Fertility (NF) strains nor on the behaviour of the fertility factor in NF × UF mixed cultures. The hypothesis is put forward that the fertility factor works as an episome in S. coelicolor, fixed to the chromosome in the NF strains, free in the IF strains and missing in the UF strains.

A spontaneous chromosomalmutation(plc A(-)) in Streptomyces coelicolor A3(2) made the inborn plasmid SCP1 susceptible to curing by UV irradiation. Lack of the SCP1 plasmid (in SCP1(-) strains) prevented the occurrence of the co-mutation process after MNNG treatment, although it left the susceptibility to the lethal effect of the mutagen virtually unaffected. SCP1(-) strains were, however, ultrasensitive to the lethal effect of UV. Curing a plc A(-) strain of its SCPI plasmid made it refractory to co-mutation by MNNG and sensitive to the lethal effect of UV; reinfected by the plasmid, the strain resumed both the co-mutation proficiency and the UV-resistance. The occurrence on the SCP1 plasmid of a gene comparable to the uvrE gene of E. coli (Nevers and Spatz 1975) was assumed.

Actinomycetes, filamentous Gram-positive bacteria, are usually exploited as bio-farms naturally producing a wide range of small biologically active metabolites, such as antibiotics, extensively used in medicine, food-industry, chemistry and bio-remediation strategies. The development of high throughput technologies, like proteomics, allows functional genomic studies aimed at shedding light on molecular mechanisms controlling the production of useful compounds and macromolecules. Differential proteomic analyses, performed by using Two Dimensional PolyAcrylamide Gel Electrophoresis (2D-PAGE) coupled to mass spectrometry (MS) procedures, revealed novel links between balhimycin production (a vancomycin-like antibiotic) and metabolic pathway regulation in Amycolatopsis balhimycina DSM5908. In particular, our investigation, performed by combining data from differential proteomic analyses carried-out using wild-type (Wt) and non-producing strains incubated in different growth conditions, s...

... Nature Biotechnology 18, 343 - 345 (2000) doi :10.1038/73810. Artificial chromosomes for antibiotic-producing actinomycetes. Margherita Sosio 1, 5 , Francesco Giusino 2, 3, 5 , Carmela Cappellano 4 , Elena Bossi 1 , Anna Maria Puglia 2 & Stefano Donadio 1. Abstract. ...

Proteomics was recently used to reveal enzymes whose expression is associated with the production of the glycopeptide antibiotic balhimycin in Amycolatopsis balhimycina batch cultivations. Combining chemostat fermentation technology, where cells proliferate with constant parameters in a highly reproducible steady-state, and differential proteomics, the relationships between physiological status and metabolic pathways during antibiotic producing and non-producing conditions could be highlighted. Two minimal defined media, one with low Pi (0.6 mM; LP) and proficient glucose (12 g/l) concentrations and the other one with high Pi (1.8 mM) and limiting (6 g/l; LG) glucose concentrations, were developed to promote and repress antibiotic production, respectively, in A. balhimycina chemostat cultivations. Applying the same dilution rate (0.03 h-1), both LG and LP chemostat cultivations showed a stable steady-state where biomass production yield coefficients, calculated on glucose consumptio...

ABSTRACT When penicillin and other classes of antibiotics were discovered and used to treat infectious diseases human morbidity and mortality due to infective microorganisms were successfully contrasted. However, almost as soon as antibacterial drugs were introduced in clinics, bacterial resistance spread. From a biological and microbiological viewpoint, antibacterial drug resistance is a fascinating aspect of molecular evolution, and resistome concept has been introduced to shed light on driving forces that control the spreading and maintenance of resistance genes within microbial populations. Despite the wide range of chemical complexity of antibiotics, there is a restricted number of modes of action and, on the other hand, bacteria may manifest resistance to antibacterial drugs through a restricted range of molecular strategies. In fact, resistant bacteria can possess molecular mechanisms acting on the antibiotic molecular structure and thus inhibiting its effect. On the other hand, antibiotic target can be altered to have low affinity for antibiotic recognition in the resistant bacterium. Finally, when the cellular target of an antibiotic is located inside cells, antibiotic–target interaction can be prevented by pumping the antibiotic out from resistant bacterial cells through efflux pumps to keep low the intracellular drug concentrations. In this chapter, resistance mechanisms are systematically described and discussed with respect to molecular strategies and modes of action. Spreading models are also discussed with highlights on two examples of intrinsic and acquired antibiotic resistance in Pseudomonas aeruginosa and Staphylococcus aureus, respectively.

Changes in expression of ribosomal protein genes during growth and stationary phase of Streptomyces coelicolor A3(2) in liquid medium were studied. Proteins being synthesized were pulse-labelled with [35S]-methionine, separated by two-dimensional polyacrylamide gel electrophoresis, and quantified using the BioImage computer software. Most of the ribosomal proteins were synthesized throughout the life cycle. Exceptions were two proteins whose synthesis drastically decreased at the approach of stationary phase. These two proteins were identified in purified ribosomes as homologues of Escherichia coli ribosomal proteins L10 and L7/L12, using antibodies raised against fusion proteins between these ribosomal proteins and Escherichia coli beta-galactosidase. The genes (rplJ and rplL) encoding the L10 and L7/L12 proteins were contained in a 1.2 kb BamHI fragment that was cloned and sequenced. The linkage and order of the genes coincide with other L10-L7/L12 operons. However, L11 and L1 genes were not present immediately upstream of the L10 gene, as is the case for E. coli and other bacteria. Instead, two open reading frames of unknown function were found immediately upstream of the L10 gene, in an adjacent 1.9 kb BamHI fragment.

In the differentiating eubacterium Streptomyces coelicolor, nutritional imbalances activate a developmental programme which involves the heat-shock stress regulon. In liquid batch cultures, the growth curve could be separated into four components: rapid growth 1 (RG1), transition (T), rapid growth 2 (RG2) and stationary (S). Patterns of gene expression in cultures subjected to heat shock in various phases were recorded on two-dimensional gels and analysed using advanced statistical methods. The responses of all heat-shock proteins (HSPs) were highly dependent upon growth phase, thus demonstrating that the four phases of growth were physiologically distinct. For many HSPs, the level of thermal induction attained were closely related to growth stage-determined levels of synthesis before heat shock, thus supporting the idea that developmental and thermal induction of this stress regulon have common control elements. Cluster analysis identified five groups of HSPs displaying similar kinetics of heat- and developmentally induced synthesis, probably reflecting the influence of major regulatory systems. Methods introduced here to analyse the response of groups of genes to multiple simultaneous stimuli should find broad applications to studies of other prokaryotic and eukaryotic regulons.

Both industry and academia have shown a growing interest in materials with antimicrobial properties suitable for food packaging applications. In this study, we prepared and characterized thin films of ethylene-co-vinyl acetate (EVA) copolymer with antimicrobial properties. The films were prepared with a film blowing process by incorporating a nisin preparation as an antimicrobial agent in the melt. Two grades of EVA containing 14 and 28% (wt/wt) vinyl acetate (EVA 14 and EVA 28, respectively) and two commercial formulations of nisin with different nominal activities were used. The effect of the nisin concentration also was evaluated. The films with the highest antimicrobial activity were those formulated with nisin at the highest activity and EVA with the highest content of vinyl acetate. The use of the commercial formulation of nisin with high activity in the EVA films allowed reduction in the amount of nisin needed to provide antimicrobial properties. Consequently, the mechanical properties of these films were only slightly inferior to those of the pure polymers. In contrast, films prepared by incorporating more of the nisin with lower activity had poor mechanical properties. The effect of different processing temperatures used in the preparation of the films on the antimicrobial properties of the films also was evaluated. The materials displayed antimicrobial properties even when they were prepared at temperatures as high as 160 °C, probably because of the very short processing time (60 to 90 s) required for preparation.

Through the screening of a Streptomyces coelicolor genomic library, carried out in a histidinol phosphate phosphatase (HolPase) deficient strain, SCO5208 was identified as the last unknown gene involved in histidine biosynthesis. SCO5208 is a phosphatase, and it can restore the growth in minimal medium in this HolPase deficient strain when cloned in a high or low copy number vector. Moreover, it shares sequence homology with other HolPases recently identified in Actinobacteria. During this work a second phosphatase, SCO2771, sharing no homologies with SCO5208 and all so far described phosphatases was identified. It can complement HolPase activity mutation only at high copy number. Sequence analysis of SCO5208 and SCO2771, amplified from the HolPase mutant strain, revealed that SCO5208 shows a mutation in a conserved amino acid, whereas SCO2771 does not show any mutation. All these results show that S. coelicolor SCO5208, recently renamed hisN, is the HolPase involved in histidine biosynthesis.

TROIA A., CARDINALE M, LA MANNA M, LO CASCIO P, PASTA S, PUGLIA AM, et al. (2006). Preliminary results of EOLIFE99, a project concerning the conservation of four endangered plant species of Aeolian Archipelago (South Tyrrhenian Sea, Italy). QUADERNI DI ...

HspR (heat shock protein regulator) acts as a negative regulator of different genes in many bacteria. In Streptomyces coelicolor hspR gene is part and the transcriptional repressor of the dnaK operon which encodes the DnaK, GrpE, DnaJ chaperone machines and HspR itself. Our experiments led us to the discovery of a second promoter, internal to dnaK operon, located upstream hspR gene. Transcription from this promoter was detected at 30 degrees C indicating that hspR could play a key physiological role.