DE4204103A1 - New gamma-amino butyric acid permease gene - for growth inhibition of microbial or plant cells or as selectable marker - Google Patents

Abstract

A gene for GABA permease is claimed (GABA = gamma-aminobutyric acid). Also claimed are: (1) GABA permease having a cytotoxic effect on cells in which it is overexpressed; (2) a plasmid contg. the gene; and (3) microorganisms (esp. E. coli), plants, plant cells and seeds contg. the plasmid. USE - Overexpression of the gene in microbial or plant cells may be used to produce cytotoxic effects in microorganisms, plants, plant cells or seeds, e.g. transforming plants with the gene under the control of a pathogen- or wound-inducible promoter produces hypersensitive resistance to pathogen attack, and transforming plants with the gene under the control of an anther-specific promoter may be used to produce male-sterile plants. The gene may also be used as a selectable marker for: (a) detecting plasmids contg. DNA inserts, since cells contg. plasmids with the insert in the GABA permease gene will not be inhibited in growth; and (b) positive selection of integrated DNA fragments in the genome of an organism, by using a cloning vector contg. a selectable DNA fragment and the GABA permease gene, and selecting clones contg. the integrated fragment without episomal or chromosomal copies of the GABA permease gene

Description

The invention relates to cytotoxic 4-aminobutyric acid (GABA) -permeas, their genes, methods for isolating these genes and the use of genes as well as specific subfragments, for the targeted growth inhibition of Microorganisms or plant cells in which the permeases or specific Subfragments are overexpressed under controlled conditions.

Controlled expression can be achieved through the use of inducible promoters be achieved. Promoters are understood here as sequences 100 bp in length and from which the transcription of the gene, d. H. the Synthesis of mRNA, starts. Promoters control the transcriptional activity of genes, in most cases immediately in the 3 'direction behind the Promoter lie on the same DNA. Promoters are usually cis However, they can also be effective in terms of location and Orientation with respect to the gene vary. We speak of weak and strong Promoters, depending on how efficiently a gene is transcribed. So-called inducible promoters can be regulated in their strength, while constitutive Promoters are not regulatable. The regulatory sequences are around binding sites for sequence-specific DNA-binding proteins. These proteins depending on the physiological state of the cells cell type and tissue specific in a positive way, the expression of the genes under the Control of the respective promoter, control. Inducible promoters are for example, the lac, tac, trp and trc as well as the tapetum promoter. Although prokaryotic and eukaryotic promoters differ in Regarding their structure, but not regarding the property to be regulated can.

So far, toxins have been used to selectively switch off cells. The Disadvantages associated with the expression of toxins are below Another is that toxins are under the control of an inducible promoter stand, even in the non-induced state always in a certain, though to be expressed constitutionally, since even a very good Inducible promoter is never completely switched off. The expression of toxins in Plants or microorganisms intended for consumption are not desirable. With the help of the present genes, which at low expression no can possess damaging and negative properties, cells under controlled conditions killed or impaired in their growth become.

The permease genes as well as their specific subfragments can also be described as Selection marker used to detect successfully performed cloning become. The presence of DNA not by an insertion of foreign DNA disrupted marker gene leads to a growth inhibition, while the Cloning occurred to inactivate the permease and to a normal one Growth of the recipients lead.

The invention also relates to those DNA fragments which have arisen by replacement, insertion or deletion of one or more nucleotides of the preferably claimed DNA ( FIG. 1) and whose overexpression under the control of an inducible promoter leads to a growth inhibition of the cells. Also, a change in the sequence in the context of the genetic code is to be understood by this.

As target cells for overexpression of the permease come microorganisms and Plant cells in question, for the appropriate transformation methods and Promoters are available. Preference is given to dicotyledonous plants, especially useful plants, starch, carbohydrates, proteins or fats in produce or store usable quantities in their organs, fruits or Produce vegetables, spices, fibers and technically usable products or  Provide medicines, dyes or waxes and also all feed plants.

GABA permeases according to the invention can be obtained not only from E. coli, but also be isolated from all organisms that have appropriate enzymes for Transport of GABA through the cell membrane.

The invention relates in particular to the permease from Escherichia coli K-12 encoding a GABA permease and its specific subfragments.

The gabP gene is part of the gab-Gluster from E. coli K-12, which contains the enzymes of the 4-aminobutyric acid (GABA) degradation pathway [Metzger and Halpern (1990), J. Bacteriol. 172: 3250; Bartsch et al. (1990), Appl. Env. Microbiol. 56: 7; Bartsch et al. (1990) J. Bacteriol. 172: 7035]. The gene was from the genome of E. coli isolated, characterized and sequenced. It has a length of 1401 nucleotides and encodes a 466 amino acid GABA-specific permease. The Permease is a highly hydrophobic membrane protein with a total of 12 Transmembrane domains. Overexpression of the permease gene under control strong, inducible promoters leads to disintegration of the cell membrane and subsequently to growth inhibition and cell death. Comparable effects not only with the complete permease, but also with specific ones Sub-fragments of the permease achieved. These are hydrophobic Polypeptides without catalytic activity, expressed by expression of 3'-truncated Fragments of the gabP gene can be generated.

Further aspects of the invention are listed in the examples and illustrates, without this being limited thereto. If nothing else is given,% means% by weight.

Examples 1. Localization of the GABA Permease Gene gabP from E. coli K-1 2, GABA  Transport kinetics

European Patent Application 89 122 939.5 described the cloning of a 5.6 kilobase (kb) Sall-HindIII fragment from E. coli K-12, which gave the genes T coding for GABA transaminase (EC 2.6.1.19), and gabD coding for succinic semialdehyde dehydrogenase (EC 1.2.1.16) ( Figure 2). This fragment was prepared by standard methods [Maniatis et al. (1982), Molecular Cloning, A Laboratory Manual, Gold Spring Harbor] into the plasmid vector pMLG12 (Table 1) and the resulting recombinant plasmid pGAB8 (Table 1) transformed into E. coli W3110.

In the transformants obtained and a control strain transformed with the vector pMLG12, the GABA uptake activities were measured in a transport assay with radioactively labeled [ ¹⁴ C] GABA as substrate. For this, the bacteria were incubated in a minimal medium consisting of 0.6% Na ₂ HPO ₄ , 0.3% KH ₂ PO ₄ , 0.05% NaCl, 0.05% MgSO ₄ , 0.001% CaCl ₂ , 0.5 % Glucose, 0.4% GABA, 0.2% glutamine, pH = 7.4 with 50 μg / ml chloramphenicol cultured at 37 ° C and in the logarithmic phase at an optical density (600 nm) of about 0.8 harvested. The cells were washed twice in uptake buffer (10 mM NaCl, 10 mM sodium phosphate, pH = 7.0) and resuspended in the same buffer. The GABA transport assays were performed at 37 ° C in a volume of 1 ml with 3.75 mg fresh cells (not older than 1 h on ice) and the desired concentration of [C ¹⁴ C] GABA (c = 2.5 , 5, 7.5, 10, 20 and 40 μM, respectively). Samples of 100 μl were taken at 2, 4, 6, 8, 10 and 12 minutes and filtered through membrane filters. The filters were washed twice with 500 μl of each receiving buffer and measured in a liquid scintillation counter. The initial rates of GABA uptake were determined from the linear regions of the time curves. The GABA transport kinetics for the different recombinant strains are shown in double reciprocal plots (1 / v vs. 1 / c) ( Figure 3).

In the pGAB8 transformants, the GABA uptake is about 3-fold higher than the  Control increased.

For precise localization of the GABA-permease gene, various sub-fragments of the 5.6-kb SalI-HindIII fragment ( Fig. 2) were subcloned into the tac expression vector pJF118u (Table 1) and the recombinant plasmids also into E. coli W3110 transformed. Only transformants containing the 1.9 kb BamHI-HindIII fragment adjacent to the 3 'end of the gabT gene (plasmid pJFPE1, Tab. 1) were shown, one with respect to the control (W3110 transformed with the vector pJF118u) approx 5-fold increased GABA uptake activity (without induction of the tac promoter) ( Figure 4). However, cloning of the 1.9 kb fragment in reverse orientation to the tac promoter (tac promoter-HindIII BamHI, plasmid pJFPE1 _R ) did not increase the GABA transport. These results indicate that the complete gabP structural gene is located on the 1.9 kb BamHI-HindIII fragment and is transcribed in the same direction as the 5 'genes gaT and gabD ( Figure 2).

2. Nucleotide sequence of GABA permease gene gabP

The DNA sequence of the 1.9 kb BamHI-HindIII fragment ( Figure 1) was determined and is shown in FIG . The sequence information obtained from the 1.9 kb fragment and subcloned partial fragments were extended by means of synthetic primers.

It contains a long open reading frame of 1401 nucleotides in length with the Translation initiation codon ATG at position 189 and stop Godon TGA in position 1589. The deduced gene product, GABA permease, is strong hydrophobic protein of 466 amino acids with 12 potential Transmembrane domains. Its calculated molecular mass is 51.1 kiloDaltons (KDa).

3. Construction of gabP expression plasmids

From the 1.9 kb BamHI-HindIII fragment, a series of exolll / S1 deletions were prepared from the HindIII end [Henikoff (1984), Gene 28: 351-359]. The truncated fragments [1601 base pair (bp), 1414-bp, 725-bp, 330-bp; see Fig. 2] as well as the unmodified 1.9 kb fragment (see above) were inserted into the tac expression vector pJF118u. The last three of the thus constructed plasmids pJFPE2-pJFPE5 (Table 1) contain 3'-truncated forms of the gabP structural gene and therefore express recombinant GABA permeases with incomplete C-terminal regions (pJFPE3: -58 amino acids, pJFPE4: 287 amino acids, pJFPE5: 419 amino acids). The catalytic activities of these permease deletion mutants were examined by [ ¹⁴ C] GABA uptake assays with the corresponding E. coli W3110 transformants (see Example 1). It was found that only transformants expressing the complete gabP structural gene (plasmids pJFPE1, pJFPE2) have an approximately 5-fold increased GABA transport, while the truncated forms of the permease (plasmids pJFPE3, pJFPE4, pJFPE5) are catalytically inactive.

4. Growth inhibition by overexpression of the GABA permease

Induction of the tac promoter on the gabP expression plasmids (pJFPE- Plasmids) by adding isopropyl-2-D-thiogalactoside (IPTG) in the Culture medium leads to a strong growth inhibition of the transformed Strain E. coli W3110.

To demonstrate this effect, the gabP transformants E. coli W3110-pJFPE1 and -pJFPE1 _R in LB (Luria-Bertani) medium (10 g / l bacto-tryptone, 5 g / l bacto-yeast extract, 10 g / l NaCl , pH = 7.5) were cultured with 50 μg / ml ampicillin at 37 ° C. with 1 mM IPTG and without IPTG. Cell growth was measured by determining the optical density at 600 nm ( Figure 5). The strain W3110-pJFPE1 _R shows a normal growth behavior both with and without an inducer. In contrast, W3110-pJFPE1 even without IPTG addition delayed growth can be detected. In the absence of 1 mM IPTG, cell growth is completely inhibited.

In another experiment, the recombinant strains W3110 and W3110 pJFPEl pJFPE1 _R in LB medium containing 50 ug / ml ampicillin were grown to an optical density at 37 ° C (600 nm) of 0.5 and cultured then by adding 1 mM IPTG induced. After 0, 2, 4, 6 and 8 h samples were taken from each of the cultures and the number of viable cells per ml determined by plating on LB agar with 50 ug / ml ampicillin at 37 ° C ( Figure 6). In the GABA permease overexpression strain W3110-pJFPE1, the titer of viable cells had decreased by more than a power of ten after the experimental period. In contrast, in the control strain W3110pJFPE1 _R the logarithmic growth was unaffected.

A cytotoxic effect can be achieved not only by overexpression of the complete permease protein but also with some of the deletion mutants described in Example 3 (Table 2). Apparently shorter fragments of a membrane protein are sufficient for a growth-inhibiting effect, as long as they contain at least some hydrophobic domains that can integrate into the cell membrane. To prove this, liquid cultures of the transformants W3110-pJFPE1 and -pJFPE1 _R , and W3110-pJFPE2-5 in LB medium were grown with 50 μg / ml ampicillin at 37 ° C. Of these aliquots were streaked on agar plates with the same medium + 1 mM IPTG and incubated overnight at 37 ° C. Growth was only observed in the control strain W3110-pJFPE1 _R and in the mutant W3110 pJFPEs with the longest deletion in the 3 'region of the gabP structural gene (330-bp fragment, -419 C-terminal amino acids). In contrast, the other permease deletion mutants (w3110-pJFPE3 and -pJFPE4) are comparable to the wild-type protein (transformants W3110-pJFPE1 and -pJFPE2) for complete growth inhibition.

5. Construction of cloning vectors with selection possibility recombinant plasmids

In many cloning processes, the problem is that only a part of the  contains clones containing recombinant plasmids with inserted foreign DNA, while the rest is transformed only with the vector plasmid. The effect growth inhibition of E. coli cells by overexpression of the gabP gene offers the possibility to construct special cloning vectors, which is a Allow selection for plasmid molecules with integrated DNA fragments.

Such a vector contains, in addition to an antibiotic or herbicide resistance marker, the gabP gene or 3'-truncated partial fragments thereof (see Example 3) Control of an IPTG Inducible Promoter (eg, Lac- or tac Promoter). A multiple cloning region with cleavage sites for various restriction enzymes (Polylinker) is inserted at a suitable position in the gabP gene, without the Changing the reading frame of the permease.

Upon integration of a DNA fragment into the polylinker, this reading frame becomes destroyed, so that the GABA-permease can no longer be expressed. To this Plasmids with DNA insertions can be selected by incubating the E. coli Transformants plated on agar medium with antibiotic and 1 mM IPTG. Under the induced conditions for the permease gene Only cell clones grow with recombinant plasmids, while the Growth of cells containing the unaltered vector is inhibited. A appropriate selection can also be done in liquid cultures, eg. B. in the amplification Genbänken be performed.

The described selection method offers, compared to other methods, z. As the lacZ complementation, the advantage that it with a variety of different E. coli strains can be performed, no genomic Mutants are needed and the recombinant clones are not biochemical Tests must be proven.

6. Use of the gabP gene as negative selection marker in integrative plasmids

When genes are generated by integrative plasmids via homologous recombination in the genome of microorganisms to be introduced, there is often the Problem, integration-positive clones of those with plasmid-encoded copies of the differentiate gene. In these cases, the GABA permease used as plasmid-encoded marker for selection against clones with plasmids become.

For this purpose, the gabP gene is under the control of an inducible promoter (eg. or tac promoter for E. coli) into an integrative plasmid vector. to Transmission by homologous recombination into the bacterial genome is particular Suitable gene to which can then be easily selected, for. B. Resistance genes such as the pat gene for phosphinothricin N-acetyltransferase or genes with mutagenized regulatory sequences. After transformation and Recombination, the cells are grown in liquid medium without antibiotic and then on agar medium with the appropriate selectable marker (eg, phosphinothricin) and the inducer for the gabP gene (eg, IPTG). Under these conditions, only clones are selected which only allow this have integrated transferring resistance genes into the genome.

The method described has the advantage that one can not complete Plasmid loss in the bacterial culture must wait to in the subsequent Selection to avoid false positive clones.

7. Expression of GABA Permease in Plants: Pathogen Resistance by Hypersensitive reaction

A mechanism of plants for protection against pathogen attack (eg by fungi or viruses) is the hypersensitive response. The die Plant cells around the infection from around, causing a spread of the pathogen is prevented. Due to the described cytotoxic Effect of GABA Permease or Hydrophobic Polypeptides Derived Therefrom can be such a hypersensitive reaction by targeted expression of the gabP gene  or 3'-truncated partial fragments thereof are caused in plants.

For this purpose, the gabP gene is incubated with a suitable pathogen or wound inducible promoter [e.g. B. promoters of P _r proteins (= pathogen related proteins)] fused and transformed the construct with known methods in tobacco plants. Subsequently, gabP-positive transformants are selected and tested for pathogen resistance in an infection assay. In this case, transformants as well as wild-type tobacco plants with a phytopathogenic virus [z. Gucumber Mosaic Virus (GMV), Tobacco Mosaic Virus (TMV)]. The symptoms of a viral infection are only detectable in the control plants; the transformants turn out to be virus-resistant.

8. Preparation of male sterile plants by tissue-specific expression the GABA permease

The production of hybrid seed in plant breeding is provided by Facilitates availability of male sterile plants or even for the first time enable. Such a phenotype is due to a tissue specific expression of the GABA permease can be produced in plants. Due to the cytotoxic effect this membrane protein, as well as hydrophobic polypeptides derived therefrom, can the affected parts of plants, z. As anthers, inhibited in their development or to die off.

For this purpose, the gabP gene or the 3'-truncated partial fragment thereof fused to a suitable anther-specific promoter and the constructs with transformed into known methods in tobacco plants. The gabP positive Transformants are inhibited in anther development and turn out to be male sterile.  

Table 1

Vectors and plasmids

Growth of W3110 transformants on agar plates in the presence of 1 mM IPTG plasmid growth pJFPE1 - pJFPE1 _R + pJFPE2 - pJFPE3 - pJFPE4 - pJFPE5 +

Legends to the pictures Illustration 1:

Nucleotide sequence of the 1.9 kb BamHI-HindIII fragment from E. coli K-12 with the complete gabP structures. Those derived from the open reading frame Amino acid sequence of the GABA permease is shown in the bottom line.

Figure 2:

Restriction map of the 5.6 kb Sall-HindIII fragment and the 1.9 kb BamHI-HindIII Fragment from the E. coli K-12 gab cluster. The location of gab genes is through Hatching, their direction of transcription indicated by arrows. The triangles at the Nucleotide scale marks the 3 'ends of four gabP deletion mutants.

Figure 3

[¹⁴C] GABA uptake kinetics of E. coli W3110 transformants (double reciprocal Plot to Lineweaver-Burk).

W3110 pGAB8 + W3110 pMLC12

Figure 4

[¹⁴C] GABA uptake kinetics of E. coli W3110 transformants (double reciprocal Plot to Lineweaver-Burk).

W3110 pJFPE1 + W3110 pJF118u

Figure 5

Growth of various E. coli transformants in LB medium with and without IPTG Additive.

W3110 pJFPE1 + W3110-pJFPE1 + mM IPTG ΔW3110-pJFPE1 _R W3110-pJFPE1 _R + 1mM IPTG

Figure 6

Cytoxic effects of GABA permease overexpression on E. coli gabP- Transformants after induction with 1 mM IPTG.

W3110 pJFPE1 + W3110-pJFPE1 _R

Claims (19)

1. A method for generating cytotoxic effects in microorganisms, plants, plant cells or seeds, characterized in that the overexpression of a GABA permease gene causes growth-inhibiting effects in a cell in which the gene is overexpressed. 2. The method according to claim 1, characterized in that the gene under is the control of an inducible promoter. 3. The method according to claim 1 or 2, characterized in that the gene by one or more deletions, additions or by exchange of Nucleotides of the sequence of a GABA permease gene has arisen and for hydrophobic polypeptides encoded with the same effect as the GABA permease. 4. The method according to one or more of claims 1 to 3, characterized in that the gene consists of a DNA sequence according to Fig. 1. 5. Gene for GABA Permease. 6. Mutated permease gene according to claim 5, characterized in that it by one or more deletions, additions or by exchange of Nucleotides has arisen and for hydrophobic polypeptides with the same Effect coded as the GABA permease. 7. gene according to claim 5 with the DNA sequence according to Fig. 1st 8. gene according to claim 5, characterized by the restriction map according to Fig. 2. 9. GABA permease, which has a cytotoxic effect on the cell in which they are overexpressed.   10. Mutant GABA permease according to claim 9, characterized in that it created by deletions, additions or by exchange of amino acids is. 11. GABA permease according to claim 9 with the amino acid sequence according to Fig. 1. 12. A plasmid containing a gene according to claims 5 to 8. 13. microorganism, plants, plant cells or seeds containing a Plasmid according to claim 12. 14. E. coli containing a plasmid according to claim 12. 15. A method for isolating a GABA permease according to claim 5, characterized characterized in that from the genome of E. coli K-12 by cutting with BAMHI and isolating a 1.9 kb BamHI-HindIII fragment. 16. Use of a gene according to claims 5 to 8 for Growth inhibition of cells by overexpression of the gene. 17. Use of a gene according to claim 16, characterized in that the permease gene under the control of a promoter selected from the group lac, tac, trc or tapetum. 18. A method of detecting plasmids carrying an insertion thereby characterized in that cell clones with recombinant plasmids containing a DNA Insertion in the GABA permease gene, not in their growth are inhibited because the insertion of the expression of the GABA permease is prevented.   19. Method for the positive selection of integrated DNA fragments of a Cloning vector into the genome of an organism, characterized in that the cloning vector comprises one or more selectable DNA fragments which to be integrated, and contains a gene according to claim 1 and according to the Transformation a selection of clones without episomal or chromosomal copy the GABA permease and with integrated DNA fragment is performed.