WO2005033322A1 - Methods and tools for the chromosomal modification of bacteria - Google Patents

Abstract

The invention relates to methods and tools for modifying the chromosome of bacteria. The invention especially relates to methods for modifying the genome of bacteria in a targeted manner, for example by inactivating or replacing a target gene. The inventive methods and tools are based on homologous recombination and on the use of a particularly effective selection and counter-selection system derived from thymidine kinase. The invention can be applied to a plurality of species of bacteria, enabling, for example, the analysis of the function of genes, the creation of new metabolic pathways, and/or the screening of gene banks.

Description

 METHODS AND TOOLS FOR CHROMOSOMIC MODIFICATION OF BACTERIA

The present application relates to methods and tools for modifying the chromosome of bacteria. It relates in particular to methods for targeted modification of the genome of bacteria, for example by inactivating or replacing a target gene. The methods and tools of the invention are based on homologous recombination and on the use of a particularly efficient selection and counter-selection system. The invention is applicable to multiple species of bacteria, making it possible, for example, to analyze the function of genes, to create new metabolic pathways and / or to screen gene banks.

The accumulation of in-depth knowledge on the metabolism and genetics of the bacterial organism Escherichia coli has been accompanied and supported by the development of patiently improved and optimized chromosome manipulation protocols. Most of these protocols are based on the properties of recombination between homologous sequences; they make it possible to generate deletions of genes, to integrate heterologous sequences, to exchange alleles, etc. Although perfectly mastered in genetic laboratories, these protocols take time, often offer disappointing performance and have certain limitations.

The protocols commonly used to obtain the deletion of a gene are based on the possibility of homologous recombination between a linear DNA strand and the chromosome, in a strain whose recombination properties are altered (recB recC sbcB strains). The selection of the recombination event is ensured by the general insertion of an antibiotic resistance cassette between the recombinant sequences, sometimes by a metabolic marker allowing the assimilation of a new nutritive source, or even by the introduction of a marker conferring a coloration or a fluorescence on the bacteria detected by suitable means. The accumulation of multiple deletions in the same strain is limited by the number of resistance genes available. The exchange of a wild allele by a modified allele may require a first step of integration into the chromosome of a circular construction (plasmid) carrying a homologous sequence, which results in the presence on the chromosome of the two alleles. This first event is selected by the integration into the construction of an antibiotic resistance cassette. Allele exchange takes place after a second recombination event between the two homologous sequences, which eliminates non-homologous sequences, including the resistance cassette. The new allele must therefore be directly selectable.

A homologous recombination method in E. coli has been described by Link (J. Bacteriol. 1997, 179: 6228-6237). This method describes the use of a positive selection marker and a negative selection marker, the sacB gene. Although this method significantly improves the state of the art, the authors nevertheless note that the negative selection marker is not absolute and can lead in certain cases to the isolation of clones not conforming to what is expected.

The constraints and limitations of these protocols require the development of methods applicable a priori to all genes and allowing the positive selection of rare recombination events as well as the direct selection of excision of the marker gene.

In addition, the development of genome exploration programs which opens access to a larger number of organisms every day, acclimatized in the laboratory and whose genomic sequences are fully known, requires methods that can be transposed from an organism to a other.

The present invention provides entirely reliable recombination methods which can be used in an automated manner. The negative selection used is based on the conditional lethality induced by thymidine kinase in the presence of a nucleoside analog. One of the advantages of this tdk marker is that it can also be a positive selection marker in the presence of certain toxicants. The functionality of the chromosomal insertion of the tdk gene can therefore be verified before proceeding to the counter-selection linked to this same gene. The present patent application describes in particular the use of the tdk gene, coding for a thymidine kinase activity and whose translation product phosphorylates nucleoside analogs such as Pazidothymidine, as a marker gene for the selection of a genetic recombination event (positive screen ) in bacteria, and the loss of which is directly selectable by the resistance of the bacteria to the nucleoside analog (negative screen). This method has been developed and validated in different species of bacteria, in particular in E. coli and in Acinetobacter ADP1.

A first aspect of the invention therefore resides in the use of a gene encoding a thymidine kinase activity as a marker for selection of recombination event in bacteria.

Another object of the invention relates to a method for targeted alteration of the genome of a bacterium by homologous double recombination, comprising: a) the introduction, into a bacterium deficient for thymidine kinase activity, of a genetic construction comprising (i) at least one region having a sequence homologous to a selected segment of the genome of the bacterium, the alteration of which is desired, said region having a length sufficient to allow homologous recombination, (ii) a positive selection marker and (iii) a negative counter-selection marker comprising a gene encoding a polypeptide having a thymidine kinase activity, b) the culture of the bacteria under conditions of selection with respect to the positive marker and the selection of the bacteria expressing the positive marker inserted into their chromosome, and c) the culture of bacteria obtained in step b) in the presence of a nucleoside analog metabolized by l a thymidine kinase, and the selection of bacteria capable of survival or growth in the presence of this analog, said bacteria having an altered genome. Preferably, the positive selection marker confers resistance to an antibiotic compound and / or the negative counter-selection marker is a tdk gene conferring toxicity on a nucleoside analog and / or the nucleoside analog is AZT . In a particular embodiment, the positive selection marker and the negative counter-selection marker are a single gene. The alteration can relate to any part of the genome of the bacteria, such as for example a gene of interest. The bacterium can be any bacterium transformable by a nucleic acid in vitro, such as for example E. coli, Acinetobacter, Pseudomonas, Bacillus, Helicobacter, Synechocystis, Methanobacterium, Methanococcus, Streptococcus, Lactobacillus, Staphylococcus, Klebsiella, Xanthomonas etc. Preferably, the genetic construct used for altering the genome does not replicate in the bacteria. This construction may be a circular or linear DNA fragment.

Another subject of the invention relates to a product comprising: - a genetic construct comprising (i) a region having a sequence homologous to a selected segment of the genome of a bacterium whose alteration is desired, said region having a length sufficient for allow homologous recombination, (ii) a positive selection marker and (iii) a negative counter-selection marker comprising a gene encoding a polypeptide having thymidine kinase activity; and - a nucleoside analog metabolized by this thymidine kinase.

The invention also relates to a recombinant genetic construction, consisting of (i) a region having a sequence homologous to a selected segment of the genome of a bacterium whose alteration is desired, said region having a length sufficient to allow homologous recombination, (ii) a positive selection marker conferring resistance to an antibiotic compound and (iii) a negative counter-selection marker comprising a gene encoding a polypeptide having thymidine kinase activity.

The invention further relates to methods for altering metabolic pathways or for creating new metabolic pathways in bacteria, including alteration, in a bacteria, a gene involved in a metabolic pathway using a method described above.

The invention also relates to methods for determining the function of genes in bacteria, comprising the alteration, in a bacterium, of a gene whose function is to be studied, using a method described above, and the analysis the phenotype of the bacteria thus obtained. Within the meaning of the invention, the phenotype broadly designates morphology, growth, mobility, division, the expression of surface markers, the secretion of factors in the environment, survival, etc.

The invention is generally applicable to the modification of any gene or region of the genome of any bacterium, is reliable and rapid, and provides new effective and powerful tools for genetic analysis, the construction of modified genomes and the creation of genetic diversity. or metabolic.

LEGEND OF FIGURES

Figure 1: Schematic representation of a genetic construction of the invention and the allelic exchange protocol, applied to the ileS gene of Pisoleucyl-fRNA synthetase from E. coli.

Figure 2: Schematic representation of a genetic construction of the invention and the gene replacement protocol, applied to the icd gene of isocitrate dehydrogenase from E. coli.

Figure 3: Schematic representation of a genetic construction of the invention and the gene deletion protocol, applied to the cysN gene of Acinetobacter ADP1 sulfurylase. DETAILED DESCRIPTION OF THE INVENTION

As indicated, the invention relates to compositions and methods making it possible to modify the genome of bacteria in a targeted manner, for example to inactivate a selected gene. The compositions and methods of the invention are based in particular on the development of particular genetic constructs and particular selection genes, ensuring high efficiency and wide application of the invention.

Genetic engineering

The term genetic construction refers to any recombinant nucleic acid molecule. It can be single-stranded or double-stranded DNA, preferably double-stranded, linear or circular. The genetic construction can be of the plasmid or phage type, or a DNA fragment. It can be constructed by conventional techniques of molecular biology, including ligation, cloning, artificial synthesis, amplification, etc.

(i) Region of Homology

The region of homology (i) is intended to allow targeted chromosomal insertion of the genetic construction (or a part of it), by homologous recombination. This region therefore comprises a sequence homologous to a selected segment of the genome of the bacterium (for example a gene or a regulatory region), the alteration of which is desired, and it has a length sufficient to allow homologous recombination.

The selected segment of the genome (and the alteration of which is desired) may be all or part of a gene (that is to say a region of the genome coding for an RNA or a protein), of a regulatory region ( e.g. promoter, terminator, etc.), from an uncharacterized region, etc. Mention may be made, for example, of a gene for a biosynthetic pathway of a metabolite, an antibiotic, an amino acid, a vitamin, an enzyme, a membrane or wall protein. bacteria, an involved gene in the virulence of a bacterium, a gene involved in a transporter system present in a membrane of the bacteria, etc.

The alteration can consist, for example, of an allelic exchange (that is to say the substitution of all or part of a gene by an altered copy of it), a replacement of a gene (i.e. say the replacement of all or part of a gene with a different gene) or a deletion of all or part of a segment of the genome.

The region of homology to a selected segment of the genome of the bacterium allows homologous recombination at the level of this segment, and thus the targeted alteration of the genome. The region of homology must be of sufficient length to allow homologous recombination. It is generally accepted that a region of homology of at least 30 bases is sufficient in bacteria to allow the realization of a recombination event. However, it is preferred to use regions of larger homologies, typically comprising from 30 to 2000 bases, for example from 50 to 1000, typically from 80 to 800. Furthermore, the degree of homology must be sufficient to allow homologous recombination. Preferably, the homology (i.e. the sequence identity) is perfect over at least a portion of the region of homology, or at least greater than 80%, preferably 85%, 90% , 95%, 97% or 98%. The degree of homology can indeed affect the selectivity of the method and, above all, its yield. It is therefore preferred that the regions of homologies have a high degree of identity, preferably greater than 95% with the targeted segment. It is understood that the length and the composition of the region of homology can be adjusted by a person skilled in the art, depending on the type of alteration sought, the bacteria considered, and the type of genetic construction.

Thus, for an allelic exchange, the region of homology typically comprises the sequence of an altered copy of at least part of the gene whose modification is sought. This altered copy preferably comprises two regions of perfect homology with the target gene, allowing double homologous recombination, framing an altered region of the target gene, for example carrying one or more mutation (s), deletion (s) and or addition ( s) one or more residues. Typically, gene alteration includes one or more mutations, occasional or not. A point mutation consists of the substitution of one base by another in the gene sequence (which can result in a codon change, for example resulting in a stop codon, nonsense, or in a codon coding a different amino acid) . A non-point mutation consists of the substitution of several consecutive bases in the gene sequence, which can result in a change of one or more consecutive codons. A deletion consists of the deletion of one or more bases, consecutive or not, in the gene sequence. This deletion can create stop or nonsense codons, for example, or introduce an offset in the reading frame. Additions of one or more bases can also be envisaged.

In the case of a replacement (partial or total) or a deletion (partial or total) of gene, the region of homology typically consists of two sub-regions of homology each having a sequence homologous to a distinct segment of the gene of the bacterium whose alteration is desired, and each having a length sufficient to allow homologous recombination. Each of these sub-regions is chosen so as to frame the sequence of the target gene whose replacement or deletion is desired. In the case of a gene replacement, the two sub-regions surround the positive selection marker (ii) or any other nucleic acid which will be inserted into the target gene, replacing the gene sequences. In the case of a gene deletion, the two sub-regions surround a set consisting of the positive selection marker (ii) and the negative counter-selection marker (iii).

The region of homology can be defined on the basis of the gene sequence or of the segment targeted in the genome of the bacterium. Once the target region has been identified and the type of alteration has been determined, a region of homology (i) can be prepared, typically by artificial synthesis, or by cloning and / or amplification.

(ii) Positive marker

The positive selection marker (ii) can be any gene (that is to say any nucleic acid) encoding an RNA product or polypeptide conferring on the bacterial cell the containing resistance to a toxic compound. It is preferably a gene encoding a product conferring resistance to an antibiotic. By way of nonlimiting examples, mention may be made of the kan gene conferring resistance to kanamycin, the neo gene conferring resistance to neomycin, the cat gene conferring resistance to chloramphenicol, the aad gene conferring resistance to spectinomycin, the Strept ^R gene, conferring resistance to streptomycin, the aac gene conferring resistance to apramycin or the tdk gene conferring resistance to trimethoprime in the presence of thymidine in a rich medium. This last positive selection marker and the conditions under which it operates constitute another particular object of the present invention. Indeed, the present application proposes to use a tdk gene both as a positive and negative marker, thus providing simplified genetic constructions.

The tdk gene coding for thymidine kinase activity constitutes, in E. coli, in the presence of thymidine, a gene for resistance to trimethoprime. Trimethoprime is a dihydrofolate reductase inhibitor; it causes secondarily, by exhaustion of the intracellular stock of methylene tetrahydrofolate (co-substrate with the dUMP of thymidilate synhthase), the blocking of the synthesis of thymidilate (dTMP) and leads to cell death by absence of the base T ("thyminless death "). In an E. coli strain whose thymidilate synthase function is deficient (in the presence of trimethoprime or by inactivation of the thyA gene), the thymidine kinase activity allows cell growth in the presence of thymidine. In the presence of trimethoprime, the bacteria resistant to the rich medium of Muller Hinton (HD) supplemented with thymidine are selected. The use of tdk as a positive selection marker is applicable to bacteria having a trimethoprime-sensitive dihydrofolate reductase and a thyA-type thymidilate synthase.

In this particular embodiment of the invention, the positive selection marker (ii) and the negative counter-selection marker (iii) are represented by a single gene, encoding a polypeptide with thymidine kinase activity, preferably a tdk gene. .

When the positive selection marker is a marker conferring resistance to a toxic compound (for example an antibiotic), step b) of the method comprises the culture of bacteria in the presence of the toxic compound and the selection of cells capable of survival and / or growth on this medium. The concentrations used for the toxic compound are concentrations which are not toxic to cells expressing resistance, but toxic to cells which do not express the marker. These concentrations are known to a person skilled in the art and can be adapted, if necessary, as illustrated in the examples.

Alternatively, the positive selection marker may be a metabolic marker allowing the assimilation of a new nutritive source, or even a marker conferring a coloration or a fluorescence or any other physical character on the bacteria which will be selected after detection by appropriate means. .

(iii) Negative marker

The negative counter-selection marker (iii) comprises a gene coding for thymidine kinase activity. A gene coding for thymidine kinase activity designates any nucleic acid molecule coding for a polypeptide having a thymidine kinase type activity which is functional in the bacterium considered. Thymidine kinase activity is mainly characterized by the ability to phosphorylate a nucleoside analog, such as azidothymidine (AZT), into the corresponding monophosphate form, in the case of AZT into azidothymidine phosphate (AZT-MP). The nucleic acid molecule can be an RNA or a DNA, typically a recombinant DNA (eg, cDNA) comprising a nucleic sequence coding for the thymidine kinase polypeptide. Different thymidine kinases have been described in the literature, such as, for example, bacterial, viral (in particular the herpes simplex virus) or mammalian (for example human, bovine, etc.) thymidine kinases. The gene used in the invention can come from viruses, bacteria, archaebacteria, and eukaryotes such as fungi, plants and animals. This gene can be a synthetic gene. It can be a wild type thymidine kinase, or possibly modified in its structure to improve its properties, for example its stability, its expression in the bacteria, its substrate specificity, its enzymatic activity, etc. Preferably, in the context of the present invention, a thymidine kinase of bacterial origin is used, in particular originating from or derived from E. coli, from Haemophilus influenzae, from Streptococus gordonii (McNab, 1996, FEMS Microbiol Lett. 135: 103- 10) of humans (Wang, 2000, Biochem Pharmacol. 59: 1583-8), of the human herpes virus (Cazaux, 1998, Cancer Gene Ther. 5: 83-91)

In a particularly preferred manner, the negative counter-selection marker used in the invention is a gene coding for a protein having a thymidine kinase activity (EC 2.7.1.21) and capable of catalyzing the phosphorylation of azidothymidine (AZT) into azidothymidine 5 phosphate (AZT-MP), preferably chosen from genes homologous to the E. coli tdk gene (Bockamp et al., Gene 1991, 101: 9-14; Black, Mol. Microbiol. 1991, 5: 373-379) or the Haemophilus influenzae tdk gene (Fleischmann, Science, 1995, 269: 496-512). layout

The marker genes (ii) and (iii) present in the genetic construction must be able to be expressed in the bacterial host in which the homologous recombination is carried out, to allow the synthesis of an active and functional product. To improve the expression of these genes, one or both are therefore generally placed, in the genetic construction, under the control of a functional transcriptional promoter in the host. A promoter, identical or preferably different, can be used for each of the two marker genes, or a single promoter directing the expression of the two marker genes, in the form of a bicistronic unit. The promoter (s) used can be constitutive or regulated, that is to say active (or activated) or inactive (or inactivated) in the presence or in the absence of a substance (eg, inducer, repressor, etc.) or a particular condition (eg, temperature, etc.).

Many functional promoters in bacterial hosts are known to those skilled in the art, and can be used in the context of the invention, such as, for example, the promoter of the lactose operon (Plac), of the tryptophan operon, hybrid promoters (e.g. Ptac) and / or synthetic, phage promoters (e.g. T5, T7), promoters of bacterial genes (e.g. ValS), etc.

The arrangement of elements (i) to (iii) in the genetic construction can be adapted by a person skilled in the art, and generally depends on the type of alteration chosen (allelic exchange, gene replacement, deletion, etc.). In general, the positive selection marker and the negative selection marker can be positioned either 5 'or 3' relative to each other, in the same transcriptional orientation or in an opposite orientation. In the case of an allelic exchange, the respective order of elements (i) to (iii) is not critical for the method of the invention. Thus, for a circular construction, a 5 '-> 3' arrangement can be used (i) - (ii) - (iii); (iii) - (ii) - (i); etc. In the case of a gene replacement (total or partial), the region of homology is generally subdivided into two portions framing the gene (ii), and the gene (iii) can be located 5 ′ or 3 'of this assembly. In the case of a gene deletion (total or partial), the region of homology is generally subdivided into two portions framing the genes (ii) and (iii), which can be arranged in any order.

In addition to the elements mentioned above, the genetic construction may, where appropriate, include other elements, such as an origin of replication, additional marker genes, etc. When it contains an origin of replication, it is preferably conditional (for example the replicon of RK6) or non-functional in the bacteria which it is desired to modify. Indeed, the absence of functional replication limits positive selection to chromosomal insertion events by homologous recombination.

In a particular embodiment of the invention, the genetic construction is a circular, double-stranded DNA, for example a plasmid. This type of construction is particularly suitable for bacteria of the genus E. coli. It can be constructed from any commercially available plasmid, such as plasmids pBR322, pUC, pTL, pSH, etc. The overall size of the plasmid can be adapted by a person skilled in the art to be preferably compatible with the transformation of bacteria. Any plasmid comprising the elements (i) to (iii) described above is compatible. In another particular embodiment of the invention, the genetic construction is a linear DNA, preferably double-stranded. Such linear DNA can be prepared in vitro by techniques known per se, such as artificial synthesis, cloning, ligation and / or amplification, etc. In a particular implementation variant, the linear DNA is produced in vitro by simultaneous amplification of one, several, or all of the elements (i) to (iii) using a mixture of specific oligonucleotide primers and appropriate matrices. Preferably, the templates are therefore used as a mixture, and the primers have appropriate covering sequences, allowing direct assembly of the genetic construction comprising the amplification products (i), (ii) and / or (iii). This aspect constitutes another particular object of the invention.

Bacteria and cultures:

As indicated, the methods of the invention are applicable to bacteria. Given the selection system used, the bacteria used in the invention are typically deficient in thymidine kinase activity, that is to say that they are not sensitive to the nucleoside analog used. Typically, such a bacterium does not express a functional endogenous tdk protein or does not express it sufficiently to confer sensitivity to the concentrations of nucleoside analog used. The bacterium can be naturally deficient for tdk activity, as is the case for example of bacteria of the genus Acinetobacter, or can be made deficient by inactivation of the endogenous tdk gene as is the case for E. coli.

The bacteria which can be used for the invention can be chosen from any bacterium of interest, which is transformable by an exogenous nucleic acid. Mention may in particular be made of cultivable bacteria such as E. coli, Pseudomonas, Acinetobacter, Pseudomonas, Bacillus, Helicobacter, Synechocystis, Methanobacterium, Methanococcus, Streptococcus, Lactobacillus, Staphylococcus, Klebsiella, Xanthomonas, Agrobacterium, etc. conditions well known to those skilled in the art, such as for example in defined media or rich media such as LB (Luria Bertani) or MH (Muller Hinton) media. Cultures are typically carried out in boxes, flasks, bags, tubes, etc., preferably in a sterile condition.

In a preferred embodiment, the bacterium is a strain of E. coli or an Acinetobacter strain, preferably Acinetobacter calcoaceticus (ADP 1).

The genetic construction can be introduced into the bacterium by any technique known to a person skilled in the art, generally gathered under the designations "transfection" or "transformation". Thus, the construction can be introduced in vitro (that is to say in culture) by electroporation, conjugative transfer, precipitation with calcium phosphate, micro-injection, etc. The introduction can also be carried out using agents which facilitate the introduction of nucleic acid into prokaryotic cells, such as cationic lipids or liposomes, cationic peptides, polymers, phages, etc. It is not essential for the method of the invention that the introduction of the constructions is carried out with very high efficiency, insofar as a selection is then applied.

After the introduction step, the second step of the method aims to select the chromosomal insertion, that is to say the cells that have integrated the genetic construction (or a part of it) in their genome . This selection is generally carried out on the basis of the expression of the positive selection marker, that is to say, depending on the type of positive marker, the resistance of the cell to a toxic compound (for example an antibiotic), the ability to use a particular nutrient source, expression of a marking, etc. For this, the bacteria are cultivated under conditions of selection with respect to the positive marker, that is to say in the presence of the toxic compound, the nutritive source, etc. and the cells expressing the selection marker are selected. In the case of a selection marker conferring resistance to a toxic compound (in particular an antibiotic), the cells are cultured in the presence of the compound and selected on the basis of their capacity for survival and / or growth in this selection medium . Insofar as the genetic construction is not capable of efficient autonomous replication in the host bacterium, the cells thus selected result indeed from chromosomal insertion. The third step of the method generally aims to excise from the genome of the bacteria the regions of the genetic construction which are not essential to the desired modification. In particular, in the case of an allelic exchange or a deletion, it is important to be able to eliminate the sequence of marker genes from the cell genome. In the case of gene replacement, it is also desirable to eliminate the sequences other than the positive marker. To do this, the invention is based on the use of the particular negative selection marker, thymidine kinase. The invention indeed shows that this marker is functional in bacteria and particularly effective for the selection of this excision event. Since the negative counter-selection marker used makes the cells which contain it and which express it sensitive to nucleoside analogues, in particular AZT, while these same cells, which do not contain this gene, are resistant to AZT, cell culture in the presence of the analog makes it possible to select the cells having lost the negative counter-selection marker.

Preferably, the nucleoside analog is AZT. However, other analogs can be considered, such as gancyclovir (GCV), acyclovir, dideoxynucleosides such as dideoxythymidine (ddT), dideoxyinosine (ddl), stavudine (d4T) etc. The toxicity of AZT and these other analogues is exerted mainly after their transformation into triphosphate derivatives by blocking the replication of DNA. AZT-triphosphate is a substrate for DNA polymerases. It is produced in E. coli from AZT by three successive phosphorylation stages: by thymidine kinase (tdk), thymidilate kinase (tmk) and nucleoside diphosphate kinase (ndp). The use of AZT as a conditional counter-selection compound in organisms other than E. coli requires that these three stages of phosphorylation actually take place. It is always possible for organisms lacking tmk activity on AZT-phosphate and / or ndk on AZT-diphosphate, to integrate on the chromosome or to provide in trans an element comprising the tmk gene and / or the E. coli ndk gene for example, in order to fulfill the conditions allowing the counter-selection of the tdk gene in the stages of homologous recombinations. In step c) of the methods of the invention, the cells are therefore cultured in the presence of the nucleoside analog, at a concentration and for a period of time sufficient to eliminate the cells containing and expressing the functional thymidine kinase. This culture can be carried out under selection pressure for the positive selection marker (that is to say for example in the presence of the antibiotic compound when the positive selection marker is an antibiotic resistance marker) or, alternatively, without selection pressure for the positive selection marker (that is to say for example in the absence of the antibiotic compound when the positive selection marker is an antibiotic resistance marker).

The ability of cells to grow or survive in the presence of the nucleoside analog can be verified by any technique known to those skilled in the art, including cell counting, morphological observation, trypan blue staining, etc.

Alternatively, the exchange of a wild allele by a modified allele or the deletion of all or part of a chromosome region (in particular of a gene) may require a first step of integration into the chromosome of a first linear construction carrying a cassette containing the positive selection and negative counter-selection markers framed by two regions of homology to extreme segments (that is to say located in the 5 'and 3' regions) of the wild-type or of the region to be deleted, which causes the presence on the chromosome of the allele interrupted by the cassette. This first event is selected by the resistance to an antibiotic brought by the integrated cassette. The allelic exchange is carried out after a second stage of integration into the chromosome of a second linear construction carrying the modified or deleted allele. This second event is selected by resistance to the pro-toxic (the nucleoside analog) activated by the negative counter-selection marker.

In a particular embodiment, the method of the invention allows the carrying out of an allele exchange or a deletion of at least part of a gene, and comprises: a) the introduction, into a deficient bacterium for thymidine kinase activity, a linear DNA comprising a positive selection marker (ii) and a negative counter-selection marker (iii) comprising a gene coding for a polypeptide having thymidine kinase activity, the assembly consisting of (ii) and (iii) being framed by two regions of homology to extreme segments of the gene to be modified or deleting, said regions of homology having a length sufficient to allow homologous recombination; b) the culture of the bacteria under selection conditions with respect to the positive marker and the selection of the bacteria expressing the positive marker, said bacteria comprising the positive marker inserted into their chromosome, and c) the culture of bacteria obtained with the step b) in the presence of a nucleoside analogue metabolized by thymidine kinase and a second linear DNA comprising the modified or deleted allele of the gene, and the selection of bacteria capable of survival or growth in the presence of this analogue , said bacteria having an altered genome.

In this mode of implementation, the modification is carried out in two stages, firstly by the insertion, by double homologous recombination, of a first construction, which is then exchanged with a second construction carrying the desired modification.

At the end of this selection step c), the cells obtained have a modification of their genome, targeted at the level of the region of interest. The modification can be controlled by any known technique, in particular PCR, in situ hybridization, etc. The bacteria obtained can be used for phenotypic analysis, the expression of proteins or other products of interest, the production of metabolites, etc.

The method according to the present application is particularly advantageous insofar as it is effective, does not generate false positives, and is applicable to a wide range of bacteria and target genes. The examples which follow serve to illustrate the method of the invention, without limiting its scope. The content of all publications, patents and patent applications cited in this description and in the examples is incorporated herein by reference. Example 1. Allelic exchange at the IS island locus. coli coding for isoleucyl-tRNA synthetase.

The technique based on the use of thymidine kinase as a genetic marker for the selection of allelic exchanges was applied to the ileS gene of the isoleucyl-tRNA synthetase IleRS. This enzyme catalyzes the formation of the covalent bond between L-isoleucine and its specific transfer RNA, tRNAile. The active site of IleRS is capable of activating isosteric amino acids with L-isoleucine, such as L-valine, and of loading them on tRNAile. In order to avoid the accumulation of defective proteins in the cell, tRNAiles carrying an amino acid other than isoleucine are recognized by a second active site of the enzyme (correction or editing site) which hydrolyzes the aminoacyl bond wrong.

To study the effects on bacterial cell viability of a high error rate during protein biosynthesis, an allele of the ileS gene of E. coli coding for a defective synthetase in its editing capacity was constructed by replacing amino acids 241 to 250 with a series of alanines (ileSalall allele, plasmid pTLH33). This mutated allele was integrated into a plasmid construct which should allow its exchange with the ileS chromosomal allele. The construction of this plasmid, pSH102, included the following steps: The tdk gene and the promoter sequence of the valS gene were amplified from the chromosomal DNA of Haemophilus influenzae; the pairs of oligonucleotides respectively used as primers in the amplification reactions of these sequences are on the one hand: tdkl.3 (5 'CCCGTCGACTTAAATTTGACCGCACTTTTCTTTA: SEQ ID NO: 1) and tdkl .5 (5' ATGGCGAAACTCTACTTCTATTAC: SEQ ID NO : 2) for the tdk gene, and secondly,

ValS 1.5 (5 'CCCCTCGAGGATAAAGGTTATCATGTTG: SEQ ID NO: 3) and ValS 1.3 (5' TAGAGTTTCGCCATTTATTTTTTCTCTTTATTATAA: SEQ ID NO: 4) for the promoter sequence of the valS gene. The DNA fragments obtained in the previous step were pooled by PCR with the oligonucleotides tdkl.3 and ValS 1.5 (described above) thus forming the Pra / S cassette _H i ^ _H i This cassette was cloned into the Xhol site of the vector pEVL497 after digestion with Xhol and Sali (plasmid pSH93). The vector pEVL497 carries the cat gene which confers resistance to chloramphenicol and the conditional replicon of the plasmid R6K which requires the expression of the protein Pi to maintain and propagate in cells (strain BW19610). The ileSalal 1a gene was inserted into the plasmid pSH93 between the EcoRI and Xmal sites, thus giving the plasmid pSH102. The plasmid pSH102 was introduced by transformation into the strain of E. coli +755 whose tdk gene coding for thymidine kinase activity is deleted (Δtdk :: kan ^r marker); transformants were selected on Mϋller Hinton medium (MH) in the presence of chloramphenicol (20 μg / ml). The +755 strain does not express the Pi protein, the colonies resistant to chloramphenicol (Cm ¹ ) carry the plasmid pSH102 integrated into their chromosome by homologous recombination at the ileS locus. These Cm ^r clones were cultured in HD medium without antibiotics to allow the second homologous recombination event. The recombinant double clones were selected on HD boxes in the presence of AZT

(30 .mu.m). The clones resistant to AZT (AZT ^{1 "} ) were tested for their sensitivity to chloramphenicol. In order to distinguish among the clones AZT ^r Cm ^s those which carry the mutated allele ileSalal 1 and those which preserved the wild allele ileS , the sensitivity to norvaline of the double recombinants was tested. The sensitivity to norvaline indicates the presence of an allele ileS whose editing is deficient. The chromosomal DNA of the clones sensitive to norvaline was extracted for carrying out amplifications. of DNA by PCR with the following two pairs of oligonucleotides: the pair Ile3 (5 'CGCCGCCGCCGCCGCCGCCGCCGC: SEQ ID NO: 5) and Ile4 (5' GATTGCGCGTGATGAATTAACC: SEQ ID NO: 6), and the pair Ile2 (5 'GTTGGCAGGCAGAGTCCACGGC : SEQ ID NO: 7) and Ile4 (5 'GATTGCGCGTGATGAATTAACC: SEQ ID NO: 6) The results of these PCR amplifications, namely the detection of a fragment amplified with the pair Ile3 and Ile4 on the one hand, and the absence of an amplified fragment with the pair Ile2 and Ile4 on the other hand, made it possible to verify that, in the four strains which had been obtained, the ileSalall gene was inserted in place of the ileSwt gene on the chromosome.

Example 2. Deletion of the icd gene from E. coli coding for Pisocitrate dehydrogenase. Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate to alpha-ketoglutarate, a reaction of the tricarboxylic cycle. Alpha-ketoglutarate is the precursor of glutamic acid; the absence of isocitrate dehydrogenase activity therefore causes an auxotrophy for glutamic acid. A partial deletion of the icd gene containing the spectinomycin resistance marker gene was obtained using the plasmid construct pEVL194. This plasmid, derived from the plasmid pEVL497 (see example 1), contained the tdk gene of E. coli preceded by the phage promoter T5 (pT5-tdk), cloned in the Kpnl site, and the aad spectinomycin resistance cassette cloned between the BamHI and Sphl sites. The resistance cassette was framed with sequences homologous to the chromosome of E. coli: in 5 ′ by the upstream region of the ATG initiation codon of the icd gene (350 base pairs), cloned between the EcoRI and BamHI sites, in 3 ′ by the sequence corresponding to nucleotides 603 to 1083 of the icd gene cloned between the Sphl and Notl sites. All the elements inserted into the plasmid pEVL497 to construct the plasmid pEVL194 were amplified by PCR from DNA templates and pairs of appropriate oligonucleotides by adding the cloning sites used.

The plasmid pEVL194 was introduced by transformation into the strain of E. coli +755 which does not express thymidine kinase activity (Δtdk :: kani marker) and recombinant transformants selected on Muller Hinton medium (HD) in the presence of spectinomycin (20 μg / ml). The bacteria of a clone resistant to spectinomycin were cultured in HD medium supplemented with spectinomycin (20 μg / ml) and glutamic acid (1 mM) at 37 ° C. with stirring until an OD600 of 0.2. The bacteria contained in 10 ml of this culture were concentrated and spread on an MH dish supplemented with spectinomycin (20 μg / ml), glutamic acid (1 mM) and AZT (30 μM). The spec ^r AZT ^r clones, obtained after incubation at 37 ° C. for 18 h, were tested for their sensitivity to chloramphenicol and for their phenotype of glutamic acid auxotrophy. The conformity of the deletion of the icd gene from Cm ^s glu "spec ^r bacteria was checked by PCR on chromosomal DNA using appropriate oligonucleotides.

Example 3. Deletion of a gene in Acinetobacter ADP1 Acinetobacter ADP1 is a gram-negative, aerobic, non-spore-forming, non-pathogenic bacteria found in soil and water. It has the genetic arsenal for multiple metabolic pathways allowing it to use very diverse carbon sources. In the laboratory, Acinetobacter ADP1 grows in a defined mineral environment with a single source of carbon and without additional growth factor. Cultivated under the same environmental conditions (mineral medium + glucose) and temperature (33-35 ° C), Acinetobacter ADP1 has a shorter generation time than Escherichia coli. In addition to these culture advantages, there are remarkable properties of competence for natural transformation (active transport in cells of linear or circular DNA molecules) and of high frequency of recombination events.

Furthermore, Acinetobacter ADP1 does not have any of the enzymes of the nucleoside recovery pathways described in E. coli and does not express thymidine kinase activity. The use of the tdk gene as a marker gene in genetic manipulations of the Acinetobacter chromosome is therefore easy, without the need for prior construction.

We used the tdk gene as a marker for selection of homologous recombination events in Acinetobacter ADP1 to obtain the deletion of the cysN gene, which codes for the sulfurylase activity essential for the assimilation of sulfur from sulfate. In a first step, the cysN gene was replaced on the chromosome by a cassette, containing the two tandem genes tdk (thymidine kinase) and kan (gene for resistance to kanamycin), introduced by homologous recombination. Recombinant bacteria, selected by their resistance to kanamycin, express the tdk gene and are therefore sensitive to AZT. In a second step, the tdk-kan cassette was eliminated from the chromosome by a homologous recombination event selected by the restoration of resistance to AZT. The tdk-kan cassette was cloned into a derivative of the vector pUC18 as follows: the sequence of the E. coli tdk gene preceded by the bacteriophage T5 promoter was cloned into the EcoRI and Sacl sites and the resistance gene to kanamycin has been cloned into the PstI and Xbal sites (plasmid pEVL385).

The construction allowing the deletion of the coding sequence of the cysN gene on the chromosome was obtained in the following way: the tdk-kan cassette was amplified by PCR from plasmid pEVL385 using oligonucleotides 1130/1131. The sequence corresponding to the first 300 nucleotides and that corresponding to the last 300 nucleotides of the cysN gene were amplified by PCR respectively using the two pairs of oligonucleotides (1150/1151) and (1152/1153) on the chromosomal DNA d 'Acinetobacter ADP1. Primers 1151 and 1152 have at one of their ends 20 bases homologous to the ends of the tdk-kan cassette, which made it possible to combine the three fragments by recombinant PCR.

15 μl of the purified PCR product (Qiaquick, Qiagen) were introduced by transformation into Acinetobacter ADP1 bacteria cultivated at 30 ° C. in a mineral medium without sulfate + succinate and supplemented with ImM thiosulfate up to an OD600 of 0.5 - 0 6. After 3 h of growth at 30 ° C. with stirring, the transforming bacteria are selected on mineral medium + succinate + thiosulfate 1 mM in the presence of kanamycin (25 μg / ml). The clones were tested for their sensitivity to AZT (800 μM). The use of sulfate and sulfite by these clones resistant to kanamycin and sensitive to AZT was then tested. The location of the cassette at the cysN locus was confirmed by PCR on the chromosomal DNA of a clone (strain +2912) expressing the sulfate- / sulfite + phenotype.

In a second step, the tdk-kan cassette was eliminated from the +2912 strain by a recombination event between the chromosome and a DNA fragment obtained by recombinant PCR amplification (primers 1150/1403, 1402/1153 then 1150/1153 ) having in tandem the first 300 and last 300 nucleotides of cysN. 15 μl of the purified PCR product (Qiaquick, Qiagen) were introduced by transformation into bacteria of the +2912 strain. The recombinant bacteria were selected on mineral medium + succinate + thiosulfate in the presence of AZT (800 μM). The cysN deletion and the elimination of the tdk-kan cassette were confirmed by PCR on the clones resistant to AZT. PRIMER TABLE

Claims

1. Method for targeted alteration of the genome of a bacterium by double homologous recombination, comprising: a) the introduction, into a bacterium deficient for thymidine kinase activity, of a genetic construction comprising (i) at least one region having a sequence homologous to a selected segment of the genome of the bacterium, the alteration of which is desired, said region having a length sufficient to allow homologous recombination, (ii) a positive selection marker and (iii) a negative marker for counter-selection comprising a gene coding for a polypeptide having a thymidine kinase activity, b) the culture of bacteria under conditions of selection with respect to the positive marker and the selection of bacteria expressing the positive marker inserted in their chromosome, and c ) the culture of bacteria obtained in step b) in the presence of a nucleoside analog metabolized by thymidine kinase, and the selection of bacteria capable of survival or growth in the presence of this analog, said bacteria having an altered genome. 2. Method according to claim 1, characterized in that the positive selection marker confers resistance to an antibiotic compound and in that step b) comprises the culture of bacteria in the presence of the antibiotic compound and the selection of bacteria capable of survival and / or growth. 3. Method according to claim 1 or 2, characterized in that the negative counter-selection marker comprises a tdk gene. 4. Method according to one of claims 1 to 3, characterized in that the positive selection marker and / or the negative counter-selection marker is placed, in the genetic construction, under the control of a constitutive or regulated transcriptional promoter . 5. Method according to claim 1, characterized in that the positive selection marker and the negative counter-selection marker are a single gene encoding a polypeptide having a thymidine kinase activity, preferably a tdk gene. 6. Method according to one of claims 1 to 5 characterized in that the nucleoside analog is AZT. 7. Method according to one of claims 1 to 6, characterized in that the bacterium is a strain of E. coli. 8. Method according to one of claims 1 to 6, characterized in that the bacterium is an Acinetobacter strain, preferably Acinetobacter calcoaceticus (ADP 1). 9. Method according to one of claims 1 to 8, characterized in that the genetic construction is a circular DNA, preferably a plasmid. 10. Method according to one of claims 1 to 8, characterized in that the genetic construction is a linear DNA. 11. Method according to any one of claims 2 to 10, characterized in that, in step c), the antibiotic compound is present in the culture medium. 12. Method according to one of claims 2 to 10, characterized in that, in step c), the antibiotic compound is absent from the culture medium. 13. Method according to any one of the preceding claims, for specifically altering the genome of a bacterium by allelic exchange. 14. Method according to claim 13, characterized in that the element (i) of the genetic construction comprises there sequence of an altered copy of at least part of a gene whose alteration is desired. 15. Method according to any one of claims 1 to 12, for specifically altering the genome of a bacterium by gene replacement. 16. Method according to claim 15, characterized in that, in the genetic construction, the positive selection marker (ii) is surrounded by two regions of homologies (i) each having a sequence homologous to a distinct segment of the gene for the bacteria whose alteration is desired, said regions each having a length sufficient to allow homologous recombination. 17. Method according to any one of claims 1 to 12, for specifically altering the genome of a bacterium by deletion of all or a functional part of the gene. 18. Method according to claim 17, characterized in that, in the genetic construction, the assembly comprising the positive selection marker (ii) and the negative counter-selection marker (iii) is surrounded by two regions of homologies ( i) each having a sequence homologous to a distinct segment of the gene of the bacterium whose alteration is desired, said regions each having a length sufficient to allow homologous recombination. 19. Method according to any one of the preceding claims, for specifically inactivating a gene in the genome of the bacterium, for example a gene involved in a biosynthesis pathway. 20. A method for specifically inactivating a gene in the genome of a bacteria of the genus Acinetobacter by double homologous recombination, comprising: a) the in vitro introduction into a bacterium of the genus Acinetobacter of a genetic construction comprising (i) the sequence d 'a mutated or altered copy of the gene whose inactivation is desired, (ii) a positive selection marker conferring resistance to an antibiotic compound and (iii) a tdk gene, b) the culture of bacteria in the presence of the antibiotic and the selection of bacteria capable of growth or survival, and c) the culture of bacteria obtained in step b) in the absence of the antibiotic and in the presence of a nucleoside analog metabolized by tdk, and the selection of bacteria capable of growth or survival, said bacteria having a genome modified by inactivation of said gene. 21. Method according to one of claims 1 to 20, for creating new metabolic pathways in bacteria. 22. Method according to one of claims 1 to 20, for analyzing the function of genes or proteins. 23. Method according to one of claims 1 to 20, for screening libraries of nucleic acids. 24. Product comprising: - a genetic construct comprising (i) a region having a sequence homologous to a selected segment of the genome of a bacterium whose alteration is desired, said region having a length sufficient to allow homologous recombination, (ii ) a positive selection marker conferring resistance to an antibiotic compound and (iii) a negative counter-selection marker comprising a tdk gene; and - a nucleoside analog. 25. Recombinant genetic construction, consisting of (i) a region having a sequence homologous to a selected segment of the genome of a bacterium whose alteration is desired, said region having a length sufficient to allow homologous recombination, (ii) a positive selection marker conferring resistance to an antibiotic compound and (iii) a negative counter-selection marker comprising a tdk gene.