AU7526500A - Method for genetic modification of lactobacillus delbrueckii - Google Patents

Description

WO 01/21819 PCT/FROO/02565 METHOD FOR GENETIC MODIFICATION OF LACTOBACILLUS DELBRUECKII The present invention relates to the genetic 5 modification of Lactobacillus delbrueckii. Lactic acid bacteria are used a great deal in the agrofoods industry, particularly for manufacturing diverse fermented products; in addition, their 10 innocuity has made their use recommended for the production, by genetic engineering, of diverse substances intended in particular for therapeutic use. The genetic modification of lactic acid bacteria makes 15 it possible to adjust their characteristics depending on the use envisaged, whether by the introduction and expression of foreign DNA, or by the overexpression or, on the contrary, the inactivation of genes naturally present in said bacteria. For a modification to be 20 stable, it must be integrated into the bacterial chromosome. With this aim, the desired modification is inserted into a nonreplicative plasmid carrying a selection marker. The vector thus obtained is introduced into the bacteria; the bacteria expressing 25 the selection marker, which are those which have integrated the vector into their chromosome, are then recovered. The modification results from 2 events which occur at 30 low frequency: 1) the introduction of the plasmid into the bacteria; 2) the integration of said plasmid into the chromosome. The modification rate which may be anticipated is the product of the probabilities of these two events; it is therefore very low, especially 35 in the case of bacteria belonging to species which are "refractory" to transformation, which is the case for many species of lactic acid bacteria. Among these, mention will be made in particular of the species - 2 Lactobacillus delbrueckii, which in particular comprises the subspecies Lb. delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. lactis, and Lb. delbrueckii subsp. delbrueckii. 5 A single example of a vector which allows integration into the chromosome of Lb. delbrueckii is described in the prior art; it is the conjugative plasmid pAM1, described as being nonreplicative in Lb delbrueckii. 10 Application EP 0603416 in the name of MEIJI MILK PRODUCTS CO LTD reports the use of this plasmid to integrate a modification into the chromosome of Lb. delbrueckii: a selection marker (erythromycin resistance) was inserted into a fragment homologous to 15 a region of the chromosome of Lb. delbrueckii, and the entire combination was introduced into the pAM01 plasmid. The integrative plasmid thus constructed was amplified in Lactococcus lactis, and then transferred by conjugation into Lb. delbrueckii; the trans 20 conjugants/integrants are then selected on the basis of the erythromycin resistance resulting from the integration, by homologous recombination, of the insert from the plasmid into the chromosomal DNA. 25 Since these modifications are obtained at very low frequency, it is desirable to have other integrative vectors which are suitable for Lb. delbrueckii and facilitate the production of the modified strains and the selection of the integrants. 30 In order to increase the modification rate and facilitate the screening of the modified bacteria, the use of conditional plasmids as vectors has been proposed. The term "conditional" is used herein to 35 describe a vector, for example a plasmid, for which at least one of the functions of replication, partition or stability is active only under certain conditions (for example of temperature, of pH, of inorganic salt concentration, of presence in the culture medium of a - 3 particular compound, etc.), either due to the fact that one or more of the proteins involved in this function comprise(s) one or more mutation(s) making the activity conditional, or due to the fact that one or more of the 5 proteins involved in this function is (are) placed under the control of an inducible promoter. BISWAS et al., J. Bacteriol., 175, 3628-3635, (1993); MAGUIN et al., J. Bacteriol., 178, 931-935, (1996) and 10 PCT application WO 93/18164 thus describe integrative vectors which comprise the pVE6002 mutant replicon replication system (CNCM 1-1179), also named pG t host, which is a thermosensitive (Ts) derivative of the plasmid pWVOl due to a mutation in the sequence 15 encoding the RepA protein. The modification may thus be carried out in two steps: in the first, the plasmid is introduced into the bacteria, at permissive temperature, which allows a 20 first selection of those in which it has installed itself; in the second, these bacteria are cultured at a temperature which is nonpermissive for the replication of the plasmid, which makes it possible to select those in which the sequences introduced are integrated into 25 the chromosome. PCT application WO 93/18164 describes the use of these plasmids in diverse species of lactic acid bacteria, including Lb. delbrueckii subsp. bulgaricus. 30 In pursuing their investigations, the inventors have presently noted that other plasmids used in lactic acid bacteria are capable of replicating in Lb. delbrueckii at temperatures possibly reaching approximately 37 0 C, 35 which allow the growth of this species, but are thermosensitive at higher temperatures, generally from 420C, which is the optimum temperature for growth of this species.

- 4 They are in particular derivatives of the plasmid pIP501; it has been shown that this plasmid may be transferred by conjugation into Lb. delbrueckii subsp. bulgaricus and replicate therein [LANGELLA and CHOPIN, 5 FEMS Microbiol. Lett., 60, 149-152, (1989)]. It has been described as thermosensitive in B. subtilis and several lactobacilli, including I,. helveticus [BHOWMICK and STEELE, J. Gen. Microbiol., 139, 1433-1439, (1993)] L. plantarum [RIXON et al., FEMS Microbiol. Lett., 71, 10 105-110, (1990)] and L. acidophilus [LUCHANSKI et al., Mol. Microbiol., 2, 637-646, (1988)], but not in Lb. delbrueckii. The inventors have established that pIP501 derivatives are thermosensitive in Lb. delbrueckii; they can in fact replicate and maintain themselves at 15 approximately 35-370C in this bacterial species, but their replication and/or their maintenance in stable form are inhibited from approximately 420C. These pIP501 derivatives have been used by the 20 inventors to transform Lb. delbrueckii and have made it possible, at nonpermissive temperature, to obtain integrants. Consequently, a subject of the present invention is the 25 use of a plasmid comprising the pIP501 theta replication system, or a related replication system, as a thermosensitive conditional vector for integration which makes it possible to introduce a modification of the genetic information of a bacterium of the species 30 Lb. delbrueckii. This modification is introduced into the chromosome via one or more transposition and/or homologous recombination events. 35 The expression "replication system related to the pIP501 theta replication system" defines herein any theta replication system which has the functional characteristics, and in particular the - 5 thermosensitivity in Lb. delbrueckii, of pIP501. This encompasses in particular any theta replication system comprising: - an origin of replication, ori, having at least 80% 5 identity, preferably at least 85% identity and advantageously at least 90% identity, at the level of the nucleotide sequence, with the origin of replication, ori, of pIP501 and/or; - a sequence encoding a rep protein, the peptide 10 sequence of which has at least 85% identity, preferably at least 90% identity, and advantageously at least 95% identity, with that of the repR protein of pIP501. 15 The sequence of the origin of replication, ori, of pIP501 is in particular described by BRANTL and BEHNKE [Molecular Microbiology, 6, 3501-3510 (1992)]; the sequence encoding the repR of pIP501 is in particular identified in the complete sequence of pGB3631 (derived 20 from pIP501), published by BRANTL et al., [Gene, 142, 155-156, (1994)], and accessible under the number X72021. The percentage identity of a sequence with a reference 25 sequence is defined herein as the percentage of residues of this sequence which are identical to those of the reference sequence over an alignment of the 2 sequences which ensures that the positions of the residues correspond to a maximum. 30 A subject of the present invention is in particular a process for modifying the genetic information carried by the chromosome of a bacterium of the species Lb. delbrueckii, characterized in that it comprises: 35 a) constructing an integrative plasmid by inserting at least one DNA sequence, capable of integrating into the bacterial chromosome, into a conditional vector comprising the pIP501 theta replication system, or a related replication system, said - 6 vector also carrying at least one selection marker; b) introducing the plasmid into said bacterium and multiplying this bacterium, under conditions 5 permissive for the replication and maintenance in stable form of said plasmid; c) multiplying the bacteria expressing at least one selection marker from the plasmid at the end of step b), under conditions nonpermissive for the 10 replication and/or maintenance in stable form of the plasmid; and, optionally, d) recovering the bacteria expressing at least one selection marker originating from the plasmid, at the end of step c). 15 According to a variant of the process in accordance with the invention, the step of multiplying the bacterium under conditions permissive for the replication and maintenance of the plasmid in stable 20 form may be omitted; in this case, after introducing the plasmid into the bacterium, the multiplication thereof is carried out directly under conditions nonpermissive for the replication and/or maintenance in stable form of the plasmid. 25 Advantageously, it is possible, after having obtained bacteria carrying the desired modification, to remove the sequences originating from the vector used in step a), in cases in which it is not desirable to keep 30 these sequences, for example in the case of bacteria intended for industrial use, in particular for the preparation of foodstuffs. In this case, the process in accordance with the 35 invention also comprises the following steps: e) excising the sequences originating from the vector, by multiplying the bacteria expressing, at the end of step c), at least one selection marker originating from the plasmid and, advantageously, - 7 f) removing the excised DNA by multiplying the bacteria obtained in step e), under conditions nonpermissive for the replication and maintenance of the vector in stable plasmid form. 5 Conditional vectors comprising the pIP501 theta replication system, or a related system, used in Lb. delbrueckii for carrying out the present invention are thermosensitive plasmids which are capable of 10 replicating and of maintaining themselves at 35-370C in this bacterial species, and the replication and/or maintenance of which in stable form are inhibited from approximately 42 0 C. 15 These plasmids also carry at least one selection marker, i.e. a gene which can be expressed in Lb. delbrueckii and the expression of which confers on the bacteria harboring it a distinctive phenotype which allows them to be selected. It may, for example, be a 20 resistance marker which confers a characteristic of resistance to a substance usually toxic for the bacterium, for example an antibiotic, or an auxotrophy marker which confers the ability to grow in the absence of a nutrient usually essential to the bacterium. The 25 bacteria expressing these markers are, for example, easily selected by virtue of their survival on a selective medium, i.e. in the presence of said toxic substance or in the absence of said nutrient. Thus, at the end of step b) of the process above, the bacteria 30 in which the plasmid is present may be selected on the basis of the expression of a selection marker originating from the plasmid. Similarly, at the end of step c), the bacteria in which the DNA of the plasmid has been integrated into the chromosome may be selected 35 on the basis of the expression of a selection marker originating from the plasmid constructed in step a). Another possibility for selection at the end of step c) consists in using a property of the strain which ensues - 8 from the integration of the plasmid and/or from the excision of the vector sequences. For example, use may be made of a conditional plasmid comprising a selection marker M (for example a resistance marker) and a DNA 5 sequence capable of integrating by homologous recombination into a bacterial gene X, said gene X being essential under certain conditions for the bacterium (for example a gene essential for the growth thereof on a given medium), the integration of the 10 plasmid inactivating the gene X, and the excision of the sequences of the vector restoring its activity. In this case, the bacteria in which the plasmid DNA has been integrated may be selected on the basis of the expression of the selection marker M, under conditions 15 in which the gene X is not essential, and the bacteria in which the sequences of the integrated vector have been excised may be selected, under conditions in which the gene X is essential, on the basis of the restoration of its activity. In addition, this method 20 of selection also makes it possible to select, in a single step, bacteria in which the integration of the plasmid DNA and the excision of the sequences of the vector have taken place; in this case, the bacteria are directly cultured under conditions in which the gene X 25 is essential, and which are also selective for the marker M. The bacteria selected will be those in which the integration of the plasmid and the excision of the sequences of the vector (including the marker M) have restored the activity of the gene X. 30 DNA sequences capable of integrating into the chromosome of a bacterium of the species Lb. delbrueckii in particular comprise: - sequences chosen on the basis of their homology 35 with a portion of the chromosome into which it is desired to introduce a modification, i.e. exhibiting a homology with this portion of the chromosome which is sufficient to be able to recombine with it; - 9 - transposable sequences, in particular transposons or insertion sequences (IS); such sequences may insert into the bacterial chromosome randomly or with a certain 5 specificity. The modifications intended to be integrated into the bacterial chromosome may be introduced by the simple integration of these sequences, this integration 10 possibly, for example, leading to the inactivation of a gene within which it occurs. It is also possible to use these sequences to integrate, into the bacterial chromosome, a DNA fragment, in particular a gene of interest of heterologous origin, or a DNA fragment from 15 Lb. delbrueckii, modified beforehand. In the case of the integration by homologous recombination, for example, it is possible to introduce, into a sequence identical to that of the 20 region of the chromosome intended to be modified, modifications by insertion, deletion or substitution which may range from a single nucleotide to several thousands of nucleotides. The sequence thus modified is inserted in step a) of the process according to the 25 invention, into a vector which is conditional in Lb. delbrueckii, and which comprises the pIP501 theta replication system, or a related system, as defined above. The integration takes place by recombination (simple crossing-over) between the cloned chromosomal 30 DNA fragment and the homologous region of the bacterial chromosome. Since this recombination event creates duplications of the region of homology, on both sides of the vector sequences, the structure integrated into the chromosome consists of the vector sequences 35 bordered on both sides by a copy of the region of homology, as shown in figure 1. The use of sequences which can integrate by homologous recombination in particular makes it possible: - 10 - to inactivate one (or more) bacterial gene(s); - to modify the expression of the genes and/or the activity of the products encoded by these genes; - to introduce, in a stable manner, new functions 5 into the chromosome; - to study and to use the expression of the genes in situ; - to mutate the chromosome by integration via random chromosomal fragments; 10 - to insert, in a stable manner, sequences intended to label the strains. These sequences will subsequently be used as tracers; - to introduce several sequential modifications into the same strain. 15 In the case of the integration via a transposable sequence, the transposable sequence will be inserted into the plasmid; the integration into the chromosome makes it possible to produce the modification, to thus 20 obtain mutants exhibiting advantageous characteristics, and to characterize more easily the mutated gene(s). The structure transposed into the chromosome may consist of the vector sequences bordered on both sides by a copy of the transposable sequence. 25 These transposable sequences may originate from bacteria belonging to the species Lb. delbrueckii, or originate from other bacterial species, in particular other lactic acid bacteria. They may be transposons or 30 insertion sequences. Transposable sequences which are functional in Lb. delbrueckii may be identified by inserting a transposable sequence to be tested (transposon or 35 insertion sequence) into a vector which replicates conditionally in Lb. delbrueckii and which comprises the pIP501 theta replication system, or a related system, as defined above, by carrying out steps b) and c) of the process in accordance with the invention and - 11 searching for the presence of transposants at the end of these steps. The inventors have thus demonstrated 2 insertion 5 sequences which are functional in Lb. delbrueckii. A subject of the present invention is also the use of either of chese sequences for modifying the chromosome of Lb. delbrueckii. 10 They are the insertion sequence named IS1233, previously demonstrated by WALKER et al. (J. Bacteriol., 176, 5330-5340, 1994) in Lb. johnsonii, and the insertion sequence named IS1201, previously demonstrated by TAILLIEZ et al. (Gene, 145, 75-79, 15 1994) in Lb. helveticus. A subject of the present invention is also any integrative plasmid resulting from the insertion of one of these 2 sequences into a vector which replicates 20 conditionally in Lb. delbrueckii, and in particular into a vector comprising the pVE6002 replication system, such as those described in PCT application WO 93/18164, or into a vector comprising the pIP501 theta replication system, or a related system, as 25 defined above. According to a preferred embodiment of the present invention, said vector is a nonconjugative vector. 30 Integrative plasmids in accordance with the invention are in particular illustrated by the pVT49 plasmid and the pVI52 plasmid. The pVI49 plasmid harbored by the E. coli strain V1209, and the pVI52 plasmid harbored by the E. coli strain V1217, were deposited, according to 35 the Treaty of Budapest, on September 17, 1999, under the respective numbers 1-2317 and 1-2318, with the CNCM (Collection Nationale de Cultures de Microorganismes [National Collection of Microorganism Cultures]), 25 rue du Docteur Roux, Paris.

- 12 In the two scenarios: integration by homologous recombination or by transposition of a mobile element, the sequences originating from the vector may be excised, during step e) of the process in accordance 5 with the invention, by recombination between the homologous sequences flanking the vector sequences. For example, in the case of the integration by homologous recombination, a second recombination event 10 (double crossing-over) may take place between the duplicated homologous regions on both sides of the vector sequences. This event leads to the excision of the vector sequences and makes it possible, for a fraction of the clones, to substitute the wild-type 15 chromosomal form with the modified plasmid form, as shown in figure 1. Legend for figure 1: A, B: chromosomal DNA cloned into the vector; 20 A: modification; CR: conditional replicon; M: selection marker; P: permissive conditions; NP: nonpermissive conditions. 25 For example, in the case of the integration using certain transposable sequences, these sequences also constitute, on both sides of the vector sequences, homologous regions which promote recombination events 30 which lead to the excision of the vector sequences and of one of the copies of the IS, the other remaining in the bacterial chromosome at the transposition site. Figure 2 represents the excision of the sequences of a 35 vector by homologous recombination between IS. Legend for figure 2: I: insertion sequence; M: selection marker; - 13 NP: nonpermissive conditions. The bacteria selected at nonpermissive temperature during step c) (>42 0 C) are cultured without any 5 selection pressure so as to allow recombination between the homologous regions flanking the vector sequences. After removal of the excised sequences, in accordance with step f) of the process in accordance with the 10 invention, the following are thus obtained: - in the case of a plasmid comprising a sequence capable of integrating by homologous recombination, a bacterium which differs from the host bacterium of origin by the presence, 15 in its chromosome, of the modification introduced by this sequence; - in the case of a plasmid comprising a transposable sequence, a bacterium which differs from the host bacterium of origin by 20 the presence, in its chromosome, of a copy of said transposable sequence. The present invention will be more fully understood with the aid of the further description which follows, 25 which refers to nonlimiting examples of the production of integrative plasmids and of their use in Lb. delbrueckii. EXAMPLE 1: SELECTION OF PLASMIDS WHICH ARE 30 THERMOSENSITIVE IN Lb. delbrueckii Production of plasmids An L. bulgaricus - E. coli shuttle plasmid consisting of pGB3631 (a sequence derivative of pIP501, (BRANTL et 35 al., Gene, 142, 155-156, (1994)]) and of the pSKII origin of E. coli was constructed. This plasmid, named pVI1055, is represented in figure 3. It carries an ery gene for erythromycin (Ery) resistance.

- 14 Another pIP501 derivative, the plasmid pGB305A [LE CHATELIER et al., Plasmid, 29, 50-61, (1995))] was also tested. 5 Demonstration of the thermosensitivity in Lb. delbrueckii Transformation of bacteria 10 Bacteria of the Lb. delbrueckii subsp. bulgaricus strain ATCC 11842 or of the strain VI104 deposited according to the Treaty of Budapest, on September 17, 1999, under the number 1-2316, with the CNCM (Collection Nationale de Cultures de Microorganismes 15 [National Collection of Microorganism Cultures]), 25 rue du Docteur Roux, Paris, are cultured in MRS medium containing 0.1% glycine (DIFCO), until the beginning of the stationary phase. They are then centrifuged and washed in electroporation buffer (0.4 M 20 sucrose, 1 mM MgCl 2 , 5 mM KH 2

PO

4 , pH 6.0) and suspended in this same buffer at a concentration corresponding to an OD 6 00 of approximately 50. The suspension is incubated for 20 minutes at 45 0 C and then cooled on ice. An 80 g1 aliquot of the suspension is mixed with 25 the plasmid DNA (-1.5 jtg) and the mixture is transferred into a 0.2 cm electroporation cuvette. The electroporation is performed at 1 kV, 800 Q, and 25 RF. Immediately after electroporation, the mixture is 30 diluted in 2 ml of expression medium (0.2 M sucrose, 5% powdered skimmed milk, 0.1% yeast extract, 1% casamino acids, 25 iM MgC1 2 ) . After incubation for 3 h at 37 0 C in this medium, the cells are plated out onto dishes containing selective medium (MRS agar with 10 pg/ml of 35 erythromycin); the dishes are incubated in anaerobic jar at 37 0 C for 48 h, and the erythromycin-resistant colonies are selected.

- 15 Thermosensitivity of pIP501 derivatives (theta replication) The thermosensitivity of pGB305A and of pVI1055 was 5 first evaluated using Lb. delbrueckii (ATCC 11842 or VI104) transformation assays according to the protocol described above, with plating out onto selective medium at 37 0 C and 420C. For both plasmids, transformants are obtained at 370C but not at 42 0 C. 10 The thermosensitivity of pVI1055 was then confirmed by comparing its stability at 37 0 C and at 440C. A culture in MRS/Ery (10 tg/ml) at 37 0 C was diluted in MRS without Ery, and then incubated in parallel at 370C and 15 at 440C. Samples are taken at various times and the cells are plated out after dilution on MRS and MRS/Ery, in order to determine the proportion of cells having lost the plasmid. 20 At 37 0 C, approximately 30% of the cells have lost the pVIl055 plasmid after 48 hours. At 44 0 C, the stability of pVI1055 is clearly affected, since ~100% of the cells have lost the plasmid after 48 hours. 25 EXAMPLE 2: DEMONSTRATION OF THE TRANSPOSITION ACTIVITY OF INSERTION SEQUENCES (IS) IN Lb. delbrueckii Insertion sequences isolated from lactic acid bacteria were cloned into pVI1055. At a temperature permissive 30 for the replication of the plasmid, the multiplication of a transformant generates a bacterial population containing the plasmid. The IS present on the plasmid in the bacterial population may transpose into the chromosome. In the event of transposition of the IS, 35 the transposed structure present in the chromosome may correspond to the plasmid vector bordered, on both sides, by a copy of the IS, as shown in figure 4. At nonpermissive temperature (440C), the plasmids containing the IS lead to the production of EryR clones - 16 with a frequency greater than that observed for pVI1055, the plasmid without IS. In the case of the production of Ery clones, structural analysis of the chromosomal DNA by Southern Blot with a probe 5 corresponding to the IS tested makes it possible to verify that the integration indeed results from a transposition event. Demonstration of the transposition: 10 The IS 1223 (WALKER and KLAENHAMMER, 1994, abovementioned reference) and 1201 (TAILLEZ et al., 1994, abovementioned reference) were cloned into the pVI1055 plasmid, generating, respectively, the plasmids pVI48, pVI49 (corresponding to the 2 orientations of 15 IS1223) and pVI52. The properties of these 2 IS and the nature of the plasmids obtained are indicated in table 1 below. TABLE 1 IS Origin Sizea (bp) Final vectorsb 1223 L. johnsonii 1502 pVI48/pVI49 1201 L. helveticus 1387 pV152 20 a: size corresponding to the IS bordered by the direct repeats (DR) b: when two plasmids are given, this indicates that the IS was cloned in both orientations 25 In order to search for the transposition activity, the strains containing the plasmids carrying the IS to be tested were cultured at 37 0 C in MRS/Ery (10 Ig/ml). The cells are then diluted in MRS and incubated at 44 0 C. Samples were taken at various times and plated out on 30 MRS dishes (to determine the viable cell count) and on MRS/Ery (EryR cell count). Results: IS1223 (pVI48/pVI49): the pVI48 or pVI49 plasmid was 35 introduced into VI104. In the presence of IS1223, the frequency of EryR cells is higher than with pVI1055 alone. After digestion, the chromosomal DNA of the EryR - 17 clones obtained are hybridized with IS1223. In all cases, the EryR clones are the result of transposition events. 5 IS1201 (pVI52): the pVI52 plasmid was introduced into VI104. The frequency of integration of pVI52 is higher than that of pVI1055 alone. All the Eryf clones analyzed by Southern are the result of transposition. 10 These results illustrate the transposition activity of IS 1223 and 1201 in Lb. delbrueckii.

Claims (10)

1. The use of a plasmid comprising the pIP501 theta replication system, or a related replication 5 system, as a thermosensitive conditional vector for integration which makes it possible to introduce a modification of the genetic information of a bacterium of the species Lb. delbrueckii. 10

2. A process for modifying the genetic information carried by the chromosome of a bacterium of the species Lb. delbrueckii, characterized in that it comprises: 15 a) constructing an integrative plasmid by inserting at least one DNA sequence, capable of integrating into the bacterial chromosome, into a conditional vector comprising the pIP501 theta replication system, or a related 20 replication system, said integrative plasmid also carrying at least one selection marker; b) introducing the plasmid into said bacterium and multiplying this bacterium, under conditions permissive for the replication and maintenance 25 in stable form of said plasmid; c) multiplying the bacteria expressing at least one selection marker from the plasmid at the end of step b), under conditions nonpermissive for the replication and/or maintenance in 30 stable form of said plasmid; and, optionally, d) recovering the bacteria expressing at least one selection marker originating from the plasmid, at the end of step c). 35

3. The process as claimed in claim 2, characterized in that it also comprises: e) excising the sequences originating from the vector, by multiplying the bacteria expressing, - 19 at the end of step c), at least one selection marker originating from the plasmid and, advantageously, f) removing the excised DNA by multiplying the 5 bacteria obtained in step e), under conditions nonpermissive for the replication and maintenance of said vector in stable plasmid form. 10

4. The process as claimed in either of claims 2 and 3, characterized in that the DNA sequence capable of inserting into the bacterial chromosome is a sequence homologous to a portion of the chromosome into which it is desired to introduce a 15 modification.

5. The process as claimed in either of claims 2 and 3, characterized in that the DNA sequence capable of inserting into the bacterial chromosome 20 is a transposable sequence.

6. The process as claimed in claim 5, characterized in that said transposable sequence is an insertion sequence. 25

7. The process as claimed in claim 6, characterized in that said insertion sequence is chosen from IS1223 of Lb. johnsonii and 11201 of Lb. helveticus. 30

8. The use of an insertion sequence chosen from 151222 from Lb. johnsonii and IS1201 of Lb. helveticus, for modifying the chromosome of Lb. delbrueckii. 35

9. An integrative plasmid for carrying out a process as claimed in any one of claims 1 to 6, characterized in that it results from the insertion of one of the sequences IS1223 or IS1201 - 20 into a plasmid chosen from: - the plasmids comprising the pVE6002 replication system; - the plasmids comprising the pIP501 replicon 5 theta replication system, or a related replication system.

10. The integrative plasmid as claimed in claim 8, chosen from the group consisting of the pVI49 10 plasmid and the pVI52 plasmid, deposited with the CNCM on September 17, 1999, under the respective numbers 1-2317 and 1-2318.