CA2385072A1 - Method for genetic modification of lactobacillus delbrueckii - Google Patents

Abstract

The invention concerns a method for modifying the chromosomal genetic information of Lactobacillus delbrueckii, using a conditional integrator plasmid. The invention also concerns integrator plasmids for use in implementing said method.

Description

GENETIC MODIFICATION PROCESS OF LACTOBACILLUS
delbrueckii The present invention relates to the genetic modification of Lactobacillus delbrueckii.
Lactic acid bacteria are widely used in food industry, particularly for manufacture of various fermented products; furthermore their safety did advocate their use for the production by genetic engineering of various substances intended in particular for therapeutic use.
Genetic modification of bacteria lactic allows to adapt their characteristics according to the intended use, whether by the introduction and expression of foreign DNA, or by overexpression or on the contrary the inactivation of genes naturally present in said bacteria. So that a modification is stable, it must be integrated into the bacterial chromosome. For this purpose, the modification desired is inserted into a non-replicating plasmid carrying a selection marker. The vector thus obtained is introduced into bacteria; we then recover bacteria expressing the selection marker, which are those who have integrated the vector into their chromosome.
The modification results from 2 events of low frequency. 1) the introduction of the plasmid into the bacteria; 2) integration of said plasmid into the chromosome. The rate of change that can be expected is the product of the probabilities of these two events;
it is therefore very low, especially in the case of bacteria belonging to species called "refractory" to the transformation, which is the case for many species lactic acid bacteria. Among these we will mention in especially the species Lactobacillus delbrueckii, which includes in particular the Lb subspecies. delbrueckii WO 01/21819

2 PCT / FR00 / 02565 subsp. bulgaricus, Lb. delbrueckii subsp. lattis, and Lb.
delbrueckii subsp. delbrueckii.
A single example of a vector allowing integration into the Lb chromosome. delbrueckii is described in the prior art. it is the plasmid conjugative pAM (31, described as non-replicative in Lb.
delbrueckii. Request EP 0603416 on behalf of MEIJI MILK
PRODUCTS CO LTD reports the use of this plasmid to integrate a modification in the chromosome of Lb. delbrueckii. a selection marker (resistance to erythromycin), was inserted into a homologous fragment to a region of the Lb chromosome. delbrueckii, and the whole was introduced into the plasmid pAM (31.
integrative plasmid thus constructed has been multiplied in Lactococcus lattis, then transferred by conjugation to Lb. delbrueckii; transconjugants / integrants are then selected based on resistance to erythromycin resulting from integration, by homologous recombination of the insert of the plasmid in chromosomal DNA.
These modifications being obtained at very low frequency it is desirable to have other integrative vectors adapted to Lb. delbrueckii and facilitating the obtaining of the modified strains and the selection of integrants.
To increase the change rate and facilitate the screening of modified bacteria it has been proposed to use plasmids as vectors Contingent. We qualify here as "conditional" a vector, for example a plasmid, at least one of the replication, partition, or stability functions is only active under certain conditions (for example temperature, pH, mineral salt concentration, presence in the culture medium of a compound particular, etc.) or because one or more of proteins involved in this function include) WO 01/21819

3 PCT / FR00 / 02565 one or more mutations) making the activity conditional, either because one or more of the proteins involved in this function are placed under control of an inducible promoter.
BISWAS et al., J. Bacteriol., 175, 3628-3635, (1993); MAGUIN et al., J. Bacteriol., 178, 931-935, (1996) and PCT Application WO 93/18164 thus describe integrative vectors which include the system of replication of the mutant replicon pVE6002 (CNCM I-1179) also called pG + host, thermosensitive derivative (Ts) of plasmid pWV01, due to a mutation in the sequence encoding the RepA protein.
The modification can thus be carried out in two step . in the first, the plasmid is introduced at permissive temperature in bacteria, which allows a first selection of those in which he established himself; in the second, these bacteria are grown at non-permissive temperature for replication of the plasmid, which allows selection those in which the introduced sequences are integrated into the chromosome.
PCT Application WO 93/18164 describes the use of these plasmids in various species of lactic acid bacteria, including Lb. delbruckii subsp.
bulgaricus.
By continuing their research, the The inventors have now found that other plasmids used in lactic acid bacteria were able to replicate in Lb. delbrueckii at temperatures up to about 37 ° C, which allow the growth of this species but were temperature sensitive at higher temperatures, usually from 42 ° C, optimal temperature for growth of this species.
These include derivatives of the plasmid pIP501; it has been shown that this plasmid can be transferred by conjugation to Lb. delbrueckii subsp.

WO 01/21819

4 PCT / FR00 / 02565 bulgaricus and replicate there [LANGELLA and CHOPIN, FEMS
Microbiol. Lett., 60, 149-152, (1989). It has been described as thermosensitive in B. subtilis and several lactobacilli including L. helveticus [BHOWMICK and STEELE, J.
Gen. Microbiol., 139, 1433-1439, (1993)], L. plantarum [RIXON et al., FEMS Microbiol. Lett., 71, 105-110, (1990)] and L. acidophilus [LUCHANSKI et al., Mol.
Microbiol., 2, 637-646, (1988)] but not in Lb.
delbrueckii. The inventors have established derivatives of pIP501 are thermosensitive at Lb. delbrueckii; they can indeed replicate and be maintained at 35-37 ° C
about in this bacterial species but their replication and / or their maintenance in stable form are inhibited from around 42 ° C.
These pIP501 derivatives have been used by Inventors to transform Lb. delbrueckii and allowed at non-permissive temperature, to obtain integrants.
The present invention therefore has for object the use of a plasmid comprising the system pIP501 theta replication system or related replication, as a conditional vector, thermosensitive, integration allowing the introduction of a modification of the genetic information of a bacteria the Lb species. delbrueckii.
This change is introduced in the chromosome via one or more events of homologous transposition and / or recombination.
We define here as "replication system related to the pIP501 theta replication system "
any theta replication system with the functional characteristics, and in particular the thermosensitivity in Lb delbrueckii, of pIP501. This includes in particular any theta replication system understanding.
- an origin of ori replication having at less 80% identity, preferably at least 85%

WO 01/21819

5 PCT / FR00 / 02565 identity, and advantageously at least 90% identity, at the nucleotide sequence level, with the origin of ori replication of pIP501 and / or;
- a sequence encoding a rep protein whose peptide sequence has at least 85% identity, preferably at least 90% identity, and preferably at minus 95% identity, with that of the folded protein pIPS0l.
The sequence of the origin of replication ori of pIP501 is described in particular by BRANTL and BEHNKE
[Molecular Microbiology, 6, 3501-3510 (1992)]; the sequence coding for the folded protein of pIP501 is identified especially in the complete sequence of pGB3631 (derived from pIP501), published by BRANTL et al., [Gene, 142, 155-156, (1994)], and available under number X72021.
The percentage identity of a sequence with a reference sequence is defined here as the percentage of residues in this sequence that are identical to those of the reference sequence on a alignment of the 2 sequences ensuring a match maximum between residue positions.
The present invention has in particular for object a process to modify genetic information carried by the chromosome of a bacteria of the species Lb delbrueckii, characterized in that it includes.
a) the construction of an integrative plasmid, by insertion at least one DNA sequence capable of integrating in the bacterial chromosome, in a vector conditional including the theta replication system of pIP501 or a related replication system, said vector additionally carrying at least one marker of selection;
b) the introduction of the plasmid into said bacteria and the multiplication thereof, in permissive conditions for replication and maintenance in stable form said plasmid;

WO 01/21819

6 PCT / FR00 / 02565 c) the multiplication of bacteria expressing at least one plasmid selection marker after step b), in non-permissive conditions for replication and / or maintaining the plasmid in stable form; and optionally, d) recovery of bacteria expressing at least one selection marker from the plasmid, upon completion from step c).
According to a variant of the process in accordance with the invention, we can omit the step of multiplying the bacteria in permissive conditions for replication and maintaining the plasmid in stable form; in this case, after the introduction of the plasmid into the bacteria, we multiply it directly by non-permissive conditions for replication and / or maintenance in stable form of the plasmid.
Advantageously, after obtaining bacteria with the desired modification, eliminate sequences from the vector used in step a), in cases where it is not desirable to keep these sequences, for example in the case of bacteria intended for industrial use, in particular for food preparation.
In this case the process according to the invention further includes the following steps.
e) excision of the sequences originating from the vector, by multiplication of bacteria expressing, after step c), at least one selection marker from of the plasmid, and advantageously, f) elimination of the excised DNA, by multiplication of the bacteria obtained in step e) under non-permissive for replication and maintenance of vector in stable plasmid form.
Conditional vectors including the pIP501 theta replication system, or a system related, used at Lb. delbrueckii for setting WO 01/21819

7 PCT / FR00 / 02565 of the present invention are plasmids thermosensitive capable of replicating and maintain at 35-37 ° C in this bacterial species, and including replication and / or maintenance in stable form are inhibited from approximately 42 ° C.
These plasmids also carry at least one selection marker, namely a gene capable of express yourself at Lb. delbrueckii and whose expression gives bacteria harboring a phenotype distinctive allowing their selection. It can be by example of a resistance marker, giving a resistance character to a substance usually toxic to bacteria, for example an antibiotic, or well of an auxotrophy marker conferring the ability to grow in the absence of a nutrient usually essential for bacteria. The bacteria expressing these markers are for example easily selected by their survival on a selective medium, namely in the presence of said toxic substance or in the absence of said nutrient.
Thus, at the end of step b) of the above process, the bacteria in which the plasmid is present can be selected based on the expression of a selection marker from the plasmid. Similarly, at after step c), the bacteria in which the plasmid DNA has been integrated into the chromosome can be selected based on the expression of a selection marker from the plasmid constructed at step a).
Another possibility of selection at the end of the stage c) consists in using a property of the strain which arises from the integration of the plasmid and / or excision vector sequences. For example, we can use a conditional plasmid comprising a marker for selection M (for example a resistance marker), and a DNA sequence capable of integrating by homologous recombination in a bacterial gene X, said WO 01/21819 g PCT / FR00 / 02565 gene X being essential under certain conditions for bacteria (for example a gene essential for its growth in a given environment), the integration of plasmid inactivating the X gene, and excision of vector sequences restoring its activity. In this case the bacteria in which the plasmid DNA has been integrated can be selected based on the expression of the selection marker M, under conditions in which the X gene is not essential, and bacteria in which the vector sequences integrated were excised can be selected, in conditions under which the X gene is essential, based on the restoration of its activity. This mode also allows you to select a single stage of bacteria in which integration of plasmid DNA and excision of vector sequences have occurred; in this case the bacteria are grown directly in conditions in which the X gene is essential, and which are also selective for marker M. Bacteria selected will be those in which integration of the plasmid and the excision of the vector sequences (y including marker M) restored the activity of the X gene.
DNA sequences capable of integrating in the chromosome of a bacteria of the species Lb. delbrueckii include.
- sequences chosen on the basis of their homology with a portion of the chromosome where we wish to introduce a modification, ie presenting a homology sufficient with this portion of the chromosome to be able to recombine with it;
transposable sequences, in particular transpose or insert sequences (IS); such sequences can insert at random or with a certain specificity in the bacterial chromosome.
The modifications that we want to integrate in the bacterial chromosome can be made by the simple integration of these sequences, which can for example lead to the inactivation of a gene at inside of which it takes place. he is also possible to use these sequences to integrate a DNA fragment, in particular an original gene of interest heterologous, or a fragment of Lb DNA. delbrueckii previously modified, in the bacterial chromosome.
In the case of integration by recombination counterpart for example we can bring inside of a sequence identical to that of the region of chromosome that we want to modify, modifications by insertion, deletion, or substitution, which may range from a single nucleotide to several thousand nucleotides.
The sequence thus modified is inserted in step a) of the method according to the invention, in a conditional vector at Lb. delbrueckii, including the system of theta replication of pIP501 or a related system, such as as defined above. Integration takes place by recombination (simple crossing-over) between the fragment of cloned chromosomal DNA and the homologous region of the bacterial chromosome. This recombination event creating duplicates of the region of homology, from and other of the vector sequences, the structure integrated into the chromosome is made up of the sequences of the vector framed on both sides by a copy of the region of homology, as shown in Figure 1.
The use of sequences that can be integrated by homologous recombination in particular allows.
- to deactivate one (or more) genes) bacteria);
- to modify the expression of genes and / or the activity of the products encoded by these genes;

- to introduce, in a stable way, new functions in the chromosome;
- to study and use the expression of genes in if you;
- to mutate the chromosome by integration via random chromosomal fragments;
- to insert, in a stable way, sequences intended to mark the strains. These sequences will be used subsequently tracers;
- introduce several modifications sequential in the same strain.
In the case of integration by through a transposable sequence, the sequence transposable will be inserted into the plasmid;
integration into the chromosome enables the modification, thereby obtaining mutants exhibiting interesting characters and characterize more easily the mutated gene (s). The structure transposed into the chromosome, can be made up of the sequences of vector framed on both sides by a copy of the transposable sequence.
These transposable sequences can come from of bacteria belonging to the Lb species. delbrueckii, or come from other bacterial species, especially other lactic acid bacteria. It can be transpose or insert sequences.
Functional transposable sequences at Lb. delbrueckii can be identified by inserting a transposable sequence to be tested (transposon or insertion sequence), in a replicating vector conditional at Lb. delbrueckii including the system thIP replication of pIP501 or a related system, as defined above, implementing the steps b) and c) of the process according to the invention, and looking for the presence of transposants at the end of these steps.

The inventors have thus highlighted 2 functional insertion sequences in Lb.
delbrueckii. The present invention also relates to the use of either of these sequences for modify the Lb chromosome. delbrueckii.
It is the insertion sequence called IS1223 previously highlighted by WALKER et al.
(J. Bacteriol., 176, 5330-5340, 1994) at Lb. johnsonii, and the insertion sequence called IS1201, previously highlighted by TAILLIEZ et al. (Uncomfortable, 145, 75-79, 1994) at Lb. helveticus.
The present invention also relates to any integrative plasmid resulting from the insertion of one of these 2 sequences in a replicating vector conditional at Lb. delbrueckii, and especially in a vector comprising the replication system of pVE6002, such as those described in PCT Application WO 93/18164 or in a vector comprising the replication system theta of pIP501 or a related system, as defined above.
According to a preferred embodiment of the present invention, said vector is a non-vector conjugative.
Integrative plasmids conforming to the invention are notably illustrated by the plasmid pVI49 and the plasmid pVI52. The plasmid pVI49 hosted by E. coli strain VI209, and the plasmid pVI52, hosted by strain VI217 of E. coli, were deposited according to the Budapest Treaty on September 17, 1999, under numbers I-2317 and I-2318, respectively, with the CNCM
(National Collection of Cultures of Microorganisms), 25 rue du Docteur Roux, in Paris.
In both cases. integration by homologous recombination or by transposition of an element mobile, excision of sequences from the vector can be done during step e) of the process WO 01/21819 cA o23a5o ~ 2 2o ï203-14 pCT ~ R00 / 02565 according to the invention, by recombination between the homologous sequences flanking the vector sequences.
For example, in the case of integration by homologous recombination, a second event of recombination (double crossing-over) can take place between the peer regions duplicated on the one hand and other vector sequences. This event leads to excision of the vector sequences, and allows, for a fraction of the clones, to substitute the wild form chromosomal by the modified plasmid form like the shows Figure 1.
Figure 1 legend.
A, B: chromosomal DNA cloned into the vector;
1. modification;
RC. conditional replicon;
M. selection marker;
P. permissive conditions NP. non-permissive conditions.
For example, in the case of integration by certain transposable sequences, these constitute also on either side of the vector sequences, homologous regions favorable to events of recombination which lead to the excision of the sequences the vector, and one copy of the SI, the other remaining in the bacterial chromosome at the site of transposition.
Figure 2 shows the excision of sequences of a vector by homologous recombination between IS.
Figure 2 legend.
I. insertion sequence;
M. selection marker;
NP. non-permissive conditions.
The bacteria selected at temperature not permissive during step c) (> 42 ° C), are cultivated without selection pressure to allow the recombination between the homologous regions flanking the vector sequences.
After elimination of the excised sequences, in accordance with step f) of the process in accordance with the invention, one thus obtains.
- in the case of a plasmid comprising a sequence capable of integrating by homologous recombination, a bacterium differing from the original host bacteria by the presence, in its chromosome, of modification made by this sequence;
- in the case of a plasmid comprising a transposable sequence, a bacterium differing from the original host bacteria by the presence, in its chromosome, of a copy of said transposable sequence.
The present invention will be better understood from using the additional description which follows, which is refers to non-limiting examples of obtaining integrative plasmids and their use che z Lb.
delbrueckii.
EXAMPLE 1. SELECTION OF THERMOSENSITIVE PLASMIDS AT
Lb. delbrueckii Obtaining plasmids A L. bulgaricus - E. coli shuttle plasmid consisting of pGB3631 (a sequenced derivative of pIP501, [BRANTL et al., Gene, 142, 155-156, (1994)]) and the origin pSKII of E. coli was built. This plasmid, called pVI1055, is shown in Figure 3. I1 door an erythromycin (Ery) resistance gene.
Another derivative of pIP501, the plasmid pGB3050 [LE CHATELIER et al., Plasmid, 29, 50-61, (1995)] a also been tested.

Demonstration of thermosensitivity in Lb. delbrueckii Transformation of bacteria Bacteria of the ATCC 11842 strain from Lb.
delbrueckii subsp. bulgaricus or strain VI104 deposited under the Budapest Treaty on September 17, 1999, under the number I-2316, at the CNCM (Collection National Culture of Microorganisms), 25 rue du Doctor Roux, in Paris, are cultivated in MRS medium, 0.1 ~
glycine (DIFCO) until the start of the stationary phase.
They are then centrifuged and washed in buffer electroporation (0.4 M sucrose, 1 mM MgCl2, 5 mM KH2P04, pH 6.0) and suspended in this same buffer at a concentration corresponding to a D06oo of approximately 50. The suspension is incubated for 20 min at 45 ° C. and then cooled on ice. An aliquot of 80 ~ 1 of the suspension is mixed with plasmid DNA (--1.5 ~ g) and the mixture is transferred to a 0.2 cm electroporation tank.
The electroporation is carried out at 1 kV, 800 S2, and 25 ~ F.
Immediately after electroporation, the mixture is diluted in 2 ml of expression medium (0.2 M
sucrose, 5% skimmed milk powder, 0.1 ~ extract of yeast, 1% casamino acids, 25 mM MgCl2). After incubation for 3 h at 37 ° C. in this medium, the cells are spread on boxes of selective medium (MRS Agar with 10 ~, g / ml erythromycin); the dishes are incubated in anaerobic jar at 37 ° C for 48 h, and the colonies resistant to erythromycin are selected.
Thermosensitivity of pIP501 derivatives (replication theta The thermosensitivity of pGB3050 and pVI1055 was first assessed by transformation trials of Lb. delbrueckii, (ATCC 11842 or VI104) according to the protocol described above, with spreading on selective medium at 37 ° C and 42 ° C. For the 2 plasmids, transformants at 37 ° C but not at 42 ° C.
The thermosensitivity of pVI1055 was then confirmed by comparison of its stability at 37 ° C and 44 ° C. A culture in MRS / Ery (10 ~ .g / ml) at 37 ° C was diluted in MRS without Ery then incubated in parallel at 37 ° C
and at 44 ° C. Samples are taken at different time, and the cells are spread out after dilution over MRS and MRS / Ery, to determine the proportion of cells that have lost the plasmid.
At 37 ° C, approximately 30% of the cells have lost plasmid pVI1055 after 48 hours. At 44 ° C, the stability of pVI1055 is clearly affected since -100% of cells lost the plasmid after 48 hours.
EXAMPLE 2. HIGHLIGHTING THE ACTIVITY OF
TRANSPOSITION OF INSERTION SEQUENCES (IS) AT Lb.
delbrueckii.
Isolated insertion sequences of bacteria lactics were cloned into pVI1055. At temperature permissive for replication of the plasmid, the multiplication of a transformant generates a population bacterial containing the plasmid. The SI presents on the plasmid in the bacterial population can transpose in the chromosome. In case of transposition of the SI, the transposed structure present in the chromosome, can correspond to the plasmid vector, framed on both sides and other by a copy of the SI, as shown in Figure 4. At non-permissive temperature (44 ° C), the plasmids containing the IS leads to obtaining EryR clones with a frequency higher than that observed for pVI1055, the plasmid without IS. In the case of obtaining EryR clones, structural analysis of chromosomal DNA
by Southern Blot with a probe corresponding to the SI
tested, verifies that integration results of a transposition event.

Highlighting the transposition IS 1223 (WALKER and KLAENHAMMER, 1994, reference cited above), and 1201 (TAILLEZ et al., 1994, above reference) were cloned into the plasmid pVI1055, respectively generating the plasmids pVI48, pVI49 (corresponding to the 2 orientations of IS1223J, and pVI52.
The properties of these 2 IS and the nature of plasmids obtained are indicated in table 1 ci after.
TABLE I
IS Origin Sizes (bp) Final vectors b 1223 L. jonshon 1502 pV148 / pV149 1201 L. helveticus 1387 pV152 a: tance corresponaant a ris encaaree its direct repentances ~ uK ~
b: The mention of two plasmids indicates that 1'S has been cloned in the two directions To find the mapping activity, strains containing plasmids carrying IS to test were cultured at 37 ° C in MRS / Ery (10 ~ g / ml).
Then the cells are diluted in MRS and incubated at 44 ° C.
At different times, samples are taken and spread on MRS boxes (to determine the count of viable cells) and on MRS / Ery (cell count EryR).
Results IS1223 (pVI48 / pVI49). Plasmids pVI48 or pVI49 were introduced into VI104. In the presence of IS1223, the frequency of EryR cells is higher than with pVI1055 alone. After digestion, the DNA
chromosomes of the EryR clones obtained are hybridized with IS1223. In all cases, the EryR clones result from transposition events.
IS1201 (pVI52). plasmid pVI52 was introduced in VI104. The integration frequency of pVI52 is higher than that of pVI1055 alone. All EryR clones analyzed by Southern result from transposition.

WO01 / 21819 cA o23a5o ~ 2 201 03-14 pCT ~ R00 / 02565 These results demonstrate the activity of transposition of IS 1223 and 1201 at Lb. delbrueckii.

Claims (8)

18 1) Use of a plasmid comprising the theta replication system of pIP501 or a system of related replication as a conditional vector, thermosensitive, of integration allowing to introduce a change in the genetic information of a bacterium of species Lb. delbrueckii. 2) Procedure for modifying the information gene carried by the chromosome of a bacterium of the Lb species. delbrueckii, characterized in that it understand :
a) the construction of an integrative plasmid, by insertion at least one DNA sequence capable of integrating in the bacterial chromosome, in a vector conditional including the theta replication system of pIP501 or a related replication system, said integrative plasmid further carrying at least one selection marker;
b) introducing the plasmid into said bacterium and multiplication of it, under permissive conditions for replication and maintenance in stable form said plasmid;
c) multiplication of bacteria expressing at least one selection marker of the plasmid at the end of the step b) under non-permissive conditions for replication and/or the maintenance in stable form of said plasmid; and optionally, d) recovering bacteria expressing at least one selection marker from the plasmid, after of step c). 3) Process according to claim 2, characterized in that it further comprises:
e) excision of the sequences originating from the vector, by multiplication of bacteria expressing at the end of step c), at least one selection marker from of the plasmid, and advantageously, f) the elimination of the excised DNA, by multiplication of the bacteria obtained in step e) under non-permissive for the replication and maintenance of said vector in stable plasmid form. 4) Process according to any of the claims 2 or 3, characterized in that the sequence of DNA capable of inserting into the bacterial chromosome is a sequence homologous to a portion of the chromosome where you want to make a change. 5) Process according to any of the claims 2 or 3, characterized in that the sequence of DNA capable of inserting into the bacterial chromosome is a transposable sequence. 6) Process according to claim 5, characterized in that said transposable sequence is an insertion sequence. 7) Process according to claim 6, characterized in that said insertion sequence is selected from IS1223 of Lb. johnsonii and the IS1201 of lbs. helveticus.

8) Using an insert sequence selected from IS1223 of Lb. johnsonii and the IS1201 of lbs. helveticus to modify the chromosome of lbs. delbrueckii.

9) Integrative Plasmid for Implementation of a method according to any one of claims 1 to 6, characterized in that it results from the insertion of one of the sequences IS1223 or IS1201 in a plasmid chosen from:

- the plasmids comprising the system of pVE6002 replication;

- the plasmids comprising the system of theta replication of replicon pIP501, or a related replication system.

10) Integrative plasmid according to claim 8, selected from the group consisting of the plasmid pVI49 and the plasmid pVI52, deposited with the CNCM on 17 September 1999, under the respective numbers I-2317 and I-2318.