CA2266190A1 - Multi-resistant bacteria, methods for obtaining them and their uses - Google Patents

Abstract

The invention concerns bacteria with improved resistance to stress with respect to the wild strain, characterised in that they contain at least one gene mutation which affects the normal activity of said gene, this gene being involved in the transport of electron, amino acids, oligopeptides, phosphate, DNA repair, the stability of RNA or of new genes and selected among: genes from GP biosynthesis, genes of the clone R1.1 (pstS), R1.4 (arl1), R1.5 (glnP), R1.7 (carB), R1.8 (glnP), R1.14 (arl2), R1.16 (glnP), R1.17 (glnQ), R1.20 (arl3), R2.6 (pstB), R2.9 (recN), R2.11 (arl4), R2.15 (arl5), R2.17 (arl7), R2.20 (arl8, yybT), pstS, pnpA, and trl1.

Description

WO 98/1010

2 PCT / FR97 / 01566 MULTI-RESISTANT BACTERIA, PROCESSES FOR OBTAINING
AND USE
The present invention relates to new strains of bacteria lactic with resistance to stress, the process for obtaining said strains as well as their use in particular in fermentation.
One of the important factors in fermentation processes is the quality of growth and survival of bacteria, as well as survival bacterial in the final product. So the strains that turned out grow 1o very well in the laboratory can quite prove to be sensitive to conditions of stress occurring during industrial fermentation, for example by acidification of the medium (which is a natural consequence of growth), increase in temperature, presence or absence of oxygen) presence of salt (especially for cheese preparation) or problems related to cold (during storage). Inadequate survival rates of bacteria can lead to poor fermentation and increased mortality in the conservation of starter cultures.
This is why the present invention relates to new strains.
bacteria that exhibit increased resistance to stress.
2o In general, bacterial growth in laboratories has been studied for decades and we know that the growth of bacteria in laboratory follows a sigmoid curve, so when inoculating a medium fresh) there is a latent period during which the bacteria prepares for the growth, followed by cell multiplication in which cells grow exponentially until the middle becomes a limiting condition for growth. It is at this point that the cells come in in what is called the stationary phase during which the multiplication East stopped and cell metabolism decreases. Survival during these three phases growth, i.e. the exponential latency phases) and stationary) can vary very significantly depending on the specific conditions of growth and type of strain used. Although the conditions of laboratory are optimal conditions for growth, in the processes of industrial fermentation one can find conditions that lead to the dead bacteria. Below are given examples in which the survival of lactic acid bacteria depends on growing conditions.
1. L. lattis, like other lactic acid bacteria, acidify the environment during their growth and are able to withstand acidic conditions going up to pH 4, pH which is reached during the stationary phase. However, if the culture medium is acidified brutally during the exponential phase, les cells die quickly. Cells can be adapted to low pH
in first exposing them to intermediate pHs, between pH S and 6 by example, before switching to lower pH. In this case, we get a better survival.
2. L. lattis cells die if exposed to high temperatures (for example 55 ~ C). However, if exposed to temperatures intermediates (37 ~ C) before a thermal shock, there is better survival of cells.

3. Agitation oxygenation is part of the fermentation, L. lattis is tolerant to oxygen during growth.
However, 2o the oxygenated cells die more quickly during the phase stationary.

4. When preparing the cheese during the ripening phase, add salt. Stationary cells are more resistant to salt treatment only in the exponential phase.
These examples show stress conditions that can lead to z5 very poor survival of L. lattis, even if in some cases the rate of survival can be increased by pre-adaptation of cells to stress.
The existence of a stress resistance mechanism has been established in all bacteria examined so far. In each case, stress particular leads to the induction of the expression of a set of genes which help the cell to survive stress. The different proteins that are synthesized in stress response are often highly conserved. Many genes required for a particular stress, for example DNA damage (induced by oxygen or ultraviolet radiation) or thermal shock, have been identified in L. lactis by homology with the genes known in E. coli or other organizations. Heat shock genes include dnaJ, grpE-dnaK,, groELS and hflB. Genes to involve in repairing damage from oxygen include including recA and sodA. Nevertheless, each bacterial species presents a 1o specific niche and must therefore be adapted to its environment particular. Under these conditions, the regulators of the response genes to stress may be different for different organizations.
There is a great deal of information concerning the control of responses to stress in different bacteria. Basically two types of control seem to exist: a type of control that induces genes into reply only to specific stress, the other type of control resulting in the expression of many genes that will protect the cell against different types of stress. In bacteria, the first type of control can be done by through activators, repressors or metabolites, while the 2o second type of control can be carried out by ~ enaymes de transcription, RNA polymerase (by factors 6).
In E. coli, the factor 6 involved in the stress response general, is as, the factor a of the stationary phase. In B. subtilis, the reply general stress seems to be regulated by a factor 6B. In L. lactis, no a specific stress factor could not be isolated. However, there is a evidence physiological of cross-protection when exposing cells to stress determined. For example by subjecting L. lacis to irradiation we obtain a improved survival rate in the presence of acid, ethanol, H2O2 or shock thermal. Other results show that the recA gene is necessary for a complete resistance to oxygen and heat. In addition, in accordance with observations on E. coli, the stationary phase cells of L. lattis are more resistant to stress than exponentially growing cells. These results suggest that a general stress response is induced in both cases insufficient growth medium or when bacteria are subjected has a particular stress. Until now, nothing was known about the regulation of these systems, or the genes involved.
The present invention relates to the demonstration of the genes involved in resistance to stress and whose mutation leads to an increase of the resistance to stress of the corresponding bacteria.
This is why the present invention relates to bacteria having improved resistance to stress compared to the parental strain, characterized in that they contain at least one mutation in a gene which alters the normal activity of said gene, this gene being involved in the transport electron, amino acid, oligopeptide, phosphate, repair of DNA, RNA stability or new genes described in this document and chosen from - the genes of the purine biosynthesis pathway: GTP, (p) ppGpp that 2o we will call GP in the rest of the text, - the genes of the clone RI.1 (pstS), R1.4 (arll), R1.5 (glnP), R1.7 (carB), R1.8 (glnP), R1.14 (arl2), R1.16 (glnP), R1.17 (gln0), R1.20 (arl3), R2.6 (pslB), R2.9 (recll ~, R2.11 (arl4), R2.15 (arlS), R2.17 (arl7 ~, R2.20 (arl8, yyb ~, pstS, pnpA and trll.
Advantageously, the gene for the GP biosynthesis pathway is chosen among deoB, hpt, g ~ uaA, relAIspoT, tktA.
As will be demonstrated in the example, an introduced mutation in these genes leads to increased resistance to stress which in the most cases will be general resistance.

It should be noted that the terminology used "alteration of activity normal of said gene "can also mean a decrease or even disappearance or an increase in the activity of the gene or a gene belonging to the same transcription unit (operon) when this leads to an improvement in the

5 resistance to stress.
The mutation in question could also be an insertion mutation of a DNA sequence, deletion of a DNA sequence and / or mutation punctual using for example a mutagen or by mutation spontaneous.
tb In the case of insertion of a DNA sequence, we will use the introduction of mobile genetic elements such as sequences insertion as will be described in the example with ISSI.
Of course, it is possible to provide other techniques ensuring the mutation in question, we can use the known mutation systems of those skilled in the art as well as DNA insertions or other modifications obtained by homologous recombination techniques.
Preferably, when the DNA is inserted, it will be DNA
from the same species, this in order to keep the acceptability on the plan agro-food of the strain thus obtained.
Among the multiple bacteria that can benefit from the advantages of present invention, it is particularly necessary to mention bacteria lactic in especially Lactobacilhcs, Lactococcus and Streptococcus.
As will be shown in the example, we were able to insert sequences of DNA in the genes that were then determined, the bacteria as well obtained have increased resistance to certain stresses or to a set of stress conditions. It is possible to consider mutations multiple on different genes. For each specific problem posed, it is better to compose a set of mutated bacteria in different genes and WO 98 / l0102 PCT / FR97 / 01566

6 to select the most appropriate mutants for stress conditions who are likely to occur during the fermentation process.
Although we prefer to use engineering methods genetic it is also possible to plan to select mutants in the same genes by traditional means of mutations by selecting in especially when we know, as will be described below, the pathways metabolic groups in which the genes involved are involved and which will have the means to act directly on these metabolic pathways via appropriate agents.
to The present invention also relates to a fermentation process using a bacterium according to the present invention and in particular the fermentation processes using iait or milk by-products. We can also consider the use of these bacteria for the production and conservation of ferments.
Among the fermentations that can benefit from bacteria depending on the present invention, there should be mentioned in particular fermented milk products such as yogurts and equivalent products as well as the preparation of cheeses.
The bacteria according to the present invention can also be used for food preservation or for purposes zo probiotics.
It is important to remember that the genes described in the context of present invention have for a large number of them been isolated from Lactococcus but that their equivalents exist in E. cols and / or in others strains. Under these conditions, the present invention is in no way limited to of lactic type fermentations but can also be considered for others bacteria and other types of fermentation.
Figure 1 illustrates the mutagenesis system via the plasmid pGh ISSI.
Figure 2 illustrates the obtaining of mutants lacking foreign DNA.

7 Figure 3 illustrates the process for identifying the mutated gene.
Figure 4 shows the biosynthetic pathway for GPs.
SEQ ID N ~ 1 to 34 represent the sequences of the mutated genes) to the origin of multi-resistance, at the junctions of plasmid insertion, the microorganisms concerned being from what has been called Selection l in the example.
SEQ ID N ~ 35 to 44 represent the sequences of the mutated genes, to the origin of multi-resistance, at the junctions of plasmid insertion, the microorganisms concerned being from what has been called Selection 2 to in the example.
SEQ ID N ~ 45 to 52 represent the sequences of the genes mutated by site-directed mutagenesis.
SEQ ID N ~ 53 to 71 represent the sequence of the mutated genes) to the origin of an acid resistance superior to the parental strain (data no shown) at the junctions of the plasmid insertion, the mutants being from the Selection 1.
Figure 1A shows a microorganism cell with its chromosome into which the plasmid pGh has been introduced by transformation ISSI which contains the pG + host replicon (Ts ori), a gene for resistance to 2o antibiotic (AbR) and an ISSI mobile element. At 30 ~ C, the plasmid is replica in the cell of the microorganism.
Figure 1B shows the population of mutant bacterial cells resulting from the integration of the plasmid into the bacterial chromosome after duplication of the ISSI. Indeed, at 37 ~ C, the plasmid replication is inactive and the plasmid is lost unless the bacteria undergoes a transposition event.
Figure 2A shows the chromosomal structure of a mutant obtained by transposition of the ISSl. The ISSI elements, duplicated, frame the plasmid pG + host. At 30 ~ C, the replication of pG + host is activated and stimulates the homologous recombination between the duplicated ISSI sequences.

WO 98I10102 PCT / FIt9710156b

8 Figure 2B shows the homologous recombination taking place between the two ISSI sequences resulting in the excision of the plasmid pGh: ISSI which East then lost when the strain is spread at the non-permissive temperature of 37 ~ C. Following the recombination event, the mutated strain contains only a copy of the ISSI which is a sequence originating from the microorganism mutated, which therefore contains no trace of foreign DNA.
Figure 3 shows the cloning of the junctions between the ISSI and the chromosome. The HindIII and EcoR 1 restriction sites are unique in the transposed structure and are located on either side of the plasmid pG + host. The lo digestion by HiodIII of the chromosomal DNA of the mutant produces a fragment consisting of the pG + host and the right junction ISSI - chromosome. After circularization, this fragment is established as a plasmid in E. coli or others bacteria. The junction is then sequenced by means of primers corresponding to the sequence of the ISSI or that of the pG + host. The left junction ISSI -t 5 chromosome is obtained by applying the same procedure after digestion with EcoR1 of the mutant's chromosomal DNA.
SEQ ID N ~ 1 to 34 represent the sequences of the mutated genes such that they could be determined in accordance with the protocol illustrated by the Figure 3. An "R" code has been given to identify each gene concerned. Each 2o sequenced junction was compared, in terms of homology, to the sequences of microorganisms already known, and when one of them has been identified, she has been mentioned. NS means that no significant homology with a gene already known could not be established. The digital data appearing (for each identified junction), corresponds to the probability that the sequence represented of z5 not be identical to the already known genes mentioned.
SEQ ID N ~ 35 to 44 represent the sequences of the mutated genes such that they were determined in accordance with the protocol illustrated in Figure (Unless Otherwise Posted).

9 SEQ ID N ~ 45 to 52 represent the internal fragment sequences of certain genes whose junction sequence had already been determined in accordance with SEQ ID N ~ 1 to 34. The internal fragment of the gene in question has was amplified by PCR and then sequenced on the two strands. These fragments have then been used to inactivate, by homologous recombination, genes correspondents in the wild strain so as to verify that the phenotypes of multidrug resistance were due to the inactivation of these genes.
SEQ ID N ~ 53 to 71 represent only the sequences of the genes of which inactivation demonstrated at least one resistance to acid t0 of the strains concerned.
The present invention will be better understood by means of the example which follows. This example is however only illustrative and does not limit in any way the invention.
EXAMPLE
Laclococcus laclis is a bacterium which, in the wild, presents the following features - on a medium at 30 ~ C, whether the pH is 7 or 5.5) it has a same spreading efficiency;
- on a medium at 37 ~ C) the spreading efficiency on pH 7 is I04 times 2o higher than that obtained on pH 5, 5 or pH 5.
Two groups of Lactococcus lattis mutants were selected.
A group of mutants, called Selection 1, comes from the wild strain MG1363 of Lactococcus lattis and consists of mutants exhibiting at 37 ~ C
an e ~ ciency spreading on a medium at 37 ~ C at pH 5.5 between 1 and 0.0I
compared to pH 7. Thirty mutants were thus isolated.
A second group of mutants, called Selection 2, comes from the wild strain MG1363 of Lactococcus lattis previously mutated in the recA gene making it sensitive to ultraviolet radiation. This strain, called VEL1122 is sensitive to temperature. It was found that at the temperature of 39.3 ~ C (with a pH of the medium of around 7 but not decisive) .
the recA strain had a spreading efficiency of less than 10-g when the wild strain MG1363 had a spreading efficiency of 1. We have selected mutants from the VEL1122 strain capable of growing at a 5 temperature of 39.3 ~ C. About twenty mutants were thus isolated.
Selection I and Selection 2 mutants were obtained after mutagenesis via the thermosensitive plasmid pGh: ISSI (cf.
reference 16). This plasmid consists of the replicon pG + host (Ts ori), of a gene antibiotic resistance (in this case erythromycin) and lo the ISSI mobile element (see Figure I). This plasmid has the peculiarity of replicate in cells at 30 ~ C, but not at 37 ~ C or higher. This plasmid can integrate into the chromosome of bacteria via a transposition event of the ISSI. During transposition, the entire plasmid, flanked by and on the other, the ISSI is integrated into the chromosome. The integration sites of transposons are random. Bacteria that have integrated into their chromosome the plasmid pGh: ISSI therefore retain resistance to erythromycin at 37 ~ C or more. Consequently, among the bacteria having undergone the above mutagenesis, one selects those still capable of growing in the presence of antibiotics on a middle at 37 ~ C or higher. These bacteria have undergone a transposition event who generates random mutations.
Relative to the number of starting bacteria containing pGh: ISSI, 1 to 5% of mutant bacteria are obtained. From this population of mutant bacteria, more stress-resistant mutants have been selected according to the methods previously indicated (Selection 1 or 2).
Mutants resulting from Selections 1 and 2 are subject to temperature of 30 ~ C which activates the replication of pG- + host and stimulates the homologous recombination between the duplicated ISSI sequences which results in excision of the plasmid pGh: ISSI. The bacteria in question do not contain therefore, inserted into their chromosome, more than a copy of the ISSI (cf. Figure 2). The final strain mutated by insertion of the ISSI is stable and does not contain not of A.DN foreign to Laclococcrrs, it therefore constitutes a food mutant (~ background-grade ~).
The Laclococcrrs lactis bacteria thus obtained (Selection 1 mutants or Selection 2), are then subjected to different stress conditions at respect whose resistance was tested compared to that of the strain parental subject to the same conditions.
Whenever a strain was found to be resistant superior to that of the parental strain, we sought to identify the gene to be responsible for this resistance; that is, the gene at which the plasmid pGh: ISS! had inserted. The junctions between the ISS! and the chromosome have therefore were cloned and then sequenced. Then, the sequences thus determined were compared to the sequences of the databases in order to find the degree of homology they presented with other known genes from bacteria lactic or other organisms (see Figure 3).
The results of these tests are collated in Table I for what relates to part of the mutants from Selection 1 and in Table II
for with regard to the mutants from Selection 2.
The genes identified in Table I correspond to SEQ ID N ~ 1 to 34.
The genes identified in Table II correspond to SEQ ID N ~ 35 to 44.
These tables collect data calculated in relation to the measurement resistance to each of the stresses involved in the parental strain which serves therefore of reference. Depending on the conditions imposed, it will be noted that the mutants survive up to about 2,500 times better than the parental strain corresponding. These results show that said mutants, that is to say the strains with an alteration in the gene concerned, are potentially able to survive better under process stress conditions industrial.

O

Selection Stress Stress Survival Stress 1 thermal acid the term ox dative ABC SSC without 55C with Glucose HzO2 1 mM ' limiting adaptationadaptation * final pH
: 4.3 eues Duration u stress2 Bures l ~ Fre 1 hr 5 min IS min 2 days 30 min Parentage (7th-5th) (6th-5th) (9th-4th) (8th-4th) (8th-4th) (1st 4th-4th) (1st 4th-4th) R1.1 pstS (ss80) 79 1 249 1 1 1 2000 R1.4 NS (arll) 372 24 224 I 1 1 800 R. glnP 1 8 1 2 I 1 1 R1.7 carB 192 9 1 l R 1. ghrP 1 11 1 I

R1.14 NS (arl2) 233 11 2 1 1 13 R 1. GlrrP 1 9 1 I

17 GlfrO 6 1 1 1 1.8 1 . Hp 11 I 46 13 1 2 ,;, R 1.19 R1.20 NS (arl3) 2 25 1 1 1 R2.2 relAlspoT l176 54 6 6 20 1225 4 R2.3 Gua. 9 364 438 352 3 107 2503 I 3 R2.6 stB 7 13 1 1 1 1 R2.9 recN 18 20 1 7 1 1 2 R2.11 NS (arhl) 288 27 1 1 2 R2.14 GrraA 1072 421 12 7 57 1330 2 R2.15 NS (arl5) 624 9 7 3 15 605 1 R2.16 deoB 19 22 24 1 1 R2.17 NS (arl7) 244 492 28 6 107 I728 5 not R2.20 ybT (arl8) 86 3 1 1 120 1 "'3 recN 22 28 1 6 1 2 grraA I028 117 12 7 48 1318 hpt 1032 28 4 11 2 820 relAls 1176 83 3 6 _ 19 132I

oT

NS:

No significant TABLE I

Legends of table I:
A: the bacteria, in exponential phase on a medium at pH 7, have been spread on a medium at pH 3.7.
B: the bacteria, in stationary phase on a medium at pH 7, were spread out on a medium at pH 3.0.
C: the bacteria, in exponential phase on a medium at pH 7, have been spread for 30 min on a medium at pH 5.5 then on a medium at pH 3.2.
~: microorganisms, cultivated at 30 ~ C, are directly subjected to stress thermal 55 ~ C.
": microorganisms, cultivated at 30 ~ C are subjected for 15 min. to a temperature of 37 ~ C then a thermal stress of 55 ~ C.
O: the bacteria were maintained in glucose deficiency and at acidic pH
during 2 days before being spread on a medium at pH 7..
the bacteria in exponential phase at neutral pH were incubated for 30 min.
in the presence of 1 mM H 2 O 2 before being spread on a medium at pH 7.
o: actual survival of the parental strain (MG1363) which serves as a reference and for which is assigned the value of 1.

TABLE II
o NOT
Selection 2 Stress Thermal Stress Oxidative Resistance to Survival 55C Survival 55C Survival H2021mM without adaptationwith adaptation * long term Duration of stress 15 minutes 1 S minutes l 5 minutes 2 days n VEL11221 (3rd-2) (Se-6) (2nd-4) (6th-3) Parental strain 1 1 1 1 gua A 10 1190 320 l60 deo B 6 120 170 8 Genes pst S 7 28 100 5 np A 6 82 10 5 p tkt A 5 140 260 5 tr! 1 1 274 90 8 ~: microorganisms, cultivated at 30 ~ C, are directly subjected to stress thermal 55 ~ C.
*: microorganisms, cultivated at 30 ~ C, are subjected for 15 minutes to a temperature of 37 ~ C
then a thermal stress of SS ~ C.
0: actual survival of the parental strain; this value is then reduced to 1.
b not H
at ..
at, at IS
SEQUENCE LIST
(1) GENERAL INFORMATION:
(i) DEPOSITOR:
(A) NAME: INRA (NATIONAL INSTITUTE OF RESEARCH
AGRONOMIC) (B) STREET: 147 STREET OF THE UNIVERSITY
(C) CITY: PARIS
(E) COUNTRY: FRANCE
(F) POSTAL CODE: 75007 (ii) TITLE OF THE INVENTION: MULTI-RESISTANT BACTERIA, METHODS
OBTAINING AND USING
(iii) NUMBER OF SEQUENCES: 71 (iv) COMPUTER-DETACHABLE FORM:
(A) TYPE OF SUPPORT: Floppy disl:
(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: PatentIn Release Q1.0, Version N1.30 (EPO) (vi) PRIOR APPLICATION DATA:
(A) REQUEST NUMBER: FR 9610926 (B) DEPOSIT DATE: 06-SEP-1996 (vi) PRIOR APPLICATION DATA:
(A) REQUEST NUMBER: FR 9611555 (B) DEPOSIT DATE: 23-SEP-1996 (2) INFORMATION FOR THE 5EQ ID N0: 1:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 317 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.1-EcoRI junction: clone SS80 L. lactis (3e-19) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N0: i:

GTTGAACATGCTTCTATGTCATATATGAAGCAAAATCCTGATGAAATTATCATGGTTCAA l80 (2) INFORMATION FOR SEQ ID NO: 2:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 379 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.1-HindIII junction: clone SS80 L. lactis (3e-97) (xi) DESCRIPTION OF THE SQUENCE: SEQ NO: 2:
ID

TCAAAGGTCG

ATGCTCAATT

TTTAAAGCCC

AGTAGCTGAG

CGATTAGCCA

TGAATCAGCA

TATTTCCGGC AGCATCACG 37g (2) INFORMATION FOR SEQ ID NO:
3:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 179 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

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(A) NAME / KEY: R1.9-HindIII junction: NS
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N0: 3:

(2) INFORMATION FOR SEQ ID N0: 4:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 482 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
{A) NAME / KEY: R1.5-EcoRI junction: GInQ.E.coli (3.9e-14) (xi) DESCRIPTION OF THE SEQUENCE: SEQ N0: 9:
ID

GTGTACATGA TTGTTCTGCT CTTCTTGATG GTCGTGTAGA AAAAAGAATG.

GGGAATCAAT

TCTAAAAGGA

AGGTTCTGGA

TGATATCATT

CGCCAAAAAC

TTTTGGGAAA

(2) INFORMATION FOR SEQ ID N0:
5:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 365 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.5-HindIII junction: GInQ.E.coli (6.6e-39) (xi) DESCRIPTION OF THE SEQUENGE: SEQ ID N0: 5:

WO 98/10102 PCTlFR9? L01566 (2) INFORMATION FOR SEQ ID N0: 6:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 489 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
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(A) NAME / KEY: R1.7-EcoRI junction: NS
(xi) DESCRIPTION OF THE SEQUENCE: SEQ N0: 6:
ID

GTTTATTTCT

GGAGGCGTGA

AGGCTACAAA

TAAATAAAGC

TTGTTGAAAG

GCTATATAAA

ATTTGGGGAC

GCAATGAGAA

(2) INFORMATION FOR SEQ ID N0:
7:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 294 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.7-HindIII junction: carbamoyl-phosphate synthase.B.caldolyticus (3.9e-11) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:

(2) INFORMATION FOR SEQ ID N0: 8:
(i) CHARACTERISTICS OF THE SEQUENCE: ' (A) LENGTH: 554 pairs of bases (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.8-EcoRI junction; GInH.E.coli (5.3e-29) (xi) FROM N0: 8:
SQUENCE DESCRIPTION:
SEQ ID

(2) INFORMATION FOR 5EQ ID NO: 9:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 929 base pairs (8) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.8-HindIII junction: GInJ.E.coli (4.2e-7) (xi) DESCRIPTION OF THE SEQUENCE: SEQ N0: 9:
ID

TTTGCCGTTA

AATCTCCGTG

AAAACCATTC

GAACAAATCG

NCAATGATTG

ANAATTGCAC

TTTATCTTCT

(2) INFORMATION FOR SEQ ID NO:

10:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 929 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.14-EcoRI junction: NS
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10:

WO 98/10102 PCT / FR97 / Oi566 (2) INFORMATION FOR SEQ ID N0: 11:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 123 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.19-HindIII junction: NS
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N0: 11:

(2) INFORMATION FOR SEQ ID NO: 12:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 522 base pairs (B) TYPE: nucleotide {C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.16-HindIII junction: GlnP.E.coli (2.3e-30) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12:

AGCCANCCGT

AGCCATTAAA

AACATTGGTT

CCGTAACTTC

TCTCTTCTNG

AACATACTAA

(2) INFORMATION FOR SEQ ID N0:
23:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 375 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.17-EcoRI junction: NS
(xi) DESCRIPTION OF THE SEQUENCE: SEQ N0: 13:
ID

AAGACTTTTT

TTATTGAAAT

ATGTTGCAAC

TAAACCAGCT

AGCAATAGAA

TAACGGAGCT

(2) INFORMATION FOR SEQ ID NO:
19:

(i) FEATURES OF THE SQUENCE:

(A} LENGTH: 435 base pairs (B} TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) CHARACTERIST:
(A) NAME / KEY: R1.17-HindIII junction: GInQ.E.coli (7e-35) (xi) DESCRIPTION OF THE SQUENCE: SEQ NO: 14:
ID

AGGATTACTC

TCGTGAGTGA

GACATCTCCG

AGGATTCATT

TCTGGCATCG

ACATGTGTAT

AAACAGTCAT

(2) INFORMATION FOR SEQ ID N0:
15:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 485 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) CHARACTERISTIC:
(A) NAME: R1.19-EcoRI junction: Hpt.L.lactis (5e-101) (xi) DESCRIPTION OF NO: 15:
SQUENCE:
SEQ ID

WO 98/10102 PCTlFR97 / 01566 (2) INFORMATION FOR SEQ ID NO: 16:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 263 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.19-HindIII junction: Hpt.L.lactis (1.9e-62) (xi) DESCRIPTION OF THE SQUENCE: NO: 16:
SEQ ID

TGAATTTGTT

TTGGGGTTGT

ATTCTCTCAG

AGGAAATATG

(2) INFORMATION FOR THE SEQ ID
NO: 17:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 903 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) FEATURE:

(A) NAME / KEY: R1.20-EcoRI junction: NS

(xi) DESCRIPTION OF THE SQUENCE: NO: 17:
SEQ ID

CAGGAGCAAT

CTACAAACTT

GAGGTTATGG GTTTGCTGTG ATGAAAGGCA GCTCGTGGATGGTTTCAATA 1.80 AAAACCAAAC

ATTATGATAA

TAACAACTTC TP ~ AP.AAAGCA ACACCGAAAA TACCATTGCCTCAGATAATA 300 AAGATGTTTA

ACAAACAATT

TCAAAACTCA

(2) INFORMATION FOR SEQ ID NO: 18:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 769 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix} CHARACTERISTIC:
(A) NAME / KEY: R2.2-EcoRI junction: ReI.S.equisimilis (1.9e-53) (xi) DESCRIPTION OF THE SQUENCE: N0: 18:
SEQ ID

NNATCTCAAC TCGGTCCCCTGTTTTTAATT GAACAGATAAAGGTTTCATA CGTCCATTTA ~ 12D

(2) INFORMATION SEQ ID N0: 19:
FOR

(i) CHARACTERISTICS
FROM THE SQUENCE:

(A) LENGTH: 698 base pairs (B} TYPE: nucleotide (C) NUMBER OF STRANDS:
simple (D) CONFIGURATION:
linear (ii) TYPE OF MOLCULE:
DNA

(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.2-HindIII junction: ReI.S.equisimilis (l.le-9) (xi) DESCRIPTION OF THE SEQUENCE: SEQ N0: 19:
ID

CCAAGTCGTG

TCTTTAAAAA

TTAATAAAGG CCGTGAAATG TTGCAAGAAG TCP, AAAAAAA ATGATAGGAA180 CTTAACGGGC

AGATTTGAAA

CCCAAAACAA

CTGCTTTTAT

GCATAAATAA

AATATTCAAC

TAGCGTGTTT

AATGATAATA

AAAAAATAAA

(2) INFORMATION FOR SEQ ID N0:
20:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 712 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.3-EcoRI junction: NS
(xi) OF THE SQUENCE: N0: 20:
DESCRIPTION SEQ ID

TTTCATTTTC TAATGTAAAT CTATTACCTT ATTATTAATT CAATTCGCTC ATA.ATTAATC 540 {2) INFORMATION FOR SEQ ID N0: 21:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 781 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.3-HindIII junction: GuaA.B.subtilis {2.9e-33) (xi) FROM NO: 21:
SQUENCE DESCRIPTION:
SEQ ID

(2) INFORMATION FOR SEQ ID NO: 22:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 807 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.6-EcoRI junction: Hypothetical ABC
transporter.B.subtilis (6.3e-22) (xi) DESCRIPTION OF THE SQUENCE: NO: 22:
SEQ ID

CAATTGAACCCTTTTTGAAANCAAAAC g07 (2) INFORMATION SEQ ID N0: 23:
FOR

(i) CHARACTERISTICS
OF THE
SQUENCE:

(A) LENGTH:
705 pairs of basics (B) TYPE:
nucleotide (C) NOP9BRE
OF BRINS:
simple (D) CONFIGURATION:
linear (ii) TYPE
OF MOLCULE:
DNA

WO 98/10102 PCT / FIt97101566 (ix) CHARACTERISTIC:
(A) NAME / KEY: R2.6-Junction HindIII: Hypothetical ABC
transporter.B.subtilis (5.6e-56) (xi) DESCRIPTION OF THE SEQUENCE: SEQ N0: 23:
ID

CCTTAAGGGC

CTTTGNAAGT

CAATTTATGA

TGGACGAGAT

ATCTTAATCA

GTGCCTTGTC

CAATTTCAAC ~.

TTATCGTGAC

GGTACCCAAT

ACGTCGTGAC

TTTCGCCAGG

TGTTAATCTA

(2) INFORMATION FOR SEQ ID N0:
29:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 269 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) FEATURE:

(A) NAME: R2.9-recN of B. subtilis (xi) DESCRIPTION OF THE SQUENCE: N0: 24:
SEQ ID

(2) INFORMATION FOR SEQ ID NO: 25:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 335 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.9- recN of B. subtilis (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25:

(2) INFORMATION FOR SEQ ID N0: 26:
(i} CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 196 base pairs (B} TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.11-NS / supE.L.lactis (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N0: 26:

TTTCAAAAAT CAAAGATTAA GCACTTAATA GAGCTTAAAA AAACGATAAG GAGTT'CGAGA 120 (2) INFORMATION FOR SEQ ID N0: 27:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 267 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.19-ecoRI junction: GuaA.B.subtilis (le-28) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 27:

(2) INFORMATION FOR SEQ ID N0: 28:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 290 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.14-HindIII junction: GuaA.B.subtilis (5th-2) (xi) FROM THE SQUENCE: N0: 28:
DESCRIPTION SEQ ID

GTATTTACTGTGAAATATTGNCAAACATCACGNACANGTCCTGAAGGAGCAAT'TTCTTCG290 (2) INFORMATION FOR SEQ ID NO: 29:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 682 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.15-EcoRI junction: NS
(xi) FROM THE SQUENCE: NO: 29:
DESCRIPTION SEQ ID

(2} INFORMATION LA SEQ ID N0: 30:
FOR

(i) CHARACTERISTICS
OF THE
SQUENCE:

(A) LENGTH:
686 pairs of basics (B) TYPE:
nucleotide (C) NUMBER
OF BRINS:
simple (D) CONFIGURATION:
linear (ii) TYPE
OF MOLCULE:
DNA

(ix} CHARACTERISTIC:
(A) NAME / KEY: R2.15-HindIII junction: NS

"
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N0: 30:

ACGACTTCTA AAAACAAGTC ATAAATATAA AGAAGCATAA AGCTAAAAGA GAGAAAGGGA ~ 600 (2) INFORMATION FOR SEQ ID N0: 31:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 137 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.16-HindIII junction: NS
(xi.} DESCRIPTION OF THE SEQUENCE: SEQ ID N0: 31:

(2) INFORMATION FOR SEQ ID NO: 32:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 6B9 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA

~ a (ix) CHARACTERISTIC:
(A) NAME / KEY: R2.17-HindIII junction: NS
(xi) DESCRIPTION OF THE SQUENCE: NO: 32:
SEQ ID

CATTGACAAT

ATTTTTGGCA

CGCTTGCATT

CCATTCAACG

TAGGTGATAA

CCAATAGTTT

GTTAATTCTT

AATGTATCTA

ACC1'GATGTT CATTACAAAT GAAGTCATGA CAGGATAGTC ACGCATAATG590 CTAGTCGCTT

AGAAAGTCTC

TTATTAATTG

(2) INFORMATION FOR THE SEQ ID
NO: 33:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 972 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA.

(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.20-EcoRI junction: hypothetical protein of B. subtilis (le-4) (xi) FROM N0: 33:
SQUENCE DESCRIPTION:

WO 9 $ / 10102 PCT / FR97 / 01566 (2) INFORMATION FROM THE SEQ
FOR ID N0:
34:

(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 332 base pairs {B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.20-HindIII junction: Hypothetical protein of B. subtilis (8.5e-12) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N0: 39:

GCCCTTGAGT TGGCTCAAGT ACGCGGTGGT GC. 332 (2) INFORMATION FOR SEQ ID N0: 35:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 300 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
{ix) CHARACTERISTIC:
(A) NAME / KEY: tkt A-HindIII junction WO 98/10102 PCT / FR9'7 / 01566 (xi) DESCRIPTION OF NO: 35:
SQUENCE:
SEQ ID

(2) INFORMATION FOR SEQ ID N0: 36:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 300 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: tkt A-EcoRI junction (xi) DESCRIPTION OF NUMBER: 36:
SQUENCE:
SEQ ID

(2) INFORMATION FOR SEQ ID N0: 37:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 380 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: guaA-EcoRI junction (xi) DESCRIPTION OF THE SQUENCE: SEQ NO: 37:
ID

CCATGAAACG

TTTCCATGAC

TCAATTGGNC

CTGAAAGTCC

TATCAATGAA

TTCTGAGCAT

(2) INFORMATION FOR SEQ ID N0:
38:

(i) FEATURES OF THE SQUENCE:

'(A) LENGTH: 1121 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

iix) CHARACTERISTIC:
(Ay NAME / KEY: partial deoB-sequence (xi) FROM NO: 38:
SQUENCE DESCRIPTION:
SEQ ID

GAACGAACATATTCACANATTT'rATAAAGTTCTTCACGAGAAATAACATCTTCATGTGCA 840 (2) INFORMATION FOR THE SEQ ID N0: 39:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 300 base pairs - (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: gpps or pnp - HindIII junction (xi) DESCRIPTION OF NUMBER: 39:
SQUENCE:
SEQ ID

CCATAACGNACAACCACAGC.TCCANTTGGTTGTGTNGCAACTTGCACCTGTNTCAACAGT 60 (2) INFORMATION FOR SEQ ID N0: 90:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A} LENGTH: 300 base pairs (B} TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: gppS or pnp - EcoRI junction (xi) FROM NO: 90:
SQUENCE DESCRIPTION:
SEQ ID

(2) INFORMATION FOR SEQ ID NO: 41:
(i) CA ~ RACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 380 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: trll-HindIII junction (xi) DESCRIPTION OF THE SQUENCE: NO: 91:
SEQ ID

GAAGGAGAGA

AAGACATGGC

TTATCACCCA

ATTTCAAGCA

TTCACAAGCA

GGACGAAAAA

(2) INFORMATION FOR THE SEQ ID
NO: 42:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 900 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) CHARACTERISTIC:
(A) NAME / KEY: trll - EcoRI junction (xi) DESCRIPTION OF THE SQUENCE: N0: 42:
SEQ ID

CCATATTTAT

GCAAAAGTA ~, GTTGCTTTTT

TTTTNGNCTT CTGTAACTCT CGTTTTNNGG ATCTGTAATT TCA. ~ 1CAGAAA290 CTTCAATATC

AAATATATCC

ATTTNTTGGA

GTNGCGATGT 'nTTANNTATN CATTGAGG (~ T 4 ATTGGCCTT '00 (2) INFORMATION FOR THE SEQ ID
NO: 93:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 300 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) CHARACTERISTIC:
(A) NAME / KEY: pstB-HindIII junction (xi) OF THE SQUENCE: N0: 93:
DESCRIPTION SEQ ID

(2) INFORMATION FOR SEQ ID NO: 99:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 300 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: pstB - EcoRI junction (xi) FROM N0: 99:
SQUENCE DESCRIPTION:
SEQ ID

(2) INFORMATION FOR SEQ ID NO: 45:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 935 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: Bene ahrC (inactive in mutant R2.9) seqaence strand 1 (xi) DESCRIPTION OF NUMBER: 45:
SQUENCE:
SEQ ID

CAGCAGTTTTGTCAGCAAGTTCTTGATAATCC3'TATCAAAATTGCCAATTCTATCCAATT 60 (2) INFORMATION FOR SEQ ID NO: 96:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 261 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: Bene ahrC (inactive in mutant R2.9) strand sequence 2 (additional) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N0: 46:

(2) INFORMATION FOR SEQ ID N0: 97:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 468 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: Bene hpt (inactive in the mutant R1.19) strand 1 sequence (xi) FROM NO: 47:
SQUENCE DESCRIPTION:
SEQ ID

TGAAGTAAAATTGATTTTAGACGTTGATACAGCGGTTAAAGGTCGTGAAATTT'rGATTGT180 (2) INFORMATION FOR SEQ ID N0: 98:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 391 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME: Bene hpt (inactive in mutant R1.19) strand 2 sequence (complementary) (xi) DESCRIPTION OF THE SQUENCE: NO: 98:
SEQ ID

CGACATATGG AAGATTACGG TAGTTTTCTT ACCAAAGCCA ACA. ~ 1CAAATT60 CGTAGTCTAA

GTTTAATTTC

CTCCACGATG

CTTCAACAAT

CTTCACCAGA

GATGACAATC

VS

(2) INFORMATION FOR THE SEQ ID
NO: 49:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 939 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear () TYPE OF MOLCULE: DNA

(ix) CHARACTERISTIC:
(A) NAME / KEY: Bene relA (inactive in mutant R2.2) strand 1 sequence (xi) DESCRIPTION OF THE SQUENCE: N0: 99:
SEQ ID

AACCA. ~ 1TAAGGCTCGAAACAAAATTAAGCAATTCTTTAAAAACCAAGACAAAGAATTGTC920 GGTTA.ATAAAGGCC g3q (2) Ii ~ INFORMATION FOR SEQ ID NO: 50:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 959 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ü) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: Bene relA (inactive in mutant R2.2) strand 2 sequence (complementary) (xi) OF THE SQUENCE: N0: 50:
DESCRIPTION SEQ ID

ATACCGTCGACCTCGAGGGGGGGCCCGG'rACCCAATTCG 459 (2) INFORMATION FOR SEQ ID NO: 51:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 530 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: guaA gene (inactive in mutants R2.3 and R2.19) strand sequence 1 (xi) DESCRIPTION OF THE SQUENCE: N0: 51:
SEQ ID

GATGAGCCAT

ACCAAACAGT

TCACCCAGAA

CATCTGTGGA

TATTCGTGAA

ATCAGTTGTG

TGACCACGGT

ATTTGGTCTT

GGCCTTTCAG

(2) INFORMATION FOR THE SEQ ID
NO: 52:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 320 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) CHARACTERISTIC:
(A) NAME / KEY: Bene guaA (inactive in mutants R2.3 and R2.14) strand 2 sequence (additional) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N0: 52:

WO 98I10102 PCT / FR97l01566 (2) INFORMATION FOR SEQ ID NO: 53:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 268 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.2-HindIII junction: DtpT.L.lactis (5.9e-91) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N0: 53:

(2) INFORMATION FOR SEQ ID N0: 59:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 735 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: Ri.6-EcoRI junction: NS
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 59:

(2) INFORMATION
FOR
THE SEQ
ID N0:
55:

(i) CHARACTERISTICS
OF THE
SQUENCE:

(A) LENGTH: 712 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: ' simple (D) CONFIGURATION:
linear (ii) TYPE
OF MOLCULE:
DNA

(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.6-HindIII junction: NS
(xi) FROM NO: 55:
SQUENCE DESCRIPTION:
SEQ ID

TTTCATTTTC TAATGTAAAT CTATTACCTT ATTATTAATT CAATTCGCTC ATA.ATTAATC 590 (2) INFORMATION FOR SEQ ID NO: 56:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 317 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.9-HindIII junction: Orotate phosphoribosyltransferase.B.subtilis (2.2e-9) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 56:

(2) INFORMATION FOR SEQ ID N0: 57:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 664 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.10-EcoRI junction: NS

(xi) FROM N0: 57:
SQUENCE DESCRIPTION:
SEQ ID

(2) INFORMATION FOR SEQ ID NO: 58:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 680 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.10-HindIII junction: NS
(xi) FROM N0: 58:
SQUENCE DESCRIPTION:
SEQ ID

(2) INFORMATION FOR SEQ ID NO: 59:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 402 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A} NAME / KEY: R1.11-EcoRI junction: NS
(xi) DESCRIPTION OF THE SQUENCE: NO: 59:
SEQ ID

GACTGACAAA

TGGTTTAGAA

CGAAAGTAGA

TAAATCTACT

CCCACTAAAA

TCCAGCAAAT

ATATGGCCTA

(2) INFORMATION FOR THE SEQ ID
NO: 60:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 687 base pairs (B) TYPE: nucleotide (C} NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) CHARACTERISTIC:
(A} NAME / KEY: R1.13-EcoRI junction: NRAMPI.Mouse (2.6e-18) WO 98/10102 PCT / FR9'7 / 01566 (xi) DESCRIPTION OF THE SQUENCE: SEQ NO: 60:
ID

TTTAAAAAGA

CCTCAATAAT

GAATAACTAG

GTAATAAAAG

TAAACAGAAG

TTGCCAGTTC

ATAGCCTGAG

AGCATTGCAA

CCTCCAGTAA

CCTGGTCCTG

P.AAAAACCAA CATTTTTAGG CACTTCTACT TTTCCTCTAA NGGATTTTCC660 GTCCCATTTA

(2) INFORMATION FOR SEQ ID N0:
61:

{i) CHARACTERISTICS OF THE SQUENCE:

(A) LENGTH: 755 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.13-HindIII junction: NRAMPI.Mouse (2e-20) (xi) DESCRIPTION OF NUMBER: 61:
SQUENCE:
SEQ ID

GCCGTGGCTC

TTATTATGGA

CTCGCCTGCT

TGAAAAGCTC

TTGCCCTTCC

GGTGC

(2) INFORMATION FOR SEQ ID NO:
62:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 629 base pairs (B) TYPE: nucleotic (C) NUMBER OF STRANDS: single '(D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.15-EcoRI junction: Hypothetical protein B. stearothermophilus (1.6e-96) (xi) DESCRIPTION OF NUMBER: 62:
SQUENCE:
SEQ ID

(2) INFORMATION FOR SEQ ID NO: 63:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 100 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R1.18-EcoRI junction: NS
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N0: 63:

(2) INFORMATION FOR SEQ ID N0: 64:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 561 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) FEATURE:

(A) NAME / KEY: R1.18-hindIII junction: Hypothetical protein B. subtilis (7th-30th) (xi) DESCRIPTION OF THE SQUENCE: SEQ ID NO: 64:

CACCGCGTAC TTGAGCCAAC TCAAGGTTAT TGAGGGCAAC TT'PTCCGATG GTAGGAAAAT920 (2} INFORMATION FOR SEQ ID N0: 65:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 720 base pairs (B) TYPE: nucleotide (C} NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.1-EcoRI junction: NS
(xi) FROM THE SQUENCE: NO: 65:
DESCRIPTION SEQ ID

(2) INFORMATION FOR SEQ ID NO: 66:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 483 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.1-HindIII junction: Dhfr.L.lactis (l0e-93) 5 ~
(xi) DESCRIPTION OF THE SQUENCE: SEQ N0: 66:
ID

TATAATTGAT

TAAAAAACAA

AGCAAAATTT

GGAATATGGG

TTACCTGCTG

ACGAAARACC

ATTTTAACTC

AGCCCTAAGG

(2) INFORMATION FOR SEQ ID NO:
67:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 338 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.12-EcoRI junction: NS
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N0: 67:

TGCTCTAGTT GTGCTAATTT ATCGGCCGCG ACTTTTTAAA GGAATTGAGT ATCATCTTTT 24d (2) INFORMATION FOR SEQ ID NO: 68:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 708 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.12-HindIII junction: NS
(xi) DESCRIPTION OF THE SQUENCE: SEQ NO: 68:
ID

TGANGATNCN

GCCCCAGTTC

ACCTTATAAA

CCGATATGAC

ATTTCTTTAG

AAAAAAACGG

TGTACTTCTT

ACCAATTACA

TGTCACTCCA

TGTCCACTAG

TANCTGAATG

ATTATAACAT

(2) INFORMATION FOR SEQ ID NO:
69:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: 779 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.13-HindIII junction: NS
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N0: 69:

CTTGCGGAAG

TAAACTGATT

GTAATCGCTA AATAGAGCGG TAAAACCGTC AATCGTTTGT P ~ AAAAAAACG360 AAAATTGTTA

NTCGAAACAA

TAACAATCCT

AATGAACTTG

GCAATAATTT

TTTTTTAATG

GGTTTTNCCC ~ CCCCTGAANT TACCTGGAAA TACNTNCCCC TAACCTANCC720 TNTNCCCCNT

ATTTTTTTNN

(2) INFORMATION FOR SEQ ID NO:
70:

(i) FEATURES OF THE SQUENCE:

(A) LENGTH: B08 base pairs (B) TYPE: nuciotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLCULE: DNA

(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.19-EcoRI junction: DeoB.subtilis (2.1e-19) (xi) DESCRIPTION OF THE SEQUENCE: N0: 70:
SEQ ID

ss ATGCCAACAC CACNTATCNA ANCTCAAAA. ~ 1 NGCGAAATCG CCNATAAAAT TCTCCTTACC 780 ANGAAATCCA CTTCCCCCAA AATTTATC gOg (2) INFORMATION FOR SEQ ID NO: 71:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 79l base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CHARACTERISTIC:
(A) NAME / KEY: R2.19-HindIII junction: DeoB.B.subtilis (5.2e-99) (xi) THE SQUENCE: SEQ ID 71:
DESCRIPTION N0:
OF

Claims (18)

59 1/ Bacteria with improved resistance to stress by compared to the wild strain, characterized in that they comprise at least a mutation in a gene which alters the normal activity of said gene or of a embarrassed belonging to the same transcription unit, this gene being involved in the transport of electrons, amino acids, oligopeptides, phosphate, DNA repair, RNA stability or new genes and chosen among - the genes of the GP biosynthetic pathway, - the genes of clone R 1.1 (pstS), R 1.4 (arll), R 1.5 (glnP), R 1.7 (carB), R 1.8 (glnP), R 1.14 (arl2), R 1.16 (glnP), R 1.17 (glnQ), R 1.20 (arl3), R2.6 (pstB), R2.9 (recN), R2.11 (arl4), R2.1S (arl5), R2.17 (arl7), R2.20 (arl8, yybT), pstS, pnpA and trll. 2 / Bacteria according to claim 1, characterized in that the gene of the GP synthesis pathway is chosen from deoB, hpt, guaA, relA,/spoT, tktA. 3 / Bacteria according to claim 1 or 2, characterized in that the mutation arises from the introduction or deletion of a DNA sequence by recombinant pathway in said gene. 4 / Bacteria according to claim 1 or 2, characterized in that the mutation is induced by a mutagen or obtained by mutations spontaneous. 5 / Bacteria according to claim 3, characterized in that the introduced DNA sequence contains at least one mobile genetic element. 6 / Bacteria according to one of claims 1 to 5, characterized in that that the introduced DNA sequence comes from the same bacterial species. 7 / Bacteria according to one of claims 1 to 6, characterized in that that the mutation inactivates or modifies the expression of said gene or of a gene belonging to the same transcription unit. 8 / Bacteria according to one of claims 1 to 7, characterized in that that the bacterium is a lactic acid bacterium. 9 / Bacteria according to claim 8, characterized in that the bacterium lactic acid is chosen from Lactococcus, Lactobacillus and Streptococcus. 10/ Process for the preparation of a bacterium exhibiting resistance to improved stress compared to the wild strain, characterized in that it consists altering by mutation a gene of said bacteria, the gene being involved in the transport of electrons, amino acids, oligopeptides, phosphate, e la DNA repair, RNA stability or new genes and chosen among:
- the genes of the GP biosynthetic pathway, - the genes of R1.1 (pstS), R1.4 (arl1}, R1.5 (glnP), R1.7 (carB), R1.8 (glnP), R1.14 (arl2), R1.16 (glnP), R1.17 (glnQ), R1.20 (arl3), R2.6 (pstB), R2.9 (recN), R2.11 (arl4), R2.15 (arl5), R2.17 (arl7), R2.20 (arl8, yybT), pstS, pnpA and trl1. 11 / Process for obtaining bacteria according to claim 10, characterized in that the alteration consists in inserting into the targeted gene the plasmid pGh:ISS1, then, optionally, excising said plasmid. 12 / Process for obtaining bacteria according to claim 10, characterized in that the alteration of the gene consists of inserting into the gene targeted a DNA sequence by homologous recombination. 13 / Fermentation process, characterized in that it implements, on the medium to be fermented, bacteria according to one of claims 1 to 9. 14 / A method according to claim 13, characterized in that the medium to to ferment is milk or a derivative of milk. 15 / Use of bacteria according to one of claims 1 to 9, in processes for the production and preservation of ferments. 16 / Use of bacteria according to one of claims 1 to 9, in fermentation processes. 17 / Use of bacteria according to one of claims 1 to 9, in food preservation processes. 18 / Use of bacteria according to one of claims 1 to 9, to probiotic purposes.