EP1266017A1

EP1266017A1 - Vectors for the genetic transformation of lactobacillus sakei

Info

Publication number: EP1266017A1
Application number: EP00926838A
Authority: EP
Inventors: Laure Gory; Marie-Christine Montel; Monique Zagorec
Original assignee: Institut National de la Recherche Agronomique INRA
Current assignee: Institut National de la Recherche Agronomique INRA
Priority date: 2000-03-23
Filing date: 2000-03-23
Publication date: 2002-12-18
Also published as: AU2000245451A1; WO2001071011A1

Abstract

The invention concerns methods and vectors allowing to obtain stable transformants of L. sakei. More specifically, the invention provides integrative vectors allowing insertion of heterologous DNA at the LacLM chromosomal locus. The invention also concerns marker genes for monitoring L. sakei strains.

Description

VECTORS FOR THE GENETIC TRANSFORMATION OF LACTOBACILLUS SAKEI

The invention relates to methods and tools for the genetic transformation of Lactobacillus sakei.

Lactobacillus sakei is widely used in the food industry, and more specifically in the production of fermented meat products.

Some Lactobacillus sakei strains inoculated as starter cultures play an important role in the development of the organoleptic qualities of fermented meat products. L. sakei, by producing lactic acid, contributes to the hygienic safety and sensorial properties of dry sausage and may also have a role in flavor development [HAMMES et al, FEMS Microbiology Reviews, 87, 165-174, (1990)]. However, the strains, which are inoculated as starters, are in competition with the natural flora of meat and fermented meat products composed of L. sakei and other Lactobacillus, Staphylococcus and Enterococcus species. Then, in the industry of fermented meat products, there is clearly a need to monitor, among the natural flora, the growth and the activity of inoculated starter cultures. Traditional techniques for microbial assessment lacked the specificity to monitor specific species. Therefore, molecular techniques, such as the use of DNA probes have recently been developed as specific methods to identify and quantify populations of microorganisms in complex environments. In situ hybridization with oligonucleotidic probes targeting rRNAs have successfully been used to detect and identify Gram-negative filamentous bacteria in sludge [WAGNER et al, Systematic and Applied Microbiology, 17, 405-417, (1994)], lactococci in milk [BEIMFOHR et al, Systematic and Applied Microbiology, 16, 450-456, (1993)] or sulfate reducing bacteria in mixed cultures AMANN et al. [Applied and Environmental Microbiology, 56, 1919-1925, (1990)]. GRANT et al. [Journal of Applied Bacteriology, 74, 260-267, (1993)] and FURRER et al, [Journal of Applied Bacteriology, 70, 372-379, (1991)] have detected bacteria in food or cheese by the amplification of specific sequences using the Polymerase Chain Reaction (PCR).

These molecular techniques are species specific and sensitive but do not allow the detection of specific strains. Furthermore, they are limited by nucleic acid extraction from the complex environment and by factors limiting PCR efficiency.

Stable marker systems with an easily detectable phenotype provide an alternative strategy to detect microorganisms in complex environments. Several genes have thus been used as reporter genes in lactic acid bacteria, such as the luciferase genes in Lactococcus lactis [CORTHIER et al, Applied and Environmental Microbiology, 64, 2721-2722, (1998); STEWART and WILLIAMS, Journal of General Microbiology, 138, 1289-1300, (1992); EATON et al, J. Gen. Microbiol, 139, 1495-1501, (1993)], the β-glucuronidase (gusA) gene in L. lactis, Leuconostoc lactis, Lactobacillus plantarum and Lactobacillus casei [PLATTEEUW et al, Appl.

Environ. Microbiol., 60, 587-593, (1994)], the nuc gene in . lactis [LE LOLR et al, J.

Bacteriol., 176, 5135-5139, (1994)], or the gfp gene in L. plantarum [GEOFFROY et al., Appl. Environ. Microbiol., 66, 383-391, (2000)] and L. lactis [SCOTT et al, FEMS Microbiol. Lett., 182, 23-27, (2000)]. The lacZ gene of Escherichia coli, encoding β-galactosidase, and the chloramphenicol acetyl transferase gene have also been widely used in bacteria.

However, although several genes have now been cloned from

L. sakei [HAMMES and HERTEL, Meat Science, 49, 125-138, (1998)] and some genetic tools are emerging [AXELSSON et al, FEMS Microbiol. Lett., 168, 137-143,

(1998); BERTHIER et al, Microbiology (Reading), 142, 1273-1279, (1996);

LANGELLA et al, FEMS Microbiol. Lett., 139, 51-56, (1996); the molecular biology techniques specific for this species are still poorly developed. For example, no reporter gene system easy to handle has yet been developed that would help in the analysis of gene regulation.

The purpose of the invention is to develop new tools for the genetic engineering of L. sakei, and more specifically tools allowing the monitoring of one or several L. sakei strain(s) in a complex environment, without altering their growth and activity of L. sakei. Accordingly, the invention provides vectors allowing a stable and strong expression of heterologous DNA sequence in L. sakei without interfering with the expression of endogenous genes.

The inventors have now found that surprisingly, derivatives of the pG+host plasmids which have been initially developed as thermosensitive vectors to generate chromosomal insertions in several Gram-positive bacteria [BISWAS et al,

Journal of Bacteriology, 175, 3628-3635, (1993); PCT WO 93/18164, CNCM 1-1179) are very stable in a replicative form in L. sakei, both in growth laboratory conditions and during experimental dry sausage production. An object of the invention is the use of said derivatives of pG+host plasmids as extrachromosomal vectors for the stable expression of a gene of interest in L. sakei.

"Derivatives of pG+host plasmids" are herein defined as plasmids comprising at least the thermosensitive replication sequences of the plasmid pVE6002

(CNCM 1-1179) disclosed in PCT WO 93/18164.

Further, the inventors have constructed an integrative vector allowing the stable insertion of an heterologous DNA sequence into the chromosomal lacLM operon, encoding L. sakei β-galactosidase.

The invention relates to said integrative vector resulting from the insertion of a portion of the lacLM operon of L. sakei comprising of at least 300 pb of the 5'-end oϊlacL and at least 300 pb of the 3'-end of lacM oϊL. sakei into the suicide vector pRV300. pRV300 is disclosed by LELOUP et al. [Appl. Environm. Microbiol, 63, 2117-2123, (1997)]. It is composed of a pBluescript SK replicon for propagation in Escherichia coli and an erythromycin resistance marker. An heterologous DNA sequence may be inserted between the lacL and the lacM sequences.

The invention also relates to a process for obtaining a stable transformant of L. sakei wherein said process comprises transforming a L. sakei host cell with a vector selected among: - an integrative vector as defined above,

- a derivative of a pG+host plasmid.

The invention also encompasses L. sakei transformants resulting from the transformation of a L. sakei host cell with a vector of the invention. This include L. sakei cells containing at least one copy of a derivative of a pG+host plasmid as defined above, as well as L. sakei cells wherein a part of the chromosomal lacLM operon is deleted, or L. sakei cells having an heterologous DNA sequence inserted into the chromosomal lacLM operon.

The invention also provides marker genes able to be expressed in L. sakei, and easily detected even in a complex environment. In preliminary experiments, the inventors tested several genes commonly used as reporter genes in lactic acid bacteria; however, no satisfactory expression was observed in L. sakei. For instance, the luciferase genes could not be expressed at a sufficient level, and the expression of gusA could not be detected.

The inventors performed further experimentations, using lacZ gene of E. coli as a reporter gene. A strain was constructed in which an internal part of the lacLM operon was deleted. A construct comprising lacZ under transcriptional control of an inducible L. sake promoter was inserted into the chromose. Under conditions allowing the induction of the promoter, β-galactosidase activity was detected in the transformed strains, showing that lacZ can be used as a reporter gene in L. sakei. However the use oilacZ as a reporter gene necessitates that the host cell has no endogenous β-galactosidase activity or that the endogenous β- galactosidase is inactivated by the insertion of the lacZ construct into lacLM.

In order to obtain a more versatile system, also usable in cells having an endogenous β-galactosidase activity, the inventors tested other reporter genes, and selected the green fluorescent protein (GFP). They constructed L. sakei strains comprising a gfp gene under the control of a strong constitutive promoter integrated into a replicative plasmid or into the chromosome of L. sakei. In both case they observed a strong and stable expression of GFP. Expression of GFP did not alter the growth of the L. sakei transformants and allowed to detect GFP-marked strains directly on plates or in dry sausage. Further, strains obtained with a replicative plasmid or by chromosomal integration were stable both in growth laboratory conditions and during experimental dry sausage production. Thus, another object of the invention is the use of a gene selected among: a gene encoding a β-galactosidase, and preferably the lacZ gene encoding the β-galactosidase of E. coli, and a gene encoding the green fluorescent protein as a reporter gene in L. sakei.

More specifically the invention provides a process for providing a detectable cell of L. sakei, wherein said process comprises providing an expression vector comprising a reporter gene selected among: a gene encoding a β-galactosidase, and preferably the lacZ gene encoding the β-galactosidase of E. coli, and a gene encoding the green fluorescent protein, under transcriptional control of a promoter active in L. sakei and transforming an host cell of L. sakei with said expression vector.

Preferably the expression vector is obtained from a replicative vector derived from a pG+host plasmid or from an integrative vector of the invention, allowing the stable insertion of an heterologous gene into the chromosomal lacLM operon. The reporter gene can be placed under transcriptional control of an exogenous promoter active in L. sakei or under transcriptional control of an endogenous L. sakei promoter. This second embodiment allows to study the regulation of said endogenous promoter under different environmental conditions.

The invention also includes detectable cells of L. sakei, obtainable by the above-defined process.

The invention can be used, for instance, to monitor the development of L. sakei in a complex medium, like in dry sausage fermentation, to estimate its competition with other flora, or to test new L. sakei starter strains in order to demonstrate their ability to develop in meat products.

These strains are thus good tools to monitor the development of

L. sakei in environmental conditions applied in dry sausage fermentation and to estimate the competition with other flora. It should be possible to use both plasmids to test new L. sakei starter strains in order to demonstrate their ability to develop in meat products.

The present invention will be further illustrated by the additional description which follows, which refers to examples of expression of reporter genes in L. sakei according to the invention. It should be understood however that these examples are given only by way of illustration of the invention and do not constitute in any way a limitation thereof.

EXAMPLES Bacterial strains and growth conditions

Bacterial strains are listed in Table 1.

E. coli strain TGI was used for cloning and subcloning experiments and for plasmid propagation. The E. coli strain GM2929 carrying a dcm mutation was used for the propagation of plasmids prior to digestions with the Bali restriction enzyme. The E. coli strain XLlBlue (Stratagene) was used for single strand DNA preparation.

E. coli TGI were grown in Luria-Bertani (LB) medium [SAMBROOK et al, Molecular cloning. A laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, NY, (1989)] at 37°C with agitation. L. sakei 23K isolated from sausage and plasmid cured was used as recipient strain for genetic constructions. L. sakei strains were grown at 30°C in the MRS medium [DE MAN et al, Journal of Applied Bacteriology, 23, 130-135, (I960)]. For the inoculation in dry sausage model, Staphylococcus carnosus was grown at 30°C with agitation in the Brain Heart Infusion (BHI) medium (Difco). The Mannitol Salt Agar medium (MSA, Difco) was used for the detection of S. carnosus on dry sausage samples. The MCD medium was used for the detection of β-galactosidase activity in lacZ marked strain [LAURET et al, Appl. Environ. Microbiol., 62, 1922-1927, (1996)].

L. sakei 23K and the GFP-marked strain RV1040 were inoculated at 2-10³ cells/g and RV2012 was inoculated at 8 TO³ cells/g. L. sakei strains were inoculated together with S. carnosus 833 at 10⁶ cells/g in dry sausage model prepared as described by MONTEL et al, [Food Microbiology., 13, 489-499, (1996)]. Dry sausage models were incubated for 3 days at 22°C, then at 14°C. The survival of the strains at 0, 3, 11 and 28 days was followed by plating diluted aliquots on MRS and MSA. Table I

A : BERTHIER et al, Microbiology (Reading)., 142, 1273-1279, (1996)

B: SAMBROOK et al., Molecular cloning, A laboratory manual, 2nd ed. Cold Spring Harbor

Laboratory, NY, (1989)

Electrotransformation procedures

E. coli and L. sαkei electrocompetent cells were prepared and transformed by the methods of DOWER et αl [Nucleic acids research., 16, 6127- 6145, (1988)] and BERTHIER et αl. [Microbiology (Reading), (1996)], respectively. E. coli transformants were selected on LB agar plates containing ampicillin (100 mgT¹) or erythromycin (150 mgT¹). L. sαkei transformants were isolated on MRS plates containing erythromycin (5 mg ¹).

X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) was added to the MRS agar medium at 32 mg ¹ for the selection of I. sαkei RV2012. Plasmid isolation and characterization

Plasmids used and constructed in this study are listed in Table II. Plasmids were purified from E. coli cultures using the alkaline lysis method [BIRNBOΓM and DOLY, Nucleic acids research., 7, 1513-1523, (1979)] or QIAGΕN plasmid MAXIPRΕP kits. Plasmids from L. sαkei were isolated following the method described by ANDERSON and McKAY [Applied and Environmental Microbiology, 46, 549-552, (1983)]. Chromosomal DNA was prepared by standard methods as previously described by STENTZ et αl, [Applied and Environmental Microbiology., 63, 211 1-2116, (1997)]. Restriction and other DNA modifying enzymes were obtained from Boehringer Mannheim, and used as recommended by the manufacturer. Specific restriction fragments of DNA used in cloning experiments were extracted from agarose gels by the use of Gene Clean kit (Ozyme, France). Plasmids were restricted in such a way as to give compatible ends and were treated with alkaline phosphatase to prevent subsequent self-ligation. Table II

A: LELOUP et al., Applied and Environmental Microbiology, 63, 21 17-2123, (19 7) B: BISWAS et at, Journal of Bacteriology, 175, 3628-3635, (1993) C: PEREGO et al., Molecular Microbiology, 2, 689-699, (1988)

EXAMPLE 1 : CONSTRUCTION OF A VECTOR FOR INTEGRATION AT THE lαcLM LOCUS OF L. sαkei

The plasmid pRV80 was obtained by the cloning in pRV300 of two

PCR amplified DNA fragments, containing respectively the 5'- and 3'-ends of lαcLM with a large internal deletion. The lαcLM operon of L. sαkei DSM 20017 was previously cloned and sequenced [OBST et αl., Microbiology (Reading), 141, 3059-

3066, (1995)].

Oligonucleotides deduced from this sequence were designed in order to amplify two fragments corresponding respectively to the 5 '-end and 3 '-end of lαcLM. A restriction site (underlined) was added at the 5 '-extremity of each primer. lacl :

5 ' -GATC AAGCTT ATGCTTT AAGGGT ACTGG lac2:

5 '-ACGTGAATTCTTGTCATCGGACGTTGAA lac4:

5'-GATCGAGCTCGC GCTTTGAACAATAGCT lac6:

5 '-ACGTGAATTCCGGTGCTGGATAATTGTT

A first PCR fragment, obtained with the lacl and lac2 primers, was 513 bp long and contained the lαcLM promoter and the 5'-end of lαcL.

The second fragment, obtained with the lac4 and lac6 primers, was

522 bp long and contained the 3 '-end of lαcM and the downstream region. The two resulting PCR fragments were cloned in the pRV300 integrative vector. In the resulting plasmid pRV80, the lacLM operon contains a 2193 bp internal deletion.

EXAMPLE 2: USE OF LACZ AS A REPORTER GENE

Construction of a lacLM mutant bv double crossover, for use as recipient strain of lacZ fusions

In order to use lacZ as a reporter gene, it was necessary to first delete the lacLM operon of L. sakei 23K encoding the L. sakei β-galactosidase. Plasmid pRV80 can be integrated by two successive crossovers at the lacLM locus allowing gene replacement of the wild type lacLM operon by the deleted operon. Plasmid pRV80 was used to transform L. sakei 23K for erythromycin resistance (see Fig.l).

The correct insertion of pRV80, by a Campbell-like recombination, at the lacLM locus was checked by a PCR experiment using the lacl and lac4 primers. One transformant, RV2001, was then kept for further experiments. Since pRV80 contains the 5 '-end and the 3 '-end of the lacLM operon, its insertion in the chromosome by a single crossover in lacLM restored one copy of the wild-type operon and was therefore not mutagenic. Additionally, the insertion of pRV80 led to the duplication of part of the lacLM operon, a structure which is unstable unless selective pressure is maintained by the addition of erythromycin. In the absence of selective pressure, the plasmid can excise, by a second crossover, and is then lost since pRV300 can not replicate in L. sakei. Since one copy of the wild type lacLM operon, and one copy of the mutated lacLM operon are present after the first recombination, the excision of the plasmid could lead to the excision either of the wild type lacLM copy, or of the AlacLM copy. The excision of the mutated copy would restore a wild type genotype, whereas the excision of the wild type copy would lead to a copy of the lacLM operon with the internal deletion (Fig. l). In order to generate the second crossover, the RV2001 transformant was grown in MRS without erythromycin. After 100 generations, diluted culture aliquots were plated on MRS containing X-Gal and no erythromycin. Among 300 clones, two were white. The structure of the lacLM operon was verified by PCR on the chromosomal DNA extracted from these two clones, with primers complementary to various parts of lacLM. The expected 2.2 kb deletion of the internal part of lacLM was demonstrated. Moreover, these two clones were erythromycin sensitive and had thus lost the pRV300 moiety. This confirmed that the two strains were resulting from the expected recombination. One of the two clones was named RV2002 and was used for further constructions. Figure 1 represents the steps of construction of an internal deletion in the lacLM operon. Transformants, selected on erythromycin can result from a crossover at position 1 or 2. In each case, one copy of the WT lacLM operon and one copy of the AlacLM operon are present. The duplication of part of the /αcZ operon is unstable. By growing one transformant without erythromycin, strains resulting from a second crossover can be isolated. The plasmid can excise at position 3, leading to replacement of lacLM by AlacLM, or at position 4, restoring a WT genotype.

Insertion of lacZ under control of the inducible promoter of the atkYB operon

Isolation of the atkYB promoter and construction of an atkYv.lacZ transcriptional fusion

The sequences located downstream from the ptsl gene of L. sakei

[STENTZ et al, Appl. Environ. Microbiol., 63, 2111-2116, (1997); and GenBank accession N° U82366] encode a putative copper efflux P-type ATPase and its negative regulator organized in an operon (atkYB operon). A 634 bp DNA fragment, containing the transcription terminator of ptsl, the putative promoter region of atkYB, and atkY, was amplified by PCR from chromosomal DNA of L. sakei using the following primers:

ATK1 :

5 '-GATCGAATTCTAGTCGAAGATTTTATGA, and ATK3:

5 '-GATCGGATCCCATCGTGTTTCATCGTTA

PCR experiments were performed on a Perkin-Elmer 9600 apparatus, with Taq DNA polymerase from Boehringer. Reactions were carried out in

100 μl mixtures containing 0.2 mM of each deoxynucleoside triphosphates, 1 μg chromosomal DNA template and 2.5 μM of each primer. Amplification was performed for 30 cycles (94°C, 1 min; 55°C, 2 min; and 72°C, 3 min).

Plasmid pJM783 contains the lacZ gene of E. coli, and a chloramphenicol resistance gene [PEREGO et al, Molecular Microbiology, 2, 689-

699, (1988)]. This plasmid has already been largely used to construct transcriptional or translational fusions in Bacillus subtilis. The 634 bp fragment was cloned in pJM783, upstream from lacZ resulting in pRV81. pRV83 was obtained by cloning the EcoRI/Ba fragment of pRV81 containing the atkYv.lacZ fusion, into the integrative plasmid pRV300.

Chromosomal integration of the at&y::/-7eZ into the chromosome ofL. sakei. pRV83 was used to transform RV2002, deficient for β-galactosidase activity. The resulting strain, RV1023 contains an insertion of the atkYv.lacZ fusion and an intact copy of the atkYB operon. The β-galactosidase activity of RV 1023 was tested on MCD plates containing X-Gal.

Fifty microliters of various solutions of metals or salts ions were added in the center of the plates and let to diffuse from the center to the periphery of the plates. Only copper allowed the expression of the atkYBv.lacZ fusion as seen from the blue color of the clones. However the color was observed only at a certain distance from the center of the plates, suggesting that the expression was induced by a precise concentration of CuSO . Furthermore, the activity was higher when plates were incubated at room temperature (21 ± 1 °C) than at 30°C. Further experimentations were performed in order to allow a more precise characterization of the β-galactosidase activity under induction by CuSO .

RV1023 was grown at 30°C in liquid MCD medium until OD₆₀₀ 0.3, then various concentrations of CuS0 were added and cultures were incubated for 90 min at room temperature. Bacteria from 10 ml culture aliquots were collected by centrifugation, and resuspended in 1 ml Z buffer (sodium phosphate, 100 mM, pH 7.0,

KC1 10 mM, MgSO₄ 1 mM, β-mercaptoethanol 50 mM) containing 20% glycerol.

Bacteria were broken with zirconium beads in a Fast-Prep bead beater (Bio 101), two times 20 sec at maximum speed with 5 min pause on ice. Cellular debris were removed by centrifugation. The β-galactosidase activity was measured in 1 ml Z buffer at 28°C. The reaction was started by the addition of 200 μl ONPG (4 mg ml ^"') and stopped with 500 μl Na?CO₃ 1M. Absorbance was measured at 420 nm. Activity was expressed in Miller Units [MILLER, Cold Spring Harbor Laboratory Press, Cold

Spring Harbor, NY., p. 72-74, (1992)]. The results are the means of at least three independent assays.

The results of these experiments are shown in Figure 2, which represents the induction of β-galactosidase activity of the atkYBv.lacZ fusion in RV1023 as a function of CuSO₄ concentration in the medium. These results show that, as expected from the observation made on plates, an optimal concentration of CuSO₄ exists that allows expression of the atkYBv.lacZ fusion. This concentration, (30-40 μM) is much lower than the concentration required to inhibit the growth of L. sakei.

The transcription level of the atkYv.lacZ fusion in different conditions allowed to identify the regulation of this operon and suggest that AtkB is a copper ATPase.

This study shows that lacZ can be used as reporter gene in L. sakei, and demonstrates that the 634 bp PCR amplified fragment comprises the copper inducible promoter of atkYB which may be used in order to expressed foreign genes in L. sakei, with an expression controlled by the addition of small amounts of CuSO .

EXAMPLE 3: USE OF GFP AS A REPORTER GENE

In order to test GFP as marker expressed in L. sakei, two vectors were constructed: one replicative vector based on the plasmid pG+host5, and one integrative vector was derived from pRV80.

Construction of a replicative plasmid comprising the sfp, v gene under control of the pldhL promoter of L. sakei

A GFPuv protein (Clontech) optimized for the maximal fluorescence when excited by UV light was used. This modified GFPuv contains three amino acid substitutions (Phe99Ser, Metl53Thr, Vail 63 Ala), and fluoresces 18 times brighter than the wild-type GFP.

The gfp_uv gene was isolated from the plasmid pGFPuv (Clontech) as an Hz^'rzdlll/EeoRI fragment. The promoter of the IdhL gene (pldhL), encoding the L-LDΗ is known to be a strong constitutive promoter in L. sakei [MALLΕRΕT et al, Microbiology, 144, 3327-3333, (1998)].

A 234 bp fragment encompassing this promoter was obtained by PCR amplification on L. sakei 23K chromosomal DNA. The primers used, designed from the known sequence of the IdhL gene (GenBank accession n° AF054624) were: forward primer (corresponding to bases 3973-3990) ldhl: 5 ' -ATGCGAATTCTACTGAGAAGTTGCTCTC-3 ' ; reverse primer (corresponding to bases 4190-4207) ldh2: 5 '-AGCTAAGCTTTCGCCGACGAGGATAACT-3 ' . Restriction sites added at the 5 '-end of each primer are underlined.

The PCR amplification was performed on a Perkin-Εlmer 9600 apparatus, for 25 cycles (94°C, 2 min; 55°C, 2 min; 72°C, 2 min) with a final elongation step at 72°C for 5 min. Reaction was carried out in 100 μl mix containing 1 μg chromosomal DNA template, 0.5 μM of each primer, 0.2 mM of each dNTP and 2.5 U of Taq DNA polymerase from Boehringer.

The amplified fragment was restricted by EcoRI and H dlll.

The pldhL promoter and gfp_uv gene were cloned into pG+host5, at the EcoRI site. In the resulting plasmid, named pRV85, the gfp_uv gene is placed downstream from the constitutive promoter pldhL (Fig. 3). The structure of pRV85 was verified by restriction analysis. The pldhL::gfp_uv fusion was verified by DNA sequencing with the primers used for the amplification of the pldhL promoter. Plasmid stability

The replicative plasmid pRV85 was used to transform L. sakei 23K for erythromycin resistance. One transformant (RV1040) containing the plasmid was grown in liquid MRS medium without erythromycin in order to test the stability of the plasmid pRV85 in L. sakei. Approximately 10³ bacteria were inoculated to 100 ml of MRS, grown for 24 hours and then diluted to inoculate a new culture. In these growth conditions, cultures reached 10⁸ bacteria/ml, which corresponds to approximately 20 generations per day. Plasmid survival was assessed duπng 100 generations by plating diluted aliquots on MRS medium with or without erythromycin and comparing duplicate colony counts on selective or non-selective MRS plates. After 100 generations under non-selective conditions, 100% of the clones were still erythromycin resistant, indicating that the plasmid pRV85 was still present. The presence of pRV85 was confirmed by fluorescence of clones and by restriction digestion of plasmids extracted from L. sakei colonies grown under non-selective conditions.

The stability of pRV85 in L. sakei 23K shows that this plasmid can be used as a valuable tool to maintain transformed strains even in the absence of antibiotic selection. Emission of fluorescence The GFP fluorescence intensity of the colonies of RV 1040 grown at

14°C, 22°C and 30°C was quantitatively determined by the use of a Fluorlmager

(Table III).

Table III

No fluorescence was detected in the control strain 23K. The maximum expression of GFP in RV1040 was obtained at 22°C. This expression was 1.5 fold and 8 fold weaker at 14°C and at 30°C, respectively, than at 22°C indicating a temperature dependent variation of the fluorescence of GFP.

Construction of an integrative plasmid comprising the p„v gene under control of the pldhL promoter of L. sakei In order to test whether L. sakei strains containing a single copy of the pldhLv.gfp_uv fusion were detectable, the integrative plasmid pRV86 was also used to transform L. sakei 23K.

The plasmid pRV86 is a derivative of pRV80. First, the multiple cloning site, upstream from lacL, in pRV80 was deleted by digestion with Bsp 120-1 and Hz«dIII. The resulting plasmid, pRV84, presents a single EcoRI site between the lacL and lacM parts. An EcoRI fragment of pRV85 comprising the pldhLv.gfp_uv fusion was cloned at the EcoRI site of pRV84, leading to pRV86 (Fig 3).

Chromosomal integration of the pldhLv.sfp_^ into the chromosome of L. sakei 23K The plasmid pRV86 which contains the fusion pldhL::gfp_uv flanked by 513 bp of the 5 '-end of lacL and 522 bp of the 3 '-end of lacM can be integrated into the chromosome of L. sakei by homologous recombination either in lacL or in lacM.

Figure 4 schematically represents the insertion of pldhLv.gfp_uv in the lacLM operon of L. sakei 23K by two successive crossovers.

The L. sakei 23K strain was transformed with pRV86 for erythromycin resistance.

Single crossover integration of pRV86, at the lacLM locus, was checked by PCR on chromosomal DNA extracted from transformants. One transformant, RV2011, containing a single copy of pRV86 was selected and used for further experiments. This transformant, resulting from the integration of pRV86 by single cross-over, had one copy of the wild type lacLM operon and one copy of the mutated lacLM operon (AlacLM replaced by the pldhLv.gfp_uv fusion , Fig. 5 A). In the absence of selective pressure, the plasmid can excise by a second, reverse, crossover. Since pRV86 is not replicative in L. sakei, the plasmid is lost after its excision. The second crossover could lead to the excision of the mutated AlacLM copy with the gfp_uv gene or the wild-type lacLM copy. The excision of the AlacLMIgfp_m gene should restore a wild-type genotype, whereas the excision of the lacLM operon, should lead to gene replacement with a copy of the gfp_uv gene, without any other heterologous DNA (Fig. 4 B). In order to generate the second crossover, the transformant RV2011 was grown in MRS without erythromycin. The presence of a wild-type lacLM operon can be detected by the blue color of colonies in the presence of X-Gal, reflecting β- galactosidase activity. After 100 generations, diluted culture aliquots were plated on MRS medium containing X-Gal. Among 400 clones, one was white suggesting that the lacLM operon was replaced by the pldhL::gfp_uv fusion. This clone, named RV2012, was erythromycin sensitive but still fluorescent. Chromosomal DNA from RV2012 was extracted and the structure of the integrated gfp_Uv gene was verified by PCR. As expected, the lacLM operon was replaced by the pldhLv.gfp_uv fusion.

Emission of fluorescence The GFP fluorescence intensity of the colonies of RV2012 grown at

14°C, 22°C and 30°C was determined in the same way as above for RV1040. The temperature dependent variation was similar to the one observed with RV1040. At all temperatures, RV1040 exhibited a higher fluorescence intensity than L. sakei RV2012, which might reflect a higher expression of GFP because of the higher copy number of the pldhL::gfp_uv fusion in RV1040.

EXAMPLE 4: PROPERTIES OF GFP-MARKED L. SAKEI TRANSFOR- MANTS

Growth of the transformed strains

The putative burden caused by the presence of a plasmid in RV1040 or by the expression of GFP in RV1040 and RV2012 was examined. The control strain L. sakei 23K and the two transformants were grown in liquid MRS medium at 22°C, the incubation temperature for fermentation period of dry sausage, and 30°C, the optimal growth temperature for L. sakei.

Figure 5 represents the growth of the wild type strain 23K (squares), the strain RV1040 transformed with replicative plasmid pRV85 (triangle) and the strain RV2012 containing an integrated copy of gfpnv (circles) grown at 22°C (open symbols) or 30°C (bold symbols).

At both temperatures, no difference was detected between the parental strain 23K and the two transformed strains. This shows that no excessive metabolic burden was caused by the presence of the constructions.

Lactate production In both transformants, a single copy of the IdhL gene is present but its promoter is duplicated. The amount of L-lactate in culture supematants of 23K and the two transformed strains RV1040 and RV2012 was measured with a Boehringer Mannheim kit, in the conditions described by the manufacturer.

Figure 6 represents the L-lactate concentration in culture supematants of the strains 23K, RV1040 and RV2012 grown in MRS medium for 10 or 24 hours at 22°C (open symbols) or 30°C (bold symbols).

The L-lactate concentration produced by RV1040 and RV2012 was similar to that produced by the wild-type strain. The production of L-lactate was thus not affected by the presence of the gfp_uv gene under the control of IdhL promoter. For the three strains, the pH of the culture supematants after 24 hours culture dropped from 5.6 in the initial medium to 4.2 at 30°C and 4.4 at 22°C.

Monitoring GFP-marked L. sakei in dry sausage

Since the expression of GFP was detectable in both GFP-marked L. sakei transformants, these strains were used in a complex environment. GFP-marked L. sakei strains were inoculated in dry sausages with

S. carnosus 833, and assessed during 28 days (Fig. 7). Bacterial flora was followed by plating diluted aliquots on MRS and MSA media, and by the detection of fluorescence.

Figure 7 represents the bacterial counts (cfu/g) observed after plating of dry sausage sample aliquots on MRS plates. The dry sausage samples were inoculated with 23K (squares), RV1040 (triangles) or RV2012 (circles).

In all dry sausage samples, the number of lactic acid bacteria increased during the first 3 days (from 8T0³ or 2T0^D to 4-7T0⁸ cfu/g), then stabilized at 4-7- 10⁸ cfu/g during 28 days.

The implantation of RV1040 and RV2010 was assessed by measuring the percentage of fluorescent colonies.

To detect GFP-tagged L. sakei strains directly in dry sausages models, samples were diluted in saline solution (NaCl 8,5 gT¹) and stomached for 1 min. For the observation of fluorescent cells by epifluorescence microscopy the cells were prepared as follows: liquid cultures or dry sausage suspensions were centrifuged, cells were washed in saline solution and smeared on microscope slides. Before observation, slides were treated by overlaying with CITIFLUOR.

An epifluorescent microscope (ZEISS) equipped with a GFP filter set (Excitation 470 nm; emission 505-530 nm) was used to visualize fluorescent cells.

To quantify the fluorescence of bacterial clones, colonies on MRS plates were observed by using a Fluorlmager (Molecular Dynamics) coupled with an image analysis software (Image Quant).

The isolates from MRS plates of dry sausage samples inoculated by the 23K strain, exhibited no fluorescence. On the contrary, 100% of colonies isolated from samples inoculated by strains RV1040 and RV2012 were fluorescent at 0, 3, and 1 1 days. At 28 days, 100% of the clones, isolated on MRS, from sausages inoculated with RV2012 were fluorescent whereas 95% of fluorescent clones were detected with sausages inoculated with RV1040. Fluorescent strains were assimilated to L. sakei RV1040 and RV2012. The non fluorescent clones, isolated from dry sausage samples inoculated by RV1040, could correspond to lactobacilli of natural flora, or to the loss of the marker plasmid pRV85 in L. sakei RV1040.

Although pRV85 was shown to be extremely stable after 100 generations in MRS medium, the plasmid might be less stable, or the strain RV1040 carrying the plasmid might grow more slowly in a complex medium such as dry sausage. When inoculated at 2-10³ cfu g with RV1040, the final counts measured in dry sausage samples reached 7T0⁸ cfu/g which corresponds to approximately 12 generations, far less than the 100 generations followed in laboratory growth conditions. The GFP-marked L. sakei strains RV1040 and RV2012 can nevertheless be detected directly in dry sausage suspension by epifluorescence microscopy.

Claims

CLAIMS 1) An integrative vector resulting from the insertion of a portion of the lacLM operon of L. sakei comprising at least 300 pb of the 5 '-end of lacL and at least 300 pb of the 3 '-end of lacM of into vector pRV300. 2) An integrative vector of claim 1 further comprising an heterologous DNA sequence inserted between the lacL and the lacM sequences.

3) An integrative vector of claim 2, wherein said heterologous DNA sequence is selected among: a gene encoding the β-galactosidase of E. coli, and - a gene encoding the green fluorescent protein.

4) A process for obtaining a stable transformant of L. sakei wherein said process comprises transforming a L. sakei host cell with a vector selected among:

- an integrative vector of any of claims 1 to 3;

- a derivative of a pG+host plasmid. 5) A transformant of L. sakei obtainable by the process of claim 4.

6) A transformant of claim 5, wherein said transformant expresses the green fluorescent protein.