WO2001071011A1 - Vecteurs pour la transformation genetique du lactobacillus sakei - Google Patents

Vecteurs pour la transformation genetique du lactobacillus sakei Download PDF

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WO2001071011A1
WO2001071011A1 PCT/EP2000/003099 EP0003099W WO0171011A1 WO 2001071011 A1 WO2001071011 A1 WO 2001071011A1 EP 0003099 W EP0003099 W EP 0003099W WO 0171011 A1 WO0171011 A1 WO 0171011A1
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sakei
plasmid
laclm
gfp
gene
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PCT/EP2000/003099
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Laure Gory
Marie-Christine Montel
Monique Zagorec
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Institut National De La Recherche Agronomique
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Priority to AU2000245451A priority Critical patent/AU2000245451A1/en
Priority to EP00926838A priority patent/EP1266017A1/fr
Priority to PCT/EP2000/003099 priority patent/WO2001071011A1/fr
Publication of WO2001071011A1 publication Critical patent/WO2001071011A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)

Definitions

  • 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.
  • 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)].
  • 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.
  • 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 ⁇ -glucuronidase (gusA) gene in L. lactis, Leuconostoc lactis, Lactobacillus plantarum and Lactobacillus casei [PLATTEEUW et al, Appl.
  • 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.
  • 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
  • the inventors have constructed an integrative vector allowing the stable insertion of an heterologous DNA sequence into the chromosomal lacLM operon, encoding L. sakei ⁇ -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 o ⁇ lacL and at least 300 pb of the 3'-end of lacM o ⁇ L. 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,
  • the invention also encompasses L. sakei transformants resulting from the transformation of a L. sakei host cell with a vector of the invention.
  • 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.
  • 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.
  • lacZ gene of E. coli was 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, ⁇ -galactosidase activity was detected in the transformed strains, showing that lacZ can be used as a reporter gene in L. sakei.
  • the use oilacZ as a reporter gene necessitates that the host cell has no endogenous ⁇ -galactosidase activity or that the endogenous ⁇ - galactosidase is inactivated by the insertion of the lacZ construct into lacLM.
  • 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.
  • GFP green fluorescent protein
  • another object of the invention is the use of a gene selected among: a gene encoding a ⁇ -galactosidase, and preferably the lacZ gene encoding the ⁇ -galactosidase of E. coli, and a gene encoding the green fluorescent protein as a reporter gene in L. sakei.
  • 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 ⁇ -galactosidase, and preferably the lacZ gene encoding the ⁇ -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.
  • a reporter gene selected among: a gene encoding a ⁇ -galactosidase, and preferably the lacZ gene encoding the ⁇ -galactosidase of E. coli, and a gene encoding the green fluorescent protein
  • 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.
  • 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.
  • 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)].
  • BHI Brain Heart Infusion
  • 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 ⁇ -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 2-10 3 cells/g and RV2012 was inoculated at 8 TO 3 cells/g.
  • L. sakei strains were inoculated together with S. carnosus 833 at 10 6 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
  • E. coli and L. s ⁇ kei electrocompetent cells were prepared and transformed by the methods of DOWER et ⁇ l [Nucleic acids research., 16, 6127- 6145, (1988)] and BERTHIER et ⁇ l. [Microbiology (Reading), (1996)], respectively.
  • E. coli transformants were selected on LB agar plates containing ampicillin (100 mgT 1 ) or erythromycin (150 mgT 1 ).
  • L. s ⁇ kei transformants were isolated on MRS plates containing erythromycin (5 mg 1 ).
  • X-gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactopyranoside) was added to the MRS agar medium at 32 mg 1 for the selection of I. s ⁇ kei 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 [BIRNBO ⁇ M and DOLY, Nucleic acids research., 7, 1513-1523, (1979)] or QIAG ⁇ N plasmid MAXIPR ⁇ P kits. Plasmids from L. s ⁇ kei 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 ⁇ l, [Applied and Environmental Microbiology., 63, 211 1-2116, (1997)].
  • EXAMPLE 1 CONSTRUCTION OF A VECTOR FOR INTEGRATION AT THE l ⁇ cLM LOCUS OF L. s ⁇ kei
  • the plasmid pRV80 was obtained by the cloning in pRV300 of two
  • Oligonucleotides deduced from this sequence were designed in order to amplify two fragments corresponding respectively to the 5 '-end and 3 '-end of l ⁇ cLM.
  • a restriction site (underlined) was added at the 5 '-extremity of each primer.
  • the lacLM operon contains a 2193 bp internal deletion.
  • 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).
  • 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.
  • 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.
  • 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 / ⁇ cZ 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.
  • Plasmid pJM783 contains the lacZ gene of E. coli, and a chloramphenicol resistance gene [PEREGO et al, Molecular Microbiology, 2, 689-
  • 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.
  • RV1023 Chromosomal integration of the at&y::/-7eZ into the chromosome ofL. sakei. pRV83 was used to transform RV2002, deficient for ⁇ -galactosidase activity.
  • the resulting strain, RV1023 contains an insertion of the atkYv.lacZ fusion and an intact copy of the atkYB operon.
  • the ⁇ -galactosidase activity of RV 1023 was tested on MCD plates containing X-Gal.
  • RV1023 was grown at 30°C in liquid MCD medium until OD 600 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,
  • AtkB is a copper ATPase
  • lacZ can be used as reporter gene in L. sakei
  • 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 .
  • 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-LD ⁇ is known to be a strong constitutive promoter in L. sakei [MALL ⁇ R ⁇ T 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 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- ⁇ lmer 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 ⁇ l mix containing 1 ⁇ g chromosomal DNA template, 0.5 ⁇ M 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.
  • 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 10 3 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 10 8 bacteria/ml, which corresponds to approximately 20 generations per day.
  • Plasmid survival was assessed du ⁇ ng 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.
  • the plasmid pRV86 is a derivative of pRV80.
  • 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).
  • 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.
  • 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).
  • 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 ⁇ - galactosidase activity.
  • RV1040 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
  • 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).
  • 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.
  • 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.
  • GFP-marked L. sakei transformants 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
  • 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).
  • RV1040 and RV2010 The implantation of RV1040 and RV2010 was assessed by measuring the percentage of fluorescent colonies.
  • samples were diluted in saline solution (NaCl 8,5 gT 1 ) and stomached for 1 min.
  • saline solution NaCl 8,5 gT 1
  • 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 equipped with a GFP filter set (Excitation 470 nm; emission 505-530 nm) was used to visualize fluorescent cells.
  • 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.
  • 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.
  • a complex medium such as dry sausage.
  • the final counts measured in dry sausage samples reached 7T0 8 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.

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Abstract

L'invention concerne des procédés et des vecteurs permettant d'obtenir des transformants stables de L. sakei. L'invention concerne plus spécifiquement des vecteurs d'intégration permettant d'insérer de l'ADN hétérologue au niveau du locus chromosomique LacLM. L'invention concerne également des gènes marqueurs pour la surveillance de souches de L. sakei.
PCT/EP2000/003099 2000-03-23 2000-03-23 Vecteurs pour la transformation genetique du lactobacillus sakei WO2001071011A1 (fr)

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AU2000245451A AU2000245451A1 (en) 2000-03-23 2000-03-23 Vectors for the genetic transformation of lactobacillus sakei
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2770536A1 (fr) * 1997-11-06 1999-05-07 Texel Nouveau plasmide non rcr apte a etre transfere dans des bact eries lactiques; utilisation comme outil de clonage et d'expression

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2770536A1 (fr) * 1997-11-06 1999-05-07 Texel Nouveau plasmide non rcr apte a etre transfere dans des bact eries lactiques; utilisation comme outil de clonage et d'expression

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LELOUP LAURENCE ET AL: "Single-crossover integration in the Lactobacillus sake chromosome and insertion inactivation of the ptsI and lacL genes.", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 63, no. 6, 1997, pages 2117 - 2123, XP002159436, ISSN: 0099-2240 *
STENTZ REGIS ET AL: "Development of genetic tools for Lactobacillus sakei: Disruption of the beta-galactosidase gene and use of lacZ as a reporter gene to study regulation of the putative copper ATPase, AtkB.", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 66, no. 10, October 2000 (2000-10-01), pages 4272 - 4278, XP000980455, ISSN: 0099-2240 *

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