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PubMed1996

The ribonucleotide reductase system of Lactococcus lactis. Characterization of an NrdEF enzyme and a new electron transport protein.

Escherichia coli contains the genetic information for three separate ribonucleotide reductases. Two of them (class I enzymes), coded by the nrdAB and nrdEF genes, respectively, contain a tyrosyl radical, whose generation requires oxygen. The NrdAB enzyme is physiologically active. The function of the nrdEF gene is not known. The third enzyme (class III), coded by nrdDG, operates during anaerobiosis. The DNA of Lactococcus lactis contains sequences homologous to the nrdDG genes. Surprisingly, an nrdD- mutant of L. lactis grew well under standard anaerobic growth conditions. The ribonucleotide reductase system of this mutant was shown to consist of an enzyme of the NrdEF-type and a small electron transport protein. The coding operon contains the nrdEF genes and two open reading frames, one of which (nrdH) codes for the small protein. The same gene organization is present in E. coli. We propose that the aerobic class I ribonucleotide reductases contain two subclasses, one coded by nrdAB, active in E. coli and eukaryotes (class Ia), the other coded by nrdEF, present in various microorganisms (class Ib). The NrdEF enzymes use NrdH proteins as electron transporter in place of thioredoxin or glutaredoxin used by NrdAB enzymes. The two classes also differ in their allosteric regulation by dATP.

Amino Acid Sequence↗
PubMed1989

Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation.

To apply recombinant DNA techniques for genetic manipulation of the industrially important lactococci, an efficient and reliable high-frequency transformation system must be available. High-voltage electric pulses have been demonstrated to enhance uptake of DNA into protoplasts and intact cells of numerous gram-negative and gram-positive microorganisms. The objective of this study was to develop a system for electroporating intact cells of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) with a commercially available electroporation unit (BTX Transfector 100; BTX, Inc., San Diego, Calif.). Parameters which influenced the efficiency of transformation included growth phase and final concentration of cells, ionic strength of the suspending medium, concentration of plasmid DNA, and the amplitude and duration of the pulse. Washed suspensions of intact cells suspended in deionized distilled water were subjected to one high-voltage electric pulse varying in voltage (300 to 900 V corresponding to field strengths of 5 to 17 kV/cm) and duration (100 microseconds to 1 s). Transformation efficiencies of 10(3) transformants per microgram of DNA were obtained when dense suspensions (final concentration, 5 x 10(10) CFU/ml) of stationary-phase cells were subjected to one pulse with a peak voltage of 900 V (field strength, 17 kV/cm) and a pulse duration of 5 ms in the presence of plasmid DNA. Dilution of porated cells in broth medium followed by an expression period of 2 h at 30 degrees C was beneficial in enhancing transformation efficiencies. Plasmids ranging in size from 9.8 to 30.0 kilobase pairs could be transformed by this procedure.

Culture Media↗
PubMed1996

Variacin, a new lanthionine-containing bacteriocin produced by Micrococcus varians: comparison to lacticin 481 of Lactococcus lactis.

A new lanthionine-containing bacteriocin, variacin, displaying a broad host range of inhibition against gram-positive food spoilage bacteria, has been identified from two strains of Micrococcus varians isolated from meat fermentations. The new bacteriocin was purified, and its amino-terminal end and total amino acid composition were determined. The structural gene was isolated and analyzed. Variacin is resistant to heat and pH conditions from 2 to 10. Its primary sequence shows significant homology to lacticin 481 to Lactococcus lactis, which is more pronounced for the probacteriocin than for the leader sequence. Variacin, like lacticin 481, contains lanthionine and beta-methyllanthionine residues, but its leader sequence clearly resembles nonlantibiotic leader sequences. In particular, the prepeptide contains glycine residues at positions -1 and -2 of the processing site.

Alanine↗
PubMed1991

Identification, cloning and sequencing of the replication region of Lactococcus lactis ssp. lactis biovar. diacetylactis Bu2 citrate plasmid pSL2.

The replication region of the 7.8 kilobase (kb) citrate plasmid pSL2 from Lactococcus lactis ssp. lactis biovar. diacetylactis Bu2 was identified. Deletion derivatives of pSL2 were introduced into plasmid-free strain Bu2-60 and tested for their ability to replicate autonomously. The region necessary for replication was identified by comparison of the pSL2 derivatives, cloned and sequenced. No homologies were detected by comparing the putative Rep protein of pSL2 with replicons of other plasmids of Gram-positive bacteria. A part of an IS-element flanking the replication region was found.

Amino Acid Sequence↗
PubMed1990

Cell Surface Characteristics of Bacteriophage-Resistant Lactococcus lactis subsp. cremoris SK110 and Its Bacteriophage-Sensitive Variant SK112.

Several cell surface characteristics of bacteriophage-resistant Lactococcus lactis subsp. cremoris SK110 were compared with those of its phage-sensitive derivative SK112. After centrifugation, SK110 cells resisted suspension more strongly than SK112 cells. SK112 was more negatively charged and had a more hydrophobic cell surface than SK110. Furthermore, SK112 was agglutinated in the presence of concanavalin A, whereas SK110 was not. The opposite was observed upon incubation of cells of either strain with a lectin from Ricinus communis. A mild alkali treatment decreased the differences in the cell surface characteristics of the two strains remarkably.

Journal Article↗
PubMed1993

Insertion of Transposon Tn917 Derivatives into the Lactococcus lactis subsp. lactis Chromosome.

Two transposition vectors, pTV32 and pLTV1, containing transposon Tn917 derivatives TV32 and LTV1, respectively, were introduced into Lactococcus lactis subsp. lactis MG1614. It was found that pTV32 and pLTV1 replicate and that TV32 and LTV1 transpose in this strain. A protocol for production of a collection of Tn917 insertions in L. lactis subsp. lactis was developed. The physical locations of TV32 on the chromosomal SmaI fragments of 62 independent transpositions were established by pulsed-field gel electrophoresis. These transpositions could be divided into at least 38 different groups that exhibited no Tn917-dominating hot spots on the L. lactis subsp. lactis chromosome. A total of 10 of the 62 transpositions resulted in strains that express beta-galactosidase. This indicates that there was fusion of the promoterless lacZ of the Tn917 derivatives to a chromosomal promoter. Thus, the Tn917-derived transposons should be powerful genetic tools for studying L. lactis subsp. lactis.

Journal Article↗
PubMed2000

Molecular and functional analyses of the metC gene of Lactococcus lactis, encoding cystathionine beta-lyase.

The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be essential for flavor development. Cystathionine beta-lyase (CBL) can convert cystathionine to homocysteine but is also able to catalyze an alpha, gamma elimination. With methionine as a substrate, it produces volatile sulfur compounds which are important for flavor formation in Gouda cheese. The metC gene, which encodes CBL, was cloned from the Lactococcus lactis model strain MG1363 and from strain B78, isolated from a cheese starter culture and known to have a high capacity to produce volatile compounds. The metC gene was found to be cotranscribed with a downstream cysK gene, which encodes a putative cysteine synthase. The MetC proteins of both strains were overproduced in strain MG1363 with the NICE (nisin-controlled expression) system, resulting in a >25-fold increase in cystathionine lyase activity. A disruption of the metC gene was achieved in strain MG1363. Determination of enzymatic activities in the overproducing and knockout strains revealed that MetC is essential for the degradation of cystathionine but that at least one lyase other than CBL contributes to methionine degradation via alpha, gamma elimination to form volatile aroma compounds.

Amino Acid Sequence↗
PubMed2001

Molecular characterization of a theta replication plasmid and its use for development of a two-component food-grade cloning system for Lactococcus lactis.

pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putative cis-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the cis-acting ori region and the repB gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in Lactococcus lactis. A two-component food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of L. lactis DNA and has no selection marker. The companion pCOM1 is a repB-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in L. lactis if trans complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of L. lactis and two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.

Amino Acid Sequence↗
PubMed1991

Construction of an IS946-based composite transposon in Lactococcus lactis subsp. lactis.

An artificial composite transposon was constructed based on the lactococcal insertion sequence IS946. A 3.0-kb element composed of the pC194 cat gene (Cmr) flanked by inversely repeated copies of IS946 was assembled on pBluescript KS+. When subcloned into the shuttle vector pSA3 (Emr), two putative transposons were created on the recombinant plasmid pTRK128: the 3.0-kb Cmr element (Tn-CmA) and an inverse 11.5-kb Emr element (Tn-EmA). pTRK128 was electroporated into the recombination-deficient strain Lactococcus lactis MMS362, which contains the self-transmissible plasmid pRS01. An MMS362 Cmr Emr transformant was used to assay for transposition events via conjugal mobilization of pTRK128-encoded Cmr or Emr to L. lactis LM2345. Transfer of either marker alone occurred at frequencies of ca. 2 x 10(-4) per input donor. Approximately 19% of the Emr transconjugants were Cms, indicating loss of the cat gene marker. No Cmr Ems transconjugants were recovered (n = 550). Plasmid analysis showed that the Cms Emr isolates contained a single large plasmid that was determined to be a cointegrate between pRS01 and the Tn-EmA element. A 32P-labeled pSA3 probe hybridized specifically to pTRK128 sequences and revealed different junction fragments within each of the cointegrate plasmids. DNA sequence analysis of the Tn-EmA::pRS01 junctions from a representative cointegrate verified transposition by Tn-EmA. This represents the first example of a functional composite transposon in the genus Lactococcus and serves as an experimental tool and model for the genetic analyses of transposons in these organisms.

Base Sequence↗
PubMed2002

Production and targeting of the Brucella abortus antigen L7/L12 in Lactococcus lactis: a first step towards food-grade live vaccines against brucellosis.

Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that infects humans and animals by entry mainly through the digestive tract. B. abortus causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of oral live vaccines against brucellosis, using food-grade lactic acid bacteria (LAB) as a carrier. The L7/L12 gene was expressed in Lactococcus lactis, the model LAB, under the nisin-inducible promoter. Using different signals, L7/L12 was produced in cytoplasmic, cell-wall-anchored, and secreted forms. Cytoplasmic production of L7/L12 gave a low yield, estimated at 0.5 mg/liter. Interestingly, a secretable form of this normally cytoplasmic protein via fusion with a signal peptide resulted in increased yield of L7/L12 to 3 mg/liter; secretion efficiency (SE) was 35%. A fusion between the mature moiety of the staphylococcal nuclease (Nuc) and L7/L12 further increased yield to 8 mg/liter. Fusion with a synthetic propeptide (LEISSTCDA) previously described as an enhancer for heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895-1903, 1998) raised the yield to 8 mg/liter and SE to 50%. A surface-anchored L7/L12 form in L. lactis was obtained by fusing the cell wall anchor of Streptococcus pyogenes M6 protein to the C-terminal end of L7/L12. The fusions described allow the production and targeting of L7/L12 in three different locations in L. lactis. This is the first example of a B. abortus antigen produced in a food-grade bacterium and opens new perspectives for alternative vaccine strategies against brucellosis.

Animals↗
PubMed1995

A 7-base-pair sequence protects DNA from exonucleolytic degradation in Lactococcus lactis.

Linear DNA molecules are subject to degradation by various exonucleases in vivo unless their ends are protected. It has been demonstrated that a specific 8-bp sequence, 5'-GCTGGTGG-3', named Chi, can protect linear double-stranded DNA from the major Escherichia coli exonuclease RecBCD. Chi protects linear replication products of rolling-circle plasmids from RecBCD degradation in vivo, in agreement with observations in vitro. A unique 7-bp sequence, 5'-GCGCGTG-3', is shown to protect similar replication products from degradation in Lactococcus lactis strains but not in more distantly related Gram-positive bacteria. The properties of this sequence in L. lactis correspond to those of a Chi site. Linear plasmid replication products have been detected in numerous prokaryotes, suggesting the widespread existence of short species-specific sequences that preserve linear DNA from extensive degradation by host cell exonucleases.

Base Composition↗
PubMed2000

Orientation specificity of the Lactococcus lactis Chi site.

BACKGROUND: In Escherichia coli, the Chi sequence modulates the activity of RecBCD, a powerful double-stranded (ds) DNA exonuclease/helicase. Chi attenuates RecBCD exonuclease activity and stimulates homologous recombination in an orientation-dependent manner. ChiEc is frequent and over-represented on its genome, which is thought to be related to its role in dsDNA break repair. We previously identified a Chi-like sequence (referred to as ChiLl) and an exonuclease/helicase in the Gram-positive bacterium Lactococcus lactis. ChiLl and RexAB are functional analogues of ChiEc and RecBCD. RESULTS: We report that ChiLl attenuates RexAB exonuclease activity and stimulates homologous recombination in an orientation-dependent manner. Analysis of ChiLl distribution on the L. lactis chromosome reveals that ChiLl is frequent, highly over-represented, and oriented with respect to the direction of replication. CONCLUSION: Our results show that a single orientation of ChiLl interacts with RexAB. The active orientation is preferentially found on the replication leading strand of the L. lactis genome, consistent with a primary role of ChiLl in repair of dsDNA breaks at the replication fork. We propose that orientation-dependence of Chi activity and over-representation of Chi sequences on bacterial genomes may be conserved properties of exonuclease/helicase-Chi couples. Other properties of the Chi sequence distribution on the genomes might reflect more specific characteristics of each couple and of the host.

Chromosome Inversion↗
PubMed1996

Production of antifungal substance by Lactococcus lactis subsp. lactis CHD-28.3.

Six of the 2100 colonies of lactic acid bacteria isolated from 4 month old Cheddar cheese and raw buffalo milk showed antifungal activity against Aspergillus flavus IARI when tested by the well agar diffusion assay on Potato Dextrose Agar containing 0.1% Triton X-100. Out of these, the most promising isolate having a broad spectrum of antifungal activity including Aspergillus flavus IARI, A. flavus NCIM 555, A. parasiticus NCIM 898 and Fusarium spp. was identified as Lactococcus lactis subsp. lactis CHD-28.3. Among the mold cultures used as indicator strains, the most sensitive towards antifungal substance produced by the test culture was A. flavus IARI. The cell-free supernatant of the test culture in Elliker's broth adjusted to pH 6.8 produced an inhibition zone of 15-19 mm against A. flavus IARI, A. flavus NCIM555 and A. parasiticus NCIM898. The isolate when grown at 30 degrees C for 48 h in Elliker's broth showed optimum antifungal activity. When the supernatant was neutralized to pH 7.0 or 7.5, there was little reduction in activity. However, after enzymatic treatment of supernatant with chymotrypsin, trypsin and pronase E, the antifungal activity disappeared which indicated the proteinaceous nature of the antifungal substance.

Antifungal Agents↗
PubMedDate not supplied

[Study of intraspecific variations of the bacterium Lactococcus lactis in adaptation to high acidity of the medium].

This paper reports on the study of acid tolerance of lactic acid bacteria as a property of cells, determining their ability to divide efficiently and retain viability under conditions of increased nutrient medium acidity during bacterial growth. The bacteria of the strain TV2, isolated from a self-soured curd, similar to the bacteria of the strain STE05 (Russian National Collection of Industrial Microorganisms), were assigned to the species Lactococcus lactis according to their G+C composition (36.7-36.5 mol %) and the high level of DNA-DNA hybridization (93%). However, these strains were essentially different in the number and size of the plasmids and the chromosomal DNA restriction fragments, as well as in the sensitivity to phages of lactic acid bacteria. It was found that bacteria of the strain TV2 were stable (i.e., they divided efficiently at a pH as low as 5.3) and tolerant to the lactic acid that they produced while growing (i.e., they retained viability at pH 4.4). Bacteria of the strain STE05 lacked acid tolerance (at pH below 6.5, growth was retarded, and pH 5.0 was the lowest value at which the cells remained viable). The acid tolerance and phage resistance of TV2 bacteria are likely to characterize their higher adaptive capacity in comparison with STE05 bacteria. Acid tolerance is inherited in a stable manner and retained by the segregants of TV2 strain obtained in the course of long-term storage of the bacteria. Specifically, the strains TV29, TV13, and TV 229, which displayed this property, had altered physiological and biochemical characteristics (accumulation of biomass and fermentation of lactose) in spite of their genetic identity to the original strain (pulsed-field gel electrophoresis of chromosomal DNA restriction fragments).

Adaptation, Physiological↗
PubMed2003

A food-grade approach for functional analysis and modification of native plasmids in Lactococcus lactis.

While plasmids from lactic acid bacteria possess many traits that are of industrial value, their exploitation is often frustrated by an inability to conduct food-grade engineering of native plasmids or to readily screen for their transfer. Here we describe a system that uses a RepA(+) temperature-sensitive helper plasmid and a RepA(-) cloning vector to overcome these problems while maintaining the food-grade status of the native plasmid. This strategy was used to precisely delete ltnA1 alone, or in conjunction with ltnA2 (encoding the structural proteins of the lantibiotic lacticin 3147), from the native 60.2-kb plasmid pMRC01 and to select for the transfer of pMRC01 between Lactococcus lactis strains.

Bacterial Proteins↗
PubMed2006

Gene targeting in the Gram-Positive bacterium Lactococcus lactis, using various delta ribozymes.

The trans-acting antigenomic delta ribozyme, isolated from the human hepatitis delta virus, was shown to be highly stable and active in vitro, as well as in mammalian cell lines. However, the stability and gene-targeting competence of this small ribozyme have not been studied previously in bacterial cells. In this paper we describe the use of two variants of the trans-acting antigenomic delta ribozyme targeting the abundant EF-Tu mRNA in the industrially important gram-positive bacterium Lactococcus lactis. These two delta ribozyme variants were expressed at significant levels and were shown to be highly stable in vivo. The half-life of the EF-Tu mRNA was slightly but consistently reduced in the presence of the classical delta ribozymes (7 to 13%). In contrast, delta ribozymes harboring a specific on/off riboswitch (SOFA-delta ribozymes) targeting the same sites on the EF-Tu mRNA considerably reduced the half-life of this mRNA (22 to 47%). The rates of catalysis of the SOFA-delta ribozymes in L. lactis were similar to the rates determined in vitro, showing that this new generation of delta ribozymes was highly efficient in these bacterial cells. Clearly, SOFA-delta ribozymes appear to be an ideal means for development of gene inactivation systems in bacteria.

Base Sequence↗
PubMed2003

Intranasal immunization with recombinant Lactococcus lactis secreting murine interleukin-12 enhances antigen-specific Th1 cytokine production.

Interleukin-12 (IL-12), a heterodimeric cytokine, plays an important role in cellular immunity to several bacterial, viral, and parasitic infections and has adjuvant activity when it is codelivered with DNA vaccines. IL-12 has also been used with success in cancer immunotherapy treatments. However, systemic IL-12 therapy has been limited by high levels of toxicity. We describe here inducible expression and secretion of IL-12 in the food-grade lactic acid bacterium Lactococcus lactis. IL-12 was expressed as two separate polypeptides (p35-p40) or as a single recombinant polypeptide (scIL-12). The biological activity of IL-12 produced by the recombinant L. lactis strain was confirmed in vitro by its ability to induce gamma interferon (IFN-gamma) production by mouse splenocytes. Local administration of IL-12-producing strains at the intranasal mucosal surface resulted in IFN-gamma production in mice. The activity was greater with the single polypeptide scIL-12. An antigen-specific cellular response (i.e., secretion of Th1 cytokines, IL-2, and IFN-gamma) elicited by a recombinant L. lactis strain displaying a cell wall-anchored human papillomavirus type 16 E7 antigen was dramatically increased by coadministration with an L. lactis strain secreting IL-12 protein. Our data show that IL-12 is produced and secreted in an active form by L. lactis and that the strategy which we describe can be used to enhance an antigen-specific immune response and to stimulate local mucosal immunity.

Administration, Intranasal↗
PubMed2005

Eukaryotic membrane protein overproduction in Lactococcus lactis.

Eukaryotic membrane proteins play many vital roles in the cell and are important drug targets. Approximately 25% of all genes identified in the genome are known to encode membrane proteins, but the vast majority have no assigned function. Although the generation of structures of soluble proteins has entered the high-throughput stage, for eukaryotic membrane proteins only a dozen high-resolution structures have been obtained so far. One major bottleneck for the functional and structural characterisation of membrane proteins is the overproduction of biologically active material. Recent advances in the development of the Lactococcus lactis expression system have opened the way for the high-throughput functional expression of eukaryotic membrane proteins.

Animals↗