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PubMed1999

Occurrence of nisin Z production in Lactococcus lactis BFE 1500 isolated from wara, a traditional Nigerian cheese product.

Screening for bacteriocin production of 500 strains of lactic acid bacteria (LAB) from various African fermented foods resulted in the detection of a bacteriocin producing Lactococcus lactis (BFE 1500) isolated from a dairy product called wara. The bacteriocin inhibited not only the closely related LAB, but also strains of Listeria monocytogenes, Listeria innocua, Clostridium butyricum, Clostridium perfringens, Bacillis cereus and Staphylococcus aureus. It was heat stable even at autoclaving temperature (121 degrees C for 15 min) and was active over a wide pH range (2-10), but highest activity was observed in the lower pH range. The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K, but not by other proteases. Growth kinetic assay indicated stronger growth inhibition by the bacteriocin produced by Lc. lactis BFE 1500 on L. monocytogenes WS 2250 and B. cereus DSM 2301 than with the nisin A producing strain DSM 20729. Polymerase chain reaction indicated the presence of the nisin operon in strain BFE 1500 and sequencing of its structural gene showed that Lc. lactis BFE 1500 produced the natural nisin variant, nisin Z, as indicated by the substitution of asparagine residue instead of histidine at position 27. The genetic determinants for bacteriocin production in strain BFE 1500 are located on a conjugative transposon. The ability of the bacteriocin produced by Lc. lactis BFE 1500 to inhibit a wide range of food-borne pathogens is of special interest for food safety, especially in the African environment with perennial problems of poor food hygiene.

Amino Acid Sequence↗
PubMed1990

Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease.

The Bacillus subtilis nprE gene lacking its own promoter sequence was inserted in the lactococcal expression vector pMG36e. Upon introduction of the recombinant plasmid into Lactococcus lactis subsp. lactis strain MG1363, neutral protease activity could be visualized by the appearance of large clearing zones around colonies grown on milk agar plates. By measuring the activities of the neutral protease and the intracellular enzyme lactate dehydrogenase in culture supernatants and cell fractions, it was demonstrated that the neutral protease was actively secreted into the growth medium. This was corroborated by using the Western blot (immunoblot) technique, which showed the presence of the mature form of the neutral protease in the culture supernatant. On the basis of these results, it is concluded that the B. subtilis neutral protease gene was expressed in L. lactis and that the gene product was secreted into the growth medium and was apparently correctly processed to produced a biologically active protein. The secretion of this particular enzyme may be helpful in achieving accelerated cheese ripening.

Bacillus subtilis↗
PubMed2002

Structural and kinetic studies of sugar binding to galactose mutarotase from Lactococcus lactis.

Galactose mutarotase catalyzes the conversion of beta-D-galactose to alpha-D-galactose in the Leloir pathway for galactose metabolism. The high resolution x-ray structure of the dimeric enzyme from Lactococcus lactis was recently solved and shown to be topologically similar to the 18-stranded, anti-parallel beta-motif observed for domain 5 of beta-galactosidase. In addition to determining the overall molecular fold of galactose mutarotase, this initial investigation also provided a detailed description of the electrostatic interactions between the enzyme and its physiologically relevant substrate, galactose. Specifically, the side chains of His-96 and His-170 were shown to be located within hydrogen bonding distance to the C-5 oxygen of the substrate, while the carboxylate of Glu-304 was positioned near the C-1 hydroxyl group of the sugar. On the basis of this initial study, a possible role for Glu-304 as the general acid/base group in catalysis was put forth. Here we describe the combined x-ray crystallographic and kinetic analyses of L. lactis galactose mutarotase complexed with D-glucose, D-fucose, D-quinovose, L-arabinose, or D-xylose. These investigations have revealed that there are several distinct binding modes for these sugars, which are dependent upon the spatial orientation of the C-4 hydroxyl group. In those sugars with the same C-4 hydroxyl group orientation as galactose, their C-1 hydroxyl groups are invariably located near Glu-304. For those sugars, which have the same C-4 hydroxyl group configuration as glucose, the C-1 hydroxyls are typically located near Asp-243. These different binding modes correlate with both the observed kinetic parameters and the presence or absence of a hydrogen bond between the guanidinium group of Arg-71 and the C-4 hydroxyl group of the sugar ligand.

Binding Sites↗
PubMed1997

Presence of additional peptidases in Streptococcus thermophilus CNRZ 302 compared to Lactococcus lactis.

Streptococcus thermophilus is widely used in the dairy industry but little is known about its peptidase system. The aim of this study was to determine the biochemical and genetic characteristics of this system, and to compare it to the well known system of Lactococcus lactis. We separated the intracellular proteins of Strep. thermophilus CNRZ 302 and L. lactis NCDO 763 by ion-exchange chromatography and we detected the activity of the different types of peptidases. In both L. lactis and Strep. thermophilus strains, we showed 13 different peptidase activities with biochemical homologies between both species. Streptococcus thermophilus also possessed two peptidases which we did not find in L. lactis: an aminopeptidase and an oligopeptidase. We performed Southern blot experiments and among the eight peptidase genes tested, only the genes encoding the general aminopeptidases, pepC and pepN, were homologous between the L. lactis and Strep. thermophilus strains. Besides biochemical and genetic similarities, the peptidase systems of Strep. thermophilus and L. lactis thus differed by the presence of additional peptidases in Strep. thermophilus.

Blotting, Southern↗
PubMed1995

The codon usage of the nisZ operon in Lactococcus lactis N8 suggests a non-lactococcal origin of the conjugative nisin-sucrose transposon.

An 11.6 kb area downstream from the structural gene of nisin Z in the conjugative nisin-sucrose transposon of Lactococcus lactis subsp. lactis N8 was cloned and sequenced. Analysis of the sequence revealed eight open reading frames, nisZBTClPRK, followed by a putative rho-independent terminator (delta G degrees = -4.7 kcal/mol). The C-terminal hydrophilic domain of the NisK protein is homologous to the C-termini of several histidine kinases of bacterial two-component regulator systems, such as SpaK from Bacillus subtilis and KdpD and RcsC of Escherichia coli. The nisin Z biosynthetic genes were highly similar with the genes of the nisin A operons having, however, a 0-3% difference in the amino acid sequences of the individual proteins. The codon usage of eleven genes within the same conjugative transposon was calculated and found to be strikingly different from that of other lactococcal genes. This, together with the low GC-content (32%) compared to the 38% (G+C) of the lactococcal chromosome in general strongly suggests a non-lactococcal origin of this transposon.

Amino Acid Sequence↗
PubMed1999

[Expression in Lactococcus lactis of catalytically active phenylalanine ammonia-lyase from parsley].

The phenylalanine ammonia-lyase (PAL) cDNA of parsley (Petroselinum crispum) was subcloned into constitutive expression vector pMG36e downstream of the P32 promoter. The resulting plasmid pMG36ePAL was introduced into Lactococcus lactis subsp. lactis MG1363 by electroporation. The recombinant strain showed its PAL activity conversing the L-phenylalanine in the culture medium into trans-cinnamic acid. A new secretory vector pXHS was constructed by recombination of pMG36e with a Lactococcal usp45 secretion leader coding sequence and a translational coupling sequence. Then the pXHSPAL was constructed and used for expression of PAL in L. lactis, the PAL activity was also detectable. The L. lactis dnaJ promoter sequence was cloned and used to construct a heat inducible vector pXHJ. PAL cDNA was cloned into pXHJ and the L. lactis IL1403 was transformed with the recombinant plasmid pXHJPAL. After a heat shock from 30 degrees C to 37 degrees C, the PAL activity of the pXHJPAL strain could increase approximately onefold. The prospect of using these engineering L. lactis strains for PKU therapy was also discussed.

Amino Acid Sequence↗
PubMed2001

Pre-inoculation enrichment procedure enhances the performance of bacteriocinogenic Lactococcus lactis meat starter culture.

Sodium nitrite and sodium chloride may inhibit growth and bacteriocinogenesis of protective starter cultures. To reduce sensitivity of a lacticin 3147-producing starter culture to nitrite, prior to production of salami, Lactococcus lactis DPC 4275 was placed in a number of pre-inoculation treatments, containing (a) 1% glucose, (b) 2.5 ppm manganese (Mn), (c) 250 ppm magnesium (Mg), (d) 2.5 ppm manganese + 250 ppm magnesium (Mn + Mg), and held at ambient temperature for 30 min and 4 degrees C for 2 h. The growth, pH reduction, and bacteriocin production was monitored in beaker sausage over a period of 10 days at 28 degrees C, corresponding to typical salami production time, and compared to untreated starter culture. The effect of 1% tryptone and inoculum level on growth and bacteriocin production was also determined. Challenge tests were performed using Listeria innocua DPC 1770 and Staphylococcus aureus MMPR3 as target strains. All treatments gave a significantly higher (P < 0.05) initial starter level than the untreated starter. Beaker sausage inoculated with either low (10(7)) or high (10(9)) levels of starter culture, treated with Mn + Mg reached significantly (P < 0.05) higher levels by day 10 than other treatments. Trends indicate that Mn + Mg also gave best pH reduction in sausage containing the low-level starter culture, sausage and significantly lower (P < 0.05) values for sausage produced with higher inoculum. Bacteriocin production was also higher in starter culture treated with Mn, or glucose. Pre-treatment with Mg gave a 2-fold increase in bacteriocin, the addition of Mn augmenting this increase further. The incorporation of tryptone gave no additional effect. In beaker sausage, both L. innocua and S. aureus populations showed significant reductions (P < 0.05) in the presence of the bacteriocinogenic strain compared to a non-bacteriocinogenic control strain.

Antibiosis↗
PubMed1992

Use of degenerate primers for polymerase chain reaction cloning and sequencing of the Lactococcus lactis subsp. lactis recA gene.

Two particularly well-conserved stretches in the RecA protein sequences were chosen as templates to synthesize degenerate oligonucleotides, which were used in polymerase chain reaction to amplify an internal recA DNA fragment of Lactococcus lactis subsp. lactis ML3. Using this fragment, we recovered and sequenced the entire lactococcal recA gene. The end of an open reading frame present upstream of the recA gene shows strong homology with formamidopyrimidine-DNA-glycosylase, a protein involved in DNA repair.

Amino Acid Sequence↗
PubMed2003

CodY-regulated aminotransferases AraT and BcaT play a major role in the growth of Lactococcus lactis in milk by regulating the intracellular pool of amino acids.

Aminotransferases, which catalyze the last step of biosynthesis of most amino acids and the first step of their catabolism, may be involved in the growth of Lactococcus lactis in milk. Previously, we isolated two aminotransferases from L. lactis, AraT and BcaT, which are responsible for the transamination of aromatic amino acids, branched-chain amino acids, and methionine. In this study, we demonstrated that double inactivation of AraT and BcaT strongly reduced the growth of L. lactis in milk. Supplementation of milk with amino acids and keto acids that are substrates of both aminotransferases did not improve the growth of the double mutant. On the contrary, supplementation of milk with isoleucine or a dipeptide containing isoleucine almost totally inhibited the growth of the double mutant, while it did not affect or only slightly affected the growth of the wild-type strain. These results suggest that AraT and BcaT play a major role in the growth of L. lactis in milk by degrading the intracellular excess isoleucine, which is responsible for the growth inhibition. The growth inhibition by isoleucine is likely to be due to CodY repression of the proteolytic system, which is necessary for maximal growth of L. lactis in milk, since the growth of the CodY mutant was not affected by addition of isoleucine to milk. Moreover, we demonstrated that AraT and BcaT are part of the CodY regulon and therefore are regulated by nutritional factors, such as the carbohydrate and nitrogen sources.

Amino Acids, Aromatic↗
PubMed1995

Locating nisin-producing Lactococcus lactis in a fermented meat system.

Antibody-linked probes were used to locate nisin in a fermented meat system. Free nisin or nisin bound to susceptible cells or food components was not detected. Colonies of nisin-producing Lactococcus lactis were stained at all times during growth. The position of nisin-producing L. lactis colonies was noted and compared with the location of spoilage organisms or the distribution of areas with a fermented meat appearance. No relationship between the distribution of starter culture and the location of spoilage organisms or areas of fermentation was observed. In addition to the presence of L. lactis, a rapidly fermentable sugar was also required to obtain a fermented appearance and to reduce the levels of spoilage organisms.

Animals↗
PubMed1990

Stacking of three different restriction and modification systems in Lactococcus lactis by cotransformation.

Four plasmids encoding restriction and modification (R/M) systems are described that are different in the specificity of their restrictive activity toward the small isometric phage p2 and prolate phage c2. The R/M plasmids were cotransformed into Lactococcus lactis MG1363 with pVS2, encoding resistance to chloramphenicol and erythromycin, to indicate successful transformation events. Analysis of cotransformants showed that three different R/M plasmids could be combined in L. lactis MG1363. The efficiency at which phage plaqued on the transformants decreased as the number of R/M plasmids increased. Some plasmid combinations were unstable suggesting replicon incompatibility.

Chloramphenicol Resistance↗
PubMed2003

Heat and DNA damage induction of the LexA-like regulator HdiR from Lactococcus lactis is mediated by RecA and ClpP.

The SOS response is a paradigm for bacterial cells response to DNA damage. Yet some bacteria lack a homologue of the SOS regulator, LexA, including the Gram-positive, Lactococcus lactis. In this organism we have identified a negative transcriptional regulator, HdiR that induces target gene expression both upon DNA damage and heat shock. Gel mobility shift assays revealed that the binding site for HdiR is located within an inverted repeat structure. HdiR is able to carry out a self-cleavage reaction in vitro at high pHs, while in vivo it undergoes RecA-dependent self-cleavage in the presence of a DNA-damaging agent. Intriguingly, the N-terminal cleavage product of HdiR retains DNA binding activity, and only when degraded by the Clp protease, is gene expression induced. Thus, the activity of HdiR in response to DNA damage is controlled by sequential proteolysis, involving self-cleavage and Clp-dependent degradation of HdiR. During heat-stress, limited self-cleavage occurs; however, recA and clpP are still required for full induction of target gene expression. Thus, our data show that common elements are involved in both the DNA damage and the heat-mediated induction of the HdiR regulon.

Adenosine Triphosphatases↗
PubMed2000

Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp. cremoris using a new shuttle vector.

Staphylococcus aureus harbors redundant adhesins mediating tissue colonization and infection. To evaluate their intrinsic role outside of the staphylococcal background, a system was designed to express them in Lactococcus lactis subsp. cremoris 1363. This bacterium is devoid of virulence factors and has a known genetic background. A new Escherichia coli-L. lactis shuttle and expression vector was constructed for this purpose. First, the high-copy-number lactococcal plasmid pIL253 was equipped with the oriColE1 origin, generating pOri253 that could replicate in E. coli. Second, the lactococcal promoters P23 or P59 were inserted at one end of the pOri253 multicloning site. Gene expression was assessed by a luciferase reporter system. The plasmid carrying P23 (named pOri23) expressed luciferase constitutively at a level 10,000 times greater than did the P59-containing plasmid. Transcription was absent in E. coli. The staphylococcal clumping factor A (clfA) gene was cloned into pOri23 and used as a model system. Lactococci carrying pOri23-clfA produced an unaltered and functional 130-kDa ClfA protein attached to their cell walls. This was indicated both by the presence of the protein in Western blots of solubilized cell walls and by the ability of ClfA-positive lactococci to clump in the presence of plasma. ClfA-positive lactococci had clumping titers (titer of 4,112) similar to those of S. aureus Newman in soluble fibrinogen and bound equally well to solid-phase fibrinogen. These experiments provide a new way to study individual staphylococcal pathogenic factors and might complement both classical knockout mutagenesis and modern in vivo expression technology and signature tag mutagenesis.

Bacterial Adhesion↗
PubMed1998

Molecular characterization of the Lactococcus lactis LlaKR2I restriction-modification system and effect of an IS982 element positioned between the restriction and modification genes.

The nucleotide sequence of the plasmid-encoded LlaKR2I restriction-modification (R-M) system of Lactococcus lactis subsp. lactis biovar diacetylactis KR2 was determined. This R-M system comprises divergently transcribed endonuclease (llaKR2IR) and methyltransferase (llaKR2IM) genes; located in the intergenic region is a copy of the insertion element IS982, whose putative transposase gene is codirectionally transcribed with llaKR2IM. The deduced sequence of the LlaKR2I endonuclease shared homology with the type II endonuclease Sau3AI and with the MutH mismatch repair protein, both of which recognize and cleave the sequence 5' GATC 3'. In addition, M. LlaKR2I displayed homology with the 5-methylcytosine methyltransferase family of proteins, exhibiting greatest identity with M. Sau3AI. Both of these proteins shared notable homology throughout their putative target recognition domains. Furthermore, subclones of the native parental lactococcal plasmid pKR223, which encode M. LlaKR2I, all remained undigested after treatment with Sau3AI despite the presence of multiple 5' GATC 3' sites. The combination of these data suggested that the specificity of the LlaKR2I R-M system was likely to be 5' GATC 3', with the cytosine residue being modified to 5-methylcytosine. The IS982 element located within the LlaKR2I R-M system contained at its extremities two 16-bp perfect inverted repeats flanked by two 7-bp direct repeats. A perfect extended promoter consensus, which represented the likely original promoter of the llaKR2IR gene, was shown to overlap the direct repeat sequence on the other side of IS982. Specific deletion of IS982 and one of these direct repeats via a PCR strategy indicated that the LlaKR2I R-M determinants do not rely on elements within IS982 for expression and that the efficiency of bacteriophage restriction was not impaired.

Amino Acid Sequence↗
PubMed2004

Nisin-controlled production of pediocin PA-1 and colicin V in nisin- and non-nisin-producing Lactococcus lactis strains.

The introduction of chimeric genes encoding the fusion leader of lactococcin A-propediocin PA-1 or procolicin V under the control of the inducible nisA promoter and the lactococcin A-dedicated secretion genes (lcnCD) into Lactococcus lactis strains, including a nisin producer, expressing the two component regulator NisRK led to the production or pediocin PA-1 or colicin V, respectively.

Bacterial Proteins↗
PubMed1993

Cloning and expression of the manganese superoxide dismutase gene of Escherichia coli in Lactococcus lactis and Lactobacillus gasseri.

The Escherichia coli sodA gene encoding the antioxidant enzyme Mn-containing superoxide dismutase (MnSOD), was cloned in the expression vector pMG36e. This vector has a multiple cloning site downstream of a promoter and Shine-Dalgarno sequences derived from Lactococcus. The protein-coding region of sodA from E. coli was amplified by the polymerase chain reaction, using a thermocycler and Taq DNA polymerase before cloning into pMG36e. When introduced into E. coli, the recombinant plasmid expressed the predicted fusion protein, both in the presence and absence of oxygen. The expression of the fusion protein in E. coli was verified by SOD assays, activity gels and Western blots. The recombinant plasmid was also introduced into Lactococcus lactis, which contains a resident SOD, and into Lactobacillus gasseri, which is devoid of SOD. Transformed lactococci expressed an active SodA fusion protein plus an active hybrid protein composed of subunits of the Lactococcus and the recombinant E. coli enzymes. Transformants of L. gasseri expressed only the fusion SodA protein, which was enzymatically active.

Base Sequence↗
PubMed1999

Structure-function analysis of multidrug transporters in Lactococcus lactis.

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. A multidrug transporter in Lactococcus lactis, LmrA, is a member of the ATP-binding cassette (ABC) superfamily and a bacterial homolog of the human multidrug resistance P-glycoprotein. Another multidrug transporter in L. lactis, LmrP, belongs to the major facilitator superfamily, and is one example of a rapidly expanding group of secondary multidrug transporters in microorganisms. Thus, LmrA and LmrP are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by the same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA represent an intriguing area of research.

ATP-Binding Cassette Transporters↗
PubMed1995

Specificity of peptide transport systems in Lactococcus lactis: evidence for a third system which transports hydrophobic di- and tripeptides.

A proton motive force-driven di-tripeptide carrier protein (DtpT) and an ATP-dependent oligopeptide transport system (Opp) have been described for Lactococcus lactis MG1363. Using genetically well-defined mutants in which dtpT and/or opp were inactivated, we have now established the presence of a third peptide transport system (DtpP) in L. lactis. The specificity of DtpP partially overlaps that of DtpT. DtpP transports preferentially di- and tripeptides that are composed of hydrophobic (branched-chain amino acid) residues, whereas DtpT has a higher specificity for more-hydrophilic and charged peptides. The toxic dipeptide L-phenylalanyl-beta-chloro-L-alanine has been used to select for a di-tripeptide transport-negative mutant with the delta dtpT strain as a genetic background. This mutant is unable to transport di- and tripeptides but still shows uptake of amino acids and oligopeptides. The DtpP system is induced in the presence of di- and tripeptides containing branched-chain amino acids. The use of ionophores and metabolic inhibitors suggests that, similar to Opp, DtpP-mediated peptide transport is driven by ATP or a related energy-rich phosphorylated intermediate.

Amino Acid Sequence↗