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PubMed2002

Bacteriophage resistance of a deltathyA mutant of Lactococcus lactis blocked in DNA replication.

The thyA gene, which encodes thymidylate synthase (TS), of Lactococcus lactis CHCC373 was sequenced, including the upstream and downstream regions. We then deleted part of thyA by gene replacement. The resulting strain, MBP71 deltathyA, was devoid of TS activity, and in media without thymidine, such as milk, there was no detectable dTTP pool in the cells. Hence, DNA replication was abolished, and acidification by MBP71 was completely unaffected by the presence of nine different phages tested at a multiplicity of infection (MOI) of 0.1. Nonreplicating MBP71 must be inoculated at a higher level than CHCC373 to achieve a certain pH within a specified time. For a pH of 5.2 to be reached in 6 h, the inoculation level of MBP71 must be 17-fold higher than for CHCC373. However, by adding a limiting amount of thymidine this could be lowered to just 5-fold the normal amount, while acidification was unaffected with MBP71 up to an MOI of 0.01. It was found that nonreplicating MBP71 produced largely the same products as CHCC373, though the acetaldehyde production of the former was higher.

Bacteriophages↗
PubMed1999

Glucose/citrate cometabolism in Lactococcus lactis subsp. lactis biovar diacetylactis with impaired alpha-acetolactate decarboxylase.

The pyruvate metabolism of a Lactococcus lactis subsp. lactis biovar diacetylactis mutant deficient in alpha-acetolactate decarboxylase and its wild-type strain was studied during batch cultivations. A chemically defined medium was used containing glucose as carbon- and energy-source. The alpha-acetolactate decarboxylase deficiency had no effect on the specific growth rate. Addition of citrate was found to increase the specific growth rate of both strains under aerobic and anaerobic conditions. The product formation was monitored throughout the cultivations. The carbon- and redox-balances were within the accuracy of the experimental data. When citrate was added, alpha-acetolactate, diacetyl, and acetoin were formed, and aeration was shown to have a positive effect on the formation of these metabolites. By omitting lipoic acid (required for a functional pyruvate dehydrogenase complex) from the growth medium, a similar stimulatory effect on alpha-acetolactate, diacetyl, and acetoin formation was observed under aerobic conditions. The strain with impaired alpha-acetolactate decarboxylase activity accumulated alpha-acetolactate which resulted in an increased diacetyl formation compared to the wild-type strain, under aerobic and anaerobic conditions.

Biotechnology↗
PubMed2000

HtrA is the unique surface housekeeping protease in Lactococcus lactis and is required for natural protein processing.

We identified an exported protease in Lactococcus lactis ssp. lactis strain IL1403 belonging to the HtrA/DegP family. Inactivation of the chromosomal gene (htrALl) encoding this protease (HtrALl) results in growth thermo-sensitivity at very high temperatures (above 37 degrees C for L. lactis). The role of htrALl in extracellular proteolysis under normal growth conditions was examined by testing the stability of different exported proteins (i.e. fusions, a heterologous pre-pro-protein or a native protein containing repeats), having different locations. In the wild-type (wt) strain, degradation products, including the C-terminal protein ends, were present in the medium, indicating that proteolysis occurs during or after export to the cell surface; in one case, degradation was nearly total. In contrast, proteolysis was totally abolished in the htrA strain for all five proteins tested, and the yield of full-length products was significantly increased. These results suggest that HtrALl is the sole extracellular protease that degrades abnormal exported proteins. In addition, our results reveal that HtrALl is needed for the pro-peptide processing of a natural pro-protein and for maturation of a native protein. We propose that in lactococci, and possibly in other Gram-positive organisms with small sized-genomes, a single surface protease, HtrA, is totally responsible for the housekeeping of exported proteins.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of a cell wall peptidase from Lactococcus lactis subsp. cremoris IMN-C12.

A peptidase from the cell wall fraction of Lactococcus lactis subsp. cremoris IMN-C12 has been purified to homogeneity by hydrophobic interaction chromatography, two steps of anion-exchange chromatography, and gel filtration. The molecular mass of the purified enzyme was estimated to be 72 kDa by gel filtration and 23 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pI of 4.0, and it has the following N-terminal sequence from the 2nd to the 17th amino acid residues: -Arg-Leu-Arg-Arg-Leu-?-Val-Pro-Gly-Glu-Ileu-Val-Glu-Glu-Leu-Leu. The peptidase is most active at pH 5.8 and at 33 degrees C with trileucine as the substrate. Reducing agents such as dithiothreitol, beta-mercaptoethanol, and cysteine strongly stimulated enzyme activity, while p-chloromercuribenzoate had an inhibitory effect. Also, metal chelators lowered the peptidase activity, which could not be restored with Ca2+ and Mg2+. The divalent cations Cu2+, Zn2+, Fe2+, and Hg2+ completely inhibited peptidase activity. The peptidase is capable of hydrolyzing tripeptides and some dipeptides, with a preference for peptides containing leucine and with the highest activity towards the tripeptides Leu-Leu-Leu, Leu-Trp-Leu, and Ala-Leu-Leu, which were hydrolyzed with Kms of 0.37, 0.18, and 0.61 mM, respectively.

Amino Acid Sequence↗
PubMed1995

The recA gene of Lactococcus lactis: characterization and involvement in oxidative and thermal stress.

The role of recA in Lactococcus lactis, a microaerophilic fermenting organism, was examined by constructing a recA-disrupted strain. This single alteration had a surprisingly pleiotropic effect. In addition to its roles in homologous recombination and DNA repair, recA is also involved in responses to oxygen and heat stresses. We found that oxygen stress induced by aeration causes reductions in growth and stationary-phase survival of the recA strain. Toxicity is a consequence of hydroxyl radical production via the Fenton Reaction and is alleviated by catalase or Ferrozine addition. These results suggest that oxygen radicals are not efficiently eliminated and accumulate in lactococcal cultures, and that RecA is needed to deal with the damage they incur. Unexpectedly, thermal stress arrested growth of the recA strain. Immunological data indicate that the recA mutant is deficient in heat-shock proteins DnaK, GroEL, and GrpE. Poor growth at elevated temperature is therefore due to a diminished heat-shock response in the recA strain. In contrast, levels of a novel heat-shock protein, HfIB, are elevated. In Escherichia coli, HfIB downregulates the heat-shock response by promoting degradation of the transcription factor sigma 32. We propose that recA regulates the heat-shock response via HfIB. This work provides the first evidence showing that two major pathways of stress response, induced by heat shock and DNA damage, are interactive.

ATP-Dependent Proteases↗
PubMed1998

Characterization of a mutant of Lactococcus lactis with reduced membrane-bound ATPase activity under acidic conditions.

A mutant of Lactococcus lactis subsp. lactis C2 with reduced membrane-bound ATPase activity was characterized to clarify its acid sensitivity. The cytoplasmic pH of the mutant was measured in reference to the parental strain under various pH conditions. At low pH, the mutant could not maintain its cytoplasmic pH near neutral, and lost its viability faster than the parental strain. The ATPase activities of cells cultured under neutral and acidic conditions using pH-controlled jar fermentors were measured. The relative ATPase activity of the mutant at pH 7.0 was 42% of the parental strain. At pH 4.5, the parental strain showed an ATPase activity 2.8-fold higher than that at pH 7.0 while the level of increase in the mutant was only 1.6. Northern and Western blot analyses found that at pH 7.0 the transcriptional level and the amount of F1 beta subunit were similar in both strains, suggesting that the mutant has a defective ATPase structural gene. On the other hand, at pH 4.5 the transcriptional level and the amount of F1 beta subunit were found to be significantly higher in both strains than those at pH 7.0. From these results, it was suggested that the mutant has a normal regulation system for ATPase gene expression. It was concluded that the mutant is acid sensitive due to its inability to extrude protons out of the cell with defective ATPase under acidic conditions.

Amino Acid Sequence↗
PubMed1998

Lactococcus lactis phage operon coding for an endonuclease homologous to RuvC.

The function of the Lactococcus lactis bacteriophage bIL66 middle time-expressed operon (M-operon), involved in sensitivity to the abortive infection mechanism AbiD1, was examined. Expression of the M-operon is detrimental to Escherichia coli cells, induces the SOS response and is lethal to recA and recBC E. coli mutants, which are both deficient in recombinational repair of chromosomal double-stranded breaks (DSBs). The use of an inducible expression system allowed us to demonstrate that the M-operon-encoded proteins generate a limited number of randomly distributed chromosomal DSBs that are substrates for ExoV-mediated DNA degradation. DSBs were also shown to occur upstream of the replication initiation point of unidirectionally theta-replicating plasmids. The characteristics of the DSBs lead us to propose that the endonucleolytic activity of the M-operon is not specific to DNA sequence, but rather to branched DNA structures. Genetic and physical analysis performed with different derivatives of the M-operon indicated that two orfs (orf2 and orf3) are needed for nucleolytic activity. The orf3 product has amino acid homology with the E. coli RuvC Holliday junction resolvase. By site-specific mutagenesis, we have shown that one of the amino acid residues constituting the active centre of RuvC enzyme (Glu-66) and conserved in ORF3 (Glu-67) is essential for the nucleolytic activity of the M-operon gene product(s). We therefore propose that orf2 and orf3 of the M-operon code for a structure-specific endonuclease (M-nuclease), which might be essential for phage multiplication.

Amino Acid Sequence↗
PubMed1998

Detection and characterization of a bacteriocin produced by Lactococcus lactis subsp. cremoris R isolated from radish.

Bacteria isolated from radish were identified as Lactococcus lactis subsp. cremoris R and their bacteriocin was designated lactococcin R. Lactococcin R was sensitive to some proteolytic enzymes (proteinase-K, pronase-E, proteases, pepsin, alpha-chymotrypsin) but was resistant to trypsin, papain, catalase, lysozyme and lipase, organic solvents, or heating at 90 degrees C for 15, 30 and 60 min, or 121 degrees C for 15 min. Lactococcin R remained active after storage at -20 and -70 degrees C for 3 months and after exposure to a pH of 2-9. The molecular weight of lactococcin R was about 2.5 kDa. Lactococcin R was active against many food-borne pathogenic and food spoilage bacteria such as Clostridium, Staphylococcus, Listeria, Bacillus, Micrococcus, Enterococcus, Lactobacillus, Leuconostoc, Streptococcus and Pediococcus spp., but was not active against any Gram-negative bacteria. Lactococcin R was produced during log phase and reached a maximum activity (1600 AU ml-1) at early stationary phase. The highest lactococcin R production was obtained in MRS broth with 0.5% glucose, at 6.5-7.0 initial pH values, 30 degrees C temperature and 18-24-h incubation times. Lactococcin R adsorbed maximally to its heat-killed producing cells at pH 6-7 (95%). Crude lactococcin R at 1280 AU ml-1 was bactericidal, reducing colony counts of Listeria monocytogenes by 99.98% in 3 h. Lactococcin R should be useful as a biopreservative to prevent growth of food-borne pathogenic and food spoilage bacteria in ready-to-eat, dairy, meat, poultry and other food products. Lactococcin R differs from nisin in having a lower molecular weight, 2.5 kDa vs 3.4 kDa, and in being sensitive to pepsin and alpha-chymotrypsin to which nisin is resistant.

Bacteriocins↗
PubMed2001

Transcriptional analysis and regulation of expression of the ScrFI restriction-modification system of Lactococcus lactis subsp. cremoris UC503.

ScrFI is a type II restriction-modification system from Lactococcus lactis which recognizes the nucleotide sequence 5'-CC downward arrow NGG-3', cleaving at the point indicated by the arrow, and it comprises an endonuclease gene that is flanked on either side by genes encoding two 5-methylcytosine methylases. An open reading frame (orfX) of unknown function is located immediately upstream of these genes. In this study Northern analysis was performed, and it revealed that orfX, scrFIBM, and scrFIR are cotranscribed as a single polygenic mRNA molecule, while scrFIAM is transcribed independently. 5' extension analysis indicated that the start site for the scrFIAM promoter was a thymine located 4 bp downstream of the -10 motif. The transcriptional start site for the orfX promoter was also found to be a thymine which is more atypically located 24 bp downstream of the -10 motif proximal to the start codon. A helix-turn-helix motif was identified at the N-terminal end of one of the methylases (M.ScrFIA). In order to determine if this motif played a role in regulation of the ScrFI locus, M.ScrFIA was purified. It was then employed in gel retardation assays using fragments containing the two promoters found on the ScrFI operon, one located upstream of orfX and the other located just upstream of scrFIAM. M.ScrFIA was found to bind to the promoter region upstream of the gene encoding it, indicating that it may have a regulatory role. In further studies the two putative promoters were introduced into a vector (pAK80) upstream of a promoterless lacZ gene, and cloned fragments of the ScrFI locus were introduced in trans with each of these promoter constructs to investigate the effect on promoter activity. These results implicated M.ScrFIA in regulation of both promoters on the ScrFI locus.

Amino Acid Sequence↗
PubMed2002

Formation and conversion of oxygen metabolites by Lactococcus lactis subsp. lactis ATCC 19435 under different growth conditions.

A semidefined medium based on Casamino Acids allowed Lactococcus lactis ATCC 19435 to grow in the presence of oxygen at a slow rate (0.015 h(-1)). Accumulation of H(2)O(2) in the culture prevented a higher growth rate. Addition of asparagine to the medium increased the growth rate, whereby H(2)O(2) accumulated only temporarily during the lag phase. H(2)O(2) is an inhibitor for several glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase being the most sensitive. Strain ATCC 19435 contained NADH oxidase (maximum specific rate under aerobic conditions, 426 nmol of NADH min(-1) mg of protein(-1)), which reduced oxygen to water, whereby superoxide was formed as a by-product. H(2)O(2) originated from the dismutation of superoxide by superoxide dismutase. Although H(2)O(2) was rapidly destroyed under high metabolic fluxes, neither NADH peroxidase nor any other enzymatic H(2)O(2)-reducing activity was detected. However, pyruvate, the end product of glycolysis, reacted nonenzymatically and rapidly with H(2)O(2) and hence was a potential alternative for scavenging of this oxygen metabolite intracellularly. Indeed, intracellular concentrations of up to 93 mM pyruvate were detected in aerobic cultures growing at high rates. It is hypothesized that self-generated pyruvate may serve to protect L. lactis strain ATCC 19435 from H(2)O(2).

Culture Media↗
PubMed1989

Conjugal transfer of plasmid pIP501 from Lactococcus lactis to Lactobacillus delbrückii subsp. bulgaricus and Lactobacillus helveticus.

Plasmid pIP501 was transferred by conjugation from Lactococcus lactis to Lactobacillus delbrückii subsp. bulgaricus and Lactobacillus helveticus. Only Lb. delbrückii subsp. bulgaricus transconjugants could act as a donor in crosses with Lc. lactis. No Lactobacillus transconjugants were detected after inter- or intra-species Lactobacillus crosses. Plasmid pIP501 has undergone no detectable deletion or rearrangement during transfer from Lc. lactis to Lactobacillus strains.

Conjugation, Genetic↗
PubMed2003

Charged casein-derived oligopeptides competitively inhibit the transport of a reporter oligopeptide by Lactococcus lactis.

AIM: To study the effect of casein-derived peptides, accumulated during growth of Lactococcus lactis in milk, on its oligopeptide transport (Opp) function. METHODS AND RESULTS: This effect was estimated by analysing the ability of casein-derived peptides to compete for the transport of a reporter peptide by whole L. lactis cells. The transport of the reported peptide was monitored by determining the intracellular concentrations of the corresponding amino acids by means of reverse-phase high-performance liquid chromatography (HPLC). Uptake of the reporter peptide was competitively inhibited by casein-derived peptides. The competition was only because of charged casein-derived peptides, including anionic peptides. The design of specific pure peptides made it possible to evidence for a positive (or negative) influence exerted by the positively (or negatively) charged side chain of the N-terminal amino acid on the competition. CONCLUSIONS: Charged casein-derived peptides impaired the oligopeptide transport function of L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate an inhibition of Opp when too many peptides are produced by the proteinase. Peptide transport by Opp therefore represents a bottleneck for increasing the growth rate of L. lactis in milk.

Animals↗
PubMed2001

Molecular differentiation of Lactococcus lactis subspecies lactis and cremoris strains by ribotyping and site specific-PCR.

Twenty-five strains of Lactococcus lactis subspecies lactis and subspecies cremoris obtained from dairy industry and environmental collections were examined by 16S RNA automated ribotyping profiles and site-specific PCR (S-PCR). By automated ribotyping, the majority of strains were classified in accordance with phenotypic characterization, with the exception of one lactis (220) and two cremoris (BO32 and 140) strains. A complete differentiation of subspecies lactis and cremoris in agreement with conventional phenotypic methods was achieved by S-PCR with a set of site-specific primer pairs (PR1, RM4, and F3) designed particularly from a deletion region found in subspecies cremoris, but not in lactis. Therefore, S-PCR with primers (PR1, RM4, and F3) is a rapid and very sensitive method for the distinction of lactis and cremoris subspecies in dairy production.

DNA Fingerprinting↗
PubMed1999

Functional analysis of glycosyltransferase genes from Lactococcus lactis and other gram-positive cocci: complementation, expression, and diversity.

Sixteen exopolysaccharide (EPS)-producing Lactococcus lactis strains were analyzed for the chemical compositions of their EPSs and the locations, sequences, and organization of the eps genes involved in EPS biosynthesis. This allowed the grouping of these strains into three major groups, representatives of which were studied in detail. Previously, we have characterized the eps gene cluster of strain NIZO B40 (group I) and determined the function of three of its glycosyltransferase (GTF) genes. Fragments of the eps gene clusters of strains NIZO B35 (group II) and NIZO B891 (group III) were cloned, and these encoded the NIZO B35 priming galactosyltransferase, the NIZO B891 priming glucosyltransferase, and the NIZO B891 galactosyltransferase involved in the second step of repeating-unit synthesis. The NIZO B40 priming glucosyltransferase gene epsD was replaced with an erythromycin resistance gene, and this resulted in loss of EPS production. This epsD deletion was complemented with priming GTF genes from gram-positive organisms with known function and substrate specificity. Although no EPS production was found with priming galactosyltransferase genes from L. lactis or Streptococcus thermophilus, complementation with priming glucosyltransferase genes involved in L. lactis EPS and Streptococcus pneumoniae capsule biosynthesis could completely restore or even increase EPS production in L. lactis.

Amino Acid Sequence↗
PubMed2001

Conversion of methionine to methional by Lactococcus lactis.

Lactic acid bacteria were screened for methional production from 4-methylthio-2-ketobutanoate. Only Lactococcus lactis IFPL730 produced high amounts of methional. It was demonstrated that production of this compound was an exclusively enzymatic reaction. The present work describes for the first time that L. lactis can convert enzymatically methionine to methional in a process mediated by aminotransferase and alpha-ketoacid decarboxylase activities. The activity seems to be strain dependent.

Aldehydes↗
PubMed2003

Immunogenicity and protective efficacy of orally administered recombinant Lactococcus lactis expressing surface-bound HIV Env.

This study investigates whether genetically modified orally administered Lactococcus lactis (L lactis) could be used as an HIV vaccine. L lactis is immunogenic and extremely safe when delivered orally. We created a recombinant L lactis vector expressing the envelope protein of HIV on its cell surface. Oral immunization with this vector induced high levels of HIV-specific serum IgG and fecal IgA antibodies. Cell-mediated immune responses also were generated in both the regional lymph nodes and the spleen. Dendritic cells are readily infected by L lactis and appear to play a potential role in mediating the development of these immune responses. The protective efficacy of this vaccine strategy was demonstrated by challenging mice intraperitoneally with an HIV Env-expressing vaccinia virus. Their viral loads were 350-fold lower than those of control mice. These findings support the further development of L lactis-based HIV vaccines.

AIDS Vaccines↗
PubMed2005

Identification and molecular characterization of the chromosomal exopolysaccharide biosynthesis gene cluster from Lactococcus lactis subsp. cremoris SMQ-461.

The exopolysaccharide (EPS) capsule-forming strain SMQ-461 of Lactococcus lactis subsp. cremoris, isolated from raw milk, produces EPS with an apparent molecular mass of >1.6 x 10(6) Da. The EPS biosynthetic genes are located on the chromosome in a 13.2-kb region consisting of 15 open reading frames. This region is flanked by three IS1077-related tnp genes (L. lactis) at the 5' end and orfY, along with an IS981-related tnp gene, at the 3' end. The eps genes are organized in specific regions involved in regulation, chain length determination, biosynthesis of the repeat unit, polymerization, and export. Three (epsGIK) of the six predicted glycosyltransferase gene products showed low amino acid similarity with known glycosyltransferases. The structure of the repeat unit could thus be different from those known to date for Lactococcus. Reverse transcription-PCR analysis revealed that the eps locus is transcribed as a single mRNA. The function of the eps gene cluster was confirmed by disrupting the priming glycosyltransferase gene (epsD) in Lactococcus cremoris SMQ-461, generating non-EPS-producing reversible mutants. This is the first report of a chromosomal location for EPS genetic elements in Lactococcus cremoris, with novel glycosyltransferases not encountered before in lactic acid bacteria.

Animals↗
PubMed2004

Lactococcus lactis uses MscL as its principal mechanosensitive channel.

The functions of the mechanosensitive channels from Lactococcus lactis were determined by biochemical, physiological, and electrophysiological methods. Patch-clamp studies showed that the genes yncB and mscL encode MscS and MscL-like channels, respectively, when expressed in Escherichia coli or if the gene products were purified and reconstituted in proteoliposomes. However, unless yncB was expressed in trans, wild type membranes of L. lactis displayed only MscL activity. Membranes prepared from an mscL disruption mutant did not show any mechanosensitive channel activity, irrespective of whether the cells had been grown on low or high osmolarity medium. In osmotic downshift assays, wild type cells survived and retained 20% of the glycine betaine internalized under external high salt conditions. On the other hand, the mscL disruption mutant retained 40% of internalized glycine betaine and was significantly compromised in its survival upon osmotic downshifts. The data strongly suggest that L. lactis uses MscL as the main mechanosensitive solute release system to protect the cells under conditions of osmotic downshift.

Amino Acid Sequence↗