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PubMed1994

Action of a cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris AM1 on bovine kappa-casein.

The specificity of the cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris AM1 in its action on bovine kappa-casein was studied. A 4-h digest (pH 6.2, 15 degrees C) of kappa-casein was made with the purified proteinase. The pH-4.6 soluble fraction, representing more than 95% of the whole hydrolysate, was ultrafiltered to obtain a high-molecular-mass (HMM) and a low-molecular-mass (LMM) fraction, which were separately further purified by electrophoretic and chromatographic techniques. Isolated HMM and LMM products were identified by amino acid analysis, end-group determination and mass spectrometry. On-line HPLC/mass spectrometry was also used for the separation of an LMM peptide mixture and the identification of its components. The HMM products formed were the fragments 1-160, 1-151, 1-95 and 1-79 of kappa-casein, whereas the main LMM products found were the 161-169 and 152-160 fragments. The enzyme specificity was concluded to be primarily directed towards the C-terminal region of the substrate molecule by cleavage of the 160-161 and 151-152 peptide bonds. Two minor LMM products were identified as the fragments 96-104 and 103-106, indicating additional cleavage at positions 102-103, 104-105 and 106-107 of the sequence. Also several peptide bonds within the 161-169 sequence were found to be subject to secondary cleavage by the proteinase. From electrophoretic and identification data it is concluded that the lactococcal CEPI, CEPIII and several mixed-type proteinases all act on the peptide bonds at positions 79-80 and 95-96.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2002

Influence of growth conditions on the production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from kimchi.

The influence of growth parameters on the fermentative production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from kimchi was studied. The bacteriocin production was greatly affected by carbon and nitrogen sources. Strain A164 produced at least 4-fold greater bacteriocin in M17 broth supplemented with lactose than other carbon sources. The amount of 3% yeast extract was found to be the optimal organic nitrogen source. While the maximum biomass was obtained at 37 degrees C, the optimal temperature for the bacteriocin production was 30 degrees C. The bacteriocin production was also affected by pH of the culture broth. The optimal pH for growth and bacteriocin production was 6.0. Although the cell growth at pH 6.0 was nearly the same level at pH 5.5 and 6.5, the greater bacteriocin activity was observed at pH 6.0. Exponential growth took place only during an initial period of the cultivation, and then linear growth was observed. Linear growth rates increased from 0.160 g(DCW) x l(-1) x h(-1) to 0.245 g(DCW) x l(-1) x h(-1) with increases in lactose concentrations from 0.5 to 3.0%. Maximum biomass was also increased from 1.88 g(DCW) x l(-1) to 4.29 g(DCW) x l(-1). However, increase in lactose concentration did not prolong the active growth phase. After 20 h cultivation, cell growth stopped regardless of lactose concentration. Production of the bacteriocin showed primary metabolic kinetics. However, bacteriocin yield based on cell mass increased greatly during the late growth phase. A maximum activity of 131x10(3) AU x ml(-1) was obtained at early stationary growth phase (20 h) during the batch fermentation in M17L broth (3.0% lactose) at 30 degrees C and pH 6.0.

Bacteriocins↗
PubMed2000

Changes in glycolytic activity of Lactococcus lactis induced by low temperature.

The effects of low-temperature stress on the glycolytic activity of the lactic acid bacterium Lactococcus lactis were studied. The maximal glycolytic activity measured at 30 degrees C increased approximately 2.5-fold following a shift from 30 to 10 degrees C for 4 h in a process that required protein synthesis. Analysis of cold adaptation of strains with genes involved in sugar metabolism disrupted showed that both the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) subunit HPr and catabolite control protein A (CcpA) are involved in the increased acidification at low temperatures. In contrast, a strain with the PTS subunit enzyme I disrupted showed increased acidification similar to that in the wild-type strain. This indicates that the PTS is not involved in this response whereas the regulatory function of 46-seryl phosphorylated HPr [HPr(Ser-P)] probably is involved. Protein analysis showed that the production of both HPr and CcpA was induced severalfold (up to two- to threefold) upon exposure to low temperatures. The las operon, which is subject to catabolite activation by the CcpA-HPr(Ser-P) complex, was not induced upon cold shock, and no increased lactate dehydrogenase (LDH) activity was observed. Similarly, the rate-limiting enzyme of the glycolytic pathway under starvation conditions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was not induced upon cold shock. This indicates that a factor other than LDH or GAPDH is rate determining for the increased glycolytic activity upon exposure to low temperatures. Based on their cold induction and involvement in cold adaptation of glycolysis, it is proposed that the CcpA-HPr(Ser-P) control circuit regulates this factor(s) and hence couples catabolite repression and cold shock response in a functional and mechanistic way.

Bacterial Proteins↗
PubMed1993

ScrFI restriction-modification system of Lactococcus lactis subsp. cremoris UC503: cloning and characterization of two ScrFI methylase genes.

Two genes from the total genomic DNA of dairy starter culture Lactococcus lactis subsp. cremoris UC503, encoding ScrFI modification enzymes, have been cloned and expressed in Escherichia coli. No homology between the two methylase genes was detected, and inverse polymerase chain reaction of flanking chromosomal DNA indicated that both were linked on the Lactococcus genome. Neither clone encoded the cognate endonuclease. The DNA sequence of one of the methylase genes (encoded by pCI931M) was determined and consisted of an open reading frame 1,170 bp long, which could encode a protein of 389 amino acids (M(r), 44.5). The amino acid sequence contained the highly characteristic motifs of an m5C methylase. Extensive regions of homology were observed with the methylases of NlaX, EcoRII, and Dcm.

Amino Acid Sequence↗
PubMed2002

Proteome analysis of a Lactococcus lactis strain overexpressing gapA suggests that the gene product is an auxiliary glyceraldehyde 3-phosphate dehydrogenase.

The sequence of the genome from the Lactococcus lactis subspecies lactis strain IL1403 shows the presence of two reading frames, gapA and gapB, putatively encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Previous proteomic analysis of the L. lactis subspecies cremoris strain MG1363 has revealed two neighbouring protein spots, GapBI and GapBII, with amino terminal sequences identical to the product of gapA from the L. lactis subspecies cremoris strain LM0230 and that of the two IL1403 sequences. In order to assign the two protein spots to their respective genes we constructed an L. lactis strain that overexpessed the gapA gene derived from MG1363 upon nisin induction. Compared to the wild-type, the overexpressing strain had a 3.4-fold elevated level of specific GAPDH activity when grown in the presence of nisin. In both MG1363 and the gapA overexpressing strain the GAPDH activity was specific for NAD. No NADP dependent activity was detected. Proteome analysis of the gapA overexpressing strain revealed two new protein spots, GapAI and GapAII, not previously detected in proteome analysis of MG1363. Results from mass spectrometry analysis of GapA and GapB and comparison with the deduced protein sequences for the GAPDH isozymes from the genome sequence of strain IL1403 allowed us to assign GapA and GapB to their apparent IL1403 homologues encoded by gapA and gapB, respectively. Furthermore, we suggest that a homologue of a gapB product, represented by GapB, is the main source of GAPDH activity in L. lactis during normal growth.

Amino Acid Sequence↗
PubMed1997

The N-terminal sequence of Lactococcus lactis phosphoglucose isomerase purified by affinity chromatography differs from the other species.

A specific monoclonal antibody, M3A, was produced to rapidly purify Lactococcus lactis phosphoglucose isomerase (PGI) for amino acid sequence analysis. M3A recognized the Lac. lactis PGI specifically and sensitively with both enzyme-linked immunosorbent assay and Western blot analysis. The enzyme was rapidly purified to a specific activity of 21.8 U/mg with a yield of 20% by a three-step procedure, including M3A-bound Sepharose chromatography. The specific activity of PGI was increased about 64.1-fold from the cell lysate. The molecular mass of Lac. lactis PGI was estimated to be about 50 kDa by SDS-PAGE. The N-terminal amino acid sequence of Lac. lactis PGI exhibited no significant similarity to other PGIs, except for a 52.6% identity to Bacillus stearothermophilus PGI A and PGI B. These results suggest that there might be some molecular types of PGI.

Amino Acid Sequence↗
PubMed1991

Lactococcin A, a new bacteriocin from Lactococcus lactis subsp. cremoris: isolation and characterization of the protein and its gene.

A new bacteriocin, termed lactococcin A (LCN-A), from Lactococcus lactis subsp. cremoris LMG 2130 was purified and sequenced. The polypeptide contained no unusual amino acids and showed no significant sequence similarity to other known proteins. Only lactococci were killed by the bacteriocin. Of more than 120 L. lactis strains tested, only 1 was found resistant to LCN-A. The most sensitive strain tested, L. lactis subsp. cremoris NCDO 1198, was inhibited by 7 pM LCN-A. By use of a synthetic DNA probe, lcnA was found to be located on a 55-kb plasmid. The lcnA gene was cloned and sequenced. The sequence data revealed that LCN-A is ribosomally synthesized as a 75-amino-acid precursor including a 21-amino-acid N-terminal extension. An open reading frame encoding a 98-amino-acid polypeptide was found downstream of and in the same operon as lcnA. We propose that this open reading frame encodes an immunity function for LCN-A. In Escherichia coli lcnA did not cause an LCN-A+ phenotype. L. lactis subsp. lactis IL 1403 produced small amounts of the bacteriocin and became resistant to LCN-A after transformation with a recombinant plasmid carrying lcnA. The other lactococcal strains transformed with the same recombinant plasmid became resistant to LCN-A but did not produce any detectable amount of the bacteriocin.

Amino Acid Sequence↗
PubMed2004

Controlled expression of CluA in Lactococcus lactis and its role in conjugation.

CluA is a 136 kDa surface-bound protein encoded by the chromosomally located sex factor of Lactococcus lactis MG1363 and is associated with cell aggregation linked to high-frequency transfer of the sex factor. To further investigate the involvement of CluA in these phenomena, the cluA gene was cloned on a plasmid, downstream from the lactococcal nisA promoter. In a sex-factor-negative MG1363 derivative, nisin-controlled CluA expression resulted in aggregation, despite the absence of the other genes of the sex factor. Therefore, CluA is the only sex factor component responsible for aggregation. The direct involvement of CluA in the establishment of cell-to-cell contact for aggregate formation was observed by electron microscopy using immunogold-labelled CluA antibodies. Inactivation of cluA in an MG1363 background led to a dramatic decrease in sex factor conjugation frequency compared to the parental strain. Increasing levels of CluA expressed in trans in the cluA-inactivated donor strain facilitated a gradual restoration of conjugation frequency, reaching that of the parental strain. In conclusion, CluA is essential for efficient sex factor transfer in conjugation of L. lactis.

Animals↗
PubMed1994

Tripeptidase gene (pepT) of Lactococcus lactis: molecular cloning and nucleotide sequencing of pepT and construction of a chromosomal deletion mutant.

The gene encoding a tripeptidase (pepT) of Lactococcus lactis subsp. cremoris (formerly subsp. lactis) MG1363 was cloned from a genomic library in pUC19 and subsequently sequenced. The tripeptidase of L. lactis was shown to be homologous to PepT of Salmonella typhimurium with 47.4% identity in the deduced amino acid sequences. L. lactis PepT was enzymatically active in Escherichia coli and allowed growth of a peptidase-negative leucine-auxotrophic E. coli strain by liberation of Leu from a tripeptide. Using a two-step integration-excision system, a pepT-negative mutant of L. lactis was constructed. No differences between the growth of the mutant and that of the wild-type strain in milk or in chemically defined medium with casein as the sole source of essential amino acids were observed.

Amino Acid Sequence↗
PubMed1997

Dual role of alpha-acetolactate decarboxylase in Lactococcus lactis subsp. lactis.

The alpha-acetolactate decarboxylase gene aldB is clustered with the genes for the branched-chain amino acids (BCAA) in Lactococcus lactis subsp. lactis. It can be transcribed with BCAA genes under isoleucine regulation or independently of BCAA synthesis under the control of its own promoter. The product of aldB is responsible for leucine sensibility under valine starvation. In the presence of more than 10 microM leucine, the alpha-acetolactate produced by the biosynthetic acetohydroxy acid synthase IlvBN is transformed to acetoin by AldB and, consequently, is not available for valine synthesis. AldB is also involved in acetoin formation in the 2,3-butanediol pathway, initiated by the catabolic acetolactate synthase, AlsS. The differences in the genetic organization, the expression, and the kinetics parameters of these enzymes between L. lactis and Klebsiella terrigena, Bacillus subtilis, or Leuconostoc oenos suggest that this pathway plays a different role in the metabolism in these bacteria. Thus, the alpha-acetolactate decarboxylase from L. lactis plays a dual role in the cell: (i) as key regulator of valine and leucine biosynthesis, by controlling the acetolactate flux by a shift to catabolism; and (ii) as an enzyme catalyzing the second step of the 2,3-butanediol pathway.

Acetoin↗
PubMed1996

Imbalance of leucine flux in Lactococcus lactis and its use for the isolation of diacetyl-overproducing strains.

Diacetyl is a by-product of pyruvate metabolism in Lactococcus lactis, where pyruvate is first converted to alpha-acetolactate, which is slowly decarboxylated to diacetyl in the presence of oxygen. L. lactis usually converts alpha-acetolactate to acetoin enzymatically, by alpha-acetolactate decarboxylase encoded by the aldB gene. We took advantage of the fact that this enzyme also has a central role in the regulation of branched-chain amino acids, to select spontaneous aldB mutants in an unbalanced concentration of leucine versus those of valine and isoleucine in the medium. Industrial dairy strains of L. lactis subsp. lactis biovar diacetylactis containing point mutations and deletions of aldB were isolated and characterized. Their growth in milk was not affected, but they rapidly accumulated a large amount of alpha-acetolactate instead of acetoin from citrate in milk. Under aerated condition, strains devoid of AldB produced about 10 times more diacetyl than did the parental strains.

Amino Acid Sequence↗
PubMed2002

Novel food-grade plasmid vector based on melibiose fermentation for the genetic engineering of Lactococcus lactis.

The alpha-galactosidase gene (aga) and a gene coding for a putative transcriptional regulator from the LacI/GalR family (galR) of Lactococcus raffinolactis ATCC 43920 were cloned and sequenced. When transferred into Lactococcus lactis and Pediococcus acidilactici strains, aga modified the sugar fermentation profile of the strains from melibiose negative (Mel(-)) to melibiose positive (Mel(+)). Analysis of galA mutants of L. lactis subsp. cremoris MG1363 indicated that the putative galactose permease GalA is also needed to obtain the Mel(+) phenotype. Consequently, GalA may also transport melibiose into this strain. We demonstrated that when aga was associated with the theta-type replicon of a natural L. lactis plasmid, it constituted the selectable marker of a cloning vector named pRAF800. Transcriptional analysis by reverse transcriptase PCR suggests that this vector is also suitable for gene expression. The alpha-galactosidase activity conferred by pRAF800 was monitored in an industrial strain grown in the presence of various carbon sources. The results indicated that the enzymatic activity was induced by galactose and melibiose, but not by glucose or lactose. The gene encoding the phage defense mechanism, AbiQ, was cloned into pRAF800, and the resulting clone (pRAF803) was transferred into an industrial L. lactis strain that became highly phage resistant. The measurements of various growth parameters indicated that cells were not affected by the presence of pRAF803. Moreover, the plasmid was highly stable in this strain even under starter production conditions. The L. raffinolactis aga gene represents the basis of a novel and convenient food-grade molecular tool for the genetic engineering of lactic acid bacteria.

Base Sequence↗
PubMed2006

Effects of Lactococcus lactis on composition of intestinal microbiota: role of nisin.

This study examined the ability of (i) pure nisin, (ii) nisin-producing Lactococcus lactis strain CHCC5826, and (iii) the non-nisin-producing L. lactis strain CHCH2862 to affect the composition of the intestinal microbiota of human flora-associated rats. The presence of both the nisin-producing and the non-nisin-producing L. lactis strains significantly increased the number of Bifidobacterium cells in fecal samples during the first 8 days but decreased the number of enterococci/streptococci in duodenum, ileum, cecum, and colon samples as detected by selective cultivation. No significant changes in the rat fecal microbiota were observed after dosage with nisin. Pearson cluster analysis of denaturing gradient gel electrophoresis profiles of the 16S rRNA genes present in the fecal microbial population revealed that the microbiota of animals dosed with either of the two L. lactis strains were different from that of control animals dosed with saline. However, profiles of the microbiota from animals dosed with nisin did not differ from the controls. The concentrations of nisin estimated by competitive enzyme-linked immunosorbent assay (ELISA) were approximately 10-fold higher in the small intestine and 200-fold higher in feces than the corresponding concentrations estimated by a biological assay. This indicates that nisin was degraded or inactivated in the gastrointestinal tract, since fragments of this bacteriocin are detected by ELISA while an intact molecule is needed to retain biological activity.

Agar↗
PubMed2004

Only one of the two annotated Lactococcus lactis fabG genes encodes a functional beta-ketoacyl-acyl carrier protein reductase.

The small genome of the Gram-positive bacterium Lactococcus lactis ssp. lactis IL1403 contains two genes that encode proteins annotated as homologues of Escherichia coli beta-hydroxyacyl-acyl carrier protein (ACP) reductase. E. coli fabG encodes beta-ketoacyl-acyl carrier protein (ACP) reductase, the enzyme responsible for the first reductive step of the fatty acid synthetic cycle. Both of the L. lactis genes are adjacent to (and predicted to be cotranscribed with) other genes that encode proteins having homology to known fatty acid synthetic enzymes. Such relationships have often been used to strengthen annotations based on sequence alignments. Annotation in the case of beta-ketoacyl-ACP reductase is particularly problematic because the protein is a member of a vast protein family, the short-chain alcohol dehydrogenase/reductase (SDR) family. The recent isolation of an E. coli fabG mutant strain encoding a conditionally active beta-ketoacyl-ACP reductase allowed physiological and biochemical testing of the putative L. lactishomologues. We report that expression of only one of the two L. lactis proteins (that annotated as FabG1) allows growth of the E. coli fabG strain under nonpermissive conditions and restores in vitro fatty acid synthetic ability to extracts of the mutant strain. Therefore, like E. coli, L. lactis has a single beta-ketoacyl-ACP reductase active with substrates of all fatty acid chain lengths. The second protein (annotated as FabG2), although inactive in fatty acid synthesis both in vivo and in vitro, was highly active in reduction of the model substrate, beta-ketobutyryl-CoA. As expected from work on the E. coli enzyme, the FabG1 beta-ketobutyryl-CoA reductase activity was inhibited by ACP (which blocks access to the active site) whereas the activity of FabG2 was unaffected by the presence of ACP. These results seem to be an example of a gene duplication event followed by divergence of one copy of the gene to encode a protein having a new function.

Alcohol Oxidoreductases↗
PubMed2004

Complete genome sequence of the Lactococcus lactis temperate phage phiLC3: comparative analysis of phiLC3 and its relatives in lactococci and streptococci.

Complete genome sequencing of the P335 temperate Lactococcus lactis bacteriophage phiLC3 (32, 172 bp) revealed fifty-one open reading frames (ORFs). Four ORFs did not show any homology to other proteins in the database and twenty-one ORFs were assigned a putative biological function. phiLC3 contained a unique replication module and orf201 was identified as the putative replication initiator protein-encoding gene. phiLC3 was closely related to the L. lactis r1t phage (73% DNA identity). Similarity was also shared with other lactococcal P335 phages and the Streptococcus pyogenes prophages 370.3, 8232.4 and 315.5 over the non-structural genes and the genes involved in DNA packaging/phage morphogenesis, respectively. phiLC3 contained small homologous regions distributed among lactococcal phages suggesting that these regions might be involved in mediating genetic exchange. Two regions of 30 and 32 bp were conserved among the streptococcal and lactococcal r1t-like phages. These two regions, as well as other homologous regions, were located at mosaic borders and close to putative transcriptional terminators indicating that such regions together might attract recombination. The conserved regions found among lactococcal and streptococcal phages might be used for identification of phages/prophages/prophage remnants in their hosts.

Base Sequence↗
PubMed2001

Effects of structural modifications on some physical characteristics of exopolysaccharides from Lactococcus lactis.

The relationship between the primary structure and the chain stiffness of exopolysaccharides (EPSs) and modified EPSs produced by two strains of Lactococcus lactis subsp. cremoris was investigated. The molar mass and radius of gyration of these exopolysaccharides were analyzed by multiangle static light scattering after size-exclusion chromatography. From these results and the chemical structure of the repeating units of the investigated EPSs, the Kuhn lengths could be calculated. We found that the initial Kuhn lengths of the two native EPSs are similar. Modification of the EPSs by removing parts of the side groups resulted in a decrease in both the absolute value and the normalized value of the Kuhn length. It is therefore concluded that partial removal of the side groups of these polysaccharides could make them less efficient as thickeners if no specific interaction with other components occurs.

Carbohydrate Conformation↗
PubMed2002

Cre-loxP recombination system for large genome rearrangements in Lactococcus lactis.

We have used a new genetic strategy based on the Cre-loxP recombination system to generate large chromosomal rearrangements in Lactococcus lactis. Two loxP sites were sequentially integrated in inverse order into the chromosome either at random locations by transposition or at fixed points by homologous recombination. The recombination between the two chromosomal loxP sites was highly efficient (approximately 1 x 10(-1)/cell) when the Cre recombinase was provided in trans, and parental- or inverted-type chromosomal structures were isolated after removal of the Cre recombinase. The usefulness of this approach was demonstrated by creating three large inversions of 500, 1,115, and 1,160 kb in size that modified the lactococcal genome organization to different extents. The Cre-loxP recombination system described can potentially be used for other gram-positive bacteria without further modification.

Chromosome Inversion↗