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PubMed1991

Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris Wg2.

An endopeptidase has been purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that includes diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, hydroxylapatite chromatography, and fast protein liquid chromatography over an anion-exchange column and a hydrophobic-interaction column. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a molecular mass of the purified enzyme of 70,000 Da. The endopeptidase can degrade several oligopeptides into various tetra-, tri-, and dipeptides. The endopeptidase has no aminopeptidase, carboxypeptidase, dipeptidase, or tripeptidase activity. It is optimally active at pH 6.0 to 6.5 and in the temperature range of 30 to 38 degrees C. The enzyme is inactivated by the chemical agents 1,10-phenanthroline, ethylenedinitrilotetraacetate, beta-mercaptoethanol, and phenylmethylsulfonyl fluoride and is inhibited by Cu2+ and Zn2+. The ethylenedinitrilotetraacetate- or 1,10-phenanthroline-treated enzyme can be reactivated by Co2+. Immunoblotting with specific antibodies raised against the purified endopeptidase indicated that the enzyme is also present in other Lactococcus spp., as well as in Lactobacillus spp. and Streptococcus salivarius subsp. thermophilus.

Amino Acid Sequence↗
PubMed1999

Characterization of the divergent sacBK and sacAR operons, involved in sucrose utilization by Lactococcus lactis.

The divergently transcribed sacBK and sacAR operons, which are involved in the utilization of sucrose by Lactococcus lactis NZ9800, were examined by transcriptional and gene inactivation studies. Northern analyses of RNA isolated from cells grown at the expense of different carbon sources revealed three sucrose-inducible transcripts: one of 3.2 kb containing sacB and sacK, a second of 3.4 kb containing sacA and sacR, and a third of 1.8 kb containing only sacR. The inactivation of the sacR gene by replacement recombination resulted in the constitutive transcription of the sacBK and sacAR operons in the presence of different carbon sources, indicating that SacR acts as a repressor of transcription.

Fructokinases↗
PubMed1994

Genetic analysis of regions of the Lactococcus lactis subsp. lactis plasmid pRS01 involved in conjugative transfer.

The genes responsible for conjugative transfer of the 48.4-kb Lactococcus lactis subsp. lactis ML3 plasmid pRS01 were localized by insertional mutagenesis. Integration of the IS946-containing plasmid pTRK28 into pRS01 generated a pool of stable cointegrates, including a number of plasmids altered in conjugative proficiency. Mapping of pTRK28 insertions and phenotypic analysis of cointegrate plasmids identified four distinct regions (Tra1, Tra2, Tra3, and Tra4) involved in pRS01 conjugative transfer. Tra3 corresponds closely to a region previously identified (D. G. Anderson and L. L. McKay, J. Bacteriol. 158:954-962, 1984). Another region (Tra4) was localized within an inversion sequence shown to correlate with a cell aggregation phenotype. Tra1 and Tra2, two previously unidentified regions, were located at a distance of 9 kb from Tra3. When provided in trans, a cloned portion of the Tra3 region complemented Tra3 mutants.

Chromosome Mapping↗
PubMed2005

Functional expression of bacterial Zymobacter palmae pyruvate decarboxylase gene in Lactococcus lactis.

A pyruvate decarboxylase (PDC) gene from bacterial Zymobacter palmae (Zymopdc) was cloned, characterized, and introduced into Lactococcus lactis via a shuttle vector pAK80 as part of a research strategy to develop an efficient ethanol-producing lactic acid bacteria (LAB). The expression levels of Zymopdc gene in the host, as measured by a colorimetric assay based on PDC catalyzed formation of (R)-phenylacetylcarbinol ((R)-PAC), appeared to be dependent on the strength of corresponding Gram-positive promoters. A constitutive, highly expressed promoter conferred the greatest PDC activity, and an acid-inducible promoter demonstrated acid-inducible expression. The metabolic production of ethanol and other products was examined in flask fermentations. More than eightfold increases in acetaldehyde concentrations were detected in two recombinant strains. However, no detectable differences for ethanol fermentation in these engineered strains were observed compared with that of the strain carrying lacZ reporter.

Base Sequence↗
PubMed1993

Two genes present on a transposon-like structure in Lactococcus lactis are involved in a Clp-family proteolytic activity.

The lactose-protease plasmid pUCL22 of Lactococcus lactis subsp. lactis strain CNRZ270 contained two inverted copies of IS 1076 flanking a region of 3.7 kb. This internal region was sequenced and found to contain two large open reading frames, ORF1 and ORFP in opposite orientations. ORF1 consists of 2289 bp; the deduced 763-amino-acid sequence is similar to the ATPases of the ClpA family. It contains two well-conserved consensus ATP-binding sites. It was named ClpL. ORFP consists of 930 bp encoding a protein of 310 amino acids. No similarity with any known protein was found in GenBank data for ORFP. Increased ATP-dependent proteolytic activity was detected in extracts from Escherichia coli cells expressing the clpL and ORFP genes.

Adenosine Triphosphatases↗
PubMed1992

Growth and activities of Lactococcus lactis in milk enriched with low mineral retentate powders.

The growth and activities of three strains of Lactococcus lactis ssp. cremoris (Wg2, E8, and HP) and their proteinase-negative variants were studied in skim milk enriched with three types of retentate powder. The performance of these strains in enriched milks was compared with that determined in reconstituted skim milk. Proteinase-positive strains of L. lactis ssp. cremoris exhibited higher maximum specific growth rates than protease-negative variants. Moreover, maximum specific growth rates of lactococci were lower in skim milk than in enriched milk with a high buffering capacity. The performance of proteinase-positive strains was better than that of proteinase-negative variants. Growth of proteinase-positive lactococci in milk media increased alpha-amino groups as determined by the increase of equivalent glutamic acid concentration. Available alpha-amino groups decreased with proteinase-negative variants. Proteinase-positive strain Wg2 exhibited the most proteolytic activity but showed the least specific overall productivity of lactic acid despite high biomass concentration in milk. Among proteinase-positive lactococci, strain E8 produced more lactic acid than other strains, and, among proteinase-negative variants, strain HP had the best specific overall productivity of lactic acid.

Animals↗
PubMed1991

Action of a cell wall proteinase from Lactococcus lactis subsp. cremoris SK11 on bovine alpha s1-casein.

The cell wall-associated proteinase from Lactococcus lactis subsp. cremoris SK11 was partially purified and incubated with alpha s1-casein for various times up to 48 h. Sixteen trifluoroacetic acid-soluble oligopeptide hydrolysis products were identified by determination of the amino acid sequence. Eleven of these oligopeptides originated from the 78-residue sequence comprising the C-terminal region of alpha s1-casein and were present among the products after the first 60 min of digestion. Three oligopeptides from the N-terminal region and two others from the central region of the alpha s1-casein sequence were also present among the early digestion products although in smaller amounts than most of the oligopeptides from the C-terminal region. No clear consensus sequence of amino acid residues surrounding the cleavage sites could be identified.

Amino Acid Sequence↗
PubMed1997

Food-grade controlled lysis of Lactococcus lactis for accelerated cheese ripening.

An attractive approach to accelerate cheese ripening is to induce lysis of Lactococcus lactis starter strains for facilitated release of intracellular enzymes involvement in flavor formation. Controlled expression of the lytic genes lytA and lytH, which encode the lysin and the holin proteins of the lactococcal bacteriophage phi US3, respectively, was accomplished by application of a food-grade nisin-inducible expression system. Simultaneous production of lysin and holin is essential to obtain efficient lysis and concomitant release of intracellular enzymes as exemplified by complete release of the debittering intracellular aminopeptidase N. Production of holin alone leads to partial lysis of the host cells, whereas production of lysin alone does not cause significant lysis. Model cheese experiments in which the inducible holinlysin overproducing strain was used showed a fourfold increase in release of L-Lactate dehydrogenase activity into the curd relative to the control strain and the holin-overproducing strain, demonstrating the suitability of the system for cheese applications.

Bacterial Proteins↗
PubMed1997

Isolation, cloning and characterisation of the abiI gene from Lactococcus lactis subsp. lactis M138 encoding abortive phage infection.

Plasmid pND852 (56 kb) encodes nisin resistance and was isolated from Lactococcus lactis ssp lactis (L. lactis) M138 by conjugation to L. lactis LM0230. It conferred strong resistance to the isometric-headed phage phi 712 and partial resistance to the prolate-headed phage phi c2. A 2.6 kb HpaII fragment encoding phage resistance was cloned into the streptococcal/Bacillus hybrid vector pGB301 to generate pND817. The mechanism of phage resistance encoded by pND817 involved abortive infection and this was illustrated by a reduction in burst size from 166 to 6 at 30 degrees C and from 160 to 90 at 37 degrees C. Partial resistance was therefore retained at 37 degrees C. DNA sequencing revealed that the abortive infection was encoded by a single open reading frame (ORF), designated abiI, encoding a 332 amino acid protein. Neither abiI nor the predicted product showed significant homology to any existing sequence in the GenBank database. Frame shift mutation at the unique EcoRI site within the ORF resulted in loss of the Abi+ phenotype, confirming that the ORF is responsible for the encoded phage resistance.

Amino Acid Sequence↗
PubMed1994

Cloning and sequencing of the Lactococcus lactis subsp. lactis dnaK gene using a PCR-based approach.

The coding region for the dnaK gene from Lactococcus lactis subsp. lactis LM0230 was isolated and sequenced. An internal 789-bp fragment was amplified by the polymerase chain reaction (PCR) using a pair of degenerate oligodeoxyribonucleotide primers designed on the basis of amino acid (aa) sequences conserved in a number of DnaK. This PCR product was cloned, sequenced and used as a Southern hybridization probe to locate the flanking regions of the gene. The sequence of this central region from dnaK was also used to design two sets of inverse PCR primers to amplify, separately, the upstream and downstream regions. The inverse PCR products were then cloned and partially sequenced. The complete nucleotide sequence was obtained from overlapping cloned fragments of the gene and found to consist of a single 1824-bp open reading frame coding for a 602-aa protein. Alignment of the deduced aa sequence with those of other bacterial DnaK showed a high degree of homology and is most similar to the Bacillus megaterium DnaK.

Amino Acid Sequence↗
PubMed2004

Mode of action of lactococcin R produced by Lactococcus lactis R.

We investigated the mode of action and factors affecting adsorption of lactoccocin R produced by Lactococcus lactis R. It was found that lactococcin R adsorbed to all Gram-positive but not to the Gram-negative bacteria tested and its adsorption was dependent on pH. It was observed that the binding of lactococcin R was prevented by anions of several salts (Cl-, PO4(-3)) and lipoteichoic acid. Pretreatments of sensitive cells and cell walls with detergents, organic solvents or enzymes did not reduce subsequent binding of lactococcin R. However, treatment of cell wall preparations with methanol:chloroform and hot 20% trichloroacetic acid (TCA) caused such walls to lose their ability to adsorb lactococcin R. Sensitive cells treated with lactococcin R lost high amounts of intracellular K+ ions, UV-absorbing materials and became more permeable to o-nitrophenol-beta-D-glactopyranoside (ONPG). In addition, different lactococcin R concentrations (0-2560 AU/mL) decreased the colony counts of Listeria monocytogenes by 99% and also a reduction in the absorbance values. These results show that the mode of action of lactococcin R is bactericidal rather than bacteriostatic.

Adsorption↗
PubMed1993

The regulation of expression of the Lactococcus lactis lactose operon.

Translational gene fusions between the Escherichia coli beta-galactosidase (lacZ) gene and the Lactococcus lactis lactose operon were constructed such that transcription from the lactose operon promoter could be assessed by measuring beta-galactosidase activity. The level of beta-galactosidase activity was up to 2.5-fold lower when MG5267 cells, which contain a chromosomal copy of the lactose operon, were grown in glucose compared to those grown in lactose. A greater degree of repression was seen in cells containing the multi-copy plasmid-encoded repressor than in those with only the single-copy chromosomal gene, indicating that the repressor protein is at least partly responsible for the reduction in expression when the cells are grown in glucose (i.e. in the absence of inducer). However, the beta-galactosidase activity was found to be 5.5-fold lower in glucose than in lactose in cells which lacked a fully functional lactose operon. The decrease in expression was shown to be due to glucose repression. The levels of expression when the cells were grown in glucose were considerably higher for MG5267 than for MG1363 suggesting perhaps that a product of the chromosomally-encoded operon in MG5267 has a positive effect on transcription.

Cloning, Molecular↗
PubMed2004

Biosurfactant from Lactococcus lactis 53 inhibits microbial adhesion on silicone rubber.

The ability of biosurfactant obtained from the probiotic bacterium Lactococcus lactis 53 to inhibit adhesion of four bacterial and two yeast strains isolated from explanted voice prostheses to silicone rubber with and without an adsorbed biosurfactant layer was investigated in a parallel-plate flow chamber. The microbial cell surfaces and the silicone rubber with and without an adsorbed biosurfactant layer were characterized using contact-angle measurements. Water contact angles indicated that the silicone-rubber surface with adsorbed biosurfactant was more hydrophilic (48 degrees) than bare silicone rubber (109 degrees). The results showed that the biosurfactant was effective in decreasing the initial deposition rates of Staphylococcus epidermidis GB 9/6 from 2,100 to 220 microorganisms cm(-2) s(-1), Streptococcus salivarius GB 24/9 from 1560 to 137 microorganisms cm(-2) s(-1), and Staphylococcus aureus GB 2/1 from 1255 to 135 microorganisms cm(-2) s(-1), allowing for a 90% reduction of the deposition rates. The deposition rates of Rothia dentocariosa GBJ 52/2B, Candida albicans GBJ 13/4A, and Candida tropicalis GB 9/9 were far less reduced in the presence of the biosurfactant as compared with the other strains. This study constitutes a step ahead in developing strategies to prevent microbial colonization of silicone-rubber voice prostheses.

Bacterial Adhesion↗
PubMed2005

Lactococcal 936-species phage attachment to surface of Lactococcus lactis.

The interactions of the 936-species phages sk1, jj50, and 64 with the cell surface of Lactococcus lactis LM0230 were analyzed. Cell envelopes (walls + plasma membrane), cell wall, or plasma membrane from L. lactis ssp. lactis LM0230 each inactivated the phages in vitro. However, other 936-species phages kh and P008, which do not infect strain LM0230, were not inactivated by any of the subcellular fractions. Treating cell walls or plasma membrane with the cell wall hydrolase mutanolysin eliminated inactivation of phage sk1. This suggested that intact cell wall fragments were required for inactivation. A role for plasma membrane in phage sk1 inactivation was further investigated. Boiling, washing in 2 M KCl, 8 M urea, or 0.1 M Na(2)CO(3)/pH 11, or treating the plasma membrane with proteases did not reduce adsorption or inactivation of phage. Adding lipoteichoic acid or antibodies to lipoteichoic acid did not reduce inactivation of phage in a mixture with membrane, suggesting that lipoteichoic acid was not involved. Inactivation by envelopes or cell wall correlated with ejection of DNA from the phage sk1 capsid. Although calcium is required for plaque formation, it was not required for adsorption, inactivation, or ejection of phage DNA by envelopes or cell wall. The results suggest that at least for phages sk1, jj50, and 64, adsorption and phage DNA injection into the host does not require a host membrane protein or lipoteichoic acid, and that cell wall components are sufficient for these initial steps of phage infection.

Adsorption↗
PubMed2002

Effect of different NADH oxidase levels on glucose metabolism by Lactococcus lactis: kinetics of intracellular metabolite pools determined by in vivo nuclear magnetic resonance.

Three isogenic strains of Lactococcus lactis with different levels of H(2)O-forming NADH oxidase activity were used to study the effect of oxygen on glucose metabolism: the parent strain L. lactis MG1363, a NOX(-) strain harboring a deletion of the gene coding for H(2)O-forming NADH oxidase, and a NOX(+) strain with the NADH oxidase activity enhanced by about 100-fold. A comprehensive description of the metabolic events was obtained by using (13)C nuclear magnetic resonance in vivo. The most noticeable results of this study are as follows: (i) under aerobic conditions the level of fructose 1,6-bisphosphate [Fru(1,6)P(2)] was lower than the level under anaerobic conditions, and the rate of Fru(1,6)P(2) depletion was very high; (ii) the levels of 3-phosphoglycerate and phosphoenolpyruvate were considerably enhanced under aerobic conditions and significantly lower in the NOX(-) strain; and (iii) the glycolytic flux decreased in the presence of saturating levels of oxygen, but it was not altered in response to changes in the NADH oxidase activity. In particular, the observation that the glycolytic flux was not enhanced in the NOX(+) strain indicated that glycolytic flux was not primarily determined by the level of NADH in the cell. The patterns of end products were identical for the NOX(-) and parent strains; in the NOX(+) strain the carbon flux was diverted to the production of alpha-acetolactate-derived compounds, and at a low pH this strain produced diacetyl at concentrations up to 1.6 mM. The data were integrated with the goal of identifying the main regulatory aspects of glucose metabolism in the presence of oxygen.

Glucose↗
PubMed2001

Genetic and functional characterization of dpp genes encoding a dipeptide transport system in Lactococcus lactis.

The genes encoding a binding-protein-dependent ABC transporter for dipeptides (Dpp) were identified in Lactococcus lactis subsp. cremoris MG1363. Two (dppA and dppP) of the six ORFs (dppAdppPBCDF) encode proteins that are homologous to peptide- and pheromone-binding proteins. The dppP gene contains a chain-terminating nonsense mutation and a frame-shift that may impair its function. The functionality of the dpp genes was proven by the construction of disruption mutants via homologous recombination. The expression of DppA and various other components of the proteolytic system was studied in synthetic and peptide-rich media and by using isogenic peptide-transport mutants that are defective in one or more systems (Opp, DtpT, and/or Dpp). In peptide-rich medium, DppA was maximally expressed in mutants lacking Opp and DtpT. DppA expression also depended on the growth phase and was repressed by tri-leucine and tri-valine. The effect of tri-leucine on DppA expression was abolished when leucine was present in the medium. Importantly, the Dpp system also regulated the expression of other components of the proteolytic system. This regulation was achieved via the internalization of di-valine, which caused a 30-50% inhibition in the expression of the proteinase PrtP and the peptidases PepN and PepC. Similar to the regulation of DppA, the repressing effect was no longer observed when high concentrations of valine were present. The intricate regulation of the components of the proteolytic system by peptides and amino acids is discussed in the light of the new and published data.

Amino Acid Sequence↗
PubMed1998

Two methods for the genetic differentiation of Lactococcus lactis ssp. lactis and cremoris based on differences in the 16S rRNA gene sequence.

The 16S ribosomal RNA gene sequences of Lactococcus lactis ssp. lactis and ssp. cremoris differ by 9-10 bp (depending on strain), within the first 200 bp of the sequence. These differences were used to develop two methods of genetically differentiating lactis and cremoris strains. Primers to conserved sequences in the 16S rRNA gene were used in a PCR reaction to amplify fragments of the 16S rRNA gene. A single base difference at position 180 of the sequence was utilised to develop a ligase chain reaction to differentiate lactis and cremoris sequences. The second method involved digestion of the amplified fragments with restriction endonucleases specific for either the lactis or cremoris sequence. Resolution of the digested fragments on an agarose gel allowed the strains to be identified as genetically lactis or cremoris. This method was used to examine lactococci isolated from raw milk. Of 31 raw milk strains examined, 21 contained the cremoris 16S rRNA sequence, however, all 31 strains exhibited the phenotypic characteristics of the lactis subspecies.

Animals↗
PubMed2006

Peptidoglycan structure analysis of Lactococcus lactis reveals the presence of an L,D-carboxypeptidase involved in peptidoglycan maturation.

Detailed structural analysis of Lactococcus lactis peptidoglycan was achieved by identification of its constituent muropeptides separated by reverse phase high-performance liquid chromatography. Modification of the classical elution buffer allowed direct and sensitive analysis of the purified muropeptides by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The structures of 45 muropeptides were assigned for L. lactis strain MG1363. Analysis of the muropeptide composition of an MG1363 dacB mutant showed that the dacB-encoded protein has l,d-carboxypeptidase activity and is involved in peptidoglycan maturation.

Base Sequence↗