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PubMed1990

Molecular characterization of promoters of the Lactococcus lactis subsp. cremoris temperate bacteriophage BK5-T and identification of a phage gene implicated in the regulation of promoter activity.

DNA fragments from the temperate lactococcal bacteriophage BK5-T were cloned into the promoter-detecting plasmid pMU1328. Five DNA fragments conferring promoter activity were selected by transformation of Streptococcus sanguis and were functional in Escherichia coli, S. sanguis, and Lactococcus lactis subspp. lactis and cremoris. The nucleotide sequences of these fragments were determined, and primer extension analysis was used to locate the site of initiation of transcription from each promoter in both E. coli and S. sanguis. Transcription was initiated from the same nucleotide in these two organisms, and the promoters contained -10 and -35 regions similar to the consensus sequence for E. coli promoters. The activities of three of the five promoters were decreased two- to threefold when a compatible plasmid containing a 3.8-kilobase-pair EcoRI fragment (EcoRI-f) of BK5-T was coresident with the promoter-containing plasmid in either L. lactis subsp. cremoris or E. coli. Data from Tn5 mutagenesis, subcloning experiments, and DNA sequence analysis indicate that this decrease in promoter activity requires a region of EcoRI-f that contains a 621-base-pair open reading frame. This region has been designated bpi (for BK5-T promoter inhibitor).

Bacteriophages↗
PubMed2002

Lactobacillus bulgaricus proteinase expressed in Lactococcus lactis is a powerful carrier for cell wall-associated and secreted bovine beta-lactoglobulin fusion proteins.

Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine beta-lactoglobulin (BLG) is considered a major allergen in cow's milk allergy. We have designed recombinant Lactococcus lactis expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as fusions with Lactobacillus bulgaricus extracellular proteinase (PrtB). In addition to constructs encoding full-length PrtB for the targeting of heterologous proteins to the cell surface, we generated vectors aiming at the release into the medium of truncated PrtB derivatives lacking 100 (PrtB partial differential, PrtB partial differential-BLG, and PrtB partial differential-T6) or 807 (PrtBdelta) C-terminal amino acids. Expression of recombinant products was confirmed using either anti-PrtB, anti-BLG, or anti-peptide T6 antiserum. All forms of the full-length and truncated recombinant products were efficiently translocated, irrespective of the presence of eucaryotic BLG sequences in the fusion proteins. L. lactis expressing PrtB partial differential-BLG yielded up to 170 microg per 10(9) CFU in the culture supernatant and 9 microg per 10(9) CFU at the bacterial cell surface within 14 h. Therefore, protein fusions relying on the use of PrtB gene products are adequate for concomitant cell surface display and secretion by recombinant L. lactis and thus may ensure maximal bioavailability of the eucaryotic antigen in the gut-associated lymphoid tissue.

Bacterial Proteins↗
PubMed2005

Anaerobic biotransformation of dinitrotoluene isomers by Lactococcus lactis subsp. lactis strain 27 isolated from earthworm intestine.

Dinitrotoluenes are widely used as solvents and are intermediates in the synthesis of dyes, explosives, and pesticides. Environmental concerns regarding DNTs have increased due to their widespread use and their discharge into the environment. In this study, the anaerobic biodegradation of four dinitrotoluene isomers, 2,3-, 2,4-, 2,6- and 3,4-DNT, was investigated using Lactococcus lactis subsp. lactis strain 27, which was isolated from the intestines of earthworms. Liquid chromatography/mass spectrometry and NMR spectroscopy showed that L. lactis strain 27 non-specifically reduced the nitro groups on the tested dinitrotoluenes to their corresponding aminonitrotoluenes. L. lactis strain 27, however, did not reduce either sequentially or simultaneously two nitro groups of the dinitrotoluenes, resulting in the formation of the corresponding diaminotoluenes. In vitro formation of dinitroazoxytoluenes suggested the presence of oxygen-sensitive hydroxylaminonitrotoluenes. L. lactis strain 27 was capable of reducing 2,4-, 2,6-, 2,3-, and 3,4-dinitrotoluenes up to 173.6, 66.6, 287.1, and 355 microM, respectively in 12 h incubation. A relatively rapid reduction was observed in the case of the 2,3-, and 3,4-dinitrotoluenes, which have vicinal nitro groups on their arene structure. Non-specific anaerobic reduction of dinitrotoluenes by the intestinal bacterium L. lactis strain 27 differentiated the extent of reduction of DNTs according to the substitutional position of the nitro groups and produced in vitro more toxic dinitroazoxytoluenes, suggesting that anaerobic biotransformation of dinitrotoluenes could increase environmental risk.

Animals↗
PubMed1993

Efficient plasmid mobilization by pIP501 in Lactococcus lactis subsp. lactis.

pIP501 is a streptococcal conjugative plasmid which can be transmitted among numerous gram-positive strains. To identify a minimal mobilization (mob) locus of pIP501, DNA fragments of pIP501 were cloned into nonconjugative target plasmids and tested for mobilization by pIP501. We show that nonmobilizable plasmids containing a specific fragment of pIP501 are transmitted at high frequencies between Lactococcus lactis subsp. lactis strains if transfer (tra) functions are provided in trans by a pIP501 derivative. Independent transfer of the mobilized plasmid was observed in up to 44% of transconjugants. A 2.2-kb segment containing mob was sequenced. This DNA segment is characterized by three palindromes (palI, palII, and palIII) and a 202-amino-acid open reading frame (ORFX) of unknown function. The smallest DNA fragment conferring high frequency mobilization was localized to a 1.0-kb region (extending from pIP501 coordinates 3.60 to 4.60 on the 30.2-kb map) which contains palI (delta G = -27 kcal/mol [ca. -110,000 J/mol]). A 26-bp sequence identical to palI is present on pIP501, upstream of the plasmid copy control region. Further homologies with the palI sequence are also found with the related Enterococcus faecalis conjugative plasmid pAM beta 1. The region containing mob maps outside the previously described segment mediating pIP501 conjugation. Our results with recA strains indicate that the mob site is a hot spot for cointegrate formation.

Amino Acid Sequence↗
PubMed2006

Heterologous protein secretion in Lactococcus lactis is enhanced by the Bacillus subtilis chaperone-like protein PrsA.

The Bacillus subtilis lipoprotein PrsA enhances the yield of several homologous and heterologous exported proteins in B. subtilis by being involved in the posttranslocational stage of the secretion process. In this work, we have studied the effect of B. subtilis PrsA on the secretion of Bacillus amyloliquefaciens alpha-amylase (AmyQ), a target protein for PrsA, and Bacillus licheniformis penicillinase (PenP) a nontarget protein for PrsA, in Lactococcus lactis. Two compatible plasmids were constructed and introduced into L. lactis strain NZ9000: one high copy plasmid, expressing the AmyQ gene (amyQ) or the PenP gene (penP), and one low copy plasmid, expressing the PrsA encoding gene (prsA). When amyQ and prsA were simultaneously expressed under the nisin-inducible promoter P( nisA ), Western blotting experiments revealed a 15- to 20-fold increase in the total yield of AmyQ and a sixfold increase in secreted AmyQ activity, compared to a control strain lacking prsA. When expressed under the same induction conditions, PrsA had no effect on the secretion or total yield of PenP. These results show that the secretion yield of some heterologous proteins can be significantly increased in L. lactis when coproduced with the B. subtilis PrsA protein.

Bacillus subtilis↗
PubMed1999

Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system.

Lactic acid bacteria (LAB) are Gram-positive bacteria and are generally regarded as safe (GRAS) organisms. Therefore, LAB could be used for heterologous protein secretion and they are good potential candidates as antigen delivery vehicles. To develop such live vaccines, a better control of protein secretion is required. We developed an efficient secretion system in the model LAB, Lactococcus lactis. Staphylococcal nuclease (Nuc) was used as the reporter protein. We first observed that the quantity of secreted Nuc correlated with the copy number of the cloning vector. The nuc gene was cloned on a high-copy number cloning vector and no perturbation of the metabolism of the secreting strain was observed. Replacement of nuc native promoter by a strong lactococcal one led to a significant increase of nuc expression. Secretion efficiency (SE) of Nuc in L. lactis was low, i.e., only 60% of the synthesized Nuc was secreted. Insertion of a synthetic propeptide between the signal peptide and the mature moiety of Nuc increased the SE of Nuc. On the basis of these results, we developed a secretion system and we applied it to the construction of an L. lactis strain which secretes a bovine coronavirus (BCV) epitopeprotein fusion (BCV-Nuc). BCV-Nuc was recognized by both anti-BCV and anti-Nuc antibodies. Secretion of this antigenic fusion is the first step towards the development of a novel antigen delivery system based on LAB-secreting strains.

Antigens↗
PubMed2001

In vivo evidence for two active nuclease motifs in the double-strand break repair enzyme RexAB of Lactococcus lactis.

In bacteria, double-strand DNA break (DSB) repair involves an exonuclease/helicase (exo/hel) and a short regulatory DNA sequence (Chi) that attenuates exonuclease activity and stimulates DNA repair. Despite their key role in cell survival, these DSB repair components show surprisingly little conservation. The best-studied exo/hel, RecBCD of Escherichia coli, is composed of three subunits. In contrast, RexAB of Lactococcus lactis and exo/hel enzymes of other low-guanine-plus-cytosine branch gram-positive bacteria contain two subunits. We report that RexAB functions via a novel mechanism compared to that of the RecBCD model. Two potential nuclease motifs are present in RexAB compared with a single nuclease in RecBCD. Site-specific mutagenesis of the RexA nuclease motif abolished all nuclease activity. In contrast, the RexB nuclease motif mutants displayed strongly reduced nuclease activity but maintained Chi recognition and had a Chi-stimulated hyperrecombination phenotype. The distinct phenotypes resulting from RexA or RexB nuclease inactivation lead us to suggest that each of the identified active nuclease sites in RexAB is involved in the degradation of one DNA strand. In RecBCD, the single RecB nuclease degrades both DNA strands and is presumably positioned by RecD. The presence of two nucleases would suggest that this RecD function is dispensable in RexAB.

Amino Acid Motifs↗
PubMed2004

Sequencing of the tyrosine decarboxylase cluster of Lactococcus lactis IPLA 655 and the development of a PCR method for detecting tyrosine decarboxylating lactic acid bacteria.

The enzymatic decarboxylation of tyrosine produces tyramine, the most abundant biogenic amine in dairy products-especially in cheeses. The screening of lactic acid bacteria isolated from different artisanal cheeses and a number of microbial collections identified 22 tyramine-producing strains belonging to different genera. The Lactococcus lactis strain IPLA 655 was selected, and the genes encoding a putative tyrosyl tRNA synthetase, a tyrosine decarboxylase (tdcA), and a tyrosine-tyramine antiporter, found together as a cluster, were sequenced. The disruption of tdcA yielded a strain unable to produce tyramine. Comparison of the L. lactis IPLA 655 tdcA gene with database tdcA sequences led to the design of two primers for use in a PCR method that identified potential tyramine-producing strains. The proposed method can use purified DNA, isolated colonies, milk, curd, and even cheese as a template. Molecular tools for the rapid detection of tyramine-producing bacteria at any time during the fermentation process could help prevent tyramine accumulation in fermented foods. The proposed technique could be of great use to the food industry.

Amino Acid Sequence↗
PubMed1989

Mechanism of Proteinase Release from Lactococcus lactis subsp. cremoris Wg2.

The procedure generally used for the isolation of extracellular, cell-associated proteinases of Lactococcus lactis species is based on the release of the proteinases by repeated incubation and washing of the cells in a Ca-free buffer. For L. lactis subsp. cremoris Wg2, as many as five incubations for 30 min at 29 degrees C are needed in order to liberate 95% of the proteinase. Proteinase release was not affected by chloramphenicol, which indicates that release is not the result of protein synthesis during the incubations. Ca inhibited, while ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) stimulated, proteinase release from the cells. The pH optimum for proteinase release ranged between 6.5 and 7.5, which was higher than the optimum pH of the proteinase measured for casein hydrolysis (i.e., 6.4). Treatment of cells with the serine proteinase inhibitor phenylmethylsulfonyl fluoride prior to the incubations in Ca-free buffer reduced the release of the proteinase by 70 to 80%. The residual proteinase remained cell associated but could be removed by the addition of active L. lactis subsp. cremoris Wg2 proteinase. This suggests that proteinase release from cells of L. lactis subsp. cremoris Wg2 is the result of autoproteolytic activity. From a comparison of the N-terminal amino acid sequence of the released proteinase with the complete amino acid sequence determined from the nucleotide sequence of the proteinase gene, a protein of 180 kilodaltons would be expected. However, a proteinase with a molecular weight of 165,000 was found, which indicated that further hydrolysis had occurred at the C terminus.

Journal Article↗
PubMed1998

Influence of reduced water activity on lactose metabolism by lactococcus lactis subsp. cremoris At different pH values

The influence of reduced water activity (aw) on lactose metabolism by Lactococcus lactis subsp. cremoris 2254 and 2272 was studied at different pH values. In control incubations (aw, 0.99) with nongrowing cells in pH-controlled phosphate buffer, the levels of carbon recovered as L-(+)-lactate were 92% at pH 6.1 and 5.3 and 78% at pH 4.5. However, the levels of recovery decreased to approximately 50% at all pH values tested when the aw was 0.88 (with glycerol as the humectant). When growing cells in broth controlled at pH 6.3 were used, a reduction in the aw from 0.99 to 0.96 resulted in a decrease in the level of lactose carbon recovered as L-(+)-lactate from 100 to 71%. Low levels of L-(+)-lactate carbon recovery (<50%) were also observed with cells resuspended in pH-uncontrolled reconstituted skim milk at aw values of 0.99 and 0. 87 and in young cheese curds. The missing lactose carbon could not be accounted for by acetate, ethanol, formate, acetaldehyde, or pyruvate. Attempts were made to determine where the missing lactose carbon was diverted to under the stress conditions used. Some of the missing lactose carbon was recovered as galactose (0.1 to 2.5 mM) in culture supernatants. Decreasing either the aw or the pH resulted in increased galactose accumulation by nongrowing cells; adjusting both environmental factors together potentiated the effect. The sensitivities of the two lactococcal strains tested were different; strain 2272 was more prone to accumulate galactose under stress conditions. A methyl pentose(s) and additional galactose were found in acid-hydrolyzed supernatants from cultures containing both growing and nongrowing cells, indicating that a saccharide(s) rich in these components was formed by lactococci under low-aw and low-pH stress conditions.

Journal Article↗
PubMed2002

Effect of reducing agents on the acidification capacity and the proton motive force of Lactococcus lactis ssp. cremoris resting cells.

Reducing agents are potential inhibitors of the microbial growth. We have shown recently that dithiothreitol (DTT), NaBH(4) and H(2) can modify the proton motive force of resting cells of Escherichia coli by increasing the membrane protons permeability [Eur. J. Biochem. 262 (1999) 595]. In the present work, the effect of reducing agents on the resting cells of Lactococcus lactis ssp. cremoris, a species widely employed in dairy processes was investigated. DTT did not affect the acidification nor the DeltapH, in contrast to the effect previously reported on E. coli. The DeltaPsi was slightly increased (30 mV) at low pH (pH 4) in the presence of 31 mM DTT or 2.6 mM NaBH(4). In the case of Na(2)S(2)O(4), small amounts (0.9 mM) drastically decreased the acidification range and this product was shown to abolish the DeltapH. These results are discussed in terms of the diversity of action of the chemical reagents and strain sensitivity.

Borohydrides↗
PubMed2003

ClpE from Lactococcus lactis promotes repression of CtsR-dependent gene expression.

The heat shock response in bacterial cells is characterized by rapid induction of heat shock protein expression, followed by an adaptation period during which heat shock protein synthesis decreases to a new steady-state level. In this study we found that after a shift to a high temperature the Clp ATPase (ClpE) in Lactococcus lactis is required for such a decrease in expression of a gene negatively regulated by the heat shock regulator (CtsR). Northern blot analysis showed that while a shift to a high temperature in wild-type cells resulted in a temporal increase followed by a decrease in expression of clpP encoding the proteolytic component of the Clp protease complex, this decrease was delayed in the absence of ClpE. Site-directed mutagenesis of the zinc-binding motif conserved in ClpE ATPases interfered with the ability to repress CtsR-dependent expression. Quantification of ClpE by Western blot analysis revealed that at a high temperature ClpE is subjected to ClpP-dependent processing and that disruption of the zinc finger domain renders ClpE more susceptible. Interestingly, this domain resembles the N-terminal region of McsA, which was recently reported to interact with the CtsR homologue in Bacillus subtilis. Thus, our data point to a regulatory role of ClpE in turning off clpP gene expression following temporal heat shock induction, and we propose that this effect is mediated through CtsR.

Adenosine Triphosphatases↗
PubMed1999

Expression of the mobM gene of the streptococcal plasmid pMV158 in Lactococcus lactis subsp. lactis.

The streptococcal plasmid pMV158 is not auto-transferable, but it can be mobilised between bacteria by the use of functions supplied by plasmids of the pIP501/pAM beta 1 family. Plasmid pMV158 encodes a protein, MobM, which is involved in its mobilisation. This process initiates when MobM specifically cleaves supercoiled pMV158 plasmid DNA at the origin of transfer, oriT. Plasmid pMV158 has been transferred to Lactococcus lactis by conjugation aided by plasmid pAM beta 1. In the lactococcal host, MobM-mediated specific pMV158-relaxed molecules were detected. The intracellular amount of MobM has been quantified by immunoblot analyses and shown to be about 3500 molecules per cell. In the same host, we have mapped the initiation point of transcription of mobM. Transcription of this gene is directed from a promoter with an extended--10 region which overlaps with the pMV158-oriT.

Bacterial Proteins↗
PubMed1994

Identification of int and attP on the genome of lactococcal bacteriophage Tuc2009 and their use for site-specific plasmid integration in the chromosome of Tuc2009-resistant Lactococcus lactis MG1363.

The DNA sequence of the int-attP region of the small-isometric-headed lactococcal bacteriophage Tuc2009 is presented. In this region, an open reading frame, int, which potentially encodes a protein of 374 amino acids, representing the Tuc2009 integrase, was identified. The nucleotide sequence of the bacteriophage attachment site, attP, and the sequences of attB, attL, and attR in the lysogenic host Lactococcus lactis subsp. cremoris UC509 were determined. A sequence almost identical to the UC509 attB sequence was found to be present in the plasmid-free Tuc2009-resistant L. lactis subsp. cremoris MG1363. This site could be used for the site-specific integration of a plasmid carrying the Tuc2009 int-attP region in the chromosome of MG1363, thereby demonstrating that the application of chromosomal insertion vectors based on bacteriophage integration functions is not limited to the prophage-cured original host strain of the phage.

Amino Acid Sequence↗
PubMedDate not supplied

[Isolation and identification of new nisin-producing Lactococcus lactis subsp. lactis from milk].

A method for isolating active nisin-producing strains of mesophilic lactococci was developed. Overall, 55 strains of mesophilic lactic acid bacteria were isolated from fresh cow's milk obtained from milk farms in various regions throughout Russia; of them, 36 displayed nisin-synthesizing activity. The three most active strains were studied according to morphological, cultural, physiological, and biochemical characteristics and identified as Lactococcus lactis subsp. lactis. The species attribution of the strains studied was confirmed by the similarity of the nucleotide sequences of the 16S rRNA gene. The nucleotide sequences of the 16S rRNA genes were deposited with the GenBank under accession numbers DQ255951-DQ255954. The distinctions between these strains in physiological and biochemical characteristics and the ranges of their bactericide action on the microorganisms capable of developing in agricultural materials and food products were determined. The isolated strains displayed considerably wider ranges of action, which differed from the nisin-producing strain MGU and the commercial nisin preparation (Nisaplin), used as a biological preserving agent.

Animals↗
PubMed2005

Identification, cloning, and characterization of a Lactococcus lactis branched-chain alpha-keto acid decarboxylase involved in flavor formation.

The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain alpha-keto acid decarboxylase (KdcA). The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains. By using a random mutagenesis approach, the gene encoding KdcA in L. lactis B1157 was identified. The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L. lactis IL1403. Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3' terminus of the ipd gene encoding a truncated nonfunctional decarboxylase. The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme. Of all of the potential substrates tested, the highest activity was observed with branched-chain alpha-keto acids. Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2003

Substrate inhibition of Lactococcus lactis cytidine 5'-triphosphate synthase by ammonium chloride is enhanced by salt-dependent tetramer dissociation.

Cytidine 5(')-triphosphate (CTP) synthase (EC 6.4.3.2) catalyzes the transfer of an amino group to the 4 position of uridine 5(')-triphosphate (UTP) to yield CTP. The reaction proceeds by activation of the base moiety of UTP by adenosine 5(')-triphosphate (ATP)-dependent phosphorylation. The activated intermediate reacts with NH(3) in the solution or is obtained by hydrolysis of glutamine. The Lactococcus lactis CTP synthase shows significant differences from the enzymes from Escherichia coli, yeast, and mammals. One is the apparent stability of the L. lactis CTP synthase tetramer in the absence of the nucleotides ATP and UTP. This condition causes the E. coli, yeast, and mammal enzymes to dissociate into dimers. However, the L. lactis CTP synthase shows substrate inhibition by NH(4)Cl that coincides with the range of NH(4)Cl concentrations that apparently dissociates tetrameric enzyme into dimers. Even though regular substrate inhibition was observed with NH(4)Cl when the ionic strength was held constant, a significant part of the inhibition could be shown to be due to the increase in ionic strength with increasing substrate concentration. Since the substrate inhibition by NH(4)Cl was relieved by increasing the equimolar ATP and UTP concentrations, it appeared that the substrate nucleotides stabilized the tetramer in a manner similar to that found in the absence of salt for other CTP synthases. In contrast to the suggested hydrophobic nature of the tetramer interactions in E. coli CTP synthase, the dissociation of the L. lactis CTP synthase tetramer in response to an increase in ionic strength suggests that the tetramer is stabilized by ionic interactions.

Adenosine Triphosphate↗
PubMed1997

Characterization of cspB, a cold-shock-inducible gene from Lactococcus lactis, and evidence for a family of genes homologous to the Escherichia coli cspA major cold shock gene.

Upon temperature downshift, the major cold shock protein CspA is highly induced in Escherichia coli. This protein being conserved in other bacteria, we used a PCR-based approach with a pair of degenerate primers derived from highly conserved regions of the CspA-related proteins to evidence the presence of at least three related genes in Lactococcus lactis. One of them, cspB, was cloned and sequenced. It encodes a 66-residue protein which possesses 60% sequence identity with E. coli CspA. Following a cold shock from 30 to 15 degrees C, the level of the cspB mRNA transcript increased, as shown by Northern blot hybridization. In addition, induction of cspB-directed beta-galactosidase activity was observed. These results indicate that the L. lactis cspB gene is cold shock inducible.

Amino Acid Sequence↗