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PubMed1991

Heat shock induces thermotolerance and inhibition of lysis in a lysogenic strain of Lactococcus lactis.

In this preliminary work, the heat shock response of lactic acid bacteria was investigated and characterized. Log-phase Lactococcus lactis cells pre-incubated at 40 degrees C before heat challenge at 52 degrees C for 30 min demonstrated increased thermotolerance as compared with cells pre-incubated at 30 degrees C. The response persisted for at least 60 min. Additionally, we demonstrated that: (i) the physiological expression of the heat shock response is temperature dependent; (ii) ethanol 4.0% (v/v) caused, to a lesser extent, a response similar to the heat shock; and (iii) hydrogen peroxide failed to induce a detectable response. Furthermore, we suggest that the induction of the heat shock response increases the resistance of a lysogenic strain of L. lactis, treated by mitomycin C (1.25 micrograms/ml), to lysis by the bacteriophage.

Ethanol↗

PubMed2004

Development of an inducible system to control and easily monitor gene expression in Lactococcus lactis.

This report describes the implementation and use of a maltose-inducible system for regulated gene expression in Lactococcus lactis. The system was established using Green Fluorescent Protein as reporter. The transcription of a gene of interest from the inducible promoter of pLS1RGFP plasmid vector can be easily monitored by fluorescence spectroscopy and microscopy. As an example, the lactococcal ribonuclease III was overproduced in an active form.

Blotting, Western↗

PubMed2000

Diacetyl and alpha-acetolactate overproduction by Lactococcus lactis subsp. lactis biovar diacetylactis mutants that are deficient in alpha-acetolactate decarboxylase and have a low lactate dehydrogenase activity.

Lactococcus lactis subsp. lactis biovar diacetylactis strains are utilized in several industrial processes for producing the flavoring compound diacetyl or its precursor alpha-acetolactate. Using random mutagenesis with nitrosoguanidine, we selected mutants that were deficient in alpha-acetolactate decarboxylase and had low lactate dehydrogenase activity. The mutants produced large amounts of alpha-acetolactate in anaerobic milk cultures but not in aerobic cultures, except when the medium was supplemented with catalase, yeast extract, or hemoglobin.

Aerobiosis↗

PubMed1994

The Lactococcus lactis sex-factor aggregation gene cluA.

A gene, cluA, was cloned from the chromosomally located sex factor of Lactococcus lactis MG1363. Sequence analysis revealed significant homology with previously described aggregation proteins in Enterococcus and Streptococcus species. The possibility that cluA was an equivalent protein involved in cell aggregation between donor and recipient bacteria during lactococcal conjugation was confirmed by its expression under the control of a heterologous promoter in L. lactis. Analysis of the homology between the CluA protein and the related proteins of Enterococcus and Streptococcus allowed a common structure for these proteins to be postulated. This consisted of five domains. Functionally conserved domains I and V act respectively as a secretory leader and C-terminal membrane anchor. Domains II and IV are conserved at the amino acid level and probably have common structural roles whereas domain III is variable and may control binding specificity.

Amino Acid Sequence↗

PubMed2002

Lactococcus lactis AbiD1 abortive infection efficiency is drastically increased by a phage protein.

Sensitivity of phage bIL66 to the AbiD1 Lactococcus lactis abortive infection mechanism was previously shown to be determined by the phage middle-time-expressed operon composed of four orfs. Using spontaneous bIL66 mutants resistant to AbiD1, we established that this sensitivity is determined by the orf1 encoded protein. Overproduction of Orf1 in trans in AbiD1(+) cells was shown to increase AbiD1 efficiency on both wild-type phage bIL66 and mutants resistant to AbiD1. Such an increase was not observed following overproduction of mutant Orf1. We propose that wild-type, but not a mutant Orf1, activates AbiD1 expression or activity.

Amino Acid Sequence↗

PubMed1992

Characterization of the Lactococcus lactis pepN gene encoding an aminopeptidase homologous to mammalian aminopeptidase N.

The nucleotide sequence of the pepN gene from Lactococcus lactis encoding a zinc-metallo aminopeptidase has been determined. The open reading frame of 2,538 base pairs encodes a protein with a calculated M(r) of 95,368, which agrees with the apparent M(r) of 95,000 of the gene product which was identified by polyclonal antibodies raised against the purified aminopeptidase. The amino acid sequence of the aminopeptidase of L. lactis was found to be similar to the corresponding enzymes of human, rat and mouse, with almost 30% of the residues identical. Also, a highly conserved area was identified which has similarity with the active site of thermolysin. A zinc-binding site, as well as the catalytic site for PepN, is predicted to lie within this conserved stretch. Putative promoter regions upstream of PepN were confirmed by primer extension analysis.

Amino Acid Sequence↗

PubMed2001

Glucose metabolism and regulation of glycolysis in Lactococcus lactis strains with decreased lactate dehydrogenase activity.

The distribution of carbon flux at the pyruvate node was investigated in Lactococcus lactis under anaerobic conditions with mutant strains having decreased lactate dehydrogenase activity. Strains previously selected by random mutagenesis by H. Boumerdassi, C. Monnet, M. Desmazeaud, and G. Corrieu (Appl. Environ. Microbiol. 63, 2293-2299, 1997) were found to have single punctual mutations in the ldh gene and presented a high degree of instability. The strain L. lactis JIM 5711 in which lactate dehydrogenase activity was diminished to less than 30% of the wild type maintained homolactic metabolism. This was due to an increase in the intracellular pyruvate concentration, which ensures the maintained flux through the lactate dehydrogenase. Pyruvate metabolism was linked to the flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase, as previously postulated for the parent strain (C. Garrigues, P. Loubière, N. D. Lindley, and M. Cocaign-Bousquet (1997) J. Bacteriol. 179, 5282-5287, 1997). However, a strain (L. lactis JIM 5954) in which the ldh gene was interrupted reoriented pyruvate metabolism toward mixed metabolism (production of formate, acetate, and ethanol), though the glycolytic flux was not strongly diminished. Only limited production of acetoin occurred despite significant overflow of pyruvate. Intracellular metabolite profiles indicated that the in vivo glyceraldehyde-3-phosphate dehydrogenase activity was no longer flux limiting in the Deltaldh strain. The shift toward mixed acid fermentation was correlated with the lower intracellular trioses phosphate concentration and diminished allosteric inhibition of pyruvate formate lyase.

Base Sequence↗

PubMed1990

Conjugal mobilization of streptococcal plasmid pMV158 between strains of Lactococcus lactis subsp. lactis.

pMV158, a non-self-transmissible plasmid encoding tetracycline resistance, was conjugally transferred from Enterococcus faecalis JH203 to Lactococcus lactis subsp. lactis IL1403. This transfer appeared to be dependent on the cotransfer of the conjugative plasmids pAM beta 1 or pIP501. Intraspecies conjugal transfer of pMV158 also occurred in strain IL1403. In contrast to the transfer from E. faecalis, transfer in IL1403 did not require the presence of a conjugative plasmid in the donor strain but, rather, appeared to be dependent on putative chromosomal functions in strain IL1403. The transfer of pMV158 from strain IL1403 required the presence of an active pMV158-encoded protein, which showed homology to the Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from Staphylococcus aureus, such as pT181.

Conjugation, Genetic↗

PubMed1995

The occurrence of two intracellular oligoendopeptidases in Lactococcus lactis and their significance for peptide conversion in cheese.

Two intracellular oligopeptide-preferring endopeptidases have been detected in Lactococcus lactis. A neutral thermolysin-like oligoendopeptidase (NOP) has been purified to homogeneity and an alkaline oligoendopeptidase has been partially purified. The specificity of the oligoendopeptidases towards important intermediary cheese peptides, produced by chymosin action on the caseins, clearly differs from that of the cell-envelope proteinase (CEP). NOP is active under conditions prevailing in cheese and contributes to initial proteolysis in a young cheese. It probably plays a crucial role in the degradation of an important bitter peptide in cheese, the beta-casein 193-209 fragment. The relatively low activity of the alkaline endopeptidase is further suppressed in cheese by the highly competitive actions of NOP and CEP.

Amino Acid Sequence↗

PubMed2001

Molecular characterization of a new abortive infection system (AbiU) from Lactococcus lactis LL51-1.

This study reports on the identification and characterization of a novel abortive infection system, AbiU, from Lactococcus lactis. AbiU confers resistance to phages from the three main industrially relevant lactococcal phage species: c2, 936, and P335. The presence of AbiU reduced the efficiency of plaquing against specific phage from each species as follows: 3.7 x 10(-1), 1.0 x 10(-2), and 1.0 x 10(-1), respectively. abiU involves two open reading frames, abiU1 (1,772 bp) and abiU2 (1,019 bp). Evidence indicates that AbiU1 is responsible for phage resistance and that AbiU2 may downregulate phage resistance against 936 and P335 type phages but not c2 type phage. AbiU appeared to delay transcription of both phage 712 and c2, with the effect being more marked on phage c2.

Amino Acid Sequence↗

PubMed2004

Mucosal vaccine made from live, recombinant Lactococcus lactis protects mice against pharyngeal infection with Streptococcus pyogenes.

A novel vaccine (LL-CRR) made from live, nonpathogenic Lactococcus lactis that expresses the conserved C-repeat region (CRR) of M protein from Streptococcus pyogenes serotype 6 was tested in mice. Nasally vaccinated mice produced CRR-specific salivary immunoglobulin A (IgA) and serum IgG. Subcutaneously vaccinated mice produced CRR-specific serum IgG but not salivary IgA. A combined regimen produced responses similar to the salivary IgA of nasally vaccinated mice and serum IgG of subcutaneously vaccinated mice. Mice vaccinated nasally or with the combined regimen were significantly protected against pharyngeal infection following a nasal challenge with S. pyogenes M serotype 14. Mice vaccinated subcutaneously were not protected against pharyngeal infection. Mice in all three LL-CRR vaccination groups were significantly protected against the lethal effects of S. pyogenes. Only 1 of 77 challenged mice that were vaccinated with LL-CRR died, whereas 60 of 118 challenged mice that were vaccinated with a control strain or phosphate-buffered saline died. In conclusion, mucosal vaccination with LL-CRR produced CRR-specific salivary IgA and serum IgG, prevented pharyngeal infection with S. pyogenes, and promoted survival.

Administration, Intranasal↗

PubMed2004

Effect of Sorghum vulgare phosphoenolpyruvate carboxylase and Lactococcus lactis pyruvate carboxylase coexpression on succinate production in mutant strains of Escherichia coli.

Sorghum vulgare phosphoenolpyruvate carboxylase (PEPC) and Lactococcus lactis pyruvate carboxylase (PYC) were overexpressed in Escherichia coli concurrently to improve the production of succinate, a valuable industrial specialty chemical. This coexpression system was also applied to E. coli mutant strains strategically designed by inactivating the competing pathways of succinate formation. The highest level of succinate production was observed in E. coli strains coexpressing both PEPC and PYC when compared with E. coli strains individually overexpressing either PEPC or PYC. Lactate production was also significantly reduced with PEPC and PYC coexpression. Lactate and acetate pathways were inactivated to eliminate the competing pathways of succinate formation. Results showed that inactivation of both the lactate and acetate pathways with the coexpression of PEPC and PYC was most effective in improving succinate production. Inactivating the lactate or acetate pathway alone only caused a majority of the carbon flux to shift to other metabolites rather than succinate. Coexpression of PEPC and PYC was also applied to an E. coli mutant strain deficient in lactate dehydrogenase and pyruvate:formate lyase that accumulated a substantial amount of the intermediate metabolite pyruvate during growth. Results showed that PEPC and PYC coexpression was effective in depleting pyruvate accumulation and increasing the production of metabolites.

Biotechnology↗

PubMed2001

Cell release from alginate immobilized Lactococcus lactis ssp. lactis in chitosan and alginate coated beads.

The effects of chitosan and alginate coatings of alginate beads with entrapped Lactococcus lactis ssp. lactis were studied in batch and continuous fermentations. Chitosan coating reduced the final concentrations of free cells, the initial release of free cells and the rate of lactate production in milk fermented batch-wise to a final pH of 4.7 in five consecutive batch fermentations. An alternative experimental system based on continuous fermentation with controlled pH and a high dilution rate was developed to better study the phenomenon of cell release. To estimate the effects of different bead coatings on cell release, alginate beads were coated with chitosan or alginate, or sequentially with chitosan/alginate or chitosan/alginate/chitosan. Chitosan coating alone seemed to reduce the rate of cell release only in the early stages of the fermentation, while sequential coatings with chitosan and alginate showed significant reduction throughout the whole test period. To examine whether the observed effects of bead coating could be explained only by a decrease in cell activity, the ratios between the rate of cell release and the rate of lactate production were examined during the fermentations for the different beads. This ratio showed qualitatively the same behavior as direct results of volumetric cell release.

Alginates↗

PubMed1991

Characterization and overexpression of the Lactococcus lactis pepN gene and localization of its product, aminopeptidase N.

The chromosomal pepN gene encoding lysyl-aminopeptidase activity in Lactococcus lactis has been identified in a lambda EMBL3 library in Escherichia coli by using an immunological screening with antiserum against a purified aminopeptidase fraction. The pepN gene was localized and subcloned in E. coli on the basis of its expression and hybridization to a mixed-oligonucleotide probe for the previously determine N-terminal amino acid sequence of lysyl-aminopeptidase (P. S. T. Tan and W. N. Konings, Appl. Environ. Microbiol. 56:526-532, 1990). The L. lactis pepN gene appeared to complement an E. coli strain carrying a mutation in its pepN gene. High-level expression of the pepN gene in E. coli was obtained by using the T7 system. The overproduction of the 95-kDa aminopeptidase N could be visualized on sodium dodecyl sulfate-polyacrylamide gels and immunoblots. Cloning of the pepN gene on a multicopy plasmid in L. lactis resulted in a 20-fold increase in lysyl-aminopeptidase activity that corresponded to several percent of total protein. Nucleotide sequence analysis of the 5' region of the pepN gene allowed a comparison between the deduced and determined amino-terminal primary sequences of aminopeptidase N. The results show that the amino terminus of PepN is not processed and does not possess the characteristics of consensus signal sequences, indicating that aminopeptidase N is probably an intracellular protein. The intracellular location of aminopeptidase N in L. lactis was confirmed by immunogold labeling of lactococcal cells.

Amino Acid Sequence↗

PubMed2002

Function of Lactococcus lactis nisin immunity genes nisI and nisFEG after coordinated expression in the surrogate host Bacillus subtilis.

Nisin-producing Lactococcus lactis strains show a high degree of resistance to the action of nisin, which is based upon expression of the self-protection (immunity) genes nisI, nisF, nisE, and nisG. Different combinations of nisin immunity genes were integrated into the chromosome of a nisin-sensitive Bacillus subtilis host strain under the control of an inducible promoter. For the recipient strain, the highest level of acquired nisin tolerance was achieved after coordinated expression of all four nisin immunity genes. But either the lipoprotein NisI or the ABC transporter-homologous system NisFEG, respectively, were also able to protect the Bacillus host cells. The acquired immunity was specific to nisin and provided no tolerance to subtilin, a closely related lantibiotic. Quantitative in vivo peptide release assays demonstrated that NisFEG diminished the quantity of cell-associated nisin, providing evidence that one role of NisFEG is to transport nisin from the membrane into the extracellular space. NisI solubilized from B. subtilis membrane vesicles and recombinant hexahistidine-tagged NisI from Escherichia coli interacted specifically with nisin and not with subtilin. This suggests a function of NisI as a nisin-intercepting protein.

ATP-Binding Cassette Transporters↗

PubMed1999

Pyruvate flux distribution in NADH-oxidase-overproducing Lactococcus lactis strain as a function of culture conditions.

The influence of growth conditions on product formation from glucose by Lactococcus lactis strain NZ9800 engineered for NADH-oxidase overproduction was examined. In aerobic batch cultures, a large production of acetoin and diacetyl was found at acidic pH under pH-unregulated conditions. However, pyruvate flux was mainly driven towards lactate production when these cells were grown under strictly pH-controlled conditions. A decreased NADH-oxidase overproduction accompanied the homolactic fermentation, suggesting that the cellular energy was used with preference to maintain cellular homeostasis rather than for NADH-oxidase overproduction. The end product formation and NADH-oxidase activity were also studied in cells grown in aerobic continuous cultures under acidic conditions. A homoacetic type of fermentation as well as a low NADH-oxidase overproduction were observed at low dilution rates. NADH-oxidase was efficiently overproduced as the dilution rate was increased and consequently metabolic flux through lactate dehydrogenase drastically decreased. Under these conditions the flux limitation via pyruvate dehydrogenase was relieved and this enzymatic complex accommodated most of the pyruvate flux. Pyruvate was also significantly converted to acetoin and diacetyl via alpha-acetolactate synthase. At higher dilution rates, acetate production declined and the cultures turned to mixed-acid fermentation. These results suggest that the need to maintain the cellular homeostasis influenced NADH-oxidase overproduction and consequently the end product formation from glucose in these engineered strains.

Aerobiosis↗

PubMed2005

Quantitative analysis of group II intron expression and splicing in Lactococcus lactis.

The group II intron Ll.ltrB is found within the ltrB relaxase gene of the conjugative element pRS01 in Lactococcus lactis. Precise splicing of the intron is essential for pRS01 transfer. The transcription regulation and in vivo splicing activity of Ll.ltrB have not been investigated thoroughly in L. lactis in the natural pRS01 context. We developed absolute quantitative real-time reverse transcription-PCR assays to quantify RNA levels of the 5' exon (ltrBE1) and the spliced relaxase (ltrB) and intron-encoded protein (ltrA) genes, as well as Ll.ltrB splicing activity under different physiological conditions. The mRNA levels for the ATP-binding protein OppD were assayed for comparison to the ltrB transcripts. The oppD mRNA ranged from 10- to 10,000-fold higher than ltrB region genes. ltrBE1 expression was growth-phase dependent. The mRNA level of ltrA was almost constant during all growth phases and in all media tested. Ll.ltrB in vivo splicing activity ranged from (6.5 +/- 2.1)% to (22.1 +/- 8.0)%. Acid challenge significantly decreased both ltrB region mRNA levels and intron splicing activity. The presence of recipient cells, different mating environments, and temperature stress had no significant effects on expression and splicing. Western blotting showed that the level of LtrB protein expressed from an intronless ltrB gene was much higher (about 20-fold) than the level of protein expressed from an intron-containing construct. Interestingly, LtrB protein showed a tendency to function in cis on its oriT target. The low level of ltrB transcript and relatively inefficient splicing of the intron may limit Ll.ltrB mobility and dissemination in nature.

Bacterial Proteins↗

PubMed2002

A distinct physiological state of Lactococcus lactis cells that confers survival against a direct and prolonged exposure to severe stresses.

When exponential phase cultures of Lactococcus lactis were directly exposed to severe stresses (acid, bile salt, heat, and hydrogen peroxide) for a prolonged period, most of the cells were quickly killed, however, a small number of the cells, approximately 0.01% of the population, was found to survive. How these 'survivor' cells might have survived the stresses, when other supposedly-the-same cells could not, was investigated. The cultures were not exposed to any mild stresses prior to the exposure to the severe stresses, and therefore adaptation can be ruled out as the cause of survival. When the survivor cells were re-cultured and re-exposed to the same severe stresses a similar pattern of survival was displayed, indicating that the survivor cells were not stress-resistant mutants. Furthermore, the survivor cells displayed typical growth kinetics once they were freed of the stresses. The survivor cells appear to be in a distinct physiological state, because when they were tested against a second stress they exhibited significantly greater survival against that stress than the normal cells exposed to the same stress. Also, cells at different time points of synchronously growing culture displayed different levels of survival against stress. It is proposed that the difference in survival of exponential phase cells is due to the difference in the protein makeup of cells at different stages of the cell cycle.

Acids↗