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PubMed2004

Catalytic activity of tripeptidase from Lactococcus lactis to which amino acid substitution was introduced according to natural mutation.

Four mutations observed between tripeptidases from Lactococcus lactis subsp. lactis and subsp. cremoris were introduced one by one to the corresponding points in wild-type tripeptidase from L. lactis subsp. lactis. The k(cat) values of four resultant mutants were analyzed and discussed in stereographical terms. Change in catalytic activity appeared to be related to the sequential and steric location of mutation point within the enzyme protein, even though no drastic change was observed with one point mutation.

Amino Acid Sequence↗
PubMed1998

Cloning of the Lactococcus lactis adhE gene, encoding a multifunctional alcohol dehydrogenase, by complementation of a fermentative mutant of Escherichia coli.

The Lactococcus lactis adhE gene, which encodes a multifunctional alcohol dehydrogenase, has been cloned and characterized. A DNA fragment encoding the putative alcohol dehydrogenase domain of the AdhE protein was cloned by screening an L. lactis genomic library in a fermentative mutant of Escherichia coli and selecting for the ability to grow anaerobically. Further analysis of the clone obtained allowed the cloning of the entire adhE gene sequence. Analysis of adhE expression in L. lactis during anaerobiosis showed induction at the transcriptional level, especially in medium containing glucose. Constructed mutant strains produced reduced amounts of ethanol under anaerobic conditions. With the L. lactis gene as a probe, adhE homologs were found in other industrially relevant lactic acid bacteria.

Alcohol Dehydrogenase↗
PubMed1994

Regulation of 2-deoxyglucose phosphate accumulation in Lactococcus lactis vesicles by metabolite-activated, ATP-dependent phosphorylation of serine-46 in HPr of the phosphotransferase system.

Lactococcus lactis takes up glucose and the nonmetabolizable glucose analogue 2-deoxyglucose (2DG) via the phosphotransferase system and extrudes the accumulated sugar phosphates in a process apparently dependent on a cytoplasmic sugar-phosphate phosphatase. Uptake of 2DG into L. lactis vesicles was shown to be dependent on an energy source, effectively provided by intravesicular phosphoenolpyruvate (PEP). 2DG phosphate (2DG-P) accumulation in these vesicles was not inhibited, and preaccumulated 2DG-P was not released from them, upon electroporation of fructose 1,6-diphosphate (FDP), gluconate 6-phosphate or 2-phosphoglycerate into the vesicles. Intravesicular but not extravesicular wild-type HPr of Bacillus subtilis alone stimulated uptake, but in the presence of any one of these metabolites, it prevented accumulation of 2DG-P. Intravesicular H15A mutant HPr inhibited uptake and allowed further inhibition of 2DG-P accumulation in the presence of the intravesicular metabolites. Intravesicular S46A mutant HPr stimulated uptake but could not promote inhibition in the presence of the phosphorylated metabolites. The S46D mutant HPr protein promoted regulation, even in the absence of a metabolite. The Vmax but not the Km value for 2DG uptake was affected. Accumulation of the natural, metabolizable substrates of the lactose, glucose, mannose and ribose permeases was inhibited by wild-type HPr in the presence of FDP or by S46D mutant HPr. The results establish that HPr serine phosphorylation by the ATP-dependent, metabolite-activated HPr kinase selectively determines the levels of sugar accumulation via the glucose and lactose permeases in L. lactis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed2005

Oral administration of recombinant Lactococcus lactis expressing bovine beta-lactoglobulin partially prevents mice from sensitization.

BACKGROUND: The use of probiotics such as Lactococcus lactis and other lactic acid bacteria (LAB) has been proposed for the management of food allergy. However, no experimental study has clearly demonstrated any preventive or therapeutic inhibition of an allergen-specific IgE response. OBJECTIVE: We aimed to study the immunomodulatory effect of recombinant L. lactis expressing bovine beta-lactoglobulin (BLG), a major cow's milk allergen, in a validated mouse model of allergy. METHODS: Six-week-old female Balb/c mice received five repeated doses of BLG, of L. lactis plus BLG, or of recombinant L. lactis by gavage. Different recombinant strains were inoculated, which corresponded to BLG doses ranging from 4 to 70 microg/mice. Mice were then sensitized by intra-peritoneal injection of BLG emulsified in incomplete Freund's adjuvant to induce high IgE concentrations. RESULTS: Pre-treatment with natural L. lactis plus BLG allowed induction of BLG-specific T-helper type 1 (Th1) response, and abrogated the oral tolerance induced by BLG alone, demonstrating the adjuvant effect of this non-colonizing LAB. Moreover, pre-treatment with some of the recombinant strains favoured the development of a Th1 response inhibiting the Th2 one: it induced a significant decrease of specific IgE response, and an intense increase of specific IgG2a and IFN-gamma productions. The most efficient strains that inhibited the IgE response were those producing the highest amounts of the BLG protein. CONCLUSION: Oral administration of some recombinant L. lactis was demonstrated to induce a specific Th1 response down-regulating a further Th2 one. Prophylaxis protocols will thus be evaluated using the most efficient strains.

Administration, Oral↗
PubMed1992

Structure of the exopolysaccharide produced by Lactococcus lactis subspecies cremoris H414 grown in a defined medium or skimmed milk.

The structure of the exopolysaccharide of Lactococcus lactis subsp. cremoris H414, isolated from a defined medium or skimmed milk, was established by linkage analysis on the native polysaccharide, and by characterisation of oligosaccharide fragments, obtained by Smith degradation and partial acid hydrolysis, using methylation analysis, FABMS, EIMS, and 1H-NMR spectroscopy. The polysaccharide has the branched-pentasaccharide repeating unit: [formula: see text]

Animals↗
PubMedDate not supplied

Influence of intracellular pH on light emission from a luxA/B derivative of Lactococcus lactis subsp. diacetylactis.

High levels of constitutive aldehyde-dependent light emission were obtained from nongrowing cells of Lactococcus lactis subsp. diacetylactis F712 transformed with IuxA/B when they were suspended in buffered solutions. Inductions of light emission was time-dependent and was not due to growth, synthesis of luciferase or stimulation of metabolism by fermentable carbohydrate. The major factor controlling light emission in such cells appears to be the intracellular pH value. Experiments with ionophores indicated that a transmembrane pH gradient was not essential for light emission.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1998

Transcription of the trp operon in Lactococcus lactis is controlled by antitermination in the leader region.

The regulatory functions of the leader region preceding the Lactococcus lactis trp operon have been studied by mutagenesis analysis. This leader presents striking similarity to 'T-box' leaders found upstream of many Gram-positive aminoacyl-tRNA synthetase genes and some amino acid biosynthesis operons, which are controlled by antitermination through interaction of the leader transcript with cognate uncharged tRNA. A region of the L. lactis leader transcript also contains a series of (G/U) AG repeats which, in Bacillus, are involved in the binding of the trp RNA-binding protein (TRAP) which controls trp transcription. A screen was developed for the isolation of regulatory mutants affected in the leader region. All spontaneous mutants contained deletions; point mutations were only obtained after UV-induced mutagenesis. All mutations affected the putative transcription terminator upstream of the trp operon, demonstrating that trp is indeed controlled by transcription antitermination.

Base Sequence↗
PubMed1990

Cloning of a chromosomal fragment from Lactococcus lactis subsp. lactis partially complementing Escherichia coli recA functions.

A recA-like gene was isolated from a gene library of Lactococcus lactis subsp. lactis by intergeneric complementation of an E. coli recA mutant. A plasmid was obtained which fully complemented the RecA response to DNA damaging agents and UV inducibility of prophage, but not P1 plating efficiency in an E. coli recA mutant. The cloned DNA fragment also partially complemented the rec mutation in Lc. lactis MMS36. Hybridization studies showed that there was no detectable sequence homology between the recA gene of E. coli and Lc. lactis subsp. lactis chromosomal DNA.

Bacteriophage lambda↗
PubMed1991

Cloning and nucleotide sequence of the major capsid protein from Lactococcus lactis ssp. cremoris bacteriophage F4-1.

The gene (mcp) coding for the major capsid protein (MCP) of the Lactococcus lactis ssp. cremoris bacteriophage F4-1 has been cloned and its nucleotide sequence determined. The mcp gene was localized, by Western blotting with rabbit antiserum against intact bacteriophage, within a 3.3-kb HindIII-Spe I fragment and the sequence of the entire region determined. The 35-kDa MCP is coded for by a 905-bp open reading frame preceded by a putative ribosome-binding site. Deletion analysis and N-terminal sequencing of the MCP confirmed the identification of the gene coding for this bacteriophage MCP.

Amino Acid Sequence↗
PubMed2003

CTP limitation increases expression of CTP synthase in Lactococcus lactis.

CTP synthase is encoded by the pyrG gene and catalyzes the conversion of UTP to CTP. A Lactococcus lactis pyrG mutant with a cytidine requirement was constructed, in which beta-galactosidase activity in a pyrG-lacLM transcriptional fusion was used to monitor gene expression of pyrG. A 10-fold decrease in the CTP pool induced by cytidine limitation was found to immediately increase expression of the L. lactis pyrG gene. The final level of expression of pyrG is 37-fold higher than the uninduced level. CTP limitation has pronounced effects on central cellular metabolism, and both RNA and protein syntheses are inhibited. Expression of pyrG responds only to the cellular level of CTP, since expression of pyrG has no correlation to alterations in UTP, GTP, and ATP pool sizes. In the untranslated pyrG leader sequence a potential terminator structure can be identified, and this structure is required for regulation of the pyrG gene. It is possible to fold the pyrG leader in an alternative structure that would prevent the formation of the terminator. We suggest a model for pyrG regulation in L. lactis, and probably in other gram-positive bacteria as well, in which pyrG expression is directly dependent on the CTP concentration through an attenuator mechanism. At normal CTP concentrations a terminator is preferentially formed in the pyrG leader, thereby reducing expression of CTP synthase. At low CTP concentrations the RNA polymerase pauses at a stretch of C residues in the pyrG leader, thereby allowing an antiterminator to form and transcription to proceed. This model therefore does not include any trans-acting protein for sensing the CTP concentration as previously proposed for Bacillus subtilis.

Base Sequence↗
PubMed1996

Cloning and sequencing of the novel abortive infection gene abiH of Lactococcus lactis ssp. lactis biovar. diacetylactis S94.

A gene which encodes resistance by abortive infection (Abi+) to bacteriophage was cloned from Lactococcus lactis ssp. lactis biovar. diacetylactis S94. This gene was found to confer a reduction in efficiency of plating and plaque size for prolate-headed bacteriophage phi 53 (group I of homology) and total resistance to the small isometric-headed bacteriophage phi 59 (group III of homology). The cloned gene is predicted to encode a polypeptide of 346 amino acid residues with a deduced molecular mass of 41 455 Da. No homology with any previously described genes was found. A probe was used to determine the presence of this gene in two strains on 31 tested.

Bacterial Proteins↗
PubMed1995

Construction of a food-grade host/vector system for Lactococcus lactis based on the lactose operon.

A plasmid-based food-grade vector system was developed for Lactococcus lactis by exploiting the genes for lactose metabolism. L. lactis MG5267 is a plasmid-free strain containing the entire lactose operon as a chromosomal insertion. The lacF gene was deleted from this strain by a double cross-over homologous recombination event. The lacF-deficient strain produced a Lac- phenotype on indicator agar. A cloned copy of the lacF gene expressed on a plasmid was capable of complementing the lacF-deficient strain resulting in a Lac+ phenotype. This stably maintained system fits the requirements of a self-selecting vector system and has the potential to be exploited in the food industry.

Base Sequence↗
PubMed2000

LldI, a plasmid-encoded type I restriction and modification system in Lactococcus lactis.

A plasmid-encoded type I restriction and modification (R-M) system, designated LldI, was identified in Lactococcus lactis biovar diacetylactis LD10-1. LldI consists of three genes encoding endonuclease, methylase and specificity subunits, respectively. RT-PCR analysis revealed that the three genes are co-transcribed as a polycistronic mRNA in L. lactis. The specificity subunit of LldI differs significantly in the target recognition domains from those of other type I R-M systems, suggesting that LldI confers a novel specificity in L. lactis.

Amino Acid Sequence↗
PubMed2006

Genetic structure and transcriptional analysis of the arginine deiminase (ADI) cluster in Lactococcus lactis MG1363.

In a recent proteomic analysis, we showed the overproduction of the ArcA and ArcB proteins in Lactococcus lactis MG1363 at low pH. The corresponding genes belong to the arcABD1C1C2TD2 cluster that encodes components of the arginine deiminase pathway. In this study, we characterized this cluster at the genetic level. Northern blot experiments showed the expression of at least seven transcripts, all induced by acidity. Transcript analysis using 5'RACE PCR (rapid amplification of cDNA ends polymerase chain reaction) in the arcB-arcD1 intergenic region. In silico analysis identified nine stem-loop structures, all located in intergenic regions. Collectively, these data suggest a role for RNA processing and (or) premature termination in the differential expression of genes within the arcABD1C1C2TD2 cluster.

Bacterial Proteins↗
PubMed1999

Nisin production by a mixed-culture system consisting of Lactococcus lactis and Kluyveromyces marxianus.

To control the pH during antimicrobial peptide (nisin) production by a lactic acid bacterium, Lactococcus lactis subsp. lactis (ATCC11454), a novel method involving neither addition of alkali nor a separation system such as a ceramic membrane filter and electrodialyzer was developed. A mixed culture of L. lactis and Kluyveromyces marxianus, which was isolated from kefir grains, was utilized in the developed system. The interaction between lactate production by L. lactis and its assimilation by K. marxianus was used to control the pH. To utilize the interaction of these microorganisms to maintain high-level production of nisin, the kinetics of growth of, and production of lactate, acetate, and nisin by, L. lactis were investigated. The kinetics of growth of and lactic acid consumption by K. marxianus were also investigated. Because the pH of the medium could be controlled by the lactate consumption of K. marxianus and the specific lactate consumption rate of K. marxianus could be controlled by changing the dissolved oxygen (DO) concentration, a cascade pH controller coupled with DO control was developed. As a result, the pH was kept constant because the lactate level was kept low and nisin accumulated in the medium to a high level compared with that attained using other pH control strategies, such as with processes lacking pH control and those in which pH is controlled by addition of alkali.

Aerobiosis↗
PubMed2003

Ability of Lactococcus lactis to export viral capsid antigens: a crucial step for development of live vaccines.

The food grade bacterium Lactococcus lactis is a potential vehicle for protein delivery in the gastrointestinal tract. As a model, we constructed lactococcal strains producing antigens of infectious bursal disease virus (IBDV). IBDV infects chickens and causes depletion of B-lymphoid cells in the bursa of Fabricius and subsequent immunosuppression, morbidity, or acute mortality. The two major IBDV antigens, i.e., VP2 and VP3, that form the viral capsid were expressed and targeted to the cytoplasm, the cell wall, or the extracellular compartment of L. lactis. Whereas VP3 was successfully targeted to the three compartments by the use of relevant expression and export vectors, VP2 was recalcitrant to export, thus confirming the difficulty of translocating naturally nonsecreted proteins across the bacterial membrane. This defect could be partly overcome by fusing VP2 to a naturally secreted protein (the staphylococcal nuclease Nuc) that carried VP2 through the membrane. Lactococcal strains producing Nuc-VP2 and VP3 in various bacterial compartments were administered orally to chickens. The chickens did not develop any detectable immune response against VP2 and VP3 but did exhibit an immune response against Nuc when Nuc-VP2 was anchored to the cell wall of lactococci.

Animals↗
PubMed2002

Energy-based dynamic model for variable temperature batch fermentation by Lactococcus lactis.

We developed a mechanistic mathematical model for predicting the progression of batch fermentation of cucumber juice by Lactococcus lactis under variable environmental conditions. In order to overcome the deficiencies of presently available models, we use a dynamic energy budget approach to model the dependence of growth on present as well as past environmental conditions. When parameter estimates from independent experimental data are used, our model is able to predict the outcomes of three different temperature shift scenarios. Sensitivity analyses elucidate how temperature affects the metabolism and growth of cells through all four stages of fermentation and reveal that there is a qualitative reversal in the factors limiting growth between low and high temperatures. Our model has an applied use as a predictive tool in batch culture growth. It has the added advantage of being able to suggest plausible and testable mechanistic assumptions about the interplay between cellular energetics and the modes of inhibition by temperature and end product accumulation.

Calibration↗
PubMed2004

Metabolic engineering of mannitol production in Lactococcus lactis: influence of overexpression of mannitol 1-phosphate dehydrogenase in different genetic backgrounds.

To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and (13)C nuclear magnetic resonance analysis revealed that small amounts (<1%) of mannitol were formed by growing cells of mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol. Moreover, the formed mannitol was not reutilized upon glucose depletion. Of the metabolic-engineering strategies investigated in this work, mtlD-overexpressing LDH-deficient L. lactis seemed to be the most promising strain for mannitol production.

Genetic Engineering↗