Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Lactococcus lactis”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 379 records · Page 21Linked to original sources
PubMed2004

Using Lactococcus lactis for glutathione overproduction.

Glutathione and gamma-glutamylcysteine were produced in Lactococcus lactis using a controlled expression system and the genes gshA and gshB from Escherichia coli encoding the enzymes gamma-glutamylcysteine synthetase and glutathione synthetase. High levels of gamma-glutamylcysteine were found in strains growing on chemically defined medium and expressing either gshA alone or both gshA and gshB. As anticipated, glutathione was found in a strain expressing gshA and gshB. The level of glutathione production could be increased by addition of the precursor amino acid cysteine to the medium. The addition of cysteine led to an increased activity of glutathione synthetase, which is remarkable because the amino acid is not a substrate of this enzyme. The final intracellular glutathione concentration attained was 358 nmol mg(-1) protein, which is the highest concentration reported for a bacterium, demonstrating the suitability of engineered L. lactis for fine-chemical production and as a model for studies of the impact of glutathione on flavour formation and other properties of food.

Cloning, Molecular↗
PubMed1997

Product formation and phosphoglucomutase activities in Lactococcus lactis: cloning and characterization of a novel phosphoglucomutase gene.

Maltose metabolism in Lactococcus lactis involves the conversion of beta-glucose 1-phosphate to glucose 6-phosphate, a reaction which is reversibly catalysed by a maltose-inducible and glucose-repressible beta-phosphoglucomutase (beta-PGM). The gene encoding beta-PGM (pgmB) was cloned from a genomic library of L. lactis using antibodies. The nucleotide sequence of a 5695 bp fragment was determined and six ORFs, including the pgmB gene, were found. The gene expressed a polypeptide with a calculated molecular mass of 24210 Da, which is in agreement with the molecular mass of the purified beta-PGM (25 kDa). A short sequence at the N-terminus was found to be similar to known metal-binding domains. The expression of beta-PGM in L lactis was found to be induced also by trehalose and sucrose, and repressed by lactose in the growth medium. This indicates that beta-PGM does not serve solely to degrade maltose, but that it is also involved in the metabolism of other carbohydrates. The specific activity of beta-PGM during fermentation was dependent on the maltose concentration in the medium. The maximum specific activity of beta-PGM increased by a factor of 4.6, and the specific growth rate by a factor of 7, when the maltose concentration was raised from 0.8 to 11.0 g l-1. Furthermore, a higher amount of lactate produced relative to formate, acetate and ethanol was observed when the initial maltose concentration in the medium was increased. The specific activity of alpha-PGM responded similarly to beta-PGM, but the magnitude of the response was lower. Preferential sugar utilization and alpha- and beta-PGM suppression was observed when L. lactis was grown on the substrate combinations glucose and maltose, or lactose and maltose; maltose was the least-preferred sugar. In contrast, galactose and maltose were utilized concurrently and both PGM activities were high throughout the fermentation.

Amino Acid Sequence↗
PubMed1994

Mutational analysis of cat-86 gene expression controlled by lactococcal promoters in Lactococcus lactis subsp. lactis and Escherichia coli.

Promoters were cloned from the chromosomal DNA of Lactococcus lactis subsp. lactis NP4510 by using promoter-probe vector pGKV210. N-Methyl-N'-nitro-N-nitrosoguanidine-induced mutagenesis of L. lactis-(pBV413), with low-level expression of the cat-86 gene, resulted in enhanced expression. Subcloning and sequencing of the mutated plasmid designated pBV415 revealed that the mutation is located within the PstI-HindIII fragment containing the coding sequence of the cat-86 gene (the 10th CTG codon was replaced by a TTG; both code for leucine). A set of otherwise identical plasmids with four combinations of CTG and TTG codons at the 10th and 46th positions in the cat-86 gene were constructed by site-directed mutagenesis. These plasmids containing cat-86 derivatives displayed a significant variation in cat expression in L. lactis and E. coli. The data suggest that cat expression is dependent on the secondary structure of the cat mRNA. New cat-86 derivatives described here can be used in lactococci, in which they provide additional flexibility for promoter cloning.

Amino Acid Sequence↗
PubMed1995

Nucleotide sequence of a plasmid pCL2.1 from Lactococcus lactis ssp. lactis ML8.

The nucleotide sequence of a small cryptic plasmid, pCL2.1, from Lactococcus lactis ssp. lactis ML8 was determined. Sequence analysis of the pCL2.1 revealed that it contained 2112 bp, 33.9% GC, and two open reading frames that encoded polypeptides of 26 and 14 kDa. In vitro transcription-translation of the pCL2.1 confirmed the existence of two polypeptides. Based on sequence homology, it is deduced that the ORF2 product functions in plasmid replication by a rolling circle mechanism.

Base Sequence↗
PubMed1993

Cloning and sequencing of pepC, a cysteine aminopeptidase gene from Lactococcus lactis subsp. cremoris AM2.

A gene coding for an aminopeptidase (PepC) from Lactococcus lactis subsp. cremoris AM2 was cloned by complementation of an Escherichia coli mutant lacking aminopeptidase activity. The nucleotide sequence was determined. A portion of the predicted amino acid sequence of PepC (436 amino acids) showed strong homology to the active site of cysteine proteases. No signal sequence was found, indicating an intracellular location of the enzyme.

Amino Acid Sequence↗
PubMed2000

[Expression and activity analysis of the human glutathione-S-transferase in Lactococcus lactis].

The glutathione-S-transferase A1 cDNA was amplified from human liver total RNAs by RT-PCR and was cloned into a Escherichia coli expression vector pET23b, then the recombinant plasmid pET23bhgst was introduced into E. coli BL21 (DE3) and induced by IPTG, the high-level expression of hGSTA1 appeared in the E. coli cells. The cDNA encoding hGSTA1 was subcloned into pMG36e, a lactococcal expression vector, and introduced into Lactococcus lactis MG1363 by electroporation. In the positive transformants, the hGSTA1 was expressed as a fusion protein which was verified by SDS-PAGE and Western blot. The hGSTA1 from both E. coli and L. lactis was purified by affinity chromatography on glutathione-agarose and all showed enzymatic activity. The potential application of the recombinant Lactococcus lactis in functional food was also discussed.

Blotting, Western↗
PubMed1995

Glucose metabolism and internal pH of Lactococcus lactis subsp. lactis cells utilizing NMR spectroscopy.

The metabolism of glucose was studied in Lactococcus lactis subsp. CNRZ 125 by 13C NMR. The initial rate of glucose utilization was higher for exponential phase cells than for stationary phase cells [150 vs 85 nmol g (dry wt)-1 s -1]. 31P NMR was used to determine changes in glycolytic phosphorylated intermediates (fructose-1,6-diphosphate, dihydroxyacetone phosphate and phosphoglycerate). The internal pHs of L. lactis subsp. lactis CNRZ 141 and CNRZ 125 were also measured by 31P NMR as a function of the external pH during growth. When the external pH was 6.8, the internal pHs of strain CNRZ 141 and CNRZ 125 were similar, 7.4. After the external pH had decreased to 5.5, the internal pH of strain CNRZ 141 had declined by 0.6 unit, whereas that of strain CNRZ 125 had decreased by only 0.2 unit of pH.

Dihydroxyacetone Phosphate↗
PubMed2006

Transcriptome analysis reveals mechanisms by which Lactococcus lactis acquires nisin resistance.

Nisin, a posttranslationally modified antimicrobial peptide produced by Lactococcus lactis, is widely used as a food preservative. Yet, the mechanisms leading to the development of nisin resistance in bacteria are poorly understood. We used whole-genome DNA microarrays of L. lactis IL1403 to identify the factors underlying acquired nisin resistance mechanisms. The transcriptomes of L. lactis IL1403 and L. lactis IL1403 Nis(r), which reached a 75-fold higher nisin resistance level, were compared. Differential expression was observed in genes encoding proteins that are involved in cell wall biosynthesis, energy metabolism, fatty acid and phospholipid metabolism, regulatory functions, and metal and/or peptide transport and binding. These results were further substantiated by showing that several knockout and overexpression mutants of these genes had strongly altered nisin resistance levels and that some knockout strains could no longer become resistant to the same level of nisin as that of the wild-type strain. The acquired nisin resistance mechanism in L. lactis is complex, involving various different mechanisms. The four major mechanisms are (i) preventing nisin from reaching the cytoplasmic membrane, (ii) reducing the acidity of the extracellular medium, thereby stimulating the binding of nisin to the cell wall, (iii) preventing the insertion of nisin into the membrane, and (iv) possibly transporting nisin across the membrane or extruding nisin out of the membrane.

Anti-Bacterial Agents↗
PubMed2002

Production of human papillomavirus type 16 E7 protein in Lactococcus lactis.

The E7 protein of human papillomavirus type 16 was produced in Lactococcus lactis. Secretion allowed higher production yields than cytoplasmic production. In stationary phase, amounts of cytoplasmic E7 were reduced, while amounts of secreted E7 increased, suggesting a phase-dependent intracellular proteolysis. Fusion of E7 to the staphylococcal nuclease, a stable protein, resulted in a highly stable cytoplasmic protein. This work provides new candidates for development of viral screening systems and for oral vaccine against cervical cancer.

Cloning, Molecular↗
PubMed1993

Gene organization, primary structure and RNA processing analysis of a ribosomal RNA operon in Lactococcus lactis.

Southern blot analysis of genomic DNA of the mesophilic lactic bacterium Lactococcus lactis subsp. lactis strain IL1403, illuminated six rRNA gene clusters. Each cluster contains one copy each of three rRNA genes, displaying the typical eubacterial organization of physically linked 16 S, 23 S and 5 S rRNA genes. Five of the six rRNA clusters were cloned into plasmid pBR322. One recombinant plasmid, pSLCM6, containing a 6500 base-pair genomic DNA fragment, was characterized by physical mapping and the sequences encoding rRNAs and tRNAs were localized by Southern hybridization. This fragment contains a single operon composed of one promoter, a leader sequence, a 16 S rRNA gene, a tRNA(Ala) gene, a 23 S rRNA gene, a 5 S rRNA gene and a tRNA(Asn) gene. S1 nuclease mapping and primer extension analysis of in vivo transcripts localized one transcriptional initiation site 150 base-pairs upstream from the start of the 16 S rRNA gene. These procedures also suggest that this transcript is processed by an RNAse III-like activity similar to Bacillus subtilis; i.e. the L. lactis nuclease might be sequence-specific. The chronology of specific cleavages occurring during the maturation process of the precursor transcript is described. One interesting observation is that the regions flanking the 16 S and 23 S rRNAs containing the primary processing sites are identical and contain sequences that could be involved in transcriptional antitermination. S1 mapping of the 3' ends of in vivo transcripts indicate that a terminator-like sequence a few base-pairs downstream from the distal tRNA(Asn) gene is inefficient in arresting transcription.

Base Sequence↗
PubMed2001

Regulation of pyruvate metabolism in Lactococcus lactis depends on the imbalance between catabolism and anabolism.

Two strains of Lactococcus lactis ssp. cremoris, MG 1820 and MG 1363, which differed by the presence or absence of the lactose plasmid, respectively, were cultivated in batch-mode fermentation on lactose as carbon substrate. A correlation between the rate of sugar consumption, the growth rate, and the type of metabolism was observed. The MG 1820 strain grew rapidly on lactose and homolactic fermentation occurred. The major regulating factor was the NADH/NAD(+) ratio proportional to the catabolic flux, which inhibited glyceraldehyde-3-phosphate dehydrogenase activity. This control led to an increase in metabolite concentration upstream of this enzyme, glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate, and inhibition of pyruvate formate lyase activity, while lactate dehydrogenase was strongly activated by the high coenzyme ratio. The contrary was observed during growth of the MG 1363 strain. Further investigation during growth of L. lactis ssp. lactis NCDO 2118 on galactose as carbon substrate and on various culture media enabling the growth rate to proceed at various rates demonstrated that the relative flux between catabolism and anabolism was the critical regulating parameter rather than the rate of glycolysis itself. In a minimal medium, where anabolism was strongly limited, the rate of sugar consumption was reduced to a low value to avoid carbon and energy waste. Despite this low sugar consumption rate, the catabolic flux was in excess relative to the anabolic capability and the NADH/NAD+ ratio was high, typical of a situation of nonlimiting catabolism leading to a homolactic metabolism.

Cell Division↗
PubMed1996

The genes for secretion and maturation of lactococcins are located on the chromosome of Lactococcus lactis IL1403.

Southern hybridization and PCR analysis were used to show that Lactococcus lactis IL1403, a plasmid-free strain that does not produce bacteriocin, contains genes on its chromosome that are highly homologous to lcnC and lcnD and encode the lactococcin secretion and maturation system. The lcnC and lcnD homologs on the chromosome of IL1403 were interrupted independently by Campbell-type integrations. Both insertion mutants were unable to secrete active lactococcin. Part of the chromosomal lcnC gene was cloned and sequenced. Only a few nucleotide substitutions occurred, compared with the plasmid-encoded lcnC gene, and these did not lead to changes in the deduced amino acid sequence. No genes homologous to those for lactococcin A, B, or M could be detected in IL1403, and the strain does not produce bacteriocin activity.

Amino Acid Sequence↗
PubMed1999

Molecular characterization of the Lactococcus lactis ptsHI operon and analysis of the regulatory role of HPr.

The Lactococcus lactis ptsH and ptsI genes, encoding the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system, HPr and enzyme I, respectively, were cloned, and the regulatory role of HPr was studied by mutational analysis of its gene. A promoter sequence was identified upstream of the ptsHI operon, and the transcription start site was mapped by primer extension. The results of Northern analyses showed the presence of two glucose-inducible transcripts, one of 0.3 kb containing ptsH and a second of 2.0 kb containing both ptsH and ptsI. Disruption of the ptsH and ptsI genes in strain NZ9800 resulted in a reduced growth rate at the expense of glucose, but no growth at the expense of sucrose and fructose, confirming the dominant role of the phosphotransferase system in the uptake of these sugars in L. lactis. Complementation of the ptsH and ptsI mutants with the intact genes under the control of a regulated promoter resulted in the restoration of the wild-type phenotype. The role of HPr(Ser-P) in the recently established CcpA-mediated control of galactose metabolism as well as glycolysis was analyzed by producing an HPr mutant carrying an aspartic acid on residue 46 which mimicks a phosphorylated serine. The results of these experiments demonstrated the role of HPr(Ser-P) as corepressor in the catabolite repression of the gal operon. Furthermore, we show for the first time that HPr(Ser-P) functions as a coactivator in the CcpA-mediated catabolite activation of the pyruvate kinase and L-lactate dehydrogenase genes.

Bacterial Proteins↗
PubMed1994

Identification and characterization of genes involved in excision of the Lactococcus lactis conjugative transposon Tn5276.

The 70-kb transposon Tn5276, originally detected in Lactococcus lactis NIZO R5 and carrying the genes for nisin production and sucrose fermentation, can be conjugally transferred to other L. lactis strains. Sequence analysis and complementation studies showed that the right end of Tn5276 contains two genes, designated xis and int, which are involved in excision. The 379-amino-acid int gene product shows high (up to 50%) similarity with various integrases, including that of the Tn916-related conjugative transposons. The xis gene product, like almost all known excisionase (Xis) proteins, is a small (68-residue), basic protein. Expression of both the Tn5276 int and xis genes is required for efficient excision of the ends of Tn5276 in Escherichia coli that appeared to be circularized in the excision process. Mutational analysis of the xis and int genes showed that excision efficiency is dependent on the integrity of the int gene but that an intact xis gene is also required for efficient excision.

Amino Acid Sequence↗
PubMed1995

Phage operon involved in sensitivity to the Lactococcus lactis abortive infection mechanism AbiD1.

Phage bIL66 is unable to grow on Lactococcus lactis cells harboring the abortive infection gene abiD1. Spontaneous phage mutants able to grow on AbiD1 cells were used to study phage-Abi interaction. A 1.33-kb DNA segment of a mutant phage allowed growth of AbiD1s phages in AbiD1 cells when present in trans. Sequence analysis of this segment revealed an operon composed of four open reading frames, designated orf1 to orf4. The operon is transcribed 10 min after infection from a promoter presenting an extended -10 consensus sequence but no -35 sequence. Analysis of four independent AbiD1r mutants revealed a different point mutation localized in orf1, implying that this open reading frame is needed for sensitivity to AbiD1. However, the sensitivity is partly suppressed when orf3 is expressed in trans on a high-copy-number plasmid, suggesting that AbiD1 acts by decreasing the concentration of an available orf3 product.

Amino Acid Sequence↗
PubMed1997

A chloride-inducible gene expression cassette and its use in induced lysis of Lactococcus lactis.

A chloride-inducible promoter previously isolated from the chromosome of Lactococcus lactis (J. W. Sanders, G. Venema, J. Kok, and K. Leenhouts, Mol. Gen. Genet., in press) was exploited for the inducible expression of homologous and heterologous genes. An expression cassette consisting of the positive-regulator gene gadR, the chloride-inducible promoter Pgad, and the translation initiation signals of gadC was amplified by PCR. The cassette was cloned upstream of Escherichia coli lacZ, the holin-lysin cassette (lytPR) of the lactococcal bacteriophage r1t, and the autolysin gene of L. lactis, acmA. Basal activity of Pgad resulted in a low level of expression of all three proteins. Growth in the presence of 0.5 M NaCl of a strain containing the gadC::lacZ fusion resulted in a 1,500-fold increase of beta-galactosidase activity. The background activity levels of LytPR and AcmA had no deleterious effects on cell growth, but induction of lysin expression by addition of 0.5 M NaCl resulted in inhibition of growth. Lysis was monitored by following the release of the cytoplasmic marker enzyme PepX. Released PepX activity was maximal at 1 day after induction of lytPR expression with 0.1 M NaCl. Induction of acmA expression resulted in slower release of PepX from the cells. The presence of the inducing agent NaCl resulted in the stabilization of osmotically fragile cells.

Artificial Gene Fusion↗
PubMed1991

Characterization of the nisin gene as part of a polycistronic operon in the chromosome of Lactococcus lactis ATCC 11454.

The location and organization of the nisin locus in Lactococcus lactis ATCC 11454 were studied. Primer extension of in vivo mRNA transcripts of the gene that encodes the nisin prepropeptide sequence indicated the presence of a promoter at least 4 kb upstream from the nisin gene and that the mRNA has several processing sites. Restriction fragment patterns using rare-cutting enzymes, orthogonal pulsed-field clamped homogeneous electric field (CHEF) agarose gel electrophoresis, and hybridization with nisin gene probes showed that the nisin prepropeptide gene was located on a megabase-size restriction fragment, which was taken as proof of a chromosomal location. This is contrary to earlier reports, which had indicated that genes for nisin production were located on plasmids. There was no evidence of more than one chromosomal location or more than one copy of the nisin gene. The restriction patterns indicated that the size of the L. lactis genome is about 2,500 kb. The previously observed (G. W. Buchman, S. Banerjee, and J. N. Hansen, J. Biol. Chem. 263: 16260-16266, 1988) downstream open reading frame (ORF) was fully sequenced to reveal an 851-amino-acid coding region, an upstream putative mRNA processing site, and a putative rho-independent terminator. The ORF was analyzed for secondary structural features, and the sequence data bases were searched for homologies. The ORF contained many amphipathic helices, a C-terminal transmembrane helix, and homologies to some membrane-associated proteins. It lacked an N-terminal membrane insertion sequence and accordingly appears to be associated with, and anchored to, the cytoplasmic side of the membrane. An additional ORF that possessed a ribosome-binding sequence and tandem promoters, indicating the beginning of a new operon, was identified still farther downstream. The results were consistent with the nisin gene being part of a polycistronic operon with a size greater than 8.5 kb.

Amino Acid Sequence↗
PubMed2002

Optimization of a cultural medium for bacteriocin production by Lactococcus lactis using response surface methodology.

The medium composition for bacteriocin production by Lactococcus lactis ATCC 11454 was optimized using response surface methodology. The selected six factors based on CM medium were sucrose, soybean peptone, yeast extract, KH(2)PO(4), NaCl, and MgSO(4).7H(2)O. Fractional factorial designs (FFD) and the path of steepest ascent were effective in searching for the main factors and approaching the optimum region of the response. By a 2(6-2) FFD, sucrose, soybean peptone, yeast extract, KH(2)PO(4) were found to be significant factors and had positive effects on cell growth, however, only soybean peptone and KH(2)PO(4) were shown to be the two significant factors for bacteriocin production and had negative and positive effects, respectively. The effects of the two main factors on bacteriocin production were further investigated by a central composite design and the optimum composition was found to be 1% sucrose, 0.45% soybean peptone, 1% yeast extract, 2.84% KH(2)PO(4), 0.2% NaCl, and 0.02% MgSO(4) x 7H(2)O. The optimal medium allowed bacteriocin yield to be doubled compared to CM medium.

Bacteriocins↗