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 1,027 records · Page 57Linked to original sources
PubMed2005

Retrotransposition strategies of the Lactococcus lactis Ll.LtrB group II intron are dictated by host identity and cellular environment.

Group II introns are mobile retroelements that invade their cognate intron-minus gene in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. Previous studies of the Lactococcus lactis intron Ll.LtrB indicated that in its native host, as in Escherichia coli, retrohoming occurs by the intron RNA reverse splicing into double-stranded DNA (dsDNA) through an endonuclease-dependent pathway. However, in retrotransposition in L. lactis, the intron inserts predominantly into single-stranded DNA (ssDNA), in an endonuclease-independent manner. This work describes the retrotransposition of the Ll.LtrB intron in E. coli, using a retrotransposition indicator gene previously employed in our L. lactis studies. Unlike in L. lactis, in E. coli, Ll.LtrB retrotransposed frequently into dsDNA, and the process was dependent on the endonuclease activity of the intron-encoded protein. Further, the endonuclease-dependent insertions preferentially occurred around the origin and terminus of chromosomal DNA replication. Insertions in E. coli can also occur through an endonuclease-independent pathway, and, as in L. lactis, such events have a more random integration pattern. Together these findings show that Ll.LtrB can retrotranspose through at least two distinct mechanisms and that the host environment influences the choice of integration pathway. Additionally, growth conditions affect the insertion pattern. We propose a model in which DNA replication, compactness of the nucleoid and chromosomal localization influence target site preference.

Bacterial Proteins↗
PubMed1998

Induced levels of heat shock proteins in a dnaK mutant of Lactococcus lactis.

The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes and proteases, including the DnaK-GrpE-DnaJ and the GroELS chaperone complexes. In order to investigate the importance of the DnaK chaperone complex for growth and heat shock response regulation in Lactococcus lactis, we have constructed two dnaK mutants with C-terminal deletions in dnaK. The minor deletion of 65 amino acids in the dnaKDelta2 mutant resulted in a slight temperature-sensitive phenotype. BK6, containing the larger deletion of 174 amino acids (dnaKDelta1), removing the major part of the inferred substrate binding site of the DnaK protein, exhibited a pronounced temperature-sensitive phenotype and showed altered regulation of the heat shock response. The expression of the heat shock proteins was increased at the normal growth temperature, measured as both protein synthesis rates and mRNA levels, indicating that DnaK could be involved in the regulation of the heat shock response in L. lactis. For Bacillus subtilis, it has been found (A. Mogk, G. Homuth, C. Scholz, L. Kim, F. X. Schmid, and W. Schumann, EMBO J. 16:4579-4590, 1997) that the activity of the heat shock repressor HrcA is dependent on the chaperone function of the GroELS complex and that a dnaK insertion mutant has no effect on the expression of the heat shock proteins. The present data from L. lactis suggest that the DnaK protein could be involved in the maturation of the homologous HrcA protein in this bacterium.

Bacterial Proteins↗
PubMed1993

Cloning, sequence and expression of the gene encoding the malolactic enzyme from Lactococcus lactis.

Many lactic acid bacteria can carry out malolactic fermentation. This secondary fermentation is mediated by the NAD- and Mn(2+)-dependent malolactic enzyme, which catalyses the decarboxylation of L-malate to L-lactate. The gene we call mleS, coding for malolactic enzyme, was isolated from Lactococcus lactis. The mleS gene consists of one open reading frame capable of coding for a protein with a calculated molecular mass of 59 kDa. The amino acid sequence of the predicted MleS gene product is homologous to the sequences of different malic enzymes. Bacterial and yeast cells expressing the malolactic gene convert L-malate to L-lactate.

Amino Acid Sequence↗
PubMed2003

Determination of the domain of the Lactobacillus delbrueckii subsp. bulgaricus cell surface proteinase PrtB involved in attachment to the cell wall after heterologous expression of the prtB gene in Lactococcus lactis.

Belonging to the subtilase family, the cell surface proteinase (CSP) PrtB of Lactobacillus delbrueckii subsp. bulgaricus differs from other CSPs synthesized by lactic acid bacteria. Expression of the prtB gene under its own promoter was shown to complement the proteinase-deficient strain MG1363 (PrtP(-) PrtM(-)) of Lactococcus lactis subsp. cremoris. Surprisingly, the maturation process of PrtB, unlike that of lactococcal CSP PrtPs, does not require a specific PrtM-like chaperone. The carboxy end of PrtB was previously shown to be different from the consensus anchoring region of other CSPs and exhibits an imperfect duplication of 59 amino acids with a high lysine content. By using a deletion strategy, the removal of the last 99 amino acids, including the degenerated anchoring signal (LPKKT), was found to be sufficient to release a part of the truncated PrtB into the culture medium and led to an increase in PrtB activity. This truncated PrtB is still active and enables L. lactis MG1363 to grow in milk supplemented with glucose. By contrast, deletion of the last 806 amino acids of PrtB led to the secretion of an inactive proteinase. Thus, the utmost carboxy end of PrtB is involved in attachment to the bacterial cell wall. Proteinase PrtB constitutes a powerful tool for cell surface display of heterologous proteins like antigens.

Amino Acid Sequence↗
PubMed1995

Isolation of nisin-producing Lactococcus lactis strains from dry fermented sausages.

A total of 4608 lactic acid bacteria (LAB) were isolated from 24 Spanish fermented sausages and screened for bacteriocin production. Two strains, BB24 and G18, produced bacteriocins that inhibited a broad spectrum of Gram-positive bacteria. BB24 and G18 were tentatively identified as Lactococcus lactis by carbohydrate fermentation patterns and other biochemical characteristics. The characterization of their bacteriocins suggested that both could be the well-known lantibiotic nisin. This was confirmed by PCR analysis of their genomic DNA. Nucleotide sequencing revealed that they produced nisin A. The fact that BB24 and G18 were isolated from sausages produced in two different regions of Spain suggests that nisin-producing L. lactis strains may be more widespread in meat products than previously thought. Nisin produced by L. lactis BB24 has been purified to homogeneity by a procedure that included ammonium sulphate precipitation and cation-exchange, hydrophobic-interaction and reverse-phase chromatography. The purification procedure was simple, rapid and reproducible.

Bacteriocins↗
PubMed1994

A Lactococcus lactis gene encodes a membrane protein with putative ATPase activity that is homologous to the essential Escherichia coli ftsH gene product.

A gene, encoding a protein homologous to an essential Escherichia coli protein, FtsH, was identified adjacent to the hpt gene and the trnA operon in the Gram-positive bacterium Lactococcus lactis. The deduced amino acid sequence of the gene product showed full-length similarity to FtsH of E. coli, Yme1p of Saccharomyces cerevisiae and a conserved region found in a new family of putative ATPases. In-frame fusions of L. lactis ftsH and phoA1 in E. coli, and immunodetection of the L. lactis FtsH protein in cell fractions using anti-E. coli FtsH serum showed that L. lactis ftsH was expressed and encodes a membrane protein. When contained on a high copy number plasmid, the L. lactis ftsH gene complemented the lethality of a delta ftsH3::kan mutation in E. coli at 37 degrees C and below, indicating that the L. lactis ftsH gene can functionally replace the E. coli ftsH gene to some extent. The resulting E. coli strain showed temperature sensitivity and salt sensitivity. A L. lactis mutant with an insertion into ftsH was salt-, heat- and cold-sensitive. These results suggest that FtsH is somehow involved in stress responses. Southern hybridization analysis indicated that genes homologous to ftsH of L. lactis were also present in Bacillus subtilis, and several Lactobacillus and Leuconostoc species, suggesting high conservation of ftsH in bacterial species.

ATP-Dependent Proteases↗
PubMed1997

Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts.

Bacterial isolates from bean-sprouts were screened for anti-Listeria monocytogenes bacteriocins using a well diffusion method. Thirty-four of 72 isolates inhibited the growth of L. monocytogenes Scott A. One, HPB 1688, which had the biggest inhibition zone against L. monocytogenes Scott A, was selected for subsequent analysis. Both ribotyping and DNA sequencing of 16S ribosomal RNA gene demonstrated that the isolate was Lactococcus lactis subsp. lactis. Polymerase chain reaction and nucleotide sequencing revealed that the genomic DNA of the bean-sprout isolates contained a nisin Z structural gene. In MRS broth, bean-sprout isolate HPB 1688 survived at 3-4.5 degrees C for at least 20 d, grew at 4 degrees C and produced anti-listerial compounds at 5 degrees C. When co-cultured with L. monocytogenes in MRS broth, the isolate inhibited the growth of L. monocytogenes at 4 degrees C after 14 d and at 10 degrees C after 2 d. When co-inoculated with 10(2) cells g-1 of L. monocytogenes on fresh-cut ready-to-eat Caesar salad, L. lactis subsp. lactis (10(8) cells g-1) was able to reduce the number of L. monocytogenes by 1-1.4 logs after storage for 10 d at 7 zero and 10 degrees C. A bacteriocin-producing Enterococcus faecium was also able to reduce the numbers of L. monocytogenes on Caesar salad, but did not act synergistically when co-inoculated with L. lactis subsp. lactis.

Anti-Bacterial Agents↗
PubMed2002

Degradation of natural phosphorylated compounds and added polyphosphates in milk by Pseudomonas fluorescens CECT378, Lactococcus lactis CECT539, and Kluyveromyces marxianus CECT10584.

The degradation of natural phosphorylated compounds (galactose-1-phosphate, N-acetyl-glucosamine-1-phosphate, glycerophosphoethanolamine, and glycerophosphocholine) and added phosphorylated compounds (diphosphate) in milk was investigated by phosphorus 31 nuclear magnetic resonance on the incubation of a sterile milk with Pseudomonas fluorescens CECT381, Lactococcus lactis CECT539, and Kluyveromyces marxianus CECT10584. This preliminary study showed that the degradation of these compounds was dependent on the compound, microorganism, and temperature of incubation. K. marxianus CECT10584 did not show any capability to degrade these compounds, and L. lactis CECT539 was only able to degrade diphosphate at its optimum growth temperature. P. fluorescens CECT381 was the most active strain and possessed more hydrolytic capabilities at 10 degrees C than at its optimum growth temperature. It is suggested that cold-induced enzymes are involved in the ability of P. fluorescens CECT381 to hydrolyze the natural phosphorylated compounds in milk. Consequent potential alterations of dairy products are discussed.

Animals↗
PubMed1999

A comparative analysis of the citrate permease P mRNA stability in Lactococcus lactis biovar diacetylactis and Escherichia coli.

The role of ribonucleases in the control of gene expression remains unknown in lactic acid bacteria. In the present work, we analysed the expression of the citP gene, which encodes the lactococcal citrate permease P, through the stability of the citQRP messenger in both Lactococcus lactis biovar diacetylactis (L. diacetylactis) and Escherichia coli. The chemical half-life for citQRP mRNA observed in L. diacetylactis wild-type strain was abnormally long for bacteria. It was even longer than that detected in E. coli RNase E or RNase III mutant strains. A model of processing and fate of RNA species containing citP gene is presented.

Bacterial Proteins↗
PubMed1995

A system to generate chromosomal mutations in Lactococcus lactis which allows fast analysis of targeted genes.

A system for generating chromosomal insertions in lactococci is described. It is based on the conditional replication of lactococcal pWV01-derived Ori+ RepA- vector pORI19, containing lacZ alpha and the multiple cloning site of pUC19. Chromosomal AluI fragments of Lactococcus lactis were cloned in pORI19 in RepA+ helper strain Escherichia coli EC101. The frequency of Campbell-type recombinants, following introduction of this plasmid bank into L. lactis (RepA-), was increased by combining the system with temperature-sensitive pWV01 derivative pVE6007. Transformation of L. lactis MG1363 (pVE6007) with the pORI19 bank of lactococcal chromosomal fragments at the permissive temperature allowed replication of several copies of a recombinant plasmid from the bank within a cell because of the provision in trans of RepA-Ts from pVE6007. A temperature shift to 37 degrees C resulted in loss of pVE6007 and integration of the pORI19 derivatives at high frequencies. A bank of lactococcal mutants was made in this way and successfully screened for the presence of two mutations: one in the monocistronic 1.3-kb peptidoglycan hydrolase gene (acmA) and one in the hitherto uncharacterized maltose fermentation pathway. Reintroduction of pVE6007 into the Mal- mutant at 30 degrees C resulted in excision of the integrated plasmid and restoration of the ability of ferment maltose. The integration plasmid (pMAL) was rescued by using the isolated plasmid content of a restored Mal+ colony to transform E. coli EC101. Nucleotide sequencing of the 564-bp chromosomal fragment in pMAL revealed an internal part of an open reading frame of which the translated product showed significant homology with ATP-binding proteins MalK of E. coli, Salmonella typhimurium, and Enterobacter aerogenes and MsmK of Streptococcus mutans. This combined use of two types of conditional replicating pWV01-derived vectors represents a novel, powerful tool for chromosomal gene inactivation, targeting, cloning, and sequencing of the labelled gene.

Amino Acid Sequence↗
PubMed2003

Proteome analysis of the purine stimulon from Lactococcus lactis.

A comparative expression proteome analysis was carried out by analyzing differential expression patterns of pulse-labelled proteins on two-dimensional gels under standard conditions and during purine nucleotide starvation, followed by mass spectrometric identification of regulated proteins. Based upon the expression patterns, three stimulons could be identified in Lactococcus lactis subsp. cremoris. The Psu proteins (purine starvation up-regulated) had increased synthesis during purine depletion in a purine auxotroph. Among these proteins were enzymes of the purine biosynthesis pathways (PurE, PurS, PurM, PurL), and enzymes involved in the generation of C1 units (GlyA, Fhs). C1 units are primarily required for purine biosynthesis. Upon analysis of the nucleotide sequence preceding the structural genes for these proteins in the L. lactis IL1403 genome sequence showed that all contained PurBox-Pribnov box structures resembling the PurR activated promoters for the purDEK and purCSQLF operons. Most, and possibly all members of the Psu stimulon are thus members of the PurR regulon. Five Psu proteins could not be identified. The second stimulon, the Psd stimulon (purine starvation decreased), whose members are down-regulated during purine depletion, contained proteins related to protein synthesis (PpsB, EF-TS, trigger factor), or to GTPases (FtsZ, EF-TS); or are involved in energy metabolism (GapB, CcpA). No common regulatory elements could be found for members of this stimulon. Two Psd proteins escaped identification. The last, Dcu (decoynine up-regulated), stimulon contained proteins whose synthesis escaped the severe general depression during inhibition of the GMP synthetase by decoynine. This regulon was comprised of mostly glycolytic enzymes (fructose bisphosphate aldolase, enolase, pyruvate kinase) and translation elongation factors (GTPases: EF-TU, EF-G). Two Dcu proteins could not be identified. Out of 28 proteins subjected to mass spectrometry, 19 could be readily identified despite the fact that only the genome sequence of a strain of L. lactis subsp. lactis was available. The two subspecies share about 85% sequence identity, comparable to the genetic distance between Escherichia coli and Salmonella typhimurium. A success rate of 68% indicates that it may be feasible to perform proteomics based upon genomic sequences of relatives outside the genus.

Adenosine↗
PubMed2005

Sulfur amino acid metabolism and its control in Lactococcus lactis IL1403.

Cysteine and methionine availability influences many processes in the cell. In bacteria, transcription of the specific genes involved in the synthesis of these two amino acids is usually regulated by different mechanisms or regulators. Pathways for the synthesis of cysteine and methionine and their interconversion were experimentally determined for Lactococcus lactis, a lactic acid bacterium commonly found in food. A new gene, yhcE, was shown to be involved in methionine recycling to cysteine. Surprisingly, 18 genes, representing almost all genes of these pathways, are under the control of a LysR-type activator, FhuR, also named CmbR. DNA microarray experiments showed that FhuR targets are restricted to this set of 18 genes clustered in seven transcriptional units, while cysteine starvation modifies the transcription level of several other genes potentially involved in oxidoreduction processes. Purified FhuR binds a 13-bp box centered 46 to 53 bp upstream of the transcriptional starts from the seven regulated promoters, while a second box with the same consensus is present upstream of the first binding box, separated by 8 to 10 bp. O-Acetyl serine increases FhuR binding affinity to its binding boxes. The overall view of sulfur amino acid metabolism and its regulation in L. lactis indicates that CysE could be a master enzyme controlling the activity of FhuR by providing its effector, while other controls at the enzymatic level appear to be necessary to compensate the absence of differential regulation of the genes involved in the interconversion of methionine and cysteine and other biosynthesis genes.

Amino Acids↗
PubMed2002

Steady-state kinetics of the glutaminase reaction of CTP synthase from Lactococcus lactis. The role of the allosteric activator GTP incoupling between glutamine hydrolysis and CTP synthesis.

CTP synthase catalyzes the reaction glutamine + UTP + ATP --> glutamate + CTP + ADP + Pi. The rate of the reaction is greatly enhanced by the allosteric activator GTP. We have studied the glutaminase half-reaction of CTP synthase from Lactococcus lactis and its response to the allosteric activator GTP and nucleotides that bind to the active site. In contrast to what has been found for the Escherichia coli enzyme, GTP activation of the L. lactis enzyme did not result in similar kcat values for the glutaminase activity and glutamine hydrolysis coupled to CTP synthesis. GTP activation of the glutaminase reaction never reached the levels of GTP-activated CTP synthesis, not even when the active site was saturated with UTP and the nonhydrolyzeable ATP-binding analog adenosine 5'-[gamma-thio]triphosphate. Furthermore, under conditions where the rate of glutamine hydrolysis exceeded that of CTP synthesis, GTP would stimulate CTP synthesis. These results indicate that the L. lactis enzyme differs significantly from the E. coli enzyme. For the E. coli enzyme, activation by GTP was found to stimulate glutamine hydrolysis and CTP synthesis to the same extent, suggesting that the major function of GTP binding is to activate the chemical steps of glutamine hydrolysis. An alternative mechanism for the action of GTP on L. lactis CTP synthase is suggested. Here the binding of GTP to the allosteric site promotes coordination of the phosphorylation of UTP and hydrolysis of glutamine for optimal efficiency in CTP synthesis rather than just acting to increase the rate of glutamine hydrolysis itself.

Allosteric Regulation↗
PubMed1999

ClpP participates in the degradation of misfolded protein in Lactococcus lactis.

ClpP proteins constitute a family of homologous proteins found in both prokaryotic and eukaryotic organisms. In Escherichia coli, ClpP is the proteolytic component of a large complex also containing either the ClpA or the ClpX ATPases. We show here that the clpP gene from the Gram-positive bacterium Lactococcus lactis encodes a 22-kDa protein that is induced by low pH and by the t-RNA analogue puromycin, which interferes with translation, resulting in the production of misfolded puromycyl-containing peptides. Northern blot and primer extension analysis showed that clpP expression is also induced by heat shock and that stress induction occurs at the transcriptional level independent of the CIRCE regulatory element often implicated in stress regulation in Gram-positive bacteria. When we disrupted the L. lactis clpP gene by insertional inactivation, the resulting mutant was more sensitive to both heat and puromycin than wild-type cells. Furthermore, cells lacking ClpP had a reduced ability to degrade puromycyl-containing peptides, and they synthesized heat shock proteins constitutively in the absence of stress. Thus, our data suggest that ClpP plays a major role in the degradation of misfolded proteins.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1999

Cloning, DNA sequence analysis, and deletion of a gene encoding diacetyl-acetoin reductase from Lactococcus lactis.

Diacetyl is produced by strains of lactic acid bacteria used in the dairy industry. Production of this important flavour compound could be increased by genetic manipulation of genes encoding enzymes involved in diacetyl metabolism. This paper reports the cloning and sequencing of the gene (dar) encoding diacetyl-acetoin reductase from Lactococcus lactis. Analysis of the DNA sequence of the dar gene and surrounding area revealed the presence of a putative operon with similarity to the family of ABC transporter systems. The dar gene has been deleted from the chromosome by double cross-over homologous recombination.

Alcohol Oxidoreductases↗
PubMed1999

PCR amplification of the gene acmA differentiates Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris.

The occurrence of the acmA gene, encoding the lactococcal N-acetylmuramidase in new lactococcal isolates from raw milk cheeses, has been determined. Isolates were genotypically identified to the subspecies level with a PCR technique. On the basis of PCR amplification of the acmA gene, the presence or absence of an additional amplicon of approximately 700 bp correlated with Lactococcus lactis subspecies. L. lactis subsp. lactis exhibits both the expected 1,131-bp product and the additional amplicon, whereas L. lactis subsp. cremoris exhibits a single 1,131-bp fragment.

Animals↗
PubMed1997

A highly efficient and stable system for site-specific integration of genes and plasmids into the phage phiLC3 attachment site (attB) of the Lactococcus lactis chromosome.

An integration vector system based on the site-specific integration apparatus of the temperate lactococcal bacteriophage phiLC3 was developed. A 1.6-kb recombinogenic DNA cassette, containing the phiLC3 integrase gene (int) and the phage attachment site (attP), mediated site-specific integration of a single marker-gene, as well as of a replication-thermosensitive (-ts) plasmid (pINT2), into the phiLC3 attB site of Lactococcus lactis subsp. lactis LM0230 chromosome after introduction of the DNA into the cells by electroporation. Both the marker gene and the pINT2 plasmid were stably inserted as single copies in an orientation-specific and integrase-dependent manner, the pINT2-ts replicon being stably maintained at temperatures both permissive and non-permissive for plasmid-directed replication. Essentially all transformants obtained with the pINT2 plasmid appeared to be integrants, demonstrating the remarkably high efficiency of the system. This high efficiency rendered possible the detection of transformation-plus-integration events using DNA directly obtained from ligase reaction mixtures, thus avoiding initial subcloning in a non-lactococcal strain and subsequent cointegration of foreign replication functions into the chromosome of L. lactis. The above results, the observation that the phiLC3 attB site appear to be conserved in L. lactis, and the fact that the int-attP cassette functions efficiently in a non-phiLC3-host strain, show that the phiLC3 site-specific integration apparatus provides an efficient and 'food grade' tool for stable integration of genetic elements into the chromosome of L. lactis.

Bacteriophages↗
PubMed2002

Two different pathways for D-xylose metabolism and the effect of xylose concentration on the yield coefficient of L-lactate in mixed-acid fermentation by the lactic acid bacterium Lactococcus lactis IO-1.

In lactic acid bacteria, pentoses are metabolized via the phosphoketolase pathway, which catalyzes the cleavage of D-xylulose-5-phosphate to equimolar amounts of glyceraldehyde 3-phosphate and acetylphosphate. Hence the yield coefficient of lactate from pentose does not exceed 1.0 mol/mol, while that of Lactococcus lactis IO-1(JCM7638) at high D-xylose concentrations often exceeds the theoretical value. This suggests that, in addition to the phosphoketolase pathway, L. lactisIO-1 may possess another metabolic pathway that produces only lactic acid from xylose. In the present study, the metabolism of xylose in L. lactisIO-1 was deduced from the product formation and enzyme activities of L. lactisIO-1 in batch culture and continuous culture. During cultivation with xylose concentrations above ca. 50 g/l, the yield coefficient of L-lactate exceeded 1.0 mol/mol while those of acetate, formate and ethanol were very low. At xylose concentrations less than 5 g/l, acetate, formate and ethanol were produced with yield coefficients of about 1.0 mol/mol, while L-lactate was scarcely produced. In cells grown at high xylose concentrations, a marked decrease in the specific activities of phosphoketolase and pyruvate formate lyase (PFL), and an increase in those of transketolase and transaldolase were observed. These results indicate that in L. lactisIO-1 xylose may be catabolized by two different pathways, the phosphoketolase pathway yielding acetate, formate and ethanol, and the pentose phosphate (PP)/glycolytic pathway which converts xylose to L-lactate only. Furthermore, it was deduced that the change in the xylose concentration in the culture medium shifts xylulose 5-phosphate metabolism between the phosphoketolase pathway and the PP/glycolytic pathway in L. lactisIO-1, and pyruvate metabolism between cleavage to acetyl-CoA and formic acid by PFL and the reduction to L-lactate by lactate dehydrogenase.

Enzymes↗