Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Lactococcus”

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 451 records · Page 25Linked to original sources
PubMed1998

A leucine repeat motif in AbiA is required for resistance of Lactococcus lactis to phages representing three species.

The abiA gene encodes an abortive bacteriophage infection mechanism that can protect Lactococcus species from infection by a variety of bacteriophages including three unrelated phage species. Five heptad leucine repeats suggestive of a leucine zipper motif were identified between residues 232 and 266 in the predicted amino acid sequence of the AbiA protein. The biological role of residues in the repeats was investigated by incorporating amino acid substitutions via site-directed mutagenesis. Each mutant was tested for phage resistance against three phages, phi 31, sk1, and c2, belonging to species P335, 936, and c2, respectively. The five residues that comprise the heptad repeats were designated L234, L242, A249, L256, and L263. Three single conservative mutations of leucine to valine in positions L235, L242, and L263 and a double mutation of two leucines (L235 and L242) to valines did not affect AbiA activity on any phages tested. Non-conservative single substitutions of charged amino acids for three of the leucines (L235, L242, and L256) virtually eliminated AbiA activity on all phages tested. Substitution of the alanine residue in the third repeat (A249) with a charged residue did not affect AbiA activity. Replacement of L242 with an alanine elimination phage resistance against phi 31, but partial resistance to sk1 and c2 remained. Two single proline substitutions for leucines L242 and L263 virtually eliminated AbiA activity against all phages, indicating that the predicted alpha-helical structure of this region is important. Mutations in an adjacent region of basic amino acids had various effects on phage resistance, suggesting that these basic residues are also important for AbiA activity. This directed mutagenesis analysis of AbiA indicated that the leucine repeat structure is essential for conferring phage resistance against three species of lactococcal bacteriophages.

Amino Acid Sequence↗
PubMed2000

The pyrH gene of Lactococcus lactis subsp. cremoris encoding UMP kinase is transcribed as part of an operon including the frr1 gene encoding ribosomal recycling factor 1.

The pyrH gene of Lactococcus lactis subsp. cremoris MG1363, encoding UMP kinase, has been sequenced and cloned. It encodes a polypeptide of 239 amino acid residues (deduced molecular weight of 25951), which was shown to complement a temperature sensitive pyrH mutation in Escherichia coli, thus establishing the ability of the encoded protein to synthesize UDP. The pyrH gene in L. lactis is flanked downstream by frr1 encoding ribosomal recycling factor 1 and upstream by an open reading frame, orfA, of unknown function. The three genes were shown to constitute an operon transcribed in the direction orfA-pyrH-frr1 from a promoter immediately in front of orfA. This operon belongs to an evolutionary highly conserved gene cluster, since the organization of pyrH on the chromosomal level in L. lactis shows a high resemblance to that found in Bacillus subtilis as well as in Escherichia coli and several other prokaryotes

Amino Acid Sequence↗
PubMed2000

The development of TnNuc and its use for the isolation of novel secretion signals in Lactococcus lactis.

We have previously used Tn917 for the identification and characterization of regulated promoters from Lactococcus lactis [Israelsen et al., Appl. Environ. Microbiol. 61 (1995) 2540-2547]. We describe here the construction of a new Tn917-transposon derivative, termed TnNuc, which includes the Staphylococcus aureus nuclease gene (nuc) as a reporter for secretion. Transposition of TnNuc into the L. lactis chromosome allows the generation of fusions in-frame with the nuc gene. TnNuc includes also lacZ, a reporter used for identification of relevant clones from the library, i.e. clones with Lac+ phenotype result from transposition of TnNuc into a functional gene on the L. lactis chromosome. The presence of a functional signal sequence at the upstream flanking region of the left repeat of the transposed element results in the detection of nuclease activity using a sensitive plate assay. TnNuc was used for the identification of novel secretion signals from L. lactis. The sequences identified included known and unknown lactococcal-secreted proteins containing either a signal peptidase-I or -II recognition sequence. In one case, the gene identified codes for a transmembrane protein. The sequences identified were used to study functionality when located in a plasmid under the control of the pH and growth phase-dependent promoter P170 [Madsen et al., Mol. Microbiol. 32 (1999) 75-87]. In all cases, concurrent secretion of nuclease was observed during induction of P170 in a fermentor.

Bacterial Proteins↗
PubMed2003

Antigen recognition by rainbow trout (Oncorhynchus mykiss) of whole cell proteins expressed by Lactococcus garvieae when obtained directly from fish and under iron limited culture conditions.

Infection by Lactococcus garvieae has become a widely recognised problem associated with intensively cultured fish. Long-term control of fish infections may be possible by vaccination providing a suitable and efficacious epitope is expressed during production of cells used for vaccine preparation. The identification of novel vaccine candidates must, therefore, consider how the host species recognises and responds to bacterial cell components. L. garvieae was cultured in iron deficient, limited and haem iron enriched media and the whole cell proteins expressed under these conditions were compared with those expressed in bacteria extracted with Percoll gradients directly from spleen tissue of infected rainbow trout (Oncorhynchus mykiss). SDS-PAGE of the cell proteins showed the existence of several different electropherotypes according to the iron status of the culture media. Only minor differences in cell protein profile were detected in bacteria obtained directly from fish spleens, but when the electropherograms were analysed by Western blots using L. garvieae hyperimmune fish sera, several proteins could be identified that were expressed only when L. garvieae was growing in vivo. Siderophore could be detected in culture supernatant of iron deficient, limited and haem iron enriched media but not in media with higher nutrient concentrations. The siderophore could not be identified as a type of catechol or hydroxymate. Rainbow trout recognise proteins in the range of approximately 50-80 kDa for bacterial cells obtained without subculture from infected fish and culture conditions can influence protein profiles for this pathogen.

Animals↗
PubMed1999

Induction, characterisation and pathogenicity in rainbow trout Oncorhynchus mykiss (Walbaum) of Lactococcus garvieae L-forms.

Difficulties with induction and cultivation of L-forms, particularly those derived from Gram positive parent cells, have constrained to some degree the ability to evaluate the pathogenicity of these morphotypes. Induction of L-forms of Lactococcus garvieae was undertaken using either charcoal or inactivated horse serum media supplemented with ampicillin, benzylpenicillin or erythromycin, the drug of choice for treatment of infections in rainbow trout, Oncorhynchus mykiss, (Walbaum), and NaCl as an osmotic stabiliser. Lysozyme treated cells could be cultured in a cell wall deficient state using media consisting of charcoal, NaCl and either ampicillin or benzylpenicillin. The influence of some amino acids for induction of L-forms was assessed by disc diffusion and combined interaction. Analysis of variance of colony counts indicated that the amino acids glycine, DL-methionine, L-threonine and L-serine (P<0.03), and the presence of charcoal were beneficial and that inactivated horse serum was detrimental to L-form development. Electron microscopy revealed that the cell wall of L-forms was missing and this cell had a greatly expanded volume compared to parent cells. Electrophoresis of whole cell proteins showed some variation of electropherotype between parent and L-form cells. L-forms expressed greater quantities of proteins with molecular mass of 36 and 66 kDa and parent cells contained greater quantities of proteins of molecular mass 29, 43 and 60 kDa. Additional proteins of molecular mass 32, 44 and 53 kDa were present in L-form extracts, and in parent cells of 34, 38, 40, 42, 85 and 123 kDa which may represent cell wall associated proteins or alterations in expression due to different growth rates. Intraperitoneal challenge of rainbow trout with L-forms failed to produce overt infection even in immune-suppressed fish, but L-forms were shown by indirect fluorescent antibody test to remain inkidney tissue. Fish were susceptible to infection when challenged with parent cells of L. garvieae.

Animals↗
PubMed1998

Lactococcus garvieae endocarditis: report of a case and review of the literature.

Bacterial endocarditis due to Lactococcus garvieae is extremely unusual, and may actually be underreported due to its morphologic and biochemical similarities with enterococci. Only three cases have been reported in the medical literature, and all involved prosthetic valves. We report a case of native valve bacterial endocarditis caused by L. garvieae.

Aged↗
PubMed2001

Distinctive features of homologous recombination in an 'old' microorganism, Lactococcus lactis.

Homologous recombination is needed to assure faithful inheritance of DNA material, especially under stress conditions. The same enzymes that repair broken chromosomes via recombination also generate biodiversity. Their activities may result in intrachromosomal rearrangements, assimilation of foreign DNA, or a combination of these events. It is generally supposed that homologous recombination systems are conserved, and function the same way everywhere as they do in Escherichia coli, the accepted paradigm. Studies in an 'older' microorganism, the gram-positive bacterium of the low GC branch Lactococcus lactis, confirm that many enzymes are conserved across species lines. However, the main components of the double strand break (DSB) repair system, an exonuclease/helicase (Exo/hel) and a short DNA modulator sequence Chi, differ markedly between bacteria, especially when compared to the gram-negative analogues. Based on our studies, a model is proposed for the functioning of the two-subunit Exo/hel of L. lactis and other gram-positive bacteria, which differs from that of the three-subunit E. coli enzyme. The differences between bacterial DSB repair systems may underlie a selection for diversity when dealing with DSB. These and other features of homologous recombination in L. lactis are discussed.

Bacteriophages↗
PubMed2002

The structural basis for catalysis and specificity of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis.

The X-prolyl dipeptidyl aminopeptidase (X-PDAP) from Lactococcus lactis is a dimeric enzyme catalyzing the removal of Xaa-Pro dipeptides from the N terminus of peptides. The structure of the enzyme was solved at 2.2 A resolution and provides a model for the peptidase family S15. Each monomer is composed of four domains. The larger one presents an alpha/beta hydrolase fold and comprises the active site serine. The specificity pocket is mainly built by residues from a small helical domain which is, together with the N-terminal domain, essential for dimerization. A C-terminal moiety probably plays a role in the tropism of X-PDAP toward the cellular membrane. These results give new insights for further exploration of the role of the enzymes of the SC clan.

Amino Acid Sequence↗
PubMed1997

The crystal structure of the flavin containing enzyme dihydroorotate dehydrogenase A from Lactococcus lactis.

BACKGROUND: . Dihydroorotate dehydrogenase (DHOD) is a flavin mononucleotide containing enzyme, which catalyzes the oxidation of (S)-dihydroorotate to orotate, the fourth step in the de novo biosynthesis of pyrimidine nucleotides. Lactococcus lactis contains two genes encoding different functional DHODs whose sequences are only 30% identical. One of these enzymes, DHODA, is a highly efficient dimer, while the other, DHODB, shows optimal activity only in the presence of an iron-sulphur cluster containing protein with which it forms a complex tetramer. Sequence alignments have identified three different families among the DHODs: the two L. lactis enzymes belong to two of the families, whereas the enzyme from E. coli is a representative of the third. As no three-dimensional structures of DHODs are currently available, we set out to determine the crystal structure of DHODA from L. lactis. The differences between the two L. lactis enzymes make them particularly interesting for studying flavoprotein redox reactions and for identifying the differences between the enzyme families. RESULTS: . The crystal structure of DHODA has been determined to 2.0 resolution. The enzyme is a dimer of two crystallographically independent molecules related by a non-crystallographic twofold axis. The protein folds into and alpha/beta barrel with the flavin molecule sitting between the top of the barrel and a subdomain formed by several barrel inserts. Above the flavin isoalloxazine ring there is a small water filled cavity, completely buried beneath the protein surface and surrounded by many conserved residues. This cavity is proposed as the substrate-binding site. CONCLUSIONS: . The crystal structure has allowed the function of many of the conserved residues in DHODs to be identified: many of these are associated with binding the flavin group. Important differences were identified in some of the active-site residues which vary across the distinct DHOD families, implying significant mechanistic differences. The substrate cavity, although buried, is located beneath a highly conserved loop which is much less ordered than the rest of the protein and may be important in giving access to the cavity. The location of the conserved residues surrounding this cavity suggests the potential orientation of the substrate.

Amino Acid Sequence↗
PubMed1997

The structure of enzyme IIAlactose from Lactococcus lactis reveals a new fold and points to possible interactions of a multicomponent system.

BACKGROUND: The bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS) is responsible for the binding, transmembrane transport and phosphorylation of numerous sugar substrates. The system is also involved in the regulation of a variety of metabolic and transcriptional processes. The PTS consists of two non-specific energy coupling components, enzyme I and a heat stable phosphocarrier protein (HPr), as well as several sugar-specific multiprotein permeases known as enzymes II. In most cases, enzymes IIA and IIB are located in the cytoplasm, while enzyme IIC acts as a membrane channel. Enzyme IIAlactose belongs to the lactose/cellobiose-specific family of enzymes II, one of four functionally and structurally distinct groups. The protein, which normally functions as a trimer, is believed to separate into its subunits after phosphorylation. RESULTS: The crystal structure of the trimeric enzyme IIAlactose from Lactococcus lactis has been determined at 2.3 A resolution. The subunits of the enzyme, related to each other by the inherent threefold rotational symmetry, possess interesting structural features such as coiled-coil-like packing and a methionine cluster. The subunits each comprise three helices (I, II and III) and pack against each other forming a nine-helix bundle. This helical bundle is stabilized by a centrally located metal ion and also encloses a hydrophobic cavity. The three phosphorylation sites (His78 on each monomer) are located in helices III and their sidechains protrude into a large groove between helices I and II of the neighbouring subunits. A model of the complex between phosphorylated HPr and enzyme IIAlactose has been constructed. CONCLUSIONS: Enzyme IIAlactose is the first representative of the family of lactose/cellobiose-specific enzymes IIA for which a three-dimensional structure has been determined. Some of its structural features, like the presence of two histidine residues at the active site, seem to be common to all enzymes no overall structural homology is observed to any PTS proteins or to any other proteins in the Protein Data Bank. Enzyme IIAlactose shows surface complementarity to the phosphorylated form of HPr and several energetically favourable interactions between the two molecules can be predicted.

Amino Acid Sequence↗
PubMed2004

Role of capsule in serotypic differences and complement fixation by Lactococcus garvieae.

Seventeen geographically distinct isolates of Lactococcus garvieae, isolated from diseased fish, were compared serologically using antiserum raised against the various isolates in rainbow trout. Sera raised against a capsule deficient isolate did not agglutinate capsulated isolates, regardless of origin. In contrast, all antisera raised against capsulated isolates cross reacted strongly with non-capsulated isolates. Antisera raised against capsulated Japanese isolates cross reacted with other capsulated Japanese isolates including isolates from geographically distinct prefectures within Japan (Ehime and Oita). However, antisera against these virulent capsulated isolates did not cross react with European capsulated isolates. Antisera raised against European capsulated isolates cross reacted with other European isolates, regardless of origin within Europe (UK, Italy, Spain), but did not cross-react with Japanese capsulated isolates. Agglutination assays performed with a range of fifteen lectins revealed differences in surface carbohydrate structure: capsule deficient isolates agglutinated with concanavalin A, Ricinis communis agglutinin, Pisum sativum agglutinin, Lens culinaris agglutinin, wheat germ agglutinin and succinylated wheat germ agglutinin. European capsulated isolates agglutinated with concanavalin A only. The Japanese capsulated isolates were not agglutinated by any of the lectins used in this study. Representative isolates from each group (Japanese capsulated and non-capsulated, European capsulated and non-capsulated) were investigated for their ability to fix complement. Non-capsulated isolates fixed complement regardless of origin, and antibody did not markedly enhance complement fixation. In contrast, the capsulated isolates were less efficient at fixing complement, but complement fixation was markedly increased by homologous antibody.

Agglutination Tests↗
PubMed2003

Food-grade expression of human glutathione S-transferase and Cu/Zn superoxide dismutase in Lactococcus lactis.

A food-grade gene expression system in Lactococcus lactis was established by the combination of a vector containing the lacF gene as the selection marker and a strain WZ103 carrying an in-frame deletion of this gene in the chromosome as the host. The human glutathione S-transferase A1-1 (hGSTA1) and Cu/Zn superoxide dismutase (hSOD) genes were respectively cloned into a food-grade vector under the control of the lactococcal inducible promoter P(lacA). The resulting expression plasmids were separately introduced into the lactose-deficient (Lac(-)) host, and the lactose-utilizing (Lac(+)) transformants were directly selected on a chemically defined medium, using lactose as the sole carbon source. The successful food-grade expression of hGSTA1 and hSOD in the L. lactis WZ103 transformed with these plasmids were analyzed by Western blotting and enzymatic activity assay, respectively.

Cloning, Molecular↗
PubMed2002

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

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

Borohydrides↗
PubMed2000

Accumulation of casein-derived peptides during growth of proteinase-positive strains of Lactococcus lactis in milk: their contribution to subsequent bacterial growth is impaired by their internal transport.

To explain the limited nutritional value of milk cultured with proteinase-positive (Prt+) strains of Lactococcus lactis for the subsequent growth of dairy lactococci, we investigated further the time courses of modifications in the free amino acid and peptide contents of cultured milk. When growing in milk for up to 24 h, Prt+ strains of Lc. lactis progressively accumulated amino acids and casein-derived peptides. The growth of proteinase-negative (Prt-) wild-type strains and peptide transport mutants of Lc. lactis in cultured milk showed that casein-derived peptides could sustain growth up to 5 x 10(8) cfu/ml, depending on the extent of casein degradation during the preliminary growth of Prt+ strains and the Prt- strains. Of the casein-derived oligopeptides, < 25% were transported into the cell and used for Lc. lactis growth. However, they played a prominent role, contributing 90% to growth. In contrast, di- and tripeptides did not contribute to growth, suggesting that either few were released from caseins or they did not supply essential amino acids.

Amino Acids↗
PubMed2000

Metabolism of lactose and citrate by mutants of Lactococcus lactis producing excess carbon dioxide.

Mutants of Lactococcus lactis producing excess carbon dioxide could be isolated on LDHA-20 agar (described by El Attar et al. Journal of Dairy Research 67 641-646 2000). The use of these mutants in the manufacture of Roquefort cheese has the potential to improve the formation of openings in this cheese. The aim of this work was to examine the stability of these mutants, their enzymic activities and their metabolism of lactose and citrate during growth in milk. They produced less L-lactate than the parent strain and their lactate dehydrogenase activity was lower. Nevertheless none of the mutants produced no L-lactate at all and the most active gas generators among them generally produced 30-50 mM-L-lactate. Unexpectedly, all the strains produced some D-lactate, some > 10 mM. We found that carbon dioxide production by the mutants could be determined indirectly by assaying acetoin, citrate and 2,3-butanediol by high-performance liquid chromatography. Generally, spontaneous mutants were more stable than those obtained after treating with nitrosoguanidine or u.v. irradiation.

Acetates↗
PubMed2000

Varying influence of the autolysin, N-acetyl muramidase, and the cell envelope proteinase on the rate of autolysis of six commercial Lactococcus lactis cheese starter bacteria grown in milk.

The autolysin, N-acetyl muramidase (AcmA), of six commercial Lactococcus lactis subsp. cremoris starter strains and eight Lc. lactis subsp. cremoris derivatives or plasmid-free strains was shown by renaturing SDS-PAGE (zymogram analysis) to be degraded by the cell envelope proteinase (lactocepin; EC 3.4.21.96) after growth of strains in milk at 30 degrees C for 72 h. Degradation of AcmA was less in starter strains and derivatives producing lactocepin I/III (intermediate specificity) than in strains producing lactocepin I. This supports previous observations on AcmA degradation in derivatives of the laboratory strain Lc. lactis subsp. cremoris MG1363 (Buist et al. Journal of Bacteriology 180 5947-5953 1998). In contrast to the MG1363 derivatives, however, the extent of autolysis in milk of the commercial Lc. lactis subsp. cremoris starter strains in this study did not always correlate with lactocepin specificity and AcmA degradation. The distribution of autolysins within the cell envelope of Lc. lactis subsp. cremoris starter strains and derivatives harvested during growth in milk was compared by zymogram analysis. AcmA was found associated with cell membranes as well as cell walls and some cleavage of AcmA occurred independently of lactocepin activity. An AcmA product intermediate in size between precursor (46 kDa) and mature (41 kDa) forms of AcmA was clearly visible on zymograms, even in the absence of lactocepin I activity. These results show that autolysis of commercial Lc. lactis subsp. cremoris starter strains is not primarily determined by AcmA activity in relation to lactocepin specificity and that proteolytic cleavage of AcmA in vivo is not fully defined.

Animals↗
PubMed1995

Immunological and electrophoretic study of the proteolytic enzymes from various Lactococcus and Lactobacillus strains.

Cell extracts of various lactobacilli and two Lactococcus strains were investigated for their immunoresponse with monoclonal and polyclonal antibodies raised against various proteolytic enzymes from Lc. lactis. Except for Lactobacillus casei SBT 2233, none of the lactobacilli proteins showed immunoresponse with the monoclonal antibodies. With polyclonal antibodies raised against aminopeptidases N and C and endopeptidase of Lc. lactis an immunoresponse was observed. However, the molecular masses of the reactive bands on the blot were considerably different from those of the corresponding lactococcal peptidases, except for the band that reacted with polyclonal antibodies against aminopeptidase C. The polyclonal antibodies raised against X-prolyl-dipeptidyl aminopeptidase and tripeptidase did not show any immunoreaction. As a control, all antibodies reacted with the lactococcal proteins on the blot, with molecular masses corresponding to those reported for the proteinases and peptidases. The results clearly showed that most of the proteolytic enzymes of lactobacilli were immunologically different from those of lactococci. The proteolytic enzymes in the cell-free extracts were separated by non-denaturing PAGE and visualized by zymogram staining. The electrophoretic pattern of the proteolytic enzymes of lactobacilli was different from that of Lc. lactis. Both experiments indicate that the enzymes of the proteolytic system of lactobacilli are different from those of lactococci.

Antibodies, Monoclonal↗