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PubMed1999

Uncoupling of growth and exopolysaccharide production by Lactococcus lactis subsp. cremoris NIZO B40 and optimization of its synthesis.

Exopolysaccharide (EPS) production by Lactococcus lactis subsp. cremoris NIZO B40 was found to be most efficient with glucose as a substrate. The optimal temperature and pH for EPS synthesis were 25 degrees C and pH 5.8, respectively. EPS production could not be identified as a stress response: increased oxygen tension and reduced water activity negatively affected both growth and EPS synthesis. It is often assumed that there is a competition between growth and EPS formation. Within the range of 0.5 and 0.1 h(-1), reducing the growth rate resulted indeed in an increase of the specific EPS production but the polymer formation decreased again at even lower growth rates. Most of the applied fermentation conditions influenced both growth and EPS formation. As the growth rate itself also influenced EPS formation, we studied the linking between growth and EPS synthesis. Interestingly, EPS production was not strictly coupled to growth. Significant de novo synthesis of EPS was observed in non-growing cultures. Consequently, the influence of different culture conditions on EPS production could be studied independent of growth.

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PubMed2000

The production of mixed cultures containing strains of Lactococcus lactis, Leuconostoc cremoris and Lactobacillus rhamnosus, on commercial starter media.

Mixed starters containing Lactococcus lactis, Leuconostoc cremoris and Lactobacillus rhamnosus strains were produced on commercial starter media (MB Complete, Thermolac, Marlac), as well as on milk. With the exception of Marlac, the starters were cultured under pH control. The effect of media and incubation temperature (22 or 32 degrees C) on population ratios, on specific acidifying activities (SAA) of the cultures as well as on their ability to produce aroma compounds in milk was studied. The starters had higher contents in lactobacilli when they were produced at 32 degrees C, whereas a tendency to obtain higher Leuconostoc populations was observed at 22 degrees C. With respect to the lactococci, there was a significant interaction between temperature and growth medium for both strains. Thus, Le. cremoris T2 reached higher populations at 32 degrees C if grown in MB complete and Thermolac, whereas in Marlac and skim milk, viable counts were higher at 22 degrees C. The lactococci represented 50% of the total population of the culture at the beginning of the incubation, but they composed between 80% and 99% of the total population following fermentation. The best medium for growth of Leuconostoc was milk, but populations of only 10(8) cfu/ml were reached. The lactobacilli did not grow well in MB Complete, and their development was best in the low-phosphate Marlac medium. The cultures grown on Marlac had the highest SAA values, whereas those grown on MB complete had the lowest. Overall, more ethanol and diacetyl were detected in the fermented milks when the starters used to inoculate them were produced at 22 degrees C.

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PubMed2002

Monitoring transfer of recombinant and nonrecombinant plasmids between Lactococcus lactis strains and members of the human gastrointestinal microbiota in vivo--impact of donor cell number and diet.

AIMS: The generation of data of real relevance to the purported risks of DNA transfer from food-borne genetically modified microorganisms (GMMOs) using the human biota associated (HBA) rat model. Plasmid transfer between Lactococcus lactis strains and between donor strains and human gut bacteria was monitored. METHODS AND RESULTS: Transfer of the recombinant plasmid pCK1 and/or the promiscuous nonrecombinant plasmid pAMbeta1 between L. lactis strains was monitored in vivo in HBA rats. No transfer of pCK1 was observed. Transfer of pAMbeta1 was observed to Enterococcus spp. present in the HBA rats. Transconjugants persisted for 30 d and were distributed throughout the gastrointestinal tract. Both HBA rat diet and donor cell numbers impacted on transconjugant numbers. Fewer transconjugants were observed in animals fed a high-fat human type diet, while high levels of plasmid transfer were only observed at doses of donor L. lactis greater than 109 cfu. CONCLUSIONS: The utility of models of the human gut in monitoring DNA transfer events within the gut microbiota was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: Such findings give some confidence for the use of GMMOs with recombinant DNA borne on nonconjugative elements in fermented foods. HBA rats are a suitable model for monitoring the fate of food-borne GMMOs.

Animals↗

PubMed2000

Citrate can partially replace carbon dioxide required for growth of Lactococcus lactis subsp. lactis biovar diacetylactis.

Lactococcus lactis subsp. lactis biovar diacetylactis was grown as batch cultures on a chemically defined medium. No growth was observed when the cultures were sparged with pure nitrogen (1.3 l l-1 min-1) whereas the cultures displayed exponential growth in the presence of minute amounts of carbon dioxide (0.035 mol-% of the inlet gas). However, in the former case, the addition of citrate restored growth. This suggested that oxaloacetate required for aspartate biosynthesis can be formed by the carboxylation of pyruvate or by citrate catabolism. When the cultures were heavily sparged with nitrogen (2.6 l l-1 min-1), no growth was observed even in the presence of citrate. This indicated that growth in these conditions was repressed by the absence of carbon dioxide required in some other biosynthetic reaction than in the carboxylation of pyruvate leading to oxaloacetate/aspartate biosynthesis.

Carbon Dioxide↗

PubMed2000

An ABC-type multidrug transporter of Lactococcus lactis possesses an exceptionally broad substrate specificity.

LmrA is a 590-amino acid membrane protein which confers multidrug resistance on Lactococcus lactis cells by extruding amphiphilic compounds from the inner leaflet of the cytoplasmic membrane at the expense of ATP hydrolysis. Its structural and functional characteristics place it in the P-glycoprotein cluster of the ATP-binding cassette transporter superfamily, making it the first prokaryotic multidrug transporter of this cluster. The number of compounds recognized and transported by LmrA is remarkably vast and includes many lipophilic cations as well as a record of eight classes of clinically relevant broad-spectrum antibiotics. Homologs of LmrA have been found in pathogenic bacteria, suggesting that these putative efflux pumps may play a crucial role in antibiotic resistance of human pathogens. Recent evidence indicates that LmrA is functional as a homodimer, consistent with the overall structure of P-glycoprotein, and mediates drug transport by an alternating two-site transport mechanism. Copyright 2000 Harcourt Publishers Ltd.

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PubMed1991

Engineering of the Lactococcus lactis serine proteinase by construction of hybrid enzymes.

Plasmids containing wild-type and hybrid proteinase genes were constructed from DNA fragments of the prtP genes of Lactococcus lactis strains Wg2 and SK11. These plasmids were introduced into the plasmid-free strain L. lactis MG1363. The serine proteinases produced by these L. lactis strains were isolated, and their cleavage specificity and rate towards alpha s1- and beta-casein was investigated. The catalytic properties of both the SK11 and Wg2 proteinases, which differ in 44 out of 1902 amino acid residues, could be changed dramatically by the reciprocal exchange of specific fragments between the two enzymes. As a result, various L. lactis strains were constructed having new proteolytic properties that differ from those of the parental strains. Furthermore, two segments in the proteinase could be identified that contribute significantly to the cleavage specificity towards casein; within these two segments, several amino acid residues were identified that are important for substrate cleavage rate and specificity. The results also indicate that the lactococcal proteinase has an additional domain involved in substrate binding compared with the related subtilisins. This suggests that the 200 kd L. lactis proteinase may be the representative of a new subclass of subtilisin-like enzymes.

Amino Acid Sequence↗

PubMed1996

Crystallization and preliminary structural studies of lactose-specific enzyme IIA from Lactococcus lactis.

Lactose-specific enzyme IIA of the phosphoenol:pyruvate-dependent sugar phosphotransferase system from Lactococcus lactis has been crystallized in phosphate buffer. The crystals belong to space group P4(1)2(1)2 or its enantiomorph P4(3)2(1)2 with unit-cell axes a = b = 90.9 and c = 82.4 A. The packing parameter (Matthews parameter) V(m) of 2.48 A(3) Da(-1) is consistent with one trimer per asymmetric unit and non-crystallographic threefold symmetry has been confirmed by calculating a self-rotation function. The crystals diffract X-rays to at least 2.3 A resolution, are stable in an X-ray beam and are therefore appropriate for structure determination. Native data to 2.3 A resolution have been collected using a MAR image-plate system at a synchrotron source. One isomorphous heavy-atom derivative has been identified and the presence of an isomorphous signal in the data has been confirmed by Patterson methods.

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PubMed1997

Crystallization and preliminary X-ray diffraction analysis of the heterotetrameric dihydroorotate dehydrogenase B of Lactococcus lactis, a flavoprotein enzyme system consisting of two PyrDB subunits and two iron-sulfur cluster containing PyrK subunits.

Dihydroorotate dehydrogenases are flavin-containing enzymes which catalyze the conversion of (S)-dihydroorotate to orotate. Dihydroorotate dehydrogenase B (DHODB) from Lactococcus lactis is a heterotetramer containing two subunits of the protein encoded by the pyrDb gene (PyrDB) and two subunits of the protein encoded by the pyrK gene (PyrK). In addition, DHODB contains two molecules of flavin mononucleotide, two molecules of flavin adenine dinucleotide and two [2Fe-2S] iron-sulfur clusters as tightly bound cofactors. Yellow crystals of this enzyme have been grown using the hanging-drop vapour-diffusion technique from solutions of 2.5 M ammonium sulfate and 0.1 M sodium acetate, pH 4.6. The crystals have been shown to contain both the PyrDB and the PyrK subunits and fluorescence measurements indicate that the two different subunits interact very closely with each other in the active-site region. Native data sets have been collected to 2.6 A with a conventional X-ray source and to 2.2 A using synchrotron radiation. The crystals are rhombohedral, space group R32, with correspondin8 hexagonal unit-cell dimensions a = b = 202.3 and c = 81.0 A. The asymmetric unit in the crystal contains one PyrDB subunit and one PyrK subunit, which suggests that the two halves of the heterotetramer are related by a crystallographic twofold axis.

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PubMed1988

Application of electroporation for transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus and Propionibacterium.

Plasmid DNA was introduced by electroporation into Bacillus, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Propionibacterium and Staphylococcus as an alternative to competent-cell or protoplast transformation. Plasmid-containing transformants were recovered in these recipients at frequencies ranging from 10(1) to 10(5) transformants micrograms-1 of pGK12. Several parameters of the protocol, including DNA concentration, voltage, plating regimen and electroporation buffers were evaluated to determine conditions that improved transformation frequencies for Lactobacillus acidophilus. Using optimized conditions, the following plasmids were introduced into L. acidophilus: pAMB1, pC194, pGB354, pGKV1, pSA3, pTRK13, pTV1 and pVA797. The ability to transfer plasmid DNA via eletroporation will greatly facilitate the application of recombinant DNA methodology and transposon technology to Gram-positive bacteria for cloning and analysis of significant genes.

Buffers↗

PubMed1988

Properties of the Cell Walls of Lactococcus lactis subsp. cremoris SK110 and SK112 and Their Relation to Bacteriophage Resistance.

Resistance of Lactococcus lactis subsp. cremoris SK110 to bacteriophage sk11G, encoded on the plasmid pSK112, is due to poor phage adsorption. Its phage-sensitive variant SK112, cured of pSK112, adsorbs phages effectively. Incubation of SK112 with concanavalin A remarkably reduced phage adsorption to this strain. This treatment also caused agglutination of SK112 that was not found with SK110, indicating different concanavalin A adsorption characteristics of cell walls of both strains. The differences between the two strains were reduced by a mild alkali treatment of cells. This resulted in a positive agglutination with concanavalin A for both strains and in parallel adsorption of phage sk11G to both. Moreover, isolated cell walls of the two strains were investigated, and both bound phage sk11G. These observations suggest the presence of phage receptor material in SK112 as well as in SK110. SK110 contained a relatively high level of bound galactose when compared with the phage-sensitive SK112. After the mild alkali treatment, however, the galactose content of SK110 was diminished such that it became comparable with that of SK112. It is hypothesized that the alkali treatment liberates a galactose-containing component from the cell wall and causes phage sensitivity in L. lactis subsp. cremoris SK110.

Journal Article↗

PubMed1988

Monoclonal Antibodies to the Cell-Wall-Associated Proteinase of Lactococcus lactis subsp. cremoris Wg2.

Twelve monoclonal antibodies directed to the cell-wall-associated proteinase of Lactococcus lactis subsp. cremoris Wg2 were isolated after immunization of BALB/c mice with a partially purified preparation of the proteinase. The monoclonal antibodies reacted with the 126-kilodalton proteinase band in a Western immunoblot. All but one of the monoclonal antibodies reacted with protein bands with a molecular weight below 126,000, possibly degradation products of the proteinase. The monoclonal antibodies could be divided into six groups according to their different reactions with the proteinase degradation products in the Western blot. Different groups of monoclonal antibodies reacted with different components of the L. lactis subsp. cremoris Wg2 proteinase. Crossed immunoelectrophoresis showed that monoclonal antibody groups I, II, and III react with proteinase component A and that groups IV, V, and VI react with proteinase component B. The isolated monoclonal antibodies cross-reacted with the proteinases of other L. lactis subspecies. Monoclonal antibodies of group IV cross-reacted with proteinase component C of other L. lactis subsp. cremoris strains. The molecular weight of the proteinase attached to the cells of L. lactis subsp. cremoris Wg2 was 200,000, which is different from the previously reported values. This could be analyzed by immunodetection of the proteinase on a Western blot. This value corresponds to the molecular weight calculated from the amino acid sequence of the cloned L. lactis subsp. cremoris Wg2 proteinase gene.

Journal Article↗

PubMed1989

Sugar Utilization and Acid Production by Free and Entrapped Cells of Streptococcus salivarius subsp. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactococcus lactis subsp. lactis in a Whey Permeate Medium.

Cells of Streptococcus salivarius subsp. thermophilus and Lactococcus lactis subsp. lactis entrapped in k-carrageenan-locust bean gum gel performed similarly to free cells in the conversion of lactose to lactic acid. Bead diameter influenced the fermentation rate. Cells entrapped in smaller beads (0.5 to 1.0 mm) showed higher release rates, higher lactose, glucose, and formic acid utilization, higher galactose accumulation, and higher lactic acid production than did cells entrapped in larger beads (1.0 to 2.0 mm). Values for smaller beads were comparable with those for free cells. Immobilization affected the fermentation rate of lactic acid bacteria, especially Lactobacillus delbrueckii subsp. bulgaricus. Entrapped cells of L. delbrueckii subsp. bulgaricus demonstrated a lower lactic acid production than did free cells in batch fermentation. The kinetics of the production of formic and pyruvic acids by L. lactis subsp. lactis and S. salivarius subsp. thermophilus are presented.

Journal Article↗

PubMed1989

Peptide Utilization Encoded by Lactococcus lactis subsp. lactis SSL135 Chromosomal DNA.

A cloned chromosomal fragment of Lactococcus lactis subsp. lactis SSL135 on plasmid p VS8 in an L. lactis subsp. lactis MG1614 background enabled proteinase-negative strain MG1614 to grow in autoclaved milk. The strain (VS230) did not, however, degrade milk proteins and did not grow in pasteurized milk. In contrast, a strain (VS150) carrying p VS9, the proteinase plasmid of SSL135, in an MG1614 background degraded beta-casein but did not grow in milk. VS230 was shown to utilize peptides produced by VS150 in growth experiments in pasteurized milk preincubated with the latter strain. The peptide utilization phenotype linked with p VS8 was further confirmed by growth of VS230 on tryptic peptide fractions, on which the plasmid-free but otherwise isogenic strain MG1614 failed to grow. Plasmid p VS8 produced 69-, 42-, 38-, and 36-kilodalton proteins, as determined by in vitro transcription-translation. At least three of these proteins affected the peptide utilization phenotype. We suggest that there could be a coupled peptidase-peptide transport system encoded by the chromosomal fragment.

Journal Article↗

PubMed1989

Localization of Separate Genetic Loci for Reduced Sensitivity towards Small Isometric-Headed Bacteriophage sk1 and Prolate-Headed Bacteriophage c2 on pGBK17 from Lactococcus lactis subsp. lactis KR2.

The mechanism of reduced sensitivity to the small isometric-headed bacteriophage sk1 encoded on a 19-kilobase (kb) HpaII fragment subcloned from pKR223 of Lactococcus lactis subsp. lactis KR2 was examined. The reduced sensitivity to phage sk1 was due to a modest restriction/modification (R/M) system that was not active against prolate-headed phage c2. The genetic loci for the R/M system against sk1 and the abortive phage infection (Abi) mechanism effective against phage c2 were then localized by restriction mapping, subcloning, and deletion analysis. The restriction gene was localized to a region of a 2.7-kb EcoRV fragment and included an EcoRI site within that fragment. The modification gene was found to be physically separable from the restriction gene and was present on a 1.75-kb BstEII-XbaI fragment. The genetic locus for the Abi phenotype against phage c2 was localized to a region containing a 1.3-kb EcoRI fragment. Attempts to clone the c2 Abi mechanism independent of the sk1 R/M system were unsuccessful, suggesting that expression of the abi genes required sequences upstream of the modification gene. Some pGBK17 (vector pGB301 plus a 19-kb HpaII insert fragment) transformants exhibited the R/M system against phage sk1 but lost the Abi mechanism against phage c2. These transformants contained a 1.2- to 1.3-kb insertion in the Abi region. The data identified genetic loci on a cloned 19-kb HpaII fragment responsible for restriction activity and for modification activity against a small isometric-headed phage and for Abi activity against prolate-headed phage c2. A putative insertion element was also found to inactivate the abi gene(s).

Journal Article↗

PubMed1989

Mechanism of Proteinase Release from Lactococcus lactis subsp. cremoris Wg2.

The procedure generally used for the isolation of extracellular, cell-associated proteinases of Lactococcus lactis species is based on the release of the proteinases by repeated incubation and washing of the cells in a Ca-free buffer. For L. lactis subsp. cremoris Wg2, as many as five incubations for 30 min at 29 degrees C are needed in order to liberate 95% of the proteinase. Proteinase release was not affected by chloramphenicol, which indicates that release is not the result of protein synthesis during the incubations. Ca inhibited, while ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) stimulated, proteinase release from the cells. The pH optimum for proteinase release ranged between 6.5 and 7.5, which was higher than the optimum pH of the proteinase measured for casein hydrolysis (i.e., 6.4). Treatment of cells with the serine proteinase inhibitor phenylmethylsulfonyl fluoride prior to the incubations in Ca-free buffer reduced the release of the proteinase by 70 to 80%. The residual proteinase remained cell associated but could be removed by the addition of active L. lactis subsp. cremoris Wg2 proteinase. This suggests that proteinase release from cells of L. lactis subsp. cremoris Wg2 is the result of autoproteolytic activity. From a comparison of the N-terminal amino acid sequence of the released proteinase with the complete amino acid sequence determined from the nucleotide sequence of the proteinase gene, a protein of 180 kilodaltons would be expected. However, a proteinase with a molecular weight of 165,000 was found, which indicated that further hydrolysis had occurred at the C terminus.

Journal Article↗

PubMed1989

High-Frequency Transformation, by Electroporation, of Lactococcus lactis subsp. cremoris Grown with Glycine in Osmotically Stabilized Media.

An efficient method for genetic transformation of lactococci by electroporation is presented. Highly competent lactococci for electrotransformation were obtained by growing cells in media containing high concentrations of glycine and 0.5 M sucrose as the osmotic stabilizers. These cells could be stored at -85 degrees C without loss of competence. With Lactococcus lactis subsp. cremoris BC101, a transformation frequency of 5.7 x 10 transformants per mug of pIL253 DNA was obtained, which represents 5% of the surviving cells. All the lactococcal strains tested could be transformed by the present method.

Journal Article↗

PubMed1989

beta-Glucose-1-Phosphate, a Possible Mediator for Polysaccharide Formation in Maltose-Assimilating Lactococcus lactis.

Homolactic fermentation of glucose and heterolactic fermentation of maltose with Lactococcus lactis 65.1 were confirmed. When moles of glucose were compared, the uptake rates of the two carbon sources were similar. The intracellular concentration of fructose-1,6-diphosphate (FDP) in maltose-assimilating cells was half of that in glucose-assimilating cells. Similarly, formation of FDP and lactate from maltose by extracts of maltose-grown cells was half of that formed from glucose by extracts of glucose-grown cells, indicating a difference in the utilization of the two carbon sources for energy metabolism. Concentrations of adenine nucleotides were similar in both types of cells. Glucose-1-phosphate was found in extracts of maltose-grown cells given maltose and, in addition, an inducible and low beta-specific phosphoglucomutase activity was observed. beta-Glucose-1-phosphate was not metabolized by cell extracts to either FDP or lactate, suggesting an alternative metabolic route. The amount of [C]maltose incorporated into the cell material of maltose-grown cells was four times greater than that of [C]glucose incorporated into the cell material of glucose-grown cells. The intracellular concentration of UTP was lower in maltose-assimilating cells than in glucose-assimilating cells. Cells grown on maltose were more spherical and less fragile than cells grown on glucose.

Journal Article↗

PubMed1989

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2.

An aminopeptidase was purified from cell extracts of Lactococcus lactis subsp. cremoris AM2 by ion-exchange chromatography. After electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulfate, one protein band was detected. The enzyme was a 300-kilodalton hexamer composed of identical subunits not linked by disulfide bridges. Activity was optimal at 40 degrees C and pH 7 and was inhibited by classical thiol group inhibitors. The aminopeptidase hydrolyzed naphthylamide-substituted amino acids, as well as dipeptides and tripeptides. Longer protein chains such as the B chain of insulin were hydrolyzed, but at a much slower rate. The Michaelis constant (K(m)) and the maximal rate of hydrolysis (V(max)) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate l-histidyl-beta-naphthylamide. Amino acid analysis showed that the enzyme contained low levels of hydrophobic residues. The partial N-terminal sequence of the first 19 residues of the mature enzyme was determined. Polyclonal antibodies were obtained from the purified enzyme, and after immunoblotting, there was no cross-reaction between these antibodies and other proteins in the crude extract.

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