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PubMed1990

pAMbeta1-Associated Mobilization of Proteinase Plasmids from Lactococcus lactis subsp. lactis UC317 and L. lactis subsp. cremoris UC205.

A combination of plasmid curing and DNA-DNA hybridization data facilitated the identification of proteinase plasmids of 75 (pCI301) and 35 kilobases (pCI203) in the multi-plasmid-containing strains Lactococcus lactis subsp. lactis UC317 and L. lactis subsp. cremoris UC205, respectively. Both plasmids were transferred by conjugation to a plasmid-free background only after introduction of the conjugative streptococcal plasmid, pAMbeta1. All Prt transconjugants from matings involving either donor contained enlarged recombinant Prt plasmids. UC317-derived transconjugants were separable into different classes based on the presence of differently sized cointegrate plasmids and on segregation of the pCI301-derived Lac and Prt markers. All UC205-derived transconjugants harbored a single enlarged plasmid that was a cointegrate between pCI203 and pAMbeta1. The identification of prt genes on pCI301 and pCI203 derivatives was achieved by a combination of restriction enzyme and hybridization analyses.

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PubMed1990

Cell Surface Characteristics of Bacteriophage-Resistant Lactococcus lactis subsp. cremoris SK110 and Its Bacteriophage-Sensitive Variant SK112.

Several cell surface characteristics of bacteriophage-resistant Lactococcus lactis subsp. cremoris SK110 were compared with those of its phage-sensitive derivative SK112. After centrifugation, SK110 cells resisted suspension more strongly than SK112 cells. SK112 was more negatively charged and had a more hydrophobic cell surface than SK110. Furthermore, SK112 was agglutinated in the presence of concanavalin A, whereas SK110 was not. The opposite was observed upon incubation of cells of either strain with a lectin from Ricinus communis. A mild alkali treatment decreased the differences in the cell surface characteristics of the two strains remarkably.

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PubMed1990

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris Wg2.

An aminopeptidase was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that included diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, gel filtration, and high-performance liquid chromatography over an anion-exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 95,000. The aminopeptidase was capable of degrading several peptides by hydrolysis of the N-terminal amino acid. The peptidase had no endopeptidase or carboxypeptidase activity. The aminopeptidase activity was optimal at pH 7 and 40 degrees C. The enzyme was completely inactivated by the p-chloromecuribenzoate mersalyl, chelating agents, and the divalent cations Cu and Cd. The activity that was lost by treatment with the sulfhydryl-blocking reagents was restored with dithiothreitol or beta-mercapto-ethanol, while Zn or Co restored the activity of the 1,10-phenantroline-treated enzyme. Kinetic studies indicated that the enzyme has a relatively low affinity for lysyl-p-nitroanilide (K(m), 0.55 mM) but that it can hydrolyze this substrate at a high rate (V(max), 30 mumol/min per mg of protein).

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PubMed1990

Properties of 2,3-Butanediol Dehydrogenases from Lactococcus lactis subsp. lactis in Relation to Citrate Fermentation.

Two 2,3-butanediol dehydrogenases (enzymes 1 and 2; molecular weight of each, 170,000) have been partially purified from Lactococcus lactis subsp. lactis (Streptococcus diacetylactis) D10 and shown to have reductase activity with either diacetyl or acetoin as the substrate. However, the reductase activity with 10 mM diacetyl was far greater for both enzymes (7.0- and 4.7-fold for enzymes 1 and 2, respectively) than with 10 mM acetoin as the substrate. In contrast, when acetoin and diacetyl were present together, acetoin was the preferred substrate for both enzymes, with enzyme 1 showing the more marked preference for acetoin. meso-2,3-Butanediol was the only isomeric product, with enzyme 1 independent of the substrate combinations. For enzyme 2, both the meso and optical isomers of 2,3-butanediol were formed with acetoin as the substrate, but only the optical isomers were produced with diacetyl as the substrate. With batch cultures of strain D10 at or near the point of citrate exhaustion, the main isomers of 2,3-butanediol present were the optical forms. If the pH was sufficiently high (>pH 5), acetoin reduction occurred over time and was followed by diacetyl reduction, and meso-2,3-butanediol became the predominant isomer. Interconversion of the optical isomers into the meso isomer did occur. The properties of 2,3-butanediol dehydrogenases are consistent with diacetyl and acetoin removal and the appearance of the isomers of 2,3-butanediol.

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PubMed1990

Purification and Characterization of a Tripeptidase from Lactococcus lactis subsp. cremoris Wg2.

A tripeptidase from a cell extract of Lactococcus lactis subsp. cremoris Wg2 has been purified to homogeneity by DEAE-Sephacel and phenyl-Sepharose chromatography followed by gel filtration over a Sephadex G-100 SF column and a high-performance liquid chromatography TSK G3000 SW column. The enzyme appears to be a dimer with a molecular weight of between 103,000 and 105,000 and is composed of two identical subunits each with a molecular weight of about 52,000. The tripeptidase is capable of hydrolyzing only tripeptides. The enzyme activity is optimal at pH 7.5 and at 55 degrees C. EDTA inhibits the activity, and this can be reactivated with Zn, Mn, and partially with Co. The reducing agents dithiothreitol and beta-mercaptoethanol and the divalent cation Cu inhibit tripeptidase activity. Kinetic studies indicate that the peptidase hydrolyzes leucyl-leucyl-leucine with a K(m) of 0.15 mM and a V(max) of 151 mumol/min per mg of protein.

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PubMed1990

Stability of Integrated Plasmids in the Chromosome of Lactococcus lactis.

Derivatives of plasmids pBR322, pUB110, pSC101, and pTB19, all containing an identical fragment of lactococcal chromosomal DNA, were integrated via a Campbell-like mechanism into the same chromosomal site of Lactococcus lactis MG1363, and the transformants were analyzed for the stability of the integrated plasmids. In all cases the erythromycin resistance gene of pE194 was used as a selectable marker. Transformants obtained by integration of the pBR322 derivatives contained a head-to-tail arrangement of several plasmid copies, which most likely was caused by integration of plasmid multimers. Single-copy integrations were obtained with the pSC101 and pTB19 derivatives. In all of these transformants no loss of the erythromycin gene was detected during growth for 100 generations in the absence of the antibiotic. In contrast, transformants containing integrated amplified plasmid copies of pUB110 derivatives were unstable under these conditions. Since pUB110 appeared to have replicative activity in L. lactis, we suggest that this activity destabilized the amplified structures in L. lactis.

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PubMed1991

Study of the Citrate Metabolism of Lactococcus lactis subsp. lactis Biovar Diacetylactis by Means of C Nuclear Magnetic Resonance.

The metabolic fate of citrate and pyruvate in four strains of Lactococcus lactis subsp. lactis biovar diacetylactis has been studied by means of C nuclear magnetic resonance, using as a substrate either [3-C]pyruvic acid or custom-synthesized citric acid that is C labeled either at carbons 2 and 4 or at carbon 3. The fermentations were carried out batchwise in modified M17 broth. For the actual conversions of the C-labeled substrates, cells at the end of their logarithmic growth phase were used to minimize the conversion to lactic acid. A mass balance of the main citric acid metabolites was obtained; the four strains produced from 50 to 70% (on a molar basis) lactic acid from either citrate or pyruvate. The remaining 50 to 30% was converted mainly to either alpha-acetolactic acid (for one strain) or acetoin (for the other three strains). One of the strains produced an exceptionally high concentration of the diacetyl precursor alpha-acetolactic acid. Another strain (SDC6) also produced alpha-acetolactic acid, but this was decarboxylated to acetoin at a high rate. The C nuclear magnetic resonance method confirmed that the biosynthesis of alpha-acetolactic acid occurs via condensation of pyruvate and "active" acetaldehyde. Diacetyl was not found as a direct metabolite of citrate or pyruvate metabolism.

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PubMed1991

Construction of a Bacteriophage-Resistant Derivative of Lactococcus lactis subsp. lactis 425A by Using the Conjugal Plasmid pNP40.

Lactococcus lactis subsp. lactis 425A is an atypical strain which excretes a high concentration of alpha-acetolactate when grown in milk. The conjugative lactococcal plasmid pNP40, which encodes phage and nisin resistance, was introduced to strain 425A by conjugation, using resistance to phage and nisin as a selection. No phage-nisin resistance mutants were encountered. Transconjugants display complete resistance at both 21 and 39 degrees C to those phage previously identified as lytic for 425A. Transconjugants lose their resistance characteristics when spontaneously cured of pNP40. The commercially important property of 425A-production of high levels of alpha-acetolactic acid-is unaffected by the presence of pNP40.

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PubMed1991

Citrate Fermentation by Lactococcus and Leuconostoc spp.

Citrate and lactose fermentation are subject to the same metabolic regulation. In both processes, pyruvate is the key intermediate. Lactococcus lactis subsp. lactis biovar diacetylactis homofermentatively converted pyruvate to lactate at high dilution (growth) rates, low pH, and high lactose concentrations. Mixed-acid fermentation with formate, ethanol, and acetate as products was observed under conditions of lactose limitation in continuous culture at pH values above 6.0. An acetoin/butanediol fermentation with alpha-acetolactate as an intermediate was found upon mild aeration in continuous culture and under conditions of excess pyruvate production from citrate. Leuconostoc spp. showed a limited metabolic flexibility. A typical heterofermentative conversion of lactose was observed under all conditions in both continuous and batch cultures. The pyruvate produced from either lactose or citrate was converted to d-lactate. Citrate utilization was pH dependent in both L. lactis and Leuconostoc spp., with maximum rates observed between pH 5.5 and 6.0. The maximum specific growth rate was slightly stimulated by citrate, in L. lactis and greatly stimulated by citrate in Leuconostoc spp., and the conversion of citrate resulted in increased growth yields on lactose for both L. lactis and Leuconostoc spp. This indicates that energy is conserved during the metabolism of citrate.

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PubMed1991

Antisense mRNA-Mediated Bacteriophage Resistance in Lactococcus lactis subsp. lactis.

Resistance to a broad class of isometric bacteriophages that infect strains of Lactococcus lactis has been engineered into a dairy starter by expression of antisense mRNA targeted against a conserved bacteriophage gene. Maximum protection is obtained only when the entire 1,654-bp coding sequence for a 51-kDa protein is positioned in the antisense orientation with respect to a promoter sequence that functions in L. lactis subsp. lactis. Expression of the antisense mRNA results in more than 99% reduction of the total number of PFU. Plaques that do form are characterized by their relatively small size and irregular shape. A variety of truncated genes, including the open reading frame expressed in the sense orientation, fail to provide any significant measure of resistance as compared with that of the intact open reading frame. Southern hybridization with probes specific for the conserved region reveal that the [ill] plasmid constructs are maintained despite the presence of a large complement of other indigenous plasmids. Strains harboring the antisense mRNA plasmid construct grow and produce acid at a rate equivalent to that of the host strain alone, suggesting that antisense expression is not deleterious to normal cellular metabolism.

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PubMed1991

Characterization of the Heat Shock Response in Lactococcus lactis subsp. lactis.

The heat shock response in Lactococcus lactis subsp. lactis was characterized with respect to synthesis of a unique set of proteins induced by thermal stress. A shift in temperature from 30 to 42 degrees C was sufficient to arrest the growth of L. lactis subsp. lactis, but growth resumed after a shift back to 30 degrees C. Heat shock at 50 degrees C reduced the viable cell population by 10; however, pretreatment of the cells at 42 degrees C made them more thermoresistant to exposure at 50 degrees C. The enhanced synthesis of approximately 13 proteins was observed in cells labeled with S upon heat shock at 42 degrees C. Of these heat shock-induced proteins, two appeared to be homologs of GroEL and DnaK, based on their molecular weights and reactivity with antiserum against the corresponding Escherichia coli proteins. Therefore, we conclude that L. lactis subsp. lactis displays a heat shock response similar to that observed in other mesophilic bacteria.

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PubMed1991

Autoproteolysis of the Extracellular Serine Proteinase of Lactococcus lactis subsp. cremoris Wg2.

The molecular masses of purified extracellular serine proteinase of a number of Lactococcus lactis strains vary significantly, and these molecular mass values do not correspond to the values estimated on the basis of genetic data. The discrepancies can only partially be explained by N-terminal processing during maturation of the precursor enzyme and by C-terminal cleaving during the release from the cell envelope. With a monoclonal antibody that binds in the active site region of the L. lactis proteinase, the processing of the released proteinase was followed. At 30 degrees C the proteinase was degraded with a concomitant loss of beta-casein hydrolytic activity. In the presence of CaCl(2), proteinase degradation was inhibited, and new degradation products were detected. The specific serine proteinase inhibitors phenylmethylsulfonyl fluoride and diisopropylfluorophosphate also inhibited proteinase degradation. Two major high-molecular-mass proteinase fragments (165 and 90 kDa) were found to have the same N-terminal amino acid sequence as the mature proteinase, i.e., [Asp-1-Ala-2-Lys-3-Ala-4-Asn-5-Ser-6, indicating that both fragments were formed by cleavage at the C terminus. The N terminus of a proteinase fragment with low molecular mass (58 kDa) started with Gln-215. In this fragment part of the active site region was eliminated, suggesting that it is proteolytically inactive. Unlike larger fragments, this 58-kDa fragment remained intact after prolonged incubations. These results indicate that autoproteolysis of the L. lactis subsp. cremoris Wg2 proteinase ultimately leads to inactivation of the proteinase by deletion of the active site region.

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PubMed1992

Purification and Partial Characterization of Lacticin 481, a Lanthionine-Containing Bacteriocin Produced by Lactococcus lactis subsp. lactis CNRZ 481.

Lacticin 481, a bacteriocin produced during the growth of Lactococcus lactis subsp. lactis CNRZ 481, was purified sequentially by ammonium sulfate precipitation, gel filtration, and preparative and analytical reversed-phase high-pressure liquid chromatography. Ammonium sulfate precipitations resulted in a 455-fold increase in total lacticin 481 activity. The entire purification protocol led to a 107, 506-fold increase in the specific activity of lacticin 481. On the basis of its electrophoretic pattern in sodium dodecyl sulfate-polyacrylamide gels, lacticin 481 appeared as a single peptide band of 1.7 kDa. However, dimers of 3.4 kDa also exhibiting lacticin activity were detected. Derivatives of the lacticin-producing strain which did not produce lacticin 481 (Bac) were sensitive to this bacteriocin (Bac) and failed to produce the 1.7-kDa band. Amino acid composition analysis of purified lacticin 481 revealed the presence of lanthionine residues, suggesting that lacticin 481 is a member of the lantibiotic family of antimicrobial peptides. Seven residues (K G G S G V I) were sequenced from the N-terminal portion of lacticin 481, and these did not shown any homology with nisin or other known bacteriocin sequences.

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PubMed1992

Stability and Specificity of the Cell Wall-Associated Proteinase from Lactococcus lactis subsp. cremoris H2 Released by Treatment with Lysozyme in the Presence of Calcium Ions.

The cell wall-associated proteinase from Lactococcus lactis subsp. cremoris H2 (isolate number 4409) was released from the cells by treatment with lysozyme, even in the presence of 50 mM calcium chloride. Cell lysis during lysozyme treatment was minimal. The proteinase activity released by lysozyme treatment fractionated on ion-exchange chromatography as three main forms, the molecular masses of which were determined by gel exclusion chromatography and polyacrylamide gel electrophoresis. Two of the enzyme forms released, 137 and 145 kDa, were the same as those released by incubation of cells in calcium-free phosphate buffer. In the presence of calcium, lysozyme treatment also resulted in the release of a 180-kDa enzyme molecule. The total proteinase activity released by lysozyme treatment (in the presence or absence of calcium) was not only greater than that released by phosphate buffer but was also greater than that initially detectable on the surface of whole cells, suggesting an unmasking of enzyme on the cell surface. The presence of calcium during release treatment resulted in increased stability of the crude enzyme preparations. For the proteinase preparation released by using lysozyme with 50 mM CaCl(2), the half-life of proteinase activity at 37 degrees C was 39 h, compared with 0.22 h for the calcium-free phosphate buffer-released preparation. In all cases, maximum stability was observed at pH 5.5. Comparison of beta-casein hydrolysis by the three forms of the enzyme showed that the products of short-term (5- to 30-min) digestions were very similar, although subtle differences were detected with the 180-kDa form.

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PubMed1992

Production of Concentrated Lactococcus lactis subsp. cremoris Suspensions in Calcium Alginate Beads.

The effect of simultaneous modification of medium composition and growth conditions on the production of Lactococcus lactis subsp. cremoris biomass in calcium alginate beads was studied by the response surface method. Statistical methods of data analysis for unbalanced experiments are illustrated. The media tested were whey, whey supplemented with yeast extract and/or meat extract, milk, and the commercial medium Gold Complete (Nordica). Fermentations were performed at 23 degrees C under pH control (5.6, 6.0, 6.4, or 6.8). In one complete series, 1% CaCO(3) was added to the growth media. There were strong interactions between CaCO(3) and media, CaCO(3) and pH level, and CaCO(3), media, and pH level. In media with CaCO(3), all first-order interactions between media, pH, and sampling time were significant. The addition of CaCO(3) increased cell counts in whey-meat extract medium, but no significant difference was found with the other media. Uncoupling between growth and acidification occurred between 16 and 22 h. Highest counts were obtained on milk and Gold Complete (6 x 10/g). In CaCO(3)-containing media, pH influenced cell counts only in whey and in Gold Complete (pH 5.6 and 6.0 giving the best results); pH also influenced the bead mass obtained at the end of the fermentation. Biomass production in alginate gels is proposed as a method of obtaining concentrated cell suspensions without centrifugation or filtration.

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PubMed1993

Insertion of Transposon Tn917 Derivatives into the Lactococcus lactis subsp. lactis Chromosome.

Two transposition vectors, pTV32 and pLTV1, containing transposon Tn917 derivatives TV32 and LTV1, respectively, were introduced into Lactococcus lactis subsp. lactis MG1614. It was found that pTV32 and pLTV1 replicate and that TV32 and LTV1 transpose in this strain. A protocol for production of a collection of Tn917 insertions in L. lactis subsp. lactis was developed. The physical locations of TV32 on the chromosomal SmaI fragments of 62 independent transpositions were established by pulsed-field gel electrophoresis. These transpositions could be divided into at least 38 different groups that exhibited no Tn917-dominating hot spots on the L. lactis subsp. lactis chromosome. A total of 10 of the 62 transpositions resulted in strains that express beta-galactosidase. This indicates that there was fusion of the promoterless lacZ of the Tn917 derivatives to a chromosomal promoter. Thus, the Tn917-derived transposons should be powerful genetic tools for studying L. lactis subsp. lactis.

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PubMed1993

Characterization of Loosely Associated Material from the Cell Surface of Lactococcus lactis subsp. cremoris E8 and Its Phage-Resistant Variant Strain 398.

Loosely associated material (LAM) was isolated by gentle extraction procedures from the cell surface of Lactococcus lactis subsp. cremoris E8 and its phage-resistant variant strain 398. LAM from both strains was chemically characterized, and its role in the adsorption of three small isometric bacteriophages, phi 618, phi 833, and phi 852, to the cell surface of the two strains was investigated. The phage-resistant strain (strain 398) produced LAM which differed significantly from the material produced by the parent strain. The total yield of LAM from strain 398 was two- to threefold higher than that from strain E8, and the material contained fivefold more rhamnose and twofold more galactose. Polyacrylamide gel electrophoretic analysis showed that LAM from strain 398 lacked a 21-kDa protein which was present in LAM from the parent strain. Inhibition studies of phage binding by using isolated LAM from two strains showed that although LAM from strain E8 reduced the titer of phi 618 and phi 852 by 53 and 82% respectively, LAM from strain 398 had no effect on the plaque-forming ability of any of the three phages tested. Treatment of LAM from strain E8 with sodium metaperiodate destroyed its ability to bind with phi 618 and phi 852. Phenotypically, strain 398 differed from its parent strain E8 in that it was more prone to cell lysis and required an osmotically adjusted buffer system for the extraction of LAM.

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PubMed1993

Two Uptake Systems for Fructose in Lactococcus lactis subsp. cremoris FD1 Produce Glycolytic and Gluconeogenic Fructose Phosphates and Induce Oscillations in Growth and Lactic Acid Formation.

Fructose transport in lactococci is mediated by two phosphotransferase systems (PTS). The constitutive mannose PTS has a broad specificity and may be used for uptake of fructose with a fructose saturation constant (K(Fru)) of 0.89 mM, giving intracellular fructose 6-phosphate. The inducible fructose PTS has a very small saturation constant (K(Fru), <17 muM), and the fructose 1-phosphate produced enters the Embden-Meyerhof-Parnas (EMP) pathway as fructose 1,6-diphosphate. Growth in batch cultures of Lactococcus lactis subsp. cremoris FD1 in a yeast extract medium with fructose as the only sugar is poor both with respect to specific growth rate and biomass yield, whereas the specific lactic acid production rate is higher than those in similar fermentations on other sugars metabolized via the EMP pathway, e.g., glucose. In fructose-limited chemostat cultures, the biomass concentration exhibits a strong correlation with the dilution rate, and starting a continuous culture at the end of a batch fermentation leads to large and persistent oscillations in the biomass concentration and specific lactic acid production rate. Two proposed mechanisms underlying this strange growth pattern follow. (i) Fructose transported via the fructose PTS cannot be converted into essential biomass precursors (glucose 6-phosphate or fructose 6-phosphate), because L. lactis subsp. cremoris FD1 is devoid of fructose 1,6-diphosphatase activity. (ii) The fructose PTS apparently produces a metabolite (presumably fructose 1-phosphate) which exerts catabolite repression of both mannose PTS and lactose PTS. Since the repressed mannose PTS and lactose PTS are shown to have identical maximum molar transport rates, the results indicate that it is the general PTS proteins which are repressed.

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