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PubMed1999

Kinetics and consequences of binding of nona- and dodecapeptides to the oligopeptide binding protein (OppA) of Lactococcus lactis.

The oligopeptide transport system (Opp) of Lactococcus lactis belongs to the class of binding protein-dependent ABC-transporters. This system has the unique capacity to mediate the uptake of peptides from 4 up to at least 18 residues. Kinetic analysis of peptide binding to the binding protein, OppA, revealed a relationship between the peptide dissociation constants and the length of the ligand. The dissociation constants varied from submicromolar for dodecapeptides to millimolar for pentapeptides. This implies that the residues 6-12 of the peptide contribute to the binding affinity, and, in contrast to the current views on peptide binding by homologous proteins, these residues must interact with OppA. Analysis of pre-steady-state kinetics of binding showed that the observed differences in the -values result primarily from variations in the dissociation rate constants. These results are discussed in relation to the affinity constant for transport of these substrates. Overall, the data suggest that the slow dissociation rate constants for the larger peptides are rate determining in the translocation of peptides across the membrane.

ATP-Binding Cassette Transporters↗
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↗
PubMed1999

Endoglucanase V and a phosphatase from Trichoderma viride are able to act on modified exopolysaccharide from Lactococcus lactis subsp. cremoris B40.

EPS B40 from Lactococcus lactis subsp. cremoris consists of a repeating unit of-->4)-beta-D-Glcp-(1-->4)-[alpha-L-Rhap-(1 -->2)][alpha-D-Galp-1-PO4-3]-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->. A phosphatase from Trichoderma viride was able to release phosphate, but only after removal of rhamnosyl and galactosyl residues by mild CF3CO2H treatment. Purified endoV from T. viride was able to act on the backbone of the polymer, but only if rhamnosyl substituents and phosphate had been removed. After complete removal of phosphate and partial removal of rhamnosyl residues by HF treatment, incubation with endoV resulted in a homologous series of oligomers. Purification of these oligomers and subsequent characterisation by NMR demonstrated that endoV was able to cleave the beta-(1-->4) linkage between two glucopyranosyl residues when the galactopyranosyl residue towards the nonreducing end is unsubstituted. The mode of action of endoV on HF-treated EPS B40 is discussed on the basis of the subsite model described for endoV [J.-P. Vincken, G. Beldman, A.G.J. Voragen, Carbohydr. Res., 298 (1997) 299-310].

Carbohydrate Sequence↗
PubMed1998

Kinetics and specificity of peptide uptake by the oligopeptide transport system of Lactococcus lactis.

To obtain amino acids for growth, Lactococcus lactis uses a proteolytic system to degrade exogenous proteins such as caseins. The extracellular cell wall-attached proteinase PrtP and the oligopeptide transport system Opp mediate the first two steps in the utilization of caseins. beta-Casein is degraded by PrtP to fragments of 5-30 amino acid residues, and only a limited number of peptides are selected from this pool for uptake via Opp. To study the specificity of Opp and the kinetics of peptide uptake in L. lactis in detail, we used the following strategy: (i) the Opp system was overexpressed; (ii) a 4-fold peptidase mutant was used that is unable to degrade KYGK; (iii) iodinated KYGK was used as the reporter peptide; (iv) libraries of peptides, in which one amino acid position is systematically varied, were used as competitive peptides; and (v) peptides were synthesized on the basis of the beta-casein degradation products, their inhibition of KYGK uptake was determined, and the uptake of these peptides was followed by high-performance liquid chromatography (HPLC). These studies indicate that (i) the Opp system can transport a broad range of peptides from 4 up to at least 18 residues with very little preference for particular side chains and (ii) the kinetics of peptide uptake differ for different substrates tested. Whereas class I peptides such as KYGK exhibit normal Michaelis-Menten kinetics, the level of uptake of the majority of peptides (class II) increases sigmoidally with concentration. Different models for explaining the apparent cooperative effects that are observed for peptide uptake are discussed.

ATP-Binding Cassette Transporters↗
PubMed1993

Cloning, nucleotide sequence, and regulatory analysis of the Lactococcus lactis dnaJ gene.

The dnaJ gene of Lactococcus lactis was isolated from a genomic library of L. lactis NIZO R5 and cloned into pUC19. Nucleotide sequencing revealed an open reading frame of 1,137 bp in length, encoding a protein of 379 amino acids. The deduced amino acid sequence showed homology to the DnaJ proteins of Escherichia coli, Mycobacterium tuberculosis, Bacillus subtilis, and Clostridium acetobutylicum. The level of the dnaJ monocistronic mRNA increased approximately threefold after heat shock. The transcription initiation site of the dnaJ gene was determined and appeared to be preceded by a typical gram-positive vegetative promoter sequence (TTGCCA-17 bp-TAAAAT). Upstream of the promoter region, an inverted repeat is located that is identical to those detected upstream of heat shock genes of other gram-positive organisms. A transcriptional fusion between the dnaJ expression signals and a usp45-amyS secretion cassette caused a significant increase in alpha-amylase activity after heat shock induction. Deletion mutagenesis showed that the inverted repeat is involved in heat shock regulation of the dnaJ gene. The conservation of this palindromic sequence in gram-positive heat shock genes suggests a common regulatory pathway distinct from the system used in gram-negative bacteria.

Amino Acid Sequence↗
PubMed1995

Purification and characterization of a small membrane-associated sugar phosphate phosphatase that is allosterically activated by HPr(Ser(P)) of the phosphotransferase system in Lactococcus lactis.

In the Gram-positive bacterium, Lactococcus lactis, nonmetabolizable cytoplasmic sugar phosphates, accumulated by the phosphoenolpyruvate:sugar phosphotransferase system, are rapidly dephosphorylated and expelled from the cell upon addition of glucose (inducer expulsion). Our recent studies have established that a metabolite-activated, ATP-dependent protein kinase that phosphorylates serine-46 in HPr of the phosphoenolpyruvate:sugar phosphotransferase system activates a sugar phosphate phosphatase, thus initiating the inducer expulsion process. A membrane-associated, HPr(Ser(P))-dependent phosphatase has been identified, solubilized from the membrane, separated from other cellular phosphatases, and purified to near homogeneity. It exhibits a low subunit molecular mass (10 kDa) and behaves on gel filtration columns like a monomeric enzyme. It has broad substrate specificity, optimal activity between pH 7.0 and 8.0, is dependent on a divalent cation for activity, and is not inhibited by fluoride. It is stimulated more than 10-fold by HPr(Ser(P)) or a mutant derivative of HPr, S46D HPr, in which the regulatory serine is changed to aspartate, which bears a permanently negative charge as does phosphate. Stimulation is due both to an increase in the maximal velocity (Vmax) and a decrease in the Michaelis-Menten kinetic constant (Km) for sugar phosphate. The enzyme exhibits a Ka for S46D HPr of 15 microM. Although the enzyme is thermally stable, activation by HPr(Ser(P)) is heat sensitive.

Allosteric Regulation↗
PubMed1998

Reconstruction of the proteolytic pathway for use of beta-casein by Lactococcus lactis.

Amino acid auxotrophous bacteria such as Lactococcus lactis use proteins as a source of amino acids. For this process, they possess a complex proteolytic system to degrade the protein(s) and to transport the degradation products into the cell. We have been able to dissect the various steps of the pathway by deleting one or more genes encoding key enzymes/components of the system and using mass spectrometry to analyse the complex peptide mixtures. This approach revealed in detail how L. lactis liberates the required amino acids from beta-casein, the major component of the lactococcal diet. Mutants containing the extracellular proteinase PrtP, but lacking the oligopeptide transport system Opp and the autolysin AcmA, were used to determine the proteinase specificity in vivo. To identify the substrates of Opp present in the casein hydrolysate, the PrtP-generated peptide pool was offered to mutants lacking the proteinase, but containing Opp, and the disappearance of peptides from the medium as well as the intracellular accumulation of amino acids and peptides was monitored in peptidase-proficient and fivefold peptidase-deficient genetic backgrounds. The results are unambiguous and firmly establish that (i) the carboxyl-terminal end of beta-casein is degraded preferentially despite the broad specificity of the proteinase; (ii) peptides smaller than five residues are not formed in vivo; (iii) use of oligopeptides of 5-10 residues becomes only possible after uptake via Opp; (iv) only a few (10-14) of the peptides generated by PrtP are actually used, even though the system facilitates the transport of oligopeptides up to at least 10 residues. The technology described here allows us to monitor the fate of individual peptides in complex mixtures and is applicable to other proteolytic systems.

Amino Acid Sequence↗
PubMed2000

Combinatorial peptide libraries reveal the ligand-binding mechanism of the oligopeptide receptor OppA of Lactococcus lactis.

The oligopeptide transport system (Opp) of Lactococcus lactis has the unique capacity to mediate the transport of peptides from 4 up to at least 18 residues. The substrate specificity of this binding protein-dependent ATP-binding cassette transporter is determined mainly by the receptor protein OppA. To study the specificity and ligand-binding mechanism of OppA, the following strategy was used: (i) OppA was purified and anchored via the lipid moiety to the surface of liposomes; (ii) the proteoliposomes were used in a rapid filtration-based binding assay with radiolabeled nonameric bradykinin as a reporter peptide; and (iii) combinatorial peptide libraries were used to determine the specificity and selectivity of OppA. The studies show that (i) OppA is able to bind peptides up to at least 35 residues, but there is a clear optimum in affinity for nonameric peptides; (ii) the specificity for nonameric peptides is not equally distributed over the whole peptide, because positions 4, 5, and 6 in the binding site are more selective; and (iii) the differences in affinity for given side chains is relatively small, but overall hydrophobic residues are favored-whereas glycine, proline, and negatively charged residues lower the binding affinity. The data indicate that not only the first six residues (enclosed by the protein) but also the C-terminal three residues interact in a nonopportunistic manner with (the surface of) OppA. This binding mechanism is different from the one generally accepted for receptors of ATP-binding cassette-transporter systems.

Amino Acids↗
PubMed1995

Identification and molecular analysis of Lactococcus lactis rpoD operon.

The complete nucleotide sequence of an open reading frame (ORF) preceding the Lactococcus lactis rpoD gene is reported. It was suggested that this ORF encodes Lactococcus lactis DNA primase and that the L. lactis rpoD operon consists of only two genes. Northern hybridization analysis showed that i) there are four mRNAs transcribing the rpoD gene (from upstream, M1-M4), ii) only the 3.7-kb transcript M1 includes the entire rpoD operon, iii) the shortest transcript M4 exists at both logarithmic and stationary phases of growth while the other three transcripts appear only at logarithmic phase, and iv) no apparent induction at the transcriptional level but transient repression of M1 was found when the growth temperature was shifted from 30 degrees C to 42 degrees C. 5'-ends of all four mRNAs were identified by primer extension analyses.

Amino Acid Sequence↗
PubMed1995

Behavior of Listeria monocytogenes in Mozzarella cheese in presence of Lactococcus lactis.

The behavior of Listeria monocytogenes (Scott A) on fully processed Italian Mozzarella cheese was examined in presence and in absence of bacteriocins produced by Lactococcus lactis ssp. lactis strains (DIP 15 and DIP 16). These strains, isolated from raw milk, produced heat stable bacteriocins that were inactivated by pronase, alpha- chymotrypsin and proteinase K, but not by pepsin, trypsin and catalase. The addition of crude bacteriocins to the growing culture of Listeria monocytogenes resulted in a significant reduction in cell number at 5 degrees C, but not at 30 degrees C. Mozzarella cheese was inoculated with the Listeria culture to obtain an initial level of approximately 30 CFU/cm2 surface of Mozzarella and approximately 10(3) CFU/ml of the surrounding fluid and then packaged in bags containing the heat-treated neutralized-cultures of Lactococcus lactis ssp. lactis in skim milk (in Italy, Mozzarella is sold in small size pieces, individually packaged in bags containing some fluid). Bags were stored at 5 degrees C up to 21 days. The presence of bacteriocins resulted in apparent death of Listeria monocytogenes after 24 h storage. After 7 days of storage, a revival of Listeria monocytogenes was observed, followed by an increase in number. However, for a storage period of 2-3 weeks the number of Listeria monocytogenes remained significantly below the number observed for Mozzarella cheese packaged in absence of the heat-treated cultures of Lactococcus lactis.

Animals↗
PubMed1999

Behaviour of L. monocytogenes in an artificially made biofilm of a nisin-producing strain of Lactococcus lactis.

The survival of Listeria monocytogenes in a binary biofilm with a bacteriocin producer (Lactococcus lactis CNRZ 150) was investigated. Two situations were simulated: in the first, L. monocytogenes was deposited on a 1-day biofilm of Lactococcus lactis (deferred adhesion); in the second, L. monocytogenes was simultaneously mixed with Lact. lactis (simultaneous adhesion). Biofilms were cultivated in tryptic soy broth supplemented with 6 g l(-1) of yeast extract (TSB-YE) and L. monocytogenes counts were followed for 48 h, both in co-culture with Lact. lactis and in pure culture. The influence of the mode of inoculation of L. monocytogenes (deferred or simultaneous adhesion) into the Lact. lactis biofilm, the size of the L. monocytogenes inoculum and the replacement of the culture medium at 20-24 h on the survival of L. monocytogenes was studied. Results showed that the antilisterial activity of the Lact. lactis started within the first 6 h of the deposition of L. monocytogenes. The log cycle reduction rate in number of L. monocytogenes in the mixed biofilm (compared to the pure biofilm) was greatly dependent on the inoculum size: when the smallest inoculum was used to colonise stainless steel coupons (10(6)-10(7) CFU ml(-1)), the log cycle reduction was greater and L. monocytogenes was not detected after t = 10 h (simultaneous adhesion) and t = 24 h (deferred adhesion) in the adherent population as well as in the planktonic population. On the other hand, in the case of a greater supply of L. monocytogenes (10(8) CFU ml(-1)), the results showed that the early reduction of L. monocytogenes counts was relatively slow and was followed by a stabilisation of the population, leading to the establishment of a great number of resident cells in the biofilm (10(5) to 106 CFU cm(-2)). This population level was maintained during the 48 h of experimentation and replacement of the culture media with fresh medium at t = 22 h (simultaneous adhesion) or t = 24 h (deferred adhesion) did not modify the level of the population of L. monocytogenes within the biofilm.

Biofilms↗
PubMed1999

Identification of four phage resistance plasmids from Lactococcus lactis subsp. cremoris HO2.

The bacteriophage-host sensitivity patterns of 16 strains of Lactococcus lactis originally isolated from a mixed strain Cheddar cheese starter culture were determined. Using phages obtained from cheese factory whey, four of the strains were found to be highly phage resistant. One of these isolates, Lactococcus lactis subsp. cremoris HO2, was studied in detail to determine the mechanisms responsible for the phage insensitivity phenotypes. Conjugal transfer of plasmid DNA from strain HO2 allowed a function to be assigned to four of its six plasmids. A 46-kb molecule, designated pCI646, was found to harbor the lactose utilization genes, while this and plasmids of 58 kb (pCI658), 42 kb (pCI642), and 4.5 kb (pCI605) were shown to be responsible for the phage resistance phenotypes observed against the small isometric-headed phage phi712 (936 phage species) and the prolate-headed phage phic2 (c2 species). pCI658 was found to mediate an adsorption-blocking mechanism and was also responsible for the fluffy pellet phenotype of cells containing the molecule. pCI642 and pCI605 were both shown to be required for the operation of a restriction-modification system.

Bacteriophage Typing↗
PubMed2000

Identification and partial characterization of lacticin BH5, a bacteriocin produced by Lactococcus lactis BH5 isolated from Kimchi.

Strain BH5 was isolated from naturally fermented Kimchi and identified as a bacteriocin producer that has bactericidal activity against Micrococcus flavus ATCC 10240. Strain BH5 was identified tentatively as Lactococcus lactis by API test. Lactococcus lactis BH5 showed a broad spectrum of activity against most of the nonpathogenic and pathogenic microorganisms tested by the modified deferred method. The activity of lacticin BH5, named tentatively as the bacteriocin produced by L. lactis BH5, was detected at the mid-log growth phase, reached its maximum during the early stationary phase, and decreased after the late stationary phase. Lacticin BH5 also showed a relatively broad spectrum of activity against nonpathogenic and pathogenic microorganisms as tested by the spot-on-lawn method. Its antimicrobial activity on sensitive indicator cells was completely destroyed by protease XIV. The inhibitory activities of lacticin BH5 were detected during treatments up to 100 degrees C for 30 min. Lacticin BH5 was very stable over a pH range of 2.0 to 9.0 and was stable with all the organic solvents examined. It demonstrated a typical bactericidal mode of inhibition against M. flavus ATCC 10240. The apparent molecular mass of lacticin BH5 was estimated to be in the region of 3 to 3.5 kDa, by the direct detection of bactericidal activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Bacterial Proteins↗
PubMed2003

Cell cycle dysregulation induced by cytoplasm of Lactococcus lactis ssp lactis in SNUC2A, a colon cancer cell line.

The anticancer effect of cytoplasmic fraction from Lactococcus lactis ssp. lactis, which had showed strong antiproliferative activity against SNUC2A human colon cancer cell line in the previous study, was investigated. The proliferation of SNUC2A was inhibited by the treatment with cytoplasmic fraction of Lactococcus lactis ssp. lactis in a dose-dependent and partially reversible manner. After exposure to the cytoplasmic fraction of Lc. lactis for 72 h, strong antiproliferative activity was efficiently induced through S-phase accumulation in SNUC2A cells. Analysis of cell cycle regulatory proteins demonstrated that the cytoplasmic fraction enhanced the levels of p21CIP1 and cyclin A, decreased cyclin E protein, and slightly reduced the activity of cyclin-dependent kinase 2 (CDK2).

CDC2 Protein Kinase↗
PubMed2006

Characterization of bacteriocins from two Lactococcus lactis subsp. lactis isolates.

In this study, bacteriocins from two Lactococcus lactis subsp. lactis isolates from raw milk samples in Turkey designated OC1 and OC2, respectively, were characterized and identified. The activity spectra of the bacteriocins were determined by using different indicator bacteria including Listeria, Bacillus and Staphylococcus spp. Bacteriocins were tested for their sensitivity to different enzymes, heat treatments and pH values. Loss of bacteriocin activities after alpha-amylase treatment suggested that they form aggregates with carbohydrates. Molecular masses of the purified bacteriocins were determined by SDS-PAGE. PCR amplification was carried out with specific primers for the detection of their structural genes. As a result of these studies, the two bacteriocins were characterized as nisin and lacticin 481, respectively. Examination of plasmid contents of the isolates and the results of plasmid curing and conjugation experiments showed that in L. lactis subsp. lactis OC1 strain the 39.7-kb plasmid is responsible for nisin production, lactose fermentation and proteolytic activity, whereas the 16.0-kb plasmid is responsible for lacticin 481 production and lactose fermentation in L. lactis subsp. lactis OC2 strain.

Animals↗
PubMed2004

Phenotypic and genetic diversity of Lactococcus lactis and Enterococcus spp. strains isolated from Northern Spain starter-free farmhouse cheeses.

To evaluate a previous phenotypic classification of lactococci, 39 presumed lactococcal strains were classified by molecular techniques. The strains were also subjected to several typing techniques to estimate the phenotypic and genetic diversity present in original populations from starter-free farmhouse cheeses. Partial Amplified rDNA Restriction Analysis (partial ARDRA) with either restriction enzyme MboII or HhaI divided these isolates into four distinctive groups. Sequencing of representative amplicons identified 29 isolates as belonging to Lactococcus lactis subsp. lactis (24) and Lactococcus lactis subsp. cremoris (5). The remaining 10 isolates were shown to be Enterococcus durans (8) and Enterococcus faecalis (2), which were misclassified by the traditional tests. Thus, partial ARDRA was successfully used to classify wild Lactococcus-like strains into Lactococcus and Enterococcus species. The technique also allowed differentiation of L. lactis strains at subspecies level. The 29 strains of L. lactis showed five different fermentation profiles, four distinct Random Amplification of Polymorphic DNA (RAPD) profiles, and 14 unrelated profiles by both Restriction Fragment Length Polymorphism analyzed by Pulsed Field Gel Electrophoresis (RFLP-PFGE) and Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Using the same techniques, the 10 enterococcal strains showed four fermentation profiles, four RADP, and six by RFLP-PFGE and SDS-PAGE, respectively. Several typing techniques, especially RFLP-PFGE and SDS-PAGE, revealed wide phenotypic and genetic variability in both the lactococcal and enterococcal isolates. Two simple, rapid and cheap techniques (partial ARDRA and SDS-PAGE) are proposed as reliable tools for the classification and typing of new lactococcal-like isolates.

Bacterial Typing Techniques↗
PubMed2000

Secretion of biologically active murine interleukin-10 by Lactococcus lactis.

We investigated the ability of Lactococcus lactis to secrete biologically active, murine interleukin-10 (mIL-10). mIL-10 was synthesized as a fusion protein, consisting of the mature part of the eukaryotic protein fused to the secretion signal of the lactococcal Usp45 protein. The secreted protein was analyzed by PAGE, ELISA and bioassay.We show that L. lactis can efficiently secrete biologically active, murine IL-10. Determination of the N-terminal amino acid sequence confirmed correct processing of the fusion polypeptide by the lactococcal signal peptidase. The amount of mIL-10, accumulating in the medium, could be increased by a factor of ten by growing the cells in an optimized medium, buffered at near-neutral pH. Under these conditions, up to 30 mg of mIL-10 was obtained from a 10-litre fermentation.

Journal Article↗
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.

Journal Article↗