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[Studies on purification and some properties of nisin from Lactococcus lactis subsp. lactis Al2].

Nisin from Lactococcus lactis subsp. lactis AL2 was extracted with n-propanol from NaCl-saturated culture and purified by ion-exchange chromotography on CM-Sephadex C-25. Nisin was purified 1.63 fold with a yield of 41.7%. The molecular weight of nisin was determined by SDS-PAGE to be about 3500. Nisin activity was stable at low pH and sensitive to digestion by a-chymotrypsin. Nisin is capable of inhibiting a broad range of gram-positive bacteria. In contrast, the gram-negative bacteria, yeasts, molds and Nip+ L. lactis subsp. lactis ATCC11454 were not inhibited.

Anti-Bacterial Agents↗

6-Phosphogluconate dehydrogenase from Lactococcus lactis: a role for arginine residues in binding substrate and coenzyme.

A gene encoding 6-phosphogluconate dehydrogenase (6-PGDH, EC 1.1.1. 44) was identified from the homofermentative lactic acid bacterium Lactococcus lactis, by complementation of Escherichia coli mutants. The cloned gene was then expressed to high levels in E. coli and the protein purified for kinetic analysis. The enzyme had a Km for 6-phosphogluconate of 15.4+/-1.4 microM and for NADP of 1.9+/-0.2 microM at pH 7.5. Sequence comparison of the L. lactis 6-PGDH with the corresponding enzyme derived from the pathogenic protozoan Trypanosoma brucei and sheep liver revealed the substrate-binding residues to be identical in all three species, although the three coenzyme-binding pockets differed slightly. A totally conserved arginine residue (Arg-447), believed to bind the 6-phosphate of substrate, was mutated to lysine, aspartate, alanine or tryptophan. In each case enzyme activity was lost, confirming an essential role for this residue on activity. A second arginine (Arg-34), believed to be critical in binding the 2'-phosphate of cofactor NADP+, was mutated to a tyrosine residue, as found in one atypical isoform of the enzyme in Bacillus subtilis. This alteration led to decrease in affinity for NADP+ of nearly three orders of magnitude. A second 6-PGDH gene has been identified from the genome of B. subtilis. This second isoform contains an arginine (Arg-34) in this position, suggesting that B. subtilis has two 6-PGDHs with different coenzyme specificities.

Amino Acid Sequence↗

Effect of heat shock or cold shock treatment on the resistance of lactococcus lactis to freezing and lyophilization

This study investigated the effect of heat shock or cold shock treatment on the resistance of commercial Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris cheese starter bacteria to freezing and freeze-drying. The ability to withstand freezing at -60 degrees C for 24 h was variable among lactococci, but could be significantly (P < 0.05) improved in most strains by a 25-min heat shock at 42 degrees C (L. lactis ssp. lactis) or 39 degrees C (L. lactis ssp. cremoris) or by a 2-h cold shock at 10 degrees C. In addition, stress treatments that improved lactococcal cryotolerance significantly (P < 0.05) enhanced the resistance of most strains to lyophilization. Greater resistance to freezing or lyophilization was not detected when stress treatments were performed in broth that contained erythromycin, which indicated that stress-induced proteins were required for cell protection. Membrane fatty acid analysis of stress-treated cells suggested that enhanced resistance to freezing and lyophilization may be related to changes in cell membrane lipid composition. Heat-shocked cells had a higher 19:0 cyclopropane fatty acid content than control cells, and cold-shocked cells contained a lower ratio of saturated to unsaturated fatty acids. Other factors must also be involved in cell protection, however, because similar changes in membrane lipid composition were detected in strains whose resistance to freezing and lyophilization was not improved by heat or cold shock treatment. Copyright 1999 Academic Press.

Journal Article↗

Differences in short peptide-substrate cleavage by two cell-envelope-located serine proteinases of Lactococcus lactis subsp. cremoris are related to secondary binding specificity.

Various chromophoric peptides have been tested as substates for two genetically related types (PI and PIII) of cell-envelope proteinases of Lactococcus lactis subsp. cremoris. The positively charged peptide methoxy-succinyl-arginyl-prolyl-tyrosyl-p-nitroanilide appeared to be cleaved with the highest catalytic efficiency by both enzymes, although in the case of PIII only at high ionic strength. A cation binding site in the PI-type proteinase that is not present in the related PIII-type appears to be mainly responsible for the difference between these enzymes with respect to the rate of conversion of this chromophoric substrate at relatively low ionic strength. This cation binding site most probably resides in the aspartic acid residue 166, which in PIII is substituted by asparagine. Substitution of the threonine residue 138 by lysine in PIII may also play a role. The binding step in the reaction pathway catalysed by PI at low ionic strength is governed mainly by an ionic interaction involving the cation binding site. In addition, hydrophobic interactions contribute to the binding process. Masking of the cation binding site only increases the Michaelis constant Km; the catalytic constant kcat is not affected. In the absence of the cation binding site (viz. in PIII) the free energy derived from the hydrophobic interactions only is too small to promote binding of the substrate effectively. High activities are measured only if a high ionic strength is introduced. Removal of electrostatic repulsion between the substrate and positively charged residues of the enzyme, among which is lysine 138, may contribute to this activation.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Butanol↗

Comparison of bovine beta-casein hydrolysis by PI and PIII-type proteinases from Lactococcus lactis subsp. cremoris [corrected].

The action of the cell-wall-associated proteinases from Lactococcus lactis subsp. cremoris strains H2 and SK112 on bovine beta-casein was compared. The proteinase from the H2 strain was characterised as a PI-type proteinase since it did not hydrolyse alpha s1-casein and the initial trifluoroacetic acid-soluble products of beta-casein hydrolysis were identical to those previously identified as hydrolysis products of PI-type lactococcal proteinase action. The time-course of product formation by the proteinase from the H2 strain indicated that the bonds Tyr193-Gln194 and Gln182-Arg183 were the first to be hydrolysed. Cleavage of the bonds Gln175-Lys176, Ser168-Lys169, Ser166-Gln167 and Leu163-Ser164 was also very rapid. Four of the five bonds in beta-casein most susceptible to hydrolysis by the PIII-type proteinase from strain SK112 were different from those cleaved by the PI-type proteinase, initial hydrolysis being at the sites Tyr193-Gln194, Leu192-Tyr193, Asp43-Glu44, Gln46-Asp47 and Phe52-Ala53. Early hydrolysis at the three sites in the N-terminal region of beta-casein, leading to cleavage of the N-terminal phosphopeptide and rapid precipitation of the residual fragment, represents a marked contrast to the action of PI-type proteinases where cleavage at sites in the N-terminal region occurs only very slowly.

Amino Acid Sequence↗

Cloning and DNA sequence analysis of a Lactococcus bacteriophage lysin gene.

A gene for the lysin of Lactococcus lactis bacteriphage phi vML3 was cloned using an Escherichia coli/bacteriophage lambda host-vector system. The gene was detected by its expression of antimicrobial activity against L. lactis cells in a bioassay. The cloned fragment was analysed by sub-cloning on to E. coli plasmid vectors and by restriction endonuclease and deletion mapping. Its entire DNA sequence was determined and an open reading frame for the lysin structural gene was identified. The sequenced lysin gene would express a protein of 187 amino acids with a molecular weight of 21,090, which is in good agreement with that of a protein detected after in vitro transcription and translation of DNA encoding the gene. Expression of the lysin gene in E. coli and B. subtilis from an adjacent bacteriophage promoter was readily detected but in L. lactis expression of lysin was found to be lethal. The bacteriophage phi vML3 lysin had sequence homology with protein 15 of B. subtilis bacteriophage PZA. This protein is involved in DNA packaging during bacteriophage maturation rather than in host cell lysis. The cloning and analysis of the phi vML3 lysin gene is of importance in further understanding lactic streptococcal bacteriophages, for the development of positive selection vectors and for biotechnological applications of relevance to the dairy industry.

Amino Acid Sequence↗

The Lactic Acid Stress Response of Lactococcus lactis subsp. lactis

The lactic acid tolerance response (LATR) of the lactic acid bacterium Lactococcus lactis subsp. lactis has been studied. A dramatic increase in survival to a severe acid stress (pH 3.9) was obtained by preexposing the cells for 30 min to a mildly acid shock at pH 5.5. Whole-cell protein extract analysis revealed that during the acid tolerance response 33 polypeptides are induced over the level of naive cells. Among these are the major heat shock proteins DnaK and GroEL. In conjunction with a previous report (Hartke et al. 1994), the results establish that L. lactis can adapt to lactic acid exposure in two different ways: a logarithmic phase LATR, which may be activated by protons, and a stationary-phase LATR, which needs no activation by protons. Both systems are independent of de novo protein synthesis.

Journal Article↗

Isolation and characterization of a purC(orf)QLF operon from Lactococcus [correction of Lactobacillus] lactis MG1614.

We have isolated genes encoding enzymes of the de novo purine nucleotide biosynthesis pathway from Lactococcus lactis MG1614 by colony hybridization using DIG-labeled DNA probes. The organization of the genes needed for the de novo biosynthesis of purine nucleotides in L. lactis differs from that found in other organisms. In L. lactis there is a gene cluster, which contains five out of the 11 genes needed for the de novo biosynthesis of IMP, namely purC, orf, purQ, purL and purF. These genes were shown to be transcribed as a single transcription unit by Northern hybridization analysis. The 5' end of the transcript of the purC(orf)QLF operon was determined by primer extension analysis using fluorescently end-labeled probes. The purC(orf)QLF operon of L. lactis is transcribed in Escherichia coli, and the gene product of the purF gene, glutamine phosphoribosylpyrophosphate amidotransferase (glutamine PRPP ATase, EC 2.4.2.14), can functionally complement the E. coli purF mutant strain TX158. We also show that the promoter of the purC(orf)QLF operon is regulated in response to exogenously added purines.

Amino Acid Sequence↗

Genome comparison of Lactococcus strains by pulsed-field gel electrophoresis.

The two restriction enzymes SmaI and ApaI were found to produce distributions of DNA fragments useful for genome analysis of some lactic acid bacteria (Lactococcus lactis and Streptococcus salivarius subsp. thermophilus) by pulsed-field gel electrophoresis. The genome size was estimated to be 1750 to 2500 kb depending on the species. Each strain displayed unique restriction patterns; nevertheless, the percentage of the comigrating fragments of two isogenic or closely related strains was about 80% and fell to 20-40% when the patterns of two non-related strains of the same species or two strains belonging to different species were compared.

DNA, Bacterial↗

Identification, cloning and sequencing of the replication region of Lactococcus lactis ssp. lactis biovar. diacetylactis Bu2 citrate plasmid pSL2.

The replication region of the 7.8 kilobase (kb) citrate plasmid pSL2 from Lactococcus lactis ssp. lactis biovar. diacetylactis Bu2 was identified. Deletion derivatives of pSL2 were introduced into plasmid-free strain Bu2-60 and tested for their ability to replicate autonomously. The region necessary for replication was identified by comparison of the pSL2 derivatives, cloned and sequenced. No homologies were detected by comparing the putative Rep protein of pSL2 with replicons of other plasmids of Gram-positive bacteria. A part of an IS-element flanking the replication region was found.

Amino Acid Sequence↗

Homology model of the multidrug transporter LmrA from Lactococcus lactis.

LmrA is an ATP dependent multidrug transporter from Lactococcus lactis conferring antibiotic resistance to 17 out of 21 most frequently administered antibiotics. Starting from the dimeric crystal structure of Vc-MsbA, we built two homology models, with NBD:NBD interfaces reflecting the nonenergized and energized state, respectively. The TMD:TMD topology of the dimer is consistent with the previously obtained substrate photoaffinity labeling pattern suggesting binding of substrates at the TMD:TMD interface involving helix 3 of one monomer and helices 5 and 6 of the other monomer.

Bacterial Proteins↗

Monitoring the cell number of Lactococcus lactis subsp. cremoris FC in human feces by real-time PCR with strain-specific primers designed using the RAPD technique.

Strain-specific PCR primers for Lactococcus lactis subsp. cremoris FC were developed using the randomly amplified polymorphic DNA (RAPD) technique. RAPD was used to generate strain-specific markers. A 1164-bp RAPD marker found to be strain-specific was sequenced, and a primer pair specific for L. lactis subsp. cremoris FC was designed. The specificity of this primer pair was tested with 23 L. lactis subsp. cremoris strains and 20 intestinal bacterial species, and was found to be strain-specific. Subsequently, this primer pair was subjected to the quantification of L. lactis subsp. cremoris FC in the feces of subjects fed fermented milk containing this strain. After administration, L. lactis subsp. cremoris FC was detected in the feces of all 7 subjects, with the maximum number being between 10(5) and 10(9) cells g(-1) of feces. Furthermore, this strain was detected in only one feces sample 2 weeks after administration was stopped. These results suggest that L. lactis subsp. cremoris FC can survive passage through the gastrointestinal tract.

Adult↗

Lactic acid bacteria as a cell factory: rerouting of carbon metabolism in Lactococcus lactis by metabolic engineering.

Lactic acid bacteria display a relatively simple metabolism wherein the sugar is converted mainly to lactic acid. The extensive knowledge of metabolic pathways and the increasing information of the genes involved allows for the rerouting of natural metabolic pathways by genetic and physiological engineering. We discuss several examples of metabolic engineering of Lactococcus lactis for the production of important compounds, including diacetyl, alanine and exopolysaccharides.

Journal Article↗

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↗

Substrate specificity of a tripeptidase as a metalloenzyme purified from Lactococcus lactis subsp. lactis biovar. diacetylactis ATCC 13675.

The peptidase purified to homogeneity from Lactococcus lactis subsp. lactis biovar. diacetylactis ATCC 13675 was considered to be an aminotripeptidase (EC 3.4.11.4) from the results of substrate specificity. The K(m) value showed a tendency to decrease with the number of alanine residues, but to increase with the number of glycine residues in the substrate tripeptide. The effects of divalent metal ions on enzyme activity were considerably different depending on the tripepride used as a substrate. In the case of Mn2+, Co2+, Ni2+, Cu2+, Zn2+ and Cd2+, there was apparent correlation between enzyme activities observed in the presence of metal ions and following metal ion replacement. The Zn2+-replaced enzyme showed almost the same K(m) and k(cat) values as the native enzyme, suggesting the enzyme to be a zinc metallopeptidase. The K(m) of the divalent metal-replaced enzyme increased in the order of Co2+, Zn2+, and Mn2+. As a result of replacement with Co2+ an enzyme having 2.3-fold higher activity compared to the native enzyme for GGF as a substrate was obtained. Thus, the change in substrate specificity observed following metal replacement may suggest a highly specific interaction between the enzyme, metal and substrate, leading to the activity expression and stability of the tripeptidase.

Journal Article↗

Effect of alpha-acetolactate decarboxylase inactivation on alpha-acetolactate and diacetyl production by Lactococcus lactis subsp. lactis biovar diacetylactis.

Strains of Lactococcus lactis subsp. lactis biovar diacetylactis deficient in alpha-acetolactate decarboxylase produce alpha-acetolactate. This unstable compound is a precursor of acetoin and an aromatic compound, diacetyl. Following random mutagenesis of strain CNRZ 483, alpha-acetolactate decarboxylase-negative mutant 483 M1 was selected. When grown in milk, its growth and acidification characteristics were similar to those of the parental strain. In anaerobic conditions, the parental strain produced 2.10 mM acetoin and less than 0.05 mM diacetyl. The mutant accumulated up to 2.11 mM alpha-acetolactate, which spontaneously degraded to acetoin and diacetyl. After 24 h of culture, the alpha-acetolactate concentration was only 0.49 mM and the acetoin and diacetyl concentrations reached 1.50 mM and 0.26 mM, respectively. Diacetyl production by both strains increased in aerobic conditions, as well as when citrate was added. In contrast to cultures of the parental strain, however, diacetyl and acetoin concentrations in mutant cultures continued to increase without reaching a plateau. The results also showed that diacetyl production by wild type L. lactis subsp. lactis biovar diacetylactis strains cannot be explained uniquely by the spontaneous decarboxylation of the alpha-acetolactate produced in the culture medium.

Journal Article↗

Continuous production of lactic acid from molasses by perfusion culture of Lactococcus lactis using a stirred ceramic membrane reactor.

A perfusion culture system was used for continuous production of lactic acid by retaining cells at a high density of Lactococcus lactis in a stirred ceramic membrane reactor (SCMR). After the cell concentration increased to 248 g/l, half of the culture broth volume was replaced with the fermentation medium. Subsequently, a substrate solution containing glucose (run 1) or molasses (run 2) was continuously supplied to the cells retained in the SCMR. Simultaneously, the culture supernatant was extracted using a ceramic filter with a pore size of 0.2 mum. The dilution rate was initially set at 0.4 h(-1) and gradually decreased to 0.2 h(-1) due to reduction in the permeability of the filter. The concentration of glucose in the substrate solution was adjusted to 60 g/l for the transition and the first period until 240 h, 90 g/l for the second period from 240 h to 440 h, and 70 g/l for the third period from 440 h to 643 h. The average concentration of lactic acid in the filtrate reached 46 g/l in the first period, 43 g/l in the second period, and 33 g/l for the third period. The productivity obtained for the first period reached 15.8 g.l(-1).h(-1), twice as much as that achieved in repeated batch fermentations. Based on the results obtained in run 1, the substrate solution containing 120 g/l of molasses was continuously supplied for 240 h in run 2. The concentration and productivity of lactic acid reached 40 g/l and 10.6 g.l(-1).h(-1), respectively, by continuously replenishing the culture medium at a dilution rate of 0.26 h(-1). These results demonstrated that the filtration capacity of the SCMR was sufficient for a continuous and rapid replenishment of molasses solution from the dense cell culture and, therefore, the perfusion culture system is considered to provide a low-cost process for continuous production of lactic acid from cheap resources.

Journal Article↗

Purification and characterization of a bacteriocin produced by Lactococcus lactis subsp. lactis H-559 isolated from kimchi.

Lactic acid bacteria were isolated from kimchi and screened for bacteriocin production. Strain H-559, identified as Lactococcus lactis subsp. lactis, exhibited the strongest antibacterial activity among them and was active against pathogenic bacteria such as Listeria monocytogenes and Staphylococcus aureus as well as many lactic acid bacteria. The antimicrobial substance produced by L. lactis subsp. lactis H-559 was inactivated by alpha-chymotrypsin, and protease type IX and XIV and was confirmed to be a bacteriocin. The bacteriocin activity was stable from pH 2.0-11.0 and up to 10 min heating at 100 degrees C. The bacteriocin was sequentially purified by ammonium sulfate precipitation, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography (HPLC). Its molecular weight was determined to be 3343.7 Da by MALDI-mass spectrometry. Isoleucine was detected as the first N-terminal amino acid residue but the remaining amino acid sequence could not be determined by the Edman degradation method. It was different from other bacteriocins in terms of pH stability, molecular weight, amino acid composition, and the partial amino acid sequences of peptides obtained by acid hydrolysis.

Journal Article↗