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PubMed1998

Monoclonal antibodies raised against native major capsid proteins of lactococcal c2-like bacteriophages.

Phage Q38, a representative member of the c2 species, was purified by CsCl gradient and used to immunize BALB/c mice. Monoclonal antibodies (MAbs) were raised and then characterized by enzyme-linked immunosorbent assay. Two MAbs of isotype immunoglobulin G2a, designated 2A5 and 6G7, reacted only with phages belonging to the c2 species and not with phages of the 936 and P335 species, with a Lactococcus lactis cell extract, or with phage DNA. Immunoelectron microscopy showed that both MAbs recognized only phage head proteins. They did not react with any denatured phage proteins in Western blot assays. However, when the nitrocellulose membranes were treated with a Triton-based buffer to assist in protein renaturation, MAbs 2A5 and 6G7 recognized the two major capsid proteins with molecular masses of 80 and 170 kDa. Competitive inhibition tests showed that the two MAbs bind to overlapping epitopes. These MAbs may be a useful tool for monitoring c2 bacteriophages during dairy fermentation and in genetic studies.

Animals↗
PubMed2000

Nucleotide sequence analysis of the lactococcal EPS plasmid pNZ4000.

The complete 42180-bp nucleotide sequence of the mobilization plasmid pNZ4000, coding for exopolysaccharide (EPS) production in Lactococcus lactis, was determined. This plasmid contains a region involved in EPS biosynthesis, four functional replicons, a region containing mobilization genes, and three origin of transfer (oriT) sequences. Sequences identical to these oriT sequences were also found on two other lactococcal plasmids and a plasmid from Lactobacillus helveticus. Several complete and partial IS elements were identified on pNZ4000, including iso-ISS1, iso-IS946, and iso-IS982 sequences. Furthermore, pNZ4000 contains a gene cluster that may encode a cobalt transport system and a gene that encodes a CorA homologue which may function as a magnesium transporter.

Amino Acid Sequence↗
PubMed2005

Nisin biosynthesis and its properties.

The antimicrobial peptide, nisin, produced by several strains of Lactococcus lactis, which belongs to the Class I bacteriocins called lantibiotics, is a small (3.4 kDa), 34-amino acid, cationic, hydrophobic peptide and has the five characteristic (beta-methyl)lanthionine rings formed by significant post-translational modification. A cluster of 11 genes has been involved in the biosynthesis of nisin and are proposed to be transcriptionally arranged as nisA(Z)BTCIP, nisRK, and nisFEG. The biosynthesis of nisin is regulated in a growth-phase-dependent manner including nisin-mediated induction which occurs via NisRK two-component regulatory system. This review outlines some of the more recent developments in the properties, regulation and applications of nisin biosynthesis.

Anti-Bacterial Agents↗
PubMed1998

Retrohoming of a bacterial group II intron: mobility via complete reverse splicing, independent of homologous DNA recombination.

The mobile group II intron of Lactococcus lactis, Ll.LtrB, provides the opportunity to analyze the homing pathway in genetically tractable bacterial systems. Here, we show that Ll.LtrB mobility occurs by an RNA-based retrohoming mechanism in both Escherichia coli and L. lactis. Surprisingly, retrohoming occurs efficiently in the absence of RecA function, with a relaxed requirement for flanking exon homology and without coconversion of exon markers. These results lead to a model for bacterial retrohoming in which the intron integrates into recipient DNA by complete reverse splicing and serves as the template for cDNA synthesis. The retrohoming reaction is completed in unprecedented fashion by a DNA repair event that is independent of homologous recombination between the alleles. Thus, Ll.LtrB has many features of retrotransposons, with practical and evolutionary implications.

Bacterial Proteins↗
PubMed1998

Characterization of lactococci isolated from minimally processed fresh fruit and vegetables.

Lactic acid bacteria isolated from minimally processed fresh fruit and vegetable products were identified as Lactococcus lactis subsp. lactis on the basis of phenotypic tests, presence of lactococcal IS elements, and partial sequence analysis of the 16S rRNA gene. Isolated bacteria were differentiated using pulsed-field gel electrophoresis of SmaI digests of genomic DNA. Sprouted seeds were the best source of strains, and lactococci appear to be the dominant microflora on these products during the period they are intended to be eaten. Although these plant strains showed many similarities to strains of L. lactis used as dairy starter cultures, their carbohydrate fermentation patterns were unusual and probably reflect their environmental origin. Most strains fermented sucrose and xylose, and some also fermented raffinose and melibiose. Most of the bacteriocin-producing strains produced nisin, and nisin genes could also be detected in strains that showed no bacteriocin activity, or that produced a different bacteriocin with a narrow spectrum of activity. One strain produced nisin but was unable to ferment sucrose, properties that have been generally regarded as linked. These strains may have uses as biopreservatives for minimally processed plant products.

Blotting, Southern↗
PubMed2001

Design of a protein-targeting system for lactic acid bacteria.

We designed an expression and export system that enabled the targeting of a reporter protein (the staphylococcal nuclease Nuc) to specific locations in Lactococcus lactis cells, i.e., cytoplasm, cell wall, or medium. Optimization of protein secretion and of protein cell wall anchoring was performed with L. lactis cells by modifying the signals located at the N and C termini, respectively, of the reporter protein. Efficient translocation of precursor (approximately 95%) is obtained using the signal peptide from the lactococcal Usp45 protein and provided that the mature protein is fused to overall anionic amino acids at its N terminus; those residues prevented interactions of Nuc with the cell envelope. Nuc could be covalently anchored to the peptidoglycan by using the cell wall anchor motif of the Streptococcus pyogenes M6 protein. However, the anchoring step proved to not be totally efficient in L. lactis, as considerable amounts of protein remained membrane associated. Our results may suggest that the defect is due to limiting sortase in the cell. The optimized expression and export vectors also allowed secretion and cell wall anchoring of Nuc in food-fermenting and commensal strains of Lactobacillus. In all strains tested, both secreted and cell wall-anchored Nuc was enzymatically active, suggesting proper enzyme folding in the different locations. These results provide the first report of a targeting system in lactic acid bacteria in which the final location of a protein is controlled and biological activity is maintained.

Antigens, Bacterial↗
PubMed1996

Positive selection, cloning vectors for gram-positive bacteria based on a restriction endonuclease cassette.

Lactococcus lactis contains numerous restriction and modification (R/M) systems of different specificities. A novel IIS type R/M system encoded by the LlaI operon has previously been characterized from the L. lactis conjugative plasmid pTR2030. The LlaI operon is composed of six genes: First, a small regulatory gene llaIC precedes the methylase gene llaIM. The following three genes, llaI.1, llaI.2, llaI.3, are all essential for restriction endonuclease activity and are designed as the restriction cassette llaIR. The forth open reading frame of unknown function follows the llaIR gene cassette. We have successfully subcloned the three llaIR genes, llaI.1, llaI.2, and llaI.3, without llaIM, as a suicide cassette into the three shuttle vectors pTRKL2, pTRKH2, and pBV5030. A promoter (P6) from Lactobacillus acidophilus ATCC4356, which is functional in E. coli, lactococci, and lactobacilli (Djordjevic and Topisirovic, unpublished) was cloned upstream of the three gene cassette. Restriction activity was evaluated in Escherichia coli and several gram-positive bacteria. The llaIR restriction cassette was not functional in E. coli, but its presence was lethal to L. lactis, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus acidophilus, Carnobacterium pisicola, Enterococcus faecalis, Bacillus subtilis, and Leuconostoc gelidum. Several novel, positive selection cloning vectors were developed that can exploit unique cloning sites within the llaIR cassette. Insertions in llaI.1 resulted in complete inactivation of restriction activity and provided unconditional selection for recombinant plasmids in surviving transformants. These positive selection cloning vectors are the first for gram-positive bacteria that are based on a restriction endonuclease cassette. Functional activity of the llaIR genes in various gram-positive bacteria would also enable use of these cloning vectors for positive selection of promoters, terminators, and regulatory sequences across these genera.

Cloning, Molecular↗
PubMed1991

Identification and characterization of the lantibiotic nisin Z, a natural nisin variant.

Lactococcus lactis strain NIZO 22186 produces an extracellular, lanthionine-containing 3.5-kDa polypeptide with antimicrobial activity. Its retention time on reversed-phase (RP) HPLC and its amino acid composition showed high similarities but no complete identity to nisin. The gene for this lantibiotic, designated nisZ, has been cloned and its nucleotide sequence was found to be identical to that of the precursor nisin gene apart from a single mutation resulting in the substitution His27Asn in the mature polypeptide. NMR studies of the natural nisin variant, which has been designated nisin Z, confirmed the His27Asn substitution and indicated that it has a similar structure to nisin.

Amino Acid Sequence↗
PubMed2004

The Lactococcal abortive phage infection system AbiP prevents both phage DNA replication and temporal transcription switch.

We describe here a new lactococcal abortive phage infection system, designated AbiP. AbiP is effective against some lactococcal phages of one prevalent group, 936, but not against phages from the other two groups (c6A and P335). It was identified in the Lactococcus lactis subsp. cremoris strain IL420, on the native plasmid pIL2614. AbiP is encoded by a single gene, expressed in an operon with a second gene. In this work, abiP is shown to affect both the replication and transcription of phage DNA. In AbiP(+) cells, phage DNA replication is arrested approximately 10 min after infection. Levels of middle and late phage transcripts are lower in AbiP(+) than in AbiP(-) cells, probably due to the smaller amount of phage DNA. By contrast, early phage transcripts are more abundant in AbiP(+) than in AbiP(-) cells, suggesting that the switch-off, which occurs 15 min after infection in AbiP(-) cells, is prevented in AbiP(+) cells.

Bacterial Proteins↗
PubMed1993

Monitoring tripeptidase activity using capillary electrophoresis. Comparison with the ninhydrin assay.

Capillary electrophoresis (CE) was used to assay the activity of a tripeptidase from a crude extract of Lactococcus lactis subsp. lactis NCDO 712 against the substrate, Gly-Gly-Phe and a comparison with a standard ninhydrin assay was made. Standard curves of the substrates and products showed a significantly variable colorimetric reaction to ninhydrin making accurate quantification of the tripeptidase problematic. The CE assay further demonstrated that the presence of contaminating enzymes in crude cell-free extracts can cause secondary reactions that are not apparent from the ninhydrin assay data. The CE assay was also able to generate enzyme kinetics data and monitor, during purification, the presence of co-eluting contaminating activities. The speed and sensitivity with CE allows routine analysis of the tripeptidase activity without any derivatization normally required for this enzyme.

Amino Acid Sequence↗
PubMed1992

A lactococcal expression system for engineered nisins.

The nisin-producing Lactococcus lactis strain FI5876 has been modified and developed for use as an expression system for engineered nisin variants. Insertional inactivation of the resident nisA gene had a polar effect on downstream genes, including those involved in nisin immunity. However, subsequent chromosomal rearrangements in this region involving a newly discovered insertion element (IS905) generated a strain that was deficient in the nisA gene product but expressed those nisin determinants necessary for prenisin maturation, secretion, and immunity. Complementation of the lesion in the nisA gene by plasmid-encoded nisA genes containing site-specific mutations resulted in the exclusive production of altered nisins containing specific amino acid substitutions.

Amino Acid Sequence↗
PubMed2004

Overexpression of Lactobacillus casei D-hydroxyisocaproic acid dehydrogenase in cheddar cheese.

Metabolism of aromatic amino acids by lactic acid bacteria is an important source of off-flavor compounds in Cheddar cheese. Previous work has shown that alpha-keto acids produced from Trp, Tyr, and Phe by aminotransferase enzymes are chemically labile and may degrade spontaneously into a variety of off-flavor compounds. However, dairy lactobacilli can convert unstable alpha-keto acids to more-stable alpha-hydroxy acids via the action of alpha-keto acid dehydrogenases such as d-hydroxyisocaproic acid dehydrogenase. To further characterize the role of this enzyme in cheese flavor, the Lactobacillus casei d-hydroxyisocaproic acid dehydrogenase gene was cloned into the high-copy-number vector pTRKH2 and transformed into L. casei ATCC 334. Enzyme assays confirmed that alpha-keto acid dehydrogenase activity was significantly higher in pTRKH2:dhic transformants than in wild-type cells. Reduced-fat Cheddar cheeses were made with Lactococcus lactis starter only, starter plus L. casei ATCC 334, and starter plus L. casei ATCC 334 transformed with pTRKH2:dhic. After 3 months of aging, the cheese chemistry and flavor attributes were evaluated instrumentally by gas chromatography-mass spectrometry and by descriptive sensory analysis. The culture system used significantly affected the concentrations of various ketones, aldehydes, alcohols, and esters and one sulfur compound in cheese. Results further indicated that enhanced expression of d-hydroxyisocaproic acid dehydrogenase suppressed spontaneous degradation of alpha-keto acids, but sensory work indicated that this effect retarded cheese flavor development.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2000

Use of hydrolyzed whey peptide to inhibit culture agglutination.

Papain was used to hydrolyze sweet whey to prepare peptides that were harvested with ultrafiltration membranes (molecular weight cutoffs of 10,000, 3000, and 1000). Insure buffer salts were added to whey peptides (ratio 40:60% solids, respectively) to prepare media that were tested for their ability to inhibit culture agglutination. Commercial Insure medium (75.7 g/l) was used as a control. Skim milk (240 ml) in 250-ml graduated cylinders was inoculated (4%) with Lactococcus lactis spp. lactis B62 or E72. Culture agglutination was determined by measuring upper center and bottom pH values of the skim milk column during 5 h of incubation. A pH differential was calculated by subtracting the bottom pH from the upper center pH. Cultures grown in media containing whey peptides agglutinated in skim milk to a lesser degree than when grown in the control medium. Culture agglutination was inhibited to a greater degree when cultures were grown in the 1000 molecular weight cutoff peptide medium than when grown in the 10,000 or 3000 molecular weight cutoff peptide medium. When culture E72 was grown in medium containing 1000 molecular weight cutoff peptides, culture agglutination was completely inhibited.

Agglutination↗
PubMed1997

The lytic enzyme of the pneumococcal phage Dp-1: a chimeric lysin of intergeneric origin.

We have localized, cloned and characterized the genes coding for the lytic system of the pneumococcal phage Dp-1. The lytic enzyme of this phage (Pal), previously identified as an N-acetyl-muramoyl-L-alanine amidase, shows a modular organization similar to that described for the lytic enzymes of Streptococcus pneumoniae and its bacteriophages. The construction of chimeric enzymes between pneumococcus and bacteria (or phages) that belong to different Gram-positive families has shown that the interchange of functional domains switches enzyme specificity. Interestingly, Pal appears to be a natural chimeric enzyme of intergeneric origin, that is the N-terminal domain was highly similar to that of the murein hydrolase coded by a gene found in the phage BK5-T that infects Lactococcus lactis, whereas the C-terminal domain was homologous to those found in the lytic enzymes of the pneumococcal system that is responsible for the binding to the choline residues present in the cell wall substrate. Biochemical analysis of Pal revealed that this enzyme shares important properties with those of the major LytA101 autolysin found in an atypical, clinical pneumococcal isolate. These peculiar characteristics have been ascribed to a modified C-terminal domain. The natural chimeric enzyme described here provides further support for the theory of modular evolution of proteins and its characteristics also furnish interesting clues on the molecular mechanisms involved in the more invasive types of atypical pneumococci.

Amino Acid Sequence↗
PubMed2000

Characterization of mesophilic mixed starter cultures used for the manufacture of aged cheddar cheese.

Seventy-one different Lactococcus lactis subsp. cremoris strains were isolated from seven mesophilic mixed starters used in the manufacture of aged Cheddar cheese in Canada. Based on plasmid profiles and growth in milk (with or without glucose, Casamino Acids or both), two mixed starters were highly heterogeneous, containing at least 18 to 24 distinct L. lactis strains. Three mixed starters were comprised of seven to nine strains, whereas two starters were relatively homogeneous, containing two or three strains. Many strains with similar plasmid profiles behaved differently during growth in milk, indicating variability in the phenotypes. Only 20% of the strains could grow in plain milk, whereas 30% could not grow in milk supplemented with glucose and Casamino Acids. Twenty-five lactococcal bacteriophages were also isolated from whey samples with single strains as hosts. Eighteen phages belonged to the 936 species and seven to the c2 species. Thirteen strains were insensitive to all 25 phages. Almost all sensitive strains were phage species-specific. The 936-like phages had a broader host range.

Bacteriophages↗
PubMed2006

Multiplex PCR for the detection and identification of dairy bacteriophages in milk.

Bacteriophage infections of starter lactic acid bacteria are a serious risk in the dairy industry. Phage infection can lead to slow lactic acid production or even the total failure of fermentation. The associated economic losses can be substantial. Rapid and sensitive methods are therefore required to detect and identify phages at all stages of the manufacture of fermented dairy products. This study describes a simple and rapid multiplex PCR method that, in a single reaction, detects the presence of bacteriophages infecting Streptococcus thermophilus and Lactobacillus delbrueckii, plus three genetically distinct 'species' of Lactococcus lactis phages commonly found in dairy plants (P335, 936 and c2). Available bacteriophage genome sequences were examined and the conserved regions used to design five pairs of primers, one for each of the above bacteriophage species. These primers were designed to generate specific fragments of different size depending on the species. Since this method can detect the above phages in untreated milk and can be easily incorporated into dairy industry routines, it might be readily used to earmark contaminated milk for use in processes that do not involve susceptible starter organisms or for use in those that involve phage-deactivating conditions.

Animals↗
PubMed2001

Changes in rRNA levels during stress invalidates results from mRNA blotting: fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels.

Regulation of gene expression can be analyzed by a number of different techniques. Some techniques monitor the level of specific mRNA directly, and others monitor indirectly by determining the level of enzymes encoded by the mRNA. Each method has its own inherent way of normalization. When results obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting experiments, in which mRNA levels routinely are normalized to a fixed amount of extracted total RNA. The cellular levels of specific mRNA species were estimated using a renormalization with the total RNA content per cell. By a combination of fluorescence in situ rRNA hybridization, which estimates the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes in the hrcA-grpE-dnaK operon was analyzed. The hybridization data suggested a complex heat shock regulation indicating that the mRNA levels continued to rise after 30 min, but after renormalization the calculated average cellular levels exhibited a much simpler induction pattern, eventually attaining a moderately increased value.

Cell Membrane Permeability↗
PubMed2003

Pediocin production by recombinant lactic acid bacteria.

Production of the anti-listerial bacteriocin, pediocin, by lactic acid bacteria (LAB) transformed with the cloning vector pPC418 (Ped+, 9.1 kb) was influenced by composition of media and incubation temperature. Maximum pediocin production, tested against Listeria innocua, by electrotransformants of Lactococcus lactis ssp. lactis was measured in tryptone/lactose/yeast extract medium after 24 h growth at 30 degrees C, while incubation at 40 degrees C was optimum for Ped+ transformants of Streptococcus thermophilus and Enterococcus faecalis. The amount of pediocin produced by S. thermophilus in skim milk and cheese whey supplemented with 0.5% yeast extract was estimated as 51,000 units ml(-1) and 25,000 units ml(-1), respectively. Pediocin production remained essentially unchanged in reconstituted skim milk or whey media diluted up to 10-fold. The results demonstrate the capacity of recombinant strains of LAB to produce pediocin in a variety of growth media including skim milk and inexpensive cheese whey-based media, requiring minimum nutritional supplementation.

Animals↗