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PubMed2002

Widespread distribution of the bacteriocin-producing lactic acid cocci in Miso-paste products.

AIMS: A survey was conducted on the ecological distribution of bacteriocin-producing lactic acid bacteria in Miso-pastes, a typical fermented food in Japan. METHODS AND RESULTS Nine Miso-pastes were sampled for isolation of bacteriocin-producers. Almost all isolated enterococcal strains produced bacteriocins but no isolated tetragenococci did so. The bacteriocin-producing isolates were divided into nine groups by phenotypic tests. As the phenotypic characters were highly diverse, these strains could not be identified to species level on the basis of their phenotypes. The nine representative strains from each group were identified by 16S rRNA analysis. These bacteriocin-producers with one exception (Lactococcus sp.) were identified as strains of the Enterococcus faecium 'species group'. The bacteriocins of the nine strains were classified into five types according to their antibacterial spectral patterns and their SDS-PAGE profiles. The bacteriocins inhibited undesirable bacteria in Miso-pastes, such as Bacillus subtilis, but did not inhibit the useful Tetragenococcus halophila. CONCLUSIONS: The bacteriocin-producing lactic acid cocci were widespread at high frequencies in Miso-pastes. They were considered to play an important role in preventing the growth of undesirable bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that bacteriocin-producers act effectively as safe biopreservatives and may contribute to the biopreservation in Miso-pastes.

Bacteriocins↗
PubMed2003

Inhibition of Staphylococcus aureus by the commensal bacteria of human milk.

AIMS: To study the bacterial diversity in expressed human milk with a focus on detecting bacteria with an antimicrobial activity against Staphylococcus aureus, known as a causative agent of maternal breast infections and neonatal infections. METHODS AND RESULTS: Random isolates (n = 509) were collected from breast milk samples (n = 40) of healthy lactating women, genotypically identified, and tested for antimicrobial activity against Staph. aureus. Commensal staphylococci (64%) and oral streptococci (30%), with Staph. epidermidis, Strep. salivarius, and Strep. mitis as the most frequent isolates, were the predominant bacterial species in breast milk. One-fifth of Staph. epidermidis and half of Strep. salivarius isolates suppressed growth of Staph. aureus. Enterococci (Ent. faecalis), isolated from 7.5% of samples, and lactic acid bacteria (LAB) (Lactobacillus rhamnosus, Lact. crispatus, Lactococcus lactis, Leuconoctoc mesenteroides), isolated from 12.5% of samples, were also effective against Staph. aureus. One L. lactis isolate was shown to produce nisin, a bacteriocin used in food industry to prevent bacterial pathogens and spoilage. CONCLUSIONS: Expressed breast milk contains commensal bacteria, which inhibit Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The strains inhibitory against the pathogen Staph. aureus have potential use as bacteriotherapeutic agents in preventing neonatal and maternal breast infections caused by this bacterium.

Antibiosis↗
PubMed2004

Characterization of closely related lactococcal starter strains which show differing patterns of bacteriophage sensitivity.

AIMS: To characterize a group of closely related Lactococcus lactis subsp. lactis casein starter strains used commercially, which differ in their sensitivity to bacteriophages isolated from the same industrial environment. METHODS AND RESULTS: Nine strains of L. lactis, six of which had been used as starter cultures for lactic casein manufacture, were shown to be closely related by pulsed-field gel electrophoresis and total DNA profiles. Nineteen phages which propagated on one or more of these starter strains were isolated from industrial casein whey samples. The phages were all small isometric-headed and could be divided into five groups on the basis of host range on the nine strains. Most of the phages did not give a PCR product with primers designed to detect the two most common lactococcal small isometric phage species (936 and P335). The hosts could be divided into six groups depending on their phage sensitivity. Plasmids encoding genes for the cell envelope associated PI-type proteinase, lactose metabolism and specificity subunits of a type I restriction/modification system were identified. CONCLUSIONS: This work demonstrates how isolates of the same starter strain may come to be regarded as separate cultures because of their different origins, and how these closely related strains may differ in some of their industrially relevant characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: This situation may be very common among lactococci used as dairy starter cultures, and implies that the dairy industry worldwide depends on a small number of different strains.

Bacterial Typing Techniques↗
PubMed1996

Dramatic decay of phage transcripts in lactococcal cells carrying the abortive infection determinant AbiB.

The abortive infection determinant AbiB prevents growth of the sensitive phage bIL170, but not of the resistant phage bIL41, on Lactococcus lactis strain IL1403. Here we show that AbiB promotes a dramatic degradation of sensitive phage transcripts, starting 10-15 min after infection. The decay of the transcripts is the probable cause of the arrest of the sensitive phage development. Mapping of the 5' end of degradation products established that they result from endonucleolytic cleavage preferentially at U/U, A/U and U/A sites. We propose that an early product of the sensitive phage either induces the synthesis or stimulates the activity of an RNase in an AbiB+ cell.

Bacterial Proteins↗
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

Regulation of intron function: efficient splicing in vivo of a bacterial group II intron requires a functional promoter within the intron.

Conjugative transfer of the Lactococcus lactis plasmid pRS01 requires splicing of a group II intron, Ll.ltrB, for accurate translation of the mRNA for the exon gene ltrB. The protein product of ltrB is a conjugative relaxase, essential for pRS01 transfer. Using a molecular technique for the identification of transcription initiation sites in bacteria, a functional promoter within Ll.ltrB was identified upstream from the gene for the intron-encoded protein (IEP) LtrA. LtrA is required for efficient splicing of Ll.ltrB in vivo. Mutation of the ltrA promoter dramatically reduced the steady-state level of ltrA mRNA, LtrA, intron splicing and conjugation in L. lactis. These effects could be relieved by expression in trans of the ltrA gene cloned under the control of an inducible promoter. These results suggest that the ltrA mRNAs are translated inefficiently. We hypothesize that this bacterial intron, in contrast to previously studied group II introns in eukaryotes, requires a promoter within the intron to regulate ltrA expression and to produce an adequate level of the protein in the cell for efficient splicing.

Amino Acid Sequence↗
PubMed2001

Regulation of immunity to the two-component lantibiotic, lacticin 3147, by the transcriptional repressor LtnR.

Lacticin 3147 is a membrane-active, two-component lantibiotic produced by Lactococcus lactis ssp. lactis DPC3147. In this study, the promoters of the lacticin 3147 gene cluster were mapped to the intergenic region between ltnR and ltnA1 (the genes encoding the regulatory protein LtnR and the first structural gene, LtnA1), and Northern analyses revealed that the biosynthetic and immunity genes are divergently transcribed in two operons, ltnA1A2M1TM2D and ltnRIFE respectively. Although the promoter controlling biosynthesis (Pbac) appears to be constitutive, characterization of a downstream beta-galactosidase (beta-gal) fusion beyond an intragenic stem-loop structure in ltnM1 confirmed that this putative transcriptional attenuator allows limited readthrough to the downstream biosynthetic genes, thus maintaining the correct stoichiometry between structural peptides and biosynthetic machinery. The promoter of the ltnRIFE operon (Pimm) was shown to be regulated by the transcriptional repressor LtnR. A mutant with a truncated ltnR gene exhibited a hyperimmune phenotype, whereas overexpression of ltnR resulted in cells with increased sensitivity to lacticin 3147. Gel mobility shift analysis indicated that LtnR binds to the Pimm promoter region, and fusion of this promoter to the beta-gal gene of pAK80 revealed that expression from Pimm is significantly reduced in the presence of LtnR. Thus, we have demonstrated that lacticin 3147 uses a regulatory mechanism not previously identified in lantibiotic systems.

Anti-Bacterial Agents↗
PubMed2002

RusA proteins from the extreme thermophile Aquifex aeolicus and lactococcal phage r1t resolve Holliday junctions.

The RusA protein of Escherichia coli is a DNA structure-specific endonuclease that resolves Holliday junction intermediates formed during DNA replication, recombination and repair by introducing symmetrically paired incisions 5' to CC dinucleotides. It is encoded by the defective prophage DLP12, which raises the possibility that it may be of bacteriophage origin. We show that rusA-like sequences are indeed often associated with prophage sequences in the genomes of several bacterial species. They are also found in many bacteriophages, including Lactococcus lactis phage r1t. However, rusA is also present in the chromosome of the hyperthermophilic bacterium Aquifex aeolicus. In this case, there is no obvious association of rusA with prophage-like sequences. Given the ancient lineage of Aquifex aeolicus, this observation provides the first indication that RusA may be of bacterial origin. The RusA proteins of A. aeolicus and bacteriophage r1t were purified and shown to resolve Holliday junctions. The r1t enzyme also promotes DNA repair in strains lacking the RuvABC resolvase. Both enzymes cleave junctions in a sequence-dependent manner, but the A. aeolicus RusA shows a different sequence preference (3' to TG) from the E. coli protein (5' to CC), and the r1t RusA has relaxed sequence dependence, requiring only a single cytosine.

Amino Acid Sequence↗
PubMed2002

Positive role of peptidoglycan breaks in lactococcal biofilm formation.

Bacterial attachment to solid matrices depends on adhesive molecules present on the cell surface. Here we establish a positive correlation between peptidoglycan (PG) breaks, rather than particular molecules, and biofilm-forming capacity in the Gram-positive bacterium Lactococcus lactis. The L. lactis acmA strain, which is defective in PG hydrolase, adhered less efficiently than the wild-type (wt) strain to different solid surfaces and was unable to form biofilms. These phenotypes were abolished by addition of lysozyme, a PG hydrolytic enzyme. Thus, the presence of PG breaks introduced by PG hydrolase, and not the AcmA protein itself, appears to be responsible for biofilm formation. Two different genetic screens confirmed the importance of PG breaks in L. lactis biofilm formation. Using the chain-forming ability of the acmA strain as a phenotypic indicator of PG integrity, we selected for insertional mutants generating short chains. Five independent mutants were all mapped to ponA, which encodes the PG synthesis enzyme PBP1A. Double acmA ponA mutants displayed increased adhesion and biofilm-forming capacity. Direct selection for strains with increased biofilm-forming capacity resulted in the isolation of another five mutations in ponA. Based on these results, we conclude that PG breaks are important for both adhesion and biofilm formation in L. lactis.

Bacterial Adhesion↗
PubMed1999

Post-translational modification of nisin. The involvement of NisB in the dehydration process.

The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis. As with all lantibiotics, nisin contains a number of dehydro-residues and thioether amino acids that introduce five lanthionine rings into the target peptide. These atypical amino acids are introduced by post-translational modification of a ribosomally synthesized precursor peptide. In certain cases, the serine residue, at position 33 of nisin, does not undergo dehydration to Dha33. With native nisin this partially processed form represents about 10% of the total peptide, whereas with the engineered variants, [Trp30]nisin A and [Lys27,Lys31]nisin A, the proportion of peptide that escapes full processing was found to be to approximately 50%. This feature of nisin biosynthesis was exploited in an investigation of the role of the NisB protein in pre-nisin maturation. Manipulation of the level of NisB was achieved by cloning and overexpressing the plasmid-encoded nisB gene in a range of different nisin-producing strains. The resulting fourfold increase in the level of NisB significantly increased the efficiency of the dehydration reaction at Ser33. The final secreted product of biosynthesis by these strains was the homogenous form of the fully processed nisin (or nisin variant) molecule. The results presented represent the first experimental evidence for the direct involvement of the NisB protein in the maturation process of nisin.

Amino Acid Sequence↗
PubMed2002

Beta-glucose 1-phosphate-interconverting enzymes in maltose- and trehalose-fermenting lactic acid bacteria.

Maltose and trehalose catabolic pathways are linked through their common enzyme, beta-phosphoglucomutase, and metabolite, beta-glucose 1-phosphate, in Lactococcus lactis. Maltose is degraded by the concerted action of maltose phosphorylase and beta-phosphoglucomutase, whereas trehalose is assimilated by a novel pathway, including the recently discovered enzyme, trehalose 6-phosphate phosphorylase, and beta-phosphoglucomutase. In the present study, 40 strains of lactic acid bacteria were investigated for utilization of metabolic reactions involving beta-glucose 1-phosphate. All genera of the low G+C content lactic acid bacteria belonging to the clostridial subbranch of Gram-positive bacteria were represented in the study. The strains, which fermented maltose or trehalose, were investigated for beta-phosphoglucomutase, maltose phosphorylase and trehalose 6-phosphate phosphorylase activity, as indications of maltose and trehalose catabolic pathways involving beta-glucose 1-phosphate interconversions. Eighty per cent of all strains fermented maltose and, of these strains, 63% were shown to use a maltose phosphorylase/beta- phosphoglucomutase pathway. One-third of the strains fermenting trehalose were found to harbour trehalose 6-phosphate phosphorylase activity, and these were also shown to possess beta-phosphoglucomutase activity. Mainly L. lactis and Enterococcus faecalis strains were found to harbour the novel trehalose 6-phosphate phosphorylase/beta-phosphoglucomutase pathway. As lower beta-glucose 1-phosphate interconverting enzyme activities were observed in the majority of glucose-cultivated lactic acid bacteria, glucose was suggested to repress the synthesis of these enzymes in most strains. Thus, metabolic reactions involving the beta-anomer of glucose 1-phosphate are frequently found in both maltose- and trehalose-utilizing lactic acid bacteria.

Culture Media↗
PubMed2002

Staphylococcus aureus clumping factor B (ClfB) promotes adherence to human type I cytokeratin 10: implications for nasal colonization.

Staphylococcus aureus is an important cause of sepsis in both community and hospital settings, a major risk factor for which is nasal carriage of the bacterium. Eradication of carriage by topical antibiotics reduces sepsis rates in high-risk individuals, an important strategy for the reduction of nosocomial infection in targeted patient populations. Understanding the mechanisms by which S. aureus adheres to nasal epithelial cells in vivo may lead to alternative methods of decolonization that do not rely on sustained antimicrobial susceptibility. Here, we demonstrate for the first time that the S. aureus surface-expressed protein, clumping factor B (ClfB), promotes adherence to immobilized epidermal cytokeratins in vitro. By expressing a range of S. aureus adhesins on the surface of the heterologous host Lactococcus lactis, we demonstrated that adherence to epidermal cytokeratins was conferred by ClfB. Adherence of wild-type S. aureus was inhibited by recombinant ClfB protein or anti-ClfB antibodies, and S. aureus mutants defective in ClfB adhered poorly to epidermal cytokeratins. Expression of ClfB promoted adherence of L. lactis to human desquamated nasal epithelial cells, and a mutant of S. aureus defective in ClfB had reduced adherence compared with wild type. ClfB also promoted adherence of L. lactis cells to a human keratinocyte cell line. Cytokeratin 10 molecules were shown by flow cytometry to be exposed on the surface of both desquamated nasal epithelial cells and keratinocytes. Cytokeratin 10 was also detected on the surface of desquamated human nasal cells using immunofluorescence, and recombinant ClfB protein was shown to bind to cytokeratin K10 extracted from these cells. We also showed that ClfB is transcribed by S. aureus in the human nares. We propose that ClfB is a major determinant in S. aureus nasal colonization.

Adhesins, Bacterial↗
PubMed2001

Characterization of lactic acid bacteria in the larval midgut of the keratinophagous lepidopteran, Hofmannophila pseudospretella.

In two of eight Hofmannophila pseudospretella specimens studied by microscopy, the larval midgut contained an unidentified micro-organism. Although not seen microscopically in midgut sections, bacteria were isolated from dissected midgut. Microscopy, carbohydrate utilization and ribosomal sequence data all separated the isolates into the same three classes. These were identified as Lactococcus lactis, Carnobacterium piscicola and, tentatively, Bacillus subtilis, the first two being facultative anaerobes and the latter, an aerobe. The bacteria were therefore not specifically adapted to the reducing conditions of the midgut.

Animals↗
PubMed2001

Heterologous expression of lacticin 3147 in Enterococcus faecalis: comparison of biological activity with cytolysin.

Lacticin 3147 is a broad-spectrum, two-component, lanthionine-containing bacteriocin produced by Lactococcus lactis DPC3147 which has widespread food and biomedical applications as a natural antimicrobial. Other two-component lantibiotics described to date include cytolysin and staphylococcin C55. Interestingly, cytolysin, produced by Enterococcus faecalis, has an associated haemolytic activity. The objective of this study was to compare the biological activity of lacticin 3147 with cytolysin. The lacticin 3147-encoding determinants were heterologously expressed in Ent. faecalis FA2-2, a plasmid-free strain, to generate Ent. faecalis pOM02, thereby facilitating a direct comparison with Ent. faecalis FA2-2.pAD1, a cytolysin producer. Both heterologously expressed lacticin 3147 and cytolysin exhibited a broad spectrum of activity against bacterial targets. Furthermore, enterococci expressing active lacticin 3147 did not exhibit a haemolytic activity against equine blood cells. The results thus indicate that the lacticin 3147 biosynthetic machinery can be heterologously expressed in an enterococcal background resulting in the production of the bacteriocin with no detectable haemolytic activity.

Amino Acid Sequence↗
PubMed2001

An empirical model for describing the effects of nitrogen sources on nisin production.

AIMS: A study on the effects of tryptone and yeast extract on nisin production by Lactococcus lactis was carried out using a second order rotatable factorial design. METHODS AND RESULTS: The results show that both ingredients increased nisin production, although a small decrease in nisin levels was noted at high tryptone concentrations. In view of the low vitamin content of tryptone, the amino acids present in both tryptone and yeast extract may be responsible for these observations. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The observed response suggests that maintaining tryptone concentrations in the centre of the domain and increasing the concentration of yeast extract would be ideal conditions for nisin production. However, when the economic aspects are considered, it appears that low concentrations of yeast extract and reasonably high concentrations of tryptone are optimal.

Anti-Bacterial Agents↗
PubMed2002

Influence of lactococcal plasmid on the specific growth rate of host cells.

AIMS: To investigate a lactococcal plasmid responsible for a reduction in growth rate of its host cell. METHODS AND RESULTS: Lactococcus lactis subsp. lactis biovar. diacetylactis DRC1 carries a high number of plasmids. The DRC1 wild-type strain was found to grow more slowly than a plasmid-free derivative of DRC1. The plasmids extracted from DRC1 together with an indicator plasmid were cotransformed into the plasmid-free strain DRC1021. A 7.4-kb cryptic plasmid, designated pDR1-1, was found to significantly affect the maximum specific growth rate ( micro max) of the host cell. Polymerase chain reaction (PCR) analysis was carried out in order to detect the presence of pDR1-1 in the other L. lactis strains. The micro max of the single pDR1-1-positive strain was determined to be the same as that of DRC1. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that pDR1-1 (or a pDR1-1-like plasmid) is a critical factor in the reduction of the micro max of DRC1, and that its effect on the micro max is significantly greater than that of any other coexisting plasmid.

Fermentation↗
PubMed2006

Single-molecule kinetics reveals signatures of half-sites reactivity in dihydroorotate dehydrogenase A catalysis.

Subunit activity and cooperativity of a homodimeric flavoenzyme, dihydroorotate dehydrogenase A (DHODA) from Lactococcus lactis, were characterized by employing single-molecule spectroscopy to follow the turnover kinetics of individual DHODA molecules, eliminating ensemble averaging. Because the enzyme-bound FMN is fluorescent in its oxidized state but not when reduced, a single DHODA molecule exhibits stepwise fluorescence changes during turnover, providing a signal to determine reaction kinetics and study cooperativity. Our results showed significant heterogeneity in the catalytic behaviors of individual dimer molecules, with only 40% interconverting between the three possible redox states: the fully fluorescent (both subunits oxidized), the half-fluorescent (one subunit oxidized and the other reduced), and the nonfluorescent (both subunits reduced). Forty percent of the single dimer traces showed turnovers between only the fully fluorescent and half-fluorescent states. The remaining 20% of the molecules interconverted only between the half-fluorescent state and the nonfluorescent state. Kinetic analysis revealed very similar reaction rates in both the reductive and oxidative half-reactions for different DHODA dimers. Our single-molecule data provide strong evidence for half-sites reactivity, in which only one subunit reacts at a time. The present study presents an effective way to explore the subunit catalytic activity and cooperativity of oligomeric enzymes by virtue of single-molecule fluorescence.

Bacterial Proteins↗
PubMed2006

Visualization of a group II intron in the 23S rRNA of a stable ribosome.

Thousands of introns have been localized to rRNA genes throughout the three domains of life. The consequences of the presence of either a spliced or an unspliced intron in a rRNA for ribosome assembly and packaging are largely unknown. To help address these questions, and to begin an intron imaging study, we selected a member of the self-splicing group II intron family, which is hypothesized to be the progenitor not only of spliceosomal introns but also of non-LTR retrotransposons. We cloned the self-splicing group II Ll.LtrB intron from Lactococcus lactis into L. lactis 23S rRNA. The 2,492-nt Ll.LtrB intron comprises a catalytic core and an ORF, which encodes a protein, LtrA. LtrA forms a ribonucleoprotein (RNP) complex with the intron RNA to mediate splicing and mobility. The chimeric 23S-intron RNA was shown to be splicing proficient in its native host in the presence of LtrA. Furthermore, a low-resolution cryo-EM reconstruction of the L. lactis ribosome fused to the intron-LtrA RNP of a splicing-defective Ll.LtrB intron was obtained. The image revealed the intron as a large, well defined structure. The activity and structural integrity of the intron indicate not only that it can coexist with the ribosome but also that its presence permits the assembly of a stable ribosome. Additionally, we view our results as a proof of principle that ribosome chimeras may be generally useful for studying a wide variety of structured RNAs and RNP complexes that are not amenable to NMR, crystallographic, or single-particle cryo-EM methodologies.

Bacterial Proteins↗