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PubMed2000

Antimicrobial peptides of lactic acid bacteria: mode of action, genetics and biosynthesis.

A survey is given of the main classes of bacteriocins, produced by lactic acid bacteria: I. lantibiotics II. small heat-stable non-lanthionine containing membrane-active peptides and III. large heat-labile proteins. First, their mode of action is detailed, with emphasis on pore formation in the cytoplasmatic membrane. Subsequently, the molecular genetics of several classes of bacteriocins are described in detail, with special attention to nisin as the most prominent example of the lantibiotic-class. Of the small non-lanthionine bacteriocin class, the Lactococcus lactococcins, and the Lactobacillus sakacin A and plantaricin A-bacteriocins are discussed. The principles and mechanisms of immunity and resistance towards bacteriocins are also briefly reported. The biosynthesis of bacteriocins is treated in depth with emphasis on response regulation, post-translational modification, secretion and proteolytic activation of bacteriocin precursors. To conclude, the role of the leader peptides is outlined and a conceptual model for bacteriocin maturation is proposed.

Amino Acid Sequence↗
PubMed1995

Characteristics of Tn5307 exchange and intergeneric transfer of genes associated with nisin production.

Transfer of the Lactococcus lactis 11454 nisin-sucrose conjugative transposon, Tn5307, was investigated to develop a methodology for conjugation of this element to other lactic acid bacteria. Tn5307 exchange was sensitive to temperature and pH but was not affected by protease or amylase treatments to donor cells. Moreover, conjugation studies demonstrated that the direct-plate method could be employed to rapidly identify LM2301 transconjugants able to transfer Tn5307 at least ten times more efficiently than 11454. Intergeneric transfer of nisin and sucrose genes between L. lactis and a dairy Enterococcus sp. was also investigated. Erythromycin-resistant Enterococcus sp. recipients were developed by electro-transformation with pGK13 or by conjugal introduction of the broad-host-range plasmid pAM beta 1. Matings between L. lactis 11454 and an Enterococcus sp. recipient that contained pAM beta 1 yielded sucrose-positive, nisin-immune transconjugants at a frequency of 2.3 x 10(-9) transconjugants per donor cfu. Agar-overlay assays for nisin production revealed that enterococcal transconjugants did not produce the bacteriocin, but DNA.DNA hybridization with a nisA-specific probe demonstrated that these bacteria had acquired the nisin structural gene.

Anti-Bacterial Agents↗
PubMed1995

Highly bioluminescent Streptococcus thermophilus strain for the detection of diary-relevant antibiotics in milk.

Inefficient translational initiation is often the cause of poor foreign gene expression in gram-positive organisms. The expression of bacterial luciferase (lux) genes in Streptococcus thermophilus (bioluminescence) was improved by addressing this problem in two ways; by ribosome-binding site (RBS) replacement, and by enhancing lux RBS access by polymerase chain reaction modification either alone or combined with translational coupling to a truncated upstream open- reading frame (orf') having its own RBS. Lactococcal expression signals were employed for plasmid-based lux expression. The same constructs were used to monitor bioluminescence in Lactococcus lactis, as well as two non-lactic bacterial strains, for comparison. High lux expression was achieved in all four organisms with a heterodimeric thermostable enzyme. Surprisingly, where ready access to the lux RBS was predicted, translational coupling to the lactococcal orf remained a prerequisite for detectable lux expression in L. lactis. In contrast, high bioluminescence in S. thermophilus was independent of coupling. Consistent with these observations, inspection of published gene sequences suggests that RBS "strength" may be a more important factor in translation in S. thermophilus than in L. lactis. Using reduced light production in highly bioluminescent S. thermophilus as an indicator of biocide presence in milk, test times could be significantly shortened compared with a commercial test utilizing the related non-bioluminescent strain. lux genes appear to be sensitive, exponential-phase reporters of gene activity in S. thermophilus, an organism with molecular biology and genetics that remain largely unstudied.

Animals↗
PubMed1993

Construction of first-generation lactococcal integrative cloning vectors.

Using a randomly-cloned, HindIII-digested, chromosomal fragment from Lactococcus lactis subsp. lactis LM0230, first-generation lactococcal integrative cloning vectors were developed. Through dideoxy DNA sequence analysis, the cloned chromosomal DNA fragment was determined to be 1026 base pairs. Southern hybridization studies demonstrated applicability of the integrative vector to other strains of L. lactis and L. lactis subsp. cremoris. Identification of a single NruI site near the middle of the chromosomal fragment allowed insertion of the erythromycin (Em)-resistance (eryr) gene obtained from L. lactis IL1837. Integration of the eryr gene into the L. lactis LM0230 chromosome was achieved by a Campbell-like recombination. The nisin (Nis)-resistance (nisr) gene from L. lactis IL1904 was inserted into the NruI site in a separate clone and integration into the L. lactis LM0230 chromosome was achieved via a replacement recombination event following electroporation of the linearized nisr fragment flanked by the cloned chromosomal DNA. Transformants grown in the absence of either Em or Nis for > 200 generations and subsequently transferred to various concentrations of the selectable agent confirmed the stability of the integrated genes. Further studies involving the Nis-resistant (NisR) transformant suggested that the integrated nisr gene may be amplifying within the host chromosome.

Base Sequence↗
PubMed1995

The lysins of bacteriophages infecting lactic acid bacteria.

This short review highlights the complete absence of literature on lysins of bacteriophages infecting species like S. salivarius subsp. thermophilus, Pediococcus and Leuconostoc species, L. helveticus, L. acidophilus, L. plantarum and L. brevis, which are also widely used in the dairy industry. The lysins described share some similar biochemical characteristics: optimal pH and temperature, site of hydrolysis inside the peptidoglycan, and some activators and inhibitors. The cloning of the genes encoding these lysins only began in the last few years and four of them have been completely sequenced. In the future, these lysin genes could be interestingly compared to the host autolysin(s) gene(s). By contrast, the passage of phage lysins through the cytoplasmic membrane of the host cell in order to reach the peptidoglycan (via a signal sequence or the presence of a holin) seems not to be clearly resolved. The presence of a second open-reading frame upstream from the gene of the lysin, enabling a putative holin to be encoded, has already been suggested. No doubt our ever increasing knowledge about bacteriophage genome organization will help to elucidate this question. Meanwhile the obtention of a Lactococcus strain with an autolytic phenotype, using a bacteriophage lysin gene, as well as the successful use of purified PL1 lysin to obtain protoplasts of L. casei encourage us to continue to explore the field of bacteriophage lysins.

Amino Acid Sequence↗
PubMed1996

DNA sequence analysis, expression, distribution, and physiological role of the Xaa-prolyldipeptidyl aminopeptidase gene from Lactobacillus helveticus CNRZ32.

Lactobacillus helveticus CNRZ32 possesses an Xaa-prolyldipeptidyl aminopeptidase (PepX), which releases amino-terminal dipeptides from peptides containing proline residues in the penultimate position. The PepX gene, designated pepX, from Lb. helveticus CNRZ32 was sequenced. Analysis of the sequence identified a putative 2379-bp pepX open-reading frame, which encodes a polypeptide of 793 amino acid residues with a deduced molecular mass of 88,111 Da. The gene shows significant sequence identity with sequenced pepX genes from lactic acid bacteria. The product of the gene contains a motif that is almost identical with the active-site motif of the serine-dependent PepX from lactococci. The introduction of pepX into Lactococcus lactis LM0230 on either pGK12 (a low-copy-number plasmid vector) or pIL253 (a high-copy-number plasmid vector) did not result in a significant increase in PepX activity, while the introduction of pepX into CNRZ32 on pGK12 resulted in a four-fold increase in PepX activity. Southern hybridization experiments revealed that the pepX gene from CNRZ32 is well conserved in lactobacilli, pediococci and streptococci. The physiological role of PepX during growth in lactobacillus MRS (a rich medium containing protein hydrolysates along with other ingredients) and milk was examined by comparing growth of CNRZ32 and a CNRZ32 PepX-negative derivative. No difference in growth rate or acid production was observed between CNRZ32 and its PepX-negative derivative in MRS. However, the CNRZ32 PepX-negative derivative grew in milk at a reduced specific growth rate when compared to wild-type CNRZ32. Introduction of the cloned PepX determinant into the CNRZ32 PepX-negative derivative resulted in a construct with a specific growth rate similar to that of wild-type CNRZ32.

Amino Acid Sequence↗
PubMed1991

Genetic analysis of a lactococcal plasmid replicon.

The sequence and genetic organization was determined of the 2508 bp lactococcal portion of pFX2, which was derived from a cryptic Lactococcus lactis subsp. lactis plasmid and used as the basis for construction of a series of lactococcal vectors. A lactococcal plasmid plus origin and two replication protein-coding regions (repA and repB) were located. RepA has a helix-turn-helix motif, a geometry typical of DNA-binding proteins. RepB shows a high degree of homology to the plasmid replication initiation proteins from other gram-positive bacteria and Mycoplasma. The transcribed inverted repeat sequence between repA and repB could form an attenuator to regulate pFX2 replication. Up-stream of the ori site, and in a region which was non-essential for replication, a 215 bp sequence identical to the staphylococcal plasmid pE194 and carrying the RSA site was identified. The genetic organization of this lactococcal plasmid replicon shares significant similarity with pE194 group plasmids.

Amino Acid Sequence↗
PubMed1996

Inducible gene expression and environmentally regulated genes in lactic acid bacteria.

Relatively recently, a number of genes and operons have been identified in lactic acid bacteria that are inducible and respond to environmental factors. Some of these genes/operons had been isolated and analysed because of their importance in the fermentation industry and, consequently, their transcription was studied and found to be regulatable. Examples are the lactose operon, the operon for nisin production, and genes in the proteolytic pathway of Lactococcus lactis, as well as xylose metabolism in Lactobacillus pentosus. Some other operons were specifically targetted with the aim to compare their mode of regulation with known regulatory mechanisms in other well-studied bacteria. These studies, dealing with the biosynthesis of histidine, tryptophan, and of the branched chain amino acids in L. lactis, have given new insights in gene regulation and in the occurrence of auxotrophy in these bacteria. Also, nucleotide sequence analyses of a number of lactococcal bacteriophages was recently initiated to, among other things, specifically learn more about regulation of the phage life cycle. Yet another approach in the analysis of regulated genes is the 'random' selection of genetic elements that respond to environmental stimuli and the first of such sequences from lactic acid bacteria have been identified and characterized. The potential of these regulatory elements in fundamental research and practical (industrial) applications will be discussed.

Amino Acids↗
PubMed1996

Genetics of subtilin and nisin biosyntheses: biosynthesis of lantibiotics.

Several peptide antibiotics have been described as potent inhibitors of bacterial growth. With respect to their biosynthesis, they can be divided into two classes: (i) those that are synthesized by a non-ribosomal mechanism, and (ii) those that are ribosomally synthesized. Subtilin and nisin belong to the ribosomally synthesized peptide antibiotics. They contain the rare amino acids dehydroalanine, dehydrobutyrine, meso-lanthionine, and 3-methyllanthionine. They are derived from prepeptides which are post-translationally modified and have been termed lantibiotics because of their characteristic lanthionine bridges (Schnell et al. 1988). Nisin is the most prominent lantibiotic and is used as a food preservative due to its high potency against certain gram-positive bacteria (Mattick & Hirsch 1944, 1947; Rayman & Hurst 1984). It is produced by Lactococcus lactis strains belonging to serological group N. The potent bactericidal activities of nisin and other lantibiotics are based on depolarization of energized bacterial cytoplasmic membranes. Breakdown of the membrane potential is initiated by the formation of pores through which molecules of low molecular weight are released. A trans-negative membrane potential of 50 to 100 mV is necessary for pore formation by nisin (Ruhr & Sahl 1985; Sahl et al. 1987). Nisin occurs as a partially amphiphilic molecule (Van de Ven et al. 1991). Apart from the detergent-like effect of nisin on cytoplasmic membranes, an inhibition of murein synthesis has also been discussed as the primary effect (Reisinger et al. 1980). In several countries nisin is used to prevent the growth of clostridia in cheese and canned food. The nisin peptide structure was first described by Gross & Morall (1971), and its structural gene was isolated in 1988 (Buchman et al. 1988; Kaletta & Entian 1989). Nisin has two natural variants, nisin A, and nisin Z, which differ in a single amino acid residue at position 27 (histidin in nisin A is replaced by asparagin in nisin Z (Mulders et al. 1991; De Vos et al. 1993). Subtilin is produced by Bacillus subtilis ATCC 6633. Its chemical structure was first unravelled by Gross & Kiltz (1973) and its structural gene was isolated in 1988 (Banerjee & Hansen 1988). Subtilin shares strong similarities to nisin with an identical organization of the lanthionine ring structures (Fig. 1), and both lantibiotics possess similar antibiotic activities. Due to its easy genetic analysis B. subtilis became a very suitable model organism for the identification and characterization of genes and proteins involved in lantibiotic biosynthesis. The pathway by which nisin is produced is very similar to that of subtilin, and the proteins involved share significant homologies over the entire proteins (for review see also De Vos et al. 1995b). The respective genes have been identified adjacent to the structural genes, and are organized in operon-like structures (Fig. 2). These genes are responsible for post-translational modification, transport of the modified prepeptide, proteolytic cleavage, and immunity which prevents toxic effects on the producing bacterium. In addition to this, biosynthesis of subtilin and nisin is strongly regulated by a two-component regulatory system which consists of a histidin kinase and a response regulator protein.

Amino Acid Sequence↗
PubMed1993

[Lantibiotics, a class of ribosomally synthesized peptide antibiotics].

Lantibiotics are defined as peptide antibiotics containing the unusual amino acids mesolanthionine, 3-methyllanthionine, dehydroalanine, and dehydrobutyrine. They are synthesized by some gram-positive bacteria. Their inhibitory effect on certain other gram-positive bacteria is explained by detergent-like damage of cytoplasmic membranes. Prominent members of the lantibiotics are nisin of Lactococcus lactis, which can be used as a food preservative, subtilin of Bacillus subtilis, which is similar to nisin, and epidermin of Staphylococcus epidermidis, which is considered in the treatment of acne. Lantibiotics are ribosomally synthesized as prepeptides, which are posttranslationally modified. Genes probably encoding these biosynthetic enzymes and regulatory factors have been identified adjacent to the structural genes of the lantibiotics subtilin, nisin, and epidermin.

Amino Acid Sequence↗
PubMed1992

Use of antisense RNA to confer bacteriophage resistance in dairy starter cultures.

The strategy and implementation of a unique system for engineering bacteriophage resistant starter cultures of Lactococcus lactis employing antisense RNA is reviewed. As a necessary prerequisite for developing this system, we have cloned and sequenced a number of bacteriophage genes coding for minor and major structural proteins. In addition, we have also identified a series of genes whose function(s) is not known but their sequences appear to be conserved in a vast number of isolates. One of these latter sequences, designated gp51C, codes for a 51-kDa protein which is extremely charged and shares some homology with yeast translation initiation factor. Resistance to a broad class of isometric bacteriophages has been achieved by expression of an antisense RNA targeted against, for example, gp51C. In the best case, expression of the antisense gp51C RNA results is a greater than 99% reduction in the total number of plaque forming units. Additional antisense RNA constructs directed against other bacteriophage genes, including the major capsid protein, also appear effective at inhibiting infection from 40-55% suggesting that this approach may prove useful for engineering a set of truly isogenic strains to be used in a starter culture rotation plan.

Bacteriophages↗
PubMed2004

Screening of lactic-acid bacteria from South African barley beer for the production of bacteriocin-like compounds.

Strains of Lactobacillus paracasei subsp. paracasei (strain ST11BR), L. pentosus (strain ST151BR), L. plantarum (strain ST13BR), and Lactococcus lactis subsp. lactis (strain ST34BR) producing bacteriocin-like peptides were isolated from barley beer produced in the Western, Northern and Eastern provinces of South Africa. The peptides (bacST11BR, bacST151BR, bacST13BR and bacST34BR) lost their activity after treatment with proteinase K, a proteinase, papain, chymotrypsin, trypsin, pepsin and pronase, but not when they were treated with alpha-amylase, suggesting that the peptides are not glycosylated. The peptides inhibited the growth of Lactobacillus casei, L. sakei, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis, but not Enterobacter cloacae, Lactobacillus bulgaricus subsp. delbrueckii, L. plantarum, L. salivarius, Listeria innocua, Staphylococcus aureus, Streptococcus uberis, S. agalactiae, S. caprinus and S. pneumoniae. Peptides bacST11BR and bacST13BR differed from the other 2 peptides by failing to kill Klebsiella pneumoniae and one of the E. coli strains. Peptides were stable after 2 h of incubation at pH 2.0-12.0, and after 90 min at 100 degrees C. When autoclaved (121 degrees C, 20 min), only bacST13BR lost its activity. The bacteriocin-like peptides were produced at a growth temperature of 30 degrees C, but not at 37 degrees C.

Bacteriocins↗
PubMed2004

Proton motive force mediates a reorientation of the cytosolic domains of the multidrug transporter LmrP.

LmrP from Lactococcus lactis is a 45-kDa membrane protein that confers resistance to a wide variety of lipophilic compounds by acting as a proton motive force-driven efflux pump. This study shows that both the proton motive force and ligand interaction alter the accessibility of cytosolic tryptophan residues to a hydrophilic quencher. The proton motive force mediates an increase of LmrP accessibility toward the external medium and results in higher drug binding. Residues Asp128 and Asp68, from cytosolic loops, are involved in the proton motive force-mediated accessibility change. Ligand binding does not modify the protein accessibility, but the proton motive force-mediated restructuring is prerequisite for a subsequent accessibility change mediated by ligand binding. Asp142 cooperates with other membrane-embedded carboxylic residues to promote a conformational change that increases LmrP accessibility toward the hydrophilic quencher. This drug binding-mediated reorganization may be related to the transition between the high- and low-affinity drug-binding sites and is crucial for drug release in the extracellular medium.

Acrylamide↗
PubMed2003

Purification and characterisation of a lactococcal aminoacylase.

The amd1-encoded aminoacylase from Lactococcus lactis MG1363 was cloned and overexpressed in Escherichia coli and purified. The assumed dimeric enzyme has a subunit molecular mass of about 42 kDa and contains 2.0+/-0.1 g-atoms of zinc and cobalt, in equimolar amounts, per subunit of Amd1. The enzyme was characterised with respect to substrate specificity, pH, temperature and metal dependence. Amd1 exhibited a broad activity range towards N-acetylated- l-amino acids with a strong preference towards those containing neutral aliphatic and aromatic side chains. It hydrolysed N-acetyl- l-alanine most efficiently, and exhibited temperature and pH optima of 30 degrees C and 7.0, respectively. The activity of Amd1 towards N-acetyl- l-alanine was enhanced by the divalent cation Co(2+), while Cd(2+ )inhibited activity. Interestingly, Amd1 was shown to catalyse the hydrolysis of several dipeptides at pH 7.0, although with reduced V(max) values as compared to hydrolysis of N-acetylated- l-amino acids. This characteristic has also biological significance since Amd1 was able to complement a growth deficiency in a L. lactis triple peptidase mutant.

Amidohydrolases↗
PubMed2004

Ferrihydrite reduction by Geobacter species is stimulated by secondary bacteria.

Geobacter species such as G. bremensis, G. pelophilus, and G. sulfurreducens are obligately anaerobic and grow in anoxic, non-reduced medium by fast reduction of soluble ferric citrate. In contrast, insoluble ferrihydrite was either only slowly or not reduced when supplied as electron acceptor in similar growth experiments. Ferrihydrite reduction was stimulated by addition of a reducing agent or by concomitant growth of secondary bacteria that were physiologically and phylogenetically as diverse as Escherichia coli, Lactococcus lactis, or Pseudomonas stutzeri. In control experiments with heat-inactivated Geobacter cells and viable secondary bacteria, no ( E. coli, P. stutzeri) or only little ( L. lactis) ferrihydrite was reduced. Redox indicator dyes showed that growing E. coli, P. stutzeri, or L. lactis cells lowered the redox potential of the medium in a similar way as a reducing agent did. The lowered redox potential was presumably the key factor that stimulated ferrihydrite reduction by all three Geobacter species. The observed differences in anoxic non-reduced medium with ferric citrate versus ferrihydrite as electron acceptor indicated that reduction of these electron acceptors involved different cellular components or different biochemical strategies. Furthermore, it appears that redox-sensitive components are involved, and/or that gene expression of components needed for ferrihydrite reduction is controlled by the redox state.

Anaerobiosis↗
PubMed2007

Sensitive and selective determination of glutathione in probiotic bacteria by capillary electrophoresis-laser induced fluorescence.

Glutathione (GSH) is a thiol with an important function in protecting tissue against the oxidative stress which has been related to carcinogenesis in the colon. For this reason the development of probiotic species producing glutathione could be of great interest. To determine the glutathione content of some probiotic bacteria of the Bifidobacterium and Lactococcus genera, a very sensitive and selective analytical method based on capillary electrophoresis coupled to laser-induced fluorescence detection has been developed. Pretreatment of cell-lysate samples is very simple--precipitation of protein with acetonitrile in 1:2 volume ratio. The fluorophore 5-iodoacetamidofluorescein (5-IAF) was chosen for glutathione derivatisation; it reacts with thiols at pH 12.5, forming a fluorescent adduct which is excited by a laser at 488 nm for detection. The reaction conditions optimised were temperature, time, and 5-IAF/GSH molar ratio. Electrophoresis was performed with a carbonate buffer (25 mmol L(-1), pH 9.8) as background electrolyte and a voltage of 30 kV; an electrophoretic run was complete in less than 7 min. There was a good linear relationship between concentration and response in the range 2.5-500 ng mL(-1) and the LOD was 0.5 ng mL(-1). The glutathione content of probiotic cells was determined by using the standard additions method to reduce matrix effects. The method was fully validated and shown to be of suitable sensitivity and selectivity for determination of GSH in probiotic cell lysates.

Acetonitriles↗
PubMed2002

A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI.

A new food-grade cloning vector for lactic acid bacteria was constructed using the nisin immunity gene nisI as a selection marker. The food-grade plasmid, pLEB 590, was constructed entirely of lactococcal DNA: the pSH 71 replicon, the nisI gene, and the constitutive promoter P45 for nisI expression. Electroporation into Lactococcus lactis MG 1614 with 60 international units (IU) nisin/ml selection yielded approximately 10(5) transformants/ micro g DNA. MG 1614 carrying pLEB 590 was shown to be able to grow in medium containing a maximum of 250 IU nisin/ml. Plasmid pLEB 590 was successfully transformed into an industrial L. lactis cheese starter carrying multiple cryptic plasmids. Suitability for molecular cloning was confirmed by cloning and expressing the proline iminopeptidase gene pepI from Lactobacillus helveticus in L. lactis and Lb. plantarum. These results show that the food-grade expression system reported in this paper has potential for expression of foreign genes in lactic acid bacteria in order to construct improved starter bacteria for food applications.

Aminopeptidases↗
PubMed2003

Diversity of L-leucine catabolism in various microorganisms involved in dairy fermentations, and identification of the rate-controlling step in the formation of the potent flavour component 3-methylbutanal.

Various microorganisms, belonging to the genera Lactococcus, Lactobacillus, Streptococcus, Leuconostoc, Bifidobacterium, Propionibacterium, Brevibacterium, Corynebacterium and Arthrobacter, used in dairy fermentations such as cheese making, were analysed for their potential to convert leucine into flavour components, most notably 3-methylbutanal. A large variation between and within species was observed for various enzyme activities involved in the conversion pathway, e.g. transaminases, alpha-hydroxy acid dehydrogenase and alpha-keto acid decarboxylase. In particular, alpha-keto acid decarboxylase activity-leading to 3-methylbutanal-was found to be present in only two of the strains tested. It is proposed that this activity is rate-controlling in the conversion pathway leading to the flavour compound 3-methylbutanal.

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