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I F Nes

Publications and source records attributed to I F Nes.

At least 55 records · Page 3Linked to original sources
PubMed1996

Novel lantibiotics and their pre-peptides.

In recent years there has been a considerable increase in studies of bactericidal peptides produced by Gram-positive bacteria, with particular emphasis upon their potential application as food preservatives. A number of these peptides contain lanthionine and other post-translationally modified amino acid residues. The lanthionine-containing molecules (lantibiotic) appear to have evolved in two quite different lineages, type A and type B. This mini-review introduces the reader to several of the more recently described type A lantibiotics for which relatively detailed biochemical and/or genetic data has already been gathered. A wider diversity of compounds of type A lantibiotics has been described in the recent years. Novel features of some of the more recently described type A lantibiotics to be reported in this review include: a) New modifications such as D-Ala and 2-hydroxypropionyl residues, both derived from serine. b) Different types of pre-lantibiotic leader sequences. c) The apparent requirement for different numbers and types of genes for synthesis of some active type A lantibiotics. d) Cytolysin functions as both a hemolysin and a bacteriocin. e) One of the newly-described lantibiotics (lactocin S) does not have any net charge at neutral pH another (carnocin UI49) is the largest of the lantibiotics discovered and the killing action of another (cytolysin) has been shown to be depend on the interaction of two peptides.

Alanine↗
PubMed1996

Identification of the streptococcal competence-pheromone receptor.

Competence for genetic transformation in certain species of streptococci has been known for many years to be induced by a secreted protease-sensitive pheromone, referred to as the competence factor or activator, which acts as a quorum-sensing signal to co-ordinate expression of late competence genes. We recently reported identification of the pheromone of Streptococcus pneumoniae strain Rx as a small unmodified peptide, which was termed competence-stimulating peptide (CSP). By identifying the gene (comC) encoding the Rx CSP we were able to show that it is synthesized as a precursor peptide containing an N-terminal double-glycine type leader. In the present work, we describe two alleles of the corresponding gene from Streptococcus gordonii strains Challis and NCTC 7865, which are strains with distinct competence pheromones and corresponding specific pheromone reactivities. In addition, the nucleic acid sequences of two genes located downstream of comC were determined; interestingly, these genes encode a two-component signal transduction system. We therefore speculated that their products, a histidine kinase (ComD) and its cognate response regulator (ComE), act downstream of the CSP in competence regulation. By tracing the CSP specificity of the competence response in these strains to strain-specific alleles of comD, we obtained evidence demonstrating that the histidine kinase ComD is the competence-pheromone receptor.

Alleles↗
PubMed1996

Comparative studies of chitinases A and B from Serratia marcescens.

Serratia marcescens produces several chitinolytic enzymes, including chitinase A (ChiA) and chitinase B (ChiB). In this study, ChiB was purified to homogeneity using a newly developed protocol based on hydrophobic interaction chromatography. Subsequently, characteristics of ChiB and of the hitherto only partly characterized ChiA were determined and compared. Pure ChiA and ChiB shared several characteristics such as a broad pH optimum around pH 5.0-6.0, and a temperature optimum between 50 and 60 degrees C. Both enzymes were fairly stable, with half-lives of more than 10 d at 37 degrees C, pH 6.1. Analyses of the degradation of various N-acetylglucosamine oligomers, fluorogenic substrates and colloidal chitin showed that both enzymes cleave chitobiose [(GlcNAc)2] from (GlcNAc)n and thus possess an exo-N,N'-diacetylchitobiohydrolase activity. Both enzymes were also capable of producing monomers from longer (GlcNAc)n substrates, indicating that they also have an endochitinase (ChiA) or exo-N,N',N"-triacetylchitotriohydrolase (ChiB) activity. Kinetic analyses with 4-methylumbelliferyl-beta-D-N,N'-diacetylchitobioside, an analogue of (GlcNAc)3, showed cooperative kinetics for ChiA, whereas for ChiB normal hyperbolic kinetics were observed. ChiA had a higher specific activity towards chitin than ChiB and synergistic effects on the chitin degradation rate were observed upon combining the two enzymes. These results, together with the results of sequence comparisons and previous studies of the cellular localization of the two chitinases in S. marcescens indicate possible roles for ChiA and ChiB in chitin breakdown.

Amino Acid Sequence↗
PubMed1996

Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins.

A new bacteriocin has been isolated from an Enterococcus faecium strain. The bacteriocin, termed enterocin A, was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and mass spectrometry analysis. By combining the data obtained from amino acid and DNA sequencing, the primary structure of enterocin A was determined. It consists of 47 amino acid residues, and the molecular weight was calculated to be 4,829, assuming that the four cysteine residues form intramolecular disulfide bridges. This molecular weight was confirmed by mass spectrometry analysis. The amino acid sequence of enterocin A shared significant homology with a group of bacteriocins (now termed pediocin-like bacteriocins) isolated from a variety of lactic acid-producing bacteria, which include members of the genera Lactobacillus, Pediococcus, Leuconostoc, and Carnobacterium. Sequencing of the structural gene of enterocin A, which is located on the bacterial chromosome, revealed an N-terminal leader sequence of 18 amino acid residues, which was removed during the maturation process. The enterocin A leader belongs to the double-glycine leaders which are found among most other small nonlantibiotic bacteriocins, some lantibiotics, and colicin V. Downstream of the enterocin A gene was located a second open reading frame, encoding a putative protein of 103 amino acid residues. This gene may encode the immunity factor of enterocin A, and it shares 40% identity with a similar open reading frame in the operon of leucocin AUL 187, another pediocin-like bacteriocin.

Amino Acid Sequence↗
PubMed1996

New biologically active hybrid bacteriocins constructed by combining regions from various pediocin-like bacteriocins: the C-terminal region is important for determining specificity.

The pediocin-like bacteriocins, produced by lactic acid bacteria, are bactericidal polypeptides with very similar primary structures. Peptide synthesis followed by reverse-phase and ion-exchange chromatographies yielded biologically active pediocin-like bacteriocins in amounts and with a purity sufficient for characterizing their structure and mode of action. Despite similar primary structures, the pediocin-like bacteriocins, i.e., pediocin PA-1, sakacin P, curvacin A, and leucocin A, differed in their relative toxicities against various bacterial strains. On the basis of the primary structures, the polypeptides of these bacteriocins were divided into two modules: the relatively hydrophilic and well conserved N-terminal region, and the somewhat more diverse and hydrophobic C-terminal region. By peptide synthesis, four new biologically active hybrid bacteriocins were constructed by interchanging corresponding modules from various pediocin-like bacteriocins. All of the new hybrid bacteriocin constructs had bactericidal activity. The relative sensitivity of different bacterial strains to a hybrid bacteriocin was similar to that to the bacteriocin from which the C-terminal module was derived and quite different from that to the bacteriocin from which the N-terminal was derived. Thus, the C-terminal part of the pediocin-like bacteriocins is an important determinant of the target cell specificity. The synthetic bacteriocins were more stable than natural isolates, presumably as a result of the absence of contaminating proteases. However, some of the synthetic bacteriocins lost activity, but this was detectable only after months of storage. Mass spectrometry suggested that this instability was due to oxidation of methionine residues, resulting in a 10- to 100-fold reduction in activity.

Amino Acid Sequence↗
PubMed1996

Characterization of the locus responsible for the bacteriocin production in Lactobacillus plantarum C11.

Lactobacillus plantarum C11 secretes a small cationic peptide, plantaricin A, that serves as induction signal for bacteriocin production as well as transcription of plnABCD. The plnABCD operon encodes the plantaricin A precursor (PlnA) itself and determinants (PlnBCD) for a signal transducing pathway. By Northern (RNA) and sequencing analyses, four new plantaricin A-induced operons were identified. All were highly activated in concert with plnABCD upon bacteriocin induction. Two of these operons (termed plnEFI and plnJKLR) each encompass a gene pair (plnEF and plnJK, respectively) encoding two small cationic bacteriocin-like peptides with double-glycine-type leaders. The open reading frames (ORFs) encoding the bacteriocin-like peptides are followed by ORFs (plnI and -L, respectively) encoding cationic hydrophobic proteins resembling bacteriocin immunity proteins. On the third operon (termed plnMNOP), a similar bacteriocin-like ORF (plnN) and a putative immunity ORF (either plnM or -P) were identified as well. These findings suggest that two bacteriocins of two-peptide type (mature PlnEF and PlnJK) and a bacteriocin of one-peptide type (mature PlnN) could be responsible for the observed bacteriocin activity. The last operon (termed plnGHSTUV) contains two ORFs (plnGH) apparently encoding an ABC transporter and its accessory protein, respectively, known to be involved in processing and export of peptides with precursor double-glycine-type leaders. Promoter structure was established. A conserved regulatory-like box encompassing two direct repeats was identified in the promoter regions of all five plantaricin A-induced operons. These repeats may serve as regulatory elements for gene expression.

Amino Acid Sequence↗
PubMed1996

Lactococcin G is a potassium ion-conducting, two-component bacteriocin.

Lactococcin G is a novel lactococcal bacteriocin whose activity depends on the complementary action of two peptides, termed alpha and beta. Peptide synthesis of the alpha and beta peptides yielded biologically active lactococcin G, which was used in mode-of-action studies on sensitive cells of Lactococcus lactis. Approximately equivalent amounts of both peptides were required for optimal bactericidal effect. No effect was observed with either the alpha or beta peptide in the absence of the complementary peptide. The combination of alpha and beta peptides (lactococcin G) dissipates the membrane potential (delta omega), and as a consequence cells release alpha-aminoisobutyrate, a non-metabolizable alanine analog that is accumulated through a proton motive-force dependent mechanism. In addition, the cellular ATP level is dramatically reduced, which results in a drastic decrease of the ATP-driven glutamate uptake. Lactococcin G does not form a proton-conducting pore, as it has no effect on the transmembrane pH gradient. Dissipation of the membrane potential by uncouplers causes a slow release of potassium (rubidium) ions. However, rapid release of potassium was observed in the presence of lactococcin G. These data suggest that the bactericidal effect of lactococcin G is due to the formation of potassium-selective channels by the alpha and beta peptides in the target bacterial membrane.

Adenosine Triphosphate↗
PubMed1996

Induction of bacteriocin production in Lactobacillus sake by a secreted peptide.

Lactobacillus sake LTH673 is known to produce a bacteriocin called sakacin P. Production of and immunity to sakacin P were found to depend on the presence of a protease-sensitive component that is produced by L. sake LTH673 itself. This component (called inducing factor [IF]) was purified from culture supernatants and shown to be a basic, nonbacteriocin peptide consisting of 19 amino acids, which in principle is capable of forming a highly amphiphilic helical structure. Circular dichroism studies showed that IF indeed could adopt a helical structure, but only in membrane-mimicking environments. Both purified IF and chemically synthesized IF induced expression of the structural gene for sakacin P and concomitant secretion of the gene product. In addition, IF induced its own production and immunity to sakacin P and related bacteriocins. These results indicate that bacteriocin production by L. sake LTH673 is controlled by an autoinduction pathway in which IF may function as a cell density signal.

Amino Acid Sequence↗
PubMed1995

Chitinase B from Serratia marcescens BJL200 is exported to the periplasm without processing.

A gene encoding a chitinase from Serratia marcescens BJL200 was cloned and expressed in Escherichia coli and S. marcescens. Nucleotide sequencing revealed an open reading frame encoding a 55.5 kDa protein of 499 amino acids without a typical signal peptide for export. The cellular localization of the chitinase was studied, using two types of cell fractionation methods of immunocytochemical techniques. These analyses showed that the chitinase is located in the cytoplasm in E. coli, whereas it is exported to the periplasm in S. marcescens. Analysis of chitinase isolated from periplasmic fractions of S. marcescens carrying the cloned gene showed that export of the enzyme is not accompanied by processing at the N-terminus. The chitinase did not show any of the characteristics that have been proposed to direct the export of other non-processed extracellular proteins such as the E. coli haemolysin and might therefore be secreted via a hitherto unknown mechanism.

Amino Acid Sequence↗
PubMed1995

A bacteriocin-like peptide induces bacteriocin synthesis in Lactobacillus plantarum C11.

In this study, we show that bacteriocin production in Lactobacillus plantarum C11 is an inducible process triggered by a secreted protein factor produced by the bacteriocin producer itself. The induction factor was identified to be plantaricin A, a bacteriocin-like peptide whose gene (plnA) is located in the same operon as a two-component regulatory system (plnBCD). When L. plantarum C11 cultures were depleted for plantaricin A, either by growing individual colonies on agar plates or by starting a new culture with a highly diluted inoculum, no bacteriocin was produced during the following growth. When chemically synthesized plantaricin A or purified bacterially produced plantaricin A was added to non-producing cultures, bacteriocin production was induced. Only 1 ng ml-1 plantaricin A is sufficient to induce the bacteriocin production in non-producing L. plantarum C11, and bacteriocin activity appears in the growth medium approximately 150 min after induction. Northern analyses, using a plnA-specific probe, demonstrated that plantaricin A is able to induce its own synthesis by transcription of the plnABCD operon, and this is observed approximately 15 min after adding plantaricin A. Furthermore, heterologous expression of the plnABCD operon in a Lactobacillus sake strain showed that the conditioned growth medium contained the active induction factor. Neither synthetic nor expressed plantaricin A from the heterologous system possesses any bacteriocin activity, suggesting that plantaricin A is primarily an induction factor and not a bacteriocin as claimed earlier.

Amino Acid Sequence↗
PubMed1995

A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export.

Lantibiotic and non-lantibiotic bacteriocins are synthesized as precursor peptides containing N-terminal extensions (leader peptides) which are cleaved off during maturation. Most non-lantibiotics and also some lantibiotics have leader peptides of the so-called double-glycine type. These leader peptides share consensus sequences and also a common processing site with two conserved glycine residues in positions -1 and -2. The double-glycine-type leader peptides are unrelated to the N-terminal signal sequences which direct proteins across the cytoplasmic membrane via the sec pathway. Their processing sites are also different from typical signal peptidase cleavage sites, suggesting that a different processing enzyme is involved. Peptide bacteriocins are exported across the cytoplasmic membrane by a dedicated ATP-binding cassette (ABC) transporter. Here we show that the ABC transporter is the maturation protease and that its proteolytic domain resides in the N-terminal part of the protein. This result demonstrates that the ABC transporter has a dual function: (i) removal of the leader peptide from its substrate, and (ii) translocation of its substrate across the cytoplasmic membrane. This represents a novel strategy for secretion of bacterial proteins.

ATP-Binding Cassette Transporters↗
PubMed1995

Production and pH-Dependent Bactericidal Activity of Lactocin S, a Lantibiotic from Lactobacillus sake L45.

The amount of lactocin S activity in a growing culture depends on the growth stage of the bacteria, the pH of the medium, the presence of ethanol, and the aeration of the culture. We observed the highest levels of bacteriocin activity in the early stationary growth phase of cultures at 30 deg C. When Lactobacillus sake L45 was grown in a fermentor at pH 5, it produced 2,000 to 3,000 bacteriocin units per ml, which represented an 8- to 10-fold increase in bacteriocin production compared with production during batch culture fermentation. Less than 10% of this level of bacteriocin activity was observed during fermentation at pH 6.0. When 1% ethanol was included in the growth medium, a two- to fourfold increase in the bacteriocin yield was observed. Aerating the culture during growth almost completely eliminated the production of active bacteriocin. Our results also showed that lactocin S-mediated killing of target cells depended on the pH of the culture. The pH had to be less than 6 in order to obtain a bactericidal effect with lactocin S-sensitive cells. At pH values greater than 6, lactocin S had no apparent effect on sensitive cells.

Journal Article↗
PubMed1995

Purification and partial amino acid sequence of plantaricin S, a bacteriocin produced by Lactobacillus plantarum LPCO10, the activity of which depends on the complementary action of two peptides.

Plantaricin S, one of the two bacteriocins produced by Lactobacillus plantarum LPCO10, which was isolated from a green-olive fermentation (R. Jiménez-Díaz, R.M. Ríos-Sánchez, M. Desmazeaud, J.L.Ruiz-Barba, and J.-C. Piard, Appl. Environ. Microbiol. 59:1416-1424, 1993), has been purified to homogeneity by ammonium sulfate precipitation, by binding to SP-Sepharose fast-flow, phenyl-Sepharose CL-4B, and C2/C18 reverse-phase chromatographies. The purification resulted in a final yield of 91.6% and a 352,617-fold increase in the specific activity. The bacteriocin activity was associated with two distinct peptides, termed alpha and beta, which were separated by C2/C18 reverse-phase chromatography. Although beta alone appeared to retain a trace of inhibitory activity, the complementary action of both the alpha and beta peptides was required for full bacteriocin activity, as judged by both the agar well diffusion and the microtiter plate assays. From the N-terminal end, 26 and 24 amino acids residues of alpha and beta, respectively, were sequenced. Further attempts at sequencing revealed no additional amino acids residues, suggesting that either modifications in the next amino acid residue blocked the sequencing region or that the C-terminal end had been reached. The amino acid sequences of alpha and beta show no apparent homology to each or to other bacteriocins purified from lactic acid bacteria.

Amino Acid Sequence↗
PubMed1995

Isolation and characterization of pediocin L50, a new bacteriocin from Pediococcus acidilactici with a broad inhibitory spectrum.

Lactic acid bacteria were isolated from Spanish dry-fermented sausages and screened for bacteriocin production. About 10% of the isolates produced antimicrobial substances when grown on solid media, but only 2% produced detectable activity in liquid media. Strain L50, identified as Pediococcus acidilactici, showed the strongest inhibitory activity and was active against members of all of the gram-positive genera tested. The strain produced a heat-stable bacteriocin when grown at 8 to 32 degrees C but not at 45 degrees C. The bacteriocin was purified to homogeneity. Its mass was determined to be 5,250.11 +/- 0.30 by electrospray mass spectrometry. The N terminus of the bacteriocin was blocked for sequencing by Edman degradation, but a partial sequence of 42 amino acids was obtained after cleavage of the peptide by cyanogen bromide. The sequence showed no similarity to those of other bacteriocins. Pediocin L50 appears to contain modified amino acids but not lanthionine or methyl-lanthionine.

Amino Acid Sequence↗
PubMed1994

Characterization of a chitinase gene (chiA) from Serratia marcescens BJL200 and one-step purification of the gene product.

The nucleotide sequence of the chiA gene from Serratia marcescens strain BJL200 was determined. The gene was found to encode a protein of 563 amino acid residues, with a typical N-terminal signal peptide of 23 residues, that is cleaved off during export. The gene exhibited striking differences with two previously characterized chiA genes of S. marcescens in the region corresponding to amino acid residues 410-467 of the gene product. Periplasmic fractions of an Escherichia coli strain harbouring the cloned gene were used as starting material for the development of a fast, one-step purification protocol for the chitinase that is based on hydrophobic interaction chromatography.

Amino Acid Sequence↗