Fourteen bacterial strains capable of producing a trypsin-dependent antimicrobial substance activ... more Fourteen bacterial strains capable of producing a trypsin-dependent antimicrobial substance active against Clostridium perfringens were isolated from human fecal samples of various origins (from healthy adults and children, as well as from adults with chronic pouchitis). Identification of these strains showed that they belonged to Ruminococcus gnavus, Clostridium nexile, and Ruminococcus hansenii species or to new operational taxonomic units, all from the Clostridium coccoides phylogenetic group. In hybridization experiments with a probe specific for the structural gene encoding the trypsin-dependent lantibiotic ruminococcin A (RumA) produced by R. gnavus, seven strains gave a positive response. All of them harbored three highly conserved copies of rumA-like genes. The deduced peptide sequence was identical to or showed one amino acid difference from the hypothetical precursor of RumA. Our results indicate that the rumA-like genes have been disseminated among R. gnavus and phylogene...
In this review we describe the most common mechanisms through which bacterial antagonisms occur i... more In this review we describe the most common mechanisms through which bacterial antagonisms occur in the GI-tract. These include depletion and:or competition for substrates; competition for receptor sites (competitive exclusion); generation of a restrictive physiological environment (role of pH, short-chain fatty acids, oxidation-reduction potential); and production of antimicrobial substances (bacteriocinand non bacteriocin-related).
The salIR and salIM genes of Streptomyces albus G encode the restriction endonuclease (ENase) and... more The salIR and salIM genes of Streptomyces albus G encode the restriction endonuclease (ENase) and DNA methyltransferase (MTase) of the SalI restriction-modification (R-M) system. In S. albus G, the genes constitute an operon that is mainly transcribed from a promoter located upstream from sallR, the first gene of the operon. In addition, a second promoter, at the 3' end of sallR, allows independent transcription of the MTase gene. Expression of sallR and sallM in Escherichia coli was investigated. The ENase gene was not expressed in the heterologous host, probably due to inactivity of the main promoter of the salI operon. In contrast to sallR, sallM was functional in E. coli. Preliminary S1 nuclease mapping experiments suggest that the alternative promoter of the MTase gene can initiate transcription in the heterologous, as well as in the homologous host.
Gene shuffling is a way of creating proteins with interesting new characteristics, starting from ... more Gene shuffling is a way of creating proteins with interesting new characteristics, starting from diverged sequences. We tested an alternative to gene shuffling based on plasmid recombination and found that Bacillus subtilis efficiently recombines sequences with 4% divergence, and Escherichia coli mutS is more appropriate for sequences with 22% divergence.
Gene shuffling is a way of creating proteins with interesting new characteristics, starting from ... more Gene shuffling is a way of creating proteins with interesting new characteristics, starting from diverged sequences. We tested an alternative to gene shuffling based on plasmid recombination and found that Bacillus subtilis efficiently recombines sequences with 4% divergence, and Escherichia coli mutS is more appropriate for sequences with 22% divergence.
Ruminococcin C (RumC) is a trypsin-dependent bacteriocin produced by Ruminococcus gnavus E1, a gr... more Ruminococcin C (RumC) is a trypsin-dependent bacteriocin produced by Ruminococcus gnavus E1, a gram-positive strict anaerobic strain isolated from human feces. It consists of at least three similar peptides active against Clostridium perfringens. In this article, a 15-kb region from R. gnavus E1 chromosome, containing the biosynthetic gene cluster of RumC was characterized. It harbored 17 open reading frames (called rum c genes) with predicted functions in bacteriocin biosynthesis and post-translational modification, signal transduction regulation, and immunity. An unusual feature of the locus is the presence of five genes encoding highly homologous, but nonidentical RumC precursors. The transcription levels of the rum c genes were quantified. The rumC genes were found to be highly expressed in vivo, when R. gnavus E1 colonized the digestive tract of mono-contaminated rats, whereas the amount of corresponding transcripts was below detection level when it grew in liquid culture medium. Moreover, the rumC-like genes were disseminated among 10 strains (R. gnavus or related species) previously isolated from human fecal samples and selected for their capability to produce a trypsin-dependant anti-C. perfringens compound. All harbored at least a rumC1-like copy, four exhibited rumC1-5 genes identical to those of strain E1.
When cultivated in the presence of trypsin, the Ruminococcus gnavus E1 strain, isolated from a hu... more When cultivated in the presence of trypsin, the Ruminococcus gnavus E1 strain, isolated from a human fecal sample, was able to produce an antibacterial substance that accumulated in the supernatant. This substance, called ruminococcin A, was purified to homogeneity by reverse-phase chromatography. It was shown to be a 2,675-Da bacteriocin harboring a lanthionine structure. The utilization of Edman degradation and tandem mass spectrometry techniques, followed by DNA sequencing of part of the structural gene, allowed the identification of 21 amino acid residues. Similarity to other bacteriocins present in sequence libraries strongly suggested that ruminococcin A belonged to class IIA of the lantibiotics. The purified ruminococcin A was active against various pathogenic clostridia and bacteria phylogenetically related to R. gnavus . This is the first report on the characterization of a bacteriocin produced by a strictly anaerobic bacterium from human fecal microbiota.
Ruminococcin A (RumA) is a trypsin-dependent lantibiotic produced by Ruminococcus gnavus E1, a gr... more Ruminococcin A (RumA) is a trypsin-dependent lantibiotic produced by Ruminococcus gnavus E1, a gram-positive strict anaerobic strain isolated from a human intestinal microbiota. A 12.8-kb region from R. gnavus E1 chromosome, containing the biosynthetic gene cluster of RumA, has been cloned and sequenced. It consisted of 13 open reading frames, organized in three operons with predicted functions in lantibiotic biosynthesis, signal transduction regulation, and immunity. One unusual feature of the locus is the presence of three almost identical structural genes, all of them encoding the RumA precursor. In order to determine the role of trypsin in RumA production, the transcription of the rum genes has been investigated under inducing and noninducing conditions. Trypsin activity is needed for the growth phase-dependent transcriptional activation of RumA operons. Our results suggest that bacteriocin production by R. gnavus E1 is controlled through a complex signaling mechanism involving ...
Fourteen bacterial strains capable of producing a trypsin-dependent antimicrobial substance activ... more Fourteen bacterial strains capable of producing a trypsin-dependent antimicrobial substance active against Clostridium perfringens were isolated from human fecal samples of various origins (from healthy adults and children, as well as from adults with chronic pouchitis). Identification of these strains showed that they belonged to Ruminococcus gnavus, Clostridium nexile, and Ruminococcus hansenii species or to new operational taxonomic units, all from the Clostridium coccoides phylogenetic group. In hybridization experiments with a probe specific for the structural gene encoding the trypsin-dependent lantibiotic ruminococcin A (RumA) produced by R. gnavus, seven strains gave a positive response. All of them harbored three highly conserved copies of rumA-like genes. The deduced peptide sequence was identical to or showed one amino acid difference from the hypothetical precursor of RumA. Our results indicate that the rumA-like genes have been disseminated among R. gnavus and phylogene...
In this review we describe the most common mechanisms through which bacterial antagonisms occur i... more In this review we describe the most common mechanisms through which bacterial antagonisms occur in the GI-tract. These include depletion and:or competition for substrates; competition for receptor sites (competitive exclusion); generation of a restrictive physiological environment (role of pH, short-chain fatty acids, oxidation-reduction potential); and production of antimicrobial substances (bacteriocinand non bacteriocin-related).
The salIR and salIM genes of Streptomyces albus G encode the restriction endonuclease (ENase) and... more The salIR and salIM genes of Streptomyces albus G encode the restriction endonuclease (ENase) and DNA methyltransferase (MTase) of the SalI restriction-modification (R-M) system. In S. albus G, the genes constitute an operon that is mainly transcribed from a promoter located upstream from sallR, the first gene of the operon. In addition, a second promoter, at the 3' end of sallR, allows independent transcription of the MTase gene. Expression of sallR and sallM in Escherichia coli was investigated. The ENase gene was not expressed in the heterologous host, probably due to inactivity of the main promoter of the salI operon. In contrast to sallR, sallM was functional in E. coli. Preliminary S1 nuclease mapping experiments suggest that the alternative promoter of the MTase gene can initiate transcription in the heterologous, as well as in the homologous host.
Gene shuffling is a way of creating proteins with interesting new characteristics, starting from ... more Gene shuffling is a way of creating proteins with interesting new characteristics, starting from diverged sequences. We tested an alternative to gene shuffling based on plasmid recombination and found that Bacillus subtilis efficiently recombines sequences with 4% divergence, and Escherichia coli mutS is more appropriate for sequences with 22% divergence.
Gene shuffling is a way of creating proteins with interesting new characteristics, starting from ... more Gene shuffling is a way of creating proteins with interesting new characteristics, starting from diverged sequences. We tested an alternative to gene shuffling based on plasmid recombination and found that Bacillus subtilis efficiently recombines sequences with 4% divergence, and Escherichia coli mutS is more appropriate for sequences with 22% divergence.
Ruminococcin C (RumC) is a trypsin-dependent bacteriocin produced by Ruminococcus gnavus E1, a gr... more Ruminococcin C (RumC) is a trypsin-dependent bacteriocin produced by Ruminococcus gnavus E1, a gram-positive strict anaerobic strain isolated from human feces. It consists of at least three similar peptides active against Clostridium perfringens. In this article, a 15-kb region from R. gnavus E1 chromosome, containing the biosynthetic gene cluster of RumC was characterized. It harbored 17 open reading frames (called rum c genes) with predicted functions in bacteriocin biosynthesis and post-translational modification, signal transduction regulation, and immunity. An unusual feature of the locus is the presence of five genes encoding highly homologous, but nonidentical RumC precursors. The transcription levels of the rum c genes were quantified. The rumC genes were found to be highly expressed in vivo, when R. gnavus E1 colonized the digestive tract of mono-contaminated rats, whereas the amount of corresponding transcripts was below detection level when it grew in liquid culture medium. Moreover, the rumC-like genes were disseminated among 10 strains (R. gnavus or related species) previously isolated from human fecal samples and selected for their capability to produce a trypsin-dependant anti-C. perfringens compound. All harbored at least a rumC1-like copy, four exhibited rumC1-5 genes identical to those of strain E1.
When cultivated in the presence of trypsin, the Ruminococcus gnavus E1 strain, isolated from a hu... more When cultivated in the presence of trypsin, the Ruminococcus gnavus E1 strain, isolated from a human fecal sample, was able to produce an antibacterial substance that accumulated in the supernatant. This substance, called ruminococcin A, was purified to homogeneity by reverse-phase chromatography. It was shown to be a 2,675-Da bacteriocin harboring a lanthionine structure. The utilization of Edman degradation and tandem mass spectrometry techniques, followed by DNA sequencing of part of the structural gene, allowed the identification of 21 amino acid residues. Similarity to other bacteriocins present in sequence libraries strongly suggested that ruminococcin A belonged to class IIA of the lantibiotics. The purified ruminococcin A was active against various pathogenic clostridia and bacteria phylogenetically related to R. gnavus . This is the first report on the characterization of a bacteriocin produced by a strictly anaerobic bacterium from human fecal microbiota.
Ruminococcin A (RumA) is a trypsin-dependent lantibiotic produced by Ruminococcus gnavus E1, a gr... more Ruminococcin A (RumA) is a trypsin-dependent lantibiotic produced by Ruminococcus gnavus E1, a gram-positive strict anaerobic strain isolated from a human intestinal microbiota. A 12.8-kb region from R. gnavus E1 chromosome, containing the biosynthetic gene cluster of RumA, has been cloned and sequenced. It consisted of 13 open reading frames, organized in three operons with predicted functions in lantibiotic biosynthesis, signal transduction regulation, and immunity. One unusual feature of the locus is the presence of three almost identical structural genes, all of them encoding the RumA precursor. In order to determine the role of trypsin in RumA production, the transcription of the rum genes has been investigated under inducing and noninducing conditions. Trypsin activity is needed for the growth phase-dependent transcriptional activation of RumA operons. Our results suggest that bacteriocin production by R. gnavus E1 is controlled through a complex signaling mechanism involving ...
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