Michel Fons

Utilisation of Mucin Glycans by the Human Gut Symbiont Ruminococcus gnavus Is Strain-Dependent

Commensal bacteria often have an especially rich source of glycan-degrading enzymes which allow them to utilize undigested carbohydrates from the food or the host. The species Ruminococcus gnavus is present in the digestive tract of ≥90% of humans and has been implicated in gut-related diseases such as inflammatory bowel diseases (IBD). Here we analysed the ability of two R. gnavus human strains, E1 and ATCC 29149, to utilize host glycans. We showed that although both strains could assimilate mucin monosaccharides, only R. gnavus ATCC 29149 was able to grow on mucin as a sole carbon source. Comparative genomic analysis of the two R. gnavus strains highlighted potential clusters and glycoside hydrolases (GHs) responsible for the breakdown and utilization of mucin-derived glycans. Transcriptomic and functional activity assays confirmed the importance of specific GH33 sialidase, and GH29 and GH95 fucosidases in the mucin utilisation pathway. Notably, we uncovered a novel pathway by which R. gnavus ATCC 29149 utilises sialic acid from sialylated substrates. Our results also demonstrated the ability of R. gnavus ATCC 29149 to produce propanol and propionate as the end products of metabolism when grown on mucin and fucosylated glycans. These new findings provide molecular insights into the strain-specificity of R. gnavus adaptation to the gut environment advancing our understanding of the role of gut commensals in health and disease.

Trypsin Mediates Growth Phase-Dependent Transcriptional Regulation of Genes Involved in Biosynthesis of Ruminococcin A, a Lantibiotic Produced by a Ruminococcus gnavus Strain from a Human Intestinal Microbiota

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 the proteolytic processing of a putative extracellular inducer-peptide by trypsin, a specific environmental cue of the digestive ecosystem.

α-Galactosidase/sucrose kinase (AgaSK), a novel bifunctional enzyme from the human microbiome coupling galactosidase and kinase activities.

Background: Raffinose, an abundant carbohydrate in plants, is degraded into galactose and sucrose by intestinal microbial enzymes. Results: AgaSK is a protein coupling galactosidase and sucrose kinase activity. The structure of the galactosidase domain sheds light onto substrate recognition. Conclusion: AgaSK produces sucrose-6-phosphate directly from raffinose. Significance: Production of sucrose-6-phosphate directly from raffinose points toward a novel glycolytic pathway in bacteria. α-Galactosides are non-digestible carbohydrates widely distributed in plants. They are a potential source of energy in our daily food, and their assimilation by microbiota may play a role in obesity. In the intestinal tract, they are degraded by microbial glycosidases, which are often modular enzymes with catalytic domains linked to carbohydrate-binding modules. Here we introduce a bifunctional enzyme from the human intestinal bacterium Ruminococcus gnavus E1, α-galactosidase/sucrose kinase (AgaSK). Sequence analysis showed that AgaSK is composed of two domains: one closely related to α-galactosidases from glycoside hydrolase family GH36 and the other containing a nucleotide-binding motif. Its biochemical characterization showed that AgaSK is able to hydrolyze melibiose and raffinose to galactose and either glucose or sucrose, respectively, and to specifically phosphorylate sucrose on the C6 position of glucose in the presence of ATP. The production of sucrose-6-P directly from raffinose points toward a glycolytic pathway in bacteria, not described so far. The crystal structures of the galactosidase domain in the apo form and in complex with the product shed light onto the reaction and substrate recognition mechanisms and highlight an oligomeric state necessary for efficient substrate binding and suggesting a cross-talk between the galactose and kinase domains.

Cloning in Escherichia coli of genes involved in the synthesis of proline and leucine in Desulfovibrio desulfuricans Norway

SummaryA library of Deusulfovibrio desulfuricans Norway genomic DNA was constructed in Escherichia coli with pBR322 as vector and plasmids able to complement the proA and leuB mutations of the host were screened. It was observed that all the plasmids studied were highly unstable, the insert DNA being rapidely lost under non-selective growth conditions. A 2.75 kb DNA fragment of D. desulfuricans Norway was found to complement E. coli ProA, ProB and ProC deficiencies. From the results of restriction analysis and Southern hybridization, it is proposed that the genes involved in proline and leucine biosynthesis are clustered on the chromosome of D. desulfuricans Norway.

Aga1, the first alpha-Galactosidase from the human bacteria Ruminococcus gnavus E1, efficiently transcribed in gut conditions.

Differential gene expression analysis was performed in monoxenic mice colonized with Ruminococcus gnavus strain E1, a major endogenous member of the gut microbiota. RNA arbitrarily primed-PCR fingerprinting assays allowed to specifically detect the in vivo expression of the aga1 gene, which was further confirmed by RT-PCR. The aga1 gene encoded a protein of 744 residues with calculated molecular mass of 85,207 Da. Aga1 exhibited significant similarity with previously characterized α-Galactosidases of the GH 36 family. Purified recombinant protein demonstrated high catalytic activity (104 ± 7 U mg(-1)) and efficient p-nitrophenyl-α-d-galactopyranoside hydrolysis [k(cat)/K(m) = 35.115 ± 8.82 s(-1) mM(-1) at 55 °C and k(cat)/K(m) = 17.48 ± 4.25 s(-1) mM(-1) at 37 °C].

Aerobic expression of the nar operon of Escherichia coli in a fnr mutant

Mutations allowing aerobic expression of the anaerobically controlled nar operon have been located in the autoregulated fnr gene. Cloning and sequencing of the mutant fnrd20 allele, and fnr mRNA quantitation by dot blot assay, revealed that the mutation was the result of an IS5 insertion into the control region of fnr that enhanced transcription of the fnr gene at least ten‐fold. Examination of the regulatory region of the negatively autoregulated fnr gene indicated that it shared homologous sequences with the positively Fnr‐controlled frd and nar operons. The increase in fnr transcription in the fnrd20 mutated allele could be partly the result of loss of autoregulation, since the IS5 separated the Fnr target site from the ‘−35’ region of the promoter.

Characterization of ISRgn1, a Novel Insertion Sequence of the IS3 Family Isolated from a Bacteriocin-Negative Mutant of Ruminococcus gnavus E1

ABSTRACT ISRgn1, an insertion sequence of the IS3 family, has been identified in the genome of a bacteriocin-negative mutant of Ruminococcus gnavus E1. The copy number of ISRgn1 in R. gnavus E1, as well as its distribution among phylogenetically E1-related strains, has been determined. Results obtained suggest that ISRgn1 is not indigenous to the R. gnavus phylogenetic group but that it can transpose in this bacterium.

Characterization and distribution of the gene cluster encoding RumC, an anti-Clostridium perfringens bacteriocin produced in the gut.

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.

ChrASO, the chromate efflux pump of Shewanella oneidensis, improves chromate survival and reduction

The chromate efflux pump encoding gene chrASO was identified on the chromosome of Shewanella oneidensis MR1. Although chrASO is expressed without chromate, its expression level increases when Cr(VI) is added. When deleted, the resulting mutant ΔchrASO exhibits a chromate sensitive phenotype compared to that of the wild-type strain. Interestingly, heterologous expression of chrASO in E. coli confers resistance to high chromate concentration. Moreover, expression of chrASO in S. oneidensis and E. coli significantly improves Cr(VI) reduction. This effect could result either from extracytoplasmic chromate reduction or from a better cell survival leading to enhanced Cr(VI) reduction.

ETUDE D’UNE ADHESINE IDENTIFIEE CHEZ RUMINOCOCCUS GNAVUS E1

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