The unusual structure of Ruminococcin C1 antimicrobial peptide confers clinical properties.

The emergence of superbugs developing resistance to antibiotics and the resurgence of microbial infections have led scientists to start an antimicrobial arms race. In this context, we have previously identified an active RiPP, the Ruminococcin C1, naturally produced by Ruminococcus gnavus E1, a symbiont of the healthy human intestinal microbiota. This RiPP, subclassified as a sactipeptide, requires the host digestive system to become active against pathogenic Clostridia and multidrug-resistant strains. Here we report its unique compact structure on the basis of four intramolecular thioether bridges with reversed stereochemistry introduced posttranslationally by a specific radical-SAM sactisynthase. This structure confers to the Ruminococcin C1 important clinical properties including stability to digestive conditions and physicochemical treatments, a higher affinity for bacteria than simulated intestinal epithelium, a valuable activity at therapeutic doses on a range of clinical pathogens, mediated by energy resources disruption, and finally safety for human gut tissues.

The General Stress Response σS Is Regulated by a Partner Switch in the Gram-negative Bacterium Shewanella oneidensis.

Here, we show that a partner-switching system of the aquatic Proteobacterium Shewanella oneidensis regulates post-translationally σS (also called RpoS), the general stress response sigma factor. Genes SO2118 and SO2119 encode CrsA and CrsR, respectively. CrsR is a three-domain protein comprising a receiver, a phosphatase, and a kinase/anti-sigma domains, and CrsA is an anti-sigma antagonist. In vitro, CrsR sequesters σS and possesses kinase and phosphatase activities toward CrsA. In turn, dephosphorylated CrsA binds the anti-sigma domain of CrsR to allow the release of σS This study reveals a novel pathway that post-translationally regulates the general stress response sigma factor differently than what was described for other proteobacteria like Escherichia coli We argue that this pathway allows probably a rapid bacterial adaptation.

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

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.

Characterization and purification of a bacteriocin from Lactobacillus paracasei subsp. paracasei BMK2005, an intestinal isolate active against multidrug-resistant pathogens.

A strain of Lactobacillus paracasei subsp. paracasei BMK2005 isolated from healthy infant faeces has shown a remarkable antibacterial activity against 32 bacterial pathogenic strains of human clinical isolates. Among them, 13 strains belonging to species of Escherichia coli, Citrobacter freundii, Citrobacter diversus, Klebsiella oxytoca, Enterobacter cloacae and Pseudomonas aeruginosa were resistant to Cefotaxime (CTX) and Ceftazidime (CAZ), and 4 strains of Staphylococcus aureus were resistant to Methicillin (MRSA). This antibacterial activity was attributed to a bacteriocin designated as Paracaseicin A. It was heat-stable up to 120°C for 5 min and active within the pH range of 2-5. Its activity was lost when treated with proteases, which reveals its proteinaceous nature. This bacteriocin was successfully purified only by two steps of reversed phase chromatography. Its molecular mass, determined by mass spectrometry analysis, was 2,462.5 Da. To our knowledge, the present study is the first report on characterization and purification of a bacteriocin, produced by a L. paracasei subsp. paracasei strain exhibiting an antibacterial activity against various multidrug-resistant species of Gram-positive and Gram-negative bacteria, which reveals its potential for use in prevention or treatment of infections caused by multidrug-resistant species especially in cases of antibiotics-associated diarrhea (AAD).

The Tol-Pal system of Escherichia coli plays an unexpected role in the import of the oxyanions chromate and phosphate.

Chromate is a toxic metal that enters bacteria by using oxyanion importers. Here, we show that each mutant of the Tol-Pal system of Escherichia coli exhibited increased chromate resistance. This system, which spans the cell envelope, plays a major role in envelope integrity and septation. The ΔtolQR mutant accumulated three-fold less chromate than the wild-type. Addition of phosphate but not sulfate to rich medium drastically reduced chromate toxicity and import in the wild-type strain. Furthermore, the intracellular concentration of free inorganic phosphate was significantly reduced for the ΔtolR mutant in comparison to the wild-type strain. Moreover, extracellular labeled phosphate was significantly less incorporated into the ΔtolR mutant. Finally, two distinct TolQR mutant complexes, specifically affected in Tol-Pal energization without affecting the TolQRA complex structure, did not complement the ΔtolQR mutant for inorganic phosphate accumulation. We thus propose that, while the Pst system is well known to import inorganic phosphate, the Tol-Pal system participates to phosphate uptake in particular at medium to high extracellular phosphate concentrations. Since mutations disabling the Tol-Pal system lead to pleiotropic effects, chromate resistance and reduced inorganic phosphate import could occur from an indirect effect of mutations in components of the Tol-Pal system.

RadA, a MSCRAMM Adhesin of the Dominant Symbiote Ruminococcus gnavus E1, Binds Human Immunoglobulins and Intestinal Mucins.

Adhesion to the digestive mucosa is considered a key factor for bacterial persistence within the gut. In this study, we show that Ruminococcus gnavus E1 can express the radA gene, which encodes an adhesin of the MSCRAMMs family, only when it colonizes the gut. The RadA N-terminal region contains an all-ß bacterial Ig-like domain known to interact with collagens. We observed that it preferentially binds human immunoglobulins (IgA and IgG) and intestinal mucins. Using deglycosylated substrates, we also showed that the RadA N-terminal region recognizes two different types of motifs, the protein backbone of human IgG and the glycan structure of mucins. Finally, competition assays with lectins and free monosaccharides identified Galactose and N-Acetyl-Galactosamine motifs as specific targets for the binding of RadA to mucins and the surface of human epithelial cells.

α-Galactosidase and Sucrose-Kinase Relationships in a Bi-functional AgaSK Enzyme Produced by the Human Gut Symbiont Ruminococcus gnavus E1.

Plant α-galactosides belonging to the raffinose family oligosaccharides (RFOs) and considered as prebiotics, are commonly degraded by α-galactosidases produced by the human gut microbiome. In this environment, the Ruminococcus gnavus E1 symbiont-well-known for various benefit-is able to produce an original RgAgaSK bifunctional enzyme. This enzyme contains an hydrolytic α-galactosidase domain linked to an ATP dependent extra-domain, specifically involved in the α-galactoside hydrolysis and the phosphorylation of the glucose, respectively. However, the multi-modular relationships between both catalytic domains remained hitherto unexplored and has been, consequently, herein investigated. Biochemical characterization of heterologously expressed enzymes either in full-form or in separated domains revealed similar kinetic parameters. These results were supported by molecular modeling studies performed on the whole enzyme in complex with different RFOs. Further enzymatic analysis associated with kinetic degradation of various substrates followed by high pressure anionic exchange chromatography revealed that catalytic efficiency decreased as the number of D-galactosyl moieties branched onto the oligosaccharide increased, suggesting a preference of RgAgaSK for RFO's short chains. A wide prevalence and abundance study on a human metagenomic library showed a high prevalence of the RgAgaSK encoding gene whatever the health status of the individuals. Finally, phylogeny and synteny studies suggested a limited spread by horizontal transfer of the clusters' containing RgAgaSK to only few species of Firmicutes, highlighting the importance of these undispersed tandem activities in the human gut microbiome.

Ruminococcin C, a promising antibiotic produced by a human gut symbiont.

A major public health challenge today is the resurgence of microbial infections caused by multidrug-resistant strains. Consequently, novel antimicrobial molecules are actively sought for development. In this context, the human gut microbiome is an under-explored potential trove of valuable natural molecules, such as the ribosomally-synthesized and post-translationally modified peptides (RiPPs). The biological activity of the sactipeptide subclass of RiPPs remains under-characterized. Here, we characterize an antimicrobial sactipeptide, Ruminococcin C1, purified from the caecal contents of rats mono-associated with Ruminococcus gnavus E1, a human symbiont. Its heterologous expression and post-translational maturation involving a specific sactisynthase establish a thioether network, which creates a double-hairpin folding. This original structure confers activity against pathogenic Clostridia and multidrug-resistant strains but no toxicity towards eukaryotic cells. Therefore, the Ruminococcin C1 should be considered as a valuable candidate for drug development and its producer strain R. gnavus E1 as a relevant probiotic for gut health enhancement.

Heterologous Biosynthesis, Modifications and Structural Characterization of Ruminococcin-A, a Lanthipeptide From the Gut Bacterium Ruminococcus gnavus E1, in Escherichia coli.

Ruminococcin A (RumA) is a lanthipeptide with high activity against pathogenic clostridia and is naturally produced by the strict anaerobic bacterium Ruminococcus gnavus E1, isolated from human intestine. Cultivating R. gnavus E1 is challenging, limiting high-quality production, further biotechnological development and therapeutic exploitation of RumA. To supply an alternative production system, the gene encoding RumA-modifying enzyme (RumM) and the gene encoding the unmodified precursor peptide (preRumA) were amplified from the chromosome of R. gnavus E1 and coexpressed in Escherichia coli. Our results show that the ruminococcin-A lanthionine synthetase RumM catalyzed dehydration of threonine and serine residues and subsequently installed thioether bridges into the core structure of a mutant version of preRumA (preRumA∗). These modifications were achieved when the peptide was expressed as a fusion protein together with green fluorescence protein (GFP), demonstrating that a larger attachment to the N-terminus of the leader peptide does not obstruct in vivo processivity of RumM in modifying the core peptide. The leader peptide serves as a docking sequence which the modifying enzyme recognizes and interacts with, enabling its catalytic role. We further investigated RumM catalysis in conjunction with the formation of complexes observed between RumM and the chimeric GFP fusion protein. Results obtained suggested some insights into the catalytic mechanisms of class II lanthipeptide synthetases. Our data further indicated the presence of three thioether bridges, contradicting a previous report whose findings ruled out the possibility of forming a third ring in RumA. Modified preRumA∗ was activated in vitro by removing the leader peptide using trypsin and biological activity was achieved against Bacillus subtilis ATCC 6633. A production yield of 6 mg of pure modified preRumA∗ per liter of E. coli culture was attained and considering the size ratio of the leader-to-core segments of preRumA∗, this amount would generate a final yield of approximately 1-2 mg of active RumA when the leader peptide is removed. The yield of our system exceeds that attainable in the natural producer by several 1000-fold. The system developed herein supplies useful tools for product optimization and for performing in vivo peptide engineering to generate new analogs with superior anti-infective properties.

Draft Genome Sequence of Shewanella algidipiscicola H1, a Highly Chromate-Resistant Strain Isolated from Mediterranean Marine Sediments.

The ability of different Shewanella spp. to convert heavy metals and toxic substances into less toxic products by using them as electron acceptors has led to their use in environmental clean-up strategies. We present here the draft genome sequence of Shewanella algidipiscicola H1, a strain resistant to high concentrations of chromates.

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.

Efficient respiration on TMAO requires TorD and TorE auxiliary proteins in Shewanella oneidensis.

Respiration on trimethylamine oxide (TMAO) allows bacterial survival under anoxia. In Shewanella oneidensis, Tor is the system involved in TMAO respiration and it is encoded by the torECAD operon. The torA and torC genes encode TorA terminal reductase and the TorC c-type cytochrome, respectively. Sequence analysis suggests that TorD is the putative specific chaperone of TorA, whereas TorE is of unknown function. The purpose of this study was to understand whether TorD and TorE are two accessory proteins that affect the efficiency of the Tor system by chaperoning TorA terminal reductase. Moreover, by deleting each gene, we established that the absence of TorD drastically affects the stability of TorA, while the absence of TorE does not affect TorA stability or activity. Since TMAO reduction was affected in the ΔtorE mutant, TorE could be an additional component of the TorC-TorA electron transfer chain during bacterial respiration. Finally, a fitness experiment indicated that the presence of TorE, as expected, confers a selective advantage in competitive environments.

Effects of a sulfonylurea herbicide on the soil bacterial community.

Sulfonylurea herbicides are widely used on a wide range of crops to control weeds. Chevalier® OnePass herbicide is a sulfonylurea herbicide intensively used on cereal crops in Algeria. No information is yet available about the biodegradation of this herbicide or about its effect on the bacterial community of the soil. In this study, we collected an untreated soil sample, and another sample was collected 1 month after treatment with the herbicide. Using a high-resolution melting DNA technique, we have shown that treatment with Chevalier® OnePass herbicide only slightly changed the composition of the whole bacterial community. Two hundred fifty-nine macroscopically different clones were isolated from the untreated and treated soil under both aerobic and microaerobic conditions. The strains were identified by sequencing a conserved fragment of the 16S rRNA gene. The phylogenetic trees constructed using the sequencing results confirmed that the bacterial populations were similar in the two soil samples. Species belonging to the Lysinibacillus, Bacillus, Pseudomonas, and Paenibacillus genera were the most abundant species found. Surprisingly, we found that among ten strains isolated from the treated soil, only six were resistant to the herbicide. Furthermore, bacterial overlay experiments showed that only one resistant strain (related to Stenotrophomonas maltophilia) allowed all the sensitive strains tested to grow in the presence of the herbicide. The other resistant strains allowed only certain sensitive strains to grow. On the basis of these results, we propose that there must be several biodegradation pathways for this sulfonylurea herbicide.

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.

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].

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.

Escherichia coli mutators: selection criteria and migration effect.

In silico, it has been shown that mutator alleles that increase mutation rate can be selected for by generating adaptive mutations. In vitro and in vivo, competition between wild-type bacteria and isogenic mutator mutants is consistent with this view. However, in vivo, the gain of the mutator seems to be reduced when migration is allowed. In vitro, the advantage of mutators has been described as frequency-dependent, leading to mutator advantage only when they are sufficiently frequent. Using an in vitro system, it is demonstrated that (i) the selection of mutators is frequency-independent, yet depends on at least one mutator bacterium bearing an adaptive mutation (its presence depends on chance, mutation rates and population size of mutator bacteria); (ii) on average, the mutator gain is always equal to the ratio of the adaptive mutation frequency of the mutator versus wild-type; (iii) when migration into an empty niche is allowed, the mutator benefit is reduced if migration occurs after fixation of the adaptive mutation into the wild-type population. It is concluded that in all cases, mutator gain depends directly on the ratio of bacteria carrying a beneficial mutation in mutator versus wild-type lineages.

[Impact of mutation rate on the adaptation of gut bacteria]. / Taux de mutation et adaptation des bactéries au tube digestif.

To study the role of mutator bacteria in the evolution of bacterial populations, we followed the impact of the mutation rate of Escherichia coli strains in the colonisation of the gut of axenic mice and the evolution of the mutation rate of bacterial populations living in the gut. We show that mutator bacteria have an advantage during the colonization. This adaptive advantage comes from their ability to generate adaptive mutations faster than wild type strains, mutations that allow their maintenance in the ecosystem. However, while mutator bacteria are becoming specialised to the environment they are living in, they accumulate mutations that may be deleterious or lethal in secondary environments. By following the evolution of the mutation rate of bacterial populations living in the gut of mice receiving antibiotics, we show that this therapy selects not only for antibiotic resistant mutants but also for mutator alleles that enhance mutation rates and are responsible for the appearance of the resistance. The costs of a high mutation rate, due to the accumulation of mutations, is seen in environments where changes are recurrent. In an ever-changing situation where every change is new, mutator bacteria might help the evolution of bacterial populations.

Trypsin mediates growth phase-dependent transcriptional tegulation 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.

Mutator bacteria as a risk factor in treatment of infectious diseases.

We show in a gnotobiotic mouse model that, in addition to direct selection of antibiotic-resistant bacteria, some antibiotic treatments also select for mutator alleles. Because of these mutator alleles' high mutation rates, the initial treatment failure increases the probability of failures in subsequent treatments with other drugs.