ETUDE D’UNE ADHESINE IDENTIFIEE CHEZ RUMINOCOCCUS GNAVUS E1

Ruminococcus gnavus E1 is a Gram positive strict anaerobic bacterium that was isolated from the dominant faecal microbiota of a healthy adult. A 6kb-long open reading fragment called radA was identified on the E1 chromosome, next to the genetic clusters involved in the biosynthesis of the RumA and RumC bacteriocins which are active against pathogenic Clostridium perfringens. radA shares a high sequence homology with genes of Staphylococcus aureus, Bacillus cereus and C. perfingens encoding adhesins of the MSCRAMMs family. RT-PCR experiments demonstrated that the radA gene was strongly expressed in vivo, when the E1 strain colonized the digestive tract of monoxenics animals, while little transcription occured in vitro. These results suggest that RadA could play an important role in the colonization of the digestive ecosystem

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

International audienceThe 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 chromate

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

International audienceThe chromate efflux pump encoding gene chrA SO was identified on the chromosome of She-wanella oneidensis MR1. Although chrA SO is expressed without chromate, its expression level increases when Cr(VI) is added. When deleted, the resulting mutant ΔchrA SO exhibits a chromate sensitive phenotype compared to that of the wild-type strain. Interestingly, heter-ologous expression of chrA SO in E. coli confers resistance to high chromate concentration. Moreover, expression of chrA SO 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

RadA, a MSCRAMM Adhesin of the Dominant Symbiote <i>Ruminococcus gnavus</i> 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-β 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

Chromate resistance and reduction by ChrA_SO in <i>E</i>. <i>coli</i>.

<p>(A) Chromate resistance due to the expression of <i>chrA</i>_<i>SO</i> in <i>E</i>. <i>coli</i> was assessed by comparing the growth of MC1061/p<i>chrA</i>_<i>SO</i> (red lines) to that of MC1061/pBAD33 (blue lines) in the presence of various concentrations of chromate (+, 0 mM; ■, 0.2 mM; ▲, 0.4 mM; ●, 0.8 mM and ×, 1.2 mM) at 30°C. Values are means from at least three experiments. (B) Chromate reduction by MC1061/p<i>chrA</i>_<i>SO</i> (red bars) and MC1061/pBAD33 (blue bars) was evaluated as the percentage of chromate reduced after 2 hours and 7 hours of challenge. [Cr(VI)], concentration of chromate added before growth expressed as mM; * indicates that the amount of chromate reduced is below detection level. Values are means ± standard deviations (error bars) from at least three experiments.</p

Expression of <i>lacZ</i> fusions in the presence of chromate.

<p>The wild-type <i>S</i>. <i>oneidensis</i> strain containing either the plasmid <i>pchrA</i>_<i>SO</i>::<i>lacZ</i> (transcriptional fusion between the promoter of <i>chrA</i>_<i>SO</i> and the <i>lacZ</i> reporter gene; light grey bars) or the plasmid p<i>mxd</i>_<i>450</i>::<i>lacZ</i> (transcriptional fusion between the promoter of <i>mxdA</i> and the <i>lacZ</i> reporter gene; dark grey bars), used as a control, was grown during 16 hours in the presence of increasing concentrations of chromate (0, 0.05, 0.1 and 0.2 mM) before β-galactosidase activity was determined. The MC1061 <i>E</i>. <i>coli</i> strain containing the plasmid <i>pchrA</i>_<i>SO</i>::<i>lacZ</i> was grown in similar conditions and β-galactosidase activity was also determined (black bars in the insert). [Cr(VI)] indicates the concentration of chromate during growth. β-galactosidase activity is expressed as Miller arbitrary units (AU). Values are means ± standard deviations (error bars) from at least three experiments.</p

ChrA_SO, the chromate efflux pump of <i>Shewanella oneidensis</i>, improves chromate survival and reduction

<div><p>The chromate efflux pump encoding gene <i>chrA</i>_<i>SO</i> was identified on the chromosome of <i>Shewanella oneidensis</i> MR1. Although <i>chrA</i>_<i>SO</i> is expressed without chromate, its expression level increases when Cr(VI) is added. When deleted, the resulting mutant Δ<i>chrA</i>_<i>SO</i> exhibits a chromate sensitive phenotype compared to that of the wild-type strain. Interestingly, heterologous expression of <i>chrA</i>_<i>SO</i> in <i>E</i>. <i>coli</i> confers resistance to high chromate concentration. Moreover, expression of <i>chrA</i>_<i>SO</i> in <i>S</i>. <i>oneidensis</i> and <i>E</i>. <i>coli</i> 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.</p></div

Impact of <i>chrA</i>_SO on chromate reduction in <i>S</i>. <i>oneidensis</i> strains.

<p>The WT/pBAD33, Δ<i>chrA</i>_SO/pBAD33 and Δ<i>chrA</i>_<i>SO</i>/p<i>chrA</i>_<i>SO</i> strains were challenged with 1 mM chromate before quantification of Cr(VI). Results were expressed as the percentage of chromate reduced after 2 hours of incubation in semi-aerobic (A) or anaerobic conditions (B). Values are means ± standard deviations (error bars) from at least three experiments.</p

Effect of <i>chrA</i>_<i>SO</i> on chromate resistance in <i>S</i>. <i>oneidensis</i> strains.

<p>Two different assays were conducted to evaluate the impact of <i>chrA</i>_<i>SO</i> on chromate resistance. (A) In the first assay, [CFU + Cr(VI)]/[CFU—Cr(VI)] (%) indicates the percentage of viable counts of the wild-type (WT) and the <i>chrA</i>_<i>SO</i> deleted mutant (Δ<i>chrA</i>_<i>SO</i>) after 5 hours of growth in semi-aerobiosis conditions and in the presence of 0.2 mM chromate, compared to that of the same strains grown in similar conditions without chromate. Values are means ± standard deviations (error bars) from at least two experiments. The mean absolute values of the number of CFU counted in the absence of chromate are 2.49 x 10⁹.mL^-1 and 2.41 x 10⁹.mL^-1 for the wild-type and the <i>chrA</i>_<i>SO</i> deleted mutant strains, respectively. (B) In the second assay, 10-fold serial dilutions of the wild-type (WT) and the <i>chrA</i>_<i>SO</i> deleted mutant (Δ<i>chrA</i>_<i>SO</i>) cultures were spotted on LB agar supplemented or not with 0.5 mM chromate before incubation at 28°C during 4 days.</p