Isolement de Shewanella sp. d'origines algériennes et caractérisation d'un systeme de detoxication de polluants

Le Cr est principalement rencontré sous deux formes stables, Cr(III) et Cr(VI). Ce dernier est hautement toxique en raison de sa forte nature oxydante et de sa grande solubilité. L'organisme modèle pour la bioremédiation, Shewanella oneidensis MR-1, a développé divers mécanismes de résistance pour faire face à la toxicité du chromate.Le premier objectif de cette thèse était d'étudier les mécanismes de résistance au chromate et de sa réduction chez cette bactérie dans des conditions semi-aérobies. Nous avons a montré que le gène chrASO est induit par le chromate et que son produit fonctionne comme une pompe d'efflux pour extruder les ions chromate du cytoplasme. Nous avons également identifié une potentielle chromate réductase DmsA2, ainsi que deux autres protéines Fdh et DmsA1 potentiellement impliquées dans la résistance au chromate et dans sa réduction. Dans la deuxième partie de ce travail, nous vous on a isolé, identifié et caractérisé deux nouvelles souches de Shewanella, S. fidelis H76 et S. algidipiscicola H111. Les deux souches se caractérisent par leur grande résistance au chromate et leur capacité à le réduire efficacement même à des concentrations élevées. En dépit de la petite taille de son génome et l'absence de plusieurs gènes codant des enzymes connues pour jouer un rôle dans la résistance au chromate ainsi que dans sa réduction, la souche H111 est la plus efficace. De façon intéressante, les biofilms de l'interface liquide-air (Pellicules) des deux souches réduisent plus efficacement le chromate que leurs cellules planctoniques et ils peuvent accumuler une quantité importante de formes réduites du chromate.Cr primarily exists in two stable forms, Cr(III) and Cr(VI). The latter is highly toxic due to its strong oxidizing nature and its high solubility. The model organism for bioremediation Shewanella oneidensis MR-1 has evolved diverse resistance mechanisms to cope with chromate toxicity. The first aim of the present thesis was to study the chromate resistance and reduction mechanisms of this bacterium under semi-aerobic conditions. We showed that chrASO gene is induced by chromate and its products functions as an efflux pump to extrude chromate ions from the cytoplasm protecting cells from chromate toxicity. We also identified a potential chromate reductase DmsA2, as well as two other proteins Fdh and DmsA1 that are potentially involved in chromate resistance and reduction. In the second part of this work, we isolated, identified and characterized two novel Mediterranean Shewanella sp. strains, S. fidelis H76 and S. algidipiscicola H111. Both strains are characterized by their great chromate resistance and their ability to reduce it efficiently even at high concentrations. Although the small size of its genome and the absence of several genes encoding enzymes known to play a role in chromate resistance and reduction, the H111 strain is the best chromate resistant strain. Interestingly, the air liquid interface biofilm (Pellicles) of both strains reduce more efficiently chromate than their free-swimming cells and can accumulate a significant amount of its reduced forms

Bacterial resistance to temperate phage is influenced by the frequency of lysogenic establishment

Temperate phages can shape bacterial community dynamics and evolution through lytic and lysogenic life cycles. In response, bacteria that resist phage infection can emerge. This study explores phage-based factors that influence bacterial resistance using a model system of temperate P22 phage and Salmonella both inside and outside the mammalian host. Phages that remained functional despite gene deletions had minimal impact on lysogeny and phage resistance except for deletions in the immI region that substantially reduced lysogeny and increased phage resistance to levels comparable to that observed with an obligately lytic P22. This immI deletion does not make the lysogen less competitive but instead increases the frequency of bacterial lysis. Thus, subtle changes in the balance between lysis and lysogeny during the initial stages of infection can significantly influence the extent of phage resistance in the bacterial population. Our work highlights the complex nature of the phage-bacteria-mammalian host triad

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

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

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

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

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

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