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

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

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

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

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