Figure 3 Effect of arsenite concentration on swarming properties in H.
arsenicoxydans wild-type and mutant strains. selleck kinase inhibitor motility assays were performed in the presence of an increased concentration of As(III). The level of motility of each strain Stattic was evaluated as the diameter of the swarming ring expressed in mm. The results are the mean value of five independent experiments. Effect of AoxR, AoxS, RpoN and DnaJ on arsenite oxidase synthesis To get further insight into the involvement of AoxR, AoxS, RpoN and DnaJ in arsenite oxidase activity, Western immunoblotting experiments were performed using antibodies raised against AoxB. The abundance of this protein was evaluated from total protein extracts of H. arsenicoxydans wild-type and mutant strains grown in the presence or not of As(III). AoxB was detected as a single band corresponding to a molecular Selleckchem TPCA-1 mass of 92 kDa in As(III)-challenged H. arsenicoxydans strain (Figure 4). This single band was not observed in the various mutant strains. Furthermore, arsenite oxidase activity on native gel was only detected in As(III)-challenged wild type total extract (data not shown). Taken together these results suggest that the lack of activity in the mutant strains is due to the absence of AoxB protein, which may result from an effect of AoxR, AoxS, RpoN and DnaJ on aoxAB expression. Figure 4 Immunodetection of AoxB protein
in total protein extracts of H. arsenicoxydans wild-type and mutant strains. Effect of AoxR, AoxS, RpoN and DnaJ on
the control of arsenite oxidase operon expression To determine the involvement of aoxR, aoxS, dnaJ and rpoN on aoxAB transcription, we performed quantitative RT-PCR experiments. For each strain, changes in aoxB transcript abundance were compared to two internal controls, i.e. the putative RNA methyltransferase gene and the peptide deformylase gene, in cultures challenged or not PRKACG by As(III). The expression of aoxB mRNA was increased by a 9.4 fold factor after As(III) exposure in the H. arsenicoxydans wild-type strain. In contrast, aoxB expression was not increased in Ha482 (aoxS), Ha483 (aoxR), Ha3109 (rpoN) and Ha2646 (dnaJ) mutant strains, suggesting that the corresponding proteins play a crucial role in aoxAB operon expression (Table 2). Table 2 aoxB relative expression in H. arsenicoxydans wild-type and mutant strains. Strain aoxB expression ratio +As(III)/-As(III) Standard error Wild type 9.406 0.630 Ha3109 (rpoN) 0.250 0.060 Ha483 (aoxR) 0.111 0.024 Ha482 (aoxS) 0.200 0.029 Ha2646 (dnaJ) 1.156 0.289 Expression ratios of aoxB in H. arsenicoxydans wild-type and mutant strains without As(III) versus an As(III) 8 hours induction (1.33 mM), as measured by quantitative RT-PCR. Expression of each gene was normalized to the expression of the two housekeeping genes HEAR0118 and HEAR2922 coding for a peptide deformylase and a putative RNA methyltransferase, respectively.