In contrast, the membrane-bound FtsH protease was only detected in the membrane fraction of both strains analyzed (not shown). Taken together, these results showed that cells displaying increased expression of σF-dependent genes accumulate this sigma factor in the cytoplasm. Figure 6 Subcellular localization of σ F . Immunoblot assays selleckchem performed with membrane and soluble fractions obtained from parental strain NA1000 (WT) and a CC3252 mutant with both cysteine residues
C131 and C181 replaced for serine (C131-181S). Aliquots were taken immediately before or after cells were treated with 55μM potassium dichromate (K2Cr2O7) for 30min. Membrane and soluble fractions were obtained as described in Methods. buy DZNeP Blots were developed using anti-σFantiserum and fluorescent CF680 Goat Anti-Rabbit IgG. σF is shown by an arrow. Neither σF nor σF-dependent genes CC2906 and
CC3255 are essential for Caulobacter resistance to metal stress To investigate the requirement of sigF for resistance of C. crescentus cells to dichromate or cadmium, the sensitivity of the parental strain and the sigF deletion mutant to exposure to these metals was monitored. Both strains displayed similar sensitivity profile to dichromate or cadmium (data not shown), suggesting that sigF is not essential for bacterial survival under this stress condition. As the deduced protein sequences of CC2906 and CC3255 are highly similar, we constructed a single deletion mutant strain in each gene (SG19 and SG20) as well as a double mutant (SG21) and tested the resistance of these strains to the metal stresses. Similar to what was found for the sigF deletion mutant, no increased sensitivity was observed for these mutant strains following Galeterone exposure to either dichromate or
cadmium, when compared to parental cells (data not shown). Together, these data suggest that σF-mediated transcriptional response to chromium or cadmium is not essential for survival of C. crescentus to exposure to these metal ions. Discussion In this report, we clearly show that C. crescentus σF is involved in the transcriptional response to chromium and cadmium in an oxidative stress independent manner. Transcriptome analysis of cells under dichromate stress revealed that σF controls a small regulon comprised of eight genes, which are distributed in three transcriptional units. Although a conserved domain was predicted for the deduced protein sequence of all σF-dependent genes, only two of these sequences could be assigned to a possible function. The protein encoded by CC2748 belongs to the group of sulfite oxidases, which catalyze the oxidation of the toxic and very reactive sulfite to the inert sulfate anion [22]. The product of CC3257 is a member of the DoxX family.