crescentus[14, 15, 30], we were not able to delete nrsF, probably due to the toxic effect of high levels of σF under no stress conditions. However, we could isolate strains in which one or both of the conserved cysteine residues of NrsF were replaced for serine. As suggested by Western blot analysis, isolation of these point mutation strains was possible probably because most of σF molecules are still directly or indirectly sequestered in an inactive state to the inner membrane by NrsF. Substitution Milciclib in vitro of the conserved cysteines might have caused structural
changes in NrsF and hence resulting in a lower capacity to bind σF. In fact, σF was found to accumulate in the soluble fraction of cells expressing NrsF mutated in both cysteine residues even when cells were cultured under unstressed conditions. The presence of σF in the soluble fraction was also detected Pifithrin-�� concentration following treatment of parental cells with dichromate. Therefore, we could observe accumulation of σF in the soluble fraction in situations in which lower affinity of NrsF for σF is expected. Interestingly, two conserved cysteine residues in ChrR, the anti-sigma factor of Caulobacter σE, were also shown to be important for the response to cadmium mediated by that sigma factor [14, 15, 30]. Furthermore, the sensor histidine
kinase PhyK, involved in the control of the anti-anti-sigma factor PhyR of Caulobacter σT, Dapagliflozin which as mentioned above responds to dichromate and cadmium, also presents a conserved cysteine that is important to PhyK activity [14, 15, 30]. Thus, cysteines in the probable sensor proteins (NrsF, ChrR and PhyK) of ECF sigma factor mediated systems seem to play a key role in triggering the response to heavy metal stress in C. crescentus. Based on the fact that dichromate and cadmium are able to directly bind thiol groups [2, 38], it is conceivable that these metals could disrupt contacts mediated by the conserved cysteines of NrsF, leading to changes in its conformation similar
to those expected in the mutant proteins with one or both of the cysteine residues substituted. However, activation of σF might also be caused by direct interaction of chromate, dichromate and cadmium with other amino acid residues in NrsF or even with another yet unknown sensory component of the system. The finding that single substitutions of the conserved cysteine residues still allows for induction of σF-dependent genes ruled out the formation of an see more intramolecular bond between Cys131 and Cys181 residues under stress conditions. Nevertheless, we could not discard the possibility that NrsF functions as a dimer/multimer using intermolecular bonds for sensing the metals in the extracytoplasmic environment. Conclusion This report deals with the role and regulation of C.