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 . The product of CC3257 is a member of the DoxX family. Although nothing is actually known about the physiological role of bacterial proteins belonging to this family, the archaeal counterparts are involved in the elemental sulfur oxidation pathway [23, 24]. Therefore, both σF-dependent genes with a putative assigned function appear to play a role in sulfate acquisition by cells. Interestingly, Hu et al. (2005) found a strong down-regulation of a Caulobacter sulfate ABC transport system under chromate and dichromate exposure. While this detoxification strategy apparently contributes to decrease the concentration of chromate and dichromate in the cells , sulfate uptake from the extracellular environment might be significantly affected. Alternative sources such as degradation of sulfur-containing amino-acids  and organosulfonate metabolism  can be used to counteract this sulfur uptake limitation [1, 27–29]. It is therefore conceivable that induction of CC2748 and CC3257 could supply cells with sulfate. This is consistent with the observation that in Arthrobacter sp. strain FB24 and Pseudomonas putida, chromate exposure also results in increased levels of proteins potentially involved in reversing the effects of cellular sulfur limitation, such as transporters of alternative sulfur sources [27, 28].
Curiously, none of the most representative functional categories up-regulated under chromate, dichromate or cadmium exposure (protection against oxidative stress and reduction of intracellular metal concentration) were found to be controlled by σF, indicating that additional molecular systems are engaged in C. crescentus response to these metals. In fact, we previously reported the involvement of the paralogous sigma factors σT and σU in the control of response to chromium and cadmium [14, 15, 30] and σE in response to cadmium [14, 15, 30]. The observation that σF, σE and σT/σU regulate distinct sets of genes indicates that each of these sigma factors make a different contribution to the C. crescentus response to metal stress. Together, σF, σE, σT and σU are responsible for the induction of 20% of the genes previously found to be up-regulated under cadmium stress and σF, σT and σU control the expression of about 12% of genes induced following Caulobacter exposure to chromate or dichromate (Additional file 1: Table S1). Therefore, transcriptional regulators other than σF, σE, σT and σU appear to be involved in the response to chromate, dichromate and cadmium. The existence of several molecular systems contributing to the transcriptional response to metal stresses could explain why the absence of sigF, CC2906 or CC3255 does not decrease the viability of Caulobacter cells under dichromate or cadmium stresses. In agreement, we previously reported that σE elicits a rapid response to cadmium, but cells lacking rpoE are not impaired in survival to this stress condition [14, 15, 30].
Interestingly, sigF is not highly induced under either chromium or cadmium stress, different from what was observed for other ECF sigma factor genes such as rpoE and sigT in C. crescentus[14, 15, 30] and rpoJ, rpoK, rpoI, cnrH and rpoQ in C. metallidurans. This indicates that sigF is obviously not strongly auto-regulated under heavy metal stress conditions. Although the experimentally determined promoter sequences of sigF and CC3254 are highly similar to each other, promoter activity analyses supported our observation that CC3254 is solely regulated by σF, while the sigF operon is transcribed under the control of σF and a still unknown transcriptional regulator. Interestingly, both σF and the additional regulator depend on sequences located from −37 to +37 relative to the transcriptional start site (+1) of sigF. An apparent competition between these proteins might be the reason why sigF promoter activity is less responsive to high levels of σF when compared to the CC3254 promoter, which is solely controlled by σF. The existence of a second regulator of the sigF operon would be important to maintain a certain basal level of σF and consequently to allow a rapid response when cells experience environments contaminated with heavy metals. In the literature, one can find various examples of ECF sigma factor genes dependent on a second ECF sigma factor [32, 33]. In the present study, we could exclude Caulobacter rpoE and sigT as possible regulators of σF, since no difference in sigF expression was observed in the absence of either one of these ECF sigma factor genes.
In most cases, the activity of ECF sigma factors is modulated by a cognate anti-sigma factor [34–36]. Here, we showed that the second gene (CC3252) in the sigF operon acts as a negative regulator of σF function, as overexpression of the putative membrane protein encoded by CC3252 abolishes the transcriptional activation of sigF and its regulon under dichromate stress. Thus, CC3252 was here denominated nrsF. An interesting question about the nature of σF inhibition came from the observation that most of the protein encoded by nrsF is predicted to lie in the inner membrane of the bacterium: six transmembrane helices separated by five linkers ranging from 6 to 19 amino acid residues and an N-terminal segment of 25 residues. Usually, anti-sigma factors bind their cognate sigma factor through an extensive surface interaction, in which a domain of the first protein is sandwiched between domains σ2 and σ4 of the sigma factor . It is possible that several of the linkers of NrsF contact σF, resulting in a more stable interaction surface. However, we cannot discard the presence of a third component in this system able to directly bind both σF and NrsF and transduce the signal leading to activation of the sigma factor, to compensate this apparent lack of sufficient cytoplasmic segments in NrsF to contact σF. Attempts to obtain soluble recombinant full-length NrsF failed, probably because the protein cannot correctly fold in the absence of the hydrophobic environment found in the membrane compartment of bacterial cells. Therefore, it was not possible to test whether the recombinant protein encoded by nrsF directly binds σF.
As previously observed for other ECF sigma factors of C. 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 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 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, 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 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.