BACKGROUND
several heavy metals play important roles as trace elements in the metabolism of all kingdoms of life.
whether a trace element is useful or harmful depends on its concentration.
particularly, chromium and cadmium are known to be much more toxic than useful for most microorganisms  <cit> .
chromium is commonly present in solutions as chromate and dichromate oxyanions ), the most redox-reactive and soluble forms of the metal  <cit> .
due to its similar chemical structure to sulfate anions, chromate crosses membranes via sulfate uptake systems  <cit> .
on the other hand, cadmium is a non-redox-reactive metal with high affinity for thiol groups  <cit> .
once inside cells, chromate, dichromate and cadmium exert their toxic effects by directly damaging cellular components and by inducing oxidative stress  <cit> .
in order to reduce the toxicity of chromate, dichromate and cadmium, some microorganisms eliminate these metals from the cytoplasm by using active transport efflux pumps  <cit> .
cadmium can also be sequestered within the cells by metal-chelating proteins, while chromate and dichromate are reduced to the less toxic and insoluble trivalent cation cr by specific nadh-dependent enzymes under aerobic conditions or in the electron transport chain of bacteria such as pseudomonas fluorescens lb <dig> in anaerobic environments  <cit> .
in addition, several enzymes work to counteract the deleterious effects of the oxidative stress induced following cell exposure to chromate, dichromate and cadmium.
caulobacter crescentus, an oligotrophic free-living α-proteobacterium, is able to grow in polluted habitats  <cit> .
not surprisingly, its genome encodes some homologues of genes involved in heavy metal resistance.
in a previous report, the set of genes responding to caulobacter exposure to chromate, dichromate and cadmium was identified  <cit> .
the main actions triggered in response to these metals are protection against oxidative stress  and reduction of intracellular metal concentration .
however, the signal transduction and control processes involved in the bacterial response to these heavy metals are still poorly characterized.
the c. crescentus genome encodes  <dig> extracytoplasmic function  sigma factors  <cit> .
two of them, the paralogous σt and σu, are involved in the response to various environmental stress conditions, including chromium and cadmium stresses  <cit> .
additionally, σe mediates a rapid transcriptional response to cadmium, organic hydroperoxide, singlet oxygen and uv-a  <cit> .
in a previous report, σf was found to be required for bacterial survival under hydrogen peroxide stress in the stationary growth phase, but no σf-mediated transcriptional response to hydrogen peroxide could be observed  <cit> .
thus, the involvement of σf in a transcriptional response to environmental stresses still needs to be characterized.
the observation that genes cc <dig>  cc <dig> and cc <dig>  previously found to be dependent on σf <cit> , are induced following c. crescentus exposure to chromate, dichromate and cadmium  <cit>  suggested to us that σf could be involved in the transcriptional response to these heavy metals.
in the present work, we demonstrate the involvement of σf in chromium and cadmium stress responses.
we also identified the set of genes regulated by σf by using global transcriptome analysis and characterized the promoter region of these genes by 5´race experiments and β-galactosidase assays.
furthermore, we investigated the role of the protein encoded by the second gene in the sigf operon , here named nrsf, and two conserved cysteine residues in this protein on the σf-mediated response to heavy metals.
RESULTS
σf is involved in chromium and cadmium responses in c. crescentus
in order to verify a possible involvement of σf in the c. crescentus response to chromium and cadmium stresses, we monitored expression of cc <dig>  previously identified as a σf-dependent gene, as well as cc <dig>  which is co-transcribed with sigf , by quantitative rt-pcr.
this analysis showed that cc <dig> is significantly induced in parental cells following exposure to either dichromate or cadmium .
in contrast, expression of cc <dig> in a sigf deletion mutant strain exposed to dichromate or cadmium was found to be quite similar to that observed in the same strain under no stress condition .
we could also observe significant induction of cc <dig> in the presence of dichromate, though the induction was modest when compared to the increase in expression of cc <dig> .
on the other hand, no significant increase in cc <dig> expression was found in sigf mutant cells following dichromate exposure .
taken together, these results confirm the involvement of σf in c. crescentus response to chromium and cadmium stresses and suggest that the operon sigf-cc <dig> is not strongly auto-regulated under these conditions.
to simplify our analyses and data presentation, we only show the expression of sigf and its target genes under dichromate stress in all subsequent experiments.
it is assumed that heavy metal ions cause oxidative stress inside cells  <cit> .
this raises the possibility that induction of σf-dependent genes by chromium and cadmium is a direct consequence of oxidative stress.
to test this hypothesis, we stressed the parental and the sigf mutant strains with hydrogen peroxide, t-butyl hydroperoxide, paraquat  or diamide .
according to qrt-pcr experiments, expression levels of cc <dig> and cc <dig> were not increased more than twofold in the parental strain during these stress conditions .
in agreement, transcript levels of cc <dig> and cc <dig> were also not influenced by any of these stressors in cells lacking sigf.
concentrations of hydrogen peroxide and t-butyl hydroperoxide used in our analyses were previously found to be sufficient to increase expression of other genes in c. crescentus <cit> .
taken together, these data suggest that chromium and cadmium are able to induce the σf regulon in an oxidative stress independent manner.
σf controls a small set of genes under chromium stress
with the aim of identifying additional genes induced during stress conditions under the control of σf, we compared the gene expression pattern of parental cells with that of a sigf mutant under dichromate stress, using microarray chips containing up to three different probes corresponding to the beginning of the coding region of each gene from c. crescentus.
by considering these probes, we found only six genes down-regulated in sigf mutant cells relative to the parental strain  .
interestingly, close inspection of probes corresponding to the upstream region from cc <dig> and cc <dig> suggested that these regions are also down-regulated in sigf mutant cells when compared to the parental strain.
the transcriptional start sites of the operons cc2906-cc <dig> and cc3255-cc3256-cc <dig> seem to be located quite distant from the translational start sites of cc <dig> and cc <dig> predicted by the tigr annotation.
genome organization suggests that cc <dig> is the first gene of the transcriptional unit cc3254-cc3255-cc3256-cc <dig> .
according to the tigr annotation, the deduced amino acid sequence of cc <dig> displays an n-terminal extension of  <dig> amino acid residues not found in orthologous sequences.
by excluding this extension, the most probable translational start site of cc <dig> is at position + <dig> relative to the translational start site of cc <dig> suggested by the tigr annotation .
thus, all probes designed to measure cc <dig> expression in microarray chips correspond to a region upstream from the translational start site of cc <dig> proposed here.
however, probes corresponding to the upstream region of cc <dig> cover the entire coding region of cc <dig>  therefore, by considering these probes, we could include cc <dig> as a σf-dependent gene .
this is in accordance with the previous observation that the complete transcriptional unit cc3254-cc3255-cc3256-cc <dig> is induced under chromium and cadmium stresses  <cit> .
a according to cmr  annotation of genome of cb <dig> strain.
b referring to the number of amino acid of the deduced protein sequence.
protein length is according to cmr annotation or prediction from our analysis.
c corresponding to the number of possible transmembrane  helices in the mature protein.
the number was determined by tmhmm tool.
d according to a re-analysis of the deduced protein sequences by using pfam and smart tools to search for conserved domains.
e sequences were compared with protein databases using blastp.
f microarray hybridization of rna samples isolated from exponential phase cells exposed to  <dig> μm potassium dichromate  for  <dig> min.
genes with m value of < − <dig>  or >  <dig>  were assumed as differentially expressed between strains analyzed.
values are the log <dig> ratio as mentioned.
results shown are the average of three independent biological experiments.
wt and Δsigf refer to the parental strain na <dig> and sigf deletion mutant, respectively.
nc refers to no significant change in gene expression.
g quantitative rt-pcr experiments performed with total rna extracted from exponentially growing cells immediately before  and following exposure during  <dig> min to  <dig> μm potassium dichromate .
results were normalized using gene cc <dig> as the endogenous control, which was constitutively expressed under the conditions analyzed.
values are the log <dig> ratio as mentioned.
data are mean values of two independent experiments.
wt and Δsigf refer to the parental strain na <dig> and sigf deletion mutant, respectively.
na corresponds to genes not analyzed in qrt-pcr experiments.
the cc <dig> gene is predicted to be transcribed divergently from cc2906-cc <dig> in the chromosome of cb <dig> strain.
however, the corresponding gene was not included during annotation of the more recent genome sequencing of c. crescentus .
in the chromosome of na <dig>  an open reading frame  was proposed to be located between genes ccna_ <dig>  and ccna_ <dig> .
nevertheless, ccna_ <dig> appears to be co-transcribed with ccna_ <dig> and ccna_ <dig>  in addition, we could observe co-occurrence of ccna_ <dig> with other σf-dependent genes.
as the nucleotide sequence between cc <dig> and cc <dig> in cb <dig> strain is identical to the region between ccna_ <dig> and ccna_ <dig> of na <dig> strain, we conclude that cc <dig> was incorrectly annotated in the genome of cb <dig> strain and this gene is the first one of the operon cc2907-cc2906-cc <dig> .
as evaluated with probes corresponding to the upstream region of cc <dig>  the entire coding region of cc <dig> is down-regulated in sigf mutant cells relative to the parental strain .
therefore, the complete transcriptional unit cc2907-cc2906-cc <dig> is controlled by σf.
a thorough re-annotation of genes regulated by σf suggested that cc <dig> codes for a putative membrane protein belonging to the doxx family, whose members are involved in sulfur metabolism.
the cc <dig> gene, which encodes the putative sulfite oxidase subunit yedy, is another protein with a potential role in sulfur metabolism.
all of the remaining σf-dependent genes  code for proteins with conserved domains of unknown functions.
interestingly, the pairs of genes cc <dig> and cc <dig>  cc <dig> and cc <dig>  as well as cc <dig> and cc <dig> are probable paralogous genes, with amino acid sequence identities of 36%, 43% and 23%, respectively.
therefore, it is possible that the gene products of both operons exert similar functions.
no other gene in the genome of c. crescentus displays significant nucleotide sequence similarity to the above mentioned pairs of paralogous genes or to the functionally annotated genes cc <dig> and cc <dig>
proteins encoded by cc <dig> and cc <dig> present a duf <dig> domain at their n-terminus.
this domain was described to be a dna-binding domain in ngo <dig> from neisseria gonorrhoeae <cit> .
ngo <dig> is involved in the transcriptional regulation of msrab, which codes for a methionine sulfoxide reductase  <cit> .
however, in our microarray experiments, we could not observe differences in the expression of msra homologs in c. crescentus .
thus, we conclude that the role of ngo <dig> in n. gonorrhoeae and cc <dig> or cc <dig> in c. crescentus is most likely different under these circumstances.
to confirm results obtained in transcriptome analysis, we investigated the expression levels of five genes supposedly dependent on σf  by qrt-pcr experiments.
these analyses showed that expression of these selected genes under dichromate stress is more than twofold higher in the parental strain relative to the sigf deletion mutant .
interestingly, induction of cc <dig> expression in the presence of dichromate was only partially dependent on σf , suggesting the involvement of an additional regulatory protein in the control of cc <dig> expression under this stress condition.
taken together, these results confirm the data obtained in global transcriptional analysis.
promoter sequence motifs of cc <dig> and cc <dig> genes are highly similar to those of sigf
to identify putative σf-dependent promoters upstream of cc <dig> and cc <dig> genes, we performed 5’race  experiments using primers that hybridize in the beginning of the coding region of the corresponding genes.
for these experiments, rna samples from cells exposed to dichromate were used, as this stress condition leads to increased expression levels of cc <dig> and cc <dig>  this approach led to the identification of a transcriptional start site  for cc <dig> at position − <dig> relative to the translational start site + <dig> proposed here .
a tss was also determined at position
− <dig> with respect to the translational start site of cc <dig> predicted here .
as expected, no tss could be observed when an additional 5´race experiment was performed using primers that hybridize to the beginning of the coding region of cc <dig> proposed by the tigr annotation.
together, these data confirmed our microarray data with respect to expression of the operons cc2907-cc2906-cc <dig> and cc3254-cc3255-cc3256-cc <dig>
the putative promoter sequences found for cc <dig> and cc <dig> were very similar to each other and also quite similar to the promoter sequence previously determined for sigf <cit>  .
additionally, analyses of the region upstream of the translational start site + <dig> of cc <dig> also revealed a putative σf-dependent sequence , suggesting a direct control of this gene by σf.
accordingly, the putative σf-dependent promoters reported here are highly similar to sequences found upstream from sigf homologs in other bacteria  <cit> .
conserved sequences upstream of cc <dig> and sigf are necessary for expression of these genes
to confirm the putative promoter sequence of the gene cluster cc3254-cc3255-cc3256-cc <dig>  transcriptional fusions containing a fragment encompassing the region upstream of the translational start site of cc <dig> predicted in this work and the lacz reporter gene  were created .
caulobacter cells harboring these different constructs were used in β-galactosidase assays.
when monitored in unstressed parental cells, a plasmid construction with the complete promoter sequence of the transcriptional unit cc3254-cc3255-cc3256-cc <dig>  resulted in higher β-galactosidase activity with respect to the empty vector placz <dig> or to the construct lacking the − <dig> promoter element  .
only basal β-galactosidase activity was observed with any of the constructions in cells of the sigf null mutant strain .
these results confirmed the data from qrt-pcr and 5’race experiments.
analyses with dichromate stressed cells showed no significant increase in β-galactosidase activity in the parental strain , probably due to the damage caused during dichromate stress to β-galactosidase in particular or to proteins in general.
as mentioned above, the promoter sequence of the operon cc3254-cc3255-cc3256-cc <dig> is highly similar to that located upstream from sigf.
to verify if sigf expression was also dependent on these putative promoter elements, we analyzed the upstream region of the sigf gene in β-galactosidase assays using two different plasmid constructs: pcklac53- <dig> containing the promoter elements upstream from sigf and construct pcklac53- <dig> that lacks the sigf promoter .
β-galactosidase activity measured in parental cells harboring the construct pck53- <dig>  was found to be quite similar to that observed in cells with the empty vector.
on the other hand, higher β-galactosidase activity was observed in the parental strain carrying construct pck53- <dig>  which contains the complete sigf promoter sequence .
cells from sigf mutant harboring the construct pcklac53- <dig> presented β-galactosidase activity slightly lower than that observed in parental cells with the same construct, but still higher than that observed in cells harboring the construct pck53- <dig> .
altogether, these data indicate that the promoter sequence upstream from sigf is necessary for expression of the sigf operon, but in a manner that is not exclusively dependent on σf.
this observation suggests that another sigma factor could also be capable of recognizing the region upstream from sigf.
thus, we have investigated the effect of two other ecf sigma factors involved in oxidative and heavy metal stresses, σt and σe, upon sigf promoter activity, but no significant decrease in β-galactosidase activity was observed in mutant strains Δsigt and Δrpoe when compared with parental cells, all harboring construct pcklac53- <dig> .
additionally, qrt-pcr experiments confirmed these results, as no change in transcription levels of the sigf gene was observed in a sigt or rpoe mutant .
these observations allowed us to rule out the participation of σt and σe in the control of sigf expression.
to further verify if the promoter region upstream of sigf is controlled by σf, we overexpressed sigf in the parental strain from an additional plasmid-encoded copy of the gene under the control of a constitutive promoter  and measured β-galactosidase activity in these cells harboring either pcklac53- <dig> or pcklac53- <dig>  overexpression of sigf in cells with the construct containing the complete sigf promoter  led to an increase in β-galactosidase activity, whereas no difference was observed in cells harboring the promoterless construct pcklac53- <dig> .
similarly, higher β-galactosidase activity was observed in sigf overexpressing cells bearing the construct containing the promoter sequence motifs upstream from cc <dig>  when compared to the parental strain carrying the same construct or sigf overexpressing cells harboring the construct containing only the − <dig> motif of the promoter sequence of cc3254-cc3255-cc3256-cc <dig>  .
therefore, these results confirm that specific and highly similar promoter sequence motifs found upstream from sigf-cc <dig> and cc3254-cc3255-cc3256-cc <dig> are required for the control of these transcriptional units by σf.
cc <dig> negatively regulates σf regulon expression
the chromosomal organization of cc <dig> and sigf in a putative operon suggests that cc <dig> could be involved in the same regulatory pathway of σf.
to test the assumption that cc <dig> could control σf activity, we monitored the expression of σf-dependent genes in parental cells overexpressing cc <dig> from a plasmid-encoded copy of the gene under the control of the constitutive lacz promoter present in vector pjs <dig>  for that, cells overexpressing cc <dig> were stressed or not with dichromate and compared in qrt-pcr experiments with cells harboring the empty vector pjs <dig> or cells without this vector under the same conditions.
according to qrt-pcr experiments, expression of genes cc <dig> and cc <dig> was slightly reduced in cells overexpressing cc <dig> under no stress conditions, when compared to cells with the empty vector pjs <dig> or cells without the vector .
however, induction of cc <dig> and cc <dig> expression under dichromate stress was clearly absent in cc <dig> overproducing cells, when compared to cells not overexpressing cc <dig> .
no difference could be found in the expression levels of two control genes  when we compared cells overexpressing cc <dig> or not .
this observation rules out a possible nonspecific effect due to overproduction of the protein.
taken together, these data indicate that cc <dig>  here denominated nrsf, acts as a negative regulator of σf function in c. crescentus.
upon dichromate stress, sigf expression was also slightly down-regulated in cc <dig> overexpressing cells, whereas under control conditions sigf expression was practically unchanged.
this once again favors the hypothesis that sigf is not strongly auto-regulated.
a further attempt to investigate the role of nrsf as a possible negative regulator of σf function was carried out by trying to construct a null mutant strain in gene nrsf.
however, it was not possible to construct a mutant strain by deleting nrsf in the parental strain .
on the other hand, nrsf could be deleted in the absence of a functional copy of sigf , suggesting that high σf activity is apparently responsible for the failure of disrupting nrsf in cells with functional sigf.
the putative protein encoded by nrsf is composed of six putative transmembrane segments separated by five short linkers  and an n-terminal segment of  <dig> residues .
alignment of the deduced amino acid sequence of cc <dig> with its orthologs from other bacteria  revealed two highly conserved cysteine residues .
the cysteine residues of the caulobacter protein  are probably directed into the periplasmic space , which favors their putative role in the signal transduction process leading to the liberation of σf from nrsf inhibition.
substitution of the conserved cysteines by serine led to two single mutants  and a double mutant .
even under unstressed conditions, all σf-regulated genes analyzed in qrt-pcr experiments, including sigf and cc <dig>  were up-regulated in the single mutant strains when compared to the parental strain .
the substitution of both cysteines by serine in nrsf resulted in the highest expression levels of the genes analyzed .
under stress conditions, both single substitution mutant strains showed an additional induction of σf-dependent genes, whereas no differences were observed for the double substitution mutant strain, compared to unstressed conditions .
therefore, these results show that substitution of one or both of the conserved cysteines  in the protein encoded by nrsf affects the ability of this protein in maintaining expression of σf-dependent genes at basal levels, further indicating the negative role of nrsf in the control of σf activity.
σf is released into the cytoplasm during chromium stress and in cells carrying point mutations in conserved cysteines of nrsf
the presence of six putative transmembrane segments in the protein coded by nrsf would imply that σf is sequestered to the inner membrane of caulobacter cells.
however, at least a portion of this sigma factor would be expected to be released into the cytoplasm following chromium and cadmium exposure.
to investigate this assumption, we monitored σf levels in the membrane and soluble fractions of caulobacter cell extracts by western blot analysis .
when extracts from parental cells under no stress condition were analyzed, σf was only detected in the membrane fraction.
although the majority of σf was still observed in the membrane fraction of extracts from parental cells exposed to dichromate, a significant portion of the sigma factor could also be detected in the soluble fraction.
additionally, we found that substitution of both conserved cysteine residues of nrsf also led to a significant accumulation of σf in the soluble fraction, even when cells were not exposed to dichromate.
in contrast, the membrane-bound ftsh protease was only detected in the membrane fraction of both strains analyzed .
taken together, these results showed that cells displaying increased expression of σf-dependent genes accumulate this sigma factor in the cytoplasm.
neither σf nor σf-dependent genes cc <dig> and cc <dig> 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 , suggesting that sigf is not essential for bacterial survival under this stress condition.
as the deduced protein sequences of cc <dig> and cc <dig> are highly similar, we constructed a single deletion mutant strain in each gene  as well as a double mutant  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 exposure to either dichromate or cadmium, when compared to parental cells .
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 cc <dig> belongs to the group of sulfite oxidases, which catalyze the oxidation of the toxic and very reactive sulfite to the inert sulfate anion  <cit> .
the product of cc <dig> 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  <cit> .
therefore, both σf-dependent genes with a putative assigned function appear to play a role in sulfate acquisition by cells.
interestingly, hu et al.
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  <cit> , sulfate uptake from the extracellular environment might be significantly affected.
alternative sources such as degradation of sulfur-containing amino-acids  <cit>  and organosulfonate metabolism  <cit>  can be used to counteract this sulfur uptake limitation  <cit> .
it is therefore conceivable that induction of cc <dig> and cc <dig> could supply cells with sulfate.
this is consistent with the observation that in arthrobacter sp.
strain fb <dig> 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  <cit> .
curiously, none of the most representative functional categories up-regulated under chromate, dichromate or cadmium exposure  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  <cit>  and σe in response to cadmium  <cit> .
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 .
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, cc <dig> or cc <dig> 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  <cit> .
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 <cit>  and rpoj, rpok, rpoi, cnrh and rpoq in c. metallidurans <cit> .
this indicates that sigf is obviously not strongly auto-regulated under heavy metal stress conditions.
although the experimentally determined promoter sequences of sigf and cc <dig> are highly similar to each other, promoter activity analyses supported our observation that cc <dig> 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 − <dig> to + <dig> relative to the transcriptional start site  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 cc <dig> 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  <cit> .
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  <cit> .
here, we showed that the second gene  in the sigf operon acts as a negative regulator of σf function, as overexpression of the putative membrane protein encoded by cc <dig> abolishes the transcriptional activation of sigf and its regulon under dichromate stress.
thus, cc <dig> 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  <dig> to  <dig> amino acid residues and an n-terminal segment of  <dig> 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 σ <dig> and σ <dig> of the sigma factor  <cit> .
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 <cit> , 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  <cit> .
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  <cit> .
thus, cysteines in the probable sensor proteins  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  <cit> , 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 cys <dig> and cys <dig> 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.
CONCLUSIONS
this report deals with the role and regulation of c. crescentus σf under stress conditions and provides new interesting information about this conserved but still poorly characterized ecf sigma factor: i) σf-dependent genes are induced in the presence of heavy metals in a manner independent of oxidative and disulfide stress; ii) σf directly controls a small regulon including genes involved in sulfur metabolism and iii) σf is negatively regulated by a putative membrane bound protein, here named nrsf, which contains two conserved cysteine residues that are important for its function and are located in the periplasmic portion of the protein.

 several heavy metals play important roles as trace elements in the metabolism of all kingdoms of life. whether a trace element is useful or harmful depends on its concentration. particularly, chromium and cadmium are known to be much more toxic than useful for most microorganisms. chromium is commonly present in solutions as chromate and dichromate oxyanions ), the most redox-reactive and soluble forms of the metal. due to its similar chemical structure to sulfate anions, chromate crosses membranes via sulfate uptake systems. on the other hand, cadmium is a non-redox-reactive metal with high affinity for thiol groups. once inside cells, chromate, dichromate and cadmium exert their toxic effects by directly damaging cellular components and by inducing oxidative stress. in order to reduce the toxicity of chromate, dichromate and cadmium, some microorganisms eliminate these metals from the cytoplasm by using active transport efflux pumps. cadmium can also be sequestered within the cells by metal-chelating proteins, while chromate and dichromate are reduced to the less toxic and insoluble trivalent cation cr by specific nadh-dependent enzymes under aerobic conditions or in the electron transport chain of bacteria such as pseudomonas fluorescens lb in anaerobic environments. in addition, several enzymes work to counteract the deleterious effects of the oxidative stress induced following cell exposure to chromate, dichromate and cadmium. caulobacter crescentus, an oligotrophic free-living α-proteobacterium, is able to grow in polluted habitats. not surprisingly, its genome encodes some homologues of genes involved in heavy metal resistance. in a previous report, the set of genes responding to caulobacter exposure to chromate, dichromate and cadmium was identified. the main actions triggered in response to these metals are protection against oxidative stress and reduction of intracellular metal concentration. however, the signal transduction and control processes involved in the bacterial response to these heavy metals are still poorly characterized. the c. crescentus genome encodes extracytoplasmic function sigma factors. two of them, the paralogous σt and σu, are involved in the response to various environmental stress conditions, including chromium and cadmium stresses. additionally, σe mediates a rapid transcriptional response to cadmium, organic hydroperoxide, singlet oxygen and uv-a. in a previous report, σf was found to be required for bacterial survival under hydrogen peroxide stress in the stationary growth phase, but no σf-mediated transcriptional response to hydrogen peroxide could be observed. thus, the involvement of σf in a transcriptional response to environmental stresses still needs to be characterized. the observation that genes cc cc and cc previously found to be dependent on σf, are induced following c. crescentus exposure to chromate, dichromate and cadmium suggested to us that σf could be involved in the transcriptional response to these heavy metals. in the present work, we demonstrate the involvement of σf in chromium and cadmium stress responses. we also identified the set of genes regulated by σf by using global transcriptome analysis and characterized the promoter region of these genes by 5´race experiments and β-galactosidase assays. furthermore, we investigated the role of the protein encoded by the second gene in the sigf operon, here named nrsf, and two conserved cysteine residues in this protein on the σf-mediated response to heavy metals.  σf is involved in chromium and cadmium responses in c. crescentus in order to verify a possible involvement of σf in the c. crescentus response to chromium and cadmium stresses, we monitored expression of cc previously identified as a σf-dependent gene, as well as cc which is co-transcribed with sigf, by quantitative rt-pcr. this analysis showed that cc is significantly induced in parental cells following exposure to either dichromate or cadmium. in contrast, expression of cc in a sigf deletion mutant strain exposed to dichromate or cadmium was found to be quite similar to that observed in the same strain under no stress condition. we could also observe significant induction of cc in the presence of dichromate, though the induction was modest when compared to the increase in expression of cc. on the other hand, no significant increase in cc expression was found in sigf mutant cells following dichromate exposure. taken together, these results confirm the involvement of σf in c. crescentus response to chromium and cadmium stresses and suggest that the operon sigf-cc is not strongly auto-regulated under these conditions. to simplify our analyses and data presentation, we only show the expression of sigf and its target genes under dichromate stress in all subsequent experiments. it is assumed that heavy metal ions cause oxidative stress inside cells. this raises the possibility that induction of σf-dependent genes by chromium and cadmium is a direct consequence of oxidative stress. to test this hypothesis, we stressed the parental and the sigf mutant strains with hydrogen peroxide, t-butyl hydroperoxide, paraquat or diamide. according to qrt-pcr experiments, expression levels of cc and cc were not increased more than twofold in the parental strain during these stress conditions. in agreement, transcript levels of cc and cc were also not influenced by any of these stressors in cells lacking sigf. concentrations of hydrogen peroxide and t-butyl hydroperoxide used in our analyses were previously found to be sufficient to increase expression of other genes in c. crescentus. taken together, these data suggest that chromium and cadmium are able to induce the σf regulon in an oxidative stress independent manner. σf controls a small set of genes under chromium stress with the aim of identifying additional genes induced during stress conditions under the control of σf, we compared the gene expression pattern of parental cells with that of a sigf mutant under dichromate stress, using microarray chips containing up to three different probes corresponding to the beginning of the coding region of each gene from c. crescentus. by considering these probes, we found only six genes down-regulated in sigf mutant cells relative to the parental strain. interestingly, close inspection of probes corresponding to the upstream region from cc and cc suggested that these regions are also down-regulated in sigf mutant cells when compared to the parental strain. the transcriptional start sites of the operons cc2906-cc and cc3255-cc3256-cc seem to be located quite distant from the translational start sites of cc and cc predicted by the tigr annotation. genome organization suggests that cc is the first gene of the transcriptional unit cc3254-cc3255-cc3256-cc. according to the tigr annotation, the deduced amino acid sequence of cc displays an n-terminal extension of amino acid residues not found in orthologous sequences. by excluding this extension, the most probable translational start site of cc is at position + relative to the translational start site of cc suggested by the tigr annotation. thus, all probes designed to measure cc expression in microarray chips correspond to a region upstream from the translational start site of cc proposed here. however, probes corresponding to the upstream region of cc cover the entire coding region of cc therefore, by considering these probes, we could include cc as a σf-dependent gene. this is in accordance with the previous observation that the complete transcriptional unit cc3254-cc3255-cc3256-cc is induced under chromium and cadmium stresses. a according to cmr annotation of genome of cb strain. b referring to the number of amino acid of the deduced protein sequence. protein length is according to cmr annotation or prediction from our analysis. c corresponding to the number of possible transmembrane helices in the mature protein. the number was determined by tmhmm tool. d according to a re-analysis of the deduced protein sequences by using pfam and smart tools to search for conserved domains. e sequences were compared with protein databases using blastp. f microarray hybridization of rna samples isolated from exponential phase cells exposed to μm potassium dichromate for min. genes with m value of < − or >  were assumed as differentially expressed between strains analyzed. values are the log ratio as mentioned. results shown are the average of three independent biological experiments. wt and Δsigf refer to the parental strain na and sigf deletion mutant, respectively. nc refers to no significant change in gene expression. g quantitative rt-pcr experiments performed with total rna extracted from exponentially growing cells immediately before and following exposure during min to μm potassium dichromate. results were normalized using gene cc as the endogenous control, which was constitutively expressed under the conditions analyzed. values are the log ratio as mentioned. data are mean values of two independent experiments. wt and Δsigf refer to the parental strain na and sigf deletion mutant, respectively. na corresponds to genes not analyzed in qrt-pcr experiments. the cc gene is predicted to be transcribed divergently from cc2906-cc in the chromosome of cb strain. however, the corresponding gene was not included during annotation of the more recent genome sequencing of c. crescentus. in the chromosome of na an open reading frame was proposed to be located between genes ccna_ and ccna_. nevertheless, ccna_ appears to be co-transcribed with ccna_ and ccna_ in addition, we could observe co-occurrence of ccna_ with other σf-dependent genes. as the nucleotide sequence between cc and cc in cb strain is identical to the region between ccna_ and ccna_ of na strain, we conclude that cc was incorrectly annotated in the genome of cb strain and this gene is the first one of the operon cc2907-cc2906-cc. as evaluated with probes corresponding to the upstream region of cc the entire coding region of cc is down-regulated in sigf mutant cells relative to the parental strain. therefore, the complete transcriptional unit cc2907-cc2906-cc is controlled by σf. a thorough re-annotation of genes regulated by σf suggested that cc codes for a putative membrane protein belonging to the doxx family, whose members are involved in sulfur metabolism. the cc gene, which encodes the putative sulfite oxidase subunit yedy, is another protein with a potential role in sulfur metabolism. all of the remaining σf-dependent genes code for proteins with conserved domains of unknown functions. interestingly, the pairs of genes cc and cc cc and cc as well as cc and cc are probable paralogous genes, with amino acid sequence identities of 36%, 43% and 23%, respectively. therefore, it is possible that the gene products of both operons exert similar functions. no other gene in the genome of c. crescentus displays significant nucleotide sequence similarity to the above mentioned pairs of paralogous genes or to the functionally annotated genes cc and cc proteins encoded by cc and cc present a duf domain at their n-terminus. this domain was described to be a dna-binding domain in ngo from neisseria gonorrhoeae. ngo is involved in the transcriptional regulation of msrab, which codes for a methionine sulfoxide reductase. however, in our microarray experiments, we could not observe differences in the expression of msra homologs in c. crescentus. thus, we conclude that the role of ngo in n. gonorrhoeae and cc or cc in c. crescentus is most likely different under these circumstances. to confirm results obtained in transcriptome analysis, we investigated the expression levels of five genes supposedly dependent on σf by qrt-pcr experiments. these analyses showed that expression of these selected genes under dichromate stress is more than twofold higher in the parental strain relative to the sigf deletion mutant. interestingly, induction of cc expression in the presence of dichromate was only partially dependent on σf, suggesting the involvement of an additional regulatory protein in the control of cc expression under this stress condition. taken together, these results confirm the data obtained in global transcriptional analysis. promoter sequence motifs of cc and cc genes are highly similar to those of sigf to identify putative σf-dependent promoters upstream of cc and cc genes, we performed 5’race experiments using primers that hybridize in the beginning of the coding region of the corresponding genes. for these experiments, rna samples from cells exposed to dichromate were used, as this stress condition leads to increased expression levels of cc and cc this approach led to the identification of a transcriptional start site for cc at position − relative to the translational start site + proposed here. a tss was also determined at position − with respect to the translational start site of cc predicted here. as expected, no tss could be observed when an additional 5´race experiment was performed using primers that hybridize to the beginning of the coding region of cc proposed by the tigr annotation. together, these data confirmed our microarray data with respect to expression of the operons cc2907-cc2906-cc and cc3254-cc3255-cc3256-cc the putative promoter sequences found for cc and cc were very similar to each other and also quite similar to the promoter sequence previously determined for sigf. additionally, analyses of the region upstream of the translational start site + of cc also revealed a putative σf-dependent sequence, suggesting a direct control of this gene by σf. accordingly, the putative σf-dependent promoters reported here are highly similar to sequences found upstream from sigf homologs in other bacteria. conserved sequences upstream of cc and sigf are necessary for expression of these genes to confirm the putative promoter sequence of the gene cluster cc3254-cc3255-cc3256-cc transcriptional fusions containing a fragment encompassing the region upstream of the translational start site of cc predicted in this work and the lacz reporter gene were created. caulobacter cells harboring these different constructs were used in β-galactosidase assays. when monitored in unstressed parental cells, a plasmid construction with the complete promoter sequence of the transcriptional unit cc3254-cc3255-cc3256-cc resulted in higher β-galactosidase activity with respect to the empty vector placz or to the construct lacking the − promoter element. only basal β-galactosidase activity was observed with any of the constructions in cells of the sigf null mutant strain. these results confirmed the data from qrt-pcr and 5’race experiments. analyses with dichromate stressed cells showed no significant increase in β-galactosidase activity in the parental strain, probably due to the damage caused during dichromate stress to β-galactosidase in particular or to proteins in general. as mentioned above, the promoter sequence of the operon cc3254-cc3255-cc3256-cc is highly similar to that located upstream from sigf. to verify if sigf expression was also dependent on these putative promoter elements, we analyzed the upstream region of the sigf gene in β-galactosidase assays using two different plasmid constructs: pcklac53- containing the promoter elements upstream from sigf and construct pcklac53- that lacks the sigf promoter. β-galactosidase activity measured in parental cells harboring the construct pck53- was found to be quite similar to that observed in cells with the empty vector. on the other hand, higher β-galactosidase activity was observed in the parental strain carrying construct pck53- which contains the complete sigf promoter sequence. cells from sigf mutant harboring the construct pcklac53- presented β-galactosidase activity slightly lower than that observed in parental cells with the same construct, but still higher than that observed in cells harboring the construct pck53-. altogether, these data indicate that the promoter sequence upstream from sigf is necessary for expression of the sigf operon, but in a manner that is not exclusively dependent on σf. this observation suggests that another sigma factor could also be capable of recognizing the region upstream from sigf. thus, we have investigated the effect of two other ecf sigma factors involved in oxidative and heavy metal stresses, σt and σe, upon sigf promoter activity, but no significant decrease in β-galactosidase activity was observed in mutant strains Δsigt and Δrpoe when compared with parental cells, all harboring construct pcklac53-. additionally, qrt-pcr experiments confirmed these results, as no change in transcription levels of the sigf gene was observed in a sigt or rpoe mutant. these observations allowed us to rule out the participation of σt and σe in the control of sigf expression. to further verify if the promoter region upstream of sigf is controlled by σf, we overexpressed sigf in the parental strain from an additional plasmid-encoded copy of the gene under the control of a constitutive promoter and measured β-galactosidase activity in these cells harboring either pcklac53- or pcklac53- overexpression of sigf in cells with the construct containing the complete sigf promoter led to an increase in β-galactosidase activity, whereas no difference was observed in cells harboring the promoterless construct pcklac53-. similarly, higher β-galactosidase activity was observed in sigf overexpressing cells bearing the construct containing the promoter sequence motifs upstream from cc when compared to the parental strain carrying the same construct or sigf overexpressing cells harboring the construct containing only the − motif of the promoter sequence of cc3254-cc3255-cc3256-cc. therefore, these results confirm that specific and highly similar promoter sequence motifs found upstream from sigf-cc and cc3254-cc3255-cc3256-cc are required for the control of these transcriptional units by σf. cc negatively regulates σf regulon expression the chromosomal organization of cc and sigf in a putative operon suggests that cc could be involved in the same regulatory pathway of σf. to test the assumption that cc could control σf activity, we monitored the expression of σf-dependent genes in parental cells overexpressing cc from a plasmid-encoded copy of the gene under the control of the constitutive lacz promoter present in vector pjs for that, cells overexpressing cc were stressed or not with dichromate and compared in qrt-pcr experiments with cells harboring the empty vector pjs or cells without this vector under the same conditions. according to qrt-pcr experiments, expression of genes cc and cc was slightly reduced in cells overexpressing cc under no stress conditions, when compared to cells with the empty vector pjs or cells without the vector. however, induction of cc and cc expression under dichromate stress was clearly absent in cc overproducing cells, when compared to cells not overexpressing cc. no difference could be found in the expression levels of two control genes when we compared cells overexpressing cc or not. this observation rules out a possible nonspecific effect due to overproduction of the protein. taken together, these data indicate that cc here denominated nrsf, acts as a negative regulator of σf function in c. crescentus. upon dichromate stress, sigf expression was also slightly down-regulated in cc overexpressing cells, whereas under control conditions sigf expression was practically unchanged. this once again favors the hypothesis that sigf is not strongly auto-regulated. a further attempt to investigate the role of nrsf as a possible negative regulator of σf function was carried out by trying to construct a null mutant strain in gene nrsf. however, it was not possible to construct a mutant strain by deleting nrsf in the parental strain. on the other hand, nrsf could be deleted in the absence of a functional copy of sigf, suggesting that high σf activity is apparently responsible for the failure of disrupting nrsf in cells with functional sigf. the putative protein encoded by nrsf is composed of six putative transmembrane segments separated by five short linkers and an n-terminal segment of residues. alignment of the deduced amino acid sequence of cc with its orthologs from other bacteria revealed two highly conserved cysteine residues. the cysteine residues of the caulobacter protein are probably directed into the periplasmic space, which favors their putative role in the signal transduction process leading to the liberation of σf from nrsf inhibition. substitution of the conserved cysteines by serine led to two single mutants and a double mutant. even under unstressed conditions, all σf-regulated genes analyzed in qrt-pcr experiments, including sigf and cc were up-regulated in the single mutant strains when compared to the parental strain. the substitution of both cysteines by serine in nrsf resulted in the highest expression levels of the genes analyzed. under stress conditions, both single substitution mutant strains showed an additional induction of σf-dependent genes, whereas no differences were observed for the double substitution mutant strain, compared to unstressed conditions. therefore, these results show that substitution of one or both of the conserved cysteines in the protein encoded by nrsf affects the ability of this protein in maintaining expression of σf-dependent genes at basal levels, further indicating the negative role of nrsf in the control of σf activity. σf is released into the cytoplasm during chromium stress and in cells carrying point mutations in conserved cysteines of nrsf the presence of six putative transmembrane segments in the protein coded by nrsf would imply that σf is sequestered to the inner membrane of caulobacter cells. however, at least a portion of this sigma factor would be expected to be released into the cytoplasm following chromium and cadmium exposure. to investigate this assumption, we monitored σf levels in the membrane and soluble fractions of caulobacter cell extracts by western blot analysis. when extracts from parental cells under no stress condition were analyzed, σf was only detected in the membrane fraction. although the majority of σf was still observed in the membrane fraction of extracts from parental cells exposed to dichromate, a significant portion of the sigma factor could also be detected in the soluble fraction. additionally, we found that substitution of both conserved cysteine residues of nrsf also led to a significant accumulation of σf in the soluble fraction, even when cells were not exposed to dichromate. in contrast, the membrane-bound ftsh protease was only detected in the membrane fraction of both strains analyzed. taken together, these results showed that cells displaying increased expression of σf-dependent genes accumulate this sigma factor in the cytoplasm. neither σf nor σf-dependent genes cc and cc 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, suggesting that sigf is not essential for bacterial survival under this stress condition. as the deduced protein sequences of cc and cc are highly similar, we constructed a single deletion mutant strain in each gene as well as a double mutant 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 exposure to either dichromate or cadmium, when compared to parental cells. 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 cc 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 cc 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. therefore, both σf-dependent genes with a putative assigned function appear to play a role in sulfate acquisition by cells. interestingly, hu et al. 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. it is therefore conceivable that induction of cc and cc could supply cells with sulfate. this is consistent with the observation that in arthrobacter sp. strain fb 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. curiously, none of the most representative functional categories up-regulated under chromate, dichromate or cadmium exposure 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 and σe in response to cadmium. 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. 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, cc or cc 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. 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 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 cc are highly similar to each other, promoter activity analyses supported our observation that cc 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 − to + relative to the transcriptional start site 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 cc 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. 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. here, we showed that the second gene in the sigf operon acts as a negative regulator of σf function, as overexpression of the putative membrane protein encoded by cc abolishes the transcriptional activation of sigf and its regulon under dichromate stress. thus, cc 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 to amino acid residues and an n-terminal segment of 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 σ and σ 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, 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. 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. thus, cysteines in the probable sensor proteins 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, 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 cys and cys 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.  this report deals with the role and regulation of c. crescentus σf under stress conditions and provides new interesting information about this conserved but still poorly characterized ecf sigma factor: i) σf-dependent genes are induced in the presence of heavy metals in a manner independent of oxidative and disulfide stress; ii) σf directly controls a small regulon including genes involved in sulfur metabolism and iii) σf is negatively regulated by a putative membrane bound protein, here named nrsf, which contains two conserved cysteine residues that are important for its function and are located in the periplasmic portion of the protein. 
substitution of two conserved cysteine residues in nrsf affects its ability to maintain the expression of σf-dependent genes at basal levels. in addition, we show that the second gene in the sigf operon acts as a negative regulator of σf function, and the encoded protein has been named nrsf. interestingly, σf-dependent genes are not induced in the presence of agents that generate reactive oxygen species. a possible mechanism for induction of the σf-dependent genes by chromium and cadmium is the inactivation of the putative anti-sigma factor nrsf, leading to the release of σf to bind rna polymerase core and drive transcription of its regulon. in this work, we show that the ecf sigma factor σf is one of the regulatory proteins involved in the control of the transcriptional response to chromium and cadmium. the α-proteobacterium caulobacter crescentus inhabits low-nutrient environments and can tolerate certain levels of heavy metals in these sites. it has been reported that c. crescentus responds to exposure to various heavy metals by altering the expression of a large number of genes. microarray experiments indicate that σf controls eight genes during chromium stress, most of which were previously described as induced by heavy metals. furthermore, we show that σf is released into the cytoplasm during chromium stress and in cells carrying point mutations in both conserved cysteines of the protein nrsf. promoter analyses revealed that a conserved σf-dependent sequence is located upstream of all genes of the σf regulon.
 the α-proteobacterium caulobacter crescentus inhabits low-nutrient environments and can tolerate certain levels of heavy metals in these sites. it has been reported that c. crescentus responds to exposure to various heavy metals by altering the expression of a large number of genes.  in this work, we show that the ecf sigma factor σf is one of the regulatory proteins involved in the control of the transcriptional response to chromium and cadmium. microarray experiments indicate that σf controls eight genes during chromium stress, most of which were previously described as induced by heavy metals. surprisingly, σf itself is not strongly auto-regulated under metal stress conditions. interestingly, σf-dependent genes are not induced in the presence of agents that generate reactive oxygen species. promoter analyses revealed that a conserved σf-dependent sequence is located upstream of all genes of the σf regulon. in addition, we show that the second gene in the sigf operon acts as a negative regulator of σf function, and the encoded protein has been named nrsf. substitution of two conserved cysteine residues in nrsf affects its ability to maintain the expression of σf-dependent genes at basal levels. furthermore, we show that σf is released into the cytoplasm during chromium stress and in cells carrying point mutations in both conserved cysteines of the protein nrsf.  a possible mechanism for induction of the σf-dependent genes by chromium and cadmium is the inactivation of the putative anti-sigma factor nrsf, leading to the release of σf to bind rna polymerase core and drive transcription of its regulon. stress responseecf sigma factor σfchromiumcadmiumcaulobacter crescentus 