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The bacterium Caulobacter crescentus and related stalk bacterial species are known for their distinctive ability to live in low-nutrient environments, a characteristic of most heavy metal-contaminated sites. Caulobacter crescentus is a model organism for studying cell cycle regulation with well-developed genetics. We have identified the pathways responding to heavy-metal toxicity in C. crescentus to provide insights for the possible application of Caulobacter to environmental restoration. We exposed C. crescentus cells to four heavy metals (chromium, cadmium, selenium, and uranium) and analyzed genome-wide transcriptional activities postexposure using an Affymetrix GeneChip microarray. C. crescentus showed surprisingly high tolerance to uranium, a possible mechanism for which may be the formation of extracellular calcium-uranium-phosphate precipitates. The principal response to these metals was protection against oxidative stress (up-regulation of manganese-dependent superoxide dismutase sodA). Glutathione S-transferase, thioredoxin, glutaredoxins, and DNA repair enzymes responded most strongly to cadmium and chromate. The cadmium and chromium stress response also focused on reducing the intracellular metal concentration, with multiple efflux pumps employed to remove cadmium, while a sulfate transporter was down-regulated to reduce nonspecific uptake of chromium. Membrane proteins were also up-regulated in response to most of the metals tested. A two-component signal transduction system involved in the uranium response was identified. Several differentially regulated transcripts from regions previously not known to encode proteins were identified, demonstrating the advantage of evaluating the transcriptome by using whole-genome microarrays.

Alternative sigma factors of the extracytoplasmic function (ECF) subfamily are important regulators of stress responses in bacteria and have been implicated in the control of homeostasis of the extracytoplasmic compartment of the cell. This work describes the characterization of sigF, encoding 1 of the 13 members of this subfamily identified in Caulobacter crescentus. A sigF-null strain was obtained and shown to be severely impaired in resistance to oxidative stress, caused by hydrogen peroxide treatment, exclusively during the stationary phase. Although sigF mRNA levels decrease in stationary-phase cells, the amount of σF protein is greatly increased at this stage, indicating a posttranscriptional control. Data obtained indicate that the FtsH protease is either directly or indirectly involved in the control of σF levels, as cells lacking this enzyme present larger amounts of the sigma factor. Increased stability of σF protein in stationary-phase cells of the parental strain and in exponential-phase cells of the ftsH-null strain is also demonstrated. Transcriptome analysis of the sigF-null strain led to the identification of eight genes regulated by σF during the stationary phase, including sodA and msrA, which are known to be involved in oxidative stress response.

Bacteria adapt to shifts from rapid to slow growth, and have developed strategies for long-term survival during prolonged starvation and stress conditions. We report the regulatory response of C. crescentus to carbon starvation, based on combined high-throughput proteome and transcriptome analyses. Our results identify cell cycle changes in gene expression in response to carbon starvation that involve the prominent role of the FixK FNR/CAP family transcription factor and the CtrA cell cycle regulator. Notably, the SigT ECF sigma factor mediates the carbon starvation-induced degradation of CtrA, while activating a core set of general starvation-stress genes that respond to carbon starvation, osmotic stress, and exposure to heavy metals. Comparison of the response of swarmer cells and stalked cells to carbon starvation revealed four groups of genes that exhibit different expression profiles. Also, cell pole morphogenesis and initiation of chromosome replication normally occurring at the swarmer-to-stalked cell transition are uncoupled in carbon-starved cells.

Extracytoplasmic function (ECF) σ factors control the transcription of genes involved in different cellular functions, such as stress responses, metal homeostasis, virulence-related traits, and cell envelope structure. The genome of Bradyrhizobium japonicum, the nitrogen-fixing soybean endosymbiont, encodes 17 putative ECF σ factors belonging to nine different ECF σ factor families. The genes for two of them, ecfQ (bll1028) and ecfF (blr3038), are highly induced in response to the reactive oxygen species hydrogen peroxide (H2O2) and singlet oxygen (1O2). The ecfF gene is followed by the predicted anti-σ factor gene osrA (blr3039). Mutants lacking EcfQ, EcfF plus OsrA, OsrA alone, or both σ factors plus OsrA were phenotypically characterized. While the symbiotic properties of all mutants were indistinguishable from the wild type, they showed increased sensitivity to singlet oxygen under free-living conditions. Possible target genes of EcfQ and EcfF were determined by microarray analyses, and candidate genes were compared with the H2O2-responsive regulon. These experiments disclosed that the two σ factors control rather small and, for the most part, distinct sets of genes, with about half of the genes representing 13% of the members of H2O2-responsive regulon. To get more insight into transcriptional regulation of both σ factors, the 5′ ends of ecfQ and ecfF mRNA were determined. The presence of conserved sequence motifs in the promoter region of ecfQ and genes encoding EcfQ-like σ factors in related α-proteobacteria suggests regulation via a yet unknown transcription factor. By contrast, we have evidence that ecfF is autoregulated by transcription from an EcfF-dependent consensus promoter, and its product is negatively regulated via protein-protein interaction with OsrA. Conserved cysteine residues 129 and 179 of OsrA are required for normal function of OsrA. Cysteine 179 is essential for release of EcfF from an EcfF-OsrA complex upon H2O2 stress while cysteine 129 is possibly needed for EcfF-OsrA interaction.

We engineered a strain of the bacterium Caulobacter crescentus to fluoresce in the presence of micromolar levels of uranium at ambient temperatures when it is exposed to a hand-held UV lamp. Previous microarray experiments revealed that several Caulobacter genes are significantly upregulated in response to uranium but not in response to other heavy metals. We designated one of these genes urcA (for uranium response in caulobacter). We constructed a reporter that utilizes the urcA promoter to produce a UV-excitable green fluorescent protein in the presence of the uranyl cation, a soluble form of uranium. This reporter is specific for uranium and has little cross specificity for nitrate (<400 μM), lead (<150 μM), cadmium (<48 μM), or chromium (<41.6 μM). The uranium reporter construct was effective for discriminating contaminated groundwater samples (4.2 μM uranium) from uncontaminated groundwater samples (<0.1 μM uranium) collected at the Oak Ridge Field Research Center. In contrast to other uranium detection methodologies, the Caulobacter reporter strain can provide on-demand usability in the field; it requires minimal sample processing and no equipment other than a hand-held UV lamp, and it may be sprayed directly on soil, groundwater, or industrial surfaces.

Many heavy metals, including nickel (Ni), cadmium (Cd), and chromium (Cr) are toxic industrial chemicals with an exposure risk in both occupational and environmental settings that may cause harmful outcomes. While these substances are known to produce adverse health effects leading to disease or health problems, the detailed mechanisms remain unclear. To elucidate the processes involved in the toxicity of nickel, cadmium, and chromium at the molecular level and to perform a comparative analysis, H4-II-E-C3 rat liver-derived cell lines were treated with soluble salts of each metal using concentrations derived from viability assays, and gene expression patterns were determined with DNA microarrays. We identified both common and unique biological responses to exposure to the three metals. Nickel, cadmium, chromium all induced oxidative stress with both similar and unique genes and pathways responding to this stress. Although all three metals are known to be genotoxic, evidence for DNA damage in our study only exists in response to chromium. Nickel induced a hypoxic response as well as inducing genes involved in chromatin structure, perhaps by replacing iron in key proteins. Cadmium distinctly perturbed genes related to endoplasmic reticulum stress and invoked the unfolded protein response leading to apoptosis. With these studies, we have completed the first gene expression comparative analysis of nickel, cadmium, and chromium in H4-II-E-C3 cells.

High temperature and other environmental stresses induce the expression of several heat shock proteins in Caulobacter crescentus, including the molecular chaperones DnaJ, DnaK, GrpE, and GroEL and the Lon protease. We report here the isolation of the rpoH gene encoding a homolog of the Escherichia coli RNA polymerase sigma32 subunit, the sigma factor responsible for the transcription of heat shock promoters. The C. crescentus sigma32 homolog, predicted to be a 33.7-kDa protein, is 42% identical to E. coli sigma32 and cross-reacts with a monoclonal antibody to E. coli sigma32. Functional homology was demonstrated by complementing the temperature-sensitive growth defect of an E. coli rpoH deletion mutant with the C. crescentus rpoH gene. Immunoblot analysis showed a transient rise in sigma32 levels after a temperature shift from 30 to 42 degrees C similar to that described for E. coli. In addition, increasing the cellular content of sigma32 by introducing a plasmid-encoded copy of rpoH induced DnaK expression in C. crescentus cultures grown at 30 degrees C. The C. crescentus rpoH gene was transcribed from either of two heat shock consensus promoters. rpoH transcription and sigma32 levels increased coordinately following heat shock, indicating that transcriptional regulation contributes to sigma32 expression in this organism. Both the rpoH gene and sigma32 protein were expressed constitutively throughout the cell cycle at 30 degrees C. The isolation of rpoH provides an important tool for future studies of the role of sigma32 in the normal physiology of C. crescentus.

Bacteria employ extracytoplasmic function (ECF) sigma factors for their responses to environmental stresses. Despite intensive research, the molecular dissection of ECF sigma factor regulons has remained a major challenge due to overlaps in the ECF sigma factor-regulated genes and the stimuli that activate the different ECF sigma factors. Here we have employed tiling arrays to single out the ECF σW regulon of the Gram-positive bacterium Bacillus subtilis from the overlapping ECF σX, σY, and σM regulons. For this purpose, we profiled the transcriptome of a B. subtilis sigW mutant under non-stress conditions to select candidate genes that are strictly σW-regulated. Under these conditions, σW exhibits a basal level of activity. Subsequently, we verified the σW-dependency of candidate genes by comparing their transcript profiles to transcriptome data obtained with the parental B. subtilis strain 168 grown under 104 different conditions, including relevant stress conditions, such as salt shock. In addition, we investigated the transcriptomes of rasP or prsW mutant strains that lack the proteases involved in the degradation of the σW anti-sigma factor RsiW and subsequent activation of the σW-regulon. Taken together, our studies identify 89 genes as being strictly σW-regulated, including several genes for non-coding RNAs. The effects of rasP or prsW mutations on the expression of σW-dependent genes were relatively mild, which implies that σW-dependent transcription under non-stress conditions is not strictly related to RasP and PrsW. Lastly, we show that the pleiotropic phenotype of rasP mutant cells, which have defects in competence development, protein secretion and membrane protein production, is not mirrored in the transcript profile of these cells. This implies that RasP is not only important for transcriptional regulation via σW, but that this membrane protease also exerts other important post-transcriptional regulatory functions.

Sigma factor sigma32, encoded by rpoH, is required for the recognition of heat shock genes during normal growth conditions and in response to heat shock and other stresses. Unlike the well-studied Escherichia coli rpoH gene, which is transcribed from four promoters recognized by either a sigma70 (sigmaD)- or sigma24 (sigmaE)-containing RNA polymerase, the Caulobacter crescentus rpoH gene is transcribed from two promoters, P1 and P2. In this study, we have examined the structure and expression of these promoters and shown that the rpoH P2 promoter is sigma32 dependent. We present evidence here that P2 is specifically recognized and transcribed by the reconstituted C. crescentus Esigma32 RNA polymerase holoenzyme. We show that site-directed mutations within either the -10 or the -35 regions of P2 have substantial effects on the levels of transcription by the Esigma32 polymerase predicted from the sigma32 promoter consensus sequence. The mutations have similar effects in vivo as assayed with rpoH-lacZ transcription fusions. Analysis of the rpoH P1 promoter provided evidence that it is sigma70 dependent. S1 nuclease protection assays of rpoH P1- and P2-specific expression after heat shock at 42 or 50 degrees C and during synchronous cell division cycles under normal growth conditions showed that the two promoters are differentially regulated. Mutations within the rpoH P2 promoter consensus sequences abolished the response to heat shock induction in C. crescentus. We conclude from these results that, unlike rpoH genes studied previously in other bacteria, the major transcriptional response of the C. crescentus rpoH gene to heat shock depends on positive autoregulation of the sigma32-dependent promoter.

We have identified the gene encoding the Caulobacter crescentus principal sigma subunit, RpoD. The rpoD gene codes for a polypeptide of 653 amino acids with a predicted molecular mass of 72,623 Da (sigma 73). The C. crescentus sigma subunit has extensive amino acid sequence homology with the principal sigma factors of a number of divergent procaryotes. In particular, the segments designated region 2 that are involved in core polymerase binding and promoter recognition were identical among these bacteria despite the fact that the -10 region recognized by the C. crescentus sigma 73 differs significantly from that of the other bacteria. Thus, it appears that additional sigma factor regions must be involved in -10 region recognition. This conclusion was strengthened by a heterologous complementation assay in which C. crescentus sigma 73 was capable of complementing the Escherichia coli rpoD285 temperature-sensitive mutant. Furthermore, C. crescentus sigma 73 conferred new specificity on the E. coli RNA polymerase, allowing the expression of C. crescentus promoters in E. coli. Thus, the C. crescentus sigma 73 appears to have a broader specificity than does the sigma 70 of the enteric bacteria.

PhyR represents a novel alphaproteobacterial family of response regulators having a structure consisting of two domains; a predicted amino-terminal extracytoplasmic function (ECF) sigma factor-like domain and a carboxy-terminal receiver domain. PhyR was first described in Methylobacterium extorquens AM1, in which it has been shown to be essential for plant colonization, probably due to its suggested involvement in the regulation of a number of stress proteins. Here we investigated the PhyR regulon using microarray technology. We found that the PhyR regulon is rather large and that most of the 246 targets are under positive control. Mapping of transcriptional start sites revealed candidate promoters for PhyR-mediated regulation. One of these promoters, an ECF-type promoter, was identified upstream of one-third of the target genes by in silico analysis. Among the PhyR targets are genes predicted to be involved in multiple stress responses, including katE, osmC, htrA, dnaK, gloA, dps, and uvrA. The induction of these genes is consistent with our phenotypic analyses which revealed that PhyR is involved in resistance to heat shock and desiccation, as well as oxidative, UV, ethanol, and osmotic stresses, in M. extorquens AM1. The finding that PhyR is involved in the general stress response was further substantiated by the finding that carbon starvation induces protection against heat shock and that this protection is at least in part dependent on PhyR.

In Caulobacter crescentus, the alternative sigma factor sigma54 plays an important role in the expression of late flagellar genes. Sigma54-dependent genes are temporally and spatially controlled, being expressed only in the swarmer pole of the predivisional cell. The only sigma54 activator described so far is the FlbD protein, which is involved in activation of the class III and IV flagellar genes and repression of the fliF promoter. To identify new roles for sigma54 in the metabolism and differentiation of C. crescentus, we cloned and characterized a gene encoding a putative sigma54 activator, named tacA. The deduced amino acid sequence from tacA has high similarity to the proteins from the NtrC family of transcriptional activators, including the aspartate residues that are phosphorylated by histidine kinases in other activators. The promoter region of the tacA gene contains a conserved sequence element present in the promoters of class II flagellar genes, and tacA shows a temporal pattern of expression similar to the patterns of these genes. We constructed an insertional mutant that is disrupted in tacA (strain SP2016), and an analysis of this strain showed that it has all polar structures, such as pili, stalk, and flagellum, and displays a motile phenotype, indicating that tacA is not involved in the flagellar biogenesis pathway. However, this strain has a high percentage of filamentous cells and shows a clear-plaque phenotype when infected with phage phiCb5. These results suggest that the TacA protein could mediate the effect of sigma54 on a different pathway in C. crescentus.

Mycobacterium tuberculosis sigL encodes an extracytoplasmic function (ECF) sigma factor and is adjacent to a gene for a membrane protein (Rv0736) that contains a conserved HXXXCXXC sequence. This motif is found in anti-sigma factors that regulate several ECF sigma factors, including those that control oxidative stress responses. In this work, SigL and Rv0736 were found to be cotranscribed, and the intracellular domain of Rv0736 was shown to interact specifically with SigL, suggesting that Rv0736 may encode an anti-sigma factor of SigL. An M. tuberculosis sigL mutant was not more susceptible than the parental strain to several oxidative and nitrosative stresses, and sigL expression was not increased in response to these stresses. In vivo, sigL is expressed from a weak SigL-independent promoter and also from a second SigL-dependent promoter. To identify SigL-regulated genes, sigL was overexpressed and microarray analysis of global transcription was performed. Four small operons, sigL (Rv0735)-Rv0736, mpt53 (Rv2878c)-Rv2877c, pks10 (Rv1660)-pks7 (Rv1661), and Rv1139c-Rv1138c, were among the most highly upregulated genes in the sigL-overexpressing strain. SigL-dependent transcription start sites of these operons were mapped, and the consensus promoter sequences TGAACC in the −35 region and CGTgtc in the −10 region were identified. In vitro, purified SigL specifically initiated transcription from the promoters of sigL, mpt53, and pks10. Additional genes, including four PE_PGRS genes, appear to be regulated indirectly by SigL. In an in vivo murine infection model, the sigL mutant strain showed marked attenuation, indicating that the sigL regulon is important in M. tuberculosis pathogenesis.

Sinorhizobium meliloti genes transcriptionally up-regulated after heat stress, as well as upon entry into stationary phase, were identified by microarray analyses. Sixty stress response genes were thus found to be up-regulated under both conditions. One of them, rpoE2 (smc01506), encodes a putative extracytoplasmic function (ECF) sigma factor. We showed that this sigma factor controls its own transcription and is activated by various stress conditions, including heat and salt, as well as entry into stationary phase after either carbon or nitrogen starvation. We also present evidence that the product of the gene cotranscribed with rpoE2 negatively regulates RpoE2 activity, and we therefore propose that it plays the function of anti-sigma factor. By combining transcriptomic, bioinformatic, and quantitative reverse transcription-PCR analyses, we identified 44 RpoE2-controlled genes and predicted the number of RpoE2 targets to be higher. Strikingly, more than one-third of the 60 stress response genes identified in this study are RpoE2 targets. Interestingly, two genes encoding proteins with known functions in stress responses, namely, katC and rpoH2, as well as a second ECF-encoding gene, rpoE5, were found to be RpoE2 regulated. Altogether, these data suggest that RpoE2 is a major global regulator of the general stress response in S. meliloti. Despite these observations, and although this sigma factor is well conserved among alphaproteobacteria, no in vitro nor in planta phenotypic difference from the wild-type strain could be detected for rpoE2 mutants. This therefore suggests that other important actors in the general stress response have still to be identified in S. meliloti.

Caulobacter crescentus is an obligate aerobe which is exposed to high concentrations of photosynthetic oxygen and low levels of nutrients in its aquatic environment. Physiological studies of oxidative and starvation stresses in C. crescentus were undertaken through a study of lacZ fusion and null mutant strains constructed from the cloned 5' end of katG, encoding a catalase-peroxidase. The katG gene was shown to be solely responsible for catalase and peroxidase activity in C. crescentus. Like the katG of Escherichia coli, C. crescentus katG is induced by hydrogen peroxide and is important in sustaining the exponential growth rate. However, dramatic differences are seen in growth stage induction. E. coli KatE catalase and KatG catalase-peroxidase activities are induced 15- to 20-fold during exponential growth and then approximately halved in the stationary phase. In contrast, C. crescentus KatG activity is constant throughout exponential growth and is induced 50-fold in the stationary phase. Moreover, the survival of a C. crescentus katG null mutant is reduced by more than 3 orders of magnitude after 24 h in stationary phase and more than 6 orders of magnitude after 48 h, a phenotype not seen for E. coli katE and katG null mutants. These results indicate a major role for C. crescentus catalase-peroxidase in stationary-phase survival and raise questions about whether the peroxidatic activity as well as the protective catalatic activity of the dual-function enzyme is important in the response to starvation stress.

Pseudomonas syringae pv. syringae B728a, a bacterial pathogen of bean, utilizes large surface populations and extracellular signaling to initiate a fundamental change from an epiphytic to a pathogenic lifestyle. Extracytoplasmic function (ECF) sigma (σ) factors serve as important regulatory factors in responding to various environmental signals. Bioinformatic analysis of the B728a genome revealed 10 ECF sigma factors. This study analyzed deletion mutants of five previously uncharacterized ECF sigma factor genes in B728a, including three FecI-type ECF sigma factors (ECF5, ECF6, and ECF7) and two ECF sigma factors placed in groups ECF11 and ECF18. Transcriptional profiling by qRT-PCR analysis of ECF sigma factor mutants was used to measure expression of their associated anti-sigma and outer membrane receptor proteins, and expression of genes associated with production of extracellular polysaccharides, fimbriae, glycine betaine and syringomycin. Notably, the B728aΔecf7 mutant displayed reduced swarming and had decreased expression of CupC fimbrial genes. Growth and pathogenicity assays, using a susceptible bean host, revealed that none of the tested sigma factor genes are required for in planta growth and lesion formation.

Pseudomonas putida encodes 20 extracytoplasmic sigma factors (ECFs). In this study, we show that one of these ECFs, known as ECF-Pp12 (PP3006), plays a role in tolerance of toluene and other organic solvents. Based on this finding, we have called the gene that encodes this new ECF rpoT. The rpoT gene forms an operon with the preceding gene and with the gene located downstream. The translated gene product of the open reading frame PP3005 is an inner membrane protein, whereas the PP3007 protein is periplasmic. A nonpolar ΔrpoT mutant was generated by homologous recombination, and survival of the mutant was tested under various stress conditions. The mutant strain was hypersensitive to toluene and other solvents but just as tolerant as the wild type of stress imposed by heat, antibiotics, NaCl, paraquat, sodium dodecyl sulfate, H2O2, and benzoate. In the ΔrpoT mutant background, expression of around 50 transcriptional units was affected: 31 cistrons were upregulated, and 23 cistrons were downregulated. This indicates that about 1% of all P. putida genes are under the direct or indirect influence of RpoT. The rpoT gene controls the expression of a number of membrane proteins, including components of the respiratory chains, porins, transporters, and multidrug efflux pumps. Hypersensitivity of the P. putida RpoT-deficient mutant to organic solvents can be attributed to the fact that in the ΔrpoT strain, expression of the toluene efflux pump ttgGHI genes is severalfold lower than in the parental strain.

Summary

A variety of mechanisms are used to signal extracytoplasmic conditions to the cytoplasm. These mechanisms activate extracytoplasmic function (ECF) sigma factors which recruit RNA-polymerase to specific genes in order to express appropriate proteins in response to the changing environment. The two best understood ECF signaling pathways regulate σE-mediated expression of periplasmic stress response genes in Escherichia coli and FecI-mediated expression of iron-citrate transport genes in E. coli. Homologues from other Gram-negative bacteria suggest that these two signaling mechanisms and variations on these mechanisms may be the general schemes by which ECF sigma factors are regulated in Gram-negative bacteria.

Extracytoplasmic function (ECF) sigma factors are known to play an important role in the bacterial response to various environmental stresses and can significantly modulate their pathogenic potential. In the genome of Porphyromonas gingivalis W83, six putative ECF sigma factors were identified. To further evaluate their role in this organism, a PCR-based linear transformation method was used to inactivate five ECF sigma factor genes (PG0162, PG0214, PG0985, PG1660, and PG1827) by allelic exchange mutagenesis. All five isogenic mutants formed black-pigmented colonies on blood agar. Mutants defective in PG0985, PG1660 and PG1827 genes were more sensitive to 0.25 mM of hydrogen peroxide compared to the wild-type strain. Isogenic mutants of PG0162 and PG1660 showed a 50 percent decrease in gingipain activity. RT-PCR analysis showed that there was no alteration in the expression of rgpA, rgpB, and kgp gingipain genes in these mutants. Hemolytic and hemagglutination activities were decreased by more than 50 percent in the PG0162 mutant compared to the wild-type. Taken together, these findings suggest that ECF sigma factors can modulate important virulence factors in P. gingivalis. ECF sigma factors encoded by the PG0162 and PG1660 genes might also be involved in the post-transcriptional regulation of the gingipains.

Life in oligotrophic environments necessitates quick adaptive responses to a sudden lack of nutrients. Secretion of specific degradative enzymes into the extracellular medium is a means to mobilize the required nutrient from nearby sources. The aquatic bacterium Caulobacter crescentus must often face changes in its environment such as phosphate limitation. Evidence reported in this paper indicates that under phosphate starvation, C. crescentus produces a membrane surface-anchored lipoprotein named ElpS subsequently released into the extracellular medium. A complete set of 12 genes encoding a type II secretion system (T2SS) is located adjacent to the elpS locus in the C. crescentus genome. Deletion of this T2SS impairs release of ElpS in the environment, which surprisingly remains present at the cell surface, indicating that the T2SS is not involved in the translocation of ElpS to the outer membrane but rather in its release. Accordingly, treatment with protease inhibitors prevents release of ElpS in the extracellular medium suggesting that ElpS secretion relies on a T2SS-secreted protease. Finally, secretion of ElpS is associated with an increase in alkaline phosphatase activity in culture supernatants, suggesting a role of the secreted protein in inorganic phosphate mobilization. In conlusion, we have shown that upon phosphate starvation, C. crescentus produces an outer membrane bound lipoprotein, ElpS, which is further cleaved and released in the extracellular medium in a T2SS-dependent manner. Our data suggest that ElpS is associated with an alkaline phosphatase activity, thereby allowing the bacterium to gather inorganic phosphates from a poor environment.

Extracytoplasmic function (ECF) sigma factors are a heterogeneous group of alternative sigma factors that regulate gene expression in response to a variety of conditions, including stress. We previously characterized a mycobacterial ECF sigma factor, SigE, that contributes to survival following several distinct stresses. A gene encoding a closely related sigma factor, sigH, was cloned from Mycobacterium tuberculosis and Mycobacterium smegmatis. A single copy of this gene is present in these and other fast- and slow-growing mycobacteria, including M. fortuitum and M. avium. While the M. tuberculosis and M. smegmatis sigH genes encode highly similar proteins, there are multiple differences in adjacent genes. The single in vivo transcriptional start site identified in M. smegmatis and one of two identified in M. bovis BCG were found to have −35 promoter sequences that match the ECF-dependent −35 promoter consensus. Expression from these promoters was strongly induced by 50°C heat shock. In comparison to the wild type, an M. smegmatis sigH mutant was found to be more susceptible to cumene hydroperoxide stress but to be similar in logarithmic growth, stationary-phase survival, and survival following several other stresses. Survival of an M. smegmatis sigH sigE double mutant was found to be markedly decreased following 53°C heat shock and following exposure to cumene hydroperoxide. Expression of the second gene in the sigH operon is required for complementation of the sigH stress phenotypes. SigH is an alternative sigma factor that plays a role in the mycobacterial stress response.

The robust induction of metallothionein-I and II (MT-I and MT-II) genes by several heavy metals such as zinc and cadmium requires the specific transcription factor metal-responsive transcription factor 1 (MTF1). Chromium (VI), a major environmental carcinogen, not only failed to activate these genes but also inhibited their induction by Zn2+ or Cd2+. The heavy metal-induced expression of another MTF1 target gene, zinc transporter 1 (ZnT-1), was also down-regulated by Cr6+. By contrast, the expression of two MTF1-independent Cd2+-inducible genes, heme oxygenase 1 (HO-1) and HSP-70, was not sensitive to Cr6+. Cr6+ did not also affect the expression of housekeeping genes such as GAPDH or β-actin. Stable cell lines overexpressing variable levels of MTF1, the key transactivator of the MT genes, demonstrated differential resistance toward the inhibitory effect of Cr6+, indicating MTF1 as a target of chromium toxicity. The basal and inducible binding of MTF1 to metal response elements was not affected by treatment of cells with Cr6+. Transient transfection studies showed that the ability of MTF1 to transactivate the MT-I promoter was significantly compromised by Cr6+. The fusion protein consisting of a Gal-4 DNA binding domain and one or more of the three transactivation domains of MTF1, namely the acidic domain, proline-rich domain, and serine-threonine rich domain, activated the GAL-4-driven luciferase gene to different degrees, but all were sensitive to Cr6+. MTF1 null cells were prone to apoptosis after exposure to Zn2+ or Cd2+ that was augmented in presence Cr6+, whereas the onset of apoptosis was significantly delayed in cells overexpressing MTF1.

The extracytoplasmic factor (ECF) sigma factor σE is one of the most studied sigma factors of Mycobacterium tuberculosis. It has been shown to be involved in virulence as well as in survival under conditions of high temperature, alkaline pH, and exposure to detergents and oxidative stress. Unlike many ECF sigma factors, σE does not directly regulate the transcription of its own gene. Two promoters have been identified upstream of the sigE gene; one is regulated by the two-component system MprAB, while the other has been shown to be σH dependent. In this paper, we further characterize the regulation of σE by identifying its anti-sigma factor and a previously unknown promoter. Finally, we show that sigE can be translated from three different translational start codons, depending on the promoter used. Taken together, our data demonstrate that σE not only is subjected to complex transcriptional regulation but is also controlled at the translational and posttranslational levels.

In response to elevated temperature, both prokaryotic and eukaryotic cells increase expression of a small family of chaperones. The regulatory network that functions to control the transcription of the heat shock genes in bacteria includes unique structural motifs in the promoter region of these genes and the expression of alternate sigma factors. One of the conserved structural motifs, the inverted repeat CIRCE element, is found in the 5' region of many heat shock operons, including the Caulobacter crescentus groESL operon. We report the identification of another C. crescentus heat shock operon containing two genes, hrcA (hrc for heat shock regulation at CIRCE elements) and a grpE homolog. Disruption of the hrcA gene, homologs of which are also found upstream of grpE in other bacteria, increased transcription of the groESL operon, and this effect was dependent on the presence of an intact CIRCE element. This suggests a role for HrcA in negative regulation of heat shock gene expression. We identified a major promoter transcribing both hrcA and grpE and a minor promoter located within the hrcA coding sequence just upstream of grpE. Both promoters were heat shock inducible, with maximal expression 10 to 20 min after heat shock. Both promoters were also expressed constitutively throughout the cell cycle under physiological conditions. C. crescentus GrpE, shown to be essential for viability at low and high temperatures, complemented an Escherichia coli delta grpE strain in spite of significant differences in the N- and C-terminal regions of these two proteins, demonstrating functional conservation of this important stress protein.

The dual toxicity/essentiality of copper forces cells to maintain a tightly regulated homeostasis for this metal in all living organisms, from bacteria to humans. Consequently, many genes have previously been reported to participate in copper detoxification in bacteria. Myxococcus xanthus, a prokaryote, encodes many proteins involved in copper homeostasis that are differentially regulated by this metal. A σ factor of the ECF (extracytoplasmic function) family, CorE, has been found to regulate the expression of the multicopper oxidase cuoB, the P1B-type ATPases copA and copB, and a gene encoding a protein with a heavy-metal-associated domain. Characterization of CorE has revealed that it requires copper to bind DNA in vitro. Genes regulated by CorE exhibit a characteristic expression profile, with a peak at 2 h after copper addition. Expression rapidly decreases thereafter to basal levels, although the metal is still present in the medium, indicating that the activity of CorE is modulated by a process of activation and inactivation. The use of monovalent and divalent metals to mimic Cu(I) and Cu(II), respectively, and of additives that favor the formation of the two redox states of this metal, has revealed that CorE is activated by Cu(II) and inactivated by Cu(I). The activation/inactivation properties of CorE reside in a Cys-rich domain located at the C terminus of the protein. Point mutations at these residues have allowed the identification of several Cys involved in the activation and inactivation of CorE. Based on these data, along with comparative genomic studies, a new group of ECF σ factors is proposed, which not only clearly differs mechanistically from the other σ factors so far characterized, but also from other metal regulators.

Author Summary

Copper exerts a dual effect on living organisms. It is essential for life, but an excess provokes cell damage, forcing cells to maintain a regulated homeostasis for this metal. These two antagonistic biological effects of copper are clearly illustrated by two human genetic disorders, Menkes syndrome and Wilson disease, caused by deficiency or accumulation of this metal, respectively. Myxococcus xanthus, a soil-dwelling bacterium, also has to cope with changes in copper concentration in its environment. The large genome of this myxobacterium encodes many genes involved in copper homeostasis, all of which are differentially regulated, indicating that many regulators participate in copper homeostasis in this prokaryote. Here, we identify one of these regulators (CorE), which belongs to the family of the extracytoplasmic function (ECF) σ factors. We demonstrate that CorE represents a novel group of ECF σ factors and of metal regulators, because its activity is modulated by the redox state of copper. This ability resides in a Cys-rich domain, which has also been found in other σ factors of different bacterial phyla. Therefore, we propose that CorE is the first member of a mechanistically new group of ECF σ factors.