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Complex regulation of the synthesis of the compatible solute ectoine in the halophilic bacterium Chromohalobacter salexigens DSM 3043T.

The synthesis of the compatible solute ectoine, mediated by the ectABC gene products, is the main mechanism used by the halophilic bacterium Chromohalobacter salexigens to cope with osmotic stress. Evidence was found that this process is regulated at the transcriptional level. S1 protection analyses performed with RNA extracted from cells grown in minimal medium at low (0.75 M NaCl) or high (2.5 M NaCl) osmolarity suggested the existence of four promoters upstream of ectA. Two of these (PectA1 and PectA2) might be recognized by the main vegetative sigma factor sigma(70), and one (PectA3) might be dependent on the general stress sigma factor sigma(S). The S1 protection assays suggest that PectA1 and PectA3 may be osmoregulated promoters. In addition, an internal promoter showing sequences homologous to promoters dependent on the heat-shock sigma factor sigma(32) was found upstream of ectB. Transcription from PectA in C. salexigens followed a pattern typical of sigma(S)-dependent promoters, and was reduced by 50 % in an E. coli rpoS background. These data strongly suggest the involvement of the general stress sigma factor sigma(S) in ectABC transcription in C. salexigens. Expression of PectA-lacZ and PectB-lacZ trancriptional fusions was very high at low salinity, suggesting that ectABC may be a partially constitutive system. Both transcriptional fusions were induced during continuous growth at high temperature and their expression was reduced in cells grown in the presence of osmoprotectants (ectoine or glycine betaine) or the DNA gyrase inhibitor nalidixic acid. Moreover, PectA-lacZ expression was negatively modulated in cells grown with an excess of iron (FeCl(3)). Measurement of ectoine levels in the presence of glycine betaine at different NaCl concentrations suggests that an additional post-transcriptional control may occur as well.

Adaptation, Physiological↗

Regulation of Erwinia carotovora hrpL(Ecc) (sigma-L(Ecc)), which encodes an extracytoplasmic function subfamily of sigma factor required for expression of the HRP regulon.

In Erwinia carotovora subsp. carotovora (Ecc) strain 71 (Ecc71), HrpL(Ecc), an alternate sigma factor of the extracytoplasmic function subfamily, plays a central role in the expression of the hrp (hypersensitive reaction and pathogenicity) regulon. We document here that sigma-54 (RpoN) is required for full expression of hrpL(Ecc) and that HrpS, in conjunction with sigma-54, activates hrpL(Ecc) transcription. We also made the novel observation that integration host factor is required for the activation of the hrpL(Ecc) promoter. Our findings reveal that the RsmA/rsmB RNA-mediated post-transcriptional system, known to control extracellular enzyme and harpin production, affects hrpL(Ecc) expression as well. For example, hrpL(Ecc) RNA levels are barely detected in an RsmB- strain. Conversely, hrpL(Ecc) mRNA levels are much higher in RsmA- bacteria than in the RsmA+ parent. This effect is due to RsmA-promoted decay of hrpL(Ecc) RNA. Moreover, the following regulators known to control the production of either RsmA, rsmB RNA, or both also affect hrpL(Ecc) expression: GacA (response regulator of a two-component system), KdgR (an IcII type repressor), HexA (a LysR type repressor), RsmC (a putative transcriptional adapter). Based upon the data now available for Ecc and extrapolating from the evidence in other systems, we propose a tentative model that depicts the Hrp regulatory system of Ecc and explains the basis for coregulation of extracellular enzyme production and expression of the Hrp regulon.

Amino Acid Sequence↗

Deletion of Mycobacterium tuberculosis sigma factor E results in delayed time to death with bacterial persistence in the lungs of aerosol-infected mice.

The stress-induced extracytoplasmic sigma factor E (SigE) of Mycobacterium tuberculosis shows increased expression after heat shock, sodium dodecyl sulfate treatment, and oxidative stress, as well as after phagocytosis in macrophages. We report that deletion of sigE results in delayed lethality in mice without a significant reduction of bacterial numbers in lungs.

Administration, Inhalation↗

The Staphylococcus aureus alternative sigma factor sigmaB controls the environmental stress response but not starvation survival or pathogenicity in a mouse abscess model.

The role of sigmaB, an alternative sigma factor of Staphylococcus aureus, has been characterized in response to environmental stress, starvation-survival and recovery, and pathogenicity. sigmaB was mainly expressed during the stationary phase of growth and was repressed by 1 M sodium chloride. A sigB insertionally inactivated mutant was created. In stress resistance studies, sigmaB was shown to be involved in recovery from heat shock at 54 degreesC and in acid and hydrogen peroxide resistance but not in resistance to ethanol or osmotic shock. Interestingly, S. aureus acquired increased acid resistance when preincubated at a sublethal pH 4 prior to exposure to a lethal pH 2. This acid-adaptive response resulting in tolerance was mediated via sigB. However, sigmaB was not vital for the starvation-survival or recovery mechanisms. sigmaB does not have a major role in the expression of the global regulator of virulence determinant biosynthesis, staphylococcal accessory regulator (sarA), the production of a number of representative virulence factors, and pathogenicity in a mouse subcutaneous abscess model. However, SarA upregulates sigB expression in a growth-phase-dependent manner. Thus, sigmaB expression is linked to the processes controlling virulence determinant production. The role of sigmaB as a major regulator of the stress response, but not of starvation-survival, is discussed.

Abscess↗

The major sigma factor (RpoD) from Helicobacter pylori and other gram-negative bacteria shows an enhanced rate of divergence.

Sequence analysis of the Helicobacter pylori major sigma factor (RpoD) shows that it is highly divergent, which may be related to the marked diversity of the H. pylori chromosome. Furthermore, the rate of divergence of RpoD among other gram-negative bacteria is much greater than that among gram-positive bacteria. This suggests that RpoD from gram-negative bacteria is functionally less constrained than that from gram-positive bacteria.

Amino Acid Sequence↗

The putative sigma factor KatF is regulated posttranscriptionally during carbon starvation.

Transcriptional and translational 'lacZ reporter fusions were constructed to the katF gene, which encodes a putative sigma factor centrally involved in starvation-mediated general resistance in Escherichia coli. Transcription of katF was found to increase ca. twofold after carbon starvation in minimal medium. The protein fusion containing the longest fragment of katF induced ca. eightfold under the same conditions, whereas fusions to shorter segments showed only a twofold increase in expression. The protein fusion was expressed at higher levels in a strain containing a katF::Tn10 mutation, indicating katF autoregulation. The posttranscriptional regulation of katF by starvation did not require a component of the spent minimal medium. katF was also posttranscriptionally regulated during entry into late log phase in complex medium. This induction was coincident with an increase in katE transcription, suggesting that the cellular concentration of KatF directly followed the induction of the katF protein fusion.

Bacterial Proteins↗

sigmaR, an RNA polymerase sigma factor that modulates expression of the thioredoxin system in response to oxidative stress in Streptomyces coelicolor A3(2).

We have identified an RNA polymerase sigma factor, sigmaR, that is part of a system that senses and responds to thiol oxidation in the Gram-positive, antibiotic-producing bacterium Streptomyces coelicolor A3(2). Deletion of the gene (sigR) encoding sigmaR caused sensitivity to the thiol-specific oxidant diamide and to the redox cycling compounds menadione and plumbagin. This correlated with reduced levels of disulfide reductase activity and an inability to induce this activity on exposure to diamide. The trxBA operon, encoding thioredoxin reductase and thioredoxin, was found to be under the direct control of sigmaR. trxBA is transcribed from two promoters, trxBp1 and trxBp2, separated by 5-6 bp. trxBp1 is transiently induced at least 50-fold in response to diamide treatment in a sigR-dependent manner. Purified sigmaR directed transcription from trxBp1 in vitro, indicating that trxBp1 is a target for sigmaR. Transcription of sigR itself initiates at two promoters, sigRp1 and sigRp2, which are separated by 173 bp. The sigRp2 transcript was undetectable in a sigR-null mutant, and purified sigmaR could direct transcription from sigRp2 in vitro, indicating that sigR is positively autoregulated. Transcription from sigRp2 was also transiently induced (70-fold) following treatment with diamide. We propose a model in which sigmaR induces expression of the thioredoxin system in response to cytoplasmic disulfide bond formation. Upon reestablishment of normal thiol levels, sigmaR activity is switched off, resulting in down-regulation of trxBA and sigR. We present evidence that the sigmaR system also functions in the actinomycete pathogen Mycobacterium tuberculosis.

Amino Acid Sequence↗

Specialized osmotic stress response systems involve multiple SigB-like sigma factors in Streptomyces coelicolor.

Whereas in Bacillus subtilis, a general stress response stimulon under the control of a single sigma factor (SigB) is induced by different physiological and environmental stresses (heat, salt or ethanol shock), in Streptomyces coelicolor, these environmental stresses induce independent sets of proteins, and its genome encodes nine SigB paralogues. To investigate possible functions of multiple sigB-like genes in S. coelicolor, Pctc, a promoter routinely used to assay SigB activity in vivo, was analysed as a heterologous reporter. The fact that Pctc was activated by osmotic shock, but not by heat or ethanol, confirmed that stress response system(s) could operate independently in S. coelicolor. Pctc was also induced transiently during growth of liquid cultures, presumably by nutritional signals. We purified an RNA polymerase holoenzyme from crude extracts that catalysed specific transcription of Pctc in vitro. Its sigma subunit was identified as a product of the sigH gene, which is co-transcribed downstream of a putative antisigma factor gene (prsH). Although the sigH function was not needed for normal colony morphology, prsH was conditionally required for both aerial hyphae formation and regulation of antibiotic biosynthesis. Levels of two different sigH-encoded proteins were growth phase dependent but not significantly changed by osmotic stress, implying the important roles of post-translational regulatory elements such as PrsH. In addition, synthesis of three other SigH-like proteins was induced by osmotic stress, but not by ethanol or heat. Two of them were genetically assigned to sigH homologous loci sigI and sigJ and shown to be independently regulated. This family of SigH-like proteins displayed different osmotic response kinetics. Thus, in contrast to many other bacteria, S. coelicolor uses an osmotic sensory system that can co-ordinate the activity of multiple paralogues to control the relative activity of promoters within the same stress stimulon. Such specialized stress response systems may reflect adaptive functions needed for colonial differentiation.

Bacterial Proteins↗

Differential expression of three plastidial sigma factors, OsSIG1, OsSIG2A, and OsSIG2B, during leaf development in rice.

We isolated and characterized two rice nuclear genes, OsSIG2A and OsSIG2B, encoding the putative sigma-factor of the plastid RNA polymerase. Deduced protein sequences predicted a plastid-localizing signal in the N-terminus and subsequent polypeptides similar to known SIG2 proteins. Gene expression analysis revealed that the OsSIG2A transcript is more abundant than the OsSIG2B transcript in all tissues tested and that both rice SIG2s are expressed from earlier stages of leaf development than that in the case of OsSIG1. These results indicate differential expression of SIG genes in leaf morphogenesis, suggesting the existence of tissue- and stage-specific functions of SIG proteins for transcriptional regulation of chloroplast genes in plant development.

Amino Acid Sequence↗

Crystallization and preliminary X-ray crystallographic analysis of the TM1442 gene product from Thermotoga maritima, a homologue of Bacillus subtilis anti-anti-sigma factors.

A 110-residue protein encoded by the TM1442 gene of Thermotoga maritima shows amino-acid sequence similarity to Bacillus subtilis anti-anti-sigma factors RsbV and SpoIIAA. It has been overexpressed in Escherichia coli and the recombinant protein exists primarily as both a monomer and a dimer in solution. The dimeric form has been crystallized using polyethylene glycol (PEG) 8000 as a precipitant. Native X-ray diffraction data have been collected at 100 K to 2.0 A resolution. The crystals are monoclinic, belonging to the space group P2(1), with unit-cell parameters a = 31.54 (13), b = 116.83 (37), c = 31.39 (7) A, alpha = 90, beta = 119.84 (9), gamma = 90 degrees. The asymmetric unit contains two monomers of the recombinant polypeptide, with a corresponding V(M) of 2.24 A(3) Da(-1) and a solvent content of 45.0%.

Bacillus subtilis↗

The Bacillus subtilis sigma(X) protein is an extracytoplasmic function sigma factor contributing to survival at high temperature.

The sigX gene, identified as part of the international effort to sequence the Bacillus subtilis genome, has been proposed to encode an alternative sigma factor of the extracytoplasmic function (ECF) subfamily. The sigX gene is cotranscribed with a downstream gene, ypuN, during logarithmic and early stationary phases of growth. We now report that strains lacking sigma(X) are impaired in the ability to survive at high temperature whereas a ypuN mutant has increased thermotolerance. We overproduced and purified sigma(X) from Escherichia coli and demonstrate that in vitro, both sigma(A) and sigma(X) holoenzymes recognize promoter elements within the sigX-ypuN control region. However, they have distinct salt optima such that sigma(A)-dependent transcription predominates at low salt while sigma(X)-dependent transcription predominates at high salt. A 54-bp region upstream of sigX suffices as a sigma(X)-dependent promoter in vivo, demonstrating that sigX is at least partially under positive autoregulatory control. Mutation of ypuN increases expression from the sigma(X)-dependent promoter in vivo, suggesting that ypuN may encode a negative regulator of sigma(X) activity.

Acclimatization↗

hrpL activates Erwinia amylovora hrp gene transcription and is a member of the ECF subfamily of sigma factors.

hrpL of Erwinia amylovora Ea321 encodes a 21.7-kDa regulatory protein, similar to members of the ECF (extra cytoplasmic functions) subfamily of eubacterial RNA polymerase sigma factors. hrpL is a single-gene operon in complementation group VI of the E. amylovora hrp gene cluster. Its product is required by Ea321 to elicit the hypersensitive response (HR) and to cause disease. HrpL controls the expression of five independent hrp loci, including hrpN, which encodes harpin, a proteinaceous elicitor of the HR. hrpL is environmentally regulated, and its expression is affected by hrpS, another regulatory gene of the hrp gene cluster of E. amylovora. pCPP1078, a multicopy plasmid carrying hrpL, is able to restore HR-eliciting ability to hrpS mutants. A conserved motif was identified upstream of the hrpI and hrpN operons, which are transcriptionally regulated by hrpL. This conserved motif shares a high degree of similarity with other biochemically defined or putative ECF-dependent promoter sequences, including sequences upstream of Streptomyces coelicolor dagA P2, Pseudomonas aeruginosa algD, Pseudomonas syringae pv. syringae 61 hrpZ, and P. syringae pv. tomato avrD. In spite of the similarity between the hrpL genes of E. amylovora and P. syringae 61, no functional cross-complementation was observed.

Amino Acid Sequence↗

The alternative sigma factor RpoH2 is required for salt tolerance in Sinorhizobium sp. strain BL3.

The objectives of this investigation were to isolate the rpoH2 gene encoding an alternative sigma factor from Sinorhizobium sp. BL3 and to determine its role in exopolysaccharide (EPS) synthesis, salt tolerance and symbiosis with Phaseolus lathyroides. The rpoH2 gene of Rhizobium sp. strain TAL1145 is known to be required for EPS synthesis and effective nodulation of Leucaena leucocephala. Three overlapping cosmid clones containing the rpoH2 gene of BL3 were isolated by complementing an rpoH2 mutant of TAL1145 for EPS production. From one of these cosmids, rpoH2 of BL3 was identified within a 3.0-kb fragment by subcloning and sequencing. The cloned rpoH2 gene of BL3 restored both EPS production and nodulation defects of the TAL1145 rpoH2 mutants. Three rpoH2 mutants of BL3 were constructed by transposon-insertion mutagenesis. These mutants of BL3 grew normally in complete or minimal medium and were not defective in EPS synthesis, nodulation and nitrogen fixation, but they failed to grow in salt stress conditions. The mutants complemented with cloned rpoH2 from either BL3 or TAL1145 showed higher levels of salt tolerance than BL3. The expression of rpoH2 in BL3 started increasing during the exponential phase and reached the highest level in the mid-stationary phase. These results indicate that RpoH2 is required for salt tolerance in Sinorhizobium sp. BL3, and it may have additional roles during the stationary phase.

Adaptation, Physiological↗

Central regulatory role for the RpoS sigma factor in expression of Salmonella dublin plasmid virulence genes.

The plasmid virulence genes spvABCD of Salmonella spp. are regulated by SpvR and the stationary-phase sigma factor RpoS. The transcription of spv genes is induced during the post-exponential phase of bacterial growth in vitro. We sought to investigate the relationship between growth phase and RpoS in spv regulation. rpoS insertion mutations were constructed in S. dublin Lane and plasmid-cured LD842 strains, and the mutants were found to be attenuated for virulence and deficient in spv gene expression. We utilized the plasmid pBAD::rpoS to express rpoS independent of the growth phase under the control of the arabinose-inducible araBAD promoter. SpvA expression was induced within 2 h after the addition of 0.1% arabinose, even when bacteria were actively growing. This suggested that the level of RpoS, instead of the growth phase itself, controls induction of the spv genes. However, RpoS did not activate transcription of spvA in the absence of SpvR protein. Using a constitutive tet promoter to express spvR, we found that the spvA gene can be partially expressed in the rpoS mutant, suggesting that RpoS is required for SpvR synthesis. We confirmed that spvR is poorly expressed in the absence of RpoS. With an intact rpoS gene, spvR expression is not dependent on an intact spvR gene but is enhanced by spvR supplied in trans. We propose a model for Salmonella spv gene regulation in which both RpoS and SpvR are required for maximal expression at the spvR and spvA promoters.

Animals↗

A new sigma factor homolog in a cyanobacterium: cloning, sequencing, and light-responsive transcripts of rpoD2 from Microcystis aeruginosa K-81.

We isolated an rpoD2 gene encoding the potential sigma factor of RNA polymerase from the cyanobacterium Microcystis aeruginosa K-81, which can perform photosynthesis. The deduced amino acid sequence of RpoD2 (sigmaA2) exhibits extensive homology to other eubacterial RpoD proteins. This gene possessed multiple 5'-end transcripts, expressed specifically under light (P(L)), dark (P(D)), or constitutively light/dark (P(C)) conditions during exponential cell growth.

Amino Acid Sequence↗

A temporally controlled sigma-factor is required for polar morphogenesis and normal cell division in Caulobacter.

The transcription of many spatially and temporally controlled flagellar structural genes in Caulobacter requires the RNA polymerase sigma 54 subunit. Like flagellar biogenesis, stalk formation is an asymmetric polar morphogenesis that occurs once each cell cycle in response to internal cell cycle signals. We have isolated the sigma 54 gene (rpoN) and describe here a novel role for this alternative sigma-factor in cell differentiation: It is required for the biogenesis of both polar structures, and the disruption of the rpoN gene results in aberrant cell division. Surprisingly, the transcription of rpoN is temporally regulated during the cell cycle; it increases 10-fold commensurate with stalk formation and just before the onset of flagellar gene expression. These results suggest that sigma 54 abundance responds to cell cycle cues and is involved in the global timing of the central events of Caulobacter development, whereas the transcriptional activators of sigma 54-dependent promoters are responsible for the refined control of the expression of individual or small groups of genes required for each specific event.

Amino Acid Sequence↗

The ssgB gene, encoding a member of the regulon of stress-response sigma factor sigmaH, is essential for aerial mycelium septation in Streptomyces coelicolor A3(2).

The Streptomyces coelicolor A3(2) gene ssgB belongs to the regulon of stress-response sigma factor sigmaH. By integrative transformation via double cross-over, a stable null mutant of ssgB was obtained. This mutation had no obvious effect on vegetative growth, but critically affected aerial mycelium septation. The S. coelicolor ssgB mutant produced aerial hyphae without any signs of septation into spore compartments. The mutation was complemented in trans by wild-type ssgB including the sigmaH-dependent ssgBp promoter. The results proved that ssgB belongs a developmental branch of the sigmaH regulon.

Bacterial Proteins↗

Cloning, sequencing, and transcriptional analysis of the gene coding for the vegetative sigma factor of Agrobacterium tumefaciens.

The sigA gene of Agrobacterium tumefaciens was cloned and sequenced. Comparison with previously analyzed sigA genes revealed a high degree of similarity in nucleotide and amino acid sequences of regions two, three, and four of vegetative sigma factors. However, the upstream regulatory region shows no sequence homology with the Escherichia coli heat shock (sigma 32) promoters. It also does not contain the hairpin-loop structure (inverted repeat sequence) that was found in the upstream region of the groE operon in A. tumefaciens. The transcription initiation site of the gene was determined and found to be at the same position during normal growth and under heat shock conditions. Furthermore, no heat shock activation was observed at the transcriptional level.

Agrobacterium tumefaciens↗