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PubMed1993

Transcription factor sigma B of Bacillus subtilis controls a large stationary-phase regulon.

Transcription factor sigma B of Bacillus subtilis is active during the stationary growth phase, but its physiological role remains unknown. Understanding the function and regulation of genes controlled by sigma B (csb genes) should provide important clues to sigma B function in stationary-phase cells. To this end, we used a genetic approach to identify six new csb genes. This strategy relies on two elements: (i) random transcriptional fusions between the Escherichia coli lacZ gene and genes on the B. subtilis chromosome, generated in vivo with transposon Tn917lacZ, and (ii) a plate transformation technique to introduce a null sigB mutation into the fusion-bearing recipients directly on indicator plates. This strategy allowed the comparison of fusion expression in strains that were isogenic save for the presence or absence of a functional sigma B protein. Beginning with 1,400 active fusions, we identified 11 that were wholly or partly controlled by sigma B. These fusions mapped to six different loci that exhibit substantial contrasts in their patterns of expression in the logarithmic and stationary growth phases, suggesting that they participate in diverse cellular functions. However, for all six loci, the sigma B-dependent component of their expression was manifest largely in the stationary phase. The high frequency of six independent csb loci detected in a random collection of 1,400 fusions screened, the fact that four of the six new loci were defined by a single fusion, and the absence of the previously identified ctc and csbA genes in the present collection strongly suggest that sigma B controls a large stationary-phase regulon.

Bacillus subtilis↗

PubMed2003

Proteome analysis of Escherichia coli W3110 expressing an heterologous sigma factor.

The recombinant plasmid pASK18 carries a Streptomyces DNA fragment which includes an open reading frame, designated psfS (putative sigma factor, Streptomyces), as its putative product showed a high degree of similarity with RNA polymerase sigma factors. Previous results showed that PsfS causes transcription initiation within the bgl operon promoter-silencer region in Escherichia coli K12. In this study a proteomic approach has been applied in order to perform a comparative analysis of E. coli K12 W3110 wild-type, W3110 (pASK18) and a W3110 Bgl(+) spontaneous mutant. Either by qualitative or quantitative analysis, no significant difference was observed between the proteomes of W3110 and its Bgl+ derivative, while W3110 (pASK18) showed an altered profile by both analyses. Fourteen out of the 37 protein spots showing a different expression level in E. coli W3110 harboring pASK18 were identified by peptide mass fingerprinting. Among the proteins identified, thiol peroxidase (Tpx) was the only one up-regulated. The possible involvement of bgl and tpx in the survival of the pathogen E. coli during infection is discussed.

Amino Acid Sequence↗

PubMed2005

Regulation of toxin and bacteriocin synthesis in Clostridium species by a new subgroup of RNA polymerase sigma-factors.

Many Clostridium species are pathogenic for humans and animals, and most of the resulting diseases, such as tetanus, botulism, gas gangrene and pseudomembranous colitis, are due to the production of potent extracellular toxins. The biochemical mechanisms of action of Clostridium toxins have been extensively studied in the past ten years. However, detailed information about the regulation of toxin gene expression has only recently emerged. TcdR, BotR, TetR and UviA are now known to be related alternative RNA polymerase sigma factors that drive transcription of toxin A and toxin B genes in C. difficile, the neurotoxin genes in C. botulinum and C. tetani, and a bacteriocin gene in C. perfringens. Although the Clostridium sigma factors have some similarity to members of the ECF sigma factor group, they differ sufficiently in structure and function so that they have been assigned to a new group within the sigma(70)-family.

Amino Acid Sequence↗

PubMed1994

Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial RNA polymerase sigma factors involved in the regulation of extracytoplasmic functions.

sigma E, an RNA polymerase sigma factor of apparent M(r) 28,000, was previously identified by its ability to direct transcription from the P2 promoter of the agarose gene (dagA) of Streptomyces coelicolor. A degenerate oligonucleotide probe, designed from the N-terminal sequence of purified sigma E, was used to isolate the sigma E gene (sigE). The predicted sequence of sigma E shows greatest similarity to sequences of seven other proteins: Myxococcus xanthus CarQ, Pseudomonas aeruginosa AlgU, Pseudomonas syringae HrpL, Escherichia coli sigma E, Alcaligenes eutrophus CnrH, E. coli FecI, and Bacillus subtilis SigX, a protein of unknown function. These eight proteins define a subfamily of eubacterial RNA polymerase factors sufficiently different from other sigma s that, in many cases, they are not identified by standard similarity searching methods. Available information suggests that all of them regulate extracytoplasmic functions and that they function as effector molecules responding to extracytoplasmic stimuli. A. eutrophus CnrH appears to be a plasmid-encoded factor.

Amino Acid Sequence↗

PubMed1987

The nucleotide sequence of the sigma factor gene ntrA (rpoN) of Azotobacter vinelandii: analysis of conserved sequences in NtrA proteins.

The nucleotide sequence of the Azotobacter vinelandii ntrA gene has been determined. It encodes a 56916 Dalton acidic polypeptide (AvNtrA) with substantial homology to NtrA from Klebsiella pneumoniae (KpNtrA) and Rhizobium meliloti (RmNtrA). NtrA has been shown to act as a novel RNA polymerase sigma factor but the predicted sequence of AvNtrA substantiates our previous analysis of KpNtrA in showing no substantial homology to other known sigma factors. Alignment of the predicted amino acid sequences of AvNtrA, KpNtrA and RmNtrA identified three regions; two showing greater than 50% homology and an intervening sequence of less than 10% homology. The predicted protein contains a short sequence near the centre with homology to a conserved region in other sigma factors. The C-terminal region contains a region of homology to the beta' subunit of RNA polymerase (RpoC) and two highly conserved regions one of which is significantly homologous to known DNA-binding motifs. In A. vinelandii, ntrA is followed by another open reading frame (ORF) which is highly homologous to a comparable ORF downstream of ntrA in K. pneumoniae and R. meliloti.

Amino Acid Sequence↗

PubMed2004

Redox-dependent changes in RsrA, an anti-sigma factor in Streptomyces coelicolor: zinc release and disulfide bond formation.

sigmaR is a sigma factor for transcribing genes to defend cells against oxidative stresses in the antibiotic-producing bacterium Streptomyces coelicolor. The availability of sigmaR is regulated by RsrA, an anti-sigma factor, whose sigmaR-binding activity is regulated by redox changes in the environment, via thiol-disulfide exchange. We found that reduced RsrA contains zinc in a stoichiometric amount, whereas oxidized form has very little: 1 mol of zinc per mol of RsrA was released upon oxidation as monitored by a chromogenic Zn-chelator, 4-(2-pyridylazo)-resorcinol (PAR). Measurement of zinc bound in several RsrA mutants of various cysteine and histidine substitutions suggested that C3, H7, C41, and C44 serve as zinc-binding sites. The zinc-binding and sigmaR-binding activities of mutant proteins did not coincide, suggesting that zinc might not be absolutely required for the anti-sigma activity of RsrA. Zn-free apo-RsrA bound sigmaR and inhibited sigmaR-dependent transcription in vitro. Compared with Zn-RsrA, the anti-transcription activity of apo-RsrA was about threefold lower and its sigmaR-binding affinity decreased by about ninefold when measured by surface plasmon resonance analysis. Apo-RsrA was more sensitive to protease, suggesting that zinc allows RsrA to maintain a more compact structure, optimized for binding sigmaR. The cysteine pairs that form disulfide bonds were determined by MALDI-TOF mass spectrometry, revealing formation of the critical disulfide bond between C11 and one of the essential cysteine residues C41 or 44, most likely C44. An improved model for the mechanism of redox-modulation of RsrA was presented.

Bacterial Proteins↗

PubMed1999

Differential expression of 10 sigma factor genes in Mycobacterium tuberculosis.

The ability of Mycobacterium tuberculosis to adapt to different environments in the infected host is essential for its pathogenicity. Consequently, this organism must be able to modulate gene expression to respond to the changing conditions it encounters during infection. In this paper we begin a comprehensive study of M. tuberculosis gene regulation, characterizing the transcript levels of 10 of its 13 putative sigma factor genes. We developed a real-time RT-PCR assay using a family of novel fluorescent probes called molecular beacons to quantitatively measure the different mRNAs. Three sigma factor genes were identified that have increased mRNA levels after heat shock, two of which also responded to detergent stress. In addition, we also identified a sigma factor gene whose mRNA increased after mild cold shock and a second that responded to conditions of low aeration.

Bacterial Proteins↗

PubMed2005

Light-induced carotenogenesis in Streptomyces coelicolor A3(2): identification of an extracytoplasmic function sigma factor that directs photodependent transcription of the carotenoid biosynthesis gene cluster.

Carotenoids are produced by a variety of organisms, but the mechanisms that regulate gene expression leading to carotenoid biosynthesis have been characterized for only a few organisms. In this study, we found that Streptomyces coelicolor A3(2), a gram-positive filamentous bacterium, produces carotenoids under blue light induction. The carotenoid fraction isolated from the cell extract contained multiple compounds, including isorenieratene and beta-carotene. The carotenoid biosynthesis gene cluster of S. coelicolor consists of two convergent operons, crtEIBV and crtYTU, as previously shown for Streptomyces griseus. The crtEIBV null mutant completely lost its ability to produce carotenoids. The crt gene cluster is flanked by a regulatory region that consists of two divergent operons, litRQ and litSAB. The lit (light-induced transcription) genes encode a MerR-type transcriptional regulator (LitR), a possible oxidoreductase (LitQ), an extracytoplasmic function sigma factor (sigmaLitS), a putative lipoprotein (LitA), and a putative anti-sigma factor (LitB). S1 protection assay revealed that the promoters preceding crtE (PcrtE), crtY (PcrtY), litR (PlitR), and litS (PlitS) are activated upon illumination. A litS mutant lost both the ability to produce carotenoids and the activities of PcrtE, PcrtY, and PlitS, which suggested that sigmaLitS directs light-induced transcription from these promoters. An RNA polymerase holocomplex containing purified sigmaLitS recombinant protein generated specific PcrtE and PcrtY transcripts in an in vitro runoff transcriptional assay. A litR mutant that had an insertion of the kanamycin resistance gene was defective both in the ability to produce carotenoids and in all of the light-dependent promoter activities. Overexpression of litS resulted in constitutive carotenoid production in both the wild type and the litR mutant. These results indicate that sigmaLitS acts as a light-induced sigma factor that directs transcription of the crt biosynthesis gene cluster, whose activity is controlled by an unknown LitR function. This is the first report to describe light-inducible gene expression in Streptomyces.

Amino Acid Sequence↗

PubMed1998

Nuclear encoding of a plastid sigma factor in rice and its tissue- and light-dependent expression.

A full-length cDNA encoding a putative sigma factor for a plastid RNA polymerase was isolated from the higher plant Oryza sativa . The nucleotide sequence of the corresponding nuclear gene, named Os-sigA ( O.sativa sigma A), predicts a polypeptide of 519 amino acids that contains a putative plastid-targeting sequence in its N-terminal region. The predicted mature protein shows extensive sequence homology to bacterial sigma factors, encompassing the conserved regions 1.2, 2.1, 2.2, 2.3, 2.4, 3, 4.1 and 4.2 implicated in binding to -10 promoter elements, promoter melting and interaction with the core RNA polymerase enzyme. RNA blot analysis revealed that the abundance of Os-sigA transcripts was markedly greater in green shoots than in roots or in dark-grown etiolated shoots of rice seedlings. Furthermore, exposure of dark-grown etiolated seedlings to light resulted in a rapid increase in the amount of Os-sigA mRNA in the shoot. These observations suggest that regulation of expression of the nuclear gene for this putative plastid RNA polymerase sigmafactor by light contributes to light-dependent transcriptional regulation of plastid genes.

Amino Acid Sequence↗

PubMed1996

SpoIIE governs the phosphorylation state of a protein regulating transcription factor sigma F during sporulation in Bacillus subtilis.

Cell-specific activation of the transcription factor sigma F during sporulation in Bacillus subtilis is controlled by a regulatory pathway involving the proteins SpoIIE, SpoIIAA, and SpoIIAB. SpoIIAB is an antagonist of sigma F, and SpoIIAA, which is capable of overcoming SpoIIAB-mediated inhibition of sigma F, is an antagonist of SpoIIAB. SpoIIAA is, in turn, negatively regulated by SpoIIAB, which phosphorylates SpoIIAA on serine 58. SpoIIAA is also positively regulated by SpoIIE, which dephosphorylates SpoIIAA-P, the phosphorylated form of SpoIIAA. Here, isoelectric focusing and Western blot analysis were used to examine the phosphorylation state of SpoIIAA in vivo. SpoIIAA was found to be largely in the phosphorylated state during sporulation in wild-type cells but a significant portion of the protein that was unphosphorylated could also be detected. Consistent with the idea that SpoIIE governs dephosphorylation of SpoIIAA-P, SpoIIAA was entirely in the phosphorylated state in spoIIE mutant cells. Conversely, overexpression of spoIIE led to an increase in the ratio of unphosphorylated SpoIIAA to SpoIIAA-P and caused inappropriate activation of sigma F in the predivisional sporangium. We also show that a mutant form of SpoIIAA (SpoIIAA-S58T) in which serine 58 was replaced with threonine was present exclusively as SpoIIAA-P, a finding that confirms previous biochemical evidence that the mutant protein is an effective substrate for the SpoIIAB kinase but that SpoIIAA-S58T-P cannot be dephosphorylated by SpoIIE. We conclude that SpoIIE plays a crucial role in controlling the phosphorylation state of SpoIIAA during sporulation and thus in governing the cell-specific activation of sigma F.

Bacillus subtilis↗

PubMed2001

Characterization of a stress-induced alternate sigma factor, RpoS, of Coxiella burnetii and its expression during the development cycle.

Coxiella burnetii is an obligate intracellular bacterium that resides in an acidified phagolysosome and has a remarkable ability to persist in the extracellular environment. C. burnetii has evolved a developmental cycle that includes at least two morphologic forms, designated large cell variants (LCV) and small cell variants (SCV). Based on differential protein expression, distinct ultrastructures, and different metabolic activities, we speculated that LCV and SCV are similar to typical logarithmic- and stationary-phase growth stages. We hypothesized that the alternate sigma factor, RpoS, a global regulator of genes expressed under stationary-phase, starvation, and stress conditions in many bacteria, regulates differential expression in life cycle variants of C. burnetii. To test this hypothesis, we cloned and characterized the major sigma factor, encoded by an rpoD homologue, and the stress response sigma factor, encoded by an rpoS homologue. The rpoS gene was cloned by complementation of an Escherichia coli rpoS null mutant containing an RpoS-dependent lacZ fusion (osmY::lacZ). Expression of C. burnetii rpoS was regulated by growth phase in E. coli (induced upon entry into stationary phase). A glutathione S-transferase-RpoS fusion protein was used to develop polyclonal antiserum against C. burnetii RpoS. Western blot analysis detected abundant RpoS in LCV but not in SCV. These results suggest that LCV and SCV are not comparable to logarithmic and stationary phases of growth and may represent a novel adaptation for survival in both the phagolysosome and the extracellular environment.

Alternative Splicing↗

PubMed1994

Identification of a putative alternate sigma factor and characterization of a multicomponent regulatory cascade controlling the expression of Pseudomonas syringae pv. syringae Pss61 hrp and hrmA genes.

The Pseudomonas syringae hrp and hrmA genes controlling pathogenicity and elicitation of the hypersensitive response and the avr genes controlling host range have been shown previously to be regulated by carbon, nitrogen, pH, osmolarity, and hypothetical plant factors. In P. syringae pv. syringae Pss61, inactivation of hrp complementation groups II and XIII reduced expression of a plasmid-borne hrmA'-lacZ fusion. The hrp regions II and XIII were cloned on separate plasmids and shown to enhance the activity of the hrmA promoter in Escherichia coli MC4100 transformants at least 100-fold. The nucleotide sequence of region XIII revealed two open reading frames (hrpR and hrpS) whose deduced products share homology with P. syringae pv. phaseolicola NPS3121 HrpS and are both related to the NtrC family of two-component signal transduction systems. HrpR and HrpS differ from most members of the protein family by lacking an amino-terminal domain which modulates the regulatory activity. A single open reading frame, hrpL, whose product shares homology with AlgU, a putative alternate sigma factor of P. aeruginosa, as well as with the related alternate sigma factors was identified within region II. Key domains are partially conserved. Inactivation of hrpS in Pss61 repressed expression of a plasmid-borne hrpL'-lacZ fusion carried by pYXPL1R, and transformation of MC4100(pYXPL1R) with a plasmid carrying hrpRS increased hrpL promoter activity at least 200-fold. Neither hrpS nor hrpR, when cloned on separate plasmids, activated the hrpL promoter activity individually. The expression of hrpL when directed by a lac promoter was sufficient to express a set of plasmid-borne hrmA'-, hrpJ'-, and hrpZ'-lacZ fusions independently of other hrp genes. The results indicate that hrpRS and hrpL are part of a regulatory cascade in which HrpR and HrpS activate expression of hrpL and HrpL, a putative sigma factor, induces expression of HrpL-responsive genes.

Amino Acid Sequence↗

PubMed2001

[Characterization of the expression and function of SigM an ECF sigma factor in mycobacteria].

The survival of M. tuberculosis within the macrophage depends on its ability to respond to oxidative stress, and the ECF subfamily of sigma factors likely play an important role. We studied SigM, a sigma factor whose gene is located near the origin of DNA replication. In both M. smegmatis and M. bovis BCG, the expression of sigM was induced at high temperature and in stationary phase. Mutants of M. smegmatis without an intact sigM were defective for survival in oxidative stress and also for the induction of thioredoxin reductase activity in oxidative stress. The thioredoxin system reduces disulfide bonds that are formed in oxidative stress. SigM thus appears to regulate thioredoxins and forms part of the bacteria's complex protective responses.

DNA Replication↗

PubMed1998

Cloning and characterization of the gene encoding the primary sigma-factor of Campylobacter jejuni.

The rpoD gene encoding the primary sigma-factor of Campylobacter jejuni was amplified from genomic DNA with degenerate oligonucleotide primers. The complete gene encodes a polypeptide of 622 amino acids and has a deduced M(r) of 72.6 kDa. This polypeptide is 40% identical to the RpoD (sigma 70) protein of Escherichia coli and has 66% identity with the Helicobacter pylori RpoD protein. A C. jejuni sigma 70 promoter, not recognized by the E. coli sigma 70 factor, could be activated in this bacterium in the presence of the cloned C. jejuni RpoD protein.

Amino Acid Sequence↗

PubMed2004

Ribosomal protein S1 specifically binds to the 5' untranslated region of the Pseudomonas aeruginosa stationary-phase sigma factor rpoS mRNA in the logarithmic phase of growth.

The rpoS gene encodes the stationary-phase sigma factor (RpoS or sigma(s)), which was identified in several gram-negative bacteria as a central regulator controlling the expression of genes involved in cell survival in response to cessation of growth (stationary phase) and providing cross-protection against various stresses. In Pseudomonas aeruginosa, the levels of sigma(s) increase dramatically at the onset of the stationary phase and are regulated at the transcriptional and posttranscriptional levels. The P. aeruginosa rpoS gene is transcribed as a monocistronic rpoS mRNA transcript comprised of an unusually long 373-bp 5' untranslated region (5' UTR). In this study, the 5' UTR and total protein extracts from P. aeruginosa logarithmic and stationary phases of growth were used in order to investigate the protein-RNA interactions that may modulate the translational process. It was observed that a 69-kDa protein, which corresponded to ribosomal protein S1, preferentially binds the 5' UTR of the rpoS mRNA in the logarithmic phase and not in the stationary phase. This is the first report of a protein-rpoS mRNA 5' UTR interaction in P. aeruginosa, and the possible involvement of protein S1 in translation regulation of rpoS is discussed.

5' Untranslated Regions↗

PubMed1989

Switch protein alters specificity of RNA polymerase containing a compartment-specific sigma factor.

During sporulation in Bacillus subtilis, expression of developmental genes spoIVCB and cotD is induced in the mother cell compartment of the sporangium at morphological stages IV and V, respectively. A 27-kilodalton RNA polymerase sigma factor called sigma K (or sigma 27) has been found that causes weak transcription of spoIVCB and strong transcription of cotD. A 14-kD protein was also discovered that changes the specificity of sigma K-containing RNA polymerase, greatly stimulating spoIVCB transcription and markedly repressing cotD transcription. Both sigma K and the 14-kD protein are products of genes known to be required for expression of specific genes in the mother cell. Thus, sigma K directs gene expression in the mother cell and it is proposed that inactivation or sequestering of the 14-kD protein switches the temporal pattern of gene expression during the transition from stages IV to V of development.

Amino Acid Sequence↗

PubMed2002

Interaction of the conserved region 4.2 of sigma(E) with the RseA anti-sigma factor.

Esigma(E) RNA polymerase transcribes a regulon of folding factors for the bacterial envelope and is induced by physical and chemical stresses. The RseA anti-sigma factor inhibits the activity of Esigma(E) RNA polymerase. It is shown here that the N-terminal portion of sigma(E), residues 1-153, binds core RNA polymerase. RseA interacts with residues 154-191 of sigma(E), a site that is homologous to region 4, the sigma factor binding site for promoter DNA. Mutations that reduce transcription of Esigma(E) RNA polymerase map to sigma(E) residues 178, 181, and 183. Variant sigma(E) proteins with amino acid substitutions at residues 178, 181, or 183 do not associate with RseA. A regulatory mechanism is proposed whereby RseA binds to a C-terminal peptide of sigma(E) and inhibits the transcription of Esigma(E) RNA polymerase by blocking promoter recognition.

Cloning, Molecular↗

PubMed2004

Mycobacterium tuberculosis ECF sigma factor sigC is required for lethality in mice and for the conditional expression of a defined gene set.

Bacterial alternative RNA polymerase sigma factors are key global adaptive response regulators with a likely role in Mycobacterium tuberculosis pathogenesis. We constructed a mutant lacking the sigma factor gene, sigC, by allelic exchange, in the virulent CDC1551 strain of M. tuberculosis and compared the resulting mutant with the isogenic wild-type strain and complemented mutant strain. In vitro, compared to the wild-type and complemented strains, the mutant was found to have similar ability to survive in both murine bone marrow-derived macrophages and activated J774 macrophages. In time-to-death experiments in the mouse model, the DeltasigC mutant was significantly attenuated, causing no death in infected mice whereas the wild-type and complemented strains caused 100% mortality within 235 days after aerosol infection with a median time to death of 170 days. Mouse organ bacterial burdens indicated that the mutant proliferated and persisted at the same level as the wild-type and complemented strains in lung tissue and was able to persist in mice without causing death for > 300 days. A complete genomic microarray study demonstrated that SigC modulates the expression of several key virulence-associated genes including hspX, senX3 and mtrA, encoding the alpha-crystallin homologue, a two-component sensor kinase and a two-component response regulator respectively. Altered expression of a subset of these genes was confirmed by quantitative RT-PCR analysis. Analysis of genes modulated by SigC also revealed a putative consensus DNA recognition sequence for SigC of SSSAAT-N(16-20)-CGTSSS (S = C or G). Promoter recognition for one of these genes was confirmed by in vitro transcription analysis after purification of recombinant SigC and reconstitution of an Esigma(C) RNA polymerase holoenzyme. These data indicate that the M. tuberculosis transcription factor SigC governs expression of an important M. tuberculosis regulon and is essential for lethality in mice, but is not required for bacterial survival in this species. These observations place the DeltasigC mutant in a class of M. tuberculosis mutants which persist in tissues but are attenuated in their ability to elicit lethal immunopathology.

Animals↗