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PubMed1995

Sequence-specific interactions between promoter DNA and the RNA polymerase sigma factor E.

In order to determine which amino acyl residues in a secondary sigma factor govern its specificity of recognition at the -35 region of promoters, we examined the effects of amino acid substitutions in sigma E in Bacillus subtilis that made the sequence of its putative -35 recognition region more similar to another sigma factor in B. subtilis, sigma K. We found that a single amino acid substitution at position 217 of sigma E resulted in a sigma factor that could direct transcription from sigma K-dependent promoters. Furthermore, we tested whether this amino acid substitution in sigma E had changed the specificity of interactions of the sigma with -35 region sequences by examining the activity of the mutant sigma E on derivatives of sigma E-dependent promoters that contained single base-pair substitutions. We found that this substitution in sigma E specifically suppressed the effect of a single base-pair substitution at position -31 in a sigma E-dependent promoter spoIIID. The amino acyl residue at another position (219) on sigma E affected the specificity of interaction with position -33 in spoIIID promoter. The amino acyl residues at the two positions in sigma E, 217 and 219, that determine the specificity of interactions between the sigma and base-pairs in the -35 region of its cognate promoters (positions -33 and -31, respectively, in the spoIIID promoter) probably closely contact these base-pairs.

Amino Acid Sequence↗

PubMed1993

Identification of genes encoding components of the swarmer cell flagellar motor and propeller and a sigma factor controlling differentiation of Vibrio parahaemolyticus.

Vibrio parahaemolyticus possesses two distinct motility systems, the polar system used for swimming in liquid environments and the lateral system used for swarming over surfaces. Growth on surfaces induces swarmer cell differentiation and expression of the lateral motility system. Mutants, created by transposon mutagenesis of a clone expressing lateral flagellin and gene disruption in V. parahaemolyticus, were unable to swarm and failed to make lateral flagellin; therefore, unlike the case for the polar system, there is one gene (lafA) encoding lateral flagellin. In addition to lafA, other genes required for swarming but not for swimming were identified by gene replacement mutagenesis. The nucleotide sequence of the clone determined open reading frames (ORFs) and deduced amino acid sequences showed similarities to flagellar components of other bacteria: flagellin, hook-associated protein (HAP2), motor components, and flagellar sigma factor (sigma 28). Many sigma 28 factors have been shown to recognize cognate promoters; however, expression of lafA in Escherichia coli required LafS, and E. coli sigma 28 did not substitute. Also, there were no sequences preceding genes encoding flagellin or HAP2 resembling the sigma 28 consensus promoter. The product of the sigma-like gene seems to be a unique member of the sigma 28 cluster. It appears the result of requiring expression for immunodetection of flagellin clones was that the sigma locus was fortuitously cloned, since the sigma and lafA loci were not contiguous in the chromosome. This work initiates identification and placement of genes in a scheme of control for swarmer cell differentiation; three levels have been identified in the transcriptional hierarchy.

Amino Acid Sequence↗

PubMed1990

Development-specific sigma-factor essential for late-stage differentiation of Myxococcus xanthus.

The gene for a developmentally expressed sigma-factor, sigB, has been isolated from Myxococcus xanthus by use of the sigA gene (formerly rpoD) of the vegetative sigma-factor as a probe. The sequence of sigB has been determined, and an open reading frame of 193 amino acid residues (Mr = 21,551) was identified. The amino-terminal region of SigB contains 69 residues, of which 35 are identical (50% identity) to the region of SigA required for core RNA polymerase binding and initiation of RNA polymerization. SigB also possesses many features commonly found in other prokaryotic sigma-factors. Analysis of an M. xanthus strain carrying a sigB-lacZ fusion gene revealed that sigB is expressed from a middle to late stage of differentiation corresponding to the period from the onset of sporulation to late development. A sigB deletion mutant displayed normal mound formation and sporulation; however, production of the ops gene product in myxospores of the delta sigB strain was shown to be blocked. Myxospores from the sigB deletion strain also exhibited severe defects in stability and viability during late development. Our data indicate that sigB encodes a sigma-factor essential for the maturation of myxospores at a late stage of M. xanthus differentiation. Our results also suggest that differentiation of M. xanthus is regulated by development-specific sigma-factors.

Amino Acid Sequence↗

PubMed1993

A new putative sigma factor of Myxococcus xanthus.

A third putative sigma factor gene, sigC, has been isolated from Myxococcus xanthus by using the sigA gene (formerly rpoD of M. xanthus) as a probe. The nucleotide sequence of sigC has been determined, and an open reading frame of 295 residues (M(r) = 33,430) has been identified. The deduced amino acid sequence of sigC exhibits the features which are characteristic of other bacterial sigma factors. The characterization of a sigC-lacZ strain has demonstrated that sigC expression is induced immediately after cells enter into the developmental cycle and is dramatically reduced at the onset of sporulation. A deletion mutant of sigC grows normally in vegetative culture and is able to develop normally. However, in contrast to the wild-type cells, the sigC deletion mutant cells became capable of forming fruiting bodies and myxospores on semirich agar plates. This suggests that sigC may play a role in expression of genes involved in negatively regulating the initiation of fruiting body formation.

Amino Acid Sequence↗

PubMed2001

Analysis of promoters recognized by PvdS, an extracytoplasmic-function sigma factor protein from Pseudomonas aeruginosa.

The alternative sigma factor PvdS is required by Pseudomonas aeruginosa for initiation of transcription from pyoverdine (pvd) promoters. Two divergent PvdS-dependent promoters (pvdE and pvdF) were characterized by deletion analysis, and the minimal promoter region for each included a sequence element, the iron starvation (IS) box, that is present in other pvd promoters. Site-directed mutagenesis showed that the IS box elements were essential for promoter activity in vivo. Band shift assays and in vitro transcription experiments showed that a complex of PvdS and core RNA polymerase required the presence of an IS box in order to bind to and initiate transcription from pvd promoters. These results indicate that IS box elements participate in sequence-specific recognition by PvdS to enable initiation of transcription from pvd promoters and are likely to represent a -35 sequence element for this sigma factor.

ATP-Binding Cassette Transporters↗

PubMed2005

The SigB sigma factor mediates high-temperature responses in the cyanobacterium Synechocystis sp. PCC6803.

The sigma factors of RNA polymerase play central roles when bacteria adapt to different environmental conditions. We studied heat-shock responses in the cyanobacterium Synechocystis sp. PCC6803 using the sigma factor inactivation strains deltasigB, deltasigD and deltasigBD. The SigB factor was found to be important for short-term heat-shock responses and acquired thermotolerance. The normal high-temperature induction of the hspA gene depended on the SigB factor. The SigD sigma factor had a role in high-temperature responses as well, and the double inactivation strain deltasigBD grew more slowly at 43 degrees C than the deltasigB and deltasigD strains.

Adaptation, Physiological↗

PubMed2000

Construction and characterization of a Mycobacterium tuberculosis mutant lacking the alternate sigma factor gene, sigF.

The alternate RNA polymerase sigma factor gene, sigF, which is expressed in stationary phase and under stress conditions in vitro, has been deleted in the virulent CDC1551 strain of Mycobacterium tuberculosis. The growth rate of the DeltasigF mutant was identical to that of the isogenic wild-type strain in exponential phase, although in stationary phase the mutant achieved a higher density than the wild type. The mutant showed increased susceptibility to rifampin and rifapentine. Additionally, the DeltasigF mutant displayed diminished uptake of chenodeoxycholate, and this effect was reversed by complementation with a wild-type sigF gene. No differences in short-term intracellular growth between mutant and wild-type organisms within human monocytes were observed. Similarly, the organisms did not differ in their susceptibilities to lymphocyte-mediated inhibition of intracellular growth. However, mice infected with the DeltasigF mutant showed a median time to death of 246 days compared with 161 days for wild-type strain-infected animals (P < 0.001). These data indicate that M. tuberculosis sigF is a nonessential alternate sigma factor both in axenic culture and for survival in macrophages in vitro. While the DeltasigF mutant produces a lethal infection of mice, it is less virulent than its wild-type counterpart by time-to-death analysis.

Animals↗

PubMed1989

Identification of the sigma E subunit of Escherichia coli RNA polymerase: a second alternate sigma factor involved in high-temperature gene expression.

The rpoH gene of Escherichia coli encodes sigma 32, the 32-kD sigma-factor responsible for the heat-inducible transcription of the heat shock genes. rpoH is transcribed from at least three promoters. Two of these promoters are recognized by RNA polymerase containing sigma 70, the predominant sigma-factor. We purified the factor responsible for recognizing the third rpoH promoter (rpoH P3) and identified it as RNA polymerase containing a novel sigma-factor with an apparent Mr of 24,000. This new sigma, which we call sigma E, is distinct from the known sigma factors in molecular weight and promoter specificity. sigma E holoenzyme will not recognize the sigma 70- or sigma 32-controlled promoters we tested, but it does transcribe the htrA gene, which is required for viability at temperatures greater than 42 degrees C. The in vivo role of sigma E is not known. The transcripts from the sigma E-controlled rpoH P3 and htrA promoters are most abundant at very high temperature, suggesting the sigma E holoenzyme may transcribe a second set of heat-inducible genes that are involved in growth at high temperature or in thermotolerance.

Bacterial Proteins↗

PubMed1996

Characterization of the primary sigma factor of Staphylococcus aureus.

RNA polymerase (RNAP) isolated from Staphylococcus aureus is deficient in sigma factor and is poorly active in transcription assays. Based on amino acid sequence homology of the Bacillus subtilis vegetative sigma factor sigmaA and the predicted product of the chromosomally located plaC gene of S. aureus, it was hypothesized that plaC could encode the vegetative sigma factor. We cloned plaC under a T7 promoter and overexpressed it in Escherichia coli strain BL21(DE3)pLysE. The overproduced protein, present in inclusion bodies, was solubilized with guanidine hydrochloride, renatured, and purified by DEAE-Sephacel and Sephadex G-75 chromatography. The purified protein, designated sigmaSA, cross-reacted with the B. subtilis anti-sigmaA antibody. E. coli core RNAP, reconstituted with sigmaSA, initiated promoter-specific transcription from the S. aureus promoters hla, sea, and sec and from the E. coli promoters rpoH P1, rpoH P4, and ColE1 RNA-1, which are recognized by the E. coli sigma70. sigmaSA, when added to the purified RNAP from S. aureus, stimulated transcriptional activity of the RNAP up to 72-fold. As determined by primer extension studies, the 5'-ends of the sigmaSA-initiated mRNAs synthesized in vitro from the agr P2 and sea promoters are in general agreement with the 5'-ends of the cellular RNAs. Disruption of the plaC gene on the S. aureus chromosome was lethal. We conclude that plaC encodes the primary sigma factor in S. aureus.

Bacillus subtilis↗

PubMed2003

DNA microarray analysis of Bacillus subtilis sigma factors of extracytoplasmic function family.

Target gene candidates of the seven extracytoplasmic function (ECF) sigma factors of Bacillus subtilis have been surveyed using DNA microarray analysis of mRNA extracted from cells grown in Luria-Bertani broth, in which an ECF sigma factor gene was placed under the control of the spac promoter on multicopy plasmid pDG148 and overexpressed. The number of target candidates for each of the sigma factors varied greatly, and a total of 278 genes were selected. Interestingly, the above target gene candidates shared only one gene out of 94 target genes of the general stress sigma B that have been reported in the literature thus far. Furthermore, lacZ-fusion experiments based on the results of DNA microarray analysis indicated that each ECF sigma factor directs transcription of its own operon, with the exception of sigZ. The DNA microarray data collected in this study are available at the KEGG Expression Database web site (http://www.genome.ad.jp/kegg/expression/).

Bacillus subtilis↗

PubMed1997

Cloning, sequencing, and expression of the rpoD gene encoding the primary sigma factor of Xanthomonas campestris.

A DNA fragment encoding the primary sigma factor from Xanthomonas campestris pv. campestris was cloned and sequenced. The gene (rpoD) encodes a polypeptide of 622 amino acids with a calculated MW of 70,700. The deduced amino acid sequence exhibits extensive sequence homology to the conserved regions of the primary sigma factors from bacteria. The gene product expressed in Escherichia coli, detected by Western blot analysis, had a MW similar to that estimated for the purified protein in SDS-PAGE. The NH2-terminal amino acid sequence determined chemically matched with that deduced from the nucleotide sequence of the rpoD gene. The calculated pI value (9.31) for the X. campestris primary sigma factor is much higher than the values observed for the analogous proteins from other bacteria.

Amino Acid Sequence↗

PubMed1994

Interactions between a Bacillus subtilis anti-sigma factor (RsbW) and its antagonist (RsbV).

The activity of sigma B, a secondary sigma factor of Bacillus subtilis, is primarily controlled by an anti-sigma factor protein (RsbW) that binds to sigma B and blocks its ability to form an RNA polymerase holoenzyme (E-sigma B). Inhibition of sigma B by RsbW is counteracted by RsbV, a protein that is essential for the activation of sigma B-dependent transcription. When crude B. subtilis extracts were fractionated by gel filtration chromatography or electrophoresis through nondenaturing polyacrylamide gels, a complex composed of RsbW and RsbV that is distinct from the previously observed RsbW-sigma B complex was detected. In analogous experiments, RsbX, an additional regulator of sigma B-dependent transcription that is thought to act independently of RsbV-RsbW, was not found to associate with any of the other sigB operon products. Two forms of RsbV were visualized when crude cell extracts of B. subtilis were subjected to isoelectric focusing (IEF), with the more negatively charged RsbV species absent from extracts prepared from RsbW- strains. In vitro, RsbV became phosphorylated when incubated with ATP and RsbW but not with ATP alone. The phosphorylated RsbV species comigrated during IEF with the RsbW-dependent form of RsbV found in crude cell extracts. These results suggest that the modified RsbV, present in crude cell extracts, is phosphorylated. When gel filtration fractions containing RsbV-RsbW complexes or RsbV alone were subjected to IEF, only the unmodified form of RsbV was found associated with RsbW. The presumed phosphorylated variant of RsbV was present only in fractions that did not contain RsbW. The data support a model whereby RsbV binds directly to RsbW and blocks its ability to form the RsbW-sigma B complex. This activity of RsbV appears to be inhibited by RsbW-dependent phosphorylation.

Adenosine Triphosphate↗

PubMed1996

Cloning, nucleotide sequence and characterization of the rpoD gene encoding the primary sigma factor of Rhodobacter capsulatus.

A synthetic oligodeoxynucleotide probe based on a highly conserved region of the sigma factors was used to identify and clone the rpoD gene encoding the principal sigma factor of R. capsulatus. The deduced polypeptide contains 674 amino acids and has a predicted molecular mass of 75,942 Da. The deduced amino acid sequence of R. capsulatus RpoD protein exhibits 46.2% and 45.7% identity with housekeeping sigma factors of Pseudomonas and E. coli, respectively. Unsuccessful attempts to inactivate the single chromosomal rpoD gene of R. capsulatus by gene replacement technique indicate that this gene is essential for cell survival, as expected for the primary sigma factor. The rpoD transcript 5'-end was mapped by primer extension analysis, 74 bp upstream of the initiation codon and DNA sequence analysis has identified a motif resembling the delta 70 E. coli consensus promoter sequences at the expected distance from this proposed transcription start site. rpoD gene expression, as measured by the activity of the phi (rpoD'-lacZ+) translational fusion, was found to be constant throughout exponential and early plateau phases, but significantly increased at later times of the stationary phase.

Amino Acid Sequence↗

PubMed2004

RsbU-dependent regulation of Staphylococcus epidermidis biofilm formation is mediated via the alternative sigma factor sigmaB by repression of the negative regulator gene icaR.

Transposon mutagenesis of rsbU leads to a biofilm-negative phenotype in Staphylococcus epidermidis. However, the pathway of this regulatory mechanism was unknown. To investigate the role of RsbU in the regulation of the alternative sigma factor sigma(B) and biofilm formation, we generated different mutants of the sigma(B) operon in S. epidermidis strains 1457 and 8400. The genes rsbU, rsbV, rsbW, and sigB, as well as the regulatory cascade rsbUVW and the entire sigma(B) operon, were deleted. Transcriptional analysis of sarA and the sigma(B)-dependent gene asp23 revealed the functions of RsbU and RsbV as positive regulators and of RsbW as a negative regulator of sigma(B) activity, indicating regulation of sigma(B) activity similar to that characterized for Bacillus subtilis and Staphylococcus aureus. Phenotypic characterization of the mutants revealed that the dramatic decrease of biofilm formation in rsbU mutants is mediated via sigma(B), indicating a crucial role for sigma(B) in S. epidermidis pathogenesis. However, biofilm formation in mutants defective in sigma(B) or its function could be restored in the presence of subinhibitory ethanol concentrations. Transcriptional analysis revealed that icaR is up-regulated in mutants lacking sigma(B) function but that icaA transcription is down-regulated in these mutants, indicating a sigma(B)-dependent regulatory intermediate negatively regulating IcaR. Supplementation of growth media with ethanol decreased icaR transcription, leading to increased icaA transcription and a biofilm-positive phenotype, indicating that the ethanol-dependent induction of biofilm formation is mediated by IcaR. This icaR-dependent regulation under ethanol induction is mediated in a sigma(B)-independent manner, suggesting at least one additional regulatory intermediate in the biofilm formation of S. epidermidis.

Bacterial Proteins↗

PubMed2001

Cloning and transcriptional characterization of two sigma factor genes, sigA and sigB, from Brevibacterium flavum.

Using a DNA fragment containing the principal sigma factor gene hrdB of Streptomyces aureofaciens, we identified two sigma70-like genes in a library of Brevibacterium flavum. Sequence analysis of the complete genes revealed two ORFs coding for gene products of 498 and 331 amino acid residues, which showed the greatest similarity to SigA and SigB sigma factors from Brevibacterium lactofermentum. We designated them similarly sigA and sigB. Transcription of B. flavum sigA and sigB has been investigated by S1-nuclease mapping by using RNA from different growth phases and after exposure to several stress conditions. Both genes are transcribed from a single promoter with transcription start points of 368 bp and 25 bp upstream from the proposed translation initiation codon of the sigA and sigB genes, respectively. Whereas sigA is transcribed almost constitutively during growth and after stress conditions, expression of sigB is significantly induced after several stress conditions, like acid stress, ethanol shock, and cold shock. Expression of both genes is significantly reduced after heat shock. Considering these transcriptional results, and also on the basis of the similarity to other principal sigma factor genes, sigA probably encodes the functional principal sigma factor, and sigB might have a function in stress response.

Amino Acid Sequence↗

PubMed1995

Amplification of the housekeeping sigma factor in Pseudomonas fluorescens CHA0 enhances antibiotic production and improves biocontrol abilities.

Pseudomonas fluorescens CHA0 produces a variety of secondary metabolites, in particular the antibiotics pyoluteorin and 2,4-diacetylphloroglucinol, and protects various plants from diseases caused by soilborne pathogenic fungi. The rpoD gene encoding the housekeeping sigma factor sigma 70 of P. fluorescens was sequenced. The deduced RpoD protein showed 83% identity with RpoD of Pseudomonas aeruginosa and 67% identity with RpoD of Escherichia coli. Attempts to inactivate the single chromosomal rpoD gene of strain CHA0 were unsuccessful, indicating an essential role of this gene. When rpoD was carried by an IncP vector in strain CHA0, the production of both antibiotics was increased severalfold and, in parallel, protection of cucumber against disease caused by Pythium ultimum was improved, in comparison with strain CHA0.

Amino Acid Sequence↗

PubMed2002

Involvement of two putative alternative sigma factors in stress response of the radioresistant bacterium Deinococcus radiodurans.

Two genes bearing similarity to alternative sigma factors were identified in the Deinococcus radiodurans genome sequence and designated sig1 and sig2. These genes were cloned and inactivated, and both were found to be important for survival during heat and ethanol stress, although the sig1 mutants displayed a more severe phenotype than the sig2 mutants. Reporter gene fusions to the groESL and dnaKJ operons transformed into these mutant backgrounds indicated that sig1 is required for the heat shock induction of groESL and dnaKJ, whereas sig2 mutants show a more moderate defect in dnaKJ induction and are not impaired for groESL induction. Essentiality tests suggested that neither sig1 nor sig2 is essential under all conditions. Sequence comparisons demonstrated that the sig1 gene product is classed distinctly with extracytoplasmic function (ECF) sigma factors, whereas Sig2 appears to be a more divergent sigma factor ortholog. These results suggest that sig1 encodes the major ECF-derived heat shock sigma factor in D. radiodurans and that it plays a central role in the positive regulation of heat shock genes. sig2, in contrast, appears to play a more minor role in heat shock protection and may serve to modulate the expression of some heat protective genes.

Amino Acid Sequence↗

PubMed2002

Detection of multiple extracytoplasmic function (ECF) sigma factors in the genome of Pseudomonas putida KT2440 and their counterparts in Pseudomonas aeruginosa PA01.

Pseudomonas putida KT2440 is highly successful in colonizing a variety habitats, including aquatic and edaphic niches. In accordance with this ability and with the need to adapt to changing environmental conditions, P. putida has developed sophisticated mechanisms of transcriptional regulation. We analysed, at the genome level, the repertoire of sigma factors in P. putida KT2440 and identified 24 sigma factors, 19 of which corresponded to the subfamily of extracytoplasmic function (ECF) sigma factors. We detected 13 ECF sigma factors that showed similarity to the Escherichia coli FecI sigma factor, which is involved in iron acquisition. In 11 cases, a fecR-like gene was found adjacent to the fecI-like gene and, in 10 cases, a gene encoding an iron receptor lies in the vicinity of the fecI/fecR cluster. This may explain the ability of P. putida KT2440 to grow under low iron availability conditions. Five fecI/fecR/iron receptor gene clusters from P. putida were also identified in the human pathogen Pseudomonas aeruginosa.

Base Sequence↗