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PubMed2000

Functional analysis of PvdS, an iron starvation sigma factor of Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, iron modulates gene expression through a cascade of negative and positive regulatory proteins. The master regulator Fur is involved in iron-dependent repression of several genes. One of these genes, pvdS, was predicted to encode a putative sigma factor responsible for the transcription of a subset of genes of the Fur regulon. PvdS appears to belong to a structurally and functionally distinct subgroup of the extracytoplasmic function family of alternative sigma factors. Members of this subgroup, also including PbrA from Pseudomonas fluorescens, PfrI and PupI from Pseudomonas putida, and FecI from Escherichia coli, are controlled by the Fur repressor, and they activate transcription of genes for the biosynthesis or the uptake of siderophores. Evidence is provided that the PvdS protein of P. aeruginosa is endowed with biochemical properties of eubacterial sigma factors, as it spontaneously forms 1:1 complexes with the core fraction of RNA polymerase (RNAP, alpha(2)betabeta' subunits), thereby promoting in vitro binding of the PvdS-RNAP holoenzyme to the promoter region of the pvdA gene. These functional features of PvdS are consistent with the presence of structural domains predicted to be involved in core RNAP binding, promoter recognition, and open complex formation. The activity of pyoverdin biosynthetic (pvd) promoters was significantly lower in E. coli overexpressing the multicopy pvdS gene than in wild-type P. aeruginosa PAO1 carrying the single gene copy, and pvd::lacZ transcriptional fusions were silent in both pfrI (the pvdS homologue) and pfrA (a positive regulator of pseudobactin biosynthetic genes) mutants of P. putida WCS358, while they are expressed at PAO1 levels in wild-type WCS358. Moreover, the PvdS-RNAP holoenzyme purified from E. coli lacked the ability to generate in vitro transcripts from the pvdA promoter. These observations suggest that at least one additional positive regulator could be required for full activity of the PvdS-dependent transcription complex both in vivo and in vitro. This is consistent with the presence of a putative activator binding site (the iron starvation box) at variable distance from the transcription initiation sites of promoters controlled by the iron starvation sigma factors PvdS, PfrI, and PbrA of fluorescent pseudomonads.

Amino Acid Sequence↗
PubMed1988

Late sigma factor of bacteriophage T4. Formation and properties of RNA polymerase-promoter complexes.

Bacteriophage T4 late gene promoters do not display sequence homology in the -35 region (Christensen, A. C., and Young, E. T. (1982) Nature 299, 369-371), suggesting an unusual geometry of RNA polymerase-promoter interaction. We have analyzed in vitro utilization of a late T4 promoter by RNA polymerase reconstituted from E. coli core enzyme (E) and bacteriophage T4 late sigma factor (sigma gp55). The E sigma gp55 holoenzyme forms a stable promoter complex which lacks protein-DNA contacts upstream from position -30 and is sensitive to direct attack by heparin. This complex is capable of reiterative oligonucleotide synthesis (abortive initiation). Kinetic analysis of complex formation reveals a rapidly forming inactive intermediate (closed complex) which is slowly isomerized into a catalytically active form (open complex). The results indicate that all components essential for promoter binding, open complex formation, and initiation of transcription are present in the "downstream" part of the RNA polymerase molecule, which is defined by the -30 to +20 footprint. On the basis of these observations and the results of others, we suggest that during transcription initiation at bipartite (Escherichia coli) promoters, an "upstream" DNA-binding domain of RNA polymerase which recognizes specific sequence elements in the -35 region plays an auxiliary role by regulating the rate of productive interactions in the downstream part of the molecule through an allosteric mechanism.

Base Sequence↗
PubMed1996

Differential expression of two sporulation specific sigma factors of Streptomyces aureofaciens correlates with the developmental stage.

In previous experiments, the Streptomyces aureofaciens (Sa) rpoZ, and sigF genes have been shown to encode putative sigma factors essential in differentiation. In an attempt to investigate expression of these genes during differentiation, we have performed S1 nuclease mapping using RNA prepared from Sa in various developmental stages. Two putative promoters were identified upstream of the rpoZ coding region. The promoters significantly differed in their strength, and were active in distinct developmental stages; the weaker, rpoZ-P1, was active only in substrate mycelium, and the stronger, rpoZ-P2, was induced at the beginning of aerial mycelium formation. Transcriptional analysis of sigF revealed two apparent transcription start points, both being detectable only in the late phase of aerial mycelium formation. No sigF transcription was detected in an rpoZ-disrupted Sa strain. Promoter-bearing DNA fragments from rpoZ and sigF were inserted into several promoter-probe vectors, to give expression patterns consistent with the results of direct RNA analysis. The results implicate temporally different expression of rpoZ and sigF during the differentiation of Sa, and direct or indirect dependence of sigF expression on the rpoZ-encoded putative sigma factor, thus indicating a cascade of sigma factors in Streptomyces development.

Amino Acid Sequence↗
PubMed2005

The Enterococcus faecalis sigV protein is an extracytoplasmic function sigma factor contributing to survival following heat, acid, and ethanol treatments.

Analysis of the genome sequence of Enterococcus faecalis allowed the identification of two genes whose protein products showed 33 and 34% identity with those of sigV and yrhM of Bacillus subtilis, respectively. These genes, named sigV and rsiV, are predicted to encode members of the extracytoplasmic function subfamily of eubacterial RNA polymerase sigma and anti-sigma factors, respectively. This group of sigma factors has been shown to regulate gene expression in response to stress conditions. sigV and rsiV were shown to be under the control of the same promoter. The transcriptional start site was determined, and the 1.5-kb mRNA transcript was shown to be overexpressed under glucose and complete starvation, as well as under physicochemical treatments. Three mutants, affected in sigV, rsiV, and both genes, were constructed by double-crossover recombination within the genome of E. faecalis strain JH2-2. Compared with the wild type and the rsiV mutant, the sigV mutants were more susceptible to heat shock, acid, and ethanol treatments and displayed decreased survival during long-term starvation. A nisin-inducible sigV gene construction used in complementation assays restored the wild phenotype of the sigV mutants, confirming the involvement of SigV in the heat shock, ethanol, and acid stress responses. Northern blot analysis carried out with the three mutant strains revealed the inhibition of sigV expression by the related anti-sigma factor gene rsiV. In addition, putative candidates of the sigV regulon determined by computer search for the sigV promoter sequence were analyzed.

Amino Acid Sequence↗
PubMed1996

Isolation and sequence of a Mycobacterium tuberculosis sigma factor.

A DNA segment from Mycobacterium tuberculosis containing a gene for a putative sigma factor was isolated and sequenced. The protein encoded by this gene is 92% similar to the Mycobacterium smegmatis sigma factor MysB, and has been designated Mtu SigB. A Mycobacterium leprae homologue of mysB and mtu sigB was identified in the database.

Amino Acid Sequence↗
PubMed1997

Expression of two alternative sigma factors of Synechococcus sp. strain PCC 7002 is modulated by carbon and nitrogen stress.

The sigB and sigC genes, encoding two alternative sigma factors of the unicellular marine cyanobacterium Synechococcus sp. PCC 7002, were cloned and characterized. Strains in which the sigB and sigC genes were insertionally inactivated were viable under standard laboratory conditions, indicating that SigB and SigC are group 2 sigma factors. Starvation for either nitrogen or carbon caused an increase in sigB mRNA levels. Transcripts for the sigC gene initially increased but then decreased during nitrogen and carbon starvation. The SigC protein could not be identified in cyanobacterial extracts using antisera to Synechococcus sp. PCC 7002 SigA or RpoD from Bacillus subtilis. The ratio of the principal vegetative sigma factor, SigA, to SigB decreased during either nitrogen starvation or carbon starvation, and the levels of SigB also increased in the sigC mutant strain. These results imply that SigB and SigC play roles in modifying transcription in response to changes in carbon and nitrogen availability in this cyanobacterium.

Amino Acid Sequence↗
PubMed1991

Cloning and analysis of the gene (rpoDA) for the principal sigma factor of Pseudomonas aeruginosa.

A gene (rpoDA) of Pseudomonas aeruginosa whose gene product has a homologous function and structure with the principal sigma factor of Escherichia coli was cloned and sequenced. The DNA region corresponding to one of the two hybridization signals found in P. aeruginosa DNA with a synthetic oligonucleotide probe (rpoD probe) was shown to be able to complement a temperature sensitive mutation of Escherichia coli rpoD gene. The amino acid sequence deduced from the nucleotide sequence of rpoDA showed an extensive homology with that of the principal sigma factor of E. coli throughout the entire region, which indicates that the two gene products have an essentially identical domain structure. A common basic structure observed among principal sigma factors of different eubacterial strains was proposed. RpoDA protein was identified in the extract of the cell carrying a plasmid clone with the rpoDA gene insert by Western blot analysis.

Amino Acid Sequence↗
PubMed1995

Mutation of the principal sigma factor causes loss of virulence in a strain of the Mycobacterium tuberculosis complex.

Tuberculosis continues to be responsible for the deaths of millions of people, yet the virulence factors of the causative pathogens remain unknown. Genetic complementation experiments with strains of the Mycobacterium tuberculosis complex have identified a gene from a virulent strain that restores virulence to an attenuated strain. The gene, designated rpoV, has a high degree of homology with principal transcription or sigma factors from other bacteria, particularly Mycobacterium smegmatis and Streptomyces griseus. The homologous rpoV gene of the attenuated strain has a point mutation causing an arginine-->histidine change in a domain known to interact with promoters. To our knowledge, association of loss of bacterial virulence with a mutation in the principal sigma factor has not been previously reported. The results indicate either that tuberculosis organisms have an alternative principal sigma factor that promotes virulence genes or, more probably, that this particular mutant principal sigma factor is unable to promote expression of one or more genes required for virulence. Study of genes and proteins differentially regulated by the mutant transcription factor should facilitate identification of further virulence factors.

Amino Acid Sequence↗
PubMed1996

The rpoD1 gene product is a principal sigma factor of RNA polymerase in Microcystis aeruginosa K-81.

We performed molecular characterization of the RpoD1 protein encoded by the rpoD1 gene isolated from a cyanobacterium, Microcystis aeruginosa K-81. The deduced amino acid sequence (416 aa, 48,871 Da) of RpoD1 exhibited extensive similarity to those of proteins of the eubacterial RpoD family (Escherichia coli sigma 70 homologs). We overproduced and purified RpoD1 (54 kDa) from E. coli. Biological and biochemical analyses suggested that RpoD1 has a function homologous to that of E. coli sigma 70 as follows: (i) the RpoD1 protein complemented an rpoD mutant of E. coli strain YN543 (rpoD285) and (ii) the heterologous RNA polymerase holoenzyme reconstituted from the E. coli core enzyme and recombinant RpoD1 was specifically transcribed from E. coli promoters. Furthermore, Western blot analysis with antiserum against Synechococcus sp. strain PCC 7942 RpoD1 (a principal sigma factor of the sigma 70 type) indicated that M. aeruginosa K-81 RpoD1 (sigma A1) is the principal sigma factor, which is a major component of the sigma subunit on exponential cell growth.

Amino Acid Sequence↗
PubMed1998

The flagellar anti-sigma factor FlgM actively dissociates Salmonella typhimurium sigma28 RNA polymerase holoenzyme.

The anti-sigma factor FlgM of Salmonella typhimurium inhibits transcription of class 3 flagellar genes through a direct interaction with the flagellar-specific sigma factor, sigma28. FlgM is believed to prevent RNA polymerase (RNAP) holoenzyme formation by sequestering free sigma28. We have analyzed FlgM-mediated inhibition of sigma28 activity in vitro. FlgM is able to inhibit sigma28 activity even when sigma28 is first allowed to associate with core RNAP. Surface plasmon resonance (SPR) was used to evaluate the interaction between FlgM and both sigma28 and sigma28 holoenzyme (Esigma28). The Kd of the sigma28-FlgM complex is approximately 2 x 10(-10) M; missense mutations in FlgM that cause a defect in sigma28 inhibition in vivo increase the Kd of this interaction by 4- to 10-fold. SPR measurements of Esigma28 dissociation in the presence of FlgM indicate that FlgM destabilizes Esigma28, presumably via an interaction with the sigma subunit. Our data provide the first direct evidence of an interaction between FlgM and Esigma28. We propose that this secondary activity of FlgM, which we term holoenzyme destabilization, enhances the sensitivity of the cell to changes in FlgM levels during flagellar biogenesis.

Bacterial Proteins↗
PubMed2006

Two novel nuclear genes, OsSIG5 and OsSIG6, encoding potential plastid sigma factors of RNA polymerase in rice: tissue-specific and light-responsive gene expression.

Two novel nuclear genes, OsSIG5 and OsSIG6, encoding potential plastid sigma factors of RNA polymerase (RNAP) were identified in Oryza sativa. The deduced amino acid sequences contain conserved regions, regions 1.2-4.2, and a novel region A/B at the N-terminus. Tissue-specific and light-responsive transcripts of OsSIG5 and OsSIG6 were observed. The N-terminal region of OsSig5 conferred import of green fluorescent protein into the chloroplast. Specific transcripts of rice psbA were synthesized in vitro by reconstituted OsSig5-RNAP holoenzymes. These results indicated that OsSig5 is a plastid sigma factor. This is the first report of the Sig5-type sigma factor in crops.

Amino Acid Sequence↗
PubMed1993

The activity of sigma E, an Escherichia coli heat-inducible sigma-factor, is modulated by expression of outer membrane proteins.

sigma E and sigma 32 are two heat- and ethanol-inducible sigma-factors in Escherichia coli. The sigma 32 regulon is also induced by unfolded and misfolded proteins in the cytoplasm, and the function of many of the proteins in the sigma 32 regulon is to bind to cytoplasmic proteins and assist them in folding or unfolding. To further understand the function of the sigma E regulon, we searched for mutants that affected sigma E activity. Our results indicate that a signal generated by expression of outer membrane proteins modulates sigma E activity. Specifically, sigma E activity is induced by increased expression of OMPs and is reduced by decreased expression of OMPs. In addition, mutations that cause misfolded OMPs induce sigma E activity. This signal is generated after the fate of OMPs and periplasmic proteins diverge in the secretory pathway and is not the result of an accumulation of OMP precursors in the cytoplasm. Our results indicate that this effect of OMPs is specific to the sigma E regulon, because none of the above mutations affect sigma 32 activity. We propose that the sigma E regulon is involved in processes that occur in extracytoplasmic compartments and that these two heat-inducible regulons may have distinct but complementary roles of monitoring the state of proteins in the cytoplasm (sigma 32) and outer membrane (sigma E).

Bacterial Outer Membrane Proteins↗
PubMed2007

The alternative sigma factor SigB of Corynebacterium glutamicum modulates global gene expression during transition from exponential growth to stationary phase.

BACKGROUND: Corynebacterium glutamicum is a gram-positive soil bacterium widely used for the industrial production of amino acids. There is great interest in the examination of the molecular mechanism of transcription control. One of these control mechanisms are sigma factors. C. glutamicum ATCC 13032 has seven putative sigma factor-encoding genes, including sigA and sigB. The sigA gene encodes the essential primary sigma factor of C. glutamicum and is responsible for promoter recognition of house-keeping genes. The sigB gene codes for the non-essential sigma factor SigB that has a proposed role in stress reponse. RESULTS: The sigB gene expression was highest at transition between exponential growth and stationary phase, when the amount of sigA mRNA was already decreasing. Genome-wide transcription profiles of the wild-type and the sigB mutant were recorded by comparative DNA microarray hybridizations. The data indicated that the mRNA levels of 111 genes are significantly changed in the sigB-proficient strain during the transition phase, whereas the expression profile of the sigB-deficient strain showed only minor changes (26 genes). The genes that are higher expressed during transition phase only in the sigB-proficient strain mainly belong to the functional categories amino acid metabolism, carbon metabolism, stress defense, membrane processes, and phosphorus metabolism. The transcription start points of six of these genes were determined and the deduced promoter sequences turned out to be indistinguishable from that of the consensus promoter recognized by SigA. Real-time reverse transcription PCR assays revealed that the expression profiles of these genes during growth were similar to that of the sigB gene itself. In the sigB mutant, however, the transcription profiles resembled that of the sigA gene encoding the house-keeping sigma factor. CONCLUSION: During transition phase, the sigB gene showed an enhanced expression, while simultaneously the sigA mRNA decreased in abundance. This might cause a replacement of SigA by SigB at the RNA polymerase core enzyme and in turn results in increased expression of genes relevant for the transition and the stationary phase, either to cope with nutrient limitation or with the accompanying oxidative stress. The increased expression of genes encoding anti-oxidative or protection functions also prepares the cell for upcoming limitations and environmental stresses.

Bacterial Proteins↗
PubMed1994

Expression in Bacillus subtilis of the Bacillus thuringiensis cryIIIA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spo0A mutant.

Expression of the Bacillus thuringiensis cryIIIA gene encoding a Coleoptera-specific toxin is weak during vegetative growth and is activated at the onset of the stationary phase. cryIIIA'-'lacZ fusions and primer extension analysis show that the regulation of cryIIIA expression is similar in Bacillus subtilis and in B. thuringiensis. Activation of cryIIIA expression was not altered in B. subtilis mutant strains deficient for the sigma H and sigma E sporulation-specific sigma factors or for minor sigma factors such as sigma B, sigma D, or sigma L. This result and the nucleotide sequence of the -35 and -10 regions of the cryIIIA promoter suggest that cryIIIA expression might be directed by the E sigma A form of RNA polymerase. Expression of the cryIIIA'-'lacZ fusion is shut off after t2 (2 h after time zero) of sporulation in the B. subtilis wild-type strain grown on nutrient broth sporulation medium. However, no decrease in cryIIIA-directed beta-galactosidase activity occurred in sigma H, kinA, or spo0A mutant strains. Moreover, beta-galactosidase activity was higher and remained elevated after t2 in the spo0A mutant strain. beta-Galactosidase activity was weak in abrB and spo0A abrB mutant strains, suggesting that AbrB is responsible for the higher level of cryIIIA expression observed in a spo0A mutant. However, both in spo0A and spo0A abrB mutant strains, beta-galactosidase activity remained elevated after t2, suggesting that even in the absence of AbrB, cryIIIA expression is controlled through modulation of the phosphorylated form of Spo0A. When the cryIIIA gene is expressed in a B. subtilis spo0A mutant strain or in the 168 wild-type strain, large amounts of toxins are produced and accumulate to form a flat rectangular crystal characteristic of the coleopteran-specific B. thuringiensis strains.

Bacillus subtilis↗
PubMed2002

Environmental response and autoregulation of Clostridium difficile TxeR, a sigma factor for toxin gene expression.

TxeR, a sigma factor that directs Clostridium difficile RNA polymerase to recognize the promoters of two major toxin genes, was shown to stimulate its own synthesis. Whether expressed in C. difficile, Clostridium perfringens, or Escherichia coli, TxeR stimulated transcription of fusions of the txeR promoter region to reporter genes. As is the case for the tox genes, txeR expression was responsive to the cellular growth phase and the constituents of the medium. That is, the level of expression in broth culture was low during the exponential growth phase, but rapidly increased as cells approached the stationary phase. In the presence of excess glucose, expression from the txeR promoter was repressed. The results support a model for toxin gene expression in which synthesis of TxeR is induced by specific environmental signals. The increased level of TxeR then permits high-level expression of the toxin genes. The study of txeR gene regulation in C. difficile was made possible by introduction of a mobilizable, replicative plasmid via conjugation with E. coli.

Bacterial Proteins↗
PubMed1998

Regulation of proteolysis of the stationary-phase sigma factor RpoS.

RpoS, the stationary-phase sigma factor of Escherichia coli, is responsible for increased transcription of an array of genes when cells enter stationary phase and under certain stress conditions. RpoS is rapidly degraded during exponential phase and much more slowly during stationary phase; the resulting changes in RpoS accumulation play an important role in providing differential expression of RpoS-dependent gene expression. It has previously been shown that rapid degradation of RpoS during exponential growth depends on RssB (also called SprE and MviA), a protein with homology to the family of response regulators, and on the ClpXP protease. We find that RssB regulation of proteolysis does not extend to another ClpXP substrate, bacteriophage lambda O protein, suggesting that RssB acts on the specific substrate RpoS rather than on the protease. In addition, the activity of RpoS is down-regulated by RssB when degradation is blocked. In cells blocked for RpoS degradation by a mutation in clpP, cells devoid of RssB show a four- to fivefold-higher activity of an RpoS-dependent reporter fusion than cells overproducing RssB. Therefore, RssB allows specific environmental regulation of RpoS accumulation and may also modulate activity. The regulation of degradation provides an irreversible switch, while the regulation of activity may provide a second, presumably reversible level of control.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1992

Construction and characterization of Streptomyces coelicolor A3(2) mutants that are multiply deficient in the nonessential hrd-encoded RNA polymerase sigma factors.

Previous studies showed that Streptomyces coelicolor A3(2) has four genes (hrdA, hrdB, hrdC, and hrdD) that appear to encode RNA polymerase sigma factors very similar to the sigma 70 subunit of Escherichia coli and that hrdC and hrdD could be individually disrupted without causing obvious phenotypic defects. Here, hrdA was cloned and stable null hrdA and hrdD mutants were constructed by gene replacement. These two mutants and a previously constructed hrdC null mutant were used in crosses to generate hrdAC, hrdAD, hrdCD, and hrdACD strains. The inability to synthesize one, two, or all three of the nonessential hrd-encoded sigma factors had no obvious phenotypic consequences.

Alleles↗