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PubMed2005

Global gene expression and the role of sigma factors in Neisseria gonorrhoeae in interactions with epithelial cells.

Like many bacterial pathogens, Neisseria gonorrhoeae must adapt to environmental changes in order to successfully colonize and proliferate in a new host. Modulation of gene expression in response to environmental signals is an efficient mechanism used by bacteria to achieve this goal. Using DNA microarrays and a tissue culture model for gonococcal infection, we examined global changes in gene expression in N. gonorrhoeae in response to adherence to host cells. Among those genes induced upon adherence to human epithelial cells in culture was rpoH, which encodes a homolog of the heat shock sigma factor, sigma(32) (RpoH), as well as genes of the RpoH regulon, groEL and groES. Attempts to construct an rpoH null mutant in N. gonorrhoeae were unsuccessful, suggesting that RpoH is essential for viability of N. gonorrhoeae. The extracytoplasmic sigma factor, RpoE (sigma(E)), while known to regulate rpoH in other bacteria, was found not to be necessary for the up-regulation of rpoH in gonococci upon adherence to host cells. To examine the role of RpoH in host cell interactions, an N. gonorrhoeae strain conditionally expressing rpoH was constructed. The results of our experiments showed that while induction of rpoH expression is not necessary for adherence of gonococci to epithelial cells, it is important for the subsequent invasion step, as gonococci depleted for rpoH invade cells two- to threefold less efficiently than a wild-type strain. Taken together, these results indicate that sigma(32), but not sigma(E), is important for the response of gonococci in the initial steps of an infection.

Bacterial Adhesion↗
PubMed2006

The cyanobacterial principal sigma factor region 1.1 is involved in DNA-binding in the free form and in transcription activity as holoenzyme.

Cyanobacterial principal sigma factor, sigma(A), includes a specifically conserved cluster of basic amino acids in the amino-terminal extension called region 1.1. We found that the sigma(A) in a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 binds DNA in the absence of the core RNA polymerase and that sigma(A) lacking region 1.1 is not able to bind DNA. This indicates that, in the cyanobacterium, region 1.1 participates in DNA-binding, rather than inhibiting the interaction between free sigma and DNA, as found in other principal sigma factors of eubacteria. The results of in vitro transcription assays with the reconstituted RNA polymerase showed that region 1.1 reduces transcription activity from the cpc promoter.

Amino Acid Sequence↗
PubMed1989

Identification of subunits of gonococcal RNA polymerase by immunoblot analysis: evidence for multiple sigma factors.

Heparin-agarose and single-stranded DNA-cellulose chromatography were used to purify RNA polymerase 25-fold from Neisseria gonorrhoeae, and the activity of the polymerase was characterized in altered assay systems. The core subunits (beta, beta', and alpha) were tentatively identified as major proteins copurifying with polymerase activity. The identification of the core subunits was confirmed by Western (immunoblot) analysis with polyclonal antisera to Escherichia coli core RNA polymerase. Gonococcal sigma factor heterogeneity was examined by Western blot analysis with polyclonal antiserum to the major E. coli sigma factor, sigma 70, to the E. coli heat shock sigma factor, sigma 32, and with a monoclonal antiserum to Salmonella typhimurium NtrA (sigma 54). Purified RNA polymerase and whole-cell extracts from type 1, type 4, heat-shocked, and anaerobically grown gonococci were examined. Four putative gonococcal sigma factors were detected in purified RNA polymerase preparations and in whole-cell extracts from all cell types. Two of these bands appeared as a doublet, which had an estimated Mr of 80,000. A single lower-Mr band, estimated to be 40,000, was also present. All three of these bands reacted with antisera to E. coli sigma 70 and to E. coli sigma 32. A fourth gonococcal protein reacted solely with a highly specific monoclonal antibody to sigma 54 and had an Mr of 90,000. We conclude that N. gonorrhoeae may contain multiple sigma factors, which it may use to regulate gene expression.

Animals↗
PubMed1998

Bacillus licheniformis sigB operon encoding the general stress transcription factor sigma B.

The general stress response of the Gram-positive soil bacterium Bacillus subtilis is controlled by the sigma B transcription factor. sigma B activity is regulated by the newly discovered partner switching mechanism of signal transduction, which integrates the two different classes of challenges which posttranslationally activate sigma B: environmental stress and energy stress. Our investigation of a possible sigma B homologue in the related soil bacterium B. licheniformis had two goals. First, this study would contribute to understanding the distribution of the sigma B general stress system among Gram-positive bacteria. Second, a phylogenetic comparison of regulatory systems can supplement genetic and biochemical analysis by revealing conserved features that are critical for function. We report here that (1) B. licheniformis cells contain a protein that closely resembles B. subtilis sigma B in size and antigenic properties; (2) the level of this potential sigma B homologue rapidly increases following environmental or energy stress; and (3) the B. licheniformis genome encodes a homologue of the sigB general stress operon, including the sigma B structural gene and seven rsb regulatory genes. Based on these results, B. licheniformis possesses a general stress system likely regulated by two coupled partner switching modules that sense and integrate the two broad classes of activating stress signals.

Amino Acid Sequence↗
PubMed1996

A stationary-phase stress-response sigma factor from Mycobacterium tuberculosis.

Alternative RNA polymerase sigma factors are a common means of coordinating gene regulation in bacteria. Using PCR amplification with degenerate primers, we identified and cloned a sigma factor gene, sigF, from Mycobacterium tuberculosis. The deduced protein encoded by sigF shows significant similarity to SigF sporulation sigma factors from Streptomyces coelicolor and Bacillus subtilis and to SigB, a stress-response sigma factor, from B. subtilis. Southern blot surveys with a sigF-specific probe identified cross-hybridizing bands in other slow-growing mycobacteria, Mycobacterium bovis bacille Calmette-Guérin (BCG) and Mycobacterium avium, but not in the rapid-growers Mycobacterium smegmatis or Mycobacterium abscessus. RNase protection assays revealed that M. tuberculosis sigF mRNA is not present during exponential-phase growth in M. bovis BCG cultures but is strongly induced during stationary phase, nitrogen depletion, and cold shock. Weak expression of M. tuberculosis sigF was also detected during late-exponential phase, oxidative stress, anaerobiasis, and alcohol shock. The specific expression of M. tuberculosis sigF during stress or stationary phase suggests that it may play a role in the ability of tubercle bacilli to adapt to host defenses and persist during human infection.

Amino Acid Sequence↗
PubMed2002

Solution structure and stability of the anti-sigma factor AsiA: implications for novel functions.

Anti-sigma factors regulate prokaryotic gene expression through interactions with specific sigma factors. The bacteriophage T4 anti-sigma factor AsiA is a molecular switch that both inhibits transcription from bacterial promoters and phage early promoters and promotes transcription at phage middle promoters through its interaction with the primary sigma factor of Escherichia coli, sigma(70). AsiA is an all-helical, symmetric dimer in solution. The solution structure of the AsiA dimer reveals a novel helical fold for the protomer. Furthermore, the AsiA protomer, surprisingly, contains a helix-turn-helix DNA binding motif, predicting a potential new role for AsiA. The AsiA dimer interface includes a substantial hydrophobic component, and results of hydrogen/deuterium exchange studies suggest that the dimer interface is the most stable region of the AsiA dimer. In addition, the residues that form the dimer interface are those that are involved in binding to sigma(70). The results promote a model whereby the AsiA dimer maintains the active hydrophobic surfaces and delivers them to sigma(70), where an AsiA protomer is displaced from the dimer via the interaction of sigma(70) with the same residues in AsiA that constitute the dimer interface.

Binding Sites↗
PubMed1998

The anti-sigma factors.

A mechanism for regulating gene expression at the level of transcription utilizes an antagonist of the sigma transcription factor known as the anti-sigma (anti-sigma) factor. The cytoplasmic class of anti-sigma factors has been well characterized. The class includes AsiA form bacteriophage T4, which inhibits Escherichia coli sigma 70; FlgM, present in both gram-positive and gram-negative bacteria, which inhibits the flagella sigma factor sigma 28; SpoIIAB, which inhibits the sporulation-specific sigma factor, sigma F and sigma G, of Bacillus subtilis; RbsW of B. subtilis, which inhibits stress response sigma factor sigma B; and DnaK, a general regulator of the heat shock response, which in bacteria inhibits the heat shock sigma factor sigma 32. In addition to this class of well-characterized cytoplasmic anti-sigma factors, a new class of homologous, inner-membrane-bound anti-sigma factors has recently been discovered in a variety of eubacteria. This new class of anti-sigma factors regulates the expression of so-called extracytoplasmic functions, and hence is known as the ECF subfamily of anti-sigma factors. The range of cell processes regulated by anti-sigma factors is highly varied and includes bacteriophage phage growth, sporulation, stress response, flagellar biosynthesis, pigment production, ion transport, and virulence.

Bacterial Outer Membrane Proteins↗
PubMed1992

Restoration of motility to an Escherichia coli fliA flagellar mutant by a Bacillus subtilis sigma factor.

The activation of additional promoter sites by production of an alternative sigma subunit for RNA polymerase is a common strategy for the coordinate regulation of gene expression. Many alternative sigma factors control genes for specialized, and often narrowly distributed, functions. For example, most of the alternative sigma factors in Bacillus subtilis control genes necessary for endospore formation. In contrast, the B. subtilis sigma D protein controls the expression of genes important for flagellar-based motility and chemotaxis, a form of locomotion very broadly distributed in the eubacteria. A homologous sigma factor, sigma F, controls a similar group of motility genes in the enteric bacteria. The conservation of both promoter specificity and genetic function in these two regulons allowed us to test the ability of a B. subtilis sigma factor to function within an Escherichia coli host. We demonstrate that expression of the B. subtilis sigD gene restores motility to an E. coli strain mutant in the fliA locus encoding the sigma F factor. This result suggests that the B. subtilis sigma D protein can bind to the E. coli core RNA polymerase to direct transcription initiation from at least four of the late operon promoters, thereby leading to the synthesis of flagellin, motor, and hook-associated proteins. Conversely, expression of sigma D protein in a normally chemotactic strain of E. coli (fliA+) leads to a hyperflagellated, nonchemotactic phenotype.

Bacillus subtilis↗
PubMed1987

Gene encoding the 37,000-dalton minor sigma factor of Bacillus subtilis RNA polymerase: isolation, nucleotide sequence, chromosomal locus, and cryptic function.

We began an analysis of rpoF, the gene encoding the cryptic, 37,000-dalton minor sigma factor (sigma-37) of Bacillus subtilis RNA polymerase. Using antibody raised against sigma-37 holoenzyme to probe a lambda gt11 expression vector library, we isolated a 901-base-pair EcoRI fragment that expressed the COOH-terminal half of sigma-37 fused to lacZ. We used this fragment as a hybridization probe to isolate the entire rpoF gene and additional flanking sequences. Identity of the cloned gene was confirmed by the size and immunological reaction of its product expressed in Escherichia coli and, after DNA sequencing, by the homology of its predicted product (264 residues; 30,143 daltons) with other sigma factors. The DNA sequence also suggested that rpoF may lie in a gene cluster. Upstream of rpoF was an open reading frame that would encode a protein of 17,992 daltons; this frame overlapped the rpoF-coding sequence by 41 base pairs. Immediately following rpoF was a reading frame that would encode a protein of at least 20,000 daltons; expression of this region may be translationally coupled to that of rpoF. By plasmid integration and PBS1 transduction, we found the chromosomal locus of rpoF linked to ddl and dal at 40 degrees on the B. subtilis map and near no known lesions affecting growth regulation or development. Further, an rpoF null mutation resulting from gene disruption had no effect on cell growth or sporulation in rich medium, suggesting that sigma-37 may partly control a regulon not directly involved in the sporulation process.

Amino Acid Sequence↗
PubMed2006

Role of the sigma factor in transcription initiation in the absence of core RNA polymerase.

Sigma factors (sigmas) are bacterial transcription factors that bind core RNA polymerase (RNAP) and direct transcription initiation at cognate promoter sites. However, most of their functions have been investigated in the context of RNAP. This has made the exact function of sigma, and the importance of core RNAP in modulating sigma function, ambiguous. Here we identify a Bacillus subtilis mutant sigma(A) that is independently capable of specific binding and melting of the promoter DNA. Interestingly, specific and independent promoter binding of sigma is sufficient for the temperature- and Mg(2+)-independent melting of promoter DNA around the transcription start site, in contrast to the temperature- and Mg(2+)-dependent melting by RNAP around the promoter -10 element. Thus core RNAP is able to negatively modulate the sigma-initiated melting of the transcription start site and, by sensing the changes in temperature and Mg(2+) concentration, to regulate the efficiency of promoter -10 melting.

Amino Acid Sequence↗
PubMed2002

Identification and structure of the anti-sigma factor-binding domain of the disulphide-stress regulated sigma factor sigma(R) from Streptomyces coelicolor.

The extracytoplasmic function (ECF) sigma factor sigma(R) is a global regulator of redox homeostasis in the antibiotic-producing bacterium Streptomyces coelicolor, with a similar role in other actinomycetes such as Mycobacterium tuberculosis. Normally maintained in an inactive state by its bound anti-sigma factor RsrA, sigma(R) dissociates in response to intracellular disulphide-stress to direct core RNA polymerase to transcribe genes, such as trxBA and trxC that encode the enzymes of the thioredoxin disulphide reductase pathway, that re-establish redox homeostasis. Little is known about where RsrA binds on sigma(R) or how it suppresses sigma(R)-dependent transcriptional activity. Using a combination of proteolysis, surface-enhanced laser desorption ionisation mass spectrometry and pull-down assays we identify an N-terminal, approximately 10kDa domain (sigma(RN)) that encompasses region 2 of sigma(R) that represents the major RsrA binding site. We show that sigma(RN) inhibits transcription by an unrelated sigma factor and that this inhibition is relieved by RsrA binding, reaffirming that region 2 is involved in binding to core RNA polymerase but also demonstrating that the likely mechanism by which RsrA inhibits sigma(R) activity is by blocking this association. We also report the 2.4A resolution crystal structure of sigma(RN) that reveals extensive structural conservation with the equivalent region of sigma(70) from Escherichia coli as well as with the cyclin-box, a domain-fold found in the eukaryotic proteins TFIIB and cyclin A. sigma(RN) has a propensity to aggregate, due to steric complementarity of oppositely charged surfaces on the domain, but this is inhibited by RsrA, an observation that suggests a possible mode of action for RsrA which we compare to other well-studied sigma factor-anti-sigma factor systems.

Bacterial Proteins↗
PubMed2005

RshA mimetic peptides inhibiting the transcription driven by a Mycobacterium tuberculosis sigma factor SigH.

SigH, an alternative sigma factor in Mycobacterium tuberculosis, is a central regulator in responses to the oxidative and heat stress. This SigH activity is specifically controlled by an anti-sigma factor RshA during expression of stress-related genes. Thus, the specific interaction (k(on)=1.15x10(5) (M(-1) s(-1)), k(off)=1.7x10(-3) (s(-1)), KD=15 nM, determined in this study) between SigH and RshA is crucial for the survival and pathogenesis of M. tuberculosis. Using phage-display peptide library, we defined three specific regions on RshA responsible for SigH binding. Three RshA mimetic peptides (DAHADHD, AEVWTLL, and CTPETRE) specifically inhibited the transcription initiated by SigH in vitro. One of them (DAHADHD) diminished the extent of binding of RshA to SigH in a dose-dependent manner. The binding affinity (KD) of this peptide to SigH was about 1.2 microM. These findings might provide some insights into the development of new peptide-based drugs for TB.

Amino Acid Sequence↗
PubMed1994

Cloning and characterization of the gene encoding the major sigma factor of Stigmatella aurantiaca.

The gene (sigA) encoding the major sigma factor of the myxobacterium, Stigmatella aurantiaca, was cloned and sequenced. The deduced polypeptide contains 706 amino acids (aa) and has a deduced M(r) of 79,910. It exhibits four different aa sequence motifs which correlate with the conserved domains of the major sigma factors of Myxococcus xanthus (sigma 80), Escherichia coli (sigma 70) and Bacillus subtilis (sigma 43). The sigma factor (sigma A) was detected in crude lysates of vegetative cells and in cells of different developmental stages from S. aurantiaca with an antiserum to M. xanthus sigma 80 by Western blot analysis. The SigA polypeptide copurified with RNA polymerase from vegetative S. aurantiaca cells. The aa sequence of its N terminus matches a sequence located 25 codons downstream from the proposed start codon. The sigA gene was expressed in E. coli and the corresponding gene product cross-reacted with the SigA antiserum as a polypeptide of 100 kDa, which is identical in size to the sigma A detected in vegetative cells of S. aurantiaca.

Amino Acid Sequence↗
PubMed2002

Novel nuclear-encoded proteins interacting with a plastid sigma factor, Sig1, in Arabidopsis thaliana.

Sigma factor binding proteins are involved in modifying the promoter preferences of the RNA polymerase in bacteria. We found the nuclear encoded protein (SibI) that is transported into chloroplasts and interacts specifically with the region 4 of Sig1 in Arabidopsis. SibI and its homologue, T3K9.5 are novel proteins, which are not homologous to any protein of known function. The expression of sibI was tissue specific, light dependent, and developmentally timed. We suggest the transcriptional regulation by sigma factor binding proteins to function in the plastids of higher plant.

Arabidopsis↗
PubMed1990

A polymerase chain reaction-based approach to cloning sigma factors from eubacteria and its application to the isolation of a sigma-70 homolog from Chlamydia trachomatis.

Taking advantage of the known sequence conservation of portions of bacterial sigma factor proteins, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction (PCR) to amplify DNA sequences from the chlamydial genome. The PCR products were used as a probe to recover the genomic fragments from a library of cloned murine Chlamydia trachomatis DNA. Sequence analysis of one of these clones revealed striking homology to the sigma-70 protein of Escherichia coli and the sigma-43 protein of Bacillus subtilis, strongly implying that this locus (sigA) encodes the major vegetative sigma factor of murine C. trachomatis. This PCR-based approach will be broadly applicable to the cloning of major sigma factors from other eubacteria.

Amino Acid Sequence↗
PubMed1987

Genetic studies of a secondary RNA polymerase sigma factor in Bacillus subtilis.

sigma B (sigma 37) is a secondary species of RNA polymerase sigma factor found in the gram-positive bacterium Bacillus subtilis. To study the function of sigma B genetically, we sought mutations that block the expression of a gene (ctc) known to be transcribed by sigma B-containing RNA polymerase in vitro. One such mutation, called crl, was found to map in or near the structural gene (sigB) for sigma B. To determine directly whether mutations in sigB would prevent transcription of ctc, we replaced sigB in the B. subtilis chromosome with insertion and deletion mutations that disrupted the sigma B coding sequence. Like crl, these in vitro-constructed mutations blocked expression of ctc, but had little or no effect on viability, sporulation, expression of the sporulation gene spoVG, or production of sporulation-associated alkaline protease. Using fusions of ctc to the reporter genes xylE and lacZ, we also identified mutations that enhanced ctc expression. One such mutation, called socB, was found to be located in an open reading frame immediately downstream of sigB.

Bacillus subtilis↗
PubMed1988

Processing of a sporulation sigma factor in Bacillus subtilis: how morphological structure could control gene expression.

Sporulation of Bacillus subtilis is a primitive example of coupling between morphological changes and timing of gene expression during development. A major early control of transcriptional activity is dependent on a new sigma factor, sigma E, which is encoded by the sigE gene and synthesized as an inactive precursor, pro-sigma E. We show that mutations in the spoIIGA gene block the processing of pro-sigma E. Moreover, synthesis of both spoIIGA and sigE products in vegetative cells leads to expression of a sigma E-controlled promoter during growth, suggesting that SpoIIGA has pro-sigma E processing activity. The SpoIIGA polypeptide, which contains five potential transmembrane domains, is synthesized during sporulation 1 hr before processing activity can be detected. We propose that SpoIIGA processing activity is triggered by the presence of the sporulation septum, which is itself dependent on the spoIIAA and spoIIE products. These proteins are normally needed for pro-sigma E processing during sporulation but can be bypassed in vegetative cells. According to this model, a morphological structure would directly control the synthesis of a developmental sigma factor and would modify gene expression.

Amino Acid Sequence↗
PubMed2002

A phylogenomic study of the general stress response sigma factor sigmaB of Bacillus subtilis and its regulatory proteins.

Regulation of expression of the general stress regulon of Bacillus subtilis is mediated by the activation of the alternative sigma factor sigmaB. Activation of sigmaB is accomplished by a complex regulatory network involving protein-protein interactions and reversible protein phosphorylation. PSI-BLAST searches were performed and phylogenetic trees for sigmaB and its regulatory proteins were constructed. Occurrence of sigmaB is restricted to a small group of gram-positive bacteria (Bacillus, Staphylococcus, Listeria). Related sigma factors also involved in stress responses are present in Mycobacterium tuberculosis, Streptomyces species and even in cyanobacteria (Synechocystis species). Putative regulatory proteins found in several other bacterial species can be broadly catagorized into three categories: Anti sigma factors, anti-anti sigma factors and phosphatases. Anti sigma factors are able to bind to sigma factors and are also kinases of anti sigma factor antagonists. Only in their nonphosphorylated state, these antagonists are able to bind to the anti sigma factor. Phosphorylated antagonists can be dephosphorylated by PP2C phosphatases. These phosphatases are of pivotal importance for activation of the sigma factor. Different phosphatases identified in this search contain a wide variety of domains found in signal transducing proteins (PAS/PAC, GAF, REC, HATase_c, HAMP). The HATPase_c domain found in several phosphatases most probably constitutes a serine/threonine kinase domain of anti sigma factors. Such proteins are most probably bifunctional anti-anti sigma factor kinases and phosphatases. The regulatory network of anti-anti sigma factors anti sigma factors and phosphatases is probably ancient and most likely evolved from a structurally similar network found in the Deinococcus radiodurans genome. In completely sequenced genomes of several bacterial species, some elements of the network are missing. The N-terminus of RsbU, a phosphatase activated in response to environmental stress exhibits similarities to a region in the beta chain of phenylalanyl-tRNA synthetases.

Amino Acid Sequence↗