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PubMed1998

Sequence and expression characteristics of a nuclear-encoded chloroplast sigma factor from mustard (Sinapis alba).

Plant chloroplasts contain transcription factors that functionally resemble bacterial sigma factors. We have cloned the full-length cDNA from mustard (Sinapis alba) for a 53 kDa derived polypeptide that contains similarity to regions 1.2-4.2 of sigma70-type factors. The amino acid sequence at the N-terminus has characteristics of a chloroplast transit peptide. An in vitro synthesized polypeptide containing this region was shown to be imported into the chloroplast and processed. The recombinant factor lacking the N-terminal extension was expressed in Escherichia coli and purified. It confers the ability on E.coli core RNA polymerase to bind specifically to a DNA fragment that contains the chloroplast psbA promoter. Transcription of the psbA template by E.coli core enzyme in the presence of recombinant SIG1 results in enhanced formation of transcripts of the size expected for correct initiation at the in vivo start site. Together, these data suggest that the mature protein acts as one of the chloroplast transcription factors in mustard. RNA gel blot hybridization reveals a transcript at approximately 1.8 kb, which is more abundant in light-grown than in dark-grown mustard seedlings.

Amino Acid Sequence↗
PubMed2000

Characterization of an ECF sigma factor protein from Pseudomonas aeruginosa.

The PvdS protein is essential for synthesis of the siderophore pyoverdine by Pseudomonas aeruginosa. PvdS has some sequence similarity to a family of alternative sigma factor proteins (the ECF [extracytoplasmic factor] family) that direct bacterial RNA polymerases to transcribe genes encoding extracytoplasmic factors. PvdS was purified as a His-tagged protein (hPvdS) and this was used to test the hypothesis that PvdS is a sigma factor protein. The purified protein caused core RNA polymerase from Escherichia coli to bind specifically to the promoters of pyoverdine synthesis genes and enabled transcription from these promoters in vitro. In addition, PvdS was found to co-purify with RNA polymerase from P. aeruginosa, indicating that PvdS associates with RNA polymerase inside the bacteria. These results show that PvdS is a sigma factor protein.

Bacterial Proteins↗
PubMed2001

Heme utilization in Bordetella avium is regulated by RhuI, a heme-responsive extracytoplasmic function sigma factor.

Efficient utilization of heme as an iron (Fe) source by Bordetella avium requires bhuR, an Fe-regulated gene which encodes an outer membrane heme receptor. Upstream of bhuR is a 507-bp open reading frame, hereby designated rhuI (for regulator of heme uptake), which codes for a 19-kDa polypeptide. Whereas the 19-kDa polypeptide had homology to a subfamily of alternative sigma factors known as the extracytoplasmic function (ECF) sigma factors, it was hypothesized that rhuI encoded a potential in-trans regulator of the heme receptor gene in trans. Support for the model was strengthened by the identification of nucleotide sequences common to ECF sigma-dependent promoters in the region immediately upstream of bhuR. Experimental evidence for the regulatory activities of rhuI was first revealed by recombinant experiments in which overproduction of rhuI was correlated with a dramatically increased expression of BhuR. A putative rhuI-dependent bhuR promoter was identified in the 199-bp region located proximal to bhuR. When a transcriptional fusion of the 199-bp region and a promoterless lacZ gene was introduced into Escherichia coli, promoter activity was evident, but only when rhuI was coexpressed in the cell. Sigma competition experiments in E. coli demonstrated that rhuI conferred biological properties on the cell that were consistent with RhuI having sigma factor activity. Heme, hemoglobin, and several other heme-containing proteins were shown to be the extracellular inducers of the rhuI-dependent regulatory system. Fur titration assays indicated that expression of rhuI was probably Fur dependent.

Bacterial Outer Membrane Proteins↗
PubMed1997

The alternative sigma factor sigma28 of Legionella pneumophila restores flagellation and motility to an Escherichia coli fliA mutant.

Gene expression in Legionella pneumophila, the etiological agent of Legionnaires' disease, can be controlled by alternative forms of RNA polymerase programmed by distinct sigma factors. To understand the regulation of L. pneumophila flagellin expression, we cloned the sigma factor (FliA) of RNA polymerase responsible for the transcription of the flagellin gene, flaA. FliA is a member of the sigma28 class of alternative sigma factors identified in several bacterial genera. The gene fliA has been isolated from an expression library of L. pneumophila isolate Corby in Escherichia coli K-12. This library was transformed into a fliA mutant of E. coli K-12 containing a plasmid carrying the L. pneumophila-specific flaA promoter fused to the reporter gene luxAB. Screening the obtained transformants for luciferase activity, we isolated the major part of the fliA gene on a 1.64-kb fragment. This fragment was sequenced and used for reverse PCR in order to recover the complete fliA gene. The resulting 1.03-kb fragment was shown to contain the entire fliA gene. L. pneumophila FliA has 55 and 43% amino acid identity with the homologous sequences of Pseudomonas aeruginosa and E. coli. Furthermore, the L. pneumophila fliA gene was able to restore the flagellation and the motility defect of an E. coli fliA mutant. This result suggests that the L. pneumophila sigma28 protein can bind to the E. coli core RNA polymerase to direct transcription initiation from the flaA-specific promoter.

Amino Acid Sequence↗
PubMed1996

Isolation and characterization of csbB, a gene controlled by Bacillus subtilis general stress transcription factor sigma B.

In the bacterium Bacillus subtilis (Bs), the alternative transcription factor sigma B is activated by environmental stresses to control the expression of a large set of unlinked genes. However, the range of physiological functions mediated by these sigma B-controlled genes is presently unknown. We report here that the newly identified gene csbB is under the dual control of a sigma B-dependent and a sigma B-independent promoter. The predicted product of csbB is a 329 residue protein containing two potential membrane-spanning segments in its C-terminal region, leading us to speculate that one class of sigma B-controlled genes acts to modify the cell envelope as part of the general stress response.

Amino Acid Sequence↗
PubMed2004

Physical evidence for the induced release of the Bacillus subtilis transcription factor, sigma(F), from its inhibitory complex.

The release of the transcription factor sigma(F) from its inhibitory complex with SpoIIAB is a key regulatory step in the control of sporulation in Bacillus subtilis as it initiates a pattern of differential gene expression in the mother cell and prespore compartments. The sigma(F).SpoIIAB complex is dissociated by the unphosphorylated form of the protein SpoIIAA, the alternative binding partner of SpoIIAB. Here, we employ fluorescence spectroscopy to examine the mechanism by which SpoIIAA acts on the sigma(F).SpoIIAB complex. We constructed a mutant of sigma(F), sigma(F)-W46L, which displayed a reproducible fluorescence response on binding to SpoIIAB. Using this mutant we were able to quantify the amount of sigma(F) bound to SpoIIAB in real time. The results provide physical evidence for the "induced release" mechanism of sigma(F) activation. We demonstrate that SpoIIAA interacts directly with the sigma(F).SpoIIAB complex, greatly decreasing the affinity of SpoIIAB for sigma(F) and thus causing the release of the latter. We also demonstrate that sigma(F) is released before SpoIIAA is phosphorylated and that release occurs on a similar time scale to the binding of SpoIIAA to SpoIIAB.

Bacillus subtilis↗
PubMed2000

Stress triggers a process that limits activation of the Bacillus subtilis stress transcription factor sigma(B).

Stress-induced activation of the Bacillus subtilis transcription factor sigma(B) is transitory. To determine whether the process that limits sigma(B) activation is itself triggered by stress, B. subtilis strains in which the stress pathway was artificially activated by the induced expression of a positive regulatory protein (RsbT) were exposed to ethanol stress and were monitored for the persistence of sigma(B) activity. Without ethanol treatment, the induced cultures displayed continuously high sigma(B) activity. Ethanol treatment restricted ongoing sigma(B) activity, but only in strains with intact rsbX and -S genes. The loss of other gene products (RsbR and Obg) known to participate in the stress activation pathway had little influence in blocking the ethanol effect. The data argue that stress upregulates the activity of the RsbX-S regulatory pair to restrict sigma(B) induction following stress.

Adult↗
PubMed1994

Mucoid-to-nonmucoid conversion in alginate-producing Pseudomonas aeruginosa often results from spontaneous mutations in algT, encoding a putative alternate sigma factor, and shows evidence for autoregulation.

The mucoid phenotype is common among strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis and is due to overproduction of an exopolysaccharide called alginate. However, the mucoid phenotype is unstable in vitro, especially when the cells are incubated under low oxygen tension. Spontaneous conversion to the nonmucoid form is typically due to mutations (previously called algS) that are closely linked to the alginate regulatory gene algT, located at 68 min on the chromosome. Our sequence analysis of algT showed that its 22-kDa gene product shares homology with several alternate sigma factors in bacteria, suggesting that AlgT (also known as AlgU) interacts directly with RNA polymerase core to activate the promoters of alginate genes. AlgT showed striking sequence similarity (79%) to sigma E of Escherichia coli, an alternate sigma factor involved in high-temperature gene expression. Our analysis of the molecular basis for spontaneous conversion from mucoid to nonmucoid, in the cystic fibrosis isolate FRD, revealed that nonmucoid conversion was often due to one of two distinct missense mutations in algT that occurred at codons 18 and 29. RNase protection assays showed that spontaneous nonmucoid strains with the algT18 and algT29 alleles have a four- to fivefold reduction in the accumulation of algT transcripts compared with the wild-type mucoid strain. Likewise, a plasmid-borne algT-cat transcriptional fusion was about 3-fold less active in the algT18 and algT29 backgrounds compared with the mucoid wild-type strain, and it was 20-fold less active in an algT::Tn501 background. These data indicate that algT is autoregulated. The spontaneous algT missense alleles also caused about fivefold-reduced expression of the adjacent negative regulator, algN (also known as mucB). Transcripts of algN were essentially absent in the algT::Tn501 strain. Thus, algT regulates the algTN cluster, and the two genes may be cotranscribed. A primer extension analysis showed that algT transcription starts 54 bp upstream of the start of translation. Although the algT promoter showed little similarity to promoters recognized by the vegetative sigma factor, it was similar to the algR promoter. This finding suggests that AlgT may function as a sigma factor to activate its own promoter and those of other alginate genes. The primer extension analysis also showed that algT transcripts were readily detectable in the typical nonmucoid strain PAO1, which was in contrast to a weak signal seen in the algT18 mutant of FRD. A plasmid-borne algT gene in PAO1 resulted in both the mucoid phenotype and high levels of algT transcripts, further supporting the hypothesis that AlgT controls its own gene expression and expression of genes of the alginate regulon.

Alginates↗
PubMed2006

YpdC determines site-1 degradation in regulated intramembrane proteolysis of the RsiW anti-sigma factor of Bacillus subtilis.

Genes of Bacillus subtilis controlled by the alternative extracytoplasmic function family sigma factor sigmaW constitute an antibiosis regulon. Its activity is modulated by RsiW, a transmembrane anti-sigma factor that sequesters and inactivates sigmaW. Upon a stress signal, RsiW is degraded by a mechanism of regulated intramembrane proteolysis. To identify genes which influence RsiW degradation, a transposon screen with a reporter fusion of the green fluorescent protein to RsiW was performed. Among several gene loci identified, the ypdC (prsW) gene displayed a strong effect on RsiW stability. In a ypdC null mutant, induction of sigmaW-controlled genes is abolished and site-1 proteolysis of RsiW is completely blocked. Transcriptional analysis revealed that ypdC is a monocistronic gene, and the defect of sigmaW induction of the null mutant was complemented by ectopically integrated ypdC under xylose control. Orthologues of YpdC can be found in a variety of different bacteria. Its membrane topology was analysed by alkaline phosphatase fusions, revealing that YpdC contains five transmembrane segments and two larger extracytoplasmic loops. In the first loop, two invariantly conserved glutamate residues can be found. In an Escherichia coli system, the cloned ypdC is the only determinant of efficient degradation of RsiW; however, YpdC does not display plain similarities to known proteases, suggesting that it either controls the activity of site-1 proteolysis of RsiW or represents a new type of protease.

Bacillus subtilis↗
PubMed2006

The sigma factors of Mycobacterium tuberculosis.

Mycobacterium tuberculosis is a remarkable pathogen capable of adapting and surviving in various harsh conditions. Correct gene expression regulation is essential for the success of this process. The reversible association of different sigma factors is a common mechanism for reprogramming bacterial RNA polymerase and modulating the transcription of numerous genes. Thirteen putative sigma factors are encoded in the M. tuberculosis genome, several being important for virulence. Here, we analyse the latest information available on mycobacterial sigma factors and discuss their roles in the physiology and virulence of M. tuberculosis.

DNA-Directed RNA Polymerases↗
PubMed1995

Transcription of the region encoding the ferric dicitrate-transport system in Escherichia coli: similarity between promoters for fecA and for extracytoplasmic function sigma factors.

We have analyzed the molecular mechanism of regulation of the ferric dicitrate transport system in Escherichia coli (Ec), by studying the transcription of the regulatory and structural genes under various environmental conditions, and by determining the location of their transcriptional start points and promoter regions. We report here that the main species observed in Northern hybridization analyses were a 2.5-kb mRNA, encoded by the outer membrane protein receptor gene fecA, and a 1.5-kb mRNA encoded by a region including the fecIR genes. The synthesis of the 2.5-kb fecA mRNA is regulated by both citrate and iron. Furthermore, transcription of fecA is dependent on the presence of FecI. The promoter region for the fecA mRNA, a likely site of action for FecI, is not related to the consensus promoter region for sigma 70 RNA polymerase in Ec K-12. However, it shows greatest similarity with promoters of genes regulated by a new sub-family of sigma factors, i.e., the extracytoplasmic function (ECF) sigma factors, which are associated with the expression of genes involved in extracytoplasmic functions, suggesting that FecI may act as a specialized sigma factor. We also show that the fecB,C,D,E transport genes are linked in operon fashion to fecA. Since the levels of the fecB,C,D,E RNAs are extremely low, as compared to the level of fecA mRNA, it is likely that processing from the 3' end must occur and stop near the end of fecA where a hairpin structure is located.

Base Sequence↗
PubMed1999

The Streptomyces aureofaciens homologue of the sporulation gene whiH is dependent on rpoZ-encoded sigma factor.

Using the method for the identification of promoters recognized by a sporulation specific sigma factor RpoZ, we identified a promoter in Streptomyces aureofaciens, directing expression of a gene having high sequence similarity (83% amino acid identity) to sporulation transcription factor WhiH of Streptomyces coelicolor. High-resolution S1-nuclease mapping using RNA prepared from S. aureofaciens from various developmental stages showed high similarity of PwhiH promoter to the consensus sequence of flagellar and chemotaxis promoters. The promoter was induced at the time of aerial mycelium formation, and was off in S. aureofaciens strain with rpoZ-disrupted gene. The results suggest that the PwhiH promoter is recognized by sigma factor RpoZ in S. aureofaciens.

Amino Acid Sequence↗
PubMed1990

Role of eukaryotic-type functional domains found in the prokaryotic enhancer receptor factor sigma 54.

E. coli sigma 54 protein confers on promoters containing its recognition sequence the ability to be activated from distant DNA sites. Its functional domains include two leucine zipper motifs, an acidic region, and a glutamine-rich domain. Several domains were disrupted and the assembly of mutant transcription complexes was probed in vivo by footprinting. Promoter recognition was seen to depend on a C-terminal region containing a prokaryotic helix-turn-helix motif. Within the resulting stable closed complex, two leucine zipper motifs assist in positioning the sigma 54 polymerase near the DNA region that must be melted upon activation. Finally, DNA opening depends on the sigma 54 acid domain. The uncoupling of promoter recognition from DNA melting, mediated by the unusual domain structure of this prokaryotic protein, may be responsible for sigma 54,s ability to mediate activation from distant sites.

Amino Acid Sequence↗
PubMed1992

Polypeptides containing highly conserved regions of transcription initiation factor sigma 70 exhibit specificity of binding to promoter DNA.

The sigma 70 subunit of E. coli RNA polymerase is required for sequence-specific recognition of promoter DNA. Genetic studies and sequence analysis have indicated that sigma 70 contains two specific DNA-binding domains that recognize the two conserved portions of the prokaryotic promoter. However, intact sigma 70 does not bind to DNA. Using C-terminal and internal polypeptides of sigma 70, carrying one or both putative DNA-binding domains, we demonstrate that sigma 70 does contain two DNA-binding domains, but that N-terminal sequences inhibit the ability of intact sigma 70 to bind to DNA. Thus, we propose that sigma 70 is a sequence-specific DNA-binding protein that normally functions through an allosteric interaction with the core subunits of RNA polymerase.

Bacterial Proteins↗
PubMed1980

Regulation of ribonucleic acid polymerase synthesis during restriction of an Escherichia coli mutant temperature sensitive for transcription factor sigma.

An Escherichia coli mutant temperature sensitive for the sigma subunit of ribonucleic acid polymerase was shifted to restrictive temperatures. In response to these restrictions the transcription of rpoBC increased markedly, and the synthesis rates of the beta and beta' subunits of ribonucleic acid polymerase increased. The ratio of the beta and beta' synthesis rates (beta/beta') decreased.

DNA-Directed RNA Polymerases↗
PubMed1994

Functional roles for the glutamines within the glutamine-rich region of the transcription factor sigma 54.

Single and multiple point mutations were introduced to change the 12 glutamine residues within a 37-amino acid region of sigma 54. Multiple changes are shown to be required in order to interfere significantly with the function of this protein which is associated with enhancer-dependent bacterial transcription. Mutation of the central 4 glutamines leads to the production of less m-RNA, caused by an inability to fully open the promoter start site. DNA binding, however, is normal. Mutation of 4 other adjacent glutamines causes the promoter start site to open more readily than wild type, although this enhanced opening is not accompanied by more mRNA. The enhanced DNA melting is not caused by enhanced promoter binding, as indicated by normal protection of the polymerase-bound promoter against dimethyl sulfate attack. The results suggest that multiple glutamines play a role in transducing the melting signal from the enhancer protein to the polymerase.

Amino Acid Sequence↗
PubMed2001

Cloning and characterization of the cDNA for a plastid sigma factor from the moss Physcomitrella patens.

We isolated a cDNA PpSig1 encoding a plastid sigma factor from the moss Physcomitrella patens. The PpSIG1 protein is composed of the conserved subdomains for recognition of -10 and -35 promoter elements, core complex binding and DNA melting. Southern blot analysis showed that the moss sig1 gene is likely a member of a small gene family. Transient expression assay using green fluorescent protein demonstrated that the N-terminal region of PpSIG1 functions as a chloroplast-targeting signal peptide. These observations suggest that multiple nuclear-encoded sigma factors regulate chloroplast gene expression in P. patens.

Amino Acid Sequence↗
PubMed1996

Transcriptional regulation of the iron-responsive sigma factor gene pbrA.

In response to the intracellular iron concentration Pseudomonas fluorescens M114 coordinately regulates the production of pseudobactin M114, its cognate receptor PbuA, and a casein protease. Transcriptional initiation of this coordinate iron-stress response requires the sigma factor PbrA. PbrA is a member of the ECF (Extracytoplasmic function) subgroup of the sigma 70 family of eubacterial RNA polymerase sigma factors. Regulatory studies of the pbrA gene utilising promoter-lacZ transcriptional fusions demonstrate that expression of pbrA dictates the cellular response to iron. pbrA is transcribed in all phases of iron-limited growth but maximally at late-logarithmic to stationary phase. pbrA expression is independent of autoregulatory control but is strictly repressed in iron-rich conditions in a Fur-dependent fashion. Constitutive expression of pbrA from an inducible tac promoter permits the induction of PbrA-dependent transcription and pseudobactin M114 biosynthesis in high-iron conditions. A PbrA consensus sequences was derived from significant DNA sequence homologies observed within the "-25 bp" and "-16 bp" regions conserved among all PbrA-dependent promoters. The predicted PbrA target promoter consensus is homologous for the promoter recognition sites for other environmentally responsive ECF sigma factors.

Amino Acid Sequence↗