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PubMed1991

Isolation and characterization of the gene encoding the principal sigma factor of the vegetative cell RNA polymerase from the cyanobacterium Anabaena sp. strain PCC 7120.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 responds to combined nitrogen deprivation by forming specialized nitrogen-fixing cells at regular intervals along the filament. Genetic and biochemical studies have indicated that regulation of gene expression during differentiation occurs at the transcriptional level. As part of a characterization of RNA polymerase during differentiation, the gene encoding the 52-kDa principal sigma factor of the Anabaena sp. strain PCC 7120 vegetative-cell RNA polymerase was isolated by using an oligonucleotide probe based on the sequence of the N-terminal seven amino acids of the purified protein. sigA codes for a 390-amino-acid polypeptide that has a predicted molecular weight of 45,641. The amino acid sequence of the polypeptide encoded by sigA contains four regions corresponding to conserved domains of the principal RNA polymerase sigma factors of Escherichia coli (sigma 70) and Bacillus subtilis (sigma 43). Thus, although the subunit composition of cyanobacterial RNA polymerase core differs from that of other eubacteria (G. J. Schneider and R. Haselkorn, J. Bacteriol. 170:4136-4140, 1988), the principal sigma factor of at least one cyanobacterium is typically eubacterial. In contrast to sigma 70 and sigma 43 operon organization, sigA is monocistronic and encodes two transcripts of 1.7 and 2.2 kb. The abundance of the 1.7-kb transcript remains constant under both nitrogen-replete and nitrogen-limiting conditions, whereas the 2.2-kb transcript is induced following the removal of combined nitrogen. Continued or enhanced transcription of sigA under nitrogen starvation conditions is consistent with the observation that the principal RNA polymerase in differentiating cells contains SigA.

Amino Acid Sequence↗
PubMed1996

Regulation of the iron uptake genes in Pseudomonas fluorescens M114 by pseudobactin M114: the pbrA sigma factor gene does not mediate the siderophore regulatory response.

The iron-regulated PbrA sigma factor dictates the production of the siderophore, pseudobactin M114, and its cognate outer membrane receptor, PbuA, in Pseudomonas fluorescens M114. However, the siderophore molecule also has a role in regulating the expression of the siderophore biosynthetic and siderophore receptor genes in P. fluorescens M114. This is based on the fact that beta-galactosidase levels from lacZ fusions of M114 siderophore promoters (biosynthetic and receptor) were reduced in M114 siderophore biosynthetic mutants compared to wild-type M114. Expression of both promoters was increased by the addition of pseudobactin M114 to the growth medium. This effect was widespread and applicable to all but one of the siderophore negative strains of M114 tested. Furthermore, it was demonstrated that transcription of the pbr A sigma factor gene was not reduced in the siderophore biosynthetic mutants. This excludes the possibility that reduced expression of the siderophore biosynthetic and receptor promoters in the siderophore biosynthetic mutants is mediated at the level of expression of the pbr A gene itself. In addition, it was noted that the siderophore regulated response was applicable to promoters with and without the DNA sequence motif, (G/C)CTAAATCCC, which is required for iron-regulated expression of some pseudomonad promoters.

Bacterial Proteins↗
PubMed2003

The FecI extracytoplasmic-function sigma factor of Escherichia coli interacts with the beta' subunit of RNA polymerase.

Transcription of the ferric citrate transport system of Escherichia coli K-12 is mediated by the extracytoplasmic-function (ECF) sigma factor FecI, which is activated by ferric citrate in the growth medium. By using a bacterial two-hybrid system, it was shown in vivo that FecI binds to the beta' subunit of RNA polymerase. The inactive mutant protein FecI(K155E) displayed reduced binding to beta', and small deletions along the entire FecI protein led to total impairment of beta' binding. In vitro, FecI was retained on Ni(2+)-nitrilotriacetic acid agarose loaded with a His-tagged beta'(1-313) fragment and coeluted with beta'(1-313). Binding of FecI to beta' and beta'(1-313) was enhanced by FecR(1-85), which represents the cytoplasmic portion of the FecR protein that transmits the inducing signal across the cytoplasmic membrane. Interaction of FecR with FecI was demonstrated by showing that isolated FecR inhibited degradation of FecI by trypsin. This is the first demonstration of binding of an ECF sigma factor of the FecI type to the beta' subunit of RNA polymerase and of binding being enhanced by the protein that activates the ECF sigma factor.

Bacterial Outer Membrane Proteins↗
PubMed2003

Antagonistic dark/light-induced SigB/SigD, group 2 sigma factors, expression through redox potential and their roles in cyanobacteria.

The expression of group 2 sigma factors is characterized in a cyanobacterium Synechocystis sp. PCC 6803 grown in culture, changing light conditions (white, red and blue light, and darkness), or the presence of drugs (rifampicin, chloramphenicol, DCMU, and DBMIB), and the roles of these sigma factors are elucidated. The expression of dark/light-induced SigB/SigD was accelerated under opposite redox (oxidation/reduction) states in an electron transport chain of photosynthesis. Expression of the dark-induced lrtA and light-induced psbA2/3 transcript was significantly reduced in the sigB and sigD knockout strains, respectively. Abundant amounts of sigB transcript and protein were observed in the sigC knockout strain, implying that SigC represses SigB expression under light. These findings clearly showed that SigB/SigD with another group 2 sigma, SigC, contribute to transcription for a subset of dark/light-responsive genes in the cyanobacterium. A possible model for SigB/SigD is presented and the potential ability for promoter recognition is also discussed.

Bacterial Proteins↗
PubMed1992

On the evolution of the bacterial major sigma factors.

The existence of internal sequence homologies between the N-terminal halves of the gram-negative bacterial major sigma factors and their C-terminal halves, which correspond to minor factors, is reported. In the case of Escherichia-Salmonella sigma-70, an apparent homology was even found between the C-terminal helix-turn-helix DNA-binding motif and the corresponding region of the peptide N half, which, however, is not directly engaged in promoter recognition. It is proposed that major sigma factors may have originated by duplication and fusion of a DNA unit related to the ancestral gene for the whole sigma family. Coevolution of major sigma structures and complex promoters is suggested.

Amino Acid Sequence↗
PubMed1999

The hydrophobic heptad repeat in Region III of Escherichia coli transcription factor sigma 54 is essential for core RNA polymerase binding.

Escherichia coli transcription factor sigma 54 contains motifs that resemble closely those used for RNA polymerase II in mammalian cells, including two hydrophobic heptad repeats, a very acidic region and a glutamine-rich region. Triple changes in hydrophobic or multiple changes in acidic residues in Region III are known to severely impair core-binding ability. To investigate whether all the changes in triple mutants are necessary for core binding, site-directed mutagenesis was performed to create single and double mutants in the leucine or isoleucine residues in the heptad repeat in Region III. Single mutants showed no discernible loss of function. Double mutants showed partial protection of the -12 promoter element of the glnAp2 promoter due to the partial loss of their ability to bind core RNA polymerase. These mutations were deleterious to the function of sigma 54, which retained only 30-40% of wild-type mRNA levels. However, double mutants retained nearly normal ability to form open complexes. Two triple mutants created during previous work lost most, if not all, of their ability to bind core RNA polymerase, to protect the -12 promoter element of the glnAp2 promoter and to open the transcription start site. The two triple mutants produced about 20% or less than 10% of the wild-type transcripts from the glnAp2 promoter. These results demonstrate that the hydrophobic heptad repeat in Region III is essential for core RNA polymerase binding. Progressive loss of hydrophobicity of the hydrophobic heptad repeat in Region III of sigma 54 resulted in a progressive loss of core-binding ability, leading to the loss of -12 promoter element recognition and mRNA production.

Bacterial Proteins↗
PubMed1997

Sequence of the Bacillus subtilis genome region in the vicinity of the lev operon reveals two new extracytoplasmic function RNA polymerase sigma factors SigV and SigZ.

Two regions with sizes 18,900 and 25,400 bp, which join previously known contigs containing levRDEFG, aadK and blt genes near 235 degrees of the Bacillus subtilis chromosome, were sequenced. Among others, two genes, which encode proteins homologous to RNA polymerase sigma-factors, were identified within this region. The gene products designated SigV and SigZ, show the highest homology with sigma-factors encoded by the gene carQ of Myxococcus xanthus and sigX (formerly orfX20) of B. subtilis, correspondingly. All sigma-factors which show statistically significant homology to SigV and SigZ, belong to the ECF (extracytoplasmic functions) subfamily. SigV and SigZ do not have N-terminal sequence which prevents such proteins from binding to DNA without RNA polymerase core enzyme.

Amino Acid Sequence↗
PubMed1998

Specific recognition of the cyanobacterial psbA promoter by RNA polymerases containing principal sigma factors.

The psbA2 gene of a unicellular cyanobacterium, Microcystis aeruginosa K-81, encodes a D1 protein homolog in the reaction center of photosynthetic Photosystem II. To clarify the promoter recognition by a sigma factor of RNA polymerase, in vivo and in vitro analyses were performed for the photosynthetic gene. Although the specific transcript from the psbA2 promoter, whose sequence is of Escherichia coli consensus type, was observed in both cyanobacterium K-81 and E. coli cells, the expression was light-dependent in K-81 whereas it was constitutive in E. coli under the conditions of light and darkness (L/D). The specific psbA2-dependent transcripts were also detected in vitro by RNA polymerases containing the principal sigma factors, E. coli sigma70 and K-81 sigmaA1 (constitutively exists in K-81 grown under L/D cycles). Furthermore, a series of promoter fragments were constructed to confirm minimal cis elements for the in vitro psbA2 transcription. A -80 to +6 or -38 to +46 region, the sequences of which consisted of a core promoter (-38 to +6), was identified as the potential minimal cis element using the RNA polymerase fraction (*EsigmaA1) containing sigmaA1 partially purified from K-81. These results suggest that the psbA2 transcription with the minimal sequence was induced by the RNA polymerase (EsigmaA1) containing the principal sigma factor, sigmaA1, under both light and dark conditions in K-81.

Base Sequence↗
PubMed1994

Genetic and molecular analyses of the interaction between the flagellum-specific sigma and anti-sigma factors in Salmonella typhimurium.

More than 50 genes are required for flagellar formation and function in Salmonella typhimurium. According to the cascade model of flagellar regulon, the flagellar operons are divided into three classes, 1, 2, and 3, with respect to transcriptional hierarchy. FliA is an alternative sigma factor specific for transcription of the class 3 operons, while FlgM is an anti-sigma factor which binds to FliA and prevents its association with RNA polymerase core enzyme. In the present study, we isolated a number of fliA mutants in which the altered FliA proteins become insensitive to inhibition by FlgM. Sequence analysis of their mutation sites revealed that most of them caused the amino acid substitutions in region 4 of the conserved amino acid sequences of sigma factors which lies near the C-terminal end of FliA. Using a set of fliA deletion mutants in a high-expression plasmid, we demonstrated that polypeptides containing the C-terminal portion of FliA could titrate the intracellular FlgM protein resulting in derepression of the class 3 operons. This result indicates that the C-terminal region of FliA contains the FlgM-binding domain. This was confirmed by a chemical cross-linking experiment with FlgM and truncated FliA proteins.

Amino Acid Sequence↗
PubMed2004

Transcription regulation by the Mycobacterium tuberculosis alternative sigma factor SigD and its role in virulence.

Mycobacterium tuberculosis, an obligate mammalian pathogen, adapts to its host during the course of infection via the regulation of gene expression. Of the regulators of transcription that play a role in this response, several alternative sigma factors of M. tuberculosis have been shown to control gene expression in response to stresses, and some of these are required for virulence or persistence in vivo. For this study, we examined the role of the alternative sigma factor SigD in M. tuberculosis gene expression and virulence. Using microarray analysis, we identified several genes whose expression was altered in a strain with a sigD deletion. A small number of these genes, including sigD itself, the gene encoding the autocrine growth factor RpfC, and a gene of unknown function, Rv1815, appear to be directly regulated by this sigma factor. By identifying the in vivo promoters of these genes, we have determined a consensus promoter sequence that is putatively recognized by SigD. The expression of several genes encoding PE-PGRS proteins, part of a large family of related genes of unknown function, was significantly increased in the sigD mutant. We found that the expression of sigD is stable throughout log phase and stationary phase but that it declines rapidly with oxygen depletion. In a mouse infection model, the sigD mutant strain was attenuated, with differences in survival and the inflammatory response in the lung between mice infected with the mutant and those infected with the wild type.

Anaerobiosis↗
PubMed1999

An intrinsic DNA curvature found in the cyanobacterium Microcystis aeruginosa K-81 affects the promoter activity of rpoD1 encoding a principal sigma factor.

The rpoD1 gene in the unicellular cyanobacterium Microcystis aeruginosa K-81 encodes a principal sigma factor of RNA polymerase and is transcribed under light and dark conditions to produce multiple monocistronic transcripts. In the 5'-upstream region from rpoD1 Promoter 2, which has a sequence of Escherichia coli type, we found a sequence-directed DNA curvature with an AT-rich sequence. Insertions of 2 to 21 base pairs introduced into the curved center changed a gross geometry of the original curved DNA structure. The rpoD1 promoter activities assayed in vivo by using transcriptional lacZ fusions were correlated with the change in the gross geometry in not only a cyanobacterium but also E. coli. In addition, RNA polymerase binding to the rpoD1 promoter region and the efficiency of the mRNA synthesis from the rpoD1 Promoter 2 were also affected in vitro by the change in the geometry. These results suggest that the tertiary structure of the curved DNA is important for the rpoD1 transcription. The deletion of the center region of the curvature resulted in a considerable reduction of the transcription from Promoter 2 in the cyanobacterium. This report demonstrates that a curved DNA plays a significant role in transcription in cyanobacteria, and that this functional curvature is located in the 5'-upstream region from the rpoD gene, which encodes a principal sigma factor in eubacteria.

Bacterial Proteins↗
PubMed2004

Sporulation phenotype of a Bacillus subtilis mutant expressing an unprocessable but active sigmaE transcription factor.

SigmaE, a sporulation-specific sigma factor of Bacillus subtilis, is formed from an inactive precursor (pro-sigmaE) by a developmentally regulated processing reaction that removes 27 amino acids from the proprotein's amino terminus. A sigE variant (sigE335) lacking 15 amino acids of the prosequence is not processed into mature sigmaE but is active without processing. In the present work, we investigated the sporulation defect in sigE335-expressing B. subtilis, asking whether it is the bypass of proprotein processing or a residual inhibition of sigmaE activity that is responsible. Fluorescence microscopy demonstrated that sigE335-expressing B. subtilis progresses further into sporulation (stage III) than do strains lacking sigmaE activity (stage II). Consistent with its stage III phenotype, and a defect in sigmaE activity rather than its timing, the sigE335 allele did not disturb early sporulation gene expression but did inhibit the expression of late sporulation genes (gerE and sspE). The Spo- phenotype of sigE335 was found to be recessive to wild-type sigE. In vivo assays of sigmaE activity in sigE, sigE335, and merodiploid strains indicate that the residual prosequence on sigmaE335, still impairs its activity to function as a transcription factor. The data suggest that the 11-amino-acid extension on sigmaE335 allows it to bind RNA polymerase and direct the resulting holoenzyme to sigmaE-dependent promoters but reduces the enzyme's ability to initiate transcription initiation and/or exit from the promoter.

Bacillus subtilis↗
PubMed2006

Identification of mycobacterial sigma factor binding sites by chromatin immunoprecipitation assays.

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for infections that cause a substantial amount of death, suffering, and loss around the world. Still, relatively little is known about the mechanisms of gene expression in these bacteria. Here, we used genome-wide location assays to identify direct target genes for mycobacterial sigma factors. Chromatin immunoprecipitation assays were performed with M. bovis BCG for Myc-tagged proteins expressed using an anhydrotetracycline-inducible promoter, and enriched DNA fragments were hybridized to a microarray representing intergenic regions from the M. tuberculosis H37Rv genome. Several putative target genes were validated by quantitative PCR. The corresponding transcriptional start sites were identified for sigma(F), sigma(C), and sigma(K), and consensus promoter sequences are proposed. Our conclusions were supported by the results of in vitro transcription assays. We also examined the role of each holoenzyme in the expression of sigma factor genes. Our results revealed that many sigma factors are expressed from autoregulated promoters.

Bacterial Proteins↗
PubMed2006

Identification and characterization of the gene encoding the Acidobacterium capsulatum major sigma factor.

Acidobacterium capsulatum is an acid-tolerant, encapsulated, Gram-negative member of the ubiquitous, but poorly understood Acidobacteria phylum. Little is known about the genetics and regulatory mechanisms of A. capsulatum. To begin to address this gap, we identified the gene encoding the A. capsulatum major sigma factor, rpoD, which encodes a 597-amino acid protein with a predicted sequence highly similar to the major sigma factors of Solibacter usitatus Ellin6076 and Geobacter sulfurreducens PCA. Purified hexahistidine-tagged RpoD migrates at approximately 70 kDa under SDS-PAGE conditions, which is consistent with the predicted MW of 69.2 kDa, and the gene product is immunoreactive with monoclonal antibodies specific for either bacterial RpoD proteins or the N-terminal histidine tag. A. capsulatum RpoD restored normal growth to E. coli strain CAG20153 under conditions that prevent expression of the endogenous rpoD. These results indicate we have cloned the gene encoding the A. capsulatum major sigma factor and the gene product is active in E. coli.

Amino Acid Sequence↗
PubMed2000

Three new nuclear genes, sigD, sigE and sigF, encoding putative plastid RNA polymerase sigma factors in Aarabidopsis thaliana.

Three new nuclear genes (sigD, sigE and sigF) of Arabidopsis thaliana, encoding putative plastid RNA polymerase sigma factors, were identified and analyzed. Phylogenetic analysis revealed that higher plant sigma factors fell into at least four distinct subgroups within a diverse protein family. In addition, Arabidopsis sig genes contained conserved chromosomal intron sites, indicating that these genes arose by DNA duplication events during plant evolution. Transcript analyses revealed two alternatively spliced transcripts generated from the sigD region, one of which is predicted to encode a sigma protein lacking the carboxy-terminal regions 3 and 4. Finally, the amino-terminal sequence of the sigF gene product was shown to function as a plastid-targeting signal using green fluorescent protein fusions.

Alternative Splicing↗
PubMed2000

Membrane-to-cytosol redistribution of ECF sigma factor AlgU and conversion to mucoidy in Pseudomonas aeruginosa isolates from cystic fibrosis patients.

The conversion to mucoid phenotype in Pseudomonas aeruginosa during chronic infections in cystic fibrosis (CF) is due to mutations in the algU mucABCD gene cluster. This cluster encodes an extreme stress response system conserved in Gram-negative bacteria. The system includes an ECF sigma factor, AlgU (sigmaE), an inner membrane protein, MucA, which inhibits AlgU activity, and MucB, a periplasmic protein that negatively controls AlgU. In this work, we investigated whether and how these factor interact to transduce signals between different cellular compartments. The mutation mucADeltaG440, which renders a large fraction of P. aeruginosa CF isolates mucoid, did not abrogate AlgU-MucA interactions, although it eliminated MucA-MucB interactions in the yeast two-hybrid system. The mucADeltaG440 truncation of the periplasmic C-terminal tail of MucA destabilized the molecule resulting in low or undetectable steady-state levels in P. aeruginosa. Somewhat reduced levels of MucA were also seen in cells with inactivated mucB or with the mucACF53 allele carrying the missense P184S mutation, which mildly affected interactions with MucB. The events downstream from MucA destabilization were also investigated. AlgU was found to associate with inner membranes in mucA+ cells. In mutants destabilizing MucA, a limited redistribution of AlgU from the membrane to the cytosol was observed. The redistribution was spontaneous in mucADeltaG440 cells, while in mucB and mucACF53 mutants it required additional signals. Despite a large reduction in MucA levels in mucADeltaG440 cells, only a small fraction of AlgU was redistributed to the cytosol and a significant portion of this sigma factor remained membrane bound and behaved as a peripheral inner membrane protein. The fraction of AlgU that depended on MucA for association with the membrane also brought RNA polymerase into this compartment. These results are consistent with a model in which MucB-MucA-AlgU-RNA polymerase interactions at the membrane allow transduction of potentially lethal stress signals with both rapid reaction times of the preassembled complexes and efficient resupply at the membrane from the prebound components.

Alleles↗
PubMed2005

The Mycobacterium tuberculosis SigD sigma factor controls the expression of ribosome-associated gene products in stationary phase and is required for full virulence.

During infection Mycobacterium tuberculosis is exposed to several environmental conditions depending on the stage and severity of the disease. To survive, M. tuberculosis uses alternate sigma factors to regulate its gene expression in response to the changing host environment. In order to better understand the way in which stress response genes are regulated, the extracytoplasmic sigma factor gene sigD was deleted and subsequently complemented in the CDC1551 strain of M. tuberculosis. The DeltasigD mutant strain exhibited an in vitro growth rate in rich medium identical to that of both the sigD-complemented and wild-type CDC1551 strains. Additionally, no differences were observed in short-term intracellular growth between the mutant, complemented, and wild-type bacteria within the J774A.1 macrophage cell line. However, tumour necrosis factor (TNF)-alpha levels in macrophages infected with the DeltasigD mutant were decreased as compared to levels observed in macrophages infected with the wild-type bacteria. In time-to-death studies, C3H mice infected with the DeltasigD mutant exhibited a mortality delay compared to those infected with either the complemented or wild-type strains. Although mice infected with the DeltasigD mutant died at a reduced rate, the bacillary loads in the lungs and spleen of these mice were comparable to those seen in mice infected with either the complemented or wild-type strains. Microarray analysis of the DeltasigD mutant relative to wild type revealed that SigD directs the expression of a small set of ribosomal genes and adenosine triphosphate transporters whose expression is normally induced during stationary phase growth in vitro. Altered expression of a subset of these genes was confirmed by quantitative reverse transcription polymerase chain reaction analysis. Promoter-like elements resembling the consensus sequence AGAAAG-N16-20-CGTTAA were found upstream of 19 of the genes underexpressed in the DeltasigD mutant suggesting this may be the recognition sequence for the M. tuberculosis SigD-holoenzyme, EsigmaD. These data indicate that the M. tuberculosis SigD sigma factor governs the expression of a small set of ribosomal genes typically expressed in stationary phase during in vitro growth and that loss of sigD reduces macrophage TNF-alpha secretion as well as the lethality of M. tuberculosis infection in mice.

Adaptation, Physiological↗
PubMed1999

Influence of a putative ECF sigma factor on expression of the major outer membrane protein, OprF, in Pseudomonas aeruginosa and Pseudomonas fluorescens.

The gene encoding OprF, a major outer membrane protein in Pseudomonas species (formerly known as type 1 pseudomonads), was thought to be constitutively transcribed from a single sigma 70 promoter immediately upstream of the gene. We now report the identification of a novel putative ECF (extracytoplasmic function) sigma factor gene, sigX, located immediately upstream of oprF in both Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens OE 28.3 and show that disruption of this gene significantly reduces OprF expression. In P. aeruginosa, Northern analysis demonstrated that this reduction was a result of an effect on transcription of monocistronic oprF combined with a polar effect due to termination of a transcript containing sigX and oprF. Comparison of sigX-disrupted and wild-type cell transcripts by primer extension indicated that monocistronic transcription of oprF occurs from two overlapping promoters, one that is SigX-dependent and resembles ECF sigma factor promoters in its minus-35 region and another promoter that is independent of SigX and is analogous to the sigma 70-type promoter previously reported. Complementation of the P. aeruginosa sigX-disrupted mutant with plasmid-encoded OprF did not resolve the phenotypes associated with this mutant, which included a markedly reduced logarithmic-phase growth rate in rich medium (compared to that in minimal medium), further reduction of the growth rate in a low-osmolarity environment, secretion of an unidentified pigment, and increased sensitivity to the antibiotic imipenem. This indicates that SigX is involved in the regulation of other genes in P. aeruginosa. Disruption of the sigX gene in P. fluorescens also had an effect on the logarithmic-phase growth rate in rich medium. A conserved sigX gene was also identified in a Pseudomonas syringae isolate and six P. aeruginosa clinical isolates. Collectively, these data indicate that an ECF sigma factor plays a role in the regulation and expression of OprF and also affects other genes.

Bacterial Proteins↗