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PubMed2002

Autoregulation of a bacterial sigma factor explored by using segmental isotopic labeling and NMR.

Bacterial sigma factors combine with the catalytic core RNA polymerase to direct the process of transcription initiation through sequence-specific interactions with the -10 and -35 elements of promoter DNA. In the absence of core RNA polymerase, the DNA-binding function of sigma is autoinhibited by its own N-terminal 90 amino acids (region 1.1), putatively by a direct interaction with conserved region 4.2, which binds the -35 promoter element. In the present work, this mechanism of autoinhibition was studied by using a combination of NMR spectroscopy and segmental isotopic labeling of a sigma70-like subunit from Thermotoga maritima. Our data argue strongly against a high-affinity interaction between these two domains. Instead we suggest that autoinhibition of DNA binding occurs through an indirect steric and/or electrostatic mechanism. More generally, the present work illustrates the power of segmental isotopic labeling for probing molecular interactions in large proteins by NMR.

Amino Acid Sequence↗
PubMed2001

Periplasmic stress and ECF sigma factors.

Envelope stress responses play important physiological roles in a variety of processes, including protein folding, cell wall biosynthesis, and pathogenesis. Many of these responses are controlled by extracytoplasmic function (ECF) sigma factors that respond to external signals by means of a membrane-localized anti-sigma factor. One of the best-characterized, ECF-regulated responses is the sigma(E) envelope stress response of Escherichia coli. The sigma(E) pathway ensures proper assembly of outer-membrane proteins (OMP) by controlling expression of genes involved in OMP folding and degradation in response to envelope stresses that disrupt these processes. Prevailing evidence suggests that, in E. coli, a second envelope stress response controlled by the Cpx two-component system ensures proper pilus assembly. The sensor kinase CpxA recognizes misfolded periplasmic proteins, such as those generated during pilus assembly, and transduces this signal to the response regulator CpxR through conserved phosphotransfer reactions. Phosphorylated CpxR activates transcription of periplasmic factors necessary for pilus assembly.

Bacterial Outer Membrane Proteins↗
PubMed2004

Efficient regulation of sigmaF, the first sporulation-specific sigma factor in B.subtilis.

Differential gene expression is established in the prespore and mother-cell compartments of Bacillus subtilis through the successive activation of a series of cell-type-specific sigma factors. Crucial to the success of this process is the control of the first prespore-specific sigma factor, sigmaF. sigmaF is regulated by the proteins SpoIIAB, SpoIIAA and SpoIIE. SpoIIAB forms an inhibitory complex with sigmaF, which can be dissociated by interaction with SpoIIAA. During this interaction SpoIIAA is phosphorylated. SpoIIE is a membrane-bound phosphatase that dephosphorylates SpoIIAA, thereby re-activating it. It is not understood how sigmaF is activated specifically in the prespore but not in the mother cell. Here, we use a recently developed fluorescence spectroscopy technique to follow in real time the formation of sigmaF.SpoIIAB complexes and their dissociation by SpoIIAA. We show that complete activation of sigmaF is induced by a tenfold increase in SpoIIE activity. This result demonstrates that relatively small changes in SpoIIE activity, which could arise from asymmetric septation, can achieve the all-or-nothing response in sigmaF activity required by the cell. For long-term sigmaF activation, we find that sustained SpoIIE activity is required to counteract the activity of SpoIIAB. Even though the continual phosphorylation and dephosphorylation of SpoIIAA by these two enzymes will expend some ATP, the formation of SpoIIAA.SpoIIAB.ADP complexes greatly diminishes the rate of the phosphorylation reaction, and thus minimizes the wastage of energy. These features provide a very efficient system for regulating sigmaF.

Adenosine Diphosphate↗
PubMed1984

The htpR gene product of E. coli is a sigma factor for heat-shock promoters.

The htpR gene of E. coli encodes a positive regulator of the heat-shock response. We have fused the htpR gene to the inducible PL promoter of phage lambda. Overproduction of HtpR following a temperature upshift resulted in the overexpression of heat-shock proteins. We describe the purification and initial in vitro characterization of the factor controlling expression of heat-shock genes. The factor was the 32 kd htpR gene product. In vitro, a mixture of HtpR and core RNA polymerase initiated transcription at heat-shock promoters. The sigma factor encoded by rpoD was not required for this reaction. Therefore, HtpR is a sigma factor that promotes transcription initiation at heat-shock promoters. We propose that htpR be renamed rpoH and that the gene product be called sigma-32.

Bacterial Proteins↗
PubMed1997

Osmostress response in Bacillus subtilis: characterization of a proline uptake system (OpuE) regulated by high osmolarity and the alternative transcription factor sigma B.

Exogenously provided proline has been shown to serve as an osmoprotectant in Bacillus subtilis. Uptake of proline is under osmotic control and functions independently of the known transport systems for the osmoprotectant glycine betaine. We cloned the structural gene (opuE) for this proline transport system and constructed a chromosomal opuE mutant by marker replacement. The resulting B. subtilis strain was entirely deficient in osmoregulated proline transport activity and was no longer protected by exogenously provided proline, attesting to the central importance of OpuE for proline uptake in high-osmolarity environments. The transport characteristics and growth properties of the opuE mutant revealed the presence of a second proline transport activity in B. subtilis. DNA sequence analysis of the opuE region showed that the OpuE transporter (492 residues) consists of a single integral membrane protein. Database searches indicated that OpuE is a member of the sodium/solute symporter family, comprising proteins from both prokaryotes and eukaryotes that obligatorily couple substrate uptake to Na+ symport. The highest similarity was detected to the PutP proline permeases, which are used in Escherichia coli, Salmonella typhimurium and Staphylococcus aureus for the acquisition of proline as a carbon and nitrogen source, but not for osmoprotective purposes. An elevation of the osmolarity of the growth medium by either ionic or non-ionic osmolytes resulted in a strong increase in the OpuE-mediated proline uptake. This osmoregulated proline transport activity was entirely dependent on de novo protein synthesis, suggesting a transcriptional control mechanism. Primer extension analysis revealed the presence of two osmoregulated and tightly spaced opuE promoters. The activity of one of these promoters was dependent on sigma A and the second promoter was controlled by the general stress transcription factor sigma B.

Amino Acid Sequence↗
PubMed1992

Characterization of a Bacillus subtilis sporulation operon that includes genes for an RNA polymerase sigma factor and for a putative DD-carboxypeptidase.

At early stages of sporulation, the spoIIA locus is transcribed as a tricistronic (1.7-kb) operon, coding for sigma F and for two proteins that modulate the activity of sigma F. The locus is transcribed as a longer (2.9-kb) transcript at the late stages of sporulation. We show here that the longer transcript contains an additional open reading frame whose product has extensive sequence homology with DD-carboxypeptidases; the corresponding gene is designated dacF. Cotranscription of a morphogene, such as dacF, with the gene for a sigma factor suggests a way to couple transcription regulation with morphogenesis. The predicted N-terminal sequence of the DacF protein and the inhibition of sporulation by a translational dacF-lacZ fusion both suggest that the protein has a signal peptide for transport into or across a membrane. Expression of a dacF-lacZ transcriptional fusion was in the forespore. The 5' end of the 2.9-kb transcript was determined by primer extension analysis. The region 5' to the end showed no homology to promoters recognized by known sigma factors but was homologous to the corresponding region of the forespore-specific 0.3-kb gene of Bacillus subtilis.

Amino Acid Sequence↗
PubMed2003

Purification of Rhodobacter sphaeroides RNA polymerase and its sigma factors.

This article summarized methods to obtain RNA polymerase and sigma factors that can be used to analyze the in vitro control of gene expression by the facultative phototroph R. sphaeroides. While not a topic of this article, these purified components also allow one to analyze R. sphaeroides promoters that use activators to stimulate transcription. We expect that these approaches will be increasingly useful as investigators continue to dissect the number of unusual signal transduction pathways that control gene expression in this and other related species.

Base Sequence↗
PubMed2005

Analysis of promoters recognized by HrpL, an alternative sigma-factor protein from Pantoea agglomerans pv. gypsophilae.

HrpL, an alternative sigma factor, activates the transcription of the Hrp regulon by its binding to a common "hrp box" promoter. Based on computational techniques, the hrp box previously was defined as a consensus bipartite cis element, 5'-GGAACC-N(15-16)-CCACNNA-3'. The present report combines a quantitative in vivo assay for measuring Hrp promoter activity with site-specific mutagenesis to analyze the effect of consensus and nonconsensus nucleotides on promoter activity. The analysis was carried out with Hop effectors of the tumorigenic bacterium Pantoea agglomerans pv. gypsophilae, in which HrpL is indispensable for gall formation. Mutational analysis indicates that the hrp box consensus can be divided into crucial and noncrucial nucleotides. The first 5 nucleotides (nt) of the--35 consensus motif (GGAAC) and the 3 nt of the--10 motif (ACNNA) are crucial, whereas other consensus and adjacent nonconsensus nucleotides exert a significant effect on the promoter's strength. With spacing of 13 or 17 nt between the two motifs, significant activity was still retained. Gel shift assays indicated that deletion of GG from the--35 consensus motif eliminated HrpL binding, whereas mutations in the--10 consensus motif or modification of the spacing, which eliminates promoter activity, did not elicit any effect. The degeneracy in Hrp promoters of four hrp and type III effector genes of P agglomerans pv. gypsophilae indicated significant differences in promoter activity, whereas increasing the promoter strength of the Hop effector, HsvG, resulted in overexpression of gall formation.

Bacterial Proteins↗
PubMed1997

Role of sigma factor RpoS in initial stages of Salmonella typhimurium infection.

The sigma factor RpoS mediates the stationary-phase expression of a large group of genes, including those involved in resistance to a variety of environmental stresses, such as starvation, oxidation, and low pH. In addition, RpoS has been shown to regulate Salmonella virulence. In Salmonella typhimurium, RpoS controls the expression of the Salmonella plasmid virulence (spv) genes, which are required for systemic infection. However, the mechanism by which RpoS affects the pathogenicity of Salmonella remains incompletely defined. In this study, we focused on the ability of rpoS to affect the early stages of the infection process of S. typhimurium. An rpoS mutant of S. typhimurium exhibited wild-type abilities to attach to and invade Int-407 cells and J774 macrophage-like cells. In addition, rpoS did not affect the intracellular survival of S. typhimurium in either J774 macrophage-like cells or rat bone marrow-derived macrophages. However, the rpoS mutant demonstrated a decreased ability to colonize murine Peyer's patches after oral inoculation than its wild-type virulent parent strain showed. In addition, virulence plasmid-cured derivatives of the rpoS mutant were recovered in lower numbers from murine Peyer's patches than were plasmid-cured derivatives of the isogenic wild-type S. typhimurium. This indicates that RpoS regulation of chromosomally encoded genes is important for colonization of the gut-associated lymphoid tissue (GALT) by S. typhimurium. Microscopic analysis of histological sections taken from Peyer's patches after peroral infection of mice showed that, unlike its wild-type virulent parent strain, the isogenic rpoS mutant did not destroy the follicle-associated epithelium of the GALT. Furthermore, the rpoS mutant demonstrated a decreased ability to adhere to histological sections of murine Peyer's patches than its wild-type parent showed. Our data provide evidence for a role of RpoS in the interaction of Salmonella with cells of the GALT, specifically the Peyer's patches. This implicates the involvement of rpoS in the initial stages of systemic infection by Salmonella as opposed to infection leading to gastroenteritis.

Animals↗
PubMed2005

Two distinct functions of ComW in stabilization and activation of the alternative sigma factor ComX in Streptococcus pneumoniae.

Natural genetic transformation in Streptococcus pneumoniae is controlled by a quorum-sensing system, which acts through the competence-stimulating peptide (CSP) for transient activation of genes required for competence. More than 100 genes have been identified as CSP regulated by use of DNA microarray analysis. One of the CSP-induced genes required for genetic competence is comW. As the expression of this gene depended on the regulator ComE, but not on the competence sigma factor ComX (sigma(X)), and as expression of several genes required for DNA processing was affected in a comW mutant, comW appears to be a new regulatory gene. Immunoblotting analysis showed that the amount of the sigma(X) protein is dependent on ComW, suggesting that ComW may be directly or indirectly involved in the accumulation of sigma(X). As sigma(X) is stabilized in clpP mutants, a comW mutation was introduced into the clpP background to ask whether the synthesis of sigma(X) depends on ComW. The clpP comW double mutant accumulated an amount of sigma(X) higher (threefold) than that seen in the wild type but was not transformable, suggesting that while comW is not needed for sigma(X) synthesis, it acts both in stabilization of sigma(X) and in its activation. Modification of ComW with a histidine tag at its C or N terminus revealed that both amino and carboxyl termini are important for increasing the stability of sigma(X), but only the N terminus is important for stimulating its activity.

Artificial Gene Fusion↗
PubMed1996

Identification of additional genes under the control of the transcription factor sigma F of Bacillus subtilis.

We describe the identification of five transcriptional units under the control of the sporulation transcription factor sigma F in Bacillus subtilis. These are csfA, csfB, csfC, csfD, and csfF, located at approximately 230 degrees, 2 degrees, 316 degrees, 205 degrees, and approximately 290 degrees, respectively, on the genetic map. Null mutations in csfA, csfB, csfC, or csfD, either alone or together, do not cause a noticeable defect in sporulation or germination.

Bacillus subtilis↗
PubMed2001

New family of regulators in the environmental signaling pathway which activates the general stress transcription factor sigma(B) of Bacillus subtilis.

Expression of the general stress regulon of Bacillus subtilis is controlled by the alternative transcription factor sigma(B), which is activated when cells encounter growth-limiting energy or environmental stresses. The RsbT serine-threonine kinase is required to convey environmental stress signals to sigma(B), and this kinase activity is magnified in vitro by the RsbR protein, a positive regulator important for full in vivo response to salt or heat stress. Previous genetic analysis suggested that RsbR function is redundant with other unidentified regulators. A search of the translated B. subtilis genome found six paralogous proteins with significant similarity to RsbR: YetI, YezB, YkoB, YojH, YqhA, and YtvA. Their possible regulatory roles were investigated using three different approaches. First, genetic analysis found that null mutations in four of the six paralogous genes have marked effects on the sigma(B) environmental signaling pathway, either singly or in combination. The two exceptions were yetI and yezB, adjacent genes which appear to encode a split paralog. Second, biochemical analysis found that YkoB, YojH, and YqhA are specifically phosphorylated in vitro by the RsbT environmental signaling kinase, as had been previously shown for RsbR, which is phosphorylated on two threonine residues in its C-terminal region. Both residues are conserved in the three phosphorylated paralogs but are absent in the ones that were not substrates of RsbT: YetI and YezB, each of which bears only one of the conserved residues; and YtvA, which lacks both residues and instead possesses an N-terminal PAS domain. Third, analysis in the yeast two-hybrid system suggested that all six paralogs interact with each other and with the RsbR and RsbS environmental regulators. Our data indicate that (i) RsbR, YkoB, YojH, YqhA, and YtvA function in the environmental stress signaling pathway; (ii) YtvA acts as a positive regulator; and (iii) RsbR, YkoB, YojH, and YqhA collectively act as potent negative regulators whose loss increases sigma(B) activity more than 400-fold in unstressed cells.

Bacillus subtilis↗
PubMed2000

Characterization of two plastid sigma factors, SigA1 and SigA2, that mainly function in matured chloroplasts in Nicotiana tabacum.

We have isolated and characterized two genes from Nicotiana tabacum, whose products function as putative sigma factors for plastid RNA polymerase. Since the amino acid sequence deduced from the DNA sequences of both genes showed highly similar to that of the SigA protein of Arabidopsis thaliana, we termed the corresponding genes sigA1 and sigA2, respectively. Transient expression assay using a green fluorescent protein (GFP) fusion construct indicated that the N-terminal region of the sigA2 gene product could function as a transit peptide for import into chloroplasts. The gel-blot analysis of RNAs revealed that the sum of the sigA1 and sigA2 transcripts fluctuated apparently with an endogenous rhythm after 12-h-light, 12-h-dark entrainment in photomixotrophically cultured tobacco cells. RT-PCR based northern analysis revealed that the sigA1 and sigA2 transcripts increased along with the cell growth in cultured cells, and were most abundant in mature leaves and shoot meristems with very young leaves in tobacco plants. Immunoblot analysis of the cell extracts of tobacco plants also supports this notion. These results suggest that the sigma factors encoded by sigA1 and sigA2 play a role in chloroplast development and regulation of gene expression in matured chloroplasts.

Amino Acid Sequence↗
PubMed1996

The positions of the sigma-factor genes, whiG and sigF, in the hierarchy controlling the development of spore chains in the aerial hyphae of Streptomyces coelicolor A3(2).

whiG and sigF encode RNA polymerase sigma factors required for sporulation in the aerial hyphae of Streptomyces coelicolor. Their expression was analysed during colony development in wild-type and sporulation-defective whi mutant strains. Each gene was transcribed from a single promoter. Unexpectedly, whiG mRNA was present at all time points, including those taken prior to aerial mycelium formation; this suggests that whiG may be regulated post-transcriptionally. Transcription of whiG did not depend upon any of the six known 'early' whi genes required for sporulation septum formation (whiA, B, G, H, I and J), placing it at the top of the hierarchy of whi loci. sigF expression appeared to be regulated at the level of transcription; sigF transcripts were detected transiently when sporulation septa were observed in the aerial hyphae. Transcription of sigF depended upon all six of the early whi genes, including whiG. The sigF promoter does not resemble the consensus sequence established for sigma WhiG-dependent promoters and E sigma WhiG did not transcribe from the sigF promoter in vitro. Consequently, the genetic dependence of sigF upon whiG is very likely to be indirect. These results show that there is a hierarchical relationship between sigma factors required for Streptomyces sporulation and also that at least five other genes are involved in this transcriptional network.

Bacterial Proteins↗
PubMed2001

Nitrogen-regulated group 2 sigma factor from Synechocystis sp. strain PCC 6803 involved in survival under nitrogen stress.

The expression of sll1689, an open reading frame from the cyanobacterium Synechocystis sp. strain PCC 6803 putatively encoding a member of the sigma(70) family of sigma factors, appears to be regulated by the nitrogen control transcription factor NtcA. Disruption of sll1689 had no noticeable effect on exponential growth, identifying its product as a member of the group 2, nonessential class of sigma(70)-like sigma factors; however, this disruption decreased the viability of the cells after long periods of nitrogen starvation. We have named this gene rpoD2-V. The expression of glnN, encoding a type III glutamine synthetase, was impaired in strains bearing an inactivated copy of the rpoD2-V gene.

Adaptation, Biological↗
PubMed1998

Characterization of the alternative sigma-factors SigD and SigE in Synechococcus sp. strain PCC 7002. SigE is implicated in transcription of post-exponential-phase-specific genes.

The sigD and sigE genes, which encode two alternative sigma-factors from the unicellular marine cyanobacterium Synechococcus sp. PCC 7002, were cloned and characterized. Strains in which the sigD and sigE genes were insertionally inactivated were viable under standard laboratory conditions, indicating that SigD and SigE are group 2 sigma-factors. When stationary-phase cells were diluted into fresh growth medium, it was observed that the sigE mutant strain required longer times to re-establish exponential growth than the wild-type strain. By monitoring the growth rates in such dilution experiments, it was observed that the lag times for the mutant strain became progressively longer as the original cultures progressed towards stationary phase. Transcripts for the sigE gene initially increased and subsequently decreased as cells grew further into stationary phase. It was determined that a functional SigE protein is required for the expression of the starvation-induced protein DpsA/PexB. The results suggest that SigE is involved in the transcription of genes specifically expressed in the post-exponential phase.

Amino Acid Sequence↗
PubMed2004

The principal sigma factor sigA mediates enhanced growth of Mycobacterium tuberculosis in vivo.

The ability of Mycobacterium tuberculosis to grow in macrophages is central to its pathogenicity. We found previously that the widespread 210 strain of M. tuberculosis grew more rapidly than other strains in human macrophages. Because principal sigma factors influence virulence in some bacteria, we analysed mRNA expression of the principal sigma factor, sigA, in M. tuberculosis isolates during growth in human macrophages. Isolates of the 210 strain had higher sigA mRNA levels and higher intracellular growth rates, compared with other clinical strains and the laboratory strain H37Rv. SigA was also upregulated in the 210 isolate TB294 during growth in macrophages, compared with growth in broth. In contrast, H37Rv sigA mRNA levels did not change under these conditions. Overexpression of sigA enhanced growth of recombinant M. tuberculosis in macrophages and in lungs of mice after aerosol infection, whereas recombinant strains expressing antisense transcripts to sigA showed decreased growth in both models. In the presence of superoxide, sense sigA transformants showed greater resistance than vector controls, and the antisense sigA transformant did not grow. We conclude that M. tuberculosis sigA modulates the expression of genes that contribute to virulence, enhancing growth in human macrophages and during the early phases of pulmonary infection in vivo. This effect may be mediated in part by increased resistance to reactive oxygen intermediates.

Animals↗