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Transcription from some sporulation-specific promoters of Bacillus subtilis is dependent on synthesis of pro-sigma E and its conversion to sigma E by proteolysis. Certain mutations in the precursor region of sigE, the gene encoding pro-sigma E, apparently allow the mutant sigE products to be active as sigma factors without being proteolysed in the normal way.
Biosynthesis of bacterial flagella involves the coordinated expression of 30 or more genes in several separate operons. We have recently shown that in Bacillus subtilis, the sigma 28 factor is essential for flagellar synthesis, suggesting that transcription of these genes is directly under the control of this alternative sigma factor. In enteric bacteria structural genes for flagellar, chemotaxis, and motility operons appear to be under coordinate control, however, the nature of the regulatory factors has not been determined. Sequence analysis of many such genes has failed to reveal plausible promoter sequences for the predominant bacterial RNA polymerase, and several such genes are not transcribed effectively in vitro by the Escherichia coli sigma 70 RNA polymerase. However, all of the sequenced flagellar, chemotaxis, and motility operons from the enteric bacteria are preceded by DNA sequences highly homologous to B. subtilis sigma 28 promoters. We propose that an alternative sigma factor controls expression of the flagellar regulon in both B. subtilis and in the enteric bacteria.
The genes specifying chemotaxis, motility, and flagellar function in Escherichia coli are coordinately regulated and form a large and complex regulon. Despite the importance of these genes in controlling bacterial behavior, little is known of the molecular mechanisms that regulate their expression. We have identified a minor form of E. coli RNA polymerase that specifically transcribes several E. coli chemotaxis/flagellar genes in vitro and is likely to carry out transcription of these genes in vivo. The enzyme was purified to near homogeneity based on its ability to initiate transcription of the E. coli tar chemotaxis gene at start sites that are used in vivo. Specific tar transcription activity is associated with a polypeptide of apparent 28-kDa molecular mass that remains bound to the E. coli RNA polymerase throughout purification. This peptide behaves as a secondary sigma factor--designated sigma F--because it restores specific tar transcription activity when added to core RNA polymerase. The sigma F holoenzyme also transcribes the E. coli tsr and flaAI genes in vitro as well as several Bacillus subtilis genes that are transcribed specifically by the sigma 28 form of B. subtilis RNA polymerase. The latter holoenzyme is implicated in transcription of flagellar and chemotaxis genes in B. subtilis. Hence E. coli sigma F holoenzyme appears to be analogous to the B. subtilis sigma 28 RNA polymerase, both in its promoter specificity and in the nature of the regulon it controls.
Thermoinduction of cells of E. coli carrying prophage lambdacI857 within the bfe gene brings about not only "escape synthesis" of core subunits of the DNA-dependent RNA polymerase (RNA nucleotidyltransferase, nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2-7-7-6), but also a striking stimulation of sigma factor synthesis. The latter phenomenon, termed sigma induction, is generally observed after lambda phage infection or prophage induction. A series of experiments with various bacterial and phage strains led us to conclude that the N gene product of lambda is directly involved to the sigma induction. These and other results obtained with mutants defective in transcription termination factor rho suggest the involvement of a rho-sensitive site in the control of sigma gene expression in E. coli.
Utilizing a polymerase chain reaction-based approach, the gene (rpoD) encoding the primary sigma factor from Borrelia burgdorferi strain B31 was cloned and sequenced. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1632 bp (543 amino acids (aa), 63.7 kDa). Comparison with Escherichia coli sigma 70 and Bacillus subtilis sigma 43 showed a high degree of similarity in the aa sequences, especially for the regions that are known to be required for promoter recognition and core binding.
The sigmaA factor of Bacteroides fragilis is the prototype of a novel subgroup of primary sigma factors that are essential for growth and ensure the initiation of transcription of the housekeeping genes. This subgroup is confined to the phyla Bacteroidetes and Chlorobi. Its members carry a specific amino acid signature and are notably characterized by a short, basic N-terminal segment instead of the typical acidic region 1.1. Using in vitro mutagenesis, we investigated the importance of this basic segment and of several residues of the signature for the function of sigmaA. We have shown that the conserved residues Phe-61 and Lys-265, located in the core binding and DNA binding subregions 2.1 and 4.2, respectively, are critical for full function of the B. fragilis holoenzyme. With respect to the unusual subregion composition of sigmaA, we have shown that truncation of the basic N-terminal segment, or reversion of its charge, strongly affects the overall transcriptional activity of B. fragilis RNA polymerase in vitro. Our results indicate that the presence of the intact basic segment is required for the formation of RNA polymerase (RNAP)-promoter open complexes, the correct architecture of the transcription bubble, and efficient promoter clearance.
Bacillus subtilis RNA polymerase (RNAP) contains a catalytic core (beta beta' alpha 2; or E) associated with one of several sigma factors, which determine promoter recognition, and delta protein, which enhances promoter selectivity. We have shown previously that specific mutations in sigma A region 2.3, or addition of delta, decrease the ability of RNAP to melt the ilv-leu promoter. Here we extend these studies to a stable RNA promoter, PtmS, which controls transcription of seven tRNA genes. KMnO4 footprinting was used to visualize DNA melting at PtmS as a function of both temperature and the protein composition of the RNAP holoenzyme. We propose that the pathway leading to productive initiation includes several intermediates: a closed complex (RPc), a complex in which DNA melting has nucleated within the conserved TATA element (RPn), and an open complex in which DNA-melting extends to at least -4 (RPo1). RNAP reconstituted with either of two mutant sigma A proteins, Y189A and W192A, was defective for both the nucleation and propagation of the transcription bubble while a third sigma A mutant, W193A, allows normal nucleation of DNA-melting, but does not efficiently propagate the melted region downstream.
Development of plastids into chloroplasts, the organelles of photosynthesis, is triggered by light. However, little is known of the factors involved in the complex coordination of light-induced plastid gene expression, which must be directed by both nuclear and plastid genomes. We have isolated an Arabidopsis mutant, abc1, with impaired chloroplast development, which results in a pale green leaf phenotype. The mutated nuclear gene encodes a sigma factor, SigB, presumably for the eubacterial-like plastid RNA polymerase. Our results provide direct evidence that a nuclear-derived prokaryotic-like SigB protein, plays a critical role in the coordination of the two genomes for chloroplast development.
The rpoD gene coding for the primary transcription sigma factor, sigma70, and its entire operon were cloned from strain mt-2 of the purple soil bacterium Pseudomonas putida. Comparison of the deduced amino acid sequence of Ppmt-2 sigma70 with that of sigma70 from P. putida strain G1 shows that the two proteins differ in their primary structure, molecular weight, and isoelectric point. The significance of this difference is discussed in terms of bacterial taxonomy and transcription regulation.
Disruption of a gene for an alternative sigma factor, designated sigF, in the freshwater, unicellular cyanobacterium Synechocystis sp. strain PCC6803 resulted in a pleiotropic phenotype. Most notably, this mutant lost phototactic movement with a concomitant loss of pili, which are abundant on the surface of wild-type cells. The sigF mutant also secreted both high levels of yellow-brown and UV-absorbing pigments and a polypeptide that is similar to a large family of extracellular proteins that includes the hemolysins. Furthermore, the sigF mutant had a dramatically reduced level of the transcript from two tandemly arranged pilA genes (sll1694 and sll1695), which encode major structural components of type IV pili. Inactivation of these pilA genes eliminated phototactic movement, though some pili were still present in this strain. Together, these results demonstrate that SigF plays a critical role in motility via the control of pili formation and is also likely to regulate other features of the cell surface. Furthermore, the data provide evidence that type IV pili are required for phototactic movement in certain cyanobacteria and suggest that different populations of pili present on the Synechocystis cell surface may perform different functions.
A modified form of Bacillus subtilis RNA polymerase (RNA nucleotidyltransferase) has been isolated that exhibits distinctive transcriptional specificity. This modified enzyme transcribes two cloned genes from the purA-cysA region of the B. subtilis chromosome whose expression in vivo is associated with the process of sporulation. Neither of these genes is transcribed by the usual form of B. subtilis RNA polymerase holoenzyme containing a sigma factor of 55,000 daltons (sigma 55). The modified RNA polymerase lacks sigma 55 but contains a newly identified subunit of 37,000 daltons termed sigma 37. A reconstitution experiment in which sigma 37 was added to core RNA polymerase strongly suggests that sigma 37 is responsible for the transcriptional specificity of the modified RNA polymerase. Sigma 37 apparently acts at the level of promoter recognition; this transcriptional determinant enabled core RNA polymerase to form stable binary and ternary ("initiation") complexes with endonuclease restriction fragments containing promoters for the cloned B. subtilis genes.
An alternative group 2 sigma factor was identified in the nitrogen-fixing, symbiotically competent cyanobacterium Nostoc punctiforme and designated sigH. Transcription of sigH was specifically induced within 1.5 h following exposure of N. punctiforme to its symbiotic plant partner, Anthoceros punctatus. A mutation in sigH resulted in a sixfold-higher initial infection of A. punctatus tissue without a parallel increase in nitrogen-fixing activity.
Hybridization to a PCR product derived from conserved sigma-factor sequences led to the identification of two Sinorhizobium meliloti DNA segments that display significant sequence similarity to the family of rpoH genes encoding the sigma(32) (RpoH) heat-shock transcription factors. The first gene, rpoH1, complements an Escherichia coli rpoH mutation. Cells containing an rpoH1 mutation are impaired in growth at 37 degrees C under free-living conditions and are defective in nitrogen fixation during symbiosis with alfalfa. A plasmid-borne rpoH1-gusA fusion increases in expression upon entry of the culture into the stationary phase of growth. The second gene, designated rpoH2, is 42% identical to the S. meliloti rpoH1 gene. Cells containing an rpoH2 mutation have no apparent phenotype under free-living conditions or during symbiosis with the host plant alfalfa. An rpoH2-gusA fusion increases in expression during the stationary phase of growth. The presence of two rpoH-like sequences in S. meliloti is reminiscent of the situation in Bradyrhizobium japonicum, which has three rpoH genes.
The Streptomyces aureofaciens sigF gene encodes a sigma factor. By integrative transformation, via double cross-over, a stable null mutant of sigF gene was obtained. This mutation appeared to have no obvious effect on vegetative growth, but affected the late stage of spore maturation. Microscopic examination showed that spores were deformed, and spore wall was thinner, compared with the wild-type spores. The spore pigment of sigF mutant was green, compared to wild-type grey-pink spore pigmentation. The plasmid-born wild-type sigF gene complemented the mutation after transformation of the mutant strain.
The gene encoding the principal sigma factor from Synechococcus sp. strain PCC 7002 was isolated and characterized. The Synechococcus sp. strain PCC 7002 sigA gene encodes a protein of 375 amino acids (43 center dot 7 kDa) that is required for viability under normal growth conditions. The SigA protein was overproduced in Escherichia coli and the purified protein was used to raise polyclonal antiserum in rabbits. This antiserum was used in immunoblot analyses of partially purified RNA polymerase from Synechococcus sp. strain PR6000. The probable in vivo translational start site was identified by a comparison of amino acid sequencing results obtained with SigA proteins overproduced in E. coli with immunoblot analyses of SigA protein in crude preparations of RNA polymerase from the cyanobacterium. The sigA gene is encoded on a transcript of 1700 bases that initiates 496 nucleotides upstream from the probable in vivo translational start site. The abundance of sigA transcripts decreases rapidly after the removal of combined nitrogen from the growth medium.
We have isolated and studied the cloned sigma factors SASIG1-3 from mustard (Sinapis alba). In functional analyses using both promoter and factor mutants, the three recombinant proteins all had similar basic properties but also revealed differences in promoter preference and requirements for single nucleotide positions. Directed muta- genesis of SASIG1 identified critical residues within the conserved regions 2.4 and 4.2 necessary for binding of the -10 and -35 promoter elements, respectively. SASIG1 and 2, but not SASIG3, each have a typical region 2.5 for binding of the extended -10 promoter element. SASIG3 has a pro-sequence reminiscent of sigma K from Bacillus subtilis, suggesting that proteolytic cleavage from an inactive precursor is involved in the regulation of plastid transcription. In addition, SASIG2 was found to be more abundant in light-grown as compared to dark-grown mustard seedlings, while the converse was true for SASIG3.
Constitutive expression of stationary phase alternative sigma factor RpoS in Escherichia coli during the exponential phase was found to partially suppress drug sensitivity associated with an acrAB mutation that inactivates the major multidrug resistance pump, suggesting that Rpos might significantly contribute to multidrug resistance, underscoring yet another role for this important stress-related transcription factor.
The ability of Pseudomonas syringae pv. tomato DC3000 to parasitize tomato and Arabidopsis thaliana depends on genes activated by the HrpL alternative sigma factor. To support various functional genomic analyses of DC3000, and specifically, to identify genes involved in pathogenesis, we developed a draft sequence of DC3000 and used an iterative process involving computational and gene expression techniques to identify virulence-implicated genes downstream of HrpL-responsive promoters. Hypersensitive response and pathogenicity (Hrp) promoters are known to control genes encoding the Hrp (type III protein secretion) machinery and a few type III effector proteins in DC3000. This process involved (i) identification of 9 new virulence-implicated genes in the Hrp regulon by miniTn5gus mutagenesis, (ii) development of a hidden Markov model (HMM) trained with known and transposon-identified Hrp promoter sequences, (iii) HMM identification of promoters upstream of 12 additional virulence-implicated genes, and (iv) microarray and RNA blot analyses of the HrpL-dependent expression of a representative subset of these DC3000 genes. We found that the Hrp regulon encodes candidates for 4 additional type III secretion machinery accessory factors, homologs of the effector proteins HopPsyA, AvrPpiB1 (2 copies), AvrPpiC2, AvrPphD (2 copies), AvrPphE, AvrPphF, and AvrXv3, and genes associated with the production or metabolism of virulence factors unrelated to the Hrp type III secretion system, including syringomycin synthetase (SyrE), N(epsilon)-(indole-3-acetyl)-l-lysine synthetase (IaaL), and a subsidiary regulon controlling coronatine production. Additional candidate effector genes, hopPtoA2, hopPtoB2, and an avrRps4 homolog, were preceded by Hrp promoter-like sequences, but these had HMM expectation values of relatively low significance and were not detectably activated by HrpL.