SummaryThe Bacillus subtilis sigW gene encodes an extracytoplasmic function (ECF) factor that is expressed in early stationary phase from a sigW-dependent autoregulatory promoter, P W . Using a consensus-based search procedure, we have identified 15 operons preceded by promoters similar in sequence to P W . At least 14 of these promoters are dependent on W both in vivo and in vitro as judged by lacZ reporter fusions, run-off transcription assays and nucleotide resolution start site mapping. We conclude that W controls a regulon of more than 30 genes, many of which encode membrane proteins of unknown function. The W regulon includes a penicillin binding protein (PBP4*) and a co-transcribed amino acid racemase (RacX), homologues of signal peptide peptidase (YteI), flotillin (YuaG), ABC transporters (YknXYZ), non-haem bromoperoxidase (YdjP), epoxide hydrolase (YfhM) and three small peptides with structural similarities to bacteriocin precursor polypeptides. We suggest that W activates a large stationary-phase regulon that functions in detoxification, production of antimicrobial compounds or both.

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The sigX gene, identified as part of the international effort to sequence the Bacillus subtilis genome, has been proposed to encode an alternative factor of the extracytoplasmic function (ECF) subfamily. The sigX gene is cotranscribed with a downstream gene, ypuN, during logarithmic and early stationary phases of growth. We now report that strains lacking X are impaired in the ability to survive at high temperature whereas a ypuN mutant has increased thermotolerance. We overproduced and purified X from Escherichia coli and demonstrate that in vitro, both A and X holoenzymes recognize promoter elements within the sigX-ypuN control region. However, they have distinct salt optima such that A -dependent transcription predominates at low salt while X -dependent transcription predominates at high salt. A 54-bp region upstream of sigX suffices as a X -dependent promoter in vivo, demonstrating that sigX is at least partially under positive autoregulatory control. Mutation of ypuN increases expression from the X -dependent promoter in vivo, suggesting that ypuN may encode a negative regulator of X activity.Alternative sigma factors provide a powerful regulatory mechanism whereby a subset of RNA polymerase (RNAP) can be reprogrammed to activate new regulons (13,14,17). This mechanism is prominently featured in the regulatory cascade governing sporulation in Bacillus subtilis (26), in the synthesis of flagellar motility systems in diverse bacteria (16), and in both stationary-phase and heat shock adaptations (12,15,23). In Escherichia coli, the heat shock response is controlled by an alternative factor, 32, which is itself activated by a second heat-inducible factor, E (12). The E protein is a highly diverged member of the 70 family of proteins (30, 31). Recently, a large family of regulatory proteins, many of which had not previously been recognized as factors, were shown to be similar in sequence to E and proposed to coordinate the expression of extracytoplasmic functions (ECF subfamily of factors) (24).The B. subtilis sigX gene (originally defined as ORFX20) was identified during the genome sequencing project as a possible regulatory locus encoding a protein 26% identical to E. coli FecI, a regulator of ferric citrate transport (38). ORFX20 is cotranscribed with a downstream gene (ORFX21; ypuN) of unknown function (2). ORFX20 was renamed sigX because the corresponding gene product is related to the ECF family of factors (24). ECF factors control a variety of functions ranging from heat shock genes in E. coli, alginate biosynthesis in Pseudomonas aeruginosa, iron uptake in E. coli and Pseudomonas spp., carotenoid biosynthesis in Myxococcus xanthus, agarase secretion in Streptomyces coelicolor, plant pathogenicity in Pseudomonas syringae, nickel and cobalt efflux in Alcaligenes eutrophus, and outer membrane protein synthesis in Photobacterium sp. strain SS9 (7,24).In this report, we demonstrate that sigX encodes a polypeptide with the predicted factor activity, X . Reconstituted X holoenzyme (E X ) initiates transcription...

The Bacillus subtilis genome encodes at least 17 distinct sigma factors, including seven members of the extracytoplasmic function (ECF) subfamily. We have investigated the expression and regulation of the ECF ς factor encoded by the sigW gene. A ςW-dependent promoter (PW) precedessigW, demonstrating that this transcription factor is positively autoregulated. Expression of sigW is regulated by both growth phase and medium composition. Maximal expression is attained in early-stationary-phase cells grown in rich medium. We previously reported that sigW mutants have elevated transcription of some ςX-controlled genes, and we now report that the converse is also true: in a sigX mutant, PW is derepressed during logarithmic growth. Thus, these two regulons are mutually antagonistic. Reconstituted ςWholoenzyme faithfully recognizes the PW precedingsigW but does not recognize the PX promoter preceding the sigX gene. Autoregulation of sigXis also highly specific: ςX holoenzyme initiates transcription from PX but recognizes PW poorly if at all. In contrast, several promoters that are at least partially under ςX control are active with both the ςX and ςW holoenzymes in vitro. This finding supports the suggestion that the ςW and ςX regulons overlap. Sequence comparisons suggest that promoters recognized by these two ς factors have similar −35 elements but are distinguished by different base preferences at two key positions within the −10 element.

Thesigmasubunit of RNA polymerase determines promoter recognition and catalyzes DNA strand separation. The -35 promoter region is recognized by a helix-turn-helix motif in region 4, while the -10 region is specified, at least in part, by an amphipathic helix in region 2. We have proposed that conserved aromatic residues insigmaregion 2.3 interact with the non-template strand of the -10 element to drive open complex formation. We now report that Bacillus subtilis sigmaA holoenzyme, but neither core nor sigmaA alone, binds with high selectivity to single-stranded (ss) DNA containing the non-template -10 consensus sequence. UV irradiation of holoenzyme-ssDNA complexes efficiently crosslinks sigmaA to DNA and protease mapping supports a primary contact site in or near region 2. Several mutations in sigmaA region 2.3, shown previously to impair promoter melting, affect ssDNA binding: Y184A decreases binding selectivity, while Y189A and W193A decrease the efficiency of photocrosslinking. These results support a model in which these aromatic amino acids are juxtaposed to ssDNA, consistent with their demonstrated role in stabilizing the open complex.