Defining the Bacillus subtilis σW regulon: a comparative analysis of promoter consensus search, run-off transcription/macroarray analysis (ROMA), and transcriptional profiling approaches

The Bacillus subtilis extracytoplasmic function (ECF) σ factor σW controls a large regulon that is strongly induced by alkali shock. To define the physiological role of σW we have sought to identify the complete set of genes under σW control. Previously, we described a promoter consensus search procedure to identify σW controlled genes. Herein, we introduce a novel method to identify additional target promoters: run-off transcription followed by macroarray analysis (ROMA). We compare the resulting list of targets with those identified in conventional transcriptional profiling studies and using the consensus search approach. While transcriptional profiling identifies genes that are strongly dependent on σW for in vivo expression, some σW-dependent promoters are not detected due to the masking effects of other promoter elements, overlapping recognition with other ECF σ factors, or both. Taken together, the consensus search, ROMA, and transcriptional profiling approaches establish a minimum of 30 promoter sites (controlling ∼60 genes) as direct targets for activation by σW. Significantly, no single approach identifies more than ∼80 % of the regulon so defined. We therefore suggest that a combination of two or more complementary approaches be employed in studies seeking to achieve maximal coverage when defining bacterial regulons. Our results indicate that σW controls genes that protect the cell against agents that impair cell wall biosynthesis but fail to reveal any connection to operons likely to function in adaptation to alkaline growth conditions. This is consistent with the observation that a sigW mutant is unaffected in its ability to survive alkali shock. We conclude that in B. subtilis sudden imposition of alkali stress activates the σW stress response, perhaps by impairing the ability of the cell wall biosynthetic machinery to function.