Characterization of transcription units dependent upon a sporulation-specific sigma factor in Bacillus subtilis

Bacillus subtilis is a Gram-positive bacterium that responds to nutritional stress by undergoing morphological differentiation to form a dormant and environmentally resistant endospore. The primary regulators of spatial and temporal events of this complex process are sigma factors which, in association with RNA polymerase, direct transcription from specific groups of promoters. One of the hallmark events of sporulation is the creation of a large mother cell and a small pre-spore compartment within the sporangium. At this intermediate stage the first mother cell specific sigma factor, {dollar}rmsigmasp{lcub}E{rcub},{dollar} is activated.; To identify genes expressed at intermediate stages of Bacillus subtilis sporulation, we screened for {dollar}rmsigmasp{lcub}E{rcub}{dollar}-dependent promoters. One promoter that we found drives expression of an operon consisting of at least five open reading frames (ORFs). The predicted products of the first three ORFs are very homologous to enzymes involved in fatty acid metabolism, including acetyl-CoA acetyltransferase (thiolase), 3-hydroxybutyrl-CoA dehydrogenase, and acyl-CoA dehydrogenase, respectively. We showed that the fourth ORF encoded a third isozyme of citrate synthase in B. subtilis. Genetic evidence and primer extension results showed that transcription of this operon is directed by the mother cell compartment-specific sigma factor, {dollar}rmsigmasp{lcub}E{rcub},{dollar} so the operon was named mmg (mother cell metabolic genes). Furthermore, we found that a sequence (mmgO) with homology to a catabolite responsive element (CRE) mediates glucose repression of mmg promoter activity during sporulation, and that this repression was lost in a ccpA mutant.; While little is known about metabolism during intermediate sporulation, it is significant that we found several other potential metabolic operons during our screen for {dollar}rmsigmasp{lcub}E{rcub}{dollar} promoters. The characterization of one of these operons, ssd, showed that the first two ORFs (ssdA and ssdB) were similar to malate dehydrogenase and threonine dehydrogenase, respectively. Characterization of the promoter provided genetic and primer extension evidence that {dollar}rmsigmasp{lcub}E{rcub}{dollar} directed its transcription. Examination of the transcription from this promoter indicates that it is also repressed in the presence of glucose.; Further, we describe the characterization of three other promoters that are also dependent on the mother-cell sigma factor, {dollar}rmsigmasp{lcub}E{rcub}.{dollar} Two of these promoters are transcribed beginning at about the second hour of sporulation from {dollar}rmsigmasp{lcub}E{rcub}{dollar}-directed promoters, while the third becomes active at the fourth hour and is controlled by the late mother-cell transcription factor, {dollar}rmsigmasp{lcub}K{rcub}.{dollar}...