Genome-wide analysis of the stationary-phase sigma factor (sigma-H) regulon of Bacillus subtilis

Abstract

Sigma-H is an alternative RNA polymerase sigma factor that directs the transcription of many genes that function at the transition from exponential growth to stationary phase in Bacillus subtilis. Twenty-three promoters, which drive transcription of 33 genes, are known to be recognized by sigma-H-containing RNA polymerase. To identify additional genes under the control of sigma-H on a genome-wide basis, we carried out transcriptional profiling experiments using a DNA microarray containing >99% of the annotated B. subtilis open reading frames. In addition, we used a bioinformatics-based approach aimed at the identification of promoters recognized by RNA polymerase containing sigma-H. This combination of approaches was successful in confirming most of the previously described sigma-H-controlled genes. In addition, we identified 26 putative promoters that drive expression of 54 genes not previously known to be under the direct control of sigma-H. Based on the known or inferred function of most of these genes, we conclude that, in addition to its previously known roles in sporulation and competence, sigma-H controls genes involved in many physiological processes associated with the transition to stationary phase, including cytochrome biogenesis, generation of potential nutrient sources, transport, and cell wall metabolism.

FIG. 1.

Logarithmic scale plots of spot intensities (arbitrary units). (A) Transcriptional profiles from sigH⁺ and sigH mutant cells at T₋₁, T₀, and T₁ of sporulation. The intensity of each spot in one channel (sigH⁺) is plotted versus the intensity of that spot in the other channel (sigH mutant). (B) Overexpression of sigH during vegetative growth. Data are presented for RNA samples taken immediately after and 15 and 60 min after addition of IPTG to induce increased expression of sigH. The intensity of each spot in one channel (−IPTG) is plotted versus the intensity of that spot in the other channel (+IPTG). Data presented are from a representative experiment and are not normalized.

FIG. 2.

Heat map indicating the expression profiles of 79 sigma-H-regulated genes. These genes were ordered by using a hierarchical clustering algorithm (J-Express v.2.1 application from MolMine AS), so that those with similar expression patterns were grouped together. The first three columns (left) display expression ratios for the wild typeversus the sigH mutant at T₋₁, T₀, and T₁. The next four columns display the expression ratios for cells harboring Pspank-hy-sigH immediately (0) and 15, 30, and 60 min after induction with IPTG. Hybridization ratios are displayed colorimetrically: shades of green indicate that a gene had a higher RNA level in wild-type cells (first three columns) or when sigH was overproduced (last four columns); shades of red indicate that a gene had less RNA in wild-type cells (first three columns) or when sigH was overexpressed. Genes identified in this work as sigma-H regulated are indicated in red. The ratios for each gene are from averages of at least three independent experiments.