Venn diagrams for the comparison of genes that were found to be downregulated in the <i>sigW</i> mutant strain with previously reported σ^W-regulated genes and genes that display similar condition-dependent transcription profiles as <i>sigW</i>.

<p>Diagram A includes only the so-called cluster C9 genes that have highly similar condition-dependent transcription profiles as defined by Nicolas <i>et al </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048471#pone.0048471-Nicolas1" target="_blank">[35]</a>. Notably, the <i>sigW</i> gene is included in cluster C9. Diagram B includes all genes that show condition-dependent expression profiles similar to that of <i>sigW</i>, including induction upon salt stress.</p

Assignment of clusters of genes with related transcript profiles across conditions to different groups of genes that are down-regulated or up-regulated in <i>sigW</i> mutant cells.

<p>The down-regulated genes are represented by groups 1 and 2 (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048471#pone-0048471-g001" target="_blank">Fig. 1</a>). Genes in group 3 were previously reported as σ^W-regulated, but our present studies provided no evidence for their proposed σ^W-dependency (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048471#pone-0048471-g001" target="_blank">Fig. 1</a>). The up-regulated genes are represented in a separate bar. Previously defined transcription clusters <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048471#pone.0048471-Nicolas1" target="_blank">[35]</a> are indicated in each bar by their C-number.</p

Up- and down-regulation of genes in <i>rasP</i>, <i>prsW</i> or <i>sigW</i> mutant strains compared to the wild-type.

<p>A, Venn diagram for down-regulated genes. B, Venn diagram for upregulated genes. Only genes with transcriptional changes that have p-values lower than 0.05 and effect values lower than −0.40 (A) or higher than 0.40 (B) are included. The genes that are considered to be σ^W-regulated are indicated between brackets.</p

Transcriptional changes of genes regulated by the CssRS two-component system.

<p>Transcriptional changes of genes regulated by the CssRS two-component system.</p

Genes that were up-regulated in the <i>sigW</i> mutant.

<p>Only the genes with Effect values higher than 0.4 and p-values lower than 0.05 are shown. Arrows behind genes in the ‘genetic organization’ column indicate whether the transcription of these genes was up- (↑) or down-regulated (↓). For each individual gene, the Table lists the function, the previously identified regulation, the genetic organization, and the condition-dependent transcription profile cluster as defined by Nicolas <i>et al</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048471#pone.0048471-Nicolas1" target="_blank">[35]</a>.</p

Down-regulated genes in <i>sigW</i> mutant cells.

<p>Only the down-regulated genes with effect values lower than −0.4 and p-values lower than 0.05 are shown. Effect values marked with * have q-values of less than 0.05. For each individual gene, the Table lists the function, the previously identified regulation, the genetic organization, the condition-dependent transcription profile cluster as defined by Nicolas <i>et al</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048471#pone.0048471-Nicolas1" target="_blank">[35]</a>, the presence of a predicted ‘σ^WXY’ promoter sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048471#pone.0048471-Nicolas1" target="_blank">[35]</a>, and our conclusion whether it belongs to the σ^W core regulon or the secondary σ^W-regulated genes. It should be noted here that the previously predicted ‘σ^WXY’ promoter sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048471#pone.0048471-Nicolas1" target="_blank">[35]</a> also covers the potential binding site for σ^M. The division between group 1 and group 2 genes is indicated by a bold line.</p

Organization of complex σ^W-regulated operons.

<p>The σ^W-regulated ORFs are indicated in black, and the σ^W-regulated ncRNAs are indicated in grey. Genes and an ncRNA on the opposite strand are indicated in white. A, The <i>yozO</i>-<i>yocM</i> operon. B, The <i>ydbST</i>-<i>acpS</i> operon.</p

<i>B. subtilis</i> strains.

<p><i>B. subtilis</i> strains.</p

Expression profiles of <i>sigW</i>, <i>sigX</i>, <i>sigY</i> and <i>sigM</i> in <i>B. subtilis</i> 168 across 104 conditions.

<p>The 269 tiling array hybridizations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048471#pone.0048471-Nicolas1" target="_blank">[35]</a> are arranged along the x-axis. Of particular interest for discriminating the activities of the encoded sigma factors are the conditions heat stress (‘heat’), ethanol stress (‘etha’) and hypersaline stress (‘salt’), which are marked by pink shading.</p

Processing phase contrast images to create segmented cells.

<p>(A) ImageJ commands for the processing of phase contrast images to create segmented cells. (B) Visualization of the image processing from the original phase contrast image, through background subtraction, convolution, setting of a threshold grey intensity, conversion of values within threshold to mask and de-speckling. Red objects in the processed image are above the threshold and counted as cells. Notably, non-separated pairs of cells as marked with the white arrow pointing at the site of their attachment will be counted as one cell.</p