Prediction and validation of novel SigB regulon members in Bacillus subtilis and regulon structure comparison to...

Sigma factor B (SigB) is the central regulator of the general stress response in Bacillus subtilis and regulates a group of genes in response to various stressors, known as the SigB regulon members. Genes that are directly regulated by SigB contain a promotor binding motif (PBM) with a previously identified consensus sequence.

In this study, refined SigB PBMs were derived and different spacer compositions and lengths (N₁₂-N₁₇) were taken into account. These were used to identify putative SigB-regulated genes in the B. subtilis genome, revealing 255 genes: 99 had been described in the literature and 156 genes were newly identified, increasing the number of SigB putative regulon members (with and without a SigB PBM) to > 500 in B. subtilis. The 255 genes were assigned to five categories (I-V) based on their similarity to the original SigB consensus sequences. The functionalities of selected representatives per category were assessed using promoter-reporter fusions in wt and ΔsigB mutants upon exposure to heat, ethanol, and salt stress. The activity of the P_rsbV (I) positive control was induced upon exposure to all three stressors. P_ytoQ (II) showed SigB-dependent activity only upon exposure to ethanol, whereas P_pucI (II) with a N₁₇ spacer and P_ylaL (III) with a N₁₆ spacer showed mild induction regardless of heat/ethanol/salt stress. P_ywzA (III) and P_yaaI (IV) displayed ethanol-specific SigB-dependent activities despite a lower-level conserved - 10 binding motif. P_gtaB (V) was SigB-induced under ethanol and salt stress while lacking a conserved - 10 binding region. The activities of P_ygaO and P_ykaA (III) did not show evident changes under the conditions tested despite having a SigB PBM that highly resembled the consensus. The identified extended SigB regulon candidates in B. subtilis are mainly involved in coping with stress but are also engaged in other cellular processes. Orthologs of SigB regulon candidates with SigB PBMs were identified in other Bacillales genomes, but not all showed a SigB PBM. Additionally, genes involved in the integration of stress signals to activate SigB were predicted in these genomes, indicating that SigB signaling and regulon genes are species-specific.

The entire SigB regulatory network is sophisticated and not yet fully understood even for the well-characterized organism B. subtilis 168. Knowledge and information gained in this study can be used in further SigB studies to uncover a complete picture of the role of SigB in B. subtilis and other species.