Home Alone: Elimination of All but One Alternative Sigma Factor in Allows Prediction of New Roles for σ.

Among four alternative σ factors, σ controls the largest regulon. As σ-dependent transcription of some genes may be masked by overlaps among regulons, and as some σ-dependent genes are expressed only under very specific conditions, we hypothesized that the σ regulon is not yet fully defined. To further extend our understanding of the σ regulon, we used RNA-seq to identify σ-dependent genes in an strain that expresses σ following rhamnose induction, and in which genes encoding the other alternative sigma factors have been deleted. Analysis of RNA-seq data with multiple bioinformatics approaches, including a sliding window method that detects differentially transcribed 5' untranslated regions (UTRs), identified 105 σ-dependent transcription units (TUs) comprising 201 genes preceded by σ-dependent promoters. Of these 105 TUs, 7 TUs comprising 15 genes had not been identified previously as σ-dependent. An additional 23 genes not reported previously as σ-dependent were identified in 9 previously recognized σ-dependent TUs. Overall, 38 of these 201 genes had not been identified previously as members of the σ regulon. These newly identified σ-dependent genes encode proteins annotated as being involved in transcriptional regulation, oxidative and osmotic stress response, and in metabolism of energy, carbon and nucleotides. In total, 18 putative σ-dependent promoters were newly identified. Interestingly, a number of genes previously identified as σ-dependent did not show significant evidence for σ-dependent transcription in our experiments. Based on promoter analyses, a number of these genes showed evidence for co-regulation by σ and other transcriptional factors, suggesting that some σ-dependent genes require additional transcriptional regulators along with σ for transcription. Over-expression of a single alternative sigma factor in the absence of all other alternative sigma factors allowed us to: (i) identify new σ-dependent functions in , such as regulation of genes involved in 1,2-propanediol utilization (LMRG_00594-LMRG_00611) and biosynthesis of pyrimidine nucleotides (LMRG_00978-LMRG_00985); and (ii) identify new σ-dependent genes involved in stress response and pathogenesis functions. These data further support that σ not only regulates stress response functions, but also plays a broad role in homeostasis and resilience.