Home Alone: Elimination of All but One Alternative Sigma Factor in Listeria monocytogenes Allows Prediction of New Roles for σ(B)

Among Listeria monocytogenes' four alternative σ factors, σ(B) controls the largest regulon. As σ(B)-dependent transcription of some genes may be masked by overlaps among regulons, and as some σ(B)-dependent genes are expressed only under very specific conditions, we hypothesized that the σ(B) regulon is not yet fully defined. To further extend our understanding of the σ(B) regulon, we used RNA-seq to identify σ(B)-dependent genes in an L. monocytogenes strain that expresses σ(B) 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(B)-dependent transcription units (TUs) comprising 201 genes preceded by σ(B)-dependent promoters. Of these 105 TUs, 7 TUs comprising 15 genes had not been identified previously as σ(B)-dependent. An additional 23 genes not reported previously as σ(B)-dependent were identified in 9 previously recognized σ(B)-dependent TUs. Overall, 38 of these 201 genes had not been identified previously as members of the L. monocytogenes σ(B) regulon. These newly identified σ(B)-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(B)-dependent promoters were newly identified. Interestingly, a number of genes previously identified as σ(B)-dependent did not show significant evidence for σ(B)-dependent transcription in our experiments. Based on promoter analyses, a number of these genes showed evidence for co-regulation by σ(B) and other transcriptional factors, suggesting that some σ(B)-dependent genes require additional transcriptional regulators along with σ(B) 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(B)-dependent functions in L. monocytogenes, 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(B)-dependent genes involved in stress response and pathogenesis functions. These data further support that σ(B) not only regulates stress response functions, but also plays a broad role in L. monocytogenes homeostasis and resilience.