Home Alone: Elimination of All but One Alternative Sigma Factor in Listeria monocytogenes Allows Prediction of New Roles for σB

Abstract

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

Transcriptome Sequencing of Listeria monocytogenes Reveals Major Gene Expression Changes in Response to Lactic Acid Stress Exposure but a Less Pronounced Response to Oxidative Stress

“…Interestingly, differential expression analyses, including the one conducted in this study, consistently show a pattern of rli47 upregulation coupled with the concurrent downregulation of lap. This pattern is present during stationary phase (Oliver et al, 2009;Toledo-Arana et al, 2009;Mujahid et al, 2012), the overexpression of σ B (Liu et al, 2017), multiplication in the gut (Toledo-Arana et al, 2009), and in macrophages (Chatterjee et al, 2006;Mraheil et al, 2011). Furthermore, lap was the highest upregulated gene (29.9fold) in an rli47 deletion mutant (Marinho et al, 2019), suggesting that Rli47 is somehow involved in lap expression.…”

“…Interestingly, an extremely high percentage of all transcriptome reads from the lactic acid replicates (averaging 28 and 38% for 6179 and R479a, respectively) mapped to the non-coding RNA rli47 (sbrE), a member of the σ B regulon (Figure 4) (Oliver et al, 2009;Toledo-Arana et al, 2009;Mujahid et al, 2012;Liu et al, 2017). Expression of rli47 significantly increased during exposure to lactic acid, with a log2fold change of 8.47 in 6179 and 9.97 in R479a.…”

Initial Transcriptomic Response and Adaption of Listeria monocytogenes to Desiccation on Food Grade Stainless Steel

“…The upregulation of sigB in Lm 08-5578 led to members of the σ B -regulon being overrepresented (p = 2.53 × 10 −16 ) during desiccation with significantly upregulation of at least 85 σ B -regulon members that were not upregulated in Lm 568 ( Figure 7A). Examples of upregulated genes in the present study ( Supplementary Table S2) include the carnitine osmolyte uptake system opuCABCD (Sue et al, 2004), pyrimidine biosynthesis genes pyrABCDEFP (Liu et al, 2017b), carbohydrate metabolism genes with the PTS-mannose operon mpoABCD, universal stress proteins uspA, central glycolytic genes regulator cggR, stress response protein csbD, general stress protein 26 (lmo2748), Listeria adhesion protein lapB and succinate-semialdehyde dehydrogenase gabD (Orsi et al, 2015).…”

“…Removal of water from the cell surface of desiccating L. monocytogenes led to increased expression of genes related to oxidative stress including several known members of the σ B -regulon. Among these were the superoxide dismutase encoding gene, sod (Liu et al, 2017b), which was threefold and fourfold upregulated in Lm 568 and Lm 08-5578, respectively (Supplementary Table S2). Superoxide dismutase (Sod) protects L. monocytogenes from reactive oxygen species (ROS) encountered during oxidative stress and is important for virulence with sod mutants having impaired intracellular survival (Archambaud et al, 2006).…”

Role and regulation of the stress activated sigma factor sigma B (σB) in the saprophytic and host-associated life stages of Listeria monocytogenes

“…In addition to the housekeeping sigma factor σ A and the general stress response regulator σ B , the genome of L. monocytogenes has up to three additional alternative sigma factors (σ C , σ H , and σ L ) (Glaser et al, 2001). While σ B is the primary regulator of the expression of general stress response genes, crucial in the survival of challenging environments (O'Byrne & Karatzas, 2008), homeostasis and resilience (Liu, Orsi, Boor, Wiedmann & Guariglia-Oropeza, 2017), the regulons controlled by other alternative sigma factors are comparatively smaller and less well defined. σ C , which is specific to L. monocytogenes lineage II strains, has a small regulon (<10 genes) that contribute to heat stress resistance (Zhang, Nietfeldt, Zhang & Benson, 2005).…”