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.
Highlights
Listeria monocytogenes is a Gram-positive foodborne pathogen that causes the serious invasive disease listeriosis, predominantly in susceptible populations such as the immunocompromised, pregnant women, and adults over 65 years old (Goulet et al, 2012)
Using RNA sequencing (RNA-seq) data from an L. monocytogenes strain with deletions in genes encoding all four alternative sigma factors ( BCHL) as well as from a corresponding isogenic strain where sigB was re-introduced under rhamnose regulation ( BCHLPrha-sigB), we employed two separate bioinformatics approaches to identify σB-dependent genes
Our analyses identified 105 σB-dependent transcription unit (TU) (i.e., TUs upregulated in the presence of σB) that are preceded by σBdependent promoters; this includes (i) 93 TUs that showed significant differential transcript levels in one or multiple open reading frame (ORF) that are part of a TU and (ii) 12 TUs that showed significant differential transcript levels in a fragment located in the 5′untranslated region (UTR)
Summary
Listeria monocytogenes is a Gram-positive foodborne pathogen that causes the serious invasive disease listeriosis, predominantly in susceptible populations such as the immunocompromised, pregnant women, and adults over 65 years old (Goulet et al, 2012). In the US, L. monocytogenes causes around 1,600 human listeriosis cases resulting in ∼260 deaths annually (Scallan et al, 2011). Alternative sigma factors represent one key regulatory mechanism that allows bacteria to adjust rapidly to different environments. Differential association between alternative sigma factors and core RNA polymerase allows the RNA polymerase to recognize specific promoter sequences and initiate transcription of targeted genes under specific conditions. The four alternative sigma factors regulate transcription of genes important for virulence and for response to various stress and growth conditions (Chaturongakul et al, 2011). The general stress response regulator σB is the most extensively studied alternative sigma factor in L. monocytogenes; σB has been shown to control a regulon of more than 180 genes (Raengpradub et al, 2008). ΣB plays important roles in virulence and stress response, including transition to stationary phase and resistance to acid, osmotic, arsenate, oxidative, and cold stresses (O’byrne and Karatzas, 2008; Mujahid et al, 2013b)
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