Post-transcriptional modulation of the SigF regulon in Mycobacterium smegmatis by the PhoH2 toxin-antitoxin.

Emma S V Andrews,Vickery L Arcus,Elizabeth Rzoska-Smith,Horacio Bach

doi:10.1371/journal.pone.0236551

Abstract

PhoH2 proteins are highly conserved across bacteria and archaea yet their biological function is poorly characterised. We examined the growth profiles of Mycobacterium smegmatis strains mc2155 and mc2155 ΔphoH2 and observed the same growth profile and growth rate in a variety of conditions. In light of the comparable growth, we used RNAseq to provide a snapshot of the differences between the transcriptomes of M. smegmatis mc2155 and M. smegmatis mc2155 ΔphoH2 during normal growth. At 48 hours, elevated expression of the sigF regulon was observed in ΔphoH2 relative to wild type. In biochemical assays, PhoH2 showed activity toward sigF mRNA insinuating a role of PhoH2 in modulating the pool of sigF mRNA in the cell during normal growth, adding further complexity to the repertoire of reported mechanisms of post-translational regulation. Multiple copies of the preferred target site of PhoH2 were identified in loops of the sigF mRNA structure, leading us to propose a mechanism for the activity of PhoH2 that is initiated after assembly on specific single-stranded loops of RNA. We hypothesise that PhoH2 is a toxin-antitoxin that contributes to the regulation of SigF at a post-transcriptional level through targeted activity on sigF mRNA. This work presents the first evidence for post-transcriptional regulation of SigF along with the biological function of PhoH2 from M. smegmatis. This has implications for the highly conserved PhoH2 toxin-antitoxin module across the mycobacteria including the important human pathogen M. tuberculosis.

Highlights

Sigma factors initiate gene expression through their interaction with RNAP [1]
To screen for differences in the growth profile of M. smegmatis mc2155 ΔphoH2 (S1 Fig) compared with its parent, M. smegmatis mc2155, strains of M. smegmatis were grown in standard rich, defined and nutrient limiting conditions and growth measured by optical density (OD600)
RNAseq reveals an upregulation of SigF regulon genes in M. smegmatis mc2155 ΔphoH2 at 48 hours

Summary

Introduction

Sigma factors initiate gene expression through their interaction with RNAP [1]. Their function directs the binding of RNAP to specific promoter sites to initiate transcription of specific subsets of genes [1]. Sigma factors SigA and SigB are responsible for the expression of essential genes [2]. Alternate sigma factors function to coordinate gene regulation in response to different environmental stresses and changing physiological conditions [3]. One such alternate sigma factor, SigF, is involved in the cell’s adaptation to stationary phase, heat, oxidative stress and antimicrobials [4,5,6].

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Results

Conclusion