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

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 mc 2 155 and mc 2 155 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 mc 2 155 and M. smegmatis mc 2 155 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 singlestranded 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.

Single START-domain protein Mtsp17 is involved in transcriptional regulation in Mycobacterium smegmatis

“…The sigma subunit ensures the transcription machinery to initiate transcription of particular genes [ 16 , 17 ]. Sigma factors are post-translationally regulated by a partner switching system (PSS) in which anti-sigma factors sequester the sigma factor from the transcription machinery and anti-sigma factor antagonists lift the repression of sigma factors [ 18 – 20 ].…”

“…MSMEG_0586, an anti-SigF antagonist, was one of the top 20 differentially expressed genes, showing an 8.9-fold decrease (log 2 fold change = -3.16, adjusted p-value = 7.3E-40) in the M0129C_T0 versus M0129C_T20 comparison, while other sigma factors or anti-sigma factors or anti-anti-sigma factors showed log 2 fold changes of no more than 1.5. MSMEG_0586 is involved in the PSS of the SigF regulatory pathway [ 20 ]. In light of significant down-regulation of the anti-SigF antagonist MSMEG_0586 and no obvious change in other SigF PSS factors in the conditional Mtsp17 knockout strain, we hypothesized that transcriptomic changes partially overlapped between the conditional Mtsp17 knockout and the SigF knockout strains.…”

“…Since MSMEG_0586 is as an activator of SigF by post-translational regulation and 70 of 79 DEGs with log 2 fold changes > 1.5 belong to the SigF regulon ( S5 Table ), transcriptional changes in these SigF regulon genes can be explained by alterations in MSMEG_0586 expression by Mtsp17 and lead to post-translational regulation of the SigF regulon by the PSS of the SigF regulatory pathway. As both SigF and Mtsp17 are conserved in the Mycobacteria and are transcriptionally regulated in response to nutrient depletion and oxidative stress [ 10 , 20 ], we propose that the regulation of the SigF regulon by Mtsp17 may operate in conjunction with the SigF regulatory pathway during stress responses.…”