Dataset Information

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 mc2155 and mc2155 ?phoH2 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 ?phoH2 during normal growth. At 48 hours, elevated expression of the sigF regulon was observed in ?phoH2 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.

SUBMITTER: Andrews ESV

PROVIDER: S-EPMC7390352 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Andrews Emma S V ESV Rzoska-Smith Elizabeth E Arcus Vickery L VL

PloS one 20200729 7

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, e ...[more]

PMID: 32726339

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Project description:Mycobacterium smegmatis SigF is a group III sigma factor. Its ortholog in M. tuberculosis is reported to have role in regulation and function of cell wall components. In present study we have created an M. smegmatis M-NM-^TsigF mutant by allele exchange method. M. smegmatis sigF mutant shows non pigmented phenotype and is more sensitive to hydrogen peroxide generated oxidative stress. DNA microarray analysis of M. smegmatis wild type and M-NM-^TsigF mutant suggests that SigF in this species controls the expression of several energy and central intermediary metabolism genes along with regulation of carotenoid biosynthesis. Gene expression patterns of M.smegmatis wild type and M-NM-^TsigF mutant strains were compared at two growth stages i.e. log (OD600 ~1.4) and stationary (OD600 ~3.0). M. smegmatis strains were grown in DifcoTM MiddleBrook 7H9 broth base (BD Biosciences, Sparks, MD, USA) with 0.2% glycerol (v/v) and 0.05% Tween-80 (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% albumen dextrose catalase (BD Biosciences) (v/v) at 37 0C with continuous shaking. Total RNA was isolated using Trizole (Invitrogen) method and labelled with cyanine 3 (Cy3) as per Agilent 1-color labelling protocol (Version 5.5, February 2007). Six hundred nanograms of each Cy3 labelled samples were fragmented and hybridized. Fragmentation of labeled cRNA and hybridization were performed using Gene Expression Hybridization kit (Agilent Technologies). Hybridization was carried out in AgilentM-bM-^@M-^Ys Surehyb Chambers at 65 0C for 16 h. The hybridized slides were washed using Agilent Gene Expression wash buffers and scanned using the Agilent Microarray Scanner G Model G2565BA at 5 micron resolution. Feature extracted data was analysed using GeneSpring GX v 7.3.1 software from Agilent. Normalization of the data was done in GeneSpring GX using the recommended one color Per Chip and Per Gene Data Transformation. Set measurements less than 0.01 to 0.01 per chip, normalize to 50th percentile per gene, and normalize to specific samples.

Project description:Toxin-antitoxin (TA) systems are ubiquitous genetic elements in prokaryotes, but their biological importance is poorly understood. Mycobacterium smegmatis contains eight putative TA systems. Previously, seven TAs have been studied, with five of them being verified as functional. Here, we show that Ms0251-0252 is a novel TA system in that expression of the toxin Ms0251 leads to growth inhibition that can be rescued by the antitoxin Ms0252. To investigate the functional roles of TA systems in M. smegmatis, we deleted the eight putative TA loci and assayed the mutants for resistance to various stresses. Deletion of all eight TA loci resulted in decreased survival under starvation conditions and altered fitness when exposed to environmental stresses. Furthermore, we showed that deletion of the eight TA loci decreased resistance to phage infection in Sauton medium compared with the results using 7H10 medium, suggesting that TA systems might have different contributions depending on the nutrient environment. Furthermore, we found that MazEF specifically played a dominant role in resistance to phage infection. Finally, transcriptome analysis revealed that MazEF overexpression led to differential expression of multiple genes, including those related to iron acquisition. Altogether, we demonstrate that TA systems coordinately function to allow M. smegmatis to adapt to changing environmental conditions. IMPORTANCE Toxin-antitoxin (TA) systems are mechanisms for rapid adaptation of bacteria to environmental changes. Mycobacterium smegmatis, a model bacterium for studying Mycobacterium tuberculosis, encodes eight putative TA systems. Here, we constructed an M. smegmatis mutant with deletions of all eight TA-encoding genes and evaluated the resistance of these mutants to environmental stresses. Our results showed that different TA systems have overlapping and, in some cases, opposing functions in adaptation to various stresses. We suggest that complementary TA modules may function together to regulate the bacterial stress response, enabling adaptation to changing environments. Together, this study provides key insights into the roles of TA systems in resistance to various environmental stresses, drug tolerance, and defense against phage infection.

Dataset Information

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

Publications

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

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