Dataset Information

The Sole Mycobacterium smegmatis MazF Toxin Targets tRNALys to Impart Highly Selective, Codon-Dependent Proteome Reprogramming.

ABSTRACT: Survival of mycobacteria, both free-living and host-dependent pathogenic species, is dependent on their ability to evade being killed by the stresses they routinely encounter. Toxin-antitoxin (TA) systems are unique to bacteria and archaea and are thought to function as stress survival proteins. Here, we study the activity of the endoribonuclease toxin derived from the MazEF TA system in Mycobacterium smegmatis, designated MazEF-ms. We first enlisted a specialized RNA-seq method, 5' RNA-seq, to identify the primary RNA target(s) of the MazF-ms toxin. Just two tRNA species, tRNALys-UUU and tRNALys-CUU, were targeted for cleavage by MazF-ms at a single site within their anticodon sequence (UU?U and CU?U) to render these tRNAs nonfunctional for protein synthesis. The 5' RNA-seq dataset also revealed hallmarks of ribosome stalling predominantly at Lys AAA codons even though both Lys tRNAs were cleaved by MazF-ms. Stalled ribosomes were then cleaved on their 5' side by one or more RNases, resulting in very selective degradation of only those mRNAs harboring ribosomes stalled at Lys codons. This highly surgical, codon-dependent degradation of mRNA transcripts was validated using quantitative mass spectrometry of proteins that were newly synthesized during MazF-ms expression. The M. smegmatis proteome was altered as predicted, Lys AAA codon-rich proteins was downregulated while Lys AAA codon deficient proteins were upregulated. Analysis of specific subsets of proteins that were upregulated or downregulated was consistent with the growth-arrested phenotype of MazF-ms expressing cells. Curiously, the tRNA target and mechanism of action of MazF-ms paralleled that of one atypical MazF toxin in M. tuberculosis, suggesting manipulation of the levels of lysine tRNAs as the preferred conduit for reprogramming the proteomes via ribosome stalling at rare AAA codons in these GC-rich mycobacteria.

SUBMITTER: Barth VC

PROVIDER: S-EPMC7033543 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The Sole Mycobacterium smegmatis MazF Toxin Targets tRNALys to Impart Highly Selective, Codon-Dependent Proteome Reprogramming.

Barth Valdir Cristovao VC Woychik Nancy A NA

Frontiers in genetics 20200214

Survival of mycobacteria, both free-living and host-dependent pathogenic species, is dependent on their ability to evade being killed by the stresses they routinely encounter. Toxin-antitoxin (TA) systems are unique to bacteria and archaea and are thought to function as stress survival proteins. Here, we study the activity of the endoribonuclease toxin derived from the MazEF TA system in Mycobacterium smegmatis, designated MazEF-ms. We first enlisted a specialized RNA-seq method, 5' RNA-s ...[more]

PMID: 32117414

Similar Datasets

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

The Sole Mycobacterium smegmatis MazF Toxin Targets tRNALys to Impart Highly Selective, Codon-Dependent Proteome Reprogramming.

Publications

The Sole <i>Mycobacterium smegmatis</i> MazF Toxin Targets tRNA<sup>Lys</sup> to Impart Highly Selective, Codon-Dependent Proteome Reprogramming.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets