Clinically Relevant Mutations of Mycobacterial GatCAB Inform Regulation of Translational Fidelity

ABSTRACT Most bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNAGln and tRNAAsn. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction in the second step of this pathway. We have previously demonstrated in mycobacteria tha...

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Autores principales:	Yang-Yang Li, Rong-Jun Cai, Jia-Ying Yang, Tamara L. Hendrickson, Ye Xiang, Babak Javid
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2021
Materias:	GatCAB translational fidelity mycobacterium transamidosome mistranslation Mycobacterium tuberculosis Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/8667954f6d2a4905887e75541bdb280d
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spelling	oai:doaj.org-article:8667954f6d2a4905887e75541bdb280d2021-11-10T18:37:50ZClinically Relevant Mutations of Mycobacterial GatCAB Inform Regulation of Translational Fidelity10.1128/mBio.01100-212150-7511https://doaj.org/article/8667954f6d2a4905887e75541bdb280d2021-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01100-21https://doaj.org/toc/2150-7511ABSTRACT Most bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNAGln and tRNAAsn. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction in the second step of this pathway. We have previously demonstrated in mycobacteria that this two-step pathway is error prone and translational errors contribute to adaptive phenotypes such as antibiotic tolerance. Furthermore, we identified clinical isolates of the globally important pathogen Mycobacterium tuberculosis with partial loss-of-function mutations in gatA, and demonstrated that these mutations result in high, specific rates of translational error and increased rifampin tolerance. However, the mechanisms by which these clinically derived mutations in gatA impact GatCAB function were unknown. Here, we describe biochemical and biophysical characterization of M. tuberculosis GatCAB, containing either wild-type gatA or one of two gatA mutants from clinical strains. We show that these mutations have minimal impact on enzymatic activity of GatCAB; however, they result in destabilization of the GatCAB complex as well as that of the ternary asparaginyl-transamidosome. Stabilizing complex formation with the solute trehalose increases specific translational fidelity of not only the mutant strains but also of wild-type mycobacteria. Therefore, our data suggest that alteration of GatCAB stability may be a mechanism for modulation of translational fidelity. IMPORTANCE Most bacteria use a two-step indirect pathway to aminoacylate tRNAGln and tRNAAsn, despite the fact that the indirect pathway consumes more energy and is error prone. We have previously shown that the higher protein synthesis errors from this indirect pathway in mycobacteria allow adaptation to hostile environments such as antibiotic treatment through generation of novel alternate proteins not coded by the genome. However, the precise mechanisms of how translational fidelity is tuned were not known. Here, we biochemically and biophysically characterize the critical enzyme of the Mycobacterium tuberculosis indirect pathway, GatCAB, as well as two mutant enzymes previously identified from clinical isolates that were associated with increased mistranslation. We show that the mutants dysregulate the pathway via destabilizing the enzyme complex. Importantly, increasing stability improves translational fidelity in both wild-type and mutant bacteria, demonstrating a mechanism by which mycobacteria may tune mistranslation rates.Yang-Yang LiRong-Jun CaiJia-Ying YangTamara L. HendricksonYe XiangBabak JavidAmerican Society for MicrobiologyarticleGatCABtranslational fidelitymycobacteriumtransamidosomemistranslationMycobacterium tuberculosisMicrobiologyQR1-502ENmBio, Vol 12, Iss 4 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	GatCAB translational fidelity mycobacterium transamidosome mistranslation Mycobacterium tuberculosis Microbiology QR1-502
spellingShingle	GatCAB translational fidelity mycobacterium transamidosome mistranslation Mycobacterium tuberculosis Microbiology QR1-502 Yang-Yang Li Rong-Jun Cai Jia-Ying Yang Tamara L. Hendrickson Ye Xiang Babak Javid Clinically Relevant Mutations of Mycobacterial GatCAB Inform Regulation of Translational Fidelity
description	ABSTRACT Most bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNAGln and tRNAAsn. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction in the second step of this pathway. We have previously demonstrated in mycobacteria that this two-step pathway is error prone and translational errors contribute to adaptive phenotypes such as antibiotic tolerance. Furthermore, we identified clinical isolates of the globally important pathogen Mycobacterium tuberculosis with partial loss-of-function mutations in gatA, and demonstrated that these mutations result in high, specific rates of translational error and increased rifampin tolerance. However, the mechanisms by which these clinically derived mutations in gatA impact GatCAB function were unknown. Here, we describe biochemical and biophysical characterization of M. tuberculosis GatCAB, containing either wild-type gatA or one of two gatA mutants from clinical strains. We show that these mutations have minimal impact on enzymatic activity of GatCAB; however, they result in destabilization of the GatCAB complex as well as that of the ternary asparaginyl-transamidosome. Stabilizing complex formation with the solute trehalose increases specific translational fidelity of not only the mutant strains but also of wild-type mycobacteria. Therefore, our data suggest that alteration of GatCAB stability may be a mechanism for modulation of translational fidelity. IMPORTANCE Most bacteria use a two-step indirect pathway to aminoacylate tRNAGln and tRNAAsn, despite the fact that the indirect pathway consumes more energy and is error prone. We have previously shown that the higher protein synthesis errors from this indirect pathway in mycobacteria allow adaptation to hostile environments such as antibiotic treatment through generation of novel alternate proteins not coded by the genome. However, the precise mechanisms of how translational fidelity is tuned were not known. Here, we biochemically and biophysically characterize the critical enzyme of the Mycobacterium tuberculosis indirect pathway, GatCAB, as well as two mutant enzymes previously identified from clinical isolates that were associated with increased mistranslation. We show that the mutants dysregulate the pathway via destabilizing the enzyme complex. Importantly, increasing stability improves translational fidelity in both wild-type and mutant bacteria, demonstrating a mechanism by which mycobacteria may tune mistranslation rates.
format	article
author	Yang-Yang Li Rong-Jun Cai Jia-Ying Yang Tamara L. Hendrickson Ye Xiang Babak Javid
author_facet	Yang-Yang Li Rong-Jun Cai Jia-Ying Yang Tamara L. Hendrickson Ye Xiang Babak Javid
author_sort	Yang-Yang Li
title	Clinically Relevant Mutations of Mycobacterial GatCAB Inform Regulation of Translational Fidelity
title_short	Clinically Relevant Mutations of Mycobacterial GatCAB Inform Regulation of Translational Fidelity
title_full	Clinically Relevant Mutations of Mycobacterial GatCAB Inform Regulation of Translational Fidelity
title_fullStr	Clinically Relevant Mutations of Mycobacterial GatCAB Inform Regulation of Translational Fidelity
title_full_unstemmed	Clinically Relevant Mutations of Mycobacterial GatCAB Inform Regulation of Translational Fidelity
title_sort	clinically relevant mutations of mycobacterial gatcab inform regulation of translational fidelity
publisher	American Society for Microbiology
publishDate	2021
url	https://doaj.org/article/8667954f6d2a4905887e75541bdb280d
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Clinically Relevant Mutations of Mycobacterial GatCAB Inform Regulation of Translational Fidelity

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