Abstract

Most bacteria, including mycobacteria, utilize a two-step indirect tRNA aminoacylation pathway to generate correctly aminoacylated glutaminyl and asparaginyl tRNAs. This involves an initial step in which a non-discriminatory aminoacyl tRNA synthetase misacylates the tRNA, followed by a second step in which the essential amidotransferase, GatCAB, amidates the misacylated tRNA to its correct, cognate form. It had been previously demonstrated that mutations in gatA can mediate increased error rates specifically of glutamine to glutamate or asparagine to aspartate in protein synthesis. However, the role of mutations in gatB or gatC in mediating mistranslation are unknown. Here, we applied a forward genetic screen to enrich for mistranslating mutants of Mycobacterium smegmatis. The majority (57/67) of mutants had mutations in one of the gatCAB genes. Intriguingly, the most common mutation identified was an insertion in the 3′ of gatC, abolishing its stop codon, and resulting in a fused GatC-GatA polypeptide. Modeling the effect of the fusion on GatCAB structure suggested a disruption of the interaction of GatB with the CCA-tail of the misacylated tRNA, suggesting a potential mechanism by which this mutation may mediate increased translational errors. Furthermore, we confirm that the majority of mutations in gatCAB that result in increased mistranslation also cause increased tolerance to rifampicin, although there was not a perfect correlation between mistranslation rates and degree of tolerance. Overall, our study identifies that mutations in all three gatCAB genes can mediate adaptive mistranslation and that mycobacteria are extremely tolerant to perturbation in the indirect tRNA aminoacylation pathway.

Highlights

  • The vast majority of bacteria lack the specific aminoacyl tRNA synthetases coding for glutaminyl-tRNA (GlnRS) and/or asparaginyl-tRNA (AsnRS) synthetases or both (Curnow et al, 1997; Sheppard and Soll, 2008)

  • We identified mutations in mycobacterial gatA that caused increased rates of mistranslation (Su et al, 2016), and showed that mutations in gatA identified from clinical isolates of M. tuberculosis caused mistranslation, and importantly, tolerance to the antibiotic rifampicin (Su et al, 2016)

  • Our results suggest that mutations in all three constituent members of gatCAB may result in adaptive mistranslation, and that alteration of the native conformation of the essential gene products GatC and GatA results in viable bacteria, but with altered translational fidelity

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Summary

INTRODUCTION

The vast majority of bacteria lack the specific aminoacyl tRNA synthetases (aaRSs) coding for glutaminyl-tRNA (GlnRS) and/or asparaginyl-tRNA (AsnRS) synthetases or both (Curnow et al, 1997; Sheppard and Soll, 2008). A non-discriminatory glutamyl-tRNA (ND-GluRS) or aspartyl-tRNA (ND-AspRS) aminoacyl-tRNA synthetase physiologically misacylates tRNAGln to Glu-tRNAGln and tRNAAsn to Asp-tRNAAsn, respectively In both cases, the misacylated tRNA complex is recognized by the products encoded by the essential heterotrimeric amidotransferase genes gatCAB. We identified mutations in mycobacterial gatA that caused increased rates of mistranslation (Su et al, 2016), and showed that mutations in gatA identified from clinical isolates of M. tuberculosis caused mistranslation, and importantly, tolerance to the antibiotic rifampicin (Su et al, 2016) This was the first identification of naturally occurring mutations from clinical bacterial isolates that supported a role for “adaptive mistranslation” (De Pouplana et al, 2014; Rathnayake et al, 2017). We identified mutations in gatC, gatA, and gatB that caused both specific mistranslation and rifampicin tolerance. Our results suggest that mutations in all three constituent members of gatCAB may result in adaptive mistranslation, and that alteration of the native conformation of the essential gene products GatC and GatA results in viable bacteria, but with altered translational fidelity

MATERIALS AND METHODS
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