Clinically Relevant Mutations of Mycobacterial GatCAB Inform Regulation of Translational Fidelity.

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.

Forward Genetics Reveals a gatC-gatA Fusion Polypeptide Causes Mistranslation and Rifampicin Tolerance in Mycobacterium smegmatis.

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.

The essential mycobacterial amidotransferase GatCAB is a modulator of specific translational fidelity.

Although regulation of translation fidelity is an essential process1-7, diverse organisms and organelles have differing requirements of translational accuracy8-15, and errors in gene translation serve an adaptive function under certain conditions16-20. Therefore, optimal levels of fidelity may vary according to context. Most bacteria utilize a two-step pathway for the specific synthesis of aminoacylated glutamine and/or asparagine tRNAs, involving the glutamine amidotransferase GatCAB21-25, but it had not been appreciated that GatCAB may play a role in modulating mistranslation rates. Here, by using a forward genetic screen, we show that the mycobacterial GatCAB enzyme complex mediates the translational fidelity of glutamine and asparagine codons. We identify mutations in gatA that cause partial loss of function in the holoenzyme, with a consequent increase in rates of mistranslation. By monitoring single-cell transcription dynamics, we demonstrate that reduced gatCAB expression leads to increased mistranslation rates, which result in enhanced rifampicin-specific phenotypic resistance. Consistent with this, strains with mutations in gatA from clinical isolates of Mycobacterium tuberculosis show increased mistranslation, with associated antibiotic tolerance, suggesting a role for mistranslation as an adaptive strategy in tuberculosis. Together, our findings demonstrate a potential role for the indirect tRNA aminoacylation pathway in regulating translational fidelity and adaptive mistranslation.