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

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.

Ribosomal RNA methylation by GidB modulates discrimination of mischarged tRNA

“…We had previously identified via forward genetic screens strains of Mycobacterium smegmatis with extremely high specific rates of mistranslation due to mutations in the essential amidotransferase genes gatCAB (Cai et al, 2020; Su et al, 2016). We hypothesized that the mycobacterial genome may encode for other ‘fidelity factors’ that may either enhance or reduce mistranslation specifically involving the indirect tRNA aminoacylation pathway.…”

“…In this study we sought to identify further translational fidelity factors in mycobacteria, which we had previously shown have high, but specific rates of mistranslation due to the indirect tRNA aminoacylation pathway required for aminoacylation of glutaminyl- and asparaginyl-tRNAs (Cai et al, 2020; Chaudhuri et al, 2018; Su et al, 2016). Our screen identified loss-of-function mutations in gidB conferring increased fidelity to errors generated by mutants in this pathway.…”

“…The critical enzyme in the second step of the indirect pathway is GatCAB, whose function is to correct the mischarged aminoacyl moiety (Curnow et al, 1997). Despite its essential function, partial loss-of-function mutations in gatCAB can be readily selected in mycobacteria, occur naturally in clinical isolates of pathogenic M. tuberculosis (Cai et al, 2020; Su et al, 2016), and these mutant strains exhibit extremely high (up to 10%/codon) but specific rates of mistranslation (Su et al, 2016). Here, we investigated whether further fidelity factors can be identified in mycobacteria.…”

The mutational signatures of poor treatment outcomes on the drug-susceptibleMycobacterium tuberculosisgenome

“…The frequency of these mutations concentrated in the range of 5%-10%, and they were predominantly found in genes whose encoded proteins are involved in cell wall and cell processes, intermediary metabolism and respiration (Figure 2B). When the genes carrying unfixed mutations were ranked according to the significance of the mutations' association with poor outcomes, the highest ranked gene was gatA, which has been associated with rifampicin tolerance (Cai et al, 2020) (Figure 2C). Gene expression patterns under stress conditions can provide important insights into the function of the gene (Bosch et al, 2021).…”

Rifamycin antibiotics and the mechanisms of their failure

“…Su et al found that M. smegmatis experiences increased levels of mistranslation with reduced activity of GatCAB, which is an enzyme that transamidates glutamine and asparagine tRNAs that had been improperly acylated with glutamate and aspartate, respectively [111]. Strains of M. smegmatis with lower GatCAB activity not only exhibited high levels of mistranslation but also high rates of phenotypic resistance to rifampicin [117]. Further analysis revealed that rifampicin phenotypic resistance was lower in strains where residue 434 of RpoB, which is normally an asparagine, was changed to a threonine [111].…”