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.

Keywords: GatCAB; Mycobacterium; amidotransferase; antibiotic tolerance; mistranslation; persisters.

FIGURE 1

A forward genetic screen identifies a GatC-GatA fusion that mediates high rates of mistranslation. (A) Cartoon schematic of the selection strategy and subsequent phenotypic experiments. A reporter strain of M. smegmatis was plated on low-dose kanamycin agar to select for high mistranslation mutants (see section “Materials and Methods”). Serving strains had gatCAB genes sequenced. A selection of mutants were further characterized to measure mistranslation rates and rifampicin phenotypic resistance. (B) Relative frequency of mutants identified in the screen (C) Cartoon illustrating the mutation that abolished the stop codon of gatC and resulted in fusion of GatC-GatA. The wild-type genes are depicted in the top and the mutant below. (D) Western blot of GatA of wild-type, GatC-GatA fusion and a GatB mutated strains. EF-Tu as loading control. (E) Relative rates of asparagine to aspartate mistranslation using an Nluc/GFP reporter system (see section “Materials and Methods”). *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t-test.

FIGURE 2

Modeling suggests GatC-GatA fusion disrupts GatCAB structure. Ribbon model of wild-type and GatC-GatA fusion M. smegmatis GatCAB and tRNA^Asn using Rosetta (see section “Materials and Methods”). The inset shows a close-up of the interaction between GatC, the beta-hairpin of GatB and the 3′-CCA of the tRNA. For clarity, the terminal amino acids ILGEPE of wild-type GatCA are not shown in the inset.

FIGURE 3

Increased Rifampicin-specific phenotypic resistance (RSPR) from GatCAB mutations. (A) Fluorescence-dilution assay: Wild-type and mutant strains of M. smegmatis were stained with NADA and grown in axenic culture + 50 μg/mL rifampicin overnight, and then loss of fluorescence measured by flow cytometry (see section “Materials and Methods”). (B) Wild-type and mutant strains of M. smegmatis were plated on 25 μg/mL rifampicin-agar and colonies counted after 5 days, compared with growth on LB agar alone. All experiments were performed with biological triplicates and data show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ns p > 0.05 by Student’s t-test.