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

Nature Microbiology volume 1, Article number: 16147 (2016) Cite this article

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Abstract

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.

In many bacteria, non-discriminatory glutamyl- and aspartyl-tRNA synthetases naturally misacylate glutamine tRNA to Glu-tRNAGln and asparagine tRNA to Asp-tRNAAsn. The GatCAB enzyme complex is involved in the transamidation of these naturally misacylated tRNAs to their cognate Gln-tRNAGln and Asn-tRNAAsn (refs 22,24). Isogenic mutants differing only in their sequence of gatA (Supplementary Fig. 3a) also showed high mistranslation rates (Fig. 1c), and complementation of the mutant strains with WT gatCA complemented the mistranslation rate phenotype (Supplementary Fig. 4c), verifying that the gatA mutations were necessary and individually sufficient to confer the phenotype. Earlier work had shown that EF-Tu discriminates against a number of misacylated tRNAs, including physiologically misacylated Glu-tRNAGln and Asp-tRNAAsn (refs 2,28), suggesting that even if GatCAB function was suboptimal, these misacylated tRNAs would not be available for translation. However, expression of a non-discriminatory AspRS in trans in E. coli led to substantial rates of mistranslation14, suggesting that the discrimination afforded by EF-Tu in vivo is possibly overwhelmed when concentrations of misacylated tRNA increase over a critical threshold. To determine whether the SNPs caused a loss or aberration of function, we decided to perform complementation of the mutant strain by the cognate mutant allele. Loss of function mutants should be complemented by increased gene dosage, whereas aberrant or gain of function mutants should have their phenotype exacerbated by a second copy of the mutated gatA gene. Complementation of the mutant strains by overexpression of the cognate mutant alleles led to a decrease in the mistranslation and growth defect phenotypes (Fig. 1d,e) suggesting that the gatA mutations probably cause a partial loss of GatCAB activity, leading to increased abundance of misacylated Glu-tRNAGln and Asp-tRNAAsn, thus resulting in increased mistranslation. We next assessed if limitation of WT gatCAB expression would have the same effect. We created a strain that allowed for regulated gatB expression (Supplementary Fig. 3b) and, in this case, depletion of gene expression resulted in increased mistranslation (Fig. 1f). We investigated the expression of gatCA in WT cells and found that cells with decreased gatCA expression showed increased mistranslation specifically of asparagine to aspartate (Fig. 1g), hence cellular variation of GatCAB abundance appears to regulate mistranslation in mycobacteria.

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Figure 1: A forward genetic screen identifies gatCAB as a mediator of specific translational fidelity.
Figure 2: Mutations in gatA result in decreased GatCAB abundance and instability of WT GatB.
Figure 3: High mistranslation from gatA mutations cause rifampicin-specific phenotypic resistance in M. tuberculosis.
Figure 4: Mistranslation of a specific residue of RpoB causes RSPR.

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Acknowledgements

This work was funded in part by the Bill and Melinda Gates Foundation (OPP1109789) and start-up funds from Tsinghua University to B.J. B.J. and T.F.Z. are Tsinghua-Janssen scholars. The authors thank S. Fortune, E. Rubin, M. Chao and P. Lehner for their critical reading of the manuscript, and C. Koser and P. Abel Zur Wiesch for helpful discussions. The authors also acknowledge technical assistance from the microbial sorting facility at Peking University, and thank the Clinical Database and Sample Bank of Tuberculosis of Beijing (D131100005313012) of the National Clinical Lab on Tuberculosis, Beijing Chest Hospital, for access to their strain collection.

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Author notes

  1. Hong-Wei Su and Jun-Hao Zhu: These authors contributed equally to this work.

Authors and Affiliations

  1. Collaboration Innovation Centre for the Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University, Beijing, 100084, China

    Hong-Wei Su, Jun-Hao Zhu, Hao Li, Rong-Jun Cai, Xun Wang, Yu-Xiang Chen & Babak Javid

  2. School of Life Sciences, Peking University, Beijing, 10087, China

    Jun-Hao Zhu

  3. Faculty of Health Sciences, DST/NRF Centre of Excellence for Biomedical TB Research, University of the Witwatersrand, National Health Laboratory Service, Johannesburg, 2193, South Africa

    Christopher Ealand & Bavesh D. Kana

  4. Center for Synthetic and Systems Biology, MOE Key Laboratory of Bioinformatics, Collaboration Innovation Centre for the Diagnosis and Treatment of Infectious Diseases, School of Life Sciences, Tsinghua University, Beijing 100084, China

    Masood ur Rehman Kayani & Ting F. Zhu

  5. Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK

    Danesh Moradigaravand

  6. National Clinical Laboratory on Tuberculosis, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing Chest Hospital, Capital Medical University, Beijing 101149, China

    Hairong Huang

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Contributions

B.J. conceived and oversaw the design and implementation of the project. H.W.S., J.H.Z., C.E., X.W. and H.L. designed and performed the research and analysed the data. R.J.C. and Y.X.C. made and provided reagents. M.K., T.F.Z. and D.M. analysed whole-genome sequencing data. H.H., B.D.K. and B.J. analysed data. H.W.S., J.H.Z. and B.J. wrote the paper with input from the other authors.

Corresponding author

Correspondence to Babak Javid.

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The editors note that one of the individuals acknowledged for critical reading of the manuscript, M.C., as well as being a former colleague of the corresponding author, is an editor on the staff of Nature Microbiology, but was not in any way involved in the journal review process.

Supplementary information

Supplementary information

Supplementary Figures 1–11, Supplementary Tables 1–5, Supplementary References. (PDF 14754 kb)

Supplementary Tables 6–8

List of primers, strains and plasmids used in this study. (XLSX 17 kb)

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Su, HW., Zhu, JH., Li, H. et al. The essential mycobacterial amidotransferase GatCAB is a modulator of specific translational fidelity. Nat Microbiol 1, 16147 (2016). https://doi.org/10.1038/nmicrobiol.2016.147

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