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

Abstract

Although regulation of translation fidelity is an essential process17, diverse organisms and organelles have differing requirements of translational accuracy815, and errors in gene translation serve an adaptive function under certain conditions1620. 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 GatCAB2125, 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.

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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.

References

  1. 1

    Bullwinkle, T. J. et al. Oxidation of cellular amino acid pools leads to cytotoxic mistranslation of the genetic code. Elife 3, e02501 (2014).

    Article  PubMed Central  Google Scholar 

  2. 2

    LaRiviere, F. J., Wolfson, A. D. & Uhlenbeck, O. C. Uniform binding of aminoacyl-tRNAs to elongation factor Tu by thermodynamic compensation. Science 294, 165–168 (2001).

    CAS  Article  PubMed  Google Scholar 

  3. 3

    Lee, J. W. et al. Editing-defective tRNA synthetase causes protein misfolding and neurodegeneration. Nature 443, 50–55 (2006).

    CAS  Article  PubMed  Google Scholar 

  4. 4

    Liu, Y. et al. Deficiencies in tRNA synthetase editing activity cause cardioproteinopathy. Proc. Natl Acad. Sci. USA 111, 17570–17575 (2014).

    CAS  Article  PubMed  Google Scholar 

  5. 5

    Lu, J., Bergert, M., Walther, A. & Suter, B. Double-sieving-defective aminoacyl-tRNA synthetase causes protein mistranslation and affects cellular physiology and development. Nat. Commun. 5, 5650 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6

    Reynolds, N. M., Lazazzera, B. A. & Ibba, M. Cellular mechanisms that control mistranslation. Nat. Rev. Microbiol. 8, 849–856 (2010).

    CAS  Article  PubMed  Google Scholar 

  7. 7

    Zaher, H. S. & Green, R. Quality control by the ribosome following peptide bond formation. Nature 457, 161–166 (2009).

    CAS  Article  PubMed  Google Scholar 

  8. 8

    Leng, T., Pan, M., Xu, X. & Javid, B. Translational misreading in Mycobacterium smegmatis increases in stationary phase. Tuberculosis 95, 678–681 (2015).

    CAS  Article  PubMed  Google Scholar 

  9. 9

    Li, L. et al. Naturally occurring aminoacyl-tRNA synthetases editing-domain mutations that cause mistranslation in Mycoplasma parasites. Proc. Natl Acad. Sci. USA 108, 9378–9383 (2011).

    CAS  Article  PubMed  Google Scholar 

  10. 10

    Ling, J., O'Donoghue, P. & Soll, D. Genetic code flexibility in microorganisms: novel mechanisms and impact on physiology. Nat. Rev. Microbiol. 13, 707–721 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11

    Netzer, N. et al. Innate immune and chemically triggered oxidative stress modifies translational fidelity. Nature 462, 522–526 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12

    Reynolds, N. M. et al. Cell-specific differences in the requirements for translation quality control. Proc. Natl Acad. Sci. USA 107, 4063–4068 (2010).

    CAS  Article  PubMed  Google Scholar 

  13. 13

    Ribas de Pouplana, L., Santos, M. A., Zhu, J. H., Farabaugh, P. J. & Javid, B. Protein mistranslation: friend or foe? Trends Biochem. Sci. 39, 355–362 (2014).

    CAS  Article  PubMed  Google Scholar 

  14. 14

    Ruan, B. et al. Quality control despite mistranslation caused by an ambiguous genetic code. Proc. Natl Acad. Sci. USA 105, 16502–16507 (2008).

    CAS  Article  PubMed  Google Scholar 

  15. 15

    Yadavalli, S. S. & Ibba, M. Selection of tRNA charging quality control mechanisms that increase mistranslation of the genetic code. Nucleic Acids Res. 41, 1104–1112 (2013).

    CAS  Article  PubMed  Google Scholar 

  16. 16

    Bezerra, A. R. et al. Reversion of a fungal genetic code alteration links proteome instability with genomic and phenotypic diversification. Proc. Natl Acad. Sci. USA 110, 11079–11084 (2013).

    CAS  Article  PubMed  Google Scholar 

  17. 17

    Fan, Y. et al. Protein mistranslation protects bacteria against oxidative stress. Nucleic Acids Res. 43, 1740–1748 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18

    Javid, B. et al. Mycobacterial mistranslation is necessary and sufficient for rifampicin phenotypic resistance. Proc. Natl Acad. Sci. USA 111, 1132–1137 (2014).

    CAS  Article  PubMed  Google Scholar 

  19. 19

    Lee, J. Y. et al. Promiscuous methionyl-tRNA synthetase mediates adaptive mistranslation to protect cells against oxidative stress. J. Cell Sci. 127, 4234–4245 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20

    Miranda, I. et al. Candida albicans CUG mistranslation is a mechanism to create cell surface variation. MBio 4, e00285-13 (2013).

  21. 21

    Bailly, M., Blaise, M., Lorber, B., Becker, H. D. & Kern, D. The transamidosome: a dynamic ribonucleoprotein particle dedicated to prokaryotic tRNA-dependent asparagine biosynthesis. Mol. Cell 28, 228–239 (2007).

    CAS  Article  PubMed  Google Scholar 

  22. 22

    Curnow, A. W. et al. Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation. Proc. Natl Acad. Sci. USA 94, 11819–11826 (1997).

    CAS  Article  PubMed  Google Scholar 

  23. 23

    Huot, J. L. et al. Gln-tRNAGln synthesis in a dynamic transamidosome from Helicobacter pylori, where GluRS2 hydrolyzes excess Glu-tRNAGln. Nucleic Acids Res. 39, 9306–9315 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24

    Nakamura, A., Yao, M., Chimnaronk, S., Sakai, N. & Tanaka, I. Ammonia channel couples glutaminase with transamidase reactions in GatCAB. Science 312, 1954–1958 (2006).

    CAS  Article  PubMed  Google Scholar 

  25. 25

    Silva, G. N. et al. A tRNA-independent mechanism for transamidosome assembly promotes aminoacyl-tRNA transamidation. J. Biol. Chem. 288, 3816–3822 (2013).

    CAS  Article  PubMed  Google Scholar 

  26. 26

    Manickam, N., Nag, N., Abbasi, A., Patel, K. & Farabaugh, P. J. Studies of translational misreading in vivo show that the ribosome very efficiently discriminates against most potential errors. RNA 20, 9–15 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27

    Wong, S. Y. et al. Functional role of methylation of G518 of the 16S rRNA 530 loop by GidB in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 57, 6311–6318 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28

    Roy, H., Becker, H. D., Mazauric, M. H. & Kern, D. Structural elements defining elongation factor Tu mediated suppression of codon ambiguity. Nucleic Acids Res. 35, 3420–3430 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29

    Adams, K. N. et al. Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism. Cell 145, 39–53 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30

    Aldridge, B. B. et al. Asymmetry and aging of mycobacterial cells lead to variable growth and antibiotic susceptibility. Science 335, 100–104 (2012).

    CAS  Article  PubMed  Google Scholar 

  31. 31

    Comas, I. et al. Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans. Nat. Genet. 45, 1176–1182 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32

    Dhar, N. & McKinney, J. D. Microbial phenotypic heterogeneity and antibiotic tolerance. Curr. Opin. Microbiol. 10, 30–38 (2007).

    CAS  Article  PubMed  Google Scholar 

  33. 33

    Fridman, O., Goldberg, A., Ronin, I., Shoresh, N. & Balaban, N. Q. Optimization of lag time underlies antibiotic tolerance in evolved bacterial populations. Nature 513, 418–421 (2014).

    CAS  Article  PubMed  Google Scholar 

  34. 34

    Lewis, K. Persister cells. Annu. Rev. Microbiol. 64, 357–372 (2010).

    CAS  Article  PubMed  Google Scholar 

  35. 35

    Maisonneuve, E. & Gerdes, K. Molecular mechanisms underlying bacterial persisters. Cell 157, 539–548 (2014).

    CAS  Article  PubMed  Google Scholar 

  36. 36

    Shah, D. et al. Persisters: a distinct physiological state of E. coli. BMC Microbiol. 6, 53 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  37. 37

    Shan, Y., Lazinski, D., Rowe, S., Camilli, A. & Lewis, K. Genetic basis of persister tolerance to aminoglycosides in Escherichia coli. MBio 6, e00078-15 (2015).

  38. 38

    Siatecka, M., Rozek, M., Barciszewski, J. & Mirande, M. Modular evolution of the Glx-tRNA synthetase family—rooting of the evolutionary tree between the bacteria and archaea/eukarya branches. Eur. J. Biochem. 256, 80–87 (1998).

    CAS  Article  PubMed  Google Scholar 

  39. 39

    Campbell, E. A. et al. Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell 104, 901–912 (2001).

    CAS  Article  PubMed  Google Scholar 

  40. 40

    van Kessel, J. C. & Hatfull, G. F. Efficient point mutagenesis in mycobacteria using single-stranded DNA recombineering: characterization of antimycobacterial drug targets. Mol. Microbiol. 67, 1094–1107 (2008).

    CAS  Article  PubMed  Google Scholar 

  41. 41

    Snapper, S. B., Melton, R. E., Mustafa, S., Kieser, T. & Jacobs, W. R. Jr Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol. Microbiol. 4, 1911–1919 (1990).

    CAS  Article  PubMed  Google Scholar 

  42. 42

    Siegrist, M. S. et al. Mycobacterial Esx-3 is required for mycobactin-mediated iron acquisition. Proc. Natl Acad. Sci. USA 106, 18792–18797 (2009).

    Article  PubMed  Google Scholar 

  43. 43

    Parish, T. & Stoker, N. G. Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology 146, 1969–1975 (2000).

    CAS  Article  PubMed  Google Scholar 

  44. 44

    Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45

    Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  46. 46

    Danecek, P. et al. The variant call format and VCFtools. Bioinformatics 27, 2156–2158 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47

    Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly 6, 80–92 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48

    Gagneux, S. et al. Variable host–pathogen compatibility in Mycobacterium tuberculosis. Proc. Natl Acad. Sci. USA 103, 2869–2873 (2006).

    CAS  Article  PubMed  Google Scholar 

  49. 49

    Koser, C. U. et al. Whole-genome sequencing for rapid susceptibility testing of M. tuberculosis. New Engl. J. Med. 369, 290–292 (2013).

    CAS  Article  PubMed  Google Scholar 

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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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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.

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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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