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A new type V toxin-antitoxin system where mRNA for toxin GhoT is cleaved by antitoxin GhoS

Abstract

Among bacterial toxin-antitoxin systems, to date no antitoxin has been identified that functions by cleaving toxin mRNA. Here we show that YjdO (renamed GhoT) is a membrane lytic peptide that causes ghost cell formation (lysed cells with damaged membranes) and increases persistence (persister cells are tolerant to antibiotics without undergoing genetic change). GhoT is part of a new toxin-antitoxin system with YjdK (renamed GhoS) because in vitro RNA degradation studies, quantitative real-time reverse-transcription PCR and whole-transcriptome studies revealed that GhoS masks GhoT toxicity by cleaving specifically yjdO (ghoT) mRNA. Alanine substitutions showed that Arg28 is important for GhoS activity, and RNA sequencing indicated that the GhoS cleavage site is rich in U and A. The NMR structure of GhoS indicates it is related to the CRISPR-associated-2 RNase, and GhoS is a monomer. Hence, GhoT-GhoS is to our knowledge the first type V toxin-antitoxin system where a protein antitoxin inhibits the toxin by cleaving specifically its mRNA.

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Figure 1: GhoT increases persistence.
Figure 2: GhoT is toxic, and GhoS reduces this toxicity.
Figure 3: GhoS adopts a ferredoxin-like fold and Arg28 is important for its cleavage activity.
Figure 4: GhoS cleavage of native and altered ghoT transcripts.

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References

  1. Gerdes, K., Christensen, S.K. & Lobner-Olesen, A. Prokaryotic toxin-antitoxin stress response loci. Nat. Rev. Microbiol. 3, 371–382 (2005).

    Article  CAS  PubMed  Google Scholar 

  2. Hayes, F. & Van Melderen, L. Toxins-antitoxins: diversity, evolution and function. Crit. Rev. Biochem. Mol. Biol. 46, 386–408 (2011).

    Article  CAS  PubMed  Google Scholar 

  3. Masuda, H., Tan, Q., Awano, N., Wu, K.-P. & Inouye, M. YeeU enhances the bundling of cytoskeletal polymers of MreB and FtsZ, antagonizing the CbtA (YeeV) toxicity in Escherichia coli. Mol. Microbiol. 84, 979–989 (2012).

    Article  CAS  PubMed  Google Scholar 

  4. Ren, D., Bedzyk, L.A., Thomas, S.M., Ye, R.W. & Wood, T.K. Gene expression in Escherichia coli biofilms. Appl. Microbiol. Biotechnol. 64, 515–524 (2004).

    Article  CAS  PubMed  Google Scholar 

  5. Kim, Y., Wang, X., Qun, M., Zhang, X.-S. & Wood, T.K. Toxin-antitoxin systems in Escherichia coli influence biofilm formation through YjgK (TabA) and fimbriae. J. Bacteriol. 191, 1258–1267 (2009).

    Article  CAS  PubMed  Google Scholar 

  6. Kim, Y. & Wood, T.K. Toxins Hha and CspD and small RNA regulator Hfq are involved in persister cell formation through MqsR in Escherichia coli. Biochem. Biophys. Res. Commun. 391, 209–213 (2010).

    Article  CAS  PubMed  Google Scholar 

  7. Dörr, T., Vulić, M. & Lewis, K. Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli. PLoS Biol. 8, e1000317 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Hu, Y., Benedik, M.J. & Wood, T.K. Antitoxin DinJ influences the general stress response through transcript stabilizer CspE. Environ. Microbiol. 14, 669–679 (2012).

    Article  CAS  PubMed  Google Scholar 

  9. Wang, X. et al. Antitoxin MqsA helps mediate the bacterial general stress response. Nat. Chem. Biol. 7, 359–366 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Wang, X. & Wood, T.K. Toxin/antitoxin systems influence biofilm and persister cell formation and the general stress response. Appl. Environ. Microbiol. 77, 5577–5583 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Amitai, S., Kolodkin-Gal, I., Hananya-Meltabashi, M., Sacher, A. & Engelberg-Kulka, H. Escherichia coli MazF leads to the simultaneous selective synthesis of both “death proteins” and “survival proteins”. PLoS Genet. 5, e1000390 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Belitsky, M. et al. The Escherichia coli extracellular death factor EDF induces the endoribonucleolytic activities of the toxins MazF and ChpBK. Mol. Cell 41, 625–635 (2011).

    Article  CAS  PubMed  Google Scholar 

  13. González Barrios, A.F. et al. Autoinducer 2 controls biofilm formation in Escherichia coli through a novel motility quorum-sensing regulator (MqsR, B3022). J. Bacteriol. 188, 305–316 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Kim, Y. et al. Escherichia coli toxin/antitoxin pair MqsR/MqsA regulate toxin CspD. Environ. Microbiol. 12, 1105–1121 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Yamaguchi, Y., Park, J.-H. & Inouye, M. MqsR, a crucial regulator for quorum sensing and biofilm formation, is a GCU-specific mRNA interferase in Escherichia coli. J. Biol. Chem. 284, 28746–28753 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Christensen-Dalsgaard, M., Jørgensen, M.G. & Gerdes, K. Three new RelE-homologous mRNA interferases of Escherichia coli differentially induced by environmental stresses. Mol. Microbiol. 75, 333–348 (2010).

    Article  CAS  PubMed  Google Scholar 

  17. Domka, J., Lee, J., Bansal, T. & Wood, T.K. Temporal gene-expression in Escherichia coli K-12 biofilms. Environ. Microbiol. 9, 332–346 (2007).

    Article  CAS  PubMed  Google Scholar 

  18. Lewis, K. Persister cells, dormancy and infectious disease. Nat. Rev. Microbiol. 5, 48–56 (2007).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  20. Brown, B.L. et al. Three dimensional structure of the MqsR:MqsA complex: a novel TA pair comprised of a toxin homologous to RelE and an antitoxin with unique properties. PLoS Pathog. 5, e1000706 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Keren, I., Shah, D., Spoering, A., Kaldalu, N. & Lewis, K. Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli. J. Bacteriol. 186, 8172–8180 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Correia, F.F. et al. Kinase activity of overexpressed HipA is required for growth arrest and multidrug tolerance in Escherichia coli. J. Bacteriol. 188, 8360–8367 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  24. Balaban, N.Q., Merrin, J., Chait, R., Kowalik, L. & Leibler, S. Bacterial persistence as a phenotypic switch. Science 305, 1622–1625 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. Hofmann, K. & Stoffel, W. TMBASE—a database of membrane spanning protein segments. Biol. Chem. Hoppe-Seyler 374, 166 (1993).

    Google Scholar 

  26. Faridani, O.R., Nikravesh, A., Pandey, D.P., Gerdes, K. & Good, L. Competitive inhibition of natural antisense Sok-RNA interactions activates Hok-mediated cell killing in Escherichia coli. Nucleic Acids Res. 34, 5915–5922 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Van Melderen, L. & Saavedra De Bast, M. Bacterial toxin–antitoxin systems: more than selfish entities? PLoS Genet. 5, e1000437 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Güntert, P. Automated NMR structure calculation with CYANA. Methods Mol. Biol. 278, 353–378 (2004).

    PubMed  Google Scholar 

  30. Herrmann, T., Guntert, P. & Wuthrich, K. Protein NMR structure determination with automated NOE-identification in the NOESY spectra using the new software ATNOS. J. Biomol. NMR 24, 171–189 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Herrmann, T., Guntert, P. & Wuthrich, K. Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA. J. Mol. Biol. 319, 209–227 (2002).

    Article  CAS  PubMed  Google Scholar 

  32. Brünger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

    Article  PubMed  Google Scholar 

  33. Holm, L., Kaariainen, S., Rosenstrom, P. & Schenkel, A. Searching protein structure databases with DaliLite v.3. Bioinformatics 24, 2780–2781 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Orengo, C.A., Jones, D.T. & Thornton, J.M. Protein superfamilies and domain superfolds. Nature 372, 631–634 (1994).

    Article  CAS  PubMed  Google Scholar 

  35. Beloglazova, N. et al. A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats. J. Biol. Chem. 283, 20361–20371 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Samai, P., Smith, P. & Shuman, S. Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 66, 1552–1556 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Beloglazova, N. et al. A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats. J. Biol. Chem. 283, 20361–20371 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Unoson, C. & Wagner, E.G.H. A small SOS-induced toxin is targeted against the inner membrane in Escherichia coli. Mol. Microbiol. 70, 258–270 (2008).

    Article  CAS  PubMed  Google Scholar 

  39. de la Hoz, A.B. et al. Plasmid copy-number control and better-than-random segregation genes of pSM19035 share a common regulator. Proc. Natl. Acad. Sci. USA 97, 728–733 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Donegan, N.P. & Cheung, A.L. Regulation of the mazEF toxin-antitoxin module in Staphylococcus aureus and its impact on sigB expression. J. Bacteriol. 191, 2795–2805 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Jayaraman, R. Bacterial persistence: some new insights into an old phenomenon. J. Biosci. 33, 795–805 (2008).

    Article  CAS  PubMed  Google Scholar 

  42. Maisonneuve, E., Shakespeare, L.J., Jørgensen, M.G. & Gerdes, K. Bacterial persistence by RNA endonucleases. Proc. Natl. Acad. Sci. USA 108, 13206–13211 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Gerdes, K. The parB (hok/sok) locus of plasmid R1: a general purpose plasmid stabilization system. Nat. Biotechnol. 6, 1402–1405 (1988).

    Article  CAS  Google Scholar 

  44. Pecota, D.C. & Wood, T.K. Exclusion of T4 phage by the hok/sok killer locus from plasmid R1. J. Bacteriol. 178, 2044–2050 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Pedersen, K. & Gerdes, K. Multiple hok genes on the chromosome of Escherichia coli. Mol. Microbiol. 32, 1090–1102 (1999).

    Article  CAS  PubMed  Google Scholar 

  46. Kwon, A.-R et al. Structural and biochemical characterization of HP0315 from Helicobacter pylori as a VapD protein with an endoribonuclease activity. Nucleic Acids Res. 40, 4216–4228 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Donegan, K., Matyac, C., Seidler, R. & Porteous, A. Evaluation of methods for sampling, recovery, and enumeration of bacteria applied to the phylloplane. Appl. Environ. Microbiol. 57, 51–56 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Fletcher, M. The effects of culture concentration and age, time, and temperature on bacterial attachment to polystyrene. Can. J. Microbiol. 23, 1–6 (1977).

    Article  Google Scholar 

  49. Barrios, A.F., Zuo, R., Ren, D. & Wood, T.K. Hha, YbaJ, and OmpA regulate Escherichia coli K12 biofilm formation and conjugation plasmids abolish motility. Biotechnol. Bioeng. 93, 188–200 (2006).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the US National Institutes of Health (R01 GM089999 to T.K.W.) and the US National Science Foundation (CAREER award MCB 0952550 to R.P.). X.W. is partially supported by the 1000-Youth Elite Program from China. We are grateful for the Keio and ASKA strains provided by the Genome Analysis Project in Japan and for the initial growth studies conducted by X. Yan and T. Benefield. We also thank S. Vitha for assistance with microscope imaging and Y. Hu for assistance with western blotting. T.K.W. is the Biotechnology Endowed Professor at the Pennsylvania State University.

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T.K.W., X.W., R.P., W.P. and M.J.B. designed the experiments. X.W., H.-Y.C., D.M.L., S.H.H., D.O.O., V.S.-T. and C.Q. performed the in vivo and in vitro assays for the functional studies of GhoT and GhoS and for the regulation of the ghoST operon. K.Z. and D.M.L. purified GhoS, and D.M.L. and W.P. completed the NMR structure with help from T.H. with the structure calculations and refinement. T.K.W. and X.W. authored the nonstructural parts of the manuscript, and R.P. and W.P. wrote the structural sections. All authors discussed the results and commented on the manuscript.

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Correspondence to Rebecca Page or Thomas K Wood.

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Wang, X., Lord, D., Cheng, HY. et al. A new type V toxin-antitoxin system where mRNA for toxin GhoT is cleaved by antitoxin GhoS. Nat Chem Biol 8, 855–861 (2012). https://doi.org/10.1038/nchembio.1062

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