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Volume 163, Issue 3

Editor's Choice MazEF toxin-antitoxin proteins alter cell morphology and infrastructure during persister formation and regrowth Free

Abstract

When exposed to antibiotics, many bacteria respond by activating intracellular ‘toxin’ proteins, which arrest cell growth and induce formation of persister cells that survive antibiotics. After antibiotics are removed, persisters can regrow by synthesizing ‘antitoxin’ proteins that sequester toxin proteins. In , MazE antitoxin sequesters the activity of MazF toxin, which extensively cleaves cellular RNAs. Although the functions of MazEF proteins are well characterized, there is surprisingly little known about their effects on cell structure. Here, using a combination of microscopy techniques, we visualized the effects of MazEF and three bactericidal antibiotics on cell morphology and infrastructure. When ectopically expressed in , MazF temporarily stalled cell growth and induced persister formation, but only mildly elevated DNA mutagenesis. Viewed by electron microscopy, MazF-expressing persister cells were arrested in cell growth and division. Their chromosomal DNAs were compacted into thread-like structures. Their ribosomes were excluded from their nucleoids. After exposure to ciprofloxacin, persister regrowth was activated by MazE. Cell division remained inhibited while cells became extraordinarily elongated, then divided multiple times during stationary growth phase. This extreme filamentation during persister regrowth was unique to ciprofloxacin-treated persisters, likely caused by inhibition of cell division during regrowth, and was not observed with kanamycin-treated persisters.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): antibiotics , electron microscopy , nucleoid , ribosomes and SOS and stringent response
© 2017 The Authors
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2017-03-01
2024-04-25
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References

  1. Gerdes K, Maisonneuve E. Bacterial persistence and toxin-antitoxin loci. Annu Rev Microbiol 2012; 66:103–123 [View Article][PubMed]
     [Google Scholar]
  2. Schifano JM, Edifor R, Sharp JD, Ouyang M, Konkimalla A et al. Mycobacterial toxin MazF-mt6 inhibits translation through cleavage of 23S rRNA at the ribosomal A site. Proc Natl Acad Sci USA 2013; 110:8501–8506 [View Article][PubMed]
     [Google Scholar]
  3. Maisonneuve E, Shakespeare LJ, Jørgensen MG, Gerdes K. Bacterial persistence by RNA endonucleases. Proc Natl Acad Sci USA 2011; 108:13206–13211 [View Article][PubMed]
     [Google Scholar]
  4. Aizenman E, Engelberg-Kulka H, Glaser G. An Escherichia coli chromosomal ‘addiction module’ regulated by guanosine [corrected] 3′,5′-bispyrophosphate: a model for programmed bacterial cell death. Proc Natl Acad Sci USA 1996; 93:6059–6063 [View Article][PubMed]
     [Google Scholar]
  5. Zhang Y, Zhang J, Hoeflich KP, Ikura M, Qing G et al. MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli. Mol Cell 2003; 12:913–923 [View Article][PubMed]
     [Google Scholar]
  6. Vesper O, Amitai S, Belitsky M, Byrgazov K, Kaberdina AC et al. Selective translation of leaderless mRNAs by specialized ribosomes generated by MazF in Escherichia coli. Cell 2011; 147:147–157 [View Article][PubMed]
     [Google Scholar]
  7. Kamada K, Hanaoka F, Burley SK. Crystal structure of the MazE/MazF complex: molecular bases of antidote-toxin recognition. Mol Cell 2003; 11:875–884[PubMed] [CrossRef]
     [Google Scholar]
  8. Pedersen K, Christensen SK, Gerdes K. Rapid induction and reversal of a bacteriostatic condition by controlled expression of toxins and antitoxins. Mol Microbiol 2002; 45:501–510 [View Article][PubMed]
     [Google Scholar]
  9. Rodionov DG, Ishiguro EE. Direct correlation between overproduction of guanosine 3′,5′-bispyrophosphate (ppGpp) and penicillin tolerance in Escherichia coli. J Bacteriol 1995; 177:4224–4229 [View Article][PubMed]
     [Google Scholar]
  10. Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ. A common mechanism of cellular death induced by bactericidal antibiotics. Cell 2007; 130:797–810 [View Article][PubMed]
     [Google Scholar]
  11. Dwyer DJ, Kohanski MA, Collins JJ. Role of reactive oxygen species in antibiotic action and resistance. Curr Opin Microbiol 2009; 12:482–489 [View Article][PubMed]
     [Google Scholar]
  12. Cohen NR, Lobritz MA, Collins JJ. Microbial persistence and the road to drug resistance. Cell Host Microbe 2013; 13:632–642 [View Article][PubMed]
     [Google Scholar]
  13. Keren I, Wu Y, Inocencio J, Mulcahy LR, Lewis K. Killing by bactericidal antibiotics does not depend on reactive oxygen species. Science 2013; 339:1213–1216 [View Article][PubMed]
     [Google Scholar]
  14. Liu Y, Imlay JA. Cell death from antibiotics without the involvement of reactive oxygen species. Science 2013; 339:1210–1213 [View Article][PubMed]
     [Google Scholar]
  15. Vázquez-Laslop N, Lee H, Neyfakh AA. Increased persistence in Escherichia coli caused by controlled expression of toxins or other unrelated proteins. J Bacteriol 2006; 188:3494–3497 [View Article][PubMed]
     [Google Scholar]
  16. Tripathi A, Dewan PC, Siddique SA, Varadarajan R. MazF-induced growth inhibition and persister generation in Escherichia coli. J Biol Chem 2014; 289:4191–4205 [View Article][PubMed]
     [Google Scholar]
  17. Mok WW, Park JO, Rabinowitz JD, Brynildsen MP. RNA futile cycling in model persisters derived from MazF accumulation. MBio 2015; 6:e01588-15 [View Article][PubMed]
     [Google Scholar]
  18. Amitai S, Yassin Y, Engelberg-Kulka H. MazF-mediated cell death in Escherichia coli: a point of no return. J Bacteriol 2004; 186:8295–8300 [View Article][PubMed]
     [Google Scholar]
  19. Kolodkin-Gal I, Sat B, Keshet A, Engelberg-Kulka H. The communication factor EDF and the toxin-antitoxin module mazEF determine the mode of action of antibiotics. PLoS Biol 2008; 6:e319 [View Article][PubMed]
     [Google Scholar]
  20. 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 2009; 5:e1000390 [View Article][PubMed]
     [Google Scholar]
  21. Erental A, Kalderon Z, Saada A, Smith Y, Engelberg-Kulka H et al. Apoptosis-like death, an extreme SOS response in Escherichia coli. MBio 2014; 5:e01426–14 [CrossRef]
     [Google Scholar]
  22. Kolodkin-Gal I, Hazan R, Gaathon A, Carmeli S, Engelberg-Kulka H. A linear pentapeptide is a quorum-sensing factor required for mazEF-mediated cell death in Escherichia coli. Science 2007; 318:652–655 [View Article][PubMed]
     [Google Scholar]
  23. Hall BM, Ma CX, Liang P, Singh KK. Fluctuation AnaLysis CalculatOR: a web tool for the determination of mutation rate using Luria-Delbruck fluctuation analysis. Bioinformatics 2009; 25:1564–1565 [View Article][PubMed]
     [Google Scholar]
  24. Stocks SM. Mechanism and use of the commercially available viability stain, BacLight. Cytometry A 2004; 61:189–195 [View Article][PubMed]
     [Google Scholar]
  25. Robinow C, Kellenberger E. The bacterial nucleoid revisited. Microbiol Rev 1994; 58:211–232[PubMed]
     [Google Scholar]
  26. Ramisetty BC, Raj S, Ghosh D. Escherichia coli MazEF toxin-antitoxin system does not mediate programmed cell death. J Basic Microbiol 2016; 56:1398–1402 [View Article][PubMed]
     [Google Scholar]
  27. Tsilibaris V, Maenhaut-Michel G, Mine N, Van Melderen L. What is the benefit to Escherichia coli of having multiple toxin-antitoxin systems in its genome?. J Bacteriol 2007; 189:6101–6108 [View Article][PubMed]
     [Google Scholar]
  28. Luria SE, Delbrück M. Mutations of bacteria from virus sensitivity to virus resistance. Genetics 1943; 28:491–511[PubMed]
     [Google Scholar]
  29. Wrande M, Roth JR, Hughes D. Accumulation of mutants in ‘aging’ bacterial colonies is due to growth under selection, not stress-induced mutagenesis. Proc Natl Acad Sci USA 2008; 105:11863–11868 [View Article][PubMed]
     [Google Scholar]
  30. El-Hajj ZW, Newman EB. How much territory can a single E. coli cell control?. Front Microbiol 2015; 6:309 [View Article][PubMed]
     [Google Scholar]
  31. Elliott TS, Shelton A, Greenwood D. The response of Escherichia coli to ciprofloxacin and norfloxacin. J Med Microbiol 1987; 23:83–88 [View Article][PubMed]
     [Google Scholar]
  32. Drlica K, Malik M, Kerns RJ, Zhao X. Quinolone-mediated bacterial death. Antimicrob Agents Chemother 2008; 52:385–392 [CrossRef]
     [Google Scholar]
  33. Kaldalu N, Hauryliuk V, Tenson T. Persisters—as elusive as ever. Appl Microbiol Biotechnol 2016; 100:6545–6553 [View Article][PubMed]
     [Google Scholar]
  34. Chowdhury N, Kwan BW, Wood TK. Persistence increases in the absence of the alarmone guanosine tetraphosphate by reducing cell growth. Sci Rep 2016; 6:20519 [View Article][PubMed]
     [Google Scholar]
  35. Miller OL Jr, Hamkalo BA, Thomas CA Jr. Visualization of bacterial genes in action. Science 1970; 169:392–395 [View Article][PubMed]
     [Google Scholar]
  36. Kellenberger E. Functional consequences of improved structural information on bacterial nucleoids. Res Microbiol 1991; 142:229–238 [View Article][PubMed]
     [Google Scholar]
  37. Makinoshima H, Aizawa S, Hayashi H, Miki T, Nishimura A et al. Growth phase-coupled alterations in cell structure and function of Escherichia coli. J Bacteriol 2003; 185:1338–1345 [View Article][PubMed]
     [Google Scholar]
  38. Eltsov M, Zuber B. Transmission electron microscopy of the bacterial nucleoid. J Struct Biol 2006; 156:246–254 [View Article][PubMed]
     [Google Scholar]
  39. Taheri-Araghi S, Bradde S, Sauls JT, Hill NS, Levin PA et al. Cell-size control and homeostasis in bacteria. Curr Biol 2015; 25:385–391 [View Article][PubMed]
     [Google Scholar]
  40. Dörr T, Vulić M, Lewis K. Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli. PLoS Biol 2010; 8:e1000317 [View Article][PubMed]
     [Google Scholar]
  41. Joseleau-Petit D, Vinella D, D'Ari R. Metabolic alarms and cell division in Escherichia coli. J Bacteriol 1999; 181:9–14[PubMed]
     [Google Scholar]
  42. Potrykus K, Cashel M. (p)ppGpp: still magical?. Annu Rev Microbiol 2008; 62:35–51 [View Article][PubMed]
     [Google Scholar]
  43. Bohrmann B, Villiger W, Johansen R, Kellenberger E. Coralline shape of the bacterial nucleoid after cryofixation. J Bacteriol 1991; 173:3149–3158 [View Article][PubMed]
     [Google Scholar]
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MazEF toxin-antitoxin proteins alter Escherichia coli cell morphology and infrastructure during persister formation and regrowth
Microbiology 163, 308 (2017); https://doi.org/10.1099/mic.0.000436
/content/journal/micro/10.1099/mic.0.000436
/content/journal/micro/10.1099/mic.0.000436
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