1887

Abstract

Invasive strains of can cause rapid host cell apoptosis by injecting the type III effector molecule ExoS. A transposon insertional mutant bank of was screened to identify genes that contribute to the ability of the bacteria to trigger host cell apoptosis. Several isolated mutants had disruptions in the gene. A mutant was unable to induce the expression of , and genes under type III inducing conditions, thus exhibiting a defect in type III protein secretion. Furthermore, this mutant was defective in twitching motility, although type IV pili were present on the bacterial surface. Complementation by a -containing cosmid clone restored both phenotypes to the wild-type levels. However, expression of the type III genes in the mutant was not restored by the introduction of a gene alone, although it restored the twitching motility. A gene downstream of , encoding a tRNA pseudouridine synthase () homologue, was able to complement the type III gene expression defect of the mutant. Thus and form an operon and mutation has a polar effect on . Indeed, a mutant is defective in type III gene expression while its twitching motility is unaffected, and a clone is able to complement the type III secretion defect. Pseudouridination of tRNAs is important for tRNA structure, thereby improving the fidelity of protein synthesis and helping to maintain the proper reading frame; thus the results imply that controls tRNAs that are critical for the translation of type III genes or their regulators.

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2004-03-01
2019-10-14
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References

  1. Aiba, H., Kawamukai, M. & Ishihama, A. ( 1983; ). Cloning and promoter analysis of the Escherichia coli adenylate cyclase gene. Nucleic Acids Res 11, 3451–3465.[CrossRef]
    [Google Scholar]
  2. Aitken, A., Jones, D., Soneji, Y. & Howell, S. ( 1995; ). 14--3 proteins: biological function and domain structure. Biochem Soc Trans 23, 605–611.
    [Google Scholar]
  3. Alm, R. A. & Mattick, J. S. ( 1995; ). Identification of a gene, pilV, required for type 4 fimbrial biogenesis in Pseudomonas aeruginosa whose product possesses a prepilin-like leader sequence. Mol Microbiol 16, 485–496.[CrossRef]
    [Google Scholar]
  4. Alm, R. A. & Mattick, J. S. ( 1997; ). Genes involved in the biogenesis and function of type-4 fimbriae in Pseudomonas aeruginosa. Gene 192, 89–98.[CrossRef]
    [Google Scholar]
  5. Asboe, D., Gant, V., Aucken, H. M., Moore, D. A., Umasankar, S., Bingham, J. S., Kaufmann, M. E. & Pitt, T. L. ( 1998; ). Persistence of Pseudomonas aeruginosa strains in respiratory infection in AIDS patients. AIDS 12, 1771–1775.[CrossRef]
    [Google Scholar]
  6. Auffinger, P. & Westhof, E. ( 1998; ). Effects of pseudouridylation on tRNA hydration and dynamics: a theoretical approach. In Modification and Editing of RNA, pp. 103–112. Edited by H. Grosjean & R. Benne. Washington, DC: American Society for Microbiology.
  7. Bodey, G. P., Bolivar, R., Fainstein, V. & Jadeja, L. ( 1983; ). Infections caused by Pseudomonas aeruginosa. Rev Infect Dis 5, 279–313.[CrossRef]
    [Google Scholar]
  8. Bork, P. & Koonin, E. V. ( 1993; ). An expanding family of helicases within the DEAD/H superfamily. Nucleic Acids Res 21, 751–752.[CrossRef]
    [Google Scholar]
  9. Bourne, H. R., Sanders, D. A. & McCormick, F. ( 1990; ). The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348, 125–132.[CrossRef]
    [Google Scholar]
  10. Charette, M. & Gray, M. W. ( 2000; ). Pseudouridine in RNA: what, where, how, and why. IUBMB Life 49, 341–351.[CrossRef]
    [Google Scholar]
  11. Coburn, J. & Gill, D. M. ( 1991; ). ADP-ribosylation of p21ras and related proteins by Pseudomonas aeruginosa exoenzyme S. Infect Immun 59, 4259–4262.
    [Google Scholar]
  12. Comolli, J. C., Hauser, A. R., Waite, L., Whitchurch, C. B., Mattick, J. S. & Engel, J. N. ( 1999; ). Pseudomonas aeruginosa gene products PilT and PilU are required for cytotoxicity in vitro and virulence in a mouse model of acute pneumonia. Infect Immun 67, 3625–3630.
    [Google Scholar]
  13. Cornelis, G. R. & Wolf-Watz, H. ( 1997; ). The Yersinia Yop virulon: a bacterial system for subverting eukaryotic cells. Mol Microbiol 23, 861–867.[CrossRef]
    [Google Scholar]
  14. Croft, L., Beatson, S. A., Whitechurch, C. B., Huang, B., Blakeley, R. L. & Mattick, J. S. ( 2000; ). An interactive web-based Pseudomonas aeruginosa genome database: discovery of new genes, pathways and structures. Microbiol 146, 2351–2364.
    [Google Scholar]
  15. Davies, J. C. ( 2002; ). Pseudomonas aeruginosa in cystic fibrosis: pathogenesis and persistence. Paediatr Respir Rev 3, 128–134.[CrossRef]
    [Google Scholar]
  16. Davis, D. ( 1995; ). Stabilization of RNA stacking by pseudouridine. Nucleic Acids Res 23, 5022–5026.
    [Google Scholar]
  17. Durand, J. M. & Bjork, G. R. ( 2003; ). Putrescine or a combination of methionine and arginine restores virulence gene expression in a tRNA modification-deficient mutant of Shigella flexneri: a possible role in adaptation of virulence. Mol Microbiol 47, 519–527.[CrossRef]
    [Google Scholar]
  18. Durand, J. M., Okada, N., Tobe, T. & 7 other authors ( 1994; ). vacC, a virulence-associated chromosomal locus of Shigella flexneri, is homologous to tgt, a gene encoding tRNA-guanine transglycolase (Tgt) of Escherichia coli K12. J Bacteriol 176, 4627–4634.
    [Google Scholar]
  19. Durand, J. M. B., Björk, G. R., Kuwae, A., Yoshikawa, M. & Sasakawa, C. ( 1997; ). The modified nucleoside 2-methylthio-N-6-isopentenyladenosine in tRNA of Shigella flexneri is required for expression of virulence genes. J Bacteriol 179, 5777–5782.
    [Google Scholar]
  20. Durant, P. C. & Davis, D. R. ( 1997; ). The effect of pseudouridine and pH on the structure and dynamics of the anticodon stem-loop of tRNA(Lys,3). Nucleic Acids Symp Ser 36, 56–57.
    [Google Scholar]
  21. Durant, P. & Davis, D. ( 1999; ). Stabilization of the anticodon stem-loop of tRNALys,3 by an A+C base-pair and by pseudouridine. J Mol Biol 258, 115–131.
    [Google Scholar]
  22. Fey, J., Weil, J. H., Tomita, K., Cosset, A., Dietrich, A., Small, I. & Maréchal-Drouard, L. ( 2001; ). Role of editing in plant mitochondrial transfer RNAs. Gene 286, 21–24.
    [Google Scholar]
  23. Fleiszig, S. M., Wiener-Kronish, J. P., Miyazaki, H., Vallas, V., Mostov, K. E., Kanada, D., Sawa, T., Yen, T. S. & Frank, D. W. ( 1997; ). Pseudomonas aeruginosa-mediated cytotoxicity and invasion correlate with distinct genotypes at the loci encoding exoenzyme S. Infect Immun 65, 579–586.
    [Google Scholar]
  24. Frank, D. W. ( 1997; ). The exoenzyme S regulon of Pseudomonas aeruginosa. Mol Microbiol 26, 621–629.[CrossRef]
    [Google Scholar]
  25. Frank, D. W., Nair, G. & Schweizer, H. P. ( 1994; ). Construction and characterization of chromosomal insertional mutations of the Pseudomonas aeruginosa exoenzyme S trans-regulatory locus. Infect Immun 62, 554–563.
    [Google Scholar]
  26. Frithz-Lindsten, E., Du, Y., Rosqvist, R. & Forsberg, A. ( 1997; ). Intracellular targeting of exoenzyme S of Pseudomonas aeruginosa via type III-dependent translocation induces phagocytosis resistance, cytotoxicity and disruption of actin microfilaments. Mol Microbiol 25, 1125–1139.[CrossRef]
    [Google Scholar]
  27. Fu, H., Coburn, J. & Collier, R. J. ( 1993; ). The eukaryotic host factor that activates exoenzyme S of Pseudomonas aeruginosa is a member of the 14-3-3 protein family. Proc Natl Acad Sci U S A 90, 2320–2324.[CrossRef]
    [Google Scholar]
  28. Galan, J. E. & Collmer, A. ( 1999; ). Type III secretion machines: bacterial devices for protein delivery into host cells. Science 284, 1322–1328.[CrossRef]
    [Google Scholar]
  29. Goehring, U. M., Schmidt, G., Pederson, K. J., Aktories, K. & Barbieri, J. T. ( 1999; ). The N-terminal domain of Pseudomonas aeruginosa exoenzyme S is a GTPase-activating protein for Rho GTPases. J Biol Chem 274, 36369–36372.[CrossRef]
    [Google Scholar]
  30. Gray, J., Wang, J. & Gelvin, S. B. ( 1992; ). Mutation of the miaA gene of Agrobacterium tumefaciens results in reduced vir gene expression. J Bacteriol 174, 1086–1098.
    [Google Scholar]
  31. Greenberg, J. T. & Vinatzer, B. A. ( 2003; ). Identifying type III effectors of plant pathogens and analyzing their interaction with plant cells. Curr Opin Microbiol 6, 20–28.[CrossRef]
    [Google Scholar]
  32. Groisman, E. A. & Ochman, H. ( 1993; ). Cognate gene cluster govern invasion of host epithelial cells by Salmonella typhimurium and Shigella flexineri. EMBO J 12, 3779–3787.
    [Google Scholar]
  33. Ha, U. & Jin, S. ( 2001; ). Growth phase-dependent invasion of Pseudomonas aeruginosa and its survival within HeLa cells. Infect Immun 69, 4398–4406.[CrossRef]
    [Google Scholar]
  34. Harrington, K. M., Nazarenko, I. A., Dix, D. B., Thompson, R. C. & Uhlenbeck, O. C. ( 1993; ). In vitro analysis of translation rate and accuracy with an unmodified tRNA. Biochemistry 32, 7617–7622.[CrossRef]
    [Google Scholar]
  35. Hauser, A. R., Fleiszig, S., Kang, P. J., Mostov, K. & Engel, J. N. ( 1998; ). Defects in type III secretion correlate with internalization of Pseudomonas aeruginosa by epithelial cells. Infect Immun 66, 1413–1420.
    [Google Scholar]
  36. Hueck, C. J. ( 1998; ). Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62, 379–433.
    [Google Scholar]
  37. Jia, J., Alaoui-El-Azher, M., Chow, M., Chambers, T. C., Baker, H. & Jin, S. ( 2003; ). c-Jun NH2-terminal kinase-mediated signaling is essential for Pseudomonas aeruginosa ExoS-induced apoptosis. Infect Immun 71, 3361–3370.[CrossRef]
    [Google Scholar]
  38. Kang, P. J., Hauser, A. R., Apodaca, G., Fleiszig, S. M., Wiener-Kronish, J., Mostov, K. & Engel, J. N. ( 1997; ). Identification of Pseudomonas aeruginosa genes required for epithelial cell injury. Mol Microbiol 24, 1249–1262.[CrossRef]
    [Google Scholar]
  39. Kaufman, M. R., Jinghwa, J., Zeng, L., Ha, U., Chow, M. & Jin, S. ( 2000; ). Pseudomonas aeruginosa mediated apoptosis requires the ADP-ribosylating activity of ExoS. Microbiol 146, 2531–2541.
    [Google Scholar]
  40. Kinscherf, T. G. & Willis, D. K. ( 2002; ). Global regulation by gidA in Pseudomonas syringae. J Bacteriol 184, 2281–2286.[CrossRef]
    [Google Scholar]
  41. Krall, R., Schmidt, G., Aktories, K. & Barbieri, J. T. ( 2000; ). Pseudomonas aeruginosa ExoT is a Rho GTPase-activating protein. Immun Infect 68, 6066–6068.[CrossRef]
    [Google Scholar]
  42. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef]
    [Google Scholar]
  43. Mattick, J. S., Whitchurch, C. B. & Alm, R. A. ( 1996; ). The molecular genetics of type-4 fimbriae in Pseudomonas aeruginosa. Gene 179, 147–155.[CrossRef]
    [Google Scholar]
  44. McBride, M. J. ( 2001; ). Bacterial gliding motility: multiple mechanisms for cell movement over surfaces. Annu Rev Microbiol 55, 49–75.[CrossRef]
    [Google Scholar]
  45. Merz, A. J., So, M. & Sheetz, M. P. ( 2000; ). Pilus retraction powers bacterial twitching motility. Nature 407, 98–102.[CrossRef]
    [Google Scholar]
  46. Miller, J. H. ( 1972; ). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  47. Pederson, K. J., Vallis, A. J., Aktories, K., Frank, D. W. & Barbieri, J. T. ( 1999; ). The amino-terminal domain of Pseudomonas aeruginosa ExoS disrupts actin filaments via small-molecular-weight GTP-binding proteins. Mol Microbiol 32, 393–401.[CrossRef]
    [Google Scholar]
  48. Pier, G. B. ( 2002; ). CFTR mutations and host susceptibility to Pseudomonas aeruginosa lung infection. Curr Opin Microbiol 5, 81–86.[CrossRef]
    [Google Scholar]
  49. Price, D. & Gray, M. ( 1998; ). Editing of tRNA. In Modification and Editing of RNA, pp. 289–305. Edited by H. Grosjean. Washington, DC: American Society for Microbiology.
  50. Ramamurthy, V., Swann, S. L., Spedaliere, C. J. & Mueller, E. G. ( 1999; ). Role of cysteine residues in pseudouridine synthases of different families. Biochemistry 38, 13106–13111.[CrossRef]
    [Google Scholar]
  51. Sage, A. E., Vasil, A. I. & Vasil, M. L. ( 1997; ). Molecular characterization of mutants affected in the osmoprotectant-dependent induction of phospholipase C in Pseudomonas aeruginosa PAO1. Mol Microbiol 23, 43–56.[CrossRef]
    [Google Scholar]
  52. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  53. Schweizer, H. P. ( 1991; ). EscherichiaPseudomonas shuttle vectors derived from pUC18/19. Gene 97, 109–112.[CrossRef]
    [Google Scholar]
  54. Schweizer, H. P. ( 1992; ). Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol Microbiol 6, 1195–1204.[CrossRef]
    [Google Scholar]
  55. Semmler, A., Whitchurch, C. B., Leech, A. J. & Mattick, J. S. ( 2000; ). Identification of a novel gene, fimV, is involved in twitching motility in Pseudomonas aeruginosa. Microbiology 146, 1321–1332.
    [Google Scholar]
  56. Stover, C. K., Pham, X. Q., Erwin, A. L. & 28 other authors ( 2000; ). Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406, 959–964.[CrossRef]
    [Google Scholar]
  57. Totten, P. A. & Lory, S. ( 1990; ). Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK. J Bacteriol 172, 7188–7199.
    [Google Scholar]
  58. Towbin, H., Staehelin, T. & Gordon, J. ( 1979; ). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76, 4350–4354.[CrossRef]
    [Google Scholar]
  59. Urbonavicius, J., Qian, Q., Durand, J. M., Hagervall, T. G. & Bjork, G. R. ( 2001; ). Improvement of reading frame maintenance is a common function for several tRNA modifications. EMBO J 20, 4863–4873.[CrossRef]
    [Google Scholar]
  60. Urbonavicius, J., Durand, J. M. B. & Björk, G. R. ( 2002; ). Three modifications in the D and T arms of tRNA influence translation in Escherichia coli and expression of virulence genes in Shigella flexneri. J Bacteriol 184, 5348–5357.[CrossRef]
    [Google Scholar]
  61. Vallis, A. J., Yahr, T. L., Barbieri, J. T. & Frank, D. W. ( 1999; ). Regulation of ExoS production and secretion by Pseudomonas aeruginosa in response to tissue culture conditions. Infect Immun 67, 914–920.
    [Google Scholar]
  62. Van Gijsegem, F., Genin, S. & Boucher, C. ( 1993; ). Conservation of secretion pathways for pathogenicity determinants of plant and animal bacteria. Trends Microbiol 1, 175–180.[CrossRef]
    [Google Scholar]
  63. Wang, J., Mushegian, A., Lory, S. & Jin, S. ( 1996; ). Large-scale isolation of candidate virulence genes of Pseudomonas aeruginosa by in vivo selection. Proc Natl Acad Sci U S A 93, 10434–10439.[CrossRef]
    [Google Scholar]
  64. Weidner, U., Geier, S., Ptock, A., Friedrich, T., Leif, H. & Weiss, H. (1993; ). The gene locus of the proton-translocating NADH : ubiquinone oxidoreductase in Escherichia coli. Organization of the 14 genes and relationship between the derived proteins and subunits of mitochondrial complex I. J Mol Biol 233, 109–122.[CrossRef]
    [Google Scholar]
  65. Wolfgang, M. C., Lee, V. T., Gilmore, M. E. & Lory, S. ( 2003; ). Coordinate regulation of bacterial virulence genes by a novel adenylate cyclase-dependent signaling pathway. Dev Cell 4, 253–263.[CrossRef]
    [Google Scholar]
  66. Yahr, T. L., Goranson, J. & Frank, D. W. ( 1996a; ). Exoenzyme S of Pseudomonas aeruginosa is secreted by a type III pathway. Mol Microbiol 22, 991–1003.[CrossRef]
    [Google Scholar]
  67. Yahr, T. L., Barbieri, J. T. & Frank, D. W. ( 1996b; ). Genetic relationship between the 53- and 49-kilodalton forms of exoenzyme S from Pseudomonas aeruginosa. J Bacteriol 178, 1412–1419.
    [Google Scholar]
  68. Yahr, T. L., Mende-Mueller, L. M., Friese, M. B. & Frank, D. W. ( 1997; ). Identification of type III secreted products of the Pseudomonas aeruginosa exoenzyme S regulon. J Bacteriol 179, 7165–7168.
    [Google Scholar]
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