1887

Abstract

Type III secretion (T3S) systems play key roles in the assembly of flagella and the translocation of bacterial effector proteins into eukaryotic host cells. Eleven proteins which are conserved among Gram-negative plant and animal pathogenic bacteria have been proposed to build up the basal structure of the T3S system, which spans both inner and outer bacterial membranes. We studied six conserved proteins, termed Hrc, predicted to reside in the inner membrane of the plant pathogen pv. vesicatoria. The membrane topology of HrcD, HrcR, HrcS, HrcT, HrcU and HrcV was studied by translational fusions to a dual alkaline phosphatase–-galactosidase reporter protein. Two proteins, HrcU and HrcV, were found to have the same membrane topology as the homologues YscU and YscV. For HrcR, the membrane topology differed from the model for the homologue from , YscR. For our data on three other protein families, exemplified by HrcD, HrcS and HrcT, we derived the first topology models. Our results provide what is believed to be the first complete model of the inner membrane topology of any bacterial T3S system and will aid in elucidating the architecture of T3S systems by ultrastructural analysis.

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2010-07-01
2020-02-27
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References

  1. Aizawa S. I.. 2001; Bacterial flagella and type III secretion systems. FEMS Microbiol Lett202:157–164
    [Google Scholar]
  2. Alexeyev M. F., Winkler H. H.. 2002; Transposable dual reporters for studying the structure–function relationships in membrane proteins: permissive sites in R. prowazekii ATP/ADP translocase. Biochemistry41:406–414
    [Google Scholar]
  3. Allaoui A., Woestyn S., Sluiters C., Cornelis G. R.. 1994; YscU, a Yersinia enterocolitica inner membrane protein involved in Yop secretion. J Bacteriol176:4534–4542
    [Google Scholar]
  4. Bertani G.. 1951; Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol62:293–300
    [Google Scholar]
  5. Blocker A., Jouihri N., Larquet E., Gounon P., Ebel F., Parsot C., Sansonetti P., Allaoui A.. 2001; Structure and composition of the Shigella flexneri ‘needle complex’, a part of its type III secreton. Mol Microbiol39:652–663
    [Google Scholar]
  6. Bogdanove A. J., Beer S. V., Bonas U., Boucher C. A., Collmer A., Coplin D. L., Cornelis G. R., Huang H. C., Hutcheson S. W.. other authors 1996; Unified nomenclature for broadly conserved hrp genes of phytopathogenic bacteria. Mol Microbiol20:681–683
    [Google Scholar]
  7. Bonas U., Stall R. E., Staskawicz B.. 1989; Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria. Mol Gen Genet218:127–136
    [Google Scholar]
  8. Bonas U., Schulte R., Fenselau S., Minsavage G. V., Staskawicz B. J., Stall R. E.. 1991; Isolation of a gene cluster from Xanthomonas campestris pv. vesicatoria that determines pathogenicity and the hypersensitive response on pepper and tomato. Mol Plant Microbe Interact4:81–88
    [Google Scholar]
  9. Boyd D., Manoil C., Beckwith J.. 1987; Determinants of membrane protein topology. Proc Natl Acad Sci U S A84:8525–8529
    [Google Scholar]
  10. Büttner D., Bonas U.. 2006; Who comes first? How plant pathogenic bacteria orchestrate type III secretion. Curr Opin Microbiol9:193–200
    [Google Scholar]
  11. Büttner D., Nennstiel D., Klüsener B., Bonas U.. 2002; Functional analysis of HrpF, a putative type III translocon protein from Xanthomonas campestris pv. vesicatoria. J Bacteriol184:2389–2398
    [Google Scholar]
  12. Calamia J., Manoil C.. 1990; lac permease of Escherichia coli: topology and sequence elements promoting membrane insertion. Proc Natl Acad Sci U S A87:4937–4941
    [Google Scholar]
  13. Coburn B., Sekirov I., Finlay B. B.. 2007; Type III secretion systems and disease. Clin Microbiol Rev20:535–549
    [Google Scholar]
  14. Cornelis G. R.. 2006; The type III secretion injectisome. Nat Rev Microbiol4:811–825
    [Google Scholar]
  15. Creasey E. A., Delahay R. M., Daniell S. J., Frankel G.. 2003; Yeast two-hybrid system survey of interactions between LEE-encoded proteins of enteropathogenic Escherichia coli. Microbiology149:2093–2106
    [Google Scholar]
  16. Deane J. E., Graham S. C., Mitchell E. P., Flot D., Johnson S., Lea S. M.. 2008; Crystal structure of Spa40, the specificity switch for the Shigella flexneri type III secretion system. Mol Microbiol69:267–276
    [Google Scholar]
  17. Dmitrova M., Younes-Cauet G., Oertel-Buchheit P., Porte D., Schnarr M., Granger-Schnarr M.. 1998; A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli. Mol Gen Genet257:205–212
    [Google Scholar]
  18. Durocher D., Jackson S. P.. 2002; The FHA domain. FEBS Lett513:58–66
    [Google Scholar]
  19. Edqvist P. J., Olsson J., Lavander M., Sundberg L., Forsberg A., Wolf-Watz H., Lloyd S. A.. 2003; YscP and YscU regulate substrate specificity of the Yersinia type III secretion system. J Bacteriol185:2259–2266
    [Google Scholar]
  20. Elofsson A., von Heijne G.. 2007; Membrane protein structure: prediction versus reality. Annu Rev Biochem76:125–140
    [Google Scholar]
  21. Fadouloglou V. E., Tampakaki A. P., Glykos N. M., Bastaki M. N., Hadden J. M., Phillips S. E., Panopoulos N. J., Kokkinidis M.. 2004; Structure of HrcQB-C, a conserved component of the bacterial type III secretion systems. Proc Natl Acad Sci U S A101:70–75
    [Google Scholar]
  22. Fan F., Ohnishi K., Francis N. R., Macnab R. M.. 1997; The FliP and FliR proteins of Salmonella typhimurium, putative components of the type III flagellar export apparatus, are located in the flagellar basal body. Mol Microbiol26:1035–1046
    [Google Scholar]
  23. Ferris H. U., Minamino T.. 2006; Flipping the switch: bringing order to flagellar assembly. Trends Microbiol14:519–526
    [Google Scholar]
  24. Fields K. A., Plano G. V., Straley S. C.. 1994; A low-Ca2+ response (LCR) secretion ( ysc) locus lies within the lcrB region of the LCR plasmid in Yersinia pestis. J Bacteriol176:569–579
    [Google Scholar]
  25. Fraser G. M., Hirano T., Ferris H. U., Devgan L. L., Kihara M., Macnab R. M.. 2003; Substrate specificity of type III flagellar protein export in Salmonella is controlled by subdomain interactions in FlhB. Mol Microbiol48:1043–1057
    [Google Scholar]
  26. Galan J. E., Wolf-Watz H.. 2006; Protein delivery into eukaryotic cells by type III secretion machines. Nature444:567–573
    [Google Scholar]
  27. Ghosh P.. 2004; Process of protein transport by the type III secretion system. Microbiol Mol Biol Rev68:771–795
    [Google Scholar]
  28. Green D. H., Cutting S. M.. 2000; Membrane topology of the Bacillus subtilis pro- σK processing complex. J Bacteriol182:278–285
    [Google Scholar]
  29. Gürlebeck D., Thieme F., Bonas U.. 2006; Type III effector proteins from the plant pathogen Xanthomonas and their role in the interaction with the host plant. J Plant Physiol163:233–255
    [Google Scholar]
  30. Guzman L. M., Belin D., Carson M. J., Beckwith J.. 1995; Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol177:4121–4130
    [Google Scholar]
  31. He S. Y., Jin Q.. 2003; The Hrp pilus: learning from flagella. Curr Opin Microbiol6:15–19
    [Google Scholar]
  32. He S. Y., Nomura K., Whittam T. S.. 2004; Type III protein secretion mechanism in mammalian and plant pathogens. Biochim Biophys Acta 1694;181–206
    [Google Scholar]
  33. Hofreuter D., Karnholz A., Haas R.. 2003; Topology and membrane interaction of Helicobacter pylori ComB proteins involved in natural transformation competence. Int J Med Microbiol293:153–165
    [Google Scholar]
  34. Huguet E., Hahn K., Wengelnik K., Bonas U.. 1998; hpaA mutants of Xanthomonas campestris pv. vesicatoria are affected in pathogenicity but retain the ability to induce host-specific hypersensitive reaction. Mol Microbiol29:1379–1390
    [Google Scholar]
  35. Ikeda M., Arai M., Lao D. M., Shimizu T.. 2002; Transmembrane topology prediction methods: a re-assessment and improvement by a consensus method using a dataset of experimentally-characterized transmembrane topologies. In Silico Biol2:19–33
    [Google Scholar]
  36. Jones D. T.. 2007; Improving the accuracy of transmembrane protein topology prediction using evolutionary information. Bioinformatics23:538–544
    [Google Scholar]
  37. Käll L., Krogh A., Sonnhammer E. L.. 2004; A combined transmembrane topology and signal peptide prediction method. J Mol Biol338:1027–1036
    [Google Scholar]
  38. Koebnik R., Krüger A., Thieme F., Urban A., Bonas U.. 2006; Specific binding of the Xanthomonas campestris pv. vesicatoria AraC-type transcriptional activator HrpX to plant-inducible promoter boxes. J Bacteriol188:7652–7660
    [Google Scholar]
  39. Kovach M. E., Elzer P. H., Hill D. S., Robertson G. T., Farris M. A., Roop R. M. II, Peterson K. M.. 1995; Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene166:175–176
    [Google Scholar]
  40. Krogh A., Larsson B., von Heijne G., Sonnhammer E. L.. 2001; Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol305:567–580
    [Google Scholar]
  41. Kubori T., Matsushima Y., Nakamura D., Uralil J., Lara-Tejero M., Sukhan A., Galan J. E., Aizawa S. I.. 1998; Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science280:602–605
    [Google Scholar]
  42. Lavander M., Sundberg L., Edqvist P. J., Lloyd S. A., Wolf-Watz H., Forsberg A.. 2002; Proteolytic cleavage of the FlhB homologue YscU of Yersinia pseudotuberculosis is essential for bacterial survival but not for type III secretion. J Bacteriol184:4500–4509
    [Google Scholar]
  43. Lorenz C., Kirchner O., Egler M., Stuttmann J., Bonas U., Büttner D.. 2008a; HpaA from Xanthomonas is a regulator of type III secretion. Mol Microbiol69:344–360
    [Google Scholar]
  44. Lorenz C., Schulz S., Wolsch T., Rossier O., Bonas U., Büttner D.. 2008b; HpaC controls substrate specificity of the Xanthomonas type III secretion system. PLoS Pathog4:e1000094
    [Google Scholar]
  45. Marlovits T. C., Kubori T., Sukhan A., Thomas D. R., Galan J. E., Unger V. M.. 2004; Structural insights into the assembly of the type III secretion needle complex. Science306:1040–1042
    [Google Scholar]
  46. McCann H. C., Guttman D. S.. 2008; Evolution of the type III secretion system and its effectors in plant–microbe interactions. New Phytol177:33–47
    [Google Scholar]
  47. McMurry J. L., Van Arnam J. S., Kihara M., Macnab R. M.. 2004; Analysis of the cytoplasmic domains of Salmonella FlhA and interactions with components of the flagellar export machinery. J Bacteriol186:7586–7592
    [Google Scholar]
  48. Melen K., Krogh A., von Heijne G.. 2003; Reliability measures for membrane protein topology prediction algorithms. J Mol Biol327:735–744
    [Google Scholar]
  49. Minamino T., Macnab R. M.. 2000a; Domain structure of Salmonella FlhB, a flagellar export component responsible for substrate specificity switching. J Bacteriol182:4906–4914
    [Google Scholar]
  50. Minamino T., Macnab R. M.. 2000b; Interactions among components of the Salmonella flagellar export apparatus and its substrates. Mol Microbiol35:1052–1064
    [Google Scholar]
  51. Minamino T., Iino T., Kutuskake K.. 1994; Molecular characterization of the Salmonella typhimurium flhB operon and its protein products. J Bacteriol176:7630–7637
    [Google Scholar]
  52. Moraes T. F., Spreter T., Strynadka N. C.. 2008; Piecing together the type III injectisome of bacterial pathogens. Curr Opin Struct Biol18:258–266
    [Google Scholar]
  53. Nguyen L., Paulsen I. T., Tchieu J., Hueck C. J., Saier M. H. Jr. 2000; Phylogenetic analyses of the constituents of type III protein secretion systems. J Mol Microbiol Biotechnol2:125–144
    [Google Scholar]
  54. Nilsson J., Persson B., von Heijne G.. 2000; Consensus predictions of membrane protein topology. FEBS Lett486:267–269
    [Google Scholar]
  55. Ohnishi K., Fan F., Schoenhals G. J., Kihara M., Macnab R. M.. 1997; The FliO, FliP, FliQ, and FliR proteins of Salmonella typhimurium: putative components for flagellar assembly. J Bacteriol179:6092–6099
    [Google Scholar]
  56. Ota K., Sakaguchi M., Hamasaki N., Mihara K.. 1998; Assessment of topogenic functions of anticipated transmembrane segments of human band 3. J Biol Chem273:28286–28291
    [Google Scholar]
  57. Pallen M. J., Beatson S. A., Bailey C. M.. 2005; Bioinformatics, genomics and evolution of non-flagellar type-III secretion systems: a Darwinian perspective. FEMS Microbiol Rev29:201–229
    [Google Scholar]
  58. Plano G. V., Straley S. C.. 1995; Mutations in yscC, yscD, and yscG prevent high-level expression and secretion of V antigen and Yops in Yersinia pestis. J Bacteriol177:3843–3854
    [Google Scholar]
  59. Plano G. V., Barve S. S., Straley S. C.. 1991; LcrD, a membrane-bound regulator of the Yersinia pestis low-calcium response. J Bacteriol173:7293–7303
    [Google Scholar]
  60. Pourcher T., Bibi E., Kaback H. R., Leblanc G.. 1996; Membrane topology of the melibiose permease of Escherichia coli studied by melBphoA fusion analysis. Biochemistry35:4161–4168
    [Google Scholar]
  61. Preston G. M.. 2007; Metropolitan microbes: type III secretion in multihost symbionts. Cell Host Microbe2:291–294
    [Google Scholar]
  62. Pühler A., Arlat M., Becker A., Göttfert M., Morrissey J. P., O'Gara F.. 2004; What can bacterial genome research teach us about bacteria–plant interactions?. Curr Opin Plant Biol7:137–147
    [Google Scholar]
  63. Rossier O., Wengelnik K., Hahn K., Bonas U.. 1999; The Xanthomonas Hrp type III system secretes proteins from plant and mammalian bacterial pathogens. Proc Natl Acad Sci U S A96:9368–9373
    [Google Scholar]
  64. Rossier O., Van den Ackerveken G., Bonas U.. 2000; HrpB2 and HrpF from Xanthomonas are type III-secreted proteins and essential for pathogenicity and recognition by the host plant. Mol Microbiol38:828–838
    [Google Scholar]
  65. Rost B., Yachdav G., Liu J.. 2004; The PredictProtein server. Nucleic Acids Res32:W321–W326
    [Google Scholar]
  66. Saijo-Hamano Y., Minamino T., Macnab R. M., Namba K.. 2004; Structural and functional analysis of the C-terminal cytoplasmic domain of FlhA, an integral membrane component of the type III flagellar protein export apparatus in Salmonella. J Mol Biol343:457–466
    [Google Scholar]
  67. Saijo-Hamano Y., Imada K., Minamino T., Kihara M., Macnab R. M., Namba K.. 2005; Crystallization and preliminary X-ray analysis of the C-terminal cytoplasmic domain of FlhA, a membrane-protein subunit of the bacterial flagellar type III protein-export apparatus. Acta Crystallogr Sect F Struct Biol Cryst Commun61:599–602
    [Google Scholar]
  68. Sekiya K., Ohishi M., Ogino T., Tamano K., Sasakawa C., Abe A.. 2001; Supermolecular structure of the enteropathogenic Escherichia coli type III secretion system and its direct interaction with the EspA-sheath-like structure. Proc Natl Acad Sci U S A98:11638–11643
    [Google Scholar]
  69. Simon R., Priefer U., Pühler A.. 1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Nat Biotechnol1:784–791
    [Google Scholar]
  70. Sorg I., Wagner S., Amstutz M., Müller S. A., Broz P., Lussi Y., Engel A., Cornelis G. R.. 2007; YscU recognizes translocators as export substrates of the Yersinia injectisome. EMBO J26:3015–3024
    [Google Scholar]
  71. Spreter T., Yip C. K., Sanowar S., André I., Kimbrough T. G., Vuckovic M., Pfuetzner R. A., Deng W., Yu A. C.. other authors 2009; A conserved structural motif mediates formation of the periplasmic rings in the type III secretion system. Nat Struct Mol Biol16:468–476
    [Google Scholar]
  72. Tamano K., Aizawa S., Katayama E., Nonaka T., Imajoh-Ohmi S., Kuwae A., Nagai S., Sasakawa C.. 2000; Supramolecular structure of the Shigella type III secretion machinery: the needle part is changeable in length and essential for delivery of effectors. EMBO J19:3876–3887
    [Google Scholar]
  73. Tampakaki A. P., Fadouloglou V. E., Gazi A. D., Panopoulos N. J., Kokkinidis M.. 2004; Conserved features of type III secretion. Cell Microbiol6:805–816
    [Google Scholar]
  74. Thieme F., Koebnik R., Bekel T., Berger C., Boch J., Büttner D., Caldana C., Gaigalat L., Goesmann A.. other authors 2005; Insights into genome plasticity and pathogenicity of the plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria revealed by the complete genome sequence. J Bacteriol187:7254–7266
    [Google Scholar]
  75. Tusnady G. E., Simon I.. 2001; The HMMTOP transmembrane topology prediction server. Bioinformatics17:849–850
    [Google Scholar]
  76. Ujwal M. L., Jung H., Bibi E., Manoil C., Altenbach C., Hubbell W. L., Kaback H. R.. 1995; Membrane topology of helices VII and XI in the lactose permease of Escherichia coli studied by lacYphoA fusion analysis and site-directed spectroscopy. Biochemistry34:14909–14917
    [Google Scholar]
  77. van Geest M., Lolkema J. S.. 1996; Membrane topology of the sodium ion-dependent citrate carrier of Klebsiella pneumoniae. Evidence for a new structural class of secondary transporters. J Biol Chem271:25582–25589
    [Google Scholar]
  78. van Geest M., Lolkema J. S.. 2000; Membrane topology and insertion of membrane proteins: search for topogenic signals. Microbiol Mol Biol Rev64:13–33
    [Google Scholar]
  79. Van Gijsegem F., Gough C., Zischek C., Niqueux E., Arlat M., Genin S., Barberis P., German S., Castello P., Boucher C.. 1995; The hrp gene locus of Pseudomonas solanacearum, which controls the production of a type III secretion system, encodes eight proteins related to components of the bacterial flagellar biogenesis complex. Mol Microbiol15:1095–1114
    [Google Scholar]
  80. Van Gijsegem F., Vasse J., De Rycke R., Castello P., Boucher C.. 2002; Genetic dissection of Ralstonia solanacearum hrp gene cluster reveals that the HrpV and HrpX proteins are required for Hrp pilus assembly. Mol Microbiol44:935–946
    [Google Scholar]
  81. von Heijne G.. 1986; The distribution of positively charged residues in bacterial inner membrane proteins correlates with the trans-membrane topology. EMBO J5:3021–3027
    [Google Scholar]
  82. von Heijne G.. 1992; Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J Mol Biol225:487–494
    [Google Scholar]
  83. von Heijne G.. 2006; Membrane-protein topology. Nat Rev Mol Cell Biol7:909–918
    [Google Scholar]
  84. Weber E., Ojanen-Reuhs T., Huguet E., Hause G., Romantschuk M., Korhonen T. K., Bonas U., Koebnik R.. 2005; The type III-dependent Hrp pilus is required for productive interaction of Xanthomonas campestris pv. vesicatoria with pepper host plants. J Bacteriol187:2458–2468
    [Google Scholar]
  85. Weber E., Berger C., Bonas U., Koebnik R.. 2007; Refinement of the Xanthomonas campestris pv. vesicatoria hrpD and hrpE operon structure. Mol Plant Microbe Interact20:559–567
    [Google Scholar]
  86. Wengelnik K., Bonas U.. 1996; HrpXv, an AraC-type regulator, activates expression of five of the six loci in the hrp cluster of Xanthomonas campestris pv. vesicatoria. J Bacteriol178:3462–3469
    [Google Scholar]
  87. Wengelnik K., Marie C., Russel M., Bonas U.. 1996a; Expression and localization of HrpA1, a protein of Xanthomonas campestris pv. vesicatoria essential for pathogenicity and induction of the hypersensitive reaction. J Bacteriol178:1061–1069
    [Google Scholar]
  88. Wengelnik K., Van den Ackerveken G., Bonas U.. 1996b; HrpG, a key hrp regulatory protein of Xanthomonas campestris pv. vesicatoria is homologous to two-component response regulators. Mol Plant Microbe Interact9:704–712
    [Google Scholar]
  89. Yip C. K., Kimbrough T. G., Felise H. B., Vuckovic M., Thomas N. A., Pfuetzner R. A., Frey E. A., Finlay B. B., Miller S. I., Strynadka N. C.. 2005; Structural characterization of the molecular platform for type III secretion system assembly. Nature435:702–707
    [Google Scholar]
  90. Yun C. H., Van Doren S. R., Crofts A. R., Gennis R. B.. 1991; The use of gene fusions to examine the membrane topology of the L-subunit of the photosynthetic reaction center and of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides. J Biol Chem266:10967–10973
    [Google Scholar]
  91. Zarivach R., Deng W., Vuckovic M., Felise H. B., Nguyen H. V., Miller S. I., Finlay B. B., Strynadka N. C.. 2008; Structural analysis of the essential self-cleaving type III secretion proteins EscU and SpaS. Nature453:124–127
    [Google Scholar]
  92. Zhu K., Gonzalez-Pedrajo B., Macnab R. M.. 2002; Interactions among membrane and soluble components of the flagellar export apparatus of Salmonella. Biochemistry41:9516–9524
    [Google Scholar]
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