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.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.039248-0
2010-07-01
2024-11-05
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/7/1963.html?itemId=/content/journal/micro/10.1099/mic.0.039248-0&mimeType=html&fmt=ahah

References

  1. Aizawa S. I. 2001; Bacterial flagella and type III secretion systems. FEMS Microbiol Lett 202:157–164
    [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. Biochemistry 41:406–414
    [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 Bacteriol 176:4534–4542
    [Google Scholar]
  4. Bertani G. 1951; Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300
    [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 Microbiol 39:652–663
    [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 Microbiol 20:681–683
    [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 Genet 218:127–136
    [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 Interact 4:81–88
    [Google Scholar]
  9. Boyd D., Manoil C., Beckwith J. 1987; Determinants of membrane protein topology. Proc Natl Acad Sci U S A 84:8525–8529
    [Google Scholar]
  10. Büttner D., Bonas U. 2006; Who comes first? How plant pathogenic bacteria orchestrate type III secretion. Curr Opin Microbiol 9:193–200
    [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 Bacteriol 184:2389–2398
    [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 A 87:4937–4941
    [Google Scholar]
  13. Coburn B., Sekirov I., Finlay B. B. 2007; Type III secretion systems and disease. Clin Microbiol Rev 20:535–549
    [Google Scholar]
  14. Cornelis G. R. 2006; The type III secretion injectisome. Nat Rev Microbiol 4:811–825
    [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. Microbiology 149:2093–2106
    [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 Microbiol 69:267–276
    [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 Genet 257:205–212
    [Google Scholar]
  18. Durocher D., Jackson S. P. 2002; The FHA domain. FEBS Lett 513:58–66
    [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 Bacteriol 185:2259–2266
    [Google Scholar]
  20. Elofsson A., von Heijne G. 2007; Membrane protein structure: prediction versus reality. Annu Rev Biochem 76:125–140
    [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 A 101:70–75
    [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 Microbiol 26:1035–1046
    [Google Scholar]
  23. Ferris H. U., Minamino T. 2006; Flipping the switch: bringing order to flagellar assembly. Trends Microbiol 14:519–526
    [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 Bacteriol 176:569–579
    [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 Microbiol 48:1043–1057
    [Google Scholar]
  26. Galan J. E., Wolf-Watz H. 2006; Protein delivery into eukaryotic cells by type III secretion machines. Nature 444:567–573
    [Google Scholar]
  27. Ghosh P. 2004; Process of protein transport by the type III secretion system. Microbiol Mol Biol Rev 68:771–795
    [Google Scholar]
  28. Green D. H., Cutting S. M. 2000; Membrane topology of the Bacillus subtilis pro- σK processing complex. J Bacteriol 182:278–285
    [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 Physiol 163:233–255
    [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 Bacteriol 177:4121–4130
    [Google Scholar]
  31. He S. Y., Jin Q. 2003; The Hrp pilus: learning from flagella. Curr Opin Microbiol 6:15–19
    [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 1694181–206
    [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 Microbiol 293:153–165
    [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 Microbiol 29:1379–1390
    [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 Biol 2:19–33
    [Google Scholar]
  36. Jones D. T. 2007; Improving the accuracy of transmembrane protein topology prediction using evolutionary information. Bioinformatics 23:538–544
    [Google Scholar]
  37. Käll L., Krogh A., Sonnhammer E. L. 2004; A combined transmembrane topology and signal peptide prediction method. J Mol Biol 338:1027–1036
    [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 Bacteriol 188:7652–7660
    [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. Gene 166:175–176
    [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 Biol 305:567–580
    [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. Science 280:602–605
    [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 Bacteriol 184:4500–4509
    [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 Microbiol 69:344–360
    [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 Pathog 4: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. Science 306:1040–1042
    [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 Phytol 177:33–47
    [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 Bacteriol 186:7586–7592
    [Google Scholar]
  48. Melen K., Krogh A., von Heijne G. 2003; Reliability measures for membrane protein topology prediction algorithms. J Mol Biol 327:735–744
    [Google Scholar]
  49. Minamino T., Macnab R. M. 2000a; Domain structure of Salmonella FlhB, a flagellar export component responsible for substrate specificity switching. J Bacteriol 182:4906–4914
    [Google Scholar]
  50. Minamino T., Macnab R. M. 2000b; Interactions among components of the Salmonella flagellar export apparatus and its substrates. Mol Microbiol 35:1052–1064
    [Google Scholar]
  51. Minamino T., Iino T., Kutuskake K. 1994; Molecular characterization of the Salmonella typhimurium flhB operon and its protein products. J Bacteriol 176:7630–7637
    [Google Scholar]
  52. Moraes T. F., Spreter T., Strynadka N. C. 2008; Piecing together the type III injectisome of bacterial pathogens. Curr Opin Struct Biol 18:258–266
    [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 Biotechnol 2:125–144
    [Google Scholar]
  54. Nilsson J., Persson B., von Heijne G. 2000; Consensus predictions of membrane protein topology. FEBS Lett 486:267–269
    [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 Bacteriol 179:6092–6099
    [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 Chem 273:28286–28291
    [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 Rev 29:201–229
    [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 Bacteriol 177:3843–3854
    [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 Bacteriol 173:7293–7303
    [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. Biochemistry 35:4161–4168
    [Google Scholar]
  61. Preston G. M. 2007; Metropolitan microbes: type III secretion in multihost symbionts. Cell Host Microbe 2:291–294
    [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 Biol 7:137–147
    [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 A 96:9368–9373
    [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 Microbiol 38:828–838
    [Google Scholar]
  65. Rost B., Yachdav G., Liu J. 2004; The PredictProtein server. Nucleic Acids Res 32: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 Biol 343:457–466
    [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 Commun 61:599–602
    [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 A 98:11638–11643
    [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 Biotechnol 1:784–791
    [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 J 26:3015–3024
    [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 Biol 16:468–476
    [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 J 19:3876–3887
    [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 Microbiol 6:805–816
    [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 Bacteriol 187:7254–7266
    [Google Scholar]
  75. Tusnady G. E., Simon I. 2001; The HMMTOP transmembrane topology prediction server. Bioinformatics 17:849–850
    [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. Biochemistry 34:14909–14917
    [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 Chem 271:25582–25589
    [Google Scholar]
  78. van Geest M., Lolkema J. S. 2000; Membrane topology and insertion of membrane proteins: search for topogenic signals. Microbiol Mol Biol Rev 64:13–33
    [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 Microbiol 15:1095–1114
    [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 Microbiol 44:935–946
    [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 J 5:3021–3027
    [Google Scholar]
  82. von Heijne G. 1992; Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J Mol Biol 225:487–494
    [Google Scholar]
  83. von Heijne G. 2006; Membrane-protein topology. Nat Rev Mol Cell Biol 7:909–918
    [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 Bacteriol 187:2458–2468
    [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 Interact 20:559–567
    [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 Bacteriol 178:3462–3469
    [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 Bacteriol 178:1061–1069
    [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 Interact 9:704–712
    [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. Nature 435:702–707
    [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 Chem 266:10967–10973
    [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. Nature 453:124–127
    [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. Biochemistry 41:9516–9524
    [Google Scholar]
/content/journal/micro/10.1099/mic.0.039248-0
Loading
/content/journal/micro/10.1099/mic.0.039248-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Supplementary material 2

PDF

Supplementary material 3

PDF

Supplementary material 4

PDF

Supplementary material 5

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error