1887

Abstract

The majority of Gram-negative plant- and animal-pathogenic bacteria employ a type III secretion (T3S) system to deliver effector proteins to eukaryotic cells. Members of the YscU protein family are essential components of the T3S system and consist of a transmembrane and a cytoplasmic region that is autocatalytically cleaved at a conserved NPTH motif. YscU homologues interact with T3S substrate specificity switch (T3S4) proteins that alter the substrate specificity of the T3S system after assembly of the secretion apparatus. We previously showed that the YscU homologue HrcU from the plant pathogen pv. interacts with the T3S4 protein HpaC and is required for the secretion of translocon and effector proteins. In the present study, analysis of HrcU deletion, insertion and point mutant derivatives led to the identification of amino acid residues in the cytoplasmic region of HrcU (HrcU) that control T3S and translocation of the predicted inner rod protein HrpB2, the translocon protein HrpF and the effector protein AvrBs3. Mutations in the vicinity of the NPTH motif interfered with HrcU cleavage and/or the interaction of HrcU with HrpB2 and the T3S4 protein HpaC. However, HrcU function was not completely abolished, suggesting that HrcU cleavage is not crucial for pathogenicity and T3S. Given that mutations in HrcU differentially affected T3S and translocation of HrpB2 and effector proteins, we propose that HrcU controls the secretion of different T3S substrate classes by independent mechanisms.

Funding
This study was supported by the:
  • Deutsche Forschungsgemeinschaft (Award BU 2145/5-1 and BU2145/1-2)
  • Collaborative Research Centre (Award SFB 648)
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.075176-0
2014-03-01
2024-05-03
Loading full text...

Full text loading...

/deliver/fulltext/micro/160/3/576.html?itemId=/content/journal/micro/10.1099/mic.0.075176-0&mimeType=html&fmt=ahah

References

  1. Agrain C., Callebaut I., Journet L., Sorg I., Paroz C., Mota L. J., Cornelis G. R. ( 2005). Characterization of a type III secretion substrate specificity switch (T3S4) domain in YscP from Yersinia enterocolitica. Mol Microbiol 56:54–67 [View Article][PubMed]
    [Google Scholar]
  2. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. (editors) ( 1996). Current Protocols in Molecular Biology New York: Wiley;
    [Google Scholar]
  3. Berger C., Robin G. P., Bonas U., Koebnik R. ( 2010). Membrane topology of conserved components of the type III secretion system from the plant pathogen Xanthomonas campestris pv. vesicatoria. Microbiology 156:1963–1974 [View Article][PubMed]
    [Google Scholar]
  4. Bertani G. ( 1951). Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300[PubMed]
    [Google Scholar]
  5. Björnfot A. C., Lavander M., Forsberg A., Wolf-Watz H. ( 2009). Autoproteolysis of YscU of Yersinia pseudotuberculosis is important for regulation of expression and secretion of Yop proteins. J Bacteriol 191:4259–4267 [View Article][PubMed]
    [Google Scholar]
  6. 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 [View Article][PubMed]
    [Google Scholar]
  7. Bolchi A., Ottonello S., Petrucco S. ( 2005). A general one-step method for the cloning of PCR products. Biotechnol Appl Biochem 42:205–209 [View Article][PubMed]
    [Google Scholar]
  8. Büttner D. ( 2012). Protein export according to schedule: architecture, assembly, and regulation of type III secretion systems from plant- and animal-pathogenic bacteria. Microbiol Mol Biol Rev 76:262–310 [View Article][PubMed]
    [Google Scholar]
  9. Büttner D., Bonas U. ( 2010). Regulation and secretion of Xanthomonas virulence factors. FEMS Microbiol Rev 34:107–133 [View Article][PubMed]
    [Google Scholar]
  10. 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 [View Article][PubMed]
    [Google Scholar]
  11. Büttner D., Gürlebeck D., Noël L. D., Bonas U. ( 2004). HpaB from Xanthomonas campestris pv. vesicatoria acts as an exit control protein in type III-dependent protein secretion. Mol Microbiol 54:755–768 [View Article][PubMed]
    [Google Scholar]
  12. Büttner D., Lorenz C., Weber E., Bonas U. ( 2006). Targeting of two effector protein classes to the type III secretion system by a HpaC- and HpaB-dependent protein complex from Xanthomonas campestris pv. vesicatoria. Mol Microbiol 59:513–527 [View Article][PubMed]
    [Google Scholar]
  13. Cornelis G. R., Agrain C., Sorg I. ( 2006). Length control of extended protein structures in bacteria and bacteriophages. Curr Opin Microbiol 9:201–206 [View Article][PubMed]
    [Google Scholar]
  14. Dangl J. L., Jones J. D. G. ( 2001). Plant pathogens and integrated defence responses to infection. Nature 411:826–833 [View Article][PubMed]
    [Google Scholar]
  15. Daniels M. J., Barber C. E., Turner P. C., Sawczyc M. K., Byrde R. J. W., Fielding A. H. ( 1984). Cloning of genes involved in pathogenicity of Xanthomonas campestris pv. campestris using the broad host range cosmid pLAFR1. EMBO J 3:3323–3328[PubMed]
    [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 [View Article][PubMed]
    [Google Scholar]
  17. Deane J. E., Abrusci P., Johnson S., Lea S. M. ( 2010). Timing is everything: the regulation of type III secretion. Cell Mol Life Sci 67:1065–1075 [View Article][PubMed]
    [Google Scholar]
  18. DePamphilis M. L., Adler J. ( 1971). Fine structure and isolation of the hook-basal body complex of flagella from Escherichia coli and Bacillus subtilis. J Bacteriol 105:384–395[PubMed]
    [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 [View Article][PubMed]
    [Google Scholar]
  20. Engler C., Kandzia R., Marillonnet S. ( 2008). A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3:e3647 [View Article][PubMed]
    [Google Scholar]
  21. Escolar L., Van Den Ackerveken G., Pieplow S., Rossier O., Bonas U. ( 2001). Type III secretion and in planta recognition of the Xanthomonas avirulence proteins AvrBs1 and AvrBsT. Mol Plant Pathol 2:287–296 [View Article][PubMed]
    [Google Scholar]
  22. Ferris H. U., Furukawa Y., Minamino T., Kroetz M. B., Kihara M., Namba K., Macnab R. M. ( 2005). FlhB regulates ordered export of flagellar components via autocleavage mechanism. J Biol Chem 280:41236–41242 [View Article][PubMed]
    [Google Scholar]
  23. Figurski D. H., Helinski D. R. ( 1979). Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76:1648–1652 [View Article][PubMed]
    [Google Scholar]
  24. Francis N. R., Sosinsky G. E., Thomas D., DeRosier D. J. ( 1994). Isolation, characterization and structure of bacterial flagellar motors containing the switch complex. J Mol Biol 235:1261–1270 [View Article][PubMed]
    [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 [View Article][PubMed]
    [Google Scholar]
  26. Ghosh P. ( 2004). Process of protein transport by the type III secretion system. Microbiol Mol Biol Rev 68:771–795 [View Article][PubMed]
    [Google Scholar]
  27. Hartmann N., Schulz S., Lorenz C., Fraas S., Hause G., Büttner D. ( 2012). Characterization of HrpB2 from Xanthomonas campestris pv. vesicatoria identifies protein regions that are essential for type III secretion pilus formation. Microbiology 158:1334–1349 [View Article][PubMed]
    [Google Scholar]
  28. 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 [View Article][PubMed]
    [Google Scholar]
  29. Jones J. B., Lacy G. H., Bouzar H., Stall R. E., Schaad N. W. ( 2004). Reclassification of the xanthomonads associated with bacterial spot disease of tomato and pepper. Syst Appl Microbiol 27:755–762 [View Article][PubMed]
    [Google Scholar]
  30. Knoop V., Staskawicz B., Bonas U. ( 1991). Expression of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria is not under the control of hrp genes and is independent of plant factors. J Bacteriol 173:7142–7150[PubMed]
    [Google Scholar]
  31. Kousik C. S., Ritchie D. F. ( 1998). Response of bell pepper cultivars to bacterial spot pathogen races that individually overcome major resistance genes. Plant Dis 82:181–186 [View Article]
    [Google Scholar]
  32. Kubori T., Matsushima Y., Nakamura D., Uralil J., Lara-Tejero M., Sukhan A., Galán J. E., Aizawa S. I. ( 1998). Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science 280:602–605 [View Article][PubMed]
    [Google Scholar]
  33. Kutsukake K., Minamino T., Yokoseki T. ( 1994). Isolation and characterization of FliK-independent flagellation mutants from Salmonella typhimurium. J Bacteriol 176:7625–7629[PubMed]
    [Google Scholar]
  34. 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 [View Article][PubMed]
    [Google Scholar]
  35. Lorenz C. ( 2009). Functional characterization of the conserved components HrcN und HrcU of the type III secretion system from Xanthomonas campestris pv. vesicatoria PhD thesis, Martin-Luther-University Halle-Wittenberg; Halle, Germany:
    [Google Scholar]
  36. Lorenz C., Büttner D. ( 2009). Functional characterization of the type III secretion ATPase HrcN from the plant pathogen Xanthomonas campestris pv. vesicatoria. J Bacteriol 191:1414–1428 [View Article][PubMed]
    [Google Scholar]
  37. Lorenz C., Büttner D. ( 2011). Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU. Mol Microbiol 79:447–467 [View Article][PubMed]
    [Google Scholar]
  38. Lorenz C., Schulz S., Wolsch T., Rossier O., Bonas U., Büttner D. ( 2008). HpaC controls substrate specificity of the Xanthomonas type III secretion system. PLoS Pathog 4:e1000094 [View Article][PubMed]
    [Google Scholar]
  39. Lorenz C., Hausner J., Büttner D. ( 2012). HrcQ provides a docking site for early and late type III secretion substrates from Xanthomonas. PLoS ONE 7:e51063 [View Article][PubMed]
    [Google Scholar]
  40. Lountos G. T., Austin B. P., Nallamsetty S., Waugh D. S. ( 2009). Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU, a regulatory switch involved in type III secretion. Protein Sci 18:467–474 [View Article][PubMed]
    [Google Scholar]
  41. Marlovits T. C., Kubori T., Sukhan A., Thomas D. R., Galán J. E., Unger V. M. ( 2004). Structural insights into the assembly of the type III secretion needle complex. Science 306:1040–1042 [View Article][PubMed]
    [Google Scholar]
  42. Matteï P. J., Faudry E., Job V., Izoré T., Attree I., Dessen A. ( 2011). Membrane targeting and pore formation by the type III secretion system translocon. FEBS J 278:414–426 [View Article][PubMed]
    [Google Scholar]
  43. Miao E. A., Mao D. P., Yudkovsky N., Bonneau R., Lorang C. G., Warren S. E., Leaf I. A., Aderem A. ( 2010). Innate immune detection of the type III secretion apparatus through the NLRC4 inflammasome. Proc Natl Acad Sci U S A 107:3076–3080 [View Article][PubMed]
    [Google Scholar]
  44. Minsavage G. V., Dahlbeck D., Whalen M. C., Kearny B., Bonas U., Staskawicz B. J., Stall R. E. ( 1990). Gene-for-gene relationships specifying disease resistance in Xanthomonas campestris pv. vesicatoria – pepper interactions. Mol Plant Microbe Interact 3:41–47 [View Article]
    [Google Scholar]
  45. Morbitzer R., Elsaesser J., Hausner J., Lahaye T. ( 2011). Assembly of custom TALE-type DNA binding domains by modular cloning. Nucleic Acids Res 39:5790–5799 [View Article][PubMed]
    [Google Scholar]
  46. Mueller C. A., Broz P., Cornelis G. R. ( 2008). The type III secretion system tip complex and translocon. Mol Microbiol 68:1085–1095 [View Article][PubMed]
    [Google Scholar]
  47. Noël L., Thieme F., Nennstiel D., Bonas U. ( 2001). cDNA-AFLP analysis unravels a genome-wide hrpG-regulon in the plant pathogen Xanthomonas campestris pv. vesicatoria. Mol Microbiol 41:1271–1281 [View Article][PubMed]
    [Google Scholar]
  48. Noël L., Thieme F., Gäbler J., Büttner D., Bonas U. ( 2003). XopC and XopJ, two novel type III effector proteins from Xanthomonas campestris pv. vesicatoria. J Bacteriol 185:7092–7102 [View Article][PubMed]
    [Google Scholar]
  49. Römer P., Strauss T., Hahn S., Scholze H., Morbitzer R., Grau J., Bonas U., Lahaye T. ( 2009). Recognition of AvrBs3-like proteins is mediated by specific binding to promoters of matching pepper Bs3 alleles. Plant Physiol 150:1697–1712 [View Article][PubMed]
    [Google Scholar]
  50. Ronald P. C., Staskawicz B. J. ( 1988). The avirulence gene avrBs1 from Xanthomonas campestris pv. vesicatoria encodes a 50-kD protein. Mol Plant Microbe Interact 1:191–198 [View Article][PubMed]
    [Google Scholar]
  51. 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 [View Article][PubMed]
    [Google Scholar]
  52. 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 [View Article][PubMed]
    [Google Scholar]
  53. Sani M., Allaoui A., Fusetti F., Oostergetel G. T., Keegstra W., Boekema E. J. ( 2007). Structural organization of the needle complex of the type III secretion apparatus of Shigella flexneri. Micron 38:291–301 [View Article][PubMed]
    [Google Scholar]
  54. Smith T. G., Pereira L., Hoover T. R. ( 2009). Helicobacter pylori FlhB processing-deficient variants affect flagellar assembly but not flagellar gene expression. Microbiology 155:1170–1180 [View Article][PubMed]
    [Google Scholar]
  55. 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 [View Article][PubMed]
    [Google Scholar]
  56. Szczesny R., Jordan M., Schramm C., Schulz S., Cogez V., Bonas U., Büttner D. ( 2010). Functional characterization of the Xcs and Xps type II secretion systems from the plant pathogenic bacterium Xanthomonas campestris pv vesicatoria. New Phytol 187:983–1002 [View Article][PubMed]
    [Google Scholar]
  57. Szurek B., Rossier O., Hause G., Bonas U. ( 2002). Type III-dependent translocation of the Xanthomonas AvrBs3 protein into the plant cell. Mol Microbiol 46:13–23 [View Article][PubMed]
    [Google Scholar]
  58. Wengelnik K., Van den Ackerveken G., Bonas U. ( 1996). 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 [View Article][PubMed]
    [Google Scholar]
  59. Wengelnik K., Rossier O., Bonas U. ( 1999). Mutations in the regulatory gene hrpG of Xanthomonas campestris pv. vesicatoria result in constitutive expression of all hrp genes. J Bacteriol 181:6828–6831[PubMed]
    [Google Scholar]
  60. Wiesand U., Sorg I., Amstutz M., Wagner S., van den Heuvel J., Lührs T., Cornelis G. R., Heinz D. W. ( 2009). Structure of the type III secretion recognition protein YscU from Yersinia enterocolitica. J Mol Biol 385:854–866 [View Article][PubMed]
    [Google Scholar]
  61. Williams A. W., Yamaguchi S., Togashi F., Aizawa S. I., Kawagishi I., Macnab R. M. ( 1996). Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium. J Bacteriol 178:2960–2970[PubMed]
    [Google Scholar]
  62. Wood S. E., Jin J., Lloyd S. A. ( 2008). YscP and YscU switch the substrate specificity of the Yersinia type III secretion system by regulating export of the inner rod protein YscI. J Bacteriol 190:4252–4262 [View Article][PubMed]
    [Google Scholar]
  63. 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 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.075176-0
Loading
/content/journal/micro/10.1099/mic.0.075176-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

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