1887

Abstract

The VirB/D4 type IV secretion system (T4SS) mediates the transfer of single-stranded DNA and protein virulence factors into plant cells, and also determines the assembly of the T-pilus, which is believed to play a role in host recognition. The T-pilus is composed of the major component VirB2 and the minor component VirB5. Using immuno-electron microscopy we detected the major component VirB2 along the entire length of detached T-pili, but not on cell-bound T-pili or on the cell surface. In contrast, the minor T-pilus component VirB5 was detected on the tips of cell-bound T-pili as well as on the ends of detached T-pili and on the cell surface. To gain further insights into the role of VirB5 we introduced changes at its C terminus. C-terminal deletions of up to four amino acids and alanine replacements did not abolish T-pilus formation and incorporation of the VirB5 variants at the tip, although they did impact the length of T-pili. Also, these changes differentially affected the ability of the T4SS to transfer DNA into plant and bacterial recipients, suggesting differential effects on host-cell specificity. The data presented here suggest that VirB5 localizes at the T-pilus tip, and provide novel insights into its role during the type IV secretion process.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2007/010462-0
2007-11-01
2019-09-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/153/11/3766.html?itemId=/content/journal/micro/10.1099/mic.0.2007/010462-0&mimeType=html&fmt=ahah

References

  1. Anthony, K. G., Sherbourne, C., Sherburne, R. & Frost, L. S. ( 1994; ). The role of the pilus in recipient cell recognition during bacterial conjugation mediated by F-like plasmids. Mol Microbiol 13, 939–953.[CrossRef]
    [Google Scholar]
  2. Backert, S. & Meyer, T. F. ( 2006; ). Type IV secretion systems and their effectors in bacterial pathogenesis. Curr Opin Microbiol 9, 207–217.[CrossRef]
    [Google Scholar]
  3. Baron, C. ( 2005; ). From bioremediation to biowarfare: on the impact and mechanism of type IV secretion systems. FEMS Microbiol Lett 253, 163–170.[CrossRef]
    [Google Scholar]
  4. Baron, C., Llosa, M., Zhou, S. & Zambryski, P. C. ( 1997; ). C-terminal processing and cellular localization of VirB1, a component of the T-complex transfer machinery of Agrobacterium tumefaciens. J Bacteriol 179, 1203–1210.
    [Google Scholar]
  5. Berger, B. R. & Christie, P. J. ( 1994; ). Genetic complementation analysis of the Agrobacterium tumefaciens virB operon: virB2 through virB11 are essential virulence genes. J Bacteriol 176, 3646–3660.
    [Google Scholar]
  6. Carle, A., Höppner, C., Aly, K. A., Yuan, Q., den Dulk-Ras, A., Vergunst, A., O`Callaghan, D. & Baron, C. ( 2006; ). The Brucella suis type IV secretion system assembles in the cell envelope of the heterologous host Agrobacterium tumefaciens and increases IncQ plasmid pLS1 recipient competence. Infect Immun 74, 108–117.[CrossRef]
    [Google Scholar]
  7. Christie, P. J., Atmakuri, K., Krishnamoorthy, V., Jakubowski, S. & Cascales, E. ( 2005; ). Biogenesis, architecture, and function of bacterial type IV secretion systems. Annu Rev Microbiol 59, 451–485.[CrossRef]
    [Google Scholar]
  8. Craig, L., Volkmann, N., Arvai, A. S., Pique, M. E., Yeager, M., Egelman, E. H. & Tainer, J. A. ( 2006; ). Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions. Mol Cell 23, 651–662.[CrossRef]
    [Google Scholar]
  9. Eisenbrandt, R., Kalkum, M., Lai, E. M., Lurz, R., Kado, C. I. & Lanka, E. ( 1999; ). Conjugative pili of IncP plasmids, and the Ti plasmid T pilus are composed of cyclic subunits. J Biol Chem 274, 22548–22555.[CrossRef]
    [Google Scholar]
  10. Fullner, K. J., Lara, J. L. & Nester, E. W. ( 1996; ). Pilus assembly by Agrobacterium T-DNA transfer genes. Science 273, 1107–1109.[CrossRef]
    [Google Scholar]
  11. Harlow, E. & Lane, D. ( 1988; ). Antibodies: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  12. Höppner, C., Liu, Z., Domke, N., Binns, A. N. & Baron, C. ( 2004; ). VirB1 orthologs from Brucella suis and pKM101 complement defects of the lytic transglycosylase required for efficient type IV secretion from Agrobacterium tumefaciens. J Bacteriol 186, 1415–1422.[CrossRef]
    [Google Scholar]
  13. Hwang, H. H. & Gelvin, S. B. ( 2004; ). Plant proteins that interact with VirB2, the Agrobacterium tumefaciens pilin protein, mediate plant transformation. Plant Cell 16, 3148–3167.[CrossRef]
    [Google Scholar]
  14. Jin, Q., Hu, W., Brown, I., McGhee, G., Hart, P., Jones, A. L. & He, S. Y. ( 2001; ). Visualization of secreted Hrp and Avr proteins along the Hrp pilus during type III secretion in Erwinia amylovora and Pseudomonas syringae. Mol Microbiol 40, 1129–1139.[CrossRef]
    [Google Scholar]
  15. Jones, A. L., Shirasu, K. & Kado, C. I. ( 1994; ). The product of the virB4 gene of Agrobacterium tumefaciens promotes accumulation of VirB3 protein. J Bacteriol 176, 5255–5261.
    [Google Scholar]
  16. Journet, L., Agrain, C., Broz, P. & Cornelis, G. R. ( 2003; ). The needle length of bacterial injectisomes is determined by a molecular ruler. Science 302, 1757–1760.[CrossRef]
    [Google Scholar]
  17. Kalkum, M., Eisenbrandt, R., Lurz, R. & Lanka, E. ( 2002; ). Tying rings for sex. Trends Microbiol 10, 382–387.[CrossRef]
    [Google Scholar]
  18. Kau, A. L., Hunstad, D. A. & Hultgren, S. J. ( 2005; ). Interaction of uropathogenic Escherichia coli with host uroepithelium. Curr Opin Microbiol 8, 54–59.[CrossRef]
    [Google Scholar]
  19. Krall, L., Wiedemann, U., Unsin, G., Weiss, S., Domke, N. & Baron, C. ( 2002; ). Detergent extraction identifies different VirB protein subassemblies of the type IV secretion machinery in the membranes of Agrobacterium tumefaciens. Proc Natl Acad Sci U S A 99, 11405–11410.[CrossRef]
    [Google Scholar]
  20. Lacroix, B., Tzfira, T., Vainstein, A. & Citovsky, V. ( 2006; ). A case of promiscuity: Agrobacterium's endless hunt for new partners. Trends Genet 22, 29–37.[CrossRef]
    [Google Scholar]
  21. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef]
    [Google Scholar]
  22. Lai, E.-M. & Kado, C. I. ( 1998; ). Processed VirB2 is the major subunit of the promiscuous pilus of Agrobacterium tumefaciens. J Bacteriol 180, 2711–2717.
    [Google Scholar]
  23. Lai, E. M. & Kado, C. I. ( 2002; ). The Agrobacterium tumefaciens T pilus composed of cyclic T pilin is highly resilient to extreme environments. FEMS Microbiol Lett 210, 111–114.[CrossRef]
    [Google Scholar]
  24. Lai, E. M., Chesnokova, O., Banta, L. M. & Kado, C. I. ( 2000; ). Genetic and environmental factors affecting T-pilin export and T-pilus biogenesis in relation to flagellation of Agrobacterium tumefaciens. J Bacteriol 182, 3705–3716.[CrossRef]
    [Google Scholar]
  25. Llosa, M., Zupan, J., Baron, C. & Zambryski, P. C. ( 2000; ). The N- and C-terminal portions of the Agrobacterium VirB1 protein independently enhance tumorgenesis. J Bacteriol 182, 3437–3445.[CrossRef]
    [Google Scholar]
  26. Maniatis, T. A., Fritsch, E. F. & Sambrook, J. ( 1982; ). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  27. McCullen, C. A. & Binns, A. N. ( 2006; ). Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer. Annu Rev Cell Dev Biol 22, 101–127.[CrossRef]
    [Google Scholar]
  28. Mota, L. J., Journet, L., Sorg, I., Agrain, C. & Cornelis, G. R. ( 2005; ). Bacterial injectisomes: needle length does matter. Science 307, 1278 [CrossRef]
    [Google Scholar]
  29. Quintero, E. J., Busch, K. & Weiner, R. M. ( 1998; ). Spatial and temporal deposition of adhesive extracellular polysaccharide capsule and fimbriae by Hyphomonas strain MHS-3. Appl Environ Microbiol 64, 1246–1255.
    [Google Scholar]
  30. Sagulenko, V., Sagulenko, E., Jakubowski, S., Spudich, E. & Christie, P. J. ( 2001; ). VirB7 lipoprotein is exocellular and associates with the Agrobacterium tumefaciens T-pilus. J Bacteriol 183, 3642–3651.[CrossRef]
    [Google Scholar]
  31. Sauer, F. G., Remaut, H., Hultgren, S. J. & Waksman, G. ( 2004; ). Fiber assembly by the chaperone-usher pathway. Biochim Biophys Acta 1694, 259–267.[CrossRef]
    [Google Scholar]
  32. Schägger, H. & von Jagow, G. ( 1987; ). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range of 1 to 100 kDa. Anal Biochem 166, 368–379.[CrossRef]
    [Google Scholar]
  33. Schmidt-Eisenlohr, H., Domke, N., Angerer, C., Wanner, G., Zambryski, P. C. & Baron, C. ( 1999a; ). Vir proteins stabilize VirB5 and mediate its association with the T pilus of Agrobacterium tumefaciens. J Bacteriol 181, 7485–7492.
    [Google Scholar]
  34. Schmidt-Eisenlohr, H., Domke, N. & Baron, C. ( 1999b; ). TraC of IncN plasmid pKM101 associates with membranes and extracellular high molecular weight structures in Escherichia coli. J Bacteriol 181, 5563–5571.
    [Google Scholar]
  35. Schmidt-Eisenlohr, H., Rittig, M., Preithner, S. & Baron, C. ( 2001; ). Biomonitoring of pJP4-carrying Pseudomonas chlororaphis with Trb protein-specific antisera. Environ Microbiol 3, 720–730.[CrossRef]
    [Google Scholar]
  36. Schröder, G. & Dehio, C. ( 2005; ). Virulence-associated type IV secretion systems of Bartonella. Trends Microbiol 13, 336–342.[CrossRef]
    [Google Scholar]
  37. Shamaei-Tousi, A., Cahill, R. & Frankel, G. ( 2004; ). Interaction between protein subunits of the type IV secretion system of Bartonella henselae. J Bacteriol 186, 4796–4801.[CrossRef]
    [Google Scholar]
  38. Stahl, L. E., Jacobs, A. & Binns, A. N. ( 1998; ). The conjugal intermediate of plasmid RSF1010 inhibits Agrobacterium tumefaciens virulence and VirB-dependent export of VirE2. J Bacteriol 180, 3933–3939.
    [Google Scholar]
  39. Ward, J. E., Dale, E. M. & Binns, A. N. ( 1991; ). Activity of the Agrobacterium T-DNA transfer machinery is affected by virB gene products. Proc Natl Acad Sci U S A 88, 9350–9354.[CrossRef]
    [Google Scholar]
  40. Winans, S. C. & Walker, G. C. ( 1985; ). Conjugal transfer system of the N incompatibility plasmid pKM101. J Bacteriol 161, 402–410.
    [Google Scholar]
  41. Yeo, H.-J. & Waksman, G. ( 2004; ). Unveiling molecular scaffolds of the type IV secretion system. J Bacteriol 186, 1919–1926.[CrossRef]
    [Google Scholar]
  42. Yeo, H.-J., Yuan, Q., Beck, M. R., Baron, C. & Waksman, G. ( 2003; ). Structural and functional characterization of the VirB5 protein from the type IV secretion system encoded by the conjugative plasmid pKM101. Proc Natl Acad Sci U S A 100, 15947–15962.[CrossRef]
    [Google Scholar]
  43. Yuan, Q., Carle, A., Gao, C., Sivanesan, D., Aly, K., Höppner, C., Krall, L., Domke, N. & Baron, C. ( 2005; ). Identification of the VirB4-VirB8-VirB5-VirB2 pilus assembly sequence of type IV secretion systems. J Biol Chem 280, 26349–26359.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2007/010462-0
Loading
/content/journal/micro/10.1099/mic.0.2007/010462-0
Loading

Data & Media loading...

Supplementary figure legends [PDF file](48 KB)

PDF

Supplementary tables [PDF file](51 KB)

PDF

Supplementary Fig. S1 [PDF file](403 KB)

PDF

Supplementary Fig. S2 [PDF file](293 KB)

PDF

Supplementary Fig. S3 [PDF file](351 KB)

PDF

Supplementary Fig. S4 [PDF file](560 KB)

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