1887

Abstract

VirB1-like proteins are believed to act as lytic transglycosylases, which facilitate the assembly of type IV secretion systems via localized lysis of the peptidoglycan. This paper presents the biochemical analysis of interactions of purified VirB1 with core components of the type IV secretion system. Genes encoding VirB1, VirB8, VirB9, VirB10 and VirB11 were cloned into expression vectors; the affinity-tagged proteins were purified from , and analyses by gel filtration chromatography showed that they form monomers or homo-multimers. Analysis of protein–protein interactions by affinity precipitation revealed that VirB1 bound to VirB9 and VirB11. The results of bicistron expression experiments followed by gel filtration further supported the VirB1–VirB9 interaction. Peptide array mapping identified regions of VirB1 that interact with VirB8, VirB9 and VirB11 and underscored the importance of the C-terminus, especially for the VirB1–VirB9 interaction. The binding sites were localized on a structure model of VirB1, suggesting that different portions of VirB1 may interact with other VirB proteins during assembly of the type IV secretion machinery.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.28326-0
2005-11-01
2019-10-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/11/3469.html?itemId=/content/journal/micro/10.1099/mic.0.28326-0&mimeType=html&fmt=ahah

References

  1. Atmakuri, K., Ding, Z. & Christie, P. J. ( 2003; ). VirE2, a type IV secretion substrate, interacts with the VirD4 transfer protein at cell poles of Agrobacterium tumefaciens. Mol Microbiol 49, 1699–1713.[CrossRef]
    [Google Scholar]
  2. Atmakuri, K., Cascales, E. & Christie, P. J. ( 2004; ). Energetic components VirD4, VirB11 and VirB4 mediate early DNA transfer reactions required for bacterial type IV secretion. Mol Microbiol 54, 1199–1211.[CrossRef]
    [Google Scholar]
  3. Balsinger, S., Ragaz, C., Baron, C. & Narberhaus, F. ( 2004; ). Replicon-specific regulation of small heat shock genes in Agrobacterium tumefaciens. J Bacteriol 186, 6824–6829.[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. Baumli, S., Hoeppner, S. & Cramer, P. ( 2005; ). A conserved mediator hinge revealed in the structure of the MED7·MED21 (Med7·Srb7) heterodimer. J Biol Chem 280, 18171–18178.[CrossRef]
    [Google Scholar]
  6. Bayer, M., Eferl, R., Zellnig, G., Terferle, K., Dijkstra, A., Koraimann, G. & Högenauer, G. ( 1995; ). Gene 19 of plasmid R1 is required for both efficient conjugative DNA transfer and bacteriophage R17 infection. J Bacteriol 177, 4279–4288.
    [Google Scholar]
  7. Bayer, M., Iberer, R., Bischof, K., Rassi, E., Stabentheiner, E., Zellnig, G. & Koraimann, G. ( 2001; ). Functional and mutational analysis of P19, a DNA transfer protein with muramidase activity. J Bacteriol 183, 3176–3183.[CrossRef]
    [Google Scholar]
  8. 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]
  9. Bhandari, P. & Gowrishankar, J. ( 1997; ). An Escherichia coli host strain useful for efficient overproduction of cloned gene products with NaCl as the inducer. J Bacteriol 179, 4403–4406.
    [Google Scholar]
  10. Blackburn, N. T. & Clarke, A. J. ( 2001; ). Identification of four families of peptidoglycan lytic transglycosylases. J Mol Evol 52, 78–84.[CrossRef]
    [Google Scholar]
  11. Burns-Hamuro, L. L., Ma, Y., Kammerer, S. & 7 other authors ( 2003; ). Designing isoform-specific peptide disruptors of protein kinase A localization. Proc Natl Acad Sci U S A 100, 4072–4077.[CrossRef]
    [Google Scholar]
  12. Cascales, E. & Christie, P. J. ( 2003; ). The versatile bacterial type IV secretion systems. Nat Rev Microbiol 1, 137–149.[CrossRef]
    [Google Scholar]
  13. Cascales, E. & Christie, P. J. ( 2004; ). Agrobacterium VirB10, an ATP energy sensor required for type IV secretion. Proc Natl Acad Sci U S A 101, 17228–17233.[CrossRef]
    [Google Scholar]
  14. Celli, J. & Gorvel, J. P. ( 2004; ). Organelle robbery: Brucella interactions with the endoplasmic reticulum. Curr Opin Microbiol 7, 93–97.[CrossRef]
    [Google Scholar]
  15. Christie, P. J. ( 2004; ). Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems. Biochim Biophys Acta 1694, 219–234.[CrossRef]
    [Google Scholar]
  16. Comerci, D. J., Martinez-Lorenzo, M. J., Sieira, R., Gorvel, J. P. & Ugalde, R. A. ( 2001; ). Essential role of the VirB machinery in the maturation of the Brucella abortus-containing vacuole. Cell Microbiol 3, 159–168.[CrossRef]
    [Google Scholar]
  17. Dang, T. A. & Christie, P. J. ( 1997; ). The VirB4 ATPase of Agrobacterium tumefaciens is a cytoplasmic membrane protein exposed at the periplasmic surface. J Bacteriol 179, 453–462.
    [Google Scholar]
  18. den Hartigh, A. B., Sun, Y. H., Sondervan, D., Heuvelmans, N., Reinders, M. O., Ficht, T. A. & Tsolis, R. M. ( 2004; ). Differential requirements for VirB1 and VirB2 during Brucella abortus infection. Infect Immun 72, 5143–5149.[CrossRef]
    [Google Scholar]
  19. 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]
  20. Fullner, K. J. ( 1998; ). Role of Agrobacterium virB genes in transfer of T complexes and RSF1010. J Bacteriol 180, 430–434.
    [Google Scholar]
  21. Harlow, E. & Lane, D. ( 1988; ). Antibodies: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  22. Higgins, D. G. ( 1994; ). clustal v: multiple alignment of DNA and protein sequences. Methods Mol Biol 25, 307–318.
    [Google Scholar]
  23. Höltje, J. V. ( 1998; ). Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiol Mol Biol Rev 62, 181–203.
    [Google Scholar]
  24. Hong, P. C., Tsolis, R. M. & Ficht, T. A. ( 2000; ). Identification of genes required for chronic persistence of Brucella abortus in mice. Infect Immun 68, 4102–4107.[CrossRef]
    [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. Knoblauch, N. T., Rudiger, S., Schonfeld, H. J., Driessen, A. J., Schneider-Mergener, J. & Bukau, B. ( 1999; ). Substrate specificity of the SecB chaperone. J Biol Chem 274, 34219–34225.[CrossRef]
    [Google Scholar]
  29. Koraimann, G. ( 2003; ). Cell wall degrading enzymes in macromolecular transport systems of Gram-negative bacteria. Cell Mol Life Sci 60, 2371–2388.[CrossRef]
    [Google Scholar]
  30. Kromayer, M., Wilting, R., Tormay, P. & Böck, A. ( 1996; ). Domain structure of the prokaryotic selenocysteine-specific elongation factor SelB. J Mol Biol 262, 413–420.[CrossRef]
    [Google Scholar]
  31. Kumar, R. B. & Das, A. ( 2001; ). Functional analysis of the Agrobacterium tumefaciens T-DNA transport pore protein VirB8. J Bacteriol 183, 3636–3641.[CrossRef]
    [Google Scholar]
  32. Kumar, R. B. & Das, A. ( 2002; ). Polar location and functional domains of the Agrobacterium tumefaciens DNA transfer protein VirD4. Mol Microbiol 43, 1523–1532.[CrossRef]
    [Google Scholar]
  33. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef]
    [Google Scholar]
  34. Lehnherr, H., Hansen, A.-M. & Ilyina, T. ( 1998; ). Penetration of the bacterial cell wall: a family of lytic transglycosylases in bacteriophages and conjugative plasmids. Mol Microbiol 30, 454–457.[CrossRef]
    [Google Scholar]
  35. Leung, A. K., Duewel, H. S., Honek, J. F. & Berghuis, A. M. ( 2001; ). Crystal structure of the lytic transglycosylase from bacteriophage lambda in complex with hexa-N-acetylchitohexaose. Biochemistry 40, 5665–5673.[CrossRef]
    [Google Scholar]
  36. Liu, J. & Rost, B. ( 2003; ). NORSp: predictions of long regions without regular secondary structure. Nucleic Acids Res 31, 3833–3835.[CrossRef]
    [Google Scholar]
  37. Livingstone, C. D. & Barton, G. J. ( 1993; ). Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation. Comput Appl Biosci 9, 745–756.
    [Google Scholar]
  38. Llanos, R., Chevrier, V., Ronjat, M. & 7 other authors ( 1999; ). Tubulin binding sites on gamma-tubulin: identification and molecular characterization. Biochemistry 38, 15712–15720.[CrossRef]
    [Google Scholar]
  39. Llosa, M. & O'Callaghan, D. ( 2004; ). Euroconference on the Biology of Type IV Secretion Processes: bacterial gates into the outer world. Mol Microbiol 53, 1–8.[CrossRef]
    [Google Scholar]
  40. 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]
  41. Lutzmann, M., Kunze, R., Buerer, A., Aebi, U. & Hurt, E. ( 2002; ). Modular self-assembly of a Y-shaped multiprotein complex from seven nucleoporins. EMBO J 21, 387–397.[CrossRef]
    [Google Scholar]
  42. Maniatis, T. A., Fritsch, E. F. & Sambrook, J. ( 1982; ). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  43. Middleton, R., Sjölander, K., Krishnamurthy, N., Foley, J. & Zambryski, P. ( 2005; ). Predicted hexameric structure of the Agrobacterium VirB4 C terminus suggests VirB4 acts as a docking site during type IV secretion. Proc Natl Acad Sci U S A 102, 1685–1690.[CrossRef]
    [Google Scholar]
  44. Mushegian, A. R., Fullner, K. J., Koonin, E. V. & Nester, E. W. ( 1996; ). A family of lysozyme-like virulence factors in bacterial pathogens. Proc Natl Acad Sci U S A 93, 7321–7326.[CrossRef]
    [Google Scholar]
  45. Needleman, S. B. & Wunsch, C. D. ( 1970; ). A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol 48, 443–453.[CrossRef]
    [Google Scholar]
  46. O'Callaghan, D., Cazevieille, C., Allardet-Servent, A., Boschiroli, M. L., Bourg, G., Foulongne, V., Frutos, P., Kulakov, Y. & Ramuz, M. ( 1999; ). A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secretion systems is essential for intracellular survival of Brucella suis. Mol Microbiol 33, 1210–1220.
    [Google Scholar]
  47. Odenbreit, S., Gebert, B., Püls, J., Fischer, W. & Haas, R. ( 2001; ). Interaction of Helicobacter pylori with professional phagocytes: role of the cag pathogenicity island and translocation, phosphorylation and processing of CagA. Cell Microbiol 3, 21–31.[CrossRef]
    [Google Scholar]
  48. Reimer, U., Reineke, U. & Schneider-Mergener, J. ( 2002; ). Peptide arrays: from macro to micro. Curr Opin Biotechnol 13, 315–320.[CrossRef]
    [Google Scholar]
  49. Reineke, U., Kramer, A. & Schneider-Mergener, J. ( 1999; ). Antigen sequence- and library-based mapping of linear and discontinuous protein-protein-interaction sites by spot synthesis. Curr Top Microbiol Immunol 243, 23–36.
    [Google Scholar]
  50. Rohde, M., Püls, J., Buhrdorf, R., Fischer, W. & Haas, R. ( 2003; ). A novel sheathed surface organelle of the Helicobacter pylori type IV secretion system. Mol Microbiol 49, 219–234.[CrossRef]
    [Google Scholar]
  51. Rost, B. ( 1996; ). phd: predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol 266, 525–539.
    [Google Scholar]
  52. Schmidt-Eisenlohr, H., Domke, N., Angerer, C., Wanner, G., Zambryski, P. C. & Baron, C. ( 1999; ). Vir proteins stabilize VirB5 and mediate its association with the T pilus of Agrobacterium tumefaciens. J Bacteriol 181, 7485–7492.
    [Google Scholar]
  53. 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]
  54. Sieira, R., Comerci, D. J., Sanchez, D. O. & Ugalde, R. A. ( 2000; ). A homologue of an operon required for DNA transfer in Agrobacterium is required in Brucella abortus for virulence and intracellular multiplication. J Bacteriol 182, 4849–4855.[CrossRef]
    [Google Scholar]
  55. Smith, T. F. & Waterman, M. S. ( 1981; ). Overlapping genes and information theory. J Theor Biol 91, 379–380.[CrossRef]
    [Google Scholar]
  56. Thunnissen, A. M., Dijkstra, A. J., Kalk, K. H., Rozeboom, H. J., Engel, H., Keck, W. & Dijkstra, B. W. ( 1994; ). Doughnut-shaped structure of a bacterial muramidase revealed by X-ray crystallography. Nature 367, 750–753.[CrossRef]
    [Google Scholar]
  57. van Asselt, E. J., Dijkstra, A. J., Kalk, K. H., Takacs, B., Keck, W. & Dijkstra, B. W. ( 1999; ). Crystal structure of Escherichia coli lytic transglycosylase Slt35 reveals a lysozyme-like catalytic domain with an EF-hand. Structure Fold Des 7, 1167–1180.[CrossRef]
    [Google Scholar]
  58. van Asselt, E. J., Kalk, K. H. & Dijkstra, B. W. ( 2000; ). Crystallographic studies of the interactions of Escherichia coli lytic transglycosylase Slt35 with peptidoglycan. Biochem 39, 1924–1934.[CrossRef]
    [Google Scholar]
  59. Ward, D., Draper, O., Zupan, J. R. & Zambryski, P. C. ( 2002; ). Peptide linkage mapping of the A. tumefaciens vir-encoded type IV secretion system reveals novel protein subassemblies. Proc Natl Acad Sci U S A 99, 11493–11500.[CrossRef]
    [Google Scholar]
  60. Winans, S. C. & Walker, G. C. ( 1985; ). Conjugal transfer system of the N incompatibility plasmid pKM101. J Bacteriol 161, 402–410.
    [Google Scholar]
  61. Yanisch-Perron, C., Vieira, J. & Messing, J. ( 1985; ). Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC18 vectors. Gene 33, 103–119.[CrossRef]
    [Google Scholar]
  62. Yeo, H.-J. & Waksman, G. ( 2004; ). Unveiling molecular scaffolds of the type IV secretion system. J Bacteriol 186, 1919–1926.[CrossRef]
    [Google Scholar]
  63. 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]
  64. 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]
  65. Zahrl, D., Wagner, M., Bischof, K., Bayer, M., Zavecz, B., Beranek, A., Ruckenstuhl, C., Zarfel, G. E. & Koraimann, G. ( 2005; ). Peptidoglycan degradation by specialized lytic transglycosylases associated with type III and type IV secretion systems. Microbiology 151, 3455–3467.[CrossRef]
    [Google Scholar]
  66. Zupan, J., Muth, T. R., Draper, O. & Zambryski, P. C. ( 2000; ). The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. Plant J 23, 11–28.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.28326-0
Loading
/content/journal/micro/10.1099/mic.0.28326-0
Loading

Data & Media loading...

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