Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Feb;179(3):583–591. doi: 10.1128/jb.179.3.583-591.1997

Characterization of membrane and protein interaction determinants of the Agrobacterium tumefaciens VirB11 ATPase.

S Rashkova 1, G M Spudich 1, P J Christie 1
PMCID: PMC178735  PMID: 9006008

Abstract

The VirB11 ATPase is a putative component of the transport machinery responsible for directing the export of nucleoprotein particles (T complexes) across the Agrobacterium tumefaciens envelope to susceptible plant cells. Fractionation and membrane treatment studies showed that approximately 30% of VirB11 partitioned as soluble protein, whereas the remaining protein was only partially solubilized with urea from cytoplasmic membranes of wild-type strain A348 as well as a Ti-plasmidless strain expressing virB11 from an IncP replicon. Mutations in virB11 affecting protein function were mapped near the amino terminus (Q6L, P13L, and E25G), just upstream of a region encoding a Walker A nucleotide-binding site (F154H;L155M), and within the Walker A motif (P170L, K175Q, and delta GKT174-176). The K175Q and delta GKT174-176 mutant proteins partitioned almost exclusively with the cytoplasmic membrane, suggesting that an activity associated with nucleotide binding could modulate the affinity of VirB11 for the cytoplasmic membrane. The virB11F154H;L155M allele was transdominant over wild-type virB11 in a merodiploid assay, providing strong evidence that at least one form of VirB11 functions as a homo- or heteromultimer. An allele with a deletion of the first half of the gene, virB11 delta1-156, was transdominant in a merodiploid assay, indicating that the C-terminal half of VirB11 contains a protein interaction domain. Products of both virB11 delta1-156 and virB11 delta158-343, which synthesizes the N-terminal half of VirB11, associated tightly with the A. tumefaciens membrane, suggesting that both halves of VirB11 contain membrane interaction determinants.

Full Text

The Full Text of this article is available as a PDF (522.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson L. B., Hertzel A. V., Das A. Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complex. Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8889–8894. doi: 10.1073/pnas.93.17.8889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baichwal V., Liu D., Ames G. F. The ATP-binding component of a prokaryotic traffic ATPase is exposed to the periplasmic (external) surface. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):620–624. doi: 10.1073/pnas.90.2.620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beijersbergen A., Dulk-Ras A. D., Schilperoort R. A., Hooykaas P. J. Conjugative Transfer by the Virulence System of Agrobacterium tumefaciens. Science. 1992 May 29;256(5061):1324–1327. doi: 10.1126/science.256.5061.1324. [DOI] [PubMed] [Google Scholar]
  4. Berger B. R., Christie P. J. Genetic complementation analysis of the Agrobacterium tumefaciens virB operon: virB2 through virB11 are essential virulence genes. J Bacteriol. 1994 Jun;176(12):3646–3660. doi: 10.1128/jb.176.12.3646-3660.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berger B. R., Christie P. J. The Agrobacterium tumefaciens virB4 gene product is an essential virulence protein requiring an intact nucleoside triphosphate-binding domain. J Bacteriol. 1993 Mar;175(6):1723–1734. doi: 10.1128/jb.175.6.1723-1734.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Binns A. N., Beaupré C. E., Dale E. M. Inhibition of VirB-mediated transfer of diverse substrates from Agrobacterium tumefaciens by the IncQ plasmid RSF1010. J Bacteriol. 1995 Sep;177(17):4890–4899. doi: 10.1128/jb.177.17.4890-4899.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen C. Y., Winans S. C. Controlled expression of the transcriptional activator gene virG in Agrobacterium tumefaciens by using the Escherichia coli lac promoter. J Bacteriol. 1991 Feb;173(3):1139–1144. doi: 10.1128/jb.173.3.1139-1144.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Christie P. J., Ward J. E., Jr, Gordon M. P., Nester E. W. A gene required for transfer of T-DNA to plants encodes an ATPase with autophosphorylating activity. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9677–9681. doi: 10.1073/pnas.86.24.9677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Christie P. J., Ward J. E., Winans S. C., Nester E. W. The Agrobacterium tumefaciens virE2 gene product is a single-stranded-DNA-binding protein that associates with T-DNA. J Bacteriol. 1988 Jun;170(6):2659–2667. doi: 10.1128/jb.170.6.2659-2667.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dang T. A., Christie P. J. The VirB4 ATPase of Agrobacterium tumefaciens is a cytoplasmic membrane protein exposed at the periplasmic surface. J Bacteriol. 1997 Jan;179(2):453–462. doi: 10.1128/jb.179.2.453-462.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Economou A., Wickner W. SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion. Cell. 1994 Sep 9;78(5):835–843. doi: 10.1016/s0092-8674(94)90582-7. [DOI] [PubMed] [Google Scholar]
  12. Fath M. J., Kolter R. ABC transporters: bacterial exporters. Microbiol Rev. 1993 Dec;57(4):995–1017. doi: 10.1128/mr.57.4.995-1017.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fernandez D., Dang T. A., Spudich G. M., Zhou X. R., Berger B. R., Christie P. J. The Agrobacterium tumefaciens virB7 gene product, a proposed component of the T-complex transport apparatus, is a membrane-associated lipoprotein exposed at the periplasmic surface. J Bacteriol. 1996 Jun;178(11):3156–3167. doi: 10.1128/jb.178.11.3156-3167.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fernandez D., Spudich G. M., Zhou X. R., Christie P. J. The Agrobacterium tumefaciens VirB7 lipoprotein is required for stabilization of VirB proteins during assembly of the T-complex transport apparatus. J Bacteriol. 1996 Jun;178(11):3168–3176. doi: 10.1128/jb.178.11.3168-3176.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Finberg K. E., Muth T. R., Young S. P., Maken J. B., Heitritter S. M., Binns A. N., Banta L. M. Interactions of VirB9, -10, and -11 with the membrane fraction of Agrobacterium tumefaciens: solubility studies provide evidence for tight associations. J Bacteriol. 1995 Sep;177(17):4881–4889. doi: 10.1128/jb.177.17.4881-4889.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Foster P. L. In vivo mutagenesis. Methods Enzymol. 1991;204:114–125. doi: 10.1016/0076-6879(91)04007-b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fullner K. J., Stephens K. M., Nester E. W. An essential virulence protein of Agrobacterium tumefaciens, VirB4, requires an intact mononucleotide binding domain to function in transfer of T-DNA. Mol Gen Genet. 1994 Dec 15;245(6):704–715. doi: 10.1007/BF00297277. [DOI] [PubMed] [Google Scholar]
  18. Garfinkel D. J., Simpson R. B., Ream L. W., White F. F., Gordon M. P., Nester E. W. Genetic analysis of crown gall: fine structure map of the T-DNA by site-directed mutagenesis. Cell. 1981 Nov;27(1 Pt 2):143–153. doi: 10.1016/0092-8674(81)90368-8. [DOI] [PubMed] [Google Scholar]
  19. Kado C. I. Promiscuous DNA transfer system of Agrobacterium tumefaciens: role of the virB operon in sex pilus assembly and synthesis. Mol Microbiol. 1994 Apr;12(1):17–22. doi: 10.1111/j.1365-2958.1994.tb00990.x. [DOI] [PubMed] [Google Scholar]
  20. Kerppola R. E., Shyamala V. K., Klebba P., Ames G. F. The membrane-bound proteins of periplasmic permeases form a complex. Identification of the histidine permease HisQMP complex. J Biol Chem. 1991 May 25;266(15):9857–9865. [PubMed] [Google Scholar]
  21. Lessl M., Balzer D., Pansegrau W., Lanka E. Sequence similarities between the RP4 Tra2 and the Ti VirB region strongly support the conjugation model for T-DNA transfer. J Biol Chem. 1992 Oct 5;267(28):20471–20480. [PubMed] [Google Scholar]
  22. Lessl M., Lanka E. Common mechanisms in bacterial conjugation and Ti-mediated T-DNA transfer to plant cells. Cell. 1994 May 6;77(3):321–324. doi: 10.1016/0092-8674(94)90146-5. [DOI] [PubMed] [Google Scholar]
  23. Osborn M. J., Gander J. E., Parisi E. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Site of synthesis of lipopolysaccharide. J Biol Chem. 1972 Jun 25;247(12):3973–3986. [PubMed] [Google Scholar]
  24. Ostrovsky P. C., Maloy S. Protein phosphorylation on serine, threonine, and tyrosine residues modulates membrane-protein interactions and transcriptional regulation in Salmonella typhimurium. Genes Dev. 1995 Aug 15;9(16):2034–2041. doi: 10.1101/gad.9.16.2034. [DOI] [PubMed] [Google Scholar]
  25. Pansegrau W., Lanka E., Barth P. T., Figurski D. H., Guiney D. G., Haas D., Helinski D. R., Schwab H., Stanisich V. A., Thomas C. M. Complete nucleotide sequence of Birmingham IncP alpha plasmids. Compilation and comparative analysis. J Mol Biol. 1994 Jun 24;239(5):623–663. doi: 10.1006/jmbi.1994.1404. [DOI] [PubMed] [Google Scholar]
  26. Pohlman R. F., Genetti H. D., Winans S. C. Common ancestry between IncN conjugal transfer genes and macromolecular export systems of plant and animal pathogens. Mol Microbiol. 1994 Nov;14(4):655–668. doi: 10.1111/j.1365-2958.1994.tb01304.x. [DOI] [PubMed] [Google Scholar]
  27. Possot O., Pugsley A. P. Molecular characterization of PulE, a protein required for pullulanase secretion. Mol Microbiol. 1994 Apr;12(2):287–299. doi: 10.1111/j.1365-2958.1994.tb01017.x. [DOI] [PubMed] [Google Scholar]
  28. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rajapandi T., Oliver D. Integration of SecA protein into the Escherichia coli inner membrane is regulated by its amino-terminal ATP-binding domain. Mol Microbiol. 1996 Apr;20(1):43–51. doi: 10.1111/j.1365-2958.1996.tb02487.x. [DOI] [PubMed] [Google Scholar]
  30. Regensburg-Tuïnk A. J., Hooykaas P. J. Transgenic N. glauca plants expressing bacterial virulence gene virF are converted into hosts for nopaline strains of A. tumefaciens. Nature. 1993 May 6;363(6424):69–71. doi: 10.1038/363069a0. [DOI] [PubMed] [Google Scholar]
  31. Sandkvist M., Bagdasarian M., Howard S. P., DiRita V. J. Interaction between the autokinase EpsE and EpsL in the cytoplasmic membrane is required for extracellular secretion in Vibrio cholerae. EMBO J. 1995 Apr 18;14(8):1664–1673. doi: 10.1002/j.1460-2075.1995.tb07155.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schneider E., Hunke S., Tebbe S. The MalK protein of the ATP-binding cassette transporter for maltose of Escherichia coli is accessible to protease digestion from the periplasmic side of the membrane. J Bacteriol. 1995 Sep;177(18):5364–5367. doi: 10.1128/jb.177.18.5364-5367.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shirasu K., Koukolíková-Nicola Z., Hohn B., Kado C. I. An inner-membrane-associated virulence protein essential for T-DNA transfer from Agrobacterium tumefaciens to plants exhibits ATPase activity and similarities to conjugative transfer genes. Mol Microbiol. 1994 Feb;11(3):581–588. doi: 10.1111/j.1365-2958.1994.tb00338.x. [DOI] [PubMed] [Google Scholar]
  34. Spudich G. M., Fernandez D., Zhou X. R., Christie P. J. Intermolecular disulfide bonds stabilize VirB7 homodimers and VirB7/VirB9 heterodimers during biogenesis of the Agrobacterium tumefaciens T-complex transport apparatus. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7512–7517. doi: 10.1073/pnas.93.15.7512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stephens K. M., Roush C., Nester E. Agrobacterium tumefaciens VirB11 protein requires a consensus nucleotide-binding site for function in virulence. J Bacteriol. 1995 Jan;177(1):27–36. doi: 10.1128/jb.177.1.27-36.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sundberg C., Meek L., Carroll K., Das A., Ream W. VirE1 protein mediates export of the single-stranded DNA-binding protein VirE2 from Agrobacterium tumefaciens into plant cells. J Bacteriol. 1996 Feb;178(4):1207–1212. doi: 10.1128/jb.178.4.1207-1212.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Thorstenson Y. R., Kuldau G. A., Zambryski P. C. Subcellular localization of seven VirB proteins of Agrobacterium tumefaciens: implications for the formation of a T-DNA transport structure. J Bacteriol. 1993 Aug;175(16):5233–5241. doi: 10.1128/jb.175.16.5233-5241.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Turner L. R., Lara J. C., Nunn D. N., Lory S. Mutations in the consensus ATP-binding sites of XcpR and PilB eliminate extracellular protein secretion and pilus biogenesis in Pseudomonas aeruginosa. J Bacteriol. 1993 Aug;175(16):4962–4969. doi: 10.1128/jb.175.16.4962-4969.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ward J. E., Akiyoshi D. E., Regier D., Datta A., Gordon M. P., Nester E. W. Characterization of the virB operon from an Agrobacterium tumefaciens Ti plasmid. J Biol Chem. 1988 Apr 25;263(12):5804–5814. [PubMed] [Google Scholar]
  40. Ward J. E., Akiyoshi D. E., Regier D., Datta A., Gordon M. P., Nester E. W. Correction: characterization of the virB operon from Agrobacterium tumefaciens Ti plasmid. J Biol Chem. 1990 Mar 15;265(8):4768–4768. [PubMed] [Google Scholar]
  41. Ward J. E., Jr, Dale E. M., Christie P. J., Nester E. W., Binns A. N. Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes. J Bacteriol. 1990 Sep;172(9):5187–5199. doi: 10.1128/jb.172.9.5187-5199.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Watson B., Currier T. C., Gordon M. P., Chilton M. D., Nester E. W. Plasmid required for virulence of Agrobacterium tumefaciens. J Bacteriol. 1975 Jul;123(1):255–264. doi: 10.1128/jb.123.1.255-264.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Weiss A. A., Johnson F. D., Burns D. L. Molecular characterization of an operon required for pertussis toxin secretion. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2970–2974. doi: 10.1073/pnas.90.7.2970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Winans S. C., Kerstetter R. A., Ward J. E., Nester E. W. A protein required for transcriptional regulation of Agrobacterium virulence genes spans the cytoplasmic membrane. J Bacteriol. 1989 Mar;171(3):1616–1622. doi: 10.1128/jb.171.3.1616-1622.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yanofsky M. F., Porter S. G., Young C., Albright L. M., Gordon M. P., Nester E. W. The virD operon of Agrobacterium tumefaciens encodes a site-specific endonuclease. Cell. 1986 Nov 7;47(3):471–477. doi: 10.1016/0092-8674(86)90604-5. [DOI] [PubMed] [Google Scholar]
  46. Zupan J. R., Zambryski P. Transfer of T-DNA from Agrobacterium to the plant cell. Plant Physiol. 1995 Apr;107(4):1041–1047. doi: 10.1104/pp.107.4.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. de Maagd R. A., Lugtenberg B. Fractionation of Rhizobium leguminosarum cells into outer membrane, cytoplasmic membrane, periplasmic, and cytoplasmic components. J Bacteriol. 1986 Sep;167(3):1083–1085. doi: 10.1128/jb.167.3.1083-1085.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES