Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Sep;177(17):4881–4889. doi: 10.1128/jb.177.17.4881-4889.1995

Interactions of VirB9, -10, and -11 with the membrane fraction of Agrobacterium tumefaciens: solubility studies provide evidence for tight associations.

K E Finberg 1, T R Muth 1, S P Young 1, J B Maken 1, S M Heitritter 1, A N Binns 1, L M Banta 1
PMCID: PMC177261  PMID: 7665464

Abstract

The eleven predicted gene products of the Agrobacterium tumefaciens virB operon are believed to form a transmembrane pore complex through which T-DNA export occurs. The VirB10 protein is required for virulence and is a component of an aggregate associated with the membrane fraction of A. tumefaciens. Removal of the putative membrane-spanning domain (amino acids 22 through 55) disrupts the membrane topology of VirB10 (J. E. Ward, E. M. Dale, E. W. Nester, and A. N. Binns, J. Bacteriol. 172:5200-5210, 1990). Deletion of the sequences encoding amino acids 22 to 55 abolishes the ability of plasmid-borne virB10 to complement a null mutation in the virB10 gene, suggesting that the proper topology of VirB10 in the membrane may indeed play a crucial role in T-DNA transfer to the plant cell. Western blot (immunoblot) analysis indicated that the observed loss of virulence could not be attributed to a decrease in the steady-state levels of the mutant VirB10 protein. Although the deletion of the single transmembrane domain would be expected to perturb membrane association, VirB10 delta 22-55 was found exclusively in the membrane fraction. Urea extraction studies suggested that this membrane localization might be the result of a peripheral membrane association; however, the mutant protein was found in both inner and outer membrane fractions separated by sucrose density gradient centrifugation. Both wild-type VirB10 and wild-type VirB9 were only partially removed from the membranes by extraction with 1% Triton X-100, while VirB5 and VirB8 were Triton X-100 soluble. VirB11 was stripped from the membranes by 6 M urea but not by a more mild salt extraction. The fractionation patterns of VirB9, VirB10, and VirB11 were not dependent on each other or on VirB8 or VirD4. The observed tight association of VirB9, VirB10, and VirB11 with the membrane fraction support the notion that these proteins may exist as components of multiprotein pore complexes, perhaps spanning both the inner and outer membranes of Agrobacterium cells.

Full Text

The Full Text of this article is available as a PDF (394.9 KB).

Selected References

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

  1. An G. Development of plant promoter expression vectors and their use for analysis of differential activity of nopaline synthase promoter in transformed tobacco cells. Plant Physiol. 1986 May;81(1):86–91. doi: 10.1104/pp.81.1.86. [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. Bolland S., Llosa M., Avila P., de la Cruz F. General organization of the conjugal transfer genes of the IncW plasmid R388 and interactions between R388 and IncN and IncP plasmids. J Bacteriol. 1990 Oct;172(10):5795–5802. doi: 10.1128/jb.172.10.5795-5802.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cangelosi G. A., Best E. A., Martinetti G., Nester E. W. Genetic analysis of Agrobacterium. Methods Enzymol. 1991;204:384–397. doi: 10.1016/0076-6879(91)04020-o. [DOI] [PubMed] [Google Scholar]
  8. Chang C. H., Winans S. C. Functional roles assigned to the periplasmic, linker, and receiver domains of the Agrobacterium tumefaciens VirA protein. J Bacteriol. 1992 Nov;174(21):7033–7039. doi: 10.1128/jb.174.21.7033-7039.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Covacci A., Rappuoli R. Pertussis toxin export requires accessory genes located downstream from the pertussis toxin operon. Mol Microbiol. 1993 May;8(3):429–434. doi: 10.1111/j.1365-2958.1993.tb01587.x. [DOI] [PubMed] [Google Scholar]
  11. Cunningham K., Lill R., Crooke E., Rice M., Moore K., Wickner W., Oliver D. SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA. EMBO J. 1989 Mar;8(3):955–959. doi: 10.1002/j.1460-2075.1989.tb03457.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dale E. M., Binns A. N., Ward J. E., Jr Construction and characterization of Tn5virB, a transposon that generates nonpolar mutations, and its use to define virB8 as an essential virulence gene in Agrobacterium tumefaciens. J Bacteriol. 1993 Feb;175(3):887–891. doi: 10.1128/jb.175.3.887-891.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Davidson A. L., Nikaido H. Overproduction, solubilization, and reconstitution of the maltose transport system from Escherichia coli. J Biol Chem. 1990 Mar 15;265(8):4254–4260. [PubMed] [Google Scholar]
  14. De Vos G., Zambryski P. Expression of Agrobacterium nopaline-specific VirD1, VirD2, and VirC1 proteins and their requirement for T-strand production in E. coli. Mol Plant Microbe Interact. 1989 Mar-Apr;2(2):43–52. doi: 10.1094/mpmi-2-043. [DOI] [PubMed] [Google Scholar]
  15. Dubnau D. Genetic competence in Bacillus subtilis. Microbiol Rev. 1991 Sep;55(3):395–424. doi: 10.1128/mr.55.3.395-424.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Gelvin S. B., Habeck L. L. vir genes influence conjugal transfer of the Ti plasmid of Agrobacterium tumefaciens. J Bacteriol. 1990 Mar;172(3):1600–1608. doi: 10.1128/jb.172.3.1600-1608.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Helenius A., Simons K. Solubilization of membranes by detergents. Biochim Biophys Acta. 1975 Mar 25;415(1):29–79. doi: 10.1016/0304-4157(75)90016-7. [DOI] [PubMed] [Google Scholar]
  20. Ishidate K., Creeger E. S., Zrike J., Deb S., Glauner B., MacAlister T. J., Rothfield L. I. Isolation of differentiated membrane domains from Escherichia coli and Salmonella typhimurium, including a fraction containing attachment sites between the inner and outer membranes and the murein skeleton of the cell envelope. J Biol Chem. 1986 Jan 5;261(1):428–443. [PubMed] [Google Scholar]
  21. Jones A. L., Shirasu K., Kado C. I. The product of the virB4 gene of Agrobacterium tumefaciens promotes accumulation of VirB3 protein. J Bacteriol. 1994 Sep;176(17):5255–5261. doi: 10.1128/jb.176.17.5255-5261.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Kim Y. J., Rajapandi T., Oliver D. SecA protein is exposed to the periplasmic surface of the E. coli inner membrane in its active state. Cell. 1994 Sep 9;78(5):845–853. doi: 10.1016/s0092-8674(94)90602-5. [DOI] [PubMed] [Google Scholar]
  24. Koukolíková-Nicola Z., Raineri D., Stephens K., Ramos C., Tinland B., Nester E. W., Hohn B. Genetic analysis of the virD operon of Agrobacterium tumefaciens: a search for functions involved in transport of T-DNA into the plant cell nucleus and in T-DNA integration. J Bacteriol. 1993 Feb;175(3):723–731. doi: 10.1128/jb.175.3.723-731.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kuldau G. A., De Vos G., Owen J., McCaffrey G., Zambryski P. The virB operon of Agrobacterium tumefaciens pTiC58 encodes 11 open reading frames. Mol Gen Genet. 1990 Apr;221(2):256–266. doi: 10.1007/BF00261729. [DOI] [PubMed] [Google Scholar]
  26. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  27. Leroux B., Yanofsky M. F., Winans S. C., Ward J. E., Ziegler S. F., Nester E. W. Characterization of the virA locus of Agrobacterium tumefaciens: a transcriptional regulator and host range determinant. EMBO J. 1987 Apr;6(4):849–856. doi: 10.1002/j.1460-2075.1987.tb04830.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Motallebi-Veshareh M., Balzer D., Lanka E., Jagura-Burdzy G., Thomas C. M. Conjugative transfer functions of broad-host-range plasmid RK2 are coregulated with vegetative replication. Mol Microbiol. 1992 Apr;6(7):907–920. doi: 10.1111/j.1365-2958.1992.tb01541.x. [DOI] [PubMed] [Google Scholar]
  31. Okamoto S., Toyoda-Yamamoto A., Ito K., Takebe I., Machida Y. Localization and orientation of the VirD4 protein of Agrobacterium tumefaciens in the cell membrane. Mol Gen Genet. 1991 Aug;228(1-2):24–32. doi: 10.1007/BF00282443. [DOI] [PubMed] [Google Scholar]
  32. Osborn M. J., Gander J. E., Parisi E., Carson J. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem. 1972 Jun 25;247(12):3962–3972. [PubMed] [Google Scholar]
  33. Pon L., Moll T., Vestweber D., Marshallsay B., Schatz G. Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins. J Cell Biol. 1989 Dec;109(6 Pt 1):2603–2616. doi: 10.1083/jcb.109.6.2603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Prinz W. A., Beckwith J. Gene fusion analysis of membrane protein topology: a direct comparison of alkaline phosphatase and beta-lactamase fusions. J Bacteriol. 1994 Oct;176(20):6410–6413. doi: 10.1128/jb.176.20.6410-6413.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schekman R. Translocation gets a push. Cell. 1994 Sep 23;78(6):911–913. doi: 10.1016/0092-8674(94)90265-8. [DOI] [PubMed] [Google Scholar]
  36. Shirasu K., Kado C. I. Membrane location of the Ti plasmid VirB proteins involved in the biosynthesis of a pilin-like conjugative structure on Agrobacterium tumefaciens. FEMS Microbiol Lett. 1993 Aug 1;111(2-3):287–294. doi: 10.1111/j.1574-6968.1993.tb06400.x. [DOI] [PubMed] [Google Scholar]
  37. Shirasu K., Kado C. I. The virB operon of the Agrobacterium tumefaciens virulence regulon has sequence similarities to B, C and D open reading frames downstream of the pertussis toxin-operon and to the DNA transfer-operons of broad-host-range conjugative plasmids. Nucleic Acids Res. 1993 Jan 25;21(2):353–354. doi: 10.1093/nar/21.2.353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Shirasu K., Morel P., Kado C. I. Characterization of the virB operon of an Agrobacterium tumefaciens Ti plasmid: nucleotide sequence and protein analysis. Mol Microbiol. 1990 Jul;4(7):1153–1163. doi: 10.1111/j.1365-2958.1990.tb00690.x. [DOI] [PubMed] [Google Scholar]
  40. Stachel S. E., Nester E. W. The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens. EMBO J. 1986 Jul;5(7):1445–1454. doi: 10.1002/j.1460-2075.1986.tb04381.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stachel S. E., Zambryski P. C. virA and virG control the plant-induced activation of the T-DNA transfer process of A. tumefaciens. Cell. 1986 Aug 1;46(3):325–333. doi: 10.1016/0092-8674(86)90653-7. [DOI] [PubMed] [Google Scholar]
  42. Steck T. L., Yu J. Selective solubilization of proteins from red blood cell membranes by protein perturbants. J Supramol Struct. 1973;1(3):220–232. doi: 10.1002/jss.400010307. [DOI] [PubMed] [Google Scholar]
  43. Steck T. R., Kado C. I. Virulence genes promote conjugative transfer of the Ti plasmid between Agrobacterium strains. J Bacteriol. 1990 Apr;172(4):2191–2193. doi: 10.1128/jb.172.4.2191-2193.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Thompson D. V., Melchers L. S., Idler K. B., Schilperoort R. A., Hooykaas P. J. Analysis of the complete nucleotide sequence of the Agrobacterium tumefaciens virB operon. Nucleic Acids Res. 1988 May 25;16(10):4621–4636. doi: 10.1093/nar/16.10.4621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Thorstenson Y. R., Zambryski P. C. The essential virulence protein VirB8 localizes to the inner membrane of Agrobacterium tumefaciens. J Bacteriol. 1994 Mar;176(6):1711–1717. doi: 10.1128/jb.176.6.1711-1717.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Traxler B., Boyd D., Beckwith J. The topological analysis of integral cytoplasmic membrane proteins. J Membr Biol. 1993 Feb;132(1):1–11. doi: 10.1007/BF00233047. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. 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]
  51. Ward J. E., Jr, Dale E. M., Binns A. N. Activity of the Agrobacterium T-DNA transfer machinery is affected by virB gene products. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9350–9354. doi: 10.1073/pnas.88.20.9350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Ward J. E., Jr, Dale E. M., Nester E. W., Binns A. N. Identification of a virB10 protein aggregate in the inner membrane of Agrobacterium tumefaciens. J Bacteriol. 1990 Sep;172(9):5200–5210. doi: 10.1128/jb.172.9.5200-5210.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. 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]
  56. Werner P. K., Saier M. H., Jr, Müller M. Membrane insertion of the mannitol permease of Escherichia coli occurs under conditions of impaired SecA function. J Biol Chem. 1992 Dec 5;267(34):24523–24532. [PubMed] [Google Scholar]
  57. Williamson M. P. The structure and function of proline-rich regions in proteins. Biochem J. 1994 Jan 15;297(Pt 2):249–260. doi: 10.1042/bj2970249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Winans S. C., Kerstetter R. A., Nester E. W. Transcriptional regulation of the virA and virG genes of Agrobacterium tumefaciens. J Bacteriol. 1988 Sep;170(9):4047–4054. doi: 10.1128/jb.170.9.4047-4054.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Winans S. C. Two-way chemical signaling in Agrobacterium-plant interactions. Microbiol Rev. 1992 Mar;56(1):12–31. doi: 10.1128/mr.56.1.12-31.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zambryski P. Basic processes underlying Agrobacterium-mediated DNA transfer to plant cells. Annu Rev Genet. 1988;22:1–30. doi: 10.1146/annurev.ge.22.120188.000245. [DOI] [PubMed] [Google Scholar]

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

RESOURCES