Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Jan;177(1):27–36. doi: 10.1128/jb.177.1.27-36.1995

Agrobacterium tumefaciens VirB11 protein requires a consensus nucleotide-binding site for function in virulence.

K M Stephens 1, C Roush 1, E Nester 1
PMCID: PMC176552  PMID: 7798144

Abstract

virB11, one of the 11 genes of the virB operon, is absolutely required for transport of T-DNA from Agrobacterium tumefaciens into plant cells. Previous studies reported that VirB11 is an ATPase with autophosphorylation activity and localizes to the inner membrane even though the protein does not contain the consensus N-terminal export sequence. In this report, we show that VirB11 localizes to the inner membrane even in the absence of other tumor-inducing (Ti) plasmid-encoded proteins. To facilitate the further characterization of VirB11, we purified this protein from the soluble fraction of an Escherichia coli extract by fusing VirB11 to the maltose-binding protein. The maltose-binding protein-VirB11 fusion was able to complement a virB11 deletion mutant of A. tumefaciens for tumor formation and also localized properly to the inner membrane of A. tumefaciens. The 72-kDa protein, purified from E. coli, exhibited no autophosphorylation, ATPase activity, or ATP-binding activity. To study the importance of the Walker nucleotide-binding site present in VirB11, mutations were generated to replace the conserved lysine residue with either alanine or arginine. Expression of the virB11K175A mutant gene resulted in an avirulent phenotype, and expression of the virB11K175R mutant gene gave rise to an attenuated virulence phenotype. Both mutant proteins were present at levels three to four times higher than that of VirB11 in the wild-type strain. The mutant genes did not exhibit a transdominant phenotype on tumor formation in bacteria that were expressing wild-type virB11. The mutant proteins also localized properly to the inner membrane of A. tumefaciens, but the VirB11K175R protein appeared to be unstable after lysis of the cells.

Full Text

The Full Text of this article is available as a PDF (605.0 KB).

Selected References

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

  1. Albright L. M., Yanofsky M. F., Leroux B., Ma D. Q., Nester E. W. Processing of the T-DNA of Agrobacterium tumefaciens generates border nicks and linear, single-stranded T-DNA. J Bacteriol. 1987 Mar;169(3):1046–1055. doi: 10.1128/jb.169.3.1046-1055.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alfano C., McMacken R. Ordered assembly of nucleoprotein structures at the bacteriophage lambda replication origin during the initiation of DNA replication. J Biol Chem. 1989 Jun 25;264(18):10699–10708. [PubMed] [Google Scholar]
  3. Ankenbauer R. G., Best E. A., Palanca C. A., Nester E. W. Mutants of the Agrobacterium tumefaciens virA gene exhibiting acetosyringone-independent expression of the vir regulon. Mol Plant Microbe Interact. 1991 Jul-Aug;4(4):400–406. doi: 10.1094/mpmi-4-400. [DOI] [PubMed] [Google Scholar]
  4. Bally M., Filloux A., Akrim M., Ball G., Lazdunski A., Tommassen J. Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase. Mol Microbiol. 1992 May;6(9):1121–1131. doi: 10.1111/j.1365-2958.1992.tb01550.x. [DOI] [PubMed] [Google Scholar]
  5. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. Caron P. R., Grossman L. Involvement of a cryptic ATPase activity of UvrB and its proteolysis product, UvrB* in DNA repair. Nucleic Acids Res. 1988 Nov 25;16(22):10891–10902. doi: 10.1093/nar/16.22.10891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. De Feyter R., Yang Y., Gabriel D. W. Gene-for-genes interactions between cotton R genes and Xanthomonas campestris pv. malvacearum avr genes. Mol Plant Microbe Interact. 1993 Mar-Apr;6(2):225–237. doi: 10.1094/mpmi-6-225. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Dums F., Dow J. M., Daniels M. J. Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria. Mol Gen Genet. 1991 Oct;229(3):357–364. doi: 10.1007/BF00267456. [DOI] [PubMed] [Google Scholar]
  15. Fry D. C., Kuby S. A., Mildvan A. S. ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins. Proc Natl Acad Sci U S A. 1986 Feb;83(4):907–911. doi: 10.1073/pnas.83.4.907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fry D. C., Kuby S. A., Mildvan A. S. NMR studies of the AMP-binding site and mechanism of adenylate kinase. Biochemistry. 1987 Mar 24;26(6):1645–1655. doi: 10.1021/bi00380a024. [DOI] [PubMed] [Google Scholar]
  17. Fry D. C., Kuby S. A., Mildvan A. S. NMR studies of the MgATP binding site of adenylate kinase and of a 45-residue peptide fragment of the enzyme. Biochemistry. 1985 Aug 13;24(17):4680–4694. doi: 10.1021/bi00338a030. [DOI] [PubMed] [Google Scholar]
  18. Garfinkel D. J., Nester E. W. Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism. J Bacteriol. 1980 Nov;144(2):732–743. doi: 10.1128/jb.144.2.732-743.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hiratsuka T. Biological activities and spectroscopic properties of chromophoric and fluorescent analogs of adenine nucleoside and nucleotides, 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) adenosine derivatives. Biochim Biophys Acta. 1982 Dec 17;719(3):509–517. doi: 10.1016/0304-4165(82)90240-9. [DOI] [PubMed] [Google Scholar]
  20. Iwamoto A., Park M. Y., Maeda M., Futai M. Domains near ATP gamma phosphate in the catalytic site of H+-ATPase. Model proposed from mutagenesis and inhibitor studies. J Biol Chem. 1993 Feb 15;268(5):3156–3160. [PubMed] [Google Scholar]
  21. Jackson A. P., Maxwell A. Identifying the catalytic residue of the ATPase reaction of DNA gyrase. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11232–11236. doi: 10.1073/pnas.90.23.11232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jin S., Song Y., Pan S. Q., Nester E. W. Characterization of a virG mutation that confers constitutive virulence gene expression in Agrobacterium. Mol Microbiol. 1993 Feb;7(4):555–562. doi: 10.1111/j.1365-2958.1993.tb01146.x. [DOI] [PubMed] [Google Scholar]
  23. Ko Y. H., Thomas P. J., Delannoy M. R., Pedersen P. L. The cystic fibrosis transmembrane conductance regulator. Overexpression, purification, and characterization of wild type and delta F508 mutant forms of the first nucleotide binding fold in fusion with the maltose-binding protein. J Biol Chem. 1993 Nov 15;268(32):24330–24338. [PubMed] [Google Scholar]
  24. Koronakis V., Hughes C., Koronakis E. ATPase activity and ATP/ADP-induced conformational change in the soluble domain of the bacterial protein translocator HlyB. Mol Microbiol. 1993 Jun;8(6):1163–1175. doi: 10.1111/j.1365-2958.1993.tb01661.x. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Lee H. S., Berger D. K., Kustu S. Activity of purified NIFA, a transcriptional activator of nitrogen fixation genes. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2266–2270. doi: 10.1073/pnas.90.6.2266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lee H. S., Narberhaus F., Kustu S. In vitro activity of NifL, a signal transduction protein for biological nitrogen fixation. J Bacteriol. 1993 Dec;175(23):7683–7688. doi: 10.1128/jb.175.23.7683-7688.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lindeberg M., Collmer A. Analysis of eight out genes in a cluster required for pectic enzyme secretion by Erwinia chrysanthemi: sequence comparison with secretion genes from other gram-negative bacteria. J Bacteriol. 1992 Nov;174(22):7385–7397. doi: 10.1128/jb.174.22.7385-7397.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Maina C. V., Riggs P. D., Grandea A. G., 3rd, Slatko B. E., Moran L. S., Tagliamonte J. A., McReynolds L. A., Guan C. D. An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. Gene. 1988 Dec 30;74(2):365–373. doi: 10.1016/0378-1119(88)90170-9. [DOI] [PubMed] [Google Scholar]
  32. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206–210. doi: 10.1016/0003-2697(78)90586-9. [DOI] [PubMed] [Google Scholar]
  33. Ninfa E. G., Atkinson M. R., Kamberov E. S., Ninfa A. J. Mechanism of autophosphorylation of Escherichia coli nitrogen regulator II (NRII or NtrB): trans-phosphorylation between subunits. J Bacteriol. 1993 Nov;175(21):7024–7032. doi: 10.1128/jb.175.21.7024-7032.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nunn D., Bergman S., Lory S. Products of three accessory genes, pilB, pilC, and pilD, are required for biogenesis of Pseudomonas aeruginosa pili. J Bacteriol. 1990 Jun;172(6):2911–2919. doi: 10.1128/jb.172.6.2911-2919.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Oh E. Y., Claassen L., Thiagalingam S., Mazur S., Grossman L. ATPase activity of the UvrA and UvrAB protein complexes of the Escherichia coli UvrABC endonuclease. Nucleic Acids Res. 1989 Jun 12;17(11):4145–4159. doi: 10.1093/nar/17.11.4145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Pazour G. J., Ta C. N., Das A. Constitutive mutations of Agrobacterium tumefaciens transcriptional activator virG. J Bacteriol. 1992 Jun;174(12):4169–4174. doi: 10.1128/jb.174.12.4169-4174.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Possot O., d'Enfert C., Reyss I., Pugsley A. P. Pullulanase secretion in Escherichia coli K-12 requires a cytoplasmic protein and a putative polytopic cytoplasmic membrane protein. Mol Microbiol. 1992 Jan;6(1):95–105. doi: 10.1111/j.1365-2958.1992.tb00841.x. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Reinstein J., Brune M., Wittinghofer A. Mutations in the nucleotide binding loop of adenylate kinase of Escherichia coli. Biochemistry. 1988 Jun 28;27(13):4712–4720. doi: 10.1021/bi00413a020. [DOI] [PubMed] [Google Scholar]
  42. Seeley T. W., Grossman L. Mutations in the Escherichia coli UvrB ATPase motif compromise excision repair capacity. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6577–6581. doi: 10.1073/pnas.86.17.6577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Seeley T. W., Grossman L. The role of Escherichia coli UvrB in nucleotide excision repair. J Biol Chem. 1990 May 5;265(13):7158–7165. [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. Sigal I. S., Gibbs J. B., D'Alonzo J. S., Temeles G. L., Wolanski B. S., Socher S. H., Scolnick E. M. Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects. Proc Natl Acad Sci U S A. 1986 Feb;83(4):952–956. doi: 10.1073/pnas.83.4.952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Story R. M., Steitz T. A. Structure of the recA protein-ADP complex. Nature. 1992 Jan 23;355(6358):374–376. doi: 10.1038/355374a0. [DOI] [PubMed] [Google Scholar]
  50. Sumner J. B. A METHOD FOR THE COLORIMETRIC DETERMINATION OF PHOSPHORUS. Science. 1944 Nov 3;100(2601):413–414. doi: 10.1126/science.100.2601.413. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. 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]
  53. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. Ueda K., McMacken R., Kornberg A. dnaB protein of Escherichia coli. Purification and role in the replication of phiX174 DNA. J Biol Chem. 1978 Jan 10;253(1):261–269. [PubMed] [Google Scholar]
  56. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  57. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. 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]
  59. 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]
  60. 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]
  61. 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]
  62. 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]
  63. Wigley D. B., Davies G. J., Dodson E. J., Maxwell A., Dodson G. Crystal structure of an N-terminal fragment of the DNA gyrase B protein. Nature. 1991 Jun 20;351(6328):624–629. doi: 10.1038/351624a0. [DOI] [PubMed] [Google Scholar]
  64. 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]
  65. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  66. Ziegelin G., Pansegrau W., Strack B., Balzer D., Kröger M., Kruft V., Lanka E. Nucleotide sequence and organization of genes flanking the transfer origin of promiscuous plasmid RP4. DNA Seq. 1991;1(5):303–327. doi: 10.3109/10425179109020786. [DOI] [PubMed] [Google Scholar]
  67. 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