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Proc Natl Acad Sci U S A. Aug 20, 1996; 93(17): 8889–8894.
PMCID: PMC38564

Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complex.

Abstract

Agrobacterium tumefaciens VirB proteins are essential for gene transfer from bacteria to plants. These proteins are postulated to form a transport pore to allow transfer of the T-strand DNA intermediate. To study the function of the VirB proteins in DNA transfer, we developed an expression system in A. tumefaciens. Analysis of one VirB protein, VirB9, by Western blot assays showed that under nonreducing conditions VirB9, when expressed alone, migrates as a approximately 31-kDa band but that it migrates as a approximately 36-kDa band when expressed with all other VirB proteins. The 36-kDa band is converted to the 31-kDa band by the reducing agent 2-mercaptoethanol. Using strains that contain a deletion in a defined virB gene and strains that express specific VirB proteins, we demonstrate that the 36-kDa band is composed of VirB9 and VirB7 that are linked to each other by a disulfide bond. Mutational studies demonstrate that cysteine residues at positions 24 of VirB7 and 262 of VirB9 participate in the formation of this complex.

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Selected References

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  • Berger BR, Christie PJ. Genetic complementation analysis of the Agrobacterium tumefaciens virB operon: virB2 through virB11 are essential virulence genes. J Bacteriol. 1994 Jun;176(12):3646–3660. [PMC free article] [PubMed]
  • Stachel SE, Timmerman B, Zambryski P. Activation of Agrobacterium tumefaciens vir gene expression generates multiple single-stranded T-strand molecules from the pTiA6 T-region: requirement for 5' virD gene products. EMBO J. 1987 Apr;6(4):857–863. [PMC free article] [PubMed]
  • Veluthambi K, Jayaswal RK, Gelvin SB. Virulence genes A, G, and D mediate the double-stranded border cleavage of T-DNA from the Agrobacterium Ti plasmid. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1881–1885. [PMC free article] [PubMed]
  • Ward JE, Akiyoshi DE, Regier D, Datta A, Gordon MP, Nester EW. Characterization of the virB operon from an Agrobacterium tumefaciens Ti plasmid. J Biol Chem. 1988 Apr 25;263(12):5804–5814. [PubMed]
  • Kuldau GA, 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. [PubMed]
  • Christie PJ, Ward JE, Jr, Gordon MP, Nester EW. 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. [PMC free article] [PubMed]
  • Ward JE, Jr, Dale EM, Nester EW, Binns AN. Identification of a virB10 protein aggregate in the inner membrane of Agrobacterium tumefaciens. J Bacteriol. 1990 Sep;172(9):5200–5210. [PMC free article] [PubMed]
  • Thorstenson YR, Kuldau GA, Zambryski PC. 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. [PMC free article] [PubMed]
  • 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]
  • Weiss AA, Johnson FD, Burns DL. Molecular characterization of an operon required for pertussis toxin secretion. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2970–2974. [PMC free article] [PubMed]
  • Stachel SE, Nester EW. 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. [PMC free article] [PubMed]
  • Yusibov VM, Steck TR, Gupta V, Gelvin SB. Association of single-stranded transferred DNA from Agrobacterium tumefaciens with tobacco cells. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):2994–2998. [PMC free article] [PubMed]
  • Beijersbergen A, Dulk-Ras AD, Schilperoort RA, Hooykaas PJ. Conjugative Transfer by the Virulence System of Agrobacterium tumefaciens. Science. 1992 May 29;256(5061):1324–1327. [PubMed]
  • Bundock P, den Dulk-Ras A, Beijersbergen A, Hooykaas PJ. Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J. 1995 Jul 3;14(13):3206–3214. [PMC free article] [PubMed]
  • Vogel AM, Das A. The Agrobacterium tumefaciens virD3 gene is not essential for tumorigenicity on plants. J Bacteriol. 1992 Aug;174(15):5161–5164. [PMC free article] [PubMed]
  • Berger BR, Christie PJ. 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. [PMC free article] [PubMed]
  • Stephens KM, 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. [PMC free article] [PubMed]
  • Jones AL, Shirasu K, Kado CI. The product of the virB4 gene of Agrobacterium tumefaciens promotes accumulation of VirB3 protein. J Bacteriol. 1994 Sep;176(17):5255–5261. [PMC free article] [PubMed]
  • Ward JE, Jr, Dale EM, Binns AN. 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. [PMC free article] [PubMed]
  • Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. [PubMed]
  • Schmidhauser TJ, Helinski DR. Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria. J Bacteriol. 1985 Oct;164(1):446–455. [PMC free article] [PubMed]
  • Ditta G, Stanfield S, Corbin D, Helinski DR. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. [PMC free article] [PubMed]
  • Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. [PMC free article] [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Pazour GJ, Ta CN, Das A. Constitutive mutations of Agrobacterium tumefaciens transcriptional activator virG. J Bacteriol. 1992 Jun;174(12):4169–4174. [PMC free article] [PubMed]
  • Das A, Xie YH. Replication of the broad-host-range plasmid RK2: isolation and characterization of a spontaneous deletion mutant that can replicate in Agrobacterium tumefaciens but not in Escherichia coli. Mol Gen Genet. 1995 Feb 6;246(3):309–315. [PubMed]
  • Das A, Stachel S, Ebert P, Allenza P, Montoya A, Nester E. Promoters of Agrobacterium tumefaciens Ti-plasmid virulence genes. Nucleic Acids Res. 1986 Feb 11;14(3):1355–1364. [PMC free article] [PubMed]
  • Mersereau M, Pazour GJ, Das A. Efficient transformation of Agrobacterium tumefaciens by electroporation. Gene. 1990 May 31;90(1):149–151. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Das A. Agrobacterium tumefaciens virE operon encodes a single-stranded DNA-binding protein. Proc Natl Acad Sci U S A. 1988 May;85(9):2909–2913. [PMC free article] [PubMed]
  • Koerner TJ, Hill JE, Myers AM, Tzagoloff A. High-expression vectors with multiple cloning sites for construction of trpE fusion genes: pATH vectors. Methods Enzymol. 1991;194:477–490. [PubMed]
  • Pazour GJ, Das A. Characterization of the VirG binding site of Agrobacterium tumefaciens. Nucleic Acids Res. 1990 Dec 11;18(23):6909–6913. [PMC free article] [PubMed]
  • Pohlman RF, Genetti HD, Winans SC. Common ancestry between IncN conjugal transfer genes and macromolecular export systems of plant and animal pathogens. Mol Microbiol. 1994 Nov;14(4):655–668. [PubMed]
  • Beijersbergen A, Smith SJ, Hooykaas PJ. Localization and topology of VirB proteins of Agrobacterium tumefaciens. Plasmid. 1994 Sep;32(2):212–218. [PubMed]
  • Spudich GM, Fernandez D, Zhou XR, Christie PJ. 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. [PMC free article] [PubMed]
  • Fernandez D, Dang TA, Spudich GM, Zhou XR, Berger BR, Christie PJ. 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. [PMC free article] [PubMed]

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