Molecular and General Genetics MGG

, Volume 228, Issue 1–2, pp 24–32

Localization and orientation of the VirD4 protein of Agrobacterium tumefaciens in the cell membrane

  • Shigehisa Okamoto
  • Akiko Toyoda-Yamamoto
  • Kenji Ito
  • Itaru Takebe
  • Yasunori Machida
Article

Summary

The virD4 gene of Agrobacterium tumefaciens is essential for the formation of crown galls. Analysis of the nucleotide sequence of virD4 has suggested that the N-terminal region of the encoded protein acts as a signal peptide for the transport of the VirD4 protein to the cell membrane of Agrobacterium. We have examined the localization and orientation of this protein in the cell membrane. When the nucleotides encoding the first 30 to 41 amino acids from the N-terminus of the VirD4 protein were fused to the gene for alkaline phosphatase from which the signal sequence had been removed, alkaline phosphatase activity was detectable under appropriate conditions. Immunoblotting with VirD4-specific antiserum indicated that the VirD4 protein could be recovered exclusively from the membrane fraction of Agrobacterium cells. Moreover, when the membrane fraction was separated into inner and outer membrane fractions by sucrose density-gradient centrifugation, VirD4 protein was detected in the inner-membrane fraction and in fractions that sedimented between the inner and outer membrane fractions. By contrast, the VirD4′/′alkaline phosphatase fusion protein with the N-terminal sequence from VirD4 was detected only in the inner membrane fraction. Treatment of spheroplasts of Agrobacterium cells with proteinase K resulted in digestion of the VirD4 protein. These results indicate that the VirD4 protein is transported to the bacterial membrane and anchored on the inner membrane by its N-terminal region. In addition, the C-terminal portion of the VirD4 protein probably protrudes into the periplasmic space, perhaps in association with some unidentified cellular factor(s).

Key words

Crown gall tumour Agrobacterium tumefaciens Ti plasmids VirD4 protein Alkaline phosphatase 

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References

  1. Achtman M, Manning PA, Edelbluth C, Herrlich P (1979) Export without proteolytic processing of inner and outer membrane proteins encoded by F sex factor tra cistrons in Escherichia coli minicells. Proc Natl Acad Sci USA 76:4837–4841Google Scholar
  2. Akiyoshi DE, Morris RO, Hinz R, Mischke BS, Kosuge T, Garfinkel DJ, Gordon MP, Nester EW (1983) Cytokinin/auxin balance in crown gall tumors is regulated by specific loci in the T-DNA. Proc Natl Acad Sci USA 80:407–411Google Scholar
  3. Albright LM, Yanofsky MF, Leroux B, Ma D, Nester EW (1987) Processing of the T-DNA of Agrobacterium tumefaciens generates border nicks and linear, single-stranded T-DNA. J Bacteriol 169:1046–1055Google Scholar
  4. Alt-Moerbe J, Rak B, Schröder J (1986) A 3.6-kb segment from vir region of Ti plasmids contains genes responsible for border sequence-directed production of T region circles in E. coli. EMBO J 5:1129–1135Google Scholar
  5. Barbas JA, Díaz J, Rodríguez-Tébar A, Vázquez D (1986) Specific location of penicillin-binding proteins within the cell envelope of Escherichia coli. J Bacteriol 165:269–275Google Scholar
  6. Barry GF, Rogers SG, Fraley RT, Brand L (1984) Identification of a cloned cytokinin biosynthetic gene. Proc Natl Acad Sci USA 81:4776–4780Google Scholar
  7. Bayer ME (1968) Areas of adhesion between wall and membrane of Escherichia coli. J Gen Microbiol 53:395–404Google Scholar
  8. Bayer MH, Bayer ME (1985) Phosphoglycerides and phospholipase C in membrane fractions of Escherichia coli B. J Bacteriol 162:50–54Google Scholar
  9. Bayer MH, Costello GP, Bayer ME (1982) Isolation and partial characterization of membrane vesicles carrying markers of the membrane adhesion sites. J Bacteriol 149:758–767Google Scholar
  10. Bevan MW, Chilton M-D (1982) T-DNA of the Agrobacterium Ti and Ri plasmids. Annu Rev Genet 16:357–384Google Scholar
  11. Buchanan-Wollaston V, Passiatore JE, Cannon F (1987) The mob and oriT mobilization functions of a bacterial plasmid promote its transfer to plants. Nature 328:172–175Google Scholar
  12. Cangelosi GA, Ankenbauer RG, Nester EW (1990) Sugars induce the Agrobacterium virulence genes through a periplasmic binding protein and a transmembrane signal protein. Proc Natl Acad Sci USA 87:6708–6712Google Scholar
  13. Christie PJ, Ward JE, Winans SC, Nester EW (1988) the Agrobacterium tumefaciens virE2 gene product is a single-stranded-DNA-binding protein that associates with T-DNA. J Bacteriol 170:2659–2667Google Scholar
  14. Christie PJ, Ward JE, Gordon MP, Nester EW (1989) A gene required for transfer of T-DNA to plants encodes an ATPase with autophosphorylating activity. Proc Natl Acad Sci USA 86:9677–9681Google Scholar
  15. Das A (1988) Agrobacterium tumefaciens virE operon encodes a single-stranded DNA-binding protein. Proc Natl Acad Sci USA 85:2909–2913Google Scholar
  16. De Maagd RA, Lugtenberg B (1986) Fractionation of Rhizobium leguminosarum cells into outer membrane, cytoplasmic membrane, periplasmic, and cytoplasmic components. J Bacteriol 167:1083–1085Google Scholar
  17. Douglas CJ, Stenloni RJ, Rubin RA, Nester EW (1985) Identification and genetic analysis of an Agrobacterium tumefaciens chromosomal virulence region. J Bacteriol 161:850–860Google Scholar
  18. Dürrenberger F, Crameri A, Hohn B, Koukolíková-Nicolá Z (1989) Covalently bound VirD2 protein of Agrobacterium tumefaciens protects the T-DNA from exonucleolytic degradation. Proc Natl Acad Sci USA 86:9154–9158Google Scholar
  19. Engström P, Zambryski P, Van Montagu M, Stachel S (1987) Characterization of Agrobacterium tumefaciens virulence proteins induced by the plant factor acetosyringone. J Mol Biol 197:635–645Google Scholar
  20. Garfinkel DJ, Simpson RB, Ream LW, White FF, Gordon MP, Nester EW (1981) Genetic analysis of crown gall: Fine structure map of the T-DNA by site-directed mutagenesis. Cell 27:143–153Google Scholar
  21. Gietl C, Koukolíková-Nicolá Z, Hohn B (1987) Mobilization of T-DNA from Agrobacterium to plant cells involves a protein that binds single-stranded DNA. Proc Natl Acad Sci USA 84:9006–9010Google Scholar
  22. Gutierrez C, Barondess J, Manoil C, Beckwith J (1987) The use of transposon TnphoA to detect genes for cell envelope proteins subject to a common regulatory stimulus. J Mol Biol 195:289–297Google Scholar
  23. Haymerle H, Herz J, Bressan GM, Frank R, Stanley KK (1986) Efficient construction of cDNA libraries in plasmid expression vectors using an adaptor strategy. Nucleic Acids Res 14:8615–8624Google Scholar
  24. Herrera-Estrella A, Chen Z-m, Van Montagu M, Wang K (1988) VirD proteins of Agrobacterium tumefaciens are required for the formation of a covalent DNA-protein complex at the 5′ terminus of T-strand molecules. EMBO J 7:4055–4062Google Scholar
  25. Hille J, van Kan J, Schilperoort R (1984) Trans-acting virulence functions of the octopine Ti plasmid from Agrobacterium tumefaciens. J Bacteriol 158:754–756Google Scholar
  26. Hirayama T, Muranaka T, Ohkawa H, Oka A (1988) Organization and characterization of the virCD genes from Agrobacterium rhizogenes. Mol Gen Genet 213:229–237Google Scholar
  27. Howard EA, Winsor BA, De Vos G, Zambryski P (1989) Activation of the T-DNA transfer process in Agrobacterium results in the generation of a T-strand-protein complex: Tight association of VirD2 with the 5′ ends of T-strands. Proc Natl Acad Sci USA 86:4017–4021Google Scholar
  28. Inzé D, Follin A, Van Lijsebettens M, Simoens C, Genetello C, Van Montagu M, Schell J (1984) Genetic analysis of the individual T-DNA genes of Agrobacterium tumefaciens; further evidence that two genes are involved in indole-3-acetic acid synthesis. Mol Gen Genet 194:265–274Google Scholar
  29. Ippen-Ihler KA, Minkley Jr EG (1986) The conjugation system of F, the fertility factor of Escherichia coli. Annu Rev Genet 20:593–624Google Scholar
  30. Jayaswal RK, Veluthambi K, Gelvin SB, Slightom JL (1987) Double-stranded cleavage of T-DNA and generation of single-stranded T-DNA molecules in Escherichia coli by a virD-encoded border-specific endonuclease from Agrobacterium tumefaciens. J Bacteriol 169:5035–5045Google Scholar
  31. Keleti G, Lederer WH (ed) (1974) Handbook of micromethods for the biological sciences. Van Nostrand Reinhold, New York, pp 74–75Google Scholar
  32. Klee HJ, Gordon MP, Nester EW (1982) Complementation analysis of Agrobacterium tumefaciens Ti plasmid mutations affecting oncogenicity. J Bacteriol 150:327–331Google Scholar
  33. Klee HJ, White FF, Iyer VN, Gordon MP, Nester EW (1983) Mutational analysis of the virulence region of an Agrobacterium tumefaciens Ti plasmid. J Bacteriol 153:878–883Google Scholar
  34. Klionsky DJ, Emr SD (1989) Membrane protein sorting: biosynthesis, transport and processing of yeast vacuolar alkaline phosphatase. EMBO J 8:2241–2250Google Scholar
  35. Koukolíkova-Nicolá Z, Shillito RD, Hohn B, Wang K, Van Montagu M, Zambryski P (1985) Involvement of circular intermediates in the transfer of T-DNA from Agrobacterium tumefaciens to plant cells. Nature 313:191–196Google Scholar
  36. Koukolíková-Nicolá Z, Albright L, Hohn B (1987) The mechanism of T-DNA transfer from Agrobacterium tumefaciens to the plant cell. Plant Gene Res 4:109–148Google Scholar
  37. Kuldau GA, De Vos G, Owen J, McCaffrey G, Zambryski P (1990) The virB operon of Agrobacterium tumefaciens pTiC58 encodes 11 open reading frames. Mol Gen Genet 221:256–266Google Scholar
  38. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
  39. Leemans J, Deblaere R, Willmitzer L, De Greve H, Hernalsteens JP, Van Montagu M, Schell J (1982) Genetic identification of functions of TL-DNA transcripts in octopine crown galls. EMBO J 1:147–152Google Scholar
  40. Machida Y, Usami S, Yamamoto A, Niwa Y, Takebe I (1986) Plant-inducible recombination between the 25 by border sequences of T-DNA in Agrobacterium tumefaciens. Mol Gen Genet 204:374–382Google Scholar
  41. Machida Y, Okamoto S, Matsumoto S, Usami S, Yamamoto A, Niwa Y, Jeong SD, Nagamine J, Shimoda N, Machida C, Iwahashi M (1989) Mechanisms of crown gall formation: T-DNA transfer from Agrobacterium to plant cells. Bot Mag Tokyo 102:331–350Google Scholar
  42. Manoil C, Beckwith J (1986) A genetic approach to analyzing membrane protein topology. Science 233:1403–1408Google Scholar
  43. Matsumoto S, Takebe I, Machida Y (1988) Escherichia coli lacZ gene as a biochemical and histochemical marker in plant cells. Gene 66:19–29Google Scholar
  44. Melchers LS, Maroney MJ, den Dulk-Ras A, Thompson DV, van Vuuren HAJ, Schilperoort RA, Hooykaas PJJ (1990) Octopine and nopaline strains of Agrobacterium tumefaciens differ in virulence; molecular characterization of the virF locus. Plant Mol Biol 14:249–259Google Scholar
  45. Minsky A, Summers RG, Knowles JR (1986) Secretion of β-lacta-mase into the periplasm of Escherichia coli: Evidence for a distinct release step associated with a conformational change. Proc Natl Acad Sci USA 83:4180–4184Google Scholar
  46. Mizuno T, Kageyama M (1978) Separation and characterization of the outer membrane of Pseudomonas aeruginosa. J Biochem 84:179–191Google Scholar
  47. Nester EW, Gordon MP, Amasino RM, Yanofsky MF (1984) Crown gall: A molecular and physiological analysis. Annu Rev Plant Physiol 35:387–413Google Scholar
  48. Niwa Y, Yamamoto A, Machida C, Takebe I, Machida Y (1988) Right-hand border regions of octopine T-DNA are recognized by RNA polymerase of Agrobacterium as well as by VirD1 and VirD2 proteins. Nucleic Acids Res 16:7647–7661Google Scholar
  49. Petit A, Tempé J, Kerr A, Holster M, Van Montagu M, Schell J (1978) Substrate induction of conjugative activity of Agrobacterium tumefaciens Ti plasmids. Nature 271:570–571Google Scholar
  50. Porter SG, Yanofsky MF, Nester EW (1987) Molecular characterization of the virD operon from Agrobacterium tumefaciens. Nucleic Acids Res 15:7503–7517Google Scholar
  51. Ream LW, Gordon MP, Nester EW (1983) Multiple mutations in the T region of the Agrobacterium tumefaciens tumor-inducing plasmid. Proc Natl Acad Sci USA 80:1660–1664Google Scholar
  52. Rodríguez-Tébar A, Barbas JA, Vázquez D (1985) Location of some proteins involved in peptidoglycan synthesis and cell division in the inner and outer membranes of Escherichia coli. J Bacteriol 161:243–248Google Scholar
  53. Schröder G, Waffenschmidt S, Weiler EW, Schröder J (1984) The T-region of Ti plasmids codes for an enzyme synthesizing indol3-acetic acid. Eur J Biochem 138:387–391Google Scholar
  54. Shimoda N, Toyoda-Yamamoto A, Nagamine J, Usami S, Katayama M, Sakagami Y, Machida Y (1990) Control of expression of Agrobacterium vir genes by synergistic actions of phenolic signal molecules and monosaccharides. Proc Natl Acad Sci USA 87:6684–6688Google Scholar
  55. Spencer PA, Towers GHN (1988) Specificity of signal compounds detected by Agrobacterium tumefaciens. Phytochemistry 27:2781–2785Google Scholar
  56. Stachel SE, Nester EW (1986) The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens. EMBO J 5:1445–1454Google Scholar
  57. Stachel SE, Timmerman B, Zambryski P (1986) Generation of single-stranded T-DNA molecules during the initial stages of TDNA transfer from Agrobacterium tumefaciens to plant cells. Nature 322:706–712Google Scholar
  58. Stachel SE, Zambryski PC (1986) Agrobacterium tumefaciens and the susceptible plant cell: A novel adaptation of extracellular recognition and DNA conjugation. Cell 47:155–157Google Scholar
  59. Stachel SE, An G, Flores C, Nester EW (1985a) A Tn3 lacZ transposon for the random generation of β-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. EMBO J 4:891–898Google Scholar
  60. Stachel SE, Messens E, Van Montagu M, Zambryski P (1985b) Identification of th signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318:624–629Google Scholar
  61. Steck TR, Close TJ, Kado CI (1989) High levels of double-stranded transferred DNA (T-DNA) processing from an intact nopaline Ti plasmid. Proc Natl Acad Sci USA 86:2133–2137Google Scholar
  62. Usami S, Okamoto S, Takebe I, Machida Y (1988) Factor inducing Agrobacterium tumefaciens vir gene expression is present in monocotyledonous plants. Proc Natl Acad Sci USA 85:3748–3752Google Scholar
  63. Veluthambi K, Jayaswal RK, Gelvin SB (1987) The virulence genes A, G and D mediate the double-stranded border cleavage of the T-DNA from the Agrobacterium tumefaciens Ti plasmid. Proc Natl Acad Sci USA 84:1881–1885Google Scholar
  64. Ward ER, Barnes WM (1988) VirD2 protein of Agrobacterium tumefaciens very tightly linked to the 5′ end of T-strand DNA. Science 242:927–930Google Scholar
  65. Ward JE, Akiyoshi DE, Regier D, Datta A, Gordon MP, Nester EW (1988) Characterization of the virB operon from an Agrobacterium tumefaciens Ti plasmid. J Biol Chem 263:5804–5814Google Scholar
  66. Ward JE, Dale EM, Nester EW, Binns AN (1990) Identification of a VirB10 protein aggregate in the inner membrane of Agrobacterium tumefaciens. J Bacteriol 172:5200–5210Google Scholar
  67. Yamamoto A, Iwahashi M, Yanofsky MF, Nester EW, Takebe I, Machida Y (1987) The promoter proximal region in the virD locus of Agrobacterium tumefaciens is necessary for the plantinducible circularization of T-DNA. Mol Gen Genet 206:174–177Google Scholar
  68. Yanofsky ME, Porter SG, Young C, Albright LM, Gordon MP, Nester EW (1986) The virD locus of Agrobacterium tumefaciens encodes a site specific endonuclease. Cell 47:471–477Google Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Shigehisa Okamoto
    • 1
  • Akiko Toyoda-Yamamoto
    • 1
  • Kenji Ito
    • 1
  • Itaru Takebe
    • 1
  • Yasunori Machida
    • 1
  1. 1.Department of Biology, Faculty of ScienceNagoya UniversityChikusa-ku, NagoyaJapan
  2. 2.Plant Cell Biology Research CentreSchool of Botany University of MelbourneParkvilleAustralia

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