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. 1994 Sep;176(17):5255–5261. doi: 10.1128/jb.176.17.5255-5261.1994

The product of the virB4 gene of Agrobacterium tumefaciens promotes accumulation of VirB3 protein.

A L Jones 1, K Shirasu 1, C I Kado 1
PMCID: PMC196708  PMID: 8071199

Abstract

The process of T-DNA transfer from Agrobacterium tumefaciens to plant cells is thought to involve passage of a DNA-protein complex through a specialized structure in the bacterial membrane. The virB operon of A. tumefaciens encodes 11 proteins, of which 9 are known to be located in the membranes and 10 have been shown to be essential for virulence. Sequence comparisons between proteins encoded by the virB operon and those encoded by operons from conjugative plasmids indicated that VirB proteins may form a structure similar to a conjugative pilus. Here, we examine the effects of mutations in virB4 on the accumulation and localization of other VirB proteins. VirB4 shares amino acid sequence similarity with the TraC protein of plasmid F, which is essential for pilus formation in Escherichia coli, and with the PtlC protein of Bordetella pertussis, which is required for toxin secretion. Polar and nonpolar virB4 mutants were examined, and all were shown to be unable to accumulate VirB3 protein to wild-type levels. A low level of VirB3 protein which was present in induced NT1RE cells harboring virB4 nonpolar mutant pBM1130 was found to associate with the inner membrane fraction only, whereas in wild-type cells VirB3 associated with both inner and outer membranes. The results indicate that for VirB3 to accumulate in the outer membrane, VirB4 must also be present, and it is possible that one role of VirB4 is in the correct assembly of a VirB protein membrane structure.

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Selected References

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

  1. 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]
  2. 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]
  3. 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]
  4. 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]
  5. Cangelosi G. A., Ankenbauer R. G., Nester E. W. Sugars induce the Agrobacterium virulence genes through a periplasmic binding protein and a transmembrane signal protein. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6708–6712. doi: 10.1073/pnas.87.17.6708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Frost L. S., Ippen-Ihler K., Skurray R. A. Analysis of the sequence and gene products of the transfer region of the F sex factor. Microbiol Rev. 1994 Jun;58(2):162–210. doi: 10.1128/mr.58.2.162-210.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gallie D. R., Novak S., Kado C. I. Novel high- and low-copy stable cosmids for use in Agrobacterium and Rhizobium. Plasmid. 1985 Sep;14(2):171–175. doi: 10.1016/0147-619x(85)90078-2. [DOI] [PubMed] [Google Scholar]
  9. Harrington L. C., Rogerson A. C. The F pilus of Escherichia coli appears to support stable DNA transfer in the absence of wall-to-wall contact between cells. J Bacteriol. 1990 Dec;172(12):7263–7264. doi: 10.1128/jb.172.12.7263-7264.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hultgren S. J., Normark S., Abraham S. N. Chaperone-assisted assembly and molecular architecture of adhesive pili. Annu Rev Microbiol. 1991;45:383–415. doi: 10.1146/annurev.mi.45.100191.002123. [DOI] [PubMed] [Google Scholar]
  11. Jiang B., Howard S. P. The Aeromonas hydrophila exeE gene, required both for protein secretion and normal outer membrane biogenesis, is a member of a general secretion pathway. Mol Microbiol. 1992 May;6(10):1351–1361. doi: 10.1111/j.1365-2958.1992.tb00856.x. [DOI] [PubMed] [Google Scholar]
  12. Kado C. I., Heskett M. G. Selective media for isolation of Agrobacterium, Corynebacterium, Erwinia, Pseudomonas, and Xanthomonas. Phytopathology. 1970 Jun;60(6):969–976. doi: 10.1094/phyto-60-969. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Kuehn M. J., Normark S., Hultgren S. J. Immunoglobulin-like PapD chaperone caps and uncaps interactive surfaces of nascently translocated pilus subunits. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10586–10590. doi: 10.1073/pnas.88.23.10586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. Lessl M., Balzer D., Weyrauch K., Lanka E. The mating pair formation system of plasmid RP4 defined by RSF1010 mobilization and donor-specific phage propagation. J Bacteriol. 1993 Oct;175(20):6415–6425. doi: 10.1128/jb.175.20.6415-6425.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lundquist R. C., Close T. J., Kado C. I. Genetic complementation of Agrobacterium tumefaciens Ti plasmid mutants in the virulence region. Mol Gen Genet. 1984;193(1):1–7. doi: 10.1007/BF00327406. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Ou J. T., Anderson T. F. Role of pili in bacterial conjugation. J Bacteriol. 1970 Jun;102(3):648–654. doi: 10.1128/jb.102.3.648-654.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pansegrau W., Lanka E. Common sequence motifs in DNA relaxases and nick regions from a variety of DNA transfer systems. Nucleic Acids Res. 1991 Jun 25;19(12):3455–3455. doi: 10.1093/nar/19.12.3455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rogowsky P. M., Powell B. S., Shirasu K., Lin T. S., Morel P., Zyprian E. M., Steck T. R., Kado C. I. Molecular characterization of the vir regulon of Agrobacterium tumefaciens: complete nucleotide sequence and gene organization of the 28.63-kbp regulon cloned as a single unit. Plasmid. 1990 Mar;23(2):85–106. doi: 10.1016/0147-619x(90)90028-b. [DOI] [PubMed] [Google Scholar]
  24. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. 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]
  29. 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]
  30. 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]
  31. 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]
  32. 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]
  33. 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]
  34. Waters V. L., Hirata K. H., Pansegrau W., Lanka E., Guiney D. G. Sequence identity in the nick regions of IncP plasmid transfer origins and T-DNA borders of Agrobacterium Ti plasmids. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1456–1460. doi: 10.1073/pnas.88.4.1456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. 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]

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