Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Feb;177(4):892–897. doi: 10.1128/jb.177.4.892-897.1995

The octopine-type Ti plasmid pTiA6 of Agrobacterium tumefaciens contains a gene homologous to the chromosomal virulence gene acvB.

V S Kalogeraki 1, S C Winans 1
PMCID: PMC176680  PMID: 7860597

Abstract

Although the majority of genes required for the transfer of T-DNA from Agrobacterium tumefaciens to plant nuclei are located on the Ti plasmid, some chromosomal genes, including the recently described acvB gene, are also required. We show that AcvB shows 50% identity with the product of an open reading frame, designated virJ, that is found between the virA and virB genes in the octopine-type Ti plasmid pTiA6. This reading frame is not found in the nopaline-type Ti plasmid pTiC58. acvB is required for tumorigenesis by a strain carrying a nopaline-type Ti plasmid, and virJ complements this nontumorigenic phenotype, indicating that the products of these genes have similar functions. A virJ-phoA fusion expressed enzymatically active alkaline phosphatase, indicating that VirJ is at least partially exported. virJ is induced in a VirA/VirG-dependent fashion by the vir gene inducer acetosyringone. Primer extension analysis and subcloning of the virJ-phoA fusion indicate that the acetosyringone-inducible promoter lies directly upstream of the virJ structural gene. Although the roles of the two homologous genes in tumorigenesis remain to be elucidated, strains lacking acvB and virJ (i) are proficient for induction of the vir regulon, (ii) are able to transfer their Ti plasmids by conjugation, and (iii) are resistant to plant wound extracts. Finally, mutations in these genes cannot be complemented extracellularly.

Full Text

The Full Text of this article is available as a PDF (326.9 KB).

Selected References

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

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [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. Breedveld M. W., Miller K. J. Cyclic beta-glucans of members of the family Rhizobiaceae. Microbiol Rev. 1994 Jun;58(2):145–161. doi: 10.1128/mr.58.2.145-161.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Charles T. C., Nester E. W. A chromosomally encoded two-component sensory transduction system is required for virulence of Agrobacterium tumefaciens. J Bacteriol. 1993 Oct;175(20):6614–6625. doi: 10.1128/jb.175.20.6614-6625.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen C. Y., Winans S. C. Controlled expression of the transcriptional activator gene virG in Agrobacterium tumefaciens by using the Escherichia coli lac promoter. J Bacteriol. 1991 Feb;173(3):1139–1144. doi: 10.1128/jb.173.3.1139-1144.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Citovsky V., Zupan J., Warnick D., Zambryski P. Nuclear localization of Agrobacterium VirE2 protein in plant cells. Science. 1992 Jun 26;256(5065):1802–1805. doi: 10.1126/science.1615325. [DOI] [PubMed] [Google Scholar]
  10. Crews J. L., Colby S., Matthysse A. G. Agrobacterium rhizogenes mutants that fail to bind to plant cells. J Bacteriol. 1990 Nov;172(11):6182–6188. doi: 10.1128/jb.172.11.6182-6188.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ditta G., Stanfield S., Corbin D., Helinski D. R. 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. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Farrand S. K., O'Morchoe S. P., McCutchan J. Construction of an Agrobacterium tumefaciens C58 recA mutant. J Bacteriol. 1989 Oct;171(10):5314–5321. doi: 10.1128/jb.171.10.5314-5321.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fischer H. M., Babst M., Kaspar T., Acuña G., Arigoni F., Hennecke H. One member of a gro-ESL-like chaperonin multigene family in Bradyrhizobium japonicum is co-regulated with symbiotic nitrogen fixation genes. EMBO J. 1993 Jul;12(7):2901–2912. doi: 10.1002/j.1460-2075.1993.tb05952.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fuqua W. C., Winans S. C. A LuxR-LuxI type regulatory system activates Agrobacterium Ti plasmid conjugal transfer in the presence of a plant tumor metabolite. J Bacteriol. 1994 May;176(10):2796–2806. doi: 10.1128/jb.176.10.2796-2806.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gay P., Le Coq D., Steinmetz M., Berkelman T., Kado C. I. Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. J Bacteriol. 1985 Nov;164(2):918–921. doi: 10.1128/jb.164.2.918-921.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gray J., Wang J., Gelvin S. B. Mutation of the miaA gene of Agrobacterium tumefaciens results in reduced vir gene expression. J Bacteriol. 1992 Feb;174(4):1086–1098. doi: 10.1128/jb.174.4.1086-1098.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huang M. L., Cangelosi G. A., Halperin W., Nester E. W. A chromosomal Agrobacterium tumefaciens gene required for effective plant signal transduction. J Bacteriol. 1990 Apr;172(4):1814–1822. doi: 10.1128/jb.172.4.1814-1822.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kaniga K., Delor I., Cornelis G. R. A wide-host-range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene. 1991 Dec 20;109(1):137–141. doi: 10.1016/0378-1119(91)90599-7. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  21. Lin T. S., Kado C. I. The virD4 gene is required for virulence while virD3 and orf5 are not required for virulence of Agrobacterium tumefaciens. Mol Microbiol. 1993 Aug;9(4):803–812. doi: 10.1111/j.1365-2958.1993.tb01739.x. [DOI] [PubMed] [Google Scholar]
  22. Long S. R., Staskawicz B. J. Prokaryotic plant parasites. Cell. 1993 Jun 4;73(5):921–935. doi: 10.1016/0092-8674(93)90271-q. [DOI] [PubMed] [Google Scholar]
  23. Machida Y., Sakurai M., Kiyokawa S., Ubasawa A., Suzuki Y., Ikeda J. E. Nucleotide sequence of the insertion sequence found in the T-DNA region of mutant Ti plasmid pTiA66 and distribution of its homologues in octopine Ti plasmid. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7495–7499. doi: 10.1073/pnas.81.23.7495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Manoil C., Beckwith J. A genetic approach to analyzing membrane protein topology. Science. 1986 Sep 26;233(4771):1403–1408. doi: 10.1126/science.3529391. [DOI] [PubMed] [Google Scholar]
  25. Mantis N. J., Winans S. C. The chromosomal response regulatory gene chvI of Agrobacterium tumefaciens complements an Escherichia coli phoB mutation and is required for virulence. J Bacteriol. 1993 Oct;175(20):6626–6636. doi: 10.1128/jb.175.20.6626-6636.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matthysse A. G. Characterization of nonattaching mutants of Agrobacterium tumefaciens. J Bacteriol. 1987 Jan;169(1):313–323. doi: 10.1128/jb.169.1.313-323.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Matthysse A. G. Role of bacterial cellulose fibrils in Agrobacterium tumefaciens infection. J Bacteriol. 1983 May;154(2):906–915. doi: 10.1128/jb.154.2.906-915.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Metts J., West J., Doares S. H., Matthysse A. G. Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes. J Bacteriol. 1991 Feb;173(3):1080–1087. doi: 10.1128/jb.173.3.1080-1087.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pazour G. J., Das A. Characterization of the VirG binding site of Agrobacterium tumefaciens. Nucleic Acids Res. 1990 Dec 11;18(23):6909–6913. doi: 10.1093/nar/18.23.6909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schwedock J. S., Long S. R. Rhizobium meliloti genes involved in sulfate activation: the two copies of nodPQ and a new locus, saa. Genetics. 1992 Dec;132(4):899–909. doi: 10.1093/genetics/132.4.899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sciaky D., Montoya A. L., Chilton M. D. Fingerprints of Agrobacterium Ti plasmids. Plasmid. 1978 Feb;1(2):238–253. doi: 10.1016/0147-619x(78)90042-2. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Summers W. C. A simple method for extraction of RNA from E. coli utilizing diethyl pyrocarbonate. Anal Biochem. 1970 Feb;33(2):459–463. doi: 10.1016/0003-2697(70)90316-7. [DOI] [PubMed] [Google Scholar]
  38. Vogel A. M., Das A. The Agrobacterium tumefaciens virD3 gene is not essential for tumorigenicity on plants. J Bacteriol. 1992 Aug;174(15):5161–5164. doi: 10.1128/jb.174.15.5161-5164.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Winans S. C., Kerstetter R. A., Ward J. E., Nester E. W. A protein required for transcriptional regulation of Agrobacterium virulence genes spans the cytoplasmic membrane. J Bacteriol. 1989 Mar;171(3):1616–1622. doi: 10.1128/jb.171.3.1616-1622.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Winans S. C., Mantis N. J., Chen C. Y., Chang C. H., Han D. C. Host recognition by the VirA, VirG two-component regulatory proteins of agrobacterium tumefaciens. Res Microbiol. 1994 Jun-Aug;145(5-6):461–473. doi: 10.1016/0923-2508(94)90095-7. [DOI] [PubMed] [Google Scholar]
  41. Winans S. C. Transcriptional induction of an Agrobacterium regulatory gene at tandem promoters by plant-released phenolic compounds, phosphate starvation, and acidic growth media. J Bacteriol. 1990 May;172(5):2433–2438. doi: 10.1128/jb.172.5.2433-2438.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wirawan I. G., Kang H. W., Kojima M. Isolation and characterization of a new chromosomal virulence gene of Agrobacterium tumefaciens. J Bacteriol. 1993 May;175(10):3208–3212. doi: 10.1128/jb.175.10.3208-3212.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yun A C, Hadley R G, Szalay A A. A plasmid sequence from Rhizobium leguminosarum 300 contains homology to sequences near the octopine TL-DNA right border. Mol Gen Genet. 1987 Oct;209(3):580–584. doi: 10.1007/BF00331166. [DOI] [PubMed] [Google Scholar]

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

RESOURCES