Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Oct;171(10):5281–5289. doi: 10.1128/jb.171.10.5281-5289.1989

Characterization of conjugal transfer functions of Agrobacterium tumefaciens Ti plasmid pTiC58.

S B von Bodman 1, J E McCutchan 1, S K Farrand 1
PMCID: PMC210363  PMID: 2551885

Abstract

Physical characterization of 13 transposon Tn5 insertions within the agrocinopine-independent, transfer-constitutive Ti plasmid pTiC58Trac identified three separate loci essential for conjugation of this nopaline/agrocinopine A + B-type Ti plasmid. Complementation analysis with relevant subcloned DNAs indicated that the three physically separated blocks of conjugal genes constitute distinct complementation groups. Two independent Tn5 insertions within the wild-type, agrocinopine-dependent, repressed pTiC58 plasmid resulted in constitutive expression of conjugal transfer. These two insertions were physically indistinguishable and could not be complemented in trans. However, the Trac phenotype resulted when the Tn5-mutated fragment cointegrated into the wild-type Ti plasmid. While the spontaneous Trac mutant Ti plasmids were also derepressed for agrocinopine catabolism, those generated by Tn5 insertions remained inducible, indicating that this apparent cis-acting site is different from that affected in the spontaneous mutants. No chromosomal Tn5 insertion mutations were obtained that affected conjugal transfer. An octopine-type Ti plasmid, resident in different Agrobacterium tumefaciens chvB mutants, transferred at normal frequencies, demonstrating that this virulence locus affecting plant cell binding is not required for Ti plasmid conjugation. None of our conjugal mutants limited tumor development on Kalanchoe diagremontiana. Known lesions in pTiC58 vir loci had no effect on conjugal transfer of this Ti plasmid. These results show that pTiC58 Ti plasmid conjugal transfer occurs by functions independent of those required for transfer of DNA to plant cells.

Full text

PDF
5284

Images in this article

5283Image
on p.5283
5286Image
on p.5286
5286Image
on p.5286

Selected References

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

  1. Bevan M. W., Chilton M. D. T-DNA of the Agrobacterium Ti and Ri plasmids. Annu Rev Genet. 1982;16:357–384. doi: 10.1146/annurev.ge.16.120182.002041. [DOI] [PubMed] [Google Scholar]
  2. Bolivar F., Rodriguez R. L., Betlach M. C., Boyer H. W. Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9. Gene. 1977;2(2):75–93. doi: 10.1016/0378-1119(77)90074-9. [DOI] [PubMed] [Google Scholar]
  3. Bomhoff G., Klapwijk P. M., Kester H. C., Schilperoort R. A., Hernalsteens J. P., Schell J. Octopine and nopaline synthesis and breakdown genetically controlled by a plasmid of Agrobacterium tumefaciens. Mol Gen Genet. 1976 May 7;145(2):177–181. doi: 10.1007/BF00269591. [DOI] [PubMed] [Google Scholar]
  4. Caplan A., Herrera-Estrella L., Inzé D., Van Haute E., Van Montagu M., Schell J., Zambryski P. Introduction of genetic material into plant cells. Science. 1983 Nov 18;222(4625):815–821. doi: 10.1126/science.222.4625.815. [DOI] [PubMed] [Google Scholar]
  5. Chilton M. D., Currier T. C., Farrand S. K., Bendich A. J., Gordon M. P., Nester E. W. Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3672–3676. doi: 10.1073/pnas.71.9.3672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Close T. J., Tait R. C., Kado C. I. Regulation of Ti plasmid virulence genes by a chromosomal locus of Agrobacterium tumefaciens. J Bacteriol. 1985 Nov;164(2):774–781. doi: 10.1128/jb.164.2.774-781.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Comai L., Schilling-Cordaro C., Mergia A., Houck C. M. A new technique for genetic engineering of Agrobacterium Ti plasmid. Plasmid. 1983 Jul;10(1):21–30. doi: 10.1016/0147-619x(83)90054-9. [DOI] [PubMed] [Google Scholar]
  8. Depicker A., De Wilde M., De Vos G., De Vos R., Van Montagu M., Schell J. Molecular cloning of overlapping segments of the nopaline Ti-plasmid pTiC58 as a means to restriction endonuclease mapping. Plasmid. 1980 Mar;3(2):193–211. doi: 10.1016/0147-619x(80)90109-2. [DOI] [PubMed] [Google Scholar]
  9. Dessaux Y., Tempé J., Farrand S. K. Genetic analysis of mannityl opine catabolism in octopine-type Agrobacterium tumefaciens strain 15955. Mol Gen Genet. 1987 Jun;208(1-2):301–308. doi: 10.1007/BF00330457. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Douglas C. J., Halperin W., Nester E. W. Agrobacterium tumefaciens mutants affected in attachment to plant cells. J Bacteriol. 1982 Dec;152(3):1265–1275. doi: 10.1128/jb.152.3.1265-1275.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Douglas C. J., Staneloni R. J., Rubin R. A., Nester E. W. Identification and genetic analysis of an Agrobacterium tumefaciens chromosomal virulence region. J Bacteriol. 1985 Mar;161(3):850–860. doi: 10.1128/jb.161.3.850-860.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ellis J. G., Kerr A., Tempé J., Petit A. Arginine catabolism: a new function of both octopine and nopaline Ti-plasmids of Agrobacterium. Mol Gen Genet. 1979 Jun 20;173(3):263–269. doi: 10.1007/BF00268636. [DOI] [PubMed] [Google Scholar]
  14. Engler G., Depicker A., Maenhaut R., Villarroel R., Van Montagu M., Schell J. Physical mapping of DNA base sequence homologies between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens. J Mol Biol. 1981 Oct 25;152(2):183–208. doi: 10.1016/0022-2836(81)90239-4. [DOI] [PubMed] [Google Scholar]
  15. Gallie D. R., Hagiya M., Kado C. I. Analysis of Agrobacterium tumefaciens plasmid pTiC58 replication region with a novel high-copy-number derivative. J Bacteriol. 1985 Mar;161(3):1034–1041. doi: 10.1128/jb.161.3.1034-1041.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Guyon P., Chilton M. D., Petit A., Tempé J. Agropine in "null-type" crown gall tumors: Evidence for generality of the opine concept. Proc Natl Acad Sci U S A. 1980 May;77(5):2693–2697. doi: 10.1073/pnas.77.5.2693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hayman G. T., Farrand S. K. Characterization and mapping of the agrocinopine-agrocin 84 locus on the nopaline Ti plasmid pTiC58. J Bacteriol. 1988 Apr;170(4):1759–1767. doi: 10.1128/jb.170.4.1759-1767.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hohn B. In vitro packaging of lambda and cosmid DNA. Methods Enzymol. 1979;68:299–309. doi: 10.1016/0076-6879(79)68021-7. [DOI] [PubMed] [Google Scholar]
  20. Holsters M., Silva B., Van Vliet F., Genetello C., De Block M., Dhaese P., Depicker A., Inzé D., Engler G., Villarroel R. The functional organization of the nopaline A. tumefaciens plasmid pTiC58. Plasmid. 1980 Mar;3(2):212–230. doi: 10.1016/0147-619x(80)90110-9. [DOI] [PubMed] [Google Scholar]
  21. Holsters M., de Waele D., Depicker A., Messens E., van Montagu M., Schell J. Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet. 1978 Jul 11;163(2):181–187. doi: 10.1007/BF00267408. [DOI] [PubMed] [Google Scholar]
  22. Jorgensen R. A., Rothstein S. J., Reznikoff W. S. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet. 1979;177(1):65–72. doi: 10.1007/BF00267254. [DOI] [PubMed] [Google Scholar]
  23. Kao J. C., Perry K. L., Kado C. I. Indoleacetic acid complementation and its relation to host range specifying genes on the Ti plasmid of Agrobacterium tumefaciens. Mol Gen Genet. 1982;188(3):425–432. doi: 10.1007/BF00330044. [DOI] [PubMed] [Google Scholar]
  24. Kerr A., Manigault P., Tempé J. Transfer of virulence in vivo and in vitro in Agrobacterium. Nature. 1977 Feb 10;265(5594):560–561. doi: 10.1038/265560a0. [DOI] [PubMed] [Google Scholar]
  25. Klapwijk P. M., Scheulderman T., Schilperoort R. A. Coordinated regulation of octopine degradation and conjugative transfer of Ti plasmids in Agrobacterium tumefaciens: evidence for a common regulatory gene and separate operons. J Bacteriol. 1978 Nov;136(2):775–785. doi: 10.1128/jb.136.2.775-785.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Klapwijk P. M., Schilperoort R. A. Negative control of octopine degradation and transfer genes of octopine Ti plasmids in Agrobacterium tumefaciens. J Bacteriol. 1979 Aug;139(2):424–431. doi: 10.1128/jb.139.2.424-431.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Knauf V. C., Nester E. W. Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid. Plasmid. 1982 Jul;8(1):45–54. doi: 10.1016/0147-619x(82)90040-3. [DOI] [PubMed] [Google Scholar]
  28. Lippincott J. A., Beiderbeck R., Lippincott B. B. Utilization of octopine and nopaline by Agrobacterium. J Bacteriol. 1973 Oct;116(1):378–383. doi: 10.1128/jb.116.1.378-383.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nester E. W., Kosuge T. Plasmids specifying plant hyperplasias. Annu Rev Microbiol. 1981;35:531–565. doi: 10.1146/annurev.mi.35.100181.002531. [DOI] [PubMed] [Google Scholar]
  30. Rees C. E., Bradley D. E., Wilkins B. M. Organization and regulation of the conjugation genes of IncI1 plasmid colIb-P9. Plasmid. 1987 Nov;18(3):223–236. doi: 10.1016/0147-619x(87)90065-5. [DOI] [PubMed] [Google Scholar]
  31. Rogowsky P. M., Close T. J., Chimera J. A., Shaw J. J., Kado C. I. Regulation of the vir genes of Agrobacterium tumefaciens plasmid pTiC58. J Bacteriol. 1987 Nov;169(11):5101–5112. doi: 10.1128/jb.169.11.5101-5112.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. STONIER T. Agrobacterium tumefaciens Conn. II. Production of an antibiotic substance. J Bacteriol. 1960 Jun;79:889–898. doi: 10.1128/jb.79.6.889-898.1960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Slota J. E., Farrand S. K. Genetic isolation and physical characterization of pAgK84, the plasmid responsible for agrocin 84 production. Plasmid. 1982 Sep;8(2):175–186. doi: 10.1016/0147-619x(82)90055-5. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Stachel S. E., Zambryski P. C. Agrobacterium tumefaciens and the susceptible plant cell: a novel adaptation of extracellular recognition and DNA conjugation. Cell. 1986 Oct 24;47(2):155–157. doi: 10.1016/0092-8674(86)90437-x. [DOI] [PubMed] [Google Scholar]
  36. Tempé J., Petit A., Holsters M., Montagu M., Schell J. Thermosensitive step associated with transfer of the Ti plasmid during conjugation: Possible relation to transformation in crown gall. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2848–2849. doi: 10.1073/pnas.74.7.2848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Van Larebeke N., Engler G., Holsters M., Van den Elsacker S., Zaenen I., Schilperoort R. A., Schell J. Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability. Nature. 1974 Nov 8;252(5479):169–170. doi: 10.1038/252169a0. [DOI] [PubMed] [Google Scholar]
  38. Van Montagu M., Schell J. The Ti plasmids of Agrobacterium. Curr Top Microbiol Immunol. 1982;96:237–254. doi: 10.1007/978-3-642-68315-2_13. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Winans S. C., Walker G. C. Conjugal transfer system of the IncN plasmid pKM101. J Bacteriol. 1985 Jan;161(1):402–410. doi: 10.1128/jb.161.1.402-410.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zaenen I., Van Larebeke N., Van Montagu M., Schell J. Supercoiled circular DNA in crown-gall inducing Agrobacterium strains. J Mol Biol. 1974 Jun 15;86(1):109–127. doi: 10.1016/s0022-2836(74)80011-2. [DOI] [PubMed] [Google Scholar]
  42. Zimmerer R. P., Hamilton R. H., Pootjes C. Isolation and morphology of temperature Agrobacterium tumefaciens bacteriophage. J Bacteriol. 1966 Sep;92(3):746–750. doi: 10.1128/jb.92.3.746-750.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES