Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Jul;178(14):4248–4257. doi: 10.1128/jb.178.14.4248-4257.1996

The conjugal transfer system of Agrobacterium tumefaciens octopine-type Ti plasmids is closely related to the transfer system of an IncP plasmid and distantly related to Ti plasmid vir genes.

J Alt-Mörbe 1, J L Stryker 1, C Fuqua 1, P L Li 1, S K Farrand 1, S C Winans 1
PMCID: PMC178183  PMID: 8763954

Abstract

We have determined the DNA sequences of two unlinked regions of octopine-type Ti plasmids that contain genes required for conjugal transfer. Both regions previously were shown to contain sequences that hybridize with tra genes of the nopaline-type Ti plasmid pTiC58. One gene cluster (designated tra) contains a functional oriT site and is probably required for conjugal DNA processing, while the other gene cluster (designated trb) probably directs the synthesis of a conjugal pilus and mating pore. Most predicted Tra and Trb proteins show relatively strong sequence similarity (30 to 50% identity) to the Tra and Trb proteins of the broad-host-range IncP plasmid RP4 and show significantly weaker sequence similarity to Vir proteins found elsewhere on the Ti plasmid. An exception is found in the Ti plasmid TraA protein, which is predicted to be a bifunctional nickase-helicase that has no counterpart in IncP plasmids or among Vir proteins but has homologs in at least six other self-transmissible and mobilizable plasmids. We conclude that this Ti plasmid tra system evolved by acquiring genes from two or three different sources. A similar analysis of the Ti plasmid vir region indicates that it also evolved by appropriating genes from at least two conjugal transfer systems. The widely studied plasmid pTiA6NC previously was found to be nonconjugal and to have a 12.65-kb deletion of DNA relative to other octopine-type Ti plasmids. We show that this deletion removes the promoter-distal gene of the trb region and probably accounts for the inability of this plasmid to conjugate.

Full Text

The Full Text of this article is available as a PDF (605.7 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. Balzer D., Ziegelin G., Pansegrau W., Kruft V., Lanka E. KorB protein of promiscuous plasmid RP4 recognizes inverted sequence repetitions in regions essential for conjugative plasmid transfer. Nucleic Acids Res. 1992 Apr 25;20(8):1851–1858. doi: 10.1093/nar/20.8.1851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beck von Bodman S., Hayman G. T., Farrand S. K. Opine catabolism and conjugal transfer of the nopaline Ti plasmid pTiC58 are coordinately regulated by a single repressor. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):643–647. doi: 10.1073/pnas.89.2.643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Cook D. M., Farrand S. K. The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders. J Bacteriol. 1992 Oct;174(19):6238–6246. doi: 10.1128/jb.174.19.6238-6246.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. De Greve H., Decraemer H., Seurinck J., Van Montagu M., Schell J. The functional organization of the octopine Agrobacterium tumefaciens plasmid pTiB6s3. Plasmid. 1981 Sep;6(2):235–248. doi: 10.1016/0147-619x(81)90069-x. [DOI] [PubMed] [Google Scholar]
  7. De Vos G., De Beuckeleer M., Van Montagu M., Schell J. Restriction endonuclease mapping of the octopine tumor-inducing plasmid pTiAch5 of Agrobacterium tumefaciens. Plasmid. 1981 Sep;6(2):249–253. doi: 10.1016/0147-619x(81)90070-6. [DOI] [PubMed] [Google Scholar]
  8. Deblaere R., Bytebier B., De Greve H., Deboeck F., Schell J., Van Montagu M., Leemans J. Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants. Nucleic Acids Res. 1985 Jul 11;13(13):4777–4788. doi: 10.1093/nar/13.13.4777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dessaux Y., Guyon P., Petit A., Tempé J., Demarez M., Legrain C., Tate M. E., Farrand S. K. Opine utilization by Agrobacterium spp.: octopine-type Ti plasmids encode two pathways for mannopinic acid degradation. J Bacteriol. 1988 Jul;170(7):2939–2946. doi: 10.1128/jb.170.7.2939-2946.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Doolittle R. F. Similar amino acid sequences: chance or common ancestry? Science. 1981 Oct 9;214(4517):149–159. doi: 10.1126/science.7280687. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Farrand S. K., Hwang I., Cook D. M. The tra region of the nopaline-type Ti plasmid is a chimera with elements related to the transfer systems of RSF1010, RP4, and F. J Bacteriol. 1996 Jul;178(14):4233–4247. doi: 10.1128/jb.178.14.4233-4247.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Farrand S. K., Tempé J., Dessaux Y. Localization and characterization of the region encoding catabolism of mannopinic acid from the octopine-type Ti plasmid pTi15955. Mol Plant Microbe Interact. 1990 Jul-Aug;3(4):259–267. doi: 10.1094/mpmi-3-259. [DOI] [PubMed] [Google Scholar]
  16. Fong S. T., Stanisich V. A. Identification and characterization of RP1 Tra1 cistrons involved in pilus function and plasmid mobilization. J Bacteriol. 1993 Jan;175(2):448–456. doi: 10.1128/jb.175.2.448-456.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Fuqua C., Burbea M., Winans S. C. Activity of the Agrobacterium Ti plasmid conjugal transfer regulator TraR is inhibited by the product of the traM gene. J Bacteriol. 1995 Mar;177(5):1367–1373. doi: 10.1128/jb.177.5.1367-1373.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fuqua C., Winans S. C. Conserved cis-acting promoter elements are required for density-dependent transcription of Agrobacterium tumefaciens conjugal transfer genes. J Bacteriol. 1996 Jan;178(2):435–440. doi: 10.1128/jb.178.2.435-440.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Fuqua W. C., Winans S. C., Greenberg E. P. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J Bacteriol. 1994 Jan;176(2):269–275. doi: 10.1128/jb.176.2.269-275.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gelvin S. B., Habeck L. L. vir genes influence conjugal transfer of the Ti plasmid of Agrobacterium tumefaciens. J Bacteriol. 1990 Mar;172(3):1600–1608. doi: 10.1128/jb.172.3.1600-1608.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Giladi H., Wang W. X., Oppenheim A. B. Isolation and characterization of the hupA gene coding for HU of Aeromonas proteolytica. Nucleic Acids Res. 1992 Aug 11;20(15):4092–4092. doi: 10.1093/nar/20.15.4092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gold L., Pribnow D., Schneider T., Shinedling S., Singer B. S., Stormo G. Translational initiation in prokaryotes. Annu Rev Microbiol. 1981;35:365–403. doi: 10.1146/annurev.mi.35.100181.002053. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Hwang I., Cook D. M., Farrand S. K. A new regulatory element modulates homoserine lactone-mediated autoinduction of Ti plasmid conjugal transfer. J Bacteriol. 1995 Jan;177(2):449–458. doi: 10.1128/jb.177.2.449-458.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hwang I., Li P. L., Zhang L., Piper K. R., Cook D. M., Tate M. E., Farrand S. K. TraI, a LuxI homologue, is responsible for production of conjugation factor, the Ti plasmid N-acylhomoserine lactone autoinducer. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4639–4643. doi: 10.1073/pnas.91.11.4639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. LaPointe G., Leblanc D., Morin A. Use of a polymerase-chain-reaction-amplified DNA probe from Pseudomonas putida to detect D-hydantoinase-producing microorganisms by direct colony hybridization. Appl Microbiol Biotechnol. 1995 Mar;42(6):895–900. doi: 10.1007/BF00191188. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Lipman D. J., Wilbur W. J., Smith T. F., Waterman M. S. On the statistical significance of nucleic acid similarities. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):215–226. doi: 10.1093/nar/12.1part1.215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Llosa M., Bolland S., de la Cruz F. Genetic organization of the conjugal DNA processing region of the IncW plasmid R388. J Mol Biol. 1994 Jan 14;235(2):448–464. doi: 10.1006/jmbi.1994.1005. [DOI] [PubMed] [Google Scholar]
  33. Lyras D., Chan A. W., McFarlane J., Stanisich V. A. The surface exclusion system of RP1: investigation of the roles of trbJ and trbK in the surface exclusion, transfer, and slow-growth phenotypes. Plasmid. 1994 Nov;32(3):254–261. doi: 10.1006/plas.1994.1064. [DOI] [PubMed] [Google Scholar]
  34. Matson S. W., Morton B. S. Escherichia coli DNA helicase I catalyzes a site- and strand-specific nicking reaction at the F plasmid oriT. J Biol Chem. 1991 Aug 25;266(24):16232–16237. [PubMed] [Google Scholar]
  35. Meagher R. B., Tait R. C., Betlach M., Boyer H. W. Protein expression in E. coli minicells by recombinant plasmids. Cell. 1977 Mar;10(3):521–536. doi: 10.1016/0092-8674(77)90039-3. [DOI] [PubMed] [Google Scholar]
  36. Nakamura K., Inouye M. Construction of versatile expression cloning vehicles using the lipoprotein gene of Escherichia coli. EMBO J. 1982;1(6):771–775. doi: 10.1002/j.1460-2075.1982.tb01244.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pansegrau W., Lanka E., Barth P. T., Figurski D. H., Guiney D. G., Haas D., Helinski D. R., Schwab H., Stanisich V. A., Thomas C. M. Complete nucleotide sequence of Birmingham IncP alpha plasmids. Compilation and comparative analysis. J Mol Biol. 1994 Jun 24;239(5):623–663. doi: 10.1006/jmbi.1994.1404. [DOI] [PubMed] [Google Scholar]
  38. Piper K. R., Beck von Bodman S., Farrand S. K. Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction. Nature. 1993 Apr 1;362(6419):448–450. doi: 10.1038/362448a0. [DOI] [PubMed] [Google Scholar]
  39. Pohlman R. F., Genetti H. D., Winans S. C. Common ancestry between IncN conjugal transfer genes and macromolecular export systems of plant and animal pathogens. Mol Microbiol. 1994 Nov;14(4):655–668. doi: 10.1111/j.1365-2958.1994.tb01304.x. [DOI] [PubMed] [Google Scholar]
  40. Poustka A., Rackwitz H. R., Frischauf A. M., Hohn B., Lehrach H. Selective isolation of cosmid clones by homologous recombination in Escherichia coli. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4129–4133. doi: 10.1073/pnas.81.13.4129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Reygers U., Wessel R., Müller H., Hoffmann-Berling H. Endonuclease activity of Escherichia coli DNA helicase I directed against the transfer origin of the F factor. EMBO J. 1991 Sep;10(9):2689–2694. doi: 10.1002/j.1460-2075.1991.tb07812.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Runser S. M., Meyer P. C. Purification and biochemical characterization of the hydantoin hydrolyzing enzyme from Agrobacterium species. A hydantoinase with no 5,6-dihydropyrimidine amidohydrolase activity. Eur J Biochem. 1993 May 1;213(3):1315–1324. doi: 10.1111/j.1432-1033.1993.tb17883.x. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Steck T. R., Kado C. I. Virulence genes promote conjugative transfer of the Ti plasmid between Agrobacterium strains. J Bacteriol. 1990 Apr;172(4):2191–2193. doi: 10.1128/jb.172.4.2191-2193.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tabata S., Hooykaas P. J., Oka A. Sequence determination and characterization of the replicator region in the tumor-inducing plasmid pTiB6S3. J Bacteriol. 1989 Mar;171(3):1665–1672. doi: 10.1128/jb.171.3.1665-1672.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Traxler B. A., Minkley E. G., Jr Revised genetic map of the distal end of the F transfer operon: implications for DNA helicase I, nicking at oriT, and conjugal DNA transport. J Bacteriol. 1987 Jul;169(7):3251–3259. doi: 10.1128/jb.169.7.3251-3259.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wang L., Helmann J. D., Winans S. C. The A. tumefaciens transcriptional activator OccR causes a bend at a target promoter, which is partially relaxed by a plant tumor metabolite. Cell. 1992 May 15;69(4):659–667. doi: 10.1016/0092-8674(92)90229-6. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. 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]
  50. 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]
  51. Winans S. C. Two-way chemical signaling in Agrobacterium-plant interactions. Microbiol Rev. 1992 Mar;56(1):12–31. doi: 10.1128/mr.56.1.12-31.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zagursky R. J., Berman M. L. Cloning vectors that yield high levels of single-stranded DNA for rapid DNA sequencing. Gene. 1984 Feb;27(2):183–191. doi: 10.1016/0378-1119(84)90139-2. [DOI] [PubMed] [Google Scholar]
  53. Ziegelin G., Pansegrau W., Lurz R., Lanka E. TraK protein of conjugative plasmid RP4 forms a specialized nucleoprotein complex with the transfer origin. J Biol Chem. 1992 Aug 25;267(24):17279–17286. [PubMed] [Google Scholar]
  54. von Bodman S. B., McCutchan J. E., Farrand S. K. Characterization of conjugal transfer functions of Agrobacterium tumefaciens Ti plasmid pTiC58. J Bacteriol. 1989 Oct;171(10):5281–5289. doi: 10.1128/jb.171.10.5281-5289.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES