Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1987 Nov;169(11):5035–5045. doi: 10.1128/jb.169.11.5035-5045.1987

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.

R K Jayaswal 1, K Veluthambi 1, S B Gelvin 1, J L Slightom 1
PMCID: PMC213905  PMID: 2822660

Abstract

The virD locus of Agrobacterium tumefaciens Ti plasmid pTiA6 was sequenced. Computer analysis of the sequence indicated five possible open reading frames (ORFs) within this locus. Two additional ORFs were identified distal to this locus. However, only two polypeptides of apparent molecular masses 16 and 56 kilodaltons, the products of ORFs 1 and 2, were detected in Escherichia coli, both in vivo and in an in vitro coupled transcription-translation system. The virD locus was cloned in expression vector pKK223.3 under control of a tac promoter and introduced into an E. coli strain harboring mini-Ti plasmid pAL1050. When induced with isopropyl-beta-D-thiogalactopyranoside, the virD gene products exhibited double-stranded T-DNA border-specific endonuclease activity. Deletion analysis demonstrated that this activity is encoded within the 5'-proximal 1.7-kilobase-pair portion of the virD locus that carries ORF 1 and most of ORF 2. Neither ORF 1 nor ORF 2 independently showed endonuclease activity; complementation studies indicated that the products of ORFs 1 and 2 together have this activity. The expression of this 1.7-kilobase-pair region of the virD locus caused double-stranded cleavage of the T-DNA at or near the borders and generated single-stranded T-DNA molecules with approximately equal frequencies in E. coli.

Full text

PDF
5045

Images in this article

5041Image
on p.5041
5042Image
on p.5042
5042Image
on p.5042
5043Image
on p.5043

Selected References

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

  1. Akiyoshi D. E., Morris R. O., Hinz R., Mischke B. S., Kosuge T., Garfinkel D. J., Gordon M. P., Nester E. W. Cytokinin/auxin balance in crown gall tumors is regulated by specific loci in the T-DNA. Proc Natl Acad Sci U S A. 1983 Jan;80(2):407–411. doi: 10.1073/pnas.80.2.407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Albright L. M., Yanofsky M. F., Leroux B., Ma D. Q., Nester E. W. Processing of the T-DNA of Agrobacterium tumefaciens generates border nicks and linear, single-stranded T-DNA. J Bacteriol. 1987 Mar;169(3):1046–1055. doi: 10.1128/jb.169.3.1046-1055.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alt-Moerbe J., Rak B., Schröder J. A 3.6-kbp segment from the vir region of Ti plasmids contains genes responsible for border-sequence-directed production of T region circles in E. coli. EMBO J. 1986 Jun;5(6):1129–1135. doi: 10.1002/j.1460-2075.1986.tb04337.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Amann E., Brosius J., Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene. 1983 Nov;25(2-3):167–178. doi: 10.1016/0378-1119(83)90222-6. [DOI] [PubMed] [Google Scholar]
  5. Baehr W., Morita E. A., Swanson R. J., Applebury M. L. Characterization of bovine rod outer segment G-protein. J Biol Chem. 1982 Jun 10;257(11):6452–6460. [PubMed] [Google Scholar]
  6. 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]
  7. Bischofberger N., Ng P. G., Webb T. R., Matteucci M. D. Cleavage of single stranded oligonucleotides by EcoRI restriction endonuclease. Nucleic Acids Res. 1987 Jan 26;15(2):709–716. doi: 10.1093/nar/15.2.709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Das A., Stachel S., Ebert P., Allenza P., Montoya A., Nester E. Promoters of Agrobacterium tumefaciens Ti-plasmid virulence genes. Nucleic Acids Res. 1986 Feb 11;14(3):1355–1364. doi: 10.1093/nar/14.3.1355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. DeVries J. K., Zubay G. DNA-directed peptide synthesis. II. The synthesis of the alpha-fragment of the enzyme beta-galactosidase. Proc Natl Acad Sci U S A. 1967 Apr;57(4):1010–1012. doi: 10.1073/pnas.57.4.1010. [DOI] [PMC free article] [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. 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. Gelvin S. B., Karcher S. J., DiRita V. J. Methylation of the T-DNA in Agrobacterium tumefaciens and in several crown gall tumors. Nucleic Acids Res. 1983 Jan 11;11(1):159–174. doi: 10.1093/nar/11.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hille J., van Kan J., Klasen I., Schilperoort R. Site-directed mutagenesis in Escherichia coli of a stable R772::Ti cointegrate plasmid from Agrobacterium tumefaciens. J Bacteriol. 1983 May;154(2):693–701. doi: 10.1128/jb.154.2.693-701.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hille J., van Kan J., Schilperoort R. trans-Acting virulence functions of the octopine Ti plasmid from Agrobacterium tumefaciens. J Bacteriol. 1984 May;158(2):754–756. doi: 10.1128/jb.158.2.754-756.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Hooykaas P. J., Hofker M., den Dulk-Ras H., Schilperoort R. A. A comparison of virulence determinants in an octopine Ti plasmid, a nopaline Ti plasmid, and an Ri plasmid by complementation analysis of Agrobacterium tumefaciens mutants. Plasmid. 1984 May;11(3):195–205. doi: 10.1016/0147-619x(84)90026-x. [DOI] [PubMed] [Google Scholar]
  18. Hooykaas P. J., Schilperoort R. A. The molecular genetics of crown gall tumorigenesis. Adv Genet. 1984;22:209–283. doi: 10.1016/s0065-2660(08)60041-3. [DOI] [PubMed] [Google Scholar]
  19. Klee H. J., White F. F., Iyer V. N., Gordon M. P., Nester E. W. Mutational analysis of the virulence region of an Agrobacterium tumefaciens Ti plasmid. J Bacteriol. 1983 Feb;153(2):878–883. doi: 10.1128/jb.153.2.878-883.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983 Mar;47(1):1–45. doi: 10.1128/mr.47.1.1-45.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  24. Melchers L. S., Thompson D. V., Idler K. B., Schilperoort R. A., Hooykaas P. J. Nucleotide sequence of the virulence gene virG of the Agrobacterium tumefaciens octopine Ti plasmid: significant homology between virG and the regulatory genes ompR, phoB and dye of E. coli. Nucleic Acids Res. 1986 Dec 22;14(24):9933–9942. doi: 10.1093/nar/14.24.9933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Montoya A. L., Chilton M. D., Gordon M. P., Sciaky D., Nester E. W. Octopine and nopaline metabolism in Agrobacterium tumefaciens and crown gall tumor cells: role of plasmid genes. J Bacteriol. 1977 Jan;129(1):101–107. doi: 10.1128/jb.129.1.101-107.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Neel J. V., Satoh C., Goriki K., Fujita M., Takahashi N., Asakawa J., Hazama R. The rate with which spontaneous mutation alters the electrophoretic mobility of polypeptides. Proc Natl Acad Sci U S A. 1986 Jan;83(2):389–393. doi: 10.1073/pnas.83.2.389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nishigaki K., Kaneko Y., Wakuda H., Husimi Y., Tanaka T. Type II restriction endonucleases cleave single-stranded DNAs in general. Nucleic Acids Res. 1985 Aug 26;13(16):5747–5760. doi: 10.1093/nar/13.16.5747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Peralta E. G., Ream L. W. T-DNA border sequences required for crown gall tumorigenesis. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5112–5116. doi: 10.1073/pnas.82.15.5112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schröder G., Klipp W., Hillebrand A., Ehring R., Koncz C., Schröder J. The conserved part of the T-region in Ti-plasmids expresses four proteins in bacteria. EMBO J. 1983;2(3):403–409. doi: 10.1002/j.1460-2075.1983.tb01437.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Slightom J. L., Blechl A. E., Smithies O. Human fetal G gamma- and A gamma-globin genes: complete nucleotide sequences suggest that DNA can be exchanged between these duplicated genes. Cell. 1980 Oct;21(3):627–638. doi: 10.1016/0092-8674(80)90426-2. [DOI] [PubMed] [Google Scholar]
  32. Slightom J. L., Jouanin L., Leach F., Drong R. F., Tepfer D. Isolation and identification of TL-DNA/plant junctions in Convolvulus arvensis transformed by Agrobacterium rhizogenes strain A4. EMBO J. 1985 Dec 1;4(12):3069–3077. doi: 10.1002/j.1460-2075.1985.tb04047.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Stachel S. E., Timmerman B., Zambryski P. Activation of Agrobacterium tumefaciens vir gene expression generates multiple single-stranded T-strand molecules from the pTiA6 T-region: requirement for 5' virD gene products. EMBO J. 1987 Apr;6(4):857–863. doi: 10.1002/j.1460-2075.1987.tb04831.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Stachel S. E., Zambryski P. C. virA and virG control the plant-induced activation of the T-DNA transfer process of A. tumefaciens. Cell. 1986 Aug 1;46(3):325–333. doi: 10.1016/0092-8674(86)90653-7. [DOI] [PubMed] [Google Scholar]
  38. Veluthambi K., Jayaswal R. K., Gelvin S. B. Virulence genes A, G, and D mediate the double-stranded border cleavage of T-DNA from the Agrobacterium Ti plasmid. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1881–1885. doi: 10.1073/pnas.84.7.1881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  40. Wang K., Herrera-Estrella L., Van Montagu M., Zambryski P. Right 25 bp terminus sequence of the nopaline T-DNA is essential for and determines direction of DNA transfer from agrobacterium to the plant genome. Cell. 1984 Sep;38(2):455–462. doi: 10.1016/0092-8674(84)90500-2. [DOI] [PubMed] [Google Scholar]
  41. Wang K., Stachel S. E., Timmerman B., VAN Montagu M., Zambryski P. C. Site-Specific Nick in the T-DNA Border Sequence as a Result of Agrobacterium vir Gene Expression. Science. 1987 Jan 30;235(4788):587–591. doi: 10.1126/science.235.4788.587. [DOI] [PubMed] [Google Scholar]
  42. Wilbur W. J., Lipman D. J. Rapid similarity searches of nucleic acid and protein data banks. Proc Natl Acad Sci U S A. 1983 Feb;80(3):726–730. doi: 10.1073/pnas.80.3.726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Winans S. C., Allenza P., Stachel S. E., McBride K. E., Nester E. W. Characterization of the virE operon of the Agrobacterium Ti plasmid pTiA6. Nucleic Acids Res. 1987 Jan 26;15(2):825–837. doi: 10.1093/nar/15.2.825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Winans S. C., Ebert P. R., Stachel S. E., Gordon M. P., Nester E. W. A gene essential for Agrobacterium virulence is homologous to a family of positive regulatory loci. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8278–8282. doi: 10.1073/pnas.83.21.8278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yadav N. S., Vanderleyden J., Bennett D. R., Barnes W. M., Chilton M. D. Short direct repeats flank the T-DNA on a nopaline Ti plasmid. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6322–6326. doi: 10.1073/pnas.79.20.6322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yamamoto A., Iwahashi M., Yanofsky M. F., Nester E. W., Takebe I., Machida Y. The promoter proximal region in the virD locus of Agrobacterium tumefaciens is necessary for the plant-inducible circularization of T-DNA. Mol Gen Genet. 1987 Jan;206(1):174–177. doi: 10.1007/BF00326554. [DOI] [PubMed] [Google Scholar]
  47. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  48. Yanofsky M. F., Nester E. W. Molecular characterization of a host-range-determining locus from Agrobacterium tumefaciens. J Bacteriol. 1986 Oct;168(1):244–250. doi: 10.1128/jb.168.1.244-250.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yanofsky M. F., Porter S. G., Young C., Albright L. M., Gordon M. P., Nester E. W. The virD operon of Agrobacterium tumefaciens encodes a site-specific endonuclease. Cell. 1986 Nov 7;47(3):471–477. doi: 10.1016/0092-8674(86)90604-5. [DOI] [PubMed] [Google Scholar]

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

RESOURCES