Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Apr;173(8):2608–2616. doi: 10.1128/jb.173.8.2608-2616.1991

The virC and virD operons of the Agrobacterium Ti plasmid are regulated by the ros chromosomal gene: analysis of the cloned ros gene.

M B Cooley 1, M R D'Souza 1, C I Kado 1
PMCID: PMC207827  PMID: 2013576

Abstract

The ros chromosomal gene is present in octopine and nopaline strains of Agrobacterium tumefaciens as well as in Rhizobium meliloti. This gene encodes a 15.5-kDa protein that specifically represses the virC and virD operons in the virulence region of the Ti plasmid. The ros gene was cloned from a genomic bank by electroporation and complementation in Agrobacterium cells. Reporter fusion to the ros gene indicates that the level of transcription is controlled in part by autoregulation. A consensus inverted repeat sequence present in the ros promoter and in the virC and virD promoters of pTiC58, pTiA6, and pRiA4b suggests that a specific Ros binding site exists in these promoters. In the virC and virD promoter region, this binding site is within a cluster of vir box consensus sequences in which the VirG protein binds. This suggests possible binding competition between Ros and VirG at the virC and virD promoters. That the Ros protein binds DNA is suggested by the presence of a 'zinc finger' consensus sequence in the protein.

Full text

PDF
2608

Images in this article

Selected References

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

  1. Berg J. M. Proposed structure for the zinc-binding domains from transcription factor IIIA and related proteins. Proc Natl Acad Sci U S A. 1988 Jan;85(1):99–102. doi: 10.1073/pnas.85.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  5. Close T. J., Rogowsky P. M., Kado C. I., Winans S. C., Yanofsky M. F., Nester E. W. Dual control of Agrobacterium tumefaciens Ti plasmid virulence genes. J Bacteriol. 1987 Nov;169(11):5113–5118. doi: 10.1128/jb.169.11.5113-5118.1987. [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. Close T. J., Zaitlin D., Kado C. I. Design and development of amplifiable broad-host-range cloning vectors: analysis of the vir region of Agrobacterium tumefaciens plasmid pTiC58. Plasmid. 1984 Sep;12(2):111–118. doi: 10.1016/0147-619x(84)90057-x. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Doherty D., Leigh J. A., Glazebrook J., Walker G. C. Rhizobium meliloti mutants that overproduce the R. meliloti acidic calcofluor-binding exopolysaccharide. J Bacteriol. 1988 Sep;170(9):4249–4256. doi: 10.1128/jb.170.9.4249-4256.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dürrenberger F., Crameri A., Hohn B., Koukolíková-Nicola Z. Covalently bound VirD2 protein of Agrobacterium tumefaciens protects the T-DNA from exonucleolytic degradation. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9154–9158. doi: 10.1073/pnas.86.23.9154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Finan T. M., Hirsch A. M., Leigh J. A., Johansen E., Kuldau G. A., Deegan S., Walker G. C., Signer E. R. Symbiotic mutants of Rhizobium meliloti that uncouple plant from bacterial differentiation. Cell. 1985 Apr;40(4):869–877. doi: 10.1016/0092-8674(85)90346-0. [DOI] [PubMed] [Google Scholar]
  14. Finan T. M., Kunkel B., De Vos G. F., Signer E. R. Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J Bacteriol. 1986 Jul;167(1):66–72. doi: 10.1128/jb.167.1.66-72.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gallie D. R., Gay P., Kado C. I. Specialized vectors for members of Rhizobiaceae and other gram-negative bacteria. Biotechnology. 1988;10:333–342. doi: 10.1016/b978-0-409-90042-2.50022-2. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Ghai J., Das A. The virD operon of Agrobacterium tumefaciens Ti plasmid encodes a DNA-relaxing enzyme. Proc Natl Acad Sci U S A. 1989 May;86(9):3109–3113. doi: 10.1073/pnas.86.9.3109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Grimsley N., Hohn B., Ramos C., Kado C., Rogowsky P. DNA transfer from Agrobacterium to Zea mays or Brassica by agroinfection is dependent on bacterial virulence functions. Mol Gen Genet. 1989 Jun;217(2-3):309–316. doi: 10.1007/BF02464898. [DOI] [PubMed] [Google Scholar]
  19. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Huang Y., Morel P., Powell B., Kado C. I. VirA, a coregulator of Ti-specified virulence genes, is phosphorylated in vitro. J Bacteriol. 1990 Feb;172(2):1142–1144. doi: 10.1128/jb.172.2.1142-1144.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jin S. G., Roitsch T., Christie P. J., Nester E. W. The regulatory VirG protein specifically binds to a cis-acting regulatory sequence involved in transcriptional activation of Agrobacterium tumefaciens virulence genes. J Bacteriol. 1990 Feb;172(2):531–537. doi: 10.1128/jb.172.2.531-537.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jin S., Roitsch T., Ankenbauer R. G., Gordon M. P., Nester E. W. The VirA protein of Agrobacterium tumefaciens is autophosphorylated and is essential for vir gene regulation. J Bacteriol. 1990 Feb;172(2):525–530. doi: 10.1128/jb.172.2.525-530.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kamoun S., Cooley M. B., Rogowsky P. M., Kado C. I. Two chromosomal loci involved in production of exopolysaccharide in Agrobacterium tumefaciens. J Bacteriol. 1989 Mar;171(3):1755–1759. doi: 10.1128/jb.171.3.1755-1759.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Leigh J. A., Signer E. R., Walker G. C. Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6231–6235. doi: 10.1073/pnas.82.18.6231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Leroux B., Yanofsky M. F., Winans S. C., Ward J. E., Ziegler S. F., Nester E. W. Characterization of the virA locus of Agrobacterium tumefaciens: a transcriptional regulator and host range determinant. EMBO J. 1987 Apr;6(4):849–856. doi: 10.1002/j.1460-2075.1987.tb04830.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mead D. A., Skorupa E. S., Kemper B. Single stranded DNA SP6 promoter plasmids for engineering mutant RNAs and proteins: synthesis of a 'stretched' preproparathyroid hormone. Nucleic Acids Res. 1985 Feb 25;13(4):1103–1118. doi: 10.1093/nar/13.4.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Melchers L. S., Regensburg-Tuïnk A. J., Schilperoort R. A., Hooykaas P. J. Specificity of signal molecules in the activation of Agrobacterium virulence gene expression. Mol Microbiol. 1989 Jul;3(7):969–977. doi: 10.1111/j.1365-2958.1989.tb00246.x. [DOI] [PubMed] [Google Scholar]
  30. Miller J., McLachlan A. D., Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985 Jun;4(6):1609–1614. doi: 10.1002/j.1460-2075.1985.tb03825.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nagai K., Nakaseko Y., Nasmyth K., Rhodes D. Zinc-finger motifs expressed in E. coli and folded in vitro direct specific binding to DNA. Nature. 1988 Mar 17;332(6161):284–286. doi: 10.1038/332284a0. [DOI] [PubMed] [Google Scholar]
  32. Pazour G. J., Das A. virG, an Agrobacterium tumefaciens transcriptional activator, initiates translation at a UUG codon and is a sequence-specific DNA-binding protein. J Bacteriol. 1990 Mar;172(3):1241–1249. doi: 10.1128/jb.172.3.1241-1249.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Powell B. S., Kado C. I. Specific binding of VirG to the vir box requires a C-terminal domain and exhibits a minimum concentration threshold. Mol Microbiol. 1990 Dec;4(12):2159–2166. doi: 10.1111/j.1365-2958.1990.tb00577.x. [DOI] [PubMed] [Google Scholar]
  34. Powell B. S., Rogowsky P. M., Kado C. I. virG of Agrobacterium tumefaciens plasmid pTiC58 encodes a DNA-binding protein. Mol Microbiol. 1989 Mar;3(3):411–419. doi: 10.1111/j.1365-2958.1989.tb00186.x. [DOI] [PubMed] [Google Scholar]
  35. Rhodes D., Klug A. An underlying repeat in some transcriptional control sequences corresponding to half a double helical turn of DNA. Cell. 1986 Jul 4;46(1):123–132. doi: 10.1016/0092-8674(86)90866-4. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Schmidhauser T. J., Ditta G., Helinski D. R. Broad-host-range plasmid cloning vectors for gram-negative bacteria. Biotechnology. 1988;10:287–332. doi: 10.1016/b978-0-409-90042-2.50021-0. [DOI] [PubMed] [Google Scholar]
  41. Shaw W. V. Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol. 1975;43:737–755. doi: 10.1016/0076-6879(75)43141-x. [DOI] [PubMed] [Google Scholar]
  42. Shimoda N., Toyoda-Yamamoto A., Nagamine J., Usami S., Katayama M., Sakagami Y., Machida Y. Control of expression of Agrobacterium vir genes by synergistic actions of phenolic signal molecules and monosaccharides. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6684–6688. doi: 10.1073/pnas.87.17.6684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Spratt B. G., Hedge P. J., te Heesen S., Edelman A., Broome-Smith J. K. Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9. Gene. 1986;41(2-3):337–342. doi: 10.1016/0378-1119(86)90117-4. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. Steck T. R., Close T. J., Kado C. I. High levels of double-stranded transferred DNA (T-DNA) processing from an intact nopaline Ti plasmid. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2133–2137. doi: 10.1073/pnas.86.7.2133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Steck T. R., Lin T. S., Kado C. I. VirD2 gene product from the nopaline plasmid pTiC58 has at least two activities required for virulence. Nucleic Acids Res. 1990 Dec 11;18(23):6953–6958. doi: 10.1093/nar/18.23.6953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tait R. C., Kado C. I. Regulation of the virC and virD promoters of pTiC58 by the ros chromosomal mutation of Agrobacterium tumefaciens. Mol Microbiol. 1988 May;2(3):385–392. doi: 10.1111/j.1365-2958.1988.tb00043.x. [DOI] [PubMed] [Google Scholar]
  49. Toro N., Datta A., Carmi O. A., Young C., Prusti R. K., Nester E. W. The Agrobacterium tumefaciens virC1 gene product binds to overdrive, a T-DNA transfer enhancer. J Bacteriol. 1989 Dec;171(12):6845–6849. doi: 10.1128/jb.171.12.6845-6849.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. 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]
  52. Young C., Nester E. W. Association of the virD2 protein with the 5' end of T strands in Agrobacterium tumefaciens. J Bacteriol. 1988 Aug;170(8):3367–3374. doi: 10.1128/jb.170.8.3367-3374.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES