Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Sep;173(17):5449–5456. doi: 10.1128/jb.173.17.5449-5456.1991

The osa gene of pSa encodes a 21.1-kilodalton protein that suppresses Agrobacterium tumefaciens oncogenicity.

S M Close 1, C I Kado 1
PMCID: PMC208257  PMID: 1832152

Abstract

The incompatibility group W plasmid pSa suppresses Agrobacterium tumefaciens oncogenicity (J. Loper and C. Kado, J. Bacteriol. 139:591-596, 1979). The oncogenic suppressive activity was localized to a 3.1-kb region of pSa by Tn5 mutagenesis and deletion analysis. Within this fragment, a 1.1-kb subclone bearing oncogenic suppressive activity was subjected to further characterization. Nucleotide sequencing of the 1.1-kb fragment revealed a 570-bp open reading frame (ORF1) that has a coding capacity for a protein of 21.1 kDa. Sequencing of flanking regions revealed a second ORF (ORF2) located 3 bp upstream of ORF1, with a coding capacity for a protein of 22.8 kDa. Gene fusions of these ORFs to a T7 phi 10 expression system in Escherichia coli resulted in the synthesis of polypeptides of the predicted sizes. An E. coli promoter consensus sequence was not found in the expected positions in the region preceding ORF1. However, several sequences with similarity to the consensus -10 sequence of the A. tumefaciens vir gene promoters were found upstream of ORF1. Potential translational start signals are upstream of ORF1 and ORF2. These sequences showed no significant similarity at the nucleotide or amino acid levels with those in available data bases. However, the C-terminal portion of the ORF1 protein is rich in hydrophobic residues. Perhaps oncogenicity suppression is effected by an association of this protein with the Agrobacterium membrane such that T-DNA transfer is blocked.

Full text

PDF

Images in this article

Selected References

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

  1. Adhya S., Gottesman M. Promoter occlusion: transcription through a promoter may inhibit its activity. Cell. 1982 Jul;29(3):939–944. doi: 10.1016/0092-8674(82)90456-1. [DOI] [PubMed] [Google Scholar]
  2. Berg D. E., Weiss A., Crossland L. Polarity of Tn5 insertion mutations in Escherichia coli. J Bacteriol. 1980 May;142(2):439–446. doi: 10.1128/jb.142.2.439-446.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  6. Close T. J., Rodriguez R. L. Construction and characterization of the chloramphenicol-resistance gene cartridge: a new approach to the transcriptional mapping of extrachromosomal elements. Gene. 1982 Dec;20(2):305–316. doi: 10.1016/0378-1119(82)90048-8. [DOI] [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. Cooley M. B., D'Souza M. R., Kado C. I. The virC and virD operons of the Agrobacterium Ti plasmid are regulated by the ros chromosomal gene: analysis of the cloned ros gene. J Bacteriol. 1991 Apr;173(8):2608–2616. doi: 10.1128/jb.173.8.2608-2616.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  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. Gallie D. R., Kado C. I. Agrobacterium tumefaciens pTAR parA promoter region involved in autoregulation, incompatibility and plasmid partitioning. J Mol Biol. 1987 Feb 5;193(3):465–478. doi: 10.1016/0022-2836(87)90260-9. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Gerlitz M., Hrabak O., Schwab H. Partitioning of broad-host-range plasmid RP4 is a complex system involving site-specific recombination. J Bacteriol. 1990 Nov;172(11):6194–6203. doi: 10.1128/jb.172.11.6194-6203.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  15. Hausler B., Somerville R. L. Interaction in vivo between strong closely spaced constitutive promoters. J Mol Biol. 1979 Jan 25;127(3):353–356. doi: 10.1016/0022-2836(79)90335-8. [DOI] [PubMed] [Google Scholar]
  16. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Kado C. I., Liu S. T. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol. 1981 Mar;145(3):1365–1373. doi: 10.1128/jb.145.3.1365-1373.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Klapwijk P. M., van Beelen P., Schilperoort R. A. Isolation of a recombination deficient Agrobacterium tumefaciens mutant. Mol Gen Genet. 1979 Jun 7;173(2):171–175. doi: 10.1007/BF00330307. [DOI] [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. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Lin B. C., Kado C. I. Studies on Agrobacterium tumefaciens. VIII. Avirulence induced by temperature and ethidium bromide. Can J Microbiol. 1977 Nov;23(11):1554–1561. doi: 10.1139/m77-229. [DOI] [PubMed] [Google Scholar]
  23. Liu S. T., Perry K. L., Schardl C. L., Kado C. I. Agrobacterium Ti plasmid indoleacetic acid gene is required for crown gall oncogenesis. Proc Natl Acad Sci U S A. 1982 May;79(9):2812–2816. doi: 10.1073/pnas.79.9.2812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Loper J. E., Kado C. I. Host range conferred by the virulence-specifying plasmid of Agrobacterium tumefaciens. J Bacteriol. 1979 Aug;139(2):591–596. doi: 10.1128/jb.139.2.591-596.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Queen C., Korn L. J. A comprehensive sequence analysis program for the IBM personal computer. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):581–599. doi: 10.1093/nar/12.1part2.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  27. Ruvkun G. B., Ausubel F. M. A general method for site-directed mutagenesis in prokaryotes. Nature. 1981 Jan 1;289(5793):85–88. doi: 10.1038/289085a0. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Shirasu K., Morel P., Kado C. I. Characterization of the virB operon of an Agrobacterium tumefaciens Ti plasmid: nucleotide sequence and protein analysis. Mol Microbiol. 1990 Jul;4(7):1153–1163. doi: 10.1111/j.1365-2958.1990.tb00690.x. [DOI] [PubMed] [Google Scholar]
  31. Tait R. C., Lundquist R. C., Kado C. I. Genetic map of the crown gall suppressive IncW plasmid pSa. Mol Gen Genet. 1982;186(1):10–15. doi: 10.1007/BF00422905. [DOI] [PubMed] [Google Scholar]
  32. Tanimoto K., Iino T. Transfer inhibition of RP4 by F factor. Mol Gen Genet. 1983;192(1-2):104–109. doi: 10.1007/BF00327654. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Ward D. F., Murray N. E. Convergent transcription in bacteriophage lambda: interference with gene expression. J Mol Biol. 1979 Sep 15;133(2):249–266. doi: 10.1016/0022-2836(79)90533-3. [DOI] [PubMed] [Google Scholar]
  35. Watanabe T., Furuse C., Sakaizumi S. Transduction of various R factors by phage P1 in Escherichia coli and by phage P22 in Salmonella typhimurium. J Bacteriol. 1968 Nov;96(5):1791–1795. doi: 10.1128/jb.96.5.1791-1795.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Willetts N., Skurray R. The conjugation system of F-like plasmids. Annu Rev Genet. 1980;14:41–76. doi: 10.1146/annurev.ge.14.120180.000353. [DOI] [PubMed] [Google Scholar]
  37. Winans S. C., Walker G. C. Fertility inhibition of RP1 by IncN plasmid pKM101. J Bacteriol. 1985 Jan;161(1):425–427. doi: 10.1128/jb.161.1.425-427.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Yusoff K., Stanisich V. A. Location of a function on RP1 that fertility inhibits Inc W plasmids. Plasmid. 1984 Mar;11(2):178–181. doi: 10.1016/0147-619x(84)90022-2. [DOI] [PubMed] [Google Scholar]

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

RESOURCES