Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Mar;178(6):1491–1497. doi: 10.1128/jb.178.6.1491-1497.1996

Nucleotide sequence and characterization of the trbABC region of the IncI1 Plasmid R64: existence of the pnd gene for plasmid maintenance within the transfer region.

N Furuya 1, T Komano 1
PMCID: PMC177830  PMID: 8626273

Abstract

A 6.72-kb DNA sequence between the exc gene and the oriT operon within the transfer region of IncI1 plasmid R64 was sequenced and characterized. Three novel transfer genes, trbA, trbB, and trbC, were found in this region, along with the pnd gene responsible for plasmid maintenance. The trbABC genes appear to be organized into an operon located adjacent to the oriT operon in the opposite orientation. The trbA and trbC genes were shown to be indispensable for R64 plasmid transfer, while residual transfer activity was detected in the case of R64 derivatives carrying the trbB++ deletion mutation. The T7 RNA polymerase-promoter system revealed that the trbB gene produced a 43-kDa protein and the trbC gene produced an 85-kDa protein. The nucleotide sequence of the pnd gene is nearly identical to that of plasmid R483, indicating a function in plasmid maintenance. The plasmid stability test indicated that the mini-R64 derivatives with the pnd gene are more stably maintained in Escherichia coli cells under nonselective conditions than the mini-R64 derivatives without the pnd gene. It was also shown that the R64 transfer system itself is involved in plasmid stability to a certain degree. Deletion of the pnd gene from the tra+ mini-R64 derivative did not affect transfer frequency. DNA segments between the exc and trbA genes for IncI1 plasmids R64, Colb-P9, and R144 were compared in terms of their physical and genetic organization.

Full Text

The Full Text of this article is available as a PDF (371.4 KB).

Selected References

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

  1. 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]
  2. Furuya N., Komano T. Surface exclusion gene of IncI1 plasmid R64: nucleotide sequence and analysis of deletion mutants. Plasmid. 1994 Jul;32(1):80–84. doi: 10.1006/plas.1994.1047. [DOI] [PubMed] [Google Scholar]
  3. Furuya N., Nisioka T., Komano T. Nucleotide sequence and functions of the oriT operon in IncI1 plasmid R64. J Bacteriol. 1991 Apr;173(7):2231–2237. doi: 10.1128/jb.173.7.2231-2237.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gerdes K., Poulsen L. K., Thisted T., Nielsen A. K., Martinussen J., Andreasen P. H. The hok killer gene family in gram-negative bacteria. New Biol. 1990 Nov;2(11):946–956. [PubMed] [Google Scholar]
  5. Ham L. M., Skurray R. Molecular analysis and nucleotide sequence of finQ, a transcriptional inhibitor of the F plasmid transfer genes. Mol Gen Genet. 1989 Mar;216(1):99–105. doi: 10.1007/BF00332236. [DOI] [PubMed] [Google Scholar]
  6. Hama C., Takizawa T., Moriwaki H., Urasaki Y., Mizobuchi K. Organization of the replication control region of plasmid ColIb-P9. J Bacteriol. 1990 Apr;172(4):1983–1991. doi: 10.1128/jb.172.4.1983-1991.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hartskeerl R. A., vd Guchte M., Zuidweg E. M., Hoekstra W. P. Physical and genetic characterization of the IncI plasmid R144-drd3. Plasmid. 1984 Nov;12(3):215–217. doi: 10.1016/0147-619x(84)90048-9. [DOI] [PubMed] [Google Scholar]
  8. Hartskeerl R., Overduin P., Hoekstra W., Tommassen J. Nucleotide sequence of the exclusion-determining locus of IncI plasmid R144. Gene. 1986;42(1):107–111. doi: 10.1016/0378-1119(86)90156-3. [DOI] [PubMed] [Google Scholar]
  9. Hiraga S. Chromosome and plasmid partition in Escherichia coli. Annu Rev Biochem. 1992;61:283–306. doi: 10.1146/annurev.bi.61.070192.001435. [DOI] [PubMed] [Google Scholar]
  10. Kim S. R., Funayama N., Komano T. Nucleotide sequence and characterization of the traABCD region of IncI1 plasmid R64. J Bacteriol. 1993 Aug;175(16):5035–5042. doi: 10.1128/jb.175.16.5035-5042.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kim S. R., Komano T. Cloning and nucleotide sequence of the ColIb shufflon. Plasmid. 1989 Sep;22(2):180–184. doi: 10.1016/0147-619x(89)90029-2. [DOI] [PubMed] [Google Scholar]
  12. Komano T., Funayama N., Kim S. R., Nisioka T. Transfer region of IncI1 plasmid R64 and role of shufflon in R64 transfer. J Bacteriol. 1990 May;172(5):2230–2235. doi: 10.1128/jb.172.5.2230-2235.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Komano T., Kim S. R., Yoshida T. Mating variation by DNA inversions of shufflon in plasmid R64. Adv Biophys. 1995;31:181–193. doi: 10.1016/0065-227x(95)99391-2. [DOI] [PubMed] [Google Scholar]
  14. Komano T., Kim S. R., Yoshida T., Nisioka T. DNA rearrangement of the shufflon determines recipient specificity in liquid mating of IncI1 plasmid R64. J Mol Biol. 1994 Oct 14;243(1):6–9. doi: 10.1006/jmbi.1994.1625. [DOI] [PubMed] [Google Scholar]
  15. Komano T., Kubo A., Nisioka T. Shufflon: multi-inversion of four contiguous DNA segments of plasmid R64 creates seven different open reading frames. Nucleic Acids Res. 1987 Feb 11;15(3):1165–1172. doi: 10.1093/nar/15.3.1165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kubo A., Kusukawa A., Komano T. Nucleotide sequence of the rci gene encoding shufflon-specific DNA recombinase in the IncI1 plasmid R64: homology to the site-specific recombinases of integrase family. Mol Gen Genet. 1988 Jul;213(1):30–35. doi: 10.1007/BF00333394. [DOI] [PubMed] [Google Scholar]
  17. Lessl M., Lanka E. Common mechanisms in bacterial conjugation and Ti-mediated T-DNA transfer to plant cells. Cell. 1994 May 6;77(3):321–324. doi: 10.1016/0092-8674(94)90146-5. [DOI] [PubMed] [Google Scholar]
  18. Nielsen A. K., Thorsted P., Thisted T., Wagner E. G., Gerdes K. The rifampicin-inducible genes srnB from F and pnd from R483 are regulated by antisense RNAs and mediate plasmid maintenance by killing of plasmid-free segregants. Mol Microbiol. 1991 Aug;5(8):1961–1973. doi: 10.1111/j.1365-2958.1991.tb00818.x. [DOI] [PubMed] [Google Scholar]
  19. Ohnishi Y., Akimoto S. I-like R plasmids promote degradation of stable ribonucleic acid in Escherichia coli. J Bacteriol. 1980 Nov;144(2):833–835. doi: 10.1128/jb.144.2.833-835.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Oishi M., Cosloy S. D. The genetic and biochemical basis of the transformability of Escherichia coli K12. Biochem Biophys Res Commun. 1972 Dec 18;49(6):1568–1572. doi: 10.1016/0006-291x(72)90520-7. [DOI] [PubMed] [Google Scholar]
  21. Ono K., Akimoto S., Ohnishi Y. Nucleotide sequence of the pnd gene in plasmid R483 and role of the pnd gene product in plasmolysis. Microbiol Immunol. 1987;31(11):1071–1083. doi: 10.1111/j.1348-0421.1987.tb01339.x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  26. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  27. 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]

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

RESOURCES