• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Mar 1989; 86(6): 1771–1775.
PMCID: PMC286786

Conjugative transfer of promiscuous IncP plasmids: interaction of plasmid-encoded products with the transfer origin.

Abstract

To characterize protein-DNA interactions involved in the initiation of conjugative transfer replication, we isolated and sequenced the transfer origins (oriT) of the promiscuous IncP plasmids RP4 and R751. The central initiating event at the transfer origin of a conjugative plasmid is the cleavage at a unique site (nic) of the strand to be transferred to a recipient cell. This process can be triggered after the assembly of "relaxosomes" (plasmid DNA-protein relaxation complexes), requiring plasmid-encoded gene products. We analyzed the nicking reaction for plasmid RP4 and demonstrated that one of the plasmid strands is specifically cleaved within oriT. The fully functional oriT of RP4 represents an intergenic DNA region of approximately 350 base pairs. Dissection of oriT revealed that a portion carrying nic and symmetric sequence repeats determines oriT specificity. This part of oriT is contiguous to a region that is essential for efficient mobilization of oriT plasmids. In addition, oriT contains potential promoter sites allowing divergent transcription of two operons flanking oriT. We over-produced gene products and, from analyzing the products of defined deletion mutants, deduced the gene arrangements. Formation of RP4 relaxosomes is likely to depend on the presence of at least two plasmid-encoded components, which act in trans. Corresponding genes map on one side of oriT. Purification of the traJ product revealed it to be an 11-kDa polypeptide that binds to oriT DNA in vitro. The protein recognizes the part of oriT that is responsible for oriT specificity.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.2M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Willetts N, Wilkins B. Processing of plasmid DNA during bacterial conjugation. Microbiol Rev. 1984 Mar;48(1):24–41. [PMC free article] [PubMed]
  • Clewell DB, Helinski DR. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. [PMC free article] [PubMed]
  • Guiney DG, Helinski DR. The DNA-protein relaxation complex of the plasmid RK2: location of the site-specific nick in the region of the proposed origin of transfer. Mol Gen Genet. 1979 Oct 3;176(2):183–189. [PubMed]
  • Yakobson E, Guiney G. Homology in the transfer origins of broad host range IncP plasmids: definition of two subgroups of P plasmids. Mol Gen Genet. 1983;192(3):436–438. [PubMed]
  • Boyer HW, 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. [PubMed]
  • 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. [PubMed]
  • Datta N, Hedges RW, Shaw EJ, Sykes RB, Richmond MH. Properties of an R factor from Pseudomonas aeruginosa. J Bacteriol. 1971 Dec;108(3):1244–1249. [PMC free article] [PubMed]
  • Jobanputra RS, Datta N. Trimethoprim R factors in enterobacteria from clinical specimens. J Med Microbiol. 1974 May;7(2):169–177. [PubMed]
  • Covarrubias L, Bolivar F. Construction and characterization of new cloning vehicles. VI. Plasmid pBR329, a new derivative of pBR328 lacking the 482-base-pair inverted duplication. Gene. 1982 Jan;17(1):79–89. [PubMed]
  • Fürste JP, Pansegrau W, Frank R, Blöcker H, Scholz P, Bagdasarian M, Lanka E. Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene. 1986;48(1):119–131. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Biggin MD, Gibson TJ, Hong GF. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. [PMC free article] [PubMed]
  • Mizusawa S, Nishimura S, Seela F. Improvement of the dideoxy chain termination method of DNA sequencing by use of deoxy-7-deazaguanosine triphosphate in place of dGTP. Nucleic Acids Res. 1986 Feb 11;14(3):1319–1324. [PMC free article] [PubMed]
  • Pansegrau W, Lanka E. Conservation of a common 'backbone' in the genetic organization of the IncP plasmids RP4 and R751. Nucleic Acids Res. 1987 Mar 11;15(5):2385–2385. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Harley CB, Reynolds RP. Analysis of E. coli promoter sequences. Nucleic Acids Res. 1987 Mar 11;15(5):2343–2361. [PMC free article] [PubMed]
  • Guiney DG, Yakobson E. Location and nucleotide sequence of the transfer origin of the broad host range plasmid RK2. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3595–3598. [PMC free article] [PubMed]
  • McDonell MW, Simon MN, Studier FW. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. [PubMed]
  • Lanka E, Lurz R, Fürste JP. Molecular cloning and mapping of SphI restriction fragments of plasmid RP4. Plasmid. 1983 Nov;10(3):303–307. [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Gene
    Gene
    Gene links
  • MedGen
    MedGen
    Related information in MedGen
  • Nucleotide
    Nucleotide
    Published Nucleotide sequences
  • Protein
    Protein
    Published protein sequences
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...