• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Aug 1995; 177(16): 4779–4791.
PMCID: PMC177245

Bacterial conjugation mediated by plasmid RP4: RSF1010 mobilization, donor-specific phage propagation, and pilus production require the same Tra2 core components of a proposed DNA transport complex.


DNA transfer by bacterial conjugation requires a mating pair formation (Mpf) system that specifies functions for establishing the physical contact between the donor and the recipient cell and for DNA transport across membranes. Plasmid RP4 (IncP alpha) contains two transfer regions designated Tra1 and Tra2, both of which contribute to Mpf. Twelve components are essential for Mpf, TraF of Tra1 and 11 Tra2 proteins, TrbB, -C, -D, -E, -F, -G, -H, -I, -J, -K, and -L. The phenotype of defined mutants in each of the Tra2 genes was determined. Each of the genes, except trbK, was found to be essential for RP4-specific plasmid transfer and for mobilization of the IncQ plasmid RSF1010. The latter process did not absolutely require trbF, but a severe reduction of the mobilization frequency occurred in its absence. Transfer proficiency of the mutants was restored by complementation with defined Tra2 segments containing single trb genes. Donor-specific phage propagation showed that traF and each of the genes encoded by Tra2 are involved. Phage PRD1, however, still adsorbed to the trbK mutant strain but not to any of the other mutant strains, suggesting the existence of a plasmid-encoded receptor complex. Strains containing the Tra2 plasmid in concert with traF were found to overexpress trb products as well as extracellular filaments visualized by electron microscopy. Each trb gene and traF are needed for the formation of the pilus-like structures. The trbK gene, which is required for PRD1 propagation and for pilus production but not for DNA transfer on solid media, encodes the RP4 entry-exclusion function. The components of the RP4 Mpf system are discussed in the context of related macromolecule export systems.

Full Text

The Full Text of this article is available as a PDF (1.1M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Achtman M, Kennedy N, Skurray R. Cell--cell interactions in conjugating Escherichia coli: role of traT protein in surface exclusion. Proc Natl Acad Sci U S A. 1977 Nov;74(11):5104–5108. [PMC free article] [PubMed]
  • Achtman M, Manning PA, Kusecek B, Schwuchow S, Willetts N. A genetic analysis of F sex factor cistrons needed for surface exclusion in Escherichia coli. J Mol Biol. 1980 Apr 25;138(4):779–795. [PubMed]
  • Allen SP, Polazzi JO, Gierse JK, Easton AM. Two novel heat shock genes encoding proteins produced in response to heterologous protein expression in Escherichia coli. J Bacteriol. 1992 Nov;174(21):6938–6947. [PMC free article] [PubMed]
  • Bachmann BJ. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev. 1972 Dec;36(4):525–557. [PMC free article] [PubMed]
  • Balzer D, Pansegrau W, Lanka E. Essential motifs of relaxase (TraI) and TraG proteins involved in conjugative transfer of plasmid RP4. J Bacteriol. 1994 Jul;176(14):4285–4295. [PMC free article] [PubMed]
  • Balzer D, Ziegelin G, Pansegrau W, Kruft V, Lanka E. KorB protein of promiscuous plasmid RP4 recognizes inverted sequence repetitions in regions essential for conjugative plasmid transfer. Nucleic Acids Res. 1992 Apr 25;20(8):1851–1858. [PMC free article] [PubMed]
  • Bamford DH, Rouhiainen L, Takkinen K, Söderlund H. Comparison of the lipid-containing bacteriophages PRD1, PR3, PR4, PR5 and L17. J Gen Virol. 1981 Dec;57(Pt 2):365–373. [PubMed]
  • Barth PT, Grinter NJ, Bradley DE. Conjugal transfer system of plasmid RP4: analysis by transposon 7 insertion. J Bacteriol. 1978 Jan;133(1):43–52. [PMC free article] [PubMed]
  • Beijersbergen A, Smith SJ, Hooykaas PJ. Localization and topology of VirB proteins of Agrobacterium tumefaciens. Plasmid. 1994 Sep;32(2):212–218. [PubMed]
  • Bolland S, Llosa M, Avila P, de la Cruz F. General organization of the conjugal transfer genes of the IncW plasmid R388 and interactions between R388 and IncN and IncP plasmids. J Bacteriol. 1990 Oct;172(10):5795–5802. [PMC free article] [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]
  • Bradley DE. Adsorption of bacteriophages specific for Pseudomonas aeruginosa R factors RP1 and R1822. Biochem Biophys Res Commun. 1974 Apr 8;57(3):893–900. [PubMed]
  • Bradley DE. Determination of pili by conjugative bacterial drug resistance plasmids of incompatibility groups B, C, H, J, K, M, V, and X. J Bacteriol. 1980 Feb;141(2):828–837. [PMC free article] [PubMed]
  • Bradley DE. Morphological and serological relationships of conjugative pili. Plasmid. 1980 Sep;4(2):155–169. [PubMed]
  • Bradley DE. Specification of the conjugative pili and surface mating systems of Pseudomonas plasmids. J Gen Microbiol. 1983 Aug;129(8):2545–2556. [PubMed]
  • Cook DM, Farrand SK. The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders. J Bacteriol. 1992 Oct;174(19):6238–6246. [PMC free article] [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]
  • Cowan P, Krishnapillai V. Tn7 insertion mutations affecting the host range of the promiscuous IncP-1 plasmid R18. Plasmid. 1982 Sep;8(2):164–174. [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]
  • Derbyshire KM, Hatfull G, Willetts N. Mobilization of the non-conjugative plasmid RSF1010: a genetic and DNA sequence analysis of the mobilization region. Mol Gen Genet. 1987 Jan;206(1):161–168. [PubMed]
  • Eberl L, Kristensen CS, Givskov M, Grohmann E, Gerlitz M, Schwab H. Analysis of the multimer resolution system encoded by the parCBA operon of broad-host-range plasmid RP4. Mol Microbiol. 1994 Apr;12(1):131–141. [PubMed]
  • Figurski DH, Young C, Schreiner HC, Pohlman RF, Bechhofer DH, Prince AS, D'Amico TF. Genetic interactions of broad host-range plasmid RK2: evidence for a complex replication regulon. Basic Life Sci. 1985;30:227–241. [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]
  • Gennity JM, Inouye M. The protein sequence responsible for lipoprotein membrane localization in Escherichia coli exhibits remarkable specificity. J Biol Chem. 1991 Sep 5;266(25):16458–16464. [PubMed]
  • Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. [PubMed]
  • Hartskeerl RA, Bergmans JE, Kamp MC, Hoekstra WP. Cloning of an exclusion-determining fragment of the IncI plasmid, R144. Plasmid. 1983 Jul;10(1):11–20. [PubMed]
  • Hedges RW, Jacob AE. Transposition of ampicillin resistance from RP4 to other replicons. Mol Gen Genet. 1974;132(1):31–40. [PubMed]
  • Jagura-Burdzy G, Ibbotson JP, Thomas CM. The korF region of broad-host-range plasmid RK2 encodes two polypeptides with transcriptional repressor activity. J Bacteriol. 1991 Jan;173(2):826–833. [PMC free article] [PubMed]
  • Johnson FD, Burns DL. Detection and subcellular localization of three Ptl proteins involved in the secretion of pertussis toxin from Bordetella pertussis. J Bacteriol. 1994 Sep;176(17):5350–5356. [PMC free article] [PubMed]
  • Kado CI. Promiscuous DNA transfer system of Agrobacterium tumefaciens: role of the virB operon in sex pilus assembly and synthesis. Mol Microbiol. 1994 Apr;12(1):17–22. [PubMed]
  • Kao SM, Olmsted SB, Viksnins AS, Gallo JC, Dunny GM. Molecular and genetic analysis of a region of plasmid pCF10 containing positive control genes and structural genes encoding surface proteins involved in pheromone-inducible conjugation in Enterococcus faecalis. J Bacteriol. 1991 Dec;173(23):7650–7664. [PMC free article] [PubMed]
  • Kornacki JA, Chang CH, Figurski DH. kil-kor regulon of promiscuous plasmid RK2: structure, products, and regulation of two operons that constitute the kilE locus. J Bacteriol. 1993 Aug;175(16):5078–5090. [PMC free article] [PubMed]
  • Kotilainen MM, Grahn AM, Bamford JK, Bamford DH. Binding of an Escherichia coli double-stranded DNA virus PRD1 to a receptor coded by an IncP-type plasmid. J Bacteriol. 1993 May;175(10):3089–3095. [PMC free article] [PubMed]
  • Kristensen CS, Eberl L, Sanchez-Romero JM, Givskov M, Molin S, De Lorenzo V. Site-specific deletions of chromosomally located DNA segments with the multimer resolution system of broad-host-range plasmid RP4. J Bacteriol. 1995 Jan;177(1):52–58. [PMC free article] [PubMed]
  • Kuldau GA, De Vos G, Owen J, McCaffrey G, Zambryski P. The virB operon of Agrobacterium tumefaciens pTiC58 encodes 11 open reading frames. Mol Gen Genet. 1990 Apr;221(2):256–266. [PubMed]
  • Lanka E, Wilkins BM. DNA processing reactions in bacterial conjugation. Annu Rev Biochem. 1995;64:141–169. [PubMed]
  • Lessl M, Balzer D, Lurz R, Waters VL, Guiney DG, Lanka E. Dissection of IncP conjugative plasmid transfer: definition of the transfer region Tra2 by mobilization of the Tra1 region in trans. J Bacteriol. 1992 Apr;174(8):2493–2500. [PMC free article] [PubMed]
  • Lessl M, Balzer D, Pansegrau W, Lanka E. Sequence similarities between the RP4 Tra2 and the Ti VirB region strongly support the conjugation model for T-DNA transfer. J Biol Chem. 1992 Oct 5;267(28):20471–20480. [PubMed]
  • Lessl M, Balzer D, Weyrauch K, Lanka E. The mating pair formation system of plasmid RP4 defined by RSF1010 mobilization and donor-specific phage propagation. J Bacteriol. 1993 Oct;175(20):6415–6425. [PMC free article] [PubMed]
  • Lessl M, Krishnapillai V, Schilf W. Identification and characterization of two entry exclusion genes of the promiscuous IncP plasmid R18. Mol Gen Genet. 1991 May;227(1):120–126. [PubMed]
  • 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. [PubMed]
  • Lyras D, Chan AW, McFarlane J, Stanisich VA. The surface exclusion system of RP1: investigation of the roles of trbJ and trbK in the surface exclusion, transfer, and slow-growth phenotypes. Plasmid. 1994 Nov;32(3):254–261. [PubMed]
  • McConlogue L, Brow MA, Innis MA. Structure-independent DNA amplification by PCR using 7-deaza-2'-deoxyguanosine. Nucleic Acids Res. 1988 Oct 25;16(20):9869–9869. [PMC free article] [PubMed]
  • Messing J, Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. [PubMed]
  • Motallebi-Veshareh M, Balzer D, Lanka E, Jagura-Burdzy G, Thomas CM. Conjugative transfer functions of broad-host-range plasmid RK2 are coregulated with vegetative replication. Mol Microbiol. 1992 Apr;6(7):907–920. [PubMed]
  • Olsen RH, Shipley P. Host range and properties of the Pseudomonas aeruginosa R factor R1822. J Bacteriol. 1973 Feb;113(2):772–780. [PMC free article] [PubMed]
  • Olsen RH, Siak JS, Gray RH. Characteristics of PRD1, a plasmid-dependent broad host range DNA bacteriophage. J Virol. 1974 Sep;14(3):689–699. [PMC free article] [PubMed]
  • Olsen RH, Thomas DD. Characteristics and purification of PRR1, an RNA phage specific for the broad host range Pseudomonas R1822 drug resistance plasmid. J Virol. 1973 Dec;12(6):1560–1567. [PMC free article] [PubMed]
  • Pansegrau W, Balzer D, Kruft V, Lurz R, Lanka E. In vitro assembly of relaxosomes at the transfer origin of plasmid RP4. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6555–6559. [PMC free article] [PubMed]
  • Pansegrau W, Lanka E, Barth PT, Figurski DH, Guiney DG, Haas D, Helinski DR, Schwab H, Stanisich VA, Thomas CM. Complete nucleotide sequence of Birmingham IncP alpha plasmids. Compilation and comparative analysis. J Mol Biol. 1994 Jun 24;239(5):623–663. [PubMed]
  • Pansegrau W, Ziegelin G, Lanka E. Covalent association of the traI gene product of plasmid RP4 with the 5'-terminal nucleotide at the relaxation nick site. J Biol Chem. 1990 Jun 25;265(18):10637–10644. [PubMed]
  • Perlman D, Halvorson HO. The MURFI linker for multiple reading frame insertion of a sense or nonsense codon into DNA. Nucleic Acids Res. 1986 Mar 11;14(5):2139–2155. [PMC free article] [PubMed]
  • Perumal NB, Minkley EG., Jr The product of the F sex factor traT surface exclusion gene is a lipoprotein. J Biol Chem. 1984 May 10;259(9):5357–5360. [PubMed]
  • Pinkney M, Diaz R, Lanka E, Thomas CM. Replication of mini RK2 plasmid in extracts of Escherichia coli requires plasmid-encoded protein TrfA and host-encoded proteins DnaA, B, G DNA gyrase and DNA polymerase III. J Mol Biol. 1988 Oct 20;203(4):927–938. [PubMed]
  • Pohlman RF, Genetti HD, Winans SC. Common ancestry between IncN conjugal transfer genes and macromolecular export systems of plant and animal pathogens. Mol Microbiol. 1994 Nov;14(4):655–668. [PubMed]
  • Pohlman RF, Genetti HD, Winans SC. Entry exclusion of the IncN plasmid pKM101 is mediated by a single hydrophilic protein containing a lipid attachment motif. Plasmid. 1994 Mar;31(2):158–165. [PubMed]
  • Roberts RC, Ström AR, Helinski DR. The parDE operon of the broad-host-range plasmid RK2 specifies growth inhibition associated with plasmid loss. J Mol Biol. 1994 Mar 18;237(1):35–51. [PubMed]
  • Sano T, Cantor CR. Expression of a cloned streptavidin gene in Escherichia coli. Proc Natl Acad Sci U S A. 1990 Jan;87(1):142–146. [PMC free article] [PubMed]
  • Sayers JR, Schmidt W, Eckstein F. 5'-3' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res. 1988 Feb 11;16(3):791–802. [PMC free article] [PubMed]
  • Scheiffele P, Pansegrau W, Lanka E. Initiation of Agrobacterium tumefaciens T-DNA processing. Purified proteins VirD1 and VirD2 catalyze site- and strand-specific cleavage of superhelical T-border DNA in vitro. J Biol Chem. 1995 Jan 20;270(3):1269–1276. [PubMed]
  • Scherzinger E, Lurz R, Otto S, Dobrinski B. In vitro cleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins. Nucleic Acids Res. 1992 Jan 11;20(1):41–48. [PMC free article] [PubMed]
  • Shirasu K, Kado CI. Membrane location of the Ti plasmid VirB proteins involved in the biosynthesis of a pilin-like conjugative structure on Agrobacterium tumefaciens. FEMS Microbiol Lett. 1993 Aug 1;111(2-3):287–294. [PubMed]
  • Smit E, Venne D, van Elsas JD. Mobilization of a Recombinant IncQ Plasmid between Bacteria on Agar and in Soil via Cotransfer or Retrotransfer. Appl Environ Microbiol. 1993 Jul;59(7):2257–2263. [PMC free article] [PubMed]
  • Stanisich VA. The properties and host range of male-specific bacteriophages of Pseudomonas aeruginosa. J Gen Microbiol. 1974 Oct;84(2):332–342. [PubMed]
  • Stoops JK, Momany C, Ernst SR, Oliver RM, Schroeter JP, Bretaudiere JP, Hackert ML. Comparisons of the low-resolution structures of ornithine decarboxylase by electron microscopy and X-ray crystallography: the utility of methylamine tungstate stain and Butvar support film in the study of macromolecules by transmission electron microscopy. J Electron Microsc Tech. 1991 Jun;18(2):157–166. [PubMed]
  • Strack B, Lessl M, Calendar R, Lanka E. A common sequence motif, -E-G-Y-A-T-A-, identified within the primase domains of plasmid-encoded I- and P-type DNA primases and the alpha protein of the Escherichia coli satellite phage P4. J Biol Chem. 1992 Jun 25;267(18):13062–13072. [PubMed]
  • Sukupolvi S, O'Connor CD. TraT lipoprotein, a plasmid-specified mediator of interactions between gram-negative bacteria and their environment. Microbiol Rev. 1990 Dec;54(4):331–341. [PMC free article] [PubMed]
  • Tabor S, Richardson CC. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. [PMC free article] [PubMed]
  • Thorstenson YR, Zambryski PC. The essential virulence protein VirB8 localizes to the inner membrane of Agrobacterium tumefaciens. J Bacteriol. 1994 Mar;176(6):1711–1717. [PMC free article] [PubMed]
  • Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. [PMC free article] [PubMed]
  • Ward JE, Akiyoshi DE, Regier D, Datta A, Gordon MP, Nester EW. Characterization of the virB operon from an Agrobacterium tumefaciens Ti plasmid. J Biol Chem. 1988 Apr 25;263(12):5804–5814. [PubMed]
  • Waters VL, Strack B, Pansegrau W, Lanka E, Guiney DG. Mutational analysis of essential IncP alpha plasmid transfer genes traF and traG and involvement of traF in phage sensitivity. J Bacteriol. 1992 Oct;174(20):6666–6673. [PMC free article] [PubMed]
  • Weidlich G, Wirth R, Galli D. Sex pheromone plasmid pAD1-encoded surface exclusion protein of Enterococcus faecalis. Mol Gen Genet. 1992 May;233(1-2):161–168. [PubMed]
  • Weiss AA, Johnson FD, Burns DL. Molecular characterization of an operon required for pertussis toxin secretion. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2970–2974. [PMC free article] [PubMed]
  • Williams DR, Thomas CM. Active partitioning of bacterial plasmids. J Gen Microbiol. 1992 Jan;138(1):1–16. [PubMed]
  • Winans SC, Walker GC. Entry exclusion determinant(s) of IncN plasmid pKM101. J Bacteriol. 1985 Jan;161(1):411–416. [PMC free article] [PubMed]
  • Yamaguchi K, Yu F, Inouye M. A single amino acid determinant of the membrane localization of lipoproteins in E. coli. Cell. 1988 May 6;53(3):423–432. [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]
  • Zatyka M, Jagura-Burdzy G, Thomas CM. Regulation of transfer genes of promiscuous IncP alpha plasmid RK2: repression of Tra1 region transcription both by relaxosome proteins and by the Tra2 regulator TrbA. Microbiology. 1994 Nov;140(Pt 11):2981–2990. [PubMed]

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


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...