Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Oct;174(20):6666–6673. doi: 10.1128/jb.174.20.6666-6673.1992

Mutational analysis of essential IncP alpha plasmid transfer genes traF and traG and involvement of traF in phage sensitivity.

V L Waters 1, B Strack 1, W Pansegrau 1, E Lanka 1, D G Guiney 1
PMCID: PMC207648  PMID: 1400217

Abstract

Although the broad-host-range IncP plasmids can vegetatively replicate in diverse gram-negative bacteria, the development of shuttle vector systems has established that the host range for IncP plasmid conjugative transfer is greater than the range of bacteria that sustain IncP replicons. Towards understanding IncP plasmid conjugation and the connection between IncP conjugation and Agrobacterium tumefaciens T-DNA transfer to plants, two sets of mutants were generated in the larger transfer region (Tra1) of the IncP alpha plasmid RK2. Mutagenesis strategies were chosen to minimize transcriptional polar effects. Mutant Tra1 clones were mapped, sequenced, and processed to reconstruct 49.5-kb Tra2-containing plasmid derivatives in order to assay for transfer activity and IncP plasmid-specific phage sensitivity. Focusing on the activities of the gene products of traF and traG in Escherichia coli, we found that mutations in traF abolished transfer activity and rendered the host cells phage resistant and mutations in traG abolished transfer activity but had no effect on phage sensitivity. Complementation of these mutant derivatives with corresponding trans-acting clones carrying traF or traG restored transfer activity and, in the case of the traF mutant, the phage sensitivity of the host cell. We conclude that in E. coli, both TraF and TraG are essential for IncP plasmid transfer and that TraF is necessary (but not sufficient) for donor-specific phage sensitivity, and sequencing data suggest that both TraF and TraG are membrane spanning.

Full text

PDF
6670

Images in this article

6671Image
on p.6671

Selected References

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

  1. Barth P. T., Grinter N. J., Bradley D. E. Conjugal transfer system of plasmid RP4: analysis by transposon 7 insertion. J Bacteriol. 1978 Jan;133(1):43–52. doi: 10.1128/jb.133.1.43-52.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beijersbergen A., Dulk-Ras A. D., Schilperoort R. A., Hooykaas P. J. Conjugative Transfer by the Virulence System of Agrobacterium tumefaciens. Science. 1992 May 29;256(5061):1324–1327. doi: 10.1126/science.256.5061.1324. [DOI] [PubMed] [Google Scholar]
  3. Biggin M. D., Gibson T. J., Hong G. F. 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. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  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. Bradley D. E. Morphological and serological relationships of conjugative pili. Plasmid. 1980 Sep;4(2):155–169. doi: 10.1016/0147-619x(80)90005-0. [DOI] [PubMed] [Google Scholar]
  7. 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. doi: 10.1016/0378-1119(82)90103-2. [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. Fürste J. P., 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. doi: 10.1016/0378-1119(86)90358-6. [DOI] [PubMed] [Google Scholar]
  10. Fürste J. P., Pansegrau W., Ziegelin G., Kröger M., Lanka E. Conjugative transfer of promiscuous IncP plasmids: interaction of plasmid-encoded products with the transfer origin. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1771–1775. doi: 10.1073/pnas.86.6.1771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Garoff H., Ansorge W. Improvements of DNA sequencing gels. Anal Biochem. 1981 Aug;115(2):450–457. doi: 10.1016/0003-2697(81)90031-2. [DOI] [PubMed] [Google Scholar]
  12. Guiney D. G., Deiss C., Simnad V., Yee L., Pansegrau W., Lanka E. Mutagenesis of the Tra1 core region of RK2 by using Tn5: identification of plasmid-specific transfer genes. J Bacteriol. 1989 Jul;171(7):4100–4103. doi: 10.1128/jb.171.7.4100-4103.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Guiney D. G., Hasegawa P., Davis C. E. Plasmid transfer from Escherichia coli to Bacteroides fragilis: differential expression of antibiotic resistance phenotypes. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7203–7206. doi: 10.1073/pnas.81.22.7203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guiney D. G., Helinski D. R. 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. doi: 10.1007/BF00273212. [DOI] [PubMed] [Google Scholar]
  15. Guiney D. G., 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. doi: 10.1073/pnas.80.12.3595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Heinemann J. A., Sprague G. F., Jr Bacterial conjugative plasmids mobilize DNA transfer between bacteria and yeast. Nature. 1989 Jul 20;340(6230):205–209. doi: 10.1038/340205a0. [DOI] [PubMed] [Google Scholar]
  18. Lanka E., Lurz R., Fürste J. P. Molecular cloning and mapping of SphI restriction fragments of plasmid RP4. Plasmid. 1983 Nov;10(3):303–307. doi: 10.1016/0147-619x(83)90047-1. [DOI] [PubMed] [Google Scholar]
  19. Lanka E., Lurz R., Kröger M., Fürste J. P. Plasmid RP4 encodes two forms of a DNA primase. Mol Gen Genet. 1984;194(1-2):65–72. doi: 10.1007/BF00383499. [DOI] [PubMed] [Google Scholar]
  20. Lazraq R., Clavel-Sérès S., David H. L., Roulland-Dussoix D. Conjugative transfer of a shuttle plasmid from Escherichia coli to Mycobacterium smegmatis [corrected]. FEMS Microbiol Lett. 1990 May;57(1-2):135–138. doi: 10.1016/0378-1097(90)90427-r. [DOI] [PubMed] [Google Scholar]
  21. Lessl M., Balzer D., Lurz R., Waters V. L., Guiney D. G., 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. doi: 10.1128/jb.174.8.2493-2500.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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. doi: 10.1007/BF00260716. [DOI] [PubMed] [Google Scholar]
  23. Lyons L. B., Zinder N. D. The genetic map of the filamentous bacteriophage f1. Virology. 1972 Jul;49(1):45–60. doi: 10.1016/s0042-6822(72)80006-0. [DOI] [PubMed] [Google Scholar]
  24. Matsunaga T., Nakamura C., Burgess J. G., Sode K. Gene transfer in magnetic bacteria: transposon mutagenesis and cloning of genomic DNA fragments required for magnetosome synthesis. J Bacteriol. 1992 May;174(9):2748–2753. doi: 10.1128/jb.174.9.2748-2753.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Meyer R., Hinds M. Multiple mechanisms for expression of incompatibility by broad-host-range plasmid RK2. J Bacteriol. 1982 Dec;152(3):1078–1090. doi: 10.1128/jb.152.3.1078-1090.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Miele L., Strack B., Kruft V., Lanka E. Gene organization and nucleotide sequence of the primase region of IncP plasmids RP4 and R751. DNA Seq. 1991;2(3):145–162. doi: 10.3109/10425179109039685. [DOI] [PubMed] [Google Scholar]
  27. 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. doi: 10.1093/nar/14.3.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Motallebi-Veshareh M., Balzer D., Lanka E., Jagura-Burdzy G., Thomas C. M. Conjugative transfer functions of broad-host-range plasmid RK2 are coregulated with vegetative replication. Mol Microbiol. 1992 Apr;6(7):907–920. doi: 10.1111/j.1365-2958.1992.tb01541.x. [DOI] [PubMed] [Google Scholar]
  29. Okamoto S., Toyoda-Yamamoto A., Ito K., Takebe I., Machida Y. Localization and orientation of the VirD4 protein of Agrobacterium tumefaciens in the cell membrane. Mol Gen Genet. 1991 Aug;228(1-2):24–32. doi: 10.1007/BF00282443. [DOI] [PubMed] [Google Scholar]
  30. Olsen R. H., Siak J. S., Gray R. H. Characteristics of PRD1, a plasmid-dependent broad host range DNA bacteriophage. J Virol. 1974 Sep;14(3):689–699. doi: 10.1128/jvi.14.3.689-699.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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. doi: 10.1073/pnas.87.17.6555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pansegrau W., Lanka E. Common sequence motifs in DNA relaxases and nick regions from a variety of DNA transfer systems. Nucleic Acids Res. 1991 Jun 25;19(12):3455–3455. doi: 10.1093/nar/19.12.3455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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. doi: 10.1093/nar/15.5.2385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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] [Google Scholar]
  35. 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]
  36. Tabor S., Richardson C. C. 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. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Thatte V., Gill S., Iyer V. N. Regions on plasmid pCU1 required for the killing of Klebsiella pneumoniae. J Bacteriol. 1985 Sep;163(3):1296–1299. doi: 10.1128/jb.163.3.1296-1299.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ubben D., Schmitt R. Tn1721 derivatives for transposon mutagenesis, restriction mapping and nucleotide sequence analysis. Gene. 1986;41(2-3):145–152. doi: 10.1016/0378-1119(86)90093-4. [DOI] [PubMed] [Google Scholar]
  39. Waters V. L., Hirata K. H., Pansegrau W., Lanka E., Guiney D. G. Sequence identity in the nick regions of IncP plasmid transfer origins and T-DNA borders of Agrobacterium Ti plasmids. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1456–1460. doi: 10.1073/pnas.88.4.1456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Williams D. R., Young D. I., Young M. Conjugative plasmid transfer from Escherichia coli to Clostridium acetobutylicum. J Gen Microbiol. 1990 May;136(5):819–826. doi: 10.1099/00221287-136-5-819. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Ziegelin G., Fürste J. P., Lanka E. TraJ protein of plasmid RP4 binds to a 19-base pair invert sequence repetition within the transfer origin. J Biol Chem. 1989 Jul 15;264(20):11989–11994. [PubMed] [Google Scholar]
  43. Ziegelin G., Pansegrau W., Strack B., Balzer D., Kröger M., Kruft V., Lanka E. Nucleotide sequence and organization of genes flanking the transfer origin of promiscuous plasmid RP4. DNA Seq. 1991;1(5):303–327. doi: 10.3109/10425179109020786. [DOI] [PubMed] [Google Scholar]

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

RESOURCES