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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Oct 25;91(22):10571–10575. doi: 10.1073/pnas.91.22.10571

KorA protein of promiscuous plasmid RK2 controls a transcriptional switch between divergent operons for plasmid replication and conjugative transfer.

G Jagura-Burdzy 1, C M Thomas 1
PMCID: PMC45063  PMID: 7937995

Abstract

The trfA and trb operons, encoding genes essential for replication and conjugative transfer of broad host range plasmid RK2, are transcribed divergently. Deletion analysis presented here indicates that trfAp and trbAp are arranged as face to face promoters. The presence of the korA gene, whose product is known to repress seven operons on RK2, including the trfA operon, is shown here to stimulate trbAp. The effect of korA on trbAp is mimicked by the trfAp-1 promoter down mutation, suggesting that a reduction in the activity of trfAp is required for derepression of trbAp activity. The trfAp-1 mutation reduces RNA polymerase binding and open complex formation at trfAp but does not stimulate melting at trbAp in vitro. Therefore, the inhibition of trbAp is most probably due to forward transcription initiated at trfAp. The simultaneous inhibition/stimulation by KorA is seen even in the presence of the other repressors KorB and TrbA, which act at this region, thus providing a dominant mode of coordinating plasmid replication and transfer. This may be one of the keys to understanding how the maintenance and spread of promiscuous plasmids are balanced in different environments.

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Selected References

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  1. Aiba H., Adhya S., de Crombrugghe B. Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem. 1981 Nov 25;256(22):11905–11910. [PubMed] [Google Scholar]
  2. 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. doi: 10.1093/nar/20.8.1851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bechhofer D. H., Figurski D. H. Map location and nucleotide sequence of korA, a key regulatory gene of promiscuous plasmid RK2. Nucleic Acids Res. 1983 Nov 11;11(21):7453–7469. doi: 10.1093/nar/11.21.7453. [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. Duval-Valentin G., Schmitt B., Ehrlich R. A second RNA-polymerase can bind specifically to the bla promoter of Tn3, repressing transcription initiation. Nucleic Acids Res. 1988 Jun 24;16(12):5277–5290. doi: 10.1093/nar/16.12.5277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Jagura-Burdzy G., Ibbotson J. P., Thomas C. M. The korF region of broad-host-range plasmid RK2 encodes two polypeptides with transcriptional repressor activity. J Bacteriol. 1991 Jan;173(2):826–833. doi: 10.1128/jb.173.2.826-833.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jagura-Burdzy G., Khanim F., Smith C. A., Thomas C. M. Crosstalk between plasmid vegetative replication and conjugative transfer: repression of the trfA operon by trbA of broad host range plasmid RK2. Nucleic Acids Res. 1992 Aug 11;20(15):3939–3944. doi: 10.1093/nar/20.15.3939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jagura-Burdzy G., Thomas C. M. kfrA gene of broad host range plasmid RK2 encodes a novel DNA-binding protein. J Mol Biol. 1992 Jun 5;225(3):651–660. doi: 10.1016/0022-2836(92)90392-w. [DOI] [PubMed] [Google Scholar]
  10. Jovanovic O. S., Ayres E. K., Figurski D. H. The replication initiator operon of promiscuous plasmid RK2 encodes a gene that complements an Escherichia coli mutant defective in single-stranded DNA-binding protein. J Bacteriol. 1992 Jul;174(14):4842–4846. doi: 10.1128/jb.174.14.4842-4846.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kahn M., Kolter R., Thomas C., Figurski D., Meyer R., Remaut E., Helinski D. R. Plasmid cloning vehicles derived from plasmids ColE1, F, R6K, and RK2. Methods Enzymol. 1979;68:268–280. doi: 10.1016/0076-6879(79)68019-9. [DOI] [PubMed] [Google Scholar]
  12. Kornacki J. A., Balderes P. J., Figurski D. H. Nucleotide sequence of korB, a replication control gene of broad host-range plasmid RK2. J Mol Biol. 1987 Nov 20;198(2):211–222. doi: 10.1016/0022-2836(87)90307-x. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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] [Google Scholar]
  15. 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. doi: 10.1128/jb.175.20.6415-6425.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lillie J. W., Green M., Green M. R. An adenovirus E1a protein region required for transformation and transcriptional repression. Cell. 1986 Sep 26;46(7):1043–1051. doi: 10.1016/0092-8674(86)90704-x. [DOI] [PubMed] [Google Scholar]
  17. McKenney K., Shimatake H., Court D., Schmeissner U., Brady C., Rosenberg M. A system to study promoter and terminator signals recognized by Escherichia coli RNA polymerase. Gene Amplif Anal. 1981;2:383–415. [PubMed] [Google Scholar]
  18. 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]
  19. Mullis K., Faloona F., Scharf S., Saiki R., Horn G., Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol. 1986;51(Pt 1):263–273. doi: 10.1101/sqb.1986.051.01.032. [DOI] [PubMed] [Google Scholar]
  20. Pinkney M., Theophilus B. D., Warne S. R., Tacon W. C., Thomas C. M. Analysis of transcription from the trfA promoter of broad host range plasmid RK2 in Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa. Plasmid. 1987 May;17(3):222–232. doi: 10.1016/0147-619x(87)90030-8. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Sasse-Dwight S., Gralla J. D. KMnO4 as a probe for lac promoter DNA melting and mechanism in vivo. J Biol Chem. 1989 May 15;264(14):8074–8081. [PubMed] [Google Scholar]
  23. Shinger V., Thomas C. M. Transcription in the trfA region of broad host range plasmid RK2 is regulated by trfB and korB. Mol Gen Genet. 1984;195(3):523–529. doi: 10.1007/BF00341457. [DOI] [PubMed] [Google Scholar]
  24. Shingler V., Thomas C. M. Analysis of the trfA region of broad host-range plasmid RK2 by transposon mutagenesis and identification of polypeptide products. J Mol Biol. 1984 May 25;175(3):229–249. doi: 10.1016/0022-2836(84)90346-2. [DOI] [PubMed] [Google Scholar]
  25. Smith C. A., Shingler V., Thomas C. M. The trfA and trfB promoter regions of broad host range plasmid RK2 share common potential regulatory sequences. Nucleic Acids Res. 1984 Apr 25;12(8):3619–3630. doi: 10.1093/nar/12.8.3619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Smith C. A., Thomas C. M. Nucleotide sequence of the trfA gene of broad host-range plasmid RK2. J Mol Biol. 1984 May 25;175(3):251–262. doi: 10.1016/0022-2836(84)90347-4. [DOI] [PubMed] [Google Scholar]
  27. Straney D. C., Crothers D. M. A stressed intermediate in the formation of stably initiated RNA chains at the Escherichia coli lac UV5 promoter. J Mol Biol. 1987 Jan 20;193(2):267–278. doi: 10.1016/0022-2836(87)90218-x. [DOI] [PubMed] [Google Scholar]
  28. Theophilus B. D., Cross M. A., Smith C. A., Thomas C. M. Regulation of the trfA and trfB promoters of broad host range plasmid RK2: identification of sequences essential for regulation by trfB/korA/korD. Nucleic Acids Res. 1985 Nov 25;13(22):8129–8142. doi: 10.1093/nar/13.22.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Theophilus B. D., Thomas C. M. Nucleotide sequence of the transcriptional repressor gene korB which plays a key role in regulation of the copy number of broad host range plasmid RK2. Nucleic Acids Res. 1987 Sep 25;15(18):7443–7450. doi: 10.1093/nar/15.18.7443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thomas C. M. Complementation analysis of replication and maintenance functions of broad host range plasmids RK2 and RP1. Plasmid. 1981 May;5(3):277–291. doi: 10.1016/0147-619x(81)90005-6. [DOI] [PubMed] [Google Scholar]
  31. Thomas C. M. Evidence for the involvement of the incC locus of broad host range plasmid RK2 in plasmid maintenance. Plasmid. 1986 Jul;16(1):15–29. doi: 10.1016/0147-619x(86)90075-2. [DOI] [PubMed] [Google Scholar]
  32. Thomas C. M., Ibbotson J. P., Wang N. Y., Smith C. A., Tipping R., Loader N. M. Gene regulation on broad host range plasmid RK2: identification of three novel operons whose transcription is repressed by both KorA and KorC. Nucleic Acids Res. 1988 Jun 24;16(12):5345–5359. doi: 10.1093/nar/16.12.5345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Thomas C. M., Meyer R., Helinski D. R. Regions of broad-host-range plasmid RK2 which are essential for replication and maintenance. J Bacteriol. 1980 Jan;141(1):213–222. doi: 10.1128/jb.141.1.213-222.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Thomas C. M., Sherlock S. Broad host range plasmid RK2 encodes a polypeptide related to single-stranded DNA binding protein (SSB) of Escherichia coli. Nucleic Acids Res. 1990 May 11;18(9):2812–2812. doi: 10.1093/nar/18.9.2812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Thomas C. M., Smith C. A. Incompatibility group P plasmids: genetics, evolution, and use in genetic manipulation. Annu Rev Microbiol. 1987;41:77–101. doi: 10.1146/annurev.mi.41.100187.000453. [DOI] [PubMed] [Google Scholar]
  36. Thomson V. J., Jovanovic O. S., Pohlman R. F., Chang C. H., Figurski D. H. Structure, function, and regulation of the kilB locus of promiscuous plasmid RK2. J Bacteriol. 1993 Apr;175(8):2423–2435. doi: 10.1128/jb.175.8.2423-2435.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Williams D. R., Motallebi-Veshareh M., Thomas C. M. Multifunctional repressor KorB can block transcription by preventing isomerization of RNA polymerase-promoter complexes. Nucleic Acids Res. 1993 Mar 11;21(5):1141–1148. doi: 10.1093/nar/21.5.1141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Williams D. R., Motallebi-Veshareh M., Thomas C. M. Multifunctional repressor KorB can block transcription by preventing isomerization of RNA polymerase-promoter complexes. Nucleic Acids Res. 1993 Mar 11;21(5):1141–1148. doi: 10.1093/nar/21.5.1141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zukowski M. M., Gaffney D. F., Speck D., Kauffmann M., Findeli A., Wisecup A., Lecocq J. P. Chromogenic identification of genetic regulatory signals in Bacillus subtilis based on expression of a cloned Pseudomonas gene. Proc Natl Acad Sci U S A. 1983 Feb;80(4):1101–1105. doi: 10.1073/pnas.80.4.1101. [DOI] [PMC free article] [PubMed] [Google Scholar]

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