Skip to main content
PMC home page
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
. 1985 Nov;82(21):7374–7378. doi: 10.1073/pnas.82.21.7374

korA function of promiscuous plasmid RK2: an autorepressor that inhibits expression of host-lethal gene kilA and replication gene trfA.

C Young, A S Prince, D H Figurski
PMCID: PMC391347  PMID: 3903752

Abstract

In broad host-range plasmid RK2, korA function prevents the lethal effect of kilA on Escherichia coli host cells and inhibits expression of trfA, the essential replication gene. From gene fusion and promoter replacement studies, we determined that control of kilA is also mediated at the level of gene expression and that the target resides in the kilA promoter region. The nucleotide sequence of this region shows the same two operator-like palindromes present in the previously sequenced promoters of trfA and korA. One of the palindromes (5'-GTTTAGCTAAAC-3') at the -10 position is sufficient to confer sensitivity to korA function. The presence of the same sequences in the korA promoter region suggested that korA might also regulate its own expression. Using the structural gene for chloramphenicol acetyltransferase (cat) fused to the korA promoter, we found that korA gene expression is indeed autoregulated. The results show that korA gene product is very likely a repressor that negatively regulates expression of at least three different genes by interacting with an operator-like sequence in their promoter regions. Coordinate regulation of host-lethal gene kilA and essential replication gene trfA by a common mechanism also supports our hypothesis that these genes are functionally related.

Full text

PDF

Selected References

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

  1. Barth P. T., Ellis K., Bechhofer D. H., Figurski D. H. Involvement of kil and kor genes in the phenotype of a host-range mutant of RP4. Mol Gen Genet. 1984;197(2):236–243. doi: 10.1007/BF00330969. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Burkardt H. J., Wohlleben W. RNA polymerase binding sites on the broad host range plasmid RP4. J Gen Microbiol. 1981 Jul;125(1):189–193. doi: 10.1099/00221287-125-1-189. [DOI] [PubMed] [Google Scholar]
  4. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Close T. J., Rodriguez R. L. Construction and characterization of the chloramphenicol-resistance gene cartridge: a new approach to the transcriptional mapping of extrachromosomal elements. Gene. 1982 Dec;20(2):305–316. doi: 10.1016/0378-1119(82)90048-8. [DOI] [PubMed] [Google Scholar]
  6. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Datta N., Hedges R. W. Host ranges of R factors. J Gen Microbiol. 1972 May;70(3):453–460. doi: 10.1099/00221287-70-3-453. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Figurski D. H., Pohlman R. F., Bechhofer D. H., Prince A. S., Kelton C. A. Broad host range plasmid RK2 encodes multiple kil genes potentially lethal to Escherichia coli host cells. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1935–1939. doi: 10.1073/pnas.79.6.1935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ingram L. C., Richmond M. H., Sykes R. B. Molecular characterization of the R factors implicated in the carbenicillin resistance of a sequence of Pseudomonas aeruginosa strains isolated from burns. Antimicrob Agents Chemother. 1973 Feb;3(2):279–288. doi: 10.1128/aac.3.2.279. [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. Matsubara K. Replication control system in lambda dv. Plasmid. 1981 Jan;5(1):32–52. doi: 10.1016/0147-619x(81)90076-7. [DOI] [PubMed] [Google Scholar]
  13. Meyer R. J., Helinski D. R. Unidirectional replication of the P-group plasmid RK2. Biochim Biophys Acta. 1977 Sep 6;478(1):109–113. doi: 10.1016/0005-2787(77)90249-0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Olsen R. H., Shipley P. Host range and properties of the Pseudomonas aeruginosa R factor R1822. J Bacteriol. 1973 Feb;113(2):772–780. doi: 10.1128/jb.113.2.772-780.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pabo C. O., Sauer R. T. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. doi: 10.1146/annurev.bi.53.070184.001453. [DOI] [PubMed] [Google Scholar]
  17. Rüther U. Construction and properties of a new cloning vehicle, allowing direct screening for recombinant plasmids. Mol Gen Genet. 1980;178(2):475–477. doi: 10.1007/BF00270503. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Schmidhauser T. J., Filutowicz M., Helinski D. R. Replication of derivatives of the broad host range plasmid RK2 in two distantly related bacteria. Plasmid. 1983 May;9(3):325–330. doi: 10.1016/0147-619x(83)90010-0. [DOI] [PubMed] [Google Scholar]
  20. Schreiner H. C., Bechhofer D. H., Pohlman R. F., Young C., Borden P. A., Figurski D. H. Replication control in promiscuous plasmid RK2: kil and kor functions affect expression of the essential replication gene trfA. J Bacteriol. 1985 Jul;163(1):228–237. doi: 10.1128/jb.163.1.228-237.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Shafferman A., Kolter R., Stalker D., Helinski D. R. Plasmid R6K DNA replication. III. Regulatory properties of the pi initiation protein. J Mol Biol. 1982 Oct 15;161(1):57–76. doi: 10.1016/0022-2836(82)90278-9. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Siebenlist U., Simpson R. B., Gilbert W. E. coli RNA polymerase interacts homologously with two different promoters. Cell. 1980 Jun;20(2):269–281. doi: 10.1016/0092-8674(80)90613-3. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Smith C. A., Thomas C. M. Molecular gentic analysis of the trfB and korB region of broad host range plasmid RK2. J Gen Microbiol. 1984 Jul;130(7):1651–1663. doi: 10.1099/00221287-130-7-1651. [DOI] [PubMed] [Google Scholar]
  26. Sompayrac L., Maaloe O. Autorepressor model for control of DNA replication. Nat New Biol. 1973 Jan 31;241(109):133–135. doi: 10.1038/newbio241133a0. [DOI] [PubMed] [Google Scholar]
  27. Stalker D. M., Thomas C. M., Helinski D. R. Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2. Mol Gen Genet. 1981;181(1):8–12. doi: 10.1007/BF00338997. [DOI] [PubMed] [Google Scholar]
  28. Søgaard-Andersen L., Rokeach L. A., Molin S. Regulated expression of a gene important for replication of plasmid F in E. coli. EMBO J. 1984 Feb;3(2):257–262. doi: 10.1002/j.1460-2075.1984.tb01794.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Thomas C. M., Hussain A. A., Smith C. A. Maintenance of broad host range plasmid RK2 replicons in Pseudomonas aeruginosa. Nature. 1982 Aug 12;298(5875):674–676. doi: 10.1038/298674a0. [DOI] [PubMed] [Google Scholar]
  30. Thomas C. M., Hussain A. A. The korB gene of broad host range plasmid RK2 is a major copy number control element which may act together with trfB by limiting trfA expression. EMBO J. 1984 Jul;3(7):1513–1519. doi: 10.1002/j.1460-2075.1984.tb02004.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  33. Young C., Bechhofer D. H., Figurski D. H. Gene regulation in plasmid RK2: positive control by korA in the expression of korC. J Bacteriol. 1984 Jan;157(1):247–252. doi: 10.1128/jb.157.1.247-252.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES