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. 1985 Sep;82(18):6090–6094. doi: 10.1073/pnas.82.18.6090

Protein RepC is involved in copy number control of the broad host range plasmid RSF1010.

V Haring, P Scholz, E Scherzinger, J Frey, K Derbyshire, G Hatfull, N S Willetts, M Bagdasarian
PMCID: PMC390705  PMID: 3898079

Abstract

Essential replication (rep) genes of the broad host range plasmid RSF1010 have been cloned onto controlled expression vectors and their protein products have been visualized, after induction, by NaDodSO4/polyacrylamide gel electrophoresis of whole cell lysates. During this induction the replication of a coresident RSF1010 replicon, pKT210, was analyzed by quantitative DNA X DNA hybridization. The initiation of pKT210 replication was stimulated 6-fold by a simultaneous overproduction of the RepA and RepC proteins compared to cells in which only the RepA protein was overproduced. An enhanced synthesis of the RepB protein resulted in a 1.6-fold stimulation of pKT210 replication, whereas an overproduction of the RepA protein alone had no effect. Purified RepC protein has been shown to bind preferentially to DNA carrying the replication origin of RSF1010. Within this segment it was bound specifically to those DNA fragments that contained the 20-base-pair direct repeats of the origin region. These results suggest that RepC protein acts as a positive replication regulator, that its concentration is rate-limiting, and that the replication rate of RSF1010 is controlled, at least in part, at the level of RepC synthesis.

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Selected References

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

  1. Amann E., Brosius J., Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene. 1983 Nov;25(2-3):167–178. doi: 10.1016/0378-1119(83)90222-6. [DOI] [PubMed] [Google Scholar]
  2. Bagdasarian M., Lurz R., Rückert B., Franklin F. C., Bagdasarian M. M., Frey J., Timmis K. N. Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene. 1981 Dec;16(1-3):237–247. doi: 10.1016/0378-1119(81)90080-9. [DOI] [PubMed] [Google Scholar]
  3. Bagdasarian M., Timmis K. N. Host: vector systems for gene cloning in Pseudomonas. Curr Top Microbiol Immunol. 1982;96:47–67. doi: 10.1007/978-3-642-68315-2_4. [DOI] [PubMed] [Google Scholar]
  4. Barth P. T., Grinter N. J. Comparison of the deoxyribonucleic acid molecular weights and homologies of plasmids conferring linked resistance to streptomycin and sulfonamides. J Bacteriol. 1974 Nov;120(2):618–630. doi: 10.1128/jb.120.2.618-630.1974. [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. Chattoraj D., Cordes K., Abeles A. Plasmid P1 replication: negative control by repeated DNA sequences. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6456–6460. doi: 10.1073/pnas.81.20.6456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Frey J., Chandler M., Caro L. Overinitiation of chromosome and plasmid replication in a dna Acos mutant of Escherichia coli K12. Evidence for dnaA-dnaB interactions. J Mol Biol. 1984 Oct 25;179(2):171–183. doi: 10.1016/0022-2836(84)90464-9. [DOI] [PubMed] [Google Scholar]
  8. Frey J., Chandler M., Caro L. The effects of an Escherichia coli dnaAts mutation on the replication of the plasmids colE1 pSC101, R100.1 and RTF-TC. Mol Gen Genet. 1979 Jul 13;174(2):117–126. doi: 10.1007/BF00268349. [DOI] [PubMed] [Google Scholar]
  9. Lin L. S., Meyer R. J. Nucleotide sequence and functional properties of DNA encoding incompatibility in the broad host-range plasmid R1162. Mol Gen Genet. 1984;194(3):423–431. doi: 10.1007/BF00425554. [DOI] [PubMed] [Google Scholar]
  10. Lugtenberg B., Meijers J., Peters R., van der Hoek P., van Alphen L. Electrophoretic resolution of the "major outer membrane protein" of Escherichia coli K12 into four bands. FEBS Lett. 1975 Oct 15;58(1):254–258. doi: 10.1016/0014-5793(75)80272-9. [DOI] [PubMed] [Google Scholar]
  11. 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. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  12. Meyer R., Hinds M., Brasch M. Properties of R1162, a broad-host-range, high-copy-number plasmid. J Bacteriol. 1982 May;150(2):552–562. doi: 10.1128/jb.150.2.552-562.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Murotsu T., Matsubara K., Sugisaki H., Takanami M. Nine unique repeating sequences in a region essential for replication and incompatibility of the mini-F plasmid. Gene. 1981 Nov;15(2-3):257–271. doi: 10.1016/0378-1119(81)90135-9. [DOI] [PubMed] [Google Scholar]
  14. Poncz M., Solowiejczyk D., Ballantine M., Schwartz E., Surrey S. "Nonrandom" DNA sequence analysis in bacteriophage M13 by the dideoxy chain-termination method. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4298–4302. doi: 10.1073/pnas.79.14.4298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Roberts J. D., McMacken R. The bacteriophage lambda O replication protein: isolation and characterization of the amplified initiator. Nucleic Acids Res. 1983 Nov 11;11(21):7435–7452. [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Scherzinger E., Bagdasarian M. M., Scholz P., Lurz R., Rückert B., Bagdasarian M. Replication of the broad host range plasmid RSF1010: requirement for three plasmid-encoded proteins. Proc Natl Acad Sci U S A. 1984 Feb;81(3):654–658. doi: 10.1073/pnas.81.3.654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Tsurimoto T., Matsubara K. Purified bacteriophage lambda O protein binds to four repeating sequences at the lambda replication origin. Nucleic Acids Res. 1981 Apr 24;9(8):1789–1799. doi: 10.1093/nar/9.8.1789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Vocke C., Bastia D. Primary structure of the essential replicon of the plasmid pSC101. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6557–6561. doi: 10.1073/pnas.80.21.6557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. de Graaff J., Crosa J. H., Heffron F., Falkow S. Replication of the nonconjugative plasmid RSF1010 in Escherichia coli K-12. J Bacteriol. 1978 Jun;134(3):1117–1122. doi: 10.1128/jb.134.3.1117-1122.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]

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