Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1985 Apr;162(1):72–77. doi: 10.1128/jb.162.1.72-77.1985

Copy mutant of mini-Rts1: lowered binding affinity of mutated RepA protein to direct repeats.

Y Terawaki, Y Itoh
PMCID: PMC218955  PMID: 3156849

Abstract

Nucleotide sequence analysis of mini-Rts1 and its copy mutant disclosed the presence of two clusters of direct-repeat sequences flanking the coding region for the 33,000-dalton RepA protein and two base substitutions on the mini-Rts1cop1 genome (Kamio et al., J. Bacteriol. 158:307-312, 1984). On subcloning of the left cluster (incI) that is located downstream from repA, the five 24-base-pair repeats expressed a stronger incompatibility toward mini-Rts1 than did the four repeats. The right cluster (incII) that contains three 21-base-pair repeats also exhibited strong incompatibility toward mini-Rts1. By separating the two base substitutions of mini-Rts1cop1, the mutation that is responsible for the copy increase was determined to be a single base change in the RepA coding region. Both clusters of the repeats, cloned separately into the vector plasmid, showed a weaker incompatibility toward mini-Rts1cop1 than to the wild-type mini-Rts1. These findings suggest a lowered binding affinity of the mutated RepA protein to the direct repeats.

Full text

PDF
76

Images in this article

Selected References

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

  1. Abeles A. L., Snyder K. M., Chattoraj D. K. P1 plasmid replication: replicon structure. J Mol Biol. 1984 Mar 5;173(3):307–324. doi: 10.1016/0022-2836(84)90123-2. [DOI] [PubMed] [Google Scholar]
  2. Baumstark B. R., Lowery K., Scott J. R. Location by DNA sequence analysis of cop mutations affecting the number of plasmid copies of prophage P1. Mol Gen Genet. 1984;194(3):513–516. doi: 10.1007/BF00425567. [DOI] [PubMed] [Google Scholar]
  3. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [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. Churchward G., Linder P., Caro L. The nucleotide sequence of replication and maintenance functions encoded by plasmid pSC101. Nucleic Acids Res. 1983 Aug 25;11(16):5645–5659. doi: 10.1093/nar/11.16.5645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clark A. J., Chamberlin M., Boyce R. P., Howard-Flanders P. Abnormal metabolic response to ultraviolet light of a recombination deficient mutant of Escherichia coli K12. J Mol Biol. 1966 Aug;19(2):442–454. doi: 10.1016/s0022-2836(66)80015-3. [DOI] [PubMed] [Google Scholar]
  7. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Danbara H., Brady G., Timmis J. K., Timmis K. N. Regulation of DNA replication: "target" determinant of the replication control elements of plasmid R6-5 lies within a control element gene. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4699–4703. doi: 10.1073/pnas.78.8.4699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Easton A. M., Rownd R. H. The incompatibility product of IncFII R plasmid NR1 controls gene expression in the plasmid replication region. J Bacteriol. 1982 Nov;152(2):829–839. doi: 10.1128/jb.152.2.829-839.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Germino J., Bastia D. Interaction of the plasmid R6K-encoded replication initiator protein with its binding sites on DNA. Cell. 1983 Aug;34(1):125–134. doi: 10.1016/0092-8674(83)90142-3. [DOI] [PubMed] [Google Scholar]
  12. Itoh Y., Kamio Y., Furuta Y., Terawaki Y. Cloning of the replication and incompatibility regions of a plasmid derived from Rts1. Plasmid. 1982 Nov;8(3):232–243. doi: 10.1016/0147-619x(82)90061-0. [DOI] [PubMed] [Google Scholar]
  13. Kado C. I., Liu S. T. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol. 1981 Mar;145(3):1365–1373. doi: 10.1128/jb.145.3.1365-1373.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kamio Y., Tabuchi A., Itoh Y., Katagiri H., Terawaki Y. Complete nucleotide sequence of mini-Rts1 and its copy mutant. J Bacteriol. 1984 Apr;158(1):307–312. doi: 10.1128/jb.158.1.307-312.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kamio Y., Terawaki Y. Nucleotide sequence of an incompatibility region of mini-Rts1 that contains five direct repeats. J Bacteriol. 1983 Sep;155(3):1185–1191. doi: 10.1128/jb.155.3.1185-1191.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kolter R., Helinski D. R. Plasmid R6K DNA replication. II. Direct nucleotide sequence repeats are required for an active gamma-origin. J Mol Biol. 1982 Oct 15;161(1):45–56. doi: 10.1016/0022-2836(82)90277-7. [DOI] [PubMed] [Google Scholar]
  17. Light J., Molin S. Post-transcriptional control of expression of the repA gene of plasmid R1 mediated by a small RNA molecule. EMBO J. 1983;2(1):93–98. doi: 10.1002/j.1460-2075.1983.tb01387.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Miller J., Manis J., Kline B., Bishop A. Nonintegrated plasmid-folded chromosome complexes: genetic studies on formation and possible relationship to plasmid replication. Plasmid. 1978 Jun;1(3):273–283. doi: 10.1016/0147-619x(78)90045-8. [DOI] [PubMed] [Google Scholar]
  19. Murotsu T., Matsubara K. Role of an autorepression system in the control of lambda dv plasmid copy number and incompatibility. Mol Gen Genet. 1980;179(3):509–519. doi: 10.1007/BF00271740. [DOI] [PubMed] [Google Scholar]
  20. NAKAYA R., NAKAMURA A., MURATA Y. Resistance transfer agents in Shigella. Biochem Biophys Res Commun. 1960 Dec;3:654–659. doi: 10.1016/0006-291x(60)90081-4. [DOI] [PubMed] [Google Scholar]
  21. Pribnow D. Nucleotide sequence of an RNA polymerase binding site at an early T7 promoter. Proc Natl Acad Sci U S A. 1975 Mar;72(3):784–788. doi: 10.1073/pnas.72.3.784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rosen J., Ryder T., Ohtsubo H., Ohtsubo E. Role of RNA transcripts in replication incompatibility and copy number control in antibiotic resistance plasmid derivatives. Nature. 1981 Apr 30;290(5809):794–797. doi: 10.1038/290794a0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Stougaard P., Molin S., Nordström K. RNAs involved in copy-number control and incompatibility of plasmid R1. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6008–6012. doi: 10.1073/pnas.78.10.6008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Tanak N., Cramer J. H., Rownd R. H. EcoRI restriction endonuclease map of the composite R plasmid NR1. J Bacteriol. 1976 Jul;127(1):619–636. doi: 10.1128/jb.127.1.619-636.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tomizawa J., Itoh T., Selzer G., Som T. Inhibition of ColE1 RNA primer formation by a plasmid-specified small RNA. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1421–1425. doi: 10.1073/pnas.78.3.1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tsutsui H., Fujiyama A., Murotsu T., Matsubara K. Role of nine repeating sequences of the mini-F genome for expression of F-specific incompatibility phenotype and copy number control. J Bacteriol. 1983 Jul;155(1):337–344. doi: 10.1128/jb.155.1.337-344.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Uhlin B. E., Nordström K. R plasmid gene dosage effects in Escherichia coli K-12: copy mutants of the R plasmic R1drd-19. Plasmid. 1977 Nov;1(1):1–7. doi: 10.1016/0147-619x(77)90003-8. [DOI] [PubMed] [Google Scholar]
  30. Womble D. D., Dong X., Wu R. P., Luckow V. A., Martinez A. F., Rownd R. H. IncFII plasmid incompatibility product and its target are both RNA transcripts. J Bacteriol. 1984 Oct;160(1):28–35. doi: 10.1128/jb.160.1.28-35.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES