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. 1982 Jun;150(3):1234–1243. doi: 10.1128/jb.150.3.1234-1243.1982

Replication of a low-copy-number plasmid by a plasmid DNA-membrane complex extracted from minicells of Escherichia coli.

W Firshein, P Strumph, P Benjamin, K Burnstein, J Kornacki
PMCID: PMC216345  PMID: 7042688

Abstract

A DNA-membrane complex was extracted from minicells of an Escherichia coli mutant harboring a "miniplasmid" derivative (11.2 kilobases) of the low-copynumber plasmid RK2 (56 kilobases). The complex contained various species of supercoiled and intermediate forms of plasmid DNA, of which approximately 20% was bound firmly to the membrane after centrifugation in a CsCl density gradient. The plasmid DNA-membrane complex synthesized new plasmid DNA without the addition of exogenous template, enzymes, or other proteins. DNA synthesis appeared to proceed semi-conservatively, was dependent on the four deoxynucleoside triphosphates, partially dependent on ribonucleoside triphosphates, and was sensitive to rifampin, an antibiotic known to inhibit initiation of replication. Novobiocin and nalidixic acid also inhibited synthesis, as did the omission of ATP, N-Ethylmaleimide, an inhibitor of DNA polymerase II and III activity, but not DNA polymerase I activity, also partially inhibited the synthetic reaction, as did chloramphenicol. The plasmid DNA synthetic product was analyzed by alkaline sucrose and dye-CsCl gradient centrifugation, as well as by agarose gel electrophoresis. In each case, the product consisted of parental and intermediate forms of plasmid DNA. Some chromosomal DNA was also synthesized by a contaminating bacterial DNA-membrane complex, but this synthesis was rifampin insensitive and could be separated from plasmid DNA 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. 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]
  2. Conrad S. E., Campbell J. L. Role of plasmid-coded RNA and ribonuclease III in plasmid DNA replication. Cell. 1979 Sep;18(1):61–71. doi: 10.1016/0092-8674(79)90354-4. [DOI] [PubMed] [Google Scholar]
  3. Diaz R., Nordström K., Staudenbauer W. L. Plasmid R1 DNA replication dependent on protein synthesis in cell-free extracts of E. coli. Nature. 1981 Jan 22;289(5795):326–328. doi: 10.1038/289326a0. [DOI] [PubMed] [Google Scholar]
  4. Dworsky P., Schaechter M. Effect of rifampin on the structure and membrane attachment of the nucleoid of Escherichia coli. J Bacteriol. 1973 Dec;116(3):1364–1374. doi: 10.1128/jb.116.3.1364-1374.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gellert M., Mizuuchi K., O'Dea M. H., Nash H. A. DNA gyrase: an enzyme that introduces superhelical turns into DNA. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3872–3876. doi: 10.1073/pnas.73.11.3872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Greene M., Firshein W. Role of deoxyribonucleic acid ligase in a doxyribonucleic acid membrane fraction extracted from pneumococci. J Bacteriol. 1976 May;126(2):777–784. doi: 10.1128/jb.126.2.777-784.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Horowitz S., Doyle R. J., Young F. E., Streips U. N. Selective association of the chromosome with membrane in a stable L-form of Bacillus subtilis. J Bacteriol. 1979 Jun;138(3):915–922. doi: 10.1128/jb.138.3.915-922.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Inuzuka M., Helinski D. R. Replication of antibiotic resistance plasmid R6K DNA in vitro. Biochemistry. 1978 Jun 27;17(13):2567–2573. doi: 10.1021/bi00606a017. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Osborn M. J., Gander J. E., Parisi E., Carson J. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem. 1972 Jun 25;247(12):3962–3972. [PubMed] [Google Scholar]
  11. Robertson W., Watkins D. The 'DNA-membrane complex' of Escherichia coli B/r. Its composition and properties and the fate of nascent and genome DNA during DNA synthesis. Eur J Biochem. 1979 Feb 15;94(1):65–75. doi: 10.1111/j.1432-1033.1979.tb12872.x. [DOI] [PubMed] [Google Scholar]
  12. Sherratt D. J., Helinski D. R. Replication of colicinogenic factor E1 in Escherichia coli. Properties of newly replicated supercoils. Eur J Biochem. 1973 Aug 1;37(1):95–99. doi: 10.1111/j.1432-1033.1973.tb02962.x. [DOI] [PubMed] [Google Scholar]
  13. Sparks R. B., Jr, Helinski D. R. Association of cellular membrane of E. coli minicells with the origins/terminus region of replication of plasmid ColEl DNA. Nature. 1979 Feb 15;277(5697):572–574. doi: 10.1038/277572a0. [DOI] [PubMed] [Google Scholar]
  14. Staudenbauer W. L. Structure and replication of the colicin E1 plasmid. Curr Top Microbiol Immunol. 1978;83:93–156. doi: 10.1007/978-3-642-67087-9_3. [DOI] [PubMed] [Google Scholar]
  15. Strätling W., Knippers R. Deoxyribonucleic acid synthesis in isolated DNA-membrane complexes. J Mol Biol. 1971 Nov 14;61(3):471–487. doi: 10.1016/0022-2836(71)90060-x. [DOI] [PubMed] [Google Scholar]
  16. Thomas C. M. Molecular genetics of broad host range plasmid RK2. Plasmid. 1981 Jan;5(1):10–19. doi: 10.1016/0147-619x(81)90074-3. [DOI] [PubMed] [Google Scholar]
  17. Tremblay G. Y., Daniels M. J., Schaechter M. Isolation of a cell membrane-DNA-nascent RNA complex from bacteria. J Mol Biol. 1969 Feb 28;40(1):65–76. doi: 10.1016/0022-2836(69)90296-4. [DOI] [PubMed] [Google Scholar]
  18. Winston S., Sueoka N. DNA-membrane association is necessary for initiation of chromosomal and plasmid replication in Bacillus subtilis. Proc Natl Acad Sci U S A. 1980 May;77(5):2834–2838. doi: 10.1073/pnas.77.5.2834. [DOI] [PMC free article] [PubMed] [Google Scholar]

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