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. 1976 Feb;125(2):635–642. doi: 10.1128/jb.125.2.635-642.1976

Isolation and characterization of Escherichia coli chromosomal mutants affecting plasmid copy number.

D E Cress, B C Kline
PMCID: PMC236125  PMID: 1107327

Abstract

We have isolated chromosomal mutants of an Escherchia coli K-12 strain that maintain higher levels of an F' plasmid. The mutants are designated as plasmid copy number (pcn) mutants. They were detected by selecting for increased lactose fermentation in bacteria deleted for the lac operon but harboring an F'lacI,P pro+ plasmid. When examined for the amount of F' plasmid deoxyribonucleic acid (DNA) by the dye-CsCl isopycnic technique, the mutants show two to seven times as much covalently closed, circular (CCC) DNA as does the parental strain. The increased plasmid level in one mutant strain (pcn-24) was confirmed by DNA-DNA hybridization; however, this latter technique indicated about a twofold lower increase when compared with the increase measured for pcn-24 by the dye-CsCl technique. In mutant pcn-24 the increased amount of F' DNA reflects a proportional increase in monomeric-size plasmid molecules because oligomeric forms are not found. Also, in mutant pcn-24 the extra CCC plasmid copies do not seem to be randomly distributed throughout the cell's cytoplasm but appear complexed in situ with their host's folded chromosome. In all pcn mutants examined to date, the classical sex factor F is maintained at normal levels, whereas the viral plasmid Pl CM is maintained at two to three times the normal level. In all 17 pcn mutants isolated, the pcn mutation maps on the chromosome and not on the plasmid. Finally, the absolute amount of CCC F' DNA detectable in lysates of the six different pcn mutants examined decreased 50 to 90% upon incubation of the lysate at 37 C. In contrast, no loss of CCC DNA occurs when lysates of the parental F' strain are incubated at 37 C.

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Selected References

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  1. Bazaral M., Helinski D. R. Circular DNA forms of colicinogenic factors E1, E2 and E3 from Escherichia coli. J Mol Biol. 1968 Sep 14;36(2):185–194. doi: 10.1016/0022-2836(68)90374-4. [DOI] [PubMed] [Google Scholar]
  2. Bazaral M., Helinski D. R. Replication of a bacterial plasmid and an episome in Escherichia coli. Biochemistry. 1970 Jan 20;9(2):399–406. doi: 10.1021/bi00804a029. [DOI] [PubMed] [Google Scholar]
  3. Brenner D. J., Fanning G. R., Johnson K. E., Citarella R. V., Falkow S. Polynucleotide sequence relationships among members of Enterobacteriaceae. J Bacteriol. 1969 May;98(2):637–650. doi: 10.1128/jb.98.2.637-650.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Collins J., Pritchard R. H. Relationship between chromosome replication and F'lac episome replication in Escherichia coli. J Mol Biol. 1973 Jun 25;78(1):143–155. doi: 10.1016/0022-2836(73)90434-8. [DOI] [PubMed] [Google Scholar]
  6. Cooper S., Helmstetter C. E. Chromosome replication and the division cycle of Escherichia coli B/r. J Mol Biol. 1968 Feb 14;31(3):519–540. doi: 10.1016/0022-2836(68)90425-7. [DOI] [PubMed] [Google Scholar]
  7. Crosa J. H., Brenner D. J., Falkow S. Use of a single-strand specific nuclease for analysis of bacterial and plasmid deoxyribonucleic acid homo- and heteroduplexes. J Bacteriol. 1973 Sep;115(3):904–911. doi: 10.1128/jb.115.3.904-911.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Curtiss R., 3rd, Caro L. G., Allison D. P., Stallions D. R. Early stages of conjugation in Escherichia coli. J Bacteriol. 1969 Nov;100(2):1091–1104. doi: 10.1128/jb.100.2.1091-1104.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cuzin F., Jacob F. Mutations de l'épisome F d'Escherichia coli K 12. II. Mutants à réplication thermosensible. Ann Inst Pasteur (Paris) 1967 Apr;112(4):397–418. [PubMed] [Google Scholar]
  10. Frame R., Bishop J. O. The number of sex-factors per chromosome in Escherichia coli. Biochem J. 1971 Jan;121(1):93–103. doi: 10.1042/bj1210093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hathaway B. G., Bergquist P. L. Temperature-sensitive mutations affecting the replication of F-prime factors in Escherichia coli K 12. Mol Gen Genet. 1973 Dec 31;127(4):297–306. doi: 10.1007/BF00267100. [DOI] [PubMed] [Google Scholar]
  12. KAHN P., HELINSKI D. R. RELATIONSHIP BETWEEN COLICINOGENIC FACTORS E1 AND V AND AN F FACTOR IN ESCHERICHIA COLI. J Bacteriol. 1964 Dec;88:1573–1579. doi: 10.1128/jb.88.6.1573-1579.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kingsbury D. T., Helinski D. R. Temperature-sensitive mutants for the replication of plasmids in Escherichia coli. I. Isolation and specificity of host and plasmid mutations. Genetics. 1973 May;74(1):17–31. doi: 10.1093/genetics/74.1.17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kline B. C., Helinski D. R. F 1 sex factor of Escherichia coli. Size and purification in the form of a strand-specific relaxation complex of supercoiled deoxyribonucleic acid and protein. Biochemistry. 1971 Dec 21;10(26):4975–4980. doi: 10.1021/bi00802a022. [DOI] [PubMed] [Google Scholar]
  15. Kline B. C. Mechanism and biosynthetic requirements for F plasmid replication in Escherichia coli. Biochemistry. 1974 Jan 1;13(1):139–146. doi: 10.1021/bi00698a022. [DOI] [PubMed] [Google Scholar]
  16. Kline B. C., Miller J. R. Detection of nonintegrated plasmid deoxyribonucleic acid in the folded chromosome of Escherichia coli: physiochemical approach to studying the unit of segregation. J Bacteriol. 1975 Jan;121(1):165–172. doi: 10.1128/jb.121.1.165-172.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kline B. C. Role of DNA transcription in the initiation of Escherichia coli sex factor (F) DNA replication. Biochem Biophys Res Commun. 1973 Jan 23;50(2):280–288. doi: 10.1016/0006-291x(73)90837-1. [DOI] [PubMed] [Google Scholar]
  18. Kool A. J., Nijkamp H. J. Isolation and characterization of a copy mutant of the bacteriocinogenic plasmid Clo DF13. J Bacteriol. 1974 Nov;120(2):569–578. doi: 10.1128/jb.120.2.569-578.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Macrina F. L., Weatherly G. G., Curtiss R., 3rd R6K plasmid replication: influence of chromosomal genotype in minicell-producing strains of Escherichia coli K-12. J Bacteriol. 1974 Dec;120(3):1387–1400. doi: 10.1128/jb.120.3.1387-1400.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Morris C. F., Hashimoto H., Mickel S., Rownd R. Round of replication mutant of a drug resistance factor. J Bacteriol. 1974 Jun;118(3):855–866. doi: 10.1128/jb.118.3.855-866.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nordström K., Ingram L. C., Lundbäck A. Mutations in R factors of Escherichia coli causing an increased number of R-factor copies per chromosome. J Bacteriol. 1972 May;110(2):562–569. doi: 10.1128/jb.110.2.562-569.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pritchard R. H., Chandler M. G., Collins J. Independence of F replication and chromosome replication in Escherichia coli. Mol Gen Genet. 1975;138(2):143–155. doi: 10.1007/BF02428118. [DOI] [PubMed] [Google Scholar]
  23. Radloff R., Bauer W., Vinograd J. A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc Natl Acad Sci U S A. 1967 May;57(5):1514–1521. doi: 10.1073/pnas.57.5.1514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rosner J. L. Formation, induction, and curing of bacteriophage P1 lysogens. Virology. 1972 Jun;48(3):679–689. doi: 10.1016/0042-6822(72)90152-3. [DOI] [PubMed] [Google Scholar]
  25. San Blas F., Thompson R., Broda P. An Escherichia coli K12 mutant apparently carrying two autonomous F-prime factors. Mol Gen Genet. 1974 May 21;130(2):153–163. doi: 10.1007/BF00269086. [DOI] [PubMed] [Google Scholar]
  26. Sharp P. A., Hsu M. T., Otsubo E., Davidson N. Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. I. Structure of F-prime factors. J Mol Biol. 1972 Nov 14;71(2):471–497. doi: 10.1016/0022-2836(72)90363-4. [DOI] [PubMed] [Google Scholar]
  27. Sutton W. D. A crude nuclease preparation suitable for use in DNA reassociation experiments. Biochim Biophys Acta. 1971 Jul 29;240(4):522–531. doi: 10.1016/0005-2787(71)90709-x. [DOI] [PubMed] [Google Scholar]
  28. Worcel A., Burgi E. On the structure of the folded chromosome of Escherichia coli. J Mol Biol. 1972 Nov 14;71(2):127–147. doi: 10.1016/0022-2836(72)90342-7. [DOI] [PubMed] [Google Scholar]
  29. Worcel A., Burgi E. Properties of a membrane-attached form of the folded chromosome of Escherichia coli. J Mol Biol. 1974 Jan 5;82(1):91–105. doi: 10.1016/0022-2836(74)90576-2. [DOI] [PubMed] [Google Scholar]

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