Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1974 Dec;120(3):1387–1400. doi: 10.1128/jb.120.3.1387-1400.1974

R6K Plasmid Replication: Influence of Chromosomal Genotype in Minicell-Producing Strains of Escherichia coli K-12

Francis L Macrina a,1, George G Weatherly a, Roy Curtiss III a
PMCID: PMC245926  PMID: 4612021

Abstract

Alkaline sucrose velocity sedimentation and cesium chloride-ethidium bromide equilibrium centrifugation have been used to determine the number of copies per chromosomal equivalent of the relaxedly replicating R6K plasmid (a conjugative plasmid conferring ampicillin and streptomycin resistance) in two minicell-producing strains of Escherichia coli K-12. In one strain, the average number of covalently closed circular R6K molecules per chromosomal equivalent is 13 in log-phase and 35 in stationary-phase cells. In the other strain, there is an average of six covalently closed circular R6K molecules per chromosomal equivalent in both log- and stationary-phase cells. Selection from this strain of spontaneously occurring mutants resistant to high concentrations of ampicillin has been accomplished and such mutants show a two- to threefold increase in the number of R6K copies per chromosomal equivalent. Relative to the parental strain, mutants display the following properties: (i) elevated streptomycin resistance, (ii) a 10-fold increase in R6K conjugal transfer, (iii) a 10-fold increase in the amount of R6K plasmid deoxyribonucleic acid segregated into minicells, and (iv) a two- to threefold increase in R6K-specified β-lactamase. The mutation(s) responsible for the increase in the number of R6K molecules per chromosomal equivalent is located on the bacterial chromosome. No R6K-linked mutations conferring the above phenotypes have been obtained. The mutations are presumed to be in chromosomal genes which play a role in the regulation of R6K replication in this strain.

Full text

PDF
1387

Selected References

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

  1. Adler H. I., Fisher W. D., Cohen A., Hardigree A. A. MINIATURE escherichia coli CELLS DEFICIENT IN DNA. Proc Natl Acad Sci U S A. 1967 Feb;57(2):321–326. doi: 10.1073/pnas.57.2.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. CURTIS S. R., 3rd CHROMOSOMAL ABERRATIONS ASSOCIATED WITH MUTATIONS TO BACTERIOPHAGE RESISTANCE IN ESCHERICHIA COLI. J Bacteriol. 1965 Jan;89:28–40. doi: 10.1128/jb.89.1.28-40.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clowes R. C. Molecular structure of bacterial plasmids. Bacteriol Rev. 1972 Sep;36(3):361–405. doi: 10.1128/br.36.3.361-405.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Curtiss R., 3rd, Charamella L. J., Stallions D. R., Mays J. A. Parental functions during conjugation in Escherichia coli K-12. Bacteriol Rev. 1968 Dec;32(4 Pt 1):320–348. [PMC free article] [PubMed] [Google Scholar]
  8. Demerec M., Adelberg E. A., Clark A. J., Hartman P. E. A proposal for a uniform nomenclature in bacterial genetics. Genetics. 1966 Jul;54(1):61–76. doi: 10.1093/genetics/54.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fenwick R. G., Jr, Curtiss R., 3rd Conjugal deoxyribonucleic acid replication by Escherichia coli K-12: effect of nalidixic acid. J Bacteriol. 1973 Dec;116(3):1236–1246. doi: 10.1128/jb.116.3.1236-1246.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Frazer A. C., Curtiss R., 3rd Production, properties and utility of bacterial minicells. Curr Top Microbiol Immunol. 1975;69:1–84. doi: 10.1007/978-3-642-50112-8_1. [DOI] [PubMed] [Google Scholar]
  11. Freifelder D. Studies on Escherichia coli sex factors. 3. Covalently closed F'Lac DNA molecules. J Mol Biol. 1968 May 28;34(1):31–38. doi: 10.1016/0022-2836(68)90232-5. [DOI] [PubMed] [Google Scholar]
  12. Goebel W. Replication of the DNA of the colicinogenic factor E 1 (Col E 1 ) at the restrictive temperature in a DNA replication mutant thermosensitive for DNA polymerase. 3. Nat New Biol. 1972 May 17;237(72):67–70. doi: 10.1038/newbio237067a0. [DOI] [PubMed] [Google Scholar]
  13. Goebel W., Schrempf H. Replication of plasmid DNA in temperature-sensitivity DNA replication mutants of Escherichia coli. Biochim Biophys Acta. 1972 Feb 23;262(1):32–41. doi: 10.1016/0005-2787(72)90216-x. [DOI] [PubMed] [Google Scholar]
  14. Goebel W., Schrempf H. Replication of the minicircular DNA of E. coli 15 is dependent on DNA polymerase I but independent of DNA polymerase 3. Biochem Biophys Res Commun. 1972 Oct 17;49(2):591–600. doi: 10.1016/0006-291x(72)90452-4. [DOI] [PubMed] [Google Scholar]
  15. Goebel W. The influence of DNA A and DNA C mutations on the initiation of plasmid DNA replication. Biochem Biophys Res Commun. 1973 Apr 16;51(4):1000–1007. doi: 10.1016/0006-291x(73)90026-0. [DOI] [PubMed] [Google Scholar]
  16. Hedges R. W., Datta N., Coetzee J. N., Dennison S. R factors from Proteus morganii. J Gen Microbiol. 1973 Aug;77(2):249–259. doi: 10.1099/00221287-77-2-249. [DOI] [PubMed] [Google Scholar]
  17. Helinski D. R., Clewell D. B. Circular DNA. Annu Rev Biochem. 1971;40:899–942. doi: 10.1146/annurev.bi.40.070171.004343. [DOI] [PubMed] [Google Scholar]
  18. Helinski D. R. Plasmid determined resistance to antibiotics: molecular properties of R factors. Annu Rev Microbiol. 1973;27:437–470. doi: 10.1146/annurev.mi.27.100173.002253. [DOI] [PubMed] [Google Scholar]
  19. Inselburg J. Segregation into and replication of plasmid deoxyribonucleic acid in chromosomeless segregants of Escherichia coli. J Bacteriol. 1970 Jun;102(3):642–647. doi: 10.1128/jb.102.3.642-647.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kingsbury D. T., Helinski D. R. DNA polymerase as a requirement for the maintenance of the bacterial plasmid colicinogenic factor E1. Biochem Biophys Res Commun. 1970 Dec 24;41(6):1538–1544. doi: 10.1016/0006-291x(70)90562-0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Kingsbury D. T., Sieckmann D. G., Helinski D. R. Temperature-sensitive mutants for the replication of plasmids in Escherichia coli. II. Properties of host and plasmid mutations. Genetics. 1973 May;74(1):1–16. doi: 10.1093/genetics/74.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kontomichalou P., Mitani M., Clowes R. C. Circular R-factor molecules controlling penicillinase synthesis, replicating in Escherichia coli under either relaxed or stringent control. J Bacteriol. 1970 Oct;104(1):34–44. doi: 10.1128/jb.104.1.34-44.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kupersztoch Y. M., Helinski D. R. A catenated DNA molecule as an intermediate in the replication of the resistance transfer factor R6K in Escherichia coli. Biochem Biophys Res Commun. 1973 Oct 15;54(4):1451–1459. doi: 10.1016/0006-291x(73)91149-2. [DOI] [PubMed] [Google Scholar]
  25. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  26. Levy S. B. Physical and functional characteristics of R-factor deoxyribonucleic acid segregated into Escherichia coli minicells. J Bacteriol. 1971 Oct;108(1):300–308. doi: 10.1128/jb.108.1.300-308.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nisioka T., Mitani M., Clowes R. C. Molecular recombination between R-factor deoxyribonucleic acid molecules in Escherichia coli host cells. J Bacteriol. 1970 Jul;103(1):166–177. doi: 10.1128/jb.103.1.166-177.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nordström K. Increased resistance to several antibiotics by one mutation in an R-factor, R 1a. J Gen Microbiol. 1971 May;66(2):205–214. doi: 10.1099/00221287-66-2-205. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Novick R. P. Extrachromosomal inheritance in bacteria. Bacteriol Rev. 1969 Jun;33(2):210–263. doi: 10.1128/br.33.2.210-263.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Odakura Y., Tanaka T., Hashimoto H., Mitsuhashi S. Mutation of R factors capable of specifying hypersynthesis of penicillinase. Antimicrob Agents Chemother. 1973 Mar;3(3):315–324. doi: 10.1128/aac.3.3.315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Roozen K. J., Fenwick R. G., Jr, Curtiss R., 3rd Synthesis of ribonucleic acid and protein in plasmid-containing minicells of Escherichia coli K-12. J Bacteriol. 1971 Jul;107(1):21–33. doi: 10.1128/jb.107.1.21-33.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rownd R. Replication of a bacterial episome under relaxed control. J Mol Biol. 1969 Sep 28;44(3):387–402. doi: 10.1016/0022-2836(69)90368-4. [DOI] [PubMed] [Google Scholar]
  34. Rubin F. A., Smith D. H. Characterization of R factor beta-lactamases by the acidimetric method. Antimicrob Agents Chemother. 1973 Jan;3(1):68–73. doi: 10.1128/aac.3.1.68. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sheehy R. J., Perry A., Allison D. P., Curtiss R., 3rd Molecular nature of R-factor deoxyribonucleic acid isolated from Salmonella typhimurium minicells. J Bacteriol. 1973 Jun;114(3):1328–1335. doi: 10.1128/jb.114.3.1328-1335.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Taylor A. L., Trotter C. D. Linkage map of Escherichia coli strain K-12. Bacteriol Rev. 1972 Dec;36(4):504–524. doi: 10.1128/br.36.4.504-524.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Veltkamp E., Nijkamp H. J. The role of DNA polymerase I, II and 3 in the replication of the bacteriocinogenic factor Clo DF 13. Mol Gen Genet. 1973 Sep 27;125(4):329–340. doi: 10.1007/BF00276588. [DOI] [PubMed] [Google Scholar]
  38. Watanabe T. Infectious drug resistance in bacteria. Curr Top Microbiol Immunol. 1971;56:43–98. doi: 10.1007/978-3-642-65241-7_3. [DOI] [PubMed] [Google Scholar]
  39. Yamagata H., Uchida H. Chromosomal mutations affecting the stability of sex-factors in Escherichia coli. J Mol Biol. 1972 Jan 28;63(2):281–294. doi: 10.1016/0022-2836(72)90375-0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES