Molecular and General Genetics MGG

, Volume 218, Issue 1, pp 50–56 | Cite as

Initiation of DNA replication in Escherichia coli after overproduction of the DnaA protein

  • Kirsten Skarstad
  • Anders Løbner-Olesen
  • Tove Atlung
  • Kaspar von Meyenburg
  • Erik Boye
Article

Summary

Flow cytometry was used to study initiation of DNA replication in Escherichia coli K12 after induced expression of a plasmid-borne dnaA+ gene. When the dnaA gene was induced from either the plac or the λpL promoter initiation was stimulated, as evidenced by an increase in the number of origins and in DNA content per mass unit. During prolonged growth under inducing conditions the origin and DNA content per mass unit were stabilized at levels significantly higher than those found before induction or in similarly treated control cells. The largest increase was observed when using the stronger promoter λpL compared to plac. Synchrony of initiation was reasonably well maintained with elevated DnaA protein concentrations, indicating that simultaneous initiation of all origins was still preferred under these conditions. A reduced rate of replication fork movement was found in the presence of rifampin when the DnaA protein was overproduced. We conclude that increased synthesis levels or increased concentrations of the DnaA protein stimulate initiation of DNA replication. The data suggest that the DnaA protein may be the limiting factor for initiation under normal physiological conditions.

Key words

Escherichia coli Initiation of DNA replication DnaA protein Flow cytometry 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Atlung T, Clausen ES, Hansen FG (1985) Autoregulation of the dnaA gene of Escherichia coli K-12. Mol Gen Genet 200:442–450Google Scholar
  2. Atlung T, Løbner-Olesen A, Hansen FG (1987) Overproduction of DnaA protein stimulates initiation of chromosome and minichromosome replication in Escherichia coli. Mol Gen Genet 206:51–59Google Scholar
  3. Boye E, Steen HB, Skarstad K (1983) Flow cytometry of bacteria: a promising tool in experimental and clinical microbiology. J Gen Microbiol 129:973–980Google Scholar
  4. Boye E, Løbner-Olesen A, Skarstad K (1988) Timing of chromosomal replication in Escherichia coli. Biochim Biophys Acta 951:359–364Google Scholar
  5. Braun RE, O'Day K, Wright A (1985) Autoregulation of the DNA replication gene dnaA in Escherichia coli K-12. Cell 40:159–169Google Scholar
  6. Churchward G, Holmans P, Bremer H (1983) Increased expression of the dnaA gene has no effect on DNA replication in a dnaA + strain of Escherichia coli. Mol Gen Genet 192:506–508Google Scholar
  7. Clark DJ, Maaløe O (1967) DNA replication and the division cycle in Escherichia coli. J Mol Biol 23:99–112Google Scholar
  8. Deuschle U, Kammerer W, Gentz R, Bujard H (1986) Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures. EMBO J 5:2987–2994Google Scholar
  9. Donachie WD (1968) Relationship between cell size and time of initiation of DNA replication. Nature 219:1077–1079Google Scholar
  10. Fuller RS, Kornberg A (1983) Purfied dnaA protein in initiation of DNA replication of the origin of Escherichia coli chromosome. Proc Natl Acad Sci USA 80:5817–5821Google Scholar
  11. Fuller RS, Funnell BE, Kornberg A (1984) The dnaA complex with the E. coli chromosomal replication origin (oriC) and other DNA sites. Cell 38:889–900Google Scholar
  12. Hansen FG, von Meyenburg K (1979) Characterization of the dnaA, gyrB and other genes in the dnaA region of the Escherichia coli chromosome on specialized transducing phages λtna. Mol Gen Genet 175:135–144Google Scholar
  13. Løbner-Olesen A, Skarstad K, Hansen FG, von Myenburg K, Boye E (1989) The DnaA protein determines the initiation mass of Escherichia coli K-12. Cell, in pressGoogle Scholar
  14. von Meyenburg K, Hansen FG (1987) Regulation of chromosome replication. In: Neidhart FG, Ingraham JL, Low KB, Magasanik B, Schaechter M, Umbarger HE (eds) Escherichia coli and Salmonella typhimurium: Cellular and molecular biology. Am Soc Microbiol. Washington DC, pp 1555–1577Google Scholar
  15. von Meyenburg K, Boye E, Skarstad K, Koppes L, Kogoma T (1987) Mode of initiation of constitutive stable DNA replication in RNaseH-deficient mutants of Escherichia coli K-12. J Bacteriol 169:2650–2658Google Scholar
  16. Pierucci O, Helmstetter CE, Rickert M, Weinberger M, Leonard AC (1987) Overexpression of the dnaA gene in Escherichia coli B/r: Chromosome and minichromosome replication in the presence of rifampicin. J Bacteriol 169:1871–1877Google Scholar
  17. Pritchard RH, Barth PT, Collins J (1969) Control of DNA synthesis in bacteria. Symp Soc Gen Microbiol 19:263–297Google Scholar
  18. Skarstad K, Boye E (1988) Perturbed chromosomal replication in recA mutants of Escherichia coli. J Bacteriol 170:2549–2554Google Scholar
  19. Skarstad K, Steen HB, Boye E (1983) Cell cycle parameters of slowly growing Escherichia coli B/r studied by flow cytometry. J Bacteriol 154:656–662Google Scholar
  20. Skarstad K, Boye E, Steen HB (1986) Timing of initiation of chromosome replication in individual Escherichia coli cells. EMBO J 5:1711–1717Google Scholar
  21. Skarstad K, von Meyenburg K, Hansen FG, Boye E (1988) Coordination of chromosome replication initiation in Escherichia coli: Effects of different dnaA alleles. J Bacteriol 170:852–858Google Scholar
  22. Sompayrac L, Maaløe O (1973) Autorepressor model for control of DNA replication. Nature New Biol 241:133–135Google Scholar
  23. Steen HB, Lindmo T (1979) Flow cytometry: a high resolution instrument for everyone. Science 204:403–404Google Scholar
  24. Xu Y-C, Bremer H (1988) Chromosome replication in Escherichia coli induced by oversupply of DnaA. Mol Gen Genet 211:138–142Google Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • Kirsten Skarstad
    • 1
  • Anders Løbner-Olesen
    • 1
    • 2
  • Tove Atlung
    • 2
  • Kaspar von Meyenburg
    • 2
  • Erik Boye
    • 1
  1. 1.Department of Biophysics, Institute of Cancer ResearchThe Norwegian Radium HospitalOslo 3Norway
  2. 2.Department of MicrobiologyThe Technical University of DenmarkLyngbyDenmark
  3. 3.Department of BiochemistryStanford University School of MedicineStanfordUSA
  4. 4.Ciba-Geigy AGBaselSwitzerland

Personalised recommendations