Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1988 Feb;170(2):852–858. doi: 10.1128/jb.170.2.852-858.1988

Coordination of chromosome replication initiation in Escherichia coli: effects of different dnaA alleles.

K Skarstad 1, K von Meyenburg 1, F G Hansen 1, E Boye 1
PMCID: PMC210732  PMID: 2828328

Abstract

The synchrony of initiation of chromosomal replication in single cells was determined in ten different dnaA(Ts) mutants. After inhibiting the initiation of replication but allowing initiated rounds of replication to terminate, we measured the number of fully replicated chromosomes per individual cell by flow cytometry. Synchronous initiation at the several independent origins (oriC) in single rapidly growing cells would give 2'' (n = 0,1,2,3,...) chromosomes per cell, whereas asynchronous initiation was indicated by the presence of a different number of chromosomes. Mutations mapping in the central part of the dnaA gene (dnaA5, dnaA46, dnaA601, dnaA602, and dnaA604) lead to a high degree of asynchrony (class I mutants), whereas mutations mapping in either of the distal parts of the gene (dnaA508, dnaA167, dnaA203, and dnaA204) yielded a low degree of asynchrony at the permissive temperature (class 2 mutants). The dnaA205 mutant exhibited an intermediate degree of asynchrony. Mutants dnaA203 and dnaA204 (promoter distal) differed from the other class 2 mutants (dnaA167, dnaA508; promoter proximal) in that asynchrony increased no more than twofold between 25 and 37 degrees C compared with the more-than-fourfold increase in the latter. The high degree of asynchrony in class 1 mutants was independent of temperature and was not due to insufficient functional DnaA protein, because overproduction of DnaA46 protein did not decrease the asynchrony. The data demonstrate that the DnaA protein has functions in addition to acting positively in the initiation process and negatively as its own repressor, namely in coordinating initiations at all oriC sites within a single cell.

Full text

PDF
852

Selected References

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

  1. Atlung T. Allele-specific suppression of dnaA(Ts) mutations by rpoB mutations in Escherichia coli. Mol Gen Genet. 1984;197(1):125–128. doi: 10.1007/BF00327932. [DOI] [PubMed] [Google Scholar]
  2. Atlung T., Clausen E. S., Hansen F. G. Autoregulation of the dnaA gene of Escherichia coli K12. Mol Gen Genet. 1985;200(3):442–450. doi: 10.1007/BF00425729. [DOI] [PubMed] [Google Scholar]
  3. Atlung T., Løbner-Olesen A., Hansen F. G. Overproduction of DnaA protein stimulates initiation of chromosome and minichromosome replication in Escherichia coli. Mol Gen Genet. 1987 Jan;206(1):51–59. doi: 10.1007/BF00326535. [DOI] [PubMed] [Google Scholar]
  4. Boye E., Steen H. B., Skarstad K. Flow cytometry of bacteria: a promising tool in experimental and clinical microbiology. J Gen Microbiol. 1983 Apr;129(4):973–980. doi: 10.1099/00221287-129-4-973. [DOI] [PubMed] [Google Scholar]
  5. Braun R. E., O'Day K., Wright A. Autoregulation of the DNA replication gene dnaA in E. coli K-12. Cell. 1985 Jan;40(1):159–169. doi: 10.1016/0092-8674(85)90319-8. [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. Donachie W. D. Relationship between cell size and time of initiation of DNA replication. Nature. 1968 Sep 7;219(5158):1077–1079. doi: 10.1038/2191077a0. [DOI] [PubMed] [Google Scholar]
  8. Eberle H. A comparison of [13C]glucose-15NH4Cl with 5-bromouracil as density label in two experiments on sequential DNA replication in Escherichia coli 15T-. J Mol Biol. 1968 Jan 14;31(1):149–152. doi: 10.1016/0022-2836(68)90065-x. [DOI] [PubMed] [Google Scholar]
  9. Fuller R. S., Funnell B. E., Kornberg A. The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites. Cell. 1984 Oct;38(3):889–900. doi: 10.1016/0092-8674(84)90284-8. [DOI] [PubMed] [Google Scholar]
  10. Fuller R. S., Kornberg A. Purified dnaA protein in initiation of replication at the Escherichia coli chromosomal origin of replication. Proc Natl Acad Sci U S A. 1983 Oct;80(19):5817–5821. doi: 10.1073/pnas.80.19.5817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hansen E. B., Atlung T., Hansen F. G., Skovgaard O., von Meyenburg K. Fine structure genetic map and complementation analysis of mutations in the dnaA gene of Escherichia coli. Mol Gen Genet. 1984;196(3):387–396. doi: 10.1007/BF00436184. [DOI] [PubMed] [Google Scholar]
  12. Hansen F. G., Koefoed S., Sørensen L., Atlung T. Titration of DnaA protein by oriC DnaA-boxes increases dnaA gene expression in Escherichia coli. EMBO J. 1987 Jan;6(1):255–258. doi: 10.1002/j.1460-2075.1987.tb04747.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hansen F. G., Rasmussen K. V. Regulation of the dnaA product in Escherichia coli. Mol Gen Genet. 1977 Oct 20;155(2):219–225. doi: 10.1007/BF00393163. [DOI] [PubMed] [Google Scholar]
  14. Helmstetter C. E. DNA synthesis during the division cycle of rapidly growing Escherichia coli B/r. J Mol Biol. 1968 Feb 14;31(3):507–518. doi: 10.1016/0022-2836(68)90424-5. [DOI] [PubMed] [Google Scholar]
  15. Kogoma T., Skarstad K., Boye E., von Meyenburg K., Steen H. B. RecA protein acts at the initiation of stable DNA replication in rnh mutants of Escherichia coli K-12. J Bacteriol. 1985 Aug;163(2):439–444. doi: 10.1128/jb.163.2.439-444.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Koppes L. J., von Meyenburg K. Nonrandom minichromosome replication in Escherichia coli K-12. J Bacteriol. 1987 Jan;169(1):430–433. doi: 10.1128/jb.169.1.430-433.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Leonard A. C., Helmstetter C. E. Cell cycle-specific replication of Escherichia coli minichromosomes. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5101–5105. doi: 10.1073/pnas.83.14.5101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Løbner-Olesen A., Atlung T., Rasmussen K. V. Stability and replication control of Escherichia coli minichromosomes. J Bacteriol. 1987 Jun;169(6):2835–2842. doi: 10.1128/jb.169.6.2835-2842.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Newman C. N., Kubitschek H. E. Variation in periodic replication of the chromosome in Escherichia coli B/rTT. J Mol Biol. 1978 Jun 5;121(4):461–471. doi: 10.1016/0022-2836(78)90394-7. [DOI] [PubMed] [Google Scholar]
  20. Ohmori H., Kimura M., Nagata T., Sakakibara Y. Structural analysis of the dnaA and dnaN genes of Escherichia coli. Gene. 1984 May;28(2):159–170. doi: 10.1016/0378-1119(84)90253-1. [DOI] [PubMed] [Google Scholar]
  21. Pierucci O., Helmstetter C. E., Rickert M., Weinberger M., Leonard A. C. Overexpression of the dnaA gene in Escherichia coli B/r: chromosome and minichromosome replication in the presence of rifampin. J Bacteriol. 1987 May;169(5):1871–1877. doi: 10.1128/jb.169.5.1871-1877.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Skarstad K., Boye E., Steen H. B. Timing of initiation of chromosome replication in individual Escherichia coli cells. EMBO J. 1986 Jul;5(7):1711–1717. doi: 10.1002/j.1460-2075.1986.tb04415.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Skarstad K., Steen H. B., Boye E. Cell cycle parameters of slowly growing Escherichia coli B/r studied by flow cytometry. J Bacteriol. 1983 May;154(2):656–662. doi: 10.1128/jb.154.2.656-662.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Skarstad K., Steen H. B., Boye E. Escherichia coli DNA distributions measured by flow cytometry and compared with theoretical computer simulations. J Bacteriol. 1985 Aug;163(2):661–668. doi: 10.1128/jb.163.2.661-668.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Steen H. B. A microscope-based flow cytophotometer. Histochem J. 1983 Feb;15(2):147–160. doi: 10.1007/BF01042283. [DOI] [PubMed] [Google Scholar]
  26. Steen H. B., Lindmo T. Flow cytometry: a high-resolution instrument for everyone. Science. 1979 Apr 27;204(4391):403–404. doi: 10.1126/science.441727. [DOI] [PubMed] [Google Scholar]
  27. Tippe-Schindler R., Zahn G., Messer W. Control of the initiation of DNA replication in Escherichia coli. I. Negative control of initiation. Mol Gen Genet. 1979 Jan 10;168(2):185–195. doi: 10.1007/BF00431444. [DOI] [PubMed] [Google Scholar]
  28. von Meyenburg K., Boye E., Skarstad K., Koppes L., Kogoma T. Mode of initiation of constitutive stable DNA replication in RNase H-defective mutants of Escherichia coli K-12. J Bacteriol. 1987 Jun;169(6):2650–2658. doi: 10.1128/jb.169.6.2650-2658.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. von Meyenburg K., Hansen F. G., Riise E., Bergmans H. E., Meijer M., Messer W. Origin of replication, oriC, of the Escherichia coli K12 chromosome: genetic mapping and minichromosome replication. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):121–128. doi: 10.1101/sqb.1979.043.01.018. [DOI] [PubMed] [Google Scholar]

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

RESOURCES