Abstract
It is widely accepted that the initiation mass of Escherichia coli is constant and independent of growth rate, and therefore is an important parameter in the regulation of initiation of DNA replication. We have used flow cytometry to measure the initiation mass of E. coli K-12 cells as a function of growth rate. The average initiation mass was determined by two methods: (i) from a mathematical relationship between average cell mass, cell age at initiation and number of origins present in the cells, and (ii) directly from the cell mass distribution. The light scattering signal from individual cells and the protein content per cell were employed as measures of cell mass. The initiation mass was found to increase monotonically with decreasing growth rate, being 1.6 times higher (light scattering) or 2.1 times higher (protein content) at 0.3 than at 2.5 doublings per hour. We conclude that the initiation mass is dependent on growth rate. This finding indicates that the control for timing of initiation is not governed by a direct connection between mass accumulation and the molecule(s) determining initiation of replication.
Full text
PDFImages in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Allman R., Schjerven T., Boye E. Cell cycle parameters of Escherichia coli K-12. J Bacteriol. 1991 Dec;173(24):7970–7974. doi: 10.1128/jb.173.24.7970-7974.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Baker T. A., Kornberg A. Transcriptional activation of initiation of replication from the E. coli chromosomal origin: an RNA-DNA hybrid near oriC. Cell. 1988 Oct 7;55(1):113–123. doi: 10.1016/0092-8674(88)90014-1. [DOI] [PubMed] [Google Scholar]
- Baker T. A., Sekimizu K., Funnell B. E., Kornberg A. Extensive unwinding of the plasmid template during staged enzymatic initiation of DNA replication from the origin of the Escherichia coli chromosome. Cell. 1986 Apr 11;45(1):53–64. doi: 10.1016/0092-8674(86)90537-4. [DOI] [PubMed] [Google Scholar]
- Boye E., Løbner-Olesen A. Bacterial growth control studied by flow cytometry. Res Microbiol. 1991 Feb-Apr;142(2-3):131–135. doi: 10.1016/0923-2508(91)90020-b. [DOI] [PubMed] [Google Scholar]
- 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]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
- Bramhill D., Kornberg A. Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome. Cell. 1988 Mar 11;52(5):743–755. doi: 10.1016/0092-8674(88)90412-6. [DOI] [PubMed] [Google Scholar]
- Bremer H., Churchward G. Deoxyribonucleic acid synthesis after inhibition of initiation of rounds of replication in Escherichia coli B/r. J Bacteriol. 1977 May;130(2):692–697. doi: 10.1128/jb.130.2.692-697.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chiaramello A. E., Zyskind J. W. Coupling of DNA replication to growth rate in Escherichia coli: a possible role for guanosine tetraphosphate. J Bacteriol. 1990 Apr;172(4):2013–2019. doi: 10.1128/jb.172.4.2013-2019.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Churchward G., Estiva E., Bremer H. Growth rate-dependent control of chromosome replication initiation in Escherichia coli. J Bacteriol. 1981 Mar;145(3):1232–1238. doi: 10.1128/jb.145.3.1232-1238.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Cooper S. Leucine uptake and protein synthesis are exponential during the division cycle of Escherichia coli B/r. J Bacteriol. 1988 Jan;170(1):436–438. doi: 10.1128/jb.170.1.436-438.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Donachie W. D. The cell cycle of Escherichia coli. Annu Rev Microbiol. 1993;47:199–230. doi: 10.1146/annurev.mi.47.100193.001215. [DOI] [PubMed] [Google Scholar]
- Fantes P. A., Grant W. D., Pritchard R. H., Sudbery P. E., Wheals A. E. The regulation of cell size and the control of mitosis. J Theor Biol. 1975 Mar;50(1):213–244. doi: 10.1016/0022-5193(75)90034-x. [DOI] [PubMed] [Google Scholar]
- 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]
- Funnell B. E., Baker T. A., Kornberg A. In vitro assembly of a prepriming complex at the origin of the Escherichia coli chromosome. J Biol Chem. 1987 Jul 25;262(21):10327–10334. [PubMed] [Google Scholar]
- Hansen F. G., Christensen B. B., Atlung T. The initiator titration model: computer simulation of chromosome and minichromosome control. Res Microbiol. 1991 Feb-Apr;142(2-3):161–167. doi: 10.1016/0923-2508(91)90025-6. [DOI] [PubMed] [Google Scholar]
- Helmstetter C., Cooper S., Pierucci O., Revelas E. On the bacterial life sequence. Cold Spring Harb Symp Quant Biol. 1968;33:809–822. doi: 10.1101/sqb.1968.033.01.093. [DOI] [PubMed] [Google Scholar]
- Hernandez V. J., Bremer H. Characterization of RNA and DNA synthesis in Escherichia coli strains devoid of ppGpp. J Biol Chem. 1993 May 25;268(15):10851–10862. [PubMed] [Google Scholar]
- Koppes L. H., Woldringh C. L., Nanninga N. Size variations and correlation of different cell cycle events in slow-growing Escherichia coli. J Bacteriol. 1978 May;134(2):423–433. doi: 10.1128/jb.134.2.423-433.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- LeBowitz J. H., McMacken R. The Escherichia coli dnaB replication protein is a DNA helicase. J Biol Chem. 1986 Apr 5;261(10):4738–4748. [PubMed] [Google Scholar]
- Mahaffy J. M., Zyskind J. W. A model for the initiation of replication in Escherichia coli. J Theor Biol. 1989 Oct 23;140(4):453–477. doi: 10.1016/s0022-5193(89)80109-2. [DOI] [PubMed] [Google Scholar]
- Nurse P. The Florey Lecture, 1990. How is the cell division cycle regulated? Philos Trans R Soc Lond B Biol Sci. 1991 Jun 29;332(1264):271–276. doi: 10.1098/rstb.1991.0055. [DOI] [PubMed] [Google Scholar]
- Ogawa T., Okazaki T. Concurrent transcription from the gid and mioC promoters activates replication of an Escherichia coli minichromosome. Mol Gen Genet. 1991 Nov;230(1-2):193–200. doi: 10.1007/BF00290668. [DOI] [PubMed] [Google Scholar]
- Ryals J., Little R., Bremer H. Control of rRNA and tRNA syntheses in Escherichia coli by guanosine tetraphosphate. J Bacteriol. 1982 Sep;151(3):1261–1268. doi: 10.1128/jb.151.3.1261-1268.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- SCHAECHTER M., MAALOE O., KJELDGAARD N. O. Dependency on medium and temperature of cell size and chemical composition during balanced grown of Salmonella typhimurium. J Gen Microbiol. 1958 Dec;19(3):592–606. doi: 10.1099/00221287-19-3-592. [DOI] [PubMed] [Google Scholar]
- Sekimizu K., Bramhill D., Kornberg A. Sequential early stages in the in vitro initiation of replication at the origin of the Escherichia coli chromosome. J Biol Chem. 1988 May 25;263(15):7124–7130. [PubMed] [Google Scholar]
- Skarstad K., Baker T. A., Kornberg A. Strand separation required for initiation of replication at the chromosomal origin of E.coli is facilitated by a distant RNA--DNA hybrid. EMBO J. 1990 Jul;9(7):2341–2348. doi: 10.1002/j.1460-2075.1990.tb07406.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]