The eclipse period of Escherichia coli - PubMed (original) (raw)
The eclipse period of Escherichia coli
U von Freiesleben et al. EMBO J. 2000.
Abstract
The minimal time between successive initiations on the same origin (the eclipse) in Escherichia coli was determined to be approximately 25-30 min. An inverse relationship was found between the length of the eclipse and the amount of Dam methyltransferase in the cell, indicating that the eclipse corresponds to the period of origin hemimethylation. The SeqA protein was absolutely required for the eclipse, and DnaA titration studies suggested that the SeqA protein prevented the binding of multiple DnaA molecules on oriC (initial complex formation). No correlation between the amount of SeqA and eclipse length was revealed, but increased SeqA levels affected chromosome partitioning and/or cell division. This was corroborated further by an aberrant nucleoid distribution in SeqA-deficient cells. We suggest that the SeqA protein's role in maintaining the eclipse is tied to a function in chromosome organization.
Figures
Fig. 1. Reinitiation in SeqA-deficient cells. Cells of strain CM742 (A) or CM742_seqA_ (ALO1417; B) were grown exponentially at 30°C. At time T = –90, cultures were shifted to 42°C, kept at this non-permissive temperature for 90 min and at time T = 0 shifted back to 30°C. At the times indicated, samples of the cultures were taken and treated with rifampicin and cephalexin for 4 h prior to flow cytometric analysis. The numbers of origins per cell were determined as described in Materials and methods. The small panels on the right hand side of the figure show selected histograms.
Fig. 2. Controlled SeqA expression. Strains CM742 and CM742 containing plasmid pMAK7 (p_lac_-seqA) were grown exponentially at 30°C in the presence of the indicated IPTG concentration. Samples were taken for western blot analysis (Materials and methods) using a polyclonal antibody towards the SeqA protein.
Fig. 3. Excess SeqA protein does not alter the eclipse. Cells of strain CM742 (A; redrawn from Figure 1) or CM742 containing plasmid pMAK7 (B and C) were grown exponentially at 30°C and subsequently treated as described in the legend of Figure 1. CM742/pMAK7 cells were grown in the presence of 0.05 (B) or 0.1 mM IPTG (C).
Fig. 4. Dam methylation and the eclipse. Cultures of CM742 (A; redrawn from Figure 1), CM742 containing plasmid pdam118 (ALO1472; B) and CM742 aroK17::cam, and containing plasmid pMS2 (ALO1480; C) were grown exponentially at 30°C and subsequently treated as described in the legend of Figure 1.
Fig. 5. DnaA protein content. The strains listed were grown as described in the legend of Figure 1. Samples for western blot analysis were taken during balanced growth at 30°C or following 90 min incubation at the non-permissive temperature (42°C). Following blotting, the filter was probed with a polyclonal DnaA antibody.
Fig. 6. Nucleoid distribution in SeqA-deficient cells. Exponentially growing ALO1266 (wt) or ALO1393 (Δ_seqA_) cells were incubated for 15 min with 300 µg/ml chloramphenicol to condense the nucleoids. In order to form filaments, cells were incubated with 20 µg/ml cephalexin for approximately three mass doublings prior to condensing the nucleoids. Cells were fixed and stained prior to microscopic analysis as described in Materials and methods.
References
- Arraj J.A., Wu,T.-H. and Marinus,M.G. (1990) Expression of a DNA methylation (dam) gene in Escherichia coli. Curr. Microbiol., 20, 133–136.
- Atlung T., Clausen,E. and Hansen,F.G. (1985) Autoregulation of the dnaA gene of Escherichia coli. Mol. Gen. Genet., 200, 442–450. - PubMed
- Begg K.J. and Donachie,W.D. (1991) Experiments on chromosome separation and positioning in Escherichia coli. New Biol., 3, 475–486. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Molecular Biology Databases