The DnaA Protein Is Not the Limiting Factor for Initiation of Replication in Escherichia coli
Fig 1
Cell cycle parameters are not changed in cells with a two-fold DnaA concentration.
Exponentially growing cells were analyzed by flow cytometry and cell cycle parameters were calculated. The first (leftmost) panels illustrate replication patterns of wild type (MG1655) cells grown in acetate medium (A), glucose medium (B) and GluCAA medium (C). Cells with chromosomes (black lines) were drawn schematically to show the number of replication forks at different stages of the cell cycle. In acetate grown cells initiation of replication occurred at one origin (red dot) and the replication period (red arrow) was completed within one generation (A). In the glucose and GluCAA grown cells initiation of replication occurred at two and four origins, respectively (B-C). The second and third panels (A-C) show representative DNA histograms of exponentially growing MG1655 and IF72 cells with run-out DNA histograms inserted for (B) and (C). The theoretical curves of the best fit simulation are shown in green. The chromosome equivalents are shown on the abscissa and the number of cells on the ordinate. 10000 cells were measured and one tick on the ordinate represents 100 cells. The calculated values for initiation age, replication period, cell mass and DNA/mass relative to the values for the wild type are shown in the bar histograms in panel four (A-C). The values are an average of three or more experiments and the error bars represent the standard deviation. For a more detailed description of the cell cycle parameters see S2 Fig. The length of the replication period was also determined with quantitative PCR of the oriC/ter ratio for the cells grown in GluCAA medium (S6 Table).