FIG 3.

Chromosome replication in HA-DnaA^WT and HA-DnaA^R328H cells under illumination. (A) To visualize replicating chromosomes by fluorescence microscopy, GFP-tagged SSB protein was expressed in DnaA^WT and DnaA^R328H cells. Mid-log-phase cultures were inoculated into fresh inorganic medium (hour 0) and cultured under illumination. Growth curves of DnaA^WT (black) and DnaA^R328H (red) cells are shown. (B) Fluorescence microscopic images showing replicating chromosomes in DnaA^WT and DnaA^R328H cells. In the upper panel, SSB-GFP was observed in living DnaA^WT and DnaA^R328H cells at hour 6. In the lower panel, newly synthesized DNA was visualized by immunofluorescence microscopy with the anti-BrdU antibody. Cells were labeled with BrdU for 1 h (hour 5 to 6) and then fixed for immunofluorescence staining. The phase-contrast and immunofluorescence images are merged. Bar, 5 µm. (C) Frequencies of cells exhibiting zero (blue), one (red), two (deep blue), or three or more (green) SSB-GFP foci in DnaA^WT and DnaA^R328H cultures at hour 6 (n ≥ 300 cells for each strain). The average number of SSB foci is indicated above the bars. (D) Histograms showing number of chromosomes per cell in DnaA^WT and DnaA^R328H cultures at hour 6. The number of chromosomes was determined based on micrographs of SYBR Green-stained cells. (E) Distribution of DnaA^WT and DnaA^R328H cell volumes at hour 6. Orange points represent volumes of single cells (n ≥ 300 cells for each strain). The black diamond (DnaA^WT) and circle (DnaA^R328H) represent the means, and the error bars represent the standard deviations (n ≥ 300 cells for each strain). (F) Distribution of DnaA^WT and DnaA^R328H cell volumes and chromosomal numbers at hour 6. Blue points represent single cells. The green diamond (DnaA^WT) and circle (DnaA^R328H) represent the means, and the error bars represent the standard deviations (n ≥ 300 cells for each strain). (G) Mean cell volume occupied by one chromosome in DnaA^WT and DnaA^R328H cells at hour 6. The black diamond (DnaA^WT) and circle (DnaA^R328H) represent the means, and error bars represent the standard deviations (n ≥ 300 cells for each strain).