FIG 5.

Effect of temperature on growth rate, number of replicating chromosomes, chromosome copy number, and cell size. (A) Growth rate of S. elongatus at 20, 30, and 40°C. A mid-log-phase SSB-GFP culture at 30°C was inoculated into fresh inorganic medium (hour 0) and then cultured at the indicated temperature under 70-µE m⁻² s⁻¹ illumination. (B) Frequency of cells exhibiting zero (blue), one (red), two (deep blue), or three or more (green) SSB-GFP foci at hour 6 (n ≥ 300 cells per condition). The average number of SSB foci is indicated above the bars. (C) Fluorescence microscopic images showing SYBR Green staining of nucleoids at hour 6. SYBR Green images are shown. Bar, 5 µm. (D) Histograms showing number of chromosomes per cell at hour 6. Number of chromosomes was determined based on micrographs of SYBR Green-stained cells (n ≥ 300 cells per condition). (E) Flow cytometry analysis showing the DNA level (upper) and relative cell volume (lower) at hour 6. DNA level was calculated by intensity of SYTOX Green fluorescence. Blue line, 20°C; black line, 30°C; red line, 40°C; A.U., arbitrary unit. (F) Distribution of cell volume and chromosomal copy number. The blue points represent single cells. The green diamond represents the mean, and the error bar represents the standard deviation (n ≥ 300 cells per condition). The schematic illustrations above the graphs show the average cell volume occupied by one chromosome. (G) Mean cell volume occupied by one chromosome at hour 6. Error bar represents the standard deviation (n ≥ 300 cells per condition). (H) Immunoblot analysis showing DnaA and SSB-GFP protein levels. Proteins extracted from cells at hour 0 and 6 were reacted with the anti-HA antibody (top) and anti-GFP antibody (middle). Coomassie brilliant blue (CBB) staining of the protein samples resolved by SDS-PAGE is shown in the bottom panel as a gel loading control. (I) ChIP-qPCR analysis showing the affinity of DnaA for the oriC region at hour 6. The value indicated is the ratio of percent recovery (oriC/1294). Error bars represent the standard deviation (n = 3). Three independent experiments yielded similar results. Results from one experiment are shown (for panels A to H).