FIG 6.

The new protocol for T starvation to ensure that dnaA(Ts) cells do not initiate or harbor preexisting forks. The strains are as follows: thyA mutant, KKW58; thyA dnaA601(Ts) mutant, SRK291. (A) TLD kinetics of the thyA dnaA601(Ts) mutant versus the thyA DnaA⁺ parent, both grown in the presence of thymidine at 28°C to an OD of 0.1 and shifted to 42°C for 2 h before being switched to the –dT medium and with continued incubation at 42°C. As a control for dnaA(Ts) viability at the nonpermissive temperature, half of the thyA dnaA601(Ts) culture was switched to fresh +dT medium and also incubated at 42°C. (B) The “awakening” protocol of thymine starvation: direct dilution of the overnight (15- to 18-h) culture into fresh medium supplemented with various concentrations of dT, from 0 to 5 µg/ml (the values marking the curves), at time zero and with shaking at 37°C. (C) ori/ter ratios in 48-h MOPS-CAA +dT cultures. Standard normalization, normalization to the average of 24-h stationary cultures in LB and cultures treated with chloramphenicol for 3 h; plasmid normalization, normalization to the “loading control” plasmid pSRK-OD that carries one copy each of the origin and terminus probes. The horizontal dotted line indicates the expected ori/ter ratio of 1 in a perfectly aligned chromosome. (D) TLD kinetics of the thyA dnaA(Ts) mutant versus the thyA single mutant, grown to saturation (for 48 h) in the presence of thymidine at 28°C, washed and diluted into thymineless medium prewarmed and with shaking at 42°C. As a control for dnaA(Ts) viability at the nonpermissive temperature, a part of the 48-h dnaA thyA culture was diluted into fresh +dT medium, also prewarmed and with shaking at 42°C. (E) Evolution of the ori/ter ratios in the 48-h +dT cultures of thyA and thyA dnaA(Ts) mutants at 28°C after they were diluted into the same –dT medium and with incubation at 42°C (“awakening protocol”). (F and G) Evolution of the origin (F) and terminus (G) amounts in the same cultures and conditions as described for panel E. (H) Schematic illustration of the current findings. Left column, degradation of the origin and terminus in a nonreplicating chromosome during T starvation; right column, the published findings, according to which only the chromosomal regions that initiate during T starvation are susceptible to an incomplete loss.