Figure 1

DNA gyrase reactivation in stationary-phase cells with low levels of the main chaperones. Cells were grown in LB-MOPS medium at 30°C. Strains used included the following: a) BB7222 (wild type) and BB7224 (ΔrpoH), and b) C600 (wild type) and CAG9310 groEL140 bearing the reporter plasmid pMS01. Strain BB7224 expresses very low levels of the main cellular chaperones, except for GroE, while CAG9310 carries the temperature-sensitive groEL140 mutation. To induce the recovery of the DNA SC level in stationary-phase cells, cell cultures were diluted in pre-warmed LB-MOPS medium. a: 1) Exponentially growing cells, 2) 48 hr stationary-phase cells, and 3) stationary-phase cells diluted 1:10 in LB-MOPS at 30°C and incubated for 5 min. b: 1) Exponentially growing cells, 2) 48 hr stationary-phase cells, 3) stationary-phase cells diluted 1:30 in LB-MOPS at 30°C and incubated for 5 min, and 4) stationary-phase cells diluted 1:30 in LB-MOPS at 43°C and incubated for 5 min. Before dilution in LP-MOPS at 43°C, SP cell cultures were incubated for 15 min at 43°C. Plasmid topoisomers were isolated and separated on 1% agarose gels containing 10 μg/mL chloroquine. Migration proceeded from top to bottom. Topoisomers more supercoiled migrated more rapidly in the gel. Similar results were obtained in at least three independent experiments.