Fig 6.

Effects on DNA supercoiling of substituting the gyrB(Ts) allele of strain SB383 [topA rnhA gyrB(Ts)] for a gyrB⁺ one. (a) NF88 (RFM475 gyrB⁺) and NF98 (SB383 gyrB⁺) cells were grown in LB with IPTG (1 mM) at 37°C to an OD₆₀₀ of 0.6, at which time spectinomycin (250 μg/ml) was added, and 15 min later the cells were transferred to 28°C for 30 min. pPH1243 DNA was extracted, and the topoisomers were resolved following electrophoresis in an agarose gel containing 7.5 μg/ml of chloroquine. At this chloroquine concentration, the relaxed topoisomers migrate more rapidly than the negatively supercoiled ones, except the hypernegatively supercoiled topoisomers, which also migrate rapidly. (b) SB383 and NF98 cells were grown in LB at 37°C to an OD₆₀₀ of 0.6, at which time pPH1243 DNA was extracted and the topoisomers were resolved as described above. The gels were hybridized with a probe to detect pPH1243 topoisomers. [--] and [rel] indicate hypernegatively supercoiled and extensively relaxed DNA, respectively.