FIG. 1.

Temperature effects on DNA supercoiling in vitro or in vivo. A. Supercoiling assays were assembled with relaxed pMP1000 plasmid DNA and either 1 U (lanes 1, 2, 5, 6, 9, and 10) or 4 U (lanes 3, 4, 7, 8, 11, and 12) of DNA gyrase containing GyrB652 subunits (odd-numbered lanes) or WT GyrB (even-numbered lanes). After 15 min of incubation at 30°C (lanes 1 to 4), 37°C (lanes 5 to 8), or 42°C (lanes 9 to 12), reactions were terminated by addition of SDS and the products were run on a 1% agarose gel. Arrows at the side indicate the position of the relaxed substrate (Rel) and supercoiled product (Sc). Intermediates in the supercoiling reaction run between these two positions. B. Plasmid DNAs (pUC19) isolated from exponential cultures of strain NH2585 (WT; lanes 1 and 2) or NH2589 (GyrB652; lanes 3 and 4) were run in an agarose gel containing Tris-borate-EDTA and 25 μM chloroquine phosphate. In lanes 1 and 3, cells were incubated continuously at 30°C, while in lanes 2 and 4, cells were shifted to 42°C for an hour before harvest. The arrows indicate the centers of the topoisomer distribution.