FIG. 3.

Chromosomal fragmentation measured by pulsed-field gel electrophoresis. Exponential-phase cultures were induced with arabinose for the expression of wild-type and mutant recombinant Y. pestis topoisomerase I. After incubation with shaking at 37°C for 30 min, cells were collected by centrifugation and processed with the Bio-Rad CHEF bacterial genomic DNA plug kit. The agarose plugs containing the genomic DNA were electrophoresed in a 1% agarose gel with 0.5× Tris-borate-EDTA buffer with the Bio-Rad CHEF-DRII pulsed-field electrophoresis system (14°C, 6 V/cm, 21 h, linear ramping from 50 to 92 s), followed by staining with ethidium bromide. Analysis was carried out for BW27784 (lanes 1 to 9) or YT101 (recA::Tn5, lanes 10 to 19) cells transformed with pAYTOP (w, lanes 1 to 4 and lanes 10 to 14) or pAYTOP128 (m, lanes 5 to 9 and lanes 15 to 19). Chromosomal DNA gel plugs were prepared after 30 min of no treatment (lanes 1, 10, and 15), treatment with 0.5 mg ciprofloxacin/liter (C, lane 4), or arabinose at the indicated concentrations. W, gel wells; L, linear chromosome; M, Saccharomyces cerevisiae chromosome standards (225 to 2,200 kb).