Title: Single-molecule imaging of DNA gyrase activity in living Escherichia coli,
Abstract: Bacterial DNA gyrase introduces negative supercoils into chromosomal DNA and
relaxes positive supercoils introduced by replication and transiently by
transcription. Removal of these positive supercoils is essential for
replication fork progression and for the overall unlinking of the two duplex
DNA strands, as well as for ongoing transcription. To address how gyrase copes
with these topological challenges, we used high-speed single-molecule
fluorescence imaging in live Escherichia coli cells. We demonstrate that at
least 300 gyrase molecules are stably bound to the chromosome at any time, with
~12 enzymes enriched near each replication fork. Trapping of reaction
intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell
times of ~2 s were observed for the dispersed gyrase molecules, which we
propose maintain steady-state levels of negative supercoiling of the
chromosome. In contrast, the dwell time of replisome-proximal molecules was ~8
s, consistent with these catalyzing processive positive supercoil relaxation in
front of the progressing replisome.