Single-molecule imaging of DNA gyrase activity in living Escherichia coli

TL;DR

This study uses live-cell single-molecule fluorescence imaging to quantify and analyze the activity and dynamics of DNA gyrase enzymes in Escherichia coli, revealing their roles in managing DNA supercoiling during replication and transcription.

Contribution

It provides the first direct in vivo measurements of gyrase binding times, enzyme distribution, and activity near replication forks in living bacteria.

Findings

At least 300 gyrase molecules are stably bound to the chromosome.

Approximately 12 gyrase enzymes are enriched near each replication fork.

Gyrase molecules near replisomes have longer dwell times (~8 s) indicating processive supercoil relaxation.

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.