Single-molecule insights into DNA gyrase in live bacteria
Molecular motors travelling along DNA introduce positive supercoils that present as barriers to replication leading to genome instability. To counter these, bacterial cells express DNA gyrase, a topoisomerase that introduces negative supercoils. While much is known about DNA gyrase from genetic and in vitro biochemical studies, the spatiotemporal dynamics of this enzyme remain a mystery. Only recently have we been able to observe the in vivo spatiotemporal dynamics down to single molecule level using advanced super-resolution microscopy techniques. We used Slimfield microscopy, a cutting-edge molecule microscopy technique to address the gap in our knowledge. We analysed a dual fluorescently labelled Escherichia coli strain expressing the replisome marker DnaN-mCherry along with mYPet-GyrB as the enzyme marker. We performed sequential Slimfield microscopy of the labelled proteins from the same strain and analysed in vivo GyrB dynamics in live E. coli cells in relation to the replisome. We find that the majority of replisomes are associated with GyrB. Inhibition of gyrase activity reduces the proportion of replisomes associated with GyrB. Interestingly, GyrB behaviour is distinct from that observed for GyrA in a previous study. Our results reveal the previously unknown dynamics of GyrB inside living bacterial cells highlighting the advantages of in vivo single molecule investigations. Our findings also demonstrate the importance of analysing all subunits of a functional enzyme complex to gain comprehensive understanding of its in vivo mechanisms. This study demonstrates the utility of single-molecule super-resolved microscopy as a valuable underpinning technology to understand in vivo behaviour of biomedically important molecules. Our insights will help impact discovery and development of novel antibiotics that interfere with gyrase function, thus contributing to tackling the growing problem of antimicrobial resistance.