Bacterial type II topoisomerases cleave DNA in a species-specific manner.

The type II topoisomerases, gyrase and topoisomerase IV, are ubiquitous enzymes in bacteria that help regulate DNA topology and are the molecular targets of fluoroquinolone antibacterials. As part of their catalytic mechanism, these enzymes transiently cleave DNA in a sequence-dependent manner. Determining the extent to which various factors influence the sequence-dependent cleavage of these enzymes, particularly across bacterial species, could help reveal important insights into their physiological functions and guide the development of new, more effective antibacterials. Here, we used our recently developed SHAN-seq method to map and compare the DNA cleavage site preferences of gyrases and topoisomerase IVs from three different pathogenic bacterial species, Escherichia coli, Bacillus anthracis, and Mycobacterium tuberculosis, in the presence of the fluoroquinolone, ciprofloxacin. We found that the enzymes' cleavage specificities vary across bacterial species, with DNA supercoil chirality, and in response to ciprofloxacin. Our findings suggest that subtle variations in the enzymes' catalytic core and C-terminal domains alter their cleavage site preferences, which could, in turn, influence their physiological activities and susceptibility to fluoroquinolone antibacterials.