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

The type II topoisomerases, gyrase and topoisomerase IV, are essential enzymes in nearly all bacteria and are the targets of fluoroquinolones, which are some of the most widely prescribed broad-spectrum antibacterials in clinical use. As part of their catalytic cycle, gyrase and topoisomerase IV transiently cleave DNA in a sequence-dependent manner. However, it is unclear whether this sequence-dependence is species-specific. Therefore, using our recently developed SHAN-seq method, we mapped and compared cleavage sites for type II topoisomerases 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 have substantially different DNA cleavage specificities that vary between gyrase and topoisomerase IV, across species, with supercoil chirality, and in response to ciprofloxacin. Our results demonstrate that bacterial species fine-tune the DNA cleavage specificity of their type II topoisomerases. This finding suggests that cleavage specificity may play important physiological roles and, in turn, may affect the susceptibility of bacteria to fluoroquinolone antibacterials. Overall design: Supercoiled pBR322 DNA was cleaved with DNA gyrase or topoisomerase IV and spiked with three DNA oligonucleotides. The DNA was subject to standard DNA-seq library preparation to label the enzyme-cleaved ends, fragmented, and subject to a second round of standard DNA-seq library preparation to label the fragmented ends. The labelled DNA was pair-end sequenced and mapped to the plasmid and spike-in oligonucleotide reference sequences. The reads from the topo-cleaved plasmid DNA ends were normalized by the reads from the spike-in ends and were used to determine the gyrase or topo IV cleavage levels at each position on the plasmid.