Topoisomerase IV (Topo IV) is the main decatenation enzyme in ; it removes catenation links that are formed during DNA replication. Topo IV binding and cleavage sites were previously identified in the genome with ChIP-Seq and NorfIP. Here, we used a more sensitive, single-nucleotide resolution Topo-Seq procedure to identify Topo IV cleavage sites (TCSs) genome-wide. We detected thousands of TCSs scattered in the bacterial genome. The determined cleavage motif of Topo IV contained previously known cleavage determinants (-4G/+8C, -2A/+6 T, -1 T/+5A) and additional, not observed previously, positions -7C/+11G and -6C/+10G. TCSs were depleted in the Ter macrodomain except for two exceptionally strong non-canonical cleavage sites located in 33 and 38 bp from the of the -site. Topo IV cleavage activity was increased in Left and Right macrodomains flanking the Ter macrodomain and was especially high in the 50-60 kb region containing the origin of replication. Topo IV enrichment was also increased downstream of highly active transcription units, indicating that the enzyme is involved in relaxation of transcription-induced positive supercoiling.