Topoisomerase I participates in chromosome segregation of the polyploid bacterium Thermus thermophilus through its decatenation activity

Thermus thermophilus , a polyploid thermophilic bacterium, lacks the canonical decatenases Topo III and Topo IV, posing a challenge for faithful chromosome segregation. This study aims to determine whether T. thermophilus topoisomerase I (TopoI Tth ) has acquired decatenation activity and participates in chromosome segregation. Through gene deletion, we found that topA is essential; only a heterozygous Δ topA::blm / WT-topA mutant could be obtained. Partial depletion of TopoI Tth caused severe Par − phenotypes, including cell filamentation, unsegregated nucleoids, anucleate cells, and DNA fragmentation, all of which were rescued by ectopic expression of wild-type topA , confirming the specificity of the defects. TopoI Tth localized to cell poles, mirroring the localization of classical segregation proteins SMC and XerD, and bacterial two-hybrid assays revealed physical interactions with both proteins. Truncation analysis mapped the interaction interfaces to the C-terminal domain of SMC and the N-terminal domain of XerD. In vitro , TopoI Tth exhibited decatenation and catenation activities, contrasting with Escherichia coli TopoI. SMC stimulated its decatenation in a dose-dependent manner, whereas XerD did not. Collectively, our results indicate that TopoI Tth acts as a decatenase integrated with the chromosome segregation machinery to compensate for the absence of Topo III and Topo IV. This study uncovers a functional expansion of type IA topoisomerases and offers a paradigm for how genome streamlining can drive the evolution of novel enzyme functions. IMPORTANCE This work refines the canonical view that bacterial topoisomerase I functions solely as a relaxase. By demonstrating that Thermus thermophilus Topo I has evolved robust decatenation activity and physically interacts with SMC to drive chromosome segregation, we uncover a functional expansion of type IA topoisomerases. This provides a paradigm for understanding how genome streamlining drives the acquisition of new functions by an existing enzyme. Beyond evolutionary significance, our findings may have translational relevance. Certain pathogens, such as Mycobacterium tuberculosis and Mycobacterium smegmatis , similarly lack canonical decatenases and depend on Topo I for survival. Although further studies are needed to validate whether this decatenase activity is widely conserved and essential in these pathogens, our work suggests that Topo I could be explored as a potential target for developing novel antibiotics against multidrug-resistant bacteria.