Ancestral Sequence Reconstruction Reveals CTP-dependent Loading of the Bacterial Centromere-binding Protein ParB as an Ancient Evolutionary Feature

In most bacteria, chromosome and low-copy plasmid segregation are mediated by the ParABS system. Its component ParB functions as a DNA sliding clamp that assembles on centromere-like parS sites to form large nucleoprotein complexes, which are subsequently positioned by the ATPase ParA. Loading of ParB onto DNA is regulated by a recently discovered conserved CTPase domain, yet the evolutionary origin of this regulatory module remains unclear. Here, we apply ancestral sequence reconstruction to resurrect ancient ParB proteins dating back to the early origins of the bacterial domain. Biochemical, structural, and cell biological analyses demonstrate that these reconstructed proteins display all core activities of their modern counterparts, suggesting that the ParABS system emerged early during bacterial evolution and has essentially remained unchanged ever since. More broadly, our findings indicate that regulatory CTPases represent an ancient molecular innovation, whose origins can be traced back to the earliest stages of life.