Role of MukBEF in Escherichia coli chromosome organization

Abstract

In E. coli cells, spatial chromosome organization reflects the genetic map, with the origin located at mid-cell and left and right replichores in either cell half. In contrast to the eukaryotic cell cycle, chromosome segregation occurs progressively as replication proceeds. The highly conserved SMC complex, MukBEF, plays a central role in chromosome organization and segregation, with null mutants having temperature sensitive growth and mispositioned chromosomal regions. However, despite much effort, their mechanism of action in vivo remains elusive.The current work aimed to shed light on MukBEF function by developing tools to investigate its mode of action in live cells. Degron derivatives of MukBEF were developed as a rapid way of depleting cells of MukBEF to observe its effects on chromosome organization. “Real-time” Muk depletion/ repletion shows that MukBEF can establish and maintain position of chromosomal loci independent of the cell cycle. Origin relocation from cell-pole to mid-cell is a slow process, preceded by the formation of MukBEF foci at various locations on the chromosome. Hence, Muk-mediated shaping of the chromosome is a replication-independent phenomenon. In addition, while ATP binding is required for focus formation, ATP hydrolysis is required for MukBEF function in the cell. ATPase mutants are inviable in vivo, highlighting the importance of MukB ATPase activity.In a complementary approach, the composition and dynamics of MukBEF foci were assessed in vivo. The stoichiometries of wild type and ATP-hydrolysis deficient (Walker B) MukBEF complexes in fluorescent foci were estimated using slimfield microscopy. These results suggest that MukBEF complexes are ATP bound in foci with a broad and heterogeneous distribution. Only 19% of total MukBEF molecules are present at a given time in foci. Furthermore, unlike wild type foci, which localize around the origin, Walker B foci colocalize with the terminus. One model proposed is that the terminus may represent the loading site for wild type complexes and that Walker B MukBEF may represent the loading complex, which can associate with the loading site, but requires ATP hydrolysis for movement to the origin.This work provides insight into the role of MukBEF in spatial chromosome organization as well as the in vivo significance of MukB ATPase activity.</p