Coordinating terminal cell cycle events in Escherichia coli

Various timing and checkpoint mechanisms are known to orchestrate cell cycle progression in eukaryotic cells. In contrast, little is known about how cell cycle events are coordinated in bacteria.;In Escherichia coli, which has a single circular chromosome, DNA replication results in formation of topologically catenated daughter chromosomes. Topoisomerase IV (Topo IV), a type II enzyme, catalyzes the unlinking of daughter chromosomes and is therefore essential for chromosome segregation. Previous studies have shown that the activity of Topo IV is confined to only a short period late in the cell cycle. However, neither the mechanism nor the significance of this regulation is understood.;By perturbing the temporal regulation of Topo IV in several ways, the data in this thesis show that unconstrained Topo IV activity results in the inhibition of cell division. Characterization of this division arrest revealed a strong correlation between cytokinesis and nucleoid condensation---cells with decondensed chromosomes were unable to divide. The data presented in this thesis suggest that this division arrest is effected by destabilizing FtsZ rings and is dependent upon the Min system that determines division-site placement in E. coli. Furthermore, bypassing this division arrest compromises cell viability. These results imply the existence of a checkpoint that preserves viability in cells with decondensed chromosomes.;Recently, the discovery of MreB, a bacterial actin homologue that forms helical structures beneath the membrane, raised the possibility that bacteria use this cytoskeletal structure to segregate their chromosomes. Previous studies showed that a dysfunctional MreB causes defects in chromosome segregation; however, the reasons behind these defects were unclear. The results in this thesis indicate that MreB participates in the temporal oscillation of Topo IV activity. The analysis of purified nucleoids suggests that mreB mutants are deficient in Topo IV activity. In addition, MreB physically interacts with the ParC subunit of Topo IV and differentially affects Topo IV activity depending on whether it is a monomer or a polymer. These results might explain how the remodeling of the cytoskeleton is coordinated with the timing of chromosome segregation.;Thus, by studying the oscillation of Topo IV activity, this work illuminates novel mechanisms that could coordinate events necessary for bacterial cell cycle progression.