Plasmid segregation by a moving ATPase gradient.

Unlike the mitotic segregation of eukaryotic sister chromatids, DNA partitioning in bacteria is still not well understood. Bacterial high–copy-number plasmids can be stably maintained by random distribution of their copies during cell division. In contrast, the faithful transmission of low–copy-number plasmids and many chromosomes depends on an active process mediated by conserved, tripartite segregation systems (1). A central component of these machineries is a nucleoside triphosphatase driving the partitioning reaction, which can be classified as an actin-like ATPase (ParM), a tubulin-like GTPase (TubZ), or a Walker-type ATPase (ParA). Systems using an actin or tubulin homolog function by means of a filamentbased pushing or pulling mechanism (2, 3). Most low–copy-number plasmids and chromosomes are, however, segregated by Walker-type ATPases. Despite extensive research, it is not yet unambiguously established how this third group of proteins harnesses the energy released during ATP hydrolysis for plasmid movement. Based on previous analyses, two competing models have been put forward. In the filament-pulling model, ParA is assumed to form polymers that move DNA by repeated polymerization/depolymerization cycles. In contrast, the diffusion-ratchet model proposes a concentration gradient of ParA dimers on the nucleoid as the driving force for DNA segregation. In PNAS, Vecchiarelli et al. (4) now provide direct evidence in support of the latter model by fully reconstituting in vitro the segregation system of the Escherichia coli F plasmid. Plasmid segregation systems based on Walker-type ATPases position plasmid copies at regular distances over the nucleoid (5–7). In addition to the ATPase component (ParA), they comprise a centromere-like DNA sequence (parS) and an adaptor protein (ParB). Binding of ParB to parSmotifs on the DNA cargo results in the formation of a socalled “partition complex” (8). This complex then dynamically interacts with ParA to drive the directed movement of the DNA cargo (Fig. 1). In the presence of ATP, ParA associates nonspecifically with DNA and exhibits a weak intrinsic ATPase activity that is stimulated synergistically by ParB and DNA (9– 11). Moreover, several biochemical studies have shown that ParA can assemble into filamentous structures upon ATP binding (6, 10, 12, 13). This finding, together with analyses of ParA localization in vivo, has provided the basis for the filament-pulling model of DNA segregation (7). However, the physiological relevance of ParA polymerization is highly controversial (14, 15). In particular, recent studies investigating the E. coli P1 and F plasmid segregation systems have cast serious doubt on a role of filament formation in the partitioning process (5, 16–18). Instead, ParA was proposed to act by a diffusion-ratchet mechanism, which includes the following steps: ParA-ATP dimers bind nonspecifically to chromosomal DNA and transiently tether plasmids to the nucleoid surface through interaction with the ParB–parS partition complex (17). In the resulting quaternary complex, ParB stimulates the ATPase activity of ParA, thereby inducing its release from the DNA. Because reactivation of ParA involves a series of slow conformational changes, it is unable to immediately reassociate with the nucleoid (17). This lag creates a ParA depletion zone in the vicinity of the partition complex, which ultimately results in the detachment of the plasmid from the nucleoid surface. After its dissociation, the plasmid diffuses in a stochastically chosen direction. As it reaches the edge of the depletion zone, it encounters an increasing number of nucleoidbound ParA dimers, which make new contacts to the plasmid partition complex. Moving along this ParA gradient, the plasmid is finally immobilized again and initiates the formation of a new depletion zone. As a central point of the model, the initial direction taken by the plasmid is reinforced by Fig. 1. (A) Partitioning (sop) locus of the E. coli F plasmid, comprising genes for an ATPase (SopA) and an adaptor protein (SopB), as well as a centromere-like region including multiple tandem repeats of the SopB binding site (sopC ). (B) The sop locus is essential for the faithful segregation of F plasmids to the future daughter-cell compartments. Wild-type plasmids are regularly positioned over the nucleoid (Upper), whereas plasmids lacking the sop locus are concentrated in the polar regions of the cell (Lower). (C ) Mechanism of sop-mediated plasmid movement. Nucleoid-associated SopA-ATP dimers bind to a SopB–sopC partition complex, thereby immobilizing a copy of the F plasmid on the nucleoid surface. After stimulation of its ATPase activity by SopB, SopA dissociates from the DNA, leaving behind a zone of low SopA concentration (Upper). As a consequence, the plasmid starts to diffuse and then reattaches to the nucleoid through association with neighboring SopA molecules (Lower). Iteration of these steps results in the directed movement of plasmid copies across the nucleoid surface.