Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division. Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes. We present here a phylogenetic analysis of par loci from plasmids and chromosomes from prokaryotic organisms. All known plasmid‐encoded par loci specify three components: a cis‐acting centromere‐like site and two trans‐acting proteins that form a nucleoprotein complex at the centromere (i.e. the partition complex). The proteins are encoded by two genes in an operon that is autoregulated by the par‐encoded proteins. In all cases, the upstream gene encodes an ATPase that is essential for partitioning. Recent cytological analyses indicate that the ATPases function as adaptors between a host‐encoded component and the partition complex and thereby tether plasmids and chromosomal origin regions to specific subcellular sites (i.e. the poles or quarter‐cell positions). Two types of partitioning ATPases are known: the Walker‐type ATPases encoded by the par/sop gene family (type I partitioning loci) and the actin‐like ATPase encoded by the par locus of plasmid R1 (type II partitioning locus). A phylogenetic analysis of the large family of Walker type of partitioning ATPases yielded a surprising pattern: most of the plasmid‐encoded ATPases clustered into distinct subgroups. Surprisingly, however, the par loci encoding these distinct subgroups have different genetic organizations and thus divide the type I loci into types Ia and Ib. A second surprise was that almost all chromosome‐encoded ATPases, including members from both Gram‐negative and Gram‐positive Bacteria and Archaea, clustered into one distinct subgroup. The phylogenetic tree is consistent with lateral gene transfer between Bacteria and Archaea. Using database mining with the ParM ATPase of plasmid R1, we identified a new par gene family from enteric bacteria. These type II loci, which encode ATPases of the actin type, have a genetic organization similar to that of type Ib loci.

The mechanism of prokaryotic chromosome segregation is not known. MreB, an actin homolog, is a shapedetermining factor in rod-shaped prokaryotic cells. Using immuno¯uorescence microscopy we found that MreB of Escherichia coli formed helical ®laments located beneath the cell surface. Flow cytometric and cytological analyses indicated that MreB-depleted cells segregated their chromosomes in pairs, consistent with chromosome cohesion. Overexpression of wildtype MreB inhibited cell division but did not perturb chromosome segregation. Overexpression of mutant forms of MreB inhibited cell division, caused abnormal MreB ®lament morphology and induced severe localization defects of the nucleoid and of the oriC and terC chromosomal regions. The chromosomal terminus regions appeared cohered in both MreBdepleted cells and in cells overexpressing mutant forms of MreB. Our observations indicate that MreB ®laments participate in directional chromosome movement and segregation.

The mechanisms responsible for prokaryotic DNA segregation are largely unknown. The partitioning locus (par) encoded by the Escherichia coli plasmid R1 actively segregates its replicon to daughter cells. We show here that the ParM ATPase encoded by par forms dynamic actin-like ®laments with properties expected for a force-generating protein. Filament formation depended on the other components encoded by par, ParR and the centromere-like parC region to which ParR binds. Mutants defective in ParM ATPase exhibited hyper®lamentation and did not support plasmid partitioning. ParM polymerization was ATP dependent, and depolymerization of ParM ®laments required nucleotide hydrolysis. Our in vivo and in vitro results indicate that ParM polymerization generates the force required for directional movement of plasmids to opposite cell poles and that the ParR±parC complex functions as a nucleation point for ParM polymerization. Hence, we provide evidence for a simple prokaryotic analogue of the eukaryotic mitotic spindle apparatus.

Uropathogenic Escherichia coli (UPEC) strains are capable of invading bladder epithelial cells (BECs) on the bladder luminal surface. Based primarily on studies in mouse models, invasion is proposed to trigger an intracellular uropathogenic cascade involving intracellular bacterial proliferation followed by escape of elongated, filamentous bacteria from colonized BECs. UPEC filaments on the mouse bladder epithelium are able to revert to rod-shaped bacteria, which are believed to invade neighboring cells to initiate new rounds of intracellular colonization. So far, however, these late-stage infection events have not been replicated in vitro. We have established an in vitro model of human bladder cell infection by the use of a flow chamber (FC)-based culture system, which allows investigation of steps subsequent to initial invasion. Short-term bacterial colonization on the FC-BEC layer led to intracellular colonization. Exposing invaded BECs to a flow of urine, i.e., establishing conditions similar to those faced by UPEC reemerging on the bladder luminal surface, led to outgrowth of filamentous bacteria similar to what has been reported to occur in mice. These filaments were capable of reverting to rods that could invade other BECs. Hence, under growth conditions established to resemble those present in vivo, the elements of the proposed uropathogenic cascade were inducible in a human BEC model system. Here, we describe the model and show how these characteristics are reproduced in vitro.

SummaryFormation of the Z ring is the first known event in bacterial cell division. However, it is not yet known how the assembly and contraction of the Z ring are regulated. Here, we identify a novel cell division factor ZapB in Escherichia coli that simultaneously stimulates Z ring assembly and cell division. Deletion of zapB resulted in delayed cell division and the formation of ectopic Z rings and spirals, whereas overexpression of ZapB resulted in nucleoid condensation and aberrant cell divisions. Localization of ZapB to the divisome depended on FtsZ but not FtsA, ZipA or FtsI, and ZapB interacted with FtsZ in a bacterial two-hybrid analysis. The simultaneous inactivation of FtsA and ZipA prevented Z ring assembly and ZapB localization. Time lapse microscopy showed that ZapB-GFP is present at mid-cell in a pattern very similar to that of FtsZ. Cells carrying a zapB deletion and the ftsZ84 ts allele exhibited a synthetic sick phenotype and aberrant cell divisions. The crystal structure showed that ZapB exists as a dimer that is 100% coiled-coil. In vitro, ZapB self-assembled into long filaments and bundles. These results raise the possibility that ZapB stimulates Z ring formation directly via its capacity to selfassemble into larger structures.