Correct positioning of the division plane is a prerequisite for the generation of daughter cells with a normal chromosome complement. Here, we present a mechanism that coordinates assembly and placement of the FtsZ cytokinetic ring with bipolar localization of the newly duplicated chromosomal origins in Caulobacter. After replication of the polarly located origin region, one copy moves rapidly to the opposite end of the cell in an MreB-dependent manner. A previously uncharacterized essential protein, MipZ, forms a complex with the partitioning protein ParB near the origin of replication and localizes with the duplicated origin regions to the cell poles. MipZ directly interferes with FtsZ polymerization, thereby restricting FtsZ ring formation to midcell, the region of lowest MipZ concentration. The cellular localization of MipZ thus serves the dual function of positioning the FtsZ ring and delaying formation of the cell division apparatus until chromosome segregation has initiated.

The chromosomal origin and terminus of replication are precisely localized in bacterial cells. We examined the cellular position of 112 individual loci that are dispersed over the circular Caulobacter crescentus chromosome and found that in living cells each locus has a specific subcellular address and that these loci are arrayed in linear order along the long axis of the cell. Time-lapse microscopy of the location of the chromosomal origin and 10 selected loci in the origin-proximal half of the chromosome showed that during DNA replication, as the replisome sequentially copies each locus, the newly replicated DNA segments are moved in chronological order to their final subcellular destination in the nascent half of the predivisional cell. Thus, the remarkable organization of the chromosome is being established while DNA replication is still in progress. The fact that the movement of these 10 loci is, like that of the origin, directed and rapid, and occurs at a similar rate, suggests that the same molecular machinery serves to partition and place many, if not most, chromosomal loci at defined subcellular sites. Bacterial chromosomes are not static structures. They undergo dynamic topological changes during DNA replication, segregation, and transcription (1-5). For bacteria with circular chromosomes, replication initiates at a single origin of replication (ori) and proceeds bidirectionally toward the terminus of replication (ter) (6). During replication, the DNA double helix is unwound locally, introducing compensatory superhelicity and entanglements that are relieved by the action of topoisomerases (1). Before newly replicated DNA can be segregated, the two sister chromosomes are unlinked by topoisomerases, resolvases, and recombinases (2,7,8). After the completion of replication and segregation, each half of the predivisional cell contains one sister chromosome. The mechanism whereby the chromosomes are moved, positioned, and finally restructured before cell division is poorly understood.Fluorescence in situ hybridization (FISH) has been used to visualize distinct chromosomal loci in fixed bacterial cells (9). In addition, a technique to label chromosomal loci in live cells has been developed by using a lac repressor GFP hybrid protein (LacI-GFP) that binds to arrays of lac operator (lacO) sequences inserted at specific sites on the chromosome (10-12). By using this labeling technique along with time-lapse fluorescence microscopy (FM), both the Escherichia coli and the Bacillus subtilis ori have been shown to move rapidly toward the cell poles once DNA replication has initiated (13-15).Chromosome replication and segregation is coordinated with other events during the cell cycle, such as polar morphogenesis and cell division. In Caulobacter, DNA replication initiates once and only once per cell cycle and proceeds bidirectionally from a single origin (16,17). Caulobacter cell division is asymmetric, yielding a replicative stalked cell and a nonreplicative swarmer cell with polar pili and a polar flagellum (18)(19...

Caulobacter crescentus is widely used as a powerful model system for the study of prokaryotic cell biology and development. Analysis of this organism is complicated by a limited selection of tools for genetic manipulation and inducible gene expression. This study reports the identification and functional characterization of a vanillate-regulated promoter (Pvan) which meets all requirements for application as a multi-purpose expression system in Caulobacter, thus complementing the established xylose-inducible system (Pxyl). Furthermore, we introduce a newly constructed set of integrating and replicating shuttle vectors that considerably facilitate cell biological and physiological studies in Caulobacter. Based on different narrow and broad-host range replicons, they offer a wide choice of promoters, resistance genes, and fusion partners for the construction of fluorescently or affinity-tagged proteins. Since many of these constructs are also suitable for use in other bacteria, this work provides a comprehensive collection of tools that will enrich many areas of microbiological research.

The cytoskeleton has a key function in the temporal and spatial organization of both prokaryotic and eukaryotic cells. Here, we report the identification of a new class of polymer-forming proteins, termed bactofilins, that are widely conserved among bacteria. In Caulobacter crescentus, two bactofilin paralogues cooperate to form a sheet-like structure lining the cytoplasmic membrane in proximity of the stalked cell pole. These assemblies mediate polar localization of a peptidoglycan synthase involved in stalk morphogenesis, thus complementing the function of the actin-like cytoskeleton and the cell division machinery in the regulation of cell wall biogenesis. In other bacteria, bactofilins can establish rod-shaped filaments or associate with the cell division apparatus, indicating considerable structural and functional flexibility. Bactofilins polymerize spontaneously in the absence of additional cofactors in vitro, forming stable ribbon-or rod-like filament bundles. Our results suggest that these structures have evolved as an alternative to intermediate filaments, serving as versatile molecular scaffolds in a variety of cellular pathways.