Low copy-number bacterial replicons occupy specific locations in their host cells. Production of a GFP-Lac repressor hybrid protein in cells carrying F or P1 plasmids tagged with a lac operator array reveals that in smaller (younger) cells these plasmids are seen mainly as a single fluorescent focus at mid-cell, whereas larger cells tend to have two foci, one at each quarter-cell position. Duplication of the central focus is presumed to represent active partition of plasmid copies. We report here our investigation by time-lapse microscopy of the subsequent movement of these copies to the quarter positions. Following duplication of the central focus, the new foci migrated rapidly and directly to their quarter-cell destinations, where they remained until the next cell cycle. The speed of movement was about five times faster than poleward migration of oriC and 50 times faster than cell elongation. Aberrant positioning of mini-F lacking its sopC centromere demonstrated the requirement for the partition system in this localization process. From the measured number of F plasmid copies per cell it appears that each migrating focus contains two or more plasmid molecules. The molecular basis of this clustering, and evidence for phasing of the partition event in the cell cycle, are discussed.

We used the LacO/GFP-LacI system to label and visualize the IncPbeta plasmid R751 fluorescently during conjugative transfer between live donor and recipient bacteria. Comparisons of R751 in conjugative and non-conjugative conditions have allowed us to identify key localizations and movements associated with the initiation of conjugative transfer in the donor and the establishment of R751 in the recipient. A survey of successful mating pairs demonstrates that close physical contact between donor and recipient bacteria is required for DNA transfer and that regions of intimate contact can occur at any location on the donor or recipient cell membrane. The transferred DNA is positioned at the characteristic centre or quarter-cell position after conversion to a double-stranded molecule in the recipient cell. Initial duplication of plasmids often results in an asymmetric distribution of plasmid foci. Symmetric localization (either at centre or at 1/4 and 3/4 cell lengths) occurs only after a significant lag, presumably reflecting the time required to synthesize the plasmid-encoded partitioning proteins.

The location of the origin-linked region of the Escherichia coli chromosome was analysed in strains lacking the core origin locus, oriC . In these strains, which initiate replication from F factors integrated at different locations around the chromosome, origin-linked DNA remains localized near the cell poles, as in wild-type cells. In contrast, minichromosomes containing 7 kb of chromosomal DNA including oriC are generally excluded from the ends of the cell. Thus, we propose that positioning of the wild-type origins at the poles is not a function of their order of replication but a sequence-specific phenomenon. It is proposed that there are centromere-like sequences, bordering the wild-type origin of replication, which are used by host mechanisms to direct the proper placement of the origin region of the chromosome. This function, combined with other host processes, may assure efficient segregation of the E. coli chromosome.

Cell polarity is a fundamental feature of all cell types and is essential for cell division, cell migration, and vectorial transport of cell fate determinants within a cell (1). In recent years, application of cytological methods to study protein, chromosome, and episome localization in chemically fixed and live bacterial cells has signaled the advent of prokaryotic cell biology and has provided us with new, sometimes startling, imagery of the spatial organization within a bacterial cell (2). Proteins are found to be positioned at specific sites in bacteria such as the cell pole, the cell equator, and the division septum. Such site-specific protein targeting appears to be central to the regulation of DNA replication, chromosome segregation, cell division, cell differentiation, and chemotaxis. Recent studies on a group of proteins in Escherichia coli , termed Min, have revealed an extraordinary oscillatory behavior that governs their cellular location. The dynamic behavior of the Min proteins creates transient domains of subcellular asymmetry in the E. coli cell and such cell polarization is critical to the ability of the Min system to ensure that a cell divides unerringly in the middle. In a recent issue of PNAS, Fu et al. (3) describe a remarkable cell polarization feature of the MinE protein that localizes as an off-center ring (E-ring) and as a polar zone (PZ) that extends from the ring to the proximal cell pole. Time-lapse microscopy of live E. coli cells expressing a MinE-green fluorescent protein fusion reveals that the membrane-associated E-ring and the PZ form a mobile unit, with the PZ shrinking as the E-ring moves toward the proximal pole. Upon reaching the polar extremity, the PZ and the ring disappear, only to reappear at the opposite pole. The sequence of MinE assembly, poleward movement, dispersion, and reassembly is repeated many times in each …

Escherichia coli cell division is restricted to the middle of the cell by the MinCDE system. Through complex interactions with MinC and MinD, the MinE protein promotes the correct placement of the cell division apparatus by specifically localizing to the cell center. The first detailed structural analysis of MinE provides key clues to help understand the critical actions of this protein.

Segregation of DNA in bacterial cells is an efficient process that assures that every daughter cell receives a copy of genomic and plasmid DNA. In this review, we focus primarily on observations in recent years, including the visualization of DNA and proteins at the subcellular level, that have begun to define the events that separate DNA molecules. Unlike the process of chromosome segregation in higher cells, segregation of the bacterial chromosome is a continuous process in which chromosomes are separated as they are replicated. Essential to separation is the initial movement of sister origins to opposite ends of the cell. Subsequent replication and controlled condensation of DNA are the driving forces that move sister chromosomes toward their respective origins, which establishes the polarity required for segregation. Final steps in the resolution and separation of sister chromosomes occur at the replication terminus, which is localized at the cell center. In contrast to the chromosome, segregation of low-copy plasmids, such as Escherichia coli F, P1, and R1, is by mechanisms that resemble those used in eukaryotic cells. Each plasmid has a centromere-like site to which plasmid-specified partition proteins bind to promote segregation. Replication of plasmid DNA, which occurs at the cell center, is followed by rapid partition protein-mediated separation of sister plasmids, which become localized at distinct sites on either side of the division plane. The fundamental similarity between chromosome and plasmid segregation-placement of DNA to specific cell sites-implies an underlying cellular architecture to which both DNA and proteins refer.

Recent studies provide evidence that bacterial chromosomes are replicated by an enzyme factory, the replisome, located at a fixed position at the center of the cell; the fixed replisome could be a major factor in determining chromosome order in the cell, and may provide the force that drives chromosome segregation.

To investigate chromosome segregation in B. subtilis, we introduced tandem copies of the lactose operon operator into the chromosome near the replication origin or terminus. We then visualized the position of the operator cassettes with green fluorescent protein fused to the LacI repressor. In sporulating bacteria, which undergo asymmetric cell division, origins localized near each pole of the cell whereas termini were restricted to the middle. In growing cells, which undergo binary fission, origins were observed at various positions but preferentially toward the poles early in the cell cycle. In contrast, termini showed little preference for the poles. These results indicate the existence of a mitotic-like apparatus that is responsible for moving the origin regions of newly formed chromosomes toward opposite ends of the cell.

We have investigated DNA segregation in E. coli by inserting multiple lac operator sequences into the chromosome near the origin of replication (oriC), in the hisC gene, a terminus marker, and into plasmids P1 and F. Expression of a GFP-LacI fusion protein allowed visualization of lac operator localization. oriC was shown to be specifically localized at or near the cell poles, and when duplicated, one copy moved to the site of new pole formation near the site of cell division. In contrast, P1 and F localized to the cell center and on duplication appeared to move rapidly to the quarter positions in the cell. Our analysis suggests that different active processes are involved in movement and localization of the chromosome and of the two plasmids during segregation.