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.

The rod-shaped Gram-negative bacterium Escherichia coli multiplies by elongation followed by binary fission. Longitudinal growth of the cell envelope and synthesis of the new poles are organized by two protein complexes called elongasome and divisome, respectively. We have analyzed the spatio-temporal localization patterns of many of these morphogenetic proteins by immunolabeling the wild type strain MC4100 grown to steady state in minimal glucose medium at 28°C. This allowed the direct comparison of morphogenetic protein localization patterns as a function of cell age as imaged by phase contrast and fluorescence wide field microscopy. Under steady state conditions the age distribution of the cells is constant and is directly correlated to cell length. To quantify cell size and protein localization parameters in 1000s of labeled cells, we developed ‘Coli-Inspector,’ which is a project running under ImageJ with the plugin ‘ObjectJ.’ ObjectJ organizes image-analysis tasks using an integrated approach with the flexibility to produce different output formats from existing markers such as intensity data and geometrical parameters. ObjectJ supports the combination of automatic and interactive methods giving the user complete control over the method of image analysis and data collection, with visual inspection tools for quick elimination of artifacts. Coli-inspector was used to sort the cells according to division cycle cell age and to analyze the spatio-temporal localization pattern of each protein. A unique dataset has been created on the concentration and position of the proteins during the cell cycle. We show for the first time that a subset of morphogenetic proteins have a constant cellular concentration during the cell division cycle whereas another set exhibits a cell division cycle dependent concentration variation. Using the number of proteins present at midcell, the stoichiometry of the divisome is discussed.

SummaryFtsZ, the essential regulator of bacterial cell division, is a dynamic cytoskeletal protein that forms helices that condense into the Z-ring prior to division. Two small coiled-coil proteins, ZapA and ZapB, are both recruited early to the Z-ring. We show here that ZapB is recruited to the Z-ring by ZapA. A direct interaction between ZapA and ZapB is supported by bacterial two-hybrid and in vitro interaction assays. Using high-resolution 3-D reconstruction microscopy, we find that, surprisingly, ZapB is located inside the Z-ring in virtually all cells investigated. We propose a molecular model in which ZapA increases lateral interactions between FtsZ proto-filaments and ZapB mediates further stabilization of this interaction by cross-linking ZapA molecules bound to adjacent FtsZ proto-filaments. Gene deletion and complementation assays show that ZapB can mitigate cell division and Z-ring assembly defects even in the absence of ZapA, raising the possibility that ZapB stimulates Z-ring assembly by two different mechanisms.

No abstract

The replication terminus region (Ter) of the unique chromosome of most bacteria locates at mid-cell at the time of cell division. In several species, this localization participates in the necessary coordination between chromosome segregation and cell division, notably for the selection of the division site, the licensing of the division machinery assembly and the correct alignment of chromosome dimer resolution sites. The genome of Vibrio cholerae, the agent of the deadly human disease cholera, is divided into two chromosomes, chrI and chrII. Previous fluorescent microscopy observations suggested that although the Ter regions of chrI and chrII replicate at the same time, chrII sister termini separated before cell division whereas chrI sister termini were maintained together at mid-cell, which raised questions on the management of the two chromosomes during cell division. Here, we simultaneously visualized the location of the dimer resolution locus of each of the two chromosomes. Our results confirm the late and early separation of chrI and chrII Ter sisters, respectively. They further suggest that the MatP/matS macrodomain organization system specifically delays chrI Ter sister separation. However, TerI loci remain in the vicinity of the cell centre in the absence of MatP and a genetic assay specifically designed to monitor the relative frequency of sister chromatid contacts during constriction suggest that they keep colliding together until the very end of cell division. In contrast, we found that even though it is not able to impede the separation of chrII Ter sisters before septation, the MatP/matS macrodomain organization system restricts their movement within the cell and permits their frequent interaction during septum constriction.