The determination and regulation of cell morphology are critical components of cell-cycle control, fitness, and development in both single-cell and multicellular organisms. Understanding how environmental factors, chemical perturbations, and genetic differences affect cell morphology requires precise, unbiased, and validated measurements of cell-shape features. Here we introduce two software packages, Morphometrics and BlurLab, that together enable automated, computationally efficient, unbiased identification of cells and morphological features. We applied these tools to bacterial cells because the small size of these cells and the subtlety of certain morphological changes have thus far obscured correlations between bacterial morphology and genotype. We used an online resource of images of the Keio knockout library of nonessential genes in the Gram-negative bacterium Escherichia coli to demonstrate that cell width, width variability, and length significantly correlate with each other and with drug treatments, nutrient changes, and environmental conditions. Further, we combined morphological classification of genetic variants with genetic meta-analysis to reveal novel connections among gene function, fitness, and cell morphology, thus suggesting potential functions for unknown genes and differences in modes of action of antibiotics. Morphometrics and BlurLab set the stage for future quantitative studies of bacterial cell shape and intracellular localization. The previously unappreciated connections between morphological parameters measured with these software packages and the cellular environment point toward novel mechanistic connections among physiological perturbations, cell fitness, and growth.

Assembly of the cell division apparatus in bacteria starts with formation of the Z ring on the cytoplasmic face of the membrane. This process involves the accumulation of FtsZ polymers at midcell and their interaction with several FtsZ-binding proteins that collectively organize the polymers into a membrane-associated ring-like configuration. Three such proteins, FtsA, ZipA, and ZapA, have previously been identified inEscherichia coli. FtsA and ZipA are essential membrane-associated division proteins that help connect FtsZ polymers with the inner membrane. ZapA is a cytoplasmic protein that is not required for the fission processper sebut contributes to its efficiency, likely by promoting lateral interactions between FtsZ protofilaments. We report the identification of YcbW (ZapC) as a fourth FtsZ-binding component of the Z ring inE. coli. Binding of ZapC promotes lateral interactions between FtsZ polymers and suppresses FtsZ GTPase activity. This and additional evidence indicate that, like ZapA, ZapC is a nonessential Z-ring component that contributes to the efficiency of the division process by stabilizing the polymeric form of FtsZ.

Formation of the FtsZ ring (Z ring) in Escherichia coli is the first step in the assembly of the divisome, a protein machine required for cell division. Although the biochemical functions of most divisome proteins are unknown, several, including ZipA, FtsA and FtsK, have overlapping roles in ensuring that the Z ring assembles at the cytoplasmic membrane, and that it is active. As shown previously, a single amino acid change in FtsA, R286W, also called FtsA*, bypasses the requirement for either ZipA or FtsK in cell division. In this study, the properties of FtsA* were investigated further, with the eventual goal of understanding the molecular mechanism behind the bypass. Compared to wild-type FtsA, the presence of FtsA* resulted in a modest but significant decrease in the mean length of cells in the population, accelerated the reassembly of Z rings, and suppressed the cell-division block caused by excessively high levels of FtsZ. These effects were not mediated by Z-ring remodelling, because FtsA* did not alter the kinetics of FtsZ turnover within the Z ring, as measured by fluorescence recovery after photobleaching. FtsA* was also unable to permit normal cell division at below normal levels of FtsZ, or after thermoinactivation of ftsZ84(ts). However, turnover of FtsA* in the ring was somewhat faster than that of wild-type FtsA, and overexpressed FtsA* did not inhibit cell division as efficiently as wild-type FtsA. Finally, FtsA* interacted more strongly with FtsZ compared with FtsA in a yeast two-hybrid system. These results suggest that FtsA* interacts with FtsZ in a markedly different way compared with FtsA.

Summary: In Escherichia coli, chemoreceptor clustering at a cell pole seems critical for signal amplification and adaptation. However, little is known about the mechanism of localization itself. Here we examined whether the aspartate chemoreceptor (Tar) is inserted directly into the polar membrane by using its fusion to green fluorescent protein (GFP). After induction of Tar–GFP, fluorescent spots first appeared in lateral membrane regions, and later cell poles became predominantly fluorescent. Unexpectedly, Tar–GFP showed a helical arrangement in lateral regions, which was more apparent when a Tar–GFP derivative with two cysteine residues in the periplasmic domain was cross‐linked to form higher oligomers. Moreover, similar distribution was observed even when the cytoplasmic domain of the double cysteine Tar–GFP mutant was replaced by that of the kinase EnvZ, which does not localize to a pole. Observation of GFP–SecE and a translocation‐defective MalE–GFP mutant, as well as indirect immunofluorescence microscopy on SecG, suggested that the general protein translocation machinery (Sec) itself is arranged into a helical array, with which Tar is transiently associated. The Sec coil appeared distinct from the MreB coil, an actin‐like cytoskeleton. These findings will shed new light on the mechanisms underlying spatial organization of membrane proteins in E. coli.