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Over the past few years, our lab has developed a method for precisely determining the shape of bacterial cells in 3D by fluorescence microscopy. We use this method to measure the position of MreB in relation to the cell surface. To ensure that our measurements of fluorescent MreB reflect untagged MreB as nearly as possible, we have integrated our fusion at the native locus. This construct has unperturbed mass doubling times and proper rod-like shape. The shape and localization measurements that we report here are measured as snapshots from hundreds to a few thousand cells per condition.MreB, a membrane-binding structural homolog of actin, is one of the key players in properly patterning growth of the bacterial cell wall and thereby the shape of the cell. In one series of experiments, we show that the helical pitch of MreB filaments in E. coli is highly correlated to the diameter of the cell. This correlation holds for E. coli whose diameter has been altered by treatment with sub-lethal doses of the MreB targeting drug A22 and for single point mutants in MreB.Additionally, MreB polymers show a clear preference for regions of negative Gaussian curvature. We hypothesize that cells use this geometric sensing mechanism to straighten their centerlines [Ursell et al., PNAS 2014]. When this curvature preference is abolished (E. coli MreB A158T), cells are more frequently branched. In the smaller, comma shaped bacterium Caulobacter crescentus, the curvature enrichment profile shows a plateau region at low positive Gaussian curvature. This plateau enables Caulobacter cells to grow with their characteristic comma shape instead of as straight rods.

A peptidoglycan cell wall determines the shape of nearly all bacteria. The cell wall, along with the shape that it adopts, is crucial to cellular physiology. The mechanical integrity and morphology of the cell wall is determined by the spatiotemporal patterning of cell wall synthesis; therefore a rod-shaped cell such as Escherichia coli faces the challenge of coordinating the nanoscale proteins responsible for peptidoglycan synthesis to construct a micron-scale sacculus. What are the principles that allow cell wall synthesis proteins to establish order over a range of length scales spanning nearly three orders of magnitude? We approached this question by examining the process of reversion in cell-wall-deficient ‘L-forms’ of E. coli, in which cell-wall synthesis has disrupted by beta-lactam antibiotics. An L-form undergoing reversion begins in a spherical shape without an intact cell wall. When cell wall synthesis inhibiting antibiotics are removed, the cell generates newrod-shaped protrusions, which eventually undergo septation and adopt the normal rod morphology of E. coli. The reversion of L-forms thus provides on opportunity to study morphogenesis in bacteria lacking an intact cell wall. We therefore investigated the morphological dynamics of the reversion process in L-forms of E. coli, and simultaneously imaged the localization of MreB, a bacterial actin required for the rod-like morphology of many bacteria. We found that MreB localizes to negatively curved regions of the cell (e.g. invaginations), and furthermore targets cell wall synthesis to those regions. Our results therefore suggest a model in which the localization of MreB in response to geometric cues is crucial to morphogenesis in E. coli.