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Bacteria have remarkably robust cell shape control mechanisms. For example, cell diameter only varies by a few percent across a given population. The bacterial actin homolog, MreB, is necessary for establishment and maintenance of rod shape although the detailed properties of MreB that are important for shape control remained unknown. In this study, we perturb MreB in two ways: by treating cells with the polymerization-inhibiting drug A22 and by creating point mutants in mreB. These perturbations modify the steady-state diameter of cells over a wide range, from 790 ± 30 nm to 1700 ± 20 nm. To determine which properties of MreB are important for diameter control, we correlated structural characteristics of fluorescently tagged MreB polymers with cell diameter by simultaneously analyzing three-dimensional images of MreB and cell shape. Our results indicate that the helical pitch angle of MreB inversely correlates with the cell diameter of Escherichia coli. Other correlations between MreB and cell diameter are not found to be significant. These results demonstrate that the physical properties of MreB filaments are important for shape control and support a model in which MreB organizes the cell wall growth machinery to produce a chiral cell wall structure and dictate cell diameter.

The rod shape of most bacteria requires the actin homolog, MreB. Whereas MreB was initially thought to statically define rod shape, recent studies found that MreB dynamically rotates around the cell circumference dependent on cell wall synthesis. However, the mechanism by which cytoplasmic MreB is linked to extracytoplasmic cell wall synthesis and the function of this linkage for morphogenesis has remained unclear. Here we demonstrate that the transmembrane protein RodZ mediates MreB rotation by directly or indirectly coupling MreB to cell wall synthesis enzymes. Furthermore, we map the RodZ domains that link MreB to cell wall synthesis and identify mreB mutants that suppress the shape defect of ΔrodZ without restoring rotation, uncoupling rotation from rod-like growth. Surprisingly, MreB rotation is dispensable for rod-like shape determination under standard laboratory conditions but is required for the robustness of rod shape and growth under conditions of cell wall stress.

Over the past few years we have developed an image-processing framework that allows us to extract precise 3D shapes of bacterial cells from fluorescence microscopy data. This active mesh approach minimizes the difference between an observed Z-stack and model shapes that have been convolved with the experimental point spread function. From these xyz coordinates, we calculate geometric parameters such as local curvatures, surface areas, and the relative enrichment of fluorescent signals. Our fluorescent signal is the concentration of the bacterial actin homolog MreB. We have integrated a fluorescent sandwich fusion of MreB with msfGFP at the native locus. This construct has unperturbed mass-doubling times and a proper rod-like shape.We have previously shown in both two and three dimensions that MreB is enriched at negative curvatures and away from positive curvatures [Ursell et al. PNAS 2014]. Here we show in the straight rod Escherichia coli and the curved rod Caulobacter crescentus that MreB senses local surface curvature and, as a result, helps ensure rod-like growth of the cell. The curvature sensed by Escherchia coli MreB is the Gaussian curvature of the surface, the product of the two principal curvatures. Using MreB point mutants that have altered curvature sensitivities, we are testing the hypothesis that cells straighten deformations by patterning growth at the proper geometry. To stably generate their comma shaped geometry, Caulobacter crescentus have developed ways to overcome this straightening mechanism. The enrichment profile of CcMreB shows a plateau in enrichment at small, positive Gaussian curvature values that depends on the protein crescentin. In addition to its structural role in bending the cell, we propose a mechanism whereby crescentin alters the curvature preference of MreB and allows the stabile propagation of the Caulobacter shape.

In virtually all bacteria, the cell wall is crucial for mechanical integrity and for determining cell shape. Escherichia coli's rod-like shape is maintained via the spatiotemporal patterning of cell-wall synthesis by the actin homologue MreB. Here, we transiently inhibited cell-wall synthesis in E. coli to generate cell-wall-deficient, spherical L-forms, and found that they robustly reverted to a rod-like shape within several generations after inhibition cessation. The chemical composition of the cell wall remained essentially unchanged during this process, as indicated by liquid chromatography. Throughout reversion, MreB localized to inwardly curved regions of the cell, and fluorescent cell wall labelling revealed that MreB targets synthesis to those regions. When exposed to the MreB inhibitor A22, reverting cells regrew a cell wall but failed to recover a rod-like shape. Our results suggest that MreB provides the geometric measure that allows E. coli to actively establish and regulate its morphology.

Cells typically maintain characteristic shapes, but the mechanisms of self-organization for robust morphological maintenance remain unclear in most systems. Precise regulation of rod-like shape in Escherichia coli cells requires the MreB actin-like cytoskeleton, but the mechanism by which MreB maintains rod-like shape is unknown. Here, we use time-lapse and 3D imaging coupled with computational analysis to map the growth, geometry, and cytoskeletal organization of single bacterial cells at subcellular resolution. Our results demonstrate that feedback between cell geometry and MreB localization maintains rod-like cell shape by targeting cell wall growth to regions of negative cell wall curvature. Pulse-chase labeling indicates that growth is heterogeneous and correlates spatially and temporally with MreB localization, whereas MreB inhibition results in more homogeneous growth, including growth in polar regions previously thought to be inert. Biophysical simulations establish that curvature feedback on the localization of cell wall growth is an effective mechanism for cell straightening and suggest that surface deformations caused by cell wall insertion could direct circumferential motion of MreB. Our work shows that MreB orchestrates persistent, heterogeneous growth at the subcellular scale, enabling robust, uniform growth at the cellular scale without requiring global organization.

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.