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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 rodlike shape determination under standard laboratory conditions but is required for the robustness of rod shape and growth under conditions of cell wall stress.bacterial cytoskeleton | bacterial cell shape | cell growth | cytoskeleton dynamics | robust rod shape B acterial cell shape is structurally determined by a rigid peptidoglycan (PG) cell wall built outside of the cytoplasmic membrane by a series of cell wall assembly enzymes (1). In many rod-shaped species these enzymes are coordinated by the actinlike protein, MreB, though the mechanism coupling this cytoplasmic protein to the extracellular cell wall enzymes and the specific functions executed by MreB have remained largely mysterious. Polymeric MreB is necessary to maintain rod-shaped cells, as inhibition of MreB polymerization or deletion of mreB cause cells to lose their rod shape. Initially, MreB was thought to form long helical structures that statically define rod shape (2, 3). Later, improved fluorescent fusion proteins and imaging methods revealed that MreB forms short polymers that dynamically rotate around the cell circumference (4-7).This circumferential rotation requires cell wall synthesis and is conserved across both Gram-negative and Gram-positive species (5-7), leading multiple groups to conclude that rotation promotes rod-shape formation. However, experimentally testing this hypothesis has proven difficult because all previous attempts to disrupt rotation have either led to cell death or massive cell shape changes, making it impossible to isolate the specific function of MreB rotation (5, 6). Furthermore, it remained difficult to explain the mechanistic link between cell wall growth and MreB rotation because of their separation in space by the cytoplasmic membrane. Here, we address both the coupling of MreB to cell wall synthesis and the function of MreB rotation. Results and DiscussionRodZ Rotates Similarly to MreB. We initially set out to identify proteins necessary for MreB rotation. In Escherichia coli, multiple proteins have been suggested to interact with MreB, including the penicillin binding protein (PBP) cell wall synthesis enzymes and RodZ, an integral membrane protein that directly binds MreB (8-11). PBP2 inhibitor...

Cell shape matters across the kingdoms of life, and cells have the remarkable capacity to define and maintain specific shapes and sizes. But how are the shapes of micron-sized cells determined from the coordinated activities of nanometer-sized proteins? Here, we review general principles that have surfaced through the study of rod-shaped bacterial growth. Imaging approaches have revealed that polymers of the actin homolog MreB play a central role. MreB both senses and changes cell shape, thereby generating a self-organizing feedback system for shape maintenance. At the molecular level, structural and computational studies indicate that MreB filaments exhibit tunable mechanical properties that explain their preference for certain geometries and orientations along the cylindrical cell body. We illustrate the regulatory landscape of rod-shape formation and the connectivity between cell shape, cell growth, and other aspects of cell physiology. These discoveries provide a framework for future investigations into the architecture and construction of microbes.

The rise of antibiotic resistance and declining discovery of new antibiotics have created a global health crisis. Of particular concern, no new antibiotic classes have been approved for treating Gram-negative pathogens in decades. Here, we characterize a compound, SCH-79797, that kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism of action (MoA) with undetectably low resistance frequencies. In an animal host model, SCH-79797 reduces pathogenesis of Acinetobacter baumannii, a drug-resistant Gramnegative pathogen. To characterize the MoA of SCH-79797 we combined quantitative imaging, proteomic, genetic, metabolomic, and cell-based assays. This pipeline shows that SCH-79797 has two independent cellular targets, folate metabolism and bacterial membrane integrity, and outperforms combination treatments with other antifolates and membrane disruptors in killing MRSA persisters. Thus, SCH-79797 represents a promising lead antibiotic and suggests that combining multiple MoAs onto a single chemical scaffold may be an underappreciated approach to target challenging bacterial pathogens..

Dynamic control of cell polarity is of critical importance for many aspects of cellular development and motility. In Myxococcus xanthus, MglA, a G protein, and MglB, its cognate GTPase-activating protein, establish a polarity axis that defines the direction of movement of the cell and that can be rapidly inverted by the Frz chemosensory system. Although vital for collective cell behaviours, how Frz triggers this switch has remained unknown. Here, we use genetics, imaging and mathematical modelling to show that Frz controls polarity reversals via a gated relaxation oscillator. FrzX, which we identify as a target of the Frz kinase, provides the gating and thus acts as the trigger for reversals. Slow relocalization of the polarity protein RomR then creates a refractory period during which another switch cannot be triggered. A secondary Frz output, FrzZ, decreases this delay, allowing rapid reversals when required. Thus, this architecture results in a highly tuneable switch that allows a wide range of reversal frequencies.