RodZ links MreB to cell wall synthesis to mediate MreB rotation and robust morphogenesis

Abstract

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...

YodL and YisK Possess Shape-Modifying Activities That Are Suppressed by Mutations in Bacillus subtilis mreB and mbl

“…If YodL does specifically target MreB activity, then these results suggest that MreB likely retains at least some of its width maintenance function. Morgenstein et al recently found that the interaction between RodZ and MreB in E. coli is required for MreB rotation but that MreB rotation was not required for rod shape or cell viability under standard laboratory conditions (26). The findings of that study are consistent with prior findings indicating that RodZ is not absolutely required for maintenance of rod shape (25).…”

“…However, in various mutant backgrounds, rodZ can be deleted without loss of rod shape or viability, indicating that RodZ is not absolutely required for MreB's function in maintaining shape (24)(25)(26). Based on these observations and others, it has been proposed that MreB-RodZ interactions may regulate some aspect of MreB activity (10,26).…”

“…Aside from RodZ (10,26), only a few proteins targeting MreB activity in vivo have been identified. In E. coli, the YeeU-YeeV prophage toxin-antitoxin system is comprised of a negative regulator of MreB polymerization, CbtA (36), and a positive regulator of MreB bundling, CbeA (37).…”

Single-molecule imaging reveals modulation of cell wall synthesis dynamics in live bacterial cells

“…The mechanistic link between cell wall synthesis and processive MreB motion, which was recently shown to depend on RodZ35, remains to be fully elucidated but has important implications for cell growth and shape determination. A motor-like enzymatic cycle similar to eukaryotic actinomyosin systems may not be required; localization of MreB to areas of negative membrane curvature could explain circumferential motion21.…”

A Periplasmic Polymer Curves Vibrio cholerae and Promotes Pathogenesis

“…Images were acquired using NIS Elements software (Nikon) on both the upright and inverted microscopes. Images for 3D reconstructions and subcellular localization were taken on an upright monolithic aluminum microscope (custom-made, (Morgenstein et al, 2015)) with a 100×/1.49 N.A. objective (Nikon), iXon DU-897 cooled EMCCD camera (Andor Technology), and a homemade LabView software package (National Instruments).…”