Key mechanistic steps coordinating ATP-driven MreB polymerization and turnover are established and provide a basis for a complete MreB assembly–disassembly cycle and for further elucidating how MreB dynamics contribute to cell-wall organization.
Abstract
MreB, a bacterial actin homolog and polymerizing ATPase, is central to cell-shape maintenance and cell-wall integrity. Its functions rely on its ability to assemble into dynamic, membrane-associated polymers. However, how nucleotide binding and hydrolysis, MreB-MreB contacts, and membrane association are coordinated to enable polymer assembly and disassembly remains unclear. Here, we combined genetics and live-cell microscopy with biochemical approaches to dissect these processes. Using a highly sensitive reporter of MreB activity, we identified, through a genetic screen, residues critical for MreB function in Bacillus subtilis. Subsequent extensive characterization of corresponding stable variants of the homologous Geobacillus stearothermophilus MreB revealed that ATP binding, but not ATP hydrolysis, is required for polymerization. Productive longitudinal intraprotofilament contacts are required for efficient ATP hydrolysis and enhance membrane association. Perturbations predicted to weaken lateral interprotofilament contacts altered membrane association and modulated ATPase activity. Together, these effects provide experimental evidence consistent with long-range functional coupling among the longitudinal and lateral protofilament interfaces, the distant nucleotide-binding site, and membrane association dynamics. Moreover, impaired ATP hydrolysis delays disassembly of lipid-associated polymers, indicating that hydrolysis promotes polymer turnover. These results establish key mechanistic steps coordinating ATP-driven MreB polymerization and turnover and provide a basis for a complete MreB assembly–disassembly cycle and for further elucidating how MreB dynamics contribute to cell-wall organization.
During endospore (spore) development in bacteria, polar cell division generates two transcriptionally distinct cellular compartments, the mother cell and future spore (forespore). Signalling between these cells leads to sequential and compartmentalized transcription, along with key morphogenetics events, including the phagocytic-like process of engulfment and the recruitment of coat proteins to the engulfing membrane. The SpoIVA ATPase is an essential sporulation protein that assembles into static filaments at the forespore surface during engulfment, where it functions as the basement layer for coat assembly. Here, using Bacillus subtilis, we reveal an additional role for SpoIVA during engulfment. Cytological analysis of a spoIVA null mutant (ΔspoIVA) revealed engulfment defects such as septal membrane bulges and asymmetric membrane migration, similar to those typically associated with impaired peptidoglycan remodelling during engulfment. Engulfment defects were exacerbated when ΔspoIVA was combined mutants known to impact engulfment progression and efficiency. Importantly, a spoIVA mutant (K30A) impaired for ATP hydrolysis and filament formation in vitro but partially functional for coat assembly in vivo, closely phenocopies the spoIVA null mutant engulfment defects. Based on these data, we propose a model whereby SpoIVA polymerisation at the spore surface, independently of coat assembly, plays a mechanical and structural role during engulfment, akin to the cytoskeletal proteins that drive phagocytosis in eukaryotic cells. IMPORTANCE Endospore formation relies on membrane remodeling to generate highly resistant dormant spores in human, animal and insect pathogens. During engulfment, the mother-cell membrane migrates around the forespore in a phagocytic-like process while the spore coat is simultaneously assembled. SpoIVA is widely recognized as the ATPase that polymerizes at the forespore surface to nucleate assembly of the multilayered spore coat. Here, we demonstrate that SpoIVA also performs a distinct function during engulfment that is independent of its role in coat formation. Cells lacking SpoIVA, or carrying a polymerization-defective SpoIVA variant, exhibit membrane deformations characteristic of impaired engulfment. We propose that SpoIVA polymers provide structural and mechanical support for membrane migration, analogous to the role of cytoskeletal systems during eukaryotic phagocytosis. These findings reveal an unexpected function for SpoIVA and demonstrate that bacterial protein polymers can provide mechanical support for membrane remodeling during complex developmental processes, analogous to cytoskeletal systems in eukaryotic cells
Mycobacteria possess a complex double-membrane cell envelope critical for survival and pathogenesis. Proper assembly of this architecture requires the biosynthesis and transport of major components, including arabinogalactan (AG) polysaccharides and mycolic acids (MAs), but how these processes are effectively coordinated is unknown. Here, we discover an essential membrane complex that serves as a regulatory node in mycobacterial envelope biogenesis. The acyltransferase TmaT and the arabinofuranosyltransferase AftD physically interact; cryo-EM structures reveal a 1:1 stoichiometry, and present a novel fold for TmaT, featuring a central channel that binds co-factor for acetylation in the periplasm. We establish that the TmaT-AftD interaction, and the catalytic activities of both enzymes, are required for MA transport across the cell envelope, as well as AG ligation to the cell wall, the final stage of AG biosynthesis. The TmaT-AftD complex coordinates the two major envelope assembly pathways, presenting a structural vulnerability for future anti-mycobacterial drug development.
The cytoskeleton, comprising intracellular filamentous structures composed of polymerized proteins, is crucial for the survival of both eukaryotes and prokaryotes. Although bacterial cytoskeletal proteins have diverged, they generally do not drive cellular motility. Spiroplasma, a genus of wall-less helical bacteria, swims by propagating a helicity-switching point (kink) along its cell axis. Unlike typical walled bacteria, whose motility depends on widespread motility machineries such as flagella and pili, Spiroplasma swimming is powered by the coordinated dynamics of five isoforms of bacterial actin MreB (SMreB1–5), which are grouped into three phylogenetic classes: SMreB1 and 4, SMreB2 and 5, and SMreB3. Despite the efforts to understand Spiroplasma swimming, its molecular mechanism remains unclear. In this review, we summarize how in vitro analyses of SMreBs have provided mechanistic insights into Spiroplasma swimming. While all SMreBs conserve the canonical actin fold, each SMreB class exhibits unique characteristics in its polymerized structures, ATPase activities, polymerization dynamics, and membrane binding. Studies of an essential SMreB subset for Spiroplasma swimming, i.e. SMreB1 and SMreB5, have revealed that SMreB1 binds to polymerized SMreB5 and disassembles it depending on the nucleotide state. These results challenge the previous model in which Spiroplasma swimming is driven by the coordinated extension and contraction of two distinct SMreB filaments. Finally, we discuss potential molecular mechanisms underlying Spiroplasma swimming and highlight key questions that must be answered to validate these models.
This work uncovered an unprecedented role of V-ATPase in shaping the macropinocytic cup in the pathogenic amoeba Entamoeba histolytica and highlights the novel role of V-ATPase in directly driving actin polymerization, in conjunction with phosphatidic acid, to shape the macropinocytic cup.
Septal peptidoglycan (sPG) biosynthesis during bacterial cell division is driven by the dynamic divisome complex. Its core components, glycosyltransferase FtsW and transpeptidase FtsI are responsible for glycan chain polymerization and crosslinking, respectively. FtsI is also the target of β-lactams. The essential membrane complex FtsQ-FtsB-FtsL regulates FtsWI enzymatic activity. However, the mechanism of FtsQBLWI-mediated sPG synthesis and β-lactam-induced conformational changes have remained elusive. Here, we present cryo-electron microscopy (cryo-EM) structures of the Pseudomonas aeruginosa FtsQBLWI complex in the apo state and bound to aztreonam or imipenem. Our work reveals intricate structural details, including the putative substrate-binding cavities of FtsW, FtsI-mediated allosteric activation of FtsW, and β-lactam-triggered conformational rearrangements. Collectively, these structural, genetic and biochemical analyses reveal the mechanism of FtsQBLWI-controlled sPG synthesis and β-lactam action on this complex, providing a molecular basis for optimizing existing β-lactams and developing novel antibiotics.
Shimin Zhu, Yanjie Hu, Rong Wang et al.· Structure· 0 citations