Peptidoglycan layer and disruption processes in Bacillus subtilis cells visualized using quick-freeze, deep-etch electron microscopy

Abstract

Peptidoglycan, which is the main component of the bacterial cell wall, is a heterogeneous polymer of glycan strands cross-linked with short peptides and is synthesized in cooperation with the cell division cycle. Although it plays a critical role in bacterial survival, its architecture is not well understood. Herein, we visualized the architecture of the peptidoglycan surface in Bacillus subtilis at the nanometer resolution, using quick-freeze, deep-etch electron microscopy (EM). Filamentous structures were observed on the entire surface of the cell, where filaments about 11 nm wide formed concentric circles on cell poles, filaments about 13 nm wide formed a circumferential mesh-like structure on the cylindrical part and a ‘piecrust’ structure was observed at the boundary. When growing cells were treated with lysozyme, the entire cell mass migrated to one side and came out from the cell envelope. Fluorescence labeling showed that lysozyme preferentially bound to a cell pole and cell division site, where the peptidoglycan synthesis was not complete. Ruffling of surface structures was observed during EM. When cells were treated with penicillin, the cell mass came out from a cleft around the cell division site. Outward curvature of the protoplast at the cleft seen using EM suggested that turgor pressure was applied as the peptidoglycan was not damaged at other positions. When muropeptides were depleted, surface filaments were lost while the rod shape of the cell was maintained. These changes can be explained on the basis of the working points of the chemical structure of peptidoglycan.

Quick-Freeze, Deep-Etch Electron Microscopy Reveals the Characteristic Architecture of the Fission Yeast Spore

“…To prepare a replica, the frozen sample was knife-fractured, and subjected to platinum and carbon rotary-shadowing in a JFDV freeze-etching device (JEOL, Akishima, Japan). The platinum replica was removed by floatation on the surface of hydrofluoric acid, cleaned in commercial bleach, rinsed in distilled water, and transferred to a 400-mesh copper grid as previously described [ 23 , 24 ]. Replicas were viewed under a transmission electron microscope (JEM1010, JEOL) at an acceleration voltage of 80 kV.…”

Movements ofMycoplasma mobilegliding machinery detected by high-speed atomic force microscopy

“…To observe the shape of immobilized cells, we adopted quick-freeze, deep-etch electron microscopy that visualizes cells under aqueous conditions with nanometer spatial resolution (36,37). The morphology of immobilized cells (Fig.…”

“…The surface structure, composed of mainly large filamentous proteins, may be too thin and/or mobile to be detected by the current scanning performance of HS-AFM on the cell membrane (12)(13)(14)18). The lack of a peptidoglycan layer should be advantageous for visualizing the inside structure, due to the lack of stiffness (36)(37)(38). Moreover, the internal structure should be sufficiently stiff and positioned beneath the cell membrane, reminiscent of cortical actin in animal cells (34).…”

Tree of motility – A proposed history of motility systems in the tree of life

“…The loss of peptidoglycan is likely related to the parasitic or commensal lifestyle of Mollicutes. Generally, in order for Bacteria to live inside animals and plants, it is necessary to deal with the stress of peptidoglycan layer decomposition by lysozyme (Kawai, Mickiewicz, & Errington, 2018;Tulum, Tahara, & Miyata, 2019). Also, peptidoglycans are a target of innate immunity (Royet & Dziarski, 2007;Royet, Gupta, & Dziarski, 2011).…”