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Free keywords:
TRANSMISSION ELECTRON-MICROSCOPY; SURFACE GLYCOPROTEIN LAYER; VOLTA
PHASE PLATE; HALOBACTERIUM-SALINARUM; HALOFERAX-VOLCANII; CRYO-EM;
CELLS; HALOBIUM; BACTERIAL; VESICLESBiochemistry & Molecular Biology; Microbiology; Halobacterium salinarum; Haloarchaea; Gas vesicles; Cryo-electron
tomography; Focused-ion-beam micromachining; Vitrification;
Abstract:
Most halophilic Archaea of the class Halobacteriaceae depend on the presence of several molar sodium chloride for growth and cell integrity. This poses problems for structural studies, particularly for electron microscopy, where the high salt concentration results in diminished contrast. Since cryo-electron microscopy of intact cells provides new insights into the cellular and molecular organization under close-to-live conditions, we evaluated strategies and conditions to make halophilic microbes available for investigations in situ. Halobacterium salinarum, the test organism for this study, usually grows at 4.3 M NaCl. Adaptation to lower concentrations and subsequent NaCl reduction via dialysis led to still vital cells at 3 M salt. A comprehensive evaluation of vitrification parameters, thinning of frozen cells by focused-ion-beam micromachining, and cryo-electron microscopy revealed that structural studies under high salt conditions are possible in situ.
