Cryo-electron microscopy of an extremely halophilic microbe: technical aspects

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00792-016-0912-0) contains supplementary material, which is available to authorized users.

Analysis of Cell–Cell Bridges in Haloferax volcanii Using Electron Cryo-Tomography Reveal a Continuous Cytoplasm and S-Layer

“…Furthermore, more cell–cell bridges would probably have been observed in a medium containing a higher salt concentration, as mating efficiency was shown to be higher at high salt concentrations (3.4 M NaCl), which is most likely caused by an altered S-layer glycosylation ( Guan et al, 2012 ; Shalev et al, 2017 ). Since previous studies in Halobacterium salinarum showed that higher salt concentrations reduced the contrast of electron micrographs ( Bollschweiler et al, 2017 ), images were obtained from cells grown in a medium containing 2.4 M NaCl where optimal growth was still observed.…”

In situ architecture and cellular interactions of PolyQ inclusions

“…A pioneering work from (Marko et al, 2006) showed the proof-of-principle that a new non-mechanical approach -using a focused-ion-beam -can be used to prepare thin vitreous sections in prokaryotes. In 2009 we developed the cryo-FIB technology for single cells on EM-grids (Bäuerlein, 2010;Rigort and Bäuerlein et al, 2012) that is now used as a routine preparation-technique for in-situ cryo-TEM studies in eukaryotes and prokaryotes (Arnold et al, 2016;Asano et al, 2015;Böck et al, 2017;Bollschweiler et al, 2017;Engel et al, 2015bEngel et al, , 2015aFukuda et al, 2017;Hagen et al, 2015;Jasnin et al, 2016;Mahamid et al, 2016;Pfeffer et al, 2017;Schaffer et al, 2015) as well as in this work (Bäuerlein et al, 2017).…”

Archaeal cell surface biogenesis

“… 2016 ), especially as technical limitations due to the high salt concentrations have been partially overcome (Bollschweiler et al. 2017 ). Moreover, the expansion of available tools for in vitro studies will aid to address basic questions related to the energetics of protein transport.…”