Cell complexity in prokaryotic cells, whether Bacteria or Archaea, is generally considered low, with a single compartment. A few examples, however, have been recently reported showing evidence for compartmentalization [e.g. 1,2,3]. These studies gained in particular from improved sample preparation methods (plunge freezing or high-pressure freezing), adequate follow-up methods, and 3D imaging of the samples, either by FIB-SEM or by electron tomography. Here we report on our analyses of the ultrastructure of exceptional Archaea of the genera Ignicoccus and Pyrococcus, which show remarkable physiological characteristics (e.g. optimal growth at T~85 up to 100°C, under anoxic conditions), and unusual ultrastructural features. Although prokaryotes, these cells have a diameter of > 1 µm, which does not allow for reasonable cryoTEM observation without sectioning. Thus, samples were harvested by gentle concentration on ultrafiltration membranes, cryo-immobilized by high-pressure freezing, freeze-substituted, and finally resin-embedded; this enabled us to perform 3D ultrastructural analysis by (S)TEM tomography and immuno-localization studies on sections of the same resin block.

So far, we are about to routinely investigate single or serial sections (ranging from 200 up to 800 nm in thickness), on a transmission electron microscope operated as TEM or STEM at 200 keV. For STEM, the illumination is adjusted to have an almost parallel beam, i.e. small convergence angle, resulting in an extended depth of focus [4]. Under these conditions, if the sample is imaged at high tilt angle (e.g. up to 70 deg), all visible image details are seen ‘in focus’, which greatly facilitates image alignment during 3D image reconstruction using IMOD (Boulder, Co, USA). In addition, the system in use allows parallel acquisition of BF and DF signal. Reconstruction and visualization is done using either WBP or SIRT in IMOD, and final manual segmentation using AMIRA®.

Our 3D data show novel and remarkable ultrastructural features: (A) Ignicoccus cells possess two membranes; in the intermembrane compartment, there is a complex endogenous membrane system. Attached to the outer cellular membrane, we have localized enzyme complexes for proton gradient generation, ATP and acetyl-CoA synthesis, and CO₂ fixation [5]. (B) Pyrococccus cells possess flagella of the archaeal type [6], i.e. extracellular filaments involved in motility and cell-cell interaction, and a cytoplasmic membrane and an S-layer, and a so far unknown layer inside the cytoplasm presumbably involved in anchoring or organizing the flagellar bundle.

[1] R Santarella-Mellwig et al 2013 PLOS Biol 11, e1001565

[2] S Schlimpert et al 2012 Cell 151, 1270

[3] B Junglas et al 2008 Arch Microbiol 190: 395

[4] AA Sousa and RD Leapman 2012 Ultramicroscopy 123, 38

[5] H Huber et al 2012 Antonie van Leeuwenhoek 102, 203

[6] D Näther-Schindler et al 2014 Frontiers Microbiol 5, 695

Figures:

Fig 1: A: two images from a BF STEM tomography tiltseries of a Pyrococcus cell, at nominal tilt angle of 0° (left) and 66° (right). Even at 66° tilt, all fiducials are perfectly in focus. Bar: 500 nm. B: Isosurface visualization of the cell after 3D reconstruction (IMOD, Boulder, Co), visualizing the cell body and the flagella.

Fig 2: A: 2nm slice from a 3D reconstruction of Ignicoccus, from a TEM tomography dataset incl. nine consecutive serial 200 nm sections; bar: 500 nm. B: 3D visualization of the inner membrane of an Ignicoccus cell, manual segmentation. Cell diameter: about 1.5 µm.

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EMC Abstracts - https://emc-proceedings.com/abstract/serial-section-stem-tomography-of-prokaryotic-cells/