The Stack: A New Bacterial Structure Analyzed in the Antarctic Bacterium Pseudomonas deceptionensis M1T by Transmission Electron Microscopy and Tomography

Abstract

In recent years, improvements in transmission electron microscopy (TEM) techniques and the use of tomography have provided a more accurate view of the complexity of the ultrastructure of prokaryotic cells. Cryoimmobilization of specimens by rapid cooling followed by freeze substitution (FS) and sectioning, freeze fracture (FF) and observation of replica, or cryoelectron microscopy of vitreous sections (CEMOVIS) now allow visualization of biological samples close to their native state, enabling us to refine our knowledge of already known bacterial structures and to discover new ones.Application of these techniques to the new Antarctic cold-adapted bacterium Pseudomonas deceptionensis M1T has demonstrated the existence of a previously undescribed cytoplasmic structure that does not correspond to known bacterial inclusion bodies or membranous formations. This structure, which we term a “stack”, was mainly visualized in slow growing cultures of P . deceptionensis M1T and can be described as a set of stacked membranous discs usually arranged perpendicularly to the cell membrane, but not continuous with it, and found in variable number in different locations within the cell. Regardless of their position, stacks were mostly observed very close to DNA fibers. Stacks are not exclusive to P . deceptionensis M1T and were also visualized in slow-growing cultures of other bacteria. This new structure deserves further study using cryoelectron tomography to refine its configuration and to establish whether its function could be related to chromosome dynamics.

Cryo-electron tomography of plunge-frozen whole bacteria and vitreous sections to analyze the recently described bacterial cytoplasmic structure, the Stack

“…In a previous article (Delgado et al, 2013), we described for the first time a bacterial cytoplasmic structure referred to as a ''Stack''. We used a variety of electron microscopy techniques to decipher this unusual structural feature, including TEM observation of high-pressure frozen and freeze-substituted (HPF-FS), Tokuyasu and freeze-fractured samples.…”

“…HPF of samples was performed on colonies resuspended in 30% dextran (Fluka) in 0.01 M phosphate buffer saline (PBS) (Delgado et al, 2013). The suspensions were introduced into 350 lm inner diameter copper tubes and ultrarapidly frozen using a Leica EMPACT High-Pressure Freezer (Leica Microsystems, Vienna, Austria).…”

“…Firstly, fiducial markers were attached to the carbon of the grids by successively floating them on drops of protein A coupled to 10-nm diameter colloidal gold particles (CMC, University of Utrecht, The Netherlands) 1:500 in 0.01 M PBS and distilled water (Delgado et al, 2013). The grids were left in a dry chamber for at least 2 h before being used.…”

“…In our previous work, we described a new bacterial structure termed a ''Stack'', which was revealed by different electron microscopy techniques and electron tomography and CEMOVIS (Delgado et al, 2013). Stacks, which were observed in bacteria of the Pseudomonas genus, were defined as piles of oval disc subunits surrounded by a membrane-like structure and localized in the cell cytoplasm.…”

Cellular structural biology as revealed by cryo-electron tomography

“…In this method, frozen hydrated cells or tissues of interest are sectioned with a diamond knife resulting in samples with a thickness of 40-100 μm (Al-Amoudi et al, 2004). Samples that have been studied by CEMOVIS thus far include mitochondria (Hsieh et al, 2006), human skin (Al-Amoudi et al, 2007;Al-Amoudi and Frangakis, 2008), microtubules (Bouchet-Marquis et al, 2007), bacteria (Delgado et al, 2013;Zuber et al, 2008) and bacterial spores (Couture-Tosi et al, 2010;Dittmann et al, 2015). However, this technique has its limitations, mainly due to compression of the thin sections in the direction of cutting, which can complicate the proper interpretation of the data (Al-Amoudi et al, 2005).…”