How advances in cryo-electron tomography have contributed to our current view of bacterial cell biology

Abstract

Advancements in the field of cryo-electron tomography have greatly contributed to our current understanding of prokaryotic cell organization and revealed intracellular structures with remarkable architecture. In this review, we present some of the prominent advancements in cryo-electron tomography, illustrated by a subset of structural examples to demonstrate the power of the technique. More specifically, we focus on technical advances in automation of data collection and processing, sample thinning approaches, correlative cryo-light and electron microscopy, and sub-tomogram averaging methods. In turn, each of these advances enabled new insights into bacterial cell architecture, cell cycle progression, and the structure and function of molecular machines. Taken together, these significant advances within the cryo-electron tomography workflow have led to a greater understanding of prokaryotic biology. The advances made the technique available to a wider audience and more biological questions and provide the basis for continued advances in the near future.

Keywords: CEMOVIS; Cryo-CLEM; Cryo-ET; Data processing; FIB-milling; Subtomogram averaging.

Fig. 1

Changing view of the bacterial cell structure. A; Traditional EM image of Pseudomonas aeruginosa (arrow indicates tightly packed material). Image reproduced from (Latino et al., 2019) with permission. B; Simplified schematic of a bacterial cell, lacking structural detail (made in Biorender.com). C; Workflow of cryo-electron tomography. D; Modern interpretation of a bacterium, containing selected structural features (schematic by Robert van Sluis).

Fig. 2

FtsZ structure; (A) comparison from the first EM visualization of the FtsZ structure within Escherichia coli cell (Bi and Lutkenhaus, 1991) with (B) the current models of asymmetric constriction of a Belliella baltica cell (white arrow indicates the asymmetric division site) (Yao et al., 2017) and (C) a visualization of a semi-atomic model of symmetric FtsZ ring constriction in a liposome (Szwedziak et al., 2014).

Fig. 3

Comparative micrographs of Mycobacteria smegmatis cell envelope for the different sectioning types; CEMOVIS (A); and classical preparation with chemical fixation and resin embedding (B). Red line indicates the cell wall thickness, whereby the inner and outer membranes are easily detectable in A compared to B. Adapted from (Bleck et al., 2010). CM, cytoplasmic membrane; MOM, mycobacterial outer membrane.

Fig. 4

A. Schematic representation of cryo-FIB milling of the bacterial cell). B & C. By thinning of Bacillus subtilis using cryo-FIB SEM, Khanna et al. were able to identify the location of FtsZ (blue) and FtsA (purple) at the division septum for vegetative (B) and sporulating cells (C). Adapted from Khanna et al. (Khanna et al., 2021).

Fig. 5

cryo-FLM workflow used in the identification of the extended T6SS in enteroaggregative E. coli. The cells were first vitrified and then imaged by cryo-FLM to identify the cells that contained the Hcp1-CouAA labeled T6SS. Subsequent cryo-ET and rotation of the tomogram in the area of interest led to the visualization of the T6SS.

Fig. 6

A, Top: Top view of a chemotaxis array in a Salmonella enterica minicell. IM: inner membrane, OM: outer membrane. Bottom: subtomogram averages of chemoreceptor arrays in diverse species, highlighting the universal receptor arrangement. Reproduced with permission from Briegel et al., 2012. B, Left: STA of the functional unit. PP: periplasmic ligand-binding domains, MH: methylation-helix bundles, GH: flexible regions containing the glycine hinge, KC: kinase control region Right: Molecular model. Red: receptors, blue: CheA, gold: CheW. Reproduced with permission from Burt et al. (2020). C. Schematic representation of the structural changes between the kinase-off (left) and kinase-on states (right). Courtesy of Dr. Alise Muok (Muok et al., 2020).

Fig. 7

Flagellar motor, showing the conserved core elements as well as the species and genera-specific motor adaptations to fulfill their needs in their specific environment. Adapted from (Carroll and Liu, 2020).