A new view into prokaryotic cell biology from electron
cryotomography
Catherine M. Oikonomou
and
Grant J. Jensen
Howard Hughes Medical Institute; Division of Biology and Biological Engineering, California
Institute of Technology, 1200 E. California Blvd., Pasadena, California 91125, USA
Abstract
Electron cryotomography (ECT) enables intact cells to be visualized in 3D in an essentially native
state to ‘macromolecular’ (~4 nm) resolution, revealing the basic architectures of complete
nanomachines and their arrangements
in situ
. Since its inception, ECT has advanced our
understanding of many aspects of prokaryotic cell biology, from morphogenesis to subcellular
compartmentalization and from metabolism to complex interspecies interactions. In this Review,
we highlight how ECT has provided structural and mechanistic insights into the physiology of
bacteria and archaea and discuss prospects for the future.
Historically, bacteria and archaea were viewed mainly as undifferentiated sacs of jumbled
enzymes (for example, see REFS
1
,
2
). Technological advances, particularly in imaging, have
given rise to a much more complicated and beautiful view of prokaryotic cells
3
. We now see
these cells as organized assemblies of macromolecular machines
4
, optimized to travel
through and interact with complex and dynamic environments. This increasing knowledge of
finer and finer details of microbial cell biology has been enabled by astounding
technological advances in imaging. In the seventeenth century, Antonie van Leeuwenhoek
constructed microscopes with magnifying powers of several hundred times, enabling the first
visualization of single-celled organisms. The development of electron microscopy in the first
half of the twentieth century enabled magnification on the order of hundreds of thousands of
times, bringing resolution from the level of microorganisms to that of atoms. This provided
an unprecedented view inside cells, and much of what we know about cellular structure has
come from studies using electron microscopy.
However, because of the vacuum that is necessary to operate an electron microscope, the
traditional preparation of biological samples for electron microscopy involved fixation and
dehydration, which can denature structures and introduce misleading artefacts
5
,
6
. This
limitation was circumvented with the discovery that thin aqueous samples can be cooled so
quickly that water molecules stop rearranging before they can crystallize, which results in
Correspondence to: G.J.J., jensen@caltech.edu.
Competing interests statement
The authors declare no competing interests.
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the formation of a thin film of ‘vitreous ice’ (an amorphous solid that preserves native
cellular structures)
7
. In electron cryotomography (ECT; also known as cryo-ET or CET),
such plunge-frozen samples are imaged in an electron microscope at different angles as they
are tilted, resulting in a series of projection images that can be computationally combined to
produce a reconstruction of the specimen in 3D. Therefore, ECT enables entire cells or parts
of cells to be viewed with macromolecular resolution (~4 nm) in 3D, in what is essentially a
native state (BOX 1; FIG. 1) (Supplementary information S1 (movie)).
Box 1
Electron cryotomography
In electron cryotomography (ECT), cells in standard aqueous media are plunge-frozen
onto electron microscope grids using an efficient cryogen, such as a mixture of ethane
and propane, which cools the sample at ~10
4
K s
−1
, leading to the formation of vitreous
(non-crystalline) ice
7
,
156
. Grids are subsequently kept at the temperature of liquid
nitrogen (~80 K). A series of images is acquired by transmission electron microscopy
(TEM) as the grid is rotated, typically one or two degrees between images. Full 180° tilt
is impossible owing to increasing sample thickness at high angles, as well as intrusion of
the sample holder. In practice, 120° to 140° are usually collected. The resulting
projection images are then digitally reconstructed into a 3D tomogram. Fiducial markers
(usually electron-dense gold beads) can be added to the sample to assist in alignment
during reconstruction. The lack of high-tilt-angle images introduces a ‘missing wedge’
artefact that decreases the resolution of the reconstruction along the z-axis (parallel to the
beam).
The fundamental limitation to resolution in ECT is radiation damage; in imaging a
unique object, the clarity of the final image is limited by how many electrons are
scattered before the sample is destroyed. In addition, the high electron-scattering cross-
section of biological material limits sample thickness to less than ~500 nm for reasonable
image quality in currently available TEMs. Therefore, the small size of some prokaryotic
cells makes them amenable to whole-cell ECT. Thicker cells can be prepared for ECT by
sectioning, for example by vitreous cryosectioning
157
, although this technique can
introduce artefacts, particularly compression artefacts from the pressure of the
blade
158
,
159
. An alternative approach, focused ion beam (FIB) milling, uses an ion
(typically gallium) source to precisely mill away material in a thick frozen sample,
leaving a thin lamella, just a few hundred nanometres thick, that is suitable for imaging
by ECT
160
–
163
. This expands the utility of ECT to larger prokaryotic and eukaryotic cells
and tissues.
The typical resolution of whole-cell ECT is a few nanometres, enabling visualization of
the shapes and organization of macromolecular complexes and bridging the gap between
the near-atomic resolution of X-ray crystallography or NMR spectroscopy and the
broader cellular-level resolution of light microscopy. However, the ongoing development
of many technologies promises to increase resolution. For example, direct detectors are
replacing phosphor charge-coupled devices. In a direct detector, electron hits are recorded
directly (rather than through photon production) and can be counted individually,
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increasing resolution
164
. Resolution is also increasing through the adoption of energy
filters, which block electrons that are inelastically scattered by thick biological samples
and contribute to imaging noise. Zernike and Volta phase plates increase contrast in
images, especially at low spatial frequencies, enabling the resolution of biological detail
with lower electron doses, and therefore less damage to samples
67
,
165
–
169
.
A major limitation of ECT is the thickness of the sample, which should be less than ~500
nm for good resolution. However, the small size of many bacterial and archaeal cells makes
them particularly amenable to ECT analysis. Indeed, ECT has enabled the observation of
many components of these cells, including: the cell envelope and cytoskeleton;
macromolecular machines involved in cell division, motility and navigation; and subcellular
compartments. This has provided new insights into various aspects of prokaryotic
physiology, including metabolism, interspecies cooperation and pathogenesis.
In this Review, we highlight the advances in our structural knowledge of prokaryotic cells
that these efforts have produced (for more focused reviews of the technical aspects of ECT,
see REFS
5
,
8
,
9
; and for a review of eukaryotic applications of ECT, see REF.
10
). In a
previous review of this subject, in 2007, we discussed all of the relevant studies at that time,
15 in total
11
. Now that there is an order of magnitude more studies, we quickly summarize
the existing published literature, highlighting what we feel are particularly interesting
examples. As a narrative tool, we organize our discussion in terms of hypothetical
challenges and opportunities that a single-celled organism might have encountered during
the course of evolution.
Separation from the environment
One of the first requirements for the development of a cell is separation from the
environment by a selectively porous envelope. As a first example of how ECT has provided
new insights into cell biology, we discuss the mycobacterial cell envelope, an understanding
of which is vital for the development of therapeutics.
Until fairly recently, the structure of pathogenic mycobacterial cell envelopes was debated,
with various models proposing either a single membrane or a double membrane. Traditional
electron microscopy of thin fixed sections could not resolve the structure of the
mycobacterial cell envelope, as lipid structures are susceptible to rearrangements that are
caused by dehydration and/or organic solvents during sample preparation. By contrast, ECT
is particularly effective at preserving lipid structures, and studies using vitreous
cryosectioning as well as whole cells have provided the first direct visualization of native
envelopes in
Mycobacterium bovis
bacillus Calmette–Guérin (BCG) and
Mycobacterium
smegmatis
, and the closely related bacterium
Corynebacterium glutamicum
. These studies
revealed that the envelope of these bacteria is composed of an inner cytoplasmic membrane
and a symmetrical outer membrane that is morphologically similar to that of Gram-negative
bacteria
12
,
13
(FIG. 2a), which rules out a fundamentally different envelope morphology for
mycobacteria.
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Structural support
Cellular membranes must be supported against turgor and environmental pressures. The
solution most bacteria use is the peptidoglycan cell wall, which is created by crosslinking
long stiff glycan strands into a mesh-like network with short peptide crosslinks. Despite the
ubiquity of peptidoglycan in microorganisms and decades of study of its structure, details of
the arrangement of the glycan strands remained unclear until fairly recently. Competing
models proposed either a circumferential arrangement of the glycan strands parallel to the
cell surface, a scaffold-like arrangement perpendicular to the surface, or coiled cables. ECT
of purified peptidoglycan sacculi revealed that strands are aligned circumferentially around
the cell in both Gram-negative bacteria (such as
Caulobacter crescentus
and
Escherichia
coli
)
14
(FIG. 2b) and Gram-positive bacteria (such as
Bacillus subtilis
)
15
, with the main
difference between these sacculi simply being the presence of multiple layers in the thicker
Gram-positive cell wall
16
. This insight eroded the notion that Gram-negative and Gram-
positive phyla are only distantly related (although some debate continues regarding the
Gram-positive architecture)
17
.
ECT also helped to clarify the related ‘chlamydial anomaly’: the long-standing debate over
whether Chlamydiae have a peptidoglycan cell wall. This debate arose from the observation
that Chlamydiae are susceptible to peptidoglycan-targeting antibiotics, but attempts to purify
sacculi from these bacteria have been unsuccessful. This contradiction was resolved when
ECT revealed a peptidoglycan cell wall in two Chlamydiae, ‘
Candidatus
Protochlamydia
amoebophila’ and
Simkania negevensis
18
.
Cytoskeleton
A reinforced cell envelope enables cells to adopt different shapes. Most cells maintain
specialized shapes by building an internal scaffold of filaments, or cytoskeleton. The
cytoskeleton, which is well understood in eukaryotes, has been the subject of much debate in
prokaryotes. Homologues of all three main classes of eukaryotic cytoskeletal elements (actin
microfilaments, microtubules and intermediate filaments) have been found in bacterial
genomes, but traditional electron microscopy methods largely failed to identify any of these
filaments in bacterial cells. However, by providing better structural preservation than
traditional electron microscopy methods, ECT has now revealed an impressive diversity of
cytoskeletal filaments in bacterial cells that mediate a range of processes.
The actin homologue MreB has a role in shape determination in many rod-shaped bacteria,
although its exact function remains unclear. Based on light microscopy of fluorescently
tagged MreB, it was thought that long helical filaments of MreB wrapped around the cell,
perhaps globally coordinating cell wall synthesis. However, long filaments were not seen in
the cryotomograms of six bacterial species (
E. coli
,
C. crescentus
,
B. subtilis
,
Vibrio
cholerae
,
Borrelia burgdorferi
and
Acetonema longum
)
19
, and such filaments were later
determined to be artefacts, at least in the case of one MreB fluorescent protein fusion
20
.
These studies highlight the power of ECT to elucidate what is not present, in addition to
what is. Subsequent fluorescence light microscopy studies showed small patches of MreB
dynamically travelling around the cell
21
,
22
. These data support the idea that MreB does not
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form long helical filaments around the cell but also highlight a major limitation of ECT: it
can only provide snapshots of cell structures, missing the dynamics that are inherent in many
cellular processes.
An intermediate filament homologue, crescentin, is known to have an important role in
defining the curved cell shape of
C. crescentus
. ECT helped to identify novel filaments of a
metabolic enzyme, CTP synthase, that seem to negatively regulate crescentin to define the
correct curvature of the cell
23
,
24
. Interestingly, the assembly of CTP synthase into filaments
seems to inhibit its activity, reflecting a more general mechanism of regulating enzymatic
activity by polymerization. Notably, this regulatory mechanism seems to be conserved
across prokaryotes and eukaryotes, and may in fact have given rise to the bacterial
cytoskeleton
25
.
ECT has also helped to identify some cytoskeletal filaments that are unique to bacteria, such
as a unique class of polymeric proteins called bactofilins, which are nearly universally
conserved in bacteria. ECT of
C. crescentus
revealed that two bactofilins form a scaffold to
recruit cell wall biosynthetic enzymes to the stalked cell pole, probably supporting the
formation of this thin adhesion structure
26
. The functions of bactofilin in other organisms are
still being discovered, but seem to reflect an intermediate-filament-like role in the bacterial
cytoskeleton.
Other cytoskeletal elements function in processes other than cell shape determination.
MamK, another protein related to MreB (and actin), is found in magnetotactic bacteria and,
as shown by ECT, forms filaments that help to align magnetosomes into a compass that
orients the bacterium in a magnetic field
27
–
31
(FIG. 2c). Two tubulin homologues, bacterial
tubulin A (BtubA) and BtubB, were identified in
Prosthecobacter
spp.
32
, but were not seen
to form microtubule-like filament bundles
in vivo
32
or
in vitro
33
by traditional electron
microscopy. However, ECT revealed that BtubA and BtubB form filament tubes that are
very similar to eukaryotic microtubules, although they comprise only five protofilaments
rather than the 13 usually found in eukaryotes
34
. These findings challenge the idea that
microtubules are a uniquely eukaryotic structure. ECT has also been helpful in
characterizing the structure and function of several other filaments that are involved in cell
division (see below).
Subcellular organization
In addition to having a cytoskeleton and a defined shape, cells benefit from organizing their
interiors in other ways. An early goal of ECT was to address the question of whether protein
components localize to specialized locations in bacterial cells. Visual proteomics, whereby
protein complexes are surveyed in individual cells using template matching algorithms that
are based on avail able structures
35
,
36
, has been used to count proteins in the cell
37
,
38
and to
determine their subcellular localization. For example, an estimated 15% of the 70S
ribosomes in
Spiroplasma melliferum
were found to be associated with the membrane in a
preferred orientation
39
, and ribosomes were seen to be similarly peripherally localized in
ultra-small archaeal Richmond Mine acidophilic nanoorganism (ARMAN) cells
40
. In
Leptospira interrogans
41
and
Mycoplasma pneumoniae
42
,
the cellular distribution of several
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molecular complexes was mapped by visual proteomics, which showed that large complexes
can be located within the cell and paved the way for future work to reveal the finer details of
proteome organization (FIG. 2d).
One way that prokaryotic cells organize their interior is by clustering functionally related
enzymes into specialized compartments, which are functional analogues of eukaryotic
organelles. For example, green photosynthetic bacteria use compartments called
chlorosomes to aggregate light-harvesting pigments, with each chlorosome containing up to
250,000 bacterio chlorophyll molecules
43
. ECT was used to reveal the structure and
distribution of chlorosomes (covering roughly 70% of the cytoplasmic membrane) and their
association with reaction centres in the membrane
44
,
45
. This enabled, for the first time, the
mapping of reaction centres, which were found to be irregularly clustered throughout the
membrane
45
. Similarly, cyanobacteria concentrate photosynthetic enzymes in the inner
membrane, expanding their energy-harvesting capabilities by elaborating invaginations of
the membrane. The extensive lamellar structure
46
,
47
, and assembly in response to light
48
, of
these intracytoplasmic membranes were characterized by ECT. The additional finding that
these membranes can bud into fully detached vesicular structures challenges the assumption
that membrane-enclosed organelles that are not attached to the plasma membrane are found
only in eukaryotic cells
49
.
In addition to photosynthetic compartments, ECT has also elucidated the structure of
carboxysomes, which are used by cyanobacteria and chemo autotrophic bacteria to
concentrate ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), probably the
most abundant protein on earth and a crucial catalyst in carbon fixation
50
. ECT revealed that
carboxysomes have irregular proteinaceous icosahedral shells and that these shells and the
internal RuBisCO lattice probably form simultaneously
51
–
53
. Interestingly, carboxysomes
were seen to associate with other subcellular structures, polyphosphate storage granules
(discussed below), which suggests a functional relationship between these two
compartments
53
. ECT also revealed the fairly heterogeneous polyhedral structure of another
bacterial microcompartment that is responsible for the utilization of propanediol. This
microcompartment also has clustered enzymes surrounded by a thin protein shell, which
indicates that this organization may represent a general structural feature of bacterial
microcompartments
54
.
ECT has even been able to reveal some information about how DNA is organized within
living cells. Similarly to eukaryotes, bacterial cells have subcellular organization of their
genetic material, packing their chromosomes into the nucleoid (the genome-containing
region of the cell). ECT of
Bdellovibrio bacteriovorus
, a small predatory bacterium, revealed
a twisted spiral compaction of the nucleoid with ribosomes located along the periphery.
Different levels of compaction were observed in different cells, including an MreB mutant,
which suggests that DNA packing may regulate transcription, similarly to what is observed
for eukaryotic chromatin
55
. ECT has shown that
Leptospira
spp. organize their DNA into
bundles of parallel filaments
56
and that ultra-small bacteria, which were recently discovered
with the help of a portable field system to plunge-freeze samples from diverse natural
environments for ECT
57
, have similar dense packing of putative DNA filaments
58
. Further
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study is required to examine how microorganisms may use DNA packing to regulate
transcription.
Another kind of subcellular differentiation occurs in
C. crescentus
, which attaches to a
surface by means of a long thin stalk. Correlated light microscopy and ECT helped to
identify four proteins (StpABCD) that form bands in the stalk, which act as general barriers
to diffusion of periplasmic, inner membrane and outer membrane proteins,
compartmentalizing the cell and further blurring the boundary between eukaryotic and
prokaryotic features
59
.
Finally, some extracellular appendages also act to spatially differentiate cellular processes.
For example, ECT revealed the stalk structures that iron-oxidizing bacteria (
Mariprofundus
ferrooxydans
and
Gallionella-
like bacteria) use to concentrate iron precipitation away from
their interior or overall surface, where it would be detrimental
60
. Similarly, ECT has
elucidated the structure of the ferric nano-aggregates that decorate the surface of iron-
reducing planktonic bacteria
61
. Gram-negative bacteria in aqueous environments frequently
secrete outer membrane vesicles for metabolic purposes, and ECT revealed a clever
adaptation of this system for the partially hydrated environment of the soil, in which a
Delftia
sp. Cs1-4 forms ‘nanopods’ that are composed of extended tubes contiguous with the
surface layer (S-layer) and that deliver vesicles some distance from the cell
62
.
Motility
Prokaryotic cells have evolved complicated mechanisms that enable them to move to new
and possibly more beneficial environments. Perhaps because motility is of such fundamental
importance, or because of the diversity of environments that microorganisms inhabit, there is
a surprising variety of ways by which these cells move.
In many cases, motility is mediated by cellular nanomachines (large, nanometre-scale multi-
protein complexes) the overall architectures of which have been solved by ECT.
Nanomachines that are involved in motility frequently span the cell envelope and contain
extra cellular, periplasmic and cytoplasmic components. The structures of the individual
pieces of nano machines can be solved by X-ray crystallography and/or NMR spectroscopy,
but it often remains unclear how the pieces fit together, because either the complex cannot
be purified intact or it cannot be assembled
in vitro
. Therefore, ECT is invaluable in
revealing the structure of the complete complex in its intact state
in vivo
. The location of
specific components can then be determined by comparing wild-type ECT structures to
those of mutants in which individual proteins are either knocked out or tagged. This
localization map can then be used to inform electron microscopy docking of atomic models
into the ECT density, ultimately enabling the construction of pseudo-atomic models of the
complete system.
One of the most common nanomachines involved in motility is the flagellum. ECT studies,
and in particular subtomogram averaging, have shown the detailed location of most of the
components of the flagellar motor
63
–
65
and its architecture across many diverse bacterial
species
66
(FIG. 3a). This has revealed a surprising number of species-specific peripheral
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adaptations around a common structural core
64
–
67
. For example, motors in some organisms,
such as
Borrelia burgdorferi
, contain collars that may function to stabilize the stator at
higher torque. ECT was used successfully to visualize key structures that were lost in
previous purified preparations, including the stator and the export apparatus, which is
responsible for self-assembly of the flagellum
63
,
68
. Furthermore, the ability of ECT to
resolve the complete macro molecular structure of the entire machine, as opposed to
individual parts, has provided insights into its mechanical properties. For instance, in species
that use periplasmic flagella to drive the rotation of the cell body (rather than an oar-like
extracellular flagellum), ECT revealed larger rings that may produce the increased torque
that is required to drive whole-cell rotation
63
. Finally, ECT also uncovered the modular
sequence of assembly of the flagellum in
B. burgdorferi
: opening of the central channel and
assembly of the rod, assembly of the hook and finally formation of the filament
68
.
In addition to the structure of flagella, ECT has helped to identify details of their function in
different motility systems. For example, in pathogenic spirochetes, such as
Treponema
denticola, Treponema pallidum
and
B. burgdorferi
, periplasmic flagella enable the cells to
penetrate tissue
64
,
65
,
69
–
71
. Whereas traditional electron microscopy preparations of
B.
burgdorferi
showed a bundle of flagella, similar to that found in other species
72
, ECT
revealed that in this case the flagella form a flat ribbon. This structure is indicative of a
mechanism to exert force on the cell cylinder and propagate a wave along the long axis of
the cell, which propels the cell forward
73
. Other ECT studies have identified the roles of
individual proteins in
B. burgdorferi
flagellar assembly
74
–
76
and have shown that motor
rotation is required for the formation of the flagellar ribbon
77
. ECT of a mutualistic
spirochete,
Treponema primitia
, lent support to a model of motility in which the rigid helical
cell cylinder rolls within the sheath of the outer membrane
78
. ECT of the magnetotactic
bacterium MO-1 identified a sheathed bundle of extracellular flagella that, along with
associated thin filaments, may rotate in an interlocking-gear configuration that can generate
tremendous power and speed
79
.
Other bacteria use non-flagellar systems for motility. For example,
Flavobacterium
johnsoniae
exhibits gliding motility but does not contain genes that encode known motility
structures. The mechanism of motility of this bacterium remained a mystery until ECT
revealed tufts of thin filaments attached to the outer membrane. These filaments were shown
to have a role in adherence-based motility
80
(FIG. 3b). Furthermore, ECT revealed long
cytoskeletal ribbons that are used by helical Mollicutes to propel themselves across surfaces;
changes in the relative length of these ribbons are thought to turn the whole cell into a
helical propeller
81
. ECT also identified that
M. pneumoniae
uses an elaborate attachment
structure that consists of a large, jointed core in the cytoplasm and associated adhesion
proteins on the surface to mediate what is thought to be inchworm-like movement
37
,
82
. In
yet another mechanism, the iron-oxidizing bacteria discussed above are thought to propel
themselves, at least in part, by the extrusion of their mineralizing stalks
60
. Finally,
Listeria
monocytogenes
can remodel the actin cytoskeleton of its host into ‘comet tails’, which ECT
has shown to comprise bundles of parallel, hexagonally packed filaments that power
movement, perhaps by pushing or squeezing the bacterium forward, highlighting the
remarkable ability of pathogens to remodel host cells to suit their needs
83
.
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Navigation
With motility comes the problem of deciding where to go. We have already mentioned one
potential orienting mechanism — magnetotaxis. ECT has revealed the structure and
formation of magnetosome chains in several species
27
–
30
,
84
,
85
, including
Magnetospirillum
magneticum
sp. AMB-1, which have vesicles loaded with ferrous crystals that are attached
to the cytoplasmic membrane
28
(FIG. 2c), and
Magnetovibrio blakemorei
, which have
magnetosomes that are fully detached from the cytoplasmic membrane
85
. Interestingly, ECT
of
Desulfovibrio magneticus
RS-1 led to the suggestion that magnetosomes in this bacterium
are not surrounded by membranes
84
.
More widespread than magnetotaxis is chemotaxis, which integrates cues about the chemical
environment into a signal transduction cascade that controls flagellar rotation or pilus
extension. Much of our knowledge about the macromolecular structure of the chemotaxis
system comes from a series of ECT studies showing that chemoreceptors are coupled
together into a hexagonal lattice of interconnected rings
71
,
86
–
93
(FIG. 3c). ECT revealed that
this architecture is conserved across bacteria
94
and archaea
95
, as well as between membrane-
bound and cytoplasmic arrays
96
, suggestive of the fundamental utility of this architecture for
chemosensory transduction. In another example of the power of subtomogram averaging and
electron microscopy docking, ECT was used to generate a pseudo-atomic model of this
extensive subcellular structure, revealing the interconnections between chemosensory
components and supporting the idea that conformational changes in one hexagon of
receptors could be transmitted to adjacent hexagons, thus amplifying the signal and
explaining the remarkable sensitivity of the system
97
,
98
(FIG. 3c). Other ECT studies have
provided insights into the mechanism of activation of the system (in which substrate binding
at the proximal end of the chemoreceptor triggers a conformational change in the kinase
bound at its distal tip)
99
and into the assembly of arrays from subunits of trimers-of-
receptor-dimers
100
.
Division
Successful cells propagate through division, a process that presents many challenges. Below,
we discuss how ECT has elucidated the molecular mechanisms of two of these processes —
DNA segregation and cytokinesis.
First, to ensure that both daughter cells are functional copies, the cell must evenly distribute
low-copy-number structures. The most obvious low-copy-number structure in the cell is the
chromosome. ECT has helped to identify a ribosome exclusion zone at the poles of
C.
crescentus
cells where PopZ tethers replicated chromosomes at either end of the cell to
facilitate segregation
101
,
102
. In addition to the chromosome, different extrachromosomal
plasmids are present at different copy numbers, sometimes as few as one per cell. ECT of
E.
coli
revealed one solution to ensure that these plasmids are equally segregated: the actin
homologue ParM, present as double helical filaments, positions the plasmids at the ends of
the nucleoid for division
103
, and each replicated plasmid pair is probably pushed apart by a
dedicated pair of antiparallel ParM doublets
104
(FIG. 4a). Other bacteria, including
Bacillus
thuringiensis
, use a tubulin homologue, TubZ, to segregate plasmids. TubZ also forms a
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double helical superstructure, which suggests the convergent evolution of bacterial filaments
for this role of plasmid segregation
105
.
Once contents are segregated, the cell must physically divide. Visualization of this process
by ECT has provided mechanistic insights for both bacteria and archaea. Most bacteria use
FtsZ, a GTPase that is homologous to eukaryotic tubulin, and FtsA, an actin homologue, to
constrict the cell. ECT has shown that FtsA forms protofilaments that tether FtsZ filaments
to the membrane
106
. However, details of the mechanism of action of FtsZ remained unclear
until the filaments and division process were visualized directly by ECT in
C. crescentus
. In
this organism, division is asymmetrical, and an early ECT study showed that the inner
membrane completes constriction before the outer membrane
107
. Later tomograms revealed
relatively few arcing FtsZ filaments positioned around the division site
108
, in contrast to
previous models of thick, complete FtsZ rings based on early fluorescence microscopy.
These observations support a model of division
109
in which the FtsZ filaments themselves
provide the force of constriction through iterative rounds of polymerization, attachment,
conformational changes gathering in the membrane and depolymerization
108
,
109
. By
contrast, later ECT work provided evidence for bundles of FtsZ filaments forming complete
rings that encircle cells during the late stages of division and proposed a different model for
constriction that was based on filament sliding
110
(FIG. 4b). The discrepancies between
these observations highlight two drawbacks of ECT imaging: the ‘missing wedge’ effect (see
BOX 1) means that features, such as filaments, cannot be tracked all the way around the
circumference of the cell; and static images, no matter how high their resolution, can only
provide hints to the mechanisms of dynamic processes. FtsZ filaments are indeed highly
dynamic and additional work is needed to resolve their constrictive mechanism.
ECT has also recently helped to show that two proteins, MinC and MinD, form copolymers
that interact with the membrane and position FtsZ filaments at the division plane
111
. The use
of ECT also led to the description of a
C. crescentus
protein, DipM, that coordinates cell
wall remodelling for constriction during cell division
112
,
113
.
Instead of using FtsZ, some archaea use endosomalsorting complexes required for transport
(ESCRT) proteins to divide. ESCRT mediates membrane scission events across many
archaea and eukaryotes, but the exact mechanism has been the subject of much debate.
However, ECT of dividing
Sulfolobus acidocaldarius
revealed a belt of filaments at the
constriction zone, supporting a model in which spiralling filaments constrict the cell
membrane
114
, although more work is required to test this.
Cytokinesis itself helps to divide some cellular features, such as the magnetosome chain in
magnetotactic bacteria mentioned above. ECT identified a mechanism in which MamK
localizes the magnetosome chain to the mid-cell, which ensures proper segregation of the
chain to both daughter cells by cytokinesis
115
.
Surviving lean times
Nutrient sources in the environment are by no means guaranteed, and cells frequently face
shortages. To prepare for such lean times, both bacteria and archaea store essential nutrients
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in storage granules during times of sufficiency, and details of these granules have been
revealed by ECT. In both Gram-positive and Gram-negative bacteria, including
C.
crescentus
, polyphosphate is stored in uniform, spherical structures that lack any membrane
boundary, indicating that they form through a mechanism of local aggregation
116
,
117
.
C.
crescentus
also stores carbon in slightly larger, less regular structures
116
.
D. magneticus
has
been shown to concentrate iron and phosphorus in a membrane-bound compartment,
although it is not clear whether this is for purposes of sequestration or storage
84
.
Cupriavidus necator
stores polyhydroxybutyrate in compartments that are not membrane
bound but instead are coated by a protein shell and form as small granules that then
coalesce
118
. ECT also revealed that the archaeon
Methanospirillum hungatei
contains
several amorphous polyphosphate bodies that are located at the ends of the cell
119
.
To deal with lean times, some bacterial species form metabolically inert and environmentally
protective spores. ECT studies of a rare Gram-negative endospore-forming member of the
Firmicutes phylum revealed that the inner membrane of the mother cell surrounding the
spore is inverted and eventually becomes the outer membrane of the germinating Gram-
negative cell
120
(FIG. 4c). This unexpected finding suggests that the sporulation process
might have given rise to the diderm cell plan. ECT has also helped to identify polyphosphate
storage granules in Gram-negative but not Gram-positive spores, which suggests that these
granules may help to maintain the outer membrane during spore outgrowth
16
. Furthermore,
ECT has also shown that membrane vesicles that are secreted by sporulating colonies of
Streptomyces coelicolor
are densely packed with proteins (including metabolic enzymes,
antioxidants and resistance factors) that may aid survival
121
.
Cooperation and competition
In nature, prokaryotic cells are surrounded by other organisms, both single-celled and
multicellular; these include conspecifics, symbionts, competitors, predators and prey. To
adapt to such an environment, cells may coat their envelopes with proteins, creating a layer
that provides protection against predation or mediates biofilm attachment. The S-layer can
represent 10–15% of the total protein in the cell
122
, but its structure was difficult to resolve
using traditional methods; although the S-layer is a crystalline lattice, it makes a poor
sample for methods such as X-ray crystallography, at least in part because of its natural
curvature around the cell. One of the first applications of ECT was to visualize the S-layer of
intact
Pyrobaculum aerophilum
archaeal cells
123
, and ECT was later applied to the Gram-
negative bacterium
C. crescentus
124
. Surprisingly, these studies revealed that the S-layer is
not a uniform lattice; instead, it has significant heterogeneity across the cell and includes
regions of double-stacked layers, suggesting assembly from multiple nucleation points.
Cooperation can occur within or between species and even kingdoms, and it often involves
specialized structures that enable physical contact between organisms. For example, studies
have used ECT to obtain images of: the vast extracellular matrix of hollow tubules (known
as cannulae) that anchors a community of
Pyrodictium abyssi
to one another
125
; interspecies
cell–cell contacts of various archaeal communities
126
,
127
; and the grappling hook structures
known as hami that the SM1 euryarchaeon uses to anchor itself in symbiotic communities
with bacteria
128
(FIG. 5a).
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Instead of cooperating, prokaryotic cells might benefit more by killing their neighbours. For
example, ECT revealed that the type VI secretion system (T6SS), a cellular nanomachine
that was first characterized in
V. cholerae
and is used by many Gram-negative bacteria to
combat bacterial and eukaryotic cells, is a spring-loaded ‘molecular dagger’ that is
structurally homologous to a contractile phage tail (BOX 2). ECT visualized the dynamic
conformations of T6SSs, including extended filled (loaded) and contracted empty (fired)
tubes, thereby uncovering a mechanism by which the force of contraction drives the
translocation of the T6SS needle into the target cell
129
(FIG. 5b). In addition, correlated
cryo-PALM and ECT enabled the identification of transient assembly and disassembly
intermediates, highlighting the utility of this technique for identifying previously
uncharacterized structures and conformations
130
. A T6SS-like structure is also responsible
for a remarkable interaction between bacteria and multicellular eukaryotes. Larvae of a
marine tubeworm select surfaces covered by bacterial biofilms of
Pseudoalteromonas
luteoviolacea
to settle and differentiate into sessile adults. ECT revealed that the signals that
the tubeworm recognizes are metamorphosis-associated contractile structures that are
formed by an interconnected web of T6SS-like pyocins that develop inside
P. luteoviolacea
cells and are released by lysis to form a micrometre-scale, well-organized extracellular spike
ball consisting of roughly 100 pyocins and associated proteins
131
(FIG. 5c). The function of
this structure for the bacterium remains unknown.
Box 2
Prokaryotic viruses
Another important aspect of prokaryotic cell biology is the interaction of prokaryotes
with viruses or phage. Electron cryotomography (ECT) studies have elucidated the
structural features of viral capsids
170
,
171
and detailed phage attachment to bacterial
cells
172
–
175
, including a particularly elegant mechanism by which phage wrap a filament
around the flagellum of
Caulobacter crescentus
, taking advantage of flagellar rotation to
move toward the cell pole where they are concentrated around their pilus entry point,
increasing the chance of infection in a dilute environment
176
(see the figure, part
a
).
Another study of a virus that attacks the conjugative F-pilus used subtomogram averaging
to reveal asymmetries in the capsid that may lead to viral entry
177
. An early application
of ECT was to show the contractile mechanism of the phage tail that injects the viral
genome into the host cell
178
, which was further elucidated in later studies
172
,
174
,
179
–
181
.
Further work probed the mechanism of phage assembly within bacterial cells
182
,
183
,
including outward expansion of the nascent capsid due to pressure from RNA genome
packaging
184
. Another study imaged host lysis, completing the infection cycle
185
.
Archaea are also prey to specific viruses, notably archaeal pyramidal viruses. ECT was
used to elucidate the entry mechanism of these viruses, which involves tracking along
pilus-like filaments
186
, as well as the striking release mechanism, in which a seven-faced
pyramidal structure penetrates the membrane and surface layer (S-layer) of the host cell
and opens to release mature virions
187
,
188
(see the figure, part
b
). Archaea can also be
infected by lemon-shaped viruses, the pleomorphic structure of which was recently
resolved by ECT
189
.
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IM, inner membrane; OM, outer membrane. Figure part
a
shows a 3D segmentation
overlaid on a volume rendering of a tomographic reconstruction of phage interacting with
an intact
C. crescentus
cell. Figure part
b
shows slices (left) and 3D segmentations (right)
of tomographic reconstructions of
Sulfolobus islandicus
cells infected with
S. islandicus
rod-shaped virus 2. Part
a
is reproduced with permission from REF.
176
, National
Academy of Sciences. Part
b
is modified with permission from REF.
188
, National
Academy of Sciences.
Alternatively, prokaryotic cells might eat their neighbours, as exemplified by
B.
bacteriovorus
, which pierces the periplasm of its prey and eats their cytoplasmic contents.
ECT provided new insights into this process by revealing an unusual flexibility of the cell
wall that probably underlies the ability of
B. bacteriovorus
to squeeze into its prey
132
.
Pathogenicity
One particularly important form of interaction between prokaryotes and their neighbouring
cells is pathogenicity to human hosts. Many of the adaptations that turn a bacterium into an
efficient pathogen involve structural changes, and ECT has provided insights into the
mechanisms of pathogenesis of several bacteria.
First, pathogens must sense and enter their host. In addition to pathogenic adaptations to the
flagellar motor that are discussed above, many structural adaptations occur in the cell tip,
which ECT has shown can be complex in pathogenic bacteria. For example, ECT of
Campylobacter jejuni
revealed an elaborate cell tip with storage granules, extensive
chemoreceptor arrays and a complex flagellar motor, all of which are thought to help
mediate invasion of host cells
133
. Similarly, ECT of
Leptospira
spp. revealed extensive
chemoreceptor arrays that are probably important for sensing host cells
56
. ECT of
T.
pallidum
and
T. denticola
identified complex conelike periplasmic structures at the cell tip,
although the function of these structures remains unknown
65
,
134
.
Once a cell enters its host, it must evade the host immune system, and studies using ECT
have provided key insights into how pathogens adapt their cell surface for this purpose. For
example,
T. pallidum
exhibits a complex and labile outer membrane that is not tethered to
the peptidoglycan layer as in most Gram-negative cells, enabling the bacterium to shed the
outer membrane (which contains multiple components that are recognized by the immune
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system) and thereby evade the host immune response
65
. Similarly, ECT of
B. burgdorferi
revealed a labile outer membrane sheath
70
. Interestingly, ECT also observed cell fusion
events in
Borrelia
spp., as well as two cells sharing a single outer membrane, suggesting that
these bacteria use this as a strategy to diversify surface antigens to evade host immunity
135
.
Pathogenic bacteria use another nanomachine that is related to the flagellar motor to
facilitate infection. ECT of
Shigella flexneri
,
Salmonella enterica
and
Yersinia entero
colitica
identified the complete structure of the type III secretion system (T3SS) injectisome.
This structure consists of a basal body and a secretion channel that delivers effector proteins
directly into a target cell. The basal body is structurally similar to the flagellar motor,
although it exhibits key differences, including a wider channel that may allow the secretion
of at least partially folded substrates (rather than unfolded flagellin sub units) and greater
elasticity
136
–
138
. Furthermore, pathogenic T3SSs in
Y. enterocolitica
were seen to cluster,
perhaps to enhance secretion into a target cell
139
, and
Chlamydia trachomatis
was also seen
to orient an array of T3SSs to contact the target cell
140
(FIG. 5d). Later, higher-resolution
ECT and subtomogram averaging of
S. flexneri
minicells revealed, for the first time, how the
flagellar C-ring has been adapted into the sorting platform of the injectisome, a cytoplasmic
complex that selects specific effector proteins for secretion
141
(FIG. 5d).
Once in a host cell, some pathogenic bacteria, such as
Chlamydia
spp., undergo
differentiation from infectious elementary bodies to metabolically active reticulate bodies.
During this process, many pathogenic adaptations that are no longer required for survival
within the host are lost. For example, ECT of
C. trachomatis
showed that T3SS-mediated
cell contact induces bacterial internalization into early vacuoles, where polarization and
T3SSs are lost
140
. Conversely, ECT also revealed the adaptations that accompany the
differentiation of
C. trachomatis
from reticulate bodies into elementary bodies, such as a
near doubling in the thickness of the outer membrane
142
. ECT of another pathogen,
Chlamydophila abortus
, showed that it has a similar T3SS structure and differentiation
strategy to
C. trachomatis
143
. ECT also identified that a previously identified Chlamydial
development stage, the crescent-shaped stage, was actually an artefact of the chemical
fixation and dehydration used in traditional electron microscopy preparations
6
, highlighting
the power of ECT to image native structures.
ECT can be useful to characterize therapeutic agents that target pathogenic bacteria. For
example, ECT was used to see how an antibody against
B. burgdorferi
lyses the cells by
inducing outer membrane projections that probably increase permeability
144
. ECT may also
be useful in identifying future therapeutic targets. For example, ECT has shown that a
known
Mycobacterium marinum
virulence factor, SecA2, helps to maintain cell wall
integrity
145
, which suggests that targeting this protein could be an effective strategy to
combat mycobacterial infections. Similarly, ECT of
Acinetobacter baumannii
identified a
pathogenic mechanism that is based on outer membrane vesicle delivery of effector
molecules. Interestingly, these vesicles were seen to form in areas with irregular
peptidoglycan, and sub-lethal concentrations of a peptidoglycan-targeting antibiotic
enhanced vesicle formation
146
.
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Finally, ECT may also help to design strategies against antibiotic-resistant pathogens. Many
bacterial cells have evolved defence mechanisms against anti biotics, including the
multidrug efflux pump, a cellular nanomachine that spans the inner and outer membranes
and selectively exports antibiotics from the cytoplasm. The structure is too unstable to be
purified, precluding most structure determination methods. ECT and subtomogram
averaging revealed part of the structure and possible assembly of a reconstituted
Pseudomonas aeruginosa
multidrug efflux pump
147
, which offers promise for solving the
full structure. This
in situ
structure would probably elucidate the mechanism of the efflux
pump, which could inform new therapeutic strategies to combat antibiotic resistance.
Outlook
Compared with the scarcity of information available before 2002, we are witnessing an
explosion of new, high-resolution information about molecular arrangements inside
prokaryotic cells. This Review highlights the impressive diversity of structural insights into
bacterial and archaeal physiology that have already arisen from ECT of whole cells in a
native state. For example, for the first time, the arrangement of polymers in the cell wall can
be directly visualized, which has enabled the observation of previously unappreciated
structural similarities between Gram-positive and Gram-negative bacteria. Similarly, ECT
revealed the interconversion of outer and inner membranes during sporulation, suggesting a
simple new hypothesis for the evolution of the diderm cell plan. Also for the first time, ECT
has directly proven that bacteria not only have a cytoskeleton, but that this cytoskeleton is
rich and varied in function. Similarly, cell compartmentalization is no longer a feature
restricted to eukaryotes; membrane-bound, even budded, compartments have been observed,
and many structures have restricted subcellular localizations. ECT has also enabled the
visualization of intact nanomachines, the macro molecular complexes that carry out diverse
functions, from motility to interspecies interactions and pathogenicity. The complexity of
these machines (which can comprise many copies of dozens of distinct proteins) and their
locations spanning cellular compartments (cytoplasm, periplasm and extracellular space)
mean that many cannot be purified or reconstituted intact. Now, by visualizing them
in situ
and creating pseudoatomic models, we can finally begin to understand them in context.
Much work remains to be done, and continuing technological developments (BOX 1) should
expand the power of ECT in the near future. A key difficulty in ECT is identifying
macromolecules of interest; the structures of many protein complexes remain undetermined
because they cannot be unambiguously identified in the crowded cellular environment. For
that reason, many ECT studies to date have focused on structures for which the morphology
or localization was already known. To identify new structures, we need improved
technologies to locate them. Correlated light microscopy and ECT has been successfully
applied to identify several structures
in vivo
, and the recent development of correlated cryo-
PALM and ECT has increased the precision of this technique, enabling the localization of
tagged proteins to within a few hundred nanometres in a cell
130
. Anticipated technological
improvements could further enhance localization to within tens of nanometres, in 3D.
However, adding a bulky fluorophore to a protein of interest can introduce structural and
localization artefacts, so the potential application of fluorescent small molecules
148
to
in
vivo
protein labelling, perhaps through the use of unnatural amino acids
149
, is intriguing.
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