Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 1, pp. 7-10, 2005

Accepted
Bacterial Cell Wall Structure and Implications for Interactions with Metal Ions and Minerals
Terrance J. Beveridge*
Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Guelph,
Ontario, N1G 2W1, Canada
Received: November 15, 2004; In Final Form: November 15, 2004
New techniques in the rapid freezing of cells, so that they are vitrified, and cryo-transmission electron microscopy(cryoTEM) of the frozen hydrated thin-sections from the vitrified cells are showing their true native structure.
Unlike other forms of TEM, frozen hydrated thin-sections cannot be contrasted by heavy-metal stains (such as U,
Pb, and Os) and their contrast is via the inherent density of the constituent molecules within the cells. Therefore,
these frozen sections show the true mass distribution within the biomatter. Another cryoTEM technique, freezesubstitution, also produces thin sections for viewing by TEM, but these are plastic sections of heavy-metal stained
cells. The heavy-metal ions of the stain complex to the available reactive sites of the biomatter. When such
images are compared to those from the frozen hydrated sections, a clear interpretation of native structure and its
metal reactivity can be made. These types of observations will be invaluable for the study of microbemetal/radionuclide interactions.

1. Introduction
Prokaryotes are the Earth’s smallest life form and, yet, havethe largest surface area-to-volume ratio of all cells.1, 2 They arealso the most ancient form of life and have persisted on Earthfor at least 3.6 Ga, even (we think) in some of the mostextreme environments imaginable, such as the deep subsurface. Most of these early primitive (and today’s modern)natural environments possessed reasonably high amounts ofmetal ions that were capable of precipitation under suitable pH
or redox conditions. Deep-seated in such geochemical situations is the likelihood of suitable interfaces that lower the local
free-energy so that interfacial metal precipitation is promoted.
Bacteria, being minute and having highly reactive surfaces(interfaces), are exquisitely efficient environmental particles
for metal ion adsorption and mineral nucleation. Metal ionsinteract with available surface reactive groups (or ligands)
available on the bacterial surface and precipitates grow as
environmental counter-ions interact with more and more metalat the site.3−7 Once formed these precipitates are under the
influence of natural geochemical and additional microbiallymediated conditions8 that instigate the development of finegrain minerals, usually via dehydration so that crystalline
phases are eventually developed.9 These minerals commence
as so-called ‘nano-mineral phases’ and grow with time tobecome larger and larger. This bacterially-induced mineralization is likely the natural phenomenon that so encases somecells in fine-grain minerals that they die and become bona fide‘microfossils’.10 In ancient times, these mineral-encased prokaryotes, enduring low-temperature metamorphic geological conditions, survived as microfossils still existent in such very old
Precambrian formations as the ~2.0 Ga Gun Flint Chert, northof Lake Superior in Canada.
It is certain that bacterial surfaces interact with environmental metals ions and can provide nucleation sites for mineral
precipitation but it has been extremely difficult to study such
systems with high precision on a cell to cell basis, even thougha wide base of techniques exists.11 This is because the cells are
extremely small and the interactive structures even smaller,*Corresponding author. E-mail: tjb@uoguelph.ca. FAX: +1-519837-1802.

and because the reactive sites responsible for adsorbing metals
retain their reactivity only over a certain range of pH and Eh,which is difficult to monitor over micro-scale distances. This
article will outline our new advances in elucidating the structure of bacterial surfaces.
2. Use of Cryo-Transmission Electron Microscopy
Using conventional fixation, embedding and thin-section
techniques, the transmission electron microscopic observationof bacteria has been a most powerful method for elucidating
the internal cytoplasmic organization and the juxtaposition ofencompassing envelope layers of cells.12, 13 Yet, there are many
drawbacks to such conventional techniques since the cells are
chemically fixed, using harsh fixatives (such as glutaraldehyde
and osmium tetroxide), and dehydrated before embedding in aplastic resin for thin sectioning.14 Essential lipids are extracted,
proteins are denatured, and nucleic acids are artificiallycondensed. The cells are a specter of their former selves.
Clearly, the images of such cells have been beneficial to our
initial perception of the structural organization of prokaryotic
cells12 but hydration (which these embedded cells no longerhave) is a necessary prerequisite for the maintenance of native
structure. With proper expertise, care and equipment, it is now
possible through the use of cryo-transmission electron
microscopy (cryoTEM) to obtain a better and more natural
view of bacteria.15−18
Freeze-substitution. One cryoTEM technique that became
popular during the 1980 – 90s was freeze-substitution. 18−21
Here, cells are rapidly frozen at ~–196 ˚C so as to vitrify themin amorphous ice, which is not crystalline and is a kind ofglass.13, 14 Hence the cells are physically ‘fixed’ since there isno time during freezing for structure to degrade; in fact,
freezing occurs within milli- to micro-seconds and all molecular motion is stopped. If the bacteria are thawed, they comeback to the living state and continue to grow and divide. Thisis a clear-cut measure of how well the cells are preserved.
Once vitrification is accomplished and cellular structure
preserved, the temperature is raised from –196 ˚C to – 80 ˚Cand the cells put into a freeze-substitution mixture. Thisconsists of a cryogenic fluid (such as acetone) containing achemical fixative (osmium tetroxide), a heavy metal stain© 2005 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 07/31/2005

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J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005
20, 21(uranyl acetate), and a molecular sieve (for trapping water).
At this low temperature, the cells do not melt and remain vitrified, and structure is preserved. The cellular and surrounding
ice (water) sublimes and is trapped in the molecular sieve.
Eventually, the specimen is thoroughly dehydrated and fixed inthis freeze-substitution mixture with the structure maintaining
many of its native features. Now, it can be embedded inplastic resin, cured and thin sectioned for viewing by TEM.
Freeze-substitution and Gram-positive (Bacillus subtilis)
cell walls. The results of freeze-substitution are breath taking,especially when examining the surface structures (Figure 1).
The fabric of Gram-positive cell walls is no longer featureless(as seen in conventional embeddings),22 but is a tripartite structure (Figure 1). The region associated with the plasma
membrane (immediately above the bilayer) is highly contrasted
due to the acquisition of large quantities of heavy metal stain.
The middle region is lightly contrasted since the wall polymers(mainly peptidoglycan) are stretched almost to the breaking
point so that the mass-to-volume ratio is much reduced
compared with other wall regions. The outermost region
consists of thin fibres that extend into the external milieu. This
tripartite cell wall structure is compatible with current models
of cell wall turnover.21, 22
More importantly for this chapter is the fact that the polymeric structure of the wall has been preserved by freeze-substitution and the available reactive sites within the wall have been
‘decorated’ with the heavy metal stain so that we obtain a clear
picture of where the reactive sites reside. Many sites are in theregion immediately apposed to the membrane since, here, newwall polymers are being extruded and compacted via penicillin-binding proteins in the membrane. As an area of de novo
wall assembly, where new polymers are being cross-linked
into the pre-existing wall fabric, many reactive groups areavailable for decoration in this region since the mass is great
and the availability of reactive groups is high. The middle
region is different though. It is the area in the cell wall that
resists turgor pressure and maintains the integrity of the cell. It
has to be highly cross-linked to hold the cell together undersuch a pressure load and is considerably stretched. Thisregion, then, has few reactive groups left available for interaction with the heavy metal stain since most have been used tocross-link the network together for strength. The outermost
region is an area where the cell wall is being broken down by
the wall’s constituent autolysins. Covalent bonds are being
broken and new reactive groups being made. This region
probably has little mass (since it is being shed during cell wall
turnover) but an excess of reactive sites that are readily decorated by the stain.
Frozen hydrated Gram-positive (B. subtilis) cell walls. A
more difficult and therefore less used cryoTEM technique isthe use of frozen hydrated sections. 16, 23 This techniquerequires skill and perseverance. As in freeze-substitution, cells

Figure 1. Thin-section image of a freeze-substituted of a Gram-positive Bacillus subtilis cell showing three distinct regions in the cellwall, the inner (#1), middle (#2) and outer (#3) regions that correspond to cell wall turnover and to the available reactive groups within
the cell wall network. Bar = 50 nm.

are vitrified but now, instead of processing the cells so thatconventional plastic thin sections are obtained, this frozenmaterial is immediately put into a cryo-ultramicrotome andthin sectioned. The cells are sectioned while vitrified and thefrozen sections immediately mounted into a cryo-specimen
holder and inserted into the cryo-chamber of a cryoTEM. We
emphasize the word ‘cryo’ because the temperature must be
maintained at –196 to –140 ˚C during all manipulations, otherwise the amorphous ice embedding the cells will become crystalline and ruin the native structure to be observed. No
chemical fixatives or heavy metal stains are used during theentire process and, since the sections remain vitrified, allcellular macromolecules and polymers remain in a hydrous
state.
One of the advantages of the cryo-sectioning technique isthat no artificial chemical fixatives or heavy metal stains need
to be used. However, implicit in the use of TEM is that the
specimen must possess enough density to efficiently scatterhigh voltage electrons from the electron gun of the microscope(i.e., the electron potential is typically ~100,000 – 200,000
electron volts). Biomaterials, once thin sectioned, rarely haveenough density to effectively scatter such high powered electrons since the thin sections are only ~50 nm thick and thebiomatter possesses only low atomic number elements (such as
H, C, O, N, etc.). This is the primary reason why conventionaland freeze-substitution thin sections use stained material; theheavy metal stains increase the density (or the mass-to-volume
ratio) of the specimen so that the contrast becomes great
enough for the cells to be easily visualized.14 Frozen hydrated
thin sections of unstained bacteria do not have this luxury
since they cannot be stained once sectioned. (The staining
fluids would immediately freeze over the sample and obliterate
the structure of the cells.)
Clearly, then, these frozen hydrated sections of bacteria are
difficult to see since their contrast is close to that of the surrounding vitrified ice. For this reason, we rely on the inherent
phase function of the lenses of the cryoTEM and use phase
contrast to help imaging by over focusing to see the bacteria.
Certain microscopes (e.g. those with energy filters) can also
derive more additional contrast for the specimen. But eventhen there are additional problems in visualizing frozen sections. The energy of the electron beam is often high enough tolocally increase the specimen’s temperature, resulting in theformation of crystalline ice (from amorphous ice) and, eventually, in ice sublimation. Since bacteria are excellent nucleation
particles (remember how efficient they are for forming finegrain minerals!), the amorphous to crystalline phase transitionof ice frequently occurs on the bacteria and their structure is
obscured. Furthermore, since the specimen is kept so cold, thefrozen sections act as ‘cold traps’ for the condensation of extraneous molecules within the high vacuum of the microscopecolumn, thereby often contaminating the structure of the specimen. With all these associated problems, it is a wonder thatfrozen hydrated sections of bacteria can be imaged, but they
can and they are extraordinary (Figure 2).
These images differ from what we see in freeze-substitution
images. Remember, now we have no heavy metal stains toassist contrast and must rely on the inherent density imparted
on the cell by the constituent atoms within its molecules.
Proteins will be more readily discerned from the surrounding
ice than, say, carbohydrates because they are usually larger,
contain nitrogen and (sometimes) sulfur, and tend to foldtighter. And, most important, all cellular constituents remain
hydrated and therefore not artificially condensed because of adehydration regimen. The ribosomes are larger and more robust
and can be seen because of their large concentration of protein
and rRNA (here the phosphorus adds additional contrast)(Figure 2). Even the bilayer of the membrane can be seen
because of the inherent contrast of the phosphorus in the phos-

Bacterial Cell Wall Structure and Implications

J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005

9

Figure 3. Conventional thin section of a B. subtilis that has been
subjected to 50 mM FeCl3 treatment for 15 min at 22˚C. The iron has
begun to precipitate from solution onto the cell wall (arrows). Noticethat most iron is associated with the wall surface and with theperiplasm. No stains other than the iron have been used on this cell.

Figure 2. Frozen hydrated thin section of the B. subtilis cell wallshowing the plasma membrane (PM) the inner wall zone (IWZ) andthe outer wall zone (OWZ). Here the IWZ corresponds to region #1
and the OWZ to region #2 of the freeze-substituted wall in Figure 1.
Region #3 is not seen. These IWZ and OWZ have different dimensions than the regions in Figure 1 and are visualized entirely due to thedensity of the constituent macromolecules. Bar = 50 nm.

pholipids. Most important for this chapter, though, is the cellwall. Here, we get a clear idea of the mass distribution within
the Gram-positive wall of B. subtilis (Figure 2). Immediately
above the bilayered membrane is a low-density space with little
contrast. Experiments have shown that this is a periplasmic
space.24 Above this is a more densely contrasted region that
represents the peptidoglycan-teichoic acid network of the wall.
Unlike freeze-substitutions, there is not an outermost fibrous
region (cf., Figures 1 and 2).
3. Correlation of Freeze-Substitution and Frozen Hydrated
ImagesHow can we reconcile the differences seen in Figures 1 and2 remembering that the cell in Figure 1 has been dehydrated
and decorated with a heavy metal stain? Since it is dehydrated, we would expect there would be a certain amount ofcontraction of the wall regions in freeze-substitution images(Figure 1) because the structures are no longer hydrated. We
would also expect reactive groups to be labeled. On the other
hand, frozen hydrated structure would not be condensed andthis is why the thickness of each wall region is greater inFigure 2 as compared to Figure 1. This same image shows aperiplasmic space whereas Figure 1 does not. The periplasm
has contracted and condensed in Figure 1, but it has beenpreserved in its natural state in Figure 2. Accordingly, theperiplasm in Figure 1 is more concentrated and (it seems) morereactive since it stains strongly. In Figure 2, the periplasm has
not condensed and it has low density. The conclusion, then, isthat the natural state of the periplasm in these cells is as a relatively low-density matrix of highly reactive biomatter occupying a definite periplasmic space defined by the membrane
and the middle region. Presumably the periplasm in this spaceconsists of new wall polymers, secreted proteins (and theirassociated chaperones), and both periplasmic enzymes andoligosaccharides.24
The region above this periplasmic space is wider in frozen
sections than in freeze-substitutions (cf., Figures 1 and 2) and,since this is the hydrated structure, the increased width showsits natural state. Both figures reveal it to be of relatively low
contrast; freeze-substitutions suggest there are few available
reactive groups and frozen sections suggest that there is little

density.25 Therefore this region, as the stress-bearing region of
the wall, has been stretched taunt (thereby reducing its mass),
and most reactive groups have been utilized to ensure that the
network is cemented firmly together. This forms a strong but
elastic fabric of wall polymers, mainly of peptidoglycan.25, 26
The outermost fibrous region, which is highly decorated
with stain in Figure 1, is not seen in Figure 2. Accordingly,
this outermost region has so little mass it cannot be seen but ishighly reactive. This is in accordance with cell wall turnover
since this is a region where autolysins are breaking down old
peptidoglycan making it soluble. This would reduce its mass
while at the same time would generate many new reactive sites
due to hydrolysis.24
4. How Does This New Interpretation of Wall Structure
Correlate with Metal Ion Interaction and Mineralization?
It is undeniable that metal ions interact strongly with bacterial surfaces and mineralize them.3, 7, 9, 27−29 Cell walls adsorb
metal ions and minerals are nucleated in Gram-positive cell
walls because of metal ion interaction with the peptidoglycan
and secondary polymers (Figure 3). 4, 5 Our new structuralobservations on Gram-positive walls now show the quantity of
hydrated biomatter that resides in the wall for metal interaction(Figure 2). They also reveal where are the most reactive andlikely regions for metal ion interaction and mineral growth(Figure 1). The outermost fibrous region would be the mostaccessible reactive region and, here, there would be little
problem of metal ion access and of mineral particle growth.
Since many wall polymers are in the act of being solubilized,
these polymers and their precipitates would be sloughed fromthe cells but could continue to grow and mature into bona fidemineral phases in the external milieu. The middle region hasless reactivity since it is highly cross-linked. Because turgorpressure stretches this region almost to the breaking point, thepeptidoglycan would be in a relatively ‘open’ configuration25
so that most metal ions could penetrate through. The inner
region (or periplasmic space) is a highly reactive loose gel ofpolymers and proteins that would both interact with andprecipitate metal ions. Here, though, since the periplasm
resides within a confined space between the plasma membrane
and the middle wall region, mineral growth would be restricted
to the accessible space.
These correlations and interpretations allow certain predictions to be made as to where environmental metal ions shouldinteract with Gram-positive bacterial surfaces. Gram-negative
surfaces are structurally more complicated12, 13, 15, 19, 21 but they
have also been imaged via freeze-substitution and frozen
hydrated sections16, 19−21, 30 and are, therefore, also able to becorrelated. These Gram-negative surfaces also interact
strongly with metal ions.27 It is therefore undeniable thatbacterial surfaces make excellent nucleation sites for the development of fine-grained minerals. Hopefully these new electron microscopy observations on bacterial walls help shedsome light on these metal ion-cell surface interactions.

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Acknowledgments. The author thanks his student V. Matias
for supplying the frozen hydrated section used in Figure 2. Amuch more extended version of this paper will be published in
an up-coming Society for General Microbiology book in late2005 entitled ‘Micro-organisms and Earth Systems–Advancesin Geomicrobiology’. The research presented in this article wasmade possible through funding provided by a Canadian
National Science and Engineering Research Council (NSERC)
Discovery grant and a United States Department of Energy
Natural and Accelerated Bioremediation Research Program(US-DOE-NABIR) grant to TJB. The electron microscopy was
performed in the Guelph Regional Integrated Imaging Facility(GRIIF), which is partially funded by an NSERC- Major
Facility Access grant to TJB.
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