matias 2005 bacillus subtilis periplasmic space
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Molecular Microbiology (2005) 56(1), 240–251 doi:10.1111/j.1365-2958.2005.04535.x
© 2005 Blackwell Publishing Ltd
Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2005? 2005561240251Original ArticleGram-positive cell walls and periplasm shown by cryo-TEMV. R. F.Matias and T.J. Beveridge
Accepted 16 December, 2004. *For correspondence. E-mail tjb@
uoguelph.ca; Tel. (+1) 519 824 4120 ext. 53366; Fax (+1) 519837 1802.
Cryo-electron microscopy reveals native polymeric cellwall structure in Bacillus subtilis
168 and the existenceof a periplasmic space
Valério R. F. Matias and Terry J. Beveridge*
Biophysics Interdepartmental Group and Department of
Microbiology, College of Biological Science, University of
Guelph, Guelph, Ontario, Canada N1G 2W1.
Summary
Ultrarapid freezing of bacteria (i.e. vitrification)
results in optimal preservation of native structure. In
this study, cryo-transmission electron microscopy of
frozen-hydrated sections was used to gain insightinto the organization of the Bacillus subtilis
168 cell
envelope. A bipartite structure was seen above the
plasma membrane consisting of a low-density 22 nm
region above which a higher-density 33 nm region or
outer wall zone (OWZ) resided. The interface between
these two regions appeared to possess the most
mass. In intact and in teichoic acid-extracted wall
fragments, only a single region was seen but the
mass distribution varied from being dense on the
inside to less dense on the outside (i.e. similar to
the OWZ). In plasmolysed cells, the inner wall zone
(IWZ)’s thickness expanded in size but the OWZ’sthickness remained constant. As the IWZ expanded it
became filled with plasma membrane vesicles indicat-
ing that the IWZ had little substance and was empty
of the wall’s polymeric network of peptidoglycan and
teichoic acid. Together these results strongly suggest
that the inner zone actually represents a periplasmic
space confined between the plasma membrane and
the wall matrix and that the OWZ is the peptidoglycan-
teichoic acid polymeric network of the wall.
Introduction
Transmission electron microscopy (TEM) of conventional
thin sections has long been a primary tool to examine the
ultrastructure of bacterial boundary layers. Based on the
response of bacteria to the Gram reaction, thin sections
have shown that Gram-negatives possess a cell wall con-
sisting of an outer membrane and a peptidoglycan layer.
This layer is found in a defined space between the plasma
and outer membrane, called the periplasmic space, and
is filled with periplasm (Hobot et al
., 1984; Beveridge and
Graham, 1991). Together the outer membrane, the pepti-
doglycan layer and the periplasm constitute the cell wall
in Gram-negative bacteria (Murray, 1963; Beveridge,
1981; 1999; Beveridge and Graham, 1991; Matias et al
.,
2003).
Gram-positive envelopes are much different; the
plasma membrane is thought to be in tight apposition toa relatively thick cell wall consisting of peptidoglycan, sec-
ondary polymers (usually teichoic or teichuronic acids;
Neuhaus and Baddiley, 2003) and proteins (Sutcliffe and
Russel, 1995; Antelmann et al
., 2001; 2002; Hyyryläinen
et al
., 2001; Vitikainen et al
., 2001; Tjalsma et al
., 2000,
2004). Although freeze-substitution revealed a periplas-
mic space in Staphylococcus aureus
(Umeda et al
.,
1992), most Gram-positive bacteria do not appear to have
a clearly defined periplasmic space (Beveridge, 1981;
1995) and it has been suggested that the periplasm of
these cells is intermixed with the polymeric network of the
wall matrix (Beveridge and Graham, 1991; Beveridge,1999; 2000). Obviously, this interdigitation of periplasmic
proteins and wall polymers could have a profound effect
on the mass distribution within the wall fabric and it could
also effect polymer conformation and distribution.
For conventional embeddings, it is well recognized that
the harsh treatment that bacteria are subjected to during
fixing, dehydration and embedding can both denature and
extract essential cell envelope constituents, thereby
inducing structural artefacts (Beveridge et al
., 2005). A
more recent cryo-technique, freeze-substitution, provides
better preservation and a more natural view of bacteria in
thin section (Beveridge, 1999; 2000). Here, bacteria are
vitrified by rapid freezing, and are chemically fixed,
stained and dehydrated at -
80
∞
C without thawing (Gra-
ham and Beveridge, 1990). They are then embedded in
plastic and thin sectioned. Thin sections of freeze-
substituted Gram-positive walls appear to show more
complexity than seen by more traditional means (Umeda
et al
., 1987; Paul et al
., 1993; Graham and Beveridge,
1994; Beveridge, 2000). This is most apparent in Bacillus
subtilis
, which has been used as a model Gram-positive
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Gram-positive cell walls and periplasm shown by cryo-TEM
241
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, 240–251
bacterium (Fig. 1), where a three-zoned wall is seen (Gra-
ham and Beveridge, 1994). Here, the wall region immedi-
ately above the plasma membrane is an electron darkzone followed by a more electron translucent zone. The
outermost (and third) zone is a fibrous layer. Each of these
zones is compatible with the concept of cell wall turnover
(Koch, 1983; Koch and Doyle, 1985; Beveridge and Gra-
ham, 1991; Archibald et al
., 1993; Beveridge, 2000), even
when the new ‘scaffold’ (Dmitriev et al
., 2003) or older
‘horizontal layer’ (Vollmer and Höltje, 2004) models for
peptidoglycan arrangement are considered. Like conven-
tional thin sections, freeze-substitutions reveal no appar-
ent periplasmic space between the plasma membrane
and the cell wall (Fig. 1). Even though modern transmis-
sion electron microscopes currently resolve some bioma-terials at better than 0.5–1.0 nm (often by selected area
electron diffraction), major questions still remain about the
organization of Gram-positive cell walls and this is mainly
due to our inability to preserve and view their fabric in its
natural state.
In this report, we use frozen-hydrated thin sections to
provide a more natural view of the B. subtilis
cell envelope
in order to differentiate the wall from the plasma mem-
brane, to more accurately determine the location of the
wall’s polymeric network, and to detect the possible exist-
ence of a periplasmic space. We chose this method
because by rapidly freezing and vitrifying the cells, molec-
ular motion and hydrolytic enzymes are rapidly stopped
thereby preserving ultrastructure in the best possible way
(Dubochet et al
., 1988; Harris, 1997). If the vitrified cells
are thawed, the vast majority of cells regains viability and
once more become active metabolic cellular units. The
difficulty of this cryo-technique, though, is that, unlike thin
sections from conventional and freeze-substitution prepa-
rations that use water to float and stretch sections during
sectioning, the production of frozen-hydrated sections is
done on a dry knife. Accordingly, compression of cells and
the development of discontinuities within the ice during
sectioning impact clarity. Yet, these difficulties can be sur-
mounted and the results are gratifying as they ensure a
re-evaluation of Gram-positive envelope structure. Unlike
traditional TEM methods, the frozen-hydrated sections
used in our study are not stained with heavy metal con-
trasting agents and imaging relies on differential mass
within the cells.
Results
General remarks on the freezing and vitrification
of bacteria
Although high-pressure freezing results in a 10-fold
increase in the vitrification depth (Sartori et al
., 1993), a
cryo-protectant was still required to ensure that deleteri-
ous cytoplasmic nucleation of ice crystals was inhibited.
In our experience, and that of others (Dubochet et al
.,
1983; 1988), the use of cryo-protectants is a necessary
requirement for efficient vitrification of large aggregates ofbacteria for cryo-sectioning. Usually dextran, sucrose or
glycerol are used as cryo-protectants (Dubochet et al
.,
1983; Matias et al
., 2003). In this present study, we chose
glycerol over dextran or sucrose because it has a lower
molecular weight, can be used at a lower concentration
(10% versus 15–20% w/w) and is satisfactory for most
Gram-positive bacteria (V.R.F. Matias and T.J. Beveridge,
unpublished). The reduction in glycerol concentration for
cryo-protection and vitrification can be attributed to the
lower freezing point of the glycerol (compared to dextran
or sucrose) at these concentrations. Furthermore, cells
tend to pack tighter when pelleted by centrifugation inglycerol, resulting in a smaller interstitial volume outside
cells. This is important as cells and their structures tend
to more readily vitrify than the interstitial fluid because of
their relative higher dry weight content.
In order to minimize osmotic effects associated with the
use of glycerol, bacteria were grown with this low molec-
ular weight cryo-protectant in the growth medium (This
was instead of shocking them by brief immersion in the
cryo-protectant immediately before high-pressure freez-
ing). Compared to growth without cryo-protectant, the only
noticeable difference with glycerol was a slight decrease
in the growth rate (
T
d
=
29 min for glycerol versus
T
d
=
20 min without glycerol). During division, cells did not
separate as rapidly during growth in glycerol (resulting in
short chains of cells) but had developed a typical single
cell phenotype by stationary phase. This same alteration
in growth pattern was also seen with growth in (20% w/
w) dextran or (15% w/w) sucrose. Admittedly, glycerol can
be a carbon source for some Gram-positive bacteria,
including B. subtilis
, but under our growth and cryo-pro-
tectant regimen, this did not alter vitr ification. It is possible,
Fig. 1.
Freeze-substituted B. subtilis
168. At high magnification, threeregions of the bacterial envelope are distinguished: 1, heavily stained,innermost region; 2, intermediate region; 3, fibrous wall, outermostregion. Bar represents 50 nm.
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© 2005 Blackwell Publishing Ltd, Molecular Microbiology
, 56
, 240–251
though, that glycerol could have had a subtle effect on
structural detail such as the size of regions within the cell
envelope.
Frozen-hydrated thin sections and associated
cutting artefacts
At low magnification, frozen-hydrated sections of B. sub-
tilis
showed cutting artefacts that are typically associatedwith the technique (Fig. 2A and Dubochet et al
., 1988). In
contrast to resin-embedded samples, where water is used
to float and stretch sections during sectioning, frozen-
hydrated sections cannot be collected on a fluid surface
as there are no liquids with high enough surface tension
that can be used at such low temperatures (i.e. -
140 to
-
180
∞
C). Accordingly, stresses created during cryo-
sectioning remain on the sections as they collect on the
dry knife edge. Cutting artefacts include knife marks, cre-
vasses and compressions associated with the cutting
direction. Contaminating ice crystals can also be found on
the surface of frozen sections because the sections act
as a cold trap for water vapour in the air.
Thin copper tubes were used in our high-pressure
freezing system for rapid energy conduction during vitrifi-
cation. The diameter of the tubes was so narrow that
capillary action on these rod-shaped bacteria aligned
them longitudinally to the tube axis. This also appeared to
be a close-packing phenomenon as the cells were highly
concentrated. Consequently, only cross-sections of the
cylindrical region of cells were obtained (Figs 2A and 3).
Some cross-sections look more oblong than circular
because of compression during sectioning, which reduces
the section length in the cutting direction with a corre-
sponding increase in section thickness (Fig. 2B).
Structure of frozen-hydrated
B. subtilis
The cytoplasm of B. subtilis
was filled with large well-
preserved ribosomes and thin DNA fibres that were dis-
persed throughout the cytosol (Fig. 3A) (Conventional
protocols using chemical fixation show small compacted
ribosomes with the DNA condensed into a fibrous central
mass in the cytoplasm; Beveridge, 1989a). In frozen-
hydrated sections, the cell wall was particularly well pre-
served in the non-deformed regions of the cells (Figs 2B
and 3A) and, strikingly, walls appeared to be bipartite, with
a 22 nm inner zone (IWZ) showing less contrast than a
33 nm outer zone (OWZ) (Fig. 3B and Table 1). As con-
Fig. 2.
Cutting artefacts in frozen-hydrated sections.A. Low-magnification energy-filtered image of a frozen-hydrated sec-tion of B. subtilis
168. Long arrows point to knife marks, short arrowsto crevasses and double arrows to compression in the cutting direc-
tion; bar represents 500 nm.B. Schematic drawing of a cross-section in the absence of compres-sion and of a highly compressed cross-section (upper and lowerpanel respectively; PM: plasma membrane, CW: cell wall). Compres-
sion along the cutting direction results in an increase in sectionthickness. Circles enclose regions of the cell envelope that are leastdeformed. This is where the most accurate measurements of thethickness of structures could be taken (Matias et al
., 2003).
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243
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, 56
, 240–251
trast is directly proportional to density in frozen-hydrated
samples (Dubochet et al
., 1983), our results showed that
the B. subtilis
wall possesses two regions of different
distinct densities. This view differs from the tripartite for-
mat seen in freeze-substituted cells, which show a heavily
stained inner zone followed by a translucent zone and a
more heavily stained fibrous zone at the outer surface of
the wall (Fig. 1; Graham and Beveridge, 1990; 1994).
Cell wall fragments
To help make correlation easier between freeze-substitu-
tion and frozen-hydrated sections of intact cells, cells were
mechanically broken with a French press and SDS boiled
so that cell wall fragments could be isolated, frozen and
cryo-sectioned (Fig. 4A; for images of freeze-substituted
cell wall fragments, see Graham and Beveridge, 1994).
Surprisingly, cross-sections of wall fragments did not
Fig. 3.
Cross-sections of frozen-hydrated B. subtilis
168.A. All cells are aligned at right angles to the plane of the sectionbecause of capillary action (see text for more details). Ribosomes
appear dispersed in the cytoplasm and the plasma membrane isbound by a bipartite wall.B. High magnification image of the envelope showing the plasmamembrane (PM) enclosed by a low-density inner wall zone (IWZ)which is bound by a high-density outer wall zone (OWZ).
Bars represent 200 (A) and 50 nm (B).
Fig. 4.
Cell wall fragments.
A. At low magnification, cell wall (CW) fragments are seen as circularbands with a shape similar to the OWZ seen on cells, indicating thatthe fragments, like the cells, were aligned along the length of thecopper tubes used for high-pressure freezing; only one wall zone is
observed on the fragments.B. At high magnification still only one zone is observed. Black arrow-heads point to ice crystal contamination.Bars represent 500 (A) and 50 nm (B).
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, 56
, 240–251
reveal them to be bipartite but instead to be monopartite
(Fig. 4B). The cell walls consisted of a 45 nm thick single-
zoned dense matrix, presumably the OWZ, which retained
the original curvature of the cell (Fig. 4B and Table 1).
Indeed, cell shape was retained by the wall fragments
(Fig. 4A) implying a certain rigidity and prepatterned con-
tour. Compared to the cell wall seen on intact cells, the
thickness of the OWZ had expanded (from 33 nm to45 nm). As hot SDS treatment solubilizes wall-associated
proteins and contaminating debris from wall fragments
(e.g. membrane, ribosomes, DNA, etc.; Hancock and
Poxton, 1988) and leaves the cell wall intact (i.e. the
peptidoglycan-teichoic acid matrix; Archibald et al
., 1993),
this is strong evidence that the OWZ is in fact the cell wall.
The cross-section diameter of the cell wall fragments
remained unchanged when measured from outer face to
outer wall face (Table 1) but the wall matrix expanded
inwards, which could be a response to the absence of
turgor pressure once the cells were broken. The absence
of the IWZ in isolated wall fragments implies that the innerregion of the wall seen on cells is composed mostly of
soluble less dense components that were washed away
during the isolation of walls. Mass distribution is not
always readily apparent in frozen-hydrated sections but
densitometry of these fragments suggested that there was
a mass decrease from inner to outer face (Fig. 6B). This
will be further discussed in a latter section.
Removal of teichoic acid
Since our cells were grown in relatively rich medium con-
taining phosphate, the walls were in the teichoic acid state
(Neuhaus and Baddiley, 2003). This was confirmed by
phosphorus analysis. As alkali removes this polymer from
the peptidoglycan network, we treated our cell wall frag-
ments with NaOH removing 92.8% of teichoic acids (also
based on phosphorus analysis) resulting in fragments
composed predominantly of peptidoglycan. This removal
of teichoic acid had a profound influence on the wall as
seen in frozen-hydrated sections because the cellular
shape and rigidity of the fragments were lost (Fig. 5A).
Higher magnifications revealed that the walls were thinner
(
~
34 nm thick; Table 1), more bendable and less dense
than untreated walls (cf. Figs 4B and 5B). As, in our 168
strain, teichoic acid accounts for approximately 50% of the
dry weight of wall fragments (Koch and Doyle, 1985;
Archibald et al
., 1993), a loss of contrast and a reduced
thickness were expected once the teichoic polymers were
removed. In an indirect way, this removal of teichoic acidand the maintenance of a monopartite structure help con-
firm that the OWZ is the actual cell wall of the cell.
It is interesting that the isolated walls showed a reten-
tion of cellular shape, which was however, lost after the
extraction of teichoic acids. This is consistent with the
irregularly shaped cells that resulted from the controlled
depletion of an important enzyme for the synthesis of
teichoic acids (Bhavsar et al
., 2001). It also points to a
substantial role for teichoic acids for the rigidity of cell
walls. It seems that interactions between both the phos-
phoryl and protonated D
-alanyl groups of teichoic acids
with the carboxyl groups of the peptidoglycan (Neuhausand Baddiley, 2003) could restrict the conformation of
peptidoglycan fibres, aiding rigidity and retention of
shape.
Polymeric differentiation of the cell wall into zones of
mass distribution
Closer examination of the OWZ at higher magnification
revealed an asymmetric distribution of mass throughout
the wall thickness (Fig. 6A). The OWZ appeared with pro-
gressively less contrast from the inner face to the outer
face, which is similar to the differentiation seen on walls
of freeze-substituted cells (between the intermediate and
outermost regions; Fig. 1). At this point, it must be empha-
sized that the contrast seen in freeze-substitution prepa-
rations is generated by heavy metal stains, thereby
making the detection of different wall zones easier. The
outermost region of the wall is especially better seen
where high hydrolysis rates generate many reactive sites
for metal binding and higher contrast. Conversely, frozen-
hydrated sections show less contrast on the outer face of
Table 1.
Measurements on structures and compartments of B. subtilis
.
a
Structure/compartment Cells Cell wall fragmentsTeichoic acid-extractedcell wall fragments Plasmolysed cells
Cell/cylinder diameter (
m
m)
b
1.04 ± 0.04 1.02 ± 0.07 na 0.95 ± 0.05Protoplast diameter (
m
m) 0.91 ± 0.03 na na 0.77 ± 0.08Plasma membrane thickness (nm) 6.6 ± 0.8 na na 7.1 ± 1.3Inner wall zone thickness (nm) 22.3 ± 4.8 na na na
c
Outer wall zone thickness (nm) 33.3 ± 4.7 44.9 ± 5.4 33.6 ± 4.0 42.7 ± 5.3
a.
Average ±
standard deviation of 12 measurements.
b.
Taken across the cell from outer face to outer face of the wall.
c.
The uneven spacing of the inner wall zone made measurement impossible.
na, not applicable.
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, 56
, 240–251
the OWZ because of its lower local density because autol-
ysins, involved in cell wall turnover, have solubilized the
polymeric network in this region (Fig. 6A). Similar wall
differentiations were also observed in native and teichoic
acids-extracted wall fragments where the wall was denser
on the inner face compared to the outer face as shown by
density tracings of the corresponding images (Fig. 6B,C).
The tracings of all preparations (Fig. 6A–C) showed a
progressive
decrease of wall mass from inner face to outer
face, which is what would be expected with wall turnover
progressing from the inside layers of polymeric network
to the outside.
Interestingly, the removal of teichoic acids affected the
wall uniformly from inside to outside, so that no change in
monopartite infrastructure was seen, suggesting that
these anionic polymers were not segregated to any spe-
cialized region within the wall, thereby confirming previous
experimentation (Neuhaus and Baddiley, 2003). However,
extraction of teichoic acids reduced the wall thickness by
10 nm (Table 1) indicating that either teichoic polymers
extend about 10 nm above the surface of the peptidogly-
can network or that their interaction with peptidoglycan
expands the entire wall fabric by this amount of extension.
Plasmolysed cells
In order to further investigate how the IWZ integrates into
the cell envelope structure of B. subtilis
, intact cells wereplasmolysed so as to artificially increase the separation
between the wall fabric and the plasma membrane. In this
case cells were grown without a cryo-protectant and then
subjected to aqueous solutions of increasing osmolarity
(10% glycerol, 20% sucrose, 20% glycerol and 20%
glycerol-5% NaCl solutions). The 20% glycerol-5% NaCl
solution caused the best plasmolysis of cells and,
because glycerol was used, vitrification could immediately
be done. These plasmolysed cells possessed compacted
cytoplasms and gaps were seen between the plasma
membrane and the cell wall (cf. Fig. 3A with Fig. 7A–C);
the protoplasts were shrinking (as the water was beingdrawn out of them) and the space between wall and mem-
brane was increasing. The density of the space resembled
the IWZ except that it was thicker. High magnifications of
this space showed it to be of low density (i.e. similar to
the ice surrounding the cells) so that there could be little
actual substance to it (Fig. 7D). Above this space a much
denser layer, the OWZ, could be seen. The OWZ now
approached the thickness of the cell wall fragments
(Table 1), which would be expected as plasmolysed cells
do not exert turgor pressure (or surface tension; Koch,
1983) on the walls (i.e. the walls would not be as stretched
and compacted as in normal cells).
Frequently, membrane vesicles extruded into the artifi-
cial gap (Fig. 7C) and these were filled with cytoplasmic
material (Fig. 7E). These resembled the ‘mesosome bod-
ies’ found in Bacillus megaterium
after plasmolysis in
sucrose solutions stronger than 1 M (Weibull, 1965). Con-
traction of the protoplast during plasmolysis resulted in a
considerable reduction in the total area of the membrane,
and hence a fraction of it was excised as membrane
vesicles. It is also possible that distinct regions of the
Fig. 5.
Teichoic acid-extracted cell wall fragments.A. Here the fragments lack a defined shape, implying that teichoicacids play a significant role in the overall structure of the cell wall.B. At higher magnification the walls remain single-zoned (as in Fig. 4)
but they also appear bendable and less dense. Black arrowheadspoint to ice crystal contamination, while white arrowheads point tocracks and loose fibres in the supporting film.Bars represent 500 (A) and 75 nm (B).
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, 240–251
membrane were strongly bound to the cell wall via peni-
cillin-binding proteins (PBPs; Blumberg and Strominger,
1972), lipoteichoic acids (Neuhaus and Baddiley, 2003)and lipoproteins (Sutcliffe and Russel, 1995; Sutcliffe and
Harrington, 2002), thereby pulling away these areas as
the protoplast retracted from the cell wall and helping to
develop the vesicles. As the protoplasts continued to
shrink during plasmolysis, these vesicles were forced out
of the cell into this space until they were confined by the
OWZ (Fig. 7E). If this space (i.e. the IWZ) consisted of
significant substance and was an essential part of the cell
wall matrix, the vesicles would not have room to develop
and be present. Whatever is in the IWZ, it is quite com-
pactable, is of low density, and can be deformed by the
action of the vesicles. These data again suggest that
the IWZ is less substantial (as it has little density) than
the OWZ, and that the actual wall is the OWZ. It is prob-
able that the IWZ is a periplasmic space, which is confined
between the plasma membrane and the cell wall and is
filled with a variable and low concentration of periplasmic
components. Even though this region has low mass, it
must still contain important components such as PBPs,
lipoteichoic acids, enzymes and secreted proteins.
Indeed, the concentration of substance in this periplasmic
region, although low, must be great enough to resist com-
pression resulting from turgor pressure exerted by the
protoplast. Additionally, lipoteichoic acids, which areembedded into the plasma membrane and extend into the
cell wall, could aid in connecting the membrane, periplas-
mic space and cell wall amalgam together.
Discussion
Existence of a periplasmic space
In this study, we present the structure of B. subtilis
168
and its cell envelope by cryo-TEM of frozen-hydrated
sections. The plasma membrane is surrounded by a low-
density 22 nm thick zone, which is enclosed by a higher-
density 33 nm thick zone. Our results strongly suggest
that the IWZ is a periplasmic space while the OWZ is the
actual cell wall consisting of a peptidoglycan-teichoic acid
polymeric matrix and associated proteins. By convention,
using the terminology employed for the Gram-negative
envelope (Beveridge and Graham, 1991; Beveridge,
1999; Matias et al ., 2003), the periplasmic space and its
constituent periplasm should be considered an essential
but less substantial part of the cell wall.
Fig. 6. Cell wall differentiation. High magnifica-tion images with corresponding digital densito-
metry scans.A. The cell envelope showing the OWZ withprogressively less contrast from its inner faceto outer face.B. In cell wall fragments, the inner face also
shows more contrast than the outer face of thewall.C. Similar to cell wall fragments, teichoic acid-
extracted fragments show a similar wall differ-entiation.
Arrows point to the inner face of the OWZ andwall fragments, while arrowheads point to thewall’s outer face. The density tracings empha-size this differentiation as well as showing that
the mass distribution progressively decreasesfrom inner face to outer face. Bar represents50 nm.
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The conclusion that the IWZ represents an extra-proto-
plasmic compartment between the plasma membrane
and the peptidoglycan-teichoic acid wall matrix comes
from a number of observations. First, only one substantivewall zone was observed above the plasma membrane with
intact cells, and this same zone was seen in wall frag-
ments (i.e. the OWZ) and to a lesser extent in teichoic
acid-extracted fragments. Second, the OWZ and both
types of wall fragments possessed a differentiated wall
structure, showing higher density at the wall inner face
compared to the outer face. The IWZ was not differenti-
ated. Third, membrane vesicles were able to extrude into
the IWZ. Fourth, after extrusion the membrane vesicles
were deformed and limited by the OWZ indicating the solid
nature of this outer layer, whereas the IWZ was non-
deforming. And, fifth, the IWZ had very low density (little
substance) and the OWZ had high density (elevated sub-
stance) as determined by their innate electron scattering
power.
The conclusions drawn from our hydrated-frozen sec-
tion data could be interpreted as diametrically opposed to
previous freeze-substitution experiments (Amako et al .,
1982; Umeda et al ., 1987; Graham and Beveridge, 1990;
1994; Beveridge and Graham, 1991; Graham et al ., 1991;
Paul et al ., 1993; Beveridge, 1995; 2000), but this is not
so. Freeze-substitution, although a cryo-technique that
accurately preserves cells, provides entirely different infor-
mation than that provided here by frozen-hydrated sec-
tions. In freeze-substitution, cells are vitrified and thenchemically substituted at -80∞C (i.e. chemically fixed,
dehydrated and embedded in plastic; Beveridge et al .,
2005). Before the bacteria can be imaged in thin section,
they must be stained by heavy metal salts (such as ura-
nium and lead) to increase the scattering power of the
biomaterial beyond that of the embedding plastic. Electron
microscopic images of freeze-substituted bacteria, then,
depend on the binding of heavy metal stains to reactive
sites in the biomaterial (primarily electronegative groups;
Beveridge, 1989b) and, accordingly, reveal where these
reactive sites are (Koval and Beveridge, 1999). This is
different from frozen-hydrated sections, which reveal
where regions of high differential mass occur (i.e. as
opposed to the density of vitrified water).
Correlation of previous freeze-substitutions of B. subti-
lis (Graham and Beveridge, 1994; also see Fig. 1) with
our current frozen-hydrated section results (e.g. Fig. 3B)
suggests a periplasmic space exists that is filled with
low-density material (frozen-hydrated sections) and that
this material (i.e. periplasm) is highly reactive with heavy
metal stains so as to produce high contrast in the region
Fig. 7. Plasmolysed cells.A. Suspension in 20% glycerol-5% NaCl solu-tion caused plasmolysis of cells; most cells
show a large separation between the protoplastand OWZ.B. Plasmolysed cells are often observed with alarger separation between the OWZ (longer
arrow) and plasma membrane (shorter arrow)at one side of the cell. About half of the
observed plasmolysed cells show no additionalstructure between the OWZ and the plasmamembrane.
C. The other half of observed plasmolysedcells are seen with ‘blebs’ (arrows) between theOWZ and plasma membrane.D. A high magnification image in an area of the
IWZ that does not contain vesicles further cor-roborates its low density with no additionalstructures between the OWZ and membrane.E. Areas containing membrane vesicles showthem to be relatively well separated from one
another.Arrowheads in A–C point to ice crystal contam-ination. Bars represent 500 (A), 200 (B and C)and 50 nm (D and E).
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248 V. R. F. Matias and T. J. Beveridge
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(freeze-substitution). This correlation is diagrammed in
Fig. 8.
Initial observations on Gram-negative cell envelopes by
freeze-substitution suggested that these periplasmic
spaces where so filled with periplasmic materials that a
gel was formed, the ‘periplasmic gel’ (Hobot et al ., 1984).
Yet, frozen-hydrated sections suggested that the Gram-
negative periplasm was a low-density material (Matias
et al ., 2003). Like the periplasm of B. subtilis seen in our
present study, the periplasms of Escherichia coli and
Pseudomonas aeruginosa also appear to be filled with
low-density but intensely reactive (staining) substance(Matias et al ., 2003). This new information adds to our
growing understanding of periplasmic spaces in prokary-
otes (Graham et al ., 1991; Beveridge, 1995; Merchante
et al ., 1995).
The existence of a periplasmic space in Gram-positive
bacteria could be advantageous; it would provide a space
to manoeuvre enzymes in between the plasma mem-
brane and cell wall, away from the highly negatively
charged wall polymers and sufficiently apart from the wall
to avoid steric crowding. In S. pneumoniae and S. aureus ,
crystal structure determination of PBPs has shown that
they could extend 13–9.7 nm above the membrane
(Pares et al ., 1996; Lim and Strynadka, 2002). This
approximates the 7.5 nm length of PBP5 in E. coli (Nicho-
las et al ., 2003; C. Davies, pers. comm.), which is close
to the distance of 9.3 nm between the plasma membrane
and the peptidoglycan layer seen in frozen-hydrated sec-
tions of this bacterium (Matias et al ., 2003). It is probable
that PBPs require a certain amount of free space within
the periplasm to catalyse the development of new wall
fabric.
Interpretation of the mass distribution within the
peptidoglycan-teichoic acid network (i.e. OWZ)
One additional feature that is not seen in the frozen-
hydrated sections is the ‘fringe’ that is seen at the top of
the wall in freeze-substitutions (Figs 1 and 8), which has
been attributed to cell wall turnover (Graham and Bever-
idge, 1994) because this is a region that is actively being
hydrolysed into soluble polymers (Mobley et al ., 1984;
Koch and Doyle, 1985; Archibald et al ., 1993). It is thought
that the hydrolysis of bonds in this region of the wall
results in the disassembly and agglomeration of wall com-ponents into a fibrillar fringe during dehydration, which is
readily seen because of the staining of many of these
exposed reactive sites with heavy metals during freeze-
substitution. This fringe is not seen in frozen-hydrated
sections because biological structures are immobilized in
their fully hydrated state during vitrification, preventing the
highly hydrolysed outermost wall from agglomerating into
a fibrillar material. Instead, frozen-hydrated sections
showed progressively less contrast (lower density)
through the wall thickness from inside to outside on cells
and isolated wall fragments (Figs 3B, 4B and 5B). This is
also in agreement with the concept of cell wall turnover
and corroborates previous freeze-substitution results on
the differentiation of the B. subtilis cell wall (Fig. 8).
The traditional view of peptidoglycan organization in
bacterial walls is that the glycan strands are laid down as
interconnected polymeric sheets (Pink et al ., 2000; Voll-
mer and Höltje, 2004). Gram-positive walls would have
multiple sheets sitting on top of and covalently linked to
one another (Koch and Doyle, 1985) in a manner that
should provide innate molecular infrastructure throughout
Fig. 8. Schematic representation of the B. subtilis cell envelope, as revealed by freeze-substitution (left) and frozen-hydrated sections (right). Thedark innermost region seen in freeze-substituted images corresponds to the IWZ of frozen-hydrated sections and accordingly is the periplasmicspace. In freeze-substitutions, we believe the periplasmic space appears much thinner than the space seen in frozen-hydrated sections as theformer has condensed in size because of dehydration and plastic embedding. The wall differentiation depicted by freeze-substitution and frozen-hydrated sections is also depicted in these diagrams and is explained in the text. Both diagrams are consistent with wall turnover progressively
occurring from inside face of the wall to the outside face. PM, plasma membrane; PS, periplasmic space; CW, cell wall.
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Acknowledgements
This work was supported by a Natural Science and Engineer-
ing Research Council of Canada (NSERC) Discovery grant
to T.J.B. V.R.F.M. is recipient of a PhD scholarship from
CNPq/Brazil. Microscopy was performed in the NSERC
Guelph Regional STEM Facility, which is partially funded by
an NSERC Major Facility Access grant to T.J.B.
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