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



Gram-positive cell walls and periplasm shown by cryo-TEM

241

© 2005 Blackwell Publishing Ltd, Molecular Microbiology

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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.



242

V. R. F. Matias and T. J. Beveridge


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, 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).




243


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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).



244



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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.




245


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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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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.



Gram-positive cell walls and periplasm shown by cryo-TEM 247

© 2005 Blackwell Publishing Ltd, Molecular Microbiology , 56, 240–251

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).



248 V. R. F. Matias and T. J. Beveridge


(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.





250 V. R. F. Matias and T. J. Beveridge


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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