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Chapter 1 : Ultrastructure of Gram-Positive Cell Walls

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Abstract:

This chapter explains the procedures used to examine ultrastructure of gram-positive cell walls by transmission electron microscopy (TEM) and discusses the results in the context of our current view of polymeric arrangements. Often today, electron microscopy is performed by institutional nonspecialized TEM centers that have little microbiological experience, and few of these have state-of-the-art cryo-units. Due to this, the chapter discusses the traditional techniques and their results in case this more modern equipment is not readily available so that the reader knows what to expect. Then, it gives a more up-to-date current viewpoint provided by cryo-TEM and atomic force microscopy (AFM) and shows how these results integrate with traditional views. The chapter also talks about general chemistry of gram-positive cell walls, cell wall turnover, mycobacterial walls, S-layered walls, and gram-positive periplasm.

Citation: Beveridge T, Matias V. 2006. Ultrastructure of Gram-Positive Cell Walls, p 3-11. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch1

Key Concept Ranking

Bacterial Diseases
0.53627205
Cell Wall
0.50281274
Scanning Electron Microscopy
0.4995817
Transmission Electron Microscopy
0.4995817
Scanning Electron Microscopy
0.4995817
Transmission Electron Microscopy
0.4995817
Cell Wall Proteins
0.4709522
0.53627205
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Figures

Image of FIGURE 1
FIGURE 1

SEM micrograph of showing the concentric circular structures of the cell wall surface (arrows) that suggest the circular arrangement of polymers in the cell wall. Bar = 250 nm. (Reproduced from 147–148 [1978] with permission.)

Citation: Beveridge T, Matias V. 2006. Ultrastructure of Gram-Positive Cell Walls, p 3-11. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch1
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Image of FIGURE 2
FIGURE 2

TEM micrograph of a freeze-fractured and freeze-etched showing a cross fracture of the cell wall (large arrow). Notice that little polymeric infrastructure is seen in the wall by this technique. The encircled arrow points out the shadow direction and the scale bar = 50 nm.

Citation: Beveridge T, Matias V. 2006. Ultrastructure of Gram-Positive Cell Walls, p 3-11. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch1
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Image of FIGURE 3
FIGURE 3

TEM freeze-etching of a showing a closing septum (small arrows) between two daughter cells. Again, cross fractures of the cell wall show little infrastructure within the wall's matrix. The large arrow with circle denotes shadow direction. Bar = 100 nm.

Citation: Beveridge T, Matias V. 2006. Ultrastructure of Gram-Positive Cell Walls, p 3-11. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch1
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Image of FIGURE 4
FIGURE 4

TEM freeze-fracture image of a cell wall septum from an wall preparation that was previously treated with trichloroacetic acid to remove all associated teichoic acid and proteins. Because this wall now contains only peptidoglycan, the concentric circular arrangement (small arrows) must be the arrangement of peptidoglycan. The large arrow with circle denotes shadow direction. Bar = 100 nm. (Reproduced from 844–850 [1982] with permission.)

Citation: Beveridge T, Matias V. 2006. Ultrastructure of Gram-Positive Cell Walls, p 3-11. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch1
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Image of FIGURE 5
FIGURE 5

TEM negative stain of the pole of a B. anthracis cell showing capsular material (arrow) above the cell wall. Underneath the capsule a moiré pattern is seen, which is the S-layer ( ) of this pathogen. Both of these structures are virulence factors. The arrow points to small fibrils that are the capsule of the bacterium. Bar = 100 nm.

Citation: Beveridge T, Matias V. 2006. Ultrastructure of Gram-Positive Cell Walls, p 3-11. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch1
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Image of FIGURE 6
FIGURE 6

TEM micrograph of a thin section of a conventionally embedded showing a growing septum (arrow) complete with a developing midline that will be used to eventually separate the two daughter cells. Note that the septum has some texture but that the older preexisting wall does not. Bar = 100 nm.

Citation: Beveridge T, Matias V. 2006. Ultrastructure of Gram-Positive Cell Walls, p 3-11. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch1
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Image of FIGURE 7
FIGURE 7

TEM micrograph of a thin section of freeze-substituted showing the tripartite fine structure in the cell wall that demonstrates cell wall turnover. Region #1 is densely stained because the newly laid down peptidoglycan is condensed and reactive to the staining reagents. Region #2 is more lightly stained because this is the stress-bearing peptidoglycan that is highly stretched and therefore not as dense or condensed as in region #1. Region #3 is undergoing hydrolysis by peptidoglycan hydrolases (autolysins) and is more fibrous. Because covalent bonds are being clipped, there are many reactive groups with which the staining reagents can interact, and this region is darkly stained. Bar = 100 nm.

Citation: Beveridge T, Matias V. 2006. Ultrastructure of Gram-Positive Cell Walls, p 3-11. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch1
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Image of FIGURE 8
FIGURE 8

(A) Cryo-TEM micrograph of a hydrated frozen section of a cell. The ribosomes are dispersed throughout the cytoplasm (arrows), and the plasma membrane (PM) is bounded by a bipartite cell wall (CW). Unlike other TEM images, this cell is unstained, and the contrast is provided by cellular mass distribution and phase contrast. Ridges and valleys can be seen in the surrounding ice and are cutting artifacts (see reference 29 for details). Bar = 200 nm. (B) High magnification of a hydrated frozen section of the cell envelope for comparison with the freeze-substitution tripartite wall image in Fig. 7 . The PM is enclosed by a low-density inner wall zone (IWZ), which is bounded by a high-density outer wall zone (OWZ). The inner surface of the OWZ (white arrow) appears to have higher contrast and therefore more innate density. Bar = 50 nm.

Citation: Beveridge T, Matias V. 2006. Ultrastructure of Gram-Positive Cell Walls, p 3-11. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch1
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Image of FIGURE 9
FIGURE 9

(A) AFM deflection image of a newly formed cell wall derived from the septum showing concentric rings surrounding the central depression of the closed septum. The rings correspond to the manner in which the new wall polymers are laid down and configured during septation and cell separation. Bar = 50 nm. (Previously published in 186:3286–3295 [2004] and reproduced with permission.) (B) High-resolution AFM image of an old region of the cell wall (well removed from a septal region) showing the topography of the wall surface. Notice how fibrous and porous it is; this is probably due to cell wall turnover. Bar = 50 nm.

Citation: Beveridge T, Matias V. 2006. Ultrastructure of Gram-Positive Cell Walls, p 3-11. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch1
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Image of FIGURE 10
FIGURE 10

TEM thin section of a cell pole of a conventionally embedded showing its S-layer (SL) above the cell wall (CW) and plasma membrane (PM). A flagellum (F) emanating from the surface can also be seen. Bar = 100 nm.

Citation: Beveridge T, Matias V. 2006. Ultrastructure of Gram-Positive Cell Walls, p 3-11. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch1
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Image of FIGURE 11
FIGURE 11

Thin section of isolated cell walls from that were processed by the freeze-substitution technique after the culture was mechanically disrupted. As with the freeze-substituted cells shown in Fig. 7 , these cell walls also have a fibrous surface (arrow) and tripartite structure. Bar = 100 nm. (Reproduced from 2482–2487 [1987] with permission.)

Citation: Beveridge T, Matias V. 2006. Ultrastructure of Gram-Positive Cell Walls, p 3-11. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch1
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Image of FIGURE 12
FIGURE 12

Thin section of a freeze-substituted that shows the complexity of this bacterium's cell envelope. C, capsule or electron-transparent region; M, mycolates; A + PG, arabinogalactan plus peptidoglycan; PM, plasma membrane. Bar = 50 nm. More details of this image can be found in references 14 and 28. (Reproduced with permission of the author.)

Citation: Beveridge T, Matias V. 2006. Ultrastructure of Gram-Positive Cell Walls, p 3-11. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch1
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Image of FIGURE 13
FIGURE 13

Freeze-etched surface of an 10155/C showing the S-layer with its square lattice (; = = 10 nm), which is made up of a glycoprotein. See reference 20 for more details. Flagella can be seen on top of the S-layer (small arrow). The large arrow with circle denotes shadow direction. Bar = 100 nm.

Citation: Beveridge T, Matias V. 2006. Ultrastructure of Gram-Positive Cell Walls, p 3-11. In Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J (ed), Gram-Positive Pathogens, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816513.ch1
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