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2 Wall Ultrastructure and Periplasm

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Abstract:

This chapter concentrates on gram-positive bacteria, such as and , that the author elucidated using new cryoTEM technology, which maintains the hydrated nature of the sample. For mycobacterial cell envelopes, more care is necessary because it is recognized that several of the wall constituents are soluble or deformable in the organic solvents that are used during dehydration. Ultrarapid freezing of cells is the central feature for cryoTEM. This freezing must occur very rapidly (less than milliseconds) so that vitrification of the sample occurs; amorphous ice, having the consistency of a glass, is formed and all macromolecular motion is stopped. In , new wall polymers enter the wall immediately above the plasma membrane where they accumulate and are (eventually) covalently bonded into the existing framework via transpeptidation of newly synthesized peptidoglycan strands to older strands. Above the periplasmic zone, there lies a darker zone of higher mass. This must be the cell wall constructed of peptidoglycan and secondary polymers. Careful densitometry of frozen hydrated walls revealed a graduation of high density to low density from the inside face of the cell wall to the outside. Mycobacteria possess an unusual cell wall consisting of an intermediate thickness of peptidoglycan and a set of unique additional components that, together, retain crystal violet within the cell when they undergo Gram staining.

Citation: J. Beveridge T. 2008. 2 Wall Ultrastructure and Periplasm, p 13-23. In Daffé M, Reyrat J, Avenir G (ed), The Mycobacterial Cell Envelope. ASM Press, Washington, DC. doi: 10.1128/9781555815783.ch2

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Figures

Image of Figure 1.
Figure 1.

(A) Thin section of a conventionally embedded showing a rather featureless gram-positive cell wall. The DNA is condensed in the center of the cell. Scale bar = 200 nm. (B) Thin section of a dividing cell through conventional fixation. Note that this cell possesses a more complex cell wall that possesses an electron dense outer layer and an electron translucent inner layer (arrow). The DNA is (again) condensed in the center of the cell. These cells are more difficult to fix than (A) and are not as well preserved. Scale bar = 100 nm. See Paul and Beveridge ( ) for more details.

Citation: J. Beveridge T. 2008. 2 Wall Ultrastructure and Periplasm, p 13-23. In Daffé M, Reyrat J, Avenir G (ed), The Mycobacterial Cell Envelope. ASM Press, Washington, DC. doi: 10.1128/9781555815783.ch2
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Image of Figure 2.
Figure 2.

(A) Thin section of a PAO1 biofilm that has been freeze-substituted. This type of bacterial consortium is among the most difficult biomaterials to preserve, and these gram-negative cells complete with their lipopolysaccharide (LPS, large arrow) O-side chains, and exopolymeric substances (EPS, small arrows) are seen at high fidelity. Scale bar = 100 nm. See Hunter and Beveridge ( ) for more details. (B) This cell has been freeze-substituted, and the cell has been preserved with high fidelity. Now, the DNA is dispersed throughout the entire cytoplasm, and the cell wall is composed of three separate regions (numbered 1 to 3). As explained in the text, these regions correspond to the way in which this gram-positive wall turns over. Scale bar = 100 nm. See Graham and Beveridge ( ) for more details.

Citation: J. Beveridge T. 2008. 2 Wall Ultrastructure and Periplasm, p 13-23. In Daffé M, Reyrat J, Avenir G (ed), The Mycobacterial Cell Envelope. ASM Press, Washington, DC. doi: 10.1128/9781555815783.ch2
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Image of Figure 3.
Figure 3.

(A) A frozen hydrated thin section of a vitrified cell. Here, the cell is unstained, is preserved exactly as it was in life, and is seen simply because of the density of its constituent mass. As in freeze-substitutions, the DNA is dispersed throughout the cytoplasm with the ribosomes appearing as dark particles. The plasma membrane (PM) and cell wall (CW) are pointed out. The large striations running from upper right to lower left are score marks left in the ice by the diamond knife used for sectioning. Scale bar = 100 nm. (B) A higher magnification of a frozen hydrated cell showing the plasma membrane and cell wall. Again, it is the actual concentration of mass in the various parts of the cell that provides us with an image. Notice how the cell wall is darker (higher mass) close to the membrane and less dark (lower mass) at the cell walls periphery, confirming the cell wall turnover detected in freeze-substitutions (cf. this Fig with Fig. 4 ). For the first time, using any thin section method, the periplasmic space (PS) is preserved. Scale bar = 20 nm. See Matias and Beveridge ( ) for more details. (C) High magnification of the plasma membrane (PM) and cell wall (CW) of a frozen hydrated cell. Because this coccoid bacterium produces most of its new wall through septation during division, it does not turnover its cell wall (like does), and therefore, the cell wall has a continuous density throughout its thickness. Like and other gram-positive bacteria processed by the frozen hydrated thin section method, a periplasmic space (PS) is seen. Scale bar = 20 nm. See Matias and Beveridge (2006b) for more details.

Citation: J. Beveridge T. 2008. 2 Wall Ultrastructure and Periplasm, p 13-23. In Daffé M, Reyrat J, Avenir G (ed), The Mycobacterial Cell Envelope. ASM Press, Washington, DC. doi: 10.1128/9781555815783.ch2
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Image of Figure 4.
Figure 4.

(A) High magnification of the cell envelope of a freeze-substituted cell showing a well preserved cell wall structure. The darkly stained peptidoglycan (PG), with a weakly stained midlayer (ML) within the electron transparent layer (the ETL is not labeled), and electron dense outer layer (OL). Scale bar = 100 nm. See Paul and Beveridge ( ) for more details. (B) A frozen hydrated thin section of a cell revealing that the septum is much more complex than originally thought. PM, plasma membrane; CW, cell wall; PS, periplasmic space; SW, septum wall; MZ, middle zone; Tip, septal tip. Scale bar = 20 nm. See Matias and Beveridge ( ) for more details.

Citation: J. Beveridge T. 2008. 2 Wall Ultrastructure and Periplasm, p 13-23. In Daffé M, Reyrat J, Avenir G (ed), The Mycobacterial Cell Envelope. ASM Press, Washington, DC. doi: 10.1128/9781555815783.ch2
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