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Cryo-transmission electron microscopy of frozen-hydrated sections of Escherichia coli and Pseudomonas aeruginosa - PubMed

Cryo-transmission electron microscopy of frozen-hydrated sections of Escherichia coli and Pseudomonas aeruginosa

Valério R F Matias et al. J Bacteriol. 2003 Oct.

Abstract

High-pressure freezing of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1 in the presence of cryoprotectants provided consistent vitrification of cells so that frozen-hydrated sections could be cut, providing approximately 2-nm resolution of structure. The size and shape of the bacteria, as well as their surface and cytoplasmic constituents, were nicely preserved and compared well with other published high-resolution techniques. Cells possessed a rich cytoplasm containing a diffuse dispersion of ribosomes and genetic material. Close examination of cells revealed that the periplasmic space was compressed during cryosectioning, a finding which provided supporting evidence that this space is filled by a compressible gel. Since the outer membrane and peptidoglycan layer are bonded together via lipoproteins, the space between them (although still part of the periplasmic space) was not as compacted. Even when this cryosectioning compression was taken into account, there was still substantial variability in the width of the periplasmic space. It is possible that the protoplast has some capacity to float freely within the periplasm.

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Figures

FIG. 1.
FIG. 1.

Compression of the bacterial envelope in gram-negative cells during cryosectioning. (A) Schematic drawing of a cross-section in the absence of compression. (B) Schematic drawing of a highly compressed cross-section. The circles at each end of the cell enclose nondeformed regions of the envelope, while rectangles enclose highly deformed regions; compression along the cutting direction corresponds to an increase in the section thickness (see edges of each diagram).

FIG. 2.
FIG. 2.

An energy-filtered image of a frozen-hydrated section of E. coli K-12 shown at low magnification. The long arrows point to knife marks (running in the direction indicated by the arrows), and the shorter arrows point to ice crystal contamination. The inset shows corresponding electron diffraction pattern. Bar, 500 nm.

FIG. 3.
FIG. 3.

A frozen-hydrated section of P. aeruginosa PAO1 at low magnification. Compression along the cutting direction is indicated by the double arrows, and crevasses are indicated by arrowheads. Bar, 500 nm.

FIG. 4.
FIG. 4.

E. coli K-12 at higher magnification. OM, PG, and PM are clearly seen around cells, and the cytoplasm has an even distribution of ribosomes and genetic material. Bar, 200 nm.

FIG. 5.
FIG. 5.

P. aeruginosa PAO1 at higher magnification. The OM, PG, and PM are clearly seen around cells, and the cytoplasm has a similar appearance to that of E. coli in Fig. 4. Bar, 200 nm.

FIG. 6.
FIG. 6.

Enlarged view of Fig. 4 showing the uneven compression of the cell wall resulting from the force of cutting the thin section (see text for more detailed explanation). Comparison between highly deformed (rectangle) and nondeformed (rectangle) regions of the envelope shows that the space between PM and PG (long arrows) is more condensed than the space between PG and OM (short arrows). Bar, 150 nm.

FIG. 7.
FIG. 7.

High magnification of the K-12 cell envelope showing the asymmetric densities of the two OM faces (short arrow) in contrast with the more symmetric PM (long arrow). Bar, 50 nm.

FIG. 8.
FIG. 8.

An image of the PAO1 envelope (similar to that of K-12 depicted in Fig. 7). The cell was grown on Trypticase soy agar and processed in 20% (wt/vol) dextran for high-pressure freezing. This high-magnification view of the envelope shows OM (short arrow) asymmetry in comparison to that of the PM (long arrow). The periplasmic space appears wider in comparison to the other images, which might be due to the growth of the cells on solid medium or to the presence of dextran as the cryoprotectant. Bar, 50 nm.

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References

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