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The Gram-Positive Bacterial Cell Wall

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.
  • Author: Manfred Rohde1
  • Editors: Vincent A. Fischetti2, Richard P. Novick3, Joseph J. Ferretti4, Daniel A. Portnoy5, Miriam Braunstein6, Julian I. Rood7
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Helmholtz Centre for Infection Research, HZI, Central Facility for Microscopy, ZEIM, Braunschweig, Germany; 2: The Rockefeller University, New York, NY; 3: Skirball Institute for Molecular Medicine, NYU Medical Center, New York, NY; 4: Department of Microbiology & Immunology, University of Oklahoma Health Science Center, Oklahoma City, OK; 5: Department of Molecular and Cellular Microbiology, University of California, Berkeley, Berkeley, CA; 6: Department of Microbiology and Immunology, University of North Carolina-Chapel Hill, Chapel Hill, NC; 7: Infection and Immunity Program, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia
  • Source: microbiolspec May 2019 vol. 7 no. 3 doi:10.1128/microbiolspec.GPP3-0044-2018
  • Received 13 September 2018 Accepted 17 September 2018 Published 24 May 2019
  • Manfred Rohde [email protected]
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  • Abstract:

    The chapter about the Gram-positive bacterial cell wall gives a brief historical background on the discovery of Gram-positive cell walls and their constituents and microscopic methods applied for studying the Gram-positive cell envelope. Followed by the description of the different chemical building blocks of peptidoglycan and the biosynthesis of the peptidoglycan layers and high turnover of peptidoglycan during bacterial growth. Lipoteichoic acids and wall teichoic acids are highlighted as major components of the cell wall. Characterization of capsules and the formation of extracellular vesicles by Gram-positive bacteria close the section on cell envelopes which have a high impact on bacterial pathogenesis. In addition, the specialized complex and unusual cell wall of mycobacteria is introduced thereafter. Next a short back view is given on the development of electron microscopic examinations for studying bacterial cell walls. Different electron microscopic techniques and methods applied to examine bacterial cell envelopes are discussed in the view that most of the illustrated methods should be available in a well-equipped life sciences orientated electron microscopic laboratory. In addition, newly developed and mostly well-established cryo-methods like high-pressure freezing and freeze-substitution (HPF-FS) and cryo-sections of hydrated vitrified bacteria (CEMOVIS, Cryo-electron microscopy of vitreous sections) are described. At last, modern cryo-methods like cryo-electron tomography (CET) and cryo-FIB-SEM milling (focus ion beam-scanning electron microscopy) are introduced which are available only in specialized institutions, but at present represent the best available methods and techniques to study Gram-positive cell walls under close-to-nature conditions in great detail and at high resolution.

  • Citation: Rohde M. 2019. The Gram-Positive Bacterial Cell Wall. Microbiol Spectrum 7(3):GPP3-0044-2018. doi:10.1128/microbiolspec.GPP3-0044-2018.

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/content/journal/microbiolspec/10.1128/microbiolspec.GPP3-0044-2018
2019-05-24
2019-09-18

Abstract:

The chapter about the Gram-positive bacterial cell wall gives a brief historical background on the discovery of Gram-positive cell walls and their constituents and microscopic methods applied for studying the Gram-positive cell envelope. Followed by the description of the different chemical building blocks of peptidoglycan and the biosynthesis of the peptidoglycan layers and high turnover of peptidoglycan during bacterial growth. Lipoteichoic acids and wall teichoic acids are highlighted as major components of the cell wall. Characterization of capsules and the formation of extracellular vesicles by Gram-positive bacteria close the section on cell envelopes which have a high impact on bacterial pathogenesis. In addition, the specialized complex and unusual cell wall of mycobacteria is introduced thereafter. Next a short back view is given on the development of electron microscopic examinations for studying bacterial cell walls. Different electron microscopic techniques and methods applied to examine bacterial cell envelopes are discussed in the view that most of the illustrated methods should be available in a well-equipped life sciences orientated electron microscopic laboratory. In addition, newly developed and mostly well-established cryo-methods like high-pressure freezing and freeze-substitution (HPF-FS) and cryo-sections of hydrated vitrified bacteria (CEMOVIS, Cryo-electron microscopy of vitreous sections) are described. At last, modern cryo-methods like cryo-electron tomography (CET) and cryo-FIB-SEM milling (focus ion beam-scanning electron microscopy) are introduced which are available only in specialized institutions, but at present represent the best available methods and techniques to study Gram-positive cell walls under close-to-nature conditions in great detail and at high resolution.

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Figures

Image of FIGURE 1
FIGURE 1

The bacterial cell wall backbone, peptidoglycan; shown are the two glycan strands (in black). Peptide stems are depicted in black (left side), and the second peptide stem, in blue. Note the cross-linking NH (in red) via the two unusual amino acids m-diaminopimelic acid (m-Dap, in red) and the presence of -alanine in the peptide stems. Two more peptide stems (green and pink) are depicted which can interact to build the next cross-linking between glycan strands.

Source: microbiolspec May 2019 vol. 7 no. 3 doi:10.1128/microbiolspec.GPP3-0044-2018
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Image of FIGURE 2
FIGURE 2

TEM image taken at an acceleration voltage of 80 kV of a peptidoglycan sacculus of after boiling for 3 h in 10% SDS. The mesh-like sacculus was negatively stained with 1% aqueous uranyl acetate, air-dried, and observed in a normal TEM.

Source: microbiolspec May 2019 vol. 7 no. 3 doi:10.1128/microbiolspec.GPP3-0044-2018
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Image of FIGURE 3
FIGURE 3

Schematic drawing of Gram-negative and Gram-positive cell walls. A characteristic of Gram-negative cell walls is the presence of two membranes, the cytoplasmic membrane and the outer membrane. Between these membranes is the periplasmic space, in which a very thin layer of peptidoglycan is found; lipopolysaccharides are attached to the outer membrane, and porins are inserted in the outer membrane. A thick layer of peptidoglycan and the lack of an outer membrane are the main characteristics of Gram-positive cell walls; instead of lipopolysaccharides, Gram-positive bacteria have lipoteichoic acid and teichoic acid localized in the cell wall. The periplasmic space is not shown since the existence of such a periplasm in Gram-positive bacteria is still being studied.

Source: microbiolspec May 2019 vol. 7 no. 3 doi:10.1128/microbiolspec.GPP3-0044-2018
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Image of FIGURE 4
FIGURE 4

Visualization of Gram-positive bacterial capsules. Cationic gold nanoparticles (lysine-coated 15-nm gold nanoparticles) label the thick capsule of after fixation with 1% formaldehyde at low pH (stars). Cryo-FESEM at close-to-nature conditions reveals the thick capsule layer of marked with white stars. The thickness is comparable to the labeled capsule in (A); samples were nitrogen-slush-frozen, freeze-fractured at –105°C, freeze-etched at –105°C for 30 sec, and sputter-coated with gold/palladium. For ultrathin sections, capsules can be preserved with the lysine-ruthenium-red osmium embedding protocol (see reference 52 ). Following embedding in LRWhite resin, is surrounded by a dense capsule layer (white stars).

Source: microbiolspec May 2019 vol. 7 no. 3 doi:10.1128/microbiolspec.GPP3-0044-2018
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Image of FIGURE 5
FIGURE 5

Good preservation of streptococcal capsules under conditions. Streptococcal capsules ( administered intravenously) are well preserved (black stars) in spleen under conditions in the mouse model even after fixation with glutaraldehyde and formaldehyde, dehydration with acetone, and embedding in epoxy resin and ultrathin sectioning. Enlargement of another bacterium depicting nicely preserved capsule (black stars). Most likely, proteins in the blood have covered and preserved the capsule and prevented loss of capsule during aldehyde fixation.

Source: microbiolspec May 2019 vol. 7 no. 3 doi:10.1128/microbiolspec.GPP3-0044-2018
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Image of FIGURE 6
FIGURE 6

Formation of membrane vesicles on the surface of M1 serotype imaged with FESEM after chemical fixation with aldehydes, dehydration with acetone, critical-point drying, and sputter-coating with gold/palladium.

Source: microbiolspec May 2019 vol. 7 no. 3 doi:10.1128/microbiolspec.GPP3-0044-2018
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Image of FIGURE 7
FIGURE 7

Schematic drawing of a mycobacterial cell wall. A thin layer of peptidoglycan and arabinogalactan, to which large amounts of mycolic acids are attached, is characteristic. An unusual compound is lipoarabinomannan, which is attached to the cytoplasmic membrane. On the outermost outside, glycolipids are attached to the mycolic acids; transport is facilitated by inserted porins. The mycobacterial outer membrane is not included in the scheme since the presence of such an outer membrane is still under discussion.

Source: microbiolspec May 2019 vol. 7 no. 3 doi:10.1128/microbiolspec.GPP3-0044-2018
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Image of FIGURE 8
FIGURE 8

Typical appearance of a triple-layer structure of the mycobacterial cell wall of ssp. after special embedding applying the osmium-thiocarbohydrazide (TCH)-osmium method; this method especially preserves lipids much better than other methods because after the first osmium tetroxide step TCH binds to the sample bound osmium, and in the second osmium step more osmium is bound to TCH, therefore stabilizing lipids. In addition bacteria were embedded by applying the progressive lowering of temperature method down to –50°C, and bacteria were then embedded in the hydrophobic Lowicryl resin HM20; this protocol makes it possible to clearly define the triple-layer structure of the mycobacterial cell, which is lost in most other embedding methods, despite the cryo-based protocols. CM, cytoplasmic membrane; CW, cell wall; OL, outer layer; OM, outer membrane.

Source: microbiolspec May 2019 vol. 7 no. 3 doi:10.1128/microbiolspec.GPP3-0044-2018
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Image of FIGURE 9
FIGURE 9

FESEM of aldehyde-fixed, acetone-dehydrated, critical-point-dried, and gold/palladium sputter-coated samples. (Gram-negative), (Gram-negative), (Gram-positive), and (Gram-positive). Since FESEM reveals only the surface structures and no information from inside the bacteria, FESEM does not distinguish between Gram-negative and Gram-positive bacteria.

Source: microbiolspec May 2019 vol. 7 no. 3 doi:10.1128/microbiolspec.GPP3-0044-2018
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Image of FIGURE 10
FIGURE 10

TEM images of negatively stained bacteria. (Gram-negative) and (Gram-positive). Negative staining with 1% aqueous uranyl acetate cannot discriminate between Gram-negative and Gram-positive bacteria because the different peptidoglycan thicknesses cannot be resolved.

Source: microbiolspec May 2019 vol. 7 no. 3 doi:10.1128/microbiolspec.GPP3-0044-2018
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Image of FIGURE 11
FIGURE 11

Typical image of a conventional embedded Gram-positive bacterium, , after aldehyde fixation, contrasting with osmium tetroxide and uranyl acetate, dehydration, and embedding in epoxy resin. In ultrathin sections the DNA region is typically aggregated and forms a translucent area within the bacterial cell. The thick peptidoglycan layers are dark featureless structures. CM, cytoplasmic membrane; CW, cell wall.

Source: microbiolspec May 2019 vol. 7 no. 3 doi:10.1128/microbiolspec.GPP3-0044-2018
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Image of FIGURE 12
FIGURE 12

Comparison of embedding in different embedding resins. embedded in low-viscosity (LV) resin. embedded in LRWhite resin. was fixed, stained with osmium and uranyl acetate during dehydration with ethanol, and embedded in the epoxy LV resin, a replacement for the widely used Spurr resin, and the aromatic acrylic resin LRWhite. Both protocols show similar features, namely, a clearly defined cyoplasmic membrane (CM) and a peptidoglycan with two distinct zones, the dark inner wall zone (IWZ) and the outer wall zone (OWZ), which is more translucent in appearance. LRWhite resin preserves the OWZ much better compared to LV resin. In addition, the cytoplasm of both identically treated bacterial cells looks quite different. LRWhite has proven to be a reliable resin for studying bacterial ultrastructures.

Source: microbiolspec May 2019 vol. 7 no. 3 doi:10.1128/microbiolspec.GPP3-0044-2018
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Image of FIGURE 13
FIGURE 13

Comparison of conventional embedded and high-pressure frozen and freeze-substituted group G streptococci. Cross-section and longitudinal section of dividing conventional embedded bacteria. Note the prominent translucent DNA region and the absence of any material attached to the outside of the bacterial cell wall. Cross-section and longitudinal section of high-pressure frozen and freeze-substituted bacteria. No translucent DNA region is detectable, and the bacterial cytoplasm appears as a uniform structure throughout. Some material attached on the outer side of the bacterial cell wall is also preserved.

Source: microbiolspec May 2019 vol. 7 no. 3 doi:10.1128/microbiolspec.GPP3-0044-2018
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Image of FIGURE 14
FIGURE 14

Cryo-electron microscopy of a vitreous ultrathin section (CEMOVIS) of . Note that the very well-preserved cytoplasmic membrane (CM) and some structural details can be detected in the peptidoglycan layers of the cell wall (CW, arrow heads) when compared to the dark appearance of cell walls in conventional embedded bacteria (compare to Fig. 11 ).

Source: microbiolspec May 2019 vol. 7 no. 3 doi:10.1128/microbiolspec.GPP3-0044-2018
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