Reduction of radiation damage in an electron microscope with a superconducting lens system

Dietrich, I.; Formanek, H.; Fox, F.; Knapek, E.; Weyl, R.

doi:10.1038/277380a0

Cited by 48 publications

(15 citation statements)

References 5 publications

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“…Their visualization is certainly a very challenging task because of the gel-like and heterogeneous nature of the wall, attested to by many high-resolution techniques (e.g., X-ray scattering, TEM of isolated walls, and atomic force microscopy [8,24,46]). Accordingly, many models have been proposed to describe the tertiary structure of peptidoglycan.…”

Section: Discussionmentioning

confidence: 99%

Native Cell Wall Organization Shown by Cryo-Electron Microscopy Confirms the Existence of a Periplasmic Space in Staphylococcus aureus

2006

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The current perception of the ultrastructure of gram-positive cell envelopes relies mainly on electron microscopy of thin sections and on sample preparation. Freezing of cells into a matrix of amorphous ice (i.e., vitrification) results in optimal specimen preservation and allows the observation of cell envelope boundary layers in their (frozen) hydrated state. In this report, cryo-transmission electron microscopy of frozen-hydrated sections of Staphylococcus aureus D 2 C was used to examine cell envelope organization. A bipartite wall was positioned above the plasma membrane and consisted of a 16-nm low-density inner wall zone (IWZ), followed by a 19-nm high-density outer wall zone (OWZ). Observation of plasmolyzed cells, which were used to artificially separate the membrane from the wall, showed membrane vesicles within the space associated with the IWZ in native cells and a large gap between the membrane and OWZ, suggesting that the IWZ was devoid of a cross-linked polymeric cell wall network. Isolated wall fragments possessed only one zone of high density, with a constant level of density throughout their thickness, as was previously seen with the OWZs of intact cells. These results strongly indicate that the IWZ represents a periplasmic space, composed mostly of soluble low-density constituents confined between the plasma membrane and OWZ, and that the OWZ represents the peptidoglycan-teichoic acid cell wall network with its associated proteins. Cell wall differentiation was also seen at the septum of dividing cells. Here, two high-density zones were sandwiched between three low-density zones. It appeared that the septum consisted of an extension of the IWZ and OWZ from the outside peripheral wall, plus a low-density middle zone that separated adjacent septal cross walls, which could contribute to cell separation during division.Staphylococcus aureus is a gram-positive pathogen often involved in nosocomial infections and food-borne diseases, and there is now growing concern about the spread of multidrugresistant strains of methicillin-resistant S. aureus (4,25,26,31,39,45). The staphylococcal cell wall plays an important role for this organism's success, as it withstands tremendous turgor pressures throughout the cell cycle (ca. 20 to 30 atm), strongly interacts with the cell's external environment, particularly in infection processes, and is intimately involved in cell division (1-3, 15, 21, 22, 33). Over the past two decades much knowledge has been accumulated on the primary structure of staphylococcal cell wall components, i.e., peptidoglycan (3, 15, 45), (lipo)teichoic acids (34,47,48), and surface proteins (33, 43), but there is still a lack of structural information at high resolution on the spatial organization of this complex cell wall (4, 9, 17, 46, 49).As with other gram-positive bacteria, the envelope of S. aureus is commonly seen in conventional thin sections by electron microscopy as composed of a plasma membrane tightly bound by a thick and undifferentiated wall (Fig. 1). This simple architect...

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Section: Discussionmentioning

confidence: 99%

Native Cell Wall Organization Shown by Cryo-Electron Microscopy Confirms the Existence of a Periplasmic Space in Staphylococcus aureus

2006

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“…Recently Formanek et al [61,62] reported new electron microscopy studies on peptidoglycan using a microscope equipped with super-conductive lenses and a very effectively cooled specimen stage. With this technique a striation with 0.45-nm periodicity was reported for peptidoglycan species on graphitic oxide supports, stained with heavy metal atom compounds such as 'platinum blue' or 2,4,6-trisacetoxymercuri-acetaminotoluene.…”

Section: Rejection Of Crystalline-chitin-based Modelsmentioning

confidence: 99%

Infrared Spectroscopy, a Tool for Probing Bacterial Peptidoglycan

Naumann

Barnickel

Bradaczek

et al. 1982

European Journal of Biochemistry

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Infrared spectroscopic measurements are used to obtain insights into the three-dimensional architecture of peptidoglycan (murein), the rigid component of almost all bacterial cell walls. The infrared spectra of various types of peptidoglycans (including all chemotypes and examples of the so called A and B groups) were compared to each other and to those obtained from crystalline chitin.All peptidoglycans investigated exhibited very similar infrared spectra. In particular the conformationally sensitive amide A, I and I1 absorption bands were found to be constantly centered around 3300 cm-', 1657 cm-' and 1534 cm-' respectively; furthermore, the spectral region between 1200 cm-' and 800 cm-', characterized by several strong absorption bands connected to complex sugar ring modes, proved to be remarkably uniform.Additionally the infrared spectra remained significantly constant between -175 ' C and + 75 "C and turned out to be rather independent of sample preparation (solvent replacement, freeze-drying and film producing).An analysis of band half-widths revealed no high crystalline state of order of peptidoglycan. On the basis of band positions and half-widths of amide bands, regular conformations like M helices of p pleated sheets could be excluded. Several distinctive, fingerprint-like spectral features of the various murein samples permitted a facile identification of individual peptidoglycans. Moreover, infrared spectroscopy seems to be very promising as an analytical tool, e.g. for tracing variations of cell wall structure, detecting conformational changes and estimating crosslinking indices in a quick and simple way.The comparative analysis of amide band positions and band half-widths yielded substantial differences between infrared spectra of chitin and murein, thus rejecting previous models based on the assumption of a nearly crystalline chitin-like structure of the glycan chains of murein.The peptidoglycan (murein) of bacterial cell walls surrounds nearly all bacteria as a protecting network, which in gram-negative bacteria is probably monolayered, while multilayered in gram-positive bacteria, thus constituting up to 60% of the cell wall weight [I -41. Its primary structure consists basically of disaccharide-pentapeptide subunits with unusual features such as the occurrence of alternating D and L-amino acids and a y-bonded D-glutamic acid residue ( Fig. 1 and 2). Because of its unique structure and its function in protecting bacteria against osmotic lysis, peptidoglycan has been proved to be an ideal target for chemotherapeutic attack. In particular the mode of action of some of the most useful antibiotics, the penicillins and cephalosporins, is closely related to the inhibition of the biosynthesis of the peptidoglycan network [4].Owing to its ubiquitous existence in bacteria and the continuous exposure of individuals to peptidoglycan a huge variety of biologically and medically important properties have been observed (for a review [5]). These include, as major immunological properties, immunogenicity [6], a...

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“…Sacculi are constantly remodeled, but they are robust enough that they can be purified from cells and still retain their original shape. Early electron microscope images of purified sacculi were difficult to interpret because the images were 2-D projections through heavy metal-stained sacculi (1,2). Nevertheless the slender appearance of peptidoglycan in thinsectioned cells (3) and the gaps perpendicular to the polar axis produced by a peptidoglycan-specific endopeptidase (4) lead to the (Circumferential) Layered model (Fig.…”

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confidence: 99%

Molecular organization of Gram-negative peptidoglycan

Gan¹,

Chen²,

Jensen

2008

Proc. Natl. Acad. Sci. U.S.A.

249

238

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The stress-bearing component of the bacterial cell wall-a multigigadalton bag-like molecule called the sacculus-is synthesized from peptidoglycan. Whereas the chemical composition and the 3-dimensional structure of the peptidoglycan subunit (in at least one conformation) are known, the architecture of the assembled sacculus is not. Four decades' worth of biochemical and electron microscopy experiments have resulted in two leading 3-D peptidoglycan models: ''Layered'' and ''Scaffold'', in which the glycan strands are parallel and perpendicular to the cell surface, respectively. Here we resolved the basic architecture of purified, frozenhydrated sacculi through electron cryotomography. In the Gramnegative sacculus, a single layer of glycans lie parallel to the cell surface, roughly perpendicular to the long axis of the cell, encircling the cell in a disorganized hoop-like fashion.cell wall ͉ Cryo-EM ͉ sacculus ͉ tomography ͉ cell shape T he organization of peptidoglycan within the sacculus is a longstanding question in microbiology. Although the sacculus completely envelopes the cell, protecting it from osmotic and mechanical lysis, it is also porous, and thus sieves nutrients and small proteins. Sacculi are constantly remodeled, but they are robust enough that they can be purified from cells and still retain their original shape. Early electron microscope images of purified sacculi were difficult to interpret because the images were 2-D projections through heavy metal-stained sacculi (1, 2). Nevertheless the slender appearance of peptidoglycan in thinsectioned cells (3) and the gaps perpendicular to the polar axis produced by a peptidoglycan-specific endopeptidase (4) lead to the (Circumferential) Layered model (Fig. 1A ) in which the glycan strands lie in the plane of the cell wall and wrap around the cell. The alternative Scaffold model (5) (Fig. 1B) was proposed to fit biochemical data such as pore size and crosslinkage. Various arguments have been made in favor of (6) and against (7) the Scaffold model, but the debate intensified after a recent NMR structure of a synthetic peptidoglycan subunit dimer was interpreted to support the Scaffold model (8). To gain further insight into this issue, we imaged unstained sacculi in 3-D by electron cryotomography (9). Results and DiscussionElectron Cryotomography of Purified Sacculi. Sacculi isolated from C. crescentus strain CB15N and E. coli strains MG1655 and XL-10 were vitrified across holey carbon grids in thin (Ͻ 100 nm) ice. Whereas the gossamer sacculi were barely discernable in the low-dose, 2-D projection images, they were clearly visible in the 3-D tomograms and resembled flattened cells [ Fig. 2 and supporting information (SI) Movie S1 and Movie S2]. In stark contrast to intact cells, the sacculi were flexible and in some positions had radii of curvature less than 20 nm, highlighting how cell shape is not determined by the ''rigidity'' of the sacculus, but rather the cytosolic turgor (osmotic pressure) pushing the cytoplasmic membrane outward against the sacc...

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Reduction of radiation damage in an electron microscope with a superconducting lens system

Cited by 48 publications

References 5 publications

Native Cell Wall Organization Shown by Cryo-Electron Microscopy Confirms the Existence of a Periplasmic Space in Staphylococcus aureus

Native Cell Wall Organization Shown by Cryo-Electron Microscopy Confirms the Existence of a Periplasmic Space in Staphylococcus aureus

Infrared Spectroscopy, a Tool for Probing Bacterial Peptidoglycan

Molecular organization of Gram-negative peptidoglycan

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