The Gram-Positive Bacterial Cell Wall

Gram Positive Pathogens 3rd Edition Chapter: The Gram-positive bacterial cell wall By Manfred Rohde Prof. Dr. Manfred Rohde Helmholtz Centre for Infection Research, HZI Central Facility for Microscopy, ZEIM Inhoffenstrasse 7 D-38124 Braunschweig, Germany e-mail: [email protected] Tel ++49 (0)531-61814413 THE GRAM-POSITIVE BACTERIAL CELL WALL 1. Historical background 2. The bacterial cell wall 2.1. Chemistry of the cell wall backbone 2.2. Biochemical synthesis of the peptidoglycan layer 2.3. Turnover of peptidoglycan 3. Lipoteichoic acids and wall teichoic acids as major constituents of the cell wall 4. Capsule 5. Extracellular vesicles of Gram-positive bacteria 6. A specialized cell wall in Mycobacteria 7. Electron microscopic techniques applied to study morphology of Grampositive bacteria cell envelopes 7.1. Heavy metal coating and shadowing 7.2. Negative-staining 7.3. Conventional embedding 7.4. Cryo-methods 7.4.1. Freeze-fracturing and freeze-etching 7.4.2. High pressure freezing and freeze-substitution (HPF-FS) 7.4.3. Cryo-sections of hydrated vitrified bacteria (CEMOVIS) and CryoFIB-SEM 7.4.4. Cryo-electron tomography 8. Outlook The Gram-positive bacterial cell wall 1. Historical background Back in 1884, the Danish bacteriologist Hans Christian Gram had developed a staining procedure to view stained bacteria under the light microscope (47). His staining method, nowadays simply called Gram-staining, discriminated between a Gram-positive and Gramnegative bacterial cell wall. He introduced a dye, gentian violet, which penetrates over the cell wall and cytoplasmic membrane, thus, staining the cytoplasm of the heat fixed bacteria. After addition of iodine an insoluble complex is formed which is retained by the Gram-positive bacterial cell wall upon addition of a decolorizer such as ethanol. Therefore, Gram-positive bacteria appear almost purple while Gram-negative bacteria retain the dye to a lesser extent or not at all and have to be counterstained with a second dye, safranin or fuchsine, appearing pink or reddish. Noteworthy, some mycobacteria showed an indifferent staining behavior when applied to Gram-staining suggesting that the cell wall of mycobacteria might be somehow different from the other two types. In the following decades, it became obvious that cell walls/cell envelopes turned out to be more diverse and Gram-staining alone often could lead to misinterpretations of the cell wall composition. Until the early 1950s, when the chemical composition of bacterial cell walls was not known, it was speculated if chitin or cellulose, polymers recognized as providing rigid structures to other organisms, represented also the building material of the bacterial cell wall. In 1951, experiments with phenol-insoluble material from Corynebacterium diphtheria (57) revealed glucosamine and diaminopimelic acid as components of the bacterial cell wall which are associated with polysaccharides. Chemical examination of streptococci cell wall layers highlighted the presence of amino acids and hexosamines in the cell wall extract as well as rhamnose as a main component in Gram-positive bacteria (83, 109). Systematic analyses of a number of Grampositive bacteria identified the two hexosamines glucosamine and muramic acid as major components together with three prevalent amino acids, namely D-alanine, lysine or diaminopimelic acid and glutamic acid. By then also a typical basic basal unit in Gram-positive cell walls was recognized in which glucosamine and muramic acid are linked with three amino acids via a peptide bond (27, 129). Gram-negative bacteria express the identical basal unit. Numerous analysis of other bacteria revealed that each bacterial genus or even species are often characterized by a distinctive pattern of amino acids, amino sugars and sugars connected to the basic basal unit. It was discussed that these differences should give a valuable pattern to discriminate between bacterial genus/species (28, 130). Over the following years other compounds of the Gram-positive cell wall were recognized as teichoic acids, which are polyribityl phosphates (8), and lipoteichoic acid. Furthermore, numerous proteins had been found linked to the cell wall. Staining methods of bacteria for light microscopic examinations have limitations since the resolution is not high enough to reveal structural details. With the advent of transmission electron microscopes (TEMs) in the 1930s and the parallel development of preparation methods for biological samples, electron microscopy imaging of ultrathin sections of embedded bacteria became the method of choice to study bacterial cell walls in detail at high resolutions (25, 65, 66, 67). With this methodology, it was possible for the first time to discriminate between the structures of Gram-positive and Gram-negative bacteria based on morphological differences in an image. Firstly, electron microscopic preparation protocols developed for eukaryotic cells or tissues were also applied for bacteria. The most fruitful era started when embedding protocols were customized for bacteria and new kinds of embedding resins became available; for example the Lowicryl resins for low temperature embedding which allowed introducing the progressive lowering of temperature (PLT) method (1, 5, 22,). This development was paralleled by technical inventions, especially cryo-methods in which bacteria are physically fixed instead of chemically, and opened up a new horizon in understanding bacterial cell walls. It should be mentioned that even today new methodologies are arising and push morphological studies towards vitrified and unstained bacteria in a fully hydrated status and therefore in a close-tonature condition. Noteworthy, major developments in electron microscopic methodology required a long time period of invention and testing before the technique was introduced to the market. For example, three-dimensional (3D) electron microscopy was developed appr. 30 years after invention of the TEM. Invention and pre-commercial development of cryo-electron tomography (CET) was brought to the market another 30 years later. Due to the rapid development of computer performance and progress in specialized software, nowadays one can estimate that new imaging techniques are introduced faster. For example, the introduction of cryo-FIB (focused ion-beam) combined with a scanning electron microscope (cryo-FIB-SEM) as a new close-to-nature approach was sold a few years after the first advent of FIB-SEM microscopes for conventional resin embedded biological samples. 2. The bacterial cell wall Bacteria are mostly unicellular organisms, which can be found in a very wide variety of different environments. Therefore, bacterial cell walls deserve special attention because they a) are the essential structure for bacterial viability by protecting against the often hostile environment, b) are composed of unique components found nowhere else in nature, c) are responsible for the shape of the bacteria, d) provide halt for ligands and proteins for adherence to host cells, e) expose receptor sites for drugs or viruses, f) represent the most important sites for attack of antibiotics, g) provide structures for immunological distinction and variation and h) can cause symptoms of disease in animals and humans. 2.1. Chemistry of the bacterial cell wall backbone The major backbone of the bacterial cell wall is the peptidoglycan, also called murein, which consists of repeating linear units of the disaccharide N-acetyl glucosamine (NAG) linked to Nacetyl muramic acid (NAM). The disaccharides are cross-linked via often flexible pentapeptide amino acid chains forming a mesh like framework (123). Chemically, the peptidoglycan consists of alternating β-1,4-linked N-acetylglucosamine (GlcNAc; NAG) and N-acetylmuramic acid (MurNAc, NAM, a variant of GlcNAc with a D-lactate attached to the C-3 by an ether bond). Termination of a peptidoglycan strand is achieved at the reducing end by a 1,6-anhydroMurNAc residue, in which the C-1 and C-6 of the sugar backbone are bound through an ether linkage. The appearance of the unusual 1,6-anhydroMurNac is used to determine the end of the strands. The peptide stems are covalently linked to the glycan strands with an amide bond to the carboxyl carbon of the D-lactyl group of MurNAc. One hallmark of the peptidoglycan is that the glycans are conserved across bacterial species whereas the peptide stem is often modified and diverse containing Damino acids. An L-alanine (L-Ala) is usually found in the first position of the pentapeptide stem from the lactyl group of MurNAc, which can be replaced by glycine or Lserine in some rare exceptions. The second amino acid is mostly occupied by a D-isoglutamic acid (D-iGlu). In Streptococcus pneumoniae this D-iGlu is amidated to yield a D-isoglutamine (D-iGln) (134). The γ-carbon of D-iGlu is bound to the third amino acid. This amino acid in the third position of the peptide stem has the highest diversity among bacteria. Generally one can summarize that in most Gram-negative bacteria and some Gram-positive bacteria, like in Bacilli and Mycobacteria, this third position is occupied by the unusual amino acid mesodiaminopimelic acid (m-Dap). In contrast, in most other Gram-positive bacteria it is usually a Llysine (L-Lys) (see Fig. 1). The peptide stem is finally terminated by two D-alanines (D-Ala), although different D-amino acids can be found in this place, too (123). In summary, one hallmark of Gram-positive bacteria is the observed differences in the types of crosslinks in which the peptides are connected to the peptidoglycan. Today more than 100 chemotypes can be distinguished and their differences are based on different linking units and substituents in the peptide chain (114). Due to its unique chemical structure, the peptidoglycan sacculus forms a very large polymer that can be isolated and viewed even in the light microscope (see Fig. 2). The difference between Gram-negative and Gram-positive bacteria is given by the thickness of the peptidoglycan layer surrounding the cytoplasmic membrane. Gram-positive bacteria exhibit a layer of peptidoglycan strands which can reach a size between 30-100 nm or even thicker, whereas Gram-negative bacteria show a layer of only a few nanometers (see Fig. 3). While the chemical composition of peptidoglycan and the family of proteins for assembling is known for a number of different bacteria, the overall arrangement of these components in Gram-positive cell walls is not fully solved. For Gram-negative bacteria it has been shown with CET (cryo electron tomography) that individual very thin densities, most probably representing glycan strands, run circumferentially around the long axis of the bacterial cell (42). In contrast, the 3-dimentional arrangement of peptidoglycan in Gram-positive bacteria is still under discussion (124). More or less three different models have been proposed over the years. The first model suggests that glycan strands run circumferentially around the long axis as in Gram-negative bacteria. This model was called the “circumferential” or “layered” model (43). In the second model the glycan strands are supposed to run perpendicular to the bacterial cell wall in a hexagonal lattice, and is called therefore “perpendicular” or “scaffold” model (31, 85). This model was proposed on the basis of NMR studies applying a synthetic 2 kDa fragment of the peptidoglycan formed of NAG-NAM (pentapeptide)-NAG-NAM(pentapeptide). NMR revealed that this fragment forms a right handed helix with a periodicity of three NAG-NAM per helix turn. The first two amino acids can adopt a limited number of different conformations (85). Atomic force studies (AFM) with gently disrupted sacculi of Bacillus subtilis established the so-called “coiled cable” model. Herein, bundles of glycan strands form thicker moieties of around 50 nm which run around the cell (53). It should be noted that a model with glycan strands running parallel to the long axis has never been considered because it was unclear how such a sacculus could elongate. In addition, the “coiled cable” model is not considered nowadays since CET did not show any cable-like structures in the thick peptidoglycan layer. These earlier observations might have been based on the fact that isolated peptidoglycan was harvested by boiling of bacteria, opened up by a French Press cell, diluted in water and air dried on mica before AFM imaging was performed. 2.2. Biochemical synthesis of the peptidoglycan layer Synthesis of the peptidoglycan is a three-step mechanism, which is localized at three different locations within a bacterium. The sequential Mur ligase pathway is involved in biosynthesis of the peptidoglycan. The early steps of synthesis start in the bacterial cytoplasm where precursors linked to undecaprenylpyrophosphate (UDP), like UDP-N-acetylmuramylpentapeptide (UDP-NAM) and UDP-N-acetylglucosamine (UDP-NAG), are formed. In a second step UDP-NAM is bound to another cytoplasmic membrane-bound UDP functioning as a transport lipid. This complex is called lipid I and is located at the inner cytoplasmic face of the membrane. Covalent attachment of the second precursor UDP-NAG forms the transport lipid complex lipid II. Then, for example in case of S. aureus, a peptide cross-bridge is attached to the third amino acid in the pentapeptide consisting of 5 glycine residues. Next, the entire lipid II complex is flipped over the cytoplasmic membrane to the extracellular side by a flippase. The precise biochemical process of the flipping mechanism is not yet fully understood. Lipid II is incorporated into nascent growing peptidoglycan by penicillin binding proteins (PBPs) on the extracellular side of the membrane. This third step involves firstly a transglycosylation and secondly a transpeptidation reaction performed by PBPs for incorporating new glycans with flexible peptides into the existing peptidoglycan layer (10, 16, 110, 111, 121, 122, 134). For detailed reading of chemical reactions and enzymes involved in this process, please refer to the review of Teo and Roper (134). 2.3. Turnover of peptidoglycan The first report describing the bacterial cell wall turnover was published for the Gram-positive bacterium Bacillus megaterium more than 50 years ago (23). Later on, pulse-chased experiments demonstrated with radioactively labeled cell wall precursors that all studied Grampositive bacteria carry out a cell wall turnover as well as Gram-negative bacteria (14, 34, 81, 107). The model for peptidoglycan growth in Gram-positive bacteria implies an inside-to-outside growth in which newly synthesized peptidoglycan is delivered to the cytoplasmic membrane face of the peptidoglycan layer in a relaxed form. With polymerization and cross-linking, peptidoglycan is moving to the outside of the cell wall and gets stretched due to the high turgor pressure within the bacterial cell (68). Once the maximally stretched peptidoglycans in the outer layers start to age, they are subsequently hydrolyzed by autolysins (117). It was estimated that around 50% of the total cell wall mass is turned over within one generation. This would have been a massive loss of resources for the bacteria and it was speculated that hydrolyzed constituents of the cell wall might be recycled by bacteria. Indeed, this was found to be the case in Gram-negative bacteria like E. coli and the biochemical pathways are well understood (64, 94). If Gram-positive bacteria would also recycle cell wall material remained unclear. It was found that high amounts of cell wall fragments could be detected in growth medium of exponentially grown Gram-positive bacteria like in Bacillus, Lactobacillus, Listeria and Staphylococcus strains (64). Thus, a robust turnover of cell wall components was established Gram-positive peptidoglycan layers. CET could serve as the method of choice to clarify the transmigration through the peptidoglycan layer since bacteria are snap-frozen and even fast events can be fixed. However, the search for such events in tomograms can be very time consuming. Nevertheless, more and more evidence is emerging pointing out that MVs of Gram-positive bacteria play an important role in pathogenesis since for S. aureus it is reported that MVs contain penicillin-binding proteins, which can block activity of ß-lactam antibiotics. Furthermore, the global regulator MsrR, which is involved in methillicin resistance, was also detected in S. aureus MVs. MVs can be considered as Trojan horses being involved in passing on resistance genes among Gram-positive bacteria (108). In addition, several toxins have been found in MVs like listerolysin O (LLO) in L. monocytogenes and pneumolysin (Ply) in S. pneumoniae. Both toxins induce pore formation in the host cells and are therefore important virulence factors for colonization and invasion (72, 93). In Group A streptococci, MVs have also been described and its content characterized in detail (see Fig. 6). In summary, not only virulence associated proteins like M1 protein, streptolysin O (SLO) and serine protease HtrA were detected, but also numerous metabolic proteins residing in the streptococcal cytoplasm were identified as well as surface exposed proteins including anchorless surface proteins, lipids and RNA. Furthermore, the involvement of the virulence associated two-component regulator CovRS was demonstrated and loss of CovRS resulted in increased vesicle formation (105). 6. A specialized cell wall in Mycobacteria Mycobacteria are classified as Gram-positive bacteria, although are also referred to as acid-fast bacteria due to the high density of lipids in the cell wall, which prevents an accurate Gramstaining. Thus, the staining is performed with Ziehl-Neelsen stain. The complexity of Mycobacteria cell walls is a distinct feature that is not found in other bacteria. Three major macromolecules, peptidoglycan, arabinogalactan and mycolic acids are the building blocks of the mycobacterial cell wall. Structural description of the mycobacterial cell wall was conducted in the 1960s and 1970s and electron microscopy played an important role in describing the unusual morphological structures of the cell wall. The current accepted model of the cell wall is based on studies which identified the mycolyl-arabino-galactan-peptidoglycan complex as the core structure of Mycobacteria (86). This unique arrangement with lipids and proteins being included is responsible for the characteristically important and very efficient permeability barrier of the mycobacterial cell wall, particularly against drugs and provide the basis for the very potent pathogenicity of mycobacteria (see Fig. 7). Due to the presence of a high amount of lipids in the cell wall, earlier studies were confronted with the difficulty of extracting the lipids during the dehydration protocol. Therefore, for a long time it was discussed whether the lipids formed a lipid bilayer in the cell wall, as suggested by Minnikin in 1982 (86). He suggested an asymmetrical membrane to which the mycolic acids are covalently attached as an inner leaflet. The presence of such a lipid bilayer was confirmed by freeze-fracture studies, which clearly defined a second fracture plane, typical for a lipid bilayer. These findings supported the hypothesis of a second bilayer outside the cytoplasmic membrane, even though these studies were performed with Corynebacteria (101, 136). However, the existence of a bilayer outside of the cytoplasmic membrane was still heavily criticized because the proposed bilayer has never been clearly identified in ultrathin sections due to artefact-producing embedding preparations like chemical fixation and dehydration with acetone. Instead, a more or less lucent zone, outer layer, most properly representing lipids and mycolic acids, was detected dividing the mycobacterial cell wall into a triple layer structure composed of the cytoplasmic membrane, cell wall and outer layer (see Fig. 8). This translucent zone is covered with a very thin stainable layer consisting of capsule and attached proteins. Freeze-substitution revealed more or less similar images even though the cell wall appeared thinner (95, 96, 97). A major step forward in elucidating the mycobacterial outer cell wall structure was performed with close-to-nature imaging applying cryo ultrathin sections and CET of fully hydrated and vitrified samples (55). These studies revealed important changes to the current model. Firstly, a lipid bilayer was detected covering the outside of the cell wall and therefore, indicating that mycobacteria express a similar outer membrane resembling Gramnegative bacteria. Secondly, no evidence was found for an asymmetrical membrane, instead, a symmetric membrane and an additional periplasmic space was postulated (136). In addition, it is clear that extractable lipids play a dominant role for mycobacterial membrane integrity and properties. The mycobacterial peptidoglycan is synthesized as observed in other bacteria, in the cytoplasm, using UDP and is then flipped over the cytoplasmic membrane and inserted in the growing peptidoglycan network by the action of hydrolases and PBPs. Nevertheless, Mycobacteria exhibit a number of differences when compared to model bacteria. Firstly, mycobacterial peptidoglycan is extremely cross-linked, secondly the cross-links are based on up to 80% of the total peptidoglycan on 3-3 peptide cross-links instead of the 4-3 peptide cross-links found in other bacteria, and thirdly the peptidoglycan backbone shows modifications such as glycolylation’s of NAM and amidation of D-Glu and m-DAP (62, 70, 74, 75, 102). Furthermore, the mycobacterial peptidoglycan is surrounded by a layer of arabinogalactan, a disaccharide, which has long arabinan polymers attached. Noteworthy, some galactans remain free of arabinan polymers and most important, the arabinan chain ends are branched. These branched ends are the binding partners for the long carbon chains of mycolic acid. These incorporated fatty acids are responsible for the extremely thick waxy coat of mycobacteria and make the mycobacterial cell wall mostly impermeable, contributing to pathogenicity. For detailed reaction and enzymes involved in the process please refer to the review of Jankute et al. (62). 7. Electron microscopic techniques applied to study morphology of Gram-positive bacteria cell envelopes Since the early 1950s transmission electron microscopy has been applied for studying the morphology and ultrastructure of Gram-positive bacterial cell walls. Surprisingly, even today a single unique method, that would allow studying the ultrastructural details of all the different Gram-positive bacteria under close-to-nature conditions in an electron microscope, is still needed. The newly developed CET might be the current method of choice, though CET bears some drawbacks and restrictions especially when bacteria, with a width of more than 0.4 to 0.5 µm, have to be imaged. The new super resolution light microscopy methods like STED (stimulated emission depletion), PALM (photoactivated localization microscopy), SIM (structured illumination microscopy) and TIRF (total internal reflection fluorescence) were all very promising, but a breakthrough was hindered by the limitations when imaging immune fluorescent labeled structures. The expression of fluorescence tags or fluorescence proteins, like GFP proteins, might alter the in vivo biological activities to a certain extent, therefore giving rise to non-accurate localizations within the bacterial cell. If the reader is interested in following this in detail, it is recommended to follow the literature regarding the MreB protein (involved in the bacterial division process) over the last years. Depending on the high resolution imaging method applied, different assumptions about its distribution, arrangement and localization in Bacillus subtilis were made, i.e., looking if MreB forms helices in the bacterial cell or not (39). Several attempts have been undertaken to elucidate the ultrastructure of Gram-positive cell walls. In early TEM imaging of embedded and ultrathin cut bacteria the preparation scheme included chemical fixation with aldehydes, introduction of heavy metals, dehydration with acetone/ethanol and embedding in suitable resins. All these preparations steps had to be done to cope with the “hostile” environment created by the electron microscope, namely high vacuum and bombardment with high-energy electrons resulting in heating up the section. Thus, it is obvious that these treatments might not result in a proper preservation of the native cell wall. To overcome some of these detrimental effects in the preparation of bacteria, cryo-methods have been introduced like freeze-substitution or hydrated cryo-ultrathin sections. With the advent of high pressure-freezing techniques preservation of bacterial cell wall structures was pushed further in the direction of close-to-nature conditions. Nowadays, CET is the best method to perform imaging in a frozen vitrified hydrated state of the bacteria (91). However, drawbacks of this technique are that it is only available in certain institutes and it needs a sophisticated infrastructure and time for performing in depth analysis. The future will show if the newly developing cryo-focused-ion-beam scanning electron microscopy micromachining (Cryo-FIBSEM) will advance the deciphering of the ultrastructural details of the bacterial cell wall since the examined bacteria are in their fully hydrated condition and physically frozen overcoming the problem of chemical fixation with aldehydes. This technique allows observing lamellas (appr. 10-20 nm in thickness) cut out of the bacterium; thus, gaining access to small ultrastructural details. It should also be mentioned that other techniques like x-ray diffraction and X-ray lithography have been unsuccessful because the bacterial cell wall is not crystalline. Atomic force microscopy (AFM) has also been implemented, but with AFM only the surface of a sample can be imaged and therefore, only limited ultrastructural information was obtainable. The same restriction holds true for scanning electron microscopy. With the advent of field emission scanning electron microscopes (FESEM) it was possible to study bacterial structures at very high magnification (up to 400,000-fold) and resolution. Nevertheless, FESEM has never been able to provide the amount of ultrastructural details observed with TEM on ultrathin sections. This is simply due to the fact that FESEM reveals only the surface topography of a bacterial cell. Noteworthy, FESEM does not allow discriminating between Gram-positive and Gram-negative bacteria (see Fig. 9). In addition, FESEM samples need to be coated to be conductive. This so-called sputter-coating is often the last step in a scanning EM preparation protocol. Samples are usually sputter coated with a thin 5-8 nm film of either gold, gold palladium or platinum. Even though these layers are very thin, it might cover some fine ultrastructural details of interest when observed at high magnifications. Surprisingly images at high resolution of the Gram-positive cell wall do more or less not exist. On the other side, FESEM has been very useful in studying pathogenic bacteria interactions with host cells. In the following a general description of most of the electron microscopic methods applied for studying the bacterial surface structures will be given. If the reader is interested in fully detailed protocols please refer to specialized text books for electron microscopic methods. 7.1. Heavy metal coating or shadowing When biological samples were examined for the first time under a TEM it became obvious that the contrast of the biological material is fundamental and that methods had to be developed to increase the contrast for TEM images. One of the first approaches applied was metal coating with heavy metals (87, 128). A shadow line behind the exposed structures appeared when the metal coating was performed under a certain angle. Knowing the coating angle and the measured length of the resulting shadow, the height of the structure could be determined. In earlier years, metal coating had become the method of choice for the ultrastructural description of regularly patterned cell wall structures, named S-layers, attached to the cell wall of bacteria (6, 60, 61, 116). 7.2. Negative-staining The metal coating approach has its restrictions when macromolecules or protein complexes have to be imaged. For such purposes the idea of embedding macromolecules into heavy metal salts like Na-K-phosphotungstate, and later on uranyl acetate or others, were considered. Advantages of negative-staining are manifold: a) reliable and repeatable, b) fast, c) avoids flattening of macromolecules on the support film when air-drying, e) stabilizes the protein in the electron beam, f) allows to determine the shape and quaternary structure of an enzyme complex at around 1.3 nm resolution and, g) usage of different heavy metal salts results in higher or lower contrasts (4, 17, 18). Noteworthy, negative stained viruses and larger enzymes opened the door for 3D microscopy and image processing beginning in the 1970s (41, 59). Negative staining was the method of choice when isolated peptidoglycan sacculi or pole caps were analyzed (13). Nevertheless, negative-staining is not suitable to differentiate between Gramnegative and Gram-positive bacteria (see Fig. 10). 7.3. Conventional embeddings Since the 1950s, the embedding technique was introduced to analyze the ultrastructural details of bacteria (25) because intact bacteria were unsuitable for these studies. Therefore, ultrathin sectioning of bacteria was needed to gain access to internal morphological structures. With the invention of ultramicrotomes, it was possible to obtain ultrathin sections of biological samples, thus facilitating detailed studies of bacterial cell walls (see Fig. 11). From then on, morphological studies of bacterial cell walls started to blossom. Ultrathin sections have a thickness of around 50-80 nm, meaning that a single bacterium measuring 1 µm in length can be cut into nearly 15 sections. Again, one was directly confronted with the problem of low contrast of biological samples. Thus, the early embedding protocols usually included fixation with aldehydes, contrasting with heavy metals like osmium tetroxide, ruthenium red and uranyl acetate, dehydration with acetone/ethanol depending of the resin used for embedding. At that time mostly epoxy or methacrylate resins were used and polymerization was carried out at 6070°C. Nowadays, many different resins are available and every class of resin offers a slightly different image of the embedded bacterial ultrastructure, depending on the embedding protocol (see Fig. 12). In addition, the counter-staining of ultrathin sections before TEM examination influences the appearance of ultrastructural details in the sections. These protocols revealed unequivocally the visible differences between Gram-positive and Gram-negative bacterial cell walls. In most of the ultrathin sections, the Gram-positive cell wall appears as an amorphous structure and, depending on the resin and applied embedding protocol; some structural details could be detected like the discussed periplasmic space in Gram-positive cell walls (12, 46). It should be clearly stated here that these embedding protocols are prone to induce artifacts in the samples and therefore influence the interpretation of the observed ultrastructural details (44). Nevertheless, these methods served as a basis for most of the description of bacterial ultrastructure and they are widely available in nearly all life science electron microscopy units. 7.4. Cryo-methods Conventional embedding approaches lack the accuracy to investigate tiny ultrastructural details considering potential adverse effects of chemical fixatives, introduction of heavy metals and dehydration during preparations. Cryo-methods began to develop from the beginning of the 1980s onwards, when vitrification of water in biological samples for electron microscopic studies was applied for the first time by Mayer and Brüggeler (82) and Dubochet et al. (35, 36, 37). Earlier on freeze-fracturing was introduced in the 1960s (88). 7.4.1. Freeze-fracturing and freeze etching One of the earliest cryo-method applied was freeze-fracturing and freeze-etching. Samples are frozen in nitrogen slush, thereby water in the samples is brought into its vitrified state. Then samples are fractured, sometimes etched and subsequently coated with metal or carbon or both. From this sample a replica is produced which exhibits the surface topography. The depth of the topographical structures depends on the etching time (58, 103). Usually, during freezefracture, the fracture line is in the hydrophobic region of a membrane, i.e., in the bacterial cytoplasmic membrane. Thus, exposing transmembrane or membrane bound proteins. Only rarely does a fracture line run across the cell wall or inside a cell wall. Areas which are exposed give a more or less featureless matrix or, as in cross fractures, show a polymeric network which could not be further resolved. These findings did not succeed in gaining considerable new understandings of the Gram-positive cell wall (11). 7.4.2. High-pressure freezing and freeze-substitution (HPF-FS) The development of freeze-substitution of quickly frozen samples, which was paralleled by the invention of low temperature embedding resins as the Lowicryl series of methacrylate resins, was started in the 1980s (1, 22). Bacteria are snap-frozen in liquid propane or ethane and then rapidly transferred into a substitution medium containing osmium and/or uranyl acetate in acetone. Remarkably, it was demonstrated that a certain water content in acetone (up to 4%) resulted in a much better visibility of membranes (125). Samples are then kept for 2 days at - 80°C, warmed up to -50°C and -20°C and left for 1 day at each step. The following embedding can be performed with low temperature resins, Lowicryl resins, or samples are brought to ambient temperature and embedded with conventional resins (44, 45, 96). During substitution, bacteria are stained and dehydrated resulting in a visibly better preservation of ultrastructural details. Most of freeze-substituted bacteria in ultrathin sections are recognizable by the fact that no distinct DNA region can be observed, whereas, in conventional embedding, DNA mostly aggregates and forms the typical lucent DNA region in the middle of the bacterial cell (see Fig. 13). Currently, hundreds of substitutions protocols do exist which are customized to fulfil the needs of the examined biological samples and to address the study purpose, e.g., for ultrastructural studies or immune cytochemical localization studies. Freeze-substitution was pushed even more forward by high-pressure freezing of bacteria. This method was developed in the 1960s (88). At ambient temperatures adequate freezing of bacteria is reached with cooling rates of more than 10.000 K/s to vitrify the water content in the sample. High pressure is a potent physical cryo-protectant because it lowers the freezing point of water considerably and thicker samples can be vitrified. 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Figure legends Fig. 1 The bacterial cell wall backbone, peptidoglycan; shown are the two glycan strands (in black) and 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 (mDap in red) and the presence of D-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. Fig. 2 Transmission electron microscopic image taken at an acceleration voltage of 80 kV of a peptidoglycan sacculus of E. coli 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. Fig. 3 Schematic drawing of Gram-negative and Gram-positive cell walls; a characteristic of Gram-negative cell walls is the presence of two membranes i) the cytoplasmic membrane and ii) the outer membrane, between both membranes the periplasmic space is found in which a very thin layer of peptidoglycan is found; attached to the outer membrane are lipopolysaccharides and in the outer membrane porins are inserted. 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 discussed periplasmic space is not drawn since the existence of such a periplasm in Gram-positive bacteria is still ongoing. Fig. 4 Visualization of Gram-positive bacterial capsules. A) cationic gold nanoparticles (lysine coated 15 nm gold nanoparticles) label the thick capsule of Streptococcus pneumoniae after fixation with 1% formaldehyde at low pH (stars), B) cryo-FESEM at close-to-nature conditions reveals the thick capsule layer of S. pneumoniae 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, C) for ultrathin sections capsules can be preserved with lysine-ruthenium-red osmium embedding protocol (see 52) followed by embedding in LRWhite resin, Streptococcus suis is surrounded by a dense capsule layer (white stars). Fig. 5 Good preservation of streptococcal capsules under in vivo conditions. A) Streptococcal capsules (Streptococcus pyogenes administered i.v.) are well preserved (black stars) in spleen under in vivo conditions in the mouse model even after fixation with glutaraldehyde and formaldehyde, dehydration with acetone and embedding in epoxy resin and ultrathin sectioning, B) enlargement of another bacterium depicting nicely preserved capsule (black stars). Most likely proteins in the blood have covered and preserved the capsule and prevent loss of capsule during aldehyde fixation. Fig. 6 Formation of membrane vesicles (MVs) on the surface of Streptococcus pyogenes M1 serotype imaged with FESEM after chemical fixation with aldehydes, dehydration with acetone, critical-point drying, and sputter coating with gold/palladium. Fig. 7 Schematical drawing of a mycobacterial cell wall; characteristic is a thin layer of peptidoglycan and arabinogalactan to which high amounts of mycolic acids are attached; another unusual compound is lipoarabinomannan which is attached to the cytoplasmic membrane, on the outer most outside glycolipids are attached to the mycolic acids, transport is facilitated by inserted porins. The “mycobacterial outer membrane” is not drawn in the scheme since the presence of such an outer membrane is still under discussion. Fig. 8 Typical appearance of a triple layer structure of the mycobacterial cell wall of Mycobacterium avium ssp. paratuberculosis after special embedding applying the OTO method (osmium-thiocarbohydrazide(TCH)-osmium); this method especially preserves lipids much better 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 applying the PLT method (progressive lowering of temperature) down to -50°C and bacteria are then embedded in the hydrophobic Lowicryl resin HM20; this protocol allows to clearly define the triple layer structure of the mycobacterial cell