J. B. Ward
National Institute for Medical Research
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BioScience | 1980
H. J. Rogers; H. R. Perkins; J. B. Ward
1 Ultrastructure of bacterial envelopes.- 1.1 Introduction.- 1.2 The Gram-positive cell wall.- 1.3 The Gram-negative cell wall.- 1.4 Membrane morphology.- 1.5 Internal membranes.- 1.6 Specialized membrane systems.- References.- 2 Isolation of walls and membranes.- 2.1 Introduction.- 2.2 Isolation of walls and membranes from Gram-positive species.- 2.3 Separation of the components of the wall from Gram-negative species.- 2.4 Preparation of specialized intracytoplasmic membranes.- References.- 3 Membrane structure and composition in micro-organisms.- 3.1 General ideas of membrane structure.- 3.2 Some physical properties of membranes.- 3.3 Composition of microbial membranes.- 3.4 Proteins in membranes.- References.- 4 Membrane functions.- 4.1 Active components and functions of bacterial cell walls.- 4.2 Functions of the cytoplasmic membrane.- 4.3 Components of the electron transport chain.- 4.4 The coupling of energy flow to phosphorylation.- 4.5 Isolation and properties of Mg2+-Ca2+ ATPase.- 4.6 Vesiculation of membranes.- 4.7 Transport of metabolites and ions.- 4.8 Binding proteins.- 4.9 Mesosomal membrane.- 4.10 Outer membrane of Gram-negative bacteria.- References.- 5 Membranes of bacteria lacking peptidoglycan.- 5.1 Introduction.- 5.2 Mycoplasmas.- 5.3 Extreme halophiles.- 5.4 Bacterial L-forms.- References.- 6 Structure of peptidoglycan.- 6.1 Introduction.- 6.2 Modification of the basic peptidoglycan structure.- 6.3 Three-dimensional structure of peptidoglycans.- 6.4 Cell walls of prokaryotes without peptidoglycan.- References.- 7 Additional polymers in bacterial walls.- 7.1 Gram-positive bacteria.- 7.2 Gram-negative bacteria.- References.- 8 Biosynthesis of peptidoglycan.- 8.1 Introduction.- 8.2 Synthesis of nucleotide sugar precursors.- 8.3 The lipid cycle.- 8.4 Formation of cross-bridge peptides.- 8.5 Polymerization of disaccharide-peptide units.- 8.6 Transpeptidation: The formation of cross-links.- 8.7 D-Alanine carboxypeptidases.- References.- 9 Antibiotics affecting bacterial wall synthesis.- 9.1 Introduction.- 9.2 Phosphonomycin (Fosfomycin).- 9.3 Antibiotics inhibiting D-alanine metabolism in peptidoglycan biosynthesis: cycloserine, O-carbamoyl-D-serine, alaphosphin (L-alanyl-L-1-aminoethyl phosphonic acid) and the haloalanines.- 9.4 Bacitracin.- 9.5 Tunicamycin.- 9.6 The vancomycin group of antibiotics: vancomycin, ristocetins, ristomycins, actinoidin.- 9.7 ?-Lactam antibiotics: the penicillins and cephalosporins.- 9.8 Antibiotics inhibiting biosynthesis of wall polymers but whose site of action is not yet established.- References.- 10 Biosynthesis of other bacterial wall components.- 10.1 Biosynthesis of teichoic acids.- 10.2 Biosynthesis of other components of the Gram-positive bacterial wall.- 10.3 Biosynthesis of the lipopolysaccharides.- 10.4 Iipoprotein from the outer membrane of Gram-negative bacteria.- References.- 11 The bacterial autolysins.- 11.1 Introduction.- 11.2 Bond specificity and distribution of bacterial autolysins.- 11.3 Purification and properties of the autolytic enzymes.- 11.4 Location of autolytic enzymes.- 11.5 Function of autolysins.- References.- 12 Cell walls of Mycobacteria.- 12.1 Wall composition.- 12.2 Adjuvant and other immunostimulant properties.- 12.3 Antitumour activity.- References.- 13 Cell walls of filamentous fungi.- 13.1 Introduction.- 13.2 Carbohydrates in the wall.- 13.3 Wall composition and dimorphism.- 13.4 Melanins and depsipeptides.- 13.5 Conclusion.- References.- 14 Biosynthesis of wall components in yeast and filamentous fungi.- 14.1 Introduction.- 14.2 Biosynthesis of chitin.- 14.3 Biosynthesis of mannan.- 14.4 Biosynthesis of glucan.- References.- 15 The cell wall in the growth and cell division of bacteria.- 15.1 Introduction.- 15.2 Growth of streptococcal cell walls.- 15.3 Growth of the walls of Gram-positive rod-shaped bacteria.- 15.4 Growth of the Gram-negative cell wall.- 15.5 Growth of cytoplasmic membranes.- 15.6 Mutants with disturbed surface growth.- 15.7 Helical growth of bacteria.- References.
Microbial cell walls and membranes | 1980
H. J. Rogers; H. R. Perkins; J. B. Ward
Among the earliest knowledge about microbes is the fact that cells, suspended and incubated under unfavourable conditions for growth and anabolism, disintegrate. Indeed autolysates of yeasts and other cells were some of the earliest sources of soluble enzymes, and did trojan work in the heroic days of the investigations of intermediary carbohydrate metabolism. With the development of the electron microscope and consequent ultrastructural awareness, cell walls were recognized, isolated and their chemistry studied. Autolysis could then be seen to be due to dissolution of the walls. As the chemical structure of the polymers making up the walls was elucidated, it became possible to designate the bonds specifically hydrolysed by various autolytic enzymes and thus to account for the solubilization of the walls and the disintegration of the cells. Since the supportive polymers in microbial cell walls are often either complex, such as, for example, are the peptidoglycans of bacteria or, consist of a number of polymers interacting to provide mechanical support for the cell as in some microfungi, autolysins with a variety of bond specificities are often to be found in the same cell. It is not always possible to distinguish which autolysin is responsible for any particular effect, or to exclude the necessity for the combined action of more than one enzyme.
Archive | 1980
H. J. Rogers; H. R. Perkins; J. B. Ward
The mechanism of biosynthesis of peptidoglycan has been elucidated in a number of organisms including Staphylococcus aureus, Micrococcus luteus, Escherichia coli and more recently several members of the Bacilli. Although these organisms differ widely in morphology and the chemical composition of their peptidoglycans, the process of biosynthesis in each shows sufficient common characteristics to establish the basic nature of the process. Clearly, such modifications as are made to the biosynthetic pathway in the individual organisms are characteristic of those organisms and probably represent the evolution of specific enzymes. Overall peptidoglycan synthesis can be divided into three distinct stages: (1) The formation of the nucleotide sugar-linked precursors, UDP-N-acetylmuramylpentapeptide and UDP-N-acetylglucosamine; (2) The transfer of phospho-N-acetylmuramyl-pentapeptide and N-acetylglucosamine to a lipophilic carrier, undecaprenyl phosphate, to yield a disaccharide-(pentapeptide)-pyrophosphate-undecaprenol and (3) The transfer of this completed sub-unit to the growing peptidoglycan. At this stage cross-bridge formation occurs, together with secondary modification of the newly-synthesized peptidoglycan.
Archive | 1980
H. J. Rogers; H. R. Perkins; J. B. Ward
Fungal walls, like those of plants and bacteria, consist of a rigid layer outside the protoplast, which they protect from osmotic and other changes in the environment. In addition they are responsible for the characteristic shape of the cell and have to be modified when the cell changes, as for instance during the growth of a hyphal tip, the initiation of a branching hypha, the change to a conidiospore, or from mycelial to yeast-like growth or vice versa. The wall is composed largely of polysaccharides, with some protein and lipid, although the latter represents only a small proportion. It has been known for a long time that one of the chief polysaccharides is chitin, the homopolymer of β-1, 4-N-acetylglucosamine that also occurs in the integument of arthropods, and another β-1, 4-glucose, ie cellulose. Where present, both chitin and cellulose have been identified by X-ray powder crystallography and shown to be the same as authentic samples from other sources [3, 37]. These two polymers form the fibrils that make the rigid component of most fungal walls, in contrast to the yeasts where other glucose polymers, along with a small amount of chitin, take over this function [4, 7]. Before looking in detail at the other polysaccharides present, we will examine the evidence for the minor components, protein and lipid, as integral parts of the wall. Walls that have been isolated and washed as thoroughly as possible [43] still contain about 5–10% of lipid, but so far this has not been well characterized [40]. It is noteworthy, however, that mycelial walls of Penicillium charlesii contained as much as 37.5% lipid [15] and the wall of a strain of Aspergillus nidulans has been reported to have half its 10% of galactose in the form of a glycolipid [47]. The residual protein, on the other hand, can sometimes be extracted by detergents as in Aspergillus nidulans [14], whereas in other instances such as Aspergillus niger and Chaetomium globosum it resists even protracted extraction with 8 M urea [33]. Analysis of the latter firmly-attached protein and its peptic digests suggested that many acidic peptide sequences were present and showed high serine and threonine contents, as often observed in the protein of yeast walls. The importance of protein in the wall was emphasized by Hunsley and Burnett [24], who used a succession of glycanases and the proteolytic enzyme pronase to ‘dissect’ fungal walls. They concluded that in the wall of Neurospora crassa, for instance, there was a glycoprotein reticulum embedded in easily-removable protein, beneath which lay a separate protein layer, which was in turn outside and possibly intermingled with the innermost layer of chitin microfibrils. It is now clear that, in some although not all fungi [46], as hyphae extend two separate kinds of wall are laid down, a primary wall consisting of chitin microfibrils covered with protein which is laid down as tip extension proceeds, and a layer of secondary wall that is later deposited on the outside. All layers may thicken as maturation occurs, except that the inner chitin layer of N. crassa appears not to change [45] (Fig. 13.1). Another process that intervenes, but is at present little understood, is rigidification, whereby the primary wall that is first deposited in a relatively plastic state becomes more rigid as it thickens and adopts the shape and diameter of the final hyphal tube. It has been suggested that rigidification may correspond either to a diminution in the activity of autolysins or to an increasing resistance to these enzymes as the wall matures [45], though no mechanism for either process is known at present.
Archive | 1980
H. J. Rogers; H. R. Perkins; J. B. Ward
The initial observations on the effect of benzylpenicillin on the bacterial wall came in the early 1940s shortly after its introduction as a therapeutic agent. Gardner [74] found that concentrations of penicillin, much lower than those required to kill a range of different bacteria, caused considerable morphological changes to the organisms with apparent damage to the cell envelope. In certain organisms, cell division also appeared inhibited. These observations were extended by Duguid [58] who showed that growth was a necessary requirement for penicillin action, and that killing of the bacteria was apparently associated with cell lysis. He suggested that ‘penicillin at these concentrations interferes specifically with the formation of the outer cell wall, while otherwise allowing growth to proceed until the organism finally bursts its defective envelope and undergoes cell lysis’. Many Gram-positive bacteria, particularly staphylococci, are extremely sensitive to the inhibitory action of penicillin, suggesting that its action is highly specific. This observation prompted the first biochemical studies of Park and Johnson [208] who described the accumulation of a labile phosphate compound by Staphylococcus aureus when treated with penicillin. The accumulated material was subsequently shown to be composed of several nucleotide compounds, all of which contained an unknown amino sugar, and some, the uncommon D-isomers of glutamic acid and alanine too [201–203] ; the function of these compounds was not known. However, when it was discovered the wall of S. aureus contained a structure analogous to part of the accumulated nucleotidepeptide, it became clear that the compounds were in fact the biosynthetic precursors of the wall [209]. At this time, considerable advances were being made in the determination of bacterial wall structure, many of which pointed to its being unique. These findings, taken in conjunction with the other studies on the mode of action of penicillin, pointed to the wall as a structure necessary for the survival of the organism and in consequence a site for the selective action of antibiotics. The biosynthesis of peptidoglycan has been discussed in detail in the previous chapter and a number of antibiotics have been mentioned where information relating to biosynthesis has resulted from investigation of their mode of action. The mode of action of these and other antibiotics will now be described in greater detail at a molecular level. This consideration will be related to their site of action in the synthesis of peptidoglycan, which may be at the level of synthesis of the nucleotide precursors, the membrane-bound stages involving the lipid carrier or the terminal stages involving transfer of the newly-synthesized unit to the growing peptidoglycan.
Archive | 1980
H. J. Rogers; H. R. Perkins; J. B. Ward
The fundamental polymer that is a common component of the cell walls of Grampositive and Gram-negative bacteria, Rickettsiae and blue-green bacteria is called peptidoglycan (formerly mucopeptide or murein). As its name implies, it consists of glycan chains with peptide substituents, and in all examples that have been studied the peptide subunits are cross-linked so that the overall structure is a network that surrounds the cell. This network seems responsible for the integrity of the shape of Gram-positive bacteria, and at least partially of Gram-negative bacteria as well. Certainly when the peptidoglycan is degraded, as for instance by lysozyme, the bacterium tends to lose its characteristic shape and to form a spherical body known as a spheroplast, which usually needs to be maintained in a hypertonic medium if it is not to burst because of the high osmotic pressure within it and the lack of external support. The chemical composition of peptidoglycan has been established over the period since the early 1950s, when Salton [40] first showed that the cell walls prepared from Gram-positive organisms were of a comparatively simple amino acid composition, although both he and Weidel [56] found that the walls of Gramnegative species were more complex.
Archive | 1980
H. J. Rogers; H. R. Perkins; J. B. Ward
In addition to peptidoglycan, the walls of most Gram-positive bacteria contain other polymers, frequently with repeating acidic groups. Often these groups are phosphodiesters, which generally occur in polymers called teichoic acids. The teichoic acids were discovered in 1958, as the result of a search for a role for the CDP-glycerol and CDP-ribitol that had been identified in Gram-positive bacteria [4]. As originally defined, they consist of polymerized polyol phosphates, often with side-chains of mono- or oligo-saccharide units, and also ester-linked D-alanine residues. These ester linkages have exceptional lability, occasioned by the presence of vicinal hydroxyl or phosphate groups [75]. In some cases, such as the membrane teichoic acids of group D streptococci, the D-alanyl groups are attached to D-glucose residues rather than to the polyol part of the molecule, and in these instances the ester linkages are appreciably more stable to alkali [86]. As more information has accumulated it has become clear that many variations on the basic structural pattern of teichoic acids exist, although the various polymers presumably serve the same function in the cell. Some characteristic structures of teichoic acids are set out in Fig. 7.1 and of related structures in Fig. 7.2. An exception to the general rule that teichoic acids occur only in Gram-positive bacteria came with the description of lipoteichoic acid in the Gram-negative rumen bacterium Butyrivibrio flbrisolvens [36]. However, ultrastructural study showed that the walls were of the Gram-positive morphological type, but exceptionally thin. The thinness was probably the cause of the apparent Gram-negativity [11].
Archive | 1980
H. J. Rogers; H. R. Perkins; J. B. Ward
In much the same way as the information available describing the biosynthesis of bacterial wall polymers has increased dramatically during the last decade, so a corresponding increase has occurred for the wall polymers of yeasts and other fungi. In view of its ready availability perhaps it is not surprising that much of this information concerns the yeast Saccharomyces cerevisiae. Less immediately obvious are the reasons underlying the relatively little information available concerning the biosynthesis of the glucans, undoubtedly the major structural polymers of the yeast wall. Thus, the detailed evidence available is concentrated on the biosynthesis of chitin, found in S. cerevisiae specifically as a component of the bud scar but as a major wall polymer in many other fungi, and on mannan, the immunodeterminant of the yeast wall. In the following sections, the biosynthesis of each of these polymers and of the glucans is considered, taking together evidence from both yeast and other fungi in an attempt to show both the similarities and differences in the various systems. Further details of the biosynthesis of fungal wall polymers are to be found in the reviews of Ballou [6, 7], Cabib [22] and Farkas [37].
Archive | 1980
H. J. Rogers; H. R. Perkins; J. B. Ward
The walls of Mycobacteria, like those of most other prokaryotes, contain peptidoglycan but characteristically they also contain glycolipid. Much glycolipid is covalently bound to the structural wall components, but there is also an unbound lipid fraction that is extractable into organic solvents representing in Mycobacterium bovis BCG, for example, about 34–40% of the wall [6, 18]. The walls of Mycobacteria have formed the subject of a valuable review [23] and a more general account of mycobacterial physiology is given by Ratledge [30]. In its amino acid composition the peptidoglycan resembles that of Bacilli, but as mentioned in Chapter 6.1, the most noticeable difference is that the acylation of the amino group of the muramic acid residue is by a glycolyl rather than the more common acetyl group [3, 7] a substitution also found in Nocardia [17]. There is evidence that oxidation of acetyl to glycolyl occurs at the level of UDP-N-acetylmuramic acid, since it has been shown that extracts of Nocardia asteroides will oxidize the latter substrate to UDP-N-glycolylmuramic acid, whence the precursor nucleotide pentapeptide presumably is synthesized in the usual way. Another major difference from most other peptidoglycans is in the type of cross-linking. Some links are made directly from the sub-terminal D-alanine of one pentapeptide side-chain to the D-centre of meso-diaminopimelic acid in another chain, just as in Bacilli or in Gramnegative species, but other links involve a direct link between the mesodiaminopimelic acid residue of one chain and that of another, or even the presence of tripeptides of diaminopimelic acid [34]. The exact linkages between the diaminopimelic acid residues (ie whether the L-centre carboxyl group of one is linked to the D-centre amino group of the next and so on) have not been established, and attempts to demonstrate a specific LD-transpeptidase that would catalyse the formation of that type of linkage have not so far succeeded [28].
Archive | 1980
H. J. Rogers; H. R. Perkins; J. B. Ward
The majority of the text of this book will be concerned with the chemistry, biochemistry and physiology of the envelopes and intracytoplasmic membranes of bacteria. As a background to this discussion, something needs to be said about the appearance and arrangement of these structures in the cell, even if this serves no other purpose than to allow people some idea of the complexity of present day aims in trying to understand the more complicated functions of bacteria, such as their ability to divide. This chapter should, however, be regarded only as a topological guide to what follows, not as a thorough review of the ultrastructure of bacteria.