H. R. Perkins

Microbial cell walls and membranes

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.

[51] Exocellular dd-carboxypeptidases-transpeptidases from Streptomyces

Publisher Summary Strains R39 and R61 are soil isolates. Their designations are arbitrary. In strain R39, the cross-link between the peptide units of the wall peptidoglycan extends from the C-terminal D-alanine of one unit to the amino group at the D-center of meso-diaminopimelic acid of another unit (peptidoglycan of chemotype I). The interpeptide bond is in position to a free carboxyl group. In strain R61, the cross-link extends from a C-terminal D-alanine of a peptide unit to a glycine residue attached to the amino group of LL-diaminopimelic acid of another peptide unit (peptidoglycan of chemotype II). The exocellular DD carboxypeptidases-transpeptidases produced by both strains catalyze hydrolysis, react with β-lactam antibiotics. This chapter explains the assay methods for DD-Carboxypeptidase activity like the standard reaction, chemical estimation of free Alanine, as well as, assay method for β-Lactamase. It also discusses the Excretion of DD-Carboxypeptidase-Transpeptidase and β -Lactamase by Streptomyces R39, Excretion of DD-Carboxypeptidase-Transpeptidase and β-Lactamase by Streptomyces R61, purification of the DD-Carboxypeptidase-Transpeptidase from Streptomyces R39 (for 500 Liters of Culture Fluid), Purification of the DD-Carboxypeptidase-Transpeptidase from Streptomyces R61 (for 400 Liters of Culture Fluid), Physicochemical Properties of DD-Carboxypeptidases-Transpeptidases from Streptomyces R39 and R61, Interaction between DD-Carboxypeptidases-Transpeptidases from Streptomyces R39 and R61 and β-Lactam Antibiotics , Titration of DD-Carboxypeptidases-Transpeptidases from Streptornyces R39 and R61 by β-Lactam Antibiotics, Hydrolysis Reactions Catalyzed by the DD-Carboxypeptidases-Transpeptidases from Streptomyces R39 and R61, Concomitant Hydrolysis and Transfer Reactions Involving Distinct Donor and Acceptor Peptides, Catalyzed by the DD-Carboxypeptidases-Transpeptidases from Streptonayces R39 and R61, Concomitant Hydrolysis and Transfer Reactions Catalyzed by the DD-Carboxypeptidases-Transpeptidases from Streptomyces R39 and R61 and in Which the Same Peptide Acts as Donor and Acceptor and Inhibition of DD-Carboxypeptidases-Transpeptidases from Streptomyces R39 and R61 by β-Lactam Antibiotics in the Presence of Substrates.

The bacterial autolysins

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.

Biosynthesis of peptidoglycan

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.

Cross-linking and O-acetylation of newly synthesized peptidoglycan in Staphylococcus aureus H.

Staphylococcus aureus H growing exponentially was labelled with N-acetyl[14C]glucosamine, which became incorporated into the peptidoglycan. The portion of peptidoglycan not linked to teichoic acid (60-75% of the whole) was degraded with Chalaropsis muramidase to yield disaccharide-peptide monomers and dimers, trimers and oligomers formed by biosynthetic cross-linking of the monomers. The degree of O-acetylation of these fragments was also examined. Pulse-chase experiments showed that the proportion of label initially in the monomer fraction immediately after the 1 min pulse declined rapidly during a 3 min chase, while the oligomer fraction (fragments greater than trimer) gained the radioactivity proportionately. The radioactivity of the dimer and trimer fractions remained virtually unchanged. At 4 min after the commencement of labelling (i.e. approx. one-tenth of a generation time) final values had been reached. The O-acetylation of all fragments had achieved final values even at 1 min, except for the monomer fraction, which showed an increase from 40% to 60% during the first 3 min of chase. Although O-acetylation was clearly a very rapid process, no O-acetylated peptidoglycan lipid-intermediates could be detected.

Biosynthesis of Peptidoglycan in Wall plus Membrane Preparations from Micrococcus luteus: Direction of Chain Extension, Length of Chains and Effect of Penicillin on Cross-linking

SUMMARY: A wall + membrane preparation from Micrococcus luteus was used to synthesize radioactively labelled peptidoglycan. The newly synthesized peptidoglycan either was cross-linked by transpeptidation to existing wall or remained associated with the membrane fraction but was not cross-linked. The average biosynthetic chain lengths, calculated from the ratio of free reducing groups of muramic acid to total muramic acid, were 66 disaccharide units for cross-linked and 26 disaccharide units for the uncross-linked material. The latter value was confirmed by the release of lactyl peptide side chains by β-elimination. Benzylpenicillin (1 μg ml−1) inhibited cross-linking but not overall synthesis of glycan whereas at concentrations above 10 μg ml−1 overall glycan synthesis was slightly inhibited. In the presence of 100 μg benzylpenicillin ml−1 the incorporation of disaccharide units to existing walls decreased to 25% of the control. This residual incorporation represented extension by transglycosylation of peptidoglycan already cross-linked to existing walls. Chains with an average length of between 30 and 45 disaccharide units were added during a 30 min incubation period. However, if incubation was continued for up to 120 min (in the presence of 100 μg benzylpenicillin ml−1) a considerable amount of the newly synthesized peptidoglycan was lost from the purified wall because autolytic enzymes were expressed in the wall + membrane preparation after the action of the antibiotic.

In Vitro Synthesis of Peptidoglycan by β-Lactam-Sensitive and -Resistant Strains of Neisseria gonorrhoeae: Effects of β-Lactam and Other Antibiotics

The synthesis in vitro of peptidoglycan by Neisseria gonorrhoeae was studied in organisms made permeable to nucleotide precursors by treatment with ether. Optimum synthesis occurred at 30°C in tris(hydroxymethyl)aminomethane-maleate buffer (0.05 M; pH 6) in the presence of 20 mM Mg2+. The incorporation from uridine 5′-diphosphate-N-acetyl-[14C]glucosamine into peptidoglycan, measured after precipitation of the cells with trichloroacetic acid, was sensitive to the β-lactam antibiotics, bacitracin, diumycin, and tunicamycin and relatively resistant to spectinomycin and tetracycline. Differences in sensitivity between preparations from a β-lactamase producer and a laboratory segregant derived from it were not great. Synthesized peptidoglycan was also fractionated into sodium dodecyl sulfate-soluble and -insoluble portions. β-Lactam antibiotics at concentrations equivalent to the minimal inhibitory concentrations for growth of the organisms did not inhibit peptidoglycan synthesis, but rather caused a small enhancement. At higher concentrations, above about 0.5 μg/ml, incorporation into sodium dodecyl sulfate-insoluble material was progressively inhibited, whereas the amount of sodium dodecyl sulfate-soluble product increased greatly, more than compensating for the loss of the precipitable fraction. Similar observations were made with three strains, and also with the β-lactam clavulanic acid, normally considered as a β-lactamase inhibitor rather than as itself an effective antibiotic.

Biosynthesis of wall-linked teichuronic acid by a wall-plus-membrane preparation from Micrococcus luteus. Effect of antibiotics.

Cell-walls of Micrococcus luteus contain two major polymers, peptidoglycan and a polysaccharide called teichuronic acid. The latter was first extracted from it!. Zuteus cell walls with trichloroacetic acid and contained D-glucose and N-acetylmannosaminuronic acid in approximately equimolar amounts [l] . It was shown [2] to consist of repeating units of D-N-acetylmannosamin-pyranuronosyl

Cell walls of filamentous fungi

( 1 ,6)-D-glucose-linked cy [ 1,4] . The in vitro biosynthesis of the polysaccharide by a particulate membrane preparation required the addition of the nucleotide precursors, UDP-glucose, UDP-N-acetyl-D-mannosaminuronic acid (UDPManNAcUA) and UDP-iV-acetylglucosamine [3]. Synthesis of new polysaccharide is now demonstrated in a wall-plus-membrane preparation of M. Zuteus. As with the synthesis of peptidoglycan in this type of preparation [4-61, newly synthesized polysaccharide was either linked to pre-existing wall or found in the soluble fraction. Total polysaccharide synthesis was inhibited by bacitracin and tunicamycin but was not affected by benzylpenicillin. It was concluded that UDP-N-acetylglucosamine was incorporated by means of a lipid carrier into a link-piece between the polysaccharide and peptidoglycan. The inhibitory actions of tunicamycin and bacitracin suggest that the lipid carrier might be a polyisoprenyl phosphate.

The direction of peptide trimer synthesis from the donor-acceptor substrate Nalpha-(acetyl)-Nepsilon-(glycyl)-L-lysyl-D-alanyl-D-alanine by the exocellular dd-carboxypeptidase-transpeptidase of Streptomyces R61

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.