J. Baddiley

Occurrence and function of membrane teichoic acids

Membrane teichoic acids, sometimes described as lipoteichoic acids, are important but not major components of nearly all Gram-positive bacteria. They appear on the outer surface of the cytoplasmic membrane and possess antigenic properties. Several functions have been ascribed to these glycerol phosphate polymers, including the binding of divalent cations required for optimal activity of membrane-bound enzymes, and the control of certain lytic enzymes. A substance that is identical or closely similar to membrane teichoic acid, lipoteichoic acid carrier, plays an important part in the biosynthesis of wall teichoic acid; it accepts polyol phosphate residues from CDP-glycerol or CDP-ribitol to form a polyol phosphate chain which is then transferred after the incorporation of a tri(glycerol phosphate) linkage unit, to the growing glycan chain of peptidoglycan.

The linkage between teichoic acid and peptidoglycan in bacterial cell walls

The major polymers in the walls of most Grampositive bacteria are peptidoglycan and teichoic acid [l-3] . Whereas peptidoglycan is insoluble because of extensive cross-linkage of glycan chains through oligopeptide bridges, teichoic acids can be extracted from walls as highly soluble polymers by the action of dilute acid or alkali under gentle conditions [4-61. The simple teichoic acids comprising chains of glycerol phosphate or ribitol phosphate units, with D-alanyl ester substituents and in some cases sugar residues, can often be removed from their association with peptidoglycan with little or no degradation of the polymer chain. Those teichoic acids and related polymers possessing sugar l-phosphate linkages in the chain are readily degraded by acid; however, even in these cases most of the polymer can be removed from the wall by acid with only partial chain degradation if conditions are chosen carefully [7] . In all cases, however, extraction is relatively slow and the association with peptidoglycan can not be explained exclusively as electrostatic attraction between acidic and basic centres in the two polymers. It seems then that attachment involves covalent linkages that are generally more labile towards acids and bases than are the inter-unit linkages in the teichoic acid chain. Much effort has been directed towards establishing the details of the chemistry of the linkage between these wall polymers, despite the considerable difficulties associated with the characterization of small amounts bf the components of the linkage region when accompanied by much larger amounts of chemically similar products of degradation of the main

Biosynthesis of the unit that links teichoic acid to the bacterial wall: Inhibition by tunicamycin

The walls of Gram-positive bacteria contain acidic polysaccharides and teichoic acids that are covalently linked through phosphodiesters to muramic acid residues in the peptidoglycan. It has been shown recently that in St@yZococcus agrees H [ 1 ] and in a mutant strain that lacks N-acetylglucosaminyl substituents on its wall teichoic acid [2] a ‘linkage unit’ that is a trimer of glycerol phosphate is interposed between the phosphate-terminal end of the teichoic acid chain and a muramic acid residue of the peptidoglycan to which it is attached. Evidence for similar linkage units has been found in Micrococcus sp. 2102 and in Bacillus subtilis W32 [J. Coley and E. Tarelli, unpublished work]. None of the bacteria examined contains a glycerol teichoic acid in its wall, and it has been demonstrated that the glycerol membrane teichoic acids are synthesized from phosphatidylglycerol [3,4]. Nevertheless, we have found [5] that membrane preparations from all of them catalyse the synthesis from CDP-glycerol of material containing a glycerol phosphate oligomer. Maximal synthesis of this product requires the addition of UDP-N-acetylglucosamine and the substrate for the synthesis of the backbone of the wall teichoic acid. The product is amphiphilic and appears to consist of the teichoic acid chain linked through the glycerol phosphate oligomer to a lipid acceptor. Bracha and Glaser [6] found that synthesis of the ribitol teichoic acid covalently linked to wall, in a wall-membrane preparation from S. aure’eus H, also required the addition of CDP-glycerol and UDP-N-acetylglucosamine. If therefore appears that teichoic acid attached to linkage unit is synthesized in

Lipoteichoic acid and lipoteichoic acid carrier in Staphylococcus aureus H

Synthesis of poly(ribito1 phosphate) by a purified polymerase from Staphylococcus aureus H has been shown to proceed by transfer of ribitol phosphate units from CDP-ribitol to a ‘lipoteichoic acid carrier’ onto which the ribitol phosphate chain is assembled [l-3] . Lipoteichoic acid carriers, active with the S. aureus polymerase, have been extracted from several Gram-positive bacteria and the material isolated from S. aureus also acts as carrier in the synthesis of poly(glycero1 phosphate) by a polymerase from Bacillus subtilis [2]. These lipoteichoic acid carriers have usually been isolated by extraction from defatted membrane preparations into buffers containing Triton. On the other hand, the lipoteichoic acids examined in this laboratory have usually been extracted with aq. phenol from de-fatted membrane or ‘cell contents’ preparations [4-61; this treatment appears to extract all of the membrane teichoic acid, essentially all of which is present as lipoteichoic [6,7]. In all lipoteichoic acids so far examined the lipid moiety is an acylated diglycosylglycerol [4,5,7,8]. In contrast the lipoteichoic acid carrier from S. aureus H, which is stated [3] to be identical to the lipoteichoic acid present in the membrane of this organism, has been reported to consist of chains of 1214 glycerol residues, one molecule of glucose and one molecule of fatty acid being present in each chain [ 1,3]. We now report that the lipoteichoic acid extracted from

Variation of polar lipid composition of Bacillus subtilis (Marburg) with different growth conditions

A previous study of the polar lipids of Bacillus subtilis (Marburg) has shown that at least five phospholipids in addition to glycolipid may be present [ 11. The report shows that by manipulation of growth conditions the lipid composition of this organism can be varied and greatly simplified. In batch cultures, under conditions of apparent phosphate starvation, a close interrelation of phospholipids and phosphate-free polar lipids is observed, the latter increasing in proportion as growth progresses. Certain chemostat cultures (magnesium limitation) are found to contain only one acidic phospholipid, phosphatidylglycerol (PC), and one neutral polar lipid, diglucosyldiglyceride (DG). In phosphate-limited cultures the proportions of phospholipids are reduced and phosphate-free polar lipids are observed in increased proportions; one of these lipids is DG, the other is an acidic peptidolipid. vessel. Lipids were extracted from lyophilized cells with chloroform-methanol (2: 1, v/v) and investigated by two-dimensional thin-layer chromatography [4] (fig. 2 for details). Qualitative identifications of lipids was made by use of specific spray reagents [2], and densitometry [6] gave an approximate measure of the relative proportions of the lipids. The lipids of B. subtilis (Marburg) grown in batch culture included the phospholipids reported in previous studies [I] i.e. diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), PG and traces of its lysine ester. DG was identified in all cultures, and in some extracts an additional phosphorous-free lipid (X) was found (see fig. 1). Preliminary investigations

The Occurrence of Lipoteichoic Acids in the Membranes of Gram-positive Bacteria

Glycerol teichoic acids have been found associated with the cytoplasmic membrane of all of the Gram-positive bacteria examined and it is believed that, unlike wall teichoic acids which are absent from some organisms, these membrane teichoic acids are characteristically present in all species (for reviews see Archibald, Baddiley & Blumsom, 1968; Baddiley, 1970, 1972). Wall teichoic acid is attached to the peptidoglycan of the wall through its terminal phosphate residue, but although it has been known for some time that membrane teichoic acid is associated with the outer surface of the membrane (Hay, Wicken & Baddiley, 1963; Shockman & Slade, 1964) the nature of this association has only recently been examined.

The alanine ester content and magnesium binding capacity of walls of Staphylococcus aureus H grown at different pH values

Abstract Recent studies have shown that teichoic acids are functionally involved in the uptake of Mg 2+ and that their ability to bind Mg is reduced by the presence of alanine ester substituents. Walls of Staphylococcus aureaus H grown at pH 5 contain more ester alanine and bind less magnesium than do walls of S. aureaus grown under similar conditions at pH 6 and 7. The differences in alanine content are largely due to its removal by base catalysed hydrolysis of the labile ester linkages during growth in media of pH 6 and 7 and the incorporation of alanine esters into the walls does not appear to be influenced by the pH of the medium in which the bacteria are grown under the conditions described. The decreased magnesium binding capacity of walls of S. aureus grown at pH 5 correlates with their increased alanine ester content but there is no proportional relationship between these two quantities.

Extraction and purification of lipoteichoic acids from Gram-positive bacteria.

Hot and cold, 80% aqueous phenol extraction procedures together with an aqueous extraction technique have been evaluated for the isolation of lipoteichoic acids from the cytoplasmic membrane of Gram-positive bacteria. Lipoteichoic acids of Staphlococcus aureus H, Micrococcus 2102, Baccillus subtilis 168, and Bacillus subtilis W-23 were examined as each of them emphasises a different problem of contamination. The purity of the lipoteichoic acids with respect to cell-wall material, nucleic acid, and protein is discussed together with the criteria of purity which enables critical structural analysis of lipoteichoic acids to be carried out.

A linkage unit joining peptidoglycan to teichoic acid in staphylococcusaureus H

Walls of many Gram-positive bacteria are composed principally of teichoic acid and peptidoglycan, and a great deal is now known about the structure and biosynthesis of these polymers. A major unsolved feature has been the way in which these two polymers are attached to each other; consequently there is little information on how they become attached during synthesis and assembly of the wall. It has long been known that teichoic acids are covalently linked to the glycan chain of peptidoglycan and that this linkage probably involves a phosphorylated muramic acid residue of the glycan. However difficulties have been encountered in studies on the precise nature of the way in which these major wall components are associated. In order to simplify such studies we have recently examined a mutant of StuphyZococcus aureus H [ 1 ] that lacks the N-acetylglucosaminyl substituents normally present on the ribitol residues of the teichoic acid [2-41. In addition to poly(ribito1 phosphate) walls of this mutant were found to contain an oligomer of glycerol phosphate [l] . This could have been present as a separate component, independently linked to the peptidoglycan, or it could have been interposed between the ribitol teichoic acid and the glycan so as to constitute a ‘linkage unit’. The glycerol phosphate oligomer isolated by alkali extraction of walls that had first been oxidized with sodium metaperiodate, so as to destroy and remove the ribitol acid, and then reduced with potassium [3H]borohydride consisted of a chain of three glycerol phosphate residues attached to which was a radioactively labelled ethylene glycol phosphate

The interrelation of polar lipids in bacterial membranes

Bacillus subtilis W23 and Bacillus cereus T, grown under conditions of apparent phosphate starvation approaching stationary phase of growth, accumulate diglucosyl diglycerides while the proportion of phosphatidylethanolamine is reduced. The latter organism also produces under these conditions an acidic glycolipid which apparently partially replaces the acidic phospholipids, phosphatidylglycerol and diphosphatidylglycerol.