The structure of a mannitol teichoic acid from Bifidobacterium bifidum ssp. Pennsylvanicum

The isolation and analysis of the cell wall and cell wall fractions of Bifidobacterium bifidum ssp. pennsylvanicum are presented. With lysozyme a solubilized cell wall fraction is obtained which contains muramic acid, glucosamine, rhamnose, glucose, mannitol, phosphate and all peptidoglycan amino acids. Its composition did not change with culture age. A glycogen-like glucose polymer which is of cytoplasmic origin is identified in the insoluble cell wall fraction. The solubilized cell wall fraction contains a glucosylated rhamnose polymer which is linked by glycosidic bonds to the peptidoglycan fragments. This polymer is a 1,2-linked or an alternating, 1,2/1,3-linked α-rhamnose chain substituted on average at every second rhamnose residue with an α-linked glucose molecule. Various experiments gave evidence that mannitol and phosphate are present in 4,6-linked mannitol phosphate oligomers which are linked by phosphodiester bonds to the glucosylated rhamnose polymer. These oligomers may fulfill the functions of the more common wall teichoic acids.     

An electron microscopic study of the location of peptidoglycan in group A and C streptococcal cell walls.

The morphological appearance of deproteinized Group A and C streptococcal walls after treatment by different procedures extracting teichoic acids and polysaccharides (formamide, hydrochloric acid, nitrous acid, trichloroacetic acid, sulphuric acid, sodium hydroxide and sodium deoxycholate) was compared with the content of teichoic acids and polysaccharides remaining in the treated walls. All procedures extracted teichoic acids almost completely, but polysaccharides were extracted to various degrees. The ultrastructural appearance of walls after these extractions still exhibited the triple-layered wall profile; only a reduction of thickness of the wall and of electron density of the layers occurred. There was no direct correlation between the reduction of rhamnose content and thickness of walls. The ultrastructural localization of peptidoglycan in the streptococcal walls was explored by means of the indirect immunoferritin technique using anti-peptidoglycan antibodies isolated from anti-Group A-variant antisera. Ferritin particles were bound predominantly to filamentous structures which protruded from both surfaces of peptidoglycan fragments and isolated walls. Peptidoglycan was also detected on the filamentous protrusions of whole cocci. These results contradict models of the streptococcal wall in which peptidoglycan forms the innermost layer and support a mosaic structure in which peptidoglycan forms a network of the peptidoglycan-polysaccharide complex.     

Transposon-induced mutations in two loci of Listeria monocytogenes serotype 1/2a result in phage resistance and lack of N-acetylglucosamine in the teichoic acid of the cell wall.

Teichoic acid-associated N-acetylglucosamine and rhamnose have been shown to serve as phage receptors in Listeria monocytogenes serotype 1/2a. We generated and characterized two single-copy Tn916DeltaE mutants which were resistant to phage A118 and several other serotype 1/2a-specific phages. In one mutant the insertion was immediately upstream of the recently identified ptsHI locus, which encodes two proteins of the phosphoenolpyruvate-dependent carbohydrate uptake system, whereas in the other the insertion was immediately upstream of an operon whose most distal gene was clpC, involved in stress responses and virulence. Transduction experiments confirmed the association of the phage-resistant phenotype of these mutants with the transposon insertion. Phage A118 resistance of the mutants could be attributed to inability of the phage to adsorb onto the mutant cells, and biochemical analysis of cell wall composition showed that the teichoic acids of both mutants were deficient in N-acetylglucosamine. Rhamnose and other teichoic acid and cell wall components were not affected.     

Nature and Origins of Phosphorus Compounds in Isolated Cell Walls of Staphylococcus aureus

Preparations of purified cell walls from Staphylococcus aureus were shown to contain small amounts of phospholipid and glycerol teichoic acid. Since these are components of the cell membrane, it is probable that the wall itself contains no lipid, but does retain fragments of membrane because of physical connections between wall and membrane. In walls of S. aureus strain 52A5, which completely lacks ribitol teichoic acid, the only phosphorylated compound identified as a genuine wall component was a phosphorylated derivative of murein that gave rise to muramic acid phosphate on acid hydrolysis. Muramic acid phosphate was also identified in hydrolysates of walls from S. aureus H and strain 52A2.     

Rapid analysis of Listeria monocytogenes cell wall teichoic acid carbohydrates by ESI-MS/MS

We report the application of electrospray ionization (ESI) mass spectrometry for compositional characterization of wall teichoic acids (WTA), a major component of gram-positive bacterial cell walls. Tandem mass spectrometry (ESI-MS/MS) of purified and chemically hydrolyzed monomeric WTA components provided sufficient information to identify WTA monomers and their specific carbohydrate constituents. A lithium matrix was used for ionization of uncharged WTA monomers, and successfully applied to analyze the WTA molecules of four Listeria strains differing in carbohydrate substitution on a conserved polyribitol-phosphate backbone structure. Carbohydrate residues such as N-acetylglucosamine or rhamnose linked to the WTA could directly be identified by ESI-MS/MS, circumventing the need for quantitative analysis by gas chromatography. The presence of a terminal N-acetylglucosamine residue tethered to the ribitol was confirmed using fluorescently labeled wheat-germ agglutinin. In conclusion, the mass spectrometry method described here will greatly facilitate compositional analysis and characterization of teichoic acids and similar macromolecules from diverse bacterial species, and represents a significant advance in the identification of serovar-specific carbohydrates and sugar molecules on bacteria.     

Cell Wall Polysaccharide Biosynthesis by Membrane Fragments from Streptococcus pyogenes and Stabilized L-Form

The formation and composition of a cell wall rhamnose-containing polysaccharide by membrane fragments from Streptococcus pyogenes and its stabilized L-form were compared. Also, the effect of prior treatment on the ability of coccal whole-cell and membrane fragments to incorporate radioactivity from thymidine diphosphate-¹⁴C-rhamnose, and the results of subsequent attempts to remove labeled polysaccharide from such membranes are given. L-form membrane fragments were capable of only 10% uptake of ¹⁴C-rhamnose from this nucleotide as compared with streptococcal membranes. However, once bound, both membrane fragments polymerized rhamnose to the same extent. These findings tend to negate the almost complete lack of polymeric rhamnose within the intact L-form as being due to the absence of membrane enzymes necessary for the transfer of rhamnose from a suitable precursor to membrane acceptor sites or enzymes responsible for rhamnose polymerization. Degradation of labeled rhamnose polysaccharide after isolation from coccal membranes by mild acid hydrolysis showed muramic acid and glucosamine to be attached. This same polysaccharide from L-form membrane fragments was devoid of amino sugars. These data suggest the possible involvement of amino sugars in the attachment of cell wall polymeric rhamnose to the streptococcal cytoplasmic membrane. The absence of attached amino sugars to rhamnose polysaccharide from L-form membrane fragments is discussed in terms of this organism's continued inability for new cell wall formation. The isolation, from streptococcal membrane fragments, of a polysaccharide containing rhamnose and amino sugars common to at least two different streptococcal cell wall-type polymers was demonstrated.     

Isolation of the Teichoic Acid of Bacillus Subtilis 168 by Affinity Chromatography

A column of insoluble concanavalin A was prepared by coupling the protein to cyanogen bromide-activated Sepharose. When autolysates of Bacillus subtilis 168 cell walls were passed over the column, the alpha glucosylated teichoic acid component of the cell wall was retained. The teichoic acid could be eluted with dilute alpha-methylglucopyranose. The teichoic acid prepared by affinity chromatography from cell wall autolysates had a higher sedimentation rate than teichoic acids obtained by conventional methods.

Several authors have shown that concanavalin A (con A) forms complexes with alpha-glucosylated teichoic acids^1–3. Doyle and Birdsell¹ found that the teichoic acid of Bacillus subtilis 168 (trp C2) would precipitate with con A at neutral pH in dilute buffer. The formation of a precipitate was inhibited by sugars which bind to the active site of con A. This observation suggested that it should be possible to purify the teichoic acid by affinity chromatography using insoluble con A as the affinity probe. Lloyd⁴ and Donnelly and Goldstein⁵ have successfully employed insoluble con A to purify polysaccharides and glycoproteins. In this communication, we describe conditions for the rapid purification of the alpha-glucosylated teichoic acid of B. subtilis 168. The teichoic acid prepared by this procedure appears to be less degraded than teichoic acids obtained by conventional methods.     

Listeria monocytogenes tyrosine phosphatases affect wall teichoic acid composition and phage resistance

Tyrosine phosphatase (PTP)-like proteins exist in many bacteria and are segregated into two major groups: low molecular weight and conventional. The latter group also has activity as phosphoinositide phosphatases. These two kinds of PTP are suggested to be involved in many aspects of bacterial physiology including stress response, DNA binding proteins, virulence, and capsule/cell wall production. By annotation, Listeria monocytogenes possesses two potential low molecular weight and two conventional PTPs. Using L.?monocytogenes wild-type (WT) strain 10403S, we have created an in-frame deletion mutant lacking all four PTPs, as well as four additional complemented strains harboring each of the PTPs. No major physiological differences were observed between the WT and the mutant lacking all four PTPs. However, the deletion mutant strain was resistant to Listeria phages A511 and P35 and sensitive to other Listeria phages. This was attributed to reduced attachment to the cell wall. The mutant lacking all PTPs was found to lack N-acetylglucosamine in its wall teichoic acid. Phage sensitivity and attachment was rescued in a complemented strain harboring a low molecular weight PTP (LMRG1707).     

Reassembly of the Regularly Arranged Subunits in the Cell Wall of Lactobacillus brevis and Their Reattachment to Cell Walls

The reassembly of tetragonally arranged subunits in the cell wall of Lactobacillus brevis and the reattachment of the subunits to cell wall fragments were investigated by electron microscopy. The subunits dissociated from the cell wall with guanidine hydrochloride (GHCl) reassembled into the same regular array as seen in the native cell wall after dialysis against neutral buffer even in the absence of specific cations. The subunits could also reattach to the cell wall fragments from which they had been removed by treatment with GHCl, sodium dodecyl sulfate or cold trichloroacetic acid but not to those treated with hot formamide. Heterologous reattachment of the subunits occurred on cell wall fragments obtained from L. fermentum but not on those from L. plantarum or L. casei subsp. casei. On the basis of these observations and chemical analyses of the cell wall fragments, the subunits of L. brevis appeared to be bound by hydrogen bonds to a neutral polysaccharide moiety in the cell wall but not to peptidoglycan or teichoic acid.     

Production and release of peptidoglycan and wall teichoic acid polymers in pneumococci treated with beta-lactam antibiotics

Autolysin-defective pneumococci treated with inhibitory concentrations of penicillin and other beta-lactam antibiotics continued to produce non-cross-linked peptidoglycan and cell wall teichoic acid polymers, the majority of which were released into the surrounding medium. The released cell wall polymers were those synthesized by the pneumococci after the addition of the antibiotics. The peptidoglycan and wall teichoic acid chains released were not linked to one another; they could be separated by affinity chromatography on an agarose-linked phosphorylcholine-specific myeloma protein column. Omission of choline, a nutritional requirement and component of the pneumococcal teichoic acid, from the medium inhibited both teichoic acid and peptidoglycan synthesis and release. These observations are discussed in terms of plausible mechanisms for the coordination between the biosynthesis of peptidoglycan and cell wall teichoic acids.     

Transposon-Induced Mutations in Two Loci of Listeria monocytogenes Serotype 1/2a Result in Phage Resistance and Lack of N-Acetylglucosamine in the Teichoic Acid of the Cell Wall

Teichoic acid-associated N-acetylglucosamine and rhamnose have been shown to serve as phage receptors in Listeria monocytogenes serotype 1/2a. We generated and characterized two single-copy Tn916ΔE mutants which were resistant to phage A118 and several other serotype 1/2a-specific phages. In one mutant the insertion was immediately upstream of the recently identified ptsHI locus, which encodes two proteins of the phosphoenolpyruvate-dependent carbohydrate uptake system, whereas in the other the insertion was immediately upstream of an operon whose most distal gene was clpC, involved in stress responses and virulence. Transduction experiments confirmed the association of the phage-resistant phenotype of these mutants with the transposon insertion. Phage A118 resistance of the mutants could be attributed to inability of the phage to adsorb onto the mutant cells, and biochemical analysis of cell wall composition showed that the teichoic acids of both mutants were deficient in N-acetylglucosamine. Rhamnose and other teichoic acid and cell wall components were not affected.     

Chemical composition and serological analysis of the cell wall of Peptostreptococcus

Bahn, Arthur N. (Northwestern University, Chicago, Ill.), Patrick C. Y. Kung, and James A. Hayashi. Chemical composition and serological analysis of the cell wall of Peptostreptococcus. J. Bacteriol. 91:1672-1676. 1966.-Chemical and serological analyses were made of the cell wall of Peptostreptococcus to characterize taxonomically this genus of anaerobic streptococci. Cell wall hydrolysates of P. putridus strains 06 and 85, P. intermedius strains 11 and 87, and P. elsdenii strain B-159 were prepared, and the cell wall sugars were measured quantitatively by paper chromatography. Strain 85 contained only glucose, whereas strain 06 contained 93% glucose and 7% mannose. Strain 87 contained only rhamnose, and strain 11 contained approximately equal amounts of glucose and rhamnose. Strain B-159 differed from all the other strains in having a low (3.1%) content of total carbohydrate, consisting of rhamnose, galactose, and glucose. Quantitative amino acid analyses showed that the major amino compounds present in the cell wall were glutamic and aspartic acids, alanine, lysine, muramic acid, glucosamine, and galactosamine. Strains 06 and 85 possessed this complement of amino compounds, but strains 11 and 87 had relatively little aspartic acid. Strain B-159 was markedly different in having a high content of glycine and diaminopimelic acid, with only traces of lysine; it was the only strain in which teichoic acid was found. Serological analyses were made with the use of cell wall extracts as antigenic material and with homologous antisera, as well as streptococcal group antisera for groups A through S. The only strong agglutination was obtained between strain 87 antigen and group C antisera; weak agglutination was obtained with 87 against N, O, and K, and between strain 11 and groups E and F. All other antisera gave negative reactions. It is concluded that strain B-159 does not belong to the genus Peptostreptococcus, that strains 06 and 85 are members of P. putridus, and that strains 11 and 87 may be members of two different genera.     

Use of bacteriophage-resistant mutants to study the nature of the bacteriophage receptor site of Staphylococcus aureus

Bacteriophage-resistant strains of Staphylococcus aureus H were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Cell walls isolated from about half of these resistant strains were incapable of inactivating phages and were shown to lack N-acetyl-d-glucosamine (GlcNAc) in their cell wall teichoic acid. Apart from the lack of GlcNAc, two of these mutant strains were deficient in cell wall phosphorus and ester-linked d-alanine. These two strains were also found to be resistant to both phage K and a host-range mutant isolated from the parent phage. These two phages could lyse the other phage-resistant mutants which lacked GlcNAc in their teichoic acid. Cell walls from the remaining phage-resistant mutant strains did inactivate phages and were found to have normal cell wall teichoic acid. Although GlcNAc in teichoic acid was required for phage inactivation, no difference in phage inactivation ability was detected with cell walls isolated from strains of S. aureus having exclusively alpha- or exclusively beta-linked GlcNAc in their cell wall teichoic acid.     

Cell wall anionic polymers of Streptomyces sp. MB-8, the causative agent of potato scab

The cell wall of Streptomyces sp. MB-8 contains a major teichoic acid, viz., 1,3-poly(glycerol phosphate) substituted with N-acetyl-alpha-D-glucosamine (the degree of substitution is 60%), a minor teichoic acid, viz., non-substituted poly(glycerol phosphate), and a family of Kdn (3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid)-containing oligomers of the following general structure: [carbohydrate structure: see text]. The composition of the oligomers was established using MALDI-TOF mass spectroscopy. The present study provides the second example of the identification of Kdn as a component of cell wall polymers of streptomycetes, which are the causative agents of potato scab.     

Peptidoglycan cross-linking and teichoic acid attachment in Streptococcus pneumoniae.

Autolysin-defective pneumococci continue to synthesize both peptidoglycan and teichoic acid polymers (Fischer and Tomasz, J. Bacteriol. 157:507-513, 1984). Most of these peptidoglycan polymers are released into the surrounding medium, and a smaller portion becomes attached to the preexisting cell wall. We report here studies on the degree of cross-linking, teichoic acid substitution, and chemical composition of these peptidoglycan polymers and compare them with normal cell walls. peptidoglycan chains released from the penicillin-treated pneumococci contained no attached teichoic acids. The released peptidoglycan was hydrolyzed by M1 muramidase; over 90% of this material adsorbed to vancomycin-Sepharose and behaved like disaccharide-peptide monomers during chromatography, indicating that the released peptidoglycan contained un-cross-linked stem peptides, most of which carried the carboxy-terminal D-alanyl-D-alanine. The N-terminal residue of the released peptidoglycan was alanine, with only a minor contribution from lysine. In addition to the usual stem peptide components of pneumococcal cell walls (alanine, lysine, and glutamic acid), chemical analysis revealed the presence of significant amounts of serine, aspartate, and glycine and a high amount of alanine and glutamate as well. We suggest that these latter amino acids and the excess alanine and glutamate are present as interpeptide bridges. Heterogeneity of these was suggested by the observation that digestion of the released peptidoglycan with the pneumococcal murein hydrolase (amidase) produced peptides that were resolved by ion-exchange chromatography into two distinct peaks; the more highly mobile of these was enriched with glycine and aspartate. The peptidoglycan chains that became attached to the preexisting cell wall in the presence of penicillin contained fewer peptide cross-links and proportionally fewer attached teichoic acids than did their normal counterparts. The normal cell wall was heavily cross-linked, and the cross-linked peptides were distributed equally between the teichoic acid-linked and teichoic acid-free fragments.     

Characterization of a novel linkage unit between ribitol teichoic acid and peptidoglycan in Listeria monocytogenes cell walls

The structure of the linkage unit between ribitol teichoic acid and peptidoglycan in the cell walls of Listeria monocytogenes EGD was studied. A teichoic-acid--glycopeptide preparation isolated from lysozyme digests of the cell walls of this strain contained mannosamine, glycerol, glucose and muramic acid 6-phosphate in an approximate molar ratio of 1:1:2:1, together with large amounts of glucosamine and other components of teichoic acid and glycopeptides. A teichoic-acid-linked sugar preparation, obtained by heating the cell walls at pH 2.5, also contained glucosamine, mannosamine, glycerol and glucose in an approximate molar ratio of 25:1:1:2. Part of the glucosamine residues were shown to be involved in the linkage unit. Thus, on mild alkaline hydrolysis, the teichoic-acid-linked sugar preparation gave a disaccharide characterized as N-acetylmannosaminyl(beta 1----4)-N-acetylglucosamine [ManNAc(beta 1----4)GlcNAc] in addition to the ribitol teichoic acid moiety, whereas the teichoic-acid - glycopeptide was separated into disaccharide-linked glycopeptide and the ribitol teichoic acid moiety by the same procedure. Furthermore, Smith degradation of the cell walls gave a characteristic fragment, EtO2-P-Glc(beta 1----3)Glc(beta 1----1/3)Gro-P-ManNAc(beta 1----4)GlcNAc (where EtO2 = 1,2-ethylenediol and Gro = glycerol). The results lead to the conclusion that in the cell walls of this organism, the ribitol teichoic acid chain is linked to peptidoglycan through a novel linkage unit, Glc(beta 1----3)Glc(beta 1----1/3)Gro-P-(3/4)ManNAc-(beta 1----4)GlcNAc.     

Chemical Composition of the Cell Walls of Clostridium botulinum Type A

Cell walls were isolated by sonic disruption of log-phase cells of Clostridium botulinum type A strain 190L and purified by treatment with sodium dodecyl sulfate (SDS) followed by digestion with proteases. Electron microscopy revealed that the cell walls thus obtained were free of both cytoplasmic membrane and cytoplasmic fragments. The purified cell wall contained 8.7% total nitrogen, 15.0% total hexosamines, 22.4% reducing groups, 8.3% carbohydrate, and 3.1% glucose. The content of total phosphorus was very low (0.02%), and therefore it was expected that teichoic acid might be absent in the cell wall. The wall peptidoglycan contained glutamic acid, alanine, diaminopimelic acid, glucosamine and muramic acid in the molar ratios of 1.00:1.85:0:85:1.06:0.67. A low amount of galactosamine was also present, but no other amino acids were found in significant quantities. The SDS-treated cell walls were not attacked by lysozyme, but after extraction with hot formamide they were completely dissolved by the enzyme and released reducing groups. The lysozyme digest was separated into two constituents, the saccharide moiety and the peptide moiety on Sephadex G-50.     

Host-Pathogen Interactions : XIX. THE ENDOGENOUS ELICITOR, A FRAGMENT OF A PLANT CELL WALL POLYSACCHARIDE THAT ELICITS PHYTOALEXIN ACCUMULATION IN SOYBEANS

An elicitor of phytoalexin accumulation (endogenous elicitor) is solubilized from purified cell walls of soybean (Glycine max [L.] Merr., cv. Wayne) by extracting the walls with hot water or by subjecting the walls to partial acid hydrolysis. The endogenous elicitor obtained from soybean cell walls binds to an anion exchange resin. The elicitor-active material released from the resin contains oligosaccharides rich in galacturonic acid; small amounts of rhamnose and xylose are also present. The preponderance of galacturonic acid in the elicitor-active fragments suggests that the elicitor is, in fact, a fragment of a pectic polysaccharide. This possibility is supported by the observation that treatment of the wall fragments with a highly purified endopolygalacturonase destroys their ability to elicit phytoalexin accumulation. This observation, together with other evidence presented in this paper, suggests that galacturonic acid is an essential constituent of the elicitor-active wall fragments. Endogenous elicitors were also solubilized by partial hydrolysis from cell walls of suspension-cultured tobacco, sycamore, and wheat cells.     

Structure of the linkage units between ribitol teichoic acids and peptidoglycan.

The structure of the linkage regions between ribitol teichoic acids and peptidoglycan in the cell walls of Staphylococcus aureus H and 209P and Bacillus subtilis W23 and AHU 1390 was studied. Teichoic acid-linked saccharide preparations obtained from the cell walls by heating at pH 2.5 contained mannosamine and glycerol in small amounts. On mild alkali treatment, each teichoic acid-linked saccharide preparation was split into a disaccharide identified as N-acetylmannosaminyl beta(1----4)N-acetylglucosamine and the ribitol teichoic acid moiety that contained glycerol residues. The Smith degradation of reduced samples of the teichoic acid-linked saccharide preparations from S. aureus and B. subtilis gave fragments characterized as 1,2-ethylenediol phosphate-(glycerolphosphate)3-N-acetylmannosaminyl beta(1----4)N- -acetylxylosaminitol and 1,2-ethylenediolphosphate-(glycerol phosphate)2-N-acetylmannosaminyl beta(1----4)N-acetylxylosaminitol, respectively. The binding of the disaccharide unit to peptidoglycan was confirmed by the analysis of linkage-unit-bound glycopeptides obtained from NaIO4 oxidation of teichoic acid-glycopeptide complexes. Mild alkali treatment of the linkage-unit-bound glycopeptides yielded disaccharide-linked glycopeptides, which gave the disaccharide and phosphorylated glycopeptides on mild acid treatment. Thus, it is concluded that the ribitol teichoic acid chains in the cell walls of the strains of S. aureus and B. subtilis are linked to peptidoglycan through linkage units, (glycerol phosphate)3-N-acetylmannosaminyl beta(1----4)N-acetylglucosamine and (glycerol phosphate)2-N-acetylmannosaminyl beta(1----4)N-acetylglucosamine, respectively.     

The function of teichoic acids in cation control in bacterial membranes

1. The effects of teichoic acids on the Mg(2+)-requirement of some membrane-bound enzymes in cell preparations from Bacillus licheniformis A.T.C.C. 9945 were examined. 2. The biosynthesis of the wall polymers poly(glycerol phosphate glucose) and poly(glycerol phosphate) by membrane-bound enzymes is strongly dependent on Mg(2+), showing maximum activity at 10-15mm-Mg(2+). 3. When the membrane is in close contact with the cell wall and membrane teichoic acid, the enzyme systems are insensitive to added Mg(2+). The membrane appears to interact preferentially with the constant concentration of Mg(2+) that is bound to the phosphate groups of teichoic acid in the wall and on the membrane. When the wall is removed by the action of lysozyme the enzymes again become dependent on an external supply of Mg(2+). 4. A membrane preparation that retained its membrane teichoic acid was still dependent on Mg(2+) in solution, but the dependence was damped so that the enzymes exhibited near-maximal activity over a much greater range of concentrations of added Mg(2+); this preparation contained Mg(2+) bound to the membrane teichoic acid. The behaviour of this preparation could be reproduced by binding membrane teichoic acid to membranes in the presence of Mg(2+). Addition of membrane teichoic acid to reaction mixtures also had a damping effect on the Mg(2+) requirement of the enzymes, since the added polymer interacted rapidly with the membrane. 5. Other phosphate polymers behaved in a qualitatively similar way to membrane teichoic acid on addition to reaction mixtures. 6. It is concluded that in whole cells the ordered array of anionic wall and membrane teichoic acids provides a constant reservoir of bound bivalent cations with which the membrane preferentially interacts. The membrane teichoic acid is the component of the system which mediates the interaction of bound cations with the membrane. The anionic polymers in the wall scavenge cations from the medium and maintain a constant environment for the membrane teichoic acid. Thus a function of wall and membrane teichoic acids is to maintain the correct ionic environment for cation-dependent membrane systems.