Structure and glycosylation of lipoteichoic acids in Bacillus strains.

The occurrence, structure, and glycosylation of lipoteichoic acids were studied in 15 Bacillus strains, including Bacillus cereus (4 strains), Bacillus subtilis (5 strains), Bacillus licheniformis (1 strain), Bacillus polymyxa (2 strains), and Bacillus circulans (3 strains). Whereas in the cells of B. polymyxa and B. circulans neither lipoteichoic acid nor related amphipathic polymer could be detected, the cells of other Bacillus strains were shown to contain lipoteichoic acids built up of poly(glycerol phosphate) backbone chains and hydrophobic anchors [gentiobiosyl(beta 1----1/3)diacylglycerol or monoacylglycerol]. The lipoteichoic acid chains of the B. licheniformis strain and three of the B. subtilis strains had N-acetylglucosamine side branches, but those of the B. cereus strains and the remaining two B. subtilis strains did not. The membranes of the B. licheniformis strain and the first three B. subtilis strains exhibited enzyme activities for the synthesis of beta-N-acetylglucosamine-P-polyprenol and for the transfer of N-acetylglucosamine from this glycolipid to endogenous acceptors presumed to be lipoteichoic acid precursors. In contrast, the membranes of the other strains lacked both or either of these two enzyme activities. The correlation between the occurrence of N-acetylglucosamine-linked lipoteichoic acids and the distribution of these enzymes is consistent with the previously proposed function of beta-N-acetylglucosamine-P-polyprenol as a glycosyl donor in the introduction of alpha-N-acetylglucosamine branches to lipoteichoic acid backbone chains.     

Occurrence of ribitol-containing lipoteichoic acid in Staphylococcus aureus H and its glycosylation

A ribitol-containing lipoteichoic acid was obtained from the 20,000 x g supernatant fraction of Staphylococcus aureus H by extraction with Triton X-100 followed by fractionation on Sepharose 6B and DEAE-cellulose columns. The purified lipoteichoic acid was composed of phosphate, glycerol, glucose, glucosamine, ribitol, and fatty acids in a molar ratio of 1 : 0.9 : 0.06 : 0.03 : 0.09 : 0.07. Based on the structural analysis of fragments from alkali and HF hydrolysis, the lipoteichoic acid appears to consist of three moieties, namely a ribitol phosphate oligomer, poly(glycerol phosphate) which has about 30 glycerol phosphate units, and beta-glucosyl-beta-glucosyl(1 leads to 1)diacylglycerol. N-Acetylglucosamine was linked to the ribitol residues. The lipoteichoic acid serves as an acceptor of glycosyl moieties from UDP-glucose and UDP-N-acetylglucosamine in the enzyme reaction catalyzed by the membrane preparation. The rate of enzymatic glycosylation was increased by prior treatment of the lipoteichoic acid with N-acetyl-beta-D-glucosaminidase. The glycosylation seems to occur at the ribitol residues of the lipoteichoic acid.     

Structure of the lipoteichoic acids from Bifidobacterium bifidum spp. pennsylvanicum

The lipoteichoic acids from Bifidobacterium bifidum spp. pennsylvanicum were extracted from cytoplasmic membranes or from disintegrated bacteria with aqueous phenol and purified by gel chromatography. The lipoteichoic acid preparations contained phosphate, glycerol, galactose, glucose and fatty acids in a molar ratio of 1.0:1.0:1.3:1.2:0.3. Chemical analysis and NMR studies of the native preparations and of products from various acid and alkaline hydrolysis procedures gave evidence for the structure of two lipoteichoic acids. The lipid anchor appeared to be 3-O-(6'-(sn-glycero-1-phosphoryl)diacyl-beta-D-galactofuranosyl)-sn-1, 2-diacylglycerol. The polar part showed two structural features not previously described for lipoteichoic acids. A 1,2-(instead of the usual 1,3-) phosphodiester-linked sn-glycerol phosphate chain is only used substituted at the terminal glycerol unit with a linear polysaccharide, containing either beta(1----5)-linked D-galactofuranosyl groups or beta(1----6)-linked D-glucopyranosyl groups.     

On the basic structure of poly(glycerophosphate) lipoteichoic acids

Poly(glycerophosphate) lipoteichoic acids from 24 Gram-positive bacteria of the genera Bacillus, Enterococcus, Lactobacillus, Lactococcus, Listeria, Staphylococcus, and the streptococcal pyogenic and oral group were analyzed. The 1,3-linked poly(glycerophosphate) structure was proved by analysis of glycerol and glycerophosphates after acid and alkaline hydrolysis. Using the molar ratios of glycolipid to phosphorus (A) and phosphomonoester to phosphorus after periodate oxidation followed by hydrazinolysis (B) or beta-elimination (C), we show that all lipoteichoic acids contain a single unbranched poly(glycerophosphate) chain and that the chain is uniformly phosphodiester-linked to C-6 of the nonreducing hexopyranosyl residue of the glycolipid moiety. On some chains minor phosphate-containing substituents were detected whose structure remains to be clarified. The lipoteichoic acids of enterococci and listeria strains were separated by hydrophobic interaction chromatography into glycolipid- and phosphatidylglycolipid-containing molecular species. The phosphatidylglycolipid moieties were structurally characterized after liberation from lipoteichoic acids with moist acetic acid. After periodate oxidation of lipoteichoic acids beta-elimination released both phosphatidic acid and the poly(glycerophosphate) chain. This indicates together with the sequence analysis of the released phosphatidylglycolipid that the phosphatidyl residue is located at C-6 of the reducing hexosyl residue of the glycolipid moiety and the poly(glycerophosphate) chain at C-6 of the nonreducing one. Together with earlier observations these results complete the evidence for the structural and possibly biosynthetic relationship between lipoteichoic acids and glycerophosphoglycolipids.     

N-acetylmannosaminyl(1----4)N-acetylglucosamine, a linkage unit between glycerol teichoic acid and peptidoglycan in cell walls of several Bacillus strains.

The structure of teichoic acid-glycopeptide complexes isolated from lysozyme digests of cell walls of Bacillus subtilis (four strains) and Bacillus licheniformis (one strain) was studied to obtain information on the structural relationship between glycerol teichoic acids and their linkage saccharides. Each preparation of the complexes contained equimolar amounts of muramic acid 6-phosphate and mannosamine in addition to glycopeptide components and glycerol teichoic acid components characteristic of the strain. Upon treatment with 47% hydrogen fluoride, these preparations gave, in common, a hexosamine-containing disaccharide, which was identified as N- acetylmannosaminyl (1----4) N-acetylglucosamine, along with large amounts of glycosylglycerols presumed to be the dephosphorylated repeating units of teichoic acid chains. The glycosylglycerol obtained from each bacterial strain was identified as follows: B. subtilis AHU 1392, glucosyl alpha (1----2)glycerol; B. subtilis AHU 1235, glucosyl beta(1----2) glycerol; B. subtilis AHU 1035 and AHU 1037, glucosyl alpha (1----6)galactosyl alpha (1----1 or 3)glycerol; B. licheniformis AHU 1371, galactosyl alpha (1----2)glycerol. By means of Smith degradation, the galactose residues in the teichoic acid-glycopeptide complexes from B. subtilis AHU 1035 and AHU 1037 and B. licheniformis AHU 1371 were shown to be involved in the backbone chains of the teichoic acid moieties. Thus, the glycerol teichoic acids in the cell walls of five bacterial strains seem to be joined to peptidoglycan through a common linkage disaccharide, N- acetylmannosaminyl (1----4)N-acetylglucosamine, irrespective of the structural diversity in the glycosidic branches and backbone chains.     

The function of beta-N-acetyl-D-glucosaminyl monophosphorylundecaprenol in biosynthesis of lipoteichoic acids in a group of Bacillus strains

Membrane preparations, obtained from Bacillus strains which have N-acetylglucosamine-linked lipoteichoic acids in their membranes, were shown to catalyze the transfer of N-[14C]acetylglucosamine (GlcNAc) from beta-[14C]GlcNAc-P-undecaprenol to endogenous polymer. In this reaction, alpha-GlcNAc-P-undecaprenol or alpha-GlcNAc-PP-undecaprenol could not substitute for beta-GlcNAc-P-undecaprenol as the N-acetylglucosamine donor. This enzyme was most active at pH 6.0 and in the presence of 40 mM MgCl2. The apparent Km for beta-GlcNAc-P-undecaprenol was 2 microM. The radioactive polymer products, solubilized by hot phenol treatment, coincided with lipoteichoic acids in chromatographic behavior. Hydrogen fluoride treatment of the polymer products gave a major fragment identical with GlcNAc(alpha 1----2)glycerol, which corresponded to the dephosphorylated repeating units of the lipoteichoic acids in the examined strains. Thus it is concluded that beta-GlcNAc-P-undecaprenol serves as the donor of N-acetylglucosamine in the biosynthesis of lipoteichoic acids in a group of Bacillus strains.     

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 galactosyl phosphorylpolyprenol in biosynthesis of lipoteichoic acid in Bacillus coagulans

Incubation of UDP-[14C]galactose with membranes of Bacillus coagulans led to the formation of a radioactive glycolipid, which was tentatively characterized as beta-galactosyl phosphorylpolyprenol (Gal-P-prenol) on the basis of its chromatographic behavior and data from structural analysis of its sugar 1-phosphate moiety. The sugar moiety of [14C]Gal-P-prenol was shown to be incorporated into a membrane-bound polymer, which coincided with the diacyl form of lipoteichoic acid in its chromatographic behavior on columns of Sephacryl S-300, DEAE-Sephacel and octyl-Sepharose. Hydrogen fluoride hydrolysis of the polymer afforded an alpha-galactoside identical with Gal(alpha 1----2)Gro obtained from lipoteichoic acids. The incorporation of galactose residues from [14C]Gal-P-prenol into the polymer was greatly enhanced by exogenous lipoteichoic acids, especially of the diacyl and monoacyl forms. The optimal pH and metal concentration for the Gal-P-prenol formation, respectively, were found to be 8.4 and 10 mM (MgCl2), whereas those for the transfer of galactose from this lipid intermediate to polymer were 4.5 and 16 mM (CaCl2). The above results lead to the conclusion that Gal-P-prenol serves as the direct galactosyl donor in the synthesis of lipoteichoic acids in B. coagulans.     

A common linkage saccharide unit between teichoic acids and peptidoglycan in cell walls of Bacillus coagulans

Teichoic acid-glycopeptide complexes were isolated from lysozyme digests of the cell walls of Bacillus coagulans AHU 1631, AHU 1634, and AHU 1638, and the structure of the teichoic acid moieties and their linkage regions was studied. On treatment with hydrogen fluoride, each of the complexes gave a hexosamine-containing disaccharide, which was identified to be glucosyl(beta 1----4)N-acetylglucosamine, in addition to dephosphorylated repeating units of the teichoic acids, namely, galactosyl(alpha 1----2)glycerol and either galactosyl(alpha 1----2)[glucosyl(alpha 1----1/3)]glycerol (AHU 1638) or galactosyl(alpha 1----2)[glucosyl(beta 1----1/3)]glycerol (AHU 1631 and AHU 1634). From the results of Smith degradation, methylation analysis, and partial acid hydrolysis, the teichoic acids from these strains seem to have the same backbone chains composed of galactosyl(alpha 1----2)glycerol phosphate units joined by phosphodiester bonds at C-6 of the galactose residues. The presence of the disaccharide, glucosyl(beta 1----4)N-acetylglucosamine, in the linkage regions between teichoic acids and peptidoglycan was confirmed by the isolation of a disaccharide-linked glycopeptide fragment from each complex after treatment with mild alkali and of a teichoic acid-linked saccharide from each cell wall preparation after treatment with mild acid. Thus, it is concluded that despite structural differences in the glycosidic branches, the teichoic acids in the cell walls of the three strains are linked to peptidoglycan through a common linkage saccharide, glucosyl (beta 1----4) N-acetylglucosamine.     

Products of phosphatidylglycerol turnover in two Bacillus strains with and without lipoteichoic acid in the cells

In order to understand the phosphatidylglycerol turnover mechanism, especially the differential turnover of diacylated and unacylated glycerol moieties of the lipid, products of phosphatidylglycerol metabolism were surveyed in vivo in Bacillus subtilis W23 and an alkalophile, Bacillus sp. strain A007. When cells of B. subtilis W23 labeled with radioactive glycerol were chased, lipoteichoic acid accumulated 90% of the radioactivity lost from the unacylated glycerol moiety of phosphatidylglycerol. Also, lipids other that phosphatidylglycerol, except diacylglycerol, and glycerol and glycerophosphate incorporated much less radioactivity. The [32P]phosphoryl group was also transferred from phosphatidylglycerol to lipoteichoic acid almost quantitatively in B. subtilis W23. A unique metabolism of phosphatidylglycerol was found in Bacillus sp. strain A007 which lacked phosphoglycolipid and lipoteichoic acid, that is, the turnover of phosphatidylglycerol of this organism was less extensive compared with that of B. subtilis W23, and both glycerol moieties of the lipid were metabolized at an identical rate. These results suggested that the major reaction involved in the turnover of phosphatidylglycerol was the transfer of glycerophosphate residue to lipoteichoic acid in a bacterium which possessed lipoteichoic acid and that several minor reactions also were involved in phosphatidylglycerol turnover.     

Lipoteichoic acid from Bacillus licheniformis 6346 MH-1. Comparative studies on the lipid portion of the lipoteichoic acid and the membrane glycolipid.

A lipoteichoic acid and a membrane glycolipid were isolated from Bacillus licheniformis 6346 MH-1. The fatty acid composition of the two preparations were similar. Most of the fatty acids were of the branched chain type. The glycolipid was shown to be a diacyl derivative of O-beta-D-glucopyranosyl-(1 leads to 6)-O-beta-D-glucopyranosyl-(1 leads to 3)-glycerol. The lipoteichoic acid contained lipid, polyglycerol phosphate, and glucosamine. The lipid was released by treatment with hydrofluoric acid and by hydrolysis in dilute acid and was shown to have a structure identical with that of the membrane glycolipid.     

Studies on glucosyltransferase and endogenous glucosyl acceptor in Bacillus cereus AHU 1030 membranes

A glucosyltransferase, extracted from the membranes of Bacillus cereus AHU 1030 with Tris-HCl buffer containing 0.1% Triton X-100 at pH 9.5, was separated from an endogenous glucosyl acceptor by chromatography on DEAE-Sepharose CL-6B subsequent to chromatography on Sepharose 6B. Structural analysis data showed that the glucosyl acceptor was a glycerol phosphate polymer linked to beta-gentiobiosyl diglyceride. The enzyme catalyzed the transfer of glucosyl residues from UDP-glucose to C-2 of the glycerol residues of repeating units of the acceptor. On the other hand, a lipoteichoic acid which contained 0.3 D-alanine residue per phosphorus was isolated from the cells by phenol treatment at pH 4.6. Except for the presence of D-alanine, this lipoteichoic acid had the same structure as the glucosyl acceptor. The rate of glucosylation observed with the D-alanine-containing lipoteichoic acid as the substrate was less than 40% of that observed with the D-alanine-free lipoteichoic acid, indicating that the ester-linked D-alanine in the lipoteichoic acid interferes with the action of the glucosyltransferase. The enzyme also catalyzed glucosylation of poly(glycerol phosphate) which was synthesized in the reaction of a separate enzyme fraction with CDP-glycerol. Thus, it is likely that the glucosyltransferase functions in the synthesis of cell wall teichoic acid.     

Function of alpha-D-glucosyl monophosphorylpolyprenol in biosynthesis of cell wall teichoic acids in Bacillus coagulans.

D-[alpha-14C]]glucosyl phosphorylpolyprenol ([ 14C]Glc-P-prenol) was formed from UDP-D-[14C]glucose in each of the membrane systems obtained from Bacillus coagulans AHU 1631 and AHU 1634 and two Bacillus megaterium strains. Membranes of these B. coagulans strains, which possess beta-D-glucosyl branches on the repeating units in their major cell wall teichoic acids, were shown to catalyze the transfer of the glucose residue from [14C]Glc-P-prenol to endogenous polymer. On the other hand, membranes of B. coagulans AHU 1366, which has no glucose substituents in the cell wall teichoic acid, exhibited neither [14C]Glc-P-prenol synthetase activity nor the activity of transferring glucose from [14C]Glc-P-prenol to endogenous acceptor. The enzyme which catalyzes the polymer glycosylation in the former two B. coagulans strains was most active at pH 5.5 and in the presence of the Mg2+ ion. The apparent Km for [14C]Glc-P-prenol was 0.6 microM. Hydrogen fluoride hydrolysis of the [14C]glucose-linked polymer product yielded a major fragment identical to D-galactosyl-alpha(1----2)(D-glucosyl-beta(1----1/3)) glycerol, the dephosphorylated repeating unit in the major cell wall teichoic acids of these B. coagulans strains. This result, together with the behavior of the radioactive polymer in chromatography on Sepharose CL-6B, DEAE-Sephacel, and Octyl-Sepharose CL-4B, led to the conclusion that [14C]Glc-P-prenol serves as an intermediate in the formation of beta-D-glucosyl branches on the polymer chains of cell wall teichoic acids in B. coagulans.     

Structural studies on lipoteichoic acids from four Listeria strains.

The lipoteichoic acids were isolated from phenol extracts of four Listeria strains representing serotypes 4a, 4b, 6a, and 6 to compare the differences in structure of amphiphilic polysaccharides from various serotypes of Listeria spp. The lipoteichoic acids from the four strains examined had the same structure in both hydrophilic chains and lipid portions. On the basis of the results of nuclear magnetic resonance spectroscopy and Smith degradation, the hydrophilic chains were shown to be 1,3-linked poly(glycerol phosphate) in which some of the glycerol residues had alpha-galactosyl substituents. The lipid portions were released by treatment with 46% hydrogen fluoride or 98% acetic acid. They were determined to be 3(1)-(2'-O-alpha-D-galactopyranosyl-alpha-D-glucopyranosyl)-1(3), 2-diacylglycerol and 3(1)-[6'-phosphatidyl-2'-O-(alpha-D-galactopyranosyl)-alpha- D-glucopyranosyl]-1(3),2-diacylglycerol. The degrees of glycosyl substitution and proportions of the two lipids varied to some extent among these four strains.     

The primary structure of teichuronic acid in Bacillus subtilis AHU 1031

Structural studies were carried out on the acidic polysaccharide fraction obtained from lysozyme digest of the cell walls of Bacillus subtilis AHU 1031. The polysaccharide fraction contained N- acetylmannosaminuronic acid ( ManNAcA ), N-acetylglucosamine (GlcNAc), glucose, glycerol and phosphorus in a molar ratio of 2:2:4:1:1, together with glycopeptide components. The results of analyses involving Smith degradation, chromium trioxide oxidation, methylation and proton magnetic resonance spectroscopy led to the conclusion that the backbone chain of the polysaccharide has the repeating unit----6)Glc(alpha 1----3/4) ManNAcA (beta 1----4)GlcNAc(beta 1----. About 50% of the N-acetylglucosamine residues in the backbone chain seem to be substituted at C-3 by the glycosidic branches, glycerol phospho-6-glucose, while the other half seem to be substituted by glucose.     

Structure and functions of linkage unit intermediates in the biosynthesis of ribitol teichoic acids in Staphylococcus aureus H and Bacillus subtilis W23

The stepwise formation and characterization of linkage unit intermediates and their functions in ribitol teichoic acid biosynthesis were studied with membranes obtained from Staphylococcus aureus H and Bacillus subtilis W23. The formation of labeled polymer from CDP-[14C]ribitol and CDP-glycerol in each membrane system was markedly stimulated by the addition of N-acetylmannosaminyl(beta 1----4)N-acetylglucosamine (ManNAc-GlcNAc) linked to pyrophosphorylyisoprenol. Whereas incubation of S. aureus membranes with CDP-glycerol and ManNAc-[14C]GlcNAc-PP-prenol led to synthesis of (glycerol phosphate) 1-3-ManNAc-[14C]GlcNAc-PP-prenol, incubation of B. subtilis membranes with the same substrates yielded (glycerol phosphate)1-2-ManNAc-[14C]GlcNAc-PP-prenol. In S. aureus membranes, (glycerol phosphate)2-ManNAc-[14C]GlcNAc-PP-prenol as well as (glycerol phosphate)3-ManNAc-[14C]GlcNAc-PP-prenol served as an acceptor for ribitol phosphate units, but (glycerol phosphate)-ManNAc-[14C]GlcNAc-PP-prenol did not. In B. subtilis W23 membranes, (glycerol phosphate)-ManNAc-[14C]GlcNAc-PP-prenol served as a better acceptor for ribitol phosphate units than (glycerol phosphate)2-ManNAc-[14C]GlcNAc-PP-prenol. In this membrane system (ribitol phosphate)-(glycerol phosphate)-ManNAc-[14C]GlcNAc-PP-prenol was formed from ManNAc-[14C]GlcNAc-PP-prenol, CDP-glycerol and CDP-ribitol. The results indicate that (glycerol phosphate)1-3-ManNAc-GlcNAc-PP-prenol and (glycerol phosphate)1-2-ManNac-GlcNAc-PP-prenol are involved in the pathway for the synthesis of wall ribitol teichoic acids in S. aureus H and B. subtilis W23 respectively.     

Biosynthesis of linkage units for teichoic acids in gram-positive bacteria: distribution of related enzymes and their specificities for UDP-sugars and lipid-linked intermediates.

The distribution and substrate specificities of enzymes involved in the formation of linkage units which contain N-acetylglucosamine (GlcNAc) and N-acetylmannosamine (ManNAc) or glucose and join teichoic acid chains to peptidoglycan were studied among membrane systems obtained from the following two groups of gram-positive bacteria: group A, including Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Staphylococcus aureus, and Lactobacillus plantarum; group B, Bacillus coagulans. All the membrane preparations tested catalyzed the synthesis of N-acetylglucosaminyl pyrophosphorylpolyprenol (GlcNAc-PP-polyprenol). The enzymes transferring glycosyl residues to GlcNAc-PP-polyprenol were specific to either UDP-ManNAc (group A strains) or UDP-glucose (group B strains). In the synthesis of the disaccharide-bound lipids, GlcNAc-PP-dolichol could substitute for GlcNAc-PP-undecaprenol. ManNAc-GlcNAc-PP-undecaprenol, ManNAc-GlcNAc-PP-dolichol, Glc-GlcNAc-PP-undecaprenol, Glc-GlcNAc-PP-dolichol, and GlcNAc-GlcNAc-PP-undecaprenol were more or less efficiently converted to glycerol phosphate-containing lipid intermediates and polymers in the membrane systems of B. subtilis W23 and B. coagulans AHU 1366. However, GlcNAc-GlcNAc-PP-dolichol could not serve as an intermediate in either of these membrane systems. Further studies on the exchangeability of ManNAc-GlcNAc-PP-undecaprenol and Glc-GlcNAc-PP-undecaprenol revealed that in the membrane systems of S. aureus strains and other B. coagulans strains both disaccharide-inked lipids served almost equally as intermediates in the synthesis of polymers. In the membrane systems of other B. subtilis strains as well as B. licheniformis and B. pumilus strains, however, the replacement of ManNAc-GlcNAc-PP-undecaprenol by Glc-GlcNAc-PP-undecaprenol led to a great accumulation of (glycerol phosphate)-Glc-GlcNAc-PP-undecaprenol accompanied by a decrease in the formation of polymers.     

Structural studies on N-acetylmannosaminuronic acid-containing and glucuronic acid-containing teichuronic acids in the cell wall of Bacillus megaterium AHU 1375

Structural studies were carried out on two kinds of teichuronic acid-glycopeptide complexes (designated as TU-GP-I and TU-GP-II) isolated from lysozyme digest of N-acetylated cell walls of Bacillus megaterium AHU 1375 by ion-exchange chromatography and gel chromatography. TU-GP-I, accounting for about 25% of the cell walls, contained N-acetylmannosaminuronic acid, N-acetylglucosamine, glucose, galactose, glycerol, and phosphorus in an approximate molar ratio of 1:1:2:1:0.5:0.5, together with small amounts of glycopeptide components. TU-GP-II, accounting for about 9% of the cell walls, contained glucuronic acid, glucose, and fucose in a molar ratio of about 2:1.5:1, together with small amounts of glycopeptide components. The results of analyses involving Smith degradation, chromium oxidation, methylation, acetolysis, and H-NMR measurement led to the conclusion that the polysaccharide chain of TU-GP-I comprised repeating units,----6) Glc(alpha 1----3)-ManNAcUA(beta 1----4)[Gal(alpha 1----3)][Glc(beta 1----6)]GlcNAc(beta 1----. About half of the repeating units were substituted by glycerophosphoryl residues at C-6 of the beta-glucosyl residues linked to the N-acetylglucosamine residues. By means of a similar procedure, the polysaccharide chain of TU-GP-II was shown to comprise repeating units,----4)GlcUA(alpha 1----3)GlcUA(alpha 1----3)Glc(alpha 1----3)Fuc(alpha 1----, of which about half were substituted by alpha-glucosyl residues at C-3 of the 4-substituted glucuronosyl residues.     

Teichoic acids and lipids associated with the membrane of a Bacillus licheniformis mutant and the membrane lipids of the parental strain.

Bacillus licheniformis 6346 MH-1 and a phosphoglucomutase-deficient poorly lytic mutant, B. licheniformis 6346 MH-5, both contain cardiolipin, phosphatidyl ethanolamine, and phosphatidyl glycerol but are devoid of phosphoglycolipids. Gentiobiosyl diglyceride is present in the parent organism but glycolipids are absent from the mutant. Lipoteichoic acid was extracted from the whole cells of MH-5 with hot aqueous phenol and contained fatty acids, glucosamine, and 1,3-polyglycerol phosphate. The fatty acids were predominantly of the branched-chain type and were esterified to hydroxyl groups of a terminal glycerol residue. The polyglycerol phosphate chains contained, on average, 32 to 40 glycerol residues, some of which were substituted at the secondary hydroxyl group with alpha-N-acylglucosaminyl residues. Phenol extraction of the supernatant fluid that remained when walls were removed from preparations of disrupted cells of MH-5 yielded membrane teichoic acid, which consisted of substituted polyglycerol phosphate but was devoid of fatty acids.     

Hydrophobic interaction chromatography fractionates lipoteichoic acid according to the size of the hydrophilic chain: a comparative study with anion-exchange and affinity chromatography for suitability in species analysis.

Hydrophobic interaction chromatography fractionated the lipoteichoic acid of Enterococcus faecalis into species of decreasing poly(glycerophosphate) chain length and decreasing extent of substitution with alpha-kojibiosyl residues (Glcp alpha 1----2Glcp alpha 1----). The chain length varied between 14 and 33 glycerophosphate residues per lipid anchor, the extent of glycosylation between 0.18 and 0.44 mol of alpha-kojibiosyl residues per mole of phosphorus, and, accordingly, the number of alpha-kojibiosyl substituents per chain between 3 and 15. Almost identical values were obtained when the same lipoteichoic acid was chromatographed on DEAE-Sephadex and concanavalin A, which separate molecular species according to increasing number of phosphate groups and alpha-kojibiosyl residues, respectively. Species from all three columns, which were identical in chain length and glycosylation, also had similar fatty acid patterns. These results prove the suitability of all three procedures for species analysis. One advantage of hydrophobic interaction chromatography over the other two procedures lies in its broader applicability since it is not dependent on negative charges or specifically binding oligosaccharide structures. Another advantage is the capacity of hydrophobic interaction chromatography to separate molecular species differing in the number of fatty acids [W. Fischer, H.U. Koch, and R. Haas (1983) Eur. J. Biochem. 133, 523-530] and render them accessible to molecular analyses.