Poly(glucosylglycerol phosphate) teichoic acid in the walls of Bacillus stearothermophilus B65.
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ABSTRACT:
SUBMITTER: Anderson AJ
PROVIDER: S-EPMC1172332 | biostudies-other | 1975 Oct
REPOSITORIES: biostudies-other
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Project description:1. Particulate enzyme preparations obtained from Bacillus stearothermophilus B65 by digestion with lysozyme were shown to catalyse teichoic acid synthesis. With CDP-glycerol as sole substrate the preparations synthesized 1,3-poly(glycerol phosphate). It was characterized by alkaline hydrolysis, by glucosylation to the alkali-stable 2-glucosyl-1,3-poly(glycerol phosphate) with excess of UDP-glucose and a Bacillus subtilis Marburg enzyme system, by degradation of this latter product with 60%HF and periodate oxidation of the resulting glucosylglycerol. The specificity of the B. subtilis system previously reported (Glaser & Burger, 1964), was confirmed in the present work. 2. Pulse-labelling experiments, followed by periodate oxidation of the product and isolation of formaldehyde from the glycerol terminus of the polymer, showed that the B. stearothermophilus enzyme system transferred glycerol phosphate units to the glycerol end of the chain. The transfer reaction was irreversible. It was not determined if these poly(glycerol phosphate) chains were synthesized de novo, but it was shown that the newly synthesized oligomers were bound to much larger molecules. 3. When the B. stearothermophilus enzyme system was supplied with both CDP-glycerol and UDP-glucose, 1-glucosyl-2,3-poly(glycerol phosphate) was synthesized in addition to the 1,3-isomer. The former polymer was characterized by acid and alkaline hydrolysis, degradation with HF and periodate oxidation of the resulting glucosylglycerol, and periodate oxidation of the intact polymer followed by mild acid hydrolysis. This latter procedure removed the glucose substituents without disrupting the poly(glycerol phosphate) chain. 4. The poly(glycerol phosphate) isomers were distinguished by glucosylation with the B. subtilis enzymes and alkaline hydrolysis, the 2,3-isomer remaining alkali-labile. The proportion of 2,3-poly(glycerol phosphate) in the product increased with increasing amounts of UDP-glucose in the incubation mixture, but the total glycerol phosphate incorporated into products remained constant. It is suggested that the synthetic pathways of the two poly(glycerol phosphate) species may share a rate-limiting step.
| S-EPMC1166238 | biostudies-other
Project description:1. A glycerol teichoic acid has been extracted from cell walls of Bacillus stearothermophilus B65 and its structure examined. 2. Trichloroacetic acid-extractable teichoic acid accounted for 68% of the total cell-wall phosphorus and residual material could be hydrolysed to a mixture of products including those characteristic of glycerol teichoic acids. 3. The extracted polymer is composed of glycerol, phosphoric acid, d-glucose and d-alanine. 4. Hydrolysis of the polymer with alkali gave glycerol, 1-O-alpha-d-glucopyranosylglycerol and its monophosphates, glycerol mono- and di-phosphate, as well as traces of a glucosyldiglycerol triphosphate and a glucosylglycerol diphosphate. 5. The teichoic acid is a polymer of 18 or 19 glycerol phosphate units having alpha-d-glucopyranosyl residues attached to position 1 of 14 or 15 of the glycerol residues. 6. The glycerol residues are joined by phosphodiester linkages involving positions 2 and 3 in each glycerol. 7. d-Alanine is in ester linkage to the hydroxyl group at position 6 of approximately half of the glucose residues. 8. One in every 13 or 12 polymer molecules bears a phosphomonoester group on position 3 of a glucose residue, the possible significance of which in linkage of the polymer to other wall constituents is discussed.
| S-EPMC1264963 | biostudies-other
Project description:When grown in a chemostat under various nutritional conditions, cells of Bacillus subtilis W23 produce walls containing teichoic acid or teichuronic acid. The binding of Mg2+ to these walls and to the isolated anionic polymers in solution was measured by equilibrium dialysis. In solution the ribitol teichoic acid bound Mg2+ in the molar ratio Mg2+/P=1:1 with an apparent association constant (Kassoc.) of 0.61 X 10(3)M-1, and the teichuronic acid bound Mg2+ in the ratio Mg2+/CO2-=1.1, Kassoc.=0.3 X 10(3)M-1. Cell walls containing teichuronic acid exhibited closely similar binding properties to those containing teichoic acid; in both cases Mg2+ was bound in the ratio Mg/P or Mg/CO2- of 0.5:1 and with a greater affinity than displayed by the isolated polymers in solution. It was concluded that Mg2+ ions are bound bivalently between anionic centres in the walls and that the incorporation of teichoic acid or teichuronic acid into the walls gives rise to similar ion-binding and charged properties. The results are discussed in relation to the possible functions of anionic polymers in cell walls.
| S-EPMC1164609 | biostudies-other
Project description:Wall teichoic acids are anionic phosphate-rich polymers that are part of the complex meshwork of carbohydrates that make up the gram-positive cell wall. These polymers are essential to the proper rod-shaped morphology of Bacillus subtilis and have been shown to be an important virulence determinant in the nosocomial opportunistic pathogen Staphylococcus aureus. Together, sequence-based studies, in vitro experiments with biosynthetic proteins, and analyses of the chemical structure of wall teichoic acid have begun to shed considerable light on our understanding of the biogenesis of this polymer. Nevertheless, some paradoxes remain unresolved. One of these involves a putative duplication of genes linked to CDP-ribitol synthesis (tarI'J' and tarIJ) as well as poly(ribitol phosphate) polymerization (tarK and tarL) in S. aureus. In the work reported here, we performed careful studies of the dispensability of each gene and discovered a functional redundancy in the duplicated gene clusters. We were able to create mutants in either of the putative ribitol phosphate polymerases (encoded by tarK and tarL) without affecting teichoic acid levels in the S. aureus cell wall. Although genes linked to CDP-ribitol synthesis are also duplicated, a null mutant in only one of these (tarI'J') could be obtained, while tarIJ remained essential. Suppression analysis of the tarIJ null mutant indicated that the mechanism of dysfunction in tarI'J' is due to poor translation of the TarJ' enzyme, which catalyzes the rate-limiting step in CDP-ribitol formation. This work provides new insights into understanding the complex synthetic steps of the ribitol phosphate polymer in S. aureus and has implications on specifically targeting enzymes involved in polymer biosynthesis for antimicrobial design.
| S-EPMC2519377 | biostudies-literature
Project description:1. The teichoic acid from walls of Staphylococcus lactis I3 was isolated by extraction with trichloroacetic acid and shown to contain glycerol, N-acetylglucosamine, phosphate and d-alanine in the molecular proportions 1:1:2:1. The alanine is attached to the polymer through ester linkages. 2. Hydrolysis with acid gave alanine, glucosamine and glycerol diphosphates. Under mild acid conditions a repeating unit was produced; this consists of glycerol diphosphate joined through a phosphodiester group to N-acetylglucosamine. 3. Hydrolysis with alkali gave glycerol diphosphates, saccharinic acid and two phosphodiesters containing glucosamine whose structures were elucidated; these both contain glucosamine 1-phosphate, and N-acetylglucosamine 1-phosphate was isolated by a degradative procedure. 4. The unusual properties of the teichoic acid are explained by a polymeric structure in which N-acetylglucosamine 1-phosphate is attached through its phosphate to glycerol phosphate. 5. The biosynthetic implications of this structure are discussed.
| S-EPMC1187384 | biostudies-other
Project description:1. Walls of Staphylococcus epidermidis I2 contain 30% (w/w) of a glycerol teichoic acid containing phosphate, d-alanine and d-glucose in the molecular proportions 1:0.25:0.50. 2. The teichoic acid was isolated by extraction with trichloroacetic acid and with dilute aqueous NN-dimethylhydrazine at pH7, and was shown to be a (1-->3)-linked poly(glycerol phosphate) containing beta-d-glucopyranosyl and d-alanyl ester substituents. 3. 2-O-beta-d-Glucopyranosylglycerol was isolated and characterized as its crystalline hexa-O-acetate. 4. Unlike that of certain other bacteria, the peptidoglycan component of the wall is not solubilized by NN-dimethylhydrazine. 5. The membrane teichoic acid is also a (1-->3)-linked poly(glycerol phosphate) but contains a smaller proportion of glucosyl substituents.
| S-EPMC1187389 | biostudies-other
Project description:A lambda-EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting beta-xylosidase activity were isolated. The clones overlap each other and together represent a 23.5-kb chromosomal segment. The segment contains a cluster of xylan utilization genes, which are organized in at least three transcriptional units. These include the gene for the extracellular xylanase, xylanase T-6; part of an operon coding for an intracellular xylanase and a beta-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of alpha-D-glucuronic acid (GlcUA). The first four genes in the potential GlcUA operon (orf1, -2, -3, and -4) code for a putative sugar transport system with characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic acid [2-O-alpha-(4-O-methyl-alpha-D-glucuronosyl)-xylotriose] (MeGlcUAXyl3). The following two genes code for an intracellular alpha-glucuronidase (aguA) and a beta-xylosidase (xynB). Five more genes (kdgK, kdgA, uxaC, uxuA, and uxuB) encode proteins that are homologous to enzymes involved in galacturonate and glucuronate catabolism. The gene cluster also includes a potential regulatory gene, uxuR, the product of which resembles repressors of the GntR family. The apparent transcriptional start point of the cluster was determined by primer extension analysis and is located 349 bp from the initial ATG codon. The potential operator site is a perfect 12-bp inverted repeat located downstream from the promoter between nucleotides +170 and +181. Gel retardation assays indicated that UxuR binds specifically to this sequence and that this binding is efficiently prevented in vitro by MeGlcUAXyl3, the most likely molecular inducer.
| S-EPMC93846 | biostudies-literature
Project description:The thick wall of gram-positive bacteria is a polymer meshwork composed predominantly of peptidoglycan (PG) and teichoic acids, both of which have a critical function in maintenance of the structural integrity and the shape of the cell. In Bacillus subtilis 168 the major teichoic acid is covalently coupled to PG and is known as wall teichoic acid (WTA). Recently, PG insertion/degradation over the lateral wall has been shown to occur in a helical pattern. However, the spatial organization of WTA assembly and its relationship with cell shape and PG assembly are largely unknown. We have characterized the localization of green fluorescent protein fusions to proteins involved in several steps of WTA synthesis in B. subtilis: TagB, -F, -G, -H, and -O. All of these localized similarly to the inner side of the cytoplasmic membrane, in a pattern strikingly similar to that displayed by probes of nascent PG. Helix-like localization patterns are often attributable to the morphogenic cytoskeletal proteins of the MreB family. However, localization of the Tag proteins did not appear to be substantially affected by single disruption of any of the three MreB homologues of B. subtilis. Bacterial and yeast two-hybrid experiments revealed a complex network of interactions involving TagA, -B, -E, -F, -G, -H, and -O and the cell shape determinants MreC and MreD (encoded by the mreBCD operon and presumably involved in the spatial organization of PG synthesis). Taken together, our results suggest that, in B. subtilis at least, the synthesis and export of WTA precursors are mediated by a large multienzyme complex that may be associated with the PG-synthesizing machinery.
| S-EPMC2258661 | biostudies-literature
Project description:The reaction of iodine with glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus was investigated. The active-site thiol group of the cysteine residue homologous with cysteine-149 in the pig muscle enzyme was protected by reaction with tetrathionate. The apoenzyme was readily inhibited by KI3 solution at pH8, but the coenzyme, NAD+, protected the enzyme against inhibition and decreased the extent of iodination. At pH 9.5, ready inhibition of both apo- and holo-enzyme was observed. Tryptic peptides containing residues iodinated at pH 8 were isolated and characterized. One of the most reactive residues in both holo- and apo-enzymes was a tyrosine homologous with tyrosine-46 in the pig muscle enzyme, and this residue was iodinated without loss of enzymic activity. Other reactive tyrosine residues in the apoenzyme were in positions homologous with residues 178, 273, 283 and 311 in the pig muscle enzyme, but they were not readily iodinated in the holoenzyme. Histidine residues in both holo- and apo-enzymes were iodinated at pH 8 in sequence positions homologous with residues 50, 162 and 190 in the pig muscle enzyme. The inhibition of the enzyme was not correlated with the iodination of a particular residue. The results are discussed in relation to a three-dimensional model based on the structure of the lobster muscle enzyme and demonstrate that conformational changes affecting the reactivity of several tyrosine residues most probably occur on binding of the coenzyme.
| S-EPMC1172874 | biostudies-other
Project description:1. The cell walls of Bacillus stearothermophilus B65 contain glucosamine, muramic acid, alanine, alphain-diaminopimelic acid (Dap), glutamic acid, aspartic acid, glycine, and serine in the molecular proportions 0.60:0.64:2.30:0.85:1.00:0.11:0.13:0.31. 2. Both d- and l-alanine are present, but glutamic acid and diaminopimelic acid are present only as the d- and meso-isomers respectively. 3. The peptide fragments Ala-Dap, Dap-Ala, and Dap-Ala-Dap have been isolated from a partial acid hydrolysate of the cell walls. 4. The major products of autolysis of the cell wall were d-alanine, a peptide mixture, peptidoglycan material and a peptidoglycan-teichoic acid complex. 5. Separation of the peptide mixture into ten major peptides was achieved by DEAE-Sephadex and paper chromatography, and paper electrophoresis. 6. The structures of these peptides have been determined and they fall into four groups, the individual members of each group differing only in number or position of carboxamide substituents. 7. The structures are I, a tripeptide l-Ala-d-Glu-meso-Dap; II, a pentapeptide made up by the tripeptide (I) linked through the in-amino group of its diaminopimelic acid residue to the carboxyterminal of the dipeptide meso-Dap-d-Ala; III, a heptapeptide made up by a similar linkage between the tripeptide (I) and the tetrapeptide l-Ala-d-Glu-meso-Dap-d-Ala; IV, a possible undecapeptide made up by a further tetrapeptide similarly linked to the heptapeptide (III) structure. 8. The structure of the peptidoglycan and the actions of the autolytic enzymes are discussed in terms of these peptide structures.
| S-EPMC1179297 | biostudies-other