Biosynthesis of the wall teichoic acid in Bacillus licheniformis.
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ABSTRACT:
SUBMITTER: Hancock IC
PROVIDER: S-EPMC1178556 | biostudies-other | 1972 Mar
REPOSITORIES: biostudies-other
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Project description:1. Particulate fractions prepared from disrupted cells of Bacillus licheniformis N.C.T.C. 6346 catalyse the uptake of radioactivity from UDP-[(14)C]glucuronic acid or UDP-N[(14)C]-acetylglucosamine. Maximal uptake requires the presence of both nucleotides and Mg(2+) ions. The reaction is inhibited markedly by high concentrations of novobiocin and, to a certain extent, by vancomycin and by methicillin. 2. The radioactive product formed is resistant to Pronase and is soluble in 5% (w/v) trichloroacetic acid. It is of high molecular weight, from its behaviour on columns of Sephadex G-50 or G-200, and behaves during paper electrophoresis in n-acetic acid and chromatography on DEAE-cellulose in a manner similar to teichuronic acid. 3. Both teichuronic acid and the synthesized material are resistant to testicular hyaluronidase and to Flavobacterium heparinum heparinase. 4. The specific activity of suspensions of broken cells or of washed particulate fractions is greatest when they are prepared from exponentially growing cells. Fractions obtained from late exponential-phase or stationary-phase cells have very low activity. 5. The galactosamine content of B. licheniformis N.C.T.C. 6346 cell walls increases during the exponential phase and decreases during the stationary phase.
| S-EPMC1270174 | biostudies-other
Project description:ABSTRACT The major clonal lineages of the human pathogen Staphylococcus aureus produce cell wall-anchored anionic poly-ribitol-phosphate (RboP) wall teichoic acids (WTA) substituted with d-Alanine and N-acetyl-d-glucosamine. The phylogenetically isolated S. aureus ST395 lineage has recently been found to produce a unique poly-glycerol-phosphate (GroP) WTA glycosylated with N-acetyl-d-galactosamine (GalNAc). ST395 clones bear putative WTA biosynthesis genes on a novel genetic element probably acquired from coagulase-negative staphylococci (CoNS). We elucidated the ST395 WTA biosynthesis pathway and identified three novel WTA biosynthetic genes, including those encoding an ?-O-GalNAc transferase TagN, a nucleotide sugar epimerase TagV probably required for generation of the activated sugar donor substrate for TagN, and an unusually short GroP WTA polymerase TagF. By using a panel of mutants derived from ST395, the GalNAc residues carried by GroP WTA were found to be required for infection by the ST395-specific bacteriophage ?187 and to play a crucial role in horizontal gene transfer of S. aureus pathogenicity islands (SaPIs). Notably, ectopic expression of ST395 WTA biosynthesis genes rendered normal S. aureus susceptible to ?187 and enabled ?187-mediated SaPI transfer from ST395 to regular S. aureus. We provide evidence that exchange of WTA genes and their combination in variable, mosaic-like gene clusters have shaped the evolution of staphylococci and their capacities to undergo horizontal gene transfer events. IMPORTANCE The structural highly diverse wall teichoic acids (WTA) are cell wall-anchored glycopolymers produced by most Gram-positive bacteria. While most of the dominant Staphylococcus aureus lineages produce poly-ribitol-phosphate WTA, the recently described ST395 lineage produces a distinct poly-glycerol-phosphate WTA type resembling the WTA backbone of coagulase-negative staphylococci (CoNS). Here, we analyzed the ST395 WTA biosynthesis pathway and found new types of WTA biosynthesis genes along with an evolutionary link between ST395 and CoNS, from which the ST395 WTA genes probably originate. The elucidation of ST395 WTA biosynthesis will help to understand how Gram-positive bacteria produce highly variable WTA types and elucidate functional consequences of WTA variation.
| S-EPMC3993852 | biostudies-literature
Project description:Both Gram-positive and Gram-negative bacteria contain bactoprenol-dependent biosynthetic pathways expressing non-essential cell surface polysaccharides that function as virulence factors. Although these polymers are not required for bacterial viability in vitro, genes in many of the biosynthetic pathways are conditionally essential: they cannot be deleted except in strains incapable of initiating polymer synthesis. We report a cell-based, pathway-specific strategy to screen for small molecule inhibitors of conditionally essential enzymes. The screen identifies molecules that prevent the growth of a wildtype bacterial strain but do not affect the growth of a mutant strain incapable of initiating polymer synthesis. We have applied this approach to discover inhibitors of wall teichoic acid (WTA) biosynthesis in Staphylococcus aureus. WTAs are anionic cell surface polysaccharides required for host colonization that have been suggested as targets for new antimicrobials. We have identified a small molecule, 7-chloro-N,N-diethyl-3-(phenylsulfonyl)-[1,2,3]triazolo[1,5-a]quinolin-5-amine (1835F03), that inhibits the growth of a panel of S. aureus strains (MIC = 1-3 microg mL(-1)), including clinical methicillin-resistant S. aureus (MRSA) isolates. Using a combination of biochemistry and genetics, we have identified the molecular target as TarG, the transmembrane component of the ABC transporter that exports WTAs to the cell surface. We also show that preventing the completion of WTA biosynthesis once it has been initiated triggers growth arrest. The discovery of 1835F03 validates our chemical genetics strategy for identifying inhibitors of conditionally essential enzymes, and the strategy should be applicable to many other bactoprenol-dependent biosynthetic pathways in the pursuit of novel antibacterials and probes of bacterial stress responses.
| S-EPMC2787957 | biostudies-literature
Project description:UTP-glucose-1-phosphate uridylyltransferases are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In Bacillus subtilis 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The B. subtilis UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and gtaB mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UTP-glucose-1-phosphate uridylyltransferases are present in B. subtilis. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated in vitro using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a B. subtilis gtaB mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase in vivo. When WT and mutant B. subtilis strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing yngB and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with glucose residues during oxygen-limited growth condition.
| S-EPMC7961091 | biostudies-literature
Project description:Background: Wall teichoic acid (WTA) genes are essential for production of cell walls in gram-positive bacteria and necessary for survival and variability in the cassette has led to recent antibiotic resistance acquisition in pathogenic bacteria. Methods: Using a pan-genome approach, we examined the evolutionary history of WTA genes in Bacillus subtilis ssp. subtilis. Results: Our analysis reveals an interesting pattern of evolution from the type-strain WTA gene cassette possibly resulting from horizontal acquisition from organisms with similar gene sequences. The WTA cassettes have a high level of variation which may be due to one or more independent horizontal transfer events during the evolution of Bacillus subtilis ssp. subtilis. This swapping of entire WTA cassettes and smaller regions within the WTA cassettes is an unusual feature in the evolution of the Bacillus subtilis genome and highlights the importance of horizontal transfer of gene cassettes through homologous recombination within B. subtilis or other bacterial species. Conclusions: Reduced sequence conservation of these WTA cassettes may indicate a modified function like the previously documented WTA ribitol/glycerol variation. An improved understanding of high-frequency recombination of gene cassettes has ramifications for synthetic biology and the use of B. subtilis in industry.
| S-EPMC8753576 | biostudies-literature
Project description:A small molecule (1835F03) that inhibits Staphylococcus aureus wall teichoic acid biosynthesis, a proposed antibiotic target, has been discovered. Rapid, parallel, solution-phase synthesis was employed to generate a focused library of analogs, providing detailed information about structure-activity relationships and leading to the identification of targocil, a potent antibiotic.
| S-EPMC2844852 | biostudies-literature
Project description:Methicillin resistance in Staphylococcus aureus depends on the production of mecA, which encodes penicillin-binding protein 2A (PBP2A), an acquired peptidoglycan transpeptidase with reduced susceptibility to beta-lactam antibiotics. Here, we show that preventing the expression of wall teichoic acids (WTAs) genetically or with a TarO inhibitor sensitizes MRSA strains to beta-lactams although PBP2A is still expressed. Using S. aureus microarrays and array data analysis protocols (NIAID's Pathogen Functional Genomics Resource Center) we have characterized the transcriptomes of S. aureus COL. in order to further understand the sensitization of strain COL to methicillin by tunicamycin we determined the tunicamycin and methicillin transcriptomes alone and in combination. Methicillin treatment of COL at 500 µg/mL had almost no effect on cell growth rate and, remarkably, the only gene in the transcriptome that showed a more than two-fold change in expression was lytM, which was downregulated. The tunicamycin transcriptome of COL, acquired at 0.4 µg/mL, shows modest changes compared to the untreated control both in terms of the total numbers of affected genes and in the degree of up- or downregulation. Several of the genes upregulated upon tunicamycin treatment are part of the cell wall stress stimulon.COL was grown with methicillin to an OD600 ~0.4, and challenged with tunicamycin for 2 hrs whereas the control culture contained methicillin alone. transcriptome for COL growing in the presence of both agents showed extensive changes in gene expression. , the cell wall stress stimulon, which was not induced by methicillin when tunicamycin was absent, was clearly induced in its presence and the changes were far more dramatic than observed with tunicamycin alone. ). vraS and vraR, which encode a two component signaling system dedicated to the cell wall regulon, were upregulated 3.8 and 3.7 fold, respectively. Other upregulated cell wall stress stimulon genes include pbp2, fmtA, mvaD (mevalonate diphosphate decarboxylase), crtN (dehydrosqualene desaturase) mvak1 (mevalonate kinase), recU, SAV1424 (methionine sulfoxide reductase A), prsA (peptidyl-prolyl cis/trans isomerase), tcaA (Tca protein) and cwrA. A considerable number of genes were also downregulated upon challenge of COL with the combination of tunicamycin and methicillin. These included sspB, lrgA, dltA, capL, SAS0988, sspA, pflB, and spa. Several of these genes have been found to be downregulated in previous studies of cell wall-active antibiotic challenge of S. aureus.
2010-10-20 | GSE24439 | GEO
Project description:Wall teichoic acids (WTAs) are phosphate-rich, sugar-based polymers attached to the cell walls of most Gram-positive bacteria. In Staphylococcus aureus, these anionic polymers regulate cell division, protect cells from osmotic stress, mediate host colonization, and mask enzymatically susceptible peptidoglycan bonds. Although WTAs are not required for survival in vitro, blocking the pathway at a late stage of synthesis is lethal. We recently discovered a novel antibiotic, targocil, that inhibits a late acting step in the WTA pathway. Its target is TarG, the transmembrane component of the ABC transporter (TarGH) that exports WTAs to the cell surface. Here we examine the effects of targocil on S. aureus using transmission electron microscopy (TEM) and gene expression profiling. We report that targocil treatment leads to multicellular clusters containing swollen cells displaying evidence of osmotic stress, strongly induces the cell wall stress stimulon, and reduces the expression of key virulence genes, including dltABCD and capsule genes. We conclude that WTA inhibitors that act at a late stage of the biosynthetic pathway may be useful as antibiotics, and we present evidence that they could be particularly useful in combination with beta-lactams.
2012-01-12 | GSE34441 | GEO
Project description:1. The biosynthesis of the wall teichoic acid in Staphylococcus lactis I3 was studied. Cell-free particulate enzyme preparations, probably representing fragmented membrane, were isolated and used for the synthesis of polymer. 2. By using appropriately labelled CDP-glycerol and UDP-N-acetylglucosamine it was shown that the former contributes a glycerol phosphate residue and the latter contributes an N-acetylglucosamine 1-phosphate residue to the repeating unit. 3. No polymer was synthesized unless both nucleotides were present, and no other substrates were required. 4. The properties of the enzyme system were studied. 5. Although attempts to fractionate the system failed, the biosynthesis is believed to be complex and its mechanism is considered.
| S-EPMC1187387 | 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