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Presented to the Graduate Council of the North Texas State University In 1A0 I CHEMICAL COMPOSITION OF THE PEPTIDOGLYCAN OF VITREOSCILLA STERCORARIA THESIS Presented to the Graduate Council of the North Texas State University in Partial Fulfillment of the Requirements For the Degree of MASTER OF SCIENCE By Gary Levit, B. A. Denton, Texas August, 1976 Copyright by Gary Levit 1976 To Dr. A. S. Kester ABSTRACT Levit, Gary, Chemical Composition of the Peptidoglycan of Vitreoscilla stercoraria. Master of Science (Biology) , August, 1976, 30 pp., 2 tables, bibliography, 25 titles. The peptidoglycan layer of Vitreoscilla stercoraria, ATCC 15218, was isolated from intact cells after treatment with sodium lauryl sulfate (SLS) and digestion with Pronase. Amino acid and amino sugar content was analyzed and 67% of the total present was made up of glutamic acid, ala nine, diaminopimelic acid (DAP), and glucosamine in a molar ratio of 1:1.7:1:0.7. Electron microscopy of the final peptidoglycan product showed a thin, delicately folded sacculus which exhibited a morphology different from that of the intact vegetative cells. Within these sacculi occurred electron-dense structures which were assayed and found to be poly- 3-hydroxybutyrate (PHB) granules. The final yield of peptidoglycan was 2.9% of the dry weight of the intact vegetative cell. TABLE OF CONTENTS Page LIST OF TABLES - - W -. iv LIST OF ILLUSTRATIONS. INTRODUCTION.S-.... MATERIALS AND METHODS* 7 RESULTS.......-.....-.. .13 DISCUSSION....-.-.-.-.. 025 LITERATURE CITED - .29 iii LIST OF TABLES Table Page I. Rf Values for Diaminopimelic Acid (DAP) Isomers and Peptidoglycan Hydrolysate for Vitreoscilla stercoraria................22 II. Amino Acid and Amino Sugar Analysis of Pep todoglycan of Vitreoscilla stercoraria . 23 iv LIST OF ILLUSTRATIONS Figure Page 1. Basic Structure of the Peptidoglycan of a Gram-negative Organism. ............. 2 2. Purification of Peptidoglycan of Vitreo scilla stercoraria..................... 9 3. Growth Curve for Vitreoscilla stercoraria in Tryptic Soy Broth (TSB)........ ........ 14 4. Electron-micrograph of Vitreoscilla stercoraria on Formvar-coated Grid (XIOOOO).................. ........ 15 5. Photomicrograph of the Gram Reaction of Vitreoscilla stercoraria (X1,000).w........16 6. Photomicrograph of a Sudan Black B stained Preparation of Vitreoscilla stercoraria (Xl,000)...............................17 7. Electron-micrograph of pellet after SLS treatment of Vitreoscilla stercoraria (X15,500).................... ........ 19 8. Electron-micrograph of Pellet after Pronase Treatment of Vitreoscilla stercoraria (X15,500)..............................20 9. Electron-micrograph of Final Peptidoglycan Purification from cells of Vitreoscilla stercoraria............... ....... 21 10. Proposed Chemical Structure of the Peptido glycan layer of Vitreoscilla stercoraria. 26 V INTRODUCTION There are many unique features in the structure of the bacterial cell wall which are of great interest and impor tance. For instance, the cell wall (a) contains surface antigens which interact with the immune system of a suitable host, (b) contains structures and chemical compounds which can be used in taxonomic differentiation of bacteria, (c) appears to have a direct influence on the staining char acteristics of an individual cell, (d) exhibits a high degree of specificity in bacteriophage adsorption, and (e) is the point of attack of some antibiotics. Contained within the cell wall is an electron-dense structural layer which is thought to be responsible for giving each bacterial cell its characteristic morphology. This rigid, covalently linked framework surrounds the bacterial cell and is actually one large sac-like molecule called the peptidoglycan layer (6). The essential features of this sacculus, found in Gram-nega tive bacteria, are a backbone of alternating N-acetylgluco samine and N-acetylmuramic acid residues in 3-1,4 linkage, a tetrapeptide side chain with alternating L and D amino acid residues, and a peptide bridge from the terminal car boxyl of one tetrapeptide to an available NH 2 -group of a neighboring tetrapeptide (Fig. 1). Included in the tetra peptide side-chain are L-alanine, D-alanine, D-glutamate, 1 2 Y Y Ala Ala D9In D-GIn X-PX.m-AP D-A D-Ala Fig. 1. Basic structure of the peptidoglycan of a Gram-negative organism. The backbones are made up of X which is an N-acetylglucosamine residue, and Y an N-acetylmuramic acid residue. The tetra peptide side-chains of each disaccharide unit are cross-linked to those of adjacent chains by a peptide bridge. 3 and either diaminopimelic acid or its decarboxylation product L-lysine. Within this framework, many variations are found; muramic acid may occur as N-glycolymuramic acid, the amino acids in the peptide bridge vary from organism to organism, and the degree of cross-linking varies in frequency (3, 12). Although other wall layers contribute to rigidity in some way, it has been shown that cells bounded only by the pep tidoglycan layer maintain their shape (14). Electron microscopy of a Gram-positive cell shows a 0 thick (200-800 A), dense outer layer which surrounds a thin ner (75 A) plasma membrane (3). The plasma membrane has a protein content of 60 to 70% with 20 to 30% lipid and small amounts of carbohydrate. The thick layer is the peptidoglycan and constitutes 30 to 70% of the total wall. It may have covalently attached to it polypeptides, polysaccharides, or teichoic acids. The latter two are believed to have antigenic significance (8). Electron microscopy of the Gram-negative cell wall shows three major layers. There is an outer membrane (60 to 180 A) which is composed mainly of phos pholipids, lipopolysaccharides, and proteins (2). Beneath this membrane lies what is called the "periplasmic space" (13). Martin describes this "space" as an enzyme-containing compartment bounded on the inside by the cytoplasmic membrane and on the outside by a "molecular sieve." Within the peri plasm is an electron-dense layer (20 to 30 A) which repre sents less than 10% of the Gram-negative cell wall. 4 This dense layer corresponds to the thick peptidoglycan of the Gram-positive bacteria. The cytoplasmic membrane lies innermost in the wall and contains approximately 30% lipid, 60% protein, and 2% carbohydrate (13). The peptidoglycan is recognized as the only cell wall polymer common to both Gram-positive and Gram-negative bacteria. Although the walls of Gram-positive bacteria reveal a great variation in the composition and structural arrangement of their peptidoglycans, all Gram-negative organisms pre viously studied for amino acid composition have shown only slight variation. The tetrapeptide ha-s been shown to contain the amino acids D-glutamic acid, meso- diaminopimelic acid, and alanine (D and L) in a molar ratio of 1:1:2 (20). Much interest has been shown in the peptidoglycan layer and work has been carried out in this area to elucidate its biosyn thesis and chemical structure as it occurs in different organisms (7, 8, 14, 171 18, 21, 22, 23, 24). It is the purpose of this work to determine the chemical composition of the peptidoglycan layer of the gliding bac terium Vitreoscilla stercoraria. According to the Eighth Edition of Bergey's Manual of Determinative Bacteriology (1), the genus Vitreoscilla belongs in the family Beggiatoacae which is located in Part 2, "The Gliding Bacteria." These organisms grow as filaments containing cells in chains. The filaments are flexible, motile by gliding, Gram-negative, and commonly exhibit poly- -hydroxybutyrate granules within 5 the cell. The genera of this family are distinguished by the presence or absence of sulfur granules deposited when cells are grown in the presence of H2 S and/or by the ability or lack of ability of the organism to form a well-defined sheath. Vitreoscilla stercoraria is a non-sheathed organism which does not deposit sulfur granules. It is further stated in Bergey's Manual that although the genera are apparently recognizable, this grouping may be precarious inasmuch as their affinities are uncertain because of the lack of criti cal information about these organisms. The only published information to date on the peptido glycan layer of the gliding bacteria has been that of Verma and Martin,who studied the genera Cytophaga and Sporocytophaga, (25) and Dworkinconcerning the genus Myxococcus xanthus (4). Verma and Martin found that peptidoglycan was a major cell component in enzyme-treated vegetative cells as well as in the microcysts of these non-fruiting myxobacteria. The amino sugar and amino acid constituents of the peptidoglycan of myxobacteria were found to be muramic acid, glucosamine, 2,6-diaminopimelic acid (DAP), glutamic acid, and alanine in molar ratios of 1:1:1:1.2. In similar experiments, Dworkin (4) found that both vegetative cells and microcysts of M. xanthus contain approximately 0.6% peptidoglycan (by weight). The overall composition of the peptidoglycan is similar in both cell types, containing muramic acid, glucosamine, DAP, glutamic acid, and alanine in a molar ratio of 6 0.75:0.75:1:1:1.7. Dworkin also found that treatment of M. xanthus vegetative cells with trypsin and sodium lauryl sulfate caused complete disaggregation of the walls, sug gesting a discontinuous peptidoglycan layer. The value of a study of the peptidoglycan layer of V. stercoraria was justified by the limited knowledge of the peptidoglycan of the gliding bacteria and the paucity of information concerning the genera of the family Beggiatoacae. MATERIALS AND METHODS Organism. Vitreoscilla stercoraria ATCC 15218, was maintained on Trypticase Soy Agar slants (Difco). Growth conditions. Large numbers of cells were obtained by the inoculation of 50 ml of Trypticase Soy Broth (TSB) (Difco) in 250-ml Erlenmeyer flasks with 5.0 ml of a sus pension of cells washed from a 48-hour-old agar slant culture. The culture was incubated at 25-28 C on an shaker (150 rev/min) until it reached the log phase of growth (approximately 48 hr). The 55-ml broth culture was then transferred into a fermenter (Fermentation Design Inc.) containing 3 L of TSB.
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