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Structure and Metabolism of the Intracellular Polysaccharide of Corynebacterium. Don D

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Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1969 Structure and Metabolism of the Intracellular Polysaccharide of Corynebacterium. Don D. Mickey Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Mickey, Don D., "Structure and Metabolism of the Intracellular Polysaccharide of Corynebacterium." (1969). LSU Historical Dissertations and Theses. 1679. https://digitalcommons.lsu.edu/gradschool_disstheses/1679 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. This dissertation has been microfilmed exactly as received 70-9079 MICKEY, Don D., 1940- STRUCTURE AND METABOLISM OF THE INTRACELLULAR POLYSACCHARIDE OF CORYNEBACTERIUM. The Louisiana State University and Agricultural and Mechanical College, Ph.D., 1969 Microbiology University Microfilms, Inc., Ann Arbor, Michigan STRUCTURE AND METABOLISM OF THE INTRACELLULAR POLYSACCHARIDE OF CORYNEBACTERIUM A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Microbiology by Don D. Mickey B. S., Louisiana State University, 1963 August, 1969 ACKNOWLEDGMENT The author wishes to acknowledge Dr. M. D. Socolofsky for his guidance during the preparation of this dissertation. He also wishes to thank Dr. H. D. Braymer and Dr. A. D. Larson and other members of the Department of Microbiology for helpful advice given during various phases of this research. Appreciation is also extended to Mrs. Joann T. Parker for the preparation of the ultrathin sections for electron microscopy and for the preparation of the figures. He wishes to thank Miss Marsha White and Miss Amy Tynes for technical help during the preparation of the dis­ sertation. A special appreciation is extended to Mrs. Lucy P. Tynes for the typing of the dissertation. This investigation was partly supported by NIH AI-2924. This dissertation is dedicated to my wife, Tecetl R. Mickey, whose encouragement and understanding made the completion of this work possible. ii TABLE OF CONTENTS Page ACKNOWLEDGMENT ii TABLE OF CONTENTS iii LIST OF TABLES vii LIST OF FIGURES viii ABSTRACT xi INTRODUCTION 1 REVIEW OF LITERATURE 3 Characteristics of the genus Corynebacterium 3 Morphology 3 Conditions for growth 3 Utilization of carbon compounds 4 Utilization of nitrogen compounds 4 Polysaccharides of microorganisms 5 General considerations 5 Location 5 Function 5 Occurrence in Gram positive bacteria 7 Occurrence in corynebacteria 8 Glycogen metabolism 9 General considerations 9 Enzymes acting uponQ^-1,4-glucosidic bonds 11 Enzymes acting upon ^t»-l, 6-glucosidic bonds 13 Specific transferases and hydrolases 13 UDPglucoseiglycogen- o^4-glucosyltransferase . 13 0( -1,4-glucan:©^-l, 4-glucan 6-glycosyltransferase . 15 Amylopectin 6-glucanohydrolase 16 iii Page MATERIALS AND METHODS 17 Organisms and culture media 17 Chemicals and reagents 17 Substrate dependency experiments 19 Preparation of cells 19 Effect of carbon source 19 Effect of length of incubation 20 Effect of nitrogen concentration 20 Radioisotope experiment 20 Isolation of polysaccharide 21 Physical procedures 21 Isolation of cell wall carbohydrate 22 Determination of dry weight of cells 23 Determination of total carbohydrate content ..... 23 Determination of reducing sugar content 23 Chromatographic analysis 24 Viability study 25 Cell viability 25 Viable cell counts 25 Polysaccharide structure analyses 25 Infrared spectra 25 Iodine-polysaccharide complex spectra 26 End group determination by periodate oxidation.... 26 Enzymatic determinations 27 Action of oL -amylase 27 Action of ft -amylase 27 Enzyme assays 28 Preparation of extracts 28 Protein determination 28 ATP:D-hexose 6-phosphotransferase 29 C* -D-glucose-1,6-diphosphate:c>£-D-glucose- 1-phosphate phosphotransferase 30 UDPglucose:o^-4-glucosyltransferase 30 oC-l, 4-glucan: o^.-l, 4-glucan 6-glycosyltransferase . 32 ©<-l,4-glucan:orthophosphate gluco^yltransferase . 33 iv Page Amylopectin 6-glucanohydrolase 34 O^-l, 4-glucan 4-glycosyltransferase (cyclizing) .... 35 Electron microscopy 35 Negative staining 35 Carbon replicas and metal shadowing 36 Freeze-etching technique 37 Fixation, thin sectioning, and post staining 38 RESULTS 40 Role of substrate in formation of polysaccharide 41 Effect of carbon source 41 Effect of length of incubation 43 Effect of nitrogen content 43 Glucose incorporation 49 Analyses of isolated polysaccharide 49 Chromatographic analysis 49 Electron microscopic analysis 51 Proposed roles for polysaccharide 59 Relationship to cell protein content 59 Relationship to cell viability 62 Structure of polysaccharide 65 Iodine-polysaccharide complex spectra 65 Infrared spectra 67 Periodate oxidation 70 Enzymatic analysis 70 oL -amylase 70 ^3 -amylase 72 Biosynthesis and degradation of the polysaccharide 73 ATP:D-hexose 6-phosphotransferase 73 -D-glucose-1, 6-diphosphate:o£-D- glucose-l-phosphate 75 UDPglucose: glycogen o^.-4-glucosyl transferase .... 75 O^-l, 4-glucan:o^-l, 4-glucan 6-glycosyltransferase . 78 o(-i» 4-glucan:orthophosphate glucosy transferase ... 87 Amylopectin 6-glucanohydrolase 89 o(-i, 4-glucan 4-glycosyltransferase (cyclizing) .... 90 v Page DISCUSSION 91 LITERATURE CITED 108 VITA 117 vi LIST OF TABLES Table Page 1. Occurrence of glycogen in microorganisms 10 2. Composition of semi-synthetic medium 18 3. Polysaccharide accumulation of C_. striatum and C_. diphtheriae type gravis grown on various carbon sources ... 42 4. Properties of the isolated and known polysaccharides 69 5. Branching enzyme activity in crude extracts of C. striatum and CJ. diphtheriae type gravis 86 6. Comparison of properties of glycogen isolated from other microorganisms to polysaccharide from C. striatum and C_. diphtheriae type gravis 93 vii LIST OF FIGURES Figure Page 1. Effect of incubation period on the carbohydrate content of cultures of (3. striatum grown in semi-synthetic medium .... 44 2. Effect of incubation period on the carbohydrate content of cultures of C\ diphtheriae type gravis grown in semi­ synthetic medium 45 3. Dry weight, carbohydrate content and non-carbohydrate content of C_. striatum cells grown on varying concentrations of (NH4)2SC>4 47 4. Dry weight, carbohydrate content and non-carbohydrate content of C_. diphtheriae type gravis cells grown on varying concentrations of (NH4)2SC>4 48 5. Electron micrograph of a carbon replica of C. striatum cells grown for 72 hr on semi-synthetic medium 52 6. Electron micrograph of a negatively stained cell of C. diphtheriae type gravis grown for 24 hr on semi-synthetic medium 53 7. Electron micrograph of a carbon replica of cells of JD. diphtheriae type gravis which have been grown on semi­ synthetic medium and subjected to freeze-etching 55 8. Electron micrograph of an ultrathin section of cells of C. diphtheriae type gravis grown on semi-synthetic medium for 24 hr 56 9. High magnification electron micrograph of an ultrathin section of cells of diphtheriae type gravis grown on semi-synthetic medium for 24 hr 57 10. Electron micrograph of an ultrathin section of cells of C. striatum grown for 72 hr on semi-synthetic medium 58 11. Electron micrograph of an ultrathin section of cells of C. diphtheriae type gravis grown on semi-synthetic medium for 72 hr 60 viii Figure Page 12. High magnification electron micrograph of an ultrathin section of cells of C. diphtheriae type gravis grown on semi-synthetic medium for 72 hr 61 13. Cell carbohydrate content and cell protein content of a resting cell suspension of (3. striatum incubated in the presence and absence of (NH4)2S04 63 14. Cell carbohydrate content and cell protein content of a resting cell suspension of C_. diphtheriae type gravis incubated in the presence and absence of (NH^SC^ 64 15. Cell carbohydrate and number of viable cells in a resting cell suspension of C_. striatum incubated in the presence and absence of (NH4)2SC>4 66 16. Comparison of the absorption spectra of the isolated poly­ saccharides to standard polysaccharides 68 17. Infrared absorption spectra of the isolated polysaccharides and of rabbit glycogen 71 18. Hexokinase activity in crude extracts of C. diphtheriae type gravis and C. striatum 74 19. Phosphoglucomutase activity in crude extracts of C_. diphtheriae type gravis and C_. striatum 76 20. Glycogen synthetase activity in crude extracts of C. diphtheriae type gravis and C_. striatum 77 21. Branching enzyme activity of crude extracts of C. striatum on various substrates 79 22. Branching enzyme activity of crude extracts of C_. diphtheriae type gravis on various substrates . 80 23. Relationship between branching enzyme activity and enzyme concentration in crude extracts of £. diphtheriae type gravis and _C_. striatum 82 ix Figure Page 24. Relationship between branching enzyme activity and pH in crude extracts of C. diphtheriae type gravis and C. striatum 83 25. Relationship between branching enzyme activity and temperature in crude extracts of C2. diphtheriae type gravis and C_. striatum 85 26. Phosphorylase activity in crude extracts of C_. diphtheriae type gravis and _C_. striatum 88 x ABSTRACT The intracellular
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