THE PHYSIOLOGY OF AZOTOBACTER VINELANDII CYSTS APPROVED: GRADUATE COMMITTEE: Ma'Yor Professor Minor jlrof/ssor onmmftee Member Comm itee Membe Crr tee Member 7,.- V / ~ 41 ~- / N ~ -~ - K- L Director ~6\f the Oepartment of Biological'~: ~ Sciences 4-4e Dean o 'the Graduate School ,7cI A'I THE PHYSIOLOGY OF AZOTOBACTER VINELANDII CYSTS DISSERTATION Presented to the Graduate Council of the North Texas State University in Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY BY Solomon L. Aladegbami, B.S. Denton, Texas December, 1980 Aladegbami, Solomon L., The Physiology of Azotobacter vinelandii cysts. Doctor of Philosophy (Biology). December, 1980, 149 pp., 18 tables, 28 figures, bibliography, 147 titles. The value of the adenylate energy charge [(ATP) + 1/2 (ADP)/(ATP) + (ADP) +(AMP)] in Azotobacter vinelandii cells was monitored during growth and germination in flask cultures. The miximal value of 0.88 was attained during mid-log phase; this declined gradually to 0.50 by late stationary phase. When these cultures were transferred to encystment media, the adenylate energy charge decreased to an average value of 0.40 as the vegetative cells encysted and remained unchanged during the next 20 days. Encysting cultures were composed of vegetative cells, encysting cells and nature cysts but the proportionate value of the energy charge could be assigned. Viability of the total population remained 95% or higher during the entire period studied. Azotobacter vinelandii cysts cultivated on phosphate- deficient media were compared to cysts grown in phosphate- sufficient media. Although cell protein and nucleic acids were unaffected by phosphate deficiency, cell wall struc- tures, oxygen uptake and encystment were significantly affected. Phosphate-limited cysts contained much larger amounts of poly-beta-hydroxybutyric acid but had a lower adenylate energy charge than did control cysts. The ATP/ADP ratio was much lower in phosphate-deficient cysts than in the control cysts. The data indicate a "substrate saving" choice of three metabolic pathways available to cells of Azotobacter under different growth conditions. TABLE OF CONTENTS LIST OF TABLES.................................... ii LIST OF ILLUSTRATIONS................................ iv Chapter I. INTRODUCTION..............................1 II. MATERIALS AND METHODS......................34 III. RESULTS................................... 50 IV. DISCUSSI0N........................ ........ 124 BIBLI0GRAPHY...................................... 138 i LIST OF TABLES Table Page I. Composition of Burk's nitrogen- free medium..................................... 34 II. Tryptic Soy Broth (TSB)......................... 35 III. Method of preparation of A. vinelandii for isoelectrofocusing on polyacrylamide gels............................. 47 IV. Distribution of adenine nucleotides (ATP, ADP, and AMP) and energy charge in A. vinelandii during growth in Burk's medium supplemented with ammonium chloride.........................................65 V. Distribution of adenine nucleotides and energy charge in A. vinelandii during growth in Tryptic Soy Broth...............66 VI. Effect of growth medium on oxygen uptake and ratio of ATP to ADP in A. vinelandii.........67 VII. Oxygen uptake and adenylate energy charge of dry cysts of A. vinelandii held on filter membranes................................ 68 VIII. Oxygen consumption and content of poly- beta-hydroxybutyric acid in cells and cysts of A. vinelandii.......................... 69 IX. Distribution of adenine nucleotides, adenylate charge, and the ratio of ATP to ADP in vegetative cells of A. vinelandii grown in phosphate-sufficient and phosphate-deficient media................... 70 X. Effect of phosphate-deficiency on cyst formation in A. vinelandii...................... 71 XI. Effect of phosphate-deficiency on oxygen uptake and ratio of ATP to ADP in A. vinelandii.......................................72 ii Table Page XII. Effect of phosphate-deficiency of poly-beta-hydroxybutyric acid storage and protein content in cysts of A. vinelandii....................................... 73 XIII. Comparative rates of cyst formation in A. vinelandii on substrates chemically similar to n-butanol.................. 74 XIV. Comparative ratio of energy charge and ATP to ADP ratio of n-butanol-related compounds inA. vinelandii cysts...............75 XV. Distribution of adenine nucleotides and energy charge in germinating cysts of A. vinelandii...... ........................ 76 XVI. Relative oxygen consumption and ATP/ADP ratio in germinating cysts of A. vinelandii...... 77 XVII. Relative protein content of A. vinelandii grown in Burk's nitrogen-free medium sup- plemented with glucose as compared to cells in different growthmedia..................... 78 XVIII. Relative protein content of A. vinelandii grown in Burk's nitrogen free medium sup- plemented with glucose as compared to cysts grown in different substrates.................... 79 iii TABLE OF ILLUSTRATIONS Figure Page 1. Degradation of glucose.......................... 20 2. Reactions involved in adenine nucleotide formation, utilization and interconversion............................. 27 3. Detection of flourescence signal................ 45 4. Adenine nucleotide concentrations as a function of culture age and adenylate energy charge in A. vinelandii. Cells were grown in Burk's nitrogen-free liquid medium to which glucose was added to a final concentration of 2% (w/v)............ 81 5. Adenine nucleotide concentration in mature cysts of A. vinelandii as a function of time. Cysts were grown in Burk's nitrogen- free media to which n-butanol was added to a final concentration of 0.3% (w/v)............. 83 6. Effect of phosphate limitation on adenine nucleotides inA. vinelandii cysts.............. 85 7. Oxygen - uptake of A. vinelandii grown in phosphate-sufficient media; and phos- phate-deficient media........................... 87 8. Effect of n-butanol-related substrates on oxygen consumption by cysts of A. vinelandii.......................................89 9. Level of NAD as a function of culture age in A. vinelandii grown in Burk's nitrogen-free media, media supplemented with ammonium chloride and in Tryptic Soy Broth....................................... 91 10. Soluble proteins from vegetative cells of A. vinelandii grown on different media........93 11. Densitometer scanning patterns of the isoelectrofocusing separations of soluble proteins shown in Figure10......................95 iv Fi gure Pag9e 12. Isoelectrofocusing patterns of proteins from cells of A. vinelandii grown in Burk's nitrogen-free media supplemented with glucose and in phosphate-limited Burk's nitrogen- free medium.......................97 13. Densitometer scanning patterns of soluble protein from cells of A. vinelandii grown in Burk's nitrogen-free medium supplemented with glucose and in phosphate-limited Burk's medium supplemented with glucose..................99 14. Soluble proteins of A. vinelandii cysts grown in Burk's medium supllemented with n-butanol instead of glucose..................... 101 15. Densitometer scanning patterns of the isoelectrofocusing separation of the soluble proteins shown in Figure 14.............. 103 16. The effect of growth medium on cell protein as determined by flow micro- fluorometry. The substrates employed were Burk's medium supplemented with glucose, Burk's medium supplemented with ammonium chloride, phosphate- limited Burk's nitrogen-free medium and Tryptic SoyBroth............................105 17. Cyst proteins of A. vinelandii determined by flow microfluorometry.............. 107 18. The effect of substrates on protein of A. vinelandii cyst as determined by fTow microfluorometry......................... 109 19. Relative DNA content of A. vinelandii cysts grown on Burk's nitrogen-free medium with n-butanol............................ 111 20. Effect of phosphate-deficiency on the morphology of A. vinelandii...................... 113 21. Scanning electron micrograph of cells of A. vinelandii grown in Tryptic Soy Brot~hI and fixed in glutaraldehyde................ 115 V Figure Pag e 22. Scanning electron micrograph of cells of A. vinelandii grown in Burk' s medium supplemented with ammonium chloride and fixed with glutaraldehyde................................. 115 23. Early precystic cells visualized by scanning electron microscopy.................. .117 24. Precystic forms observed on day three by scanning electron microscopy............. 117 25. Inner core of matured cysts grown on n-butanol and observed by scanning electron microscopy after glutaldehyde treatment........... 119 26. Inner core of matured cysts grown on n-butanol and observed by scanning electron microscopy after glutaraldehyde treatment......... 119 27. Alternate pathway for substrate energy utilization in A. vinelandii....................121 28. Distribution of energy charge during morphogenetic stages in cells of A. vinelandii ... ........... 123 vi Chapter I INTRODUCTION Members of the family Azotobacteraceae are said to form large gram-negative cells which are capable of fixing nitrogen in a nitrogen-free medium with an organic carbon source. They produce rod-shaped cells which become coccoid when mature. The Azotobacter are characterized physiologi- cally by an aerobic mode of life and metabolically by growth in media free of combined nitrogen. The other heterotrophic bacteria that utilize atmospheric nitrogen non-symbiotically, belong to the genus Clostridium and these are anaerobic