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Pathways and Enzymes Involved in Glucose Catabolism by Lactic Streptococci

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Pathways and Enzymes Involved in Glucose Catabolism by Lactic Streptococci AN ABSTRACT OF THE THESIS OF RAJIVA NANDAN for the DOCTOR OF PHILOSOPHY (Name) (Degree) in MICROBIOLOGY presented on Mc-\\Ì 11)\q/e7 (Major) tt (Date) Title: PATHWAYS AND ENZYMES INVOLVED IN GLUCOSE CATABOLISM BY LACTIC STREPTOCOCCI Abstract approved: Dr. William E. Sandine The enzymes and pathways involved in the catabolism of glucose by several strains of Streptococcus diacetilactis, Strepto- coccus cremoris, and Streptococcus lactis, commonly called the lactic streptococci, were studied. The presence of aldolase, triosephosphate isomerase and alcohol dehydrogenase in these organisms provided evidence for the operation of the Embden- Meyerhof- Parnas (EMP) pathway for the catabolism of glucose, resulting in the formation of ethanol in addition to large amounts of lactic acid. Results of manometric experiments showed that the lactic streptococci had the capability to carry out the oxidative decarboxylation of pyruvate and other alpha -keto acids (alpha -keto- butyric, alpha -ketovaleric, alpha -ketoisovaleric, alpha -keto- caproic and alpha -ketoisocaproic acid) possibly by NAD- lipoate linked pyruvate dehydrogenase catalyzed reactions. The existance of operative hexosemonophosphate (HMP) path- way in the lactic streptococci was demonstrated as a result of the discovery of glucose -6- phosphate dehydrogenase and 6- phospho- gluconate dehydrogenase in these organisms. This pathway may provide the lactic streptococci with pentoses and dihydronicotinamide adenine dinucleotide phosphate (NADPH), which may be used in biosynthetic reactions. Acetate kinase was found to be present in the lactic strepto- cocci. The presence of this enzyme provided evidence for the occurrence of phosphoroclastic reaction involving phosphotrans- acetylase and acetate kinase in these organisms. This reaction sequence converts acetyl coenzyme A to acetate via the intermediate formation of acetyl phosphate resulting in the ultimate generation of one molecule of adenosine triphosphate from one molecule of acetyl coenzyme A. The presence of acetate kinase also provided evidence for the possible operation of the phosphoketolase catalyzed phosphorolytic cleavage of xylulose -5- phosphate, an intermediate of the HMP pathway, to form triose phosphate and acetyl phosphate Acetyl phosphate may then form acetate and ATP through the acetate kinase catalyzed reaction. The radiorespirometric pattern for the catabolism of specifically labeled glucose by the various species and strains of lactic streptococci was found to be essentially the same and showed the actual operation of both EMP and HMP pathways for the catabolism of glucose by these organisms. The EMP pathway was however found to be the main route of the catabolism of glucose by these organisms. The radiorespirometric experiments also demonstrated the absence of operative tricarboxylic acid cycle in these organisms. This investigation of the enzymes and pathways . involved in the catabolism of glucose by lactic streptococci helped in obtaining a proper understanding of the processes by which glucose is utilized for biosynthesis and energy production in these organisms. This investigation also provided evidence against the classification of S. diacetilactis, S. cremoris, and S. lactis in a strictly homofer, mentative group. The classification of these organisms in the microaerophilic group, which includes organisms that grow best in the presence of very limited amount of oxygen, has also become questionable because of the presence of an active oxidative decar- boxylation mechanism of pyruvate decarboxylation in these organisms. Also the presence of excess oxygen does not inhibit in any way the growth of S. diacetilactis M- 8- 224(a). Pathways and Enzymes Involved in Glucose Catabolism by Lactic Streptococci by Rajiva Nandan A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy June 19 67 APPROVED: Professor of Microbiology in charge of major Chairman of Department of Microbiology Dean of Graduate School ! f b 7 Date thesis is presented 1 ` Typed by Gwendolyn Hansen for Rajiva Nandan TO MY PARENTS ACKNOWLEDGMENTS I wish to express my sincere thanks to Dr. W. E. Sandine and Dr. P. R. Elliker for their guidance and help throughout the course of this investigation. I would also like to thank Dr. K. S. Pilcher and Dr. R. R. Becker, the members of my program committee, for their advice and suggestions. Grateful acknowledgment is made to Dr. C. H. Wang for permitting me to perform the radiorespirometric experiments in his laboratory. TABLE OF CONTENTS Page INTRODUCTION 1 REVIEW OF LITERATURE 4 Pathways of Glucose Catabolism 4 Methodology of Pathway Analysis 13 Pathways of Carbohydrate Catabolism in Lactic Streptococci 15 Fermentation of Lactose 15 Lactose Fermentation by Homofermentative Lactic Acid Bacteria 15 Lactose Fermentation by Heterofermentative Lactic Acid Bacteria 19 Decarboxylation of Alpha -keto Acids 21 MATERIALS AND METHODS 30 Microbiological Methods 30 Cultures Used 30 Enzymatic Methods 31 Preparation of Cell -Free Extracts 31 Streptophotometric Analysis 31 Aldolase 32 Triosephosphate Isomerase 34 Alcohol Dehydrogenase 36 Glucose -6- phosphate Dehydrogenase 38 6- Phosphogluconate Dehydrogenase 39 Acetate Kinase (acetokinase) 41 Protein Determinations 43 Manometric Experiments 44 Substrates for Manometric Experiments 44 Radiorespirometric Experiments 45 RESULTS 47 Enzyme Experiments 47 Manometric Experiments 81 Radiorespirometry 121 Page DISCUSSION 134 EMP Pathway in Lactic Streptococci 134 Hexose Monophosphate Pentose Pathway 138 Calculations for the Percent Pathway Participation 151 SUMMARY 159 BIBLIOGRAPHY 164 LIST OF FIGURES Figure Page 1. The Embden- Meyerhof -Parnas (EMP) Pathway 6 2. The Pentose Cycle 8 3. The Entner Doudoroff (E. D.) Pathway 9 4. The Pentose cycle and Entner- Doudoroff pathways: fate of individual carbon atoms. 12 5. Triosephosphate isomerase activity observed in cell -free extracts of Streptococcus diacetilactis 18 -16 51 6. Triosephosphate isomerase activity observed in cell -free extracts of Streptococcus cremoris M1 -W1 -3 52 7. Triosephosphate isomerase activity observed in cell -free extracts of Streptococcus lactis C2F 53 8. Alcohol dehydrogenase activity observed in cell - free extracts of Streptococcus diacetilactis M -8- 224(a) 55 9. Alcohol dehydrogenase activity observed in cell - free extracts of Streptococcus cremoris M1 -W1 -3 56 10. Alcohol dehydrogenase activity observed in cell- free extracts of Streptococcus: cremoris 144F 57 11. Alcohol dehydrogenase activity observed in cell - free extracts of Streptococcus lactis E 58 12. Alcohol dehydrogenase activity observed in cell - free extracts of Streptococcus lactis R -24(e) 59 13. Alcohol dehydrogenase activity observed in cell- free extracts of Streptococcus lactis 144 -54 60 Figure Page 14. Alcohol dehydrogenase activity observed in cell -free extracts of Streptococcus lactis ML -8 61 15. Glucose -6- phosphate dehydrogenase activity observed in cell- free - extracts of Streptococcus diacetilactis M- 8- 224(a) 63 16. Glucose -6- phosphate dehydrogenase activity observed in cell -free extracts of Streptococcus diacetilactis 18 -16 64 17. Glucose -6- phosphate dehydrogenase activity observed in cell -free extracts of Streptococcus cremoris Ml -W1 -3 65 18. Glucose -6- phosphate dehydrogenase activity observed in cell -free extracts of Streptococcus cremoris 144F 66 19. Glucose -6- phosphate dehydrogenase activity observed .. in cell -free extracts of Streptococcus lactis M -9 -924 67 20. Glucose -6- phosphate dehydrogenase activity observed in cell -free extracts of Streptococcus 68 lactis C 2F 21. 6- Phosphogluconate dehydrogenase activity observed in cell -free extracts of Streptococcus diacetilactis M- 8- 224(a) 71 22. 6- Phosphogluconate dehydrogenase activity observed in cell -free extracts of Streptococcus cremoris Ml -W1 -3 72 23. 6- Phosphogluconate dehydrogenase activity observed in cell -free - extracts of Streptococcus cremoris 144F 73 24. 6- Phosphogluconate dehydrogenase activity observed in cell -free extracts of Streptococcus lactis E 74 Figure Page 25. 6- Phosphogluconate dehydrogenase activity observed in cell -free extracts of Streptococcus lactis R -24(e) 75 26. 6- Phosphogluconate dehydrogenase activity observed in cell -free extracts of Streptococcus lactis 144 -54 76 27. 6- Phosphogluconate dehydrogenase activity observed in cell -free - extracts of Streptococcus lactis ML -8 77 28. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus diacetilactis M- 8- 224(a) metabolizing pyruvate. 85 29. Uptake of oxygen and evolution of CO2 by resting ,, cells of Streptococcus diacetilactis 18 -16 metabolizing pyruvate. 87 30. Uptake of oxygen and evolution. of CO2 by resting cells. of Streptococcus cremoris Ml -W1 -3 metabolizing pyruvate. 89 31. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus cremoris: 144F metabolizing pyruvate. 91 32. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus lactis E metabolizing pyruvate. 93 33. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus lactis M -9 -924 metabolizing pyruvate. 95 34. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus diacetilactis M- 8- 224(a) metabolizing alpha- ketobutyric acid. 98 35. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus diacetilactis 18 -16 metabolizing alpha -ketobutyric acid. 100 Figure Page 36. Uptake of oxygen and evolution
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