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 of CO2 by resting cells of Streptococcus cremoris Ml -W1 -3 metabolizing alpha -ketobutyric acid. 102

37. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus cremoris 144F metabolizing alpha -ketobutyric acid. 104

38. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus lactis E metabolizing alpha - ketobutyric acid. 106

39. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus lactis M -9 -924 metabolizing alpha- ketobutyric acid. 108

40. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus diacetilactis M- 8- 224(a) metabolizing alpha- ketovaleric acid. 111

41. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus diacetilactis M- 8- 224(a) metabolizing alpha- ketoisovaleric acid. 114

42. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus diacetilactis -8- 224(a) metabolizing alpha -ketocaproic acid. 117

43. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus diacetilactis M- 8- 224(a) metabolizing alpha- ketoisocaproic acid. 120 44. Radiorespirometric pattern exhibited by Streptococcus diacetilactis M- 8- 224(a) metabolizing specifically labeled glucose. 122 45. Radiorespirometric pattern exhibited by Streptococcus diacetilactis 18 -16 metabolizing specifically labeled glucose. 123 46. Radiorespirometric pattern exhibited by Streptococcus cremoris Ml -W1 -3 metabolizing specifically labeled glucose. 124 Figure Page 47. Radiorespirometric pattern exhibited by Streptococcus cremoris 144F metabolizing specifically labeled glucose. 125

48. Radiorespirometric pattern exhibited by Streptococcus lactis E metabolizing specifically labeled glucose. 126

49. Radiorespirometric pattern exhibited by Streptococcus lactis C2F metabolizing specifically labeled glucose. 127

50. Radiorespirometric pattern exhibited by Streptococcus lactis R -24(e) metabolizing specifically labeled glucose. 128

51. Radiorespirometric pattern exhibited by Streptococcus lactis 144 -54 metabolizing specifically- labeled glucose. 129

52. Glucose catabolism in lactic streptococci via the EMP pathway. 145

53. Glucose catabolism in lactic streptococci via the HMP pathway. 146 LIST OF TABLES

Table Page 1. Experimental design for the assay of aldolase. 33

2. Experimental design for the assay of Triosephos- phate isomerase. 36

3. Experimental design for the assay of alcohol dehydrogenase. 37

4. Experimental design used in assays for glucose - 6- phosphate dehydrogenase. 39

5. Protocal for assay of 6- phosphogluconate dehydrogenase. 40

6. Typical experimental design for acetate kinase assay. 42

7. Specific activity of aldolase in lactic streptococci. 48

8. Specific activity of triosephosphate isomerase in lactic streptococci. 50

9. Specific activity of alcohol dehydrogenase in lactic streptococci. 54

10. Specific activity of glucose -6- phosphate dehydrogenase in lactic streptococci. 62

11. Specific activity of 6- phosphogluconate dehydrogenase in lactic streptococci. 70

12. Specific activity of acetate kinase in lactic streptococci. 79

13. Affect of continuous aeration on the growth of S. 80 diacetilactis M- 8- 224(a) .

14(a). Uptake of oxygen by cells of S. diacetilactis M -8- 224(a) metabolizing pyruvate. 84

14(b). Evolution of CO2 by cells of S. diacetilactis M -8- 224(a) metabolizing pyruvate. 84 Table Page 15(a). Uptake of oxygen by cells of S. diacetilactis 18 -16 metabolizing pyruvate. 86

15(b). Evolution of CO2 by cells of S. diacetilactis 18 -16 metabolizing pyruvate. 86

16(a). Uptake of oxygen by cells of S. cremoris Ml -W1- 3 metabolizing pyruvate. 88

16(b). Evolution of CO2 by cells of S. cremoris Ml -W1- 3 metabolizing pyruvate. 88

17(a). Uptake of oxygen by cells of S. cremoris 144F metabolizing pyruvate. 90

17(b). Evolution of CO2 by cells of S. cremoris 144F metabolizing pyruvate. 90

18(a). Uptake of oxygen by cells of S. lactis E metabolizing pyruvate. 92

18(b). Evolution of CO2 by cells of S. lactis E metabolizing pyruvate. 92

19(a). Uptake of oxygen by cells of S. lactis M -9 -924 metabolizing pyruvate. 94

19(b). Evolution of CO2 by cells of S. lactis M -9 -924 metabolizing pyruvate. 94

20(a). Uptake of oxygen by cells of S. diacetilactis M-8- 224(a) metabolizing alpha -ketobutyric acid. 96

20(b). Evolution of CO2 by cells of S. diacetilactis M -8- 224(a) metabolizing alpha -ketobutyric acid. 97

21(a). Uptake of oxygen by cells of S. diacetilactis 18 -16 metabolizing alpha -ketobutyric acid. 99

21(b). Evolution of CO2 by cells of S. diacetilactis 18 -16 metabolizing alpha- ketobutyric acid. 99 Table Page 22(a). Uptake of oxygen by cells of S. cremoris Ml -W1 -3 metabolizing alpha -ketobutyric acid. 101

22(b). Evolution of CO2 by cells of S. cremoris Ml -W1 -3 metabolizing alpha -ketobutyric acid. 101

23(a). Uptake of oxygen by cells of S. cremoris 144F metabolizing alpha -ketobutyric acid. 103

23(b). Evolution of CO2 by cells of S. cremoris 144F metabolizing alpha- ketobutyric acid. 103

24(a). Uptake of oxygen by cells of S. lactis E metabolizing alpha -ketobutyric acid. 105

24(b). Evolution of CO by cells of S. lactis E metabolizing alpha- ketobutyric acid. 105

25(a). Uptake of oxygen by cells of S. lactis M -9 -924 metabolizing alpha- ketobutyric acid. 107

25(b). Evolution of CO2 by cells of S. lactis M -9 -924 metabolizing alpha- ketobutyric acid. 107

26(a). Uptake of oxygen by cells of S. diacetilactis M-8- 224(a) metabolizing alpha -ketovaleric acid. 109

26(b). Evolution of CO2 by cells of S. diacetilactis M -8- 224(a) metabolzing alpha- ketovaleric acid. 110

27(a). Uptake of oxygen by cells of S. diacetilactis M -8- 224(a) metabolizing alpha -ketoisovaleric acid. 112

27(b). Evolution of CO2 by cells of S. diacetilactis M -8- 224(a) metabolizing alpha -ketoisovaleric acid. 113

28(a) . Uptake of oxygen by cells of S. diacetilactis M-8- 224(a) metabolizing alpha -ketocaproic acid. 115

28(b). Evolution of CO2 by cells of S. diacetilactis M -8- 224(a) metabolizing alpha- ketocaproic acid. 116 Table Page 29(a). Uptake of oxygen by cells of S. diacetilactis M -8- 224(a) metabolizing alpha -ketoisocaproic acid. 118

29(b). Evolution of CO2 by cells of S. diacetilactis M -8- 224(a) metabolizing alpha -ketoisocaproic acid. 119

30. Incorporation of substrate into cells, medium, and CO2 by S. diacetilactis M- 8- 224(a) cells metabolizing glucose specifically labeled with 14C. 130

31. Incorporation of substrate into cells, medium, and CO2 by S. diacetilactis 18 -16 cells metabolizing glucose specifically labeled with 14C. 130

32. Incorporation of substrate into cells, medium, and CO2 by S. cremoris M1 -W1 -3 cells metabolizing glucose specifically labeled with 14C. 131

33. Incorporation of substrate into cells, medium, and CO2 by S. cremoris 144F cells metabolizing glucose specifically labeled with 14C. - 131

34. Incorporation of substrate into cells, medium, and CO2 by S. lactis E cells metabolizing glucose specifically labeled with 14C. 132

35. Incorporation of substrate into cells, medium, and CO2 by S. lactis C2F cells metabolizing glucose specifically labeled with 14C. 132

36. Incorporation of substrate into cells, medium, and CO2 by S. lactis R -24(e) cells metabolizing glucose specifically labeled with 14C. 133

37. Incorporation of substrate into cells, medium, and CO2 by S. lactis 144 -54 cells metabolizing glucose specifically labeled with 14C 133

38. Percent Pathway Participation in Lactic Streptococci. 158 PATHWAYS AND ENZYMES INVOLVED IN GLUCOSE CATABOLISM BY LACTIC STREPTOCOCCI

INTRODUCTION

Catabolism of carbohydrates in living cells results in the production of energy and intermediates for biosynthetic functions. Numerous investigators in recent years have helped to elucidate the complex nature of the various pathways involved in the carbohydrate catabolism. Several valuable tools have been developed in recent years to study the nature of these catabolic pathways. Among the research methods are the modern techniques for enzyme assay and enzyme purification, ion exchange resin and column chromatography, and manometric methods. Recent advances in radioactive tracer methodology also have been invaluable in the study of carbohydrate catabolism.

Glucose may be regarded as the most important carbon source

since other carbohydrates often are converted . to glucose or a derivative thereof prior to catabolism. The present investigation deals with the study of the enzymes and pathways involved in the catabolism of glucose by Streptococcus diacetilactis, Streptococcus cremoris, and Streptococcus lactis. These organisms are often called the lactic streptococci and have been classified as the lactic acid bacteria because they produce lactic acid as the major 2 end -product of carbohydrate catabolism.

On the basis of the pathways involved in the catabolism of glucose, the lactic acid bacteria have been divided into the homo- fermentative and the heterofermentative groups Homofermentative lactic acid bacteria have been defined as those which utilize glucose via the exclusive operation of the Embden- Meyerho f - Parnas (EMP) pathway and produce lactic acid almost exclusively as the end - product of carbohydrate catabolism. In contrast the heterofermenta- tive species catabolize glucose by way of the hexosemonophosphate

(HMP) pathway and produce, in addition to lactic acid, considerable quantities of other metabolic end products, including carbon dioxide, ethanol, and acetic and formic acids. S. diacetilactis, S. cremoris,

and S . lactis have been classified into the homofermentative group.

As a result of recent reports by various investigators, considerable evidence has accumulated to show that many homo- fermentative lactic streptococci produce some or all of the following

compounds in addition .. to lactic acid: acetic acid, formic acid, carbon dioxide, ethanol, diacetyl, acetoin, and 2 :3 butanediol.

The presence of highly active alcohol dehydrogenase has also been reported in these organisms. Also, some evidence has accumulated that at least a few strains of homofermentative lactic streptococci have the ability to utilize the HMP pathway. 3

The present investigation of the enzymes and pathways involved in the catabolism of glucose by the various strains of S. diacetilactis, S. cremoris, and S. lactis was carried out in order to obtain a proper understanding of the processes by which glucose is utilized for biosynthesis and energy production in these organisms and to reexamine and clarify the vague classification of these organisms into the homofermentative group. 4

REVIEW OF LITERATURE

Pathways of Glucose Catabolism

Buchner in 1897 (32) demonstrated that a cell -free extract obtained from yeast cells was able to ferment sugar. Since that time, numerous investigations have revealed that two major metabolic pathways exist for glucose utilization in microorganisms, plants and animal cells. These are recognized today as the Embden-

Meyerhof- Parnas (EMP) pathway, shown in Figure 1 and the hexose monophosphate (HMP) pathway, shown in Figure 2. Recently a third pathway has been demonstrated in some microorganisms (22,

29, 63, 99, 100, 108) known as the Entner -Doudoroff (ED) pathway, shown in Figure 3.

Both the EMP and ED pathways are responsible for the degradation of glucose with formation of two molecules of pyruvate; the carboxyl groups of each molecule of pyruvate formed via the

EMP pathway is derived from the third (C -3) and fourth (C -4) carbons of glucose. On the other hand, the carboxyl groups of pyruvate formed via the ED pathway are derived from C -1 and C -4 of glucose (22, 26). The key step in. the catabolism of glucose by both the pathways is the aldol cleavage of a C6 intermediate to yield two C3 fragments. In the EMP pathway, fructose 1 :6 diphosphate is Figure 1. The Embden- Meyerhof -Parnas (EMP) Pathway

Phosphate (P) Coenzyme A (CoA) Adenosine Triphosphate (ATP) Adenosine Diphosphate (ADP) Nicotinamide Adenine Dinucleotide (NAD or DPN) Dihydronicotinamide Adenine Dinucleotide (NADH or DPNH) Thiamine pyrophosphate (TPP) THE EMBOEN - MEYERHOF- PARNAS (E MP) PATHWAY x-ó-ó-ó -N 00x O z M N C z o N 4 ñ ó m o r o m o O ó a O Y o ó E

m o o D o a' z o z x N 9 aOtD Ü-Ú-Ú-T O S O T T x 0 á T M co Glucose -6-P Fructose -6 -P Fructose- I,6 -di -P Q 0 Q. `) GlyceraIdehyde-3-P 1,3-di-P-Glycerate - f + 15 ; o V, 11-1,faoydsoyd faSOUiv a I- a d4V 4.6W -NMV41n Ú-Ü-Ú-Ú-Ú-Ú 20x002 I IAdapÁ16 d1V x o x x O x N u- 6-P-Gluconate ... o o ó

w 3-P-Glycerate M a 0+ O (V M op= Pentose JAJaoAINAJOydsoyd

Cycle c.) in S Ü-Ú OT_ N o x ó ta z M Alb asojnW -.d-in--ç' Acetaldehyde _-7, Z 0=0-(J-U 0=U-U-Ú M .r U N f + ó IQ o0x X PC.) ar 0 x M Q (enclose, Mg+ +) +O=U-U

oxidase) N t 01 x ln (pyruvic x M N in 0 N N - á x á Q U S N In CO V l M T. C. A. Cycle - á Ì- M o T P-Enoipyruvate 2-F-Glycerate 6 Figure 2. The Pentose Cycle

Nicotinamide Adenine Dinucleotide Phosphate (NADP or TPN) Dihydronicotinamide Adenine Dinucleotide Phosphate (NADPH or TPNH) Glucose -6- phosphate (G -6 -P) 6- phosphogluconate (6 -P -G) Ribulose -5- phosphate (Ru -5 -P) Ribose -5- phosphate (R -5 -P) Xylulose -5- phosphate (Xu -5 -P) Glyceraldehyde -3- phosphate (G -3 -P) Sedoheptulose -7- phosphate (S -7 -P) Erythrose -4- phosphate (E -4 -P) Fructose -6- Phosphate (F -6 -P) Fructose -l:6- diphosphate (F- l:6 -di P) THE PENTOSE CYCLE

2 5 2 3 4 5 6 1 2 3 4 5 6 2 3 4 5 6 I 34 6

1 1 1 1 O I I I 1 0 O II 10 C-C-CH2OP CO2+ C-C-C-C-CH2OP HC-C-C-C-CH2OP - HC-C-C-C-C-CH2OP HO-C-C-C- 2 3 4 5 6 4 2 3 4 5 6 2 3 4 5 6 merase) 1 2 3 5 6 I O I 1 1 10 1 O 1 I 1011 I»HC-C-C-C-CH2OP - 6TPN 6TPNH2 H O-C-C- C-C- 6TPN 6TPNH2 CO2 + C-C-C-C-CH2OP HC-C-C-C-C-CH2OP C-CH2OP R-5-P 1 -6- 6-P- (glucose 6-P-gluconic 2 3 4 6 1 5 2 3 4 5 6 J 2 3 4 5 6 I 2 4 5 6 dehyrogenase I O 1 II dehyrogenase,) 0 1 I I 1011 3 HC-C-C-C-C-CH2OP HO-C-C-C-C-C-CH2OP .,_ CO2 C-C-C-C-CH2OP rC-C-C-C-CH20P the lactone I thru s- 1 Mg" or Mn" 4 5 2 3 4 5 2 3 4 5 6 1 2 3 4 5 6 1 2 3 6 I 6 01 II 01 II 1011 I 0 I CO2+ C-C-C-C-CH2OP C-C-C-C-CH2OP - HC-C-C-C-C-CH2OP HO-C-C-C-C-C-CH2OP (Xu-5-P-isomerase) I 4 2 3 4 5 6 1 2 3 4 5 2 3 5 6 1 2 3 4 5 6 6

I I I 1 1 1 1 1 O 1 I 0 0 10 HC-C-C-C-C-CH2OP HO-C-C-C-C-C-CH2OP CO2 + C-C-C-C-CH2OP »C-C-C-C-CH2OP - ! } 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 I 2 3 4 5 6

I I O 1 II 01 II 1011 C-C-C-C-CH2OP -.C-00 HC-C-C-C-C-CH2OP HO-C-C-C-C-GCH2OP CO2+ -C-C-CH2OP G-6-P 6-P-G Ru-5-P Xu-5-P

2 3 2 4 5 6 2 3 2 4 5 6 2 3 2 3 4 5 6 O 1 II IO 1 10 III -- HC-C-C-C-C- CH2OP -C-C-C-C-C-CH2OP .- 1 C-C-C-C-C-C-CH2OP ( p xoi a 2 3 2 4 5 6 hos phohe somer se) 2 3 2 4 5 6 2 3 2 3 4 5 6

O 1 I 1 I I 1 0 +- 10 III C-C-C-C-C-CH2OP { ^- HC-C-C-C-C-CH2OP 1 C-C-C-C-C-C-CH2OP G-6-P F-6-PP S-7-P (transketolose) (transaldolose) < 4 5 6 TPP, HC-C-CH2OP 3 4 5 6 - 4 5 6 O I 1 011 HC-C-CH2OP C-C-C-CH2CP 1 I 2 3 3 4 5 6 2 3 3 4 5 6 G-3-P 3 4 5 6 O 1 I I 1 0 I i I 1 -^ HC-C-C-C-C-CH2OP C-C-C-CH2OP I 1 (phosphohexoisomerase) -C-C-C-C-C-CH2OPT E-4-P 3 3 5 6 . 2 3 4 5 6 20 1 4 - 3 -- HC-C-C-C-C-CH2OP C-C-Ç-C-C-CH2OP (tronsketolase) G-6-P F-6-P 1

4 5 6 TPP, Mg" 01 aldolase, HC-C-CH2OP -- (phosphohexoiso.merase) (Fructose diphosphatase) triose-P-isomeros ) 4 5 6 6 5 4 4 5 6 6 5 4 4 5 6 6 5 4 4 5 6 01 01 1I 10 1I 0 11 I -, ( J HC-C-CH2OP HC-C-C-C-C-CH2OP C-C-C-C-C-CH2OP P0H2C-C-C-C-C-CH2OP I 1 Mg G-3-P + T. C. A. G-6-P F-6-P6-P F-1,6-diP ---°

00 9

fTHE ENTNER DOUDOROFF ( E. D. ) PATHWAY]

I/O I/O 0

I I OH C-OH I HC =0 I C-OH Ç=0 2 HCOH 2 HCQH ATP ADP 2 HCOH 2 3 3 HOCH (glucose oxidase) 3 HOCH \------j ' 3 HOCH ( 6-P-G dehydrase) CH2 _ I I - I glucono- ' 4 HCOH 4 HCOH ( kinase 4 HCOH 4 HCOH 5 HCOH 5 HCOH 5 HCOH 5 HCOH I 6 CH2OH 6 CH2OH 6 HC-OP03H2 6 HGOP03H2 H H Glucose Gluconate 6-P -Gluconate 2-keto-3-deoxy- 6-P- Gluconate

0 ó

4 HC-0H 4 HC=O I C-OH

I I I + T.C.A. cycle °--5 HC=O d 5 HCOH 2 C=0 ----,T.C.A. cycle I I I 6 CH3 6 HC-OP03H2 3 CH3

Pyruvate H Pyruvate !! Glyceraldehyde--3-P --

Figure 3. The Entner Doudoroff (E. D.) Pathway. 10 cleaved to yield dihydroxy acetone phosphate and glyceraldehyde-3- phosphate. In the ED pathway 2- keto -3- deoxy -6- phosphogluconate is cleaved to yield pyruvate and glyceraldehyde -3- phosphate. These

C3 intermediates for each pathway are then converted to pyruvate.

The HMP pathway is a direct oxidative pathway for glucose catabolism which does not involve a cleavage of hexose into two triose molecules. This pathway involves instead an oxidation of glucose -6- phosphate to form 6- phosphogluconate, which may undergo phosphogluconate decarboxylation (PGD) to yield pentose phosphate or be further catabolized via the ED pathway. The first reaction of the HMP pathway, the direct oxidation of glucose -6- phosphate, was discovered by Warburg, Christian, and Griese in 1935 (112). Later efforts by investigators such as Dickens (19), Lipmann (57), and

Dische (20) and the more recent work by Scott and Cohen (84, 85, 86),

Horecker (35, 39), and Racker (76) have made it possible to establish a detailed scheme which results in the conversion of pentose phosphate to hexose.

The pentose phosphate resulting from PGD, may be catabolized by pentose cleavage reactions or be metabolized via the reactions of the pentose cycle pathway (PC). If the enzymes of the pentose cycle are present, the pentose phosphate, formed from phosphogluconate decarboxylation becomes essentially a mixture of xylulose phosphate and ribose phosphate. These pentose moieties are formed through 11

an,epimerase and an is orne rase - enzyme acting upon ribulose phosphate, which is the first intermediate of phosphogluconate decarboxylation. Through a series of enzymatic reactions, involving the enzymes transketolase and transaldolase, the pentose phosphate is converted to fructose -6- phosphate and triose phosphate. Triose phosphate, by the action of aldolase, may also finally be converted to fructose -6- phosphate. In either case, the fructose -6- phosphate thus formed may be further catabolized via the reactions of the pentose cycle or those of the EMP pathway. The relative positions of carbon atoms in fructose -6- phosphate, derived from the gluconate catabolized via the pentose cycle pathway, have been determined by

Beevers (7). These relationships are illustrated in Figure 4. Alternatively glyceraldehyde -3- phosphate can be metabolized via phosphoglycerate and phosphoenolpyruvate to pyruvic acid.

Some other pathways related to the pentose phosphate cycle pathway have been found in biological systems. Studies with a mutant of Acetobacter xylinum (83) grown on glucose have shown the presence of an enzyme which, under anaerobic conditions, carried out a phosphorolytic cleavage of fructose -6- phosphate, as follows:

fructose -6- phosphate + inorganic phosphate acetyl phosphate + erythrose -4- phosphate + H2O

This enzyme also splits xylulose -5- phosphate to form acetyl 12

IGLUCONATE

IçooH 2C 3Ç

45 GLUCOSE I ENTNER- DOUDOROFF PATHWAY 45 t

ICHO I CHO 6 CH2OH ICOOH ICOOH 2C 26 2? 2C0 36 ------3C 302 4C 4V 46 4Ç 56 5Ç 5C 5C 6 CH2OH 6CH2oP 6CH2OP I CO2 6 CH2OP Glucose-6-P 6-P-Gluconote 2- keto- 3- deoxy Labeling Pottern -6-P-Gluconote \ in Reformed Hexose IC 2Ç 2Ç 6Ç

2 CO= 3C0 3 CO 5Ç0 2C 3C ` - 2C 3Ç 4C 36 Pyruvote 4 6 4C 4Ç 4C 46 5C 5C 5C 56 5C IçoOH 6 CH2OP 6C 6C 6C 6 CH2OP 2C=0 --` Fructose -6 -P Pntose Phosphate 3613 PENTOSE CYCLIC - \ PATHWAY 4CH0 5CHOH 4 CHO 6CH2OP 5CHOH Glycero!dehyde-3-P 6 CH2OP Glycera ldehyde - 3-P

Dihydroxyocetone - P ( ICH2OP I CH2OP 0 2C=0 2C=0 4 CH3 -C- COON `-` -s___ 3 CH2OH -- 4C + 4CH0 IPYRUVATEI 56 5 CHOH 6 CH2OP 6CH2OP Fructose -I, 6 -P Glyceroldehyde- 3-P to- EMBDEN- MEYERHOF PATHWAY

Figure 4. The Pentose cycle and Entner- Doudoroff pathways: fate of individual carbon atoms. 13 phosphate and glyceraldehyde -3- phosphate, a reaction also carried out by an enzyme from Lactobacillus pentosus (34). Leuconostoc mesenteroides converts one molecule of glucose to one molecule each of CO2, ethanol, and lactic acid, according to the following scheme:

1 C 1 CO2 2

2 C 2 CH3 ( ethanol) 3 3 C H2 OH

4 C 4 COOH

5 C 5 CHOH (lactate)

6C 6 CH3

The glucose degradation appears to proceed via the pentose cycle pathway to CO2 and xylulose -5- phosphate, which is in turn cleaved to give rise to C2 and C3 fragments (29).

Methodology of Pathway Analysis

In the concurrent operation of two or more metabolic pathways for a given substrate, it is essential to know the relative contribution of each of the pathways so that the function of the respective path- ways can be fully evaluated. Several approaches have been employed in the detection of specific pathways of carbohydrate catabolism in microorganisms. Isolation of a particular enzyme or enzyme system responsible for the specific conversion of a given 14 pathway intermediate has formed the basis for most of the earlier work in this connection. Another method, which has been success- fully applied in many studies, is the kinetic relationship between supplied precursors and product formation. Several methods have been devised for estimating the relative contribution of individual pathways to the overall catabolism of glucose (1, 2, 10, 11, 12, 13,

18, 44, 47, 53, 54, 95, 109).

The radiorespirometric method of Wang et al. (107, 110) is basically a study of the kinetics of concurrent pathways. This method has been tested in fungi, yeast, bacteria, insects, plants, and fruit (19, 21, 97, 110). In applying the radiorespirometric method to the study of pathway of carbohydrate catabolism in micro- organisms, important information is gained with regard to the quantitative importance of concurrent catabolic pathways through an analysis of the relative rates of combustion to CO2 of specifically 14C labeled carbohydrates or organic acid intermediates. One advantage of this method is that quantitative results regarding path- way distributions are obtained by utilizing intact systems where the organization of enzymes remains undisturbed. The nature of this method permits one to trace the routes of carbohydrate catabolism by an analysis of the rates and extents of respiratory CO2 evolved during successive time intervals. 15

Pathways of Carbohydrate Catabolism in Lactic Streptococci

Fermentation of Lactose

The initial step in the fermentation of lactose by lactic streptococci involves its hydrolysis to yield glucose and galactose.

Glucose is then susceptible to further catabolism, but galactose, if it is fermented, must first be converted to glucose -l- phosphate as outlined by Kandler (42):

1. galactose galactose - l- phosphate galactokinase + + adenosine triphosphate adenosine diphosphate

2. galatose- l-phosphate glucose -l- phosphate galactose-) -phos-

+ phate transurydilase Uridine diphosphate UDP- galactose (UDP)- glucose

UDP- galactose- 3. UDP -galactose UDP- glucose -4- epimerase

Lactose Fermentation by Homofermentative Lactic Acid Bacteria

Homofermentative utilization of glucose, produced from lactose as outlined above, proceeds via the EMP pathway. The theoretical yield of lactic acid via this route is two molecules per molecule of glucose fermented. By definition, homofermentative 16 microorganisms are those which utilize glucose via the EMP pathway and produce lactic acid almost exclusively as a terminal product of the fermentation.

The distribution of hexose carbon atoms in the lactic acid molecules after fermentation via the EMP scheme has been deter- mined by means of radioactive tracer methods (42) . The results of such studies are shown below:

1 CHO 1 CH

2 CHOH 2 CHOH

I I 3 CHOH 3 COOH 1 4 CHOH 4 COOH

I ( 5 CHOH 5 CHOH

( 6 CH2 OH 6 CH3 glucose L( +) lactic acid

Lactic streptococci, which would be classified as homofermentative, are S. lactis, S. diacetilactis, S. cremoris, S. faecalis, S. durans,

S. thermophilus, and S. liquefaciens (74), L. casei, L. plantarum, and L. helveticus (23, p. 17) .

It has been demonstrated that the homofermentative organisms once believed to produce lactic acid exclusively as an end product of glucose metabolism, produce small amounts of other compounds.

For example, Friedman (24) demonstrated the production of formic acid, acetic acid and ethanol by S. faecalis in peptone medium 17 enriched with glucose. Gunsalus and Niven (30) showed that at an alkaline pH, homofermentative organisms can produce up to 40% formic acid, acetic acid, and ethanol in the ratio of 2 :1:1. In addition to lactic acid Platt and Foster (74) reported that certain strains of S. faecalis (homofermentative) produce acetic acid, formic acid, carbon dioxide, ethanol, diacetyl, acetoin, and 2 :3 butanediol. Platt and Foster also found (74) that a strain of S. cremoris produced acetic acid, formic acid, carbon dioxide, and ethanol, while a strain of S. lactis produced acetic acid, carbon dioxide, ethanol, and acetoin. S. faecalis, S. cremoris, and S. lactis produced 7. 4, 6. 4 and 2.8 mmoles of ethanol per 100 mmoles of glucose fermented respectively. The approximate molar equiva- lence of ethanol and CO2 yields led Platt and Foster to speculate that carbon dioxide arises from the conversion of pyruvic acid to ethanol. Homofermentative streptococci were reported by Gunsalus and Wood (31) to produce highly active alcohol dehydrogenase. The presence of acetaldehyde, the immediate product of decarboxylation of pyruvic acid, has only recently been demonstrated in cultures of homofermentative lactic streptococci. Harvey (33) has reported that in milk medium strains of S. lactis, S. cremoris, and S. diacetilactis produced acetaldehyde.

Galesloot (25) has suggested that under conditions of low 18 oxygen tension where reduced pyridine nucleotides accumulate, the conversion of acetyl phosphate to ethanol would afford L. citrovorum a means of regenerating pyridine nucleotides. In this regard,

Lindsay et al. (55) demonstrated that added acetaldehyde was stimulatory to the growth of L. citrovorum, and they postulated that acetaldehyde was reduced to ethanol thereby regenerating the pyridine nucleotides.

Pyruvic acid is a key intermediate in the catabolism of glucose. Marth (68) summarized the reactions which pyruvic acid can undergo during the course of homofermentative catabolism.

Pyruvic acid can be reduced to lactic acid, and this is the pre- dominant reaction which takes place. Pyruvic acid can also be converted to alanine by means of a reductive amination. Malic acid can be produced by the carboxylation and reduction of pyruvic acid, carbon dioxide being fixed in this reaction. Malic acid can then be converted to oxaloacetic acid, an important precursor of other amino acids.

Another possible fate of pyruvic acid is its conversion to phosphoenolpyruvic acid which may then be carboxylated to form phosphoenoloxaloacetic acid and then oxaloacetic acid. The forma- tion of oxaloacetic acid by this route is necessary to the lactic acid bacteria since they are unable to utilize the citric acid cycle for the formation of this acid. Acetaldehyde, ethanol, alpha - acetolactic 19 acid, and acetoin may also arise from pyruvic acid. As mentioned by Reiter and M$ller- Madsen (79), there is some evidence to indicate that some homofermentative lactic streptococci are also able to utilize glucose via the ED pathway. This route would provide another mode of synthesis of ethanol and carbon dioxide which are known to be metabolic end products of these organisms.

Lactose Fermentation By Heterofermentative Lactic Acid Bacteria

Heterofermentative utilization of lactose results in a consider- able amount of end products other than lactic acid. Heterofermenta- tive lactic acid bacteria produce predominantly D( -) lactic acid in contrast to the production of predominantly L( +) lactic acid by the homofermentative organisms (25). Glucose is considered to be utilized by heterofermentative organisms by way of the hexose monophosphate pathway as outlined by Kandler (42). Heterofermenta- tive bacteria are forced to use this pathway for glucose metabolism because they lack the enzyme aldolase. Aldolase is required to catalyse the conversion of fructose 1 :6 diphosphate to dihydroxy- acetone phosphate and glyceraldehyde -3- phosphate in the EMP scheme. Typical heterofermentative lactic streptococci are L. citrovorum and L. brevis.

Reiter and M$ller- Madsen (79) have suggested that in addition to homofermentative and heterofermentative organisms there should 20 be a third class of lactic acid bacteria, the facultative homofermenta- tive types. This class includes those organisms that have the en- zymes necessary for glucose fermentation via either the EMP or HMP scheme, but utilize the EMP pathway nearly exclusively. Some evidence has accumulated to indicate that some strains, at least of

S. lactis and S. cremoris, have the ability to utilize the HMP path- way. Shahani and coworkers (92, 93, 94) have demonstrated key enzymes involved in both schemes for one strain of S. lactis. Also, at low glucose concentrations, L. casei appears to preferentially utilize the HMP pathway, and growing cells metabolize more glucose this way than by the EMP scheme (79).

The production of acetone by strains of S. lactis, S. cremoris, and S. diacetilactis has been demonstrated by Keenan (45). Harvey

(33) reported that S. cremoris and S. lactis produced acetone, while

S. diacetilactis was only able to utilize acetone. A known route for synthesis of acetone involves a condensation of acetyl phosphate and active acetate to yield beta -ketobutyricacid, which is then decar- boxylated to yield acetone, Pyruvic acid serves as the precursor of both acetyl phosphate and active acetate. Clostritium butylicum is an example of an organism which utilizes this pathway (75, p. 340-

344).

The biosynthesis of diacetyl has been reviewed by Seitz (87, p.

95). Several of the precursors in the synthesis of diacetyl, as 21 outlined by Seitz, have been isolated from Chedder cheese. This list of precursors includes oxaloacetic acid, pyruvic acid, alpha acetolactic acid (4), acetaldehyde, and acetoin (73).

Decarboxylation of Alpha -keto Acids

Decarboxylation of alpha -keto acids has been studied in yeast since the discovery of Neuberg and Hildesheimer (69) that pyruvic acid was converted by this organism to ethanol and carbon dioxide.

Acetaldehyde was identified as the fermentable intermediate in this reaction (70). The enzymatic nature of this process was proved when the same reaction was achieved with acetone killed yeast, and toluene- treated cells. Until this time all decarobxylation by micro- organisms was thought to be limited to the formation of amines from amino acids by putrefactive bacteria.

Testing of additional keto acids revealed that alpha -keto acids were especially susceptible to decarboxylation, giving support to Ehrlich's suggestion that amino acids are metabolized by way of their respective keto acids (71). Neuberg and Karczag postulated that the metabolism of alpha -keto acids in the absence of sugar

resulted in aldehydes which could be used - further in synthesis, condensation, and catabolism.

Lohman (59) was the first to show the requirement for magnesium ions for the alcoholic fermentation. Auhagen (3) obtained 22 organic cocarboxylase which was essential in the alcohol fermentation. This cocarboxylase was also discovered in liver, heart and kidney tissues. Lohman and Schuster (60, 61) obtained a crystalline substance which was necessary for the decarboxylation of pyruvate +2 in combination with carboxylase and Mg and identified the sub- stance as thiamine pyrophosphate (TPP) hydrochloride. Green,

Herbert, and Subrahmanyan (28) isolated and purified carboxylase from yeast as a diphosphothiamine -magnesium -protein. Alpha -keto acids other than pyruvic acid were decarboxylated by this enzyme, among which were alpha -ketoisovaleric acid, alpha -ketoisocaproic acid, as well as the dicarboxylic acids such as oxaloacetic and alpha - ketoglutaric acids. Alpha -ketoisovaleric acid was utilized almost as well as pyruvic acid but alpha -ketoisocaproic acid was attacked at only one twenty -fifth the rate. Oxaloacetic acid was about one - third as active as pyruvic, but the next higher homologue, alpha - ketoglutaric acid, was almost inactive.

King and Cheldelin (46) showed that Acetobacter suboxydans possessed a yeast type of decarboxylase forming acetaldehyde from pyruvate and that this intermediate was then oxidized to acetate.

Carboxylase from A. suboxydans was more specific than the yeast preparation because this decarboxylated only pyruvic and alpha - keto butyric acids.

Suomalainen and Oura (102) demonstrated that the 23 decarboxylation order with baker's, brewer's, and pure yeast carboxylase could be expressed as follows: the velocity of decarboxylation varied inversely with the carbon number of the keto acid. Alpha -ketoisovaleric acid was the only branched chain keto acid investigated and it was decarboxylated slower than any other keto acid tested when disintegrated or intact yeast was used. Juni

(41) studied the mechanism for decarboxylation of alpha -keto acids using cell -free extracts of baker's yeast. He also found that the relative rate of decarboxylation was inversely proportional to the carbon number of the keto acid. The order from most to least rapid was pyruvic, alpha -ketobutyric, alpha -ketovaleric, alpha -keto- isovaleric.

Sentheshanmuganathan (91) studied the transaminating and decarboxylating capacities of crude cell- free extracts of Saccharomyces cerevisiae using aspartic acid, leucine, norleucine, isoleucine, valine, norvaline, methionine, phenylalanine, tyrosine, and tryptophane. Carbon dioxide and the corresponding aldehyde with one less carbon atom was formed from leucine, valine and their nor derivatives when added to a suspension of yeast and alpha - ketoglutarate. Cell -free extracts and purified yeast carboxylase rapidly decarboxylated alpha -ketoisovaleric, alpha -ketovaleric and alpha -ketoisocaproic acids, forming carbon dioxide and the corresponding aldehyde with one less carbon atom. Cell -free extracts 24 reduced butyraldehyde, isobutyraldehyde, valeraldehyde, isovaleraldehyde and hydroxyphenyl acetaldehyde in the presence of dihydronicotinamide adenine dinucleotide (NADH) producing the corresponding alcohol; the rate of reduction decreased with increasing chain length. Thus, according to these reactions, the corresponding alcohol was produced from alpha -keto acids through the intermediate formation of the aldehyde. Sasaki (82) found a dec arboxylas e enzyme in Proteus vulgaris which converted alpha -ketoisocaproate to 3- methyl butanal and carbon dioxide. This enzyme decarboxylated alpha- ketoisocaproic acid but did not react with pyruvic or alpha- ketoglutaric acid.

In his studies, Lipmann (56) used Lactobacillus delbrueckii due to its powerful pyruvic acid dehydrogenation system which was stable during acetone drying. Five components were found to be essential for decarboxylation and dehydrogenation of pyruvate with oxygen as the ultimate hydrogen acceptor; these components were TPP, flavin +2, adenine dinucleotide (FAD), Mn +2 or Mg +2 or Co protein(s) and inorganic phosphate. Acetyl phosphate was the product of the reaction which was later shown to arise from a phosphotransacetyla - tion between acetyl coenzyme A and inorganic phosphate (98).

Although Colstridium kluyveri was the main organism investigated by

Stadtman and coworkers (98), the presence of phosphotransacetylase was also demonstrated in L. delbrueckii, Proteus morganii, 25

Clostridium tetanamorphum, Clostridium butylicum, and Escherichia coli but was not detected in yeast.

Stumpf (101) demonstrated an enzyme from partially purified cell -free extracts of Proteus vulgaris which catalyzed the oxidative decarboxylation of pyruvate in the presence of TPP and a bivalent metal ion. O'Kane and Gunsalus (72) studied the metabolism of pyruvate by S. faecalis -10C1 when grown on a synthetic medium and identified lipoic acid as the factor present in yeast extract which activated the pyruvic oxidase apoenzyme present in the cell. The product of this reaction was acetyl coenzyme A. Koike, Reed and

Carrol (48) and Koike and Reed (49) obtained purified coenzyme A

(CoA) and nicotinamide adenine dinucleotide (NAD) linked pyruvate and alpha- ketoglutarate dehydrogenation systems from Escherichia coli extracts as multienzyme units. Protein bound lipoic acid and

FAD were present as separate complexes but both were necessary for the two substrates.

The multienzyme NAD- lipoate- linked pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to acetyl coenzyme A in aerobic microorganisms. This complex is associated with the cell membranes of bacteria and has been isolated in pure form by Jagannathan, Scheweet, Reed, Koike, and collaborators (67) and characterized as multicomponent aggregates of definite composition. PDC from E. coli has been separated by 26

Reed and Koike (50) into the following three subunits, each exhibiting a separate set of enzymatic activities: a. Sixteen molecules of pyruvate decarboxylase containing enzyme -bound TPP; b. One aggregate of lipoic reductase -transacetylase containing lipoyl residues linked in amide linkage to a -amino groups of lysine resi- dues on the protein; and c. Eight molecules of dihydrolipoate dehydrogenase containing enzyme -bound FAD, two per molecule. The PDC can be reconstituted as the enzymic and morphologically intact form from its completely dissociated subunits. The reactions catalyzed by the PDC are the following:

Mg+2 CH3COCOOH + Ea. TPP _ _ _ _ ` Ea. TPP -CHOH -CH3+ CO2

OH Ea. TPP-Ç-CH3 + Eb NH-CO-( CH2) .- H S S

Ea. TPP + Eb NHCO( CH2) 5 f.) CH3COS SH

EbNHCO( CH2) + CoASH 5 CH3COS SH

EbNHCO(CH2) + CH3COSCoA 5 1 SH SH 27

EbNH-CO(CH2)5 + Ec. FAD SH SH

EbNH-CO(CH2)5 ( + Ec. FADH2

Ec. FADH2 + NAD+ Ec. FAD + NADH + H+

Net. PDC (Ea, b, c) CH3COCOOH + NAD+ + CoASH Mg+2 TPP-lip ,

CoASCOCH3 + NADH + H+ + CO2

Standard free energy change a Go = -9.38 Kcal /mole

Jackson and Morgan in 1954 (40) reported that the malty aroma produced in milk by S. lactis var. maltigenes was due mainly to 3- methylbutanal. Investigation of the mechanism of formation of this aldehyde (65, 66) revealed that leucine in the presence of alpha - ketoglutaric acid and resting cells of S. lactis var. maltigenes was transaminated to alpha -ketoisocaproic acid and that this keto acid was decarboxylated to 3- methylbutanal. Although both S. lactis and

S. lactis var. maltigenes posessed a transaminase system, the 28 decarboxylase enzyme was thought to be lacking in S. lactis. Experi- ments with acetone powders of both these organisms confirmed this hypothesis and established a requirement for Mg +2 and thiamine pyrophosphate when dialyzed acetone powders were used for manometric determination of the CO2 evolved during the decarboxyla- tion of alpha -ketoisocaproic acid. The authors pointed out the similarity of the S. lactis var. maltigenes decarboxylating systems to that of yeast. Resting cell studies of the conversion of isoleucine, valine, methionine, and phenylalanine by S. lactis, S. lactis var. maltigenes, and S. cremoris indicated that only traces of the respec- tive aldehydes were produced by organisms other than S. lactis var. maltigenes (64). A comprehensive study of possible differentiating characteristics between malty and non -malty strains of S. lactis

(27) led Gordon et al. to conclude that the most outstanding useful characteristic for separating these organisms was the production of aldehyde from an alpha -keto acid by the malty strain.

Tucker (103) studied the decarboxylation of alpha -keto acids by

Streptococcus lactis var. maltigenes. Alpha -keto acids with 5 to 6 carbon atoms were non - oxidatively decarboxylated by resting cells and cell free extracts of S. lactis var. maltigenes at rates most rapid for branched chain substrates and particularly those branched at the penultimate carbon atom. Unbranched alpha -keto acids were utilized at rates directly proportional to their carbon number. The rate of 29 decarboxylation of alpha- ketoisocaproate was more rapid than alpha - ketoisovalerate when resting cells were used. Pyruvate and alpha - keto butyrate were oxidatively decarboxylated by resting cells at

rates faster than and as fast as that for alpha -ketoisocaproate respectively. Decarboxylation but not oxidation of pyruvate occurred

with extracts in the presence of ferricyanide. A mixed substrate experiment suggested that the same enzyme was involved in

decarboxylation of alpha- ketoisocaproate and alpha- ketoisovalerate

but that a different mechanism was probably involved in decarboxyla-

tion of pyruvate. Decarboxylation of pyruvate appeared to be an important reaction for the formation of CO2 in cultures of both

malty and non -malty S. lactis. 30

MATERIALS AND METHODS

Microbiological Methods

Cultures Used

Single- strain organisms were available from the stock culture collection in the Department of Microbiology, Oregon State Univer- sity. These were Streptococcus lactis strains E, C2F, 144 -54,

ML -8 and R- 24(e), Streptococcus cremoris strains Ml -W1 -3 and

144F and Streptococcus diacetilactis strains M- 8- 224(a) and 18 -16.

All cultures were maintained in 10 ml of sterile (1210 C for 12 minutes) inhibitor -free ten percent nonfat milk by transfer of one percent inoculum every other day followed by incubation at 18 hours at 21° C; the mature cultures were held at 2° C between weekly maintenance transfers. Lactic broth was used for the mass production of the cells of lactic streptococci and this medium had the following composition: tryptone 20 g, yeast extract 5 g, gelatin 2.5 g, glucose 5 g, lactose

5 g, sucrose 5 g, sodium chloride 4 g, sodium acetate 1.5 g, tween -80 0.5 ml, and distilled water 1, 000 ml. The final pH of the medium was adjusted to 7.0. One percent inoculum in lactic broth was used for the large scale production of cells. The cultures were incubated for 16 to 18 hours at 21° C which allowed the cells to 31 traverse the log growth phase and achieve a maximum population.

The cells were harvested by centrifugation at 5, 000 rpm for 10 min

in a refrigerated centrifuge, washed twice and resuspended either in 0.1 M pH 7.0 phosphate buffer or when radiorespirometric experiments were performed, in carbohydrate free lactic broth at pH 7.0.

Enzymatic Methods

Preparation of Cell -Free Extracts

Cell -free extracts were prepared by disrupting washed cells for 15 to 20 minutes in a 10 -KC sonic oscillator. Cell debris was

removed by centrifugation at 15, 000 rpm for 20 min in a refrigerated centrifuge set at 2° C. Cell -free extracts were also prepared using an Eaton press and the cell debris removed in the same way as described above.

Spectrophotometric Analysis

Absorption spectra were determined using a Beckman model

DU spectrophotometer equipped with an automatic Gilford continuous recorder. All analysis and enzyme assays were carried out at room temperature (25 °C). 32

Aldolase

Total triose phosphate produced by aldolase activity in crude cell extracts was measured by the use of carbonyl trapping agent such as hydrazine. Hydrazine reacts with the triose phosphates quantitatively, thus preventing further enzymatic transformation and at the same time serves to displace the reaction equilibrium.

The triose phosphate hydrazone formed .. then was assayed in deproteinized filtrates by the method of Sibley and Lehninger (96) as modified by Beck (5, 6). Aldolase catalyzes the following reaction:

H OPO3H2 H-C OPO3H2

:I ` C=O C=O I CHI OH HO-C-H aldolase H--OH dihydroxyacetone phosphate I H-C-OH + ( H- OPO3H2 Hi=O O H-C-OH I fructose 1:6 diphosphate H-CA-OPO3H2

glyceraldehyde-3-phosphate

The reagents used were as follows:

(a) Fructose 1:6 diphosphate (FDP) - 0.05 M, pH 8.6

(b) Hydrazine sulfate - 0.22 M, pH 8.6 33

(c) Tris (hydroxymethyl) aminomethane (Tris) buffer - 0. 1 M,

pH 8.6

(d) Sodium hydroxide - 0.75 N

(e) 2, 4- dinitrophenylhydrazine (DNP) - one gram was dissolved

in and diluted to one liter with 2 N HC1

(f) Enzyme:Cell -free extract was used

(g) Trichloroacetic acid (TCA) - 20% aqueous solution (w /v)

Table 1 shows the experimental design used in carrying out the aldolase assays.

Table 1. Experimental design for the assay of aldolase. Experimental Control Additions (ml) (ml)

Tris buffer 1.0 1.0

Fructose 1 :6 diphosphate 0.25 Hydrazine sulfate 0.25 0.25 Enzyme 0.5 0.5

Total Volume 2.0 1. 75a

a See text.

Tubes containing buffer, substrate, hydrazine, and enzyme were incubated for 15 minutes at 38o C. The reaction was then terminated by the addition of 2 ml of 20% Trichloroacetic (TCA) acid and the 34

precipitated protein was removed by centrifugation. The control

sample was treated in an identical manner except that fructose 1 :6-

diphosphate was not added until after the addition of TCA. One

tenth ml of the protein -free filtrate was transferred to a cuvette and

one ml of 0. 75 N NaOH was added with mixing. The tubes were

allowed to stand at room temperature for ten min. One ml of 2,4

dinitrophenylhydrazine was added to all the tubes which were then

incubated for 60 min at 38° C. At the end of the incubation period,

7.0 ml of the NaOH was added and after ten min the optical density

was read at 550 mp. using the control as an instrument blank.

One unit of activity was defined as the amount of enzyme necessary to cause the increase of 0.001 absorbancy above the

control at 550 mµ under standard assay conditions. Specific activity was defined as the units of enzyme activity per mg of protein.

Triosephosphate Isomerase

The measurement of triosephosphate isomerase activity was determined by a coupled reaction with alpha- glycerophosphate dehydrogenase using glyceraldehyde -3- phosphate as the substrate

(8, 78). The following reactions were catalyzed by the enzymes: 35

CHO CH OH 410H \ triosephosphate isomerase L-20 CH2OPO3H21 CH2OPO3H2

D- glyceraldehyde -3- phosphate dihydroxyacetone phosphate

NADH + H+ NAD+ CH2OH HOCH \ alpha -glycerophosphate dehydrogenase CH2OPO3H2 alpha -glycerophosphate

The activity of triosephosphate dehydrogenase was measured by following changes in absorbancy at 340 mµ caused by the oxidation

of dihydronicotinamide adenine dinucleotide (NADH) using a Beckman model DU spectrophotometer supplied with a Gilford continuous recorder.

The following reagents, prepared in triethanolamine - HC1 buffer at pH 7. 9, were used:

(a) NADH - 2.56 x 10-4 M -3 (b) D- glyceraldehyde -3- phosphate - 1.17 x 10 M

(c) Alpha -glycerophosphate dehydrogenase - (One mg of protein

reduced approximately 60 p. moles of dihydroxyacetone

phosphate per min; 0.02 of this enzyme (0.2 mg) was used for assay) -2 (d) Triethanolamine -HC1 buffer - 2 x 10 M, pH 7. 9 36

Crude cell -free extract was assayed for triosephosphate isomerase activity using the experimental design shown in Table 2.

Table 2. Experimental design for the assay of Triosephos- phate isomerase.

Experimental Control Additions (ml) (ml)

NADH 1.0 1.0 Glyceraldehyde- phosphate 1.0 1.0

Alpha - glyce rophosphate dehydrogenase 0.02 Buffer 1.0 1.02 Triosephosphate isomerase (cell extract) 0.5 0.5 Total Volume 3.52 3.52

The reaction was started by the addition of substrate to the reaction mixture.

Alcohol Dehydrogenase

Alcohol dehydrogenase was assayed spectrophotometrically by measuring the change in absorption spectrum of NADH with respect to time, as a result of its oxidation to nicotinamide adenine dinucleotide (NAD) in the presence of acetaldehyde (77). The 37 following biochemical reaction was followed:

CH3CHO + NADH + H+ CH3CH2OH + NAD+ alcohol acetaldehyde dehydrogenase ethanol

The reagents used in the assay were as follows:

(a) Acetaldehyde - 0.01 M

(b) Phosphate buffer - 0.1 M, pH 7.2

(c) NADH - 2.56x10 -4M Cell -free extract was used to measure the alcohol dehydrogenase activity using the experimental design shown in Table 3.

Table 3. Experimental design for the assay of alcohol dehydrogenase.

Experimental Control Additions (ml) (ml)

Buffer 0.0 0.5

NADH 1.0 1.0 Cell -free extract 1.0 1.0 Acetaldehyde 0.5 0.0

Total Volume 2.5 2.5

All the reagents were prepared in phosphate buffer and the reaction was started by the addition of acetaldehyde. 38

Glucose -6- phosphate Dehydrogenase

The activity of this enzyme was measured by following the change in absorbancy with time at 340 mµ caused by the reduction of nicotinamide adenine dinucleotide phosphate (NADP) in the presence of glucose -6- phosphate (36, 38, 51, 52). This enzyme catalyzes the following reaction:

CHO COOH H çi OH NADP+ NADPH + H+ HC OH through the act HOCH HOCH S- lactone / 1 HCOH HCOH glucose -6- phosphate dehydrogenase HCOH HCOH CH2OPOH2 CH2OPO3H2 glucose -6- phosphate 6-phosphogluconate

The reagents used were prepared as follows:

(a) NADP+ - 2.8 x 10-3 M (2.8 µ. moles /ml)

(b) MgCl2 - 0.1 M

(c) Glycylglycine buffer - 0.25 M, pH 7.5 -2 (d) Glucose -6- phosphate - disodium salt at 1.5 x 10-2 M

The experimental protocal used to assay the crude enzyme extract for glucose -6- phosphate dehydrogenase was as shown in Table 4. 39

Table 4. Experimental design used in assays for glucose- 6- phosphate dehydrogenase.

Experimental Control Additions (ml) (ml)

Glycylglycine buffer 1.0 1.4

MgC12 0.1 0.1

Glucose -6- phosphate dehydrogenase (cell -free extract) 1.0 1.0

Glucose -6- phosphate 0.4 0.0

NADP 0.2 0.2

Total Volume 2.7 2.7

The reaction was initiated by the addition of substrate and the control was run without glucose -6- phosphate.

6-Phosphogluconate Dehydrogenase

The activity of 6- phosphogluconate dehydrogenase was measured by following the changes in absorbancy at 340 mµ caused by the reduction of NADP with respect to time (37, 111). This enzyme catalyzes the following reaction: 40

+2 M g 6 -phosphogluconate ' 3-keto-6-phosphogluconate

NADP+ ÑNADPH + H+

ribulose -5- phosphate + CO2

The reagents used to assay the cell -free extract were each prepared in 0.4 M, pH 7.5 glycylglycine buffer at the strengths indicated below:

(a) 6- phosphogluconic acid - 0. 1 M (potassium salt)

(b) NADP+ - 3.0 x 10-3 M

(c) Magnesium chloride - 0.02 M

Table 5 shows the experimental design used .. for assay of

6- phosphogluconate dehydrogenase.

Table 5. Protocal for assay of 6- phosphogluconate dehydrogenase.

Experimental Control Additions (ml) (ml)

Glycylglycine buffer 0.2 0. 3

NADP+ 0.1 0.1

MgC12 0. 1 0. 1

Enzyme 1. 0 1. 0

6- phosphogluconic acid 0.1 0.0

Total Volume 1.5 1.5 41

The reaction was started by the addition of 6- phosphogluconic acid to the experimental cuvette.

One unit of activity of triosephosphate isomerase, alcohol dehydrogenase, glucose -6- phosphate dehydrogenase, and 6- phosphogluconate dehydrogenase, was defined as the amount of enzyme necessary to cause 0.0001 change in absorbancy per minute under standard assay conditions. Specific activity was defined as the units of enzyme activity per mg of protein.

Acetate Kinase (acetokinase)

The method of Rose (81) was followed in measurement of this enzyme. This method depends on the ability of acyl phosphates to react with hydroxylamine to rapidly form hydroxamic acid which in turn is converted to a colored complex in the presence of ferric iron under acid conditions (58). The following reaction is catalyzed by the enzyme:

O CH3COOH CH3COPO3H2 acetate acetyl phosphate + adenosine triphosphate adenosine diphosphate

The reagents employed in the kinase assay were prepared as indicated below: 42

(a) Potassium Acetate - 3.2 M

(b) Tris (hydroxymethyl) amino methane (Tris) buffer - 1.0 M

(c) Magnesium chloride - 1.0 M

(d) Potassium hydroxide - 4.0 M

(e) Hydroxylamine hydrochloride - 28%

(f) Adenosine triphosphate (ATP) - 0.1 M

(g) Trichloroacetic acid (TCA) - 10%

(h) Ferric chloride - 1.25% in 1 N HC1 The stock substrate solution, prepared fresh as needed, consisted of potassium acetate, Tris buffer and magnesium chloride in a volume ratio of 25 :5 :1. A neutral solution of hydroxylamine was made by mixing equal volumes of the hydroxylamine hydrochloride and potassium hydroxide. Table 6 shows the experimental protocal used for the acetate kinase assays. The control reaction tube had

0.1 ml of buffer instead of 0.1 ml of ATP.

Table 6. Typical experimental design for acetate kinase assay.

Additions Volume (ml)

Stock substratea 0.3 Neutral hydroxylamine 0.35 Adenosine triphosphate 0.1 Tris -HC1 buffer 0.25

Total Volume 1.0 asee text. 43

The tubes were placed in a 29° C water bath, and 0.025 ml of cell- free extract was added to each one. After seven min the reaction was stopped by the addition of one ml of TCA; the precipitated protein was removed by centrifugation and the color was developed by addition of 4.0 ml of ferric chloride.

It was necessary to remove coenzyme A from the bacterial extracts assayed for acetate kinase. This was accomplished by treating the crude extract with 200 mesh Dowex ion exchange resin in chloride form. One half the volume of resin was mixed with one volume (10 ml) of bacterial extract and the mixture held in an ice bath for 30 minutes (15). The resin was then removed by centrifuga- tion; the final pH of the solution was 6.0.

One unit of this enzyme was defined as the amount necessary to cause an increase of 0.001 absorbancy above the control at

540 mµ under standard assay conditions. Specific activity was defined as units of enzyme activity per mg of protein.

Protein Determinations

Protein in the cell -free bacterial extracts was determined according to the method of Lowry (62). 44

Manometric Experiments

A Gilson Medical Electronics (GME) Warburg Respirometer

set at a temperature of 30o C was used in these experiments. The

direct method of Warburg (104) was used for measurement of CO2

evolution. Each Warburg flask contained 3. 2 ml of solution which

included 2. 5 ml of the resting cell suspension in 0. 1 M pH 7.0 phosphate buffer, and 0.5 ml of the substrate in the same buffer.

The center well of the Warburg flask, measuring oxygen uptake,

contained 0.2 ml of 20% KOH solution; this was replaced by

water in the flask being used for CO2 measurement. After a 15 min temperature equilibration period, the reaction was started by tipping the substrate into the main chamber of the flask. Results obtained were plotted as microliters (µl) of 02 uptake or CO2 evolution verses time using an appropriate scale. Control endogenous values for cells in the absence of substrate also were plotted.

Substrates for Manometric Experiments

Alpha -ketobutyric, alpha- ketocaproic, alpha- ketoisocaproic, alpha- ketovaleric, and alpha- ketoisovaleric acid were purchased from the California Corporation for Biochemical Research (Calbiochem); sodium pyruvate was obtained from the same source.

Stock solutions of the keto acids were prepared by weighing the 45 amount of each required to make a concentration of 750 µg of acid per 0.5 ml of distilled water. Stock solutions were frozen in screw -cap tubes until needed. Each Warburg flask contained 75Q µg of the alpha -keto acid.

Radiorespirometric Experiments

Cells grown in lactic broth for 12 to 14 hours to the logarith- mic stage of growth, were harvested by centrifugation and immedi- ately resuspended in the same carbohydrate -free medium at pH 7.0.

Glucose 1, 2, 3 4, and 6 -14C were made available by Dr. C. H.

Wang, Oregon State University and experiments were performed according to the method of Wang et al. (106, 110). A series of incubation flasks (50 ml capacity) were placed in a 30° C water bath, each flask containing cell suspension to which was added, from

a side arm of the incubation .. flask, the specifically labeled glucose substrate. Each flask contained 10 mg of the radioactive glucose and approximately 18 mg (dry weight) of cells in 10 ml of the medium. The shaking flasks were aerated with air at the rate of

60 ml per minute and metabolic CO2 was collected in the trap containing 10 ml of a mixture of ethanol and ethanolamine (2:1).

The trap solution was replaced every thirty minutes and processed for liquid scintillation counting of the radioactivity.

The trap solution containing the CO2 was diluted to 15 ml with 46 absolute ethanol. A 5 -ml aliquot was taken from the resulting solution and mixed with 10 ml of toluene containing p- terphenyl

(3 g per liter) and 1, 4-bis-Z' (5' - phenyloxazolyl) benzene (30 mg per liter) in a 20 ml glass counting vial. The scintillation mixture was counted by means of a Packard Tri -Carb model 314 -DC liquid scintillation counter. The photomultipler voltage set at 1150 volts and the pulse discriminater set at 10 to 100 volts (red channel) and

10 to infinity volts (green channel) in the two channels. The reading in the 10 to 100 volt channel was independent of minor quenching effects caused by color or other contaminants in the counting sample.

Counting was carried out to a standard deviation of no greater than

2 %.

At the end of the experiment, the cells and incubation media were separated from each other by centrifugation and aliquots of each were counted as thixotropic gel preparations according to the

method of White and Helf (113) . 47

RESULTS

Enzyme Experiments

The study of the key enzymes involved in. the EMP, HMP, and

ED pathways was carried out in order to evaluate the pathways

involved in the catabolism of glucose by lactic streptococci. Aldolase, triosephosphate isomerase, and alcohol dehydrogenase were

selected as the key enzymes of the EMP pathway and alcohol fermenta-. tion. Glucose -6- phosphate dehydrogenase was selected as the enzyme

representing both HMP and ED pathways and 6- phosphogluconate

dehydrogenase was selected as an enzyme exclusive to the HMP pathway. Evidence for the occurrence of phosphoroclastic

reaction in lactic streptococci was provided by studies of acetate kinase in these organisms.

Aldolase was found to be present in all the lactic streptococci

studied for the quantitative determination of the activity of this

enzyme. The results of the aldolase experiments are given in

Table 7.

The activity of triosephosphate isomerase was calculated from the initial rate of NADH oxidation measured at 340 mµ. The decrease

in absorbancy with respect to time as a result of NADH oxidation by

cell -free extracts of S. diacetilactis 18 -16, S. cremoris Ml -W1 -3, Table 7. Specific activity of aldolase in lactic streptococci.

Volume of cell -free Units/O. 1 ml of Units /4.0 ml of extract having Absorbancy deproteinized deproteinized one mg protein Specific Organism above control supernatant supernatant (ml) Activity

S. diacetilactis M-8-224( a) 0.175 175 7000 0.125 1750

S. diacetilactis 18-16 0. 140 140 5600 0. 151 1691

S. cremoris M1-W1 3 0.140 140 5600 0. 158 1769

S. cremoris 144F 0. 180 180 7200 0. 138 1987

S. lactis M-9-924 0. 150 150 6000 0. 138 1656

S. lactis C2F 0.170 170 6800 0.181 2461

S. lactis R24( e) 0.170 170 6800 0.135 1836

S. lactis 144-54 0. 130 130 5200 0. 106 1102

S. lactis ML-2 0. 195 195 7800 0.111 1731

S. lactis ML-8 0.430 430 17200 0. 111 3818

S. lactis BR-4 0. 145 145 5800 0. 206 2389 49

and S. lactis C2F is shown in Figures 5, 6, and 7, respectively.

The specific activity of triosephosphate isomerase in these organisms

is shown in Table 8. The change in absorbancy in the endogenous control may be due to the presence of endogenous alpha- glycero- phosphate dehydrogenase in lactic streptococci.

The activity of alcohol dehydrogenase was also calculated

from the initial rate of NADH oxidation measured at 340 mµ. The decrease in absorbancy with respect to time as a result of NADH oxidation by the cell -free extracts of the lactic streptococci is

shown in Figures 8, 9, 10, 11, 12, 13, and 14. The specific

activity of alcohol dehydrogenase in these organisms is shown in

Table 9. The endogenous experiment (control) showed no change in

absorbancy at the concentration of the cell -free extract used in these experiments.

The activity of glucose -6- phosphate dehydrogenase was cal-

culated from the initial rate of NADP reduction measured at 340 mµ.

The increase in absorbancy with respect to time as a result of NADP

reduction by the cell -free extracts of lactic streptococci is shown

in Figures 15, 16, 17, 18, 19, and 20. The specific activity of glucose -6- phosphate dehydrogenase in these organisms is shown in

Table 10. The endogenous (control) experiment did not show any

change in absorbency at the concentration of the cell -free extract used in these experiments. Table 8. Specific activity of triosephosphate isomerase in lactic streptococci.

Initial rate of change in absorbancy Units/0.5 ml of ml of cell -free extract per minute cell -free extract Specific activity having 1 mg Organism experimental endogenous experimental endogenous of protein experimental endogenous

S. diacetilactis 18-16 0.228 0.030 2280 300 0.151 688.5 90

S. cremoris M1-W1-3 0. 176 0.033 1760 330 0. 158 557.6 104

S. lactis C2F 0.133 0.033 1330 330 0,181 481.4 119 Figure Ó 0 A- 1 A OD at 340 mN.. 0.2 0.3 0.4 0.5 0.6 0.7 0. 5. 0 1 0 cell 18 Triosephosphate -16 -free assayed extracts 1 at isomerase 25° of Streptococcus C 2 Time as explained ( min) activity 3 diacetilactis observed in the text. in 4 51 Figure COD at 340 mil 0. 0.4 0.6 0.2 0.3 0.5 0. 0 1 7 6. o Ml.- cell Triosephosphate -free W1 -3 1 j extracts assayed isomerase at of Streptococcus 25° 2 1 Time C ( min) as activity explained 3 cremoris observed in the 4 in text. 52 Figure a 0 A OD at 340 mF. 0.1 0.2 0. 0.4 0.5 0.6 0. 0 3 7 7. - o assayed cell Triosephosphate -free at 1 i extracts 25° C isomerase as of explained 2 1 Time Streptococcus ( min) activity in 3 t the lactis observed text. C2F 4 in 53 Table 9. Specific activity of alcohol dehydrogenase in lactic streptococci.

Initial rate of Volume of cell -free extract change in absorbancy Units per ml of having 1 mg of protein Specific Organism per minute cell -free extract (ml) Activity

S. diacetilactis M-8-224( a) 0. 152 1520 0. 095 144. 4

S. cremoris M1-W1-3 0.235 2350 0. 125 293. 7

S. cremoris 144F 0. 105 1050 0.085 89.2

S. lactis E 0. 121 1210 0. 105 129. 0

S. lactis R-24(e) 0. 184 1840 0. 166 305.4

S. lactis 144-54 0. 190 1900 0. 114 216.6

S. lactis ML-8 0. 113 1130 0. 066 64. 6

4Vt 55

0.7

0.6

0.5

0.4 3. g

Va ó d 0.3

0.2

0. 1

0 ( ) ) 0 1 2 3 4 5 Time (min)

Figure 8. Alcohol dehydrogenase activity observed in cell - free extracts of Streptococcus diacetilactis M -8- 224(a) assayed at 25 C as explained in the text. 56

0.7

0.6

0.5

± 0. 4 o m aO 0. 3

0.2

0. 1

t 1 1 2 3 4 5 Time (min)

Figure 9. Alcohol dehydrogenase activity observed in cell- free extracts of Streptococcus cremoris Ml -W1 -3 assayed at 25° C as explained in the text. 57

0. 7

0.6

0.5

0.2

0. 1

t 1 I 1 I 1 2 3 4 5 Time ( min)

Figure 10. Alcohol dehydrogenase activity observed in cell - free extracts of Streptococcus cremoris 144F assayed at 25° C as explained in the text. Figure A OD at 340 m N., 0.6 0.7 0.4 0.5 0. 0.2 0.3 0 1 0 11. free at Alcohol 25° extracts 1 C dehydrogenase as explained of Streptococcus 2 in Time activity the (mint :text. 3 1 lactis observed E assayed in 4 I cell - 58 5 l 59

0.7 7

0.6

0.5

0.4

0.3 r

0.2

0. 1

i L i I I i 2 3 4 5 Time (min)

Figure 12. Alcohol dehydrogenase activity observed in cell - free extracts of Streptococcus lactis R -24(e) assayed at 25° C as explained in the text. 60

0.7

0.6

0.5

E 0.

G O

0.3

0, 2

0. 1

0 4 5 0 1 2 3 Time (min)

Figure 13. Alcohol dehydrogenase activity observed in cell- free extracts of Streptococcus lactis 144 -54 assayed at 25° C as explained in the text. AODat340mp, Figure 0.7 0.5 0.6 0.2 0.3 0.4 0. 0 1 o 14. assayed cell Alcohol 1 -free dehydrogenase at extracts 25° C 2 as :of Time explained Streptococcus activity ( min) 3 in observed the lactis text. 4 in ML -8 61 5 Table 10. Specific activity of glucose -6- phosphate dehydrogenase in lactic streptococci.

Initial rate of Volume of cell -free extract change in absorbancy Units per ml of having 1 mg of protein Specific Organism per minute cell -free extract (ml) Activity

S. diacetilactis M-8-224( a) 0. 580 5800 0. 125 725

1099 S. diacetilactis 18-16 0.728 7280 0. 151

1580 S. cremoris M1-W1-3 0. 500 5000 x 0. 158 2 = 10000 919 S. cremoris 144F 0. 333 3330 x 0. 138 2 = 6660 138 1407 S. lactis M-9-924 1. 020 10200 0.

S. lactis C2F 0.772 7720 0, 181 1397 Figure LOD at 340 mµ 0. 0.2 0.3 0.4 0.5 0.6 1 7 0 15. M- in Glucose cell 8- 224(a) 0.5 -free -6- I phosphate assayed extracts at Time of dehydrogenase 1. I 25° 0 Streptococcus ( min) C as explained activity 1.5 I diacetilactis in the observed text. 2.0 63 rX ggk Figure at 340 ni dOD N., 0.1 0.2 0.3 0.4 0.6 0.5 0.7 0 - 16. in Glucose assayed cell 0.5 -free -6- at phosphate 25° extracts C as Time explained of dehydrogenase 1.0 4 Streptococcus ( min) in the text. activity 1.5 l diacetilactis observed 2. 18 0 64 -16 Figure

A OD at 340 mp. 0. 0.2 0.3 0.4 0.5 0.6 0.7 17. 1 0 Ml- in Glucose cell W1 -free -3 -6- assayed 0.5 phosphate extracts at 25° of dehydrogenase Streptococcus Time C 1.0 as (min) explained activity cremoris in 1.5 the observed text. 65 2.0 Figure AOD at 340 miA, 0. 0.2 0.3 0.4 0. 0.6 0.7 1 18. 0 assayed in Glucose cell -free -6- at 0,5 25° phosphate extracts C as explained Time of dehydrogenase Streptococcus (min) 1.0 i- in the text. activity cremoris 1.S observed 144F 66 2.0 Figure O A OD at 340 m p. 0. 0.3 0.4 0.5 0.6 0.2 1 19. IMP 0 assayed in Glucose cell -free -6- at 0.5 phosphate 25° - extracts C as explained of dehydrogenase Streptococcus Time 1.0 l ( min) in the text. activity lactis 1.5 M observed -9 -924 2. 67 0 Figure 340 AOD at mi,l., 0. 0.2 0.3 0.4 0.5 0.6 0.7 1 20. 0 r r assayed in Glucose cell -free -6- at 0.5 25° phosphate extracts C as Time explained of dehydrogenase (min) Streptococcus 1.0 in the text. activity lactis 1.5 C2F observed 68 2.0 69

The cell -free extracts of S. cremoris M1 -W1 -3 and 144 F showed a high turbidity therefore only 0.5 ml of the cell -free extract of these organisms was taken for the above experiment. The units of activity per ml for both the above strains of S. cremoris is cal- culated by multiplying the units per 0.5 ml obtained in the experi- ments by a factor of 2 as shown in Table 10.

The activity of 6- phosphogluconate dehydrogenase was also calculated from the initial rate of NADP reduction measured at 340 mµ. The increase in absorbancy with respect to time as a result of

NADP reduction by the cell -free extracts of lactic streptococci is shown in Figures 21, 22, 23, 24, 25, 26, and 27. The specific activity of 6- phosphogluconate dehydrogenase in these organisms is shown in Table 11. The endogenous experiment showed no change in absorbancy at the concentration of cell -free extract used in these experiments. Both glucose -6- phosphate dehydrogenase and 6- phosphogluconate dehydrogenase in lactic streptococci were found to be NADP specific.

Acetate kinase was found to be present in all the lactic streptococci examined quantitatively for this enzyme. Before performing the assay for acetate kinase, the cell -free extract was treated with anion exchange resin to remove coenzyme A since the acetate activating enzyme gives rise to aceto- hydroxamic acid in the Table 11. Specific activity of 6- phosphogluconate dehydrogenase in lactic streptococci.

Initial rate of Volume of cell -free extract change in absorbancy Units per ml of having 1 mg of protein Specific Organism per minute cell -free extract (ml) Activity

S. diacetilactis M-8-224( a) 0. 150 1500 0. 095 142. S

S. cremoris M1-W1-3 0.071 710 0.125 89

S. cremoris 144F 0.140 1400 0.085 119

S. lactis E 0.050 500 0. 105 52

S. lactis R 24(e) 0.130 1300 0. 166 216

S. lactis 144-54 0. 150 1500 0. 114 171

S. lactis ML-8 0. 160 1600 0. 066 106

0 71

0.6

0.5

0.4 i

S 0.3 G

0.2

0. 1

i r o I 2 3 4 Time ( min)

Figure 21. 6- Phosphogluconate dehydrogenase activity observed in cell extracts of Streptococcus diacetilactis M- 8- 224(a) assayed at 25° C as explained in the text. Figure

22, A OD at 340 m p. 0.5 0.6 0.4 0.2 0.3 0. Ml in 6- 1 Phosphogluconate o cell -W1 -free -3 assayed extracts 1 at dehydrogenase 25° of Streptococcus Time C 2 as ( min) explained activity 3 cremoris in the observed text, 4 72 Figure

L OD at 340 m N., 23. assayed in 6- Phosphogluconate cell -free at 25° extracts C as explained dehydrogenase of Streptococcus in the activity text. cremoris observed 144F 73 Figure

LSOD 24. at 340 mµ 0. 0.2 0.3 0.4 0.5 0.6 at in 6- 1 Phosphogluconate low 25° cell -free C as explained extracts dehydrogenase of in Streptococcus Time the 1 2 (min) text. activity 3 1 lactis observed E assayed I 4 74 Figure

25. AODat340mp.. 0. 0.2 0.3 0.4 0.5 0.6 assayed in 1 6- Phosphogluconate cell -free at 25° extracts 1 C as explained dehydrogenase of Time Streptococcus 2 ( min) in the activity text. 3 lactis observed R -24(e) 4 75 Figure

26. A OD at 340 mp, 0.6 0.3 0.4 0.5 0. 0.2 assayed in 6- 1 0 IIMI Phosphogluconate cell -free at 250 extracts 1 C as explained dehydrogenase of Time Streptococcus 2 1 ( min) in the activity text. 3 1 lactis observed 144 -54 4 1 76 Figure

27. A OD at 340 m Ft 0.S 0.6 0. 0.2 0.4 in assayed 6- 1 Phos.phogluconate r 0 cell -free at 15° extracts 1 C as explained dehydrogenase of Streptococcus Time 2 ( min) in the activity text. 3 lactis observed ML -8 4 77 78 presence of catalytic concentrations of CoA. The results of acetate kinase experiments are given in Table 12. Lactic streptococci have been classified in the past as micro -

aerophils (80), because they grow best in the presence of low con-

centrations of oxygen. An experiment was performed to study the

effect of continuous aeration on the growth of these organisms.

S. diacetilactis M- 8- 224(a) was selected for this experiment. This

organism was inoculated into two two -liter flasks, each containing

one liter of lactic broth, using a one percent inoculum in each case.

One of these two flasks was equipped with a device that bubbled filtered air constantly through the medium. The other flask, which

did not have air bubbling through its medium, was used as a control.

Both these flasks were incubated in a water bath set at 210 C. Samples were aseptically withdrawn, every two hours, simultaneously

from each of the two flasks. Appropriate dilutions of the sample

were plated on lactic agar and the number of colonies on each plate

counted after incubating the plates at 30o C for 48 hours. The results

of this experiment, given in Table 13, suggested that the continuous

aeration of the medium did not alter appreciably the growth of

S. diacetilactis M- 8- 224(a) in lactic broth. Table 12. Specific activity of acetate kinase in lactic streptococci.

Volume of cell -free extract Absorbancy Units/0. 025 ml of Units per ml of having 1 mg protein Specific Organism above control cell -free extract cell -free extract (ml) Activity

§.. diacetilactis M-8-224( a) 0. 595 595 23, 800 0. 095 2261

S. cremoris M1-W1-3 0. 105 105 4, 200 0. 125 525

S. cremoris 144F 0. 565 565 22, 600 0. 085 1921

S. lactis E 0. 300 300 12, 000 0. 105 1260

S. lactis R-24(e) 0.445 445 17, 800 0. 166 2955

S. lactis 144-54 0. 620 620 24, 800 0. 114 2827

S. lactis ML-8 0. 630 630 25, 200 0. 006 1663 80

Table 13. Affect of continuous aeration on the growth of S. diacetilactis M- 8- 224(a)

Counts per ml

Time in hours Experimental Control

0 0.44x107 0.42x1Ó

2 1.89x.107 2.05x107

4 5.01x107 4.-.99x107

6 19.6 x 107 25.4 x 107

8 56.0 x 107 55.0 x 107

10 63.0 x 107 76.0 x 107

12 68.0 x lÓ 70.0 x 107

14 67.0 x 107 74.0 x 107

16 78.0 x 107 80.0 x 107

18 60.0 x 107 67.0 x 107

20 74.0 x 107 126.0 x 107

24 89.0 x 107 96.0 x SÓ 81

Manometric Experiments

The rate of decarboxylation of pyruvate and other alpha -keto

acids was determined by manometric measurement of oxygen uptake

and carbon dioxide evolution according to the direct method of

Warburg using 750 µg of the keto acids as substrate in each experi- ment. Each vessel contained 2.3 ml of resting cell suspension in

0.1 M pH 7.0 phosphate buffer (approximately 115 mg dry wt per

flask). The reaction was initiated by tipping neutralized keto acid from the side arm. Substrate controls containing water in place of

cells and endogenous controls containing water in place of substrate were included in each run for both oxygen uptake and CO2 evolution.

The substrate control values were negligible in all the cases. The endogenous control values were plotted together with the experimental

values. At the end of the experiment, 0.5 ml of 0.1 M sulfuric acid

was tipped in from the second side arm of the flask in order to

release the absorbed CO2. All the procedures used can be found in

the fourth edition of Manometric Techniques (104). The lactic streptococci used for the study of the oxidative

decarboxylation of pyruvic and alpha- ketobutyric acids were S.

diacetilactis strains M- 8- 224(a) and 18 -16, S. cremoris Ml -W1 -3

and 144F, and S. lactis E and M -9 -924. The oxidative decarboxyla-

tion of alpha-ketovaleric, alpha-ketoisovaleric, alpha-ketocaproic 82 and alpha -ketoisocaproic acids was studied with the resting cells of S. diacetilactis M- 8- 224(a).

The results of the keto acid experiments are shown graphically in figures 28 through 43.in which the micro liters of oxygen uptake and CO2 evolution were plotted against time. Data, from which these curves were obtained, are presented in Tables 14 through 29. The theoretical oxygen uptake and CO2 evolution was calculated as follows: pyruvic acid was selected as an example to show the details of the calculations.

Molecular wt of pyruvic acid = 88

Amount of pyruvic acid used in the experiments = 750 µg

88 µg of pyruvic acid = 1 µ mole

therefore 750 µg of pyruvic acid will be equal to

750 moles .= µmoles 88 µ 8,52 therefore 8.52 µ moles of pyruvic acid was taken for the experiment.

According to the oxidative decarboxylation reaction of pyruvate to form acetyl Co A, one p. mole of pyruvic acid should take up one - half µ mole of oxygen. Therefore 8.52 µ moles of pyruvic acid should take up 4. 26 µ moles of oxygen. One µ mole of oxygen under standard temperature and pressure = 22.4 p. liters. Therefore, 22.4 x 4.26 =

95.4 will be the µl of oxygen that should theoretically be taken up when 750 p.g of pyruvic acid is used for the experiment. The 83 theoretical value for the CO2 evolution was obtained in the same way by making the assumption that one micro mole of pyruvic acid yields one p. mole of CO2. The theoretical oxygen uptake and CO2 evolution values for all the rest of the keto acids used was calculated in exactly the same manner. Percent recovery of 02 and CO2 was calculated by assuming the theoretical values to be 100 %. 84

Table 14 (a). Uptake of oxygen by cells of S. diacetilactis M -8 -224( a) metabolizing pyruvate.

Time Oxygen Oxygen Cumulative Theoretical Percent Experimental Endogenous Recovery Oxygen Recovery (Exp -Endo) Uptake min p.1 41 N.,1 µ 1

10 42 16 26 20 68 28 40 30 86 41 45 47 100 40 97 53 44 95.4 x SO 106 61 45 60 118 70 48 = 49.5 70 124 77 47 80 130 83 47 95.4

Table 14 (b). Evolution of CO2 by cells of S. diacetilactis M -8 -224( a) metabolizing pyruvate.

Time CO2 CO2 Cumulative Theoretical Percent Experimental Endogenous Recovery CO2 Recovery (Exp -Endo) Evolution min µ, l p, 1 µ 1 N., 1

10 54 11 43 20 88 18 70 30 108 26 82 84 x 100 40 115 34 81 190.85 50 122 41 81 _ 44 60 130 45 85 70 135 51 84 80 138 54 84 190.85 85 140

120

i /

100

/

20 40 60 80 Time (min)

Figure 28. Uptake of oxygen and evolution of CO222 by resting cells of Streptococcus diacetilactis M- 8- 2Z4(a) metabolizing pyruvate. The experiment was carried out at 30° C as explained in the text. Oxygen experimental------Oxygen endogenous-...... _ CO2 experimental CO2 endogenous -- -- 86

Table 15 (a). Uptake of oxygen by cells of S. diacetilactis 18 -16 metabolizing pyruvate.

Time Oxygen Oxygen Cumulative Theoretical Percent Experimental Endogenous Recovery Oxygen Recovery (Exp -Endo) Uptake min p.1 pA µl p.1

10 37 19 18 20 71 33 38 49 71 30 93 44 100 40 110 53 57 95.4 50 126 63 63 60 137 69 68 = 74.4 70 145 75 70 80 153 82 71 95.4

Table 15 (b). Evolution of CO2 by cells of S. diacetilactis 18 -16 metabolizing pyruvate.

Time CO2 CO2 Cumulative Theoretical Percent Experimental Endogenous Recovery CO Recovery (Exp -Endo) Evolution min µ1 p.1 µ 1 p,1

10 43 12 31 81 21 60 20 106 30 105 27 78 x 100 190. 85 40 121 32 89 50 134 38 96 = 55. 5 60 144 41 103 70 150 45 105 80 155 49 106 190.85 87

140 / i / / / /

120

/

ó.. 100 > ON U /

o 20 40 60 80 Time (min) Figure 29. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus diacetilactis 18 -16 metabolizing pyruvate. The experiment was carried out at 30° C as explained in the text. Oxygen experimental------Oxygen endogenous--- CO2 experimental CO2 endogenous -_ 88

Table 16 (a). Uptake of oxygen by cells of S. cremoris Ml -W1 -3 metabolizing pyruvate.

Time Oxygen Oxygen Cumulative Theoretical Percent Experimental Endogenous Recovery Oxygen Recovery (Exp -Endo) Uptake min µ1 p,1 µ,l µ,1

10 9 1 8 14 20 16 2 74 x100 30 26 9 17 95.4 40 32 9 23 50 39 10 29 = 77.5 60 43 9 33 70 46 7 39 150 79 16 63 160 86 20 66 180 86 15 71 200 87 13 74 95.4

Table 16 (b). Evolution of CO2 by cells of S. cremoris M1 -W1 -3 metabolizing pyruvate.

Time CO2 CO2 Cumulative Theoretical Percent Experimental Endogenous Recovery CO2 Recovery (Exp -Endo) Evoluion min µ 1 µl µl µl

10 9 0 9 20 18 +1 17 30 24 -3 27 93 40 33 -2 35 100 50 40 3 37 190.85x 60 46 3 43 70 53 4 49 = 48.7 150 89 9 80 160 96 11 85 180 101 10 91 200 104 11 93 190.85 89

140

120

100

.,.o o r - v 0`V U 80 0

.1

/

b0 / 60 / ó /

J._

40 / /

/

20 ..,. mamma.; ---

0 40 80 120 160 200 Time ( min) Figure 30. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus cremoris Ml-W1 L3 metabolizing pyruvate. The experiment was carried out at 30° C as explained in the text. Oxygen experimental - -- -- Oxygen endogenous --- -® CO2 experimental - CO2 . endogenous - 90

Table 17 (a). Uptake of oxygen by cells of S. cremoris 144F metabolizing pyruvate.

Time Oxygen Oxygen Cumulative Theoretical Percent Experimental Endogenous Recovery Oxygen Recovery (Exp -Endo) Uptake min µl µl N,1 µ,1

41 82 10 47 6 x 100 20 84 12 72 95.4 92 15 77 30 = 85.9 40 101 21 80 50 102 21 81 60 106 24 82 95.4

Table 17 (b). Evolution of CO2 by cells of S. cremoris 144F metabolizing pyruvate.

Time CO2 CO Cumulative Theoretical Percent Experimetal Endogenous Recovery CO Recovery (Exp -Endo) Evolution min fi.1 µl µ,1 1,1

10 56 3 53 144 x 100 20 113 7 106 190.85 30 121 10 111 40 125 13 112 = 75.4 50 126 13 113 60 129 15 114 190.85 91

140

120

100 /

I

1

I

I I

I

I 40

I

I

I

I 20 I .

I I 1 I 20 40 60 80 Time ( min) Figure 31. Uptake of oxygen and evolution of -CO2 by resting cells of Streptococcus cremoris 144F metabolizing pyruvate. The experiment was carried out at 30° C as explained

in . the text. Oxygen experimental------Oxygen endogenous- - CO2 experimental CO2 endogenous - - 92

Table 18 (a). Uptake of oxygen by cells of S. lactis E metabolizing pyruvate.

Time Oxygen Oxygen Cumulative Theoretical Percent Experimental Endogenous Recovery Oxygen Recovery (Exp -Endo) Uptake min µ1 p, 1 111 µ1

10 13 5 8 20 27 10 17 30 38 14 24 40 48 19 29 50 54 21 33 60 64 26 38 51 x 100 70 69 30 39 95.4 80 78 35 43 90 85 39 46 100 90 40 50 = 53.4 110 100 50 50 120 105 54 51 130 112 62 SO 140 116 65 51 95.4

Table 18 (b). Evolution of CO2 by cells of S. lactis E metabolizing pyruvate.

Time CO2 CO2 Cumulative Theoretical Percent Experimental Endogenous Recovery CO Recovery (Exp -Endo) Evolution min p,1 P.1 l p,1

10 14 3 11 20 29 6 23 30 40 9 31 40 50 10 40 50 S8 14 44 60 68 17 51 76 70 75 22 53 x 100 80 84 24 60 190.85 64 90 91 27 = 39.8 100 98 29 69 110 107 37 70 120 115 40 75 130 121 47 74 140 125 49 76 190.85 93 140

40 :0 20 160 Time (min) Figure 32, Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus lactis E metabolizing pyruvate. The experiment was carried out at 30° C as explained in the text. Oxygen experimental------Oxygen endogenous--- CO2 experimental CO2 endogenous - -- 94

Table 19 (a). Uptake of oxygen by cells of S. lactis M -9 -924 metabolizing pyruvate.

Time Oxygen Oxygen Cumulative Theoretical Percent Experimental Endogenous Recovery Oxygen Recovery (Exp -Endo) Uptake min µl µ1 Ill 1

10 34 12 22 20 67 20 47 30 91 28 63 72 102 x 100 40 33 69 95.4 50 108 39 69

60 112 42 _ 70 = 75.4 70 116 44 72 150 139 66 73 160 142 70 72 180 142 70 72 95.4

Table 19 (b). Evolution of CO2 by cells of S. lactis M -9 -924 metabolizing pyruvate.

Time CO CO Cumulative Theoretical Percent Experimental Endogenous Recovery CO2 Recovery (Exp -Endo) Evolution min p, 1 µl µl 1-1' l

10 44 6 38 20 89 12 77 30 115 17 98 103 40 125 104 x 100 21 190.85 50 129 26 103 60 133 30 103 = 53.9 70 139 34 105 150 153 50 103 160 156 53 101 180 156 55 101 190.85 95

160

140

100 / /

o / ó I oN so U o m I

,x 1 , 60 bo

o 1 .

1 /. . .i

1 40 1 Z. 1 / 1 / ¡1 / /

jI 20 1 / //' //iI//

0 I I 0 40 80 120 160 Time (min) Figure 33. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus lactis M -9 -924 metabolizing pyruvate. The experiment was carried out at 30° C as explained in the text. Oxygen experimental______-_ Oxygen. endogenous------CO2 experimental CO2 ,endogenous 96

Table 20 (a). Uptake of oxygen by cells of S. diacetilactis M -8 -224( a) metabolizing alpha- ketobutyric acid.

Time Oxygen Oxygen Cumulative Theoretical Percent Experimental Endogenous Recovery Oxygen Recovery (Exp -Endo) Uptake

min µ 1 µ,1 p l p,1

15 98 40 58 30 137.5 59 78 45 171 82 89 60 197 101 96 75 216 116 100 90 231 124 107 105 243 133 110 120 253 143 110 135 267 151 116 150 274 156 118 165 279 160 119 165 120 132 180 285 100 x 195 288 167 121 82.2 210 294 175 119 225 300 172 128 161 240 302 174 128 255 307 176 131 270 312 179 133 285 312 180 132 82.2 97

Table 20 (b). Evolution of CO2 by cells of S. diacetilactis M -8 -224( a) metabolizing alpha - ketobutyric acid

Time CO2 CO2 Cumulative Theoretical Percent Experimental Endogenous Recovery CO2 Recovery (Exp -Endo) Evolution min 41 p,1 N,1 µ,1

15 118 31 87 30 161 46 115 45 189 62 127 60 206 79 127 75 227 91 136 90 239 119 120 105 248 105 143 120 256.5 112 144 135 267 120 147 150 275 123 152 164 165 278 123 155 x 100 164.4 180 283 131 152 195 286 132 154 = 99.7 210 291 140 151 225 296 136 160 240 297 139 158 255 302 140 162 270 307 143 164 285 310 146 164 164.4 98 420

390

360

330

300

M0

o 270

N o 240

m

!a2b0 v

00 , ó 180

150

120

90

60

30

0 30 60 90 120 150 180 210 240 270 300 Time ( min) Figure 34. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus diacetilactis M- 8- 224(a) metabolizing alpha -ketobutyric acid. The experiment was carried out at 30° C as explained in the text. Oxygen experimental Oxygen. endogenous-- - CO2 experimental CO2 endogenous -- 99

Table 21 (a). Uptake of oxygen by cells of S. diacetilactis 18 -16 metabolizing alpha -keto- butyric acid.

Time Oxygen Oxygen Cumulative Theoretical Percent Experimental Endogenous Recovery Oxygen Recovery (Exp -Endo) Uptake min N,1 ill µ,l N,1

10 SO 19 31 20 89 33 56 30 113 44 69 79 100 40 129 53 76 82.2 x 50 139 63 76 = 96 60 148 69 79 70 154 75 79 80 161 82 79 82.2

Table 21 (b). Evolution of CO2 by cells of S. diacetilactis 18 -16 metabolizing alpha-keto- butyric acid.

Time CO2 CO2 Cumulative Theoretical Percent Experiental Endognous Recovery CO Recovery (Exp -Endo) Evolution min µ,l µ1 N,1 µl

10 59 7 52 20 103 12 91 30 129 17 112 135 x 100 40 143 21 122 164.4 50 152 25 127 60 158 28 130 = 82 70 163 30 133 80 168 33 135 164.4 100

160 i i

140

100

Ó .I I 60 I Ì

I

o /

/ I

I 1 /,

1 40

1 /

j

Ir. 20 ./ /

20 40 60 80 Time (min) Figure 35. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus diacetilactis 18 -16 metabolizing alpha - ketobutyric acid. The experiment was carried out at 30° C as explained in the text. Oxygen experimental Oxygen endogenous - CO2 experimental - CO2 endogenous - 101

Table 22 (a). Uptake of oxygen by cells of S. cremoris Ml -W1 -3 metabolizing alpha -keto- 1 butyric acid.

Time Oxygen Oxygen Cumulative Theoretical Percent Experimental Endogenous Recovery Oxygen Recovery (Exp -Endo) Uptake min N.1 µ l N.1 p.1

15 34 7 27 47 30 53 11 42 x 100 82.2 45 61 13 48 60 65 17 48 = 57 75 66 19 47 82.2

Table 22 (b). Evolution of CO2 by cells of S. cremoris M1 -W1 -3 metabolizing alpha -keto- butyric acid.

Time CO2 CO2 Cumulative Theoretical Percent Experimntal Endognous Recovery CO2 Recovery (Exp -Endo) Evolution min N,1 N,1 N,1 N,1

15 37 4 33 60 x 100 30 59 6 53 164.4 45 66 7 59 = 36.5 60 69 9 60 75 71 11 60 164.4 102

120

100

o u

- a 60

40

/ / I

/ 20 /

...0""" ------

-i I E J 0 20 40 60 80 100 Time (min) Figure 36. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus cremoris Ml - W -3 metabolizing alpha - ketobutyric acid. The experiment was carried out at 30° C as explained in the text. Oxygen. experimental------Oxygen .endogenou.s--- CO2 experimental CO2 endogenous 2 -- 103

Table 23 (a). Uptake of oxygen by cells of S. cremoris 144F metabolizing alpha -keto- butyric acid.

Time Oxygen Oxygen Cumulative Theoretical Percent Experimental Endogenous Recovery Oxygen Recovery (Exp -Endo) Uptake min ill l µl N,1 N-

10 42 6 36 20 79 12 67 84 x 100 30 90 15 75 82.2 40 100 21 79 50 104 21 83 = 102 60 108 24 84 82.2

Table 23 (b). Evolution of CO2 by cells of S. cremoris 144F metabolizing alpha-keto- butyric acid.

Time CO CO2 Cumulative Theoretical Percent Experimental Endognous Recovery CO2 Recovery (Exp- Endo) Evoluion

1 min N, ill N,1 ill

10 68 3 65 20 117 7 110 127 30 130 10 120 x 100 164.4 40 136 13 123 50 139 13 126 = 77 60 142 15 127 164.4 104

140

120

100

/ / / 80 /

/ 60

40

I / 20 ¡ ` / ` 1 // 1 ,.j/ . % l r 1 0 20 40 60 80 Time (min) Figure 37. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus cremoris 144F metabolizing alpha -keto- butyric acid. The experiment was carried out at 30° C as explained in the text. Oxygen experimental__- __ -___ Oxygen endogenous-.- -

CO2 experimental - CO2 endogenous 105

Table 24 (a). Uptake of oxygen by cells of S. lactis E metabolizing alpha-ketobutyric acid.

Time Oxygen Oxygen Cumulative Theoretical Percent Experimental Endogenous Recovery Oxygen Recovery (Exp -Endo) Uptake min µl µl µ1 µ1

10 17 5 12 20 37 10 27 30 51 14 37 40 66 19 47 50 77 21 56 60 88 26 62 83 100 70 96 30 63 82.2 x 80 109 35 74 90 118 39 79 = 100.2 100 123 40 83 110 133 50 83 82.2

Table 24 (b). Evolution of CO2 by cells of S. lactis E metabolizing alpha-ketobutyric acid.

Time CO CO2 Cumulative Theoretical Percent Experimental Endogenous Recovery CO Recovery (Exp -Endo) Evolution min PA µl µl µl

10 26 3 23 20 53 6 47 30 72 9 63 40 92 10 82 137 50 107 14 93 100 164.4x 60 121 17 104 70 132 22 110 = 83.3 80 146 24 122 90 156 27 129 100 164 29 135 110 174 37 137 164.4 106

240

200

ó 120 i

/ / / / / / 80 / /

/ / / / i

40 % / //

/

.

40 80 120 160

Time ( min) Figure 38. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus lactis E metabolizing alpha -ketobutyric acid. The experiment was carried out at 30° C as explained in the text. Oxygen experimental------Oxygen endogenous-- - CO2 experimental CO2 endogenous 107

Table 25 (a). Uptake of oxygen by cells of S. lactis M -9 -924 metabolizing alpha- ketobutyric acid.

Time Oxygen Oxygen Cumulative Theoretical Percent Experimental Endogenous Recovery Oxygen Recovery (Exp -Endo) Uptake min µ 1 p,l µl 11.1

30 59 24 35 60 75 33 42 90 90 46 44 82 x 100 2 120 100 54 46 150 106 59 47 = 58.4 180 111 64 47 270 126 76 50 300 128 80 48 82.2

Table 25 (b). Evolution of CO2 by cells of S. lactis M -9 -924 metabolizing alpha-ketobutyric acid.

Time CO2 CO2 Cumulative Theoretical Percent Experiental Endoenous Recovery CO2 Recovery (Exp -Endo) Evolution min p,1 p,1 µ,1 1-1.1

30 75 9 66 60 91 18 73 90 99 25 74 78 100 120 108 30 78 164,4 150 111 33 78 180 115 37 78 = 47.4 270 123 43 80 300 124 46 78 164.4 108

240

200

80

i

40 / ' / / /

0 I t 1 I I 30 60 90 110 150 180 210 240 270 300'

Figure 39. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus lactis M -9 -924 metabolizing alpha -keto- butyric acid. The experiment was carried out at 30° C as explained in the text. Oxygen experimental- - - -- Oxygen endogenous-. - CO2 experimental CO2 endogenous 109

Table 26 (a). Uptake of oxygen by cells of S. diacetilactis M -8 -224( a) metabolizing alpha -keto- valeric acid.

Time Oxygen Oxygen Cumulative Theoretical Percent Experimental Endogenous Recovery Oxygen Recovery (Exp -Endo) Uptake min µ,l µ l µl µ,l

15 33 25 8 30 61 41 20 45 82 51 31 60 95 58 37 75 106 65 41 90 113 70 43 105 121 76 45 120 124 77 47 135 130 80 50 150 136 84 52 165 138 86 52 180 143 89 54 195 146 91 55 210 149 93 56 225 150 94 56 240 153 95 58 59 -x 100 255 155 97 58 72.35 270 155 96 59 285 158 99 59 = 81.5 300 159 100 59 375 170 110 60 72.35 110

Table 26 (b). Evolution of CO2 by cells of S. diacetilactis M- 8- 224(a) metabolizing alpha -keto- valeric acid.

Time CO CO2 Cumulative Theoretical Percent Experimental Endogeous Recovery CO2 Recovery (Exp -Endo) Evolution min µl µl µl µl

15 50 18 32 30 85 29 56 45 113 40 73 60 128 50 78 75 140 56 84 90 149 64 85 105 158 71 87 120 165 77 88 135 172 81 91 150 179 86 93 165 183 90 93 180 189 95 94 195 195 99 96 210 199 102 97 101 225 203 106 97 x 100 240 208 110 98 114.7 255 212 113 99 115 101 270 216 = 88 28S 219 120 99 300 223 124 99 375 239 138 101 114.70 111

280

240 r

200

.,.o

v 160 N ----.- O - I ..- ' p td '// : - , j 120 /- q b°10 i // / // / j. / / 80 % / / / / 40 / / /

120 150 180 210 240 270 300 Time (min) Figure 40. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus diacetilactis M- 8- 224(a) metabolizing alpha -ketovaleric acid. The experiment was carried out at 30° C as explained in the text. Oxygen experimental- - - - - Oxygen endogenous-- - CO2 experimental CO2 endogenous - - - - 112

Table 27 (a). Uptake of oxygen by cells of S. diacetilactis M- 8- 224(a) metabolizing alpha-kelo- isovaleric acid.

Time Oxygen Oxygen Cumulative Theoretical Percent Experimental Endogenous Recovery Oxygen Recovery (Exp -Endo) Uptake min µ1 p. 1 µ1 p. 1

15 24 23 1 30 48 38 10 45 67 49 18 60 88 60 28 75 111 73 38 90 126 80 46 105 145 90 55 120 158 96 62 135 171 101 70 150 193 112 81 165 205 118 87 180 221 126 95 195 232 131 101 210 240 138 102 124 225 253 145 108 100 240 261 148 113 72.35 x 255 269 153 116 = 171 270 277 158 119 285 285 164 121 300 293 169 124 72.35 113

Table 27 (b). Evolution of CO2 by cells of S. diacetilactis M -8 -224 (a) metabolizing alpha -keto- isovaleric acid.

Time CO2 CO2 Cumulative Theoretical Percent Experiental Endognous Recovery CO Recovery (Exp -Endo) Evolution min µl µ1 µl µ,1

15 45 14 31 30 84 24 60 45 112 32 80 60 136 39 97 75 154 46 108 90 170 54 116 105 184 58 126 120 197 64 133 135 209 70 139 150 225 75 150 165 235 80 155 180 248 86 162 195 259 92 167 188 100 210 266 97 169 114.7 x 225 275 103 172 240 284 106 178 = 164 255 290 108 182 270 297 112 185 285 301 116 185 300 308 120 188 114.7 114

280

/

240

/ 200 .PIó '5 ó / > / óa, i U i á 160 r !

cd rn a 00 X 120 / / - f V l / / V

80 / /

/ / 40 "- // / / /

f j 1 1 f ' t 0 3b 60 90 120 150 180 210 240 270 300 Figure 41. Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus diacetilactis M- 8- 224(a) metabolizing alpha- ketoisovaleric. acid. The experiment was carried out at 30° C as explained in the text. Oxygen experimental -- Oxygen endogenous------CO2 experimental CO2 endogenous -- 115

Table 28 (a). Uptake of oxygen by cells of S. diacetilactis M -8 -224 (a) metabolizing alpha -veto - caproic acid.

Time Oxygen Oxygen Cumulative Theoretical Percent Experimental Endogenous Recovery Oxygen Recovery (Exp -Endo) Uptake min µi µi 111 µ1

15 26 25 1 30 51 41 10 45 73 51 22 60 89 58 31 75 104 64 40 90 117 70 47 105 131 76 55 120 140 77 63 135 150 80 70 150 161 84 77 165 169 8S 84 180 178 89 89 195 186 91 95 210 193 92 101 133 x 100 225 202 93 109 64.5 240 207 95 112 255 214 97 117 = 206 270 221 96 125 285 228 99 129 300 233 100 133 64.5 116

Table 28 (b). Evolution of CO2 by cells of S. diacetilactis M -8 -224 (a) metabolizing alpha -keto- caproic acid.

Time CO2 CO Cumulative Theoretical Percent Experiental Endogenous Recovery CO Recovery (Exp -Endo) Evolution min 1-11 µl µ1 µ l

15 36 18 18 30 66 29 37 45 94 40 54 60 114 50 64 75 132 56 76 90 148 64 84 105 164 71 93 120 178 77 101 135 190 81 109 150 202 86 116 165 212 90 122 180 223 95 128 195 233 99 134 210 241 102 139 225 254 106 148 171 x 100 240 261 110 151 129 255 269 113 156 270 279 11S 164 = 132.5 285 288 120 168 300 295 124 171 129 117

280

240

200 / /i 80

40 /

I 1 I I t 0 30 60 90 120 150 1$0 2 0 240 270 3 Time ( min) Figure 42, Uptake of oxygen and evolution of CO2 by resting cells of Streptococcus diacetilactis M-8-224(a) metabolizing alpha -ketocaproic acid. This experiment was carried out at 30° C as explained in the text. Oxygen experimental- - - - - Oxygen endogenous-._ CO2 experimental CO2 endogenous -- 118

Table 29 (a). Uptake of oxygen by cells of S. diacetilactis M -8 -224 (a) metabolizing alpha keto- isocaproic acid.

Time Oxygen Oxygen Cumulative Theoretical Percent Experimental Endogenous Recovery Oxygen Recovery (Exp -Endo) Uptake min 41 µ,l µ,l N,1

15 23 23 0 30 38 37 1 45 49 49 0 60 62 60 2 75 75 73 2 90 83 80 3 105 94 89 5 120 101 96 5 135 109 101 8 150 122 112 10 165 129 118 11 180 139 126 13 195 146 131 15 210 153 138 15 225 161 145 16 148 18 240 166 17 x 100 255 169 153 16 64.5 270 175 158 17 285 181 164 17 = 26.3 300 186 169 17 64.5 119

Table 29 (b). Evolution of CO by cells of S. diacetilactis M -8 -224 (a) metabolizing alpha-keto- isocaproic acid?

Time CO CO Cumulative Theoretical Percent 2 Experimental Endogenous Recovery CO Recovery (Exp-Endo) Evolution min µl µl µl µ1

15 23 14 9 30 34 24 10 45 47 32 15 60 54 40 14 75 56 46 10 90 60 54 6 105 71 58 13 120 78 64 14 135 98 70 18 150 109 75 34 165 114 80 34 180 122 86 36 195 130 92 38 210 136 97 39 225 143 103 40 148 106 42 42 240 x 100 255 149 108 41 129 270 154 112 42 = 32.5 285 158 116 42 300 162 120 42 129 120

240

200

q Mo Ñ o óN160 U /' , ' /,// rt. 120 /', ,%/ ó . / . i / 80

40

30 60 0 20 1 0 Time (min)

Figure 43. Uptake of oxygen and evolution of CO7 by resting cells of Streptococcus diacetilactis M- 8- 224(a) metabolizing alpha -ketoisocaproic acid. The experiment was carried out at 30o C as explained in the text. Oxygen experimental------Oxygen endogenous-.--.-- CO2 experimental CO2 endogenous -- 121

Radiorespirometry

Enzyme and manometric experiments on lactic streptococci gave evidence for the pathways involved in the catabolism of glucose by these organisms. The radiorespirometric experiments were carried out in order to evaluate the pathways actually operative in the catabolism of glucose by the intact cells of lactic streptococci.

The radiorespirometric data for the utilization of glucose by the various strains of lactic streptococci studied are given in Figures

44 through 51. The complete inventory of the labeled carbon atoms in the respiratory CO2, cells, and incubation media, observed at the end of each experiment is presented in Tables 30 through 37.

The radiochemical recovery data on cells and fermentation products inthe medium are less precise than those on respiratory CO2 samples, mainly due to the difficulties encountered in the preparation of counting samples with either cells or incubation medium. This fact largely accounts for the deviation of total substrate inventory from the theoretical value, 100 %. 122 20

18

13 16

0 14

12 - - 3,4 11 8

6

10 4 1 2 6 9 3,4 30 60 90 120 150 180 210 Ó Time (min) U 8 o

o 7 u s.

6 a ...á a 1 5

4

3

2 i

1

0 30 60 90 120 150 210 Time (min) Figure 44. Radiorespirometric pattern exhibited by Streptococcus diacetilactis M- 8- 224(a) metabolizing specifically labeled glucose. Glucose- ____ _- Glucose -i -14e - - - -- 2- 14C - -- 14 Glucose-3,4- C - - Glucose -6 -14C Figure . w U ON v . > U 0 a, O U. w F N 10 11 12 0 2 3 4 5 6 9 7 8 0 45. / . / /° / diacetilactis Glucose-3, Glucose glucose. Radiorespirometric / ' f - / / 30 3, --°'\ 1 \ 4 -1 \ -14C 2 4 \ -14C 18 ` s--..` ---- -16 \ 60 - metabolizing Time pattern

Percent cumul. recovery;; u o > (min) 12 20 10 14 16 18 2 4 6 8 exhibited 30 90 ` 60 Glucose Glucose-2-14C------.-o specifically 90 Time _. 120 by _. -6 (min) Streptococcus 150 120 -14C .. 180 labeled 6 ' 210 2 3, 1 4 150 123 210 124

18

12 16 14

11 ó 12

2-) 10 -3,4 0 10 8 / 6

4 a- -- -__-. 1 6 2 O _ _ V 30 60 90 120 150 18'_10 Time (min)

7

6

d 3, 4 ` á 5 /

/ / 1 . 1 ,/"', 6 2 - p 30I 90 120 150 210 60 Time (min)

Figure 46. Radiorespirometric pattern exhibited by Streptococcus cremoris Ml-W1-3 metabolizing specifically labeled glucose. 14 Glucose -1-14C -. Glucose-2-14C------Glucose-3, 4 -14C - - - Glucose -6 -j4C. 125

36

32 - 3, 4 > 28 o Jr 1, 24 1 20 u 16 /

v 12 ái a 8 3. 4 / 1 .2 6 30 60 90 120 15 180 210 240 270 Time (min) 4

12 /

f0

/

0 2 0 30 60 , 90 120 150 18 Time (min) Figure 47. Radiorespirometric pattern exhibited by Streptococcus cremoris 144F metabolizing specifically labeled glucose. Glucose- 14C - - -- Glucose -2 -14C Glucose -3, 4 -14C Glucose -6 -14C It should be noted that the scale on this figure is condensed since this strain gave a higher recovery of14CO2 from glucose 3,4-14C than other strains tested. Figure a PercentC) interval recovery of 10 11 12 6 7 48. Glucose Glucose lactis Radiorespirometric / 30 E -3, -1 metabolizing -14C 4 -14C 60 - 1 Time pattern specifically (min) - -- a+ o G1 q 0 °: U > 12 14 16 18 4 6 8 2 3, 1 exhibited 90 -Glucose ---..._ 4 30 Glucose- 60 6 . labeled 90 Time I' by 120 " -6 (min) Streptococcus 120 2 150 glucose. -14C -14C- 180 v 210 3, 2 4 150 126 210 Figure 12 13 10 11 0 49. lactis Glucose-3, Glucose Radiorespirometric f 2 3, Ì 30 4 C2F -1 \ , -14C metabolizing 4 -14C -. II 60 -.- Time pattern - ..; Pere( n ^10 0 ( specifically 12 14 16 18 min) 4 8 - exhibited 90 30 ' i ' / 60 Glucose Glucose Time 90 labeled 120 by ( min) .. -6 -2- 120 15s Streptococcus .--1 -C 6 14 180 glucose. 3, 2 C 4 - - --- 150 127 180 128

13 18

16

12 14 o u ;; 12 3,4

11 3,4 £ ú 8

10 6 v a 4 / 2. ' 2 6 / 30 60 90 120 150 180 Time (min)

£

2

0 30 60 90 120 15 180 Time (min)

Figure 50. Radiorespirometric pattern exhibited by Streptococcus lactis R -24(e) metabolizing specifically labeled glucose. 14 Glucose -1 -14C - - - Glucose-2- C------14 Glucose -3, 4 -14C - Glucose -6 -14C _- Figure .1 N i+ U cu a N % i 9 td o o 10 12 11 13 o 2 4r 1 3 5 6 7 8 9 , 0 51. / L_-_-?-4 Glucose-3,4- Glucose lactis Radiorespirometric / '\ 3,4 30 1 155 -1 -54 -14C 14 metabolizing C 60 Time

Percent cumul. recovery pattern (min) 20 18 10 14 12 16 - 6 30 specifically - exhibited 90 60 Time 90 Glucose- Glucose 12015S`' (min) by labeled -6 120 2- Streptococcus 80 -14C 3, 14C--- 2 4 glucose. -- - - 150 129 - -- 130

Table 30. Incorporation of substrate into cells, medium, and CO2 by S. diacetilactis M -8 -224( a) cells metabolizing glucose specifically labeled with 14 C.

Dry Wt =25mg

Substrate Inventories Substrate CO2 Cells Medium Total % % % %

Glucose -1 -14C 3.0 4.0 89.0 96.0 Glucose -2 -14C 0.5 5.0 88.0 93.5 Glucose -3, 4 -14C 11.0 4.0 83.0 98.0 Glucose -6- C 0.3 4.0 92.0 96.3

Table 31. Incorporation of substrate into cells, medium, and CO2 by S. diacetilactis 18 -16 cells metabolizing glucose specifically labeled with 14C.

Dry Wt = 22 mg

Substrate Inventories Substrate CO2 Cells Medium Total

96 96 96 96

Glucose -1 -14C 2. 1 3.0 91.0 96. 1 Glucose -2 -14C 0.3 3.0 89.0 92.3 Glucose -3, 4 -14C 7.0 3.0 83.0 93.0 Glucose -6 -14C 0.3 3.3 92.0 95.6 131

Table 32. Incorporation of substrate into cells, medium, and CO2 by S. cremoris M1 -W1 -3 cells metabolizing glucose specifically labeled with 14C.

Dry Wt = 18 mg

Substrate Inventories

Substrate CO2 Cells Medium Total 2 o,' %

Glucose -1 -14C 2.0 4.0 90.0 96.0 Glucose -2 -14C 0.2 4.0 92.0 96.2 Glucose -3, 4-14C 10.0 4.0 81.0 95.0 Glucose-6-14C 0.0 4.0 80.0 84.0

33. Incorporation of substrate into cells, medium, la4nd CO by S. cremoris 144 F cells Table 2 metabolizing glucose specifically labeled with C.

Dry Wt = 17 mg

Substrate Inventories

Substrate CO2 Cells Medium Total 2

Glucose -1 -14C 2. 0 3. 0 87.0 92.0 Glucose -2 -14C 0.4 4.0 97.0 101. 4 Glucose -3, 4 -14C 30.0 3.0 59.0 92.0 Glucose -6 -14C 0.2 4.0 87.0 91.2 132

Table 34, Incorporation of substrate into cellis4 medium, and CO2 by S. lactis E cells metabolizing glucose specifically labeled with C.

Dry Wt = 16 mg

Substrate Inventories Substrate CO Cells Medium Total 2

Glucose -1 -14C 3.0 3.0 90.0 96.0 Glucose-2-14C 0.0 3.0 92.0 95.0 Glucose -3, 4 -14C 3.0 3.0 86.0 92.0 14 Glucose -6- C 0.0 4.0 91.0 95.0

Table 35. Incorporation of substrate into cells, medium, and CO2 by S. lactis C2F cells metabolizing glucose specifically labeled with 14C.

Dry Wt = 17 mg

Substrate Inventories Substrate CO2 Cells Medium Total %

14 Glucose -l- C 2.0 5.0 87.0 94.0 Glucose -2 -14C 0.0 5.0 78.0 83.0 14 Glucose -3, 4- C 15.0 3.0 72.0 90.0 Glucose -6 -14C 0.0 5.0 89.0 94.0 133

substrate into cells, medium, and CO by S. lactis R -24 (e) cells Table 36. Incorporation of 2 metabolizing glucose specifically labeled with 14C.

Dry Wt = 19 mg

Substrate Inventories

Substrate CO2 Cells Medium Total % % %

Glucose-1-14C 4.0 4.0 83.0 91.0 Glucose-2-14C 0.5 5.0 86.0 91.5 91. 0 Glucose -3, 4-14C 12.0 4.0 75.0 Glucose -6 -14C 0.2 5.0 88.0 93.2

into cells, medium, and CO by S. lactis 144 -54 cells Table 37. Incorporation of substrate 2 metabolizing glucose specifically labeled with 14C.

Dry Wt = 14 mg

Substrate Inventories

Substrate CO2 Cells Medium Total % % % %

14 90.5 Glucose -l- C 2.5 3. 0 85.0 14 Glucose-2-14C 0.0 3.4 91.0 94.4 Glucose -3, 4 -14C 6.0 3.2 85.0 94.2 14 96.0 Glucose -6- C 0.0 4.0 92.0 134

DISCUSSION

EMP Pathway in Lactic Streptococci

Aldolase and triosephosphate isomerase were selected as enzymes characteristic of the EMP pathway. Aldolase performs the unique C -C bond scission reaction converting the ketohexose diphosphate into two molecules of triose phosphate. Triosephosphate isomerase is responsible for the interconversion of these two aldo and keto phosphate molecules so that both the triose phosphate molecules formed as a result of aldolase reaction can be channeled into the EMP pathway.

The results of the enzyme experiments indicated that aldolase and triosephosphate isomerase were both present in the lactic streptococci. The presence of these enzymes demonstrated the existance and also gave evidence for the possible operation of the

EMP pathway for the catabolism of glucose by these organisms.

The homolactic acid fermentation is carried out by a large number of microorganisms. Among these are the various species of the genera Lactobacillus, Bacillus, Streptococcus, and Clostridium. This fermentation yields lactate as essentially the sole end product of glucose catabolism by the EMP pathway. The reversible reduction of pyruvate to lactate, with NADH as a 135 reducing agent, is the terminal step that characterizes glycolysis in these organisms.

CH3CO COOH + NADH + H+, CH3CHOHCOOH + NAD+ pyruvate lactate

In certain heterofermentative microorganisms such as Saccharomyces cerevisiae, the catabolism of pyruvate differs from that described above. These organisms carry out the non - oxidative decarboxylation of pyruvate, leading to the formation of acetaldehyde and then ethanol. The first step in this reaction is catalyzed by pyruvate decarboxylase which carries out the virtual irreversible decarboxylation of pyruvate to yield acetaldehyde. This reaction +2 requires thiamine pyrophosphate (TPP, cocarboxylase) and Mg as indispensable cofactors. In the final step of alcohol formation, the aldehyde is reduced to ethanol by the alcohol dehydrogenase catalyzed reaction requiring NADH.

CH3CHO + NADH + H+ ' CH3 Co - OH + NAD+ H

acetaldehyde ethanol

Enzyme experiments to measure alcohol dehydrogenase indicated that this enzyme was also present in all the lactic streptococci studied. Thus the so- called homofermentative lactic streptococci 136 were producing ethanol in addition to lactic acid and demonstrating a heterofermentative type reaction.

The manometric experiments with lactic streptococci in the presence of alpha -keto acids as substrate resulted in the uptake of approximately one half of a mole of oxygen and evolution of one molecule of CO2 per molecule of the alpha -keto acid consumed.

These results suggested the oxidative decarboxylation of pyruvate and other alpha -keto acids,possibly by NAD- lipoate linked pyruvate dehydrogenase. It was assumed that all of the alpha -keto acids studied were decarboxylated by the same enzyme (pyruvate

dehydrogenase) . This reaction has been known to occur in aerobic microorganisms in which pyruvate is oxidatively decarboxylated to acetyl CoA. This reaction is mediated by the multienzyme pyruvate dehydrogenase complex (PDC) associated with the cell membranes of bacteria. The net reaction catalyzed by this enzyme system is the following:

PDC(Ea, b, c) CH3CO COOH + NAD+ + CoASH S +2 pyruvic acid TPP-lipI , Mg \S CoAS COCH3 + NADH + H+ + CO2 acetyl coenzyme A

FAD is also involved in this reaction. The same reaction can be written with oxygen as follows: 137

Same enzyme complex CH CO COOH + 1/2 O + CoASH 3 2 plus electron transport

CoASCOCH3 + H2O + CO2

The results of the enzyme and manometric experiments suggested that pyruvate was being metabolized in lactic streptococci by the following three reactions:

1. The reversible reduction of pyruvate to lactate with NADH

as the reducing agent. This reaction is catalyzed by lactic dehydrogenase.

2. The non - oxidative decarboxylation of pyruvate leading to

the formation of ethanol. These reactions are catalyzed

by pyruvate decarboxylase and alcohol dehydrogenase.

3. The oxidative decarboxylation of pyruvate leading to the

formation of acetyl CoA as catalyzed by NAD- lipoate linked pyruvate dehydrogenase.

The NADH formed in the lactic streptococci during the operation of EMP pathway could be reoxidized to NAD by lactic and alcohol dehydrogenase catalyzed reactions in which part of the pyruvate formed via EMP pathway was reduced to lactate and ethanol respectively. NADH formed in the oxidative decarboxylation of pyruvate could be reoxidized to NAD possibly by an electron trans- port system involving oxygen as the ultimate acceptor of electrons. 138

This was suggested by the manometric experiments with pyruvate and other alpha -keto acids which showed the uptake of oxygen occurring together with the decarboxylation of the keto acids by lactic streptococci. Operation of such an electron transport system in the lactic streptococci, however was not studied in this work.

Experiments to detect the presence of peroxidase in these organisms were attempted but without any success.

Hexose Monophosphate Pentose Pathway

The first oxidative step in the HMP pathway catalyzed by glucose -6- phosphate dehydrogenase was discovered and studied by

Warburg (67, p. 450) . This enzyme has been found to be specific for NADP, though Leuconostoc mesenteroides has been shown to utilize NAD (67, p. 450). Glucose -6- phosphate dehydrogenase and 6- phosphogluconate dehydrogenase characterize the HMP pathway, and they catalyze reactions 1 and 3 shown below.

NADP+ NADPH + H+

GH2OPO3H2 \ / CH_OPO H 2 1. 0

D- glucose -6- phosphate D- glucono- ó - lactone -6- phosphate 139

COOH + CO2 NADP NADPH + H+ HO H-C-OH CH OH

I HO-C-H C=0 I ---)HMP ( H-C-OH H-C-OH

H-C-OH H-C-OH

I CH2OPO3H2 CH2OPO3H2

6- phospho -D- gluconate D- ribulos e- 5 -phosphate

1. Glucose -6- phosphate dehydrogenase

2. Gluconolactonase

3. 6- phosphogluconate dehydrogenase

Results of the enzyme and radiorespirometric experiments provided evidence for the presence of both of these enzymes in the lactic streptococci; they were found to be NADP specific. The presence of glucose -6- phosphate dehydrogenase enzyme in these organisms also gave evidence for the possible operation of the HMP pathway and /or the Entner Doudoroff pathway for glucose catabolism.

While the results of the enzyme experiments . did not rule out the operation of the ED pathway in lactic streptococci, the presence of

6- phosphogluconate dehydrogenase provided further evidence for the 140 operation of the HMP pathway since this enzyme functions exclusively in the HMP pathway and not in the Entner Doudoroff pathway. In the ED scheme 6- phosphogluconate is transformed to

2- keto -3- deoxy- 6- phosphogluconate. This reaction is catalyzed by

6- phosphogluconate dehydrase. The operation of the HMP pathway in lactic streptococci may serve the following functions:

1. To provide the organisms with pentoses for the synthesis

of ribose and deoxyribo- nucleotide derivatives. Pentoses

also may be used for coenzyme, polysaccharide and especially polynucleotide biosynthesis.

Z. To provide for the formation of NADPH by the glucose -6-

P- dehydrogenase and phosphogluconate dehydrogenase

catalyzed reactions. These two dehydrogenases furnish

the bulk of extra mitochondrial NADPH which serves as

the source of reducing power for the above mentioned

anabolic reactions and other biosynthetic processes

(fatty acids, steroids, etc.), most of the biosynthetic

processes are known to be localized in just such extra mitochondrial compartments. Anabolism is known to

require and consume NADP almost exclusively (67, p. 413).

3. To provide a means of interconverting and reshuffling the

carbon atoms of hexoses and pentoses. 141

The manometric and radiorespirometric experiments all gave very strong evidence for the presence of enzymes in lactic strepto- cocci which carry out the oxidative decarboxylation of pyruvate leading to the formation of acetyl CoA. Since the citric acid cycle

(TCA) is lacking in these organisms, acetyl CoA cannot be further utilized by this cycle. Acetyl CoA can therefore undergo the phos- phoroclastic reaction in which it can be converted to acetyl phosphate and then to acetate and ATP. Acetyl CoA, however, can also remain as the metabolically active form of acetate itself, participating in a large number of additional reactions. A large number of micro- organisms growing under anaerobic conditions have been shown to follow this route, e. g. Enterobacteriaceae (coliform) and Clostridium species.

Enzyme experiments gave evidence for the presence of acetate kinase in all the lactic streptococci studied. The presence of this enzyme in lactic streptococci explained the formation of acetyl phosphate through the phosphoroclastic reaction shown to occur in the following two steps:

O O

0 00 CH3CSCoA + Pi \ CH3C -OPO3H2 + HSCoA 1, acetyl coenzyme A acetyl phosphate

11 CH3C- OPO3H2 + ADP CH3COOH + ATP 2 142

1. Phosphotransacetylase

2. Acetate kinase or acetokinase

Acetyl coenzyme A as an acyl thioester has a high - 6G° (standard free energy change) of hydrolysis ( -7. 7 k cal /mole) (67); it can therefore be equilibrated with the ATP systems by reactions 1 and

2. Acetyl CoA undergoes phosphotransacetylase reaction involving an inorganic phosphate pickup and resulting in the formation of acetyl phosphate. Acetyl phosphate in the presence of ADP undergoes a kinase reaction shown in equation 2 resulting in the formation of a molecule of acetate and one molecule of ATP from each molecule of acetyl CoA metabolized. Therefore from each molecule of acetyl CoA formed in lactic streptococci there should be one molecule of ATP generated via the above reactions. Since two molecules of pyruvate are formed from one molecule of glucose via the EMP pathway, and if all the pyruvate was involved in the forma- tion of acetyl CoA, two molecules of ATP should be generated via the phosphoroclastic reaction. The experimental results indicated, however, that all the pyruvate was not converted to acetyl CoA because part of it was involved in the formation of lactate and another part in the formation of ethanol. Nevertheless lactic streptococci studied were found to have a route for the formation of ATP in addi- tion to the EMP pathway. This is the first known report of this fact in these bacteria. 143

In addition to the functions of the HMP pathway in lactic streptococci as mentioned before, it can also serve in the formation of additional ATP by carrying on the pentose fermentation shown to occur in heterofermentative lactic acid bacteria such as the various

Lactobacilli and Leuconostoc me sente roides. Pentose fermentation in Lactobacilli has been shown to occur as follows (67, p. 455):

ADP ATP CH C 3 CH3 I __ Ç OPO3H2 Z COOH II Ó 2. CH ,OH acetyl phosphate O P.i SH, TPP HO-C-H 1. H-C-OH

CH2OPO3H2CI I-C=0 COOH H+-OH( D-xylulose-5- H- OH phosphate CH2OPO3H2 CH3

D-glycer aldehyde - L- lactate 3- phosphate

1. Phosphoketolase

2. Acetate kinase 144

The enzymes concerned in the fermentative degradation of several different pentoses by L. plantarum or L. arabinosus have been isolated and separated from each other by Horecker and his colleagues. Phosphoketolase, the enzyme catalyzing the phosphoro- lytic cleavage of a ketopentose phosphate to triose phosphate and acetyl phosphate plays a pivotal role. This reaction has been shown to be virtually irreversible (LG °:-- -13, 000 cal /mole) (67). The D- xylulose- 5- phosphate formed via the HMP pathway in lactic streptococci studied, can undergo the above mentioned phospho- ketolase cleavage forming acetyl phosphate and D- glyceraldehyde-

3- phosphate. Acetyl phosphate can then form acetate and ATP through the acetate kinase catalyzed reaction. D- glyceraldehyde phosphate can in turn be converted to pyruvic acid and finally to lactate through the EMP pathway.

The catabolism of glucose, operative in lactic streptococci via the EMP and HMP pathway, has been shown in Figures 52 and

53 respectively.

Bacteria produce acetoin (67, p. 436) by the following reactions:

E. TPP (1) 2 CH3CO COOH > 2 CO2 + CH3COCHOHCH3 +2 Mg pyruvate acetoin 145

D- lucg ose ATP D- glue ose -6--phosphate -3 ADP D- fructose -6- phosphate ATP V D- fructose- 1 :6- diphosphate > ADP (1) T

Dihydroxyacetone-phosphate ` D glyceraldehyde -3- phosphate (2) P1 1:3 diphosphogl cerate 2ADP 3hosphoglycerate ) 2ATP Phi phoenol pyruvate 2ADP Lactate Ethanol Pyruvate > 2ATP

5) 1 (4) (6) Acetaldehyde Acetyl CoA l CO2 i (7) Acetyl phosphate ADP (8) ATP Acetate

1. Aldolase 2. Triosephosphate isomerase 3. Lactic dehydrogenase 4. Pyruvate decarboxylase 5. Alcohol dehydrogenase 6. Pyruvate dehydrogenase

7. Phosphotransacetylase 8. Acetate kinase

Figure 52. Glucose catabolism in .lactic. streptococci via the EMP pathway. 146

NADP+ NADPH + H+

Glucose -6- phosphate \ D-glucono- 6- lactone -6- phosphate H20 Acetate 6-phospho-D-gluconate ATP NADP (S) (2) ADP NADPH+ H Acetyl phosphate D-ribulose-5-phosphate Pi (3)1 (4) ) D- xylulose -5- phosphate -SH, TPP

D- glyceraldehyde phosphate

1Pyiuvic acid

(6) L- lactate

1. Glucose -6- phosphate dehydrogenase 2. 6- phosphogluconate dehydrogenase 3. Ribulose phosphate- 3- epimerase 4. Phosphoketolase 5. Acetate kinase 6. Lactic dehydrogenase

Figure 53. Glucose catabolism in lactic streptococci via the HMP pathway. 147

TPP (2) CH3COCOOH + CH3CHO + H+ E' ) acetaldehyde Mg

CH3CHOHCOCH3 + CO2 acetoin

(3) Aerobacter aerogenes and Proteus vulgaris carry out yet another variant reaction:

E. 2 CH3COCOOH +PP ) CO2 + acetolactate Mg

In this case, the acetoin formation requires the intervention

of a second and entirely separate enzyme, a specific aceto- lactate decarboxylase catalyzing the following reaction:

OH CH-3 3 6(CO2)COCH3 + H+--, CH3CHOHCOCH3 + CO2 acetoin

The acetolactate produced in this reaction is a key intermediate in the biosynthesis of valine.

Streptococcus diacetilactis has been shown (87, 88, 89, 90) to carry out the formation of acetoin from alpha -acetolactic acid.

Acetoin so formed, in the presence of an appropriate dehydrogenase, can act as an alternative electron acceptor and can therefore provide means for the reoxidation of NADH by the following reaction: 148

CH3COCHOHCH3 + NADH + H ` CH3CHOHCHOHCH3 + NAD+ acetoin 2:3-butanediol

The radiorespirometric pattern for the catabolism of glucose by the various species and strains of lactic streptococci was found to be essentially the same. The extensive conversion of C3 of glucose to CO2 in the early phase of incubation indicated the important role played by the EMP pathway in these organisms. In addition, the preferential oxidation of C -1 to CO2 over that of C -6 provided evidence for the operation of the HMP pathway. After 60 to 120 minutes of incubation, depending upon the organism used, the exhaustion of glucose from the medium marked the end of the assimilation phase which was immediately followed by the depletion phase. At this point a sizable amount of substrate was presumably converted mainly via the EMP pathway to the biosynthetic inter- mediates such as pyruvate, lactate, and acetate. The inability of glucose carbon atoms 2 and 6 to be converted to CO2 therefore provided evidence for the absence of operative tricarboxylic acid cycle in these organisms.

It was noted that the rate of assimilation as reflected by the conversion pattern of C3,4 of glucose to CO2 via the EMP pathway varied greatly between the various species and strains. The rate of assimilation amongst the lactic streptococci was in the following 149 order:

S. cremoris 144 F highest

S. lactis C2F

S. lactis R-24(e)

S. diacetilactis M-8-224(a)

S. cremoris Ml-W -3 1 S. diacetilactis 18-16

S. lactis 144-54

S. lactis E lowest

The assimilation data also indicated indirectly that the lactic streptococci with the higher rate of assimilation also had higher pyruvate decarboxylase and pyruvate dehydrogenase activity and therefore accumulated a lower amount of lactic acid; conversely, those with a lower rate of assimilation also had a lower pyruvate decarboxylase and pyruvate dehydrogenase activity and therefore accumulated a higher amount of lactic acid. These data were supported by the radioactive inventory findings in the respiratory

and medium. The results obtained with S. diacetilactis CO2,2' cells, M- 8- 224(a) were typical of those found with all the strains tested and these data provided support of the above suggestion; the recovery of only 11% of radioactive CO2 from glucose labeled in C3,4 positions in this organism showed that most of the pyruvate formed from 150 glucose 3, 4 was not being decarboxylated to yield CO2. This pyruvate in turn was most probably being converted to lactic acid and excreted in the medium. This conclusion was also supported by the high recovery of radioactivity in the medium containing cells specifically labeled with glucose -3, 4 -14C. The high recovery in the medium could be attributed to excretion of lactic acid and also the excretion of the products of pyruvate decarboxylase, alcohol dehydrogenase, and pyruvate dehydrogenase (acetaldehyde, ethanol and acetyl coenzyme A respectively).

Qualitatively, one can therefore conclude that no basic dif- ferences exist in the mechanism for glucose assimilation among the various lactic streptococci. The radiorespirometric data on the utilization of glucose

confirmed the occurrence of the HMP pathway but did not provide

information of the nature of this pathway in lactic streptococci. Investigations of other workers have suggested several possibilities as to the fate of pentose produced in bacteria by the HMP pathway. For example, in studies with Sarcina lutea, Dawes and Holms (17) postulated that the pentose phosphate, derived from substrate glucose via the HMP pathway, is first converted by way of pentose

cycle reactions . to fructose -6- phosphate which in turn is catabolized in a manner identical with that of substrate fructose; that is, by way

of the concurrent operation of the EMP and HMP pathways. Also possible is the catabolic sequence converting pentose phosphate to 151 fructose -6- phosphate which is isomerized to glucose -6- phosphate.

Further catabolism of the glucose -6- phosphate so formed, via the pentose cycle pathway exclusively, results in the formation of three moles of CO2 and one mole of triose phosphate from each mole of substrate glucose that traverses the route. It is equally possible that the fructose -6- phosphate derived from pentose phosphate, can be catabolized exclusively by way of the EMP scheme via the intermediary formation of fructose -1:6 - diphosphate. The effect of recycling of the pentose cycle pathway on the rearrangement of the glucose skeleton and the estimation of pathway participation with radiotracer methods has been recently examined by Katz and

Wood (43, 114). The fate of pentose formed in lactic streptococci may follow any one or more of the above possibilities.

Calculations for the Percent Pathway Participation

The oxidation of glucose exclusively at carbon -1 was indicative of phosphogluconate cleavage. Any entrance of carbon -1 into CO2 via the EMP route followed by the TCA cycle should also be reflected in equal C6 oxidation, because carbons 1 and 6 are identical in EMP pathway. So the difference between the C1 and C6 yield in respiratory CO2 should give a measure of the extent of phospho- gluconate cleavage. The percent of the total catabolism of glucose that the pentose phosphate pathway supports on the other hand would 152 be chiefly influenced by the appearance of C3,4 in CO2.

The calculation of the fraction of the total metabolism of glucose that travels via the pentose phosphate pathway, with and without recycling, is measured as (16):

G1 - G6 G = (1) P G,I,

G is the fraction of glucose participating in phosphogluconate decarboxylation. G1 and G6 are the total activity in respiratory

CO2 from cells utilizing equal amounts of C1 or C6 labelled glucose and GT is the total activity of the administered substrate (always taken as 100% or unity).

The fraction of glucose catabolized via glycolysis G(e) is:

G(e) = 1 - G (2)

This equation assumes that Entner Doudoroff pathway does not simultaneously operate. This assumption seems generally safe because when the ED pathway is present, glycolysis is seen generally to have subsided (16). According to Wang (105) there exists no evidence that Entner Doudoroff pathway is operative concurrently with the EMP pathway; this is understandable in view of the fact that either the EMP pathway or the ED pathway can function as the mechanism for the conversion of glucose to two molecules of pyruvate. Consequently there is really no need for the 153 concurrent operation of these two pathways in a given biological system.

The Entner Doudoroff pathway can be demonstrated conclu- sively by using radioactive glucose labeled exclusively in C -3 position. The general availability of glucose 3, 4 -14C dictated its routine use over glucose -3 -14C which is not available at the present time and therefore glucose -3 -14C could not be used in these experiments.

Busse (14) in his experiments with S. diacetilactis used specifically labeled glucose in C -1 and C -6 positions. He found that the lactic acid produced, as a result of glucose catabolism by this organism, contained the radioactive label only on the methyl group. Consequently he concluded that glucose in these organisms was catabolized mainly via the EMP pathway. Busse obtained labeled CO2 and formic acid from glucose- 1 -14C, which led him to conclude that the ED pathway was operative in these organisms.

He ruled out the involvement of the pentose cycle pathway because in this pathway, the COOH group of pyruvate is exclusively derived from C -4 of glucose.

The validity of Busse's experiments seem questionable for the following reasons:

1. Use of specifically labeled glucose only on C -1 and C -6

positions does not give a complete picture of its catabolism. 154

The simultaneous operation of the pentose cycle pathway

and /or the ED pathway together with the EMP pathway, results in the formation of pyruvate and lactate via the

intermediate formation of glyceraldehyde -3- phosphate.

O O II II 4 H-C-O 4 C- OH 4 C--OH

f EMP- ( 5 H-C-OH 7-- 5C=-O ' 5 H-C-OH

I I I 6 H-C-OPO3H2 6 CH3 6 CH3 H glyc e raldehyde -3 Pyruvate Lactate phosphate

The methyl groups of pyruvate and lactate formed via this route are therefore also derived from the sixth carbon of

glucose, shown in Figures 1, 2 and 3. Busse's experi-

ments therefore do not rule out the simultaneous operation

of the EMP and HMP pathways in S. diacetilactis. 14 2. The recovery of radioactivity in CO2 from glucose -1 -14C

can be attributed to the operation of the HMP and /or ED pathway(s). Carbon dioxide formed from glucose -1 -14C

via the operation of the HMP pathway can in turn be

reduced to formate in the presence of NADH.

14CO2 + H14COOH + NAD+ formate 155

This reaction, catalyzed by formate dehydrogenase, may provide the lactic streptococci with yet another means of reoxidizing the

NADH.

In the present experiments, the presence in lactic strepto- cocci of 6- phosphogluconate dehydrogenase, an enzyme exclusive to the HMP pathway, supported the actual operation of the HMP pathway in these organisms. Also the recovery of only 2 to 3% of CO2 from glucose -1 -14C may be attributed to the fixation of carbon dioxide in the presence of NADH to produce formic and oxaloacetic acids.

Wang (16, 107) has developed the following modification of equation 1:

G - (G6 - A6Gp) G = (3) p G T - G T,

where GT = total radioactivity of each administered substrate

GT' = fraction of the labeled substrate engaged in anabolism

G1, G6 = percent total yields of 14CO2 from systems

metabolisms equal amounts of glucose label in these positions

A6 = percent total 14CO2 yield from systems metabolizing

equal amounts of gluconate labeled in these positions; 156

A6 represents the CO2 yield from C6 of glucose by

way of phosphogluconate

G = fraction of the administered glucose catabolized via P the phosphogluconate cleavage

Glucose degradation by the EMP - TCA pathway and the pentose phosphate cycle result in the formation of triose phosphate by both the pathways. Equal amounts of glucose carbons 1 and 6 appear in the triose formed by glycolysis but the triose formed by pentose cycle pathway will contain only carbons 4, 5 and 6. The total triose pool will therefore be enriched in carbon 6, and if there is active TCA cycle operation, the carbon 6 yields in CO2 will be too high. Thus the term G6 in reality will represent the total conversion of C6 of glucose to CO2 via both EMP and pentose cycle pathways whereas the derivation of equation 1 calls for the consideration of CO2 production from C -6 of glucose via the glycolysis exclusively. Therefore (G6 - A6G ) represents the net P production of CO2 from C6 of glucose via glycolysis -TCA pathway which is theoretically identical to the production of CO2 from C -1 of glucose via the same metabolic route. A6Gp represents the

CO2 production from C6 of glucose via the pentose cycle reactions since G represents the fraction of glucose routed into the pentose P cycle and A6 represents CO2 yield from C6 of glucose via phospho- gluconate. 157

Equation 3 can not be used in the case of lactic streptococci because these organisms do not have an operative TCA cycle, thus even if the total triose pool is enriched in carbon 6 both via EMP and pentose cycle pathway, this would not be reflected in CO2 because very little if any of CO2 from C6 will be evolved by these organisms. Therefore G6 in equation 1 does not need to be corrected. GT, correction also does not need to be applied since the fraction of labeled substrate engaged in anabolism in the case of lactic streptococci is very small and will therefore not affect the calculations.

In the lactic streptococci, equation 1 is therefore the most practical equation for the calculation of the fraction of total

metabolism of glucose that travels . via the pentose phosphate pathway, with and without recycling.

Table 38 gives the percent pathway participation in lactic streptococci calculated on the basis of equation 1 and 2. G1 and G6 values were taken at the end of the time course which was the same as the end of the assimilation phase. GT was taken as unity. 158

Table 38. Percent Pathway Participation in Lactic Streptococci.

EMP PP TCA Organism %

S. diacetilactis M-8-224(a) 97 3 0

S. diacetilactis 18-16 98 2 0

S. cremoris M1 -W1-3 98 2 0

S. cremoris 144F 98 2 0

S. lactis E 98 2 0

S. lactis C2F 98 2 0

S. lactis R-24(e) 97 3 0

S. lactis 144-54 98 2 0 159

SUMMARY

The investigation of the enzymes and pathways involved in the catabolism of glucose by several strains of Streptococcus diaceti- lactis, Streptococcus cremoris, and Streptococcus lactis was carried out in order to obtain a proper understanding of the processes by which glucose is utilized for biosynthesis and energy production in these organisms. Aldolase, triosephosphate isomerase and alcohol dehydrogenase were found to be present in the lactic streptococci. The presence of these enzymes provided evidence for the operation of the EMP pathway for the catabolism of glucose by these organisms, which

resulted in the formation of ethanol in addition to large amounts of lactic acid. These so -called homofermentative lactic streptococci therefore were also carrying out the reactions involved in the alcoholic fermentation.

The manometric experiments with lactic streptococci using pyruvate and other alpha -keto acids as substrate suggested that the NAD- lipoate linked pyruvate dehydrogenase catalyzed an oxidative decarboxylation of these keto acids, possibly resulting in the forma- tion of acetyl CoA. Pyruvate was therefore being metabolized by lactic streptococci by the following three reactions:

(a) The lactic dehydrogenase catalyzed reversible reduction 160

of pyruvate to lactate with NADH serving as the reducing agent.

(b) The pyruvate decarboxylase and alcohol dehydrogenase

catalyzed non - oxidative decarboxylation of pyruvate leading

to the production of ethanol via the intermediate formation

of acetaldehyde.

(c) NAD- lipoate linked pyruvate dehydrogenase catalyzed

oxidative decarboxylation of pyruvic acid - leading to the

formation of acetyl CoA.

Reoxidation of NADH formed during the operation of EMP pathway could be accomplished by the lactic and alcohol dehydrogenase catalyzed reactions. The NADH formed during the oxidative decar- boxylation of pyruvate could be reoxidized to NAD possibly by some electron transport mechanism in which oxygen served as the ultimate electron acceptor.

The presence of glucose -6- phosphate dehydrogenase and

6- phosphogluconate dehydrogenase in lactic streptococci demonstrated the existence and possible operation of the HMP pathway in these organisms. The operation of the HMP pathway in lactic streptococci may serve to provide these organisms with pentoses, which may be used in ribose and deoxyribonucleotide, coenzyme, polysaccharide and polynucleotide biosynthesis. The operation of this pathway leads to the formation of NADPH, which may be utilized in the biosynthetic 161 reactions.

The presence of acetate kinase in lactic streptococci gave evidence for the formation of acetate from acetyl CoA by way of the intermediate formation of acetyl phosphate through the phosphoro- clastic reaction involving the enzymes phosphotransacetylase and acetate kinase. This reaction mechanism generates one molecule of ATP from one molecule of acetyl CoA metabolized.

The operation of the HMP pathway in lactic streptococci and the presence of acetate kinase in these organisms also provided evidence for the formation of another molecule of ATP through the pentose fermentation in which xylulose -5- phosphate, which is formed from glucose via the operation of the HMP pathway, may undergo a phosphoketolase catalyzed phosphorolytic cleavage to form triose phosphate and acetyl phosphate. Acetyl phosphate in the presence of ADP and acetate kinase may then form acetate resulting in the generation of one molecule of ATP. Acetoin, which is formed from alpha acetolactic acid by S. diacetilactis, in the presence of appropriate dehydrogenase may act as an electron acceptor and may therefore provide yet another means for the reoxidation of NADH by producing 2:3 butanediol. Radiorespirometric experiments with lactic streptococci using specifically labeled glucose as substrate confirmed the actual operation of both EMP and HMP pathway for the catabolism 162

of glucose by these organisms. The radiorespirometric pattern for the catabolism of glucose by the various species and strains of lactic streptococci was found to be essentially the same, and showed the important role played by the EMP pathway in these organisms. These experiments also demonstrated the absence of an operative tricarboxylic acid cycle in lactic streptococci. The radioactive

inventory data and the assimilation data as reflected by the conver-

sion of C3,4 of glucose to CO2, indicated that lactic streptococci

with higher rates of assimilation also had greater pyruvate decarboxylase and pyruvate dehydrogenase activity and therefore

accumulated a lower amount of lactic acid; those with a lower rate

of assimilation which also had a lower pyruvate decarboxylase and pyruvate dehydrogenase activity accumulated a higher amount of lactic acid. Radiorespirometric data were also used for the calculation of

the percent pathway participation in lactic streptococci which showed that glucose was catabolized mainly via the EMP pathway by these organisms. This investigation therefore provided evidence against the

classification of S. diacetilactis, S. cremoris, and S. lactis in a strictly homofermentative group. The classification of these lactic

streptococci in the microaerophilic group, which includes organisms

that grow best in the presence of very limited amount of oxygen has 163

also become questionable because these organisms were found to have a very active oxidative decarboxylation mechanism operative

which catalyzed the conversion of pyruvic acid to acetyl CoA. Also the presence of excess oxygen did not inhibit in any way the growth

of S. diacetilactis M- 8- 224(a). 164

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