LACTOSE AND “D-GALACTOSE- MEIABOUSM 1N . STAPHYLOCOCCUSE AUREUS: V ELuOIDAnON OF THE ” PATHWAY .OF D -GALACTOSE 6_- PHOSPHATE j _ _ DEGRADATION AND PUR‘IHGAHO‘NANDA ; » CHARACTERIZATION OF THE ENZYMES INVOLVED Dissertation for the Degree Of Ph. D. ' MICHIGANSTATE UNIVERSITY * DONALD LYNN BISSEIT ' ' 197-5, I- '4!" .. G. M W LACTOSE AND D-GALACTOSE METABOLISM IN STAPHYLOCOCCUS AUREUS: ELUCIDATION OF THE PATHWAY OF D-GALACTOSE 6-PHOSPHATE DEGRADATION AND PURIFICATION AND CHARACTERIZATION OF THE ENZYMES INVOLVED By Donald Lynn Bissett Others have shown that D-galactose 6—phosphate is formed intracellularly when Staphylococcus aureus metabolizes lactose or D—galactose. I have determined by enzymatic, chemical, chromato- graphic, and genetic methods that D—galactose 6-phosphate is metabolized by a previously undescribed pathway, the D—tagatose 6-phosphate pathway, which is outlined in the following reactions: (1) D-Galactose 6—phosphate isomerase D-galactose 6-phosphate +_____ D—tagatose 6-phosphate (2) D-Tagatose 6-phosphate kinase D-tagatose 6-phosphate D—tagatose 1,6-diphosphate + ——+ + adenosine 5'-triphosphate adenosine 5'-diphosphate (3) D-Tagatose 1,6-diphosphate aldolase. dihydroxyacetone phosphate D—tagatose l,6-diphosphate-—-+ + D-glyceraldehyde 3-phosphate # Donald Lynn Bissett The enzymes catalyzing these reactions were induced only by growth of §, aureus on lactose and D-galactose, and were chromatographi— cally separable from the corresponding enzymes of the Embden— Meyerhof pathway (D-glucose 6-phosphate isomerase, D-fructose 6-phosphate kinase, and D—fructose 1,6-diphosphate aldolase) of D-glucose 6—phosphate metabolism. The enzymes of the D—tagatose 6-phosphate pathway have been purified and some of their proper- ties determined. These properties include substrate specificities, Km values, pH optima, molecular weights, S values, and monovalent and divalent metal ion requirements. In addition, the products of the reactions they catalyze have been conclusively identified. Mutants which were defective in each of the three enzymes were isolated and were found to be specifically unable to utilize lac- tose or D—galactose. These mutants accumulated the substrate of the missing enzyme intracellularly, as would be predicted. Spon- taneous revertants, which regained the ability to utilize lactose and D—galactose, regained the missing enzyme and no longer accumu- lated intermediates of the D-tagatose 6-phosphate pathway. These studies indicate that the pathway is physiologically significant in lactose and D—galactose metabolism in_§. aureus. The absence of f other possible reactions for the modification of D-galactose 6-phosphate and the mutant studies indicate it is the sole pathway for the utilization of these two sugars. The enzymes of the D-tagatose 6-phosphate pathway comprise a heretofore undiscovered route of lactose and D—galactose metabolism that functions instead of, rather than in addition to, pathways previously established in other organisms. LACTOSE AND D-GALACTOSE METABOLISM IN STAPHYLOCOCCUS AUREUS: ELUCIDATION OF THE PATHWAY OF D-GALACTOSE 6-PHOSPHATE DEGRADATION AND PURIFICATION AND CHARACTERIZATION OF THE ENZYMES INVOLVED By Donald Lynn Bissett A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1975 ACKNOWLEDGMENTS I acknowledge the invaluable assistance of Dr. Richard L. r Anderson, my friend, teacher, and advisor. For their assistance, I thank Drs. W. C. Deal, P. K. Kindel, C. C. Sweeley, and R. N. Costilow. The helpful suggestions and valuable discussions of J. P. Markwell, R. A. Simkins, and G. T. Shimamoto are greatly appreciated. This research was supported by the National Institutes of Health. ; 11 ' '71?- Q ‘1 in. L n TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . LIST OF ABBREVIATIONS . . . . . . . . . . . GENERAL INTRODUCTION . . . . . . . . . .' . . LITERATURE REVIEW OF LACTOSE AND D—GALACTOSE METABOLISM SECTION 1 ELUCIDATION OF THE PATHWAY OF D-GALACTOSE 6-PHOSPHATE DEGRADATION INTRODUCTION . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . Bacterial Strain, Cell Growth and Storage, and Preparation of Cell Extracts . . . . Bacterial Strain . . . . . . . . . . Media . . . . . . . . . . . Sterilization Technique . . . . . . . . Growth Conditions. . . . . . . . . . Centrifugation . . . . . . . . . . . Storage of Bacteria . . . . . . . . Harvesting of Cells . . . . . . . . Preparation of Buffer Solutions . . . . . Preparation of Glass Beads . . . . . . . Preparation of Cell Extracts . . . . . . Colorimetric Determinations . . . . . . . Protein Determination . . . . . . . . Phosphate Determination . . . . . . . . Ketohexose Determination . . . . . . . Aldohexose Determination . . . . . . . Preparation of Substrates . . . . . . . . Preparation of D-Galactose . . . . Removal of Barium From Sugar Phosphates . Preparation of D-Galactose 6-Phosphate . . . Preparation of D—Tagatose 6-Phosphate . . . iii Page viii Gas-Liquid Chromatography . . . . . . . . . Acid Hydrolysis . . . . . . . . . . . . Enzymatic Assays . . . . . . . . . . Galactose 6-Phosphatase . . . . . . Galactose 6— —Phosphate Dehydrogenase . Galactose Dehydrogenase . . . . . . Glucose 6—Phosphate Dehydrogenase Galactose 6-Phosphate Reductase . . . . . . Galactose Reductase . . . . . . . . . . Galactose 6—Phosphate 4-Epimerase . . . . . Galactose 4-Epimerase . . . . . . . . Galactose l—Phosphate 4-Epimerase . . . . . Galactose 6—Phosphate Kinase . . . . . . . Galactose l—Phosphate Kinase . . . . . . . Galactokinase . . . . Glucokinase . . . . . . . Galactose l-Phosphate Uridylyltransferase . . . UDP- Galactose 4— —Epimerase . . . . . . . . Galactose Isomerase . . . . . . . . . . Galactose 6-Phosphate Isomerase . . . . . . Glucose 6-Phosphate Isomerase . . . . . . . Fructose 6—Phosphate Kinase . . . . . . . Fructose 1,6—Diphosphate Aldolase . . . . . Preparation of Ion Exchange Resins . . . . . . Enzyme Purification Procedures . . . . . . . Bentonite Treatment . . . . . . . . . Protamine Sulfate Treatment . . . . . . . Ammonium Sulfate Precipitation . . . . . . Protein Chromatography . . . . . . . . . DEAE- Cellulose Chromatography . . . . . . . Sephadex G-100 Chromatography . . . . . . . Sources of Materials . . . . . . . RESULTS . . . . . . . . . . . . . . . . . Enzymatic Reactivity of D—Galactose 6-Phosphate . Enzymatic Reactivity of D-Tagatose 6—Phosphate . . Assays for the D—Tagatose 6-Phosphate Pathway Enzymes Induction of the D-Tagatose 6-Phosphate Pathway Enzymes . . . DEAE-Cellulose Chromatography of the D-Tagatose 6-Phosphate Pathway and Embden—Meyerhof Pathway Enzymes . . . . . . . DISCUSSION . . . . . . . . . . . . . . . iv 58 66 SECTION 2 PURIFICATION AND CHARACTERIZATION OF THE ENZYMES OF THE D—TAGATOSE 6—PHOSPHATE PATHWAY INTRODUCTION . MATERIALS AND METHODS Cell Growth and Preparation of Cell Extracts Media . . . . . . . . . Growth Conditions . . . . . . Harvesting of Cells . . Preparation of Cell Extracts . . . Enzymatic Assays . . . . . . . . Determinations . . . D-Galactose 6-Phosphate Isomerase Assays D— -Tagatose 6- -Ph03phate Kinase Assays D-Tagatose 1, 6- -Diphosphate Aldolase Assays Alkaline Phosphatase (E. coli) Assays Alkaline Phosphatase (Calf Intestine) Assays . Fructose l-Phosphate Kinase Assays Hexokinase Assays . . . . . . Peroxidase Assays . . . . . . Ovalbumin Assays . . . . Enzyme Purification PhOSphocellulose and CM-Cellulose Chromatography Hydroxyapatite Chromatography . . Sephadex G-15 and G-25 Chromatography Dialysis . . . . . Paper Chromatography . . . . . . . Gas-Liquid Chromatography . . . Polyacrylamide Gel Electrophoresis . . Native Polyacrylamide Gel Electrophoresis . Elution of Protein From Native Gels . Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Molecular Weight and Sedimentation Coefficient Determination . . . Sucrose Gradient Centrifugation Molecular Weight Determination on Sephadex G-100 Ketohexose Differentiation . . . . Nucleoside Phosphate Determination . . Effect of NEM . . . Dephosphorylation of Sugar Phosphates . Removal of Cations . . . Removal of Monovalent Cations . . . Removal of Divalent Cations . . . Effect of NaBHa . . . . . . . . Sources of Materials . . . . . . . Page “SULTS I I I I I I O I I C I C O O I I I I D—Galactose 6—Phosphate Isomerase (D—Galactose 6-Phosphate Ketol—Isomerase) . . . . . . . . Purification . . . . . . . . . . . . . Stability . . . . . . . . . . . Product Identification . . . . . . . . . . Properties . . . . . . . . . . . . . . Discussion . . . . D-Tagatose 6-Phosphate Kinase (ATP: D-Tagatose 6- -Phosphate 1-Phosphotransferase) . . . . . Purification . . . . . . . . . . . . . Stability . . . . . . . . . . . . Product Identification . . . . . . . . . . Properties . . . . . . . . . . . . . . Discussion . . . . D—Tagatose 1, 6- —Diphosphate Aldolase (D-Tagatose 1,6- Diphosphate D-Glyceraldehyde 3-Phosphate-Lyase) . . Purification . . . . . . . . Stability . . . . . . . . . . . . Product Identification . . . . . . . . . . Properties . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . SECTION 3 GENETIC EVIDENCE FOR THE PHYSIOLOGICAL SIGNIFICANCE OF THE D-TAGATOSE 6-PHOSPHATE PATHWAY INTRODUCTION . . . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . Bacterial Strains, Cell Growth, and Preparation of Cell Extracts . . . . . . . . . . . . . Organisms . . . . . . . . . . . . . . Media . . . . . . . . . . . . Preparation of Cell Extracts . . . . . . Cell Growth and Enzyme Induction Procedures . . . Total Sugar Determination . . . . . . . . . . Enzymatic Assays . . . . . . . . . . . . . Isolation of Mutants . . . . . . . Isolation of Streptomycin—Resistant Mutant (Strain SR7) . . . Isolation of a Mutant (Strain L2) Constitutive for the Enzymes of the D—Tagatose 6-Phosphate Pathway . . Isolation of Mutants (GBZ7, .GD29, and GE26) Defi- cient in Enzymes of the D-Tagatose 6-Phosphate Pathway . . . . . . . . . . . . vi Page 85 143 168 174 175 175 175 175 177 177 177 178 178 178 178 179 ‘r——_'——_——‘ ' Page Isolation of Spontaneous Revertants GBZ7R, g GD29R, and GE26R . . . . . 179 . Determination of Intracellular Metabolite Levels . . 180 Sources of Materials . . . . . . . . . . . . 182 1 RESULTS . . . . . . . . . . . . . . . . . . 183 j Mutant Isolation . . . . . . . . . . . . 183 Carbohydrate Utilization . . . . . . . . . . 188 Intracellular Metabolite Levels . . . . . 189 Common Genetic Control of D—Tagatose 6—Phosphate Pathway Enzymes . . . . . . . . . 194 ' DISCUSSION . . . . . . . . . . . . . . . . . 197 GENERAL DISCUSSION . . . . . . . . . . . . . . 200 REFERENCES . . . . . . . . . . . . . . . . . 203 LIST OF TABLES Table Page 1. Conversion of D—galactose 6-phosphate to triose phosphates . . . . . . . . . . . . . 47 2. Phosphorylation of D-tagatose 6-phosphate with ATP . . . . . . . . . . . 50 3. Cleavage of D—tagatose 1, 6— —diphosphate to triose phosphates . . . . . . . . . . . 51 4. Enzyme levels in cell extracts of_§. aureus . . . . 59 5. Levels of D-fructose 1,6-diphosphate aldolases in cell extracts of S, aureus . . . . . . . . . 64 6. Purification of D-galactose 6-phosphate isomerase . . 88 7. Time-dependent reactivation of D—galactose 6-phosphate isomerase by BME . . . . . . . . 107 8. Purification of D-tagatose 6-phosphate kinase . . . 114 9. Stoichiometric formation of ADP and D-tagatose 1, 6- -diphosphate by D-tagatose 6—phosphate kinase . . . . . . . . . . 126 10. Effect of monovalent cations on D—tagatose 6-phosphate kinase activity . . . . . . . . 135 11. Time-dependent reactivation of D—tagatose 6-phosphate kinase by BME . . . . . . . . . 138 12. Purification of D—tagatose 1, 6- -diphosphate aldolase . . . . . . . . . . 144 13. Enzymatic identification of the D-tagatose 1,6— diphosphate aldolase cleavage products . . . . . 152 14. Stoichiometric formation of dihydroxyacetone phosphate and D-glyceraldehyde 3—phosphate by D-tagatose 1,6-diphosphate aldolase . . . . . 154 viii Table 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. Competition between D-tagatose 1,6-diphosphate and D-fructose l, 6- -diphosphate for D-tagatose l, 6- -diphosphate aldolase . . . . . . Enzymatic identification of the D—tagatose 1,6- diphosphate aldolase condensation reaction product . . . . . . . . . . . . . Time-dependent reactivation of D-tagatose 1,6- diphosphate aldolase by BME . . . . . Comparison of Class I and Class II aldolases Genealogy and phenotype of the S, aureus strains Enzymes of the D-tagatose 6—phosphate pathway and corresponding enzymes of the Embden-Meyerhof pathway in various strains of S, aureus . . Utilization of carbohydrates by various strains of S. aureus as determined in bromcresol purple broth . . . . . . . . . . . . . . Utilization of lactose and D—galactose by various strains of S, aureus as determined by the anthrone test . . . . . . . . . . . Effect of carbohydrate on the colony size of several strains of S, aureus . . . . . . Intracellular accumulation of intermediates of the D-tagatose 6—phosphate pathway . . . . . ix Page 158 161 164 171 176 184 190 191 192 193 ‘Tfirw—v— 10. 11. 12. 13. LIST OF FIGURES Scheme for the chemical synthesis of 6-phosphate . . . . . . . D-tagatose Gas—liquid chromatography of the trimethylsilyl derivative of D-galactose 6—phosphate The D—tagatose 6—phosphate pathway as elucidated in this study . . . . . Linearity of the forward D-galactose isomerase reaction with respect to Linearity of the forward D—galactose isomerase reaction with respect to concentration . . . . . . . Linearity of the reverse D-galactose isomerase reaction with respect to Linearity of the reverse D-galactose isomerase reaction with respect to concentration . . . . . . . 6-phosphate time . . 6-phosphate enzyme a O o o 6-phosphate time . . 6-ph03phate enzyme Linearity of the D-tagatose 6-phosphate kinase assay with respect to time . . . Linearity of the D—tagatose 6-phosphate kinase assay with respect to enzyme concentration . Linearity of the D—tagatose 1,6-diphosphate aldolase assay with respect to time . . . Linearity of the D-tagatose 1,6—diphosphate aldolase ‘ o O . assay with resPect to enzyme concentration Chromatography of D—galactose 6—phosphate and D—glucose 6-phosphate isomerases on DEAE- cellulose . . . . . . . . Chromatography of D-tagatose 6-phosphate and D-fructose 6-phosphate kinases on DEAE- cellulose . . . . . Page 29 45 48 53 53 55 55 56 56 57 57 60 61 Figure Page 14. Chromatography of D—tagatose l, 6— —diphosphate and D-fructose l, 6— —diphosphate aldolases on DEAE— cellulose . . . . . . . . . . . . . 63 15. Chromatography of D-galactose 6-phosphate isomerase on DEAE- cellulose . . . . . . . . . . 89 16. Chromatography of D—galactose 6-phosphate isomerase on DEAE-cellulose . . . . . . . . . . . 91 l7. Chromatography of D-galactose 6-phosphate isomerase on Sephadex G—100 . . . . . . . . . . . . 93 18. Gas-liquid chromatography of the trimethylsilyl derivative of the forward D-galactose 6—phosphate isomerase reaction product . . . . . . . . 95 19. Gas-liquid chromatography of the trimethylsilyl derivative of the reverse D—galactose 6—phosphate isomerase reaction product . . . . . . . . . 98 20. Effect of pH and buffer composition on D-galactose 6-ph03phate isomerase activity . . . . . . 100 21. Lineweaver—Burk plots showing the effect of substrate concentration on the D—galactose 6-ph03phate isomerase reaction velocity . . . . . . . 101 22. Equilibrium of D-galactose 6-phosphate isomerase- catalyzed reaction . . . . . . . . . . . 102 23. Gas-liquid chromatography of the trimethylsilyl derivative of the reverse D-galactose 6—phosphate isomerase-catalyzed reaction equilibrium mixture . 104 24. Plot of elution volume against log MW of standards and D—galactose 6—phosphate isomerase from a chromatographic run on Sephadex G—100 . . . . . 108 25. Sedimentation pattern of calf intestine alkaline phosphatase and D-galactose 6-phosphate isomerase in a sucrose density gradient . . . . 108 , 26. Chromatography of D-tagatose 6-phosphate kinase on I DEAE-cellulose . . . . . . . . . . . . 116 h 27. Chromatography of D—tagatose 6-phosphate kinase on DEAE-cellulose . . . . . . . . 117 xi r———_ Figure 28. 29. 30. 31. 32. 33. 34. 35. 36» 37. 38. 39. 40. 41. 42. Chromatography of D—tagatose 6-phosphate kinase on hydroxyapatite . . . . . . . . . Chromatography of D-tagatose 6-phosphate kinase on Sephadex G-100 . . . . . . . . . Polyacrylamide gel electrophoresis of purified D— —tagatose 6-phosphate kinase . . . . . . . Chromatography of the D-tagatose 6-phosphate kinase reaction product on Dowex l-X8 . . . . . . . Effect of pH and buffer composition on D—tagatose 6-phosphate kinase activity . . . . . . . . Lineweaver—Burk plots showing the effect of substrate concentration on the D-tagatose 6—phosphate kinase reaction velocity . . . . . . . . . . . Lineweaver—Burk plots showing the effect of phosphoryl donor concentration on the D—tagatose 6—phosphate kinase reaction velocity Effect of magnesium to ATP ratio on D-tagatose 6-phosphate kinase activity . . . . . . . . Plot of elution volume against log MW of standards and D—tagatose 6—phosphate kinase from a chromatographic run on Sephadex G—100 . . . . . Plot of migration distance against log subunit molecular weight of standards and D—tagatose 6-phosphate kinase in a polyacrylamide gel in the presence of dodecyl sulfate . . . . . . Sedimentation pattern of standards and D-tagatose 6-phosphate kinase in a sucrose density gradient . . . . . . . . . . . . . . . Chromatography of D—tagatose 1,6—diphosphate aldolase on phosphocellulose . . . . . . . . Chromatography of D-tagatose 1, 6— —diphosphate aldolase on phosphocellulose . . . . . Chromatography of D— —tagatose l, 6- -diphosphate aldolase on DEAE- cellulose . . . . . . . . Polyacrylamide gel electrophoresis of purified D-tagatose 1,6-diph03phate aldolase . . . . . . xii Page 119 120 121 123 127 129 131 136 139 139 141 147 148 149 151 r——+——‘ Figure Page 43. Effect of pH and buffer composition on D-tagatose 1,6-diphosphate aldolase activity . . . . . . 155 44. Lineweaver-Burk plots showing the effect of substrate concentration on the D-tagatose 1,6-diphosphate aldolase reaction velocity . . . . . . . . . 157 45. Thermal inactivation of D—tagatose l, 6— —diphosphate aldolase . . . . . . . . . . . . . 159 46. Plot of elution volume against log MW of standards and D-tagatose 1,6—diphosphate aldolase from a chromatographic run on Sephadex G—100 . . . . . 166 47. Plot of migration distance against log subunit molecular weight of standards and D—tagatose 1,6—diphosphate aldolase in a polyacrylamide gel in the presence of dodecyl sulfate . . . . . 165 48. Sedimentation pattern of horseradish peroxidase and D-tagatose 1,6-diphosphate aldolase in a sucrose density gradient . . . . . . . . . . . . 167 49. Effect of penicillin upon the growth of SR7 on D-galactose . . . . . . . 186 50. Effect of D-cycloserine upon the growth of SR7 on D-galactose . . . . . . . . . . 187 51. Time course of induction of the enzymes of the D-tagatose 6-phosphate pathway in S. aureus NCTC 8511 . . . . . . . . . . 195 52. Coordinate induction of the enzymes of the D-tagatose 6—phosphate pathway in S, aureus NCTC 8511 . . . 196 xiii ONPG IPTG ATP PEP GTP ITP TTP GTP NAD NADH NADP NADPH LIST OF ABBREVIATIONS ortho-nitrophenyl-B-D—galactoside thio-methyl-B-D-galactoside isopropylthio-B-D—galactoside molecular weight adenosine 5'-triphosphate adenosine 5'-diphosphate phosphoenol pyruvate uridine 5'—triphosphate uridine 5'-diphosphate guanosine 5'—triphosphate inosine 5'-triphosphate thymidine 5'-triphosphate cytidine 5'—triphosphate nicotinamide adenine dinucleotide reduced nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate reduced nicotinamide adenine dinucleotide phosphate 2,6-dichlorophenol indophenol acceleration of gravity gram milligram microgram xiv r—_____———_. 1 liter m1 . milliliter ul microliter M molar mu millimolar HM micromolar cm centimeter mm millimeter um micrometer ' nm nanometer i EDTA ethylenediamine tetraacetic acid } DEAE- diethylaminoethyl- CM- carboxymethyl- NEM N-ethylmaleimide t TEMED N,N,N',N'-tetramethylethylenediamine i Tris tris (hydroxymethyl) aminomethane Bicine N,N—bis (2-hydroxyethyl) glycine ' HEPES N-2-hydroxyethylpiperazine-N'-2—ethane-su1fonic acid B-mercaptoethanol; 2—thioethanol absorbance sedimentation coefficient (Svedberg; 1 X 10'-13 second) at 20°C in water GENERAL INTRODUCTION This study was undertaken to discover the pathway of lac- tose and D-galactose metabolism in Staphylococcus aureus. Others have shown that the metabolism of lactose is initiated by phos- phorylation at carbon 6 of the D-galactosyl moiety by PEP (see Literature Review). The resulting lactose phosphate is then cleaved by a phospho—B-galactosidase to yield D—glucose plus D-galactose 6-phosphate. D—Galactose metabolism is also initiated by phosphorylation at position 6 by PEP. Since no route was known for the further metabolism of D—galactose 6-phosphate, this inves- tigation was designed to elucidate the pathway for its degradation. The results are divided into three sections. The first section deals with those experiments which led to the discovery of the D-tagatose 6-phosphate pathway for D-galactose 6-phosphate netabolism in S. aureus. The results are presented in a manner such that the rationale used in proposing the pathway is revealed. The second section summarizes the purification of the three enzymes of the D—tagatose 6-phosphate pathway, the determination of some of their properties, and the identification of their reaction products. The data establish the existence of the pathway: D-galactose 6-phosphate'—-+ D-tagatose 6—phosphate-——+ D—tagatose 1,6- diphosphate--+ dihydroxyacetone phosphate + D-glyceraldehyde 3—phosphate. The results presented in the third section deal with genetic evidence for the physiological role of the pathway. The results demonstrate that the D~tagatose 6-phosphate pathway is physiologically significant in the metabolism of lactose and D—galactose in S. aureus. LITERATURE REVIEW OF LACTOSE AND D-GALACTOSE METABOLISM The most common route for the metabolism of lactose (4-O—B-D-ga1actopyranosyl-D—glucopyranose) is via hydrolytic cleav- age to D—glucose plus D-galactose. This reaction is catalyzed by the enzyme B-galactosidase (l). D—Galactose is most commonly metabolized by the Leloir pathway (2): D-galactose--—+ D-galactose l-phosphate-—-+ D—glucose l-phosphate--—+ D-glucose 6-phosphate. In adult, but not in infant, humans D-galactose can be metabolized by an alternate route (3): D-galactose-———+ D—galactose l—phosphate -—-+ UDP-galactose-—-+ UDP-glucose-——+ D—glucose 1-phosphate-——‘+ D—glucose 6-phosphate. Thus, an adult galactosemic who is lacking galactose 1-phosphate uridylyltransferase can metabolize D-galactose by utilizing the enzyme UDP-galactose pyrophosphorylase, which also functions as a UDP-glucose pyrophosphorylase (4). Several publications reported the oxidation of lactose at carbon 1 of the D—glucose moiety to form lactobionic acid (4-O-B-D- galactopyranosyl—D-gluconic acid). The reaction was performed by a variety of organisms: a number of Pseudomonas strains (5-9), some paracolon bacteria (10), the bacterium Alcaligenes faecalis (ll-13), the bacterium Cogynebacterium simplex (14), the red alga Iridophycus flaccidum (15), the mold Penicillium chgysogenum (16), and one strain of Streptococcus lactis (17, 18). Lactobionic acid F——————_ was detected in the culture medium after growth of the organisms in media containing lactose, and some of these organisms were found to utilize lactobionic acid that was added to the culture medium (6, 14, 16-19). Bernaerts and DeLey (ll—l3) reported that in addition to the conversion of lactose to lactobionic acid they observed the conversion of lactose to 4—0-8-3—keto-D-galacto- pyranosyl-D-glucopyranose and of lactobionic acid to 4-0-B—3-keto- D—galactopyranosyl-D-gluconic acid; no further metabolism of these compounds was reported. Nishizuka and Hayaishi (9) found that lactose was converted to lactobiono—G—lactone by a dehydrogenase and that the lactone was then hydrolyzed to lactobionic acid by a lactonase. They purified both enzymes from cell extracts: the dehydrogenase was purified from the particulate fraction and was found not to be a pyridine nucleotide-linked enzyme, and the lactonase was purified from the soluble fraction. Bernaerts and DeLey (l4) and Vakil and Shahani (17, 18) reported that cell extracts of their strains could cleave lactobionic acid to D-galactose plus D—gluconic acid. The cleavage of lactobionic acid occurred at a much slower rate than the cleavage of lactose, but the rate of hydrolysis was sufficient to support the growth that occurred on lactobionic acid. There are two mammalian enzymes, an aldose reductase (20- 22) and an L-hexonate dehydrogenase (23, 24), which reduce D-galactose to galactitol, but only at high concentrations of I D-galactose, such as those that occur in galactosemic individuals; A however, the galactitol was found to accumulate (25-27) since there ‘r——_—____—— is apparently no pathway for its metabolism (28). Insects (the butterfly Pieris brassicae and the boll weevil Anthonomus grandis) also possess an aldose reductase which converts D—galactose to galactitol (29, 30); the galactitol accumulates since it is not metabolized (30). Leaves of Euonymus japonica also reduce D—galactosetxygalactitol (31), and the galactitol is used in the synthesis of structural carbohydrates (32). A pathway for D-galactose metabolism involving oxidation was reported to occur in mammalian liver (33): D—galactose-————+ D—galactonic acid-—+ 3-keto-D-galactonic acid--—+ D-xylulose -—-+ D—xylulose 5-phosphate. Partial product identification for all reactions was done. The galactose dehydrogenase was purified (34) and characterized (35) in later work. However, the occurrence of this pathway has been denied by other researchers who were unable to demonstrate galactose dehydrogenase activity (36). These researchers reported that if all the ethanol was removed from the commercial D—galactose used, the galactose dehydrogenase activity was not observed,suggestingthe enzyme was actually alco— hol dehydrogenase (37). A galactose dehydrogenase is involved in an oxidative pathway in Pseudomonas saccharophila (38—40): D-galactose--+ D-galactonic acid--—+ 2-keto-3—deoxy-D-galactonic acid-——+ 2-keto-3-deoxy-D—galactonic acid 6—phosphate--—+ pyruvic acid + D-glyceraldehyde 3—phosphate. The reactions of this pathway have been conclusively established. This pathway has also been found to occur in Pseudomonas fluorescens (41). Oxidation of D-galactose is also performed by a galactose oxidase from the mold A... 515 Polyporus circinatus (42). This enzyme converts D—galactose to the 1,6-dialdehyde (D-galactohexodialdose) by oxidation of carbon 6 with molecular oxygen (42, 43). The enzyme is believed to function naturally in the oxidation of polysaccharides which contain terminal D—galactose since such saccharides are oxidized more rapidly and with higher affinity than free D-galactose (44). The metabolism of lactose and D-galactose in Staphylococcus aureus is unlike that in any of the organisms cited above, as the following discussion will indicate. Initially, efforts to demon- strate B-galactosidase in cell-free extracts were unsuccessful (45, 46), but whole cells did hydrolyze ONPG (45, 47, 48), a metabolizable analog of lactose. The enzyme was erroneously con- sidered to be extremely unstable in cell-free extracts. In 1965 Egan and Morse (46) reported the isolation of several mutants of S, aureus which simultaneously lost their ability to utilize and to accumulate D—fructose, D—mannitol, D~galactose, -D—ribose, lactose, maltose, sucrose, and trehalose; revertants regained the ability to metabolize and to accumulate all eight carbohydrates, indicating that a single genetic locus had been affected. These mutants were designated £257. Similar mutants of S. aureus had been isolated previously by Korman (49), and she also noted that they reverted to the wild-type phenotype. However, she did no further characterization. Egan and Morse (48) found that the transport defect (the inability to accumulate carbohy- drates) was not associated with the cell wall since spheroplasts still displayed the car- phenotype, and the defect was restricted ‘PnlIFr_—_—_________—___—___________—_———___'___——_____—"IIIWII to carbohydrates since whole cells were still able to transport other classes of compounds. In addition, transport of carbohy- drates (except sucrose) was absent in the Egg? mutant. In agree- ment with the theory of sugar transport (50, 51), Egan and Morse (48, 52) concluded that the £357 mutant lacked the common carrier responsible for the transport of several carbohydrates into the cell, and suggested that an inducible, carbohydrate—specific per- mease functioned to couple the carbohydrate and the carrier. Evidence presented by several groups indicated that lac- tose transport in S. aureus involved phosphorylation. A phosphoenolpyruvate:carbohydrate phosphotransferase system (PEP: carbohydrate PTS) was discovered in Escherichia 221$ by Kundig, Ghosh, and Roseman (53). It consists of two enzymes (I and II, II being membrane associated) and a histidine—containing protein (HPr). The following sequence of reactions occurs during trans— port by this system: PEP HPr carbohydrate phosphate EI EII pyruvate phospho-HPr carbohydrate In 1967 Kennedy and Scarborough (54) postulated that ONPG (and therefore lactose) was transported by this system in S, aureus, and that the resulting ONPG phosphate was hydrolyzed by a phospho—B- galactosidase. To support their contention, Kennedy and Scarborough reaffirmed that whole cells, but not cell-free extracts, of lactose- induced S, aureus hydrolyzed ONPG. The hydrolysis by whole cells was inhibited by NaF, which was consistent with the hypothesis that PEP, provided via the enolase reaction, was necessary for uptake and indirectly for hydrolysis. In addition, cell—free extracts hydrolyzed ONPG when supplied with PEP, but not with ATP. In 1966, Egan and Morse (52) had found that 14C-lactose accumulated in cells of S, aureus in the form of a derivative. In 1967, Hengstenberg, Egan, and Morse (55) identified the derivative as a phosphate. Using whole cells of a lactose-nonmetabolizing strain of S. aureus, which formed but could not utilize the derivative, the derivative was prepared in quantity from 14C-lactose (14C was in the glucose moiety; lactose with 14C in the galactose moiety was not commercially available). During electrophoresis the derivative migrated as a negatively-charged species; treatment with alkaline phosphatase destroyed the derivative and converted it to 14C—lactose. If the cells were provided additionally with 32P- phosphate, the derivative was labeled with both 32P and 14C. Treat- ment of this derivative with a cell-free extract of lactose-grown wild-type S, aureus resulted in formation of l4C-D—glucose and 32P-hexose, suggesting the presence of a phospho-B—galactosidase. Purified S, 221; B-galactosidase did not hydrolyze the derivative. Further study (56) revealed that the derivative had a phosphate to lactose ratio of 1. To determine the site of phosphorylation in lactose phosphate, ONPG 6—phosphate was synthesized chemically (57). The ONPG 6-phosphate served as substrate for the staphylo- coccal B—galactosidase (a phospho-B-galactosidase), but not for purified_S. coli B—galactosidase (56, 57). Laue and MacDonald (58) continued this line of work. They incubated cells of lactose-grown S, aureus with l4C-TMG (a nonmetabolizable analog of lactose) and isolated a single 14C-derivative from the cells. Upon electro— phoresis, the derivative was found to be negatively charged, and treatment with alkaline phosphatase converted it to ll'C-TMG. When double labeling experiments were done with 14C—TMG and 32P- phosphate, the derivative was labeled with both isotopes. Elemental analysis identified the derivative as TMG phosphate. Using chemi- cally synthesized TMG 6-phosphate as a standard, nuclear magnetic resonance and X-ray powder diffraction pattern data identified the derivative as TMG 6-phosphate. Later work by Laue and MacDonald (59) revealed that TMG 6-phosphate was the only intracellularly accumulated form of TMG. In addition, the accumulated compound was not subject to counterflow. Laue and MacDonald also found that in cell-free extracts PEP was the phosphoryl donor for forma- tion of TMG 6-phosphate, which was in agreement with the results obtained by Kennedy and Scarborough (54) with ONPG phosphorylation. Therefore, S, aureus accumulates lactose solely as lactose phos- phate via the PEP:lactose PTS, and a phospho-B-galactosidase cleaves lactose phosphate to D-glucose plus D—galactose 6-phosphate. A simple and rapid chemical synthesis of ONPG 6-phosphate was reported by Hengstenberg and Morse (60) in 1969, and the availability of this substrate allowed more study to be done. In 1970, Hengstenberg, Penberthy, and Morse (61) described the purifi- cation and some of the properties of the staphylococcal phospho-B- galactosidase. The enzyme has a molecular weight of 50,000, is 10 composed of a single polypeptide chain, has a pH optimum of 7.0, and has a Km of 3mM for ONPG 6-phosphate. The conclusive demonstration of the existence of the staphylococcal phosphotransferase system for lactose and D-galactose was accomplished by Roseman's group. The system was resolved into four proteins (62). In addition to EI, EII, and HPr [the components as identified in the PEstugar PTS of S, 321; (53)], an additional factor, termed FIII, was identified. Both EII and FIII were induced by D—galactose while EI and HPr were constitutive. In a later report (63), Roseman's group showed that EII and FIII were induced also by lactose. Extracts from D—galactose-induced cells of S. aureus were used as a source to isolate the four components (62): EII was associated with the membrane fraction, and EI, HPr, and FIII were separated from one another by DEAE-cellulose chromatography of the soluble fraction. Using 14C-TMG as sub- strate, formation of 14C-TMG 6-phosphate required PEP, EI, EII, FII, and HPr. In addition, using 32P—PEP (63) they were able to obtain 32P-phospho—FIII in the presence of El and HPr; there was an absolute requirement for El and HPr. The phosphoryl group from 32P-phospho-HPr (isolated from an incubation containing 32P-PEP, EI, and HPr) was transferred to FIII, and no other protein appeared necessary for the transfer. In the presence of EII, the phosphoryl group of 32P—phospho-FIII was transferred to TMG to form 32P—TMZG 6-ph08phate. Since EII and FIII were induced by either lactose or D-galactose, this system was presumed to be involved in the phos- phorylation of both sugars. ll Roseman's group has purified and characterized the four com- ponents of the S, aureus PTS for lactose. HPr was purified to homogeneity; it has a molecular weight of 8600, and is species specific (64), in contrast to S, £21$_and Salmonella Eyphimnrium HPr's which substitute freely for one another in enzymatic assays (65). El was purified to a limited extent, but it eluted from DEAE-cellulose as multiple peaks (64), as does EI from S, EZRElf murium (an unreferenced statement by Roseman; 64). Due to the existence of multiple peaks and instability, extensive charac- terization was not done, but the enzyme has a molecular weight of about 80,000. EII was partially purified from the membrane frac- tion, but it was difficult to purify, was somewhat unstable, and could not be solubilized under a multitude of conditions (64). Hengstenberg (66) and Korte and Hengstenberg (67) reported that they could solubilize EII with Triton X—100, and after purification, they obtained a molecular weight of 36,000. Even though Roseman's results (64) argued against those of Korte and Hengstenberg, no mention was made of the discrepancy. FIII was purified to homo- geneity (68) and was found to be a trimer of identical subunits (subunit molecular weight of 12,000). FIII accepted and transferred three phosphoryl groups. The molecular weight of 36,000 agreed with the value of 33,000 determined by Schrecker and Hengstenberg (69), but they obtained a subunit molecular weight of 8000, indi— cating thatFIIIwas a tetramer; however, only partially purified protein was used in that study. 12 The complete system (EI, EII, FIII, and HPr) transferred the phosphoryl group of PEP to TMG, IPTG, ONPG, lactose, and D-galactose to form the corresponding phosphate ester (in which the 6 position of the D-galactosyl moiety was substituted) (64). All four proteins were necessary for phosphorylation, an observation that had been made earlier by Roseman's group (62) and by Hengstenberg et a1. (70) using crude preparations of the four proteins. Therefore, FIII and EII are involved in the phosphorylation of both lactose and D—galactose. The facts that lactose and D-galactose induce and are phos- phorylated by the same system might have been predicted from previous induction data. Creaser (45) measured lactose metabolism by determining ONPG hydrolysis by whole cells, and he noted that lactose metabolism was inducible in S, aureus, and that D—galactose was a more efficient inducer than lactose. McClatchy and Rosenblum (47) verified Creaser's results and further found that IPTG, TMG, and ONPG did not induce but did competitively inhibit induction by D—galactose. Morse et a1. (71) found that D-galactose 6-phosphate induced lactose metabolism to eight-fold higher levels than did D-galactose. The Egg: mutant [determined to lack EI (62)], which could not concentrate or phosphorylate lactose or D—galactose, was not induced by D-galactose; however, D—galactose 6-phosphate .induced as well as D-galactose did in the wild-type organism. These results suggested that D-galactose and lactose are trans- ported by the same system and indicated that D—galactose 6-phos- phate, not D—galactose or lactose, is the inducer of the lactose system. 13 Roseman's group (72) demonstrated the phosphorylation of HPr with 32P-PEP by El and presented data to suggest the existence of a phospho-EI intermediate. Using 32P-PEP, Hengstenberg's group (73) demonstrated the transfer of the phosphoryl group of PEP to E1, and isolated the 32P-phospho-EI intermediate. They were then able to use 32P-phospho—EI as a phosphoryl donor to quantitatively convert HPr to 32P—phospho-HPr, which served as a phosphoryl donor to convert IPTG to 32P-IPTG 6—phosphate in the presence of E11 and FIII. In similar experiments, Roseman's group (72) obtained quantitative phosphorylation of HPr with 32P-PEP in the presence of El; HPr accepted one phosphoryl group per protein molecule. The phosphoryl group in phospho-HPr was found (62) to have stability similar to that in phospho-HPr from S, £211, in which the phosphoryl group is attached to position N—l in the imidazole ring of a histi- dine residue (65); Hengstenberg's group (74) found that the phosphoryl group in the S. aureus phospho-HPr is attached to the N-l position. 32P-Phospho-HPr was shown to transfer reversibly its 32P-phosphoryl group to FIII to form 32P3-phospho-FIII (72); the phosphoryl groups in P3—phospho—FIII were found to be attached to the N-3 in the imidazole ring of the histidine residues (68). In the presence of EII, 32P3-phospho-FIII quantitatively donated its phosphoryl groups to TMG to form 32P-TMG 6-phosphate (72). This last result indicated that all three phosphoryl groups in phospho- FIII are enzymatically active. No phospho—EII intermediate was detected. Also, EII bound 1[’C-lactose with 8 RD of 0.25 pH, and l4 binding did not result in any alteration in the lactose. There— fore, the phosphorylation of lactose and D—galactose follows the scheme: 3 PEP 3 El 3 P—HPr FIII 3 sugar phosphate E11 3 pyruvate 3 P-Ei 3 HPr p3-FIII 3 sugar Hengstenberg et a1. (70) performed studies to determine the physiological significance of the PEP:1actose PTS in S. aureus. Using partially purified preparations of El, EII, FIII, and HPr, they noted that all four components were necessary for ONPG hydrolysis in the presence of phospho-B-galactosidase and PEP. Using whole cells of mutants missing either EI, EII, FIII, or HPr, they found that significant 14C-IPTG accumulation occurred only in those strains with all four components intact. Simoni and Roseman (75) also isolated several mutants to demonstrate the physiological significance of the PEP:1actose PTS in S, aureus. A strain con- stitutive for lactose metabolism was isolated, and it, like the wild-type strain, accumulated 14C-TMG as 14C-TMG 6-phosphate. Mutants lacking EI, EII, or FIII were isolated from the consti- tutive strain and were found to display negligible initial rates of uptake of 14C-TMG, and the internal concentration did not even equilibrate with the external concentration. These results indi- cated that a significant rate of uptake occurred only if there was an intact PTS. If a membrane carrier were responsible for trans- port prior to phosphorylation, the initial rates of uptake and 15 equilibration would not be effected by mutations in the PTS. Therefore, the PEP:1actose PTS is the permease system and not a "t ap" which phosphorylates lactose subsequent to transport. Some genetic analysis of the lactose operon of S, aureus was performed (71, 76). By phage transduction, it was determined that at least four genes are involved: a gene coding for the synthesis of phospho-B-galactosidase, a gene coding for EII, a gene coding for FIII, and a gene regulating the first three struc- tural genes. All four genes are closely linked; since constitutive point mutants had been isolated (75), the genes most likely comprise an operon. The metabolism of lactose and D—galactose by a number of other organisms has been found to be similar to that just described for S, aureus. The B-galactosidases of all but one strain (7962) of Streptococcus lactis were reported to be unstable upon disrup- tion of whole cells (77). Lactose utilization was investigated more intensively in two strains, 7962 and CZF’ of S, lactis in a later publication (78). Lactose uptake by whole cells of S. lactis CZF was inhibited by NaF whereas uptake by strain 7962 was not. ONPG hydrolysis by whole cells was similarly affected by NaF. Cell—free extracts of CZF could hydrolyze ONPG or lactose only if PEP, but not ATP, was present, whereas PEP was not required for hydrolysis by extracts of strain 7962. When whole cells of CZF were incubated with 14C-TMG, a l('C-TMG derivative was isolated from the cells. The derivative behaved as a negatively-charged species upon electrophoresis, and it was destroyed and converted to 14C-TMG 16 by treatment with phOSphatase. The system in C2F converting TMG to TMG phosphate was found to consist of EI, EII, FIII, and HPr; EII and FIII were induced by both lactose and D—galactose, which was a better inducer than lactose. Further study (79) revealed that the apparent lability of B—galactosidase was due to the absence of this enzyme and the presence of phospho-B—galactosidase activity. The levels of both hydrolyses were reported for several strains of S, lactis, Streptococcus cremoris, and Streptococcus diacetilactis (79, 80). All strains had both activities, but phospho-B- galactosidase activity predominated in all but S, lactis 7962. It thus appeared that these lactic streptococci have the enzymatic capability to utilize lactose via two pathways: initial cleavage to D—glucose plus D-galactose, which is probably utilized via the Leloir pathway; and initial phosphorylation with PEP, followed by cleavage to D-glucose plus D—galactose 6-phosphate. Preliminary data was presented by Elliker's group (81) to suggest the operation of a PEcharbohydrate PTS for lactose, but not for D—galactose, utilization in one strain of S, lactis. Using KF to inhibit enolase, they found that ATP stimulated the uptake of D-galactose by toluene-treated whole cells to a much greater extent than did PEP, whereas the opposite was true for lactose uptake. Analysis of extracts of cells incubated with D-galactose indicated that they accumulated D—galactose l-phosphate, D—glucose l-phosphate, and D-glucose 6-phosphate (intermediates of the Leloir pathway), but did not accumulate D-galactose 6-phosphate. In addition, UDP-galactose 4-epimerase activity was detected in 3’“ 17 extracts of these cells. These data were interpreted to indicate that the Leloir pathway is the only route for D-galactose metabo- lism in S, lactis, whereas lactose is primarily utilized via D-galactose 6-phosphate and to a minor extent via the Leloir pathway. However, the data are not sufficient to allow this determination of the relative participation of D—galactose 1-phosphate (an intermediate of the Leloir pathway) and D-galactose 6-phosphate in the metabolism of lactose and D-galactose. The phospho-B—galactosidase from one strain of S, cremoris was purified and characterized (82). The enzyme has a molecular weight of 68,000, has a broad pH optimum (5.5 to 7.0), has a Km for ONPG 6-phosphate of 0.6 mM, and is competitively inhibited by D—galactose 6—phosphate (K1 = 7.5 mM). The inhibition by D-galactose 6-phosphate may play a role in regulation of lactose metabolism. A survey of 13 species of the lactobacilli revealed that several strains possess phospho—B-galactosidase activity (83). Three categories were observed: lactose-nonfermenting strains had neither B-galactosidase nor phospho—B-galactosidase activity, most strains had both activities, and some strains had only the latter activity. Apparently, most strains can utilize lactose via two pathways, as can the streptococci, and some strains utilize lactose solely via the PEP:1actose PTS, as does S. aureus. The partially purified phospho-B-galactosidase from L, Eggs; has a molecular weight of 130,000, has a pH optimum at 5.0, and has a Km of 1.6 mM for ONPG 6—phosphate. .— 18 Recently, D-galactose has been reported to be utilized via the PEP:sugar PTS in Vibrio cholerae (84-86), Vibrio parahaemolyticus (87), and Streptococcus mutans (88). Presumably, these strains accumulate D—galactose as D-galactose 6-phosphate. Phospho-B— galactosidase activity has been observed in extracts of several strains of Propionibacterium shermanii (89); B—galactosidase activity was also detected in these extracts. Thus, these strains appear to be similar to the streptococci and lactobacilli in their lactose catabolic pathways. The metabolism of D—galactose 6-phosphate in S, aureus or in anyotherorganism has not been investigated to any significant extent. Galloway and Krauss (90) reported the occurrence of D-galactose 6-phosphate in extracts of the algae Chlorella vulgaris and Scenedesmus obliguus. Buck and Obaidah (91) identified D-galactose 6-phosphate as a hydrolytic product from the cell wall of the fungus Fusicoccum amygdali. D-Galactose 6—phosphate also has been found to occur in galactosemic erythrocytes (92); it was probably formed from D-galactose by hexokinase-catalyzed phosphoryla- tion with ATP since D-galactose, at high concentrations, serves as a substrate (93). Musick and Wells (94) observed that D—galactose 6-phosphate accumulated in mammalian tissues that were incubated with high concentrations of D-galactose, which mimicked the galacto- semic state. The nonspecific utilization of D-galactose 6-phosphate by glucose 6-phosphate dehydrogenase (95-98), phosphoglucoisomerase (99), phosphoglucomutase (100, 101), NADP L-hexonate dehydrogenase f———————' 19 (102), and UDP-glucose:a—l,4-a-4-glucosy1transferase (103) has been reported; the maximal velocities were low and the Km's were high. Beutler (36, 104, 105) detected a liver hexose 6-phosphate dehy—H drogenase which used either NAD+ or NADP+ as cofactor to oxidize D—galactose 6-phosphate and D—glucose 6-phosphate to 6-phospho—D— galactonate and 6-phospho-D—gluconate, respectively. A specific galactose 6-phosphate dehydrogenase also has been reported to occur in liver (106). The enzyme was specific for D-galactose 6-phosphate and NAD+, and the product was preliminarily stated as having an intact aldehydic group, indicating the formation of a keto group at one of the four secondary alcohol groups. However, since D-galactose 6-phosphate does not normally arise in liver and since the products of these two dehydrogenases are not further metabolized, the reactions must not be physiologically significant. These reports on the metabolism of D-galactose 6-phosphate involve apparent "dead-end" reactions and probably are not important. Simoni and Roseman (75) presented evidence to indicate that the first step in the metabolism of D—galactose 6—phosphate in S, aureus is the oxidation to 6-phospho-D-galactonate. Either NAD+ or NADP+ served as cofactor for the dehydrogenation reaction in a cell- free system. The reaction product chromatographed as a more highly negatively-charged material than D-galactose 6-phosphate, but fur- ther characterization of the unknown compound was not done. The authors also proposed that D—galactose 6-phosphate may be mutated to D-galactose l-phosphate, but did not report any investigation of this reaction. The evidence presented in this thesis will indicate fi— that neither the dehydrogenase nor the mutase reaction occurs in the metabolism of D-galactose 6-phosphate by S, aureus. ’ As indicated by this literature review, the metabolism of D-galactose or lactose by S, aureus involves formation of D—galactose 6-phosphate intracellularly. The further metabolism A of D-galactoSe 6-phosphate had not been determined in this or in any other organism, and the elucidation of this catabolic pathway is the subject of my Ph.D. thesis research. SECTION 1 ELUCIDATION OF THE PATHWAY 0F D-GALACTOSE 6-PHOSPHATE DEGRADATION ‘21 fifiwfi ._7' V——— INTRODUCTION As indicated in the Literature Review section, the pathway for the catabolism of D-galactose 6-phosphate in Staphylococcus aureus or in any other organism was not known. DeGalactose 6—phosphate was known to be an intermediate in the metabolism of lactose and D—galactose in S, aureus and was presumed to arise during the metabolism of lactose and/or D-galactose by numerous strains of Streptococcus, Lactobacillus, Vibrio, and Propionibac- terium. The preliminary experimental results which are presented in this section indicate the rationale used in proposing a pathway for D—galactose 6-phosphate degradation, termed the D—tagatose 6-phosphate pathway, in S, aureus: D-galactose 6-phosphate-—-* D-tagatose 6-phosphate ———+ D-tagatose l,6—diphosphate-———-* dihydroxyacetone phosphate + D-glyceraldehyde 3—phosphate. MATERIALS AND METHODS Bacterial Strain, Cell Growth and Storage, and Preparation of Cell Extracts Bacterial Strain The organism used was Staphylococcus aureus NCTC 8511. Eadie. Routine liquid cultivation was done in media containing 2% (w/v) peptone, 0.2% (w/v) KZHPOQ, and 0.1% (w/v) yeast extract. When S, aureus was grown on carbohydrates, the peptone concentration was lowered to 1%, and 1% (w/v) carbohydrate was added (47). For short-term storage (3 to 6 months), cells were maintained on an agar slant, which contained 1.5% (w/v) agar in addition to the com- ponents of the 2% peptone medium. Sterilization Technique All media were sterilized for 20 minutes at 121°C in 3 Castle autoclave, model Thermomatic 60; carbohydrates were auto- claved separately from the other components of the medium. Growth Conditions Cells were grown aerobically by agitation on a New Brunswick Scientific Co. gyrotory shaker, model G10, at 37°C in the dark. Cells were grown either in culture tubes (18 X 150 mm) containing 7 m1 of medium or in Fernbach flasks containing 0.3 to 1.0 l of 23 24 medium, in which case the inoculum was 7 ml of an overnight culture in the 2% peptone medium. Centrifpgation Unless stated otherwise, all centrifugation was done in a Sorvall refrigerated centrifuge, model RC-ZB, for 10 minutes at 0 to 4°C. The speed of centrifugation and the rotor used will be indi- cated in each instance. Storage of Bacteria For short-term storage (3 to 6 months), cells were main- tained on an agar slant at 5°C in the dark. For long-term storage, cells were maintained as freeze-dried cultures and as glycerol suspensions. To prepare freeze—dried cultures, cells were grown overnight in 500 ml of 2% peptone medium. One-half of the culture was poured into a sterile centrifuge bottle, and the cells were collected by centrifugation (14,000 x‘g in the GSA rotor). The cells were suspended in 2 ml of sterile non-fat milk, and 0.2 ml portions were placed in sterile capped vials. The slurries were frozen in a dry ice-acetone bath and freeze-dried on a VirTis freeze-mobile, model 10-147CAS, in vacuum bottles. The dried cultures were then capped and stored at —20°C. To prepare glycerol suspensions, the second half of the previously mentioned culture was collected as described, and the cells were suspended in 2 ml of mineral medium (107) containing 10% (v/v) glycerol. The cells were dispensed in 0.5 ml portions into sterile capped vials and stored at '20°C 0 -— _——, “mr,e~v_ _.—~— 25 Harvesting of Cells Incubation of cells was ceased when the pH of the medium fell below 6, which occurred after about 8 hours. Cells were har- vested by centrifugation (14,000 X g_in the GSA rotor). The cells were suspended in 0.85% (w/v) NaCl and centrifuged at 12,000 X‘g in the 88-34 rotor. These cells were eitherusedimmediately or stored at -20°C. Preparation of Buffer Solutions All buffers were prepared at room temperature; room tempera- ture varied slightly from day to day but was approximately 21°C. The pH was measured with a Beckman pH meter, model 9600 Zeromatic, using a Sargent-Welch combination electrode, model S-30070-10. If a buffer was to contain BME, this reagent was added subsequent to adjusting the pH; erroneous pH readings are obtained with many electrodes when the solution contains BME (108). Preparation of Glass Beads ‘ _ Commercially obtained glass beads (88 to 125 um diameter) were washed with 3 N HCl by stirring with a Teflon stir bar. Small quantities of iron filings were present in the beads, and they adhered to the stir bar and were thus removed. The beads were then washed free of acid with distilled water and dried prior to use. Prior to reuse, the beads were washed free of whole cells and cellular debris with water, and then washed as before. 26 Preparation of Cell Extracts Cell extracts were prepared by exposing buffered [20 mM K HPO4 (pH 7.5) containing 0.2% (v/v) BME] cell suspensions to 2 sonic vibration (10,000 Hertz) in a Raytheon sonic oscillator, model DFlOl, for 20 minutes at 0°C in the presence of five times the packed-cell volume of glass beads; glass beads have been used in the past (56, 109, 110) to disrupt S, aureus. The broken-cell suspension was centrifuged at 40,000 X g_in the 88-34 rotor, and the resulting supernatant solution was used as the cell extract. Colorimetric Determinations Those procedures for colorimetric determinations involving heating were done in a Precision Scientific Co. sero-utility bath, model lO-S-l. The optical density of the solutions was measured with a Gilford micro-sample spectrophotometer, model 300-N. Protein Determination Protein was determined by the method of Lowry et al. (111) or by the measurement of the absorbance at 210 nm or 220 nm (112) using bovine serum albumin as standard. Phosphate Determination Inorganic phosphate was determined by a modification of the Fiske-SubbaRow procedure (113) as described by Clark (114). Total phosphate was determined by the method described by Umbreit, Burris, and Stauffer (115). NaZHPO4 was used as standard. 27 Ketohexose Determination Ketohexose was determined by the method of Roe (116) as described by Ashwell (117). In some cases, the carbazole method (118) was also used. D—Fructose, D-fructose 6-phosphate, and D-fructose 1,6-diphosphate were used as standards. Aldohexose Determination Aldohexose was determined by a method employing 2-aminobiphenyl (119). D-Galactose and D-galactose 6-phosphate were used as standards Preparation of Substrates Preparation of D-Galactose Commercial D-galactose was recrystallized (120). Removal of Barium From Spgar Phosphates Barium salts of sugar phosphates were converted to their corresponding sodium salts by treatment with a slurry of Dowex 50W-X8 (H+ form) and titration of the supernatant solution to pH 7.0 with NaOH. The solution was then diluted to the desired volume. Preparation of D-Galactose 6-Phosphate Preliminary studies using the Roe test for ketohexose (117) revealed that there was ketohexose (D-tagatose 6-phosphate) present in the commercial product. This contamination was removed by treatment of 0.6 mmole of the commercial D-galactose 6-phosphate 28 with 0.6 unit of rabbit muscle fructose 6-phosphate kinase [which phosphorylates D—tagatose 6-phosphate (121)] in the presence of 0.06 mmole of MgZATP at pH 7.0 at 25°C in a total volume of 3 ml. The pH of the reaction mixture was adjusted to pH 8.5 with KHC03, and the solution was applied to a Dowex 1-X8 column (1.2 X 10 cm) (H003- form) (122), and eluted.with 150 ml of water and of 0.15 M KHC03. Fractions containing aldohexose, which eluted with 0.15 M KHCO3, were combined and treated with Dowex 50W-X8 (H+ form) until the pH was 2.0. The solution was freeze—dried and dissolved in 10 ml of water, and the pH of the solution was adjusted to 7.0 with NaOH. The resulting sugar phosphate was free of ketohexose. Preparation of D-ngatose 6-Phosphate Since the procedure involves the combination of three pub- lished syntheses (123-125) and since these procedures are somewhat vague, a detailed description will be presented. All steps for the removal of solvents were done at the minimum practical temperature; unless stated otherwise, solvents were removed using a Buchi rotary evaporator, model KRv 65/45. The synthesis is outlined in Figure 1. Ten grams of D-galacturonic acid (I) were dissolved in 2500 ml of cold water. CaO (2.9 g) was added with stirring until all solids had dissolved; the pH was slightly basic. The solution, which turned yellow, was allowed to stand at room temperature for four days, during which time crystals of the calcium salt of D—tagaturonic acid (11) precipitated. The crystals were collected coon oo'CaH' 0" 010 I acetone "I H D-Galacturonic D-Tagaturonic 1,2;5,h-di-0-iso- acid (I) seid (II) propylidene-DQ (calcium salt) tagaturonic acid (calcium salt) (III) 0" CH 0" 0 CMfla III 2 "' cnzuz "‘ mm, 1,233,4-d1-o-iso- 1,2;5,4-di-0-iso- 1,2;3,4-di-O- prepylidene-D- propylidene-D- isopropylidene- tagaturonic acid (IV) tagaturonic acid D-tagatose (VI) (methyl ester) (V) i 01%“ u m m m diphonyl- > "z i chloro’hosmomto Ptoz 1,2;3,4-di-0-iso- l,2;3,4-di-O- propylidene-D- _ isoprOpylidene-D- tagatose 6- tagatose 6-phosphate (VIII) diphenylphosphate (VII) A fifl—ga 0 0H 0 BI(OH)2 . fl hut “1°" D-Tagatose 6-phosphate (Ix) D-Tagatose 6-phosphate (barium salt) (X) Figure 1. Scheme for the chemical synthesis of D-tagatose 6- phosphate. Symbols: m, methyl; p, phenyl. 30 by suction filtration, washed with cold water, and dried over CaSO4. Yield: 10.2 3. For each gram of II, 19.5 ml of dry acetone (dried with N32804) and 0.8 ml of H2804 were mixed and stirred with II for four hours at room temperature. The solution was rapidly neutralized with anexcess of CaO suspended in water. Since the solution became slightly basic, the pH was adjusted to 7.0 with H2804. After suction filtration, which removed acetone by evaporation, the solution was freeze-dried to a.white solid, the calcium salt of 1,2;3,4-di-O-isopropylidene- D—tagaturonic acid (III). Yield: 9.2 g. The free acid of III was obtained by addition of III to a solution of 100 ml of cold water, 100 ml of ethyl ether, and 32.8 ml of 10% (w/v) H2804 with stirring. The ether layer was washed four times with 100 ml portions of cold water, dried with NaZSOA, and evaporated to dryness. Yield: 5.9 g of 1,2;3,4-di-O-isopropylidene- D-tagaturonic acid (IV). Ethereal diazomethane, for use in the next step in the synthesis, was prepared from.Diazald (N-methyl-N—nitrosofipf toluenesulfonamide) as described on the reagent bottle obtained from Aldrich Chemical Co. I will quote the procedure, in which only smooth, polished glassware should be used. All work with diazomethane shouldlnacarried out behind safety shields in efficient hoods. Ethanol [25 ml of 95% (v/v)] is added to a solution of potassium hydroxide (5 g) in water (8 ml) in a 100 ml distilling flask fitted with drOpping funnel and an efficient condenser set downward for distillation. The condenser is connected to two receiving flasks in series, the second of which contains 20-30 ml ether. The inlet tube of the second receiver dips below the surface of the ether and both receivers are cooled to 0°C. The flask containing the alkali solution is heated in a water bath to 65°C, and a. solution of 21.5 g (0.1 mole) of Diazald in about 200 ml of 31 ether is added through the dropping funnel in about two hours. The rate of distillation should about equal the rate of addi- tion. When the dropping funnel is empty, another 40 m1 of ether is added slowly and the distillation is continued until the distilling ether is colorless. The combined ethereal distillate contains about 3 g of diazomethane. The diazomethane solution is not stable to storage and should be used the same day. IV was taken up ina small volume of ethyl ether, esterified with anexcess of ethereal diazomethane (the end point was detected when the solution turned yellow), and allowed to stand for 30 minutes. The esterified solution was concentrated to a syrup of the methyl ester of 1,2;3,A-di-O-isopropylidene-D—tagaturonic acid (V). The syrup was taken up in 150 ml of ether, stirred during the gradual addition (to prevent vigorous bubbling) of 3.7 g of LiAlH4, and stirred for five hours. Excess LiAlH4 was destroyed by the gradual addition of ethyl acetate; the end point was detected as the cessation of bub- bling. Water (150 ml) was added, and the organic solvents were removed. The solution was neutralized with 10% (v/v) acetic acid, filtered, re-neutralized, and extracted with an equal volume of C8013. The CHCl3 layer was washed with an equal volume of water, dried with Na2804, filtered, and evaporated to a syrup. Two recrystallizations at 490 from petroleum ether gave white needles of 1,2;3,4-di-0- isopropylidene-D-tagatose (VI). Yield: 2.8 3. VI was dissolved in 36.4 ml of cold, dry pyridine (dried with KOH). With constant stirring at 0°C, 2.3 ml of cold diphenylchloro- phosphonate were added dropwise; a precipitate formed, indicating excess reagent had been added. After 30 minutes at 0°C, the solution was stored at 4°C for 24 hours. The mixture was poured onto 300 m1 of 32 finely crushed, distilled-water ice with stirring. The slurry was stirred for 30 minutes and then extracted with 200 m1 of CHC13. The CHCl3 layer was washed with 40 ml of 10% (w/v) HCl and then washed four times with 30 ml of water. The CHCl3 layer was dried with Nazsot‘, filtered, and concentrated to a syrup. The syrup was taken up in 200 ml of ethanol, and water (about an equal volume) was slowly added until the solution became turbid. The mixture was stored at 4°C for 48 hours, and the precipitate was filtered from the solution and dried over CaSOa. Yield: 3.4 g of 1,2;3,4-di-0-isopropylidene-D-tagatose 6-dipheny1phosphate (VII). The phosphate (VII) was dissolved in 65 ml of absolute ethanol, and 0.31 gof PtO2 was added. The mixture was shaken under hydrogen gas (20 pounds per square inch) in aParr pressure reaction apparatus, model 1518, for five hours, at which time no more hydrogen was being consumed. The catalyst (Pt02) was removed by centrifugation (12,000X gin the» SS-34 rotor). The solution was concentrated to asyrup, and 100 ml of pentane and afew drops of water were added. Crystals were obtained after storage for 24 hours at 4°C. The product (VIII; 1,2;3,4- di—O-isopropylidene-D-tagatose 6-phosphate) was heated in 15 m1 of water for 3minutes onasteam bath to give asolution of IX (D-tagatose 6-phosphate) . Barium hydroxide solution (CO2 free) was added untilthe solution had a pH of 10.2. After filtering, the solution was poured into 320 mlof 95% (v/v) ethanol and stored at 4°C for 24 hours. The solution was decanted and centrifuged (14,000 X g in the GSA rotor). The product was washed with absolute ethanol, absolute ethanol:ethy1 ether (4:1, 1:1, 1:4), and ethyl ether (twice). Yield: 1.5 g of the barium salt of D-tagatose 6-phosphate (X). 33 Gas-Liquid Chromatography Trimethylsilyl derivatives of sugar phosphates were prepared as described by Musick and wells (94). Gas-liquid chromatography was performed on a Hewlett and Packard gas chromatograph, model 402, employing a 6-foot column of 3% SE-30 at 190°C. Acid Hydrolysis Sugar phosphates were treated with 1 N HCl in a boiling water bath. Samples were withdrawn at timed intervals for determination of inorganic phosphate (114). Enzymatic Assays One unit of enzymatic activity is defined as the amount of enzyme that catalyzes the formation of l umole of product per minute. Activity is expressed in units per ml, and specific activity is expressed in units per mg protein. Assays that involved the oxidation or reduction of pyridine nucleotides were done in microcuvettes with a 1.0 cm light path at 340 nm.using¢ithermostated (30°C) Gilford absorbance-recording instrument, model 220. Those assays involving the dye DIP were done at 600 nm with the same instrument. The incu- bation portions of assays not involving pyridine nucleotides or DIP were done at 30°C in a Precision Scientific Co. sero-utility bath, model 10-R-3. The concentration of substrate added to an assay was determined on a weight basis of the added substrate. Galactose 6-Phosphatase Assays (0.2 ml) contained 10 umoles of glycylglycine buffer (pH 7.5) or 5 pmoles of potassium phosphate buffer (pH 6.0), 1.0 34 uncle of M3012, 2.0 umoles of D-galactose 6-phosphate, and cell extract. After incubation for a timed period, the reaction mix- ture was assayed for inorganic phosphate (114). Galactose 6-Phosphate Dehydrogenase Assays (0.15 ml) contained 10 umoles of glycylglycine buffer (pH 7.5 or 9.0); 1.0 umole of M3012; 0.1 umole of NADP+, 0.05 umole of NAD+, or 0.01 umole of DIP; 1.0 umole of D—galactose 6¥phosphate; and cell extract. In assays containing DIP, controls to correct for DIP reductase activity were minus D-galactose 6-phosphate. Galactose Dehydrogenase Assays were identical to those just described except that they contained 1.0 umole of D-galactose instead of D-galactose 6-phosphate as substrate. Glucose 6-Phosphate Dehydrogenase Assays (0.15 ml) contained 10 umoles of glycylglycine buffer (pH 7.5), 1.0 umole of MgClz, 0.1 umole of NADP+, 0.25 pmole of D-glucose 6-phosphate, and cell extract. Galactose 6—Phosphate Reductaser Assays (0.15 ml) contained 10 umoles of glycylglycine buffer (pH 7.5) or 5 umoles of potassium phosphate buffer (pH 6.0), 1.0 umole of M3012, 0.05 umole of NADH or NADPH, 1.0 umole of D—galactose 6-phosphate, and cell extract. Controls to correct for NADH oxidase or NADPH oxidase were minus D-galactose 6-phosphate. 35 Galactose Reductase Assays were identical to those just described except that they contained 1.0 umole of D—galactose instead of D-galactose 6-phosphate as substrate. Galactose 6-Phosphate 4—Epimerase Assays (0.15 ml) contained 10 umoles of glycylglycine buf- fer (pH 7.5), 1.0 umole of M3012, 0.1 umole of NADP+, 1.0 umole of D-galactose 6-phosphate, nonlimiting amounts of glucose 6-phosphate dehydrogenase, and cell extract. Controls to correct for the low level of glucose 6-phosphate dehydrogenase-catalyzed oxidation of D-galactose 6-phosphate were minus cell extract. Galactose 4-Epimerase Assays were identical to those just described except that they contained 1.0 umole of D-galactose instead of D—galactose 6-phosphate as substrate and that they additionally contained 0.5 mmole of ATP and nonlimiting anounts of hexokinase. Galactose l-Phosphate 4-Epimerase Assays were identical to those for galactose 6-phosphate 4-epimerase except that they contained 1.0 umole of D-galactose 1-phosphate instead of D-galactose 6-ph08phate as substrate and that they additionally contained nonlimiting amounts of phospho- glucomutase. 36 Galactose 6-Phosphate Kinase Assays (0.15 ml) contained 10 umoles of glycylglycine buffer (pH 7.5), 1.0 umole of MgClz, 0.5 mmole of ATP, 0.5 uncle of PEP, 0.05 umole of NADH, 1.0 mmole of D—galactose 6-phosphate, nonlimiting amounts of pyruvate kinase and lactate dehydrogenase, and cell extract. Controls to correct for NADH oxidase and ATPase were minus D-galactose 6-phosphate. Galactose l-Phosphate Kinase Assays were identical to those just described except that they contained 0.5 umole of D-galactose l-phosphate instead of D-galactose 6-phosphate as substrate. Galactokinase Assays were identical to those just described except that they contained 0.5 umole of D-galactose instead of D—galactose 1-phosphate as substrate. Glucokinase Assays were identical to those just described except that they contained 0.5 umole of D-glucose instead of D-galactose as substrate. Galactose l-Phosphate Uridylyltransferase Assays (0.15 ml) contained 10 umoles of glycylglycine buf- fer (pH 7.5), 1.0 umole of MgClz, 0.1 mmole of NADP+, 0.1 umole of UDP-glucose, 0.1 umole of D-galactose l-phosphate, nonlimiting 37 amounts of phosphoglucomutase and glucose 6-phosphate dehydrogenase, and cell extract. UDP-Galactose 4-Epimerase Assays were done by the two-step procedure of Maxwell et al. (126). Galactose Isomerase Assays (0.2 m1) contained 10 umoles of glycylglycine buffer (pH 7.5), 1.0 mmole of MgClz, 0.5 umole of D-galactose, and cell extract. After incubation for a timed period, the reaction mixtures were assayed for ketohexose by either of two methods (117, 118). Galactose 6-Phosphate Isomerase Assays were identical to those just described except that they contained 1.0 umole of D-galactose 6-phosphate instead of D-galctose as substrate. Glucose 6-Phosphate Isomerase Two assays were used. The first (0.15 ml) contained 10 umoles of glycylglycine buffer (pH 7.5), 1.0 umole of M3012, 0.1 umole of NADP+, 0.45 umole of D-fructose 6-phosphate, non- limiting amounts of glucose 6-phosphate dehydrogenase, and cell extract. The second (0.15 ml) contained 10 umoles of glycyl- glycine buffer (pH 7.5), 1.0 umole of MgCl 0.5 umole of ATP, 2’ 0.5 umole of PEP, 0.05 umole of NADH, 0.15 umole of D—glucose 6-phosphate, nonlimiting amounts of fructose 6-phosphate kinase, pyruvate kinase, and lactate dehydrogenase, and cell extract. 38 Controls to correct for NADH oxidase and ATPase were minus D-glucose 6-phosphate. Fructose 6-Phosphate Kinase Assays (0.15 ml) contained 10 umoles of glycylglycine buf- fer (pH. 7.5), 1.0 mmole of MgClz, 0.5 umole of ATP, 0.5 umole of PEP, 0.05 pmole of NADH, 0.15 umole of D-fructose 6-phosphate, nonlimiting amounts of pyruvate kinase and lactate dehydrogenase, and cell extract; this enzyme was found to be inhibited by high D—fructose 6-phosphate concentrations (greater than 1 mM), so the substrate concentration was fixed at 1 mM to give maximal activity. Controls to correct for NADH oxidase and ATPase were minus D-fructose 6-phosphate. Controls to correct for possible fructose 6-phosphate reductase were minus ATP; no such reductase was observed. Fructose 1,6-Diphosphate Aldolase Assays (0.15 ml) contained 10 umoles of glycylglycine buffer (pH 7.5), 1.0 umole of MgClz, 0.05 umole Of NADH, 0.03 umole of D-fructose 1,6-diphosphate, nonlimiting amounts of triose phosphate isomerase and arglycerol phosphate dehydrogenase, and cell extract. Controls to correct for NADH oxidase were minus D-fructose 1,6-diphosphate. Preparation of Ion Exchange Resins Commercial resins were stirred overnight in 2 N NaOH, washed free of base with water, washed with 4 N HCl, and washed 39 free of acid with water. The desired ionic forms of the resins were obtained by washing with the appropriate acid or base. The resins were stored in the dry state. This is a modification of a published procedure (127). Enzyme Purification Procedures Bentonite Treatment The enzyme solution (5 mg protein per ml) was stirred at 4°C, and bentonite (50 mg per ml) was added. After 10 minutes, the suspension was centrifuged at 12,000 X g_in the 88-34 rotor, and the precipitate was discarded. Protamine Sulfate Treatment The enzyme solution (10 mg protein per ml) was stirred at 4°C, and 2.6% (w/v) ammonium sulfate was added. To this solution was added 20% (v/v) of 2% (w/v) protamine sulfate (pH 7.5). After 10 minutes, the suspension was centrifuged at 12,000 X.g in the 88-34 rotor, and the precipitate was discarded. Ammonium Sulfate Precipitation The enzyme solution (10 mg protein per ml) was stirred at 0°C, and solid, ground ammonium.su1fate was added to the desired percent of saturation (128). After all the ammonium sulfate had dissolved, the suspension was centrifuged at 12,000 X.g in the SS-34 rotor. The supernatant solution was treated as above to the next desired percent of saturation, and the precipitate was dis- solved in buffer. 40 Protein Chromatography A11 chromatography for the separation of proteins was done at 4°C. Fractions were collected with a Gilson linear frac- tionator, model VL, using a Gilson dropcounter, model DCT. DEAE-Cellulose Chromatography. The DEAE-cellulose was washed with NaOH, HCl, and NaOH as reported by Peterson and Sober (129) prior to use. When not in use, the cellulose was stored in 1.M NaCl at 4°C. After a column had been poured, it was washed with the desired buffer until the effluent had the same pH as the buffer. The sample was then loaded, and the column was washed with buffer until no more pro- tein washed off the column. The salt gradient (10 times the column volume) was then run. Prior to reuse, the column was washed with one volume of 2 M NaCl to remove tightly bound material. Sephadex G-100 Chromatography The gel was swollen by heating in water on a steam.bath for 12 hours. When not in use, the gel was stored in 0.02% (w/v) sodium azide at 4°C. After the column had been poured, it was washed with five column volumes of the desired buffer before application of the sample. After use, the column was washed free of buffer by elution with 0.02% (w/v) sodium azide. Sources of Materials §, aureus NCTC 8511 was from the National Collection of Type Cultures, London, England. D-Fructose, D-glucose, D-galactose, 41 D-galacturonic acid, D-glucose 6-phosphate, D-fructose 1,6- diphosphate, D—galactose l-phosphate, D—galactose 6-phosphate, UDP-galactose, UDP-glucose, ATP, solid bovine serum albumin, crystalline (type III) rabbit muscle fructose 6-phosphate kinase (EC 2.7.1.11), crystalline (grade I) rabbit muscle fructose 1,6- diphosphate aldolase (EC 4.1.2.13), crystalline (type C-302) hexokinase (EC 2.7.1.1), crystalline phosphoglucomutase (EC 2.7.5.1), solid (type III) UDP-glucose dehydrogenase (EC 1.1.1.22), crystalline (grade III) phosphoglucoisomerase (EC 5.3.1.9), crystalline (type III) triose phosphate isomerase (EC 5.3.1.1)- a—glycerol phosphate dehydrogenase (EC 1.1.1.8) mixture, and DIP were from Sigma Chemical Co., St. Louis, Mo. D-Fructose 6-phosphate was from Boehringer Mannheim GmbH, Mannheim, W. Germany. NADH, PEP, crystalline (Argrade) glucose 6-phosphate dehydrogenase (EC 1.1.1.49), crystalline (Argrade) pyruvate kinase (EC 2.7.1.40), and crystalline (Argrade) lactate dehydrogenase (EC 1.1.1.27) were from Calbiochem, Los Angeles, Calif. NADP+, NAD+, and NADPH were from.P-L Biochemicals, Inc., Milwaukee, Wis. Diazald was from Aldrich Chemical Co., Inc., Milwaukee, Wis. Glass beads (88 to 125 um diameter) were from LaPine Scientific Co., Chicago, Ill. Bentonite was from Fisher Scientific Co., Fair Lawn, N.J. RESULTS Enzymatic Reactivity of D-Galactose 6-Phosphate The approach used to elucidate the pathway of D-galactose 6-phosphate degradation in S, aureus was to postulate and test several possible modifications that might occur to the sugar phos- phate. These possibilities included the following: epimerization to D-glucose 6-phosphate, reduction to D-galactitol 6-phosphate, oxidation to 6-phospho-D—galactonate, or mutation to D—galactose l-phosphate. If D-galactose were formed by the dephosphorylation of D-galactose 6-phosphate, the following modifications of D-galactose might occur: phosphorylation to D-galactose l-phosphate, epimerization to D-glucose, reduction to galactitol, oxidation to D-galactonate, or isomerization to D—tagatose. If D-galactose 1-phosphate was formed from D-galactose or D-galactose 6-phosphate (by phosphorylation or by mutation, respectively), D-galactose l-phosphate might be modified by epimerization to D-glucose 1-ph08phate, phosphorylation to D-galactose 1,6- diphosphate, or converstion to UDP-galactose, as in the Leloir pathway. For the following studies, extracts were prepared from freshly harvested cells. To ensure that cell disruption had occurred and that active enzymes were present in the cell extracts used to test the 42 43 postulated reactions, glucokinase and glucose 6-phosphate dehy- drogenase specific activities were measured; the average values were 0.09 and 0.03 units per mg protein, respectively. The postu- lated reductase reactions were tested using either NADH or NADPH as cofactor at both acidic and neutral pH values; the dehydrogenase reactions were tested using NAD+, NADP+, or the artificial oxidant DIP as cofactor at both neutral and basic pH values. I was not able to detect (< 0.2 nmole Xminute-l leg protein-1) those redox reactions or any of the other postulated modifications using cell extracts from D-galactose- or lactose-grown g. aureus. These included the Leloir pathway enzymes galactokinase, galactose l-phosphate uridylyltransferase, and UDP-galactose 4-epimerase. Therefore, many possible routes of D-galactose, D-galactose l-phosphate, and D-galactose 6-phosphate metabolism, including the Leloir pathway, could not be demonstrated in cell extracts. However, there were reactions which I could detect. These were dephosphorylation of D—galactose 6-phosphate, isomerization of D-galactose 6-phosphate to ketohexose, and apparent phosphoryla- tion of D-galactose 6-phosphate (D-galactose 6-phosphate-dependent conversion of ATP to ADP). Only a low level (0.010 unit per mg protein) of dephosphorylation of D—galactose 6-phosphate was observed; since no modification of D-galactose was observed, this reaction was most likely due to a nonspecific phosphatase. The isomerization of D-galactose 6—phosphate to ketohexose was also found to be low (0.004 unit per mg protein); both the Roe test (117) and the carbazole test (118) were used to measure ketohexose. 44 Initially, the activity was considered so low as to be insignifi- cant; however, as much as a five-fold increase in activity was obtained in later experiments. The formation of ketohexose remained linear with time for only a short time period, and then the rate declined rapidly to zero; the percent conversion of D—galactose 6-phosphate to ketohexose was about 8%. Adding more enzyme at the start or at the finish of the reaction did not cause an increase in the percent conversion; longer incubation times also did not result in an increase in the formation of ketohexose. The results sug- gested that the enzyme catalyzing the reaction used a minor anomer that was present in the D-galactose 6-phosphate, and that anomerization did not occur under the conditions of the assay. Multiple anomers of D-galactose 6-phosphate were observed when gas- liquid chromatography of the trimethylsilyl derivative of the sugar was done (Figure 2; also, see references 94 and 130). This aspect of the isomerization reaction was not investigated further at the time, but was delayed for later experimentation (see Section 2, Results, D-Galactose 6-Phosphate Isomerase). The additional observation of D-galactose 6-phosphate- dependent conversion of ATP to ADP (apparent galactose 6-phosphate kinase activity; 0.010 unit per mg protein) suggested a possible pathway for metabolism involving isomerizatidn of D-galactose 6-phosphate to ketohexose, followed by phosphorylation of the ketohexose with ATP. This prompted an investigation to determine if triose phosphates were formed from D-galactose 6-phosphate in 45 1 r I l n 53 a 83 :3 as a n E I l I l- O 5 5 20 10 l RETENTION TIME (minutes) Figure 2. Gas-liquid chromatography of the trimethylsilyl derivative of D-galactose 6-phosphate. The areas of the peaks were determined using the equation area ='§ X base X height; the peak areas in terms of percent of the total area for the four peaks were (from left to right) 18, 8, 20, 54. 46 the presence of ATP. The results obtained from such an experiment are shown in Table l. The data suggest the presence of a pathway involving isomerization of D—galactose 6-phosphate to D-tagatose 6-phosphate, which is phosphorylated with ATP to D-tagatose 1,6- diphosphate, which is then cleaved to triose phosphates (Figure 3). Enzymatic Reactivity of D—Tagatose 6-Phosphate D-Tagatose 6-phosphate, an intermediate of the proposed pathway (Figure 3), termed the D-tagatose 6-phosphate pathway, is not commercially available; therefore, it was synthesized chemi- cally. This synthesis was accomplished by a combination of procedures (123—125) in which D—galacturonic acid was converted to the barium salt of D-tagatose 6-phosphate, as shown in Figure 1. The synthesis was effected with an overall yield of 7.4%. The product had a phosphate (114) to ketohexose (117) ratio of 1 and had a half-life in l N HCl at 100°C of 68 minutes, which was identical to that of D—fructose 6-phosphate and which agreed well with the published value of 70 minutes-for D-fructose 6-phosphate (131). The product was phosphorylated with ATP by rabbit muscle fructose 6-phosphate kinase, which has been described by Lardy's group (121). Gas-liquid chromatography of the trimethylsilyl derivative of the product revealed that it consisted of only one component (retention time of 10.3 minutes) which was distinct from that of D-fructose 6-phosphate (retention time of 10.6 minutes). In this manner, the product was verified to be D-tagatose 6—phosphate. 47 TABLE 1. Conversion of D-galactose 6-phosphate to triose phos- phates. Specific activity Reaction mixture (units per mg protein) Completea 0.006b Complete minus ATP 0.000 Complete minus D-galactose 6-phosphate 0.000 Complete minus TPI and a—GPDH 0.000 Complete plus phosphoglucoisomerase 0.006 Complete plus fructose 1,6-diphosphate aldolase 0'006 8The complete reaction mixture (0.15 ml) contained 10 umoles of glycylglycine buffer (pH 7.5), 1.0 umole of MgClz, 0.5 umole of ATP, 0.05 umole of NADH, 1.0 umole of D-galactose 6-phosphate, nonlimiting amounts of triose phosphate isomerase (TPI) and arglycerol phosphate dehydrogenase (a—GPDH), and cell extract from lactose-grown cells. bThe specific activity of NADH oxidase was 0.005, and this value was subtracted from all specific activities reported. 48 .msoum abonmuone am “egos—Em .55: 3:» a.“ voyages—Ho no unsound ougmaonmum ououumuunn one .m 05.qu fiémofiA manganese Eggnog ”538$ 3438?“ N . . n—o U .m a umoaéfid um eucafifiun .w mmoauflawnn aouzo aomzo aouzo .. J . .. ohm... .. .22 .:.< r .. sagas—Ban A _._o~.._o 0H gonzo aouzo :ouzo o.._o 49 The synthesized D—tagatose 6-phosphate was then used to determine if a D—tagatose 6-phosphate kinase was present in a cell extract of lactose-grown §, aureus. The results of this experi- ment are shown in Table 2. The data indicate that there was a D-tagatose 6-phosphate kinase present. To determine its activity, corrections for both NADH oxidase and ATPase activities were neces— sary. The fact that assays lacking ATP displayed no more activity than assays for NADH oxidase indicated the absence of a possible tagatose 6-phosphate reductase. The value for the D-tagatose 6-phosphate kinase activity was 0.017 unit per mg protein. In addition, the data indicated that triose phosphates were being formed from D-tagatose 6-phosphate in the presence of ATP, sug- gesting the presence of a D-tagatose 1,6-diphosphate aldolase. Thus, these data supported the proposed pathway for D-galactose 6-phosphate metabolism (Figure 3). To clearly demonstrate the existence of a D-tagatose 1,6-diphosphate aldolase, the experiment outlined in Table 3 was done. The data indicate that D—tagatose 1,6-diphosphate aldolase activity was present in the cell extract. Thus, all the activities necessary for the D—tagatose 6-phosphate pathway (Figure 3) were present in cell extracts of lactose-grown §, aureus. The presence of these reactions and the absence of the enzymes of the Leloir pathway (galactokinase, galactose l-phosphate uridylyltransferase, and UDP-galactose 4—epimerase) indicated that a previously undescribed pathway for the metabolism of the D-galactosyl moiety of lactose was operating. 50 TABLE 2. Phosphorylation of D-tagatose 6—ph03phate with ATP. Specific activity Reaction mixture (units per mg protein) Completea 0.046b Complete minus ATP 0.000 Complete minus PEP 0.000 Complete minus D—tagatose 6-phosphate 0.029 Complete minus PK and LDH 0.000 Complete minus PEP, PK, and LDH plus TPIc and a-GPDHc 0.009 8The complete reaction mixture (0.15 ml) contained 10 umoles of glycylglycine buffer (pH 7.5), 1.0 pmole of Mgc12, 0.5 umole of ATP, 0.5 mmole of PEP, 0.05 umole of NADH, 0.2 umole of D-tagatose 6-phosphate, nonlimiting amounts of pyruvate kinase (PK) and lactate dehydrogenase (LDH), and cell extract from lactose-grown cells. bThe specific activity of NADH oxidase was 0.013, and this value was subtracted from all specific activities reported. cAbbreviations: see Table l. 51 TABLE 3. Cleavage of D-tagatose 1,6—diphosphate to triose phos— phates. Specific activity Reaction mixture (units per mg protein) Completea 0.026b Complete minus ATP 0.000 Complete minus D-tagatose 6-phosphate 0.000 Complete minus F6PK 0.010 Complete minus TPI and a-GPDH 0.000 8The complete reaction mixture (0.15 ml) contained 10 umoles of glycylglycine buffer (pH 7.5), 1.0 umole of MgClz, 0.5 umole of ATP, 0.05 umole of NADH, 0.2 umole of D-tagatose 6-phosphate, nonlimiting amounts of rabbit muscle fructose 6-phosphate kinase (F6PK), triose phosphate isomerase (TPI), and arglycerol phosphate dehydrogenase (a-GPDH), and cell extract from lactose-grown cells; rabbit muscle fructose 6-phosphate kinase will phosphorylate. D—tagatose 6-phosphate to D-tagatose 1,6-diphosphate (121). bThe specific activity of NADH oxidase was 0.007, and this value was subtracted from all specific activities reported. 52 Assays for the D-Tagatose 6—Phosphate Pathway_Enzymes Quantitative assays for the three enzymatic activities were then developed. The D-galactose 6-phosphate isomerase reaction (0.15 ml) contained 10 umoles of glycylglycine buffer (pH 7.5), 1.0 uncle of MgClz, 0.5 umole of ATP, 0.5 umole of PEP, 0.05 umole of NADH, X umole of D-galactose 6-phosphate, nonlimiting amounts of rabbit muscle fructose 6-phosphate kinase, pyruvate kinase, and lactate dehydrogenase, and cell extract from lactose-grown cells; rabbit muscle fructose 6-phosphate kinase will phosphorylate D—tagatose 6-phosphate (121), the product of the isomerase reac- tion. Examination of the effect of substrate concentration on the velocity of the reaction revealed that the Km of the enzyme for D-galactose 6-phosphate was approximately 12 mM. Using 12 mM sub- strate, the assay for the isomerase was found to be linear with both time (Figure 4) and enzyme concentration (Figure 5). An . assay to measure the isomerase activity in the reverse reaction, using D-tagatose 6-phosphate as the substrate, was also developed. The assay involved use of a chemical method which is specific for aldohexoses (119). The reaction (0.10 ml) contained 6.8 umoles of glycylglycine buffer (pH 7.5), 0.6 umole of MgClz, X umole of D-tagatose 6-phosphate, and cell extract from lactose-grown cells; after incubation for a timed period, the reaction mixture was assayed for aldohexose (119). Examination of the effect of sub- strate concentration on the velocity of the reaction revealed that the Km of the enzyme for D-tagatose 6-phosphate was approximately 3mM. Using 6 mM substrate, the assay for the isomerase was fl \ f \\.. \o \. E I D-TAGAIOSE 6-PHOSPHATE FORMED (nmoles) 10L/0//o /. /./00.5L11 _ . //7/, ’,/’. ‘,4v"”'. . C C C /. 0.25111 //-/// o”” _.—o-*"° ././ /./. .I. l ’ l I 05 , 5 10 15 20 TIME (minutes) Figure 4. Linearty of the forward D-galactose 6-phosphate isomerase reaction with respect to time. The pl volumes for each curve indicate the volume of cell extract used in that assay; the nmoles of product formed during the course of the continuous assays were plotted against the respective times. \n E: I I 205 b /. q 0 1 2 3 4 5 VOLUME OF CELL EXTRACT (“1) D-TAGATOSE 6-PHDSPHATE FORMED (nmoles per minute) Figure 5. Linearity of the forward D-galactose 6-phosphate isomerase reaction with respect to enzyme concentration. The rates (in nmoles per minute) of the reactions in Figure 4 were plotted against the volume of cell extract used in the respective assays. 54 found to be linear with both time (Figure 6) and enzyme concentra- tion (Figure 7). The D-tagatose 6-phosphate kinase reaction (0.15 ml) con- tained 10 umoles of glycylglycine buffer (pH 7.5), 1.0 umole of MgClz, 0.5 pmole of ATP, 0.5 Hmole of PEP, 0.05 umole of NADH, X umole of D-tagatose 6-phosphate, nonlimiting amounts of pyruvate kinase and lactate dehydrogenase, and cell extract from lactose- grown cells. Examination of the effect of substrate concentration on the velocity of the kinase reaction revealed that the Km of the enzyme for D-tagatose 6-phosphate was approximately 25 DM. Using 330 uM substrate, the assay for the kinase was found to be linear with both time (Figure 8) and enzyme concentration (Figure 9). The D-tagatose 1,6—diphosphate aldolase reaction (0.15 ml) contained 10 umoles of glycylglycine buffer (pH 7.5), 1.0 pmole of MgClz, 0.5 Dmole of ATP, 0.05 umole of NADH, X umole of D-tagatose 6-phosphate, nonlimiting amounts of rabbit muscle fructose 6-phosphate kinase, triose phosphate isomerase, and a-glycerol phosphate dehydrogenase, and cell extract from lactose- grown cells; rabbit muscle fructose 6-phosphate kinase will phos- phorylate D-tagatose 6-phosphate (121) to effect the synthesis of D—tagatose 1,6-diphosphate, the substrate for the aldolase. Examination of the effect of substrate concentration on the velocity of the aldolase reaction revealed that the Km of the enzyme for D—tagatose 1,6-diphosphate was approximately 1.4 mM. Using 1.7 mM substrate, the assay for the aldolase was found to be linear with both time (Figure 10) and enzyme concentration (Figure 11). D-GAIACI‘OSE 6-PHOSPHATE FORMED (nmoles) .. 74/1 I O 5 10 15 20 TIME (minutes) Figure 6. Linearity of the reverse D-galactose 6-phosphate isomerase reaction with respect to time. The ul volumes for each curve indicate the volume of cell extract used in those assays; the nmoles of product formed in the end-point assays were plotted against the respective times of reaction. 50" 0y 1 1 1 0 5 10 15 20 VOLUME OF CELL EXTRACT (01) D-GALACTOSE 6-PHOSPHATE FORMED (nmoles per minute) Figure 7. Linearity of the reverse D-galactose 6-phosphate isomerase reaction with respect to enzyme concentration. The rates (in nmoles per minute for a five minute reaction) of the reactions in Figure 6 were plotted against the volume of cell extract used in the respective assays. D-TAGATOSE 1,6-DIPHOSPHATE FORMED (nmoles) Figure 8. respect to time. times. D-TAGATOSE 1,6-DIPHOSPHATE FORMED (nmoles per minute) Figure 9. respect to enzyme concentration. The rates (in nmoles per minute of the reactions in Figure 8 were plotted against the volume of cell extract used in the respective assays. 56 15 20 TIME (minutes) Linearity of the D-tagatose 6-phosphate kinase assay with The ul volumes for each curve indicate the volume of cell extract used in that assay; the nmoles of product formed during the course of the continuous assays were plotted against the respective l I l 2 5 h 5 VOLUME OF CELL EXTRACT (pl) Linearity of the D-tagatose 6-phosphate kinase assay with 57 7.5 F .5111 " CLEAVED (nmoles) 2.5 --//C ////' ’,,z°”/’ " D-TAGATOSE 1,6-DIPHOSPHATE E” O I O\ O 0 ~\\\. “\\\\\:\\. O H T.’ H all o o O a”’ o""."" 0 :/"T l 1 0 5 10 15 20 TIME (minutes) Figure 10. Linearity of the D-tagatose 1,6-diphosphate aldolase assay with respect to time. The ul volumes for each curve indicate the volume of cell extract used in that assay; the nmoles of substrate cleaved during the course of the continuous assays were plotted against the respective times. / O l l l l l l I I 03?: 1 2 5 h 5 6 7 VOLUME OF CELL EXTRACT (p1) D-TAGATOSE 1,6-DIPHOSPHATE CLEAVED (nmoles per minute) H 1 Figure 11. Linearity of the D-tagatose 1,6-diphosphate aldolase assay with respect to enzyme concentration. The rates (in nmoles per minute) of the reactions in Figure 10 were plotted against the volume of cell extract used in the respective assays. 58 Induction of the D-Tagatose 6-Phosphate Pathway Enzymes These quantitative assays were then used to determine whether the enzymes of the D—tagatose 6-phosphate pathway are distinct from those of the Embden-Meyerhof pathway for D-glucose 6-phosphate metabolism, and whether they are constitutive or inducible. The two determinations were made by measuring the specific activities of the enzymes of the two pathways in extracts of cells grown on several sugars (Table 4). The data indicate that the three enzymes of the Embden-Meyerhof pathway are constitutive and are distinguishable from those of the Dhtagatose 6-phosphate pathway which are specifi- cally induced by growth of the organism on lactose or D-galactose. DEAR-Cellulose Chromatography of the D-Tagatose 6-Phosphate Pathgay and Embden—Meyerhof Pathway_Enzymes I determined that the corresponding enzymes of the two path- ways were separable by DEAE-cellulose chromatography. This separa- tion was required in order to perform product identification of the reactions of the D-tagatose 6-phosphate pathway. Figure 12 shows the use of DEAE-cellulose to separate D-galactose 6-phosphate isomerase from D—glucose 6-phosphate isomerase. A single pass through the column was sufficient to Obtain the former enzyme free of the latter. Figure 13 shows the use of DEAE-cellulose to sepa- rate D-tagatose 6-phosphate kinase from D-fructose 6-phosphate kinase. Some separation of the former from.the latter was obtained by one passage through the column (Figure 13A). The combined frac- tions of D-tagatose 6-phosphate kinase activity (Figure 13A) were ofiflhflfiflfla mw3 flOHUUQQH ”mahflaomfi 0mHfl>0H ”fl?“ oUOHflwflfla OM? GOfiUUNNH GGMHQBOMfl UHMRHOH NERO .NN ou woman“ on: sowumuuomosoo Odoueoe mean .000: ones as 000 no mos=Ho>o 59 00H.0 ~00.0 m~.H 50.H 000.0 H00.0 HH0.0 000.0 mmouamz 0NH.0 000.0 0H.H 00.H 000.0 H00.0 ~H0.0 000.0 omouoom HOH.0 0N0.0 «05.0 0H.H 000.0 000.0 000.0 000.0 mmosomz 000.0 000.0 000.0 000.0 0N0.0 000.0 005.0 000.0 omouosa 00H.0 000.0 050.0 005.0 000.0 000.0 505.0 ~0N.0 mmouomamu 00H.0 000.0 000.0 0~.H 000.0 H00.0 500.0 000.0 omouosum NOH.0 000.0 H0.H H0.H 000.0 H00.0 0H0.0 000.0 Cmoooau 000.0 000.0 0000.0 0000.0 000.0 000.0 0000.0 0000.0 noooz ommaova< ammoam ommumaomH ommHo0H< ommofiu omsuoaomH panacea moahnoo mosaume mmahuso 5manus0 suaouw ofi monum5ostoveam oumadmozel0 mmOumwmaln Ououvmnonumo Anamuoua ma non muwosv hua>wuon oamqooem .am. .W mo pooch—No HHCO a.“ nao>oa gen .0 as 60 0003080 003 50 no 00000000 .000: z 0.0 00 o 0 «00.300000 :33 00000.3 0000 000 000.0.» A0300 039001000003 0503 000.500 Ag A33 $0.0 000 000000.00 A33 *3 0000000000 3.0. 00v 0E5. ZS 0+3 00900000 0 003 90300 0:0 00 00.3000 30.000 00....“ ADV 00090800.“ 00000000010 00000000010 000 on 00000800.“ 00000020010 00003010 ”0.30600 000.3300 10000 no 0000008000 00050005010 00003010 000 00000000010 00000000010 mo 0000000003005 .9 0000.3.— mflgz zen—”Hug 0 00 On 00 000 . jr. 1 Add \oVo . \fl /. X. o\. 2 . ./ / - A ./‘ '\. x O O MIAIIDV ZSVHZNOSI ELVHdSOHJ'9 HSOOfl'IS-(I D-CALACTOSE 6-PHOSPHATE ISOMERASE ACTIVITY . : 61 “0 00 00 000000000000000 000 00000000 0003 0_00 m: 00 00 000000000 .00000000 003 000 00 00000000 000000 2 0.0 00 0.0 0 .00000000 003.000 00 00000000 000000 2 m.0 00 0 0 “00010 00000000 00 000000000000000 000 A00000u0000 $00 00 000 0000000 00000000 0003 000000000000 0000 000 00003 “00000 0300010000000 00000 0000000 A020 A>\>v mm.o 000 00000000 A>\>v mom 0000000000 Am.~.00v 000000000.s00000000 =0 omv 00000000 0 003.0.000000 00 0000000 000000 000 mmmznz ZOHHU¢MM 10 04.... \0. 0/0 O - . .1 -. / l o o\0 ._ 1 \ 13 .\. n F n - .AOQ 000003 00000000010 0000000010 000_on 000003 00000000010 0000000010 "0000000 10000 00 0000000 00000000010 0000000010 000 00000000010 0000000010 00 00000000000000 .000000000 .nH «0:000 000000 20000000 on mm 11.1. \\\mollhuw //. \ . - /. /./. . .\ 1Lom0 ALIAIIDV’ESVNIX HIVHJSOHJ'9 HSOIVSVE'G HO HSVNIX filVHdSOHd-Q HSOIDHHH‘G 62 then rechromatographed, employing a shallower salt gradient, to completely separate it from the D-fructose 6-phosphate kinase activity (Figure 133, page 61). However, this separation could only be obtained when a significant loss in D-tagatose 6-phosphate kinase was tolerated (note the combined fractions in Figure 13A). Figure 14 shows the use of DEAF-cellulose to separate D-tagatose 1,6-diphosphate aldolase from D-fructose 1,6-diphosphate aldolase. One pass through the column (Figure 14A) resolved two peaks of D—fructose 1,6-diphosphate aldolase activity and one peak of D—tagatose 1,6-diphosphate aldolase activity, which coincided with the second peak of D-fructose 1,6-diphosphate aldolase. The second peak was rechromatographed (Figure 143) employing a shal- lower salt gradient, and a similar pattern resulted. Since the second peak of D-fructose 1,6-diphosphate aldolase activity coincided with the D-tagatose 1,6-diph05phate aldolase activity on both columns and was not observed when extracts from glucose- grown cells were chromatographed, D-tagatose 1,6-diphosphate aldo- lase must not be specific for its substrate. ,The Km for D-fructose 1,6-diphosphate is approximately'Zumh whereas the constitutive D-fructose 1,6-diphosphate aldolase has a Km of approximately 15 MM. This difference in Km values is sufficient to allow determination of the indicibility of the high Km aldolase. It was induced by growth of §, aureus only on lactose or D-galactose (Table 5), fur- ther suggesting its identity as D-tagatose 1,6-diphosphate aldolase. This pr0perty of the aldolase will be discussed further in 63 00000060 003 000 00 00000000 000000 00 0.0 00 0.0 0 mm 00 00 00000003000000 000 00000000 0003 0 00 mm 00 00 000000000 00000000 003 000 .00 00000000 000000 00 m.0 00 0 0 “000000000 000 0000000 000600000 0003 0000000 0000 000 00003 “00000 0300010000000 0000v 0000000 “020 A>\>v $0.0 000 00000000 A>\>v $00 0000000000 Am.» 0000 000000000 000000000 SE 03 00000000 0 003 0 000000 00 0000000 000000 00.0. .AOV 00000000 000000000001000 0000000010 000 on 00000000 000000000001000 0000000010 “0000000 .00000000010000 00 000000000 000000000001000 0000000010 000 000000000001000 0000000010 00 00000000000000 .00 000000 000252 ZOHHUooo¢o .wa ca ma cwououmn .Ha cw ma masao>o on ~.ma a.ma m~.H o.m ooa.o xmvmgamm mm 05.5 o.mm oo.m 0.0 HH omoasaaoolmoomm huw>wuom muw>auom Adamuoua . oa=Ho> aofiuomum o oawwuonm Hmuoh HauoH m .mmduofioma mumnnmonmlo omouomamwln mo doaumoamuusm .o mqm 85%) of activity in 20 minutes. The addition of BME [to 0.2% (v/v)] partially (to 30%) reversed the inhibition by NEM. BME also stabilized the isomerase during puri- fication. When BME was removed from the enzyme by dialysis, the recovery (about 20%) was considerably less than the quantitative 106 recovery obtained when dialysis was done in the presence of BME. Reactivation was readily achieved by adding back BME (Table 7); reactivation was complete in about 12 hours. Molecular weight and sedimentation coefficient.--The behavior of the isomerase on Sephadex G-100 against several stan- dards of known molecular weight was determined and was plotted as a function of log molecular weight (Figure 24). The standards used and their molecular weights on Sephadex follow: rabbit muscle fructose 1,6-diphosphate aldolase, MW - 149,000 (159); yeast 81“! cose 6-phosphate dehydrogenase, MW = 128,000 (152)3.§3.2211 alka- line phosphatase, MW - 86,000 (163); and A, aerogenes fructose l-phosphate kinase, MW - 75,000 (146). The isomerase chromato- graphed as a 99,000 molecular weight protein. The sedimentation behavior of the isomerase in a sucrose density gradient is shown in Figure 25. The standard used was calf intestine alkaline phosphatase which has a sedimentation coefficient (820,w) of 6.20 and a molecular weight of 100,000 (158). The sedimentation coefficient and the approximate molecular weight of the isomerase were calculated (157) using the following equations: 2/3 distance 1 traveled 3.51. a d El_= Mwl distance 2 traveled 82 n 82 MW2 The coefficient was 6.22 using alkaline phosphatase as a standard, and 6.22 corresponded to a molecular weight of 100,000. 107 TABLE 7. Time-dependent reactivation of D-galactose 6-phosphate isomerase by BME. A sample of the isomerase was dialyzed against 40 mM Tris buffer (pH 7.5) containing 15% (v/v) glycerol and 0.2 mM EDTA. The sample was incubated at 4°C in the absence or presence of 0.2% (v/v) BME, and portions were withdrawn at intervals and assayed for isomerase activity. Experiment Time of incubation8 Activity Without BME 0 0.119 12 0.119 With BME 0 0.119 2 0.335 4 0.400 8 0.444 12 0.450 8Time was measured in hours. 200,000 90,000 MDLECULAR.WEIGHT H .8 3 § 70,000 60,000 Figure 24. I ll, ' I l 60 65 70 75 FRACTION NUMBER Plot of elution volume against log MW of standards and D- galactose 6-phosphate isomerase from a chromatographic run on Sephadex G-100. Abbreviations: FDRA, fructose 1,6-diphosphate aldolase; G6PDH, glucose 6-phosphate dehydrogenase; Ga16PI, D-galactose 6-phosphate isomerase; AP, alkaline phosphatase; and FlPK, fructose l-phosphate kinase. I'D ENZYME ACTIVITY (arbitrary units) p—I Figure 25. . ox. ' 0’ a. _ / .\.\ O (y’l/o 0 .\\\° .l" “'\." ‘\“O I ("I I I °‘0 I 25 30 35 40 “5 FRACTION NUMBER Sedimentation pattern of calf intestine alkaline phosphatase and D-galactose 6-phosphate isomerase in a sucrose density gradient. Symbols: phosphatase (0) and isomerase (O). 109 The data indicate that D—galactose 6-phosphate isomerase has a molecular weight of 100,000 and a sedimentation coefficient of about 6.2. Discussion The data presented here establish that D—galactose 6-phosphate is reversibly isomerized to D—tagatose 6-phosphate in S, aureus. The isomerase that catalyzes this reaction is specific for these two sugar phosphates. The studies revealed that only a small percentage (8%) of the D—galactose 6-phosphate was isomer- ized to D-tagatose 6-phosphate but that a much larger percentage (40%) of D-tagatose 6-phosphate was present at equilibrium'when D-tagatose 6-phosphate was the substrate. The discrepancy was due to the specificity of the isomerase for only one anomer of D-galactose 6-phosphate and to the lack of spontaneous anomerization under the assay conditions. The particular anomer utilized by the isomerase has been tentatively designated a-D-galactopyranose 6-phosphate by Harving and Horning (130); the assignment of0um 0cu aoum maqamm 0nu mo wamslmco haao macaw vmumaommuuxm 0000 ppm: mmsam> ommnhm .N ca ma >H0>oommo .08 ca ma afimuoumn :1: CH EH gHOPm cam n.aa ew.ma ema.o N.m coauo xmemaamm mm «0.0 ~.~m 50.x H.m muwumnmhxouvhm Nu 0H.H N.~¢ n.m0 ma HH omoaaaamolm<00 00 005.0 0.5m «.mn 00 H wwoaoaamolmoomm ”NWMHWMM . hwwwwwom nammwwme mmabao> cofiuomum .mmmcwx mumnamosmle omoumwwuln Ho ooaumuwmwusm .0 0A0 tum" (mn‘l) E Amoaoas you ousoaav H-sanoogm> 0.2 0.15 0.1 Ema" um“) 0.05 O -0.0S F Amoaoaa you ousowav a-»auooam> 0.2 0.15 0.1 [TTPJ-l (mM'l) 0.05 -o.os 135 TABLE 10. Effect of monovalent cations on D-tagatose 6-phosphate kinase activity. The standard assay, from which mono- valent cations were excluded, was used. The cations were added to the assays as their chloride salts. Activitya at a cation concentration of Monovalent cation 1.33 mm 6.67 mm 33.3 mM Ammonium 215 319 413 Rubidium 211 288 392 Potassium 211 286 342 Cesium 115 165 304 Sodium 96 85 69 Lithium, 91 77 35 8The activities are expressed as a percentage of that (activity - 0.254) observed with no monovalent cation added. 136 63.2; was coauouuooooou 03.330 5:253 05 unsu unooxo van: no: human Boon—3n one .huazuoo anon: 33:23-0 ououuwmuun no and» mad ca .533on no uoommm .mn shaman 3.5a mafia»: 06 o.m 0.: . o.n 0d 04 o . . _ . . a a . n I. I. no.0 m 0 S E 9 . m m I O l OH.O m 1 3 O 8 r1 I 3.0 s V / D m C /94 u .I- 1 1.0-III J 8.0 — — r — P 137 could be replaced partially by several other divalent cations with the following relative rates: MgClz, 100; CoClz, 54; MnClz, 27; NiCl 15; CdSOa, 8; CaClz, 3; ZnClz, 2; Fe804, BaClZ, Cusoé, and 2’ none, 0. Sulfhydryl requirement.--The thiol-blocking reagent NEM inhibited the kinase. At a concentration of 0.2 mM, the reagent caused almost complete loss (> 85%) of activity in 30 minutes. The addition of BME [to 0.2% (v/v)] partially (to 302) reversed the inhibition by NEM. BME also stabilized the kinase during puri- fication. When BME was removed from the enzyme by dialysis, the recovery (about 60%) was appreciably less than the quantitative recovery obtained when dialysis was done in the presence of BME. Reactivation was readily achieved by adding back BME (Table 11); reactivation was complete in about 12 hours. Mblecular weight and sedimentation coefficient.--The behavior of the kinase on Sephadex G-100 against several standards of known molecular weight was determined and was plotted as a function of log molecular weight (Figure 36). The standards used and their molecular weights on Sephadex follow: yeast glucose 6-phosphate dehydrogenase, MM - 128,000 (162); E, ggli_a1kaline phosphatase, MW - 86,000 (163); A, aerogenes fructose 1-phosphate kinase, MW - 75,000 (146); and yeast hexokinase, MW - 51,000 (161). The kinase chromatographed as an 82,000 molecular weight protein. The behavior of the kinase on polyacrylamide gels in the presence of dodecyl sulfate against several standards of known 138 TABLE 11. Time-dependent reactivation of D-tagatose 6—phosphate kinase by BME. A sample of the kinase was dialyzed against 20 mM potassium phosphate buffer (pH 7.5) con- taining 20% (v/v) glycerol. The sample was incubated at 4°C in the absence or presence of 0.2% (v/v) BME, and portions were withdrawn at intervals and assayed for kinase activity. ‘ Experiment Time of incubation8 Activity Without BME 0 0.077 12 0.077 With BME 0 0.077 2 0.098 4 0.107 8 0.116 12 0.120 8Time was measured in hours. 139 150,000 I I I I ‘\\0 G6PDH a 100,000 I- u- E 90,000 - .AP I-i 80:000 - \.\T336PK " g . 1px 70,000 - d a 60,000 - I- - . IIK - 50,000 \\\ I I I I 90,000 as 50 55 6O FRACTION NUMBER Figure 56. Plot of elution volume against log MW of standards and D- tagatose 6-phosphate kinase from a chromatographic run on Sephadex G-100. Abbreviations: G6PDH, glucose 6-phosphate dehydrogenase; AP, alkaline phosphatase; Tag6PK, D-tagatose 6-ph0sphate kinase; FlPK, fructose l-phosphate kinase; and HK, hexokinase. I I I I 70,000 - \, BSA 3 60:000 - \O{ h! E 50,... — - {P g; 10 Oval :3 “0,000 In \5\\\ 2 ‘ “ £1 £3 30,000 r- " g; 0‘3:C 20,000 I I I I O 10 20 30 40 MIGRATION DISTANCE (um) Figure 37. Plot of migration distance against log subunit molecular weight of standards and D-tagatose 6-phosphate kinase in a poly- acrylamide gel in the presence of dodecyl sulfate. Abbreviations: BSA, bovine serum.albumin; Cat, catalase; Tag6PK, D-tagatose 6-phos- phate kinase; Oval, ovalbumin; ADH, alcohol dehydrogenase; and 050, abchymotrypsinogen. 140 subunit molecular weight was determined and was plotted as a function of log subunit molecular weight (Figure 37, page 139). The standards used and their subunit molecular weights (157) follow: bovine serum albumin, MW = 68,000; beef liver catalase, MW = 60,000; ovalbumin, MW - 43,000; yeast alcohol dehydrogenase, MW - 37,000; and bovine a-chymotrypsinogen A, MW - 25,700. The kinase had a subunit molecular weight of 52,000. The sedimentation behavior of the kinase in a sucrose density gradient is shown in Figure 38.- The standards used were rabbit muscle fructose 1,6-diphosphate aldolase, $20,w - 7.35 and MW - 149,000 (159), and calf intestine alkaline phosphatase, S - 6.20 and MW . 100,000 (158). The sedimentation coefficient 20,w and the approximate molecular weight of the kinase were calculated (157) using the following equations: distance 1 traveled -.El 1 1 distance 2 traveled 82 $2 MW2 The coefficient was 6.78 and 6.85, using aldolase and phosphatase, respectively, as standard. The coefficients corresponded to molecular weights of 132,000 and 112,000, respectively. The data indicate that D-tagatose 6-ph08phate kinase has a molecular weight of about 100,000, is a dimer of 52,000 molecular weight subunits, and has a sedimentation coefficient of about 6.8. 141 ALKALINE PHOSPHATASE ACTIVITY um ououowouun «ADV omofiopao muonnuoeoaVImaa ououooum «House.uannou “muonshm (arbitrary units) .Adv «mouoeoaono ooaaoxao ooguaouoa mass was «on oooofix cameouosa .uoowvouw huaanop ououonu u a“ unnoax ouonouonouw ououuwouun was avuovnwua.uo nuouuoa ooquouooaqvom .mn ouswam 0H ma ON mm. Mumzzz ZOHBUoommo .mfi oH mu ofimuoum a .HE 6H ma mauao>m on m.aa H.aq Nm.~ s.m mmoaaaeoosmooom >ua>wuon mue>wuoo naaououa oasao> sowuomum uawaumam deuce Hausa m .mmoHova muoemmoenavlo.d omouommuln mo nowumuwmaknm .NH mamsa 145 pH 5.2, and purification similar to that obtained with phosphocel- lulose (see Table 12) resulted. The aldolase bound to hydroxyapa- tite at pH 7.0 and could be quantitatively eluted from.the adsorbent. There was also quantitative recovery after chromatog- raphy on Sephadex G-100. The initial experiments indicated that the aldolase was fairly stable for several days in cell extracts prepared in potas- sium phosphate buffer (20 mM, pH 7.5) or in sodium acetate buffer (20 mM, pH 5.2). The enzyme was completely stable for several days when 20% (v/v) glycerol and 0.22 (v/v) BME were added to the phos- phate buffer, but was still only fairly stable in the acetate buffer. The enzyme was stable to freezing in either buffer. Cell extracts prepared in 20 mM sodium acetate buffer (pH 5.2) containing 20% (v/v) glycerol and 0.2% (v/v) BME were found to have a pH of about 6.0. The pH was therefore adjusted to pH 5.2 with acetic acid prior to centrifugation to remove cellular debris; this adjustment was a pH precipitation step and resulted in the removal of some protein. This procedure was used to prepare an extract from 68 g of D-galactose-grown S, aureus. The aldolase was purified from this extract as described below. All steps were performed at 4°C; a summary of the purification appears in Table 12. Phosphocellulose chromatography I.e-A phosphocellulose column (3.1 X 22 cm) was equilibrated with the cell extract buffer. The cell extract was applied to the column, which was then washed with 500 ml of the same buffer. The protein was eluted with a —. 146 linear gradient of 1660 m1 of 0 to 0.4 M KCl in the same buffer. The elution pattern for this column is shown in Figure 39. A total of 110 fractions of 15 ml each were collected, and the fractions (numbers 41 to 50) containing most of the aldolase activity were combined. Phosphocellulose chromatography II.--A phosphocellulose column (1.6 X 17 cm) was equilibrated with the same buffer. The combined fractions from the previous step were diluted to 300 ml with the same buffer and were applied to the column, which was then washed with 100 m1 of the same buffer. The protein was eluted with a linear gradient of 320 m1 of 0 to 0.35 M.KC1 in the same buffer containing 1 mM D—fructose 1,6-diphosphate. The elution pattern for this column is shown in Figure 40. A total of 44 fractions of 7.2 ml each were collected, and the fractions (numbers 10 to 19) containing most of the aldolase activity were combined. DEAE-Cellulose chromatography.--A DEAE-cellulose column (1.2 X 7.5 cm) was equilibrated with 20 mM potassium phosphate buffer (pH 7.5) containing 20% (v/v) glycerol and 0.2% (v/v) BME. The combined fractions from the previous step were adjusted to pH 7.5 with 0.4 M.K2HP04, diluted to 300 ml with the pH 7.5 buffer, and applied to the column, which was then washed with 50 m1 of the same buffer. The protein was eluted with a linear gradi- ent of 90 m1 of 0 to 0.4 M ROI in the same buffer. The elution pattern for this column is shown in Figure 41. A total of 64 fractions of 1.4 mi each were collected, and the fractions 147 .on odououa can «ADV oooaovHo unannoonowvuwed ooouosumun ”AOW ouoaovao ouosoaonmavumqa ououowou In nuaooahm .0u0aoaaooosonoeo no ouoaovuu muonmoosmwvumaa ooouo noun «0 Mandamouoaoumo .mm enough MNfiZfiz ZOHHU \\\ D-TAGATOSE 1,6-DIPHDSPHATE ALDOLASE ACTIVITY 0.5 F- '- __ .1— (:I- I I l I ‘1- 6.0 7.0 8.0 9.0 pH Figure 43. Effect of pH and buffer composition on D-tagatose 1,6- diphosphate aldolase activity. The standard assay was used except that the pH and buffer (0.15 M) were varied. Symbols: HEPES (0), sodium cacodylate (O), Tris-maleate (A), potassium phosphate (A), glycylgly- cine (l), and Tris (D). The activities were measured at two aldolase concentrations, and the pH of each reaction mixture was measured at the completion of the reaction. 156 From Lineweaver-Burk plots (Figure 44), the Km values for D-tagatose 1,6-diphosphate and D-fructose 1,6-diphosphate were determined to be 1.5 mM and 2.5 mM, respectively. At saturating levels of substrate, D—fructose 1,6-diphosphate was cleaved at 472 the rate of D-tagatose 1,6-diphosphate. Several lines of evidence, in addition to that presented in Section 1, indicate that the cleavage of the two substrates is performed by the same enzyme. Firstly, the enzyme is homogeneous. Even if the constitutive D-fructose 1,6-diphosphate aldolase were present, the cleavage of D-fructose 1,6-diphosphate could not be attributed to that enzyme since its Km for the substrate is about 15 uM. Secondly, the activities are not additive (Table 15), i.e., the activity measured in the presence of saturating levels of both substrates is less than that measured in the presence of D—tagatose 1,6-diphosphate alone. This indicates that the activities are not additive, but are competitive. In addition, the two activities decay coincidentally when the enzyme is heated at 50°C (Figure 45). The data establish that D-tagatose 1,6;diphosphate aldolase cleaves both substrates. In addition, the aldolase catalyzed the cleavage of D-sorbose 1,6-diph08phate and D-psicose 1,6-diphosphate, as the following data demonstrates. A reaction (5.0 ml) containing 200 nmoles of HEPES buffer (pH 7.0), 100 nmoles of DL-glyceraldehyde 3-phosphate, triose phosphate isomerase, and D-tagatose 1,6- diphosphate aldolase was incubated at 25°C. Samples were withdrawn at timed intervals and assayed for ketohexose (117) to follow the 157 VELOCITY"1 (minute per nmoles) . I1: 1 I __I.J -0.5 0 0.5 1.0 1.5 2.0 [D-TAGATOSE 1,6-DIPHOSPHATE1-1 (mM-l) I r I T VELOCITT'l (minute per mmoles) _.I_/ I I L I -O.5 O 0.5 1.0 1.5 2-0 [D-FRUCI‘OSE 1,6-DIPHOSPHATEJ-1 (mM-l) Figure 44. Lineweaver-Burk plots showing the effect of substrate con- centration on the D-tagatose 1,6-diphosphate aldolase reaction velocity. The standard assay was used except that the substrate (D- tagatose 1,6-diphosphate in A and D-fructose 1,6-diphosphate in B) concentrations were varied. 158 TABLE 15. Competition between D—tagatose 1,6—diphosphate and D—fructose 1,6-diphosphate for D-tagatose 1,6-diphosphate aldolase. The standard assay was used except that the substrate concentration and composition was varied as indicated. Substratea b D-Tagatose D—Fructose Activity 1,6-diphosphate 1,6-diphosphate 0 0 0 15 0 100 0 25 47 15 25 82 aThe substrate concentrations are expressed in mM. bThe activities are expressed as a percentage of that (activity - 0.580) observed in the presence of D-tagatose 1,6~ diphosphate alone. ' \ 159 90"9 '- 80- -‘ 70_ 9 .. 60 I- " E H C a O Eso- - it p: i2. ‘3 E; n. 0 El 30- g 0 O 20 'I -' l l l O 10 2O 30 MINUTES AT 50°C Figure 45. Thermal inactivation of D-tagatose 1,6-diphosphate aldo- lase. The purified aldolase was heated at 50°C, and samples were withdrawn at intervals and assayed with both D-tagatose 1,6-diphos- phate (O) and D-fructose 1,6-diphosphate (0) as substrate. 160 course of the reaction. When the reaction was complete, the pH of the solution was adjusted to 8.5 with NaOH, and the solution was applied to a Dowex 1-X8 bicarbonate column (1.2 X 10 cab, and eluted with a stepwise gradient (150 ml each) of water and 0.15 M, 0.30 M, and 0.45 MIKHCO3 (122). Preliminary experiments indicated that monophosphates and diphosphates eluted with 0.15 M and 0.30 M KHCOS, respectively. Fractions containing ketohexose (117), which eluted with 0.30 M KHCOB, were combined and treated with Dowex 50W-X8 (H+ form) until the pH was 2.0. The sample was then freeze- dried and dissolved in 10 m1 of water, and the pH of the solution was adjusted to 7.0 with NaOH. All of the ketohexose product eluted from.the Dowex column in the position of a diphOSphate, and it had a phosphate (114) to ketohexose (117) ratio Of 2.0. In separate reactions, the product reacted quantitatively through the sequences: ketohexose diphosphate--*-dihydroxyacetone phosphae + D-glyceraldehyde 3—phosphate--+'2 dihydroxyacetone phosphate'-—-* 2 arglycerol phosphate through the sequential partiCipation of D—tagatose 1,6- diphosphate aldolase, triose phosphate isomerase, and a—glycerol phosphate dehydrogenase; and ketohexose diphosphate--+ dihydroxy- acetone phosphate + D-glyceraldehyde 3—phosphate--+ 2 D-glyceral- dehyde 3-phosphate--+ 2 3-phosphog1ycerate through the sequential participation of D-tagatose 1,6-diphosphate aldolase, triose phosphate isomerase, and D-glyceraldehyde 3-phosphate A dehydrogenase (Table 16). This indicated that only the D-isomer TABLE 16. 161 Enzymatic identification of the D-tagatose 1,6- diphosphate aldolase condensation reaction product. Reactions A and B (0.15 ml) contained 10 nmoles of HEPES buffer (pH 7.0), 0.05 Hmole of NADH, 3.3 nmoles of reac- tion product, a-glycerol phosphate dehydrogenase, and D-tagatose 1,6-diphosphate aldolase; B also contained triose phosphate isomerase. Reactions C and D (0.15 ml) contained 10 nmoles of HEPES buffer ( H 7.0), 0.5 nmole of sodium arsenate, 0.05 nmole of NAD , 3.3 nmoles of reaction product, glyceraldehyde 3-phosphate dehydrogen- ase, and D-tagatose 1,6-diphosphate aldolase; D also contained triose phosphate isomerase. ""A..' -"h‘ I nu “I'D. I. ”I... k Reaction Product Enzyme presenta nmoles Identityb a-GPDH + TPI 6.6 DHAP-PG3P G3PDH ,. 3. 3 G3P G3PDH + TPI 6.6 G3P +'DHAP aAbbreviations: see Table 13. bAbbreviations: see Table 13. 162 of the commercial DL-glyceraldehyde 3-phosphate was utilized in the condensation reaction. The product was dephosphorylated enzymatically for further identification. This dephosphorylated product possessed components which chromatographed on paper as each of the four ketohexoses (149): tagatose, fructose (R . 1.14), sorbose (R - tagatose tagatose 0.96), and psicose (Rtagatose - 1.22); the components were located using an orcinol spray for ketohexoses (149), and the locations were verified on a duplicate chromatogram with a silver nitrate procedure for total carbohydrates (151). The presence of sorbose, which chromatographs closely with tagatose, was verified with the cysteine-H2804 reaction (164, 165), which yields a distinctive absorption spectrum.for sorbose, as evidenced by the A412 to A605 ratio after 20 hours: the ratio for fructose, psicose, and tagatose, alone or in mixtures, was 3.5, whereas the ratio for sorbose was 0.5; the ratio for the dephosphorylated reaction product was 2.5. These data indicate that dihydroxyacetone phos- phate and D—glyceraldehyde 3-phosphate were condensed to a mixture of the four D—ketohexose 1,6-diphosphates. Effect of cations.--There was no effect on the aldolase activity by addition of 5 mM concentrations of monovalent cations to standard assays. In assays from which monovalent cations were excluded, there was also no effect on the activity by the addition of NaCl, KC1,'NH4C1, LiCl, RbCl, or CsCl at 5 mM concentrations. Monovalent cations, therefore, do not modulate the enzyme activity. 163 An EDTA (10 mM)-treated sample of the aldolase did not display any loss in activity. At 1 mM.concentrations, MgClz, C0012, CaClz, and Cd804 did not affect this enzyme; however, BaCl2 and NiCl caused about 20% inhibition while FeSOA, MhClz, 2 and CuSO4 caused about 50% inhibition. Thus, a divalent metal ion requirement could not be demonstrated. Effect of NaBH4.--NaBH4 caused severe inhibition of the aldolase in the presence of substrate (25 mM D-fructose 1,6- diphosphate). At 10 mM.NaBH4, 90% of the activity was lost in less than 5 minutes; at 100 mM‘NaBHa, all of the activity was lost in the same time period. In the absence of substrate, 100% and 672 of the activity were recovered in the presence of 10 mM and 100 mM NaBHa, respectively. Since there was no divalent metal ion require- ment but there was inhibition by NaBH4, D—tagatose 1,6-diphosphate aldolase is a Class I aldolase (180, 181). Sulfhydryl requirenent.--The thiol-blocking-reagent NEM inhibited the aldolase. At a concentration of 0.1 mM, the reagent caused almost complete loss (> 902) of activity in 30 minutes. The addition of BME [to 0.22 (v/v)] partially (to 252) reversed the inhibition by NEM. BME also stabilized the aldolase during puri- fication. When BME was removed from the enzyme by dialysis, the recovery (about 40%) was considerably less than the quantitative recovery obtained when dialysis was done in the presence of BME. Reactivation was readily achieved by adding back BME (Table 17); reactivation was complete in about 12 hours. 164 TABLE 17. Time-dependent reactivation of D-tagatose 1,6-diphosphate aldolase by BME. A sample of the aldolase was dialyzed against 20 mM potassium phosphate buffer (pH 7.5) con- taining 20% (v/v) glycerol. The sample was incubated at 4°C in the absence or presence of 0.2% (v/v) BME, and portions were withdrawn at intervals and assayed for aldolase activity. ' Experiment Time of incubation8 Activity Without BME 0 0.280 12 0.280 With BME 0 0.280 2 0.454 4 0.517 8 0.569 12 0.585 8Time was measured in hours. 165 Mblecular weight and sedimentation coefficient.--The behavior of the aldolase on Sephadex G-100 against several stand— ards of known molecular weight was determined and was plotted as a function of log molecular weight (Figure 46). The standards used and their molecular weights on Sephadex follow: (E, 221; alkaline phosphatase, MW - 86,000 (163); A, aerogenes fructose l-phosphate kinase, MW - 75,000 (146); yeast hexokinase, MW - 51,000 (161); and ovalbumin, MW = 43,000 (162). The aldolase chromatographed as a 50,000 molecular weight protein. The behavior of the aldolase on polyacrylamide gels in the presence of dodecyl sulfate against several standards of known sub- unit molecular weight was determined and was plotted as a function of log subunit molecular weight (Figure 47). The standards used and their subunit molecular weights (155) follow: bovine serum albumin, MW - 68,000; beef liver catalase, MW = 60,000; ovalbumin, MW - 43,000; yeast alcohol dehydrogenase, MW = 37,000; and bovine pancreas archymotrypsinogen A, MW'I 25,700. The aldolase had a subunit molecular weight of 37,000. . 'The sedimentation behavior of the aldolase in a sucrose density gradient is shown in Figure 48. The standard used was horseradish peroxidase, 820,w - 3.48 and MW = 40,000 (160). The sedimentation coefficient and the approximate molecular weight of the aldolase were calcuaated (157) using the following equations: - -- and --— 2/3 distance 1 traveled S1 .51., MN1 distance 2 traveled 2 $2 MW2 166 100,000 I- I I l I 90,000 - \. p 5,; 80,000 I- K H ,FIPK g 70,000 - é 60,000 ,_ a 50,000 — ‘ - 5K TagDPA SI .Oval 110,000 - \ I I I I 58 61 6h 67 FRACTION NUMBER Figure 46. Plot of elution volume against log MW of standards and D-tagatose 1,6-diphosphate aldolase from a chromatographic run on Sephadex G-100. Abbreviations: AP, alkaline phosphatase; FlPK, fructose l-phosphate kinase; HK, hexokinase; TagDPA, D-tagatose 1,6- diphosphate aldolase; Oval, ovalbumin. 80,000 ' ' 1 I 70,000 \eBSA fig \\\\\.Cat ,_. 60,000 9 50,000 g; .Oval 5 40,000 '\.Ann 6 TagDPA E O 000 §§ 5 i 1.060 ‘\. I l I l 2O’OOCO ' 20 ho 60 80 MIGRATION DISTANCE (m) Figure 4?. Plot of migration distance against 10g subunit molecular weight of standards and D-tagatose 1,6-diphosphate aldolase in a poly- acrylamide gel in the presence of dodecyl sulfate. Abbreviations: BSA, bovine serum albumin; Cat, catalase; Oval, ovalbumin; ADH, alcohol de- hydrogenase; TagDPA, D-tagatose 1,6-diphosphate aldolase; 050, 0b chymotrypsinogen. 167 *1 I l I . o 4 - -d E H E. 53 < N 3 O L. a 5. 4 In] HA fie FIII-I a: *4 85 p, '33? 6:: Fry: In '3 2 " 'I as {-0 5 :5 o “o‘ a} o a 11- .J g o E: / 94 o ‘ 0 ° 0 \. 0 I l I I, 30 55 1+0 1+5 50 FRACTION NUMBER Figure 48. Sedimentation pattern of horseradish peroxidase and D. tagatose 1 ,6-diphosphate aldolase in a sucrose density gradient. Symbols: horseradish peroxidase (O) and D-tagatose 1, 6- diphosphate aldolase (o). 168 The coefficient was 3.37, using peroxidase as a standard, which corresponded to a molecular weight of 38,100. The molecular weight of 50,000 obtained on Sephadex G-100 is most likely due to anomalous behavior of the aldolase on the gel since the subunit molecular weight and the molecular weight (deter- mined in a sucrose density gradient) values are almost identical (about 37,000). Thus, the aldolase is most likely not a dimer, as might be suggested by the value of 50,000 obtained from.the Sephadex G-100 data, since its molecular weight would then be 74,000 (2 X 37,000), which leaves a difference of 24,000. However, the difference (50,000 - 37,000 = 13,000) between the molecular weight obtained by Sephadex G-100 chromatography and sucrose density gradient centrifugation can be explained as anomalous behavior on Sephadex G-100; such behavior has been observed for peroxidase, whose molecular weight is 40,000 by sucrose density gradient centri- fugation and amino acid analysis (160) but which behaves like a 49,000 molecular weight protein on Sephadex G-100 (161). Therefore, D-tagatose 1,6-diphosphate aldolase has a molecular weight of 37,000, is composed of one subunit, and has a sedimentation coefficient of about 3.4. Discussion The data presented here establish that D-tagatose 1,6- diphosphate is cleaved enzymatically in S, aureus to yield equimolar amounts of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate. D-Tagatose 1,6-diphosphate has an erythro configuration 169 of hydroxyls at the site of cleavage. An aldolase that requires the egythro configuration has been found in E, 221;; it functions to cleave both Lffuculose l-phosphate and D-ribulose 1-phosphate in the metabolism of L-fucose (182) and D-arabinose (183), respec- tively. The D-tagatose 1,6-diphosphate aldolase does not require «it '1 the erythro configuration since it also catalyzes the cleavage of D—fructose 1,6-diphosphate, a compound which possesses the threo «‘fiv‘r .' configuration. The demonstration that the enzyme catalyzes the condensation of dihydroxyacetone phosphate and D-glyceraldehyde flu! an I 3-phosphate to a mixture of the four D—ketohexose 1,6-diphosphates indicates that the aldolase also catalyzes the cleavage of D-sorbose 1,6—diphosphate and D-psicose 1,6-diphosphate. However, the aldo- lase does not cleave ketohexose l- or 6-phosphates and does not utilize L-glyceraldehyde 3-phosphate in the condensation reaction. These data demonstrate that D-tagatose 1,6-diphosphate aldolase is specific for carbon atoms 1, 5, and 6 of the ketohexose molecule, but is nonspecific for carbons 3 and 4. Such unique specificity has not been previously reported for an aldolase. However, similar nonspecificity at carbon 4 has been observed for 2-keto-4- hydroxyglutarate aldolase from liver (184), Acetobacter (185), and .§:.£2l£ (186); for 2-keto-4-hydroxypimelate aldolase from Acineto- bacter (187); and for 2-keto-4—hydroxy-4-methylglutarate aldolase from peanut cotyledons (188) and Pseudomonas (189). Although these examples provide precedent for nonspecificity at carbon 4, I know of no report of an aldolase which is nonspecific at carbon 170 3 or at both carbons 3 and 4. D-Tagatose 1,6-diphosphate aldo- lase is therefore unique among aldolases. Other distinguishing characteristics of this enzyme include its lack of a divalent metal ion requirement and its inhibition by NaBHA. These properties make D-tagatose 1,6- diphosphate aldolase a Class I aldolase (180, 181). The properties of a number of Class I and Class II (divalent metal ion requiring) aldolases have been determined and a pattern has emerged (Table 18). The only property, besides its sensitivity to NaBHA, that D-tagatose 1,6-diphosphate aldolase has in common with the typical Class I aldolase is its lack of activation by K+; its sharp pH optimum.and its sulfhydryl requirement are reminiscent of a Class II aldolase. The most diagnostic property, inhibition by NaBHa, places the enzyme in Class 1, although it occurs in a typically Class II organism. However, the occurrence of Class I aldolases in bacteria is more widespread than once thought (190-195). The Class I fructose 1,6-diphosphate aldolase from.Micrococcus aerogenes was considered unique among these enzymes since it was a monomer (MW - 33,000) (193). The D-tagatose 1,6-diphosphate aldolase from S. aureus constitutes the second example of a monomeric Class I aldolase, and both of these enzymes are of bacterial origin. The use of substrate elution has been reported for the purification of fructose 1,6-diphosphate aldolase (196) and of fructose 1,6-diphosphatases (196-198). A similar procedure was 171 .oumAmmvoIH mucuoouwln .mam moumAQmOAaHvlo.H omouonumlo .mnm "moowumH>OHAA< A .Hma one cow ouodoumwou 00mm . unoaoufiooou Hfivhfigm . m .omokuaommonumo ou o>auamoomoH .m .anawuoo mo aqum .5 .+M mA woum>fiuo< .A a .2 m.o u mHh How M .m A a .23 m.o a page Com M .e .ooo.nm u a: usesesm .ueuuafia .m 3. .m.m a cum “coo.oe u a: .N .wuamoom .w .sfiawumo we omoum .5 .+M hA wouo>wuom 002 .o .E .23 H u mHm How M .m A .z: 3 u ease new as .e .ooo.oe u a: usesese .efieeaeuuma .m 3. .o.w I cum "coo.oeH u a: .N .emmez he eeueeeeeH .H AowuouomA .omwao soouwlmaaA .Hwnnm .ummo>v momoaovaw HH momao Aomwam some» .moquona .musmam HOAwHA .mamaaomv mommaovflm H mmmHo .momoaovao HH mmeflu sea H sense we encasemeaoo. .ma mamas 172 used in this study to purify D-tagatose 1,6-diphosphate aldolase. The enzyme eluted from phosphocellulose at about 0.2 M KCl. In the presence of substrate (1 mM D-fructose 1,6-diphosphate), it eluted from phosphocellulose at about 0.12 M KCl, providing an excellent separation from the remainder of the protein, which apparently did not interact with the substrate. Homogeneous enzyme was then obtained by elution from DEAE-cellulose. SECTION 3 GENETIC EVIDENCE FOR THE PHYSIOLOGICAL SIGNIFICANCE OF THE D-TAGATOSE 6-PHOSPHATE PATHWAY 173 INTRODUCTION The previous two sections of this thesis have demonstrated that lactose- or D-galactose-grown cells of S, aureus possess enzymes for the degradation of D-galactose 6-phosphate via phos- phate esters of D-tagatose. That the three enzymes were induced by growth Of the organism only on lactose or D-galactose suggested that the D-tagatose 6-ph03phate pathway does play a role in the metabolism of these two sugars. The genetic studies presented in this section provide conclusive proof that the D-tagatose 6-phos- phate pathway functions in the metabolism of the two sugars and supports the conclusion that this is the only pathway of D-galactose 6-phosphate catabolism in S, aureus. 174 1 MATERIALS AND METHODS All materials and methods not described here were presented in the Materials and Methods of Sections 1 and 2. Bacterial Strains, Cell Growth, and Preparation of Cell Extracts Organisms The wild-type organism was §_. aureus NCTC 8511. The genealogy (199) and description of the mutants (isolated as described below) are given in Table 19. Edge Routine liquid cultivation was done in the 2% peptone broth described in Section 1 (routine broth). Induction of the enzymes of D-galactose metabolismuwas achieved by incubation of the cells in 1% peptone broth (47) containing 12 (w/v) D-galactose (induction broth). Carbohydrate utilization was determined in bromcresol purple broth (46). D-Galactose-negative mutants were isolated on the agar medium described by Mbrse and Alire (200) or modifications of it as specified below. Spontaneous revertants ‘ were isolated on the modified eosine-methylene blue agar described by McClatchy and Rosenblum (47). 175 176 .ommaovam oumAamOAafivlc.H omoumwmuln .<~me.amoy momma“: oaMAomOAaIA mmouowmula .xmowme mommumaomw mumAQMOAQIA mucuomamwln .Hmoamu “mucuoma ONHHOAmuoa moo moov .Iomq Mancuoma moufiHOAmuoa . 0mg momOuomHmen oNHHOAmuoa uo: + moom .IHou momouomamwln mouwHOAmuma .+Hmu “He won wumwaow owomaounouum wo w: mm 00 unmumemou .Nmuluum “Ha pom oumwasm :eomfiouoouum mo mm mm on m>HuHmoom mummnuum "maowumw>muAA< .omhuoomw mo ucuaowfimmm mAu cw pom: mums Ammav A .Hm um oouuaon mo mcowumvooaaooou OAHN +<~ee.awme +eaewea +Heeaeo “+664 +Heo AmmAIOOm “nuns emmu aeumu I¢~me.awma +eEewms +Hmeamo “-664 name Ammuuuum sawmm.emem Nam ewes +<~me.awes +emewes +Hmeaeu A+ueu +Hmo Ammuueum emem ammo memes +aeaueume66 “+664 +Hmo mumeuuum Hmew atmmm mam Nu +OH Qan— .voo:vdw woo woo AuouA oofiunou a“ noon» on: AoaAa .NA museum umoono mucuomamwln Anus consume mums mawonum Adda «Gd 0m0.0 0~0.0 nm¢.0 00H.0 ~0.N NA 000.0 N~0.0 00¢.0 0m0.0 n~0.0 MH.H Mommu 0m0.0 ~m0.0 wee.0 000.0 0H0.0 0m.0 Ammo meo.0 ma0.0 nm¢.0 050.0 0e0.0 ~0.H amumu hm0.0 HH0.0 hmm.0 «no.0 000.0. no.0 Rune mm0.0 mm0.0 «0N.0 wm0.0 m~0.0 mc.a Momma ac0.0 HN0.0 caH.0 m00.0 NM0.0 00.0 mmnw nqo.0 0N0.0 0~¢.0 000.0 nm0.0 0N.H mam 000.0 n~0.0 «no.0 «00.0 «no.0 0N.H Hana oauz ommHooH< ommowm AommuoaomH ommHovH< omooaM AommuoaomH moahuao hosAuoa moahnoo mannose awouum monu05021aoomem oumnowonmlo omoumwoaln Aofiououa we won muwosv mufi>wuum owwflooam nummmmmm.mm.wo mowmuum msouuo> ow mmszuoa nonhquZIaoweam OAu mo moahnoo woavnommmuuoo was hasnuma oumAamonaIA omoumwmuua 0A0 mo mofihuam .om MAA he moumuvhnonumo mo coaumnwawua .HN mAm’ a _. _ I . ,x /\ _ g g g 0.02 ./ / ‘/A A\‘\ 0.50 a ['4 a a ' / I ‘5 i -\./ /‘ ‘ 0.01 _ / . -0.25 kr‘ ‘/ 0 ’ 1 1 1 0 0 60 120 180 TIME (minutes) . Figure 51. Time course of induction of the enzymes of the D-tagatose. 6-phosphate pathway in S. aureus NCTC 8511. Cells harvested from one liter of routine broth were suspended in one liter of induction broth. Samples were withdrawn at intervals, extracts prepared, and enzyme specific activities were determined. Symbols: Dégalactose 6-phosphate isomerase (I), D-tagatose 6-phosphate kinase (A), and D-tagatose 1,6- diphosphate aldolase (I). 196 003030 0093023303 00000935 «$3on 000 "00003 30%—00:06 0000093-: gamma“. “00000803. 009300506 0000030qu «Havana £33000 330000 «mm "00033550.? 0030005 005500000 305000800 00 0.3: 03030 00 00000.3 «H03 am can»; a.“ 030:0 300 can. .Smw 0.82 050.50 ..ml 5 503500 cuwnauosoum 000009319 23 mo 008.930 05 mo 00.30203 3003.300 .mm was»; 23 gamma :3 Emma. 23 Emma... 80.0 80.0 00 $0.0 $06 0 No.0 8.0 o . _ Q _ . jo . _ u- o \ . O I \ I mood I I mN.O I I mm.o . . O O l I 08.0 n I . I 8.0 r . 1 Rd my 0 .w/ m m» 0 J 0 M; 0 d H I H I I m~0.0 m I I I WNWO WI I I MF.O m 0 l.\ O \O l o 1 omod l . I 84 r. . l 84 o .\ m .\ < _ b 80.0 L . mm; \P . mm; DISCUSSION The data presented in Sections 1 and 2 established that §, aureus has the enzymatic potential to metabolize D-galactose 6-phosphate through a new pathway termed the D-tagatose 6-phosphate pathway (Figure 3). The isomerase, kinase, and aldolase that catalyze the reactions were found to be induced by growth of the organism on lactose or D—galactose, both of which are known to be metabolized to D—galactose 6-phosphate. Since no alternative metabolic route could be demonstrated, I proposed that the D-tagatose 6-phosphate pathway is the physiologically significant route for the metabolism of D-galactose and the D-galactosyl moiety of lactose in S. aureus. The data presented in Section 3 provide confirmatory evidence for this pr0posal. Since mutants of S. aureus lacking either D-galactose 6-phosphate isomerase, D-tagatose 6-phosphate kinase, or D—tagatose 1,6-diphosphate aldolase were specifically unable to metabolize D-galactose or lactose, each of these enzymes must be essential in the metabolism of these two sugars. The demonstrated intracellular accumulation of the substrates of the missing enzymes indicated that the enzymes were indeed nonfunctional _i_t_1_ 1133 as well as in .gitrg, being undetectable in cell extracts. The isolation of spontaneous revertants that simultaneously regained the deficient enzymes and the ability to metabolize lactose and D-galactose, and 197 198 no longer accumulated intermediates of the D—tagatose 6-phosphate pathway, confirmed that the D-galactose-negative mutants were the results of single point mutations rather than deletions or multiple point mutations. The results obtained with the mutants and their spontaneous revertants demonstrate that the D-tagatose 6-phosphate pathway is physiologically significant in the metabo- lism of lactose and D-galactose in S, aureus. The mutants that were missing the D-tagatose 6-phosphate pathway enzymes nevertheless retained the corresponding enzymes of the Embden-Meyerhof pathway (D—glucose 6-phosphate isomerase, D—fructose 6-phosphate kinase, and D-fructose 1,6—diphosphate aldolase) and metabolized all carbohydrates tested except lactose and D-galactose. This is consistent with the data presented in Section 1 which indicated that the enzymes of the two pathways are distinct and separable, and that the activities of the D-tagatose 6-phosphate pathway are not merely manifestations of nonspecificity of the glycolytic enzymes. This is of interest because it has been reported that a bacterial D—glucose 6-phosphate isomerase can isomize D-galactose 6-phosphate (99), that mammalian D-fructose 6-phosphate kinase can phosphroylate D-tagatose 6-phosphate (121, 125), and that mammalian D-fructose 1,6-diphosphate aldolase can cleave D-tagatose 1,6-diphosphate (121, 125). The isolation of a mutant (strain L2) that is constitutive for the enzymes of the D-tagatose 6-phosphate pathway indicated that the three enzymes are under common genetic control. This was con- firmed by demonstrating the coordinate induction of the three enzymes 199 in the wild-type strain. The data suggest that the genes coding for the synthesis of these enzymes reside in a single operon, but this would have to be verified by genetic mapping. Genetic mapping has been done in S. aureus for lactose metabolism (71, 76), and the genes coding for the synthesis of the three proteins, EII, FIII, and phospho-B-galactosidase, are closely linked. Simoni and Roseman (75) isolated a mutant which constitutively synthesized these three proteins and was able to constitutively metabolize lactose. The existence of this and my constitutive strain, and the genetic mapping results, suggest that the genes coding for both transport and subsequent catabolism of lactose are under common genetic con- trol and are possibly linked. Thus, six proteins, EII, FIII, phospho-B-galactosidase, D-galactose 6-phosphate isomerase, D-taga- tose 6-phosphate kinase, and D-tagatose 1,6-diphosphate aldolase, are under common genetic control and may reside in a single operon. The genetic symbol gal has been used previously for enzymes of the Leloir pathway of D-galactose metabolism (208). To dis- tinguish between the genes of the Leloir pathway and of the D-tagatose 6-phosphate pathway, and bearing in mind that some organisms possess enzymes for both of these pathways (145), I propose that £§g_be used as the genetic symbol for the D—tagatose 6-phosphate pathway of D-galactose metabolism. Furthermore, I pro— pose the gene symbols EagI_for D-galactose 6-phosphate isomerase, £335 for D—tagatose 6-phosphate kinase, and Eagé for D-tagatose 1,6-diphosphate aldolase. GENERAL DISCUSSION 200 GENERAL DISCUSSION It had previously been reported that D-galactose 6-phosphate is an intermediate in the metabolism of lactose and D—galactose in Staphylococcus aureus. The data presented in this dissertation document that in S. aureus D-galactose 6-phosphate is further catabolized through a previously unknown pathway as follows: D-galactose 6-phosphate -—+-D-tagatose 6-phosphate--+ D-tagatose 1,6-diphosphate--+ dihydroxyacetone phosphate + D-glyceraldehyde 3—phosphate. This conclusion is based on (i) the demonstration of and purification of D-galactose 6-phosphate isomerase, D-tagatose 6-phosphate kinase, and D-tagatose 1,6—diphosphate aldolase; (ii) the identification of the reaction products of these enzymes; (iii) the inducibility of these enzymes by lactose and D-galactose; and (iv) the isolation of lactose- and D-galactose-negative mutants which lack these enzymes. The existence of these mutants and the apparent lack of enzymes that could function in alternative path- ways of D-galactose 6-phosphate metabolism indicate that the D-tagatose 6-phosphate pathway is the sole pathway for lactose and D—galactose metabolism in S. aureus. I also obtained data (145), which was not reported in this dissertation, indicating that several strains of Streptococcus possess enzymes of the D-tagatose 6-phosphate pathway when grown on lactose or D—galactose. 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