Carbohydrate Metabolism in Lactic Streptococci with Special Reference to Galactose
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AN ABSTRACT OF THE THESIS OF Don Royce Lee for the Doctor of Philosophy (Name) (Degree) in Microbiology presented on / 7 ; (Major) /(Dafe) Title: CARBOHYDRATE METABOLISM INLACTIC STREPTO- COCCI WITH SPECIAL REFERENCE TO GALACTOSE Redacted for Privacy Abstract approved: Dr. W. E. Sandine Phosphorylation of free galactose by lactic streptococci was mediated by an adenosine triphosphate (ATP)dependent kinase which was repressed by glucose.The phosphoenolypyruvate (PEP) phos- photransferase system (PTS) was not involved in transportof galactose, The conversion of free galactose to glucose wasalso demonstrated. A key Leloir pathway enzyme, uridinediphosphogalactose -4- epimerase, was found present with atleast 0, 66 units/mg protein in cells grown in milk or broth containing galactose orlactose.A lower level of the enzyme (0, 14units/mg protein) was found in glucose-grown cells. A modification of the standard techniquefor the assay of uridine diphosphogalactose-4-epimerase was introduced.Undialyzed crude extracts of lactic streptococcal cellscould be used for the assay but only after incorporation of 0.2 Msemicarbazide in the glycine buffer. In cell dry weight yield studies, it wasshown that glucose was the best carbohydrate for growth, andgalactose the poorest,It was, however, very difficult to show differences in growth rates of lactic streptococci using five carbohydrates.Also, in this group of organisms diauxic growth on a mixture of glucose and galactose could not be shown.Efforts were made to determine if lactic streptococci contain- ing p-D-phosphogalactoside galactohydrolase ((3 -Pgal) could utilize glucose and galactose simultaneously.Results indicated that galactose- induced cells of Streptococcus diacetilactis 18-16 could catabolize both sugars at the same time.In Streptococcus lactis 7962, however, an organism which possesses p-galactosidase (p-gal), growth curves employing both glucose and galactose revealed two distinct slopes with no lag between the time of glucose exhaustionand the time when galac- tose was utilized. In differential respirometry studies involving washed cells of S. diacetilactis 18-16, it was shown that less CO2 (< 170p,1/100 mg cell dry wt/hr. ) was evolved from galactose by glucose-induced cells, compared to that (916 p. 1 / 100 mg cell dry wt/hr.) from glucose.The inability of glucose-induced cells to catabolize galactose and lactose to the same degree as glucose was apparently due to classical catabolite repression.Lactose-induced cells evolved CO2 equally well from lactose or glucose, but the evolution of CO2 from galactose and a mixture of glucose and galactose was somewhat repressed.Galactose- induced cells required more oxygen (>1500 Ill /100 mg cell dry wt/hr.) than did glucose or lactose-induced cells (<850 [11/100 mg cell dry wt/hr.). Paper, thin layer, and column chromatographic techniques were employed in a search for galactose-6-phosphate from several wild type lactic streptococci.The compound was never detected nor was it possible to isolate it from a reaction mixture of o-nitrophenyl,3 -D, galactopyranoside-6-phosphate (ONPG-6-P) and crude cellular extracts.A search for a mutant which transported and phosphorylated lactose but was defective in cleaving the phosphorylated derivative was undertaken.Of more than 60 lactose negative mutants examined, no such mutant was found.All mutants examined lacked any detectable 3 -P -gal and also the ability to transport and phosphorylate lactose. In metabolism studies involving galactose-6-PO4, it was found that whole cells of lactic streptococci could not utilize the compound as a carbon source.Possible routes for the metabolism of this com- pound were hypothesized, and experiments were designed to test each route.The only pathway which appeared to function in its catabolism was the pentose pathway.However, it was later found that the galac- tose-6-PO4 used in these studies was contaminated with at least 0. 2 percent glucose-6-PO4 and observed activity was believeddue to the contaminant. Involvement of the PTS of transport in sugar utilization was investigated using 14C- labeled substrates andtoluene-acetone-treated cells.Substrates and phosphorylated products were separated by ion-exchange chromatography and quantitated by liquid scintillation counting.The PTS was involved in the utilization of glucose, lactose, and mannose.In controlled experiments it was determined that ATP also acted as a phosphate donor for glucose, lactose and mannose. ATP was the exclusive phosphate donor for galactose,Two transport systems appeared to act for glucose, lactose and mannose. In a survey conducted on representatives of the four major groups of streptococci, it was shownthat all lactic streptococci examined cleaved ONPG-6-P except S. lactis 7962, which cleaved 0-nitrophenyl D-galactopyranoside (ONPG).This survey also showed that all Trernbe.rs of the viridans streptococci examined possessed 6 -gal and not 13-Pgal activity. Carbohydrate Metabolism in Lactic Streptococci with Special Reference to Galactose by Don Royce Lee A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy June 1974 APPROVED: Redacted for Privacy Professor of Microbiology in charge of major Redacted for Privacy Head of Department of Microbiology Redacted for Privacy Dean of Graduate School Date thesis is presented Typed by Cheryl E. Curb for Don Royce Lee DEDICATION This thesis is dedicated to my closest friends, ACKNOWLEDGEMENTS Special gratitude is expressed to Dr. W.E. Sandine, my major professor, for his mature, Christian attitudes toward his students. Without his interest and continued help this research would never have been possible.Appreciation is also expressed to: Dr. P. R. Elliker, other staff members of the Microbiology Department, and fellow students for their knowledge and helpfulness; Members of my family for their interest and concern in my career; Faculty members of the Biology Department and Administration at East Texas State University, Commerce, Texas, for leave of absence time in which to complete this research; Tennyson and Emerson for what they left us to read and ponder; Mr. A. C. Patton for first stimulating my interest in science. Gratitude is also expressed to the Department of Microbiology, Oregon State University and the Public Health Service for assistance while completing this research.The National Science Foundation through a grant to East Texas State University partially supported the author in the beginning stages of this research. TABLE OF CONTENTS Page INTRODUCTION 1 REVIEW OF LITERATURE 6 Sugar Transport 6 The Staphylococcus aureus System 15 The Galactose Operon 19 Transport and Metabolism of Galactose 21 Galactose-6-Phosphate Metabolism 25 MATERIALS AND METHODS 29 Microorganisms 29 Chemicals 30 Media and Growth Conditions 31 Disappearance of Galactose 31 Conversion of Galactose to Glucose 32 Chromatography of Sugar Phosphates 33 Inhibition of Galactose Uptake by 6-deoxygalactose. 34 Galactose Uptake 35 Competition by Carbohydrate 35 Effect of Energy Sources 35 Effect of Metabolic Inhibitors 36 Sugar Phosphorylation Studies 36 Simultaneous Use of Glucose and Galactose by Galactose-adapted Cells of Streptococcus diacetilactis 18-16. 37 Galactose-6-Phosphate Dehydrogenase Assay 38 Galactose Oxidase Assay 39 Lactose Dehydrogenase Assay 40 Uridine Diphosphogalactose-4-Epimerase Assay 40 Inorganic Phosphate Assay 42 Assay for Galactose-l-Phosphate Uridyl Transferase 42 Differential Respirometry 43 Growth Curves 44 Mutagene sis 45 Ultraviolet Irradiation 45 Ethylmethanesulfonate (EMS) Treatment 45 Acriflavin Treatment 45 TABLE OF CONTENTS (Cont. ) Page RESULTS 47 Assay for Enzymes of Lactose Hydrolysis 47 Growth Studies 52 Differential Respirometry 65 Galactose Metabolism 75 Galactose Utilization in S. lactis C2 75 Conversion of Galactose to Glucose 76 Uridine Diphosphogalactose-4-Epimerase (UDP-gal-4-Epimerase) 80 Galactose-I-Phosphate Uridyl Transferse in Lactic Streptococci 89 Galactose Transport in S. lactis C2 89 Phosphorylation Studies 95 Metabolism of Galactose-6-Phosphate 103 DISCUSSION 105 involvement of the PTS in Carbohydrate 105 Metabolism in the Lactic Streptococci Metabolism any Transport of Free Galactose Growth Studies and Differential Respirometry Metabolism of Galactose-6-Phosphate SUMMARY 122 BIBLIOGRAPHY 124 LIST OF TABLES Table Page 1 Specific activity of p-galactosidase (p-gal) and 3, D- phosphogalactoside galactohydrolase (p-P-gal) in various streptococci. 49 2 Characteristics of selected streptococci as determined using the API system for characterizing the lactic acid bacteria. 51 3 Cell dry weight yields of S. lactis G2 grown in five different carbohydrates. 55 4 The specific activity of CO2 evolved by galactose adapted cells of S. diacetilactis 18-16 over a 30 minute period at 30 C in different carbohydrates. 64 5 C O7 evolved and 02 absorbed by cells of S. diacetilactls 13-16 when induced in galactose and returned to four different substrates. 74 Galactose oxidase activity of S. diacetilactis 18-16 in various carbohydrates. 75 7 The influence of PEP, ATP and KF on the utilization of galactose by resting, toluene-acetone-treated, galactose-grown cells of S. lactis C2. 76 8 Compounds formed from galactose by toluene- acetone-treated, resting cells of S. lactis G2 grown (six hour) on galactose. 77 9 Activity of UDP- galactose -4- epimerase in S. lactis C2 when grown in different carbohydrates to late log phase. 86 10 Activity of UDP-galactose-4-epimerase from various lactic streptococci grown to late log phase in galactose broth. 88 LIST OF TABLES (Cont. ) Table Page 11 Galactose transport in S. lactis C2 and the effect of competing carbohydrates. 90 12 Galactose transport in S. lactis C2 and the effect of metabolic inhibitors. 92 13 Galactose transport in S. lactis C2 and the effect of energy sources, 93 14 Role of ATP and PEP in phosphorylation of various sugars by S. lactis C2 induced on five different carbon sources. 96 15 Phosphorylation of four growth substrates by S cremoris Hp and a S. cremoris Hp Lac- mutant. 99 16 Percent phosphorylation of various carbohydrates by S. lactis C2 mutants. 100 17 Characteristics of some selected lac- mutants of S.