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Carbohydrate transport and metabolism in resting suspension of clostridium perfringens type A Groves, David John 1968

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CARBOHYDRATE TRANSPORT AND METABOLISM IN RESTING f SUSPENSIONS OF CLOSTRIDIUM PERFRINGENS TYPE A BY DAVID JOHN GROVES B.Sc. (Biochemistry) , Un ivers i ty of B r i t i s h Columbia, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Microbio logy We accept th is thes is as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1968 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C olumbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and Study. i f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department or by nils r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f M icrobiology The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada 6th September 1968 ABSTRACT Su s p e n s i o n s o f C. p e r f r i n g e n s , when grown on a peptone-f r e e , s e m i - d e f i n e d medium, have been shown t o remain r e s i s t a n t t o a u t o l y s i s f o r extended p e r i o d s o f t i m e . The s t a b i l i t y o f t h e s e s u s p e n s i o n s has been compared w i t h t h a t o f c e l l s grown on complex media. E x t r a c t s o f c e l l s grown on t h i s s e m i - d e f i n e d medium were found t o c o n t a i n a l l o f t h e enzymes o f the Embden-Meyerhof pathway o f g l y c o l y s i s , i n a d d i t i o n t o l a c t i c a c i d dehydrogenase and t h e p y r u v a t e - c l a s t i c s y s t e m , but no e v i d e n c e o f g l u c o s e - 6 -phosphate dehydrogenase a c t i v i t y c o u l d be d e m o n s t r a t e d . E v i d e n c e has been p r e s e n t e d f o r t h e i m p l i c a t i o n o f t h e Embden-Meyerhof pathway as t h e major pathway o f g l u c o s e d e g r a d a t i o n by t h i s o r g a n i s m . R e s t i n g s u s p e n s i o n s o f C. p e r f r i n g e n s were shown t o t r a n s p o r t r a d i o a c t i v e g l u c o s e and mannose, but not o t h e r c a r b o h y d r a t e s by a common mechanism and a c c u m u l a t e g l u c o s e t o c o n c e n t r a t i o n s s e v e r a l hundred t i m e s t h o s e found i n t h e e x t e r n a l medium. The t r a n s p o r t system was found t o be an e n z y m a t i c , e nergy-dependent, t e m p e r a t u r e - s e n s i t i v e , and h i g h l y s p e c i f i c mechanism wh i c h was s a t u r a t e d a t h i g h s u b s t r a t e concentrat ions. The carbohydrate was found to be accumulated as an equi l ibr ium mixture of phosphorylated hexoses. The phosphorylation mechanism involved in accumulation was demonstrated to be other than the soluble hexo-kinase. TABLE OF CONTENTS Page INTRODUCTION LITERATURE REVIEW . . . 2 I. Metabolism of Carbohydrates and Amino Acids . . . 2 1 . Metabolism of carbohydrates 2 2 . Metabolism of amino acids . 7 I I . The Transport of Metabol i tes . 9 1 . Metabol i te transport in mammals . . . . . . 9 2 . Metaboli te transport in bacter ia 9 a. Amino acid transport 9 b. Carbohydrate transport 1 0 i . Galactose and galactos ides. . . . . . 1 0 i i . Glucose 1 1 i i i . Carbohydrate accumulation mechanisms and components. 1 2 3 . Carbohydrate transport in yeast'and . fungi . . . 1 3 MATERIALS AND METHODS . . . . . 1 6 I. Organisms and Media 1 6 1 . Complex medium . . . 1 6 2 . Semi-defined medium 16 3 . Stock cultures 1 8 h. Growth of inoculum. . . 1 8 II. Growth Conditions . 1 9 Table of Contents (Continued) Page I I I . Stab i 1 i ty of Ce l1 Suspens ions 2 0 IV. Enzyme Assays . . . 2 1 1 . Ce l l - f r ee extracts . . 2 1 2 . Enzyme assays . . . . . . . . . . . 2 3 V. Carbohydrate Transport 2 9 1 . Preparation of anaerobic c e l l suspensions . . 2 9 \k 2 . Assays for C-carbohydrate incorporation . . 3 0 3 . Determination of the nature of the accumulated C-carbohydrate . . . . . . 3 2 VI. Assay of Radioact iv i ty . . . . . . . . . 3k 1 . Mi 1 1 ipore f i I ters . . 3k 2 . Chromatograms and electrophoretograms . . . 3k VI I. Chromatography and Electrophoresis . . . . . 3 5 VII I . Ana ly t ica l and Preparative Techniques . . . . 3 6 1 . Dry weight . . . . 3 6 2 . Protein . . 3 6 3 . Optical density measurements 3 6 k. Dephosphorylation of carbohydrates . . . . 3 7 5 . Glucose . . . . . . . . . . . . . 3 7 IX. Chemicals, Enzymes and Substrates. . . . . . 3 7 RESULTS AND DISCUSSION 3 9 I. Growth and Resting Cel l Suspensions . . . . . 3 9 1 . Growth curves . . kO vi Table of Contents (Continued) Page 2. Stab i 1 i ty of eel 1 suspens ions 43 I I. G l yco l y t i c Enzyme Assays. 54 -.III. Carbohydrate Transport . . . . . . . . . 64 1. U t i l i z a t i o n of carbohydrates by C_. perf r i ngens 65 14 2. Transport of C-carbohydrates. . . . . . 65 (i) Transport of ^C-g lucose . 6 5 ( i i ) Transport of C-carbohydrates other than glucose 6 7 ( i i i ) Temperature dependence of C-glucose transport . . . . . . . . . 69 (iv) Energy requirement of the transport of glucose . . . . . . . . . . . 7 1 (v) Kinet ics of accumulation of C-glucose and C-mannose. . . . . . . lb (vi) S p e c i f i c i t y of the transport of glucose and mannose . . . . . . . . 82 3. The pooling of C-glucose . . . . . . . 92 ( i) Pool capacity : . . . . . . . . . 92 ( i i ) Internal concentration . . . . . . . 97 14 ( i i i ) Nature of the pooled C . . . . . . 99 4. Mechanism of the accumulation of glucose . . 102 GENERAL DISCUSSION. . . 1 0 4 Table of Contents (Continued) Page LITERATURE CITED . . . . . . . . . . . . . 108 v i i i LIST OF TABLES Page T a b l e I. The a u t o l y s i s o f e e l 1 s u s p e n s i o n s o f C. p e r f r i n g e n s under s t a r v a t i o n c o n d i t i o n s 49 T a b l e I I . The s t a b i l i t y o f c e l l s u s p e n s i o n s o f £. p e r f r i n g e n s under s t a r v a t i o n c o n d i t i o n s when p r e v i o u s l y grown i n a c h e m i c a l l y d e f i n e d medium 50 T a b l e I I I . S p e c i f i c a c t i v i t i e s o f g l y c o l y t i c enzymes i n e x t r a c t s o f C_. p e r f r i n g e n s . The e x t r a c t s were p r e p a r e d from c e l l s grown on s e m i -d e f i n e d medium p l u s 0.6% g l u c o s e , e x c e p t where i n d i c a t e d o t h e r w i s e 57 T a b l e IV. Growth o f £. p e r f r i n g e n s w i t h v a r i o u s c a r b o h y d r a t e s as t h e s o u r c e o f c a r b o n 66 T a b l e V. S a t u r a t i o n k i n e t i c s o f t h e g l u c o s e -mannose t r a n s p o r t system. E s t i m a t e d v a l u e s o f and V m from t r i a l s i n F i g u r e s 15 and 17 81 T a b l e V I . A c c u m u l a t i o n o f c a r b o h y d r a t e s and c o m p e t i t i v e i n h i b i t i o n o f t h e r a t e o f u p take o f ' ^ C - g l u c o s e (8 x 10"'? mM, 1.4 uc/umole) and ^C-mannose (1.6 x 10"2 mM, 1.4 u c / u m o l e ) , by a 100-fold e x c e s s o f t h e s e c a r b o -h y d r a t e s 88 ix LIST OF FIGURES Page F ig . 1. Growth of C. perfringens in complex medium plus 0.7% glucose. 41 F ig . 2. Growth of £ . per f r i ngens i n complex medium plus 0.7% glucose 42 F ig . 3. Growth of £ . per f r i ngens in secondary cul ture in semi-defined medium plus 0.7% glucose 44 F ig . 4. Growth of C. perfringens in semi-defined medium plus 0.7% glucose 45 F i g . 5. S t a b i l i t y of eel 1 suspensions of C. perfringens grown with 0.7% glucose 47 F i g . 6. The rate of l ys i s of suspensions <i>f c e l l s grown in complex medium 52 F i g . 7. The a c t i v i t y of NADH^-oxidase in aerobic and anaerobic eel 1-free extracts of C. perfringens 58 F i g . 8. The a c t i v i t y of 3-phospho-glyceraldehyde dehydrogenase in eel 1-free extracts of £ . perfr? ngens < 60 F ig . 9. A c t i v i t y of the pyruvate-c las t ic system in c e l l - f r e e extracts of C_. perf r i ngens 62 F i g . 10. Ear ly time course of uptake by whole ce 11 s of C_. perf r i ngens 68 F i g . 11. Total uptake of rad ioac t i v i t y by c e l l s of C. per f r i ngens 70 F i g . 12. Total incorporation of rad ioac t i v i t y by c e l l suspensions 72 14 F i g . 13. Saturation k ine t ics of C-glucose incorporation by C. per f r i ngens 75 X Li st of Figures (Continued) Page 1 4 F i g . 14.. Saturation k inet ics of C-glucose incorporation by C. per f r i hgens 76 14 F i g . 15- Saturation k inet ics of C-glucose incorporation by C. per f r i hgens 77 14 F i g . 16. Saturation k ine t ics of C-mannose incorporation by C_. perf r i ngens 79 14 F ig . 17. Saturation k ine t ics of C-mannose incorporation by C_. perf r i ngens 80 F i g . 18. Competition for glucose uptake in C_. per f r i hgens A and B 83 F i g . 18. Competition for glucose uptake in C^ _ perfringens C and D 84 F i g . 19. Competition for mannose uptake in C_. per f r i ngens A and B 86 F i g . 19. Competition for mannose uptake in C_. perf r i tigens C and D 87 F ig . 20. Formation of a cold TCA so lub le , radioact ive poo l , by c e l l suspensions 93 F ig . 21. Formation of a cold TCA so lub le , radioact ive pool , by c e l l suspensions 94 F i g . 22. Total incorporation of rad ioac t i v i t y by c e l l suspensions 96 ACKNOWLEDGEMENTS My s i n c e r e g r a t i t u d e i s extended t o Dr. A.F. G r o n l u n d f o r h e r s u p e r v i s i o n and encouragement o f t h e r e s e a r c h , and f o r her h e l p and c o n s t r u c t i v e c r i t i c i s m i n the p r e p a r a t i o n o f the m a n u s c r i p t . I would l i k e t o thank Dr. J.J.R. Campbell f o r h i s encouragement and f o r e d i t i n g t h e t h e s i s . I would a l s o l i k e t o thank my w i f e H a l l i e f o r her c o n s i d e r a t i o n and s u p p o r t d u r i n g t h e r e s e a r c h , and p a r t i c u l a r l y f o r her many hours s p e n t i n the p r e p a r a t i o n o f t h e t h e s i s . L a s t l y , I o f f e r my thanks t o my f e l l o w - s t u d e n t s , e s p e c i a l l y Dr. W.W. Kay, f o r h e l p i n my e x p e r i m e n t s , and t o Mrs. R i t a Rosbergen f o r the t y p i n g o f t h e t h e s i s . INTRODUCTION The m e t a b o l i s m o f g l u c o s e , the major s o l u b l e c a r b o h y d r a t e i n mamma 1 ian s y s t e m s , by an o r g a n i s m p a t h o g e n i c t o man, i s o f o b v i o u s i n t e r e s t . W h i l e t h e s e r i o u s n e s s o f the i n f e c t i o n by C . p e r f r i n g e n s has been g r e a t l y d i m i n i s h e d i n r e c e n t y e a r s by improved c l i n i c a l t e c h n i q u e , c a s e s o f " g a s - g a n g r e n e " have not y e t become unknown. T h i s d a n g e r , p l u s t h e i n c r e a s i n g r e c o g n i t i o n o f £ . p e r f r i n g e n s as a c a u s a t i v e agent i n f o o d p o i s o n i n g , have m o t i v a t e d numerous a t t e m p t s t o d e f i n e the p h y s i o l o g y and m e t a b o l i s m o f t h i s o r g a n i s m . In a d d i t i o n , the c h a r a c t e r i z a t i o n o f t h i s o r g a n i s m as m i c r o - a e r o t o l e r a n t in a genus t h a t c o n t a i n s o n l y o b l i g a t e a n a e r o b e s w a r r a n t s i n t e n s i v e i n v e s t i g a t i o n o f i t ' s m e t a b o l i s m . An i n t e g r a l p a r t o f t h e d e g r a d a t i o n o f a c a r b o h y d r a t e i s t h e i n i t i a l p a s s a g e o f the c a r b o h y d r a t e t h r o u g h t h e s e m i -permeab le membrane t o e n t e r the c e l l . However , f o r m e a n i n g f u l s t u d i e s o f t r a n s p o r t , u n i f o r m , r e s t i n g s u s p e n s i o n s o f the o r g a n i s m a r e e s s e n t i a l . An i n s i g h t i n t o the p r e p a r a t i o n o f such s t a b l e s u s p e n s i o n s c o u l d a l s o p r o v i d e i n f o r m a t i o n about t h e w i d e s p r e a d s u r v i v a l o f t h e o r g a n i s m in the a u s t e r e e n v i r o n m e n t o f the s o i 1 . A knowledge o f the m e t a b o l i c f a t e o f the t r a n s p o r t e d g l u c o s e i s o f g r e a t t h e o r e t i c a l i n t e r e s t and p o s s i b l y o f prac t ica l value, due to the pathogenic nature of the organism. It was the object of th is invest igat ion to devise a method of preparing rest ing c e l l suspensions of C. perfr ingens, an anaerobic saccharo ly t ic pathogen, to study the metabolism of glucose by th is organism, and to elucidate the system of entry of carbohydrates into the c e l l s . 2 LITERATURE REVIEW The i m p o r t a n c e o f C l o s t r i d i u m p e r f r i n g e n s as a p a t h o g e n , both in gas gangrene and i n f o o d p o i s o n i n g , and i t s g e n e r a l i m p o r t a n c e as a G r a m - p o s i t i v e a n a e r o b e , have r e s u l t e d i n f a i r l y e x t e n s i v e s t u d i e s o f t h e m e t a b o l i s m o f the o r g a n i s m . I. M e t a b o l i s m o f C a r b o h y d r a t e s and Amino A c i d s 1. M e t a b o l i s m o f c a r b o h y d r a t e s The d e g r a d a t i o n o f g l u c o s e by C . p e r f r i hgens has been s t u d i e d by many w o r k e r s . F o r r e a s o n s t h a t w i l l become a p p a r e n t , much o f t h i s work on c a r b o h y d r a t e d i s s i m i l a t i o n was a s s o c i a t e d w i t h the m e t a l l i c - i o n r e q u i r e m e n t s f o r growth and f o r t o x i n p r o d u c t i o n . It has been known s i n c e 1932 ( H a s t i n g s and McCoy) t h a t F e + + must be added t o m i l k medium i n o r d e r t o i n s u r e the gaseous s tormy f e r m e n t a t i o n t h a t i s c h a r a c t e r i s t i c o f the o r g a n i s m . £ . p e r f r i ngens has l o n g been r e c o g n i z e d as b e i n g a s a c c h a r o l y t i c , h e t e r o f e r m e n t a t i v e o r g a n i s m p r o d u c i n g l a c t a t e , a c e t a t e , b u t y r a t e , e t h a n o l , CO^ and H^ f rom g l u c o s e (Fr iedemann and K m i e c i a k , 1932). Pappenheimer and Shaskan (1944) l o o k e d more c l o s e l y i n t o the f e r m e n t a t i o n p r o d u c t s o f g l u c o s e , and the e f f e c t s o f the F e + + c o n c e n t r a t i o n s . As t h e F e + + i on c o n c e n t r a t i o n o f the medium a p p r o a c h e d z e r o , l a c t i c a c i d p - roduct ion a p p r o a c h e d 2 moles o f l a c t i c a c i d per mole o f g l u c o s e 3 fermented. Thus low Fe concentration shi f ted the normal hetero-fermentation to a homofermentative production of l a c t i c ac id . These workers also demonstrated the coincidence of optimum Fe concentration for minimum lactate production, maximum growth, ++ and maximum toxin production. Thus, Fe is required for the production of acetate and butyrate from pyruvate, the las t precursor common to both the v o l a t i l e fa t ty acids and to l a c t i c a c i d . Pappenheimer and Shaskan (I3kk) a lso demonstrated that while i ron-def ic ien t medium gave homofermentative g l y c o l y s i s , i ron- f ree medium produced by aa ' - d i p y r i d y l treatment, would not support growth. On th is basis they assumed that iron played a ro le in the actual g l y co l y t i c pathway to pyruvate as wel1 as in the hetero-fermentative steps beyond pyruvate. Bacon (19^9) indicated that carbon monoxide would a lso cause a sh i f t from heterofermentation to homofermentation, and other workers (Lerner and P icke t t , 19^5; Kubowitz, 193^ ) showed that high cyanide concentrations caused an inh ib i t i on of gas production of other C l o s t r i d i a . It is known that aa ' - d i p y r i d y l a f fects inorganic i ron , whi le CO and CN bind heme i ron. While invest igat ing the dual function of i ron , Bard and Gunsalus (1950) found that free ionic iron was required for fructose-1,6-di-phosphate a ldo lase. Removal of F e + + from eel 1 free extracts ++ ++ completely inhib i ted aldolase a c t i v i t y and addit ion of Fe or Co reactivated the enzyme. They concluded that the presence of aldolase a c t i v i t y indicated that the Embden-Meyerhof pathway of glucose degradation was present. In add i t ion , they suggested that the 4 enzymes 3-phosphoglyceraIdehyde dehydrogenase, triose-phosphate isomerase, and ethanol dehydrogenase were operative In the organism; however, they did not present de f i n i t i ve evidence. In a ser ies of pub l ica t ions , Shankar and Bard (1952, 1955a, 1955b) studied the ef fects of Mg and Co on the growth of £ . per- f r i ngens. By adding various ions to deionized medium (1952), they demonstrated, as Webb (1948) had done, that Mg def ic iency (1955a) produced filamentous c e l l s . They also showed that , with Mg + + -de f i c i en t c e l l s , there was a considerable decrease in gas evolut ion during growth. In an unsuccessful attempt to demonstrate a def ic iency of Embden-Meyerhofr pathway enzymes, they qua 1 i ta t ive ly indicated the existence of hexokinase, phosphohexoseisomerase, phosphofructokinase, a ldo lase, and 3-phosphoglyceraldehyde dehydrogenase. In add i t ion , they showed a sh i f t to homolactate fermentation from heterofermentation upon addit ion of excess C o + + , thus indicat ing that C o + + interfered with formation of the heterofermentative system (1955b). Ivanov (1954) examined the hexokinase of C. perfringens in c e l l - f r e e extracts and c e l l suspensions. In ex t rac ts , the pH optimum ++ ++ was shown to range from 7.0 - 8.0, and Mg and Co were found to act ivate the enzyme. The a b i l i t y of £ . perfringens to grow under higher oxygen tensions than other C l o s t r i d i a is wel l documented (Fredette, P lan ts , and Roy, 1967; Pr£vot, 1966). Hi rano e_t al_. (1954) compared the aerobic and anaerobic degradation of glucose by £ . perfringens and Escherichia  co1 i , and found that while sodium azide stimulated aerobic degradation of glucose by £ . co1 i , i t prevented the accumulation of pyruvate 5 f rom g l u c o s e by £ . p e r f r i n g e n s . Bard (1952) a l s o l o o k e d a t t h e r e s p i r a t i o n o f g l u c o s e and found t h a t whole c e l l s degraded g l u c o s e i n the p r e s e n c e o f oxygen by a mechanism showing a d o u b l e dependence on i r o n , s i m i l a r t o t h a t o f t h e f e r m e n t a t i o n o f g l u c o s e t o a c e t a t e . A t t e m p t s t o d e f i n e t h e ma jor pathways o f hexose and p e n t o s e m e t a b o l i s m , u s i n g r a d i o a c t i v e c a r b o h y d r a t e s l a b e l l e d a t s p e c i f i c c a r b o n a t o m s , have been c a r r i e d o u t . P a e g e , G i b b s , and Bard (1956) 14 used C - g l u c o s e p r e p a r a t i o n s l a b e l l e d a t the C - 1 , C-2, C-6 and C-3 and C4 p o s i t i o n s r e s p e c t i v e l y and d e g r a d e d t h e end p r o d u c t s o f g l u c o s e d e g r a d a t i o n t o show the f a t e o f each c a r b o n . They c o n c l u d e d t h a t the ma jor r o u t e o f g l u c o s e d i s s i m i l a t i o n was indeed v i a the Embden-Meyerhof pathway. However , t h e s p e c i f i c a c t i v i t y o f the e t h a n o l p r o d u c e d was lower than t h a t o f a c e t a t e , i n d i c a t i n g t h a t 12 C - e t h a n o l f rom some o t h e r s o u r c e had d i l u t e d the l a b e l f rom the C - 1 , C-2 and C-6 o f g l u c o s e . L a t e r , however , C y n k i n and G i b b s (1958) used c e l l s grown on p e n t o s e s , and were a b l e t o show t h a t t h e e t h a n o l and a c e t a t e p r o d u c t s had n e a r l y i d e n t i c a l s p e c i f i c a c t i v i t i e s . E i t h e r t h e Embden-Meyerhof pathway i s the major pa thway , 12 o r growth on a p e n t o s e " t u r n s o f f " t h e a d d i t i o n a l s o u r c e o f C -e t h a n o l . C y n k i n and D e l w i c h e (1958) s t u d i e d t h e enzymes o f r i b o s e d i s s i m i l a t i o n i n c e l l - f r e e e x t r a c t s o f £ . p e r f r i h g e n s . It had p r e v i o u s l y been shown ( C y n k i n and G i b b s , 1957) t h a t t h e o r g a n i s m c o u l d f e rment r i b o s e , but not x y l o s e o r a r a b i n o s e , t o p r o d u c t s s i m i l a r t o t h o s e o f g l u c o s e f e r m e n t a t i o n . C y n k i n and D e l w i c h e were a b l e t o d e m o n s t r a t e t h e e x i s t e n c e o f r i b o k i n a s e , p h o s p h o p e n t o i s o m e r a s e and 6 the p r o d u c t i o n o f hexose -monophospha te f rom r i b o s e-5 - p h o s p h a t e . The p r o d u c t i o n o f hexose -monophospha te f rom the p h o s p h o r y l a t e d p e n t o s e i n d i c a t e d , they c l a i m e d , the e x i s t e n c e o f t r a n s - a l d o l a s e and t r a n s - k e t o l a s e a c t i v i t y . No g l u c o s e-6 - p h o s p h a t e d e h y d r o g e n a s e a c t i v i t y , e i t h e r d i - o r t r i - p h o s p h o - p y r i d i n e n u c l e o t i d e (NAD o r NADP) l i n k e d was found in e i t h e r g l u c o s e o r p e n t o s e grown c e l l s , i n d i c a t i n g t h a t the hexose -monophospha te pathway o f g l u c o s e d e g r a d a t i o n was not p r e s e n t (Wood 1961). In a r e l a t e d o r g a n i s m , C . t e t a n i , w h i c h i s not n o r m a l l y c o n s i d e r e d t o be s a c c h a r o l y t i c , an i n d u c i b l e g l u c o k i n a s e a c t i v i t y and the r e q u i r e d enzymes t o reduce NAD w i t h f r u c t o s e 1,6 -diphosphate as s u b s t r a t e were found and t h i s i m p l i c a t e d t h e p r e s e n c e o f t h e Embden-Meyerho f / pathway. O x i d a t i o n o f reduced NAD (NADh^) by c e l l - f r e e e x t r a c t s w i t h p y r u v a t e as s u b s t r a t e s u g g e s t e d t h a t e i t h e r a l a c t i c d e h y d r o g e n a s e o r a p y r u v a t e d e c a r b o x y l a s e was o p e r a t i v e a l s o ( M a r t i n e z and R i t t e n b e r g , 1959). In the s a c c h a r o l y t i c , t h e r m o p h i l i c , o b i i g a t e a n a e r o b e , C_. t h e r m o - s a c c h a r o l y t i c u m , a l l o f the enzymes o f the Embden-Meyerhof pa thway , as w e l l as t h e p y r u v a t e - c l a s t i c s y s t e m , have been d e m o n s t r a t e d ; ( L e e and O r d a l , 1967). The mechanism o f the h e t e r o f e r m e n t a t i v e breakdown o f p y r u v a t e has been s t u d i e d e x t e n s i v e l y and rev iewed by s e v e r a l w o r k e r s ( K o e p s e l l and J o h n s o n , 19^ 3; M o r t l o c k , V a l e n t i n e , and W o l f e , 1959; V a l e n t i n e , 1964). The non-heme i r o n c o n t a i n i n g component o f the p y r u v a t e - c l a s t i c o r p h o s p h o r o - c l a s t i c s y s t e m , f e r r e d o x i n , has been i d e n t i f i e d i n and p u r i f i e d f rom s e v e r a l d i f f e r e n t C l o s t r i d i a 7 and o t h e r a n a e r o b e s wh ich p r o d u c e m o l e c u l a r h y d r o g e n . F e r r e d o x i n has a l s o been found in C_. a c i d i - u r i c i w h i c h does not p r o d u c e h y d r o g e n d u r i n g growth ( V a l e n t i n e , 1964). The p h y s i c a l and c h e m i c a l p r o p e r t i e s o f f e r r e d o x i n a r e w e l l e s t a b l i s h e d and have been e x c e l l e n t l y rev iewed ( M a l k i n and R a b i n o w i t z , 1967). The p y r u v a t e - c l a s t i c sys tem i s c o n s i d e r e d t o be the g e n e r a l mechanism o f p y r u v a t e c l e a v a g e Tii ' C l o s t r i d i a - and p r o d u c e s c a r b o n d i o x i d e , hydrogen and a c e t y l - C o A , the l a t t e r b e i n g used s u b s e q u e n t l y f o r s y n t h e s i s o f a c e t a t e and b u t y r a t e ( V a l e n t i n e and W o l f e , 1963). N e i t h e r the p y r u v a t e - c l a s t i c s y s t e m , nor the p r e s e n c e o f f e r r e d o x i n have been d e m o n s t r a t e d in C_. p e r f r i n g e n s . A NADH^ o x i d a s e has been i s o l a t e d and c h a r a c t e r i z e d f rom C . p e r f r i n g e n s ( D o ! i n , 1959a) and found t o be c y a n i d e i n s e n s i t i v e and i n d e p e n d e n t o f ^2^2 as an i n t e r m e d i a t e . The enzyme was shown t o c a t a l y z e a f o u r e l e c t r o n r e d u c t i o n o f U 2 ~ ^ 2 ^ u s ' n 9 NADr^. A l t h o u g h C l o s t r i d i a have not been shown to c o n t a i n c y t o c h r o m e s , D o l i n showed cy tochrome c r e d u c t a s e a c t i v i t y (Dolin,1959b) i n the p u r i f i e d N A D r ^ - o x i d a s e p r e p a r a t i o n . 2 . M e t a b o l i s m o f amino a c i d s Most p r o t e o l y t i c C l o s t r i d i a fe rment amino a c i d s in a c o u p l e d -o x i d a t i o n - d e a m i n a t i o n r e a c t i o n known as t h e S t i c k l a n d r e a c t i o n (N isman, 1954). However , most s a c c h a r o l y t i c C l o s t r i d i a , and p a r t i c u l a r l y C . p e r f r ? h g e n s , do not use the S t i c k l a n d mechanism (Nisman, 1954). 8 Woods and T r i m (19^2) showed t h a t C . p e r f r i n g e n s was a b l e t o degrade o n l y 5 amino a c i d s . S e r i n e , c y s t i n e , c y s t e i n e and t h r e o n i n e were m e t a b o l i z e d t o p r o d u c e C C ^ , NH^ and H^, w h i l e a r g i n i n e p r o d u c e d NH^ and H^, but no C 0 2 > In 1952, T y t e l1 d e m o n s t r a t e d the d e g r a d a t i o n o f a r g i n i n e t o o r t h i n i n e , CO^ and NH^ by t h e o r g a n i s m . The g l u t a m i n a s e o f C_. p e r f rihgens has been p u r i f i e d and c h a r a c t e r i z e d (Hughes and W i l l i a m s o n , 1952). E v i d e n c e f o r t r a n s a m i n a s e a c t i v i t y has been p r e s e n t e d by H i c k s (195*0, who found t h a t a s p a r t a t e and ct -keto g l u t a r a t e p r o d u c e d a l a n i n e and C O ^ , but no •y-ami n o b u t y r a t e as would be e x p e c t e d i f a s i m p l e t r a n s a m i n a t i o n , f o l l o w e d by d e c a r -b o x y l a t i o n had o c c u r r e d . It was c o n c l u d e d t h a t a s p a r t a t e and a - k e t o g l u t a r a t e formed o n l y c a t a l y t i c amounts o f o x a l o a c e t a t e and g l u t a m i c a c i d , and t h a t a s p a r t a t e and p y r u v a t e r e s u l t e d from d e -c a r b o x y l a t i o n o f the t r a n s a m i n a t e d o x a l o a c e t a t e . T h i s p r o v i d e d o x a l o a c e t a t e , t o keep t h e s y s t e m in o p e r a t i o n , as w e l l as t h e ma jor p r o d u c t , a l a n i n e . The r e d u c t i o n o f n i t r a t e t o n i t r i t e by washed c e l l s u s p e n s i o n s , w i t h g l u c o s e o r e t h a n o l as s u b s t r a t e , has been shown to o c c u r and t o be i n h i b i t e d by KCN, i o d o a c e t a m i d e , and u rea ( H i c k s , 1965). Fuchs and Bonde (1957) s t u d i e d the s u l p h u r m e t a b o l i s m o f C . p e r f r i n g e n s and have found t h a t n e i t h e r s u l p h a t e , s u l p h i t e o r t h i o s u l p h a t e c o u l d s u p p l y the o r g a n i s m ' s s u l p h u r r e q u i r e m e n t s . C y s t e i n e , c y s t i n e o r h o m o c y s t e i n e were d e m o n s t r a t e d t o s a t i s f y t h e s e r e q u i r e m e n t s , d e p e n d i n g on the s t r a i n u s e d , w h i l e s u l p h a t e , t h i o s u l p h a t e , c y s t e i n e , c y s t i n e and g l u t a t h i o n e were shown to be degraded by the o r g a n i s m s t o p r o d u c e H S . 9 I I. The T r a n s p o r t o f M e t a b o l i t e s The i m p o r t a n c e o f the p r o c e s s by wh ich m i c r o o r g a n i s m s pass m e t a b o l i t e s t h r o u g h t h e i r membranes, and the p r o c e s s e s by which t h e s e membranes can m a i n t a i n t h e c e l l ' s i n t e r n a l e n v i r o n m e n t , has met w i t h i n c r e a s i n g r e c o g n i t i o n w i t h i n the l a s t f i f t e e n y e a r s . Systems have been d e s c r i b e d w h i c h a r e h i g h l y s p e c i f i c and w h i c h a r e a b l e t o c o n c e n t r a t e m e t a b o l i t e s t o l e v e l s s e v e r a l thousand t i m e s t h a t o f the e x t e r n a l e n v i r o n m e n t . 1. M e t a b o l i t e t r a n s p o r t in mammals The numerous s t u d i e s o f p e r m e a t i o n o f membranes o f v a r i o u s mammalian c e l l s by amino a c i d s , i o n s , and c a r b o h y d r a t e s have been most a d e q u a t e l y rev iewed ( W i l b r a n d t and R o s e n b e r g , 1961; Q u a s t e l , 1965; A l b e r s , 1967). 2. M e t a b o l i t e t r a n s p o r t in b a c t e r i a a . Amino a c i d t r a n s p o r t The t r a n s p o r t o f amino a c i d s by m i c r o o r g a n i s m s has been e x t e n s i v e l y s t u d i e d and r e v i e w e d ( H o l d e n , 1962; Kepes and C o h e n , 1962; B r i t t e n and M c C l u r e , 1962). The w o r k e r s o f the P a s t e u r I n s t i t u t e (Cohen and R i c k e n b e r g , 1956) p a r a l l e l e d t h e i r s t u d i e d on the t r a n s p o r t o f g - g a l a c t o s i d e s w i t h i n v e s t i g a t i o n s i n t o t h e n a t u r e o f amino a c i d t r a n s p o r t . They d e v e l o p e d a model t h a t a c c o u n t e d f o r the c o n c e n t r a t i o n o f amino a c i d s in a s p e c i f i c , e n e r g y - d e p e n d e n t p r o c e s s . B r i t t e n and M c C l u r e (1962) d e f i n e d s e v e r a l s p e c i f i c sys tems f o r amino a c i d c o n c e n t r a t i o n by E.: c o l i . R e c e n t l y , the t r a n s p o r t and a c c u m u l a t i o n o f amino a c i d s by Pseudomonas a e r u g i n o s a was shown t o be m e d i a t e d by s e v e r a l permeases s p e c i f i c f o r s t r u c t u r a l " f a m i l i e s " o f amino a c i d s (Kay , 1968). As w e l l , t h e movement o f amino a c i d s a c r o s s t h e c e l l membrane was shown t o be e n e r g y - i n d e p e n d e n t , w h i l e t h e a c c u m u l a t i o n p r o c e s s on the i n s i d e o f the membrane i s found t o be an e n e r g y - d e p e n d e n t p r o c e s s . b. C a r b o h y d r a t e t r a n s p o r t i . G a l a c t o s e and g a l a c t o s i d e s The g a l a c t o s i d e p e r m e a t i o n sys tem o f E_. c o l i i s one o f the most w e l l d e f i n e d , bo th as t o s p e c i f i c i t y , c o n t r o l , and mechanism. The w o r k e r s o f the P a s t e u r I n s t i t u t e ( R i c k e n b e r g , C o h e n , B u t t i n and Monod, 1956) showed the e x i s t e n c e o f an i n d u c i b l e s y s t e m 14 t h a t s e l e c t i v e l y t r a n s p o r t e d C - t h i o m e t h y l - g - D - g a l a c t o p y r a n o s i d e 14 ( C-TMG) i n t o the c e l l , i n a p r o c e s s t h a t f o l l o w e d s a t u r a t i o n k i n e t i c s and was l a b i l e t o enzyme i n h i b i t o r s . Because o f t h e s e e n z y m e - l i k e p r o p e r t i e s , they c o i n e d t h e word " p e r m e a s e " t o d e s c r i b e the t r a n s p o r t f u n c t i o n . Kepes (1957) d e m o n s t r a t e d t h e i e n e r g y -dependence o f t h e p r o c e s s and d i d k i n e t i c s t u d i e s ( K e p e s , 1960) on t h e - e x i t ' : and e n t r a n c e p r o c e s s e s , u s i n g the r a p i d M i l l i p o r e f i l t r a t i o n t e c h n i q u e . In l a t e r work by Koch (1964), c e l l s p r e l o a d e d w i t h TMG a t a low t e m p e r a t u r e , t h e n warmed t o a l l o w e x i t , were shown t o c o n t a i n an e x i t p r o c e s s t h a t was f a c i l i t a t e d by energy i n h i b i t i o n , and an e n e r g y - i n d e p e n d e n t e n t r a n c e p r o c e s s . F u r t h e r k i n e t i c s t u d i e s d e monstrated t h a t energy was u t i l i z e d t o a l t e r t he r a t e c o n s t a n t o f e x i t (K^) i n a manner such t h a t e x i t was p r e v e n t e d , thus a l l o w i n g c o n c e n t r a t i o n o f the s u b s t r a t e w i t h i n the c e l l s ( W i n k l e r and W i l s o n , 1966). The s p e c i f i c i t y o f the v a r i o u s systems f o r g a l a c t o s e and g a l a c t o s i d e s have been s t u d i e d f a i r l y e x t e n s i v e l y . Ganeson and Rotman (I966) r e v i e w e d t h e e v i d e n c e f o r two TMG permeases i n E. c o l i , as w e l l as a permease f o r a m e t h y l - g - D - g a l a c t o s e . TMG-permease I , t h e o r i g i n a l permease s t u d i e d by t h e P a s t e u r group ( R i c k e n b e r g , Cohen, B u t t i n and Monod, 1956) t r a n s p o r t e d a- and 3-D - g a l a c t o p y r a n o s i d e s and i t was induced by compounds c o n t a i n i n g an u n s u b s t i t u t e d g a l a c t o - p y r a n o s e r i n g . The second system was induced by g a l a c t i n o l and t r a n s p o r t e d TMG but not l a c t o s e . The t h i r d g a l a c t o s i d e permease t r a n s p o r t e d m e t h y l - 3 - D - g a l a c t o p y r a n o s i d e but not TMG. A system f o r t h e t r a n s p o r t o f D - g a l a c t o s e by E_. c o l i has a l s o r e c e i v e d much a t t e n t i o n ( H o r e c k e r , Thomas, and Monod, 1960a; 1960b; Osborn, M c L e l l a n and H o r e c k e r , 1961) and found t o e x h i b i t k i n e t i c s o f e x i t and u p t a k e v e r y s i m i l a r t o t h o s e o f t h e TMG permease i i . G l u c o s e In c o n t r a s t t o t h e g a l a c t o s i d e permease s y s t e m , a permease f o r g l u c o s e i n E_. c o l i has been d e s c r i b e d where removal o f t h e energy s u p p l y was shown t o cause an a c c u m u l a t i o n o f a - m e t h y l -glucoside (otMG), a non-metabolizable analogue of glucose (Hoffee, Englesberg and Lamy, 1964). The glucose transport system in E. c o l i had e a r l i e r been shown, by competition and k ine t i c studies (Cohen and Monod, 1957; Kessler and Rickenberg, 1963), to accumulate aMG. Hagih i ra , Wilson and Lin (1963) have shown further that mutants defect ive in glucose uptake cannot concentrate aMG. In competition studies with subst i tuted der ivat ives of aMG, the same workers showed the s p e c i f i c i t y of the system depended upon the substi tuents on C-2, C-3, and C-6 of the aMG. Addit ion of an exogenous energy source by Hoffee et a l . (1964) resulted in a depression of accumulation. These resul ts were presented as evidence for the existence of an energy-requir ing ex i t react ion in glucose transport . The s p e c i f i c i t y of aMG uptake has also been studied by these workers and i t was shown that a ten- fo ld excess in concentration of gMG or glucose competed very strongly with aMG uptake, whi le maltose allowed 68% of normal accumulation and mannose, deoxy-glucose, and sucrose each allowed 80% of normal a c t i v i t y . Other carbohydrates had e i ther 1 i t t l e ef fect or stimulated aMG uptake. These resul ts have been confirmed by Halpern and Lupo (1966). Studies of a galactose-negative s t ra in of E. co l i showed that galactose was entering v ia the glucose permeation system (Rogers and Yu, 1962). Although the s t ra in lacked galactokinase, as much as50% of the galactose was phosphorylated in the pool , suggesting an accumulation mechanism based on phosphorylation during transport. I I I . Carbohydrate accumulation mechanisms and components The study of carbohydrate transport in Gram-positive o r g a n i s m s has been l i m i t e d t o the c o c c u s S t a p h y l o c o c c u s a u r e u s (Egan and M o r s e , 1966). E x t e n s i v e c o m p e t i t i o n e x p e r i m e n t s d e f i n e d permease f u n c t i o n s , w h i l e c o u n t e r f l o w e x p e r i m e n t s and g e n e t i c e v i d e n c e i n d i c a t e d a common c a r r i e r p r o t e i n f o r a number o f c a r b o h y d r a t e s . S t u d i e s w i t h a mutant o f S . a u r e u s i n d i c a t e d t h a t t h e a b i l i t y t o t r a n s p o r t e i g h t c a r b o h y d r a t e s c o u l d be l o s t by a s i n g l e m u t a t i o n (Egan and M o r s e , 1965). It was a l s o found t h a t w i t h l a c t o s e , m a l t o s e , s u c r o s e and aMG, p h o s p h o r y l a t i o n o f the c a r b o h y d r a t e s was c o i n c i d e n t w i t h t r a n s p o r t ( H e n g s t e n b e r g , Egan and M o r s e , 1967; 1968). Wang and Morse (1968) c h a r a c t e r i z e d s i m i l a r p l e i o t r o p i c c a r r i e r mutants f o r E . c o l i and A e r o b a c t e r a e r o g e n e s . T h e s e mutants have been d e m o n s t r a t e d t o l a c k e i t h e r o f two p r o t e i n components o f the p h o s p h o t r a n s f e r a s e sys tem (Tanaka and L i n , 1967; T a n a k a , F r a e n k e l and L i n , 1967; S imoni e_t a j_ . , 1-9'67). A t t e m p t s t o i s o l a t e and c h a r a c t e r i z e components o f c a r b o h y d r a t e t r a n s p o r t sys tems have been r e l a t i v e l y s u c c e s s f u l (Fox and Kennedy, 1965; K o l b e r and S t e i n , 1966). 3. C a r b o h y d r a t e t r a n s p o r t in y e a s t and f u n g i The mechanism and s p e c i f i c i t y o f the t r a n s p o r t o f c a r b o h y d r a t e s by y e a s t has r e c e n t l y r e c e i v e d a g r e a t d e a l o f a t t e n t i o n . It has l o n g been r e c o g n i z e d t h a t C o b a l t o u s , N i c k e l o u s and U r a n y l ions c o u l d i n h i b i t c a r b o h y d r a t e m e t a b o l i s m by y e a s t . H u r w i t z and R o t h s t e i n . (1-951).; Van S t e v e n i n c k (1966) and Van S t e v e n i n c k and Dawson (1967) have i m p l i c a t e d p o l y p h o s p h a t e s as t h e e n e r g y s o u r c e f o r c a r b o h y d r a t e t r a n s p o r t in y e a s t s , as N i c k e l o u s i o n s i n t e r f e r e d with polyphosphate s t ructure, and inhib i ted transport . In add i t ion , these workers have claimed the existence of an ac t i ve , induced, energy-dependent transport system for galactose, using the same ca r r i e r mechanism as that used in non-induced, f a c i l i t a t e d d i f fus ion. The s p e c i f i c i t y of the carbohydrate transport system in yeast has been studied to a considerable extent (Scharff and Kraemer, 1962; C i r i l l o , 1962; C i r i l l o , 1968). From competition studies on the uptake of L-sorbose and D-xylose using 25 d i f fe rent carbo-hydrates, C i r i l l o (1968) has defined the spec i f i c s t ructura l requirements of the const i tu t ive yeast monosaccharide transport system. He demonstrated that the conformation and substi tuents of every carbon except C-2 contr ibute to the carbohydrates' a b i l i t y to compete with the transport of the non-metabolized sugars, sorbose and xy lose. In add i t ion , C i r i l l o (1968) demonstrated that the sugar must be in the pyranose form and that whi le removal of the anomeric l-OH has l i t t l e ef fect on t ransport , subst i tu t ion at th is pos i t ion completely destroyed competitive a c t i v i t y . Mul t ip le a l tera t ions at carbon atoms other than C-2 had an ef fect greater than the sum of the i r indiv idual e f f ec t s . A study of the mechanism of transport of carbohydrate by yeast c e l l s under anaerobic condi t ions, by k ine t i c ana lys i s , and a tentat ive model, based on ca r r i e r mediated d i f f u s i o n , for anaerobic carbohydrate transport in general , has been published (Scharff and Kraemer, 1962). From a comparison of rate constants for transport and for pur i f ied hexokinase, these workers concluded that hexokinase did not play an important, d i rec t part in the anaerobic transport of sugars by yeast. They have suggested that metabolism plays essen t ia l l y no part in anaerobic sugar t ransport . Further, ca r r i e r f a c i l i t a t e d d i f f us i on , being the mechanism of transport in erythrocytes and anaerobic yeast, is the method of transport for a l l c e l l s capable of anaerobic metabolism. MATERIALS AND METHODS 1. 0 rgah ? sms arid Med i a Clost r id i urn perf r i ngens (BP6K) was used throughout th is study. 1. Complex mediurn The composition of the complex medium was as fo l lows: yeast ex t rac t , 2 gm; proteose peptone, 5 gm; sodium ascorbate, 0.2 gm; and FeSO^.yH^ (0.5% so lu t i on ) , 1.0 ml per l i t e r . These were dissolved in d i s t i l l e d water, the solut ion was neutral ized with 1N NaOH and 160 ml of 0.5 M phosphate buffer (pH 7-2) were added. The volume was adjusted to 1 l i t e r , and the medium was autoclaved. S te r i l e solut ions of glucose and MgSO^^r^O were added to 0.7% and 0.02% respect ive ly . 2. Semi-defined medium This medium was a modif icat ion of that described by Boyd, Logan and Ty te l l (1948). Stock solut ions were prepared as fol lows 2.0 mg of b io t i n were dissolved in 100 ml of d i s t i l l e d water, 10 mg of r i bo f l av in and 20 mg of Ca-d-pantothenate were dissolved in 100 ml of d i s t i l l e d water, and 50 mg of u rac i l and 87 mg of a d e n i n e s u l f a t e were d i s s o l v e d in 50 ml o f 0.2N HG1. In a d d i t i o n , 20 mg o f p y r i d o x i n e m o n o h y d r o c h l o r i d e were d i s s o l v e d i n 100 ml o f d i s t i l l e d w a t e r , and a s o l u t i o n c o n t a i n i n g 10 g m o f MgS0^.7H20 and 0 . 5 gm each o f F e S 0 ^ . 7 H 2 0 , NaCl a n r j M n S O ^ H ^ , per 100 ml o f d i s t i l l e d w a t e r , was p r e p a r e d . A l l s t o c k s o l u t i o n s were s t o r e d a t 4 C . To r e p l a c e the i n d i v i d u a l amino a c i d s o f t h e B o y d , Logan and T y t e l 1 (1948) medium, 16 .7 gm o f a c i d h y d r o l y z e d c a s e i n and 0.83 gm o f L - t r y p t o p h a n were used per l i t e r . T h e s e , t o g e t h e r w i t h 0 .25 gm o f sodium a s c o r b a t e , were d i s s o l v e d in d i s t i l l e d w a t e r by u s i n g low heat and a g i t a t i o n . A f t e r c o o l i n g , t h e pH was a d j u s t e d t o 7.2 w i t h 1N.NaOH. F i v e ml o f the s a l t s s o l u t i o n , 11 .25 ml o f the u r a c i l and a d e n i n e s o l u t i o n , 5.0 ml o f the r i b o f l a v i n and p a n t o t h e n a t e s o l u t i o n , 2 . 5 ml o f the p y r i d o x i n e s o l u t i o n , and 0 .25 ml o f t h e b i o t i n s o l u t i o n were m i x e d , d i l u t e d t o 100 ml w i t h . d i s t i 1 l e d w a t e r and t h e pH a d j u s t e d t o 7 .2 w i t h 1N NaOH. T h i s m i x t u r e was added t o t h e n e u t r a l i z e d amino a c i d s o l u t i o n , 100 ml o f 0.5 M p h o s p h a t e b u f f e r (pH 7.2) were a d d e d , and the t o t a l volume was a d j u s t e d t o 1000 ml w i t h d i s t i l l e d - w a t e r . D u r i n g a u t o c l a v i n g , a heavy p r e c i p i t a t e formed and t h i s was a l l o w e d t o s e t t l e o u t f o r 12 h r s a t 4 C . The s u p e r n a t a n t f l u i d was then d e c a n t e d i n t o one l i t e r Na lgene b o t t l e s , b u b b l e d w i t h N^ f o r 15 m i n , then f r o z e n and s t o r e d a t - 2 0 C . B e f o r e u s e , t h e medium was t h a w e d , f i l t e r e d t h r o u g h a 0.3 u M i l l i p o r e f i l t e r and d i s p e n s e d i n t o s t e r i l e , w a t e r - j a c k e t e d reaction vessels . S t e r i l e solut ions of glucose or other substrates were added asep t i ca l l y . 3. Stock cultures Cultures were grown in the complex medium supplemented with 0.5% acid hydrolyzed case in , unt i l an opt ica l density (O.D.) at 660 my of 1.2 was reached, then ster i1e glycerol was added to 15%. The mixture was dispensed asep t i ca l l y in 2.0 ml amounts into s t e r i l e tubes, frozen and held for as long as several months at -20 C (Pivnick et aj_., 1964). Cultures were rout inely checked for pur i ty by streaking onto blood agar plates which had been previously spread with gas-gangrene ant i tox in (Connaught Medical Research Lab . , Toronto). Control p la tes , without an t i t ox in , were also streaked with the cu l tu res . Plates were incubated for 2k hrs at 37 C, under and a lso aerob ica l l y . The plates were then examined for colony morphology and par t ia l i nh ib i t i on of haemolysis in the presence of an t i t ox in . k. Growth of inoculum A stock cul ture was thawed by slowly warming i t and 0.5 ml was added to a tube of Roger's meat medium with the desired carbohydrate substrate, and incubated at 37 C. The cul ture was used as an inoculum when i t was ac t i ve ly gassing (2.5 hrs incubat-ion) . II. Growth Conditions Cultures were usual ly grown in 100 or 500 ml water-jacketed reaction vessels heated to 37 C by a temperature contro l led c i r cu la t i ng waterpump. S t e r i l e nitrogen was passed over the surface of the medium at 50 ml/min to keep the cultures anaerobic. S t e r i l e was bubbled through the medium for 20 min pr ior to the addi t ion of a 5% inoculum from an ac t i ve ly gassing meat tube cu l tu re . With the semi-defined medium, to ensure a rapid ly growing cul ture with a minimum of carry over from the meat medium, the cul tures were grown, with s t i r r i n g , to an O.D. of 1.0 at 660 mu (approximately 3 hrs) . A 5% inoculum from th is cul ture was transferred to a second f lask of semi-defined medium prepared as the f i r s t , and grown to the desired O.D. at 660 my. On occasion, the inoculated meat tube was held in ice in an insu lated, water-jacketed reaction vessel unt i l a t imer-control led c i r cu la t i ng waterpump warmed the reaction vessel to 37 C and i n i t i a t ed ac t ive growth of the cu l tu re . Af ter 2.5 hrs growth, a t imer-contro l led air-pump asep t i ca l l y forced approximately 2 ml of the supernatant f l u i d of the ac t i ve ly gassing meat tube into 100 ml of the semi-defined medium in a water-jacketed react ion vesse l . The medium was maintained under anaerobic condit ions by bubbling s t e r i l e through i t . Simultaneous w i th . inocu la t ion , the medium was warmed to 37 C by a timer-contro l led c i r cu la t i ng waterpump. When the growing cu l ture reached an O.D. of 1.0 at 660 mu, i t was used to inoculate a second reaction vessel of the semi-defined medium. No s ign i f i can t d i f ference was observed between the c e l l s resu l t ing from manual or automatic subculture. During growth of the second cul ture in the semi-defined medium or the cul ture in complex medium, a l iquots were removed asep t i ca l l y and the O.D. at 660 my was measured. In add i t ion , the pH of the medium was determined and an al iquot was frozen for the quant i ta t ive determination of glucose. I I I . Stabi1i ty of Cel1 Suspens ions Cultures were grown to an O.D. at 660 my of 1.75 in the complex medium and to 0.75 in the semi-defined medium and harvested by centr i fugat ion at room temperature at 5,000 x £. The c e l l s were washed by resuspending them in 0.85% NaCl (pH 7.2) and centr i fuging again at 5,000 x £. This washing procedure was repeated and then the c e l l s were resuspended to the desired O.D. at 660 my in 0.05 M tris(hydroxymethyl)amino methane-HCl(tris-HCl) buffer (pH 7.2) and held under s t e r i l e N^ in a water-jacketed reaction vessel at 37 C. The surface of the suspension was flushed with s t e r i l e at a rate of 50 ml per min and samples of the c e l l suspension were removed at vari>oustime in te rva ls . Al iquots of the samples were d i lu ted with t r i s -HCl buffer (pH 7.2) for the determination of O.D. at 660 my. Other al iquots were centri fuged at k C for 10 min at 10,000 x £ and u l t r a - v i o l e t (UV) adsorption spectra from 200 to 300 my were determined on the resul t ing supernatant f l u i d with a Beckmann model DBG recording spectrophotometer. IV. Enzyme Assays 1. C e l l - f r e e extracts Cultures were grown to an O.D. at 660 my of 0.6 in the semi-defined medium with 0.7% glucose. The cultures were then trans-ferred to a screw-capped 300 ml p l as t i c centr i fuge bo t t le . The surface of the cul ture was flushed with for 5 min and the bot t le was sealed and centri fuged at room temperature at 5,000 x £ for 7 min. The pe l le t was resuspended in the fol lowing so lu t i on , which had previously been bubbled with to remove oxygen: 0.2 M t r i s -HCl (pH 7.5) , 10~3 M MgCl and 6 x 10"^ M cysteine-HC1. The suspension was t ransfer red, with a 5.0 ml , l i gh t l y greased, glass syringe f i t t ed with a 3 _ inch #21 needle, to a th ick -wa l led , 16 mm centr i fuge tube with a rubber stopper. The tube had previously been made anaerobic by f lushing i t with N^,, using two #20 needles inserted in the rubber stopper (anaerobic centr i fuge tube). The suspension was centri fuged at room temperature, at 5,000 x £ for 7 min, the supernatant f l u i d was removed, and the pel le t was resuspended in the anaerobic buf fer, Mg + + , cystei ne so lu t ion . Al iquots were transferred by a syringe and needle, as described previously , to several anaerobic centr i fuge tubes. Af ter centr i fugat ion for 7 min at 5000 x g_, the supernatant f l u ids were removed and the pel 1ets were flushed with N 2 for 5 min. The needles were removed from the stopper and the tubes were stored at -70 C. C e l l - f r e e extracts were prepared from these c e l l s wi th in a maximum time interval of one week. There was no apparent loss of enzyme a c t i v i t y during th is storage period at -70 C. As required, the ce l l pe l le ts were thawed and resuspended to 50 times the growth concentration (approximately 12 mg dry weight/ml) by the addi t ion of the anaerobic buffer-Mg -cyste ine so lu t ion . The suspension was subjected to 2.5 min of sonic o s c i l l a t i o n at 88 watts (Bronwill B P - l i , 1/8 i n . d i a . probe, Bronwil l Industr ies, Rochester, N .Y. ) . Sonication was performed in anaerobic centr i fuge tubes which were maintained in an ice-water bath. Nitrogen was flushed over the surface of the suspensions at 50 ml per min, and sonicat ion was applied for 1/2 min i n te rva ls , with a l ternate 1/2 min in tervals for coo l ing. The sonicate was centri fuged in the anaerobic centr i fuge tube for 7 min at 10,000 x g_ and the supernatant anaerobic extract was transferred by means of a 5.0 ml l i gh t l y greased, glass syringe f i t t ed with a 3 inch #21 needle to a second anaerobic centr i fuge tube, where i t was kept under at O X . A sample of each eel 1-free extract was frozen and subsequently assayed for protein content. 2. Enzyme assays In the fol lowing ser ies of assays, with the exception of enolase and the pyruvate-c las t ic system, the reduction of NADP or NAD, or the oxidat ion of NADH^ were followed spectrophoto-met r ica l ly by observing the change in O.D. at 3^0 my. A l l assays, except that for the pyruvate-c last ic system, were carr ied out at 37 C. Anaerobic assays were prepared in 2.5 or 1.5 ml to ta l volumes in 3.0 ml, 1 cm l igh t -pa th , quartz cuvettes (Beckmann Instruments Inc. , Fu l le r ton , Ca l i fo rn ia ) with c i r c u l a r openings sealed with a rubber serum stopper. To remove oxygen, the react ion mixtures were equi l ibrated with nitrogen by f lushing the surface of the solut ions with at the rate of 20 ml per min, using two #21 hypodermic needles inserted through the rubber serum stoppers. A l l so lu t ions , which were added anaerobica l ly , were equi l ibrated under N^ at 0 C and added through the serum stopper v ia a l i g h t l y greased, 1.0 ml glass syringe f i t t e d with a 3 _ inch #21 needle. Aerobic assays were set up in 1.0 or 0.5 ml total volume systems in quartz cuvettes with 1 cm l ight-path of the appropriate s i z e . Anaerobic c e l l - f r e e extracts were used for a l l assays in order to l im i t enzyme degradation by protein ox idat ion. The buffer used in a l l assays, except the pyruvate-c las t ic system, was t r i s -HCl (pH 7.5). Control react ion mixtures were prepared for each enzyme assay to ensure that the observed reactions were dependent upon the presence of the various substrates employed. Enzyme a c t i v i t i e s were corrected for the control values and expressed 2 as ymoles of substrate u t i l i z e d x 10 /min/mg of prote in . a. Hexokinase was assayed in an aerobic system of 1.0 ml tota l volume contain ing: buf fer , 80 ymoles; glucose ( f ructose, mannose or galactose) , 5 umoles; adenosine .5 tr iphosphate (ATP)[, k ymoles; NADP, 0.1 ymoles ; and commercial glucose-6-phosphate dehydrogenase, 5-35 yg. The reaction was i n i t i a ted by the addi t ion of the anaerobic ext ract . b. Glucose-6-phosphate dehydrogenase was assayed in an anaerobic, 2.5 ml system contain ing: buf fer , 2k0 ymoles; glucose-6-phosphate, 1.0 ymoles; and MgCl^, 20 ymoles. Af ter equ i l i b ra t i on at 37 C with for 7 min,anaerobic extract was added and the react ion started by the addi t ion of anaerobic NADP or NAD. c. Phosphohexose isomerase was assayed in an anaerobic system of 1.0 ml total volume,containing: buf fer , 60 ymoles; fructose-6-phosphate, 5 ymoles; glucose-6-phosphate dehydrogenase 0.3 yg; NADP, 0.125 ymoles; and anaerobic ex t rac t . d. Phospho-fructokinase was measured in a 2.5 ml tota l volume under anaerobic condit ions contain ing: buf fer , 200 ymoles; ATP, 10 ymoles; a -g lycero l phosphate dehydrogenase, 20 yg; a ldo lase, 20 yg; MgC l 2 , 30 ymoles; and NAD.H , 0.25 ymoles. Af ter equ i l i b ra t -ion at room temperature under N 2 for 7 min, the reaction was started by the addit ion of anaerobic ex t rac t . e. Fructose-1,6-diphosphate aldolase was assayed by a modified procedure of Groves e_t aj_. (1966) in an anaerobic system of 2.5 ml tota l volume containing: buf fer , 120 ymoles; potassium acetate, 240 ymoles; cobalt ch lo r ide , 1.68 ymoles; cys te ine-HCl , 0.24 ymoles; a-glycerophosphate dehydrogenase, 20 yg; and t r i ose -phosphate isomerase, 20 yg. Af ter equ i l i b ra t ion for 7 min at 37 C, under anaerobic extract and 0.25 ymoles of NADH^ were added and the reaction was started by the addit ion of 10 ymoles of fructose-1,6-phosphate. f. Triose-phosphate isomerase was measured in an anaerobic system of 2.5 ml to ta l volume contain ing: buf fer , 240 ymoles; iodoacetamide, 10 ymoles; and 20 yg of a -g lycero l phosphate dehydrogenase. The mixture was equi l ibrated at room temperature under for 7 min, then anaerobic extract and 0.25 ymoles of NADH^ were added. The react ion was started by the addit ion of 7.5 ymoles of anaerobic 3~phospho-glyceraldehyde. g. Three-phosphoglyceraldehyde dehydrogenase was measured by a modif icat ion of Krebs' (1955) procedure in an anaerobic, 2.5 ml system containing: buf fer , 240 ymoles; cys te ine-HCl , 1.5 ymoles; sodium arsenate, 30 ymoles; sodium f l uo r i de , 30 ymoles; and 3~phosphoglyceraldehyde, 7.5 ymoles. Af ter equ i l i b ra t ion at room temperature for 7 min with N^, anaerobic extract was added and the reaction mixture equi l ibrated under for a further 5 min at room temperature and 2 min at 37 C. The reaction was i n i t i a ted by the addit ion of 0.25 ymoles of NAD. In a pa ra l l e l experiment, 10 ymoles of iodoacetamide was added to the reaction mixture pr ior to the f i r s t equ i l i b ra t ion with N 2 (Krebs, 1955). h. Three-phospho-glyceric acid kinase was measured in a 1.5 ml to ta l volume system under anaerobic condi t ions. The reaction mixture contained: buf fer , 60 ymoles; 3-phosphoglycer-aldehyde dehydrogenase, 50 yg; ATP, 1.5 ymoles; M g C ^ , 12.5 ymoles; cysteine-HCl , 9 ymoles; and 7.5 ymoles of 3 -phospho-glycerate. Af ter equ i l i b ra t ion at room temperature under for 7 min, 0.15 ymoles of anaerobic NADH^ were added and the assay started by the addit ion of the anaerobic ext ract . i . Mutase. The a c t i v i t y of th is enzyme was assayed in an anaerobic system with 1.5 ml tota l volume containing: buf fer , 60 ymoles; MgCl^, 15 ymoles; ATP, 0.75 ymoles; 2 ,3 _ diphosphoglyceric ac i d , 0.625 ymoles; 2-phosphoglyceric a c i d , 7.5 ymoles; cyste ine-HCl , 9 ymoles; 3-phosphoglyceraldehyde dehydrogenase, 20 yg; and 3-phosphoglycerate k inase, 20 yg. Af ter 7 min under at room temperature, 0.15 ymoles of anaerobic NADH^ were added, and the reaction in i t i a ted by the addit ion of anaerobic ex t rac t . j . Pyruvate kinase was assayed in an anaerobic 2.5 ml system by a modif icat ion of the procedure of BUcher and P f le iderer (1955). The reaction mixture contained the fo l lowing: buf fer , 100 ymoles; KC1 , 187 ymoles; M g C ^ , 20 ymoles; l a c t i c dehydrogenase, 1 yg; and ADP, 0.125 ymoles. Af ter equ i l i b ra t ing with N2 for 7 min at room temperature, anaerobic extract and 0.5 ymoles of NADH2 were added and the reaction started by the addit ion of 2.5 moles of anaerobic phosphoenol pyruvate. k. Lac t i c dehydrogenase was assayed in a 1.5 ml system under U^, that contained the fo l lowing: buf fer , 40 ymoles; NADh^, 0.15 ymoles; and sodium pyruvate, 5 ymoles. Af ter 7 min at room temperature, the reaction was started by the addi t ion of anaerobic ext ract . 1. NADH2 oxidase was measured under both aerobic and anaerobic condi t ions. 28 (i) The aerobic a c t i v i t y of NADH^ oxidase was measured in a 2.5 ml system containing 30 ymoles of buffer and anaerobic ex t rac t . The reaction mixture was mixed in a i r and warmed to 37 C, a f ter which the reaction was started by the addit ion of 0.45 ymoles of aerobic NADH^. ( i i ) The a c t i v i t y of the oxidase was measured in the absence of molecular oxygen by using a 2.5 ml system containing 250 ymoles of buffer equi l ib ra ted under for 7 min at 37 C. Anaerobe extract was added, and the reaction was started by the addit ion of 0.45 ymoles of anaerobic NADH2. m. Enolase was measured by fol lowing the change in O.D. at 230 my, according to the method of Westhead (1966). An aerobic system of 0.5 ml total volume was used and i t contained the fo l lowing: buf fer , 20 ymoles; MgCl^, 1 ymole; and 2-phospho-. 1 1 . glycerate, 2.5 ymoles. Af ter warming to 37 C, the assay was i n i t i a ted by the addit ion of d i lu ted anaerobic ex t rac t . n. Pyruvate-c las t ic system. This enzyme system was assayed by a modif icat ion of the method of Lovenberg e_t a_l_. (I963), which was based on the assay of Lipmann and Tut t le (19^5) for acetyl phosphate. A 10 ml react ion mixture containing the fo l lowing: phosphate buffer (pH 6.5) , 250 ymoles; sodium pyruvate, 100 ymoles; and coenzyme A, 0.35 mg; was bubbled with for 5 min at room temperature, and then for 2 min at 30 C. The reaction was started by the addi t ion of the anaerobic extract and was flushed over the surface of the reaction mixture during the experiment. One ml samples were removed at various time intervals and added to a mixture of 100 ymoles of acetate buffer (pH 5.4) and-14 mg of neutral ized hydroxy lamine-HC1 in a tota l volume of 2.0 ml. Af ter mixing, the suspension was allowed to stand at room temperature for 10 min, a f ter which 1.0 ml of 3N HCI, 1.0 ml of 10% t r i ch lo roace t i c acid (TCA) and 1.0 ml of 5% FeCl^.SH^O in 0.1N HCI were added with vigorous mixing. The mixtures were centr i fuged for 5 min at 12,000 x £ and the supernatant f l u i d was examined for O.D. at 540 my. A molar ex t inc t ion coe f f i c ien t was calculated from a standard curve obtained by measuring the O.D. at 540 my of solut ions of the complex of f e r r i c ions and the hydroxamate of succ in ic anhydride (Lipmann and Tu t t l e , 1945). V. Carbohydrate Transport .• 1.'; Preparation :of anaerobic c e l l suspensions Cultures were grown in the semi-defined medium with 0.6% sodiurn pyruvate and 0.1% glucose unless otherwise indicated. The c e l l s were harvested and washed twice with semi-defined medium plus 0.6% pyruvate under N^, as described for the preparation of eel 1-free ex t rac ts . Af ter the f i na l washing, the c e l l s were resuspended to an O.D. at 660 my of 0.28 (0.078 mg dry weight/ml) in the semi-defined medium plus 0.6% sodium pyruvate, held under N 2 and at 18 C in a water-jacketed reaction vesse l . The O.D. at 660 my was monitored during the course of the experiment to ensure that the c e l l density remained constant. 14 2. Assays for C-carbohydrate incorporation A small reaction vessel consis t ing of a shortened 18 mm test- tube was f i t t e d with a rubber stopper, a #18 hypodermic needle for i n l e t , and an open port for sampling, addi t ion of substrates and c e l l s , and e x i t . The reaction vessel contained a small magnetic s t i r r i n g bar driven by an underwater magnetic-s t i r r e r (Bronwill Industr ies, Rochester, N .Y . ) , was flushed with at 50 ml per min, and was warmed to 30 C in a water-bath. 14 C- label led carbohydrate substrates were prepared in 1.0 ml volumes of the semi-defined medium and placed in a small tube which was f i t t ed with a rubber stopper, with a in le t needle and an open port . The substrate solut ion was held in th is tube on i ce , under ni t rogen, for 5 min and then warmed to 30 C for 3 min. Four ml of the anaerobic c e l l suspension was transferred with a 5.0 ml syringe f i t t ed with a 3-inch #21 hypodermic needle, to the anaerobic, 30 C react ion vesse l . The c e l l suspension was warmed to 30 C, s t i r r ed for 3 min and the react ion was started by the addi t ion of the 30 C, anaerobic substrate so lu t i on , using a 1.0 ml syringe f i t t ed with a 3 - i nch #21 needle. At time in te rva ls , 1.0 ml a l iquots of the reaction mixture were removed with a s im i la r 1.0 ml syringe and needle. The samples were analyzed for incorporation of C-label led carbohydrates into the whole eel Is by the method of Br i t ten and McClure (1962). Ce l l s were f i1 tered onto a Tracer lab E8B prec ip i ta t ion apparatus (Tracer lab, Waltham, Mass. ) , containing a 0.45 y M i l l i po re f i l t e r , and quick ly washed with 2.0 ml of semi-defined medium containing 0.6% sodium pyruvate. Samples were a 1 so added to test-tubes containing equal volumes of ice-co ld 5% TCA, held at 0 C for 15 min and f i l t e r e d on the p rec ip i ta t ion apparatus containing a 0.45 y M i l l i po re as above. The tubes were rinsed twice with ice-co ld 5% TCA and the r inse solut ions were added to the f i1 te r (Br i t ten and McClure, 1962). This procedure was used to determine the incorporation 1 4 of the C-label into the cold TCA insoluble proteins and nucleic acids (Roberts et_ a)_., 1955). To study the competitive inh ib i t i on of carbohydrate t ransport , 12 a 100-fold excess of the C possible competitor was added to the 14 C- label led carbohydrate so lut ion and the rate of total 14 incorporation of C into the whole eel Is was compared to 14 12 the rate of incorporation of C in the absence of the C-carbohydrate. 14 In cases where the C-carbohydrate did not accumulate in t he .ce l l s against a concentration gradient, extreme caution in f i I t e r i n g was necessary in order to prevent the retention of high levels of background rad ioac t i v i t y in the f i1 tered samples, which would obscure the re la t i ve l y smal1 changes in rad ioac t iv i t y b e i n g measured. The M i l l i p o r e f i l t e r s were pre-washed w i t h 12 2.0 ml o f s e m i - d e f i n e d medium c o n t a i n i n g t h e C - s u b s t r a t e a t 14 t h e same c o n c e n t r a t i o n as t h e C - l a b e l l e d s u b s t r a t e i n t h e r e a c t i o n m i x t u r e t o reduce s p e c i f i c a d s o r p t i o n t o t h e f i l t e r . The danger o f r e t a i n i n g s m a l l volumes o f t h e r e a c t i o n m i x t u r e i n t h e f i l t e r was reduced by the use o f h i g h e r than normal s u c t i o n r a t e s , and washing t h e f i l t e r e d c e l l s 3 t i m e s w i t h 1.0 ml volumes o f t h e wash s o l u t i o n , i n s t e a d o f once w i t h 2.0 m l , as i n normal i n c o r p o r a t i o n e x p e r i m e n t s . Care was t a k e n t o keep t h e f i l t e r s on the p r e c i p i t a t i o n a p p a r a t u s f o r equal l e n g t h s o f t i m e b e f o r e t h e y were removed f o r d r y i n g and c o u n t i n g . C o n t r o l v a l u e s f o r n o n - s p e c i f i c r e t e n t i o n o f l a b e l by t h e f i l t e r s were d e t e r m i n e d by p e r f o r m i n g p a r a l l e l e x p e r i m e n t s f o r t o t a l i n c o r p o r a t i o n u s i n g 5.0 ml o f s e m i - d e f i n e d medium c o n t a i n i n g 0.6% sodium p y r u v a t e , r a d i o a c t i v e s u b s t r a t e , and no c e l l s u s p e n s i o n . The f i l t e r s were c a r e f u l l y washed and d r i e d as above, and t h e v a l u e s t h a t were o b t a i n e d were s u b t r a c t e d from t h e v a l u e s f o r t o t a l i n c o r p o r a t i o n i n t o e e l I s . 14 C - l a b e l l e d c a r b o h y d r a t e s were added a t t h e v a r i o u s c o n c e n t r a t -i o n s and s p e c i f i c a c t i v i t i e s i n d i c a t e d i n t h e R e s u l t s and D i s c u s s i o n . 14 3. D e t e r m i n a t i o n o f t h e n a t u r e o f t h e a c c u m u l a t e d C-c a r b o h y d r a t e A r e a c t i o n m i x t u r e was p r e p a r e d as d e s c r i b e d f o r t h e s t u d y of tota l incorporation of C into c e l l s . The ent i re reaction mixture was removed with a 5.0 ml syringe f i t t e d with a #21 hypodermic needle at 0.5 min af ter addi t ion of the C- label led substrate. The mixture was injected onto a 0.45 y M i l l i po re f i l t e r , and washed once with 2.0 ml of semi-defined medium containing 0.6% sodium pyruvate. The f i l t r a t e from the reaction mixture was recovered and held at 0 C for ana lys is . The f i l t e r was quickly removed and immersed in a beaker containing 3.0 ml of ice-co ld 5% TCA. The f i l t e r and TCA solut ion were placed in an 18 mm test - tube, the beaker was rinsed with 2.0 ml of ice-co ld 5% TCA, and the r inse so lut ion was added to the tes t -tube. Af ter vigorous ag i ta t ion of the f i l t e r in the TCA so lu t i on , the so lut ion was removed and the f i l t e r was further extracted with two washings of 2.0 ml of ice-co ld 5% TCA, which were added to the o r ig ina l so lu t ion . The combined solut ions were c e n t r i -fuged at 12,000 x £ for 15 min at 4 C, to remove the precip i tated material and the supernatant f l u i d was extracted four times with equal volumes of cold ethyl ether to remove the TCA. The 14 remaining aqueous so lu t i on , containing the pooled C-carbohydrate, was concentrated to a small volume by warming to 30 C in a f l ash -evaporator (Laboratory Glass and Instruments Corp. , New York, N.Y.) with the condenser cooled in an ice-water bath. The concentrated solut ion was subjected to analysis by electrophoresis and paper chromatography. An a l iquot of the material was fract ionated by preparative paper electrophoresis and the separated f ract ions were eluted with d i s t i l l e d water and de-phosphorylated with bacter ia l a l ka l ine phosphatase. The de-phosphorylated preparations were concentrated and subjected to analyses by paper electrophoresis and paper chromatography. The f i l t r a t e of the o r ig ina l reaction mixture was assayed for tota l rad ioac t i v i t y and then concentrated to a small volume and the nature of the radioact ive compounds was determined by the same procedures as used for the accumulated radioact ive compounds. The recovery of the rad ioac t iv i t y in a l l of the samples was monitored during the preparative and ana ly t ica l procedures, and neg l ig ib le losses occurred. VI . Assay of Radioact iVi ty 1. M i l l i p o r e f i I t e r s Dried M i l l i po re f i l t e r s were placed in the v i a l s containing 5 ml of s c i n t i 1 l a t i o n f l u i d (L iqu i f l uo r , New England Nuclear Corporation) and the v i a l s were assayed for r ad ioac t i v i t y in a Nuclear Chicago 1iquid s c i n t i 1 l a t i o n spectrometer model 725. 2. Chromatograms and electrophoretograms Radioactive chromatograms were scanned by running 1-inch s t r i ps through a Nuclear Chicago model C 100 B Actigraph II with a gas flow counter and a model 1620 B Ana ly t ica l Count ratemeter equipped with a chart recorder. The rad ioac t i v i t y of compounds from electrophoretograms and chromatograms were quant i ta t ive ly determined by cutt ing out the areas where label had been detected, drying under an in f ra -red lamp, and counting in the 1iquid s c i n t i 1 l a t i o n counter. VI I . Chromatography and Electrophores?s Paper chromatography was rout inely performed on substrate solut ions and extracted pool materials by spott ing samples on Whatman No. k paper, and running them by the descending technique with the solvent system of Grado and Ballou (1961) as fo l lows: e thy l -acetate/pyr id ine/saturated aqueous bor ic acid (60/25/20). Electrophoresis was carr ied out with a water-cooled apparatus s im i la r to a Resco model E-800-2B equipped with a Resco model 1911 power supply. The buffer system rout inely employed was 0.1 M ammonium carbonate (NH^HCO^.NH^COONH^-Analar) (pH 8.6) and samples were spotted onto e i ther Whatman No. k paper or Whatman No. 3 paper for preparative e lect rophores is . The maximum voltage of 750 vol ts was applied for 1.5 to 2.0 hrs. Af ter drying chromatograms overnight at kO C and e lec t ro -phoretograms for 20 min at 100 C, they were developed in the fol lowing manner. Carbohydrates were detected by dipping chromatograms in 0.12% m-periodate in acetone and spraying with 0.18% benzidine in acetone ( C i f o n e l l i , 195*0. Reducing sugars were detected by dipping chromatograms in 0.5% AgNO^ in acetone and developing in 0.5N NaOH in 70% N a ^ O ^ S H ^ (Smith, I960). 36 VIII . A h a l y t i c a l arid P r e p a r a t i v e T e c h n i q u e s 1. Dry w e i g h t The d r y w e i g h t o f c e l l s u s p e n s i o n s was d e t e r m i n e d by c e n t r i f u g i n g a known volume o f the s u s p e n s i o n , r e s u s p e n d i n g t h e p e l l e t in d i s t i l l e d w a t e r and r e - c e n t r i f u g i n g . The p e l l e t was r e s u s p e n d e d in o n e - t e n t h o f the o r i g i n a l v o l u m e , i_n d i s t i l l e d w a t e r . A measured a l i q u o t o f t h i s c o n c e n t r a t e d s u s p e n s i o n was p l a c e d i n a p r e - d r i e d , p r e - w e i g h e d aluminum d i s h . The d i s h and sample were d r i e d a t 95 C f o r 2.5 h r s , then p l a c e d in a vacuum d e s s i c a t o r a t room t e m p e r a t u r e f o r 2k h r s . The pans were w e i g h e d , and a g a i n p l a c e d under vacuum a t room t e m p e r a t u r e . T h i s f i n a l p r o c e d u r e was r e p e a t e d u n t i l t h e w e i g h t o f t h e samples was c o n s t a n t . 2. P r o t e i n P r o t e i n c o n t e n t o f samples was d e t e r m i n e d a c c o r d i n g t o the method o f Lowry e_t aj_. . ( 1 9 5 1 ) . The p r o t e i n s t a n d a r d used was egg a l b u m i n f i v e t imes r e c r y s t a l 1 i z e d . 3. O p t i c a l d e n s i t y measurements O p t i c a l d e n s i t i e s in the v i s i b l e range were d e t e r m i n e d u s i n g a Beckman model B spectrophotometer. Values for op t ica l densi t ies in the u l t r a - v i o l e t range were taken from spectra obtained using a Spectronic 505 or Spectronic 600 spectrophoto-meter (Bausch and Lomb). To record the change in O.D. with t ime, for enzyme assays, a G i l f o rd recording spectrophotometer, model 2000, was used. 4. Dephosphorylation of carbohydrates The removal of phosphate from phosphorylated intermediates was carr ied out by incubation with a commercial bacter ia l a l ka l i ne phosphatase in 0.1 M t r i s -HCl buffer (pH 8.0) at 37 C. 5. Glucose Samples were analyzed for glucose content with the enzymatic Glucostat procedure (Worthington Biochemical Corp. , Freehold, N . J . ) . IX. Chemicals, Enzymes and Substrates A l l chemicals, enzymes and substrates were purchased from commercial sources. Where necessary the substrates were neutral ized with NaOH. Barium was removed from substrates by treatment with Dowex-50 H + and the resul t ing free acids were neutral ized with NaOH. C-U-maltose, C-U-D-r ibose, C-1-D-galactose, C-14 1-lactose and C-U-aMG were obtained from the Nuclear 14 Chicago Corp. (Des P la ines , 111.); Me- C-thio-D-galactoside 14 and C-lhglucose were purchased from Schwarz Bioresearch Corp. (Orangeburg, N.Y. ) . A l l label led and unlabel led carbohydrates were found to be chromatographically homogeneous by the ethyl acetate /pyr id ine/ saturated aqueous bor ic acid solvent system. RESULTS AND DISCUSSION. I. Growth and Resting Cel l Suspensions The tendency of Gram-positive organisms, both anaerobic and facu l ta t i ve l y anaerobic, to undergo auto lys is has long been recognized (Jones, Stacey, and Webb, 1 9 4 9 ) . This rapid auto lys is occurs in both stat ionary phase cultures and rest ing c e l l suspensions and has prevented extensive metabolic studies from being carr ied out with C_. perfr?ngens and s im i la r micro-organisms. Jones, Stacey, and Webb ( 1 9 4 9 ) defined the system of au to ly t i c enzymes in C. per f r i hgens and in Staphylococcus c i t r eus , as consist ing of a ribonuclease and two pro teo ly t ic enzymes. It has been noted that , when in a magnesium-deficient, enriched medium, C_. perfringens formed filamentous c e l l s which were res is tant to auto lys is (Webb, 1 9 4 8 ) . Boyd, Logan, and Ty te l l ( 1 9 4 8 ) demonstrated that the production of toxins by C_. perf r i ngens could be repressed, by growing the organism in a defined medium. It was assumed that possibly a cor re la t ion existed between the production of exotoxins and au to ly t i c enzymes. A modif icat ion of Boyd, Logan, and T y t e l l ' s medium was employed and the s t a b i l i t y of c e l l s grown in th is semi-defined medium was compared with the s t a b i l i t y of c e l l s grown in the complex medium. To prevent loss by p rec ip i t a t i on , magnesium was added to the complex medium a f te r autoclav? ng. 1. Growth curves Cultures grown in the enriched and in the semi-defined media, with glucose as the energy source, produced somewhat d i f ferent growth curves. Af ter a considerable lag , rapid growth to a high c e l l density (O.D.^Q, approximately 4.5) was evident in the enriched medium (F ig . 1). The hydrogen ion concentration rose very rapidly during logarithmic growth (F ig. 1) and, as there was excess glucose present during the stat ionary phase, low pH was most l i k e l y the factor which l imited growth. Calculat ions from a logarithmic plot of the c e l l density (opt ical density at 660- my) versus time indicated that the generation time was approximately 48 min (F ig . 2) . Microscopic observations of Gram-stained preparations of the c e l l s from the complex medium demonstrated the typ ica l square-ended, r e l a t i ve l y short th ick , Gram-positive b a c i l l i , : described by other workers (Breed, Murray, and Smith, 1957). During log phase growth, the cultures produced considerable quant i t ies of gas and gave of f the rancid odor charac te r i s t i c of a mixture of acet ic and butyr ic ac ids . Neither high c e l l densi ty , high acid concentrat ion, nor low glucose concentration l imited growth in the semi-defined medium 41 F i g . 1. Growth.of C. p e r f r i n g e n s i n complex medium p l u s 0.7% g l u c o s e . o o, O.D. a t 660m u ; A — A , pH. 5.0 HOURS F i g . 2. Growth.of C. p e r f r i n g e n s i n complex medium p l u s 0.7% g l u c o s e . L o g a r i t h m i c p l o t o f O.D.,, v e r s u s t i m e . (F ig . 3). Rather, depletion of some required growth factor from the medium was more l i k e l y . When compared with the 2-3 hrs lag in growth, rout inely observed in the complex medium, the lag in both the primary and secondary cul ture in the semi-defined medium was neg l ig ib le . The generation time of the secondary cul ture in the semi-defined medium was Ml min, which was;, s i g n i f i c a n t l y shorter than the 51 min generation time of the primary cul ture (F ig. k). Microscopic observation of logarithmic phase c e l l s from the second cul ture in semi-defined medium revealed d ras t i ca l l y al tered c e l l u l a r morphology, although the Gram-reaction of the c e l l s was s t i l l pos i t i ve . The majority of the c e l l s were filamentous and much narrower than the c e l l s from the complex medium. The lengths of the fi laments varied considerably. In add i t i on , the gas produced by cultures grown in the semi-defined medium was great ly reduced, and the rancid odor was not detectable. 2. S t a b i l i t y of c e l l suspensions Resting c e l l suspensions prepared from c e l l s grown in the semi-defined medium, and suspended in 0.05 M t r i s -HCl (pH 7.4) _ o _|__|_ and 10 M Mg were stable to auto lys is as determined by measuring the O.D. of the suspensions at 660 mu. Ce l l s which had been grown in the enriched medium lysed rapid ly and a 60% decrease in O.D. 660 occurred during the course of a 3 hrs experiment (F ig. 5) . In prel iminary experiments, harvesting and resuspending of the kk 0 2 4 6 8 HOURS F i g . 3 . Growth.of £ . perfringens in secondary cul ture in semi-defined medium.plus 0 . 7 % glucose, o o, O .D .^^ Q ; A- —A, pH; Q '•—Q , glucose concentrat ion. k. ' G r o w t h o f C . ~ : p e r f r i n g e n s i n s e m i - d e f i n e d medium p l u s 0 . 7 % g l u c o s e . L o g a r i t h m i c p l o t o f O.D.g, 0 v e r s u s , t i m e , o- o , p r i m a r y c u l t u r e ; A - - A , s e c o n d a r y c u l t u r e . c e l l s from the complex medium were carr ied out under various condit ions of anaerobiosis, ag i ta t i on , and temperature. In add i t i on , the c e l l s were resuspended in various concentrations of phosphate and t r i s -HCl buf fers , as well as in 0.05 M t r i s -HCl (pH 7.4). with 10 - 2 M M g + + , 5% mannitol , 0.5% s o r b i t o l , or 3.5% sucrose. The rate of c e l l l y s i s was not affected by any of these attempts to produce stable c e l l suspensions. Suspensions of these c e l l s in 0.05 M t r i s -HCl (pH 7.**) containing 5% galactose or 0.05% glucose remained re la t i ve l y s tab le , however the pH of the medium decreased in both cases, demonstrating that galactose and glucose were being metabolized. In the absence of galactose or glucose, c e l l l y s i s commenced immediately, and th is indicated that the a c t i v i t y of the au to ly t i c enzymes were, essen t i a l l y , completely repressed by the presence of an adequate energy source. The concentration of casein hydrolysate in the semi-defined medium was the l im i t ing factor of growth, i f the primary cul ture of the semi-defined medium was allowed to reach the stat ionary phase. A tendency to form carbonaceous storage products has been observed in other organisms (Dawes and Ribbons, 1962; Doudoroff and Stan ier , 1959), when grown under condit ions of carbon excess and nitrogen l im i t a t i on . No d i rect evidence of such a product has been demonstrated in C. per f r i hgens. U t i l i z a t i o n of a carbonaceous storage product could have served the same purpose in repressing the a c t i v i t y of au to l y t i c enzymes as the addi t ion of low levels of glucose did in suspensions of c e l l s grown on complex medium. 2 3 F ig . 5. .Stabi1j ty of eel 1 suspens ions of C_." perfringens grown wi th 0.7% gl ucose i n : o o, complex medium; ©———©, semi-defined medium. As the c e l l s in the suspensions lysed and released the i r i n t r ace l l u l a r mater ia l , the u l t r av io le t absorption of the suspending f l u i d increased s i g n i f i c a n t l y . The O.D. at 260 my in the supernatant f l u i d s , increased by a factor of 4.68, over the zero-hour value with the c e l l s grown in the complex medium (Table I ) , and by a factor of 4.5 for the c e l l s grown in the semi-defined medium during a 3 hrs period (Table I I ) . However, the zero-time rat ios of the concentration of u l t r av io le t absorbing material (O.D. at 260 my) to the c e l l densi t ies (O.D. at 660 my) were found to be 0.0652 for the ce l l s grown on semi-defined medium (Table I ) , and 0.314 for the suspensions of c e l l s from the complex medium. This high i n i t i a l concentration of u l t r av io le t absorbing material in the suspensions of c e l l s from the complex medium demonstrated that a s ign i f i can t amount of l y s i s had occurred during the washing and resuspension procedures. This tendency to lyse immediately upon resuspension indicated that the c e l l s were induced to synthesize the au to l y t i c enzymes during growth on the complex medium. The fact that the suspension of c e l l s grown in the semi-defined medium did not have th is high i n i t i a l O.D .^^Q suggested that the synthesis of the au to ly t i c enzymes had been repressed, by some fac to r , during growth in th is medium. The i n i t i a l concentration of the c e l l suspensions could not have conferred s t a b i l i t y on the c e l l s grown in the semi-defined medium 49 T a b l e I. The a u t o l y s i s o f c e l l s u s p e n s i o n s o f C . p e r f r i hgens under s t a r v a t i o n c o n d i t i o n s . INCUBATION TIME (HR) OPTICAL DENSITY 1 660 my 260 my c e l l s u s p e n s i o n s u p e r n a t a n t f l u i d 5.10 1.60 5.10 2.60 1 4 ..85 5.40 2 3.85 5.70 3 2.30 7.50 3 hr /0 hr 0.45 4.68 Table II. The s t a b i l i t y of eel 1 suspensions of C. perfringens under starvat ion condit ions when previously grown in a chemically defined medium. STARVATION MEDIUM3 NO ADDITION 1 0 ~ 3 M MgCl 2 10~2 M MgCl 2 INCUBATION OPTICAL DENSITY TIME b 660 my 260 myC 660 my 260 my 660 my 220 my 0 2.91 0.19. 2.70 0.20 2.76 0.16 1 2.85 0.82 2.79 0.59 2.80 0.49 2 2.67 1.80 2.87 0.71 2.90 0.66 3 2.53 2.70 2.88 0.90 2.90 0.80 6 2.28 4.20 2.96 1.40 2.90 0.95 18 2.16 9.10 3.34 3.05 3.12 1.95 3 hr/0 hr 0.87 14.2 1,07 4.50 1.05 5.0 18 hr/0 hr 0.74 83.5 1.24 15.2 1.13 12.2 a - 0.05 in T r i s buffer (pH 7.4) ; ^ - O.D.ggg.of c e l l suspension; - O.D < 2gQ of supernatant f l u i d . as the rate of decrease in O . D . ^ Q , with time, for c e l l suspensions grown on complex medium, increased l i near l y with increasing i n i t i a l c e l l density ( O . D . ^ ) (F ig . 6). One of the major di f ferences between the complex and the semi-defined media was the subst i tu t ion of ac id-casein hydrolysate, for the proteose peptone as the source of amino ac ids . It has been demonstrated that exotoxins were not produced when the C_. per fri hgens was grown on the semi-defined medium (Boyd, Logan, and T y t e l l , 1948) and the importance of peptides for toxinogenesis by C. perfrihgens type D has been demonstrated (HauschiId, 1965)-If a cor re la t ion existed between the production of the exotoxins, ex t race l l u la r p ro teo ly t i c enzymes, and au to ly t i c enzymes (Jones, Stacey, and Webb, 1949), a l l three classes of enzymes may have been coordinately induced by the presence of the peptides in the complex medium. The absence of these substrates of p ro teo ly t i c enzymes from the semi-defined medium, could have repressed the synthesis of the three classes of enzymes, and thus produced stable eel 1 suspensions. Attempts were made to improve the stabi1 i ty of suspensions of c e l l s grown in the semi-defined medium by the addi t ion of various concentrations of Mg to the buffer used to wash and resuspend the c e l l s . S ign i f i can t changes in the rate of decrease in O.D .^^Q and increase in 0- u ' 260 w e r e observed (Table I I ) . SIight auto lys is of the suspension in 0.05 M t r i s -HCl (pH 7.2).was demonstrated with I of the i n i t i a l O . D . ^ Q remaining,after 3 hrs. During the same 6. The rate of l ys i s of suspensions of eel Is grown in complex medium, as a function of the i n i t i a l c e l l concentration of the suspensions. per iod, the O . D . ^ g was increased to 14.2 times the i n i t i a l -2 ++ value. The addi t ion of 10 M Mg to the rest ing suspension resulted in 105% of the i n i t i a l O.D.^Q at the end of 3 hrs , with only a f i ve - f o l d increase in 260 my absorbing mater ia l . -3 ++ The addi t ion of 10 M Mg gave resul ts nearly ident ica l to those found with the addi t ion of 10 M Mg (Table I I ) . , j | The addi t ion of the Mg ions may have served to s t a b i l i z e the eel 1 suspensions in e i ther of two ways, for which no evidence is presented. The existence of low levels of au to ly t i c enzymes in the suspensions of c e l l s grown in the semi-defined medium was evident from the slow, but d e f i n i t e , rate of l y s i s of the.eel 1 .suspensions.' Magnesium ions, in the concentrations used, may have d i r ec t l y inhib i ted the a c t i v i t y of the au to ly t i c enzymes. However, M g + + is a lso known to s t a b i l i z e the structure of ribosomes in v i t r o and in vivo (McCarthy, 1962; T iss ie res e_t aj_., 1959) and therefore the magnesium may have prevented the d issoc ia t ion of the ribosomes and the release of r ibonucle ic acid and proteins which would have served as substrates for the ribonuclease and pro teo ly t i c enzymes which have been demonstrated to be involved in auto lys is (Jones, Stacey, and Webb, 19^9). The u l t rav io le t absorbing material released by the stable suspensions of C. per f r i hgens (Table II) may have been the degradation products of the endogenous metabolism of the organism, as has been observed in other microorganisms (Campbell, Gronlund, and Duncan, 1963; Strange e_ta_l_., 1963). The gradual increase in c e l l numbers (O.D.ggg) ' n the stable suspensions (F ig . 2) may have resulted from th is u t i l i z a t i o n of an endogenous storage product. The lower concentration of Mg , 10 M_, conferred adequate s t a b i l i t y on the eel 1 suspensions, and was considered to be more su i tab le for use in routine studies of rest ing suspensions. II. G l yco l y t i c Enzyme Assays The Embden-Meyerhof pathway has often been implicated as being the major pathway of g lyco lys is in the C l o s t r i d i a (Bard and Gunsalus, 1950; Shankar and Bard, 1955a), however the enzymes of th is pathway have been demonstrated de f i n i t e l y in only one C los t r i d i urn, C. thermosaccharo1yticum (Lee and Ordal , 1967). One major reason for the lack of a successful demonstration of the complete pathway in C_. perfringens has undoubtedly been the existence of a strong NADH2-oxidase (Dol in, 1959a, 1959b), which interferes with the most e f f i c i e n t procedure for assaying g l yco l y t i c enzymes, that of fol lowing the oxidat ion or reduction of pyr id ine nucleotides spectrophotometr ical ly. The NADH2~oxidase a c t i v i t y in c e l l - f r e e extracts was considerably greater than the a c t i v i t y of phosphohexose isomerase, phosphofructokinase, and triosephosphate isomerase and more than ten- fo ld greater than hexokinase, 3-phosphoglyceraldehyde dehydrogenase, pyruvate k inase, and l a c t i c dehydrogenase. In prel iminary experiments, oxidase a c t i v i t y prevented the detection of a l l enzymes except those which could be l inked to NADP reduction v ia a commercial source of glucose-6-phosphate dehydrogenase. Attempts were made to decrease the oxidase a c t i v i t y by various procedures. Extended periods of i r rad ia t ion of c e l l -free extracts with l igh t at 365 my (Dol in, 1959b), using the monochrometer of a Bausch and Lomb Spectronic 20 spectrophoto-meter, f a i l ed to reduce the oxidase a c t i v i t y s u f f i c i e n t l y . -3 -h A combination of 10 M atabrine and 10 M H2O2 reduced the NADh^-oxidase a c t i v i t y to a low l e v e l , however, the r i sk of destroying other enzymes by oxidat ion with 1^ 02 and a lso the very high absorption at 3^ 0 my of a tabr ine, combined to severely l im i t the use of these compounds. Centr i fugat ion at 25,000 x g_ for 30 min caused p rac t i ca l l y no reduction in the oxidase a c t i v i t y and confirmed the reported soluble nature of the enzyme (Dol in, 1959a). Because of the enzyme's dependence on molecular oxygen as an electron acceptor for the oxidat ion of NADh^ (Dol in, 1959a), the possible usefulness of the exclusion of oxygen by preparing c e l l - f r e e extracts under . l ^ , and of assaying the enzymes anaerobical ly was examined. This technique reduced the NADr^ -oxidase a c t i v i t y of the extracts to less than \% of the a c t i v i t y 56 evident under aerobic condit ions (Table I I I , F ig . 7). That molecular oxygen was not the physio logical e lectron acceptor for the NADr^-oxidase has been demonstrated by the lack of involvement of H^O^ as an intermediate in the react ion, and by the fact that other enzymes of the organism produced tox ic quant i t ies of r^C^ when exposed to oxygen (Dol in, 1961). With the exception of 3-phosphoglyceraldehyde dehydrogenase, each of the enzymes of the Embden-Meyerhof pathway was measured spectrophotometrical ly by using the product of each react ion, as a substrate for one of the fo l lowing>. commercial ly prepared enzymes-, glucose-6-phosphate dehydrogenase, a-glycerophosphate dehydrogenase, or l a c t i c acid dehydrogenase. When required, the product of the enzyme being assayed was converted to the appropriate substrate by the use of a second commercial enzyme preparat ion. The commercial enzyme preparations were always added in excess, therefore, the enzyme in the c e l l - f r e e extract was ra te - l im i t i ng . A l l of the enzymes of the Embden-Meyerhof pathway were independently shown to be present in the extracts 2 and s p e c i f i c ' . ^ a c t i v i t i e s , expressed as umoles x 10 of product formed per min per mg of p ro te in , were calculated (Table I I I ) . Prel iminary growth studies demonstrated that the organism would grow readi ly on mannose, fructose and glucose. Kinase a c t i v i t y with mannose could not be demonstrated, however, the phosphohexose isomerase was act ive with mannose-6-phosphate.(Table I 11). The f a i l u re to detect the mannose kinase even in the presence of high concentrations of the substrate Table I I I . Spec i f i c a c t i v i t i e s of g l yco l y t i c enzymes in extracts of C. per f r i hgens. The extracts were prepared from eel 1s grown on semi-defi ned medi urn pi us 0.6% glucose, except where indicated otherwise. ORGANISM a ENZYME C_. perf r i hgens C_. thermosaccharolyticum spec i f i c a c t i v i t y Hexokinase glucose 5.87 ' 8.69 fructose 0.210 mannose 0 Phospho-hexose isomerase fructose-6-phosphate 21.4 13.4 mannose-6-phosphate 13.95 Glucose-6-phosphate dehydrogenase in extracts from semi-defined medium, log phase 0 semi-defined medium, s ta t . phase 0 complex medium, log phase 0 Phospho-fructo kinase 72.5 9.6 Fructo 1,6-diphosphate aldolase 3,940 1,84 Triose-phosphate isomerase 37.7 192 3-phospho-g1ycera1dehyde dehydrogenase 7.9 1.6 3-phospho-glycerate kinase 726 20.8 Mutase 210 840 Enolase 1 ,416 56,000 Pyruvate, kinase 5.85 1,28 Lac t ic acid dehydrogenase 8.5 Pyruvate-c las t ic system in extracts from enriched medium 16.3 88 semi-defined medium 4.1 semi-defined medium plus 5 mg Fe /ml 6.5 b 2 Lee and Ordal (1967); - . (TO.) umoles/min/mg prote in. 58 F i g . 7. The a c t i v i t y of NADH -oxidase in aerobic and c e l l - f r e e extracts or C. per f r i hgens. anaerobic and ex t rac t , indicated that e i ther the assay system was inadequate or a d i f ferent system for ac t iva t ing th is hexose was operative in the organism. A phosphoenol-pyruvate dependent phospho-transferase system has been demonstrated to phosphorylate several carbohydrates in E_. col ? and Aerobacter aerogenes (Kundig and Roseman, 1966). The resul ts from mannose transport studies suggest that a phospho-transferase system o r , at least a very s im i la r system, is probably the agent for ac t iva t ing mannose for metaboli sm in C. per fri hgens. Three-phosphoglyceraldehyde dehydrogenase was measured d i r e c t l y by recording the reduction of NAD in the presence of excess 3-phosphoglyceraldehyde. The enzyme was sens i t i ve to iodoacetamide (Krebs, 1955) and k um/ml of the inh ib i to r caused 100% i nh ib i t i on of a c t i v i t y . It was necessary to add 12 umoles/ml of sodium arsenate and 30 umoles/ml of sodium f luor ide to the assay mixture to obtain measurable a c t i v i t y of the enzyme (F ig. 8). The arsenate ion rendered the reaction i r revers ib le by sub-s t i t u t i ng for phosphate and subsequently forming the unstable product, 1-arseno-3-phosphoglycerate. This product decomposed to 3-phosphoglyceric acid and therefore, prevented the accumulation of the inh ib i tory end products of the dehydrogenase react ion (Krebs, 1955). Fluoride and arsenate ions inhib i ted enolase a c t i v i t y by forming a complex wi th Mg (BUcher, 1955), and consequently, th is prevented the oxidat ion of the accumulated 60 Fig. 8. 0.5 3 E O < 0.2 o a . O 0.1 0 TEST CONTROL - FLUORIDE _ ARSENATE + I0D0ACETAMIDE 2 S C U T E S The a c t i v i t y of 3-phospho-glyceraldehyde dehydrogenase in c e l l - f r e e extracts of C. perfr ingens. Control minus 3-phospho-glyceraldehyde. lodoacetamide (k mM) or the omission of f luor ide and arsenate from the reaction mixture inh ib i ts a c t i v i t y completely. NADH2 by the subsequent act ion of l a c t i c acid dehydrogenase. The p o s s i b i l i t y of interference by 3-phosphoglyceraldehyde dehydrogenase a c t i v i t y in other spectrophotometric assays was neg l ig ib le when f luor ide and arsenate were not present in the react ion mixture (F ig. 8). A comparison was made of the spec i f i c a c t i v i t i e s of the g l yco l y t i c enzymes in C. perfringens with those values determined by Lee and Ordal (1967) for glucose-grown cul tures of C_. thermosacchafblyti cum (Table I I I). In addi t ion to the g l yco l y t i c enzymes from glucose to pyruvate, l a c t i c acid dehydrogenase and the pyruvate-c last ic system were present in the organism. The method of assaying the pyruvate-c las t ic system had some undesirable charac te r i s t i cs which should be considered when interpret ing the resu l t s . The rate of acetyl-phosphate production in the react ion mixture decreased over the duration of the experiment (F ig . 9). This decrease in the rate of product accumulation may have been due to e i ther the conversion of acetyl phosphate to acet ic and butyr ic ac ids , two of the normal end-products of the metabolism of pyruvate in C. per f r i ngens or to the end-product i nh ib i t i on . Recently, Biggins and Ditworth (1968) demonstrated a control of the pyruvate-c last ic system in C.pasteurianum extracts by ADP and acetyl phosphate concentrat ions. Acetyl phosphate was found to.be a "p roduc t : i nh i b i t o r " of the c l a s t i c react ion. High ADP 62 5 1 0 M I N U T E S 15 ' F i g . 9. A c t i v i t y of the pyruvate-c last ic system iri c e l l - f r e e extracts of C_. perf r i hgens. The role of accumulation of acetyl-phosphate is extrapolated from the i n i t i a l rate. 63 concentrations stimulated t h e a c t i v i t y of the c l a s t i c system, probably by lowering the acetyl-phosphate concentration through st imulat ion of acetate kinase. The non-1inear accumulation of acetyl-phosphate wi th time precluded exact measurements of the a c t i v i t y of the pyruvate-c l a s t i c system, however, 1inear extrapolat ions from the i n i t i a l rates of product accumulat ion gave comparable estimates of the a c t i v i t y of the system under various condit ions (F ig. 9, Table I I I ) . The pyruvate-c last ic system was more act ive in c e l l s grown in the enriched medium than in the.semi-defined medium (Table M l ) . Attempts to st imulate the a c t i v i t y of th is system by the addi t ion of excess ferrous iron to the semi-defined medium were only p a r t i a l l y successful and resulted in an increase in a c t i v i t y of 60%. This increased a c t i v i t y however, was only k0% of the value obtained with eel Is grown in the enriched medium. This indicated that some factor other than F e + + (required for the synthesis of ferredoxin) was lacking in the semi-defined medium. The presence of both l a c t i c acid dehydrogenase and the pyruvate-e last ic system explains the heterofermentative production of l a c t i c , ace t i c , and butyr ic ac ids , as we11 as the production of carbon dioxide and hydrogen from glucose by cul tures of £ . per fri hgens. The f a i l u r e to demonstrate the existence of glucose"-6-phosphate dehydrogenase a c t i v i t y , e i ther NAD- or NADP-1inked, in the ex t rac ts , argued against the hexose-monophosphate pathway as a major route of g l yco lys is in th is organism. The f a i l u r e of glucose-grown c e l l s to accumulate acetate under condit ions of low F e + + concentrat ion, probably indicated the absence of the phosphoketolase system of g l yco lys i s (De V r i es , Gerbrandy, and Stouthamer, 1967). Five-carbon skeletons, for the synthesis of nucleic ac ids , are probably generated by a reversal of the t ransketolase-transaldolase system which was demonstrated in C_. perfringens by Cynkin and Delwiche (1958). I I I . Carbohydrate Transport An integral step in the u t i l i z a t i o n of carbohydrates by any c e l l is the transport ing of the carbohydrate across the semi-permeable membrane into the in te r io r of the c e l l . The a b i l i t y to transport carbohydrates s p e c i f i c a l l y and rapidly across the membrane and, to a greater extent, the a b i l i t y to accumulate metabolites wi th in the c e l l at concentrations many times above that of the external concentrat ion, confer a de f in i te b io log ica l advantage upon a microorganism. 14 The mechanism of transport of C-glucose by C. perfringens was studied with regard to i ts k i ne t i c s , s p e c i f i c i t y and energy dependence. The a b i l i t y to accumulate radioact ive carbohydrates and the nature of the accumulated product were also invest igated. As a resul t of the var iety of techniques used for studying metabolite uptake and for segregating the component functions of "membrane passage and metabolite accumulation", the term " t ranspor t " has come to have several meanings (Hengstenberg, Egan, and Morse, 1968). In these s tud ies , „the term transport has been considered to encompass the two funct ions, although, as w i l l be discussed, attempts were made to separate them in th is organism. 1. U t i1 iza t ion of carbohydrates by C. perfringens Growth of C. perfringens with various carbohydrates as the source of carbon was studied. Flasks with anaerobic semi-defined medium containing 0.5% of the appropriate carbohydrate were inoculated as for growth s tud ies , and the increase in O.D. at 660 my was observed over 5.5 hrs , two hours beyond the beginning of stat ionary phase of glucose-grown cul tures (Table IV). Carbohydrates which fa i l ed to support growth were added to 3 sequential subcultures of organisms growing on glucose, but no inducible u t i l i z a t i o n of these carbohydrates was observed. 14 2. Transport of C-carbohydrates (i) Transport of ^C -g lucose . When suspensions of eel Is grown with 0.5% pyruvate plus 0.1% glucose as the energy sources (pyruvate-grown eel 1s) , -2 and resuspended to 7.8 x 10. mg dry weight/ml (1.4 ycur ie / ymole) 14 in the presence of pyruvate and the tota l C incorporated into 14 the c e l l s was determined, the C entered the c e l l s at a l inear 66 Table IV. Growth of C. perfringens with various carbohydrates as the source of carbon. Data obtained from two separate t r i a l s . CARBOHYDRATE INITIAL FINAL Control 0.17 0.36 Sorbose 0.165 0.31 Mannose 0.2 1.11 Control 0.06 0.137 1-Arabinose 0.125 0.16 Xylose 0.125 0.44 Ribose 0.04 0.952 Trehalose 0.125 0.44 Sucrose 0.09 0.45 Lactose 0.12 0.465 Maltose 0.05 0.45 Fructose 0.105 0.435 Galactose 0.08 0.22 Glucose 0.12 1.38 rate for 1.5 min, and then the rate decreased during the remainder of the experiment (F ig. 10). As w i l l be demonstrated, 12, the concentration of C in the c e l l s at 1.5 min was several hundred times greater than in the external environment. ( i i ) Transport of ^C-carbohydrates other than glucose. Of the seven radioact ive compounds examined, only glucose and mannose were concentrated in detectable amounts (Table VI ) . Ribose, maltose, aMG, lactose and galactose were not concentrated in the c e l l s during the intervals studied. Growth of the organism on lactose did not induce an accumulation mechanism for th is carbohydrate. Attempts to study the process of equ i l i b ra t ion of these sugars across the c e l l membrane, or to detect concentrations only s l i g h t l y above the ex t race l lu la r l e v e l , were interfered with by the non-speci f ic retention of rad ioac t i v i t y by the membrane f i l t e r s even in the absence of 14 c e l l s . The amount of C retained was of the same order of magnitude as one would expect to enter the c e l l i f a concentration gradient was not establ ished. The amount of the retained rad ioac t i v i t y was s u f f i c i e n t l y e r r a t i c to obscure changes in the pool s i z e . This retained rad ioac t i v i t y could not be removed by washing the f i l t e r s twice with medium, by pre- t reat ing the f i l t e r with unlabel led substrate at the same 14 concentration as the C-substrate, or by varying the I 2 3 4 5 M I N U T E S -6 14 1.0. Early time course of uptake of 8 x 10 M C-lhglucose (1.4 yc/ymole) by whole c e l l s of C. perfr ingens. temperatures of the f i l t e r holder and wash medium. In prel iminary experiments, the organism was shown to grow readi ly with any of these carbohydrates as the carbon source, except aMG, which was not tested. Therefore, i t is l i k e l y that these compounds entered the c e l l by f a c i l i t a t e d d i f f us ion . However, the l imi ta t ions discussed above make studies o f . t h i s type of transport process a technical imposs ib i l i t y at present. 1 /, ( i i i ) Temperature dependence of C-glucose transport . The rate of transport was dependent on the temperature of the react ion mixture as determined by observing the tota l 14 accumulation of C at 37 C and 30 C. While the maximum s ize of the pool remained nearly constant, the i n i t i a l rate of uptake was found to be considerably less at the lower temperature (F ig. 11). Although the c e l l s were grown at 37 C, i t was found necessary to rout inely observe the uptake of carbohydrates at 30 C to obtain adequate periods of l inear uptake. The i n i t i a l rate of uptake was found to increase by 81% over the seven degree change in temperature (F ig . 11). The O^Q of the transport system was estimated from these data to be 2.54. This value compared well with the theoret ical value of 2.0 for chemical react ions, and with the value of 2.2 found for components of the glucose transport system'in E. co l i (Hoffee et a l . , 1963). TOTAL UPTAKE 11. Total uptake of rad ioac t i v i t y by cells of X . with 1.25 x 10-5 M 1^C-U-glucose (1.4 yc/ymoleT o- o, at 37 C; A— 4 , at 30 C. Ingens (iv) Energy requirement of the transport of glucose. The processes of accumulation have been shown to be energy dependent for $-galactosides (Kepes, 1957), galactose (Horecker, Thomas, and Monod, 1-9'60a), and amino acids (Kay, 1968). Hoffee, Englesberg and Lamy . (1-963)' demonstrated that a lack of energy caused an increase in accumulation by the glucose transport system in suspensions of E. co1 i . Attempts to demonstrate energy dependence of transport in £ . perfringens were hampered by the fact that the normal uncouplers of energy for transport systems, such as 2 ,4 -d i n i -trophenol (Winkler and Wilson, 1966; Horecker, Thomas, and Monod, 1960a), are of l i t t l e use in l im i t i ng the energy y ie ld ing reactions of an obl igate anaerobe. This could be due to the fact that th is organism undoubtedly re l i es largely on substrate level phosphorylation. The addi t ion of pyruvate to the c e l l suspension was demonstrated to be necessary for the achievement of reproducible rates of accumulation (F ig . 12). However, i t s omission from the resuspension medium did not el iminate accumulation, but did reduce the i n i t i a l rate of uptake by approximately 50%. Af ter the f i r s t 0.5 min of the experiment, the rate of accumulation Ik of C was demonstrated to increase rap id ly . Addit ion of 1.5 mM iodoacetamide to the solut ion without pyruvate during resuspension 72 +PYRUVATE -PYRUVATE -PYRUVATE + IODOACETAMIDE 0.5 M I N U T E S .5 F i g . 12. Total incorporation of rad ioac t i v i t y by c e l l suspension with 8 x 10"^ M.glucose . (1.4 yc/ymole). o o, with 0.6% pyruvate; A — : A». minus pyruvate, Q rj, minus pyruvate, plus 1,5 x 10_3 M iodoacetamide. of the c e l l s did not af fect the i n i t i a l rate of glucose accumulation without pyruvate, but prevented the increase in the rate observed when iodoacetamide was not present (F ig. 12). The a b i l i t y of th is organism to metabolize f i ve of the amino acids present in the medium (Woods and Trim, 19^2), probably accounted for the base level of uptake observed in the absence of added pyruvate. Attempts were made to s impl i fy the resuspension medium in order to define more c lose ly the source of energy for accumulation. Various buffer solut ions and combinations of the components of the medium with and without added pyruvate, were used to wash and resuspend the c e l l s but only the complete semi-defined medium with 0.6% pyruvate was found to y i e l d reproducible rates of accumulation. The increase in the rate of transport with time in the pyruvate-less c e l l s was probably due to the supply of energy from metabolism of the glucose which had entered the c e l l s during the ear ly time course of the experiment. Iodoacetamide has been shown to inh ib i t the production of energy by g l y c o l y s i s , through inh ib i t ing 3~phosphoglyceraldehyde dehydrogenase a c t i v i t y (Krebs, 1955). Act ive transport in cer ta in systems has been shown to occur by the counter-transport of a s im i la r compound using the same membrane ca r r i e r (Wilbrandt, and Rosenberg, 1961). It was d i f f i c u l t to demonstrate that pyruvate or some metabolic product of pyruvate was not involved in counter-transport with glucose, thus providing the energy required for accumulation. However, i t is un l ike ly that a system capable of d i f fe ren t ia t ing between sugars epimeric in one hydroxyl , as w i l l be shown, would a lso recognize a low molecular weight a c i d . Therefore, i t would appear that the accumulation of glucose was dependent on energy gained, at least in par t , by the metabolism of exogenously added pyruvate. (v) K inet ics of accumulation of ^C-g lucose and 14_ C-mannose. 14 Exposure of pyruvate-grown c e l l s to C-U-glucose resulted in typ ica l saturat ion k ine t ics for to ta l uptake. The rate of uptake of glucose was found to increase 1inearly with concentration of substrate unt i l 0.01 mM, then the rate of change in ve loc i ty with increasing substrate concentration decreased as the ve loc i ty approached a maximum (F ig . 13). When the rates were plotted in the Lineweaver-Burke fashion, a l inear re la t ionship between the reciprocal of the ve loc i t y of uptake and the reciprocal of the substrate concentration was' found (F ig . 14). Three separate t r i a l s were performed for qlucose (F ig. 15) and the values of K and V for the three y t max t r i a l s were estimated and averaged (Table V) . A s im i la r response of the rate of uptake of mannose to increasing external concen-t ra t ion of mannose was observed with saturat ion of the uptake mechanism becoming apparent af ter 0.016 mM mannose was present (F ig . 16). Values of K and V were estimated from the t max reciprocal plot of these data (F ig. 17), and compared to those values for glucose uptake (Table V) . 0 0.01 0.02 GLUCOSE CONC mM 0.03 F ig . 13. Saturation k ine t i cs .o f C-glucose (1.4 yc/ymole) incorporation by £ . perfr ingens. Ce l ls at 30 C were exposed for 1 min to ' increas ing concentrations of l 4 C - g l ucose. The rate of to ta l uptake is plotted against concentration of glucose. 2 w G L U C O S E C O M C x IO"° M F i g . 14. Saturation k ine t i cs .o f O g l u c o s e (1.4 yc/ymole) incorporation by C. perfr ingens.• Liheweaver-Burke plot of rate of uptake against glucose concentration from t r i a l in Figure 13. 4LCELLS) -- v. V . ' ^ 1 o * ' • i ' 1 0 I 2 3 4 §  G L U C O S E C O N C x 10" U F i g . 15. Saturation k inet ics of 1 ^C-glucose (1.4 yc/ymole) incorporation by C . perfr ingens. Lineweaver-Burke plot of rate of uptake against glucose concentration for three d i f ferent t r i a l s performed as in Figure 13. The change from l inear response of the rate of uptake to substrate concentration was demonstrated to occur over a very narrow range of substrate concentrat ion, a phenomenon which has been shown to be typ ica l of carbohydrate transport systems (Hoffee, Englesberg, and Lamy, 1964; Egan and Morse, 1966; Horecker, Thomas, and Monod, 1960). This abrupt saturat ion of the transport system caused technical d i f f i c u l t i e s in accurately determining the changes in the rate of uptake as the transport system approached saturat ion. The s l i gh t var ia t ion in the slope of the three reciprocal plots of ve loc i ty versus substrate concentration for glucose and the subsequent var ia t ion in the estimates of the K and V values resulted from th is t max uncertainty. While the K value for mannose transport was over three times as large as the average value for glucose transport, the V for • ft m a x mannose was only 33% larger than the average value for glucose (Table V). The values, while not absolute measures of a f f i n i t y for the substrate, served to indicate that the accumulation mechanism had a much higher a f f i n i t y for glucose than for mannose. The di f ference between the V values for the transport of the max r sugars was small enough to indicate that both carbohydrates were accumulated at approximately the same maximum rate. It has been demonstrated that glucose and mannose each inhib i ted the accumulation of the other, and thus were both accumulated by the same mechanism, probably at the same maximum ve loc i t y . Values of K of the order of 10 ^ M have been demonstrated to be typ ica l of microbial carbohydrate transport mechanisms (Egan, 79 o.oso F ig . .'16. 0 . 0 2 0 0.025 Saturation k inet ics of ' 'C-mannose (1.4 uc/umole) incorp-orat ion by C. : per f r ingens. Ce l l s at 30 C were exposed for 1 min to increasing concentrations of C-mannose. The rate of total uptake is plotted against the concentration df mannose. i.O o 0 0.2 0.4 0.6 0.8 I MAM.MOSE CONG % 80-% . 17. Saturation k inet ics of C-mannose (1.4 yc/ymole) incorporation by C. perfr ingens. Lineweaver-Burke plot of the rate of uptake against mannose concentration from Figure 16. 81 Table V. Saturation k ine t ics of the glucose-mannose transport system. Estimated values of K t and ^ x ' f r o m t r i a l s in Figures 15 and 17. TRIAL K (M) V a t max Glucose A 2.0 x 10~5 38.9 B 1.67 x 10~5 31.3 C 2.38 x 10~5 29.5 Average 2.02 x 10~5 33.2 Mannose 7.70 x 10~5 kk.k - ymoles/min/mg dry weight. 82 and Morse, 1966; Hoffee, Englesberg, and Lamy, 1964; and Horecker, Thomas, and Monod, 1960). It should be noted that th is exh ib i t ion of saturat ion k inet ics did not d i f fe ren t ia te between enzymatic and adsorptive processes of accumulation (Egan, and Morse, 1966). (vi) S p e c i f i c i t y of the transport of glucose and mannose. As previously mentioned, the demonstration of 14 saturat ion k ine t ics for the accumulation of C-carbohydrates did not d i f f e ren t ia te between adsorptive and enzymatic mechanisms. In order to demonstrate that the concentrative mechanism was enzymatic, i t was necessary to ascerta in that the accumulation of carbohydrates was s p e c i f i c , a property that adsorption would not have exh ib i ted. 14 14 When the eel 1s were exposed to C-U-glucose, or to C-U-14 mannose, and the tota l uptake of C observed over 1.5 min, a 14 l inear concentration of C with time occurred for both sugars (F ig. 18a, 19a). In para l le l experiments, unlabel led carbo-14 hydrates were added to 100 times the concentration of the C-substrate, and the rate of tota l incorporation was compared to 12 the rate observed in the absence of added C-carbohydrate. The demonstration that for both glucose (F ig. 18) and mannose (Fig. 19) most carbohydrates did not competi t ively inh ib i t transport indicated that not al1 carbohydrates metabolized by the c e l l s were transported 0 B O 60 90 0 10 S O 9 0 SECONDS F ig . 18. Competition for glucose uptake in C. perfr?hgens. The rate of incorporat ion o f .8 x 10~ b , H C - g l ucose (1.4 yc/urnole) in the presence or absence of 8 x 10~ M C-carbohydrates. A. o—, C-glucose o n l y ; © — , '^C-pentoses added (r ibose, xy lose, arabinose). B . 0 — , 2 C-d isacchar ides except maltose added ( lac tose, sucrose, t reha lose) ;A— , C-maltose. ± so 0 3 0 S E C O N D S Competition for glucose uptake in C. perfr ingens. The rate of incorporation of 8 x 10" 6 ^C-g lucose Cl . 4'uc/ymol e) in the presence or absence.of C-carbohydrates. C. A-galactose; 0 — , 1 ^C-mannose; ®—, C-glucose only : A-—. ' T - m a H n i glucose control . C-f ructose; g-D. C-glucpse control —, C-mannose; fc into the ce l l by a common mechanism, but rather that glucose and mannose were accumulated by mechanisms of a highly spec i f i c nature. The mutual inh ib i t i on of mannose and glucose concentration by each other (Table VI) demonstrated that they were accumulated or transported by the same mechanism (Wilbrandt, and Rosenberg, 1961). It was demonstrated that f ruc tose, r ibose, and sucrose, a l l readi ly metabolized by th is organism for growth, did not inh ib i t uptake of e i ther mannose or glucose, while the disaccharide maltose did reduce the rate of accumulation (Table V I ) . Thus, the s p e c i f i c i t y of the accumulation mechanism was not determined so le ly by the s ize or number of r ings , but was probably determined by the conf igurat ion of the substi tuents on the carbohydrate r i ng . At th is point i t is necessary to consider the d i s t i nc t i on between transport and accumulation. These functions have been separated in several metabolite transport systems (Winkler, and Wilson, 1966; Osborn, McLel lan , and Horecker, 1-961; and Kay, 1968) by observing the s p e c i f i c i t y of displacement of preloaded 14 pools of one C-carbohydrate when a second, unlabel led carbo-hydrate was added. Competitive inh ib i t i on of i n i t i a l uptake was considered to act on e i ther the transport mechanism or the accumulation mechanism. However, the displacement of an accumulated pool was only possible i f the two compounds shared the same accumulation mechanism. Stable pools of metabolites were found necessary for th is type of study, and techniques for generating these pools are 0 2 0 4 0 S O SO 0 20 4 0 6 0 S O S E C O N D S F i g . 19. C o m p e t i t i o n f o r mannose up take in £ . p e r f r i n g e n s . The r a t e o f i n c o r p o r a t i o n o f 1.5 x 10"5 M 1 ^ C - m a n n o s e (1.4 y c / y m o l e ) in the p r e s e n c e o r a b s e n c e o f 1.6 x 10"3 M 1 2 C - c a r b o h y d r a t e s . A . 0—, ' ^C-mannose a l o n e ; A — , 1 2 C - s u c r o s e ; t r e h a l o s e , 1 a c t o s e ; iH - , 1 2 C - m a 1 t o s e a d d e d ; 0 — , l 2 C - g l u c o s e a d d e d ; © — , 1 2 C - m a n n o s e c o n t r o l . B . Q — 1 2 C - f r u c t o s e ; /S—, 1 2 C - a M G a d d e d . oo A S E C O N D S 19- Competition for mannose uptake in C. perfr ingens. The rate of incorporation of 1.5 x 10"-^  M ^C^mannose (1.4 yc/ymole) in the presence or absence of 1.6 x 10 1 2 C-carbohydrates. C, A- , 1 2 C - x y l o s e added;D—j Sc-arab inose added; f— , C-ribose added. 88 Table VI. Accumulation of carbohydrates and competitive inh ib i t ion of the rate of up take^ f C-glucose (8 x 10"? mM, 1.k uc/umole) and C-mannose (1.6 x 10" 2 mM, 1 A uc/umole), by a 100-fold excess of these carbohydrates. 12 C-carbohydrate Accumulation by % Inhib i t ion of added in 100-fold C_. perfringens 14C -uptake C-glucose ^C-mannose excess , , , 1 '14, 1 ^ r - n I n r n c p f Lactose 0 0 0 Trehalose - 0 0 Sucrose - . 0 0 Maitose 0 33 52 Ribose 0 0 0 Xylose - 0 0 Arabinose - 0 0 aMG 0 0 0 Galactose 0 0 0 Fructose 0 0 0 Glucose + 100 100 Mannose + 79 100 known for other organisms (Kay, 1968; Hoffee, and Englesberg, 1962). The techniques used depended upon the use of non- or slowly metabolizable substrates to form the pools, or the use of mutant s t ra ins of the organism that did not immediately degrade the pooled mater ia l . Neither non-metabolizable analogues which could be accumulated, nor adequate mutant se lect ion techniques, were ava i lab le for the study of transport in C. perfr ingens. Studies of the transport mechanism alone are possible i f analogues of carbohydrates that are transported, but not accumulated, are ava i lab le . The competitive inh ib i t ion of the '•14' transport of C-U-aMG was attempted, but, as mentioned prev ious ly , non-speci f ic retention of the rad ioac t i v i t y on the membrane f i l t e r obscured the re la t i ve l y small changes in 14 incorporated C leve ls . The f i na l possible method for segre-gating transport from accumulation was the use of metabolic poisons to remove the energy supply for those accumulation mechanisms which are energy dependent (Wilbrandt, and Rosenberg, 1961; Winkler, and Wilson, 1966; Hoffee et a_j_., 1963). As w i l l be discussed, i t was not possible to completely destroy accumulation through energy l im i t a t i on . Had i t been possible to remove accumulation by th is technique, the technical d i f f i c u l t i e s mentioned previously in observing simple equ i l i b ra t ion of substrates across the membrane would have in ter fered. Therefore, as i t proved impossible to d i f fe ren t ia te between the functions of trans-membrane passage and accumulation of carbohydrates in C. per f r i hgens, the terms transport and accumulation w i l l be used interchangeably. Based on the s p e c i f i c i t y of inh ib i t i on of the uptake of mannose and glucose, the st ructura l requirements of a substrate of the accumulation process were e luc idated. The transport mechanism was shown to have the higher a f f i n i t y for glucose (Table V ) , with substi tuents or with any modif icat ion in structure reducing i t s a c t i v i t y . In compounds with any changes other than at the C-2 pos i t i on , accumulation was completely el iminated. Epimerization of the -OH at the C-2, to produce mannose, raised the K value by a factor of three, but s t i l l allowed appreciable accumulation and approximately the same V m a x as for glucose accumulation (Table V) . As a resul t of th is lowered a f f i n i t y , the competitive a c t i v i t y of mannose was found to be only 79% that of glucose, for glucose uptake (Table VI ) . Replacement of the C-1 hydroxyl with any subst i tuents, as in aMG, t rehalose, or sucrose, completely destroyed the transport a c t i v i t y . The sugar a l l o s e , which is the epimer of glucose at the C-3 hydroxyl , was not ava i lab le for study of i t ' s inh ib i tory a c t i v i t y . Epimerization of the C-k hydroxyl group to form galactose, completely el iminated a c t i v i t y as a competitor for the glucose-mannose accumulation system, and was not i t s e l f accumulated by the system. However, subst i tu t ion of the 4-hydroxyl in the (3-conf igurat ion, by the formation of maltose, a 1 lowed 33% of the inh ib i tory a c t i v i t y to remain, while accumulation was found to be-el iminated. The s im i la r subst i tu t ion with galactose at the k carbon of glucose in the B-configuration to form lactose, completely el iminated both forms of a c t i v i t y in the accumulation system. Therefore, on the basis of these resu l t s , the s p e c i f i c i t y of the glucose-mannose accumulation system was found to be only p a r t i a l l y dependent upon the conf igurat ion about C-2, and very sens i t i ve to changes in the conf igurat ion about C-k. The system was a lso found to be very sens i t i ve to substi tuents at C-1 , and subst i tu t ions at C-k resulted in varying degrees of reduction of competitive a c t i v i t y . Studies by Hagih i ra, Wilson, and L in (1963) on the s p e c i f i c i t y of glucose uptake by E. cbl i , using der ivat ives of aMG, demonstrated that substi tuents on C-2 af fected the a c t i v i t y of the carbohydrates. Other workers have demonstrated 30% i nh ib i t i on of aMG concentration by £. cbl ? by the addit ion of maltose, whi le mannose and sucrose each caused a 20% i nh ib i t i on of a c t i v i t y (Hoffee, Englesberg, and Lamy, 1963). The s p e c i f i c i t y of the glucose concentration system in E. cb l i appeared therefore to d i f f e r great ly from that demonstrated in s us pens'? ons of C. perf r i hgens, 1 n that the conf igurat ion about the C-2 in the substrate or competitor of the C. perfringens mechanism was not found to be c r i t i c a l , whi le changes at th is posi t ion reduced the rate of accumulation in suspensions of E. cb1 i . The "anaerob ic transport mechanism" for carbohydrates in yeast, as described by Scharff and Kramer (1962), did not appear in C. perf r l hgens as fructose was shown to be transported by the yeast system, while i t had no a c t i v i t y in the glucose-mannose accumulation system studied. The "cons t i tu t i ve monosaccharide transport system" of yeast ( C i r i l l o , 1968) is more s im i la r to the system described for C. perfr ingens. Subst i tut ion of the anomeric.hydroxyl completely destroyed a c t i v i t y , and, substi tuents or conf igurat ional changes at a l l other carbons except C-2 decreased the a c t i v i t y of the glucose r ing in both systems. However, the carbohydrates were compared as competitive inh ib i tors of the transport of d-xylose, or of ^-sorbose, which are not metabolized by yeast. D-xylose was found to be inact ive in the system for the accumulation of glucose by C. perfringens l im i t i ng the value of comparisons between the two systems. Studies of the s p e c i f i c i t y of the carbohydrate accumulation system of the Gram-positive S. aureus, have indicated that carbohydrate accumulation is very s p e c i f i c , with very l i t t l e competition for entry between the carbohydrates. Maltose had no detectable ef fect on the rate of concentration of glucose, and mannose was not tested for a c t i v i t y in the system (Egans, and Morse, 1966), making comparisons with the s p e c i f i c i t y of C_. perfr?hgens uptake d i f f i c u l t . 14 3. The pooling of C-glucose (i) Pool capaci ty. In experiments in which both the total incorporation and the Jncorporation into the cold TCA insoluble materia 1 were measured over 40 min, the amount of C in the soluble pool rapidly reached a maximum value (F ig. 20, 21), a f ter which the s ize of 14 the pool remained re la t i ve l y constant as the C of both the total 93 suspensions with 8 x 10" .M 1 C-U-glucose (1.4 uc/umole) 0-0, to ta l incorporat ion; A—A, soluble radioact ive pool ; El—Q, cold TCA prec ip i ta te . . 94 TOTAL 0 10 2 0 3 0 4 0 i y i i N U T . e s 21. Formation of cold TCA so lub le , radioact ive pool by eel 1 suspensions in the 1.82 x l O " ' ' M 1 ^C-U-g l ucose (1.4 yc/ymole). 0-0, to ta l incorporat ion; A—A, cold TCA p rec ip i ta te ; D—0, soluble radioact ive pool . 95 and cold TCA insoluble f ract ions increased at the same rate. The pool s i ze decreased towards the end of the experiment, presumably due to the metabolism of glucose (F ig . 21). The decrease in the rate of uptake a f ter the i n i t i a l 1.5 min, may have been the resul t of any of several events. The transport system may have become l imited by the deplet ion of exogenous glucose, by the rate of energy production or by the 14 equi l ibr ium constant of the transport process. The C-glucose may have been rapidly metabolized by the c e l l to intermediates or end-products which were released to the medium, making the system g lucose- l im i t ing . When the pool s ize was maximal (F ig . 10), the f i l t r a t e of the suspension was concentrated and passed through a combined column of Dowex-50-H+ and Dowex-1-formate ion-exchange res ins . Essen t ia l l y a l l of the rad ioac t i v i t y was eluted f ron ta l l y with d i s t i l l e d water. When the eluate was chromatographed, i t was found to co-chromatograph with the glucose standard. Thus, glucose had not been converted to a degradation product and the system had not become g lucose- l im i t ing . When the density of the c e l l suspension was reduced by one-half in an attempt to prolong the time required to form the maximum pool s i z e , the 14 rate of uptake and the level of the C-pool were also decreased by one-half (F ig. 22). This demonstrated that the rate of accumulation, as well as the total accumulation, was proportional to c e l l mass and that the transport enzymes were saturated under these experimental condi t ions. The decrease in maximum pool s i z e , proportional to the decrease 96 T O T A L 20 40 60 80 SECONDS FIG. 22. Total incorporation of rad ioac t i v i t y by c e l l suspensions with2.5 x 10"5 M 1^C-U-glucose.(1.4 yc/ymole).A-A, 7.8 x 10"2 mg dry weight of c e l l s / m l ; 0-0, 3.9 x 10 dry weight of c e l l s / m l . in c e l l mass, indicated that some property of the c e l l l imi ted the s ize of the pool of accumulated carbohydrate. Assuming that the concentration of the carbohydrate was f a c i l i t a t e d by an energy-requir ing system, then the s ize of the pool may have been determined by the energy supply or by the rate of energy production. As the c e l l s were resuspended in 0.6% pyruvate at the beginning of the experiments, and as the rate of accumulation of C remained constant over several hours of sequential experiments, the rate of energy production, rather than the amount of substrate ava i l ab le , may have been the l im i t ing fac tor . An a l ternat ive explanation is that the equi1 ibrium constant of the transport process, a measure of the a f f i n i t y of the transport mechanism for the carbohydrate, may have l imi ted the pool s i ze by def in ing the maximum ra t io of internal to external concentrations of the carbohydrate. As w i l l be shown l a te r , the C inside the c e l l was in the form of a charged der ivat ive of glucose, and therefore was not in a form d i rec t l y in equi l ibr ium across the membrane with the glucose. However, the ra t io of the concentration of the der ivat ive to the concentration of free exogenous glucose determined by the rate constant of the accumulation mechanism, could have defined the maximum pool s i ze poss ib le . ( i i ) Internal concentrat ion. During the ear ly time course of transport the C entered the c e l l s at a l inear ra te , and a f ter 1 min, 32% of the to ta l label was present in the soluble pool (F ig. 20). 14 There was a 15 to 20 sec lag in the incorporation of the C into the cold TCA insoluble f rac t ion and th is was followed by increasing rate of incorporation for the remainder of the experiment. 14 The s i ze of the soluble pool of C was estimated from the 14 level of soluble C at 1.5 min (F ig . 20), a f ter which time the rate of incorporation decreased as the pool capacity was reached (F ig . 10). Based on the assumption that 80% of the c e l l weight was water (Lur ia , 1966), and that 10% of th is c e l l was in ter -14 c e l l u l a r , the concentration of the C in the i n t race l l u l a r water at 1.5 min was calculated to be 396 times the concentration of 14 14 14 the ex t race l l u la r C. Losses of C as C 0^ from the system over the course of the experiments were neg l ig ib le . A comparison of th is concentration ra t io with values obtained with other microorganisms was not pa r t i cu la r l y valuable because of the wi ld range of published ra t i os . 4 Galactose was concentrated by 10 - f o l d in E. cb l i (Horecker, 3 Thomas, and Monod, 1960a). Lactose was accumulated 7.2 x 10 times above the external concentration in S. aureus whi le the same organism was found to concentrate ma 1tose by a factor of 700, sucrose by a factor of 520, and aMG by a factor of 370 over the extracel1ular concentration (Egan, and Morse, 1966). These la t te r values are of a magnitude comparable with the level of accumulation demonstrated in C. per f r i ngens. Higher concentration factors have been demonstrated for the accumulation of amino acids by microorganisms, with values running into the tens of thousands (Br i t ten and McClure, 1962). ( i i i ) Nature of the pooled C. The accumulation of pools of soluble C which occurred 14 when suspens ions of C. perfr? hgens were exposed to C-U-glucose has been described as the accumulation of glucose, even though the nature of the pooled material was not known. In S . aureus, however, i t has been demonstrated that maltose, sucrose, aMG, lac tose, and isopropyl - th io galactosides were accumulated as phosphorykated der ivat ives (Hengstenberg, Egan, and Morse, 1968). In add i t i on , the impl icat ion of the phosphoenolpyruvate-dependent phospho-transferase system for the phosphorylation of carbohydrates, in the accumulation of carbohydrates by E. col? and S. typhimuriurn has been demonstrated (Tanaka and L i n , 1967; Tanaka, Fraenkel, and L i n , 1967; Simoni et_ aj_., 1967). An attempt to e luc idate the 14 nature of the C material accumulated wi th in the c e l l indicated 14 that in £ . perfr ingens, the C from glucose and mannose was concentrated as a der iva t i ve . 14 When the C material present in the cold TCA soluble pool at 0.5 min was isolated and concentrated, chromatography of 14 the pool demonstrated no detectable C which chromatographed as free glucose or as free mannose. Rather, in both cases two peaks containing a l l of the rad ioac t i v i t y were shown to remain near the o r i g i n . When al iquots of the pool were separated by e lect rophores is , some free carbohydrate was present near the o r i g i n of the electrophoretogram, probably as a resul t of hydrolysis during preparation or during the actual e lec t ro -phoresis. The two charged peaks were observed to move the same distance as glucoses-phosphate and fructose 1,6-di -phosphate standards with approximately 65% of the label in the singly-charged f rac t i on . The remainders of the concentrated pools were separated by preparative e lect rophores is , and the f ract ions were recovered and concentrated. Al iquots of these 14 14 f ract ions from both C-glucose and C-mannose pools were dephosphorylated with commercial bacter ia l a l ka l i ne phosphatase. The dephosphorylated f ract ions were concentrated, and then co-chromatographed with f ructose, glucose and mannose standards. Upon dephosphorylation, a l l of the pooled materials chromatographed 14 as uncharged hexoses. The pool of C-glucose was then found to contain monophosphorylated glucose, monophosphorylated fructose 14 and diphosphorylated f ructose. The accumulated pool of C from 14 C-mannose contained monophosphorylated der ivat ives of mannose, glucose and f ructose, in addi t ion to a diphosphorylated der ivat ive of f ructose. The pooled material isolated af ter 4 min exposure 14 to C-glucose was found by electrophoresis to be largely in the highly charged f r ac t i on , probably as a diphosphorylated der ivat ive of f ructose. It has been demonstrated that c e l l membranes which are re l a t i ve l y impermeable to charged der ivat ives of carbohydrates, al low the transport of the free carbohydrates (Hengstenberg, Egan, and Morse, 1968). Thus, an e f f i c i e n t method of concentrating carbohydrates in the c e l l would be to phosphorylate them on the inside of the membrane. The uncharged carbohydrate would be transported in by f a c i l i t a t e d d i f f us i on , and the charged product of phosphorylation would be unable to leave the c e l l , thus forming a concentration gradient of carbohydrate. Although an act ive soluble hexokinase could have phosphorylated the glucose and mannose during accumulation by th is organism, evidence has been accumulated for a phospho-transferase or k inase, of the membrane-bound type described for E. co l i (Kundig, Ghosh, and Roseman, 1964). Comparisons were made of the s p e c i f i c i t y of the accumulation mechanism with that determined for the hexokinase a c t i v i t y in c e l l - f r e e ext rac ts . The hexo-kinase was found to phosphorylate glucose and f ructose, with fructose being phosphorylated at a rate 10% that of glucose (Table I I I ) . No a c t i v i t y of hexokinase for mannose was observed, even when excess quant i t ies of mannose and extract were added. While the f a i l u r e to detect th is enzyme did not completely disprove i t ' s existence, there remained 1 i t t l e probabi1ity that mannose-kinase was involved in the accumulation of mannose. 14 Studies of the s p e c i f i c i t y of C-carbohydrate accumulation mechanism have demonstrated the opposite e f fec t . Fructose i f neither accumulated nor has any a c t i v i t y in the inh ib i t ion of glucose accumulation, whi le mannose is accumulated and inh ib i ts the rate of glucose uptake by 70%. It therefore appeared that the phosphorylation of the carbohydrates during accumulation was performed by a mechanism which d i f fered from that of the soluble hexoki nase. The absence of detectable free glucose or mannose in the pooled material indicated that these carbohydrates were phosphorylated upon transport , impl icat ing a phosphotrans-ferase or kinase associated with the membrane. This mechanism of accumulation techn ica l ly cannot be cor rec t ly described as a glucose-concentration mechanism, because a der ivat ive of glucose and not free glucose was demonstrated to be accumulated. k. Mechanism of the accumulation of glucose The accumulation of glucose and mannose in suspensions of C. perfr?ngens has been demonstrated to occur by a system display ing saturat ion k i n e t i c s , temperature s e n s i t i v i t y , and energy dependence. In add i t i on , by i t s high degree of s p e c i f i c i t y the reaction appeared to be enzymatic in nature. The mechanism was demonstrated to accumulated glucose to concentrations several hundred times the external concentrat ion, and th is pooled material was shown to be in the form of phos-phorylated der iva t ives . The method of phosphorylation was not the soluble hexokinase, but rather was probably due to a membrane-bound phosphotransferase or kinase. Metabolism of exogenously-added pyruvate or previously accumulated glucose probably supplied the high energy inter-mediates necessary for phosphorylation. The l imi ted soluble pool s i ze might well have resulted from e i ther the deplet ion of the reserve'of the high energy intermediate, or from the attainment of an equi1 ibrium across the phosphorylating mechanism. This mechanism as proposed bears more than super f i c ia l resemblance to the phosphorylation mechanisms of accumulation by :S. aureus (Egan, and Morse, 1966) and the phosphoenol-pyruvate dependent phospho-transferase system of accumulation of carbohydrates in E. col? (Simoni et a l . , 1967). The advantages of possession of such an accumulation mechanism to the c e l l are severa l . The carbohydrate which served as the most common energy source in the organism's normal environment was e f f i c i e n t l y scavenged from the external medium and the energy expended by the accumulation mechanism simultaneously served to .ac t iva te the carbohydrate for metabolism by g l yco l ys i s . GENERAL DISCUSSION Necrot ic mammalian t issue during pathological condi t ions, the lower region of the mammalian digest ive system under normal condi t ions, and s o i l , are known to be the three major ecological systems of C. perfr?ngens (Breed, Murray, and Smith, 1957). There are two major mechanisms by which th is organism may survive extended periods of exposure to the def ic iency of nutr ients in s o i l . C_. perfringens is known to form spores, and as cer ta in s t ra ins are important causative agents of food poisoning, extensive studies of the formation, s u r v i v a l , and germination of the organism's spores have been published (Col lee, Knowlden, and Hobbs, 1961; Weiss, and Strong, 1967). Studies in cu l tu ra l condit ions have indicated that only under a lka l ine condi t ions, with a source of carbohydrate present, w i l l spores be formed (Wi11 i s , 1964). These condit ions occur in the lower region of the mammalian d igest ive system, so considerable numbers of spores may be excreted in wastes, to u l t imately be d is t r ibu ted in the s o i l . The maintenance of the in teg r i t y , over 18 hrs , of the vegetative c e l l s grown on the peptone-less, semi-defined medium, would seem to provide an a l te rnat ive explanation for the surv iva l of £ . perfringens in the austere condit ions of the s o i l . In the absence of substrates for the pro teo ly t ic exo-enzymes, the formation of the au to ly t i c enzyme system would be repressed, and the magnesium present in the so i l d would repress the small ^amounts of au to ly t i c a c t i v i t y present. The c e l l s could u t i l i z e any carbonaceous or nitrogenous endogenous supply of energy that they may possess, and the v i a b i l i t y and in tegr i ty of the vegetative c e l l s could be maintained for extended periods of time. Any glucose or mannose present in the s o i l , could be immediately accumulated to levels far above the external concentrat ion. Other carbohydrates which would support growth could not be accumulated, but rather could be rapidly transported into the c e l l by f a c i l i t a t e d d i f f us ion . During growth in necrot ic mammalian t i ssues , the supply of glucose and nutr ients would al low rapid growth. Peptides would be present, and the production of the toxins upon which the pathogenicity of £ . perfr ingens depends, would be induced. Induction of the au to ly t i c enzymes by the presence of peptides would not destroy the cu l tu re , as the presence of an energy source has been shown to repress the a c t i v i t y of the au to ly t i c enzymes. The accumulated glucose would be metabolized v ia the Embden-Meyerhof pathway, with the heterofermentative production of l a c t i c , ace t i c , and butyr ic ac ids , in addi t ion to the carbon-dioxide and molecular hydrogen which produce the gaseous disrupt ion of t issue in "gas-gangrene". Five-carbon sugars for the production of nucle ic acids may be produced by the t ransketolase/ t ransaldolase system, from fructose-6-phosphate and from 3-phospho-glyceraldehyde, in the absence of a hexose-monophosphate pathway or complete pentose cyc le . To provide the energy required for the rapid growth, the maintenance of a low oxidat ion po ten t ia l , and the extensive production of exo- tox ins, required for the patho-genic s ta te , the organism could concentrate the ava i lab le glucose from the t issue by the economical means of ac t iva t ing the glucose, through phosphorylation, for g l yco l ys i s . Thus, in one step the organism would simultaneously reta in the carbohydrate molecule wi th in the c e l l , and prepare i t for further metabolism. This system is twice as e f f i c i e n t as that which has been proposed for the accumulation of g-galactosides in E_. co 1 i , by the use of one mole of ATP to concentrate one mole of carbohydrate through a l te r ing the a f f i n i t y of the transport mechanism (Kepes, 1960). An organism which used th is system, would then be required to expend another mole of ATP to phosphorylate the carbohydrate during g l y c o l y s i s . The other Gram-positive organism which has been studied for carbohydrate t ransport , S. aureus, accumulated eight d i f ferent sugars v ia permeases highly spec i f i c for each carbohydrate, coupled to a general membrane ca r r i e r mechanism (Egan, and Morse, 1966). The evolut ion of C. perfringens "'into a form which, in nature, grows rapidly only in mammalian systems, where the major soluble carbohydrate is glucose, may have been related to the loss of the accumulation mechanisms for carbohydrates other than glucose. A study of the s p e c i f i c i t y of the transport mechanism for non-accumulated carbohydrates in C. perfringens would serve to c l a r i f y th is concept, but has proven to be techn ica l ly d i f f i c u l t . Perhaps a comparative study of the s p e c i f i c i t y of transport and accumulation mechanisms of related pathogens and non-pathogens would serve to support evidences of evolut ionary trends in microorganisms. 108 LITERATURE CITED 1. A lbers , R.W. 1967. Biochemical aspects of act ive transport . Ann. Rev. Bioch. 36_: 727-756. 2. Anraku, Y. 1967. The reduction and restorat ion of galactose transport in osmotical ly shocked c e l l s of Escherichia c o l i . J . B i o l . Chem. 242: 793-800. 3. Bacon,. S. .''1-949. Unpublished data. In Stephenson, M. 1949, Bacter ia l metabolism. Longmans, Green & Co . , New York. 4. Bard, R .C . , and I.C. Gunsalus. 1950. Glucose metabolism of Clostr idium perfr ingens: existence of a metal1o-aldo lase. J . Bac te r i o l . 59: 387-400. 5a. Bard, R.C. 1952. Respirat ion of Clostr id ium perfr ingens. Indiana Academy of Science Proceedings 61: 62-63. 5b. Biggens, D.R., and M.J . Di lworth. 1968. Control of the pyruvate phosphoroclastic ac t i v i t y in extracts of Clostr idium pasteuranium by ADP and acetyl phosphate. Biochim. Biophys. Acta 156: 285-296. 6. Boyd, M . J . , M.A. Logan, and A.A. T y t e l l . 1948. The growth requirements of Clostr id ium perfringens (welchi i) BP6K. J . B i o l . Chem. 174:~1013-1025. 7a. Breed, R .S . , E.G.D. Murray, and H.R. Smith. 1957. Bergey's manual of determinative bacter io logy, p. 666-667. Williams & Wi lk ins , Balt imore. 7b. B r i t t en , R . J . , and F.T. McClure. 1962. The amino acid pool in Escherichia c o l i . Bac te r i o l . Rev. 26: 292-335. 8. Bucher, J . , and G. P f le ide re r . 1955. Pyruvate kinase from muscle, p. 435-440 _T_n_ Colowick, S . P . , and M.0. Kaplan (ed.) , Methods in Enzymology Vo l . I. Academic Press, Inc. , New York. 109 9. Campbell, J . J . R . , A . F . Gronlund, and M.G. Duncan. 1963. Endogenous metabolism of Pseudomonas. Ann. N.Y. Acad, of Science 102: 669-677. 10. C i f o n e l l i , J . A . , and F. Smith. 1954. Detection of glycosides and other carbohydrate compounds on paper chromatograms. Anal. Chem. 26_: 1132-1134. 11. C i r i l l o , V.P. 1961. Sugar transport in microorganisms. Ann. Rev. Microbiol ..J_5_: 197-218. 12. C i r i l l o , V.P. 1962. Mechanism of glucose transport across the yeast cel l membrane. J . B a c t e r i d . 84: 485-491. 13- C i r i l l o , V.P. 1968, Relationship between sugar structure and competition for the sugar transport system in baker's yeast. J . Bacteriol . 95: 603-611. 14a. Cohen, G.N. , and J . Monod. 1957. Bacterial permeases. Bacter i o l . Rev. 21_: 169-194. 14b. Cohen, G.N. , and M.V. Rickenberg. 1956. Concentration specifique reversible des amino acides chez Escherichia c o l i . Ann. Inst. Pasteur 84: 937"945. 15'.. Cynkin, M.A., and E.A. Delwiche. 1958. Metabolism of pentoses by Clostr id ia . I. Enzymes of ribose dissimilation in extracts of Clostridi.uni perfr?ngens. J . Bacteriol . 75: 331-334. 16. Cynkin, M.A., and M. Gibbs. 1957. Pentose metabolism in the Clostr id ia . Bacteriol . Proc. 1957: 122. 17. Cynkin, M.A., and M. Gibbs. 1958. Metabolism of pentoses by Clostr id ia . II. The fermentation«of C-1abel1ed pentoses by Clost rid ium perfri hgens, Clostridium beijerihcki i and Clostridium butylicum. J . Bacteriol 75: 335-338. 18. Dawes, E .A. , and D.W. Ribbons. 1962. Effect of environ-mental conditions on the endogenous metabolism of Escherichia col?. Biochem. J . 84: 97"98. 110 19. De V r i es , W. , J . Gerbrandy, and A.N. Stouthamer. 1967-Carbohydrate metabolism in Bif idobacterium  bif idum. Biochim. Biophys. Acta 136: 414-425. 20. Do l in , N.I. 1959a. Oxidation of reduced diphospho-pyr idine nucleotide by Clostr idium per f r i hgens. I. Relat ion of paroxide to the over -a l l react ion. J . Bac te r i o l . 77: 383-392. 21. Do l in , M.I. 1959b. Oxidation of reduced dipho.spho-pyr id ine nucleotide by Clostr id ium perfr ingens. I I P u r i f i c a t i o n - o f . the oxidase; re la t ion to cytochrome-reductase. J . Bac te r i o l . 77_: 392-402. 22. Doudoroff, M. , and R.Y. Stanier . 1959. Role of poly-3 -hydroxybutyr ic acid in the ass imi la t ion of organic carbon by bac ter ia . Nature 183: 1440. 23. Egan, J . B . , and M.L. Morse. 1965- Carbohydrate transport in Staphylococcus aureus. II. Character izat ion of the defect of a p le io t rop ic transport mutant. Biochim. Biophys. Acta 109: 172-183. 24. Egan, J . B . , and M.L. Morse. 1966. Carbohydrate transport in Staphylococcus aureus. I I I . Studies of the transport process. Proc. Na t l . Acad. Science 55: 63-71. • 25. Fox, C . F . , and E.P. Kennedy. 1965. S p e c i f i c label 1ing and par t ia l pu r i f i ca t i on of the M pro te in , a component of the $-galactoside transport system of Escher i ch ia ' co l? . Proc. Na t l . Acad. Science 5±: 891-899. 26. Fredette, V . , C. P lante, and A. Roy. 1967. Numerical data concerning s e n s i t i v i t y of anaerobic bacter ia to oxygen. J . Bac te r i o l . 94: 2012-2017. •27. Friedemann, T . E . , and T.C. Kmieciak. 1932. Metabolism of pathogenic C los t r i d i a in complex carbohydrate r ich cul ture media. Proc. Soc. Exp. V i o l , and Med. 47: 84-87. 111 28. Fuchs, A . R . , and G . J . Bonde. 1957. The ava i lab i1 i t y of sulphur for C lbs t r id ium per f r i hgens and an examination of hydrogen sulphide product ion. 1 J . Gen. M ic rob io l . Jl_6: 330-340. 29. Ganesan, A . K . , and B. Rotman. 1966. Transport systems for galactose and galactosides in Escherichia  cbl i . J . Mol. B i o l . 16_: 42-50. 30. Grado, C , and C.E. Ba l lou. 1961. Myo- inosi to l phosphates obtained by a lka l i ne hydrolysis of beef-brain phosphoinosit ides. J . B i o l . Chem. 236: 54-59. 31. Groves, V . E . , J . Calder, and W .J. Rutter. 1966. Fructose diphosphate a ldo lase. 11. Clostr id ium  perfr ingens. pp. 486-491 j_n Wood, \CKl (ed. ) , Methods in Enzymology Vo l . IX. Academic Press, Inc. , N.Y. 32. Magih i ra, H. , T.H. Wilson, and E.C.C. L i n . 1963. Studies on the glucose-transport system in Esther ichia c b l i . With a-methyl-glucoside as substrate. Biochim. Biophys. Acta 78: 505-515. 33. Halpern, Y . S . , and M. Lupo. 1966. Effects of glucose and other carbon compounds on the transport of a-methyl-glucoside in Escherichia c b l ? ; K12. Biochim. Biophys. Acta 126: 163-167. 34. Hastings, E .G . , and E. McCoy. 1932. The use of reduced iron in the cu l t i va t i on of anaerobic bac ter ia . J . Bac te r i o l . 23: 54-56. 35. Hauschi ld, A.H.W. 1965. Peptides for toxinogenesis of C lbs t r id ium per f r i hgens type D. J . Bac te r i o l . 90: 1793-1794. 36. Hengstenberg, W., J . B . Egan, and M.L. Morse. 1967. Carbohydrate transport ih Staphylococcus aureus. V. The accumulation of phosphorylated der iva t i ves , and evidence for a new s p l i t t i n g lactose phosphate. Proc. Na t l . Acad. Science 58: 274-279. 112 37. Hengstenberg, W., J . B . Egan, and M.L. Morse. 1968. Carbohydrate transport ih Staphylococcus aureus. VI. The nature of the der ivat ives accumulated. J . B i o l . Chem. 243: 1881-1885. 38. H i c k s , H.R. 1952. Transaminase a c t i v i t y i n C l o s t r i d i u m w e l c h i ?. Biochem. J . 60: ? i ?-1y. 39. H i r a n o , S., J . Somejma, and M. Kuni m i . 1954. S t u d i e s on the m e t a b o l i s m o f anaerobe s . D i f f e r e n c e s o f m e t a b o l i s m between E s c h e r i c h i a c o l ? and C l o s t r i d i u m welch? i . I I I . E f f e c t s o f a z i d e on th e g l u c o s e m e t a b o l i s m . Japanese J . B a c t e r i o l . 9: 755-760. 40. H o f f e e , P., and E. E n g l e s b e r g . 1962. E f f e c t o f m e t a b o l i c a c t i v i t y on the g l u c o s e permease o f b a c t e r i a l c e l l s . P r o c . ' N a t l . Acad. S c i e n c e 48: 1795-1765. 41. H o f f e e , P., E. E n g l e s b e r g , and F,' Lamy. 1964. The g l u c o s e permease i n b a c t e r i a . B i o c h i m . B i o p h y s . A c t a 79.: 337-350. 42. H o l d e n , J.T. 1962. T r a n s p o r t and a c c u m u l a t i o n o f amino a c i d s by m i c r o o r g a n i s m s , pp. 566-594. In Ho l d e n , J.T. ( e d . ) , Amino a c i d p o o l s . E l s e v i e r P u b l i s h i n g Co., Amsterdam. 43. H o r e c k e r , B.L., J . Thomas, and J . Monod. 1960a. G a l a c t o s e t r a n s p o r t i n E s c h e r i c h i a c o l ? . I. G e n e r a l p r o p e r t i e s as s t u d i e d i n a g a l a c t o -k i n a s e - l e s s mutant. J . B i o l . Chem. 235: 1580-1585. 44. H o r e c k e r , B.L., J . Thomas, and J . Monod. 1960b. G a l a c t o s e t r a n s p o r t ?n E s c h e r i ch i a c b l ? . I I . C h a r a c t e r i s t i c s o f t h e e x i t p r o c e s s . J . B i o l . Chem. 235: 1586-1590. 45. Hughes, D.E., and H. Wil l iamson. 1952. Some propert ies of the glutaminase of Clbst r id ium welch i ?. Biochem. J . 51: 45~54. 113 4 6 . Hurwitz, L . , and A. Rothstein. 1951. The re la t i on -ship of the c e l l surface to metabolism. VI I . The k ine t ics and temperature charac te r i s t i c of uranium-inhib i t ion. J . C e l l , and Comp. Phys io l . 38: 437-450. 47. Ivanov, V . J . , and A.V. Tobanova. 1954. Hexokinase of Baci1lus per f r i ngens and some of i t s propert ies. Biokhimia J_9_: 257-260. 4 8 . Jones, A . S . , M. Stacey, and M. Webb. 1949. Studies on the au to ly t i c enzyme system of Gram-positive microorganisms. I. The l y t i c system of Staphylococci . Biochim. Biophys. Acta 3_: 383-399. 49. Katsura, T . , H. I to, T. Hojima, M. Hemoto, and F. Egami. 1954. N i t ra te reduction by C. welchi i . J . Biochem. (Tokyo) 4j_: 745-756. 50. Kay,.W.W. 1 9 6 8 . Amino acid transport and pool formation in Pseudorhonas aerugihosa. PhD. Thesis. Department of Microbiology, Univers i ty of B r i t i sh Columbia, Vancouver, B .C . , Canada. 51. Kepes, A. 1957- Metabolism oxydati f l i e a u functionnent de la galactoside-permease d 'Escher ich ia c o l i . Compt. rend. 2 4 4 : 809-811. 52. Kepes, A. i960. Etudies cinetiques sur la galactoside permease d 'Escher ich ia c o l i . Biochim. Biophys. Acta 4 0 : 70-84. 53. Kepes, A . , and G.H. Cohen. 1962. Permeation 179-221. j_n Gunsalus, I .C., and R.Y. Stanier (ed. ) , The Bacter ia , Vo l . 4. Academic Press, Inc. , N.Y. 54. Kess ler , D.P. , and W.V. Rickenberg. 1963- The competitive inh ib i t ion of a-methyl glucoside uptake in Escherichia co l ? . Biochem. Biophys. Res. Comm. 10: 4 8 2 - 4 8 6 . 114 5 5 . Koch, A .L . 1964. The role of permease in t ransport . Biochim. Biophys. Acta 79_: 177-200. 5 6 . Koepsel l , J . H . , and M.J . Johnson. : 1942. D iss imi la t ion of pyruvic acid by eel 1-free preparations of Clostr idium butylicum. J . B i o l . Chem. 2 1 6 : 7 3 7 - 7 4 8 . 5 7 . Kolber, A . R . , and W.D. S te in . 1966. Ident i f i ca t ion of a component of a transport " c a r r i e r " system: i so la t ion of the permease expression of the lac operon of Escherichia cbl ?. Nature 2 0 9 : 6 9 1 - 6 9 4 . 58. Krebs, E.G. 1955. Glyceraldehyde-3 -phosphate dehydrogenase from yeast .p . 407-411 .J_n_ Colowick, S .P . and N.O. Kaplan (ed. ) , Methods in Enzymology, Vo l . 1. Academic Press, Inc. , N-Y. 5 9 . Kubowitz, F. 1934 . Ueber die Hemmung der Buttersauregarung durch Kohlenoxyd. Biochem. Z.27_4_: 285-298. 6 0 a . Kundig, W., S. Ghosh, and S. Roseman. 1964 . Phosphate bound to h i s t i d ine in a protein as an intermediate in a novel phospho-transferase system. Proc. Na t l . Acad. Science 5 2 : 1067-1072. 60b. Kundig, W., and S. Roseman. 1966. Phosphotransferase system, p. 3 9 6 - 4 0 3 . In Colowick, S . P . , N.O. Kaplan and A.W. Wood Ted.), Methods in Enzymology V o l . , 9 , Academic Press, Inc. , N.Y. 6 1 . Kundig, W.F. , D. Kundig, B. Anderson, and S. Roseman. 1966. Restoration of act ive transport of glycosides in E_. cbl i by a component of the phospho-transferase system. J . B i o l . Chem. 241 3 2 4 3 - 3 2 4 6 . 6 2 . Lee, C .K . , and Z . J . Ordal. 1967- Regulatory e f fect of pyruvate on the glucose metabolism of C lbs t r i d ium thermosaccharolyt icum. J . Bac te r io l . 9 4 : 530-536. n'5 63. Lerner, E .M. , and M.J. P icke t t . 1949. The fermentation of glucose by Clostr id ium te tan i . Arch. Biochem. 8: 183-196. 64. Lipmann, F . , and L.C. Tu t t le . 1-945. A spec i f i c micromethod for the determination of acyl phosphates. J . B i o l . Chem. 159: 21-28. 65. Lovenberg, W., B.B. Buchanan, and J . C . Rabinowitz. 1963. Studies on the chemical nature of c l o s t r i d i a l ferredoxin. J . B i o l . Chem. 283: 3899-3913. 66. Lowry, O .H. , N .J . Rosebrough, A .L . Far r , and R . J . Randal l . 1951. Protein measurement with the Fo l in phenol reagent. J . B i o l . Chem. 193: 265-271. 67. Lu r i a , S .E. I960. The bacter ia l protoplasm: composition and organizat ion.p. 1 -31 , J_nGunsalus , I .C . and R.Y. Stanier (ed.) , The Bacteria Vo l . 1. Academic Press, Inc. , N.Y. 68. McCarthy, B . J . 1962. The ef fects of magnesium starvat ion upon the ribosome content of Escherichia cb l? . Biochim. Biophys. Acta 55: 880-883. 69. Ma lk in , R., and J . C . Rabinowitz. 1967. Nonheme iron e lect ron- t ransfer prote ins. Ann. Rev. Biochem. 36: 113-148. 70. Mart inez, R . J . , and S.C. Rit tenberg. 1959. Glucose d i ss im i la t i on by C lbs t r i d ium te tan i . J . B a c t e r i o l , 77_: 156-163. 71. Mort lock, R . P . , R.C. Valent ine, and R.S. Wolfe. 1959. Carbon dioxide ac t iva t ion in the pyruvate-c last ic system of Clbstridiurn butyricum. J . B i o l . Chem. 234: 1653-1656. 72. Nisman, B. 1954. The St ickland react ion. Bac te r i o l . Rev. j8_: 17-42. 73. Nossal , N.G. , and L.A. Heppel. 1966. The release of enzymes by osmotic shock from E. cblI i in exponential phase. J . B i o l . Chem. 24l_: 3055-306T. 74. Osborn, J . J . , W.L. McLennan, and B.L. Horecker. 1961. Galactose transport ?h Escherichia col u •' ' I I I. The ef fect of 2 ,4-dihitrophenol on entry and accumulation. J . B i o l . Chem. 236: 2585-2589. 116 75. Paege, I.M.. M. Gibbs, and R.C. Bard. 1956. Fermentation of l 4 6 - l a b e l l e d gl ucose by Clost r Id I urn per f r i ngens. J . B a c t e r i d . 7_2_: 65-67. 76. Pappenheimer, A.W., and E. Shaskan. 1944. Ef fect of Iron on carbohydrate metabolism of Clostr id ium welchi i . J . B i o l . Chem. 155: 265-275. 77. P ivn ick , H. , A. Habeeb, B. Gorenstein, P.F. Stuar t , and A.H.W. Hauschi ld. 1964. Effect of pH on toxinogenisis by Clostridiurn perfrIhgens type C. Can. J . M ic rob io l . Kh 329-344T 78. Pre*vot, A.R. 1966. Manual for the c l a s s i f i c a t i o n and determination of anaerobic bac ter ia . Lea and Febiger, Ph i lade lph ia . 79. Quastel , J .Hi .' 1-965. Molecular transport at eel 1 membranes. Proc. Royal Soc. London Ser. B. 163: 169-196. 80. Rickenberg, H.V., G.N. Cohen, G. Bu t t in , and J . Monod. 1956. La galactoside permease d 1 Escher ich ia c o l i . Ann. Inst. Pasteur 91: 829-857. 81. Roberts, R .B . , P.H. Abelson, D.B. Cowie, E.T. Bolton, and R . J . B r i t t en . 1955. Studies of biosynthesis in E. c b l i . Carnegie Inst. Wash. Publ . No. 607. 82. Rogers, D., and Shon-Hua Yu. 1962. Substrate s p e c i f i c i t y of a glucose permease of Escherichia c b l i . J . Bac te r i o l . 84: 877-881. 83. Scharf f , T.G. , and E.H. Kremer. 1962. A tentat ive mechanism for the anaerobic transport of glucose, fructose and mannose in yeast. Arch. Biochem. Biophys. 97: 192-198. 84. Shankar, K., and R.C. Bard. 1952. The ef fect of meta l l i c ions on the growth and morphology of Clostr id ium  perfr ingens. J . B a c t e r i o l . 6 3 : 279-290. 85. Shankar, K., and R.C. Bard. 1955a. Effect of metal l i e ions on the growth, morphology and metabolism of. Clostr id ium perfr ingens. I. Magnesium. J . Bac te r i o l . 69: 436-444. 86. Shankar, K., and R.C. Bard. 1955b. Effect of meta l l i c ions on the growth, morphology and metabolism of Clostr id ium  perfr ingens. II. Cobalt. J . B a c t e r i o l . 6 9 : 444-448. 117 87. Simoni, R.D., M. Levi'nthal ,. F.D. Kundig, W. Kundig, B. Anderson, P.E. Hartman, and S. Roseman.. 1967. Genetic evidence for the role of a bacter ia l phospho-transferase system in sugar t ransport . Proc. Na t l . Acad. Science 58_: 1963-1970. 88. Smith, I. 1960. Chromatographic and elect rophoret ic techniques, Vo l . 1. 2nd ed. Interscience Publ ishers , Inc. , New York. 89. Strange, R . E . , H.E. Wade, and A.G. Ness. 1963. The catabolism of proteins and nucle ic acids in starved Aerobacter aerogenes. Biochem. J . 86: 147-203. 90. Stephenson, M.. 1949. Bacter ia l metabolism. Longmans, Green, and Co . , New York. 91. Tanaka, S . , and E.C.C. L i n . 1967. Two classes of p le io t rop ic mutants of Aerobacter aerogenes lacking components of a phosphoenolpyruvate-dependent phosphotransferase system. Proc. Na t l . Acad. Science 5_7_: 913-919. 92. Tanaka, S . , D.G. Fraenkel, and E.C.C. L i n . 1967. The enzymatic les ion of s t ra in MM-G, a p le io t rop ic carbohydrate negative mutant of Escherichia co l? ; Biochem. Biophys. Res. Comm. 27: 63-67. 93. T i ss i& res , A. , J .D . Watson, D. Schlesinger, and B.R. Hoi 1ingsworth, 1959- Ribonucleoprotein par t i c les from Escherichia col i . J . MoT. B i o l . J_: 221-233. 94. T y t e l l , A.A. 1952. The degradation of arginine by Clostr id ium perfringens (BP6K). J . B i o l . Chem. 198: 771-783. 95. Van Steveninck, J . 1966. The influence of nickelous ions on carbohydrate transport in yeast c e l l s . Biochim. Biophys. Acta 126: 154-162. 96. Van Steveninck, J . , and E.C. Dawson. I968. Act ive and passive galactose transport in yeast. Biochim. Biophys. Acta 150: 47-55. 97. Valent ine, R.C. 1964. Bacter ia l ferredoxin. Bac te r i o l . Rev. 28: 497-517. 1 T8 98. Valent ine, R . C , and R.S. Wolfe. 1963. Role of ferredoxin in the metabol isin of molecular hydrogen, J . Bac te r io l . 85_: 1114-1120. 99. Wang, R . J . , and M.L. Morse. 1968. Carbohydrate accumulation and metabolism in Escherichia c b l i . I. Descript ion of p le io t rop ic mutants. J . M o l . B i o l . 32: 59-66. 100. Webb. M. 1948. The influence of magnesium on c e l l d i v i s i o n . I. The growth of Clostr id ium welch? i in complex media def ic ien t in magnesium. J . Gen. M ic rob io l . 2; 275-287. 101. Westhead, E.W. 1966. Enolase from yeast and rabbit muscle, p. 670-679, in Wood, W.A. (ed. ) , Methods in Enzymology, Vo l . IX, Academic Press, Inc. , N.Y. 102. Wilbrandt, W. 1963- Transport through b io log ica l membranes. Ann. Rev. Phys io l . 25: 601-630. 103. Wilbrandt, W. , and T. Rosenberg. 1-961.'. The concept of ca r r i e r transport and i t s co ro l l a r i es in pharmacology. Pharmacol. Rev. 13:'••109-183.'. 104. W i l l i s , T. 1964. Anaerobic bacteriology in c l i n i c a l medicine. Butterworth, London. 105. Winkler, H.H., and T.H. Wilson. 1-966. The role of energy coupling in the transport of 0-galactosides by Escherichia c o l i . J . B i o l . Chem. 241: 2201-2211. 106. Wood, W.A. 1961• Fermentation of carbohydrates, p. 59-149. J_n_ Gunsalus, I .C . , and R.Y. Stanier (ed.) , The Bacter ia , Vo l . 2. Academic Press, Inc. , N.Y. 107. Woods, D.D. , and A.R. Trim. 1942., Studies in the.metabolism of s t r i c t anaerobes. 8. The metabolism of amino acids by C: welch? i . Biochem. J . 36: 501-512. 

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