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Succinate metabolism and tricarboxylic acid cycle activity Tiwari, Narayan Prasad 1969

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SUCCINATE METABOLISM AND TRICARBOXYLIC ACID CYCLE ACTIVITY IN PSEUDOMONAS AERUGINOSA by NARAYAN PRASAD TI WAR I B . V . S c , U n i v e r s i t y o f J a b a l p u r , 1957 M . S c , Panjab U n i v e r s i t y , 19&3 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t he Department o f M i c r o b i o l o g y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1969 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 Columbia, 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 purposes may be g r a n t e d by the Head o f my Department o r by h i s 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 o r p u b l i c a t i o n of t h i s thes.is 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 Microbiology The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date 30 Apri l 1969 i i ABSTRACT A l t h o u g h t h e impo r t a n c e o f t r i c a r b o x y l i c a c i d c y c l e a c t i v i t y i n the m e t a b o l i s m o f a e r o b i c b a c t e r i a i s w e l l e s t a b l i s h e d , d e t a i l e d s t u d i e s on the u t i l i z a t i o n o f i n t e r m e d i a t e s o f the c y c l e d e s i g n e d t o a s s e s s t h e n a t u r e , importance and c o n t r o l o f the enzymes concerned have not been performed w i t h pseudomonads. R e s u l t s o f t h i s i n v e s t i g a t i o n have shown t h a t Pseudomonas a e r u g i n o s a ATCC 9027 l a c k s NAD o r NADP l i n k e d L-mal?c dehydrogenase. S t u d i e s w i t h c e l l f r a c t i o n s have shown t h a t an NAD and NADP i n d e p e n d e n t , p a r t i c u l a t e L - m a l i c dehydrogenase c a t a l y s e s t he o x i d a t i o n o f L - m a l i c a c i d t o o x a l a c e t i c a c i d . The l a b e l l i n g p a t t e r n s o f c i t r a t e o b t a i n e d from 12, -|Z| s u c c i n a t e - 1 , 4 - C and s u c c i n a t e - 2 , 3 - C have demonstrated t h e i n v o l v e m e n t o f t h e p a r t i c u l a t e m a l i c dehydrogenase and have e x c l u d e d any o t h e r p o s s i b i l i t y . P h o s p h o f r u c t o k i n a s e c o u l d not be d e t e c t e d i n the c e l l - f r e e e x t r a c t p r e p a r a t i o n s and thus a c c o u n t i n g f o r the n o n - f u n c t i o n a l Embden-Meyerhof pathway i n t h i s o r g a n i s m . C e l l s grown i n s u c c i n a t e medium e i t h e r do not have o r have e x t r e m e l y low l e v e l s o f g l u c o s e m e t a b o l i z i n g enzymes. The g l u c o s e e f f e c t on t r i c a r b o x y l i c a c i d c y c l e enzymes was not o b s e r v e d . F u r t h e r , t he a d d i t i o n o f a - k e t o -g l u t a r a t e and g l u t a m a t e t o the medium d i d not r e p r e s s t h e s e enzymes. These o b s e r v a t i o n s s u g g e s t t h a t t r i c a r b o x y l i c a c i d c y c l e a c t i v i t y i s o f s p e c i a l i m p o r t a n c e f o r growth and m e t a b o l i s m i n pseudomonads. The d a t a have a l s o i n d i c a t e d t h a t d u r i n g growth i n a s u c c i n a t e medium, pe n t o s e s y n t h e s i s must o c c u r by t h e a c t i o n of t r a n s k e t o l a s e upon compounds d e r i v e d from t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s . I t has been shown t h a t both t h e g l u c o s e permease and t h e g l u c o s e m e t a b o l i z i n g enzymes were induced s i m u l t a n e o u s l y on s h i f t from s u c c i n a t e t o g l u c o s e medium. The g l u c o s e permease was found t o be v e r y s p e c i f i c s i n c e g l u c o s e u ptake was not i n h i b i t e d even i n the p r e s e n c e o f 1 0 0 - f o l d . e x c e s s o f a - C H ^ - g l u c o s i d e , 2 - d e o x y g l u c o s e , g a l a c t o s e , f r u c t o s e o r mannose. P a r t i c u l a t e m a l i c dehydrogenase was i n h i b i t e d by a d e n i n e n u c l e o t i d e s w h i l e i t was a c t i v a t e d by GTP and GDP. The mechanism o f t h i s r e g u l a t i o n i s not c l e a r , however, i t i s o b v i o u s l y i m p o r t a n t i n t h e c o n t r o l o f t r i c a r b o x y l i c a c i d c y c l e a c t i v i t y . A d d i t i o n o f g l u t a m a t e t o t h e medium r e p r e s s e d t h e s y n t h e s i s o f g l u t a m i c dehydrogenase. High l e v e l s o f m a l i c enzyme were m a i n t a i n e d on growth i n g l u c o s e and s u c c i n a t e media, whereas t h e l e v e l s were low i n a c e t a t e medium. These o b s e r v a t i o n s d e m o n s t r a t e d th e c a p a c i t y o f t h e o r g a n i s m t o r e g u l a t e t h e s y n t h e s i s o f enzymes i n r e sponse t o the p a r t i c u l a r e n v i r o n m e n t . T r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s and g l y o x y l a t e have been found t o e x e r t " f i n e c o n t r o l " o v e r t h e a c t i v i t i e s o f i s o -- c i t r a t e dehydrogenase and i s o c i t r a t e l y a s e i n such a way t h a t f l o w o f i s o c i t r a t e t h r o u g h the t r i c a r b o x y l i c a c i d c y c l e and t h e g l y o x y l a t e c y c l e i s p r e c i s e l y r e g u l a t e d t o s u i t t h e needs o f the e e l 1 . V TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW • . . 3 I. G e n e r a l P a t t e r n o f C a r b o h y d r a t e M e t a b o l i s m i n Pseudomonads 3 I I . D i s t r i b u t i o n o f T r i c a r b o x y l i c A c i d C y c l e Enzymes and M e t a b o l i s m o f D i c a r b o x y l i c A c i d I n t e r m e d i a t e s i n Pseudomonads . . . . • . . . £ I I I . T r i c a r b o x y l i c A c i d C y c l e A c t i v i t y and Enzymes o f G l u c o s e O x i d a t i o n i n C e l l s Grown on Media C o n t a i n i n g G l u c o s e o r a T r i c a r b o x y l i c A c i d C y c l e I n t e r m e d i a t e . . • . 8 IV. C o n t r o l o f T r i c a r b o x y l i c A c i d C y c l e and G l y o x y l a t e C y c l e A c t i v i t y i n M i c r o o r g a n i s m s . . . 11 V. M o l a r Growth Y i e l d as a Tool i n t h e Study o f M e t a b o l i c Pathways . . . . . . . . . . . 16 V I . S u c c i n a t e U t i l i z a t i o n s as D i s t i n c t from O t h e r D i c a r b o x y l i c A c i d s o f the T r i c a r b o x y l i c A c i d C y c l e . . . . . . . • . . . . • . . . . 17 VI I. M e t a b o l i c P a t t e r n and E v o l u t i o n . . . . . . . 18 .MATERIALS AND METHODS I. Organisms and Growth Media . . . . . . . . 23 v i T a b l e o f C o n t e n t s ( C o n t i n u e d ) Page I I . P r e p a r a t i o n o f R e s t i n g C e l l S u s p e n s i o n s and C e l l - F r e e E x t r a c t s . . . . . . . • . . . . . 24 I I I . D e t e r m i n a t i o n o f M o l a r Growth Y i e l d on D i f f e r e n t S u b s t r a t e s 25 IV. I s o l a t i o n o f Mutants o f P. a e r u g i hosa 25 V . M a n o m e t r i c Methods . 27 VI . Enzyme A s s a y s . . 27 V I I . A n a l y s i s o f R e a c t i o n P r o d u c t s . . . . . . . 31 V I I I . T h i n L a y e r C h r o m a t o g r a p h y , A u t o r a d i o g r a p h y and R a d i o a c t i v e M e a s u r e m e n t s . 31 IX. Uptake o f L a b e l l e d S u b s t r a t e s . 32 X . A n a l y t i c a l Methods . 33 XI . C h e m i c a l s . . . . . . . . 34 RESULTS AND DISCUSSION I. O x i d a t i o n o f T r i c a r b o x y l i c A c i d C y c l e and R e l a t e d I n t e r m e d i a t e s by Pseudomonas a e r u g i n o s a . 35 I I. Growth Y i e l d s o f P. a e r u g i nosa w i t h Some T r i c a r b -o x y l i c A c i d C y c l e I n t e r m e d i a t e s . 41 I I I . S t u d i e s w i t h Mutants . 44 IV. L a c k o f NAD o r NADP Dependent L - M a l i c Dehydrogenase i n P. a e r u g i n O s a 44 T a b l e o f C o n t e n t s ( C o n t i n u e d ) Page V. D e t e c t i o n and S i t e o f Ma l a t e O x i d i z i n g A c t i v i t y . . . 45 14 V I . L a b e l l i n g o f C i t r a t e from S u c c i n a t e - 1 , 4 - C 14 and S u c c i n a t e - 2 , 3 - C . 52 V I I . O x i d a t i o n o f S u c c i n a t e , Fumarate and M a l a t e by a C e l l - F r e e E x t r a c t o f A. a e r o g e n e s . . . . . . 59 V I I I . The Enzymes o f C a r b o h y d r a t e Metabol ism-in-P_. a e r u g i n o s a Grown i n S u c c i n a t e o r G l u c o s e Media . . . 61 1. G l u c o s e d e g r a d i n g enzymes . . . . . . . . . 68 2. T r i c a r b o x y l i c a c i d c y c l e and r e l a t e d enzymes. .• . 76 a. T r i c a r b o x y l i c a c i d c y c l e a c t i v i t y . . .• . . 76 b. G l u t a m i c dehydrogenase a c t i v i t y . . . . . . 79 |iX. L e v e l o f M a l i c Enzyme i n C e l l s Grown i n S u c c i n a t e , G1ucose o r A c e t a t e Med i a . . . . . . . . . . 80 X. I n d u c t i o n o f t h e Enzymes o f G l u c o s e U t i l i z a t i o n i n C e l l s H a r v e s t e d from S u c c i n a t e Medium . . . . . 83 X I . C o n t r o l o f T r i c a r b o x y l i c A c i d C y c l e A c t i v i t y . . . . 89 1. P a r t i c u l a t e m a l i c dehydrogenase a c t i v i t y . . . . 89 2. C o n t r o l o f t h e f l o w o f i s o c i t r a t e v i a the t r i c a r b o x y l i c a c i d c y c l e and g l y o x y l a t e c y c l e , i n P_. a e r u g i hosa . • . . . . • . . . . . . . 94 a. I s o c i t r a t e dehydrogenase a c t i v i t y . . . . . 97 VI I I T a b l e o f C o n t e n t s ( C o n t i n u e d ) Page b. I s o c i t r a t e l y a s e a c t i v i t y 101 c. A c o n i t a s e a c t i v i t y . - . .• . .• . .• . . 105 GENERAL DISCUSSION . . 108 BIBLIOGRAPHY . . . . . . . . . . . . . . . 117 i x LIST OF TABLES T a b l e I T a b l e I I . T a b l e I I I . T a b l e IV. T a b l e V. T a b l e V I . T a b l e V I I . T a b l e V I I I . T a b l e IX. T a b l e X, T a b l e XI A c c u m u l a t i o n o f k e t p a c i d s d u r i n g t h e o x i d a t i o n o f s u c c i n a t e , fumarate and ma l a t e i n t h e p r e s e n c e o f 1 mM sodium a r s e n i t e Growth y i e l d o f P_. a e r u g i nosa from v a r i o u s c a r b o n s o u r c e s P a r t i c u l a t e m a l i c dehydrogenase and m a l i c enzyme i h P_. a e r u g i n o s a E f f e c t o f EDTA and r i b o n u c l e a s e t r e a t m e n t on t h e d i s t r i b u t i o n o f p a r t i c u l a t e m a l i c dehydrogenase o f P_. a e r u g i n o s a The t r i c a r b o x y l i c a c i d c y c l e and r e l a t e d enzymes o f P_. a e r u g ? n o s a M32 S p e c i f i c a c t i v i t y o f r a d i o a c t i v e c i t r a t e formed from s u c c i n a t e - 1 , 4 - ^ a n d s u c c i n a t e - 2 , 3 - 1 ^ C A c t i v i t y o f some enzymes o f c a r b o h y d r a t e metabol ism i n P_. a e r u g i n o s a ' grown i n s u c c i n a t e o r g l u c o s e media G l u c o s e - 6 - p h o s p h a t e f o r m i n g a c t i v i t y from r i b o s e - 5 - p h o s p h a t e and r i b o s e phosphate isomerase a c t i v i t y S p e c i f i c a c t i v i t i e s o f t h e t r i c a r b o x y l i c a c i d c y c l e and r e l a t e d enzymes i n P_. a e r u g i nosa grown w i t h d i f f e r e n t c a r b o n s o u r c e s E f f e c t o f c a r b o n s o u r c e s on t h e s p e c i f i c a c t i v i t i e s o f m a l i c enzyme and i s o c i t r a t e l y a s e i ri P_. a e r u g i nosa I n d u c t i o n o f some enzymes o f g l u c o s e o x i d a t i o n by g l u c o s e Page 40 43 48 51 55 57 69 72 78 82 84 X L i s t o f T a b l e s ( C o n t i n u e d ) Page T a b l e X I I . E f f e c t o f n u c l e o t i d e phosphates on t h e p a r t i c u l a t e m a l i c dehydrogenase i n 105,000 x £ p e l l e t 92 T a b l e X I I I . P a r t i a l p u r i f i c a t i o n o f i s o c i t r a t e dehydrogenase and i s o c i t r a t e l y a s e from c e l l e x t r a c t s o f P. a e r u g i n o s a 96 T a b l e XIV. I n h i b i t i o n o f i s o c i t r a t e dehydrogenase a c t i v i t y by v a r i o u s o r g a n i c a c i d s and r e l a t e d compounds 98 T a b l e XV. I n h i b i t i o n o f i s o c i t r a t e l y a s e a c t i v i t y by o r g a n i c a c i d s and r e l a t e d compounds 102 T a b l e X V I . A c o n i t a s e i n h i b i t i o n i n c e l l - f r e e e x t r a c t o f P. a e r u g i riosa 106 x i LIST OF FIGURES Page F i g . 1. O x i d a t i o n o f s u c c i n a t e , fumarate and m a l a t e by e e l 1 s u s p e n s i o n o f P. a e r u g i n o s a 36 F i g . 2. O x i d a t i o n o f s u c c i n a t e , fumarate and m a l a t e by c e l l - f r e e e x t r a c t o f P. a e r u g i n o s a 37 F i g . 3 . O x i d a t i o n o f a c e t a t e and p y r u v a t e by c e l l s and c e l l - f r e e e x t r a c t o f P. a e r u g i n o s a 38 F i g . 4. O x i d a t i o n o f s u c c i n a t e , fumarate and m a l a t e by r e s t i n g c e l l s u s p e n s i o n o f P_. a e r u g i n o s a i n the p r e s e n c e o f 1 mM sodium a r s e n i t e 39 F i g . 5. O x i d a t i o n o f L - m a l i c a c i d by c e l l - f r e e e x t r a c t and 100,000 x g_ p e l l e t o f P. a e r u g i n o s a 49 F i g . 6. A u t o r a d i o g r a m o f t h e t h i n - l a y e r chromatograph from t h e r e a c t i o n m i x t u r e c o n t a i n i n g c e l l - f r e e e x t r a c t o f P. a e r u g i n o s a w i I d t y p e and r a d i o -a c t i v e s u c c i n a t e 53 F i g . 7. A u t o r a d i o g r a m o f t h e t h i n - l a y e r chromatograph from t h e r e a c t i o n m i x t u r e c o n t a i n i n g c e l l - f r e e e x t r a c t o f P. a e r u g ? h o s a M32 and r a d i o a c t i v e s u c c i n a t e 54 14 F i g . 8 a . C i t r a t e f o r m a t i o n from s u c c i n a t e - 1 , 4 - C. 58' 14 F i g . 8b . C i t r a t e f o r m a t i o n from s u c c i n a t e - 2 , 3 - C 58 F i g . 9 . O x i d a t i o n o f s u c c i n a t e , fumarate and m a l a t e by c e l l - f r e e e x t r a c t o f A. aerogenes 60 F i g . 10. Growth o f s u c c i n a t e grown inoc u l u m i n the media c o n t a i n i n g s u c c i n a t e , g l u c o s e o r s u c c i n a t e p l u s g l u c o s e 63 F i g . 1 1 . Growth o f g l u c o s e grown inoculum i n media c o n t a i n i n g g l u c o s e , s u c c i n a t e o r g l u c o s e p l u s s u c c i n a t e 64 XI L i s t o f F i g u r e s ( C o n t i n u e d ) Page F i g . 12. O x i d a t i o n o f s u c c i n a t e , g l u c o s e o r a m i x t u r e o f s u c c i n a t e and g l u c o s e by e e l 1s h a r v e s t e d from a s u c c i n a t e minimal medium 65 F i g . .13. O x i d a t i o n o f s u c c i n a t e , g l u c o s e o r a m i x t u r e o f s u c c i n a t e and g l u c o s e by c e l l s h a r v e s t e d from a g l u c o s e medium 66 F i g . 14. O x i d a t i o n o f g l u c o s e and s u c c i n a t e by the c e l l -f r e e e x t r a c t s from t h e c e l l s grown i n g l u c o s e o r s u c c i n a t e media 67 F i g . 15. G l u c o s e - 6 - p h o s p h a t e f o r m a t i o n from r i b o s e - 5 -phosphate by P_. a e r u g i nosa grown i n s u c c i n a t e o r g l u c o s e media 73 F i g . 16. I n c r e a s e i n t h e l e v e l o f 3 - p h o s p h o g l y c e r a l d e -hyde dehydrogenase and g l u c o s e - 6 - p h o s p h a t e f o r m i n g a c t i v i t y from r i b o s e - 5 - p h o s p h a t e , on s h i f t from s u c c i n a t e t o g l u c o s e medium 74 F i g . 17. Uptake o f r a d i o a c t i v i t y by g l u c o s e grown e e l Is o f P.. a e r u g i n o s a w i t h 10 "5 M g l u c o s e - U - 1 4 C and w i t h 10"i> M a - m e t h y l - g l u c o p y r a n o s i d e 86 F i g . 18. I n c o r p o r a t i o n o f g l u c o s e - U - ^ C i n the p r e s e n c e o r absence o f o t h e r s u g a r s by t h e whole c e l l s o f P. a e r u g i n o s a h a r v e s t e d from the g l u c o s e m i n i m a l medium 87 F i g . 19. G l u c o s e - U - 1 / | C i n c o r p o r a t i o n by t h e whole c e l l s o f P. a e r u g i n o s a w i I d type and i t s mutant s t r a i n M5, h a r v e s t e d from s u c c i n a t e m i n i m a l med i um 88 F i g . 20 . I n c r e a s e i n t h e l e v e l o f p a r t i c u l a t e m a l i c dehydrogenase a c t i v i t y on s h i f t t o g l u c o s e med i um 90 F i g . 21 . L i n e w e a v e r - B u r k p l o t f o r p a r t i c u l a t e m a l i c dehydrogenase i n the absence o r p r e s e n c e o f 1 mM ATP 93 XI I I L i s t o f F i g u r e s ( C o n t i n u e d ) Page F i g . 2 2 . E f f e c t o f a m i x t u r e o f t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s on i s o c i t r a t e dehydrogenase a c t i v i t y 9 9 F i g . 2 3 . L i n e w e a v e r - B u r k p l o t f o r i s o c i t r a t e dehydrogenase o f P_. a e r u g i n o s a 100 F i g . 24. E f f e c t o f a m i x t u r e o f t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s on i s o c i t r a t e l y a s e a c t i v i t y 103 F i g . 2 5 . L i n e w e a v e r - B u r k p l o t f o r i s o c i t r a t e l y a s e o f P. a e r u g i n o s a 104 ACKNOWLEDGEMENTS I would l i k e t o e x p r e s s my s i n c e r e g r a t i t u d e t o Dr. J.J.R. Campbell f o r h i s i n t e r e s t , s u p e r v i s i o n and c o n s t r u c t i v e c r i t c i s m d u r i n g t h e c o u r s e o f t h i s s t u d y and w r i t i n g o f t h e t h e s i s . I would a l s o l i k e t o thank Dr. A.F. G r o n l u n d f o r her h e l p f u l s u g g e s t i o n s and c r i t i c i s m . My s i n c e r e a p p r e c i a t i o n i s extended t o my w i f e Kamla f o r h e r f o r b e a r a n c e d u r i n g t he c o u r s e o f t h i s work and f o r t y p i n g a p a r t o f t h e t h e s i s . I a l s o w i s h t o thank Mrs. R i t a Rosbergen f o r the f i n a l t y p i n g o f t h i s m a n u s c r i p t . INTRODUCTION Pseudomonas aerug i hosa does not p o s s e s s a f u n c t i o n a l Embden-Meyerhof pathway (Campbel1 and N o r r i s , 1950; S t e r n , Wang and G i l m o u r , 1960), and when organisms a r e grown on t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s , the enzymes o f t h e E n t n e r - D o u d o r o f f pathway and the o x i d a t i v e p o r t i o n o f p e n t o s e phosphate pathway a r e a b s e n t o r e x t r e m e l y low ( H a m i l t o n and Dawes, I960; Von T i g e r s t r o m and C a m p b e l l , 1966b). I t has been proposed t h a t when 6-phosphogluconate dehydrogenase a c t i v i t y i s a b s e n t , t h i s o r g a n i s m s y n t h e s i z e s p e n t o s e by the c o n d e n s a t i o n o f and C^ f r a g m e n t s (Wang, S t e r n and G i l m o u r , 1959; L e s s i e and N e i d h a r d t , 1967a), w h i c h c o u l d be d e r i v e d from t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s . Thus, under t h e s e c o n d i t i o n s , t h e t r i c a r b o x y l i c a c i d c y c l e meets t h e major m e t a b o l i c and b i o s y n t h e t i c demands i n P. a e r u g i hosa. However, o n l y a weak s u c c i n i c dehydrogenase a c t i v i t y has been r e p o r t e d i n t h i s o r g a n i s m ( C a m p b e l l , Hogg and S t r a s d i n e , 1962; Von T i g e r s t r o m and C a m p b e l l , 1966b), a l t h o u g h i n c e l l s u s p e n s i o n s s u c c i n a t e was u t i l i z e d a t a r a p i d ' r a t e comparable t o o t h e r d i c a r b o x y l i c a c i d s s u g g e s t i n g a u n i q u e mode o f u t i l i z a t i o n o f t h i s compound. S u c c i n a t e has a l s o been found t o r e p r e s s t h e i n d u c t i o n o f c e r t a i n d e g r a d a t i v e enzymes ( L e s s i e and N e i d h a r d t , 2 1967b; R o s e n f e l d and F e i g e l s o n , 1 969 ) . The u t i l i z a t i o n o f s u c c i n a t e and o t h e r t r i c a r b o x y l i c a c i d c y c l e members has not been s t u d i e d i n d e t a i 1 i i i P. a e r u g i n o s a and no:, at t e m p t has been made t o c o r r e l a t e t h e a c t i v i t y o f the enzymes t o the u t i l i z a t i o n o f c y c l e i n t e r m e d i a t e s . W h i l e t h e r e have been e x t e n s i v e s t u d i e s on r e g u l a t i o n o f t r i c a r b o x y l i c a c i d c y c l e a c t i v i t y i n c o l i f o r m s (Gray, Wimpenny and Mossman, 1966b; Wimpenny and Warmsley, 1968) and i n B a c i 1 l u s s p e c i e s (Hanson e t a l , 1964; Hanson and Cox, 1967 ), v e r y l i t t l e i s known about the r e g u l a t i o n o f the a c t i v i t y o f t h i s c y c l e i n pseudomonads. I t was the o b j e c t o f t h i s i n v e s t i g a t i o n t o s t u d y t h e mode o f u t i l i z a t i o n o f d i c a r b o x y l i c a c i d members o f t h e t r i c a r b o x y l i c a c i d c y c l e w i t h s p e c i a l r e f e r e n c e t o . s u c c i n a t e i n P. a e r u g i n o s a ATCC 9027 . The l e v e l o f t h e enzymes o f t h e t r i c a r b o x y l i c a c i d c y c l e , p e n t o s e phosphate c y c l e and t h e E n t n e r - D o u d o r o f f pathway i n c e l l s grown i n s u c c i n a t e and g l u c o s e media has been compared i n o r d e r t o a s s e s s t h e r e l a t i v e r o l e o f t h e s e pathways i n s u c c i n a t e m e t a b o l i s m . F u r t h e r , an a t t e m p t has been made t o e l u c i d a t e t h e mechanism c o n t r o l 1ing t r i c a r b o x y l i c a c i d c y c l e and g l y o x y J a t e c y c l e a c t i v i t i e s i n t h i s o r g a n i s m . LITERATURE REVIEW I . G e n e r a l P a t t e r n o f C a r b o h y d r a t e M e t a b o l ism i t i Pseudomonads A l t h o u g h B e r g e y ' s manual (1957) 1 i s t s 149 s p e c i e s o f Pseudomonas , a f a c t w h i c h i s i n d i c a t i v e o f t h e w i d e range o f m e t a b o l i c c a p a b i l i t i e s o f t h i s g e n u s , o n l y a few have been s t u d i e d i n d e t a i l . In p a r t i c u l a r o u r k n o w l e d g e o f t h e pathways o f c a r b o h y d r a t e m e t a b o l i s m o f t h i s genus has l a r g e l y come f r o m s t u d i e s o f Pseudomonas a e r u g ? n o s a , Pseudomonas f l u o r e s c e n s and Pseudomonas p u t i d a . S t r a i n A 3 . 1 2 o f P . f 1 u o r e s c e n s used by W.A . Wood and c o w o r k e r s i n e x h a u s t i v e m e t a b o l i c s t u d i e s has now been c l a s s i f i e d as a s t r a i n o f P . p u t i d a ( S t a n i e r e t a l , 1 966 ) . I t has been e s t a b l i s h e d t h a t t h e E n t n e r - D o u d o r o f f pathway p l a y s an i m p o r t a n t r o l e i n t h e u t i l i z a t i o n o f g l u c o s e i n pseudomonads . Pseudomonas s a c c h a r o p h ? 1 a d e g r a d e s g l u c o s e e x c l u s i v e l y v i a t h i s scheme w h i l e P_. a e r u g ? n o s a and P. r e p t i 1 i v o r a m e t a b o l i z e 71% and 72% o f added g l u c o s e r e s p e c t i v e l y v i a t h i s pathway and t h e r e m a i n d e r v i a t h e p e n t o s e - p h o s p h a t e pathway ( S t e r n , Wang and G i l m o u r , I 9 6 0 ) . The E n t n e r - D o u d o r o f f pathway has a l s o been shown t o be t h e m a j o r d e g r a d a t i v e pathway f o r g l u c o s e i n P. f l u o r e s c e n s (Wang, S t e r n and G i l m o u r , 1 959 ) . Mos t o f t h e Pseudomonas s p e c i e s have been found t o l a c k a c o m p l e t e E m b d e n - M e y e r h o f scheme. The f i r s t i n d i c a t i o n 4 o f t h i s was p r o v i d e d by a s t u d y o f t h e d i s t r i b u t i o n o f p h o s p h o r y l a t e d c a r b o h y d r a t e s i n P. a e r u g i h o s a , on t h e b a s i s o f wh i c h i t was c o n c l u d e d t h a t t h i s o r g a n i s m l a c k e d the Embden-Meyerhof pathway (Campbell and N o r r i s , 1950 ) . Wood and Schwerdt (1954) f u r t h e r showed t h a t p h o s p h o f r u c t o k i n a s e was a b s e n t i n P. f l u o r e s c e n s and thus the Embden-Meyerhof pathway was not o p e r a t i o n a l . A s i m i l a r o b s e r v a t i o n was r e p o r t e d i n Pseudomonas 1ihdrier? (Zymomonas m b b i l i s ) by Raps and DeMoss ( 1 962 ) . U s i n g r a d i o r e s p i r o m e t r i c e x p e r i m e n t s , S t e r n e t a l ( i 9 6 0 ) showed the absence o f t h e Embden-Meyerhof pathway i n t h r e e s p e c i e s o f pseudomonads. P r o d u c t i o n o f g l u c o n a t e and 2 - k e t o g l u e o n a t e from g1ucose has been o b s e r v e d i n pseudomonads by many w o r k e r s (DeLey,.1960 ). However, the s i g n i f i c a n c e o f t h i s o b s e r v a t i o n f i r s t became c l e a r when N o r r i s and Campbell (1949) found t h a t P_. a e r u g i n o s a o x i d i z e d g l u c o s e t o carb o n d i o x i d e and w a t e r v i a g l u c o n a t e and 2 - k e t o g l u c o n a t e . T h i s d i s c o v e r y t o g e t h e r w i t h the f i n d i n g t h a t t h i s o r g a n i s m l a c k s t h e Embden-Meyerhof pathway e n a b l e d the a u t h o r s t o d e s c r i b e t h e o p e r a t i o n o f a new pathway, t h e non-phosphory,l;ated pathway o f g l u c o s e d e g r a d a t i o n . T h i s has s i n c e been found i n P. f l u o r e s c e n s ( Narrod and Wood, 1956; Frampton and Wood, 1 961 ) . G 1uconokinase and 2 - k e t o -g l u c o n o k i n a s e have been d e m o n s t r a t e d and the p r o d u c t s , 6 - p h o s p h o g l u c o n a t e and 2 - k e t o - 6 - p h o s p h o g l u c o n a t e have been i d e n t i f i e d . A r e d u c t a s e c o n v e r t i n g 2 - k e t o - 6 - p h o s p h o g l u c o n a t e t o 6 - p h o s p h o g l u c o n a t e has been d e m o n s t r a t e d . I t has been proposed f u r t h e r t h a t 6 - p h o s p h o g l u c o n a t e 5 and 2 - k e t o - 6 - p h o s p h o g l u c o n a t e a r e m e t a b o l i z e d t o p y r u v a t e v i a t h e E n t n e r - D o u d o r o f f pathway. E v i d e n c e has been p r e s e n t e d t h a t 2-keto-g l u c o n a t e f o l lows a s i m i l a r m e t a b o l i c p a t t e r n i n P. a e r u g i riosa. (Kay, 1965). The e a r l i e s t a t t e m p t s t o de m o n s t r a t e the p r e s e n c e o f a complete t r i c a r b o x y l i c a c i d c y c l e i n m i c r o o r g a n i s m s met w i t h f a i l u r e . C e l l s u s p e n s i o n s were used i n t h e enzyme s t u d i e s and t h e s e s u s p e n s i o n s a p p a r e n t l y l a c k e d t h e a b i l i t y t o o x i d i z e c i t r i c a c i d o r some o f t h e d i c a r b o x y l i c a c i d s w i t h o u t a p e r i o d o f i n d u c t i o n . Campbell and Stokes (1951) o b s e r v e d t h a t a l t h o u g h a e e l 1 suspens i o n o f P_. a e r u g i hbsa f a ? l e d t o o x i d i z e a number o f the members o f t h e t r i c a r b o x y l i c a c i d c y c l e w i t h o u t a p r i o r p e r i o d o f i n d u c t i o n , d r i e d c e l l s o f t h e same p r e p a r a t i o n o x i d i z e d a l l o f the i n t e r m e d i a t e s w i t h o u t a l a g . They c o n c l u d e d " A p p a r e n t l y t h e c e l l membrane o f a e r u g i n o s a was i n i t i a l l y impermeable t o the compounds m e n t i o n e d , and t h e p e r i o d o f a d a p t a t i o n e n c o u n t e r e d w i t h r e s t i n g c e l l s was the tim e n e c e s s a r y f o r t h e e l a b o r a t i o n o f t h e system f o r t r a n s f e r r i n g t h e s u b s t r a t e s a c r o s s t h e membrane". S u b s e q u e n t l y , B a r r e t t , L a r s o n and K a l l i o (1953) u s i n g P, f l u o r e s c e n s showed t h a t d u r i n g p r o l o n g e d l a g s b e f o r e t h e o x i d a t i o n o f c e r t a i n t r i c a r b o x y l i c a c i d c y c l e members, p r o t e i n e s s e n t i a l t o t h e t r a n s p o r t o f t h e s e compounds a c r o s s the c e l l membrane was b e i n g s y n t h e s i z e d . F o r m a t i o n o f g l y o x y l a t e and s u c c i n a t e from c i t r a t e o r c i s -a c o n i t a t e was d e s c r i b e d by C a m p b e l l , Smith and E a g l e s (1953) i n 6 P_. a e r u g i n o s a . D i s c o v e r y o f t h i s r e a c t i o n t o g e t h e r w i t h t h a t o f m a l a t e s y n t h a s e by Wong and A j 1 (195°) l e d t o the f o r m u l a t i o n o f d i f f e r e n t s t e p s o f g l y o x y l a t e c y c l e ( K o r n b e r g and K r e b s , 1957) w h i c h i s e s s e n t i a l f o r growth on a c e t a t e i n pseudomonads and v a r i o u s o t h e r o r g a n i s m s . I t has been found t h a t g l y c o l l a t e , w h i c h i s a t a h i g h e r o x i d a t i o n l e v e l t h a n a c e t a t e , c a n n o t e n t e r t h e t r i c a r b o x y l i c a c i d o r g l y o x y l a t e c y c l e d i r e c t l y and i s m e t a b o l i z e d v i a a n o t h e r scheme named the " g l y c e r a t e pathway" ( K o r n b e r g , 1961) i n w h i c h two m o l e c u l e s o f g l y o x y l a t e , d e r i v e d from the o x i d a t i o n o f g l y c o l l a t e , condense t o g i v e t a r t r o n i c s e m i a l d e h y d e and c a r b o n d i o x i d e . T a r t r o n i c s e m i -a l d e h y d e i s reduced t o g l y c e r i c a c i d w h i c h i s p h o s p h o r y l a t e d t o p h o s p h o g l y c e r i c a c i d and f u r t h e r e n t e r s t h e Embden-Meyerhof scheme. I I. D i s t r i b u t i o n o f T r i c a r b o x y l i c Ac?d C y c l e Enzymes and M e t a b o l i s m  Of D i c a r b o x y l i c A c i d I htermedi a t e s i n Pseudomonads The i m p o r t a n c e o f the t r i c a r b o x y l i c a c i d c y c l e i n a e r o b i c m e t a b o l i s m i n b a c t e r i a i s w e l l e s t a b l i s h e d . In an e x c e l l e n t r e v i e w t h e f o r m u l a t i o n o f d i f f e r e n t s t e p s o f t r i c a r b o x y l i c a c i d c y c l e i n mammalian t i s s u e and m i c r o o r g a n i s m s has been d e s c r i b e d ( K r a m p i t z , 1961 ) . U s i n g d i f f e r e n t e x p e r i m e n t a l a p p r o a c h e s , K r a m p i t z a l s o p r e s e n t e d e v i d e n c e a g a i n s t t h e o p e r a t i o n o f d i c a r b o x y l i c a c i d c y c l e as proposed e a r l i e r by Thunberg (1920) and by A j 1 (1950; 1951 ) . Kogut and Podo s k i (1953) s t u d i e d t he o x i d a t i o n o f s u c c i n a t e 7 by whole c e l l s and c e l l - f r e e e x t r a c t s o f a s t r a i n o f P_. f l u o r e s c e n s . On t h e growth medium r i c h i n s u c c i n a t e and low i n ammonia, a c c u m u l a t i o n o f a - k e t o g l u t a r a t e was o b s e r v e d . C e l l - f r e e e x t r a c t s were shown t o po s s e s s t h e t r i c a r b o x y l i c a c i d c y c l e enzymes. From t h i s s t u d y i t was proposed t h a t s u c c i n a t e was b e i n g o x i d i z e d v i a the t r i c a r b o x y l i c a c i d c y c l e , a l t h o u g h t h e y c o u l d not e l i m i n a t e o t h e r p o s s i b i l i t i e s . Campbell and Smith (1956) showed t h e p r e s e n c e o f t r i c a r b o x y l i c a c i d c y c l e enzymes i n P_. a e r u g i n o s a , thus c o n f i r m i n g t h e i r e a r l i e r o b s e r v a t i o n o f the p r e s e n c e o f t h e c y c l e i n t h i s o r g a n i s m ( C a m p b e l l , and S t o k e s , 1951 ) . C a m p b e l l , Hogg.and S t r a s d i n e (1962) s t u d i e d t h e d i s t r i b u t i o n o f many i m p o r t a n t enzymes i n d i f f e r e n t f r a c t i o n s o f P_. a e r u g i n o s a and found t h a t s u c c i n i c dehydrogenase was a s s o c i a t e d w i t h t h e c e l l membrane whereas fumarase, i s o c i t r a t e dehydrogenase and i s o c i t r a t a s e were i n t h e s o l u b l e c y t o p l a s m . S t a n i e r , G u n s a l u s and Gunsalus (1953) r e p o r t e d t h a t t he m a l a t e o x i d i z i n g a c t i v i t y i n P. f l u o r e s c e n s was p r e s e n t i n t h e p a r t i c u l a t e f r a c t i o n . However, they d i d not attempt t o e l u c i d a t e t h e n a t u r e and l o c a t i o n o f t h i s enzyme. W h i l e s t u d y i n g o x i d a t i o n o f L-malate i r i Pseudomonas B? aba and Pseudomonas ova 1 i s C h e s t e r , F r a n c i s e_t a j _ (1963) found t h a t the f o r m e r o r g a n i s m p o s s e s s e d s o l u b l e L - m a l i c dehydrogenase and i t s c e l l - f r e e e x t r a c t u t i 1 i z e d m a l a t e v e r y s l o w l y and i n c o m p l e t e l y w h i l e e x t r a c t s from t h e l a t t e r o r g a n i s m o x i d i z e d t h i s s u b s t r a t e r a p i d l y and c o m p l e t e l y . T h i s i n v e s t i g a t i o n showed t h a t P. o v a l i s C h e s t e r p o s s e s s e s an NAD, NADP independent 8 p a r t i c u l a t e L - m a l i c dehydrogenase. I I I . T r i c a r b o x y l i c A c i d C y c l e A c t i v i t y and Enzymes o f G l u c o s e Oxi d a t i o n i h C e l 1 s Grown oh Med i a Conta? h i ng G1ucose o r a T r i c a r b o x y l i c A c i d C y c l e I n t e r m e d i a t e The g l u c o s e e f f e c t w h i c h i s d e f i n e d as the a b i 1 i t y o f g l u c o s e t o i n h i b i t t h e s y n t h e s i s o f c e r t a i n d e g r a d a t i v e enzymes was f i r s t r e c o g n i z e d by Epp and G a l e (1942) when th e y o b s e r v e d t h e i n h i b i t i o n o f t h e f o r m a t i o n o f amino a c i d deaminases by g l ucose i i i E.' c o l i ; These w o r k e r s e l i m i n a t e d t h e l o w e r i n g o f t h e pH o f the medium as the f a c t o r r e s p o n s i b l e f o r t h i s e f f e c t , I t has been s u g g e s t e d t h a t the g l u c o s e e f f e c t i s e x e r t e d by t h e c a t a b o l i t e s formed from g l u c o s e and hence i t i s s i m i l a r t o c a t a b o l i t e r e p r e s s i o n ( Magasanik, 1961). The g l u c o s e e f f e c t on t r i c a r b o x y l i c a c i d c y c l e enzymes has been r e c o g n i z e d i n v a r i o u s b a c t e r i a . C o l l i n s and L a s c e l l e s (1962) found t h a t when grown on n u t r i e n t b r o t h S. a u r e u s , o x i d i z e d g l u c o s e , a c e t a t e and i n t e r m e d i a t e s o f t r i c a r b o x y l i c a c i d c y c l e . However, when grown i n a g l u c o s e medium, th e o r g a n i s m f a i l e d t o o x i d i z e t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s and t h i s i n a b i l i t y was c o r r e l a t e d w i t h a low a c t i v i t y o f enzymes o f t h i s c y c l e i n e x t r a c t s o f t h e o r g a n i s m . S i m i l a r o b s e r v a t i o n s were r e p o r t e d by S t r a s t e r s and W i n k l e r (1963) i n the same o r g a n i s m . G l u c o s e has 9 a l s o been o b s e r v e d t o r e p r e s s the s y n t h e s i s o f t r i c a r b o x y l i c a c i d c y c l e enzymes i n B a c i 1 l u s s u b t i l i s and B a c i 1 l u s c e r e u s (Hanson, S r i n i v a s a n and H a l v o r s o n , 1963a; 1963b; Hanson e t a l , 1964; Hanson and Cox, 1 967 ) . An i n c r e a s e i n the s p e c i f i c a c t i v i t y o f t h e s e enzymes o c c u r r e d a f t e r d e p l e t i o n o f g l u c o s e from t h e medium. In an e x h a u s t i v e s t u d y on t h e r e g u l a t i o n o f m e t a b o l i s m o f E s c h e r i c h i a c o l i , G r a y , Wimpenny and Mossman (1966) showed t h a t g l u c o s e r e p r e s s e d t r i c a r b o x y l i c a c i d c y c l e enzymes. However, t h i s r e p r e s s i o n was p a r t i a l l y reduced when t h e c e l l s were grown i n a s y n t h e t i c medium c o n t a i n i n g g l u c o s e i n w h i c h s i t u a t i o n t h e c y c l e must be used f o r s y n t h e s i s . The g l u c o s e e f f e c t on enzymes o f t r i c a r b o x y l i c a c i d c y c l e has not been r e p o r t e d i n pseudomonads w i t h the e x c e p t i o n o f one s p e c i e s , i . e . Pseudomonas h i t r i e g e n s (Cho and Eagon, 1 967 ) . But t h i s s p e c i e s i s so a t y p i c a l t h a t i t p o s s e s s e s an a c t i v e Embden-Meyerhof pathway, can grow a n a e r o b i c a l l y , does not show E n t n e r -Doudorof f pathway a c t i v i t y when grown on g l u c o s e (Eagon and Wang, 1962) and does not p o s s e s s NADPH o x i d a s e o r t r a n s h y d r o g e n a s e (Eagon, 1963). Von T i g e r s t r o m and Campbell (T966b) compared the a c t i v i t y o f t r i c a r b o x y l i c a c i d c y c l e enzymes i ri P. a e r u g ? n o s a grown on g l u c o s e , d - k e t o g l u t a r a t e and a c e t a t e and found t h a t t h e l e v e l s o f t h e enzymes i n t h e c e l l s h a r v e s t e d from t h e t h r e e media were not s t r i k i n g l y d i f f e r e n t . I t has been r e p o r t e d t h a t t r i c a r b o x y l i c a c i d c y c l e enzymes may be c o n s t i t u t i v e i n pseudomonads but permeases f o r the c y c l e i n t e r m e d i a t e s a r e i n d u c i b l e (Campbell and S t o k e s , 1951 ; B a r r e t t et_ aj_, 1953). In c o n t r a s t i t appears t h a t t h e enzymes o f g l u c o s e c a t a b o l i s m i n pseudomonads a r e i n d u c i b l e . H a m i l t o n and Dawes (1960) found t h a t c u l t u r e s o f P. aerugiriosa:wh1ch have been grown on a g l u c o s e medium p o s s e s s g l u c o s e and g l u c o n i c dehydrogenases, h e x o k i n a s e , g l u c o n o k i n a s e , 2 - k e t o g l u c o n o k i n a s e , g l u c o s e - 6 - p h o s p h a t e and 6-phosphog1uconate dehydrogenases and the enzymes o f t h e E n t n e r -Doudorof f pathway. However, organisms grown on media c o n t a i n i n g members o f t h e t r i c a r b o x y l i c a c i d c y c l e showed e x t r e m e l y low l e v e l s o f t h e s e enzymes and t h e y were i n d u c e d on i n c u b a t i o n o f such c e l l s w i t h g l u c o s e . L a t e r i t was shown t h a t a l l o f t h e s e enzymes were induced on t r a n s f e r o f c i t r a t e grown c e l l s t o a medium c o n t a i n i n g g l u c o s e (Ng and Dawes, 1967). Von T i g e r s t r o m and Campbell (1966b) a l s o found t h a t when grown on a - k e t o g l u t a r a t e and a c e t a t e P. aerug?riosa was e i t h e r d e v o i d o f o r p o s s e s s e d v e r y low l e v e l s o f t h e enzymes r e q u i r e d f o r g l u c o s e o x i d a t i o n . S u c c i n a t e grown c e l l s were shown t o behave s i m i l a r l y ( L e s s i e and N e i d h a r d t , 1967a). Growth on t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s p r o b a b l y resembles t h a t on a complex medium s i n c e when grown i n such a medium P. f 1 u o r e s c e n s has been shown t o l a c k t h e enzymes o f the E n t n e r -Doudorof f pathway ( E i s e n b e r g and Dobrogosz, 1967)• 11 IV. C o n t r o l o f T r i c a r b o x y l i c A c i d C y c l e arid G l y o x y l a t e C y c l e  A c t i v i t y i n M i c r o o r g a n i s m s The t o p i c o f r e g u l a t i o n o f t r i c a r b o x y l i c a c i d c y c l e and g l y o x y l a t e c y c l e a c t i v i t y i s a r e c e n t s u b j e c t . Most o f the s t u d y has been w i t h c o l i f o r m b a c t e r i a and B a c i 1 l u s s p e c i e s . The e f f e c t o f a e r o b i o s i s and a n a e r o b i o s i s a p a r t from " c o a r s e " and " f i n e " c o n t r o l has been s t u d i e d . I n t e r e s t i n g o b s e r v a t i o n s were made by Amarsingham and Davi s (1965) on t h e r e g u l a t i o n o f a-keto-g l u t a r a t e dehydrogenase ?h E. c o l i . T h i s enzyme was found t o be c o m p l e t e l y r e p r e s s e d i n a n a e r o b i c a l l y grown c e l l s w h i l e i n a e r o b i c c u l t u r e s grown on g l u c o s e o r l a c t a t e i t was not formed u n t i l a s u b s t a n t i a l a c c u m u l a t i o n o f m e t a b o l i t e s (presumably a-keto-g l u t a r a t e o r a c e t a t e ) had o c c u r r e d . They proposed t h a t a - k e t o g l u t a r a t e dehydrogenase s e r v e s as a c o n n e c t i n g l i n k between the r e d u c t i v e b r a n c h l e a d i n g t o s u c c i n a t e and t h e b i o s y n t h e t i c branch l e a d i n g t o a - k e t o g l u t a r a t e . Under a n a e r o b i c c o n d i t i o n s t h i s c o n n e c t i o n i s not r e q u i r e d and i s a b s e n t . Under a e r o b i c c o n d i t i o n s t h e second h a l f o f the c y c l e can s e r v e a c a t a b o l i c r o l e and a - k e t o g l u t a r a t e dehydrogenase i s s y n t h e s i z e d t o c o m p l e t e l y o x i d i z e s u b s t r a t e s by r e c y c l i n g . Gray et_a_l_ (1966b) have r e p o r t e d t h a t t h e l e v e l o f t r i c a r b o x y l i c a c i d c y c l e enzymes i s g r e a t l y reduced i n a n a e r o b i c a l l y grown E. c o 1 i . The c o a r s e c o n t r o l o f the enzymes o f t h i s c y c l e i s e x e r t e d i n such a way t h a t the a c t i v i t y i s m o d i f i e d a c c o r d i n g t o the c a t a b o l i c and a n a b o l i c need. When g l u t a m a t e must be s y n t h e s i z e d , the enzymes l e a d i n g t o i t s f o r m a t i o n a r e i n c r e a s e d even i n the p r e s e n c e o f g l u c o s e , but t h e o t h e r enzymes o f the c y c l e a r e not d e r e p r e s s e d p r o p o r t i o n a t e l y . The a u t h o r s s u g g e s t t h a t the enzymes o f t h i s a m p h i b o l i c pathway ( D a v i s , 1961) cannot be r e g a r d e d as c o n s t i t u t i v e and can be d i v i d e d i n t o a t l e a s t t h r e e groups w h i c h a r e under independent c o n t r o l , i . e . the enzymes c a t a l y z i n g : a. t h e t r i c a r b o x y l i c a c i d s ; b. t h e 5-carbon d i c a r b o x y l i c a c i d s , a-keto-g l u t a r a t e and g l u t a m a t e ; and c. t h e 4-carbon d i c a r b o x y l i c a c i d s . On complex media w i t h o u t g l u c o s e , a l 1 enzymes e x c e p t g l u t a m i c dehydrogenase a r e d e r e p r e s s e d so t h a t t h e c y c l e may o p e r a t e as an energy g e n e r a t i n g pathway. On s y n t h e t i c media w i t h g l u c o s e t h e a and c groups o f enzymes a r e induced w h i l e a - k e t o g l u t a r a t e dehydrogenase remains r e p r e s s e d s i n c e a - k e t o g l u t a r a t e i s now needed f o r g l u t a m a t e b i o s y n t h e s i s . The p r e s e n c e o f g l u t a m a t e , a - k e t o g l u t a r a t e o r compounds g i v i n g r i s e t o g l u t a m a t e i n g l u c o s e m i n i m a l medium caused complete r e p r e s s i o n o f a c o n i t a s e i n B. s i i b t i 1 i s and B. 1 i c h e n ? f o r m i s (Hanson and Cox, 1967), but fumarase, s u c c i n i c dehydrogenase, m a l i c dehydrogenase and i s o c i t r i c dehydrogenase were not a f f e c t e d . These w o r k e r s s u g g e s t e d t h a t i n d i v i d u a l c o n t r o l mechanisms o p e r a t e t o r e g u l a t e c a t a b o l i c and a n a b o l i c s e c t i o n s o f t h e t r i c a r b o x y l i c a c i d c y c l e a c t i v i t y i n t h e b a c i l l i , and t h i s c o n t r o l mechanism i s d i f f e r e n t • from-Ej-'col i s i n c e i n the l a t t e r o r g a n i s m a c t i v i t y o f both the a n a b o l i c and t h e c a t a b o l i c s i d e o f t h e c y c l e was r e p r e s s e d under the same c o n d i t i o n s o f growth. R e c e n t l y , S h i i o and Oz a k i (1968) have r e p o r t e d i n t e r e s t i n g o b s e r v a t i o n s on c o n c e r t e d i n h i b i t i o n o f N A D P - s p e c i f i c i s o c i t r a t e dehydrogenase a c t i v i t y i n B r e v i b a c t e r i u m f l a v u m w h i c h was a l s o o b s e r v e d w i t h t h i s enzyme from E. c o l i , B. s u b t ? l i s and p i g h e a r t . These w o r k e r s found t h a t the s i m u l t a n e o u s a d d i t i o n o f s m a l l amount o f g l y o x y l a t e and o x a l a c e t a t e caused s t r o n g i n h i b i t i o n o f t h i s enzyme w h i l e t h e i r a d d i t i o n s i n g l y i n e q u i m o l a r amounts d i d n ot r e s u l t i n s i g n i f i c a n t i n h i b i t i o n . K i n e t i c a n a l y s i s showed t h a t the enzyme when a l r e a d y bound t o one o f t h e s e i n h i b i t o r s combines about 60 ,000 t i m e s s t r o n g e r w i t h the o t h e r i n h i b i t o r than when i t i s f r e e . The c o n d e n s a t i o n p r o d u c t o f g l y o x y l a t e and o x a l a c e t a t e ( o x a l m a l a t e ) was not found t o be a s t r o n g i n h i b i t o r o f t he i s o c i t r a t e dehydrogenase from t h e above s o u r c e s , a l t h o u g h i t was a l s o shown t h a t the c o n d e n s a t i o n p r o d u c t i s not formed under p h y s i o l o g i c a l c o n d i t i o n s and i s t h e r e f o r e not i m p o r t a n t i n the r e g u l a t i o n o f t r i c a r b o x y l i c a c i d c y c l e a c t i v i t y as was proposed by R u f f b e t _ a l _ (1967) i n p i g h e a r t t i s s u e . On t h e b a s i s o f f u r t h e r o b s e r v a t i o n t h a t the combined e f f e c t o f t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s was more i n h i b i t o r y t o . i s o c i t r a t e l y a s e than t o t h e i s o c i t r a t e dehydrogenase o f B. f l a v u m , i t was proposed t h a t a h i g h c o n c e n t r a t i o n o f t r i c a r b o x y l i c a c i d c y c l e members i n h i b i t s t h e a c t i v i t y o f t h e g l y o x y l a t e c y c l e by r e p r e s s i n g i s o c i t r a t e l y a s e (Ozaki and S h i i o , 7968). On t h e o t h e r hand, e x c e s s g l y o x y l a t e produced as a r e s u l t o f i s o c i t r a t e l y a s e a c t i v i t y i n h i b i t s i s o c i t r a t e dehydrogenase i n c o n c e r t w i t h o x a l a c e t a t e . O x a l -a c e t a t e i s presumed t o be p r e s e n t i n the c e l l a t low l e v e l s a t a l l t i m e s . I t has been shown t h a t when E. c o l i o r pseudomonads a r e grown, i n g l u c o s e , p y r u v a t e o r m e t a b o l i z a b l e i n t e r m e d i a t e s o f t h e t r i c a r b o x y l i c a c i d c y c l e , i s o c i t r a t e l y a s e s y n t h e s i s i s r e p r e s s e d ( K o r n b e r g , 1965; 1966). I t has been proposed t h a t i n E. c O l i and Achromobacter s p e c i e s , t he g l y o x y l a t e c y c l e i s not induced by a c e t a t e o r a c e t y l - C o A d i r e c t l y , but t h a t t h e s e compounds induce the c y c l e i n d i r e c t l y by.removing t h e r e p r e s s e r w h i c h has been i d e n t i f i e d as p h o s p h o e n o l p y r u v a t e . The f i n e c o n t r o l o f i s o c i t r a t e l y a s e has been proposed t o be e x e r t e d a g a i n by phospho-e n o l p y r u v a t e i h E_. col_ i _ and t h i s mechanism seems t o be d i f f e r e n t than t h a t found i n B. f l a v u m , j n whi c h i s o c i t r a t e l y a s e a c t i v i t y i s u n a f f e c t e d by p h o s p h o e n o l p y r u v a t e (Ozaki and S h i i o , 1968). Pseudomonads a r e known t o u t i 1 i z e and grow on a g r e a t v a r i e t y o f o r g a n i c compounds and, t h e r e f o r e , they must p o s s e s s a w e l l d e v e l o p e d r e g u l a t o r y mechanism. However, r e f e r e n c e s on the c o n t r o l o f t h e • t r i c a r b o x y l i c -acid c y c l e and g l y o x y l a t e c y c l e a c t i v i t y i n pseudomonads a r e not a v a i l a b l e . Smith and Gunsalus (1957) s t u d i e d t h e r e a c t i o n mechanism o f i s o c i t r a t e l y a s e i n P. a e r u g i n o s a and found t h a t i t was n o n - c o m p e t i t i v e l y i n h i b i t e d by i t s p r o d u c t g l y o x y l a t e and s u c c i n a t e . In P. i r i d i g o f e r a , s u c c i n a t e i s a "mixed" t y p e i n h i b i t o r o f t h i s enzyme (McFadden and Howes, 1963) as was a l s o o b s e r v e d w i t h B. f l a v u m (Ozaki and S h i i o , 1 9 68 ) . There have been a few r e p o r t s on the e f f e c t o f n i t r a t e on the t r i c a r b o x y l i c a c i d c y c l e a c t i v i t y o f pseudomonads. C e r t a i n s p e c i e s such as P. d e r i ? t r i f i c a n s , P. aerug?nosa and P. s t u t z e r i grow a n a e r o b i c a l l y i f n i t r a t e i s i n c l u d e d i n the medium. T h e • t r i c a r b o x y l i c a c i d c y c l e was shown t o f u n c t i o n i n P. s t u t z e r i grown u n d e r . t h e s e c o n d i t i o n s ( S pangIer and G i l m o u r , 1 966 ) . S i m i l a r l y , Wimpenny and Warms l e y (1968) found t h a t fumarase and a e o n i t a s e were u n a f f e c t e d i n P_. a e r u g i nosa, P: s t u t z e r i • and o t h e r aerobes such as B. s u b t i 1 i s and B. megatherium when grown a n a e r o b i c -a l l y w i t h n i t r a t e . However, i n f a c u l t a t i v e organisms such as A. a e r o g e n e s , E. c o l i and B. marcerans t h e s e enzymes f e l l t o u n d e t e c t a b l e l e v e l s under t h e s e c o n d i t i o n s due t o a c c u m u l a t i o n o f h i g h l e v e l s o f n i t r i t e i n the medium. These o b s e r v a t i o n s thus s u g g e s t d i f f e r e n t t y p e s o f c o n t r o l o f t r i c a r b o x y l i c a c i d c y c l e a c t i v i t y i n aerobes and f a c u l t a t i v e a n a e r o b e s . -16 V. M o l a r Growth Y i e l d as a T o o l i n t h e Study o f M e t a b o l i c Pathways M o l a r growth y i e l d (MGY) i s d e f i n e d as t h e d r y w e i g h t o f c e l l s i n gms o b t a i n e d per mole o f car b o n s o u r c e used d u r i n g g r o wth. An-a e r o b i c b a c t e r i a , i n w h i c h assessment o f energy p r o d u c t i o n i s r e l a t i v e l y e a s y , were f i r s t t e s t e d f o r d e v i a t i o n s from the Embden-Meyerhof pathway u s i n g MGY d a t a . DeMoss, Bard and . Guns a l u s (1951)'• compared the MGY o f S t r e p t o c o c c u s f a e c a l i s and LeuconOstoc m e s e n t e r o i d e s grown on g l u c o s e and c o n c l u d e d t h a t L. m e s e n t e r o i d e s fermented g l u c o s e by a pathway w h i c h y i e l d e d l e s s e n e r g y than the Embden-Meyerhof pathway used by S_. f a e c a l i s . F u r t h e r work l e d t o t h e d i s c o v e r y o f the mechanism o f h e t e r o l a c t i c f e r m e n t a t i o n i n L, m e s e n t e r o i d e s (Gunsalus and G i b b s , 1 952 ) . Bauchop and E l s d e n (1960) showed t h a t v ^ T p , t h e y i e l d i n gms o f d r y w e i g h t per mole o f ATP i s s i m i l a r f o r d i f f e r e n t m i c r o o r g a n i s m s u s i n g t h e same pathway o f c a r b o h y d r a t e m e t a b o l i s m . T h i s o b s e r v a t i o n i s o f g r e a t i m p o r t a n c e i n c o r r e l a t i n g t h e o b t a i n e d on a l i m i t i n g c a r b o n s o u r c e i n t h e medium w i t h t h e m e t a b o l i c r o u t e used. Under a e r o b i c c o n d i t i o n s a l a r g e p a r t (20 t o 50%) o f t h e energy s o u r c e i s c o n v e r t e d t o c e l l u l a r m a t e r i a l (Hernandez and Jo h n s o n , V967) w h i c h makes t h e c a l c u l a t i o n o f energy b a l a n c e i m p o s s i b l e . M o r e o v e r , i t i s w i d e l y a c c e p t e d t h a t under a e r o b i c c o n d i t i o n s , e nergy i s never l i m i t i n g and t h e r e f o r e , Y^.p v a l u e s have no s i g n i f i c a n c e . Hernandez and Johnson Q967) found t h a t under a e r o b i c c o n d i t i o n s ATP i s not the f a c t o r l i m i t i n g growth and the Y^-p v a l u e i s not a c o n s t a n t . I t has been o b s e r v e d t h a t under a e r o b i c c o n d i t i o n s the growth y i e l d i s p r o p o r t i o n a l t o the amount o f carbon s u p p l i e d by t h e s e s u b s t r a t e s (Monod, 1942; Campbel1, L i n n e s and E a g l e s , 1952 ) . However, i t can be argued t h a t two s u b s t r a t e s o f e q u i v a l e n t c a r b o n c o n t e n t c o u l d g i v e d i f f e r e n t c e l l y i e l d s i f t h e y a r e m e t a b o l i z e d by d i f f e r e n t pathways, one o f w h i c h i n v o l v e s an advantageous s t e p o f g a i n i n g c a r b o n , i ; e . CC^ f i x a t i o n . R e c e n t l y , M a y b e r r y , P r o c h a z k a and Payne (1967) made an i n t e r e s t i n g s t u d y on growth y i e l d s o f b a c t e r i a under a e r o b i c c o n d i t i o n s and found t h a t t h e v a l u e s o f y i e l d s p er mole o f s u b s t r a t e ; p e r gm atom o f s u b s t r a t e c a r b o n ; and per mole o f oxygen consumed v a r i e d o v e r a v e r y wide range. However, t h e y found t h a t the growth y i e l d p e r e q u i v a l e n t o f a v a i l a b l e e l e c t r o n s . i n t h e s u b s t r a t e u t i l i z e d was c o n s t a n t , and was a p p r o x i m a t e l y 3.14 gm o f c e l l s p er e q u i v a l e n t o f a v a i 1 a b l e e l e c t r o n s . V I . S u c c i n a t e . U t ? 1 i z a t i o n as Di s t ? net from O t h e r Di c a r b o x y l i c A c i d s o f t h e T r i c a r b o x y l i c Ac?d C y c l e In s e v e r a l s t u d i e s , i t was found t h a t i n r e l a t i o n t o o t h e r t r i c a r b o x y l i c a c i d c y c l e enzymes, a low l e v e l o f s u c c i n i c dehydrogenase was p r e s e n t i n P. a e r u g i n o s a (Campbell e t a l , 1962; Von T i g e r s t r o m and C a m p b e l l , 1 9 6 6 b ) , but i n s p i t e o f t h i s t he o r g a n i s m o x i d i z e d s u c c i n a t e a t r a t e s comparable t o t h o s e o f o t h e r c y c l e i n t e r m e d i a t e s . Thus, on the b a s i s o f t h e s e s t u d i e s , i t was d i f f i c u l t t o c o n c l u d e t h a t s u c c i n a t e was u t i l i z e d s o l e l y by the a c t i o n o f s u c c i n i c dehydrogenase. R e c e n t l y , s u c c i n a t e w h i c h was p r e v i o u s l y c o n c l u d e d t o be a f e e d b a c k i n h i b i t o r o f h i s t i d a s e i h v i v o i n P. a e r u g i hosa (Less i e and N e i d h a r d t , 1967b) has been found t o i nh ?b? t u r o c a n a s e i n P. p u t i d a (Hug, Roth and H u n t e r , 1968). U r o c a n a t e a c c u m u l a t e d due t o i n h i b i t i o n o f u r o c a n a s e w h i c h i n t u r n i n h i b i t e d h i s t i d a s e . Thus, s u c c i n a t e i n d i r e c t l y i n h i b i t e d h i s t i d a s e by i n h i b i t i n g u r o c a n a s e . However, o t h e r compounds wh i c h s u p p o r t r a p i d growth such as c i t r a t e a l s o s u p p r e s s h i s t i d a s e s y n t h e s i s presumably by t h e same mechanism as s u c c i n a t e . I t w ould have been r e l e v a n t t o have o b s e r v e d the i n h i b i t i o n by f u m a r a t e and m a l a t e , but t h e i r e f f e c t was not s t u d i e d . V I I . M e t a b o l i c P a t t e r n and E v o l u t 1 on There a r e two major forms o f a v a i l a b l e b i o c h e m i c a l e n e r g y . One i s ATP and the o t h e r the r e d u c i n g power o f NADPH (or NADH) whi c h i s used t o d r i v e t h e r e a c t i o n s i n t h e d i r e c t i o n o f r e d u c t i o n , f o r m i n g compounds w h i c h y i e l d more energy on c o m b u s t i o n . T h i s r e d u c i n g power may be c a l l e d the " m e t a b o l i c h y d r o g e n " w h i c h i n t h e modern form o f l i f e r e p l a c e s the m o l e c u l a r hydrogen t h a t m a i n t a i n e d t h e m e t a b o l i t e s i n reduced form i n p r i m i t i v e l i f e forms ( H o r e c k e r , 1965). H o r e c k e r (1965) has d e s c r i b e d t h e r e l a t i o n s h i p o f NADH and NADPH w i t h oxygen and c e l l u l a r b i o s y n t h e s i s as f o l l o w s : S u b s t r a t e NADH f a s t S u b s t r a t e I r NADPH sl o w N o ~ P R e d u c t i o n r e a c t i o n s There w o u l d be no need f o r two coenzymes i n the absence o f m o l e c u l a r oxygen, s i n c e a s i n g l e coenzyme c o u l d a c t bo t h as t h e coenzyme f o r f e r m e n t a t i o n and as a s o u r c e o f m e t a b o l i c hydrogen. T h i s i s what i s seen i n the p r e s e n t day s t r i c t anaerobes such as C l o s t r i d i a . In t h e s e s p e c i e s f e r m e n t a t i o n f o r t he p r o d u c t i o n o f energy i s s t i l l t h e major m e t a b o l i c a c t i v i t y . T h i s a l s o i m p l i e s t h a t anaerobes d e v e l o p e d f i r s t i n e v o l u t i o n a r y h i s t o r y s i n c e i t i s b e i i e v e d t h a t t h e atmosphere o f t h e p r i m i t i v e e a r t h was h i g h l y r e d u c i n g c o n t a i n i n g hydrogen compounds such as methane, ammonia and m o l e c u l a r hydrogen i t s e l f . L a t e r , i n t h e p r e s e n c e o f oxygen, p r e e x i s t i n g s u b u n i t s i n the h y d r o s p h e r e were c o n v e r t e d t o more o x i d i z e d forms. S i n c e NADH was now u t i l i z e d f o r t he p r o d u c t i o n o f energy i n o x i d a t i v e p h o s p h o r y l a t i o n , i t was not a v a i l a b l e f o r r e d u c t i v e r e a c t i o n s and a second coenzyme, NADP was e v o l v e d from NAD when r e s p i r a t i o n a p p e a r e d . NADPH p r o v i d e d a s o u r c e o f m e t a b o l i c hydrogen w h i c h was not i n e q u i l i b r i u m w i t h oxygen. The hexose monophosphate pathway has been shown t o be r a t e -l i m i t e d p r i m a r i l y by the r a t e o f NADP s u p p l y i n a v a r i e t y o f animal t i s s u e s such as l i v e r (Cahi 1 1 et_ a l _ , 1958) and i n mammary t i s s u e (McLean, I 9 6 0 ) . Eagon (1962) showed t h a t t h e s u p p l y o f NADP was r a t e l i m i t i n g i n the o p e r a t i o n o f the hexose monophosphate pathway i i i P. l i i t r i e g e n s . T h i s o r g a n i s m p o s s e s s e s enzymes o f t h i s pathway but l a c k s NADH o x i d a s e and t r a n s h y d r o g e n a s e . A s t r o n g c o r r e l a t i o n between t h e s i m u l t a n e o u s p r e s e n c e o f NADPH o x i d a s e p l u s t r a n s -hydrogenase w i t h t h e u t i l i z a t i o n o f t h e hexose monophosphate pathway and the E n t n e r - D o u d o r o f f pathway when grown on g l u c o s e has been shown i n d i f f e r e n t m i c r o o r g a n i s m s (Eagon, 1963) -A l t h o u g h c o mplete t r i c a r b o x y l i c a c i d c y c l e a c t i v i t y i s a s s o c i a t e d w i t h a e r o b i c l i f e , two p o r t i o n s o f the c y c l e s e r v i n g b i o s y n t h e t i c p u rposes o p e r a t e under a n a e r o b i c c o n d i t i o n s . I t i s now becoming i n c r e a s i n g l y common t o f i n d t h e a n a e r o b i c r o u t e v i z . the c i t r a t e s y n t h a s e , a c o n i t a s e and i s o c i t r a t e dehydrogenase a c t i v i t y l e a d i n g t o b i o s y n t h e s i s o f a - k e t o g l u t a r a t e i n a n a e r o b e s . I t has been r e p o r t e d i n a v a r i e t y o f anaerobes such as C l o s t r i d i u m k l u y v e r i , Methanobaci 1 l u s onie 1 i a i i s k ? i , P e p t o s t r e p t o c o c c u s e l s d e n i 1 and Ch 1oropseudomohas e t h y l icum ( S t e r n and Bambers, 1 9 6 6 ; G o t t s c h a l k and B a r k e r , 1 9 6 7 ; S o m e r v i l i e , 1 9 6 8 ; C a l l e l y and F u l l e r , 1 9 6 7 ) . In cytochrome c o n t a i n i n g anaerobes such as B a c t e r o i d e s ' r u m i n i c o l a , ' s u c c i n a t e has been shown t o be formed from p y r u v a t e o r i t s d e r i v a t i v e t h r o u g h the r e d u c t i v e branch o f t h e t r i c a r b o x y l i c a c i d c y c l e ( W h i t e , B r y a n t and C a l d w e l l , 1 962 ) . The s h i f t from a n a e r o b i c t o f a c u l t a t i v e o r a e r o b i c l i f e i n v o l v e d some m e t a b o l i c changes i n m i c r o o r g a n i s m s . The coenzyme FAD was p r o b a b l y o r i g i n a l l y e v o l v e d i n anaerobes f o r c a t a l y s i n g r e d u c t i v e s t e p s i n f a t t y a c i d s y n t h e s i s , but the f l a v o p r o t e i n o x i d a s e system t o c a t a l y s e t r a n s f e r o f e l e c t r o n s from s u b s t r a t e s d i r e c t l y t o oxygen must have been t h e f i r s t s t e p towards a d a p t a t i o n t o a e r o b i o s i s ( D o l i n , 1 9 61 ) . I t has been o b s e r v e d t h a t l a c t i c a c i d b a c t e r i a w h i c h have no c y t o c h r o m e s , c a r r y out c e r t a i n o x i d a t i o n r e a c t i o n s i n t h i s f a s h i o n . R e c e n t l y , London (1968) has shown t h a t 1 ti S. f aec i um an L (+) s p e c i f i c l a c t a t e o x i d i z i n g enzyme i s induced on a e r o b i c g r o wth. T h i s o x i d a s e w h i c h has FAD as the coenzyme a t t a c h e d t o the apoenzyme e n a b l e s t h e o r g a n i s m t o grow under a e r o b i c c o n d i t i o n s on l a c t a t e . I t was su g g e s t e d t h a t t h i s enzyme c o n f e r s on t h e o r g a n i s m the a b i 1 i t y t o . s u r v i v e ' i n a c a r b o h y d r a t e d e p l e t e d a e r o b i c e n v i r o n m e n t c o n t a i n i n g L(+) l a c t a t e . F a c u l t a t i v e o r g a n i s m s a r e c a p a b l e o f d e v e l o p i n g b o t h a n a e r o b i c and a e r o b i c m e t a b o l i s m depending upon the c o n d i t i o n s p r o v i d e d . The e x t e n s i v e s t u d y by Gray e t a l _ ( 1966a; 1966b) and by Wimpenny and Warms l e y (1968) p r o v i d e good i n s i g h t i n t o t h i s phenomenon. The metabol i c p a t t e r n o f E_. co]_i_, when grown under a e r o b i c • c o n d i t i o n s , ' r e s e m b l e s ' t h a t - o f an aerobe w h i l e i t s i m u l a t e s an anaerobe when grown i n the absence o f oxygen. A u t o t r o p h s , w h i c h a r e c o n s i d e r e d a s t e p beyond h e t e r o t r o p h s i n e v o l u t i o n , a r e c o m p r i s e d o f a e r o b i c and a n a e r o b i c s p e c i e s and have d e v e l o p e d d i f f e r e n t m e t a b o l i c p a t t e r n s . I t has been proposed t h a t o b l i g a t e chemoautotrophs have e v o l v e d from h e t e r o t r o p h i c a n c e s t o r s by e l i m i n a t i o n o f c e r t a i n enzymes r e s u l t i n g i n t h e l o s s o f h e t e r o t r o p h i c m e t a b o l i s m . S e v e r a l s p e c i e s o f a u t o t r o p h i c t h i o b a c i l l i and b l u e - g r e e n a l g a e l a c k NADH o x i d a s e (thus c o n s e r v i n g r e d u c i n g power o f NADH) and a - k e t o g l u t a r a t e dehydrogenase a c t i v i t y , w h i c h r e s u l t s i n the d e s t r u c t i o n o f t h e a b i l i t y o f t h e o r g a n i s m t o use o r g a n i c compounds f o r growth and p r o d u c t i o n o f energy ( S m i t h , London and S t a n i e r , 1967). However, l o s s o f NADH o x i d a s e and a - k e t o g l u t a r a t e dehydrogenase i s not always t h e cause o f a u t o t r o p h y . S t u d i e s oh N i t r o c y s t i s oceanus f a i l e d t o show t h a t o b i i g a t e a u t o t r o p h y was s o l e l y due t o t h i s m e t a b o l i c d e f e c t ( W i l l i a m s and Watson, 1968). The a u t h o r s c o n c l u d e d t h a t an o r g a n i s m was a u t o t r o p h i c because i t e i t h e r l a c k e d o r had low l e v e l s o f one o r more enzymes o r i t s membrane was not permeable t o o r g a n i c m o l e c u l e s a t a r a t e s u f f i c i e n t f o r g r o w t h . A c o m b i n a t i o n o f two o r more o f t h e s e d e f e c t s may be e x p e c t e d . I t i s thus o b v i o u s from t h i s d i s c u s s i o n t h a t a v a s t amount o f i n f o r m a t i o n c o u l d be g a t h e r e d about the b e h a v i o u r and c o n t r o l mechanisms i n the o r g a n i s m by t h e s t u d y o f i t s m e t a b o l i c a c t i v i t y vand the n a t u r e and l e v e l o f enzymes p r e s e n t , when grown on a v a r i e t y o f c a r b o n s o u r c e s and e n v i r o n m e n t a l c o n d i t i o n s . MATERIALS AND METHODS I . 0 rgari ? srfis arid Growth • Med i a Pseudomonas aeruginosa ATCC 9027 and Aerobacter aerogenes (departmental stock) were grown in Roux flasks without shaking at 30 C and 37 C respectively. The growth medium consistedof NH!jH2P0Zf, 0.3%; K 2 H P 0 ^ , 0.2%; iron as FeSO^H^, 0.5 p.p.m; MgS0^.7H20, 0.05% and carbon source (minimal medium). The pH was adjusted to 7.0 and sterile MgSO^^H^O and the carbon source were added to the autoclaved medium before inoculation., This medium without the carbon source will be referred to as the basal salts medium. The organisms were grown with 0.2% carbon source for manometric experiments and with 0.3% when used for enzyme assays. The carbon sources used and their concentration as weir as-.pH of the medium when different than mentioned above wi11 be specified. In some growth experiments the organisms were grown in250 ml side arm flasks in a shaking water bath and density of the culture was recorded with a Klett-Summerson colorimeter. Cultures were routinely checked for purity by'. streak?ng on suitable media. P. aeruginosa produces pyocyanine on King's medium (King, Ward and Raney, 1954) which was used to check the p u r i t y o f t h i s o r g a n i s m . I I . P r e p a r a t i o n o f Rest i hg Cel 1 Suspens ions and C e l 1 - F r e e E x t r a c t s The o r ganisms were h a r v e s t e d by c e n t r i f u g a t i o n a t room t e m p e r a t u r e i n e a r l y s t a t i o n a r y phase f o r manometric s t u d i e s o r i n the l o g a r i t h m i c phase o f growth f o r enzyme a s s a y s . . They were washed t w i c e w i t h 0.01 M t r i s (hydroxymethyl)aminomethane ( T r i s ) c h l o r i d e b u f f e r , pH 7 - 0 . In some e x p e r i m e n t s n e u t r a l i z e d 0.3% sodium c h l o r i d e s o l u t i o n was used f o r w a s h i n g . R e s t i n g c e l l s u s p e n s i o n s were o b t a i n e d by r e s u s p e n d i n g t h e p e l l e t i n 0.1 M T r i s b u f f e r , pH J.k, t o a p p r o x i m a t e l y 5 mg d r y w e i g h t o f c e l l s / m l . , For p r e p a r a t i o n o f c e l l - f r e e e x t r a c t s t he p e l l e t was resuspended i n 0 .2 M T r i s b u f f e r , pH 1.k, t o a s u i t a b l e c o n c e n t r a t i o n and 1 drop o f d e o x y r i b o n u c l e a s e (1 mg/ml aqueous s o l u t i o n ) was added f o r e v e r y 2.0 ml o f t h e s u s p e n s i o n . The c e l l s were d i s r u p t e d i n a French p r e s s u r e c e l l and c e n t r i f u g e d a t 8,000 x g_ o r 20,000 x £ f o r 15 min t o remove whole c e l l s and d e b r i s . The s u p e r n a t a n t , r e f e r r e d t o as t h e c e l l - f r e e e x t r a c t , was used f o r r e s p i r o m e t r y e x p e r i m e n t s and f o r enzyme a s s a y s r e s p e c t i v e l y . When f u r t h e r f r a c t i o n a t i o n was d e s i r e d t he c e l l - f r e e e x t r a c t was c e n t r i fuged f o r 30 min a t 25 ,000 x g_ and f o r 90 min a t 100,000 x £ i n a S p i n c o Model L U l t r a c e n t r i f u g e . The 100,000 x £ p e l l e t was washed once wrt-th 0.1 M T r i s b u f f e r , pH 7 . 0 , and resuspended a t a s u i t a b l e c o n c e n t r a t i o n i n 0 .2 M T r i s b u f f e r , pH 7 . 4 , u s i n g a " P o t t e r " g l a s s homogenizer. When m o d i f i c a t i o n s were made i n t h i s scheme they have been s p e c i f i e d . I I I . Deterrhi hat i o n o f M o l a r Growth Y i e l d on Di f f e r e h t S u b s t r a t e s P. a e r u g i n o s a 9027 was grown w i t h s h a k i n g i n t h e medium a t pH 7.4 w i t h 0 .2% g l u c o s e (11.1 umole/ml o f t h e medium). When o t h e r c a r b o n s o u r c e s were used, the c o n c e n t r a t i o n added was e q u i v a l e n t t o t h e carbon c o n t e n t o f g l u c o s e and t h e i n i t i a l pH o f the medium was 6 .7 i n o r d e r t o keep t h e f i n a l pH from g o i n g above 7 . 7 . C e l l s were h a r v e s t e d a t room t e m p e r a t u r e and washed t w i c e w i t h 0 .025 M T r i s b u f f e r , pH 7 . 0 . The p e l l e t was resuspended i n t h e same b u f f e r t o a d e f i n i t e volume i n a v o l u m e t r i c f l a s k and d r y w e i g h t s were d e t e r m i n e d on a s u i t a b l e volume o f t h i s s u s p e n s i o n by d r y i n g d u p l i c a t e samples t o a c o n s t a n t w e i g h t a t 95 t o 97 C; For c a l c u l a t i o n o f growth y i e l d s t h e v a l u e a t t h e peak o f t h e growth c u r v e was t a k e n . IV. I s o l a t i o n o f Mutants o f P_. a e r u g i n o s a The method recommended by A d e l b e r g , Mandel and Chen (1965) was f o l l o w e d . W i l d t y p e l o g a r i t h m i c phase c e l l s were h a r v e s t e d from ^fumarate m i n i m a l medium by c e n t r i f u g a t i o n a t room t e m p e r a t u r e . The c e l l p e l l e t was washed t w i c e w i t h 0.01 M c i t r a t e b u f f e r , pH 5.5 and resuspended i n t h e same b u f f e r a t t h e o r i g i n a l c o n c e n t r a t i o n and shaken a t 30 C f o r 1 h r . N - m e t h y l - N ' - n i t r o - N - n i t r o s o g u a n i d i n e was added t o g i v e a f i n a l c o n c e n t r a t i o n o f 100 Ug/ml and i t was f u r t h e r shaken f o r 45 min. T h i s s u s p e n s i o n was then c e n t r i f u g e d a t 3 t o 4 C and t h e p e l l e t , a f t e r w a s h i n g t w i c e w i t h b a s a l s a l t s medium, was resuspended t o the o r i g i n a l volume i n n u t r i e n t b r o t h . A g a i n , t h i s c e l l s u s p e n s i o n was i n c u b a t e d f o r 4 t o 6 h r t o e n r i c h f o r t h e mutants. The c e l l s u s p e n s i o n was then d i l u t e d and s p r e a d on s u c c i n a t e o r fumarate minimal medium c o n t a i n i n g a l i m i t i n g c o n c e n t r a t i o n (0.0\%)- o f a-ketog 1 u t a r a t e f o r a c q u i r i n g mutants b l o c k e d i n some s t e p i n t h e t r i c a r b o x y l i c a c i d c y c l e l e a d i n g t o b i o s y n t h e s i s o f a - k e t o g l u t a r a t e . For i s o l a t i o n o f mutants b l o c k e d i n fumarase a c t i v i t y , the s u s p e n s i o n was s p r e a d on f u m a r a t e minimal medium c o n t a i n i n g a l i m i t i n g c o n c e n t r a t i o n o f g l u c o s e . The s m a l l c o l o n i e s were p i c k e d and checked by p a t c h i n g on p l a t e count a g a r and minimal media c o n t a i n i n g d i f f e r e n t c a r b o n s o u r c e s . I t was found t h a t mutants unable t o grow on a - k e t o g l u t a r a t e and on g l u c o s e w h i c h grew s l o w e r t h a n w i l d t y p e e e l Is , were a l s o i s o l a t e d i n t h e s e e x p e r i m e n t s . F u r t h e r c h a r a c t e r -i z a t i o n was a t t e m p t e d by manometric e x p e r i m e n t s and enzyme a s s a y s . V. Manometric Methods U t i l i z a t i o n o f d i f f e r e n t s u b s t r a t e s by whole c e l l s , c e l l - f r e e e x t r a c t s and c e l l f r a c t i o n s was i n v e s t i g a t e d by c o n v e n t i o n a l manometric t e c h n i q u e s ( U m b r e i t , B u r r i s and S t a u f f e r , 1957) a t 30 C u s i n g Warburg r e s p i r o m e t e r s . C o r r e c t i o n s were made f o r endogenous v a l u e s . V I . Enzyme A s s a y s S o l u b l e L - m a l i c dehydrogenase was assayed a c c o r d i n g t o , Ochoa (1955) u s i n g T r i s b u f f e r , pH 7.6 and NADH o r NADPH as coenzymes. E t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA), 5 mM was used i n t h e r e a c t i o n m i x t u r e t o e l i m i n a t e t h e i n t e r f e r e n c e due t o m a l i c enzyme a c t i v i t y . M a l i c enzyme was assayed a t pH 8 . 5 , u s i n g t h e method o f J a c o b s o n e t a l (1966). A c o n t r o l c o n t a i n i n g 5 mM EDTA but w i t h o u t MgC.^ was used t o check i f t h e a c t i v i t y measured was s o l e l y due t o m a l i c enzyme. P a r t i c u l a t e L - m a l i c dehydrogenase was measured s p e c t r o p h o t o m e t r i c a l l y using' 2 , 6 - d i c h l o r o p h e n o l indophenol a c c o r d i n g t o F r a n c i s e t _ a j _ (1963). A g a i n , the a d d i t i o n o f 5 mM EDTA was used t o c o n f i r m t h e e l i m i n a t i o n o f m a l i c enzyme a c t i v i t y w h i c h might show up due t o t h e p r e s e n c e o f endogenous coenzymes i n the c e l l - f r e e e x t r a c t s . For i s o c i t r a t e dehydrogenase t h e r e a c t i o n m i x t u r e c o n t a i n e d T r i s b u f f e r , pH 7 . 8 , 50 mM; MgC.l 10. mM; NADP, 0 .25 mM; D L - i s o c i t r a t e , 3 .0 mM and the enzyme. The r e d u c t i o n o f coenzyme was r e c o r d e d a t 3^0 my. A c o n i t a s e and fumarase were as s a y e d by t h e method o f Racker ( 1950 ) . D L - i s o c i t r i c a c i d ( p o t a s s i u m s a l t ) was p r e p a r e d from i t s l a c t o n e a c c o r d i n g t o the p r o c e d u r e g i v e n by Hanson e t a l ( 1963b ) . a - K e t o g l u t a r a t e dehydrogenase was assayed by t h e p r o c e d u r e o f Amarsingham and D a v i s (1965) u s i n g a c e t y l p y r i d i n e - N A D as t h e coenzyme. For s u c c i n i c dehydrogenase t h e p h e n a z i n e m e t h o s u l f a t e method d e s c r i b e d by K i n g (1967) was used. G l u t a m a t e dehydrogenase was ass a y e d i n a 1.0 ml r e a c t i o n m i x t u r e w i t h NADH o r NADPH as t h e coenzyme and e x c e s s NH^Cl and a - k e t o g 1 u t a r a t e (Von T i g e r s t r o m and C a m p b e l l , 1 966a ) . When the a s s a y was i n t h e d i r e c t i o n o f a - k e t o g l u t a r a t e 1 ml o f the r e a c t i o n m i x t u r e c o n t a i n e d p o t a s s i u m phosphate b u f f e r , pH 8 . 0 , 50 mM; sodium L - g l u t a m a t e , 25 mM; NAD o r NADP, 0.25 mM and t h e enzyme. I s o c i t r a t a s e was a s s a y e d by t h e d e t e r m i n a t i o n o f g l y o x y l a t e formed from DL-i s o c i t r a t e by the a c t i o n o f t h e enzyme (Ozaki and S h i i o , 1 968 ) . In some p r e l i m i n a r y e x p e r i m e n t s , however, t h e a c t i v i t y was d e t e r m i n e d by the method o f Dixon and Kornberg ( 1959). S u c c i n y l CoA s y n t h e t a s e was a s s a y e d by the hydroxamate a s s a y method as d e s c r i b e d by G i b s o n , Upper and Gunsalus ( 1 967 ) . For t he a s s a y o f g l u c o s e - 6 - p h o s p h a t e dehydrogenase t h e r e a c t i o n m i x t u r e c o n t a i n e d T r i s b u f f e r , pH 8 . 0 , 50 mM; M g C l 2 , 5.0 mM; NADP, 0 .25 mM; g l u c o s e - 6 - p h o s p h a t e , 2.5 mM and t h e c e l l - f r e e e x t r a c t . For 6 - p h o s p h o g l u c o n a t e dehydrogenase a s s a y , t h e r e a c t i o n m i x t u r e was t h e same e x c e p t t h a t 6 - p h o s p h o g l u c o n a t e was s u b s t i t u t e d f o r g l u c o s e - 6 - p h o s p h a t e . For g l u c o k i n a s e t h e r e a c t i o n m i x t u r e had T r i s b u f f e r , pH 8 . 0 , 50 mM; M g C l 2 , 10 mM; ATP, 2.5 mM; g l u c o s e , 5 mM; NADP, 0.5 mM; t h e c e l 1 - f r e e e x t r a c t and e x c e s s commercial g l u c o s e - 6 - p h o s p h a t e dehydrogenase. G l u c o s e dehydrogenase a c t i v i t y was d e t e r m i n e d u s i n g t h e method o f Hauge ( 1 966 ) . P y r u v a t e f o r m a t i o n from 6 - p h o s p h o g l u c o n a t e by t h e combined a c t i o n o f 6-phosph g l u c o n a t e dehydrase and 2 - k e t o - 3 - d e o x y - 6 - p h o s p h o g l u c o n a t e a l d o l a s e was measured u s i n g commercial l a c t i c dehydrogenase (Von T i g e r s t r o m and C a m p b e l l , 1 966b ) . The f o r m a t i o n o f 3 - p h o s p h o g l y c e r a l d e h y d e from f r u c t o s e - 1 , 6 - d i p h o s p h a t e by f r u c t o s e d i p h o s p h a t e a l d o l a s e a c t i v i t y was measured u s i n g commercial t r i o s e phosphate isomerase and 3 - p h o s p h o g l y c e r a l d e h y d e dehydrogenase (Bock and N e i d h a r d t , I966) Phosphohexose isomerase and f r u c t o s e d i p h o s p h a t a s e were a s s a y e d a c c o r d i n g t o G a l e and Beck (1967). The method d e s c r i b e d by L i n g e t _ a j _ (1966) was f o l l o w e d f o r the a s s a y o f p h o s p h o f r u c t o -k i n a s e a c t i v i t y and t h a t o f D e V r i e s , Gerbrandy and Stouthamer (1967) f o r f r u c t o s e - 6 - p h o s p h a t e p h o s p h o k e t o l a s e . For 3-phospho-g l y c e r a l d e h y d e dehydrogenase t h e r e a c t i o n m i x t u r e c o n t a i n e d T r i s b u f f e r , pH 7 . 4 , 50 mM; D L - 3 _ p h o s p h o g l y c e r a l d e h y d e , 6 mM; c y s t e i n e h y d r o c h l o r i d e , 10. mM; sodium a r s e n a t e , 17 mM; sodium f l u o r i d e , 20 mM; NADP o r NAD, 0 .25 mM and t h e enzyme. The f o l l o w i n g enzymes were measured by the methods d e s c r i b e d i n the r e f e r e n c e s g i v e n a f t e r t h e name o f each enzyme: t r i o s e phosphate isomerase and 3 - p h o s p h o g l y c e r i c a c i d k i n a s e ( C a m p b e l l , H e l l e b u s t and Watson, 1966 ) ; e n o l a s e (Westhead, 1966) as f o r the y e a s t enzyme but w i t h T r i s b u f f e r ; p y r u v a t e k i n a s e ( V a l e n t i n e and Tanaka, 1 966 ) ; t r a n s -a l d o l a s e ( T c h o l a and H o r e c k e r , 1966 ); and r i b o s e p h o s p h a t e " i s o m e r a s e (Axel rod and J a n g , 1 954 ) . T r a n s k e t o l a s e a c t i v i t y , t o g e t h e r w i t h t h e a c t i v i t i e s o f r i b o s e phosphate isomerase and r i b u l o s e - 5 - p h o s p h a t e e p i m e r a s e was assayed i n a system c o n t a i n i n g T r i s b u f f e r , phi 8 . 0 , 50 mM; r i b o s e - 5 - p h o s p h a t e , 1 mM; t h i a m i n e p y r o p h o s h a t e , 1 mM; c y s t e i n e h y d r o c h l o r i d e , 1.5 mM; NADH, 0 .25 mM; enzyme and e x c e s s commercial t r i o s e phosphate isomerase and a - g l y c e r o p h o s p h a t e dehydrogenase. The a b i l i t y o f t h e c e l l - f r e e e x t r a c t s t o t r a n s f o r m r i b o s e - 5 - p h o s p h a t e t o g l u c o s e - 6 - p h o s p h a t e ( i . e . g l u c o s e - 6 - p h o s p h a t e f o r m i n g a c t i v i t y f r o m r i b o s e - 5 - p h o s p h a t e ) w h i c h i n v o l v e s t h e c o u p l e d a c t i o n o f s e v e r a l endogenous pentose phosphate pathway enzymes such as r i b o s e phosphate i s o m e r a s e , r i b u l o s e - 5 _ p h o s p h a t e e p i m e r a s e , t r a n s -k e t o l a s e and t r a n s a l d o l a s e was as s a y e d a c c o r d i n g t o t h e p r o c e d u r e o f G a l e and Beck ( 1967 ) . S p e c t r o p h o t o m e t r i c a s s a y s were performed e i t h e r i n a Beckman s p e c t r o p h o t o m e t e r model DB-G o r G i l f o r d model 2000 a t 30 C. The s p e c i f i c ; a c t i v i t i e s a r e e x p r e s s e d as mymoles o f t h e s u b s t r a t e u t i l i z e d p e r min p e r mg o f p r o t e i n . 31 V I I . A n a l y s i s o f R e a c t i o n P r o d u c t s For t h e i d e n t i f i c a t i o n o f k e t o a c i d s one ml p o r t i o n s o f t h e spe n t r e a c t i o n m i x t u r e s from Warburg v e s s e l s o r from c u v e t t e s were poured i n t o one ml o f a 1 umole/ml s o l u t i o n o f 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e i n 2 N H C l . The p r e c i p i t a t e d p r o t e i n was removed by c e n t r i f u g a t i o n . The s u p e r n a t a n t was i n c u b a t e d a t 37 C f o r 30 min. The hydrazohes were e x t r a c t e d i n t o e t h y l a c e t a t e and r e -e x t r a c t e d i n t o 1 M T r i s b u f f e r , pH 11 from e t h y l a c e t a t e . The T r i s e x t r a c t was a c i d i f i e d w i t h 5 N HCl and t h e hydrazohes were f i n a l l y e x t r a c t e d i n t o e t h y l a c e t a t e and chromatographed on Whatman no. 4 paper u s i n g n - b u t a n o l - e t h a n o l - 0 . 5 N NH^OH (70:10:20) as d e s c r i b e d by Smith (1960). St a n d a r d s were run s i m u l t a n e o u s l y and samples were c o n c l u s i v e l y i d e n t i f i e d by co-chromatography. In t h e ca s e o f r a d i o a c t i v e s a m p l e s , s t r i p s o f t h e chromatograms were c u t and scanned i n an a c t i g r a p h ( N u c l e a r C h i c a g o ) . V I M . T h i n L a y e r Chromatography, A u t o r a d i o g r a p h y and R a d i o a c t i v e Measurements R e a c t i o n i n the Warburg v e s s e l s was stoppe d by t h e a d d i t i o n o f 0.12 ml o f 5.8 M HC10. per ml o f r e a c t i o n m i x t u r e . The p r e c i p i t a t e d p r o t e i n was removed by c e n t r i f u g a t i o n and the s u p e r n a t a n t f l u i d was n e u t r a l i z e d by the a d d i t i o n o f 0.24 ml o f 2.9 M p o t a s s i u m b i c a r b o n a t e and the p r e c i p i t a t e d p e r c h l o r a t e was removed by c e n t r i f u g a t i o n ( W e i s s , 1 9 67 ) . A s u i t a b l e volume o f the s u p e r n a t a n t f l u i d was chromatographed t w o - d i m e n s i o n a l l y on t h i n l a y e r chromatography (TLC) p l a t e s c o a t e d w i t h e e l l u l o s e CC-41 a c c o r d i n g t o the method o f Myers and Huang . (1966 ). I d e n t i f i c a t i o n o f s t a n d a r d s on t h i n l a y e r chromatograms was a c h i e v e d by s p r a y i n g w i t h brom c r e s o l p u r p l e o r by a n i 1 i n e - r i b o s e ( H i g g i n s and Von Brand, 1 966 ) . To l o c a t e t h e r a d i o a c t i v e s p o t s on TLC p l a t e s , t he a u t o r a d i o g r a m s were d e v e l o p e d on Kodak m e d i c a l X-ray f i l m s f o r 7 t o 10 d a y s . The c o n t e n t s c o r r e s p o n d i n g t o the d e s i r e d r a d i o a c t i v e s p o t s on the p l a t e s were s c r a p e d and p l a c e d i n the s c i n t i l l a t i o n v i a l s and counted i n a N u c l e a r C h i c a g o s c i n t i l l a t i o n s p e c t r o m e t e r model 725 , u s i n g t o l u e n e c o n t a i n i n g PPO, P0P0P and C a b - 0 - S i l a c c o r d i n g t o the p r o c e d u r e o f Snyder and Stephens ( 1 962 ) . IX. Uptake o f L a b e l l e d S u b s t r a t e s 14 The i n c o r p o r a t i o n o f the l a b e l l e d s u b s t r a t e s , g l u c o s e - U - C 14 and a - m e t h y l - g 1 u c o s i d e - U - C i n t o whole c e l l s was s t u d i e d by t h e 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 o f B r i t t e n and M c C l u r e . ( 1 962 ) . The l o g a r i t h m i c phase c e l l s were h a r v e s t e d a t room t e m p e r a t u r e and washed once w i t h b a s a l s a l t s medium. The p e l l e t was r e s u s p e n d e d i n s u c c i n a t e min imal medium. T h i s c e l 1 s u s p e n s i o n was added t o the i n c u b a t i o n m i x t u r e s t o g i v e a f i n a l o p t i c a l d e n s i t y o f 0 .3 a t 660 my. The i n c u b a t i o n m i x t u r e s were s t i r r e d on a Mag-j e t s u b m e r g e a b l e m a g n e t i c s t i r r e r ( B r o n w i 1 1 , S c i e n t i f i c , R o c h e s t e r , New York) f o r 10 min in a 30 C w a t e r ba th p r i o r t o the a d d i t i o n o f r a d i o a c t i v e s u b s t r a t e . The r e a c t i o n was s t a r t e d by the a d d i t i o n o f l a b e l l e d s u b s t r a t e and was t e r m i n a t e d a t t h e d e s i r e d i n t e r v a l s by c o l l e c t i n g the c e l l s f rom 1 ml i n c u b a t i o n m i x t u r e on a T r a c e r l a b E8B p r e c i p i t a t i o n a p p a r a t u s ( T r a c e r l a b , Wal tham, M a s s . ) and w a s h i n g w i t h 2 ml o f min imal medium. M i l l i p o r e f i l t e r s o f 25 mm d i a m e t e r and 0.45 y p o r e s i z e were u s e d . The f i l t e r s were d r i e d under the i n f r a - r e d s o u r c e and p l a c e d i n v i a l s c o n t a i n i n g 10 ml o f s c i n t i l l a t i o n f l u i d ( l i q u i f l u o r , New E n g l a n d N u c l e a r C o r p . , B o s t o n , M a s s . ) f o r c o u n t i n g i n a 1 i q u i d s c i n t i 1 l a t i o n s p e c t r o m e t e r . X . A n a l y t i c a l Methods P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d by the method o f Lowry et_ a_l_ (1951 ) . C i t r a t e was e s t i m a t e d by the method out 1 ined by S t e r n ( 1 957 ) . P y r u v a t e was a s s a y e d u s i n g l a c t i c d e h y d r o g e n a s e and NADH in a s y s t e m c o n t a i n i n g T r i s b u f f e r , pH 7 . 4 , 40 mM; M g C l 2 , 5 mM; NADH, 0 .25 mM; commerc ia l l a c t i c d e h y d r o g e n a s e and the s a m p l e . a - K e t o g l u t a r a t e was e s t i m a t e d by a l l o w i n g the r e a c t i o n t o p r o c e e d i n t h e d i r e c t i o n o f g l u t a m a t e s y n t h e s i s i n th e p r e s e n c e o f g l u t a m i c dehydrogenase and e x c e s s ammonia. The r e a c t i o n m i x t u r e c o n t a i n e d T r i s b u f f e r , pH 8.0, 40 mM; NH^Cl, 200 mM;.NADH, 0.25 mM; commercial g l u t a m i c dehydrogenase and the sample. S u c c i n a t e was assayed m a n o m e t r i c a l l y u s i n g c r u d e s u c c i n i c o x i d a s e p r e p a r a t i o n from beef h e a r t (Umbreit £t_ aj_, 1957). Fumaric a c i d was a l s o a s s a y e d m a n o m e t r i c a l l y i n a c o u p l e d a s s a y system u s i n g commercial fumarase; L a c t b b a c i 1 l u s a r a b i n o s u s - c u l t u r e as a s o u r c e o f NAD, m a l i c enzyme and l a c t i c dehydrogenase. S e m i c a r b a z i d e was a l s o i n c l u d e d i n th e system (Sanger and L u s t y , T 9 6 3 ) . M a l a t e was d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y u s i n g NAD and commercial m a l i c dehydrogenase ( H o h o r s t , 1 9 6 3 ) . X I . Chemi c a l s 14 14 S u c c i n a t e - 1 , 4 - C and s u c c i n a t e - 2 , 3 - C were t h e p r o d u c t s o f 14 I n t e r n a t i o n a l Chemical and N u c l e a r C o r p o r a t i o n . G l u c o s e - U - C was o b t a i n e d from Schwarz B i o r e s e a r c h I n c . a-Methy1-D-gluco-14 p y r a n o s i d e - U - C was th e p r o d u c t o f N u c l e a r C h i c a g o Corp. Other c h e m i c a l s and enzymes used t h r o u g h o u t t h i s s t u d y were s i m i l a r l y o b t a i n e d from commercial s o u r c e s . RESULTS AND DISCUSSION I . O x i d a t i o n o f T r i c a r b o x y l i c A c i d C y c l e arid R e l a t e d I ri termed i a t e s by Pseudomonas ae rug i hosa S i n c e i t was p ropo sed t o s t u d y t h e u t i l i z a t i o n o f s u c c i n a t e and o t h e r d i c a r b o x y l i c a c i d s o f t h e t r i c a r b o x y l i c a c i d c y c l e , c u l t u r e s were n o r m a l l y grown on s u c c i n a t e medium. Washed c e l l s u s p e n s i o n s and c e l l e x t r a c t s p r e p a r e d f rom such c u l t u r e s o x i d i z e d s u c c i n a t e , f u m a r a t e and m a l a t e a t a r a p i d r a t e ( F i g s . 1 and 2). The p e r c e n t a g e o f t h e t h e o r e t i c a l oxygen u p t a k e was h i g h e r w i t h t h e c e l l - f r e e e x t r a c t and was s i m i l a r f o r t he t h r e e d i c a r b o x y l i c a c i d s . Comparab le r e s u l t s were o b t a i n e d w i t h a - k e t o g l u t a r a t e as s u b s t r a t e . Washed c e l l s u s p e n s i o n s o x i d i z e d c i t r a t e o n l y a f t e r a l a g o f 60 m i n , w h i l e c e l l - f r e e e x t r a c t s u t i l i z e d t h i s s u b s t r a t e i m m e d i a t e l y . A l t h o u g h who le c e l l s u t i l i z e d a c e t a t e and p y r u v a t e r a p i d l y , c e l l - f r e e e x t r a c t s d i d no t o x i d i z e t h e s e s u b s t r a t e s ( F i g . 3). These o b s e r v a t i o n s i n d i c a t e d t h a t t h e enzymes o r c o f a c t o r s , r e q u i r e d f o r t h e o x i d a t i o n o f a c e t a t e and p y r u v a t e , were d e s t r o y e d d u r i n g t h e p r e p a r a t i o n o f c e l l - f r e e e x t r a c t , w h i l e t h o s e r e q u i r e d f o r t h e o x i d a t i o n o f t h e t r i c a r b o x y l i c a c i d c y c | e i n t e r m e d i a t e s were r e l a t i v e l y undamaged. o ' ' ' , • t 0 30 60 90 120 150 M I N U T E S F ig . 1. Oxidation of succinate, fumarate. and malate by. ce l1 suspensions • of P. -aeruginosa. The Warburg vessels contained T r i s . b u f f e r , . pH 7.4, 50 ymoles; c e l l suspension (5 mg dry weight) ; 7.5 ymoles of substrate in a total.-volume-of 3.0 ml . Center wel 1 contained 0.15 ml of 20% KOH.c: Symbols: •., succinate; A , malate; 0., fumarate I 0 60 120 180 240 M I N U T E S F i g . 2. O x i d a t i o n o f s u c c i n a t e , fumarate and m a l a t e by c e l l f r e e e x t r a c t o f P. a e r u g i n o s a . The Warburg f l a s k s c o n t a i n e d T r i s b u f f e r pH 7 . 4 , 150 ymoles; c e l l f r e e e x t r a c t approx. 30 mg p r o t e i n ; 7 . 5 ymoles s u b s t r a t e i n a t o t a l volume o f 3.0 ml. C e n t e r w e l l c o n t a i n e d 0 . 15 ml o f 20% K0H. Symbols: • , s u c c i n a t e ; A , m a l a t e ; 6 , fumarate.-M I N U T E S . O x i d a t i o n o f a c e t a t e and p y r u v a t e by c e l l s arid c e l l - f r e e e x t r a c t o f P. a e r u g i hosa. The Warburg v e s s e l s were s e t up as f o r F i g s . 1 and 2. Symbols: open, c e l l s ; c l o s e d , ' , c e l l - f r e e e x t r a c t ; •, a c e t a t e ; O, p y r u v a t e . 3 9 200 r 0 30 60 90 120 M I N U T E S Fig', k. O x i d a t i o n o f s u c c i n a t e , , fumarate and malate;; by r e s t i n g c e l l s u s p e n s i o n o f P. aerug? nosa i n t h e . p r e s e n c e o f 1 mM sodium . a r s e n i t e . E x p e r i m e n t a l c o n d i t i o n s ; w e r e s i m i l a r t o t h o s e i n -• F i g . 1. Symbols, •, s u c c i n a t e ; A, m a l a t e ; O, f u m a r a t e . T a b l e I. A c c u m u l a t i o n o f k e t o a c i d s d u r i n g the o x i d a t i o n o f s u c c i n a t e , f u m a r a t e and m a l a t e i n the p r e s e n c e o f 1 mM sodium a r s e n i t e . Carbon s o u r c e i n Keto a c i d , , a c c u m u l a t e d t h e Warburg v e s s e l /., r . • , \ P y r u v a t e a - k e t o g l u t a r a t e (7.5 ymole) 1 . , ... ymoles ymoles ... S u c c i n ate Fumarate M a l a t e 6.9 6.5 5.1 <0.2 <0.2 <0.2 In a subsequent e x p e r i m e n t a r s e n i t e was employed t o p r e v e n t t h e o x i d a t i o n o f the f i r s t i n t e r m e d i a t e k e t o a c i d i n the pathway o f o x i d a t i o n o f t h e d i c a r b o x y l i c a c i d s ( F i g . k). C a l c u l a t i o n s showed t h a t t h e s e compounds were o x i d i z e d t o e i t h e r o x a l a c e t a t e o r p y r u v a t e . Paper chromatography showed the a c c u m u l a t i o n o f p y r u v a t e i n e v e r y c a s e . T a b l e I shows the r e l a t i v e a c c u m u l a t i o n o f p y r u v a t e and a - k e t o g l u t a r a t e . From t h e s e r e s u l t s i t appeared t h a t under t h e c o n d i t i o n s employed, s u c c i n a t e was o x i d i z e d s o l e l y by way o f p y r u v a t e as was f u m a r a t e and m a l a t e . I I . Growth Y i e l d s o f P_. a e r u g i nosa w i t h Some T r i c a r b o x y l i c A c i d C y c l e I n t e r m e d i a t e s In a f u r t h e r a t t e m p t t o see i f s u c c i n a t e was degraded d i f f e r e n t l y than o t h e r d i c a r b o x y l i c a c i d s , growth y i e l d s w i t h s u c c i n a t e , f u m a r a t e , m a l a t e and a - k e t o g l u t a r a t e as s u b s t r a t e s were d e t e r m i n e d . G l u c o s e and p y r u v a t e were i n c l u d e d f o r comparison ( T a b l e I I ) . To e n s u r e t h a t a l l o f t h e s u b s t r a t e s i n the media were u t i l i z e d , s u p e r n a t a n t s from t h e growth media were a n a l y z e d . I t was found t h a t no s u b s t r a t e remained i n any i n s t a n c e . Under s i m i l a r c o n d i t i o n s o f growth g l u c o s e was found t o be c o m p l e t e l y used (Campbel1 e t a l , 1956) and i t s e s t i m a t i o n was not a t t e m p t e d i n t h e p r e s e n t e x p e r i m e n t s . I f one a c c e p t e d the s t a t e m e n t t h a t energy i s never l i m i t i n g i n a e r o b i c m e t a b o l i s m t h e n one would e x p e c t t h a t s u c c i n a t e , f u m a r a t e and m a l a t e would g i v e i d e n t i c a l growth y i e l d s f o r i t would be t h e amount o f c a r b o n t h a t would d e t e r m i n e the growth y i e l d . On t h i s b a s i s s u c c i n a t e must be c a p a b l e o f e n t e r i n g pathways o f m e t a b o l i s m t h a t a r e not s h a r e d by fumarate and m a l a t e ( T a b l e I I ) . However, i f energy i s l i m i t i n g i n an a e r o b i c system then growth y i e l d w i l l be a f u n c t i o n o f t h e s t a t e o f r e d u c t i o n o f the s u b s t r a t e . T h i s l a t t e r s i t u a t i o n would appear t o be o p e r a t i v e i n t h e p r e s e n t s i t u a t i o n f o r t h e growth y i e l d s a r e s i m i l a r f o r a l 1 s u b s t r a t e s t e s t e d when e x p r e s s e d on t h e b a s i s o f y i e l d per e q u i v a l e n t o f a v a i l a b l e e l e c t r o n s . The a v e r a g e v a l u e f o r gms o f c e l l s per e q u i v a l e n t o f a v a i l a b l e e l e c t r o n s was 3.26 ± 0.1 ( T a b l e l l ) . T h e r e f o r e , under t h e s e c o n d i t i o n s t h e o b s e r v a t i o n s a r e i n agreement w i t h t h o s e o f Mayberry et_ a l _ (1967) , who r e p o r t e d t h a t i n the c a s e o f b a c t e r i a grown a e r o b i c a l l y , y i e l d per mole o f the s u b s t r a t e o r p e r gm atom o f the s u b s t r a t e c a r b o n o r per mole o f t h e oxygen consumed v a r i e d o v e r a wide range w h i l e t h e y i e l d p e r e q u i v a l e n t o f a v a i l a b l e e l e c t r o n s was a c o n s t a n t and was c l o s e t o 3.14 gm o f c e l l s per e q u i v a l e n t o f a v a i l a b l e e l e c t r o n s . I t would appear t h a t growth y i e l d i s a f u n c t i o n o f t h e energy a v a i l a b l e from t h e s u b s t r a t e . S i n c e t h i s i s e s s e n t i a l l y c o n s t a n t r e g a r d l e s s o f t h e r o u t e o f d e g r a d a t i o n (assuming t h a t t h e end p r o d u c t s a r e C t ^ and H^O) , one i s n o t g o i n g t o l e a r n a n y t h i n g about p o s s i b l e d i f f e r e n c e s i n r o u t e s o f m e t a b o l i s m i n - a e r o b i c b a c t e r i a by m e a s u r i n g growth y i e l d s . T a b l e I I . Growth y i e l d o f P. a e r u g i n o s a from v a r i o u s c a r b o n s o u r c e s . Carbon E q u i v a l e n t t o M o l a r growth G m / e q u i v a l e n t Gm/gm s o u r c e a v a i l a b l e e l e c t r o n s y i e l d (gm/mole) o f a v a i l a b l e atom p e r mole e l e c t r o n s o f c a r b o n G l u c o s e 24 7 9 . 0 3.29 13.2 S u c c i n a t e 14 44.2 3.16 11.0 Fumarate 12 40.3 3.36 10.1 M a l a t e 12 40.3 3.36 10.1 a - K e t o g l u t a r a t e 16 51.1 3.19 10.2 P y r u v a t e 10 32.0 3.20 10.7 I I I . S t u d i e s w i t h Mutants A l l a t t e m p t s t o i s o l a t e mutants o f P. a e r u g i n o s a 9027 u n a b l e t o grow on fumarate but c a p a b l e o f growth on s u c c i n a t e met w i t h f a i l u r e . M utants unable t o grow on fumarate were a l s o i n c a p a b l e o f growth on s u c c i n a t e . The r e s u l t s p r e s e n t e d above d i d not p r o v i d e any e v i d e n c e t o . s u p p o r t t h e s u g g e s t i o n t h a t t h e r e was a unique pathway o f s u c c i n a t e u t i l i z a t i o n . Hence, f u r t h e r s t u d i e s were f o c u s s e d on.the mode o f u t i l i z a t i o n o f t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s w i t h s u c c i n a t e as t h e example t o e l u c i d a t e t h e r o l e and c o n t r o l o f t h e t r i c a r b o x y l i c a c i d c y c l e w h i c h o b v i o u s l y seems t o be d i f f e r e n t i n pseudomonads than i n f a c u l t a t i v e o r ganisms and a e r o b i c s p o r e f o r m i n g b a c i l l i as o u t l i n e d e a r l i e r . IV. Lack o f NAD o r NADP Dependent L - M a l i c Dehydrogenase i n . P. a e r u g i nosa P r e l i m i n a r y s t u d i e s on the u t i l i z a t i o n o f s u c c i n a t e and o t h e r d i c a r b o x y l i c a c i d s o f t h e t r i c a r b o x y l i c a c i d c y c l e i n d i c a t e d t h a t c e l l - f r e e e x t r a c t s , w i t h o u t t h e a d d i t i o n o f coenzymes, o x i d i z e d s u c c i n a t e , fumarate and m a l a t e a t a r a p i d r a t e ( F i g . 2) and a l m o s t t o c o m p l e t i o n . However, s e v e r a l a t t e m p t s t o d e t e c t NAD o r NADP dependent L - m a l i c dehydrogenase i n t h e c e l l -f r e e e x t r a c t s and i n t h e 100,000 x g_ s u p e r n a t a n t were u n s u c c e s s f u l . S i n c e a e r o b i c organisms p o s s e s s a r e l a t i v e l y l a r g e number o f p a r t i c u l a t e ' o r membrane bound enzymes ( M a r r , 1960; S m i t h , 1961), 25,000 x g_ and 100,000 x g_ p e l l e t s were a l s o s c r e e n e d f o r t h e p r e s e n c e o f t h i s enzyme w i t h t h e same n e g a t i v e r e s u l t s . To e n s u r e t h a t t h e p r o c e d u r e o f p r e p a r a t i o n o f c e l l - f r e e e x t r a c t s and the a s s a y o f the enzyme were not a t f a u l t , a e e l 1 - f r e e e x t r a c t was p r e p a r e d from A e r o b a c t e r aerogenes and a s s a y e d f o r NAD l i n k e d L - m a l i c dehydrogenase under t h e same c o n d i t i o n s . A. aerogenes showed a v e r y h i g h a c t i v i t y o f NAD dependent L - m a l i c dehydrogenase ( s p e c i f i c a c t i v i t y , 3410) c o n f i r m i n g t h a t the p r o c e d u r e b e i n g f o l l o w e d was r e l i a b l e . V. D e t e c t i b h and S ? t e o f M a l a t e O x i d i z i n g A c t i v i t y S i n c e the f o r m a t i o n o f o x a l a c e t i c a c i d i s e s s e n t i a l f o r the f u n c t i o n i n g o f t h e t r i c a r b o x y l i c a c i d c y c l e , m a l a t e must be o x i d i z e d t o o x a l a c e t a t e . To show t h a t o x a l a c e t a t e i s indeed a p r o d u c t d u r i n g o x i d a t i o n o f m a l a t e by t h e c e l l s o f P_. a e r u g i n o s a , a " c a r r i e r t y p e " e x p e r i m e n t was c a r r i e d o u t ( K r a m p i t z , 1961). A washed c e l l s u s p e n s i o n and c o n v e n t i o n a l Warburg t e c h n i q u e were 14 .employed. S u c c i n a t e - 2 , 3 - C was t i p p e d i n a t z e r o t i m e , w h i l e o x a l a c e t a t e was t i p p e d i n from t h e second s i d e arm a few minutes l a t e r , when a p p r o x i m a t e l y 60 u4 o f oxygen had been consumed as a r e s u l t o f s u c c i n a t e o x i d a t i o n . F i v e t o 10 min a f t e r t h e a d d i t i o n o f u n l a b e l l e d o x a l a c e t a t e , t h e r e a c t i o n m i x t u r e s were removed, hyd pr e p a r e d and chromatographed. R a d i o a c t i v e o x a l a c e t a t e was i d e n t i f i e d i n a l l t h e f l a s k s r e c e i v i n g r a d i o a c t i v e s u c c i n a t e as t h e i n i t i a l s u b s t r a t e . I f 3-3 mM EDTA (an i n h i b i t o r o f m a l i c enzyme) was i n c l u d e d i n t h e r e a c t i o n m i x t u r e s , h i g h e r a c c u m u l a t i o n o f r a d i o a c t i v e o x a l a c e t a t e c o u l d be d e m o n s t r a t e d . There a r e s e v e r a l a l t e r n a t i v e r o u t e s by which o x a l a c e t a t e c o u l d be formed from L - m a l i c a c i d ( F r a n c i s e t a l , 1963)• S i n c e pseudomonads p o s s e s s t h e m a l i c enzyme ( T a b l e I I I ) and p y r u v a t e c a r b o x y l a s e ( S e u b e r t and Remberger, 1 9 61), an a t t e m p t was made t o d e t e r m i n e i f t h i s sequence o f o x i d a t i v e d e c a r b o x y l a t i o n then r e c a r b o x y l a t i o n was u t i l i z e d f o r o x a l a c e t a t e f o r m a t i o n as o b s e r v e d i n a Chroma Hum s p e c i e s ( F u l l e r and K o r n b e r g , I 96 I ) o r whether a d i r e c t o x i d a t i o n by a p a r t i c u l a t e m a l i c dehydrogenase was i n v o l v e d . D u r i n g s t u d i e s on t h e o x i d a t i o n o f L - m a l i c a c i d by two s p e c i e s o f Pseudomonas, F r a n c i s ' e t ' a l (1963) found t h a t e x t r a c t s o f Pseudomonas B^ , a b a, p o s s e s s i n g s o l u b l e NAD dependent L - m a l i c d e hydrogenase, o x i d i z e d L-malate v e r y s l o w l y and i n c o m p l e t e l y w h i l e e x t r a c t s o f P. o v a l i s C h e s t e r , p o s s e s s i n g p a r t i c u l a t e L - m a l i c dehydrogenase, c a t a l y z e d the r a p i d and a l m o s t c o m p l e t e o x i d a t i o n o f t h i s s u b s t r a t e . T h i s s u g g e s t e d a s i m i l a r i t y i n the enzyme complement o f P. a e r u g i n o s a 9027 and P. ova 1 i s C h e s t e r s i n c e t h e c e l l - f r e e e x t r a c t p r e p a r a t i o n s o f t h e former a l s o o x i d i z e d m a l a t e a t a r a p i d r a t e . And indeed i t was found t h a t P. a e r u g i n o s a does p o s s e s s t h e p a r t i c u l a t e L - m a l i c dehydrogenase ( T a b l e I I I ) . O x a l a c e t i c a c i d and p y r u v i c a c i d were i d e n t i f i e d i n t h e a s s a y r e a c t i o n m i x t u r e s f o r p a r t i c u l a t e m a l i c dehydrogenase and m a l i c enzyme r e s p e c t i v e l y . To reduce i n t e r f e r e n c e due t o endogenous NAD o r NADP whi c h might be p r e s e n t i n cr u d e c e l l - f r e e e x t r a c t s , m a l a t e o x i d i z i n g a c t i v i t y was a l s o assayed i n c h a r c o a l t r e a t e d c e l l - f r e e e x t r a c t s ( H o c h s t e r and S t o n e , 1956) and comparable a c t i v i t y was d e m o n s t r a t e d . In a n o t h e r a t t e m p t t o a s s e s s the impo r t a n c e o f t h e p a r t i c u l a t e m a l a t e o x i d i z i n g s y s t e m , L-malate o x i d a t i o n i n the c e l l - f r e e e x t r a c t and the 100,000 x g_ p e l l e t was s t u d i e d ( F i g . 5 ) . The i n i t i a l c e l l - f r e e e x t r a c t a c t i v e l y o x i d i z e d m a l a t e and consumed 74.6% o f the t h e o r e t i c a l oxygen u p t a k e , whereas t h e resuspended p e l l e t o x i d i z e d i t s l o w l y and consumed o n l y 17.8% o f t h e amount o f oxygen r e q u i r e d f o r complete o x i d a t i o n . C a l c u l a t i o n showed t h a t when the o x i d a t i o n c u r v e s t a r t e d l e v e l l i n g o f f , a p p r o x i m a t e l y 0 .5 umole o f oxygen per umole o f m a l a t e was t a k e n up. T h i s i n d i c a t e d t h a t m a l a t e was o x i d i z e d e i t h e r t o o x a l a c e t i c a c i d o r p y r u v i c a c i d by t h e p e l l e t . A n a l y s i s o f t h e r e a c t i o n m i x t u r e f o r k e t o a c i d s r e v e a l e d o x a l a c e t a t e as t h e main p r o d u c t and p y r u v a t e as a minor p r o d u c t . T h i s amount o f p y r u v a t e c o u l d be t h e r e s u l t o f the non-enzymatic breakdown o f o x a l a c e t a t e . T a b l e I I I . P a r t i c u l a t e m a l i c dehydrogenase and m a l i c enzyme i n P. a e r u g i n o s a P a r t i c u l a t e m a l i c NADP m a l i c NAD m a l i c P r e p a r a t i o n . dehydrogenase enzyme enzyme S p e c i f i c . A c t i v i t y C e l l - f r e e e x t r a c t 100,000 x £ p e l l e t 100,000 x g_ s u p e r n a t a n t C e n t r i f u g e d a t 100,000 x £ f o r 90 min t w i c e . I f c e n t r i f u g a t i o n was c a r r i e d o u t o n l y once, about 10% dehydrogenase a c t i v i t y r e mained- i n the s u p e r n a t a n t . 17.1 574.0 133.0 24.3 n i l n i l n i l 972.0 194.0 400 3OO < h-z 200 LU O >-X ° 100 60 90 M I N U T E S 120 F i g . 5. ' O x i d a t i o n o f L-mal i c a c i d by (A) e e l 1-free e x t r a c t and .(B) 100,00Q\ x £ p e l l e t o f P. a e r u g i n o s a . / The r e a c t i o n m i x t u r e • c o n t a i n e d 250 ymoles o f p o t a s s i u m phosphate buffer,\ :.pR 7.2; . 2.0 ml c e l l - f r e e e x t r a c t o r p e l l e t s u s p e n s i o n ; 7.5 ymoles o f m a l a t e i n a t o t a l / v o l u m e o f 3.0 ml. The c e n t e r wel1 o f t h e Warburg v e s s e l c o n t a i n e d 0.15 ml of. 20% KOH. These o b s e r v a t i o n s show t h a t t h e m a l i c dehydrogenase i s p a r t i c u l a t e and i s p r o b a b l y s i m i l a r i n d i s t r i b u t i o n t o t h a t i n P. Oval i s C h e s t e r ( F r a n c i s e t _ a j _ , 1963). However, the d a t a d i d not e x c l u d e t h e p o s s i b i l i t y t h a t the enzyme.was a s s o c i a t e d w i t h r ibosomes. To r e s o l v e t h i s q u e s t i o n a 25 ,000 x g_ s u p e r n a t a n t o f the c e l l - f r e e e x t r a c t , w h i c h c o n t a i n e d much h i g h e r t o t a l a c t i v i t y o f t h e enzyme than the c o r r e s p o n d i n g p e l l e t , was t r e a t e d w i t h 5 x 10\ J H_ EDTA and a l l o w e d t o s t a n d a t 0 C f o r 15 min. I t was then c e n t r i f u g e d a t 100,000 x g_ f o r 90 min. The p e l l e t was resuspended i n a s u i t a b l e volume o f 0 .25 M_ T r i s b u f f e r , pH 7 . 4 . " 3 P o r t i o n s o f t h i s p e l l e t s u s p e n s i o n were t r e a t e d w i t h 5 x 10 M_ EDTA and 500 ug p a n c r e a t i c r i b o n u c l e a s e ( W o r t h i n g t o n ) per ml a t 15 C f o r 4 h r . The low t e m p e r a t u r e o f i n c u b a t i o n was used t o a v o i d i n a c t i v a t i o n o f t h e enzyme. At the end o f the r e a c t i o n p e r i o d , the m i x t u r e was c e n t r i f u g e d a t 100,000 x g_ f o r 90 min and t h e p e l l e t was washed once w i t h 0 .25 M_ T r i s b u f f e r , pH 7 - 4 . The p e l l e t so o b t a i n e d was resuspended i n the same b u f f e r . T a b l e IV shows t h a t t h i s t r e a t m e n t was q u i t e e f f e c t i v e i n removing most o f t h e r i b o n u c l e i c a c i d from t h e p e l l e t i n d i c a t i n g e x t e n s i v e ribosome d e g r a d a t i o n . I t i s seen t h a t t h e r e i s no i n d i c a t i o n o f the enzyme h a v i n g t h e same d i s t r i b u t i o n as r i b o n u c l e i c a c i d o r b e i n g s o l u b i l i z e d . T h e r e f o r e , i t i s c o n c l u d e d t h a t t h i s enzyme i s not a s s o c i a t e d w i t h r i b o s o m e s . T a b l e IV. E f f e c t o f EDTA and r i b o n u c l e a s e t r e a t m e n t on t h e d i s t r i b u t i o n o f p a r t i c u l a t e m a l i c dehydrogenase o f f_. a e r u g i nosa .j. i ^ ... Enzyme a c t i v i t y O.D. 280/260 T o t a l p r o t e i n 7 . 7 -1 I I I P31 IO No. F r a c t i o n (mg) sp. a c t . t o t a l a c t . (1) 100,000 x g_ p e l l e t 26.6 24.3 646.0 0.67 (2) (1) a f t e r EDTA and RNAse t r e a t m e n t 8.1 16.1 T30.5 0 .688 (3) 100,000 x £ p e l l e t o f (2) 5.8 19.85 115.0 0.966 (4) 100,000 x £ 5 V s u p e r n a t a n t o f (2) 2 . 5 " 2.9 7-3 0 .607 A f t e r c o r r e c t i n g f o r t h e q u a n t i t y o f RNAse added. U l t r a v i o l e t s p e c t r a from 340 t o 230 mu o f a l l the f r a c t i o n s ; were r e c o r d e d i n Beckman S p e c t r o p h o t o m e t e r model DB-G. 52 VI . L a b e l 1ing o f C ? t r a t e from S u c c i nate-1 ,4- C and S u c c i nate-2,3~ C The above e x p e r i m e n t s i n d i c a t e d t h e i n v o l v e m e n t o f p a r t i c u l a t e m a l i c dehydrogenase i n t h e d i r e c t o x i d a t i o n o f m a l a t e t o o x a l a c e t a t e , but d i d not e x c l u d e o x i d a t i v e d e c a r b o x y l a t i o n and r e c a r b o x y l a t i o n as a sequence o f r e a c t i o n s f o r the f o r m a t i o n o f o x a l a c e t a t e from m a l a t e . To c o n c l u s i v e l y show t h a t p a r t i c u l a t e m a l i c dehydrogenase a c t i v i t y i s t h e i m p o r t a n t system i n t h e f o r m a t i o n o f o x a l a c e t a t e from L - m a l i c a c i d a n o t h e r e x p e r i m e n t was f o r m u l a t e d (see f o o t n o t e o f T a b l e V I ) . The r e a c t i o n was c a r r i e d out i n the Warburg v e s s e l s w i t h NaHCO^ and s u c c i n a t e - 1 , 4 - C o r s u c c i n a t e - 2 , 3 - C as s u b s t r a t e s . The f l a s k s were gassed w i t h 35% oxygen - 5% c a r b o n d i o x i d e m i x t u r e t o m a i n t a i n the pH (Umbre?t e t a l , 1957). Under t h e s e c o n d i t i o n s , c i t r a t e was found t o a c c u m u l a t e i n the r e a c t i o n m i x t u r e ( F i g . 6). To e l i m i n a t e t h e p o s s i b i l i t y o f s l i g h t r e c y c l i n g o f c i t r a t e , t h e mutant o f P. a e r u g i n o s a l a c k i n g O y k e t o g l u t a r a t e d ehydrogenase, d e s i g n a t e d P. a e r u g i n o s a M32, was a l s o used ( F i g . 7). The mutant c e l l s o x i d i z e d s u c c i n a t e i n c o m p l e t e l y and a n a l y s i s o f the s u p e r n a t a n t s o f the r e a c t i o n m i x t u r e s f o r k e t o a c i d s showed a c c u m u l a t i o n o f a - k e t o g l u t a r a t e . T a b l e V shows t h a t t h i s mutant does not p o s s e s s a - k e t o g 1 u t a r i c dehydrogenase and o t h e r t r i c a r b o x y l i c a c i d c y c l e and r e l a t e d enzymes a r e a t a l ower l e v e l than i n w i l d t y p e c e l l s citrate I- ETHER-FORMIC ACID-WATER F i g . 6. A u t o r a d i o g r a m o f th e t h i n - l a y e r chromatograph from t h e r e a c t i o n m i x t u r e c o n t a i n i n g e e l 1-free e x t r a c t o f P_. ' a e r u g i n o s a - w i l d t y p e and r a d i o a c t i v e s u c c i n a t e . E x p e r i m e n t a l c o n d i t i o n s were the same as i n T a b l e VI. 54 • C - k - g l citrate ETHER-FORMIC ACID-WATER F i g . 7. A u t o r a d i o g r a m o f the t h i n - l a y e r chromatograph from t he r e a c t i o n m i x t u r e c o n t a i n i n g c e l l - f r e e e x t r a c t o f R. a e r u g ? n o s a M32 and r a d i o a c t i v e s u c c i n a t e . E x p e r i m e n t a l c o n d i t i o n s were the same as i n T a b l e V I . a - k - g l = a - k e t o g l u t a r a t e . T a b l e V. The t r i c a r b o x y l i c P. a e r u g i n o s a M32 a c i d c y c l e and r e l a t e d enzymes o f Enzyme S p e c i f i c A c t i v i t y Fumarase 3 8 0 . 0 P a r t i c u l a t e m a l i c dehydrogenase 6 . 9 S o l u b l e m a l i c dehydrogenase n i l A c o n i t a s e 3 5 . 5 I s o c i t r a t e dehydrogenase 1 4 6 . 0 a - K e t o g l u t a r a t e dehydrogenase n i l NADP m a l i c enzyme 2 5 9 . 0 NAD m a l i c enzyme 5 7 . 0 mumoles s u b s t r a t e u t i l i z e d p e r min p e r mg o f p r o t e i n . ( T a b l e I X ) . C i t r a t e a c c u m u l a t i o n w i t h s u c c i n a t e as t h e s u b s t r a t e o c c u r r e d t o a much l e s s e r e x t e n t i n M32. D e t e r m i n a t i o n s o f a c o n i t a s e and i s o c i t r i c dehydrogenase i n d i c a t e d t h a t a c o n i t a s e was a l m o s t c o m p l e t e l y i n h i b i t e d i n the p r e s e n c e o f 21 mM NaHCO^ (th e c o n c e n t r a t i o n used i n t h e Warburg v e s s e l s ) i n both w i l d type and t h e mutant c e l l - f r e e e x t r a c t s , i f b i c a r b o n a t e was i n c o n t a c t w i t h t h e enzyme f o r 15 t o 20 min b e f o r e s t a r t i n g the a s s a y . A p p a r e n t l y t h i s was the c a u s e o f c i t r a t e a c c u m u l a t i o n . The mechanism o p e r a t i n g i n the mutant s t r a i n , w h i c h r e s u l t s i n much lower a c c u m u l a t i o n o f c i t r a t e , i s not c l e a r . The s p e c i f i c a c t i v i t y o f c i t r i c a c i d formed from s u c c i n a t e -14 14 2 , 3 " C was c l o s e t o d o u b l e t h a t o b t a i n e d from s u c c i n a t e - 1 , 4 - C ( T a b l e V l ) . The s p e c i f i c a c t i v i t y o f c i t r a t e o b t a i n e d from 14 s u c c i n a t e - 1 , 4 - C was c l o s e t o t h a t o f s t a r t i n g s u b s t r a t e , v a r y i n g from 84 t o 87 p e r c e n t o f t h a t v a l u e . These r e s u l t s c o u l d be e x p e c t e d i n the p r e s e n c e o f e x c e s s b i c a r b o n a t e o n l y i f m a l i c a c i d was d i r e c t l y o x i d i z e d t o o x a l a c e t a t e and then u t i l i z e d f o r c i t r a t e f o r m a t i o n as shown i n F i g s . 8a and 8b. These d a t a e l i m i n a t e o x i d a t i v e d e c a r b o x y l a t i o n and r e c a r b o x y l a t i o n as a mechanism o f any i m p o r t a n c e f o r o x a l a c e t a t e f o r m a t i o n , s i n c e i n t h i s s i t u a t i o n c i t r a t e o b t a i n e d from s u c c i n a t e - 1 , 4-^C would have one h a l f t h e s p e c i f i c a c t i v i t y o f s t a r t i n g s u b s t r a t e w h i l e c i t r a t e o b t a i n e d from 14 s u c c i n a t e - 2 , 3 - C would show d o u b l e the s p e c i f i c a c t i v i t y o f s t a r t i n g s u b s t r a t e w h i c h i s f o u r t i m e s t h a t o f c i t r a t e formed 14 from s u c c i n a t e - 1 , 4 - C. Thus, the d a t a p r e s e n t e d h e r e have demonstrated t h e i n v o l v e m e n t Table VI. Specific activity of radioactive citrate formed from succinate-1,4- C and succinate-2,3 - C T e of Cell- Citrate formed £ . . c , . per Warburg Sp. Act. Sp.Act. as % of Sp. free extract Substrate ^ . • 3 , v , , - K vessel (cpm/ymole Act. of succinate (ymoles) x.10'5) . at zero time 6.85 84.0 11.85 145.5 7.07 87.O 13.60 162.0 The reaction mixture in Warburg vessels contained Tris buffer, pH 7.5, 50 mM; c e l l -free extract equivalent to approximately 30 mg protein; NaHCO,, 21 mM; succinate-1,4-^C or succinate -2,3 - 1^C, 7.5 ymoles (5 yc) in a total volume of 3.15 ml. KOH was not put in the center well. Atmosphere^ a mixture of 95% O2 and 5% CO2. The reaction was stopped after 120 min. Zero time samples were prepared by addition of HC10. before adding succinate to the reaction mixture. P_. aeruginosa ... 9027 W+ succinate-1,4- C 2.91 14 succinate-,2,3- C 3.18 P_. aerug?nosa 9027 M32 succinate-1,4- C 0.70 14 succinate-2,3 - c 0.76 COOH H.C-COOH H2C-C OOH HC-COOH II HC-COOH 0=C-COOH I H 2 C-COOH COOH I HOOC-HoC-C -OH 2 I H 2 C-COOH H2 C-COOH COOH I HOOC-H 2C-C-OH H 2 C-COOH 8a. C i t r a t e f o r m a t i o n from s u c c i n a t e - 1 ,h- C R a d i o a c t i v e c a r b o n i s i n d i c a t e d by a s o l c i r c l e (•). H 2 C - COOH HC-COOH OH r 0 COOH H C - C O O H y _ . i . = 0 H 2C-COOH HC-COOH 0=-C —COOH H, C —COOH H, C-COOH / ' C H 3 c^o„ CH,-CO~S -CoA • 3 • COOH • • J HOOC-H 2 C - C - O H HP C-COOH F i g . 8b. C i t r a t e f o r m a t i o n from s u c c i n a t e - 2 , 3 - C. R a d i o a c t i v e c a r b o n i s i n d i c a t e d by a s o l i d cTrcle (•). VJI oo o f the NAD, NADP independent p a r t i c u l a t e m a l i c dehydrogenase i n t h e o x i d a t i o n o f m a l a t e t o o x a l a c e t a t e and t h e p a t t e r n o f l a b e l l i n g o f c i t r a t e o b t a i n e d from s u c c i nate-1 ,4-^C and 14 s u c c i n a t e - 2 , 3 " C has e l i m i n a t e d any o t h e r p o s s i b i 1 i t y . V I I . O x i d a t i o n o f S u c c i n a t e , Fumarate and M a l a t e by a C e l 1 - F r e e E x t r a c t o f A. aerogenes , S i n c e t h e r e a r e d i f f e r e n c e s between the m a l a t e o x i d i z i n g systems o f P. a e r u g i n o s a and A. a e r o g e n e s , the o x i d a t i o n o f d i c a r b o x y l i c a c i d s i n c e l l - f r e e e x t r a c t s o f t h e l a t t e r o r g a n i s m was a l s o s t u d i e d ( F i g . 9). A c e l l - f r e e e x t r a c t o f A. aerogenes o x i d i z e d these, compounds o n l y v e r y s l o w l y . I n i t i a l l y m a l a t e and f u m a r a t e were u t i l i z e d a t a s l o w e r r a t e than s u c c i n a t e w h i c h i s j u s t t he o p p o s i t e t o t h e s i t u a t i o n w i t h P. a e r u g i n o s a . These f i n d i n g s a r e i n agreement w i t h the o b s e r v a t i o n s o f . Jones and K i n g (1968) who have r e p o r t e d such d i f f e r e n c e s between pseudomonads and the c o l i f o r m group. D e t e r m i n a t i o n o f m a l a t e i n the Warburg v e s s e l s r e c e i v i n g s u c c i n a t e showed t h a t a t 60 min, 4.1 ymoles o f m a l a t e accumulated i n t h e A_. aerogenes r e a c t i o n m i x t u r e . A l t h o u g h P. a e r u g i n o s a c e l l - f r e e e x t r a c t a l s o showed some m a l a t e a c c u m u l a t i o n , the amount was o n l y 0.57 ymoles per v e s s e l . The p a t t e r n o f o x i d a t i o n o f t h e s e d i c a r b o x y l i c a c i d s i n d i c a t e s t h a t P. a e r u g i hosa has an advantage o v e r A. aerogenes because 60 0 60 120 180 2 4 0 M I N U T E S , F i g . 9. O x i d a t i o n o f s u c c i n a t e , fumarate and m a l a t e by c e l l - f r e e e x t r a c t s . o f A..aerogenes. The r e a c t i o n m i x t u r e s were s e t up as i n F i g . 2. Symbols; •, s u c c i n a t e ; A, m a l a t e ; v •'. O, f u m a r a t e . • t h e l a t t e r o r g a n i s m depends on t h e a v a i l a b i l i t y o f f r e e NAD f o r th e o x i d a t i o n o f m a l a t e . The u t i 1 i z a t i o n o f m a l a t e by A_. aerogenes o b v i o u s l y i s a l i m i t i n g s t e p . O x i d a t i o n o f s u c c i n a t e i n pseudomonads and c o l i f o r m s i s c a t a l y z e d by t h e p a r t i c u l a t e s u c c i n i c o x i d a s e system. T h i s c o u l d e x p l a i n the r a p i d o x i d a t i o n o f s u c c i n a t e by the c e l l - f r e e e x t r a c t s o f both groups o f organisms The m a l a t e o x i d a s e system has been shown t o c o n t a i n FAD as t h e p r o s t h e t i c group i n P. o v a l i s C h e s t e r ( P h i z a c k e r l e y and F r a n c i s , 1966) and A c e t o b a c t e r x y l i h u m (Benziman and G a l a n t e r , 1 964 ) . The i n i t i a l , r a p i d r a t e o f o x i d a t i o n o f m a l a t e and fumarate o b s e r v e d i n the c e l l - f r e e e x t r a c t o f P. a e r u g i n o s a and not seen i n the c e l l f r e e e x t r a c t o f A_. aerogenes can be a s c r i b e d t o the p o s s e s s i o n o f t h e p a r t i c u l a t e m a l a t e o x i d a s e system. Thus, organisms p o s s e s s i n g p a r t i c u l a t e L - m a l i c dehydrogenase a r e b e t t e r e quipped f o r u t i l i z a t i o n o f d i c a r b o x y l i c a c i d s s i n c e t h e i r a c t i v i t y on t h e s e compounds i s not r a t e l i m i t e d by t h e l a c k o f a c o n s t a n t s u p p l y o f f r e e NAD. V I I I . The Enzymes o f C a r b o h y d r a t e Metabol ism i h P_. a e r u g i n o s a  Grown i n S u c c i n a t e o r G l u c o s e Media The pseudomonads p o s s e s s a g r e a t c a p a c i t y t o adapt t o t h e i r n u t r i t i o n a l e n v i r o n m e n t . That t h e t r i c a r b o x y l i c a c i d c y c l e a c t i v i t y i s c o n s t i t u t i v e and i s o f s p e c i a l i m p o r t a n c e i n the m e t a b o l i s m i n t h e s e organisms i s s u p p o r t e d by the s t u d y o f the p a t t e r n o f u t i l i z a t i o n o f s u c c i n a t e and g l u c o s e . The organisms grown on s u c c i n a t e d i d so w i t h o u t a l a g , w h i l e they showed a lo n g l a g i n g l u c o s e medium ( F i g . 10). In the medium c o n t a i n i n g both s u c c i n a t e and g l u c o s e , the d i a u x i c growth was o b t a i n e d and i n i t i a l l y t h e growth p a t t e r n resembled t h a t o b s e r v e d i n s u c c i n a t e medium. F i g u r e 11 shows t h e growth p a t t e r n i n g l u c o s e , g l u c o s e p l u s s u c c i n a t e and s u c c i n a t e media o f organisms p r e v i o u s l y grown on g l u c o s e . There was no pronounced l a g on s u c c i n a t e medium and no d i a u x i c growth on the medium c o n t a i n i n g g l u c o s e p l u s s u c c i n a t e . Very s i m i l a r p a t t e r n s were o b t a i n e d when r e s t i n g c e l l s u s p e n s i o n s , h a r v e s t e d from g l u c o s e o r s u c c i n a t e media, o x i d i z e d t h e s e compounds ( F i g s . 12 and 1 3 ) . The e x t r a c t o f the c e l l s grown i n g l u c o s e medium o x i d i z e d s u c c i n a t e r a p i d l y consuming 65% o f t h e t h e o r e t i c a l oxygen uptake a t 240 min, but d i d not o x i d i z e g l u c o s e beyond t h e s t a g e where a p p r o x i m a t e l y 1 Umole oxygen was consumed pe r umole o f g l u c o s e ( F i g . 1 4 ) . T h i s b e h a v i o u r o f the c e l l - f r e e e x t r a c t s i n t h e absence o f added c o f a c t o r s , however, was t o be e x p e c t e d s i n c e under t h e s e c o n d i t i o n s g l u c o s e i s o x i d i z e d o n l y t o the 2-keto-g l u c o n a t e l e v e l (Campbel1 e t a l , 1956). The e x t r a c t o f t h e c e l l s grown i n s u c c i n a t e medium o x i d i z e d g l u c o s e v e r y s l o w l y s u g g e s t i n g t h e weaker a c t i v i t y o f g l u c o s e o x i d a s e system. The above r e s u l t s i n d i c a t e d t h a t t h e enzymes n e c e s s a r y f o r t h e u t i l i z a t i o n o f s u c c i n a t e were p r e s e n t i n c e l l s grown on e i t h e r 63 I O H « • 0 120 240 360 480 M i n u t e s • F i g . 10. Growth o f s u c c i n a t e grown inoculum i n the media c o n t a i n i n g ' s u c c i n a t e , g l u c o s e o r s u c c i n a t e + g l u c o s e . Log/phase • e e l Is from s u c c i n a t e m i n i m a l medium were h a r v e s t e d , washed once and i n o c u l a t e d i n the media. ! Incubation'.'was. a t 37 C w i t h shaking.. Symbols: O, 0 . 2 % . s u c c i n a t e ; #,0.2% g l u c o s e ; • , 0.04% s u c c i n a t e + 0.2% g l u c o s e . . 10 « — » > i 0 120 240 360 480 M i n u t e s \, 11.' Growth o f g l u c o s e grown i n o c u l u m ' i n media c o n t a i n i n g g l u c o s e , s u c c i n a t e o r g l u c o s e + s u c c i n a t e . E x p e r i m e n t a l c o n d i t i o n s were the same as i n F i g . 10.. Symbols: O, 0.2% s u c c i n a t e ; . : •,' 0.2% g l ucose; •, 0.04% g l u c o s e + 0.2% s u c c i n a t e . 60 120 I N U T E S 180 240 F i g . 12. O x i d a t i o n o f s u c c i n a t e , g l u c o s e o r a m i x t u r e o f s u c c i n a t e and g l u c o s e by c e l l s h a r v e s t e d from a s u c c i n a t e m i n i m a l medium. The Warburg v e s s e l s were s e t up as i n F i g . .1. Symbols: • , s u c c i n a t e , 7 .5 pmoles; A, g l u c o s e , 5 .0 y m o l e s ; O, s u c c i n a t e , 3.75 ymoles + g l u c o s e , 2.5 y m o l e s . 66 0 20 40 60 80 100 120 140 M I N U T E S F i g . 13'.;V O x i d a t i o n o f s u c c i n a t e , g l u c o s e o r a: m i x t u r e o f . s u c c i nate'+ g l u c o s e by c e l l s h a r v e s t e d from a g l u c o s e medium. The e x p e r i m e n t a l c o n d i t i o n s and the symbols used a r e s i m i l a r t o th o s e i n F i g . 12. 4 0 0 r 0 60 . 120 180 240 M I N U T E S . 1 4 . O x i d a t i o n o f g l u c o s e ;and s u c c i n a t e by.the e e l 1 - f r e e e x t r a c t s from t h e . c e l l s grown i n g l u c o s e (o) o r s u c c i n a t e (•) media. The Warburg v e s s e l s were s e t up as i n F i g . 2. Symbols: open.,, r e c e i v i ng 7 i5 . ymoles s u c c i n a t e ; c l o s e d , r e c e i v i n g 5.0 ymoles g l u c o s e . g l u c o s e o r s u c c i n a t e medium, whereas t h e enzymes o f g l u c o s e o x i d a t i o n were e i t h e r low o r not d e t e c t a b l e i n c e l l s h a r v e s t e d from the s u c c i n a t e medium. The absence o r p r e s e n c e o f o n l y a low l e v e l o f t h e enzymes o f g l u c o s e o x i d a t i o n i n pseudomonads, when grown on t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s , has been r e p o r t e d e a r l i e r ( H a m i l t o n and Dawes, 1960; Von T i g e r s t r o m and C a m p b e l l , 1966b; Ng and Dawes, 1967; L e s s i e and N e i d h a r d t , 1967a), but t h e d e t a i l e d s t u d y o f the l e v e l o f the enzymes r e g u l a t i n g the p a t t e r n o f m e t a b o l i s m has not been c a r r i e d o u t . T h e r e f o r e , the d e t e r m i n a t i o n o f the l e v e l s o f the enzymes o f c a r b o h y d r a t e m e t a b o l i s m i n c e l l s grown i n s u c c i n a t e o r g l u c o s e medium was a t t e m p t e d . 1. G l u c o s e d e g r a d i n g enzymes In s u c c i n a t e grown c e l l s t h e enzymes o f t h e E n t n e r - D o u d o r o f f pathway and t h e o x i d a t i v e p o r t i o n o f pentose phosphate pathway were e i t h e r a b s e n t o r a t a v e r y low l e v e l ( T a b l e V I I ) . Hence, t h e s e pathways would be u n a v a i l a b l e f o r t h e o x i d a t i o n o f s u b s t r a t e s by r e c y c l i n g . I t has been r e p o r t e d t h a t t h e Embden-Meyerhof pathway i s not o p e r a t i v e i n P. a e r u g i n o s a but t h e a c t u a l r e a s o n has not been d e f i n e d . T h i s i n v e s t i g a t i o n has shown t h a t t h e o r g a n i s m l a c k s p h o s p h o f r u c t o k i n a s e , thus p r e v e n t i n g t h e f u n c t i o n i n g o f t h e Embden-Meyerhof pathway. V a l i d i t y o f p h o s p h o f r u c t o k i n a s e assay was checked by p r e p a r i n g a c e l l - f r e e Table VI I. Activity of some in P. aerugi nosa enzymes of carbohydrate metabolism grown in succinate or glucose media. Speci f ? c Act ? vi ty  Enzyme Succinate medium Glucose medium Glucokinase 16.4 98.7 G1ucose-6-phosphate dehydrogenase 2.4 224.0 , 6-Phosphog1uconate dehydrogenase nil 26.0 6-Phosphogluconate dehydrase + 2-keto-3_deoxy-6-phospho-gluconate aldolase <1 49.2 Phosphohexose isomerase 35.7 43.1 Phosphofructokinase nil nil Fructose diphosphatase 36.0 39.0 Fructose-1,6-diphosphate aldolase 74.6 93.0 Triosephosphate isomerase 1780.0 1145.0 NADP 3-phosphoglyceraldehyde dehydrogenase 76.0 346.0 NAD 3-phosphoglyceraldehyde dehydrogenase 15-0 81.0 3-Phosphoglycerate kinase 435.0 375.0 Enolase 408.0 635.0 Pyruvate kinase .61'.8 71.6 Transketolase 33'.3 ' 41.0 Transaldolase 12.0 12.0 Specific activity = mymoles substrate utilized per min per mg of protei e x t r a c t o f A. aerogenes and a s s a y i n g under the s i m i l a r c o n d i t i o n s . A. aerogenes showed h i g h a c t i v i t y o f t h i s enzyme ( s p e c i f i c a c t i v i t y o f 292.0). The a s s a y s f o r t h i s enzyme i n a c e l l - f r e e e x t r a c t o f P. a e r u g i n o s a were a l s o c a r r i e d out i n the p r e s e n c e o f p h o s p h o e n o l p y r u v a t e w i t h the same n e g a t i v e r e s u l t s . Lack o f p h o s p h o f r u c t o k i n a s e i s a s s o c i a t e d w i t h absence o f f u n c t i o n i n g Embden-Meyerhof pathway i n P;. f 1 uo r e s c e n s (Wood and Schwerdt, 1954); Z_. mobi 1 i s (Raps and DeMoss, 1962) and s e v e r a l s p e c i e s o f R h o d o t o r u l a (Brady and Chamb1iss, 1967). However, L e s s i e and N e i d h a r d t (1967a) found t h a t t h e i r s t r a i n o f P. a e r u g i n o s a l a c k e d f r u c t o s e d i p h o s p h a t e a l d o l a s e a c t i v i t y . I t i s seen t h a t i n P_. a e r u g i n o s a 9027 hexose phosphates c o u l d be s y n t h e s i z e d by t h e r e v e r s a l o f Embden-Meyerhof scheme, but t h i s w i l l not be p o s s i b l e i n the s t r a i n s t u d i e d by L e s s i e and N e i d h a r d t . T h r e e - p h o s p h o g l y c e r a l d e h y d e dehydrogenase showed h i g h e r a c t i v i t y w i t h NADP as the coenzyme. The l e v e l o f t h i s enzyme was much h i g h e r i n g l u c o s e grown c e l l s than i n c e l l s grown on s u c c i n a t e . The enzymes whose l e v e l f l u c t u a t e s w i t h n u t r i t i o n a l c o n d i t i o n s have been s u g g e s t e d t o be r e g u l a t o r y . T h i s enzyme i s i n v o l v e d i n t he a c t i v i t i e s o f the E n t n e r - D o u d o r o f f pathway, t h e Embden-Meyerhof pathway and p e n t o s e phosphate c y c l e and may have s p e c i a l i m p o r t a n c e i n the r e g u l a t i o n o f m e t a b o l i s m . T h i s a s p e c t w i l l be d i s c u s s e d i n l a t e r s e c t i o n s . A c o n s i d e r a t i o n o f p e n t o s e phosphate c y c l e a c t i v i t y i n g l u c o s e grown and s u c c i n a t e grown c e l l s b r i n g s out a v e r y i n t e r e s t i n g p a t t e r n o f m e t a b o l i s m o f p e n t o s e s ( T a b l e V I I , V I M and F i g . 15'). When grown i n a g l u c o s e medium c e l l s e x h i b i t e d good a c t i v i t i e s o f a l l t h e enzymes a s s a y e d i n d i c a t i n g t h a t t h e p e n t o s e phosphate pathway c o u l d o p e r a t e as a c y c l e f o r the breakdown o f t h e s u b s t r a t e s . However, i n s u c c i n a t e grown c e l l s , w h i l e t h e a c t i v i t i e s o f r i b o s e p h o s p h a t e i s o m e r a s e , t r a n s k e t o l a s e and t r a n s -a l d o l a s e were i d e n t i c a l t o t h o s e i n g l u c o s e c e l l s , t h e a c t i v i t i e s o f t h e enzymes o f o x i d a t i v e p o r t i o n o f p e n t o s e phosphate c y c l e , i . e . g l u c o s e - 6 - p h o s p h a t e dehydrogenase and 6 - p h o s p h o g l u c o n a t e dehydrogenase i n d i c a t e d t h a t hexoses cannot be u t i l i z e d v i a t h i s c y c l e i n t h e s e c e l l s . F r u c t o s e - 6 - p h o s p h a t e p h o s p h o k e t o l a s e a c t i v i t y , w h i c h i s i m p o r t a n t i n some b a c t e r i a f o r d e g r a d a t i o n o f hexose phosphates ( D e V r i e s e t a l , 1 967 ) , was found t o be i n s i g n i f i c a n t . A l s o , t h e o v e r a l l a c t i v i t y o f t h e a n a e r o b i c p o r t i o n o f t h e p e n t o s e phosphate c y c l e measured by t r a n s f o r m a t i o n o f p e n t o s e phosphate t o g l u c o s e - 6 - p h o s p h a t e was low i n the organisms grown i n s u c c i n a t e medium, a g a i n i n d i c a t i n g t h a t r e a c t i o n s o f t h i s c y c l e a r e not o f importance i n d e g r a d i n g c a r b o h y d r a t e s i n t h e s e c e l l s . In an e f f o r t t o d e t e r m i n e i f the a b i l i t y t o form g l u c o s e -6-phosphate from r i b o s e - 5 - p h o s p h a t e i s i n d u c e d by g l u c o s e , an a d d i t i o n a l e x p e r i m e n t was u n d e r t a k e n . S i n c e 3 - p h o s p h o g l y c e r -a l d e h y d e dehydrogenase was a l s o found t o be a r e g u l a t o r y enzyme, i t s l e v e l was a s s a y e d i n t h i s e x p e r i m e n t ( F i g . 1 6 ) . I t i s a p p a r e n t t h a t on the t r a n s f e r o f s u c c i n a t e c e l l s t o t h e g l u c o s e T a b l e V I M . G1 u c o s e - 6 - p h o s p h a t e f o r m i n g a c t i v i t y from r i b o se - 5-phosphate and r i bosephosphate isomerase a c t i v i t y . Growth medium G l u c o s e S u c c i n a t e R i b o s e p h o s p h a t e isomerase 0 .33 0.30 G 1ucose - 6-phosphate f o r m i n g a c t i v i t y from r i b o s e - 5 -phosphate 1" 28 .0 2.5 The a c t i v i t y o f r i b o s e p h o s p h a t e isomerase i s e x p r e s s e d as A O.D. 520 my per min p e r mg o f p r o t e i n . The r e a c t i o n was a l l o w e d t o p r o c e e d f o r 20 min. The a c t i v i t y i s e x p r e s s e d as mymoles NADP reduced p e r min p e r mg o f p r o t e i n . 0 5 10 15 20 M I N U T E S 15. G1ucose^6-phosphate f o r m a t i o n from r i b o s e - 5 - p h o s p h a t e by P. a e r u g i n o s a grown i n s u c c i n a t e (•, 1.2 mg p r o t e i n ; • , 0.6 mg p r o t e i n ) o r g l u c o s e (o, 0.315 mg p r o t e i n ) media. One ml r e a c t i o n m i x t u r e c o n t a i n e d T r i s b u f f e r , pH 8.0, 50 mM; t h i a m i n e - p y r o p h o s p h a t e , . 0.5 mM; r i b o s e - S ^ p h o s p h a t e , 2.0 mM; NADP, 1 mM; commercial g l u c o s e - 6 - p h o s p h a t e dehydrogenase and c e l l - f r e e e x t r a c t . In A, NADP t o s t a r t t h e r e a c t i o n was added a f t e r 40 min i n c u b a t i o n o f t h e . a s s a y ; r e a c t i o n m i x t u r e a t room t e m p e r a t u r e . 74 H O U R S F i g . 16. I n c r e a s e i n the l e v e l o f 3 - p h o s p h o g l y c e r a l d e h y d e dehydrogenase and g I u c o s e - 6 - p h o s p h a t e f o r m i n g a c t i v i t y from r i b o s e - 5 - p h o s p h a t e , oh - s h i f t : from s u c c i n a t e t o g l u c o s e medium. Log phase c e l l s f r o m . s u c e i n a t e medium were h a r v e s t e d , washed, i n b a s a l . s a l t s medium and i n o c u l a t e d i n t o 0 .3% g l u c o s e medium i n ' E r l e n m e y e r f l a s k s . . I n c u b a t i o n was a t 30 C w i t h s h a k i n g . Samples were t a k e n out a t d e s i r e d i n t e r v a l s and used f o r enzyme a s s a y s . Symbols: o, o p t i c a l d e n s i t y ; •, 3 - p h o s p h o g l y c e r a l d e h y d e dehydrogenase; A, G-6-P f o r m a t i o n from R-5-P (mymoles/ min/mg o f p r o t e i n ) . medium these a c t i v i t i e s are great ly increased. The increase in 3-phosphoglyceraldehyde dehydrogenase in glucose medium can be e a s i l y r a t i o n a l i z e d f o r 3-phosphoglyceraldehyde is a product of Entner-Doudoroff pathway a c t i v i t y on glucose . In order to increase the capaci ty to u t i l i z e t h i s substrate and d i r e c t i t to the t r i c a r b o x y l i c ac id c y c l e , h i g h a c t i v i t y of t h i s enzyme is necessary. This is not so in a succinate medium where 3 -phosphoglyceraldehyde dehydrogenase is u t i l i z e d only f o r l i m i t e d reversal of Embden-Meyerhof reactions for the purpose of synthe-s i z i n g pentoses and hexoses for c e l l b i o s y n t h e s i s . From the above observations i t could be concluded that in order to grow on succinate or any other t r i c a r b o x y l i c a c i d cyc le intermediate , t h i s organism must u t i l i z e the t ransketolase r e a c t i o n , u s i n g compounds which could be derived from t r i c a r b o x y l i c a c i d c y c l e intermediates , to synthesize pentoses by one or more of the several a l t e r n a t i v e reactions (Racker, 1954; Sable , 1 966 ) . These mechanisms could be summarized as f o l l o w s : -1) Fructose-6 -phosphate Pentose; phosphate + + Triose-phosphate Erythrose-4 -phosphate 2) Triose-phosphate Pentose phosphate + An " a c t i v e g l y c o l a l d e h y d e " donor ( e . g . Hydroxypyruvate) E v i d e n c e o f such r e a c t i o n s i n pseudomonads has come from many l a b o r a t o r i e s . The work o f DeLey and D o u d o r o f f as quoted by DeLey (1960) showed t h a t t r a n s k e t o l a s e - t r a n s a l d o l a s e r e a c t i o n s were i n v o l v e d i n pentose phosphate s y n t h e s i s i n P_. saccharoph? l a , an o r g a n i s m l a c k i n g 6-phosphogluconate dehydrogenase. T h i s was c o n f i r m e d by the work o f F o s s i t t and B e r n s t e i n (1963). Wang e t a l _ (1959) proposed t h a t g l u c o n a t e grown P. a e r u g i hosa l a c k e d t he a c t i v i t y o f the o x i d a t i v e p o r t i o n o f t h e pentose phosphate pathway and t h e r e f o r e , must s y n t h e s i z e p e n t o s e s by a l t e r n a t e schemes. A s i m i l a r c o n c l u s i o n was drawn by L e s s i e and N e i d h a r d t (1967a) i n d i s c u s s i n g s u c c i n a t e grown P. a e r u g i n o s a . S t u d i e s oh S t r e p t o c o c c u s f a e c a 1 i s and A l c a l i g e n e s f a e c a l i s a l s o showed i n v o l v e m e n t o f such r e a c t i o n s i n b i o s y n t h e s i s o f pentoses ( S a b l e , 1966). 2. T r i c a r b o x y l i c a c i d c y c l e and r e l a t e d enzymes a. T r i c a r b o x y l i c a c i d c y c l e a c t i v i t y The r e s u l t s o f s t u d i e s on t h e enzymes o f g l u c o s e o x i d a t i o n d i d not acc o u n t f o r t h e growth and o x i d a t i o n p a t t e r n o f g l u c o s e and s u c c i n a t e grown c e l l s i n media w i t h one o f t h e s e o r a m i x t u r e o f t h e s e s u b s t r a t e s . The c e l l s h a r v e s t e d from g l u c o s e medium u t i l i z e d s u c c i n a t e w i t h o u t a l a g . T h e r e f o r e , a s t u d y o f the l e v e l s o f t r i c a r b o x y l i c a c i d c y c l e enzymes i n the two t y p e s o f c e l l s was c o n s i d e r e d e s s e n t i a l . M o r e o v er, the d e t a i l e d s t u d y o f t r i c a r b o x y l i c a c i d c y c l e a c t i v i t y becomes i m p o r t a n t because t h i s i s t h e o n l y pathway l e f t a t t h e d i s p o s a l o f P. aerug1nosa grown on s u c c i n a t e s i n c e , d u r i n g growth i n t h i s medium, th e a c t i v i t i e s o f t h e E n t n e r - D o u d o r o f f pathway and t h e p e n t o s e phosphate pathway are not adequate f o r the o x i d a t i o n o f the compounds, by r e c y c l i n g . T a b l e IX shows the a c t i v i t y o f t h e s e and r e l a t e d enzymes f o r c o m p arison i n t h e c e l l s grown on g l u c o s e , s u c c i n a t e , s u c c i n a t e + a - k e t o g l u t a r a t e and s u c c i n a t e + g l u t a m a t e media. H i g h e r growth r a t e s on s u c c i n a t e media supplemented w i t h a - k e t o g l u t a r a t e o r g l u t a m a t e i n d i c a t e d t h a t t h e s e compounds were u t i l i z e d s i m u l t a n e o u s l y w i t h s u c c i n a t e f o r g r o wth. The a c t i v i t i e s o f most o f the t r i c a r b o x y l i c a c i d c y c l e enzymes were comparable and no s t r i k i n g d i f f e r e n c e s were seen. The v a r i a t i o n s o b s e r v e d i n the l e v e l s o f a c o n i t a s e and a - k e t o g l u t a r a t e dehydrogenase were found t o b e ' r e p r o d u c i b l e . The l e v e l o f p a r t i c u l a t e m a l i c dehydrogenase was about t w i c e as g r e a t i n g l u c o s e c e l l s as i n c e l l s grown on s u c c i n a t e . A g a i n , t h i s enzyme c o u l d be r e g u l a t o r y and d e s e r v e d f u r t h e r s t u d y . These r e s u l t s a l s o show t h a t the c o n t r o l mechanism on t h e t r i c a r b o x y l i c a c i d c y c l e a c t i v i t y i s not s i m i l a r t o the p a t t e r n o b s e r v e d i n s p o r e f o r m i n g b a c i l l i (Hanson and Cox, 1967) and E. c o l ? (Gray e t a l , 1 966b ) , s i n c e a - k e t o g l u t a r a t e and g l u t a m a t e p r e s e n t i n t h e media do not s h u t o f f t h e a c t i v i t y o f enzymes l e a d i n g t o a - k e t o g l u t a r a t e f o r m a t i o n . These o b s e r v a t i o n s i n d i c a t e t h a t t h e a c t i v i t y o f t h e t r i c a r b o x y l i c a c i d c y c l e i n T a b l e IX. S p e c i f i c a c t i v i t i e s o f the t r i c a r b o x y l i c a c i d c y c l e and r e l a t e d enzymes, i n P. a e r u g i n o s a grown w i t h d i f f e r e n t c a r b o n s o u r c e s Growth Medium S u c c i n a t e + S u c c i n a t e Enzyme a - k e t o - + G l u c o s e S u c c i nate g l u t a r a t e G1utamate A c o n i t a s e 44.0 31.6 44.8 30. 0 NADP i s o c i t r a t e dehydrogenase 438 464 600 483 NAD i s o c i t r a t e dehydrogenase n i l n i l - -a - K e t o g l u t a r a t e dehydrogenase 50.4 91.7 93.3 80. 8 S u c c i n i c dehydrogenase 128 102 - -Fumarase 1260 1165 1230 1185 P a r t i c u l a t e m a l i c dehydrogenase 28.2 15.0 16.5 14. 2 S u c c i n y l CoA s y n t h e t a s e 270 294 - -NADPH G l u t a m i c dehydrogenase 343 300 273 163 NADH G l u t a m i c dehydrogenase 75.0 62.0 57.0 48. 0 NADP G l u t a m i c dehydrogenase 76.0 70.0 53.0 28. 0 NAD G l u t a m i c dehydrogenase n i l n i l n i l n i l NADP M a l i c enzyme 438 524 481 471 NAD M a l i c enzyme 117 141 153 125 S p e c i f i c a c t i v i t y = mymoles o f s u b s t r a t e u t i l i z e d p e r min per mg o f p r o t e i n . a - K e t o g l u t a r a t e and g l u t a m a t e a d d i t i o n s , when s p e c i f i e d , were a t ; 0.3% l e v e l . pseudomonads i s not o n l y e s s e n t i a l f o r energy p r o d u c t i o n when growth o c c u r s i n g l u c o s e medium, but i s o f s p e c i a l i m p o r t a n c e f o r t he d e g r a d a t i o n o f i n t e r m e d i a t e s o f t h i s c y c l e when t h e organisms a r e growing on such compounds. Under any c i r c u m s t a n c e s t h e enzymes o f t h e t r i c a r b o x y l i c a c i d c y c l e can be c o n s i d e r e d c o n s t i t u t i v e . Good a c t i v i t y o f s u c c i n i c dehydrogenase i n bo t h g l u c o s e and s u c c i n a t e grown c e l l s was o b t a i n e d i n d i c a t i n g t h a t t h e method used i n the p r e s e n t i n v e s t i g a t i o n was s u p e r i o r t o t h e one used by p r e v i o u s w o r k e r s who found v e r y low a c t i v i t y o f t h i s enzyme i r i P. a e r u g i n o s a (Campbel 1 e_t a l _ , 1962; Von T i g e r s t r o m and C a m p b e l l , 1966b). The i n f l u e n c e o f t h e k i n d o f as s a y method used on d e t e c t i n g a d i f f e r e n c e i n a c t i v i t y o f s u c c i n i c dehydrogenase was p o i n t e d o ut by S i n g e r and Kearny (1963). S u c c i n y l CoA s y n t h e t a s e , w h i c h has been found t o i n c r e a s e about 8 - f o l d i n E_. c o l j _ grown on s u c c i n a t e as compared t o t h e c e l l s grown i n g l u c o s e medium ( G i b s o n , Upper and G u n s a l u s , 1967), showed l i t t l e d i f f e r e n c e i n a c t i v i t y i n P. a e r u g i n o s a d u r i n g growth i n s u c c i n a t e o r g l u c o s e media. Thus, t h i s enzyme d i d not seem t o be r e g u l a t o r y . b. G l u t a m i c dehydrogenase a c t i v i t y The v a r i a t i o n i n t h e a c t i v i t y o f g l u t a m i c dehydrogenase i n d i f f e r e n t growth media i s i n t e r e s t i n g t o n o t e . The enzyme a c t i v i t y i n the d i r e c t i o n o f g l u t a m a t e s y n t h e s i s was about f i v e t i m e s as g r e a t as i n t h e d i r e c t i o n o f o x i d a t i o n . A l s o , w i t h NADP (H) the a c t i v i t y was h i g h i n both d i r e c t i o n s . NADH gave some a c t i v i t y i n t h e d i r e c t i o n o f g l u t a m a t e s y n t h e s i s , but t h e r e was no a c t i v i t y w i t h NAD i n the o p p o s i t e d i r e c t i o n . Such r e s u l t s were a l s o r e p o r t e d i n Ch1oropseudomonas e t h y l i c u m ( S t e r n , 1968) and p r o b a b l y i n d i c a t e t h e p r e s e n c e o f two g l u t a m i c dehydrogenases as has been shown i n some f u n g i and p l a n t s ( H o l z e r and S c h n e i d e r , 1957; Sanwal and L a t a , 1961; Leech and K i r k , 1 9 68 ) . The h i g h a c t i v i t y o f t h e enzyme i n the d i r e c t i o n o f g l u t a m a t e f o r m a t i o n i n d i c a t e s t h a t i t s p r i m a r y f u n c t i o n i s ammonia i n c o r p o r a t i o n w h i c h i s i n agreement w i t h the o b s e r v a t i o n s made i n o t h e r systems (Vender, Jayaraman and R i c k e n b e r g , 1965; Leech and K i r k , 1968). In s u p p o r t o f t h i s s u g g e s t i o n i t was found t h a t the p r e s e n c e o f g l u t a m a t e i n the medium r e p r e s s e d t h e a c t i v i t y o f t h e enzyme and the l e v e l s f e l l t o l e s s than h a l f i n both d i r e c t i o n s . R e p r e s s i o n o f t h e a c t i v i t y o f g l u t a m i c dehydro-genase i n g l u t a m a t e medium ha:s been found i n E_. c o l ? (Vender e t a l , 1965) and i n Rhizobium j a p o n i c u m (Mooney and F o t t r e l l , 1968 ) . IX. L e v e l o f M a l i c Enzyme i n C e l l s Grown i n S u c c i n a t e , G l u c o s e  o r A c e t a t e Med ? a T h i s enzyme was found t o e x h i b i t much h i g h e r a c t i v i t y w i t h NADP than w i t h NAD as t h e coenzyme ( T a b l e I X ) . The l e v e l s were h i g h i n both the s u c c i n a t e and t h e g l u c o s e grown c e l l s but t h e a c t i v i t y dropped t o about k0% o f t h i s v a l u e when the organisms were grown i n a c e t a t e medium ( T a b l e X ) . Such r e s u l t s were a l s o r e p o r t e d i n P. p u t i d a (Jacobson e t a l , 1966) . T h i s t y p e o f " c o a r s e c o n t r o l " on m a l i c enzyme i n d i c a t e s t h a t i t s a c t i v i t y i s i m p o r t a n t i n the m e t a b o l i s m o f both t he g l u c o s e and the t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s . When org a n i s m s a r e grown on a t r i c a r b o x y l i c a c i d c y c l e member,, e.g. s u c c i n a t e , i t has t o be c a t a l y s e d m a i n l y by t h e r e a c t i o n s o f t h i s c y c l e as d i s c u s s e d e a r l i e r . Hence, a c e t y l CoA i n a d d i t i o n t o o x a l a c e t a t e has t o be c o n s t a n t l y d e r i v e d from s u c c i n a t e . T h i s i s a c h i e v e d by the o x i d a t i o n o f s u c c i n a t e t o p y r u v a t e v i a m a l a t e by the a c t i v i t y o f m a l i c enzyme and t h u s , t h e h i g h l e v e l o f t h i s enzyme i s m a i n t a i n e d . A c e t y l CoA can then be d e r i v e d from t h e f u r t h e r o x i d a t i o n o f p y r u v a t e . In pseudomonads t h e a c t i v i t y o f the. t r i c a r b o x y l i c a c i d c y c l e i s a l s o i m p o r t a n t f o r the co m p l e t e o x i d a t i o n o f g l u c o s e and hence a g a i n a h i g h l e v e l o f m a l i c enzyme i s m a i n t a i n e d d u r i n g growth i n a g l u c o s e medium. However, when t h e s e o r g a n i s m s a r e grown i n a c e t a t e medium, a c e t y l CoA i s o b t a i n e d by t h e d i r e c t a c t i v a t i o n o f a c e t a t e . T h i s c o u l d be a c h i e v e d e i t h e r by a system s i m i l a r t o t h a t i n E_. c o l i c a t a l y s e d by a c e t a t e k i n a s e and p h o s p h o t r a n s a c e t y l a s e , s i n c e p h o s p h o t r a n s a c e t y l a s e a c t i v i t y has been demonstrated i n f_. a e r u g i n o s a grown on v a l i n e ( S o k a c t h , 1967 ) ; o r by a c e t i c t h i o k i n a s e w h i c h has been r e p o r t e d i n P. f 1uorescens (Kornberg Table X. Effect of carbon sources on the specific activities of malic enzyme and isocitrate lyase in P_. aeruginosa. Growth Medium Glucose Succinate Acetate Enzyme speci f i c act i vi ty' NADP Malic enzyme 435 407 174 Isocitrate lyase 24.3 11.3 450 mymoles substrate utilized per min per mg of protein and Madsen, 1 9 58 ) . Thus, h i g h l e v e l s o f m a l i c enzyme a r e no l o n g e r n e c e s s a r y f o r t h e g e n e r a t i o n o f a c e t y l CoA from a c i d s e i t h e r f o r m a i n t a i n i n g t h e a c t i v i t y o f g l y o x y l a t e c y c l e o r o f t h e t r i c a r b o x y l i c a c i d c y c l e . I t i s th u s seen t h a t t h e organisms p o s s e s s a c a p a c i t y t o r e g u l a t e t h e f l o w o f m e t a b o l i t e s a l o n g s e l e c t e d pathways o f m e t a b o l i s m a c c o r d i n g t o t h e i r need by a l t e r i n g t h e s y n t h e s i s o f t h e n e c e s s a r y enzymes. The commonly made o b s e r v a t i o n o f t h e g r e a t v a r i a t i o n i n the l e v e l o f i s o c i t r a t e l y a s e a c c o r d i n g t o the growth medium (Tabl e X) i s y e t a n o t h e r example t o s u p p o r t t h i s . X. I tiduct i o n o f the Enzymes o f Gl ucose U t i 1 i z a t i o n ' i t i ' C e l l s  H a r v e s t e d from S u c c i hate Med ium To d e t e r m i n e i f the i n i t i a l enzymes o f g l u c o s e m e t a b o l i s m i n P. a e r u g i n o s a a r e induced f o r g l u c o s e u t i l i z a t i o n i n s u c c i n a t e grown c e l l s , i n c u b a t i o n . m i x t u r e s i d e n t i c a l t o t h o s e f o r Warburg e x p e r i m e n t s were s e t up i n two Er l e n m e y e r f l a s k s . Each f l a s k c o n t a i n e d 0.1 M _ T r i s b u f f e r , pH l.k, 10 m l ; s u c c i n a t e grown c e l l s u s p e n s i o n (5 mg d r y w e i g h t / m l ) , 10 m l ; and H^ O t o 27 ml . One o f t h e s e f l a s k s c o n t a i n e d 15 mg c h l o r a m p h e n i c o l . These s u s p e n s i o n s were shaken a t 30 C f o r 30 min and the n 3.0 ml o f g l u c o s e s o l u t i o n (75 umole) was added t o each f l a s k . They were f u r t h e r shaken f o r 2 h r a t wh i c h time t h e c e l l s were h a r v e s t e d , T a b l e X I . I n d u c t i o n o f some enzymes o f g l u c o s e o x i d a t i o n by g l u c o s e . G l u c o s e G l u c o - G l u c o s e - 6 - p h o s p h a t e dehydrogenase k i n a s e dehydrogenase Source o f c e l l s • S p e c i f i c a c t i v i t y " G l u c o s e medium S u c c i n a t e medium C e l I s t r a n s f e r r e d from s u c c i n a t e medium t o g l u c o s e s u s p e n s i o n c o n t a i n i n g c h l o r -amphenicol 32 .0 98 .7 224.0 1.5 16.4 2.5 3.0 13.8 1.0 C e l l s t r a n s f e r r e d from s u c c i n a t e medium t o g l u c o s e s u s p e n s i o n c o n t a i n i n g no c h l o r a m p h e n i c o l 10.0 23.0 9.0 mumoles s u b s t r a t e u t i l i z e d p er min per mg o f p r o t e i n . c e l l e x t r a c t s p r e p a r e d and a s s a y e d f o r glucose-6-phosphate de-h y d r o g e n a s e , g l u c o s e dehydrogenase and g l u c o k i n a s e . The r e s u l t s ( T a b l e X l ) showed t h a t a l 1 o f t h e s e enzymes were in d u c e d i n t h e c e l l s not i n c u b a t e d w i t h c h l o r a m p h e n i c o l , w h i l e t h e i r s y n t h e s i s was s u p p r e s s e d by c h l o r a m p h e n i c o l . The v a l u e s f o r g l u c o s e and s u c c i n a t e c e l l s a r e i n c l u d e d f o r c o m p a r i s o n . A l t h o u g h the enzymes o f g l u c o s e m e t a b o l i s m a r e i n d u c e d when a s h i f t from s u c c i n a t e t o g l u c o s e i s made, i t s t i l l remained t o be seen i f s u c c i n a t e grown c e l l s p o s s e s s t h e permease f o r g l u c o s e o r i f i t t o o has t o be i n d u c e d . I f the t r a n s p o r t system f o r g l u c o s e has t o be i n d u c e d , how long does i t t a k e f o r the 14 i n d u c t i o n ? To answer t h e s e q u e s t i o n s g l u c o s e - U - C t r a n s p o r t was s t u d i e d . F i r s t l y , c e l l s grown i n g l u c o s e medium were used and s u c c i n a t e s e r v e d as the energy s o u r c e i n t h e s e e x p e r i m e n t s . Under t h e s e c o n d i t i o n s g l u c o s e u p t a k e c u r v e l e v e l l e d o f f a t about 5 min ( F i g . 1 7 ) . I t was i n t e r e s t i n g t o f i n d t h a t a n a l o g u e s o f g l u c o s e , i . e . a - C H ^ - g l u c o s i d e and 2 - d e o x y g l u c o s e d i d not r e t a r d the g l u c o s e i n c o r p o r a t i o n , even when added a t 1 0 0 - f o l d t h e c o n c e n t r -14 a t i o n o f g l u c o s e - U - C. To f u r t h e r see t h e s p e c i f i c i t y o f g l u c o s e permease sy s t e m , c o m p e t i t i o n w i t h g a l a c t o s e , f r u c t o s e and mannose was t r i e d ( F i g . 1 8 ) . None.of t h e s e s u g a r s competed w i t h g l u c o s e i n d i c a t i n g a h i g h l y s p e c i f i c mechanism o f g l u c o s e t r a n s p o r t i n P. a e r u g ? h b s a . I t was a l s o found t h a t g l u c o s e grown c e l l s d i d not a c c u m u l a t e a - C H ^ - g l u c o s i d e w h i c h i s i n c o n t r a s t t o the o b s e r v a t i o n w i t h E'. c o l i system and i s i n agreement w i t h the f i n d i n g o f H a m i l t o n 86 6-Or M I N U T E S F i g . 17. Uptake of with 10'-r a d i o a c t i v i t y by glucose grown c e l l s of P . . aerugi •U-1 4C (0.5yc/ymole) and with 10 5 M nosa M glucose-a -methyl-D-glucopyranoside (0.5yc/ymole). Symbols: 0-0 , g lucose-U- C; • - • ,_glucose-U- C + 250 mM chloramphenicol; o - o , g lucpse -U -^C with 10'"* M, 2-deoxygl ucose; • - •, g1ucose-U-1^C with 10"3 M a -methyl -D-glucopyroside, A- A, a -methyl -D-glucopyranoside-U- C. M I N U T E S F i g . 18. I n c o r p o r a t i o n o f g l u c o s e - U - 1 4 C i n t h e p r e s e n c e o r absence o f o t h e r s u g a r s by t h e whole c e l l s o f P. a e r u g i n o s a h a r v e s t e d from the g l u c o s e m i n i m a l medium. The e x t e r n a l g l u c o s e c o n c e n t r a t i o n was 1 0 ~ 5 M; s p e c i f i c a c t i v i t y , 0 . 5 uc/umole. Symbols: • - • , no a d d i t i o n ; o - o , w i t h 10"-5 M f r u c t o s e ; • - •, w i t h ,10"3 M g a l a c t o s e ; • - • , w i t h 10~3 M mannose. •8.8.: MINUTES f i g . 19 . G l u c o s e - U - C i n c o r p o r a t i o n by the whole c e l I s o f PI a e r u g i hosa w i l d t y p e and i t s mutant s t r a i n M5, h a r v e s t e d from s u c c i n a t e m i n i m a l medium. E x t e r n a l g l u c o s e c o n c e n t r a t i o n was 1 0 " ^ M; s p e c i f i c a c t i v i t y 0.5 uc/ymole. Symbols : A - A , w i l d t y p e c e l l s ; o-o, w i l d type c e l l s w i t h 250 mM c h l o r a m p h e n i c o l ; w i 1 d t y p e c e l 1 s . w i t h 30 mM sodium a z i d e + 1 mM i o d o a c e t a m i d e ; • - • , M5 c e l l s . :. '•'•^r::[ • • ' ' • V ' *• • and Dawes ( 1 9 6 0 ) , who made s i m i l a r o b s e r v a t i o n s i n t h e i r s t r a i n o f P. a e r u g i n o s a . The a b i l i t y t o ac c u m u l a t e 2 - d e o x y g l u c o s e was not t e s t e d . S i n c e t h e c e l l s grown i n g l u c o s e medium a c t i v e l y t r a n s p o r t e d g l u c o s e a c r o s s t h e membrane i n the system used i n t h i s s t u d y , the t r a n s p o r t o f g l u c o s e i n the c e l l s grown i n s u c c i n a t e medium was f u r t h e r s t u d i e d w i t h t h i s system i n w i l d t y p e P. aerug?nosa» and i t s mutant s t r a i n M 5 . T h i s mutant i s unable t o grow on g l u c o s e , g l u c o n a t e o r 2 - k e t o g l u c o n a t e but does grow on t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s . The r e s u l t s o f t h e s e e x p e r i m e n t s a r e p r e s e n t e d i n F i g u r e 19. The s t u d y c l e a r l y r e v e a l e d t h a t g l u c o s e permease i s induced s l o w l y o v e r a long p e r i o d o f t i m e . I t was c o n c l u d e d from t h e above d a t a t h a t on s h i f t from s u c c i n a t e t o g l u c o s e medium both t h e s p e c i f i c g l u c o s e permease as w e l l as g l u c o s e o x i d a t i o n enzymes have t o be s y n t h e s i z e d s i m u l t a n e o u s l y i n o r d e r t h a t t h e organisms can adapt t o growth on g1ucose. X I . C o n t r o l o f Tr? c a r b o x y l ? c A c i d C y c l e Act? v i t y 1. P a r t i c u l a t e m a l i c dehydrogenase a c t i v i t y The l e v e l o f p a r t i c u l a t e m a l i c dehydrogenase was . i n t h e c e l l s grown i n g l u c o s e medium ( T a b l e I X ) . The t o d e t e r m i n e t h e i n c r e a s e i n t h e l e v e l o f t h i s enzyme much h i g h e r e x p e r i m e n t on s h i f t t o 90 HOURS F i g . 20. Increase in the level of par t i cu la te malic dehydrogenase a c t i v i t y on s h i f t to glucose medium. The experimental condit ions were the same as in F i g . 16, except for the higher rate of shaking at 37 C. Symbols, O , opt ica l densi ty ; • , par t i cu la te malic dehydrogenase a c t i v i t y . g l u c o s e medium showed the e x p e c t e d r e s u l t s ( F i g . 2 0 ) . I t was t h e r e f o r e t hought d e s i r a b l e t o o b s e r v e t h e c o n t r o l o f m a l a t e o x i d a s e a c t i v i t y . S u b s t r a t e s l i k e a c e t a t e , c i t r a t e , p y r u v a t e and s u c c i n a t e a t a c o n c e n t r a t i o n o f 3 mM d i d not i n f l u e n c e the a c t i v i t y . When ATP was added t o the r e a c t i o n m i x t u r e i n 1 mM c o n c e n t r a t i o n , about h a l f o f t h e a c t i v i t y was l o s t ( T a b l e X I l ) . T h i s i n h i b i t i o n was not c o m p e t i t i v e w i t h t h e s u b s t r a t e , a s t h e a d d i t i o n o f h i g h e r c o n c e n t r a t i o n s o f ATP d i d not cause any f u r t h e r i n h i b i t i o n . E s s e n t i a l l y s i m i l a r r e s u l t s were o b t a i n e d w i t h ADP and AMP ( T a b l e X I I ) . C u m u l a t i v e i n h i b i t i o n o r c o n c e r t e d i n h i b i t i o n w i t h two o r more o f t h e s e n u c l e o t i d e s was not o b s e r v e d . However, GTP was found t o a c t i v a t e t h e enzyme and t h i s a c t i v a t i o n i n c r e a s e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f GTP. A d d i t i o n o f an e q u i v a l e n t c o n c e n t r a t i o n o f ATP and GTP i n the as s a y r e a c t i o n m i x t u r e p a r t i a l l y overcame the ATP i n h i b i t i o n . W h i l e GDP was as s t i m u l a t o r y as GTP; GMP, CTP and ITP d i d not i n f l u e n c e t h e a c t i v i t y . A d e n o s i n e , w h i c h i s common t o t h e s t r u c t u r e o f ATP, ADP and AMP, was w i t h o u t any e f f e c t . The k i n e t i c a n a l y s i s showed t h a t t h i s enzyme has a Km f o r m a l a t e o f 3.77 x 10 m o l e / l i t e r and t h i s i s i n c r e a s e d t o 8.33 x 10 ^ m o l e / l i t e r i n the p r e s e n c e o f 1 mM ATP. From t h e L i n e w e a v e r Burk p l o t i t i s seen t h a t ATP i s a "mixed" t y p e o f i n h i b i t o r , i . e . i t i s n e i t h e r a c o m p e t i t i v e nor a n o n - c o m p e t i t i v e i n h i b i t o r o f t h e enzyme a c t i v i t y ( F i g . 2 1 ) . The r e s u l t s o f the p r e s e n t i n v e s t i g a t i o n do not a l l o w t h e p r e c i s e i n t e r p r e t a t i o n o f t h e s e o b s e r v a t i o n s but such r e s u l t s have been r e p o r t e d w i t h NAD-T a b l e X I I . E f f e c t o f n u c l e o t i d e phosphates on t h e p a r t i c u l a t e m a l i c dehydrogenase i n 105,000 x g_ p e l l e t " . . Inn i b i t o r Concent r a t ion S p e c i f i c a c t i v i t y (mM) (mumoles/min/mg p r o t e i n ) - - 29?0 ATP 0.5 18.4 1.0 15.6 2.0 15.6 3.0 15.6 ADP 0.5 15.6 1.0 14 . 2 2.0 14 .2 AMP 0.5 17.0 1.0 17.0 2.0 15.6 GTP 0.5 34.0 1.0 41 . 0 2.0 53.0 GDP 1 .0 39.6 GMP 1.0 29 . 7 ATP+AMP 1 . 0 a 15.6 ATP+ADP 1 . 0 8 17.0 ATP+ADP+AMP 1 . 0 a 15.6 ATP+GTP i . o a 22.4 CTP 1 .0 29 .7 ITP 1.0 28.9 2 x 10 J M EDTA was added t o 20,000 x g_.supernatant b e f o r e c e n t r i f u g a t i o n a t 105,000.x g_ f o r 90 min.. The p e l l e t was washed ;;with 0 .2 M T r i s b u f f e r , pH 7.2 c o n t a i n i n g 10 "3 M EDTA and resuspended i n 0 .2 M T r i s , pH 7 . 2 . 'Each n u c l e o t i d e phosphate was added a t a c o n c e n t r a t i o n o f 1 mM. - 4 0 0 20 40 60 80 , 100 [Sl ( mM M A L A T E ) F i g . 21. Lineweaver-Burk plot for par t i cu la te malic dehydrogenase in the absence (A) or presence (B) of 1 mM ATP. l i n k e d p a r t i c u l a t e ( m i t o c h o n d r i a l ) m a l i c dehydrogenase from p i g h e a r t i n w h i c h ATP, ADP and AMP were found t o be i n h i b i t o r y when assa y e d i n t h e d i r e c t i o n o f NADH o x i d a t i o n ; and w i t h t h e enzyme from peas i n w h i c h t h e i n h i b i t i o n was ob s e r v e d i n both d i r e c t i o n s ( K u r a m i t s u , 1966; K u r a m i t s u , 1968). T h i s p a t t e r n o f i n h i b i t i o n by a d e n i n e n u c l e o t i d e s was presumed t o mimic t h e c o n t r o l e x e r t e d by y e t a n o t h e r compound r e g u l a t i n g t he enzyme a c t i v i t y . However, i n the absence o f a r e g u l a t o r y mechanism f o r i s o c i t r a t e dehydrogenase by a d e n i n e n u c l e o t i d e s i n P. a e r u g i n o s a t h e s i g n i f i c a n c e o f such an o b s e r v a t i o n i s v e r y g r e a t . S i n c e a c o n s t a n t s u p p l y o f o x a l a c e t i c a c i d i s n e c e s s a r y t o e n s u r e the a c t i v i t y o f t h e t r i c a r b o x y l i c a c i d c y c l e , a c o n t r o l a t t h i s s t e p on m a l a t e o x i d a s e system can thus r e g u l a t e the a c t i v i t y o f t h e c y c l e . 2. C o n t r o l o f t h e f l o w o f i s o c i t r a t e v i a t h e t r i c a r b o x y l i c a c i d c y c l e and g l y o x y l a t e c y c l e i n P_. aer u g ? h o s a The i n h i b i t o r y and r e p r e s s i v e e f f e c t s o f t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s and p h o s p h o e n o l p y r u v a t e on i s o c i t r a t e l y a s e o f E. c o l i have been r e p o r t e d ( K o r n b e r g , 1966 ) . T h i s t y p e o f s t u d y on t h e c o n t r o l o f t h i s enzyme from pseudomonads has not been made. R u f f o and cow o r k e r s (1959, 1962, 1963, 1967) have r e p o r t e d t h a t o x a l m a l a t e , a c o n d e n s a t i o n p r o d u c t o f g l y o x y l a t e and o x a l a c e t a t e , i n h i b i t s mammalian a e o n i t a s e and i s o c i t r a t e dehydrogenase and i t has been proposed t h a t t h i s c o n d e n s a t i o n p r o d u c t may be i m p o r t a n t in the r e g u l a t i o n o f t r i c a r b o x y l i c a c i d c y c l e a c t i v i t y . However , more r e c e n t l y S h i i o and Ozak i (1968) have found t h a t o x a l m a l a t e i s not formed f rom g l y o x y l a t e and o x a l a c e t a t e under p h y s i o l o g i c a l c o n d i t i o n s . They r e p o r t e d s t r o n g c o n c e r t e d i n h i b i t i o n by t h e s e two compounds o f NADP s p e c i f i c i s o c i t r a t e d e h y d r o g e n a s e f rom B r e v i b a c t e r i u m f l a v u m and r e l a t i v e l y lower i n h i b i t i o n o f t h i s enzyme when o b t a i n e d from E_. c o l i , B_. s u b t i 1 i s o r p i g h e a r t . It was o f i n t e r e s t t o look f o r such c o n t r o l o f i s o c i t r a t e d e h y d r o g e n a s e i n P. a e r u g i n o s a , s i n c e i t shows a c t i v i t y o n l y w i t h NADP (Von T i g e r s t r o m and C a m p b e l l , 1966; T a b l e IX ) . It was a l s o found i n the p r e s e n t s t u d y t h a t u n l i k e the N A D - s p e c i f i c i s o c i t r a t e d e h y d r o g e n a s e o f N e u r o s p o r a and y e a s t ( A t k i n s o n , 1966) the N A D P - s p e c i f i c enzyme o f P. a e r u g i n o s a was not i n f l u e n c e d by AMP, ADP o r A T P . P r e l i m i n a r y r e s u l t s w i t h the e e l 1-free e x t r a c t s showed t h a t t h e enzyme i s s t r o n g l y i n h i b i t e d by s i m u l t a n e o u s a d d i t i o n o f g l y o x y l a t e and o x a l a c e t a t e . T h e r e f o r e , f o r d e t a i l e d s t u d y , i s o c i t r a t e d e h y d r o g e n a s e and i s o c i t r a t e l y a s e f rom a c e t a t e grown c e l l s o f P. a e r u g i n o s a were p a r t i a l l y p u r i f i e d by ammonium s u l f a t e f r a c t i o n a t i o n p r o c e d u r e ( T a b l e X l l l ) as o u t l i n e d by Ozak i and S h i i o (1968). M a l i c enzyme which i s l i k e l y t o i n t e r f e r e w i t h the i s o c i t r a t e d e h y d r o g e n a s e a s s a y when the e f f e c t o f m a l a t e i s s t u d i e d , was low i n Pk and P5 f r a c t i o n s . The P4 f r a c t i o n was used f o r the a s s a y o f i s o c i t r a t e d e h y d r o g e n a s e and PS f o r i s o c i t r a t e l y a s e . T a b l e XIV and XV show the e f f e c t o f d i f f e r e n t t r i c a r b o x y l i c a c i d c y c l e and r e l a t e d compounds on i s o c i t r a t e d e h y d r o g e n a s e and i s o c i t r a t e l y a s e a c t i v i t y r e s p e c t i v e l y . 'Table X I I I . P a r t i a l p u r i f i c a t i o n o f i s o c i t r a t e dehydrogenase and i s o c i t r a t e l y a s e from t h e c e l l e x t r a c t s o f P_. a e r u g i n o s a ; Enzyme I s o c i t r a t e I s o c i t r a t e NADP m a l i c T o t a l .dehydrogenase l y a s e enzyme p r o t e i n ; — : — : — : (mg) sp. a c t . t o t . a c t . s p . a c t . t o t . a c t . s p . a c t . t o t . a c t . 105,000 x £ s u p e r n a t a n t (A) 169. 2 1090. 184,300 877 148 ,300 P r e c i p i t a t e between 0.5 and 0.8 s a t u r a t i o n o f (A) P1 68. 1200 82,000 672 45,900 . 210 14,350 P r e c i p i t a t e from.PI between 0.5 and 0.6 s a t u r a t i o n P2 31. 2 2140 66,700 880 27,450 37.5 1,170 P r e c i p i t a t e from P2 a t 0 .55 s a t u r a t i o n P4 . 5. 68 3290 18 ,700 1265 7,180 14.8 . 8 4 P r e c i p i t a t e from P2 between 0 .55 and 0.65 s a t u r a t i o n P5 6. 16 3480 21,450 1695 10,450 18.9 116 (NH^)2^^ZJ F r a c t i o n a t i o n p r o c e d u r e F r a c t i o n a. I s o c i t r a t e dehydrogenase a c t i v i t y The a n a l y s i s o f t h e r e s u l t s showed t h a t i s o c i t r a t e d ehydrogenase, a l t h o u g h i n h i b i t e d by a l l t h e compounds t o some e x t e n t , i s v e r y s e n s i t i v e t o c o n c e r t e d i n h i b i t i o n by low c o n c e n t r a t i o n s o f g l y o x y l a t e p l u s o x a l a c e t a t e ( T a b l e X I V ) . When added s i n g l y , o x a l a c e t a t e caused some i n h i b i t i o n o f t h e enzyme, but g l y o x y l a t e a c t u a l l y a c t i v a t e d t h e enzyme. The c o n d e n s a t i o n p r o d u c t was not found t o be as s t r o n g an i n h i b i t o r as o x a l a c e t a t e and g l y o x y l a t e t o g e t h e r . M o r e o v e r , i t was found t h a t a l m o s t a l l o f t h e g l y o x y l a t e c o u l d be r e c o v e r e d a t t h e end o f the a s s a y i n the r e a c t i o n m i x t u r e r e c e i v i n g g l y o x y l a t e p l u s o x a l a c e t a t e . T h i s i n d i c a t e d t h a t a c o n d e n s a t i o n p r o d u c t was not formed and thus was not i m p o r t a n t i n the r e g u l a t i o n o f enzyme a c t i v i t y . The m i x t u r e o f t r i c a r b o x y l i c a c i d c y c l e a c i d s added t o t h e r e a c t i o n m i x t u r e i n e q u i m o l a r c o n c e n t r a t i o n d i d not cause a s t r i k i n g i n h i b i t i o n but i f g l y o x y l a t e (1 mM) was i n c l u d e d i n the r e a c t i o n m i x t u r e , the a c t i v i t y dropped s h a r p l y t o almo s t z e r o a t 1.5 mM c o n c e n t r a t i o n o f the m i x t u r e o f a c i d s ( F i g . 2 2 ) . S t r i k i n g i n h i b i t i o n was a l s o o b s e r v e d even when 0.1 mM g l y o x y l a t e was added t o the a s s a y m i x t u r e s . L i n e w e a v e r - B u r k p l o t ( F i g . 23) showed t h a t the Km.of the T a b l e XIV. I n h i b i t i o n o f i s o c i t r a t e dehydrogenase a c t i v i t y by v a r i o u s o r g a n i c a c i d s and r e l a t e d compounds. 0.2 mM D L - i s o c i t r a t e was used i n the a s s a y r e a c t i o n m i x t u r e . A d d i t i o n s C o n c e n t r a t i o n % I n h i b i t i o n * G l y o x y l a t e r e c o v e r e d (mM) C i t r a t e 10 57.7 C i s - a c o n i t a t e 10 66 . 7 a - K e t o g l u t a r a t e 10 60.0 S u c c i n a t e 10 46 . 7 Fumarate 10 33.3 M a l a t e 10 35.6 O x a l a c e t a t e 10 88.5 1.0 42 . 5 "»0.1 11.4 G l y o x y l a t e 10 -33.5 1 .0 -16 .7 0.1 n i l O x a l a c e t a t e + g l y o x y l a t e 10 each 100 1.0 each 100 0.1 each 91 . 3 C o n d e n s a t i o n p r o d u c t 10 100 1 .0 77.8 0.1 35.7 94.0 105.0 n i l n i l The c o n d e n s a t i o n p r o d u c t was p r e p a r e d by t h e method o u t l i n e d by S h i i o and Ozaki (1968). 99 O-O n r , g 0 2«0 4*0 6*0 CONCENTRATION( mM) F i g . 22. E f f e c t o f a m i x t u r e o f t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s on i s o c i t r a t e dehydrogenase a c t i v i t y . A s s a y s were c a r r i e d o u t i n 1.0 ml r e a c t i o n m i x t u r e c o n t a i n i n g 6.8 yg enzyme p r o t e i n and i n d i c a t e d c o n c e n t r a t i o n o f D - i s o c i t r a t e ( o - o ) ; an e q u i m o l a r m i x t u r e o f D - i s o c i t r a t e , c i t r a t e , cis-acon4tate, a - k e t o g l u t a r a t e , s u c c i n a t e , f u m a r a t e , L-malate and o x a l -a c e t a t e ( • - • ) . G l y o x y l a t e , 1 mM (• - •) o r 0.1 mM (•- •) was added t o t h e a s s a y m i x t u r e s c o n t a i n i n g t h e m i x t u r e o f o r g a n i c a c i d s . 100 IS] (mM DL-ISOCITRATE ) F i g . 23. Lineweaver-Burk p lot for i s o c i t r a t e dehydrogenase of P. aerug?hosa. 4-' enzyme f o r D - i s o c i t r a t e i s 2.3 x 10 m o l e / l i t e r , b. I s o c i t r a t e l y a s e a c t i v i t y S u c c i n a t e and g l y o x y l a t e p r e v i o u s l y were shown t o be non-c o m p e t i t i v e i n h i b i t o r s o f t h i s enzyme i n P. a e r u g i n o s a (Smith and G u n s a l u s , 1 9 57 ) . The p r e s e n t r e s u l t s show t h a t s u c c i n a t e was t h e most p o t e n t i n h i b i t o r o f a l l t h e compounds t r i e d . The c o n d e n s a t i o n p r o d u c t was not a s t r o n g i n h i b i t o r n o r was p h o s p h o e n o l p y r u v a t e , w h i c h i s known t o be a s t r o n g i n h i b i t o r o f t h i s enzyme from E. c o l i . A n o t h e r i m p o r t a n t o b s e r v a t i o n was t h a t o x a l a c e t a t e p l u s g l y o x y l a t e d i d not s t r o n g l y i n h i b i t t h i s enzyme. T h i s p a t t e r n i s a l s o seen i n F i g . 2k. The a c t i v i t y o f the enzyme i n c r e a s e d w i t h i n c r e a s i n g c o n c e n t r a t i o n o f D - i s o c i t r a t e u n t i l about 1.5 mM c o n c e n t r a t i o n , but when o t h e r 7 a c i d s o f t h e t r i c a r b o x y l i c a c i d c y c l e were a l s o i n c l u d e d i n the r e a c t i o n m i x t u r e i n e q u i m o l a r c o n c e n t r a t i o n , the i n h i b i t o r y a c t i o n o f t h e s e a c i d s was v e r y s t r o n g on.the enzyme and the a c t i v i t y remained low. I f 0.1 mM g l y o x y l a t e was added a l o n g w i t h t h e m i x t u r e o f a c i d s i n the r e a c t i o n m i x t u r e , i t d i d not f u r t h e r i n h i b i t t he a c t i v i t y . The Km o f the enzyme f o r D - i s o c i t r i e . a c i d was found t o be 2.5 x 10 ^ m o l e s / l i t e r ( F i g . 2 5 ) . Thus, the a f f i n i t y o f i s o c i t r a t e l y a s e f o r D - i s o c i t r i c a c i d i s about one t e n t h t h a t o f i s o c i t r a t e dehydrogenase. 102 T a b l e XV. I n h i b i t i o n o f i s o c i t r a t e l y a s e a c t i v i t y by o r g a n i c a c i d s and r e l a t e d compounds. - 3.0 mM D L - i s o c i t r a t e was used i n t h e a s s a y r e a c t i o n m i x t u r e s . A d d i t i o n s C o n c e n t r a t i o n .% I n h i b i t i o n (mM) C i t r a t e 10 13.5 C i s - a c o n i t a t e 10 42 . 4 a - K e t o g l u t a r a t e 10 57.0 S u c c i n a t e 10 81 .0 Fumarate 10 16.0 M a l a t e 10 39.0 O x a l a c e t a t e 10 55.0 G l y o x y l a t e 0 .5 40.0 0.1 31.0 O x a l a c e t a t e p l u s g l y o x y l a t e 0 .5 each 32.0 C o n d e n s a t i o n p r o d u c t o f g l y o x y l a t e and o x a l a c e t a t e 1.0 16.0 P h o s p h o e n o l p y r u v a t e 1.0 5.0 103 l - 5 p 6 w O E w 0 - 5 u < o. 1-0 2 - 0 CONCENTRATION (mM) 3 - 0 F i g . " 2 4 . E f f e c t o f a m i x t u r e o f t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s on i s o c i t r a t e l y a s e a c t i v i t y . A s s a y s were c a r r i e d o u t i n 2 . 0 ml r e a c t i o n m i x t u r e s c o n t a i n i n g 48 y g o f enzyme p r o t e i n and i n d i c a t e d c o n c e n t r a t i o n o f D - i s o c i t r a t e (O) o r an e q u i m o l a r m i x t u r e o f D - i s o c i t r a t e , c i t r a t e , c i s - a c o n i t a t e , a - k e t o g l u t a r a t e , s u c c i n a t e , f u m a r a t e , m a l a t e and o x a l a c e t a t e (•). 0 . 1 mM g l y o x y l a t e was added t o t h e a s s a y m i x t u r e c o n t a i n i n g t h e m i x t u r e o f o r g a n i c a c i d s , when i n d i c a t e d (•). A f t e r s t o p p i n g t h e r e a c t i o n , t h e m i x t u r e was d i l u t e d t e n t i m e s b e f o r e c o l o r d e v e l o p m e n t t o m i n i m i z e t h e i n t e r f e r e n c e due t o t h e added o r g a n i c a c i d s . .104 c. Aeon i t a s e a c t i v i t y A c o n i t a s e i n h i b i t i o n w i t h g l y o x y l a t e p l u s o x a l a c e t a t e and w i t h c o n d e n s a t i o n p r o d u c t was a l s o t e s t e d t o see i f t h i s c o u l d be i m p o r t a n t i n t h e r e g u l a t i o n o f t r i c a r b o x y l i c a c i d c y c l e a c t i v i t y . T h i s enzyme was found t o be v e r y s e n s i t i v e t o i n h i b i t i o n by the c o n d e n s a t i o n p r o d u c t ( T a b l e X V I ) . However, as shown p r e v i o u s l y the condensed p r o d u c t i s not formed under p h y s i o l o g i c a l c o n d i t i o n s and i s u n i m p o r t a n t . The c o n c e r t e d i n h i b i t i o n w i t h o x a l a c e t a t e p l u s g l y o x y l a t e was not so pronounced on a c o n i t a s e and i t was c o n c l u d e d t h a t t h i s enzyme i s not i m p o r t a n t i n the r e g u l a t i o n o f the a c t i v i t y o f t r i c a r b o x y l i c a c i d and g l y o x y l a t e c y c l e . I n t e r p r e t a t i o n o f t h i s s t u d y on i s o c i t r a t e d ehydrogenase, i s o c i t r a t e l y a s e and a c o n i t a s e a c t i v i t y i n P. a e r u g i n o s a b r i n g s out a p a t t e r n o f c o n t r o l o f t r i c a r b o x y l i c a c i d c y c l e and g l y o x y l a t e c y c l e w h i c h i s s i m i l a r t o t h a t d e s c r i b e d i n B r e v i b a c t e r i u m f l a v u m . At low l e v e l s o f t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s t h e i s o c i t r a t e formed from a c e t a t e must e n t e r t h e g l y o x y l a t e c y c l e i n o r d e r t h a t the organisms c o u l d grow on t h i s c a r b o n s o u r c e . The i s o c i t r a t e l y a s e i s induced by growth i n a c e t a t e medium (Tabl e X ) . The g l y o x y l a t e formed as a r e s u l t o f i s o c i t r a t e l y a s e a c t i v i t y , i n h i b i t s i s o c i t r a t e dehydrogenase a c t i v i t y c o n c e r t e d l y w i t h o x a l -106 T a b l e X V I . A c o n i t a s e i n h i b i t i o n i n c e l l - f r e e e x t r a c t o f P_. a e r u g i n o s a . Add! t i o n s C o n c e n t r a t i o n ; % I n h i b i t i o n O x a l a c e t i c a c i d 0.1 17-5 1.0 30.0 G l y o x y l i c a c i d 0.1 14.0 1.0 30.0 O x a l a c e t a t e p i us g l y o x y l a t e 0.1 each 14.0 1 .0 each 41.8 Condensat i o n p r o d u c t 0.1 100 1 .0 100 a c e t i c a c i d . Small amounts of oxalacetate are presumably always present in the c e l l . However, even when i s o c i t r a t e dehydrogenase a c t i v i t y is weak, some i s o c i t r a t e enters the t r i c a r b o x y l i c a c i d c y c l e , because of the high a f f i n i t y of i s o c i t r a t e dehydrogenase for t h i s s u b s t r a t e , thus ensuring a supply of a - k e t o g l u t a r a t e . When large amounts of organic acids have accumulated due to the high a c t i v i t y of the g lyoxyla te c y c l e or when these t r i c a r b o x y l i c a c i d cyc le members serve as the carbon source in the medium, they suppress the a c t i v i t y of i s o c i t r a t e lyase more e f f e c t i v e l y than they do the i s o c i t r a t e dehydrogenase a c t i v i t y ( F i g s . 22 and 2k). This r e s u l t s in a lowering of glyoxyl'ate production and thus i s o c i t r a t e dehydrogenase is released from the i n h i b i t i o n . The complete t r i c a r b o x y l i c a c i d c y c l e now functions and the g lyoxyla te cycle a c t i v i t y remains suppressed. GENERAL DISCUSSION Pseudomonas a e r u g i nosa l i k e o t h e r members o f t he genus Pseudomonas . u t i l i z e s a w i d e v a r i e t y o f compounds f o r g r o w t h . I t w o u l d be a d i s a d v a n t a g e t o t h e c e l l t o m a i n t a i n a t a l l t i m e s t h e enzymes f o r the d e g r a d a t i o n o f t h i s l a r g e number o f compounds and hence b a c t e r i a have d e v e l o p e d a s y s t em by w h i c h t h e y can i n d u c t i v e l y s y n t h e s i z e enzymes when r e q u i r e d . Thu s , t he i n t e r m e d i a t e compounds n e c e s s a r y f o r t h e s y n t h e s i s o f e s s e n t i a l c e l l m a t e r i a l a r e no t d i v e r t e d toward s t h e s y n t h e s i s o f u n n e c e s s a r y enzymes . However, t h e r e a r e o t h e r enzymes , i . e . t h e c o n s t i t u t i v e enzymes , w h i c h a r e p roduced by t he o r g a n i s m a t r e l a t i v e l y c o n s t a n t l e v e l s i ndependen t o f n u t r i t i o n a l c o n d i t i o n s . The phenomenon o f enzyme i n d u c t i o n has been e x t e n s i v e l y s t u d i e d but v e r y l i t t l e a t t e n t i o n has been g i v e n t o t h e s t u d y o f c o n s t i t u t i v e enzymes. I t has been p ropo sed t h a t the i n a b i l i t y o f an o r g a n i s m t o c o n t r o l t h e s y n t h e s i s , i . e . c o n s t i t u t i v i t y o f c e r t a i n enzymes , g i v e s i t a s e l e c t i o n a l advan tage o v e r i t s n e i g h b o u r s w h i c h po s se s s i n d u c i b l e c o u n t e r -p a r t s o f such enzymes (Pa rdee and B e c k w i t h , 1 9 6 3 ; Moses and S h a r p , 1 9 6 8 ) . The r e s u l t s o f t h i s i n v e s t i g a t i o n a r e i n agreement w i t h o t h e r r e p o r t s t h a t i n P. a e r u g i nosa t he enzymes o f g l u c o s e oxidation are inducible while those of the t r i c a r b o x y l i c acid cycle are const i tu t i ve (Von Tigerstrom and Campbell, 1966; Ng and Dawes, 1 9 6 7 ) . Lack of phosphofructokinase a c t i v i t y has been found to be the reason for a non-functional Embden-Meyerhof pathway in th is organism. When the c e l l s were grown in succinate medium,.the enzymes of glucose oxidation were e i ther at a low level or were absent. Repression of these enzymes, i . e . the enzymes of the Entner-Doudoroff pathway and the oxidat ive portion of the pentose phosphate pathway is a very important regulatory mechanism avai lab le to the c e l l when the organisms are grown on any t r i c a r b o x y l i c acid cycle intermediate. Under these conditions s u f f i c i e n t energy is avai lable from the t r i c a r b o x y l i c acid cycle and therefore, the breakdown of glucose and related compounds which are synthesized in l imited quant i t ies by the reversal of Embden-Meyerhof pathway reactions and are required for biosynthesis of s t ructura l components, is stopped. P. aeruginosa does not accumulate any special storage product which could serve as a readi ly ava i lab le source of hexose (MacKelvie, Campbell and Gronlund, 1968) and hence i t w i l l be a disadvantage for the organism to catabol ize a l1 of the avai lable hexoses in the c e l l . It has been reported that at low concentrations of g1ucose-6-phosphate, ATP strongly inh ib i ted the glucose-6-phosphate dehydrogenase of P . aeruginosa, while at high concentrations of th i s substrate the inh ib i t i on was ins ign i f i cant (Lessie and Neidhardt, 1 9 6 7 a ) . These o b s e r v a t i o n s c o u l d be m e a n i n g f u l l y r a t i o n a l i z e d i n . t h i s c o n t e x t s i n c e even a minor a c t i v i t y o f t h i s enzyme, p r e s e n t i n t he c e l l s grown i n s u c c i n a t e medium, may d e p l e t e the low l e v e l s o f g l u c o s e - 6 - p h o s p h a t e w h i c h may be d e r i v e d from s u c c i n a t e . Thus, s t r o n g i n h i b i t i o n o f g1ucose - 6-phosphate dehydrogenase by ATP a t low l e v e l s o f g1ucose - 6-phosphate e n s u r e s the s u p p l y o f t h i s compound f o r c e l l u l a r b i o s y n t h e s i s . When g l u c o s e - 6 - p h o s p h a t e i s p r e s e n t i n e x c e s s f o r example d u r i n g growth i n g l u c o s e medium, ATP i n h i b i t i o n i s r e l e a s e d and the enzyme i s p e r m i t t e d t o o x i d i z e t h e compound w i t h f a c i l i t y . The r e p r e s s i o n o f t h e s y n t h e s i s o f g l u c o s e o x i d a t i o n enzymes i n P_. a e r u g ? h o s a upon growth i n medium w i t h t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s i s i n c o n t r a s t t o t h e o b s e r v a t i o n s made w i t h B a c i 1 l u s s p e c i e s . In one r e p o r t where the s t u d y on the r e g u l a t i o n o f g1ucose - 6-phosphate dehydrogenase, 6-phosphogluconate dehydro-genase and h e x o k i n a s e was c a r r i e d out w i t h B. s u b t i 1 i s , i t was found t h a t t h e l e v e l s o f t h e s e t h r e e enzymes i n c e l l s grown on s u c c i n a t e o r g l u t a m a t e medium d i d not d e c r e a s e . T h e r e f o r e , t h e s e c e l l s were c a p a b l e o f m e t a b o l i z i n g g l u c o s e a t r a t e s comparable t o t h o s e o f c e l l s grown i n a g l u c o s e medium (Moses and Shar p , 1968). As a m a t t e r o f f a c t t h e organisms grown i n a s u c c i n a t e medium showed more t h a n t h r e e t i m e s the r a t e o f s y n t h e s i s o f h e x o k i n a s e than organisms grown i n a g l u c o s e medium. T h i s i s c o n t r a r y vto what would be p r e d i c t e d s i n c e g l u c o s e would be e x p e c t e d t o be the i n d u c e r o f t h e enzyme. No e x p l a n a t i o n was o f f e r e d f o r t h i s b e h a v i o u r o f a s e e m i n g l y c o n s t i t u t i v e enzyme. P a r t i c u l a t e m a l i c dehydrogenase o f P. a e r u g i n o s a has been found t o behave s i m i l a r l y , i n t h a t i t showed h i g h e r r a t e s o f s y n t h e s i s i n g l u c o s e medium r a t h e r t h a n i n t h e p r e s e n c e o f a l i k e l y i n d u c e r , namely s u c c i n a t e ( T a b l e IX, F i g . 2 0 ) . A l t h o u g h the r e s u l t s o f Moses and Sharp were not c o n c l u s i v e , i t appeared t h a t g l u c o s e m e t a b o l i z i n g enzymes a r e c o n s t i t u t i v e i n B. s u b t i 1 i s . A l t h o u g h no such s t u d y has been made on g l u c o s e o x i d a t i o n enzymes i n c o l i f o r m s , they appear t o resemble b a c i l l i i n t h i s c o n t r o l mechanism s i n c e A_. aerogenes c e l l s grown i i i c i t r a t e medium have been shown t o o x i d i z e g l u c o s e w i t h o u t / a l a g (Dagley and Dawes, 1 953 ) . Indeed, g l u c o k i n a s e has been c o n c l u d e d t o be a c o n s t i t u t i v e enzyme i i i A. aerogenes (Kamel, A l l i s o n and An d e r s o n , 1 966 ) . The r e s u l t s p r e s e n t e d here a l s o s u p p o r t the s u g g e s t i o n o f H o r e c k e r (1965) t h a t i n most i n s t a n c e s t h e pentose phosphate pathway does not f u n c t i o n as a c y c l e , but r a t h e r as two mechanisms f o r c e l l u l a r b i o s y n t h e s i s . The o x i d a t i v e p o r t i o n o f t h e pathway o p e r a t e s when NADPH i s r e q u i r e d by the c e l l w h i l e n o n - o x i d a t i v e t r a n s f o r m a t i o n s a r e u t i l i z e d when pe n t o s e phosphates a r e r e q u i r e d . I t i s o n l y when the r e q u i r e m e n t o f t h e c e l l f o r NADPH exceeds i t s r e q u i r e m e n t f o r the pen t o s e phosphates t h a t i t o p e r a t e s as a c y c l e t o r e t u r n e x c e s s p e n t o s e phosphates t o the m e t a b o l i c p o o l o f v c a r b o h y d r a t e s . I t would a p p e a r , t h e r e f o r e , t h a t t h e demand f o r NADPH o f t h e c e l l s o f P. a e r u g i n o s a grown i n s u c c i n a t e medium i s met by the r e a c t i o n s o f t h e t r i c a r b o x y l i c a c i d c y c l e and hence, t h e o x i d a t i v e p o r t i o n s o f the pe n t o s e phosphate c y c l e i s u n n e c e s s a r y and does not o p e r a t e . For the same r e a s o n s , i t s c o n t r i b u t i o n t o pe n t o s e s y n t h e s i s i n the c e l l s grown i n g l u c o s e medium may a l s o be low i n pseudomonads. The a n a l y s i s o f the r e s u l t s a l s o i n d i c a t e s t h a t d u r i n g growth i n s u c c i n a t e medium the demand f o r p e n t o s e phosphate must be met by the t r a n s k e t o l a s e r e a c t i o n between compounds whi c h c o u l d be d e r i v e d from t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s . T h i s f i n d i n g i s i n agreement w i t h t h e p r o p o s a l s made by s e v e r a l o t h e r w o r k e r s (DeLey, 1960; S a b l e , 1966; L e s s i e and N e i d h a r d t , 1967a). F a c u l t a t i v e b a c t e r i a and some s p e c i e s o f a e r o b i c s p o r e f o r m i n g b a c i l l i w h i c h can l i v e and grow w i t h o u t the a c t i v i t y o f the t r i c a r b o x y l i c a c i d c y c l e under c e r t a i n c o n d i t i o n s have been found t o show a s t r i k i n g g l u c o s e e f f e c t on t h e enzymes o f t h i s c y c l e (Col 1 i n s and L a s c e l l e s , 1962; Gray e _ t a l _ , 1966b; Hanson e t a l _ , 1963a; 1963b; 1964; Hanson and Cox, 1967). On t h e o t h e r hand, the g l u c o s e e f f e c t on t h e t r i c a r b o x y l i c a c i d c y c l e enzymes i s not seen i n P. a e r u g i n o s a , i n d i c a t i n g t h a t t h e a c t i v i t y o f t h i s c y c l e i s e s s e n t i a l f o r growth on g l u c o s e . The a d d i t i o n o f g l u t a m a t e and a - k e t o g l u t a r a t e t o t h e growth medium d i d not r e p r e s s t h e s y n t h e s i s o f the enzymes o f t h e t r i c a r b o x y l i c a c i d c y c l e , l e a d i n g t o a - k e t o g l u t a r a t e s y n t h e s i s . In a d d i t i o n , growth i n a < s u c c i n a t e medium r e p r e s s e d t h e s y n t h e s i s o f e n z y m e s o f g l u c o s e o x i d a t i o n a s a l r e a d y d i s c u s s e d a b o v e . T h u s , t h e t r i c a r b o x y l i c a c i d c y c l e f u l f i l s t h e m a j o r m e t a b o l i c a n d b i o s y n t h e t i c n e e d s an d t h e r e f o r e , i s o f s p e c i a l i m p o r t a n c e t o t h e s e b a c t e r i a . S u c c i n a t e i s known t o r e p r e s s h i s t i d i n e d e g r a d a t i o n more e f f e c t i v e l y t h a n d o e s g l u c o s e i n P. a e r u g i n o s a ( L e s s i e a n d N e i d h a r d t , 1967b). In P. a c i d o v o r a n s s u c c i n a t e h a s r e c e n t l y b e e n shown t o p r e v e n t t h e i n d u c t i o n o f t r y p t o p h a n o x y g e n a s e ( R o s e n f e l d a n d F e i g e l s o n , 1969). T h u s , i n p s e u d o m o n a d s , w h i c h p o s s e s s c o n s t i t u t i v e t r i c a r b o x y l i c a c i d c y c l e a c t i v i t y , s u c c i n a t e , a c o m p o n e n t o f t h i s c y c l e a p p e a r s t o e x e r t c a t a b o l i t e r e p r e s s i o n o n e n z y m e s , t h e a c t i v i t i e s o f w h i c h g i v e r i s e t o more o f t h e c y c l e i n t e r m e d i a t e s s i n c e t h e y a r e n o t r e q u i r e d . W h e r e a s i n E_. c o l i a n d A. a e r o g e n e s w h i c h p o s s e s s a c t i v e E m b d e n - M e y e r h o f p a t h w a y s and u t i l i z e g l u c o s e as t h e s o l e s o u r c e o f c a r b o n a n d e n e r g y f o r g r o w t h e v e n i n t h e a b s e n c e o f c o m p l e t e t r i c a r b o x y l i c a c i d c y c l e , g l u c o s e h a s b e e n shown t o l e a d t o e f f e c t i v e c a t a b o l i t e r e p r e s s i o n o n v a r i o u s d e g r a d a t i v e p a t h w a y s ( M a g a s a n i k , 1961). T h e s e f a c t s a g a i n s u g g e s t t h e d i f f e r e n c e i n m e t a b o l i c c o n t r o l p a t t e r n i n t h e t w o g r o u p s o f o r g a n i s m s . The s t u d y o f t h e i n d u c t i o n o f t h e s y s t e m s f o r g l u c o s e t r a n s p o r t a n d o x i d a t i o n i n P. a e r u g i n o s a g r o w n i n s u c c i n a t e medium s u g g e s t e d t h a t b o t h t h e i n d u c t i o n o f g l u c o s e m e t a b o l i z i n g e n z y m e s a n d t h e - g l u c o s e p e r m e a s e w e r e r e s p o n s i b l e f o r t h e l o n g l a g p e r i o d s r e q u i r e d b e f o r e c e l l s d e v e l o p e d an a c t i v e system f o r g l u c o s e u t i l i z a t i o n . The f i n d i n g t h a t g j u c o s e permease i s i n d u c i b l e i n t h i s o r g a n i s m i s unique s i n c e i n E. c O l i a - m e t h y l - g l u c o s i d e permease w h i c h i s a l s o r e s p o n s i b l e f o r g l u c o s e u p t a k e has been shown t o be c o n s t i t u t i v e (Cohen and Monod, 1 957 ) . Many o r g a n i s m s , i n c l u d i n g a n i m a l s and p l a n t s , p o s s e s s b o t h a p a r t i c u l a t e m i t o c h o n d r i a l and a s o l u b l e c y t o p l a s m i c m a l i c dehydrogenase. In y e a s t , i t has been shown t h a t t h e s o l u b l e m a l i c dehydrogenase, which i s r e q u i r e d f o r g l u c o n e o g e n e s i s from a c e t a t e , i s r e p r e s s e d by g l u c o s e w h i l e t h e m i t o c h o n d r i a l enzyme n e c e s s a r y f o r c a t a b o l i s m v i a t h e t r i c a r b o x y l i c a c i d c y c l e i s not i n f l u e n c e d by growth i n a g l u c o s e medium ( H o i z e r , 1 966 ) . I t i s t h e m i t o c h o n d r i a l enzyme o f p i g h e a r t , peas and o t h e r p l a n t s t h a t has been shown t o be i n f l u e n c e d by aden i n e n u c l e o t i d e s w h i l e the s o l u b l e enzyme i s u n a f f e c t e d ( K u r a m i t s u , 1966; 1968). T h e r e f o r e , t h e p a r t i c u l a t e m a l i c dehydrogenase o f P. a e r u g i n o s a resembles t h e m i t o c h o n d r i a l enzyme o f h i g h e r o rganisms i n i t s c o n t r o l p a t t e r n . I t has been found t h a t the i s o c i t r a t e dehydrogenase o f P_. a e r u g i n o s a i s NADP s p e c i f i c and shows no a c t i v i t y w i t h NAD. G l u t a m i c dehydrogenase i n the d i r e c t i o n o f a-ketog 1 u t a r a t e f o r m a t i o n shows a c t i v i t y o n l y w i t h NADP and NADPH i s the major coenzyme u t i l i z e d i n t h e d i r e c t i o n o f g l u t a m a t e s y n t h e s i s . M a l i c dehydrogenase i s p a r t i c u l a t e and has e l i m i n a t e d the need f o r f r e e NAD, r e q u i r e d by o t h e r b a c t e r i a a t t h i s s t e p . T h e r e f o r e , a l t h o u g h t h i s o r g a n i s m r e l i e s on NADPH f o r a n a b o l i c r e a c t i o n s , i t does not r e l y on f r e e NAD f o r many c a t a b o l i c r e a c t i o n s . The o n l y s t e p s where t h e o r g a n i s m o b v i o u s l y needs NAD a r e p y r u v a t e dehydrogenase and a - k e t o g l u t a r a t e dehydrogenase c a t a l y z e d r e a c t i o n s where t h i s coenzyme i s c a r r i e d as apoenzyme i n a complex. P_. a e r u g i nosa p o s s e s s e s a c t i v e t r a n s h y d r o g e r i a s e (Eagon, 1963; Von T i g e r s t r o m and C a m p b e l l , 1966b ) . O t h e r a e r o b i c o rganisms p o s s e s s i n g t r a n s -h y d r o g e n a s e , e.g. P. f 1 u o r e s c e n s and A. v i he1 aridi i a r e known t o pos s e s s o n l y NADP l i n k e d i s o c i t r a t e dehydrogenase ( K a p l a n , T '963)'. Thus, t h e r e seems t o be a g e n e r a l p a t t e r n i n the a e r o b i c o r g a n i s m s p o s s e s s i n g a c t i v e t r a n s h y d r o g e n a s e i n t h a t t h e y a r e l e s s dependent on f r e e NAD f o r t h e m e t a b o l i c r e a c t i o n s and NADH f o r the purpose o f energy g e n e r a t i o n c o u l d be d e r i v e d from e x c e s s NADPH t h r o u g h the t r a n s h y d r o g e n a s e r e a c t i o n . T h i s p r o p o s a l a g rees w i t h t h e p o s t u l a t e d r o l e s o f NADH whi c h i s r a p i d l y o x i d i z e d by t h e oxygen w i t h t h e f o r m a t i o n o f h i g h energy phosphate and o f NADPH w h i c h i s not r e a d i l y o x i d i z e d by m o l e c u l a r oxygen and i s thus a v a i l a b l e f o r b i o s y n t h e t i c r e a c t i o n s ( H o r e c k e r , 1965; K a p l a n , 1963). Pseudomonads a r e known f o r t h e i r g r e a t c a p a c i t y o f b i o s y n t h e s i s and i t i s an advantage t o the c e l l t o produce a pool o f NADPH r a t h e r than r a p i d l y o x i d i z a b l e NADH f o r t h i s p urpose. A l t h o u g h P_. a e r u g i n o s a appears t o have c o n s t i t u t i v e t r i -c a r b o x y l i c a c i d c y c l e a c t i v i t y , t h e c o n t r o l on key s t e p s i s - e x e r t e d by m e t a b o l i t e s t o r e g u l a t e the f l o w o f compounds t h r o u g h the c y c l e . P a r t i c u l a t e m a l i c dehydrogenase w h i c h i s i n h i b i t e d by a d e n i n e n u c l e o t i d e s and a c t i v a t e d by GTP and GDP p r o v i d e s a mechanism by w h i c h the c e l l c o u l d c o n t r o l t h e t r i c a r b o x y l i c a c i d c y c l e a c t i v i t y . S i m i l a r l y , i s o c i t r a t e dehydrogenase and i s o c i t r a t e l y a s e a c t i v i t i e s a r e c o n t r o l l e d by t h e members o f t h e t r i c a r b o x y l i c a c i d c y c l e and t h e g l y o x y l a t e c y c l e i n ; s u c h a way t h a t t h e f l o w o f compounds t h r o u g h t h e s e c y c l e s does not proceed i n a haphazard manner, but i s r e g u l a t e d i n a p r e c i s e f a s h i o n t o b e s t s u i t the needs o f t h e o r g a n i s m . The l a c k ' o f . s i m i l a r i t i e s i n the c o n t r o l o f t r i c a r b o x y l i c a c i d c y c l e a c t i v i t y and t h e a c t i v i t y o f g l u c o s e o x i d a t i o n enzymes between P. a e r u g i n o s a and e n t e r i c and a e r o b i c s p o r e f o r m i n g b a c t e r i a s u g g e s t s t h a t d u r i n g t h e p r o c e s s o f e v o l u t i o n the o r g anisms have d e v e l o p e d c h a r a c t e r i s t i c m e t a b o l i c and c o n t r o l mechanisms s u i t i n g t h e environment w h i c h t h e y found t h e m s e l v e s i n . BIBLIOGRAPHY A d e l b e r g , E.A., Mandel , M. and Chen, G .C .C. 1965. O p t i m a l c o n d i t i o n s f o r m u t a genesis by N-methy1-N 1-ni t r o - N - n i t r o s o g u a n i d i ne ?n E s c h e r i ch i a c o l i K l 2 . Biochem. B i o p h y s . Res. Commun. : J_8_: 7 8 8 - 7 9 5 . Aj 1, S . J . 1950. 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