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Regulation of carbohydrate-catabolizing enzymes in Pseudomonas aeruginosa ATCC 9027 Lynch, William Henry Walter 1973

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REGULATION OF CARBOHYDRATE-CATABOLIZING ENZYMES IN PSEUDOMONAS AERUGINOSA ATCC 9027 by WILLIAM HENRY WALTER LYNCH B.Sc. U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in t h e Department o f MICROBIOLOGY 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 to t he r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A u g u s t , 1973 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of M i c r o b i o l o g y  The University of British Columbia Vancouver 8 , Canada i ! ABSTRACT A l t h o u g h g rowth w i t h s i x - c a r b o n c a r b o h y d r a t e s , as w e l l as w i t h g l y c e r o l o r g l y c e r a t e , has been shown to i nduce the enzymes o f g l u c o s e c a t a b o l i s m i n Pseudomonas a e r u g i n o s a , t he ac tua1 i n d u c e r ( s ) o f t h e s e enzymes has not been i d e n t i f i e d . Low l e v e l s o f t h e s e enzymes a r e p r e s e n t d u r i n g g rowth w i t h a c e t a t e , p y r u v a t e , o r 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 . By e x a m i n i n g t he i n d u c t i o n o f g 1 u c o s e - c a t a b o 1 i z i n g enzymes i n t h e w i l d - t y p e and g l u c o s e - n e g a t i v e mutant s o f P_. a e r u g i n o s a ATCC 9027 by g l u c o s e , g l u c o n a t e , 2 - k e t o g l u c o n a t e , o r g l y c e r o l , t h e c o m p l e x i t y o f t h e r e g u l a t i o n o f t h e s e enzymes was d e m o n s t r a t e d , _i_.e_. o f the seven enzymes examined i n d e t a i l , no l e s s t han f o u r i n d u c t i v e e v e n t s were r e q u i r e d t o cau se t h e i r s y n t h e s i s . G l u c o s e dehyd rogena se was i nduced by g l u c o s e - 6 -pho spha te (and p o s s i b l y g l u c o s e ) ; g l u c o n a t e dehyd rogena se by 2 - k e t o -g l u c o n a t e ; g l u c o k i n a s e , and g l u c o s e - 6 - p h o s p h a t e dehyd rogena se by 6 -pho spho -g l u c o n a t e ; and t he E n t n e r - D o u d o r o f f enzymes by 6 - p h o s p h o g l u c o n a t e o r some d e r i v a t i v e t h e r e o f . G l u c o n o k i n a s e and 3 _ p h o s p h o g 1 y c e r a 1 d e h y d e d e h y d r o g e n -a se were i nduced by g l u c o n a t e . However, a weak i n d u c t i o n o f bo th g l u c o n o -k i n a s e ( p o s s i b l y by 2 - k e t o g l u c o n a t e ) and 3 - p h o s p h o g l y c e r a l d e h y d e d e h y d r o -genase ( p o s s i b l y by 6 -pho sphog1ucona te ) was a l s o o b s e r v e d and d i d not appea r t o i n v o l v e g l u c o n a t e . G l y c e r o k i n a s e and L - a - g l y c e r o p h o s p h a t e d e -hyd rogena se were s p e c i f i c a l l y i nduced by g l y c e r o l and not by e i t h e r p y r u v a t e o r g l u c o s e m e t a b o l i s m . The i m p o r t a n c e o f t he o x i d a t i v e n o n - p h o s p h o r y l a t e d pathway f o r g l u c o s e o r g l u c o n a t e d i s s i m i l a t i o n i n JP. a e r u g i n o s a was d e m o n s t r a t e d . D u r i n g g rowth o f t h i s o r g a n i s m w i t h g l u c o s e o r g l u c o n a t e , t he m a j o r i t y o f bo t h s u b s t r a t e s was r a p i d l y o x i d i z e d t o 2 - k e t o g l u c o n a t e p r i o r t o b e i n g p h o s p h o r y l a t e d and f u r t h e r c a t a b o l i z e d by t he E n t n e r - D o u d o r o f f pathway enzymes. T h e r e f o r e , the ma jo r amount o f g r o w t h , when e i t h e r g l u c o s e o r g l u c o n a t e was added as t he c a r b o n s o u r c e , a c t u a l l y t o o k p l a c e w i t h 2 - k e t o g l u c o n a t e s e r v i n g as t h e s o u r c e o f c a r b o n . The mutant s o f P_. a e r u g i n o s a , i s o l a t e d f o r t h e i r i n a b i l i t y t o g r ow : w i t h g l u c o s e as a c a r b o n s o u r c e , were a l s o i n c a p a b l e o f g rowth when e i t h e r g l u c o n a t e o r 2 - ke tog1uconate was the s o l e s o u r c e o f c a r b o n . Growth o f t he mutant s w i t h p y r u v a t e , a c e t a t e , o r 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 i n h i b i t e d by t he p r e s e n c e o f 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 . The i n h i b i t i o n o f g rowth o f the g l u c o s e - n e g a t i v e mutant s o f P_. a e r u g i n o s a w i t h p y r u v a t e was due t o an a c c u m u l a t i o n o f h i g h i n t e r n a l c o n c e n t r a t i o n s o f 6 - p h o s p h o g l u c o n a t e f rom the m e t a b o l i s m o f e i t h e r 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 . T h i s a c c u m u l a t i o n o f 6 - p h o s p h o g l u c o n a t e cau sed a r e p r e s s i o n ( e i t h e r d i r e c t o r i n d i r e c t ) o f p y r u v i c dehyd rogena se s y n t h e s i s and c o n s e q u e n t l y r e s u l t e d i n g rowth s t a s i s . The e x a m i n a t i o n o f t h e s e mutant s a l s o i n d i c a t e d t h a t 6 - p h o s p h o g l u c o n a t e dehyd rogena se was ab s en t and a 6 - p h o s p h o g l u c o n a t e p h o s p h a t a s e was p r e s e n t i n t h i s s t r a i n o f P_. a e r u g i n o s a . i V TABLE OF CONTENTS PAGE INTRODUCTION ' LITERATURE REVIEW 3 I. Pathways f o r the d i s s i m i l a t i o n o f g l u c o s e i n Pseudomonas s p e c i e s 3 I I . R e g u l a t i o n o f g l u c o s e c a t a b o l i s m ^ I I I . The i n d u c t i o n o f c a r b o h y d r a t e - c a t a b o l i z i n g enzymes i n Pseudomonas a e r u g i n o s a  IV. The r o l e o f t he o x i d a t i v e n o n - p h o s p h o r y l a t e d pathway i n g l u c o s e d i s s i m i l a t i o n i n P_. a e r u g i n o s a . . . . . . . . 9 V. Othe r c a t a b o l i c pathways i n Pseudomonas s p e c i e s . . . . 10 V I . C a t a b o l i c mutan t s 11 MATERIALS AND METHODS I . Organ i sms 13 ! I I . Growth and Med ia 1 I I I . 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 15 IV. Enzyme a s s a y s 16 1. G l u c o s e - c a t a b o l i z i n g enzymes 16 2. G l y c o l y t i c enzymes 17 3. TCA c y c l e enzymes 18 k. G l y c e r o l enzymes 19 V. I s o l a t i o n o f p r o d u c t s o f g l u c o s e , g l u c o n a t e , 2-KG and g l y c e r o l m e t a b o l i s m 20 V PAGE 1. I s o l a t i o n o f p r o d u c t s o f g l u c o s e , g l u c o n a t e , and 2-KG m e t a b o l i s m f rom c u l t u r e s o f g l u c o s e -n e g a t i v e mutant s 20 14 2. S e p a r a t i o n o f p r o d u c t s o f g l u c o s e - U - C c a t a b o l i s m i n t he c e l l w a t e r and medium o f g l u c o s e - n e g a t i v e mutant s 22 14 3- I s o l a t i o n o f p r o d u c t s o f g l y c e r o l - U - C m e t a b o l i s m i n t he w i l d - t y p e and g l u c o s e -n e g a t i v e mutant X l l l j _ ^ 22 14 4. I s o l a t i o n o f p r o d u c t s o f g l u c o s e - U - C m e t a b o l i s m i n t h e p a r e n t s t r a i n d u r i n g g r owth w i t h g l u c o s e 23 V I . I d e n t i f i c a t i o n o f p r o d u c t s i s o l a t e d by i o n - e x c h a n g e ch romatog raphy 23 1 2 V I I . I d e n t i f i c a t i o n o f C - p r o d u c t s i n the g rowth medium . . 25 V I I I . C h e m i c a l s 26 RESULTS AND DISCUSSION I. C h a r a c t e r i z a t i o n o f g l u c o s e - n e g a t i v e mutan t s o f P_. a e r u g i nosa 28 1. Growth o f g l u c o s e - n e g a t i v e mutant s 28 2. I d e n t i f i c a t i o n o f the p r o d u c t s o f g l u c o s e c a t a b o l i s m i n g l u c o s e - n e g a t i v e mutant s 33 PAGE 3- E x a m i n a t i o n o f g l u c o s e - c a t a b o l i z i n g enzymes in g l u c o s e - n e g a t i v e mutant s 38 11. The i n h i b i t o r y e f f e c t o f g l u c o s e i n g l u c o s e - n e g a t i v e mutants hi 1. P r o d u c t s o f g l u c o s e - U - C c a t a b o l i s m i n t he c e l l w a t e r and medium o f g l u c o s e - n e g a t i v e mutan t s . . ^8 2. A s s ay o f TCA c y c l e and g l y c o l y t i c enzymes. . . . 52 3- E f f e c t o f g l u c o s e on p y r u v a t e d i s a p p e a r a n c e w i t h g l u c o s e - n e g a t i v e mutant s 58 k. E f f e c t o f 6-PG on p y r u v i c dehyd rogena se a c t i v i t y 61 I I I . A n a l y s i s o f o t h e r g l u c o s e - n e g a t i v e mutant s o f P_. ae rug 1 nosa . . 63 1. I d e n t i f i c a t i o n o f p r o d u c t s o f g l u c o s e , g l u c o n a t e , o r 2-KG m e t a b o l i s m i n the mutant s t r a i n s . . . . 63 2. E x a m i n a t i o n o f g l u c o s e - c a t a b o l i z i n g enzymes. . . 67 IV. G l u c o s e and g l u c o n a t e dehyd rogena se s and t h e i r r o l e i n g l u c o s e d e g r a d a t i o n by P_. a e r u g i nosa 75 1. A c c u m u l a t i o n o f p r o d u c t s f r om g l u c o s e , g l u c o n a t e , o r 2-KG d e g r a d a t i o n . . . . 76 2. L e v e l s o f g l u c o s e and g l u c o n a t e dehyd rogena se s d u r i n g g rowth 82 PAGE 3. I n d u c t i o n o f g l u c o s e and g l u c o n a t e d e h y d r o -genases by g l u c o s e , g l u c o n a t e , and 2-KG 85 V. R e g u l a t i o n o f c a r b o h y d r a t e - c a t a b o l i z i n g enzymes i n P_. a e r u g i nosa by g l u c o s e , g l u c o n a t e , and 2-KG 88 1. L e v e l s o f c a r b o h y d r a t e - c a t a b o l i z i n g enzymes p r e s e n t d u r i n g g r owth o f P_. a e r u g i n o s a w i t h g l u c o s e , g l u c o n a t e , and 2-KG 88 2. I n d u c t i o n o f c a r b o h y d r a t e - c a t a b o l i z i n g enzymes by g l u c o s e , g l u c o n a t e , and 2-KG i n P_. a e r u g i n o s a 93 V I . The r e g u l a t i o n o f c a r b o h y d r a t e - c a t a b o l i z i n g enzymes i n P_. a e r u g i n o s a by g l y c e r o l 99 1. Growth o f w i l d - t y p e c e l l s 101 2. L e v e l s o f c a r b o h y d r a t e - c a t a b o l i z i n g enzymes d u r i n g g rowth w i t h g l y c e r o l 101 3. I n d u c t i o n o f c a r b o h y d r a t e - c a t a b o 1 i z i n g enzymes by g l y c e r o l 106 1 k 4. F a t e o f u n i f o r m l y l a b e l l e d C - g l y c e r o l d u r i n g m e t a b o l i s m by w i l d - t y p e P_. a e r u g i n o s a and g l u c o s e -n e g a t i v e mutant X l l l j _ ^ 112 V I I . R e g u l a t i o n o f the i n d u c t i o n o f c a r b o h y d r a t e - c a t a b o l i z i n g enzymes as e v i d e n c e d by the p a t t e r n s o f enzyme i n d u c t i o n and p r o d u c t a c c u m u l a t i o n f r om g l y c e r o l m e t a b o l i s m . . . . 117 v i i i PAGE 1. G l u c o k i n a s e , G-6-P d e h y d r o g e n a s e , and t h e enzymes o f t he ED pathway 118 2. G l u c o n o k i n a s e and g l u c o n a t e dehyd rogena se . . . . 118 3- G e n e r a t i o n t i m e and amounts o f 3~PG and g l u c o s e dehyd rogena se s 119 4. I n d u c t i o n o f 3"PG dehyd rogena se 123 5. Pathways o f g l y c e r o l d i s s i m i l a t i o n 124 GENERAL DISCUSSION 128 BIBLIOGRAPHY 136 APPENDIX 146 I. C o n t r i b u t i o n s o f t h i s t h e s i s t o t he u n d e r s t a n d i n g o f t h e r e g u l a t i o n o f c a r b o h y d r a t e c a t a b o l i sm i n P_. ae rug i nosa (ATCC 9027) 146 1. The o x i d a t i v e n o n - p h o s p h o r y l a t e d pathway 146 2. I n d u c t i o n o f g l u c o s e - c a t a b o l i z i n g enzymes . . . . 146 3. S i m i l a r i t i e s i n the r e g u l a t i o n o f c e r t a i n g l u c o s e -c a t a b o l i z i n g enzymes 147 4. R e g u l a t i o n o f g 1 u c o s e - c a t a b o l z i n g enzymes by g l y c e r o l 148 5- G 1 y c e r o l - c a t a b o l i z i n g enzymes 148 6. G l u c o s e t o x i c i t y i n g l u c o s e - n e g a t i v e m u t a n t s . . . 149 I I . L i s t o f a b b r e v i a t i o n s 150 i x L IST OF TABLES TABLE PAGE I. I d e n t i t y o f r a d i o a c t i v e i n t e r m e d i a t e s a c c u m u l a t e d f r om 1 k g l u c o s e - U - C c a t a b o l i s m i n mutant s X I I I ^ and XV. . . . . 36 I I . D e g r a d a t i o n p r o d u c t s o f g l u c o s e - U - C m e t a b o l i s m i n medium and c e l l s o f g l u c o s e - n e g a t i v e mutant s o f P_. ae r u g i nosa 37 I I I . I n d u c t i o n o f g l u c o s e d e g r a d i n g enzymes i n w i l d - t y p e and g l u c o s e - n e g a t i v e mutant s o f _P. ae r u g i nosa 41 IV. I n d u c t i o n o f g l u c o s e - c a t a b o l i z i n g enzymes i n r e v e r t a n t s o f g l u c o s e - n e g a t i v e mutant s XVj and XI I I^  kG V. C o n c e n t r a t i o n s o f p r o d u c t s o f g l u c o s e - U - C c a t a b o l i s m i n g l u c o s e - n e g a t i v e mutant X I l l j c e l l w a t e r and medium . kS V I . C o n c e n t r a t i o n s o f p r o d u c t s o f g l u c o s e - U - C c a t a b o l i s m i n g l u c o s e - n e g a t i v e mutant XV^ c e l l w a t e r and medium . . 50 V I I . A c t i v i t i e s o f TCA c y c l e enzymes i n w i l d - t y p e P_. ae r u g i nosa 53 V I I I . A c t i v i t i e s o f TCA c y c l e enzymes i n P_. a e r u g i n o s a g l u c o s e - n e g a t i v e mutant XI I I ^ 55 IX. A c t i v i t i e s o f TCA c y c l e enzymes i n P. a e r u g i n o s a g l u c o s e - n e g a t i v e mutant XV^ 56 X. A c t i v i t i e s o f g l y c o l y t i c enzymes i n P_. a e r u g i n o s a w i l d - t y p e and g l u c o s e - n e g a t i v e mutan t s X I I I . and XV, . . 57 X TABLE PAGE X I . P r o d u c t s a c c u m u l a t i n g i n medium and c e l l s o f g l u c o s e -n e g a t i v e mutant s o f P_. a e r u g i n o s a f rom g l u c o s e , g l u c o n a t e , and 2 - k e t o g l u c o n a t e m e t a b o l i s m . . . . . . . . 66 X I I . G l u c o s e - c a t a b o l i z i n g enzymes i n g l u c o s e - n e g a t i v e mutant s o f P_. a e r u g i nosa 68 X I I I . Compar i s on o f g l u c o s e dehyd rogena se a c t i v i t y and g e n e r a t i o n t i m e i n P_. a e r u g i n o s a g l u c o s e - n e g a t i v e mutant s 73 X IV. P o s s i b l e i n d u c e r s o f g 1 u c o s e - c a t a b o l i z i n g enzymes i n P_. a e r u g i n o s a as d e t e r m i n e d f rom d a t a o b t a i n e d w i t h g l u c o s e - n e g a t i v e mutant s 7h 1 k XV. D e g r a d a t i o n p r o d u c t s o f g l y c e r o l - U - C m e t a b o l i s m i n med ium and e e l Is o f wi I d - t y p e P_. ae rug i n o s a and g l u c o s e - n e g a t i v e mutant X l l l ^ _ ^ . 116 XV I . Compar i son o f s p e c i f i c a c t i v i t i e s and g e n e r a t i o n t i m e s o f P_. a e r u g i nosa d u r i n g d i f f e r e n t s t a g e s o f g rowth w i t h d i f f e r e n t s u b s t r a t e s 120 X V I I . I nduce r s o f c a r b o h y d r a t e - c a t a b o l i z i n g enzymes i n P_. a e r u g i n o s a as d e t e r m i n e d f rom the e x a m i n a t i o n o f g l u c o s e - n e g a t i v e mutant s and the p a r e n t s t r a i n 127 x i L IST OF FIGURES FIGURE PAGE 1. The i n h i b i t o r y e f f e c t o f g l u c o s e , g l u c o n a t e , and 2-KG on t h e g rowth o f g l u c o s e - n e g a t i v e mutant s o f P_. a e r u g i n o s a . . 29 2. The e f f e c t o f g l u c o s e c o n c e n t r a t i o n on t he i n h i b i t i o n o f g rowth o f g l u c o s e - n e g a t i v e mutant XV. i n p y r u v a t e m i n i m a l medium 31 3- The e f f e c t o f g l u c o s e on t he g rowth o f g l u c o s e - n e g a t i v e mutan t s o f JP. a e r u g i nosa wi t h a c e t a t e , c i t r a t e , i s o c i t r 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 , and m a l a t e 32 1 k k. E l u t i o n p r o f i l e o f C compounds f rom a Dowel 1-X8 f o r m a t e a n i o n exchange co lumn f o l l o w i n g g l u c o s e - U - C c a t a b o l i s m i n g l u c o s e - n e g a t i v e mutan t s o f P_. a e r u g i nosa 35 5- Growth o f w i l d - t y p e and g l u c o s e - n e g a t i v e mutant s o f P_. a e r u g i nosa 39 6. Growth o f w i l d - t y p e and r e v e r t a n t s o f g l u c o s e - n e g a t i v e mutant s o f P_. ae rug i nosa i n g l u c o s e m i n i m a l medium k$ 7- Dec rea se i n t he s p e c i f i c a c t i v i t y o f p y r u v i c dehyd rogena se i n g l u c o s e - n e g a t i v e mutan t s o f P. a e r u g i nosa f o l l o w i n g t he a d d i t i o n o f g l u c o s e 59 8. E f f e c t o f g l u c o s e on g rowth and on p y r u v a t e d i s a p p e a r a n c e i n g l u c o s e - n e g a t i v e mutant XI I I j 60 9- E f f e c t o f g l u c o s e on the g rowth o f g l u c o s e - n e g a t i v e mutant 3 i n p y r u v a t e m i n i m a l medium. 6k FIGURE PAGE 10. Growth o f P_. a e r u g i n o s a w i l d - t y p e w i t h g l u c o s e , g l u c o n a t e , and 2-KG as s o l e c a r b o n s o u r c e s 77 14 11. Appea rance o f C - l a b e l l e d g l u c o n a t e and 2-KG d u r i n g ]Li g rowth o f P_. a e r u g i n o s a on 0.4% C - g l u c o s e 79 12. D i s a p p e a r a n c e o f c a r b o n s o u r c e d u r i n g g rowth o f P_. a e r u g i n o s a on (A) g l u c o n a t e and (B) 2-KG 80 13- Changes i n t he l e v e l s o f g l u c o s e and g l u c o n a t e d e h y d r o -genases d u r i n g g rowth o f P_. a e r u g i n o s a on g l u c o s e ( A ) , g l u c o n a t e (B) , and 2-KG (C) 83 14. I n d u c t i o n o f g l u c o s e dehyd rogena se (A) and g l u c o n a t e dehyd rogena se (B) by g l u c o s e , g l u c o n a t e , and 2-KG i n P_. a e r u g i nosa g r ow ing i n p y r u v a t e m i n i m a l medium 87 15- S p e c i f i c a c t i v i t i e s o f g l u c o k i n a s e , G-6-P d e h y d r o g e n a s e , and t he ED pathway enzymes d u r i n g g rowth w i t h g l u c o s e ( A ) , g l u c o n a t e (B) , and 2-KG (C) 90 16. S p e c i f i c a c t i v i t i e s o f g 1 u c o n o k i n a s e and 3"PG d e h y d r o -genase d u r i n g g r owth w i t h g l u c o s e ( A ) , g l u c o n a t e ( B ) , and 2-KG (C) 91 17- I n d u c t i o n o f g l u c o k i n a s e ( A ) , G-6-P dehyd rogena se ( B ) , and t h e ED pathway enzymes (C) 9** 18. I n d u c t i o n o f g l u c o n o k i n a s e (A) and 3"PG dehyd rogena se (B) . 96 19. S p e c i f i c a c t i v i t i e s o f g l u c o s e dehyd rogena se and g l u c o n a t e dehyd rogena se d u r i n g g rowth o f P_. a e r u g i nosa w i t h 0.043 M g l y c e r o l 102 x i i i FIGURE PAGE 20. S p e c i f i c a c t i v i t i e s o f g l u c o k i n a s e , G -6 -P d e h y d r o g e n a s e , ED pathway enzymes, 3~PG d e h y d r o g e n a s e , and g l u c o n o -k i n a s e d u r i n g g rowth o f P_. a e r u g i n o s a w i t h 0.043 M g l y c e r o l 104 21. S p e c i f i c a c t i v i t i e s o f g l y c e r o l k i n a s e and L-a-GP d e h y d r o -genase d u r i n g g rowth o f P_. a e r u g i nosa wi t h 0.043 M g l y c e r o l . 105 22. S p e c i f i c a c t i v i t i e s o f g l u c o s e dehyd rogena se and g l u c o n a t e dehyd rogena se f o l l o w i n g the a d d i t i o n o f 0.022 M g l y c e r o l t o P_. a e r u g i n o s a g r ow ing w i t h 0 .045 M p y r u v a t e 107 23. S p e c i f i c a c t i v i t i e s o f g l u c o k i n a s e , G-6-P d e h y d r o g e n a s e , ED pathway enzymes, 3 _ PG d e h y d r o g e n a s e , and g l u c o n o -k i n a s e f o l l o w i n g the a d d i t i o n o f 0.022 M g l y c e r o l t o P_. a e r u g i nosa g r o w i n g w i t h 0.045 M p y r u v a t e 109 24. S p e c i f i c a c t i v i t i e s o f g l y c e r o l k i n a s e and L-a-GP d e h y d r o -genase f o l l o w i n g the a d d i t i o n o f 0.022 M g l y c e r o l t o P_. ae rug i nosa g r ow ing w i t h 0.045 M p y r u v a t e 110 25. Growth o f g l u c o s e - n e g a t i v e mutant X l l l j _ ^ w i t h 0.045 M p y r u v a t e and w i t h 0.045 M p y r u v a t e p l u s 0.022 M g l y c e r o l . . 113 14 26. F a t e o f u n i f o r m l y l a b e l l e d C - g l y c e r o l d u r i n g m e t a b o l i s m by w i l d - t y p e P_. a e r u g i nosa and g l u c o s e - n e g a t i v e mutant XI I 11 _ A 115 27. Pathways o f s i x - c a r b o n c a r b o h y d r a t e and g l y c e r o l m e t a b o l i s m i n P. a e r u g i n o s a ATCC 9027 126 ACKNOWLEDGEMENTS I wou ld l i k e t o e x p r e s s my s i n c e r e a p p r e c i a t i o n t o Dr. A. F. G r o n l u n d f o r he r i n t e r e s t , s u p e r v i s i o n , and h e l p f u l c r i t i c i s m d u r i n g the c o u r s e o f t h i s s t u d y and t he w r i t i n g o f t he t h e s i s . I w i s h f u r t h e r t o a cknow ledge t h e t e c h n i c a l a s s i s t a n c e o f Mrs . D. B lew f o r c e r t a i n p a r t s o f the e x p e r i m e n t a l work . My s i n c e r e a p p r e c i a t i o n i s e x t ended t o my w i f e Edna f o r her f o r e b e a r a n c e d u r i n g the 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 . 1 INTRODUCTION The o x i d a t i v e n o n - p h o s p h o r y l a t e d pathway o f g l u c o s e d i s s i m i l a t i o n i n Pseudomonas a e r u g i n o s a ATCC 9027, was e s t a b l i s h e d by N o r r i s and Campbe l l (88). Both g l u c o n a t e and 2 - k e t o g l u c o n a t e were i d e n t i f i e d as i n t e r m e d i a t e s o f g l u c o s e c a t a b o l i s m , d u r i n g g rowth o f the o r g a n i s m when g l u c o s e was t he ca rbon s o u r c e . The p r e s e n c e o f the E n t n e r - D o u d o r o f f pathway has a l s o been d e m o n s t r a t e d i n t h i s m i c r o o r g a n i s m (38,50,68,118). The enzymes o f g l u c o s e c a t a b o l i s m i n P. a e r u g i n o s a have been shown to be i nduced by g rowth w i t h s i x - c a r b o n c a r b o h y d r a t e s such as g l u c o s e , g l u c o n -a t e , f r u c t o s e , and m a n n i t o l (19,38,50,5^,68,80,115,118), as w e l l as w i t h g l y c e r o l (50,68,80).and g l y c e r a t e (50). However, v e r y l i t t l e i s known c o n c e r n i n g t he r e g u l a t i o n o f t he enzymes, by t h e s e s u b s t r a t e s , and t h e a c t u a l i n d u c e r s have not been i d e n t i f i e d . 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 was t o examine the r e l a t i v e c o n t r i b u -t i o n o f the o x i d a t i v e n o n - p h o s p h o r y l a t e d pathway t o t he d e g r a d a t i o n o f g l u c o s e and g l u c o n a t e i n P_. a e r u g i n o s a and a l s o t o examine t he i n d u c t i o n o f g 1 u c o s e - c a t a b o l i z i n g enzymes by g l u c o s e and i t s o x i d a t i o n p r o d u c t s , g l u c o n a t e and 2 - k e t o g l u c o n a t e . I t was f e l t t h a t a s t u d y o f the i n d u c t i o n o f t h e s e enzymes by g l u c o s e , g l u c o n a t e , and 2 - k e t o g l u c o n a t e , i n t he w i l d -t ype s t r a i n and i n g l u c o s e - n e g a t i v e m u t a n t s , wou ld p r o v i d e i n f o r m a t i o n c o n c e r n i n g the r e g u l a t i o n o f the enzymes, by t h e s e s u b s t r a t e s , a s . w e l l as t he a c t u a l i n d u c e r s . F u r t h e r , due t o t he s e e m i n g l y g r a t u i t o u s i n d u c t i o n o f g 1 u c o s e - c a t a b o l i z i n g enzymes by t h r e e - c a r b o n compounds such as g l y c e r o l 2 and g l y c e r a t e , t h e i n d u c t i o n o f t h e s e enzymes by g l y c e r o l and t he pathways o f g l y c e r o l d i s s i m i l a t i o n i n P_. a e r u g i n o s a were i n v e s t i g a t e d . The pathways o f g l u c o s e , g l u c o n a t e , 2 - K G , and g l y c e r o l c a t a b o l i s m i n P.. a e r u g i n o s a (ATCC 9 0 2 7 ) a r e i l l u s t r a t e d i n F i g . 2 7 (p . 1 2 6 ) . 3 LITERATURE REVIEW I. Pathways f o r t h e d i s s i m i l a t i o n o f g l u c o s e i n Pseudomonas s p e c i e s . The pathways f o r t he d i s s i m i l a t i o n o f g l u c o s e i n Pseudomonas a e r u - g i n o s a ATCC 9027 have been examined (8,36,55,88,109,115,118) and found t o be s i m i l a r t o t h o s e i n o t h e r s t r a i n s o f the same m i c r o o r g a n i s m (38, 50,68) and i n r e l a t e d m i c r o o r g a n i s m s such as Pseudomonas p u t i d a A.3-12 (31,63,64,85,121,]22) ( f o r m e r l y d e s i g n a t e d as Pseudomonas f l u o r e s c e n s A.3-12 (107)), Pseudomonas m u l t i v o r a n s (70), and P_. f 1 u o r e s c e n s (95). The use o f t he E n t n e r - D o u d o r o f f (ED) enzymes (24), 6 -phosphog1uconate (6-PG) d e h y d r a t a s e and 2 - k e t o - 3 _ d e o x y - 6 - p h o s p h o g 1 u c o n a t e (KDGP) a l d o l a s e , as t he ma jo r r o u t e o f g l u c o s e c a t a b o l i s m i n pseudomonads (16,5^,59,78,79, 108,119) i s c h a r a c t e r i s t i c o f a l l t h e s e m i c r o o r g a n i s m s as i s t he ab sence o f a f u n c t i o n a l Embden-Meyerhof pathway (8,68,70,97,115,122). G l u c o s e may e n t e r t h e ED pathway by t h r e e r o u t e s i n P_. a e r u g i nosa and P_. mu 11 i v o r an s and by two r o u t e s i n P_. f 1 u o r e s c e n s and P_. put i da . G l u c o s e may be phosphory 1 a ted d i r e c t l y by P_. ae r u g i nosa and P_. mul t i v o r a n s o r o x i d i z e d t o g l u c o n a t e and 2 - k e t o g l u c o n a t e (2-KG) p r i o r t o p h o s p h o r y l a t i o n a t t he l e v e l o f e i t h e r o f t h e s e o x i d a t i o n p r o d u c t s (88,109). P_. f 1 u o r e s c e n s and IP. p u t i d a l a c k g l u c o k i n a s e and t h e r e f o r e , g l u c o s e i s o x i d i z e d t o g l u c o n a t e o r 2-KG p r i o r t o p h o s p h o r y l a t i o n (85,95)- C a t a b o l i s m o f t he p r o d u c t s o f the ED pathway (3 - phosphog1yce ra1dehyde (3 - PG) and p y r u v a t e ) o c c u r s v i a t h e Embden-Meyerhof pa thway, a t t he t r i o s e - p h o s p h a t e l e v e l , and v i a t he t r i c a r b o x y l i c a c i d (TCA) c y c l e . The re a r e , however , s e v e r a l 4 g roups o f n o n - f l u o r e s c e n t Pseudomonas s p e c i e s w h i c h a r e u n a b l e t o u t i l i z e g l u c o s e , g l u c o n a t e o r 2-KG as t h e s o l e c a r b o n and energy s o u r c e ( 107 ) . I I. R e g u l a t i o n o f g l u c o s e c a t a b o l i s m . From the s t u d i e s o f enzyme a c t i v i t i e s i n m i c r o o r g a n i s m s grown under d i f f e r e n t n u t r i t i o n a l c o n d i t i o n s , i t has become a p p a r e n t t h a t o n l y a s m a l l f r a c t i o n o f the t o t a l c a t a b o l i c c a p a c i t y i s e x p r e s s e d a t any one t i m e . A l t h o u g h some enzymes do not appea r t o v a r y c o n s i d e r a b l y under d i f f e r e n t c o n d i t i o n s and may be termed c o n s t i t u t i v e , many c a t a b o l i c enzymes a r e s u b -j e c t t o i n d u c t i o n and r e p r e s s i on ( 1 2 ) . The g 1 uco se e f f e c t _i_.e_. t h e a b i l i t y o f g l u c o s e t o r e p r e s s the s y n t h e s i s o f c e r t a i n c a t a b o l i c enzymes was f i r s t o b s e r v e d by Epps and G a l e (26) i n E s c h e r i c h i a c o l i . T h i s e f f e c t r e s u l t s i n t he p r e f e r e n t i a l u t i l i z a t i o n o f g l u c o s e and c o n s e q u e n t l y a d i a u x i c p a t t e r n o f g rowth when t he o r g a n i s m i s grown i n a medium c o n t a i n i n g g l u c o s e and c e r t a i n o t h e r s u b s t r a t e s (8l) as g l u c o s e i n h i b i t s o r r e p r e s s e s the c a t a b o l i s m o f t h e s e s u b s t r a t e s . T h i s t o p i c has been r e v i e w e d ( 7 7 , 9 2 ) . The g l u c o s e e f f e c t has been shown to c a u s e t he r e p r e s s i o n o f c e r t a i n TCA c y c l e enzymes i n a number o f b a c t e r i a and t h e c o n s t i t u t i v e n a t u r e o f 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 t h e s e m i c r o o r g a n i s m s has been r e p o r t e d . These b a c t e r i a i n c l u d e E_. c o l i ( 2 1 , 3 5 , 1 0 6 ) , S a l m o n e l l a t y p h i m u r i u m ( 3 0 ) , A e r o b a c t e r ae rogene s ( 1 5 , 5 3 ) , S t a p h y l o c o c c u s a u r e u s ( 1 3 , 1 1 1 ) , S t r e p t o c o c c u s  f a e c a l i s ( 1 05 ) , B a c i 1 l u s c e r e u s ( 4 2 , 4 3 ) , B a c i l l u s s u b t i 1 i s (41 ,82), and B a c i l l u s 1 i c h e n i f o r m i s ( 4 l ) . However, JE. c o l i ( 2 1 ) , :S_. t y p h i m u r i u m ( 21 , 3 0 ) , /\. ae rogene s ( 2 1 ) , and S_. f a e c a 1 i s (105) do po s se s s an i n d u c i b l e ED pathway 5 w h i c h i s used d u r i n g g rowth w i t h g l u c o n a t e and r e l a t e d compounds. A d i f f e r e n t s i t u a t i o n c o n c e r n i n g t he r e g u l a t i o n o f g l u c o s e c a t a b o l i s m a p p e a r s t o e x i s t i n a number o f Gram n e g a t i v e , a e r o b i c m i c r o o r g a n i s m s t h a t have been exam ined . In t h e s e b a c t e r i a , a l t h o u g h d i a u x i c g rowth has been o b s e r v e d under c e r t a i n c o n d i t i o n s w i t h a m i x t u r e o f g l u c o s e and some TCA c y c l e i n t e r m e d i a t e s , as i n P_. a e r u g i n o s a (37 ,50 ,115) and A r t h r o - b a c t e r c r y s t a l l o p o i e t e s (65), i t i s t he TCA c y c l e i n t e r m e d i a t e t h a t i s u t i l i z e d p r e f e r e n t i a l l y o v e r g l u c o s e . In P_. a e r u g i n o s a , t h e enzymes o f the TCA c y c l e have been r e p o r t e d as b e i n g c o n s t i t u t i v e ( 3 8 , 1 1 5 , 1 1 8 ) , a l t h o u g h a r e c e n t r e p o r t by Ng and Dawes s u g g e s t s t h a t t h i s may not a c t u a l l y be t he c a s e (87)• The enzymes o f g l u c o s e c a t a b o l i s m i n t h e Gram n e g a t i v e , a e r o b i c m i c r o -o r g a n i s m s t h a t have been examined a r e not c o n s t i t u t i v e and have been shown to be i nduced by g rowth w i t h g l u c o s e i n P_. ae rug i nosa ( 3 8 ) , P_. f 1 u o r e s c e n s ( 2 1 , 9 5 ) , P_. m u l t i v o r a n s ( 7 0 ) , P_. p u t i d a ( 2 5 , 8 5 , 1 0 0 ) , A. c r y s t a l l o p o i e t e s ( 6 5 ) , Th i o b a c i 1 1 us i ntermed i us ( 7 8 ) , and Th i obac i11 us p e r o m e t a b o l i s (79)• These b a c t e r i a a l l c a t a b o l i z e g l u c o s e by an i n d u c i b l e ED pathway w i t h the e x c e p t i o n o f A_. c r y s t a l l o p o i e t e s , w h i c h K r u l w i c h and E n s i g n have shown t o po s s e s s an i n d u c i b l e Embden-Meyerhof pathway ( 6 5 ) . The r e p r e s s i o n o f t he enzymes o f g l u c o s e c a t a b o l i s m by the p r e s e n c e o f TCA c y c l e i n t e r m e d i a t e s has been s t u d i e d by Dawes and h i s c o - w o r k e r s i n P_. ae rug i nosa ( 38 - 40 , 86 , 87 ) and e v i d e n c e has been p r e s e n t e d w i t h b o t h P_. a e r u g i nosa (84) and A_. c r y s t a l l o p o i e t e s (65) t h a t t h i s r e p r e s s i o n may, a t l e a s t i n p a r t , be m a n i f e s t a t the l e v e l o f g l u c o s e t r a n s p o r t . The 6 e f f e c t o f TCA c y c l e i n t e r m e d i a t e s on r e p r e s s i o n o r i n h i b i t i o n a p p e ars to e x t e n d t o o t h e r c a t a b o l i c pathways such as the s u c c i n a t e e f f e c t on t h e amidase system o f P_. a e r u g i nosa (4) and the h i s t i d i n e d e g r a d i n g pathway i n P_. a e r u g i nosa (69) and P. p u t i d a (49). I I I . The i n d u c t i o n o f c a r b o h y d r a t e - c a t a b o 1 i z i n g enzymes i n Pseudomonas  a e r u g i nosa. C a r b o h y d r a t e - c a t a b o l i z i ng enzymes i n P_. aerug i nosa have been shown t o be induced by growth w i t h s i x - c a r b o n c a r b o h y d r a t e s , as w e l l as w i t h g l y c e r o l o r g l y c e r a t e , but o n l y low l e v e l s o f t h e s e enzymes a r e p r e s e n t d u r i n g growth w i t h a c e t a t e , p y r u v a t e , o r TCA c y c l e i n t e r m e d i a t e s . H a m i l t o n and Dawes (38) r e p o r t e d t h a t growth w i t h g l u c o s e , but not w i t h o r g a n i c a c i d s , r e s u l t e d i n h i g h l e v e l s o f g l u c o s e , g l u c o n a t e , g1ucose - 6-phosphate (G - 6-P), and 6-PG dehydrogenases, g l u c o s e , g l u c o n a t e , and 2-KG k i n a s e s , and t he enzymes o f the ED pathway i n P_. aerug i nosa 2 F 3 2 . S i m i l a r r e s u l t s were o b t a i n e d by M i d g l e y and Dawes (80) w i t h t h i s same s t r a i n , grown w i t h e i t h e r g l u c o n a t e o r g l y c e r o l . L e s s i e and N e i d h a r d t (68), u s i n g a d i f f e r e n t s t r a i n o f P_. aerug i nosa, o b s e r v e d t h a t g l u c o k i n a s e , G-6-P and 6-PG de-hyd r o g e n a s e s , and the ED pathway enzymes were induced d u r i n g growth w i t h f r u c t o s e , g l u c o s e , g l u c o n a t e , g l y c e r o l , o r a m i x t u r e o f p y r u v a t e and g l u c o s e but not d u r i n g growth w i t h p y r u v a t e o r s u c c i n a t e . In y e t a n o t h e r s t r a i n o f P_. aerug i nosa, growth w i t h m a n n i t o l , f r u c t o s e , g l u c o s e , g l y c e r o l , o r g l y c e r a t e but not w i t h a c e t a t e , p y r u v a t e , o r s u c c i n a t e r e s u l t e d i n h i g h l e v e l s o f g l u c o s e , G-6-P, and 6-PG dehy d r o g e n a s e s , g l u c o k i n a s e , and KDGP a l d o l a s e (19,50,9*0 • 7 P_. aeruginosa ATCC 9027 appears to be similar to the other strains of this microorganism in that growth with glucose, but not with acetate, a-ketoglutarate or succinate, caused the induction of glucose, G-6-P, 6-PG, and 3-PG dehydrogenases, glucokinase, and the enzymes of the ED pathway ( 1 1 5 , 1 1 8 ) . Although growth with six-carbon carbohydrates and glycerol or glycer-ate has been shown to induce the glucose-catabol izing enzymes in P_. aerug i nosa, the actual inducer(s) of these enzymes has not been identified. Hylemon and Phibbs (50) have reported that the induction of glucose transport in P_. aeruginosa can be separated from the induction of hexose-catabolizing enzymes. Glucose transport was induced by the glucose ana-logues, a-methylglucoside and 2-deoxyg1ucose, in the absence of the induction of the catabolic enzymes. The enzymes were induced by growth with glycerol or glycerate in the absence of the induction of glucose transport activity. Since a-methylglucoside was not metabolized and 2-deoxyg1ucose was meta-bolized only to 2-deoxyg1uconate, evidence was provided that glucose per se may induce glucose transport activity but does not cause the induction of hexose-catabolizing enzymes. Some studies on the induction of the enzymes of glucose catabolism have been reported with P_. put ida and P_. f 1 uorescens. Narrod and Wood (85) observed that growth of P_. put i da A. 3 • 12 with gluconate or 2-KG resulted in the induction of both gluconokinase and 2-KG kinase, although the activity of g1uconokinase was somewhat lower when 2-KG was the carbon source. They suggested that the induction of g1uconokinase during growth with 2-KG may be due to the fact that 2-KG acts as a weak inducer of this 8 enzyme. P_. f 1 u o r e s c e n s l a c k s g l u c o k i n a s e and Quay, F r iedman and E i s e n -be rg ( 9 5 ) , s t u d y i n g the i n d u c t i o n o f g 1 u c o s e - c a t a b o l i z i n g enzymes by g l u c o s e and g l u c o n a t e i n g l u c o s e and g l u c o n a t e d e h y d r o g e n a s e - d e f i c i e n t m u t a n t s , have a s c e r t a i n e d t h a t g l u c o n a t e o r some d e r i v a t i v e ( s ) t h e r e o f , bu t not g l u c o s e o r 2 -KG, caused t he i n d u c t i o n o f g l u c o n o k i n a s e and the enzymes o f t he ED pathway. F u r t h e r , g l u c o n a t e o r some m e t a b o l i t e ( s ) d e r i v e d f rom g l u c o n a t e , but not g l u c o s e , cau sed t he i n d u c t i o n o f g l u c o n a t e d e h y d r o g e n a s e . The e v i d e n c e p r e s e n t e d w i t h P_. put i da and P_. f 1 u o r e s c e n s , as w i t h P_. a e r u g i n o s a , s u g g e s t s t h a t g l u c o s e per se does not cau se t he i n d u c t i o n o f the g 1 u c o s e - c a t a b o l i z i n g enzymes. The o b s e r v a t i o n t h a t g rowth w i t h g l y c e r o l o r g l y c e r a t e c au se s the i n d u c t i o n o f c e r t a i n g 1 u c o s e - c a t a b o l i z i ng enzymes i n P_. ae rug ? nosa (50,68) has l e d t o t h e s u g g e s t i o n by Hylemon and P h i b b s (50) t h a t the a c t u a l i n d u c e r ( s ) o f t h e s e enzymes may be a t r i o s e - p h o s p h a t e o r some d e r i v a t i v e t h e r e o f . Quay, F r i e d m a n , and E i s e n b e r g ( 9 5 ) , who have shown t h a t g l u c o n a t e o r some d e r i v a t i v e ( s ) o f g l u c o n a t e i nduce s t h e s e enzymes, o b t a i n e d s i m i l a r r e s u l t s a f t e r g r owth o f P_. f l u o r e s c e n s w i t h g l y c e r o l and s u g g e s t e d t h a t g l y c e r i c a c i d p roduced f rom g l y c e r o l may a c t as a g r a t u i t o u s i n d u c e r , i f g l u c o n a t e i s a c t u a l l y t he t r u e i n d u c e r . I t has been r e p o r t e d t h a t g l y c e r o l i s c a t a b o l i z e d by g l y c e r o k i n a s e and a - g 1 y c e r o p h o s p h a t e (a-GP) dehyd rogena se t h r o u g h 3"PG i n P_. a e r u g i nosa (116) and P_. put i da (100) a n d , as s u g g e s t e d by Hylemon and P h i b b s ( 5 0 ) , t h i s wou ld i n d i c a t e t h a t the enzymes o f g l u c o s e c a t a b o l i s m i n c e r t a i n o f the Pseudomonas s p e c i e s a r e s y n t h e s i z e d g r a t u i -t o u s l y d u r i n g g rowth w i t h g l y c e r o l . An a n a l o g y has been made (50) w i t h t h e B -ketoad j p a t e pathway i n P_. a e r u g i nosa , P_. put i da and P_. mu 11 i v o r a n s 9 i n w h i c h O r n s t o n (89) d e m o n s t r a t e d t h a t the b r anch p o i n t m e t a b o l i t e , 3 - k e t o a d i p a t e , i nduced t he two p r e c e e d i n g enzymes o f t he p r o t o c a t e c h u a t e b r a n c h . When t he o r gan i sms were grown w i t h s u b s t r a t e s c a t a b o l i z e d by the c a t e c h o l b r a n c h , t h e s e two enzymes i n t h e p r o t o c a t e c h u a t e b r anch were s y n t h e s i z e d g r a t u i t o u s l y . IV. The r o l e o f the o x i d a t i v e n o n - p h o s p h o r y l a t e d pathway i n g l u c o s e d i s s i m i l a t i o n i n P_. ae r u g i nosa . The a b i l i t y o f c e r t a i n Pseudomonas s p e c i e s t o o x i d i z e g l u c o s e t o 2-KG has been known f o r some t i m e (71)- In IP. put i d a , w h i c h l a c k s g l u c o k i n a s e (122), g l u c o s e i s c a t a b o l i z e d by t h i s pathway (25,62) and p h o s p h o r y l a t i o n o c c u r s e i t h e r a t the l e v e l o f g l u c o n a t e o r 2-KG (85). P_. f 1 u o r e s c e n s i s s i m i l a r t o P_. put i da i n t h a t i t a l s o l a c k s g l u c o k i n a s e and t he i m p o r t a n c e o f t h e o x i d a t i v e n o n - p h o s p h o r y l a t e d pathway has been i n d i c a t e d by t he f a c t t h a t g l u c o s e a d d i t i o n t o b a s a l medium had no e f f e c t on t he g rowth r a t e o f g l u c o s e d e h y d r o g e n a s e - d e f i c i e n t m u t a n t s . The p a r e n t s t r a i n grew t w i c e as f a s t when g l u c o s e was added (95). The i m p o r t a n c e o f t h i s o x i d a t i v e pathway i s somewhat more o b s c u r e i n P. a e r u g i n o s a as t h i s o r g a n i s m p o s s e s s e s an i n d u c i b l e g l u c o k i n a s e (19, 38,68,118) and t h e o x i d a t i v e n o n - p h o s p h o r y l a t e d pathway f o r g l u c o s e d i s s i m i l a t i o n does not appea r t o be r e q u i r e d f o r g rowth w i t h g l u c o s e (80). The d i r e c t pathway f o r the o x i d a t i o n o f g l u c o s e t o 2-KG i n P_. a e r u g i n o s a was d e m o n s t r a t e d by S t o k e s and Campbe l l (109). I t s i m p o r t a n c e i n t h e d i s s i m i l a t i o n o f g l u c o s e was s u gge s t ed by t h e s e w o r k e r s (88), because bo th g l u c o n a t e and 2-KG were p r e s e n t i n the medium f o r a t l e a s t e i g h t h o u r s , 1 0 d u r i n g g rowth wi t h . g l u c o s e , and t r a c e amounts o f g l u c o n a t e and 2-KG were p r e s e n t i n t h e medium when g l u c o s e had c o m p l e t e l y d i s a p p e a r e d . V. Othe r c a t a b o l i c pathways i n Pseudomonas s p e c i e s . E v i d e n c e t o d a t e s u g g e s t s t h a t the gene a r r angement i n Pseudomonas s p e c i e s may be q u i t e d i f f e r e n t f rom t h a t o b s e r v e d i n t he more w i d e l y s t u d i e d E n t e r o b a c t e r i a c e a e , i n t h a t r e l a t e d genes i n a g i v e n pathway appea r t o be a r r a n g e d l e s s c o n t i g u o u s l y a l o n g t he genome, b e i n g a g g r e g a t e d i n t o c l u s t e r s o f o n l y two o r t h r e e genes ( f o r r e v i e w see 4 8 ) . The m e t a b o l i c d i v e r s i t y o f Pseudomonas s p e c i e s (107) endows them w i t h a l a r g e number o f c a t a b o l i c pathways t h a t a r e s u b j e c t t o comp lex r e g u l a t i o n . T h i s s u b j e c t has been r e v i e w e d r e c e n t l y (90). The l e s s c o n t i g u o u s a r rangement o f genes w i t h r e l a t e d f u n c t i o n , i .e_. t h o s e c o d i n g f o r a l l t he enzymes o f a g i v e n c a t a b o l i c pa thway, i s r e f l e c t e d i n t h e r e g u l a t i o n o f t he enzymes i n t h e c a t a b o l i c pa thway s . T h i s has been d e m o n s t r a t e d i n the 3 ~ k e t o a d i p a t e pathway o f P_. ae r u g i n o s a (58) and P_. put i da (89) i n w h i c h t h e r e g u l a t i o n o f t he pathway i n bo th o r gan i sms has been shown t o v e r y s i m i l a r . Of t he t w e n t y - f i v e enzymes c o n v e r g i n g i n t o t h i s pa thway , t e n i n d u c t i v e e v e n t s o c c u r t o r e g u l a t e t h e i r s y n t h e s i s . Some enzymes a r e i nduced by t h e i r s u b s t r a t e , o t h e r s by t h e i r p r o d u c t a n d , on an a v e r a g e , each e f f e c t o r r e g u l a t e s t h e s y n t h e s i s o f o n l y two o r t h r e e enzymes. The r e g u l a t i o n o f t h e s e pathways was e s t a b l i s h e d by a number o f w o r k e r s and has been r e v i e w e d (90,120). Bo th the number o f i n d u c e r s i n -v o l v e d and t h e d e g r e e o f c o o r d i n a t e c o n t r o l a r e c o n s i d e r a b l y l e s s i n o t h e r m i c r o o r g a n i s m s t h a t have been examined (10,11,89). 11 V I . C a t a b o l i c mutants. The i s o l a t i o n , c h a r a c t e r i z a t i o n , and e x a m i n a t i o n o f mutants b l o c k -ed i n a g i v e n c a t a b o l i c pathway has p r o v i d e d u s e f u l i n f o r m a t i o n c o n c e r n i n g t h e r e g u l a t i o n o f t h a t pathway i n Pseudomonas s p e c i e s ( f o r r e v i e w see 9 0 ) . I t has a l s o become a common o b s e r v a t i o n t h a t m u t a n t s , b l o c k e d i n the m e t a b o l i s m o f a g i v e n c a r b o h y d r a t e , o f t e n a c c u m u l a t e a p h o s p h o r y l a t e d i n t e r m e d i a t e from t h i s compound as a r e s u l t o f the m u t a t i o n and, t h a t t h i s a c c u m u l a t i o n i n h i b i t s growth w i t h c e r t a i n o t h e r s u b s t r a t e s , o r i n some manner i s t o x i c t o the o r g a n i s m . T h i s phenomenon has been r e p o r t e d i n a w ide v a r i e t y o f m i c r o o r g a n i s m s i n c l u d i n g E_. c o l i (3 ,1^,17,18,22 ,28,29, 45,^7,66,103 ,112,123), S a l m o n e l l a t y p h o s a ( 2 3 ) , S_. t y p h i m u r i u m (52), and Saccharomyces c e r e v i s i a e (27,113) and i n v o l v e s a l a r g e number o f d i f f e r e n t p h o s p h o r y l a t e d i n t e r m e d i a t e s . I t a l s o a p p ears to be the c a use o f g l u c o s e t o x i c i t y i n HeLa c e l l s w i t h b l o c k e d g l y c o l y s i s ( 3 * 0 , mannose t o x i c i t y i n honey bees ( 1 0 4 ) , and the b i o c h e m i c a l b a s i s f o r g a l a c t o s e m i a ( 1 0 1 ) . E x p l a n a t i o n s f o r t h i s t o x i c i t y o r growth i n h i b i t i o n , as a r e s u l t o f the a c c u m u l a t i o n o f a p h o s p h o r y l a t e d i n t e r m e d i a t e have been s u g g e s t e d , i . e . d e p l e t i o n o f i n t r a c e l l u l a r ATP or i n o r g a n i c phosphate, a c t i o n on some e s s e n t i a l c e l l p r o c e s s , o r a c t i o n on the a c t i v i t y o f some enzyme ( 3 , 1 ^ , 27,29 ,52,66). However, from the c a s e s examined, i t does not appear t h a t t h e i n h i b i t i o n o f growth o f b a c t e r i a w i t h c e r t a i n s u b s t r a t e s , as a r e s u l t o f the a c c u m u l a t i o n o f a p h o s p h o r y l a t e d i n t e r m e d i a t e , i s due t o t h e de-p l e t i o n o f i n t r a c e l l u l a r ATP o r i n o r g a n i c phosphate ( 3 , 1 ^ , 2 7 ) . I t has been r e p o r t e d t h a t an a c c u m u l a t i o n o f f r u c t o s e - 1 , 6 - d i p h o s p h a t e i n E_. c o l i i n h i b i t s the a c t i v i t y o f g l y c e r o l k i n a s e and r e p r e s s e s i t s 12 s y n t h e s i s by p r e v e n t i n g t h e s y n t h e s i s o f t h e i n d u c e r , L -a-GP ( 3 , 2 0 , 1 2 4 ) . Growth o f mutant s o f E_. c o l i , w h i c h a r e d e f i c i e n t i n phosphog1ucose i s omera se and G-6-P dehyd rogena se and a c c u m u l a t e G-6-P f rom g l u c o s e as a r e s u l t o f t h e s e m u t a t i o n s , i s i n h i b i t e d w i t h c e r t a i n o t h e r c a r b o n s o u r c e s i n t he p r e s e n c e o f g l u c o s e . I t has been shown t h a t f r u c t o s e d i p h o s p h a t a s e a c t i v i t y i n t h i s o r g a n i s m i s i n h i b i t e d i n v i t r o by G-6-P and t h i s may e x p l a i n t he i n h i b i t o r y e f f e c t o f G-6-P i n v i v o ( 2 9 ) - In a n o t h e r r e p o r t , a mutant o f S_. t yph imur iurn, l a c k i n g mann i t o 1 - 1 -phosphate d e h y d r o g e n a s e , was f ound t o a c c u m u l a t e m a n n i t o l - 1 -phosphate f rom m a n n i t o l m e t a b o l i s m . A c e t a t e i n c o r p o r a t i o n i n t o l i p i d s was i n h i b i t e d v e r y e a r l y a f t e r e x p o s u r e o f t he mutant c e l l s t o m a n n i t o l and t h i s was p r i o r to t he i n h i b i t i o n o f the i n -c o r p o r a t i o n o f p r e c u r s o r s i n t o n u c l e i c a c i d s , p r o t e i n , and c e l l w a l l m a t e r i a l ( 5 2 ) . T h e r e f o r e , i t a p p e a r s t h a t i n h i b i t i o n o f g rowth w i t h c e r t a i n s u b s t r a t e s as a r e s u l t o f t h e a c c u m u l a t i o n o f a p h o s p h o r y l a t e d i n t e r m e d i a t e due t o a m e t a b o l i c b l o c k i n the d i s s i m i l a t i o n o f some o t h e r s u b s t r a t e , i s due t o some e f f e c t on an enzyme(s ) o r c e l l u l a r f u n c t i o n ( s ) e s s e n t i a l t o g rowth w i t h t h a t s u b s t r a t e . The r e g u l a t i o n o f c a t a b o l i c enzymes i n the pseudomonads a p p e a r s t o d i f f e r - s i g n i f i c a n t l y f r om t h a t o f t he E n t e r o b a c t e r i a c e a e . C o n s e q u e n t l y , a s t u d y o f the i n d u c t i o n o f c a r b o h y d r a t e - c a t a b o 1 i z i n g enzymes d u r i n g g rowth w i t h d i f f e r e n t c a rbon s o u r c e s was c a r r i e d ou t w i t h P_. a e r u g i n o s a ATCC 9027. C a t a b o l i c mutant s o f t h i s m i c r o o r g a n i s m were a l s o employed i n an e f f o r t t o d e t e r m i n e t he a c t u a l i n d u c e r ( s ) o f t he enzymes o f c a r b o h y d r a t e c a t a b o l i s m . 13 MATERIALS AND METHODS I. Organ i sms. Pseudomonas a e r u g i n o s a ATCC 9027 and g l u c o s e - n e g a t i v e m u t a n t s , d e -r i v e d f rom the p a r e n t s t r a i n , were used t h r o u g h o u t t h i s s t u d y . G l u c o s e -n e g a t i v e mutant s were o b t a i n e d by the n a t u r a l s e l e c t i o n p r o c e d u r e o f O r n s t o n , O r n s t o n and Chou ( 9 1 ) , m o d i f i e d by t he a d d i t i o n o f h% g l y c i n e t o the l y t i c c y c l e (73,7M and f o l l o w e d by f o u r o r more e n r i c h m e n t c y c l e s . I s o l a t e d c l o n e s o f t he p a r e n t s t r a i n were s e l e c t e d and grown to s t a t i o n a r y phase i n m i n i m a l medium ( see s e c t i o n I l ) , c o n t a i n i n g 0.8 mM p y r u v a t e . A d d i t i o n a l p y r u v a t e (5 mM) was t hen added t o r e i n s t a t e g r owth i n t h e s t a t i o n a r y phase c u l t u r e . A f t e r i n c u b a t i o n a t 30 C f o r 30 m i n , 10 mM g l u c o s e was added and a f u r t h e r i n c u b a t i o n p e r i o d o f 60 min a t 30 C was emp loyed . The c e l l s were then h a r v e s t e d a t room t e m p e r a t u r e by c e n t r i f u g a t i o n a t 6,000 x g_ f o r 10 m i n , washed and r e su spended i n l y t i c medium c o n t a i n i n g m i n i m a l medium w i t h 10 mM g l u c o s e , 0.1 mg D - c y c l o s e r i n e / m l , 1 x 10 u n i t s p e n i c i l l i n G/ml, and 0.0k g g l y c i n e / m l . The c e l l s were t hen i n c u b a t e d i n t he l y t i c medium a t 30 C f o r 5 h o u r s . T h i s p r o c e d u r e was c a r r i e d o u t a minimum o f f i v e t i m e s . A f t e r e n r i c h m e n t , samples were p l a t e d o n t o t he i n i t i a l s e l e c t i o n medium and i n c u b a t e d a t 30 C. Mutan t s were s e l e c t e d as m i n u t e c o l o n i e s on m i n i m a l aga r p l a t e s c o n t a i n i n g 11 mM g l u c o s e (0.2%) and 5-7 mM p y r u v a t e ( 0 . 05% ) - The mutant s were then examined f o r t h e i r a b i l i t y t o grow w i t h p y r u v a t e and t h e i r i n a b i l i t y t o grow w i t h g l u c o s e as t he s o l e c a r b o n s o u r c e . Mu tan t s were i s o l a t e d by D. B lew. Mutant 3 was o b t a i n e d by W.W. Kay f o l l o w i n g N - m e t h y 1 - N - n i t r o - N 1 -n i t r o s o g u a n i d i ne (NTG)muTtag.ehe.sis as p r e v i o u s l y r e p o r t e d (56) and f o l l o w i n g i s o l a t i o n as a m i n u t e c o l o n y a f t e r p l a t i n g on m in ima l a g a r c o n t a i n i n g 0.2% g l u c o s e and 0.01% p y r u v a t e as c a r b o n s o u r c e s . The p a r e n t s t r a i n was m a i n t a i n e d on g l u c o s e (0.2%) m i n i m a l a ga r s l a n t s and t he g l u c o s e - n e g a t i v e mutan t s were m a i n t a i n e d on p y r u v a t e (0.2%) m i n i m a l a ga r s l a n t s . The mutant s t r a i n s were r e g u l a r l y checked f o r p u r i t y and f o r the a p p e a r a n c e o f r e v e r t a n t s t r a i n s by p l a t i n g samples on bo th g l u c o s e and p y r u v a t e m i n i m a l aga r p l a t e s . Both c e l l u l a r and c o l o n i a l morpho logy were e xam ined . The p a r e n t s t r a i n was a l s o examined r e g u l a r l y by p l a t i n g on s u i t a b l e med i um,_i_. e_. K i n g ' s and a c e t a m i d e a ga r p l a t e s . I I. Growth and M e d i a . The g rowth medium (m in ima l medium) c o n s i s t e d o f NH^h^PO^, 0.3%; K ^ P O ^ , 0.2%; i r o n as F e S O ^ h ^ O , 0.5 ug/ml ; MgSO^-yh^O, 0.5%; c a r b o n s o u r c e as i n d i c a t e d , d i s s o l v e d i n d i s t i l l e d w a t e r and a d j u s t e d t o pH 7.k w i t h 5N K0H. S t e r i l e c a r b o n s o u r c e s and MgSO^•7H_0 were added t o the a u t o c l a v e d medium b e f o r e i n o c u l a t i o n . Carbon s o u r c e s and MgSO^ were g e n e r a l l y p r e p a r e d as 10% s o l u t i o n s and s t e r i l i z e d by f i l t r a t i o n t h r ough 0.3 um membrane f i l t e r s ( M i l l i p o r e C o r p . , B e d f o r d , M a s s . ) . Growth e x p e r i m e n t s were c a r r i e d ou t i n 250 ml s i d e - a r m f l a s k s w i t h 25 ml m i n i m a l medium o r i n 125 ml s i d e - a r m f l a s k s w i t h 12.5 ml m i n i m a l medium, g e n e r a l l y c o n t a i n i n g 0.2% o r 0.k% c a r b o n s o u r c e , o r w i t h f u r t h e r a d d i t i o n s as i n d i c a t e d i n t h e T a b l e s and F i g u r e s , on a m e t a b o l i t e - s h a k i n g w a t e r b a t h (New B r u n s w i c k S c i e n t i f i c C o . , New B r u n s w i c k , N . J . ) a t 30 C 15 and 200 rpm. Growth was measured w i t h a K l e t t - S u m m e r s o n p h o t o e l e c t r i c c o l o r i m e t e r e q u i p p e d w i t h a No. 66 f i l t e r . A p p r o p r i a t e c o r r e c t i o n s were c a r r i e d o u t t o e s t a b l i s h l i n e a r i t y between K l e t t r e a d i n g s and o p t i c a l d e n s i t y a t 660 nm. Growth f o r enzyme a s s a y s was c a r r i e d o u t i n 2 1 E r l e n m e y e r f l a s k s c o n t a i n i n g 300 o r kSO ml o f m i n i m a l medium a t 30 C and 300 rpm on a C o n t r o l l e d Env i r onment I n c u b a t o r Shaker o r on an Aquatherm Water Ba th Shaker (New B r u n s w i c k S c i e n t i f i c Co. I n c . , New B r u n s w i c k , N . J . ) . The c o n c e n t r a t i o n o f t he c a r b o n s o u r c e was 0.k% i n each c a s e o r w i t h f u r t h e r a d d i t i o n s as i n d i c a t e d i n the T a b l e s and F i g u r e s . Growth was measured a t an o p t i c a l d e n s i t y o f 660 nm w i t h a model B s p e c t r o p h o t o m e t e r (Beckman I n s t r umen t s I n c . , F u l l e r t o n , C a l i f . ) . In a l l i n s t a n c e s , f l a s k s were i n o c u l a t e d w i t h 1 t o k% o f a l o g phase c u l t u r e p r e v i o u s l y grown i n t he same medium as t h a t used i n t he sub sequen t e x p e r i m e n t s , e x c e p t when g l u c o s e - n e g a t i v e mutant s were t e s t e d f o r g rowth w i t h g l u c o s e , g l u c o n a t e , 2-KG, o r g l y c e r o l . In t h e s e l a t t e r c a s e s , the i n ocu l um was grown i n p y r u v a t e m i n i m a l medium. I I I . 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 . A t v a r i o u s t ime s d u r i n g g rowth as i n d i c a t e d i n t he T a b l e s and F i g -u r e s , 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 by c e n t r i f u g a t i o n a t 10,000 x £ f o r 10 m i n , washed t w i c e w i t h 0.1 M t r i s ( h y d r o x y m e t h y 1 ) a m i n o -m e t h a n e - C l ( t r i s ) b u f f e r (pH 7.k) and t he r e s u l t i n g c e l l p e l l e t s were s t o r e d a t -70 C. S t o r a g e o f packed c e l l s a t -70 C f o r as l ong as t h r e e month s , d i d not e f f e c t t he a c t i v i t y o f the enzymes exam ined . C e l l p e l l e t s 16 were re su spended i n 0.1 M t r i s b u f f e r (pH 7-^0 t o an o p t i c a l d e n s i t y a t 660 nm o f 50 f o l l o w e d by t h e a d d i t i o n o f 100 ug o f d e o x y r i b o n u c l e a s e (2 mg/ml aqueous s o l u t i o n ) pe r 2.0 ml o f c e l l 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 F r ench p r e s s u r e c e l l a t 17,000 p s i and c e n t r i f u g e d a t 10,000 x £ f o r 10 min a t 6 C, t o remove w h o l e c e l l s and d e b r i s . The r e s u l t i n g c e l l - f r e e e x t r a c t s were s t o r e d i n i c e . When compa r i n g t h e a c t i v i t y o f 6-PG d e h y d r a t a s e p l u s KDGP a l d o l a s e i n the p a r e n t s t r a i n and g l u c o s e - n e g a t i v e m u t a n t s , c e l l - f r e e e x t r a c t s were s u b j e c t e d t o a f u r t h e r c e n t r i f u g a t i o n a t 25,000 x £ f o r 30 min a t 6 C t o remove e x c e s s reduced n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e (NADH) o x i d a s e a c t i v i t y ( 6 ) . I V. Enzyme a s s a y s . 1. G l u c o s e - c a t a b o l i z i n g enzymes. G l u c o s e dehyd rogena se i n c e l l - f r e e e x t r a c t s was measured by a s l i g h t m o d i f i c a t i o n o f the method o f Hauge (hk). The i n c u b a t i o n m i x t u r e c o n t a i n e d 20 mM p o t a s s i u m pho spha te b u f f e r (pH 6 . 0 ) , 0.14 mM 2 , h - d i c h l o r o -pheno l i n d o p h e n o l , 2.5 mM g l u c o s e and c e l l - f r e e e x t r a c t . The r a t e o f d e -c r e a s e i n o p t i c a l d e n s i t y a t 600 nm was measu red . G l u c o n a t e dehyd rogena se i n c e l l - f r e e e x t r a c t s was a s s a y e d i n t he same manner e x c e p t t h a t 5 mM p o t a s s i u m g l u c o n a t e r e p l a c e d g l u c o s e . G l u c o k i n a s e , G-6-P dehyd rogena se , and 6-PG d e h y d r a t a s e p l u s KDGP a l d o l a s e were a l l measured by a s l i g h t m o d i f i c a t i o n o f t he methods o f Von T i g e r s t r o m and Campbe l l ( 1 18 ) . The r e a c t i o n m i x t u r e f o r g l u c o k i n a s e c o n t a i n e d 40 mM N - 2 - h yd roxye thy1 p i p e r a z i n e -N ' - 2 - e t h a n e s u l f o n i c a c i d (Hepes) b u f f e r (pH 7-h), 10 mM M g C l ^ H - O , 2.5 mM a d e n o s i n e t r i p h o s p h a t e (ATP ) , 0.2 mM n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e pho spha te (NADP), 2 .5 mM g l u c o s e , e x ce s s c ommerc i a l G - 6 - P d e h y d r o g e n a s e , and c e l l - f r e e e x t r a c t . The r e a c t i o n m i x t u r e f o r the G - 6 - P dehyd rogena se a s s a y c o n t a i n e d kO mM t r i s b u f f e r (pH 1 .k) , 1 mM M g C l . ' G H - O , 0.2 mM NADP, 2.5 mM G - 6 - P , and c e l l - f r e e e x t r a c t . In the 6-PG d e h y d r a t a s e p l u s KDGP a l d o l a s e a s s a y , the d e h y d r a t a s e was r a t e l i m i t i n g (55) and t he r e a c t i o n m i x t u r e c o n s i s t e d o f kO mM Hepes b u f f e r (pH 8.0), 0.16 mM NADH, 1.25 mM 6 - P G , e x c e s s c ommerc i a l l a c t i c d e h y d r o g e n a s e , and c e l l - f r e e e x t r a c t . The a s s a y m i x t u r e f o r g l u c o n o k i n s e c o n t a i n e d kO mM t r i s b u f f e r (pH 7-h), 10 mM MgCl 2-6H_0, 1.0 mM NADP, 2.5 mM ATP, 5 mM p o t a s s i u m g l u c o n a t e , e x ce s s c ommerc i a l 6-PG dehyd rogena se , and c e l l - f r e e e x t r a c t . Exces s ammonium s u l f a t e , w h i c h i n t e r f e r e s w i t h the k i n a s e a s s a y s was removed f r om the commerc i a l 6-PG d e h y d r o g e n a s e . The enzyme s u s p e n s i o n was f i l t e r e d t h r ough a 0 .3 urn membrane f i l t e r ( M i l l i p o r e C o r p . , B e d f o r d , M a s s . ) , t he f i l t r a t e was d i s c a r d e d and the f i l t e r was re su spended and washed i n an equa l vo lume o f 0.05 M t r i s b u f f e r (pH 1.k). The 2-KG k i n a s e p l u s 2 - ke to-6 - pho spho -g l u c o n a t e (2-K-6-PG) r e d u c t a s e a s s a y , i n w h i c h 2-KG k i n a s e was the r a t e l i m i t i n g enzyme, was a m o d i f i c a t i o n o f t he p r o c e d u r e o f W.W. Kay (55). The s y s tem c o n s i s t e d o f kO mM t r i s and 5 mM g1ycy1 - g1yc i ne b u f f e r (pH 7 . 4 ) , 5 mM MgCl 2-6H_0, 0.2 mM NADPH, 2.5 mM ATP, 10 mM i o d o a c e t a m i d e , 2.5 mM 2-KG and c e l l - f r e e e x t r a c t . For t h e l a t t e r 5 enzymes t h e r a t e o f change i n o p t i c a l d e n s i t y a t 3^0 nm was r e c o r d e d . 2. G l y c o l y t i c enzymes. The enzymes 3~PG dehyd rogena se , 3 ~ p h o s p h o g1 y c e r a t e k i n a s e , 2 , 3 " d i p h o s p h o g l y c e r a t e muta se , e n o l a s e , and p y r u v i c k i n a s e were measured by 18 p r o c e d u r e s m o d i f i e d f r om t h e methods o f R.G. Von T i g e r s t r o m (117): 3-PG dehyd rogena se : kO mM t r i s b u f f e r (pH 8.5) , 0.2 mM NADP, 5 mM DL-3-PG, and c e l l - f r e e e x t r a c t ; 3 ~ p h o s p h o g l y c e r a t e k i n a s e : hO mM t r i s b u f f e r (pH 8.5) , 0.2 mM NADH, 2.5 mM ATP, 2.5 mM MgCl_.6r.-0, 2.5 mM 3" p h o s p h o - D - g l y c e r a t e , e x c e s s c ommerc i a l 3~PG d e h y d r o g e n a s e , and c e l l -f r e e e x t r a c t ; 2 , 3 ~ d i p h o s p h o g l y c e r a t e mutase was t h e same as f o r 3~phos-p h o g l y c e r a t e k i n a s e e x c e p t t h a t 2.5 mM 2 - p h o s p h o g l y c e r a t e r e p l a c e d 3 -p h o s p h o - D - g l y c e r a t e ; e n o l a s e : kO mM t r i s b u f f e r (pH 7.h), 0.2 mM NADH, 2.5 mM ADP, 10 mM M g C l - ^ H - O , 2.5 mM 2 - p h o s p h o g l y c e r a t e , e x c e s s c ommerc i a l p y r u v i c k i n a s e and l a c t i c d e h y d r o g e n a s e , and c e l l - f r e e e x t r a c t ; p y r u v i c  k i n a se : was t he same as f o r e n o l a s e e x c e p t t h a t c ommerc i a l p y r u v i c k i n a s e was o m i t t e d , 50 mM KC1. was a d d e d , and 2 - p h o s p h o g l y c e r a t e was r e p l a c e d by 2.5 mM 2 - p h o s p h o e n o I p y r u v a t e ; p y r u v i c dehyd rogena se c o m p l e x : was m o d i f i e d f r om t h a t o f Mukhe r j ee e_t aj_. (83) and c o n t a i n e d kQ mM p o t a s s i u m pho spha te b u f f e r (pH 7 - z 0 , 0.2 mM t h i a m i n e p y r o p h o s p h a t e , 1 mM NAD, 1 mM M g C l - ^ H - O , 0.1 mM f l a v i n a d e n i n e d i n u c l e o t i d e (FAD) , 0.25 mM coenzyme A i n 0.5 mg d i t h i o t h r e i t o l / m l , 5 mM p o t a s s i u m p y r u v a t e , and c e l l - f r e e e x t r a c t . A t t e m p t s t o i n h i b i t p y r u v i c dehyd rogena se a c t i v i t y w i t h 6-PG were p e r -fo rmed by a d d i n g up t o 50 umoles o f 6-PG/ml t o t he r e a c t i o n m i x t u r e and p r e -i n c u b a t i n g f o r 15 min b e f o r e a d d i n g p y r u v a t e t o i n i t i a t e the r e a c t i o n . For the above f i v e enzymes the r a t e o f change i n o p t i c a l d e n s i t y a t 3^0 nm was r e c o r d e d . 3- TCA c y c l e enzymes. A c o n i t a s e and i s o c i t r i c dehyd rogena se were a s s a yed a c c o r d i n g t o S i e b e r t (102) e x c e p t t h a t c ommerc i a l i s o c i t r i c dehyd rogena se was o m i t t e d 19 f r om the a c o n i t a s e r e a c t i o n m i x t u r e as t h e r e was an e x c e s s o f t h i s enzyme i n the c e l l - f r e e e x t r a c t . The a s s a y f o r the a - k e t o g l u t a r a t e dehyd rogena se complex was m o d i f i e d f r om the p r o c e d u r e o f Amaras ingham and Dav i s ( l ) and was composed o f 100 mM t r i s b u f f e r (pH 8 . 5 ) , 1 mM NAD, 5 mM e t h y l e n e -d i a m i n e t e t r a a c e t a t e (EDTA), 0.25 mM coenzyme A i n 0.5 mg d i t h i o t h r e i t o l / m l , 1 mM a - k e t o g l u t a r i c a c i d , and c e l l - f r e e e x t r a c t . M a l i c enzyme (NAD and NADP) was measured a t pH 8.5 by t he method o f J a c o b s e n e_t a_l_. ( 5 1 ) . For the above enzymes t he r a t e o f change i n o p t i c a l d e n s i t y a t 3^0 nm was r e c o r d e d . C i t r a t e s y n t h e t a s e was a s s a y e d as d e s c r i b e d i n the B i o c h e m i c a c a t a l o g u e ( B o e h r i n g e r Mannheim C o r p . , New Y o r k , N . Y . ) , s u c c i n y l - C o A s y n t h e t a s e was measured a c c o r d i n g t o G i b s o n __________ (33) e x c e p t t h a t 3 - m e r c a p t o e t h a n o l was o m i t t e d and coenzyme A was d i s s o l v e d i n 0.5 mg d i t h i o t h r e i t o l / m l , s u c c i n i c dehyd rogena se was by the p r o c e d u r e d e s c r i b e d by K i n g ( 6 0 ) , f umara se was d e t e r m i n e d by t h e method o f Racker ( 9 6 ) , and m a l i c dehyd rogena se was a s s a yed u s i n g 2 , 6 - d i c h l o r o p h e n o l i n dopheno l a c c o r d i n g t o F r a n c i s (32) a t pH 6 . 0 , e x c e p t t h a t KCN was o m i t t e d and 5 mM EDTA was added ( 114 ) . k. G l y c e r o l enzymes. G l y c e r o l k i n a s e was a s s a yed by a s l i g h t m o d i f i c a t i o n o f the method o f B u b l i t z and Wei l a nd ( 5 ) . The c ommerc i a l g l y c e r o k i n a s e was o m i t t e d a n d , due t o t he s e n s i t i v i t y o f the g l y c e r o l k i n a s e i n t he c e l l -f r e e e x t r a c t t o s u l f a t e , t he commerc i a l L -a-GP dehyd rogena se was f i l t e r e d i n the manner p r e v i o u s l y d e s c r i b e d f o r 6-PG dehyd rogena se . The change i n o p t i c a l d e n s i t y a t 3^0 nm was r e c o r d e d f o l l o w i n g the a d d i t i o n o f g l y c e r o l t o the r e a c t i o n m i x t u r e . The method f o r a s s a y i n g L -a-GP dehyd rogena se was t h a t o f K i s t l e r and L i n ( 6 l ) . A l l enzyme a s s a y s were c a r r i e d ou t i n 1.0 vo lumes a t 35 C i n q u a r t z c u v e t t e s w i t h a 1 cm l i g h t p a t h , i n a model 2000 m u l t i p l e sample a b s o r b -ance r e c o r d e r ( G i l f o r d I n s t r ument L a b o r a t o r i e s I n c . , O b e r l i n , O h i o ) . P r o t e i n a s s a y s were pe r f o rmed a f t e r t he method o f Lowry (72) w i t h c r y s t a l l i n e egg a l b u m i n as t h e s t a n d a r d p r o t e i n . Enzyme 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 nanomoles o f s u b s t r a t e c o n v e r t e d per m i n u t e per m i l l i g r a m o f p r o t e i n . V. I s o l a t i o n o f p r o d u c t s o f g l u c o s e , g l u c o n a t e , 2-KG, and g l y c e r o l  m e t a b o l i sm. 1. I s o l a t i o n o f p r o d u c t s o f g l u c o s e , g l u c o n a t e , and 2-KG m e t a b o l i s m f r om c u l t u r e s o f g l u c o s e - n e g a t i v e m u t a n t s . D u r i n g g rowth o f g l u c o s e - n e g a t i v e mutant s w i t h p y r u v a t e i n the p r e -14 sence o f u n i f o r m l y l a b e l l e d C - g l u c o s e , g l u c o n a t e , o r 2-KG o r f o l l o w i n g t he a d d i t i o n o f t h e s e r a d i o a c t i v e l y l a b e l l e d s i x - c a r b o n c a r b o h y d r a t e s to c u l t u r e s g r o w i n g i n p y r u v a t e medium, samples were removed a t the t i m e s i n d i c a t e d i n t h e T a b l e s and F i g u r e s and t r e a t e d w i t h t r i c h l o r o a c e t i c a c i d t o a f i n a l c o n c e n t r a t i o n o f 5% (w/v) . The samples were kep t on i c e f o r 20 min a f t e r w h i c h t i m e the i n s o l u b l e m a t e r i a l was removed by c e n t r i -f u g a t i o n and t he t r i c h l o r o a c e t i c a c i d was e x t r a c t e d f rom the s u p e r n a t a n t f l u i d w i t h 2 vo lumes o f e t h y l e t h e r . The s u p e r n a t a n t f l u i d was then washed o n t o a Dowex 1-x8 (100-200 mesh) f o r m a t e co lumn (0 .9 x 25 cm) and 21 e l u t e d w i t h 500 ml o f a f o r m i c a c i d g r a d i e n t (0 - 1.0 N) and t hen w i t h 200 ml o f 4 N f o r m i c a c i d and 0.4 N ammonium f o r m a t e a f t e r t h e method o f Kay and G r o n l u n d ( 57 ) . F r a c t i o n s (10 ml) were c o l l e c t e d w i t h a model 1205 a u t o m a t i c f r a c t i o n c o l l e c t o r ( W a r n e r - C h i 1 c o t t L a b o r a t o r i e s , I n s t r umen t s D i v . , R i chmond, C a l i f . ) o r a model 7000 f r a c t i o n c o l l e c t o r (LKB P r o d u k t e r , Bromma, Sweden) and a s s a y e d w i t h a model 43^2 a u t o m a t i c low backg round p l a n c h e t c o u n t i n g s y s t e m , o r a model 725 l i q u i d s c i n t i l l a t i o n s p e c t r o -meter ( N u c l e a r C h i c a g o C o r p . , Des P l a i n e s , 1 11 . ) . In t h e f o rme r c a s e samples were p l a t e d o n t o s t a i n l e s s s t e e l p l a n c h e t s a t i n f i n i t e t h i n n e s s and i n t h e l a t t e r c a s e one hundred u l o f each o f t he f r a c t i o n s were p i p e t t e d i n t o v i a l s u s i n g a m i c r o p i p e t t e r (Ox f o rd L a b o r a t o r i e s , San Ma teo , C a l i f . ) and coun ted i n 10 ml o f a b s o l u t e m e t h a n o l - t o l u e n e (40:60) c o n -t a i n i n g 42 ml o f L i q u i f 1 u o r / 1 i t e r (New Eng l and N u c l e a r , B o s t o n , M a s s . ) . F r a c t i o n s i n c l u d e d i n t he r a d i o a c t i v e peaks f rom the e l u t i o n p r o f i l e (eg. F i g u r e 4) were p o o l e d and t he f o r m a t e was removed by r e p e a t e d e v a p -o r a t i o n t o d r y n e s s . When r e q u i r e d , the e l u a n t s c o n t a i n i n g ammonium f o r m a t e were a l s o t r e a t e d w i t h Dowex-50 H + t o remove ammonium i o n s . These e x p e r i m e n t s were pe r f o rmed i n 125 ml s i d e - a r m f l a s k s c o n t a i n -ing 12.5 ml o f m i n ima l medium o r 250 ml s i d e - a r m f l a s k s c o n t a i n i n g 25 ml m i n i m a l medium, under t h e c o n d i t i o n s d e s c r i b e d f o r g rowth e x p e r i m e n t s . The c o n c e n t r a t i o n o f p y r u v a t e and t he c o n c e n t r a t i o n s and s p e c i f i c a c t i -v i t i e s o f t he r a d i o a c t i v e l y l a b e l l e d s i x - c a r b o n c a r b o h y d r a t e s were as d e s c r i b e d i n t he T a b l e s and F i g u r e s . 22 2. S e p a r a t i o n o f p r o d u c t s o f g l u c o s e - U - C c a t a b o l i s m i n t he c e l l w a t e r and medium o f g l u c o s e - n e g a t i v e m u t a n t s . C u l t u r e s o f mutant s t r a i n s XI I Ij and XVj were grown i n 125 ml s i d e -arm f l a s k s c o n t a i n i n g 12.5 ml o f m i n i m a l medium as d e s c r i b e d f o r g r owth c o n d i t i o n s w i t h c o n c e n t r a t i o n s o f c a r b o n s o u r c e s as l i s t e d i n T a b l e V. C e l l s 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 t he t imes i n d i c a t e d . The s u p e r -n a t a n t f l u i d ( c a l l e d the c e l l - f r e e medium) was removed. The r e m a i n i n g c e l l p e l l e t was washed once w i t h 10 ml o f m i n ima l medium minus s u b s t r a t e and then re su spended i n 5 ml o f a 5% t r i c h l o r o a c e t i c a c i d s o l u t i o n (w/v ) , s e t on i c e f o r 20 m i n , and c e n t r i f u g e d . The t r i c h l o r o a c e t i c a c i d s o l u b l e s u p e r n a t a n t ( c a l l e d t he c e l l w a t e r ) was removed and e x t r a c t e d w i t h 2 vo lumes o f e t h y l e t h e r . The c e l l w a t e r vo lume was d e t e r m i n e d by the method o f R o b e r t s e_t_ aj_. (98). A l l c e n t r i f u g a t i on s were c a r r i e d ou t a t 10,000 x £ f o r 10 m in a t 6 C. P r o d u c t s o f g l u c o s e m e t a b o l i s m i n the c e l l - f r e e medium and the c e l l w a t e r were i s o l a t e d by i o n - e x c h a n g e ch romatog raphy as p r e -v i o u s l y d e s c r i b e d . 14 3. I s o l a t i o n o f p r o d u c t s o f g l y c e r o l - U - C m e t a b o l i s m i n t h e w i l d -t y p e and g l u c o s e - n e g a t i v e mutant X l l l | _ ^ . To d e t e r m i n e t h e f a t e o f g l y c e r o l d u r i n g m e t a b o l i s m w i t h t he p a r e n t s t r a i n and mutant XI I I ^ , c u l t u r e s o f each o r g a n i s m were grown as d e -s c r i b e d f o r g rowth c o n d i t i o n s i n 250 ml s i d e - a r m f l a s k s c o n t a i n i n g 25 ml m i n i m a l medium and p y r u v a t e as t he c a r b o n s o u r c e . The c u l t u r e s were h a r v e s t e d a t an o p t i c a l d e n s i t y a t 660 nm o f a p p r o x i m a t e l y 1.0, washed w i t h 25 ml m i n i m a l medium minus s u b s t r a t e , and re su spended t o an o p t i c a l 23 d e n s i t y a t 660 nm, as r e p o r t e d i n T a b l e XV, i n 250 ml s i d e - a r m f l a s k s 14 c o n t a i n i n g 25 ml m i n i m a l medium and g l y c e r o l - U - C. The c u l t u r e s were i n c u b a t e d as d e s c r i b e d and samples were removed a t t he t i m e s i n d i c a t e d ( F i g . 25 , T a b l e XV ) . The samples were t r e a t e d w i t h a f i n a l c o n c e n t r a t i o n o f 5% t r i c h l o r o a c e t i c a c i d (w/v) and t h e p r o d u c t s o f g l y c e r o l m e t a b o l i s m were i s o l a t e d by i o n - e x c h a n g e ch romatog raphy as d e s c r i b e d . The p y r u v a t e 14 c o n c e n t r a t i o n and t he c o n c e n t r a t i o n and s p e c i f i c a c t i v i t y o f g l y c e r o l - U - C a r e r e p o r t e d i n F i g . 26. 14 4 . I s o l a t i o n o f p r o d u c t s o f g l u c o s e - U - C m e t a b o l i s m i n the p a r e n t s t r a i n d u r i n g g rowth w i t h g l u c o s e . A c u l t u r e o f the p a r e n t s t r a i n was grown as d e s c r i b e d f o r g rowth c o n d i t i o n s i n a 250 ml s i d e - a r m f l a s k c o n t a i n i n g 35 ml o f m i n i m a l medium 14 w i t h g l u c o s e - U - C as t he c a r b o n s o u r c e . D u r i n g g rowth 5 ml samples were removed a t a p p r o p r i a t e t i m e i n t e r v a l s . The samples were t r e a t e d w i t h t r i c h l o r o a c e t i c a c i d ( f i n a l c o n c e n t r a t i o n 5% w/v) and t h e p r o d u c t s o f g l u c o s e m e t a b o l i s m were i s o l a t e d by i o n - e x c h a n g e ch romatog raphy as p r e v i o u s l y d e s c r i b e d . The c o n c e n t r a t i o n and s p e c i f i c a c t i v i t y o f g l u c o s e -14 U- C i s r e p o r t e d i n F i g . 11. V I . I d e n t i f i c a t i o n o f p r o d u c t s i s o l a t e d by i o n - e x c h a n g e c h r o m a t o g r a p h y . G l u c o s e , g l u c o n a t e , 2-KG, and 6-PG were i d e n t i f i e d by paper e l e c t r o -p h o r e s i s u s i n g Whatman #4 f i l t e r paper (8 x 20 .5 in) and a w a t e r - c o o l e d e l e c t r o p h o r e s i s a p p a r a t u s s i m i l a r t o a Resco model E-800-2B e q u i p p e d w i t h a Resco model 1.11 power s u p p l y a t 750 v o l t s f o r 1.5 hour s i n 0.1 M ammonium c a r b o n a t e (NH.HC0-.NH-COONH,-Ana 1ar) b u f f e r (pH 8.6). A l l paper e l e c t r o p h o r e t o g r a m s were c a r r i e d ou t i n d u p l i c a t e and d e v e l o p e d i n bo th s i l v e r n i t r a t e r eagen t (2) and a n i 1 i n e - p h t h a l i c a c i d r e a g e n t (93) w h i c h a l l o w e d d i f f e r e n t i a t i o n between g l u c o n a t e and 2-KG w h i c h a r e s e p a r a t e d by co lumn ch romatog raphy but not by paper e l e c t r o -p h o r e s i s . R a d i o a c t i v e a r e a s on paper e l e c t r o p h o r e t o g r a m s were d e t e c t e d w i t h an A c t i g r a p h I I I model 1002 ( N u c l e a r - C h i c a g o C o r p . , Des P l a i n e s , 111 . ) . G l u c o s e was f u r t h e r i d e n t i f i e d by the G l u c o s t a t a s s a y ( W o r t h i n g t o n B i o c h e m i c a l C o r p . , F r e e h o l d , N . J . ) . G l u c o n a t e and 2-KG were f u r t h e r i d e n t i f i e d by coch romatog r aphy o f t h e i s o l a t e d peaks w i t h u n l a b e l l ed g l u c o n a t e o r 2-KG r e s p e c t i v e l y o v e r Dowex l -x8 f o r m a t e co l umns . S i x -p h o s p h o g l u c o n a t e was f u r t h e r i d e n t i f i e d w i t h commerc i a l 6-PG d e h y d r o g e n -a se and by d e p h o s p h o r y l a t i o n w i t h commerc i a l b a c t e r i a l a l k a l i n e pho spha -t a s e and coch romatog ramming w i t h u n l a b e l l e d g l u c o n a t e o v e r a Dowex I-x8 f o r m a t e co l umn . For t h e 6-PG a s s a y , 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 kO mM t r i s b u f f e r (pH l . h ) , 0.5 mM NADP, 0 - 0 . 1 umole 6-PG s t a n d a r d o r an a p p r o p r i a t e vo lume o f s amp le . The change i n o p t i c a l d e n s i t y a t 3^0 nm was f o l l o w e d . For dephosphory1 a t i o n , an a p p r o p r i a t e vo lume o f sample was i n c u b a t e d w i t h 0.2 M t r i s b u f f e r (pH 8.5) and e x ce s s c ommerc i a l b a c t e r i a l a l k a l i n e p h o s p h a t a s e a t 37 C f o r 30 m i n . G l y c e r o l was i d e n t i f i e d by t he method o f B u b l i t z and Wei l and (5)• D i h y d r o x y a c e t o n e pho spha te was i d e n t i f i e d w i t h commerc i a l L -a-GP d e h y d r o -genase i n t he p r e s e n c e o f kO mM t r i s b u f f e r (pH 7-*0 and 0.14 mM NADH 25 by f o l l o w i n g the change i n o p t i c a l d e n s i t y a t 3^0 nm. A s s a y s f o r 6-PG, g l y c e r o l , and d i h y d r o x y a c e t o n e pho spha te were c a r r i e d o u t under t h e c o n d i t i o n s p r e v i o u s l y d e s c r i b e d f o r enzyme a s s a y s . 1 2 V I I . I d e n t i f i c a t i o n o f C - p r o d u c t s i n the g rowth medium. G l u c o s e was d e t e r m i n e d by the G l u c o s t a t p r o c e d u r e ( W o r t h i n g t o n B i o c h e m i c a l C o r p . , F r e e h o l d , N . J . ) . W i th c u l t u r e s u s i n g g l u c o n a t e as a c a r b o n s o u r c e , g l u c o n a t e d i s -a p p e a r a n c e was measured e n z y m a t i c a 1 1 y w i t h a c r u d e p r e p a r a t i o n o f g l u c o n a t e dehyd rogena se f rom a c e l l - f r e e e x t r a c t o f g l u c o s e - g r o w n P_. ae rug 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 0.05 M p o t a s s i u m pho spha te b u f f e r (pH 6 . 0 ) , 0 .15 mM 2 , 6 - d i c h l o r o p h e n o l i n d o p h e n o l , a p p r o x i m a t e l y 0.15 mg p r o t e i n c o n t a i n i n g e x ce s s g l u c o n a t e dehyd rogena se , and 0 - 0.125 umole o f g l u c o n a t e s t a n d a r d o r an a p p r o p r i a t e vo lume o f s a m p l e , i n a t o t a l vo lume o f 1.0 m l . The change i n o p t i c a l d e n s i t y a t 600 nm was f o l l o w e d . The o - p h e n y 1 e n e d i a m i n e p r o c e d u r e o f L ann i ng and Cohen (67) was used t o q u a n t i t a t e 2-KG i n the v a r i o u s c u l t u r e s . Samples i n a t o t a l vo lume o f 2.0 ml were mixed w i t h 1.0 ml o f o - p h e n y 1 e n e d i a m i n e - 2 H C l (25 mg/ml ) , hea ted a t 100 C f o r 30 m i n , and o p t i c a l d e n s i t y was measured a t 335 nm. S t a n d a r d s f o r g l u c o s e , g l u c o n a t e , 2-KG, p y r u v a t e , and a - k e t o g l u t a r a t e were p r e p a r e d , and s p e c t r a o f t he d e r i v a t i v e s fo rmed were r e c o r d e d w i t h a model DBG r e c o r d i n g s p e c t r o p h o t o m e t e r (Beckman I n s t r u m e n t s , F u l l e r t o n , C a l i f . ) . G l u c o n a t e d i d not fo rm a d e r i v a t i v e w h i c h a b s o r b e d l i g h t between 300 and hOO nm, 720 yg 2 - K G , 50 yg p y r u v a t e and 50 yg a - k e t o g l u t a r i c a c i d 26 formed d e r i v a t i v e s w i t h a peak a t 335 nm. S t a n d a r d c u r v e s f o r bo th g l u c o s e and 2-KG were c a r r i e d ou t each t i m e samples were a s s a y e d , w h i c h c o n t a i n e d bo th o f t h e s e compounds, and a p p r o p r i a t e c o r r e c t i o n s f o r i n t e r -f e r e n c e f rom g l u c o s e were made. P y r u v i c a c i d and a - k e t o g l u t a r i c a c i d were a s s a y e d f o r u s i n g t h e a p p r o p r i a t e c o m m e r c i a l enzymes. The a s s a y f o r p y r u v i c a c i d c o n t a i n e d 0.10 M p o t a s s i u m pho spha te b u f f e r (pH 7-6), 0.26 ymo le NADH, 0.10 mg com-m e r c i a l l a c t i c dehyd rogena se , and 0 - 0.28 umole o f s t a n d a r d p o t a s s i u m p y r u v a t e o r 300 y l o f s amp le . The a s s a y f o r a - k e t o g l u t a r i c a c i d c o n t a i n e d 0.10 M p o t a s s i u m pho spha te b u f f e r (pH 7.6), 20 ymo le s N H ^ C l , 0.26 ymo le NADH, 0.20 mg L - g l u t a m i c d e h y d r o g e n a s e , and 0 - 0.26 ymo le o f s t a n d a r d a -k e t o g l u t a r i c a c i d o r 100 - 300 y l o f s amp le . The d e c r e a s e i n o p t i c a l d e n s i t y a t 3^0 nm was f o l l o w e d . A s s a y s f o r g l u c o n a t e , p y r u v a t e , and a - k e t o g 1 u t a r a t e were p e r f o r m e d under t h e c o n d i t i o n s d e s c r i b e d f o r enzyme a s s a y s . V I I I . C h e m i c a l s . P o t a s s i u m p y r u v a t e , p o t a s s i u m g l u c o n a t e , L - a - G P , and the c ommerc i a l enzymes d e o x y r i b o n u c l e a s e 1, g l y c e r o k i n a s e , L - a - G P d e h y d r o g e n a s e , 3~PG d e h y d r o g e n a s e , p y r u v i c k i n a s e , and L - g l u t a m i c dehyd rogena se were o b t a i n e d f rom C a l b i o c h e m , Los A n g e l e s , C a l i f . L a c t i c d e h y d r o g e n a s e , 6-PG d e h y d r o -gena se , G-6-P d e h y d r o g e n a s e , and 6-PG were o b t a i n e d f rom Sigma Chemica l C o . , S t . L o u i s , Mo., and b a c t e r i a l a l k a l i n e p h o s p h a t a s e was f rom W o r t h i n g -t on B i o c h e m i c a l C o r p . , F r e e h o l d , N . J . U n i f o r m l y l a b e l l e d g l u c o s e , g l u c o n a t e , and g l y c e r o l were pu r cha sed f rom Amer sham/Sea r l e , Des P l a i n e s , 111. T w o - k e t o g l u c o n a t e was a generous g i f t f r om K. S m i l e y , P e o r i a , 1 11 . , and f u r t h e r p u r i f i e d by R.P. L o n g l e y . U n i f o r m l y l a b e l l e d 2-KG was p r e -1 k pa red f rom g l u c o s e - U - C as p r e v i o u s l y r e p o r t e d (55). O the r b i o c h e m i c a l were f rom the u s ua l c ommerc i a l s u p p l i e r s . 28 RESULTS AND DISCUSSION I. Character iza t ion of g lucose-negat ive mutants of P_. aeruginosa. In order to examine the regulat ion of g lucose -ca tabo l i z ing enzymes, a number of mutants were i s o l a ted , by natural s e l e c t i o n , for the i r i n a b i l i t y to grow with glucose as the sole carbon source whi le r e t a i n -ing the i r a b i l i t y to grow with pyruvate. These mutants were not only incapable of growth with g lucose, but a l so with gluconate and 2-KG, which suggested that the metabolic block was in some enzyme common to the catabol ism of a l l three carbohydrates. It was a l so observed that growth of the mutants with pyruvate was inh ib i ted by the presence of glucose in the medium. To determine the nature of th i s i nh ib i to ry e f f e c t of g lucose, two mutants, XI I I^  and XV ^ , which exhib i ted somewhat d i f f e r e n t i nh ib i to ry responses to g lucose, were se lected and examined in deta i1 . 1. Growth of g lucose-negat ive mutants. The growth of both mutant s t ra in s in pyruvate minimal medium was normal j_.e_. s im i l a r to the wi ld - type (F ig . 1). Growth of XIII- and XV^ in pyruvate medium was inh ib i ted by the presence of g lucose, g l u -conate, or 2-KG. When s t r a in XV^ was grown with pyruvate in the presence of these s ix-carbon carbohydrates, i n h i b i t i o n of growth was eventual ly overcome a f t e r 20 to 40 hours incubat ion, when the growth rate approach-ed that observed when pyruvate was the sole carbon source (F ig . 1). This growth was found not to be due to revertants by comparing p late 1.0 E c 0.8 O CD 0.6 CD ITY 0.4 00 •z: • . L-J Q 0.2 < U h-CL O 0.11 10 15 20 HOURS 25 30 35 F ig . 1. The i nh ib i t o ry e f fec t of g lucose, gluconate, and 2-KG on the growth of g lucose-negative mutants of f_. aeruginosa. Symbols: XI I 11 or XVj with 0.023 M pyruvate, ( O ) ; XV.j with 0.023 M pyruvate plus 2.8 mM glucose, ( © ) ; plus 2.6 mM g luconate, ( • ); or plus 2.6 mM 2-KG, ( • ). ro vo c o u n t s on p y r u v a t e and g l u c o s e minimal p l a t e s . Growth i n h i b i t i o n on p y r u v a t e by t h e s e compounds i n s t r a i n XV| was i n the o r d e r o f g l u c o s e > g l u c o n a t e > 2-KG. When s t r a i n XI I I was grown under t h e s e same c o n d i -t i o n s , growth s t a s i s was co m p l e t e f o r a t l e a s t 60 hours a t wh i c h time i n c u b a t i o n was t e r m i n a t e d . The d u r a t i o n o f growth s t a s i s i n XV| was a l s o dependant on the c o n c e n t r a t i o n o f g l u c o s e i n the medium ( F i g . 2), j_.e_. t he h i g h e r the g l u c o s e c o n c e n t r a t i o n t h e l o n g e r t h e p e r i o d o f growth s t a s i s . In the case o f XI I I | , complete growth s t a s i s was a g a i n o b s e r v e d -h f o r a t l e a s t 60 hours w i t h as l i t t l e as 2.2 x 10 M g l u c o s e ( l o w e s t con-c e n t r a t i o n t e s t e d ) . Growth o f both mutants on o t h e r u t i l i z a b l e c a r b o n s o u r c e s ( a c e t a t e , c i t r a t e , i s o c i t r 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 , and malate) i n the absence and p r e s e n c e o f g l u c o s e was a l s o examined ( F i g . 3)- In the absence o f g l u c o s e , the growth r a t e s and p a t t e r n s o f both mutants were e s s e n t i a l l y i d e n t i c a l w i t h each o f t h e s e c a r b o n s o u r c e s and were s i m i l a r t o t h o s e o b t a i n e d w i t h the p a r e n t s t r a i n . However, i n the p r e s e n c e o f g l u c o s e , growth w i t h t h e s e s u b s t r a t e s showed a much d i f f e r e n t p a t t e r n than d i d growth w i t h p y r u v a t e p l u s g l u c o s e . W i t h s t r a i n X V j , the c a r b o n s o u r c e s were s i m i l a r i n t h a t t h e r e was no p r o l o n g e d p e r i o d o f growth s t a s i s however, the growth r a t e d i d e v e n t u a l l y t a p e r o f f t o a v e r y slow r a t e and s t a s i s was o b s e r v e d b e f o r e t h e normal s t a t i o n a r y phase was reached ( F i g . 3). These p a t t e r n s o f growth were a l m o s t i d e n t i c a l t o t h a t found w i t h p y r u v a t e i f g l u c o s e was not added u n t i l t he c u l t u r e had reached an o p t i -c a l d e n s i t y a t 660 nm (O.D.) o f 0.30 (see F i g . 5 ) . From t h e s e d a t a , i t F i g . 2. The e f f e c t o f g l u c o s e c o n c e n t r a t i o n on t he i n h i b i t i o n o f g r owth o f g l u c o s e -n e g a t i v e mutant XV 1 i n p y r u v a t e m i n i m a l medium. S ymbo l s : g r owth w i t h 0 .023 M p y r u v a t e , (o); 0 .023 M p y r u v a t e p l u s 2.2 x 10~ M g l u c o s e , ( • ) ; p l u s 2 .8 x 1 0 " 3 M g l u c o s e , ( • ) ; p l u s 5-5 x 1 0 ~ 3 M g l u c o s e , ( o ) ; and p l u s l . l x 1 0 ~ 2 M g l u c o s e , ( A ) . i—I—r E c O CD CO 1.5 1.0 0.8 >-H 0.6 co z Q 0.4 < o fc 0.2 n l I I I ! I I !_ 8 !0 12 14 16 i r i r i — i — r A, I I I A ± L • 8 12 HOURS 16 20 24 Fig. 3. The effect of glucose on the growth of glucose-negative mutants of P. aeruginosa with acetate, c i trate, isocitrate, a-ketoglutarate, succinate, fumarate, and malate. Substrate concentration was 0 . 2 % . Symbols: strain XV. in the absence, (•); and presence of 2 . 8 x 1 0 ~ ^ M glucose, (o); strain XIII in the absence, ( * ) ; and presence of 2 . 8 x 1 0 M glucose, ( A ) . i s a p p a r e n t t h a t d u r i n g g rowth o f XV^ w i t h t h e s e o t h e r u t i l i z a b l e s u b -s t r a t e s , g l u c o s e was u n a b l e t o e x e r t i t s i n h i b i t o r y e f f e c t n e a r l y as r a p i d l y as when p y r u v a t e was t he c a rbon s o u r c e . Growth o f X I11^ w i t h t h e s e d i f f e r e n t s u b s t r a t e s i n the p r e s e n c e o f g l u c o s e was s i m i l a r i n each ca se and was i n h i b i t e d s i g n i f i c a n t l y . The c u l t u r e e x h i b i t e d a p e r i o d o f g rowth s t a s i s o f a p p r o x i m a t e l y 15 hour s b e f o r e g rowth commenced ( F i g . 3 )• However, t h e r e was much l e s s i n h i b i t i o n o f g rowth w i t h t h e s e s u b s t r a t e s than found w i t h p y r u v a t e p l u s g l u c o s e where g rowth s t a s i s c o n t i n u e d f o r 60 hour s o r more. These r e s u l t s show t h a t w i t h X1 I I and XV^, g l u c o s e i s u n a b l e t o e x e r t as s e v e r e an i n h i b i t o r y e f f e c t on g rowth w i t h a c e t a t e o r TCA c y c l e i n t e r m e d i a t e s as i t i s w i t h p y r u v a t e . The d i f f e r e n c e s i n t h e deg ree o f t he i n h i b i t o r y e f f e c t o f g l u c o s e on t h e s e mutan t s grown w i t h p y r u v a t e v e r s u s a c e t a t e and TCA c y c l e i n t e r m e d -i a t e s was not examined f u r t h e r . However, i t has been r e p o r t e d t h a t i n F\ a e r u g i n o s a , a c e t a t e and TCA c y c l e i n t e r m e d i a t e s i n t e r f e r e w i t h o r i n h i b i t t he t r a n s p o r t o f g l u c o s e whereas p y r u v a t e does not ( 8 4 ) . F u r t h e r , o r g a n i c a c i d s have been shown to be u t i l i z e d p r e f e r e n t i a l l y as g rowth s u b s t r a t e s o v e r c a r b o h y d r a t e s such as g l u c o s e ( 37 ,38 ,39 ,115 ) and the s y n t h e s i s o f c a r b o h y d r a t e - c a t a b o l i z i n g enzymes i s r e p r e s s e d by t h e s e o r g a n i c a c i d s (hO, 8 6 , 8 7 ) . The enzymes a r e i nduced d u r i n g g rowth w i t h a m i x t u r e o f p y r u v a t e and g l u c o s e ( 6 8 ) . For t h i s r e a s o n , f u r t h e r e x p e r i m e n t s t o e l u c i d a t e t h e i n h i b i t o r y e f f e c t o f g l u c o s e i n t h e s e mutant s were c a r r i e d o u t w i t h p y r u v a t e as the c a r b o n s o u r c e . 2. I d e n t i f i c a t i o n o f t he p r o d u c t s o f g l u c o s e c a t a b o l i s m i n g l u c o s e -n e g a t i v e m u t a n t s . P r o d u c t s a c c u m u l a t i n g f rom g l u c o s e - U - C d e g r a d a t i o n were i d e n t i f i e d i n an e f f o r t t o d e t e r m i n e t he l o c a t i o n o f t h e m e t a b o l i c b l o c k i n t h e mutant s ( F i g . 4 , T a b l e l ) . P y r u v a t e grown c u l t u r e s o f X l l l | and XVj were used t o i n o c u l a t e m i n i m a l medium c o n t a i n i n g p y r u v a t e and g l u c o s e -14 14 U- C o r j u s t g l u c o s e - U - C. Samples were removed a t t he t i m e s i n d i c a t e d ( T a b l e I I ) and t h e t r i c h l o r o a c e t i c a c i d s o l u b l e m a t e r i a l was e l u t e d o ve r a Dowex l - x 8 co lumn (a t y p i c a l e l u t i o n p r o f i l e i s shown in F i g . 4 ) . On l y 4 d i f f e r e n t compounds were i s o l a t e d f rom the v a r i o u s samples 14 a s s a y e d and t hey a c c o u n t e d f o r e s s e n t i a l l y 100% o f t he g l u c o s e - U - C added ( T a b l e s I and I I ) . The r e s u l t s i n d i c a t e d t h a t g l u c o s e was f i r s t o x i d i z e d t o g l u c o n a t e and then t he g l u c o n a t e was c o n v e r t e d t o 2-KG and 6-PG. A t t he t e r m i n a t i o n o f t he e x p e r i m e n t , 2-KG r e p r e s e n t e d a t l e a s t 85% o f the t o t a l g l u c o s e added t o t he c u l t u r e and t he r e m a i n i n g r a d i o a c t i v i t y was i n 6-PG. The o x i d a t i v e r o u t e o f g l u c o s e t o 2-KG has been d e s c r i b e d p r e v i o u s l y ( 8 8 , 1 0 9 ) . A s i g n i f i c a n t a c c u m u l a t i o n o f 2-KG i n n i t r o g e n d e -f i c i e n t , g l u c o s e grown c u l t u r e s o f the p a r e n t s t r a i n o f P_. a e r u g ? nosa has been r e p o r t e d (76) a n d , t h e r e f o r e , such an a c c u m u l a t i o n i s not unexpec ted i n mutant s b l o c k e d i n g l u c o s e c a t a b o l i s m . The p a t t e r n o f l i m i t e d g l u c o s e d i s s i m i l a t i o n i n bo th mutant s was g e n e r a l l y s i m i l a r , however , t he r a t e o f c a t a b o l i s m was much more r a p i d i n XI I I| than i n XV|, even though g rowth was much more r e s t r i c t e d . The a c c u m u l a t i o n o f s i g n i f i c a n t amounts o f 6 - P G , i f r o m g l u c o s e c a t a b o l i s m i n bo th mutant s s u gge s t ed t he m e t a b o l i c b l o c k was a t t he l e v e l o f 6-PG d e h y d r a t a s e ; the f i r s t enzyme common t o t h e c a t a b o l i s m o f g l u c o s e , g l u c o n a t e , and 2-KG. T h i s wou ld e x p l a i n t he i n a b i l i t y o f t he mutant s t o grow i n medium c o n t a i n i n g any o f t h e s e 3 s i x - c a r b o n c a r b o h y d r a t e s as the s o l e c a r b o n and ene rgy s o u r c e . 25 CD - Q _ (/> __ o £ 3 LU 2Q 15 T •0-1 N FORMIC ACID o3 O o / \ ____ 4 N FORMIC ACID* 0.4N AMMONIUM FORMATE ? 4 10 50 60 70 F i g . h. 20 30 40 FRACTION NO. E lut ion p r o f i l e of C-compounds from a Dowex 1-Xo formate anion exchange ]h column fo l lowing g lucose-U- C catabol ism in g lucose-negat ive mutants of P. aerugi nosa. T a b l e I. I d e n t i t y o f r a d i o a c t i v e i n t e r m e d i a t e s a c c u m u l a t e d f rom g l 14 U- C c a t a b o l i s m i n mutant s XI I I and X V j . Peak N o . 3 Compound 1 G l u c o s e 2 G l u c o n a t e 3 2 - k e t o g l u c o n a t e 4 6 -pho sphog1ucona te From the e l u t i o n p r o f i l e i n F i g u r e 4. T a b l e I I . D e g r a d a t i o n p r o d u c t s o f g l u c o s e - U - C m e t a b o l i s m i n medium and c e l l s o f g l u c o s e - n e g a t i v e mutants o f P. a e r u g i n o s a . Mutant Ca r b o n a Sou rce Time h a r v e s t e d (hour s ) 14 O.D. a t P e r c e n t o f l a b e l r e c o v e r e d f rom g l u c o s e - U - C 660 nm G1ucose G l u c o n a t e 2-KG 6-PG XV^ P y r u v a t e p l u s g 1 u c o s e - U - 1 ^C 12.5 37-75 42.5 0.08 0.38 0.87 94 0 0 6.0 0 0 0 85 -7 85.1 0 14.3 14.9 14 G l u c o s e - U - C 32 29- 18.2 50.4 2.3 X I I I P y r u v a t e p i us g l u c o s e - U - l 4 c 12.5 37-75 42 .5 0.01 0.09 0.09 36 .7 0 0 36.9 0 0 24 .7 93-3 94 .3 1.7 6.7 5.7 14 G l u c o s e - U - C 45. 4.3 7.1 87 .0 1.6 P y r u v a t e c o n c e n t r a t i o n was 0.023 M and g1ucose -U-14_ c o n c e n t r a t i o n was 2.8 mM ( s p e c i f i c a c t i v i t y 0 .143 y C i / u m o l e ) . 14 No d e t e c t a b l e o p t i c a l d e n s i t y a t 660 nm. The s l ow r a t e o f g l u c o s e - U - C d e g r a d a t i o n was c a r r i e d o u t by t he i n o cu l um. 38 3. E x a m i n a t i o n o f g 1 u c o s e - c a t a b o l i z i ng enzymes i n g l u c o s e - n e g a t i v e mutants. To d e t e r m i n e c o n c l u s i v e l y the p o i n t o f the m e t a b o l i c b l o c k i n t h e g l u c o s e - n e g a t i v e mutants o f P_. aerug i nosa, t h e enzymes o f g l u c o s e c a t a -bol ism were a s s a y e d i n mutants X I I 1 ^ and XVj and compared t o the a c t i v i t i e s p r e s e n t i n the p a r e n t s t r a i n . The w i l d - t y p e o r g a n i s m was grown i n m i n i -mal medium w i t h p y r u v a t e o r g l u c o s e t o an O.D. o f a p p r o x i m a t e l y 1.0. When d e t e r m i n i n g t h e i n f l u e n c e o f g l u c o s e on c u l t u r e s growing w i t h p y r u v a t e , g l u c o s e was added to the c u l t u r e when i t had reached an O.D. o f a p p r o x i -m a t e l y 0.30 and growth was a l l o w e d t o c o n t i n u e f o r 2 hours b e f o r e h a r v e s t i n g ( F i g . 5). S i m i l a r l y , both mutant s t r a i n s were grown i n minimal medium w i t h p y r u v a t e , and a l s o w i t h p y r u v a t e t o an O.D. o f a p p r o x i m a t e l y 0.30 and then g l u c o s e was added. Growth was a l l o w e d t o c o n t i n u e and c e l l s were h a r v e s t e d 2 and 8 hours a f t e r g l u c o s e a d d i t i o n ( F i g . 5 ) . Growth o f the g l u c o s e - n e g a t i v e mutants began to t a p e r o f f s l o w l y a f t e r t h e a d d i t i o n o f g l u c o s e , i n c o n t r a s t t o t h e p a r e n t s t r a i n where the a d d i t i o n o f g l u c o s e i n c r e a s e d the growth r a t e . In s t r a i n X V j , the r a t e o f d e c r e a s e i n growth c o n t i n u e d t o i n c r e a s e u n t i l f i n a l l y o n l y a v e r y s l o w growth r a t e was e v i d e n t . However, i n s t r a i n XI I I | , t h e d e c r e a s e i n growth r a t e was s i g n i -f i c a n t l y f a s t e r and c o m p l e t e growth s t a s i s o c c u r r e d a p p r o x i m a t e l y 3 hours a f t e r the a d d i t i o n o f g l u c o s e . T h i s was i n agreement w i t h p r e v i o u s r e s u l t s w h i c h showed t h a t the i n h i b i t o r y e f f e c t o f g l u c o s e was more s e v e r e i n mutant XI I I| than i n mutant X V j . The s p e c i f i c a c t i v i t i e s o f the enzymes d e t e c t e d i n the w i l d - t y p e i—i—i—r 2.0 E c l A — B Ji L I I I i I I I r J Ss I I I I I I I I I I I " i — i — i — i — i — i — i — r c P-o ^L_l I i i i i i 8 10 4 6 8 10 12 14 H O U R S 4 6 8 10 12 F i g . 5. Growth o f w i l d - t y p e and g l u c o s e - n e g a t i v e mutan t s o f P_. a e r u g i n o s a . G l u c o s e was added t o c u l t u r e s g r o w i n g i n p y r u v a t e medium as i n d i c a t e d by a r r o w s . S y m b o l s : A. w i l d - t y p e w i t h 0 .045 M p y r u v a t e , ( o ) ; 0.045 M p y r u v a t e p l u s 0.011 M g l u c o s e , ( A ) ; 0.022 M g l u c o s e , ( • ) . B. C. XI 11^ symbol s as i n B. XV1 w i t h 0.045 M p y r u v a t e , ( o ) ; 0.045 M p y r u v a t e p l u s 0.011 M g l u c o s e , ( • ) s t r a i n ( T a b l e I I I ) a r e c o m p a t i b l e w i t h t h o s e o b t a i n e d p r e v i o u s l y w i t h t h i s (115,118) and o t h e r s t r a i n s o f P_. ae rug i nosa ( 3 8 , 5 0 , 6 8 ) , i n t h a t the enzymes o f g l u c o s e c a t a b o l i s m were p r e s e n t a t h i g h l e v e l s d u r i n g g rowth w i t h g l u c o s e o r w i t h a m i x t u r e o f p y r u v a t e and g l u c o s e . Low amounts o f t h e s e enzymes were d e t e c t e d i n c u l t u r e s grown w i t h a c e t a t e , p y r u v a t e , o r TCA c y c l e i n t e r m e d i a t e s . The ab sence o f any d e t e c t a b l e enzyme a c t i v i t y i n t he 6-PG d e h y d r a -t a s e p l u s KDGP a l d o l a s e a s s ay (ED pathway enzymes) i n bo th mutant s c o n f i r m e d the p r e v i o u s r e s u l t s , t h a t t he m u t a t i o n i n s t r a i n XI I I| and XVj was a t t h e l e v e l o f 6-PG d e h y d r a t a s e . A l t h o u g h t he p o s s i b i l i t y t h a t bo th 6-PG d e h y d r a t a s e and KDGP a l d o l a s e a r e m i s s i n g o r a r e n o n - f u n c t i o n a l canno t be e x c l u d e d (KDGP a l d o l a s e was not a s s a y e d d i r e c t l y due t o t he i n a v a i l -a b i l i t y o f t he s u b s t r a t e KDGP), t he r e v e r s i o n f r e q u e n c y o f bo th mutant s - 7 - 8 t o g rowth on g l u c o s e , g l u c o n a t e , o r 2-KG was 1 x 1 0 - 10 as wou ld be e x p e c t e d f o r a s i n g l e g e n e t i c l e s i o n . S e v e r a l u nexpec ted d i f f e r e n c e s were no ted i n t he mutant s t r a i n s when enzyme a c t i v i t i e s were compared t o t h o s e o f t he w i l d - t y p e c u l t u r e s . I n d u c t i o n o f g l u c o k i n a s e and G-6-P dehyd rogena se o c c u r r e d w i t h i n 2 hour s a f t e r t h e a d d i t i o n o f g l u c o s e t o c u l t u r e s o f the p a r e n t s t r a i n g r ow ing i n m i n i m a l p y r u v a t e medium. The re was a 4 - f o l d i n c r e a s e i n t he s p e c i f i c a c t i v i t y o f the f o r m e r enzyme and a 4 2 - f o l d i n c r e a s e i n the l a t t e r enzyme. When mutant XV| was grown w i t h p y r u v a t e , G-6-P dehyd rogena se a c t i v i t y was s i g n i f i c a n t l y g r e a t e r than i n w i l d - t y p e grown w i t h p y r u v a t e , however , i n d u c t i o n o f t h i s enzyme and o f g l u c o k i n a s e d i d not o c c u r a f t e r t he Table III. Induction of glucose degrading enzymes in wi ld-type and g lucose-negat ive mutants of P. aeruginosa. Organ i sms Carbon 3 ENZYME ACTIVITY b source Glucose dehydrogenase Gluconate dehydrogenase Gluco- G-6-P kinase dehydrogenase Glucono-ki nase ED pathway enzymes 3-PG dehydrogenase W+ Pyruvate 19 61 26 2 3 8 4 Pyruvate plus glucose 2 hours 230 613 94 84 64 119 129 Glucose 49 1560 196 163 123 160 232 XV, Pyruvate 4 19 23 12 145 0 C 202 Pyruvate plus glucose 2 hours 9 128 21 15 161 0 254 Pyruvate plus glucose 8 hours 15 602 19 15 136 0 366 XI 1 1 j Pyruvate 13 19 49 11 154 0 164 Pyruvate plus glucose 2 hours 180 492 59 21 138 0 201 Pyruvate plus glucose 8 hours 148 1175 51 21 122 0 245 a Cultures grown with 0.045 M pyruvate, 0.022 M glucose, or 0.045 M pyruvate with 0.011 M glucose added at an O.D. of approximately 0.30 (F ig. 5). Enzyme a c t i v i t y is expressed as nanomoles of substrate converted per min per mg p ro te in . No detectable a c t i v i t y . 42 a d d i t i o n o f g l u c o s e . When mutant XI11^ was grown w i t h p y r u v a t e , bo th g l u c o k i n a s e and G-6-P dehyd rogena se a c t i v i t i e s were c o n s t i t u t i v e l y g r e a t e r than i n t h e w i l d - t y p e p y r u v a t e grown c e l l s , bu t a g a i n , a s i g n i f i c a n t i n d u c t i o n o f t h e s e enzymes was not e v i d e n t f o l l o w i n g t he a d d i t i o n o f g l u c o s e . I t c anno t be a rgued t h a t g l u c o s e was not g e t t i n g i n t o the mutant s t r a i n s under t h e s e e x p e r i m e n t a l c o n d i t i o n s as i t i s a p p a r e n t f rom F i g . 5 t h a t g l u c o s e , o r a m e t a b o l i t e o f g l u c o s e , b e g i n s t o e x e r t an i n h i b i t o r y e f f e c t on g rowth o f t h e s e c u l t u r e s w i t h i n 1 hour a f t e r g l u c o s e a d d i t i o n . G l u c o n o k i n a s e and 3 _ PG dehyd rogena se were a l s o i nduced i n the w i l d -t y p e c u l t u r e s o f P_. a e r u g i n o s a by t h e p r e s e n c e o f g l u c o s e . The p r e s e n c e o f g l u c o s e f o r 2 hou r s i n p y r u v a t e g r o w i n g c e l l s r e s u l t e d i n a 2 1 - f o l d 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 g l u c o n o k i n a s e and 3 2 - f o l d i n c r e a s e i n 3 - P G dehyd rogena se . In bo th m u t a n t s , g l u c o n o k i n a s e was c o n s t i t u t i v e and the s p e c i f i c a c t i v i t y o f t he enzyme i n p y r u v a t e grown c u l t u r e s was a p p r o x i m a t e l y t he same as t h a t f ound i n the p a r e n t s t r a i n when grown w i t h g l u c o s e as the s o l e c a r b o n s o u r c e . S i m i l a r l y , i n bo th m u t a n t s , 3 _ PG dehyd rogena se was c o n s t i t u t i v e l y h i g h and t he s p e c i f i c a c t i v i t y o f the enzyme i n p y r u v a t e grown c u l t u r e s was 51 and 41 t ime s g r e a t e r f o r mutant s XVj and X I l l j r e s p e c t i v e l y , than i n t he p a r e n t s t r a i n grown w i t h p y r u v a t e . However, the p r e s e n c e o f g l u c o s e f o r 8 hou r s i n t he c u l t u r e s o f the mutant s g r o w i n g w i t h p y r u v a t e r e s u l t e d i n a s m a l l , but s i g n i f i c a n t i n c r e a s e i n t he s p e c i f i c a c t i v i t y o f t h i s enzyme j_.e_. f r om 51 and 41 t i m e s g r e a t e r t o 91 and 61 t ime s g r e a t e r t han t h a t i n t he w i l d - t y p e , p y r u v a t e grown c u l t u r e s . 43 These r e s u l t s s t r o n g l y s u gge s t t h a t t h e s e two m u t a n t s , w h i c h a r e d e f e c t i v e i n 6-PG d e h y d r a t a s e , have a l s o l o s t t h e i r normal r e g u l a t o r y c o n t r o l o v e r 4 o t h e r enzymes i n v o l v e d i n the c a t a b o l i s m o f s i x - c a r b o n c a r b o h y d r a t e s . A l t h o u g h t he e x a c t n a t u r e o f t h i s c o n t r o l i s not c l e a r a t t h i s p o i n t , t he d a t a i n d i c a t e s t h a t t he m u t a t i o n i n t h e 6-PG d e h y d r a -t a s e may a l s o be r e s p o n s i b l e , e i t h e r d i r e c t l y o r i n d i r e c t l y , f o r a l t e r i n g t h e normal c o n t r o l g e n e r a l l y e x e r t e d o v e r g l u c o k i n a s e , G-6-P d e h y d r o g e n -a s e , g l u c o n o k i n a s e , and 3 - P G dehyd rogena se . In t he c a s e o f the f o r m e r two enzymes, i t may be t h a t t h e i n d u c e r i s not p roduced by t h e s e mutan t s J_.e_. KDGP o r some d e g r a d a t i o n p r o d u c t t h e r e o f . The d a t a a l s o s u g ge s t t h e r e may be one mechanism f o r the c o n t r o l o f the f o rme r two enzymes and a n o t h e r mechanism f o r t he c o n t r o l o f t he l a t t e r two enzymes. The o n l y ma jo r d i f f e r e n c e s i n t h e enzyme s p e c i f i c a c t i v i t i e s d e t e c t e d between mutant s X I I I ^ and XVj were i n the g l u c o s e and g l u c o n a t e d e h y d r o -gena se s . The a d d i t i o n o f g l u c o s e t o s t r a i n XI I I^  g r ow ing w i t h p y r u v a t e r e s u l t e d i n an i n d u c t i o n o f bo th o f t h e s e enzymes t o s i m i l a r l e v e l s found i n the p a r e n t s t r a i n . However, i n mutant XV|, a l t h o u g h the a d d i t i o n o f g l u c o s e t o t he p y r u v a t e g r ow ing c u l t u r e appea red to c au se i n d u c t i o n o f t h e s e two enzymes, the deg ree o f i n d u c t i o n was not as s i g n i f i c a n t as i n e i t h e r mutant XI I I| o r the w i l d - t y p e o r g a n i s m , p a r t i c u l a r l y i n the c a s e o f g l u c o s e d e h y d r o g e n a s e . The " i n d u c e d " g l u c o s e dehyd rogena se i n XVj had , i n f a c t , t he same s p e c i f i c a c t i v i t y as the r e p r e s s e d enzyme i n bo th the p a r e n t s t r a i n and mutant X I I I . . R e v e r t a n t s t r a i n s o f bo th mutant s XI I I| and XV. , w h i c h had r e g a i n e d the a b i l i t y t o grow w i t h g l u c o s e as the s o l e c a r b o n s o u r c e , were o b t a i n e d a t a f r e q u e n c y s u g g e s t i n g a s i n g l e g e n e t i c l e s i o n -7 -8 f o r t he i n a b i l i t y t o grow w i t h g l u c o s e _i_.e_. 1 x 10 - 10 . R e v e r t a n t s o f mutant XI I I j appea red t o grow n o r m a l l y , _i_.e_. as the w i l d - t y p e w i t h e i t h e r p y r u v a t e o r g l u c o s e as the c a rbon s o u r c e . A l t h o u g h they grew n o r m a l l y i n p y r u v a t e m i n i m a l medium, r e v e r t a n t s o f mutant XV e x h i b i t e d a s l o w r a t e o f g rowth when g l u c o s e was p r e s e n t as the s o l e s o u r c e o f c a r b o n ( F i g . 6 ) . G l u c o s e - c a t a b o l i z i n g enzymes were examined i n one r e v e r t a n t o f each mutant ( T a b l e I V ) . In bo th c a s e s , normal 6-PG d e h y d r a t a s e p l u s KDGP a l d o l a s e a c t i v i t y had been r e g a i n e d as w e l l as normal c o n t r o l o f g l u c o -k i n a s e , G-6-P dehyd rogena se , g l u c o n o k i n a s e , and 3 - P G d e h y d r o g e n a s e , t h a t i s , s p e c i f i c a c t i v i t i e s were v e r y s i m i l a r t o t h o s e found i n the p a r e n t s t r a i n grown w i t h p y r u v a t e o r w i t h g l u c o s e ( T a b l e I I I ) . In t he r e v e r t a n t o f X l l l j , normal c o n t r o l was a l s o o b s e r v e d f o r bo th g l u c o s e and g l u c o n a t e dehyd rogena se as found i n t he o r i g i n a l mutant s t r a i n . However, i n the r e v e r t a n t o f mutant X V j , t h e s e two enzymes e x h i b i t e d a s i m i l a r p a t t e r n t o t h a t found i n the o r i g i n a l mutant s t r a i n j_.e_. l ower amounts o f bo t h enzymes than i n t h e p a r e n t s t r a i n i n bo th p y r u v a t e and g l u c o s e grown c u l t u r e s , p a r t i c u l a r l y i n t he c a s e o f g l u c o s e dehyd rogena se . These r e s u l t s s u g -g e s t e d t h a t mutant XI I I| had a s i n g l e m u t a t i o n a t t he l e v e l o f 6-PG d e h y d r a t a s e but t h a t mutant XV| had a second m u t a t i o n w h i c h r e s u l t e d i n l ower amounts o f bo th g l u c o s e and g l u c o n a t e d e h y d r o g e n a s e s , p a r t i c u l a r l y g l u c o s e dehyd rogena se . The d a t a p r e s e n t e d i n T a b l e II show t h a t g l u c o s e was m e t a b o l i z e d t o g l u c o n a t e , 2 -KG, and 6-PG a t a much f a s t e r r a t e i n X I I I . 2 6 1 0 1 4 HOURS g. 6. Growth of w i ld - type and revertants of g lucose-negative mutants of P_. aeruginosa in glucose minimal medium. Glucose concentrat ion was 0.022 Symbols: w i ld - type or XIII, revertant , ( o ) ; XV, rever tant , ( e ) . T a b l e IV. I n d u c t i o n o f g l u c o s e - c a t a b o l i z i n g enzymes in r e v e r t a n t s o f g l u c o s e - n e g a t i v e mutants o f XV. and X I I I . . Organ i sm Carbon ENZYME ACT IV I TY 3 s o u r c e G l u c o s e dehydrogenase G l u c o n a t e dehyd rogena se 6-PG d e h y d r a t a s e + KDGP a l d o l a s e R e v e r t a n t o f mutant XV^ P y r u v a t e 3 ]k k G l u c o s e 19 518 212 R e v e r t a n t o f mutant X I11 ] P y r u v a t e 21 112 k G l u c o s e 121 2417 179 Enzyme a c t i v i t y e x p r e s s e d as nanomoles o f s u b s t r a t e c o n v e r t e d pe r min pe r mg o f p r o t e i n a t 35 C. than i n XV,. The e x a m i n a t i o n of g l u c o s e - c a t a b o l i z i n g enzymes s t r o n g l y s u g ge s t ed t h a t t he s l o w e r r a t e of g l u c o s e m e t a b o l i s m i n XV, was due t o the l ower amounts of t h e s e 2 enzymes. T h i s was s u p p o r t e d by t he f a c t t h a t r e v e r t a n t s of XV, r e t a i n t h e s e low l e v e l s of g l u c o s e and g l u c o n a t e dehyd rogena se s and a r e a b l e t o grow o n l y s l o w l y w i t h g l u c o s e . The r e -s u l t s w i t h XV, and i t s r e v e r t a n t a l s o s u g ge s t t h a t t he o x i d a t i v e non -p h o s p h o r y l a t e d pathway f o r g l u c o s e d i s s i m i l a t i o n (88,109) may not be r e q u i r e d f o r g rowth w i t h g l u c o s e , but may be r e q u i r e d i n o r d e r t o m a i n t a i n a maximal r a t e of g l u c o s e d i s s i m i l a t i o n i n t h i s m i c r o o r g a n i s m . M i d g e l y and Dawes (80) have a l s o r e p o r t e d t h a t mutant s of P_. a e r u g i n o s a , b l o c k e d i n t h i s pa thway, a r e s t i l l c a p a b l e o f g r owth w i t h g l u c o s e . The second m u t a t i o n i n XV, , r e s u l t i n g i n low l e v e l s o f g l u c o s e and g l u c o n a t e d e h y d r o g e n a s e s , may be e x p e c t e d t o a r i s e d u r i n g s e l e c t i o n o f mutan t s w i t h a l t e r e d a b i l i t y t o grow w i t h g l u c o s e . That i s , a mutant c a p a b l e o f o n l y s l ow g rowth w i t h g l u c o s e wou ld be s e l e c t e d f o r o v e r the w i l d - t y p e w i t h a much f a s t e r r a t e o f g rowth and a second m u t a t i o n a l e ven t m i gh t then o c c u r , l e a d i n g t o c o m p l e t e i n a b i l i t y t o grow w i t h g l u c o s e . I I . The i n h i b i t o r y e f f e c t o f g l u c o s e i n g l u c o s e - n e g a t i v e m u t a n t s . T o x i c i t y f rom t h e a c c u m u l a t i o n o f c e r t a i n p h o s p h o r y l a t e d i n t e r m e d -i a t e s , due t o a m u t a t i o n a l b l o c k has been r e p o r t e d i n a v a r i e t y o f o r g a n -isms (17,18,27-29,52,103,113) and s e v e r a l o f t h e s e r e p o r t s have been r e -v i ewed (3). In each c a s e , t he mutant o r g a n i s m was no t o n l y u n a b l e t o grow w i t h c a r b o h y d r a t e s u t i l i z i n g the m e t a b o l i c pathway l e a d i n g t o t he pho s -p h o r y l a t e d i n t e r m e d i a t e , but g rowth w i t h c e r t a i n o t h e r c a r b o n s o u r c e s i n 48 t he p r e s e n c e o f t h e s e c a r b o h y d r a t e s r e s u l t e d i n g rowth s t a s i s o r v e r y s l ow g rowth r a t e s . In each i n s t a n c e , t h i s i n h i b i t o r y e f f e c t on g rowth has been a t t r i b u t e d t o t he a c c u m u l a t i o n o f t he p h o s p h o r y l a t e d i n t e r m e d i a t e . In s e v e r a l o f t h e s e r e p o r t s (3,14,18,22,23,29,52,66,103,112,123), t he p a t t e r n s o f g rowth i n h i b i t i o n o r g rowth s t a s i s have been shown and a r e r e m a r k a b l y s i m i l a r t o t h e e f f e c t s o f g l u c o s e , g l u c o n a t e , o r 2-KG on t he g rowth o f mutant s XI I I, and XV, w i t h p y r u v a t e , as found h e r e . As w i t h the p r e v i o u s c a s e s , i t i s s t r o n g l y f e l t t h a t t he i n h i b i t o r y e f f e c t o f t h e s e s i x - c a r b o n c a r b o h y d r a t e s on t h e g rowth o f the 6-PG d e h y d r a t a s e - d e f i c i e n t mutant s w i t h p y r u v a t e was t he r e s u l t o f an a c c u m u l a t i o n o f a p h o s p h o r y l a t e d i n t e r m e d i a t e , 6 -PG. I t i s not f e l t t h a t t h i s was due t o an a c c u m u l a t i o n o f 2-KG as t h i s compound i s s i g n i f i c a n t l y l e s s e f f e c t i v e than g l u c o s e i n p r o d u c i n g g rowth s t a s i s in mutant XV| ( F i g . 1). In a d d i t i o n , g l u c o s e , g l u c o n a t e , and 2-KG a l l s e r v e as g rowth s u b s t r a t e s f o r the p a r e n t s t r a i n . 14 1. P r o d u c t s o f g l u c o s e - U - C c a t a b o l i s m i n the c e l l w a t e r and medium o f g l u c o s e - n e g a t i v e m u t a n t s . G l u c o s e - n e g a t i v e mutant s X I I I , and XV, were grown i n m i n i m a l medium w i t h p y r u v a t e as the c a r b o n s o u r c e t o an O.D. o f a p p r o x i m a t e l y 0.30 14 when g l u c o s e - U - C was added . Growth was a l l o w e d t o c o n t i n u e f o r 2 and 4 hou r s a f t e r g l u c o s e a d d i t i o n a t w h i c h t i m e s c e l l s were h a r v e s t e d . The p r o d u c t s a c c u m u l a t i n g f r om g l u c o s e m e t a b o l i s m were i d e n t i f i e d and t h e i r c o n c e n t r a t i o n s d e t e r m i n e d f o r bo th t he c e l l w a t e r and the c e l l - f r e e medium ( T a b l e V and V I ) . A t o t a l o f 4 compounds were d e t e c t e d i n the f r a c t i o n s -g l u c o s e , g l u c o n a t e , 2-KG and 6 -PG. 49 14 T a b l e V. C o n c e n t r a t i o n o f p r o d u c t s o f g l u c o s e - U - C c a t a b o l i s m i n g l u c o s e - n e g a t i v e mutant X l l l ^ c e l l w a t e r and medium. Carbon s o u r c e Compound f r o m 14 g l u c o s e - U - C M o l a r i t y Cel1 w a t e r x 10 3 med i urn Cel1 w a t e r / ^led i urn P y r u v a t e p l u s 3 G1ucose 1.3 2.8 0.46 g l u c o s e 2 hour s G l u c o n a t e 1.5 6.8 0.22 2-KG Ob 1.9 -6-PG 13.0 0.1 130 P y r u v a t e p l u s G l u c o s e 2.1 0.2 10.5 g l u c o s e 4 hour s G l u c o n a t e 3.1 0.5 6.2 2-KG 0 11.0 -6-PG 11.3 0.6 18.9 Resuspended i n G l u c o s e 1.2 0.2 6.0 g l u c o s e 4 hour s G l u c o n a t e 0.9 3-6 0.25 2-KG 0 6.5 -6-PG 9-3 0.5 19.6 C u l t u r e s were grown w i t h 0.045 M p y r u v a t e to an O.D. o f a p p r o x i m a t e l y 0.30 when g l u c o s e - U - 14c was added t o a f i n a l c o n c e n t r a t i o n o f 0.011 M ( s p e c i f i c a c t i v i t y 0.72 u C i / u m o l e ) . No d e t e c t a b l e 2-KG i n the c e l l w a t e r under the c o n d i t i o n s emp loyed . C u l t u r e s were grown w i t h 0.045 M p y r u v a t e , h a r v e s t e d a t an O.D. o f a p p r o x i m a t e l y 1.0, washed, and re su spended i n 0.011 M g l u co se -U - ^4c ( s p e c i f i c a c t i v i t y 0.72 uC i /umo l e ) m i n i m a l medium. No g rowth was o b s e r v e d . T a b l e V I . C o n c e n t r a t i o n s o f p r o d u c t s o f g l u c o s e - U - C c a t a b o l i s m i n g l u c o s e - n e g a t i v e mutant XV. c e l l w a t e r and medium. Carbon s o u r c e 7 , c M o l a r i t y x ICr Compound f rom g 1 u c o s e - U - ' . . . t ,. 3 Ce l1 w a t e r mediurn Ce l 1 w a t e r Med i urn P y r u v a t e p l u s g l u c o s e 2 hou r s G l u c o s e G1ucona t e 2-KG 6-PG 1.4 1-7 0 11.1 10.8 0.7 0.3 0. 1 0.13 2.4 111 P y r u v a t e p l u s g l u c o s e k hour s G l u c o s e G l u c o n a t e 2-KG 6-PG 3.4 4 .3 0 9-7 7-5 1.1 2.6 0.5 0.45 3-9 19.4 Resuspended i n g l u c o s e 4 hour s G l u c o s e G1uconate 2-KG 6-PG 2.2 1.5 0 8.6 6.6 2.6 1 .7 0.3 0.33 0.58 29 .0 C o n d i t i o n s as d e s c r i b e d i n T a b l e V. I t was d e m o n s t r a t e d t h a t 6-PG a c c u m u l a t e d t o s i g n i f i c a n t l y h i g h e r i n t e r n a l c e l l poo l c o n c e n t r a t i o n s than e i t h e r g l u c o s e o r g l u c o n a t e a t t he t i m e i n t e r v a l s measured and no 2-KG was d e t e c t e d i n t he i n t e r n a l c e l l p o o l , a l t h o u g h i t was the ma jo r p r o d u c t o f g l u c o s e m e t a b o l i s m i n t he c e l l - f r e e medium. The e x t e r n a l c o n c e n t r a t i o n o f 6-PG i n c r e a s e d 6 - f o l d i n mutant X I I I , and 5~fo ld i n mutant XV , , f rom 2 t o 4 hou r s a f t e r g l u c o s e a d d i t i o n and t he c o n c e n t r a t i o n s i n t h e c e l l w a t e r d e c r e a s e d s l i g h t l y d u r i n g t he same t i m e . i n t e r v a l . The r e v e r s e was t r u e f o r t he g l u c o s e and g l u c o n a t e c o n c e n t r a t i o n s w h i c h i n c r e a s e d i n t h e c e l l wa te r and d e c r e a s e d i n t he c e l l - f r e e medium d u r i n g t he p e r i o d between 2 and k hours a f t e r g l u c o s e was a d d e d . The i n t e r n a l c o n c e n t r a t i o n o f 6-PG was h i g h e r i n mutant X I I I , a t 2 and k hou r s a f t e r g l u c o s e a d d i t i o n than i n mutant XV, ( a p p r o x i m a t e l y 17% h i g h e r a t each t i m e ) . However, t he i n t e r n a l c o n c e n t r a t i o n s o f g l u c o s e and g l u c o n a t e were h i g h e r i n mutant XV, than i n mutant X I I I , . S i n c e no 2-KG was d e t e c t e d i n the c e l l w a t e r and f o r the rea sons d i s c u s s e d p r e v i o u s l y , i t i s not f e l t t h a t t h i s i s t he i n h i b i t o r y compound. S i m i l a r l y , i t i s not f e l t t h a t e i t h e r g l u c o s e o r g l u c o n a t e i s t he i n h i b i t o r y compound s i n c e h i g h e r c o n c e n t r a t i o n s o f bo th o f t h e s e compounds were p r e s e n t i n t he c e l l w a t e r o f mutant. XV, than i n mutant X I I I , and XV, i s l e s s r e s p o n -s i v e t o the i n h i b i t o r y e f f e c t o f g l u c o s e . G l u c o s e and g l u c o n a t e a l s o s e r v e as g r owth s u b s t r a t e s f o r the p a r e n t s t r a i n . The c o n c e n t r a t i o n o f 6-PG i n the c e l l was s i g n i f i c a n t l y h i g h e r t han the c o n c e n t r a t i o n o f g l u -c o s e o r g l u c o n a t e and i t was h i g h e r i n mutant X I I I , than i n mutant XV, . T h e r e f o r e , i t i s s t r o n g l y f e l t t h a t t he a b i l i t y o f g l u c o s e t o cau se g rowth s t a s i s o f t h e 6-PG d e h y d r a t a s e - d e f i c i e n t mutant s i s due t o a l a r g e i n t r a -c e l l u l a r a c c u m u l a t i o n o f 6-PG. The h i g h e r c o n c e n t r a t i o n o f 6-PG i n mutant XII I j than i n mutant XV^ and t h e o b v i o u s f a s t e r r a t e o f g l u c o s e m e t a b o l i s m i n XI I I| l i k e l y e x p l a i n why s t r a i n X I I I . was more r e s p o n s i v e to the i n h i b i t o r y e f f e c t o f g l u c o s e . 2. Assay of TCA c y c l e and g l y c o l y t i c enzymes. G l y c o l y t i c and TCA c y c l e enzyme a c t i v i t i e s ( e x c e p t t h o s e p r e v i o u s l y a s s a y e d , T a b l e I I I ) were examined i n an a t t e m p t t o e l u c i d a t e the p o i n t o f t o x i c i t y r e s u l t i n g from the a c c u m u l a t i o n o f 6-PG i n t h e s e mutants. For enzyme a s s a y s , c u l t u r e s o f the p a r e n t s t r a i n and g l u c o s e - n e g a t i v e mutants X I I I . and XVj were grown i n a medium w i t h p y r u v a t e as t h e c a r b o n s o u r c e . S i m i l a r l y , t h e p a r e n t s t r a i n was grown i n a medium w i t h g l u c o s e as t h e s o u r c e o f carbon and g l u c o s e was added to c u l t u r e s o f the w i l d - t y p e and mutants growing l o g a r i t h m i c a l l y i n a medium w i t h p y r u v a t e (as F i g . 5)• The s p e c i f i c a c t i v i t i e s o f the TCA c y c l e enzymes i n the p a r e n t s t r a i n were s i m i l a r when th e m i c r o o r g a n i s m was grown on p y r u v a t e , g l u c o s e o r p y r u v a t e p l u s g l u c o s e ( T a b l e V I l ) . However, some d e c r e a s e i n t h e a c t i v i t y o f c i t r a t e s y n t h e t a s e , 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 was noted as a r e s u l t o f g l u c o s e a d d i t i o n f o r 2 hours t o the p a r e n t s t r a i n g rowing i n p y r u v a t e minimal medium. Only a d e c r e a s e i n a - k e t o g 1 u t a r a t e dehydrogen ase was a p p a r e n t i n the w i l d - t y p e s t r a i n grown i n minimal medium w i t h g l u c o s e as the c a r b o n s o u r c e . The s i g n i f i c a n c e o f the g l u c o s e - m e d i a t e d d e c r e a s e s i n t h e a c t i v i t i e s o f c e r t a i n TCA c y c l e enzymes has not been d e t e r m i n e d . A l l o t h e r a c t i v i t i e s appeared t o be c o n s t i t u t i v e under t h e s e e x p e r i m e n t a l c o n d i t i o n s . These r e s u l t s were s i m i l a r t o t h o s e o b t a i n e d by Table VII. A c t i v i t i e s of TCA cyc le enzymes in wi ld - type P. aerug?nosa. Carbon source Pyruvate Pyruvate + Glucose glucose 2 hr. C i t r a t e synthetase 30 18 28 Aconitase 14 6 16 I soc i t ra te dehydrogenase 603 451 679 a-Ketog lutarate dehydrogenase 97 46 55 Succinyl-CoA synthetase 227 160 197 Succinate dehydrogenase 161 150 105 Fumarase 653 822 750 Ma late enzyme NAD 45 39 46 Ma late enzyme NADP 230 231 230 Ma la te dehydrogenase 41 39 47 Enzyme a c t i v i t i e s expressed as nanomoles of substrate converted per min per mg p ro te in . Cultures were grown with 0.045 M pyruvate or 0.022 M glucose and har-vested at an O.D. of approximately 1.0. To determine the inf luence of g lucose, cu l tures were grown with 0.045 M pyruvate to an O.D. of approximately 0.30 when 0.011 M glucose was added and the cu l ture was harvested 2 hours l a t e r . 54 T i w a r i and Campbe l l who compared the a c t i v i t i e s o f c e r t a i n o f t h e s e enzymes p r e s e n t i n c u l t u r e s o f the p a r e n t s t r a i n grown i n medium w i t h s u c c i n a t e o r g l u c o s e as the c a r b o n s o u r c e ( i n -c o m p a r a b l e r e s u l t s were o b t a i n e d w i t h bo th mutant s t r a i n s i n t h a t t he a c t i v i t i e s o f the TCA c y c l e enzymes appea red t o be c o n s t i t u t i v e under t h e c o n d i t i o n s measured ( T a b l e s VI I I and I X ) . However, the g l u c o s e -m e d i a t e d d e c r e a s e i n a c t i v i t y o f some o f t he TCA c y c l e enzymes no ted i n t he p a r e n t s t r a i n was not a p p a r e n t i n t he mutant s t r a i n s . F u r t h e r , many o f t he a c t i v i t i e s i n the mutant s t r a i n s were somewhat h i g h e r than i n t he p a r e n t s t r a i n . The s i g n i f i c a n c e o f t h e s e h i g h e r a c t i v i t i e s has not been d e t e r m i n e d , but they may a c c o u n t f o r t he f a s t e r r a t e o f g rowth w i t h p y r u -v a t e o b s e r v e d i n bo th mutant s ( g e n e r a t i o n t i m e o f a p p r o x i m a t e l y 55 min) when compared t o t h a t o f the p a r e n t s t r a i n ( g e n e r a t i o n t ime o f a p p r o x i -m a t e l y 69 m i n ) . The r e s u l t s o b t a i n e d w i t h bo th t he p a r e n t and mutant s t r a i n s o f P_. a e r u g i n o s a i n d i c a t e d t h a t , a l t h o u g h the l e v e l s o f TCA c y c l e enzymes do not v a r y c o n s i d e r a b l y under t h e c o n d i t i o n s used h e r e , t h e enzymes may not be c o n s t i t u t i v e and may be s u b j e c t t o some r e g u l a t i o n . The n a t u r e o f t h i s r e g u l a t i o n i s not a p p a r e n t . S i m i l a r r e s u l t s , c o n c e r n i n g t h e r e g u l a t i o n o f c e r t a i n TCA c y c l e enzymes i n P_. a e r u g i n o s a , have been r e p o r t e d by Ng and Dawes (87)-The g l y c o l y t i c enzymes i n the mutant s t r a i n s appea red t o be v e r y s i m i l a r t o t h o s e i n t he p a r e n t s t r a i n w i t h the o b v i o u s e x c e p t i o n o f p y r u v i c dehyd rogena se ( T a b l e X ) . P y r u v i c dehyd rogena se a c t i v i t y was a l m o s t i d e n t i c a l i n the p a r e n t s t r a i n under t h e t h r e e c o n d i t i o n s t e s t e d . However, w h i l e bo th mutant s t r a i n s grown i n p y r u v a t e medium po s s e s s ed T a b l e V I I I . A c t i v i t i e s o f TCA c y c l e enzymes i n P. a e r u g i n o s a g l u c o s e -n e g a t i v e mutant X l l l . . a Enzyme Carbon b s o u r c e P y r u v a t e P y r u v a t e + g l u c o s e 2 hr P y r u v a t e + g l u c o s e 4 h r C i t r a t e s y n t h e t a s e 33 36 72 A c o n i t a s e 10 12 13 I s o c i t r a t e dehyd rogena se 1580 1240 1520 a - K e t o g l u t a r a t e dehyd rogena se 216 214 157 S u c c i n y l - C o A s y n t h e t a s e 236 203 254 S u c c i n a t e dehyd rogena se 168 167 141 Fumarase 1290 1350 1190 Ma l a t e enzyme NAD 37 53 47 Ma l a t e enzyme NADP 150 166 164 M a l a t e dehyd rogena se 66 60 58 Enzyme a c t i v i t i e s e x p r e s s e d as nanomoles o f s u b s t r a t e c o n v e r t e d pe r min pe r mg p r o t e i n . C u l t u r e s were grown w i t h 0.045 M p y r u v a t e and h a r v e s t e d a t an O.D. o f a p p r o x i m a t e l y 1.0. To d e t e r m i n e t he e f f e c t o f g l u c o s e , c u l t u r e s were grown t o an O.D. o f a p p r o x i m a t e l y 0.30 when 0.011 M g l u c o s e was added and samples o f t he c u l t u r e were h a r v e s t e d 2 and 4 hou r s l a t e r . T a b l e IX. A c t i v i t i e s o f TCA c y c l e enzymes i n P_. a e r u g i n o s a g l u c o s e -n e g a t i v e mutant X V . . a Enzyme Carbon s o u r c e P y r u v a t e P y r u v a t e + g l u c o s e 2 h r P y r u v a t e + g l u c o s e 4 h r C i t r a t e s y n t h e t a s e 62 68 63 A c o n i t a s e 13 11 22 I s o c i t r a t e dehyd rogena se 1510 I960 2060 a - K e t o g l u t a r a t e d e h y d r o -genase 248 322 391 S u c c i n y l - C o A s y n t h e t a s e 240 245 328 S u c c i n a t e dehyd rogena se 142 160 255 Fumarase 1970 1740 1830 Ma l a t e enzyme NAD 45 52 61 Ma l a t e enzyme NADP 204 171 182 Ma l a t e dehyd rogena se 87 71 68 a C o n d i t i o n s a s d e s c r i b e d f o r T a b l e V I I I . T a b l e X. A c t i v i t i e s o f g l y c o l y t i c enzymes i n P. ae rug i nosa w i l d - t y p e and g l u c o s e - n e g a t i v e mutant s X I I I , and X V , . a Enzyme Organ i sm Carbon s o u r c e G l u c o s e P y r u v a t e P y r u v a t e w i t h g l u c o s e 2 hour s P y r u v a t e w i t h g l u c o s e 4 hours 3 - P h o s p h o g l y c e r a t e W + 138 110 122 c X I I I , d 210 168 198 k i nase XV, d 166 176 160 2 , 3 ~ D i p h o s p h o g l y c e r a t e w+ 58 39 65 c X I I I ; d 62 44 51 mutase XV, d 62 90 73 E n o l a s e w+ 207 241 242 c X I I I , d 300 252 289 XV] d 287 298 369 P y r u v i c k i n a s e w+ 276 289 368 c X I I I ] d 336 219 239 XV] d 476 514 500 P y r u v i c dehydrogenase w+ 76 65 92 c X I I I , d 63 4 6 XV] d 82 19 16 3 Enzyme a c t i v i t i e s e x p r e s s e d as nanomoles o f s u b s t r a t e c o n v e r t e d pe r min per mg p r o t e i n . As d e s c r i b e d i n T a b l e s VI 1 and V I I I . Not done, c u l t u r e had reached s t a t i o n a r y phase . No g rowth o f mutants w i t h g l u c o s e as s o l e c a rbon s o u r c e . 58 p y r u v i c dehyd rogena se a c t i v i t y s i m i l a r t o t he p a r e n t o r g a n i s m , t h e a d d i t i o n o f g l u c o s e t o bo th mutant s g r ow ing l o g a r i t h m i c a l l y i n p y r u v a t e medium r e s u l t e d i n a s i g n i f i c a n t d e c r e a s e i n t h e a c t i v i t y o f t h i s enzyme _j_.e_. a t e n - f o l d d e c r e a s e i n mutant X I I I , and a f i v e - f o l d d e c r e a s e i n mutant XV, a t k hou r s a f t e r g l u c o s e a d d i t i o n . As m i gh t be e x p e c t e d f rom the r e spon se o f the two d i f f e r e n t mutant s t o the i n h i b i t o r y e f f e c t o f g l u c o s e , t he d e c r e a s e i n p y r u v i c dehyd rogena se a c t i v i t y was more s e v e r e i n mutant X I I I . than i n XV. a f t e r g l u c o s e a d d i t i o n . The d e c r e a s e i n t he I I 3 a c t i v i t y o f t h i s enzyme was measured i n bo th mutant s f o r k hour s a f t e r g l u c o s e was added t o t he c e l l s g r o w i n g i n p y r u v a t e medium. A t t h a t t i m e , o n l y 10% o f t he p y r u v i c dehyd rogena se a c t i v i t y p r e s e n t i n the p y r u v a t e grown c u l t u r e rema ined i n mutant X I I I , and o n l y 30% rema ined i n mutant XV, ( F i g . 7). 3- E f f e c t o f g l u c o s e on p y r u v a t e d i s a p p e a r a n c e w i t h g l u c o s e - n e g a t i v e m u t a n t s . To d e t e r m i n e t h e e f f e c t o f g l u c o s e on t he u t i l i z a t i o n o f p y r u v a t e by t he g l u c o s e - n e g a t i v e m u t a n t s , c u l t u r e s o f s t r a i n X I I I , were grown under t he c o n d i t i o n s d e s c r i b e d f o r enzyme a s s a y s and p y r u v a t e d i s a p p e a r a n c e f r om t he medium was f o l l o w e d ( F i g . 8 ) . W i t h mutant X I I I , i t can be seen t h a t p y r u v a t e d i s a p p e a r e d as g rowth p r o ceeded and was e s s e n t i a l l y gone when the c u l t u r e reached s t a t i o n a r y phase . However, t h e a d d i t i o n o f g l u c o s e t o the c u l t u r e r e s u l t e d i n a d e c r e a s e d r a t e o f p y r u v a t e u t i l i z a -t i o n w h i c h became a l m o s t s t a t i c a t the t i m e g l u c o s e - m e d i a t e d g rowth i n h i b i t i o n was i n s t i t u t e d . 1 2 3 4 HOURS AFTER GLUCOSE ADDED F i g . 7> Decrease i n the s p e c i f i c a c t i v i t y o f p y r u v i c dehydrogenase i n g l u c o s e - n e g a t i v e mutants o f I P . a e r u g i n o s a f o l l o w i n g t he a d d i t i o n o f g l u c o s e . G l u c o s e was added a t a f i n a l c o n cen-t r a t i o n o f 0.011 M, t o c u l t u r e s a t an O.D. o f 0.30 i n p y r u v a t e (0.045 M) mini m a l medium. S p e c i f i c a c t i v i t y i s e x p r e s s e d as a p e r c e n t a g e o f the v a l u e o b t a i n e d i m m e d i a t e l y p r i o r t o a d d i n g o f g l u c o s e . Symbols: XIII,, (o); XV , (©) 60 HOURS F i g . 8. E f f e c t o f g l u c o s e on g r owth and on p y r u v a t e d i s a p p e a r a n c e i n g l u c o s e - n e g a t i v e mutant X I 1 1^ . S ymbo l s : g r owth w i t h 0 .045 M p y r u v a t e , ( • ) ; p y r u v a t e d i s a p p e a r a n c e , Growth w i t h 0 .045 M p y r u v a t e and 0.011 M g l u c o s e added as i n d i c a t e d by t h e a r r o w , (o); p y r u v a t e d i s a p p e a r a n c e , ( A ) . 61 k. E f f e c t o f 6-PG on p y r u v i c dehyd rogena se a c t i v i t y . A t t e m p t s t o i n h i b i t p y r u v i c dehyd rogena se a c t i v i t y i n v i t r o i n c e l l - f r e e e x t r a c t s w i t h up t o 50 umoles o f 6-PG/ml met w i t h l i t t l e s u c c e s s , as i n h i b i t i o n v a r i e d f rom 0% to a maximum o f 20% i n e x t r a c t s o f w i l d - t y p e and mutant s t r a i n s . To t e s t t h e e f f e c t o f 6-PG on p y r u v i c dehyd rogena se a c t i v i t y i n v i v o , c u l t u r e s o f the mutant s t r a i n s were grown t o an O.D. o f a p p r o x i m a t e l y 1.0 i n p y r u v a t e medium, h a r v e s t e d , washed, and r e su spended i n b u f f e r and i n medium c o n t a i n i n g g l u c o s e as the s o l e c a r b o n s o u r c e . The c u l t u r e s were t hen i n c u b a t e d f o r k h o u r s , h a r v e s t e d , and a s s a y e d f o r p y r u v i c dehyd rogena se a c t i v i t y . Under t h e s e c o n d i t i o n s , the a c t i v i t y o f t h i s enzyme was c o m p a r a b l e to the a c t i v i t y o b t a i n e d i n the p y r u v a t e grown mutant c u l t u r e s . A s m a l l d e c r e a s e i n p y r u v i c dehyd rogena se a c t i v i t y was no ted between t he c o n t r o l c u l t u r e s r e su spended i n b u f f e r and the c u l t u r e s r e su spended i n g l u c o s e medium f o r k h o u r s , J_.e_. a 25% d e c r e a s e i n a c t i v i t y i n mutant XI I I| and a 13% d e c r e a s e i n mutant XV|. Under the same c o n d i t i o n s , the mutant s t r a i n s r e su spended and i n c u b a t e d f o r h hours i n medium c o n t a i n i n g g l u c o s e - U - C a c c u m u l a t e d 6-PG i n the c e l l w a t e r a t c o n c e n t r a t i o n s a p p r o a c h i n g t h o s e o b t a i n e d when g l u c o s e was a l l o w e d t o i n s t i t u t e g rowth s t a s i s i n mutant c u l t u r e s g r ow ing l o g a r i t h m i c a l l y i n p y r u v a t e medium ( T a b l e s V and V I ) . These r e s u l t s s u g g e s t e d , t h a t a l t h o u g h 6-PG may have caused some i n h i b i t i o n o f p y r u v i c dehyd rogena se a c t i v i t y , c o n t i n u e d g rowth f o r a l i m i t e d p e r i o d was r e q u i r e d b e f o r e any s i g n i f i c a n t e f f e c t was o b s e r v e d and t h a t the a c c u m u l a t i o n o f 6-PG i n t h e s e g l u c o s e -n e g a t i v e mutant s o f P_. a e r u g i nosa appea red t o c au se a r e p r e s s i o n o f the s y n t h e s i s o f t he enzyme. I t i s p o s s i b l e t h a t 6-PG i n h i b i t s o r i n a c t i v a t e s t he enzyme o n l y i n t h e p r e s e n c e o f p y r u v a t e o r some d e g r a d a t i o n p r o d u c t t h e r e o f , however , t h i s c o u l d not be d e m o n s t r a t e d i n v i t r o wi t h c r u d e e x t r a c t s i n t he p r e s e n c e o f p y r u v a t e and 6-PG. T h e r e f o r e , i t i s s t r o n g l y f e l t t h a t t he g l u c o s e - m e d i a t e d g rowth s t a s i s o r i n h i b i t i o n , i n 6-PG d e h y d r a t a s e - d e f i c i e n t mutant s X I I I , and XV, g r ow ing i n p y r u v a t e medium, was due t o an a c c u m u l a t i o n o f 6-PG f rom g l u c o s e m e t a b o l i s m . A l s o , t h i s a c c u m u l a t i o n o f 6-PG caused a r e p r e s -s i o n o f the s y n t h e s i s o f p y r u v i c d e h y d r o g e n a s e , e i t h e r d i r e c t l y o r i n d i r e c t l y , w h i c h i n t u r n , d e c r e a s e d t he r a t e o f p y r u v a t e u t i l i z a t i o n by t he c e l l s and c o n s e q u e n t l y i n h i b i t e d g r o w t h . The f a c t t h a t mutant X I I I , a c c u m u l a t e d h i g h e r i n t e r n a l c o n c e n t r a t i o n s o f 6 -PG, e x h i b i t e d a more s i g n i f i c a n t d e c r e a s e i n p y r u v i c dehyd rogena se a c t i v i t y , a n d , i n d e e d , m e t a b o l i z e d g l u c o s e a t a much f a s t e r r a t e than mutant XV, , l i k e l y e x p l a i n s why mutant X I I I , was more r e s p o n s i v e t o t he i n h i b i t o r y e f f e c t o f g l u c o s e t han mutant XV, . I t i s not c e r t a i n t h a t t h i s r e g u l a t o r y s y s tem o p e r a t e s i n t h e p a r e n t s t r a i n . However, t h e r e i s some e v i d e n c e t h a t p y r u v i c d e h y d r o -genase a c t i v i t y may be under some s o r t o f r e g u l a t o r y c o n t r o l i n t he w i l d - t y p e P_. a e r u g i n o s a . Von T i g e r s t r o m and Campbe l l r e p o r t e d t h a t t h e a c t i v i t y o f t h i s enzyme was l e s s ( a t l e a s t 70%) i n c e l l s grown w i t h a c e t a t e than i n t h o s e grown w i t h a - k e t o g 1 u t a r a t e o r g l u c o s e ( 118 ) . The p o s s i b i l i t y t h a t t he a c c u m u l a t i o n o f 6-PG i n t he mutant s d e s c r i b e d may i n t e r f e r e w i t h some o t h e r e s s e n t i a l c e l l u l a r f u n c t i o n c anno t be e x c l u d e d . I t has been s u g g e s t e d t h a t 6-PG i s t he e f f e c t o r o f g l u c o s e -i n i t i a t e d c a t a b o l i t e r e p r e s s i o n o f h i s t i d a s e i n P. a e r u g i n o s a ( 4 6 ) . I I I . A n a l y s i s o f o t h e r g 1 u c o s e - n e g a t i v e mutants o f P_. aerug i nosa. In an e f f o r t t o e l u c i d a t e the r e g u l a t i o n o f c a r b o h y d r a t e - c a t a b o l i z i n g enzymes, s e v e r a l o t h e r g l u c o s e - n e g a t i v e mutants o f P_. a e r u g i n o s a were examined and c h a r a c t e r i z e d ( T a b l e s XI and X I l ) . Mutant s t r a i n s I , , 11^, l l ^ , M l , , XI I I j , XIV,, and XV, were o b t a i n e d by n a t u r a l s e l e c t i o n , mutant X l l l , _ ^ was a s p o n t a n e o u s l y o c c u r r i n g d e r i v a t i v e of mutant X I I I , , and mutant 3 was i s o l a t e d f o l l o w i n g NT6 m u t a g e n e s i s by W.W. Kay ( 5 6 ) . The above mutants were s e l e c t e d f o r t h e i r i n a b i l i t y t o grow w i t h g l u c o s e as the s o l e s o u r c e o f c a r b o n . However, they were a l s o found t o be i n -c a p a b l e o f growth when e i t h e r g l u c o n a t e o r 2-KG was p r e s e n t as the o n l y c a r b o n s o u r c e . W i t h the e x c e p t i o n o f mutant 3, growth o f t h e s e mutants w i t h p y r u v a t e , w h i c h was s i m i l a r t o the w i l d - t y p e s t r a i n , was i n h i b i t e d by t h e p r e s e n c e o f g l u c o s e , g l u c o n a t e , o r 2-KG i n a manner a n a l o g o u s t o t h a t p r e v i o u s l y d e s c r i b e d f o r mutants X I I I , and XV,. Growth o f mutant 3 w i t h p y r u v a t e was i n h i b i t e d by the p r e s e n c e o f g l u c o s e but much l e s s s e v e r e l y than the o t h e r mutants t e s t e d and growth was not i n h i b i t e d by the p r e s e n c e o f g l u c o n a t e o r 2-KG ( F i g . 9 ) • 1. I d e n t i f i c a t i o n o f p r o d u c t s o f g l u c o s e , g l u c o n a t e , o r 2-KG m e t a b o l i s m i n the mutant s t r a i n s . C u l t u r e s o f the mutants ( w i t h the e x c e p t i o n of s t r a i n s I I . and I I I , w hich were not examined) were grown i n p y r u v a t e m i n i m a l medium to an 14 14 O.D. o f a p p r o x i m a t e l y 0.30 then e i t h e r C - g l u c o s e , C - g l u c o n a t e , o r 14 C-2-KG was added ( T a b l e X I ) . Samples were removed a t 2 and 3 hours 14 a f t e r t h e a d d i t i o n o f the C-compound and the t r i c h l o r o a c e t i c a c i d s o l u b l e m a t e r i a l was e l u t e d o v e r a Dowex l-x8 column. In each mutant 64 r....—j—^.^—u.^..^,, M i — . . . • • 1 ) p _ T 1 | - n r i | | , 11 im || | | | m i 4 t IIM'IT.'iTK""' »""-aiiakmim.,-i,< 1 2 6 1 0 1 4 1 8 2 2 HOURS F i g . 9 . E f f e c t o f g l u c o s e on t he g r owth o f g l u c o s e - n e g a t i v e mutant 3 i n p y r u v a t e m i n i m a l medium. S ymbo l s : 0 .045 M p y r u v a t e , ( • ) ; p l u s 3.4 x 10 - / * M g l u c o s e , ( A ) ; p l u s 1.1 x 1 0 _ 2 M g l u c o s e , ( D ) . l i s t e d ( T a b l e X I ) , e s s e n t i a l l y 100% o f t he r a d i o a c t i v e m a t e r i a l added t o the c u l t u r e s was r e c o v e r e d i n t he t r i c h l o r o a c e t i c a c i d s o l u b l e f r a c t i o n as some c o m b i n a t i o n o f g l u c o s e , g l u c o n a t e , 2 -KG,o r 6 -PG. In each m u t a n t , 6-PG a c c u m u l a t e d i n s i g n i f i c a n t q u a n t i t i e s , s u g g e s t i n g t h a t a l l t h e s e mutant s were s i m i l a r t o mutant s X I I I , and XVj i n t h a t t he m u t a t i o n l e a d i n g t o t he i n a b i l i t y t o grow w i t h g l u c o s e was p r o b a b l y a t t he l e v e l o f 6-PG d e h y d r a t a s e . The a c c u m u l a t i o n o f 6-PG wou ld a l s o e x p l a i n why g rowth o f t h e s e mutant s w i t h p y r u v a t e was i n h i b i t e d by g l u c o s e , g l u c o n a t e , o r 2-KG a n d , i n t he c a s e o f mutant 3, by g l u c o s e . When g l u c o s e was the compound added t o mutant s I,, IV, , and X I I I , ^ , bo th g l u c o s e and g l u c o n a t e were d e t e c t e d i n the 2 hour s amp le . However, i n t he 8 hour s amp le , o n l y 2-KG and 6-PG were d e t e c t e d . S i m i l a r l y , when g l u c o n a t e was added t o mutant s l l ^ , X I V , , and X l l l , _ ^ , g l u c o n a t e was i d e n t i f i e d i n t h e 2 hour sample but o n l y 2-KG and 6-PG were p r e s e n t i n t h e 8 hour s amp le . When 2-KG was added t o s t r a i n X I I I , , o n l y 2-KG and 6-PG were d e t e c t e d i n bo th s amp l e s . In t he 8 hour sample i n each o f t he above m u t a n t s , a l l o f the r a d i o a c t i v e m a t e r i a l was p r e s e n t as 2-KG and 6-PG and i n a p p r o x i m a t e l y t he same c o n c e n t r a t i o n j_.e_. a p p r o x i m a t e l y 90% as 2-KG and 10% as 6-PG In mutant 3, no 2-KG was d e t e c t e d i n e i t h e r t he 2 o r the 3 hour sample s u g g e s t i n g t h a t t he m e t a b o l i s m o f g l u c o s e t o 2-KG was somehow i m p a i r e d i n t h i s mu tan t . A l s o , no g l u c o n a t e o r 6-PG was d e t e c t e d i n t he 2 hour samp le and o n l y v e r y s m a l l amounts were i s o l a t e d i n the 8 hour sample _i_.e_. i n the 8 hour sample e s s e n t i a l l y 100% o f the r a d i o a c t i v e l a b e l was r e c o v e r e d as 87 -5% u n r e a c t e d g l u c o s e , 9% g l u c o n a t e , and 3-5% 6-PG. I t T a b l e X I . P r o d u c t s a c c u m u l a t i n g i n medium and c e l l s o f g l u c o s e - n e g a t i v e mutant s o f P_. a e r u g i n o s a f rom 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 m e t a b o l i s m . Mutant 14 C - s u b s t r a t e a d d e d 3 14 P r o d u c t s f r om C - s u b s t r a t e G l u c o n a t e 2-KG 6-PG M 2 I V 1 XIV XI I I G l u c o s e G l u c o n a t e G1ucose G l u c o n a t e 2-KG n.d. XI I I 1-A G l u c o s e G l u c o n a t e G1ucose + + n.d. + + C u l t u r e s grown w i t h 0.045 M p y r u v a t e t o an O.D. o f a p p r o x i m a t e l y 0.30 a t w h i c h t i m e e i t h e r 0.011 M g l u c o s e - U - 1 4 r ; , 0 .010 M g 1 u c o n a t e - U - 1 4 c , o r 0.010 M 2 - K G - U - 1 4 c was added ( s p e c i f i c a c t i v i t y 0 .143 y C i / y m o l e ) . C u l t u r e s were i n c u b a t e d and samples were removed a t 2 and 8 hou r s a f t e r t he a d d i t i o n . Compound d e t e c t e d . Compound added . Compound not d e t e c t e d . i s a p p a r e n t t h a t t h e r e was a v e r y s l o w r a t e o f g l u c o s e m e t a b o l i s m i n mutant s t r a i n 3 and t h a t o n l y a s m a l l amount o f 6-PG a c c u m u l a t e d , w h i c h was no t d e t e c t e d u n t i l t he 8 hour s amp le . In c o n t r a s t , an a c c u m u l a t i o n o f 6-PG was found i n t h e 2 hour sample w i t h a l l o t h e r mutant s e xam ined . These r e s u l t s may e x p l a i n why mutant 3 was much l e s s r e s p o n s i v e t o the i n h i b i t o r y e f f e c t s o f g l u c o s e . 2. E x a m i n a t i o n o f g l u c o s e - c a t a b o l i z i n g enzymes. G l u c o s e - c a t a b o l i z i n g enzymes were measured i n some o f t h e g l u c o s e -n e g a t i v e mutant s o f P_. a e r u g i n o s a . C u l t u r e s o f t he mutant s t r a i n s were grown i n p y r u v a t e medium t o an O.D. o f a p p r o x i m a t e l y 0.30 a t w h i c h t i m e e i t h e r g l u c o s e , g l u c o n a t e o r 2-KG was added as i n d i c a t e d ( T a b l e s XI I and XI I l ) . The c u l t u r e s were i n c u b a t e d f o r an a d d i t i o n a l k hour s and t hen h a r v e s t e d f o r enzyme a s s a y s . C o n t r o l c u l t u r e s were grown w i t h p y r u v a t e as the s o l e c a r b o n s o u r c e and h a r v e s t e d a t an O.D. o f a p p r o x i m a t e l y h.0. A l l mutan t s e xam ined , e x c e p t s t r a i n s 11^ and l l l j , were d e f i c i e n t i n 6-PG d e h y d r a t a s e , c h a r a c t e r i z e d by the l a c k o f d e t e c t a b l e a c t i v i t y o f t h i s enzyme and by the a c c u m u l a t i o n o f 6-PG f rom g l u c o s e , g l u c o n a t e o r 2-KG m e t a b o l i s m . I t was t hough t t h a t mutant s 11^ and 111^ a l s o a c c u m u l a t e 6-PG, as g rowth o f t h e s e mutant s w i t h p y r u v a t e was i n h i b i t e d by the p r e s e n c e o f the s i x - c a r b o n c a r b o h y d r a t e s and no d e t e c t a b l e 6-PG d e h y d r a -t a s e p l u s KDGP a l d o l a s e a c t i v i t y was f ound i n s t r a i n M l j and o n l y v e r y low l e v e l s were p r e s e n t i n s t r a i n 11^. Mutant 3 had m u l t i p l e m u t a t i o n s c h a r a c t e r i z e d by t he l a c k o f d e t e c t a b l e a c t i v i t y f o r g l u c o s e and g l u c o n a t e d e h y d r o g e n a s e s , g l u c o n o k i n a s e , 3~PG d e h y d r o g e n a s e , 6-PG d e h y d r a t a s e , 2-KG T a b l e X I I . G l u c o s e - c a t a b o l i z i n g enzymes i n g l u c o s e - n e g a t i v e mutan t s o f P. a e r u g ? n o s a . ENZYME ACTIV ITY b G l u c o - G-6-P G l u c o n o - 3~PG ED pathway k i n a s e dehydrogenase k i n a s e dehyd rogena se enzymes No a d d i t i o n 47 14 157 161 od G l u c o s e 4 hours 42 12 177 276 0 No a d d i t i o n 2k 11 0 104 0 G l u c o s e 4 hours 35 29 0 104 0 No a d d i t i o n 13 1 0 0 0 2-KG 4 hours 18 13 0 0 0 No a d d i t i o n 12 4 0 0 0 G l u c o s e 4 hour s 145 192 0 0 0 2-KG 4 hou r s 155 202 0 0 12 Mutant Carbon s o u r c e C u l t u r e s were grown w i t h 0.045 M p y r u v a t e . C o n t r o l c u l t u r e s were h a r v e s t e d a t an O.D. o f a p p r o x i m a t e l y 1.0 (No a d d i t i o n ) . The t e s t c u l t u r e s r e c e i v e d 0.011 M g l u c o s e , 0.010 M g l u c o n a t e , o r 0 .010 M 2-KG as i n d i c a t e d and were h a r v e s t e d 4 hou r s l a t e r . b As f o r T a b l e I I I . Enzyme a c t i v i t i e s were v e r y s i m i l a r i n mutants X l l l j , l j , I I - , I V j , X IV^, XMy ^ No d e t e c t a b l e a c t i v i t y . co k i n a s e and 2-K-6-PG r e d u c t a s e . These l a t t e r two enzymes were a s s a y e d i n t h e p a r e n t s t r a i n and i n mutant s X I I I , and XV, , and were p r e s e n t a t s i m i -l a r l e v e l s i n a l l t h r e e o r g a n i s m s . The p r e s e n c e o f g l u c o k i n a s e and G-6-P dehyd rogena se and the ab sence o f any d e t e c t a b l e a c t i v i t y f o r g l u c o n o -k i n a s e and 2-KG k i n a s e i n mutant 3 e x p l a i n i t s s e n s i t i v i t y t o i n h i b i t i o n by g l u c o s e and not by g l u c o n a t e o r 2-KG. W i t h t h i s r e s t r i c t e d m e t a b o l i s m , o n l y two i n t e r m e d i a t e s a c c u m u l a t e d f o l l o w i n g a d d i t i o n o f g l u c o s e - U - C t o c u l t u r e s g r ow ing i n p y r u v a t e medium, j_.e_. g l u c o n a t e and 6-PG and t h e s e a c c o u n t e d f o r e s s e n t i a l l y a l l t he g l u c o s e m e t a b o l i z e d . However, t he a d d i -t i o n o f g l u c o s e t o t h i s mutant g r o w i n g i n p y r u v a t e medium s t i l l r e s u l t e d i n normal i nduced l e v e l s o f bo th g l u c o k i n a s e and G-6-P dehyd rogena se . R e v e r t a n t s o f mutant 3, w h i c h have r e g a i n e d the a b i l i t y t o u t i l i z e g l u c o s e as t he s o l e c a rbon s o u r c e , o n l y r e c o v e r e d a c t i v i t y o f 6-PG d e h y d r a t a s e w h i c h was induced t o w i l d - t y p e l e v e l s when g l u c o s e was p r e s e n t i n t h e medium. T h e r e f o r e , i t i s s t r o n g l y f e l t t h a t t h e r e s u l t s w i t h mutant 3 r u l e ou t any r o l e f o r 2-KG o r 2-K-6-PG as i n d u c e r s f o r g l u c o k i n a s e , G-6-P dehyd rogena se and t he ED pathway enzymes (6-PG d e h y d r a t a s e and KDGP a l d o -l a s e ) . The f a c t t h a t f u l l y i nduced amounts o f g l u c o k i n a s e and G-6-P dehyd rogena se a r e found i n mutant 11^, f o l l o w i n g the a d d i t i o n o f 2-KG, s u g g e s t s t h a t t h e i n d u c e r f o r t h e s e two enzymes i s some m e t a b o l i t e o f 2-KG. However, f r om mutant 3 i t does not appear t o be 2-KG o r 2-K-6-PG and i t i s not r e q u i r e d t o be m e t a b o l i z e d p a s t t h e l e v e l o f 6-PG. These r e s u l t s w o u l d s t r o n g l y i n d i c a t e t h a t t he i n d u c e r o f t h e s e two enzymes i s 6-PG. A l l g l u c o s e - n e g a t i v e mutant s examined appea red t o have c o n c u r r e n t l y l o s t d e t e c t a b l e a c t i v i t y o f 6-PG d e h y d r a t a s e and c o n t r o l o v e r 2 o r k o f 70 the o t h e r enzymes a s s a yed ( T a b l e X I I ) . R e v e r t a n t s o f t h e s e m u t a n t s , w i t h t he e x c e p t i o n o f mutant 3, o c c u r r e d a t a f r e q u e n c y s u g g e s t i n g a s i n g l e m u t a t i o n and a t l e a s t mutant s XI I I, and XV^ r e g a i n e d w i l d - t y p e c o n t r o l o ve r t h e s e f i v e enzymes. E x a c t l y what t he r e l a t i o n s h i p i s between a m u t a t i o n i n 6-PG d e h y d r a t a s e and l o s s o f normal c o n t r o l o v e r s e v e r a l o t h e r c a r b o h y d r a t e - c a t a b o l i z i n g enzymes i s u n c l e a r a t the p r e s e n t t i m e . The d a t a showed, however , t h a t a ma jo r c o n t r o l p o i n t may e x i s t a t t he l e v e l o f 6-PG d e h y d r a t a s e . They a l s o showed t h a t a s i m i l a r mechanism may e x i s t f o r the c o n t r o l o f t h e i n d u c t i o n o f bo th g l u c o k i n a s e and G-6-P d e h y d r o -genase and a second s i m i l a r c o n t r o l mechanism f o r t he i n d u c t i o n o f bo th g l u c o n o k i n a s e and 3"PG dehyd rogena se . Mutant X I I I , and r e l a t e d t ype s had c o n s t i t u t i v e l y low s p e c i f i c a c t i v i t i e s f o r bo th g l u c o k i n a s e and G-6-P dehyd rogena se a n d , a l t h o u g h r e t a i n i n g the a b i l i t y t o m e t a b o l i z e g l u c o s e , g l u c o n a t e , and 2-KG t o the l e v e l o f 6 -PG, they had l o s t t he a b i l i t y t o i nduce s i g n i f i c a n t l y e i t h e r o f t h e s e enzymes. These same mutant s had c o n -s t i t u t i v e l y h i g h s p e c i f i c a c t i v i t i e s f o r g l u c o n o k i n a s e and 3"PG d e h y d r o g e n -a s e , t h a t i s , s i m i l a r t o f u l l y i nduced l e v e l s i n t he p a r e n t s t r a i n ( T a b l e I I I ) . The p r e s e n c e o f g l u c o s e , g l u c o n a t e , o r 2-KG i n t h e medium d i d not r e s u l t i n any 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 g l u c o n o k i n a s e , a l t h o u g h some i n c r e a s e i n t h e s p e c i f i c a c t i v i t y o f 3~PG dehyd rogena se was o b t a i n e d c o n s i s t e n t l y . Mu tan t s I I I , , 11^, and 3 had no d e t e c t a b l e a c t i v i t y f o r g l u c o n o k i n a s e o r 3"PG d e h y d r o g e n a s e , whe the r g l u c o s e , g l u c o n a t e , o r 2-KG were p r e s e n t o r a b s e n t i n the medium. Mutant M l | , had c o n s t i t u t i v e l y low s p e c i f i c a c t i v i t i e s f o r bo th g l u c o k i n a s e and G-6 -P d e h y d r o g e n a s e , whereas mutant I I . and 3 had a p p a r e n t l y r e t a i n e d w i l d - t y p e c o n t r o l o v e r 71 b o t h o f t h e s e enzymes. These r e s u l t s s t r o n g l y s u gge s t ed t h a t w i t h g l u c o k i n a s e and G-6-P dehyd rogena se , a l o s s o f c o n t r o l o v e r one enzyme was accompan ied by a s i m i l a r l o s s o f c o n t r o l o v e r t he o t h e r . The same s i m i l a r i t y o f c o n t r o l appea red t o e x i s t f o r g l u c o n o k i n a s e and 3~PG dehyd rogena se . S i n c e a l l mutant s examined were d e f i c i e n t i n 6-PG d e -h y d r a t a s e , v e r y l i t t l e i n f o r m a t i o n r e g a r d i n g r e g u l a t i o n o f t h i s enzyme was o b t a i n e d . Mutant X I I I , and r e l a t e d t y p e s c o n t a i n e d c o n s t i t u t i v e l y h i g h amounts o f g l u c o n o k i n a s e and 3 - PG dehyd rogena se d u r i n g g rowth w i t h p y r u v a t e ( T a b l e X I I ) . The c o n s t i t u t i v e l y h i g h l e v e l s o f g l u c o n o k i n a s e were the same as t he maximum i nduced l e v e l s p r e s e n t i n t h e p a r e n t s t r a i n and d i d not i n c r e a s e f o l l o w i n g t he a d d i t i o n o f g l u c o s e , w h i c h has been shown t o be r a p i d l y m e t a b o l i z e d t o g l u c o n a t e , 2-KG and 6-PG. However, t he c o n s t i t u t i v e l y h i g h amounts o f 3"PG dehyd rogena se i n t he mutant s was l e s s ( a p p r o x i m a t e l y 100 u n i t s ) t han the maximum amount o b t a i n e d i n the w i l d - t y p e c e l l s and an i n c r e a s e i n s p e c i f i c a c t i v i t y o f 3"PG dehyd rogena se ( a p p r o x i m a t e l y 100 u n i t s ) was o b t a i n e d c o n s i s t e n t l y f o l l o w i n g t h e a d d i t i o n o f g l u c o s e . A s p o n t a n e o u s l y o c c u r r i n g mutant ( X l l l , _ ^ ) d e r i v e d f rom s t r a i n X I I I , l o s t a l l d e t e c t a b l e g l u c o n o k i n a s e a c t i v i t y but r e t a i n e d a l ower c o n s t i t u t i v e amount o f 3"PG dehyd rogena se ( a p p r o x i m a t e l y 100 u n i t s ) t han t h a t f ound i n X I I I , . T h i s a c t i v i t y d i d not i n c r e a s e f o l l o w i n g t h e a d d i t i o n o f g l u c o s e . These r e s u l t s s u g g e s t e d t h a t , a l t h o u g h t h e r e appea red t o be one mechanism f o r t he c o n t r o l o f g l u c o n o k i n a s e and 3"PG d e h y d r o g e n a s e , t h e r e may a l s o be a second i ndependan t mechanism w h i c h r e g u l a t e s t he low l e v e l o f i n -d u c t i o n o f 3~PG dehyd rogena se . The i n d u c t i o n o f g l u c o s e and g l u c o n a t e dehyd rogena se s was a l s o examined i n t h e s e m u t a n t s . G l u c o n a t e dehyd rogena se was a s s a yed i n mutant s I .j , I I . , IV^, X M I ^ , XIV ^ , and XV ^ , and was i nduced by t he a d d i t i o n o f e i t h e r g l u c o s e , g l u c o n a t e , o r 2-KG. The most l i k e l y e x p l a n a t i o n i s t h a t some common i n t e r m e d i a t e i n the m e t a b o l i s m o f a l l t h r e e c a r b o h y d r a t e s , t h a t i s no t m e t a b o l i z e d p a s t t he l e v e l o f 6 -PG, cau sed t h e i n d u c t i o n o f t h i s enzyme j_.e_. 2-KG, 2 -K -6 -PG o r 6 -PG. Some d i f f e r e n t i a t i o n i n t he p a t t e r n o f i n d u c t i o n o f g l u c o s e d e h y d r o -genase was o b t a i n e d w i t h t h o s e mutant s w h i c h had r e t a i n e d w i l d - t y p e i n -d u c i b l e l e v e l s o f t h i s enzyme J_.e_. I ^ , XI I I ^ , and XI I I ^ I n t h e s e m u t a n t s , the a d d i t i o n o f g l u c o s e r e s u l t e d i n t h e i n d u c t i o n o f g l u c o s e d e h y d r o g e n a s e , whereas t he a d d i t i o n o f g l u c o n a t e o r 2-KG d i d not ( T a b l e XI I l ) . These r e s u l t s s t r o n g l y s u g g e s t e d t h a t g l u c o s e dehyd rogena se was i nduced by g l u c o s e o r some m e t a b o l i t e o f g l u c o s e (eg . G -6 -P) but not by g l u c o n a t e o r 2-KG o r t h e i r m e t a b o l i t e s . The r e l a t i o n s h i p between t he i n d u c t i o n o f t h i s enzyme and the g e n e r a t i o n t i m e w i l l be. d i s c u s s e d l a t e r . A summary o f t h e p o s s i b l e i n d u c e r s o f g 1 u c o s e - c a t a b o 1 i z i n g enzymes i n P_. a e r u g i n o s a , as d e t e r m i n e d f r om the e x a m i n a t i o n o f c a t a b o l i c mutant s o f t h i s m i c r o o r g a n i s m , i s p r e s e n t e d i n T a b l e XIV. The pathways o f g l u c o s e c a t a b o l i s m have been e s t a b l i s h e d i n the p a r e n t s t r a i n ( F i g . 27). Two o t h e r 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 d u r i n g the e x a m i n a t i o n o f the p r o d u c t s o f g l u c o s e , g l u c o n a t e , and 2-KG m e t a b o l i s m i n t h e 6-PG d e h y d r a t a s e - d e f i c i e n t m u t a n t s . In a l l o f the mutant s e xam ined , no g rowth was o b s e r v e d when l a b e l l e d g l u c o s e , g l u c o n a t e , o r 2-KG was p r e s e n t as t he T a b l e X I I I . Compar i s on o f g l u c o s e dehyd rogena se a c t i v i t y and g e n e r a t i o n t i m e i n P. a e r u g i n o s a g l u c o s e - n e g a t i v e m u t a n t s . Mutant Carbon s o u r c e 3 G e n e r a t i o n S p e c i f i c a c t i v i t y o f b t i m e (min) g l u c o s e dehyd rogena se X I I I . P y r u v a t e 55 5 P y r u v a t e + g l u c o s e f o r k h r ^ 0 C 74 P y r u v a t e + 2-KG f o r 4 h r M) 7 XI I 1 1_ A P y r u v a t e 55 14 P y r u v a t e + g l u c o s e f o r 4 h r ^0 62 P y r u v a t e + g l u c o n a t e f o r 4 hr M) 13 3 As f o r T a b l e XI I b As f o r T a b l e I I I c Growth s t a s i s . T a b l e X IV. P o s s i b l e i n d u c e r s o f g l u c o s e - c a t a b o l i z i n g enzymes i n P_. a e r u g i n o s a as d e t e r m i n e d f rom d a t a o b t a i n e d w i t h g l u c o s e - n e g a t i v e m u t a n t s . Enzyme P o s s i b l e i n d u c e r ( s ) G l u c o s e dehydrogenase G l u c o s e , G-6-P G l u c o n a t e dehydrogenase 2-KG, 2 - K - 6 - P G , 6-PG G l u c o k i nase 6-PG G-6-P dehydrogenase 6-PG G l u c o n o k i nase a 3-P-G dehydrogenase a ED pathway enzymes G l u c o s e , g l u c o n a t e , 6-PG o r some d e g r a d a t i o n p r o d u c t o f 6-PG a These two enzymes were e i t h e r c o n s t i t u t i v e o r ab sen t i n a l l mutan t s e x a m i n e d . 75 s o l e s o u r c e o f c a r b o n and no l o s s o f r a d i o a c t i v i t y as CO^ was found w i t h any o f t h e s e t h r e e c a r b o h y d r a t e s . None o f t h e s e c a r b o h y d r a t e s was m e t a b o l -i z e d beyond the l e v e l o f 6-PG and the mutant s r e v e r t e d t o g rowth w i t h g l u c o s e a t a f r e q u e n c y s u g g e s t i n g a s i n g l e g e n e t i c l e s i o n . These r e s u l t s s u g g e s t e d t he p a r e n t s t r a i n o f P_. a e r u g i nosa does no t po s s e s s a f u n c t i o n a l 6-PG dehydrogenase and s ub sequen t e x p e r i m e n t s c o n f i r m e d t h i s s u g g e s t i o n (D. B l ew, W.H. L y n c h , and A . F . G r o n l u n d . M a n u s c r i p t s u b m i t t e d f o r p u b l i -c a t i o n ) . The ab sence o f 6-PG dehyd rogena se has a l s o been r e p o r t e d i n Pseudomonas s a c c h a r o p h i 1 a ( 1 6 ) . The second o b s e r v a t i o n was t h a t mutant 3, a l t h o u g h e x h i b i t i n g no d e t e c t a b l e a c t i v i t y f o r g l u c o s e and g l u c o n a t e d e h y d r o g e n a s e s , a c c u m u l a t e d s i g n i f i c a n t q u a n t i t i e s o f g l u c o n a t e and 6-PG f rom g l u c o s e m e t a b o l i s m . These r e s u l t s s u g g e s t e d t h a t g l u c o n a t e was not formed f rom t h e d i r e c t o x i d a t i o n o f g l u c o s e but pe rhaps by t he a c t i o n o f a pho spha t a s e on the 6-PG t h a t a c c u m u l a t e d f rom g l u c o s e m e t a b o l i s m . Subsequent e x p e r i m e n t s s t r o n g l y i n d i c a t e d t h a t a p h o s p h a t a s e , w h i c h appea red t o be s p e c i f i c f o r 6 -PG, was p r e s e n t i n P_. ae rug i nosa (D. B l e w , W.H. L y n c h , and A . F . G r o n l u n d . M a n u s c r i p t s u b m i t t e d f o r p u b l i c a t i o n ) . IV. G l u c o s e and g l u c o n a t e dehyd rogena se s and t h e i r r o l e i n g l u c o s e d e g r a d a t i o n by P_. a e r u g i n o s a . The a c c u m u l a t i o n o f 2-KG d u r i n g g l u c o s e c a t a b o l i s m under a v a r i e t y o f c o n d i t i o n s , has been r e p o r t e d (76,88) i n P_. a e r u g i no sa . The i m p o r t a n c e o f g l u c o n a t e and 2-KG as i n t e r m e d i a t e s o f g l u c o s e c a t a b o l i s m was s u g g e s t e d by N o r r i s and Campbe l l (88) because o f the p r e s e n c e o f bo th t h e s e m e t a b o l i t e s 76 o ve r a t l e a s t an 8 hour p e r i o d d u r i n g g rowth on. g l u c o s e . S i m i l a r r e s u l t s have been r e p o r t e d i n P_. f l u o r e s c e n s (62) and P_. put i da ( 2 5 ) . However, t h e r e l a t i v e c o n t r i b u t i o n o f t he o x i d a t i v e n o n - p h o s p h o r y l a t e d pathway f rom g l u c o s e to 2-KG, t o t he o v e r a l l d e g r a d a t i o n o f g l u c o s e has not been e s t a b l i s h e d . The s t u d i e s w i t h g l u c o s e - n e g a t i v e mutant s s ugge s ted t h a t g l u c o s e dehyd rogena se was i nduced by g l u c o s e o r G -6 -P . G l u c o n a t e d e h y d r o -genase was i nduced f o l l o w i n g the a d d i t i o n o f g l u c o s e , g l u c o n a t e , o r 2-KG and e i t h e r 2-KG, 2 - K - 6 - P G , o r 6-PG was t he i n d u c e r . The i n d u c t i o n and i n t r a c e l l u l a r l e v e l s o f t h e s e two o x i d a t i v e enzymes were examined i n t h e w i l d - t y p e o r g a n i s m when e i t h e r g l u c o s e , g l u c o n a t e , o r 2-KG was t he c a r b o n s o u r c e . 1. A c c u m u l a t i o n o f p r o d u c t s f rom g l u c o s e , g l u c o n a t e , o r 2-KG d e g r a d a t i o n . When P^ . a e r u g i n o s a was grown w i t h e i t h e r g l u c o s e , g l u c o n a t e o r 2-KG as t he c a r b o n and energy s o u r c e , i n each i n s t a n c e , two d i s t i n c t r a t e s o f g rowth were a p p a r e n t ( F i g . 10 ) . Growth p a t t e r n s w i t h g l u c o s e and g l u c o n a t e were s i m i l a r w i t h an i n i t i a 1 g e n e r a t i o n t i m e o f 63 - 64 min f o l l o w e d by a s l o w e r g rowth r a t e w i t h a g e n e r a t i o n t i m e o f 73 m i n . However, the s l o w e r r a t e o f g rowth o c c u r r e d e a r l i e r when g l u c o n a t e was the g rowth s u b s t r a t e . When 2-KG.was t he c a rbon s o u r c e , t h e r e was an i n i t i a l c h a r a c t e r i s t i c a l l y s l o w r a t e o f . g r o w t h ( g e n e r a t i o n t i m e 96 min) f o l l o w e d by a more r a p i d r a t e ( g e n e r a t i o n t ime Ik m i n ) . Of s i g n i f i c a n c e was t h a t f a c t t h a t w i t h each c a r b o n s o u r c e , t he g e n e r a -t i o n t i m e d u r i n g the second r a t e o f g rowth was s i m i l a r . T h i s s u g g e s t e d p r o v i s i o n a l l y t h a t the c a rbon s o u r c e , a t t h i s t i m e , was i d e n t i c a l i n each i n s t a n c e . 2 4 6 8 H O U R S F i g . 10. Growth o f P_. a e r u g i n o s a w i l d - t y p e w i t h g l u c o s e ( o ) , g l u c o n a t e ( A ) , and 2-KG ( © ) as s o l e c a r b o n s o u r c e s . C o n c e n t r a t i o n o f c a r b o n s o u r c e s was 0.k%. 78 The d i s a p p e a r a n c e o f C - g l u c o s e d u r i n g g rowth o f P_. a e r u g i n o s a and t he appea r ance o f d e g r a d a t i o n p r o d u c t s were measu red . A t no t ime c o u l d e i t h e r p y r u v a t e o r a - k e t o g l u t a r a t e be d e t e c t e d . G l u c o n i c a c i d was not a s s a y e d f o r w i t h g l u c o n a t e dehyd rogena se f rom c r u d e c e l l - f r e e e x t r a c t s due t o the i n t e r f e r e n c e f rom g l u c o s e and g l u c o s e dehyd rogena se . When l e s s than 15% o f the t o t a l g rowth y i e l d was a t t a i n e d , o n l y 16% o f the o r i g i n a l g l u c o s e remained i n the c u l t u r e and 54% o f the g l u c o s e had been c o n v e r t e d to 2-KG. The 2-KG c o n c e n t r a t i o n i n c r e a s e d as g l u c o s e d i s a p p e a r e d and s u b s e q u e n t l y was 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 . To d e t e r m i n e i f g l u c o n a t e a l s o a c c u m u l a t e d i n t h e s e c u l t u r e s , t h e 14 same e x p e r i m e n t was r e p e a t e d u s i n g g l u c o s e - U - C. L a b e l l e d g l u c o n a t e appea red i n t he c u l t u r e f l u i d d u r i n g e a r l y g rowth but was r a p i d l y c o n v e r t e d t o 2-KG ( F i g . 11 ) . The b reak i n t he g rowth c u r v e found w i t h g l u c o s e grown c e l l s c o r r e s p o n d e d v e r y c l o s e l y t o t he d i s a p p e a r a n c e o f g l u c o s e and the u t i l i z a t i o n o f 2-KG as the c a r b o n s o u r c e . S i m i l a r e x p e r i m e n t s were c o n d u c t e d on c u l t u r e s grown w i t h g l u c o n a t e and w i t h 2-KG as c a r b o n s o u r c e s ( F i g . 12 ) . G l u c o n i c a c i d d i s a p p e a r e d v e r y r a p i d l y f rom the medium d u r i n g the e a r l y l o g a r i t h m i c phase o f g rowth and t he 2-KG t h a t a c c u m u l a t e d r e p r e s e n t e d as much as 72% o f the o r i g i n a l g l u c o n a t e . A g a i n , the s e c o n d , s l o w e r r a t e o f g rowth c o r r e l a t e d w i t h the e x h a u s t i o n o f g l u c o n a t e f rom the medium and t he u t i l i z a t i o n o f 2-KG as the c a rbon and energy s o u r c e ( F i g . 12A). N e i t h e r p y r u v a t e nor a - k e t o -g l u t a r a t e c o u l d be d e t e c t e d i n t he c u l t u r e medium. The e a r l y s l ow r a t e o f 2-KG d i s a p p e a r a n c e f rom 2-KG g row ing c u l t u r e s c o r r e l a t e d w i t h the E c O CO to >-CO UJ O o Q_ O 4 HOURS l l 14, A p p e a r a n c e o f C - l a b e l l e d g l u c o n a t e and 2-KG 14 d u r i n g g r owth o f P_. a e r u g i nosa wi t h Q.h% C-g l u c o s e . S p e c i f i c a c t i v i t y o f g l u c o s e was 0.013 y C i / y m o l e . S ymbo l s : o p t i c a l d e n s i t y , ( O ) ; g l u c o s e , ( A ) ; g l u c o n a t e , ( © ) ; 2 -KG, G l u c o s e , g l u c o n a t e , and 2-KG c o n c e n t r a -t i o n s were d e t e r m i n e d by D. B l ew. F i g . 12. D i s a p p e a r a n c e o f c a rbon s o u r c e d u r i n g g rowth o f P. a e r u g i n o s a on (A) g l u c o n a t e and (B) 2-KG. Symbols as F i g . 11. C o n c e n t r a t i o n o f c a r b o n s o u r c e s was Q.k%. G l u c o n a t e and 2-KG c o n c e n t r a t i o n s were d e t e r m i n e d by D. B l ew. a a 81 i n i t i a l l ong g e n e r a t i o n t i m e ( F i g . 12B). T h i s g rowth p a t t e r n on 2-KG i s c h a r a c t e r i s t i c f o r t h i s m i c r o o r g a n i s m . Under c o n d i t i o n s o f v i g o r o u s a e r a t i o n , as used h e r e , i t i s a p p a r e n t t h a t t h e o x i d a t i v e non -pho spho r y1a ted pathway p l a y s a majo r r o l e i n g l u c o s e and g l u c o n a t e d i s s i m i l a t i o n i n P_. a e r u g i n o s a . In f a c t , more t han t w o - t h i r d s o f the o r i g i n a l s u b s t r a t e becomes c o n v e r t e d t o 2-KG. As bo th g l u c o s e and g l u c o n a t e dehyd rogena se a r e l o c a t e d i n t he c e l l membrane (6), t h i s l i k e l y a c c o u n t s f o r the r a p i d o x i d a t i o n o f g l u c o s e and g l u c o n a t e . O x i d a t i o n may o c c u r a t the membrane s u r f a c e and t r a n s p o r t o f g l u c o s e o r g l u c o n a t e may not be a p r e r e q u i s i t e t o the i n i t i a t i o n o f c a t a b o l i s m v i a t h i s r o u t e . F u r t h e r e v i d e n c e f o r the i m p o r t a n c e o f t h i s d i r e c t o x i d a t i v e pathway f o r g l u c o s e c a t a b o l i s m was p r e s e n t e d e a r l i e r w i t h r e v e r t a n t s o f mutant XVj, w h i c h were d e f e c t i v e i n g l u c o s e dehyd rogena se ( T a b l e IV ) . These r e v e r t a n t s d i s p l a y e d a much l o n g e r g e n e r a t i o n t i m e d u r i n g g rowth w i t h g l u c o s e than d i d the p a r e n t s t r a i n ( F i g . 6) . T h e r e f o r e , a l t h o u g h t he o x i d a t i v e non -phosphory l a t e d r o u t e f o r g l u c o s e c a t a b o l i s m i s not r e q u i r e d f o r g rowth w i t h g l u c o s e by P_. ae rug i n o s a , i t i s o b v i o u s l y i m p o r t a n t f o r the r a p i d g rowth r a t e n o r m a l l y found w i t h g l u c o s e . M i d g e l y and Dawes have a l s o r e -p o r t e d t h a t t h i s pathway does not appear to be a r e q u i r e m e n t f o r g r owth o f P_. a e r u g i n o s a w i t h g l u c o s e (80) . The p r e s e n c e o f g l u c o s e and g l u c o n a t e dehyd rogena se s i n t h i s m i c r o -o r g a n i s m endows P_. a e r u g i n o s a w i t h two a l t e r n a t e r o u t e s f o r g l u c o s e c a t a -b o l i s m ( see F i g . 27) o t h e r t h a n d i r e c t p h o s p h o r y l a t i o n . I t i s c o n c e i v a b l e t h a t the p o s s e s s i o n o f t h e s e t h r e e a l t e r n a t e r o u t e s f rom g l u c o s e t o 6-PG may impa r t a b i o l o g i c a l a d van t a ge t o the o r g a n i s m i n i t s n a t u r a l e n v i r o n -82 ment f rom two p o i n t s o f v i e w . F i r s t l y , n a t u r a l l y a r i s i n g m u t a t i o n s w i t h a d e f e c t i n any one o f s i x enzymes ( g l u c o s e , g l u c o n a t e and G-6-P d e h y d r o -genases o r g l u c o s e , g l u c o n a t e and 2-KG k i n a s e s ) o r i n c e r t a i n c o m b i n a t i o n s , wou ld not r e s u l t i n the i n a b i l i t y o f P_. a e r u g i nosa t o u t i l i z e g l u c o s e . S e c o n d l y , under c o n d i t i o n s where g l u c o s e was i n e x c e s s f o r a l i m i t e d p e r i o d o f t i m e , t he r a p i d o x i d a t i o n o f g l u c o s e t o 2-KG c o u l d a c t as a mechanism o f c o n s e r v i n g a c a r b o n and energy s o u r c e , as has been s u g g e s t e d e l s e w h e r e ( 5 5 ) . 2. L e v e l s o f g l u c o s e and g l u c o n a t e dehyd rogena se s d u r i n g g r o w t h . The s y n t h e s i s o f b o t h g l u c o s e and g l u c o n a t e dehyd rogena se s was i nduced when P_. a e r u g i n o s a ATCC 9027 was grown w i t h g l u c o s e as t he c a r b o n s o u r c e (6,118), w h i c h i s s i m i l a r t o o t h e r s t r a i n s o f t h i s o r g a n i s m ( 38 , 50 , 86). I t was o f i n t e r e s t , t h e r e f o r e , t o examine t h e l e v e l s o f the two enzymes d u r i n g g r owth o f J P . a e rug i nosa on g l u c o s e , g l u c o n a t e and 2-KG. D u r i n g t he e a r l y l o g a r i t h m i c phase o f g rowth on g l u c o s e , the s p e c i f i c a c t i v i t y o f g l u c o s e dehyd rogena se was f o u r t i m e s g r e a t e r t han t h a t f ound i n p y r u v a t e grown c e l l s . T h i s v a l u e s u b s e q u e n t l y d e c r e a s e d by a f a c t o r o f two a t mid l o g a r i t h m i c phase and then i n c r e a s e d u n t i l a t s t a t i o n a r y phase t h e e a r l y l o g a r i t h m i c phase v a l u e had been r e g a i n e d ( F i g . 13A). The i n i t i a l d e c r e a s e i n s p e c i f i c a c t i v i t y o f g l u c o s e dehyd rogena se c o r -r e l a t e d w i t h t he d i s a p p e a r a n c e o f g l u c o s e f rom the medium and the f o l l o w i n g i n c r e a s e was c o i n c i d e n t w i t h t he app r oach o f s t a t i o n a r y phase . G l u c o n a t e dehyd rogena se showed a r e l a t i v e l y s t e a d y i n c r e a s e f rom e a r l y l o g a r i t h m i c t o s t a t i o n a r y phase J_.e_. an i n c r e a s e i n s p e c i f i c a c t i v i t y f r om 700 t o 2400 ( F i g . 13A). T h i s l a t t e r v a l u e i s f o r t y t ime s g r e a t e r t han t h a t f ound i n OPTICAL DENSITY 660 nm o p o o — ro o> — ro b> b b o £2 84 p y r u v a t e grown c e l l s . When g l u c o n a t e was the carb o n s o u r c e , g l u c o n a t e dehydrogenase i n -c r e a s e d as growth p r o c e e d e d , but g l u c o s e dehydrogenase e x h i b i t e d a l a g b e f o r e a s i g n i f i c a n t i n c r e a s e i n a c t i v i t y was noted. G l u c o s e dehydrogenase i n c r e a s e d by a f a c t o r o f 4.5 whereas g l u c o n a t e dehydrogenase i n c r e a s e d by a f a c t o r o f 2 ( F i g . 13B). The s p e c i f i c a c t i v i t y o f the l a t t e r enzyme was 30 time s g r e a t e r than t h a t found i n p y r u v a t e grown c e l l s . The e a r l y l o g a r i t h m i c phase v a l u e f o r g l u c o s e dehydrogenase was a l m o s t f o u r t i m e s lower than t h a t found i n g l u c o s e grown c e l l s d u r i n g the same phase o f growth and s i m i l a r t o the low l e v e l s found i n p y r u v a t e grown c e l l s . In 2-KG grown c e l l s , g l u c o s e dehydrogenase was f u l l y induced i n e a r l y l o g a r i t h m i c phase but d e c r e a s e d as growth c o n t i n u e d ( F i g . 13C). G l u c o n a t e dehydrogenase, w h i l e showing s l i g h t changes i n s p e c i f i c a c t i v i t y , was e s s e n t i a l l y f u l l y induced d u r i n g t h e e n t i r e growth c y c l e . These d a t a s u g g e s t e d t h a t , i n P_. aerug i nosa, g l u c o s e dehydrogenase i s induced by g l u c o s e o r a d e g r a d a t i o n p r o d u c t o f g l u c o s e t h a t i s not r e a d i l y produced from g l u c o n a t e o r 2-KG (eg. G-6-P) and a l s o by 2-KG o r a degrada-t i o n p r o d u c t o f 2-KG but o n l y a f t e r a s i g n i f i c a n t l a g p e r i o d , t h a t i s , d u r i n g s t a t i o n a r y phase w i t h g l u c o s e grown c e l l s o r l a t e l o g a r i t h m i c phase w i t h g l u c o n a t e grown c e l l s . G l u c o n a t e , on t h e o t h e r hand, does not appear t o a c t as an i n d u c e r . The i n c r e a s e i n s p e c i f i c a c t i v i t y found i n g l u c o n a t e grown c e l l s was v e r y l i k e l y t h e r e s u l t o f the c o n v e r s i o n o f g l u c o n a t e t o 2-KG. I t i s a p p a r e n t t h a t the l a t e i n d u c t i o n o f g l u c o s e dehydrogenase d u r i n g growth w i t h g l u c o s e o r g l u c o n a t e o c c u r r e d a t a time when the ca r b o n s o u r c e p r e s e n t i n the medium was e s s e n t i a l l y 2-KG and a l s o a t a time when 85 the g r owth r a t e had s l owed down c o n s i d e r a b l y . I t i s a l s o a p p a r e n t t h a t t h e s p e c i f i c a c t i v i t y o f t h i s enzyme d u r i n g g r owth w i t h 2-KG d e c r e a s e d as t h e g rowth r a t e i n c r e a s e d . I t i s not known w h e t h e r t he i n d u c t i o n o f g l u c o s e dehyd rogena se i n t he p r e s e n c e o f 2-KG as t h e c a r b o n s o u r c e has any r e l a t i o n s h i p w i t h a d e c r e a s e i n the g r owth r a t e . From t h e d a t a p r e s e n t e d i n T a b l e X I I I i t a p p e a r s t h a t 2-KG must be m e t a b o l i z e d p a s t t he l e v e l o f 6-PG i n o r d e r t o e l u c i d a t e t h e i n d u c t i o n o f t h i s enzyme, r e g a r d l e s s o f t h e g rowth r a t e . Two - ke tog1ucona te o r p o s s i b l y a d e g r a d a t i o n p r o d u c t o f t h i s compound, a p p e a r s t o i n duce s y n t h e s i s o f g l u c o n a t e dehyd rogena se . I n d u c t i o n o f enzyme s y n t h e s i s by t h e p r o d u c t o f t he r e a c t i o n i s not uncommon, and s e v e r a l examples o f t h i s have been r e v i e w e d (90). The i n d u c t i o n o f g l u -c o n a t e dehyd rogena se i n the p r e s e n c e o f g l u c o s e o r g l u c o n a t e was no doubt t h e r e s u l t o f t he c o n v e r s i o n o f t h e s e compounds t o 2-KG and was c o i n c i d e n t w i t h an a c c u m u l a t i o n o f 2-KG i n t he g rowth m e d i a . The r e s u l t s c o n f i r m t h o s e o b t a i n e d w i t h t he g l u c o s e - n e g a t i v e mutant s w h i c h s u g g e s t e d t h a t 2-KG o r i t s d e g r a d a t i o n p r o d u c t s 2 -K -6 -PG o r 6-PG cau sed t he i n d u c t i o n o f t h i s enzyme. 3. I n d u c t i o n o f g l u c o s e and g l u c o n a t e dehyd rogena se s by g l u c o s e , g l u c o n a t e , and 2-KG. In an e f f o r t t o examine the i n d u c t i o n o f t h e s e enzymes more c l o s e l y , g l u c o s e , g l u c o n a t e , 2-KG o r c o m b i n a t i o n s o f t h e s e s u b s t r a t e s were added t o l o g a r i t h m i c phase c u l t u r e s g r ow ing w i t h p y r u v a t e as t he c a r b o n s o u r c e and t he i n c r e a s e s i n t h e s p e c i f i c a c t i v i t i e s o f t he two enzymes were f o l l o w e d 86 ( F i g . 14). The a d d i t i o n o f g l u c o s e caused a r a p i d i n d u c t i o n o f g l u c o s e dehyd rogena se w i t h a 4.5 f o l d i n c r e a s e i n s p e c i f i c a c t i v i t y . The d e c r e a s e i n s p e c i f i c a c t i v i t y o b s e r v e d a t 2 i hou r s a f t e r g l u c o s e a d d i t i o n was l i k e l y a d i r e c t r e s u l t o f t h e d e p l e t i o n o f g l u c o s e f rom the medium. A much s l o w e r and l e s s s i g n i f i c a n t i n c r e a s e i n t h i s enzyme was found when e i t h e r g l u c o n a t e o r 2-KG was added t o t he c u l t u r e s and t h e r e appea red t o be a l a g b e f o r e i n d u c t i o n o f the enzyme o c c u r r e d ( F i g . 14A). S i m i l a r p a t t e r n s o f i n d u c t i o n were o b s e r v e d when t h e s e compounds were added i n v a r i o u s c o m b i n a t i o n s eg . 0.1% g l u c o s e p l u s 0.4% g l u c o n a t e . That i s , i n each i n s t a n c e , t he a d d i t i o n o f g l u c o s e promoted i n d u c t i o n o f the enzyme and n e i t h e r g l u c o n a t e nor 2-KG, even when p r e s e n t i n a c o n c e n t r a t i o n 4 - f o l d i n e x c e s s o f g l u c o s e , p r e v e n t e d o r a c c e n t u a t e d t h i s i n d u c t i o n . When g l u c o n a t e o r 2-KG was added i n d i v i d u a l l y o r t o g e t h e r , s l i g h t i n d u c t i o n o f t h e enzyme was e v i d e n t but o n l y a f t e r a l a g and when the g rowth r a t e was d e c r e a s i n g as s t a t i o n a r y phase was a p p r o a c h e d . From t h e s e d a t a , i t can be c o n c l u d e d t h a t g l u c o s e (o r p o s s i b l y G-6-P) i nduce s g l u c o s e dehyd rogena se w i t h some e f f e c t shown by 2-KG o r a d e g r a d a t i o n p r o d u c t o f 2-KG. The s l i g h t i n d u c t i o n o f t h i s enzyme by g l u c o n a t e i s p r o b a b l y t he r e s u l t o f g l u c o n a t e b e i n g o x i d i z e d t o 2-KG. The r a t e and e x t e n t o f i n d u c t i o n o f g l u c o n a t e dehyd rogena se o c c u r r e d w i t h the t h r e e s u b s t r a t e s i n t h e f o l l o w i n g o r d e r 2-KG > g l u c o n a t e > g l u c o s e ( F i g . 14B). The i n c r e a s e i n s p e c i f i c a c t i v i t y o b s e r v e d i n t he p r e s e n c e o f g l u c o s e and g l u c o n a t e was l i k e l y due t o the c o n v e r s i o n o f t h e s e compounds t o 2-KG. When g l u c o s e was added t o g e t h e r w i t h , and a t f o u r t ime s the c o n c e n t r a t i o n o f g l u c o n a t e o r 2-KG, bo th t h e r a t e and e x t e n t o f g l u c o n a t e / Q dehyd rogena se i n d u c t i o n were s i g n i f i c a n t l y d e c r e a s e d and re semb led t he p a t t e r n seen w i t h g l u c o s e a l o n e . G l u c o s e e i t h e r e x h i b i t s a s l i g h t i n h i b i -t o r y e f f e c t on i n d u c t i o n o r p o s s i b l y competes w i t h t h e s e two compounds f o r u p t a k e . V. R e g u l a t i o n o f c a r b o h y d r a t e - c a t a b o l i z i n g enzymes i n Pj_ a e rug i nosa by g l u c o s e , g l u c o n a t e , and 2-KG. G l u c o s e - c a t a b o l i z i n g enzymes i n P. a e r u g i n o s a ATCC 9027 (115,118) and o t h e r s t r a i n s o f t h i s m i c r o o r g a n i s m ( 1 9 , 38 , 50 , 68 , 80 , 94 ) have been shown t o be i nduced by g rowth w i t h g l u c o s e and o t h e r s i x - c a r b o n c a r b o h y d r a t e s . However , l i t t l e i s known c o n c e r n i n g the r e g u l a t i o n o f t h e s e enzymes. From the s t u d i e s o f g l u c o s e - n e g a t i v e mutant s i t appea red t h a t t he i n d u c e r o f g l u c o k i n a s e and G-6-P dehyd rogena se was 6-PG but no i n f o r m a t i o n was o b t a i n e d c o n c e r n i n g the i n d u c e r ( s ) o f g l u c o n o k i n a s e o r 3"PG dehyd rogena se and v e r y l i t t l e i n f o r m a t i o n was o b t a i n e d c o n c e r n i n g the i n d u c e r ( s ) o f t he ED pathway enzymes e x c e p t t h a t 2-KG o r 2 - K - 6 - P G were not l i k e l y i n v o l v e d . The r e g u l a t i o n o f t h e s e enzymes was s t u d i e d i n the p a r e n t s t r a i n by mea su r i t h e l e v e l s o f t he enzymes p r e s e n t d u r i n g g rowth w i t h g l u c o s e , g l u c o n a t e , and 2-KG and by f o l l o w i n g t h e i n d u c t i o n o f t h e s e enzymes by t h e s e t h r e e c a r b o h y d r a t e s . 1. L e v e l s o f c a r b o h y d r a t e - c a t a b o l i z i n g enzymes p r e s e n t d u r i n g g rowth o f P_. a e r u g i n o s a wi t h g l u c o s e , g l u c o n a t e , and 2-KG. Due t o t h e s i g n i f i c a n t amounts o f g l u c o n a t e and 2-KG t h a t a c -c u m u l a t e d i n t h e medium f rom g l u c o s e m e t a b o l i s m and 2-KG t h a t a c c u m u l a t e d i n t he medium f rom g l u c o n a t e m e t a b o l i s m ( F i g . 11, 1 2 ) , t he e f f e c t o f t h e s e 89 t h r e e c a r b o h y d r a t e s on the s p e c i f i c a c t i v i t i e s o f enzymes o f s i x - c a r b o n c a r b o h y d r a t e c a t a b o l i s m was examined. The s p e c i f i c a c t i v i t i e s o f enzymes induced d u r i n g growth w i t h g l u c o s e , g l u c o n a t e , o r 2-KG were d e t e r m i n e d a t d i f f e r e n t s t a g e s o f growth. I t was thought t h a t p o s s i b l y the r e s u l t s o b t a i n e d , by f o l l o w i n g the enzyme a c t i v i t i e s d u r i n g c o n v e r s i o n o f g l u c o s e t o g l u c o n a t e and then t o 2-KG, would i n d i c a t e how the enzymes were con-t r o l l e d by t h e s e t h r e e c a r b o h y d r a t e s . The f i v e enzymes measured were s e p a r a t e d i n t o two g r o u p s , based on the p a t t e r n o f t h e i r i n d u c t i o n d u r i n g growth i n medium c o n t a i n i n g e i t h e r g l u c o s e , g l u c o n a t e , or 2-KG as the o r i g i n a l c a r b o n s o u r c e . The f i r s t group c o n s i s t e d o f g l u c o k i n a s e , G-6-P dehydrogenase, and the enzymes o f the ED pathway ( F i g . 15). Each o f t h e s e enzymes was s i g n i f i c a n t l y induced by growth i n medium c o n t a i n i n g any o f the t h r e e s i x - c a r b o n c a r b o -h y d r a t e s and h i g h l e v e l s were p r e s e n t e a r l i e r i n the 2-KG grown c e l l s , f o l l o w e d by g l u c o n a t e grown c e l l s and then g l u c o s e grown c e l l s . R epressed l e v e l s o f the enzymes, J_.e_. s p e c i f i c a c t i v i t i e s o b t a i n e d d u r i n g growth i n medium c o n t a i n i n g p y r u v a t e as the s o l e c a r b o n s o u r c e , a r e shown i n T a b l e I I I . These r e p r e s s e d l e v e l s o f enzyme a c t i v i t i e s were m a i n t a i n e d d u r i n g growth w i t h p y r u v a t e from e a r l y l o g a r i t h m i c t o s t a t i o n a r y phase. The r e s u l t s s u g g e s t e d t h a t some common i n t e r m e d i a t e ( s ) i n the m e t a b o l i s m o f g l u c o s e , g l u c o n a t e , o r 2-KG caused the i n d u c t i o n o f t h e s e enzymes. The second group o f enzymes c o n s i s t e d o f g l u c o n o k i n a s e and 3"PG dehydrogenase ( F i g . 16). These two enzymes were induced s i g n i f i c a n t l y d u r i n g growth w i t h g l u c o s e , i n c r e a s i n g i n s p e c i f i c a c t i v i t y u n t i l t he c u l t u r e reached an O.D. o f a p p r o x i m a t e l y 1.0, a f t e r which time the HOURS F i g . 15. S p e c i f i c a c t i v i t i e s o f g l u c o k i n a s e , G-6-P d e h y d r o g e n a s e , and t he ED pathway enzymes d u r i n g g r owth w i t h g l u c o s e (A), g l u c o n a t e (B) , and 2-KG ( C ) . S ymbo l s : g r o w t h , ( • ) ; g l u c o k i n a s e , (o); G-6-P d e h y d r o g e n a s e , (A); and ED pathway enzymes, ( o ) . C o n c e n t r a t i o n o f c a r b o n s o u r c e s was 0.k%. o 1 3 5 7 1 3 5 7 2 3 5 7 9 H O U R S F i g . 1 6 . S p e c i f i c a c t i v i t i e s o f g l u c o n o k i n a s e and 3 _ P G dehyd rogena se d u r i n g g r owth w i t h g l u c o s e ( A ) , g l u c o n a t e ( B ) , and 2-KG (c). S ymbo l s : g r o w t h , ( © ) ; g l u c o n o k i n a s e , ( A ) ; 3 " P G d e h y d r o g e n a s e , ( • ) . C o n c e n t r a t i o n o f c a r b o n s o u r c e s was 0.k%. U 3 92 a c t i v i t i e s l e v e l l e d o f f and then d e c r e a s e d as s t a t i o n a r y phase was r eached ( F i g . 16A). D u r i n g g rowth w i t h g l u c o n a t e , t he enzymes were i nduced t o h i g h l e v e l s f rom the f i r s t measurement a t ear . ly l o g a r i t h m i c phase and remained a t a r e l a t i v e l y c o n s t a n t l e v e l u n t i l mid l o g a r i t h m i c phase was r e a c h e d . The s p e c i f i c a c t i v i t i e s s u b s e q u e n t l y d e c r e a s e d s i g n i f i c a n t l y and then l e v e l l e d o f f t oward s t a t i o n a r y phase ( F i g . 16B). The l e v e l s o f t he a c t i v i t i e s rema ined r e l a t i v e l y c o n s t a n t d u r i n g g rowth w i t h 2 -KG and were s i m i l a r t o the low l e v e l s o b t a i n e d d u r i n g t he l a t t e r s t a g e s o f g rowth w i t h g l u c o n a t e ( F i g . 1 6 c ) . The r e s u l t s showed t h a t , d u r i n g g rowth w i t h g l u c o s e , t he l e v e l s o f g l u c o n o k i n a s e and 3 - P G dehyd rogena se i n c r e a s e d as g l u c o n a t e was p roduced i n t he medium, l e v e l l e d o f f as g l u c o n a t e d i s a p p e a r e d f rom the medium, and t hen d e c r e a s e d w i t h i n 1 t o 2 hour s a f t e r g l u c o n a t e d i s a p p e a r a n c e ( F i g . 11). D u r i n g g rowth w i t h g l u c o n a t e , the enzymes were i nduced t o h i g h l e v e l s w h i c h rema ined r e l a t i v e l y c o n s t a n t and then d e c r e a s e d s i g n i f i c a n t l y w i t h i n 1 t o 2 hour s a f t e r g l u c o n a t e had d i s a p p e a r e d f rom the medium ( F i g . 1 2 A ) and w h i l e t he c u l t u r e was s t i l l g r ow ing l o g a r i t h m i c a l l y . When s t a t i o n a r y phase was r eached t he s p e c i f i c a c t i v i t i e s t hen l e v e l l e d o f f t o t he low i n -duced l e v e l s f ound d u r i n g g rowth w i t h 2 - K G . These r e s u l t s s t r o n g l y s u g -g e s t e d t h a t g l u c o n a t e i nduced the s y n t h e s i s o f g l u c o n o k i n a s e and 3 - P G dehyd rogena se . The low l e v e l o f i n d u c t i o n o f t h e s e two enzymes, a p p a r e n t d u r i n g g rowth w i t h 2 - K G , may have r e s u l t e d f rom the f o r m a t i o n o f s m a l l amounts o f g l u c o n a t e f rom 2 -KG m e t a b o l i s m by the a c t i o n o f a 6-PG pho spha -t a s e , w h i c h has been d i s c u s s e d p r e v i o u s l y . Due t o i t s s t r u c t u r a l s i m i l a r i t y t o g l u c o n a t e , t h e p o s s i b i l i t y t h a t 2 -KG l e s s e f f e c t i v e l y caused the i n d u c t i o n 93 o f t h e s e two enzymes c anno t be r u l e d o u t . T h i s l a t t e r s u g g e s t i o n was f i r s t made by N a r r o d and Wood (85) who o b t a i n e d s i m i l a r r e s u l t s o f a low l e v e l o f i n d u c t i o n o f g l u c o n o k i n a s e d u r i n g g rowth o f P_. p u t i d a wi th 2-KG. 2. I n d u c t i o n o f c a r b o h y d r a t e - c a t a b o l i z i n g enzymes by g l u c o s e , g l u c o n a t e , and 2-KG i n P_. ae rug ?nosa. To examine f u r t h e r t h e i n d u c t i o n o f s i x - c a r b o n c a r b o h y d r a t e -m e t a b o l i z i n g enzymes, c u l t u r e s o f P_. a e r u g i n o s a were grown i n a medium c o n t a i n i n g p y r u v a t e as t he s o l e c a r b o n s o u r c e . When t he c u l t u r e s had r eached an O.D. o f a p p r o x i m a t e l y 0.45, g l u c o s e , g l u c o n a t e , 2-KG, o r com-b i n a t i o n s o f t h e s e s u b s t r a t e s were added . Growth was f o l l o w e d and samples were h a r v e s t e d a t 1/2, 1, 1 3/4, and 2 1/2 hour s a f t e r t h e a d d i t i o n o f the s i x - c a r b o n c a r b o h y d r a t e s and a s s a yed f o r t he a p p r o p r i a t e enzymes. The enzymes c o u l d a g a i n be s e p a r a t e d i n t o t he same two g roups based on the p a t t e r n o f t h e i r i n d u c t i o n f o l l o w i n g t he a d d i t i o n o f g l u c o s e , g l u c o -n a t e , o r 2-KG. A l t h o u g h t h e r e was some s l i g h t d i f f e r e n c e i n t he r a t e and e x t e n t o f i n c r e a s e i n s p e c i f i c a c t i v i t y between g l u c o k i n a s e , G-6-P dehyd rogena se , and the ED pathway enzymes, they were v e r y s i m i l a r i n each o f t he c u l t u r e s ( F i g . 17)- The r a t e and e x t e n t o f i n d u c t i o n f o r each i n d i v i d u a l enzyme was v e r y s i m i l a r whe the r t h e s i x - c a r b o n c a r b o h y d r a t e used was g l u c o s e , g l u c o n a t e o r 2-KG c o m b i n a t i o n s o f t h e s e s u b s t r a t e s . The f a c t t h a t g l u c o s e , g l u c o n a t e , and 2-KG cau sed a s i g n i f i c a n t i n d u c t i o n o f g l u c o k i n a s e , G-6-P d e h y d r o g e n a s e , and t he enzymes o f t he ED pathway a t s i m i l a r r a t e s and t o s i m i l a r l e v e l s s u g g e s t e d t h a t some common i n t e r m e d i a t e ( s ) i n t he m e t a b o l i s m o f t h e s e t h r e e s i x - c a r b o n c a r b o h y d r a t e s was t h e i n d u c e r . 1 3 5 7 1 3 5 7 1 3 5 7 HOURS F i g . 17- I n d u c t i o n ' o f g l u c o k i n a s e ( A ) , G -6 -P d e h y d r o g e n a s e , ( B ) , and t h e ED pathway enzymes ( C ) . E i t h e r g l u c o s e , g l u c o n a t e , o r 2-KG ( f i n a l c o n c e n t r a t i o n 0.1%) were added t o t h e c u l t u r e s g r ow ing w i t h 0.h% p y r u v a t e as i n d i c a t e d by t h e a r r o w s . S ymbo l s : g r o w t h , ( • ) ; i n d u c t i o n o f t h e enzymes f o l l o w i n g t he a d d i t i o n o f g l u c o s e , ( o ) ; g l u c o n a t e , ( A ) ; 2 -KG, ( • ) . -e -95 The a d d i t i o n o f g l u c o s e t o c u l t u r e s o f 6-PG d e h y d r a t a s e - d e f i c i e n t mutant 3, g r o w i n g i n medium w i t h p y r u v a t e as the c a r b o n s o u r c e , r e s u l t e d i n f u l l y i nduced l e v e l s o f g l u c o k i n a s e and G-6-P dehyd rogena se ( T a b l e X I I ) . G l u c o n a t e and 6-PG were t h e o n l y d e t e c t a b l e i n t e r m e d i a t e s w h i c h a c c u m u l a t e d f r om g l u c o s e m e t a b o l i s m ( T a b l e X I ) . Of t h e s e two i n t e r m e d i a t e s , o n l y 6-PG ( t h e f i r s t common i n t e r m e d i a t e i n t h e m e t a b o l i s m o f g l u c o s e , g l u c o n a t e , o r 2-KG) appea r s t o f i l l t he r o l e o f i n d u c e r o f t h e s e two enzymes. I f g l u c o n -a t e was t he i n d u c e r , one m i gh t e x p e c t t he i n d u c t i o n o f t h e s e two enzymes t o f o l l o w t h e p a t t e r n o b s e r v e d f o r g l u c o n o k i n a s e and 3~PG d e h y d r o g e n a s e . S i n c e r e v e r t a n t s o f mutant 3, w i t h r e g a i n e d a b i l i t y t o grow w i t h g l u c o s e (but no t w i t h g l u c o n a t e o r 2 -KG) , o n l y r e c o v e r e d w i l d - t y p e a c t i v i t y and c o n t r o l o f t h e ED pathway enzymes, t h e same l o g i c s u g g e s t e d t h a t 6-PG o r some m e t a b o l i c p r o d u c t ( s ) o f t h i s common i n t e r m e d i a t e a c t s as t he i n d u c e r o f a t l e a s t 6-PG d e h y d r a t a s e (KDGP a l d o l a s e was not a s s a y e d due t o t h e u n a v a i l a b i l i t y o f KDGP). I t i s a l s o l o g i c a l t h a t a common i n t e r m e d i a t e i n t he m e t a b o l i s m o f g l u c o s e , g l u c o n a t e , and 2-KG wou ld c au se t h e i n d u c t i o n o f t he ED pathway enzymes, s i n c e t h e enzymes a r e r e q u i r e d f o r g r owth on a l l t h r e e s u b s t r a t e s as e v i d e n c e d by t he e x a m i n a t i o n o f g l u c o s e - n e g a t i v e m u t a n t s , and a l s o due t o t he r a p i d o x i d a t i o n o f g l u c o s e o r g l u c o n a t e t o 2-KG d u r i n g g r o w t h . S i g n i f i c a n t d i f f e r e n c e s i n t h e r a t e and e x t e n t o f i n c r e a s e i n s p e c i f i c a c t i v i t y o f g l u c o n o k i n a s e and 3~PG dehyd rogena se were o b s e r v e d , depend i n g on w h e t h e r g l u c o s e , g l u c o n a t e , o r 2-KG was used t o e l i c i t t h e i n d u c t i o n , a l t h o u g h t he p a t t e r n s o f i n d u c t i o n f o r t he two enzymes were v e r y s i m i l a r ( F i g . 18). The f a s t e s t r a t e o f i n c r e a s e i n s p e c i f i c a c t i v i t y f o r bo th enzymes was o b s e r v e d f o l l o w i n g t he a d d i t i o n o f g l u c o n a t e t o c u l t u r e s 96 97 g r o w i n g i n medium w i t h p y r u v a t e as the c a r b o n s o u r c e . A s l i g h t l y s l o w e r r a t e was o b s e r v e d f o r bo th enzymes f o l l o w i n g t he a d d i t i o n o f g l u c o s e . However, bo th g l u c o n a t e and g l u c o s e cau sed a s i m i l a r e x t e n t o f i n d u c t i o n f o r bo th enzymes. The d i f f e r e n c e s i n t he r a t e s o f i n c r e a s e i n s p e c i f i c a c t i v i t y c au sed by g l u c o n a t e and g l u c o s e l i k e l y i s due t o a r e q u i r e m e n t f o r g l u c o s e t o be m e t a b o l i z e d t o g l u c o n a t e . S i n c e t he i n c r e a s e i n s p e c i f i c a c t i v i t y o f g l u c o s e dehyd rogena se was v e r y r a p i d f o l l o w i n g t he a d d i t i o n o f g l u c o s e t o c u l t u r e s ( F i g . 14 ) , one wou ld not e x p e c t a s i g n i f i c a n t l a g b e f o r e g l u c o n a t e was p roduced f rom g l u c o s e m e t a b o l i s m . T h e r e f o r e , t he l a g b e f o r e i n d u c t i o n o f enzymes by g l u c o n a t e a l s o wou ld not be s i g n i f i c a n t . The r a t e o f i n c r e a s e i n s p e c i f i c a c t i v i t y o f g l u c o n o k i n a s e and 3 - PG dehyd rogena se was s l o w e r and t he l e v e l a t t a i n e d was much l ower f o l l o w i n g 2-KG a d d i t i o n . No s i g n i f i c a n t e f f e c t was no ted on t h e r a t e and e x t e n t o f i n c r e a s e i n s p e c i f i c a c t i v i t y o f t h e s e enzymes by g l u c o n a t e whethe r g l u c o s e o r 2-KG were p r e s e n t (even a t 4 t i m e s t he c o n c e n t r a t i o n o f g l u c o n a t e ) o r a b s e n t . These r e s u l t s c o n f i r m e d t h e e a r l i e r f i n d i n g s t h a t g rowth w i t h g l u c o n a t e cau sed a f a s t e r r a t e o f i n d u c t i o n o f t he enzymes than g rowth w i t h g l u c o s e but maximum l e v e l s o f a c t i v i t y were s i m i l a r . Low l e v e l s o f t h e s e two enzymes were p r e s e n t o n l y d u r i n g g rowth w i t h 2-KG ( F i g . 16 ) . I t a g a i n appea red t h a t t h e s e two enzymes were i nduced by t he p r e s e n c e o f g l u c o n a t e . The p r e s e n c e o f g l u c o n a t e i n t he g rowth medium was r e q u i r e d f o r f u l l y i nduced l e v e l s o f g l u c o n o k i n a s e and 3~PG dehyd rogena se t o be e x p r e s s e d . The s p e c i f i c a c t i v i t i e s o f t h e s e two enzymes i n c r e a s e d c o n s i d e r a b l y as g l u c o n a t e was p roduced i n t h e medium f r om g l u c o s e m e t a b o l i s m and d e c r e a s e d s i g n i f i c a n t l y soon a f t e r i t d i s a p p e a r e d f rom the medium. G l u c o n a t e (and 98 g l u c o s e due t o i t s a b i l i t y to be o x i d i z e d t o g l u c o n a t e ) promoted a r a p i d r a t e o f i n c r e a s e i n s p e c i f i c a c t i v i t i e s o f t h e s e two enzymes and t o h i g h l e v e l s . T h e r e f o r e , i t i s s t r o n g l y f e l t t h a t g l u c o n a t e a c t s as t he i n d u c e r o f bo t h g l u c o n o k i n a s e and 3~PG dehyd rogena se . When g l u c o n a t e was ab s en t o r had d i s a p p e a r e d f r om the medium under c o n d i t i o n s where 2-KG was s t i l l p r e s e n t , o n l y s l o w r a t e s and low l e v e l s o f i n d u c t i o n o f t h e s e two enzymes were o b s e r v e d . I t i s p o s s i b l e , t h a t when 2-KG was p r e s e n t i n t h e medium, s m a l l amounts o f g l u c o n a t e were formed f rom the a c t i o n o f 6-PG p h o s p h a t a s e on some o f the 6-PG p roduced f rom 2-KG m e t a b o l i s m . I t i s a l s o p o s s i b l e t h a t some s t r u c t u r a l s i m i l a r i t y between g l u c o n a t e and 2-KG may have a l l o w e d 2-KG t o a c t but l e s s e f f i c i e n t l y , as an i n d u c e r o f t h e s e enzymes. The r e s u l t s p r e s e n t e d s t r o n g l y s u p p o r t t he s e p a r a t i o n o f t he f i v e enzymes i n t o two g roups - one c o n s i s t i n g o f g l u c o k i n a s e , G-6-P d e h y d r o g e n a s e , and t he ED pathway enzymes; the o t h e r c o n s i s t i n g o f g l u c o n o k i n a s e and 3"PG dehyd rogena se ; w i t h 6-PG and g l u c o n a t e a c t i n g as t he i n d u c e r o f t h e i r r e s p e c t i v e g r oup . T h i s i s f u r t h e r s u p p o r t e d by t he f a c t t h a t t he e x a m i n a -t i o n o f s e v e r a l g l u c o s e - n e g a t i v e m u t a n t s , d e f i c i e n t i n 6-PG d e h y d r a t a s e , a l s o s t r o n g l y s u g g e s t e d t he p r e s e n c e o f two s e p a r a t e r e g u l a t o r y mechan i sms; one i n v o l v i n g g l u c o k i n a s e and G-6-P d e h y d r o g e n a s e , and t he o t h e r i n v o l v i n g g l u c o n o k i n a s e and 3"PG dehyd rogena se , and t h a t 6-PG a c t e d as the i n d u c e r o f t he f o r m e r two enzymes ( T a b l e X I l ) . As p r e v i o u s l y s t a t e d , i t has not been d e t e r m i n e d whe the r t he ED pathway enzymes a r e i nduced by 6-PG o r a d e g r a d a t i o n p r o d u c t o f t h i s compound. In P_. put i d a , i t has been shown t h a t g l u c o n o k i n a s e and ED pathway enzymes a r e i nduced by g l u c o n a t e o r some p r o d u c t ( s ) o f g l u c o n a t e m e t a b o l i s m 99 (95). In t h i s r e s p e c t , t he r e s u l t s o b t a i n e d w i t h IP. a e r u g i n o s a a r e i n agreement w i t h t h o s e found i n P_. pu t i d a . The r e s u l t s have shown t h a t w i t h c a r b o h y d r a t e - c a t a b o l i z i n g enzymes i n P_. a e r u g i n o s a , some enzymes a r e s u b s t r a t e i nduced and some a r e p r o d u c t i nduced w h i c h i s not u n l i k e t he r e g u l a t i o n o f o t h e r c a t a b o l i c pathways i n Pseudomonas t h a t have been r e v i e w e d (90). In one r e s p e c t , t he c o n t r o l o f the c a r b o h y d r a t e - c a t a b o l i z i n g enzymes by 6-PG i s v e r y s i m i l a r t o the c o n t r o l o f some o f the enzymes o f t he 3 ~ k e t o a d i p a t e pathway by 3 " k e t o a d i p a t e i n P_. a e r u g i n o s a (58). The b r a n c h p o i n t i n t e r m e d i a t e (6-PG) appea r s not o n l y t o i n d u c e the enzyme m e t a b o l i z i n g i t (ED pathway enzymes) but a l s o t he two enzymes i n one b ranch l e a d i n g t o i t s f o r m a t i o n ( g l u c o k i n a s e and G-6-P d e h y d r o g e n a s e ) . When t he b r a n c h p o i n t compound i s fo rmed f rom a d i f f e r e n t b r a n c h i n t he pathway ( f rom g l u c o n a t e o r 2-KG) t he l a t t e r two enzymes appear t o be s y n t h e s i z e d g r a t u i t o u s l y . V I . The r e g u l a t i o n o f c a r b o h y d r a t e - c a t a b o l i z i ng enzymes i n P_. ae rug i nosa  by g l y c e r o l . I t has been r e p o r t e d p r e v i o u s l y t h a t t h e s i x - c a r b o n c a r b o h y d r a t e - c a t a -bo l i z i n g (CC) enzymes i n P_. a e r u g i n o s a a r e p r e s e n t a t low l e v e l s d u r i n g g rowth w i t h a c e t a t e , p y r u v a t e , o r 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 , but t h a t t h e s e enzymes a r e i nduced t o s i g n i f i c a n t l y h i g h e r l e v e l s d u r i n g g rowth w i t h s i x - c a r b o n c a r b o h y d r a t e s , g l y c e r o l , o r g l y c e r a t e (19,38,50, 68,80,94,115,118). The c o n t r o l e x e r t e d o v e r t he CC enzymes by t he s i x -c a r b o n c a r b o h y d r a t e s ; g l u c o s e , g l u c o n a t e , and 2-KG, i n t h i s s t r a i n o f P_. a e r u g i nosa has a l r e a d y been d i s c u s s e d . However, v e r y l i t t l e i s known 100 c o n c e r n i n g the r e g u l a t i o n o f t h e s e enzymes by g l y c e r o l . L e s s i e and N e i d h a r d t have shown t h a t h i g h i nduced l e v e l s o f g l u c o -k i n a s e , G-6-P d e h y d r o g e n a s e , 6-PG dehyd rogena se and t he ED pathway enzymes were p r e s e n t d u r i n g g rowth o f t h e i r s t r a i n o f P_. a e r u g i n o s a w i t h g l y c e r o l (68). S i m i l a r r e s u l t s , i n c l u d i n g t h e i n d u c t i o n o f g l u c o s e dehyd rogena se , were r e p o r t e d by Hylemon and P h i b b s u s i n g a d i f f e r e n t s t r a i n o f P_. ae rug i nosa (50). These w o r k e r s a l s o showed t h a t g rowth w i t h g l y c e r a t e a c t e d i n a manner a n a l o g o u s t o g rowth w i t h g l y c e r o l and s u g g e s t e d that t he a c t u a l i n d u c e r ( s ) o f c e r t a i n CC enzymes i n P_. a e r u g i nosa may be a t r i o s e - p h o s p h a t e o r some d e r i v a t i v e t h e r e o f , due t o t he i n d u c t i o n o f t h e s e enzymes by g rowth w i t h t h r e e - c a r b o n compounds such as g l y c e r o l o r g l y c e r a t e . M i d g l e y and Dawes, u s i n g y e t a n o t h e r s t r a i n o f P_. a e r u g i n o s a , r e p o r t e d s i m i l a r r e s u l t s d u r i n g g rowth w i t h g l y c e r o l (80). The p r e s e n c e o f h i g h amounts o f CC enzymes d u r i n g g rowth w i t h g l y c e r o l has a l s o been r e p o r t e d i n r e l a t e d o r gan i sms such as _P. put i d a (100) and P_. f 1 u o r e s c e n s (95)-E v i d e n c e , p r e s e n t e d p r e v i o u s l y , s t r o n g l y s u g g e s t e d t h a t the a c t u a l i n d u c e r s o f CC enzymes i n P. a e r u g i n o s a ATCC 9027 were s i x - c a r b o n c a r b o -h y d r a t e s , J_.e_. g l u c o s e o r G-6-P as the i n d u c e r o f g l u c o s e d e h y d r o g e n a s e , g l u c o n a t e as the i n d u c e r o f g l u c o n o k i n a s e and 3"PG dehyd rogena se , 2-KG as the i n d u c e r o f g l u c o n a t e d e h y d r o g e n a s e , 6-PG as the i n d u c e r o f g l u c o -k i n a s e and G-6-P d e h y d r o g e n a s e , and 6-PG o r some d e r i v a t i v e t h e r e o f as t h e i n d u c e r o f t he ED pathway enzymes. Due t o the a p p a r e n t c o n t r o l e x e r t e d o v e r t he i n d u c t i o n o f CC enzymes by s i x - c a r b o n c a r b o h y d r a t e s i n t h i s s t r a i n o f P_. a e r u g i n o s a and r e p o r t s i n t h e l i t e r a t u r e c o n c e r n i n g t he p r e s e n c e o f h i g h l e v e l s o f c e r t a i n CC enzymes d u r i n g g rowth o f v a r i o u s s t r a i n s o f 101 P_. a e r u g i n o s a w i t h g l y c e r o l , t h e i n d u c t i o n o f CC enzymes by g l y c e r o l and the pathways o f g l y c e r o l d i s s i m i l a t i o n i n P_. a e r u g i n o s a ATCC 9027 were i n v e s t i g a t e d . 1. Growth o f w i l d - t y p e c e l l s . Growth o f w i l d - t y p e c e l l s i n g l y c e r o l m i n i m a l medium i s shown i n F i g . 19- The g e n e r a t i o n t i m e (150 min) i s c o n s i d e r a b l y s l o w e r than w i t h p y r u v a t e ( see F i g . 22), o r w i t h s i x - c a r b o n c a r b o h y d r a t e s g l u c o s e , g l u c o n a t e , o r 2-KG ( F i g . 10), and i s s i m i l a r t o t h a t r e p o r t e d f o r o t h e r s t r a i n s o f P_. ae r u g i nosa (50,68). When g l y c e r o l was added t o c u l t u r e s growing i n p y r u v a t e m i n i m a l medium, g rowth c o n t i n u e d a t a r a t e s i m i l a r t o t h a t w i t h p y r u v a t e a l o n e u n t i l t he p y r u v a t e was e x h a u s t e d f rom the medium. A t t h i s t ime t he g rowth r a t e d e c r e a s e d c o n s i d e r a b l y u n t i l i t r eached a r a t e more t y p i c a l o f t h a t f ound w i t h g l y c e r o l a l o n e ( see F i g . 22). 2. L e v e l s o f c a r b o h y d r a t e - c a t a b o 1 i z i n g enzymes d u r i n g g rowth w i t h g l y c e r o l . The l e v e l s o f CC enzymes p r e s e n t d u r i n g g rowth o f w i l d - t y p e c e l l s w i t h g l y c e r o l were f o l l o w e d f rom e a r l y l o g a r i t h m i c to s t a t i o n a r y phase . G l u c o s e dehyd rogena se was f u l l y i nduced a t the f i r s t measurement t h a t was made and the a c t i v i t y remained r e l a t i v e l y c o n s t a n t w i t h o n l y a s l i g h t d e c r e a s e by s t a t i o n a r y phase ( F i g . 19). T h i s was s i m i l a r t o t h e p a t t e r n o f i n d u c t i o n e x h i b i t e d by t h i s enzyme d u r i n g g rowth w i t h 2-KG as the c a r b o n s o u r c e ( F i g . 13C). G l u c o n a t e d e h y d r o g e n a s e , w h i c h was s t r o n g l y s u gge s t ed as b e i n g i nduced by 2-KG, showed no d e t e c t a b l e i n c r e a s e i n s p e c i f i c a c t i -v i t y o v e r t h e low un i nduced amounts , a t any s t a g e d u r i n g g rowth w i t h g l y c e r o l ( F i g . 19). HOURS F i g . 19- S p e c i f i c a c t i v i t i e s o f g l u c o s e dehyd rogena se ( O ) and g l u c o n a t e d e h y d r o g e n a s e ( A ) d u r i n g g rowth o f P. a e r u g i n o s a ( © ) w i t h 0 .043 M g l y c e r o l . o 103 G l u c o k i n a s e , G-6-P dehyd rogena se , and t he ED pathway enzymes were a l s o f u l l y i nduced a t the f i r s t measurement and the s p e c i f i c a c t i v i t i e s r ema ined r e l a t i v e l y c o n s t a n t , bu t d e c r e a s e d somewhat as s t a t i o n a r y phase was r eached ( F i g . 2 0 ) . For a l l t h r e e enzymes the p a t t e r n o f i n d u c t i o n r e semb led t h a t f ound d u r i n g g rowth w i t h 2-KG ( F i g . 15C) . The s p e c i f i c a c t i v i t y o f 3~PG dehyd rogena se rema ined a t a c o n s t a n t low i nduced amount d u r i n g g rowth w i t h g l y c e r o l ( F i g . 2 0 ) , and t h i s was a l s o s i m i l a r t o t h e s p e c i f i c a c t i v i t y p r e s e n t d u r i n g g rowth w i t h 2-KG but a p p r o x i m a t e l y 50% l o w e r . The l e v e l o f t he enzyme was c o n s i d e r a b l y lower t han t h a t f ound d u r i n g c e r t a i n s t a g e s o f g rowth w i t h g l u c o s e o r g l u c o n a t e ( F i g . 16 ) . No d e t e c t a b l e 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 g l u c o n o k i n a s e , w h i c h was s i m i l a r t o t h a t amount p r e s e n t i n p y r u v a t e grown c e l l s , was o b s e r v e d d u r i n g g rowth w i t h g l y c e r o l ( F i g . 2 0 ) . Both g l y c e r o l k i n a s e and L - a - G P dehyd rogena se were p r e s e n t i n t h i s s t r a i n o f P_. a e r u g i n o s a and h i g h amounts o f bo th enzymes were d e t e c t e d d u r i n g g rowth w i t h g l y c e r o l . The s p e c i f i c a c t i v i t i e s f ound d u r i n g g rowth w i t h p y r u v a t e o r g l u c o s e were < 1 but a 2 8 - f o l d i n c r e a s e i n t he s p e c i f i c a c t i v i t y o f g l y c e r o l k i n a s e and a 1 5 - f o l d 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 L - a - G P dehyd rogena se were found i n g l y c e r o l grown c e l l s ( F i g . 2 1 ) . The s p e c i f i c a c t i v i t y o f L - a - G P dehyd rogena se d e c r e a s e d i n t h e s t a t i o n a r y phase o f g l y c e r o l grown c u l t u r e s . These d a t a d e m o n s t r a t e d t h a t , as w i t h o t h e r s t r a i n s o f the same (50,68,80) and r e l a t e d s p e c i e s (95,100) o f Pseudomonas, c e r t a i n CC enzymes were p r e s e n t a t h i g h l e v e l s d u r i n g g rowth o f P_. a e r u g i n o s a ATCC 9027 w i t h g l y c e r o l . The l e v e l s o f some o f the CC enzymes i n w i l d - t y p e c e l l s grown w i t h g l y c e r o l were v e r y s i m i l a r t o t he 5 1 0 1 5 1 9 HOURS F i g . 2 1 . S p e c i f i c a c t i v i t i e s o f g l y c e r o l k i n a s e ( • ) and L -a -GP d e h y d r o g e n a s e ( O ) d u r i n g g rowth o f P_. a e r u g i n o s a ( © ) w i t h 0 .043 M g l y c e r o l . o 106 l e v e l s f ound d u r i n g g rowth w i t h 2-KG J_.e_. f u l l y i nduced l e v e l s o f g l u c o s e d e h y d r o g e n a s e , g l u c o k i n a s e , G-6-P dehyd rogena se , and t he ED pathway enzymes, and low i nduced amounts o f 3 _ PG dehyd rogena se (but 50% l ower i n g l y c e r o l t han i n 2-KG grown c e l l s ) . However, the amounts o f g l u c o n a t e dehyd rogena se and g l u c o n o k i n a s e d e t e c t e d d u r i n g g rowth w i t h g l y c e r o l were the same as the r e p r e s s e d amounts o f t h e s e two enzymes p r e s e n t d u r i n g g rowth w i t h p y r u v a t e . In t h i s r e s p e c t , g l y c e r o l grown c e l l s d i f f e r g r e a t l y f rom 2-KG grown c e l l s w h i c h have f u l l y i nduced l e v e l s o f g l u c o n a t e d e h y d r o -genase and low i nduced l e v e l s o f g l u c o n o k i n a s e . 3- I n d u c t i o n o f c a r b o h y d r a t e - c a t a b o l i z i n g enzymes by g l y c e r o l . To examine f u r t h e r the i n d u c t i o n o f CC enzymes, g l y c e r o l was added t o c u l t u r e s o f P_. a e r u g i n o s a w h i c h were g r ow ing l o g a r i t h m i c a l l y i n p y r u v a t e m i n i m a l medium and samples o f the c u l t u r e were removed f o r enzyme a s s a y s . No d e t e c t a b l e i n c r e a s e i n t h e s p e c i f i c a c t i v i t y o f g l u c o s e d e h y d r o -genase was o b s e r v e d f o l l o w i n g t he a d d i t i o n o f g l y c e r o l u n t i l a f t e r a p p r o x i -m a t e l y 3 hou r s i n c u b a t i o n . A t t h i s t i m e a marked i n c r e a s e o c c u r r e d , u n t i l a p p r o x i m a t e l y k\ hou r s a f t e r g l y c e r o l a d d i t i o n ( F i g . 2 2 ) . The t i m e o f t he i n d u c t i o n o f g l u c o s e dehyd rogena se c o i n c i d e d w i t h t he p o i n t i n t he g rowth c u r v e a t w h i c h p y r u v a t e had been e x h a u s t e d f rom the medium and t he g rowth r a t e was d e c r e a s i n g c o n s i d e r a b l y to a r a t e w h i c h was more t y p i c a l o f g l y c e r o l grown c e l l s . T h i s was s i m i l a r t o t he p a t t e r n o f i n d u c t i o n o f g l u c o s e dehyd rogena se o b s e r v e d f o l l o w i n g t h e a d d i t i o n o f g l u c o n a t e o r 2-KG t o c e l l s g r o w i n g i n p y r u v a t e medium. Tha t i s , t h e i n c r e a s e i n s p e c i f i c a c t i v i t y was not s i g n i f i c a n t u n t i l s e v e r a l hou r s a f t e r t he a d d i t i o n and was a t a p o i n t when t h e g rowth r a t e was d e c r e a s i n g ( F i g . 14A). N O i n c r e a s e 107 1 4 7 1 0 HOURS F i g . 22. S p e c i f i c a c t i v i t i e s o f g l u c o s e dehyd rogena se ( O ) , and g l u c o n a t e dehyd rogena se (A) f o l l o w i n g t h e a d d i t i o n o f 0.022 M g l y c e r o l (as i n d i c a t e d by the a r r ow ) to P_. a e r u g i n o s a g r o w i n g w i t h 0 .045 M p y r u v a t e ( ® ) . Growth w i t h 0 .045 M p y r u v a t e a l o n e i s a l s o shown ( D ) . 108 i n t he s p e c i f i c a c t i v i t y o f g l u c o n a t e dehyd rogena se , o v e r the low amount p r e s e n t p r i o r t o g l y c e r o l a d d i t i o n , was d e t e c t e d d u r i n g 6 hours i n c u b a t i o n a f t e r g l y c e r o l had been added ( F i g . 22). An i n d u c t i o n o f g l u c o k i n a s e , G-6-P dehyd rogena se , and the ED pathway enzymes o c c u r r e d i m m e d i a t e l y f o l l o w i n g the a d d i t i o n o f g l y c e r o l . Both the r a t e and t he e x t e n t o f i n c r e a s e i n s p e c i f i c a c t i v i t i e s were c o n s i d e r -a b l e ( F i g . 23), and were s i m i l a r t o t h e e f f e c t s p r e v i o u s l y f ound a f t e r the a d d i t i o n o f g l u c o s e , g l u c o n a t e o r 2-KG ( F i g . 17 ) . The r a t e o f i n c r e a s e i n t he s p e c i f i c a c t i v i t y o f 3~PG dehyd rogena se and the l e v e l o f i n d u c t i o n were much l ower f o l l o w i n g g l y c e r o l a d d i t i o n ( F i g . 23) t han were found f o l l o w i n g g l u c o s e o r g l u c o n a t e a d d i t i o n . In t h e s e r e s p e c t s , the i n d u c t i o n somewhat r e semb led t he p a t t e r n m e d i a t e d by 2-KG a d d i t i o n ( F i g . 1 8 ) , w i t h t h e e x c e p t i o n t h a t t he r a t e o f i n c r e a s e i n s p e c i f i c a c t i v i t y w i t h g l y c e r o l was s l o w e r and t he amount o f enzyme was abou t 1/2 o f t h a t f ound w i t h 2-KG. G l u c o n o k i n a s e d i d not e x h i b i t any i n -c r e a s e i n s p e c i f i c a c t i v i t y , o v e r the low amounts p r e s e n t i n p y r u v a t e grown c e l l s , d u r i n g t he 6 hour i n c u b a t i o n p e r i o d a f t e r g l y c e r o l a d d i t i o n ( F i g . 23). When g l y c e r o l was added t o the c e l l s i n p y r u v a t e medium, the s p e c i f i c a c t i v i t i e s o f g l y c e r o l k i n a s e and L - a - G P dehyd rogena se i n c r e a s e d r a p i d l y and t o amounts s i m i l a r t o t h o s e p r e s e n t i n w i l d - t y p e c e l l s grown w i t h g l y c e r o l as the s o l e c a r b o n s o u r c e . A g a i n , t he s p e c i f i c a c t i v i t y o f L - a - G P dehyd rogena se d e c r e a s e d r a p i d l y as s t a t i o n a r y phase was r eached ( F i g . 2k). A c o n s t a n t low s p e c i f i c a c t i v i t y was f ound f o r bo th enzymes d u r i n g g rowth w i t h p y r u v a t e and no i n c r e a s e i n t he a c t i v i t y o f e i t h e r enzyme was no ted f o l l o w i n g g l u c o s e a d d i t i o n t o c e l l s g r o w i n g i n p y r u v a t e med i urn. 1 4 7 1 0 HOURS g. 23- S p e c i f i c a c t i v i t i e s o f g l u c o k i n a s e G -6 -P dehyd rogena se ( • ) , ED pathway enzymes ( • ) , 3-PG dehyd rogena se ( v ) , and g l u c o n o k i n a s e ( • ) f o l l o w i n g t he a d d i t i o n o f 0 .022 M g l y c e r o l (as i n d i c a t e d by the a r r ow ) t o P_. a e r u g i n o s a g r o w i n g w i t h 0 .045 M. p y r u v a t e ( • ) . 1 4 7 1 0 H O U R S g.' 24. S p e c i f i c a c t i v i t i e s o f g l y c e r o l k i n a s e ( • ) and L - a -GP dehyd rogena se ( O ) f o l l o w i n g the a d d i t i o n o f 0 .022 M g l y c e r o l (as i n d i c a t e d by t he a r r ow ) t o P_. a e r u g i n o s a g r o w i n g w i t h 0 .045 M p y r u v a t e ( © ) . 111 The r e s u l t s c o n f i r m e d t he p r e v i o u s f i n d i n g s w i t h g l y c e r o l grown c e l l s , i n t h a t a d d i t i o n o f g l y c e r o l t o c e l l s g r ow ing i n p y r u v a t e medium r e s u l t e d i n an i n d u c t i o n o f g l u c o k i n a s e , G-6-P dehyd rogena se , and t he ED pathway enzymes a t a r a t e and e x t e n t v e r y s i m i l a r t o t h a t caused by the a d d i t i o n o f g l u c o s e , g l u c o n a t e , o r 2-KG. P r e v i o u s work w i t h bo th the p a r e n t s t r a i n and g l u c o s e - n e g a t i v e mutant s s t r o n g l y s u g g e s t e d t h a t 6-PG was the i n d u c e r o f t he f o rmer two enzymes and t h a t t h i s compound o r some p r o d u c t d e r i v e d f rom i t was the i n d u c e r o f the ED pathway enzymes. The p r o d u c t i o n o f 6-PG f rom g l y c e r o l m e t a b o l i s m was not o b v i o u s a n d , a l t h o u g h the pathways o f g l y c e r o l m e t a b o l i s m have not been e s t a b l i s h e d i n t h i s s t r a i n o f P_. a e r u g i n o s a , d a t a p r e s e n t e d h e r e s t r o n g l y s u g g e s t e d the e x i s t e n c e o f a t l e a s t one r o u t e 2_.e_. t h r ough g l y c e r o l k i n a s e and L-a-GP dehyd rogena se . T h i s was s u p p o r t e d by t h e f a c t t h a t bo th o f t h e s e enzymes appea red t o be s p e c i f i c a l l y i nduced by g rowth w i t h g l y c e r o l o r by g l y c e r o l a d d i t i o n t o c e l l s g r ow ing w i t h p y r u v a t e , but not by g rowth w i t h p y r u v a t e o r g l u c o s e o r by g l u c o s e a d d i t i o n t o c e l l s g r o w i n g w i t h p y r u v a t e . T h i s r o u t e f o r g l y c e r o l m e t a b o l i s m has been r e p o r t e d f o r P_. a e r u g i nosa (116) and P_. put i da ( 100 ) . P r e v i o u s r e s u l t s i n d i c a t e d t h a t g l u c o s e dehyd rogena se was i nduced by g l u c o s e , o r some p r o d u c t o f g l u c o s e m e t a b o l i s m (eg . G - 6 - P ) , t h a t i s not r e a d i l y o b t a i n e d f rom g l u c o n a t e o r 2 -KG, and a l s o by some p r o d u c t o f 2-KG m e t a b o l i s m t h a t a p p a r e n t l y has t o be m e t a b o l i z e d p a s t the l e v e l o f 6 -PG. The i n d u c t i o n p a t t e r n o f t h i s enzyme d u r i n g g l y c e r o l m e t a b o l i s m c l o s e l y r e semb led t h a t caused by 2-KG m e t a b o l i s m and s u g g e s t e d t h a t the i n d u c t i o n o f g l u c o s e dehyd rogena se by g l y c e r o l o r 2-KG d e g r a d a t i o n was cau sed by some common i n t e r m e d i a t e o f t h e s e two compounds. S i m i l a r l y , e a r l i e r r e s u l t s have s t r o n g l y i n d i c a t e d t he g l u c o n o k i n a s e and 3 _PG dehyd rogena se were i nduced by g l u c o n a t e and t h a t g l u c o n a t e d e -hyd rogena se was i nduced by 2-KG o r i t s d e g r a d a t i o n p r o d u c t s , 2-K-6-PG o r 6-PG. No d e t e c t a b l e i n d u c t i o n o f e i t h e r g l u c o n o k i n a s e o r g l u c o n a t e dehyd rogena se and o n l y a v e r y s l o w r a t e and a v e r y low l e v e l o f i n d u c t i o n o f 3"PG dehyd rogena se was o b s e r v e d f o l l o w i n g g l y c e r o l m e t a b o l i s m . k. F a t e o f u n i f o r m l y l a b e l l e d C - g l y c e r o l d u r i n g m e t a b o l i s m by w i l d - t y p e P_. a e r u g i n o s a and g l u c o s e - n e g a t i v e mutant X l l l ^ _ ^ . Due t o t h e i n d u c t i o n o f some, but no t o t h e r enzymes, o f g l u c o s e c a t a b o l i s m d u r i n g g rowth o f P_. a e r u g i n o s a w i t h g l y c e r o l and t o the f a c t t h a t a l l g l u c o s e - c a t a b o l i z i n g enzymes measured a p p e a r e d , f r om p r e v i o u s r e s u l t s , t o be i nduced a t t he s i x - c a r b o n c a r b o h y d r a t e l e v e l , t he f a t e o f g l y c e r o l d u r i n g m e t a b o l i s m was examined i n bo th t he p a r e n t o r g a n i s m and mutant s t r a i n X l l l j _ ^ . T h i s mutant p o s s e s s e d no d e t e c t a b l e g l u c o n o k i n a s e a c t i v i t y and o n l y low c o n s t i t u t i v e amounts o f 3 -PG dehyd rogena se ( T a b l e X l l ) . These enzyme l e v e l s were s i m i l a r t o t ho se found i n t he w i l d - t y p e c e l l s grown w i t h g l y c e r o l ( F i g . 20). Growth o f mutant s t r a i n X l l l ^ _ ^ w i t h p y r u v a t e was n o r m a l , _i_.e_. s i m i l a r t o t he w i l d - t y p e s t r a i n , however , g rowth w i t h p y r u v a t e i n the p r e s e n c e o f g l y c e r o l was somewhat i n h i b i t e d . No d e t e c t a b l e g rowth was o b s e r v e d a f t e r 31 hou r s i n c u b a t i o n when g l y c e r o l was t he s o l e c a r b o n s o u r c e ( F i g . 25). In o r d e r t o examine the p r o d u c t s o f g l y c e r o l m e t a b o l i s m , t he above s t r a i n s were grown i n p y r u v a t e med ium.to m i d - l o g a r i t h m i c pha se , h a v e s t e d , 1 4 7 1 0 H O U R S F i g . 25- Growth o f g l u c o s e - n e g a t i v e mutant XI I I^  ^ w i t h 0 .045 M p y r u v a t e ( o ) and w i t h 0.045 M p y r u v a t e p l u s 0 .022 M g l y c e r o l ( © ) . 114 re su spended i n u n i f o r m l y l a b e l l e d C - g l y c e r o l medium and i n c u b a t e d f o r 7 hour s ( F i g . 2 6 ) . Samples were removed f rom each c u l t u r e a f t e r 3 i and 7 hour s i n c u b a t i o n and t h e t r i c h l o r o a c e t i c a c i d s o l u b l e f r a c t i o n o f each sample was e l u t e d f r om a Dowex I-x8 f o r m a t e co l umn . In t h e w i l d - t y p e c e l l s , g r owth w i t h g l y c e r o l had commenced by 3 i hour s and i n t he sample t a k e n a t t h i s t i m e two peaks were d e t e c t e d . One c o n t a i n e d a l m o s t a l l o f t he r a d i o a c t i v i t y p r e s e n t i n t h e t r i c h l o r o a c e t i c a c i d s o l u b l e f r a c t i o n and was i d e n t i f i e d as u n r e a c t e d g l y c e r o l , t he o t h e r c o n t a i n e d 0.3% o f t he l a b e l i n t he t r i c h l o r o a c e t i c a c i d s o l u b l e f r a c t i o n and was i d e n t i f i e d as d i h y d r o x y a c e t o n e p h o s p h a t e . Both o f t h e s e compounds were ab sen t f r om t h e 7 hour s amp le , a t w h i c h t i m e t he c u l t u r e was i n s t a t i o n a r y pha se . D u r i n g i n c u b a t i o n w i t h t he w i l d - t y p e c e l l s , t h e g l y c e r o l c o n c e n t r a t i o n i n the c u l t u r e medium d e c r e a s e d as t he g rowth p roceeded and was u n d e t e c t -a b l e a t t he t i m e the c u l t u r e reached s t a t i o n a r y phase ( T a b l e X V ) . In mutant X l l l j _ ^ c u l t u r e s a t 3 i h o u r s , two s i m i l a r peaks were d e t e c t e d i n t h e e l u t i o n p r o f i l e f r om the c o l u m n : one c o n t a i n i n g most o f the r a d i o -a c t i v i t y p r e s e n t i n t he t r i c h l o r o a c e t i c a c i d s o l u b l e f r a c t i o n was i d e n t i f i e d as u n r e a c t e d g l y c e r o l , t he second c o n t a i n i n g 5% o f the t r i c h l o r o a c e t i c a c i d s o l u b l e r a d i o a c t i v i t y , was i d e n t i f i e d as 33% 6-PG w i t h the r e m a i n d e r as d i h y d r o x y a c e t o n e p h o s p h a t e . In t he 7 hour sample o f X l l l | _ ^ c u l t u r e s , e s s e n t i a l l y the same r e s u l t s were o b t a i n e d e x c e p t t h a t t he f i r s t peak e l u t e d f rom the co lumn c o n t a i n i n g u n r e a c t e d g l y c e r o l had d e c r e a s e d somewhat and t he second peak e l u t e d f rom the co lumn had a p p r o x i m a t e l y d o u b l e d i n s i z e ( T a b l e XV ) . A l t h o u g h no v i s i b l e g rowth o c c u r r e d d u r i n g the 7 hour 115 2 4 6 8 H O U R S 14 F i g . 26. Fate of uniformly l abe l l ed C-g lycero l during metabolism by w i ld - type JP. aerug i nosa (A) and gl ucose.-negat ive mutant X l l l , _ ^ (B). Cultures were grown in 0.045 M pyruvate and resuspended 14 in 8 mM g l y c e r o l - U - C ( s p e c i f i c a c t i v i t y 0.125 uCi/umole). Symbols: o p t i c a l density ( • ) . ' G lycero l disappearance ( A ) and CO^ evo lut ion ( A ) (measured as loss of r a d i o a c t i v i t y from the cu l tu re med i urn). T a b l e XV. D e g r a d a t i o n p r o d u c t s o f g l y c e r o l - U - C m e t a b o l i s m i n medium and c e l l s o f w i l d - t y p e P_. a e r u g i n o s a and the g l u c o s e - n e g a t i v e mutant. X l l l , . . a Organ ism 1. W + 2. W + 3. X I I I 1-A 4. X M I ^ O.D.at Hours i n c u b a t e d P r o d u c t s ' 3 f rom **C-g 1 y c e r o l m e t a b o l i s m ( m o l a r i t y x 10^) 660 nm w i t h g l y c e r o l -U-14C G l y c e r o l 6-PG D i h y d r o x y a c e t o n e pho spha te 0.96 0 80 0 0 1.10 3-5 56 0 _c 1 .35 d 7 0 0 0 2.15 0 80 0 0 2.48 4 9-2 0 0.24 2 . 2 5 d 12.5 0 0 0 1.14 0 80 0 0 1.09 3.5 68 0.2 -1 .05 c 7 56 0.44 -3.2 1 0 80 0 0 3 . 0 e 12.5 0 2.0 0.28 E x p e r i m e n t a l c o n d i t i o n s as F i g . 26. 14c a s s i m i l a t e d by the c e l l s and l o s t as ^ C 0 2 i s not r e c o r d e d h e r e . I n s u f f i c i e n t m a t e r i a l p r e s e n t to i d e n t i f y p o s i t i v e l y . A v e r y s m a l l r a d i o a c t i v e peak was e v i d e n t w i t h t he w i l d - t y p e . ' ( 1 ) . W i t h w i l d - t y p e c e l l s a t a h i g h e r O.D. (2) t he peak was i d e n t i f i e d as b e i n g 100% d i h y d r o x y a c e t o n e pho spha te . 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 mutant X l l l , _ ^ ( 3 ) , a t the h i g h e r O.D. (4) 93% o f the r a d i o a c t i v i t y i n t h i s peak was 6-PG and 7% was d i h y d r o x y a c e t o n e p h o s p h a t e . C u l t u r e s were i n s t a t i o n a r y phase. No g rowth was e v i d e n t . 117 i n c u b a t i o n o f mutant XI I I ^ , t h e r e was a p p r o x i m a t e l y a 30% d e c r e a s e i n t he g l y c e r o l c o n c e n t r a t i o n , a 12% l o s s o f r a d i o a c t i v i t y as c a r b o n d i o x i d e , and an i n c o r p o r a t i o n o f 10% o f the r a d i o a c t i v i t y i n t o t r i c h l o r o a c e t i c a c i d i n s o l u b l e m a t e r i a l ( F i g . 2 6 ) . These r e s u l t s i n d i c a t e d t h a t a l t h o u g h v i s i b l e g r owth o f mutant X l l l , _ ^ w i t h g l y c e r o l was c o m p l e t e l y i n h i b i t e d , p r o b a b l y due t o t h e a c c u m u l a t i o n o f 6 -PG, a s l ow r a t e o f g l y c e r o l m e t a b o l i s m to c a r b o n d i o x i d e and o t h e r c e l l p r o d u c t s s t i l l t ook p l a c e . The a c c u m u l a -t i o n o f 6-PG f rom g l y c e r o l m e t a b o l i s m l i k e l y e x p l a i n s the i n h i b i t e d r a t e o f g rowth o f s t r a i n X l l l , _ ^ w i t h p y r u v a t e i n t h e p r e s e n c e o f g l y c e r o l . The much l e s s s e v e r e i n h i b i t o r y r e s p o n s e o f mutant X I I I , ^ t o the p r e s e n c e o f g l y c e r o l i s p r o b a b l y due t o the l ower amounts o f a c c u m u l a t i o n o f 6-PG f rom g l y c e r o l m e t a b o l i s m than t h o s e o b t a i n e d p r e v i o u s l y d u r i n g g l u c o s e m e t a b o l i s m . V I I . R e g u l a t i o n o f t h e i n d u c t i o n o f c a r b o h y d r a t e - c a t a b o l i z i n g enzymes as e v i d e n c e d by the p a t t e r n s o f enzyme i n d u c t i o n and p r o d u c t a c c u m u l a t i o n  f r om g l y c e r o l m e t a b o l i s m . . I n d u c t i o n o f c a r b o h y d r a t e - c a t a b o 1 i z i n g enzymes d u r i n g g rowth w i t h g l y c e r o l and the pathways o f g l y c e r o l d i s s i m i l a t i o n i n P_. a e r u g i n o s a (ATCC 9027) were i n v e s t i g a t e d i n an e f f o r t t o e l u c i d a t e t he r e g u l a t o r y r o l e o f g l y c e r o l o v e r enzymes n o r m a l l y i nduced by s i x - c a r b o n m e t a b o l i t e s . The r e s u l t s o b t a i n e d p r o v i d e d i n f o r m a t i o n w h i c h a l l o w e d a c o r r e l a t i o n t o be made between t h e s e and the p r e v i o u s r e s u l t s d e r i v e d f rom the s t u d y o f g l u c o s e c a t a b o l i c mutan t s and t h e i n d u c t i o n o f c a r b o h y d r a t e - c a t a b o l i z i n g enzymes by s i x - c a r b o n c a r b o h y d r a t e s i n the p a r e n t s t r a i n . 118 1. G l u c o k i n a s e , G-6-P d e h y d r o g e n a s e , and t he enzymes o f t he ED pathway. P r e v i o u s r e s u l t s p r e s e n t e d e v i d e n c e t h a t v e r y s t r o n g l y s u g g e s t e d 6-PG was t he i n d u c e r o f g l u c o k i n a s e and G-6-P d e h y d r o g e n a s e , and t h a t t h i s m e t a b o l i c i n t e r m e d i a t e , o r some d e r i v a t i v e ( s ) t h e r e o f , was t he t h e i n d u c e r o f t he ED pathway enzymes i n IP. a e r u g i no sa . These enzymes were a l s o i n -duced by g rowth w i t h g l y c e r o l and t o l e v e l s s i m i l a r t o t h o s e found d u r i n g g r owth w i t h g l u c o s e , g l u c o n a t e , o r 2-KG. The e x a m i n a t i o n o f p r o d u c t s o f g l y c e r o l m e t a b o l i s m i n mutant X l l l | _ ^ d e m o n s t r a t e d a s i g n i f i c a n t a c c u m u l a -t i o n o f 6-PG w h i c h wou ld e x p l a i n t he i n d u c t i o n o f t h e s e enzymes d u r i n g g l y c e r o l m e t a b o l i s m i n t he w i l d - t y p e c e l l s . No a c c u m u l a t i o n o f 6-PG f rom g l y c e r o l m e t a b o l i s m was d e t e c t e d i n t h e p a r e n t s t r a i n , however , t h i s wou ld be e x p e c t e d because o f t h e h i g h i nduced l e v e l s o f the ED pathway enzymes i n t h e s e c e l l s d u r i n g g l y c e r o l d e g r a d a t i o n . 2. G l u c o n o k i n a s e and g l u c o n a t e dehyd rogena se . I t was p r e v i o u s l y s t a t e d t h a t g l u c o n o k i n a s e was i nduced by g l u c o n a t e and t h a t g l u c o n a t e dehyd rogena se was i nduced by 2-KG o r i t s d e g r a d a t i o n p r o d u c t s , 2 -K -6 -PG o r 6 -PG. No i n d u c t i o n o f e i t h e r o f t h e s e enzymes was o b s e r v e d d u r i n g g rowth w i t h g l y c e r o l o r f o l l o w i n g t he a d d i t i o n o f g l y c e r o l t o c e l l s g r o w i n g w i t h p y r u v a t e and no a c c u m u l a t i o n o f g l u c o n a t e o r 2-KG was d e t e c t e d f rom g l y c e r o l m e t a b o l i s m i n the p a r e n t s t r a i n o r mutant X l l l | _ ^ . Both m e t a b o l i t e s a r e ma jo r p r o d u c t s o f g l u c o s e m e t a b o l i s m and were e a s i l y d e t e c t e d i n c u l t u r e s o f bo th t he w i l d - t y p e c e l l s and mutant X l l l | _ ^ when g l u c o s e was p r e s e n t i n t h e medium. The i n d u c t i o n o f enzymes by 6-PG d u r i n g g r owth w i t h g l y c e r o l i n t he ab sence o f any i n d u c t i o n o f g l u c o n a t e 119 d e h y d r o g e n a s e , p l u s t he f a c t t h a t s i g n i f i c a n t amounts o f 6-PG a c c u m u l a t e d f r om g l y c e r o l m e t a b o l i s m i n 6-PG d e h y d r a t a s e - d e f i c i e n t mutant X I I I . , i n d i c a t e s t h a t 6-PG does not a c t as an i n d u c e r o f g l u c o n a t e d e h y d r o g e n a s e . I t i s f e l t t h a t 2 -K -6 -PG does not a c t as t he i n d u c e r o f g l u c o n a t e d e h y d r o -genase as t h i s o r g a n i s m p o s s e s s e s an a c t i v e and r e v e r s i b l e 2 -K -6 -PG r e d u c t a s e (55) and t h i s i n t e r m e d i a t e has been shown t o a c c u m u l a t e f rom 6-PG i n c u b a t e d i n t he p r e s e n c e o f c e l l - f r e e e x t r a c t s o f P_. a e r u g i n o s a (D. B l e w , W.H. L y n c h , and A . F . G r o n l u n d , M a n u s c r i p t s u b m i t t e d f o r p u b l i c a t i o n ) . These r e s u l t s s t r o n g l y s u g ge s t t h a t 2-KG i nduce s g l u c o n a t e dehyd rogena se and t h a t 2 -K -6 -PG o r 6-PG do n o t . 3. G e n e r a t i o n t i m e and amounts o f 3 - PG and g l u c o s e d e h y d r o g e n a s e s . A l t h o u g h i t c anno t be s t a t e d d e f i n i t e l y t h a t t he amount o f 3"PG dehyd rogena se i n w i l d - t y p e c e l l s i s t he g rowth l i m i t i n g f a c t o r , t h e r e appea red t o be some c o r r e l a t i o n between the g e n e r a t i o n t i m e , w i t h s u b s t r a t e s p a s s i n g t h r ough 3~PG as a c a t a b o l i c i n t e r m e d i a t e , and the amount o f 3 _ PG dehyd rogena se p r e s e n t i n the c e l l s ( T a b l e X V I ) . S h o r t e r g e n e r a t i o n t ime s were o b s e r v e d when t he s p e c i f i c a c t i v i t y o f the 3"PG dehyd rogena se was r e l a t i v e l y h i g h , J_.e_. e a r l y and mid l o g a r i t h m i c phase g rowth w i t h g l u c o s e and e a r l y l o g a r i t h m i c phase g rowth w i t h g l u c o n a t e . When the g e n e r a t i o n t ime s i n c r e a s e d , as i n mid and l a t e l o g a r i t h m i c phases o f g rowth w i t h g l u c o n a t e o r d u r i n g g rowth w i t h 2-KG o r g l y c e r o l , t he amount o f 3~PG dehyd rogena se had e i t h e r d e c r e a s e d s i g n i f i c a n t l y o r was p r e s e n t a t low i nduced l e v e l s . The l o n g e s t g e n e r a t i o n t i m e s c o r r e l a t e d w i t h t he l o w e s t enzyme l e v e l s . T a b l e XV I . Compar i son o f s p e c i f i c a c t i v i t i e s and g e n e r a t i o n t i m e s o f f_. a e r u g i nosa d u r i n g d i f f e r e n t s t a g e s o f g rowth w i t h d i f f e r e n t s u b s t r a t e s . Growth S t a ge o f G e n e r a t i o n S u b s t r a t e g rowth t i m e (min) S p e c i f i c a c t i v i t y 3-PG dehyd rogena se G1ucose dehyd rogena se G l u c o s e e a r l y l o g mid l o g l a t e l o g 69 69 81 178 280 255 73 39 45 G l u c o n a t e e a r l y l o g mid l o g l a t e l o g 65 83 90 289 143 132 20 28 90 2-KG e a r l y l o g mid l o g l a t e l o g 110 90 80 95 129 122 107 72 68 G l y c e r o l ea r1y 1og mid l o g l a t e l o g 150 150 162 54 57 47 198 189 164 P y r u v a t e e a r l y l o g mid l o g l a t e l o g 69 81 108 16 19 26 121 There a l s o appeared t o be a somewhat i n v e r s e r e l a t i o n s h i p between t h e s e two f a c t o r s and the amount o f g l u c o s e dehydrogenase p r e s e n t d u r i n g growth w i t h s u b s t r a t e s p a s s i n g through 3~PG as a c a t a b o l i c i n t e r m e d i a t e ( T a b l e X V I ) . When g l u c o s e was p r e s e n t i n the medium a r a p i d i n c r e a s e i n th e s p e c i f i c a c t i v i t y o f g l u c o s e dehydrogenase was o b s e r v e d , w h i c h appeared t o be independant o f the g e n e r a t i o n t i m e and a r a p i d d e c r e a s e was o b s e r v e d as g l u c o s e d i s a p p e a r e d from the medium ( F i g . 13A). However, i n the absence o f g l u c o s e , j_.e_. d u r i n g growth w i t h g l u c o n a t e , 2-KG, o r g l y c e r o l , o r f o l -l o w i n g the a d d i t i o n o f t h e s e s u b s t r a t e s t o c e l l s g rowing i n p y r u v a t e medium, h i g h amounts o f g l u c o s e dehydrogenase were found o n l y when t h e s p e c i f i c a c t i v i t y o f 3"PG dehydrogenase d e c r e a s e d s i g n i f i c a n t l y o r when t h i s enzyme was p r e s e n t a t a low induced amount and when the g e n e r a t i o n t i m e was s l o w . T h i s was o b s e r v e d d u r i n g e a r l y s t a t i o n a r y phase growth w i t h g l u c o s e , l a t e -l o g a r i t h m i c phase growth w i t h g l u c o n a t e o r d u r i n g growth w i t h 2-KG o r g l y c e r o l ( T a b l e X V I ) . T h i s was a l s o found a f t e r t he a d d i t i o n o f g l u c o n a t e , 2-KG o r g l y c e r o l t o c e l l s g rowing i n p y r u v a t e medium, but o n l y a f t e r a s i g n i f i c a n t l a g and when the growth r a t e had slowed down c o n s i d e r a b l y ( F i g s . 14A, 2 2 ) . As an e x p l a n a t i o n f o r t h e s e o b s e r v a t i o n s , i t i s s u g g e s t e d t h a t g l u c o s e dehydrogenase i s induced by G-6-P but t h a t a s i g n i f i c a n t amount o f s u b s t r a t e i s r e q u i r e d t o pass through t h i s i n t e r m e d i a t e p r i o r t o i n d u c t i o n . T h i s would p r o b a b l y o c c u r when g l u c o s e i s p r e s e n t i n t h e medium as i t would l i k e l y s a t u r a t e the system i n v o l v i n g g l u c o k i n a s e and G-6-P dehydrogenase and i n d u c e g l u c o s e dehydrogenase t o o f f e r a second r o u t e o f g l u c o s e meta-b o l i s m t o the o r g a n i s m . However, w i t h g l u c o n a t e , 2-KG, o r g l y c e r o l , 122 the i n d u c t i o n o f g l u c o s e dehyd rogena se wou ld o c c u r o n l y d u r i n g s l o w e r g e n e r a t i o n t i m e s when the amount o f 3~PG dehyd rogena se was low a n d , t h e r e -f o r e , t he r a t e o f g l u c o n e o g e n e s i s wou l d be f a s t e r than t h a t r e q u i r e d f o r c e l l u l a r s y n t h e s i s . T h i s wou l d a l l o w a s i g n i f i c a n t amount o f s u b s t r a t e t o pass t h r o u g h G - 6 -P . G l y c e r o l a d d i t i o n t o w i l d - t y p e c e l l s r e s u l t e d i n an e a r l y i n d u c t i o n o f t h o s e enzymes i nduced by 6 -PG, but no i n d u c t i o n o f g l u c o s e dehyd rogena se u n t i l t he g rowth r a t e had s l owed down c o n s i d e r -a b l y . T h i s s u g g e s t s t h a t s i g n i f i c a n t l y h i g h e r amounts o f s u b s t r a t e were r e q u i r e d t o pass t h r ough G -6 -P , p r i o r t o i n d u c t i o n o f g l u c o s e d e h y d r o g e n -a s e , t han were r e q u i r e d t o pass t h r ough 6-PG b e f o r e i n d u c t i o n o f enzymes i nduced by 6-PG o c c u r r e d . T h i s i s based on t he a s s u m p t i o n t h a t a c o n s i d -e r a b l e s l o w i n g down o f bo th t he g e n e r a t i o n t i m e and t he removal o f p r o d u c t s o f g 1 u c o n e o g e n e s i s f o r c e l l u l a r s y n t h e s i s wou ld a l l o w l a r g e r amounts o f s u b s t r a t e t o be m e t a b o l i z e d a t the h e x o s e - p h o s p h a t e l e v e l . Some e v i d e n c e t o s u p p o r t t h i s c o n t e n t i o n i s p r e s e n t e d i n T a b l e X I I I . In mutan t s X I I I , and X l l l , _ ^ , the a d d i t i o n o f g l u c o s e , g l u c o n a t e , o r 2-KG t o c e l l s g r ow ing i n p y r u v a t e medium r e s u l t e d i n g rowth s t a s i s w i t h i n k h o u r s , p re sumab l y due t o the a c c u m u l a t i o n o f 6 -PG. G l u c o s e dehyd rogena se i n t h e s e mutant s was i nduced by the a d d i t i o n o f g l u c o s e bu t not by the a d d i t i o n o f g l u c o n a t e o r 2-KG. S i n c e none o f t he s i x - c a r b o n c a r b o h y d r a t e s was m e t a b o l i z e d pas sed t h e l e v e l o f 6-PG i n t h e mutant s and s i n c e g l u c o s e , g l u c o n a t e , and 2-KG a l l caused g rowth s t a s i s , t h e n , g l u c o n a t e and 2-KG must be m e t a b o l i z e d p a s t t h e l e v e l o f 6-PG i n o r d e r t o i n d u c e g l u c o s e d e h y d r o g e n a s e . I t can be seen f rom F i g . 27 , t h a t a m e t a b o l i c b l o c k a t t h e l e v e l o f 6-PG wou ld e f f e c t -i v e l y p r e v e n t g l u c o n a t e o r 2-KG f rom e n t e r i n g a g l u c o n e o g e n i c r o l e and f rom r e a c h i n g G-6-P as a m e t a b o l i c i n t e r m e d i a t e . I t has a l s o been r e -p o r t e d t h a t g l u c o s e dehyd rogena se was i nduced d u r i n g g rowth o f P_. a e r u g i n o s a w i t h g l u c o s e , f r u c t o s e o r m a n n i t o l and the f i r s t i n t e r m e d i a t e common t o the m e t a b o l i s m o f t h e s e 3 c a r b o h y d r a t e s was G-6-P (50,94). How-e v e r , t h e p o s s i b i l i t y t h a t g l u c o s e a l s o a c t s as an i n d u c e r o f t h i s enzyme canno t be r u l e d o u t . k. I n d u c t i o n o f 3~PG dehyd rogena se . R e s u l t s o b t a i n e d w i t h w i l d - t y p e P_. a e r u g i n o s a , d u r i n g g rowth w i t h g l u c o s e , g l u c o n a t e , o r 2 -KG, s t r o n g l y s u g g e s t e d t h a t g l u c o n a t e a c t s as an i n d u c e r o f g l u c o n o k i n a s e and 3 - PG dehyd rogena se and c au se s the s y n t h e s i s o f the maximum l e v e l s o f bo t h enzymes ( s p e c i f i c a c t i v i t y o f a p p r o x i m a t e l y 130 f o r g l u c o n o k i n a s e and 300 f o r 3~PG dehyd rogena se ( F i g . 16)). In t he w i l d - t y p e c e l l s , g l y c e r o l m e t a b o l i s m r e s u l t e d i n a low i nduced amount o f 3"PG dehyd rogena se , w h i c h was s y n t h e s i z e d i n the ab sence o f any i n d u c t i o n o f g l u c o n o k i n a s e ( F i g s . 20, 23). S i g n i f i c a n t amounts o f 6-PG a c c u m u l a t e d f r om g l y c e r o l m e t a b o l i s m i n mutant X l l l | _ ^ i n t he ab sence o f t h e a c c u m u l a t i o n o f any d e t e c t a b l e amounts o f g l u c o n a t e . The p a t t e r n o f low l e v e l i n d u c t i o n o f 3"PG dehyd rogena se i n t he p a r e n t s t r a i n r e semb led the p a t t e r n s o f i n d u c t i o n e x h i b i t e d by g l u c o k i n a s e , G-6-P d e h y d r o g e n a s e , and the ED pathway enzymes f o l l o w i n g t he a d d i t i o n o f g l y c e r o l t o c e l l s g r ow ing i n p y r u v a t e medium ( F i g . 23). These d a t a s u g g e s t e d t h a t two mechanisms e x i s t w h i c h c o n t r o l t he i n d u c t i o n o f 3 _ PG d e h y d r o g e n a s e : one i n v o l v i n g g l u c o n a t e , w h i c h a l l o w s t he i n d u c t i o n o f bo th g l u c o n o k i n a s e and 3-PG dehyd rogena se ; and a second w h i c h c au se s a low l e v e l o f i n d u c t i o n o f 124 3 _ PG d e h y d r o g e n a s e , i n t he ab sence o f any i n d u c t i o n o f g l u c o n o k i n a s e , and does not appea r t o i n v o l v e g l u c o n a t e . T h i s s u g g e s t i o n was s u p p o r t e d by t he e x a m i n a t i o n o f two c l a s s e s o f 6-PG d e h y d r a t a s e - d e f i c i e n t m u t a n t s . The f i r s t c l a s s p o s s e s s e d c o n s t i t u t i v e l y h i g h l e v e l s o f bo th g l u c o n o k i n a s e and 3"PG dehyd rogena se and o n l y t h e l a t t e r enzyme was s u b j e c t to i n d u c t i o n f o l l o w i n g t he a d d i t i o n o f g l u c o s e , w h i c h was no t m e t a b o l i z e d p a s t the l e v e l o f 6-PG (mutant X I I I , and r e l a t e d t y p e s T a b l e X I l ) . The second c l a s s had l o s t d e t e c t a b l e a c t i v i t y f o r t h e c o n -s t i t u t i v e l y h i g h l e v e l s o f b o t h g l u c o n o k i n a s e and 3~PG dehyd rogena se but p o s s e s s e d a c o n s t i t u t i v e l y l ow, i nduced l e v e l o f 3"PG dehyd rogena se (mutant X I I I , T a b l e X I I ) . In t he w i l d - t y p e c e l l s , t h e p a t t e r n s o f i n d u c t i o n o b s e r v e d f o r the low l e v e l s y s tem o f 3"PG dehyd rogena se r e semb led t h a t o f t he enzymes a p p a r e n t l y i nduced by 6-PG a n d , a l t h o u g h somewhat s p e c u l a t i v e a t t h i s p o i n t , i t i s s u g ge s t ed t he low l e v e l s y s tem i s i nduced by 6-PG. I t may be t h a t , due t o i t s s t r u c t u r a l s i m i l a r i t y w i t h g l u c o n a t e , 6-PG a c t s , but l e s s e f f i c i e n t l y , t o i nduce t h i s enzyme. 5. Pathways o f g l y c e r o l d i s s i m i l a t i o n . The pathways o f g l y c e r o l d i s s i m i l a t i o n i n P. a e r u g i n o s a were i n -v e s t i g a t e d . G 1 y c e r o k i n a s e and L - a - g l y c e r o p h o s p h a t e dehyd rogena se were s p e c i f i c a l l y i nduced by g l y c e r o l but not by p y r u v a t e o r g l u c o s e m e t a b o l i s m . F u r t h e r s u p p o r t f o r t h i s r o u t e o f g l y c e r o l d i s s i m i l a t i o n was o b t a i n e d d u r i n g g l y c e r o l m e t a b o l i s m in the p a r e n t s t r a i n and mutant XI I 1 , ^ f rom the d e t e c t i o n o f t he a c c u m u l a t i o n o f s m a l l amounts o f d i h y d r o x y a c e t o n e p h o s p h a t e , t h e p r o d u c t o f t he above two enzymes. T h i s pathway f o r g l y c e r o l 125 m e t a b o l i s m has a l s o been d e s c r i b e d i n a n o t h e r s t r a i n o f P_. ae rug i n o s a ( 1 16 ) . From d i h y d r o x y a c e t o n e p h o s p h a t e , t w o r o u t e s o f g l y c e r o l d e g r a d a t i o n appea r p o s s i b l e ( F i g . 2 7 ) : one to 3~PG and g l y c o l y s i s t o t he TCA c y c l e , and t he o t h e r v i a t he r e v e r s a l o f c e r t a i n Embden-Meyerhof pathway enzymes and g l u c o n e o g e n e s i s . Both r o u t e s appea red t o be u t i l i z e d as e v i d e n c e d by g l y c e r o l m e t a b o l i s m i n mutant XI I I. /\» ' n w n ' c n s i g n i f i c a n t amounts o f 6-PG a c c u m u l a t e d and l o s s o f r a d i o a c t i v i t y as CO. o c c u r r e d . The r e s u l t s o b t a i n e d f rom the e x a m i n a t i o n o f the i n d u c t i o n o f CC enzymes by g l y c e r o l and t he pathways o f g l y c e r o l d i s s i m i l a t i o n , c o n f i r m e d e a r l i e r f i n d i n g s on t he c o n t r o l e x e r t e d o v e r t h e CC enzymes a t t h e s i x - c a r b o n l e v e l . These CC enzymes may be e s s e n t i a l f o r g rowth w i t h g l y c e r o l , i n o r d e r t o p r e v e n t an a c c u m u l a t i o n o f s i x - c a r b o n p h o s p h o r y l a t e d i n t e r m e d i a t e s , r e s u l t i n g f rom a f a s t e r r a t e o f g 1uconeogene s i s t han t h a t r e q u i r e d f o r c e l l u l a r s y n -t h e s i s . T h i s i s s u p p o r t e d by the f a c t t h a t no g rowth o f the 6-PG d e h y d r a -t a s e - d e f i c i e n t mutant ( X l l l | _ ^ ) was o b s e r v e d i n g l y c e r o l medium and the f a c t t h a t the p r e s e n c e o f g l y c e r o l w i t h t h i s mutant g r ow ing i n p y r u v a t e medium r e s u l t e d i n an i n h i b i t e d r a t e o f g rowth ( F i g . 2 5 ) . The summary o f t h e pathways o f c a r b o h y d r a t e c a t a b o l i s m i n P_. a e r u g i nosa i s shown i n F i g . 27 and the i n d u c e r s o f t he enzymes examined a r e l i s t e d i n T a b l e XVI I. Glucose "> Glucose-6-P ,4-Gluconolactone GluconoIactone-6-P Gluconate 2-Ketogluconate 2-Ketogluconate-6-P >Gluconate-6-P 2-Keto-3-Deoxygluconate-6-P Pyruvate Glyceraldehyde-3-P^ Glycerol •$>Glycerol-3-P«& 1—-fe'Dihydroxyacetone-P Fructose-16-DiP V F i g , 2 7 - Pathways o f s i x - c a r b o n c a r b o h y d r a t e and g l y c e r o l m e t a b o l i s m i n JP. a e r u g i n o s a ATCC 9 0 2 7 . T a b l e X V I I . I nduce r s o f c a r b o h y d r a t e - c a t a b o l i z i n g enzymes i n P, a e r u g i nosa as d e t e r m i n e d f rom the e x a m i n a t i o n o f g l u c o s e -n e g a t i v e mutant s and t he p a r e n t s t r a i n . Enzyme Inducer G l u c o s e dehyd rogena se G-6-P (and p o s s i b l y g l u c o s e ) G l u c o n a t e dehyd rogena se 2-KG G l u c o k i n a s e G-6-P dehyd rogena se G l u c o n o k i n a s e 3 _ PG dehyd rogena se ED pathway enzymes G l y c e r o k i n a s e a-GP dehyd rogena se 6-PG 6-PG G l u c o n a t e (and p o s s i b l y 2-KG as a weak i n d u c e r ) G l u c o n a t e (and p o s s i b l y 6-PG as a weak i n d u c e r ) 6-PG o r some d e g r a d a t i o n p r o d u c t o f 6-PG G l y c e r o l o r some d e g r a d a t i o n p r o d u c t , eg .a -GP G l y c e r o l o r some d e g r a d a t i o n p r o d u c t , eg .a -GP GENERAL DISCUSSION The numerous c a t a b o l i c pathways p r e s e n t i n Pseudomonas s p e c i e s (107) endows them w i t h m e t a b o l i c d i v e r s i t y . That t h e enzymes i n t h e s e pathways a r e s u b j e c t t o i n d u c t i o n and r e p r e s s i o n (90), i s an a d v a n t a g e t o t he economy o f t h e m i c r o o r g a n i s m s , e s p e c i a l l y i f t h e i r n a t u r a l e n v i r o n m e n t does not p r o v i d e a c o n s t a n t s u p p l y o f t he s u b s t r a t e s c a t a b o l i z e d by t h e s e pa thways . The r e s u l t s o b t a i n e d d u r i n g 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 t h o s e p r e s e n t e d i n o t h e r r e p o r t s , t h a t i s , t he enzymes o f g l u c o s e c a t a -b o l i s m i n f_. a e r u g i n o s a (19,38,50,55,68,80,94,115,118), o t h e r r e l a t e d Pseudomonas s p e c i e s (21,25,70,85,95,100), and a t l e a s t s e v e r a l o t h e r Gram n e g a t i v e , a e r o b i c m i c r o o r g a n i s m s (65,78,79), a r e s u b j e c t t o i n d u c t i o n and r e p r e s s i o n . However, t he enzymes o f t he TCA c y c l e , a l t h o u g h p o s s i b l y s u b j e c t t o some t y p e o f r e g u l a t i o n (87), appea r t o be c o n s t i t u t i v e (115). C a t a b o l i t e r e p r e s s i o n o r i n h i b i t i o n may be cau sed by TCA i n t e r m e d i a t e s s uch as s u c c i n a t e and c i t r a t e (4,49,69,86,87). These r e s u l t s a r e i n c o n t r a s t w i t h t h e r e g u l a t i o n p r e s e n t i n t he e n t e r i c and t h e 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 and s u g ge s t some e v o l u t i o n a r y d i v e r g e n c e i n t h e deve lopment o f t h e s e d i f f e r e n t g roups o f m i c r o o r g a n i s m s . The i m p o r t a n c e o f t he o x i d a t i v e n o n - p h o s p h o r y l a t e d pathway f o r g l u c o s e c a t a b o l ism was d e m o n s t r a t e d i n P_. a e r u g ? n o s a . A l t h o u g h i t i s not c l e a r whe the r ene r gy i s g a i n e d f r om t h e d i r e c t o x i d a t i o n o f g l u c o s e t o 2-KG 129 ( 7 , 9 , 7 5 , 1 1 0 ) , i t i s a p p a r e n t t h a t l a r g e amounts o f 2-KG a c c u m u l a t e d f rom g l u c o s e o r g l u c o n a t e m e t a b o l i s m d u r i n g g r owth o f the p a r e n t s t r a i n o r when t h e m e t a b o l i s m o f t h e s e c a r b o h y d r a t e s was r e s t r i c t e d , _i_.e_. w i t h 6-PG d e h y d r a t a s e - d e f i c i e n t m u t a n t s . I t a l s o has been shown t h a t l a r g e amounts o f 2-KG a c c u m u l a t e d under c o n d i t i o n s o f c a r b o n e x c e s s ( g l u c o s e ) and n i t r o g e n l i m i t a t i o n ( 7 6 ) . In f a c t , under t h e s e c o n d i t i o n s o f c a r b o n e x c e s s and n i t r o g e n l i m i t a t i o n , s t o r a g e o f i n t r a c e l l u l a r c a r b o n a c e o u s m a t e r i a l s c o u l d not be d e m o n s t r a t e d ( 7 6 ) . The f a c t t h a t P_. ae rug i nosa does not s t o r e e x c e s s c a r b o n and ene rgy i n t he f o rm o f g l y c o g e n , t r e h a l o s e , o r p o l y - B - h y d r o x y b u t y r a t e , but e x c r e t e s v e r y l a r g e amounts o f 2-KG i n t o t he medium f rom g l u c o s e o r g l u c o n a t e o x i d a t i o n , i n d i c a t e s t h a t 2-KG may s e r v e as a mechanism f o r the c o n s e r -v a t i o n o f c a r b o n and e n e r g y . T h i s a p p e a r s t o o c c u r under c o n d i t i o n s when c a r b o h y d r a t e s such as g l u c o s e o r g l u c o n a t e a r e p r e s e n t i n e x c e s s o f t h e a v a i l a b l e n i t r o g e n s o u r c e o r when t he enzymes i n v o l v e d w i t h d i r e c t p h o s p h o r y l a t i o n o f t he s u b s t r a t e s i s s a t u r a t e d . T w o - k e t o g l u c o n a t e wou ld p r o v i d e more ene rgy and wou ld s u p p o r t more c e l l u l a r s y n t h e s i s t han a m e t a b o l i t e such as a - k e t o g l u t a r a t e and a l s o i t s u t i l i z a t i o n by m i c r o -o r g a n i s m s i s more r e s t r i c t e d . The p o s s i b i l i t y t h a t o x i d a t i o n t o 2-KG may s e r v e as a mechanism f o r t he c o n s e r v a t i o n o f c a r b o n and energy has p r e v i o u s l y been s u g g e s t e d ( 55 ) -The r e g u l a t i o n o f g l u c o s e - c a t a b o l i z i n g enzymes i n _P_. a e r u g i nosa a p p e a r s t o be somewhat c o m p l e x , _i_.e_. o f t h e seven enzymes examined i n 130 detail, no less than four inductive events occurred to bring about their synthesis. This indicates that, on an average, each inducer controls the synthesis of two enzymes, with some enzymes being induced by their substrate and others by their product. These results are compatible with the regulation determined for other catabolic pathways of Pseudomonas species (90). Glucose dehydrogenase appears to be induced by G-6-P (and possibly glucose). This form of inductive control would provide the organism with an alternative route for glucose catabolism when the pathway initiated by glucokinase becomes saturated in the presence of excess glucose. This is emphasized by the fact that significant amounts of the inducer appeared to be required before induction of the enzyme took place. The induction of gluconate dehydrogenase by its product, 2-KG, might ensure that this carbohydrate accumulates only when excess amounts of glucose or gluconate are present. Under these conditions, saturation of the direct phosphorylation pathways for glucose or gluconate would allow sufficient quantities of 2-KG to accumulate to cause induction of the enzyme. Thus, the wasteful synthesis of this enzyme would not occur when the external concentration of glucose and gluconate were such that an accumulation of 2-KG would not occur. Therefore, it appears that the regulation of the enzymes of the oxidative non-phosphorylated pathway, glucose and gluconate dehydrogenase, is such that they are not optimally induced unless significant concen-trations of glucose or gluconate are present. This may lend some support t o t he t h e o r y t h a t o x i d a t i o n to 2-KG s e r v e s as a mechanism f o r the c o n -s e r v a t i o n o f c a r b o n and e n e r g y . However, the r e s u l t s o b t a i n e d d u r i n g t h i s i n v e s t i g a t i o n s t r o n g l y s u g ge s t t h a t t he f u n c t i o n o f t he o x i d a t i v e non -phosphory 1 a t e d r o u t e f o r g l u c o s e d i s s i m i l a t i o n may be t w o - f o l d J_.e_. i t may not o n l y p r o v i d e t h e o r g a n i s m w i t h a mechanism f o r c o n s e r v i n g c a r b o n d u r i n g p e r i o d s o f c a r b o n e x c e s s but a l s o p r o v i d e s t h r e e a c c e s s r o u t e s t o 6 -PG, w h i c h appea r t o be r e q u i r e d f o r an o p t i m a l r a t e o f g rowth w i t h g l u c o s e . The re i s an a b s o l u t e r e q u i r e m e n t f o r ED pathway enzymes f o r g r owth o f P_. a e r u g i nosa wi t h g l u c o s e , g l u c o n a t e , o r 2-KG, as e v i d e n c e d by the e x a m i n a t i o n o f g l u c o s e - n e g a t i v e m u t a n t s . T h e r e f o r e , i n o r d e r f o r t he o r g a n i s m t o grow w i t h a l l t h r e e s i x - c a r b o n c a r b o h y d r a t e s , i t i s e s s e n t i a l t h a t t h e s e two ED pathway enzymes be i nduced d u r i n g g r owth w i t h each o f t he t h r e e s u b s t r a t e s . T h i s i s f u r t h e r emphas i zed by t he f a c t t h a t d u r i n g g rowth w i t h g l u c o s e o r g l u c o n a t e , a m a j o r amount o f e i t h e r s u b s t r a t e was o x i d i z e d t o 2-KG. The i n d u c e r o f the ED pathway enzymes was f ound t o be 6-PG ( t h e f i r s t i n t e r m e d i a t e common t o t h e m e t a b o l i s m o f g l u c o s e , g l u c o n -a t e , o r 2-KG) o r p o s s i b l y some d e r i v a t i v e t h e r e o f . The i n d u c t i o n o f t h e s e two enzymes by t h e i r p r o d u c t , 6-PG (or a d e r i v a t i v e ) , w o u l d e n s u r e t h a t t he o r g a n i s m wou ld grow w i t h any c a r b o h y d r a t e c a t a b o l i z e d by t h i s pathway _i_.e_. f r u c t o s e o r m a n n i t o l ( 1 9 , 9 4 ) , i n a d d i t i o n t o g l u c o s e , g l u c o n a t e o r 2-KG The i n d u c e r o f g l u c o k i n a s e and G-6-P dehyd rogena se was 6 -PG, t he f i n a l p r o d u c t o f t h e s e two enzymes. T h i s wou ld e n s u r e t h a t the o r g a n i s m wou ld grow on any s u b s t r a t e e n t e r i n g t he ED pathway v i a t h e s e two enzymes, J_.e_. g l u c o s e , f r u c t o s e , o r m a n n i t o l ( 1 9 , 9 4 ) . However, i t a l s o s u g g e s t e d t h a t 132 t h e s e two enzymes were s y n t h e s i z e d g r a t u i t o u s l y . d u r i n g g r owth w i t h g l u -c o n a t e o r 2-KG. T h i s i s s i m i l a r t o t he c o n t r o l e x e r t e d o v e r the 3 ~ k e t o -a d i p a t e pathway by 3 ~ k e t o a d i p a t e , the b r anch p o i n t i n t e r m e d i a t e . When c u l t u r e s o f P_. ae rug i n o s a , P_. put i d a , o r I P . m u l t i v o r a n s were grown w i t h s u b s t r a t e s c a t a b o l i z e d by t he c a t e c h o l b r anch o f t h e pathway, two enzymes i n the p r o t o c a t e c h u a t e b r a n c h , i nduced by 3 _ k e t o a d i p a t e , were s y n t h e s i z e d g r a t u i t o u s l y ( 8 9 ) . The i n d u c t i o n o f g l u c o k i n a s e and G-6-P dehyd rogena se by 6 -PG, formed f r om the m e t a b o l i s m o f g l u c o n a t e o r 2 -KG, wou ld s u gge s t t h a t P_. a e r u g ? nosa ma i n t a i ns t he ab i 1 i t y t o metabo l i ze g 1 uco se r a p i d l y , even i n t h e p r e s e n c e o f e x ce s s amounts o f g l u c o n a t e o r 2-KG. The i n d u c e r o f g l u c o n o k i n a s e was f ound t o be g l u c o n a t e . S i n c e g l u c o n a t e was o n l y a t r a n s i e n t i n t e r m e d i a t e i n t h e o x i d a t i o n o f g l u c o s e t o 2-KG, t he i n d u c t i o n o f t h i s enzyme by i t s s u b s t r a t e wou ld appear t o p r e v e n t w a s t e f u l s y n t h e s i s o f t he enzyme when a l l t he g l u c o s e and g l u c o n a t e had been c o n v e r t e d t o 2-KG, o r when the g rowth s u b s t r a t e s were c a t a b o l i z e d by t he ED pathway but d i d not pass t h r ough g l u c o n a t e as an i n t e r m e d i a t e , eg . f r u c t o s e o r m a n n i t o l . The i n d u c t i o n o f 3"PG dehyd rogena se by g l u c o n a t e and pe rhaps w e a k l y by 6-PG wou ld appear somewhat l e s s advan tageou s t o t he m i c r o o r g a n i s m . H i gh i nduced amounts o f t he enzyme were s y n t h e s i z e d i n the p r e s e n c e o f g l u c o s e (due t o i t s o x i d a t i o n t o g l u c o n a t e ) , o r i n t h e p r e s e n c e o f g l u c o n a t e . However, when a l l t he g l u c o s e and g l u c o n a t e had been c a t a b o l -i z e d , o r o x i d i z e d t o 2-KG, o n l y low i nduced l e v e l s o f t he enzyme were p r e s e n t and t he d e c r e a s e i n 3~PG dehyd rogena se a c t i v i t y was accompan ied by a d e c r e a s e i n t he g rowth r a t e . S i m i l a r r e s u l t s were o b s e r v e d w i t h 133 g l y c e r o l as t he c a r b o n s o u r c e , e x c e p t t h a t even l ower amounts o f the enzyme and s l o w e r g rowth r a t e s were o b s e r v e d . I f t he amount o f 3~PG dehyd rogena se p r e s e n t i n c e l l s g r ow ing w i t h s u b s t r a t e s t h a t a r e c a t a -b o l i z e d t h r o u g h 3 - P G i s a g rowth l i m i t i n g f a c t o r , t hen t he low l e v e l o f i n d u c t i o n o f t h i s enzyme by some m e t a b o l i t e such as 6-PG wou ld e n s u r e t h a t g rowth wou ld o c c u r w i t h s u b s t r a t e s such as 2-KG o r g l y c e r o l but o n l y a t s l o w r a t e s . P_. a e r u g i n o s a ATCC 9027 does not po s se s s a f u n c t i o n a l Embden-Meyer-hof pathway as i t l a c k s d e t e c t a b l e a c t i v i t y f o r p h o s p h o f r u c t o k i n a s e ( 115 ) . I t a l s o a p p e a r s t o l a c k a c o m p l e t e l y f u n c t i o n a l hexose mono-phosphate shun t as e v i d e n c e d by t he ab sence o f any d e t e c t a b l e a c t i v i t y f o r 6-PG dehyd rogena se (D. B l e w , W.H. L y n c h , and A . F . G r o n l u n d . M a n u s c r i p t s u b -m i t t e d f o r p u b l i c a t i o n ) . T h e r e f o r e , t h e demand f o r p e n t o s e pho spha te s wou l d appea r t o be met by t r a n s k e t o l a s e and t r a n s a 1 d o l a s e r e a c t i o n s as has been s u g g e s t e d f o r s e v e r a l s p e c i e s o f Pseudomonas ( 1 6 , 6 8 , 1 1 9 ) - These d a t a s u gge s t t h a t the demand f o r NADPH f o r t he numerous b i o s y n t h e t i c c a p a b i l -i t i e s o f P_. a e r u g i nosa i s met by r e a c t i o n s o t h e r than 6-PG dehyd rogena se J_.e_. 3 - P G d e h y d r o g e n a s e , G-6-P d e h y d r o g e n a s e , i s o c i t r i c d e h y d r o g e n a s e , o r m a l i c enzyme. The e q u i l i b r i u m o f the 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 r e a c t i o n s has been r e p o r t e d t o be s t r o n g l y i n the d i r e c t i o n o f f r u c t o s e - 6 - p h o s p h a t e ( F - 6 - P ) and G-6-P i n P_. s a c c h a r o p h i l a ( 1 6 ) . DeLey has s u g g e s t e d t h a t t h i s i s advan tageou s t o t he o r g a n i s m as i t wou l d p r e v e n t any l e a k a g e f rom the G-6-P poo l t o be l o s t as u s e l e s s end p r o d u c t s and t h a t the e q u i l i b r i u m wou ld o n l y be d i s p l a c e d when g r o w i n g c e l l s draw o f f p e n t o s e pho spha te s f o r t he s y n t h e s i s o f n u c 1 e o p r o t e i n s and r i b o s e c o n t a i n i n g c o f a c t o r s ( 1 6 ) . A s i m i l a r e q u i l i b r i u m o f 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 r e a c t i o n s i n the d i r e c t i o n o f G-6-P and F-6-P has been d e m o n s t r a t e d i n P_. a e r u g ? nosa (D. B l ew, W.H. L y n c h , and A . F . G r o n l u n d . M a n u s c r i p t s u b m i t t e d f o r p u b l i -c a t i o n ) . An e q u i l i b r i u m between a n a b o l i s m ( f o r t he s y n t h e s i s o f p e n t o s e pho s -pha te s and hexose s ) and c a t a b o l i s m wou ld appea r t o e x i s t i n P_. a e r u g i nosa and pe rhaps i s r e g u l a t e d by t h e energy c h a r g e o f the c e l l d u r i n g g rowth w i t h s u b s t r a t e s such as a c e t a t e , p y r u v a t e , o r TCA c y c l e i n t e r m e d i a t e s . However, d u r i n g g rowth w i t h g l u c o s e , g l u c o n a t e , 2-KG, o r g l y c e r o l some im-b a l a n c e i n t he e q u i p a r t i t i o n o f a n a b o l i s m and c a t a b o l i s m appea r s to o c c u r . W i t h g l u c o s e o r g l u c o n a t e , t h i s i m b a l a n c e i s o n l y a p p a r e n t when t h e s e s u b -s t r a t e s have been c o n v e r t e d to t h e i r o x i d a t i v e p r o d u c t , 2-KG. The i n d u c t i o n o f g l u c o s e d e h y d r o g e n a s e , when 2-KG o r g l y c e r o l were p r e s e n t as t he g rowth s u b s t r a t e i n t he medium, s u gge s t ed t h a t G-6-P was b e i n g formed f rom g1uconeogene s i s a t a r a t e f a s t e r t han t h a t r e q u i r e d f o r c e l l u l a r s y n t h e s i s . The i n d u c t i o n o f enzymes i nduced by 6 -PG , J_.e_. g l u c o k i n a s e , G-6-P d e h y d r o g e n a s e , and the ED pathway enzymes, d u r i n g g rowth o f the p a r e n t s t r a i n w i t h g l y c e r o l , t o g e t h e r w i t h t he a c c u m u l a t i o n o f 6-PG f rom g l y c e r o l m e t a b o l i s m i n the 6-PG d e h y d r a t a s e - d e f i c i e n t mutant c o n f i r m e d t h i s s u g g e s t i o n . Two o t h e r e v e n t s appea red t o be r e l a t e d w i t h t h i s i m b a l a n c e o f a n a -b o l i s m and c a t a b o l i s m ; t he f i r s t was t he low induced l e v e l s o f 3"PG dehyd rogena se when 2-KG o r g l y c e r o l were p r e s e n t as the g rowth s u b s t r a t e , and the second was t he s l o w r a t e o f g rowth w i t h e i t h e r o f t h e s e s u b s t r a t e s . The l ower amounts o f 3 - P G d e h y d r o g e n a s e , d u r i n g g r owth w i t h t h e s e s u b -s t r a t e s w h i c h a r e c a t a b o l i z e d t h r ough 3~PG, may r e s u l t i n t he s l o w e r g rowth r a t e s o b s e r v e d w i t h t h e s e s u b s t r a t e s . Due to t h e e q u i 1 i b r i u r n o f t he 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 r e a c t i o n s i n t he d i r e c t i o n o f G-6-P and F-6-P and t he a p p a r e n t c o n s t i t u t i v e n a t u r e o f t h e s e enzymes ( 1 15 ) , t h e lower amounts o f 3"PG dehyd rogena se may a l s o r e s u l t i n a more e f f i -c i e n t c o m p e t i t i o n f o r 3"PG i n t he d i r e c t i o n o f g l u c o n e o g e n e s i s . D u r i n g s l o w g rowth r a t e s , when i n t e r m e d i a t e s wou ld be drawn o f f f o r c e l l u l a r s y n t h e s i s a t a s l ow r a t e , t h i s i m b a l a n c e i n the d i r e c t i o n o f g l u c o n e o -g e n e s i s wou ld r e s u l t i n the f o r m a t i o n o f e x ce s s G - 6 - P , and i n i t s c a t a -bo l ism a t t he h e x o s e - p h o s p h a t e l e v e l . 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Wood, W.A. 1955- Pathways o f c a r b o h y d r a t e d e g r a d a t i o n i n Pseudomonas f l u o r e s c e n s . B a c t e r i o l . Rev. j_9_: 222-233-122. Wood, W.A., and R.F. S c h w e r d t . 1954. C a r b o h y d r a t e o x i d a t i o n by Pseudomonas f l u o r e s c e n s . I I . Mechanism o f hexose pho spha te o x i -d a t i o n . J . B i o l . Chem. 206: 625-635-123- Y a r m o l i n s k y , M.B. , H. Wiesmeyer , H.M. K a l c k a r , and E. J o r d a n . 1959-H e r e d i t a r y d e f e c t s i n g a l a c t o s e m e t a b o l i s m i n E s che r i c h i a c o l i m u t a n t s , I I . G a l a c t o s e - i n d u c e d s e n s i t i v i t y . P r o c . Na t . A c a d . S c i . 45: 1786-1791 124. Z w a i g , N . , W.S. K i s t l e r , and E.C.C. L i n . 1970. G l y c e r o l k i n a s e , t h e pacemaker f o r t he d i s s i m i l a t i o n o f g l y c e r o l i n E s c h e r i c h i a c o l i . J . B a c t e r i o l . 102: 753 -759-APPENDIX I. C o n t r i b u t i o n s o f t h i s t h e s i s t o t he u n d e r s t a n d i n g o f t he r e g u l a t i o n o f c a r b o h y d r a t e c a t a b o l i s m i n _P. a e r u g i n o s a (ATCC 9027 ) . The pathways o f g l u c o s e , g l u c o n a t e , 2-KG, and g l y c e r o l c a t a b o l i s m i n f_. a e r u g i n o s a (ATCC 9027) a r e i l l u s t r a t e d i n F i g . 27 (p. 126 ) . 1. The o x i d a t i v e n o n - p h o s p h o r y 1 a t e d pathway. The o x i d a t i v e n o n - p h o s p h o r y l a t e d pathway f o r t h e d i s s i m i l a t i o n o f g l u c o s e was e s t a b l i s h e d by N o r r i s and Campbe l l (88,109) i n P_. a e r u g i n o s a (ATCC 9027 ) , however , the i m p o r t a n c e o f t h i s pathway t o t h e o v e r a l l d e g r a d a t i o n o f g l u c o s e had not been e s t a b l i s h e d . I t has been r e p o r t e d t h a t t h i s pathway i s not e s s e n t i a l f o r g rowth o f P_. a e r u g i n o s a w i t h g l u -c o s e ( 8 0 ) . I t was d e m o n s t r a t e d , d u r i n g t he c o u r s e o f t h i s i n v e s t i g a t i o n , t h a t d u r i n g g rowth o f P_. a e r u g i n o s a w i t h g l u c o s e o r g l u c o n a t e , more than t w o - t h i r d s o f t he o r i g i n a l s u b s t r a t e was c a t a b o l i z e d by t he o x i d a t i v e n o n - p h o s p h o r y l a t e d pathway t o 2-KG p r i o r t o b e i n g p h o s p h o r y l a t e d and f u r t h e r c a t a b o l i z e d by t he ED pathway enzymes. A l t h o u g h t he e x a m i n a t i o n o f mutant s d e f i c i e n t i n one o r more enzymes o f t h i s pathway d e m o n s t r a t e d t h a t t h e o x i d a t i v e n o n - p h o s p h o r y l a t e d pathway was not r e q u i r e d f o r g rowth w i t h g l u c o s e , i t appea red t o be r e q u i r e d t o m a i n t a i n t he maximal r a t e o f g rowth n o r m a l l y o b s e r v e d w i t h t h i s s u b s t r a t e . 2. I n d u c t i o n o f g l u c o s e - c a t a b o l i z i n g enzymes. Growth o f _P. a e r u g i n o s a w i t h s i x - c a r b o n c a r b o h y d r a t e s as w e l l as w i t h g l y c e r o l o r g l y c e r a t e has p r e v i o u s l y been shown to c au se t h e i n d u c t i o n o f 147 g l u c o s e - c a t a b o l i z i n g enzymes (19,38,50,54,68,80,115,118). However, t he a c t u a l i n d u c e r s o f t h e s e enzymes had not been i d e n t i f i e d . By e x a m i n i n g the i n d u c t i o n o f c a r b o h y d r a t e - c a t a b o l i z i n g enzymes by g l u c o s e , g l u c o n a t e , 2-KG, and g l y c e r o l i n t he w i l d - t y p e and g l u c o s e - n e g a t i v e mutant s o f P_. a e r u g i n o s a , i t was shown h e r e t h a t : a . g l u c o s e dehyd rogena se was i nduced by G-6-P (and p o s s i b l y g l u c o s e ) b. g l u c o n a t e dehyd rogena se by 2-KG c . g l u c o k i n a s e and G-6-P dehyd rogena se by 6-PG d. t h e ED pathway enzymes by 6-PG o r some d e r i v a t i v e t h e r e o f e . g l u c o n o k i n a s e and 3 - PG dehyd rogena se by g l u c o n a t e . A weak i n d u c t i o n o f bo th g l u c o n o k i n a s e ( p o s s i b l y by 2-KG) and 3 - PG dehyd rogena se ( p o s s i b l y by 6-PG) was a l s o o b s e r v e d and a p p a r e n t l y d i d no t i n v o l v e g l u c o n a t e . 3- S i m i l a r i t i e s i n t he r e g u l a t i o n o f c e r t a i n g l u c o s e - c a t a b o l i z i n g enzymes. The e x a m i n a t i o n o f s e v e r a l 6-PG d e h y d r a t a s e - d e f i c i e n t mutant s d e m o n s t r a t e d t h a t a s i m i l a r mechanism e x i s t e d f o r t h e r e g u l a t i o n o f t he s y n t h e s i s o f g l u c o k i n a s e and G-6-P dehyd rogena se J_.e_. a l o s s o f the normal c o n t r o l o v e r one o f t h e s e enzymes was accompan ied by a s i m i l a r l o s s o f c o n t r o l o v e r t h e o t h e r . T h i s s i m i l a r i t y o f c o n t r o l was a l s o o b s e r v e d f o r g l u c o n o k i n a s e and 3"PG d e h y d r o g e n a s e . The e x a m i n a t i o n o f the i n d u c t i o n o f t h e s e enzymes c o n f i r m e d t he above r e s u l t s i n t h a t g l u c o k i n a s e and G -6 -P dehyd rogena se were i nduced by t he same compound .- 6 -PG. S i m i l a r l y , g l u c o n o k i n a s e and 3 - PG dehyd rogena se were i nduced by t he same compound -g l u c o n a t e . 148 These r e s u l t s a l s o s u g g e s t e d t h a t a ma jo r c o n t r o l p o i n t o f s i x - c a r b o n c a r b o h y d r a t e c a t a b o l i s m may e x i s t a t t he l e v e l o f 6-PG d e h y d r a t a s e j_.e_. a m u t a t i o n r e s u l t i n g i n the l o s s o f 6-PG d e h y d r a t a s e a c t i v i t y a l s o appea red t o p romote a l o s s o f normal w i l d - t y p e c o n t r o l o v e r 2 o r 4 o f t h e o t h e r enzymes o f g l u c o s e c a t a b o l i s m . R e v e r t a n t s , o c c u r r i n g a t a f r e q u e n c y s u g g e s t i n g a s i n g l e g e n e t i c l e s i o n , r e g a i n e d normal c o n t r o l o v e r 6-PG d e h y d r a t a s e as w e l l as g l u c o k i n a s e , G-6-P d e h y d r o g e n a s e , g l u c o n o k i n a s e and 3 - P G d e h y d r o g e n a s e . 4. R e g u l a t i o n o f g 1 u c o s e - c a t a b o l i z i n g enzymes by g l y c e r o l . Due t o t h e h i g h l e v e l s o f g l u c o s e - c a t a b o l i z i n g enzymes p r e s e n t i n P_. a e r u g i n o s a d u r i n g g rowth w i t h t h r e e - c a r b o n compounds such as g l y c e r o l o r g l y c e r a t e , i t has been s u g g e s t e d p r e v i o u s l y t h a t t h e s e enzymes a r e a c t u a l l y i nduced by a t r i o s e - p h o s p h a t e o r some d e r i v a t i v e t h e r e o f ( 5 0 ) . However, i t was shown he re t h a t t he i n d u c t i o n o f t h e s e enzymes i n g l u c o s e -n e g a t i v e mutant s was m e d i a t e d a t the s i x - c a r b o n c a r b o h y d r a t e l e v e l . The a c c u m u l a t i o n o f a s i g n i f i c a n t amount o f 6-PG d u r i n g g l y c e r o l m e t a b o l i s m w i t h a 6-PG d e h y d r a t a s e - d e f i c i e n t mutant and the p a t t e r n s o f i n d u c t i o n o f g 1 u c o s e - c a t a b o l i z i n g enzymes d u r i n g g rowth o f t he p a r e n t s t r a i n w i t h g l y c e r o l c o n f i r m e d t h e above f i n d i n g s t h a t t h e s e enzymes were i nduced a t the s i x - c a r b o n c a r b o h y d r a t e l e v e l even d u r i n g g rowth w i t h t h r e e - c a r b o n compounds such as g l y c e r o l . 5. G l y c e r o l - c a t a b o l i z i n g enzymes. D u r i n g t h i s s t u d y g l y c e r o k i n a s e and L - a - G P dehyd rogena se were s p e c i f i -c a l l y i nduced by g l y c e r o l o r a d e g r a d a t i o n p r o d u c t t h e r e o f ( e . g . L - a - G P ) and no t by e i t h e r g l u c o s e o r p y r u v a t e m e t a b o l i s m i n P_. a e r u g i n o s a . T h i s pathway f o r g l y c e r o l m e t a b o l i s m has been r e p o r t e d i n a n o t h e r s t r a i n o f P_ a e r u g i n o s a (116) as w e l l as i n r e l a t e d o r g a n i s m s such as P_. p u t i d a (100) 6. G l u c o s e t o x i c i t y i n g l u c o s e - n e g a t i v e m u t a n t s . An e x a m i n a t i o n o f 6-PG d e h y d r a t a s e - d e f i c i e n t mutant s d e m o n s t r a t e d t h a t g rowth o f t h e s e mutant s w i t h p y r u v a t e , a c e t a t e , o r TCA c y c l e i n t e r -m e d i a t e s was i n h i b i t e d by t he p r e s e n c e o f g l u c o s e , g l u c o n a t e , 2-KG o r g l y c e r o l . The i n h i b i t i o n o f g rowth o f g l u c o s e - n e g a t i v e mutant s w i t h p y r u v a t e was shown to be due t o an a c c u m u l a t i o n o f h i g h i n t e r n a l c o n c e n -t r a t i o n s o f 6 -PG. T h i s a c c u m u l a t i o n o f 6-PG caused a r e p r e s s i o n ( e i t h e r d i r e c t o r i n d i r e c t ) o f p y r u v i c dehyd rogena se s y n t h e s i s and c o n s e q u e n t l y r e s u l t e d i n g r owth s t a s i s . T h i s phenomenon o f g rowth i n h i b i t i o n o r t o x i c i t y due t o t he a c c u m u l a t i o n o f some p h o s p h o r y 1 a t e d i n t e r m e d i a t e has become a common o b s e r v a t i o n ( see p. 11 ) . However, t h i s i s a p p a r e n t l y t he f i r s t c a s e r e p o r t e d t o i n v o l v e e i t h e r P. a e r u g i n o s a o r 6 -PG. 150 L i s t o f a b b r e v i a t i o n s . a d e n o s i n e d i p h o s p h a t e ADP a d e n o s i n e t r i p h o s p h a t e ATP c a r b o h y d r a t e - c a t a b o l i z i n g CC E n t n e r - D o u d o r o f f ED e t h y l e n e d i a m i n e t e t r a a c e t a t e EDTA f l a v i n a d e n i n e d i n u c l e o t i d e FAD f r u c t o s e - 6 - p h o s p h a t e F -6 -P g l u c o s e - 6 - p h o s p h a t e G-6-P L - a - g l y c e r o p h o s p h a t e L - a - G P N -2 - hyd roxye thy1 p i p e r a z i n e - N 1 - 2 - e t h a n e s u l f o n i c a c i d . . . . Hepes 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 KDGP 2 - k e t o g l u c o n a t e 2-KG 2- k e t o - 6 - p h o s p h o g l u c o n a t e 2 -K -6 -PG N - m e t h y 1 - N - n i t r o - N 1 - n i t r o s o g u a n i d i n e NTG n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e NAD n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e ( r educed ) NADH n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e pho spha te NADP n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e pho spha te ( r e d u c e d ) . . . NADPH o p t i c a l d e n i s t y a t 660 nm- • O.D. 6 -phosphog1uconate 6-PG 3- p h o s p h o g l y c e r a l d e h y d e 3 - PG t r i c a r b o x y l i c a c i d TCA t r i s ( h y d r o x y m e t h y 1 ) a m i n o m e t h a n e - C l t r i s 

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