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Observations on the control of biosynthesis of valine and isoleucine in Escherichia coli Stapleton, Joyce Alice 1970

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' Observations on the Control of Biosynthesis of Valine and Isoleucine in Escherichia c o l i by Joyce Alice Stapleton B.Sc, Honors, University of Alberta, 19 68 A thesis submitted i n partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in the Department of Biochemistry We accept this thesis as conforming to the required standard: The University of. British Columbia Ap r i l , 19 70 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 it freely available for reference and study. I further agree tha 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. Depa rtment The University of British Columbia Vancouver 8, Canada i ABSTRACT P r e v i o u s work i n many l a b o r a t o r i e s has e s t a b l i s h e d t h a t t h e a d d i t i o n o f L - v a l i n e t o a c u l t u r e o f E s c h e r i c h i a c o l i K-12 g r o w i n g e x p o n e n t i a l l y on m i n i m a l medium causes i n h i b i t i o n r e s u l t i n g i n p e r s i s t e n t l i n e a r growth. The i n h i b i t i o n can be removed by a d d i t i o n o f i s o l e u c i n e . Growth o f o t h e r s t r a i n s o f E. c o l i e.g. E. c o l i B and two mutants o f K-12 (E. c o l i AB1020 and AB1005) was n o t a f f e c t e d by a d d i t i o n o f v a l i n e . E. c o l i L L 5 , a n o t h e r mutant o f K-12, showed no i n h i b i t i o n o f growth by v a l i n e added i n c o n c e n t r a t i o n s up t o 1 x 10 _ I*M. However, t h e a d d i t i o n o f 1 x 10" 3M v a l i n e c a u sed a d e c r e a s e i n t h e l o g a r i t h m i c growth r a t e , and 1 x 1 0 - 2 M v a l i n e c a u sed p e r s i s t e n t l i n e a r growth. The i n h i b i t i o n o f growth by v a l i n e i n LL5 was a n t a g o n i z e d by L - i s o l e u c i n e (as i s t h e c a s e w i t h w i l d - t y p e E. c o l i K -12), b u t n o t by a - k e t o b u t y r a t e , t h r e o n i n e o r p y r u v a t e . K i n e t i c s t u d i e s showed t h a t t h e maximal i n h i b i t i o n o f a c e t o h y d r o x y a c i d s y n t h e t a s e (AHAS) by 1.5 x 10" 3M v a l i n e was 84% f o r E. c o l i K-12, 55% f o r E. c o l i B, 75% f o r E. c o l i AB1005, 77% f o r E. c o l i AB1020 and o n l y 20% f o r E. c o l i L L 5 . I t i s p r o p o s e d t h a t t h e p e r s i s t e n c e o f l i n e a r growth o f K-12 i n v a l i n e - c o n t a i n i n g medium i s t h e r e s u l t o f i n c o m p l e t e (84%) feed-back i n h i b i t i o n o f AHAS. A n a l y s i s o f t h e d a t a f o r v a l i n e i n h i b i t i o n o f AHAS was c a r r i e d o u t by t h e method o f L e v i t s k y and K o s h l a n d (1969) u s i n g H i l l P l o t s . E. c o l i s t r a i n s K-12, AB1005 and AB10 20 showed "positive cooperativity" between i n h i b i t o r (valine) binding s i t e s at low valine concentrations, and "negative cooperativity" between i n h i b i t o r binding s i t e s at high valine concentrations. The AHAS of E. c o l i s trains E and LL5, however, showed only negative cooperativity between binding s i t e s for i n h i b i t o r , which could be the mechanism f o r incomplete i n h i b i t i o n of AHAS by v a l i n e . Preliminary k i n e t i c analyses using Michaelis-Menten plots were c a r r i e d out with strains K-12 and LL5. The l e v e l s of threonine deaminase (TD) and AHAS were also examined i n four of the E. c o l i s t r a i n s , K-12, B, AB1020 and LL5. AHAS was repressed and TD derepressed i n E. c o l i K-12 grown (li n e a r l y ) with 10"3M v a l i n e . In E. c o l i s trains B and AB1020, growth with 10"3M valine had no e f f e c t on the level s of AHAS or (derepressed) TD. In E. c o l i s t r a i n LL5, growth with lO'^M valine did not change the AHAS l e v e l but caused s i g n i f i c a n t derepression of TD. S e n s i t i v i t y of growth to valine has been correlated with three properties of the organism: 1) Feed-back s e n s i t i v i t y of acetohydroxy acid synthetase, to L-valine; 2) The l e v e l of acetohydroxy acid synthetase i n the c e l l ; 3) The l e v e l of biosynthetic threonine deaminase - the regulatory enzyme f o r isoleucine biosynthesis. i i i ACKNOWLEDGEMENTS The author would like to thank Dr. W. J . Polglase for the guidance, suggestions and encouragement which he offerred during the course of the research; and for his inspiration and patience during the writing of this thesis. Thanks also to Mrs. Tanis Matheson for her encouragement and technical assistance. This work was supported by a University of British Columbia Graduate Fellowship and a Medical Research Council Grant. i v TABLE OF CONTENTS I n t r o d u c t i o n M a t e r i a l s and Methods I I I I I I V V R e s u l t s I I I Organisms (a) W i l d - t y p e c u l t u r e s (b) V a l i n e r e s i s t a n t c u l t u r e s 11 11 Growth o f C u l t u r e s and P r e p a r a t i o n o f E x t r a c t s 12 (a) Media (b) E s t i m a t i o n o f growth (c) P r o c e d u r e s f o r growing and h a r v e s t i n g c u l t u r e s i ) Growth i n m i n i m a l medium i i ) Growth i n supplemented medium P r e p a r a t i o n o f Crude C e l l E x t r a c t s C h e m i c a l A n a l y s i s and Enzyme A s s a y s (a) C h e m i c a l a n a l y s i s (b) Enzyme a s s a y s i ) A c e t o h y d r o x y a c i d s y n t h e t a s e i i ) T h r e o n i n e deaminase Treatment o f R e s u l t s 14 14 15 17 18 Growth C h a r a c t e r i s t i c s o f t h e B a c t e r i a l S t r a i n s 18 (a) Growth o f b a c t e r i a l s t r a i n s on m i n i m a l medium w i t h o r w i t h o u t s u p p l e m e n t a t i o n w i t h L - v a l i n e . (b) Antagonism o f v a l i n e i n h i b i t i o n 23 i ) E f f e c t o f L - i s o l e u c i n e i i ) Lack o f e f f e c t o f a - k e t o b u t y r a t e i i i ) Lack o f e f f e c t o f sodium p y r u v a t e and L - t h r e o n i n e (c) Growth on a l t e r n a t e c a r b o n s o u r c e s 34 Enzyme K i n e t i c s (a) I n h i b i t i o n o f t h e a c t i v i t y o f a c e t o h y d r o x y a c i d s y n t h e t a s e by v a l i n e . (b) H i l l p l o t s (c) M i c h a e l i s - M e n t e n p l o t s o f a c e t o h y d r o x y a c i d s y n t h e t a s e a c t i v i t y from E. c o l i s t r a i n s K-12 and L L 5 . D e t e r m i n a t i o n o f K r (d) xm D i s c u s s i o n 37 37 50 51 58 66 S u g g e s t i o n s f o r F u t u r e Work 78 B i b l i o g r a p h y 81 V LIST OF TABLES I H i l l Coefficients for E. c o l i strains K-12, AB1020, AB1005, B and LL5. II % decrease in fin a l reaction velocity of AHAS, caused by valine. III Calculation of Km. IV Ratio of [valine] giving 46% inhibition in E. c o l i strains K-12 and LL5. V Specific Activity of AHAS VI Specific Activity of Threonine Deaminase VII Characteristics of E. c o l i strains. LIST OF SCHEMES 1. The biosynthetic pathway to isoleucine, valine, leucine and pantothenate in E. c o l i . 2. The formation of a-acetolactate and a-aceto a-hydroxybutyrate in E. c o l i . v i LIST OF FIGURES Growth o f E. c o l i K-12 on s a l t s - g l u c o s e medium w i t h and w i t h o u t added v a l i n e . Growth o f E. c o l i B on s a l t s - g l u c o s e medium w i t h and w i t h o u t added v a l i n e . Growth o f E. c o l i AB1020 on s a l t s - g l u c o s e medium w i t h and w i t h o u t added v a l i n e . Growth o f E. c o l i AB1005 on s a l t s - g l u c o s e medium w i t h and w i t h o u t added v a l i n e . Growth o f E. c o l i LL5 on s a l t s - g l u c o s e medium w i t h and w i t h o u t added v a l i n e . Growth o f E. c o l i K-12 on s a l t s - g l u c o s e medium w i t h and w i t h o u t added v a l i n e . I s o l e u c i n e a ntagonism o f v a l i n e i n h i b i t i o n o f growth i n E. c o l i L L 5 . Lack o f antagonism by a - k e t o b u t y r a t e o f v a l i n e i n h i b i t e d E. c o l i L L 5 . Lack o f antagonism by a - k e t o b u t y r a t e o f v a l i n e i n h i b i t e d E. c o l i L L 5 . Lack o f antagonism o f v a l i n e i n h i b i t i o n by sodium p y r u v a t e and L - t h r e o n i n e i n E. c o l i L L 5 . V a l i n e i n h i b i t i o n o f t h e a c t i v i t y o f AHAS from E. c o l i K-12 V a l i n e i n h i b i t i o n o f t h e a c t i v i t y o f AHAS from E. c o l i B. V a l i n e i n h i b i t i o n o f t h e a c t i v i t y o f AHAS from E. c o l i AB1020. v i i 14. V a l i n e i n h i b i t i o n o f t h e a c t i v i t y o f AHAS from E. c o l i AB1005. 15. V a l i n e i n h i b i t i o n o f t h e a c t i v i t y o f AHAS from E. c o l i L L 5 . 16. I n h i b i t i o n by h i g h [ v a l i n e ] o f t h e a c t i v i t y o f AHAS from E. c o l i L L 5 . 17. H i l l p l o t s f o r E. c o l i s t r a i n s ' K - 1 2 , AB1020 and AB1005. 18. Comparison o f H i l l p l o t s f o r E. c o l i K-12 and B. 19. Comparison o f H i l l p l o t s f o r E. c o l i K-12 and L L 5 . 20. M i c h a e l i s - M e n t e n p l o t f o r AHAS from E. c o l i K-12 21. M i c h a e l i s - M e n t e n p l o t f o r AHAS from E. c o l i L L 5 . 1. INTRODUCTION In 1939, Gladstone, working with B a c i l l u s anthracis, found that the growth of t h i s organism was i n h i b i t e d by the addition of any one of isoleucine, valine or leucine. Simultaneous addition of the three amino acids, however, stimulated growth. An extension of t h i s work was done on Escherichia c o l i K-12 by D. Bonner i n 19 46 who showed that while valine alone i n h i b i t e d growth, isoleucine addition would reverse t h i s i n h i b i t i o n . The next related observation came i n 1955 from Umbarger and Brown who had undertaken a study of the metabolism of isoleucine and valine i n E^ c o l i . They found that isoleucine v/as a non-competitive antagonist of valine which indicated that i t was e i t h e r a d i r e c t or i n d i r e c t product of the i n h i b i t e d reaction, i e . , valine i n h i b -i t e d the synthesis of isoleucine rather than i t s incorporation i n t o protein. Studies on the pathways and enzymes involved i n the synthesis of v a l i n e , isoleucine, leucine and also pantothenate were subsequently c a r r i e d out by a number of workers. The results are summarized by Umbarger and Davis (1962). The pathways for the synthesis of v a l i n e , i s o l e u c i n e , leucine and pantothenate are given i n Scheme 1. I t should be noted that the reaction sequences for valine and isoleucine formation are p a r a l l e l , the same set of enzymes operating i n both pathways. Furthermore, the f i r s t step i n the valine pathway i s the second step i n the isoleucine pathway. The f i r s t step i n the isoleucine pathway i s the deamination of threonine. threonine a-Keto-butyrate-pyruvate pyruvate a-aceto-a -hydroxy--o butyrate -a-aceoo--> l a c t a t e _ I Acetohydroxy Acid Synthetase I I Reductoisomerase .III Dihydroxy Dehydrase IV Transaminase B . I I a/S-dihydroxy-g-methyl-. > valerate a-ksto-3-methyl-valerate I I I a ,S--dihydroxy--> isovalerate -a-keto--oisovalerate a -keto-isocaproate L-l e u c i n e -» L - i s o l e u c i n e rv L - v a l i n e a-keto-pantoate pantoate pantothenate Scheme 1 The b i o s y n t h e t i c pathway to i s o l e u c i n e , v a l i n e , — . leucine and pantothenate i n E s c h e r i c h i a c o l i * 3. The e x p l a n a t i o n o f t h e p r e l i m i n a r y o b s e r v a t i o n s o f G l a d s t o n e , Bonner and Umbarger l i e s i n t h e p a t t e r n o f c o n t r o l o f t h e s y n t h e s i s o f the f o u r p r o d u c t s ( l e u c i n e , v a l i n e , i s o l e u c i n e and p a n t o t h e n i c a c i d ) o f t h e branched pathway wh i c h o r i g i n a t e s a t pyruvate.' The f i r s t enzyme i n t h e p a t h -way, c a l l e d c o n d e n s i n g enzyme (Umbarger and Brown, 1958) o r ac e t o h y d r o x y a c i d s y n t h e t a s e (AHAS) ( B a u e r l e e t a l , 1964) has been found t o be one o f t h e key c o n t r o l p o i n t s . A l t h o u g h t h i s enzyme i s v e r y l a b i l e and a l l work w i t h i t must be c a r r i e d o u t u s i n g a crude e x t r a c t , t h e mechanism o f a c t i o n seems t o i n v o l v e t h e f o r m a t i o n o f an " a c t i v e a l d e h y d e " by c o n d e n s a t i o n o f p y r u v a t e and t h i a m i n e p y r o p h o s p h a t e (TPP) t o form an a c e t a l d i p h o s p h o t h i a m i n e complex (Scheme 2 ) . The a c e t a l group i s t h e n t r a n s f e r r e d t o a n o t h e r p y r u v a t e m o l e c u l e f o r t h e v a l i n e pathway o r t o t h e t h r e o n i n e d e a m i n a t i o n p r o d u c t , a - k e t o - b u t y r a t e , i n t h e i s o l e u c i n e pathway. Stermer and Umbarger i n 1964 r e p o r t e d t h a t AHAS r e q u i r e s f l a v i n e a d enine d i n u c l e o t i d e (FAD) as w e l l as TPP and M g + + f o r maximum a c t i v i t y . The r e a s o n f o r t h i s i s s t i l l n o t u n d e r s t o o d . I n 1958, Georges Cohen c a l l e d a t t e n t i o n t o t h e f a c t t h a t t h e a d d i t i o n o f v a l i n e t o an e x p o n e n t i a l l y g r owing c u l t u r e does n o t s t o p g r o w t h , b u t r a t h e r a l l o w s growth t o c o n t i n u e o n l y l i n e a r l y . He s u g g e s t e d t h a t t h i s was a r e s u l t o f t h e f o r m a t i o n o f " f a l s e p r o t e i n " where v a l i n e was i n c o r p o r a t e d i n p l a c e o f i s o l e u c i n e . However, Cohen advanced h i s h y p o t h e s i s p r i o r t o t h e d e t a i l e d s t u d i e s o f r e g u l a t i o n o f b i o s y n t h e s i s 0 CH3-CH2-C-COOH a-ke tobutyrate 0 a CH^-C-COCH-pyruvate •• + DPTH - C O 2 CH^-C-DPT H 0 n CH-j-C-CCOH pyruvate 0 OH CH3-C-C-COCH CH^—CH2 a-acetoa-hydrcxy-butyrate Acetohydroxy Acid Synthetase + FAD + Mg* 0 OH -> C H - . - C - C - C 0 0 H cc-acetolaetate -aceto a -hydroxy-5. o f a l i p h a t i c amino a c i d s . Umbarger and Brown (1958) r e p o r t e d t h a t AHAS a c t i v i t y was i n h i b i t e d by L - v a l i n e and t h a t t h i s i n h i b i t i o n was compet-i t i v e w i t h p y r u v a t e (the s u b s t r a t e ) . S i n c e AHAS c a t a l y z e s t h e f o r m a t i o n o f b o t h a - a c e t o l a c t a t e and a - a c e t o h y d r o x y b u t y r a t e , i t was l o g i c a l t h a t L e a v i t t and Umbarger (1962) s h o u l d o b s e r v e t h a t v a l i n e i n h i b i t e d t h e f o r m a t i o n o f b o t h compounds t o t h e same e x t e n t . They p r o p o s e d t h a t v a l i n e i n h i b i t e d growth o f E. c o l i K-12 by l i m i t i n g t he s u p p l y o f a c e t o h y d r o x y b u t y r a t e and t h u s o f i s o l e u c i n e f o r p r o t e i n s y n t h e s i s by e x e r t i n g f e e d -back c o n t r o l o v e r AHAS. I n a c o m p a r a t i v e s t u d y o f t h e s e n s i t -i v i t y o f AHAS t o v a l i n e i n t h r e e E ^ c o l i s t r a i n s , i t was found ( L e a v i t t and Umbarger, 1962) t h a t t h e AHAS o f E^ c o l i K-12 was much more s e n s i t i v e t o i n h i b i t i o n by L - v a l i n e t h a n were t h e AHAS's o f E ^ c o l i W and E ^ c o l i K-12/V.R., two s t r a i n s whose growth was n o t i n h i b i t e d by v a l i n e . S i n c e i t had been de m o n s t r a t e d t h a t a - k e t o b u t y r a t e , t h e t h r e o n i n e d e a m i n a t i o n p r o d u c t and s u b s t r a t e f o r AHAS i n t h e i s o l e u c i n e pathway, d i d no t r e l i e v e t h e i n h i b i t i o n , (Umbarger and Brown, 1955) i t was c o n c l u d e d t h a t t h e s i t e o f v a l i n e s e n s i t i v i t y must come a f t e r t h e t h r e o n i n e d e a m i n a t i o n s t e p , w h i c h would s u p p o r t t h e i d e a o f AHAS as a c o n t r o l p o i n t . I n d i r e c t c o n t r a s t t o t h e s e r e s u l t s were t h o s e p r e s e n t e d by Ramakrishnan and A d e l b e r g (1964, 1965). They found t h a t t h e enzymes i n t h e " i l v a " * pathway c o u l d be grouped i n t o * i l v a = i s o l e u c i n e , l e u c i n e , v a l i n e 6. t h r e e o p e r o n s , each c o n t r o l l e d by a s i n g l e o p e r a t o r l o c u s . The genes f o r t h r e o n i n e deaminase, t r a n s a m i n a s e B and dehydrase a r e c o n t r o l l e d by o p e r a t o r l o c u s A (opr A ) , w h i l e AHAS i s c o n t r o l l e d by o p r B. The e x i s t e n c e o f a t h i r d o p e r a t o r c o n t r o l l i n g t h e s y n t h e s i s o f r e d u c t o i s o m e r a s e was i n f e r r e d . T h e i r r e s u l t s i n d i c a t e d t h a t t h e r e were two s i t e s o f v a l i n e s e n s i t i v i t y , AHAS, and a l s o t h r e o n i n e deaminase (TD) wh i c h t h e y t h o u g h t was t h e p r i m a r y s i t e . Two t y p e s o f v a l i n e r e s i s t a n t mutants were i s o l a t e d . One w h i c h was r e s i s t a n t t o 2 x 10_I*M v a l i n e was found t o have d e r e p r e s s e d l e v e l s o f AHAS; w h i l e a n o t h e r w h i c h was r e s i s t a n t t o 10~ 2M v a l i n e was found t o have d e r e p r e s s e d l e v e l s o f t h r e o n i n e deaminase. The m u t a t i o n s were i n t h e o p e r a t o r l o c i , o p r B f o r AHAS and o p r A f o r TD. From t h e s e r e s u l t s t h e y c o n c l u d e d t h a t t h r e o n i n e deaminase i s t h e main r a t e l i m i t i n g enzyme f o r i s o l e u c i n e b i o s y n t h e s i s i n E. c o l i K-12 i n t h e p r e s e n c e o f v a l i n e , b u t t h a t AHAS a l s o p l a y s a p a r t . Thus, i s o l e u c i n e b i o s y n t h e s i s can be i n c r e a s e d and v a l i n e s e n s i t i v i t y overcome e i t h e r by d e r e p r e s s i n g t h r e o n i n e deaminase o r by d e r e p r e s s i n g AHAS; t h e for m e r c o n f e r s r e s i s t a n c e t o h i g h l e v e l s o f v a l i n e , and t h e l a t t e r r e s i s t a n c e t o low l e v e l s o f v a l i n e . Umbarger and F r e u n d l i c h (1965), however, s t u d i e d p a t t e r n s o f d e r e p r e s s i o n o f t h e i l v a enzymes i n E. c o l i s t r a i n s W and w i l d - t y p e K-12 and i n S^ t y p h i m u r i u m . T h e i r p r e v i o u s r e p o r t o f m u l t i v a l e n t r e p r e s s i o n ( F r e u n d l i c h , Burns and Umbarger, 1962) had i n d i c a t e d t h a t a l l t h r e e o f v a l i n e , l e u c i n e and i s o l e u c i n e 7. were r e q u i r e d t o r e p r e s s t h e s y n t h e s i s o f t h e i l v a enzymes. I f any one o f t h e t h r e e was m i s s i n g , a d e r e p r e s s i o n o f t h e enzyme s y n t h e s i s s h o u l d o c c u r , and t h i s would be e x p e c t e d t o overcome v a l i n e i n h i b i t i o n . I n v e s t i g a t i o n showed t h a t i n E. c o l i W and S. ty p h i m u r i u m a l l t h e enzymes were d e r e p r e s s e d when any one o f t h e amino a c i d s was l i m i t i n g , b u t t h a t t h e AHAS o f E. c o l i K-12 was n o t d e r e p r e s s e d on l i m i t i n g i s o l e u c i n e . Thus F r e u n d l i c h , e t a l (1962) c o n c l u d e d t h a t t h e r e were two f a c t o r s d i s t i n g u i s h i n g E. c o l i K-12 from t h e two v a l i n e r e s i s t a n t b a c t e r i a , E. c o l i W and S. t y p h i m u r i u m , t h e f i r s t b e i n g an enzyme w i t h a g r e a t e r v a l i n e s e n s i t i v i t y i n E. c o l i K-12, and t h e second b e i n g a d i f f e r e n t p a t t e r n o f d e r e p r e s s i o n o f t h e i l v a enzymes, e s p e c i a l l y AHAS. More r e c e n t s t u d i e s by P o l g l a s e (1966 a) w i t h s e v e r a l s t r a i n s o f E. c o l i r e v e a l e d t h a t t h e r e p r e s s i o n o f AHAS was g e n e r a l l y g r e a t e r i n t h e p r e s e n c e o f t h e f o u r e n d - p r o d u c t s ( v a l i n e , l e u c i n e , i s o l e u c i n e and p a n t o t h e n a t e ) t h a n i n t h e p r e s e n c e o f v a l i n e a l o n e ; however t h e degree o f r e p r e s s i o n was n o t l a r g e and t h e r e were d i f f e r -ences between s t r a i n s . The h y p o t h e s i s p u t f o r t h by Cohen (1958) t h a t l i n e a r growth was t h e r e s u l t o f t h e f o r m a t i o n o f f a l s e p r o t e i n , was d i s p r o v e n by Temple e t a l (19 65) who showed t h a t s e v e r a l r e p r e s e n t a t i v e enzymes o f E. c o l i K-12 can i n f a c t be s y n t h e -s i z e d i n a c t i v e form d u r i n g v a l i n e i n h i b i t i o n i f c a t a b o l i t e r e p r e s s i o n i s e l i m i n a t e d . T h i s t h e n was f u r t h e r e v i d e n c e t o s u p p o r t t h e view t h a t v a l i n e e x e r t s i t s i n h i b i t o r y e f f e c t on 8. t h e growth o f E. c o l i K-12 s o l e l y by p r e v e n t i n g t h e b i o s y n -t h e s i s o f i s o l e u c i n e . They a l s o s u g g e s t e d t h a t l i n e a r growth c o u l d be an a r t i f a c t , w h i c h i s i n f a c t , d e c r e a s i n g l o g a r i t h m i c growth w h i c h cannot be a d e q u a t e l y graphed w i t h p r e s e n t methods. A new avenue o f approach was opened when Bragg and P o l g l a s e (1962) r e p o r t e d t h a t s t r e p t o m y c i n - d e p e n d e n t s t r a i n s o f E. c o l i K-12 s e c r e t e d v a l i n e when grown i n t h e p r e s e n c e o f adequate a n t i b i o t i c ; and f u r t h e r s t u d i e s ( P o l g l a s e , 1965) i n d i c a t e d t h a t one o f t h e e f f e c t s o f t h e a n t i b i o t i c appeared t o be d e r e p r e s s i o n o f t h e s y n t h e s i s o f AHAS. Growth o f s t r e p t o m y c i n - d e p e n d e n t E. c o l i K-12 (DK-12) w i t h v a l i n e showed no d e c r e a s e o f l o g a r i t h m i c g r o w t h , b u t t h e AHAS enzyme o f t h e mutant was found t o be as s e n s i t i v e t o v a l i n e as t h a t o f t h e w i l d - t y p e s t r a i n . The e x p l a n a t i o n f o r t h i s r e s i s t a n c e o f DK-12 t o v a l i n e t h e n was g i v e n as t h e d e r e p r e s s i o n o f AHAS s y t h e s i s , a r e s u l t a g r e e i n g w i t h t h a t o f Umbarger and F r e u n d l i c h (1965) . D e s a i and P o l g l a s e (1966) a s s a y e d t h r e o n i n e deaminase i n s e v e r a l s t r a i n s o f E. c o l i . W i l d - t y p e E. c o l i K-12 was found t o have a v e r y low l e v e l o f t h r e o n i n e deaminase when compared w i t h s t r a i n s o f E. c o l i such as B, B/r and E w h i c h were v a l i n e i n s e n s i t i v e . Thus i t seemed t h a t t h e r e were i n f a c t t h r e e p r o p e r t i e s w h i c h c o u l d c o n t r i b u t e t o v a l i n e r e s i s t a n c e : (1) an AHAS w h i c h i s r e l a t i v e l y i n s e n s i t i v e t o v a l i n e . (2) d e r e p r e s s e d l e v e l s o f AHAS. (3) d e r e p r e s s e d l e v e l s o f t h r e o n i n e deaminase. 9. I n 1969, C o u k e l l and P o l g l a s e r e p o r t e d work w i t h E. c o l i B w h i c h i n d i c a t e d t h a t AHAS was s u b j e c t t o c a t a b o l i t e r e p r e s s i o n , such t h a t t h e n a t u r e and c o n c e n t r a t i o n o f t h e carb o n s o u r c e had more e f f e c t on enzyme f o r m a t i o n t h a n t h e known e n d - p r o d u c t s , v a l i n e , i s o l e u c i n e , l e u c i n e and p a n t o t h -e n a t e . Thus AHAS may be under t h e c o n t r o l o f two t y p e s o f c o - r e p r e s s o r m o l e c u l e s ; a c a t a b o l i t e c o - r e p r e s s o r d e r i v e d from t h e c a r b o n s o u r c e , and a b i o s y n t h e t i c e n d - p r o d u c t c o - r e p r e s s o r d e r i v e d from t h e v a r i o u s amino a c i d e n d - p r o d u c t s . I t was a l s o found t h a t t h r e o n i n e deaminase, and t h e o t h e r enzymes o f opr A a r e n o t under t h e c o n t r o l o f a c a t a b o l i t e c o - r e p r e s s o r . T h i s r e v i e w o f c o n t r o l o f t h e i l v a pathway s e r v e s t o emphasize t h a t t h e d e t a i l s o f c o n t r o l a r e n o t c o m p l e t e l y e l u c i d a t e d . U n p u b l i s h e d o b s e r v a t i o n s i n t h i s L a b o r a t o r y ( C o u k e l l and P o l g l a s e ) i n d i c a t e d t h a t i n h i b i t i o n by v a l i n e o f t h e AHAS o f s e v e r a l s t r a i n s o f E. c o l i d i d n o t r e a c h 10 0% r e g a r d l e s s o f t h e c o n c e n t r a t i o n o f s u b s t r a t e and v a l i n e employed. I t was found t h a t a d d i t i o n o f a l l f o u r known en d - p r o d u c t s ( v a l i n e , i s o l e u c i n e , l e u c i n e and p a n t o t h e n a t e ) d i d n o t enhance ( o r r e l i e v e ) t h i s i n h i b i t i o n , w h i c h would r u l e o u t t h e p o s s i b i l i t y o f t h e need f o r t h e c o n c e r t e d a c t i o n o f more t h a n one i n h i b i t o r . M o d i f i c a t i o n s i n t h e method o f o b t a i n i n g c r u d e e x t r a c t s o f AHAS d i d n o t a f f e c t t h e degree o f i n h i b i t i o n . 10. The a c t i v i t y o f AHAS from E. c o l i K-12 was found t o be i n h i b i t e d t o t h e e x t e n t o f 87 - 90%, w h i l e t h a t o f E. c o l i B c o u l d be i n h i b i t e d o n l y t o t h e e x t e n t o f 55 - 65%. The s i g n i f i c a n c e o f i n c o m p l e t e feedback i n h i b i t i o n o f AHAS by v a l i n e had n o t p r e v i o u s l y been t a k e n i n t o c o n s i d e r a t i o n i n e v a l u a t i n g t h e e f f e c t o f v a l i n e on growth. The purpose o f th e work p r e s e n t e d i n t h i s t h e s i s was t o a s s e s s t h e s i g n i f i -cance o f i n c o m p l e t e i n h i b i t i o n by comparing t h e e f f e c t o f v a l i n e on t h e AHAS a c t i v i t y o f s e v e r a l s t r a i n s o f E. c o l i , i n c o n j u n c t i o n W i t h o t h e r known p r o p e r t i e s o f t h e i l v a pathway. D u r i n g t h e c o u r s e o f t h e i n v e s t i g a t i o n , i t became a p p a r e n t t h a t t h e k i n e t i c s o f i n h i b i t i o n o f AHAS by v a l i n e r e q u i r e d a more d e t a i l e d s t u d y t h a n p r e v i o u s l y had been a c c o r d e d t o t h i s s u b j e c t . 11. MATERIALS AND METHODS I . Organisms (a) W i l d - t y p e c u l t u r e s Two s t r a i n s o f E. c o l i v/ere used i n t h i s work; E. c o l i B(ATCC11303) and E. c o l i K-12, t h e l a t t e r o b t a i n e d o r i g i n a l l y from t h e Department of B a c t e r i o l o g y and Immunology, 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 . (b) V a l i n e r e s i s t a n t c u l t u r e s Two s t r a i n s o f v a l i n e r e s i s t a n t E. c o l i , E. c o l i AB10 20 and E. c o l i AB1005, were o b t a i n e d from Dr. E. A. A d e l b e r g , Department o f M i c r o b i o l o g y , Y a l e U n i v e r s i t y , New Haven, C o n n e c t i c u t . A spontaneous v a l i n e r e s i s t a n t mutant was i s o l a t e d from E. c o l i K-12 by t h e f o l l o w i n g p r o c e d u r e . A volume o f 5 mis o f g l u c o s e ( 0 . 4 % ) - s a l t s medium (see Methods I I , (a)) was i n o c u l a t e d w i t h a c u l t u r e o f E. c o l i K-12 w h i c h had been s t o r e d on a s a l t s - a g a r s l o p e a t 5°. T h i s c u l t u r e was i n c u b a t e d f o r 24 h r s . a t 37°C and t h e n used t o i n o c u l a t e 50 mis o f g l u c o s e ( 0 . 4 % ) - s a l t s medium. T h i s c u l t u r e was i n t u r n grown f o r 24 h r s . a t 37° w i t h o u t a g i t a t i o n and used t o i n o c u l a t e 500 mis o f g l u c o s e ( 0 . 4 % ) - s a l t s medium. T h i s c u l t u r e was i n c u b a t e d a t 37° f o r 24 h r s . w i t h o u t a g i t a t i o n and t h e r e s u l t i n g growth t r a n s f e r r e d a s e p t i c a l l y t o two 300 m l , s t e r i l e , c e n t r i f u g e b o t t l e s and c e n t r i f u g e d a t 2000 rpm f o r 1 h r . The p e l l e t was suspended i n 40 mis o f s t e r i l e 0.85% N a C l , and t h i s was used t o i n o c u l a t e 300 mis o f g l u c o s e ( 0 . 4 % ) -s a l t s medium c o n t a i n i n g 12 ug/ml L - v a l i n e . T h i s c u l t u r e was 12. i n c u b a t e d a t 37° w i t h o u t a g i t a t i o n f o r 48 h r s . The growth was t h e n t r a n s f e r r e d a s e p t i c a l l y t o c e n t r i f u g e b o t t l e s and c e n t r i f u g e d a t 2000 rpm f o r 1 h r . The p e l l e t was resuspended i n 10 mis o f s t e r i l e 0.85% N a C l . P e t r i p l a t e s c o n t a i n i n g b a s a l s a l t s a g ar and L - v a l i n e a t a c o n c e n t r a t i o n o f 1 x 10 - 1*M were s t r e a k e d w i t h 0.5 ml o f t h e c e l l s l u r r y . The p l a t e s were i n c u b a t e d f o r 48 h r s . a t 37°. A l a r g e number o f v a l i n e r e s i s t a n t c o l o n i e s d e v e l o p e d . S e v e r a l o f t h e s e were s e l e c t e d and s t r e a k e d o n t o p l a t e s c o n t a i n i n g b a s a l s a l t s agar and 1 x 10 _ I ,M v a l i n e . These were i n c u b a t e d f o r 48 h r s . a t 37°C and a s i n g l e c o l o n y from each p l a t e was s e l e c t e d and p l a t e d a g a i n i n t h e manner d e s c r i b e d . A f t e r i n c u b a t i o n a t 37° f o r 48 hrs.,. a s i n g l e c o l o n y was s e l e c t e d from each o f two o f t h e p l a t e s and t r a n s f e r r e d t o n u t r i e n t a gar s l o p e s and i n c u b a t e d a t 37° f o r 24 h o u r s . The s t r a i n used i n t h i s s t u d y was d e s i g n a t e d E. c o l i LL5 ( " l o w - l e v e l " v a l i n e r e s i s t a n t ) . I I . Growth o f c u l t u r e s and p r e p a r a t i o n o f e x t r a c t s (a) Media Throughout t h i s work a m i n i m a l medium was u s e d , based on t h a t d e s c r i b e d by D a v i s and M i n g i o l i (1950). The compos-i t i o n i s as f o l l o w s : K 2HPO^ (0 .7%) , KH 2 PC\ (0.3%) , MgS0i»-7Ha0 ( 0 . 0 2 % ) , (NHij) 2SOj, (0.1%) and sodium c i t r a t e ( 0 . 0 5 % ) . The pH was a d j u s t e d t o 7.4. The c a r b o n s o u r c e , g l u c o s e , was a u t o c l -aved s e p a r a t e l y i n a c o n c e n t r a t e d s o l u t i o n and added t o g i v e a f i n a l c o n c e n t r a t i o n o f 0.4%. 13. (b) E s t i m a t i o n o f growth Growth o f b a c t e r i a l c u l t u r e s was d e t e r m i n e d t u r b i d i m e t r i -c a l l y by m e a suring t h e change i n absorbance o f t h e c u l t u r e a t 420 my ( A i ^ o ) i n a Beckman B s p e c t r o p h o t o m e t e r ( l i g h t p a t h , 1.0 cm). T u r b i d i t y was measured a g a i n s t a b l a n k c o n t a i n i n g d i s t i l l e d w a t e r . (c) P r o c e d u r e s f o r g r owing and h a r v e s t i n g c u l t u r e s ( i ) Growth i n m i n i m a l medium. An i n o c u l u m (50-100 ml) was grown a e r o b i c a l l y on a s h a k i n g w a t e r b a t h f o r 1 8 - 2 2 h r s . a t 37° i n g l u c o s e ( 0 . 4 % ) - s a l t s medium. The c u l t u r e was d i l u t e d i n f r e s h , warm (37°) g l u c o s e -s a l t s medium (500 - 2000 ml) t o an A t ) 2o o f a p p r o x i m a t e l y 0.1. The c u l t u r e was i n c u b a t e d a e r o b i c a l l y ( s p a r g e r ) a t 37° u n t i l t h e absorbance r e a c h e d 0.9 - 1.0. The c e l l s were c h i l l e d i n an i c e - b a t h , 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 4° (16,300 g f o r 10 m i n ) , washed once i n 0.1 M-potassium phosphate b u f f e r , pH 7.4 and s t o r e d as packed c e l l s a t 0°. N o r m a l l y t h e packed c e l l s were s t o r e d no l o n g e r t h a n 36 h r s . b e f o r e they were d i s r u p t e d by u l t r a s o n i c t r e a t m e n t and a s s a y e d f o r enzymic a c t i v i t y . ( i i ) Growth i n supplemented medium. Growth of t h e c e l l s i n t h e p r e s e n c e o f v a l i n e was c a r r i e d o u t ( w i t h one e x c e p t i o n ) by p r e p a r i n g t h e c e l l s as i n p a r t (a) t o t h e p o i n t o f i n o c u l a t i n g f r e s h medium t o an A 4 2 0 = 0.1. The c e l l s were t h e n a l l o w e d t o grow t o an Ai»2o of 0.3, a t w h i c h time t h e c u l t u r e was s u b d i v i d e d by volume i n t o f l a s k s 14. c o n t a i n i n g v a l i n e o r o t h e r compounds (e.g. p y r u v a t e , a - k e t o b u t -y r a t e , i s o l e u c i n e ) i n a c o n c e n t r a t e d s o l u t i o n . O ther a d d i t i o n s made d u r i n g growth a r e i n d i c a t e d i n t h e e x p e r i m e n t s . The e x c e p t i o n was one s e t o f e x p e r i m e n t s where growth was c a r r i e d o u t w i t h v a l i n e added a t z e r o time ( A ^ 2 0 = 0.1). E x c e p t where i n d i c a t e d , t h e c e l l s grown i n t h e p r e s e n c e o f added compounds were n o t used f o r enzyme a s s a y s . I I I . P r e p a r a t i o n o f crude c e l l e x t r a c t s S o n i c t r e a t m e n t The crude c e l l e x t r a c t s were p r e p a r e d by d i s r u p t i o n o f th e c e l l s w i t h u l t r a s o n i c o s c i l l a t i o n . The wet, packed c e l l s were suspended homogeneously i n i c e - c o l d 0.1 M-potassium phosphate b u f f e r , pH 8.0. The c o n c e n t r a t i o n o f c e l l s was 1 g o f c e l l s (wet wt.)/15 ml o f b u f f e r f o r t h e a c e t o h y d r o x y a c i d s y n t h e t a s e a s s a y , and 1 g o f c e l l s (wet wt.)/7.5 ml o f b u f f e r f o r t h e t h r e o n i n e deaminase a s s a y . F o r s m a l l volumes, 3 - 5 m i s , t h e d i s r u p t i o n was c a r r i e d o u t u s i n g a p r o b e - t y p e Branson s o n i f i e r a t s e t t i n g 1 f o r 30 seconds. F o r l a r g e r volumes, 12 - 20 m i s , a b a t h - t y p e B r o n w i l l 20-K.C. s o n i c o s c i l l a t o r was used and t h e c e l l s were d i s r u p t e d f o r 4 m i n u t e s . IV. C h e m i c a l a n a l y s i s and enzyme a s s a y s (a) C h e m i c a l a n a l y s i s : P r o t e i n P r o t e i n was e s t i m a t e d by t h e F o l i n - P h e n o l method as d e s c r i b e d by Lowry e t . a l . (1951). B o v i n e serum a l b u m i n 15. (Armour Pharmaceutical Company, Chicago, I l l i n o i s ) was used as a standard. Protein samples were generally d i l u t e d (with d i s t i l l e d water) to contain 25 to 250 ug of protein/ml. (b) Enzyme assays A l l spectrophotometric determinations were made on a Beckman B spectrophotometer at 25°C. S p e c i f i c a c t i v i t i e s c were calculated as units of enzyme/mg. of protein: (i) Acetohydroxy acid synthetase (AHAS) Although AHAS i s stable for several days i n stored, whole packed c e l l s at 0°, i t i s extremely l a b i l e i n c e l l extracts (Desai and Polglase, 1965). Therefore, AHAS a c t i v i t y was determined only on freshly prepared crude extracts. When ca r r i e d out without the addition of valine, each assay tube contained, i n a t o t a l volume of 1.0 ml: potassium phosphate buffer, pH 8.0, 100 umoles; sodium pyruvate, 0.25 mmoles; MgCl 2, 0.5 umoles; thiamine pyrophosphate (TPP), 0.3 umoles and 0.5 ml of crude extract (3.0 - 4.0 mg pr o t e i n ) . When AHAS was assayed i n the presence of valine , the amount of sodium pyruvate was reduced to .025 mmoles, and valine was added i n varying concentrations depending on the experiment. The amounts of the other additives remained the same. The Michaelis k i n e t i c s of AHAS from E. c o l i s t r a i n s K-12 and LL5 were ca r r i e d out using the method described above with the exception of pyruvate concentrations which ranged from 2.5 mM to 250 mM, and valine concentrations ranging from 1 x 10_,tM to 1 x 10~2M. 16. A f t e r i n c u b a t i o n f o r 15 min. a t 37°, t h e r e a c t i o n was s t o p p e d by the. a d d i t i o n o f 0.1 ml o f 40% (w/v) t r i c h l o r o a c e t i c a c i d . T h i s was f o l l o w e d by i n c u b a t i o n a t 60° f o r 15 min. t o c o n v e r t t h e a c e t o l a c t a t e t o a c e t o i n . The r e a c t i o n m i x t u r e was d i l u t e d 100 f o l d and t h e r e s u l t i n g s o l u t i o n was a n a l y z e d f o r a c e t o i n by t h e method o f W e s t e r f e l d (1945). To a 1.0 ml a l i q u o t o f t h e d i l u t e d r e a c t i o n m i x t u r e was added 1.0 ml o f 0.5% c r e a -t i n e i n w a t e r and 1.0 ml o f f r e s h l y p r e p a r e d 5% a - n a p h t h o l i n 2.5N sodium h y d r o x i d e . The s o l u t i o n was mixed v i g o r o u s l y and c o l o r was a l l o w e d t o d e v e l o p f o r 55 min. i n t h e d a r k . The r e s u l t i n g c o l o r was r e a d a t 540 my and c o n v e r t e d t o pmoles o f a c e t o i n by means o f a s t a n d a r d c u r v e . One u n i t o f a c t i v i t y i s e q u i v a l e n t t o t h e f o r m a t i o n o f 1 umole o f a c e t o i n / h r a t 37°. ( i i ) T h r e o n i n e deaminase The assay system c o n s i s t e d o f t h e f o l l o w i n g i n a t o t a l volume o f 1.0 m l : p o t a s s i u m phosphate b u f f e r , pPI 8.0, 100 umoles; L - t h r e o n i n e , 80 umoles; p y r i d o x a l p h o s p h a t e , 100 mumoles and 0.5 mis o f crude c e l l e x t r a c t (1.5 - 2 mg p r o t e i n ) . The p y r i d o x a l phosphate and t h e crude c e l l e x t r a c t were p r e -i n c u b a t e d a t 37° f o r 5 minutes and t h e n L - t h r e o n i n e was added. The m i x t u r e was i n c u b a t e d a t 37° f o r 10 minutes and t h e r e a c t i o n was s t o p p e d by t h e a d d i t i o n o f 0.1 ml o f 40% (w/v) t r i c h l o r o a c e t i c a c i d . F o l l o w i n g a 1 0 - f o l d d i l u t i o n w i t h d i s t i l l e d w a t e r t h e p r e c i p i t a t e was removed by c e n t r i f u g a t i o n a t 3200 rpm f o r 10 m i n u t e s , and t h e s u p e r n a t a n t was a n a l y z e d f o r a - k e t o b u t y r i c a c i d by t h e method o f Friedemann (1957). 17. To 1.0 ml o f t h e s u p e r n a t a n t s o l u t i o n was added 2.0 ml o f d i s t i l l e d w a t e r and 1.0 ml o f 0.1% 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e i n 2N H C l . A f t e r 5 min. a t room t e m p e r a t u r e , 3.0 ml o f d i s t i l l e d benzene and 1.0 ml o f 95% e t h a n o i were added and the aqueous phase was e x t r a c t e d by a g i t a t i n g t h e c o n t e n t s o f the tube w i t h a s t r e a m o f a i r f o r 3 min. The two l a y e r s were s e p a r a t e d by low speed c e n t r i f u g a t i o n and t h e aqueous l a y e r was removed and d i s c a r d e d . The o r g a n i c phase was t h e n e x t r a c t e d w i t h 6.0 ml o f 10% sodium c a r b o n a t e f o r 3 min. E x a c t l y 2.0 ml o f t h e aqueous c a r b o n a t e l a y e r were removed and added t o 2.0 ml o f 1.5 N sodium h y d r o x i d e . C o l o r was a l l o w e d t o d e v e l o p f o r 5 min. a t room t e m p e r a t u r e . The a b s o r b -ance o f t h e s o l u t i o n was r e a d a t 435 my and c o n v e r t e d by means o f a s t a n d a r d c u r v e t o ymoles o f a - k e t o b u t y r i c a c i d . One u n i t o f t h r e o n i n e deaminase a c t i v i t y i s e q u i v a l e n t t o t h e amount o f enzyme r e q u i r e d t o form 1 ymole o f a - k e t o b u t y r a t e / h r a t 37°. V. Treatment o f R e s u l t s The d a t a from t h e a s s a y s o f a c e t o h y d r o x y a c i d s y n t h e t a s e a c t i v i t y v/ere p l o t t e d i n a number of d i f f e r e n t ways: (a) I n h i b i t i o n C u r v e s : % I n h i b i t i o n vs [ V a l i n e ] (b) M i c h a e l i s - M e n t e n C u r v e s : V e l o c i t y vs [ S u b s t r a t e ] (c) H i l l p l o t s by t h e method o f L e v i t s k y and K o s h l a n d (1969) u s i n g t h e e q u a t i o n l o g (v/v^-1) = l o g 1/K^ + n l o g [ V a l i n e ] where n = H i l l c o e f f i c i e n t . The d e t a i l s a r e shown i n F i g u r e s 1 - 2 1 i n c l u s i v e . 18. RESULTS I . Growth c h a r a c t e r i s t i c s o f t h e b a c t e r i a l s t r a i n s (a) Growth o f b a c t e r i a l s t r a i n s on m i n i m a l medium w i t h o r w i t h o u t s u p p l e m e n t a t i o n w i t h L - v a l i n e . F i g u r e s 1 - 5 i n c l u s i v e show t h e growth c u r v e s f o r t h e f i v e s t r a i n s o f E. c o l i . L - v a l i n e was added as i n d i c a t e d . F o r E. c o l i s t r a i n s B, AB10 20 and AB1005, f i v e v a l i n e c o n c e n t -r a t i o n s were used: 1 x 1 0 ~ 2 M , 1 x 1 0 ~ 3 M , 1 x l O ' ^ M , 1 x 1 0 " 5 M and 1 x 1 0 " 6 M . Because a l l c o n c e n t r a t i o n s gave c u r v e s w h i c h d i d n o t d i f f e r s i g n i f i c a n t l y from the c o n t r o l , o n l y t h e c u r v e f o r 1 x 1 0 ~ 2 M v a l i n e was p l o t t e d . F o r E. c o l i L L 5 , t h e s e f i v e c o n c e n t r a t i o n s o f v a l i n e were a l s o added. C o n c e n t r a t i o n s o f 1 x 1 0 ~ 2 M and 1 x 1 0 " 3 M v a l i n e p r o d u c e d t h e e f f e c t shown i n F i g u r e 6 , w h i l e c u l t u r e s w i t h 1 x 10 - 1*, 1 x 10" 5 and 1 x 1 0 _ 6 M v a l i n e added grew a t t h e same r a t e as t h e c o n t r o l and t h e r e f o r e a r e n o t p l o t t e d . E. c o l i K-12 ( F i g . 1) gave l i n e a r growth a t a v a l i n e c o n c e n t r a t i o n o f 2.5 x 10" ^ and h i g h e r c o n c e n t r a t i o n s o f v a l i n e (5 x 10" 5 M and 1 x 1 0 - l 4 M ) d i d n o t d e c r e a s e t h e l i n e a r growth r a t e , as o b s e r v e d p r e v i o u s l y by L e a v i t t and Umbarger (1962) . E. c o l i s t r a i n s B, AB1020 and AB1005 were r e s i s t a n t t o v a l i n e c o n c e n t r a t i o n s as h i g h as 1 x 1 0 _ 2 M ( F i g s . 2 - 4 i n c l u s i v e ) . W i t h E. c o l i L L 5 , a d i f f e r e n t e f f e c t was f o u n d . Low l e v e l s o f v a l i n e , e.g. 1 x 1 0 ~ 6 M , 1 x 10" 5 M and 1 x 1 0 - I * M showed no e f f e c t on g r o w t h , w h i l e 1 x 1 0 " 3 M v a l i n e caused a s l i g h t d e c r e a s e i n t h e r a t e o f l o g a r i t h m i c growth and 1 x 1 0 " 2 M v a l i n e 19. Fig. 1. Growth of E. c o l i K-12 (wild-type) on salts-glucose medium. (•): no additions. (o) : ^ -valine, 2.5xlO~5M, 5x10" Ixl0-I*M added as indicated by the arrow. The addition of L-valine to E. c o l i K-12 was followed immediately by inhibition of growth. The linear growth rate in the presence of valine was maintained, and did not give any indication of becoming logarithmic. 20. F i g . 2. Growth of E. c o l i B on s a l t s - g l u c o s e medium. (•): no a d d i t i o n s . (•): l x l O ~ 2 M v a l i n e as i n d i c a t e d by the arrow. F i g . 3. Growth of E. c o l i AB10 20 on s a l t s - g l u c o s e medium. (•): no a d d i t i o n s . (n): l x l O ~ 2 M v a l i n e as i n d i c a t e d by the arrow. F i g . 4. Growth of E. c o l i AB1005 on s a l t s - g l u c o s e medium. (•): no a d d i t i o n s . ( a ) : l x l O ~ 2 M v a l i n e as i n d i c a t e d by the arrow. V a l i n e d i d not a f f e c t the growth of E. c o l i s t r a i n s B, AB1020 and AB1005. T F i g u r e 2 F i g u r e 3 F i g u r e 4 caused a marked d e c r e a s e i n t h e growth r a t e w h i c h a l s o ceased t o be l o g a r i t h m i c . Growth o f t h i s s t r a i n (LL5) t h u s i s r e s i s t a n t t o low b u t s e n s i t i v e t o h i g h l e v e l s o f v a l i n e . The e f f e c t on growth of w i l d - t y p e E. c o l i K-12 on s a l t s -g l u c o s e medium supplemented w i t h v e r y low c o n c e n t r a t i o n s o f L - v a l i n e i s shown i n F i g u r e 6. T h i s e x p e r i m e n t i n d i c a t e s t h a t t h e m i n i m a l c o n c e n t r a t i o n o f v a l i n e f o r s u s t a i n e d i n h i b i t i o n o f e x p o n e n t i a l growth was 1 x 10~ 7M. A t t h i s and h i g h e r c o n c e n t r a t i o n s o f L - v a l i n e growth was l i n e a r ( F i g . 1 and 6 ) . (b) Antagonism o f v a l i n e i n h i b i t i o n ( i ) E f f e c t o f L - i s o l e u c i n e The r e l i e f o f v a l i n e i n h i b i t i o n o f e x p o n e n t i a l growth o f E. c o l i K-12 i n t h e p r e s e n c e o f L - i s o l e u c i n e , has been documented (Umbarger and Brown 1955, L e a v i t t and Umbarger 1962, B a u e r l e , F r e u n d l i c h , Stormer and Umbarger 1964). The i n h i b i t i o n by v a l i n e o f t h e growth of s t r a i n LL5 was found t o be a n t a g o n i z e d by L - i s o l e u c i n e ( F i g . 7 ) . V a l i n e (1 x 10 - 2M) was added a t 0 t ime and t h i s caused l i n e a r growth. I s o l e u c i n e (1 x 10~ 2M) was added a t A l t 2 0 = 0.3, and v a l i n e i n h i b i t i o n was r e l i e v e d a f t e r a l a g o f 60 m i n u t e s . ( i i ) Lack o f e f f e c t o f a - k e t o b u t y r a t e F i g u r e 8 shows t h e r e s u l t when s t r a i n LL5 was grown w i t h 1 x 1 0 - 2 M L - v a l i n e added a t 0 time and 0.4% a - k e t o b u t y r a t e added l a t e r . An i d e n t i c a l c u r v e was found when 1 x 10" 3M L - v a l i n e and 0.4% a - k e t o b u t y r a t e were used i n a s i m i l a r 24. F i g . 5. The growth o f E. c o l i LL5 on s a l t s - g l u c o s e medium. (•): no a d d i t i o n s . (o): l x l O ~ 3 M v a l i n e , (o): Ixl0 - 2M v a l i n e as i n d i c a t e d by t h e arrow. A d d i t i o n o f 1x10"3M v a l i n e d e c r e a s e d the l o g a r i t h m i c growth r a t e o f E. c o l i LL5 t o a s m a l l b u t r e p r o d u c i b l e e x t e n t . W ith t h e a d d i t i o n o f Ixl0 - 2M v a l i n e , t h e growth r a t e was d e c r e a s e d even f u r t h e r , and ceased t o be l o g a r i t h m i c . i Fig. 6. Growth of E. c o l i K-12 on saits-glucose medium supplemented with varying amounts of L.-valine. (•): no additions. (A): .0625xlO"7M valine, (A): .125x10"7M valine (n): .25x10"7M valine, (a): .5x10"7M valine, and (o): 1x10" valine. Added as indicated by the arrow. The minimal concentration of valine for sustained inhibition of exponential growth was 1x10"7M. 2 7 . 28. Fig. 7. Isoleucine antagonism of valine inhibition of growth in E. c o l i LL5 grown on salts-glucose medium. (•): no additions, (a): lxlO~ 2M valine added at 0 time. («): lxlO - 2M valine added at 0 time. lxlO~ 2M isoleucine added at the time indicated by the arrow, (a) . 2 9 . 30. Fig. 8. Lack of antagonism by a-ketobutyrate of valine inhibited E. c o l i LL5 grown on salts-glucose medium. (•): no additions. (D) 1X10~3M (or lxlO~2M) valine added at 0 time. (•): 0.4% a-ketobutyrate added as indicated by the arrow. 32. F i g . 9. Lack of antagonism by a-ketobutyrate of valine i n h i b i t e d E. c o l i LL5 grown on salts-glucose medium. (•): no additions. (0) lxlO~ 2M valine added as indicated by the f i r s t arrow. («): 0.4% a-ketobutyrate added as indicated by the second arrow. Addition of 10 _ 2M valine a f t e r i n i t i a t i o n of exponential growth i n h i b i t e d E. c o l i LL5, and this i n h i b i t i o n was not antagonized by a-ketobutyrate. 34. e x p e r i m e n t . F i g u r e 9 shows t h e r e s u l t s o b t a i n e d when v a l i n e was added a f t e r t h e c u l t u r e had r e a c h e d an A ^ o o f 0.3. I n n e i t h e r case d i d a - k e t o b u t y r a t e r e l i e v e t h e i n h i b i t i o n o f e x p o n e n t i a l growth imposed by v a l i n e , i n agreement w i t h Umbarger and Brown (1955) w o r k i n g w i t h E. c o l i K-12. ( i i i ) Lack o f e f f e c t o f sodium p y r u v a t e and L - t h r e o n i n e As shown i n F i g u r e 10, n e i t h e r sodium p y r u v a t e a l o n e , o r i n c o m b i n a t i o n w i t h L - t h r e o n i n e showed any antagonism o f t h e i n h i b i t i o n o f e x p o n e n t i a l growth o f E. c o l i LL5 caused by v a l i n e . S i m i l a r r e s u l t s were o b t a i n e d when a - k e t o b u t y r a t e and sodium p y r u v a t e were used i n c o m b i n a t i o n . The s l i g h t e f f e c t on l i n e a r growth o f a d d i t i o n s o f a - k e t o b u t y r a t e , t h r e o n i n e and p y r u v a t e ( F i g s . 9 and 10) was r e p r o d u c i b l e , (c) A l t e r n a t i v e c a r b o n s o u r c e s E. c o l i s t r a i n s K-12 and LL5 f a i l e d t o grow a t a l l when 0.4% a - k e t o b u t y r a t e was used as a car b o n s o u r c e . Both s t r a i n s grew l o g a r i t h m i c a l l y w i t h 0.4% p y r u v a t e as a car b o n s o u r c e . 35. Fig. 10. Lack of antagonism of valine i n h i b i t i o n by sodium pyruvate and L-threonine of E. c o l i LL5 i n salts-glucose medium. (•): no additions. ( Q ) : l x l 0 - 2 M valine added at the f i r s t arrow. (») : l x l O ~ 2 M valine plus 0.2% pyruvate added at the f i r s t arrow. (A) : L_-threonine added as indicated by the second arrow to the culture containing sodium pyruvate and v a l i n e . 36. I I . Enzyme K i n e t i c s (a) I n h i b i t i o n o f the a c t i v i t y o f a c e t o h y d r o x y a c i d s y n t h e t a s e by v a l i n e . A c e t o h y d r o x y a c i d s y n t h e t a s e (AHAS) was as s a y e d w i t h v a r y i n g c o n c e n t r a t i o n s o f v a l i n e u s i n g each o f the E. c o l i s t r a i n s (E. c o l i B, K-12, AB1020, AB1005 and L L 5 ) . The r e s u l t s a r e shown i n F i g u r e s 11 - 15 i n c l u s i v e . The AHAS o f w i l d - t y p e E. c o l i K-12 gave a h y p e r b o l i c c u r v e r e l a t i n g % i n h i b i t i o n t o i n h i b i t o r ( v a l i n e ) c o n c e n t r a -t i o n . However t h e maximum i n h i b i t i o n o b s e r v e d was 84%. E. c o l i B ( F i g . 12) showed a v a l i n e i n h i b i t i o n c urve o f a s i g n i f i c a n t l y d i f f e r e n t shape t o t h a t o f K-12. The i n i t i a l s l o p e was l o w e r and t h e maximum i n h i b i t i o n was 55%. F o r b o t h K-12 and B, h a l f - m a x i m a l i n h i b i t i o n o f AHAS o c c u r r e d a t a c o n c e n t r a t i o n o f 2.2 x 10"''M v a l i n e . E. c o l i s t r a i n s AB1020 and AB1005 ( F i g s . 13 & 14) gave v a l i n e i n h i b i t i o n c u r v e s o f AHAS w h i c h v/ere q u i t e s i m i l a r t o each o t h e r b u t d i f f e r e n t t o E. c o l i B and K-12 i n i n i t i a l s l o p e and i n maximum i n h i b i t i o n by v a l i n e w h i c h was 77% f o r E. c o l i AB1020 and 75% f o r E. c o l i AB1005. The AHAS o f E. c o l i s t r a i n LL5 was o n l y s l i g h t l y i n h i b i t e d (maximum 15%) by v a l i n e a t c o n c e n t r a t i o n s up t o 1.5 x 10" 3M ( F i g . 1 5 ) . A v e r y h i g h c o n c e n t r a t i o n o f v a l i n e (5 x 10" 2M) was r e q u i r e d t o i n h i b i t t h e AHAS o f s t r a i n LL5 a p p r e c i a b l y ( F i g . 1 6 ) . 38. F i g . 11. V a l i n e i n h i b i t i o n o f t h e a c t i v i t y o f AHAS from E. c o l i K-12. The h y p e r b o l i c i n h i b i t i o n c u r v e showed a maximum o f 84% i n h i b i t i o n o f AHAS from E. c o l i K-12. H a l f maximal i n h i b i t i o n o f AHAS o c c u r r e d a t a c o n c e n t r a t i o n o f 2.2xlO~'*M v a l i n e . 39. N O I 1 I 9 1 H N S % Fig. 12. Valine inhibition of the activity of AHAS from E. c o l i B. The maximal inhibition of AHAS from E. co l i B was 55 Half-maximal inhibition of the enzyme occurred at a concentration of 2.2x10"**M valine, as was found for the AHAS of E. co l i K-12. 41. 42. Fig. 13. Valine inhibition of the activity of AHAS from E. c o l i AB1020. This bacterial strain showed a much lower i n i t i a l slope than the parent strain (E. col i K-12) and the maximal inhibition of AHAS by valine was 77%. F i g . 14. V a l i n e i n h i b i t i o n o f t h e a c t i v i t y o f AHAS from E. c o l i AB1005. The l a c k o f d e t e c t a b l e s e n s i t i v i t y t o low l e v e l s o f v a l i n e o f t h e AHAS from E. c o l i AB1005 was i l l u s t r a t e d i n t h i s e x p e r i m e n t . The maximal i n h i b i t i o n o f AHAS by v a l i n e was 75%. 4 6 . Fig. 15. Valine inhibition of the activity of AHAS from E. c o l i LL5. The relative insensitivity of the AHAS from E. co l i LL5 was indicated by the low degree of inhibition of enzyme activity by 1.5x10"3M valine. 5 0 1 0 0 1 5 0 [VALINE] xlO_5M F i g u r e 15 48. Fig. 16. Valine inhibition of the activity of AHAS from E. c o l i LL5. When extremely high valine concentrations were used, the AHAS from E. c o l i LL5 could be inhibited approximately 86%. 49. (b) H i l l p l o t s The d a t a from t h e v a l i n e i n h i b i t i o n c u r v e s ( S e c t i o n II I, P a r t (a)) were a n a l y z e d by means o f H i l l p l o t s e m p l o y i n g t h e method o f L e v i t s k y and K o s h l a n d (1969) u s i n g t h e e q u a t i o n Log (v/v^ - 1) = l o g l/K-j_ + n l o g [ V a l ] (where v = a c t i v i t y i n the absence o f v a l i n e and v^ = a c t i v i t y i n t h e p r e s e n c e o f v a l i n e ) . The r e s u l t s a r e shown i n F i g u r e s 17, 18 and 19. The I n h i b i t i o n o f AHAS o f w i l d - t y p e E. c o l i K-12 gave, a t v e r y low v a l i n e c o n c e n t r a t i o n s , a H i l l c o e f f i c i e n t l e s s t h a n u n i t y ( F i g . 17) whi c h i n c r e a s e d t o a v a l u e g r e a t e r t h a n 1 a t i n t e r m e d i a t e v a l i n e c o n c e n t r a t i o n s and t h e n d e c l i n e d a t h i g h c o n c e n t r a t i o n s o f v a l i n e (Table I ) . The AHAS o f s t r a i n s AB1020 and AB1005 ( F i g . 17) showed no b i n d i n g a t v e r y low v a l i n e c o n c e n t r a t i o n s b u t a t i n t e r m e d i a t e c o n c e n t r a t i o n s o f v a l i n e , H i l l c o e f f i c i e n t s g r e a t e r t h a n u n i t y were o b s e r v e d . A t h i g h v a l i n e c o n c e n t r a t i o n s , t h e H i l l c o e f f i c i e n t s d e c l i n e d ( F i g . 17 and T a b l e I ) . F i g u r e 18 compares t h e H i l l p l o t f o r t h e i n h i b i t i o n o f t h e AHAS o f E. c o l i B w i t h t h a t f o r K-12. A t low v a l i n e c o n c e n t r a t i o n s the H i l l c o e f f i c i e n t f o r t h e E. c o l i B enzyme i s c o n s i d e r a b l y lower t h a n t h a t f o r t h e K-12 enzyme. A t i n t e r m e d i a t e and a t h i g h v a l i n e c o n c e n t r a t i o n s t h e H i l l c o e f f i c i e n t f o r i n h i b i t i o n o f AHAS o f E. c o l i B i n c r e a s e d b u t remained l e s s t h a n u n i t y i n c o n t r a s t t o t h a t o f K-12 wh i c h was g r e a t e r t h a n 1 a t i n t e r m e d i a t e v a l i n e c o n c e n t r a t i o n s ( F i g . 18 and T a b l e I ) . The H i l l p l o t f o r v a l i n e i n h i b i t i o n o f AHAS o f E. c o l i LL5 i s compared w i t h t h a t o f K-12 i n F i g u r e 19. Two s l o p e s were o b s e r v e d f o r the enzyme from LL5 c o r r e s p o n d i n g w i t h H i l l c o e f f i c i e n t s o f l e s s t h a n 0.5 a t low and i n t e r m e d i a t e v a l i n e c o n c e n t r a t i o n s and, a p p r o a c h i n g u n i t y a t v e r y h i g h v a l i n e c o n c e n t r a t i o n s (Table I ) . TABLE I H i l l C o e f f i c i e n t s E. c o l i s t r a i n V a l u e s o f n K-12 0 .84 1.32 0 .39 AB1020 1.56 0.63 AB1005 1.50 0.38 B 0 .44 0.75 LL5 0.39 0.87 Thus t h e H i l l p l o t s show f o u r d i f f e r e n t p a t t e r n s o f v a l i n e i n h i b i t i o n amongst t h e f i v e s t r a i n s o f E. c o l i . (c) M i c h a e l i s - M e n t e n p l o t s o f a c e t o h y d r o x y a c i d s y n t h e t a s e a c t i v i t y from E. c o l i s t r a i n s K-12 and L L 5 . F i g u r e 20 g i v e s t h e M i c h a e l i s - M e n t e n p l o t o f d a t a f o r v a l i n e i n h i b i t i o n o f AHAS from E. c o l i K-12. Wi t h 1 x 10"Hi L - v a l i n e , t h e r e was a d e c r e a s e o f 11.3% i n t h e maximal r e a c t i o n v e l o c i t y , and h i g h e r v a l i n e c o n c e n t r a t i o n s r e d u c e d t h e v e l o c i t y i r r e g u l a r l y w i t h r e s p e c t t o i n c r e a s e s i n v a l i n e c o n c e n t r a t i o n F i g . 17. H i l l p l o t o f d a t a o f v a l i n e i n h i b i t i o n o f AHAS f o r E. c o l i s t r a i n s K-12 (•); AB1020 ( A ) ; and AB1005 (a). The c u r v e s are p l o t t e d a c c o r d i n g t o t h e e q u a t i o n Log (v/v^-1) = l o g 1/K^ + n l o g [ V a l ] where n i s t h e H i l l c o e f f i c i e n t . A l s o shown on t h e graph are t h e t h e o r e t i c a l H i l l P l o t s f o r n = 0.5, 1.0 and 2.0. " N e g a t i v e c o o p e r a t i v i t y " i s i n d i c a t e d when n <1; " p o s i t i v e c o o p e r a t i v i t y " i s i n d i c a t e d when n >1. S t r a i n K-12 shows t h r e e v a l u e s f o r n (n = 0.84, 1.32 and 0.39) as v a l i n e c o n c e n t r a t i o n i n c r e a s e s , whereas t h e K-12 mutants, AB10 20 and AB1005 show o n l y two d i f f e r e n t v a l u e s f o r n (1.56 and .63; and 1.5 and .38, r e s p e c t i v e l y ) . F i g u r e 17 F i g . 18. Comparison o f H i l l P l o t s o f d a t a f o r v a l i n e i n h i b i t i o n o f AHAS o f E. c o l i s t r a i n s K-12 (•) and B (a). The c u r v e s a re p l o t t e d a c c o r d i n g t o the e q u a t i o n l o g ( v / v ^ l ) = l o g 1/K± + n l o g [ V a l ] where n i s the H i l l c o e f f i c i e n t . A l s o shown are t h e t h e o r e t i c a l H i l l P l o t s f o r n = 0.5, 1.0 and 2.0. E. c o l i B d i f f e r s from K-12 i n showing o n l y n e g a t i v e c o o p e r a t i v i t y (n <1 f o r each o f t h e two v a l u e s o f n ) . 56. F i g . 19. Comparison o f H i l l P l o t o f d a t a f o r v a l i n e i n h i b i t i o n o f AHAS o f E. c o l i s t r a i n s K-12 (•); and LL5 (o) . The c u r v e s a r e p l o t t e d a c c o r d i n g t o t h e e q u a t i o n l o g (v/v^ -= l o g 1/K^ + n l o g [ V a l ] where n i s t h e H i l l c o e f f i c i e n t . A l s o shown a r e t h e t h e o r e t i c a l H i l l P l o t s f o r n = 0.5, 1.0 and 2.0. E. c o l i L L 5 , d i f f e r s from K-12 i n showing o n l y n e g a t i v e c o o p e r a t i v i t y (n <1 f o r each o f the two v a l u e s o f n ) . 58. such t h a t 1 x 1 0 - 2 M v a l i n e reduced t h e maximal v e l o c i t y by more t h a n 70% (Table I I ) . W i t h E. c o l i L L 5 , ( F i g . 21) t h e r e was a l e s s pronounced d e c r e a s e i n t h e maximal r e a c t i o n v e l o c i t y f o r v a l i n e i n h i b i t i o n o f AHAS, p l o t t e d a c c o r d i n g t o t h e method of M i c h a e l i s and Menten, i n comparison w i t h E. c o l i K-12, such t h a t 1 x 1 0 - 2 M v a l i n e showed o n l y a 32.5% d e c r e a s e i n t h e r e a c t i o n v e l o c i t y from t h e v a l u e f o r t h e c o n t r o l ( T able I I I ) . TABLE I I % Decrease i n F i n a l R e a c t i o n V e l o c i t y E. c o l i s t r a i n No a d d i t i o n s 1 x 10" \M V a l 1 x 10 ~ 3M V a l 1 x 10~ 2M V a l K-12 0 1 1 . 3 65 .0 71.4 LL5 0 5. 2 14 .3 32.5 (d) D e t e r m i n a t i o n o f K m The L i n e w e a v e r - B u r k e p l o t s o f t h e d a t a from t h e M i c h a e l i s -Menten c u r v e s were p l o t t e d , and from t h e s e a K m f o r each o f t h e enzymes was c a l c u l a t e d , (Table I I I ) where -1/K m = - i n t e r c e p t on t h e 1/[S] a x i s . TABLE I I I C a l c u l a t i o n o f K m E. c o l i s t r a i n I n t e r c e p t on 1/[S] a x i s Km K-12 -0.040 2. 5 x 10" 2M LL5 -0.060 1. 66 x 10~ 2M 59. F i g . 20. M i c h a e l i s - M e n t e n P l o t o f v v s . [S] f o r (the a c e t o h y d r o x y a c i d ) s y n t h e t a s e o f E. c o l i K-12. (•); no a d d i t i o n s ; (n) : 1x10"''M L - v a l i n e added; (a): 1x10" 3M L - v a l i n e added; ( A ) : 1x10" 2M L - v a l i n e added. A d d i t i o n o f 1x10"^M v a l i n e gave an 11.3% r e d u c t i o n i n th e f i n a l r e a c t i o n v e l o c i t y , w h i l e l x l 0 _ 3 M v a l i n e and l x l O ~ 2 M v a l i n e r e d u c e d t h e v e l o c i t y 65% and 71.4% r e s p e c t i v e l y . 60. T o o A Fig. 21. Michaelis-Menten Plot of v vs [S] for the acetohydroxy acid synthetase of E. co l i LL5. (•): no additions; (Q) : 1x10"14M L-valine added; ( a ) : lxlO~ 3M L-valine added; (A) : lxlO"~2M L-valine added. Although addition of L-valine did decrease the fi n a l reaction velocity, this was not to the extent observed with E. c o l i K-12. With lxlO~ 2M valine, a reduction in velocity of 32.5% occurred. The K m f o r t h e E. c o l i K-12 enzyme was o n l y s l i g h t l y h i g h e r t h a n t h a t f o r t h e enzyme from E. c o l i L L 5 . These doub l e r e c i p r o c a l p l o t s o f 1/v v s . 1 / [ p y r u v a t e ] , i n t h e pr e s e n c e o f v a l i n e , i n d i c a t e d t h a t v a l i n e was a c o m p e t i t i v e i n h i b i t o r w i t h r e s p e c t t o s u b s t r a t e . However, t h i s was found t o be m i s l e a d i n g and due m a i n l y t o t h e i m p r e c i s e n a t u r e o f t h e L i n e w e a v e r - B u r k e p l o t s . O ther methods o f p l o t t i n g t h e d a t a i n d i c a t e d t h a t t h e k i n e t i c s o f i n h i b i t i o n were r a t h e r more c o m p l i c a t e d , and p o s s i b l y a m i x t u r e o f compet-i t i v e and n o n - c o m p e t i t i v e . To g i v e some i d e a o f t h e d i f f e r e n c e s i n K^ f o r AHAS from t h e two s t r a i n s , a comparison o f t h e v a l i n e c o n c e n t r a t i o n r e q u i r e d t o g i v e 46% i n h i b i t i o n w i t h 25 mM p y r u v a t e as subs-t r a t e was made (Table IV) . TABLE IV E. c o l i s t r a i n % I n h i b i t i o n [ V a l i n e ] R a t i o o f [ V a l i n e ] g i v i n g 46% i n h i b i t i o n LL5/K-12 K-12 46% 2.1 x 10" "M 47.6 LL5 46% 1 x 10~ 2M The AHAS enzyme from E. c o l i K-12 i s a l m o s t 50 tim e s more s e n s i t i v e t o v a l i n e t h a n t h e enzyme from E. c o l i L L 5 . 64. I I I . F o r m a t i o n o f a c e t o h y d r o x y a c i d s y n t h e t a s e i n £,. g_o_li_ s t r a i n s -The s p e c i f i c a c t i v i t y (S.A. = ymoles ac e t o i n / m g . p r o t e i n / h r . ) o f a c e t o h y d r o x y a c i d s y n t h e t a s e (AHAS) was d e t e r m i n e d f o r each s t r a i n o f b a c t e r i a , grown w i t h and w i t h o u t v a l i n e a d d i t i o n , and t h e r e s u l t s a r e summarized i n T a b l e V. TABLE V S p e c i f i c A c t i v i t y t o f AHAS E. c o l i S t r a i n Grown w i t h no a d d i t i o n s 1 x 1 0" 3M V a l i n e added K-12 5.3 1.65 B 3.67 3.59 AB1020 6.25 4.75 LL5 3.38 ! 3.11 t S p e c i f i c A c t i v i t y = ymoles acetoin/mg. p r o t e i n / h r . The l e v e l o f AHAS was found t o be h i g h e s t i n E. c o l i AB1020. An approximate 3 - f o l d r e p r e s s i o n o f AHAS i n E. c o l i K-12 was seen when t h e b a c t e r i a were grown w i t h 1x10" 3M v a l i n e added. S l i g h t r e p r e s s i o n i n t h e o t h e r t h r e e s t r a i n s was o b s e r v e d , b u t n o t o f the magnitude o b s e r v e d i n E. c o l i K-12. 65. IV. F o r m a t i o n o f t h r e o n i n e deaminase i n E. c o l i s t r a i n s The s p e c i f i c a c t i v i t y o f t h r e o n i n e deaminase (T.D.) was d e t e r m i n e d f o r E. c o l i s t r a i n s K-12, B, AB1020 and L L 5 , each grown w i t h and w i t h o u t added v a l i n e . The r e s u l t s are summarized i n T a b l e V I . TABLE V I i S p e c i f i c A c t i v i t y t o f T h r e o n i n e Deaminase E. c o l i . S t r a i n Grown w i t h no a d d i t i o n s 1x10" 3M V a l i n e added K-12 1.9 7.0 B 17.4 12 .0 AB1020 10.0 5.8 LL5 2.4 5.7 t S p e c i f i c A c t i v i t y = umoles a - k e t o b u t y r a t e / m g . p r o t e i n / h r . The low l e v e l o f T.D. i n E. c o l i K-12 and the h i g h l e v e l i n E. c o l i B have been p r e v i o u s l y r e p o r t e d , ( D e s a i and P o l g l a s e , 1966). E. c o l i LL5 was found t o have a T.D. a c t i v i t y o n l y s l i g h t l y h i g h e r t h a n t h a t f o r E. c o l i K-12, w h i l e E. c o l i AB1020 showed a 5 - f o l d i n c r e a s e i n T.D. a c t i v i t y from the l e v e l i n E. c o l i K-12, n o t however, r e a c h i n g the l e v e l o f T.D. i n E. c o l i B. Growth o f t h e s e s t r a i n s w i t h added v a l i n e showed t h a t t h e r e i s a d e r e p r e s s i o n o f T.D. i n E. c o l i s t r a i n s K-12 and LL5, and a r e p r e s s i o n o f T.D. i n E. c o l i s t r a i n s B and AB1020. DISCUSSION P r e v i o u s w o r k e r s (Umbarger and Brown 1955 and 1958; L e a v i t t and Umbarger 1962; and Temple, Umbarger and Magasanik 1965) have p r o p o s e d t h a t i n h i b i t i o n o f growth o f E. c o l i K-12 by L - v a l i n e i s c a u s e d by s t a r v a t i o n f o r i s o l e u c i n e due t o feedback i n h i b i t i o n by v a l i n e o f a c e t o h y d r o x y a c i d s y n t h e t a s e (AHAS) an enzyme common t o t h e b i o s y n t h e t i c pathways f o r b o t h o f t h e s e amino a c i d s . S u p p o r t f o r t h i s h y p o t h e s i s comes from th e o b s e r v a t i o n t h a t L - i s o l e u c i n e overcomes i n h i b i t i o n by L - v a l i n e . I m p l i c i t i n t h i s p r o p o s a l i s t h e p r e d i c t i o n t h a t r e s i s t a n c e t o v a l i n e w o u l d be accompanied by a l o s s o f feedback s e n s i t i v i t y t o t h i s amino a c i d . However, growth o f most s t r a i n s o f E. c o l i , o t h e r t h a n K-12, i s n o t i n h i b i t e d by v a l i n e even though v a l i n e b i o s y n t h e s i s i s under feedback c o n t r o l . F u r t h e r m o r e , even i n K-12, i n h i b i t i o n o f growth by v a l i n e i s i n c o m p l e t e s i n c e l i n e a r growth p e r s i s t s even a t h i g h c o n c e n t r a t i o n s o f v a l i n e . I t has been p r o p o s e d (Ramakrishnan and A d e l b e r g 1964, 1965) t h a t r e s i s t a n c e t o v a l i n e r e s u l t s from d e r e p r e s s i o n o f TD, o r o f AHAS, o r a l t e r n a t i v e l y , ( L e a v i t t and Umbarger, 1962; F r e u n d l i c h and Umbarger, 1965) i n s e n s i t i v i t y o f AHAS t o en d - p r o d u c t i n h i b i t i o n by v a l i n e . The f o r e g o i n g h y potheses are n o t adequate t o p r o v i d e a c o m p l e t e l y s a t i s f a c t o r y e x p l a n a t i o n f o r t h e l i n e a r growth o f E. c o l i K-12 and t h e e x p o n e n t i a l growth o f o t h e r s t r a i n s o f E. c o l i (e.g. B, AB1020, AB1005, LL5) i n m i n i m a l medium supplemented w i t h v e r y h i g h c o n c e n t r a t i o n s o f v a l i n e . The d a t a p r e s e n t e d h e r e from s t u d i e s o f t h e k i n e t i c s o f i n h i b i t i o n o f AHAS by v a l i n e i n t h e two w i l d - t y p e E. c o l i s t r a i n s K-12 and B, however, i n d i c a t e d t h a t a l t h o u g h t h e i n i t i a l r e s p o n s e o f t h i s enzyme t o v a l i n e was a p p r o x i m a t e l y t h e same i n b o t h s t r a i n s , t h e maximum i n h i b i t i o n o f AHAS a t t a i n a b l e i n E. c o l i K-12 was 84%, w h i l e i n E. c o l i B i t was o n l y 55%. T h i s s u g g e s t e d t h a t i n E. c o l i B, growing i n v a l i n e - s u p p l e m e n t e d medium, a p p r o x i m a t e l y 45% o f t h e AHAS a c t i v i t y remained p e r m i t t i n g adequate i s o l e u c i n e t o be s y n t h e s i z e d t o s u p p o r t a l e v e l o f p r o t e i n s y n t h e s i s consonant w i t h an e x p o n e n t i a l growth r a t e . I n E. c o l i K-12, however, where h i g h v a l i n e c o n c e n t r a t i o n s can d e c r e a s e t h e i l v a pathway c a p a b i l i t y t o a t most 16% o f i t s u n i n h i b i t e d l e v e l , e x p o n e n t i a l growth i s n o t p o s s i b l e because o f r e s t r i c t e d i s o l e u c i n e s y t h e s i s . How-e v e r , s i n c e t h e enzyme cannot be c o m p l e t e l y i n h i b i t e d , enough i s o l e u c i n e can be s y n t h e s i z e d t o a l l o w l i m i t e d ( l i n e a r ) growth. S i n c e b o t h t h e s e n s i t i v i t y o f E. c o l i K-12, and t h e r e s i s t a n c e o f E. c o l i B t o v a l i n e a re n a t u r a l c h a r a c t e r i s t i c s o f t h e w i l d - t y p e s t r a i n s , i t was o f i n t e r e s t t o examine t h e p r o p e r t i e s o f t h r e e v a l i n e - r e s i s t a n t mutants o f E. c o l i K-12, E. c o l i s t r a i n s AB10 20, AB1005 and LL 5 . E. c o l i LL5 showed d i s t i n c t i v e growth c h a r a c t e r i s t i c s i n t h a t v a l i n e c o n c e n t r a t i o n s up t o 1 x 10 _ l fM had no e f f e c t on l o g a r i t h m i c g r o w t h , w h i l e 1 x 10" 3M v a l i n e caused a s l i g h t d e c r e a s e i n t h e r a t e o f l o g a r i t h m i c g r o w t h , and 1 x 10~ 2M v a l i n e caused an even f u r t h e r d e c r e a s e such t h a t t h e growth r a t e was no l o n g e r e x p o n e n t i a l . 68. Analysis of the k i n e t i c s of valine i n h i b i t i o n of AHAS from t h i s s t r a i n showed that the enzyme was very i n s e n s i t i v e to valin e , with a maximum of approximately 20% i n h i b i t i o n of a c t i v i t y with 1.5 x 10"3M v a l i n e . Higher valine concentr-ations were found to i n h i b i t the enzyme to a much greater' extent. Thus, 1 x 10 - 2M gave 46% i n h i b i t i o n , and 5 x 10~2M valine gave 87% i n h i b i t i o n . The growth of the two E. c o l i s trains AB1020 and AB1005 was found to be unaffected by valin e concentrations as high as 1 x 10~2M. However, instead of a v a l i n e - i n s e n s i t i v e enzyme which might be expected, the s e n s i t i v i t y to i n h i b i t i o n of AHAS by valine was intermediate between E. c o l i s t r a i n s K-12 and B. Maximal i n h i b i t i o n of E. c o l i AB1020 was 77%, while that of E. c o l i AB1005 was 75%. Further analysis of the valine i n h i b i t i o n curves was c a r r i e d out by the method of Levitsky and Koshland (1969) employing the equation log (v/v i~l) = log 1/Kj^ + n log [Val] where v i s the v e l o c i t y i n the absence of the i n h i b i t o r , (valine) and Vj_ i s the v e l o c i t y i n the presence of v a l i n e . The value of n i s the H i l l c o e f f i c i e n t . A value of n approaching 0.5 was interpreted to indicate "negative cooperativity" between i n h i b i t o r s i t e s , while a value of n approaching 2.0 indicated "positive cooperativity" between binding s i t e s for i n h i b i t o r . A value of n=l would indicate no "cooperativity" between i n h i b i t o r binding s i t e s . The data for E. c o l i K-12 indicated three stages of binding of valine to AHAS. At very low valine concentrations, the H i l l c o e f f i c i e n t of n=0.84 suggested that binding of one molecule of valine would make i t more d i f f i c u l t for subsequent molecules to bind ("negative c o o p e r a t i v i t y " ) . At i n t e r -mediate valine concentrations, however, where n=1.32, binding of one valine molecule would be thought to f a c i l i t a t e the binding of subsequent valin e molecules to other i n h i b i t o r s i t e s on AHAS ("positive c o o p e r a t i v i t y " ) . Again at high valine concentrations, interactions between i n h i b i t o r binding s i t e s became negatively cooperative. The H i l l plots for E. c o l i s trains AB1020 and AB1005 were very s i m i l a r to that for E. c o l i K-12 with the exception that there was no evidence for binding of valine at low valine concentrations with the r e s u l t that there were only two slopes shown, rather than three, and these were s h i f t e d to higher concentrations of v a l i n e . The same pattern of p o s i t i v e cooperativity at intermediate valine concentrations, tending to negative cooperativity at high concentrations of i n h i b i t o r e xists here (AB1005, AB1020) as i t does i n E. c o l i K-12. The tendency to negative cooperativity ( i . e . decreasing enzyme-inhibitor a f f i n i t y ) with increasing valine concentrations i n the E. c o l i strains K-12, AB1005 and AB10 20 presents a p l a u s i b l e explanation for the mechanism of incomplete i n h i b -i t i o n of AHAS i n these s t r a i n s . The advantage to the organism of incomplete feed-back i n h i b i t i o n by valine i s that isoleucine biosynthesis i s never completely shut o f f as would be the 70. case i f t h e enzyme c o u l d be i n h i b i t e d 100% by v a l i n e . The H i l l p l o t s f o r E. c o l i s t r a i n s B and LL5 were b o t h d i f f e r e n t t o t h o s e f o r E. c o l i s t r a i n s K-12, AB1020 and AB1005 i n t h a t t h e y d i d not show p o s i t i v e c o o p e r a t i v i t y f o r v a l i n e b i n d i n g o v e r t h e range o f v a l i n e c o n c e n t r a t i o n s used. Both a t low and a t i n t e r m e d i a t e c o n c e n t r a t i o n s o f v a l i n e , t h e H i l l c o e f f i c i e n t f o r E. c o l i B was 0.44 i n d i c a t i n g neg-a t i v e c o o p e r a t i v i t y between t h e b i n d i n g s i t e s f o r v a l i n e . A l t h o u g h t h e H i l l c o e f f i c i e n t i n c r e a s e d a t h i g h v a l i n e con-c e n t r a t i o n s , t h e maximum was n=0.75. The H i l l P l o t f o r E. c o l i LL5 i s s i m i l a r t o t h a t o f E. c o l i B o v e r t h e range i n c l u d i n g low and i n t e r m e d i a t e v a l i n e c o n c e n t r a t i o n s i n t h a t n=0.39. However, E. c o l i LL5 v/as d i f f e r e n t from E. c o l i B because even a t h i g h v a l i n e c o n c e n t r a t i o n s (1.5 x 10" 3M) t h i s s l o p e p e r s i s t e d , and i t was n o t u n t i l t h e v a l i n e con-c e n t r a t i o n was r a i s e d t o 1 x 10" 2M v a l i n e t h a t n i n c r e a s e d t o 0.87. T h i s n e g a t i v e c o o p e r a t i v i t y t h a t was c a r r i e d t h r o u g h o u t t h e range o f v a l i n e c o n c e n t r a t i o n s , n o t o n l y can e x p l a i n t h e l a c k o f complete i n h i b i t i o n o f AHAS by v a l i n e as. i n E. c o l i s t r a i n s K-12, AB10 20 and AB1005, b u t a l s o a i d s i n t h e u n d e r s t a n d i n g o f t h e re d u c e d s e n s i t i v i t y t o v a l i n e o f t h e AHAS from t h e s e two s t r a i n s , E. c o l i B and L L 5 . The o b s e r v a t i o n o f a d e c r e a s e d s e n s i t i v i t y o f AHAS t o low v a l i n e c o n c e n t r a t i o n s d i d n o t seem s u f f i c i e n t t o e x p l a i n t h e a b i l i t y o f s t r a i n s AB10 20 and AB1005 t o grow e x p o n e n t i a l l y i n m i n i m a l medium c o n t a i n i n g h i g h c o n c e n t r a t i o n s o f v a l i n e w h i l e growth o f s t r a i n LL5 was d e c r e a s e d by h i g h v a l i n e c o n c e n t r a t i o n s even though t h e AHAS o f t h i s s t r a i n was r e l a t i v e l y r e s i s t a n t t o v a l i n e . I t seemed c l e a r t h a t more t h a n t h e one f a c t o r o f end-p r o d u c t i n h i b i t i o n must be i n v o l v e d i n t h e change from v a l i n e s e n s i t i v i t y t o r e s i s t a n c e . P r e v i o u s w o r k e r s , Ramakrishnan and A d e l b e r g , 1964, 1965; L e a v i t t and Umbarger 1962; F r e u n d l i c h and Umbarger 1965, have s u g g e s t e d t h a t t h e l e v e l s o f AHAS and TD i n t h e b a c t e r i a l s t r a i n s p l a y a r o l e i n v a l i n e s e n s i t i v i t y . I f TD were t h e key enzyme i n d e t e r m i n i n g v a l i n e s e n s i t i v i t y , t h e n s u p p l y i n g t h e p r o d u c t o f TD a c t i v i t y ( a - k e t o b u t y r a t e ) s h o u l d p r o v i d e v a l i n e r e s i s t -ance i n E. c o l i K-12. However, Umbarger and Brown (19 55) found t h a t t h e r e was no d e c r e a s e i n s e n s i t i v i t y on t h e a d d i t i o n o f a - k e t o b u t y r a t e t o v a l i n e i n h i b i t e d c u l t u r e s o f K-12. S i m i l a r l y i n t h e p r e s e n t work i t was o b s e r v e d t h a t t h e s e n s -i t i v i t y o f s t r a i n LL5 t o h i g h v a l i n e c o n c e n t r a t i o n s was n o t d e c r e a s e d by t h e a d d i t i o n o f a - k e t o b u t y r a t e , w i t h o r w i t h o u t added p y r u v a t e . L i k e w i s e , t h r e o n i n e w i t h o r w i t h o u t added p y r u v a t e f a i l e d t o overcome v a l i n e i n h i b i t i o n o f s t r a i n L L 5 . However, i t was a l s o o b s e r v e d t h a t E. c o l i s t r a i n s K-12 and LL5 c o u l d n o t grow w i t h a - k e t o b u t y r a t e as s o l e c a r b o n s o u r c e , even though t h e y would grow e x p o n e n t i a l l y on p y r u v a t e , i t s l o w e r homologue. I t would t h e r e f o r e seem p o s s i b l e t h a t e i t h e r a - k e t o b u t y r a t e does n o t p e n e t r a t e t h e c e l l , o r i t cannot be m e t a b o l i z e d i f i t does c r o s s t h e membrane. 72. When t h e l e v e l s o f AHAS and TD were d e t e r m i n e d , from c e l l s grown w i t h o u t added v a l i n e , i t was n o t e d t h a t E. c o l i AB1020 had t h e h i g h e s t l e v e l o f AHAS f o l l o w e d n e x t by E. c o l i K-12, and t h e n by E. c o l i s t r a i n s LL5 and B. Wi t h r e s p e c t t o TD, E. c o l i B showed v e r y h i g h l e v e l s , r e f e r r e d t o as " d e r e p r e s s e d " l e v e l s by Ramakrishnan and A d e l b e r g (1965) . S t r a i n AB1020 a l s o showed a d e r e p r e s s e d l e v e l o f TD. Both E. c o l i s t r a i n s K-12 and LL5 showed v e r y low, " r e p r e s s e d 1 1 l e v e l s o f TD. When t h e b a c t e r i a l s t r a i n s were grown on m i n i m a l s a l t s -g l u c o s e medium w i t h 1 x 10" 3M L - v a l i n e added, an i n t e r e s t i n g p a t t e r n o f r e p r e s s i o n and d e r e p r e s s i o n was o b s e r v e d . E. c o l i K-12 showed a marked r e p r e s s i o n o f AHAS, b u t an e q u a l l y s i g n i f i c a n t d e r e p r e s s i o n o f TD. The o t h e r E. c o l i s t r a i n s B, AB1020 and LL5 showed a v e r y s l i g h t t o moderate r e p r e s s i o n o f AHAS, and two s t r a i n s B, and AB1020 showed a r e p r e s s i o n o f TD from t h e o r i g i n a l , h i g h l e v e l s . Growth o f s t r a i n LL5 w i t h added v a l i n e d e r e p r e s s e d TD. These r e s u l t s , t o g e t h e r w i t h t h o s e d i s c u s s e d e a r l i e r ( feed-back i n h i b i t i o n k i n e t i c s ) p r o v i d e a p o s s i b l e e x p l a n a t i o n f o r t h e s e n s i t i v i t y t o v a l i n e o f E. c o l i K-12, and t h e r e s i s t a n c e o f E. c o l i s t r a i n s B, AB1020 and L L 5 . The r e p r e s s i o n o f AHAS by v a l i n e i n E. c o l i K-12 c o u p l e d w i t h t h e v e r y h i g h maximal i n h i b i t i o n (84%) o f AHAS by v a l i n e w o uld make i t much more d i f f i c u l t f o r K-12 t o s y n t h e s i z e adequate i s o l e u c i n e t o m a i n t a i n p r o t e i n s y n t h e s i s a t a l e v e l 73. which would allow logarithmic growth. The derepression of TD i n response to the lack of isoleucine would tend to compensate for t h i s by increasing the l e v e l of a-ketobutyrate to act as substrate for AHAS. However, t h i s excess substrate could not be used since AHAS i s both repressed and i n h i b i t e d by va l i n e . In E . c o l i B, although the l e v e l of AHAS was not extremely high, there was no s i g n i f i c a n t repression of thi s l e v e l by growth with 1 x 10"3M valine and coupled with the fact that maximal i n h i b i t i o n by valine of the enzyme was only 55%, t h i s s t r a i n probably retained as much or almost as much of i t s isoleucine synthesizing a b i l i t y i n the presence of added valine as i n i t s absence. E . c o l i AB1020, showing a maximum of 77% i n h i b i t i o n of AHAS by v a l i n e , had the highest levels of AHAS a c t i v i t y , and although there v/as a s l i g h t repression r e s u l t i n g from growth on 1 x 10" 3M val i n e , the repressed l e v e l v/as not much lower than the l e v e l of AHAS i n E . c o l i K-12 grown with no additions. Thus i t seems l i k e l y that the higher AHAS level s would compen-sate for the r e l a t i v e l y high maximal i n h i b i t i o n by va l i n e , and the s t r a i n would therefore r e t a i n a good part of i t s isoleucine synthesizing a b i l i t y even when grown with high concentrations of v a l i n e . In both E . c o l i s trains B and AB1020, the derepressed leve l s of TD would also contribute to the valine resistance by maintaining a high concentration of a-ketobutyrate as subtrate for AHAS i n the isoleucine pathway. The s l i g h t 74. repression of TD by val i n e , although not anticipated, would not be expected to starve the c e l l s of these str a i n s (B, AB1020) for i s o l e u c i n e . The reason for the s l i g h t s e n s i t i v i t y to 1 x 10~ 3M valine of E. c o l i LL5 i s also evident from these findings. Even though the AHAS enzyme of t h i s s t r a i n was i n s e n s i t i v e to moderate concentrations of valine , the l e v e l of AHAS was not e x t r a o r d i n a r i l y high, and the TD l e v e l was very low. On the addition of 1 x 10" 3M valine to a growing culture of LL5, the l e v e l of AHAS did not change s i g n i f i c a n t l y , but TD was derepressed. Thus the low i n i t i a l l e v e l of TD was probably inadequate to provide the amount of a-ketobutyrate needed as substrate for AHAS when thi s enzyme was subject to i n h i b i t i o n by v a l i n e . However, once t h i s deficiency was corrected, adequate a-ketobutyrate would have been supplied to increase isoleucine synthesis and thus overcome growth i n h i b i t i o n . Thus i t would seem that a combination of factors deter-mine whether a b a c t e r i a l s t r a i n i s r e s i s t a n t or sensi t i v e to valine: 1) TD l e v e l i n the presence of added v a l i n e . 2) The s e n s i t i v i t y of AHAS to i n h i b i t i o n by va l i n e : a) Extent of maximal i n h i b i t i o n b) Cooperativity of valine binding (positive or negative) 3) R e p r e s s i b i l i t y of AHAS by va l i n e . These r e s u l t s seem t o i n d i c a t e t h a t the c o m b i n a t i o n o f a n o n - r e p r e s s e d l e v e l o f TD, p l u s e i t h e r one o f t h e o t h e r two f a c t o r s f a v o r i n g r e s i s t a n c e w i l l r e n d e r growth o f a b a c t e r i a l s t r a i n r e s i s t a n t t o v a l i n e , whether o r n o t TD d e r e p r e s s i o n alon<^ i s adequate t o c o n f e r r e s i s t a n c e t o v a l i n e cannot be c o m p l e t e l y d e t e r m i n e d from t h i s s t u d y because t h e r e s i s t a n t b a c t e r i a l s t r a i n s used a l l showed two f a c t o r s f a v o r i n g r e s i s t a n c e . However, s i n c e TD l e v e l s o f E. c o l i K-12 are d e r e p r e s s e d on t h e a d d i t i o n o f v a l i n e , and t h e i n h i b i t i o n o f growth i s s t i l l n o t overcome, i t would appear t h a t h i g h TD l e v e l s are n o t s u f f i c i e n t a l o n e t o overcome i n h i b i t i o n . I t s h o u l d be n o t e d however, t h a t one i n s t a n c e o f an i n c r e a s e i n t h e r a t e o f growth was o b s e r v e d when E. c o l i K-12 was b e i n g grown w i t h 1 x 10" 3M v a l i n e . TD l e v e l i n t h i s case was found t o be v e r y h i g h , i . e . s p e c i f i c a c t i v i t y = 19 umoles a - k e t o b -u t y r a t e / m g p r o t e i n / h r . F u r t h e r i n v e s t i g a t i o n i n t o t h i s q u e s t i o n would have t o be c a r r i e d o u t t o d e t e r m i n e t h e s i g n -i f i c a n c e o f t h i s e v e n t . S i m i l a r l y , a s t u d y o f enzyme l e v e l s i n s t r a i n LL5 grown w i t h 1 x 10~ 2M v a l i n e added would a l s o g i v e i n f o r m a t i o n on t h i s p r o b l e m . I t seems p o s s i b l e t h a t i f t h e s t r a i n s were grown w i t h 1 x 10~ 2M v a l i n e f o r a l o n g p e r i o d o f t i m e , TD m ight be s u f f i c i e n t l y d e r e p r e s s e d t o o v e r -come t h e i n h i b i t i o n o f growth by v a l i n e . I n f a c t , Temple e t a l (1965) found t h a t l i n e a r growth o f E. c o l i K-12 w i t h added v a l i n e was accompanied by e x t e n s i v e d e r e p r e s s i o n o f TD, and t h a t l i n e a r growth was n o t a permanent c o n d i t i o n b u t c o u l d 76. change t o l o g a r i t h m i c growth. They d i d n o t c o r r e l a t e t h e d e r e p r e s s i o n o f TD and t h e r e l i e f o f growth i n h i b i t i o n , b u t i t seems r e a s o n a b l e t h a t t h i s c o r r e l a t i o n c o u l d be made. The c o m b i n a t i o n o f f a c t o r s d e t e r m i n i n g s e n s i t i v i t y o r r e s i s t a n c e t o v a l i n e f o r each o f t h e f o u r E. c o l i s t r a i n s s t u d i e d i s shown i n T a b l e V I I . TABLE V I I C h a r a c t e r i s t i c s o f E. c o l i S t r a i n s E. c o l i S t r a i n Growth w i t h V a l i n e Maximum* % I n h i b i t i o n AHAS L e v e l w i t h o u t v a l i n e o f TD | w i t h v a l i n e | L e v e l o f AHAS on v a l i n e a d d i t i o n K-12 s e n s i t i v e 84 r e p r e s s e d d e r e p r e s s e d r e p r e s s e d B r e s i s t a n t 55 d e r e p r e s s e d d e r e p r e s s e d no change AB1020 r e s i s t a n t 77 d e r e p r e s s e d d e r e p r e s s e d s l i g h t r e p r e s s i o n LL5 r e s i s t a n t 20 r e p r e s s e d d e r e p r e s s e d no change t o low [ v a l i n e ] *Maximum % i n h i b i t i o n i s g i v e n as a measure o f AHAS s e n s i t i v i t y t o v a l i n e , and r e f e r s t o i n h i b i t i o n by 1.5 x 10" 3M v a l i n e . The i n s e n s i t i v i t y o f AHAS from E. c o l i LL5 was s t u d i e d by comparing t h e M i c h a e l i s - M e n t e n k i n e t i c p l o t s o f v e l o c i t y vs [ v a l i n e ] . The d i f f e r e n c e i n s e n s i t i v i t y t o v a l i n e o f LL5 and K-12 was c l e a r l y shown by t h e d i f f e r e n c e i n maximal v e l o c i t i e s a t d i f f e r e n t s p e c i f i e d v a l i n e c o n c e n t r a t i o n s ( R e s u l t s , S e c t i o n C ) . Thus, 1 x 10~ 2M v a l i n e r e d u c e d t h e maximal r e a c t i o n v e l o c i t y o f AHAS 71.4% i n E. c o l i K-12, b u t o n l y 32.5% i n E . c o l i L L 5 . The v a l u e s o f K m were o f t h e same o r d e r o f magnitude f o r t h e two s t r a i n s , and a l t h o u g h t h e L i n e w e a r - B u r k e double r e c i p r o c a l p l o t s i n d i c a t e d t h a t t h e v a l u e s o f Kj_ would be v e r y d i f f e r e n t , t h e i m p r e c i s e n a t u r e o f t h e s e p l o t s made a c c u r a t e c a l c u l a t i o n s o f t h e Kj_ i m p o s s i b l e . An i n d i c a t i o n o f t h e v a r i a n c e c a n , however, be o b t a i n e d by a c omparison o f t h e v a l i n e c o n c e n t r a t i o n r e q u i r e d t o g i v e 46% i n h i b i t i o n o f AHAS. F o r E . c o l i K-12 t h i s c o n c e n t r a t i o n was c a l c u l a t e d t o be 2.1 x 10 _ 1 * M v a l i n e , w h i l e f o r E . c o l i LL5 i t was o b s e r v e d t o be 1 x 1 0 ~ 2 M v a l i n e . The K-12 enzyme i s t h u s 48 f o l d more s e n s i t i v e t o v a l i n e t h a n t h a t o f LL5 i n t h i s c a s e . Thus i t would seem t h a t t h e d i f f e r e n c e i n K^'s would p r o b a b l y be q u i t e s i g n i f i c a n t . 7 8 . SUGGESTIONS FOR FURTHER WORK 1. I n v e s t i g a t i o n o f t h e l e v e l s o f AHAS and TD i n E. c o l i LL5 grown w i t h 1 x 10 - 2M v a l i n e added w o u l d , i t i s hoped, g i v e an e x p l a n a t i o n o f why i n h i b i t i o n by v a l i n e i n t h i s case causes l i n e a r g rowth, s i n c e w i t h 1 x 10" 3M v a l i n e t h e r e was o n l y a s h o r t p e r i o d o f d e c r e a s e d growth r a t e f o l l o w e d by l o g a r i t h m i c growth. I t would be e x p e c t e d , i n v i e w o f t h e p r e s e n t r e s u l t s , t h a t r e l i e f o f growth i n h i b i t i o n c aused by 1 x 10 - 2M v a l i n e would be a c c o m p l i s h e d by e x t e n s i v e d e r e p r -e s s i o n o f TD. T h i s c o u l d be t e s t e d i f t h e b a c t e r i a c o u l d be grown i n a medium wh i c h w o u l d cause d e r e p r e s s i o n o f TD ( f o r example growth on l o w e r v a l i n e c o n c e n t r a t i o n s 1 x 10" 5M). I f t h e TD l e v e l was h i g h i n i t i a l l y , one might e x p e c t e i t h e r a s h o r t e r l i n e a r growth p e r i o d , o r a t r a n s i e n t r e s i s t a n c e t o v a l i n e as seen by Umbarger & F r e u n d l i c h (1965). 2. I f E. c o l i K-12 were grown w i t h v a l i n e f o r e x t e n d e d p e r i o d s o f t i m e , one m i g h t a g a i n see t h e r e l i e f o f l i n e a r growth t h a t was o b s e r v e d i n one e x p e r i m e n t . E x a m i n a t i o n o f enzyme l e v e l s i n t h i s e v e n t would be v e r y i n f o r m a t i v e . I t w o u l d be e x p e c t e d t h a t r e l i e f from i n h i b i t i o n by v a l i n e would be accompanied by a marked d e r e p r e s s i o n o f TD as r e p o r t e d by Temple e t a l . (1965). A t r a n s i e n t r e s i s t a n c e t o v a l i n e m i g h t be demonstrated i f t h e c e l l s were f i r s t grown i n a medium c o n t a i n i n g 1 x 10" 6M v a l i n e so t h a t t h e TD l e v e l was i n i t i a l l y h i g h e r t h a n u s u a l . 79. 3. I t would be o f i n t e r e s t t o d e t e r m i n e i f t h e pH vs enzyme a c t i v i t y c u r v e f o r t h e AHAS from E. c o l i LL5 were t h e same as t h a t f o r t h e AHAS from E. c o l i K-12. S i n c e growth i s c a r r i e d o ut i n medium a t pH.= 7.2 (which becomes more a c i d i c as t h e c e l l s grow), and t h e enzyme i s a s s a y e d a t pH 8, i t i s p o s s i b l e t h a t an amino a c i d s u b s t i t u t i o n n e a r a s i t e o f i n h i b i t o r b i n d i n g has changed t h e optimum pH f o r b i n d i n g v a l i n e . Thus a l t h o u g h a t pH 8, 1 x 10 - 3M v a l i n e i n h i b i t s AHAS o n l y 20%, a t pH 7.2 o r l o w e r i t i s p o s s i b l e t h a t v a l i n e causes a much more s i g n i f i c a n t i n h i b i t i o n o f AHAS and t h i s , c o u p l e d w i t h t h e low l e v e l o f TD i n t h e s t r a i n , a c c o u n t s f o r t h e i n h i b i t i o n o f e x p o n e n t i a l growth o b s e r v e d w i t h 1 x 10 - 3M v a l i n e . 4. To d e t e r m i n e c o n c l u s i v e l y whether t h e c e l l s a r e permeable t o a - k e t o b u t y r a t e and i f t h e y a r e , t o see whether t h i s a - k e t o b u t y r a t e i s m e t a b o l i z e d , i t would be n e c e s s a r y t o do r a d i o t r a c e r e x p e r i m e n t s w i t h C11* a - k e t o b u t y r a t e . 5. V a r i o u s r e p o r t s (e.g. F r e u n d l i c h & C l a r k e (1968)) have i n d i c a t e d t h a t a - k e t o b u t y r a t e and a-amino b u t y r a t e a r e them-s e l v e s i n h i b i t o r y t o growth o f some s t r a i n s o f b a c t e r i a , and t h a t t h i s i n h i b i t i o n i s r e v e r s e d by v a l i n e . I n v e s t i g a t i o n o f t h i s f i n d i n g w i t h r e s p e c t t o t h e s t r a i n s used i n t h i s work c o u l d add more i n f o r m a t i o n on t h e mechanism o f c o n t r o l o f t h e " i l v a " pathway. H o r v a t h e t a l . (1961, 1964) r e p o r t e d t h a t growth on a - k e t o b u t y r a t e d e r e p r e s s e d t h e l e v e l o f AHAS. 8 0 . T h i s c o u l d p o s s i b l y be used as a mechanism f o r i n c r e a s i n g t h e i n i t i a l amount o f AHAS i n c e l l s t o be grown w i t h v a l i n e , t h u s a i d i n g i n t h e u n d e r s t a n d i n g : o f the f u l l c o n t r i b u t i o n o f t h e l e v e l o f AHAS t o v a l i n e r e s i s t a n c e . 81. BIBLIOGRAPHY B a u e r l e , R . H . , F r s u n d l i c h , M., S t e r m e r , F.C. and Umbarger, H.E. (1964) B i o c h i m . B i o p h y s . A c t a 92: 142-149 Bonner, D. (1946) J . B i o l . Chem. 166: 545 B r a g g , P.D. and P o l g l a s e , W.J. (1962) J . B a c t e r i o l . 84: 370 B r a g g , P.D. and P o l g l a s e , W.J. (1965) J . B a c t e r i o l . 89: 1158-1159 Cohen, G.N. (1958) Ann. I n s t . P a s t e u r 94: 15-30 C o u k e l l , M.B.. and P o l g l a s e , W.J. (1965) Canadian J . 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