Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
DSpace
UBCV
Coukell, Milton Barrie
Doctor of Philosophy - PhD
University of British Columbia
Biosynthetic reactions in micro-organisms normally are under precise control, usually by a feed-back mechanism in which the end product inhibits the activity and/or represses the formation of the enzyme which initiates the biosynthetic pathway. Streptomycin (Sm)-dependent mutants of Escherichia coli, unlike the parent wild-type strains excrete the amino acid, L-valine, thereby indicating a loss of precise regulation in the biosynthesis of this amino acid (Tirunarayanan, Vischer and Renner, 1962; Bragg and Polglase, 1962). The initiating enzyme for valine biosynthesis, acetohydroxy acid (AHA) synthetase, is derepressed in Sm-dependent E. coli (Coukell and Polglase, 1965), thus providing an explanation for the excretion of valine by this mutant. However, it was apparent that the extent and cause of regulatory insufficiency in Sm-dependent E. coli required further investigation. A study of the effect of the carbon source used for growth on AHA synthetase formation revealed that in wild-type E. coli B this enzyme was subject to catabolite repression. End-product inhibition of AHA synthetase by L-valine in E. coli B attained a maximum at 60-70% inhibition. These previously unreported properties of AHA synthetase (sensitivity to catabolite repression and incomplete end-product inhibition) are significant in the regulation of the biosynthetic pathway leading to the aliphatic amino acids and pantothenate. In Sm-dependent E. coli B, growing with non-limiting antibiotic, catabolite repression of AHA synthetase was relaxed. Additional evidence for relaxation of catabolite repression in Sm-dependent E. coli was provided by the observation that Sm-dependent mutants of E. coli (strains B and E) were inducible for β-galactosidase in the presence of glucose Furthermore, several glucose-sensitive enzymes of wild-type E. coli B (citrate synthase, fumarase, aconitase and iso-citrate dehydrogenase) were found to be insensitive to variation in the nature of the carbon source in a Sm-dependent mutant. Cell yield experiments revealed that aerobic glucose metabolism in the Sm-dependent mutant was one-third less efficient than in the Sm-sensitive strain, although the two strains were equally efficient under anaerobic conditions. Moreover, the rate of ATP synthesis in the Sm-dependent mutant was less than that of the wild-type parent organism. Therefore, relaxation of catabolite repression in Sm-dependent E. coli B appears to result from an impairment of aerobic energy metabolism in this mutant. Catabolite repression in Sm-dependent E. coli B under conditions of antibiotic-limitation was investigated. The growth rate of Sm-dependent E. coli B on limiting concentrations of dihydrostreptomycin (DHSm) was evaluated by means of a constant (K DHSm ) relating half-maximal growth rate to antibiotic concentration. K DHSm varied with the nature of the carbon source being highest with energy-rich compounds (e.g. gluconate) and lowest with energy-poor compounds (e.g. lactate). Glucose-sensitive enzymes of Sm- dependent E. coli B were specifically repressed by antibiotic-limitation and exhibited specific activities lower than those observed for the same enzymes in glucose-grown extracts of wild-type E. coli B. Parallelism was observed between decreasing antibiotic concentration, decreasing growth rate, and increasing catabolite repression of certain glucose-sensitive enzymes (notably AHA synthetase and fumarase). The decreased efficiency of aerobic glucose metabolism of Sm-dependent E. coli B was not affected by variation in the concentration of antibiotic. Thus, it is improbable that carbohydrate metabolism is the antibiotic dependent site in the Sm-dependent mutant. The results are compatible with the hypothesis of Spotts and Stanier (1961) that the primary site of action of DHSm in the Sm-dependent organism is the ribosome (i.e . , protein synthesis). However, the growth-limiting effect of antibiotic deprival appears to be augmented by catabolite repression. Additionally, the Sm-dependent mutant is deficient in energy metabolism which can explain the relaxation of control by catabolite repression when antibiotic is present in non-limiting concentration.
CATABOLITE REPRESSION IN STREPTOMYCIN-DEPENDENT ESCHERICHIA COLI by MILTON BARRIE COUKELL B . S c , 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 , 1963 M.Sc., U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e Department of BIOCHEMISTRY We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d T/flE UNIVERSITY OF A p r i l , BRITISH COLUMBIA 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C olumbia, I ag r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and Study. I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department of // C L ^ O e y - C ^ K ^ K ^ L - ^ X / S-y The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date i ABSTRACT B i o s y n t h e t i c r e a c t i o n s i n m i c r o - o r g a n i s m s n o r m a l l y a re under p r e c i s e c o n t r o l , u s u a l l y by a feed-back mechanism i n whi c h t h e end p r o d u c t i n h i b i t s t he a c t i v i t y and/or r e p r e s s e s the f o r m a t i o n o f t h e enzyme which i n i t i a t e s t he b i o s y n t h e t i c pathway. S t r e p t o m y c i n (Sm)-dependent mutants o f Escherichia c o l i , u n l i k e t h e p a r e n t w i l d - t y p e s t r a i n s e x c r e t e the amino a c i d , L - v a l i n e , t h e r e b y i n d i c a t i n g a l o s s o f p r e c i s e r e g u -l a t i o n i n t h e b i o s y n t h e s i s o f t h i s amino a c i d ( T i r u n a r a y a n a n , V i s c h e r and Renner, 1962; Bragg and P o l g l a s e , 1962). The i n i t i a t i n g enzyme f o r v a l i n e b i o s y n t h e s i s , a c e t o h y d r o x y a c i d (AHA) s y n t h e t a s e , i s d e r e p r e s s e d i n Sm-dependent E. c o l i ( C o u k e l l and P o l g l a s e , 1965), thu s p r o v i d i n g an e x p l a n a t i o n f o r t h e e x c r e t i o n of v a l i n e by t h i s mutant. However, i t was app a r e n t t h a t the e x t e n t and cause o f r e g u l a t o r y i n s u f f i c i e n c y i n Sm-dependent E. c o l i r e q u i r e d f u r t h e r i n v e s t i g a t i o n . A s t u d y o f the e f f e c t o f t h e carbon s o u r c e used f o r growth on AHA s y n t h e t a s e f o r m a t i o n r e v e a l e d t h a t i n w i l d - t y p e E. c o l i B t h i s enzyme 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 . End-p r o d u c t i n h i b i t i o n of AHA s y n t h e t a s e by L - v a l i n e i n E. c o l i B a t t a i n e d a maximum a t 60-70% i n h i b i t i o n . These p r e v i o u s l y u n r e p o r t e d p r o p e r t i e s o f AHA s y n t h e t a s e ( s e n s i t i v i t y t o c a t a -b o l i t e r e p r e s s i o n and i n c o m p l e t e end-product i n h i b i t i o n ) a r e s i g n i f i c a n t i n t h e r e g u l a t i o n o f t h e b i o s y n t h e t i c pathway l e a d -i n g t o the a l i p h a t i c amino a c i d s and p a n t o t h e n a t e . I n Sm-dependent E. c o l i B, growing w i t h n o n - l i m i t i n g a n t i b i o t i c , c a t a b o l i t e r e p r e s s i o n o f AHA s y n t h e t a s e was r e l a x e d ! A d d i t i o n a l e v i d e n c e f o r r e l a x a t i o n o f c a t a b o l i t e r e p r e s s i o n i n Sm-dependent E. coli was p r o v i d e d by the o b s e r -v a t i o n t h a t Sm-dependent mutants o f E. coli ( s t r a i n s B and E) were i n d u c i b l e f o r 8 - g a l a c t o s i d a s e i n t h e p r e s e n c e o f g l u c o s e F u r t h e r m o r e , s e v e r a l g l u c o s e - s e n s i t i v e enzymes o f w i l d - t y p e E. coli B ( c i t r a t e s y n t h a s e , f u m a r a s e , a c o n i t a s e and i s o -c i t r a t e dehydrogenase) were found t o be i n s e n s i t i v e t o v a r i a -t i o n i n the n a t u r e o f t h e c a r b o n s o u r c e i n a Sm-dependent m u t a n t . C e l l y i e l d e x p e r i m e n t s r e v e a l e d t h a t a e r o b i c g l u c o s e m e t a b o l i s m i n t h e Sm-dependent mutant was o n e - t h i r d l e s s e f f i c i e n t t h a n i n t h e S m - s e n s i t i v e s t r a i n , a l t h o u g h t h e two r s t r a i n s were e q u a l l y e f f i c i e n t under a n a e r o b i c c o n d i t i o n s . M o r e o v e r , the r a t e o f ATP s y n t h e s i s i n the Sm-dependent mut-a n t was l e s s t h a n t h a t o f the w i l d - t y p e p a r e n t o r g a n i s m . T h e r e f o r e , r e l a x a t i o n o f c a t a b o l i t e r e p r e s s i o n i n Sm-depen-d e n t E. coli & a p p e a r s t o r e s u l t from an i m p a i r m e n t o f a e r o b i c energy m e t a b o l i s m i n t h i s m u t a n t . C a t a b o l i t e r e p r e s s i o n i n Sm-dependent E.' coli B under c o n d i t i o n s o f a n t i b i o t i c - l i m i t a t i o n was i n v e s t i g a t e d . The growth r a t e o f Sm-dependent E. coli B on l i m i t i n g c o n c e n -t r a t i o n s o f d i h y d r o s t r e p t o m y c i n (DHSm) was e v a l u a t e d by means o f a c o n s t a n t ( K ^ . , ) r e l a t i n g h a l f - m a x i m a l growth r a t e t o a n t i b i o t i c c o n c e n t r a t i o n . K „ v a r i e d w i t h the. DHSm n a t u r e o f t h e c a r b o n s o u r c e b e i n g h i g h e s t w i t h e n e r g y - r i c h compounds ( e . g . g l u c o n a t e ) and l o w e s t w i t h e n e r g y - p o o r compounds ( e . g . l a c t a t e ) . G l u c o s e - s e n s i t i v e enzymes o f Sm-dependent E. c o l i B were s p e c i f i c a l l y r e p r e s s e d by a n t i b i o t i c -l i m i t a t i o n and e x h i b i t e d s p e c i f i c a c t i v i t i e s l o wer t h a n t h o s e o b s e r v e d f o r t h e same enzymes i n glucose-grown e x t r a c t s o f w i l d - t y p e E. c o l i B. P a r a l l e l i s m was obser v e d between de-c r e a s i n g a n t i b i o t i c c o n c e n t r a t i o n , d e c r e a s i n g growth r a t e , and i n c r e a s i n g c a t a b o l i t e r e p r e s s i o n of c e r t a i n g l u c o s e -s e n s i t i v e enzymes ( n o t a b l y AHA s y n t h e t a s e and f u m a r a s e ) . The d e c r e a s e d e f f i c i e n c y o f a e r o b i c g l u c o s e m e t a b o l i s m o f Sm-dependent E. ooli B was not a f f e c t e d by v a r i a t i o n i n t h e c o n c e n t r a t i o n o f a n t i b i o t i c . Thus, i t i s im p r o b a b l e t h a t c a r b o h y d r a t e m e t a b o l i s m i s the a n t i b i o t i c dependent s i t e i n the Sm-depehdent mutant. The r e s u l t s a re c o m p a t i b l e w i t h t h e h y p o t h e s i s o f S p o t t s and S t a n i e r (1961) t h a t the p r i m a r y s i t e of a c t i o n o f DHSm i n t h e Sm-dependent o r g a n i s m i s the ribosome• ( i . e . , p r o t e i n s y n t h e s i s ) . However, t h e growth-l i m i t i n g e f f e c t of a n t i b i o t i c d e p r i v a l appears t o be aug-mented by c a t a b o l i t e r e p r e s s i o n . A d d i t i o n a l l y , the Sm-dependent mutant i s d e f i c i e n t i n energy m e t a b o l i s m w h i c h can e x p l a i n the r e l a x a t i o n o f c o n t r o l by c a t a b o l i t e r e p r e s s i o n when a n t i b i o t i c i s p r e s e n t i n n o n - l i m i t i n g c o n c e n t r a t i o n . i v TABLE 0F; CONTENTS Page No. INTRODUCTION 1 I. Genetics of Streptomycin Mutants. . .. 1 I I . Proposed Sites of Streptomycin Action............. 2 I I I . E f f e c t of Streptomycin on Pyruvate Metabolism 7 IV. Biosynthesis of A l i p h a t i c Amino Acids and Regu-latory Mechanisms 9 V. E f f e c t of Streptomycin on Enzyme Formation... I 4 VI. Object of this Investigation 16 METHODS AND MATERIALS . •. . 18 I. Organisms 18 (i) Wild-type cultures •••• 18 ( i i ) Streptomycin-dependent mutants . 18 I I . Growth of Cultures and Preparation of Extracts.... 19 (i) Media 19 ( i i ) Estimation of growth and c e l l mass 20 ( i i i ) Procedures for growing and harvesting cultures . 21 (a) Streptomycin-sensitive and supplemented streptomycin-dependent c e l l s 21 (b) A n t i b i o t i c - l i m i t e d , streptomycin-dependent c e l l s . . 21 (c) Antibiotic-depleted, streptomycin-dependent c e l l s ; 22 (iv) Preparation of crude c e l l extracts and permeabilized c e l l s 23 (a) Sonic treatment 2 3 (b) Disruption i n a French pressure c e l l . . . 23 (c) Disruption by grinding with alumina.... 24 (d) Toluene treatment 24 II I . B-Galactosidase Induction Experiments 25 (i) Diauxie growth. 25 ( i i ) Determination of.the d i f f e r e n t i a l rate of 8-galactosidase synthesis. .. . 25 ( i i i ) 8-Galactosidase induction, i n pseudo-resting c e l l s i . . 26 V Page No. IV. C e l l Y i e l d E x p e r i m e n t s . 27 (1) D e f i n i t i o n . . . . . . 27 ( i i ) A e r o b i c c e l l y i e l d from g l u c o s e . 27 ( i i i ) A n a e r o b i c c e l l y i e l d from g l u c o s e , 28 ( i v ) A e r o b i c c e l l y i e l d from g l u c o n a t e . . . . . 30 V. E s t i m a t i o n o f the Rate o f ATP F o r m a t i o n and t h e ATP P o o l 30 ( i ) Method I 30 ( i i ) Method I I . . . . . 31 V I . C h e m i c a l A n a l y s e s .' 32 ( i ) P r o t e i n • • • • 32 ( i i ) G l u c o s e . . ; 32 ( i i i ) ATP. 33 (a) R e c o n s t i t u t i o n of t h e f i r e - f l y l a n t e r n e x t r a c t . ; . . . . 33 (b) Assay system . . . . ^ 33 ( i v ) V a l i n e . . . . 34 V I I . Enzyme A s s a y s . . . . ; 35 ( i ) B - G a l a c t o s i d a s e 35 ( i 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 . 36 ( i i i ) T h r e o n i n e d e h y d r a t a s e ^ 37 ( i v ) C i t r a t e s y n t h a s e 38 (v) Fumarase ;.. ; 39 ( v i ) A c o n i t a s e . 39 ( v i i ) I s o c i t r i c dehydrogenase 39 ( v i i i ) G l u c o k i n a s e 40 ( i x ) ! G l u c o s e 6-phosphate dehydrogenase 40 PART A: R e p r e s s i o n by G l u c o s 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 i n Escherichia coli- B 41 RESULTS 41 I . E f f e c t o f Carbon Source on 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 F o r m a t i o n 41 I I . E v i d e n c e f o r S i n g l e A c e t o l a c t a t e - F o r m i n g System i n Escherichia c o l i B. 43 I I I . K i n e t i c s o f V a l i n e 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 o f Escherichia c o l i B 55 IV. R e l a t i o n s h i p between G l u c o s e R e p r e s s i o n and End-Product R e p r e s s 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 o f Escherichia c o l i B 57 v i Page No, DISCUSSION .... • 60 PART B: R e l a x a t i o n o f C a t a b o l i t e R e p r e s s i o n i n Streptomycin-Dependent Escherichia c o l i 70 RESULTS . ... 70 I . R e l a x a t i o n o f t h e G l u c o s e E f f e c t on Acetohy-d r o x y A c i d S y n t h e t a s e i n Streptomycin-Depen-dent Escherichia c o l i B 70 I I . M o d i f i c a t i o n o f D i a u x i e Growth i n S t r e p t o -mycin-Dependent Mutants . 70 I I I . D i f f e r e n c e s between S t r e p t o m y c i n - S e n s i t i v e and Streptomycin-Dependent Escherichia c o l i i n th e G l u c o s e E f f e c t on B - G a l a c t o s i d a s e S y n t h e s i s . . . 75 IV. 8 - G a l a c t o s i d a s e I n d u c t i o n i n P s e u d o - R e s t i n g C e l l s . 78 V. R e l a x a t i o n o f C a t a b o l i t e R e p r e s s i o n i n Pathways of C a r b o h y d r a t e M e t a b o l i s m 81 V I . C e l l Y i e l d from G l u c o s e o f S t r e p t o m y c i n - S e n s i t i v e and Streptomycin-Dependent S t r a i n s o f Escherichia c o l i B. . . . . . . . 84 V I I . ATP S y n t h e s i s i n S t r e p t o m y c i n - S e n s i t i v e and Streptomycin-Dependent S t r a i n s o f Escherichia c o l i B 88 VIII. E f f e c t o f Carbon Source on V a l i n e E x c r e t i o n by S t r e p t o m y c i n - S e n s i t i v e and Streptomycin-Dependent c u l t u r e s o f Escherichia c o l i B 93 DISCUSSION .... 96 PART C: .. Enhancement o f C a t a b o l i t e R e p r e s s i o n by A n t i b i o t i c - L i m i t a t i o n o f S t r e p t o m y c i n -Dependent Escherichia coli' B . ... . 108 RESULTS i 108 I . A n t i b i o t i c - R e q u i r e m e n t f o r Growth o f S t r e p t o -mycin-Dependent Escherichia c o l i B. on V a r i o u s Carbon Sources 10 8 v i i Page No. I I . E f f e c t o f Growth w i t h L i m i t i n g - D i h y d r o s t r e p t o -m ycin on t h e F o r m a t i o n o f Enzymes o f Carbohy-d r a t e M e t a b o l i s m 114 I I I . Response of C a t a b o l i t e R e p r e s s i o n - S e n s i t i v e Enzymes o f A n t i b i o t i c - D e p l e t e d C e l l s t o D i h y d r o s t r e p t o m y c i n S u p p l e m e n t a t i o n . . . . . 12 3 IV. R e l a t i o n s h i p between Growth Rate and Enzyme S y n t h e s i s i n Streptomycin-Dependent C e l l s Grown w i t h L i m i t i n g - A n t i b i o t i c . . . 125 V. E f f e c t o f A n t i b i o t i c C o n c e n t r a t i o n on the Y i e l d o f Streptomycin-Dependent C e l l s from G l u c o s e . . . . . . 125 V I . E f f e c t o f A n t i b i o t i c - L i m i t a t i o n on the ATP P o o l of Streptomycin-Dependent C e l l s 129 DISCUSSION ., i , • . 131 GENERAL DISCUSSION 140 SUGGESTIONS FOR FUTURE WORK 146 BIBLIOGRAPHY . ... 147 v i i i LIST OF TABLES I E f f e c t o f i n c r e a s i n g g l u c o s e c o n c e n t r a t i o n on enzyme s p e c i f i c a c t i v i t i e s i n Escherichia c o l i B . 42 I I E f f e c t o f t h e , f o u r end p r o d u c t s of the v a l i n e -i s o l e u c i n e pathway on t h e a c e t o l a c t a t e - f o r m i n g a c t i v i t y , .of •Escherichia 'coli B.... 48 I I I V a l i n e i n h i b i t i o n o f a c e t o l a c t a t e - f o r m i n g a c t i -v i t y i n c e l l s o f Escherichia c o l i B d i s r u p t e d o r p e r m e a b i l i z e d by v a r i o u s methods 50 IV L a b i l i t y o f a c e t o l a c t a t e - f o r m i n g a c t i v i t y d u r i n g s t o r a g e ; 53 V D e s e n s i t i z a t i o n o f AHA s y n t h e t a s e t o v a l i n e i n h i b i t i o n . 54 V I E f f e c t o f b i o s y n t h e t i c end p r o d u c t s and c a r b o n s o u r c e on t h e f o r m a t i o n o f AHA s y n t h e t a s e i n Escherichia c o l i B. 58 V I I S p e c i f i c a c t i v i t i e s o f enzymes from s t r e p t o -m y c i n - s e n s i t i v e and 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 Escherichia c o l i B grown on g l u c o s e o r g l y c e r o l 82 V I I I C e l l y i e l d s of s t r e p t o m y c i n s e n s i t i v e and 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 Escherichia c o l i B d u r i n g a e r o b i c and a n a e r o b i c growth w i t h g l u c o s e . 87 IX Growth r a t e s o f Escherichia c o l i s t r a i n s . . . 90 X V a l i n e e x c r e t i o n by s t r e p t o m y c i n - s e n s i t i v e and 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 Escherichia c o l i ' B . .... 94 XI E f f e c t o f g l u c o s e c o n c e n t r a t i o n on the d i h y -d r o s t r e p t o m y c i n r e q u i r e m e n t f o r t h e growth of s t r e p t o m y c i n - d e p e n d e n t Escherichia c o l i B 112 X I I R e l a t i o n s h i p between the c a r b o n so u r c e and t h e d i h y d r o s t r e p t o m y c i n r e q u i r e m e n t f o r t h e growth of s t r e p t o m y c i n - d e p e n d e n t Escherichia c o l i B 113 i x No. Page No. X I I I S p e c i f i c a c t i v i t i e s o f enzymes from s t r e p t o -mycin-dependent Escherichia c o l i B grown on v a r i o u s c a r b o n s o u r c e s and e i t h e r a growth-r a t e - l i m i t i n g o r n o n - l i m i t i n g c o n c e n t r a t i o n o f d i h y d r o s t r e p t o m y c i n . . 116 XIV R e p r e s s i o n o f AHA s y n t h e t a s e by g l u c o s e and g l u c o n a t e i n c u l t u r e s o f s t r e p t o m y c i n s e n s i -t i v e and s t r e p t o m y c i n - d e p e n d e n t Escherichia c o l i B . . i 121 XV E f f i c i e n c y o f g l u c o s e and g l u c o n a t e u t i l i z a -t i o n by c u l t u r e s o f s t r e p t o m y c i n - s e n s i t i v e and s t r e p t o m y c i n - d e p e n d e n t Escherichia c o l i B 122 XVI S p e c i f i c a c t i v i t i e s o f v a r i o u s enzymes i n d i h y d r o s t r e p t o m y c i n - d e p l e t e d and supplemented e x t r a c t s of s t r e p t o m y c i n - d e p e n d e n t Escherichia c o l i B 124 X V I I E f f e c t o f a n t i b i o t i c c o n c e n t r a t i o n on t h e c e l l y i e l d from g l u c o s e of s t r e p t o m y c i n - d e p e n d e n t Escherichia c o l i B 128 X LIST OF FIGURES No. Page No. 1 The b i o s y n t h e t i c pathway t o i s o l e u c i n e , v a l i n e , l e u c i n e and p a n t o t h e n a t e i n Escherichia c o l i . . . . . . • 10 2 The f o r m a t i o n o f 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 n . Escherichia c o l i • 12 3 E f f e c t o f i n c r e a s i n g i n i t i a l c o n c e n t r a t i o n s o f carb o n s o u r c e on' the f o r m a t i o n o f AHA s y n t h e -t a s e i n Escherichia c o l i B 44 4 E f f e c t of pH on t h e a c e t o l a c t a t e - f o r m i n g system from c u l t u r e s o f Escherichia c o l i B grown on d i f f e r e n t c a r b o n s o u r c e s 46 5 V a l i n e i n h i b i t i o n o f a c e t o l a c t a t e - f o r m i n g a c-t i v i t y i n e x t r a c t s o f Escherichia c o l i B grown on d i f f e r e n t c a r b o n s o u r c e s 51 6 V a l i n e i n h i b i t i o n of AHA s y n t h e t a s e i n Escheri-chia c o l i B. . . . 56 7. E f f e c t o f i n c r e a s i n g i n i t i a l c o n c e n t r a t i o n s o f c a r b o n s o u r c e on t h e f o r m a t i o n o f AHA syn-t h e t a s e i n s t r e p t o m y c i n - d e p e n d e n t Escherichia c o l i B j 71 8 G l u c o s e - l a c t o s e d i a u x i e growth o f w i l d - t y p e Escherichia c o l i B a n d a s t r e p t o m y c i n - d e p e n d e n t mutant o f t h i s o rganism. . . . . . . . i . . 73 9 G l u c o s e - l a c t o s e d i a u x i e growth, o f w i l d - t y p e Escherichia c o l i E and a s t r e p t o m y c i n - d e p e n d e n t mutant o f t h i s o r g a n i s m . . . . . . 74 10 E f f e c t o f t h e a d d i t i o n o f g l u c o s e on the d i f -f e r e n t i a l r a t e o f 8 - g a l a c t o s i d a s e i n d u c t i o n i n c u l t u r e s of w i l d - t y p e Escherichia c o l i B and a s t r e p t o m y c i n - d e p e n d e n t mutant o f t h i s o r g a n i s m 76 11 E f f e c t o f .the a d d i t i o n of g l u c o s e on the d i f f e r -e n t i a l r a t e o f 8 - g a l a c t o s i d a s e i n d u c t i o n i n c u l -t u r e s o f w i l d - t y p e Escherichia c o l i E and a s t r e p t o m y c i n - d e p e n d e n t mutant o f t h i s o r g a n i s m 77 x i No. Page No. 12 E f f e c t o f t h e a d d i t i o n o f g l u c o s e on (3-galac-t o s i d a s e i n d u c t i o n i n " p s e u d o - r e s t i n g " c u l t u r e s o f w i l d - t y p e Escherichia c o l i E and a s t r e p t o -mycin-dependent mutant o f t h i s o rganism. 80 13 Y i e l d o f c e l l s from g l u c o s e d u r i n g a e r o b i c growth o f s t r e p t o m y c i n - s e n s i t i v e and s t r e p t o m y c i n - d e p e n -d ent s t r a i n s o f Escherichia c o l i B 85 14 E f f e c t o f a change from a e r o b i c t o a n a e r o b i c growth on the y i e l d o f s t r e p t o m y c i n - s e n s i t i v e and 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 of Escherichia c o l i B w i t h g l u c o s e as the carbon s o u r c e 86 15 Rate o f ATP f o r m a t i o n i n c u l t u r e s of s t r e p t o m y c i n -s e n s i t i v e and s t r e p t o m y c i n - d e p e n d e n t Escherichia c o l i B growing a e r o b i c a l l y on g l u c o s e - s a l t s medium. 91 16 The ATP p o o l o f s t r e p t o m y c i n - s e n s i t i v e and s t r e p -tomycin-dependent s t r a i n s o f Escherichia c o l i B d u r i n g a e r o b i c growth on g l u c o s e - s a l t s medium...... 92 17 Growth o f a s t r e p t o m y c i n - d e p e n d e n t mutant o f Escherichia c o l i . B on r a t e - l i m i t i n g c o n c e n t r a -t i o n s o f d i h y d r o s t r e p t o m y c i n 109 18 R e l a t i o n s h i p between t h e growth r a t e o f Escherichia c o l i s t r a i n DB and the d i h y d r o s t r e p t o m y c i n con-c e n t r a t i o n o f t h e medium. HO 19 E f f e c t o f a n t i b i o t i c - l i m i t a t i o n on the f o r m a t i o n o f AHA s y n t h e t a s e i n c u l t u r e s of s t r e p t o m y c i n -dependent Escherichia c o l i B grown on v a r i o u s c arbon s o u r c e s . ^ 2 0 R e l a t i o n s h i p between the d e c r e a s e i n growth r a t e and the r e p r e s s i o n of AHA s y n t h e t a s e and fumarase i n d i h y d r o s t r e p t o m y c i n - l i m i t e d c u l t u r e s o f s t r e p t o -mycin-dependent Escherichia c o l i B 126 21 E f f e c t o f d i h y d r o s t r e p t o m y c i n c o n c e n t r a t i o n on t h e ATP p o o l o f s t r e p t o m y c i n - d e p e n d e n t Escherichia c o l i B . I 3 0 x i i ABBREVIATIONS AHA a c e t o h y d r o x y a c i d ATP a d e n o s i n e t r i p h o s p h a t e DHSm d i h y d r o s t r e p t o m y c i n DNA d e o x y r i b o n u c l e i c a c i d 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 mRNA messenger r i b o n u c l e i c a c i d NAD + n i c o t i n a m i d e adenine d i n u c l e o t i d e NADH reduced n i c o t i n a m i d e adenine d i n u c l e o t i d e NADP + n i c o t i n a m i d e adenine d i n u c l e o t i d e phosphate NADPH reduced n i c o t i n a m i d e adenine d i n u c l e o t i d e phosphate RNA r i b o n u c l e i c a c i d Sm s t r e p t o m y c i n x i i i ACKNOWLEDGEMENTS The a u t h o r w i s h e s t o e x p r e s s h i s a p p r e c i a t i o n t o Dr. W.J. P o l g l a s e f o r h i s guidance and encouragement d u r i n g the co u r s e of t h i s work and t o Dr s . Audrey G r o n l u n d , P.D. Bragg and S.H. Zbarsky f o r t h e i r a d v i c e d u r i n g t h e p r e p a r a t i o n o f the m a n u s c r i p t . Thanks a r e a l s o due Mrs. T a n i s Matheson and Mr. J . W i t h a a r f o r t h e i r g e n e r a l c o o p e r a t i o n i n t h e l a b o r a t o r y and t e c h n i c a l a s s i s t a n c e w i t h c e r t a i n e x p e r i m e n t s . F i n a n c i a l s u p p o r t i n the form o f a N a t i o n a l R e s e a r c h C o u n c i l S t u d e n t s h i p and an H.R. M c M i l l a n F a m i l y F e l l o w s h i p i s g r a t e f u l l y acknowledged. - 1 -INTRODUCTION I . G e n e t i c s of S t r e p t o m y c i n Mutants A n t i b i o t i c s o f the s t r e p t o m y c i n f a m i l y i n c l u d e s t r e p t o -mycin (Sm), m a n n o s i d o s t r e p t o m y c i n and h y d r o x y s t r e p t o m y c i n and t h e i r c h e m i c a l l y r e d u c e d d e r i v a t i v e s of which d i h y d r o s t r e p t o -m ycin (DHSm) i s t h e most i m p o r t a n t t h e r a p e u t i c a l l y . A m i c r o -o r g a n i s m s e l e c t e d f o r r e s i s t a n c e t o any one o f t h e s e compounds i s a l s o r e s i s t a n t t o o t h e r members of t h i s f a m i l y of a n t i b i o t i c s . ' The e x p r e s s i o n o f r e s i s t a n c e t o the s t r e p t o m y c i n a n t i b i o t i c s i s complex. In a d d i t i o n t o m u t a t i n g t o modest l e v e l s o f r e s i s -t a n c e , v a r i o u s m i c r o - o r g a n i s m s g i v e r i s e , i n s i n g l e s t e p s , t o h i g h l y r e s i s t a n t mutants and a l s o t o dependent mutants w h i c h r e q u i r e the a n t i b i o t i c f o r growth. S i n c e Sm-dependent mutants have been i s o l a t e d from many G r a m - p o s i t i v e and Gram-negative m i c r o b i a l s p e c i e s , t h e r e i s good r e a s o n t o b e l i e v e t h a t mu-t a t i o n t o an a b s o l u t e dependence on t h e s e compounds i s a w i d e - s p r e a d g e n e t i c p o t e n t i a l i t y o f b a c t e r i a " . I n Escherichia c o l i 3 g e n e t i c a n a l y s e s have shown t h a t s e n s i t i v i t y , dependence and s i n g l e - s t e p r e s i s t a n c e ( i n d i f f e r e n c e ) t o a h i g h c o n c e n t r a -t i o n o f Sm a r e d e t e r m i n e d by m u l t i p l e a l l e l e s a t a s i n g l e l o c u s , known as t h e "Sm" l o c u s (Newcombe and Nyholm, 1950; Lennox, 1955; Hashimoto, 1960). T h i s g e n e t i c f a c t i m p l i e s t h a t t h e t h r e e ph'anotypic s t a t e s i n q u e s t i o n a r e a l l d e t e r m i n e d by a l t e r n a t i v e s t r u c t u r a l m o d i f i c a t i o n s of a s i n g l e c h e m i c a l s u b s t a n c e w i t h i n the c e l l . L o w - l e v e l S m - r e s i s t a n c e ( r e s i s t a n c e t o a n t i b i o t i c c o n c e n t r a t i o n s up t o a p p r o x i m a t e l y 25 y g . / m l . ) , - 2 -on t h e o t h e r hand, i s due t o m u l t i p l e l o c i a t s i t e s u n l i n k e d t o t he h i g h - l e v e l Sm-* r e s i s t a n c e l o c u s (Watanabe and Watanabe, 1959a; 1959b). I n a d d i t i o n , l o w - l e v e l S m - r e s i s t a n t mutants have been i s o l a t e d w h i c h p o s s e s s an episome f o r m u l t i p l e drug r e s i s t a n c e . I n ; t h i s c a s e , the or g a n i s m was found t o be r e s i s -t a n t t o Sm because o f a change i n t h e p e r m e a b i l i t y o f t h e c e l l membrane t o t h e a n t i b i o t i c (Rosenkranz, 1964) o r t o the pres e n c e of an enzyme wh i c h i n a c t i v a t e s the Sm m o l e c u l e (Umezawa, et al., 1967). I I . P roposed S i t e s o f S t r e p t o m y c i n A c t i o n A t n e u t r a l pH, b o t h Sm and DHSm are c a t i o n i c m o l e c u l e s and as such p r e c i p i t a t e in vitro a wide v a r i e t y o f p o l y -a n i o n i c m a c r o m o l e c u l e s , presumably by c r o s s - l i n k i n g a d j a c e n t polymers and b u i l d i n g up i n s o l u b l e l a t t i c e s (see B r o c k , 1964). Sm, however, i s a p p r o x i m a t e l y t e n t i m e s more e f f e c t i v e t h a n DHSm a t p r e c i p i t a t i n g most p o l y a n i o n s (Brock, 1964), des-p i t e t he f a c t t h a t b o t h m o l e c u l e s p o s s e s s the same n e t charge (+3) ( P o l g l a s e , 1965). S i n c e Sm and DHSm are e q u a l l y a c t i v e as a n t i b a c t e r i a l a g e n t s , t h e n o n - s p e c i f i c b i n d i n g o f t h e s e compounds t o RNA, DNA, p h o s p h o l i p i d s , e t c . (Rybak and G r o s , 1947; Waksman, 1949) and t h e i r c a t i o n i c e f f e c t s on the a c t i v i t i e s of a number o f u n r e l a t e d enzymes ( Z e l l e r , et al. . 19 51; Darrow and C r e v e l i n g , 196 4) p r o b a b l y a r e not i n v o l v e d i n the p r i m a r y a c t i o n o f t h e s e a n t i b i o t i c s i n the b a c t e r i a l c e l l . T h i s n o n - s p e c i f i c b i n d i n g o f Sm (and DHSm) t o c e l l u l a r components, however, may be r e s p o n s i b l e f o r many o f t h e secon-- 3 -d a r y e f f e c t s w h i c h appear t o accompany Sm a c t i o n (see D a v i s and F e i n g o l d , 1962). I n 1961, S p o t t s and S t a n i e r proposed a u n i t a r y h y p o t h e s i s o f Sm a c t i o n which was d e v e l o p e d i n a l o g i c a l manner from g e n e t i c , b i o c h e m i c a l and p h y s i o l o g i c a l d a t a o b t a i n e d p r i m a r i l y from s t u d i e s on Sm-dependent mutants. They su g g e s t e d t h a t Sm-s e n s i t i v i t y , dependence and h i g h - l e v e l r e s i s t a n c e were u l t i -m a t e l y d e t e r m i n e d by s t r u c t u r a l m o d i f i c a t i o n s of a s p e c i f i c p r o t e i n a t a s i n g l e i n t r a c e l l u l a r s i t e . I t was proposed t h a t t h i s p r o t e i n r e s i d e d i n the ribosome. The s e n s i t i v e r i b o -some was p i c t u r e d as p o s s e s s i n g a s t r u c t u r e t h a t c o n f e r r e d on i t a v e r y h i g h a f f i n i t y f o r Sm. The r e s u l t i n g Sm-ribosome complex would p r e v e n t the attachment o f mRNA, thus i n h i b i t i n g p r o t e i n s y n t h e s i s . The c o r r e s p o n d i n g s t r u c t u r e s o f t h e r e -s i s t a n t arid dependent ribosomes were such t h a t Sm had no e f f e c t on the former and i t s p r e s e n c e was o b l i g a t o r y f o r t h e l a t t e r t o f u n c t i o n n o r m a l l y . T h i s p r e d i c t i o n t h a t t h e ribosome was the S m - s e n s i t i v e s i t e i n the c e l l was s u p p o r t e d by e a r l y s t u d i e s in v i t r o on p r o t e i n s y n t h e s i s u s i n g v a r i o u s c o m b i n a t i o n s o f crude s u p e r -n a t a n t f l u i d s and ribosomes from S m - s e n s i t i v e and S m - r e s i s t a n t E. ooli (Speyer: e t al. 3 1962; F l a x et al. , 1962). S i m i l a r l y , u s i n g v a r i o u s c o m b i n a t i o n s o f 50S and 30S r i b o s o m a l s u b u n i t s from S m - s e n s i t i v e and S m - r e s i s t a n t E. c o l i , D a v i e s (1964) and Cox et al. (1964) showed t h a t the S m - s e n s i t i v e s i t e r e s i d e d on t h e 30S s u b u n i t o f the 70S ribosome. R e c e n t l y , Traub and Nomura (196 8) r e p o r t e d t h a t the a l t e r a t i o n i n d u c e d by the - 4 -S m - r e s i s t a n c e m u t a t i o n i s i n a p a r t i c u l a r p r o t e i n ("core") o f the 30S s u b u n i t and i s n o t i n t h e RNA o r o t h e r p r o t e i n f r a c t i o n s ( " s p l i t " ) o f t h i s p a r t i c l e . D a v i e s (1964) demonstrated t h a t Sm d i d not i n t e r f e r e w i t h the b i n d i n g o f 1 ^ C - p o l y u r i d y l i c a c i d ( p o l y U) t o S m - s e n s i t i v e ribosomes (as sug g e s t e d by S p o t t s and S t a n i e r , 1961) even though p o l y U - d i r e c t e d p o l y p h e n y l a l a n i n e s y n t h e s i s was markedly i n h i b i t e d by the a n t i b i o t i c . T h e r e f o r e , t h e Sm e f f e c t appeared t o be l o c a l i z e d somewhere between codon r e c o g n i t i o n and amino a c i d p o l y m e r i z a t i o n . Recent s t u d i e s by L u z z a t t o . et al. , (196 8) i n d i c a t e d t h a t Sm had no e f f e c t on Sm-s e n s i t i v e ribosomes w h i c h were beyond th e i n i t i a t i o n s t e p of the t r a n s l a t i o n p r o c e s s . However, as t h e s e ribosomes com-p l e t e d t h e t r a n s l a t i o n o f one s t r a n d o f messenger and a t t a c h e d t h e m s e l v e s t o a new mRNA m o l e c u l e , Sm bound the ribosome t o the i n i t i a t i o n s i t e . T h i s u l t i m a t e l y r e s u l t e d i n the f o r -m a t i o n o f the "70S i n i t i a t i o n complexes" (70S ribosomes bound t o the i n i t i a t i o n s i t e o f mRNA m o l e c u l e s ) . F u r t h e r m o r e , they showed t h a t a S m - s e n s i t i v e , c e l l - f r e e p r o t e i n - s y n t h e s i z i n g system d i r e c t e d by messengers p o s s e s s i n g t h e normal i n i t i a t i n g codon, AUG (e . g . , phage RNA o r p o l y AUG) was c o n s i d e r a b l y more s e n s i t i v e t o Sm th a n was the same system d i r e c t e d by a syn-t h e t i c messenger such as p o l y U whi c h f u n c t i o n e d v i a an abnormal i n i t i a t i o n mechanism (Nakamoto and K o l a k o f s k y , 1966; L u z z a t t o et al. 3 1968). A l t h o u g h p o l y U p r e d o m i n a t e l y s t i m u l a t e s t h e i n c o r p o r a -t i o n of p h e n y l a l a n i n e i n t o p r o t e i n i n c e l l - f r e e e x t r a c t s , t he a d d i t i o n o f Sm t o a S m - s e n s i t i v e system can i n d u c e the i n c o r -- 5 -p o r a t i o n o f s u b s t a n t i a l q u a n t i t i e s o f l e u c i n e , i s o l e u c i n e o r o t h e r amino a c i d s i n t o p r o t e i n (Davies et al., 196 4; Van Knippen-b e r g et al., 1965). T h i s e f f e c t has been termed " m i s c o d i n g " or " m i s r e a d i n g " and can be i n d u c e d n o n - s p e c i f i c a l l y by a number o f e n v i r o n m e n t a l f a c t o r s (as w e l l as Sm) i n c l u d i n g t e m p e r a t u r e , magnesium c o n c e n t r a t i o n , o r g a n i c s o l v e n t s and pH (Friedman et al., 1968). M i s c o d i n g s t i m u l a t e d by Sm was s m a l l or n e g l i g i b l e i n c e l l - f r e e systems c o n t a i n i n g * S m - r e s i s t a n t ribosomes (Davies e t ' a l . , 1964). T h e ' o b s e r v a t i o n o f Sm-i n d u c e d t r a n s l a t i o n a l e r r o r s i n c e l l - f r e e p r e p a r a t i o n s from S m - s e n s i t i v e c e l l s l e d t o the i n f e r e n c e t h a t Sm may k i l l t h e s e n s i t i v e o r g a n i s m by f l o o d i n g t h e c e l l w i t h n o n - f u n c t i o n a l p r o t e i n (Davies et al., 1964). Subsequent s t u d i e s , however, s u g g e s t e d t h a t t h i s was h i g h l y u n l i k e l y s i n c e c e l l s o f c e r -t a i n s t r a i n s can s u r v i v e and grow i n t h e p r e s e n c e o f Sm w h i l e making ss much as 80% f a u l t y p r o t e i n ( G o r i n i and K a t a j a , 1964b). Thus, Sm can e x h i b i t two d i s t i n c t and e x p e r i m e n t a l l y s e p a r a b l e e f f e c t s on p r o t e i n s y n t h e s i s i n S m - s e n s i t i v e c e l l s ; f i r s t , i n h i b i t i o n of p e p t i d e bond f o r m a t i o n and second, s t i m u l a t i o n o f m i s c o d i n g . Both o f th e s e e f f e c t s am v i r t u a l l y absent i n c e l l - f r e e systems p r e p a r e d from S m - r e s i s t a n t o r g anisms. U n t i l r e c e n t l y a t t e m p t s were u n s u c c e s s f u l a t d e m o n s t r a t i n g s t i m u l a t i o n by Sm (or DHSm) o f p r o t e i n s y n t h e s i s i n v i t r o u s i n g ribosomes from Sm-dependent organisms. T h i s was p r o b a b l y due t o the f a c t t h a t Sm, bound t o Sm-dependent r i b o s o m e s , i s not removed by s t a n d a r d w ashing p r o c e d u r e s - 6 -( K u r l a n d , 1966); thus f u r t h e r a d d i t i o n o f a n t i b i o t i c would have l i t t l e o r no e f f e c t on t h e a c t i v i t y o f the Sm-dependent rib o s o m e s . When Sm-dependent ribosomes were p r e p a r e d from c e l l s s t a r v e d o f a n t i b i o t i c and were e x t e n s i v e l y washed w i t h h i g h c o n c e n t r a t i o n s o f s a l t , Sm was shown t o s t i m u l a t e p o l y -p e p t i d e s y n t h e s i s ( L i k o v e r and K u r l a n d , 1967a). F u r t h e r m o r e , the Sm e f f e c t was shown t o be a s p e c i f i c p r o p e r t y o f the 30S s u b u n i t . Thus, the Sm phenotype o f an o r g a n i s m can be c o r r e -l a t e d w i t h t h e phenotype i n v i t r o o f a p a r t i c u l a r component (30S s u b u n i t ) o f t h e ribosomes o b t a i n e d from the c o r r e s p o n d -i n g S m - s e n s i t i v e , S m - r e s i s t a n t o r Sm-dependent c e l l s . These r e s u l t s s t r o n g l y s u p p o r t t h e h y p o t h e s i s o f S p o t t s and S t a n i e r (1961) t h a t the ribosome i s a p r i m a r y s i t e of Sm a c t i o n i n b a c t e r i a . S t u d i e s on p r o t e i n s y n t h e s i s i n S m - s e n s i t i v e and Sm-dependent E. c o l i s u g g e s t t h a t the Sm phenotype o f an org a n i s m p r o b a b l y r e s u l t s from the i n h i b i t o r y e f f e c t o r the s t i m u l a t o r y e f f e c t o f Sm on t h e r a t e o f p r o t e i n s y n t h e s i s r a t h e r t h a n from the a b i l i t y o f t h i s compound t o i n d u c e o r suppress m i s c o d i n g ( L i k o v e r and K u r l a n d , 1967b). I n a d d i t i o n t o the e f f e c t s o f Sm on t h e ribosome, e a r l y s t u d i e s s u g g e s t e d t h a t Sm a l s o may a c t on t h e c e l l membrane. Roth et al. (1960) r e p o r t e d t h a t t h e a d d i t i o n o f Sm t o S m - s e n s i t i v e c u l t u r e s r e s u l t e d i n the e x c r e t i o n o f 5 ' - r i b o n u c l e o t i d e s . S i n c e the i n t r a c e l l u l a r p o o l o f n u c l e o -t i d e s was n o t e l e v a t e d under t h e s e c o n d i t i o n s e x c r e t i o n must be due t o membrane damage. T h i s c o n c l u s i o n was v e r i f i e d by - 7 -t h e d e m o n s t r a t i o n t h a t the a d d i t i o n of Sm t o S m - s e n s i t i v e c e l l s caused i n c r e a s e d e x c r e t i o n o f amino a c i d s (Anand and D a v i s , 1960), i n c r e a s e d a c c e s s i b i l i t y , of 6 - g a l a c t o s i d a s e i n a c r y p t i c mutant (Brock.and B r o c k , 1959) and an i n c r e a s e i n K + e f f l u x (Dubin and D a v i s , 1961). F u r t h e r m o r e , s i n c e e l e c t r o n -t r a n s p o r t components i n v o l v e d i n t e r m i n a l o x i d a t i v e p r o c e s s e s a r e b e l i e v e d t o be l o c a t e d i n t h e c y t o p l a s m i c membrane (see S m i t h , 1961), the e f f e c t o f Sm on r e s p i r a t i o n (Umbreit, 1950) and on o x i d a t i v e p h o s p h o r y l a t i o n (Bragg and P o l g l a s e , 19 6 3b) ar e c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t Sm a c t s on t h e c e l l membrane. D e s p i t e t h e e x t e n s i v e e v i d e n c e t h a t the membrane i s a s i t e o f Sm a c t i o n , however, the i n a b i l i t y o f t h e s e s t u d i e s t o p i n p o i n t a s i n g l e S m - s e n s i t i v e s i t e c o u p l e d w i t h t h e enormous s u c c e s s o f r e l a t e d s t u d i e s on the ribosome has l e d t o a l e s s e n i n g o f i n t e r e s t i n t h i s a r e a o f i n v e s t i g a t i o n . I l l . E f f e c t o f S t r e p t o m y c i n on P y r u v a t e M e t a b o l i s m Two groups o f i n v e s t i g a t o r s ( T i r u n a r a y a n a n et al., 1962; Bragg and P o l g l a s e , 1962) r e p o r t e d i n d e p e n d e n t l y t h a t Sm-dependent m i c r o - o r g a n i s m s grown on g l u c o s e - s a l t s medium e x c r e t e r e l a t i v e l y l a r g e amounts o f L - v a l i n e and a l e s s e r amount o f L - l e u c i n e . Each group, however, i n t e r p r e t e d t h e s e r e s u l t s d i f f e r e n t l y . T i r u n a r a y a n a n and co-workers (1962) a t t r i b u t e d t h e e x c r e t i o n o f t h e s e amino a c i d s t o a " p a r t i a l b l o c k a g e " o f p r o t e i n s y n t h e s i s . S i n c e the Sm-dependent s t r a i n s were a b l e t o grow and m u l t i p l y i n s p i t e o f the p a r t i a l - 8 -b l o c k i n p r o t e i n s y n t h e s i s , they s u g g e s t e d t h i s phenomenon c o n s t i t u t e d o n l y the p r e l i m i n a r y f i x a t i o n of Sm t o the c e l l and t h a t t h e a n t i m i c r o b i a l e f f e c t was due t o subsequent meta-b o l i c changes. E x p e r i m e n t s c a r r i e d out. by Bragg and P o l g l a s e (196 2) s u g g e s t e d t h a t the e x c r e t i o n o f v a l i n e r e s u l t e d from an a l t e r a t i o n i n t h e pathway o f p y r u v a t e d i s s i m i l a t i o n . E x t r a c e l l u l a r m e t a b o l i t e s were not n o r m a l l y d e t e c t a b l e i n the s u p e r n a n t a n t f l u i d s o f S m - s e n s i t i v e c u l t u r e s or Sm-r e s i s t a h t c u l t u r e s grown w i t h o u t a n t i b i o t i c supplement. However, a S m - r e s i s t a n t mutant, i n t h e p r e s e n c e of a n t i b i o t i c , e x c r e t e d s i g n i f i c a n t q u a n t i t i e s o f b o t h l a c t a t e and p y r u v a t e (Bragg and P o l g l a s e , 1962). These r e s u l t s s u g g e s t e d t h a t t h e r e s i s t a n t mutant used pathways of a n a e r o b i c m e t a b o l i s m i n a medium c o n t a i n i n g t h e a n t i b i o t i c b u t i n t h e absence of the a n t i b i o t i c employed a pathway of p y r u v a t e d i s s i m i l a t i o n s i m i l a r t o t h a t employed by s e n s i t i v e o r g a nisms. The Sm-dependent mutant d i f f e r e d from b o t h the s e n s i t i v e and r e s i s -t a n t organisms i n i t s p r o d u c t i o n o f s u b s t a n t i a l amounts of v a l i n e . As much as 10% o f the g l u c o s e carbon c o u l d be a c c o u n t e d f o r i n t h i s p r o d u c t (Bragg and P o l g l a s e , 1962). Subsequent work (Bragg and P o l g l a s e , 196 4a) i n d i c a t e d t h a t a n t i b i o t i c - d e p l e t e d , Sm-dependent c e l l s , produced l a r g e q u a n t i t i e s o f l a c t a t e b u t no v a l i n e w h i l e the same c e l l s supplemented w i t h n o n - r a t e - l i m i t i n g c o n c e n t r a t i o n s o f Sm, e x c r e t e d o n l y v a l i n e . When Sm-dependent c e l l s were grown a n a e r o b i c a l l y t h e i r m e t a b o l i s m r e s e m b l e d t h a t o f a e r a t e d , a n t i b i o t i c - d e p l e t e d c e l l s ( p r o d u c t i o n o f l a c t a t e ) even i n t h e - 9 -p r e s e n c e of t h e o p t i m a l Sm c o n c e n t r a t i o n . Thus, the n a t u r e o f the e x t r a c e l l u l a r p r o d u c t s o f Sm-dependent organisms appears t o be r e g u l a t e d by b o t h the c o n c e n t r a t i o n o f oxygen and o f a n t i -b i o t i c i n the growth medium. These workers proposed t h a t t h e f o r m a t i o n o f v a l i n e was a secondary a e r o b i c pathway o f g l u -cose m e t a b o l i s m e x i s t i n g i n Sm-dependent mutants (Bragg and P o l g l a s e , 1964a). S t u d i e s from the same l a b o r a t o r y (Bragg and P o l g l a s e , 1963a) on t h e e f f e c t of DHSm on e l e c t r o n t r a n s -p o r t i n E. c o l i , s u g g e s t e d t h a t v a l i n e may f u n c t i o n as a n e u t r a l hydrogen a c c e p t o r i n c a r b o h y d r a t e m e t a b o l i s m . I f t h i s were t r u e , t h e n , i n Sm-dependent c e l l s , t h e a n t i b i o t i c m ight a c t i v a t e a mechanism e n a b l i n g a b i o s y n t h e t i c pathway t o f u n c t i o n c a t a b o l i c a l l y as a major r o u t e of p y r u v a t e m e t a b o l i s m . IV. B i o s y n t h e s i s o f A l i p h a t i c Amino A c i d s and R e g u l a t o r y Mechanisms The b i o s y n t h e t i c pathways l e a d i n g t o L - v a l i n e , L - i s o l e u -c i n e , L - l e u c i n e and p a n t o t h e n a t e i n E. c o l i have been i n v e s t i -g a t e d t h r o u g h i s o t o p e s t u d i e s on s e l e c t e d a u x o t r o p i c mutants. T h i s work has been r e v i e w e d by Umbarger and D a v i s (1962) ( F i g . 1 ) . V a l i n e and t h e p r e c u r s o r s o f l e u c i n e are s y n t h e s i z e d from two m o l e c u l e s of p y r u v a t e w h i l e the p r e c u r s o r s f o r i s o -l e u c i n e r e s u l t from th e c o n d e n s a t i o n o f one m o l e c u l e each of p y r u v a t e and a - k e t o b u t y r a t e . Umbarger and Brown (1958) r e -p o r t e d t h a t the enzymes c a t a l y z i n g t h e l a s t t h r e e r e a c t i o n s i n t h e b i o s y n t h e t i c pathway l e a d i n g t o i s o l e u c i n e a l s o c a t a -l y z e t h e c o r r e s p o n d i n g r e a c t i o n s i n v a l i n e s y n t h e s i s . T h i s threonine pyruvate pyruvate a-aceto-a -hydroxy-* butyrate — a-aceto-> lactate I Acetohydroxy Acid Synthetase II Reductoisomerase .III Dihydroxy Dehydrase _ IV Transaminase B . II a,-g-dihydroxy-•g-methyl-> valerate a -keto-g-methyl-valerate III a , g- -dihydroxy-H> isovalerate — a-keto--» .Isovalerate a -keto-•isocaprcate L-leucine -» L-ispleucine IV L-valine a - k e t o -pantoate pantoate pantothenate F i g . 1 The biosynthetic pathway to isoleucine, val ine , leucine and pantothenate i n Escherichia c o l i . - 11 -f i r s t became e v i d e n t when i t was found t h a t a u x o t r o p h i c mutants l a c k i n g an enzyme on the v a l i n e pathway g e n e r a l l y l a c k e d the c o r r e s p o n d i n g enzyme on the i s o l e u c i n e pathway. Thus " s i n g l e - s i t e " mutants o c c u r which a r e m u l t i - a u x o t r o p h i c f o r v a l i n e , l e u c i n e and i s o l e u c i n e . These f i n d i n g s have been s u p p o r t e d by k i n e t i c s t u d i e s (Umbarger and Brown, 1958 ; L e a v i t t and Umbarger, 1 9 6 1 ) . I t s h o u l d be n o t e d , however, t h a t the enzyme w h i c h c a t a l y z e s the f i r s t s t e p i n t h e v a l i n e pathway, namely, the c o n d e n s a t i o n o f two m o l e c u l e s o f p y r u -v a t e t o one m o l e c u l e o f a - a c e t o l a c t a t e , a l s o c a t a l y z e s t h e second s t e p i n t h e i s o l e u c i n e pathway, the c o n v e r s i o n o f a - k e t o b u t y r a t e t o a - a c e t o h y d r o x y b u t y r a t e ( L e a v i t t and Umbarger, 1961). T h i s enzyme complex ( F i g . 2) has been d e s i g n a t e d the condensing enzyme o r acetolactate-forming system (Umbarger and Brown, 1958) and more r e c e n t l y as acetohydroxy acid (AHA) synthetase ( B a u e r l e et al.3 1 9 6 4 ) . T h i s enzyme i s e x t r e m e l y l a b i l e and has not y e t been p u r i f i e d from b a c t e r i a l s o u r c e s . An e a r l y i n v e s t i g a t i o n i n crude e x t r a c t s o f E. c o l i o f t h e mechanism o f a c t i o n o f AHA s y n t h e t a s e s u g g e s t e d t h a t two r e a c -t i o n s were i n v o l v e d . The f i r s t c o n s i s t s o f t h e g e n e r a t i o n o f an " a c t i v e a c e t a l d e h y d e " , presumably as an a c e t a l - d i p h o s p h o -t h i a m i n e (DPT) complex. The second r e a c t i o n i s the a c t u a l , t r a n s f e r o f the a c e t a l group t o t h e a c c e p t o r m o l e c u l e , e i t h e r " p y r u v a t e o r a - k e t o b u t y r a t e . T h i s r e a c t i o n proceeds o p t i m a l l y o n l y i n t h e pr e s e n c e o f a d i v a l e n t c a t i o n such as M g + + o r M h + + . CH3-CH2-C-COOH a-ketobutyrate 0 OH CH3-C-C-COOH CH^ a-aceto a - hydroxy-butyrate 0 CH3-C-COOK pyruvate + D P T H - C0 2 CH--C-DPT i l Acetohydroxy + ypj) Acid ' + Mg^ Synthetase 0 CH3-C-COOH pyruvate 0 OH > CH-3-C-C-COOH CH. a-acetolactate F i g . 2 The formation of a-acetolactate and a-acetoarhydroxy-butyrate i n Escherichia c o l i . - 13 -B a u e r l e et al., (1964) r e p o r t e d t h a t AHA-synthetase a c t i v i t y i s g r e a t l y s t i m u l a t e d by the p r e s e n c e of t h e coenzyme f l a v i n a denine d i n u c l e o t i d e (FAD). The f u n c t i o n o f t h i s u nexpected c o f a c t o r i s as y e t not u n d e r s t o o d . I t has been r e p o r t e d (Umbarger and Brown, 195 8 ) . t h a t t h e AHA s y n t h e t a s e of E. c o l i , l i k e i n i t i a l enzymes of o t h e r b i o s y n t h e t i c pathways, i s s u b j e c t t o end-product i n h i b i t i o n when assay e d i n the p r e s e n c e of L - v a l i n e . R e g u l a t i o n o f the formation o f AHA s y n t h e t a s e , however, has been the s u b j e c t o f a number of r e p o r t s . Umbarger and Brown (1958) p r e s e n t e d e v i d e n c e t h a t i n E. c o l i t h e enzyme was r e p r e s s e d by growth i n the p r e s e n c e of v a l i n e . However, i n l a t e r work w i t h " d e r e p r e s s e d " mutants of Salmonella typhimurium and E. c o l i i t was found t h a t r e p r e s s i o n was m u l t i v a l e n t ( F r e u n d l i c h et al., 1962) and r e q u i r e d the p r e s e n c e of l e u c i n e and i s o -l e u c i n e i n a d d i t i o n t o v a l i n e . I n w i l d - t y p e o r g a n i s m s , how-e v e r , m u l t i v a l e n t r e p r e s s i o n o f t h e enzyme was not o b s e r v e d (Armstrong et al., 1963). F r e u n d l i c h and Umbarger (1963) r e p o r t e d t h a t m u l t i v a l e n t r e p r e s s i o n of AHA s y n t h e t a s e was o b s e r v e d i f p a n t o t h e n a t e was s u p p l i e d i n a d d i t i o n t o t h e t h r e e b r a n c h e d - c h a i n a l i p h a t i c amino a c i d s . More r e c e n t s t u d i e s by P o l g l a s e (1966a) 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 the r e p r e s s i o n o f AHA s y n t h e t a s e was g e n e r a l l y g r e a t e r i n the p r e s e n c e of t h e f o u r end 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 ) than i n the p r e s ence o f v a l i n e a l o n e ; however, the degree o f r e p r e s s i o n was not l a r g e - 14 -and t h e r e were d i f f e r e n c e s between s t r a i n s . The o b v i o u s c o n c l u s i o n t h a t can be drawn from t h e s e r e p o r t s i s t h a t t h e r e g u l a t i o n o f AHA-synthetase f o r m a t i o n i n t h e s e organisms i s complex. T h i s c o n c l u s i o n i s not s u r p r i s i n g i n view o f t h e i n t r i c a t e r o l e p l a y e d by t h i s enzyme i n t h e b i o s y n t h e s i s o f f o u r i m p o r t a n t p r o d u c t s . I f v a l i n e o r any o f t h e o t h e r end p r o d u c t s a l o n e c o u l d r e p r e s s the f o r m a t i o n of AHA s y n t h e t a s e , the p r o d u c t i o n o f the r e m a i n i n g p r o d u c t s would be i m p a i r e d . T h e o r e t i c a l l y , t h i s p roblem s h o u l d be e l i m i n a t e d by 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 et al. 3 1962). However, the e x p e r i m e n t a l e v i d e n c e a v a i l a b l e s u g g e s t s t h a t t h i s mechanism does nottplay a major r o l e i n the r e g u l a t i o n o f AHA-syn-t h e t a s e f o r m a t i o n i n w i l d - t y p e m i c r o - o r g a n i s m s . V. E f f e c t o f S t r e p t o m y c i n on Enzyme F o r m a t i o n F i t z g e r a l d et al. (1948) r e p o r t e d t h a t Sm i n h i b i t e d the s y n t h e s i s o f an a d a p t i v e ( i n d u c i b l e ) b e n z o i c a c i d - o x i d i z i n g -enzyme i n two S m - s e n s i t i v e s p e c i e s o f Mycobacteria. T h i s e f f e c t of Sm on i n d u c i b l e enzyme f o r m a t i o n i n S m - s e n s i t i v e organisms was c o n f i r m e d by a s e r i e s o f s t u d i e s on E. c o l i (Roote and P o l g l a s e , 19S55; P o l g l a s e et al., 19 56; P e r e t z and P o l g l a s e , 1957). I n a d d i t i o n , t h e s e a u t h o r s showed t h a t the p r e s e n c e o r absence of a n t i b i o t i c had no e f f e c t on 3 -g a l a c t o s i d a s e i n d u c t i o n i n S m - r e s i s t a n t ( i n d i f f e r e n t ) mutants and t h a t Sm was o b l i g a t o r y f o r s i g n i f i c a n t B - g a l a c t o s i d a s e p r o d u c t i o n i n the Sm-dependent organism. I n an attempt t o e x p l a i n the e x c r e t i o n o f v a l i n e by Sm-dependent mutants, the s p e c i f i c a c t i v i t y o f AHA s y n t h e t a s e was d e t e r m i n e d i n e x t r a c t s o f S m - s e n s i t i v e , S m - r e s i s t a n t and Sm-dependent c e l l s o f 4 d i f f e r e n t s t r a i n s of E.\>coli ( C o u k e l l and P o l g l a s e , 1965). The r e s u l t s o f t h i s s t u d y i n d i c a t e d t h a t AHA-synthetase f o r m a t i o n was d e r e p r e s s e d i n a l l Sm-dependent mutants grown i n the pre s e n c e o f ex c e s s DHSm when compared t o e i t h e r t he S m - s e n s i t i v e o r g a n i s m o r t h e Sm-r e s i s t a n t mutant grown i n t h e p r e s e n c e o r t h e absence o f a n t i b i o t i c . The s p e c i f i c a c t i v i t i e s o f two o t h e r enzymes, g l u c o k i n a s e and g l u t a m i c dehydrogenase, however, remained c o n s t a n t i n a l l e x t r a c t s . Subsequent work ( P o l g l a s e , 1966a; 1966b) r e v e a l e d t h a t growth o f Sm-dependent mutants on g r o w t h - r a t e - l i m i t i n g c o n c e n t r a t i o n s of a n t i b i o t i c r e p r e s s e d AHA s y n t h e t a s e t o a . l e v e l l e s s t h a n t h a t o b s e r v e d i n t h e c o r r e s p o n d i n g w i l d - t y p e organism. Upon t h e a d d i t i o n o f a n t i b i o t i c , however, AHA-synthetase f o r m a t i o n was r a p i d l y and s p e c i f i c a l l y d e r e p r e s s e d . Thus, DHSm has a p o s i t i v e r e g u l a -t o r y e f f e c t on AHA-synthetase f o r m a t i o n i n Sm-dependent mutants. An e x a m i n a t i o n of m u l t i v a l e n t end-product r e p r e s s i o n o f AHA s y n t h e t a s e i n S m - s e n s i t i v e and i n Sm-dependent organisms of s e v e r a l s t r a i n s of E. c o l i i n d i c a t e d t h a t a l t h o u g h r e p r e s s i o n was s l i g h t , t he AHA s y n t h e t a s e o f t h e Sm-dependent mutants was j u s t as r e s p o n s i v e t o t h i s form o f c o n t r o l as was the AHA s y n t h e t a s e from t h e r e s p e c t i v e p a r e n t S m - s e n s i t i v e organisms ( P o l g l a s e , 1966a). However, d u r i n g t h e cour s e o f t h i s s t u d y , - 16 -i t was no t e d t h a t h i g h c o n c e n t r a t i o n s o f g l u c o s e i n the growth medium c o n s i s t e n t l y r e s u l t e d i n a d i m i n i s h e d AHA-synthetase a c t i v i t y i n e x t r a c t s of w i l d - t y p e E. c o l i B. S i n c e i t has l o n g been known t h a t the s y n t h e s i s o f 6 - g a l a c t o s i d a s e as w e l l as many o t h e r i n d u c i b l e enzymes i s r e p r e s s e d by growth on g l u c o s e (Monod, 1947), t h e ob s e r v e d e f f e c t o f g l u c o s e on AHA s y n t h e t a s e s u g g e s t e d t h a t t h e a n t i b i o t i c - d e p e n d e n t d e r e p r e s s i o n o f enzymes i n Sm-dependent mutants may be the r e s u l t o f i n t e r f e r e n c e o f DHSm w i t h g e n e r a l c a t a b o l i t e r e p r e s s i o n (Magasanik, 1961). V I . O b j e c t o f t h i s I n v e s t i g a t i o n The o b j e c t o f the p r e s e n t i n v e s t i g a t i o n was t o d e t e r -mine whether the d e r e p r e s s e d f o r m a t i o n of AHA s y n t h e t a s e and 3 - g a l a c t o s i d a s e by DHSm i n Sm-dependent mutants was due t o a d i r e c t e f f e c t o f the a n t i b i o t i c on t r a n s c r i p t i o n o r t r a n s -l a t i o n o r whether d e r e p r e s s i o n was mediated through an e f f e c t o f the a n t i b i o t i c on some g e n e r a l r e g u l a t o r y p r o c e s s such as c a t a b o l i t e r e p r e s s i o n . S i n c e p r e l i m i n a r y e x p e r i m e n t s i n d i c a t e d t h a t the f o r -m a t i o n o f AHA s y n t h e t a s e ( l i k e B - g a l a c t o s l d a s e ) was i n h i b i t e d by growth on g l u c o s e , i t was n e c e s s a r y i n i t i a l l y t o d e t e r -mine whether o r not t h e AHA s y n t h e t a s e o f w i l d - t y p e E. c o l i B 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 . S u b s e q u e n t l y , when i t had been e s t a b l i s h e d t h a t t h e f o r m a t i o n o f AHA s y n t h e t a s e was r e g u l a t e d by t h i s - mechanism, c a t a b o l i t e r e p r e s s i o n o f o t h e r enzyme systems was i n v e s t i g a t e d i n Sm-dependent mutants grown on g r o w t h - r a t e - l i m i t i n g and n o n - l i m i t i n g c o n c e n t r a t i o n s - 17 -o f a n t i b i o t i c . As a r e s u l t s o f t h e s e s t u d i e s an attempt was made t o c o r r e l a t e the c o n c e n t r a t i o n of DHSm on w h i c h a Sm-dependent mutant was grown w i t h the growth r a t e o f t h e organ-ism and t h e degree of c a t a b o l i t e r e p r e s s i o n i n the c e l l . I n a d d i t i o n , i t was hoped t h a t t h i s i n v e s t i g a t i o n would c l a r i f y the m e t a b o l i c s i g n i f i c a n c e o f v a l i n e e x c r e t i o n by Sm-dependent m i c r o - o r g a n i s m s . - 18 -METHODS AND MATERIALS I . Organisms ( i ) 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 Escherichia coli were used i n t h i s work; E. coli B (ATCC 11303) and E. coli E, the 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 o f B a c t e r i o l o g y , U n i v e r s i t y o f L a v a l , M o n t r e a l . I n t h i s r e p o r t , t h e s e two S m - s e n s i t i v e s t r a i n s were d e s i g n a t e d SB and SE, r e s p e c t i v e l y . ( i i ) S t r e p t o m y c i n - d e p e n d e n t mutants Spontaneous Sm-dependent mutants were i s o l a t e d from each S m - s e n s i t i v e s t r a i n by the f o l l o w i n g p r o c e d u r e . A volume o f a p p r o x i m a t e l y 500 ml. o f g l u c o s e ( 0 . 4 % ) -s a l t s medium (see METHODS I I , ( i ) ) was i n o c u l a t e d w i t h a Sm-s e n s i t i v e c u l t u r e w h i c h had been s t o r e d on a h e a r t i n f u s i o n agar 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 w i t h o u t a g i t a -t i o n f o r 20^-24 h r . a t 37°. The r e s u l t i n g growth was 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 , g l a s s b o t t l e s and c e n t r i f u g e d a t 1,000 g f o r 1 h r . a t 4°. The p e l l e t was r e -suspended i n 6.0 ml. o f b u f f e r (0.05 M-potassium phosphate, pH 7.4). E x a c t l y 0.5 ml. o f t h e resuspended c e l l s (^10" c e l l s ) were p i p e t t e d on t o t h e s u r f a c e o f s e v e r a l P e t r i p l a t e s c o n t a i n i n g h e a r t i n f u s i o n agar [ -heart i n f u s i o n b r o t h s u p p l e -mented w i t h 1.5% agar and 1 mg./ml. o f d i h y d r o s t r e p t o m y c i n (DHSm)]. The s l u r r y o f c e l l s was e v e n l y d i s t r i b u t e d o v er t h e agar s u r f a c e w i t h t h e a i d o f a g l a s s s p r e a d e r , and t h e p l a t e s - 19 -were i n c u b a t e d a t 37° f o r 24 h r . The c o l o n i e s w h i c h formed were s u b c u l t u r e d i n t o 5 ml. o f s t e r i l e g l u c o s e - s a l t s medium, e i t h e r d e v o i d o f a n t i b i o t i c o r supplemented w i t h 1 mg./ml. o f DHSm, and i n c u b a t e d as d e s c r i b e d above. S m - r e s i s t a n t mutants grew i n b o t h t u b e s , whereas, Sm-dependent mutants grew o n l y i n the p r e s e n c e o f DHSm. To ensure t h a t growth i n the absence o f a n t i b i o t i c was i n f a c t due t o the S m - r e s i s t a n t mutant and n o t t o growth o f a Sm-dependent c u l t u r e r e s u l t i n g from a c a r r y -o v e r o f DHSm from t h e p l a t e , a l o o p f u l o f t h i s c u l t u r e was f u r t h e r t r a n s f e r r e d b o t h t o a n t i b i o t i c supplemented and un-supplemented medium i n t u b e s . Sm-dependent mutants o b t a i n e d i n t h i s way, were m a i n t a i n e d b o t h on h e a r t i n f u s i o n agar s l o p e s a t 5°, and as l y o p h i l i z e d p r e p a r a t i o n s . A l l c u l t u r e s s t o r e d on h e a r t i n f u s i o n agar s l o p e s were s u b c u l t u r e d monthly. Sm-dependent mutants d e r i v e d i n t h i s way from E. c o l i s t r a i n B and s t r a i n E were d e s i g n a t e d DB and DE, r e s p e c t i v e l y . 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 ( i ) Media Throughout t h i s work a m i n i m a l medium was used, w h i c h i s 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) w i t h t h e o m i s s i o n o f c i t r a t e . The c o m p o s i t i o n i s as f o l l o w s : K 2 H P O 4 ( 0 . 7 % ) , KHzPCU ( 0 . 3 % ) , MgSCH .7H 20 (0.02%) and (NH 4)2SG> ( 0 . 1 % ) . The carb o n s o u r c e s ( g l u c o s e , g l y c e r o l , l a c t o s e , l a c t a t e o r g l u c o n a t e ) were a u t o c l a v e d s e p a r a t e l y i n a con-c e n t r a t e d s o l u t i o n and were added as i n d i c a t e d i n each e x p e r i -- 20 -ment. I n c e r t a i n e x p e r i m e n t s , t h e m i n i m a l medium was s u p p l e -mented w i t h s p e c i f i c amino a c i d s o r c a l c i u m p a n t o t h e n a t e as s p e c i f i e d . U n l e s s i n d i c a t e d o t h e r w i s e , Sm-dependent c u l t u r e s were r o u t i n e l y grown on m i n i m a l medium supplemented w i t h 1 mg. of a n t i b i o t i c / m l . (supplemented Sm-dependent c e l l s ) . DHSm ( s e s q u i s u l f a t e ) (Merck, Sharp and Dohme, M o n t r e a l , Canada) was used i n t h i s work. F o r c u l t u r e s w h i c h were t o be grown o v e r n i g h t w i t h o u t a g i t a t i o n , t h e pH o f t h i s medium was a d j u s t e d t o 7.6 by the a d d i t i o n o f sodium h y d r o x i d e s o l u t i o n . ( i i ) E s t i m a t i o n of growth and c e l l mass Growth o f b a c t e r i a l c u l t u r e s was de 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 measuring t h e change i n absorbancy o f t h e c u l -t u r e s a t 420 my ( A 4 2 0 ) 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 con-t a i n i n g e i t h e r d i s t i l l e d w a t e r , f o r c u l t u r e s grown on m i n i m a l medium, o r f r e s h medium when the c u l t u r e s were grown on m i n i m a l medium supplemented w i t h o t h e r c o n s t i t u e n t s . S t a n d a r d c u r v e s r e l a t i n g t o t a l c e l l u l a r p r o t e i n o r c e l l mass (dry w t . ) / m l . o f c u l t u r e t o A 4 2 0 were p r e p a r e d f o r each organism. T o t a l c e l l u l a r p r o t e i n was e s t i m a t e d (see METHODS V I , ( i ) ) on a l i q u o t s ~ b f c e l l s from e x p o n e n t i a l c u l t u r e s . The c e l l s were washed t w i c e i n d i s t i l l e d w a t e r and resuspended i n water t o g i v e a b s o r b a n c i e s r a n g i n g from 0.1 t o 1.0. To deter m i n e c e l l mass, 20 ml. volumes o f t h e washed, resuspended c e l l s were added t o d r y , pre-weighed sample b o t t l e s . The - 21 -b o t t l e s were h e a t e d i n an oven a t 85° f o r 40 h r . , c o o l e d i n a d e s s i c a t o r and r e - w e i g h e d . C e l l mass i s e x p r e s s e d as yg. o f d r y c e l l s / m l . Under t h e c o n d i t i o n s employed f o r t h e growth o f c u l t u r e s , absorbancy was found t o be d i r e c t l y p r o p o r t i o n a l t o t o t a l c e l l u l a r p r o t e i n and t o c e l l mass up t o an A ^ i o o f 1.0. ( i i i ) 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 (a) S t r e p t o m y c i n - s e n s i t i v e and supplemented s t r e p t o m y c i n - d e p e n d e n t c e l l s An i n o c u l u m (50-200 ml.) was grown a e r o b i c a l l y f o r 18 h r . a t 37° under c o n d i t i o n s (medium and a n t i b i o t i c s u p p l e m e n t a t i o n ) s i m i l a r t o t h o s e employed i n t h e f i n a l growth e x p e r i m e n t . 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°) medium (200-1000 ml.) t o an Ai*2o o f a p p r o x i m a t e l y 0.1, and a c a r b o n s o u r c e was added t o g i v e the d e s i r e d i n i t i a l c o n c e n t r a t i o n . 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 a t 37° u n t i l the absorbancy r e a c h e d 0.8-0.9. Growth was t h e n t e r m i n a t e d by c h i l l i n g t h e c u l t u r e . The c e l l s were h a r v e s t e d by c e n t r i f u g a t i o n (16,300 g f o r 20 min.) 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°. A l l c e n t r i f u g a t i o n s were performed a t 4°. 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 24 h r . b e f o r e t h e y were d i s r u p t e d and a s s a y e d f o r enzymic a c t i v i t y . (b) A n t i b i o t i c - l i m i t e d , s t r e p t o m y c i n - d e p e n d e n t c e l l s The Sm-dependent c u l t u r e was t r a n s f e r r e d from a h e a r t i n -f u s i o n agar s l o p e t o a p p r o x i m a t e l y 50 m l . o f m i n i m a l s a l t s - 22 -medium c o n t a i n i n g 30 ug. o f a n t i b i o t i c / m l . The c u l t u r e was 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 u n t i l heavy growth r e s u l t e d (24-48 h r . ) . A p o r t i o n (2-4 ml.) o f t h i s c u l t u r e .-was t r a n s -f e r r e d t o a n o t h e r 50 m l . of t h e same medium and the p r o c e d u r e was r e p e a t e d . A f t e r t h r e e o r f o u r such t r a n s f e r s ? , t h e e n t i r e c u l t u r e (50 ml.) was added t o 500 m l . o f the same medium and t h e c e l l s were grown a e r o b i c a l l y f o r 18 h r . a t 37°. The r e s u l t i n g c e l l s were c e n t r i f u g e d a t 16,300 g f o r 2 0 min. and r e suspended i n 6 1. o f f r e s h , warm (37°) medium t o g i v e an Ai,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 d i v i d e d i n t o s i x 1 - l i t e r p o r t i o n s , and supplemented w i t h the r e q u i r e d amount o f DHSm u s u a l l y i n the range 3-25 ug./ml. A carbon s o u r c e was added and the c u l t u r e s were grown a e r o b i c a l l y a t 37° t o an Al* 20 o f 0.9-1.0. The c e l l s were h a r v e s t e d and s t o r e d as d e s c r i b e d above. C e l l s grown i n t h i s way a r e r e f e r r e d t o as " a n t i b i o t i c - l i m i t e d , Sm-dependent c e l l s " . (c) A n t i b i o t i c - d e p l e t e d , s t r e p t o m y c i n - d e p e n d e n t c e l l s A c u l t u r e (50 ml.) o f Sm-dependent c e l l s was grown a e r o -b i c a l l y f o r 18 h r . a t 37° on m i n i m a l medium supplemented w i t h 0.4% g l u c o s e and 100 ug. o f DHSm/ml. The c e l l s were c e n t r i -f u ged a t 16,300 g f o r 20 min., washed once w i t h f r e s h , warm (37°) a n t i b i o t i c - f r e e medium ( g l u c o s e o m i t t e d ) and r e s u s -pended i n 1 l i t e r o f t h e same medium t o g i v e an A* 20 o f 0.10-0.15. G l u c o s e (0.4%) was added and t h e c u l t u r e was i n c u b a t e d a t 37° w i t h a e r a t i o n . A f t e r 6 h r . t h e c u l t u r e was c e n t r i -f u ged a t 16,300 g f o r 20 min. and t h e c e l l s were resuspended - 23 -i n the same volume o f a n t i b i o t i c - f r e e medium t o an A i t 2 o o f 0.35-0.40. 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 a t 37° f o r a f u r t h e r 6 h r . I f t h e c u l t u r e e x h i b i t e d a r i t h m e t i c growth d u r i n g t h e f i n a l 3 h r . t h e c e l l s were h a r v e s t e d as p r e v i o u s l y d e s c r i b e d . I f a r i t h m e t i c growth d i d n o t o c c u r d u r i n g t h i s p e r i o d , t h e time o f i n c u b a t i o n was extended 1-2 h r . p r i o r t o h a r v e s t i n g . C e l l s s t a r v e d f o r a n t i b i o t i c i n t h i s way a r e r e f e r r e d t o as " a n t i b i o t i c - d e p l e t e d , Sm-dependent c e l l s " . ( i v ) 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 and permea-b i l i z e d c e l l s (a) S o n i c t r e a t m e n t To d i s r u p t t h e c e l l s by s o n i c o s c i l l a t i o n , t h e wet, packed c e l l s were resuspended homogeneously i n . i c e - c o l d 0.1 M-p o t a s s i u m phosphate b u f f e r , pH 8.0, a t a c o n c e n t r a t i o n o f 1 g. o f c e l l s (wet wt.)/15 ml. o f b u f f e r . Twelve-20 ml. o f s u s p e n s i o n were p l a c e d i n a 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 and t h e c e l l s were d i s r u p t e d f o r 4 min. (b) D i s r u p t i o n i n a F r e n c h p r e s s u r e c e l l A p p r o x i m a t e l y 20 ml. o f the c e l l s u s p e n s i o n d e s c r i b e d above (see METHODS I I , ( i v ) ! , ( a ) ) were added t o a f r e s h l y g r e a s e d , c h i l l e d (5°) F r e n c h p r e s s u r e c e l l (American I n s t r u m e n t Co., S i l v e r S p r i n g , M a r y l a n d , U.S.A.). The c e l l s were d i s r u p t e d by a s i n g l e passage t h r o u g h t h e c e l l w i t h a p r e s s u r e d i f -f e r e n t i a l o f 15,000 p . s . i . E n z y m a t i c a c t i v i t i e s o f e x t r a c t s p r e p a r e d by s o n i c - 24 -t r e a t m e n t o r by t r e a t m e n t i n a F r e n c h p r e s s u r e c e l l were d e t e r m i n e d e i t h e r i n t h e crude c e l l e x t r a c t s o r i n the s u p e r -n a t a n t f l u i d s f o l l o w i n g c e n t r i f u g a t i o n a t 12,000 g f o r 10 min. Crude e x t r a c t s and s u p e r n a t a n t f l u i d s were s t o r e d a t -20°. (c) D i s r u p t i o n by g r i n d i n g w i t h a l u m i n a To d i s r u p t t h e c e l l s by a l u m i n a g r i n d i n g , a p p r o x i m a t e l y 1 g. (wet wt.) o f packed c e l l s was p l a c e d i n a p r e c h i l l e d (5°) p o r c e l a i n m o r t a r and 1 g. o f c h i l l e d a l u m i n a (Merck and Co. L t d . ) was added. The c e l l s were ground v i g o r o u s l y t o a t h i c k p a s t e (5 min.) and an a d d i t i o n a l 1 g. o f a l u m i n a was added. G r i n d i n g was c o n t i n u e d f o r a f u r t h e r 10 min. The p a s t e was t h e n suspended e v e n l y i n 15 m l . o f 0.1 M-potassium phosphate b u f f e r , pH 8.0, t r a n s f e r r e d t o a c e n t r i f u g e tube and c e n t r i -f u ged a t 12,000 g f o r 10 min. The r e s u l t i n g s u p e r n a t a n t f l u i d was e i t h e r a s s a y e d i m m e d i a t e l y o r s t o r e d a t -20°. (d) Toluene t r e a t m e n t P e r m e a b i l i z e d c e l l s were p r e p a r e d by t r e a t i n g the r e s u s -pended c e l l s (see METHODS I I , ( i v ) , ( a ) ) w i t h t o l u e n e i n the f o l l o w i n g manner. D i s t i l l e d t o l u e n e was added t o 3.0 m l . o f c e l l s 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 e i t h e r 5 o r 10% v/v and t h e s u s p e n s i o n was a g i t a t e d v i g o r o u s l y on.a V o r t e x m i x e r f o r 1 min. and t h e n p l a c e d i n a 37° w a t e r b a t h . A f t e r t h e d e s i r e d i n c u b a t i o n time (2 o r 6 m i n . ) , t h e c e l l s were t r a n s -f e r r e d t o i c e and a s s a y e d i m m e d i a t e l y . Under t h e s e c o n d i t i o n s l e s s t h a n 15% o f t h e t o t a l c e l l u l a r p r o t e i n was r e l e a s e d from t h e c e l l . - 25 -I I I . g - G a l a c t o s i d a s e I n d u c t i o n E x p e r i m e n t s ( i ) D i a u x i e growth The c e l l s were grown i n a G i l s o n (Omni) s h a k i n g w a t e r b a t h f o r 18 h r . a t 37° i n m i n i m a l medium supplemented w i t h 0.2% o f g l u c o s e . To remove t h e r e s i d u a l c o n s t i t u e n t s o f the medium, t h e c u l t u r e s were h a r v e s t e d by c e n t r i f u g a t i o n (12,000 g f o r 10 min.) and resuspended i n o n e - h a l f t h e o r i g i n a l c u l -t u r e volume o f 0.1 M-potassium phosphate b u f f e r , pH 7.4 a t 37°. The s u s p e n s i o n s were a g a i n c e n t r i f u g e d and the washed c e l l s were resuspended i n f r e s h m i n i m a l medium t o g i v e an A 4 2 . 0 of a p p r o x i m a t e l y 0.1. A f t e r t h e a d d i t i o n o f g l u c o s e (0.025%) and l a c t o s e (0.20%) t h e c u l t u r e s were v i g o r o u s l y a e r a t e d by means o f a s p a r g e r d u r i n g a g i t a t i o n i n t h e s h a k i n g w a t e r b a t h a t 37°. The Ait 2 0 o f t h e c u l t u r e was f o l l o w e d d u r i n g growth and, a t i n t e r v a l s , 1.0 ml. samples o f c u l t u r e were removed f o r d e t e r m i n a t i o n o f B - g a l a c t o s i d a s e a c t i v i t y (see METHODS V I I , ( i ) ) . Growth was g e n e r a l l y e x p r e s s e d as a change i n the ug. o f t o t a l c e l l u l a r p r o t e i n / m l . o f c u l t u r e . P r e l i m i n a r y e x p e r i m e n t s e s t a b l i s h e d t h a t under t h e s e c o n d i -t i o n s , a l l t he g l u c o s e was consumed when t h e p r o t e i n concen-t r a t i o n r e a c h e d 85-95 ug./ml. (see F i g s . 8 and 9 ) . ( i i ) D e t e r m i n a t i o n o f t h e d i f f e r e n t i a l r a t e o f 6-g a l a c t o s i d a s e s y n t h e s i s The i n o c u l u m was grown a e r o b i c a l l y f o r 18 h r . a t 37° i n m i n i m a l medium supplemented w i t h 0.2% o f g l y c e r o l . The c e l l s were washed once (see METHODS I I I , ( i ) ) and resuspended i n - 26 -f r e s h m i n i m a l medium t o g i v e an A^zo o f 0.10-0.15. G l y c e r o l (0.1%) and l a c t o s e (0.2%) were added and the c u l t u r e was shaken v i g o r o u s l y a t 37°. Samples (1.0 ml.) o f c u l t u r e were t a k e n a t f r e q u e n t i n t e r v a l s a f t e r the i n i t i a t i o n o f growth and assayed f o r B - g a l a c t o s i d a s e a c t i v i t y (see METHODS V I I , ( i ) ) . U n i t s o f enzyme a c t i v i t y / m l . o f c u l t u r e were p l o t t e d a g a i n s t t h e t o t a l p r o t e i n c o n c e n t r a t i o n . The s l o p e o f the r e s u l t i n g l i n e i n d i c a t e s t h e r a t e o f B - g a l a c t o s i d a s e s y n t h e -s i s r e l a t i v e t o the r a t e o f t o t a l p r o t e i n s y n t h e s i s i n the c e l l ; i . e . the d i f f e r e n t i a l r a t e o f B - g a l a c t o s i d a s e s y n t h e s i s . ( i i i ) B - G a l a c t o s i d a s e s y n t h e s i s i n p s e u d o - r e s t i n g c e l l s Owing t o t h e extreme s e n s i t i v i t y o f B - g a l a c t o s i d a s e i n d u c t i o n t o c a t a b o l i t e r e p r e s s i o n , the e f f e c t s o f d i f f e r e n t c a r b o n s o u r c e s on B - g a l a c t o s i d a s e i n d u c t i o n c o u l d n o t be e v a l u a t e d r e p r o d u c i b l y i n c e l l s s t a r v e d f o r n i t r o g e n . To overcome t h i s problem a " p s e u d o - r e s t i n g " c e l l system was de-v i s e d i n w h i c h b o t h a carbon s o u r c e and a n i t r o g e n s o u r c e were added t o c e l l s p r e v i o u s l y s t a r v e d f o r b o t h o f t h e s e compounds. C u l t u r e s grown a e r o b i c a l l y on g l y c e r o l (0.2%) at' 37° were h a r v e s t e d by c e n t r i f u g a t i o n a t an A 4 2 0 o f 1.0 and were washed once i n warm (37°) 0.1 M-potassium phosphate b u f f e r , pH 7.4. The washed c e l l s were resuspended i n b u f f e r o f the same c o m p o s i t i o n and were shaken v i g o r o u s l y a t 37° f o r 30 min. A t t h e end o f t h i s i n c u b a t i o n p e r i o d , t h e absorbancy o f the c u l t u r e was a d j u s t e d t o 1.0 w i t h b u f f e r and the c e l l s were t r a n s f e r r e d t o a 30° water b a t h . A f t e r 5 min., a c a r b o n s o u r c e ( g l y c e r o l , 0.05%), a n i t r o g e n s o u r c e (ammonium s u l -f a t e , 0.1%) and t h e i n d u c e r , l a c t o s e (0.2%) were added and 3 - g a l a c t o s i d a s e f o r m a t i o n was f o l l o w e d d u r i n g a 2 h r . i n d u c -t i o n p e r i o d . C e l l s u s p e n s i o n s t r e a t e d i n t h i s manner, showed les^s t h a n 15% i n c r e a s e i n A i ^ o d u r i n g t h e 2 h r . i n d u c t i o n p e r i o d and were r e f e r r e d t o as " p s e u d o - r e s t i n g c e l l s " . IV. C e l l Y i e l d E x p e r i m e n t s ( i ) D e f i n i t i o n C e l l y i e l d on a m i n i m a l ammonium s u l f a t e medium can be d e f i n e d (Monod, 1942) by t h e f o r m u l a : where Y i s t h e c e l l y i e l d c o n s t a n t ; G i s t h e t o t a l change i n c e l l mass; and C i s t h e t o t a l amount o f energy s o u r c e consumed. I n t h i s s t u d y , Y i s e q u i v a l e n t t o yg. d r y wt. o f c e l l s formed/ yg. d r y wt. o f c a r b o n ' s o u r c e consumed. ( i i ) A e r o b i c c e l l y i e l d from g l u c o s e C e l l s g r o wing e x p o n e n t i a l l y a t 37° on m i n i m a l medium ( w i t h o r w i t h o u t a n t i b i o t i c ) supplemented w i t h 0.4%-glucose were h a r v e s t e d by c e n t r i f u g a t i o n , washed once w i t h 0.1 M-p o t a s s i u m phosphate b u f f e r , pH 7.0, and resuspended i n f r e s h , warm (37°) g l u c o s e - f r e e medium t o g i v e an A i+ 2 o o f a p p r o x i m a t e l y 0.1. The washed c e l l s were i n c u b a t e d a e r o b i c a l l y a t 37° f o r 30 min. A f t e r t h i s t r e a t m e n t , the absorbancy was r e a d j u s t e d t o 0.1 w i t h warm medium and a known amount of g l u c o s e ( u s u a l l y s u f f i c i e n t 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 400 - 2 8 -yg./ml.) was added. A 2 . 0 ml. p o r t i o n o f c u l t u r e was i m m e d i a t e l y t a k e n f o r g l u c o s e d e t e r m i n a t i o n (see METHODS V I ( i i ) ) and t h e c u l t u r e was shaken v i g o r o u s l y a t 3 7 ° . The i n c r e a s e i n a b s o r -bancy o f t h e c u l t u r e was f o l l o w e d c l o s e l y u n t i l growth ceased. A t t h i s t i m e a n o t h e r sample of c u l t u r e was removed f o r g l u c o s e d e t e r m i n a t i o n . To ensure t h a t growth was compl e t e , the A i ^ o o f t h e c u l t u r e was checked a t 1 0 min. i n t e r v a l s f o r a f u r t h e r 4 0 - 6 0 min. Some e x p e r i m e n t s concerned a stu d y o f t h e r a t e o f uptake of g l u c o s e i n t o t h e c e l l s r e l a t i v e t o t h e r a t e o f f o r m a t i o n of c e l l mass. I n t h e s e e x p e r i m e n t s , samples f o r g l u c o s e e s t i m a t i o n were t a k e n a t more f r e q u e n t i n t e r v a l s d u r i n g t h e growth o f t h e c u l t u r e . P o r t i o n s of c u l t u r e t o be assaye d f o r g l u c o s e were im-m e d i a t e l y p l a c e d i n 1 5 m l . t h i c k - w a l l e d , g l a s s c e n t r i f u g e tubes p r e v i o u s l y c h i l l e d i n an i c e - s a l t b a t h . Under t h e s e c o n d i t i o n s the temp e r a t u r e o f t h e sample dropped from 3 7 ° t o 8 ° i n 1% min. A f t e r 5 - 1 0 min. i n t h e i c e - s a l t b a t h , t h e c u l t u r e s were c e n t r i f u g e d a t 1 7 , 0 0 0 g f o r 1 2 min. and t h e s u p e r n a t a n t was t r a n s f e r r e d t o a c l e a n , d r y tube and s t o r e d a t - 2 0 ° . A l l samples were assay e d f o r g l u c o s e (see METHODS V I , ( i i ) ) w i t h i n 7 days. ( i i i ) A n a e r o b i c c e l l y i e l d from g l u c o s e To e s t i m a t e t h e a n a e r o b i c c e l l y i e l d from g l u c o s e washed, e x p o n e n t i a l c e l l s s t a r v e d f o r g l u c o s e were p r e p a r e d - 29 -as d e s c r i b e d i n METHODS I V , ( i i ) . The washed c e l l s were r e -suspended i n f r e s h , warm (37°) m i n i m a l medium (150 ml.) t o g i v e an Ait 20 o f 0.1. G l u c o s e was added t o g i v e a f i n a l con-c e n t r a t i o n o f 900 ug./ml. The c u l t u r e was grown a e r o b i c a l l y and p o r t i o n s o f t h e c u l t u r e were removed f o r g l u c o s e d e t e r -m i n a t i o n as p r e v i o u s l y d e s c r i b e d (see METHODS IV, ( i i ) ) . When the Al* 20 o f t h e c u l t u r e r e a c h e d a p p r o x i m a t e l y 0.4, t h e e n t i r e c u l t u r e was r a p i d l y t r a n s f e r r e d t o a 250 ml. g r a d u a t e d c y l i n d e r p a r t i a l l y immersed i n a water b a t h a t 37°. The c y l i n d e r was q u i c k l y capped w i t h a r u b b e r s t o p p e r f i t t e d w i t h t h r e e p i e c e s o f g l a s s t u b i n g and s e a l e d around t h e r i m w i t h p l a s t i c i n e and t a p e . S p e c t r o s c o p i c grade n i t r o g e n (Canadian L i q u i d A i r L t d . , Vancouver, Canada) was bu b b l e d v i g o r o u s l y beneath t h e s u r f a c e o f t h e c u l t u r e t h r o u g h one tub e . A f t e r 5 min., the tube s u p p l y i n g n i t r o g e n was withdrawn from t h e c u l t u r e and e l e v a t e d t o a p o s i t i o n a p p r o x i m a t e l y 4 cm. above the s u r f a c e o f t h e l i q u i d . The f l o w o f n i t r o g e n was d e c r e a s e d t o a r a t e w h i c h would m a i n t a i n a n a e r o b i c c o n d i t i o n s w h i l e c a u s i n g m i n i -mal e v a p o r a t i o n . Samples o f c u l t u r e c o u l d be removed w i t h o u t d i s t u r b i n g a n a e r o b i o s i s by c l o s i n g t h e n i t r o g e n e x h a u s t tube c a u s i n g t h e i n c r e a s e d p r e s s u r e t o d r i v e t h e c u l t u r e up t h e t h i r d tube i n t o a c o l l e c t i n g f l a s k . The i n i t i a l p o r t i o n o f c u l t u r e (5-10 ml.) from each s a m p l i n g was d i s c a r d e d , w h i l e subsequent samples were used t o de t e r m i n e t h e c e l l mass and g l u c o s e c o n t e n t o f t h e c u l t u r e . G l u c o s e uptake was f o l l o w e d c l o s e l y u n t i l growth ceased. -The a n a e r o b i c c e l l y i e l d from - 30' -g l u c o s e was c a l c u l a t e d from t h e i n c r e a s e i n c e l l mass (dry wt.) and g l u c o s e consumption o c c u r r i n g a f t e r c h a n g i n g from a e r o b i c t o a n a e r o b i c c o n d i t i o n s . ( i v ) A e r o b i c c e l l y i e l d from g l u c o n a t e The e f f i c i e n c y o f g l u c o n a t e u t i l i z a t i o n f o r c e l l s y n t h e -s i s was d e t e r m i n e d i n washed, g l u c o n a t e - g r o w n c e l l s p r e p a r e d as d e s c r i b e d i n METHODS I V , ( i i ) . The c e l l s were suspended i n f r e s h , warm ( 3 7 ° ) m i n i m a l medium t o g i v e an A 1*20 o f 0 . 1 , and were supplemented w i t h a known amount o f g l u c o n a t e ( u s u a l l y 4 0 0 y g . / m l . ) . The c u l t u r e was grown a e r o b i c a l l y a t 3 7 ° u n t i l t he i n c r e a s e i n absorbancy o f t h e c u l t u r e ceased. The change i n c e l l mass o f t h e c u l t u r e was o b t a i n e d from a s t a n d a r d c u r v e r e l a t i n g A i t 2 o t o yg. d r y c e l l s / m l . V. E s t i m a t i o n o f t h e Rate o f ATP F o r m a t i o n and t h e ATP P o o l The r a t e of ATP f o r m a t i o n and t h e c o n c e n t r a t i o n o f the ATP p o o l i n e x p o n e n t i a l c e l l s were s t u d i e d i n c u l t u r e s t r e a t e d i n two d i f f e r e n t ways. ( i ) Method I C e l l s growing e x p o n e n t i a l l y on g l u c o s e ( 0 . 4 % ) - s a l t s medium a t 3 7 ° were d i l u t e d i n f r e s h , warm medium of t h e same c o m p o s i t i o n t o g i v e an Ai,2o o f a p p r o x i m a t e l y 0 . 2 5 . The d i -l u t e d c u l t u r e was p l a c e d on a shaker a t 3 7 ° and a g i t a t e d v i g o r o u s l y . Samples ( 2 . 0 ml.) o f c u l t u r e were removed f o r ATP d e t e r m i n a t i o n (see METHODS V I , ( i i i ) ) a t A k 2 o i n c r e m e n t s o f 0 . 0 7 - 0 . 1 0 from 0 . 3 t o 1 . 0 . - 31 -A l i q u o t s o f c u l t u r e t o be assay e d f o r ATP were i m m e d i a t e l y added t o 0.2 m l . p o r t i o n s o f i c e - c o l d 30% (w/v) p e r c h l o r i c a c i d i n c e n t r i f u g e t u b e s . A f t e r 10 min. on i c e , the e x t r a c t s were shaken w e l l and t h e n n e u t r a l i z e d w i t h 0.8 ml. o f 2M-K0H (the f i n a l pH was 7.0-7.8). The p r e c i p i t a t e was removed by cen-t r i f u g a t i o n and t h e s u p e r n a t a n t f l u i d s were e i t h e r a s s a y e d i m m e d i a t e l y o r s t o r e d a t -20°. A l l e x t r a c t s were assayed, w i t h -i n 4 8 h r . ( i i ) Method I I In some e x p e r i m e n t s the i n i t i a l c o n c e n t r a t i o n o f t h e ATP p o o l o f t h e c e l l s was d e c r e a s e d below t h e l e v e l n o r m a l l y o b s e r v e d i n e x p o n e n t i a l c u l t u r e s by t r e a t i n g t h e c e l l s i n th e f o l l o w i n g manner. E x p o n e n t i a l c u l t u r e s growing a e r o b i -c a l l y on g l u c o s e ( 0 . 1 % ) - s a l t s medium a t 37° were r a p i d l y h a r -v e s t e d by c e n t r i f u g a t i o n (12,000 g f o r 10 min.) a t 4°. The c e l l s were washed once w i t h c o l d (0°) 0.1 M-potassium phos-phate b u f f e r , pH 7.4 and resuspended i n warm (37°) g l u c o s e -f r e e m i n i m a l medium t o g i v e an A 4 2 o o f 0.20-0.25. The f l a s k was s t o p p e r e d t i g h t l y and i n c u b a t e d as a s t a t i o n a r y c u l t u r e a t 37° f o r 30 min. I t was found t h a t t h i s t r e a t m e n t r e s u l t e d i n a 50-75% r e d u c t i o n i n the i n i t i a l c o n c e n t r a t i o n o f t h e ATP p o o l . A t t h e end o f t h i s p e r i o d , g l u c o s e (0.4%) was added and the c u l t u r e was grown a e r o b i c a l l y t o an A 4 2 0 o f 1.0. P o r t i o n s o f c u l t u r e were removed and t r e a t e d w i t h p e r c h l o r i c a c i d as d e s c r i b e d i n t h e p r e v i o u s s e c t i o n . - 32 -V I . C h e m i c a l A n a l y s i s ( i ) 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 des-c r i b e d by Lowry et at. (1951) . B o v i n e Y-<?l°kulin (Calbiochem, Los A n g e l e s , C a l i f . , U.S.A.) was used as a s t a n d a r d . P r o t e i n samples were g e n e r a l l y d i l u t e d ( w i t h d i s t i l l e d water) t o c o n t a i n 25 t o 250 yg. o f p r o t e i n / m l . ( i i ) G l u c o s e G l u c o s e was d e t e r m i n e d by the commercial p r e p a r a t i o n , G l u c o s t a t (Worthington B i o c h e m i c a l Corp., F r e e h o l d , N.J., U.S.A.) as f o l l o w s : t o 0.5 ml. a l i q u o t s c o n t a i n i n g 0-300 yg. o f g l u c o s e / m l . were added 2.5 m l . of 0.1 M-potassium phosphate b u f f e r , pH 7.0 and 2.0 ml. o f t h e G l u c o s t a t r e a g e n t (see b e l o w ) . T h i s m i x t u r e was i n c u b a t e d a t 37° u n t i l t he r e a c t i o n had r e a c h e d c o m p l e t i o n (30 m i n . ) . Two drops o f 6 N HCl were added t o each t u b e , f o l l o w e d by 5.0 ml. o f d i s t i l l e d w a t e r . The c o n t e n t s o f the tube were mixed v i g o r -o u s l y on a V o r t e x m i x e r and l e f t a t room t e m p e r a t u r e f o r 3—5 min. T h e - 2 0 o f the r e s u l t i n g s o l u t i o n s was measured 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 i n t o yg. o f g l u c o s e . A f t e r the s o l u t i o n i s a c i d i f i e d ( w i t h 6 N HCl) t h e c o l o r i s s t a b l e a t room t e m p e r a t u r e f o r s e v e r a l h r . The G l u c o s t a t r e a g e n t was p r e p a r e d by d i s s o l v i n g one v i a l of l y o p h i l i z e d enzyme p r e p a r a t i o n and one v i a l o f chromogen i n 50 m l . o f d i s t i l l e d w a t e r . The pH was a d j u s t e d , i f neces-- 33 -sary, t o 7.0 and the s o l u t i o n was s t o r e d a t 5° i n the d a r k . As t h e r e c o n s t i t u t e d G l u c o s t a t r e a g e n t i s extremely u n s t a b l e , i t was p r e p a r e d i m m e d i a t e l y b e f o r e use. ( i i i ) ATP ATP was d e t e r m i n e d e n z y m a t i c a l l y by a l u c i f e r a s e a s s a y system based on a method d e s c r i b e d by C o l e et al. (1967). (a) R e c o n s t i t u t i o n o f t h e f i r e f l y l a n t e r n e x t r a c t A l y o p h i l i z e d p r e p a r a t i o n o f f i r e f l y l a n t e r n s (250 mg.) (Sigma FLE-250) was d i s s o l v e d i n 25 m l . o f d i s t i l l e d w a t e r . A c c o r d i n g t o t h e s u p p l i e r (Sigma), a f t e r r e c o n s t i t u t i o n , the s u s p e n s i o n c o n t a i n e d 0.05 M-potassium a r s e n a t e and 0.02 M-magnesium s u l f a t e a t pH 7.4. The s u s p e n s i o n was s t o r e d a t 0° f o r 24 h r . , c l a r i f i e d by c e n t r i f u g a t i o n a t 5,000 g f o r 10 min.. and t h e n s t o r e d i n 1.0 m l . a l i q u o t s a t -20°. (b) Assay system The f o l l o w i n g components were added t o a c l e a n , d r y s c i n t i l l a t i o n v i a l : 0.2 m l . o f 0.2 M ^ g l y c y l g l y c i n e b u f f e r , pH 7.4; 0.7 m l . o f d i s t i l l e d w a t e r ; 0.05 ml.of r e c o n s t i t u t e d f i r e f l y l a n t e r n e x t r a c t and 0.02 m l . o f a s o l u t i o n c o n t a i n i n g ATP ( s t a n d a r d or sample). The v i a l was i m m e d i a t e l y shaken, capped and p l a c e d i n a N u c l e a r - C h i c a g o Mark I s c i n t i l l a t i o n c o u n t e r w h i c h p r e v i o u s l y had been programmed t o t h e most s e n s i t i v e s e t t i n g . F o l l o w i n g a s t a n d a r d d e l a y time o f 45 s e c . a f t e r t h e a d d i t i o n o f the ATP s o l u t i o n , f i v e 12 s e c . c o u n t s - 34 -were t a k e n a t 15 s e c . i n t e r v a l s . Under t h e s e c o n d i t i o n s , t h e l u m i n e s c e n c e decays e x p o n e n t i a l l y . T h e r e f o r e , i t was p o s s i b l e t o e s t i m a t e t h e maximum luminescence by e x t r a p o l a t i n g back t o z e r o t i m e . The background c o u n t , under t h e s e c o n d i t i o n s , was 8-12 counts/12 s e c , whereas, sample c o u n t s g e n e r a l l y ranged from 1 0 3 t o 1 0 6 over the same time p e r i o d . A s t o c k s o l u t i o n o f 1 mM ATP was. p r e p a r e d i n d i s t i l l e d w a t er and f r o z e n a t -20°. Each day f r e s h d i l u t e ATP s t a n d a r d s c o n t a i n i n g 2-2 5 uumoles/0.02 ml. were p r e p a r e d from t h e s t o c k s o l u t i o n . . The ATP c o n t e n t o f t h e p e r c h l o r i c a c i d - e x t r a c t e d c e l l s i s e x p r e s s e d as mumoles o f ATP/ml. of c u l t u r e . The ATP p o o l i s d e f i n e d as mumoles o f ATP/mg. d r y wt. o f b a c t e r i a l c e l l s . ( i v ) V a l i n e The v a l i n e c o n t e n t o f c u l t u r e s u p e r n a t a n t f l u i d s was e s t i m a t e d c h r o m a t o g r a p h i c a l l y by t h e method of Bode (1955). Samples (50 ml.) o f s u p e r n a t a n t f l u i d s from c u l t u r e s c e n t r i -f u ged a t 16,000 g f o r 20 min. were c o n c e n t r a t e d 10 or 2 0 - f o l d by e v a p o r a t i o n t o d r y n e s s under d i m i n i s h e d p r e s s u r e a t 50° and r e s u s p e n d i n g the r e s i d u e i n d i s t i l l e d w a t e r . E x a c t l y 0.10 ml. o f each c o n c e n t r a t e d sample and a s e r i e s o f v a l i n e s t a n d a r d s (0.1 t o 1.0 mM) were s p o t t e d on a s h e e t o f Whatman No. 1 chromatography paper. The chromatogram was r u n by d e s c e n d i n g chromatography f o r 16 h r . i n a s o l v e n t composed of b u t a n o l - a c e t i c a c i d - w a t e r (4:1:1 v / v ) . The d r i e d chroma-togram was d e v e l o p e d by immersing i t i n a n i n h y d r i n s o l u t i o n - 3 5 -(ninhydrin, 5 0 0 mg.; acetone, 9 0 ml.; water, 5.0 ml. and acetic acid, 5.0 ml.) followed by heating at 90° for 1 5 min. The r e s u l t i n g spots were fixed by treating the chromatogram with a copper n i t r a t e solution (saturated aqueous CUNO3, 1.0 ml; concentrated n i t r i c acid, 0.02 ml. and acetone, 99 ml.). To-measure valine q u a n t i t a t i v e l y , the spots were excised from the chromatogram, shredded, and eluted with 1.0 ml. of methanol. The absorbancy of the eluant was determined at 5 3 0 my. The ymoles of valine present was determined from a standard curve prepared i n an i d e n t i c a l manner. VII. Enzyme Assays A l l spectrophotometric determinations were made with a Cary 1 5 recording spectrophotometer at 25°. S p e c i f i c a c t i -v i t i e s were calculated as units of enzyme/mg. of protein. (i) g-Galactosidase 3-Galactosidase was determined i n samples (1.0 ml.) of culture by the method of Pardee et al. (1959). Toluene (1 drop) and 3-mercaptoethanol ( f i n a l concentration 1 5 mM) were added to the c e l l suspensions. The c e l l s were lysed by agit a t i o n on a Vortex mixer for 3 0 sec. followed by incubation at 37° for 3 0 min. The contents of the tubes were then brought to 28°; 0.2 ml. of a solution of 0.02 M-o-nitrophenyl 3-D galactopyranoside i n 0.25 M-sodium phosphate buffer, pH 7.0, was added, and the tubes were incubated a measured period of time, u n t i l the desired i n t e n s i t y of color had developed. The - 36 -r e a c t i o n was stopped by t h e a d d i t i o n o f 0.5 ml. o f 1 M-sodium c a r b o n a t e , and t h e absorbancy was d e t e r m i n e d a t 4 20 my. A c o r r e c t i o n f o r t u r b i d i t y o f the c e l l s c o u l d be made by mul-t i p l y i n g t h e A 5 5 o o f t h e s o l u t i o n by 1.60 and s u b t r a c t i n g t h i s v a l u e from t h e A 4 2 0 of t h e same s o l u t i o n . Under t h e above c o n d i t i o n s , 1 mymole o f o - n i t r o p h e n o l / m l . has an A 4 2 0 o f 0.00 75 (Pardee et al.3 1959). One u n i t o f enzyme i s de-f i n e d as t h e amount o f enzyme t h a t c a t a l y z e s t h e h y d r o l y s i s o f 1 mymole o f o - n i t r o p h e n y l 3 - D - g a l a c t o p y r a n o s i d e / m i n . a t 28° and pH 7.0. ( i 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 A l t h o u g h AHA s y n t h e t a s e i s s t a b l e f o r s e v e r a l days i n s t o r e d , whole packed c e l l s a t 0°, i t i s e x t r e m e l y l a b i l e i n c e l l e x t r a c t s (Desai and P o l g l a s e , 1965) . T h e r e f o r e , AHA-s y n t h e t a s e a c t i v i t y was d e t e r m i n e d o n l y on f r e s h l y p r e p a r e d c r u d e c e l l e x t r a c t s . Each a s s a y tube c o n t a i n e d , 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 , pH 8.0, 100 ymoles; sodium p y r u -v a t e , 0.25 m-mole; M g C l 2 r 0.5 ymole; f l a v i n a denine d i n u -c l e o t i d e (FAD), 10 mymoles; t h i a m i n e pyrophosphate (TPP), 0.3 ymole and 0.5 m l . o f crude e x t r a c t (2.5-3.0 mg. o f p r o t e i n ) . 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 stopped by t h e a d d i t i o n o f 0.1 m l . of 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 f o r 15 min. a t 60° t o c o n v e r t t h e a - a c e t o l a c t a t e i n 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 1 0 0 - f o l d and a p o r t i o n o f t h e r e s u l t i n g s o l u t i o n was - 37 -a n a l y z e d f o r a c e t o i n by the method of W e s t e r f e l d (1945). To a 1.0 m l . 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 m l . 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 2.5 M-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 the c o l o r a l l o w e d t o d e v e l o p f o r 1 h r . i n the 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 ymoles of a c e t o i n (or a - a c e t o l a c t a t e ) by means of 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 ymole of a c e t o i n / h r . at-37°. - ( i i i ) T h r e o n i n e d e h y d r a t a s e The a s s a y system c o n s i s t e d o f the f o l l o w i n g i n a t o t a l volume of 1.0 m l . : p o t a s s i u m phosphate b u f f e r , pH 8.0, 100 ymoles; L - t h r e o n i n e , 80 ymoles; p y r i d o x a l phosphate, 10 0 mymoles and 0.5 m l . o f crude c e l l e x t r a c t (2-3 mg. o f p r o t e i n ) . 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 min. and t h e r e a c -t i o n s t opped 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 ter 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 2,000 g f o r 5 min. and the 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). To 1.0 ml. o f the 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 2 N 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 l were added and t h e aqueous phase was e x t r a c t e d f o r 3 min. by a g i t a t i n g t h e c o n t e n t s - 38 -of the tube w i t h a stream o f a i r . The two l a y e r s were se-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 e x t r a c t e d a g a i n (3 min.) w i t h 6.0 ml. o f 10% sodium c a r b o n a t e . E x a c t l y 2.0 ml. o f the 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 absorbancy 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 of a s t a n -d a r d c u r v e i n t o ymoles of 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 d e h y d r a t a s e 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 of a - k e t o b u t y r a t e / h r . a t 37°. ( i v ) C i t r a t e s y n t h a s e The c o n d e n s i n g enzyme, c i t r a t e s y n t h a s e , was d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y by measuring t h e r a t e o f change i n A 2 3 3 ( S r e r e and K o s i c k i , 1961). Each a s s a y c o n t a i n e d i n a volume of 1.0 ml.: t r i s - H C l b u f f e r , pH 8.0, 100 ymoles; o x a l o -a c e t i c a c i d , 0.40 ymole and a c e t y l - C o A , 0.16 ymole. The r e a c t i o n was i n i t i a t e d by t h e a d d i t i o n o f crude e x t r a c t (25-35 yg. o f p r o t e i n ) . The change i n A 2 3 3 was r e c o r d e d c o n t i n u -o u s l y a g a i n s t a b l a n k c o n t a i n i n g a l l t h e i n g r e d i e n t s e x c e p t e x t r a c t . The d e c r e a s e i n A 2 3 3 i s due b o t h t o c l e a v a g e o f the t h i o l e s t e r bond and t o t h e u t i l i z a t i o n of o x a l o a c e t i c a c i d . The combined molar e x t i n c t i o n c o e f f i c i e n t o f 5.4 x 1 0 3 de-t e r m i n e d by S r e r e and K o s i c k i (1961) was used i n t h i s work. - 39 -A u n i t o f enzyme a c t i v i t y i s d e f i n e d as the f o r m a t i o n o f 1 mymole of c i t r a t e / m i n . (v) Fumarase Fumarase was d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y a t 240 my as d e s c r i b e d by Hanson and Cox (1967). Each c u v e t t e - c o n -t a i n e d 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 , pH 7.2, 50 ymoles; m a l i c a c i d , 17.5 ymoles and ex-t r a c t (80-120 yg. o f p r o t e i n ) . The r a t e of i n c r e a s e i n absorbancy due t o t h e f o r m a t i o n o f f u m a r i c a c i d was f o l l o w e d c o n t i n u o u s l y 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 ter and e x t r a c t . One u n i t o f fumarase a c t i v i t y i s d e f i n e d as an A 2 i i o change o f 0.001/min. ( v i ) A c o n i t a s e A c o n i t a s e a c t i v i t y was d e t e r m i n e d by measuring a t 240 my t h e r a t e o f f o r m a t i o n of c i s ^ a c o n i t i c a c i d from i s o c i t r i c a c i d as d e s c r i b e d by Hanson and Cox (196 7 ) . Each a s s a y mix-t u r e c o n t a i n e d i n a volume o f 1.0 ml.: p o t a s s i u m phosphate b u f f e r , pH 7.2, 50 ymoles; D L - i s o c i t r i c a c i d , 15 ymoles and c e l l e x t r a c t (80-120 yg. o f p r o t e i n ) . A u n i t o f a c o n i t a s e 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 of enzyme which p r o -duces an A 2 I J O change o f 0.001/min. ( v i i ) I s o c i t r i c dehydrogenase I s o c i t r i c dehydrogenase was d e t e r m i n e d by measuring a t 340 my t h e r a t e o f r e d u c t i o n o f n i c o t i n a m i d e a denine d i n u -c l e o t i d e phosphate (NADP +). Each r e a c t i o n m i x t u r e c o n t a i n e d - 40 -i n a volume o f 1.0 m l . : t r i s - H C l b u f f e r , pH 7.5, 50 ymoles; MgCl2, 10 ymoles; sodium D L - i s o c i t r a t e , 10 ymoles; NADP +, 0.3 ymole and e x t r a c t (50-70 yg. o f p r o t e i n ) . A 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 the f o r m a t i o n o f 1 mymole o f reduced NADP +/ min. ( v i i i ) G l u c o k i n a s e G l u c o k i n a s e a c t i v i t y was d e t e r m i n e d s p e c t r o p h o t o m e t r i -c a l l y by measuring the r a t e o f r e d u c t i o n o f NADP + i n a r e a c -t i o n system c o u p l e d t o g l u c o s e 6-phosphate dehydrogenase.. The r e a c t i o n system c o n t a i n e d t h e f o l l o w i n g i n a volume o f 1.0 m l . : t r i s - H C l b u f f e r , pH 7.0, 100 ymoles; g l u c o s e , 4 ymoles; ATP, 2 ymoles; MgSCK, 4.5 ymoles; NADP +, 10 0 mymoles; g l u c o s e - 6 - p h o s p h a t e dehydrogenase (C.F. B o e h r i n g e r and Soehne, Mannheim, Germany), 1 u n i t , and crude e x t r a c t (0.25-0.35 mg. o f p r o t e i n ) . One g l u c o k i n a s e u n i t i s d e f i n e d as t h a t amount o f enzyme t h a t forms 1 mymole of NADPH/min. ( i x ) G l u c o s e 6-phosphate dehydrogenase The r e a c t i o n m i x t u r e used f o r the d e t e r m i n a t i o n o f g l u c o s e 6-phosphate dehydrogenase c o n t a i n e d i n 1.0 ml.: g l u c o s e 6-phosphate, 5 ymoles; M g C l 2 , 10 ymoles; NADP +, 0.14 ymole; g l y c y l g l y c i n e b u f f e r , pH 7.5, 50 ymoles and c e l l e x t r a c t (0.25-0.35 mg. o f p r o t e i n ) . The change i n A 3 h 0 was r e c o r d e d as a f u n c t i o n o f t i m e . A u n i t o f a c t i v i t y i s d e f i n e d as t h e f o r m a t i o n o f 1 mymole o f NADPH/min. In the absence of exogenous s u b s t r a t e s , NADP + was not r e -duced by t h e crude c e l l e x t r a c t s under the as s a y c o n d i t i o n s d e s c r i b e d . - 41 -PART A; R e p r e s s i o n by G l u c o s 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 i n E s c h e r i c h i a c o l i B RESULTS I . E f f e c t o f Carbon Source on 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 F o r m a t i o n When E. c o l i B was grown on g l u c o s e - s a l t s medium, the s p e c i f i c a c t i v i t y o f AHA s y n t h e t a s e d e c r e a s e d w i t h i n c r e a s i n g g l u c o s e c o n c e n t r a t i o n . The r e s u l t s o f a t y p i c a l e x p e r i -ment a r e p r e s e n t e d i n T a b l e I . A l t h o u g h t h e s p e c i f i c a c -t i v i t i e s o f AHA s y n t h e t a s e f l u c t u a t e d s l i g h t l y from one expe r i m e n t t o t h e n e x t , a d e c r e a s e o f 35-40% i n s p e c i f i c a c -t i v i t y d u r i n g a 2 0 - f o l d i n c r e a s e i n g l u c o s e c o n c e n t r a t i o n i n v a r i a b l y was o b s e r v e d . E v i d e n c e t h a t a g e n e r a l r e p r e s s i o n by g l u c o s e o f a l l enzymes i n t h e e x t r a c t d i d not o c c u r under t h e s e c o n d i t i o n s was p r o v i d e d by the c o n s t a n c y o f t h e spe-c i f i c a c t i v i t i e s of two o t h e r enzymes, g l u c o k i n a s e and t h r e o n i n e d e h y d r a t a s e (Table I ) . I t has l o n g been known t h a t t h e f o r m a t i o n o f a number of c a t a b o l i c enzymes i n c e r t a i n m i c r o - o r g a n i s m s i s r e p r e s s e d by growth on ah e n e r g y - r i c h c a r b o n s o u r c e such as g l u c o s e b u t n o t by growth on poor c a r b o n s o u r c e s such as g l y c e r o l or l a c t a t e . T h i s phenomenon, w h i c h was o r i g i n a l l y termed t h e " g l u c o s e e f f e c t " (Epps and G a l e , 1942) has been shown t o be a p r o p e r t y n ot e x c l u s i v e t o g l u c o s e m e t a b o l i s m and as a r e s u l t i s now g e n e r a l l y r e f e r r e d t o as " c a t a b o l i t e - 42 -T a b l e I . Effect, of increasing glucose concentration on enzyme s p e c i f i c a c t i v i t i e s in E s c h e r i c h i a c o l i B I n i t i a l concn. g l u c o s e , i n the medium (%) 0 .05 0.10 0.20 0.40 0.60 1.00 AHA s y n t h e t a 7.45 6.50 5.55 5.40 4.90 4.55 S p e c i f i c a c t i v i * G l u c o k i n a s e ^ 38.5 39.7 38.4 37.5 37.5 38.5 Thr e o n i n e d e h y d r a t a s e * 14.0 15.2 14.2 14.4 13.7 13.0 The c u l t u r e s were grown a e r o b i c a l l y from an A ^ o o f 0.1 t o 0.8, h a r v e s t e d , d i s r u p t e d by s o n i c t r e a t m e n t and assayed f o r enzymic a c t i v i t i e s as d e s c r i b e d i n the METHODS. *umoles of p r o d u c t formed/mg. o f p r o t e i n / h r . a t 37° mumoles of NADP + reduced/mg. o f p r o t e i n / m i n . a t 25° - 43 -r e p r e s s i o n " (Magasanik, 19 6 1 ) . We d e c i d e d , t h e r e f o r e , t o i n v e s t i g a t e t h e e f f e c t o f g l y c e r o l as c arbon s o u r c e on AHA-s y h t h e t a s e formation... The r e s u l t s shown i n F i g . 3, i n d i -c a t e t h a t i n c r e a s i n g c o n c e n t r a t i o n s - o f g l y c e r o l have no s i g -n i f i c a n t e f f e c t on AHA-synthetase f o r m a t i o n . I n o t h e r e x p e r i m e n t s s l i g h t r e p r e s s i o n was o c c a s i o n a l l y o b s e r v e d a t t h e h i g h e s t g l y c e r o l c o n c e n t r a t i o n s employed ( 1 % ) . F u r t h e r e v i d e n c e t h a t the f o r m a t i o n of AHA s y n t h e t a s e i n E. c o l i B i s 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 i s p r o v i d e d i n PARTS B and C o f t h i s t h e s i s . I I . E v i d e n c e f o r a S i n g l e A c e t o l a c t a t e - F o r m i n g System i n Escherichia .coli B I n E. c o l i K-12 the p r e s e n c e of two a c e t o l a c t a t e -f o r m i n g systems has been r e p o r t e d (Radhakrishnan and S n e l l , 1960): a b i o s y n t h e t i c , pH 8 enzyme w h i c h i s i n h i b i t e d by t h e u l t i m a t e p r o d u c t o f t h e pathway ( L - v a l i n e ) b u t i s r e -s i s t a n t t o r e p r e s s i o n by g l u c o s e , and a c a t a b o l i c pH 6 enzyme, whi c h i s r e s i s t a n t t o i n h i b i t i o n by v a l i n e b u t i s r e p r e s s e d by growth on g l u c o s e . T h e r e f o r e , i t was e s s e n t i a l t o e s t a b l i s h whether a s i n g l e o r m u l t i p l e a c e t o l a c t a t e - f o r m i n g system e x i s t e d i n E. c o l i B under the c o n d i t i o n s used f o r g r owing t h e s e c u l t u r e s . Owing t o the extreme l a b i l i t y o f a c e t o l a c t a t e -f o r m i n g a c t i v i t y i n c e l l l y s a t e s , i t was n e c e s s a r y t o l i m i t t h i s i n v e s t i g a t i o n t o an e x a m i n a t i o n o f crude c e l l e x t r a c t s . I n o r d e r t o d i s t i n g u i s h between c a t a b o l i c (pH 6) and a n a b o l i c - 44 -F i g . 3. E f f e c t o f i n c r e a s i n g ( i n i t i a l ) c o n c e n t r a t i o n s o f c a r -bon s o u r c e on t h e f o r m a t i o n o f AHA s y n t h e t a s e i n Escherichia c o l i B. The b a s a l - s a l t s medium v/as supplemented w i t h e i t h e r g l u c o s e (©.) o r g l y c e r o l (o.) a t v a r i o u s i n i t i a l c o n c e n t r a t i o n s as i n d i c a t e d . C u l t u r e s were h a r v e s t e d a t an Ai, 2o o f 0.8 and s o n i c e x t r a c t s were p r e p a r e d as d e s c r i b e d i n the METHODS. S p e c i f i c a c t i v i t y i s d e f i n e d as ymoles of a c e t o i n formed/mg. o f p r o t e i n / h r . a t 37°. - 45 -(pH 8) a c e t o l a c t a t e - f o r m i n g a c t i v i t i e s we took advantage of t h e d i s t i n c t i v e r e g u l a t o r y p r o p e r t i e s r e p o r t e d f o r each system, i . e . s e n s i t i v i t y t o r e p r e s s i o n by g l u c o s e and s e n s i t i v i t y t o i n h i b i t i o n by v a l i n e , r e s p e c t i v e l y . I n F i g . 4 a r e pH c u r v e s f o r t h e f o r m a t i o n of a c e t o l a c t a t e i n c r u d e e x t r a c t s o f c e l l s grown on m i n i m a l medium supplemented w i t h g l u c o s e o r g l y c e r o l . A l t h o u g h the s p e c i f i c a c t i v i t y o f the g l y c e r o l - g r o w n c e l l s was n e a r l y double t h a t o f t h e g l u -cose-grown c e l l s t h e pH optima of b o t h e x t r a c t s was between 7.8 and 8.0. A d d i t i o n o f v a l i n e t o the a s s a y system con-t a i n i n g t h e g l y c e r o l - g r o w n e x t r a c t s t r o n g l y i n h i b i t e d a c e t o -l a c t a t e f o r m a t i o n (lower c u r v e ) , a l t h o u g h t h e p o s i t i o n o f t h e maximum o f t h e pH c u r v e was not s i g n i f i c a n t l y a l t e r e d . These r e s u l t s s uggest t h a t the pH 6 enzyme i s a b s e n t , s i n c e under t h e s e c o n d i t i o n s (grown on g l y c e r o l and a s s a y e d i n the p r e s e n c e o f v a l i n e ) the a c t i v i t y p r o f i l e o f a pH 6 enzyme s h o u l d be p r ominent. D u r i n g an i n v e s t i g a t i o n of feedback i n h i b i t i o n o f a c e t o l a c t a t e - f o r m i n g a c t i v i t y i n crude e x t r a c t s of g l y c e r o l -grown c e l l s , i t was o b s e r v e d t h a t i n h i b i t i o n by v a l i n e d i d n o t exceed 60-70%, 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 sub-s t r a t e and v a l i n e employed. I n o r d e r t o i n v e s t i g a t e the problem o f i n c o m p l e t e i n h i b i t i o n t h r e e a l t e r n a t e hypotheses were c o n s i d e r e d : (1) complete i n h i b i t i o n may r e q u i r e t h e c o n c e r t e d a c t i o n o f more th a n one i n h i b i t o r , (2) i n c o m p l e t e i n h i b i t i o n by v a l i n e may be an a r t i f a c t o f t h e method by - 46 -T i i r 1 1 1 T 8 . 0 -5.0 6.0 7 0 8 .0 9.0 10.0 pH F i g . 4. E f f e c t o f pH on the a c e t o l a c t a t e - f o r m i n g system from c u l t u r e s o f Escherichia c o l i B grown on d i f f e r e n t carbon s o u r c e s . Crude e x t r a c t s were p r e p a r e d from c e l l s grown on e i t h e r 0.4% g l u c o s e ( ) o r on 0.4% g l y c e r o l ( ). Assay m i x t u r e s were p r e p a r e d i n e i t h e r 0.1 M - T r i s - K C l (o) o r p o t a s -sium phosphate ( o ) . The c o m p o s i t i o n of the assay m i x t u r e s was as d e s c r i b e d i n the METHODS w i t h the e x c e p t i o n o f L - v a l i n e (bottom curve) w h i c h was added a t a c o n c e n t r a t i o n of 1 mM. The c e l l s were d i s r u p t e d by s o n i c t r e a t m e n t i n d i s t i l l e d w a t e r and as s a y e d i m m e d i a t e l y . S p e c i f i c a c t i v i t y i s e x p r e s s e d as ymoles o f a c e t o i n formed/mg. o f p r o t e i n / h r . a t 37°. - 47 -w h i c h t h e crude c e l l e x t r a c t s were p r e p a r e d , and (3) two pH 8 enzymes may be p r e s e n t under t h e s e c o n d i t i o n s , one which i s r e s p o n s i b l e f o r 60-70% o f t h e t o t a l a c t i v i t y and i s 100% i n h i b i t e d by v a l i n e and a second enzyme which c o n t r i b u t e s o n l y 30-^40% o f the a c t i v i t y b u t i s v a l i n e i n s e n s i t i v e . To examine the f i r s t p o s s i b i l i t y , a c e t o l a c t a t e - f o r m i n g a c t i v i t y was d e t e r m i n e d i n e x t r a c t s p r e p a r e d from g l y c e r o l - g r o w n c e l l s i n an as s a y system c o n t a i n i n g 25 mM s u b s t r a t e and i n t h e same system supplemented w i t h v a l i n e o r w i t h the f o u r known end p r o d u c t s o f t h e i s o l e u c i n e - v a l i n e ( " i l v a " ) pathway ( 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 ) . The r e s u l t s a r e p r e -s e n t e d i n T a b l e I I . The a d d i t i o n o f f o u r end p r o d u c t s t o the as s a y system d i d n o t enhance (or r e l i e v e ) t h e i n h i b i t i o n w hich r e s u l t e d from the pr e s e n c e o f v a l i n e a l o n e . The f o l l o w i n g compounds a l s o f a i l e d t o i n h i b i t (or s t i m u l a t e ) a c e t o l a c t a t e -f o r m i n g a c t i v i t y when added t o t h e r e a c t i o n m i x t u r e a t a f i n a l c o n c e n t r a t i o n of 1 mM: c i t r a t e , a c e t a t e , l a c t a t e , phospho-e n o l p y r u v a t e , 2 - p h o s p h o g l y c e r a l d e h y d e , NAD , NADH, NADP , NADPH and ATP. Thus, w h i l e c o n c e r t e d feedback c o n t r o l o f t h i s a c -t i v i t y cannot be r u l e d o u t , i t seems h i g h l y u n l i k e l y t h a t t h i s mechanism i s r e s p o n s i b l e f o r the l i m i t e d i n h i b i t i o n o b s e r v e d . To a s c e r t a i n whether i n c o m p l e t e i n h i b i t i o n . b y v a l i n e i s an t a r t i f a c t r e s u l t i n g i n v i t r o from u l t r a - s o n i c t r e a t m e n t o f the c e l l s , v a l i n e i n h i b i t i o n of a c e t o l a c t a t e - f o r m i n g a c t i v i t y was compared i n crude e x t r a c t s and s u p e r n a t a n t f l u i d s o f g l y c e r o l -grown c e l l s d i s r u p t e d i n v a r i o u s ways. The r e s u l t s o f t h i s - 48 -T a b l e I I . Effect of the four end products of the v a l i n e - i s o -leucine pathway on the acetolactate-forming a c t i v i t y of E s c h e r i c h i a c o l i B A d d i t i o n s None V a l i n e 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 ymoles of a c e t o i n I n h i b i t i o n formed/mg. o f p r o t e i n / h r . 4.71 1.76 62.6 1.76 62.6 The c e l l s were grown on g l y c e r o l ( 0 . 4 % ) - s a l t s medium, h a r v e s t e d and crude s o n i c e x t r a c t s were p r e p a r e d as des-c r i b e d i n t h e METHODS. The c e l l e x t r a c t was assay e d f o r AHA-s y n t h e t a s e a c t i v i t y i n a r e a c t i o n system c o n t a i n i n g 25 mM s u b s t r a t e . End p r o d u c t s when p r e s e n t , were 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 of 0.5 mM each. - 49 -expe r i m e n t a r e summarized i n T a b l e I I I . The degree o f i n -h i b i t i o n by v a l i n e o f a c e t o l a c t a t e - f o r m i n g a c t i v i t y remained c o n s t a n t (59-67%) r e g a r d l e s s . o f whether t h e c e l l s were d i s -r u p t e d by s o n i c o s c i l l a t i o n , a l u m i n a g r i n d i n g o r passage t h r o u g h a F r e n c h p r e s s u r e c e l l . E v i d e n c e t h a t i n c o m p l e t e i n h i b i t i o n a l s o o c c u r s i n p e r m e a b i l i z e d whole c e l l s i s p r o -v i d e d by the r e s u l t s o f the e x p e r i m e n t s w i t h t o l u e n e - t r e a t e d c e l l s (Table I I I ) . I t was found t h a t when c e l l s u s p e n s i o n s were b r i e f l y exposed a t 37° t o d i l u t e s o l u t i o n s o f t o l u e n e , t h e p e r m e a b i l i t y o f t h e c e l l membrane i n c r e a s e d w i t h o u t c e l l l y s i s , p e r m i t t i n g s m a l l m o l e c u l e s such as p y r u v a t e and a c e t o -l a c t a t e t o e n t e r and l e a v e t h e c e l l r a p i d l y . P e r m e a b i l i z e d c e l l s a s s a y e d f o r a c e t o l a c t a t e f o r m a t i o n i n t h e p r e s e n c e and absence o f v a l i n e e x h i b i t e d h i g h a c t i v i t y and i n c o m p l e t e v a l i n e i n h i b i t i o n comparable t o t h a t o b s e r v e d i n c e l l e x t r a c t s (65%) (Table I I I ) . P r o l o n g e d exposure t o , o r h i g h e r concen-t r a t i o n s o f t o l u e n e r e s u l t e d i n d e c r e a s e d enzymic a c t i v i t y and caused c e l l l y s i s . Even under t h e s e c o n d i t i o n s , however, the degree o f i n h i b i t i o n by v a l i n e remained c o n s t a n t (Table I I I ) . The p o s s i b i l i t y t h a t e x t r a c t s o f g l y c e r o l - g r o w n c e l l s c o n t a i n two pH 8 a c e t o l a c t a t e - f o r m i n g enzymes p o s s e s s i n g d i f -f e r e n t v a l i n e s e n s i t i v i t i e s (0 o r 100%) was i n v e s t i g a t e d by measuring a c e t o l a c t a t e f o r m a t i o n i n e x t r a c t s from g l u c o s e and g l y c e r o l - g r o w n c e l l s i n t h e pr e s e n c e o f i n c r e a s i n g c o n c e n t r a -t i o n s of v a l i n e . A l t h o u g h t h e s p e c i f i c a c t i v i t i e s o f t h e a c e t o l a c t a t e - f o r m i n g systems of t h e g l y c e r o l and g l u c o s e -T a b l e I I I . Valine i n h i b i t i o n of acetolactate-f'orming a c t i v i t y in c e l l s of E s c h e r i c h i a c o l i B disrupted or permeaVilizsd by various methods. Treatment Crude e x t r a c t S u p e r n a t a n t P r o t e i n ymoles o f a c e t o i n I n h i b i t i o n P r o t e i n ymoles o f a c e t o i n I n h i b i t i o n (mg./ml.) /mg. o f p r o t e i n / h r . (%) (mg. /ml.) /mg. o f p r o t e i n / h r . (%) - V a l i n e + V a l i n e ' - V a l i n e + V a l i n e Whole c e l l s 5. 75 o l - - 0.10 - -S o n i c o s c i l l a t i o n 5. 80 5.11 2.06 60 4.93 5.23 1.87 64 Alum i n a g r i n d i n g - - - - 2.96 1.17 0.48 59 F r e n c h p r e s s u r e c e l l 5.88 2.74 0.94 66 4.40 3.00 0.98 67 i T o l u e n e * 5.56 6.21 2.19 65 0 .16 Ot 0? 0 o t Toluene 5.32 3.55 1.21 66 0.80 0? o l 0 1 The c e l l s were grown a e r o b i c a l l y on g l y c e r o l ( 0 . 4 % ) - s a l t s medium, h a r v e s t e d by c e n t r i f u -g a t i o n and washed w i t h 0.1 M - p o t a s s i u m phosphate b u f f e r , pH 7.4. The washed c e l l s were t r e a t e d as d e s c r i b e d i n the METHODS. A H A - s y n t h e t a s e a c t i v i t y was d e t e r m i n e d i n the c r u d e e x -t r a c t s and i n the 12,000 g 1 ( 1 0 m i n . ) s u p e r n a t a n t f l u i d s . The a s s a y s y s t e m was as d e s c r i b e d i n the METHODS w i t h t h e e x c e p t i o n o f t h e c o n c e n t r a t i o n o f p y r u v a t e w h i c h was r e d u c e d t o 25 mM. V a l i n e , when p r e s e n t , was added t o g i v e a f i n a l c o n c e n t r a t i o n o f 1 mM. *5% (v/v) t o l u e n e f o l l o w e d by i n c u b a t i o n a t 37° f o r 2 m i n . t 10% (v/v) t o l u e n e f o l l o w e d by i n c u b a t i o n a t 37° f o r 6 m i n . Ino a c t i v i t y was d e t e c t e d . - 51 -100-DQ X 6s VALINE, M x 1 0 ' F i g . 5. V a l i n e i n h i b i t i o n o f a c e t o l a c t a t e - f o r m i n g a c t i v i t y i n e x t r a c t s o f Escherichia coli B grown on d i f f e r e n t carbon s o u r c e s . Crude s o n i c e x t r a c t s were p r e p a r e d from c e l l s grown on e i t h e r 0.4% g l u c o s e (•) or on 0.4% g l y c e r o l ( o ) . A c e t o l a c t a t e f o r m a t i o n was d e t e r m i n e d i n t h e assay system de-s c r i b e d i n t h e METHODS e x c e p t f o r t h e c o n c e n t r a t i o n o f sub-s t r a t e w hich was reduced t o 25 mM. V a l i n e was added a t t h e c o n c e n t r a t i o n s i n d i c a t e d . -The s p e c i f i c a c e t o l a c t a t e - f o r m i n g a c t i v i t i e s on g l u c o s e and on g l y c e r o l were 4.6 and 8.1, r e s p e c t i v e l y . - 52 -grown c e l l s were d i f f e r e n t (8.1 and 4.6, r e s p e c t i v e l y ) , t h e p e r c e n t a g e e n d - p r o d u c t i n h i b i t i o n a t each v a l i n e c o n c e n t r a -t i o n was the same ( F i g . 5 ) . R e s u l t s such as t h e s e would not be e x p e c t e d i f t h e i n c r e a s e d a c e t o l a c t a t e - f o r m i n g a c t i v i t y o f e x t r a c t s from g l y c e r o l - g r o w n c e l l s had been due t o the de-r e p r e s s i o n o f a second enzyme ( s e n s i t i v e t o g l u c o s e r e p r e s s i o n and r e s i s t a n t t o i n h i b i t i o n by v a l i n e ) . T h e r e f o r e , t h e i n c r e a s e d pH 8 a c e t o l a c t a t e - f o r m i n g a c t i v i t y o f g l y c e r o l -grown c e l l s ( F i g . 4) appeared t o r e s u l t from t h e d e r e p r e s s i o n o f a s i n g l e enzyme wh i c h i s p a r t i a l l y s e n s i t i v e t o v a l i n e . R a d h a k r i s h n a n and S n e l l (1960) r e p o r t e d t h a t i n E. coli K-12, t h e pH 6 enzyme c o u l d be r e a d i l y d i s t i n g u i s h e d from the pH 8 enzyme by t h e g r e a t e r s t a b i l i t y o f t h e former a c t i v i t y d u r i n g s t o r a g e and p u r i f i c a t i o n . S i n c e t h e pH 6 enzyme was a l s o r e s i s t a n t t o v a l i n e i n h i b i t i o n (Radhakrishnan and S n e l l , 1960) , an. e x p e r i m e n t was d e s i g n e d t o det e r m i n e i f a change i n v a l i n e s e n s i t i v i t y o c c u r r e d d u r i n g s t o r a g e o f a cru d e e x t r a c t o f E. coli B. The r e s u l t s of t h i s e x p e r i m e n t a r e g i v e n i n T a b l e IV. The c o n c e n t r a t i o n s of. p y r u v a t e and v a l i n e employed i n t h i s s t u d y (see l a t e r ) r e s u l t e d i n a 62.5% i n h i -b i t i o n of a c e t o l a c t a t e f o r m a t i o n i n the f r e s h e x t r a c t s from g l y c e r o l - g r o w n c e l l s . F o l l o w i n g s t o r a g e f o r 29 h r . a t 0°, the e x t r a c t l o s t a p p r o x i m a t e l y 70% of i t s a c e t o l a c t a t e -f o r m i n g a c t i v i t y . V a l i n e s e n s i t i v i t y , however, remained r e l a t i v e l y c o n s t a n t d u r i n g s t o r a g e , i n d i c a t i n g the p r e s e n c e o f a s i n g l e a c e t o l a c t a t e - f o r m i n g system w h i c h , a f t e r r e l e a s e from - 53 -T a b l e IV. L a b i l i t y of acetolactate-forming a c t i v i t y during storage Time a t 0° ymoles o f a c e t o i n formed I n h i b i t i o n (hr.) /mg. o f p r o t e i n / h r . (%) - V a l i n e + V a l i n e 0 5.93 2.23 62.5 6 3.78 1.25 66.9 12 3.17 1.00 68.5 22 2.47 0.89 64.0 29 1.92 0.72 62.5 C e l l s were grown on m i n i m a l medium supplemented w i t h 0.4% g l y c e r o l . The crude s o n i c e x t r a c t was s t o r e d i n an i c e b a t h a t 0°. A t the tim e s i n d i c a t e d , p o r t i o n s (0.5 ml.) were as s a y e d f o r enzymic a c t i v i t y . The c o m p o s i t i o n o f t h e r e a c -t i o n m i x t u r e was i d e n t i c a l t o the system d e s c r i b e d i n the METHODS e x c e p t f o r the c o n c e n t r a t i o n of p y r u v a t e which was de-c r e a s e d t o 2 5 mM. V a l i n e , when p r e s e n t , was added t o g i v e a f i n a l c o n c e n t r a t i o n o f 1 mM. - 54 -T a b l e V. Desensitization of AHA synthetase to valine -Inhibition Time a t 25° ymoles o f a c e t o i n formed I n h i b i t i o n (hr.) /mg. o f p r o t e i n / h r . (%) - V a l i n e + V a l i n e 0 4.02 1.46 63.6 1 2.45 0 .89 63.7 2 1.8.8 0.76 59.6 3 1.65 0.84 49 .2 5 1.49 0.90 39 .7 7 1.30 0.95 27.0 The c u l t u r e was grown on g l y c e r o l ( 0 . 4 % ) - s a l t s medium, h a r v e s t e d and a crude s o n i c e x t r a c t was p r e p a r e d as d e s c r i b e d i n the METHODS. The e x t r a c t was i m m e d i a t e l y p l a c e d i n a water b a t h a t 25° and p o r t i o n s (0.5 ml) were removed a t t h e times i n d i c a t e d f o r enzyme d e t e r m i n a t i o n . AHA s y n t h e t a s e was as s a y e d i n a r e a c t i o n system c o n t a i n i n g 2 5 mM p y r u v a t e and when p r e s e n t , 0.5 mM L - v a l i n e . I - 55 -t h e c e l l by u l t r a - s o n i c t r e a t m e n t , showed a p a r a l l e l l o s s b o t h i n c a t a l y t i c a c t i v i t y and i n s e n s i t i v i t y t o feedback i n h i b i t i o n . When crud e c e l l e x t r a c t s were i n c u b a t e d a t 25° f o r 2 h r . o r l o n g e r , AHA s y n t h e t a s e was p a r t i a l l y d e s e n s i t i z e d t o v a l i n e i n h i b i t i o n (Table V ) . A l t h o u g h t h e d e c r e a s e i n v a l i n e s e n s i -t i v i t y c aused by warming t h e e x t r a c t was accompanied by a d e c r e a s e i n enzymic a c t i v i t y , i t s h o u l d be noted t h a t t h e l a t t e r l o s s was d i s p r o p o r t i o n a t e w i t h t h e former. Thus p y r u v a t e and v a l i n e p r o b a b l y b i n d t o t h e enzyme a t s e p a r a t e s i t e s . I l l . K i n e t i c o f V a l i n e I n h i b i t i o n of 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 Escherichia c o l i B As a r e s u l t o f the o b s e r v a t i o n ( F i g . 5) t h a t v a l i n e , even a t h i g h c o n c e n t r a t i o n s c o u l d o n l y p a r t i a l l y (60-70%) i n -h i b i t AHA s y n t h e t a s e a c t i v i t y of E. c o l i B, a p r e l i m i n a r y i n v e s t i g a t i o n was u n d e r t a k e n i n crude e x t r a c t s o f t h e i n f ' h i b i t i o n k i n e t i c s o f t h i s system. E x a m i n a t i o n o f a p l o t o f enzyme v e l o c i t y v e r s u s s u b s t r a t e c o n c e n t r a t i o n i n t h e presence o r i n t h e absence o f v a l i n e ( F i g . 6A) s u g g e s t e d t h a t v a l i n e i n h i b i t i o n was n o n - c o m p e t i t i v e . However, a dou b l e r e c i p r o c a l p l o t o f t h e same d a t a ( F i g . 6B) i n d i c a t e d t h a t t h e t y p e o f i n h i b i t i o n i n v o l v e d h e r e was n e i t h e r t r u e n o n - c o m p e t i t i v e n or t r u e c o m p e t i t i v e i n h i b i t i o n . A t v a l i n e c o n c e n t r a t i o n s l e s s t h a n t h a t r e q u i r e d t o produce maximum i n h i b i t i o n (<0.5 mM) (see F i g . 5 ) , i n h i b i t i o n a t low s u b s t r a t e c o n c e n t r a t i o n s appeared n o n - c o m p e t i t i v e ( F i g . 6B) b u t a t v e r y h i g h s u b s t r a t e F i g . 6. V a l i n e i n h i b i t i o n o f AHA s y n t h e t a s e i n Escherichia c o l i B. AHA-synthetase a c t i v i t y was d e t e r m i n e d i n a crude s o n i c e x t r a c t p r e p a r e d from c e l l s grown a e r o b i c a l l y a t 37° on g l y c e r o l ( 0 . 4 % ) - s a l t s medium. V a l i n e was added t o g i v e a f i n a l c o n c e n t r a t i o n o f 0.25 mM. S, i n i t i a l c o n c e n t r a t i o n o f s u b s t r a t e ( p y r u v a t e ) . V, umoles of p r o d u c t formed/mg. of p r o t e i n / h r . . a t 37°. A. A p l o t o f s u b s t r a t e c o n c e n t r a t i o n (S) v e r s u s r e a c t i o n v e l o c i t y (V). B. A double r e c i p r o c a l p l o t o f s u b s t r a t e c o n c e n t r a t i o n (1/S) v e r s u s r e a c t i o n v e l o c i t y (1/V). - 57 -c o n c e n t r a t i o n s i n h i b i t i o n became s l i g h t l y c o m p e t i t i v e ( F i g . 6B). When t h e v a l i n e c o n c e n t r a t i o n exceeded 0.5 mM, changes i n s u b s t r a t e c o n c e n t r a t i o n had l i t t l e o r no e f f e c t on the degree o f i n h i b i t i o n ( n o n - c o m p e t i t i v e ) . F o r example, a t a v a l i n e c o n c e n t r a t i o n o f 0.5 mM a 1 0 - f o l d d e c r e a s e i n p y r u v a t e c o n c e n t r a t i o n (250 t o 25 mM) i n c r e a s e d i n h i b i t i o n by l e s s than 10%. T h e r e f o r e , i n v a l i n e i n h i b i t i o n e x p e r i m e n t s the p y r u -v a t e c o n c e n t r a t i o n used was 25 mM e n s u r i n g t h a t v a l i n e i n -h i b i t i o n was maximal. IV. R e l a t i o n s h i p between G l u c o s e R e p r e s s i o n and End-Product R e p r e s s 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 o f Escheri- chia c o l i B The r e s u l t s o f an i n v e s t i g a t i o n o f the r e l a t i o n s h i p between g l u c o s e r e p r e s s i o n and end-product r e p r e s s i o n o f AHA s y n t h e t a s e i n E. c o l i B a r e shown i n T a b l e V I . P r e v i o u s r e p o r t s have p r e s e n t e d e v i d e n c e t h a t i n E. c o l i , AHA s y n t h e -t a s e i s r e p r e s s e d by growth i n a m i n i m a l medium supplemented w i t h v a l i n e (Umbarger and Brown, 1958) or a m i x t u r e o f 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 ( F r e u n d l i c h and Umbarger, 1963). The e f f e c t o f t h e s e f o u r end p r o d u c t s o r v a l i n e a l o n e was examined i n t h e pre s e n c e o f g l y c e r o l o r g l u c o s e o r b o t h . Of the t o t a l r e p r e s s i o n o b s e r v e d i n t h e presence o f g l u c o s e and end p r o d u c t s , t h e g r e a t e s t p r o p o r t i o n , i n a l l e x p e r i m e n t s was due t o g l u c o s e (or g l u c o s e and g l y c e r o l ) . When g l y c e r o l was t h e o n l y carbon s o u r c e p r e s e n t , the s p e c i f i c a c t i v i t i e s i n t he pre s e n c e o f end-products ( g l y c e r o l and end p r o d u c t s ) - 58 -T a b l e V I . Effect of biosynthetic end •products and carbon source on the formation of AHA synthetase in E s c h e r i c h i a c o l i B E x p t . No. A d d i t i o n s t o b a s a l medium Gl u c o s e G l u c o s e + L - v a l i n e G l y c e r o l G l y c e r o l + L - v a l i n e G l y c e r o l G l y c e r o l + g l u c o s e G l y c e r o l + end p r o d u c t s G l y c e r o l + g l u c o s e + end p r o d u c t s G l u c o s e G l u c o s e + end p r o d u c t s S p e c i f i c a c t i v i t y (ymoles o f a c e t o i n formed/ mg. o f p r o t e i n / h r . ) 5.30 6.03 9.30 10.30 8.73 5.27 8.40*, 8.83 5.64 6 .16 5.28*, 5.68" t The i n o c u l u m was grown f o r 16 h r . i n a s h a k i n g w a t e r b a t h on b a s a l medium supplemented w i t h 0.4% g l y c e r o l . A f t e r h a r -v e s t i n g , the c e l l s ( s t a t i o n a r y phase c e l l s ) were washed once i n phosphate b u f f e r and resuspended i n t h e same medium p l u s g l u -cose ( 0 . 4 % ) , g l y c e r o l ( 0 . 4 % ) , L - v a l i n e (0.5 mM) or end p r o d u c t s as i n d i c a t e d . End p r o d u c t s when p r e s e n t were added t o g i v e the f o l l o w i n g c o n c e n t r a t i o n s : L - v a l i n e , L - l e u c i n e , L - i s o l e u c i n e , + 0.5 mM each; and c a l c i u m p a n t o t h e n a t e , 0.1 mM* o r 0.001 mM . The c u l t u r e s were grown, h a r v e s t e d , d i s r u p t e d by s o n i c t r e a t -ment and as s a y e d f o r enzymic a c t i v i t y as d e s c r i b e d i n the METHODS. - 59 -were as h i g h as o r h i g h e r than i n the c o n t r o l ( g l y c e r o l ) (Table V I , e x p t . 2). The presence o f v a l i n e a l o n e (Table V I , e x p t . 1) c o n s i s t e n t l y r e s u l t e d i n a., s l i g h t d e r e p r e s s i o n o f AHA-synthetase a c t i v i t y whether the c e l l s were grown on g l u c o s e o r on g l y c e r o l . - 60 -DISCUSSION The r e s u l t s r e p o r t e d here i n d i c a t e t h a t i n Escherichia c o l i s t r a i n B, grown a e r o b i c a l l y on.minimal s a l t s medium, a s i n g l e a c e t o l a c t a t e - f o r m i n g system e x i s t s w hich i s s u b j e c t t o g l u c o s e r e p r e s s i o n . D e r e p r e s s e d f o r m a t i o n o f t h i s enzyme system (AHA s y n t h e t a s e ) o c c u r s when t h i s s t r a i n i s grown e i t h e r on a v e r y low c o n c e n t r a t i o n o f g l u c o s e o r on any c o n c e n t r a t i o n of a poor carbon s o u r c e such as g l y c e r o l . A l t h o u g h t h i s enzyme i s s e n s i t i v e t o c a t a b o l i t e r e p r e s s i o n , i t s pH optimum (7.8-8.0) and i t s s e n s i t i v i t y . t o . feedback i n h i -b i t i o n , by. v a l i n e , a r e in., a c c o r d w i t h t h e p r e v i o u s l y e s t a b -l i s h e d p r o p e r t i e s o f t h e b i o s y n t h e t i c AHA s y n t h e t a s e . V a l i n e . i n h i b i t i o n o f AHA.synthetase a c t i v i t y has been r e p o r t e d t o be c o m p e t i t i v e w i t h r e s p e c t t o s u b s t r a t e i n E. c o l i K-12 (Umbarger and Brown, 1958) b u t n o n - c o m p e t i t i v e f o r the e q u i v a l e n t a c t i v i t i e s i n A. aerogenes ( H a l p e r n and Umbarger, 1959) and S. typhimurium ( B a u e r l e et al., 1964). I n t h e p r e s e n t s t u d y , AHA s y n t h e t a s e o f E. c o l i B i s incom-p l e t e l y i n h i b i t e d by v a l i n e (60-70%), even a t v e r y h i g h con-c e n t r a t i o n s , and e x h i b i t s i n h i b i t i o n k i n e t i c s which a r e n e i t h e r p u r e l y , c o m p e t i t i v e nor n o n - c o m p e t i t i v e w i t h s u b s t r a t e ( F i g . 6 ) . S e v e r a l r e p o r t s o f i n c o m p l e t e feedback i n h i b i t i o n have appeared, i n the l i t e r a t u r e . Magee and de R o b i c h o n - S z u l m a j s t e r (1968) found t h a t t h e pH 7.2, AHA-synthetase a c t i v i t y i n crude l y s a t e s of,Saccharomyces cerevisiae was i n h i b i t e d t o a maxi-mum of 60% by the a d d i t i o n o f e x c e s s L - v a l i n e . Subsequent - 61 -ex p e r i m e n t s r e v e a l e d t h a t , i n t h i s c a s e , i n c o m p l e t e i n h i b i t i o n r e s u l t e d from t h e s e l e c t i v e d e s e n s i t i z a t i o n of the v a l i n e f e e d -back i n h i b i t i o n s i t e d u r i n g the p r e p a r a t i o n o f c e l l e x t r a c t s . When the enzyme was assay e d i n b e n z e n e - t r e a t e d , p e r m e a b i l i z e d , i n t a c t c e l l s , v a l i n e i n h i b i t i o n approached 100%. The i n v e s t i -g a t i o n r e p o r t e d h e r e i n of t h e e f f e c t o f v a r i o u s methods o f c e l l u l a r d i s r u p t i o n on t h e v a l i n e s e n s i t i v i t y o f AHA s y n t h e -t a s e of E. c o l i B i n d i c a t e d t h a t i n c o m p l e t e feedback i n h i b i -t i o n i n t h i s s t r a i n was n o t an a r t i f a c t o f c e l l l y s i s s i n c e t h i s enzyme demonstrated t h e same p a r t i a l r e s p onse t o v a l i n e i n t he i n t a c t c e l l (Table I I I ) . S t u d i e s on t h e c o n t r o l o f a s p a r t o k i n a s e a c t i v i t y i n v a r i o u s m i c r o - o r g a n i s m s ( D a t t a and G e s t , 1964; P a t t e et al., 1965) uncovered a n o t h e r example o f i n c o m p l e t e feedback i n h i b i t i o n . A s p a r t o k i n a s e i s r a t h e r un-u s u s a l i n t h a t t h i s enzyme r e g u l a t e s t h e f l o w o f m e t a b o l i t e s i n t o a b r a n c h e d , b i o s y n t h e t i c pathway. The pr e s e n c e o f h i g h c o n c e n t r a t i o n s o f a s i n g l e end p r o d u c t o n l y p a r t i a l l y r e duces enzymic a c t i v i t y . Complete i n h i b i t i o n o f a s p a r t o k i n a s e a c-t i v i t y i n .Rhodoyseudomonas capsulatus i s a c h i e v e d o n l y by the c o n c e r t e d a c t i o n o f two o r more p r o d u c t s o f t h i s complex p a t h -way. The o b v i o u s s i m i l a r i t y between t h e r o l e o f a s p a r t o k i n a s e and t h a t o f AHA s y n t h e t a s e i n r e g u l a t i n g a m u l t i - p r o d u c t b i o -s y n t h e t i c system s u g g e s t e d t h a t 100% i n h i b i t i o n o f the l a t t e r enzyme might be a c h i e v e d by t h e c o o p e r a t i v e a c t i o n o f more tha n one end p r o d u c t . R e s u l t s of such an experiment o b t a i n e d i n t h i s i n v e s t i g a t i o n w i t h AHA s y n t h e t a s e o f E. c o l i B - 62 -i n d i c a t e d t h a t the degree of p a r t i a l i n h i b i t i o n of t h i s enzyme by v a l i n e was n e i t h e r enhanced nor l e s s e n e d by t h e presence o f t h e o t h e r known end p r o d u c t s ( 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 ) o f t h e " i l v a " pathway. I n many m i c r o - o r g a n i s m s , b i o s y n t h e t i c pathways l e a d i n g t o t h e f o r m a t i o n of more than one end p r o d u c t a r e c o n t r o l l e d by s e v e r a l enzymes which c a t a l y z e t h e same i n i t i a l r e a c t i o n , b u t which a r e r e g u l a t e d by d i f f e r e n t end p r o d u c t s . E. c o l i K-12, f o r example, p o s s e s s e s t h r e e a s p a r t o k i n a s e s each o f w h i c h i s s p e c i f i c a l l y i n h i b i t e d by e i t h e r l y s i n e , t h r e o n i n e or homoserine (Stadtman, 196 3 ) . S i n c e t h e s e enzymes d i f f e r somewhat i n p r i m a r y s t r u c t u r e t h e i r p r e s e n c e can g e n e r a l l y be d e t e c t e d by changes i n t h e i n h i b i t i o n k i n e t i c s o f the crude a c t i v i t y d u r i n g s t o r a g e , h e a t o r a c i d t r e a t m e n t o r p r e l i m i n a r y f r a c t i o n a t i o n . V a l i n e i n h i b i t i o n and enzyme s t a b i l i t y s t u d i e s performed on c r u d e e x t r a c t s of ff. c o l i B i n the p r e s e n t i n -v e s t i g a t i o n s u ggest t h a t a s i n g l e , pH 8, AHA s y n t h e t a s e i s p r e s e n t i n t h i s o rganism. I n E. c o l i K-12 (Radhakrishnan and S n e l l , 1960) and i n A. aerogenes ( H a l p e r n and Umbarger, 1959) two a c e t o l a c t a t e -f o r m i n g systems have been r e p o r t e d : (1) a c a t a b o l i c system f u n c t i o n i n g a t an optimum of pH 6 and (2) a pH 8 system i n -v o l v e d i n the b i o s y n t h e s i s o f t h e a l i p h a t i c amino a c i d s and of p a n t o t h e n a t e . The f o r m a t i o n of t h e pH 6 enzyme i n A. aerogenes can o c c u r o n l y i n an a c i d i c environment .(<pH 6) ( H a l p e r n and Umbarger, 1959). S i n c e the c o n d i t i o n s employed by R a d h a k r i s h n a n - 63 -and S n e l l t o grow E. c o l i K-12 can a l s o r e s u l t i n an a c i d i c medium ( u n p u b l i s h e d o b s e r v a t i o n ) perhaps t h e pH 6 a c t i v i t y ob-s e r v e d i n the s t u d y w i t h s t r a i n K-12 b u t n o t i n t h e p r e s e n t s t u d y w i t h s t r a i n B can be a t t r i b u t e d t o d i f f e r e n c e s i n growth c o n d i t i o n s o f the c u l t u r e s . An e a r l i e r s t u d y i n t h i s l a b o r a -t o r y ( D e s a i and P o l g l a s e , 1965) on the p r o p e r t i e s o f t h e a c e t o l a c t a t e - f o r m i n g system from a s t r e p t o m y c i n - d e p e n d e n t mutant o f E, c o l i , a l s o f a i l e d t o d e t e c t a c t i v i t y a t pH 6. Hence, a l t h o u g h the o c c u r r e n c e of a pH 6 enzyme under c e r t a i n c o n d i t i o n s cannot be e x c l u d e d i n E. c o l i B, t h e r e s u l t s p r e -s e n t e d here i n d i c a t e t h a t t h i s enzyme i s not r e s p o n s i b l e f o r the augmented AHA s y n t h e t a s e a c t i v i t y o b s e r v e d i n g l y c e r o l -grown c u l t u r e s o f t h i s o r g a n i s m . Many p r o p e r t i e s o f t h e feedback system o b s e r v e d f o r the AHA s y n t h e t a s e of E. c o l i B i n d i c a t e t h a t t h i s enzyme b e l o n g s t o the c l a s s o f a l l o s t e r i c p r o t e i n s d e s c r i b e d by Monod et al. (1963). F i r s t , t h e s e n s i t i v i t y of t h e enzyme t o v a l i n e i s markedly a f f e c t e d by changes i n pH ( F i g . 4 ) , as i s t h e case i n the i n h i b i t i o n of t h r e o n i n e d e h y d r a t a s e by i s o l e u c i n e (Changeux, 1961) and p h o s p h o r i b o s y l - A T P p y r o p h o s p h o r y l a s e by h i s t i d i n e ( M a r t i n , 1962) . Second, t h e s e l e c t i v e removal o f s e n s i t i v i t y t o v a l i n e can be e f f e c t e d by heat t r e a t m e n t (Table V ) . S i m i l a r r e s u l t s have been r e p o r t e d f o r t h r e o n i n e dehy-d r a t a s e (Changeux, 1961) , a s p a r t a t e t r a n s c a r b a m y l a s e ( G e r h a r t and Pardee, 1962) and homoserine dehydrogenase ( P a t t e et al.3 - 64 -1963). I n t h e p r e s e n t s t u d y , however, d e s e n s i t i z a t i o n of AHA s y n t h e t a s e , even by m i l d h e a t t r e a t m e n t (25°), r e s u l t s i n a r a -p i d l o s s of a c t i v i t y . N e v e r t h e l e s s , t h e a s s u m p t i o n t h a t t h e enzyme p o s s e s s e s a s e p a r a t e s i t e f o r v a l i n e b i n d i n g ( i n h i b i -t o r s i t e ) , t h e i n t e g r i t y o f w h i c h i s i n d e p e n d e n t l y d e s t r o y e d by h e a t t r e a t m e n t , seems v a l i d due t o t h e d i s p r o p o r t i o n a l i t y between t h e l o s s of v a l i n e s e n s i t i v i t y and t h e l o s s o f c a t a -l y t i c a c t i v i t y under c e r t a i n c o n d i t i o n s (Table V ) . F i n a l l y , the complex i n h i b i t i o n k i n e t i c s o f t h i s enzyme ( F i g . 6) a r e c o n s i s t e n t w i t h t h e i d e a o f a d i s t i n c t i n h i b i t o r s i t e (Monod et al., 1963). I n a d d i t i o n t o p r o p e r t i e s of the feedback system, the a l l o s t e r i c n a t u r e o f AHA s y n t h e t a s e o f E. c o l i B i s s u g g e s t e d by t h e extreme l a b i l i t y o f t h i s enzyme i n c e l l e x t r a c t s . A common p r o p e r t y o f a l l o s t e r i c enzymes i s the r a p i d l o s s o f c a t a l y t i c a c t i v i t y f o l l o w i n g r e l e a s e from the c e l l (Monod et al., 1965). A l l o s t e r i c p r o p e r t i e s have been r e p o r t e d f o r the AHA s y n t h e t a s e o f E. c o l i K-12 (Umbarger and Brown, 1958) and of S. typhimurium ( B a u e r l e et al., 1964). The f a c t t h a t t h r e o n i n e d e h y d r a t a s e , th e enzyme t h a t i n i t i a t e s t h e b i o s y n t h e s i s of i s o l e u c i n e (Changeux, 1961), does not show a g l u c o s e e f f e c t (Table I) i n d i c a t e s t h a t r e -p r e s s i b i l i t y by c a t a b o l i t e r e p r e s s i o n i s not a g e n e r a l p r o -p e r t y o f the r e g u l a t o r y enzymes i n v o l v e d i n t h e b i o s y n t h e s i s o f the b r a n c h e d , a l i p h a t i c amino a c i d s . I n E. c o l i K-12 t h e s t r u c t u r a l gene g o v e r n i n g AHA s y n t h e t a s e (condensing enzyme) - 65 -i s c o n t r o l l e d by an o p e r a t o r l o c u s (opr. B) e n t i r e l y i n d e p e n -dent o f t h a t o f t h e o t h e r enzymes of the pathway (Ramhakrishnan and A d e l b e r g , 1965). T h r e o n i n e d e h y d r a t a s e , however, a l o n g w i t h t r a n s a m i n a s e B and t h e d i h y d r o x y a c i d d ehydrase, i s under the c o n t r o l o f a second o p e r a t o r l o c u s d e s i g n a t e d o p r . A. I t has been r e p o r t e d t h a t the AHA s y n t h e t a s e i n v a l i n e -i s o l e u c i n e a u x o t r o p h s o f E. a o l i i s s u b j e c t t o " m u l t i - v a l e n t r e p r e s s i o n " by t h e f o u r end 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 (Freundlich and Umbarger, 1963; F r e u n d l i c h , 1967) . R e s u l t s o f t h e p r e s e n t s t u d y (Table VI) i n d i c a t e t h a t , i n t h e w i l d - t y p e o r g anism, whether i n the p r e s e n c e or absence of added end p r o d u c t s , the AHA s y n t h e t a s e i s r e p r e s s e d t o a l e v e l d e t e r m i n e d by t h e c a r b o n s o u r c e on which t h e c e l l s were grown. Hence, growth on an e n e r g y - r i c h c a r b o n s o u r c e such as g l u c o s e can r e p r e s s f u r t h e r t h e l e v e l o f AHA s y n t h e t a s e i n c e l l s p r e v i o u s l y grown on a poor c a r b o n s o u r c e p l u s f o u r end p r o d u c t s (Table V I ) . These d a t a suggest t h a t t h i s enzyme may be under the c o n t r o l o f two d i s t i n c t 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 which i s d e r i v e d from the 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 end-product c o - r e p r e s s o r which i n t h i s case may be d e r i v e d from a number o f end p r o d u c t s . S i m i l a r i t y e x i s t s between the r e g u l a t o r y mechanism des-c r i b e d h e r e f o r AHA s y n t h e t a s e and t h e r e g u l a t i o n o f the K r e b s - c y c l e i n v a r i o u s m i c r o - o r g a n i s m s . Hanson and Cox (1967) r e p o r t e d t h a t w h i l e g l u c o s e a l o n e c o u l d p a r t i a l l y - 66 -(40-60%) r e p r e s s many o f t h e K r e b s - c y c l e enzymes i n B a c i l l u s s u b t i l i s j B a c i l l u s lichenifovmis and E. c o l i , complete r e -p r e s s i o n was a c h i e v e d o n l y when the c e l l s were grown i n t h e p r e s e n c e o f a r a p i d l y m e t a b o l i z a b l e energy so u r c e and c e r t a i n b i o s y n t h e t i c end p r o d u c t s o f t h e c y c l e , p a r t i c u l a r l y g l u t a -mate. Growth on end p r o d u c t s p l u s a poor c a r b o n s o u r c e (e.g. a c e t a t e ) f a i l e d t o r e p r e s s enzyme l e v e l s . The r e q u i r e -ment f o r d u a l c o n t r o l o f t h i s c y c l e was a t t r i b u t e d t o i t s am-p h i b o l i c r o l e i n i n t e r m e d i a r y m e t a b o l i s m . F or AHA s y n t h e t a s e , t h e o b s e r v a t i o n of d u a l c o n t r o l s u g g e s t s t h a t t h i s enzyme a l s o may p l a y an a m p h i b o l i c r o l e . The r e q u i r e m e n t o f t h i s enzyme f o r FAD s u p p o r t s t h i s s u g g e s t i o n . A l t e r n a t i v e l y , the s e n s i t i v i t y of" AHA s y n t h e t a s e t o c a t a b o l i t e r e p r e s s i o n a l o n g w i t h i n c o m p l e t e feedback i n h i b i -t i o n by v a l i n e and t h e h i g h Km o f t h i s enzyme f o r s u b s t r a t e may be p r o p e r t i e s of a r e g u l a t o r y system p o s s e s s e d by t h i s o r g a n i s m t o c o n t r o l the f u n c t i o n o f t h e branched " i l v a " p a t h -way. AHA s y n t h e t a s e i s i n v o l v e d i n the s y n t h e s i s o f 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 i n a d d i t i o n t o v a l i n e , t h e r e f o r e complete i n h i b i t i o n of AHA s y n t h e t a s e a c t i v i t y by v a l i n e would i m p a i r the s y n t h e s i s o f the o t h e r product's o f t h e p a t h -way. I n E. c o l i B t h i s problem appears t o be a l l e v i a t e d by t h e p r e s e n c e of an AHA s y n t h e t a s e which i s o n l y p a r t i a l l y s e n s i t i v e t o feedback i n h i b i t i o n ( F i g . 5 and T a b l e I I ) . A d d i t i o n a l c o n t r o l o f t h i s pathway may be p r o v i d e d by c a t a b o l i t e - 67 -r e p r e s s i o n which r e g u l a t e s the c o n c e n t r a t i o n of AHA s y n t h e t a s e i n the c e l l . F o r example, when the w i l d - t y p e o r g a n i s m i s growing on an e n e r g y - r i c h c a r b o n s o u r c e such as g l u c o s e , p y r u v a t e and o t h e r m e t a b o l i c i n t e r m e d i a t e s a r e r a p i d l y formed and accumulate (Magasanik, 1961). C a t a b o l i t e r e p r e s s i o n i s s t r o n g under t h e s e c o n d i t i o n s and t h e s y n t h e s i s o f AHA s y n -t h e t a s e i s r e p r e s s e d ( F i g . 3 ) . However, s i n c e growth on g l u c o s e r e s u l t s i n t h e e l e v a t i o n of t h e i n t r a c e l l u l a r con-c e n t r a t i o n o f p y r u v a t e , the i n c r e a s e d a c t i v i t y o f the r e -m a i n i n g enzyme w i l l be s u f f i c i e n t t o s a t i s f y t h e b i o s y n t h e t i c r e q u i r e m e n t s of t h e c e l l . When g l u c o s e i s r e p l a c e d by a carbon s o u r c e w h i c h i s m e t a b o l i z e d more s l o w l y (e.g. g l y c e r o l ) , the c o n c e n t r a t i o n o f t h e p y r u v a t e , p o o l d e c r e a s e s . Due t o the h i g h Km o f t h i s enzyme f o r s u b s t r a t e (^20 mM) ( F i g . 6 ) , t h i s d e c r e a s e i n p y r u v a t e c o n c e n t r a t i o n i n t h e c e l l , r educes AHA-synthetase a c t i v i t y and s u b s e q u e n t l y t h e a c t i v i t y o f the e n t i r e " i l v a " pathway. However, s i n c e c a t a b o l i t e r e p r e s s i o n i s a l s o r e l a x e d d u r i n g growth on a poor energy s o u r c e , AHA s y n -t h e t a s e i s d e r e p r e s s e d ( F i g . 3 ) . Thus, t h e c e l l compensates f o r t h e d e c r e a s e i n t h e c o n c e n t r a t i o n of t h e p y r u v a t e p o o l by i n c r e a s i n g t h e c o n c e n t r a t i o n o f AHA s y n t h e t a s e i n t h e c e l l . C o n t r o l o f AHA s y n t h e t a s e a c t i v i t y by a c o m b i n a t i o n of c a t a -b o l i t e r e p r e s s i o n and p a r t i a l feedback i n h i b i t i o n e n a b l e s t h e organism t o s a t i s f y i t s 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 r e q u i r e m e n t s under v a r i o u s growth c o n d i t i o n s , w i t h o u t syn-t h e s i z i n g e x c e s s i v e q u a n t i t i e s of v a l i n e . F u r t h e r e v i d e n c e i n - 68' -s u p p o r t o f t h i s h y p o t h e s i s w i l l be p r e s e n t e d i n PART B o f t h i s t h e s i s . A l t h o u g h i t appears anomalous, t h a t AHA s y n t h e t a s e , an enzyme i n v o l v e d i n t h e r e g u l a t i o n , o f a b i o s y n t h e t i c pathway, sh o u l d . b e 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 , t h i s c a s e i s not un i q u e . G o r i n i and Gundersen (1961) have r e p o r t e d t h a t t h e o r n i t h i n e t r a n s c a r b a m y l a s e of.E. c o l i B (but n o t o f s e v e r a l o t h e r s t r a i n s o f E. c o l i examined) was s u b j e c t t o a g l u c o s e e f f e c t . . R e p r e s s i o n o f o r n i t h i n e t r a n s c a r b a m y l a s e by t h e carb o n s o u r c e appears t o p l a y a major r o l e i n t h e r e g u l a t i o n o f t h e a r g i n i n e pathway i n t h i s s t r a i n ( B ) , s i n c e o r n i t h i n e t r a n s c a r b a m y l a s e i s n o t r e g u l a t e d by end-product r e p r e s s i o n . Thus, a l t h o u g h r e g u l a t i o n o f t h e v a l i n e - i s o l e u c i n e and o f the a r g i n i n e pathways appears t o d i f f e r i n many r e s p e c t s i n E. c o l i B ( G o r i n i and Gundersen, 1961), t h e i n v o l v e m e n t o f c a t a b o l i t e r e p r e s s i o n i n the c o n t r o l of t h e s e two a n a b o l i c pathways r e p r e s e n t s an i n t e r e s t i n g d e v i a t i o n from t h e g e n e r a l hypotheses c o n c e r n i n g r e g u l a t i o n of b i o s y n t h e t i c systems. G o r i n i (196 3) s u g g e s t e d t h a t the s e n s i t i v i t y t o c a t a b o l i t e r e p r e s s i o n o f o r n i t h i n e t r a n s c a r b a m y l a s e i n E. c o l i B may r e p r e s e n t a v e s t i g i a l c o n t r o l mechanism from a p e r i o d i n t h e p a s t when t h e a r g i n i n e pathway of t h i s organism f u n c t i o n e d c a t a b o l i c a l l y as w e l l as a n a b o l i c a l l y . The p r e s e n t - d a y s t r a i n B t h e n may have l o s t the d e g r a d a t i v e use of t h e p a t h -way w h i l e r e t a i n i n g t h e " c a t a b o l i t e r e p r e s s i o n " t y p e o f con-t r o l . A l t h o u g h t h i s h y p o t h e s i s may e x p l a i n t h e o r i g i n o f - 69 -t h i s unexpected c o n t r o l mechanism i n the " i l v a " pathway o f E. coli. B, i t i s d i f f i c u l t t o u n d e r s t a n d how t h i s mechanism c o u l d w i t h s t a n d n a t u r a l s e l e c t i o n u n l e s s i t can bestow on a c e l l a g r e a t e r s u r v i v a l v a l u e t h a n t h a t p r o v i d e d by t h e more common end-product r e p r e s s i o n o b s e r v e d i n o t h e r a n a b o l i c pathways. - 70 -PART B; R e l a x a t i o n of C a t a b o l i t e R e p r e s s i o n i n S t r e p t o -mycin-Dependent EschericHid c o l i RESULTS I . R e l a x a t i o n o f t h e G l u c o s e E f f e c t oh 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 Streptomycin-Dependent Escherichia c o l i I n PART A, e v i d e n c e was p r e s e n t e d (Table I and F i g . 3) whic h i n d i c a t e d t h a t the b i o s y n t h e t i c a c e t o l a c t a t e - f o r m i n g system (AHA s y n t h e t a s e ) o f w i l d - t y p e E. c o l i B, i s r e p r e s s e d by growth on a r a p i d l y m e t a b o l i z a b l e c a r b o n s o u r c e such as g l u c o s e . The r e s u l t s o f a s i m i l a r e x periment u s i n g a Sm-dependent mutant of E. c o l i B a r e p r e s e n t e d i n F i g . 7. A f t e r a s l i g h t d e c r e a s e i n s p e c i f i c a c t i v i t y a t c o n c e n t r a t i o n s of g l u c o s e o r g l y c e r o l g r e a t e r t h a n 0.05% the a c t i v i t y remained c o n s t a n t d u r i n g a 1 0 - f o l d i n c r e a s e i n c a r b o n - s o u r c e concen-t r a t i o n . U n l i k e the AHA s y n t h e t a s e o f t h e w i l d - t y p e o r g a n i s m ( F i g . 3 ) , t h e s p e c i f i c a c t i v i t y o f t h i s enzyme i n the Sm-dependent mutant was not s i g n i f i c a n t l y a l t e r e d by c h a n g i n g the c a r b o n s o u r c e from g l u c o s e t o g l y c e r o l ( F i g . 7 ) . These r e s u l t s s u ggest t h a t i n the Sm-dependent mutant, i n c o n t r a s t w i t h t h e w i l d - t y p e organism, t h e r e i s a t l e a s t p a r t i a l r e l a x a -t i o n o f r e p r e s s i o n by the carbon s o u r c e o f AHA s y n t h e t a s e . I I . M o d i f i c a t i o n o f D i a u x i e Growth i n Streptomycin-Dependent Mutants F o r a more d e t a i l e d i n v e s t i g a t i o n o f t h e mechanism o f - 71 ->-O < u. o UJ CL CARBON SOURCE % Ficj. 7. E f f e c t of i n c r e a s i n g ( i n i t i a l ) concentrations of car-bon source on the formation of AHA synthetase i n streptomycin-dependent Escherichia coli B. The minimal s a l t s medium was supplemented with e i t h e r glucose ( 0 ) or g l y c e r o l (o). The r e s u l t s of an i d e n t i c a l experiment ( ) w i t h the wi l d - t y p e parent of t h i s mutant, grown on glucose, were taken from F i g . 3. S p e c i f i c a c t i v i t y i s expressed as ymoles of a c e t o i n formed/mg. of p r o t e i n / h r . at 37°. - 72 -r e l a x a t i o n of c a t a b o l i t e r e p r e s s i o n i n Sm-dependent mutants, t h e B - g a l a c t o s i d a s e system was chosen as the e x p e r i m e n t a l t o o l (Jacob and Monod, 1961). The c l a s s i c a l e x p e r i m e n t t h a t demonstrates the " g l u c o s e e f f e c t " (Epps and G a l e , 1942) i s the o c c u r r e n c e of a, b i p h a s i c growth c u r v e . i n a b a c t e r i a l c u l t u r e growing on a m i x t u r e o f g l u c o s e and l a c t o s e . T h i s b i p h a s i c growth p a t t e r n was named the " d i a u x i e e f f e c t " by Monod (1947). W i l d - t y p e E. c o l i B ( F i g . 8A) e x h i b i t s a c l a s s i c a l d i a u x i e growth c u r v e r e s u l t i n g from t h e complete r e p r e s s i o n o f 3 - g a l a c t o s i d a s e d u r i n g growth on g l u c o s e . The c o r r e s p o n d i n g Sm-dependent c u l t u r e (DB), however, showed o n l y a s l i g h t d i a u x i e e f f e c t ( F i g . 8B). The p a r t i a l e l i m i n a t i o n o f d i a u x i e growth i n t h e Sm-dependent mutant appeared t o r e s u l t from i n i t i a t i o n o f i n d u c t i o n of 3-g a l a c t o s i d a s e a t l e a s t lh h r . before the e x h a u s t i o n o f g l u c o s e (3h h r . ) . V a r y i n g the l a c t o s e : g l u c o s e r a t i o d i d not p r e v e n t e a r l y i n d u c t i o n nor r e s t o r e t y p i c a l d i a u x i e growth. To c o n f i r m t h e d i f f e r e n c e s i n t h e d i a u x i e response of S m - s e n s i t i v e and Sm-dependent o r g a n i s m s , a n o t h e r s t r a i n o f E. c o l i was examined. The' growth c u r v e s a r e g i v e n i n F i g . 9 f o r w i l d - t y p e E. c o l i E ( s t r a i n SE) and t h e Sm-dependent d e r i -v a t i v e .of t h i s s t r a i n (DE). S t r a i n SE, l i k e s t r a i n SB, e x h i -b i t e d d i a u x i e growth ( F i g . 9A), whereas t h e Sm-dependent mutant DE ( F i g . 9 B ) , d i d not show even the s l i g h t d i a u x i e e f f e c t o b t a i n e d w i t h mutant DB. Thus growth on g l u c o s e c o m p l e t e l y f a i l e d t o r e p r e s s 3 - g a l a c t o s i d a s e i n d u c t i o n i n TIME, HOURS F i g . .8. G l u c o s e - l a c t o s e d i a u x i e growth o f w i l d - t y p e Escherichia c o l i B (SB) and a s t r e p t o m y c i n -dependent mutant (DB) of t h i s o r g a n i s m . Growth (o) and 3 - g a l a c t o s i d a s e f o r m a t i o n (©) were f o l -lowed i n c u l t u r e s g r owing a e r o b i c a l l y a t 37° on m i n i m a l medium supplemented w i t h 0.025% g l u -cose and 0.2% l a c t o s e . See METHODS f o r f u r t h e r e x p e r i m e n t a l d e t a i l s . A, S t r a i n SB; B, s t r a i n DB. • T 1 1 1 1 1 I 1 I ( 1 1 ' ' ' ' ' r F i g . 9. G l u c o s e - l a c t o s e d i a u x i e growth of w i l d - t y p e Escherichia c o l i E (SE) and a s t r e p t o m y c i n -dependent mutant (DE) o f t h i s o r g a n i s m . Growth (o) and B - g a l a c t o s i d a s e f o r m a t i o n (o) were f o l -lowed i n c u l t u r e s g r o w i n g a e r o b i c a l l y a t 37° on m i n i m a l medium supplemented w i t h 0.025% g l u c o s e and 0.2% l a c t o s e . See METHODS, f o r f u r t h e r e x p e r i m e n t a l d e t a i l s . A, S t r a i n SE; B, s t r a i n DE. - 75 -s t r a i n DE. T h i s was c o n f i r m e d by an e x a m i n a t i o n o f t h e i n d u c t i o n c u r v e ( F i g . 9B). I I I . D i f f e r e n c e s b e t w e e n . S t r e p t o m y c i n - S e n s i t i v e and Streptomycin-Dependent Escherichia coli' i n the G l u c o s e E f f e c t on g - G a l a c t o s i d a s e S y n t h e s i s A more d i r e c t approach t o t h e measurement of c a t a b o l i t e r e p r e s s i o n i s t o s t u d y t h e e f f e c t o f v a r i o u s carbon s o u r c e s on t h e d i f f e r e n t i a l r a t e o f enzyme s y n t h e s i s (Cohn and H o r i b a t a , 1959). F i g . 10A shows the l a c t o s e - i n d u c e d f o r m a t i o n o f B-g a l a c t o s i d a s e i n c u l t u r e s o f s t r a i n SB growing e x p o n e n t i a l l y on g l y c e r o l , a c a r b o n s o u r c e t h a t e x e r t s l i t t l e c a t a b o l i t e r e p r e s s i o n owing t o i t s r e l a t i v e l y low r a t e o f m e t a b o l i s m (Magasanik et al., 1959). The c o n c e n t r a t i o n o f enzyme/ml. i s p l o t t e d as a f u n c t i o n o f t o t a l c e l l u l a r p r o t e i n ( F i g . 10A). The a d d i t i o n o f g l u c o s e ( f i n a l c o n c e n t r a t i o n , 0.05%) r e s u l t e d i n a r a p i d d e c r e a s e i n the d i f f e r e n t i a l r a t e o f B-g a l a c t o s i d a s e s y n t h e s i s . I n t h i s s t r a i n , t h e r a t e of enzyme s y n t h e s i s on g l u c o s e was a p p r o x i m a t e l y 70% l e s s t h a n th e r a t e on the g l y c e r o l c o n t r o l . The r e s u l t s o f an i d e n t i c a l e x periment w i t h s t r a i n DB a r e g i v e n i n F i g . 10B. I n t h i s c a s e , the a d d i t i o n o f g l u -cose evoked a sharp b u t s h o r t - l i v e d r e p r e s s i o n o f B - g a l a c -t o s i d a s e s y n t h e s i s , which r a p i d l y gave way t o a d i f f e r e n t i a l r a t e o n l y 15% l e s s t h a n t h a t o f t h e c o n t r o l . Experiments" " i d e n t i c a l w i t h t h o s e d e s c r i b e d f o r s t r a i n s PROTEIN, fig /mi F i g . .10. E f f e c t o f t h e a d d i t i o n o f g l u c o s e on the d i f f e r e n t i a l r a t e o f 8 - g a . l a c t o s i d a s e i n d u c t i o n i n c u l t u r e s o f w i l d - t y p e Escherichia c o l i B (SB) and a s t r e p t o m y c i n - d e p e n d e n t mutant (DB) c f t h i s o rganism. C e l l s were i n d u c e d w i t h l a c t o s e (0.2%) w h i l e g rowing expo-n e n t i a l l y on g l y c e r o l ( 0 . 1 % ) - s a l t s medium (o) . G l u c o s e (0.05%, f i n a l c o n c e n t r a t i o n ) was added as i n d i c a t e d by t h e arrows (©). F u r t h e r e x p e r i m e n t a l d e t a i l s a r e g i v e n i n the METHODS. A, S t r a i n SB; B, s t r a i n DB. T 1 1 1 1 ! 1 1 I 1 1 1 1 r 0 . 40 80 120 160 0 4 0 8 0 i 2 0 160 P R O T E I N , f i g / m l F i g . 11. E f f e c t of t h e a d d i t i o n o f g l u c o s e on the d i f f e r e n t i a l r a t e o f frgalactosidase i n d u c t i o n i n c u l t u r e s of w i l d - t y p e Escherichia c o l i E (SE) and a s t r e p t o m y c i n - d e p e n d e n t mutant (DE) of t h i s o r g a n i s m . C e l l s were i n d u c e d w i t h l a c t o s e (0.2%) w h i l e g r o w i n g ex-p o n e n t i a l l y on g l y c e r o l ( 0 . 1 % ) - s a l t s medium ( o ) . G l u c o s e (0.05%, f i n a l c o n c e n t r a t i o n ) was added as i n d i c a t e d by t h e arrows (©). F u r t h e r e x p e r i m e n t a l d e t a i l s a r e g i v e n i n t h e METHODS. A, S t r a i n SE; B, s t r a i n DE. - 78 -SB and DB were c a r r i e d " out on w i l d - t y p e s t r a i n E and i t s Sm- • dependent d e r i v a t i v e , DE. A d d i t i o n o f g l u c o s e t o c u l t u r e s o f s t r a i n SE growing e x p o n e n t i a l l y on g l y c e r o l caused an i n i t i a l s h a rp r e p r e s s i o n o f enzyme s y n t h e s i s t h a t was t r a n s i e n t and gave way t o a s t e a d y - s t a t e r a t e about 40% l e s s than the r a t e o b s e r v e d on g l y c e r o l a l o n e ( F i g . 11A). I n c o n t r a s t , a d d i t i o n o f g l u c o s e t o s t r a i n DE had no e f f e c t on the r a t e o f 3 - g a l a c -t o s i d a s e s y n t h e s i s ( F i g . 11B). B o th of t h e Sm-dependent s t r a i n s t h u s showed r e s i s t a n c e toward c a t a b o l i t e r e p r e s s i o n when compared t o the p a r e n t Sm-s e n s i t i v e s t r a i n s . However, t h e r e were q u a n t i t a t i v e d i f f e r e n c e s between the two dependent s t r a i n s , b o t h i n d i a u x i e growth ( F i g s . 8B and 9B, r e s p e c t i v e l y ) and i n 3 - g a l a c t o s i d a s e i n d u c -t i o n ( F i g s . 10B and 11B, r e s p e c t i v e l y ) . Thus s t r a i n DE appeared c o m p l e t e l y r e s i s t a n t t o c a t a b o l i t e r e p r e s s i o n , b u t s t r a i n DB was somewhat l e s s r e s i s t a n t t o t h i s e f f e c t . IV. 3 - G a l a c t o s i d a s e I n d u c t i o n i n P s u e d o - R e s t i n g C e l l s The q u e s t i o n now may be asked: do Sm-dependent mutants l a c k the mechanism f o r the f o r m a t i o n o f t h e c a t a b o l i t e co-r e p r e s s o r , as s u g g ested by r e s u l t s w i t h s t r a i n DE> o r i s the c o - r e p r e s s o r s y n t h e s i z e d a t a r a t e i n s u f f i c i e n t t o cause r e -p r e s s i o n , t h e s i t u a t i o n i n d i c a t e d by r e s u l t s w i t h s t r a i n DB? To answer t h i s q u e s t i o n , 3 - g a l a c t o s i d a s e i n d u c t i o n was examined i n " p s e u d o - r e s t i n g " c e l l s u s p e n s i o n s of s t r a i n s SE and DE p r e -p a r e d as d e s c r i b e d i n the METHODS s e c t i o n . These e x p e r i m e n t a l - 79 -c o n d i t i o n s r e s u l t e d i n a l i m i t a t i o n o f a n a b o l i s m w i t h o u t l i m i t i n g t o t h e same degree th e c o r r e s p o n d i n g c a t a b o l i c p r o -c e s s e s , t h u s p e r m i t t i n g an a c c u m u l a t i o n o f c a t a b o l i t e c o - r e p r e s -s o r m o l e c u l e s (Mandelstam, 1961). R e s u l t s o f a t y p i c a l ex-p e r i m e n t a r e p r e s e n t e d i n F i g . 12. When g l u c o s e a t a f i n a l c o n c e n t r a t i o n o f 0.075% was added t o c u l t u r e s o f s t r a i n SE o r s t r a i n DE a f t e r 90 min. o f l a c t o s e - i n d u c e d s y n t h e s i s o f 3-g a l a c t o s i d a s e , enzyme s y n t h e s i s was r a p i d l y i n h i b i t e d i n b o t h c u l t u r e s . Hence, the mechanism f o r f o r m a t i o n o f c a t a b o l i t e c o - r e p r e s s o r was o p e r a t i v e in.Sm-dependent mutants, b u t under the c o n d i t i o n s of e x p o n e n t i a l growth, a p p a r e n t l y the r a t e o f f o r m a t i o n of c o - r e p r e s s o r i n s t r a i n DE was i n s u f f i c i e n t t o cause a " g l u c o s e e f f e c t " . Only w h e n . c o n d i t i o n s were m o d i f i e d so as t o f a v o u r t h e a c c u m u l a t i o n o f h i g h c o n c e n t r a t i o n s o f c a t a b o l i t e c o - r e p r e s s o r was c a t a b o l i t e r e p r e s s i o n demonstrable i n t h i s Sm-dependent organism. I n a d d i t i o n t o l a c t o s e , t h e g r a t u i t o u s i n d u c e r s , . m e t h y l 3 - D - t h i o g a l a c t o p y r a n o s i d e (TMG) and i s o p r o p y l 3 - D - t h i o g a l a c t o -p y r a n o s i d e (IPTG) were used as i n d u c e r s o f 3 - g a l a c t o s i d a s e i n many of t h e s e s t u d i e s . R e s u l t s w i t h TMG g e n e r a l l y agreed w i t h t h o s e o b t a i n e d w i t h l a c t o s e . IPTG, however, was un-s a t i s f a c t o r y i n t h i s s t u d y ; 3 - g a l a c t o s i d a s e i n d u c t i o n by t h i s p o w e r f u l i n d u c e r (Jacob and Monod, 1961; Moses and P r e v o s t , 1966) was c o m p l e t e l y r e s i s t a n t t o r e p r e s s i o n by g l u c o s e under the c o n d i t i o n s used. TIME , MINUTES F i g . 12. E f f e c t o f the a d d i t i o n of g l u c o s e on 3 - g a l a c t o s i d a s e i n d u c t i o n i n " p s e u d o - r e s t i n g c u l t u r e s o f w i l d - t y p e Escherichia c o l i E (SE) and a s t r e p t o m y c i n - d e p e n d e n t mutant (DE)- o f t h i s organism. E x p e r i m e n t a l c o n d i t i o n s a r e d e s c r i b e d i n the METHODS. G l u c o s e (0.075%, f i n a l c o n c e n t r a t i o n ) was added t o one f l a s k as i n d i c a t e d by the a r r o w s . G l y c e r o l c o n t r o l ( o ) ; g l u c o s e added (©). A, S t r a i n SE; B, s t r a i n DE. - 81 -V. R e l a x a t i o n of C a t a b o l i t e R e p r e s s i o n i r i Pathways o f C a r b o h y d r a t e M e t a b o l i s m I f t h e d e r e p r e s s i o n o f AHA s y n t h e t a s e i n t h e Sm-dependent mutant ( F i g . 7) was the r e s u l t .-of ? a g e n e r a l d e c r e a s e i n t h e c o n c e n t r a t i o n o f c a t a b o l i t e c o - r e p r e s s o r m o l e c u l e s i n t h e c e l l s , t h e n o t h e r enzymes s u b j e c t t o r e p r e s s i o n by c a r b o n s o u r c e s h o u l d a l s o r e f l e c t t h i s l o s s of c o n t r o l . Many o f the enzymes of i n t e r m e d i a r y m e t a b o l i s m a r e known t o be s e n s i -t i v e t o c a t a b o l i t e r e p r e s s i o n (Magasanik, 1961; G o r i n i and Gundersen, 1961; Hanson and Cox, 1967). F o r example, t h e K r e b s - c y e l e enzymes a r e l o w e r i n m i c r o - o r g a n i s m s grown on a s a l t s medium c o n t a i n i n g g l u c o s e t h a n i n c e l l s grown on a poor c a r b o n s o u r c e such as g l y c e r o l o r l a c t a t e (Gray et al. , 1966; Hanson and Cox, 1967). To d e t e r m i n e whether or not the a p p a r e n t d e c r e a s e i n c o n c e n t r a t i o n o f c a t a b o l i t e c o - r e p r e s s o r i n Sm-dependent mutants i n f l u e n c e s the c o n t r o l o f o t h e r enzymes, the s p e c i f i c a c t i v i t i e s o f s e v e r a l K r e b s - c y c l e enzymes i n v o l v e d i n c a r b o h y d r a t e m e t a b o l i s m were d e t e r m i n e d i n crude e x t r a c t s o f s t r a i n s SB and DB grown on g l u c o s e and g l y c e r o l . The r e s u l t s a r e summarized i n T a b l e V I I . E x a m i n a t i o n o f t h e r e -s u l t s f o r the w i l d - t y p e o r g a n i s m ( s t r a i n SB) i n d i c a t e s t h a t t h e f o u r K r e b s - c y c l e enzymes i n v e s t i g a t e d as w e l l as AHA s y n t h e -t a s e e x h i b i t a " g l u c o s e e f f e c t " as demonstrated by g l y c e r o l / g l u c o s e s p e c i f i c a c t i v i t y r a t i o s g r e a t e r t h a n u n i t y (Table V I I , column b / a ) : t h i s e f f e c t was p a r t i c u l a r l y marked i n t h e case o f fumarase and a c o n i t a s e (Table V I I ) . I n t h e c o r r e s -T a b l e V I I . S p e c i f i c a c t i v i t i e s of enzymes from streptomycin-sensitive (SB) dependent (DB) s t r a i n s of E s c h e r i c h i a c o l i B grown on glucose or glycerol and streptomycin-Enzyme SB G l u c o s e G l y c e r o l (a) (b) b/a DB Gl u c o s e G l y c e r o l (c) (d) d/c DB/SB G l u c o s e G l y c e r o l c/a d/b AHA s y n t h e t a s e 4.42 9.32 2.11 9.64 9.52 0.99 2.18 1. 02 C i t r a t e s y n thase 172 440 2.56 493 349 0.71 2.87 0. 80 Fumarase 396 2070 5.23 2545 1885 0.74 6.42 0. 91 A c o n i t a s e 111 460 4.14 789 1317 1.67 7.10 2- 86 I s o c i t r i c dehydrogenase 365 629 1.73 880 943 1.07 2.41 1. 49 G l u c o s e 6-phosphate dehydrogenase 130 109 0. 85 137 137 1.00 1.05 1. 25 G l u c o k i n a s e 55.4 68.9 1.22 67.0 64.2 0.96 1.20 0. 93 G l u c o s e and g l y c e r o l were added t o the m i n i m a l medium t o g i v e an i n i t i a l c o n c e n t r a t i o n of 0.4%. Growth of the c u l t u r e s , p r e p a r a t i o n o f e x t r a c t s and enzyme assays a re d e s c r i b e d i n the METHODS. S p e c i f i c a c t i v i t i e s (Units/mg. of p r o t e i n ) r e p o r t e d a r e mean v a l u e s of a c t i v i t i e s ob-t a i n e d i n two o r more, s e p a r a t e e x p e r i m e n t s . - 83 -p o n d i n g Sm-dependent mutant, t h e r e p r e s s i o n of g l u c o s e - s e n s i -t i v e enzymes o b s e r v e d i n t h e w i l d - t y p e o r g a n i s m was ab s e n t (Table V I I ) , p e r m i t t i n g many of t h e enzymes t o r e a c h s p e c i -f i c a c t i v i t i e s e q u a l t o o r even e x c e e d i n g l e v e l s o b t a i n e d by growth o f t h e w i l d - t y p e p a r e n t s t r a i n on g l y c e r o l . The r e -l a t i o n s h i p between carbon s o u r c e and Sm phenotype w i t h r e s p 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 i s summarized by t h e r a t i o s o f t h e g l u c o s e s e n s i t i v e enzymes i n s t r a i n DB t o t h o s e i n s t r a i n SB (Table V I I ) . A f t e r growth o f c e l l s on g l u c o s e , t h e r a t i o s f o r a l l f i v e g l u c o s e - s e n s i t i v e enzymes exceeded 2 and f o r fumarase and a c o n i t a s e DB/SB r a t i o s g r e a t e r t h a n 6 were ob-s e r v e d (Table V I I , column c / a ) . I n e x t r a c t s o f c e l l s grown on g l y c e r o l , t h e DB/SB r a t i o s of most enzymes approached u n i t y (Table V I I , column d/b) e x c e p t t h e r a t i o f o r a c o n i t a s e , which was r e l a t i v e l y h i g h . The s p e c i f i c a c t i v i t i e s o f two o t h e r enzymes, g l u c o s e 6-phbsphate dehydrogenase and g l u c o k i n a s e were e s s e n t i a l l y the same i n t h e S m - s e n s i t i v e and Sm-dependent organisms and were n o t a f f e c t e d by cha n g i n g t h e carbon s o u r c e ( g l u c o s e o r g l y c e r o l ) (Table V I I ) . These r e s u l t s i n d i c a t e t h a t o n l y t h o s e enzymes t h a t respond t o changes i n the c o n c e n t r a t i o n o f c a t a b o l i t e c o - r e p r e s s o r m o l e c u l e s a r e e l e v a t e d i n Sm-dependent E.'coli. - 84 -V I . C e l l Y i e l d from G l u c o s e o f S t r e p t o m y c i n - S e n s i t i v e and Streptomycin-Dependent S t r a i n s o f Escherichia c o l i B To d e t e r m i n e i f c o n t r o l by c a t a b o l i t e r e p r e s s i o n was r e -l a x e d i n Sm-dependent mutants because o f i m p a i r e d c a r b o h y d r a t e m e t a b o l i s m , t h e e f f i c i e n c y o f g l u c o s e u t i l i z a t i o n f o r c e l l mass s y n t h e s i s was d e t e r m i n e d i n S m - s e n s i t i v e and Sm-depen-dent c u l t u r e s ofE. c o l i B. P r e l i m i n a r y e x p e r i m e n t s i n d i c a t e d t h a t i n E. c o l i B, g l u c o s e i s c o m p l e t e l y o x i d i z e d as soon as i t i s t a k e n up by t h e c e l l , s i n c e growth (determined by the i n c r e a s e i n c e l l mass) ceases a l m o s t i m m e d i a t e l y a f t e r t h e d i s a p p e a r a n c e o f g l u c o s e from t h e medium. T h e r e f o r e , i n c u l t u r e s o f E. c o l i B, the r a t e a t wh i c h g l u c o s e i s removed from t h e medium i s a measure of the r a t e of g l u c o s e u t i l i z a ^ t i o n by the c e l l . I n F i g . 13, t h e d r y wt. o f S m - s e n s i t i v e and Sm-dependent c e l l s o b t a i n e d d u r i n g a e r o b i c growth on l i m i t i n g g l u c o s e was p l o t t e d as a f u n c t i o n o f the d e c r e a s e i n g l u c o s e c o n c e n t r a t i o n o f t h e medium. The y i e l d o f Sm-dependent c e l l s was a p p r o x i m a t e l y 37% lower t h a n f o r the w i l d - t y p e organ-ism ( F i g . 1 3 ) . To d e t e r m i n e whether t h e d e c r e a s e i n e f f i c i e n c y o f c a r b o h y d r a t e m e t a b o l i s m i n the Sm-dependent mutant o c c u r r e d a n a e r o b i c a l l y as w e l l as a e r o b i c a l l y , t h e c e l l y i e l d from g l u c o s e of each o r g a n i s m was e s t i m a t e d i n t h e pr e s e n c e and i n the absence o f oxygen. The e f f e c t o f a r a p i d s h i f t from a e r o b i c t o a n a e r o b i c growth c o n d i t i o n s , on t h e r a t e o f g l u c o s e consumption r e l a t i v e to> c e l l mass f o r m a t i o n i s shown i n F i g . 14. - 85 -300 250 -I L_ I U J 0 100 200 3 00 4 0 0 DECREASE IN GLUCOSE , u.g/ml F i g . 13. Y i e l d o f c e l l s from g l u c o s e d u r i n g a e r o b i c growth o f s t r e p t o m y c i n - s e n s i t i v e (SB) and s t r e p t o m y c i n - d e p e n d e n t (DB) s t r a i n s o f Escherichia c o l i B. E x p o n e n t i a l c e l l s were washed and resuspended i n m i n i m a l s a l t s medium c o n t a i n i n g 400 yg. o f g l u c o s e / m l . D u r i n g a e r o b i c growth, p o r t i o n s o f c u l t u r e were removed, r a p i d l y c o o l e d , c e n t r i f u g e d (16,000 g f o r 10 min.) .and the s u p e r n a t a n t s o l u t i o n s were assay e d f o r g l u c o s e by t h e G l u c o s t a t method. S t r a i n S B ' ( I D ) ; s t r a i n DB (o) . - 86 -250r 0 I '. : L : J 1 1 1 0 200 400 600 800 1000 D E C R E A S E IN G L U C O S E , /xg/ml F i g . 14. E f f e c t o f a change from a e r o b i c t o a n a e r o b i c growth on the y i e l d o f c e l l s from g l u c o s e of s t r e p t o m y c i n - s e n s i t i v e (SB) and s t r e p t o m y c i n - d e p e n d e n t (DB) s t r a i n s o f Escherichia coli B. P o r t i o n s of a e r o b i c and a n a e r o b i c c u l t u r e were r e -moved f o r g l u c o s e d e t e r m i n a t i o n as d e s c r i b e d i n the l e g e n d o f F i g . 13. The arrows i n d i c a t e t h e ' p o i n t s a t which the growth c o n d i t i o n s were made a n a e r o b i c . Other e x p e r i m e n t a l d e t a i l s a re g i v e n i n the METHODS. S t r a i n SB, (©) ; s t r a i n DB, ( o ) . - 87 -Tab l e V I I I . Cell y i e l d s from glucose of streptomycin-sensitive and streptomycin-dependent s t r a i n s of E s c h e r i c h i a c o l i B during aerobic and anaerobic growth Y * S t r a i n A e r o b i c A n a e r o b i c S m - s e n s i t i v e 0.423 0.161 Sm-dependent 0.261 0.16 4 *Y i s t h e c e l l y i e l d c o n s t a n t d e f i n e d as the number o f yg. dry wt. o f c e l l s 'formed/yg. o f g l u c o s e consumed. V a l u e s of Y were c a l c u l a t e d from the d a t a o f F i g . 14. - 88 -Under a e r o b i c c o n d i t i o n s , the Sm-dependent mutant (DB), ex-h i b i t e d t h e same d e c r e a s e d e f f i c i e n c y o f g l u c o s e u t i l i z a t i o n d e s c r i b e d p r e v i o u s l y ( F i g . 1 3 ) . When growth c o n d i t i o n s were made a n a e r o b i c , however (at the a r r o w s ) , b o t h t h e Sm-sensi-t i v e and t h e Sm-dependent c u l t u r e s showed a d i m i n i s h e d and s i m i l a r e f f i c i e n c y o f g l u c o s e m e t a b o l i s m ( F i g . 1 4 ) . T h i s o b s e r v a t i o n was s u p p o r t e d by the c e l l y i e l d c o n s t a n t s (Y) c a l c u l a t e d f o r each o r g a n i s m d u r i n g a e r o b i c and a n a e r o b i c growth (Table V I I I ) . A l t h o u g h t h e a e r o b i c c e l l y i e l d c o n s t a n t f o r t h e Sm-dependent mutant was 38% lower t h a n f o r the w i l d -t y p e organism under the same c o n d i t i o n s , a n a e r o b i c a l l y , t h e c e l l y i e l d c o n s t a n t s f o r each o r g a n i s m were the same (Table V I I I ) . The d e c r e a s e d e f f i c i e n c y o f c a r b o h y d r a t e m e t a b o l i s m o b s e r v e d i n t h e Sm-dependent mutant thus appears t o be t h e r e s u l t of a s p e c i f i c impairment o f o x i d a t i v e p r o c e s s e s i n t h i s o rganism. V I I . ATP S y n t h e s i s - i n S t r e p t o m y c i n - S e n s i t i v e and S t r e p t o - mycin-Dependent S t r a i n s of Escherichia c o l i B The e x i s t e n c e o f a d i r e c t r e l a t i o n s h i p between the de-gree o f c a t a b o l i t e r e p r e s s i o n and t h e energy c o n t e n t o f an organis m o r between c a t a b o l i t e r e p r e s s i o n and the growth r a t e of an organi s m on a v a r i e t y o f energy s o u r c e s i s w e l l docu-mented i n t h e l i t e r a t u r e (Magasanik, 19 61; Mandelstam, 1961; T u s t a n o f f and B a r t l e y , 1964; Okinaka and Dobrogosz, 1967). I n f a c t , r e s u l t s from i n d i r e c t s t u d i e s have l e d t o t h e p r o p o s a l t h a t ATP (Mandelstam, 1961; Moses and P r e v o s t , 1966) - 89 -o r compounds m e t a b o l i c a l l y r e l a t e d t o ATP (Dobrogosz, 196 8; Cox and.Hanson, 1968) may be the c a t a b o l i t e c o - r e p r e s s o r . The o b s e r v a t i o n s t h a t c e l l y i e l d ( a e r o b i c ) from g l u c o s e ( F i g s . 13 and 14) and growth r a t e s on g l u c o s e , g l y c e r o l o r l a c t a t e (Table IX) o f Sm-dependent mutants a r e lower than f o r t h e c o r r e s p o n d i n g w i l d - t y p e p a r e n t s t r a i n s s u ggest t h a t t h e l o s s o f c o n t r o l by c a t a b o l i t e r e p r e s s i o n i n t h e s e mutants may r e -s u l t from an impairment o f g e n e r a l energy m e t a b o l i s m . To i n v e s t i g a t e t h i s p o s s i b i l i t y , t h e r a t e o f ATP s y n t h e s i s and t h e c o n c e n t r a t i o n o f t h e ATP p o o l were measured i n exponen-t i a l c u l t u r e s o f s t r a i n s SB and DB growing a e r o b i c a l l y on g l u c o s e s a l t s medium. R e s u l t s o f i n i t i a l e x p e r i m e n t s i n d i -c a t e d t h a t under t h e s e c o n d i t i o n s the ATP p o o l s o f b o t h the S m - s e n s i t i v e and the Sm-dependent organisms o s c i l l a t e d d u r i n g growth. T h e r e f o r e , a d i r e c t c omparison o f the ATP c o n c e n t r a -t i o n i n the two organisms a t a p a r t i c u l a r c e l l d e n s i t y was m e a n i n g l e s s . As a r e s u l t o f f u r t h e r e x p e r i m e n t a t i o n , , i t was found t h a t i f the i n i t i a l ATP p o o l was d e c r e a s e d by h a r v e s t i n g and p r e - i n c u b a t i n g t h e c e l l s i n the absence of an energy s o u r c e (see METHODS), the subsequent r a t e o f ATP s y n t h e s i s was ex-p o n e n t i a l d u r i n g a 3 - f o l d i n c r e a s e i n c e l l mass. The r e s u l t s o f a t y p i c a l e x p e r i m e n t a r e shown i n F i g . 15. The d o u b l i n g t i m e s f o r growth and f o r ATP f o r m a t i o n i n the S m - s e n s i t i v e c u l t u r e , were 52 and 36 min., r e s p e c t i v e l y ( F i g . 15A) , and i n t h e Sm-dependent c u l t u r e were b o t h a p p r o x i m a t e l y 70 min. ( F i g . 15B). Thus, ATP f o r m a t i o n p a r a l l e l e d growth r a t e i n t h e - 90 -T a b l e IX. Growth rates of E s c h e r i c h i a c o l i s t r a i n s S t r a i n Carbon so u r c e D o u b l i n g time (min.) S B ( S m - s e n s i t i v e ) G l u c o s e 56 G l y c e r o l 60 L a c t a t e 80 DB(Sm-dependent) G l u c o s e 85 G l y c e r o l 102 L a c t a t e 124 S E ( S m - s e n s i t i v e ) G l u c o s e 54 G l y c e r o l 66 DE(Sm-dependent) Glu c o s e 85 G l y c e r o l 110 A l l c u l t u r e s were grown a e r o b i c a l l y on the b a s a l s a l t s medium supplemented w i t h a ca r b o n s o u r c e a t an i n i t i a l c o n c e n t r a t i o n of 0.4%. 100 0 20 40 TIME , MINUTES vo F i g . 15. Rate of ATP f o r m a t i o n i n c u l t u r e s of s t r e p t o m y c i n - s e n s i t i v e (SB) and s t r e p t o -mycin-dependent (DB) Es cherichia c o l i B growing a e r o b i c a l l y on g l u c o s e - s a l t s medium. Growth (o) and ATP c o n c e n t r a t i o n (o) were d e t e r m i n e d on p o r t i o n s o f c u l t u r e removed a t the t i m e s i n d i c a t e d . The i n o c u l u m v/as p r e p a r e d by method I I (see METHODS) . A, S t r a i n 33; B, s t r a i n DB. The d o u b l i n g t i m e s of c e l l s f o r SB and DB were 52 min. and 71 min., r e -s p e c t i v e l y and o f ATP f o r m a t i o n were 36 min.-and 70 min., r e s p e c t i v e l y . - 92 -5.0 r i r- r to _ l _J 0 0.05 0.10 0A5 0 2 0 0.25 0.30 C E L L MASS , mg/ml F i g . 16. The ATP p o o l o f s t r e p t o m y c i n - s e n s i t i v e (SB) and s t r e p t o -mycin-dependent (DB) s t r a i n s of Escherichia c o l i B d u r i n g a e r o b i c growth on g l u c o s e - s a l t s medium. The p o o l was c a l c u l a t e d from t h e d a t a o f F i g . 15. S t r a i n SB ( o ) ; s t r a i n DB ( o ) . - 93 -Sm-dependent mutant whereas i n the S m - s e n s i t i v e organism t h e r a t e o f energy p r o d u c t i o n exceeded t h e r a t e of growth. T h i s o b s e r v a t i o n was s u p p o r t e d by a p l o t o f t h e ATP p o o l v e r s u s c e l l mass d u r i n g the growth of t h e s e c u l t u r e s ( F i g . 1 6 ) . The average c o n c e n t r a t i o n o f t h e ATP p o o l was n o t s i g n i f i c a n t l y d i f f e r e n t i n the S m - s e n s i t i v e and the Sm-dependent c u l t u r e s . However, the p o o l c o n c e n t r a t i o n remained r e l a t i v e l y c o n s t a n t i n the Sm-dependent mutant d u r i n g growth w h i l e i t i n c r e a s e d s l o w l y i n t h e w i l d - t y p e p a r e n t s t r a i n . T h e r e f o r e , a l t h o u g h t h e r a t e o f ATP s y n t h e s i s i s d e c r e a s e d i n t h e Sm-dependent or g a n i s m , the a c t u a l c o n c e n t r a t i o n o f the ATP p o o l i n t h i s mutant does not d i f f e r - . g r e a t l y from t h a t o f t h e S m - s e n s i t i v e s t r a i n . V I I I . E f f e c t o f Carbon Source on V a l i n e E x c r e t i o n by S t r e p - t o m y c i n - S e n s i t i v e and Streptomycin-Dependent C u l t u r e s of Es• cherichia c o l i B AHA s y n t h e t a s e i s d e r e p r e s s e d t o t h e same e x t e n t i n s t r a i n SB grown on g l y c e r o l as i n s t r a i n DB grown on g l u c o s e or on g l y c e r o l (Table V I I ) . Sm-dependent mutants growing a e r o b i c a l l y on g l u c o s e - s a l t s medium are known t o e x c r e t e L - v a l i n e (Bragg and P o l g l a s e , 1962, T i r u n a r a y a n a n et al. 3 1962). T h e r e f o r e , i f d e r e p r e s s i o n of AHA s y n t h e t a s e a l o n e i s s u f f i c i e n t t o cause v a -l i n e e x c r e t i o n , v a l i n e s h o u l d be p r e s e n t i n s u p e r n a t a n t f l u i d s o f c u l t u r e s o f s t r a i n s SB and DB grown on m i n i m a l medium supplemented w i t h g l y c e r o l . The r e s u l t s o f an e x p e r i m e n t - 94 -T a b l e X. Valine excretion by streptomycin-dependent s t r a i n s F l a s k No. S t r a i n 1 S B ( S m - s e n s i t i v e ) 2 DB(Sm-dependent) 3 SB 4 DB 5 SB treptomycin-sensitive and of E s c h e r i c h i a c o l i B Carbon so u r c e V a l i n e (ymoles/1) G l u c o s e 4.2 G l u c o s e 8.0 G l y c e r o l 0 G l y c e r o l 0 G l y c e r o l + G l u c o s e . 6.2 C u l t u r e s o f s t r a i n s SB and DB were grown, f o r 16 h r . on a s h a k e r a t 37° i n m i n i m a l medium supplemented w i t h 0.4% of e i t h e r g l u c o s e o r g l y c e r o l . The c e l l s were d i l u t e d w i t h f r e s h medium t o g i v e an A i t 2 o o f 0.1. F l a s k s nos. 1-4 r e c e i v e d e i t h e r g l u c o s e o r g l y c e r o l ( f i n a l concn., 0.4%) as i n d i c a t e d and the c u l t u r e s were grown w i t h v i g o r o u s a e r a t i o n t o an A 4 2 0 o f 1.6; The c e l l s were removed by c e n t r i f u g a t i o n and p o r t i o n s of the s u p e r n a t a n t f l u i d were assa y e d f o r v a l i n e (see METHODS). In f l a s k no. 5, d i l u t e d , g l y c e r o l - g r o w n c e l l s o f s t r a i n SB were supplemented w i t h 0.4% g l y c e r o l and grown a e r o b i c a l l y t o an Ai* 2 0 o f 0.8. G l u c o s e (0.4%) was th e n added and growth con-t i n u e d t o an Ai* 2 0 o f 1.6. - 95 -d e s i g n e d t o i n v e s t i g a t e the r o l e of the carbon s o u r c e i n v a l i n e e x c r e t i o n by S m - s e n s i t i v e and Sm-dependent c u l t u r e s o f E. e o l i B a r e p r e s e n t e d i n T a b l e X. W i l d - t y p e B ( s t r a i n SB) u n l i k e most S m - s e n s i t i v e s t r a i n s (Bragg and P o l g l a s e , 1962; T i r u n a r -ayanan et al. , 1962) e.x^creted a s i g n i f i c a n t amount o f v a l i n e d u r i n g a e r o b i c growth on g l u c o s e even though AHA s y n t h e t a s e was r e p r e s s e d under t h e s e c o n d i t i o n s ( F i g . 3 and T a b l e I ) . The c o r r e s p o n d i n g Sm-dependent mutant ( s t r a i n DB), however, produced a p p r o x i m a t e l y t w i c e as much v a l i n e under t h e same c o n d i t i o n s (Table X ) . When g l y c e r o l r e p l a c e d g l u c o s e as the ca r b o n and energy s o u r c e , v a l i n e was not d e t e c t e d i n t h e s u p e r n a t a n t f l u i d s of e i t h e r t h e S m - s e n s i t i v e or t h e Sm-dependent c u l t u r e s . However, when g l u c o s e was added t o a. c u l t u r e o f s t r a i n SB d e r e p r e s s e d f o r AHA s y n t h e t a s e by growth on g l y c e r o l , a r a p i d e x c r e t i o n o f v a l i n e r e s u l t e d (Table X, l i n e 5 ) . - 96 -DISCUSSION The r e s u l t s of e x p e r i m e n t s p r e s e n t e d i n PART B i n d i c a t e t h a t m e t a b o l i c c o n t r o l by c a t a b o l i t e r e p r e s s i o n i s r e l a x e d i n Sm-dependent mutants of Escherichia c o l i growing expo-n e n t i a l l y on a g l u c o s e - s a l t s medium. T h i s d e c r e a s e i n c a t a -b o l i t e r e p r e s s i o n appears to.be r e s p o n s i b l e f o r t h e d e r e p r e s s e d f o r m a t i o n o f the AHA s y n t h e t a s e of Sm-dependent mutants o f E. c o l i ( C o u k e l l and P o l g l a s e , 1965; T a b l e V I I ) . S t u d i e s on c a t a b o l i t e r e p r e s s i o n of the i n d u c i b l e 3 - g a l a c t o s i d a s e system i n E. c o l i B and i n an a d d i t i o n a l s t r a i n E. c o l i E i n d i c a t e t h a t c o n t r o l o f t h i s enzyme by the ca r b o n s o u r c e a l s o i s r e -l a x e d i n Sm-dependent mutants ( F i g s . 8-11). F u r t h e r m o r e , c e r t a i n enzymes o f the K r e b s - c y c l e w h i c h a r e s e n s i t i v e t o c a t a b o l i t e r e p r e s s i o n i n w i l d - t y p e s t r a i n B a r e d e r e p r e s s e d i n t h e Sm-dependent mutant (Table V I I ) . I n p a r t i c u l a r , t h e a c t i v i t i e s o f a c o n i t a s e , fumarase, c i t r a t e s y n t h a s e and i s o -c i t r a t e dehydrogenase a r e h i g h e r i n t h e Sm-dependent mutant than i n the p a r e n t s t r a i n o f E. c o l i B. Thus, r e l a x a t i o n o f c a t a b o l i t e r e p r e s s i o n appears t o be a g e n e r a l phenomenon i n t h e s e mutants s i n c e a l l , o f t h e g l u c o s e - s e n s i t i v e enzymes i n v e s t i g a t e d e x h i b i t e d t h i s l o s s o f c o n t r o l . An o b v i o u s q u e s t i o n t o ask a t t h i s p o i n t i s whether a s i n g l e compound i s r e s p o n s i b l e f o r the r e p r e s s i o n o f a l l g l u c o s e - s e n s i t i v e enzymes in.E. c o l i o r whether each enzyme responds o n l y t o i t s own s p e c i f i c c o - r e p r e s s o r m o l e c u l e . - 97 -D u r i n g a s e r i e s o f s t u d i e s by N e i d h a r d t and Magasanik (19 57) on the mechanism o f c a t a b o l i t e r e p r e s s i o n o f h i s t i d a s e i n -d u c t i o n i n Aevobactev aerogenes3 i t was found t h a t c e r t a i n growth c o n d i t i o n s which p e r m i t t e d t h e s y n t h e s i s of h i s t i d i n e , s t i l l r e p r e s s e d t h e f o r m a t i o n of o t h e r g l u c o s e - s e n s i t i v e enzymes. They c o n c l u d e d t h a t more than one c o - r e p r e s s o r m o l e c u l e must be i n v o l v e d i n t h e r e g u l a t i o n o f g l u c o s e - s e n s i -t i v e enzymes. F o r example, a s i n g l e n i t r o g e n - c o n t a i n i n g co-r e p r e s s o r may be r e s p o n s i b l e f o r the r e p r e s s i o n o f a l l enzymes a c t i n g on n i t r o g e n o u s compounds, w h i l e a n o t h e r c o - r e p r e s s o r may r e g u l a t e t h e s y n t h e s i s o f enzymes wh i c h degrade sugars and r e l a t e d m o l e c u l e s (Magasanik, 1961). R e c e n t l y , Moses and P r e v o s t (1966) p o s t u l a t e d a mechanism f o r g e n e r a l c o n t r o l by c a t a b o l i t e r e p r e s s i o n which r e q u i r e s o n l y a s i n g l e co-r e p r e s s o r m o l e c u l e . A c c o r d i n g t o t h i s h y p o t h e s i s , t h e v a r i o u s g l u c o s e - s e n s i t i v e enzymes i n the c e l l e x h i b i t d i f f e r e n t s e n s i -t i v i t i e s t o the same c a t a b o l i t e c o - r e p r e s s o r . F u r t h e r m o r e , the i n t r a c e l l u l a r c o n c e n t r a t i o n o f t h i s c o - r e p r e s s o r f l u c -t u a t e s i n r e s p o n s e t o changes i n t h e r a t e s o f t h e v a r i o u s c a t a b o l i c and a n a b o l i c p r o c e s s e s . Thus, when the c o - r e p r e s s o r c o n c e n t r a t i o n i n t h e c e l l i s low, enzymes wh i c h a r e v e r y s e n s i t i v e t o t h e c o - r e p r e s s o r remain r e p r e s s e d , w h i l e enzymes whi c h a r e l e s s s e n s i t i v e t o t h i s m o l e c u l e a r e d e r e p r e s s e d t o v a r y i n g d e g r e e s . A l t h o u g h the i d e n t i t y o f t h e c o - r e p r e s s o r m o l e c u l e remains o b s c u r e , a number of independent s t u d i e s s u g g e s t t h a t ATP (Mandelstam, 1961; Moses and P r e v o s t , 1966) - 98 -or compounds m e t a b o l i c a l l y r e l a t e d t o ATP (Dobrogosz, 1968; Cox and Hanson, 196 8) may be t h e e l u s i v e c o - r e p r e s s o r . S i n c e t h e g e n e r a l r e l a x a t i o n o f c o n t r o l by c a t a b o l i t e r e p r e s s i o n i n s t r a i n DB c o r r e l a t e s w i t h a d e c r e a s e d r a t e o f ATP s y n t h e s i s i n t h i s mutant ( F i g . 1 5 ) , t h e s i n g l e c a t a b o l i t e c o - r e p r e s s o r h y p o t h e s i s i n i t i a l l y appears t h e most a t t r a c t i v e ; However, i t i s p o s s i b l e t h a t a number o f d i f f e r e n t c o - r e p r e s s o r mole-c u l e s a r e i n v o l v e d , a l l o f w h i c h depend upon ATP f o r t h e i r s y n t h e s i s . Recent s t u d i e s , p r i m a r i l y by T y l e r et al. (1967) i n d i c a t e t h a t t h e r e i s more t h a n one t y p e o f g l u c o s e e f f e c t on g - g a l a c -t o s i d a s e i n d u c t i o n . I n a d d i t i o n t o t h e e f f e c t o f g l u c o s e on t h e t r a n s p o r t o f i n d u c e r a c r o s s t h e c e l l membrane, two t y p e s o f c a t a b o l i t e r e p r e s s i o n have been d e s c r i b e d w h i c h can be d i s t i n g u i s h e d from one a n o t h e r as b e i n g transient o r permanent. T r a n s i e n t r e p r e s s i o n i s a s h o r t - l i v e d r e p r e s s i o n ( u s u a l l y l e s s t h a n one g e n e r a t i o n ) w h i c h o c c u r s when g l u c o s e o r an a n a l o g o f g l u c o s e i s added t o a c u l t u r e g rowing e x p o n e n t i a l l y on a car b o n s o u r c e such as g l y c e r o l , l a c t a t e o r s u c c i n a t e . S i n c e o n l y g l u c o s e and g l u c o s e analogues can evoke t r a n s i e n t r e -p r e s s i o n , i t i s thought t h a t t h e c o - r e p r e s s o r i s e i t h e r g l u c o s e i t s e l f o r a c l o s e l y r e l a t e d m e t a b o l i t e ( g l u c o s e 6-phosphate, NADPH, e t c . ) ( P a i g e n , 1966; H s i e and R i c k e n b e r g , 1967; P r e v o s t and Moses; 1967). Permanent c a t a b o l i t e r e -p r e s s i o n , however, can be e x e r t e d by any carbon s o u r c e p r o -v i d e d t h a t the r a t e o f energy m e t a b o l i s m i n t h e c e l l exceeds - 99 -t h e b i o s y n t h e t i c r e q u i r e m e n t o f t h e c e l l (Magasanik, 1961; Mandelstam, 1961). F u r t h e r m o r e , permanent r e p r e s s i o n i s e l i -c i t e d by c a t a b o l i t e s w h i c h a r e m e t a b o l i c a l l y d i s t a n t from g l u c o s e and t h e degree o f r e p r e s s i o n remains r e l a t i v e l y con-s t a n t i n e x p o n e n t i a l . c u l t u r e s as l o n g as the c a r b o n s o u r c e i s p r e s e n t (Moses and P r e v o s t °,, 1966) . The c o n d i t i o n s employed i n t h i s work t o s t u d y t h e e f f e c t o f g l u c o s e on the d i f f e r e n t i a l r a t e o f 3 - g a l a c t o s i d a s e s y n t h e s i s ( a d d i t i o n o f g l u c o s e t o c e l l s g rowing e x p o n e n t i a l l y on g l y c e r o l ) would be e x p e c t e d t o r e s u l t i n t r a n s i e n t as w e l l as permanent c a t a b o l i t e r e p r e s s i o n ( F i g s . 10 and 1 1 ) . G l u c o s e does not b l o c k t h e uptake of i n d u c e r i n t o t h e s e c e l l s s i n c e an i n c r e a s e i n l a c t o s e c o n c e n t r a t i o n accompanied by a d e c r e a s e i n the c o n c e n t r a t i o n o f g l u c o s e had no e f f e c t on d i a u x i e growth o r 3 - g a l a c t o s i d a s e i n d u c t i o n (see Loomis and Magasanik, 1967). T r a n s i e n t r e p r e s s i o n i s p r o b a b l y r e s p o n s i b l e f o r t h e b i p h a s i c i n d u c t i o n k i n e t i c s w hich o c c u r r e d f o l l o w i n g t h e a d d i t i o n o f g l u c o s e t o e x p o n e n t i a l c u l t u r e s of s t r a i n SE ( F i g . 11A). In s t r a i n SB ( F i g . 10A), t h e s e v e r e permanent r e p r e s s i o n produced by g l u c o s e must "mask" a s i m i l a r t r a n s i e n t e f f e c t s i n c e t r a n s -i e n t r e p r e s s i o n i s o b s e r v e d i n t h e Sm-dependent mutant o f t h i s s t r a i n (DB) ( F i g . 10B). The f a i l u r e t o d e t e c t a r e s i d u a l t r a n s i e n t e f f e c t i n . s t r a i n DE ( F i g . 11B) may be due t o t h e l i m i t e d s e n s i t i v i t y o f t h i s e x p e r i m e n t a l system.to s l i g h t changes i n c a t a b o l i t e r e p r e s s i o n . i n s t r a i n s which n o r m a l l y e x h i b i t weak t r a n s i e n t and permanent r e p r e s s i o n . These d a t a - 100 -suggest, t h a t t h e d e c r e a s e d g l u c o s e . e f f e c t o b s e r v e d i n Sm-dependent mutants r e s u l t s from an impairment o f permanent and not o f t r a n s i e n t c a t a b o l i t e r e p r e s s i o n . F u r t h e r s u p p o r t f o r t h i s c o n c l u s i o n i s p r o v i d e d by the s p e c i f i c a c t i v i t i e s of enzymes i n v o l v e d i n the i n i t i a l s t e p s o f g l u c o s e m e t a b o l i s m . The s p e c i f i c a c t i v i t i e s o f g l u c o k i n a s e and g l u c o s e 6-phosphate dehydrogenase (Table V I I ) o f s t r a i n DB do n o t d i f f e r s i g n i -f i c a n t l y from t h o s e of the w i l d - t y p e p a r e n t s t r a i n (SB). S i n c e t h e o c c u r r e n c e of t r a n s i e n t c a t a b o l i t e r e p r e s s i o n i s dependent upon a r a p i d c o n v e r s i o n of g l u c o s e t o t h e l e v e l o f t h e s u g a r -phosphates ( P r e v o s t and Moses, 1967; T y l e r et al., 1967), e q u i v a l e n t g l u c o k i n a s e and g l u c o s e 6-phosphate dehydrogenase a c t i v i t i e s i n the two organisms t e n d t o r u l e out d i f f e r e n c e s i n t h i s form of c a t a b o l i t e r e p r e s s i o n . S t u d i e s on t h e r e g u l a t i o n o f AHA s y n t h e t a s e i n Sm-dependent E. c o l i B (DB) and o f t h e i n d u c i b l e B - g a l a c t o s i d a s e o f s t r a i n DB and o f an a d d i t i o n a l Sm-dependent s t r a i n (DE) i n d i c a t e t h a t a l t h o u g h c o n t r o l by c a t a b o l i t e r e p r e s s i o n i s r e l a x e d i n t h e s e mutants i t i s n o t c o m p l e t e l y a b s e n t . F o r example, B - g a l a c t o s i d a s e i n d u c t i o n i n Sm-dependent s t r a i n DE was e x t r e m e l y s e n s i t i v e t o g l u c o s e r e p r e s s i o n under c o n d i t i o n s where t h e a n a b o l i c r a t e o f t h i s mutant was l i m i t e d ( F i g . 1 2 ) . Thus, th e mechanism, concerned w i t h c a t a b o l i t e c o - r e p r e s s o r s y n t h e s i s i s o p e r a t i v e i n Sm-dependent mutants but under c o n d i t i o n s o f e x p o n e n t i a l growth, t h e c o n c e n t r a t i o n o f co-r e p r e s s o r i n the c e l l a p p a r e n t l y i s not m a i n t a i n e d a t a h i g h - 101 -enough l e v e l t o r e g u l a t e enzyme s y n t h e s i s . N e i d h a r d t (1960) i n v e s t i g a t e d c a r b o h y d r a t e m e t a b o l i s m i n a mutant o f A. aerogenes l a c k i n g g l u c o s e r e p r e s s i o n . As a r e s u l t o f t h i s s t u d y he c o n c l u d e d t h a t any mutant t h a t has l o s t i t s s e n s i t i v i t y t o g l u c o s e f o r a number of g l u c o s e -s e n s i t i v e enzymes s h o u l d have e i t h e r (1) an- . i m p a i r e d g l u c o s e d i s s i m i l a t i o n o r (2) an improved o v e r a l l a n a b o l i c c a p a c i t y . S i n c e t h e growth r a t e o f Sm-dependent E. c o l i B on g l u c o s e -s a l t s medium i s lower t h a n th e growth r a t e of t h e w i l d - t y p e s t r a i n (Table I X ) , the d e c r e a s e i n c a t a b o l i t e r e p r e s s i o n ob-s e r v e d i n t h i s mutant must r e s u l t . - f r o m an impairment of g l u -cose metabolism" because a s t i m u l a t i o n o f a n a b o l i s m (which i s t h e r a t e - l i m i t i n g p r o c e s s under t h e s e c o n d i t i o n s ) would increase t h e growth r a t e o f t h e dependent mutant. T h i s de-d u c t i o n was c o n f i r m e d by t h e d i s c o v e r y t h a t t h e e f f i c i e n c y o f a e r o b i c g l u c o s e u t i l i z a t i o n i n Sm-dependent E. c o l i B was o n l y t w o - t h i r d s t h a t o f t h e p a r e n t s t r a i n ( F i g . 1 3 ) . F u r t h e r s u p p o r t f o r the h y p o t h e s i s t h a t the d e c r e a s e i n c a t a b o l i t e r e p r e s s i o n i n the Sm-dependent mutant r e s u l t s from an e f f e c t on c a t a b o l i c r a t h e r t h a n on a n a b o l i c p r o c e s s e s was o b t a i n e d from an i n v e s t i -g a t i o n o f ATP f o r m a t i o n i n t h i s organism. The r e s u l t s i n d i -c a t e t h a t a l t h o u g h t h e ATP p o o l i n the Sm-dependent mutant was a p p r o x i m a t e l y e q u a l t o t h a t o f the s e n s i t i v e o r g a n i s m ( F i g . 1 6 ) , t h e r a t e cf ATP s y n t h e s i s i n the mutant was d i m i n i s h e d and p a r a l l e l e d , t h e growth r a t e o f the Sm-dependent c u l t u r e ( F i g . 15).... T h i s s u g g e s t s t h a t t h e growth r a t e of the; Sm-dependent - 102 -mutant ( u n l i k e t h a t o f t h e w i l d - t y p e p a r e n t s t r a i n ) i s l i m i t e d by t h e r a t e o f energy p r o d u c t i o n i n t h e c e l l . F u r t h e r m o r e , t h i s i n v e s t i g a t i o n o f ATP f o r m a t i o n s u g g e s t s t h a t i n expo-n e n t i a l c u l t u r e s , o f S m - s e n s i t i v e and Sm-dependent o r g a n i s m s , c a t a b o l i t e r e p r e s s i o n may be r e l a t e d more c l o s e l y t o the r a t e o f energy p r o d u c t i o n t h a n t o t h e c o n c e n t r a t i o n of energy i n t h e c e l l . G o r i n i and K a t a j a (1964a) d e s c r i b e d a c l a s s o f Sm-r e s i s t a n t , a r g i n i n e a u x o t r o p h s o f E. ooli w h i c h would grow o n l y i f a r g i n i n e o r Sm were added t o t h e m i n i m a l medium. These mu-t a n t s were termed c o n d i t i o n a l l y s t r e p t o m y c i n - d e p e n d e n t (CSD) because of t h e i r dependence on a n t i b i o t i c f o r growth i n the absence o f t h e r e q u i r e d amino a c i d . Subsequent s t u d i e s r e -v e a l e d t h a t one of t h e s e CSD-mutants p o s s e s s e d a d e f e c t i v e s t r u c t u r a l " , gene f o r t h e enzyme o r n i t h i n e t r a n s c a r b a m y l a s e (OTCase) and t h i s m u t a t i o n was p h e n o t y p i c a l l y c o r r e c t e d by Sm a t the l e v e l o f t r a n s l a t i o n when t h i s mutant was grown i n t h e p r e s e n c e of a n t i b i o t i c . E x a m i n a t i o n of the s p e c i f i c a c t i v i t i e s o f a number of enzymes.in t h e s e "Sm-suppressed" mutants i n d i -c a t e d t h a t w h i l e the c o n c e n t r a t i o n o f OTCase i n CSD-mutants was e l e v a t e d s u f f i c i e n t l y t o p e r m i t growth, th e f o r m a t i o n o f c e r t a i n o t h e r enzymes was i m p a i r e d ( G o r i n i and K a t a j a , 1964b). T h i s " o v e r - s u p p r e s s i o n " by Sm l e d t o t h e f o r m a t i o n , o f non-f u n c t i o n a l p r o t e i n i n the c e l l and.a r e d u c t i o n i n t h e growth r a t e s o f t h e s e mutants ( G o r i n i and K a t a j a , 1964b).. I n t h e p r e s e n t s t u d y , many of t h e p r o p e r t i e s o b s e r v e d f o r t h e Sm-- 103 -dependent mutant o f E.^coli B (DB) a r e analogous t o p r o p e r -t i e s d e s c r i b e d f o r t h e CSD-mutants. For example, b o t h mutants r e q u i r e a n t i b i o t i c f o r growth b u t grow more s l o w l y t h a n t h e i r r e s p e c t i v e w i l d - t y p e p a r e n t s t r a i n s ; b o t h mutants e x h i b i t i r -r e g u l a r i t i e s i n the c o n c e n t r a t i o n s o f a number of m e t a b o l i -c a l l y u n r e l a t e d enzymes and b o t h mutants appear l e s s e f f i c i e n t a t c e l l s y n t h e s i s t h a n t h e c o r r e s p o n d i n g a n t i b i o t i c independent o r g a n i s m s . D e s p i t e t h e s e s i m i l a r i t i e s , however, r e s u l t s o f e x p e r i m e n t s d e s c r i b e d i n t h i s s e c t i o n i n d i c a t e t h a t the Sm-dependent mutant of E. c o l i B and t h e CSD-mutants d e s c r i b e d by G o r i n i a r e u n r e l a t e d . F i r s t , the d e r e p r e s s e d f o r m a t i o n o f c e r t a i n enzymes i n s t r a i n DB does not o c c u r randomly, as would be e x p e c t e d i f Sm were m e r e l y a l t e r i n g t h e f i d e l i t y o f t r a n s l a -t i o n . Only g l u c o s e - s e n s i t i v e enzymes appear t o be a f f e c t e d , s i n c e g l u c o k i n a s e and g l u c o s e 6-phosphate dehydrogenase w h i c h a r e r e s i s t a n t t o c a t a b o l i t e r e p r e s s i o n e x h i b i t e q u i v a l e n t s p e c i f i c a c t i v i t i e s i n t h e S m - s e n s i t i v e and i n t h e Sm-depen-dent o r g a n i s m s . F u r t h e r m o r e , w h i l e the c o n c e n t r a t i o n s o f a number o f enzymes a r e e l e v a t e d i n t h e Sm-dependent mutant (DB), no enzymes have been d e t e c t e d w h i c h a r e s y n t h e s i z e d a t a de-c r e a s e d c o n c e n t r a t i o n . Second, w h i l e a e r o b i c c u l t u r e s o f s t r a i n DB e x h i b i t a d e c r e a s e d e f f i c i e n c y o f g l u c o s e u t i l i z a -t i o n f o r c e l l s y n t h e s i s , a n a e r o b i c a l l y , the Sm-dependent mutant i s e q u a l l y as e f f i c i e n t as the w i l d - t y p e p a r e n t s t r a i n (SB) (Table V I I I ) . A change, such as t h i s , i n oxygen t e n s i o n would n o t be e x p e c t e d t o a l t e r t h e c o d i n g p r o p e r t i e s o f CSD-- 1 0 4 -mutant ri b o s o m e s . T h e r e f o r e , most of t h e abnormal p r o p e r -t i e s of s t r a i n DB appear t o r e s u l t from i m p a i r e d energy meta-b o l i s m r a t h e r than from a d i r e c t e f f e c t o f DHSm on p r o t e i n s y n t h e s i s . I n PART A, i t was s u g g e s t e d t h a t the c a p a c i t y o f an o r g a n i s m t o c o n t r o l AHA-synthetase f o r m a t i o n by c a t a b o l i t e r e p r e s s i o n may a s s i s t i t i n r e g u l a t i n g t h e a c t i v i t y o f the complex, branched " i l v a " pathway. T h i s h y p o t h e s i s i s suppor-t e d by the i n v e s t i g a t i o n (Table X) o f the e f f e c t o f t h e n a t u r e of t h e c a r b o n s o u r c e on v a l i n e e x c r e t i o n by c u l t u r e s o f w i l d -t y p e E. c o l i B and a Sm-dependent d e r i v a t i v e ( s t r a i n DB) o f t h i s o r g a n i s m . When s t r a i n SB o r DB i s . grown on a m i n i m a l medium supplemented w i t h a s l o w l y m e t a b o l i z a b l e c a r b o n s o u r c e such as g l y c e r o l , no e x t r a c e l l u l a r v a l i n e i s produced (Table X) d e s p i t e t h e f a c t t h a t AHA s y n t h e t a s e i s d e r e p r e s s e d under t h e s e c o n d i t i o n s (Table V I I ) . T h i s r e s u l t i s c o n s i s t e n t w i t h t h e r e p o r t t h a t d u r i n g e x p o n e n t i a l growth on g l y c e r o l , i n t e r -m e d i a tes such as p y r u v a t e do not accumulate s i g n i f i c a n t l y s i n c e t h e y a r e s y n t h e s i z e d s l o w l y and m e t a b o l i z e d r a p i d l y (Magasanik et al., 1959). The i m p o r t a n t r o l e p l a y e d by t h e carbon s o u r c e d u r i n g v a l i n e e x c r e t i o n i s f u r t h e r emphasized by an e x a m i n a t i o n o f t h e s u p e r n a t a n t f l u i d s o f glucose-grown c u l t u r e s . S t r a i n SB, f o r example, e x c r e t e d a s m a l l b u t s i g -n i f i c a n t q u a n t i t y o f v a l i n e (Table X) even though AHA s y n t h e -t a s e was r e p r e s s e d by growth o n . g l u c o s e (Table V I I ) . The enhanced amount o f e x t r a c e l l u l a r v a l i n e by t h e Sm-dependent - 105 -mutant (DB) , under the same growth c o n d i t i o n s , c o r r e l a t e s w i t h t h e d e r e p r e s s e d l e v e l o f AHA s y n t h e t a s e i n t h i s organism (Table V I I ) . The most d r a m a t i c p r o d u c t i o n o f e x t r a c e l l u l a r v a l i n e from g l u c o s e , however, r e s u l t e d when g l u c o s e was added t o a S m - s e n s i t i v e c u l t u r e growing e x p o n e n t i a l l y on g l y c e r o l . A d d i t i o n o f g l u c o s e caused a r a p i d i n c r e a s e i n the p y r u v a t e c o n c e n t r a t i o n i n the c e l l (Magasanik et- al. , 1959) . S i n c e t h e AHA s y n t h e t a s e o f t h e s e c e l l s was a l r e a d y d e r e p r e s s e d as a r e s u l t o f growth on g l y c e r o l , v a l i n e c o u l d be r a p i d l y s y n -t h e s i z e d and e x c r e t e d b e f o r e t h e enzyme c o u l d be r e p r e s s e d (Table X ) . Thus, i n E. c o l i B, w h i l e a s l i g h t e x c r e t i o n o f v a l i n e can r e s u l t s i m p l y from growth on a r a p i d l y m e t a b o l i z a b l e c a r b o n s o u r c e such as g l u c o s e , maximum v a l i n e p r o d u c t i o n r e -q u i r e s both a h i g h c o n c e n t r a t i o n o f i n t r a c e l l u l a r p y r u v a t e and a d e r e p r e s s e d l e v e l o f AHA s y n t h e t a s e . These c o n d i t i o n s a r e p r e s e n t i n the Sm-dependent mutant of E. c o l i B (DB) growing e x p o n e n t i a l l y on g l u c o s e . I t has been r e p o r t e d t h a t v a l i n e i s e x c r e t e d when Sm (or DHSm) i s added t o growing c u l t u r e s o f S m - s e n s i t i v e b u t not h i g h -l e v e l S m - r e s i s t a n t m i c r o - o r g a n i s m s ( T i r u n a r a y a n a n et al. , 1962; Bragg and P o l g l a s e , 1962). I n v i e w of the r e l a t i o n s h i p between p y r u v a t e a c c u m u l a t i o n and v a l i n e e x c r e t i o n o b s e r v e d i n E. c o l i B, i t i s now p o s s i b l e t o s p e c u l a t e on the p r o b a b l e mechanism of t h i s phenomenon. When Sm i s added t o a s u s c e p t i b l e c u l t u r e , p r o t e i n s y n t h e s i s i s i n h i b i t e d (Erdos and Ullmann, 1959). T h i s impairment of a n a b o l i c a c t i v i t y w i l l r e s u l t i n a r a p i d and - 106 -d r a m a t i c i n c r e a s e i n the c o n c e n t r a t i o n of m e t a b o l i c i n t e r m e -d i a t e s such as p y r u v a t e . I f feedback i n h i b i t i o n o f AHA s y n -t h e t a s e by v a l i n e i s i n c o m p l e t e , as i t i s i n E. c o l i B ( F i g . 5 ) , v a l i n e s y n t h e s i s w i l l n o t be shut o f f as the c o n c e n t r a t i o n o f the amino a c i d i n c r e a s e s i n t h e c e l l . T h i s w i l l r e s u l t i n an o v e r p r o d u c t i o n and e v e n t u a l e x c r e t i o n o f v a l i n e . P r o t e i n s y n t h e s i s i n h i g h - l e v e l S m - r e s i s t a n t mutants i s not i n h i b i t e d by Sm (Erdos and Ullman n , 1959), thus the p y r u v a t e c o n c e n t r a -t i o n i n the c e l l w i l l n o t i n c r e a s e s i g n i f i c a n t l y upon the a d d i t i o n o f a n t i b i o t i c . S i n c e AHA s y n t h e t a s e i s a l s o r e p r e s s e d under th e s e c o n d i t i o n s ( C o u k e l l and Polglase, 1965) v a l i n e w i l l n o t be produced i n e x c e s s . Support f o r t h i s h y p o t h e s i s comes from a s t u d y by Bragg and P o l g l a s e (1962) on t h e f o r m a t i o n of e x t r a c e l l u l a r m e t a b o l i t e s by Sm-mutants o f E. c o l i . They found t h a t the a d d i t i o n o f DHSm t o s e n s i t i v e c u l t u r e s r e s u l t e d i n a p r o g r e s s i v e change i n t h e c o m p o s i t i o n o f the growth medium. A p p r o x i m a t e l y 2 h r . a f t e r t he a d d i t i o n of a n t i b i o t i c , p y r u v a t e was d e t e c t e d . The amount o f p y r u v a t e produced by the c e l l s i n c r e a s e d as a f u n c t i o n o f the c o n c e n t r a t i o n o f DHSm added t o the c u l t u r e and was r e l a t e d t o the v i a b l e - c e l l count of the DHSm-treated c u l t u r e . T h i s was f o l l o w e d d u r i n g the n e x t 1-2 h r . by a g r a d u a l d e c r e a s e i n t h e c o n c e n t r a t i o n of e x t r a c e l l u l a r p y r u v a t e and an i n c r e a s e i n t h e c o n c e n t r a t i o n o f l a c t a t e , a l a n i n e and p a r t i c u l a r l y o f v a l i n e . I t was p o s s i b l e t o a c c o u n t , a l m o s t q u a n t i t a t i v e l y , f o r t h e dec r e a s e i n e x t r a c e l l u l a r p y r u v a t e by t h e f o r m a t i o n o f t h e secondary p r o d u c t s , a l l o f - 107 -whic h a re s y n t h e s i z e d from p y r u v a t e . Growth of t h e c o r r e s -p onding S m - r e s i s t a n t mutant on a h i g h c o n c e n t r a t i o n o f DHSm (1 mg./ml.) r e s u l t e d i n the e x c r e t i o n o f a s m a l l q u a n t i t y o f p y r u v a t e and some l a c t a t e b u t o n l y a t r a c e o f v a l i n e . - 108 -PART C: Enhancement o f C a t a b o l i t e R e p r e s s i o n by D i h y d r o -s t r e p t o m y c i n - L i mi t a11on o f Streptomycin-Dependent Escherichia c o l i B RESULTS I . A n t i b i o t i c - R e q u i r e m e n t f o r Growth of S t r e p t o m y c i n - Dependent Escherichia c o l i B on V a r i o u s Carbon Sources P r e l i m i n a r y e x p e r i m e n t s i n d i c a t e d t h a t t h e Sm-dependent mutant (DB) whi c h was i s o l a t e d on medium c o n t a i n i n g 1 mg./ml. of DHSm, had comparable growth r a t e s a t e i t h e r 1 mg./ml. o r 100 ug./ml. o f a n t i b i o t i c . As t h e c o n c e n t r a t i o n o f DHSm i n the medium was d e c r e a s e d below 50 ug./ml., however, the growth r a t e o f t h i s mutant was markedly d i m i n i s h e d . Growth c u r v e s f o r c u l t u r e s o f s t r a i n DB on g l u c o s e - s a l t s medium c o n t a i n i n g r a t e - l i m i t i n g c o n c e n t r a t i o n s o f DHSm are shown i n F i g . 17. When a n t i b i o t i c was not added t o the c u l t u r e , a s l i g h t i n -c r e a s e i n absorbancy was ob s e r v e d d u r i n g t h e p e r i o d o f i n c u b a -t i o n . T h i s i n c r e a s e was accompanied by the f o r m a t i o n o f f i l a -mentous forms from c e l l s i n the o r i g i n a l i n o c u l u m , a c h a r a c t e r -i s t i c r e s p onse o f Sm-dependent organisms t o a n t i b i o t i c s t a r v a -t i o n ( D e l a p o r t e , 1949). The c u l t u r e s which were supplemented w i t h 5 t o 100 ug./ml. o f DHSm e x h i b i t e d e x p o n e n t i a l growth b u t the growth r a t e s d e c r e a s e d as t h e a n t i b i o t i c c o n c e n t r a t i o n i n the medium d e c r e a s e d . When t h e growth r a t e s o f th e s e c u l t u r e s were p l o t t e d a g a i n s t the c o n c e n t r a t i o n o f a n t i b i o t i c on which th e y were grown, a h y p e r b o l i c , s a t u r a t i o n c u r v e was o b t a i n e d - 109 -0 1 1 1 1 1 — — i • i i i i i— 0 2 4 6 8 10 TIME .HOURS F i g . 17. Growth of a s t r e p t o m y c i n - d e p e n d e n t mutant o f Esoherioh c o l i B on r a t e - l i m i t i n g c o n c e n t r a t i o n s o f d i h y d r o s t r e p t o m y c i n . The i n o c u l u m (600 ml.) was grown a e r o b i c a l l y f o r IS h r . on g l u -cose ( 0 . 4 % ) - s a l t s medium supplemented w i t h 30 yg./ml. o f d i h y d r o s t r e p t o m y c i n . The r e s u l t i n g c e l l s were h a r v e s t e d by c e n t r i f u g a -t i o n and resuspended i n 6 1. o f the same medium t o g i v e an Ai»2o o f 0.1. The c u l t u r e was d i v i d e d i n t o s i x 1-1. volumes and supplemented w i t h a n t i b i o t i c t o g i v e the f i n a l c o n c e n t r a t i o n s (yg./ml.) i n d i c a t e d . The i n c r e a s e i n A M 2 0 o f each c u l t u r e was f o l l o w e d d u r i n g a e r o b i c growth a t 37°. F i g . 18. R e l a t i o n s h i p between the growth r a t e o f Escherichia c o l i s t r a i n DB and the d i -h y d r o s t r e p t o m y c i n c o n c e n t r a t i o n o f t h e medium. The d a t a were o b t a i n e d from th e e x p e r i m e n t shown i n F i g . 17. Growth r a t e i s d e f i n e d as the r e c i p r o c a l o f t h e d o u b l i n g t i m e i n h r . A. A p l o t of growth r a t e v e r s u s d i h y d r o s t r e p t o m y c i n c o n c e n t r a t i o n . B. A p l o t o f the r e c i p r o c a l o f t h e growth r a t e v e r s u s the r e c i p r o c a l o f t h e d i -h y d r o s t r e p t o m y c i n c o n c e n t r a t i o n . - I l l -( F i g . 18A) . The c o r r e s p o n d i n g double r e c i p r o c a l p l o t of t h e s e d a t a i s shown i n F i g . 18B. From t h i s g r a p h , two c o n s t a n t s may be c a l c u l a t e d , K^TTr, and V , r e l a t i n g t h e c o n c e n t r a t i o n o f DHSm max' ^ DHSm i n t h e medium t o the growth r a t e . These c o n s t a n t s may be i n t e r p r e t e d i n t h e c o n v e n t i o n a l manner so t h a t K D H S m i - s e q u i v a l e n t t o the c o n c e n t r a t i o n o f DHSm whic h w i l l p e r m i t one-h a l f t h e maximum growth r a t e , and V i s t h e growth r a t e a t a • max J n o n - l i m i t i n g c o n c e n t r a t i o n o f DHSm. F o r t h i s e x p e r i m e n t w i t h g l u c o s e as t h e carbon s o u r c e t h e v a l u e s f o r K n T J „ - and V „„„ 3 DHSm max were 5.3 yg./ml. and 0.6 45 r e s p e c t i v e l y (growth r a t e i s ex-p r e s s e d as the r e c i p r o c a l of the d o u b l i n g t i m e i n h r . ) . E x p e r i m e n t s were c a r r i e d out t o i n v e s t i g a t e t he e f f e c t o f a change i n b o t h t h e n a t u r e and the i n i t i a l c o n c e n t r a t i o n o f the carbon s o u r c e i n the medium on the v a l u e s o f K r.„ 0 and DHSm V . A 1 0 - f o l d i n c r e a s e i n i n i t i a l g l u c o s e c o n c e n t r a t i o n had max ^ no s i g n i f i c a n t e f f e c t on t h e v a l u e o f b u t d i d i n c r e a s e 3 DHSm the maximum growth r a t e of t h e c u l t u r e as i n d i c a t e d by the 40% i n c r e a s e i n V (Table X I ) . The e f f e c t o f a change i n t h e max • n a t u r e o f t h e carbon s o u r c e on t h e DHSm-requirement o f t h i s mutant was i n v e s t i g a t e d i n a d d i t i o n a l e x p e r i m e n t s s i m i l a r t o the one d e s c r i b e d i n F i g s . 17 and 18 b u t employing d i f f e r e n t carbon s o u r c e s . The K^TT„ and V v a l u e s o b t a i n e d f o r each carbon DHSm max sou r c e a r e l i s t e d i n T a b l e X I I . Average K D H S m v a l u e s ranged from a h i g h o f 12.6 when g l u c o n a t e was t h e carbon s o u r c e t o a low o f 1.5 when l a c t a t e was the carbon s o u r c e . Thus, i n o r d e r t o a t t a i n o n e - h a l f maximal growth r a t e s , Sm-dependent c u l t u r e s - 112 -Ta b l e X I . Effect of glucose concentration on the dihydrostrep mycin requirement for the growth of streptomycin-dependent E s c h e r i c h i a c o l i B + I n i t i a l g l u c o s e concn. Vj.-:. * K„ T T r, ^ 'max DHSm (%) 0.1 0.527 5.40 0.4 O.SSlX 5.30 1.0 0.741 5.00 V;cu ;<. and K_„_ v a l u e s a t each g l u c o s e c o n c e n t r a t i o n were max DHSm 3 o b t a i n e d from double r e c i p r o c a l p l o t s of growth r a t e s v e r s u s d i h y d r o s t r e p t o m y c i n c o n c e n t r a t i o n . D e t a i l s of t h e experimen-t a l p r o c e d u r e a re g i v e n i n the le g e n d o f F i g . 17 and i n the METHODS. *V..; i s the growth r a t e a t the o p t i m a l c o n c e n t r a t i o n o f max 3 c a n t i b i o t i c . Growth r a t e i s d e f i n e d as the r e c i p r o c a l o f the d o u b l i n g time i n h r . ^KDHSm '''s t* i e d i h y d r o s t r e p t o m y c i n c o n c e n t r a t i o n r e q u i r e d t o p e r m i t o n e - h a l f t h e maximal growth r a t e . $Average v a l u e o f t h r e e s e p a r a t e e x p e r i m e n t s . - 113 -Tabl e X I I . Relationship between the carbon source and the dihydrostreptomycin requirement for the growth of streptomycin-dependent E s c h e r i c h i a c o l i B + Carbon Source V' > * K^ T T O max DHSm Gl u c o n a t e 0.914 12.6 Glu c o s e 0.661 5.3 G l y c e r o l 0.558 2.0 L a c t a t e 0.4 85 1.5 Growth e x p e r i m e n t s were performed as d e s c r i b e d i n t h e le g e n d o f F i g . 17. The carbon s o u r c e was added t o g i v e an i n i t i a l c o n c e n t r a t i o n o f 0.4%. Vfv-^s and K^TT„ v a l u e s a r e aver-max DHSm ages o f a t l e a s t two s e p a r a t e e x p e r i m e n t s . *Vf$crfi i s t h e growth r a t e a t the o p t i m a l c o n c e n t r a t i o n o f max 3 c d i h y d r o s t r e p t o m y c i n . KDHSm "*"S t* i e d i h y d r o s t r e p t o m y c i n c o n c e n t r a t i o n r e q u i r e d t o p e r m i t o n e - h a l f the maximal growth r a t e . - 114 -of s t r a i n DB required 8.4 times the concentration of DHSm when gluconate was the carbon source as when lactate was used. The order of -increasing growth rate was found to be lactate < gl y c e r o l < glucose < gluconate. KoHSm v a-'- u e s o n these carbon sources followed the same order (Table XII). The values obtained for K_TT_ and V were found to be highly DHSm max ^ 2 reproducible. Cultures grown on the energy-rich, rapidly metabolizable carbon sources, gluconate and glucose (see DISCUSSION) exhibited a r e l a t i v e l y high DHSm-requirement while cultures grown on a poor energy source such as gly c e r o l or lactate rquired less a n t i b i o t i c . I I . E f f e c t of Growth with Limiting-Dihydrostreptomycin on the Formation of Enzymes of Carbohydrate Metabolism Previous studies with Sm-dependent mutants of E. coli. have shown that certain enzymes are s p e c i f i c a l l y repressed by ant i b i o t i c - d e p r i v a t i o n . For example, constitutive 3-galacto-sidase (Spotts, 1962), i s o c i t r a t e dehydrogenase (Bragg and Polglase, 1964b) and AHA synthetase (Polglase, 1966a; 1966b) were repressed at least 80% i n ant i b i o t i c - s t a r v e d c e l l s com-pared to the s p e c i f i c a c t i v i t i e s observed i n the corresponding antibiotic-supplemented, Sm-dependent organisms. These en-zymes have also been shown to be subject to catabolite re-pression (Table VII) (for c o n s t i t u t i v e 3-galactosidase see Mandelstam, 1962). Therefore, i t was anticipated that an i n -vestigation of catabolite repression i n a n t i b i o t i c - l i m i t e d - 115 -c u l t u r e s o f s t r a i n DB might h e l p t o e x p l a i n t h e d i v e r s e DHSm-r e q u i r e m e n t s o f t h i s mutant when grown on d i f f e r e n t carbon s o u r c e s . C a t a b o l i t e r e p r e s s i o n as a f u n c t i o n o f a n t i b i o t i c c o n c e n t r a t i o n was s t u d i e d by d e t e r m i n i n g t h e s p e c i f i c a c t i v i -t i e s of s e v e r a l c a t a b o l i t e r e p r e s s i o n - s e n s i t i v e enzymes o f c a r b o h y d r a t e m e t a b o l i s m (Table V I I ) d u r i n g growth on v a r i o u s carbon s o u r c e s i n t h e presence o f g r o w t h - r a t e - l i m i t i n g o r non-l i m i t i n g c o n c e n t r a t i o n s o f DHSm. As a c o n t r o l , t h e s p e c i f i c a c t i v i t i e s o f two g l u c o s e - i n s e n s i t i v e enzymes, g l u c o k i n a s e and g l u c o s e 6-phosphate dehydrogenase (Table V I I ) were d e t e r -mined i n the same e x t r a c t s . The r e s u l t s o f t h e s e e x p e r i m e n t s a r e summarized i n T a b l e X I I I . I n g e n e r a l , t he s p e c i f i c a c t i v i t i e s of t h e c a t a b o l i t e r e p r e s s i o n - s e n s i t i v e enzymes (AHA s y n t h e t a s e , i s o c i t r a t e dehydrogenase, a c o n i t a s e , fumarase and c i t r a t e s y n t h a s e ) from c e l l s grown i n the pre s e n c e o f 100 yg./ml. o f DHSm, were l o w e s t when t h e K D H S m v a l u e s f o r the v a r i o u s carbon s o u r c e s were h i g h e s t . M orever, when the a n t i b i o t i c c o n c e n t r a t i o n of the medium was s e v e r e l y l i m i t i n g (3 yg./ml.) t h i s i n v e r s e r e l a t i o n s h i p between enzyme, a c t i v i t y and K D H S m w a s d r a m a t i c a l l y expanded f o r a l l g l u c o s e - s e n s i t i v e enzymes e x c e p t i s o c i t r a t e dehydrogenase (see DISCUSSION). F o r any p a r t i c u l a r carbon s o u r c e e x c e p t g l u c o n a t e , the p e r c e n t a g e r e p r e s s i o n o f the g l u c o s e - s e n s i t i v e enzymes ( i s o c i t r a t e dehy-drogenase e x c l u d e d ) from c e l l s grown on 3 yg./ml. o f DHSm com-pa r e d w i t h c e l l s grown on 100 yg./ml. o f a n t i b i o t i c was g r e a t e r the h i g h e r t h e v a l u e o f K D H S m « G l u c o n a t e e x h i b i t e d a - 116 -Tabl e X I I I . S p e c i f i c a c t i v i t i e s of enzymes from streptomycin-dependent E s c h e r i c h i a c o l i B grown on various carbon sources and either a growth-rate-limiting or non-limiting concentration of dihydrostreptomycin DHSm (yg./ml.): Carbon Source -Gluconate G l u c o s e G l y c e r o l L a c t a t e KDHSm <yg-/ml.) 12.6 5.3 2.0 1.5 AHA s y n t h e t a s e S p e c i f i c 100 6.0 11.4 11.0 12.9 a c t i v i t y 3 1.5 2.5 7.5 11.4 % R e p r e s s i o n 75 78 32 12 I s o c i t r i c dehydrogenase S p e c i f i c 100 564 547 800 817 a c t i v i t y 3 607 462 883 815 % R e p r e s s i o n -8 16 -11 0 A c o n i t a s e S p e c i f i c 100 270 216 478 434 a c t i v i t y 3 61 128 530 638 % R e p r e s s i o n 77 41 -11 -47 Fumarase S p e c i f i c 100 552 1942 2260 2170 a c t i v i t y 3 134 670 1590 1935 % R e p r e s s i o n 76 65 30 11 C i t r a t e s y n t h a s e S p e c i f i c 100 197 320 523 365 a c t i v i t y 3 84 89 426 287 % R e p r e s s i o n 57 72 19 21 . c o n t i n u e d on f o l l o w i n g page - 117 -TABLE X I I I . ( C o n t i n u e d from p r e c e d i n g page) DHSm (yg./ml. )• Carbon Source G l u c o n a t e G l u c o s e G l y c e r o l L a c t a t e KDHSm ( ug./ml.) 12.6 5.3 2.0 1.5 G l u c o s e 6-phosphate dehydrogenase S p e c i f i c a c t i v i t y % R e p r e s s i o n G l u c o k i n a s e S p e c i f i c a c t i v i t y % R e p r e s s i o n 100 3 100 3 125 118 6 79 77 3 109 109 0 115 92 20 120 122 -2 81 79 2 79 70 11 75 76 -1 Growth e x p e r i m e n t s were performed as d e s c r i b e d i n the l e g e n d o f F i g . 17. The carb o n s o u r c e was added t o g i v e an i n i t i a l c o n c e n t r a t i o n o f 0.4%. H a r v e s t i n g o f c u l t u r e s , p r e p a r a t i o n o f s o n i c e x t r a c t s and enzyme assays were c a r r i e d out as d e s c r i b e d i n the METHODS. KDHSm """S *~^e c o n c e n t r a t i o n o f d i h y d r o s t r e p t o m y c i n r e q u i r e d f o r the h a l f maximal growth r a t e (see T a b l e X I I ) . S p e c i f i c a c t i v i t i e s a r e e x p r e s s e d as u n i t s o f enzyme/mg. b a c t e r i a l p r o t e i n . - 118 -r e l a t i v e l y h i g h l e v e l o f r e p r e s s i o n even when t h e DHSm con-, c e h t r a t i o n was n o n - l i m i t i n g (100 yg./ml.) (Table X I I I ) . G l u c o k i n a s e and g l u c o s e 6-phosphate dehydrogenase f o r -m a t i o n were r e l a t i v e l y u n a f f e c t e d by D H S m - l i m i t a t i o n i n s t r a i n DB. A l t h o u g h the s p e c i f i c a c t i v i t i e s o f b o t h enzymes v a r i e d s l i g h t l y on t h e d i f f e r e n t carbon s o u r c e s , t h e s e d i f f e r e n c e s showed no c o r r e l a t i o n w i t h e i t h e r the a n t i b i o t i c r e q u i r e m e n t s o r the growth r a t e s o f t h e Sm-dependent c u l t u r e s (Table X I I I ) . Under c e r t a i n c o n d i t i o n s , growth o f s t r a i n DB on a r a t e - l i m i t i n g c o n c e n t r a t i o n o f a n t i b i o t i c l e d t o an i n c r e a s e i n t he f o r m a t i o n o f a number o f the g l u c o s e - s e n s i t i v e enzymes. A c o n i t a s e was r e p r e s s e d when Sm-dependent c e l l s were grown on g l u c o s e o r g l u c o n a t e a t a c o n c e n t r a t i o n o f 3 ug./ml. o f DHSm bu t was d e r e p r e s s e d when c e l l s were grown on g l y c e r o l o r l a c -t a t e a t t h e same a n t i b i o t i c c o n c e n t r a t i o n (Table X I I I ) . When the DHSm c o n c e n t r a t i o n o f Sm-dependent c u l t u r e s growing on l a c t a t e o r g l y c e r o l was d e c r e a s e d from 100 yg./ml. t o 15-25 yg./ml. t h e f o r m a t i o n o f AHA s y n t h e t a s e was a l s o s i g n i f i c a n t l y d e r e p r e s s e d . F u r t h e r r e d u c t i o n i n the a n t i b i o t i c c o n c e n t r a t i o n , however, r e s u l t e d i n a sharp r e p r e s s i o n o f AHA s y n t h e t a s e ( F i g . 19) . When t h e same organis m was grown on g l u c o s e o r on g l u c o n a t e , d e r e p r e s s i o n o f AHA s y n t h e t a s e was n o t observ e d a t any r a t e - l i m i t i n g c o n c e n t r a t i o n o f a n t i b i o t i c ( F i g . 1 9 ) . The s t r o n g r e p r e s s i o n o f g l u c o s e - s e n s i t i v e enzymes i n ex-t r a c t s of a n t i b i o t i c - s u p p l e m e n t e d . c e l l s grown on g l u c o n a t e was unexpected s i n c e p r e v i o u s s t u d i e s (Table V I I ) e s t a b l i s h e d t h a t , - 119 -20 40 60 80 100 D H S m IN M E D I U M , ug /ml F i g . 19. E f f e c t o f a n t i b i o t i c - l i m i t a t i o n on the f o r m a t i o n o f AHA s y n t h e t a s e i n c u l t u r e s o f s t r e p t o m y c i n - d e p e n d e n t Es c h e r i c h i a c o l i B (DB) grown on v a r i o u s carbon s o u r c e s . C e l l s were grown as d e s c r i b e d i n the le g e n d o f F i g . 17 on m i n i m a l medium supplemented w i t h t h e * . f o l l o w i n g carbon s o u r c e s a t an i n i t i a l c o n c e n t r a t i o n of 0.4%: g l u c o n a t e , (•') ; g l u c o s e ( o ) ; g l y c e r o l , (•) or l a c t a t e , ( A ) . P r e p a r a t i o n o f s o n i c e x t r a c t s and d e t e r m i n a t i o n o f AHA-synthetase a c t i v i t i e s were performed as d e s c r i b e d i n t h e METHODS. S p e c i f i c a c t i v i t y i s e x p r e s s e d as ymoles o f a c e t o i n formed/mg. o f . p r o t e i n / h r . a t 37°. - 120 -i n t h i s mutant, t h e s e enzymes were r e l a t i v e l y r e s i s t a n t t o r e -p r e s s i o n when grown on g l u c o s e (a v e r y good s o u r c e o f c a t a b o l i t e c o - r e p r e s s o r ) . To d e t e r m i n e whether t h e enhanced c a t a b o l i t e r e p r e s s i o n r e s u l t i n g from growth on g l u c o n a t e was a g e n e r a l c h a r a c t e r i s t i c o f t h i s s t r a i n ( B ) , or a s p e c i f i c p r o p e r t y o f the Sm-dependent mutant (DB), the s p e c i f i c a c t i v i t i e s o f AHA s y n t h e t a s e were d e t e r m i n e d i n e x t r a c t s o f S m - s e n s i t i v e (SB) and Sm-dependent c u l t u r e s of E. c o l i B grown on g l u c o s e and on g l u c o n a t e (Table X I V ) . As shown p r e v i o u s l y ( F i g . 7 and T a b l e V I I ) , the AHA s y n t h e t a s e o f t h i s Sm-dependent mutant was de-r e p r e s s e d i n e x t r a c t s o f glucose-grown c e l l s , when compared w i t h the w i l d - t y p e organism. In e x t r a c t s o f t h e Sm-dependent mutant grown on g l u c o n a t e , however, AHA s y n t h e t a s e was r e -p r e s s e d a l m o s t as s t r o n g l y as i t was i n t h e w i l d - t y p e organism (SB) grown on e i t h e r g l u c o s e o r g l u c o n a t e . F u r t h e r m o r e , the d o u b l i n g time o f t h e Sm-dependent c u l t u r e was c o n s i d e r a b l y l e s s on g l u c o n a t e t h a n on g l u c o s e , w h i l e the S m - s e n s i t i v e c u l t u r e grew a t t h e same ..rate on e i t h e r carbon s o u r c e (Table IX) . I t was shown p r e v i o u s l y ( F i g s . 13 and 1 4 ) , t h a t a e r o b i c m e t a b o l i s m i s 35-40% l e s s e f f i c i e n t i n t h e Sm-dependent mutant o f E. c o l i B than i n the p a r e n t s t r a i n . S i n c e t h e r e s u l t s of T a b l e IX su g g e s t e d t h a t g l u c o n a t e m e t a b o l i s m by t h i s mutant may be more e f f i c i e n t t h a n g l u c o s e m e t a b o l i s m , a c e l l y i e l d ex-p e r i m e n t was c a r r i e d out t o e s t i m a t e t h e e f f i c i e n c y of u t i l i -z a t i o n o f each energy s o u r c e . T a b l e XV, gives the d r y wt. of - 121 -T a b l e XIV. Repression of AHA synthetase by glucose and glucon-ate in cultures of streptomycin-sensitive and streptomycin-dependent E s c h e r i c h i a c o l i B S t r a i n Carbon Source D o u b l i n g time S p e c i f i c a c t i v i t y (min.) S B ( S m - s e n s i t i v e ) G l u c o s e 54 5.00 G l u c o n a t e 5 4 5.90 DB(Sm-dependent) G l u c o s e 91 14,1 G l u c o n a t e 74 7.85 I n o c u l a were grown 18 h r . on m i n i m a l medium c o n t a i n i n g g l u c o s e o r g l u c o n a t e . The c u l t u r e s were grown a e r o b i c a l l y from an A 4 2 p o f 0.1 t o 0.8, h a r v e s t e d , d i s r u p t e d by s o n i c a t i o n and as s a y e d f o r AHA-synthetase a c t i v i t y as d e s c r i b e d i n t h e METHODS. S p e c i f i c a c t i v i t y i s e x p r e s s e d as ymoles of a c e t o i n formed/mg. of p r o t e i n / h r . a t 37°. - 122 -T a b l e XV. Efficiency of glucose and gluconate utilization by cultures of streptomycin-sensitive and streptomycin-dependent E s c h e r i c h i a c o l i B S t r a i n Carbon Source yg. d r y c e l l s formed/ ymole o f car b o n s o u r c e consumed S B ( S m - s e n s i t i v e ) G l u c o s e G l u c o n a t e 94.2 93.7 DB(Sm-dependent) G l u c o s e G l u c o n a t e 59.7 59.3 C u l t u r e s o f s t r a i n s SB and DB were grown 18 h r . on m i n i m a l s a l t s medium supplemented w i t h e i t h e r g l u c o s e o r g l u c o n a t e . The c e l l s were washed once by c e n t r i f u g a t i o n and the y i e l d o f c e l l s from each carbon s o u r c e was d e t e r m i n e d as d e s c r i b e d i n the METHODS. C e l l mass was o b t a i n e d from a s t a n d a r d c u r v e r e l a t i n g A ^ o t o yg. d r y c e l l s / m l . - 123 -S m - s e n s i t i v e and Sm-dependent c e l l s formed from e q u a l m olar q u a n t i t i e s o f g l u c o s e and' g l u c o n a t e . The r e s u l t s i n d i c a t e t h a t g l u c o n a t e m e t a b o l i s m l i k e g l u c o s e m e t a b o l i s m , i s l e s s e f f i c i e n t (by 38%) i n s t r a i n DB t h a n i n the w i l d - t y p e organism. T h e r e f o r e , th e enhanced growth r a t e and t h e i n c r e a s e d cata.-b o l i t e r e p r e s s i o n o b s e r v e d i n t h e Sm-dependent mutant growing on g l u c o n a t e does not r e s u l t from an i n c r e a s e d y i e l d o f energy from t h i s c arbon s o u r c e . I l l . Response.of C a t a b o l i t e R e p r e s s i o n - S e n s i t i v e Enzymes of A n t i b i o t i c - D e p l e t e d C e l l s t o D i h y d r o s t r e p t o m y c i n S upp1ementat i o n F o l l o w i n g a l e n g t h y p e r i o d (10-12 hr.) o f growth i n medium c o n t a i n i n g g l u c o s e b u t no a n t i b i o t i c , the Sm-dependent c e l l s e x h i b i t e d t h e f i l a m e n t o u s growth which i s c h a r a c t e r i s t i c of a n t i b i o t i c - d e p l e t i o n . The a d d i t i o n of DHSm (1 mg./ml.) t o a n t i b i o t i c - s t a r v e d c u l t u r e s r e s u l t e d i n a p r o g r e s s i v e b r e a k -down o f the e l o n g a t e d c e l l s w h i c h was u s u a l l y complete w i t h i n 3 h r . The e f f e c t o f DHSm-?supplementation o f a n t i b i o t i c - d e p l e t e d c e l l s on the f o r m a t i o n o f c e r t a i n g l u c o s e - s e n s i t i v e enzymes i n s t r a i n DB i s shown i n T a b l e XVI. The d i f f e r e n c e i n s p e c i f i c a c t i v i t i e s o f t h e s e enzymes i n e x t r a c t s o f a n t i b i o t i c - d e p l e t e d and a n t i b i o t i c - s u p p l e m e n t e d c e l l s a f t e r 3 h r . i n d i c a t e s t h a t the s y n t h e s i s o f t h e s e enzymes undergoes a r a p i d and d r a m a t i c d e r e p r e s s i o n d u r i n g t h i s p e r i o d . The g l u c o s e - i n s e n s i t i v e en-zymes, g l u c o k i h a s e and g l u c o s e 6-phosphate dehydrogenase were T a b l e XVI. S p e c i f i c a c t i v i t i e s of various enzymes in dihydrostreptomycin-depleted and supplemented extracts of streptomycin-dependent E s c h e r i c h i a c o l i B Enzyme S p e c i f i c A c t i v i t y Supplemented/Depleted (Units/mg. o f p r o t e i n ) D e p l e t e d Supplemented AHA s y n t h e t a s e 1.6 8 Fumarase 200 A c o n i t a s e 34.4 I s o c i t r i c dehydrogenase 183 C i t r a t e s y n t h a s e 118 G l u c o s e 6-phosphate dehydrogenase 75.2 G l u c o k i n a s e 6 4.3 11.7 7.0 1622 8.1 182 5.3 490 2.7 426 3.6 81.4 1.1 91.6 1.4 P r e p a r a t i o n o f a n t i b i o t i c - d e p l e t e d c e l l s and enzyme assays a r e d e s c r i b e d i n the METHODS. Supplemented c e l l s r e c e i v e d 1 mg./ml. o f d i h y d r o s t r e p t o m y c i n 3 h r . p r i o r t o h a r v e s t i n g . - 125 -r e l a t i v e l y u n a f f e c t e d by DHSm-depletion o r by t h e subsequent a n t i b i o t i c s u p p l e m e n t a t i o n (Table X V I ) . IV. R e l a t i o n s h i p between Growth Rate and Enzyme S y n t h e s i s i n Streptomycin-Dependent C e l l s . Grown on L i m i t i n g -A n t i b i o t i c E x a m i n a t i o n o f the growth r a t e s and t h e s p e c i f i c enzyme a c t i v i t i e s i n a number of growth e x p e r i m e n t s i n which DHSm was l i m i t i n g i n d i c a t e d t h a t a d i r e c t r e l a t i o n s h i p e x i s t e d between the d i m i n i s h e d growth r a t e i n t h e p r e s e n c e of l i m i t i n g -a n t i b i o t i c and t h e degree o f r e p r e s s i o n o f c e r t a i n g l u c o s e -s e n s i t i v e enzymes. T h i s r e l a t i o n s h i p i s i l l u s t r a t e d i n F i g . • 20 f o r AHA s y n t h e t a s e and fumarase a c t i v i t i e s i n e x t r a c t s o f g l u c o s e and g l u c o n a t e - g r o w n c e l l s . I n g e n e r a l , b o t h growth r a t e and enzyme f o r m a t i o n were lower on g l u c o n a t e t h a n on g l u -cose a t the same a n t i b i o t i c c o n c e n t r a t i o n . Not a l l g l u c o s e -s e n s i t i v e enzymes e x h i b i t e d t h i s r e l a t i o n s h i p , however.. W h i l e t h e degree o f r e p r e s s i o n o f c i t r a t e s y n t h a s e under t h e s e c o n d i -t i o n s o f t e n c o r r e l a t e d w i t h t h e growth r a t e o f t h e c u l t u r e , a c o n i t a s e and i s o c i t r i c dehydrogenase r a r e l y d i s p l a y e d t h i s p a r a l l e l i s m . E x c e p t f o r fumarase, t h i s r e l a t i o n s h i p was seldom o b s e r v e d i n c u l t u r e s grown on poor carbon s o u r c e s ( g l y c e r o l and l a c t a t e ) . V. E f f e c t of A n t i b i o t i c C o n c e n t r a t i o n oh the Y i e l d o f Streptomycin-Dependent C e l l s f r o m G l u c o s e P r e v i o u s r e s u l t s showed t h a t r e l a x a t i o n o f c a t a b o l i t e 100 IE o fr o 8 0 5 6 0 4 0 ^ 2 0 0 T 1 1 1 r ~i 1 r A _1 I • I I I I I L . 100 I—' C A 0 2 0 4 0 6 0 8 0 100 0 .20 4 0 6 0 8 0 100 SPECIFIC ACTIVITY F i g . 20. R e l a t i o n s h i p between the decrease i n growth ra t e and the r e p r e s s i o n of AHA syn-thetase and fumarase i n d i h y d r o s t r e p t o m y c i n - l i m i t e d c u l t u r e s of streptomycin-dependent Escherichia coli B. Growth experiments were c a r r i e d out as described i n the legend of F i g . 17. Glucose (o) or'gluconate (o) were added to give an i n i t i a l c o n c e n t r a t i o n of 0.4% (w/v). Growth r a t e s and s p e c i f i c a c t i v i t i e s are expressed as percentages of the values obtained'with c u l t u r e s grown on 100 yg./ml. of dihydrostreptomycin. The a n t i b i o -t i c c oncentrations employed were 25, 15, 10, 5 and 3 yg./ml., r e s p e c t i v e l y . A, AHA synthetase; B, fumarase. - 127 -r e p r e s s i o n i n t h e Sm-dependent mutant growing on g l u c o s e w i t h n o n - l i m i t i n g a n t i b i o t i c c o r r e l a t e d w i t h a lowered e f f i c i e n c y o f c a r b o h y d r a t e m e t a b o l i s m ( F i g . 1 3 ) . C e l l y i e l d s t u d i e s w i t h g l u c o n a t e as the ca r b o n s o u r c e gave s i m i l a r r e s u l t s t o t h o s e o b t a i n e d w i t h g l u c o s e , i . e . a p p r o x i m a t e l y o n e - t h i r d l e s s t h a n t h e w i l d - t y p e o r g a n i s m (SB) (Table XV). T h e r e f o r e , i f t h e impairment o f c a r b o h y d r a t e m e t a b o l i s m i n t h e DHSm-sup-plemented, Sm-dependent c e l l s growing on a n o n - l i m i t i n g con-c e n t r a t i o n o f a n t i b i o t i c were due t o a d i r e c t i n t e r a c t i o n o f DHSm w i t h c a t a b o l i c p r o c e s s e s o f t h e s e c e l l s , t h e n d e c r e a s i n g t h e a n t i b i o t i c c o n c e n t r a t i o n would be e x p e c t e d t o r e s u l t i n the r e s t o r a t i o n o f e f f i c i e n t c a r b o h y d r a t e m e t a b o l i s m and en-hanced c a t a b o l i t e r e p r e s s i o n . To d e t e r m i n e whether t h e e f -f i c i e n c y o f c a r b o h y d r a t e m e t a b o l i s m o f s t r a i n DB i s dependent on a n t i b i o t i c c o n c e n t r a t i o n , t h e y i e l d o f Sm-dependent c e l l s from g l u c o s e was e s t i m a t e d i n c u l t u r e s growing e x p o n e n t i a l l y on 10,100 and 1000 yg./ml. of a n t i b i o t i c . The r e s u l t s a r e p r e s e n t e d i n T a b l e X V I I . A l t h o u g h t h e growth r a t e o f s t r a i n DB was s i g n i f i c a n t l y l o wer on 10 yg./ml., t h e e f f i c i e n c y o f g l u c o s e u t i l i z a t i o n a t t h i s a n t i b i o t i c c o n c e n t r a t i o n was i d e n -t i c a l t o t h e e f f i c i e n c i e s c a l c u l a t e d f o r c u l t u r e s growing on s u p r a - c r i t i c a l c o n c e n t r a t i o n s o f a n t i b i o t i c (100 o r 1000 y g . / m l . ) . T h e r e f o r e , the i n e f f i c i e n c y o f c a r b o h y d r a t e m e t a b o l i s m o f the Sm-dependent mutant a p p e a r s . t o be independent o f DHSm-concentration. - 128 -T a b l e X V I I . Effect of a n t i b i o t i c concentration on the c e l l yie from glucose of streptomycin-dependent E s c h e r i c h i a c o l i B A n t i b i o t i c concn. D o u b l i n g time Y* (ug./ml.) 1000 9 3 0 0.341 100 87 0.344 10 120 0.344 The i n o c u l u m was grown a e r o b i c a l l y f o r 16 h r . on g l u c o s e -s a l t s medium supplemented w i t h 10 yg./ml. o f d i h y d r o s t r e p t o -m ycin. The c e l l s were washed once by c e n t r i f u g a t i o n and t h e c e l l y i e l d from g l u c o s e was d e t e r m i n e d as d e s c r i b e d i n the METHODS i n media c o n t a i n i n g 10, 100 or 1000 yg./ml. o f a n t i -b i o t i c . C e l l mass was o b t a i n e d from a s t a n d a r d c u r v e r e l a t i n g A 4 2 0 t o d r y wt. o f c e l l s / m l . o f c u l t u r e . *Y i s the c e l l y i e l d c o n s t a n t d e f i n e d as t h e number of yg. o f d r y c e l l s formed/yg. o f g l u c o s e consumed. - 129 -V I . E f f e c t o f A n t i b i o t i c - L i m i t a t i o n on t h e ATP P o o l o f Streptomycin-Dependent C e l l s R e s u l t s o f the--experiment shown i n F i g . 15 sug g e s t e d t h a t the growth r a t e o f DHSm-supplemented (1 mg./ml.) c u l t u r e s o f s t r a i n DB'was l i m i t e d by t h e r a t e o f ATP s y n t h e s i s i n t h i s o r ganism. S i n c e growth o f t h i s mutant on low c o n c e n t r a t i o n o f a n t i b i o t i c (<100 yg./ml.) r e s u l t e d i n a f u r t h e r r e d u c t i o n of growth r a t e ( F i g . 1 7 ) , DHSm-starvation c o u l d c o n c e i v a b l y be i n t e r f e r i n g w i t h ATP s y n t h e s i s by removing DHSm from some a n t i b i o t i c - d e p e n d e n t e n e r g e t i c p r o c e s s . To examine t h i s pos-s i b i l i t y , t h e ATP p o o l o f Sm-dependent c e l l s was d e t e r m i n e d f o l l o w i n g t he a d d i t i o n o f 1 mg./ml. o f DHSm t o a c u l t u r e grow-i n g e x p o n e n t i a l l y on a g l u c o s e - s a l t s medium c o n t a i n i n g o n l y 10 yg./ml. o f DHSm ( F i g . 2 1 ) . A l t h o u g h the e x p e r i m e n t a l c o n d i t i o n s (see METHODS) caused the ATP p o o l s of b o t h t h e a n t i b i o t i c - s u p p l e m e n t e d and the a n t i b i o t i c - l i m i t e d c e l l s t o o s c i l l a t e d u r i n g growth, t h e r e s u l t s i n d i c a t e d t h a t t h e a d d i -t i o n o f a h i g h c o n c e n t r a t i o n o f DHSm t o a n t i b i o t i c - l i m i t e d c e l l s d i d not enhance the c o n c e n t r a t i o n of t h e ATP p o o l o f the Sm-dependent mutant. I n f a c t , t h e ATP p o o l o f the a n t i -b i o t i c - l i m i t e d c e l l s (10 yg./ml.) was always g r e a t e r t h a n the ATP p o o l o f t h e a n t i b i o t i c - s u p p l e m e n t e d c e l l s (1 mg.'/ml.) d u r i n g a 4 - f o l d i n c r e a s e i n c e l l mass. The d o u b l i n g t i m e s o f a n t i b i o t i c - l i m i t e d and a n t i b i o t i c - s u p p l e m e n t e d c u l t u r e s , however, were 100 and.78 min., r e s p e c t i v e l y . - 130 -0 0.05 0.10 0.15 0.20 0.25 CELL MASS , mg/ml F i g . 21. E f f e c t o f d i h y d . r o s t r e p t o m y c i n c o n c e n t r a t i o n , on the ATP p o o l o f s t r e p t o m y c i n - d e p e n d e n t Escherichia c o l i B. The i n o c u l u m was grown a e r o b i c a l l y f o r 16 h r . on g l u c o s e - s a l t s medium s u p p l e -mented w i t h 10 yg./ml. o f d i h y d r o s t r e p t o m y c i n . The c u l t u r e s t o be a s s a y e d f o r ATP were p r e p a r e d by method I (see METHODS) and c o n t a i n e d e i t h e r 10 (o) o r 1000 (©) yg./ml. o f a n t i b i o t i c . The d o u b l i n g t i m e s o f t h e c u l t u r e s were 100 and 78 min.., r e s p e c t i v e l y . - 131 -. DISCUSSION The Sm-dependent.mutant o f E. c o l i B ( s t r a i n DB) can grow on g l u c o s e - s a l t s medium i n t h e pre s e n c e o f DHSm concen-t r a t i o n s as low as 3 yg./ml. C u l t u r e s o f t h i s mutant grow e x p o n e n t i a l l y on r a t e - l i m i t i n g c o n c e n t r a t i o n s of a n t i b i o t i c (<100 yg./ml.) and t h e growth r a t e s o f t h e s e c u l t u r e s a r e d i r e c t l y r e l a t e d t o the c o n c e n t r a t i o n o f a n t i b i o t i c i n t h e medium ( F i g s . 17 and 18). The growth r a t e as a f u n c t i o n o f DHSm c o n c e n t r a t i o n can be e v a l u a t e d r e p r o d u c i b l y by d e t e r -m i n i n g a c o n s t a n t ( K DHSm^ w n i c n i - s e q u i v a l e n t t o t h e concen-t r a t i o n o f DHSm p e r m i t t i n g o n e - h a l f t h e maximal growth r a t e ( F i g . 1 8 ) . The v a l u e o b t a i n e d f o r K D H S m depends on t h e n a t u r e of t he carbon s o u r c e , b e i n g h i g h e s t f o r e n e r g y - r i c h , r a p i d l y m e t a b o l i z a b l e compounds such as g l u c o n a t e and g l u c o s e (Table X I I ) . The v a l u e o f K D j j g m i s independent o f v a r i a t i o n s i n the i n i t i a l c o n c e n t r a t i o n of t h e ca r b o n s o u r c e i n t h e medium (Table X I ) . I n a r e l a t e d s t u d y by S j i o t t s (1962) on Sm-depen-dent mutants o f E. c o l i Kl-12, the growth r a t e s o f t h e s e mutants on g l y c e r o l were a d i r e c t f u n c t i o n o f the Sm c o n c e n t r a t i o n up to a p p r o x i m a t e l y 100 yg./ml. a t which v a l u e ( " c r i t i c a l concen-t r a t i o n " ) Sm was no l o n g e r the r a t e - l i m i t i n g f a c t o r . When g l u c o s e was t h e carb o n s o u r c e , Sm remained r a t e - l i m i t i n g up t o about 250 yg./ml. A t r a t e - l i m i t i n g c o n c e n t r a t i o n s o f a n t i -b i o t i c , however, growth r a t e s were independent, o f t h e n a t u r e of t he carbon s o u r c e ( S p o t t s , 1962). Thus, w h i l e t h e c r i t i c a l - 132 -a n t i b i o t i c c o n c e n t r a t i o n o f the medium was dependent on the n a t u r e o f t h e carb o n s o u r c e , t h e growth r a t e s o f the c u l t u r e s on s u b - c r i t i c a l c o n c e n t r a t i o n s o f a n t i b i o t i c were independent o f c a r b o n s o u r c e . These r e s u l t s o b t a i n e d by S p o t t s (1962) were n o t c o n f i r m e d i n the p r e s e n t work where i t was found t h a t t h e n a t u r e o f t h e . c a r b o n s o u r c e had a marked e f f e c t on growth r a t e s when t h e . c o n c e n t r a t i o n o f DHSm was l i m i t i n g . Growth o f t h e Sm-dependent mutant (DB) i n the pre s e n c e of l i m i t i n g c o n c e n t r a t i o n s o f a n t i b i o t i c r e s u l t e d i n an enzyme imbalance i n th e s e c e l l s ( T ables X I I I and X V I ) . G l u c o s e -s e n s i t i v e enzymes were s p e c i f i c a l l y r e p r e s s e d i n d i c a t i n g a s u b s t a n t i a l i n c r e a s e i n c a t a b o l i t e r e p r e s s i o n . F u r t h e r m o r e , DHSm s u p p l e m e n t a t i o n of a n t i b i o t i c - s t a r v e d c e l l s r e s u l t e d i n a r a p i d and s p e c i f i c d e r e p r e s s i o n of the c a t a b o l i t e r e p r e s s i o n -s e n s i t i v e enzymes (Table X V I ) . R e c e n t l y , Goodman and S p o t t s (1967) r e p o r t e d t h a t t h e s y n t h e s i s of b o t h i n d u c i b l e and con-s t i t u t i v e 3 - g a l a c t o s i d a s e and o f c o n s t i t u t i v e a l k a l i n e phos-ph a t a s e were i n h i b i t e d d u r i n g the growth o f Sm-dependent s t r a i n s o f E. c o l i t r a n s f e r r e d t o medium c o n t a i n i n g no added a n t i -b i o t i c . They a t t r i b u t e d some, b u t n o t a l l , o f t h i s i n h i b i t i o n t o an i n c r e a s e i n c a t a b o l i t e r e p r e s s i o n i n the a n t i b i o t i c -d e p r i v e d c e l l s . They s u g g e s t e d t h a t i n a d d i t i o n t o i n h i b i t i o n o f enzyme s y n t h e s i s by c a t a b o l i t e r e p r e s s i o n , i n h i b i t i o n o f enzyme s y n t h e s i s c o u l d o c c u r randomly due t o the m i s r e a d i n g o f c e r t a i n codons by t h e abnormal. S m - d e f i c i e n t , dependent r i b o -somes. However, they were u n a b l e t o d e t e c t t h e presence of - 133 -c r o s s - r e a c t i n g m a t e r i a l by s e r o l o g i c a l methods o r t o f i n d any o t h e r e v i d e n c e f o r i n f i d e l i t y i n t r a n s l a t i o n in vivo, e i t h e r i n the p r e s e n c e o r absence of Sm. Thus, whereas t h e s e a u t h o r s a t t r i b u t e d t h e enzyme imbalance i n a n t i b i o t i c - d e p r i v e d Sm-dependent c e l l s t o a random i n h i b i t i o n o f enzyme s y n t h e s i s a f f e c t i n g a l l c l a s s e s o f enzymes, the r e s u l t s o f t h e p r e s e n t i n v e s t i g a t i o n s u ggest t h a t the i n h i b i t i o n i s not random b u t , i n f a c t , s p e c i f i c a l l y a f f e c t s enzymes wh i c h a r e 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 . The s u s c e p t i b i l i t y o f many of t h e s e enzymes t o c a t a b o l i t e r e p r e s s i o n may not be r e a d i l y a p p a r e n t i n t h e w i l d - t y p e organism o r i n the a n t i b i o t i c - s u p p l e m e n t e d , Sm-dependent mutant because of t h e moderate v a r i a t i o n i n con-c e n t r a t i o n o f c a t a b o l i t e c o - r e p r e s s o r n o r m a l l y o c c u r r i n g i n t h e s e c e l l s and the r e l a t i v e i n s e n s i t i v i t y o f t h e s e enzymes t o c a t a b o l i t e r e p r e s s i o n . F o r example, the s y n t h e s i s of i s o -c i t r i c dehydrogenase by w i l d - t y p e E. c o l i B was o n l y s l i g h t l y s e n s i t i v e t o c a t a b o l i t e r e p r e s s i o n (Tables V I I and X I I I ) b u t i n a n t i b i o t i c - d e p l e t e d c e l l s o f s t r a i n DB t h i s enzyme was s i g n i f i c a n t l y r e p r e s s e d (63%) (Table X V I ) . The f o r m a t i o n of s e v e r a l o f t h e g l u c o s e - s e n s i t i v e en-zymes of s t r a i n DB (AHA s y n t h e t a s e , a c o n i t a s e and i s o c i t r a t e dehydrogenase) was d e r e p r e s s e d when t h i s mutant was grown on a poor c a r b o n s o u r c e (e.g. g l y c e r o l or l a c t a t e ) i n the p r e s e n c e of c e r t a i n r a t e - l i m i t i n g c o n c e n t r a t i o n s of a n t i b i o -t i c (Table X I I I and F i g . 1 9 ) . However, when c a t a b o l i t e r e -p r e s s i o n was. f u r t h e r i n c r e a s e d i n the c e l l , - (by growth on a - 134 -b e t t e r carbon s o u r c e or.by a r e d u c t i o n i n t h e i n t r a c e l l u l a r c o n c e n t r a t i o n . o f a n t i b i o t i c ) , t h e s e enzymes were s t r o n g l y r e -p r e s s e d (Table XVI and F i g . 1 9 ) . These r e s u l t s s uggest t h a t the s y n t h e s i s o f t h e s e g l u c o s e - s e n s i t i v e enzymes i n the Sm-dependent mutant growing on r a t e - l i m i t i n g c o n c e n t r a t i o n s o f DHSm i s r e g u l a t e d by more th a n one f a c t o r . Gray et al. (196 6) s t u d i e d t h e e f f e c t s o f a e r o b i o s i s , a n a e r o b i o s i s and n u t r i t i o n on t h e f o r m a t i o n o f t h e K r e b s - c y c l e enzymes i n E. coli-. As a r e s u l t o f t h i s i n v e s t i g a t i o n , t h e y c o n c l u d e d t h a t t h e K r e b s -c y c l e can be d i v i d e d i n t o a t l e a s t t h r e e s e c t o r s each of w h i c h i s under independent c o n t r o l . These a r e enzymes m e t a b o l i z i n g ( a ) , t h e t r i c a r b o x y l i c a c i d s ; ( b ) , the 5-carbon d i c a r b o x y -l i c a c i d s and ( c ) , t h e 4-carbon d i c a r b o x y l i c a c i d s . D u r i n g a e r o b i c growth on a m i n i m a l s a l t s medium, t h e Group (a) enzymes (which i n c l u d e a c o n i t a s e and i s o c i t r i c dehydrogenase) c o n s t i t u t e an a m p h i b o l i c pathway w h i c h i s r e g u l a t e d by b o t h c a t a b o l i t e r e p r e s s i o n and end-product r e p r e s s i o n . Under the same growth c o n d i t i o n s , t h e Group (c) enzymes (which i n c l u d e fumarase) a r e p r i n c i p a l l y c a t a b o l i c i n f u n c t i o n . These en-zymes a r e s e n s i t i v e t o c a t a b o l i t e r e p r e s s i o n b u t are r e l a t i v e l y r e s i s t a n t t o end-product r e p r e s s i o n (Gray et al. , 1966). Re-s u l t s of t h e p r e s e n t s t u d y w i t h t h e Sm-dependent mutant o f E. c o l i B i n d i c a t e t h a t fumarase f o r m a t i o n i s not d e r e p r e s s e d under growth c o n d i t i o n s w h i c h d e r e p r e s s a c o n i t a s e and i s o c i t r a t e dehydrogenase (see T a b l e X I I I ) . S i m i l a r r e s u l t s have been r e p o r t e d f o r t h e r e g u l a t i o n . o f the K r e b s - c y c l e enzymes i n - 135 -B. s u b t i l u s and i n B. lichenformis (Hanson and Cox, 1967). AHA-synthetase f o r m a t i o n i n E. c o l i a l s o appears t o be r e g u l a -t e d by more th a n a s i n g l e mechanism. I n a d d i t i o n t o c o n t r o l by c a t a b o l i t e r e p r e s s i o n which i s d e s c r i b e d i n PART A o f t h i s t h e s i s f o r E. c o l i B, m u l t i v a l e n t end-product r e p r e s s i o n has been r e p o t t e d f o r t h i s enzyme ( F r e u n d l i c h et al. , 1962) . Thus, th e d e r e p r e s s i o n of c e r t a i n g l u c o s e - s e n s i t i v e enzymes d u r i n g growth on l i m i t i n g - a n t i b i o t i c appears t o be the r e s u l t o f i n t e r f e r e n c e by i n c r e a s i n g c a t a b o l i t e r e p r e s s i o n w i t h : e n d -p r o d u c t c o n t r o l (or some o t h e r mechanism) of enzyme s y n t h e s i s . S t r o n g c a t a b o l i t e r e p r e s s i o n which o c c u r s when t h i s mutant i s s e v e r e l y s t a r v e d o f a n t i b i o t i c c o m p l e t e l y a b o l i s h e s de-r e p r e s s i o n (Table X V I ) . The enhanced p r o d u c t i o n o f e x t r a c e l l u l a r v a l i n e by a n t i -b i o t i c - s u p p l e m e n t e d , Sm-dependent mutants when compared t o the c o r r e s p o n d i n g w i l d - t y p e p a r e n t s t r a i n s was a t t r i b u t e d t o the d e r e p r e s s e d l e v e l o f AHA s y n t h e t a s e i n t h e s e organisms (see PART B, DISCUSSION). P r e v i o u s s t u d i e s by Bragg and P o l g l a s e (1964a) i n d i c a t e d t h a t v a l i n e was not a major e x c r e t i o n p r o d u c t o f Sm-dependent mutants when th e y were grown i n a n t i b i o t i c -f r e e medium. An e x p l a n a t i o n o f t h i s o b s e r v a t i o n i s s u g g ested by e x a m i n a t i o n o f the AHA-synthetase a c t i v i t i e s o f a n t i b i o t i c -d e p l e t e d and supplemented c u l t u r e s o f s t r a i n . D B (Table X V I ) . The AHA-synthetase a c t i v i t y o f t h e DHSm-starved c e l l s i s 86% l o w e r than i n the c e l l s supplemented w i t h a n t i b i o t i c . F u r t h e r -more, the s p e c i f i c AHA-synthetase a c t i v i t y of t h e d e p l e t e d , - 136 -Sm-dependent c e l l s (1.67) i s 6 3% lower than t h e l o w e s t a c t i -v i t y o b s e r v e d f o r t h e c o r r e s p o n d i n g S m - s e n s i t i v e o r g a n i s m (4.5) grown on g l u c o s e . Sm-dependent c e l l s w i t h AHA s y n t h e -t a s e r e p r e s s e d t h i s . s t r o n g l y would n o t be e x p e c t e d t o e x c r e t e s i g n i f i c a n t q u a n t i t i e s o f v a l i n e , even i f the p y r u v a t e con-c e n t r a t i o n i n t h e Sm-dependent c e l l s was e l e v a t e d d u r i n g growth i n a n t i b i o t i c - f r e e medium. G l u c o s e g e n e r a l l y . i s c o n s i d e r e d . t o be t h e most e f f e c t i v e s o u r c e of c a t a b o l i t e c o - r e p r e s s o r (Magasanik, 1961). There-f o r e , i t was s u r p r i s i n g t o f i n d t h a t growth o f t h e Sm-dependent mutant (but not o f the w i l d - t y p e p a r e n t s t r a i n ) on g l u c o n a t e r e s u l t e d i n a h i g h e r r a t e o f growth and s t r o n g e r c a t a b o l i t e r e p r e s s i o n t h a n o b s e r v e d when the same or g a n i s m was grown on g l u c o s e (Table X I V ) . T h i s r e s p o n s e was not due to; ehihigher y i e l d o f energy from g l u c o n a t e s i n c e the y i e l d o f Sm-dependent c e l l s was the same from b o t h carbon s o u r c e s (Table XV). G l u c o n a t e , t h e r e f o r e , must be m e t a b o l i z e d more r a p i d l y t h a n g l u c o s e by t h i s mutant. E i s e n b e r g and Dobrogosz (1967) have r e c e n t l y r e p o r t e d t h a t i n E. c o l i , g l u c o n a t e i s m e t a b o l i z e d v i a t h e i n d u c i b l e E n t n e r - D o u d o r o f f pathway r a t h e r than by the hexose monophosphate pathway, the major a e r o b i c r o u t e of g l u c o s e d i s s i m i l a t i o n . S i n c e p y r u v a t e i s an e a r l y p r o d u c t o f the E n t n e r - D o u d o r o f f pathway, th e r a t e o f a e r o b i c p y r u v a t e - c o u p l e d energy f o r m a t i o n may be h i g h e r i n t h e a n t i -b i o t i c - d e p e n d e n t mutant growing on g l u c o n a t e t h a n i n t h e same mutant growing on g l u c o s e . An i n c r e a s e such as t h i s i n t h e - 137 -r a t e o f energy f o r m a t i o n c o u l d e x p l a i n b o t h t h e h i g h e r r a t e o f growth and.the s t r o n g e r c a t a b o l i t e r e p r e s s i o n o b s e r v e d i n g l u c o n a t e - g r o w n c u l t u r e s o f the.Sm-dependent mutant. N e i d h a r d t (1960) has s t u d i e d a mutant o f A. aevogenes w h i c h l a c k e d g l u c o s e r e p r e s s i o n b u t not g l u c o n a t e r e p r e s s i o n . T h i s mutant (JF-4) grew as w e l l as t h e w i l d - t y p e p a r e n t s t r a i n on a l l c a r b o n s o u r c e s e x c e p t g l u c o s e . An i n v e s t i g a t i o n o f g l u c o s e m e t a b o l i s m by s t r a i n JF-4 r e v e a l e d t h a t t h i s mutant was unable t o m e t a b o l i z e g l u c o s e by a d i r e c t o x i d a t i o n . T h i s l o s s i n g l u c o s e o x i d a s e a c t i v i t y d i m i n i s h e d t h e o v e r a l l r a t e o f g l u c o s e d i s s i m i l a t i o n by t h e c e l l (but not t h e y i e l d o f energy from g l u c o s e ) t h e r e b y v i r t u a l l y e l i m i n a t i n g g l u c o s e r e p r e s s i o n . I n c o n t r a s t , t h e Sm-dependent mutant of E. c o l i B (DB), w h i c h a l s o l a c k s g l u c o s e r e p r e s s i o n f o r a.number of c a t a b o l i t e . . r e p r e s s i o n s - s e n s i t i v e enzymes, grew more s l o w l y on a l l c a r b o n s o u r c e s i n v e s t i g a t e d t h a n d i d t h e S m - s e n s i t i v e p a r e n t s t r a i n ( T ables IX and X I V ) . F u r t h e r m o r e , t h i s mutant was a p p r o x i m a t e l y o n e - t h i r d l e s s e f f i c i e n t t h a n the w i l d -t y p e o r g a n i s m i n u t i l i z i n g g l u c o s e o f g l u c o n a t e a e r o b i c a l l y f o r c e l l s y n t h e s i s (Table XV). These d a t a suggest t h a t l o s s o f g l u c o s e r e p r e s s i o n by s t r a i n DB, u n l i k e s t r a i n J F - 4 , r e -s u l t s from a g e n e r a l impairment o f energy m e t a b o l i s m p r o b a b l y a t t h e l e v e l o f t e r m i n a l o x i d a t i o n . W h i l e energy f o r m a t i o n i s i m p a i r e d i n . t h e Sm-dependent mutant, t h i s c a t a b o l i c d e f i c i e n c y appears t o be independent o f a n t i b i o t i c c o n c e n t r a t i o n (Table X V I I ) . C a t a b o l i t e r e p r e s -- 138 -s i o n , however, was enhanced i n c u l t u r e s o f the same Sm-dependent mutant growing on l i m i t i n g - D H S m and t h i s e f f e c t was dependent on the c o n c e n t r a t i o n o f a n t i b i o t i c i n t h e medium ( F i g . 1 9 ) . Moreover, t h e i n c r e a s e i n c a t a b o l i t e r e p r e s s i o n c o r r e l a t e d w i t h b o t h an i n c r e a s e i n the c o n c e n t r a t i o n o f the ATP p o o l ( F i g . 21) and a d e c r e a s e i n the growth r a t e o f t h e mutant. Thus, the p h y s i o l o g i c a l changes obse r v e d i n s t r a i n DB d u r i n g growth on l i m i t i n g DHSm ar e c o n s i s t e n t w i t h t he h y p o t h e s i s t h a t t he ribosome i s t h e a n t i b i o t i c - d e p e n d e n t s i t e i n Sm-dependent mutants ( S p o t t s and S t a n i e r , 1961; L i k o v e r and K u r l a n d , 1967a). The e x p e r i m e n t a l r e s u l t s p r e s e n t e d i n t h i s s e c t i o n sug-g e s t t h a t a r e l a t i o n s h i p e x i s t s between t h e growth r a t e o f t h e Sm-dependent mutant on l i m i t i n g - c o n c e n t r a t i o n s o f a n t i b i o t i c and t h e degree o f c a t a b o l i t e r e p r e s s i o n i n the a n t i b i o t i c -d e p r i v e d c e l l . F o r example, the n a t u r e o f the carbon s o u r c e d e t e r m i n e s the c o n c e n t r a t i o n o f DHSm r e q u i r e d f o r t h e dependent c u l t u r e t o a t t a i n t h e h a l f - m a x i m a l growth r a t e (Table X I I ) , and t h e r e i s a d i r e c t r e l a t i o n s h i p between t h e growth r a t e o f the •mutant :.-on l i m i t i n g - a n t i b i o t i c and the degree o f r e p r e s s i o n o f c e r t a i n . g l u c o s e - s e n s i t i v e enzymes ( F i g . 2 0 ) . These r e s u l t s i n d i c a t e t h a t c a t a b o l i t e r e p r e s s i o n may p l a y an i m p o r t a n t " r o l e i n e s t a b l i s h i n g the q u a n t i t a t i v e r e q u i r e m e n t f o r a n t i b i o t i c f o r growth o f ,.Sm-dependent 2?..' coli. I n o r d e r t o i n c o r p o r a t e t h e s e r e s u l t s i n t o t h e h y p o t h e s i s w h i c h i n d i c a t e s p r o t e i n s y n t h e s i s t o be the p r i m a r y p r o c e s s a f f e c t e d by Sm (or DHSm) one may - 139 -p o s t u l a t e t h a t , in vivo, t h e e f f e c t o f the a d d i t i o n o f the a n t i b i o t i c on p r o t e i n s y n t h e s i s i s augmented by t h e s e c o n d a r y e f f e c t o f c a t a b o l i t e r e p r e s s i o n . In t h e Sm-dependent c e l l , t h e l i m i t a t i o n o f a n t i b i o t i c i n i t i a l l y may i m p a i r r i b o s o m a l f u n c t i o n w i t h o u t i m m e d i a t e l y a f f e c t i n g c a t a b o l i c p r o c e s s e s . The r e s u l t o f l i m i t a t i o n o f a n a b o l i s m w i t h o u t a c o r r e s p o n d i n g d e c r e a s e i n t h e r a t e o f c a t a b o l i s m would be s e v e r e r e p r e s s i o n o f the g l u c o s e - s e n s i t i v e enzymes,. u l t i m a t e l y r e s u l t i n g i n a d e c r e a s e d r a t e o f b o t h c a t a b o l i c and a n a b o l i c r e a c t i o n s . In Sm-dependent c e l l s i t would a p p e a r t h a t c a t a b o l i t e r e p r e s s i o n i s , q u a n t i t a t i v e l y , an i m p o r t a n t f a c t o r i n l i m i t i n g growth a t low c o n c e n t r a t i o n s o f a n t i b i o t i c s i n c e t h e n a t u r e o f t h e c a r b o n s o u r c e p l a y s a major r o l e i n d e t e r m i n i n g t h e c o n c e n -t r a t i o n o f DHSm r e q u i r e d f o r g r o w t h . - 140 -GENERAlr DISCUSSION M e t a b o l i c c o n t r o l by c a t a b o l i t e r e p r e s s i o n was relaxed i n a Sm-dependent mutant o f E. c o l i B ( s t r a i n DB) grown on a n o n - l i m i t i n g c o n c e n t r a t i o n o f DHSm (>100 ug./ml.). The ob-s e r v a t i o n s w h i c h l e d t h i s c o n c l u s i o n were t h a t a number o f . g l u c o s e - s e n s i t i v e enzymes were d e r e p r e s s e d i n the Sm-dependent mutant compared w i t h w i l d - t y p e E. c o l i B (Table V I I ) . The r e l a x a t i o n o f c a t a b o l i t e r e p r e s s i o n i n t h i s mutant was a t t r i -b u t e d t o a reduced e f f i c i e n c y i n a e r o b i c c a r b o h y d r a t e metabo-l i s m s i n c e i t was found ( T a b l e s V I I I and XV) t h a t t h e y i e l d o f Sm-dependent c e l l s from g l u c o s e o r g l u c o n a t e was about o n e - t h i r d l e s s t h a n the y i e l d o f w i l d - t y p e E. c o l i B from the same c a r b o n s o u r c e s . A d i r e c t measurement o f ATP s y n t h e -s i s i n e x p o n e n t i a l c u l t u r e s of s t r a i n s SB and DB ( F i g . 15) c o n f i r m e d the s u p p o s i t i o n t h a t energy m e t a b o l i s m i s i m p a i r e d i n the Sm-dependent mutant. The d e c r e a s e d r a t e o f growth o f t h e Sm-dependent mutant o f E. c o l i E ( s t r a i n DE) on g l u c o s e and on g l y c e r o l compared t o t h e w i l d - t y p e p a r e n t (Table.IX) as w e l l as the d e c r e a s e d c a t a b o l i t e r e p r e s s i o n i n t h i s mutant ( F i g s . 9 and 11) suggest t h a t a s i m i l a r d e f e c t i n energy meta-b o l i s m may e x i s t i n t h i s a d d i t i o n a l s t r a i n o f E.coli. When s t r a i n DB was grown on a r a t e - l i m i t i n g c o n c e n t r a t i o n o f a n t i b i o t i c (<100 ug./ml.), however, c a t a b o l i t e r e p r e s s i o n (as d e t e r m i n e d b y . t h e s p e c i f i c a c t i v i t i e s o f a number o f g l u c o s e -s e n s i t i v e enzymes1)'" was enhanced (Tables X I I I and XVI) . The - 141 -degree o f i n c r e a s e i n c a t a b o l i t e r e p r e s s i o n was i n v e r s e l y r e -l a t e d t o t h e l i m i t i n g c o n c e n t r a t i o n of a n t i b i o t i c on which the Sm-dependent mutant was grown ( F i g . 1 9 ) . T h i s i n c r e a s e i n c a t a b o l i t e r e p r e s s i o n c o r r e l a t e d w i t h the s l i g h t i n c r e a s e i n t he c o n c e n t r a t i o n o f th e ATP p o o l o b s e r v e d i n a n t i b i o t i c -l i m i t e d c u l t u r e s ( F i g . 2 1 ) . Thus, t h e r e a r e two d i s t i n c t and c o n t r a s t i n g o b s e r v a t i o n s on Sm-dependent E. c o l i t o be un d e r s t o o d . F i r s t , t he i m p a i r e d c a r b o h y d r a t e m e t a b o l i s m which decreases c a t a b o l i t e r e p r e s s i o n i n c e l l s grown on n o n - l i m i t i n g c o n c e n t r a t i o n s o f a n t i b i o t i c and second, t h e e f f e c t o f a n t i -b i o t i c - l i m i t a t i o n w h i c h increases c a t a b o l i t e r e p r e s s i o n . W h i l e the l a t t e r o b s e r v a t i o n can be u n d e r s t o o d i n terms o f a de c r e a s e d r a t e o f p r o t e i n s y n t h e s i s r e s u l t i n g from i m p a i r e d r i b o s o m a l f u n c t i o n (Goodman and S p o t t s , 1967; L i k o v e r and K u r l a n d , 1967b), the former o b s e r v a t i o n can be u n d e r s t o o d o n l y i f one p o s t u l a t e s a sec o n d . m u t a t i o n r e s u l t i n g i n a d e c r e a s e d e f f i c i e n c y i n energy m e t a b o l i s m . A l t e r n a t i v e l y , t h e pre s e n c e of DHSm (or Sm), w h i l e p e r m i t t i n g maximal a n a b o l i c a c t i v i t y i n t he Sm-dependent c e l l s may s i m u l t a n e o u s l y i n t e r f e r e w i t h c a r b o h y d r a t e m e t a b o l i s m . C e l l y i e l d s t u d i e s i n the p r e s e n c e of l i m i t i n g and n o n - l i m i t i n g c o n c e n t r a t i o n s of DHSm i n d i c a t e d t h a t t h e d e c r e a s e d e f f i c i e n c y of c a r b o h y d r a t e m e t a b o l i s m i n s t r a i n DB was independent of a n t i b i o t i c c o n c e n t r a t i o n (Table X V I I ) . T h e r e f o r e , w h i l e the i m p a i r e d energy m e t a b o l i s m o f s t r a i n DB appears t o be the r e s u l t of a second m u t a t i o n , f u r t h e r s t u d y w i l l be n e c e s s a r y t o e s t a b l i s h t h e e x a c t n a t u r e of t h i s -.142 -c a t a b o l i c d e f e c t . The f a c t t h a t t h e f o r m a t i o n o f AHA s y n t h e -t a s e was d e r e p r e s s e d i n Sm-dependent mutants o b t a i n e d from 5 d i f f e r e n t s t r a i n s o f E. c o l i ( C o u k e l l and P o l g l a s e , 1965; F i g . 5) and t h a t a l l Sm-dependent mutants, examined e x c r e t e d v a l i n e s u g g e s t t h a t i m p a i r e d c a r b o h y d r a t e m e t a b o l i s m may be a g e n e r a l p r o p e r t y o f Sm-dependent mutants (see l a t e r d i s c u s s i o n ) . R e s u l t s o f a number o f ex p e r i m e n t s performed w i t h s t r a i n DB growing on r a t e - l i m i t i n g c o n c e n t r a t i o n s o f DHSm i n d i c a t e d t h a t c a t a b o l i t e r e p r e s s i o n was an i m p o r t a n t f a c t o r i n l i m i t i n g growth a t low c o n c e n t r a t i o n o f a n t i b i o t i c (Table X I I and F i g . 20). T h i s f i n d i n g s u g g e s t s t h a t c a t a b o l i t e r e p r e s s i o n may a l s o p l a y a r o l e i n t h e a c t i o n o f DHSm (or Sm) on a n t i b i o t i c -s e n s i t i v e c e l l s . E x p e r i m e n t a l r e s u l t s which suggest t h a t c a t a b o l i t e r e p r e s s i o n may i n c r e a s e s i g n i f i c a n t l y i n Sm-sensi-t i v e c e l l s t r e a t e d w i t h Sm, were p r o v i d e d by t h e r e p o r t o f E n g e l b e r g and Artman (196 4) t h a t when Sm was added t o sus -c e p t i b l e c u l t u r e s o f E. c o l i , t h e s y n t h e s i s o f t h e g l u c o s e -s e n s i t i v e B - g a l a c t o s i d a s e was p r e f e r e n t i a l l y and c o m p l e t e l y (96%) i n h i b i t e d by a c o n c e n t r a t i o n o f a n t i b i o t i c w h i c h o n l y p a r t i a l l y (66%) i n h i b i t e d p r o t e i n s y n t h e s i s . S i m i l a r r e s u l t s w i t h c h l o r a m p h e n i c o l (Palmer and Moses, 1967) and 5 - f l u o r o -. u r a c i l ( H orowitz and K o h l m e i e r , 1967) have been a t t r i b u t e d t o i n c r e a s e d c a t a b o l i t e r e p r e s s i o n r e s u l t i n g from a r e d u c t i o n i n th e r a t e o f p r o t e i n s y n t h e s i s . .In s u p p o r t o f t h e con c e p t t h a t c a t a b o l i t e r e p r e s s i o n may augment the d i r e c t a c t i o n o f Sm on the s e n s i t i v e ribosome, one may. c i t e t he o b s e r v a t i o n s (Lightbown, 143 -1957) t h a t i n h i b i t i o n of growth by Sm i s l e s s . e f f e c t i v e under a n a e r o b i c c o n d i t i o n s where c a t a b o l i t e r e p r e s s i o n a l s o i s d i m i n i s h e d (Cohn and H o r i b a t a , 1959). F u r t h e r m o r e , th e a d d i t i o n o f Sm t o g r owing s e n s i t i v e c e l l s does not r e s u l t i n an immedi-a t e d e c r e a s e i n v i a b i l i t y ( E n g e l b e r g and Artman, 1964) even though i t has been e s t a b l i s h e d t h a t t h e a n t i b i o t i c i s t a k e n up by t h e c e l l e x t r e m e l y r a p i d l y ( S z y b a l s k i and Mashima, 19 59; E n g e l b e r g and Artman, 19 6 4 ) . The t ime l a g between t h e uptake of Sm by t h e c u l t u r e and the d e c r e a s e i n v i a b i l i t y c o u l d be t h e p e r i o d d u r i n g w h i c h th e secondary e f f e c t o f accummulation o f c a t a b o l i t e c o - r e p r e s s o r t a k e s p l a c e . Rosenblum and B r y s o n (1953) s t u d i e d t h e e f f e c t o f t h e r a t e o f b a c t e r i a l m e t a b o l i s m on t h e a c t i o n o f Sm i n w i l d - t y p e E. c o l i B and c o n c l u d e d t h a t m e t a b o l i c a l l y a c t i v e c e l l s , were more s e n s i t i v e . t h a n n o n - a c t i v e c e l l s t o the b a c t e r i c i d a l a c t i o n o f Sm. Of p a r t i c u l a r s i g -n i f i c a n c e t o the p r e s e n t work was t h e i r o b s e r v a t i o n t h a t washed c e l l s suspended i n s a l i n e were r e l a t i v e l y r e s i s t a n t t o t h e b a c t e r i c i d a l a c t i o n o f Sm b u t were s e n s i t i v e when g l u c o s e was p r e s e n t i n t h e s u s p e n s i o n . As a r e s u l t o f t h e extreme v a r i a b i l i t y o f c a t a b o l i t e r e p r e s s i o n - i n s t r a i n DB, t h e c o n c e n t r a t i o n s o f a number of g l u c o s e - s e n s i t i v e enzymes i n v o l v e d i n g e n e r a l i n t e r m e d i a r y m e t a b o l i s m , changed d r a m a t i c a l l y d u r i n g growth on d i f f e r e n t c o n c e n t r a t i o n s o f a n t i b i o t i c . ( T ables X I I I and X V I ) . One o f t h e s e enzymes, AHA s y n t h e t a s e , was o f p a r t i c u l a r i n t e r e s t i n t h e p r e s e n t s t u d y because o f i t s proposed r o l e i n t h e phenomenon - 144 -o f v a l i n e e x c r e t i o n by Sm-dependent mutants (Bragg and P o l -g l a s e , 1965; C o u k e l l and P o l g l a s e , 1965). An i n v e s t i g a t i o n o f t h e r e g u l a t i o n , of AHA-r s y n t h e t a s e f o r m a t i o n and a c t i v i t y i n E. c o l i B r e v e a l e d t h a t two unexpected p r o p e r t i e s o f t h i s enzyme were r e s p o n s i b l e f o r e x t r a c e l l u l a r v a l i n e p r o d u c t i o n by t h e a n t i b i o t i c - d e p e n d e n t mutant. F i r s t , AHA s y n t h e t a s e was found t o be s e n s i t i v e t o c a t a b o l i t e r e p r e s s i o n ( F i g . 3) and second, t h e r e was i n c o m p l e t e feedback i n h i b i t i o n o f t h i s enzyme by v a l i n e ( F i g . 5 ) . C a t a b o l i t e r e p r e s s i o n o f AHA s y n t h e t a s e i s r e l a x e d i n t h e Sm-dependent mutant of E. c o l i B (DB) g r owing e x p o n e n t i a l l y on g l u c o s e and on a n o n - l i m i t i n g c o n c e n t r a t i o n o f DHSm ( F i g . 7 ) . A c o n s i d e r a b l e p a r t o f t h e p y r u v a t e produced from g l u c o s e m e t a b o l i s m w i l l t h e r e f o r e be c o n v e r t e d t o v a l i n e and u l t i m a t e l y w i l l l e a d t o v a l i n e ex-c r e t i o n (Table X ) . S t a r v a t i o n o f t h e same mutant f o r a n t i -b i o t i c , however, s e v e r e l y r e p r e s s e s AHA-synthetase f o r m a t i o n (Table XVI) and i n t h i s way i n h i b i t s the p r o d u c t i o n o f e x t r a -c e l l u l a r v a l i n e (see Bragg and P o l g l a s e , 1964a). W h i l e c o n t r o l o f AHA-synthetase f o r m a t i o n by c a t a b o l i t e r e p r e s s i o n has been s t u d i e d o n l y i n E. c o l i B, i n d i r e c t s t u d i e s s u ggest t h a t t h i s p r o p e r t y o f AHA s y n t h e t a s e may not be r e s t r i c t e d t o t h i s s t r a i n . F o r example, a . s u r v e y of AHA-s y n t h e t a s e a c t i v i t y i n S m - s e n s i t i v e , r e s i s t a n t and dependent organisms o f 4 d i f f e r e n t s t r a i n s o f E. c o l i d i s c l o s e d t h a t the f o r m a t i o n o f t h i s enzyme was d e r e p r e s s e d i n a l l Sm-dependent mutants when compared t o t h e a c t i v i t i e s i n t h e - 145 -a n t i b i o t i c - s e n s i t i v e and r e s i s t a n t c e l l s ( C o u k e l l and P o l g l a s e , 196 5) . I n t h e p r e s e n t work, c a t a b o l i t e r e p r e s s i o n has been examined i n o n l y one o f t h e s e p r e v i o u s l y s t u d i e d Sm-dependent mutants ( s t r a i n DE) b u t t h e r e s u l t s i n d i c a t e t h a t c a t a b o l i t e r e p r e s s i o n i n t h i s s t r a i n i s a l s o " r e l a x e d " as i n E. c o l i s t r a i n DB ( F i g s . 9 and 1 1 ) . F u r t h e r m o r e , s i n c e v a l i n e e x c r e -t i o n by s t r a i n DB was a t t r i b u t e d t o b o t h the d e r e p r e s s i o n o f AHA s y n t h e t a s e i n t h i s mutant and the i n a b i l i t y . o f t h i s enzyme t o be. c o m p l e t e l y i n h i b i t e d by v a l i n e , t h e n th e p r o d u c t i o n of e x t r a c e l l u l a r v a l i n e by a Sm-dependent mutant o f a.' Mycobac-terium sp. ( T i r u n a r a y a n a n et al. 1962) s u g g e s t s t h a t the AHA s y n t h e t a s e of t h i s o r g a n i s m may a l s o . p o s s e s s the u n u s u a l p r o p e r t i e s d e s c r i b e d f o r the enzyme o f E. c o l i B. I t may be c o n c l u d e d from the p r e s e n t s t u d y t h a t many of the b i o c h e m c i a l and p h y s i o l o g i c a l changes which a r e ob-s e r v e d i n Sm-dependent mutants o f E. c o l i f o l l o w i n g a n t i b i o -t i c - s t a r v a t i o n o r s u p p l e m e n t a t i o n a r e m e d i a t e d t h r o u g h the a c t i o n o f the a n t i b i o t i c on c a t a b o l i t e r e p r e s s i o n r a t h e r t h a n t h r o u g h a d i r e c t e f f e c t o f the a n t i b i o t i c on p r o t e i n s y n t h e s i s . - 146 -SUGGESTIONS FOR FUTURE WORK D u r i n g the c o u r s e o f t h i s s t u d y a number o f q u e s t i o n s a r o s e w h i c h were e i t h e r not examined s i n c e t h e y were o f f the main l i n e o f t h e o r i g i n a l problem o r were examined b u t were l e f t unanswered a t the c o n c l u s i o n o f t h e p r e s e n t work. I t i s the a u t h o r ' s b e l i e f t h a t u n t i l t h e s e q u e s t i o n s a re answered, t h e s i g n i f i c a n c e o f t h e f i n d i n g s d e s c r i b e d here cannot be e v a l u a t e d f u l l y . 1. What i s t h e n a t u r e o f the impairment i n energy m e t a b o l i s m i n s t r a i n DB? I s energy m e t a b o l i s m i m p a i r e d i n a l l Sm-dependent mutants and i f s o , what i s t h e r e l a t i o n s h i p between d e f e c t i v e energy m e t a b o l i s m and Sm-dependence? 2. Can s e v e r e c a t a b o l i t e r e p r e s s i o n a l o n e i n h i b i t t h e growth" of c e r t a i n m i c r o - o r g a n i s m s and i f s o , by what mechanism? 3. 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Biochemistry and Molecular Biology
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Catabolite repression in Streptomycin-dependent Escherichia coli
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