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An investigation of modified metabolic regulation in streptomycin-dependent Escherichia coli Coukell, M.B. 1966

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AN INVESTIGATION OF MODIFIED METABOLIC REGULATION IN  STREPTOMYCIN-DBPENDENT  ESCHERICHIA COLI M. B. COUKELL A Thesis Submitted In P a r t i a l Fulfilment Of The Requirements For The Degree Of MASTER OF SCIENCE i n the Department of BIOCHEMISTRY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , I966 I n 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 of the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r -m i s s i o n f o r e x t e n s i v e c o p y i n g 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 granted by the Head of 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 understood t h a t c o p y i n g or p u b l i -c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada. ( i ) ABSTRACT The acetohydroxy a c i d synthetase l e v e l s i n s t r e p t o m y c i n - s e n s i t i v e - d e p e n d e n t and - r e s i s t a n t mutants have been s t u d i e d i n four d i f f e r e n t s t r a i n s of E s c h e r i c h i a  c o l i . The a c t i v i t y of the «><:-acetolactate-f orming system was found to be gr e a t e r both at pH 6.0 and a t pH 8.0 i n streptomycin-dependent mutants than i n the corresponding s t r e p t o m y c i n - s e n s i t i v e c u l t u r e s . In ge n e r a l , s t r e p t o m y c i n - r e s i s t a n t mutants demonstrated enzyme a c t i v i t i e s w i t h i n the range found f o r streptomycin-s e n s i t i v e organisms r e g a r d l e s s of whether they were grown i n the presence or absence of a n t i b i o t i c . The acetohydroxy a c i d synthetase a c t i v i t y of s t r e p t o m y c i n - s e n s i t i v e and - r e s i s t a n t r e v e r t a n t s was observed to be lower than t h a t of the dependent E s c h e r i c h i a c o l i c u l t u r e from which they were d e r i v e d by back-mutation. Mutation to streptomycin-resistance or -dependence had no e f f e c t on glucokinase and glutamic dehydrogenase a c t i v i t i e s . The a d d i t i o n of the coenzyme f l a v i n adenine d i n u c l e o t i d e to the i n c u b a t i o n mixtures markedly s t i m u l a t e d the a c t i v i t i e s of a l l the e x t r a c t s . This enhancement of acetohydroxy a c i d synthetase a c t i v i t y had l i t t l e or no e f f e c t on the r a t i o of a c t i v i t i e s of t h i s enzyme i n the dependent and s e n s i t i v e E s c h e r i c h i a c o l i s t r a i n s i n v e s t i g a t e d . «<-Acetohydroxybutyrate formation was found to be g r e a t e r i n e x t r a c t s from the streptomycin-dependent ( i i ) organism than i n e x t r a c t s prepared from the same s t r a i n of s e n s i t i v e and r e s i s t a n t E s c h e r i c h i a c o l i . The degree of e l e v a t i o n of oC-acetohydroxybutyrate p a r a l l e l e d t h a t of o<-acetolactate formation i n the dependent mutant. I t was concluded from these observations that e x c r e t i o n of L - v a l i n e by streptomycin-dependent E s c h e r i c h i a c o l i was a consequence of the elevated acetohydroxy a c i d synthetase a c t i v i t y of these mutants. In the dependent organism, i t was p o s t u l a t e d that streptomycin functioned as a w d e - r e p r e s s o r w of aceto-hydroxy a c i d synthetase thus p e r m i t t i n g the b i o s y n t h e t i c pathway l e a d i n g t o L - v a l i n e to serve as an important route of pyruvate d i s s i m i l a t i o n . ( i i i ) ACKNOWLEDGEMENTS The author wishes to express his appreciation to Dr. W.J. Polglase f o r his guidance and encouragement during the course of t h i s work and to Dr. I.D. Desai for his many h e l p f u l suggestions. (iv) TABLE OF CONTENTS Page No. A. INTRODUCTION 1 I. Metabolic Effects of Streptomycin in Microorganisms 1 II. Biosynthesis of Aliphatic Amino Acids and Regulatory Mechanisms k III. Objective of the Investigation of Streptomycin Mutants 9 B. METHODS AND MATERIALS 12 I. Original Cultures 12 II. Isolation of Streptomycin Mutants 12 (i) From streptomycin-sensitive parent cultures 12 (ii) Isolation of revertants from a streptomycin-dependent culture 14 III. Growth of Cultures and Preparation of Extracts lk (i) Medium lk (ii) Procedure for growing and harvesting cultures 15 (iii) Bacterial extracts 16 IV. Enzyme Assays 16 (i) Measurement of o<-acetolactate-forming activity 16 (a) Method I 16 (b) Method II 18 (ii) Measurement of <<-acetohydroxy-butyrate-forming activity 19 ( v ) Page No. (a) R e v e r s a l o f L - v a l i n e i n h i b i t i o n b y L - i s o l e u c i n e i n E . o o l i K-12 19 (b) P r e p a r a t i o n o f a s s a y p l a t e s 19 ( c ) E n z y m a t i c f o r m a t i o n o f < X - a c e t o h y d r o x y b u t y r a t e 20 (d) E s t i m a t i o n o f b a c t e r i a l g r o w t h 21 (e) P r e p a r a t i o n o f a s t a n d a r d c u r v e 22 ( i i i ) R e f e r e n c e enzymes 22 (a) G l u c o k i n a s e 22 (b) G l u t a m i c d e h y d r o g e n a s e 22 C. RESULTS 23 I . S t r e p t o m y c i n S e n s i t i v i t y o f t h e C u l t u r e s 23 I I . S p e c i f i c i t y o f t h e Method f o r < < - A c e t o l a c t a t e D e t e r m i n a t i o n 23 I I I . A c t i v i t y o f t h e «<-Acetolactate-forming S y s t e m o f t h e M u t a n t s 26 IV. A c t i v i t y o f t h e o < - A c e t o h y d r o x y b u t y r a t e -f o r m i n g S y s t e m o f t h e M u t a n t s 26 V. A c t i v i t y o f t h e R e f e r e n c e Enzymes 30 D. DISCUSSION 36 I . G e n e t i c C h a r a c t e r i s t i c s o f t h e S t r e p t o m y c i n M u t a n t s 36 I I . F o r m a t i o n o f «*-Acetolactate by S t r e p t o m y c i n M u t a n t s k0 I I I . F o r m a t i o n o f «<-Acetohydroxybutyrate by S t r e p t o m y c i n M u t a n t s kS IV. S i g n i f i c a n c 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 D e - r e p r e s s i o n i n D e p e n d e n t M u t a n t s o f E s c h e r i c h i a c o l i 50 E . REFERENCES 56 (vi) LIST OF TABLES Page No, I Relative S e n s i t i v i t i e s of Escherichia  c o l i Sensitive Strains to Dihydrostreptomycin 2k II Comparison of Methods for Determining <<-Acetolactate i n Extracts of Escherichia o o l i 25 I I I Acetohydroxy Acid Synthetase A c t i v i t i e s i n Extracts of Streptomycin-Sensitive (S) and -Dependent (D) Escherichia c o l i 27 IV Acetohydroxy Acid Synthetase A c t i v i t i e s i n Extracts of Streptomycin-Resistant Escherichia c o l i 28 V E f f e c t of F l a v i n Adenine Dinucleotide on Acetohydroxy Acid Synthetase A c t i v i t i e s i n Sensitive (S) and Dependent (D) Extracts of Escherichia c o l i 29 VI S e n s i t i v i t y of Escherichia c o l i K-12 to L-Valine and Reversal of I n h i b i t i o n by L-Isoleucine 32 VII S p e c i f i c i t y of the *:-Acetohydroxybutyrate Assay System for the Six-Carbon Inter-mediate 33 VIII Formation of <*-Acetolactate and «*-Acetohydroxybutyrate by Sensitive (S), Resistant (R) and Dependent (D) Extracts of Escherichia c o l i 3k IX Reference Enzyme A c t i v i t i e s of Streptomycin Mutants of Escherichia c o l i A. 35 ( v i i ) LIST OF FIGURES Page No. 1. Products of Catabolism of Glucose by Streptomycin-Dependent E s c h e r i c h i a c o l i 3 2. B i o s y n t h e t i c Pathway to I s o l e u c i n e , V a l i n e , Leucine, and Pantothenate i n E s c h e r i c h i a c o l i 5 3. Formation of <?<-Acetolactate and «<-Acetohydroxybutyrate i n E s c h e r i c h i a c o l i 7 4 . Growth Response of E s c h e r i c h i a c o l i K-12 to In c r e a s i n g Concentration of L - I s o l e u c i n e 31 A. INTRODUCTION I. Metabolic E f f e c t s of Streptomycin In Microorganisms The primary s i t e of the a n t i b a c t e r i a l action of streptomycin has yet to be elucidated. However, several divergent hypotheses have been proposed: (1) i n h i b i t i o n of s p e c i f i c enzyme reactions (Umbreit, 1953; Rosanoff and Sevag, 1953; Barkulis, 1953); (2) a l t e r e d permeability due to membrane damage (Anand, et a l , I960; Landman and Burchard, 1962); and (3) i n h i b i t i o n of protein synthesis (Erdos and Ullman, 1959; Mager, et a l , 1962; Speyer, et a l , 1962, Flax, et a l , 1962a, 1962b; Cox, et a l , 1964). Two groups of Investigators (Tirunarayanan, et a l , I 9 6 2 , and Bragg and Polglase, 1962) reported, independently, that streptomycin-dependent microorganisms grown on glucose-salts medium excreted r e l a t i v e l y large amounts of L-valine and a l e s s e r amount of L-leucine. Each group however, interpreted these r e s u l t s d i f f e r e n t l y . Tirunarayanan and co-workers (1962) a t t r i b u t e d the excretion of L-valine and L-leucine to a " p a r t i a l blockage" of protein synthesis. Since the streptomycin-dependent stra i n s were able to grow and multiply i n spite of the p a r t i a l block i n protein synthesis, they suggested th i s phenomenon constituted only the preliminary f i x a t i o n of streptomycin to the c e l l and that the antimicrobial e f f e c t was due to subsequent metabolic changes. Experiments c a r r i e d out by Bragg and Polglase (1962) suggested that the excretion of L-valine resulted from an - 1 -- 2 -a l t e r a t i o n i n the pathway of pyruvate 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 metabolites are not normally detectable i n the supernatant f l u i d s of streptomycin-sensitive or - r e s i s t a n t organisms growing i n the medium without a n t i b i o t i c supplement. However, a streptomycin-resistant mutant i n the presence of a n t i b i o t i c excretes s i g n i f i c a n t quantities of both l a c t a t e and pyruvate. These r e s u l t s suggest that the r e s i s t a n t mutant uses pathways of anaerobic metabolism i n a n t i b i o t i c containing medium but i n the absence of the a n t i b i o t i c employs a pathway of pyruvate d i s s i m i l a t i o n s i m i l a r to that employed by sensitive organisms. The streptomycin-dependent mutant d i f f e r s from both the sensitive and r e s i s t a n t organisms i n i t s production of substantial amounts of L-valine. As much as 10% of the glucose carbon could be accounted f o r i n t h i s product. Subsequent work (Bragg and Polglase, 1964a) c l e a r l y indicated that depleted streptomycin-dependent c e l l s , produced large quantities of l a c t a t e while the same c e l l s supplemented with streptomycin, at a concentration i n excess of that essential f o r growth, excreted only L-valine. When streptomycin-dependent c e l l s were grown anaerobically, t h e i r metabolism resembled that of aerated, a n t i b i o t i c depleted c e l l s , (production of l a c t i c acid) even i n the presence of the optimal streptomycin concentration. For the streptomycin-dependent mutant, the re l a t i o n s h i p between aerobic metabolism and the requirement for the a n t i b i o t i c i s summarized diagrammatl-c a l l y i n F i g . 1. The primary products of the aerobic catabolism - 3 -PRIMARY PRODUCTS C0 2 or ACETATE A GLUCOSE Requires Streptomycin and oxygen VALINE Streptomycin or oxygen deprivation V LACTIC ACID (and ALANINE) ALTERNATE SECONDARY PRODUCTS PIG. 1. Products of catabolism of glucose by streptomycin-dependent Escherichia  c o l i (Taken from Bragg and Polglase, 1964a). _ 4 -of glucose i n streptomycin-dependent E. c o l i are carbon dioxide or acetate. The secondary products are L-valine (aerobic and supplemented with a n t i b i o t i c ) or l a c t a t e and L-alanine (either anaerobic or deprived of a n t i b i o t i c ) . The small quantities of alanine probably a r i s e from pyruvate by transamination. These workers proposed that the formation of L-valine appeared to be a secondary aerobic pathway of glucose metabolism e x i s t i n g i n streptomycin-dependent mutants. Studies from the same laboratory (Bragg and Polglase, 1963b) on the e f f e c t of dihydrostreptomycin on electron transport i n E. c o l i . suggested that L-valine may function as a neutral hydrogen acceptor i n carbohydrate metabolism. I f t h i s were true, then, i n streptomycin-dependent c e l l s , the a n t i b i o t i c might activate a mechanism enabling a biosynthetic pathway to function c a t a b o l i c a l l y as a major route of pyruvate d i s s i m i l a t i o n . I I . Biosynthesis of A l i p h a t i c Amino Acids and Regulatory  Mechanisms. The biosynthetic pathways leading to L-isoleucine, L-valine, L-leucine and pantothenate i n E. c o l i have been investigated through isotope studies on selected auxotrophic mutants. A review of t h i s work has been given by Umbarger and Davis (I962) (see F i g . 2 ) . L-valine and L-leucine are synthesized from two moles of pyruvate while the precursors f o r L-isoleucine r e s u l t from the condensation of one mole each of pyruvate and oC-ket'obutyrate. Umbarger and Brown (1958b) reported that the enzymes catalysing the l a s t three reactions i n the biosynthetic threonine °<-keto-butyrate-pyruvate pyruvate «*-aceto-«<-hydroxy--» butyrate — •c-aceto-.» lactate I Acetohydroxy Acid Synthetase I I Reductoisomerase I I I Dihydroxy Dehydrase IV Transaminase B I I % 0-dihydroxy-' <$-methyl-• valerate — oi. -keto--methyl-valerate L-isoleucine I I I IV 4 ,6-dihydroxy-_» isovalerate — oc -keto-•isovalerate -» L-valine «-keto-isocaproate L-leucine x-keto-pantoate pantoate pantothenate i I F i g . 2 The biosynthetic pathway to isoleucine, valine, leucine and pantothenate i n Escherichia c o l i . - 6 -pathway leading to L-isoleucine also catalyse the corresponding reactions i n L-valine synthesis. This f i r s t became evident when i t was found that auxotrophic mutants lacking an enzyme on the L-valine pathway generally lacked the corresponding enzyme on the L-isoleucine pathway. Thus "single-step" mutants occur, multi-auxotrophic f o r L-valine, L-leucine and L-isoleucine. These findings have been supported by enzyme k i n e t i c studies (Umbarger and Brown, 1958b; L e a v i t t and Umbarger, I 9 6 I ) . I t should be noted however, that the enzyme which catalyses the f i r s t step i n the L-valine pathway, v i z . the condensation of two moles of pyruvate to one mole of o(-acetolactate, also catalyses the second step i n the L-isoleucine pathway, the ac e t y l a t i o n of oC-ketobutyrate to c<-acetohydroxybutyrate (Leavitt and Umbarger, 1961). This enzyme complex (see F i g . 3) has been designated by various authors as the condensing enzyme. <<-acetolactate-forming  system (Umbarger and Brown, 1958bj and recently as acetohydroxy  aci d synthetase (Bauerle, et a l , 1964). Although the enzyme complex has not been fractionated, early investigations (Umbarger and Brown, 1958b) suggest two reactions are involved. The f i r s t consists of the generation of an "active acetaldehyde", presumably as an acetal-diphosphothiamine (DPT) complex. The second reaction i s the actual transfer of the acetal group to the acceptor molecule, either pyruvate or «<-ketobutyrate. This reaction proceeds optimally only i n the presence of a divalent cation such as Mg"**" or Mh*" . Bauerle, et a l (1964) recently reported that 0 II DPTH C0 2 CH3-CH2-C-COOH «<-ketobutyrate CH3-C-DPT H 0 CH3-C-COOH pyruvate 0 O H C H 3 - C - C - C O O H C H 3 — ( J H2 << -aceto»<-hydroxy-butyrate 0 OH * CH3-C-C-COOH CHo <<-acetolactate F i g . 3 The formation of o<-acetolactate and <*-aceto«<-hydroxy-butyrate i n E s c h e r i c h i a c o l i . - 8 -acetohydroxy ac i d synthetase a c t i v i t y i s greatly stimulated by the presence of the coenzyme f l a v i n adenine dinucleotide (FAD). The function of t h i s unexpected cofactor i s as yet undefined. The pH optimum of the acetohydroxy ac i d synthetase system i n E. o o l i has involved considerable r e - i n v e s t i g a t i o n . Halpern and Umbarger (1959) c l e a r l y demonstrated the presence of two d i s t i n c t acetolactate forming systems i n A.aerogenes. one at pH 6.0 and the other at pH 8 . 0 . However, they could f i n d enzymic a c t i v i t y only at the higher pH i n E. c o l l . A l a t e r paper (Radhakrishnan and S n e l l , i960) described two pH optima i n E. c o l i corresponding to the pH values reported f o r A. aerogenes. Recently, a thorough k i n e t i c study on the acetohydroxy acid synthetase a c t i v i t y of a streptomycin-dependent E. c o l i mutant (Desai and Polglase, I965) under optimal conditions (including supplementation with FAD), strongly supported the o r i g i n a l p r e d i c t i o n of a single pH 8 .0 enzyme system i n t h i s organism. I t has been reported (Umbarger and Brown, 1958b) that the acetohydroxy ac i d synthetase complex, l i k e i n i t i a l enzymes of other biosynthetic pathways, i s subject to end-product i n h i b i t i o n when assayed i n the presence of L-valine. Repression studies, (Freundlich, et a l , I962) however, indicate that regulation of the enzymes associated with branched chain amino a c i d bio-synthesis involves a control mechanism thus far unique i n b i o l o g i c a l systems. Since L-valine and L-isoleucine are formed d i r e c t l y by a common sequence of enzymes, repression of the pathway - 9 -by the elevation of one product, could seriously a f f e c t the synthesis of the other product. In addition, L-leucine synthesis would also be impaired since the i n i t i a l reaction leading to L-leucine biosynthesis Involves an intermediate of the valine pathway. This problem i s overcome i n E. o o l i by the requirement that a l l three end-products (also possibly pantothenate) must be i n excess for repression to occur. This has been termed "multi-valent repression" (Freundlich, et a l , 1962; Umbarger and Preundlich, 1965). Although the mechanism of t h i s repression i s not completely understood i n regard to acetohydroxy a c i d synthetase, recent studies with streptomycin-dependent mutants of E. c o l i indicate that t h i s enzyme i s d e f i n i t e l y repressible by the end-products (Polglase, i n press). Therefore, i t appears to be well established that the acetohydroxy a c i d synthetase system not only catalyses the i n i t i a l step i n the biosynthesis of L-valine and L-leucine from pyruvate but also controls the production of these amino acids. Since t h i s enzyme complex i s intimately involved with L-isoleucine biosynthesis, an a l t e r a t i o n i n acetohydroxy a c i d synthetase a c t i v i t y or a change i n enzyme l e v e l would be expected to influence the synthesis of L-isoleucine as well as L-valine. I I I . Objective of the Investigation of Streptomycin Mutants I t was suggested (Bragg and Polglase, 1962) that i n streptomycin-dependent E. c o l i mutants, the a n t i b i o t i c may evoke - l o -an a l t e r a t i o n i n aerobic carbohydrate metabolism r e s u l t i n g i n amino acid excretion. This may be further interpreted as an a l t e r a t i o n i n the control of branched chain amino acid biosynthesis. Preliminary studies (Bragg and Polglase, 1964a) on acetohydroxy a c i d synthetase a c t i v i t i e s i n depleted and supplemented streptomycin-dependent E. c o l i , indicated that maximal ot-aceto-la c t a t e formation occurred only i n the presence of a n t i b i o t i c . Streptomycin appeared to de-repress the l e v e l of t h i s enzyme i n supplemented dependent mutants regardless of whether or not the c e l l s were grown i n the presence of the end-products. Since t h i s suggests that streptomycin functions at the genetic l e v e l , i t i s e s s e n t i a l to e s t a b l i s h whether t h i s i s a general phenomenon or merely the c h a r a c t e r i s t i c response of a p a r t i c u l a r streptomycin-dependent mutant. The objective of t h i s i n v e s t i g a t i o n was therefore to determine through a study of several strains of E. c o l i whether consistent differences e x i s t i n streptomycin mutants i n the l e v e l of acetohydroxy a c i d synthetase (the regulatory enzyme for L-valine biosynthesis). I t was assumed at the outset that the generality of the phenomenon of L-valine excretion by streptomycin-dependent mutants had been established by previous work (Tirunara-yanan, et a l , 1962; Bragg and Polglase, I962). In i t s i n i t i a l phase, the i n v e s t i g a t i o n required the i s o l a t i o n of several new E. c o l i mutants. The second phase of the study involved assays for enzymatic a c t i v i t i e s of b a c t e r i a l - 11 -extracts. Subsequently, when adequate evidence had been adduced to es t a b l i s h that streptomycin-dependent mutants of E. c o l i do indeed d i f f e r q u a n t i t a t i v e l y to sensitive and res i s t a n t strains i n enzyme content, an attempt was made to explain the advantage to the streptomycin-dependent organism of this difference. B. METHODS AND MATERIALS I. O r i g i n a l Cultures Four str a i n s of E. c o l i were used i n t h i s work. E. c o l i "A" as previously described by Roote and Polglase (1955) was o r i g i n a l l y obtained as the streptomycin-dependent culture. This mutant has been designated DA. A streptomycin-sensitive (SA), and streptomycin-resistant (RA) mutant was derived by "back-mutation" from the dependent culture. The s t r a i n designated E. c o l i "C" was obtained from the Laboratory of Hygiene, Ottawa, Ontario and s t r a i n E. c o l i "E" was i s o l a t e d at the Department of Bacteriology, University of Laval, Montreal. These were supplied to us as streptomycin-sensitive s t r a i n s . An a d d i t i o n a l sensitive s t r a i n was obtained from the American Type Culture C o l l e c t i o n (ATCC 1 2 4 0 7 ) . This s t r a i n i s a spontaneous r a d i a t i o n -r e s i s t a n t mutant of E. c o l i "B" and w i l l be referred to, herein-a f t e r , as E. c o l i B/r (Witkin, 1 9 ^ 7 ) . I I . I s o l a t i o n of Streptomycin Mutants (i ) From streptomycin-sensitive parent cultures. A volume of approximately 500 ml. of glucose-salts medium (composition described under "Medium") was innoculated with one or two loopfuls of streptomycin-sensitive culture stored i n heart infusion broth (25 gm. of heart infusion broth per l i t e r ) at 5°C. This culture was incubated without a g i t a t i o n for 20 - 24 hours at 3 7°C The r e s u l t i n g growth was transferred - 12 -- 13 -a s e p t i c a l l y to two 300 ml. s t e r i l e bottles and .centrifuged at 2000 RPM for 1 hour at 4°C (International Refrigerated Centrifuge). The p e l l e t was resuspended i n 6.0 ml. of buffer (0.05M potassium phosphate, pH 7.4), c a l l e d the "Standard Inoculum" (SI). Q Exactly 0 . 5 ml. of the SI (-~10 c e l l s ) were pipetted on to the surface of several P e t r i plates containing heart i n f u s i o n agar (heart i n f u s i o n broth f o r t i f i e d with 1 .5$ agar) and 1000 units per ml. (1 unit i s equivalent to 1 jxg of free streptomycin base) of either dihydrostreptomycin (DHSM) or streptomycin (SM). The sl u r r y of c e l l s was evenly d i s t r i b u t e d over the agar surface and the plates were incubated at 37°C f o r 24 hours. The colonies which formed were subcultured into 5 nil* of s t e r i l e glucose-salts medium, either devoid of a n t i b i o t i c or supplemented with 1000 jxg per ml. and incubated as described above. Dihydrostreptomycin-r e s i s t a n t mutants would grow i n both tubes, whereas, dependent mutants would grow only i n the presence of dihydrostreptomycin. To ensure that growth i n the absence of a n t i b i o t i c was i n fact due to the resis t a n t mutant and not to growth of a dependent culture r e s u l t i n g from a carry-over of dihydrostreptomycin from the plate, a l o o p f u l of t h i s culture was further transferred both to a n t i b i o t i c supplemented and to unsupplemented medium i n tubes. Pure r e s i s t a n t and dependent cultures of each s t r a i n were stored at 5°C on both heart i n f u s i o n agar slopes and i n heart i n f u s i o n broth. A l l cultures were subcultured monthly. - 14 -( l i ) I s o l a t i o n of revertants from a streptomycin-dependent culture. To 100 ml. of glucose-salts medium supplemented with 100 jig of dihydrostreptomycin per ml. was added 10 ml. of the E. c o l i dependent s t r a i n grown overnight at the same a n t i b i o t i c concentration. The culture was incubated at 37°C f o r 20 - 24 hours and centrifuged a s e p t i c a l l y at 2000 RPM for 1 hour. The p e l l e t was washed twice i n 0.05M potassium phosphate buffer, pH 7.4, added to 1000 ml. of heart infusion broth ( a n t i b i o t i c -free) and incubated a further 48 hours at 3 7 ° C The c e l l s were harvested as previously described. The p e l l e t was suspended i n 10 ml. of buffer. One loop f u l of t h i s c e l l suspension was streaked on glucose-salts agar medium (glucose-salts broth containing 1 . 5 $ agar) i n such a manner as to produce i s o l a t e d colonies. Colonies were subcultured to l i q u i d glucose-salts medium to y i e l d pure sensitive (growth only i n the absence of DHSM) and r e s i s t a n t ( i n d i f f e r e n t to the presence of DHSM) cultures. A l l cultures were stored as indicated. I I I . Growth of Cultures and Preparation of Extracts (i ) Medium The basal medium was of the composition previously described by Davis and Ming i o l i (1950) and consisted of the following: KgHPO^(0.7$), KH 2PO^(0.3$) sodium c i t r a t e ( 0 . 0 5 $ ) , MgSO^(0.02$), (NH^) 2S0^ ( 0 . 1 $ ). Glucose was autocli&yed separately and added to the basal medium to give a f i n a l concentration of 0.4$. - 15 Rather than streptomycin, the more stable analog, dihydro-streptomycin (sesqui sulfate) (Merck, Sharp and Dohme, Montreal, Canada) was generally used i n t h i s work. When streptomycin sulfate was used, i t was s t e r i l i z e d by passage through m i l l i p o r e f i l t e r s . In a l l cases the medium was adjusted to a f i n a l pH of 7.0. ( i i ) Procedure f o r growing and harvesting cultures. P r i o r to growth, the mutants involved were transferred at l e a s t three times on glucose-salts medium, under conditions (temperature and a n t i b i o t i c concentration) s i m i l a r to those employed i n the f i n a l growth experiment. The glucose-salts medium (900 ml.) was inoculated with 100 ml. of culture previously grown as a stationary culture over-night at 37°C. The two-l i t e r flask was incubated at the same temperature i n a water bath with moderate aeration provided as follows. A i r was supplied by glass tubing f i t t e d through a cork stopper, running below the surface of the medium and connected by rubber tubing to an a i r l i n e . Rate of growth i n the flask was observed by recording o p t i c a l densities hourly at 420 mu. The cultures were generally grown for 5 to 6 hours and were harvested during the l a t t e r h a l f of the exponential growth phase (usually at an O.D.^Q np o f approximately 1 . 4 ) . Streptomycin-sensitive organisms were grown i n a n t i b i o t i c - f r e e medium, while streptomycin-dependent cultures were routinely grown on the same medium f o r t i f i e d with 1,000 units per ml. of dihydrostreptomycin. The r e s i s t a n t mutants were grown on both supplemented (R + c e l l s ) and unsupplemented (R~ c e l l s ) medium. The c e l l s were harvested immediately by centrifugation at 6 , 0 0 0 x g for 20 minutes i n a r e f r i g e r a t e d centrifuge (4°C) and were then washed by centrifugation with 0.05M potassium phosphate buffer, pH 7 . 0 . ( i i i ) B a c t e r i a l extracts. The p e l l e t was resuspended i n the same buffer i n a r a t i o of 1 gm. of c e l l s to 15 ml. of buffer. The suspensions were then treated i n a Bronwill 20-kc sonic o s c i l l a t o r f o r 3 minutes followed by centrifugation at 1 0 , 0 0 0 x g for 15 minutes. The supernatant solutions were either assayed immediately or stored at -20°C and assayed within 20 - Zk hours. IV. Enzyme Assays ( i ) Measurement of oc-acetolactate-forming a c t i v i t y . Early studies on t h i s enzyme complex (Radhakrishnan and S n e l l , i 9 6 0 ) suggested a c t i v i t y maxima at both pH 6 . 0 and 8 . 0 . Therefore, i n i t i a l assays were ca r r i e d out at both pH values. Later experiments (Desai and Polglase, 1965) established the existence of only one optimal pH (pH 8 . 0 ) f o r t h i s enzyme system and thereafter pH 6 . 0 assays were discontinued. During the course of t h i s work, two assay methods were employed, (a) Method I The e a r l i e r method was modified from the procedure of Umbarger and Brown, ( 1 9 5 8 b ) . Each tube contained i n 2 . 6 ml: - 17 -potassium phosphate, pH 8.0 and pH 6 . 0 , 100 ^umoles; sodium pyruvate, 50yumoles; MgClg, 5^umoles; thiamine pyrophosphate, 0.3/Jmoles; 0 .5 ml. of E. c o l i extract (protein; 2-4 mg. per ml.) and when indicated f l a v i n adenine dinucleotide 10 mumoles. Assay tubes were generally incubated at 37°C fo r 30 minutes (longer periods were used f o r extracts of very low a c t i v i t y or shorter periods for high a c t i v i t y ) . The reaction was stopped by the addition of 0 .5 ml. of 10$ t r i c h l o r o a c e t i c a c i d and the tubes were centrifuged to c l a r i f y the reaction mixtures. To 1.0 ml. of the supernatant solu t i o n was added 0 .05 ml. of 36 N sulphuric acid, and the sample was autoclaved at 10 p . s . i . for 10 minutes to convert the oC-acetolactate to acetoin. The solution was then d i l u t e d to 10 ml. To a 5 ml* aliquot of t h i s solution was added 1.0 ml. of 0.5% creatine In water and 1.0 ml. of f r e s h l y prepared 5% oC-naphthol i n 2 .5 N sodium hydroxide. The solu t i o n was mixed vigorously and the color allowed to develop for one hour i n the dark. The o p t i c a l density at 5^0 mu was read on a Beckman B spectrophotometer. Since the enzyme acetolactate decarboxylase i s not present i n extracts of E. c o l i (Juni, 1952; Umbarger and Brown, 1958b; Radhakrishnan and S n e l l , i960) acetoin production can be a t t r i b u t e d e n t i r e l y to chemical decarboxylation of ^-aceto-l a c t a t e and provides a measure of acetohydroxy a c i d synthetase a c t i v i t y . Determination of the acetoin content of untreated extracts of E. c o l i (not a c i d i f i e d and heated) was accomplished by stopping the reaction mixtures with 0.1 ml. of 10$ zinc sulphate - 18 -and 0.1 ml. of 1 N sodium hydroxide as described by Umbarger and Brown (1958b). Acetoin was determined immediately i n one al i q u o t and a second aliquot was heated following addition of aci d and treated as usual (See Table I I ) . (b) Method II The second procedure used to assay acetohydroxy ac i d synthetase a c t i v i t y was described by Desai and Polglase, (19^5). I t d i f f e r s from the former method primarily i n the concentration of cer t a i n components of the incubation mixture and i n the regular supplementation with PAD. Each tube contained i n 1.0 ml: potassium phosphate, pH 8 . 0 , 100 pnoles; sodium pyruvate, 0 .5 mmoles; MgCl^, 0 . 5 yumole; thiamine pyrophosphate, 45 mumoles; f l a v i n adenine dinucleotide, 10 mumoles; 0 . 5 ml. of E. c o l i extract (prepared i n pH 8 . 0 , 0 .5 M potassium phosphate b u f f e r ) . A f t e r incubation for 15 minutes at 37°C the reaction was stopped by the addition of 0.1 ml. of 50$ t r i c h l o r o a c e t i c a c i d . This was followed by incubation for 15 minutes at 60°C to convert the *<-acetolactate to acetoin. An aliquot of the r e s u l t i n g solution was analysed f o r acetoin by the method of Westerfeld (19^5). Protein was determined by the method of Lowry et a l , (1951) . Results by both methods were expressed i n micromoles of *<-acetolactate formed per mgm. of protein per hour. - 1 9 -( I i ) Measurement of *<-acetohydroxybutyrate-forming a c t i v i t y . <*r-Acetohydroxybutyrate was determined i n a micro-b i o l o g i c a l assay based on the fact that growth i n h i b i t i o n of s t r a i n K - 1 2 of E. c o l i by L-valine i s reversed by L-isoleucine (Tatum, 1 9 4 6 ) or any six-carbon precursor of L-isoleucine (Umbarger, 1 9 5 8 ) . Since <?6-acetohydroxybutyrate i s decarboxylated by b o i l i n g for 5 minutes, the assay was rendered s p e c i f i c for t h i s compound by tes t i n g the extracts before and a f t e r heat treatment. (a) Reversal of L-valine i n h i b i t i o n by L-isoleucine i n E. c o l i K - 1 2 . A series of tubes ( 1 2 x 2 0 0 mm.) were prepared con-taining, i n glucose-salts medium, L-valine, 0.42 ^ umoles per ml. ( 5 0 yagm), and L-isoleucine i n s e r i a l d i l u t i o n s ranging from 0 . 3 8 to 0 . 0 1 2 paoles ( 5 0 to 1 . 5 ^gm) per ml., i n a f i n a l volume of 5 . 0 ml. A control tube containing only L-valine ( 0 . 4 2 ^ umoles per ml.) was also prepared. A l l tubes were inoculated with two loopfuls of E. c o l i K - 1 2 previously grown overnight i n amino acid-free basal medium at 3 7 ° C . The inoculated tubes were then incubated at 3 7 ° C f or three days. Relative t u r b i d i t i e s were recorded at various i n t e r v a l s during t h i s period (See Table VII). (b) Preparation of assay plates. The method employed was modified from the procedure o r i g i n a l l y described by Le a v i t t and Umbarger ( i 9 6 0 ) . The assay organism (E. c o l i K - 1 2 ) was grown overnight on glucose-salts medium. The culture was d i l u t e d a s e p t i c a l l y with l i q u i d medium - 2 0 -to an op t i c a l density (O.D. ^ 2Q mu^  o f a P P r o x i m a t e l y 1 . 0 and stored at 5°C. This culture could be maintained at that temperature for upto 5 days with consistent r e s u l t s . The s o l i d medium (L-valine agar) contained 1 . 5 $ agar and was supplemented with 0.42 ^ umoles ( 5 0 jigm) of L-valine per ml. To seed the agar 0 . 1 ml. of the culture was mixed with 1 0 ml. of the melted valine agar previously cooled to 4 5 ° C . The seeded agar was poured into a p l a s t i c P e t r i dish ( 2 0 x 6 0 mm.), rotated to d i s t r i b u t e the organisms, and allowed to s o l i d i f y . A second layer of seeded agar i d e n t i c a l l y prepared was then poured on top of the f i r s t l ayer and a porcelain assay cylinder (Penicylinder, 8 x 1 0 mm., Fisher S c i e n t i f i c Co. Ltd., Edmonton, Canada) was dropped into the upper l i q u i d l a y e r . A f t e r the second layer had s o l i d i f i e d 0 . 1 ml. of the test sample was placed i n the cup. The assay plates were incubated at 3 7°C for 1 6 hours. When a reaction mixture was assayed the cylinders were modified by i n s e r t i n g into the bottom of each a disk of Whatman # 3 f i l t e r paper s l i g h t l y larger i n diameter than the cylinder. These disks serve to l o c a l i z e any pre c i p i t a t e d protein or b a c t e r i a l contamination introduced along with the reaction mixture, (c) Enzymatic formation ofc<-acetohydroxybutyrate. B a c t e r i a l extracts were prepared as previously described. Each incubation tube contained i n a f i n a l volume of 1 . 0 ml; sodium pyruvate, 1 0 ^ umoles; o<-ketobutyrate, 5 ,umoles; MgCl^, 1 0 ^umoles; thiamine pyrophosphate, 1 7 5 ^mumoles; f l a v i n adenine - 21 -dinucleotide, 10 mjumoles; potassium phosphate 100 ^umoles (pH 8.0) and 0.5 ml. of extract protein (prepared i n pH 8.0 b u f f e r ) . A f t e r 15 minutes incubation at 37°C, the reaction was stopped by the addition of 0.1 ml. each of 10$ ZnSO^ and N NaOH. The p r e c i p i t a t e d protein was removed by low speed centrifugation and the supernatant solution (or an aliquot thereof) was placed i n the cup of the assay plate. Incubation mixtures were generally assayed i n duplicate or i n t r i p l i c a t e . (d) Estimation of b a c t e r i a l growth. At the end of the incubation period zones of growth surrounded those cups which had contained <?<-acetohydroxybutyrate. The cylinders were removed and the agar which contained growth was cut out with a cork bore of a diameter s l i g h t l y l a r g e r than the zones of growth. The agar plug was dropped i n t o a 12 ml. tapered centrifuge tube containing 0.5 ml. of d i s t i l l e d water. The contents of the tube were heated above 96 C for 90 seconds and then treated with 0.7 ml. of 0.5 N hydrochloric a c i d . The tube was then heated f o r an add i t i o n a l 60 seconds to hydrolyse the agar. Before cooling, the tubes were centrifuged (Servall) at 3^00 x g f o r 8 minutes. The supernatant was discarded and the p r e c i p i t a t e was washed i n 2.0 ml. of d i s t i l l e d water. The washed protein was dissolved i n 0.5 ml* of 5% sodium carbonate i n 0.1 N sodium hydroxide and estimated by the method of Lowry, et a l , (1951). - 22 -(e) Preparation of a standard curve. L-Isoleucine was used as a standard rather than the intermediate, << -acetohydroxybutyrate. Points on the standard curve were derived by averaging values obtained from several independent experiments (See Table IV). (iii) Reference enzymes. (a) Glucokinase Glucokinase activity was determined spectrophoto-metrically at room temperature by observing the change in optical density at 340 mu (Cary 15 spectrophotometer, Applied Physics Corporation, Monrovia, California). The system contained the following in a volume of l.o ml: glucose, 4 jumoles; adenosine triphosphate, 2 yumoles; MgCl^ j 4.5yumoles; nicotinamide adenine dinucleotide phosphate (NADP+) 100 ^umoles; glucose-6-phosphate dehydrogenase (C.F. Boehringer and Soehne, Mannheim, Germany), 1 unit; tris (hydroxymethyl) aminomethane pH 7.0, 100 /imoles. The reaction was initiated by the addition of 0.1 ml. of cell extract (prepared as described above, pH 7*0). Activities are expressed as millimicromoles of coenzyme reduced per milligram of protein per minute. (b) Glutamic dehydrogenase For the determination of glutamic dehydrogenase the following solution was prepared: tris (hydroxymethyl) amino-methane, 50 yumoles (pH 7&) i ^ -ketoglutarate, 3 jumoles; ammonium sulfate, 40 ^umoles; reduced nicotinamide adenine dinucleotide phosphate (NADPH) 150 mumoles. To 1.0 ml. of this solution in - 23 -a cuvette was added 0.1 ml. of b a c t e r i a l c e l l extract and the decrease i n o p t i c a l density at 3^0 mp. was recorded at 25°C i n the Cary 15 spectrophotometer. S p e c i f i c a c t i v i t i e s are expressed as millimicromoles of coenzyme oxidized per milligram of protein per minute. C. RESULTS I« Streptomycin S e n s i t i v i t y of the Cultures The s e n s i t i v i t y of streptomycin-sensitive (S) strains to the a n t i b i o t i c i s shown i n Table I. The revertant obtained from the dependent parent (DA) i s s l i g h t l y l e s s sensitive than wild type s t r a i n s . I I . S p e c i f i c i t y of the Method for <<-Acetolaotate Determination Since c<-acetolactate formation by c e l l free b a c t e r i a l extracts i s determined c o l o r i m e t r i c a l l y by measuring the concen-t r a t i o n of the decarboxylated product, acetoin, i t should be established that i n E. c o l i , acetoin i s not a normal end-product. Table II gives the r e s u l t s of <?<-acetolactate determination on the streptomycin-dependent mutant DE when the incubation mixtures are stopped under d i f f e r e n t conditions. I t should be noted that acetoin produced a f t e r a c i d i f i c a t i o n and heating appears to represent the t o t a l e<-acetolactate formed during the enzyme reaction (See Discussion). This supports data published on other strains of E. c o l i (Juni, 1952; Radhakrishnan and S n e l l , I960; Umbarger and Brown, 1958b). For t h i s reason, a l l ^ - a c e t o l a c t a t e - 24 -TABLE I R e l a t i v e S e n s i t i v i t i e s of E s c h e r i c h i a C o l i S e n s i t i v e S t r a i n s to Dihydrostreptomycin. S t r a i n Growth-*" i n TDHSMI yugm per ml. i t i 1x2. L° 1 2 ^ 11 20 SA ++++ ++ t r 0 0 0 SC ++++ 0 0 0 0 SE ++++ 0 0 0 0 SB/r ++++ + 0 0 0 Derived from the dependent mutant (DA) by back-mutation. + Each tube contained 5 ml. of g l u c o s e - s a l t s medium and a constant inoculum. Growth was estimated a f t e r i n c u b a t i o n a t 37°C f o r 24 hours. R e l a t i v e growth i s represented by the s c a l e 0 to ++++ wi t h M t r w i n d i c a t i n g a tr a c e of growth. - 25 -TABLE I I Comparison of Methods f o r Determining <X-Acetolactate i n E x t r a c t s of E s c h e r i c h i a c o l i . R e a c t i o n stopped with TCA , not heated * + TCA , heated ZnSO^, NaOH^ not heated Z h s o ^ , NaOH, a c i d i f i c a t i o n and heated * T r i c h l o r o a c e t i c a c i d , f i n a l c o n c e n t r a t i o n 1.6$. + Heated f o r 10 minutes i n an a u t o c l a v e a t 10 p . s . i . f o l l o w i n g a c i d i f i c a t i o n w i t h 0.05 ml. of 36 N HgSO^ $ As d e s c r i b e d i n " M a t e r i a l s and Methods". umoles of a c e t o i n per  mg. p r o t e i n per hour PH 6.0 pH 8.0 0.319 0.677 0.207 0.582 0.075 0.056 0.235 0.620 - 26 -a s s a y s d e s c r i b e d i n t h i s s t u d y employ a t o t a l a c e t o i n d e t e r m i n a t i o n on r e a c t i o n m i x t u r e s f o l l o w i n g a c i d i f i c a t i o n a n d h e a t t r e a t m e n t . I I I . A c t i v i t y o f t h e g < - A c e t o l a c t a t e - f o r m i n g S y s t e m o f t h e M u t a n t s R e l a t i v e a c e t o h y d r o x y a c i d s y n t h e t a s e a c t i v i t i e s f o r s t r e p t o m y c i n - s e n s i t i v e (S) a n d s t r e p t o m y c i n - d e p e n d e n t (D) m u t a n t s o f E . c o l i a r e g i v e n i n T a b l e I I I . I t c a n be s e e n t h a t a t b o t h pH 6.0 a n d 8.0 t h e D/S r a t i o i s g r e a t e r t h a n u n i t y . I n c o n t r a s t t o t h i s a r e t h e r e s u l t s i n T a b l e I V f o r s t r e p t o m y c i n - r e s i s t a n t m u t a n t s grown i n t h e a b s e n c e o f a n t i b i o t i c (R~ c e l l s ) o r i n a medium s u p p l e m e n t e d w i t h 1000 u n i t s p e r m l . o f d i h y d r o s t r e p t o m y c i n ( R + c e l l s ) . I n t h i s c a s e , t h e R+/R~" r a t i o b o t h a t pH 6.0 a n d 8.0 d e v i a t e s o n l y s l i g h t l y f r o m u n i t y . The e f f e c t o f f l a v i n a d e n i n e d i n u c l e o t i d e (FAD) 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 a c t i v i t y a t pH 8.0 i s g i v e n i n T a b l e V. I t was o b s e r v e d t h a t a l t h o u g h a s u b s t a n t i a l ( s i x f o l d ) i n c r e a s e i n enzyme a c t i v i t y h a d o c c u r r e d i n e x t r a c t s c o n t a i n i n g FAD, t h e D/S r a t i o s o f t h e s e m u t a n t s r e m a i n e d c o n s t a n t . A s a r e s u l t o f t h e e l e v a t e d a c t i v i t y i n t h e p r e s e n c e o f FAD, a l l f u r t h e r 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 d e t e r m i n a t i o n s i n c l u d e d t h i s coenzyme. IV. A c t i v i t y o f t h e o < - A c e t o h y d r o x y b u t y r a t e - f o r m i n g  S y s t e m o f t h e M u t a n t s . I n h i b i t i o n o f t h e g r o w t h o f E . c o l l s t r a i n K-12 by L - v a l i n e a n d r e v e r s a l o f t h i s i n h i b i t i o n by L - i s o l e u c i n e i s - 27 -TABLE III Acetohydroxy a c i d Synthetase A c t i v i t i e s i n Extracts of Streptomysin-Sensitive (S) and -Dependent (D) Escherichia c o l i . + ^amoles of ©(-acetolactate formed per mg. protein per hour. pH 6.0 pH 8.0 S t r a i n Sensitive Dependent D/S Sensitive Dependent D/S B/r 0.286 0.407 1.4 O.256 0.714 2.8 E 0.125 0.207 1.6 0.104 0.582 5.6 G 0.065 0.477 7.3 0.239 0.542 2.3 A 0.355 0.477 1.3 0.334 0.688 2.1 * D/S i s the r a t i o of a c t i v i t i e s i n dependent (D) and sensitive (S) extracts. + Determined by Method I. - 28 -TABLE IV Acetohydroxy Acid Synthetase Activities i n Extracts of Streptomycin-Resistant Escherichia c o l i . umoles of o^-acetolactate formed per mg. protein per hour pH 6 . 0 pH 8 . 0 Strain i T E* R + / R ~ B~_ R * R V R " B/r 0 .251 0 .139 0 . 6 0 .321 0 . 2 8 2 0 . 9 E 0 .167 0 . 183 1.1 0 .192 0 . 1 4 1 0 . 7 C 0 .179 0 . 1 4 0 0 . 8 0 .152 0 . 1 0 8 0 . 7 A 0 .117 0 . 1 1 8 1 .0 0 .152 0 .218 1 .4 R~ indicates extracts from cells grown without added antibiotic. R + indicates extracts from cells grown in medium containing 1,000 units per ml. of dihydrostreptomycin. * Determined by Method I. - 29 -TABLE V E f f e c t of F l a v i n Adenine Dinucleotide on Acetohydroxy acid Synthetase A c t i v i t i e s i n Sensitive (S) and Dependent (D) Extracts of Escherichia c o l i . -' ^umoles wf-acetolactate formed per mg. protein per hour.  Mutant - FAD D/S + FAD* D/S SA 0.438 1.8 2.601 1.7 DA 0.793 4.500 SG 0.188 2.4 1.165 2.5 DC 0.456 2.960 * F l a v i n adenine dinucleotide (FAD) was added to give a f i n a l concentration of 2 ^ gm.per ml. + Determined by Method I - 30 -shown on Table VI. The extreme s e n s i t i v i t y of t h i s i n h i b i t i o n to s p e c i f i c reversal, and only by L-isoleucine or any six-carbon precursor of t h i s amino ac i d , permits the quantitative determination of <?(-acetohydroxybutyrate by estimating r e l a t i v e b a c t e r i a l growth. Table VII shows that growth of E. c o l i s t r a i n K-12 i s due e n t i r e l y to the presence of aC-acetohydroxybutyrate and not to the straight chain five-carbon decarboxylation product ( i n confirmation of the observations of L e a v i t t and Umbarger, i 9 6 0 ) . A standard curve of t o t a l protein versus L-isoleucine concentration as determined by the microbiological assay method previously described i s given i n F i g . 4. Due to the poor r e p r o d u c i b i l i t y of t h i s system each point was derived by averaging values of several experiments. The r e s u l t i n g curve compared favorably with the L-isoleucine standard curve of Umbarger and L e a v i t t ( i 9 6 0 ) . A comparison of£<-acetolactate and 0^-acetohydroxybutyrate formation by c e l l - f r e e extracts prepared from streptomycin-s e n s i t i v e , -dependent and - r e s i s t a n t mutants of E. c o l i i s given i n Table VIII. I t i s i n t e r e s t i n g to note that there was formed approximately 35% lessrt-acetohydroxybutyrate than^-acetolactate i n a l l mutants. V. A c t i v i t y of the Reference Enzymes In Table IX, glucokinase and glutamic dehydrogenase a c t i v i t i e s are shown for mutants of E. c o l i A. The a c t i v i t i e s of both enzymes appeared to remain constant i n a l l mutants. - 31 -0.12 o in H W 3 W Q < V H O 0.10 -0.06 0.04 -0.02 -10 20 muM O L E S 30 40 50 L - I S O L E U C I N E FIG. 4. Growth response of Escherichia c o l i K-12 to increasing concentrations of L-isoleucine. Each point was derived by averaging the re s u l t s of f i v e separate experiments. - 32 -TABLE VI S e n s i t i v i t y of Escherichia o o l i K-12 to L-Valine and Reversal of I n h i b i t i o n by L-Isoleucine. Concentration of L-Isoleucine Relative Turbidity umoles per ml. of medium 20 hours 72 hours 0.380 ++++ ++++ 0 . 1 9 0 +++ ++++ 0 . 0 9 5 +++ ++++ 0.048 +++ +++ 0.024 ++ +++ 0.012 ++ ++ 0.000 0 ' t r A l l tubes contained L-vallne (0.42 umoles per ml.) Growth was estimated a f t e r incubation at 37°G for the period of time indicated. Relative growth i s represented by the scale 0 to ++++ with w t r " i n d i c a t i n g a trace of growth. - 3 3 -TABLE VII S p e c i f i c i t y of the o(-Acetohydroxybutyrate Assay System for the Six-Carbon Intermediate. ^umoles of o<-Acetohydroxybutyrate Reaction stopped with formed per mg. protein per hour ZnSO^, Na0H+ 2.150 TCA, Heated + 0.050 * Calculated from the L-isoleucine standard curve F i g . 4. Extracts assayed at pH 8.0 only. + As described i n "Materials and Methods". $ The reaction was stopped by the addition of. 0.2 ml. of t r i c h l o r o a c e t i c a c i d followed by b o i l i n g for 5 minutes. - 3^ -TABLE VIII Formation of c< -Acetolactate and^-Acetohydroxy butyrate by Sensitive (S), Resistant (R) and Dependent (D) Extracts of Escherichia c o l i . Culture o( -Acetolactate Ratio ^umoles of acetoin formed per mg. +. protein per hour D/X g< -Acetohydroxy butyrate ^umoles of isoleucine formed per mg. protein per hour Ratio D/X DA SA RA" RAH 5.50 3.7^ 2.67 3.64 1.5 2.1 1.5 3.62 2.36 1.85 2.48 1.5 2.0 1.5 DE SE 5.63 2.73 2.1 3.52 1.52 2.3 Determined by Method II (pH 8.0 only). + Calculated from the L-isoleucine standard curve. F i g . 4. $ X refers to cultures other than streptomycin-dependent mutants. BA~ represents r e s i s t a n t c e l l s grown i n the absence of a n t i b i o t i c , while RA + represents the same r e s i s t a n t culture grown i n a medium supplemented with 1000 units of dihydrostreptomycin per ml. - 35 -TABLE IX Reference Enzyme A c t i v i t i e s of Streptomycin Mutants of Escherichia c o l i A. # Glutamic Extract Glucokinase Dehydrogenase Sensitive (SA) 51 120 Dependent (DA) 50 120 Resistant (RA~) + 58 112 Resistant ( R A + ) + 67 122 * A c t i v i t i e s are expressed as millimicromoles of coenzyme (NADP+ or NADPH, respectively) changed per minute per milligram of protein. f RA"" refers to re s i s t a n t c e l l s grown i n a n t i -b i o t i c - f r e e medium, while RA c e l l s were grown with 1,000 units per ml. of dihydrostreptomycin. - 3 6 -D. DISCUSSION I. Genetic Charac t e r i s t i c s of the Streptomycin Mutants Pla t i n g methods employed i n t h i s work readi l y yielded spontaneous mutants exhibiting complete indifference to streptomycin or dihydrostreptomycin although the parent wild types were sensitive to low concentrations (above 3 . 0 yug per ml.) of e i t h e r a n t i b i o t i c (Table I I I ) . While i n d i v i d u a l mutation rates were not calculated, i t became apparent during t h i s work that streptomycin i n d i f f e r e n t colonies were occurring at a r e l a t i v e l y constant frequency from the various sensitive s t r a i n s . Values ranging from 10*" (Newcombe and Hawirko, 19^9) to 10~* (Demerec, 1951) have been suggested for rates of mutation to resistance. These values include the formation of both strepto-mycin r e s i s t a n t and dependent organisms. The occurrence of single step dependent mutants, unlike i n d i f f e r e n t mutants, appeared to fluctuate with the s t r a i n of E. c o l i and form of a n t i b i o t i c employed. E. c o l i s t r a i n B (obtained from the National Research Council of Canada), f o r example, refused to produce dependent mutants on medium containing eith e r streptomycin or the dihydro derivative despite repeated attempts. The same s t r a i n , however, readi l y formed r e s i s t a n t mutants on either form of the a n t i b i o t i c . I t was generally found that the reduced form of the a n t i b i o t i c (dihydrostreptomycin) was consistently more e f f e c t i v e i n i s o l a t i n g dependent mutants than was streptomycin i t s e l f . Once obtained, the mutants responded equally well to eithe r form of the a n t i b i o t i c . I t was c l e a r l y shown by Scott (19^9) that both streptomycin and dihydrostreptomycin are non-mutagenic. Resistant and dependent forms w i l l appear with a c h a r a c t e r i s t i c frequency i n susceptible populations i r r e s p e c t i v e of the presence of the a n t i b i o t i c . At the present time with the l i m i t e d under-standing of the mechanism of action of streptomycin i t i s d i f f i c u l t to explain the enhanced a b i l i t y of the dihydro form to select dependent mutants. A single step back-mutation (reversion) from high l e v e l streptomycin-dependence to streptomycin-sensitivity can be demonstrated i n many strains of dependent E. c o l i . Hashimoto ( i 9 6 0 ) has shown that mutation from dependence to s e n s i t i v i t y i s i n fact not a true reversion but i s mediated by a suppressor  mutation. This suppressor maps close to the locus governing a n t i b i o t i c dependence and high l e v e l resistance and i s capable of modifying the expression of either a l l e l e . I t i s obvious from Table I that the revertant (SA) i s not quite as sensi t i v e to dihydrostreptomycin as are the three wild type sensitive cultures. This suggests that suppression of the dependent locus i s incomplete and the r e s u l t i n g sensitive progeny acquire a low l e v e l r e s i s t a n t phenotype as demonstrated by t h e i r a b i l i t y to grow on s l i g h t l y elevated streptomycin concentrations. Charac t e r i s t i c s of the corresponding r e s i s t a n t mutant (RA) w i l l be discussed l a t e r . The rate of back-mutation has been studied by Bertani( 1 9 5 1 ) and his r e s u l t s indicate that f o r a given s t r a i n the mutation rate i s constant and ranges as high as 10*" per bacterium per d i v i s i o n . Various s t r a i n s , however, d i f f e r markedly i n t h e i r a b i l i t y to revert. In addition to reversion from dependence to s e n s i t i v i t y , c e r t a i n strains of dependent E. c o l i apparently are capable of converting to high l e v e l resistance i f stored under unfavorable conditions i n the presence of a n t i b i o t i c . This a l t e r a t i o n i n phenotype i s spontaneous and probably i s induced by a suppressor mutation (Hashimoto, i960) or a modifier mutation (Matney, et a l , i 9 6 0 ) . Due to the i n s t a b i l i t y of many dependent mutants a l l strains were subcultured monthly and the state of each was determined p r i o r to experimentation. * n E. c o l i . genetic analysis has shown that s e n s i t i v i t y , dependence and single-step high l e v e l resistance are determined by multiple a l l e l e s at a single locus known as the "Sm locus" (Newcombe and Nyholm, 1950; Hashimoto, i 9 6 0 ) . Consideration of t h i s genetic fact, along with numerous physiological and bio-chemical observations, l e d Spotts and Stanier ( I 9 6 I ) to propose a unitary hypothesis of streptomycin action. They suggested that the three phenotypes were ultimately determined by s t r u c t u r a l modification of a " s p e c i f i c " protein at a " s i n g l e " i n t r a c e l l u l a r s i t e . Streptomycin would bind r e v e r s i b l y /.with t h i s protein and depending on the structure of the receptor s i t e may or may not influence the function of the c e l l . The ribosomes were - 39 -proposed as the streptomycin binding s i t e s . The sensitive ribosome was pictured as possessing a structure that conferred on i t a very high a f f i n i t y f o r streptomycin. The r e s u l t i n g combination prevented the attachment of m-RNA, thus i n h i b i t i n g protein synthesis. The corresponding structures of the r e s i s t a n t and dependent ribosomes were such that streptomycin had no e f f e c t on the former and i t s presence was obligatory f o r the l a t t e r to function normally. This hypothesis emphasized the fact that only a single s i t e , the ribosome, was al t e r e d during mutation and that the streptomycin dependent c e l l i n the presence of a supra-c r i t i c a l l e v e l of a n t i b i o t i c (> 250 units per ml.) did not d i f f e r s i g n i f i c a n t l y from the corresponding sensitive s t r a i n grown under optimal conditions (Spotts, 1962). The discovery of ninhydrin-p o s i t i v e material present i n culture supernatants of streptomycin-dependent mutants but absent from the corresponding f l u i d s of -se n s i t i v e and -re s i s t a n t cultures l e d Bragg and Polglase (1962) to an inv e s t i g a t i o n of metabolic differences i n the three pheno-types. Their r e s u l t s (Bragg and Polglase, 1964a; 1964b) indicated that streptomycin-dependent mutants, unlike the -s e n s i t i v e and -r e s i s t a n t forms, underwent s i g n i f i c a n t changes at the l e v e l of pyruvate metabolism i n response to a l t e r a t i o n s i n streptomycin concentrations and aerobic conditions. This suggests an impair-ment i n r e s p i r a t i o n or streptomycin action at a s i t e other than the ribosome. Recently, (Cox, et a l , 1964; Davies, 1964) i t has been shown by means of sucrose gradient centrifugation that streptomycin does not prevent the attachment of m-RNA to the sensitive ribosome. Data presented in this thesis, as well as more current work from this laboratory lend further support to the steadily growing pool of evidence that the unitary hypothesis of Spotts and Stanier i s less than adequate to explain the physiological and biochemical responses to streptomycin. I I . Formation ofo<-Acetolactate by Streptomycin Mutants The synthesis of -acetolactate by cell-free bacterial preparations was determined colorimetrically by estimating the decarboxylated product, acetoin, by the method of Westerfeld ( 1 9 4 5 ) . The original color reaction was shown by Voges and Proskauer (I898) to be due to the reaction between diacetyl or acetoin and a guanidino group in the presence of a l k a l i . Attempts to increase the sensitivity of this reaction led to the addition of creatine and *-naphthol. The standard curve was linear for concentrations as high as <10 jugm-per ml. but deviated slightly from this relationship at greater concentrations. The Westerfeld method i s nearly specific for acetoin and diacetyl. Related 5-°arbon ketols give a somewhat similar color, while the 6-carbon ketols give a light olive-green color within the time limi t employed. The sensitivity of these analogs, however, i s 1 0 to 1 0 0 fold lower than either diacetyl or acetoin (Green, et a l , 1 9 4 2 ) . Color contributed by diacetyl can be distinguished from acetoin by i t s rate of formation. - 41 -Color development due to d i a c e t y l i s generally complete within 10-15 minutes, while the color complex due to acetoin reaches maximum i n t e n s i t y only a f t e r one hour. I t should be noted that under the conditions required to decarboxylatec>£-acetolactate (acid and heat) i t i s possible to oxidize small quantities of acetoin to d i a c e t y l (Westerfeld, 1 9 ^ 5 ) , hence early color formation does not necessarily indicate endogenous d i a c e t y l . With the exception of assay mixtures containing high concen-trations o f X - a c e t o l a c t a t e early color development was not observed i n t h i s work. Therefore, i t seems safe to conclude that the t o t a l color derived from these reaction mixtures i s due to e< -acetolactate production. Although the enzyme acetolactate decarboxylase i s not present i n extracts of E. c o l i (Juni, 1 9 5 2 ) , enzyme reaction mixtures s t i l l showed a low but s i g n i f i c a n t acetoin l e v e l , even when the reactions were stopped under conditions chosen to prevent chemical decarboxylation (Table I I , l i n e 3 ) . I t i s probable that t h i s acetoin arose from a spontaneous decomposition of ^ - a c e t o l a c t a t e caused by incubation at 37°C since -aceto-l a c t a t e i s known to be r e l a t i v e l y heat l a b i l e even at t h i s temperature (Umbarger and Brown, 1 9 5 8 h ). Consequently, values obtained under these conditions were not subtracted from values for t o t a l acetoin. Reaction mixtures treated with both a c i d and heat (Method I) generally resulted i n acetoin values 10 - 15$ lower than mixtures treated, with acid alone (Table I I , l i n e s 1 and 2) - 4 2 -and 5 - 1 0 $ lower than mixtures treated with heat i n the presence of zinc sulphate and sodium hydroxide ( l i n e 4 ) . This strongly suggests that the severe conditions (autoclaved for 10 minutes at 10 p . s . i . at an a c i d i c pH) employed for complete decarboxy-l a t i o n also destroyed a small quantity of the color complexing material i n the reaction mixture. This observation l e d to the use of milder decarboxylation conditions as described under Method I I . I t i s evident from the data of Tables III and IV, that at pH 8 . 0 the sensitive and r e s i s t a n t mutants (grown with or without dihydrostreptomycin supplementation) of a l l strains exhibited s i m i l a r acetohydroxy a c i d synthetase a c t i v i t i e s whereas, the corresponding dependent mutants possess a c t i v i t i e s two to f i v e times greater. At pH 6 . 0 t h i s r e l a t i o n s h i p was l e s s j dramatic but the D/S r a t i o exceeded the R /R r a t i o i n a l l s t r a i n s . The degree of de-repression of acetohydroxy a c i d synthetase i n the various dependent strains was not i d e n t i c a l and t h i s difference may be explained s o l e l y by s t r a i n v a r i a b i l i t y i n i t s response to streptomycin. The enzyme a c t i v i t i e s of the sensitive and dependent mutants of E. c o l i s t r a i n A require p a r t i c u l a r note (Table I I I , l i n e 4 , and Table V, l i n e s 1 and 2 ) since the sensitive s t r a i n (SA) was derived as a revertant of the dependent s t r a i n . In t h i s case back-mutation from streptomycin-dependence to - s e n s i t i v i t y was accompanied by a decrease i n acetohydroxy acid synthetase a c t i v i t y at pH 6 . 0 and at pH 8 . 0 . This repression of enzyme a c t i v i t y r e s u l t i n g from reversion appears to be incomplete and leaves the sensitive revertant with p a r t i a l streptomycin-dependent properties as Indicated by the s l i g h t l y elevated acetohydroxy a c i d synthetase a c t i v i t y and the low D/S r a t i o s (Table III and Table V). I t was previously pointed out that the growth of t h i s mutant (SA) i n the presence of low a n t i b i o t i c concentrations (Table I) suggested i t might also possess streptomycin-resistant tendencies. The fact that the acetohydroxy a c i d synthetase l e v e l i s de-repressed upon mutation from streptomycin s e n s i t i v i t y to dependence and r e v e r s i b l y re-repressed during back-mutation to the s e n s i t i v e state, strongly suggests that genetic control of t h i s enzyme i s li n k e d to the locus of streptomycin dependence. Umbarger and Brown ( 1 9 5 8 b ) reported a peculiar lack of l i n e a r response i n the formation of ^-acetolactate as a junction of extract concentration. A f t e r the completion of the present work, a cofactor, the addition of which corrected t h i s anomalous behavior (Leavitt, 1 9 6 4 ) was i s o l a t e d and i d e n t i f i e d as f l a v i n adenine dinucleotide (Bauerle, et a l , 1 9 6 4 ) . Further experiments were therefore immediately ca r r i e d out to determine whether or not the addition of FAD to the reaction system al t e r e d the enzyme a c t i v i t y and/or the r e l a t i o n s h i p between the sensitive and dependent s t r a i n s . The data of Table V c l e a r l y indicates that although c<-acetolactate-formation i s stimulated six to seven f o l d i n the presence of t h i s cofactor, the D/S r a t i o i n the presence or absence of FAD remained unchanged. The r o l e which FAD plays i n t h i s reaction sequence i s unknown. However, recently Hogg, et a l , (1965) have reported finding i r o n (Fe"**) associated with, t h i s enzyme complex and suggest that i t s function may involve the coenzyme. In order to eliminate the p o s s i b i l i t y that the elevated l e v e l of acetohydroxy acid synthetase was due to a general stimulation of enzyme formation i n the dependent mutant, other enzymes were studied i n the three mutants of s t r a i n A. I t i s obvious from the data of Table IX that mutation from a n t i b i o t i c dependence to s e n s i t i v i t y or dependence to resistance has no e f f e c t on glucokinase or glutamic dehydrogenase a c t i v i t y . Hence, while the involvement of other enzymes i s not excluded, the increased acetohydroxy a c i d synthetase a c t i v i t y cannot be a t t r i -buted to a general de-repression of enzymic a c t i v i t y i n the dependent mutant. In t h i s connection, i t should also be noted that c a l c u l a t i o n of s p e c i f i c a c t i v i t i e s per mgm. of protein excludes the p o s s i b i l i t y that de-repression of acetohydroxy a c i d synthetase i s a non-specific event. Concurrent work from t h i s laboratory suggests that the enzymes reductoisomerase (Lau, I966) and Transaminase B (Unpublished Observation) which catalyse subsequent steps i n the biosynthetic pathway are not de-repressed i n the dependent organism. The dihydroxy a c i d dehydrase has not yet been studied. Therefore, the evidence accumulated thus f a r i n f e r s that the - 45 -excretion of L-valine (and L-leucine) from streptomycin-dependent mutants of E. c o l i r e s u l t s s o l e l y from a de-repression of the control enzyme, acetohydroxy a c i d synthetase and not of the subsequent biosynthetic enzymes. The fact that only trace quantities of L-leucine are detected i n the supernatant f l u i d s (Bragg and Polglase, 1 9 6 2 ; Tirunarayanan, et a l , 1 9 6 2 ) suggests some feedback mechanism controls the drain of e <-ketois oval era te to the 6-carbon product (Preundlich, et a l , 1 9 6 2 ) . I I I . Formation of o<.-Acetohydroxybutyrate by Streptomycin Mutants As indicated i n F i g . 2 and F i g . 3 the enzyme aceto-hydroxy a c i d synthetase i s involved not only with the synthesis of L-valine and L-leucine but also with the synthesis of L-isoleucine v i a the intermediate ^ -acetohydroxybutyrate. Since there i s no evidence for the excretion of L-isoleucine by dependent mutants, i t was important to determine whether an elevated ^-acetohydroxybutyrate l e v e l i s formed i n response to the de-repression of acetohydroxy a c i d synthetase. I f aceto-hydroxy acid synthetase stimulation i s due to an increased rate of enzyme formation rather than to an e f f e c t on the k i n e t i c s of the enzyme, then the D/S r a t i o f o r <<-acetolactate production should correspond to the D/S r a t i o for synthesis of *<-aceto-hydroxybutyrate. As mentioned i n the "Materials and Methods", the only quantitative assay method sensitive enough to measure the l e v e l - 4 6 -of ©c-acetohydroxybutyrate formed i n t h i s system was the micro-b i o l o g i c a l assay described by L e a v i t t and Umbarger ( i 9 6 0 ) . This procedure measures the degree to which i n h i b i t i o n of E. c o l i s t r a i n K-12 by L-valine can be reversed by L-isoleucine or precursors of t h i s compound. Umbarger and Brown (1955) noted that L-isoleucine was a non-competitive antagonist of L-valine, as would be expected i f L-isoleucine served to restore a deficiency created by interference with i t s biosynthesis. Furthermore, while the 6-carbon precursors of L-isoleucine, eK-keto ^-methylvalerate and ©<, (3dihydroxy (3-methylvalerate reversed i n h i b i t i o n as well as did L-isoleucine i t s e l f , a 4-carbon precursor, <*.-ketobutyrate was i n e f f e c t i v e . This suggested that the pathway was blocked at the acetohydroxy a c i d synthetase step. Since the acetohydroxy a c i d synthetase of s t r a i n K-12 i s extremely sensitive to feedback by L-valine (Umbarger and Brown, 1958a; L e a v i t t and Umbarger, 1962) the addition of L-valine i n the absence of L-isoleucine would simultaneously i n h i b i t the synthesis of both oi-acetolactate and *<-acetohydroxybutyrate and ultimately would prevent the growth of the organism as a consequence of starvation for L-isoleucine. To establish the s e n s i t i v i t y of the E. c o l i K-12 culture to L-valine and the r e v e r s i b i l i t y of t h i s i n h i b i t i o n by L-isoleucine, a growth experiment was c a r r i e d out, the r e s u l t s of which are given i n Table VI. In the absence of L-isoleucine growth was n e g l i g i b l e even a f t e r 72 hours. However, the addition of L-isoleucine at concentrations considerably lower (35 fold) than the i n h i b i t o r , L-valine, permitted s i g n i f i c a n t growth. Technical problems associated with a microbiological assay of t h i s type on occasion gave e r r a t i c r e s u l t s p a r t i c u l a r l y during the determination of the standard curve. For t h i s reason, the standard curve employed i n t h i s work was a composite p l o t of values obtained i n several separate experiments each c a r r i e d out i n duplicate. The standard curve i s shown i n F i g . 4 . I t was reported by L e a v i t t and Umbarger ( i 9 6 0 ) that at low concen-trations (upto 0.02^umoles)c<-acetohydroxybutyrate and L-isoleucine stimulate the growth of i n h i b i t e d E. c o l i to the same extent. Therefore, the end'-product rather than the intermediate was used as a standard and the reaction mixtures were d i l u t e d to give concentrations of ©<-acetohydroxybutyrate which would f a l l on the l i n e a r portion of the standard curve. In addition, extracts were routinely assayed i n t r i p l i c a t e ( i n i t i a l l y i n duplicate) i n order to reduce systematic error. Sinceu-acetohydroxybutyrate i s decarboxylated by b o i l i n g f o r 5 minutes, the assay was rendered s p e c i f i c f o r t h i s compound by testing each sample before and a f t e r heat treatment. I t can be seen from Table VII that the growth stimulated by o<-acetohydroxybutyrate i s 5 0-fold greater than that stimulated by the remainder of the intermediates and by L-isoleucine i t s e l f . These data are supported by the work of Umbarger, et a l ( i 9 6 0 ) . They found that even with crude extracts which were capable of - 48 -converting ©(-acetohydroxybutyrate to l a t e r intermediates i n the L-isoleucine pathway, no other L-isoleucine precursors were detected as products unless NADPH had also been added to the reaction system. Table VIII c l e a r l y indicates that the D/X r a t i o s of acetohydroxy acid synthetase are constant, regardless of whether the a c t i v i t y i s determined by the formation of «<-acetolactate or *f-acetohydroxybutyrate. I t i s i n t e r e s t i n g to note that the re s i s t a n t mutant of s t r a i n A i n the presence of dihydrostreptomycin consistently exhibited a higher enzyme l e v e l than did the same mutant i n the absence of a n t i b i o t i c (see Tables IV and VI I I ) . Although t h i s phenomenon, which has already been mentioned i n regard to the sensitive (SA) revertant, cannot be explained e n t i r e l y , i t probably a r i s e s as a consequence of "incomplete suppression" of the dependent locus during reversion (Hashimoto, i 9 6 0 ) . Enzyme a c t i v i t i e s as determined by the formation of ^-acetohydroxybutyrate were lower by 30-36$ for s t r a i n A and 37-44$ for s t r a i n E than enzyme a c t i v i t i e s determined by the formation of the shorter-chained intermediate, *<-acetolactate. Umbarger and Brown (1958b) reported s i m i l a r diminished enzyme a c t i v i t y foro<-acetohydroxybutyrate formation i n extracts of E. c o l i s t r a i n K-12. I t i s d i f f i c u l t to evaluate the sign i f i c a n c e of these figures with respect to r e l a t i v e substrate s p e c i f i c i t y , due to the unique substrate requirements of t h i s enzyme complex. Since -acetolactate formation involves the condensation of two moles of pyruvate and <?<f-acetohydroxybutyrate formation requires one mole each of ^ "-ketobutyrate and pyruvate, i t i s not possible even i n the presence of a high *C-ketobutyrate concentration to estimate the proportion of pyruvate entering each product. Wagner, et a l , (1965) n a v e studied the i n v i t r o synthesis of L-valine and L-isoleucine by p a r t i c u l a t e f r a c t i o n s of Neurospora crassa. Their r e s u l t s indicate that pyruvate and <*-ketobutyrate d e f i n i t e l y compete f o r the c a t a l y t i c s i t e on the condensing enzyme. In addition, L-isoleucine formation increases l i n e a r l y with increasing <*-ketobutyrate concentration but never exceeds 70$ of the L-valine forming capacity of the preparation. They in t e r p r e t t h i s as meaning that the "active acetaldehyde" (from pyruvate) i s synthesized and reacts with the two keto-acids at a common active s i t e on the enzyme. Hence, supra-c r i t i c a l concentrations of^-ketobutyrate would i n h i b i t the formation of the active acetaldehyde and eventually the synthesis of both L-valine and L-isoleucine. For t h i s reason they suggest there must be a regulatory mechanism i n vivo c o n t r o l l i n g the o<-ketobutyrate l e v e l . The evidence accumulated thus far suggests the enzyme threonine dehydratase (Umbarger and Brown, 1958a; Changeux, I 9 6 I ) . Therefore, i t seems r e l a t i v e l y safe to conclude that although acetohydroxy acid synthetase i s de-repressed with respect to K-acetohydroxybutyrate formation i n the dependent mutant, L-isoleucine does not accumulate i n vivo due to the control of - 50 -the *<-ketobutyrate l e v e l a t a second p o i n t . I f enzyme de-r e p r e s s i o n i s not accompanied by increased amounts of *<-keto-butyrate the elevated pyruvate l e v e l (Bragg and P o l g l a s e , 1962; 1964a) w i l l r a p i d l y be converted to e<-acetolactate and u l t i m a t e l y to L - v a l i n e . IV. S i g n i f i c a n c e of Acetohydroxy A c i d Synthetase De-repression  i n Dependent Mutants of E s c h e r i c h i a c o l i . Previous s t u d i e s i n t h i s l a b o r a t o r y (Bragg and P o l g l a s e , 1962; 1963a; 1963b; 1963c) concerning the involvement of s t r e p -tomycin w i t h the metabolism of streptomycin mutants, have i m p l i c a t e d t h i s a n t i b i o t i c w i t h two phenomena: (1) the e x c r e 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 ; and (2) the impairment of c e r t a i n o x i d a t i v e processes. I t i s i n t e r e s t i n g t h e r e f o r e to consider the r e s u l t s of the present work i n terms of a p o s s i b l e e x p l a n a t i o n f o r the abnormal metabolic behavior e x h i b i t e d by the v a r i o u s streptomycin mutants. I n general, streptomycin s e n s i t i v e and r e s i s t a n t s t r a i n s °f E. c o l i demonstrate no metabolic i r r e g u l a r i t i e s when grown a e r o b i c a l l y on glucose i n the absence of a n t i b i o t i c . The a d d i t i o n of streptomycin to e x p o n e n t i a l l y growing s e n s i t i v e c u l t u r e s however, i s u s u a l l y accompanied by an immediate c e s s a t i o n of exponential growth followed by the l i b e r 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 . The major e x c r e t i o n product i s pyruvate w i t h l e s s e r q u a n t i t i e s of L-alanine and L - v a l i n e (Bragg and P o l g l a s e , 1962). - 51 -The formation of the amino acids can be d i r e c t l y related to the increased amounts of pyruvate, and L-alanine by d i r e c t amina-t i o n and L-valine v i a <X-acetolactate and reduction. Streptomycin also reportedly i n h i b i t s oxidative phosphorylation i n sensitive organisms (Bragg and Polglase, 1963d). The r e s i s t a n t mutant, when grown i n the presence of a n t i b i o t i c (1000 units per ml.), produced elevated l e v e l s of both pyruvate and lactate (Bragg and Polglase, 1962). This observation supported e a r l i e r observations by Rosanoff and Sevag (1953). This suggests that the res i s t a n t organism u t i l i z e s pathways of anaerobic metabolism when grown i n the presence of a n t i b i o t i c . However, no i n h i b i t i o n of oxidative phosphorylation could be demonstrated i n the re s i s t a n t extracts. As previously pointed out, neither the sensitive nor re s i s t a n t organisms exhibited elevated acetohydroxy acid synthetase a c t i v i t i e s whether i n the presence or absence of streptomycin. The streptomycin dependent mutant accumulates and excretes L-valine during oxidation of glucose i f two environmental requirements are s a t i s f i e d . F i r s t , streptomycin or dihydrostrep-tomycin must be present and second aeration i s e s s e n t i a l . Growth under oxygen or a n t i b i o t i c deprivation r e s u l t s i n the production of l a c t a t e from glucose instead of L-valine (Bragg and Polglase, 1964a). Subsequent work showed that the addition of streptomycin to depleted c e l l s i n i t i a t e d a rapid formation of L-valine with a concurrent decrease i n pyruvate and lactate accumulation. I t has - 52 -recently been shown that the l e v e l of acetohydroxy acid synthetase i n streptomycin-depleted-dependent c e l l s i s repressed but increases l i n e a r l y upon the addition of a n t i b i o t i c to a l e v e l several f o l d greater than that of the corresponding sensi t i v e and r e s i s t a n t strains (Polglase, i n press). Therefore, i n t h i s mutant, streptomycin appears to stimulate the de-repression of acetohydroxy a c i d synthetase i n order to a l l e v i a t e the accumulation of metabolites which a r i s e as a r e s u l t of an a l t e r a t i o n i n aerobic metabolism. Although the actual s i t e of the metabolic impairment remains unknown, Bragg and Polglase ( 1 9 6 3 c ; 1963d) have presented data which advocates the electron-transport chain as a s i t e of action of streptomycin. In the dependent mutant, streptomycin (or dihydrostreptomycin) may act to maintain the i n t e g r i t y of an "alternate electron-transport system" which u t i l i z e s l a c t a t e , under conditions of a n t i b i o t i c or oxygen starvation or L-valine, under aerobic, a n t i b i o t i c -supplemented conditions as terminal hydrogenacceptors. In such a scheme de-repression of acetohydroxy a c i d synthetase and hence stimulation of L-valine synthesis could serve two functions. F i r s t , pyruvate which may accumulate due to modifications i n terminal oxidation can be e f f i c i e n t l y removed (two moles of pyruvate per mole of «<-acetolactate) and converted to a near neutral end-product. Second, since the formation of L-valine requires NADPH, the stimulation of this pathway w i l l help to maintain the l e v e l of oxidized coenzyme essential for oxidative - 53 -metabolism. I t has been reported (Bragg and Polglase, 1964b) that the i s o c i t r i c dehydrogenase (NADPH enzyme) l e v e l i n supple-mented-dependent c e l l s was several f o l d greater than i n the depleted c e l l s . At the present stage of experimental in v e s t i g a -t i o n , i t i s d i f f i c u l t to ascertain the primary function of t h i s stimulated pathway, but i t i s known that the i n t r a c e l l u l a r accumulation of "catabolites" of glucose metabolism can impair the normal control of cert a i n inducible enzymes (Monod, 1947; Neidhardt and Magasanik, 1956; Mandelstam, 1961; 1962), a s i t u a t i o n which may become deleterious to the organism. Related studies (Hepner, 1966) on streptomycin-sensitive, - r e s i s t a n t and -dependent mutants of Aerobacter aerogenes, have demonstrated a corresponding de-repression of the acetohydroxy acid synthetase i n dependent organisms of t h i s species. However, since t h i s organism possesses the enzyme acetolactate decarboxylase (June, 1952) the terminal products are acetoin and 2,3 butylene g l y c o l rather than L-valine. Rosenkranz (1963) has reported de-repression of a l k a l i n e phosphatase i n streptomycin-dependent Escherichia c o l i . Although h i s i n t e r p r e t a t i o n of t h i s phenomenon d i f f e r s somewhat from the one presented here, i t i s consistent with the idea that one role of the a n t i b i o t i c i n the dependent organism i s that of an enzyme "de-repressor" (Jacob and Monod, 1961). The regions of the b a c t e r i a l c e l l concerned with carbo-hydrate metabolism are not the only areas presently being studied - 5 4 -i n an attempt to elucidate the primary s i t e of streptomycin act i o n . Evidence (Flax, et a l , 1 9 6 2 a ; Speyer, et a l , 1 9 6 2 ; Davies, 1 9 6 4 ; Pestka, et a l , 1 9 6 5 ) accumulated over the past f i v e years p a r t i a l l y supports the "unitary hypothesis" of Spotts and Stanier ( 1 9 6 2 ) that the ribosome i s the region of the sensitive c e l l p r i n c i p a l l y affected by streptomycin. The addition of low l e v e l s of a n t i b i o t i c to an i n v i t r o protein synthesizing system containing sensitive ribosomes generally leads to a misincorpora-14 t i o n of C -amino acids with the r e s u l t i n g formation of "nonsense  protein" (Davies, et a l , 1 9 6 4 ) . I t was suggested that i n the streptomycin-sensitive organism t h i s protein floods the c e l l and i r r e v e r s i b l y i n h i b i t s c e l l d i v i s i o n . Streptomycin has no. e f f e c t on the r e s i s t a n t ribosome under the same conditions (Flax, et a l , 1 9 6 2 b ) . I t i s important to note that ribosomes prepared from streptomycin-dependent c e l l s have never been shown to demonstrate a functional dependence on streptomycin. In f a c t , amino a c i d incorporation and streptomycin binding studies indicate that the 3 0 S subunit of the r e s i s t a n t and dependent ribosomes are i d e n t i c a l (Flax, et a l , 1 9 6 2 b ; Cox, et a l , 1 9 6 4 ) . At the present time i t i s d i f f i c u l t to derive a single hypothesis capable of explaining the numerous genetic, physio-l o g i c a l and biochemical observations which have been reported on streptomycin mutants. Since a number of chemical lesions at divergent s i t e s of the b a c t e r i a l genome can give r i s e to various streptomycin-resistant phenotypes and at l e a s t one dependent phenotype and since many workers inadequately describe the c h a r a c t e r i s t i c s of t h e i r mutants and the conditions under which they were obtained, i t i s d i f f i c u l t to ascertain whether equivalent mutants have been studied. I t i s highly probable that not only do the phenotype of the r e s i s t a n t mutants d i f f e r but also the biochemical mechanisms of resistance controlled by these mutants, ; (Watanabe and Watanabe, 1959&; 1959h; Brock, 1964). Hence, i f progress i s to be made towards the understanding of streptomycin action by comparing the properties of mutants, i t i s essential that the genetic and physiological c h a r a c t e r i s t i c s of the organisms under i n v e s t i g a t i o n be c l e a r l y defined,. - 56 -E. 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