UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1966_A6_7 C68.pdf [ 3.44MB ]
Metadata
JSON: 831-1.0093662.json
JSON-LD: 831-1.0093662-ld.json
RDF/XML (Pretty): 831-1.0093662-rdf.xml
RDF/JSON: 831-1.0093662-rdf.json
Turtle: 831-1.0093662-turtle.txt
N-Triples: 831-1.0093662-rdf-ntriples.txt
Original Record: 831-1.0093662-source.json
Full Text
831-1.0093662-fulltext.txt
Citation
831-1.0093662.ris

Full Text

AN INVESTIGATION OF MODIFIED METABOLIC REGULATION IN STREPTOMYCIN-DBPENDENT ESCHERICHIA COLI  M. B. COUKELL  A T h e s i s Submitted In P a r t i a l F u l f i l m e n t Of The  Requirements For The Degree Of MASTER OF SCIENCE i n the Department of BIOCHEMISTRY  We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA April,  I966  the  In presenting  this thesis i n partial fulfilment  of  r e q u i r e m e n t s f o r an  a d v a n c e d d e g r e e a t the U n i v e r s i t y  of  B r i t i s h Columbia, I agree that a v a i l a b l e f o r r e f e r e n c e and mission for extensive p u r p o s e s may his  be  L i b r a r y s h a l l make i t f r e e l y  study.  I f u r t h e r agree that  copying of t h i s t h e s i s f o r  g r a n t e d by  representatives.,  the  the Head o f my  c a t i o n of t h i s t h e s i s for f i n a n c i a l gain w i t h o u t my  written  Department  of  permission.  The 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 , V a n c o u v e r 8, Canada.  scholarly  Department or  I t i s understood that  c o p y i n g or  s h a l l not  per-  be  by publi-  allowed  (i)  ABSTRACT The a c e t o h y d r o x y a c i d s y n t h e t a s e 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 f o u r d i f f e r e n t s t r a i n s o f E s c h e r i c h i a coli.  The a c t i v i t y  o f the «><:-acetolactate-f orming system was  found t o be g r e a t e r b o t h a t pH 6.0 and a t pH 8.0 i n s t r e p t o m y c i n dependent mutants t h a n i n t h e c o r r e s p o n d i n g s t r e p t o m y c i n - s e n s i t i v e cultures.  I n g e 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 t h e range found f o r s t r e p t o m y c i n s e n s i t i v e organisms r e g a r d l e s s o f whether t h e y were grown i n t h e presence o r absence o f a n t i b i o t i c .  The a c e t o h y d r o x y a c i d  s y n t h e t a s e a c t i v i t y o f 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 t o be l o w e r t h a n t h a t o f t h e 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 backmutation.  M u t a t i o n t o s t r e p t o m y c i n - r e s i s t a n c e o r -dependence  had no e f f e c t on g l u c o k i n a s e and g l u t a m i c dehydrogenase  activities.  The a d d i t i o n o f t h e coenzyme f l a v i n adenine d i n u c l e o t i d e t o t h e i n c u b a t i o n m i x t u r e s markedly s t i m u l a t e d t h e a c t i v i t i e s o f a l l the  extracts.  T h i s enhancement o f a c e t o h y d r o x y a c i d s y n t h e t a s e  a c t i v i t y had l i t t l e  o r no e f f e c t on t h e r a t i o o f a c t i v i t i e s o f  t h i s enzyme i n t h e dependent and s e n s i t i v e E s c h e r i c h i a  coli  s t r a i n s i n v e s t i g a t e d . «<-Acetohydroxybutyrate f o r m a t i o n was found t o be g r e a t e r i n e x t r a c t s from t h e s t r e p t o m y c i n - d e p e n d e n t  (ii)  organism than i n e x t r a c t s p r e p a r e d from the same s t r a i n o f 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 o f oC-acetohydroxybutyrate p a r a l l e l e d t h a t of o < - a c e t o l a c t a t e f o r m a t i o n i n the dependent mutant.  I t was  c o n c l u d e d from t h e s e o b s e r v a t i o n s t h a t e x c r e t i o n o f 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  o f the e l e v a t e d 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 t h e s e mutants.  of  I n the dependent organism, i t was p o s t u l a t e d  t h a t s t r e p t o m y c i n f u n c t i o n e d as a d e - r e p r e s s o r w  w  of aceto-  hydroxy a c i d s y n t h e t a s e t h u s 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 t o s e r v e as an i m p o r t a n t r o u t e of pyruvate d i s s i m i l a t i o n .  (iii)  ACKNOWLEDGEMENTS  The author wishes t o express h i s a p p r e c i a t i o n to 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 course o f t h i s work and t o Dr. I.D. Desai f o r h i s many h e l p f u l s u g g e s t i o n s .  (iv) TABLE OF CONTENTS Page No. A.  INTRODUCTION I. II.  B.  1  Metabolic Effects of Streptomycin in Microorganisms  1  Biosynthesis of Aliphatic Amino Acids and Regulatory Mechanisms  k  III. Objective of the Investigation of Streptomycin Mutants  9  METHODS AND MATERIALS  12  I.  Original Cultures  12  II.  Isolation of Streptomycin Mutants  12  (i) (ii)  From streptomycin-sensitive parent cultures  12  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  (iii) IV.  harvesting cultures  15  Bacterial extracts  16  Enzyme Assays (i)  (ii)  16  Measurement of o<-acetolactateforming activity  16  (a) Method I Measurement of <<-acetohydroxy(b) Method II butyrate-forming activity  16 18 19  (v) Page (a)  Reversal of L-valine inhibition by L - i s o l e u c i n e i n E. ooli  K-12 (b)  P r e p a r a t i o n of assay  (c)  Enzymatic formation of <X-acetohydroxybutyrate  (d)  Estimation of  (e)  Preparation of a  plates  bacterial  C.  Reference  growth  22  22  (b)  Glutamic  22  Streptomycin  S p e c i f i c i t y of <<-Acetolactate  V.  Activity  of  of  Activity  Activity  Sensitivity  the  the of  the  Cultures  the  the  «<-Acetolactate-forming 26  o<-Acetohydroxybutyrateof  the  26  Mutants  Reference  30  Enzymes  36  I.  Genetic  II.  Streptomycin Mutants F o r m a t i o n o f «*-Acetolactate Streptomycin Mutants  IV.  Characteristics  of  the 36 by  Formation of «<-Acetohydroxybutyrate Streptomycin Mutants  k0 by  S i g n i f i c a n c e of Acetohydroxy Acid Synthetase D e - r e p r e s s i o n i n Dependent Mutants of E s c h e r i c h i a c o l i  REFERENCES  23 23  Mutants  System of  of  the Method f o r Determination  DISCUSSION  III.  E.  dehydrogenase  23  forming  D.  22  enzymes  Glucokinase  I.  IV.  21  (a)  II.  System  19  standard  RESULTS  III.  19  20  curve (iii)  No.  kS  50 56  (vi)  LIST OF TABLES Page I  II  III  IV  V  VI  VII  VIII  IX  Relative S e n s i t i v i t i e s c o l i Sensitive Strains Dihydrostreptomycin  of E s c h e r i c h i a to  2k  Comparison of Methods f o r Determining <<-Acetolactate i n E x t r a c t s of Escherichia ooli  25  Acetohydroxy A c i d Synthetase A c t i v i t i e s i n E x t r a c t s of S t r e p t o m y c i n - S e n s i t i v e (S) and -Dependent (D) E s c h e r i c h i a c o l i  27  Acetohydroxy A c i d Synthetase A c t i v i t i e s i n E x t r a c t s of S t r e p t o m y c i n - R e s i s t a n t Escherichia coli  28  E f f e c t of F l a v i n Adenine D i n u c l e o t i d e on Acetohydroxy A c i d Synthetase A c t i v i t i e s i n Sensitive (S) and Dependent (D) E x t r a c t s of E s c h e r i c h i a c o l i  29  S e n s i t i v i t y of E s c h e r i c h i a c o l i K-12 to L - V a l i n e and R e v e r s a l of I n h i b i t i o n by L - I s o l e u c i n e  32  S p e c i f i c i t y of the *:-Acetohydroxybutyrate Assay System f o r the Six-Carbon I n t e r mediate  33  Formation of <*-Acetolactate and «*-Acetohydroxybutyrate by S e n s i t i v e ( S ) , R e s i s t a n t (R) and Dependent (D) E x t r a c t s of E s c h e r i c h i a c o l i  3k  Reference Enzyme A c t i v i t i e s Streptomycin Mutants of E s c h e r i c h i a c o l i A.  35  of  No,  (vii)  LIST OF FIGURES Page No. 1.  2.  3.  4.  Products of Catabolism of Glucose by Streptomycin-Dependent Escherichia c o l i  3  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 in Escherichia c o l i  5  F o r m a t i o n o f <?<-Acetolactate and «<-Acetohydroxybutyrate i n Escherichia c o l i  7  Growth Response o f E s c h e r i c h i a c o l i K-12 t o I n c r e a s i n g C o n c e n t r a t i o n of L - I s o l e u c i n e  31  A. I.  Metabolic The  streptomycin  INTRODUCTION  E f f e c t s o f Streptomycin In Microorganisms primary s i t e o f the a n t i b a c t e r i a l a c t i o n o f has y e t t o be e l u c i d a t e d .  hypotheses have been proposed: (1) reactions 1953);  (Umbreit, 1953;  (2)  et a l , I960;  divergent  i n h i b i t i o n o f s p e c i f i c enzyme  Rosanoff and Sevag, 1953;  altered permeability  Barkulis,  due t o membrane damage (Anand,  Landman and Burchard, 1 9 6 2 ) ;  of p r o t e i n synthesis 1962;  However, s e v e r a l  and (3)  (Erdos and Ullman, 1959;  inhibition  Mager, e t a l ,  Speyer, e t a l , 1962, F l a x , e t a l , 1962a,  1962b;  Cox,  et a l , 1964). Two groups o f I n v e s t i g a t o r s I 9 6 2 , and Bragg and P o l g l a s e ,  (Tirunarayanan, e t a l ,  1962) r e p o r t e d ,  independently,  that streptomycin-dependent microorganisms grown on g l u c o s e - s a l t s medium e x c r e t e d  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 of L - l e u c i n e .  Each group however, i n t e r p r e t e d these  results differently.  Tirunarayanan and co-workers  a t t r i b u t e d the e x c r e t i o n o f L - v a l i n e and L - l e u c i n e blockage" o f p r o t e i n s y n t h e s i s .  (1962) to a " p a r t i a l  Since the streptomycin-dependent  s t r a i n s were a b l e to 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 block i n p r o t e i n s y n t h e s i s , they suggested t h i s phenomenon c o n s t i t u t e d only the p r e l i m i n a r y  f i x a t i o n o f streptomycin  to the  c e l l and t h a t the a n t i m i c r o b i a l e f f e c t was due t o subsequent m e t a b o l i c changes. (1962)  Experiments c a r r i e d out by Bragg and P o l g l a s e  suggested t h a t the e x c r e t i o n o f L - v a l i n e r e s u l t e d 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 m e t a b o l i t e s a r e not n o r m a l l y d e t e c t a b l e i n the supernatant f l u i d s of s t r e p t o m y c i n - s e n s i t i v e o r - 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 s t r e p t o m y c i n - r e s i s t a n t mutant i n the presence o f a n t i b i o t i c e x c r e t e s s i g n i f i c a n t q u a n t i t i e s 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 o f a n a e r o b i c metabolism i n a n t i b i o t i c c o n t a i n i n g 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 t o that employed by s e n s i t i v e  organisms.  The streptomycin-dependent mutant d i f f e r s from both 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 L - 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  accounted f o r i n t h i s p r o d u c t .  Subsequent  work (Bragg and  P o l g l a s e , 1964a) c l e a r l y i n d i c a t e d t h a t d e p l e t e d s t r e p t o m y c i n dependent  c e l l s , produced l a r g e q u a n t i t i e s of l a c t a t e w h i l e the  same c e l l s supplemented  with s t r e p t o m y c i n , a t a c o n c e n t r a t i o n  i n excess of t h a t e s s e n t i a l f o r growth, When streptomycin-dependent  e x c r e t e d only L - v a l i n e .  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 metabolism resembled 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 cells,  ( p r o d u c t i o n of l a c t i c a c i d ) even i n the presence o f the  optimal 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 . dependent  For the s t r e p t o m y c i n -  mutant, the r e l a t i o n s h i p between a e r o b i c metabolism  and the requirement f o r the a n t i b i o t i c i s summarized c a l l y i n F i g . 1.  diagrammatl-  The primary products o f the a e r o b i c c a t a b o l i s m  -  3 -  PRIMARY PRODUCTS C0  2  o r ACETATE  A  GLUCOSE  Requires Streptomycin and oxygen  Streptomycin o r oxygen deprivation  V LACTIC ACID (and ALANINE)  VALINE  ALTERNATE  PIG. 1.  SECONDARY  PRODUCTS  Products o f c a t a b o l i s m o f g l u c o s e by streptomycin-dependent coli  Escherichia  (Taken from Bragg and P o l g l a s e ,  1964a).  _  of glucose  4  -  i n streptomycin-dependent E. c o l i are carbon d i o x i d e  or acetate.  The  secondary products are L - v a l i n e  supplemented with a n t i b i o t i c ) or l a c t a t e and a n a e r o b i c or d e p r i v e d  of a n t i b i o t i c ) .  The  (aerobic  L-alanine  (either  s m a l l q u a n t i t i e s of  a l a n i n e p r o b a b l y a r i s e from pyruvate by t r a n s a m i n a t i o n . workers proposed t h a t the formation  (Bragg and  Polglase,  be  metabolism e x i s t i n g i n  Studies  from the same l a b o r a t o r y  1963b) on the e f f e c t of  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 .  These  of L - v a l i n e appeared to  a secondary a e r o b i c pathway of glucose streptomycin-dependent mutants.  and  dihydrostreptomycin  suggested t h a t L - v a l i n e  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  may  i n carbohydrate metabolism.  I f t h i s were t r u e , then, i n streptomycin-dependent c e l l s ,  the  a n t i b i o t i c might 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 to f u n c t i o n c a t a b o l i c a l l y as a major route  of pyruvate  dissimilation.  II.  Biosynthesis Mechanisms. The  of A l i p h a t i c Amino A c i d s and  Regulatory  b i o s y n t h e t i c pathways l e a d i n g to L - i s o l e u c i n e ,  L - v a l i n e , L - l e u c i n e and  pantothenate i n E. c o l i have been  i n v e s t i g a t e d through i s o t o p e s t u d i e s on s e l e c t e d mutants.  A review of t h i s work has been g i v e n by Umbarger  Davis ( I 9 6 2 ) from two  auxotrophic  (see F i g . 2 ) .  L - v a l i n e and  L - l e u c i n e are  moles of pyruvate w h i l e the p r e c u r s o r s  synthesized  for L-isoleucine  r e s u l t from the condensation of one mole each of pyruvate oC-ket'obutyrate.  Umbarger and  and  Brown (1958b) r e p o r t e d  enzymes c a t a l y s i n g the l a s t three r e a c t i o n s i n the  that  and the  biosynthetic  threonine  °<-ketobutyrate-  II  pyruvate  pyruvate  I II  •c-aceto.» l a c t a t e  Acetohydroxy Acid Synthetase  oi. -keto-methylvalerate  % 0-dihydroxy' <$-methyl• valerate —  «*-aceto«<-hydroxy-» butyrate —  IV  III  4 ,6-dihydroxy_» i s o v a l e r a t e —  L-isoleucine  i  oc -keto•isovalerate  -» L-valine  «-ketoisocaproate  x-ketopantoate  L-leucine  pantoate  Reductoisomerase  III  Dihydroxy Dehydrase  IV  Transaminase B  pantothenate  Fig. 2  The biosynthetic pathway t o i s o l e u c i n e , v a l i n e , leucine and pantothenate i n Escherichia c o l i .  I  -  6  -  pathway l e a d i n g to L - i s o l e u c i n e a l s o c a t a l y s e the c o r r e s p o n d i n g reactions i n L-valine  synthesis.  T h i s 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 L - v a l i n e pathway g e n e r a l l y l a c k e d the L - i s o l e u c i n e pathway. multi-auxotrophic  the corresponding enzyme on  Thus " s i n g l e - s t e p " mutants occur,  f o r L - v a l i n e , L - l e u c i n e and L - i s o l e u c i n e .  These f i n d i n g s have been supported by enzyme k i n e t i c (Umbarger and Brown, 1958b;  L e a v i t t and Umbarger,  studies  I96I).  It  should be noted however, t h a t the enzyme which c a t a l y s e s the first  step i n the L - v a l i n e pathway, v i z . the condensation of two  moles of pyruvate t o one mole of o ( - a c e t o l a c t a t e , a l s o  catalyses  the second step i n the L - i s o l e u c i n e pathway, the a c e t y l a t i o n of oC-ketobutyrate to c<-acetohydroxybutyrate ( L e a v i t t and 1961).  Umbarger,  T h i s enzyme complex (see F i g . 3) has been d e s i g n a t e d by  v a r i o u s a u t h o r s as the condensing enzyme. <<-acetolactate-forming system (Umbarger and Brown, 1958bj and r e c e n t l y as acetohydroxy a c i d synthetase (Bauerle,  et a l , 1964).  Although the enzyme  complex has not been f r a c t i o n a t e d , e a r l y i n v e s t i g a t i o n s and  Brown, 1958b) suggest two r e a c t i o n s a r e i n v o l v e d .  c o n s i s t s o f the g e n e r a t i o n  The  first  of an " a c t i v e acetaldehyde", 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. is  (Umbarger  The second r e a c t i o n  the a c t u a l t r a n s f e r of the a c e t a l group to the a c c e p t o r  molecule, e i t h e r pyruvate or «<-ketobutyrate. proceeds o p t i m a l l y as Mg"**" or Mh*" .  This  reaction  only i n the presence o f a d i v a l e n t c a t i o n such B a u e r l e , e t a l (1964) r e c e n t l y r e p o r t e d  that  0  0  II  CH3-CH2-C-COOH  OH  CH3-C-C-COOH CH3—(JH2  << -aceto»<-hydroxybutyrate  «<-ketobutyrate  DPTH C0  CH3-C-DPT  H  2  0 OH  0 CH3-C-COOH  *  CH3-C-C-COOH CHo  pyruvate  Fig. 3  <<-acetolactate  The f o r m a t i o n o f o < - a c e t o l a c t a t e and <*-aceto«<-hydroxybutyrate i n E s c h e r i c h i a c o l i .  - 8 acetohydroxy a c i d 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 presence of the coenzyme f l a v i n adenine d i n u c l e o t i d e The f u n c t i o n of t h i s unexpected  (FAD).  c o f a c t o r i s as y e t u n d e f i n e d .  The pH optimum of the acetohydroxy a c i d synthetase system i n E . o o l i has i n v o l v e d c o n s i d e r a b l e r e - i n v e s t i g a t i o n . and Umbarger (1959) c l e a r l y demonstrated  Halpern  the presence of two  d i s t i n c t a c e t o l a c t a t e forming systems i n A.aerogenes. pH 6.0  and the o t h e r a t pH 8 . 0 .  one a t  However, they c o u l d f i n d  a c t i v i t y only a t the h i g h e r pH i n E. c o l l .  A later  enzymic  paper  (Radhakrishnan and S n e l l , i 9 6 0 ) d e s c r i b e d two pH optima i n E. c o l i c o r r e s p o n d i n g t o the pH v a l u e s r e p o r t e d f o r A.  aerogenes.  R e c e n t l y , a thorough k i n e t i c study on the acetohydroxy a c i d synthetase a c t i v i t y of a streptomycin-dependent E. c o l i mutant (Desai and P o l g l a s e , I965) under optimal c o n d i t i o n s supplementation w i t h FAD),  (including  s t r o n g l y supported the o r i g i n a l  p r e d i c t i o n o f a s i n g l e pH 8 . 0  enzyme system i n t h i s  organism.  I t has been r e p o r t e d (Umbarger and Brown, 1958b) the acetohydroxy a c i d synthetase complex, l i k e i n i t i a l  that  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 to end-product i n h i b i t i o n when assayed i n the presence of L - v a l i n e . studies,  Repression  ( F r e u n d l i c h , e t a l , I962) however, i n d i c a t e t h a t r e g u l a t i o n  of the enzymes a s s o c i a t e d w i t h branched c h a i n amino a c i d  bio-  s y n t h e s i s i n v o l v e s a c o n t r o l mechanism thus f a r unique i n b i o l o g i c a l systems.  Since L - v a l i n e and L - i s o l e u c i n e a r e  d i r e c t l y by a common sequence  formed  of enzymes, r e p r e s s i o n of the pathway  - 9 -  by the e l e v a t i o n of one synthesis  product, c o u l d s e r i o u s l y a f f e c t  of the other p r o d u c t .  would a l s o be impaired  pathway.  In a d d i t i o n , L - l e u c i n e  s i n c e the i n i t i a l  L - l e u c i n e b i o s y n t h e s i s Involves  the synthesis  reaction leading  an i n t e r m e d i a t e  to  of the v a l i n e  T h i s problem i s overcome i n E. o o l i by  the  requirement  t h a t a l l three end-products ( a l s o p o s s i b l y pantothenate) must be i n excess f o r r e p r e s s i o n to o c c u r . valent repression" Preundlich, not  1965).  ( F r e u n d l i c h , e t a l , 1962;  Umbarger  and  Although the mechanism of t h i s r e p r e s s i o n i s  completely understood i n r e g a r d  recent  T h i s has been termed " m u l t i -  to acetohydroxy a c i d  synthetase,  s t u d i e s w i t h streptomycin-dependent mutants of E.  coli  i n d i c a t e t h a t t h i s enzyme i s d e f i n i t e l y r e p r e s s i b l e by the products (Polglase, i n p r e s s ) .  Therefore,  end-  i t appears to be  well  e s t a b l i s h e d t h a t the acetohydroxy a c i d synthetase system not c a t a l y s e s the i n i t i a l L-leucine  step i n the b i o s y n t h e s i s  of L - v a l i n e  from pyruvate but a l s o c o n t r o l s the p r o d u c t i o n  amino a c i d s .  Since  t h i s enzyme complex i s i n t i m a t e l y  only  and  of these  involved  with L - i s o l e u c i n e b i o s y n t h e s i s , 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 i n f l u e n c e the s y n t h e s i s  of L - i s o l e u c i n e as w e l l  as  L-valine. III.  Objective I t was  of the I n v e s t i g a t i o n of Streptomycin Mutants 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 a e r o b i c amino a c i d e x c r e t i o n .  carbohydrate metabolism r e s u l t i n g i n  T h i s may  be  f u r t h e r i n t e r p r e t e d as  a l t e r a t i o n i n the c o n t r o l of branched c h a i n amino a c i d Preliminary  studies  (Bragg and  Polglase,  a c i d synthetase a c t i v i t i e s i n d e p l e t e d  an  biosynthesis.  1964a) on acetohydroxy  and  supplemented  streptomycin-dependent E. c o l i , i n d i c a t e d t h a t maximal ot-acetol a c t a t e f o r m a t i o n occurred  only i n the presence of a n t i b i o t i c .  Streptomycin appeared to d e - r e p r e s s  the l e v e l of t h i s enzyme i n  supplemented dependent mutants r e g a r d l e s s  of whether or not  c e l l s were grown i n the presence of the end-products.  the  Since  this  suggests t h a t s t r e p t o m y c i n f u n c t i o n s a t the g e n e t i c 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 g e n e r a l  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 s t r e p t o m y c i n dependent mutant. The  o b j e c t i v e 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 s e v e r a l s t r a i n s of E. c o l i whether c o n s i s t e n t d i f f e r e n c e s e x i s t i n s t r e p t o m y c i n mutants i n the of acetohydroxy a c i d synthetase (the r e g u l a t o r y L-valine biosynthesis).  I t was  assumed a t the  enzyme f o r outset  g e n e r a l i t y of the phenomenon of L - v a l i n e e x c r e t i o n by dependent mutants had yanan, et a l ,  1962;  been e s t a b l i s h e d by p r e v i o u s Bragg and  Polglase,  that  E. c o l i mutants.  the  streptomycin-  work  (Tirunara-  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 r e q u i r e d i s o l a t i o n of s e v e r a l new  level  The  the  second phase of  the study i n v o l v e d assays f o r 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 t o e s 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 t o 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 i n enzyme content, an attempt was made t o e x p l a i n the advantage of t h i s  difference.  t o the streptomycin-dependent  organism  B. I.  METHODS AND  MATERIALS  O r i g i n a l Cultures Four s t r a i n s of E. c o l i were used i n t h i s work.  E. c o l i was  "A" as p r e v i o u s l y d e s c r i b e d by Roote and  Polglase  (1955)  o r i g i n a l l y obtained as the streptomycin-dependent c u l t u r e .  T h i s mutant has been designated (SA), and  DA.  A  s t r e p t o m y c i n - r e s i s t a n t (RA)  streptomycin-sensitive mutant was  d e r i v e d by  "back-mutation" from the dependent c u l t u r e .  The  E. c o l i  of Hygiene, Ottawa,  "C" was  O n t a r i o and  obtained  from the Laboratory  s t r a i n E. c o l i  "E" was  s u p p l i e d to us as s t r e p t o m y c i n - s e n s i t i v e s t r a i n s . obtained  C o l l e c t i o n (ATCC 1 2 4 0 7 ) .  II.  "B" and w i l l be r e f e r r e d t o , h e r e i n 19^7).  (Witkin,  Mutants  From s t r e p t o m y c i n - s e n s i t i v e parent c u l t u r e s . 500 ml. of g l u c o s e - s a l t s  A volume of approximately medium (composition with one  d e s c r i b e d under "Medium") was  innoculated  or two l o o p f u l s of 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  stored i n heart i n f u s i o n broth per l i t e r ) a t 5°C. for  An a d d i t i o n a l  from the American Type C u l t u r e  I s o l a t i o n of Streptomycin (i)  These were  T h i s 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 a f t e r , as E. c o l i B/r  designated  i s o l a t e d a t the Department  of B a c t e r i o l o g y , U n i v e r s i t y of L a v a l , Montreal.  s e n s i t i v e s t r a i n was  strain  (25  gm.  T h i s c u l t u r e was  20 - 24 hours a t 3 7 ° C  The  - 12 -  of h e a r t i n f u s i o n b r o t h  incubated without a g i t a t i o n  r e s u l t i n g growth was  transferred  - 13 a s e p t i c a l l y t o two 300 ml. s t e r i l e b o t t l e s and .centrifuged a t 2000 RPM f o r 1 hour a t 4°C ( I n t e r n a t i o n a l R e f r i g e r a t e d The  Centrifuge).  p e l l e t was resuspended i n 6.0 ml. o f b u f f e r (0.05M potassium  phosphate, pH 7.4), c a l l e d the "Standard Inoculum" (SI). Q  E x a c t l y 0 . 5 ml. o f the SI (-~10 surface  c e l l s ) were p i p e t t e d on t o the  of 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 broth  f o r t i f i e d with 1 . 5 $ agar) and 1000 u n i t s  per ml. (1 u n i t i s e q u i v a l e n t  t o 1 jxg o f f r e e s t r e p t o m y c i n base)  o f e i t h e r d i h y d r o s t r e p t o m y c i n (DHSM) o r streptomycin (SM).  The  s l 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 s u r f a c e and the p l a t e s were incubated  a t 37°C f o r 24 hours.  which formed were s u b c u l t u r e d  The c o l o n i e s  i n t o 5 nil* o f s t e r i l e  glucose-salts  medium, e i t h e r devoid  o f a n t i b i o t i c or supplemented with 1000 jxg  per ml. and incubated  as d e s c r i b e d 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 o f  dihydrostreptomycin.  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 r e s i s t a n t mutant and not t o growth of a 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  of d i h y d r o s t r e p t o m y c i n from  the p l a t e , a l o o p f u l of 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 both to a n t i b i o t i c supplemented and t o unsupplemented medium i n tubes.  Pure r e s i s t a n t and dependent c u l t u r e s of each s t r a i n were  s t o r e d a t 5°C on both h e a r t i n f u s i o n agar s l o p e s and i n h e a r t i n f u s i o n broth.  A l l c u l t u r e s were s u b c u l t u r e d  monthly.  - 14 (li)  I s o l a t i o n o f r e v e r t a n t s from a dependent c u l t u r e .  streptomycin-  To 100 ml. o f g l u c o s e - s a l t s medium supplemented with 100 jig o f dihydrostreptomycin  per ml. was added 10 ml.  o f the  E. c o l i dependent s t r a i n grown overnight a t the same a n t i b i o t i c concentration.  The c u l t u r e was incubated  a t 37°C f o r 20 - 24  hours and c e n t r i f u g e d a s e p t i c a l l y a t 2000 RPM f o r 1 hour.  The  p e l l e t was washed twice i n 0.05M potassium phosphate b u f f e r , pH 7.4, added t o 1000 f r e e ) and incubated harvested  ml.  of heart i n f u s i o n broth  a f u r t h e r 48 hours a t 3 7 ° C  as p r e v i o u s l y d e s c r i b e d .  10 ml. o f b u f f e r .  (antibioticThe c e l l s were  The p e l l e t was suspended i n  One l o o p f u l o f t h i s c e l l suspension was s t r e a k e d  on g l u c o s e - s a l t s agar medium ( g l u c o s e - s a l t s b r o t h c o n t a i n i n g 1 . 5 $ agar) i n such a manner as t o produce i s o l a t e d c o l o n i e s . were s u b c u l t u r e d sensitive  Colonies  t o l i q u i d g l u c o s e - s a l t s medium t o y i e l d pure  (growth only i n the absence o f DHSM) and r e s i s t a n t  ( i n d i f f e r e n t t o the presence o f DHSM) c u l t u r e s .  A l l cultures  were s t o r e d a s i n d i c a t e d . III.  Growth o f C u l t u r e s and P r e p a r a t i o n  (i)  of Extracts  Medium The  b a s a l medium was o f the composition p r e v i o u s l y  d e s c r i b e d by Davis and M i n g i o l i ( 1 9 5 0 ) and c o n s i s t e d of the following:  KgHPO^(0.7$), K H P O ^ ( 0 . 3 $ ) sodium c i t r a t e 2  MgSO^(0.02$), ( N H ^ ) S 0 ^ ( 0 . 1 $ ) . 2  Glucose was autocli&yed  (0.05$), separately  and added t o the b a s a l medium t o g i v e a f i n a l c o n c e n t r a t i o n  o f 0.4$.  - 15 Rather  than streptomycin,  streptomycin  the more s t a b l e analog,  dihydro-  ( s e s q u i s u l f a t e ) (Merck, Sharp and Dohme, Montreal,  Canada) was g e n e r a l l y used i n t h i s work.  When streptomycin  s u l f a t e was used, i t was s t e r i l i z e d by passage through filters.  millipore  I n a l l cases the medium was a d j u s t e d t o a f i n a l pH o f  7.0.  (ii)  Procedure  f o r growing and h a r v e s t i n g c u l t u r e s .  P r i o r t o growth, the mutants i n v o l v e d were t r a n s f e r r e d at  l e a s t three times on g l u c o s e - s a l t s medium, under c o n d i t i o n s  (temperature  and a n t i b i o t i c c o n c e n t r a t i o n ) s i m i l a r t o those  employed i n the f i n a l growth experiment. The g l u c o s e - s a l t s medium (900 ml.) was i n o c u l a t e d with 100  ml. o f c u l t u r e p r e v i o u s l y grown as a s t a t i o n a r y c u l t u r e over-  n i g h t a t 37°C.  The t w o - l i t e r f l a s k was i n c u b a t e d a t the same  temperature i n a water bath w i t h moderate a e r a t i o n p r o v i d e d as follows.  A i r was s u p p l i e d by g l a s s t u b i n g f i t t e d  through a cork  stopper, running below the s u r f a c e o f the medium and connected by rubber t u b i n g t o an a i r l i n e .  Rate o f growth i n the f l a s k  was observed by r e c o r d i n g o p t i c a l d e n s i t i e s h o u r l y a t 4 2 0 mu. The  c u l t u r e s were g e n e r a l l y grown f o r 5 t o 6 hours and were  h a r v e s t e d d u r i n g the l a t t e r h a l f o f the e x p o n e n t i a l growth phase ( u s u a l l y a t an O . D . ^ Q np  o  f  approximately  1.4).  Streptomycin-  s e n s i t i v e organisms were grown i n a n t i b i o t i c - f r e e medium, streptomycin-dependent  while  c u l t u r e s were r o u t i n e l y grown on the same  medium f o r t i f i e d with 1 , 0 0 0 u n i t s p e r 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 i s t a n t mutants were grown on both supplemented  (R  +  cells)  and unsupplemented (R~ c e l l s ) medium. The c e l l s were h a r v e s t e d immediately by c e n t r i f u g a t i o n a t 6 , 0 0 0 x g f o r 20 minutes i n a r e f r i g e r a t e d c e n t r i f u g e (4°C) and were then washed by c e n t r i f u g a t i o n with 0.05M potassium phosphate b u f f e r , pH 7 . 0 . (iii)  Bacterial  extracts.  The p e l l e t was resuspended  i n the same b u f f e r i n a  r a t i o o f 1 gm. of c e l l s t o 15 ml. of b u f f e r .  The suspensions  were then t r e a t e d i n a B r o n w i l l 20-kc s o n i c o s c i l l a t o r f o r 3 minutes f o l l o w e d by c e n t r i f u g a t i o n a t 1 0 , 0 0 0 x g f o r 15  minutes.  The supernatant s o l u t i o n s were e i t h e r assayed immediately or s t o r e d a t -20°C and assayed w i t h i n 20 - Zk hours. IV.  Enzyme Assays (i)  Measurement of o 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 y . E a r l y s t u d i e s on t h i s enzyme complex (Radhakrishnan  and S n e l l , i 9 6 0 ) 8.0.  suggested a c t i v i t y maxima a t both pH 6 . 0 and  T h e r e f o r e , i n i t i a l assays were c a r r i e d out a t both pH  values.  L a t e r experiments  (Desai and P o l g l a s e , 1965)  the e x i s t e n c e o f only one optimal pH (pH 8 . 0 ) f o r t h i s  established enzyme  system and t h e r e a f t e r pH 6 . 0 assays were d i s c o n t i n u e d . D u r i n g 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 m o d i f i e d from the procedure of Umbarger and Brown, ( 1 9 5 8 b ) .  Each tube c o n t a i n e d i n 2 . 6 ml:  - 17 potassium phosphate,  pH 8.0  -  and pH 6 . 0 ,  100 ^umoles;  pyruvate, 50yumoles; MgClg, 5^umoles; thiamine 0.3/Jmoles;  0.5  sodium  pyrophosphate,  ml. o f E . c o l i e x t r a c t ( p r o t e i n ; 2-4  mg.  per  and when i n d i c a t e d f l a v i n adenine d i n u c l e o t i d e 10 mumoles.  ml.) Assay  tubes were g e n e r a l l y i n c u b a t e d a t 37°C f o r 30 minutes  ( l o n g e r p e r i o d s were used f o r e x t r a c t s of very low a c t i v i t y shorter periods f o r high a c t i v i t y ) . the a d d i t i o n of 0 . 5  The r e a c t i o n was  of the supernatant s o l u t i o n was a c i d , and the sample was  added 0 . 0 5  To 1.0  ml. o f 0.5%  5% oC-naphthol  tubes ml.  ml. of 36 N s u l p h u r i c  The  s o l u t i o n was  To a 5 ml* a l i q u o t of t h i s s o l u t i o n was  c r e a t i n e In water and 1.0  i n 2.5  by  a u t o c l a v e d a t 10 p . s . i . f o r 10 minutes  to convert the oC-acetolactate to a c e t o i n .  1.0  stopped  ml. o f 10$ t r i c h l o r o a c e t i c a c i d and the  were c e n t r i f u g e d to c l a r i f y the r e a c t i o n m i x t u r e s .  d i l u t e d to 10 ml.  or  then added  ml. of f r e s h l y prepared  N 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 to develop f o r one hour i n the dark.  The o p t i c a l d e n s i t y a t 5^0 mu was  spectrophotometer.  S i n c e the enzyme a c e t o l a c t a t e  i s not present i n e x t r a c t s of E . c o l i Brown, 1958b;  read on a Beckman B  Radhakrishnan  decarboxylase  ( J u n i , 1952;  Umbarger and  and S n e l l , i 9 6 0 ) a c e t o i n p r o d u c t i o n  can be a t t r i b u t e d e n t i r e l y to chemical d e c a r b o x y l a t i o n of ^ - a c e t o l a c t a t e and p r o v i d e s a measure of acetohydroxy activity.  acid  synthetase  Determination of the a c e t o i n content of u n t r e a t e d  e x t r a c t s of E. c o l i  (not a c i d i f i e d and heated) was  by s t o p p i n g the r e a c t i o n mixtures w i t h 0.1  accomplished  ml. of 10$ z i n c s u l p h a t e  -  and 0.1  18  -  ml. of 1 N sodium hydroxide as d e s c r i b e d by Umbarger  and Brown ( 1 9 5 8 b ) .  A c e t o i n was determined immediately i n one  a l i q u o t and a second a l i q u o t was heated f o l l o w i n g a d d i t i o n o f a c i d and t r e a t e d as u s u a l (See Table I I ) . (b)  Method I I  The second procedure used to assay acetohydroxy a c i d synthetase a c t i v i t y was d e s c r i b e d by Desai and P o l g l a s e ,  (19^5).  I t d i f f e r s from the former method p r i m a r i l y i n the c o n c e n t r a t i o n of c e r t a i n components of the i n c u b a t i o n mixture and i n the r e g u l a r supplementation with  PAD.  Each tube c o n t a i n e d i n 1.0  ml:  potassium phosphate,  pH 8 . 0 ,  100 p n o l e s ;  sodium pyruvate, 0 . 5 mmoles;  yumole;  thiamine pyrophosphate, 45 mumoles;  d i n u c l e o t i d e , 10 mumoles; i n pH 8 . 0 ,  0.5  f l a v i n adenine  0 . 5 ml. of E. c o l i e x t r a c t (prepared  0 . 5 M potassium phosphate b u f f e r ) .  f o r 15 minutes a t 37°C the r e a c t i o n was of 0.1  MgCl^,  After incubation  stopped by the a d d i t i o n  ml. of 50$ 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 minutes a t 60°C to c o n v e r t the *<-acetolactate to a c e t o i n .  An a l i q u o t of the r e s u l t i n g s o l u t i o n was a n a l y s 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 (19^5). determined by the method of Lowry e t a l , ( 1 9 5 1 ) . both methods were expressed i n micromoles formed per mgm.  of p r o t e i n per hour.  Protein  was  R e s u l t s by  of *<-acetolactate  - 1 9(Ii)  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 f a c t that growth i n h i b i t i o n o f s t r a i n K - 1 2 of E . c o l i by L - v a l i n e i s r e v e r s e d by L - i s o l e u c i n e (Tatum, 1 9 4 6 ) o r any s i x - c a r b o n p r e c u r s o r  of L - i s o l e u c i n e  (Umbarger, 1 9 5 8 ) . Since <?6-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 t e s t i n g the e x t r a c t s before and a f t e r heat treatment. (a)  Reversal of L - v a l i n e i n h i b i t i o n by L - i s o l e u c i n e i n E. c o l i K - 1 2 .  A s e r i e s of tubes ( 1 2 x 2 0 0 mm.)  were prepared con-  t a i n i n g , i n g l u c o s e - s a l t s medium, L - v a l i n e , 0 . 4 2 ^umoles p e r ml. ( 5 0 yagm), and L - i s o l e u c i n e i n s e r i a l d i l u t i o n s ranging t o 0 . 0 1 2 paoles 5 . 0 ml.  ( 5 0 t o 1 . 5 ^gm)  p e r ml., i n a f i n a l volume of  A c o n t r o l tube c o n t a i n i n g only L - v a l i n e ( 0 . 4 2 ^umoles  per ml.) was a l s o prepared.  A l l tubes were i n o c u l a t e d with  l o o p f u l s of E . c o l i K - 1 2 p r e v i o u s l y grown overnight a c i d - f r e e b a s a l medium a t 3 7 ° C . incubated recorded  from 0 . 3 8  at 3 7 ° C  f o r three days.  The  Preparation  i n amino  The i n o c u l a t e d tubes were then R e l a t i v e t u r b i d i t i e s were  a t various i n t e r v a l s during t h i s period (b)  two  (See Table V I I ) .  of assay p l a t e s .  method employed was m o d i f i e d  from the procedure  o r i g i n a l l y d e s c r i b e d by L e 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 medium.  on g l u c o s e - s a l t s  The c u l t u r e 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  - 20 to an o p t i c a l d e n s i t y stored a t 5°C.  ( O . D . ^ Q mu^ 2  o  f  a  PP  r o x  i m a t e l y 1 . 0 and  T h i s c u l t u r e c o u l d be m a i n t a i n e d a t t h a t  temperature f o r upto 5 days w i t h c o n s i s t e n t  results.  The s o l i d  medium ( L - v a l i n e agar) c o n t a i n e d 1 . 5 $ agar and was supplemented w i t h 0.42 ^umoles ( 5 0 jigm) o f L - v a l i n e p e r ml. To seed the agar 0 . 1 ml. of the c u l t u r e was mixed w i t h 1 0 ml. o f the melted v a l i n e agar p r e v i o u s l y  cooled to 4 5 ° C .  The seeded agar was poured i n t o a p l a s t i c P e t r i d i s h  ( 2 0 x 6 0 mm.),  r o t a t e d t o d i s t r i b u t e the organisms, and a l l o w e d t o s o l i d i f y . A second l a y e r o f seeded agar i d e n t i c a l l y prepared was then poured on top o f the f i r s t l a y e r and a p o r c e l a i n assay c y l i n d e r (Penicylinder,  8 x 1 0 mm.,  F i s h e r S c i e n t i f i c Co. L t d . , Edmonton,  Canada) was dropped i n t o the upper l i q u i d l a y e r .  A f t e r the  second l a y e r had s o l i d i f i e d 0 . 1 ml. o f the t e s t sample was p l a c e d i n the cup. The assay p l a t e s were i n c u b a t e d a t 3 7 ° C f o r 1 6 hours.  When a r e a c t i o n mixture was assayed the c y l i n d e r s were  m o d i f i e d by i n s e r t i n g i n t o the bottom o f each a d i s k o f Whatman # 3 f i l t e r paper s l i g h t l y l a r g e r i n diameter than the c y l i n d e r . These d i s k s serve t o l o c a l i z e any p r e c i p i t a t e d p r o t e i n o r b a c t e r i a l contamination i n t r o d u c e d a l o n g w i t h the r e a c t i o n mixture, (c)  Enzymatic f o r m a t i o n o f c < - a c e t o h y d r o x y b u t y r a t e .  B a c t e r i a l e x t r a c t s were prepared as p r e v i o u s l y  described.  Each i n c u b a t i o n tube c o n t a i n e d i n a f i n a l volume o f 1 . 0 ml; sodium p y r u v a t e , 1 0 ^umoles; o<-ketobutyrate, 5 ,umoles; 1 0 ^umoles;  thiamine pyrophosphate, 1 7 5 ^mumoles;  MgCl^,  f l a v i n adenine  - 21 dinucleotide, (pH 8.0)  10  and 0.5  buffer).  mjumoles;  potassium phosphate  ml. of e x t r a c t p r o t e i n  A f t e r 15 minutes  incubation  stopped by the a d d i t i o n of 0.1 The p r e c i p i t a t e d p r o t e i n was  100 ^umoles  (prepared i n pH  8.0  a t 37°C, the r e a c t i o n  ml. each of 10$ ZnSO^ and N NaOH.  removed by low speed  centrifugation  and the supernatant s o l u t i o n ( o r an a l i q u o t t h e r e o f ) was i n the cup of the assay p l a t e .  Estimation  placed  I n c u b a t i o n mixtures were  g e n e r a l l y assayed i n d u p l i c a t e or i n (d)  was  triplicate.  of b a c t e r i a l growth.  A t the end of the i n c u b a t i o n p e r i o d zones o f growth surrounded those cups which had c o n t a i n e d <?<-acetohydroxybutyrate. The c y l i n d e r s were removed and the agar which c o n t a i n e d growth was  c u t out w i t h a cork bore of a diameter s l i g h t l y  than the zones of growth.  The agar p l u g was  12 ml. tapered c e n t r i f u g e tube c o n t a i n i n g water.  The tube was  h y d r o l y s e the agar. ( S e r v a l l ) a t 3^00 discarded water.  dropped  into a  ml. of d i s t i l l e d  The contents of the tube were heated above 96 C f o r  90 seconds and then t r e a t e d w i t h 0.7 acid.  0.5  larger  ml. of 0.5  hydrochloric  then heated f o r an a d d i t i o n a l 60 seconds Before c o o l i n g , the tubes were  x g f o r 8 minutes.  The washed p r o t e i n was  d i s s o l v e d i n 0.5  to  centrifuged  The supernatant  and the p r e c i p i t a t e was washed i n 2.0  carbonate i n 0.1  N  was  ml. of d i s t i l l e d ml* of 5% sodium  N sodium hydroxide and estimated by the method  of Lowry, e t 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). following in a volume of l.o ml:  The system contained the  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 c u v e t t e was  added 0.1  ml. of b a c t e r i a l c e l l  decrease i n o p t i c a l d e n s i t y a t 3^0 mp. was the Cary 15 spectrophotometer.  e x t r a c t and  the  recorded a t 25°C i n  S p e c i f i c a c t i v i t i e s are  expressed as m i l l i m i c r o m o l e s of coenzyme o x i d i z e d per m i l l i g r a m of  p r o t e i n per minute. C.  I«  Streptomycin The  to  S e n s i t i v i t y of the C u l t u r e s  s e n s i 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 (S) s t r a i n s  the a n t i b i o t i c i s shown i n Table I .  from the dependent parent w i l d type II.  RESULTS  (DA)  The r e v e r t a n t obtained  i s slightly less sensitive  than  strains.  Specificity  of the Method f o r <<-Acetolaotate  Since c<-acetolactate f o r m a t i o n by c e l l e x t r a c t s i s determined  Determination free  bacterial  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 d e c a r b o x y l a t e d product, a c e t o i n , i t should be e s t a b l i s h e d t h a t i n E. c o l i , a c e t o i n i s not a normal  end-product.  Table I I g i v e s the r e s u l t s of <?<-acetolactate d e t e r m i n a t i o n on the streptomycin-dependent  mutant DE when the i n c u b a t i o n mixtures  a r e stopped under d i f f e r e n t c o n d i t i o n s . a c e t o i n produced  I t should be noted t h a t  a f t e r a c i d i f i c a t i o n and h e a t i n g appears  to  r e p r e s e n t the t o t a l e<-acetolactate formed d u r i n g the enzyme r e a c t i o n (See D i s c u s s i o n ) . s t r a i n s of E. c o l i  T h i s supports data p u b l i s h e d on other  (Juni, 1952;  Umbarger and Brown, 1 9 5 8 b ) .  Radhakrishnan  and S n e l l ,  I960;  For t h i s reason, a l l ^ - a c e t o l a c t a t e  - 24 -  TABLE I Relative 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 Sensitive S t r a i n s to Dihydrostreptomycin.  Strain  Growth *" i n TDHSMI yugm p e r m l . -  iti  1x2.  L°  SA  ++++  ++  t r  0  0  SC  ++++  0  0  0  0  SE  ++++  0  0  0  0  SB/r  ++++  +  0  0  0  1 2 ^  11  20 0  D e r i v e d from the dependent mutant (DA) by b a c k - m u t a t i o n . +  Each tube c o n t a i n e d 5 m l . o f g l u c o s e - s a l t s medium and a constant inoculum. Growth was e s t i m a t e d a f t e r i n c u b a t i o n a t 37°C f o r 24 h o u r s . R e l a t i v e growth i s r e p r e s e n t e d by the s c a l e 0 t o ++++ w i t h t r indicating a trace of growth. M  w  - 25 -  TABLE I I  Comparison o f Methods f o r D e t e r m i n i n g <X-Acetolactate i n E x t r a c t s o f Escherichia coli.  Reaction  TCA  TCA  stopped  umoles o f a c e t o i n p e r mg. p r o t e i n p e r h o u r PH 6.0 pH 8.0  with  , n o t heated *  , heated  +  ZnSO^, NaOH^ n o t h e a t e d Zhso^,  NaOH, a c i d i f i c a t i o n  and heated  final  0.319  0.677  0.207  0.582  0.075  0.056  0.235  0.620  concentration  1.6$.  *  Trichloroacetic acid,  +  H e a t e d f o r 10 m i n u t e s i n a n 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 m l . o f 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".  - 26 assays on  described  reaction  III.  i n this  mixtures  Activity  following  E.  coli  pH  6.0  and  to  this  mutants medium (R  +  8.0  8.0  the  the  grown  In  FAD,  D/S  further  IV.  the  case,  of  I t can  IV  for  units  the  heat  per ml.  ratios  of  these  elevated  acetohydroxy  the  (D)  that  unity.  Mutants for mutants  at  In  cells)  both  contrast  or  in  both  at  pH  6.0  and  at  pH  8.0  (FAD)  i s given  substantial  occurred i n extracts  mutants  remained  i n the  of  i n  (six fold) containing  constant.  presence  on  As  FAD, a l l  synthetase determinations  included  coenzyme.  A c t i v i t y of the o<-Acetohydroxybutyrate-forming System of the Mutants. Inhibition  L-valine  and  reversal  of of  the  growth  this  a  of dihydrostreptomycin  dinucleotide  although a  activity  acid  of  unity. adenine  had  seen  (R~  +  that  treatment.  streptomycin-resistant  R /R~" r a t i o  from  activity  be  of a n t i b i o t i c  flavin  observed  i n enzyme  of  and  determination  synthetase a c t i v i t i e s  synthetase a c t i v i t y  increase  result  acetoin  streptomycin-dependent III.  1000  slightly  acid  acid  i n Table  with  effect  I t was  the  total  i s g r e a t e r than  absence  this  deviates only  acetohydroxy  ratio  results  i n the  The  this  D/S  supplemented  V.  and  given i n Table  cells).  Table  (S)  are  are  a  acidification  acetohydroxy  streptomycin-sensitive f  employ  of the g<-Acetolactate-forming System  Relative  o  study  of E.  inhibition  coll by  strain  K-12  L-isoleucine  by i s  a  - 27 -  TABLE I I I Acetohydroxy a c i d Synthetase A c t i v i t i e s i n E x t r a c t s of S t r e p t o m y s i n - S e n s i t i v e (S) and -Dependent (D) E s c h e r i c h i a c o l i .  + ^amoles of ©(-acetolactate formed per mg. p r o t e i n per hour. pH 6.0 Strain  pH 8.0  S e n s i t i v e Dependent D/S  S e n s i t i v e 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 o f a c t i v i t i e s i n dependent (D) and s e n s i t i v e (S) e x t r a c t s .  +  Determined by Method I .  -  28  -  TABLE 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 .  umoles of o^-acetolactate  formed per mg. protein per hour  pH 6 . 0 Strain  pH 8 . 0 B~_  R*  0.6  0.321  0.282  0.183  1.1  0.192  0.141  0.7  0.179  0.140  0.8  0.152  0.108  0.7  0.117  0.118  1.0  0.152  0.218  iT  E*  B/r  0.251  0.139  E  0.167  C  A  R /R~ +  RVR" 0.9  1.4  R~  indicates extracts from c e l l s grown without added a n t i b i o t i c .  R  indicates extracts from c e l l s grown i n medium containing 1,000 units per ml. of dihydrostreptomycin.  *  +  Determined by Method I.  - 29 -  TABLE V E f f e c t o f F l a v i n Adenine D i n u c l e o t i d e on Acetohydroxy a c i d Synthetase A c t i v i t i e s i n S e n s i t i v e (S) and Dependent (D) E x t r a c t s o f E s c h e r i c h i a c o l i . -'  ^umoles wf-acetolactate formed per mg. p r o t e i n p e r hour. Mutant  - FAD  D/S  + FAD*  D/S  SA  0.438  1.8  2.601  1.7  DA  0.793  SG  0.188  DC  0.456  4.500  2.4  1.165  2.5  2.960  *  F l a v i n adenine d i n u c l e o t i d e (FAD) 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 2 ^gm.per ml.  +  Determined by Method I  - 30 shown on Table V I . to  The  extreme s e n s i t i v i t y of t h i s  inhibition  s p e c i f i c r e v e r s a l , and only by L - i s o l e u c i n e or any  six-carbon  p r e c u r s o r of t h i s amino a c i d , permits the q u a n t i t a t i v e d e t e r m i n a t i o n of <?(-acetohydroxybutyrate  by e s t i m a t i n g r e l a t i v e b a c t e r i a l growth.  Table V I I shows t h a t growth of E. c o l i to  the presence  s t r a i n K-12  of aC-acetohydroxybutyrate  c h a i n f i v e - c a r b o n d e c a r b o x y l a t i o n product  and not t o the  entirely straight  ( i n c o n f i r m a t i o n of the  o b s e r v a t i o n s of L e a v i t t and Umbarger, i 9 6 0 ) . of  i s due  A standard  t o t a l p r o t e i n versus L - i s o l e u c i n e c o n c e n t r a t i o n as  curve  determined  by the m i c r o b i o l o g i c a l assay method p r e v i o u s l y d e s c r i b e d i s g i v e n i n F i g . 4. each p o i n t was  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  d e r i v e d by a v e r a g i n g v a l u e s of s e v e r a l  experiments.  The r e s u l t i n g curve compared f a v o r a b l y w i t h the L - i s o l e u c i n e standard curve of Umbarger and L e a v i t t  (i960).  A comparison of£<-acetolactate and 0^-acetohydroxybutyrate f o r m a t i o n by c e l l - f r e e e x t r a c t s 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 n Table V I I I . approximately  I t i s i n t e r e s t i n g to note t h a t t h e r e was  i s given formed  35% lessrt-acetohydroxybutyrate t h a n ^ - a c e t o l a c t a t e  i n a l l mutants. V.  Activity  of the Reference  Enzymes  In Table IX, g l u c o k i n a s e and glutamic dehydrogenase a c t i v i t i e s are shown f o r mutants of E. c o l i A.  The  activities  of both enzymes appeared t o remain constant i n a l l mutants.  -  31 -  0.12  o in  0.10  -  H W  3  W Q  < V  0.06  0.04  -  0.02  -  H  O  10  FIG.  20  30  40  50  muM  O L E S  L - I S O L E U C I N E  4.  Growth response o f E s c h e r i c h i a c o l i to i n c r e a s i n g c o n c e n t r a t i o n s  K-12  of L-isoleucine.  Each p o i n t was d e r i v e d by a v e r a g i n g t h e r e s u l t s o f f i v e separate experiments.  - 32 -  TABLE VI S e n s i t i v i t y o f E s c h e r i c h i a o o l i K-12 to L - V a l i n e and R e v e r s a l o f I n h i b i t i o n by L - I s o l e u c i n e .  Concentration  of L-Isoleucine  umoles p e r ml. o f medium  All  Relative Turbidity 20 hours  72 hours  0.380  ++++  ++++  0.190  +++  ++++  0.095  +++  ++++  0.048  +++  +++  0.024  ++  +++  0.012  ++  ++  0.000  0  tubes c o n t a i n e d  L-vallne  (0.42 umoles p e r ml.)  Growth was e s t i m a t e d a f t e r i n c u b a t i o n a t 37°G f o r the p e r i o d o f time i n d i c a t e d . Relative growth i s r e p r e s e n t e d by the s c a l e 0 t o ++++ with t r " i n d i c a t i n g a t r a c e o f growth. w  ' tr  -  33  -  TABLE V I I S p e c i f i c i t y of the o(-Acetohydroxybutyrate Assay System f o r the Six-Carbon I n t e r m e d i a t e .  R e a c t i o n stopped w i t h ZnSO^, Na0H +  TCA,  Heated +  ^umoles of o<-Acetohydroxybutyrate formed per mg. p r o t e i n per hour  2.150 0.050  *  C a l c u l a t e d from the L - i s o l e u c i n e standard curve F i g . 4. E x t r a c t s assayed a t pH 8.0 only.  +  As d e s c r i b e d i n " M a t e r i a l s and Methods".  $  The r e a c t i o n was stopped by the a d d i t i o n of. 0.2 ml. o f 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 e d by b o i l i n g f o r 5 minutes.  - 3^ -  TABLE V I I I  Formation of c< - A c e t o l a c t a t e a n d ^ - A c e t o h y d r o x y b u t y r a t e by S e n s i t i v e ( S ) , R e s i s t a n t (R) and Dependent (D) E x t r a c t s of E s c h e r i c h i a c o l i .  Culture  o( - A c e t o l a c t a t e  Ratio  ^umoles of a c e t o i n formed p e r mg. +. p r o t e i n per hour D/X  g< -Acetohydroxy b u t y r a t e ^umoles of i s o l e u c i n e formed p e r mg. p r o t e i n p e r hour  Ratio D/X  3.62  DA  5.50  SA  3.7^  1.5  2.36  1.5  RA"  2.67  2.1  1.85  2.0  RA  3.64  1.5  2.48  1.5  DE  5.63  SE  2.73  H  3.52 2.1  Determined by Method I I (pH 8.0  1.52  2.3  only).  +  C a l c u l a t e d from the L - i s o l e u c i n e standard  $  X r e f e r s t o c u l t u r e s other than dependent mutants.  BA~  r e p r e s e n t s 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 r e p r e s e n t s the same r e s i s t a n t c u l t u r e grown i n a medium supplemented with 1000 u n i t s of d i h y d r o s t r e p t o m y c i n p e r ml. +  curve.  F i g . 4.  streptomycin-  - 35 -  TABLE IX Reference Enzyme A c t i v i t i e s of Streptomycin Mutants of E s c h e r i c h i a c o l i A.  # Extract  Glucokinase  Glutamic Dehydrogenase  Sensitive  (SA)  51  120  Dependent  (DA)  50  120  Resistant  (RA~)  +  58  112  Resistant  (RA )  +  67  122  +  *  A c t i v i t i e s a r e expressed as m i l l i m i c r o m o l e s of coenzyme (NADP+ or NADPH, r e s p e c t i v e l y ) changed p e r minute per m i l l i g r a m of p r o t e i n .  f  RA"" r e f e r s t o r e 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, w h i l e RA c e l l s were grown with 1,000 u n i t s p e r ml. of dihydrostreptomycin.  -  36 -  D. DISCUSSION 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 the Streptomycin  Mutants  P l a t i n g methods employed i n t h i s work r e a d i l y  yielded  spontaneous mutants e x h i b i t i n g complete i n d i f f e r e n c e t o streptomycin or d i h y d r o s t r e p t o m y c i n although the parent w i l d types were s e n s i t i v e to low c o n c e n t r a t i o n s (above 3 . 0 yug per of  either antibiotic  (Table I I I ) . While i n d i v i d u a l  ml.)  mutation  r a t e s were not c a l c u l a t e d , i t became apparent d u r i n g t h i s work t h a t streptomycin i n d i f f e r e n t c o l o n i e s were o c c u r r i n g a t a r e l a t i v e l y constant frequency V a l u e s r a n g i n g from 10*" (Demerec, 1951) resistance.  from the v a r i o u s s e n s i t i v e  (Newcombe and Hawirko, 19^9)  have been suggested  strains.  to 10~*  f o r r a t e s of mutation  to  These v a l u e s i n c l u d e the f o r m a t i o n of both s t r e p t o -  mycin r e s i s t a n t and dependent  organisms.  The occurrence of s i n g l e s t e p dependent mutants, u n l i k e i n d i f f e r e n t mutants, appeared E . c o l i and  to f l u c t u a t e w i t h the s t r a i n of  form of a n t i b i o t i c employed.  (obtained from the N a t i o n a l Research  E. c o l i  strain B  C o u n c i l of Canada), f o r  example, r e f u s e d to produce dependent mutants on medium c o n t a i n i n g e i t h e r s t r e p t o m y c i n or the dihydro d e r i v a t i v e d e s p i t e repeated attempts.  The  same s t r a i n , however, r e a d i l y formed r e s i s t a n t  mutants on e i t h e r form of the a n t i b i o t i c . t h a t the reduced was  form o f the a n t i b i o t i c  I t was  g e n e r a l l y found  (dihydrostreptomycin)  c o n s i s t e n t l y 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  e q u a l l y w e l l to e i t h e r form o f the a n t i b i o t i c . I t was c l e a r l y shown by S c o t t (19^9) t h a t both streptomycin and d i h y d r o s t r e p t o m y c i n  a r e non-mutagenic.  R e s i s t a n t 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 of  i n s u s c e p t i b l e p o p u l a t i o n s i r r e s p e c t i v e o f the presence  the a n t i b i o t i c .  A t the present time with the l i m i t e d under-  s t a n d i n g o f the mechanism o f a c t i o n o f streptomycin i t i s difficult  to e x p l a i n the enhanced a b i l i t y  o f the d i h y d r o form to  s e l e c t dependent mutants. A s i n g l e step back-mutation ( r e v e r s i o n ) from h i g h l e v e l streptomycin-dependence t o s t r e p t o m y c i n - s e n s i t i v i t y can be demonstrated i n many s t r a i n s o f dependent E . c o l i . (i960)  has shown t h a t mutation  Hashimoto  from dependence t o s e n s i t i v i t y  i s i n f a c t n o t a t r u e r e v e r s i o n but i s mediated by a suppressor mutation.  T h i s suppressor maps c l o s e to the l o c u s  governing  a n t i b i o t i c dependence and h i g h l e v e l r e s i s t a n c e and i s capable of m o d i f y i n g the e x p r e s s i o n o f e i t h e r a l l e l e .  I t i s obvious  from Table I t h a t the r e v e r t a n t (SA) i s not q u i t e as s e n s i t i v e to d i h y d r o s t r e p t o m y c i n as a r e the three w i l d type cultures.  T h i s suggests  i s incomplete  sensitive  t h a t s u p p r e s s i o n o f the dependent l o c u s  and the r e s u l t i n g s e n s i t i v e progeny a c q u i r e 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 on s l i g h t l y e l e v a t e d streptomycin c o n c e n t r a t i o n s . of  the corresponding  later.  t o grow  Characteristics  r e s i s t a n t mutant (RA) w i l l be d i s c u s s e d  The r a t e o f back-mutation has been s t u d i e d by  Bertani(1951)  and  h i s r e s u l t s i n d i c a t e that f o r a g i v e n  r a t e i s constant division.  and  Various  s t r a i n the mutation  ranges as h i g h as 10*"  per bacterium  per  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 r e v e r t . In a d d i t i o n to r e v e r s i o n from dependence to c e r t a i n s t r a i n s of dependent E. c o l i a p p a r e n t l y converting  sensitivity,  are capable of  to h i g h l e v e l r e s i s t a n c e i f s t o r e d under u n f a v o r a b l e  c o n d i t i o n s i n the presence of a n t i b i o t i c . phenotype i s spontaneous and  This a l t e r a t i o n i n  probably i s induced by a suppressor  mutation (Hashimoto, i 9 6 0 ) or a m o d i f i e r mutation (Matney, et a l , i960).  Due  to the i n s t a b i l i t y of many dependent mutants a l l  s t r a i n s were s u b c u l t u r e d  monthly and  the s t a t e of each  was  determined p r i o r to e x p e r i m e n t a t i o n . *  n  E. c o l i . g e n e t i c a n a l y s i s has  dependence and  shown t h a t  s i n g l e - s t e p high l e v e l r e s i s t a n c e are  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 the (Newcombe and Nyholm, 1950; t h i s genetic  Hashimoto, i 9 6 0 ) .  sensitivity, determined  "Sm  locus"  Consideration  f a c t , a l o n g with numerous p h y s i o l o g i c a l and  chemical o b s e r v a t i o n s ,  l e d Spotts and  a u n i t a r y h y p o t h e s i s of streptomycin  Stanier (I96I) action.  of  bio-  to propose  They suggested t h a t  the three phenotypes were u l t i m a t e l y determined by s t r u c t u r a l m o d i f i c a t i o n 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 site. and  Streptomycin would b i n d r e v e r s i b l y  /.with t h i s p r o t e i n  depending on the s t r u c t u r e of the r e c e p t o r  not i n f l u e n c e the f u n c t i o n of the c e l l .  The  s i t e may  or  ribosomes were  may  - 39 proposed  as the s t r e p t o m y c i n b i n d i n g s i t e s .  The s e n s i t i v e  ribosome 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 very h i g h a f f i n i t y combination  f o r streptomycin.  The r e s u l t i n g  prevented the attachment o f m-RNA, thus  protein synthesis.  inhibiting  The corresponding s t r u c t u r e s o f the r e s i s t a n t  and dependent ribosomes were such t h a t s t r e p t o m y c i n had no e f f e c t on the former and i t s presence was o b l i g a t o r y f o r the l a t t e r to function normally.  T h i s h y p o t h e s i s emphasized the f a c t t h a t only  a s i n g l e s i t e , the ribosome, was a l t e r e d d u r i n g mutation and t h a t the s t r e p t o m y c i n dependent c e l l i n the presence critical  l e v e l of a n t i b i o t i c  significantly  o f a supra-  ( > 250 u n i t s p e r ml.) d i d not d i f f e r  from the corresponding s e n s i t i v e s t r a i n grown under  optimal conditions (Spotts, 1962).  The d i s c o v e r y of n i n h y d r i n -  p o s i t i v e m a t e r i a l p r e s e n t i n c u l t u r e supernatants o f s t r e p t o m y c i n dependent mutants but absent  from the corresponding f l u i d s o f  - s e n s i t i v e and - r e s i s t a n t c u l t u r e s l e d Bragg and P o l g l a s e (1962) to an i n v e s t i g a t i o n o f m e t a b o l i c d i f f e r e n c e s i n the t h r e e phenotypes.  Their results  (Bragg and P o l g l a s e , 1964a; 1964b)  t h a t streptomycin-dependent  indicated  mutants, u n l i k e 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 a t the l e v e l o f pyruvate metabolism i n response  t o a l t e r a t i o n s i n streptomycin  c o n c e n t r a t i o n s and a e r o b i c c o n d i t i o n s .  T h i s suggests an i m p a i r -  ment i n r e s p i r a t i o n o r streptomycin a c t i o n a t a s i t e other than the ribosome.  R e c e n t l y , (Cox, e t a l , 1964;  D a v i e s , 1964) i t  has been shown by means o f sucrose g r a d i e n t c e n t r i f u g a t i o n t h a t  streptomycin does not prevent the attachment of m-RNA to the sensitive ribosome.  Data presented i n t h i s 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 l e s s than adequate to explain the physiological and biochemical responses to II.  streptomycin.  Formation ofo<-Acetolactate by Streptomycin The synthesis of  Mutants  -acetolactate by c e l l - f r e e b a c t e r i a l  preparations was determined c o l o r i m e t r i c a l l y by estimating the decarboxylated (1945).  product, acetoin, by the method of Westerfeld  The o r i g i n a l color reaction was  shown by Voges and  Proskauer (I898) to be due to the reaction between d i a c e t y l or acetoin and a guanidino group i n the presence of a l k a l i .  Attempts  to increase the s e n s i t i v i t y of this reaction l e d to the a d d i t i o n of creatine and *-naphthol.  The standard curve was  l i n e a r for  concentrations as high as <10 jugm-per ml. but deviated s l i g h t l y from this r e l a t i o n s h i p at greater concentrations. The Westerfeld method i s nearly s p e c i f i c for acetoin and d i a c e t y l .  Related 5-°arbon ketols give a somewhat s i m i l a r  color, while the 6-carbon ketols give a l i g h t olive-green color within the time l i m i t employed.  The s e n s i t i v i t y of these  analogs, however, i s 1 0 to 1 0 0 f o l d lower than e i t h e r d i a c e t y l or acetoin (Green, et a l ,  1942).  Color contributed by d i a c e t y l  can be distinguished from acetoin by i t s rate of formation.  - 41 C o l o r development due 10-15  -  to d i a c e t y l i s g e n e r a l l y complete  minutes, while the c o l o r complex due  maximum i n t e n s i t y only a f t e r one  hour.  within  to a c e t o i n reaches  I t should be noted t h a t  under the c o n d i t i o n s r e q u i r e d t o decarboxylatec>£-acetolactate ( a c i d and  heat) i t i s p o s s i b l e to o x i d i z e small q u a n t i t i e s of  a c e t o i n to d i a c e t y l ( W e s t e r f e l d , formation  1 9 ^ 5 ) , hence e a r l y c o l o r  does not n e c e s s a r i l y i n d i c a t e endogenous d i a c e t y l .  With the exception  of assay mixtures c o n t a i n i n g h i g h  trations ofX-acetolactate  concen-  e a r l y c o l o r development was  not  observed i n t h i s work.  Therefore,  the t o t a l c o l o r d e r i v e d  from these r e a c t i o n mixtures i s due  e< - a c e t o l a c t a t e  i t seems safe to conclude t h a t to  production.  Although the enzyme a c e t o l a c t a t e decarboxylase i s not present  i n e x t r a c t s of E. c o l i  mixtures s t i l l  showed a low but  (Juni, 1952),  enzyme r e a c t i o n  significant acetoin l e v e l ,  when the r e a c t i o n s were stopped under c o n d i t i o n s prevent chemical d e c a r b o x y l a t i o n  even  chosen to  (Table I I , l i n e 3 ) .  It is  probable t h a t t h i s a c e t o i n arose from a spontaneous decomposition of ^ - a c e t o l a c t a t e caused by i n c u b a t i o n a t 37°C s i n c e  -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 a t  this  temperature (Umbarger and obtained for total  Brown, 1 9 5 8 h ) .  under these c o n d i t i o n s were not  Consequently, subtracted  values  from  values  acetoin. Reaction  mixtures t r e a t e d w i t h both a c i d and  (Method I) g e n e r a l l y r e s u l t e d i n a c e t o i n values  10 -  heat  15$  lower  than mixtures treated, w i t h a c i d alone (Table I I , l i n e s 1 and  2)  and 5 -  42-  1 0 $ lower than mixtures t r e a t e d w i t h heat i n the  of z i n c sulphate and sodium hydroxide  (line 4 ) .  presence  This strongly  suggests t h a t the severe c o n d i t i o n s ( a u t o c l a v e d f o r 10 minutes a t 10 p . s . i . a t an a c i d i c pH)  employed f o r complete decarboxy-  l a t i o n a l s o d e s t r o y e d a small q u a n t i t y of the c o l o r m a t e r i a l i n the r e a c t i o n mixture.  complexing  T h i s o b s e r v a t i o n l e d t o the  use of m i l d e r d e c a r b o x y l a t i o n c o n d i t i o n s as d e s c r i b e d under Method I I . I t i s e v i d e n t from the data of Tables I I I and  IV,  t h a t a t pH 8 . 0 the s e n s i t i v e and r e s i s t a n t mutants (grown w i t h or without d i h y d r o s t r e p t o m y c i n supplementation) e x h i b i t e d s i m i l a r acetohydroxy  a c i d synthetase  of a l l s t r a i n s activities  whereas, the corresponding dependent mutants possess two  to f i v e times g r e a t e r .  A t pH  activities  6 . 0 t h i s r e l a t i o n s h i p was  less  j  dramatic but the D/S strains.  The  r a t i o exceeded the R /R  ratio i na l l  degree of d e - r e p r e s s i o n of acetohydroxy  synthetase i n the v a r i o u s dependent s t r a i n s was and t h i s d i f f e r e n c e may i n i t s response The  not  variability  to s t r e p t o m y c i n .  enzyme a c t i v i t i e s of the s e n s i t i v e and dependent  l i n e 4 , and Table V, l i n e s 1 and 2 )  (Table I I I ,  s i n c e the s e n s i t i v e  d e r i v e d as a r e v e r t a n t of the dependent s t r a i n .  case back-mutation was  identical  be e x p l a i n e d s o l e l y by s t r a i n  mutants of E . c o l i s t r a i n A r e q u i r e p a r t i c u l a r note  (SA) was  acid  from streptomycin-dependence  accompanied by a decrease  i n acetohydroxy  strain In t h i s  to - s e n s i t i v i t y acid  synthetase  a c t i v i t y at pH  6 . 0 and  a t pH  8.0.  T h i s r e p r e s s i o n of enzyme  a c t i v i t y r e s u l t i n g from r e v e r s i o n appears to be incomplete leaves  the s e n s i t i v e r e v e r t a n t w i t h p a r t i a l  p r o p e r t i e s as I n d i c a t e d by synthetase a c t i v i t y and Table V ) .  I t was  t h i s mutant (SA)  The  streptomycin-dependent  the s l i g h t l y e l e v a t e d acetohydroxy a c i d  the low D/S  r a t i o s (Table I I I  previously pointed  and  out t h a t the growth of  i n the presence of low a n t i b i o t i c  (Table I) suggested i t might a l s o possess tendencies.  concentrations  streptomycin-resistant  f a c t t h a t the acetohydroxy a c i d synthetase  l e v e l i s de-repressed upon mutation from streptomycin to dependence and  r e v e r s i b l y re-repressed  during  sensitivity  back-mutation  to the s e n s i t i v e s t a t e , s t r o n g l y suggests t h a t g e n e t i c of t h i s enzyme i s l i n k e d to the l o c u s of streptomycin Umbarger and  Brown  control dependence.  reported a p e c u l i a r lack  (1958b)  o f l i n e a r response i n the formation  of ^ - a c e t o l a c t a t e as  j u n c t i o n of e x t r a c t c o n c e n t r a t i o n .  A f t e r the completion of  present  work, a  and  a  c o f a c t o r , the a d d i t i o n of which c o r r e c t e d  anomalous b e h a v i o r ( L e a v i t t , 1 9 6 4 ) was as f l a v i n adenine d i n u c l e o t i d e  i s o l a t e d and  (Bauerle,  et a l ,  the this  identified  1 9 6 4 ) .  Further  experiments were t h e r e f o r e immediately c a r r i e d out to determine whether or not  the a d d i t i o n of FAD  to the r e a c t i o n system a l 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 s e n s i t i v e and  dependent s t r a i n s .  The  data of Table V c l e a r l y i n d i c a t e s  t h a t although c<-acetolactate-formation seven f o l d i n the presence of t h i s  i s stimulated  c o f a c t o r , the D/S  s i x to ratio in  the presence  or absence of FAD  remained unchanged.  The  which FAD p l a y s i n t h i s r e a c t i o n sequence i s unknown.  role However,  r e c e n t l y Hogg, e t a l , (1965) have r e p o r t e d f i n d i n g i r o n  (Fe"**)  a s s o c i a t e d with, t h i s enzyme complex and suggest t h a t i t s f u n c t i o n may  i n v o l v e the coenzyme. In order t o e l i m i n a t e the p o s s i b i l i t y t h a t the e l e v a t e d  l e v e l of acetohydroxy  a c i d synthetase was  due  to a general  s t i m u l a t i o n of enzyme f o r m a t i o n i n the dependent mutant, o t h e r enzymes were s t u d i e d i n the three mutants of s t r a i n A. obvious  from the data of Table IX t h a t mutation  from  It is  antibiotic  dependence to s e n s i t i v i t y or dependence to r e s i s t a n c e has  no  e f f e c t on g l u c o k i n a s e or glutamic dehydrogenase a c t i v i t y . while the involvement i n c r e a s e d acetohydroxy  of o t h e r enzymes i s not excluded,  Hence,  the  a c i d synthetase a c t i v i t y cannot be  attri-  buted to a g e n e r a l d e - r e p r e s s i o n of enzymic a c t i v i t y i n the dependent mutant.  In t h i s c o n n e c t i o n , i t should a l s o be  t h a t 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.  noted  of p r o t e i n  excludes the p o s s i b i l i t y t h a t d e - r e p r e s s i o n of acetohydroxy  acid  synthetase i s a n o n - s p e c i f i c event. Concurrent work from t h i s l a b o r a t o r y suggests t h a t the enzymes reductoisomerase  (Lau, I966) and Transaminase B  (Unpublished Observation) which c a t a l y s e subsequent steps i n the b i o s y n t h e t i c pathway a r e not de-repressed i n the dependent organism.  The dihydroxy a c i d dehydrase has not y e t been s t u d i e d .  T h e r e f o r e , the evidence accumulated  thus f a r i n f e r s t h a t the  - 45 e x c r e t i o n of L - v a l i n e (and L - l e u c i n e ) from s t r e p t o m y c i n dependent mutants of E. c o l i r e s u l t s s o l e l y from a d e - r e p r e s s i o n o f the c o n t r o l enzyme, acetohydroxy the subsequent  b i o s y n t h e t i c enzymes.  a c i d synthetase and not of The  f a c t t h a t only t r a c e  q u a n t i t i e s of L - l e u c i n e are d e t e c t e d i n the supernatant  fluids  (Bragg and P o l g l a s e , 1 9 6 2 ;  suggests  Tirunarayanan, et a l , 1 9 6 2 )  some feedback mechanism c o n t r o l s the d r a i n of e < - k e t o i s o v a l era te to the 6-carbon product ( P r e u n d l i c h , e t a l , 1 9 6 2 ) . III.  Formation o f o<.-Acetohydroxybutyrate  by Streptomycin Mutants  As i n d i c a t e d i n F i g . 2 and F i g . 3 the enzyme a c e t o hydroxy  a c i d synthetase i s i n v o l v e d not only w i t h the s y n t h e s i s  of L - v a l i n e and L - l e u c i n e but a l s o with the s y n t h e s i s o f Li s o l e u c i n e v i a the i n t e r m e d i a t e ^ - a c e t o h y d r o x y b u t y r a t e .  Since  t h e r e i s no evidence f o r the e x c r e t i o n of L - i s o l e u c i n e by dependent mutants, i t was  important to determine  whether an  e l e v a t e d ^ - a c e t o h y d r o x y b u t y r a t e l e v e l i s formed i n response the d e - r e p r e s s i o n of acetohydroxy a c i d s y n t h e t a s e . hydroxy  to  I f aceto-  a c i d synthetase s t i m u l a t i o n i s due t o an i n c r e a s e d r a t e  of enzyme f o r m a t i o n r a t h e r than t o 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 p r o d u c t i o n  should correspond t o the D/S  r a t i o f o r s y n t h e s i s of *<-aceto-  hydroxybutyrate. As mentioned i n the " M a t e r i a l s and Methods", the only q u a n t i t a t i v e assay method s e n s i t i v e enough t o measure the l e v e l  -  46  -  of ©c-acetohydroxybutyrate formed i n t h i s system was b i o l o g i c a l assay d e s c r i b e d  by L e a v i t t and  Umbarger  the m i c r o (i960).  T h i s procedure measures the degree to which i n h i b i t i o n E. c o l i  s t r a i n K-12  or p r e c u r s o r s  by L - v a l i n e can be r e v e r s e d  of t h i s compound.  t h a t L - i s o l e u c i n e was  Umbarger and  reversed  6-carbon p r e c u r s o r s  ^-methylvalerate  to r e s t o r e  a  of L - i s o l e u c i n e ,  and ©<, (3dihydroxy  (3-methylvalerate 4-carbon  i n h i b i t i o n as w e l l as d i d L - i s o l e u c i n e i t s e l f , a  p r e c u r s o r , <*.-ketobutyrate was the pathway was Since  of L - v a l i n e ,  by i n t e r f e r e n c e w i t h i t s b i o s y n t h e s i s .  Furthermore, while the eK-keto  L-isoleucine  Brown (1955) noted  a non-competitive a n t a g o n i s t  as would be expected i f L - i s o l e u c i n e served d e f i c i e n c y created  by  of  ineffective.  T h i s suggested that  b l o c k e d a t the acetohydroxy a c i d synthetase  the acetohydroxy a c i d synthetase of s t r a i n K-12  s e n s i t i v e to feedback by L - v a l i n e L e a v i t t and  Umbarger, 1962)  (Umbarger and  i s extremely  Brown, 1958a;  the a d d i t i o n of L - v a l i n e i n the  absence of L - i s o l e u c i n e would simultaneously of both oi-acetolactate  step.  i n h i b i t the  and *<-acetohydroxybutyrate and  synthesis  ultimately  would prevent the growth of the organism as a consequence of starvation for L-isoleucine. To e s t a b l i s h the c u l t u r e to L - v a l i n e and  s e n s i t i v i t y of the E. c o l i  the r e v e r s i b i l i t y  L - i s o l e u c i n e , a growth experiment was of which are g i v e n i n Table VI. growth was  K-12  of t h i s i n h i b i t i o n  c a r r i e d out,  by  the r e s u l t s  In the absence of L - i s o l e u c i n e  n e g l i g i b l e even a f t e r 72  hours.  However, the addition  (35  of L - i s o l e u c i n e a t c o n c e n t r a t i o n s c o n s i d e r a b l y lower  fold)  than the i n h i b i t o r , L - v a l i n e , p e r m i t t e d s i g n i f i c a n t growth. T e c h n i c a l problems a s s o c i a t e d with a m i c r o b i o l o g i c a l assay of t h i s type on o c c a s i o n gave e r r a t i c r e s u l t s d u r i n g the d e t e r m i n a t i o n of the standard curve. the standard curve employed i n t h i s work was of  was  For t h i s  a composite  v a l u e s obtained i n s e v e r a l separate experiments  out i n d u p l i c a t e .  The  particularly reason, plot  each c a r r i e d  standard curve i s shown i n F i g . 4 .  r e p o r t e d by L e a v i t t and Umbarger ( i 9 6 0 )  t h a t a t low  t r a t i o n s (upto 0.02^umoles)c<-acetohydroxybutyrate s t i m u l a t e the growth of i n h i b i t e d E. c o l i T h e r e f o r e , the end'-product  It  concen-  and L - i s o l e u c i n e  to the same e x t e n t .  r a t h e r than the i n t e r m e d i a t e was  used  as a standard and the r e a c t i o n mixtures were d i l u t e d to g i v e c o n c e n t r a t i o n s of ©<-acetohydroxybutyrate which would f a l l l i n e a r p o r t i o n of the standard curve. r o u t i n e l y assayed to  reduce  in triplicate  I n a d d i t i o n , e x t r a c t s were  ( i n i t i a l l y i n d u p l i c a t e ) i n order  systematic e r r o r . Sinceu-acetohydroxybutyrate  b o i l i n g f o r 5 minutes,  the assay was  i s d e c a r b o x y l a t e d by  rendered s p e c i f i c f o r t h i s  compound by t e s t i n g each sample b e f o r e and a f t e r heat It  on the  treatment.  can be seen from Table V I I t h a t the growth s t i m u l a t e d by  o<-acetohydroxybutyrate by the remainder  i s 5 0 - f o l d g r e a t e r than t h a t s t i m u l a t e d  of the i n t e r m e d i a t e s and by L - i s o l e u c i n e i t s e l f .  These data a r e supported by the work of Umbarger, e t a l  (i960).  They found that even with crude e x t r a c t s which were capable of  - 48 c o n v e r t i n g ©(-acetohydroxybutyrate t o l a t e r i n t e r m e d i a t e s i n the L - i s o l e u c i n e pathway, no other L - i s o l e u c i n e p r e c u r s o r s were d e t e c t e d as products u n l e s s NADPH had a l s o been added t o the r e a c t i o n system. Table V I I I c l e a r l y i n d i c a t e s t h a t the D/X r a t i o s o f acetohydroxy a c i d synthetase  a r e c o n s t a n t , r e g a r d l e s s o f whether  the a c t i v i t y i s determined by the f o r m a t i o n of «<-acetolactate o r *f-acetohydroxybutyrate. I t i s i n t e r e s t i n g t o note t h a t the r e s i s t a n t mutant o f s t r a i n A i n the presence of d i h y d r o s t r e p t o m y c i n c o n s i s t e n t l y e x h i b i t e d a h i g h e r enzyme l e v e l than d i d the same mutant i n the absence o f a n t i b i o t i c  (see T a b l e s IV and V I I I ) .  Although  this  phenomenon, which has a l r e a d y been mentioned i n r e g a r d t o the sensitive  (SA) r e v e r t a n t , cannot be e x p l a i n e d e n t i r e l y , i t  probably a r i s e s as a consequence o f "incomplete the dependent l o c u s d u r i n g r e v e r s i o n (Hashimoto,  suppression" of  i960).  Enzyme a c t i v i t i e s as determined by the f o r m a t i o n o f ^-acetohydroxybutyrate  were lower by  30-36$ f o r s t r a i n A and  37-44$ f o r s t r a i n E than enzyme a c t i v i t i e s determined by the f o r m a t i o n o f the s h o r t e r - c h a i n e d i n t e r m e d i a t e , *<-acetolactate. Umbarger and Brown (1958b) r e p o r t e d s i m i l a r d i m i n i s h e d enzyme activity E. c o l i of due  these  foro<-acetohydroxybutyrate formation i n e x t r a c t s o f s t r a i n K-12.  I t i sdifficult  t o e v a l u a t e the s i g n i f i c a n c e  f i g u r e s with respect t o r e l a t i v e substrate  t o the unique s u b s t r a t e requirements  specificity,  of t h i s enzyme complex.  Since  - a c e t o l a c t a t e f o r m a t i o n i n v o l v e s the condensation o f  two moles o f pyruvate and <?<f-acetohydroxybutyrate  formation  r e q u i r e s one mole each of ^"-ketobutyrate and p y r u v a t e , i t i s not p o s s i b l e even i n the presence of a h i g h *C-ketobutyrate c o n c e n t r a t i o n t o estimate the p r o p o r t i o n of pyruvate each p r o d u c t .  Wagner, e t a l , (1965)  n a v  e  entering  s t u d i e d the i n v i t r o  s y n t h e s i s of L - v a l i n e and L - i s o l e u c i n e by p a r t i c u l a t e  fractions  of Neurospora c r a s s a . T h e i r r e s u l t s i n d i c a t e t h a t 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 a d d i t i o n , L - i s o l e u c i n e f o r m a t i o n i n c r e a s e s  l i n e a r l y w i t h i n c r e a s i n g <*-ketobutyrate c o n c e n t r a t i o n but never exceeds 70$ o f the L - v a l i n e forming c a p a c i t y o f the p r e p a r a t i o n . They i n t e r p r e t t h i s as meaning t h a t the " a c t i v e a c e t a l d e h y d e " (from pyruvate) i s s y n t h e s i z e d and r e a c t s with the two k e t o a c i d s a t a common a c t i v e s i t e on the enzyme.  Hence, supra-  c r i t i c a l c o n c e n t r a t i o n s o f ^ - k e t o b u t y r a t e would i n h i b i t  the  f o r m a t i o n o f the a c t i v e acetaldehyde and e v e n t u a l l y the s y n t h e s i s of both L - v a l i n e and L - i s o l e u c i n e .  For t h i s reason they suggest  t h e r e must be a r e g u l a t o r y mechanism i n v i v o c o n t r o l l i n g the o<-ketobutyrate l e v e l .  The evidence accumulated  the enzyme t h r e o n i n e dehydratase  thus f a r suggests  (Umbarger and Brown, 1958a;  Changeux, I 9 6 I ) . T h e r e f o r e , i t seems r e l a t i v e l y s a f e t o conclude  that  although acetohydroxy a c i d synthetase i s d e - r e p r e s s e d w i t h r e s p e c t to K-acetohydroxybutyrate  f o r m a t i o n i n the dependent mutant,  L - i s o l e u c i n e does not accumulate i n v i v o due t o the c o n t r o l o f  - 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 i n c r e a s e d amounts of *<-ketobutyrate  the e l e v a t e d p y r u v a t e l e v e l  1964a) w i l l r a p i d l y be c o n v e r t e d  1962;  (Bragg and P o l g l a s e ,  t o 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 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 Dependent Mutants of E s c h e r i c h i a c o l i . Previous  1962;  De-repression  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  1963a; 1963b; 1963c) c o n c e r n i n g  the i n v o l v e m e n t of s t r e p -  t o m y c i n w i t h the m e t a b o l i s m of s t r e p t o m y c i n  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) o f 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 o x i d a t i v e processes.  (2)  the  consider  the r e s u l t s of the p r e s e n t  work i n terms of a p o s s i b l e  for  behavior  streptomycin  excretion  the impairment of c e r t a i n  I t i s i n t e r e s t i n g therefore to  the abnormal m e t a b o l i c  Polglase,  e x h i b i t e d by the  explanation  various  mutants.  In general, streptomycin °f E. c o l i demonstrate no m e t a b o l i c a e r o b i c a l l y on g l u c o s e of streptomycin  s e n s i t i v e and  resistant strains  i r r e g u l a r i t i e s when grown  i n the absence of a n t i b i o t i c .  The  addition  t o 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 o f e x p o n e n t i a l growth f o l l o w e d by the l i b e r a t i o n of metabolites.  The  extracellular  major e x c r e t i o n p r o d u c t i s p y r u v a t e w i t h l e s s e r  q u a n t i t i e s of L - a l a n i n e and L - v a l i n e (Bragg and  P o l g l a s e , 1962).  - 51 The formation o f the amino a c i d s can be d i r e c t l y r e l a t e d t o the i n c r e a s e d amounts o f pyruvate, and L - a l a n i n e by d i r e c t aminat i o n and L - v a l i n e v i a <X-acetolactate and r e d u c t i o n .  Streptomycin  also reportedly i n h i b i t s oxidative phosphorylation i n sensitive organisms  (Bragg and P o l g l a s e ,  1963d).  The r e s i s t a n t mutant, when grown i n the presence o f (1000  antibiotic  u n i t s p e r ml.), produced  both pyruvate and l a c t a t e  elevated l e v e l s of  (Bragg and P o l g l a s e ,  1962).  This  o b s e r v a t i o n supported e a r l i e r o b s e r v a t i o n s by Rosanoff and Sevag  (1953).  T h i s suggests t h a t the r e s i s t a n t organism  utilizes  pathways o f anaerobic metabolism when grown i n the presence o f antibiotic.  However, no i n h i b i t i o n o f o x i d a t i v e p h o s p h o r y l a t i o n  c o u l d be demonstrated  i n the r e s i s t a n t e x t r a c t s .  p o i n t e d out, n e i t h e r the s e n s i t i v e nor r e s i s t a n t  As p r e v i o u s l y organisms  e x h i b i t e d e l e v a t e d acetohydroxy a c i d synthetase a c t i v i t i e s whether i n the presence o r absence  o f streptomycin.  The streptomycin dependent mutant accumulates and e x c r e t e s L - v a l i n e d u r i n g o x i d a t i o n o f glucose i f two environmental requirements a r e s a t i s f i e d .  First,  streptomycin or d i h y d r o s t r e p -  tomycin must be present and second a e r a t i o n 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 d e p r i v a t i o n r e s u l t s i n the p r o d u c t i o n o f l a c t a t e from glucose i n s t e a d o f L - v a l i n e (Bragg and P o l g l a s e , 1964a).  Subsequent work showed that the a d d i t i o n o f streptomycin  to d e p l e t e d c e l l s i n i t i a t e d a r a p i d f o r m a t i o n o f L - v a l i n e with a concurrent decrease i n pyruvate and l a c t a t e accumulation.  I t has  - 52 r e c e n t l y been shown t h a t the l e v e l o f acetohydroxy synthetase i n streptomycin-depleted-dependent  acid  c e l l s i s repressed  but i n c r e a s e s l i n e a r l y upon the a d d i t i o n of a n t i b i o t i c t o a l e v e l s e v e r a l f o l d g r e a t e r than t h a t of the 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  (Polglase, i n press).  i n t h i s mutant, streptomycin appears of acetohydroxy accumulation  corresponding Therefore,  to s t i m u l a t e the d e - r e p r e s s i o n  a c i d synthetase i n order t o a l l e v i a t e  the  of m e t a b o l i t e s 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 a e r o b i c metabolism.  Although  the a c t u a l s i t e of  the metabolic impairment remains unknown, Bragg and P o l g l a s e (1963c;  1963d) have presented data which advocates  the e l e c t r o n -  t r a n s p o r t c h a i n as a s i t e of a c t i o n of streptomycin.  I n the  dependent mutant, streptomycin (or dihydrostreptomycin) may  act  to maintain the i n t e g r i t y of an " a l t e r n a t e e l e c t r o n - t r a n s p o r t system" which u t i l i z e s l a c t a t e , under c o n d i t i o n s of a n t i b i o t i c or oxygen s t a r v a t i o n or L - v a l i n e , under a e r o b i c , a n t i b i o t i c supplemented c o n d i t i o n s as t e r m i n a l h y d r o g e n a c c e p t o r s . a scheme d e - r e p r e s s i o n of acetohydroxy  In such  a c i d synthetase and hence  s t i m u l a t i o n of L - v a l i n e s y n t h e s i s c o u l d serve two f u n c t i o n s . F i r s t , pyruvate which may  accumulate due t o m o d i f i c a t i o n s i n  t e r m i n a l o x i d a t i o n 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 n e u t r a l end-product.  Second, s i n c e the f o r m a t i o n of L - v a l i n e  r e q u i r e s NADPH, the s t i m u l a t i o n of t h i s pathway w i l l h e l p t o m a i n t a i n the l e v e l o f o x i d i z e d coenzyme e s s e n t i a l f o r o x i d a t i v e  - 53 metabolism.  I t has been r e p o r t e d (Bragg and P o l g l a s e , 1964b)  t h a t the i s o c i t r i c dehydrogenase (NADPH enzyme) l e v e l i n s u p p l e mented-dependent c e l l s was depleted c e l l s .  s e v e r a l f o l d g r e a t e r than i n the  At the p r e s e n t stage of experimental  tion, i t i s d i f f i c u l t  investiga-  to a s c e r t a i n the primary f u n c t i o n o f t h i s  s t i m u l a t e d pathway, but i t i s known t h a t the  intracellular  accumulation of " c a t a b o l i t e s " of glucose metabolism  can i m p a i r  the normal c o n t r o l of c e r t a i n i n d u c i b l e enzymes (Monod, Neidhardt and Magasanik, 1956; s i t u a t i o n which may  Mandelstam, 1961;  become d e l e t e r i o u s to the  R e l a t e d s t u d i e s (Hepner, 1966)  1947;  1962), a  organism.  on s t r e p t o m y c i n - s e n s i t i v e ,  - r e s i s t a n t and -dependent mutants of A e r o b a c t e r aerogenes, demonstrated  a c o r r e s p o n d i n g d e - r e p r e s s i o n of the  a c i d synthetase i n dependent organisms  acetohydroxy  of t h i s s p e c i e s .  s i n c e t h i s organism possesses the enzyme a c e t o l a c t a t e (June, 1952)  have  However,  decarboxylase  the t e r m i n a l products are a c e t o i n and 2,3  butylene  g l y c o l r a t h e r than L - v a l i n e . Rosenkranz (1963) has r e p o r t e d d e - r e p r e s s i o n of a l k a l i n e phosphatase  i n streptomycin-dependent  Escherichia  coli.  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 c o n s i s t e n t w i t h the i d e a t h a t one r o l e of the a n t i b i o t i c i n the dependent organism  i s t h a t of  an enzyme " d e - r e p r e s s o r " (Jacob and Monod, 1961). The r e g i o n s of the b a c t e r i a l c e l l concerned with carbohydrate metabolism  are not the only areas p r e s e n t l y b e i n g s t u d i e d  - 5 4 i n an attempt to e l u c i d a t e the primary action. Davies,  Evidence  s i t e of  streptomycin  ( F l a x , e t a l , 1 9 6 2 a ; Speyer, et a l , 1 9 6 2 ;  1 9 6 4 ; Pestka, e t a l , 1 9 6 5 ) accumulated over the p a s t  y e a r s p a r t i a l l y supports the " u n i t a r y h y p o t h e s i s " of Spotts S t a n i e r ( 1 9 6 2 ) that the ribosome i s the r e g i o n of the c e l l p r i n c i p a l l y a f f e c t e d by s t r e p t o m y c i n .  five  and  sensitive  The a d d i t i o n 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 p r o t e i n s y n t h e s i z i n g system c o n t a i n i n g s e n s i t i v e ribosomes g e n e r a l l y l e a d s to a m i s i n c o r p o r a 14 t i o n of C  -amino a c i d s w i t h the r e s u l t i n g f o r m a t i o n of "nonsense  p r o t e i n " (Davies, e t a l , 1 9 6 4 ) . I t was  suggested  t h a t i n the  s t r e p t o m y c i n - s e n s i t i v e organism t h i s p r o t e i n f l o o d s the c e l l irreversibly inhibits cell division.  Streptomycin  and  has no. e f f e c t  on the r e s i s t a n t ribosome under the same c o n d i t i o n s ( F l a x , e t a l , 1 9 6 2 b ) . I t i s important streptomycin-dependent  to note t h a t ribosomes prepared  c e l l s have never been shown t o demonstrate  a f u n c t i o n a l dependence on s t r e p t o m y c i n . i n c o r p o r a t i o n and  from  In f a c t , amino a c i d  s t r e p t o m y c i n b i n d i n g s t u d i e s i n d i c a t e t h a t the  3 0 S subunit of the r e s i s t a n t and dependent ribosomes are ( F l a x , e t a l , 1 9 6 2 b ; Cox,  identical  et a l , 1 9 6 4 ) .  A t the present time i t i s d i f f i c u l t  to d e r i v e a s i n g l e  h y p o t h e s i s capable of e x p l a i n i n g the numerous g e n e t i c , p h y s i o l o g i c a l and b i o c h e m i c a l o b s e r v a t i o n s which have been r e p o r t e d on streptomycin mutants.  Since a number of chemical l e s i o n s a t  d i v e r g e n t s i t e s of the b a c t e r i a l genome can g i v e r i s e to v a r i o u s s t r e p t o m y c i n - r e s i s t a n t phenotypes and a t l e a s t one  dependent  phenotype and s i n c e many workers inadequately  describe  c h a r a c t e r i s t i c s of t h e i r mutants and the c o n d i t i o n s which they were obtained,  i t is difficult  e q u i v a l e n t mutants have been s t u d i e d .  the  under  t o a s c e r t a i n whether  I t i s h i g h l y probable  t h a t not only do the phenotype of the r e s i s t a n t mutants but a l s o the b i o c h e m i c a l  mechanisms of r e s i s t a n c e c o n t r o l l e d  by these mutants, ; (Watanabe and Watanabe, 1959&; Brock, 1 9 6 4 ) .  differ  1959h;  Hence, i f progress i s t o be made towards the  understanding of streptomycin  a c t i o n by comparing the p r o p e r t i e s  o f mutants, i t i s e s s e n t i a l t h a t the g e n e t i c and p h y s i o l o g i c a l 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.  REFERENCES  Anand, N., D a v i s , B.D., and Armitage, A.K. i 9 6 0 Nature  18£, 2 2 .  B a r k u l l s , I.L. 1953 J . Bact 6£, 3 3 7 . B a u e r l e , R.H., F r e u n d l i c h , M., St^rmer, F.C. and Umbarger, H.E. 1964 Biochim. Biophys. A c t a 9_2, 142. B e r t a n i , G. 1951 Genetics £ 6 , 5 9 8 . Bragg,  P.D. and P o l g l a s e , W.J. 1962 J . B a c t . 84, 3 7 0 .  Bragg, P.D. and P o l g l a s e , W.J. 1963a J . Bact. 8£, 5 9 0 . Bragg, P.D. and P o l g l a s e , W.J. 1963b J . Bact. 8JI, 7 9 5 . Bragg, P.D. and P o l g l a s e , W.J. 1963c J . Bact. 8 6 , 5 4 4 . Bragg, P.D. and P o l g l a s e , W.J. 1963d J . Bact. 8 6 , 1236. Bragg, P.D. and P o l g l a s e , W.J. 1964a J . Bact. 8 8 , 1006. Bragg, P.D. and P o l g l a s e , W.J. 1964b J . Bact. 8 8 , 1399* Brock, T.D. 1964 F e d e r a t i o n Proc. 2^, 9 6 5 . Changeux, J.P. I96I C o l d S p r i n g Harb. Symp. Quant. B i o l . 26, 3 1 3 . Cox,  E.C., White, J.R. and F l a x , J.G. 1964 P r o c . N a t l . Acad. S c i . U.S. £ 1 , 7 0 3 .  Davies, J . E . 1964 P r o c . N a t l . Acad. S c i . U.S. ^1, 6 5 9 . Davies, J . E . , G i l b e r t , W. and G o r i n i , L. 1964 P r o c . N a t l . Acad. S c i . U.S. £ 1 , 8 8 3 . D a v i s , B.D. and M i n g i o l i , E.S. 1950 J . Bact. 6 0 , 17. Demerec, M. 1951 G e n e t i c s j[6, 5 8 5 . D e s a i , I.D. and P o l g l a s e , W.J. I965 Biochim. Biophys. A c t a . 110, 181. E r d o s , T. and Ullman, A. 1959 Nature  19J2, 6 3 3 . '  F l a x , J.G., Cox, E.C. and White, J.R. 1962a Biochem. Biophys. Res. Commun. £, 385* F l a x , J.G., Cox, E . C , W i t t i n g , M.L. and White, J.R. 1962b Biochem. Biophys. Res. Commun. £, 3 9 0 .  -  57  -  F r e u n d l i c h , M., Burns, R.O. and Umbarger, H.E. 1 9 6 2 P r o c . N a t l . Acad. S c i . U.S. 48, 1804. Green, D.E., W e s t e r f e l d , W.W., Vennesland, 1942 J . B i o l . Chem. 145, 6 9 .  B. and Knox, W.E.  Halpern, Y.S. and Umbarger, H.E. 1 9 5 9 J . B i o l . Chem. 2 J 4 , 3 0 6 7 . Hashimoto, K. i 9 6 0 G e n e t i c s 4 £ , 4 9 . Heptner, A.S. I 9 6 6 B. Sc. T h e s i s (Honours), U n i v e r s i t y o f B r i t i s h Columbia, Vancouver, Canada. Hogg, R.W.,  C h i t r a , A.B. and B r o q u i s t , H.P. I 9 6 5 J . Bact. 2£» 1 2 6 5 .  Jacob, F. and Monod, J . I 9 6 I J . Mol. B i o l . 3_, 3 1 8 . J u n i , E . 1 9 5 2 J . B i o l . Chem. 1 9 5 , 7 1 5 . Landman, O.E. and Burchard, W. 1 9 6 2 Proc. N a t l . Acad. S c i . U.S. 48, 2 1 9 . Lau, D. 1 9 6 6 M. Sc. T h e s i s , U n i v e r s i t y o f B r i t i s h Vancouver, Canada.  Columbia,  L e a v i t t , R., and Umbarger, H.E. i 9 6 0 J . B a c t . 8 0 , 18. L e a v i t t , R., and Umbarger, H.E. I 9 6 I J . B i o l . Chem. 2 J 6 , 2486. L e a v i t t , R., and Umbarger, H.E. 1 9 6 2 J . Bact. 8J_, 6 2 4 . L e a v i t t , R., 1 9 6 4 J . Bact. 8 8 , 1 7 2 . Lowry, O.H., Rosebrough, N.J., F a r r , A.L. and R a n d a l l , R.J. 1 9 5 1 J . B i o l . Chem. 1 2 2 , 2 6 5 . Mager, J . , B e n e d i c t , M. and Artman, M. 1 9 6 2 Biochim. Acta. 6 2 , 2 0 2 .  Biophys.  Mandelstam, J . I 9 6 I Biochem. J . Z£, ^ 8 9 . Mandelstam, J . 1 9 6 2 Biochem. J . 82, 4 8 9 . Matney, T., Goldschmidt, E.P. and Bausum, H.T. i 9 6 0 B a c t e r i d . P r o c , p. 186. Monod, J . 1 9 4 7 Growth 1 1 , 2 2 3 . Neidhardt, F.C. and Magasanik, B. 1 9 5 6 Biochim. Acta. 2 1 , 3 2 4 .  Biophys.  - 58 Newcombe, H.B. and Hawirko, R. 1949 J . Bact. 57,  565.  Newcombe, H.B. and Nyholm, M.H. 1950 G e n e t i c s 2S» 6°3» Pestka, S., Marshall., R. and N i r e n b e r g , M. I965 P r o c . N a t l . Acad. S c i . U.S. 639. P o l g l a s e , W.J. Can. J . Biochem. i n p r e s s . Radhakrishnan, A.N. and S n e l l ,  E.E. i960 J . B i o l . Chem. 2J£, 2316.  Roote, S.M. and P o l g l a s e , W.J. 1955 Can. J . Biochem. P h y s i o l . Rosanoff, E . I . and Sevag, M.G. 1953 A n t i b i o t . Chemotherapy 3_, 495. Rosenkranz, H.S. I963 Biochem. 2, 122. S c o t t , G.W.  1949 B r i t i s h J . Expt. Path. ^Q, 501.  Speyer, J.P. L e n g y e l , P. and B a s i l i o , Acad. S c i . U.S. 48, 684. S p o t t s , C.R. and S t a n i e r , R.Y.  I96I  C. 1962 P r o c . N a t l .  Nature 192. 633.  S p o t t s , C.R. 1962 J . Gen. M i c r o b i o l . 28, 3^7. Tatum, E.L. 1946 Cold S p r i n g Harb. Symp. ^uant. B i o l . 2,  278.  Tirunarayanan, M.O., V i s c h e r , W.A. and Renner, U. I962 A n t i b i o t . Chemotherapy 12, 117. Umbarger, H..E. and Brown, B. 1955 J . B a c t . 20., 241. Umbarger, H.E. 1958 B a c t e r i o l . P r o c . 1958,  111.  Umbarger, H.E. and Brown, B. 1958a J . B i o l . Chem. 233.  415.  Umbarger, H.E. and Brown, B. 1958b J . B i o l . Chem.  II56.  Umbarger, H.E., Brown, B. and E y r i n g , E . J . i960 J . B i o l . Chem. 235,  1425.  Umbarger, H.E. and D a v i s , B.D. 1962 I n The B a c t e r i a V o l . I l l , I67. E d i t e d by I.C. Gunsalus and R.Y. S t a n i e r , New York: Academic P r e s s , I n c . Umbarger, H.E. and F r e u n d l i c h , M. I965 Biochem. Biophys. Res. Commun. 18, 889.  792.  59 -  U m b r e i t , W.W. 1953 J . B a c t . 66, 7 4 . Voges, 0 . and P r o s k a u e r ,  B. I 8 9 8 Hyg. u. I n f e k t i o u s k r 2 8 , 2 0 .  Wagner, R.P., B e r g q u i s t , A. and Baree, T. 1965 B i o p h y s . A c t a . 100, 4 4 4 .  Biochim.  Watanabe, T. and Watanabe, M. 1959a J . Gen. M i c r o b i o l . 21_, 16. Watanabe, T. and Watanabe, M. 1959h J . Gen. M i c r o b i o l . 2 1 , 3 0 . W e s t e r f e l d , W.W.  1945 J . B i o l . Chem. l 6 l , 4 9 5 .  W i t k i n , E.M. 19^7 G e n e t i c s ^ 2 , 2 2 1 .  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0093662/manifest

Comment

Related Items