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A study of the intermediate metabolism of Pseudomonas aeruginosa Ney, Phyllis Winifred 1948

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££3 /vj  37  SX eo/o. /  A STUDY OF THE INTERMEDIATE METABOLISM OF PSEUDOMONAS AERUGINOSA  - by P h y l l i s Winifred Ney  A Thesis Submitted In P a r t i a l Fulfilment of the Requirements for the Degree of MASTER OF SCIENCE IN AGRICULTURE i n the DEPARTMENT OF DAIRYING  THE UNIVERSITY OF BRITISH COLUMB May, 1948.  ABSTRACT  The i n t e r m e d i a t e metabolism  of P, a e r u g i n o s a  (A.T.C. 9027) has been s t u d i e d m a n o m e t r i c a l l y t o - determine the pathway o f g l u c o s e breakdown. found t o respond  S i n c e t h e organism was  t o i n c r e a s e d amounts o f i r o n and s i n c e no  p o s i t i v e evidence o f an a n a e r o b i c r e s p i r a t o r y mechanism c o u l d be o b t a i n e d i t was c o n c l u d e d t h a t t h i s organism was an o b l i g a t e aerobe. Among those compounds r a p i d l y a t t a c k e d by r e s t i n g suspensions  o f c e l l s h a r v e s t e d f r o m a glucose-ammonium  s u c c i n a t e medium were s u c c i n i c a c i d , g l u c o s e , g l u c o n i c a c i d and 2 - k e t o g l u c o n i c a c i d . a c i d was n o t a t t a c k e d . as 5 x 10  However, 5 - k e t o g l u c o n i c  C o n c e n t r a t i o n s of malonate as h i g h  M had no e f f e c t on t h e o x i d a t i o n o f g l u c o s e o r  s u c c i n a t e by these c e l l s , however they d i d markedly r e t a r d the r a t e of a d a p t a t i o n t o s u c c i n a t e by c e l l s  harvested  from a g l u c o s e ammonium phosphate medium. By growing  t h e c e l l s on a c o m p l e t e l y i n o r g a n i c  medium p l u s s p e c i f i c s u b s t r a t e , i t was p o s s i b l e t o show t h a t g l u c o s e grown c e l l s r a p i d l y o x i d i z e d g l u c o n i c and 2 - k e t o g l u c o n i c a c i d s but s h o r t p e r i o d s of a d a p t a t i o n were r e q u i r e d f o r the u t i l i z a t i o n of s u c c i n i c a c i d (20 - 30 mins.) Moreover i t appeared t h a t c e l l s h a r v e s t e d f r o m s u c c i n a t e and lactate-ammonium phosphate media were n o t pre-adapted  - 2 to t h e breakdown of g l u c o s e .  The r a t e o f o x i d a t i o n was  i n i t i a l l y slow, p r o g r e s s i v e l y i n c r e a s i n g throughout the experimental period. To c o n f i r m t h e presence of a d a p t i v e enzymes f o r the d i s s i m i l a t i o n of g l u c o s e , a comparison was made of t h e a b i l i t y of c e l l s grown i n t h e presence o f e i t h e r g l u c o s e o r acetate t o attack glucose.  Whereas c e l l s h a r v e s t e d from a  g l u c o s e medium o x i d i z e d t h i s s u b s t r a t e a t a r a p i d and cons t a n t r a t e , c e l l s grown i n t h e presence o f a c e t a t e showed no a c t i v i t y u n t i l a f t e r one hour of i n c u b a t i o n . S i n c e g l u c o s e was a t t a c k e d by an a d a p t i v e  enzyme  system i t was p o s s i b l e t o s t u d y i n t e r m e d i a t e g l u c o s e metab o l i s m by means of t h e simultaneous of S t a n i e r .  adaptation  technique  A comparison of t h e o x i d a t i v e a c t i v i t y of  g l u c o s e and a c e t a t e c e l l s showed t h a t w h i l e g l u c o s e a t t a c k e d g l u c o s e , g l u c o n i c a c i d and 2 - k e t o g l u c o n i c r a p i d l y and d i r e c t l y ,  cells acid  a c e t a t e c e l l s r e q u i r e d a p e r i o d of  a d a p t a t i o n o f a t l e a s t one hour b e f o r e - t h e y c o u l d o x i d i z e these s u b s t r a t e s .  I t was t h e r e f o r e concluded  t h a t the  breakdown of g l u c o s e by t h i s a e r o b i c organism proceeded by way o f g l u c o n i c a c i d and 2 - k e t o g l u c o n i c  a c i d , and t h a t  s u c c i n i c a c i d was not an i n t e r m e d i a t e m e t a b o l i t e .  ACKNOWLEDGEMENT  I wish to express my sincere appreciation to Dr. J. J. R. Campbell for his interest i n this work. I am also grateful to the National Research Council of Canada f o r f i n a n c i a l assistance received f o r the period A p r i l 1, 1947 to March 31, 1948, and to The University of B r i t i s h Columbia f o r a research grant during the summer of 1946. P. W. N.  TABLE OF CONTENTS 1  INTRODUCTION PART 1 IRON REQUIREMENTS Introduction Methods Bacteriological  4  .-«•«.•••©.i.  9 .....o  Chemical  Experimental and Discussion Summary ©.•..•©»••©. PART II THE PREPARATION OF RESTINGCEELLS Introduction ................ o Methods Bacteriological Experimental and Discussion Nitrogen and Carbon .......... o  •  11  14 21  • •• 23 © 25  . . . . . . . . . © . 29 Minerals . . . . . . Q . c . © . . . © . . . o . e . « . . . • • • • • 32 0  Age o •»..«.• 34 Aeration ......... ...........© ........ 36 Warburg studies . . . . . . . . . © . . . . . . . o . e . . . . . . . . © 38 Summary ••••• •• 4 0 0  PART III METABOLISM Introduction © ..«.•.©. • 42 Methods Bacteriological ......©........ ......•© 45 Chemical • 48 A. Studies on c e l l s grown with ammonium succinate as nitrogen source. Anaerobic .©...•.........©....•.49 Significance of the endogenous respiration 54 Oxidation of the postulated intermediates 56 The presence of 2-ketogluconic acid i n growing cultures .... 60 Anaerobic d i s s i m i l a t i o n of • 2-ketogluconic a-e acid 64 Anaerobic d i s s i m i l a t i o n of oxalacetate ••• 65 Malonate • \. 67 i Bo Studies on c e l l s grown with ammonium phosphate as nitrogen source. Introduction •©« 74 Malonate ......©.. .©•• 77 Oxygen uptake of glucose, succinate and •lactate ammonium phosphate c e l l s ......... 78 Synthesis i n h i b i t i o n 88 C. Adaptation. Introduction .... © a • ••••••*©•..•<••• © 92 Experimental and Discussion Simultaneous adaptation ..©.©.........• 96  )  Summary •»••••••••«•••,•••€•••••••••••••••.••.•• 105 BIBLIOGRAPHY •••••••••••••••••••••••••••••••••••••••••  107  - L. _  D u r i n g t h e l a s t f i f t y y e a r s a tremendous f u n d o f knowledge r e l a t i n g t o t h e mechanisms o f b i o l o g i c a l o x i d a t i o n has been accumulated.  By f a r t h e g r e a t e s t amount o f t h i s  knowledge p e r t a i n s t o t h e a n a e r o b i c breakdown o f g l u c o s e t o a l c o h o l o r l a c t i c a c i d , and a more complete u n d e r s t a n d i n g o f t h e s e two  systems has been a c h i e v e d t h a n o f any  energy y i e l d i n g p r o c e s s e s .  comparable  As a r e s u l t o f t h e e f f o r t s o f  Lipmann, Meyerhof, C o r i , Warburg and o t h e r s , t h e step by s t e p d e g r a d a t i o n o f g l u c o s e and o t h e r c a r b o h y d r a t e s t o t h e end p r o d u c t s g l y c e r o l , l a c t i c a c i d and e t h y l a l c o h o l has been shown t o be c o u p l e d w i t h a s e r i e s o f w e l l - d e f i n e d p h o s p h o r y l a t i o n s and d e p h o s p h o r y l a t i o n s by means o f which a l a r g e p o r t i o n o f t h e energy o f t h e s u b s t r a t e i s made a v a i l a b l e t o t h e c e l l (Meyerhof  194S).  Much l e s s i s known o f t h e mechanisms w h i c h f u n c t i o n i n aerobic r e s p i r a t i o n .  S z e n t - G y o r g y i (Sumner and Somers  1943)  u s i n g p i g e o n b r e a s t muscle s u s p e n s i o n s f o r m u l a t e d an a e r o b i c hydrogen t r a n s p o r t i n g system i n v o l v i n g d l c a r b o x y l i c a c i d s . By means o f t h i s system, t h e two a v a i l a b l e hydrogens from g l y c e r a l d e h y d e a r e passed by way o f o x a l a c e t a t e , m a l a t e , coenzyme I , and t h e y e l l o w enzyme t o fumarate t o y i e l d succinic acid.  By v i r t u e o f s u c c i n i c dehydrogenase, hydrogen  i s t h e n d i r e c t l y t r a n s f e r r e d t h r o u g h cytochrome t o oxygen. Although therrao-dynamically considerable c r i t i c i s m .  sound, t h e t h e o r y has met  with  I t i s d o u b t f u l ' t h a t s u c c i n i c de-  hydrogenase can pass hydrogen d i r e c t l y t h r o u g h cytochrome without  a mediator.  F u r t h e r m o r e , as P o t t e r (1940) has  out, t h e i n c l u s i o n o f t h e o x a l a c e t a t e - m a l a t e  pointed  system i n t h e  c y c l e seems u n j u s t i f i e d s i n c e , by i t s u s e , a c c u m u l a t i o n  of  reduced coenzyme I i s o b t a i n e d . On t h e b a s i s o f t h i s and a d d i t i o n a l work, K r e b s (Evans 1942)  has f o r m u l a t e d h i s c i t r i c a c i d c y c l e f o r muscle  metabolism.  Initially,  to y i e l d . c i t r i c a c i d .  pyruvate The  condenses w i t h o x a l a c e t a t e  o x i d a t i o n t h e n proceeds by way  of  <T< k e t o - g l u t a r a t e , s u c c i n a t e , fumarate, and malate t o o x a l a c e t a t e which i n t u r n can condense w i t h a second m o l e c u l e of pyruvic a c i d . K r e b s has  In contrast t o the theory of  Szent-Gyorgyi,  suggested t h a t g r e a t e r t h a n h a l f t h e hydrogen can  be t r a n s f e r r e d t o oxygen o v e r t h e o x a l a c e t a t e - m a l a t e The  system.  d i c a r b o x y l i c a c i d s , o r i g i n a l l y thought o f as hydrogen  c a r r i e r s , a r e shown t o be i n t e r m e d i a t e s i n t h e o x i d a t i v e scheme. R e l a t i v e l y l i t t l e i s known o f t h e a e r o b i c systems functioning i n bacterial oxidation.  With f a c u l t a t i v e  organisms, i t i s g e n e r a l l y agreed t h a t under e i t h e r a n a e r o b i c or aerobic 'conditions carbohydrate pyruvate  i s d i s s i m i l a t e d to the  stage by the Meyerhof-Embden pathway.  The  subsequent  1  3 -3-  a e r o b i c stages o f t h e r e a c t i o n a r e s t i l l o b s c u r e .  Although  t h e K r e b s c y c l e f u n c t i o n s as an a e r o b i c system i n muscle, and a l t h o u g h many b a c t e r i a can u t i l i z e most o f t h e i n t e r m e d i a t e s o f t h i s c y c l e , n e v e r t h e l e s s no b a c t e r i a l c e l l h a s ever been shown t o p o s s e s s t h i s t r i c a r b o x y l i c a c i d scheme.  Recently  Lockwood (1940: 1946) I s o l a t e d k e t o g l u c o n i c and k e t o g l u t a r i c a c i d s from a c u l t u r e o f Pseudpmonas, t h u s p o i n t i n g t o an o x i d a t i v e system f o r a e r o b i c o r g a n i s m s .  S i n c e h i s work was  c a r r i e d out under u n p h y s i o l o g i c a l c o n d i t i o n s such as h i g h p r e s s u r e s , i n t e n s e a e r a t i o n and h i g h c o n c e n t r a t i o n s o f subs t r a t e , i t must be v e r i f i e d i n a more n a t u r a l  environment  b e f o r e i t can be c o n s i d e r e d o f m e t a b o l i c i m p o r t a n c e . The p r e s e n t t h e s i s i s an attempt t o u n c o v e r t h e pathway by w h i c h t h i s o r g a n i s m o b t a i n s energy f r o m g f u c o s e .  The f i r s t  p a r t o f t h e s t u d y i s concerned w i t h t h e i r o n r e q u i r e m e n t s of' t h e organism, and t h e e f f e c t o f i r o n d e f i c i e n c y on t h e a c t i v i t y of the c e l l s .  The second p a r t d e a l s w i t h t h e method  o f p r e p a r a t i o n o f r e s t i n g c e l l s u s p e n s i o n s and t h e t h i r d p a r t i s devoted t o a s t u d y o f t h e m e t a b o l i s m o f t h e c e l l s w i t h emphasis on enzymic a d a p t a t i o n .  -4PART I IRON REQUIREMENTS  t C o m p a r a t i v e l y l i t t l e i s known o f t h e i r o n r e q u i r e m e n t s o f t h e h e t e r o t r o p h i c b a c t e r i a , and t h i s l a c k o f i n f o r m a t i o n i s undoubtedly due l a r g e l y t o t h e absence o f s u i t a b l e p u r i f i c a t i o n techniques.  C o - p r e c i p i t a t i o n methods, ( E l v e h j e m  1931; S t e i n b e r g 1935) e x h a u s t i o n p r o c e d u r e s , ( M o l i s c h 1892; Roberg 1928; M o l l i a r d 1929}' and t h e u s e o f r e c r y s t a l i i z e d c h e m i c a l s (Burk and Horner 1934) have been employed. A l though t i m e consuming, t h e mold p u r i f i c a t i o n method o f M o l i s c h has y i e l d e d e x c e l l e n t r e s u l t s w i t h A. i n d o l o g e n e s (Waring and Werkman 1942).  The r e m a i n i n g p r o c e d u r e s , w h i l e  s u i t e d f o r work w i t h organisms r e q u i r i n g h i g h amounts o f i r o n , a r e i n a p p l i c a b l e t o a s t u d y o f t h e more f a s t i d i o u s bacteria. The e x t r a c t i o n methods advanced  r e c e n t l y by Waring and  Werkman (1942) employing c h l o r o f o r m and 8 - h y d r o x y q u i n o l i n e , and by Perlman  (1943T) u s i n g c a t i o n i c exchange..materials have  f a c i l i t a t e d t h e study o f t h e m i n e r a l r e q u i r e m e n t s o f b a c t e r i a at l e v e l s v e r y much l o w e r t h a n was p r e v i o u s l y p o s s i b l e .  With  a knowledge o f t h e s e r e q u i r e m e n t s i t i s now p o s s i b l e t o e s t a b l i s h t h e v a r i o u s f u n c t i o n s and r e l a t i v e importance o f inorganic ions i n c e l l u l a r  metabolism.  The r e l a t i o n s h i p o f i r o n t o growth h a s been o b s e r v e d i n many organisms  (Fulmer and Buchanan  ii3o  ) Horner and Burk  (1934) u s i n g h i g h l y p u r i f i e d c h e m i c a l s o b t a i n e d maximum /  growth o f a z o t o b a c t e r w i t h i r o n c o n c e n t r a t i o n s o f 0.025 0.055 ppm.  More r e c e n t l y W a r i n g and Werkman (194<B) have  e s t a b l i s h e d the i r o n requirements  o f s t r a i n s o f P.  aeruginosa.  A. i n d o l o g e n e s . J L . aerogenes. K . pneumoniae and E. c o l i . s t i m u l a t i o n from 6 t o 100$ was w i t h i n t h e range 0 - 0.09  ppm  o b t a i n e d f o r P. Fe.  Lily  aeruginosa  (1945) o b t a i n e d a  growth response w i t h R. t r i f o l i i from 0 - 0.t25 }igm Fe ml.  B u r t o n et a l (1947) o b s e r v e d t h a t 0.1 ;ugm  gave no pyocyanine  A  per  Fe per ml.  f o r m a t i o n but abundant growth and  pro-  d u c t i o n o f t h e f l u o r e s c e n t pigment i n c u l t u r e s o f P.a e r u g i n o s a 90^7.  Pappenheimer (1936) and Feeny, M u e l l e r and  M i l l e r (1943), i n s t u d i e s on t h e r e l a t i o n o f i r o n t o t h e t o x i n p r o d u c t i o n o f G. d i x t h t h e r i a e and C. t e t a n i .  respectively,  9  have shown t h a t maximum growth o f t h e s e organisms i s obt a i n e d w i t h amounts o f i r o n i n excess o f t h e optimum concentration f o r the production of t o x i n .  The  synthesis of  r i b o f l a v i n by Candida i s c o n t r o l l e d by t h e i r o n content  of  t h e growth medium ( B u r k h o l d e r 1943, 1944j Saunders and McLung 1943; Tanner 1945). 0.5  F o r example, i n t h e presence  of  - 1.0 4igm Fe per 100 ml., t h e h i g h e s t y i e l d s o f  r i b o f l a v i n were o b t a i n e d , w h i l e t h e a d d i t i o n o f lO^igm  per  m l . s h a r p l y reduced t h e f o r m a t i o n o f t h e v i t a m i n .  cell  The  c r o p h a r v e s t e d a t t h e optimum i r o n c o n c e n t r a t i o n f o r r i b o f l a v i n p r o d u c t i o n was  40 - 80$ o f t h a t o b t a i n e d at t h e  peak o f t h e growth c u r v e (Tanner 1945).  Because o f t h e importance o f i r o n c a t a l y s t s i n a e r o b i c r e s p i r a t i o n , t h e e f f e c t s o f i r o n d e f i c i e n c y on t h e metabolism o f t h e c e l l a r e - o f g r e a t i n t e r e s t .  Kubowitz  (1934) showed t h a t when g l u c o s e f e r m e n t a t i o n by washed c e l l suspensions  o f C. b u t v r l c u m was a l l o w e d t o t a k e p l a c e i n an  atmosphere o f c a r b o n monoxide, t h e normal a c e t i c and b u t y r i c a c i d p r o d u c t i o n was r e p l a c e d by a pure l a c t i c a c i d fermentation.  R e l a t i v e l y h i g h c o n c e n t r a t i o n s o f cyanide  (10"^M) tended t o cause t h e same s h i f t i n m e t a b o l i s m . Simon (1947) c o n f i r m e d t h e s e r e s u l t s u s i n g r e s t i n g o f C. a c e t o b u t v l i c u m . 1944  cells  Pappenheimer and Shaskan (1944;  )Agrown w i t h h i g h and l o w c o n c e n t r a t i o n s o f i r o n ,  were a b l e t o c o n f i r m and extend t h e work o f K u b o w i t z .  They  found t h a t t h e s e organisms, w h i c h p r e v i o u s l y had been cons i d e r e d t o have no i r o n systems, would produce almost s o l e l y l a c t i c a c i d when grown on t h e i r o n - d e f i c i e n t medium. t h e h i g h i r o n medium, t h e u s u a l combination  On  o f b u t y r i c and  a c e t i c a c i d s was o b t a i n e d . S i m i l a r changes c o u l d be produced i n t h e a c e t i c - f o r m i c a c i d - e t h y l a l c o h o l f e r m e n t a t i o n o f A. aerogenes 1945)  (Perlman  by growing t h e c e l l s i n a.metal d e f i c i e n t medium.  P a r t i a l r e s t o r a t i o n o f t h e n o r m a l f e r m e n t a t i o n c o u l d be o b t a i n e d by a d d i n g back s m a l l amounts o f i r o n ,  although  d e f i c i e n c i e s i n o t h e r metals made i t i m p o s s i b l e t o e s t a b l i s h t h e importance o f t h i s element. E l v e h j e m (1931) observed  t h a t growth i n l o w i r o n media  decreased t h e i r o n c o n t e n t o f y e a s t c e l l s and  significantly  reduced t h e amount o f cytochrome i n t h e organisms.  This i s  i n agreement w i t h t h e f i n d i n g s o f Waring and Werkman concerning i r o n d e f i c i e n c y (1944).  U s i n g c e l l s o f A. i n d o l o g e n e s  t h e s e w o r k e r s have shown t h a t growth i n l o w i r o n media produces c e l l s l o w i n c a t a l a s e , p e r o x i d a s e , cytochrome, f o r m i c dehydrogenase, hydrogenase and h y d r o g e n l y a s e .  Succinic  dehydrogenase, m a l i c dehydrogenase and fumarase systems were also present i n a depleted condition.  M u e l l e r (1941) has  suggested t h a t £. d i p h t h e r i a e e l a b o r a t e s t o x i n under cond i t i o n s o f i r o n d e f i c i e n c y as a compensatory mechanism t o f u n c t i o n i n t h e absence o f normal i r o n - c o n t a i n i n g enzyme systems.  The supply o f i r o n a v a i l a b l e t o c e l l s d u r i n g  growth appears t o d e t e r m i n e t h e r a t i o a t w h i c h c e r t a i n o f t h e enzyme systems e x i s t w i t h i n t h e c e l l .  That i s ,  i r o n medium, enzymes w h i c h c o n t a i n t h e element i r o n  i n a low (catalase,  p e r o x i d a s e , v e r d o - p e r o x i d a s e , cytochromes, cytochrome o x i d a s e , cytochrome p e r o x i d a s e ) o r a r e dependent on i r o n f o r t h e i r f o r m a t i o n , may be p r e s e n t i n d e p l e t e d amounts i n r e l a t i o n t o t h e amount o f c e l l p r o t o p l a s m . The p r e s e n t work i n c l u d e s a study o f t h e i r o n r e q u i r e ments o f our organism grown i n a p u r i f i e d s y n t h e t i c medium. I t has been p o s s i b l e t o e s t a b l i s h an i r o n - g r o w t h c u r v e between 0 and 0.5oigm o f i r o n p e r m l . , and t o show t h a t  cells  h a r v e s t e d from low and h i g h i r o n media do not d i f f e r s i g n i f i c a n t l y i n t h e i r o x i d a t i v e a c t i v i t y f o r g l u c o s e and  l a c t i c and a c e t i c a c i d s . On t h e b a s i s o f t h i s i n f o r m a t i o n we have c o n c l u d e d t h a t t h e o x i d a t i v e mechanisms f u n c t i o n i n g i n t h e r e s p i r a t i o n o f t h e c e l l s are s t r i c t l y aerobic i n nature.  METHODS  Bacteriological;  The c u l t u r e o f Pseudomonas a e r u g i n o s a  ATC 9027 employed throughout i n a l l respects.  t h i s work was a c t i v e and t y p i c a l  No d i f f i c u l t y w i t h d i s s o c i a t i o n o r any  o t h e r v a r i a t i o n was encountered. 'Stab c u l t u r e s o f t h e organism i n l i v e r e x t r a c t were refrigerated  agar  after growth was i n i t i a t e d at 30°C.  Before  b e i n g u s e d i n e x p e r i m e n t a l work, t h e c u l t u r e was t r a n s f e r r e d 2-3 F§  t i m e s a t 24 h r . i n t e r v a l s i n b a s a l medium p l u s 0.05 jugm p e r m l . To m a i n t a i n a v i g o r o u s u n d i s s o c i a t e d c u l t u r e a  f r e s h t r a n s f e r was t a k e n each week from a r e f r i g e r a t e d s t o c k . The  c o m p o s i t i o n o f t h e s t o c k agar was 1.0$ t r y p t o n e , 0.3$  v  K^HPO. 0,1% g l u c o s e , 0,3% g l y c e r o l , 10% l i v e r ^ 4  extract,  0,5% a g a r , 2,0% g e l a t i n e a d j u s t e d t o pH 7.2 b e f o r e a u t o c l a v i n g The  b a s a l medium u s e d i n . e x p e r i m e n t s on t h e i n f l u e n c e o f  i r o n on growth and a l s o i n t h e p r o d u c t i o n o f r e s t i n g c o n s i s t e d o f 1,0% g l u c o s e , 0.3$ N H  4  cells  s u c c i n a t e , 0.1$ K^HP0 , 4  0.2$ MgSO^^H^O and 0.1 ml s a l t s o l u t i o n p e r 500 ml d i l u t e medium.  The components were made t o volume w i t h water  r e d i s t i l l e d through The  glass.  c o m p o s i t i o n o f t h e s a l t s o l u t i o n was as f o l l o w s :  0.044$ Z n S 0 - 7 i y ) , 0.04$ CuS0 '5EgO and 0.041$ MnS0 '4H 0. 4  4  4  s  F o r t h e i r o n assay t h e inoculum was p r e p a r e d from ah 18-20  h r . c u l t u r e o f t h e organism i n t h e b a s a l medium + ++ 0.05 ;ugm Fe p e r m l . These c e l l s were h a r v e s t e d by  -10-  c e n t r i f u g a t i o n , and were washed t w i c e w i t h water r e d i s t i l l e d from g l a s s .  They were resuspended t o g i v e a b a r e l y  t u r b i d i t y (approximately One  one m i l l i o n organisms per  drop o f t h i s s u s p e n s i o n was  visible ml.).  employed as i n o c u l u m f o r  10  ml. o f medium, g i v i n g an i n t i a l p o p u l a t i o n o f about 5000 c e l l s per  ml.  I n t e s t i n g t h e i n f l u e n c e o f i r o n on t h e enzyme complement o f t h e c e l l s , r e s t i n g c e l l s u s p e n s i o n s were employed. Adequate a e r a t i o n o f t h e c u l t u r e s was 100 ml. o f medium per R o u x . f l a s k Kolle  ensured by  using  o r 50 ml. o f medium per  flask. The  c u l t u r e s were h a r v e s t e d  at 30°C.  The  incubation,  c e l l s were washed t w i c e i n h a l f t h e growth  volume w i t h a 0*9% t o 1/10  a f t e r 20-22 h r s .  NaCl s o l u t i o n , and were f i n a l l y made up  t h e o r i g i n a l volume, t h u s g i v i n g a c o n c e n t r a t i o n  t i m e s t h a t o f t h e growing c u l t u r e . manner had  an e n d o g e n o u s - r e s p i r a t i o n  oxygen per hour per 0.5  Organisms p r e p a r e d i n t h i s of  l e s s t h a n 1 0 0 j i 1.  ml c e l l s u s p e n s i o n , a r a t e w h i c h d i d  not I n t e r f e r e w i t h t h e i n t e r p r e t a t i o n o f d a t a o b t a i n e d oxidizable  ten  for  substrates.  For t h e d e t e r m i n a t i o n  o f t h e a b i l i t y o f t h e organisms  t o o x i d i z e the v a r i o u s s u b s t r a t e s , the conventional t e c h n i q u e was  used ( D i x o n 1943;  suspended i n M/15 compartment and  Warburg  Umbreit et a l 1945).  Cells  phosphate b u f f e r were p l a c e d i n the main s u b s t r a t e i n t h e sidearm o f t h e f l a s k .  After  a 10 minute e q u i l i b r a t i o n p e r i o d t h e s t o p c o c k s were c l o s e d  and t e s t r e a d i n g s were t a k e n a t 5 minute i n t e r v a l s u n t i l the columns appeared t o move a t a c o n s t a n t r a t e .  S u b s t r a t e was  t h e n t i p p e d i n and r e a d i n g s were r e c o r d e d a t 10 m i n u t e i n t e r v a l s throughout t h e e x p e r i m e n t a l p e r i o d . Depending upon t h e n a t u r e o f t h e experiment, r e a c t i o n t i m e was from 1-2 h r s .  the  A t t h e end o f 1 n r . ,  5^uM  of g l u c o s e were c o m p l e t e l y u t i l i z e d by t h e c e l l s and a cons t a n t r a t e o f oxygen uptake p a r a l l e l i n g t h a t o f t h e endogenous r e s p i r a t i o n was Chemicalt  maintained.  Pyrex g l a s s w a r e was used e x c l u s i v e l y I n t h e  p r e p a r a t i o n o f t h e medium and i n the growth which f o l l o w e d . g l a s s w a r e was  experiments  To ensure complete removal of t r a c e s of i r o n ,  c l e a n e d by s u c c e s s i v e t r e a t m e n t w i t h soap and  w a t e r , s a t u r a t e d a l c o h o l i c KOH,  d i s t i l l e d w a t e r , aqua r e g i a ,  d i s t i l l e d water and g l a s s r e d i s t i l l e d w a t e r .  No c o t t o n  p l u g s , c o r k or r u b b e r s t o p p e r s were employed a t any t i m e . The c h e m i c a l s t o be e x t r a c t e d were Merck C.P.  or  r e a g e n t grade; t h o s e t o be added f o l l o w i n g p u r i f i c a t i o n were of Merck C.P.  quality.  The n u t r i e n t s o f t h e b a s a l medium were d i s s o l v e d i n a minimum volume of water and were r e p e a t e d l y e x t r a c t e d w i t h c h l o r o f o r m and 8 - h y d r o x y q u i n o l i n e u n t i l no g r e e n c o l o r c o u l d be d e t e c t e d i n t h e c h l o r o f o r m washings. t h a t a t pH's  I t has been found  r a n g i n g ; from 6 t o 8 (Waring and Werkman, 1942)  t h i s method removes trsLce amounts o f Cu, F e , Mn and  Zn.  -12-  A d d i t i o n s o f t h e s a l t s o l u t i o n and MgS04«7H 0 were 2  made f o l l o w i n g t h e e x t r a c t i o n p r o c e d u r e . t a m i n a t i o n from u n e x t r a c t e d s a l t s was g r e a t e r t h a n 0.002 ;ugm  The t o t a l i r o n con-  c a l c u l a t e d t o be n o t  o f Pe p e r m l .  A s t a n d a r d i r o n s o l u t i o n c o n t a i n i n g . 100 ugm F e was p r e p a r e d i n water r e d i s t i l l e d t h r o u g h g l a s s PeS0 »7H 0 made t o 100 m l . ) . 4  2  + +  per ml.  (498  p e r ml mg.  Prom t h i s s t o c k s o l u t i o n  d i l u t i o n s were p r e p a r e d t o c o n t a i n 10 ;ugm, Pe  + +  1 .ugm and 0.1 >ugm  To a v o i d t h e p r e c i p i t a t i o n o f i r o n h y d r a t e s  the s o l u t i o n s were s t e r i l i z e d by passage t h r o u g h s t e r i l e sintered glass  filters.  B a c t e r i a l n i t r o g e n was determined by the m i c r o - K j e l d a h l procedure.  To 1 m l . o f t h e c e l l s u s p e n s i o n were added 1 m l .  o f c o n c e n t r a t e d H S04 and 2 drops o f 30$ H 2 0 . 2  2  The sample  was d i g e s t e d s l o w l y f o r 15-20 minutes, over a m i c r o - b u r n e r u n t i l a l l o r g a n i c m a t t e r was decomposed l e a v i n g a c l e a r solution .  A f t e r c o o l i n g , t h e d i g e s t was c a r e f u l l y d i l u t e d  w i t h 6 m l . w a t e r , and was t r a n s f e r r e d q u a n t i t a t i v e l y t o t h e d i s t i l l a t i o n apparatus.  An e x c e s s o f base (10 m l . of  c a r b o n a t e - f r e e 10 N NaOH) was r u n i n t o t h e sample, and 100 ml. o f t h e steam d i s t i l l a t e was over a 30 minute p e r i o d . w i t h 0.01N  c o l l e c t e d i n 10 m l . 0.01  N HCl  The excess a c i d was t h e n t i t r a t e d  NaOH t o the e n d - p o i n t o f m e t h y l r e d i n d i c a t o r .  The amount o f n i t r o g e n i n the sample was c a l c u l a t e d f r o m t h e e q u i v a l e n t s o f a c i d n e u t r a l i z e d by ammonia. Sodium p y r u v a t e f o r use i n r e s p i r a t o r y s t u d i e s  was  p r e p a r e d a c c o r d i n g t o t h e method o f R o b e r t s o n the f o l l o w i n g g m o d i f i c a t i o n .  (  ) with  The a l c o h o l i c s o l u t i o n o f  p y r u v i c a c i d was g r a d u a l l y n e u t r a l i z e d , and each s u c c e s s i v e crop o f c r y s t a l s was f i l t e r e d o f f as i t was p r e c i p i t a t e d . T h i s n e u t r a l i z a t i o n procedure was c o n t i n u e d u n t i l p o l y m e r i z a t i o n , as e v i d e n c e d by t h e appearance o f t h e y e l l o w condensat i o n product, occurred.  Sodium p y r u v a t e p r e p a r e d i n t h i s  manner has been f o u n d more a v a i l a b l e t o organisms o b t a i n e d by o t h e r p r o c e d u r e s  ( KVl-nif^ky < UatW^^x  than t h a t  -14-  EXPERIMENTAL Experiments were d e s i g n e d t o determine t h e i n f l u e n c e o f the  c o n c e n t r a t i o n o f i r o n i n the*, growth medium on t h e  amount o f c e l l m a t e r i a l formed.  B a c t e r i a l nitrogen, which  i s t h e most u n i v e r s a l l y r e c o g n i z e d s t a n d a r d f o r e x p r e s s i n g amount o f c e l l m a t e r i a l , was used as t h e measure o f b a c t e r i a l multiplication. The c o n c e n t r a t e d b a s a l medium was d i s p e n s e d i n f i v e m l . amounts i n t o 125 m l . Erlenmeyer f l a s k s .  To each was added  water r e d i s t i l l e d t h r o u g h g l a s s so t h a t upon t h e a d d i t i o n o f i r o n s o l u t i o n a f i n a l volume o f 10 m l . was o b t a i n e d . the  After  f l a s k s were capped w i t h b e a k e r s , t h e medium was a u t o -  c l a v e d a t 15 l b s . p r e s s u r e f o r 15 m i n u t e s .  Aseptic additions  of f i l t e r e d i r o n s o l u t i o n s were made t o each a s s a y f l a s k , and 1 drop o f t h e washed c e l l s u s p e n s i o n was i n t r o d u c e d a s inoculum. A t t h e end o f 4 days i n c u b a t i o n a t 30°C, t h e c e l l c r o p of each f l a s k was h a r v e s t e d by c e n t r i f u g a t i o n , washed t w i c e w i t h g l a s s r e d i s t i l l e d water and resuspended i n 2/5 t h e growth volume.  Duplicate 1 ml. a l i q u o t s of t h i s suspension  were used f o r theu d e t e r m i n a t i o n o f b a c t e r i a l n i t r o g e n . As shown i n T a b l e I , growth o f t h e organism i s a f u n c t i o n o f t h e i r o n c o n t e n t o f t h e medium.  A response o f  from 6% t o 100$ was o b t a i n e d i n t h e range 0-0.5jugm P e per  + +  m l . w i t h t h e maximum e f f e c t apparent from 0 t o 0.01  ;ugm P e  + +  p e r m l . Throughout.cthis range a l i n e a r r e l a t i o n s h i p  -15-  Table I Response of P. aeruginosa to Iron  ;ugm F e / m l .  {  0  :  .0182  .001  !  .0364  .005  i  .0883  .01  !  .1352  .05  i!'  .1765  .1  !  .1625  .5  j  .2858  1.0  s  .2578  5.0  i  .2606 '  10.0  s  .2592  50.0  :  .0210  ++  mg. b a c t e r i a l N/ml.  -17existed. The i r o n r e q u i r e m e n t s o f P. a e r u g i n o s a 9027 on g l u c o s e ammonium s u c c i n a t e medium a r e s i g n i f i c a n t l y h i g h e r t h a n t h o s e of P. a e r u g i n o s a 2F5 as employed by Waring and Werkman (1943). Whereas maximum growth o f our organism was o b t a i n e d w i t h 0.5,ugm F e  + +  p e r m l . , maximum r e s p o n s e o f P. a e r u g i n o s a  2F3 r e q u i r e d o n l y 0.09 Aigm Fe p e r m l . The v a r i a t i o n i n t h e i r o n r e q u i r e m e n t s o f t h e s e two s t r a i n s may be a r e s u l t o f t h e d i f f e r e n t n i t r o g e n s o u r c e s employed i n t h e two growth media.  I n o r g a n i c ammonium  s u l f a t e , w h i c h was used by Waring and Werkman, c o u l d n o t d i r e c t t h e pathway o f g l u c o s e breakdown.  Since c e l l s of  P. a e r u g i n o s a appear t o c o n t a i n enzymes n e c e s s a r y f o r t h e a e r o b i c d i s s i m i l a t i o n o f s u c c i n a t e , t h e use o f ammonium s u c c i n a t e as t h e n i t r o g e n source would p r o v i d e as w e l l a n a l t e r n a t i v e carbon s o u r c e .  The development o f a more com-  p l e t e s u c c i n i c a c i d mechanism c o u l d account f o r t h e g r e a t e r i r o n r e q u i r e m e n t s o f our organism. I r o n d e f i c i e n t c e l l s o f A. i n d o l o g e n e s have been r e p o r t e d by Waring and Werkman (1944) t o c o n t a i n d e p l e t e d  dehydrogen-  ase systems f o r s u c c i n i c and m a l i c a c i d s , and t o show a decreased a b i l i t y t o o x i d i z e l a c t a t e , p y r u v a t e and a c e t a t e . To determine t h e e f f e c t o f I r o n d e f i c i e n c y  on t h e  enzymic c o n s t i t u t i o n o f c e l l s , a comparison was made o f t h e r a t e s o f oxygen uptake by d e f i c i e n t and normal r e s t i n g suspensions.  C u l t u r e s were grown i n t h e b a s a l medium a t  cell  t h r e e l e v e l s o f i r o n c o n c e n t r a t i o n - 0.005 ;ugm, 0.5 Aigm and 5.0 Mm  p e r m l . - r e p r e s e n t i n g l o w , optimum and h i g h  i r o n environments.  C e l l s produced on t h e l o w I r o n medium  were resuspended t o a t u r b i d i t y a p p r o x i m a t e l y  equal t o t h a t  of t h e l O x c o n c e n t r a t i o n o f optimum i r o n organisms. The  contents  o f t h e Warburg f l a s k s and t h e m i c r o l i t r e s  of oxygen absorbed by t h e o x i d a t i o n o f t h e s u b s t r a t e s i n a 45 minute p e r i o d a r e r e c o r d e d  i n Table I I .  Prom t h i s experiment I t appears t h a t t h e o x i d a t i o n o f p y r u v i c a c i d i s d e c r e a s e d by growth o f t h e organism i n t h e i r o n d e f i c i e n t medium.  Low i r o n c e l l s take up o n l y one-  h a l f as much oxygen as h i g h i r o n c e l l s when p y r u v a t e i s substrate.  A corresponding'loss  i n a c t i v i t y I s not observed  when g l u c o s e , l a c t a t e o r a c e t a t e i s t h e s u b s t r a t e  being  oxidized. The  d e c r e a s e d o x i d a t i o n o f p y r u v i c a c i d by low I r o n  organisms i s n o t l i k e l y t o be t h e r e s u l t o f d e p l e t e d enzyme systems w i t h i n t h e s e c e l l s .  I n t h e p r e s e n c e o f an e a s i l y  a t t a c k a b l e s u b s t r a t e i . e . g l u c o s e r e s p i r a t i o n o f l o w and h i g h i r o n s u s p e n s i o n s i s o f t h e same o r d e r .  S i n c e t h e break-  down o f g l u c o s e almost c e r t a i n l y I n v o l v e s t h e o x i d a t i o n o f p y r u v i c a c i d , i t c a n be c o n c l u d e d t h a t low i r o n c e l l s cont a i n t h e e s s e n t i a l enzymes and coenzymes f o r t h e o x i d a t i o n of t h e k e t o - a c i d . The mechanisms r e s p o n s i b l e , f o r t h e o x i d a t i o n o f p y r u v i c a c i d by b a c t e r i a a r e known t o be e x t r e m e l y s e n s i t i v e .  Table II The Influence of Iron i n the Growth Medium on the Oxidative A b i l i t y of Resting Cells of Fseudamonas aeruginosa In f l a s k : 20 h r . c e l l s - 2 0 x M/15 pH 7.4 phosphate buffer • M/15 pH 6 . 0 phosphate buffer Water to In side-arm:  'A 0 . 5 ml 1 . 5 ml 3 . 0 ml to  substrate  B 0 7 5 ml 1 . 5 ml 3 . 0 ml substrate  In centre w e l l : 20%  .15 ml  K0H  .15 ml  >uT 02 uptake in.45 minutes* : .005 >ugm • . 5 ;ugm . 5.0 -ugm. : Pe /ml .: Pe /ml .• . Pe /ml ++  ++  Glucose  (ljuM) (A)  :  70 ... :  ++  64  70  . 53  (2 JUM) (B) :  25  !• 6 2  Na lactate (2 ;uM) (A)  :  55  ]:  50  :  71  Na acetate (3 AIM) (A)  :  90  t  80  :  85  Glycerol (2>uM) (A)  :  20  ::  20  i  21  Na pyruvate  * endogenous r e s p i r a t i o n subtracted  -20-  By use o f ' c e l l - f r e e extracts of E. c o l l . Kalnitsky and ¥/erkman (1943) were able to show that pyruvic acid neutralized d i r e c t l y was  less active than that which had been  prepared by d i l u t i n g p r i o r to n e u t r a l i z a t i o n . Changes i n c e l l permeability may  account f o r the de-  creased a c t i v i t y of these i r o n deficient organisms on pyruvate.  Using acetone preparations of M. l y s o d e i k t i c u s .  Krampitz and Werkman (1941) were able to demonstrate the formation of pyruvic a c i d and carbon dioxide from oxalacetate. Whole c e l l s were not permeable to oxalacetate i n the absence of oxygen.  L i c h s t e i n and Umbreit (1947) have shown that  oxalacetate i s decarboxylated by E. c o l i as  phosphorylated  oxalacetic a c i d and i t i s probable that pyruvic a c i d also i s u t i l i z e d by the c e l l s i n the form of a phosphorylated tive.  deriva-  The growth of the organism i n low i r o n solutions might  reduce the permeability of the c e l l membranes to pyruvic a c i d . If such i s the case, the low oxygen uptake observed on pyruvate i s not incompatible with the higher oxygen consumption obtained using glucose. The growth response of the organism to i r o n i s of considerable significance i n view of the functions of i r o n In b i o l o g i c a l oxidation.  A l l aerobic organisms contain a  group of porphyrin proteins - the cytochromes - as part of their hydrogen transport system.  By means of these enzymes  hydrogen i s passed out to oxygen to y i e l d water.  Since  anaerobic systems of obtaining' energy, do not function by of tcyi;ochrome, anaerobic bacteria do not require i r o n f o r  way  -21respiration. Both aerobic and anaerobic bacteria contain the hydrogen c a r r i e r flavoprotein.  I f , as i n aerobic r e s p i r a t i o n , the  flavoprotein passes hydrogen to atmospheric oxygen, hydrogen peroxide i s formed.  The destruction of t h i s toxic product  i s accomplished by the i r o n containing enzyme catalase. Since aerobic organisms must obtain their energy by means of cytochrome or flavoprotein, i t i s apparent that they require i r o n f o r r e s p i r a t i o n .  Conversely, anaerobic systems  for getting energy would not be dependent on the presence of iron. Since we have shown that growth of the organism Is d i r e c t l y proportional to the Iron content of the medium, i t can be concluded that t h i s organism has no anaerobic means of d i s s i m i l a t i n g carbohydrates.  Moreover i t has been shown  that growing the organism i n the presence of inadequate amounts of i r o n does not foster the elaboration of an enzyme system which can function without the a i d of i r o n .  I t would  appear therefore that there i s no p o s s i b i l i t y that t h i s organism can grow anaerobically.  Iron deficiency r e s u l t s i n  the formation of fewer c e l l s , a l l of which contain the normal complement of aerobic i r o n enzymes. SUMMARY By adding increasing amounts of FeSO^RgO to a p u r i f i e d medium, a growth response has been obtained over a range of  -22from 0 to 0.5;ugm F e  + +  per ml.  Resting c e l l suspensions  of i r o n deficient c e l l s showed  a decreased a b i l i t y to oxidize pyruvic acid. Enzymes f o r the oxidation of glucose, lactate and acetate were not influenced by the concentration of i r o n i n the growth medium.  Concentrations of 0.005, 0.5 and 5.0yugm  of iron per ml. were used.  •23-  PART I I THE PREPARATION OF RESTING CELLS  ''Resting'  8  c e l l s may be p r e p a r e d by w a s h i n g b a c t e r i a  free  o f n u t r i e n t s and suspending them i n a c o n c e n t r a t i o n g r e a t e r t h a n c o u l d be a c h i e v e d by growth,=. Q u a s i e l . (19384) has d e s c r i b e d t h e s e ^resting™ b a c t e r i a a s organisms f u l l y e n dowed w i t h m e t a b o l i c p o t e n t i a l i t i e s , y e t u n a b l e t o m u l t i p l y because o f t h e l a c k o f e s s e n t i a l growth s u b s t a n c e s .  Cells  i n t h i s n o n - p r o l i f e r a t i n g s t a t e a r e w e l l s u i t e d f o r use i n m e t a b o l i c s t u d i e s , as t h e i r r e s p i r a t o r y mechanisms may be e v a l u a t e d i n t h e absence o f i n t e r f e r i n g g r o w t h r e a c t i o n s . Few changes have been made s i n c e t h e o r i g i n a l methods f o r t h e p r o d u c t i o n o f r e s t i n g b a c t e r i a were e v o l v e d . h a r v e s t e d f r o m media f a v o r a b l e f o r t h e p r o d u c t i o n o f  Cells the  enzymes t o be s t u d i e d , were p r e p a r e d f o r m e t a b o l i c work by washing them f r e e o f t h e n u t r i e n t s o f t h e growth medium, and concentrating the suspension.  The washed o r g a n i s m s .were  then aerated i n order t o destroy o x i d l z a b l e storage products w h i c h c o n t r i b u t e d t o t h e endogenous r e s p i r a t i o n . S i n c e t h e f i r s t work w i t h r e s t i n g c e l l s , t h e i m p o r t a n t , r e l a t i o n s h i p o f t h e c o m p o s i t i o n o f t h e growth medium and t h e age o f c e l l s a t t h e t i m e o f h a r v e s t i n g t o t h e  respiratory  a c t i v i t y o f t h e organisms o b t a i n e d , has been r e c o g n i z e d . t h e use o f a growth medium h i g h i n n i t r o g e n and l o w i n carbohydrate^ Kraip^r&a and Werkman  0.943  ) and Wood and  By  -24Gunsalus (1941 ) have o b t a i n e d good c r o p s o f c e l l s w i t h h i g h a c t i v i t y towards s u b s t r a t e s ,  but w i t h a v e r y l o w endogenous  r e s p i r a t i o n , t h u s a l l o w i n g a much more a c c u r a t e e v a l u a t i o n o f t h e i n f l u e n c e o f t h e added s u b s t r a t e .  The l a t t e r w o r k e r s  have a l s o p o i n t e d out t h e i m p o r t a n c e o f h a r v e s t i n g t h e during the height of t h e i r p h y s i o l o g i c a l a c t i v i t y .  cells  By so  d o i n g , the n i g h s t a t e o f r e a c t i v i t y developed during the l o g a r i t h m i c growth phase was r e t a i n e d i n t h e c e l l  suspensions  The p o s s i b i l i t y o f u s i n g S - 4 - d I n I t r o p h e n o l t o reduce t h e endogenous a c t i v i t y o f t h e s e b a c t e r i a m e r i t e d i n v e s t i g a t i o n . C l i f t o n (1937 ) , B e r n s t e i n (  1 9 4 4  ) and o t h e r s have employed  t h i s compound t o s t i m u l a t e t h e complete o x i d a t i o n o f es by r e s t i n g o r g a n i s m s .  substrat  By a e r a t i n g c e l l s u s p e n s i o n s i n .  d i l u t e s o l u t i o n s o f t h e r e a g e n t , i t s h o u l d be p o s s i b l e t o promote t h e o x i d a t i o n o f s t o r a g e p r o d u c t s , and t h e r e b y t o o b t a i n c e l l s h a v i n g a l o w endogenous r e s p i r a t i o n . By v a r y i n g t h e components o f t h e growth medium and by e x p e r i m e n t i n g w i t h t h e method o f c e l l p r e p a r a t i o n ,  active  r e s t i n g s u s p e n s i o n s o f t h e o r g a n i s m have been o b t a i n e d w h i c h a r e s u i t a b l e f o r use i n r e s p i r a t o r y s t u d i e s .  - u  -  METHODS Bacterlological;  Stock c u l t u r e s o f P . a e r u g i n o s a (ATC 90S7)  were c a r r i e d i n t h e manner d e s c r i b e d i n P a r t I . A f t e r  six  months r e f r i g e r a t i o n , s u b - c u l t u r e s grew v i g o r o u s l y i n b r o t h e x h i b i t i n g the pigmentations c h a r a c t e r i s t i c  of the  strain.  The c o m p o s i t i o n o f t h e g l u c o s e ammonium s u c c i n a t e medium u s e d f o r c a r r y i n g t h e o r g a n i s m was m o d i f i e d by r a i s i n g t h e c o n c e n t r a t i o n o f i r o n t o 0 . 5 ; a g m per m l . succinate,  To p r e p a r e ammonium  an amount o f s u c c i n i c a c i d e q u i v a l e n t t o  0.3$  ammonium s u c c i n a t e was n e u t r a l i z e d w i t h ammonium h y d r o x i d e . Media f o r t h e p r o d u c t i o n o f r e s t i n g c e l l s were i n o c u l a t e d w i t h 1.0$  o f a 20-24 h r . c u l t u r e o f t h e o r g a n i s m I n t h i s c a r r y i n g  medium. A m i n e r a l s o l u t i o n c o n t a i n i n g 0.1$ KgHEO^, 0.2$ THgO, 0.1>ugm. i r o n p e r m l . (as FeS0 *7HgO) 4  4  and 0 . 1  s o l u t i o n p e r 500 m l . was u s e d i n t h e p r o d u c t i o n o f cells.  MgS0 « cc  salt  resting  To t h i s b a s a l medium a d d i t i o n s o f g l u c o s e and  n i t r o g e n s o u r c e were made, and where r e q u i r e d , d i l u t i o n s o f 2,4  dinitrophenol.  D u r i n g t h e experiment on t h e e f f e c t  l o w M g S 0 * 7 H 0 and l o w K H P 0 4  s  0 . 3 $ ammonium s u c c i n a t e  s  4  on t h e a c t i v i t y o f  and 0.5$  of  cells,  g l u c o s e were employed, and  s u i t a b l e a d j u s t m e n t s were made i n t h e c o m p o s i t i o n o f  the  b a s a l medium. Werkman s medium f o r t h e p r o d u c t i o n o f gum-free 1  o f ]S. c o l i was as f o l l o w s :  0.4$  cells  each o f b e e f e x t r a c t and  • -  2£  2& -  peptone, 0.2$ each o f y e a s t e x t r a c t and N a C l , and 10$ t a p water. C e l l s were h a r v e s t e d h r s . at 30°C.  from t h e t e s t media a f t e r :20-£2  They were washed t w i c e and resuspended i n  s a l i n e i n twenty t i m e s t h e growth  concentration.  Unless otherwise stated, aeration o f the c e l l was a c c o m p l i s h e d i n t h e f o l l o w i n g manner.  suspensions  C e l l s washed once  i n l O x c o n c e n t r a t i o n were t r a n s f e r r e d t o s u i t a b l y s i z e d a e r a t i o n t u b e s , and a i r was drawn t h r o u g h them by means o f a s u c t i o n pump f o r a p e r i o d o f '2 h r s . no p r e c a u t i o n s  Throughout t h e procedure,  were t a k e n t o o b s e r v e a s e p t i c t e c h n i q u e .  At  t h e end o f t h e a e r a t i o n p e r i o d t h e c e l l s were t h r o w n down and washed once i n s a l i n e .  They were p r e p a r e d f o r u s e by  r e s u s p e n s i o n i n o n e - t w e n t i e t h t h e growth volume. C l i f t o n (1938) has employed 2,4 d i n i t r o p h e n o l t o inhibit  s y n t h e t i c r e a c t i o n s and t o i n c r e a s e o x i d a t i o n i n  s u s p e n s i o n s o f E. c o l i .  By i n c u b a t i n g t h e c e l l s w i t h 2,4  d i n i t r o p h e n o l d u r i n g t h e a e r a t i o n p e r i o d , I t was hoped t h a t more complete d e s t r u c t i o n o f o x i d i z a b l e s t o r a g e  products  would be o b t a i n e d t h e r e b y r e d u c i n g t h e endogenous a c t i v i t y of the c e l l suspensions.  The washed organisms were r e -  suspended i n l O x c o n c e n t r a t i o n dinitrophenol solution.  i n M/2000 n e u t r a l i z e d 2,4  A f t e r 1 h r . a e r a t i o n a t 30°C, t h e y  were c e n t r i f u g e d down and washed t h o r o u g h l y  I n h a l f the  growth volume o f s a l i n e .  A 2 0 x c e l l s u s p e n s i o n was p r e p a r e d  f o r u s e i n Warburg cups.  To ensure t h e p u r i t y o f t h e  s u s p e n s i o n , a l l a e r a t e d c e l l p r e p a r a t i o n s were Gram s t a i n e d p r i o r t o t h e i r u s e i n e x p e r i m e n t a l work. The a c t i v i t y o f t h e v a r i o u s c e l l suspensions was determined by measuring  t h e a b i l i t y o f each t o dehydrogenate  g l u c o s e i n t h e presence o f methylene b l u e .  The s t a n d a r d  Thunberg method as o u t l i n e d by "©mbreit^i (1945) was employed. Measurements were made i n v a c u o .  I n t h e tube'were p l a c e d  ;  2 ml;;. M/15 phosphate b u f f e r , pH 7.0, 2 ml. M/20 g l u c o s e and 1 ml... 1/10,000 methylene b l u e , and i n t h e s i d e arm b u l b 1 m l . of  20x c e l l s u s p e n s i o n .  A s t a n d a r d was i n c l u d e d c o n t a i n i n g  a l l o f t h e components o f t h e above system  (cells  inactivated  by b o i l i n g 20 minutes) but w i t h t h e methylene b l u e a t onet e n t h t h e normal c o n c e n t r a t i o n . T h i s tube r e p r e s e n t e d 90$ r e d u c t i o n o f t h e methylene b l u e and was used as t h e end p o i n t o f r e d u c t i o n . Thunberg t u b e s were evacuated w i t h a s t r o n g s u c t i o n pump f o r t h r e e minutes b e f o r e t h e y were sealed.  F o l l o w i n g a 10 minute e q u i l i b r a t i o n p e r i o d a t 30°C,  s u b s t r a t e was t i p p e d i n and t h e end p o i n t o f t h e r e a c t i o n was  r e c o r d e d as t h e t i m e r e q u i r e d f o r t h e c o l o r i n t e n s i t y o f  t h e e x p e r i m e n t a l t u b e s t o be reduced t o t h a t o f t h e b o i l e d c e l l standard.  At t h e end o f t h e r e d u c t i o n p e r i o d t h e s e a l s  were checked t o c o n f i r m t h e p r e s e n c e o f a vacuum throughout the  experiment. A c o n v e n t i o n a l Warburg a p p a r a t u s was u s e d t o f o l l o w t h e  oxygen uptake o f t h e c e l l s u s p e n s i o n s . t i o n s were a c c o r d i n g t o Dixon ( 1 9 4 3 ) .  Procedure  and c a l c u l a -  0.5 m l . o f a 20x c e l l  2%  ~  28 -  suspension was employed In a f i n a l volume of 3.0 ml. were run f o r a period of 1 nr. at 30 C. U  Tests  RESTING CELL PREPARATION  N i t r o g e n and Carbon:  P r e l i m i n a r y e x p e r i m e n t s have i n d i c a t e d  t h a t t h e n i t r o g e n source ammonium s u c c i n a t e w i t h g l u c o s e as carbon source gave t h e most abundant y i e l d o f p i g m e n t i n g cells.  D u r i n g t h e e a r l y growth phase i n t h e s e media, and  coincident with t h e formation of the fluooescent a gummy m a t e r i a l was e l a b o r a t e d by t h e c e l l s .  pigment,  The s u b s t i t u t i o n  o f ammonium c h l o r i d e f o r ammonium s u c c i n a t e , w h i l e p a r t i a l l y e f f e c t i v e i n r e d u c i n g gum f o r m a t i o n , p r e v e n t e d ance, o f t h e y e l l o w pigment.  t h e appear-  G l y c i n e gave p o o r e r y i e l d s o f  non-pigmenting gum-free c e l l s , but t h e s e c o u l d be h a r v e s t e d o n l y a f t e r 48 h r s . growth.  P h e n y l a l a n i n e has p r e v i o u s l y been  found t o enhance s l i m e f o r m a t i o n (White, t h e s i s ) .  Fairly  heavy y i e l d s o f non-pigmenting c e l l s o f average gumminess were o b t a i n e d u s i n g i n o r g a n i c ammonium s u l f a t e . The  methylene b l u e r e d u c t i o n t i m e s f o r media c o n t a i n i n g  t h e n i t r o g e n sources ammonium s u c c i n a t e , ammonium s u l f a t e and g l y c i n e , combined w i t h two c o n c e n t r a t i o n s o f g l u c o s e a r e g i v e n i n t a b l e I . Owing t o t h e slow growth o f t h e c e l l s w i t h g l y c i n e , t h e r e d u c t i o n v a l u e s f o r t h i s medium were o b t a i n e d u s i n g 48 h r . c e l l  preparations.  _ '30 TABLE I The  E f f e c t o f N i t r o g e n Source and  G l u c o s e C o n c e n t r a t i o n on t h e Dehydrogenase A c t i v i t y o£ t h e C e l l s  nitrogen source  NH  : nitrogen : source : %  succinate : :  4  o.i o.i  :  (NH ) 4  g  S0 ti «  glycine  4  0.3  I ; ;  . methylene blue r e d u c t i o n : glucose < '. g l u c o s e : endogenous (mins.) : (mins.) % ". _ « —  ; !  0.5  : ;  I.'O 0.5  :  1.5 2.0  2.o  ! ',!  0.087 : 0.087 . o.26 : 0.26  0.5  ;  i.o 0.5 i.o  :  IO.O 15.0  : :  i8.o  :  :  11.5  o.3  0.5  1  s  ]:  : :  0.6  :  ;  0.5  :  :  1.5 • 8.5 20 17.5 17.5 25.0 15.5  :  3.0  !  9.0  ;  4.0 12.5  amount o f N e q u i v a l e n t t o 0.1$ NR. s u c c i n a t e amount o f N e q u i v a l e n t t o 0.3$ NIT s u c c i n a t e  From t h e above d a t a i t i s a p p a r e n t t h a t g l y c i n e grown organisms a r e u n s u i t a b l e f o r r e s t i n g c e l l work.  These c e l l s  *  require approximately  t h r e e - q u a r t e r s o f t h e endogenous  r e d u c t i o n t i m e t o reduce methylene b l u e i n t h e p r e s e n c e o f glucose.  Moreover t h e r e l a t i v e l y l o n g p e r i o d r e q u i r e d f o r  t h e i r p r o d u c t i o n makes them i m p r a c t i c a l f o r u s e i n r e s p i r a t o r y studies. Ammonium s u l f a t e c e l l s can ,be o b t a i n e d i n good y i e l d i n  S 0 - 2 S h r s . , but t h e dehydrogenase a c t i v i t y o f s u c h p r e p a r a tions i s low.  T w o - t h i r d s o r more o f t h e endogenous r e d u c t i o n  t i m e i s r e q u i r e d f o r t h e r e d u c t i o n o f methylene b l u e i n t h e presence of  substrate.  The a c t i v i t y o f ammonium s u c c i n a t e c e l l s i s suitable. and 0.5$  entirely  Organisms h a r v e s t e d f r o m 0.3$ ammonium s u c c i n a t e g l u c o s e r e d u c e d methylene b l u e i n t h e p r e s e n c e o f  g l u c o s e i n l e s s t h a n o n e - t e n t h t h e t i m e r e q u i r e d by endogenous p r e p a r a t i o n s .  S i n c e growth was r a p i d on t h i s medium,  arid s i n c e e x c e l l e n t y i e l d s o f c e l l s were o b t a i n e d , ammonium s u c c i n a t e was s e l e c t e d as t h e most- s u i t a b l e n i t r o g e n  source  f o r t h e p r o d u c t i o n o f r e s t i n g c e l l s o f pseudomonas... The a c t i v i t y o f t h e c e l l s u s p e n s i o n s was  consistently  h i g h e r when t h e organisms were h a r v e s t e d from t h e medium c o n t a i n i n g 0.3$ n i t r o g e n . s o u r c e was t h e r e f o r e medium.  This concentration of nitrogen  selected f o r use i n t h e r e s t i n g c e l l  G r e a t e r amounts have been f o u n d t o produce l i t t l e  d i f f e r e n c e i n t h e dehydrogenase a c t i v i t y o f t h e  cells.  From t h e d a t a o b t a i n e d i t c a n . b e seen t h a t t h e c o n c e n t r a t i o n o f g l u c o s e i n t h e growth medium has but e f f e c t on t h e a c t i v i t y o f t h e c e l l s p r o d u c e d . presence of 0.5$  little  In the  glucose, the substrate reduction time i s  0 . 5 7 and 0.72 t i m e s t h a t o f t h e endogenous f o r 0 . 1 $ and ammonium s u l f a t e r e s p e c t i v e l y ; i n t h e p r e s e n c e o f g l u c o s e , 0.36 and 0 . 7 4 t i m e s r e s p e c t i v e l y .  0.3$  1.0$  Since the sub-  s t r a t e c o n c e n t r a t i o n appeared t o be o f l i t t l e  significance,  t h e s m a l l e r amount - 0.5% - was s e l e c t e d f o r f u t u r e use i n media f o r t h e p r o d u c t i o n o f r e s t i n g  cells.  M i n e r a l s I n her study of the m i n e r a l metabolism o f the fluorescent  p i g m e n t , K i n g ( t h e s i s ) has o b s e r v e d t h a t  concentrations  higher  of sulfate (greater than.0.5$) contributed to  t h e f o r m a t i o n of, a s l i m e y m e t a b o l i c p r o d u c t when c e l l s were grown on ammonium s u c c i n a t e and g l y c e r o l .  Previously i t  has  been found t h a t h i g h c o n c e n t r a t i o n s o f i n o r g a n i c phosphate t e n d t o s h i f t t h e e q u i l i b r i u m e x i s t i n g . b e t w e e n s y n t h e t i c and breakdown p r o c e s s e s  of the c e l l i n favor of the  r e a c t i o n (Lipmann, 1 9 4 2 ) ,  The p o s s i b i l i t y , t h a t  synthetic either  or  b o t h o f t h e s e f a c t o r s might c o n t r i b u t e t o t h e , endogenous r e s p i r a t i o n o f c e l l suspensions n e c e s s i t a t e d  a b r i e f study o f  t h e e f f e c t s o f t h e s e m i n e r a l c o n s t i t u e n t s , on c e l l a c t i v i t y . TABLE I I The E f f e c t o f Low M i n e r a l s on. t h e Dehydrogenase A c t j y . l t v o f t h e  M e t h y l e n e b l u e r e d u c t i o n £ime glucose endogenous mlns. mins.  W>4 0.1  0.-2  0.1  0.01  00.01 0.01  Cell.  0,01  5.0  10.0  11.5  24.0  3 hr.  3 hr.  3 hr.  3 hr.  Table I I  shows t h a t t h e c o n c e n t r a t i o n o f HgSp^*7EgO i n :  t h e growth medium does n o t a l t e r t h e r a t i o o f endogenous t o s u b s t r a t e r e d u c t i o n t i m e s , a n d t h e r e f o r e i t cannot be c o n s i d e r e d a f a c t o r c o n t r i b u t i n g t o t h e endogenous a c t i v i t y o f the c e l l s * A l t h o u g h i t i s i m p o s s i b l e t o draw c o n c l u s i o n s r e g a r d i n g t h e i n f l u e n c e o f phosphate on t h e r a t i o o f endogenous t o  sub  s t r a t e r e d u c t i o n t i m e s i t appears t h a t by d e c r e a s i n g t h e phosphate f r o m 0 . 1 $ t o , 0 . 0 1 $ , t h e a c t i v i t y o f t h e c e l l s p r o duced i s so d i m i n i s h e d t h a t  a three hour p e r i o d i s i n s u f f i c -  i e n t f o r t h e r e d u c t i o n o f methylene b l u e i n t h e p r e s e n c e o f substrate.  F u r t h e r m o r e , t h e c o n c e n t r a t i o n o f phosphate i n  t h e medium s h a r p l y l i m i t s t h e amount o f g r o w t h . In view of these observations, the o r i g i n a l  concentra-  t i o n s o f 0 . 2 $ MgS0 *7HgO and 0.1$ K g H P 0 were employed i n 4  4  media f o r t h e p r o d u c t i o n o f r e s t i n g o r g a n i s m s . The e f f e c t o f i r o n on t h e dehydrogenase a c t i v i t y o f t h e c e l l s was next s t u d i e d .  As might be e x p e c t e d , i r o n was  s i g n i f i c a n t o n l y i n so f a r as c e l l growth was dependent upon it.  With decreasing concentrations of i r o n , lower y i e l d s of  c e l l s were o b t a i n e d , but t h e dehydrogenase a c t i v i t y o f t h e s e c e l l s f o r g l u c o s e remained e s s e n t i a l l y unchanged.  r  TABLE  III  f h e E f f e c t p£ I r o n C o n c e n t r a t i o n on t h e D e h y a r ^ g ^ s e A c t i v i t y o f £he> C e l l s  Methylene blue r e d u c t i o n time Fe  Age;  per m l . >ugms.  glucose (mins.)  + +  0.01  !  0.05  s[  1.5  0.1  ::  1.5  endogenous (mins.) :  3  : ,  i  To o b t a i n a c t i v e l y r e s p i r i n g c e l l s ,  2.5  i t i s important  t h a t t h e organisms s h o u l d be h a r v e s t e d i n a n a c t i v e b e f o r e t h e onset o f t h e v  by Buchanan ( 1 9 1 8 ) . that  s t a t i o n a r y growth phase a s  state described  Much d a t a has been p r e s e n t e d t o  indicate  c e l l s o b t a i n e d i n t h e e a r l y phases o f t h e c u l t u r e  cycle  were s i g n i f i c a n t l y more a c t i v e m e t a b o l i c a l l y t h a n t h o s e o b t a i n e d f r o m o l d e r cultures.(WJ<*>°< » G o ^ i w tmsC). S i n c e t h e c o m p o s i t i o n o f t h e medium i s a c o n t r o l l i n g f a c t o r i n t h e r a t e o f g r o w t h o f t h e c e l l s , i t was  necessary  t o d e t e r m i n e t h e t i m e at w h i c h maximum a c t i v i t y o f organisms o c c u r r e d i n g l u c o s e ammonium s u c c i n a t e  broth.  T a b l e IV r e c o r d s t h e dehydrogenase a c t i v i t y o f t h e s u s p e n s i o n s at SO h r s . , 48 h r s . and 72 h r s .  the  cell  C e l l s were  h a r v e s t e d f r o m t h e medium c o n t a i n i n g 0 . 5 $ ammonium s u c c i n a t e and 0 . 5 $  glucose.  ;35> „  TABLE IV IM  PATec,t g£ Age, p j C j u l t n r ^ on. t h e Dehy^rogenas.e A g ^ ^ v i t y  Methylene blue r e d u c t i o n age hrs. 20  glucose (mins.) i!  48 72  i  endogenous (mins.)  7.5  it  7.5  18.0  ii  24.0  - 51.0  f  J:  § reduced i n 1 h r .  i  As t h e c u l t u r e s , were a l l o w e d t o grow beyond 20 h r s . , dehydrogenase a c t i v i t y o f t h e c e l l s d e c l i n e d i n t h e o r absence o f s u b s t r a t e .  the  presence  A c o n s i d e r a t i o n o f t h e endogenous  r e d u c t i o n t i m e s shows t h a t p r o l o n g i n g t h e growth p e r i o d t o 48 h r s . d e c r e a s e d c e l l a c t i v i t y by o n e - t h i r d , w h i l e e x t e n d i n g i t t o 72 h r s . l e f t t h e c e l l s w i t h l e s s t h a n o n e - s i x t e e n t h their original activity.  of  W h i l e t h i s i n c r e a s e d methylene b l u e  r e d u c t i o n t i m e f o r c e l l s i n t h e absence o f s u b s t r a t e might be i n t e r p r e t e d t o mean t h a t t h e c e l l s m e t a b o l i z e a l a r g e p o r t i o n o f t h e i r storage products during the l a t t e r part  of  t h e i r growth phase, t h e c o r r e s p o n d i n g d e c r e a s e i n t h e a c t i v i t y o f t h e organisms i n t h e presence o f g l u c o s e  suggests  t h a t t h e dehydrogenase a c t i v i t y o f t h e c e l l f a l l s o f f  rapidly  a f t e r a p e r i o d o f maximum a c t i v i t y .  The a b n o r m a l l y l o n g  r e d u c t i o n t i m e o b s e r v e d f o r 20 h r . c e l l s on g l u c o s e was not  repeated i n succeeding work.  I n t h e l i g h t o f t h i s experiment^  organisms f o r t h e p r e p a r a t i o n o f r e s t i n g c e l l were h a r v e s t e d a f t e r 2 0 - 2 4 h r s . growth a t Aeration?  suspensions  30°C,  An attempt was made t o r e d u c e t h e endogenous  s t o r a g e p r o d u c t s o f t h e c e l l s by means o f a e r a t i o n .  The  organisms were c u l t u r e d on t h e b a s a l m i n e r a l s o l u t i o n t o w h i c h was added 0 . 3 $ ammonium s u c c i n a t e and 0.5$  glucose.  S i n c e t h e most e f f e c t i v e c o n c e n t r a t i o n o f t h e c e l l s d u r i n g the  p r o c e d u r e was not known s e v e r a l c o n c e n t r a t i o n s o f •••••  s u s p e n s i o n s were a e r a t e d . TABLE V TJl§ l £ £ S S k M A e r a t i o n on ^ x A c ^ v J & r o f Eejtiag  Qs3M  Methylene b l u e r e d u c t i o n endogenous (mins.)  aeration (hrs.) 0  2  9  1  2  4  S..5  6  3  - 37l. „  TABLE V I T j & Ef^eqfr QL C o n c e n t r a t i o n , og C e l l s During Aeration  Concentration of cells during aeration  Aeration (hrs.) 0  'i  IB  ii  9  growth v o l ,  t  1.5  i  i i  5x  8  1.5  !t  £  ^BOx  s  :  3  •  S  •  i  • » m  —  Methylene b l u e r e d u c t i o n * glucose endogenous (mins.) (mins.)  >  i  > >  P I  #  * *  *  The substances c o n t r i b u t i n g t o t h e endogenous r e s p i r a t i o n o f t h e s e c e l l s do not a p p e a r t o be u t i l i z e d during a e r a t i o n .  As a r e s u l t of t h i s procedure, the  endogenous dehydrogenase a c t i v i t y was I n c r e a s e d 2 . 5 - 4 t i m e s The c o n c e n t r a t i o n o f t h e c e l l s d u r i n g a e r a t i o n was w i t h o u t s i g n i f i c a n t e f f e c t on t h e r e l a t i o n s h i p e x i s t i n g between endogenous and s u b s t r a t e r e d u c t i o n t i m e s .  •38-  Warburg studies:  The o r i g i n a l mineral solution to which was  added 0.3% ammonium succinate and 0.5% glucose seemed most suited to the production of active organisms.  Low endog-  enous c e l l suspensions were obtained by harvesting the c e l l s at 20-24 hours an.d employing them d i r e c t l y without aeration. To confirm the s u i t a b i l i t y of these c e l l s for respiratory studies, the oxygen consumed i n the oxidation of one and' f i v e micromoles of glucose was measured.  As the results i n Tables  VII i l l u s t r a t e , a clear interpretation of oxidative data  was  obtained when 5 micromoles of glucose were employed per Warburg cup.  Whereas the endogenous respiration approached  one-half of the t o t a l oxygen uptake i n the presence of 1 micromole of glucose, i t was less than one f i f t h of the t o t a l i f 5 micromoles of glucose were employed. To confirm the previous observation that washed c e l l suspensions had a lower endogenous a c t i v i t y before than after aeration, the oxygen consumption of aerated and organisms was  compared.  non-aerated  Data obtained on Werkman s medium 1  and on ammonium succinate glucose medium with and  without  2-4 dinitrophenol are included i n the following table. The results on aeration are i n complete agreement with those obtained during Thunberg studies.  Untreated  cells,  harvested from the 0.5% glucose and 0.3% ammonin succinate medium without 2-4-dinitrophenol gave the most active preparations.  cell  Aeration i n the presence of 2,4 di-nitrophenol  decreased the t o t a l oxygen consumption of the organisms and  -39-  Table VII The Oxidation of Glucose by Resting C e l l s  In f l a s k : 0.5 ml. 20x c e l l s 1.5 ml. M/15 phosphate buffer pH Water 3.0 ml.  7.4  In side-arm: 0, luM or 5 juM glucose  In Centre veil: 0.15 ml. 26$ KOH  substrate  ;  j endogenous lyuM glucose 5 juM glucose  >ul 0£ uptake i n 1 hr. 93.5  ; \  197.0 522.0  increased the iratio of endogenous to substrate  respiration.  A l l attempts to r i d the c e l l s of their stored products seemed to be of no a v a i l .  Since the procedures used are recognized  workable methods, i t would appear that we are dealing with an endogenous respiration which i s different by organisms such as E. c o l i .  from that exhibited  -40-  Table VIII growth medium  treatment of c e l l suspension  Ail Og uptake i n 1 hr.  endogenous  1 mM glucose  ,5% glucose, .3% NH succ.  93.5  197.0  .2^ glucose, • 3J£ NH^ succ.  61.3  144.5  aerated 4 h r s .  130.0  191.5  aerated 1 h r . with M/2000 2-4 DNP  127.5  185.5  »5j£. glucose, .3^NH succ. - 2-4 DNP  57.5  123.0  Werkman's  88.2  147.0  64.5  152.0  4  ,b% glucose, •3/£ NH^ succ. • 5/£ glucose, .2% NH  4  BUCC.  4  Workman's  aerated 4 hrs.  SUMMARY Active resting c e l l s have been prepared from the mineral medium to which was added 0.3$ ammonium succinate and -0.5$ glucose. Aerating the c e l l suspensions did not decrease t h e i r endogenous respiration or the ratio of endogenous to substrate methylene blue reduction times. Incubation of resting c e l l s f o r 1 hour with M/2000 2,4 d i nitrophenol f a i l e d to decrease the oxidizable storage products.  Although aeration of the :eells harvested from Werkman's medium reduced oxygen consumption  i n t he absence of glucose,  t h i s treatment also decreased the oxidative a c t i v i t y of the c e l l s when glucose was present.  -42PART  III  METABOLISM  L i t t l e i s known o f t h e pathways o f a e r o b i c dissimilation i n bacteria. that  carbohydrate  At t h e p r e s e n t t i m e i t i s assumed  organisms such as E . c o l i . w h i c h a r e f a c u l t a t i v e ,  use  t h e Meyerhof-Embden system under b o t h a e r o b i c and a n a e r o b i c c o n d i t i o n s , t h e s e p a r a t i o n o c c u r r i n g beyond t h e p y r u v i c acid stage.  Under a e r o b i c c o n d i t i o n s , p y r u v i c a c i d i s  oxid-  i z e d t o a c e t i c a c i d and carbon d i o x i d e , and t h e n t h e a c e t a t e i s f u r t h e r broken down t o carbon d i o x i d e and w a t e r .  Under  a n a e r o b i c c o n d i t i o n s , l a c t i c and a c e t i c a c i d s and e t h y l a l c o h o l are formed. An a l t e r n a t i v e mechanism, and one w h i c h would appear t o o c c u r i n some o b l i g a t e aerobes such a s m o l d s , y e a s t s , pseudomonas and r e l a t e d o r g a n i s m s (Moyer et a l 1940; W i l l i a m s 1945; Burk 1939;  Stubbs et a l 1940;  Lockwood et a l 1 9 4 1 ) , i s  pathway t h r o u g h g l u c o n i c , k e t o g l u c o n i c and acids.  I t has g e n e r a l l y been assumed t h a t  the  ketoglutaric beyond t h i s p o i n t  t h e i n t e r m e d i a t e breakdown i s s i m i l a r t o t h a t o f t h e K r e b s cycle, i . e .  s u c c i n i c , f u m a r i c , m a l i c , and o x a l a c e t i c  acids.  The h i g h e r i n t e r m e d i a t e s o f t h i s scheme have been i s o l a t e d by numerous w o r k e r s .  A l s b e r g (1911) o b t a i n e d y i e l d s o f g l u c o n i c  a c i d amounting t o 85$ o f t h e g l u c o s e u t i l i s e d by £ . stanoli.  savan-  No r e d u c i n g a c i d s were d e t e c t e d i n t h e s e  fermentations,  ( i n c o n t r a s t , A u b e l ( l 9 2 l ) has i s o l a t e d  -43-  a l c o h o l , a c e t i c and f o r m i c a c i d s , but none o f the o x i d i z e d a c i d s , from f e r m e n t a t i o n s o f g l u c o s e by B. pyocyaneus. f r u c t o s e was  employed as s u b s t r a t e , l a c t i c a c i d was  i n a d d i t i o n t o t h e p r o d u c t s p r e v i o u s l y mentioned.)  When  obtained Per-  v o z a n s k i i (1940) has r e p o r t e d h i g h y i e l d s o f g l u c o n i c and ketogluconic a c i d s i n c u l t u r e s of f l u o r e s c i n g b a c t e r i a .  By  the use o f i n t e n s e a e r a t i o n , p r e s s u r e , a g i t a t i o n and h i g h c o n c e n t r a t i o n s o f s u b s t r a t e i t has been p o s s i b l e t o b l o c k the e a r l i e r phases o f t h e normal a e r o b i c m e t a b o l i s m t h e r e b y a l l o w i n g l a r g e q u a n t i t i e s o f o x i d i z e d a c i d s t o accumulate i n the f e r m e n t a t i o n s .  Thus Stubbs (1940) has o b t a i n e d  y i e l d s o f 5 - k e t o g l u c o n i c a c i d I n 33 h r s . w i t h A.  90$  suboxvdons  and 82$ y i e l d s o f 2 - k e t o g l u c o n i c a c i d i n 25 h r s . w i t h an unnamed b a c t e r i u m . Lockwood, T a b e n k i n and Ward (1941) have compared t h e a b i l i t y o f a v a r i e t y o f s p e c i e s o f phytomonas and pseudomonas t o produce g l u c o n i c and 2 - k e t o g l u c o n i c a c i d s .  W h i l e amounts  r a n g i n g from 58$ t o 96$ g l u c o n i c a c i d and g r e a t e r t h a n 2 - k e t o g l u c o n i c a c i d were f o u n d i n f e r m e n t a t i o n s  70$  conducted  under commercial c o n d i t i o n s , c u l t u r e s i n w h i c h t h e n o r m a l p h y s i o l o g i c a l c o n d i t i o n s were m a i n t a i n e d produced o n l y t r a c e s o f t h e s e compounds, A f u r t h e r s t e p i n the breakdown o f g l u c o s e was by Lockwood and S t o d o l a (1946).  indicated  These w o r k e r s have shown  t h a t i f 2 - k e t o g l u c o n i c a c i d f e r m e n t a t i o n s by P. f l u o r e s c e n s were a l l o w e d t o c o n t i n u e u n t i l no r e d u c i n g power remained i n the medium, 16-17$ y i e l d s o f  k e t o g l u t a r i c a c i d were o b t a i n e d .  S i n c e the c o n d i t i o n s t o which the organism i s s u b j e c t e d d u r i n g commercial f e r m e n t a t i o n procedures are so f a r removed from the normal p h y s i o l o g i c a l environment, i t i s i m p o s s i b l e t o conclude from t h i s d a t a a l o n e t h a t the a c i d s produced are normal i n t e r m e d i a t e s i n g l u c o s e d i s s i m i l a t i o n .  However  t h e y are i n d i c a t i v e of the p o t e n t i a l mechanisms a v a i l a b l e to the c e l l . To s t u d y the metabolism o f t h i s organism, the r e s p i r a t o r y a c t i v i t y was surveyed i n a v e r y g e n e r a l manner u s i n g p o s t u l a t e d i n t e r m e d i a t e s o f both a e r o b i c and a n a e r o b i c systems.  I n t h i s e a r l y phase o f t h e work, g l u c o s e ammonium  s u c c i n a t e c e l l s were employed.  As i t l a t e r became apparent  t h a t the o x i d a t i o n o f carbon compounds by t h i s organism  was  c o n t r o l l e d by a d a p t i v e enzymes, c e l l s grown on t h e double s u b s t r a t e were abandoned, and organisms h a r v e s t e d from a p u r e l y m i n e r a l medium p l u s s u b s t r a t e were employed.  By  the use of these c e l l s , we have'been a b l e t o show t h a t the breakdown o f g l u c o s e proceeds by way o f g l u c o n i c and 2 - k e t o - gluconic acids.  Beyond these i n t e r m e d i a t e s the pathway  o f c a r b o h y d r a t e metabolism i s s t i l l o b s c u r e .  -45-  METHODS  Bacterlologleali  The c u l t u r e u s e d , £ . a e r u g i n o s a ( AT C 9 0 2 7 ) ,  has a l r e a d y been d e s c r i b e d i n P a r t s I and I I o f t h i s  thesis.  By employing t h e same methods f o r t h e c u l t i v a t i o n and s t o r a g e o f t h e o r g a n i s m , f u l l m e t a b o l i c a c t i v i t y has been m a i n t a i n e d . k b r o t h , c o n t a i n i n g g l u c o s e and ammonium s u c c i n a t e ,  was  o r i g i n a l l y used f o r c a r r y i n g t h e o r g a n i s m and f o r t h e p r e - paration of r e s t i n g b a c t e r i a . ,  C o m p o s i t i o n and s u i t a b i l i t y  o f t h i s medium f o r t h e p r o d u c t i o n o f r e s t i n g c e l l s has discussed i n the previous section.  been-  F o r s t u d i e s employing  ammonium phosphate c e l l s , t h e b a s a l medium, was m o d i f i e d t o c o n t a i n o n l y 0.1$ KC1.  MgS0 »7HgO, and K ^ H 1 0 was r e p l a c e d by 4  4  A l l growth s u b s t r a t e s were u s e d a t 0.5$  0.1$  concentrationi.  By employing 100 m l . o f t h e medium p e r Roux b o t t l e o r 50 m l . per K o l l e f l a s k ,  s u f f i c i e n t a e r a t i o n was o b t a i n e d t o a l l o w  good growth o f t h e c e l l s .  T h e . i n o c u l u m f o r t h e s e f l a s k s was  10 d r o p s and 5 d r o p s , r e s p e c t i v e l y ,  o f a 24 h r . c u l t u r e o f  t h e organism i n t h e medium b e i n g used i n t h e p r o d u c t i o n o f the resting  bacteria.  A c t i v e c e l l s were h a r v e s t e d a t 20-^22 h r s .  Washing t h e  packed organisms t w i c e i n h a l f t h e growth volume o f ^ s a l i n e gave r e s t i n g c e l l s w h i c h c o u l d be s a t i s f a c t o r i l y employed i n respiratory studies.  D i r e c t l y before t h e i r use, a  concen-  t r a t e d s u s p e n s i o n o f t h e washed organisms was p r e p a r e d , 0 . 5 m l . o f w h i c h was added t o each Warburg c u p .  I n order to  -46-  m a i n t a i n a h i g h degree o f enzymic a c t i v i t y t h r o u g h o u t t h i s s e r i e s o f e x p e r i m e n t s , n o c e l l s were s t o r e d l o n g e r t h a n o n e - h a l f hour p r i o r t o t h e i r use i n metabolic s t u d i e s . T o determine t h e a b i l i t y o f t h e c e l l s t o o x i d i z e t h e v a r i o u s p o s t u l a t e d i n t e r m e d i a t e compounds, t h e Warburg t e c h n i q u e was employed ( D i x o n 1943j U m b r e i t 1945)... buffer,  Phosphate  ( M / 1 5 ) , water and c e l l s were p l a c e d i n t h e main com-  partment o f t h e f l a s k , w h i l e s u b s t r a t e ,  i n h i b i t o r s and  a c t i v a t o r s were added t o t h e s i d e a r m s .  The c a r b o n d i o x i d e  produced d u r i n g r e s p i r a t i o n was a b s o r b e d by 20% KOH p l a c e d i n the centre w e l l *  E a c h experiment i n c l u d e d a p p r o p r i a t e  endogenous and o t h e r c o n t r o l s w h i c h were r u n i n c o n j u n c t i o n w i t h the substrate containing f l a s k s . When f e r m e n t a t i o n measurements were t o be made, a b i c a r b o n a t e s o l u t i o n was u s e d from w h i c h carbon d i o x i d e was r e l e a s e d i n p r o p o r t i o n t o t h e amount o f a c i d p r o d u c e d .  The  r e a c t i o n m i x t u r e o f each f l a s k was b u f f e r e d at pH 7 . 0 5 by u s i n g a f i n a l c o n c e n t r a t i o n o f 0.01M NaHCOg under a 5$ carbon d i o x i d e - 95$ n i t r o g e n atmosphere.  Sodium b i c a r b o n a t e  (0«1H),  w a t e r and c e l l s were i n t r o d u c e d i n t o t h e main compartment o f the respiratory v e s s e l .  I n one s i d e a r m was p l a c e d 5 uM o f  g l u c o s e , and i n t h e o t h e r , 3 N H C l f o r t h e f i n a l r e l e a s e o f carbon d i o x i d e .  The c e n t r e w e l j c o n t a i n e d a c h i p o f y e l l o w  phosphorus. B e f o r e c l o s i n g t h e s t o p c o c k s , t h e f l a s k s were f l u s h e d w i t h t h e gas m i x t u r e , t h e r a t e o f f l o w b e i n g so a d j u s t e d t h a t  -47<  a p p r o x i m a t e l y one l i t r e o f gas p a s s e d t h r o u g h each cup i n a 10-15  minute p e r i o d .  A f t e r s e t t i n g t h e columns, a 10 minute  e q u i l i b r a t i o n p e r i o d was a l l o w e d t o e n s u r e s u f f i c i e n t  time  f o r t h e l a s t t r a c e s o f oxygen t o be a b s o r b e d by t h e phosphorus.  S u b s t r a t e was i n t r o d u c e d , and t h e r e s p i r a t i o n  was f o l l o w e d f o r a p e r i o d o f 65 m i n u t e s .  A t t h e end o f  t h a t t i m e , H C l was t i p p e d i n t o l i b e r a t e r e t a i n e d  carbon  d i o x i d e , and t h e amount o f t h i s gas was r e c o r d e d .  Suitable  c o n t r o l s were a l w a y s i n c l u d e d t o d e t e r m i n e - t h e i n i t i a l  carbon  d i o x i d e content o f t h e r e a c t i o n m i x t u r e s . T o f o l l o w t h e phosphorus u p t a k e o f t h e c e l l s ,  i t was c o n -  v e n i e n t t o I n c u b a t e t h e suspensions i n Warburg cups t o the shaking apparatus.  attached  T h i s p r o c e d u r e not o n l y f a c i l i t a t e d  t h e a p p l i c a t i o n o f a n a e r o b i c c o n d i t i o n s , but a l s o e n s u r e d t h e c o n t i n u o u s c o n t a c t o f phosphate w i t h t h e c e l l s .  A series of  i d e n t i c a l f l a s k s were set up i n a manner s i m i l a r t o  that  used i n t h e f e r m e n t a t i o n s t u d y , except t h a t H C l was e n t i r e l y omitted. - 1.14  A s t a n d a r d amount o f phosphate s o l u t i o n , 0 . 5 m l .  mg. KHgP0 p e r m l . * ISOjugm. P p e r c u p , was u s e d i n  each f l a s k .  4  A n a e r o b i c c o n d i t i o n s were o b t a i n e d by f l u s h i n g  t h e f l a s k s w i t h t h e p r e v i o u s l y d e s c r i b e d gas m i x t u r e . A t zero t i m e and a t 10 minute i n t e r v a l s t h e r e a f t e r , endogenous and one s u b s t r a t e  f l a s k were removed f r o m t h e  one bath,  and t h e m e t a b o l i c a c t i v i t y o f t h e c e l l s was i m m e d i a t e l y stopped by t h e a d d i t i o n o f 1 m l . 1:3 d i l u t i o n o f sulfuric acid.  concentrated  H a v i n g ensured a t h o r o u g h m i x t u r e o f t h e a c i d  -48with the contents of the f l a s k , the bulk of the sample was transferred to a centrifuge tube.  The c e l l s were then  separated from the suspending medium, and duplicate.0.5 ml. aliquots of the supernatent were analysed f o r phosphorus. The a b i l i t y of the c e l l s to u t i l i z e glucose and fructose anaerobically was determined by allowing the c e l l s to r e s p i r e In a nitrogen atmosphere i n the presence of 10 jaM of the substrate.  Pour cups were prepared f o r each substrate,  duplicates to be removed at zero time and after 1 h r . respiration.  At the end of that time, the f l a s k s were  a c i d i f i e d as previously described, the c e l l s were centrighiged down and aliquots of the supernatent were used for the reducing sugar determination. Chemical:  Phosphorus was determined according to the method  of King (1932) and reducing sugar by the method of Johnson (1947).  Analyses f o r 2-keto-gluconic a c i d were made using  the polarimetric method of Stubbs et a l (1940). Intermediate compounds: Dr. H.L.A. Tarr.  Ca gluconate was obtained from  S amples of Ca 2 and 5-ketogluconate were  kindly supplied by Dr. C.E. Georgi and Dr. L.B. Lockwood. Oxalacetic a c i d was prepared according to Werkman, 1943. Small quantities of the acid were dissolved and neutralized immediately before use. The preparation of sodium pyruvate has been described i n the previous section.  Standard  solutions of malate, fumarate, l a c t a t e and malonate were made up by n e u t r a l i z i n g equivalent amounts of the free acids with 10 N NaOH.  The remainder of the intermediate compounds were  -49sstandard l a b o r a t o r y reagents  A.  STUDIES ON CELLS GROWN WITH AMMONIUM SUCCINATE AS NITROGEN SOURCE  Anaerobic:  Conflicting evidence exists concerning the possible  anaerobiosis of the pseudomonas organisms. growth experiments, many workers (Tanner  As a result of ; Bergey 1948)  have considered that these bacteria are f a c u l t a t i v e l y aerobic. This observation has been confirmed by i s o l a t i n g l a c t i c acid,  -50a normal end-product of g l y c o l y s i s , i n fermentations by B. pyocyaneus (Aubel 1921) and P. lendneri (Schreder 1953). However others have found that growth i n the absence of oxygen i s i n s u f f i c i e n t evidence f o r assuming the presence of an anaerobic system.  WorHing on such a theory, Seleen and  St ark (1943) were able to make a l l 199 representative strains of pseudomonas studied not to grow i n the absence of oxygen, under conditions which otnerwise were optimum f o r t h e i r activity.  As a r e s u l t of this observation, they have con-  cluded that these organisms are obligately aerobic. In view of the contradictory reports obtained with growth experiments, we have resorted to the use of r e s p i r a tory studies to determine the r e l a t i o n s h i p of these  organisms  to oxygen. Anaerobic r e s p i r a t i o n may be evaluated manometrically by measuring the p roduction of acid and/or gas by c e l l s metabolizing i n a nitrogen atmosphere.  Under these conditions,  the organism must obtain energy by means of the MeyerhofEmbden system, the end products of which are l a c t i c and acetic acids, carbon dioxide and ethyl alcohol.  While the  production of alcohol i s usually associated with the format i o n of v o l a t i l e acids, i t i s necessarily accompanied by the l i b e r a t i o n of carbon dioxide.  Therefore, irrespective of  the nature of the fermentation, the end effect of anaerobic r e s p i r a t i o n i s the production of acid and gas.  -51Table I The Anaerobic Fermentation of Glucose by P. aeruginosa In cup:  endogenous substrate 0.5 ml. 0.5 ml. 0.3 ml. 0.3 ml. to 3.0 ml. to 3.0 ml.  c e l l s 10x concentration 0.1 M NaHC0 water 3  In  side-arms: (1) glucose 25juM/ml. (2) 3N HCl  In centre well: Atmosphere:  0.2 ml. 0.3 ml.  0.3 ml.  Yellow phosphorus  5$ COg : 95$ N ! : : :  endogenous (HCl at end)  i :«  glucose (HCl at zero time)  ! i  . glucose (HCl at end)  i :  2  C0 produced : GO2 l i b e r a t e d during r e s - : by HCl piration -u.l. ju.l. J 2  j  —  mm  -  82.0 :  70.0  i  70.2  Wo i n d i c a t i o n of metabolic a c t i v i t y was obtained when the c e l l s were incubated with glucose under a nitrogen atmosphere.  These r e s u l t s have been confirmed by the i n -  a b i l i t y of the c e l l s to absorb phosphorus and to u t i l i z e glucose or fructose under the same conditions., (Stanier (1948) has suggested that fructose was more suitable than glucose f o r fermentation.)  Table I I The Uptake of Inorganic phosphorus by Resting Cells  In cup:  endogenous c e l l s 10x concentration 0.1 M NaH CO3 water  to  In side-arm: 11) glucose 25;uM/ml. (2) KH0PO4 0.114$  0,5 ml. 0.5 ml. 0.6 ml. 0.6 ml. 3.0 ml. to 3.0 ml.  0.5 ml.  (.5 ml. =130 ;ugm P) In centre well: Atmosphere:  yellow phosphorus  5$ COg : 95$ Ng  Time (mins,)  :  0 10  Amount of P per ml. : substrate endogenous t Ougm.) Cugm.) 76.2  :  20  :  72.0  :  78.4  :  72.6  SO  :  73.0  :  74.6  40  :  -  :  76.2  :  73.0  :  72.0  50 60  76.2  substrate  0.2 ml. 0.5 ml.  Table I I I The- Anaerobic U t i l i z a t i o n of Glucose and Fructose by Resting Cells In cup: c e l l s lOx concentration M/15 : phosphate buffer, pH 7.2 water to  0.5 ml. 1.5 ml. 3,0 ml.  In side-arm: substrate 25-uM/ml.  0.4 ml.  In centre w e l l : Atmosphere:  nitrogen. :  Time (mins.)  :  |  Amount of substrate per cup glucose • fructose (mgs.) ; (mgs.)  0  :  1.19  :  1.4  60  :  1.69  :  1.35  The results of our studies are i n complete agreement with the growth experiments of Seleen and Stark.. By r e l a t i i g t h i s data to that obtained on i r o n , we have concluded that the organism i s o b l i g a t e l y aerobic.  - 5* -  PART B.  III  STUDIES ON- CELLS GROWN WITH AMMONIUM SUCCINATE AS NITROGEN SOURCE  S i g n i f i c a n c e o f t h e endogenous r e s p i r a t i o n :  P r i o r t o under-  t a k i n g a study o f t h e a e r o b i c d i s s i m i l a t i o n o f g l u c o s e , i s n e c e s s a r y t o d e t e r m i n e t h e degree t o w h i c h t h e  it  endogenous  r e s p i r a t i o n c o n t r i b u t e s t o t h e t o t a l oxygen u p t a k e d u r i n g substrate  oxidation.  Since the Influence of o x i d i z a b l e sub-  s t r a t e s on endogenous r e s p i r a t i o n has not been it  established,  i s u s u a l t o c o n s i d e r e i t h e r t h a t t h e endogenous u p t a k e  unchanged i n t h e p r e s e n c e o f o x i d i z a b l e s u b s t r a t e , it i s negligible.  Is  or that  The d e c i s i o n t o I n c l u d e i n t o t o o r t o  i g n o r e t h e endogenous r e s p i r a t i o n I s o f t e n made w i t h o u t s u f f i c i e n t experimental basis f o r the choice.  Much o f  the  c o n f u s i o n a r i s i n g f r o m t h e use o f t h i s approach can be a v o i d e d by t h e a p p l i c a t i o n o f a t e c h n i q u e employed by Stanier  (1948).  S t a n i e r has suggested t h a t t h e t r u e amount o f oxygen consumed i n t h e o x i d a t i o n o f one micromole o f s u b s t r a t e  is  o b t a i n e d by o b s e r v i n g t h e d i f f e r e n c e i n t h e t o t a l oxygen uptake of c e l l s r e s p i r i n g i n the presence of d i f f e r e n t centrations  of that  substrate.  Thus, the  con-  difference  between t h e t o t a l oxygen consumption on t h r e e and f o u r , o r f o u r and f i v e micromoles i s t h e amount o f oxygen r e q u i r e d f o r t h e o x i d a t i o n o f one micromole o f t h e s u b s t r a t e .  By t h e  -55-  <0 it!  600\  >  400  > >  t  zoo  ^ 20  40  T  n</oyet?b({s  60  20  MINUTES Figure l i  D i f f e r e n t i a l method o f d e t e r m i n i n g oxygen uptake f o r La*of glucose. I n cups: g l u c o s e , V 3 0 phosphate b u f f e r , pH 0 . 5 ml. 20x c e l l s .  7.4,  5^ "  56' ~  use o f a m o d i f i c a t i o n o f t h i s t e c h n i q u e ,  i t has  been  p o s s i b l e t o show t h a t t h e endogenous oxygen u p t a k e may be disregarded i n i n t e r p r e t i n g r e s p i r a t o r y data f o r organism ( f i g u r e  this  1),  Whereas f i v e micromoles o f g l u c o s e a b s o r b e d 540 m i c r o l i t r e s o f oxygen f o r o x i d a t i o n , f o u r micromoles and t h r e e micromoles u t i l i z e d 410 and 320 m i c r o l i t r e s i.e.  respectively,  each micromole r e q u i r e d s l i g h t l y g r e a t e r t h a n 100  m i c r o l i t r e s o f oxygen f o r o x i d a t i o n .  T h i s f i g u r e was c o n -  f i r m e d by n o t i n g t h e d i f f e r e n c e i n oxygen u p t a k e between f i v e microMdnir-es and t h r e e m i c r o M i s t e s o f g l u c o s e (110 m i c r o l i t r e s o f oxygen p e r micromole o f  glucose).  D u r i n g t h e same r e s p i r a t o r y p e r i o d endogenous absorbed a p p r o x i m a t e l y 100 m i c r o l i t r e s o f o x y g e n .  suspensions If  this  amount were s u b t r a c t e d from t h e t o t a l u p t a k e o f t h e c e l l s each o f t h e s u b s t r a t e  in  f l a s k s , t h e o x i d a t i o n o f one m i c r o -  mole o f g l u c o s e would r e q u i r e 8 8 , 79 o r 74 m i c r o l i t r e s  of  oxygen depending upon t h e c o n c e n t r a t i o n o f t h e sugar employed.  S i n c e t h e o x i d a t i o n o f one micromole o f g l u c o s e s h o u l d  R e q u i r e t h e same amount o f oxygen ( w i t h i n t h e l i m i t s o f b i o l o g i c a l e r r o r ) under t h e same c o n d i t i o n s , i t was t h e r e f o r e c o n c l u d e d t h a t t h e endogenous r e s p i r a t i o n was a n e g l i g i b l e factor during substrate  oxidation.  H e n c e f o r t h no  a l l o w a n c e was made f o r t h i s a c t i v i t y when i n t e r p r e t i n g respiratory  data.  O x i d a t i o n o f the p o s t u l a t e d Intermediate.compounds;  The use  31  51 -57o f manometric t e c h n i q u e s i s w e l l s u i t e d t o a s t u d y o f t h e intermediate metabolism o f g l u c o s e .  By o b s e r v i n g t h e  o x i d a t i v e a c t i v i t y of c e l l s r e s p i r i n g i n the presence o f postulated intermediates,  the  one can determine whether o r not  t h e s e compounds a r e p o s s i b l e i n t e r m e d i a t e s I n t h e breakdown of the  sugar.  Two i m p o r t a n t f a c t o r s must be c o n s i d e r e d when i n t e r preting respiratory data.  S i n c e no m e t a b o l i c system c a n -  f u n c t i o n more r a p i d l y t h a n t h e slowest o f i t s  intermediates,  a l l i n t e r m e d i a t e compounds must be o x i d i z e d a t l e a s t r a p i d l y as the i n i t i a l s u b s t r a t e .  as  Secondly, the extent t o  w h i c h an i n t e r m e d i a t e i s o x i d i z e d must be t h e same as t h a t o f t h e parent compound.  F o r example, i f c e l l s r e s p i r i n g . . I n  t h e presence o f 5 micromoles o f g l u c o s e , 15 micromoles o f ^ sodium a c e t a t e o r 2 . 5 micromoles o f c a l c i u m g l u c o n a t e c o n sume 540, it  620 and 540 m i c r o l i t r e s o f oxygen r e s p e c t i v e l y , -  i s possible that gluconic a c i d i s intermediate i n the  breakdown o f g l u c o s e ( b o t h t h e sugar and i t s a c i d a r e o x i d i z e d t o t h e s a m e - r e l a t i v e e x t e n t , i . e . 83$ o f  the  t h e o r e t i c a l v a l u e ) , but sodium a c e t a t e c o u l d not be a p a r t o f t h i s o x i d a t i v e scheme.  ( C e l l s r e s p i r i n g i n i t s presence  consume an amount o f oxygen a p p r o a c h i n g t h e o r e t i c a l . )  Be-  cause t h e degree o f s y n t h e s i s o b t a i n e d d u r i n g t h e o x i d a t i o n o f g l u c o s e and a c e t a t e i s not t h e same, i t i s apparent t h e two compounds a r e b e i n g d i s s i m i l a t e d by means o f r e s p i r a t o r y mechanisms.  that  different  Zo  40  60  Mf/VUTES Figure 2 :  Oxygen uptake of glucose ammonium succinate  cells.  I 5 * M g l u c o s e ; II 2.5>MCa gluconate; III 2,5^*  Ca-2-ketogluconate; IV 2 . 5 A M Ca 5-ketogluconate; V 12/U.m Na oxalacetate; VI I^MM Na malate; VII 7>« Ka succinate; VIII IACH Na fumarate; IX 5^- Na c i t r a t e ; X endogenous. In cups: substrate, M / 3 0 phosphate buffer, pH 1 ,h> 0 . 5 ml. 2 0 x c e l l s . A1  -59-  £0  4-0  GO  M/A/UTtzJ Figure 3s  Oxygen uptake of glucose ammonium succinate  cells.  In cups: substrate (5>* glucose, 1 5 ^ « N a acetate, 10/<M Na pyruvate, Na l a c t a t e , Na glycerate, glycerol) M/30 phosphate buffer pH 7 . 4 (pH 6 . 0 pyruvate), 0 . 5 ml. 2 0 x c e l l s . M  -60F i g u r e s S and 3 show t h e r a t e s and amounts o f oxygen u p t a k e o f c e l l s r e s p i r i n g i n t h e presence o f v a r i o u s i n t e r mediate compounds.  In keeping w i t h the p r i n c i p l e s p r e v i o u s l y  o u t l i n e d , i t appears t h a t g l u c o n a t e , 2 - k e t o g l u c o n a t e , ate,  m a l a t e , furoarate and p y r u v a t e a r e p o s s i b l e  , i n t h e a e r o b i c breakdown o f g l u c o s e .  succin-  intermediates  Since acetate,  lactate,  g l y c e r a t e , g l y c e r o l and 5 - k e t © g l u c o n a t e a r e e a c h o x i d i z e d t o a g r e a t e r o r l e s s e r degree t h a n t h e parent s u b s t r a t e t h e y a r e e x c l u d e d f r o m t h e system.  No a b i l i t y t o d i s s i m i l a t e c i t r i c  .  a c i d was o b s e r v e d . It  i s i n t e r e s t i n g t o contrast the metabolism of these  c e l l s with that of the acetobacter.  A l t h o u g h b o t h organisms  a r e o b l i g a t e l y a e r o b i c , t h e breakdown o f g l u c o s e by t h e a c e t i c a c i d b a c t e r i a proceeds by way o f 5 - k e t o g l u c o n i c a c i d (Stubbs 1940)  w h i l e t h e o x i d a t i o n o f t h e sugar by t h e  pseudomonas i n v o l v e s t h e S - k e t o d e r i v a t i v e .  A p p a r e n t l y no  use can be made o f t h e 5 - k e t o g l u c o n a t e by t h e l a t t e r group o f organisms. The presence o f S - k e t o g l u c o n l c a c i d i n growing c u l t u r e s : From t h e p r e v i o u s r e s p i r a t o r y s t u d i e s , i t would appear t h a t  the  o x i d a t i o n o f g l u c o s e proceeded by way o f g l u c o n i c and g - k e t o gluconic a c i d s .  S i n c e t h e s e substances might be a t t a c k e d by  t h e c e l l s even a l t h o u g h t h e y d i d not n o r m a l l y f u n c t i o n i n g l u c o s e d i s s i m i l a t i o n , i t appeared advantageous t o d e t e r m i n e t h e g e n e r a l d i r e c t i o n o f g l u c o s e breakdown by i s o l a t i n g one o f t h e i n t e r m e d i a t e compounds. R e c e n t l y , by t h e use o f u n p h y s i o l o g i c a l c o n d i t i o n s ,  -61i . e . high concentrations  of substrate,  p r e s s u r e a e r a t i o n and  a g i t a t i o n , e x c e l l e n t y i e l d s o f g l u c o n i c , 2 - and 5 - k e t o g l u c o n i c and  k e t o g l u t a r i e a c i d s have been o b t a i n e d i n f e r m e n t a -  t i o n s by s p e c i e s o f phytomonas, pseudomonas and a c e t o b a c t e r (Stubbs 1937,  1940;  Lockwood, T a b e n k i n and Ward 1941;  Lockwood et a l 1 9 4 6 ) .  By a l l o w i n g t h e o r g a n i s m t o grow i n  the presence of calcium carbonate,  it  s h o u l d be p o s s i b l e  demonstrate t h e a c c u m u l a t i o n o f 2 - k e t o g l u c o n i c a c i d as sparingly soluble calcium s a l t .  One c o u l d t h e r e f o r e  to  the  conclude  t h a t t h e d i s s i m i l a t i o n o f g l u c o s e proceeded by way o f t h e s e oxidized acids. A growth experiment was set up t o demonstrate t h e p r o duction of 2-ketogluconic a c i d .  The medium and methods o f  a n a l y s i s were t h o s e employed by Stubbs d u r i n g s t u d i e s o f t h e commercial f e r m e n t a t i o n ( 1 9 4 0 ) .  To o b t a i n a normal breakdown  o f t h e s u g a r , t h e c u l t u r e s were g e n t l y a e r a t e d throughout  the  i n c u b a t i o n p e r i o d and no p r e s s u r e o r v i g o r o u s a g i t a t i o n o f any k i n d was employed. At i n t e r v a l s / samples o f t h e c u l t u r e were removed and d e p r o t e i n i z e d , and t h e i r o p t i c a l r o t a t i o n was d e t e r m i n e d . Knowing t h e t o t a l amount o f copper r e d u c e d and t h e  specific  r o t a t i o n o f t h e s o l u t i o n , i t was p o s s i b l e t o c a l c u l a t e  the  percentage g l u c o s e and 2 - k e t o g l u c o n i c a c i d i n t h e f e r m e n t a t i o n liquors.  The j u s t i f i c a t i o n f o r t h i s method o f a n a l y s i s  based on t h e f a c t t h a t  i n mixed s o l u t i o n s o f g l u c o s e ,  and 2 - k e t o g l u c o n i c a c i d s , t h e observed r o t a t i o n i s  is  gluconic  essentially  '  -62due t o t h e o p t i c a l a c t i v i t y o f t h e f i r s t and l a s t  components.  S i n c e g l u c o s e i s s t r o n g l y d e s t r o - r o t a r y and 2 - k e t o g l u c o n a t e i s even more l e v o - r o t a r y , Stubbs e t a l (1940) have been a b l e t o e s t a b l i s h a r e l a t i o n s h i p between t h e s p e c i f i c r o t a t i o n o f t h e s o l u t i o n and t h e c o n c e n t r a t i o n s o f g l u c o s e and S2-ketogluconic a c i d present. The d a t a p r e s e n t e d ( T a b l e i v ) show t h a t t h e d i s a p p e a r ance o f 1% g l u c o s e from t h e growth medium i s accompanied by an i n c r e a s e o f a p p r o x i m a t e l y 0 . 5 - 0 . 3 5 $ I n t h e amount o f Ca 2 - k e t o g l u c o n a t e .  However, t h i s r a t i o o f t h e two m e t a b o l -  i t e s cannot be used t o d e t e r m i n e t h e e f f i c i e n c y o f t h e c o n v e r s i o n of glucose through these channels,  since, as p r e -  v i o u s l y mentioned, the m a j o r i t y o f the substrate i s  oxidized  t o c o m p l e t i o n ( i . e . CO*,, BgO and s t o r a g e p r o d u c t s ) .  A l -  though t h e r e s u l t s o b t a i n e d by t h i s method a r e not q u a n t i t a t i v e , t h e y do i n d i c a t e t h e pathway by w h i c h g l u c o s e i s d i s s i r a i l a t e d t o i t s end p r o d u c t s .  It  i s interesting to  observe  that the glucose metabolism of t h i s a e r o b i c bacterium ressembles t h a t o f t h e f u n g i i n t h a t t h e o x i d a t i o n p r o c e e d s by way o f g l u c o n i c and k e t o g l u c o n i c a c i d s .  Porges e t  al  ( 1 9 4 0 ) , R o b e r t s and Murphy ( 1 9 4 4 ) , W i l l i a m s ( 1 9 4 5 ) , W e l l s et a l ( 1 9 3 9 ) , and Moyer et a l (1940) have shown t h a t l y high y i e l d s of these Intermediates  sufficient-  can be o b t a i n e d i n  c u l t u r e s o f A . n i e e r t o w a r r a n t i t s u s e i n commercial fermentations.  —63«»  Table IV The Production of 2-ketogluconic Acid . Glucose Age of ,Specific ,Reducing ,Reduced [ 2-Ketoculture ,rotation 'substances ', copper ' gluconate o .mg./ml. . mg./ml. \ * K  !+2.0  4 days  +1.65  7 days  + .23  ! 53.3 ; 38.4 . 34.9  -  j111.9 80.6 ! 73.3  !  1. 5.4 .17  1 4.83  .90  ! 2.78  -64Anaerobic d i s s i m i l a t i o n of 2-ketogluconic a c i d i  Lipmann  (1936) has shown t h a t t h e a e r o b i c o x i d a t i o n o f g l u c o s e p r o ceeds by means o f a s e r i e s o f s t e p - w i s e u n t i l a t r i o s e molecule i s o b t a i n e d .  decarboxylations  By a p p l y i n g t h i s  t o t h e o x i d a t i o n o f k e t o g l u c o n i c a c i d , i t might be  theory  expected  t h a t p e n t o s e and carbon d i o x i d e would be produced i n e q u i m o l e c u l a r amounts d u r i n g t h e d e c o m p o s i t i o n o f t h e a c i d . R u f f o et a l (1945) have suggested ( a l t h o u g h w i t h o u t s u f f i c i e n t e x p e r i m e n t a l e v i d e n c e ) t h a t t h e o x i d a t i o n o f g l u c o s e by k i d n e y and l i v e r p r e p a r a t i o n s f o l l o w s t h e g e n e r a l p a t t e r n g l u c o s e —^ g l u c o n i c  2 ketogluconic  GOg + p e n t o s e —^ p e n t o n i c , I f such i s t h e case w i t h P . a e r u g i n o s a , i t  etc.  s h o u l d be p o s s i b l e  t o o b t a i n carbon d i o x i d e from the anaerobic decomposition of S£-ketogluconic a c i d . Under t h e c o n d i t i o n s o f o u r e x p e r i m e n t , t h e l i b e r a t i o n o f c a r b o n d i o x i d e from k e t o g l u c o n i c a c i d c o u l d not demonstrated.  be  T h i s observation i s i n s u f f i c i e n t evidence  from w h i c h t o c o n c l u d e t h a t t h e a e r o b i c breakdown o f does n o t p r o c e e d i n t h i s s t e p - w i s e manner.  It  glucose  i s p o s s i b l e that  a n o x i d a t i v e d e c a r b o x y l a t i o n might be i n v o l v e d ; t h u s  the  o x i d a t i o n o f 2 - k e t o g l u c o n i c a c i d through pentose t o p e n t o n i c a c i d might be r e p l a c e d by t h e s i n g l e r e a c t i o n . -2H 2 - k e t o g l u c o n i c a c i d + H g O — p e n t o n i c acid+CO^ decarboxylase I n t h e absence o f a s u i t a b l e hydrogen a c c e p t o r ,  -65TABLE V The F o r m a t i o n o f gQg from S - k e t o g l u c o n a t e under A n a e r o b i c C o n d i t i o n s In flask:  0 . 5 m l . c e l l s (SOx c o n c e n t r a t i o n ) 1 . 5 m l . M/15 pH 7 . 4 phosphate b u f f e r water t o 3 . 0 m l .  In side-arm:  ( l ) S . 5 J U M Ca S - k e t o g l u c o n a t e (S) 0 . 3 m l . 3N H C l .  In centre w e l l : Atmosphere:  (omit i n endogenous)  y e l l o w phosphorus  nitrogen  CQg e v o l v e d :  Total i n 70 m i n s . JU.l.  a t 70 minutes ;u.l.  a t zero time  :ji.l.  *  endogenous  !  s  *  » •  *• #  '2-ketogluconate :  i.e.  1.3  *  1.4  l  0.1  2.3  :  0.2  o x y g e n , t h e k e t o - a c i d c o u l d n o t be  decarboxylated.  Lipmann (1939) has shown t h a t a s i m i l a r t y p e o f r e a c t i o n involved i n the aerobic acidifleans  is  d i s s i m i l a t i o n o f p y r u v a t e by Bac,t.  longissimum.  Anaerobic d i s s i m i l a t i o n of oxalacetate: t r e a t e d c e l l s o f M . l y s p d e i k t j.cu,s  r  U s i n g acetone  K r a m p i t z and Werkman (1941) •  O b t a i n e d p y r u v i c a c i d and carboia d i o x i d e f r o m o x a l a c e t i c a c i d . S i m i l a r r e a c t i o n s have been observed by B e r n s t e i n  (1944) and  L i c h s t e i n and Umbreit (1947) employing a n a e r o b i c  suspensions  -66-  300  40  60  I2LO  MINUTES Figure 4 :  Anaerobic  decarboxylation of oxalacetate  In cups: 12,*c*iNa oxalacetate, / 3 0 phosphate buffer, pH 7 . 4 , 0 . 5 ml. 20x c e l l s . In side arm: 3N HCl. In centre well: phosphorus. Atmosphere: nitrogen. w  -67o f P.. s a c c h a r o p h l l a and g . c o l i r e s p e c t i v e l y .  To d e t e r m i n e  whether o r not P.- a e r u g i n o s a a t t a c k s t h i s s u b s t r a t e  in a  s i m i l a r manner, a 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 was made o f t h e carbon d i o x i d e produced f r o m t h e a n a e r o b i c d e c o m p o s i t i o n o f oxalacetate. S i n c e one micromole, ("22.4 m i c r o - l i t r e s ) o f carbox d i o x i d e i s formed f r o m t h e a n a e r o b i c breakdown o f one micromole o f oxalacetate,  a t h e o r e t i c a l y i e l d o f 168 m i c r o l i t r e s o f  d i o x i d e might be e x p e c t e d f r o m t h e 12 m i c r o m o l e s o f i n i t i a l l y supplied t o the c e l l s .  carbon  substrate  ( F i g u r e 4 shows t h a t  4.5  micromoles o f o x a l a c e t a t e were l o s t by spontaneous d e c o m p o s i t i o n d u r i n g t h e c o u r s e o f t h e experiment - see b o i l e d c e l l curve). substrate  However, i n p r a c t i c e , o n l y 84$ u t i l i z a t i o n o f i s obtained, (Figure 2 ) ,  therefore the  the  complete  conversion of oxalacetate t o pyruvate should r e s u l t i n t h e e v o l u t i o n o f o n l y 141 m i c r o l i t r e s o f c a r b o n d i o x i d e . By s u b s t r a c t i n g t h e amount o f c a r b o n d i o x i d e s p o n t a n e o u s l y p r o d u c e d , f r o m t h e amount o f carbon d i o x i d e l i b e r a t e d by t h e c e l l s , i t can be seen t h a t 152 m i c r o l i t r e s ( 6 . 8  micromoles)  o f carbon d i o x i d e were a c t u a l l y o b t a i n e d f r o m t h e 6 . 3 m i c r o moles o f o x a l a c e t a t e u t i l i z e d .  T h i s amount i s w i t h i n  e x p e r i m e n t a l e r r o r o f r e p r e s e n t i n g a complete c o n v e r s i o n o f oxalacetate t o pyruvate.  I t was t h e r e f o r e  concluded t h a t  o x a l a c e t a t e f u n c t i o n e d I n t h e r e s p i r a t i o n o f t h e s e organisms by v i r t u e o f a n a e r o b i c d e c o m p o s i t i o n t o p y r u v i c a c i d . Malonate:  The c o m p e t i t i v e i n h i b i t i o n o f  succinate  -68-  60d  y  JUS.  -  -  400  -  /// yT  Zoo  /// /// ///  c  J/7  40  60  So  MINUTES Figure 5  The effect of malonate on the oxidation of glucose. -2 I glucose; II glucose + 10 Mmalonate; I I I glucose + 5 x 1 0 ~ M malonate; IV endogenous; V endogenous + 10 Mmalonate. In cups: 5-"*v glucose, malonate, w/$Q phosphate buffer, pH 7.4, 0.5 ml. 20x c e l l s . 2  -69-  20 Figure  6:  40  60  80  Ml NO T£S  The e f f e c t succinate.  o f malonate  on t h e o x i d a t i o n  In cups: 7/<.MNa s u c c i n a t e , b u f f e r , 10~ M m a l o n a t e , 0.5 2  of  M/30 p h o s p h a t e m l . 15 x c e l l s .  "0  -70-  20  60  40  Q>0  MINUTES Figure 7:  The effect of malonate on the oxidation of succinate. In cups: 7/"^i Na succinate, M / 3 0 phosphate buffer, 5 x 1 0 ~ M malonate, 0 . 5 ml. 15 x c e l l s . Substrate added simultaneously or at 30 minutes, 2  -71-  © x i d a t l o n 'by m a l o n i c a c i d has been observed by many i n v e s t i g a t o r s (Krebs 1937; S z e n t - G y o r g y i 1935, 1936; Q u a s t e l e t a l 1928 and o t h e r s ) .  Green (1941) and Ivans, (1942) have  summarized t h e e s s e n t i a l d e t a i l s o f t h i s r e a c t i o n i n t h e i r reviews o f r e s p i r a t i o n .  By v i r t u e o f i t s s t r u c t u r a l  s i m i l a r i t y t o s u c c i n i c a c i d , malonate may be u s e d a s a s p e c i f i c i n h i b i t o r o f s u c c i n i c dehydrogenase,  thereby  b l o c k i n g t h e o x i d a t i v e c y c l e and r e s u l t i n g i n t h e a c c u m u l a t i o n of succinic a c i d . Since the i n h i b i t i o n i s a competition f o r the a v a i l a b l e enzyme, t h e e f f e c t i v e  c o n c e n t r a t i o n s o f malonate a r e r e l a t i v e  r a t h e r t h a n a b s o l u t e amounts, t h a t  i s , the r a t i o of succinic  a c i d t o malonic a c i d i s t h e important f a c t o r .  Varying  degrees o f i n h i b i t i o n have been r e p o r t e d u s i n g s o l u t i o n s r a n g i n g from 1 0 ~  4  to 1(T  S  molar.  C o n c e n t r a t i o n s o f malonate from 5 x 1 0 " ^ molar t o 1QT% molar d i d n o t i n h i b i t t h e o x i d a t i o n o f g l u c o s e o r s u c c i n i c a c i d by t h e s e organisms ( F i g u r e s 5 , 6 , 7,  ).  ( ater L  experiments demonstrated t h a t t h e p e r i o d o f a d a p t a t i o n t o s u c c i n a t e by c e l l s grown i n s u c c i n i c a c i d - f r e e media was l e n g t h e n e d by t h e a d d i t i o n o f 5 x Iff® molar malonate (Figure 10)).  T h i s was t r u e i f t h e c e l l s were a l l o w e d t o  r e s p i r e i n t h e p r e s e n c e o f m a l o n i c a c i d f o r t h i r t y minutes p r i o r t o the addition of substrate,  or i f additions of  s u c c i n a t e and malonate were made s i m u l t a n e o u s l y .  By e x t e n d i n g  t h e d u r a t i o n o f t h e e x p e r i m e n t a l p e r i o d t o 2 h r s . i t was  -72-  ^ 1000.  5  800  r  '  600 J>-M  400  y  Of  * / \' y *  ><  y  y i ^ 4 30  60  SO  /20  150  MINUTES Figure 8;  The effect of malonate on the oxidation of glucose - showing adaptation to malonate. I glucose + malonate; II glucose; I I I endogenous • malonate; IV endogenous. In cups: 5 ^ H _ glucose, M / 3 0 phosphate buffer, pH 7«4, 10 M malonate, 0.5 ml. 20x c e l l s . 2  7'-\ -73-  BOO  a  1  e  2T  A< y  y  5  200  y  i  y  /  60  30  SO  /20  /so  MINUTES Figure 9:  The effect of malonate on the oxidation of succinate - showing adaptation to malonate. I succinate + malonate; II succinate; III endogenous + malonate; IV endogenous. In cups: 7><M Na succinate, M./30 phosphate buffer, pH 7.4, 1 0 ~ M malonate, 0.5 ml. 20x c e l l s . 2  •74-  p o s s i b l e t o demonstrate t h e p r e s e n c e o f a d a p t i v e enzymes f o r the o x i d a t i o n o f malonic a c i d (Figures 8,  9).  S i m i l a r r e s u l t s have been o b t a i n e d by o t h e r w o r k e r s . B e r n s t e i n (1943) observed a d e p r e s s i o n i n t h e r a t e but n o t i n t h e t o t a l amount o f oxygen u p t a k e o f c e l l s o f when employing 1 0 "  S  molar s o l u t i o n s o f m a l o n a t e .  saccharouhila Concentra-  t i o n s a s h i g h a s O . S molar were r e q u i r e d t o r e d u c e t h e t o t a l oxygen consumption o f t h e s e o r g a n i s m s .  The o x i d a t i o n o f  m a l o n i c a c i d h a s p r e v i o u s l y been r e p o r t e d by B u t t e r w o r t h i n s t u d i e s on t h e d e g r a d a t i o n o f c i t r a t e , by B.. pvocvanea ( 1 9 2 9 ) . M a l o n i c a c i d was d e c a r b o x y l a t e d t o g i v e a c e t i c a c i d , w h i c h i n t u r n was o x i d i z e d t o g l y c o l i c and g l y o x a l i c a c i d s .  Since  t h e o x i d a t i o n o f g l u c o s e was n o t i n h i b i t e d by m a l o n i c a c i d , i t would appear t h a t  s u c c i n i c a c i d d i d not f u n c t i o n i n  bacterial respiration In either of the previously established aerobic metabolic B.  cycles.  STUDIES ON CRTT.S flRQWN WITH. AMMONIUM PHOSPHITE AS f  NITROGEN SOURCE  <  As a r e s u l t o f t h e s t u d i e s on malonate i n h i b i t i o n , i t appeared p o s s i b l e t h a t t h e u s e o f ammonium s u c c i n a t e a s t h e n i t r o g e n s o u r c e i n t h e g l u c o s e growth medium might a l t e r t h e enzymic c o n s t i t u t i d n o f t h e organisms i n _ s u c h a  manner.that  t h e normal l i n e o f g l u c o s e breakdown would be o b s c u r e d . S i m i l a r l y , growth o f t h e c e l l s I n t h e p r e s e n c e o f g l u c o s e might s t i m u l a t e t h e p r o d u c t i o n o f c e r t a i n enzymes w h i c h c o u l d be u s e d a s a n a l t e r n a t i v e pathway t o t h e n o r m a l system  -75of succinate o x i d a t i o n I . e .  i n the presence o f  concentrations of malonic a c i d .  inhibitory  To o b t a i n c e l l s s u i t a b l e f o r  t h e study o f i n t e r m e d i a t e m e t a b o l i s m , a s e a r c h was made f o r a n i t r o g e n s o u r c e , o t h e r t h a n ammonium s u c c i n a t e , w h i c h w o u l d y i e l d a c t i v e r e s t i n g organisms.  TABLE V I The. I n f l u e n c e o f N i t r o g e n Source o a £ h e a c t i v i t y o f 18. h£i. Suspensions o f t h e Organism I n cup:  0 . 5 m l . c e l l s (20 x c o n c e n t r a t i o n ) 1 . 5 m l . M/15 phosphate b u f f e r , pH 7 . 4 . water t o 3.0 m l .  In side-arm:  0 . 2 m l . g l u c o s e (25^uM/ml) (omit i n endogenous)  In centre w e l l :  0.15 m l . 20$ KOH  nitrogen source  %  nitrogen source  oxygen uptake i n 40 m i n u t e s endogenous glucose (n.l.) (u.l.)  0.3  8.4  98.0  0.3  25.4  90.5  0.3  46.2  423  0.6  50.8  490  urea  0.3  19.4  345  NH^ s u c c i n a t e  0.3  46.1  383  NR4CI  <  r a  4V°4-:  From t h e d a t a p r e s e n t e d i n T a b l e  7 1  , i t appeared t h a t  ammonium phosphate was e n t i r e l y s u i t a b l e f o r use i n l a r g e  -76-  5-  0~JE  i  too  K  y y^y  A y .  y  '9-..7T  •—  ^s*  Or.  ^  77  400  //  //  // i  7 /  7  ^  200  // y  /  /  / ,  y y.  y  2<9  Figure 10:  y  y*  y ^  £  .  -  fy-  "  .—  40 MINUTES  60  SO  The effect of malonate on glucose and succinate oxidation by glucose ammonium phosphate c e l l s . I glucose; II glucose + malonate; III succinate; IV succinate + malonate; V endogenous; VI endogenous + malonate. In cups: 5x^glucose, 7 A M Na succinate, 5 x 1 0 ~ M malonate, M / 3 0 phosphate buffer, 0.5 ml. 15x c e l l s . 2  -77scale c e l l production.  W h i l e t h e endogenous a c t i v i t y o f  t h e s e organisms remained unchanged, t h e oxygen u p t a k e o f c e l l s h a r v e s t e d f r o m b o t h phosphate media exceeded t h a t  of  c e l l s u s p e n s i o n s h a r v e s t e d f r o m g l u c o s e ammonium s u c c i n a t e media.  O t h e r n i t r o g e n s o u r c e s were l e s s e f f e c t i v e ,  due t o t h e s l o w e r r a t e o f growth o f t h e o r g a n i s m .  perhaps I n order  t o a v o i d t h e e x c e s s i v e p r e c i p i t a t i o n o f phosphates o f  the  i n o r g a n i c n u t r i e n t s o f t h e medium, 0 . 3 $ o f NH^H^PO^ was emp l o y e d i n a l l subsequent growth m e d i a .  By t h e use o f t h i s  c o m p l e t e l y i n o r g a n i c b a s a l medium i t was p o s s i b l e t o  further  reduce t h e number o f enzymes which a p p e a r e d t o be c o n s t i t u t i v e f o r t h e o r g a n i s m , and t h e r e f o r e t h e a d d i t i o n o f a s p e c i f i c s u b s t r a t e as energy source r e s u l t e d i n t h e f o r m a t i o n o f  those  enzymes n e c e s s a r y f o r t h e d e g r a d a t i o n o f t h e added compound. Malonatet  E x p e r i m e n t s on t h e e f f e c t o f malonate on t h e  o x i d a t i o n o f g l u c o s e and s u c c i n a t e were r e p e a t e d u s i n g g l u c o s e ammonium phosphate c e l l s ( F i g u r e 1 0 ) .  As was p r e v i o u s l y o b -  s e r v e d w i t h t h e o r i g i n a l ammonium s u c c i n a t e s u s p e n s i o n s , breakdown o f g l u c o s e was not a f f e c t e d by t h e p r e s e n c e  the  of  a c c e p t e d i n h i b i t o r y c o n c e n t r a t i o n s o f m a l o n i c a c i d (5 x 10 M o r e o v e r , i t was shown t h a t  M).  s u c c i n i c a c i d was not a normal  i n t e r m e d i a t e i n t h e breakdown o f g l u c o s e .  C e l l s produced on  t h i s sugar i n t h e absence o f s u c c i n i c a c i d o x i d i z e d g l u c o s e d i r e c t l y ( i r r e s p e c t i v e o f the presence of malonic a c i d ) w h i l e they required a period of adaptation to succinate, the length o f w h i c h was e f f e c t i v e l y I n c r e a s e d by t h e a d d i t i o n o f malonate inhibitor.  -789 Oxygen uptake o f g l u c o s e , s u c c i n a t e and lactate ammonium phosphate c e l l s :  By o b s e r v i n g t h e o x i d a t i v e a c t i v i t y o f c e l l s  grown i n t h e p r e s e n c e o f s p e c i f i c s u b s t r a t e s ,  it  s h o u l d be  p o s s i b l e t o determine the f u n c t i o n of the p o s t u l a t e d i n t e r mediates i n t h e o x i d a t i o n o f t h o s e s u b s t r a t e s ,  and t o  relate  t h i s information to the oxidation of glucose.  The r e s p i r a -  t o r y a c t i v i t y o f g l u c o s e , s u c c i n a t e and l a c t a t e c e l l suspens i o n s a r e r e c o r d e d i n f i g u r e s ii-rt . I n t h e l i g h t o f o u r p r e s e n t knowledge, i t I s  impossible  t o e x p l a i n t h e s i g n i f i c a n c e o f much o f t h i s o x i d a t i v e d a t a . F o r example, i t would appear t h a t u t i l i z e the oxalacetate-malate,  s u c c i n a t e grown c e l l s  succinate-fumarate  mechanism  o f t h e Szent G y o r g y i system, ( a l l o f t h e s e compounds a r e r a p i d l y a t t a c k e d by s u c c i n a t e c e l l s ) , y e t 5 x 10  Molar  s o l u t i o n s o f m a l o n i c a c i d do not depress t h e r a t e o r e x t e n t of succinate o x i d a t i o n .  I t i s p o s s i b l e t h a t t h e pathway o f  l a c t i c a c i d d i s s i m i l a t i o n by l a c t a t e grown organisms i n v o l v e s p y r u v a t e , but l a c t i c a c i d i s o n l y 45$ o x i d i z e d w h i l e p y r u v i c a c i d t a k e s up 75$ o f t h e t h e o r e t i c a l amount o f oxygen r e q u i r e d f o r complete o x i d a t i o n .  I f on t h e o t h e r h a n d , l a c t a t e were  degraded f o l l o w i n g a c a r b o x y l a t i o n t o m a l i c a c i d , t h e  rates  of. o x i d a t i o n o f m a l a t e and l a c t a t e s h o u l d be i d e n t i c a l . F i g u r e 18 shows t h a t t h i s i s not  so.  However, s e v e r a l i m p o r t a n t c o n c l u s i o n s can be drawn c o n c e r n i n g t h e mechanism o f g l u c o s e d e g r a d a t i o n .  Since  the  o x i d a t i o n o f s u c c i n a t e and fumarate by c e l l s h a r v e s t e d from g l u c o s e media r e q u i r e d an a d a p t a t i o n p e r i o d o f 10 t o 2 0  -79-  Qlucosc ~~ -G/uconate  ^gluconate  5-Ketogluconak' 'Endogenous  2o  40  60  MINUTES Figure 11:  80  /oo  Oxygen uptake of glucose ammonium phosphate cells. In cups: substrate (5/&M glucose, 2,5AW Ca gluconate, Ca-2-ketogluconate, Ca 5-ketogluconate), M / 3 0 pho-sphate buffer, pH 7 . 4 > 0 . 5 ml. 15x c e l l s .  -80-  £0  '  40  60  SO  /OO  MINUTES Figure 1 2 :  Oxygen uptake of glucose ammonium phosphate cells. In cups: substrate (5/tM glucose, 8.6/^MNa succinate, 10,«.M Na fumarate and Na malate, 15-*-M Na oxalacetate) H / 3 0 phosphate buffer, pH 7 . 4 0 . 5 ml. 1 5 x c e l l s .  -81-  €0  40  60  SO  loo  M/A/OTE5 Figure 1 3 :  Oxygen uptake of glucose ammonium phosphate c e l l s . In cups: substrate glucose, 1 0 A M Na lactate, 1 5 ^ M Na acetate and Na malonate, 1 2 A M Na pyruvate) M / 3 0 phosphate buffer, pH 7 . 4 ( 6 . 0 pyruvate), 0 . 5 ml. 1 5 x c e l l s .  -82-  ao  to  eo  so  loo  M/A/UT£j Figure 1 4 : "Oxygen uptake of succinate ammonium phosphate cells. In cups: substrate (8.6^*i Na succinate, 5-«.^i glucose, 10>MNa l a c t a t e , 2.5>«-*<Ca gluconate, Ca 2-ketogluconate, Ca 5-ketogluconate) w/30 phosphate buffer, pH 7 . 4 , 0 . 5 ml. 15 x c e l l s .  -83'  4o  (bo  SO  MINUTES Figure 15:  Oxygen uptake o f s u c c i n a t e ammonium phosphate cells. I n cups: s u b s t r a t e (8,6-u.n Na s u c c i n a t e , 15MH Na o x a l a c e t a t e , 10/<n Na malate and Na fumarate) M/30 phosphate b u f f e r , pH 7 . 4 , 0.5 m l . 15x c e l l s ,  -84-  1°  ZOO  4-0  GO  80  M/NUTES Figure 16:  Oxygen uptake of succinate ammonium phosphate cells. In cups: substrate (8.6-AH Na succinate, 15-^-M Na acetate and Na malonate, \2M*A Na pyruvate), M/3Q phosphate buffer, pH 7.4, 0.5 ml. 15x cells.  -85-  £0  4C  <oO  SO  /OO  /WNUTEJ Figure 1 7 :  Oxygen uptake of lactate ammonium phosphate cells. In cups: substrate ( I Q ^ M Na l a c t a t e , 5 ^ * ^ glucose, 2 . 5 A M Ca gluconate, Ca 2-ketogluconate, Ca 5-ketogluconate) M / 3 0 phosphate buffer, pH.7.4, 0 . 5 ml. 1 5 x , c e l l s .  -86-  £0  4C  60  BO  /OO  AWVUT5S Figure 18:  Oxygen uptake of lactate ammonium phosphate cells. In cup: substrate ( I O ^ H Na lactate, Na fumarate, Na malate, 12^tM Na oxalacetate, 8.6^-iNa succinate), M / 3 0 phosphate buffer, pH 7 . 4 , 0.5 ml. 15x c e l l s .  -87-  ^Pyruvate  o Acetate -lactate  Ma/on ate  Endogenous 44  60  ao  /oo  MINUTES Figure 19:  Oxygen uptake of l a c t a t e ammonium phosphate cells. In cups: substrate (10>LM Na l a c t a t e , 12-«.MNa pyruvate, 1 5 x ^ Na acetate and Na malonate), M/30 phosphate buffer, pH 7.4 (6.0 pyruvate), 0.5 ml. 15x c e l l s .  -88n&nutes ( F i g u r e I S )  our previous observation t h a t  succinic  dehydrogenase does not f u n c t i o n i n t h e d i s s i m i l a t i o n o f glucose  has been c o n f i r m e d .  g l u c o s e grown b a c t e r i a ,  Moreover i n t h e r e s p i r a t i o n o f  o x a l a c e t a t e does not f u n c t i o n by  means o f i t s r e d u c t i o n t o m a l i c a c i d , as i t does i n t i s s u e m e t a b o l i s m (Sumner and Somers 1943) carboxylation to pyruvic a c i d . b o t h oxaloacetic  but by v i r t u e o f i t s  de-  ( F i g u r e s 1M and 13 show t h a t  and p y r u v i c a c i d s a r e o x i d i z e d a t t h e same  r a t e i n t h e s e c e l l s whereas m a l a t e i s a t t a c k e d much more slowly.)  F u r t h e r m o r e , whereas b o t h s u c c i n a t e  and l a c t a t e  grown organisms r e q u i r e a p e r i o d o f a d a p t a t i o n b e f o r e t h e y can a t t a c k g l u c o s e ,  g l u c o n i c a c i d and - 2 - k e t o g l u c o n i c a c i d ,  g l u c o s e c e l l s o x i d i z e t h e s e compounds d i r e c t l y w i t h o u t a lag period.  It  can t h e r e f o r e be c o n c l u d e d t h a t  and 2 - k e t o g l u c o n i c a c i d s a r e i n t e r m e d i a t e s  gluconic  i n the  aerobic  breakdown o f g l u c o s e , a n d t h a t t h e enzymes r e s p o n s i b l e f o r o x i d a t i o n o f t h i s hexose a r e a d a p t i v e r a t h e r t h a n i n n a t u r e ( F i g u r e s 1 1 , 14, Synthesis  inhibitions  the  constitutive  17).  The use o f sodium a z i d e and 1 , 4 - d i  n i t r o p h e n o l as r e s p i r a t o r y i n h i b i t o r s has been known f o r some t i m e ( L i c h s t e i n and T o u l e 1944J C l i f t o n 1 9 3 8 ) . employing c r i t i c a l c o n c e n t r a t i o n s  By  o f t h e s e compounds, i t  has  been p o s s i b l e t o r e v e r s e t h e i r i n h i b i t o r y e f f e c t , t h a t i s , stimulate respiration.  T h u s , C l i f t o n (1937; 1938)  t a i n e d complete o x i d a t i o n o f a c e t a t e by P_.  has  ob-  calco-acetlca  u s i n g M/600 - M/1000 s o l u t i o n s o f sodium a z i d e and 2 , 4 , n i t r o p h e n o l , and of g l u c o s e ,  to  di  a c e t a t e , p y r u v a t e and g l y c e r o l  '. 20  40  60  SO  /OO  MINUTES Figure 2 0 :  The effect of sodium azede on the oxidation of glucose. In cups: 5 o glucose, M / 3 0 phosphate buffer, pH 7 . 4 , l O " ^ * NaNo, 0 . 5 ml. 1 5 x c e l l s . Substrate added after 10 minutes' incubation of c e l l s and i n h i b i t o r . aM  •90-  £0  4-0  (aO  80  loo  Ml MUTE 5 Figure 2 1 :  The effect of 2 , 4 dinitrophenol on the oxidation of glucose. I glucose; II glucose • 10 M D N P ; III glucose + 1 0 ~ ^ M D N P ; IV glucose + 1 0 " * D N P ; V endogenous VI endogenous + 1 0 " ^ M D N P ; VII endogenous + 1 0 " ^ and 1 0 " * M D N P . In cups: glucose, M / 3 0 phosphate buffer, pH 7 . 4 , 0 . 5 ml. 15x c e l l s . J  5  fey 1* c o l i e m p l o y i n g M/SOO - M/400 sodium a z i d e o r M/2000 2,4 - d i n i t r o phenol.  The e x a c t way i n w h i c h t h e s e i n h i b i t -  o r s a t t a c k t h e s y n t h e t i c mechanism i s unknown, but i t been o b s e r v e d t h a t t h e i n c r e a s e  i n oxygen u p t a k e i s  has frequent-  l y accompanied by d e p r e s s i o n i n t h e r a t e o f o x i d a t i o n . C o n c e n t r a t i o n s o f 10"*^ M sodium a z i d e and  lo"*  5  2,4  M  d i n i t r o p h e n o l have been f o u n d t o i n c r e a s e t h e t o t a l oxygen uptake o f t h e c e l l s on g l u c o s e ,  at t h e same t i m e l e a v i n g t h e i r  endogenous a c t i v i t y u n a l t e r e d .  Whereas d u r i n g t h e n o r m a l  r e s p i r a t i o n of the c e l l s ,  570jal o f oxygen were used  (84$  —3  o f t h e t h e o r e t i c a l v a l u e ) , i n t h e p r e s e n c e o f 10 and 1 0 ~ . M DNP 616 ; u l and 610 ; u l were a b s o r b e d . 5  amounts r e p r e s e n t  M NaNg These  92$ and 90$ r e s p e c t i v e l y o f t h e  theoretical  amounts o f oxygen r e q u i r e d f o r complete o x i d a t i o n . smaller concentrations  o f t h e s e compounds have not  Since been  employed, i t i s i m p o s s i b l e t o determine t h e p o i n t a t w h i c h synthesis i s completely e l i m i n a t e d . t h e e f f e c t s produced by 1 0 " suggests t h a t  4  However a comparison o f  M and 10"*  5  M 2 , 4 - d i n i t r o phenol  complete o x i d a t i o n would be o b t a i n e d i n t h e —6  presence o f a p p r o x i m a t e l y 10~ I n any c a s e , t h e e f f e c t i v e  M s o l u t i o n s of the i n h i b i t o r .  concentrations  of these i n h i b i t o r s  a r e s u b s t a n t i a l l y l o w e r t h a n t h o s e employed by C l i f t o n ( 1937) D o u d o r o f f (1940) and B e r s t e i n  (1943).  C e r t a i n w o r k e r s ( B e r n s t e i n 1943;  D o u d o r o f f 1940)  have  c o n s i d e r e d t h a t t h e endogenous r e s p i r a t i o n f u n c t i o n e d d u r i n g synthesis i n h i b i t i o n . phenomenon.  We have o b t a i n e d no e v i d e n c e o f  this  I f t h i s were s o , t h e amount o f oxygen a b s o r b e d  -92by c e l l s i n t h e presence o f g l u c o s e + i n h i b i t o r would be t h e sum o f t h e oxygen u p t a k e o b t a i n e d on g l u c o s e (no i n h i b i t o r ) and t h e endogenous r e s p i r a t i o n i n t h e p r e s e n c e o f i n h i b i t o r . ( P r e v i o u s l y i t has been shown t h a t t h e endogenous  mechanism  does not f u n c t i o n i n t h e p r e s e n c e o f o x i d i z a b l e s u b s t r a t e see F i g u r e 1)  .  -  I h i l e a s l i g h t depression i n the rate of  oxygen consumption was o b t a i n e d u s i n g 10"" M s o l u t i o n s o f 3  2,4 DNP, t h e c u r v e f o r t h e o x i d a t i o n o f g l u c o s e + 10™ M 5  N a N , p a r a l l e l e d t h a t o f g l u c o s e and a t n o t i m e d i d i t s  t o be t h e summation o f b o t h a c t i v i t i e s .  appear  I t was t h e r e f o r e  con-  c l u d e d t h a t t h e endogenous mechanism was e q u a l l y i n a c t i v e d u r i n g n o r m a l and a c t i v a t e d r e s p i r a t i o n , and t h a t t h e i n c r e a s e d oxygen u p t a k e o b t a i n e d was due t o t h e i n h i b i t i o n o f t h e synthetic processes of t h e c e l l s . C.  ADAPTATION As a r e s u l t of the work o f Karstrom (1937) i t has been  recognized that two types of enzyme systems function during b a c t e r i a l metabolism; - the constitutive - those present a t a l l times regardless of the composition of the medium, and the adaptive - those produced i n response to stimulation by a s p e c i f i c substrate.  Although Spiegelman (1947) has since  shown that the presence of substrate does not necessarily maintain the adaptive systems at the peak of t h e i r a c t i v i t y , the concept of adaptation has remained e s s e n t i a l l y  unchanged.  In so f a r as we are able to determine, no conclusive evidence has yet been presented to show that glucose i s d i s similated by the action of adaptive enzymes.  The observation  made by Karstrom  (1937) that the fermentation of glucose  proceeded i r r e s p e c t i v e of the composition of the growth medium has since been confirmed by many workers. 1947).  (Spiegelman  Although Stevenson and Gale (1937) have produced re-  l a t i v e changes i n the glucozymase a c t i v i t y of B. c o l l . at no time have they obtained c e l l s incapable of attacking the substrate glucose. Previous studies of our organism have indicated that glucose i s dissimilated by an adaptive enzyme system.  When  the organism was grown i n an inorganic medium containing either succinate or l a c t a t e as the sole source of carbon, the c e l l s obtained oxidized glucose at a rate which became increasingly rapid as the incubation period lengthened (Figures 14 and 17). In order to demonstrate true adaptation of the organism to glucose, i t was f i r s t necessary to obtain c e l l s low i n the apparently constitutive enzymes.  Since the degradation  of a simple substrate would probably give r i s e to fewer intermediates, fewer enzymes would be elaborated by the organism when grown on such a substrate. An experiment  was  therefore set up to determine the carbon compound of least structural complexity which would support gpor-wth of the organism.  -94-  TABLE V I The U t i l i z a t ion of Carbon Compounds by Growing Cultures i n Ammonium Phosphate  substrate  Media  utilization 11 hrs. 17 hrs,  glucose  yellow • pigment present  ++++  Ca-gluconate  ++++  ++++  Ca-2-ketogluconate  ++  ++  Na c i t r a t e  +++  ++++  Na malate  +++  ++++  Na malonate  +++  +  Na fumarate  +++  ++++  +  Na succinate  +++  +++  +  Na acetate  +  +  +  Ma formate  +  Na lactate  +++  Ca-5-ketogluconate  OH  •  + +++  -  Table V I shows that glucose, gluconate, c i t r a t e , malate, lactate, fumarate, malonate, 2-ketogluconate and succinate supported good to excellent growth i n 17 hours.  However  l i g h t e r y i e l d s of c e l l s were obtained during the same period from acetate and formate media.  By extending the time of  incubation to 3-3 days, or by increasing the amount of  -95-  3  •Glucose -G/acose  *-.G lucona-te •Q--+-:.Acelate  1  Glucose  \ Endogeaous  £0  4-0  <bO  MINUTES Figure 2 2 :  BO  loo  Comparative a c t i v i t y of glucose, gluconate and acetate grown c e l l s on glucose. glucose c e l l s ; ++-^ gluconate c e l l s ; acetate c e l l s . In cups: substrate (5-* i glucose, 2.5-A» iCa gluconate, 1 5 ^ M Na acetate), M / 3 0 phosphate buffer, pH 7 . 4 , 0 . 5 ml. 1 5 x glucose and gluconate c e l l s or 2 0 x acetate cells. tlp  x  -96inoculum to 5$, i t was possible to increase the y i e l d of acetate c e l l s s u f f i c i e n t l y to permit t h e i r use as enzymic&lly  deficient r e s t i n g c e l l  suspensions.  A comparison- of the enzymic a c t i v i t y of acetate and glucose grown c e l l s shows that the organism dissimilates glucose by means of an adaptive enzyme system.  Whereas the  i n i t i a l phase of glucose oxidation by glucose and gluconate grown c e l l s i s characterized by a rapid and constant rate of oxygen uptake, the corresponding stage i n the oxidation by acetate grown organisms i s a l a g phase, not less than 1 h r . i n length, during which the c e l l s become adapted to glucose (Figure 23). Following t h i s period of adaptation the rate of substrate u t i l i z a t i o n can be seen to increase gradually and progressively as the time of incubation of c e l l s and substrate i s increased. Simultaneous adaptation;  Since i t has been shown that the  oxidation of glucose i s dependent upon the a c t i v i t y of adaptive enzyme system,s the cycle of glucose breakdowi i s well suited to analysis by means of the simultaneous adaptation technique.  By applying t h i s procedure to the  benzoic acid o x i d i z i n g system of P. fluorescens, Stanier (1947) has been able to e s t a b l i s h c e r t a i n postulated i n t e r mediates as functional units i n the breakdown of t h i s compound. A b r i e f summary of the basic p r i n c i p l e s of the method follows.  I t i s f i r s t recognized that the d i s s i m i l a t i o n of  the substrate i s the net r e s u l t of a series of well-defined,  —97«*  stepwise, chemical reactions (KLuyver 3 ^ 3 1 ),  By adapting  c e l l s to the primary substrate, a simultaneous adaptation i s effected only to those intermediates taking part i n the d i s sociation of that substrate (Karstrom 1937).  Postulated  Intermediates which a c t u a l l y are outside of the system under study, w i l l not be attacked by these c e l l s .  Furthermore,  growth of the organism on an intermediate substrate w i l l produce c e l l s more l i k e l y to adapt to those compounds above i t which are nearer i n the oxidative system. The l i m i t a t i o n s of this method of analysis are apparent. F i r s t the primary substrate must be dissimilated by adaptive enzymes.  Secondly, by i t s use one cannot determine whether  or not postulated intermediates, which are attacked by the constitutive mechanisms, are a c t u a l l y functional components of the oxidative system.  However valuable information can be  obtained concerning the importance of c e r t a i n intermediates i n the breakdown of the parent compound. To investigate the pathway of glucose metabolism by the simultaneous adaptation technique, the oxidative a c t i v i t y of the organism was  compared when the c e l l s were grown on acetate  and on glucose.media.  Since i t has been shown that acetate  c e l l s must adapt to glucose, they must therefore adapt to each of the intermediates taking part i n glucose d i s s i m i l a t i o n providing of course, that none of those intermediates i s oxidized by the constitutive enzyme systems.  Cells previously  adapted to glucose by growth i n the presence ,of that substrate, w i l l oxidize d i r e c t l y a l l intermediates i n the glucose  • Figure 2 3 :  GO  4*>  £0  &0  /OO  MINUTES  Oxygen uptake of glucose and acetate c e l l s . In cups: substrate ( 5 g l u c o s e , 2.5-^MCa gluconate and Ca -ketogluconate), M / 3 0 phosphate buffer, pH 7 . 4 , 0.5 ml. c e l l s . 2  -  Glucose c e l l s ;  Acetate c e l l s .  -99-  (QOO  4oo  £09  30  Figure 2 4 :  40  60  80  /oo  Oxygen uptake of glucose and acetate c e l l s . In cups: substrate (12/<M Da pyruvate, 15-^M Na oxalacetate, 10>MNa malate), M / 3 0 phosphate buffer, pH 7 . 4 ( 6 . 0 pyruvate), 0 . 5 ml. c e l l s . (See Figure 23 for endogenous and glucose curves of glucose c e l l s and endogenous curve of acetate c e l l s ; Figure 26 for acetate curve of acetate cells.) Glucose c e l l s ;  Acetate  cells.  •100-  3D  46  60  SO  IOO  M//VC/TEJ Figure 2 5 :  Oxygen uptake of glucose and acetate c e l l s . In cups: substrate ( 8 . 6 ^ t M N a succinate, I Q A M Na fumarate and Na l a c t a t e ) , M / 3 0 phosphate buffer, pH 7 . 4 , 0 . 5 ml. c e l l s . (See Figure 23 for endogenous and glucose curves of glucose c e l l s and endogenous curve of acetate c e l l s ; Figure 26 for acetate curve of acetate c e l l s . ) Glucose c e l l s ;  Acetate  cells.  -101-  ao  40  60  So  ioo  M MUTES Figure 26:  Oxygen uptake of glucose and acetate c e l l s . In cups: substrate (15/UMNa acetate, 15,-4M Na malonate, Ca 5-ketogluconate), M / 3 0 phosphate buffer, pH 7 . 4 , 0 . 5 ml. c e l l s . (See Figure 23 f o r endogenous and glucose curves of glucose grown c e l l s , and endogenous curve of acetate grown c e l l s . Glucose c e l l s ;  Acetate c e l l s .  -102"breakdown system.  To be an active unit i n glucose  metabolism, a compound must therefore be rapidly oxidized by glucose grown c e l l s , and at the same time, i t must i n duce a period of adaptation i n acetate c e l l s , the length of which i s approximately equal to that obtained when glucose i s the substrate being oxidized. The immediate response  of glucose c e l l s to gluconic  and 2-ketogluconic acids coupled with the prolonged adaptation period required by acetate c e l l s before they are able to attack these compounds i s adequate proof that both gluconic and 2-ketogluconic acids are intermediates i n the oxidation of glucose.  On the other hand 5-ketogluconic a c i d can be  .':•.  eliminated as a possible intermediate since c e l l s previously adapted to glucose are unable to attack this compound. On the basis of the above data i t i s impossible to determine the importance of pyruvate, oxalacetate, malate and lactate i n the breakdown of glucose since these compounds are equally active f o r glucose and. acetate grown cells'.  They  may be a part of the constitutive system of the organism or intermediates, common to both glucose and acetate oxidizing mechanisms.  On the other hand, the oxidation of these  postulated intermediates may be controlled by enzyme,systems which can adapt with ease regardless of the i n i t i a l enzymic constitution of the c e l l .  The present state of knowledge  does not j u s t i f y the inclusion or exclusion of these compounds from the possible intermediates of glucose d i s s i m i l a t i o n .  -103*  Growing the cells i n the presence of either glucose or acetate does not stimulate the production of enzymes necessary for the immediate d i s s i m i l a t i o n of succinic acid. Under the condition s of our experiment this compound necessitated shorter periods of adaptation (20-30 minutes) during wfiich the dells became f u l l y equipped to u t i l i z e the added substrate. It is interesting to compare the types of adaptation obtained using acetate c e l l s on glucose or glucose and acetate c e l l s on succinic acid.  Whereas succinate was  attacked at a rapid and constant rate following a b r i e f induction phase (20-30 minutes), glucose, gluconic acid and 2-ketogluconic acid required long periods of adaptation (approximately 1 hour) after which a slow and gradual in the oxidative a c t i v i t y was observed.  rise  In view of the  existing theory of enzymic adaptation, succinate i s not . an intermediate i n the breakdown of glucose or'acetate. Similarly, glucose and the gluconic acids are not intermediates in the oxidation of acetate.  Perhaps the two types of adapt-  ation may be explained on the basis of the r e l a t i v e nearness of the adapting systems.to the apparently constitutive enzymes of the c e l l s .  Thus i t would appear that the*  dicarboxylic acid cycle, though not functional i n glucose or acetate metabolism, i s closely related to both of these systems, since c e l l s adapt rapidly to succinate while the cycle of glucose oxidation i s apparently far removed from the normal pathways of acetate metabolism since long periods of adaptation to glucose are required.  104-  By applying the simultaneous  adaptation method of  analysis to other compounds and other c e l l s or c e l l preparations, i t should be possible to determine the importance of additional intermediates i n the glucose metabolism of these aerobic bacteria.  -105SUMMARY The meohanism of glucose oxidation i s adaptive.  Cells  grown on a completely inorganic medium plus l a c t a t e or succinate attacked glucose at g r e a t l y reduced r a t e s , while c e l l s harvested from acetate media oxidized t h i s substrate only after an i n i t i a l lag period of one hour. Gluconic and 2-ketogluconic acids are intermediates i n glucose d i s s i m i l a t i o n .  The l a t t e r compound has also been  detected i n fermentations;  by growing cultures.  no use could be made of 5-ketogluconic  Apparently  acid.  Anaerobically, oxalacetate was decarboxylated to pyruvi acid, one mole of carbons dioxide being released f o r each mole of substrate completely oxidized.  By comparing the  curves of oxygen uptake, i t was concluded that oxalacetate was not reduced to malate during the breakdown of glucose. In the presence of 5 x 10"*M malonate the oxidation of 2  glucose proceeded normally, suggesting that succinic dehydrogenase does not function i n glucose metabolism.  How-  ever the rate of adaptation to succinate by glucose grown c e l l s was retarded by t h i s concentration of i n h i b i t o r . The normal oxidation of both succinate and fumarate occurred after the c e l l suspensions had been incubated f o r 20-30 minutes i n the presence of the substrate. The oxygen uptake of glucose, l a c t a t e , succinate and acetate grown c e l l s has been recorded.  It has been shown  that the endogenous r e s p i r a t i o n may be ^disregarded during  -106substrate oxidation. No evidence of an anaerobic respiratory mechanism has been obtained.  Resting cells incubated with substrate under  a nitrogen atmosphere f a i l e d to produce acid or gas, absorb phosphorus or u t i l i z e the glucose or fructose supplied.  -107-  EIBLIOGRAPHY Alsberg, C. L., ( 1 9 1 1 ) , The formation of d-gluco#ic acid byBacterium savastanoii, J. B i o l . Chem. £, 1 . Annau, E., Banga, I., Gozsy, B.., Huzak, St., Laki, K., Straub, F. B., and A. Szent-Gyorgyi, ( 1 9 3 5 ) , The significance of fumaric acid for the respiration of animal tissue, Z. Physiol. Chem. 2 3 6 . 1. Annau, E., Banga, I., Blazer, A., Bruckner, V., Laki, K., Straub, F. 6., and Szent-Gyorgyi, A., ( 1 9 3 6 ) , Uber die Bedeutung der Fumarsaure fur die Tierisch^ Hoppe-Seylers z. fur physiol. chemie 2 2 4 . 1 0 5 . Aubel, E., ( 1 9 2 1 ) , Attaque du glucose et leyulose par l e b a c i l l e pyocyanique, Compt. rend. 1 7 3 . 1493* Bergy, ( 1 9 4 8 ) , Manual of Determinative Bacteriology, The Williams and Wilkins Company, Baltimore. Bernstein, D. E., ( 1 9 4 4 ) , Studies on the assimilation of dicarboxylic acids by _Ps. saccharophila, Arch. Bioch. 2., 4 4 5 . Buchanan and Fulmer, ( 1 9 3 0 ) , Physiology and Biochemistry of Bacteria, The Williams and Wilkins Company, Baltimore. Burk, D., ( 1 9 3 9 ) , A c o l l o q u i a l consideration of the Pasteur and Keo-Pasteur effects, Cold Springs Harbor Symp. Quamt. B i o l . 2, 4 4 9 . Burkholder, P. R., ( 1 9 4 3 ) , Synthesis of vitamin B Proc. Nat. Acad. S c i . 2 £ , 1 6 6 . Burkholder, P. R. ( 1 9 4 4 ) , Arch. Bioch.  2  by yeasts,  121.  Burton, M. 0 . , ( 1 9 4 7 ) , Thesis, The c l a s s i f i c a t i o n and n u t r i t i o n of the Pseudomonas. Butterw-orth, J . and Walker, T. K., ( 1 9 2 9 ) , A study of the mechanism of the degradation of c i t r i c acid by B_« pvocyaneus. Bioch. J« 23_, 9 2 6 | ~~ C l i f t o n , C. E., ( 1 9 3 7 ) , On the p o s s i b i l i t y of preventing assimilation i n respiring c e l l s , Enzymologia 4_> 2 4 6 . C l i f t o n , C. E., ( 1 9 3 9 | , On the relation between assimilation and respiration i n suspension and i n cultures of E_. c o l i , J. Bact. 22, 5 2 3 .  -108-  Dixon, M., (1943) > Manometric Methods, Cambridge UniversityPress, Doudoroff, M., ( 1 9 4 0 ) , The oxidative assimilation of sugars and related substances by _P, saccharophila. Enzymologia 9 . 59* Elvehjem, C, A. J , , ( 1 9 3 1 ) , The role of iron and copper i n the -growth and metabolism of yeast, J . B i o l . Chem, 9.0, 1 1 1 , Evans, E. A. J r . , ( 1 9 4 2 ) , Metabolic cycles and decarboxylation, A Symp. on Resp. Enz,, 1 9 7 , University of Wisconsin Press, Feeney, R. E., Mueller, J . H. and M i l l e r , P. A., ( 1 9 4 3 ) , Growth requirements of Clostridium t e t a n i . I l l A synthetic medium, J . Bact. 46m 5630 Green, D. E., ( 1 9 4 1 ) , The Mechanisms of Biological Oxidations, Cambridge University Press. Horner, D. K., and Burk, D., ( 1 9 3 4 ) , Magnesium ciba«cium and iron requirements for growth of Azotobacter i n free and fixed nitrogen, J . Agric, Res. A_8, 981. Johnson, , ( 1 9 4 7 ) , Reducing sugar determination, Wisconsin Dept. of Biochemistry Laboratory manual. Kalnitsky, G. and Werkman, C. H., ( 1 9 4 3 ) , The anaerobic d i s s i m i l a t i o n of pyruvate by a c e l l - f r e e extract of Escherichia c o l i . Arch. Bioch. ,2, 1 1 3 « Karstrom, H., ( 1 9 3 7 ) , Enzymatische Ergeb Enzymforch. £, 3 5 0 .  Adaptation bei Mikroorganismen,  King. J . V., ( 1 9 4 8 ) , Thesis, A study of the fluorescent pigment of the Pseudomonas. Kluyver, A. ( 1 9 3 1 ) , The chemical a c t i v i t i e s of microorganisms, University of London Press, London. Krampitz, L. C , and Werkman, C. H., ( 1 9 4 1 ) , The enzymatic decarboxylation of oxalacetate, Bioch. J.' 3 5 5 9 5 , Kubowitz, F., ( 1 9 3 4 ) , Inhibition of butyric acid fermentation by carbon monoxide, Bioch. Z. 2 7 4 . 2 8 5 , Lichstein, H. C , and Soule, M. H., (1943), Studies on the effect of sodium azide on microbic growth and r e s p i r a t i o n . I I I . The action of sodium azide on b a c t e r i a l catalase, J. Bact. 42, 2 3 1 . Lichstein, H. C , and Umbreit, W. W., ( 1 9 4 7 ) , A function of b i o t i n , J . B i o l . Chem. 17_0, 3 2 9 .  -109-  L i l y , ©. G., and Leonian L. H., ( 1 9 4 5 ) , The interrelationships of iron and certain accessory growth ^factors i n the growth of Rh. t r i f o l i i , strain 205 •, J. fy*e*-., g-o 3i3r  }  Lipmann, F., £ 1 9 3 6 ) , Fermentation of phosphogluconic acid, Nature 138. 5 5 8 .  -  Lipmann, F., ( 1 9 3 9 ) , Ananalysis of the pyruvic acid oxidation system, Symp. on Quant. Biol.' 7_, 248.  Lockwood, L. B., Tabenkin, B., and Ward, G. E., ( 1 9 4 0 ) , The production of gluconic acid and 2-ketogluconic acid from glucose by species of Pseudomonas and Phytomonas, J . Bact. 4_2, 51. Lockwood, L. B., Ward, G. E., S t u b b s , J . 3., Roe, E. T. and Tabenkin, B. ( 1 9 4 1 ) , Fermentation process for the production of 2-ketogluconic acid, U. S. 2 , 2 2 7 , 7 1 6 , ^far, 3 1 . 1  Lockwood, L. B. and Stodola, F. H., ( 1 9 4 6 ) , Preliminary studies on the production o f c ^ k e t o g l u t a r i c acid by _P. fluorescens. J . B i o l . Chem. 1 6 4 . 81. Meyerhof, 0 . , ( 1 9 4 2 ) , Intermediate carbohydrate metabolism, Symp. on Resp. Enz. _3_, University of Wisconsin Press, Madison. Molisch, H., (1892), Die Pflanze i n ihren Beziehungen zum Eisen, Jena. Molliard, M., ( 1 9 2 9 ) , Physiological characteristics shown by Sterigmat cyst i s nigra i n the absence, of zinc and i r o n , Compt. rend. 189. 4 1 7 . Meyer, A. J . , Umberger, E. J . and Stubbs, J . J., ( 1 9 4 0 ) , Fermentation of concentrated solutions of glucose to gluconic acid, Ind. and Eng. Chem. 1379. M©yer, A. J . , Umberger, E. J., and Stubbs, J . J., ( 1 9 4 0 ) , Glucose fermentation to gluconic acid i n concentrated solutions. Improved process, Ind. and Eng. Chem. ,3_2, 1 3 7 9 . Mueller; J . H., ( 1 9 4 1 ) , The influence of iron on the production of diphtheria toxin, J . Immuniol. l±2, 343* Pappenheimer, A. M., ( 1 9 3 6 ) , Studies i n diphtheria toxin -p production, 1 . The effect of iron and copper, B r i t . J . of Exptl. Path. 17_, 3 3 5 . Pappernheimer, A. M. and Shaskan, E. M. ( 1 9 4 4 ) , The effect of iron on the carbohydrate metabolism of Clostridium perfringens, J . Bact. 4_Z» 4 1 3 .  -110-  Pappenheimer, A. M. and Shaskan,. E., ( 1 9 4 4 ) , The effect of iron on the carbohydrate metabolism of CI, welchii, J. B i o l . Chem. 15_5_, 2 6 5 . Perlman, D., ( 1 9 4 5 ) , Some effects of metallic ions on the metabolism of Aerobacter aerogenes, J . Bact. Zfc9_, 1 6 7 . Pervozanskii, V. V., ( 1 9 4 0 ) , Formation of gluconic acid during the oxidation of glucose by bacteria, Microbiology (U.S.S.R.), 8, 1 4 7 . Porges, N., Clark, T. F. and Gastrock, E. A., ( 1 9 4 0 ) , Repeated use of submerged A_. niger for semi-continuous production, Ind. and Eng. Chem. _3_2, 1 0 7 . Potter, V. R., ( 1 9 4 0 ) , The mechanism of hydrogen transport i n animal tissues, Medicine lj>, 2 1 7 . Quastel, E. H. and Whetham, M. D., ( 1 9 2 4 ) , The e q u i l i b r i a existing between succinic, fumaric, and malic acids i n the presence of resting bacteria, Biochem. J._18 5 1 9 * Robert M., "(1928), 'Zentr. Ba'kt. Parasitenk Infekt II_7_4,333. Roberts, 0 . and Murphy, D., ( 1 9 4 4 ) , A rapid fermentation method for the production of calcium c i t r a t e and calcium gluconate from beet molasses, S c i . Proc. Roy. Dublin Soc. 21, 307.  Ruff©, A. and Imperato C , B i o l o g i c a l oxidation of d-glucose and d-gluconic acid, B o l l . soc. i t a l . sper. _20, 2 3 9 . Saunders, A., and McClung, L.S., ( 1 9 4 3 ) , The effect of various concentrations of iron on the production of r i b o f l a v i n by certain C l o s t r i d i a , J . Bact. Zfc6, 5 7 5 . Schreder, K. R., Brunner, And Hampe ( 1 9 3 3 ) , Pseudomonas l i n d n e r i Kluyver Seine aerobe und anaerobe Gerung mit besonderer Berucksechtigung seiner Alkoholbildung, Wochschr. Brau. j[0, 4 3 , 2 3 3 , 2 4 3 , i i , 2 4 1 , 2 4 9 . Seleen, W. A. and Stark, C. N., ( 1 9 4 3 ) , Some c h a r a c t e r i s t i c s of green fluorescent pigment producing bacteria, J . Bact. 4_6> 4 9 1 .  Simon, E., ( 1 9 4 7 ) , The formation of l a c t i c acid by Clostridium acetobutilicum (Weizmann), Arch. Biochem. 13_, 2 3 7 . Spiegelman, S. and Dunn, ( 1 9 4 7 ) , Interactions between enzymeforming systems during adap|ation, J . Gen. Phys^ 1 1 , 1 5 3 *  -111Stanier, R. Y., (1948), Personal  communication.  Steinberg, R. A., ( 1 9 3 5 ) , Nutrient solution p u r i f i c a t i o n f o r removal of heavy metals i n d e f i c i e n c y investigations with A_. niger. J . Agric, Res. j>l, 413. Stevenson, M. J . and Gale, E. F., (1937), The adaptability of glucozymase and galactozymase i n Bacterium c o l i , Bioch. J. 1 1 , 1911. Stubbs, J . J., Lockwood, L. B., et a l . , (1940), Bacterial production of ketogluconic acids from glucose, Ind. and Eng. Chem. ^ 2 , 1626. Sumner and Somers, ( 1 9 4 3 ) , Chemistry and Methods of Enzymes, Academic press, Tanner, F. ¥., (1918), A study of green fluorescent bacteria from- water, J . Bact. 1, 6 3 . Tanner, F. W., Vojnovich, C. and Van Lanen, J . F., ( 1 9 4 5 ) , Riboflavin production by Candida species, Science 101. 180. Umbreit, W. W., Burris, R. H. And Stauffer, J . F., ( 1 9 4 5 ) , Manometric Techniques and related methods for the study of tissue metabolism, Burgess Publishing Company, Minneapolis. Waring, W. S. and Werkman, C. H., ( 1 9 4 2 ) , Growth of bacteria in an iron-free medium Arch. Biochem.ig, 303» Waring, W. S., and Werkman, C. H., (1943), Iron requirements of heterotrophic bacteria, Arch. Biochem. J., %2$ Waring, W. S. and Werkman, C. H., ( 1 9 4 4 ) , Iron deficiency • i n b a c t e r i a l metabolism, Arch. Biochem. 4_, 75. White, M. J . , (1948), Unpublished data. Williams, A. K., (1945), The production of gluconic acid by fermentation, Mfg. Chemist 16, 239« «  Wood, A. J . and Gunsalus, I. C , ( 1 9 4 1 ) , The production of active resting c e l l s of streptococci, J . Bact. 4^, 3 3 1 *  

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