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Metabolic activities of Pseudomonas Aeruginosa when grown on a 2-ketogluconic acid medium Hill, Robert William 1952

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I  METABOLIC ACTIVITIES OF PSEUDOMONAS  AERUGINOSA  WHEN GROWN ON A 2-KET0GLUC0NIC ACID MEDIUM by ROBERT WILLIAM HILL  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE i n the Department of Dairying  We accept t h i s t h e s i s as conforming t o t h e standard r e q u i r e d from candidates f o r t h e degree o f MASTER OF SjDIENCE IN AGRICULTURE  Members o f the Department o f  THE UNIVERSITY OF BRITISH October 1952  COLUMBIA  - i -  ABSTRACT The  c u r r e n t concept  bohydrate  o f t h e pathway o f t h e i n t e r m e d i a t e c a r -  metabolism o f P. aeruginosa ATCC 9027 i s t h a t g l u c o s e  i s o x i d i z e d t o carbon d i o x i d e and water by way o f g l u c o s e , g l u c o n i c , 2 - k e t o g l u c o n i c and p y r u v i c a c i d s .  The pathway o f t e r m i n a l  r e s p i r a t i o n i s the c o n v e n t i o n a l Kreb's t r i c a r b o x y l i c a c i d  cycle.  However, t h e mechanism by which 2 - k e t o g l u c o n i c a c i d i s o x i d i z e d to  p y r u v i c a c i d has not been e l u c i d a t e d .  The f i r s t  the present work was t o e s t a b l i s h the accumulation  concern i n of 2-ketoglu-  conic a c i d d u r i n g g l u c o s e o x i d a t i o n by P. aeruginosa ATCC 9027 and  a l s o t o produce enough c a l c i u m 2-ketogluconate  t o serve as  substrate i n studies of i t s oxidation. The  i n i t i a l problem on t h e metabolism o f 2 - k e t o g l u c o n i c a c i d  grown c e l l s o f P. a e r u g i n o s a was t o compare t h e enzymic p a t t e r n of  these c e l l s w i t h c e l l s grown on a glucose medium.  The data  o b t a i n e d under these c o n d i t i o n s , c o n f i r m e d the p r e v i o u s c o n c l u s i o n s t h a t 2 - k e t o g l u c o n i c a c i d i s i n t h e d i r e c t pathway o f g l u c o s e oxidation. A d d i t i o n a l evidence f o r the minor r o l e o f t h e Embden-Meyerh o f f pathway i n t h i s organism was obtained when i t was shown t h a t c e l l s grown on a 2 - k e t o g l u c o n i c a c i d medium contained o n l y t r a c e s of  t h e enzyme a l d o l a s e .  The amount o f t h i s enzyme d e t e c t e d would  be s u f f i c i e n t t o permit t h e g l y c o l y t i c pathway t o f u n c t i o n as a s y n t h e s i z i n g mechanism f o r pentoses o r perhaps h e p t u l o s e s but would not be s u f f i c i e n t t o account  f o r any major amount o f g l u c o s e .  - i i  The major problem o f e s t a b l i s h i n g the presence o f i n t e r mediates o c c u r r i n g between 2-ketogluconic and p y r u v i c a c i d s was approached from two p o i n t s o f view:  f i r s t , by attempting t o pro-  duce a c e l l p r e p a r a t i o n which had a l i m i t e d a b i l i t y t o o x i d i z e 2-ketogluconic a c i d and which would accumulate i n t e r m e d i a t e s  ina  manner s i m i l a r t o t h a t o f c e l l p r e p a r a t i o n s which accumulate 2-ketogluconic a c i d d u r i n g glucose  o x i d a t i o n , and second, by t h e  b l o c k i n g of t h e enzyme system o f 2-ketogluconic a c i d grown c e l l s by an i n h i b i t o r at a p o i n t i n t e r m e d i a t e a c i d and p y r u v i c a c i d . Further  between 2-ketogluconic  N e i t h e r approach y i e l d e d p o s i t i v e r e s u l t s .  s t u d i e s were made on t h e b a s i s o f an e a r l y observa-  t i o n t h a t c e l l s which had been heated showed an impaired a c i d and 2-ketogluconic a c i d o x i d i z i n g a c t i v i t y .  gluconic  I t was found  subsequently t h a t l i v e c e l l s heated t o 55°C. f o r a t l e a s t t e n minutes would o x i d i z e glucose  but not g l u c o n i c a c i d o r 2-keto-  g l u c o n i c a c i d and t h a t f u r t h e r h e a t i n g n e a r l y completely the a b i l i t y o f the c e l l s t o assimulate preparations  glucose.  destroyed  Lyophilized  o f such heat t r e a t e d c e l l s s t i l l r e t a i n e d the a b i l i t y  t o o x i d i z e glucose  o r g l u c o n i c a c i d t o 2-ketogluconic a c i d .  This  can be i n t e r p r e t e d as i n d i c a t i n g t h a t 2-ketogluconic a c i d i s on the d i r e c t o x i d a t i v e pathway o f glucose  oxidation.  ACKNOWLEDGMENT  I wish t o express my s i n c e r e thanks t o Dr. J.J.R. Campbell f o r h i s d i r e c t i o n o f t h i s work and f o r h i s i n v a l u a b l e a s s i s t a n c e and i n t e r e s t . I a l s o wish t o thank the N a t i o n a l Research C o u n c i l f o r t h e grant t o c a r r y out t h i s i n v e s t i g a t i o n .  R.W.H  TABLE OF CONTENTS  Abstract  '  1  Introduction I.  P r o d u c t i o n o f calcium  i  2-ketogluconate.  (a) I n t r o d u c t i o n (b) Experimental i . Production i i . I s o l a t i o n arid p u r i f i c a t i o n  3 6 10  II.  The comparison o f t h e m e t a b o l i c a c t i v i t i e s of glucose grown and '2-ketogluconic a c i d grown c e l l s .  13  III.  Determination of the presence o f the enzyme a l d o l a s e .  14  IV.  Study o f d r i e d c e l l p r e p a r a t i o n s . (a) I n t r o d u c t i o n (b) Methods (c) R e s u l t s  17 18 22  V.  I n f l u e n c e of metabolic i n h i b i t o r s on the o x i d a t i o n o f glucose and 2 - k e t o g l u c o n i c ' acid.  24  VI.  , E f f e c t of heat shocking on the enzyme s t r u c t u r e of c e l l s grown on 2 - k e t o g l u c o n i c acid.  28  Addendum 1  30  V I I I . Addendum 2  32  IX.  35  VII.  Bibliography  INTRODUCTION  In the past few years i t has been e s t a b l i s h e d t h a t the o x i d a t i o n o f glucose by Pseudomonas aeruginosa ATCC 9027 proceeds by way a c i d s (12,  of glucose, g l u c o n i c , 2 - k e t o g l u c o n i c and p y r u v i c  39,  48 and 51).  I t has a l s o been shown t h a t the  system  o f t e r m i n a l r e s p i r a t i o n i s the t r i c a r b o x y l i c a c i d c y c l e (11, and 14).  13  There i s no evidence o f p h o s p h o r y l a t i o n between glucose  and 2 - k e t o g l u c o n i c a c i d and so glucose-6-phosphate i n t e r m e d i a t e (12  and 21).  the Warburg-Dickens system  an  The p r o p o s a l o f t h i s pathway has been  greeted by a great deal o f s k e p t i c i s m (21) almost u n i v e r s a l presence  i s not  l a r g e l y because o f the  i n a e r o b i c t i s s u e and microorganisms (18)  o f glucose o x i d a t i o n . When  glucose i s o x i d i z e d v i a t h i s pathway i t i s through phosphate, 6-phosphogluconolactone,  glucoses-  a p o s t u l a t e d 6-phospho-2-  k e t o g l u c o n o l a c t o n e , r i b u l o s e - 5 - p h o s p h a t e , ribose-5-phosphate, e v e n t u a l l y t o a c e t i c a c i d , C0  2  of  and water.  and  The opponents o f the  glucose, g l u c o n i c , 2 - k e t o g l u c o n i c a c i d scheme have maintained t h a t the two l a t e r compounds are formed merely i n s i d e r e a c t i o n s from members of the Warburg-Dickens pathway.  However, u s i n g c e l l  f r e e e x t r a c t s o f P. aeruginosa 9027 and P. f l u o r e s c e n s , Wood and Schwerdt (55,  55a)'  have been able to show t h a t these organisms do  have many o f the enzymes o f the Warburg-Dickens pathway, but t h a t they l a c k hexokinase, and t h e r e f o r e cannot o x i d i z e glucose by o f t h i s phosphorylated scheme.  I t would appear t h a t the  schemes are o p e r a t i v e i n the metabolism  o f these  two  organisms.  way  -  Glucose i s o x i d i z e d by way to t r i o s e s .  2  -  o f 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  A phosphorylated t r i o s e then condenses w i t h p y r u v i c  acid, t o g i v e 6-phosphogluconic  a c i d which i n t u r n can be o x i d i z e d  and d e c a r b o x y l a t e d t o y i e l d ribose-5-phosphate  (55a).  The Warburg-  Dickens enzymes thus serve as the pentose forming pathway o f these organisms.  The great h i a t u s i n our knowledge o f the  glucose-2-  k e t o g l u c o n i c a c i d scheme at the present time i s the immediate f a t e of 2-ketogluconic a c i d .  It i s possible that i t i s d i r e c t l y d i s -  s i m i l u l a t e d t o pyruvate, but i t i s e q u a l l y p o s s i b l e t h a t i t may a stepwise degradation through 5 carbon and 4 carbon  be  compounds.  There i s a l s o the p o s s i b i l i t y o f p h o s p h o r y 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 , d e c a r b o x y l a t i o n t o a pentose and then a 3-2  s p l i t o f the  pentose. As a r e s u l t o f t h i s s i t u a t i o n t h i s t h e s i s i s concerned  with  attempts t o i d e n t i f y the i n t e r m e d i a t e s , i f any, between 2-ketog l u c o n i c a c i d and p y r u v i c a c i d .  - 3 -  PART I . a.  PRODUCTION OF CALCIUxM 2-KETOGLUCONATE  Introduction Although the evidence a v a i l a b l e at t h e present time  indicates  t h a t many b a c t e r i a o x i d i z e glucose by way o f 2 - k e t o g l u c o n i c i t was not u n t i l 1935 first  t h a t Bernhauer and G o r l i c k (3,  demonstrated t h a t t h i s a c i d was produced  simulation of glucose. Acetobacter suboxydans.  4,  acid  and 5)  during b a c t e r i a l d i s -  The organism which they s t u d i e d was The enzyme which o x i d i z e s the i n t e r m e d i a t e ,  2 - k e t o g l u c o n i c a c i d , Is destroyed by i n t e n s i v e a e r a t i o n , and so t h i s a c i d accumulates under c o n d i t i o n s which f a v o u r the o x i d a t i o n o f t h e 2 - k e t o g l u c o n i c a c i d d i s s i m u l a t i n g enzyme.  In the e x p e r i -  ments o f Bernhauer and G o r l i c k (3) the d e s t r u c t i o n o f t h e 2-ketog l u c o n i c a c i d o x i d i z i n g enzyme and t h e r e f o r e the enhancement o f the accumulation  o f the a c i d was accomplished  c u l t u r e at r e l a t i v e l y low temperatures days.  by i n c u b a t i n g the  f o r p e r i o d s o f 30 t o 60  S i m i l a r work was done by U t k i n (57)  who r e p o r t e d t h e accumu-  l a t i o n o f 5 - k e t o g l u c o n i c a c i d from a c u l t u r e o f Acetobacter.  How-  ever, h i s data would i n d i c a t e t h a t 2 - k e t o g l u c o n i c a c i d r a t h e r than 5-ketogluconic a c i d was the end product.  P e r v o z v a n s k i i (41,  42)  p l a c e d 2 - k e t o g l u c o n i c a c i d p r o d u c t i o n on a f i r m e r b a s i s when he demonstrated t h a t " o n i c " or " k e t o n i c the s p e c i f i c b a c t e r i a l s t r a i n used.  n  a c i d s were a f u n c t i o n o f He suggested the i n d u s t r i a l  p o s s i b i l i t i e s o f the process by showing t h a t C a , a e r a t i o n and + +  a g i t a t i o n were n e c e s s a r y f o r r a p i d and near complete c o n v e r s i o n o f glucose t o 2 - k e t o g l u c o n i c Ward (53)  1939,  acid.  working on the g l u c o n i c a c i d f e r m e n t a t i o n  - 4 -  process i s o l a t e d a contaminating organism which 2-ketogluconic a c i d . process (54)  accumulated  Using t h e p r e v i o u s l y p u b l i s h e d p i l o t p l a n t  w i t h which the P e o r i a group had done much work,  Ward and h i s co-workers (30, 31,  34, 49,  54) were a b l e t o demon-  s t r a t e t h a t 2 - k e t o g l u c o n i c a c i d accumulation was a two step process from glucose t o g l u c o n i c a c i d and f i n a l l y t o 2 - k e t o g l u c o n i c acid.  Lockwood, Ward, Stubbs, Roe and Tabenkin  (30)  1940,  sub-  sequently o b t a i n e d a patent on 2 - k e t o g l u c o n i c a c i d p r o d u c t i o n u s i n g a Pseudomonas s p e c i e s grown w i t h i n t e n s e a e r a t i o n i n an a l k a l i n e medium w i t h e i t h e r glucose or c a l c i u m gluconate -as carbon course.  Blaisten  (8) 1947,  an Argentine worker,  produced  2 - k e t o g l u c o n i c a c i d from glucose i n 80$ y i e l d u s i n g Lockwood's method.  The 2 - k e t o g l u c o n i c a c i d was used i n the p r o d u c t i o n o f a  potential antioxidant, arabinoascorbic acid. A s a i and Ikada  (1)  1950,  Ikeda  (26)  1948,  and  o b t a i n e d s i m i l a r r e s u l t s u s i n g shake  c u l t u r e s o f P. f l u o r e s c e n s . S e r r a t i a marcescens, and  Gluconobacter  They obtained 75-&0$ 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  from  glucose and from t r a c e s t o 83$ 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  from  species.  potassium  gluconate.  The occurrence o f 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 as normal i n t e r m e d i a t e s i n glucose o x i d a t i o n has been demonstrated by N o r r i s and  Campbell (39) 1949, u s i n g P. aeruginosa ATCC 9027 i n s t i l l  culture. . De Ley and Cornut  (17) 1951, used P: p u t i d a . o b t a i n e d 90-97$  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 s t a r t i n g w i t h c a l c i u m gluconate. 1951  (43)  In  a B r i t i s h patent was g i v e n f o r the p r o d u c t i o n o f 2-keto-  hexonic a c i d from hexonic a c i d i n 90-95$ y i e l d u s i n g an organism  - 5 -  c a l l e d Cyanococcus I t was  chromospirans.  thus f e l t t h a t t h e demonstration  under i n t e n s e a e r o b i c  c o n d i t i o n s o f 2 - k e t o g l u c o n i c a c i d accumulation  u s i n g P.  aeruginosa  ATCC 9027 would add t o our knowledge o f the metabolism o f t h i s s t r a i n and would provide us w i t h a supply o f 2 - k e t o g l u c o n i c a c i d f o r use i n f u r t h e r experimental work.  b.  Experimental  i.  Production The d e f i n i t e i d e n t i f i c 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 a  b a c t e r i a l end-product Gorlick  was f i r s t  c a r r i e d out by Bernhauer and  (3) 1935, Bernhauer and Knoblock (4, 5) 1940, and  Knoblock and T i e t z e (27) 1941, w i t h Acetobacter suboxydans u s i n g calcium gluconate o r glucose p l u s calcium carbonate as s t a r t i n g materials.  With t h e i r system they could show, depending on con-  d i t i o n s , an accumulation 5-ketogluconate.  o f c a l c i u m 2-ketogluconate  T h i s has been subsequently  or calcium  s e r i o u s l y questioned  and a t t r i b u t e d by other workers t o the use o f impure c u l t u r e s as no known organism  has s i n c e been shown t o form both i n t e r m e d i a t e s .  Lockwood et a l (54,and 31) 1937, developed  a p i l o t plant  o p e r a t i o n u s i n g P. f l u o r e s c e n s which accumulated a c i d from glucose or g l u c o n i c a c i d .  2-ketogluconic  Lockwood's method has been  used by o t h e r s (1, 8, 17, 26 and 28) f o r the l a b o r a t o r y p r o d u c t i o n of  2 - k e t o g l u c o n i c a c i d because, at t h e present time, t h e market  price i s prohibitive. The procedure  used i n the present work i s a m o d i f i c a t i o n o f  the method o f Lockwood.  P. aeruginosa ATCC 9027 and a m o d i f i e d  medium o f N o r r i s and Campbell (11) w i t h glucose as a carbon and urea as a n i t r o g e n source was used.  T h i s s y n t h e t i c medium  f a c i l i t a t e d the p u r i f i c a t i o n w i t h no apparent  loss i n yield  Lockwood's medium which contained crude corn steep l i q u o r . p r e v e n t i o n o f foaming d u r i n g t h e i n i t i a l  source  over The  stages o f f e r m e n t a t i o n  was p a r t i a l l y c o n t r o l l e d by a d d i t i o n s o f s a t u r a t e d m i n e r a l o i l and  - 7 -  octadecanol.  Corn o i l and 3$ Alkaterge C helped  c o n t r o l the  foaming d u r i n g the l a t e r stages o f f e r m e n t a t i o n . ly  Foaming  apparent-  i s the l i m i t i n g f a c t o r i n t h a t i t determines the amount o f a i r  flow and a g i t a t i o n p o s s i b l e i n a system i n which a i r f l o w the fermentor  i s induced by vacuum r a t h e r than by  d i f f e r e n t i a l as i s u s u a l . produce approximately 2-ketogluconate  through  pressure  Using t h i s method i t was  possible to  1000 grams o f commercial grade calcium  and 300 grams o f pure calcium  2-ketogluconate,  c o n t a i n i n g no d e t e c t a b l e contamination w i t h glucose o r g l u c o n i c acid.  Paper chromatography u s i n g ethanol-ammonium hydroxide  ascending  solvent and ortho-phenylendiame or ammoniacal  as  silver  n i t r a t e as developer f a i l e d to show any contamination.  Manometric  s t u d i e s u s i n g g l u c o s e and g l u c o n i c a c i d o x i d i z i n g c e l l  preparations  showed t r a c e s o f fermentable The organism used was  contaminating  substances.  Pseudomonas.aeruginosa ATCC 9027 c a r r i e d  on stock agar slopes * at 0-5°C. a f t e r i n i t i a l 48 hour growth at 30 C. G  These stocks were s u b - c u l t u r e d at monthly i n t e r v a l s .  stock c u l t u r e slope was glucose b r o t h *. was  used as an.inoculum f o r 10 ml. o f yeast  A f t e r 48 hours at 30°C. the 10 ml. o f c u l t u r e  t r a n s f e r r e d to 100 ml. o f yeast glucose b r o t h i n a 500  Florence f l a s k .  The f l a s k was  f o r a s m a l l s c a l e fermentor  p r o d u c t i o n medium *.. fermentor  by way  The  ml.  incubated 72 hours on a r o t a r y  shaker or u n t i l heavy t u r b i d i t y appeared. as inoculum  A  inoculum was  T h i s c u l t u r e was  used  c o n t a i n i n g one l i t e r i n t r o d u c e d i n t o the  o f the o i l a d d i t i o n port or tube.  The  of  small  temperature  i was *  c o n t r o l l e d by p a r t i a l submersion of the fermentor Addendum 1  in a  constant  - 8 -  temperature water bath.  A e r a t i o n was accomplished by r e d u c i n g t h e  p r e s s u r e w i t h i n t h e fermentor w i t h a t r a p p e d water j e t vacuum pump. The incoming a i r was f i l t e r e d and s t e r i l i z e d by passage through a 4" x 1" tube o f s t e r i l e two porous was  c o t t o n wool.  The a i r was then l e d through  stone b l o c k s a t t h e bottom o f t h e fermentor.  accomplished by a e r a t i o n a l o n e .  Agitation  The p r o d u c t i o n medium* was  composed o f g l u c o s e , calcium carbonate, potassium a c i d magnesium s u l f a t e , i r o n s u l f a t e and urea.  phosphate,  The urea was s t e r i l i z e d  s e p a r a t e l y by f i l t r a t i o n through u l t r a f i n e s i n t e r e d g l a s s  filter  and added through t h e o i l port t o the s t e r i l i z e d medium i n t h e f e r mentor j u s t a f t e r i n o c u l a t i o n .  The g l u c o s e , c a l c i u m carbonate and  other m i n e r a l s a l t s were s t e r i l i z e d f o r one hour at 1Z\ l b s . / i n . . 2  Prior to s t e r i l i z a t i o n  a 1/8" t o l / 4 " l a y e r o f s a t u r a t e d m i n e r a l  o i l and octadecanol,.which had been s t e r i l i z e d p r e v i o u s l y , was added t o t h e main batch. enough water t o f l o a t 12g l b s . / i n . . 2  The m i n e r a l o i l and octadecanol w i t h  t h e o i l were s t e r i l i z e d f o r t h r e e hours at  During f e r m e n t a t i o n t h e foaming was p a r t i a l l y  i :  c o n t r o l l e d by the a d d i t i o n o f s i m i l a r l y s t e r i l i z e d 3% A l k a t e r g e C.  corn o i l and  T h i s o i l was added by means o f a s t e r i l e p i p e t t e  to the s m a l l fermentor o r by a s t e r i l e vented b o t t l e t o t h e l a r g e fermentor ( P l a t e 2 ) . The one l i t e r  fermentor was i n c u b a t e d f o r 48-72 hours and  then t r a n s f e r r e d as inoculum t o t h e l a r g e fermentor c o n t a i n i n g 6000 ml. o f p r o d u c t i o n medium i n a 12 l i t e r f l a s k  (Plate 1).  The  t r a n s f e r was done by r e d u c i n g t h e pressure on t h e l a r g e r v e s s e l w h i l e they were connected v i a t h e i r r e s p e c t i v e o i l a d d i t i o n p o r t s . *  Addendum 1  -  - 8a -  PRODUCTION  FERME.NTOB  P L A T E NO.  /  SAMPLING  DEV/CE  C L A M P T U B E .  ^i_-vv'teAPpe.o V A C U U M C O T T O N  AIR.  LINE. O U T Vtfooi  \N-rAvCE_  V  A  & Oii__ AODI-TION I  R . O B B E . R  C  U  U  C O V E R . M  O  L I M E .  f-^-rHTii/CoTTOM  u  n  -  c  r  W O O L  M£.DI A  L _ E L V E . 1 _  COM3T&NT BA.TH  Ca CO,  -'8b -  OIL PLATE  NO.  COTTON  WOOL'  ADD\TION  FLASK  X  - R U B B E R . TUBING,  GLASS  TU&IINGI  £5iu  av-s5CO ML.  ElRi_t.NM&YElR.  rv_A.SK  LEVEL.  WATER.  S t e r i l e urea was added immediately a f t e r i n o c u l a t i o n and a i r f l o w g r a d u a l l y i n c r e a s e d u n t i l s t a b l e c o n d i t i o n s were m a n i f e s t . ing  Foam-  was t h e l i m i t i n g f a c t o r to. adequate a e r a t i o n and a g i t a t i o n  e s p e c i a l l y toward the end o f the r u n .  The temperature  c o n t r o l and  o t h e r f a c t o r s were s i m i l a r t o the s m a l l s c a l e fermentor. mentation was complete  The f e r -  i n 3 t o 5 days a t which time t h e c a l c i u m  carbonate, some o f which s e t t l e s t o the bottom, no l o n g e r went into solution.  The end.point o f the f e r m e n t a t i o n was checked by  two d i f f e r e n t methods.  The f i r s t method was t o stop the run on  the l e v e l l i n g o f f o f the l e v o r o t a t o r y r e a d i n g s o f the p o l a r i m e t e r . The second method, used t o check the f i r s t , was t o make p i l o t q u a n t i t a t i v e i s o l a t i o n s o f 2-ketogluconic a c i d u n t i l these a l s o l e v e l l e d o f f , at which time the run was stopped.  The percentage  c o n v e r s i o n v a r i e d from r u n t o r u n so t h a t a r b i t r a r y v a l u e s c o u l d not be used to stop the runs.  -  b. ii.  10  -  Experimental I s o l a t i o n and  Purification  ______________________________  y  The y i e l d of crude product was 1120  between 4 0 0 and 7 0 0 grams per  grams o f i n i t i a l glucose or a 3 6 t o 6 2 % y i e l d . The  e s s e n t i a l steps i n the i s o l a t i o n and p u r i f i c a t i o n were  filtration, On  c o n c e n t r a t i o n , d e c o l o r i z a t i o n , and  crystallization. mixed with 1 t o  completion o f f e r m e n t a t i o n the b r o t h was  s u p e r c e l and f i l t e r e d through a s u p e r c e l precoated f i l t e r a Buchner f u n n e l or a s i m i l a r l y t r e a t e d basket r e s u l t i n g c l e a r l i q u o r was  t i o n was  The  a g i t a t i n g , then c o o l i n g the mixture  l i q u o r was  and f i l t e r i n g .  a v e r y p a l e y e l l o w green c o l o r .  The  on  The  t r e a t e d with 6 0 t o 1 0 0 gram batches  of  decoloriza-  c a r r i e d , out by h e a t i n g the l i q u o r and washings to  c l e a r l i q u o r was  cloth  centrifuge.  decolorizing charcoal u n t i l e s s e n t i a l l y colorless.  2%  60°C,  resultant  The d e c o l o r i z e d  then vacuum d i s t i l l e d batchwise t o o n e - t h i r d volume o r  t o a t h i n syrup depending on the r e l a t i v e amounts o f c a l c i u m 2-ketogluconate  and g l u c o s e p r e s e n t .  The d i s t i l l a t i o n was  done  from a c o n t r o l l e d water bath at 4 5 - 5 5 ° C . -under vacuum. I f n e c e s s a r y the concentrated b r o t h was refiltered.  The  c o n c e n t r a t e d b r o t h was  at t h i s temperature white  vacuum f i l t e r e d  hot 80% a l c o h o l and r e f i l t e r e d .  The was  and  and left  The p a l e y e l l o w -  and then s l u r r i e d with  T h i s procedure  removed most o f the  glucose.  s o l u b i l i t y o f p u r i f i e d c a l c i u m 2-ketogluconate i n water  1 9 grams per 1 0 0 ml. at 3 0 ° C . and 1 7  It was  c o o l e d to 0 ° - 5 ° C .  o v e r n i g h t to c r y s t a l l i z e .  c r y s t a l l i n e mass, was  contaminating  d e c o l o r i z e d again  o n l y very s l i g h t l y s o l u b l e i n 95%  grams per 1 0 0 ml. at alcohol.  20°C.  I t i s because o f  - 11 -  these s o l u b i l i t y c h a r a c t e r i s t i c s t h a t a l c o h o l washing, r a t h e r than r e c r y s t a l l i z a t i o n , i s emphasized.  The  s l i g h t l y moist  c r y s t a l s are  very s e n s i t i v e to h e a t i n g i n excess o f 45°C. To remove the l a s t t r a c e s o f glucose the c r y s t a l s o b t a i n e d from the above procedure were d i s s o l v e d at 80-90°C. i n a minimum o f water, d e c o l o r i z e d w i t h c h a r c o a l ; cooled, f i l t e r e d and s t a l l i z e d out at 0-5°C.  The f i l t e r e d c r y s t a l s were s l u r r i e d with.  70$ e t h y l a l c o h o l , f i l t e r e d and r e s l u r r i e d with 95$ e t h y l filtered,  cry-  and d r i e d below 4 5 ° C  alcohol,  The r e s u l t i n g c r y s t a l s were a f i n e  white c r y s t a l l i n e mass which, on d r y i n g , y i e l d e d an  essentially  pure product. The p u r i t y o f the product was  checked  chromatographically  u s i n g ethanol 80 p a r t s water,16 p a r t s and ammonium hydroxide p a r t s on ascending and descending Whatman No. 1 f i l t e r  paper.  Ammoniacal s i l v e r n i t r a t e , ortho and meta phenylenediamine brom c r e s o l green and 2,6  dichlorobenzenone-indophenol  used as spray d e v e l o p e r s .  There was  There was  (29),  (7) were  no evidence o f g l u c o n i c a c i d  o r o f glucose or o f other contaminating r e d u c i n g or a c i d i c present.  4  products  a t r a c e o f glucose and some c o l o r i n the  crude  product, however. Manometric d e t e r m i n a t i o n s u s i n g 18 oxygen e q u i v a l e n t s o f cium 2-ketogluconate  as s u b s t r a t e y i e l d e d 329  oxygen uptake (as the average  T h i s i n d i c a t e s the presence and may  micro l i t e r s o f  value f o r twenty r u n s ) .  lower than the glucose c o n t r o l s by an average o f 2.4$  cal-  This  was  of 8 m i c r o l i t e r s .  o f •.. n o n - o x i d i a a b l e  substances  be due to incomplete water removal or contaminating  salts.  Using a d r i e d p r e p a r a t i o n of P. aeruginosa and 18 oxygen e q u i v a l e n t s  - 12 -  o f c a l c i u m 2-ketogluconate as s u b s t r a t e the average d i f f e r e n c e  be-  tween endogenous r e s p i r a t i o n and calcium 2-ketogluconate f o r n i n e runs was  1.7  m i c r o l i t e r s o f oxygen.  The c o n t r o l g l u c o s e f l a s k s  c o n t a i n e d 18 oxygen e q u i v a l e n t s and took up 72 m i c r o l i t e r s o f oxygen.  Assuming the fermentable contamination was  would be 2% g l u c o s e p r e s e n t .  glucose there  However, t h i s d i f f e r e n c e o f 1.7  micro-  l i t e r s i s w e l l w i t h i n the l i m i t s o f experimental e r r o r w i t h the equipment and procedure  used.  - 13 -  PART I I .  THE COMPARISON OF THE METABOLIC ACTIVITIES OF GLUCOSE GROWN AND 2-KETOGLUCONIC ACID GROWN CELLS  C e l l s o f P. aeruginosa harvested from the glucose medium* r e q u i r e d a p e r i o d o f a d a p t a t i o n before a t t a c k i n g 2 - k e t o g l u c o n i c at a maximum r a t e ( 3 9 ) . light  acid  S t a n i e r (21 and 44) has argued t h a t i n  o f h i s theory o f simultaneous  a d a p t a t i o n , 2-ketogluconate can  t h e r e f o r e not be c o n s i d e r e d an i n t e r m e d i a t e i n glucose o x i d a t i o n . However, i f 2 - k e t o g l u c o n i c a c i d i s an i n t e r m e d i a t e i n glucose o x i d a t i o n then c e l l s grown on e i t h e r glucose o r 2 - k e t o g l u c o n i c should e x h i b i t the same spectrum to  o f adaptive enzymes.  check t h i s assumption the enzymic a c t i v i t y  from media c o n t a i n i n g g l u c o s e * as s o l e carbon  acid  In an e f f o r t  o f c e l l s harvested source and media con-  t a i n i n g 2-ketogluconic a c i d * as s o l e carbon source were compared. The compounds compared were glucose, g l u c o n i c , 2 - k e t o g l u c o n i c , p y r u v i c , e C k e t o g l u t a r i c , fumaric, s u c c i n i c , c i t r i c and a c e t i c acids.  A l l o w i n g f o r t h e normal v a r i a t i o n s between runs the two  types o f c e l l s showed no d i f f e r e n c e s i n t h e i r enzymic make-up as  i i n d i c a t e d by t h e i r a b i l i t i e s t o a t t a c k the s u b s t r a t e s t e s t e d (Plates 3 t o 11).  As a f u r t h e r comparison,  glucose and 2-ketoglu-  conic a c i d grown c e l l s which were grown on a r o t a t i n g shaker f o r IB hours a t 30°C. were compared ( P l a t e s 12 t o 1 4 ) .  The enzymic  p a t t e r n i s the same as t h a t f o r the c e l l s h a r v e s t e d from s t a t i o n a r y medium and again no d i f f e r e n c e s can be detected between the c e l l s grown on glucose and those grown on 2 - k e t o g l u c o n i c *  Addendum 2  acid.  GUOCOSE. STILL. GROWN  3001PLATE.  A  NO.  B  •+  C  Z- K_TO GLUCONIC  GLUCOSE. .  GLUCONIC  ACID  2-KETOGLOCONIC  ACID  TIME (MINUTES)  AciD  STILL  GROWN  GLUCOSE.  300 PLATE. NO. 5  STILL.  A  GLUCOSE.  B C  GLUCONIC ACID E-KETOGLUCONIC  2- KCTOGLUCONIC GROWN  Geowis  ACID  STILL  ACID  A  c  c  200U  CL  o 100  20  40  60  TIME (MINUTES)  ZO  AO  60  - 13d -  C/°0 3MVJLdO  z  0  GLUCOSE. STIUL  300l PLATE: NO.  A  7  K.E.TOGLUTAR.IC  B  FUMARJC  ACID  c  PYRUVIC  ACID  GROWN  2.- K E . T O G L U C O N I C  A C I O  S T I L L  GROWN  A C I D  a  • A zoo  •  3  -»  •  a o  IOO  20  I  I  40  I-  I 60  I  I  I  I  60  ICO  O  TIME  (MINUTES)  zo  AO  j  l  SO  i  l  ICO  B  C  300 PLATE. NO.  GLUCOSE  STIUL.  A <<- K E . T O O i _ U T - A R . I C . E> F'UMAR.tC ACIO C PYR-OVIC ACID  B  2 - KELTOGLOCONIC ACIO GftOWN  GBOWM  SflLL.  ACID  A C  200l—  Id  M O 100  20  40  60  L TIME  O (MINUTES)  20  40  60  K  GLUCOSE.  30O PLATL NO. 7  A  A C E - T I C  E>  SUCCIISIC  STILL  2,-KETOGL.UCONIC ACIO GROWN  GeowM  STILL  A C I D ACID  ZOO I  bJ  _: <  <x o N  o  B  too  20  40  0  €0 TIME.  (MINUTES)  20  40  60  GLUCOSE. PLATE. NO.  10  3  A  ACETIC  e>  S U C C I N I C  C  CITRIC  STILL  GROWN  £ - KETOGLOCONIC ACID • GROWN  STILL  ACID ACID  ACID  200  hi  < 0D  «  B  IOO  20  40  60  TIME  O (MINUTES)  20  40  60  GUUCOSE. STICL.  -  300 PLATE NO. //  A  ACELTIC ACID  B  SOOC-INIC  c  CITRIC,  2 - Kero GUUCON IC ACID ™ GROWN  GROWN  STII_L  ACID ACID  •  e A  A  —0  ZOO  UPTAKE  3.  O  J  m *~—"™  g% ™  # g,  *  100  f  B  - c ———~~*  0  i 20  i  i. -JL 40  1  Go  /  — • I  1  TIME  0  (MINUTES)  20  40  1  60  1  C  G L U C O S E  .3001 PL. AXEL NO.  /  A -  Z,  SHAKELR.  Geowrs  2.-  KETOGLOCONIC ACID SHAKCER. GROWN  G L U C O S E  B  - GLoeor-fiC  C  - 2-KETOGLUCOMIC  A c i o ACID  A c  StOOl  _:  _ a "too  l IO  Z . O 30  4 0  AO  60  V O  OO 90  I O O  TIME  O  'O  (MINUTES)  i ZO  l  t 30  AO  SO  < S O T O  SO  SO I O O  GLUCOSE  SHAKER.  J£-  GROWN  KETOGLOCONIC  PLATE.  A  - °C  NO.  B  - FUMAR.IC  /  3  ACID  SHAKER.  GROWN  300 KETOGLUTARIC.ACIO  c -PYRUVIC  ACID ACID  c  ZOO  id  _ O  J O  KD  20  «30  I I -40  SO  I  60  • 70  J  » • 1 ao  90  loo  o  lO  Z.O  TIME CMINUTES)  -SO  I -40  I L_l 50  60  70  L l_L So  90.  IOO  GU-UCOSE.  300 PLATE.  A  NO.  B  -  C  -  ACE1TIC SUCCINIC  CiTf5»o  SHAKER  A C I D  E - k_E_TO G L U C O N I C  GROWN  AciO  SHAxKELie.  .  A C I D  ACID  ZOO I  bJ  _  O (4  O  lOOl  IO  -30  -40  «50  60  7 0  So  9 0  TIME  IOO  o  »Q  (MINUTES)  2 d  -30  -SO  6 0 7 0  80  SO l o o  - 14 -  PART I I I .  DETERMINATION OF THE PRESENCE OF THE ENZYME ALDOLASE  In c o n s i d e r i n g t h e r e s u l t s so f a r o b t a i n e d  i nt h i s laboratory  (11, 12, 13, 14, 3#, 39, 48 and 51) i t would be c o n s i s t e n t t o expect P. a e r u g i n o s a phpsphorylated  t o have a r e s t r i c t e d aldolase-enzyme s i n c e t h e  Embden-Meyerhoff i n t e r m e d i a t e s  c o u l d n o t be d e t e c t e d  and do n o t appear t o f u n c t i o n t o a measurable e x t e n t i n t h e p a t h way o f g l u c o s e  o x i d a t i o n ( 1 2 ) . To demonstrate i f t h i s concept i s  correct the aldolase determination was f o l l o w e d .  o f Dounce and Beyer (20) 194#,  T h i s t e s t was chosen because i t i s c o n s i d e r e d i n -  d i c a t i v e o f an Embden-Meyerhoff system i n t h a t i t measures t h e t r i o s e s formed from fructose-lrr6-,diphosphate. c a r r i e d o u t on s e v e r a l d i f f e r e n t ATCC 9027.  Streptococcus  T h i s procedure was  c e l l p r e p a r a t i o n s o f P.  aeruginosa  f a e c a l i s B18 was used as a s t a n d a r d  s i n c e i t was known t o have an a c t i v e a l d o l a s e system ( 4 7 ) . The S. f a e c a l i s c e l l s were used as an acetone p r e p a r a t i o n .  The p r e -  p a r a t i o n s o f pseudomonas c e l l s were so weak i n t h e enzyme a l d o l a s e (Table 1) t h a t 10 mg. o f c e l l s were used f o r comparison a g a i n s t 0.1 t o 1.0 mg. o f S. f a e c a l i s .  Ten mg. o f c e l l s was t h e l i m i t  beyond w h i c h t h e b l a n k r e a d i n g became h i g h enough t o i m p a i r t h e s e n s i t i v i t y of the test. The r e s u l t s o b t a i n e d  (Table 1) a r e c o n s i s t e n t w i t h t h o s e o f  Campbell and N o r r i s (12) i n which t h e y found no members o f t h e Embden-Meyerhoff pathway p r e s e n t . P. f l u o r e s c e n s , Wood and Schwerdt  Working w i t h P. a e r u g i n o s a and (55) demonstrated t h e p r e s e n c e  o f glucose-6-phosphate o x i d i z i n g enzyme and a 6-phosphogluconic oxidizing  enzyme.  However, n e i t h e r organism had h e x o k i n a s e and  15 -  t h e r e f o r e had no means o f c o n v e r t i n g g l u c o s e t o glucose-6-phosphate. They a l s o found t r a c e s o f a l d o l a s e a c t i v i t y which might mean t h a t the  Warburg-Dickens  thetic  scheme (18) e x i s t s i n our organism as a syn-  system f o r p r o d u c t i o n o f n e c e s s a r y i n t e r m e d i a t e s .  Horecker and S m y r n i o t i s (25a) have d e s c r i b e d a system which causes t h e accumulation o f sedoheptulose from pentose  phosphate.  A l d o l a s e was r e q u i r e d presumably t o s p l i t the pentose, t r i o s e and a 2 carbon fragment.  The two d i o s e s combine t o form t e t r o s e which  i n t u r n combines w i t h p r e v i o u s l y formed t r i o s e t o y i e l d tulose.  sedohep-  T h i s c o u l d c o n c e i v a b l y account f o r t h e a l d o l a s e a c t i v i t y  which we found although the i n t e r m e d i a t e s r e q u i r e d by t h i s r e a c t i o n have not been formed.  - 16 -  TABLE  I  Results of Aldolase  Determination  Organism  Preparation of Cells  S. f a e c a l i s  Acetone p r e p a r a t i o n  1000  P. aeruginosa  Acetone p r e p a r a t i o n  1+2  P. aeruginosa  Acetone p r e p a r a t i o n  32  P. aeruginosa  Alumina ground d e b r i s  66  P. aeruginosa  Sloppy  33  P. aeruginosa  Lyophilized  Based on 1 mg.  dried  Activity *  3  o f S. f a e c a l i s equals 1000 u n i t s .  -  PART IV. a.  17  -  STUDY OF DRIED CELL PREPARATIONS  Introduction I t i s known t h a t when c e l l s o f P.. aeruginosa a r e  d r i e d under vacuum w i t h phosphorous pentoxide as d e s i c c a n t , they r e t a i n the a b i l i t y t o q u a n t i t a t i v e l y o x i d i z e glucose or g l u c o n i c a c i d t o 2 - k e t o g l u c o n i c a c i d hoped t h a t by modifying t h i s treatment  (48).  I t was  c e l l s o f pseudomonas  could be d r i e d t o a s t a b l e form which would r e t a i n the a b i l i t y t o q u a n t i t a t i v e l y convert 2 - k e t o g l u c o n i c a c i d t o further intermediates.  The q u a n t i t a t i v e conversion i s  p o s s i b l e because d r i e d c e l l p r e p a r a t i o n s have no synt h e t i c a b i l i t i e s and t h e r e f o r e no o x i d a t i v e a s s i m u l a t i o n . Using sloppy d r i e d c e l l s as a comparitive standard, a method p r e v i o u s l y used i n t h i s l a b o r a t o r y on glucose grown c e l l s did  ( 4 8 ) , i t was found t h a t the treatments  not preserve t h e a b i l i t y o f the organism  2-ketogluconic  acid.  tried  to oxidize  - 1. -  b.  Methods I t was  (CMC)  e s t a b l i s h e d manometrlcally that  could not be  raonas and  c e l l s , harvested  t o give t e n 20 mg. T h i s was  o x i d i z e d a p p r e c i a b l y by our  so t h i s compound was  enough l i v e  added i n 20 and  s t r a i n o f pseudo40 mg.  amounts to  from a 2-ketogluconic a c i d medium*,  batches o f d r i e d c e l l s a f t e r l y o p h i l i z a t i o n .  done on the  c o a t i n g around the  carboxymethylcellulose  assumption t h a t CMC  i n d i v i d u a l c e l l and  would form a p r o t e c t i v e  prevent the o x i d a t i v e des-  t r u c t i o n o f the 2-ketogluconic a c i d o x i d i z i n g enzyme. (37)  Cysteine  could not be used as a p r o t e c t i v e agent since these b a c t e r i a  oxidize i t . i t has  For the  same reason g e l a t i n could not be used although  been used by other workers (35,  It has  been demonstrated (36)  10)  f o r various  t h a t s u b s t r a t e bound enzymes are  more s t a b l e than those not bound to the s u b s t r a t e , 2-ketogluconic a c i d grown c e l l s * * were sloppy sence of 0.05%  calcium  2-ketogluconate.  sodium t h i o g l y c o l a t e at 0.3$  "SH"  was  d r i e d i n the  was  used i n an  Sucrose at a  (35)  as was  0.9$  NaCl, yeast  0.05$, mineral medium o f N o r r i s and Campbell (11), b u f f e r at pH 7.4,  and  attempt  concentration  used as a p o s s i b l e means o f p r o t e c t i n g the  enzyme system during d r y i n g  pre-  groups on the enzyme d u r i n g  d r y i n g by a c t i n g as an a n t i - o x i d a n t . of 0.5$  therefore,  In another experiment  concentration  to prevent the o x i d a t i o n o f the  organisms.  M/30  labile extract phosphate  d i s t i l l e d water.  Carbowax has been used as an embedding m a t e r i a l i n t i s s u e studies * **  (25),  and  Addendum 1 Addendum 2  under these c o n d i t i o n s  i t appeared to  help  slice  - 19 -  preserve the enzymes o f t h e p l a n t t i s s u e .  I t was t h e r e f o r e  first  t e s t e d manometrically as a p o s s i b l e p r o t e c t i v e c o a t i n g f o r b a c t e r i a during drying.  P. aeruginosa was found t o a t t a c k t h i s compound  w i t h v i g o r and so could not be used. In the s t u d i e s on l y o p h i l i z a t i o n  (22)  use was made o f both  bulk l y o p h i l i z a t i o n , i n which t h e r e s u l t a n t d r i e d c e l l s were ground i n a i r , and s t o r e d i n bulk at 0-5°C., and o f a procedure s u i t e d t o s m a l l e r q u a n t i t i e s i n which the 2-ketogluconic grown c e l l s * were d r i e d i n i n d i v i d u a l v i a l s .  20 mg.  Each v i a l contained  of d r i e d c e l l s which i s enough f o r one Warburg cup.- On completion o f drying, the v i a l s were s e a l e d o f f under vacuum.  Three methods  were used t o a r r i v e at a low enough dew p o i n t o r vapour  pressure  to maintain the c e l l s below t h e t e r t i a r y p o i n t o f water  during  drying.  In the bulk l y o p h i l i z i o n the water was trapped from t h e  vacuum system by d r y i c e and acetone.  The i n d i v i d u a l v i a l s were  d r i e d u s i n g Freon 23 i n a mechanical r e f r i g e r a t i o n system. t h i r d method made use o f the chemical d e s i c c a n t , oxide i n a l a r g e d e s i c c a t o r under h i g h vacuum.  The  phosphorous pentThe P2O5 was  l a y e r e d between g l a s s wool t o i n c r e a s e the e f f e c t i v e area o f the desiccant.  Under these c o n d i t i o n s t h e vapour pressure  was reduced  almost t o t h a t o f l i q u i d a i r and t h e c e l l s which were i n t h e d e s i c c a t o r d r i e d by s u b l i m a t i o n . Sloppy d r i e d c e l l s the d e s i c c a n t  (4$) i s a method again u t i l i z i n g P2O5 as  but i n t h i s case a t h i n l a y e r was used on the bottom  o f a d e s i c c a t o r and o n l y a water j e t vacuum was drawn on the desiccator.  Under these c o n d i t i o n s the t h i n paste o f 2-ketogluconic  * • Addendum 2  i - 20 -  a c i d grown c e l l s *  took i n excess o f 48 hours t o dry and the  d r y i n g was from the l i q u i d in  state: r a t h e r than the f r o z e n s t a t e as  lyophilization. Sloppy d r i e d c e l l s under n i t r o g e n were prepared from  2-keto-  g l u c o n i c a c i d grown c e l l s * which were t r e a t e d s i m i l a r l y t o sloppy d r i e d c e l l s except t h a t the d e s i c c a t o r was f l u s h e d w i t h n i t r o g e n , evacuated and r e f l u s h e d w i t h n i t r o g e n f i v e to s i x times b e f o r e evacuating f o r the f i n a l time.  The n i t r o g e n was added i n an e f f o r t  t o reduce the p o s s i b i l i t y o f o x i d a t i o n o f the l a b i l e 2 - k e t o g l u c o n i c a c i d enzyme d u r i n g d r y i n g . Acetone d r i e d c e l l s  (52)  were prepared by p i p e t t i n g a s l u r r y  o f washed, 2-ket6gluc6riic a c i d grown c e l l s * i n t o 25 volumes o f dry acetone at 0-5°C. c e l l s had s e t t l e d .  The supernatant was decanted o f f a f t e r t h e The c e l l s were r e p i p e t t e d i n t o 10 volumes o f  d r y . c o l d e t h e r which was decanted o f f and the remaining c e l l s r e p i p e t t e d i n t o 10 volumes o f f r e s h c o l d dry e t h e r .  The r e s u l t a n t  s l u r r y was f i l t e r e d on a buchner f u n n e l and was then d r i e d vacuum t o remove t h e e t h e r .  under  The c e l l p r e p a r a t i o n from t h i s pro-  cedure was l i g h t and f r i a b l e and c o n t a i n e d no a p p r e c i a b l e water. Alumina ground  c e l l s (32)  c e l l s were prepared by adding  t h r e e times the.weight o f washed d r i e d alumina g r i n d i n g powder t o the c e l l paste from one l i t e r o f 2 - k e t o g l u c o n i c a c i d medium*.  The  g r i n d i n g was c a r r i e d out f o r t h r e e minutes u s i n g maximum p r e s s u r e i n a c h i l l e d mortar and p e s t l e .  The mixture was e x t r a c t e d t h r e e  times w i t h c o l d 40 ml. q u a n t i t i e s o f W/lOO phosphate b u f f e r at pH 7.4. *  The alumina was c e n t r i f u g e d o f f at s u f f i c i e n t l y low speed  Addendum 2  1  - 21 -  t h a t the c e l l s o f pseudomonas would not come down i n a r e a s o n a b l time.  The c e l l d e b r i s was brought down at 6000 R.P.M.  The supe  natant and t h e c e l l d e b r i s were t e s t e d f o r t h e i r a c t i v i t y on 2-ketogluconic  acid.  - 22 -  c.  Results • Carboxymethylcellulose a p p a r e n t l y o f f e r e d no p r o t e c t i o n  the  p r e p a r a t i o n had no a b i l i t y t o o x i d i z e  of the a d d i t i o n s  tried  used i n Warburg s t u d i e s influence  2-ketogluconate.  assisted i n preserving  a c i d o x i d i z i n g enzyme (Table I I ) .  since None  any 2 - k e t o g l u c o n i c  V a r i a t i o n i n pH o f the b u f f e r  on a l y o p h i l i z e d p r e p a r a t i o n f a i l e d t o  the i n a b i l i t y t o o x i d i z e  phosphate b u f f e r was used at pH 5, Veranol b u f f e r at pH 7.4.  2 - k e t o g l u c o n i c a c i d . S/rensen's 6.6,  7.4  and 8.2 and a l s o  L y o p h i l i z a t i o n , a f t e r the methods des-  c r i b e d , f a i l e d t o preserve t h e enzyme.  2-ketogluconic a c i d  cells  grown at a temperature o f 15°C. f o r one week were prepared i n t h e and a f t e r l y o p h i l i z a t i o n f a i l e d to show any 2-keto-  normal way* gluconic the  oxidizing a b i l i t y .  o b s e r v a t i o n s o f Gale (23)  T h i s was done on the s u p p o s i t i o n might be a p p l i c a b l e  that  i n t h i s case.  This worker noted t h a t amino a c i d decarboxylases were formed t o a l a r g e r extent when t h e organisms were c u l t i v a t e d at a lower than i optimum temperature.  Acetone d r i e d c e l l s showed poor a c t i v i t y  glucose and none on 2 - k e t o g l u c o n i c a c i d .  Sloppy d r i e d c e l l s and  sloppy d r i e d c e l l s prepared under n i t r o g e n 2 - k e t o g l u c o n i c o x i d i z i n g enzyme. c e l l debris to o x i d i z e  i n an attempt t o f i n d found.  N e i t h e r the supernatant nor the ability  Although many methods were t r i e d  a method o f p r e s e r v i n g  t h i s enzyme none..were  Howe.ver, t h i s does not mean t h a t the problem has been ex-  hausted o r that *  contained no d e t e c t a b l e  o f alumina ground c e l l s showed any d i s c e r n i b l e 2-ketogluconic a c i d .  on  Addendum 2  a s o l u t i o n i s not p o s s i b l e .  - 23 -  TABLE I I  Cell  Preparations Activity  Method o f P r e p a r a t i o n  Glucose  2-ketogluconic  Sloppy d r i e d  Normal  None  Sloppy d r i e d c e l l s under n i t r o g e n  Normal  None  Lyophilized c e l l s (aJ bulk - d r y i c e and acetone (b) bulk - P 0 (c) s e a l e d i n vacuum i n d i v i d u a l v i a l s (d) i n presence o f CMC  Normal Normal Normal Normal  None None None None  Sloppy d r i e d i n presence o f : (a) 0.9$ NaCl (b) 0.05$ yeast e x t r a c t  Normal Normal  None None ( i n c r e a s e d endogenous) None None None None  cells  2  (c) (d) (e) (f)  5  m i n e r a l media phosphate b u f f e r pH 7.4 d i s t i l l e d water 2-ketogluconic acid  Normal Normal Normal Normal Slight: Activity  None  Alumina ground (a) d e b r i s (b) supernatant  Normal None  None None  Lyophilized  Normal  None  Acetone d r i e d  c e l l s grown at 15°C.  - 24 -  PART V.  THE INFLUENCE OF METABOLIC INHIBITORS ON THE OXIDATION OF GLUCOSE AND 2-KETOGLUCONIC ACID  A study o f enzyme i n h i b i t o r s was  i n s t i g a t e d i n the hope o f  f i n d i n g one which would act on the enzyme system o f P. at some p o i n t beyond the 2-ketogluconic a c i d stage.  aeruginosa  The f o l l o w i n g  i n h i b i t o r s were t e s t e d : sodium azide (16) sodium arsenate (50) 2,4 d i n i t r o p h e n o l (19) octyl alcohol (46) caffeine (46) acriflavine ( 6) The l a t t e r two  methylene blue iodoacetate hydrazine s u l f a t e sodium f l u o r i d e sodium a r s e n i t e sodium s e l e n i t e  (45) (33) (20) (24) (15) (40)  compounds i n h i b i t e d to some degree the oxida'  t i o n o f the s u b s t r a t e s while the remainder showed no  inhibition  over the range o f c o n c e n t r a t i o n s used. N e u t r a l i z e d i n h i b i t o r s were p l a c e d i n the Warburg cups w i t h the b u f f e r and  c e l l s i n the main compartment and-incubated  f o r 30  minutes at 30°C. i n the Warburg bath p r i o r to t i p p i n g i n the substrate.  The i n f l u e n c e o f the i n h i b i t o r on the r a t e o f endogenous  r e s p i r a t i o n was  determined  i n each  experiment.  The g e n e r a l i z e d r e s u l t s can be seen i n the accompanying t a b l e (Table I I I ) . The f i r s t run r e s u l t s with sodium s e l e n i t e at a c o n c e n t r a t i o n o f 0.00065 M i n d i c a t e d t h a t t h i s c o n c e n t r a t i o n caused a decrease in r a t e of oxygen uptake but not i n amount. s e l e n i t e was  i n c r e a s e d t o 0.0013 M and the i n h i b i t i o n o f 2-keto-  g l u c o n i c a c i d o x i d i z a t i o n was was  The c o n c e n t r a t i o n o f  unimpaired  ( P l a t e No.  50$ while the o x i d a t i o n o f glucose  15).  From these data i t would appear t h a t o x i d a t i o n o f 2-ketog l u c o n i c a c i d was  being i n t e r f e r e d - w i t h at some i n t e r m e d i a t e stage.  - 24a -  J  6  CL Z  - 25  I f t h i s was  -  t r u e i t s h o u l d have been p o s s i b l e t o d e t e c t the  mediate compound accumulated at the p o i n t o f i n h i b i t i o n .  inter-  In order  t o i n c r e a s e the p o s s i b i l i t y o f d e t e c t i n g , o r even i s o l a t i n g , t h i s p o s t u l a t e d i n t e r m e d i a t e , a l a r g e Warburg f l a s k c o n t a i n i n g 30 o f r e a c t i o n m i x t u r e and t h e s u b s t r a t e was  i n c r e a s e d t o 36 oxygen  e q u i v a l e n t s per 3 ml. o r t w i c e t h e normal c o n c e n t r a t i o n o f gluconic acid.  ml.  2-keto-  The r e s u l t s ( P l a t e No. 16) i n d i c a t e d a decrease  r a t e o f oxygen uptake o n l y and not the 50$ i n h i b i t i o n t o be i f t h e enzyme had been poisoned.  ;  The experiment  u s i n g s e l e n i u m a t a c o n c e n t r a t i o n o f 0.0026 M.  was  in  expected  repeated  This r e s u l t e d i n a  61+% i n h i b i t i o n o f 2 - k e t o g l u c o n i c a c i d o x i d a t i o n .  The  contents of  t h e l a r g e Warburg v e s s e l s c o n t a i n i n g endogenous p l u s s e l e n i u m  and  j  a l s o 2 - k e t o g l u c o n i c a c i d p l u s s e l e n i u m were each c e n t r i f u g e d t o remove t h e c e l l s and the s u p e r n a t a n t  c o n c e n t r a t e d t o 5 ml.  This  was passed t h r o u g h an IRA 40 r e s i n column t o remove Na, K, and ions.  Mg  These i o n s i n t e r f e r e w i t h the movement o f 2 - k e t o g l u c o n i c  a c i d on a chromatograph by s l o w i n g i t down. mask some o f t h e chromatographic  developers.  They a l s o tended  to  The d e i o n i z e d  l i q u i d s , i n c l u d i n g washings, were a g a i n concentrated, t o 0.5  ml.  and run c h r o m a t o g r a p h i c a l l y on Whatman #1 paper w i t h an endogenous c o n t r o l p l u s s e l e n i u m , and c a l c i u m 2 - k e t o g l u c o n a t e The a s c e n d i n g  system was  as a s t a n d a r d .  used w i t h e t h a n o l 86 p a r t s , w a t e r 16 p a r t s ,  and ammonium h y d r o x i d e 4 p a r t s , as a s o l v e n t m i x t u r e . d e v e l o p e r s used were: o r t h o phenylendiamine {2% i n 80$ e t h a n o l ) meta phenylendiamine {2% i n 80$ e t h a n o l ) ammoniacal s i l v e r n i t r a t e brom e r e s o l green (1$ i n 95$ e t h a n o l a d j u s t e d t o pH  The  7.0)  2- K E T O G u o o o i s i o  ACID  STI_.U_  G R O W I N  3<i OXVGEJM EJCiOIVM-E-NTS O 0026 M SOD"OM SCUENITE.  PLATE, NO.  /6>  ZOO  UJ  03  N  09  O  TIME  (MINUTES)  - 26 -  orcinol (9) phloroglucinol (9) resorcinol (9) napthol (9) n a p t h o r e s o r c i n o l (9) ninhydrin Hanes' and Isherwood's phosphate reagent (2) 2,6 dichlorobenzenoneindophenol (7) The o n l y r e d u c i n g spots observed were those due t o 2-ketog l u c o n i c a c i d from the 2-ketogluconic p l u s selenium cup and c a l c i u m 2-ketogluconate  standard.  A l l other compounds d e t e c t e d were  present i n the endogenous p l u s selenium as w e l l as i n the 2-ketog l u c o n i c a c i d p l u s selenium f l a s k s .  I t would thus appear t h a t  selenium d i d not a c t on t h e enzyme w i t h i n t h e c e l l but combined w i t h the 2 - k e t o g l u c o n i c a c i d and prevented cell.  i t from e n t e r i n g the  When 2-ketogluconic a c i d d i d c r o s s t h e c e l l  a p p a r e n t l y i t was converted t o G0  2  membrane  and water.  The i n h i b i t i o n which was o b t a i n e d w i t h a c o n c e n t r a t i o n o f 0.0001 M. sodium a r s e n i t e a p p a r e n t l y r e s u l t e d  i n the accumulation  of 2-ketogluconic a c i d s i n c e a n e a r l y t h e o r e t i c a l oxygen uptake o f 68 m i c r o l i t e r s o f oxygen was used by the c e l l s i n the presence o f a r s e n i t e and w i t h glucose as s u b s t r a t e while o n l y 4 m i c r o l i t e r s o f oxygen was taken up by 2-ketogluconic a c i d under the same conditions  ( P l a t e No. 1 7 ) .  The t h e o r e t i c a l oxygen uptake i n the o x i d a -  t i o n o f 18 oxygen e q u i v a l e n t s o f glucose t o 2-ketogluconic a c i d i s 67.2 m i c r o l i t e r s o f oxygen.  In a c o n f i r m a t o r y experiment t h e  oxygen uptake o f glucose and o f 2 - k e t o g l u c o n i c a c i d was 80 microl i t e r s and 24 m i c r o l i t e r s  respectively  ( P l a t e No. 17).  appear t h a t sodium a r s e n i t e i n h i b i t e d the 2-ketogluconic  I t would acid  o x i d i z i n g enzyme w i t h the same e f f e c t as e x c e s s i v e a e r a t i o n .  2 -KE_-TO GLUCONIC  ACID  STIUL.  GROWIN  CLLLS  0}  - 27 -  TABLE I I I Summary o f I n h i b i t i o n S t u d i e s Compound  Concentration W i t h i n the Warburg Cup  Sodium  0.015 M, 0.12M  azide  Inhibition Glucose 2-ketogluconate ...Nil  Nil  Nil  Nil  Sodium arsenate  0.013 M, 0.026 M, 0.054 M  2,4 d i n i t r o p h e n o l  1x10" 5m,  5 x 1 0 " 1 x 1 0 - %! N i l  Octyl alcohol  Saturated  solution  Complete  Nil Complete  0.016 M, 0.032 M, 0.16 M  Nil  Nil  Caffeine  0..025 M  Nil  Nil  Acriflavine  0.035 M, 0.07 M, 0.35 M  Nil  Nil  Methylene Blue  5xl0-A-M, 5X10-5m  Nil  Nil  Iodoacetate  0.001 M, 0.005 M  Nil  Nil  Hydrazine S u l f a t e  0.0037 M, 0.0148 M  Nil  Nil  Sodium A r s e n i t e *  (See  text)  70$  100$  Sodium S e l e n i t e * *  (See  text)  40-50$  50-60$  ' Sodium f l u o r i d e  *  P l a t e 17  ** P l a t e s 15 and 16  _ 28 -  PART VI.  EFFECT OF HEAT SHOCKING ON THE ENZYME STRUCTURE OF CELLS GROWN ON 2-KETOGLUCONIC ACID  During t h e h i g h speed c e n t r i f u g i n g o f a c u l t u r e , unavoidable h e a t i n g o c c u r r e d which reduced the r a t e o f o x i d a t i o n o f 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 not t h e r a t e o f g l u c o s e o x i d a t i o n . Since t h i s might be i n t e r p r e t e d as i n d i c a t i n g t h a t glucose i s not o x i d i z e d by way o f 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 i t was decided t o explore the o b s e r v a t i o n f u r t h e r .  The r e s u l t s w i t h l i v e  c e l l s which had been heated t o 4,5°C. f o r one hour i n d i c a t e d a s i m i l a r reduced r a t e o f o x i d a t i o n o f 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 ( P l a t e 18, 1 9 ) . However, 55°C. f o r 30 minutes  almost com-  p l e t e l y a b o l i s h e d the a b i l i t y t o o x i d i z e g l u c o n i c a c i d and 2-ketog l u c o n i c a c i d whereas t h e a b i l i t y to a t t a c k glucose was unimpaired. When these heat t r e a t e d c e l l s were d r i e d i n vacuum they then possessed t h e a b i l i t y t o o x i d i z e gluconate t o 2-ketogluconate ( P l a t e 20).  acid  T h i s treatment t h e r e f o r e merely d e s t r o y e d the a b i l i t y  of gluconate t o c r o s s t h e membrane o f the heat t r e a t e d c e l l but not t h e a b i l i t y o f t h i s c e l l t o o x i d i z e g l u c o n i c a c i d . phenomenon might p a r t i a l l y  be e x p l a i n e d by Dicken's l a t e s t  (1.8) i n which he p o s t u l a t e s the presence o f and 6-phospho-2-ketogluconolactone a c i d and 6-phospho-2-ketogluconic cose o x i d a t i o n .  This scheme  6-phosphogluconolactone  r a t h e r than  6-phosphogluconic  a c i d s as Intermediates o f g l u -  The presence o f a heat l a b i l e  enzyme t o convert  g l u c o n i c a c i d t o gluconolactone would then be i n d i c a t e d except t h a t t h i s would not account f o r t h e a b i l i t y o f the l y o p h i l i z e d c e l l t o oxidize gluconic acid. Heat t r e a t e d 2 - k e t o g l u c o n i c a c i d grown c e l l s  either lyophilized  - 2_a -  U * 1  He. a t TteELATELD SS°C  Jor  /O MIIV.  2-KE.TOGLOCONJC  ACID  GROWN  300 PLATE.  No.  /«?  200  bJ ro  f a  3 100  - 29 -  or  sloppy d r i e d are comparable t o normal 2 - k e t o g l u c o n i c a c i d grown  c e l l s l y o p h i l i z e d o r sloppy d r i e d when t e s t e d on glucose o r g l u conic a c i d ( P l a t e 2 0 ) . C e l l s heated f o r one-half hour a t 55°C. manifest t h e c h a r a c t e r i s t i c r e s u l t s o f c e l l s heated f o r only 10 minutes at t h i s temperat u r e ( P l a t e 18 and 19).  The glucose curve was e s s e n t i a l l y  to t h a t o f u n t r e a t e d c e l l s .  similar  The c e l l s t r e a t e d f o r one hour a t  55°C. appear t o have l o s t t h e a b i l i t y t o assimulate glucose but not the a b i l i t y t o o x i d i z e i t .  Nearly t h e o r e t i c a l oxygen uptake,  403 m i c r o l i t e r s , was achieved w i t h glucose as a s u b s t r a t e .  This  technique may be u s e f u l as a means o f p r e p a r i n g c e l l s f o r i n h i b i t i o n s t u d i e s s i n c e c e l l s so t r e a t e d would tend t o accumulate h i g h e r y e i l d s o f i n t e r m e d i a t e s than i s u s u a l because normally i n l i v e c e l l s l / 2 t o l / 3 o f the a v a i l a b l e s u b s t r a t e i s a s s i m u l a t e d and does not take part i n the o x i d a t i v e p r o c e s s .  HEAT TREATED 2 -KETOGLUCONIC ACID STILL. GROWN LYOPHILIZED CELLS  O  30  60  SO  120  ~ 0  "  TIME (MINUTES)  3  0  6  ©  90  1*3  - 30 -  PART V I I . 1.  ADDENDUM I  Stock  C u l t u r e Agar Medium  Tryptone K HP0 2  ,  1 $ 0.3$  4  Glucose  0.1$  Glycerol  0.3$  Liver Extract  10$ by volume  Agar  0.5$  Gelatin  2$  Adjust t o pH 7.2.  2.  Yeast  - Glucose  . Medium (49)  5$ glucose 0.5$ yeast e x t r a c t Adjust t o pH 7.2.  3.  Production  Medium 16 $  Glucose CaC0  2.7$  3  K HP0^  0.3$  2  MgS0^.7H 0  0.1$  FeS0 .7H 0  0.05$  Urea  0.2$ ( s t e r i l i z e d  2  4  2  separately)  - 31 -  M i n e r a l Medium o f Campbell and N o r r i s (11) NH^H P0^  0.3$  K H P0^  0.3$  2  2  MgS0^.7H 0  0.1$ ( s t e r i l i z e d  separately)  Glucose  0.1$ ( s t e r i l i z e d  separately)  2  Fe as F e C l  2  Adjust t o 7.4.  0.5 ppm  PART V I I I .  ADDENDUM 2  METHODS USED WHICH ARE Throughout  COMMON TO PHYSIOLOGICAL STUDIES  t h i s study the methods used have been those out-  l i n e d by Umbreit  B u r r i s and S t a u f f e r (56).  The organism used was  Pseudomonas aeruginosa ATCC 9027 u n l e s s  otherwise noted. In the p r e p a r a t i o n o f c e l l s the m i n e r a l media* o f and N o r r i s (11) was as carbon source. autoclaving.  used w i t h 1$ glucose or sodium The glucose was  sterilized  The sodium 2-ketogluconate was  sterilized  2-ketogluconate  as a 10$ s o l u t i o n by sterilized  s o l u t i o n through a s i n t e r e d u l t r a f i n e g l a s s f i l t e r . s u l f a t e was  Campbell  as a  10$  Magnesium  s e p a r a t e l y by a u t o c l a v i n g as a 10$  solution.  The two phosphates and i r o n were s t e r i l i z e d a f t e r n e u t r a l i z a t i o n i n 9° ml. q u a n t i t i e s at the i n d i c a t e d s t r e n g t h i n Roux b o t t l e s by autoclaving.  The medium was  One ml. o f a 18-20  compounded j u s t p r i o r t o i n o c u l a t i o n .  hour o l d c u l t u r e was  used as inoculum.  The sodium 2-ketogluconate was made up as a 50$ weight volume s o l u t i o n from c a l c i u m 2-ketogluconate by replacement o f C a calcium s u l f a t e .  + +  w i t h sodium  by  stoichiometric  s u l f a t e and f i l t e r i n g o f f the  T h i s s o l u t i o n was  s t o r e d at 0-5°C. and  redis-  solved p r i o r to use. The inoculum was  s u b - c u l t u r e d monthly on stock c u l t u r e agar  s l o p e s * incubated f o r 48 hours at 30°C..... and s t o r e d at 0-5°C. stock c u l t u r e slope was *  Addendum 1  used as an inoculum f o r 10 ml. o f  A  - 33 -  2-ketogluconic  a c i d m i n e r a l medium a f t e r i n c u b a t i o n f o r 24 hours  at 30°C. The tube o f media was 2-ketogluconic  used to i n o c u l a t e a s i m i l a r tube o f  a c i d mineral medium.  T h i s was  t o i n s u r e an a c t i v e l y growing c u l t u r e .  2-ketogluconic  ml. Roux b o t t l e s o f  a c i d m i n e r a l medium which were incubated at 30°C.  f o r 18 to 20 hours and harvested.' pended i n 1/25  A f t e r t h r e e such t r a n s f e r s  used as inoculum f o r 100  the c u l t u r e was  repeated three times  volume o f 0.9$  s u l t i n g , c e l l paste was  The harvested  c e l l s were re sus-  s a l i n e and r e c e n t r i f u g e d . . The  used immediately or was  re-  s t o r e d at 0-5°C.  f o r p e r i o d s up to t h r e e days. The washed c e l l s were made up i n M/30 b u f f e r at pH 7.4  S^rensen's phosphate  to a c o n c e n t r a t i o n such t h a t a 1:50  a 60-70$ l i g h t a b s o r p t i o n r e a d i n g on a F i s h e r  d i l u t i o n gave  electrophotometer  with a 525 ytt f i l t e r u s i n g d i s t i l l e d water as a blank. The Warburg cups were made up to a f i n a l volume o f 3.15 c o n s i s t i n g of: 0.15  ml. o f 20$ K0H  s t a n d a r d i z e d c e l l suspension, b u f f e r at pH 7.4, partment.  1.5  i n the center w e l l ; 0.5 ml. o f M/30  ml.  ml. of  SpVensen's phosphate  and water to make up to 3 nil. i n the main com-  The volume o f s u b s t r a t e or i n h i b i t o r was  f o r by an a p p r o p r i a t e r e d u c t i o n i n volume o f water. t r a t i o n o f s u b s t r a t e was  such t h a t 0.2  compensated The  concen-  ml. gave 18 oxygen equi-  v a l e n t s o r 403.2 m i c r o l i t e r s o f oxygen uptake on complete o x i d a t i o n o f the s u b s t r a t e to CO2 The Warburg was t u r e o f the bath was The  at 120  and water. o s c i l l a t i o n s per minute.  The  tempera-  30 * 0.1°C.  c e l l s used i n the metabolic  s t u d i e s were grown on a medium  - 34 -  containing  sodium 2-ketogluconate as the s o l e carbon source i n  s t a t i o n a r y Roux b o t t l e s . o f the medium was 100 ml.  A f t e r a d d i t i o n o f inoculum t h e volume  PART IX.  BIBLIOGRAPHY  1.  A s a i , Toshinobu, and Ikeda, Y o n o s u i k i , Manufacture o f i s o a s c o r b i c a c i d . I . 2 - k e t o g l u c o n i c a c i d forming s t r a i n s . Jour. Agr. Chem. S o c , Japan. 22, 50-1, 1948 (CA /£, 6191 i ) .  2.  Bandurski, R.S., and Axelrod, B., The chromatographic identif i c a t i o n o f some b i o l o g i c a l l y important phosphate e s t e r s , Jour. B i o l . Chem. 123, 405, 1951.  3.  Bernhauer, K., and G o r l i c k , B., O x i d a t i o n s w i t h a c e t i c a c i d b a c t e r i a . IV. Formation o f 2-ketogluconic a c i d by B. g l u c o n i cum. Biochem. Z. 280, 367-74, 1935 (CA 30, 1 0 8 5 ) . 3  4.  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