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

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££3 3 7 / v j S X eo/o. / A STUDY OF THE INTERMEDIATE METABOLISM OF PSEUDOMONAS AERUGINOSA - by -Phyllis Winifred Ney A Thesis Submitted In Partial Fulfilment of the Requirements for the Degree of MASTER OF SCIENCE IN AGRICULTURE in the DEPARTMENT OF DAIRYING THE UNIVERSITY OF BRITISH COLUMB May, 1948. ABSTRACT The intermediate metabolism of P, aeruginosa (A.T.C. 9027) has been studied manometrically to - determine the pathway of glucose breakdown. Since the organism was found to respond to increased amounts of i r o n and since no p o s i t i v e evidence of an anaerobic r e s p i r a t o r y mechanism could be obtained i t was concluded 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 attacked by r e s t i n g suspensions of c e l l s harvested from a glucose-ammonium succinate medium were s u c c i n i c a c i d , glucose, gluconic a c i d and 2-ketogluconic a c i d . However, 5-ketogluconic a c i d was not attacked. Concentrations of malonate as high as 5 x 10 M had no e f f e c t on the o x i d a t i o n of glucose or succinate by these c e l l s , however they d i d markedly r e t a r d the rate of adaptation t o succinate by c e l l s harvested from a glucose ammonium phosphate medium. By growing the c e l l s on a completely inorganic medium plus 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 that glucose grown c e l l s r a p i d l y o x i d i z e d gluconic and 2-ketogluconic acids but short periods of adaptation were re 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 harvested from succinate and lactate-ammonium phosphate media were not pre-adapted - 2 -to the breakdown of glucose. The ra t e of 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 p e r i o d . To confirm the presence of adaptive enzymes f o r the d i s s i m i l a t i o n of glucose, a comparison was made of the a b i l i t y of c e l l s grown i n the presence of e i t h e r glucose or acetate t o att a c k glucose. Whereas c e l l s harvested from a glucose medium o x i d i z e d t h i s substrate at a r a p i d and con-stant r a t e , c e l l s grown i n the presence of acetate 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 . Since glucose was attacked by an adaptive enzyme system i t was p o s s i b l e to study intermediate glucose meta-bolism by means of the simultaneous adaptation technique of S t a n i e r . A comparison of the o x i d a t i v e a c t i v i t y of glucose and acetate c e l l s showed that while glucose c e l l s attacked glucose, gluconic a c i d and 2-ketogluconic a c i d r a p i d l y and d i r e c t l y , acetate c e l l s r e q u i r e d a period of adaptation of at l e a s t one hour before-they 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 the r e f o r e concluded that the breakdown of glucose by t h i s aerobic organism proceeded by way of gluconic a c i d and 2-ketogluconic a c i d , and that s u c c i n i c a c i d was not an intermediate 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 in this work. I am also grateful to the National Research Council of Canada for financial assistance received for the period April 1, 1947 to March 31, 1948, and to The University of British Columbia for a research grant during the summer of 1946. P. W. N. TABLE OF CONTENTS INTRODUCTION 1 PART 1 IRON REQUIREMENTS Introduction .-«•«.•••©.i. 4 Methods Bacteriological 9 Chemical . . . . . o 11 Experimental and Discussion 14 Summary ©.•..•©»••©. 21 PART II THE PREPARATION OF RESTINGCEELLS Introduction ................ o • • •• 23 Methods Bacteriological © 25 Experimental and Discussion Nitrogen and Carbon .......... 0 o . . . . . . . . . © . 29 Minerals . . . . . . Q . c . © . . . © . . . o . e . « . . . • • • • • 32 Age o •»..«.• 34 Aeration ......... ...........© ........ 36 Warburg studies . . . . . . 0 . . . © . . . . . . . o . e . . . . . . . . © 38 Summary ••••• •• 4 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 in growing cultures .... 60 Anaerobic dissimilation of • 2-ketogluconic a-e acid 64 Anaerobic dissimilation 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 cells ......... 78 Synthesis inhibition 88 C. Adaptation. Introduction .... © a • ••••••*©•..•<••• © 92 Experimental and Discussion Simultaneous adaptation ..©.©.........• 96 Summary •»••••••••«•••,•••€•••••••••••••••.••.•• 105 BIBLIOGRAPHY ••••••••••••••••••••••••••••••••••••••••• 107 - L. _ During the l a s t f i f t y years a tremendous fund o f knowledge r e l a t i n g t o the mechanisms of 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 the greatest amount of t h i s knowledge p e r t a i n s t o the anaerobic breakdown of glucose t o a l c o h o l or l a c t i c a c i d , and a more complete understanding of these two systems has been achieved than of any comparable energy y i e l d i n g processes. As a r e s u l t of the e f f o r t s of Lipmann, Meyerhof, C o r i , Warburg and others, the step by step degradation o f glucose and other carbohydrates t o the end products 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 coupled w i t h a s e r i e s of w e l l - d e f i n e d phosphorylations and dephosphorylations by means of which a l a r g e p o r t i o n o f the energy of the substrate i s made a v a i l a b l e to the c e l l (Meyerhof 194S). Much l e s s i s known of the mechanisms which f u n c t i o n i n aerobic r e s p i r a t i o n . Szent-Gyorgyi (Sumner and Somers 1943) u s i n g pigeon breast muscle suspensions formulated an aerobic 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 of t h i s system, the two a v a i l a b l e hydrogens from glyceraldehyde are passed by way of o x a l a c e t a t e , malate, coenzyme I , and the y e l l o w enzyme to fumarate t o y i e l d s u c c i n i c a c i d . By v i r t u e o f s u c c i n i c dehydrogenase, hydrogen i s then d i r e c t l y t r a n s f e r r e d through cytochrome t o oxygen. Although therrao-dynamically sound, the theory has met w i t h considerable c r i t i c i s m . 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 through cytochrome without a mediator. Furthermore, as P o t t e r (1940) has pointed out, the i n c l u s i o n of the oxalacetate-malate system i n the 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 use, accumulation o f reduced coenzyme I i s obtained. On the b a s i s of t h i s and a d d i t i o n a l work, Krebs (Evans 1942) has formulated h i s c i t r i c a c i d c y c l e f o r muscle metabolism. I n i t i a l l y , pyruvate condenses w i t h o x a l a c e t a t e to y i e l d . c i t r i c a c i d . The o x i d a t i o n then 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 oxalacetate which i n t u r n can condense w i t h a second molecule of p y r u v i c a c i d . I n contrast t o the theory of Szent-Gyorgyi, Krebs has suggested that greater than h a l f the hydrogen can be t r a n s f e r r e d t o oxygen over the oxalacetate-malate system. The 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 of as hydrogen c a r r i e r s , are shown t o be intermediates i n the 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 of the a e r o b i c systems f u n c t i o n i n g i n b a c t e r i a l o x i d a t i o n . 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 that under e i t h e r anaerobic or aerobic 'conditions carbohydrate i s d i s s i m i l a t e d t o the pyruvate stage by the Meyerhof-Embden pathway. The subsequent 1 3 - 3 -aerobic stages o f the r e a c t i o n are s t i l l obscure. Although the Krebs c y c l e f u n c t i o n s as an aerobic system i n muscle, and although many b a c t e r i a can u t i l i z e most of the intermediates of t h i s c y c l e , nevertheless no b a c t e r i a l c e l l has ever been shown t o possess 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, thus p o i n t i n g t o an o x i d a t i v e system f o r aerobic organisms. Since 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 hi g h pressures, i n t e n s e a e r a t i o n and h i g h concentrations of sub-s 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 before i t can be considered o f metabolic importance. The present t h e s i s i s an attempt t o uncover the pathway by which t h i s organism obtains energy fromgfucose. The f i r s t p a r t o f the study i s concerned w i t h the i r o n requirements of' the organism, and the e f f e c t o f i r o n d e f i c i e n c y on the a c t i v i t y of the c e l l s . The second part deals w i t h the method of p r e p a r a t i o n o f r e s t i n g c e l l suspensions and the t h i r d part i s devoted t o a study of the metabolism of the c e l l s w i t h emphasis on enzymic adaptation. -4-PART I IRON REQUIREMENTS t Comparatively l i t t l e i s known o f the i r o n requirements of the 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 in f o r m a t i o n i s undoubtedly due l a r g e l y t o the absence of 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, (Elvehjem 1931; Steinberg 1935) exhaustion procedures, ( M o l i s c h 1892; Roberg 1928; M o l l i a r d 1929}' and the use of r e c r y s t a l i i z e d chemicals (Burk and Horner 1934) have been employed. A l -though time consuming, the mold p u r i f i c a t i o n method of 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. indologenes (Waring and Werkman 1942). The remaining procedures, 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 , are i n a p p l i c a b l e to a study of the more f a s t i d i o u s b a c t e r i a . 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 chloroform and 8-hydroxyquinoline, and by Perlman (1943T) using c a t i o n i c exchange..materials have f a c i l i t a t e d the study of the mineral requirements of b a c t e r i a at l e v e l s very much lower than was p r e v i o u s l y p o s s i b l e . With a knowledge of these requirements i t i s now p o s s i b l e t o e s t a b l i s h the 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 ino r g a n i c i o n s i n c e l l u l a r metabolism. The r e l a t i o n s h i p of i r o n t o growth has been observed 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 chemicals obtained maximum / growth of azotobacter w i t h i r o n concentrations of 0.025 -0.055 ppm. More r e c e n t l y Waring and Werkman (194<B) have e s t a b l i s h e d the i r o n requirements of s t r a i n s of P. aeruginosa. A. indologenes. J L . aerogenes. K . pneumoniae and E. c o l i . A s t i m u l a t i o n from 6 t o 100$ was obtained f o r P. aeruginosa w i t h i n the range 0 - 0.09 ppm Fe. L i l y (1945) obtained a growth response w i t h R. t r i f o l i i from 0 - 0.t25 }igm Fe per ml. Burton et a l (1947) observed that 0.1 ;ugm Fe per ml. gave no pyocyanine formation but abundant growth and pro-d u c t i o n of the f l u o r e s c e n t pigment i n c u l t u r e s of P.-aeruginosa 90^7. Pappenheimer (1936) and Feeny, Mueller and M i l l e r (1943), i n studies on the r e l a t i o n of i r o n t o the t o x i n production of G. dixththeriae and C. t e t a n i . r e s p e c t i v e l y , 9 have shown that maximum growth of these organisms i s ob-t a i n e d w i t h amounts of i r o n i n excess of the optimum concentration f o r the production of t o x i n . The synthesis o f 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 the i r o n content o f the growth medium (Burkholder 1943, 1944j Saunders and McLung 1943; Tanner 1945). For example, i n the presence of 0.5 - 1.0 4igm Fe per 100 ml., the highest y i e l d s of r i b o f l a v i n were obtained, w h i l e the a d d i t i o n of lO^igm per ml. sharply reduced the formation of the v i t a m i n . The c e l l crop harvested at the 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 production was 40 - 80$ of that obtained at the peak of the growth curve (Tanner 1945). Because of the importance o f i r o n c a t a l y s t s i n aerobic r e s p i r a t i o n , the e f f e c t s of i r o n d e f i c i e n c y on the metabolism o f the c e l l are-of great i n t e r e s t . Kubowitz (1934) showed that when glucose fermentation by washed c e l l suspensions of C. butvrlcum was allowed t o take place i n an atmosphere of carbon monoxide, the normal a c e t i c and b u t y r i c a c i d production was replaced 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 concentrations of cyanide (10"^M) tended t o cause the same s h i f t i n metabolism. Simon (1947) confirmed these r e s u l t s u s i n g r e s t i n g c e l l s of C. acetobutvlicum. Pappenheimer and Shaskan (1944; 1944 )Agrown w i t h h i g h and low concentrations of i r o n , were able t o confirm and extend the work of Kubowitz. They found that these organisms, which p r e v i o u s l y had been con-sidered 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 the i r o n - d e f i c i e n t medium. On the h i g h i r o n medium, the u s u a l combination of b u t y r i c and a c e t i c a c i d s was obtained. S i m i l a r changes could be produced i n the 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 fermentation of A. aerogenes (Perlman 1945) by growing the 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 of the normal fermentation could be obtained by adding back small amounts of i r o n , although d e f i c i e n c i e s i n other metals made i t impossible t o e s t a b l i s h t he importance o f t h i s element. Elvehjem (1931) observed t h a t growth i n low i r o n media decreased the i r o n content of yeast c e l l s and s i g n i f i c a n t l y reduced the amount of cytochrome i n the organisms. T h i s i s i n agreement w i t h the f i n d i n g s of Waring and Werkman con-cerning i r o n d e f i c i e n c y (1944). Using c e l l s of A. indologenes these workers have shown th a t growth i n low i r o n media produces c e l l s low i n c a t a l a s e , peroxidase, cytochrome, formic dehydrogenase, hydrogenase and hydrogenlyase. S u c c i n i c dehydrogenase, malic dehydrogenase and fumarase systems were also present i n a depleted c o n d i t i o n . Mueller (1941) has suggested t h a t £. d i p h t h e r i a e elaborates t o x i n under con-d i t i o n s of 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 the absence of normal i r o n - c o n t a i n i n g enzyme systems. The supply of i r o n a v a i l a b l e t o c e l l s during growth appears t o determine the r a t i o at which c e r t a i n o f the enzyme systems e x i s t w i t h i n the c e l l . That i s , i n a low i r o n medium, enzymes which contain the element i r o n ( c a t a l a s e , peroxidase, verdo-peroxidase, cytochromes, cytochrome oxidase, cytochrome peroxidase) or are dependent on i r o n f o r t h e i r formation, may be present i n depleted amounts i n r e l a t i o n t o the amount of c e l l protoplasm. The present work i n c l u d e s a study of the i r o n r e q u i r e -ments of 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 iron-growth curve between 0 and 0.5oigm of i r o n per ml., and t o show tha t c e l l s harvested 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 glucose and l a c t i c and a c e t i c a c i d s . On the b a s i s o f t h i s i n f o r m a t i o n we have concluded t h a t the o x i d a t i v e mechanisms f u n c t i o n i n g i n the r e s p i r a t i o n o f the c e l l s are s t r i c t l y a e r o b i c i n nature. METHODS B a c t e r i o l o g i c a l ; The c u l t u r e of Pseudomonas aeruginosa ATC 9027 employed throughout t h i s work was a c t i v e and t y p i c a l i n a l l r e s p e c t s . No d i f f i c u l t y w ith d i s s o c i a t i o n or any other v a r i a t i o n was encountered. 'Stab c u l t u r e s of the organism i n l i v e r e x t r a c t agar were refrigerated after growth was ini t i a t e d at 30°C. Before being used i n experimental work, the c u l t u r e was t r a n s f e r r e d 2 - 3 times at 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 F§ per ml. To maintain a vigorous 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 taken each week from a r e f r i g e r a t e d stock. The composition of the stock agar was 1.0$ tr y p t o n e , 0.3$ v K^HPO. 0,1% glucose, 0,3% g l y c e r o l , 10% l i v e r e x t r a c t , ^ 4 0,5% agar, 2,0% g e l a t i n e adjusted t o pH 7.2 before a u t o c l a v i n g The basal medium u s e d in.experiments on the i n f l u e n c e o f i r o n on growth and a l s o i n the production of r e s t i n g c e l l s c o n s isted of 1,0% glucose, 0.3$ NH 4 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 per 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 g l a s s . The composition of the s a l t s o l u t i o n was as f o l l o w s : 0.044$ ZnS 0 4 - 7 i y ) , 0.04$ CuS04'5EgO and 0.041$ MnS0 4'4H s0. For the i r o n assay the inoculum was prepared from ah 18-20 h r . c u l t u r e of the organism i n t h e basal medium + ++ 0.05 ;ugm Fe per ml. These c e l l s were harvested by -10-c e n t r i f u g a t i o n , and were washed twice 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 give a b a r e l y v i s i b l e t u r b i d i t y (approximately one m i l l i o n organisms per ml.). One drop of t h i s suspension was employed as inoculum f o r 10 ml. of medium, g i v i n g an i n t i a l p o p u l a t i o n of about 5000 c e l l s per ml. I n t e s t i n g the i n f l u e n c e of i r o n on the enzyme com-plement of the c e l l s , r e s t i n g c e l l suspensions were employed. Adequate a e r a t i o n of the c u l t u r e s was ensured by using 100 ml. of medium per Roux.flask or 50 ml. of medium per K o l l e f l a s k . The c u l t u r e s were harvested a f t e r 20-22 h r s . i n c u b a t i o n , at 30°C. The c e l l s were washed twice i n h a l f the growth volume w i t h a 0*9% NaCl s o l u t i o n , and were f i n a l l y made up t o 1/10 the o r i g i n a l volume, thus g i v i n g a c o n c e n t r a t i o n t e n times t h a t of the growing c u l t u r e . Organisms prepared i n t h i s manner had an endogenous-respiration of l e s s than 100ji 1. oxygen per hour per 0.5 ml c e l l suspension, a r a t e which d i d not I n t e r f e r e w i t h the i n t e r p r e t a t i o n of data obtained f o r o x i d i z a b l e s u b s t r a t e s . For the determination of the a b i l i t y of the 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 Warburg technique was used (Dixon 1943; Umbreit et a l 1945). C e l l s suspended i n M/15 phosphate b u f f e r were placed i n the main compartment and substrate i n the sidearm of the f l a s k . A f t e r a 10 minute e q u i l i b r a t i o n p e r i o d the stopcocks were cl o s e d and t e s t readings were taken at 5 minute i n t e r v a l s u n t i l the columns appeared to move at a constant r a t e . Substrate was then t i p p e d i n and readings were recorded at 10 minute i n t e r -v a l s throughout the experimental p e r i o d . Depending upon the nature of the experiment, the r e a c t i o n time was from 1-2 h r s . At the end of 1 n r . , 5^uM of glucose were completely u t i l i z e d by the c e l l s and a con-stant r a t e of oxygen uptake p a r a l l e l i n g t h a t of the endogen-ous r e s p i r a t i o n was maintained. Chemicalt Pyrex glassware was used e x c l u s i v e l y I n the p r e p a r a t i o n of t h e medium and i n the growth experiments which f o l l o w e d . To ensure complete removal of t r a c e s of i r o n , glassware was cleaned by successive treatment w i t h soap and water, saturated a l c o h o l i c KOH, d i s t i l l e d water, 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 water. No c o t t o n p l u g s , cork or rubber stoppers were employed at any time. The chemicals to be e x t r a c t e d were Merck C.P. or reagent grade; those 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. q u a l i t y . The n u t r i e n t s of the 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 repeatedly e x t r a c t e d w i t h chloroform and 8-hydroxyquinoline u n t i l no green c o l o r could be detected i n the chloroform washings. I t has been found that at pH's ranging; from 6 to 8 (Waring and Werkman, 1942) t h i s method removes trsLce amounts of Cu, Fe, Mn and Zn. -12-A d d i t i o n s of the s a l t s o l u t i o n and MgS04«7H20 were made f o l l o w i n g the e x t r a c t i o n procedure. The t o t a l i r o n con-tamination from unextracted s a l t s was c a l c u l a t e d to be not greater than 0.002 ;ugm of Pe per ml. A standard i r o n s o l u t i o n containing. 100 ugm F e + + per ml was prepared i n water r e d i s t i l l e d through g l a s s (498 mg. PeS0 4»7H 20 made to 100 m l . ) . Prom t h i s stock s o l u t i o n d i l u t i o n s were prepared to c o n t a i n 10 ;ugm, 1 .ugm and 0.1 >ugm P e + + per ml. To a v o i d t h e p r e c i p i t a t i o n of i r o n hydrates the s o l u t i o n s were s t e r i l i z e d by passage through s t e r i l e s i n t e r e d g l a s s f i l t e r s . 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 ml. of t h e c e l l suspension were added 1 ml. of concentrated H 2S04 and 2 drops of 30$ H20 2. The sample was digested slowly f o r 15-20 minutes, over a micro-burner u n t i l a l l organic matter was decomposed l e a v i n g a c l e a r s o l u t i o n . A f t e r c o o l i n g , the 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 ml. water, 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 to the d i s t i l l a t i o n apparatus. An excess of base (10 ml. of carbonate-free 10 N NaOH) was run i n t o the sample, and 100 ml. of the steam d i s t i l l a t e was c o l l e c t e d i n 10 ml. 0.01 N HCl over a 30 minute p e r i o d . The excess a c i d was then t i t r a t e d w i t h 0.01N NaOH to the end-point of methyl r e d i n d i c a t o r . The amount of n i t r o g e n i n the sample was c a l c u l a t e d from the equivalents of a c i d n e u t r a l i z e d by ammonia. Sodium pyruvate f o r use i n r e s p i r a t o r y studies was prepared according to the method of Robertson ( ) w i t h the f o l l o w i n g g m o d i f i c a t i o n . The a l c o h o l i c s o l u t i o n of 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 successive crop of 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 . This n e u t r a l i z a t i o n procedure was continued u n t i l polymeriza-t i o n , as evidenced by the appearance of the y e l l o w condensa-t i o n product, occurred. Sodium pyruvate prepared i n t h i s manner has been found more a v a i l a b l e to organisms than t h a t obtained by other procedures ( KVl-nif^ky < UatW^^x -14-EXPERIMENTAL Experiments were designed to determine the i n f l u e n c e of the c o n c e n t r a t i o n of i r o n i n the*, growth medium on the amount of c e l l m a t e r i a l formed. B a c t e r i a l n i t r o g e n , which i s the most u n i v e r s a l l y recognized standard f o r expressing amount of c e l l m a t e r i a l , was used as the measure of b a c t e r i a l m u l t i p l i c a t i o n . The concentrated b a s a l medium was dispensed i n f i v e ml. amounts i n t o 125 ml. Erlenmeyer f l a s k s . To each was added water r e d i s t i l l e d through g l a s s so t h a t upon the a d d i t i o n of i r o n s o l u t i o n a f i n a l volume of 10 ml. was obtained. A f t e r the f l a s k s were capped w i t h beakers, the medium was auto-claved at 15 l b s . pressure f o r 15 minutes. A s e p t i c a d d i t i o n s 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 assay f l a s k , and 1 drop of the washed c e l l suspension was introduced as inoculum. At the end of 4 days i n c u b a t i o n at 30°C, the c e l l crop of each f l a s k was harvested by c e n t r i f u g a t i o n , washed twice 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 the growth volume. Du p l i c a t e 1 ml. a l i q u o t s of t h i s suspension were used f o r theu determination of b a c t e r i a l n i t r o g e n . As shown i n Table I , growth of the organism i s a f u n c t i o n of the i r o n content of the medium. A response of from 6% t o 100$ was obtained i n the range 0-0.5jugm P e + + per ml. w i t h the maximum e f f e c t apparent from 0 to 0.01 ;ugm P e + + per ml. 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 Fe + +/ml. { mg. bacterial N/ml. 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 -17-e x i s t e d . The i r o n requirements of P. aeruginosa 9027 on glucose ammonium succinate medium are s i g n i f i c a n t l y higher than those of P. aeruginosa 2F5 as employed by Waring and Werkman (1943). Whereas maximum growth of our organism was obtained w i t h 0.5,ugm F e + + per ml., maximum response of P. aeruginosa 2F3 r e q u i r e d only 0.09 Aigm Fe per ml. The v a r i a t i o n i n the i r o n requirements of these two s t r a i n s may be a r e s u l t of the d i f f e r e n t n i t r o g e n sources employed i n the two growth media. Inorganic ammonium s u l f a t e , which was used by Waring and Werkman, could not d i r e c t the pathway of glucose breakdown. Since c e l l s of P. aeruginosa appear t o co n t a i n enzymes necessary f o r the aerobic d i s s i m i l a t i o n of su c c i n a t e , the use of ammonium succinate as the n i t r o g e n source would provide as w e l l an a l t e r n a t i v e carbon source. The development of a more com-p l e t e s u c c i n i c a c i d mechanism could account f o r the greater i r o n requirements of our organism. Iro n d e f i c i e n t c e l l s of A. indologenes have been r e p o r t e d by Waring and Werkman (1944) t o c o n t a i n depleted 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 to o x i d i z e l a c t a t e , pyruvate and ac e t a t e . To determine the e f f e c t of I r o n d e f i c i e n c y on the enzymic c o n s t i t u t i o n of c e l l s , a comparison was made of the ra t e s of oxygen uptake by d e f i c i e n t and normal r e s t i n g c e l l suspensions. Cultures were grown i n the b a s a l medium at three l e v e l s of 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 per ml. - r e p r e s e n t i n g low, optimum and h i g h i r o n environments. C e l l s produced on the low I r o n medium were resuspended to a t u r b i d i t y approximately equal t o t h a t of the lOx con c e n t r a t i o n of optimum i r o n organisms. The contents of the Warburg f l a s k s and the micro l i t r e s of oxygen absorbed by the o x i d a t i o n of the substrates i n a 45 minute p e r i o d are recorded i n Table I I . Prom t h i s experiment I t appears that the o x i d a t i o n of pyr u v i c a c i d i s decreased by growth of the organism i n the-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 only one-h a l f as much oxygen as h i g h i r o n c e l l s when pyruvate i s sub-s t r a t e . A corresponding'loss i n a c t i v i t y I s not observed when glucose, l a c t a t e or acetate i s the substrate being o x i d i z e d . The decreased o x i d a t i o n of p y r u v i c a c i d by low I r o n organisms i s not l i k e l y t o be the r e s u l t of depleted enzyme systems w i t h i n these c e l l s . I n the presence of an e a s i l y a t t a c k a b l e substrate i . e . glucose r e s p i r a t i o n of low and hi g h i r o n suspensions i s of the same order. Since the break-down of glucose almost c e r t a i n l y Involves the o x i d a t i o n of pyr u v i c a c i d , i t can be concluded t h a t low i r o n c e l l s con-t a i n the e s s e n t i a l enzymes and coenzymes f o r the o x i d a t i o n of the k e t o - a c i d . The mechanisms r e s p o n s i b l e , f o r the o x i d a t i o n of pyruvic a c i d by b a c t e r i a are known to be extremely s e n s i t i v e . Table II The Influence of Iron i n the Growth Medium on the Oxidative Ab i l i t y of Resting  Cells of Fseudamonas aeruginosa In flask: 'A B 2 0 hr. cells - 2 0 x 0 . 5 ml 0 7 5 ml M/15 pH 7.4 phosphate buffer 1 . 5 ml • M/15 pH 6 . 0 phosphate buffer - 1 . 5 ml Water to 3 .0 ml to 3 .0 ml In side-arm: substrate substrate In centre well: 20% K 0 H .15 ml .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 .. : 70 64 Na pyruvate (2 JUM) (B) : 25 ! • 6 2 . 53 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 * endogenous respiration subtracted -20-By use of'cell-free extracts of E. c o l l . Kalnitsky and ¥/erkman (1943) were able to show that pyruvic acid neutral-ized directly was less active than that which had been prepared by diluting prior to neutralization. Changes in c e l l permeability may account for the de-creased activity of these iron deficient organisms on pyruvate. Using acetone preparations of M. lysodeikticus. Krampitz and Werkman (1941) were able to demonstrate the formation of pyruvic acid and carbon dioxide from oxalacetate. Whole cells were not permeable to oxalacetate in the absence of oxygen. Lichstein and Umbreit (1947) have shown that oxalacetate is decarboxylated by E. c o l i as phosphorylated oxalacetic acid and i t i s probable that pyruvic acid also i s u t i l i z e d by the cells i n the form of a phosphorylated deriva-tive. The growth of the organism i n low iron solutions might reduce the permeability of the c e l l membranes to pyruvic acid. If such i s the case, the low oxygen uptake observed on pyruvate i s not incompatible with the higher oxygen con-sumption obtained using glucose. The growth response of the organism to iron i s of considerable significance i n view of the functions of iron In biological 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 is passed out to oxygen to yield water. Since anaerobic systems of obtaining' energy, do not function by way of tcyi;ochrome, anaerobic bacteria do not require iron for -21-respiration. Both aerobic and anaerobic bacteria contain the hydrogen carrier flavoprotein. If, as i n aerobic respiration, the flavoprotein passes hydrogen to atmospheric oxygen, hydrogen peroxide i s formed. The destruction of this toxic product is accomplished by the iron containing enzyme catalase. Since aerobic organisms must obtain their energy by means of cytochrome or flavoprotein, i t i s apparent that they require iron for respiration. 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 directly proportional to the Iron content of the medium, i t can be concluded that this organism has no anaerobic means of dissimilating carbohydrates. Moreover i t has been shown that growing the organism i n the presence of inadequate amounts of iron does not foster the elaboration of an enzyme system which can function without the aid of iron. It would appear therefore that there i s no possibility that this organism can grow anaerobically. Iron deficiency results i n the formation of fewer cel l s , a l l of which contain the normal complement of aerobic iron enzymes. SUMMARY By adding increasing amounts of FeSO^RgO to a purified medium, a growth response has been obtained over a range of -22-from 0 to 0.5;ugm F e + + per ml. Resting c e l l suspensions of iron deficient cells showed a decreased a b i l i t y to oxidize pyruvic acid. Enzymes for the oxidation of glucose, lactate and acetate were not influenced by the concentration of iron in 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 ' ' R e s t i n g ' 8 c e l l s may be prepared by washing b a c t e r i a f r e e 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,=. Quas 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 as 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 , yet 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 subs tances . C e l l s 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 metabol 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 growth 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 the 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 . C e l l s harves ted from 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 the enzymes t o be s t u d i e d , were prepared 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 c o n c e n t r a t i n g t h e s u s p e n s i o n . The washed organisms .were t h e n aera ted i n o r d e r t o d e s t r o y o x i d l z a b l e s torage p r o d u c t s 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 ince 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 important , 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 r e s p i r a t o r y 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 . By 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 -24-Gunsalus (1941 ) have o b t a i n e d good crops 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 workers have a l s o p o i n t e d out t h e importance o f h a r v e s t i n g t h e c e l l s d u r i n g the h e i g h t o f 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 . By so d o i n g , t h e n i g h s t a t e o f r e a c t i v i t y developed d u r i n g t h e 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 these b a c t e r i a mer i ted 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 s u b s t r a t es by r e s t i n g organisms . By a e r a t i n g c e l l suspensions i n . d i l u t e s o l u t i o n s o f the r e a g e n t , i t should 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 torage p r o d u c t s , and thereby t o o b t a i n c e l l s h a v i n g a low 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 exper iment ing w i t h t h e method o f c e l l prepara t i o n , a c t i v e r e s t i n g suspensions o f t h e organism have been o b t a i n e d which 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 B a c t e r l o l o g i c a l ; Stock c u l t u r e s o f P . aerug inosa (ATC 90S7) were c a r r i e d i n the manner d e s c r i b e d i n P a r t I . A f t e r s i x 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 t h e p igmenta t ions c h a r a c t e r i s t i c o f the s t r a i n . The c o m p o s i t i o n o f t h e g lucose ammonium s u c c i n a t e medium used f o r c a r r y i n g the organism 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 ;agm per m l . To prepare ammonium s u c c i n a t e , 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 organism 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$ MgS04« THgO, 0.1>ugm. i r o n per m l . (as FeS0 4*7HgO) and 0 .1 cc s a l t s o l u t i o n per 500 m l . was used i n the p r o d u c t i o n o f r e s t i n g c e l l s . To t h i s b a s a l medium a d d i t i o n s o f g lucose and n i t r o g e n source 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 d i n i t r o p h e n o l . Dur ing t h e experiment on the e f f e c t o f low MgS0 4 *7H s 0 and low K s H P 0 4 on t h e a c t i v i t y o f c e l l s , 0 .3$ ammonium s u c c i n a t e and 0.5$ g lucose were employed, and s u i t a b l e adjustments were made i n t h e c o m p o s i t i o n o f t h e b a s a l medium. Werkman 1s medium f o r t h e p r o d u c t i o n o f gum-free c e l l s o f ]S. c o l i was as f o l l o w s : 0 .4$ each o f beef e x t r a c t and • 2£ - 2& -peptone, 0.2$ each of yeast e x t r a c t and NaCl, and 10$ t a p water. C e l l s were harvested from the t e s t media a f t e r :20-£2 hrs . at 30°C. They were washed twice and resuspended i n s a l i n e i n twenty times t he growth c o n c e n t r a t i o n . Unless otherwise s t a t e d , a e r a t i o n of the c e l l suspensions was accomplished i n the f o l l o w i n g manner. C e l l s washed once i n lOx concentration 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 tubes, and a i r was drawn through them by means of a s u c t i o n pump f o r a pe r i o d of '2 h r s . Throughout the procedure, no precautions were taken t o observe a s e p t i c technique. At the end of the a e r a t i o n p e r i o d the c e l l s were thrown down and washed once i n s a l i n e . They were prepared f o r use by resuspension i n one-twentieth the 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 i n h i b i t s y n t h e t i c r e a c t i o n s and t o increase o x i d a t i o n i n suspensions of E. c o l i . By i n c u b a t i n g the c e l l s w i t h 2,4 d i n i t r o p h e n o l during the a e r a t i o n p e r i o d , I t was hoped th a t more complete d e s t r u c t i o n of o x i d i z a b l e storage products would be obtained thereby reducing the endogenous a c t i v i t y o f the c e l l suspensions. The washed organisms were r e -suspended i n lOx concentration i n M/2000 n e u t r a l i z e d 2,4 d i n i t r o p h e n o l s o l u t i o n . A f t e r 1 h r . a e r a t i o n at 30°C, they were c e n t r i f u g e d down and washed thoroughly I n h a l f the growth volume of s a l i n e . A 20x c e l l suspension was prepared f o r use i n Warburg cups. To ensure the p u r i t y of the suspension, a l l aerated c e l l preparations were Gram s t a i n e d p r i o r t o t h e i r use i n experimental work. The a c t i v i t y o f the v a r i o u s c e l l suspensions was determined by measuring the a b i l i t y o f each t o dehydrogenate glucose i n the presence o f methylene blue. The standard Thunberg method as o u t l i n e d by "©mbreit^i (1945) was employed. Measurements were made i n vacuo. I n the tube'were placed ; 2 ml;;. M/15 phosphate b u f f e r , pH 7.0, 2 ml. M/20 glucose and 1 ml... 1/10,000 methylene blue, and i n the side arm bulb 1 ml. of 20x c e l l suspension. A standard was in c l u d e d containing a l l of the components of the above system ( c e l l s i n a c t i v a t e d by b o i l i n g 20 minutes) but w i t h the methylene blue at one-t e n t h the normal concentration. T h i s tube represented 90$ red u c t i o n of the methylene blue and was used as the end point of r e d u c t i o n . Thunberg tubes were evacuated w i t h a strong s u c t i o n pump f o r three minutes before they 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 at 30°C, substrate was t i p p e d i n and the end point of the r e a c t i o n was recorded as the time r e q u i r e d f o r the c o l o r i n t e n s i t y of the experimental tubes to be reduced t o t h a t of the b o i l e d c e l l standard. At the end of the re d u c t i o n p e r i o d the s e a l s were checked t o confirm the presence of a vacuum throughout the experiment. A conventional Warburg apparatus was used t o f o l l o w the oxygen uptake of the c e l l suspensions. Procedure and c a l c u l a -t i o n s were according t o Dixon (1943). 0.5 ml. of a 20x c e l l 2% ~ 28 -suspension was employed In a final volume of 3.0 ml. Tests were run for a period of 1 nr. at 30UC. RESTING CELL PREPARATION Nitrogen and Carbon: P r e l i m i n a r y experiments have i n d i c a t e d that the n i t r o g e n source ammonium succinate with glucose as carbon source gave the most abundant y i e l d o f pigmenting c e l l s . During the e a r l y growth phase i n these media, and coincident w i t h the formation of the fluooescent pigment, a gummy m a t e r i a l was elaborated by the c e l l s . The s u b s t i t u t i o n of ammonium c h l o r i d e f o r ammonium succinate, 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 reducing gum formation, prevented the appear-ance, of the y e l l o w pigment. G l y c i n e gave poorer y i e l d s of non-pigmenting gum-free c e l l s , but these could be harvested only a f t e r 48 h r s . growth. Phenylalanine has p r e v i o u s l y been found t o enhance slime formation (White, t h e s i s ) . F a i r l y heavy y i e l d s of non-pigmenting c e l l s of average gumminess were obtained u s i n g i n o r g a n i c ammonium s u l f a t e . The methylene blue r e d u c t i o n times f o r media c o n t a i n i n g the n i t r o g e n sources ammonium succinate, ammonium s u l f a t e and g l y c i n e , combined with two concentrations of glucose are given i n t a b l e I . Owing t o the slow growth of the c e l l s w i t h g l y c i n e , the reduction v a l u e s f o r t h i s medium were obtained using 48 hr . c e l l p r e p a r a t i o n s . _ '30 -TABLE I The E f f e c t o f Nitrogen Source and  Glucose Concentration on the Dehydrogenase  A c t i v i t y o£ the C e l l s n i t r o g e n source : n i t r o g e n : source : % : glucose < ; % " . methylene blue r e d u c t i o n '. glucose : endogenous (mins.) : (mins.) . _ « — NH 4 succinate : o . i : o . i : 0 . 3 ! 0 . 5 : I.'O ; 0 . 5 : 1 . 5 : 2 . 0 ', : 2.o ! 1.5 • ! 8.5 20 ( N H 4 ) g S 0 4 ti « I 0.087 1 ; 0.087 ; o.26 s : 0.26 : 0 . 5 ; . i . o : 0.5 i . o : I O . O : 1 5 . 0 : i 8 . o : 1 1 . 5 : 17.5 17.5 25.0 15.5 g l y c i n e ] : o . 3 : 0.6 ; 0.5 : 0 . 5 : 3.0 ! 9 . 0 ; 4.0 12.5 amount of N equivalent t o 0.1$ NR. succinate amount o f N equivalent t o 0.3$ NIT succinate From the above data i t i s apparent that g l y c i n e grown organisms are 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 * r e q u i r e approximately three-quarters o f the endogenous re d u c t i o n time t o reduce methylene blue i n the presence o f glucose. Moreover the r e l a t i v e l y long p e r i o d r e q u i r e d f o r t h e i r production makes them i m p r a c t i c a l f o r use i n r e s p i r a t o r y s t u d i e s . Ammonium s u l f a t e c e l l s can ,be obtained i n good y i e l d i n S0-2S h r s . , but t h e dehydrogenase a c t i v i t y o f such p r e p a r a -t i o n s i s l o w . 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 o f s u b s t r a t e . 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 e n t i r e l y s u i t a b l e . Organisms h a r v e s t e d f rom 0.3$ ammonium s u c c i n a t e and 0.5$ g lucose reduced methylene b l u e i n the presence o f g lucose i n l e s s t h a n one- tenth t h e t i m e r e q u i r e d by endogen-ous 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 the 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 suspensions was c o n s i s t e n t l y 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 . T h i s c o n c e n t r a t i o n o f n i t r o g e n source was t h e r e f o r e s e l e c t e d f o r use i n t h e r e s t i n g c e l l medium. Greater amounts have been found 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 c e l l s . From t h e d a t a o b t a i n e d i t can .be seen t h a t t h e c o n -c e n t r a t i o n o f g lucose i n the growth medium has but l i t t l e e f f e c t on the a c t i v i t y o f t h e c e l l s produced. I n t h e presence o f 0 .5$ g l u c o s e , t h e s u b s t r a t e r e d u c t i o n t i m e i s 0 .57 and 0.72 t i m e s t h a t o f t h e endogenous f o r 0 . 1 $ and 0.3$ 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 presence o f 1 . 0 $ g l u c o s e , 0.36 and 0 .74 t i m e s r e s p e c t i v e l y . S i n c e t h e 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 s i g n i f i c a n c e , 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 c e l l s . M i n e r a l s I n h e r study of t h e m i n e r a l metabol ism o f t h e f l u o r e s c e n t pigment , K i n g ( t h e s i s ) has observed t h a t h i g h e r c o n c e n t r a t i o n s o f s u l f a t e ( g r e a t e r t h a n . 0 . 5 $ ) c o n t r i b u t e d t o the f o r m a t i o n of, a s l imey m e t a b o l i c product 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 . P r e v i o u s l y 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 processes o f t h e c e l l i n f a v o r o f t h e s y n t h e t i c r e a c t i o n (Lipmann, 1942) , The p o s s i b i l i t y , t h a t e i t h e r o r b o t h o f these f a c t o r s might c o n t r i b u t e t o the , 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 s tudy 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 C e l l . W > 4 Methylene b lue r e d u c t i o n £ime g l u c o s e endogenous m l n s . mins . 0 .1 0.-2 5 .0 1 0 . 0 0 .1 0.01 11 .5 2 4 . 0 00.01 3 h r . 3 h r . 0 .01 0 ,01 3 h r . 3 h r . T a b l e 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 the growth medium does not 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 , and 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 t h e 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 the 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 s u b r s t r a t e r e d u c t i o n t imes i t appears t h a t by decreas ing t h e phosphate f rom 0.1$ t o , 0 . 0 1 $ , t h e a c t i v i t y o f the 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 t h r e e 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 presence o f s u b s t r a t e . Furthermore , t h e c o n c e n t r a t i o n o f phosphate i n the 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 . I n v i e w o f these o b s e r v a t i o n s , t h e o r i g i n a l c o n c e n t r a -t i o n s o f 0 .2$ MgS04*7HgO and 0.1$ KgHP0 4 were employed 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 organisms . 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 expec ted , 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 i t . W i t h decreas ing c o n c e n t r a t i o n s o f i r o n , lower y i e l d s o f 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 lucose remained e s s e n t i a l l y unchanged. 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 b l u e r e d u c t i o n t i m e F e + + per m l . >ugms. g lucose (mins . ) endogenous (mins . ) 0 .01 ! : 3 0 .05 s [ 1 .5 : 0 . 1 : : 1 .5 , i 2 . 5 Age; 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 , i t i s important t h a t t h e organisms should be h a r v e s t e d i n an a c t i v e s t a t e before t h e onset o f v t h e s t a t i o n a r y growth phase as d e s c r i b e d by Buchanan (1918) . Much da ta has been presented t o i n d i c a t e t h a t 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 c y c l e 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 from 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 growth o f t h e c e l l s , i t was necessary t o determine t h e t i m e at w h i c h maximum a c t i v i t y o f t h e 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 b r o t h . 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 c e l l suspensions at SO h r s . , 48 h r s . and 72 h r s . C e l l s 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 .5$ ammonium s u c c i n a t e and 0 .5$ g l u c o s e . ;35> „ TABLE IV I M PATec,t g£ Age, p j C ju l tnr^ on. t h e Dehy^rogenas.e A g ^ ^ v i t y Methylene b l u e r e d u c t i o n age g lucose endogenous h r s . (mins.) (mins . ) 20 i ! 7.5 i t 7 .5 48 18 .0 i i 24 .0 72 i - 51.0 J f i : § reduced i n 1 h r . 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 . , t h e dehydrogenase a c t i v i t y o f the c e l l s d e c l i n e d i n t h e presence o r absence o f s u b s t r a t e . 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 . decreased 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 o f t h e i r o r i g i n a l a c t i v i t y . 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 s torage p r o d u c t s d u r i n g the l a t t e r p a r t o f t h e i r growth phase, t h e corresponding decrease i n t h e a c t i v i t y o f t h e organisms i n the presence o f g lucose 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 r a p i d l y a f t e r a p e r i o d o f maximum a c t i v i t y . The abnormal ly l o n g r e d u c t i o n t i m e observed f o r 20 h r . c e l l s on glucose 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 the p r e p a r a t i o n o f r e s t i n g c e l l suspensions were h a r v e s t e d a f t e r 20-24 h r s . growth at 30°C, A e r a t i o n ? An attempt was made t o reduce t h e endogenous s torage products 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 which was added 0 .3$ ammonium s u c c i n a t e and 0.5$ g l u c o s e . S ince 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 the c e l l s d u r i n g the procedure 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 ••• suspensions were a e r a t e d . TABLE V TJl§ l £ £ S S kM 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 a e r a t i o n ( h r s . ) endogenous (mins . ) 0 2 9 1 2 4 S..5 6 3 - 37l. „ TABLE V I T j & Ef^eqfr QL Concentrat ion , og C e l l s D u r i n g A e r a t i o n C o n c e n t r a t i o n Methylene b l u e r e d u c t i o n o f c e l l s * A e r a t i o n ( h r s . ) d u r i n g a e r a t i o n g lucose ( m i n s . ) endogenous ( m i n s . ) 0 ' i — • IB i i 9 i growth v o l , t • P 1 .5 i m i » I i 5x 8 • # 1 .5 ! t £ S i > ^BOx s > * > * * : 3 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 these c e l l s do not appear t o be u t i l i z e d d u r i n g a e r a t i o n . A s a r e s u l t o f t h i s p r o c e d u r e , t h e endogenous dehydrogenase a c t i v i t y was Increased 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 original 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 cells at 20-24 hours an.d employing them directly without aeration. To confirm the suitability of these cells for respiratory studies, the oxygen consumed in the oxidation of one and' five micromoles of glucose was measured. As the results in 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 total oxygen uptake in the presence of 1 micromole of glucose, i t was less than one f i f t h of the total i f 5 micromoles of glucose were employed. To confirm the previous observation that washed c e l l suspensions had a lower endogenous activity before than after aeration, the oxygen consumption of aerated and non-aerated organisms was compared. Data obtained on Werkman1s medium and on ammonium succinate glucose medium with and without 2-4 dinitrophenol are included in the following table. The results on aeration are in 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 c e l l preparations. Aeration in the presence of 2,4 di-nitrophenol decreased the total oxygen consumption of the organisms and -39-Table VII The Oxidation of Glucose by  Resting Cells In flask: 0.5 ml. 20x cells 1.5 ml. M/15 phosphate buffer pH 7.4 Water 3.0 ml. In side-arm: 0, luM or 5 juM glucose In Centre veil: 0.15 ml. 26$ KOH substrate ; >ul 0£ uptake j i n 1 hr. endogenous 93.5 lyuM glucose ; 197.0 5 juM glucose \ 522.0 increased the iratio of endogenous to substrate respiration. A l l attempts to rid the c e l l s of their stored products seemed to be of no avail. Since the procedures used are recognized workable methods, i t would appear that we are dealing with an endogenous respiration which is different from that exhibited by organisms such as E. c o l i . -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% NH4 succ. .2^ glucose, • 3J£ NH^ succ. ,b% glucose, •3/£ NH^ succ. • 5/£ glucose, .2% NH4 BUCC. »5j£. glucose, .3^NH4 succ. - 2-4 DNP Werkman's Workman's aerated 4 hrs. aerated 1 hr. with M/2000 2-4 DNP aerated 4 hrs. 93.5 61.3 130.0 127.5 57.5 88.2 64.5 197.0 144.5 191.5 185.5 123.0 147.0 152.0 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 their endogenous respiration or the ratio of endogenous to substrate methylene blue reduction times. Incubation of resting cells for 1 hour with M/2000 2,4 -di nitrophenol failed to decrease the oxidizable storage products. Although aeration of the :eells harvested from Werkman's medium reduced oxygen consumption in t he absence of glucose, this treatment also decreased the oxidative activity of the cells when glucose was present. -42-PART I I I 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 carbohydrate d i s s i m i l a t i o n i n b a c t e r i a . At t h e present t ime i t i s assumed t h a t 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 a c i d s t a g e . 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 o x i d -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 a r e 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 occur i n some o b l i g a t e aerobes such as molds , y e a s t s , pseudo-monas and r e l a t e d organisms (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 1941) , i s t h e 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 k e t o g l u t a r i c a c i d s . I t has g e n e r a l l y been assumed t h a t beyond t h i s p o i n t the 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 Krebs c y c l e , 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 a c i d s . 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 lucose u t i l i s e d by £ . savan-s t a n o l i . 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 these f e r m e n t a t i o n s , ( 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 of the o x i d i z e d a c i d s , from fermentations of glucose by B. pyocyaneus. When fr 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 obtained i n a d d i t i o n to the products p r e v i o u s l y mentioned.) Per-v o z a n s k i i (1940) has reported h i g h y i e l d s of 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 of intense a e r a t i o n , pressure, a g i t a t i o n and h i g h concentrations of substrate 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 of the normal aerobic metabolism thereby a l l o w i n g l a r g e q u a n t i t i e s of o x i d i z e d a c i d s to accumulate i n the fermentations. Thus Stubbs (1940) has obtained 90$ y i e l d s of 5-ketogluconic a c i d I n 33 h r s . w i t h A. suboxvdons and 82$ y i e l d s of 2-ketogluconic a c i d i n 25 h r s . w i t h an unnamed bacterium. Lockwood, Tabenkin and Ward (1941) have compared the a b i l i t y of a v a r i e t y of species of phytomonas and pseudomonas to produce g l u c o n i c and 2-ketogluconic a c i d s . While amounts ranging from 58$ to 96$ g l u c o n i c a c i d and g r e a t e r than 70$ 2-ketogluconic a c i d were found i n fermentations conducted under commercial c o n d i t i o n s , c u l t u r e s i n which the normal p h y s i o l o g i c a l c o n d i t i o n s were maintained produced only t r a c e s of these compounds, A f u r t h e r step i n the breakdown of glucose was i n d i c a t e d by Lockwood and Stodola (1946). These workers have shown that i f 2-ketogluconic a c i d fermentations by P. f l u o r e s c e n s were allowed to continue u n t i l no reducing power remained i n the medium, 16-17$ y i e l d s of k e t o g l u t a r i c a c i d were obtained. Since the c o n d i t i o n s to which the organism i s subjected during commercial fermentation 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 impossible to conclude from t h i s data alone that the a c i d s produced are normal intermediates i n glucose d i s s i m i l a t i o n . However they 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 study the metabolism of 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 very general manner using postulated intermediates of both aerobic and anaerobic systems. In t h i s e a r l y phase of the work, glucose ammonium succinate c e l l s were employed. As i t l a t e r became apparent that the o x i d a t i o n of carbon compounds by t h i s organism was c o n t r o l l e d by adaptive enzymes, c e l l s grown on the double substrate were abandoned, and organisms harvested from a purely mineral medium plus substrate were employed. By the use of these c e l l s , we have'been able to show that the breakdown of glucose proceeds by way of gluconic and 2-keto--gluconic a c i d s . Beyond these intermediates the pathway of carbohydrate metabolism i s s t i l l obscure. -45-METHODS B a c t e r l o l o g l e a li The c u l t u r e u s e d , £ . a e r u g i n o s a ( AT C 9027) , 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 t h e s i s . 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 storage o f t h e organism, 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 lucose 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 organism and 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 b a c t e r i a . , Compos i t ion 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 b e e n -d i s c u s s e d i n t h e p r e v i o u s s e c t i o n . F o r s t u d i e s employing ammonium phosphate c e l l s , the 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$ MgS04»7HgO, and K ^ H 1 0 4 was r e p l a c e d by 0.1$ K C 1 . A l l growth s u b s t r a t e s were used at 0 .5$ c o n c e n t r a t i o n i . 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 drops 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 t h e r e s t i n g b a c t e r i a . A c t i v e c e l l s were h a r v e s t e d at 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 which c o u l d be s a t i s f a c t o r i l y employed i n r e s p i r a t o r y s t u d i e s . D i r e c t l y b e f o r e t h e i r u s e , a concen-t r a t e d suspension o f t h e washed organisms was p r e p a r e d , 0 . 5 m l . o f which was added t o each Warburg cup . I n order t o -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 throughout t h i s s e r i e s o f exper iments , no 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 m e t a b o l i c s t u d i e s . To 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, the Warburg technique was employed (Dixon 1943j Umbreit 1945)... Phosphate b u f f e r , (M/15) , water and c e l l s were p l a c e d i n t h e main com-partment o f the 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 absorbed by 20% KOH p l a c e d i n t h e c e n t r e w e l l * Each 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 which were run i n c o n j u n c t i o n w i t h t h e s u b s t r a t e c o n t a i n i n g 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 -carbonate s o l u t i o n was used 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 produced . 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 7.05 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), water 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 t h e r e s p i r a t o r y v e s s e l . I n one s idearm 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 be ing so a d j u s t e d that -47< a p p r o x i m a t e l y one l i t r e o f gas passed through 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 ensure s u f f i c i e n t t i m e f o r t h e l a s t t r a c e s o f oxygen t o be absorbed 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 . At 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 . S u i t a b l e c o n t r o l s were always i n c l u d e d t o de termine- the 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 . To f o l l o w t h e phosphorus uptake o f t h e c e l l s , i t was con-v e n i e n t t o Incubate t h e suspensions i n Warburg cups a t t a c h e d t o t h e shaking a p p a r a t u s . T h i s procedure 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 ensured the cont inuous contact o f phosphate w i t h t h e c e l l s . A s e r i e s o f 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 t h a t used i n t h e f e r m e n t a t i o n s tudy , except t h a t H C l was e n t i r e l y o m i t t e d . A s tandard amount o f phosphate s o l u t i o n , 0 .5 m l . - 1 .14 mg. KHgP0 4 per m l . * ISOjugm. P per cup, was used i n each f l a s k . Anaerob ic 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 the 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 . At zero t ime and a t 10 minute i n t e r v a l s t h e r e a f t e r , one endogenous and one s u b s t r a t e f l a s k were removed f rom t h e b a t h , 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 immediate ly 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 concentra ted s u l f u r i c a c i d . Having ensured a thorough m i x t u r e o f t h e a c i d -48-with the contents of the flask, the bulk of the sample was transferred to a centrifuge tube. The cells were then separated from the suspending medium, and duplicate.0.5 ml. aliquots of the supernatent were analysed for phosphorus. The a b i l i t y of the cells to u t i l i z e glucose and fructose anaerobically was determined by allowing the cells to respire In a nitrogen atmosphere i n the presence of 10 jaM of the substrate. Pour cups were prepared for each substrate, duplicates to be removed at zero time and after 1 hr. respiration. At the end of that time, the flasks were acidified as previously described, the cells 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 for 2-keto-gluconic acid were made using the polarimetric method of Stubbs et al (1940). Intermediate compounds: Ca gluconate was obtained from Dr. H.L.A. Tarr. S amples of Ca 2 and 5-ketogluconate were kindly supplied by Dr. C.E. Georgi and Dr. L.B. Lockwood. Oxalacetic acid 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, lactate and malonate were made up by neutralizing equivalent amounts of the free acids with 10 N NaOH. The remainder of the intermediate compounds were -49-sstandard 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. As a result of growth experiments, many workers (Tanner ; Bergey 1948) have considered that these bacteria are facultatively aerobic. This observation has been confirmed by isolating lactic acid, -50-a normal end-product of glycolysis, in 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 insufficient evidence for 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 for their activity. As a result 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 respira-tory studies to determine the relationship of these organisms to oxygen. Anaerobic respiration may be evaluated manometrically by measuring the p roduction of acid and/or gas by cells metabolizing in a nitrogen atmosphere. Under these conditions, the organism must obtain energy by means of the Meyerhof-Embden system, the end products of which are lac t i c and acetic acids, carbon dioxide and ethyl alcohol. While the production of alcohol is usually associated with the forma-tion of volatile acids, i t is necessarily accompanied by the liberation of carbon dioxide. Therefore, irrespective of the nature of the fermentation, the end effect of anaerobic respiration i s the production of acid and gas. -51-Table I The Anaerobic Fermentation of Glucose  by P. aeruginosa In cup: cells 10x concentration 0.1 M NaHC03 water endogenous substrate 0.5 ml. 0.5 ml. In side-arms: (1) glucose 25juM/ml. (2) 3N HCl In centre well: Yellow phosphorus Atmosphere: 5$ COg : 95$ N 2 0.3 ml. to 3.0 ml. 0.3 ml. 0.3 ml. to 3.0 ml. 0.2 ml. 0.3 ml. ! C02 produced : : during res-: piration : j u . l . J GO2 liberated : by HCl -u.l. endogenous i (HCl at end) : « — j 82.0 glucose ! (HCl at zero time) i mm : 70.0 . glucose i (HCl at end) : - i 70.2 Wo indication of metabolic activity was obtained when the cells were incubated with glucose under a nitrogen atmosphere. These results have been confirmed by the i n -abil i t y of the cells 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 for fermentation.) Table II The Uptake of Inorganic phosphorus by  Resting Cells In cup: endogenous substrate cells 10x concentration 0.1 M NaH CO3 water In side-arm: 11) glucose 25;uM/ml. (2) KH0PO4 0.114$ (.5 ml. =130 ;ugm P) In centre well: yellow phosphorus Atmosphere: 5$ COg : 95$ Ng 0,5 ml. 0.5 ml. 0.6 ml. 0.6 ml. to 3.0 ml. to 3.0 ml. 0.5 ml. 0.2 ml. 0.5 ml. Time (mins,) : Amount of endogenous Cugm.) P per ml. : substrate t Ougm.) 0 76.2 : 72.0 10 : : 78.4 20 : 72.6 SO : 73.0 : 74.6 40 : - : 76.2 50 : 73.0 60 76.2 : 72.0 Table III The- Anaerobic Utilization of Glucose and Fructose by Resting Cells In cup: cells lOx concentration 0.5 ml. M/15 : phosphate buffer, pH 7.2 1.5 ml. water to 3,0 ml. In side-arm: substrate 25-uM/ml. 0.4 ml. In centre well: Atmosphere: nitrogen. : Amount of substrate per cup Time : glucose • fructose (mins.) | (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 relatiig this data to that obtained on iron, we have concluded that the organism is obligately aerobic. - 5* -PART I I I B. 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 t i s necessary t o determine the degree t o which t h e 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 uptake d u r i n g s u b s t r a t e o x i d a t i o n . S i n c e t h e I n f l u e n c e o f 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 e s t a b l i s h e d , i t 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 uptake I s unchanged i n t h e presence o f o x i d i z a b l e s u b s t r a t e , o r t h a t i t i s n e g l i g i b l e . 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 the 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 e x p e r i m e n t a l b a s i s f o r t h e c h o i c e . Much o f the c o n f u s i o n a r i s i n g from 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 S t a n i e r (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 i s 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 the t o t a l oxygen uptake o f c e l l s r e s p i r i n g i n t h e presence o f d i f f e r e n t c o n -c e n t r a t i o n s o f t h a t s u b s t r a t e . T h u s , t h e d i f f e r e n c e 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 the -55-<0 i t ! 600\ 400 zoo -> t > > ^ Tn</oyet?b({s 20 40 MINUTES 60 20 Figure l i D i f f e r e n t i a l method of determining oxygen uptake f o r La*of glucose. I n cups: glucose, V 3 0 phosphate b u f f e r , pH 7.4, 0 . 5 ml. 2 0 x c e l l s . 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 uptake may be d i s r e g a r d e d 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 da ta f o r t h i s organism ( f i g u r e 1 ) , Whereas f i v e micromoles o f g lucose absorbed 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 r e s p e c t i v e l y , i . e . 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 the d i f f e r e n c e i n oxygen uptake 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 lucose (110 m i c r o -l i t r e s o f oxygen per micromole o f g l u c o s e ) . Dur ing t h e same r e s p i r a t o r y p e r i o d endogenous suspensions 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 oxygen. I f t h i s amount were s u b t r a c t e d from t h e t o t a l uptake o f the c e l l s i n each o f the s u b s t r a t e 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 lucose would r e q u i r e 88 , 79 or 74 m i c r o l i t r e s o f 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 employ-e d . S ince t h e o x i d a t i o n o f one micromole o f g lucose should Require the 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 the same c o n d i t i o n s , i t was t h e r e -f o r e concluded t h a t the endogenous r e s p i r a t i o n was a n e g l i g -i b l e f a c t o r d u r i n g s u b s t r a t e o x i d a t i o n . H e n c e f o r t h no a l lowance 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 r e s p i r a t o r y d a t a . O x i d a t i o n o f t h e p o s t u l a t e d Intermediate .compounds; The use 31 51 -57-o f manometric t echniques i s w e l l s u i t e d t o a s tudy o f t h e i n t e r m e d i a t e metabolism o f g l u c o s e . By o b s e r v i n g the o x i d a t i v e a c t i v i t y o f c e l l s r e s p i r i n g i n t h e presence o f the p o s t u l a t e d i n t e r m e d i a t e s , one can determine whether or not these 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 o f the sugar . Two important 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 -p r e t i n g r e s p i r a t o r y d a t a . S i n c e no m e t a b o l i c system can-f u n c t i o n more r a p i d l y than t h e slowest o f i t s i n t e r m e d i a t e s , 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 at l e a s t as r a p i d l y as t h e i n i t i a l s u b s t r a t e . Secondly , t h e 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 the same as t h a t o f t h e parent compound. For 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 of^ sodium a c e t a t e o r 2 . 5 micromoles o f c a l c i u m gluconate c o n -sume 540, 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 t i s p o s s i b l e t h a t g l u c o n i c a c i d i s i n t e r m e d i a t e i n t h e breakdown o f g lucose (both t h e sugar and i t s a c i d are 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 approaching t h e o r e t i c a l . ) B e -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 lucose and a c e t a t e i s not t h e same, i t i s apparent t h a t t h e two compounds are be ing d i s s i m i l a t e d by means of d i f f e r e n t r e s p i r a t o r y mechanisms. Zo 40 60 Mf/VUTES Figure 2 : Oxygen uptake of glucose ammonium succinate cells. I 5*Mglucose; II 2.5>MCa gluconate; III 2,5^* Ca-2-ketogluconate; IV 2 . 5AM Ca 5-ketogluconate; V 12/U.m Na oxalacetate; VI I^MM Na malate; VII 7>« Ka succinate; VIII IACH Na fumarate; IX 5^- A 1 Na citrate; X endogenous. In cups: substrate, M/30 phosphate buffer, pH 1 ,h> 0 . 5 ml. 2 0 x c e l l s . -59-£0 4-0 M/A/UTtzJ GO Figure 3s Oxygen uptake of glucose ammonium succinate cells. In cups: substrate (5>*M glucose, 15^«Na acetate, 10/<M Na pyruvate, Na lactate, Na glycerate, glycerol) M/30 phosphate buffer pH 7 . 4 (pH 6 . 0 pyruvate), 0 . 5 ml. 20x cells. -60-F i g u r e s S and 3 show t h e r a t e s and amounts o f oxygen uptake of c e l l s r e s p i r i n g i n the presence o f v a r i o u s i n t e r -mediate compounds. I n 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 , s u c c i n -a t e , malate , furoarate and p y r u v a t e are 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 a e r o b i c breakdown o f g l u c o s e . S i n c e a c e t a t e , l a c t a t e , g l y c e r a t e , g l y c e r o l and 5-ket©gluconate a r e each o x i d i z e d t o a g r e a t e r or 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 exc luded from 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 . I t i s i n t e r e s t i n g t o c o n t r a s t the metabol ism of these c e l l s w i t h t h a t o f the a c e t o b a c t e r . A l t h o u g h both organisms are o b l i g a t e l y a e r o b i c , t h e breakdown of g lucose by the 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 the 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 -ke tog luconate 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 that t h e o x i d a t i o n o f g lucose proceeded by way o f g l u c o n i c and g - k e t o -g l u c o n i c a c i d s . S ince these 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 they d i d not n o r m a l l y f u n c t i o n i n g lucose d i s s i m i l a t i o n , i t appeared advantageous t o determine t h e genera 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 the 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 , -61-i . e . 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 , 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, Tabenkin and Ward 1941; Lockwood et a l 1946) . By a l l o w i n g the organism t o grow i n the presence o f c a l c i u m carbonate , i t should be p o s s i b l e t o demonstrate the accumula t ion o f 2 - k e t o g l u c o n i c a c i d as the s p a r i n g l y s o l u b l e c a l c i u m s a l t . One c o u l d t h e r e f o r e conclude t h a t the d i s s i m i l a t i o n o f g lucose proceeded by way o f these o x i d i z e d a c i d s . A growth experiment was set up t o demonstrate the p r o -d u c t i o n o f 2 - k e t o g l u c o n i c a c i d . The medium and methods o f a n a l y s i s were those 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 (1940) . To o b t a i n a normal breakdown o f the sugar , the 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 or 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 the 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 determined. Knowing t h e t o t a l amount o f copper reduced and 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 , i t was p o s s i b l e t o c a l c u l a t e t h e percentage g lucose 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 ' l i q u o r s . 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 i s based on t h e f a c t that i n mixed s o l u t i o n s o f g l u c o s e , 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 , the observed r o t a t i o n i s e s s e n t i a l l y -62-due t o t h e o p t i c a l a c t i v i t y o f the f i r s t and l a s t components. S ince g lucose 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 et a l (1940) have been able 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 the 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 lucose and S2-keto-g l u c o n i c a c i d p r e s e n t . The da ta presented (Table i v ) show t h a t the d i s a p p e a r -ance o f 1% g lucose 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 .35$ 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 determine 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 o f g lucose t h r o u g h these c h a n n e l s , s i n c e , as p r e -v i o u s l y mentioned, the m a j o r i t y o f t h e s u b s t r a t e i s o x i d i z e d t o comple t ion ( i . e . CO*,, BgO and s torage 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 lucose 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 . I t i s i n t e r e s t i n g t o observe t h a t t h e g lucose metabol ism o f t h i s a e r o b i c b a c t e r i u m ressembles t h a t o f the f u n g i i n t h a t t h e o x i d a t i o n proceeds 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 et a l (1940), Rober t s and Murphy (1944), W i l l i a m s (1945), W e l l s et a l (1939), and Moyer et a l (1940) have shown t h a t s u f f i c i e n t -l y h i g h y i e l d s o f these In termedia tes 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 warrant i t s use i n commercial f e r m e n t a t i o n s . —63«» Table IV The Production of 2-ketogluconic Acid Age of culture ,Specific ,rotation o ,Reducing 'substances mg./ml. ,Reduced ', copper .mg./ml. . [ 2-Keto-' gluconate . Glucose \ * K !+2.0 ! 53.3 j111.9 - 1. 5.4 4 days +1.65 ; 38.4 80.6 .17 1 4.83 7 days + .23 . 34.9 ! 73.3 ! .90 ! 2.78 -64-A n a e r o b i c d i s s i m i 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 i Lipmann (1936) has shown that t h e a e r o b i c o x i d a t i o n o f g lucose 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 d e c a r b o x y l a t i o n s u n t i l a t r i o s e molecule i s o b t a i n e d . By a p p l y i n g t h i s t h e o r y t o the 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 expected that pentose and carbon d i o x i d e would be produced i n e q u i -molecular amounts d u r i n g t h e decomposi t ion o f the a c i d . R u f f o et a l (1945) have suggested ( a l t h o u g h wi thout s u f f i c i e n t exper imenta l evidence) t h a t t h e o x i d a t i o n o f g lucose by k idney 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 genera l p a t t e r n g lucose —^ g l u c o n i c 2 k e t o g l u c o n i c GOg + pentose —^ p e n t o n i c , e t 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 should 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 f rom t h e a n a e r o b i c decomposi t ion o f S£-ketogluconic a c i d . Under t h e c o n d i t i o n s o f our exper iment , t h e l i b e r a t i o n o f carbon 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 be demonstrated. T h i s o b s e r v a t i o n i s i n s u f f i c i e n t ev idence from which t o conclude t h a t t h e a e r o b i c breakdown o f g l u c o s e does not proceed i n t h i s s t e p - w i s e manner. I t i s p o s s i b l e that an 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 t h e 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 , -65-TABLE V The Format ion 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 I n f l a s k : 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 . I n s i d e - a r m : ( l ) S . 5 J U M Ca S - k e t o g l u c o n a t e (omit i n endogenous) (S) 0 .3 m l . 3N H C l . I n centre w e l l : y e l l o w phosphorus Atmosphere: n i t r o g e n CQg e v o l v e d : a t zero t i m e : j i . l . a t 70 minutes ; u . l . T o t a l i n 70 m i n s . J U . l . endogenous ! * 1.3 s * * 1.4 l 0 . 1 '2 -ketogluconate : » *• • # 2 . 3 : 0 . 2 i . e . oxygen, t h e k e t o - a c i d c o u l d not be d e c a r b o x y l a t e d . 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 i s i n v o l v e d i n 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 pyruvate by Bac,t. a c i d i f l e a n s long iss imum. A n a e r o b i c d i s s i m i l a t i o n o f o x a l a c e t a t e : U s i n g acetone 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,sr K r a m p i t z and Werkman (1941) • Obtained p y r u v i c a c i d and carboia d i o x i d e f rom 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 -66-3 0 0 40 6 0 I2LO M I N U T E S Figure 4 : Anaerobic decarboxylation of oxalacetate In cups: 12,*c*iNa oxalacetate, w / 3 0 phosphate buffer, pH 7 . 4 , 0 . 5 ml. 20x cells. In side arm: 3N HCl. In centre well: phosphorus. Atmosphere: nitrogen. -67-o 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 determine 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 i n 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 from t h e a n a e r o b i c decomposi t ion o f o x a l a c e t a t e . 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 from t h e a n a e r o b i c breakdown o f one micromole o f o x a l a c e t a t e , 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 carbon d i o x i d e might be expected f rom t h e 12 micromoles o f s u b s t r a t e i n i t i a l l y s u p p l i e d t o t h e c e l l s . ( 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 decomposi-t i o n d u r i n g the course o f t h e experiment - see b o i l e d c e l l c u r v e ) . 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 t h e s u b s t r a t e i s o b t a i n e d , ( F i g u r e 2 ) , t h e r e f o r e t h e complete c o n v e r s i o n o f o x a l a c e t a t e t o pyruvate s h o u l d 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 carbon 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 carbon d i o x i d e spontaneous-l y produced, from 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 rom 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 o x a l a c e t a t e t o p y r u v a t e . 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 these organisms by v i r t u e o f anaerobic decompos i t ion t o p y r u v i c a c i d . M a l o n a t e : The c o m p e t i t i v e i n h i b i t i o n o f s u c c i n a t e -68-60d 400 Zoo -y JUS. --/// yT /// /// /// J/7 c 40 MINUTES 60 So Figure 5 The effect of malonate on the oxidation of glucose. -2 I glucose; II glucose + 10 Mmalonate; III glucose + 5 x 1 0 ~ 2 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 cells. -69-20 40 60 80 Ml NO T£S F i g u r e 6: The e f f e c t of malonate on the o x i d a t i o n of s u c c i n a t e . In cups: 7/<.MNa s u c c i n a t e , M/30 phosphate b u f f e r , 10~ 2M malonate, 0.5 ml. 15 x c e l l s . -70-"0 20 40 60 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 ~ 2 M malonate, 0 . 5 ml. 15 x ce l l s . Substrate added simultaneously or at 30 minutes, -71-©xidat lon 'by malon ic 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 et 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 rev iews 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 used as 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, t h e r e b y 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 accumula-t i o n o f s u c c i n i c a c i d . S i n c e t h e i n h i b i t i o n i s a c o m p e t i t i o n f o r t h e 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 than a b s o l u t e amounts, t h a t i s , t h e r a t i o o f s u c c i n i c a c i d t o malonic a c i d i s t h e important f a c t o r . V a r y i n g 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 t o 1 ( T S m o l a r . C o n c e n t r a t i o n s o f malonate from 5 x 10"^ molar t o 1QT% molar d i d not i n h i b i t the o x i d a t i o n o f g lucose o r s u c c i n i c a c i d by these organisms ( F i g u r e s 5 , 6 , 7, ) . ( L a t e r experiments demonstrated that 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 lengthened by t h e a d d i t i o n o f 5 x Iff® molar malonate ( F i g u r e 1 0 ) ) . 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 presence o f malonic a c i d f o r t h i r t y minutes p r i o r t o t h e a d d i t i o n o f s u b s t r a t e , o r i f a d d i t i o n s o f 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 extending t h e d u r a t i o n o f t h e exper imenta l p e r i o d t o 2 h r s . i t was -72-^ 1000. 5 800 600 400 >< ' r J>-M y y * * / \' y Of y i ^ 4 30 60 SO MINUTES /20 150 Figure 8; The effect of malonate on the oxidation of glucose - showing adaptation to malonate. I glucose + malonate; II glucose; III endogenous • malonate; IV endogenous. In cups: 5 ^ H _ 2 glucose, M / 3 0 phosphate buffer, pH 7«4, 10 M malonate, 0.5 ml. 20x cells. - 7 3 -7'-\ 5 BOO 200 a 1 A y < e 2T y y y i / 30 6 0 SO MINUTES /20 /so 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 ~ 2M malonate, 0.5 ml. 20x cells. •74-p o s s i b l e t o demonstrate t h e presence 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 ( F i g u r e s 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 the r a t e but not i n t h e t o t a l amount o f oxygen uptake o f c e l l s o f s a c c h a r o u h i l a when employing 1 0 " S molar s o l u t i o n s o f malonate . C o n c e n t r a -t i o n s as h i g h as O.S molar were r e q u i r e d t o reduce the t o t a l oxygen consumption o f t h e s e organisms . The o x i d a t i o n o f malonic a c i d has 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 (1929) . 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 , which 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 . S i n c e t h e o x i d a t i o n o f g lucose was not i n h i b i t e d by malonic 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 b a c t e r i a l r e s p i r a t i o n I n e i t h e r o f t h e p r e v i o u s l y e s t a b l i s h e d a e r o b i c m e t a b o l i c c y c l e s . B . STUDIES ON CRTfT.S flRQWN WITH. AMMONIUM PHOSPHITE AS  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 tha t 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 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 lucose breakdown would be obscured . S i m i l a r l y , growth o f the c e l l s I n t h e presence o f g lucose 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 which c o u l d be used as an a l t e r n a t i v e pathway t o t h e normal system -75-o f s u c c i n a t e o x i d a t i o n I . e . i n t h e presence o f 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 malonic a c i d . To o b t a i n c e l l s s u i t a b l e f o r the study o f i n t e r m e d i a t e metabol i sm, a search was made f o r a n i t r o g e n s o u r c e , o ther t h a n ammonium s u c c i n a t e , w h i c h would 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 £he a c t i v i t y o f 18. h£i. Suspensions o f the 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 . I n s i d e - a r m : 0 . 2 m l . g lucose (25^uM/ml) (omit i n endogenous) I n centre w e l l : 0 .15 m l . 20$ KOH n i t r o g e n source % n i t r o g e n source oxygen uptake i n 40 minutes endogenous g lucose ( n . l . ) ( u . l . ) N R 4 C I 0 . 3 8 .4 98 .0 < r a 4 V ° 4 - : 0 . 3 2 5 . 4 90.5 0 . 3 46 .2 423 0.6 50 .8 490 urea 0 . 3 1 9 . 4 345 NH^ s u c c i n a t e 0 . 3 4 6 . 1 383 From t h e data presented 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- too K Or. ^ 400 ^ 200 0~JE i 77 A y . y ^s* y^y y •— ' 9-..7T // // // 7 / i 7 / / // y / , y .— y y y y. ^ £ y* . - fy- " 2<9 40 MINUTES 60 SO Figure 10: The effect of malonate on glucose and succinate oxidation by glucose ammonium phosphate ce 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 ~ 2 M malonate, M / 3 0 phosphate buffer, 0.5 ml. 15x c e l l s . -77-s c a l e c e l l p r o d u c t i o n . W h i l e the endogenous a c t i v i t y o f these organisms remained unchanged, t h e oxygen uptake o f c e l l s h a r v e s t e d from b o t h phosphate media exceeded t h a t o f c e l l suspensions h a r v e s t e d f rom glucose ammonium s u c c i n a t e media . Other n i t r o g e n sources were l e s s e f f e c t i v e , perhaps due t o t h e s lower r a t e o f growth o f t h e organism. I n o r d e r 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 t h e 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 em-ployed i n a l l subsequent growth media . By the use o f t h i s complete ly 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 f u r t h e r reduce t h e number o f enzymes which appeared t o be c o n s t i t u t i v e f o r the organism, 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 necessary f o r the d e g r a d a t i o n o f t h e added compound. Malonatet Exper iments on the 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 lucose and s u c c i n a t e were repeated u s i n g g lucose 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 -served w i t h the 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 , t h e breakdown o f g lucose was not a f f e c t e d by t h e presence o f 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 malonic a c i d (5 x 10 M). Moreover , i t was shown t h a t 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 the 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 glucose d i r e c t l y ( i r r e s p e c t i v e o f the presence o f malon ic a c i d ) w h i l e t h e y 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 t o s u c c i n a t e , t h e l e n g t h o f which was e f f e c t i v e l y Increased by t h e a d d i t i o n o f malonate i n h i b i t o r . -78-9 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 of ce l ls grown i n t h e presence o f s p e c i f i c s u b s t r a t e s , i t should be p o s s i b l e t o determine t h e f u n c t i o n o f t h e 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 r e l a t e t h i s i n f o r m a t i o n t o t h e o x i d a t i o n o f g l u c o s e . 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 suspen-s i o n s are recorded i n f i g u r e s ii-rt . I n the l i g h t o f our present knowledge, i t I s i m p o s s i b l e 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 s u c c i n a t e grown c e l l s u t i l i z e the o x a l a c e t a t e - m a l a t e , s u c c i n a t e - f u m a r a t e mechanism o f t h e Szent G y o r g y i system, ( a l l o f these 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 malonic a c i d do not depress t h e r a t e o r extent o f s u c c i n a t e 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 ther hand, 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 r a t e s of. o x i d a t i o n o f malate and l a c t a t e should 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 s o . However, s e v e r a l important 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 lucose d e g r a d a t i o n . S i n c e 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 lucose 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 20 -79-Qlucosc ~~ -G/uconate ^gluconate 5-Ketogluconak' 'Endogenous 2 o Figure 11: 40 60 M I N U T E S 80 /oo Oxygen uptake of glucose ammonium phosphate cells. In cups: substrate (5/&M glucose, 2,5AW Ca gluconate, Ca-2-ketogluconate, Ca 5-keto-gluconate), M/30 pho-sphate buffer, pH 7 . 4 > 0 . 5 ml. 15x ce 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. 15x c e l l s . -81-€0 40 60 SO loo M/A/OTE5 Figure 1 3 : Oxygen uptake of glucose ammonium phosphate cells. 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. 15x 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 lactate, 2.5>«-*<Ca gluconate, Ca 2-ketogluconate, Ca 5-ketogluconate) w/30 phosphate buffer, pH 7 . 4 , 0 . 5 ml. 15 x ce l l s . -83' 4o (bo SO MINUTES Figure 1 5 : Oxygen uptake of succinate ammonium phosphate c e l l s . In cups: substrate (8,6-u.n Na succinate, 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 ml. 15x c e l l s , -84-1 ° ZOO 4-0 GO M/NUTES 80 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 S O /OO /WNUTEJ Figure 1 7 : Oxygen uptake of lactate ammonium phosphate cells. In cups: substrate ( I Q ^ M Na lactate, 5^*^ glucose, 2 . 5 A M Ca gluconate, Ca 2-ketogluconate, Ca 5-ketogluconate) M/30 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 MINUTES ao /oo Figure 19: Oxygen uptake of lactate ammonium phosphate cells. In cups: substrate (10>LM Na lactate, 12-«.MNa pyruvate, 15x^ Na acetate and Na malonate), M/30 phosphate buffer, pH 7.4 (6.0 pyruvate), 0.5 ml. 15x c e l l s . -88-n&nutes ( F i g u r e I S ) our p r e v i o u s o b s e r v a t i o n t h a t s u c c i n i c 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 g lucose has been c o n f i r m e d . Moreover i n t h e r e s p i r a t i o n o f g lucose grown b a c t e r i a , 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 metabol ism (Sumner and Somers 1943) but by v i r t u e o f i t s de -c a r b o x y l a t i o n t o p y r u v i c a c i d . ( F i g u r e s 1M and 13 show t h a t both oxaloacetic and p y r u v i c a c i d s a r e o x i d i z e d at t h e same r a t e i n these c e l l s whereas malate i s a t t a c k e d much more s l o w l y . ) Fur thermore , 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 they 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 -ke tog luconic a c i d , g lucose c e l l s o x i d i z e these compounds d i r e c t l y w i t h o u t a l a g p e r i o d . I t can t h e r e f o r e be concluded t h a t 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 are i n t e r m e d i a t e s i n t h e a e r o b i c breakdown of g l u c o s e , and that t h e enzymes 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 t h i s hexose are a d a p t i v e r a t h e r t h a n c o n s t i t u t i v e i n n a t u r e ( F i g u r e s 1 1 , 14, 1 7 ) . S y n t h e s i s i n h i b i t i o n s The use o f sodium a z i d e and 1 , 4 - d i n i t r o phenol 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 Toule 1944J C l i f t o n 1938) . By employing c r i t i c a l c o n c e n t r a t i o n s o f these 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 , t o s t i m u l a t e r e s p i r a t i o n . Thus , C l i f t o n (1937; 1938) has o b -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_. c a l c o - a c e t l c a 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 , d i n i t r o p h e n o l , and of g l u c o s e , a c e t a t e , pyruvate and g l y c e r o l '. 20 40 60 SO /OO M I N U T E S Figure 2 0 : The effect of sodium azede on the oxidation of glucose. In cups: 5o a M glucose, M/ 3 0 phosphate buffer, pH 7 . 4 , lO"^* NaNo, 0 . 5 ml. 15x c e l l s . Substrate added after 10 minutes' incubation of cells and inhibitor. •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 J M D N P ; III glucose + 1 0 ~ ^M D N P ; IV glucose + 1 0 " 5 * D N P ; V endogenous VI endogenous + 1 0 " ^ M D N P ; VII endogenous + 10"^ 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 . fey 1* c o l i employing M/SOO - M/400 sodium a z i d e or M/2000 2 , 4 - d i n i t r o p h e n o l . The exact way i n which these i n h i b i t -ors a t t a c k the s y n t h e t i c mechanism i s unknown, but i t has been observed t h a t t h e i n c r e a s e i n oxygen uptake i s f r e q u e n t -l y accompanied by d e p r e s s i o n i n the 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 M 2 , 4 d i n i t r o phenol have been found 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 the same t ime 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 normal r e s p i r a t i o n o f t h e c e l l s , 570jal o f oxygen were used (84$ — 3 of the t h e o r e t i c a l v a l u e ) , i n the presence o f 10 M NaNg and 1 0 ~ 5 . M DNP 616 ; u l and 610 ; u l were a b s o r b e d . These amounts represent 92$ and 90$ r e s p e c t i v e l y o f the t h e o r e t i c a l 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 . S i n c e s m a l l e r c o n c e n t r a t i o n s o f t h e s e compounds have not 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 at w h i c h s y n t h e s i s i s complete ly e l i m i n a t e d . However a comparison o f t h e e f f e c t s produced by 1 0 " 4 M and 10"*5 M 2 , 4 - d i n i t r o phenol suggests t h a t 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 approx imate ly 10~ M s o l u t i o n s o f the i n h i b i t o r . I n any case , 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 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 lower t h a n those employed by C l i f t o n ( 1937) Doudoroff (1940) and B e r s t e i n (1943) . C e r t a i n workers ( B e r n s t e i n 1943; Doudorof f 1940) have cons idered t h a t the 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 s y n t h e s i s i n h i b i t i o n . We have obta ined no evidence o f t h i s phenomenon. I f t h i s were so , t h e amount o f oxygen absorbed -92-by c e l l s i n t h e presence o f g lucose + i n h i b i t o r would be t h e sum o f t h e oxygen uptake o b t a i n e d on g lucose (no i n h i b i t o r ) and the endogenous r e s p i r a t i o n i n the presence 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 presence 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 d e p r e s s i o n i n the r a t e o f oxygen consumption was o b t a i n e d u s i n g 10"" 3 M s o l u t i o n s o f 2 ,4 DNP, t h e curve 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™ 5 M N a N s , p a r a l l e l e d t h a t o f g l u c o s e and at no t i m e d i d i t appear t o be t h e summation o f b o t h a c t i v i t i e s . I t was t h e r e f o r e c o n -c luded 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 normal 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 uptake 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 s y n t h e t i c processes o f t h e c e l l s . C. ADAPTATION As a result of the work of Karstrom (1937) i t has been recognized that two types of enzyme systems function during bacterial 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 specific substrate. Although Spiegelman (1947) has since shown that the presence of substrate does not necessarily maintain the adaptive systems at the peak of their activity, the concept of adaptation has remained essentially unchanged. In so far as we are able to determine, no conclusive evidence has yet been presented to show that glucose i s dis-similated by the action of adaptive enzymes. The observation made by Karstrom (1937) that the fermentation of glucose proceeded irrespective of the composition of the growth medium has since been confirmed by many workers. (Spiegelman 1947). Although Stevenson and Gale (1937) have produced re-lative changes i n the glucozymase activity of B. c o l l . at no time have they obtained cells 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 in an inorganic medium containing either succinate or lactate as the sole source of carbon, the cells 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 in the apparently constitutive enzymes. Since the degradation of a simple substrate would probably give rise 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 VI The Ut i l i z a t ion of Carbon Compounds by Growing  Cultures i n Ammonium Phosphate Media substrate u t i l i z a t i o n 11 hrs. 17 hrs, yellow • pigment present glucose ++++ Ca-gluconate ++++ ++++ Ca-2-ketogluconate ++ ++ Ca-5-ketogluconate Na citrate +++ ++++ Na malate +++ ++++ Na malonate +++ + Na fumarate +++ ++++ Na succinate +++ +++ Na acetate + + Ma formate + • + Na lactate +++ +++ OH -+ + + Table V I shows that glucose, gluconate, citrate, malate, lactate, fumarate, malonate, 2-ketogluconate and succinate supported good to excellent growth in 17 hours. However lighter yields of cells 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 1 •Glucose -G/acose *-.G lucona-te •Q--+-:.Acelate Glucose \ Endogeaous £0 4-0 <bO MINUTES BO loo Figure 2 2 : Comparative activity of glucose, gluconate and acetate grown cells on glucose. glucose cells; acetate ce l l s . ++-^  gluconate cells ; In cups: substrate (5-*tlpi glucose, 2.5-A»xiCa gluconate, 1 5 ^M Na acetate), M / 3 0 phosphate buffer, pH 7 . 4 , 0 . 5 ml. 15x glucose and gluconate cells or 20x acetate cells. -96-inoculum to 5$, i t was possible to increase the yield of acetate cells sufficiently to permit their use as enzymic&lly deficient resting c e l l suspensions. A comparison- of the enzymic activity of acetate and glucose grown cells 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 cells i s characterized by a rapid and constant rate of oxygen uptake, the corresponding stage in the oxidation by acetate grown organisms is a lag phase, not less than 1 hr. in length, during which the cells become adapted to glucose (Figure 23). Following this 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 is increased. Simultaneous adaptation; Since i t has been shown that the oxidation of glucose is dependent upon the activity 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 this procedure to the benzoic acid oxidizing system of P. fluorescens, Stanier (1947) has been able to establish certain postulated inter-mediates as functional units in the breakdown of this com-pound. A brief summary of the basic principles of the method follows. It i s f i r s t recognized that the dissimilation of the substrate is the net result of a series of well-defined, —97«* stepwise, chemical reactions (KLuyver 3^31 ), By adapting cells to the primary substrate, a simultaneous adaptation i s effected only to those intermediates taking part i n the dis-sociation of that substrate (Karstrom 1937). Postulated Intermediates which actually 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 cells more l i k e l y to adapt to those compounds above i t which are nearer i n the oxidative system. The limitations of this method of analysis are apparent. First 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 actually functional components of the oxidative system. However valuable information can be obtained concerning the importance of certain intermediates in the breakdown of the parent compound. To investigate the pathway of glucose metabolism by the simultaneous adaptation technique, the oxidative activity of the organism was compared when the cells were grown on acetate and on glucose.media. Since i t has been shown that acetate cells must adapt to glucose, they must therefore adapt to each of the intermediates taking part i n glucose dissimilation 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, wi l l oxidize directly a l l intermediates i n the glucose £0 4*> GO &0 /OO • MINUTES Figure 2 3 : Oxygen uptake of glucose and acetate ce l l s . In cups: substrate ( 5 g l u c o s e , 2.5-^MCa gluconate and Ca 2-ketogluconate), M / 3 0 phosphate buffer, pH 7 . 4 , 0 .5 ml. cells. - Glucose cells; Acetate ce l l s . -99-(QOO 4oo £09 30 40 60 80 /oo Figure 2 4 : Oxygen uptake of glucose and acetate ce 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. ce l l s . (See Figure 23 for endogenous and glucose curves of glucose cells and endogenous curve of acetate cells; Figure 26 for acetate curve of acetate cells.) Glucose cells; Acetate cells. •100-3D 46 60 SO IOO M//VC/TEJ Figure 2 5 : Oxygen uptake of glucose and acetate ce l l s . In cups: substrate (8 . 6 ^ t M N a succinate, I Q A M Na fumarate and Na lactate), M/30 phosphate buffer, pH 7 . 4 , 0 . 5 ml. cel l s . (See Figure 23 for endogenous and glucose curves of glucose cells and endogenous curve of acetate cells; Figure 26 for acetate curve of acetate cells.) Glucose cell s ; Acetate ce l l s . -101-ao 40 60 S o 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. cel l s . (See Figure 23 for endogenous and glucose curves of glucose grown cells, and endogenous curve of acetate grown cells. Glucose cells; Acetate cells. -102-"breakdown system. To be an active unit in 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 in-duce a period of adaptation in acetate ce l l s , the length of which is approximately equal to that obtained when glucose is the substrate being oxidized. The immediate response of glucose cells to gluconic and 2-ketogluconic acids coupled with the prolonged adapt-ation period required by acetate cells before they are able to attack these compounds is adequate proof that both gluconic and 2-ketogluconic acids are intermediates in the oxidation of glucose. On the other hand 5-ketogluconic acid can be .':•. eliminated as a possible intermediate since cells 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 in the breakdown of glucose since these compounds are equally active for 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 oxidiz-ing 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 justify the inclusion or exclusion of these compounds from the possible intermediates of glucose dissimilation. -103* Growing the cells in the presence of either glucose or acetate does not stimulate the production of enzymes necessary for the immediate dissimilation 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 fu 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 ce l l s on glucose or glucose and acetate cells on succinic acid. Whereas succinate was attacked at a rapid and constant rate following a brief 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 rise in the oxidative activity was observed. In view of the existing theory of enzymic adaptation, succinate i s not . an intermediate in 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 relative nearness of the adapting systems.to the apparently constitutive enzymes of the cells. Thus i t would appear that the* dicarboxylic acid cycle, though not functional in glucose or acetate metabolism, is closely related to both of these systems, since cells 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 cells or c e l l prep-arations, i t should be possible to determine the importance of additional intermediates in the glucose metabolism of these aerobic bacteria. -105-SUMMARY The meohanism of glucose oxidation is adaptive. Cells grown on a completely inorganic medium plus lactate or succinate attacked glucose at greatly reduced rates, while cells harvested from acetate media oxidized this 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 dissimilation. The latter compound has also been detected i n fermentations; by growing cultures. Apparently no use could be made of 5-ketogluconic acid. Anaerobically, oxalacetate was decarboxylated to pyruvi acid, one mole of carbons dioxide being released for 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"*2M malonate the oxidation of glucose proceeded normally, suggesting that succinic de-hydrogenase does not function in glucose metabolism. How-ever the rate of adaptation to succinate by glucose grown cells was retarded by this concentration of inhibitor. The normal oxidation of both succinate and fumarate occurred after the c e l l suspensions had been incubated for 20-30 minutes i n the presence of the substrate. The oxygen uptake of glucose, lactate, succinate and acetate grown cells has been recorded. It has been shown that the endogenous respiration may be ^ disregarded during -106-substrate oxidation. No evidence of an anaerobic respiratory mechanism has been obtained. Resting cells incubated with substrate under a nitrogen atmosphere failed to produce acid or gas, absorb phosphorus or u t i l i z e the glucose or fructose supplied. - 1 0 7 -EIBLIOGRAPHY Alsberg, C. L., ( 1 9 1 1 ) , The formation of d-gluco#ic acid by-Bacterium savastanoii, J. Biol. Chem. £, 1 . Annau, E., Banga, I., Gozsy, B.., Huzak, St., Laki, K., Straub, F. B., and A. 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