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The metabolism of organic acids in Pseudomonas Aeruginosa A.T.C. 9027 Whitaker, Judith Frances Moore 1951

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THE METABOLISM OF ORGANIC ACIDS BI FSEUDQMONAS AERUGINOSA A.T.C. 9027  JUDITH FRANCES MDORE WHITAKER  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i a the Departments of Biology and Botany and Dairying  We accept t h i s t h e s i s as conforming t o the standard required f o r candidates f o r the degree of M&STER OF ARTS.  Members tf£ the Department of Biology and Botany*  Hemo<3i7s—or the i/epartment of Dairying. THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1951.  ABSTRACT The present study has been concerned with the oxidation of the 4-carbon dicarboxylic acids: succinate, fumarate and malate, i n the striot aerobe, Pseudomonas aeruginosa A.T.C. 9027  e  -Hesting oells harvested  from a mineral medium i n 'which any one of these compounds was the sole carbon source, metabolized a l l three acids immediately and at a constant and rapid rate©  This observation has suggested the operation of the  enzymatic inter-relationship: Succinate  f umarate<r— malate  It is very likely that the oxidation of these substrates follows the familiar tricarboxylic acid cycle, which has already been shown to occur in P. aeruginosa 9027* Questionable quantitative data for the oxidation of the dicarboxylic acids by dried cell preparations has been obtained. These preparations did not require either cytochrome c or pyocyanine as a carrier system for succinate oxidation. The optimum pH range of succinic dehydrogenase in succinate-grown and acetate-grown dried cells lay between pH 7.0 and 8.0, an observation which coinoides with values found for other species© The effect of the two metabolic inhibitors, malonate and uranyl nitrate, on the respiration of both resting and dried cells of P. aeruginosa has been investigated.  Malonate v/as not inhibitory i n moderately high  concentration at pH 7.0, but when the pH was lowered, maximum inhibition was effected by low concentrations. Apparently, permeability functions as a governing factor i n malonate inhibition, a suggestion which was borne out i n experiments with dried cells, i n which the c e l l membrane has been altered during the drying process. These cells were strongly inhibited at pH 7.0 by low, concentrations of malonate* An interesting though  complicating feature of the malonata experiments with resting cells' has "been that P. aeruginosa possesses an adaptive enzyme system for metabolizing the inhibitor.  The inhibitory effect of uranyl nitrate  on the oxidation of Krebs intermediates or of glucose varied with the substrate - maximum inhibition being obtained with isocitrate oxidation, and none with malate oxidation, while tho inhibition of other oxidations f o i l between these two extremes. The use of this non-specific inhibitor in metabolic studies on whole cells has been discussed and, on the basis of the present and other studios, i t s use has been precluded*  AC KNCJl'/LEDG-EMEJHT  Without the unfailing assistance and encouragement of  Dr» J« J . R» Campbell, the enthusiastic interest  of Dean B. A. Eagles, and the financial support of the Research Council of Ontario, this thesis could never have been written*  TABLE OF CONTENTS  INTRODUCTION PART I  ..  .  1  WARBURG STUDIES WITH RESTING CELLS Introduction 10 Methods B a c t e r i o l o g i c a l ».... 11 Chemical ...... ««e«.o«.........«.«.«............ 12 Experimental 14 Discussion ........................................» 19  PART I I THE METABOLISM OF DRIED CELLS  Introduction •••.••••••.••••••••••..•••«««..•«.•««.. 20 Methods 21 Experimental ....................................... 23  Discussion ••••  •.«•••.••...•.•.  ••••...« 32  PART I I I INHIBITOR STUDIES WITH MALONATE AND URANYL NITRATE  Introduction ....................................... 34 Methods 36 Experimental...... A. Malonate Studies ..........«••.............. 38 B. I n h i b i t i o n by Uranyl Nitrate •• 52 Discussion ......................................... 61 SUMMARY  64  65  - 1 -  DTTRODUCTION  Since carbohydrate i s the main source of energy f o r l i v i n g c e l l s , the mechanism of i t s degradation has been extensively investigated.  In  general, carbohydrate breakdown occurs i n two phases: the anaerobic phase which ends i n pyruvio acid formation, and the aerobic phase which involves the u t i l i z a t i o n of pyruvate v i a tha oxidative pathway.  Some aspects of  t h i s second phase have been studied i n the present t h e s i s . The f i r s t attempts to formulate a scheme of carbohydrate oxidation are  found i n the papers of Thunberg (149),and, of Knoop (69).  These workers  suggested that pyruvate was f i r s t converted t o acetate, two molecules of  iriiich condensed to give succinate (the Thunberg-WIeland condensation).  The succinate was then c y c l i c a l l y re-converted to acetate, v i a fumarate, malate, oxalacetate and pyruvate.  The chief weakness of t h i s scheme lay  i n the complete lack of evidence i n support of the condensation. A s i m i l a r scheme was proposed by Toeniessen and Brinkman (151), except that the condensation involved two molecules of pyruvate, instead of acetate, an unknown polymerization product being formed, which was then transformed t o succinic a c i d . The f i r s t r e a l evidenoe that organic acids function i n r e s p i r a t i o n was contained i n the work of Szent-Gyorgyi and his associates (7,8,49), who found thatsuocinate (or i t s four-oarbon oxidation products)) could stimulate c a t a l y t i c a l l y the r e s p i r a t i o n of pigeon breast muscle b r e i , and that t h i s o a t a l y t i c effect was i n h i b i t e d by malonate.  Szent-^Gyorgyi suggested that  the  main, function of succinate, fumarate, malate and oxalacetate i n muscle  " i s not t o serve as f u e l , but to serve as a c a t a l y s t j as a c a t a l y t i c hydrogen c a r r i e r between foodstuff (e.g. pyruvate) ard cytochrome."(147)•  The  Szent-Gyorgyi scheme has met with considerable c r i t i c i s m * f i r s t , because i t r e s t s on the assumption that the succinate systaa i s the. only one i n animal tissues which can act d i r e c t l y with the cytochromes, and, second, beoause the  c a r r i e r function of oxalacetate~=-^malate merely results i n the accumul-  a t i o n of reduced coenzyme I, with no e f f e c t i v e transport of hydrogen taking place. Shortly a f t e r Szent-Gyorgyi had proposed t h i s scheme, Krebs announced the  discovery that, i n addition to succinate, fumarate, malate and oxalacetate,  o6-ketog!utaric and c i t r i c acids also act c a t a l y t i c a l l y on the r e s p i r a t i o n of minced muscle, and that t h e i r e f f e c t s are also inhibited by malonate. On the basis of further investigations, Krebs ( 72) proposed that pyruvate and oxalacetate might react together to form a 7-carbon compound, from which c i t r a t e , "C-ketoglutarate, and the 4-carbon dicarboxylic acids would then be re-formed.  What had formerly been considered simply as a chain or series of  reactions now became i n effect a c y c l e , the ' c i t r i c acid c y c l e ' .  The 7-carbon  condensation product (oxalocitraconio acid) has never been isolat ed, but Krebs was able to demonstrate the formation of substantial amounts of c i t r i c a c i d when pyruvate and oxalaoatata condensed i n an anaerobic system (73, 75). He therefore suggested that pyruvate condensed with oxalacetate t o give c i t r a t e , which was oxidized v i a cis-aconitate, i s o c i t r a t e , oxalosuccinate, oC -ketoglutarate and the 4-carbon dicarboxylic acids, with the r e s u l t i n g oxalacetate becoming available t o condense again with pyruvate araid t o repeat the c y c l e .  On the basis of t r a c e r studies, W od et al.(l60) proposed 0  - 3 -  that ois-aconitate, and not c i t r a t e , i s the i n i t i a l condensation product. They showed that isotopic carbon, introduced as carbon 'dioxide together with pyruvate, l e d to the formation of <<-ketoglutarate which .contained i s o t o p i c carbon only i n the carboxyl group next to the ketone group; i f °c-ketoglutarate was derived from a symmetrical molecule ( c i t r a t e ) , the f i x e d carbon would be equally d i s t r i b u t e d i n the two carboxyl groups. Therefore, they concluded t h a t ^ - k e t o g l u t a r a t e arose from cis-aoonitate, and that c i t r a t e was outside the cycle but i n equilibrium with cis-aconitate through the mediation of aconitase. Recently, Ogston (103) on purely t h e o r e t i c a l grounds, has suggested that the asymmetrical, occurrence of an isotope i n a product cannot be taken as conclusive evidence against i t s having arisen from a symmetrical preoursor. He maintains that an asymmetric  enzyme (aconitase) can indeed distirg uish  between the i d e n t i c a l groups (end carboxyls) of the compound (citrate) which i t attacks.  This conclusion i s based on the fact that enzyme and symmetrical  substrate combine at three points, and that two of the three combination s i t e s are c a t a l y t i c a l l y d i f f e r e n t .  Stern & Ochoa (l38) have now shown  that c i t r a t e i s the i n i t i a l product of condensation between oxalacetate and some " a c t i v e " 2-carbon compound (derived from pyruvate, and not pyruvate i t s e l f ) , the condensation being catalysed by condensing enzyme, Coenzyme A, Hg*" or MET* and adenosine triphosphate. The "active" 2-carbon compound maybe acetic acid or the acetyl r a d i c a l or possibly acetyl phosphate (55). By a series of hydrations, dehydrpgenations, and decarboxylations, c i t r a t e i s dissimilated i n turn to the following compounds; cis-aconitate,  isocitrata, oxalosuccinate,<<-ketoglutarate, succinate, fumarate, malate and thence back to oxalacetate. The modern concept of the tricarboxylic acid cycle i s shown in Figure I. Thescheme from oxalacetate back to succinate corresponds to the catalytic system of Szent-Gyorgyio For each complete cycle, one molecule of acetate is completely oxidized to carbon dioxide and wab er.  A l l the steps i n the cycle may be reversible under  aerobic conditions, but the only hydrogenations which appear to proceed with any rapidity under these conditions are those connecting the Szent-GyBrgyi system from oxalacetate to succinate. The experimental work upon which the Krebs cyole i s based was performed largely with minced pigeon breast muscle (72,73,75).  Ochoa  (99, 100,101,140), Wood (158), Barron (126), Vennesland, Peters and others have demonstrated and discussed the occurrence of the various reactions i n other animal tissue, including kidney, heart and l i v e r . D.S. Green et a l . (50, 51) have obtained a preparation, the "cyolophorase system"', which they have defined as a " c omplex of enzymes which catalyses the oomplete oxidation of pyruvic acid by way of the Krebs oitrio acid cycle."  Microscopic examination of cyolophorase has indicated that i t  is essentially a suspension of intaot mitoohondria, and the enzyme activity has been shown to be related to the structure of these maoro-granules (141). - Evidenoe that pyruvate i s oxidized through the tricarboxylic acid cycle in plant tissues comes from three sources:  (a) a l l the acids which  are intermediates i n the Krebs cycle are present inyplantsj (b) a number of the enzymes catalyzing the reactions of the cycle have been found; (c) the few investigations of pyruvate oxidation i n vivo (15,19,21,78) have presented evidence conforming with the four criteria originally set forth  -2H (CHg.COOH) CH3.CO' (CH .C0~P0 ) 3  COOH.CO.CHg.COOH Oxalacetate  4  - 2H COOH.C(OH).CH2.COOH CHg.COOH  COOH.CHOH.CH2.COOH  A Malate  Citrate H0 2  C00H.C.CH .C00H CH.COOH 2  Cis-aconitate + H0  COOH.CH:CH.COOH  A  Fumarate  -2H  2  V  COOH.CH.CHg.COOH CH(OH).COOH Isocitrate  COOH.CHg.CHg.COOH Succinate  -2H -CO2  - 2H  +H 0 2  COOH.CH.CHg.COOH CO.COOH Oxalosucc inate  Figure I. The t r i c a r b o x y l i c acid  CH2.CH2.COOH CO.COOH od-ketoglutarate  cycle  by Krebs i n support of the cycle (72:) • The general picture of the Krebs cycle i n plant tissue has been reviewed by Bonner (20) and by Goddard & Meeuse(48)» Scattered information has appeared during the past five years which indicates that the Krebs cycle i s present i n microorganisms, A j l & Workman (4,5) i n studies on the replacement of oarbon dioxide by various intermediates of the cycle^ suggested that at least parts of the scheme were operative i n Escherichia ooli and Aerobaoter aerogenes,  Baskett & Hinshelwood  (16,17) on the baas of growth experiments, have proposed that the cycle oocurs in Bacterium lactis aerogenes.  However, Karlsson & Barker (62) were not  able to demonstrate the presence of this system i n Azotobacter agilis , either by the teohnique of simultaneous adaptation or by radioactive traoer experiments,  Umbreit (l53),in a review of this data, suggested that part of the  cycle i s present, while Stern & Oohoa (139) using extracts of Barker's organism obtained the condensation of oxalaoetate with "active " aoetate to citrate.  They were able to showithat E, ooli (139) and Streptococcus  faecalis (70) also possess this condensation enzyme.  Indirect evidence for  the oxalacetate-"active" acetate condensation i n B,coli was given by Oginsky et a l . (102) Oand by Umbreit (154) i n studies on the bactericidal effects of streptomycin. Further evidenoe for the participation of acetate in the cycle has been provided by the action of fluoroacetate i n inhibiting the conversion of aoetate to citrate i n yeast and baoteria (59)• Hovelli & Lipmann (96) have found acetyl phosphate to be more effective than acetate plus adenosine triphosphate in the formation of oitrate by extracts of E, c o l i .  Using radioactive carbon as a traoer, Saz & Krampitz (128)  - 7demonstrated the same condensation i n a lysate of Micrococcus lysodeikticus. Campbell & Stokes (29) have presented evidence for the tricarboxylic acid cycle i n cells of gseudoraonas aeruginosa, harvested from either a glucose or an aoetate medium* Several years ago, I»ockwood & Stodola (84) found that P, fluoresoens produced considerable quantities of<<-ketoglutarate, one of the key intermediates i n the cycle, when cells were grown on a glucose medium under conditions of intense aeration* In addition to the condensation enzyme, bacterial preparations have been made of the following enzymes i n the Krebs oycle:  succinic  dehydrogenase from Corynebaoterium diphtheriae (106), Salmonella aertryoke (77) and B,ooli (2,57); fumarase from Propionibacterium Shermanii (74); malio dehydrogenase from a stenothermophilio bacterium (87); oxalaoetio decarboxylase from M, lysodeiktious (7l), and Azotobaoter vinelandii (108); pyruvic oxidase from Proteus vulgaris (145)• Barron et a l , (13) have investigated the occurrence of the Krebs cycle i n yeast, and have given a comprehensive review of previous work* The cycle may also be operative i n the metabolism of molds suoh as Aspergillus niger (30), Neurospora orassa (82) and Streptomyees species (31), Working with protozoans, Speck et_ a l , found the Krebs cycle present i n the malaria parasite, Plasmodium gallinaoeum (136), while Harvey obtained no evidence for i t s occurrence in Trypanosoma hippicum (56), Some reoent investigations of acetate oxidation have suggested that an alternative scheme to the tricarboxylic acid oycle may function i n oertain microorganisms.  Entrance into this cycle, the dicarboxylic aoid  cycle (12,42) is made by the Thunberg-lfieland reaction i n which two moles of acetate (possibly phosphorylated acetate) condense to form succinate, or by the Wood-Werkman reaction of carbon dioxide fixation by pyruvate*  °^  - 8Using radioactive carbon as a tracer, Slade & Werkman (132) and Kalnitsky et_ a l , (60) have obtained evidence for the condensab ion of two 2-carbon molecules to succinic acid, with cell-free preparations of Aerobacter indologenes ,and B.ooli respectively.  Further swpport for this  scheme of acetate oxidation has been given by A j l (3), working with A, aerogenes and B.ooli, and by Lenti, al so employing B.ooli (81)• Ten years ago, Wood & Workman (159) suggested that the Thunberg-Wieiand condensation might occur i n the organism, Propionibaoteriurn pentosaoeum, but Krebs & Eggleston (74) rejected suoh a possibility i n the allied species, P» Shermanii,  On the basis of inhibitor and ^adaptation experiments,  Barron et a l . (12) have concluded that the dicarboxylic acid oycle, and not the tricarboxylic acid cycle, i s operative i n Corynebacterium oreatinovorans. Foster & Carson (42) i n studies on the mechanism of fumaric acid formation by Rhizopus nigricans observed the four steps fitting the pattern of a dicarboxylic acid oycle, entrance to the cycle being made by the ThunbergWieiand condensation^ The experiments of Thimann, Bonner, Albaum and others (6,148), on the action of iodoacetate and organic acids on the growth and protoplasmic streaming in Avena coleoptiles and whole seedlings, have suggested that "a respiratory process, which accounts for only a small fraction of the total respiration, and which involves the 4-carbon acids, i s i n conb rol of growth. This particular respiratory process i s the one promoted by auxin," (148).  Similarly, Ryan et a l , (127) deduced that a respiratory  process involving the 4-carbon dicarboxylic acids "supplies energy for growth"' in Heurospora.  r  Investigations carried out i n this laboratory have shown that  Pseudomonas aeruginosa A.T.C. 9027 oxidizes glucose by way of gluconio and 2-ketogluconic acids (95,142) and that i t does not possess an EmbdenMeyerhof scheme of metabolism (27)•  Both pyruvate and acetate have been  isolated as intermediates i n glucose breakdown (28,157). Recently, Campbell & Stokes (29) presented evidence for the presence cf a tricarboxylic acid oycle*  It i s therefore probable that glucose i s broken down by the  following pathway:Glucose v  Gluconate v  ^ 2-ketogluconate  ——  .pyruvate  ^acetate  The intermediary steps between 2-ketogluconate and pyruvate may involve a stepwise degradation of Cg to C , C4 and Cg, with alternate phosphorylative g  oxidation and decarboxylation); as proposed originally, by Dickens  (36,3.7).  It is likely that the C3 compound i s pyruvate, whioh undergoes oxidative decarboxylation to "active" acetate, at which point the Krebs cycle i s entered. The present study has centred about the oxidation of the 4-carbon dioarboxylio acids, succinate, fumarate and malate, a l l well-established constituents of the tricarboxylic acid cycle.  By using whole resting oells  and dried c e l l preparations of P. aeruginosa, the interrelationship: Succinate  Fumarate ^. _  Malate  has been demonstrated. Strong evidence for the occurrence of the enzyme succinic dehydrogenase, which catalyses the reversible step between succinate and fumarate, has been obtained i n experiments with the competitive inhibitor, malonate.  - 10 PART I WARBURG STUDIES WITH RESTING CELLS Numerous studies i n b a c t e r i a l chemistry have been made with "resting"' c e l l s *  The bacteria are grown i n pure culture,  centrifuged  and washed, and suspended i n water or saline i n a concentration than could be achieved by growth*  greater  According to Quastel & Whetham (117),  these "resting" baoteria are f u l l y endowed with metabolic p o t e n t i a l i t i e s * 1  yet unable t o multiply because of the lack of essential growth nutrients* C e l l s i n t h i s non-proliferating state are well suited f o r use i n metabolic studies, as t h e i r respiratory mechanisms may of i n t e r f e r i n g growth reactions  be evaluated i n the absence  (91).  The pathway of glucose oxidation i n P* aeruginosa as recognized was  arrived at on the basis of resting c e l l studies  now (91,92)*  Campbell & Stokes (29) used both glucose- and acetate-grown c e l l s to obtain evidence f o r a t r i c a r b o x y l i c acid cycle i n the same organism* In the present study, c e l l s have been grown with  succinate,  fumarate, malate or acetate as the sole carbon source, end the metabolic patterns  of the c e l l s investigated, with p a r t i c u l a r reference to the  oxidation of the dicarboxylic acids*  - 11 METHODS Bacteriological:  The culture of P. aeruginosa A.T.C. 9027 employed v/as a  typical strongly pigmenting strain.  Stocks were maintained in a l i v e r  extract agar of the following composition: 1.0$ tryptone, 0.25$ K HP0 , 2  0.15& glucose, 0.3$ CaC03, 10.0$ liver extract, 1.5$ agar, pH 7.2.  4  After  growth was initiated at 30° C , cultures were refrigerated. Fresh transfers of the stock culture were made every month. Medium for growth of the organism was prepared as follows: 0.3$ (NH )H P0 , 0.3$ K HP0 , and 0.5 p.p.m. iron (as FeS0 .4H 0) were 4  2  4  2  4  4  2  adjusted to pH 6.8, dispensed i n 100 ml. quantities i n Roux flasks, and sterilized by autoclaving at 15 l b . pressure for 15 min.  Before flasks  were inoculated, the carbon source that had been sterilized through sintered glass as a 10$ solution was added aseptically to a final concentration of 0.3$ to 0.5$, and MgS04.7H 0 that had been sterilized by 2  autoclaving as a 10$ solution was added to a final concentration of 0.1$. When a dicarboxylic aoid was used as carbon souroe, i t was neutralized to pH 6.5 with N/l HaOH before being made up to volume. A 1$ inoculum of a 24 hr. culture, grown i n ammonium phosphate medium with appropriate carbon souroe, was employed. After subculture from the refrigerated stock into Sullivan's medium (146), the culture was always transferred at least twice at 24 hr. intervals in ammonium phosphate medium with appropriate carbon source, before use as inoculum. The cells for Warburg experiments were harvested after 21 to 24 hours incubation at 30° C , washed with half the growth volume of 0.9$ saline and f i n a l l y made up to the desired concentration. This concentratio n of oells gave a light transmission of 65$ to 75$ when 0.2 ml. of the  - 12 suspension was mad© 10.0 ml., and read i n the Fisher Electrophotometer. It i s equivalent to 0.30 mg. bacterial nitrogen per ml. (92). A conventional Warburg apparatus (155) was used to follow the oxygen uptake, a l l experiments being run at 31° C , pH 7.2.  Warburg  cups contained 1.5 ml. Sorensen's phosphate buffer (M/l5), pH 7.2, and 0.5 ml. c e l l suspension i n the main compartment. The substrate to be oxidiged was tipped i n from the side-arm after equilibration.  The centre  well contained 0.15 ml. of 20$ K0H for the absorption of carbon dioxide. Total volume of fluid i n the cups was 3.15 ml. A l l substrates were neutralized where necessary and added at suoh a concentration that 0.2 ml. substrate was equivalent to 18 uM oxygen.  The endogenous respiration  was always measured (94). Oxygen uptake due to substrate dissimilation was determined by subtracting the endogenous oxygen uptake from the total uptake. Chemicalt  With the exception of sodium pyruvate and cis-aconitic acid,  a l l substrates were commercial preparations.  The pyruvate was prepared  in this laboratory by the method of Robertson (122), while cis-aconitate was prepared from the trans- isomer by the procedure of Malachowski & Maslowski (86). Warburg vessels were cleaned by soaking overnight i n detergent solution, then rinsed and treated with hot 10$ M O 3 for an hour. Finally they were washed with water and dried at 80° C.  - 13 EXPERIMENTAL -  Easting oells- of P. aoruginosa harvested from a mineral medium  in which succinate, fumarate or malate was the sole carbon source, metabolize these dicarboxylic acids immediately and at a constant and rapid rate (Figures II, III and IV). Other intermediates i n the Krebs cycle are oxidized more slowly or only after a period of adaptation. This suggests that cells grown on one dicarboxylio acid («.g» succinate) hava developed the enzymes for metabolizing not only that particular acid but also fumarate and malate.  On the other hand, i f the calls are grown  on acetate (Figure V), or on gluoose or pyruvate (as carried out i n tha present and other investigations, but not shorn), they metabolize the dicarboxylic acids only after a lag period. According to Karstrom's terminology (63), cells grown on any one of the 4-carbon dicarboxylio acids possess "constitutive" enzymes for oxidizing a l l these compounds, while calls grown on some other carbon source must f i r s t develop "adaptive" enzymes for attacking these substrates. The sharp division of enzymes into "'constitutive" and "adaptive" has been questioned during recent years (161, 164). It may well be that it  the lag phase, characteristic of an "adaptive  curve, represents the time  necessary for the development of the enzyme systems which w i l l transport the substrate across the c e l l membrane. It i s therefore possible that cells whioh have been grown on any one of the 4-oarbon dicarboxylio acids may use the same system for transporting a l l these substrates across the c e l l . Similarly, cells grown on another carbon souroe, would be expected to show the same adaptive pattern for each of the 4-carbon acids. Results obtained (29) with acetate oells or with glucose cells do nob indicate  -.14 -  •SULCIMAT 'ALATE  ot-  XET0GLUTA1Z47E  TYKL VATE  IS f  <7b  MINUTES Figure I I . O x i d a t i o n o f s u c c i n a t e , fumarate, m a l a t e , ° ^ - k e t o g l u t a r a t e , p y r u v a t e and a c e t a t e by 22 h r . c e l l s h a r v e s t e d from a s u c c i n a t e ammonium phosphate medium. C i t r a t e and i s o c i t r a t e were o x i d i z e d a t a l o w , r a t e , w h i l e c i s - a c o n i t a t e was not m e t a b o l i z e d . ^ Warburg cups c o n t a i n e d 0.5 m l . c e l l s u s p e n s i o n , 1.5 m l . M/15 phosphate b u f f e r pH 7 . 2 , 0 . 2 m l . s u b s t r a t e . F i n a l volume 3 . 0 m l . T h e o r e t i c a l oxygen uptake f o r complete o x i d a t i o n of any s u b s t r a t e was 403 u l .  - 15 -  SUCCINATE PUMARATE  •8  441  3  s  MINUTES Figure I I I . Oxidation of succinate, fumarate, malate, o ^ k e t o g l u t a r a t e , pyruvate and acetate by 23 hr. c e l l s harvested from a fumarate ammonium phosphate medium. Citrate and cis-aconitate were metabolized at a low rate. Warburg cups contained 0.5 ml. c e l l suspension, 1.5 ml. M/15 phosphate buffer pH 7.2, 0.2 ml. substrate. F i n a l volume was 3.0 ml. f Theoretical oxygen uptake f o r complete oxidation of any substrate was 403 u l .  - 16 -  MALATE  20 o  ACETATE VCWTA&.TE  OMLACETATE  6b  MINUTES Figure IV. Oxidation of malate, succinate, fumarate, oxalacetate, °<-ketoglutarate, pyruvate and acetate by 22 hr. c e l l s harvested from a malate ammonium phosphate medium. Citrate and i s o c i t r a t e were oxidized at a low rate, while cis-aconitate was not metabolized. Warburg cups contained 0.5 ml. c e l l suspension, 1.5 ml; M/l5 phosphate buffer pH 7.2, 0.2 ml. substrate. F i n a l volume was 3.0 ml. Theoretical oxygen uptake f o r complete oxidation of any substrate was 403.ul.  - 17 -  Sao  ACETATE o  I*  MINUTES. Figure V. Oxidation of acetate, succinate, fumarate, malate and i s o c i t r a t e by c e l l s harvested from an acetate ammonium phosphate medium. Citrate and cis-acoriitate were oxidized at a low rate. Warburg cups contained 0.5 ml. c e l l suspension, 1.5 ml. phosphate buffer pH 7.2, 0.2 ml. substrate. F i n a l volume was 3.0 ml. Theoretical oxygen uptate f o r complete oxidation of any substrate was 403.ul.  - 13 the operation of such a transport mechanism.  The effect of uranyl  nitrate, vdiich inhibits the transport of substrate across the cell membrane, has been studied i n Part I I I , and evidence has been obtained that the same transport mechanism i s not used by a l l the 4-carbon dicarboxylic acids.  - 19 ' •  DISCUSSION  It i s suggested that the substrates succinate, fumarate and malate are metabolized at a constant and rapid rate by cells of P.aeruginosa A.T.C. 9027 whioh have been grown on any one of these compounds, because of an enzymatic relationship among themj i . e . : Succinate v  ~ Fumarate ^ Malate succinic fumarafce dehydrogenase 1  Such a relationship, "vfoioh w s f i r s t proposed by Quastel and coworkers (114,115,116,117jl56) in studies with B. ooli and B.pyooyaneus, is probably operative i n other bacteria, including Azotobacter agilis (62) and P.saocharophila.(l8)«  The enzyme succinio dehydrogenase has been  obtained i n cell-free extracts from E.ooli (2,57), S. aertryoke (77), and C. diphtheriae{106).  Conclusive proof of i t s presence i n P.aeruginosa  A.T.C. 9027 i s presented i n Part III, while suggestive evidence has been •given for i t s occurrence i n a number of other bacteriaj Azotobacter agilis **•  '  n i-  r  I I  --.  •  (62), B.laotis aerogenes (16), P. saccharophila (18), Moraxella lweffi mutant S (85), B. typhosa (52), and certain stenothemophilic bacteria (47). The enzyme fumarase has been prepared from Propionibacterium Shermanii (74). In addition to succinate, fumarate and malate,(Ney (91) has shown that oxalacetate i s metabolized at a constant and rapid rate in succinategrown cells, and that this substrate is decarboxylated anaerobioally, no doubt to pyruvate.  It i s very likely, therefore, that succinate oxidation  follows the familiar tricarboxylic acid cycle, the existence of which i n P. aeruginosa A.T.C. 9027 has already been proposed by Campbell & Stokes (29).  - 20 PART II THE METABOLISM OF DRIED CELLS It has been observed i n Part I that whole resting oells of P. aeruginosa metabolize sudcinate, fumarate and malate at approximately the same rate, regardless of which of these three substrates has been used for growth. This suggests that there is an enzymatically controlled relationship between the three compounds. Other intermediates i n the Krebs cycle are metabolized only after a lag phase or period of adaptation, whioh may represent the time necessary for the elaboration of a system for transporting the compounds across the cell membrane. By using dried cell preparations of P. aeruginosa, Campbell & Stokes (29) were able to increase the range of substrates attacked.  Ia  the absence of the complicating factor of permeability, a clear quantitative picture was obtained.  In the present study, dried cell preparations  of succinate- sad aoetate- grown cells were used i n the hope of finding a quantitative relationship amongst the 4-carbon dicarboxylic acids. The possible carrier systems and optimum pH of the succinateoxidizing enzyme in these dried cells were also investigated.  METHODS To obtain oells for driad preparations, 20 l i t r e s of ammonium phosphate medium containing succinate or acetate as sole carbon source, were dispensed i n Roux flasks and inooulated with a 24 hour culture of P. aeruginosa A.T.CV 9027.  The cells were collected at the end of 24  hours with a Sharpies centrifuge at 48,000 r.p.m. They were washed once with 0.9$ saline and re-oentrifuged i n a Servall High Speed centrifuge at 5,000 r.p.m. The resulting pink cell paste was suspended i n 50 ml. d i s t i l l e d water and spread i n a thin layer on a large Petri plate. The cells were dried slowly (3 to 5 days) i n vacuo over phosphoric  pentoxide  at room temparature. The resulting tan glassy residue was ground to a r  fine powder i n a mortar, and stored i n a i r i n the freezing compartment of a refrigerator. The cells were used after a storage period of one week or more. Two preparations of succinate cells were made, weighing 4.8 and 7.7 grams respectively. One preparation of acetate cells was made, 20 litres of medium yielding 6.2 grams of c e l l s . For Warburg studies, the dried cells were weighed accurately and suspended i n d i s t i l l e d water so that; each ml. of suspension contained 20 mgm. of c e l l s .  An even milky suspension of cells, which could be pipetted  accurately, was obtained after the dried cells had stood for 10 to 15 minutes. Each Warburg vessel contained 1.0 ml. of this suspension.  In  addition, a l l Warburg vessels contained 2 uM MgS04.7H20, 2 uM MnCIg^ 0.4 mMAlCl3, 0.4 mM CaCl , 107 ug. DPN, and 5 uM substrate, neutralized, 2  to pH 7.  A l l runs were made at 31° C , and pH 7.2 or 7.4.  In experiments  on possible oarrier systems, 1 and 4 mg. of cytochrome o, and 0.001$ and 0.01$ final concentration of pyocyanine were used. The cytochrome c was  - 22 the produot of the Sigma Chemical Company, while the pyocyanine had been prepared in the laboratory by the method of Elema & Sanders (39). The optimum pH for succinate oxidation was found by the standard Thunberg method (155).  In the tube were placed 1.0 ml. buffer (phthalate  for pH 4 to 5, phosphate for pH 5.5 to 8, and borate for pH 8 to 10), 1.0 ml. cell suspension (containing 20 mg. dried c e l l s ) , and 0.3 ml. of  0.01M  malonate. .To the side-bulb were added 0.5 ml. ljlOOO methylene blue, and 0.5 ml. 0.05M sodium succinate. A standard was included which contained a l l the components of the above system (cells inactivated by boiling for 20 minutes) but with methylene blue at l/lO normal concentration.  This  tube represented 90$ reduction of the methylene blue and was used as the end point of reduction. An endogenous control was also run.  Thunberg  tubes were evacuated with a strong water suction pump for 3 minutes before they were sealed. Following a 10 minute equilibration period at 31° C, the substrate and methylene blue were tipped i n . The end point of the reaction was recorded as the time required for the colour intensity of the experimental tube to be reduced to that of the boiled cell standard. At the end of the reduction period, the seals were checked to confirm the presence of a vacuum throughout the experiment.  - 23 EXPERIMENTAL Dried oell preparations ,o"£ bacteria have been used for many years.  These preparations have a high endogenous respiration for 4 or  5 days, which drops off to a negligible value when the cells are stored in the refrigerator.  The enzymatic activity remains constant for  several months thereafter. These observations were made forty years ago by Shattock & Dudgeon (130) and later by Callow (25)• confirmed by Sleeper et a l . (134) with P. fluoresoens.  They have been  In this laboratory,  Stokes & Campbell (142) obtained quantitative data on glucose oxidation by P. aeruginosa, while Campbell & Stokes (29) have presented evidence for a Krebs cycle i n the same organism, both studies being made with dried oell preparations. With a view to obtaining a quantitative picture of the oxidation of the 4-carbon dicarboxylic acids i n P.aeruginosa, cells were grown on succinate and on acetate and dried in vacuo.  In Warburg experiments  (Figures VI,VII,VIII), the oxygen consumption was low i n comparison to that obtained by Campbell & Stokes (29) with glucose- or aoetate - grown c e l l s . In Figure VI, although the values for oxygen consumption are approximately half the theoretical, a definite relationship i s seen, i . e . 24 ul oxygen are required for the step succinate to fumarate, 28 ul for the step succinate to malate (theoretical in each case i s 56 u l ) , while 56 ul are used in the oxidation of succinate to oxalacetate (theoretical 112 u l ) . The succinate preparation was also able to oxidize citrate, cis-aconitate, isocitrate, and ^-ketoglutarate, while pyruvate and aoetate were not metabolized (Figure VII). The enzymes for oxidizing pyruvate and acetate were probably destroyed during the drying process. ' Similarly, Campbell & Stokes (29) found that the acetate enzyme was inactivated i f drying took  Bo MINUTES Figure VI. Oxidation of succinate, fumarate, malate and oxalacetate by dried c e l l s harvested from a succinate,ammonium phosphate medium. Warburg cups contained 20 mg. dried c e l l s , 0.5 ml. M/5 phosphate buffer pH 7.2, 2 uM MgSe^HgO, 2 uM MnClg, 0.4 mM A1C1 , 0.4 mM CaClg, 107 ug. DPN, and 5 uM substrate. F i n a l volume was 3.0 ml. 3  - 25 -  Figure VII, Oxidation of succinate, fumarate, i s o c i t r a t e , 'X'-ketoglutarate, cis-aconitate and c i t r a t e by dried c e l l s harvested from a succinate ammonium phosphate medium. Pyruvate and acetate were not metabolized. Warburg cups contained 20 mg. dried c e l l s , 0.5 ml. M/5 phosphate buffer pH 7.2, 2 uM MgSO^HgO, 2 uM MnClg, 0.4 mM AICI3, 0.4 mM CaCl2, 107 ug DPN, and 5 ui£ substrate. F i n a l volume was 3.0 ml.  - 26 longer than 48 hours, an observation whioh was also made by Barron et a l . (12) with dried cells of C. oraatinovorans. With aoetate-grown cells, the quantitative picture for the stepwise oxidation of the 4-carbon dicarboxylic acids more closely approached the theoretical (Figure VIII).  Here the differences i n oxygen uptake betweeen  succinate and fumarate and between succinate and malate are 45 and 433 ul respectively (theoretical i n both cases, 56 u l ) . With succinate as substrate, dried cells take up approximately one-third of the oxygen consumed by resting c e l l s .  Since the latter have  their oarrier systems and oof actors intact, i t i s possible that the low oxygen uptake charaoteristio of dried cells may arise from the absence of these necessary constituents. The system for succinate oxidation, known as the succinoxidase system, has been studied mainly with animal tissue homogenates (65,106,133) where i t i s generally supposed to consist of the following steps: Succinate  ....  Sucoinic dehydrogenase . ... cytochrome b —> cytochrome c —•^•cytochrome a  > cytochrome oxidase (ag)  oxygen.  Of the cytochromes, only "c" has been isolated as a soluble pure protein (64), evidence for the others being based on spectrophotometry observation. In addition, succinic dehydrogenase and cytochrome oxidase are closely associated and form an integral part of the large granules or mitochondria i n certain animal cells (58,129). Although the evidence for the existenoe of cytochrome as a oarrier system i n certain species of bacteria is controversial (137,163) and no large granules are present as sites of succinic dehydrogenase and cytochrome oxidase activity, i t i s probable that P.aeruginosa possesses the various constituents of the cytochrome system (45,137).  - 27 -  SUCCINATE  1b MINUTES Figure  VXII. Oxidation o f succinate, fumarate and malate b y dried c e l l s harvested from an acetate ammonium phosphate medium.  Warburg cups contained 20 mg. dried c e l l s , 0.5 ml. M/5 phosphate buffer pH 7.2, 2 uM MgSO^HgO, 2 uM MnClg 0.4 ml A1C1 0.4 ml CaClg, 107 ug. DPN, and 5 uM substrate. F i n a l volume was 3.0 ml. 3  - 28. There i s also the possibility of pyocyanine acting as a carrier sinoe i t is produced copiously by F.aeruginosa on a succinate ammonium phosphate medium, can be reversibly oxidized and reduced, and has been found capable of acting as a carrier system with various dehydrogenase systems i n vitro (38). In addition, a number of metal ions are required as cofactors for dehydrogenase systems. Ca and A l succinoxidase (112,113), while Mg  t+  enhance the activity of animal  and Mn  ++  are important cofactors for  a large number of enzymes i n the glycolytic; and Krebs cycles i n both animal and bacterial tissues (80). These four ions, therefore, were included in a l l experimental systems. The effeot of various concentrations of cytochrome c and of pyocyanine on oxygen uptake are given i n Tables I and II.  No significant  increase in oxygen consumption is evident. In these particular experiments there was no endogenous respiration. However, i n another experiment where the oells did oxidize their storage produots, no increase i n oxygen uptake occurred i n the presence of added cytochrome o or pyocyanine* Table I The effeot of cytochrome o on the oxygen uptake of dried succinate-grown oells.  Substrate: 5VuM succinate.  pH 7.2 . Oxygen uptake (ul) Time (minutes) 60 100 140 180  No cytochrome  1 mg. cytochrome  48 73 91 133  54 75 101 137  4 mg. cytochrome 43 79 86 131  - 29 Table II The effeot of pyooyanine on the oxygen uptake of dried suooinate-grown cells*  Substrate; 5 uM succinate. pH 7.4 • Oxygen uptake (ul)  T j j n 9  (minutes) 30 60 90 120 150  No pyocyanine 30 ' 70 90 113 131  0.001$ pyooyanine 37 69 87 105 124  0.01$ pyocyanine 37 76 97 121 142  The optimum pH of succinic dehydrogenase has been determined by ;  the Thunberg method for both succinate and acetate dried cells of F.aeruginosa,(Figures IX and X). With succinate cells, the optimum lies between pH 7.0 and 7.5,, while with acetate cells i t occurs at pH 8.0 .  -  30  -  Figure IX. Effect of pH on reduction of M.B. by dried succinate-grown cells, (2 preparations).  -  Figure  X.  E f f e c t  of  pH  on  reduction  of  31  M.B.  -  by  d r i e d  acetate-grown  c e l l s .  - 32 DISCUSSION The non-stimulatory effect of cytochrome c on succinate oxidation by P. aeruginosa may be the result of one or more factors*  F i r s t , there i s  the p o s s i b i l i t y that cytochrome o i s not a l i m i t i n g factor- i n t h i s oxidation. Or, i f we assume that the cytochromes are l i m i t i n g i n t h i s preparafc ion, the non-stimulatory e f f e c t may r e s u l t either from the i n a c t i v a t i o n of cytochrome oxidase during the drying process, or from the requirement f o r some cytochrome component other than "c" . Discussion i s r e s t r i c t e d to the l a t t e r two :  possibilities. "Very l i t t l e work has bean done with b a c t e r i a l cytochrome oxidase since Kubowitz & Haas (76) f i r s t obtained evidence f o r i t s occurrence.However, Borei (22) found that the cytochrome oxidase of horse heart became inactive with increasing age, due to aL terations i n the c o l l o i d a l struoture. Such an a l t e r a t i o n may also occur i n dried c e l l s of P. aeruginosa.  Support  f o r t h i s p o s s i b i l i t y i s contained i n the work of M i l i t z e r et a l . (88) who found cytochrome oxidase t o be the l i m i t i n g enzyme f o r the cytochrome chain i n thermophile No.  2184.  The evidenoe f o r the cytochrome system (a,b and c ) i n P.aeruginosa (45,137) i s based solely on spectrophotometric observation. There i s , therefore, no guarantee that the b a c t e r i a l components are s t r u c t u r a l l y i d e n t i c a l with those i n animal t i s s u e ( 4 5 ) .  Support f o r t h i s view may  be  found i n the fact that neither of the succinoxidase preparations made thus f a r from b a c t e r i a l o e l l s (77,106) require cytochrome c as a c a r r i e r . As noted i n the preceding section, pyocyanine does not inorease succinate oxidation by dried c e l l s of P. aeruginosa.  Actually the r.'.v  evidence f o r pyocyanine as a c a r r i e r system i s fragmentary.  Friedheim (43)  _ 33 observed an increased oxygen consumption when pyocyanine was added to suspensions of B. pyooyaneus, an observation which was later confirmed by Norris (93), Reed & Boyd (121) also comment on the respiratory significance of the pigment i n B. pyocyaneus cultures, while Dickens & Mcllwain (38) found i t capable of acting as a carrier with the hexosemonophosphate system of yeast juice and red blood c e l l s .  However, oontrary results were obtained  by Barron et a l . with C. creatinovorans (12), and by Keilin & Hartree (65) with a heart muscle preparation. <In these cases, pyocyanine was inhibitory. The optimum pH for suooinio dehydrogenase i n suocinate-grown dried oells lies between pH 7.0 and 7.5, while with aoetate cells i t occurs at pH 8.0.  These values coincide with others i n the literatures  Ohlsson (104)  in the original study on bacterial succinic dehydrogenase found. pH 9.0 to be optimum, while Quastel & Whetham (117), three years later, found a pH of 7.4 to be the most suitable for the reduction of methylene blue by resting oells of B.coli.  Subsequently, Cook & Aloook (34) found pH 7.6  to 8.0 to be optimum for the aerobic oxidation of succinate by E. c o l i . In more recent work, Kun & Abood (77) showed that oell-free preparations of succinoxidase from S« aertrycke have their optimum activity at pH 7.4, while Pappenheimer & Hendee (106) found pH 8.0 to be optimum for suocinio dehydrogenase from C. diphtheriae. The anaerobic reduction of methylene blue by resting oells of P. aeruginosa was studied by Randies & Birkeland (120). Guggenheim (52,53) investigated the same property in a number of pathogens (S. typhosa,. CI. butylioum, CI. parabotulinumi, and CI. welohii.)  - 34 PART III INHIBITOR STUDIES WITH MALONATE AND URANH, NITRATE Certain specific information concerning the pathways of cellular oxidation may be obtained by studying the effects of metabolic inhibitors* In general, these compounds f a l l into two main groups, depending on the manner in which they effect inhibition*  The first and by far the larger  group act by inhibiting the activity of enzyme systems directly*  They  may do so by combining with the enzyme system through one of the following: the activating protein, the prosthetic group or the carrier system, or by competing with or otherwise reduoing the effective concentration of substrate* Malonate, which inhibits the enzyme suecinio dehydrogenase by competing with the normal substrate for a position on the enzyme surface, has been chosen from this group* The second group of inhibitors act indirectly by altering one or more of the varied meohanisms which regulate the rate and direction of enzymatic reactions i n the living o e l l .  One of these regulating mechanisms  is the state of the oell surface, which i s important not only because of its property of selective permeability, but also because of i t s more direct role i n the "active" transfer of materials into the c e l l *  That this "active"  transfer i s oonnected with phosphorylation has been suggested by Barron (90) and by Rothstein (123,124) i n studies on the metabolism of glucose in yeast and bacteria, using uranyl nitrate as an inhibitor*  An investigation of  the effect of this inhibitor on the metabolism of organic acids and glucose in P* aeruginosa was undertaken in the present study*  In recent years,  ample evidence has appeared for a coupling between the oxidation of organic  acids or glucose and phosphorylation (10,11)• Uranyl nitrate, therefore, appeared to be a useful tool -whose action might provide more concrete evidence for this connection between aerobic metabolism and phosphorylation*  - 36 METHODS The methods of maintaining stock cultures were similar to those described i n Part I. Growth medium for the preparation of resting cell suspensions was similar to that used previously. Resting oell suspensions of 21 to 24 hours cultures were prepared as in Part I. Dried c e l l preparations were the same as described i n Part I I . Warburg' experiments were conducted as i n Part I. In malonate studies, Sorensen's phosphate buffer was used, while M/7 veronal buffer was employed xvith uranyl nitrate.  0.3 ml. of inhibitor was added at ten  times the f i n a l concentration desired. Thirty to forty minutes contact between inhibitor and cells was allowed before tipping i n the substrate from the side-arm. In malonate inhibition studies at pH's 5.0 to 5.6, substrate and mal onate were both neutralized with N/l NaOH to the required pH using a Beckman pH meter. Uranyl nitrate was also neutralized with N/i NaOH before being added to the Warburg cups. Percentage inhibition has been expressed in terms of the % 2 ( u ) values. Malonate inhibition of dried oell preparations was studied by both the Warburg method and by the Thunberg technique.  In the latter  instance, 0.3 ml. of ten times the desired concentration of malonate was l e f t i n contact with the c e l l suspension in the main tube for 30 minutes before adding succinate and methylene blue from the side-bulb. Chemically-clean glassware was used for determining the influence of magnesium on growth. This was prepared by soaking overnight i n 10$ n i t r i c acid, washing ten times with tap water, three times with d i s t i l l e d water, three times with re-distilled (glass) water, and finally autoclaving for 15 minutes at 15 pounds pressure.  The basal medium, consisting of  - 37 0.11$ KH 0H, 0.11$ K HP0 , 0.5 p.p.m. Fe^* (as FeS0 .4H 0), and 0.33$ 4  2  4  4  2  sodium succinate, was dispensed i n 9 ml. amounts into 125 ml. Erlenmeyer flasks, and autoclaved.  MgS0 .7H 0 \ms made up in concentrations ranging 4  2  from 0.001$ to 10$i and autoolaved separately.  1 ml. of the various Mg  ++  solutions was added aseptically to the basal medium. Final concentrations of various constituents were: 0.3$ sodium succinate, and Mg  0.1$ HH^OH, 0.1$ K HP0 , 0.5 p.p.m. Fe 2  ++  4  ,  ranging from 0.0001$ to 1$.  The inoculum was prepared from a' 21 hour culture of the organism in basal medium •+• 0.05$ Hg  ++  . These cells w ere harvested by centrifug-  ation, and washed twice with water re-distilled from glass. They were re-suspended to give a barely visible turbidity.  One drop of this suspension  was employed as inoculum for 10 ml© of medium. Following incubation for 24 hours, the turbidity of the suspension was read i n a Fisher Eleotrophotometer using F i l t e r 525 B.  - 38 EXPERIMENTAL A. Malonate Studies The inhibition of the anzyme succinic dehydrogenase by malonate was f i r s t reported by Quastel & Whet ham (118).  Subsequent work (34,49,  116,119) has confirmed their observation and demonstrated a high degree of specificity for the action of this inhibitor.  Szent-Gyorgyi and coworkers  (8,49) observed that malonate inhibited the respiration of pigeon breast muscle and that succinate was formed oxidatively from fumarate in the presence of malonate. Further investigations of these effects by Krebs and his associates (72,73,75) have furnished some of the strongest evidence for the oxidation of pyruvate by the tricarboxylic acid cycle. Malonate i s assumed to inhibit succinic dehydrogenase by competing with the enzyme's normal metabolite, sucoinate, for the "hot spots'" or active centres on the enzyme surface. These centres are thought to be oriented specifically to pick up succinate as a preliminary to i t s dehydrogenation, but may also adsorb closely related molecules such as malonate. This phenomenon of competitive inhibition, f i r s t observed by Qjuastel (115), results i n a more or less complete blocking of the enzymatic reaction©  The  effective concentrations of malonate are relative rather than absolite i.e. the ratio of suocinate to malonate is the important factor - since the affinity of the inhibitor for the enzyme is considerably greater than that of the substrate. Potter & DuBois ( i l l ) obtained 50$ inhibition with, a v substrate/inhibitor ratio as low as 50/l, while i n earlier work Krebs & Eggleston (73) required a ratio of 9.5/1 • I n i t i a l experiments were conducted with dried c e l l preparations. A preliminary study of the effect of pH on malonate inhibition was made according to the Thunberg teohnique.  The tubes contained 20 mg. dried cells  (suspended in 1 ml.), 1.0 ml. phthalate, phosphate or borate buffer  (depending on the desired pH), and 0.3 ml* of either web er or 0*01 M malonate*  In the side-bulb were placed 0*5 ml* of 0.05M suooinate and  0*5 ml* of 1*1000 methylene blue* The ganeral procedure was carried out as i n Part I*  Results are given in Tables III and IV. Table III The effect of pH on malonate inhibition of two  different dried preparations of suocinate-grown c e l l s Preparation A Reduction time (minutes) Succ. 4.1 4.6 5.5 5.9 6*5 7.0 7.5 8*0 9.0 10.0  165 108 57 56 40 23 14 17 20 35 Note:  Succ. 165 87 129 90 91 55 50 60 40 60  Preparation B Inhibition (per cent)  Mei l . 0 0 55 39 56 58 72 72 50 42  Reduction t ime (minutes)  Inhibition (per  Succ.  Succ.-+-Mal.  160 115 40  160 110 67  0 0 40  35 16 18 20 20 35  65 43 50 55 44 75  46 63 64 64 55 53  Endogenous controls showed no reduction at 180 minutes.  Table IV The effect of pH on malonate inhibition of succinic dehydrogenase activity i n acetate-grown dried cells pH 4.1 5.1 6.0 7.0 7.2 7.5 8.0 9.0 10.0 Note:  Reduction time (min.) Succ . •+• Malon* Succ. 180 63 37 27 27 26 24 31 48  180 57 59 47 42 41 45 46 68  Inhibition (per oent) 0 0 37 43 36 37 47 33 29  Endogenous controls showed no reduction at 170 minutes.  - 40 It may be concluded that malonate exerts i t s inhibitory effect in the pH range of 7.0 to 8.0. This i s also the optimum pH range for succinate dehydrogenation i n dried c e l l preparations, as found in Part II« Qoiastel & Yfooldridge (119) carried out their experiments at pH 7.4, using normal resting cells and toluene-treated cells of B. ooli. previous studies with P. aeruginosa  Since a l l  have been done at pH 7.2 and 7.4, i t  was decided to use pH 7.4 for further investigation of malonate inhibition. Warburg studies of the effect of malonate on succinate oxidation were carried put as i n Part II, with both succinate- and acetate-grown dried cells.  The results are summarised in Table V. Table V. The effect of malonate on the oxygen uptake by dried cells. Warburg vessels, contained 20 mg, cells, 0.5 ml. M/5 phosphate buffer pH 7.4, 2 uM. MgS0 .7H20, 2 uM MnCl , 0.4 mM AICI3, 0.4 mM CaCl2, 5 ^ 10 ^ succinate i n side-arm. Malonate was i n contact with cells 30 minutes before -.tipping i n substrate. 4  2  o r  Sxpt. No. I  Dried Cells  III  Malonate  Oxygen uptake (ul) Succ. SuccMale  60 120  ' 5 uM «  0.001 M  ,51 83 98  12 18 34  45 59 69  0 0 0  100 100  62 89  0 43  100 51  0.005 M  .62 89  0 14  100 84  94 135  47 87  50 36  94 135  9 41  1  8  Q  ,1  11  n  Succinate " "  66 120 180  5 uM: " "  0.005 M  Acetate «  60 120  5 uM  0.001 M  60 120  " "  11  " " 17  Suocinate.  Succinate " ti  II  Time  Aoetate "  11  n tt  11  60 120  10 uM "  0.001 M  60 120  '» '«  0.005M  ti  11  Inhibition (per c 77 78 65  loo  90 60  It is apparent that malonate in concentrations ranging from 0.001M to 0,005M is able to inhibit the oxidation of suooinate by dried c e l l preparations offf,aeruginosa, harvested from either succinate- or acetate- ammonium phosphate medium, Table V,  Since Krebs (72) considers  that i n these concentrations malonate acts exclusively upon the enzyme succinic dehydrogenase, we may conclude that both types of cells contain this enzyme. The fact that at a concentration of 0.001 M, malonate produces a greater inhibitory effect on sucoinate-grown oells than on acetate-grown cells would t i e i n with earlier observations that cells which have been grown on succinate contain a higher relative concentration of succinic dehydrogenase than those grown on acetate. In studies with 0.05M malonate at pH 7,4, Ney (91) was unable to inhibit either the rate or the extent of oxidation of succinate or glucose by glucose-arhmonium phosphate resting cells of P, aeruginosa. These experiments were repeated in the present study, using both 0,01M and 0.05M malonate at pH 7.2 and pH 7.4 • The cells were harvested from a medium in which sodium succinate constituted the sole oarbon source. The effect of 0.01M malonate on the oxidation of suocinate, fumarate and glucose, is given i n Table 71.  This concentration of malonate  inhibited the rate of oxidation of both succinate and fumarate, but not that of glucose. The inhibitory effect of malonate, i n concentrations 0.01M and 0.05M, on the oxidation of suocinate and fumarate has been computed in the form of ^ 2(K) )o  v a l , U 9 S  i  n  ^^ &  9  VII.  Experiment 1 is shown in Figure XI.  It i s  apparent that 0.01M and 0.05M malonate inhibit the rate of oxidation of succinate, and to a lesser extent that of fumarate.  - 42 Table 71 Oxidations by suooinate-grown resting c e l l s , i a the presence of 0.01M malonate»  pH 7>2 . •  Oxygen uptake ( u l ) Warburg substrate  Time (minutes)  Endog. •+• Malonate  Substrate  Endog, + Malonate* Substrate  Total  Inhibition (per oent)  20 40 60  18 38 70  174 219 221  192 247 291  145 230 267  25 7 8  Fumarate  20 40 60  18i. 38 70  164 209 218  182 247 288  131 192 228  29 22 21  Glucose n  20 40 60  18 38 . 70  29 50 73  47 88 143  55 112 190  0 0 0  Succinate it  II  II II  it  .  Table 711 Oxidations by auooinate-grown resting  cells,  i n the presence of O.OlM.and 0,05M malonate Experiment Number  pH  Warburg Substrate  7.2  Succinate  it  ti it  it  it  7.4: It  Succinate it  2(u)  Inhibition (per cent)  3220 2040 1190  0.01 0.05  2740 2740 1605  0.01 0.05  2222 1900 1105  14 50  0.01 0.05  2395 1940 1255  19 48  Fumarate tt  9:D  0.01 0.05  Fumarate  ti  II  Malonate (Molar)  37 63  0  41  - 43 -  FUMARATE  +0OS  MAJJOHAU-  \TE_ MALONATE SUCCINATE  HATE CCINATEU001H MALtbUATE  SUCCINATE +005~H MALONATE  12 Q  3 _MINUTES  4o  Figure X L . Oxidation of succinate and fumarate by 21 hr. c e l l s harvested from succinate-ammonium phosphate medium, i n the presence of 0.O1M and 0.05M malonate. Warburg cups contained 0.5 ml. c e l l suspension^!.. 5. ml. M/15 phosphate b u f f e r pH 7.2, 0.3 ml. malonate, o.2/substrate. F i n a l volume was 3.0 ml. Theoretical oxygen uptake f o r complete oxidation of any substrate was 403 u l .  - 44 Similar results have been obtained by other workers. Bernstein (18) observed a decrease i n the rate of oxygen uptake by cells of P. saccharophila when employing 0.01M  solutions of malonate. With 0.05M malonate, he  obtained a 71$ inhibition of amount of succinate oxidation and a 59$. inhibition in the case of fumarate.  In studies with a suooinoxidase preparation from  S. aertrycke endotoxin, Kun & Abood (77) obtained 100$ inhibition of succinate oxidation i n the presence of 0.01M malonate.  Quastel and his associates  (116,118,119) worked with concentrations of 0.02M malonate and higher to obtain inhibitory effects in their Thunberg studies with B. c o l i , B.prodigiosus, and B. proteus. Quastel & Wheatley (116) reported a 44$ inhibition; of succinate oxidation i n the presence of 0.067M malonate, over a period of 2 hours.  In the same study, succinate oxidation by fresh cells of B. c o l i  was inhibited 24$, while with cells whioh had been stored at 0° C. for several days a 90$ inhibition was observed*  The increase in inhibition  may have been due to an increase of cellular permeability.  Most  experiments with animal tissue have been carried out with lower concentrations of malonate than the above (1,111), although Stoppani (143) required 0.01M malonate to give a 99$ Inhibition of his succinoxidase preparation from heart muscle or l i v e r .  As to inhibition of glucose oxidation by malonate, Harvey  (56) noted that this compound exerted no effeot at 0.01M  i n experiments  with Trypanosoma hippicum. A l l these investigations have been made using malonate concentrations higher than those recommended by Krebs (72) . Das (35) suggested that at higher concentrations malonate may interfere with enzymes other than suocinio dehydrogenase. In this connection, Pardee & Potter (107) claim that malonat<a inhibits the oxidations of the Krebs cycle by at least two mechanismss  low concentrations (0.004M) completely block suocinate oxidation, -while higher concentrations (0»02M) effect inhibition of oxalacetate oxidation, presumably by forming a' complex with free or bound magnesium, a necessary oofactor for the oxidative decarboxylation of oxalacetate, Evans et al.(41) also noted that 0,01M  malonate completely inhibits the decarboxylation of  oxalacetate. Further evidence that oxalacetate oxidation i s the point of inhibition by malonate i n higher concentrations was provided by Potter (110), He found that malate oxidation i s not inhibited when oxalacetate i s removed from the system by transamination,  Lwoff & Cailleau (85) working with the  S mutant of Moraxella Lwoffi observed that oxalacetate oxidation was  completely  inhibited by malonate at a ratio of malonio acid j oxalacetio aoid of 10, An interesting though complicating feature of the present studies on malonate inhibition has been that P, aeruginosa possesses an adaptive enzyme system for metabolizing the inhibitor,  Karlsson & Barker (62) and  Karlsson (61) have found that Azotobacter agilis also is able to adapt to malonate, .Similarly, Baskett & Hinshelwood (16) observed that cells of B,lactis aerogenes became adapted to malonate after training on glucose, the normal growth medium. The original report on the bacterial oxidation of malonate was made by Butterworth & Walker employing B, pyooyaneus (24)• To provide conclusive proof for the existence of such an adaptive enzyme system i n P, aeruginosa, the following experiment was conducted. Cells which had been grown on suocinate and on aoetate mineral media for 21 hours were divided into two parts. One was set aside as a control non-adapted cells; the other was incubated for 1 hour with malonate (in the same concentration as the original growth substrates - v i a , 0»25$Q added to the medium - adapted oells.  In subsequent Warburg studies, the succinate  " - 46' -  enzyme of both groups of cells was saturated with i t s substrate i n a concentration of 5 uM, and with a high concentration of malonate (o»011l). . The resulting curves (Figures XII and XIII) indicate that there are indeed two separate enzyme systems present - one for metabolizing succinate, the other for oxidizing malonate. In the case of the non-adapted succinategrown cells, malonate had l i t t l e inhibitory effect,  The endogenous curves  indicate that the organism i s able to adapt to malonate. With the malonateadapted, succinate-grown  oells, two distinot curves are apparent. The curve  for oxidation of suocinate (5 uM) by the" adapted cells may be an example of the "uiauxie" effect of Monod (89), i n which a double growth cycle consisting of two exponential phases i s operative. Each curve i n this instance would correspond to the exolusive utilization of one of the constituents in the growth medium (i.e. either succinate or malonate) due to the inhibitory effect of one of the compounds on the formation of the enzyme for attacking the other. The phenomenon and i t s explanation would appear to be valid only i n the oase of compounds whioh are closely related i n structure. Cells whioh have been grown on acetate show adaptive curves fbrboth suocinate and malonate. In previous experiments at pH 7 . 2 and 7 , 4 , inhibition by malonate i n low concentrations was obtained with dried c e l l preparations, while high concentrations -were necessary to inhibit whole resting cells*  This suggests  that permeability i s involved} i . e . dried oells, i n which the cell membrane has been destroyed or altered, are inhibited, while whole cells, with the membrane intact, show no inhibition. Turner & Hanly (152) i n experiments with carrot slices found that potassium malonate at pH 4 , 0 caused "inhibition of that fraction of respiration which is inhibited by cyanide,"  They conoluded  that although the actual inhibitor is the malonic ion, only undissociated malonio acid serves as cell penetrant, so that a low pH is essential. Similar  - 47 -  to^H SUCCINATE•_ J  ^u/f  SUCCINATE  /0-0fM  MALONATE  MINUT£& Figure XII. Oxidation of succinate and malonate by non-adapted succinate-grown c e l l s (unbroken l i n e s ) and by c e l l s which have been adapted to malonate f o r 1 hour (broken l i n e s ) . Warburg cups contained 0.5 ml. c e l l suspension, 1.5 ml. M/15 phosphate buffer pH 7.2, O.g. ml. substrate. F i n a l volume was 3.0 ml.  - 48  -  SUCCINATE  SUCCINATE  SUCCINATE: ACETATE  SUCCWATB •fO-OIH MALONATE  SUCC/NAT1  / 0.01 M  MALONATE  MINUTES Figure XIII. Oxidation of succinate and malonate by non-adapted acetate cells (unbroken lines) and by acetate cells which have been adapted to malonate for 1 hour (broken lines). Warburg cups contained 0.5 ml. c e l l suspension, 1.5 ml. M/15 phosphate buffer, pH 7.2, 6.2 ml. substrate. Final volume was 3.0 ml.  results were obtained by Bonner & Wildman (21), Bonner (19), and Laties (78), . with, spinaoh leaves, oat ooleoptiles and barley roots respectively,  Gunsalus  (54) working with B, subtilis noted good inhibition at low pH's while none ocourred at pH 7.0 • The same effect of pH was observed by Barron et a l , with C, creatinovorans (12) and with baker's yeast (13), Results obtained with P, aeruginosa are given in Tables VIII, EC and X, and in Figure XIV,  A l l experiments were conducted at pH 5,0 to  5,6, since at lower values (i.e. pH 3,7 and pH 4,5) no oxidation of substrate took place. Maximum inhibition of rate and extent of oxygen consumption on succinate, with succinate-grown cells, was- found at pH 5,0 i n the presence of 0•005M malonate, Malonate affected only the rate of oxidation of other Krebs intermediates studied, v i z . fumarate, malate and V-katoglutarate. There was no inhibitory effect on,pyruvate oxidation. Table VIII  '  The influence of pH and concentration on malonate inhibition of succinate oxidation,using succinate-grown c e l l s . Experiment Number I  pH 5*6 " M  II  5.0  III  5.0 n  Malonate (molar)  Q  Q 2  ( )  — 0.001 0.005  895 747 494  ~ 0.001 0.005  975 812 283  0.002 0.003  1845 1 6 4 0 12  45  N  .  Inhibition Final Og of rate % uptake(ul) (at 60«) 200 16 170 45 110  17 71  11 33  Inhibition of amount % 15 45  (at 80') 275 275 80  0 71  (at 60') 235 225 180  4 23  - 50 Table IX Inhibition, of succinate and fumarate oxidation by 0*00511 malonate using fumarate-grown oells at pH 5*1 Experimental Conditions  Inhibition of 0 uptake .."Inhibition rate $ at 90' (ul) of amt. % 2  °2( ) N  Fumarate Fumarate + malonate  880 706  20  230 225  Succinate Succinate 4-malonate  840 350  58  220 130  ;  41  Table X Inhibition by 0.005M malonate of the oxidation of malate,  -ketoglutarate and pyruvate,  using oells grown on each of these substrates. Experimental Conditions  PH  °2(N)  Inhibition of rate %  Og uptake at 90' u l *  Malate Malate + malonate  5.1 n  1310 940  28  215 220  -ketoglutarate c<-ketoglutarate + malonate  5.5 "  1170 985  16  210 195  Pyruvate' Pyruvate 4 malonate  5*5 "  1590 1470  Inhibition of amt, %  260 262  Quastel & Wheatley (116) used 0*07M malonate to obtain 30$ inhibition of fumarate oxidation by B* pyooyaneus over 2 hours, aconcentration which did not affeot B, c o l i *  These results were typical for organisms which oxidize  fumarate faster than succinate; those with slower fumarate oxidation were inhibited to the extent of about 90$. Using the same concentration of malonate, they observed a 6$ inhibition of the amount of oxygen uptake with malate over  - 51  SUCCINATE SUCCINATE +0001H MALONATE SUCCINATE +0-00$ M  MALONATE  SUCCINATE + 0OOS M MALONATE  MINUTES Figure XIV. Inhibition of succinate oxidation by various of malonate on succinate-grown c e l l s .  concentrations  Warburg cups contained 0.5 ml. c e l l suspension, 1.5 ml. M/l5 phosphate buffer pH 5.0, 0.3 ml. malonate, 0.2 ml. substrate. F i n a l volume was 3.0 ml. Theoretical oxygen uptake f o r complete oxidation of any substrate was 403 u l .  a period of 2 hours, an amount which i s but slightly significant. Although only one experiment on inhibition was carried out, pyruvate'oxidation by P, aeruginosa was.found not to be inhibited significantly.  This finding  agrees with Lenti's .(81) observation that malonate affects the succinate but not the pyruvate oxidation of B, c o l i .  On the other hand, Barron et a l .  (13) found that malonate completely inhibited the oxidation of pyruvic acid , by yeast, a finding confirmed by Speck et_ a l , (136) with the malaria parasite. Similar results were obtained by Szent-Gyorgyi aid his coworkers (8,49) and by Krebs et a l , (73) using pigeon breast muscle brei,  Pardee & Potter (107)  found that 0.004M malonate strongly inhibited pyruvate oxidation in homogenates of rat heart, kidney and brain, but not in l i v e r ,  Lehninger ( 7 9 ) has  noted that liver homogenates can oxidize pyruvate to acetoacetate in the presence of malonate, B,  Inhibition by Uranyl Hitrate Barron (14,90) and Rothstein (123,124) have shown that uranyl  nitrate in low concentrations inhibits metabolism' in whole cells of yeast and bacteria by combining with the protein of the c e l l surface. uranium-protein  This  complex can be reversed by a number of compounds, including  phosphate, whioh form more strongly associated complexes with the uranyl ion than does protein (131). I n i t i a l experiments were designed to obviate any complication by phosphate either in the form of an insoluble precipitate in the culture medium, or as inorganio phosphate attached to the cell surface. To avoid precipitation of magnesium ammonium phosphate from the mineral medium, the concentrations of KH"*" and P0 """ were reduced to 0,1$ while that of Mg*  +  4  was lowered to 0,01$  4  or 0,005$, depending on the carbon source present.  - 53 At this concentration, optimal growth of the bacterium wis maintained, as shown i n Figure XV, which confirms the previous work of Burton et a l . (23). It was found that by washing the cells three times with 0.9$ saline, the concentration of phosphate attached to the cell surfaoe could be lowered to a negligible value. A l l experiments were oonducted with veronal as buffer©  The oxygen consumption of P. aeruginosa at pH 7.2 i s the same  whether veronal or phosphate i s used as buffer. The effect of uranyl nitrate in concentrations ranging from 7.5 M to 1 x 10  x 10  _<i  M has been investigated.  Concentrations of 1 x 10  M  to 4 x 10"^ M reduced the endogenous respiration of P. aeruginosa, as shown with gluoose oells i n Table XI.  The higher concentrations, 1 x 10  M and  2 1 x 10  M, caused agglutination of the c e l l s .  The inhibition of endogenous  respiration by lower concentrations i s probably due to a combination of the uranyl ion with the proteinaceous storage material of this organism (26), while at higher concentrations, uranium may be aoting as a general protein precipitant (131). Table XI The effect of ^ 2 ( ^ 3 ) 2 on the endogenous respiration of cells harvested from a gluoose ammonium phosphate medium. Duration of experiment was 70 minutes. Experiment Number. I  Experimental Conditions Control 1 x lO" 2.5 x lO" 4 x 10" Control 1 x lO" 1 x 10" 1 x 10-2  4  4  4  II  3  4  M M M M  Oxygen Uptake (ul) UJ  U U  U U M U  M  65 35 41 28 81 63 © 0  Inhibition (per cent) 46 37 57 22 100 100  - 54  Figure XV.  -  The influence of magnesium on the growth of F. aeruginosa  - .55The effect of uranyl nitrate on the oxidation of the Krebs. intermediates and of glucose by cells which have been grown on these compounds, is shown i n Tables<XII to XV, and Figure XVI.  Uranyl nitrate i n concentrat-  ions up to 4 x 10"* M did not effect a significant inhibition of glucose 4  oxidation by P. aeruginosa 9027*  (Table XII). In contrast to this finding,  Barron (14,90), employing the same species found 1 x lO  - 4  ! U0  + + 2  , and of 69$ with 4 x H T  M  4  U0  an inhibition of 25$ with ++ 2  •  Table XII The effect of U02(N03) on the oxidation of gluoose 2  by 22 hour cells harvested from a gluoose mineral medium. Inhibition (Molar)  *(»  — 1 x 10"* 2.5 x 10" 4 x 10"*  (per cent)  1050 1000 '925 875  4  4  4  5 13 17  T h e inhibitory action of uranyl nitrate on the oxidation of the dicarboxylio acids, succinate, fumarate and malate is shown in Table XIII* Succinate and fumarate oxidations were decreased only -when a relatively high concentration of U0  was used, while malate was completely unaffected •  2  Barron (14) found i t necessary to employ 1 x 10"^ M uranyl nitrate to inhibit succinate oxidation of E. c o l i , but at this concentration P.aeruginosa was agglutinated.  The absence of inhibition with malate may be explained  by the fact that this and other hydroxypolycarboxylic acids are able to reverse uranium inhibition of isolated enzyme systems (131).  Barron et a l ,  (14) also observed no inhibition of malate oxidation i n yeast, when they used concentrations of U 0  + 2  completely toxic to glucose oxidation*  Table XIII The effect of U0 (NQ3) on the oxidation of succinate, fumarate 2  2  and malate by 21 hour o e l l s harvested from an ammonium phosphate medium containing one of these compounds as sole carbon souroe. Experiment Humber®  Experimental Conditions  U0 (N0 ) (molar) 2  Succinate it it n  I  III  Fumarate ti  IV  2 4  ° (N)  Inhibition (per cent)"  807  Fumarate tt it  II  3  Malate n tt  1 x 10" 2.5 x l O " 4 x 10"  4  eo4  4  7853 616  0 3 24  7.5 x 10~ 1 x lO"  5  510 518 480  0 6  4 xlO"  4  522 320  39  1 x 10" 4 x 10"  4  1100 1200 1000  0 9  Higher concentrations of uranyl n i t r a t e  4  4  4  (2.5 x 10  M and 4 x 10" M) 4  were required f o r s i g n i f i c a n t i n h i b i t i o n cf the oxidation of pyruvate and oc -ketoglutarate (Table XIV), and of acetate (Table XV). The most s t r i k i n g data were obtained with isooitrate (Figure XVI), i n which even the lowest concentration (7.5 x 10"^ M) caused greater than 50$ i n h i b i t i o n , and higher concentrations ( l x 10*" M and 2.5 x 10" M) were almost 4  inhibitory.  4  completely  C i t r a t e oxidation, on the .other hand, was but s l i g h t l y  affected by these conoentrations (Table XV).  - &7 ;  Table XIV The effect of U02(N03) on the oxidation of pyruvate and 2  <-ketoglutarate by 22 hour oells harvested from an ammonium phosphate medium containing either of these compounds as C souroe Experiment Number.  Experimental Conditions  I  Pyruvate  UO (N0 ) (molar) 2  11  Q  11  «* -ketoglutarat e it 11  Inhibition (per cent)  1485 1251 10E8 1034  16 31 30  7.5 x 10" 1 x 10"  538 488 437  9 19  1375 1115 920  19 33  5  it  02(N)  1 x lO** 2.5 x 10-4 4 x lO  - 4  <*. -ket oglut ar at e  III  2  4  tt n II  ?  4  1 x 10" 4 x lO""  4 4  Table XV The effect of 1102(^03)2 °n the oxidation of acetate and citrate by 24 hour oells harvested from an ammonium phosphate medium containing either of these compounds as sole C souroe. Experimental Conditions Aoetate u ti  tt  U0 (NO,) (molar) 2  °2 Uptake (ul)  2  1 x 10"* 2.5 x l O 4 x 10** - 4 4  Citrate  tt ti n  1 x 10~* 2.5 xlO" 4 x lO" 4  4  (at 120') • 280 248 193 170 (at 105') 158 160 142 112  Inhibition (per cent)  11 31 39 0 10 29  - 58 -  Figure XVI. The i n h i b i t o r y e f f e c t of U0 (N03) on the oxidation of i s o c i t r a t e by 22 hour c e l l s harvested from an i s o c i t r a t e ammonium phosphate medium. 2  2  Warburg cups contained 0.5 ml. c e l l suspension, 1.5 ml. M/7 veronal buffer pH 7.2, 0.3 ml. U02(N0 ) , 0.2 ml. substrate.. F i n a l volume was 3.0 ml. 3  2  - 59 Attempts were made to reverse the inhibition of uranyl nitrate, by the addition of phosphate, which forms a strongly associated complex with the toxio ion. The oxidizable substrate was added .to the main oomparfcment of the Warburg cup (instead of to the side-arm), together with veronal buffer, cells and water to volume, Uranyl nitrate was added to one side-arm, and phosphate buffer to the other (0.3 ml. of each).  The UOg  ++  was added  from the f i r s t side-arm when the cells had been metabolizing their substrate at a constant rate for 30 minutes. added from the second sidefearm. ate  Fifty minutes later, phosphate was  In experiments with glucose and with succin-  oells, no significant decrease in oxidation was observed when the  uranium'was added i n this manner, and hence, no reversal of inhibition was effected by phosphate. Apparently, i t i s necessary to have the uranium and oells i n oontact for well over an hour to obtain measurable inhibition. This finding i s i n contradiction to the observation^ of Rothstein (123,124) and Barron (14,90), who obtained an instantaneous uranium uptake by yeast c e l l s .  In addition, Barron (14) always used 38° C.  for bacterial studies, while a l l experiments with P. aeruginosa ATC 9027 have been oonducted at 31° C. At the latter temperature, a much slower rate of reaction would be operative. Uranyl nitrate as an inhibitor on dried cell preparations provided additional evidence that the cell membrane has been altered duringthe drying process. Concentrations of 4 x 10"" M and 5 x 10"" M which had effected a 4  4  25$ inhibition with whole resting cells, showed no toxic effect on dried cells,-in fact, a slight stimulation was observed (Table XVI).  -  60  -  Table XVI The effeot of UOgCNOjOg  o n  ^he oxidation of succinate by dried  oells harvested from a succinate ammonium phosphate medium. Experiment Number  II  UOgCNOg^  Oxygen Uptake  (molar)  At 60»  At 120*  £ x 10-4  17 21  65 75.  — S x 10"  30 33  65: 70  4  % Inhibition At 60*  0  At 120'  0  DISCUSSION From studies of malonate inhibition i n P. aeruginosa i t may  be  concluded that this organism possesses the enzyme succinic dehydrogenase* Additional evidence for a close relationship amongst the compounds succinate, fumarate and malate was also provided.  While malonate strongly inhibits  both the rate and extent of oxidation of suocinate, only rate i s affected in the case of both fumarate and malate. The rate of «W:etoglutarate oxidation was also inhibitadj an observation which indicates an enzymatio relationship between this compound and succinates  Non-inhibition of  pyruvate oxidation might suggest that this intermediate is not oxidized via the same meohanism (the Krebs cycle) as those listed above. However, such a conclusion seems unlikely i n the light of other findings (157), nor can i t be drawn on the basis of a single experiment. Turning now to uranium, i t is believed to inhibit cellular metabolism indirectly by combining with the c e l l surface,, thus causing decreased permeability to the passage of oxidizable substrates.  According  to Knaysi (66,67,68) and others (135), the c e l l membrane consists of a lipoid-protein system, with associated ribonucleic acid. Apparently, uranium complexes with the protein constituents of the membrane, through certain "active" groups, which Barron (90) and Rothstein (123,124) suggest may be phosphates, phosphate esters or polyphosphates. Moreover, this uranium-protein complex can be reversed by the addition of inorganic phosphate at low concentrations.  These facts point to some relationship between  phosphorylation and the "active" transfer of substrates across the c e l l membrane. Additional support for this suggestion was found by Gale (46) in that the passage of. glutamic acid into the cell of Streptococcus ffigpalis is dependent on an exergonic metabolic reaction involving phosphate.  - 62 It also seemed possible that the energy for transport might arise from a coupling between phosphorylation at the "active" sites in the oell surface, and the oxidation of substrate - once the reaction had been primed by energy from some other coupled reaction (44)•  That there i s a coupling  between phosphorylation and the oxidation of glucose and of certain Krebs intermediates has been shown by a number of investigators (9,11,32,97,98, 109,144), i n studies made mainly with aaimal tissue.  Evidence for the  oxidative formation of energy-rich phosphate bonds in bacterial metabolism has been presented by Lipmann (83),with Lactobaoillus delbruckij by TissiSres (150) with Aerobaoter aerogenes, and by Hersey & Agl (57) with cell-free preparations of E,coli. When the present study was undertaken, i t was hoped that some association might be uncovered between uranium inhibition and the "active" (possibly phosphorylative) transfer of substrate across the oell membrane. However, i t is very d i f f i c u l t to interpret;, the results of the experiments, since the concentration of uranyl nitrate required for significant inhibition was not the same in each case.  For example, isocitrate oxidation was strongly  inhibited by a l l concentrations used, while malate showed no inhibition whatsoever.  Inhibition falling between these two extremes was observed with the  other substrates. Similar findings were reported by Rothstein & Meier (125) in work on molybdate inhibition of surface phosphatases i n yeast. that the protein with which the uranium complexes  It may be  is not only "active" i n 1  the transport of substrate, but is itself the enzyme for metabolizing the substrate.  Thus, different enzymes would show different sensitivities  to uranium. Such an hypothesis i s , however, far beyond the present bounds of experimental verification.  - 63 -  The greater part of the work on metabolic i n h i b i t o r s has dealt with the i n h i b i t i o n of i s o l a t e d systems.  The application  of i n h i b i t i o n  studies to the metabolism of whole o e l l s would appear t o be open t o question, especially when so l i t t l e i s known of a c e l l ' s actual metabolic pathways,  A reduction i n r e s p i r a t i o n may imply that an i n h i b i t o r i s  acting on one or more of the enzymes concerned with carbohydrate metabolism (162).  In addition, an i n h i b i t o r may cause an organism to use an alternative  metabolic pathway.  U n t i l more i s known of the direct and  alternative  pathways i n b a c t e r i a l metabolism, the use of a non-specific i n h i b i t o r such as uranium i s precluded.  - m -  SUMMARY  <  Resting cells of P. aeruginosa harvested from a mineral medium, in which succinate or fumarate or malate was the sole carbon source, metabolized a l l three compounds immediately and at a constant and rapid rate, thus indicating an enzymatic inter-relationship. The oxygen consumption of dried oells was considerably lower than that observed with resting c e l l s .  These preparations did not  require cytochrome c or pyocyanine as a carrier system. Optimum pH for suocinic dehydrogenase i n succinate and acetate dried cells lay between pH 7.0 and 8.0 • Moderately high concentrations of malonate were neoessary to inhibit succinate oxidation by resting cells at pH 7.0, but when the pH was lowered, maximum inhibition v/as effected by low concentrations. P. aeruginosa possesses an adaptive enzyme system for metabolizing the inhibitor.  Dried cell preparations were strongly inhibited by low  concentrations of malonate at pH.7.0, thus indicating that permeability is an important factor i n malonate inhibition with P. aeruginosa. Low concentrations of uranyl nitrate caused varying degrees of inhibition with resting cells i n the presence of the Krebs intermediates or glucose, the most striking effect being observed i n the case of isocitrate oxidation. No reversal of uranium inhibition by phosphate was obtained.  -  65  -  BIBLIOGRAPHY Ackermann, W. W. and Potter, V.R. Enzyme inhibitors i n relation to chemotherapy. Proc. Soc. Exper. B i o l . & Med. 72: 1-9. 1949. A j l , S.J. Biotin i n succinic acid oxidation. Enzymologxa 14: 1. 1950. A j l , S.J. Acetic acid oxidation by Escherichia coli and Aerobaoter aerogene3, J . Baot* 59: 499-507. 1950. A j l , S.J. and Workman, C.H. Replacement of carbon dioxide i n heterotrophic metabolism. 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