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Oxidative assimilation of glucose by Pseudomonas aeruginosa Duncan, Margaret Grace 1962

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OXIDATIVE ASSIMILATION OF GLUCOSE BY FSEUDQMOMAS AERUGINOSA by MARGARET GRACE DUNCAN B.Sc, University of British Columbia, 1960 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURAL MICROBIOLOGY in the division of Animal Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Ap r i l , 1962 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n permission. Department The U n i v e r s i t y of B r i t i s h Columbia, Vancouver S, Canada. i . ABSTRACT Manometrie observations of the oxidation of glucose by washed cell suspensions of Pseudomonas aeruginosa grown on a glucose medium suggested that one-third to one-half of the. substrate carbon was assimilated into cellular material. However, using uniformly-labelled 14 glucose-C as the substrate i t was found that during the period of rapid glucose oxidation only a small percentage of the C^ -4 was assimi-lated into the cells, whereas a large quantity of t^-ketoglutaric acid accumulated in the supernatant. A portion of the <<-ketoglutaric acid was gradually oxidized after the disappearance of glucose, while the remainder was incorporated as ammonia became available from the break-down of endogenous reserves. Most of the products of assimilation were nitrogenous and the addition of ammonia greatly increased the 14 amount of C assimilated and prevented the accumulation of <^-keto-glutaric acid; The cold TCA-soluble fraction and the lipid fraction appeared to be important during the early stages of assimilation, while the protein contained the largest amount of the incorporated radioactivity. The presence of azide increased the total oxygen consumption during glucose oxidation by the organism and this was accompanied by a significant decrease in the amount of radioactivity incorporated into the cells. The specific action of azide is most probably the uncoupling of oxidative phosphorylation for in the presence of high concentrations of azide there was l i t t l e change in the relative amounts of the various products of assimilation, but the rate of formation was slower and the amounts synthesized were smaller; In contrast, chloramphenicol specifically inhibited the synthesis of i i . protein while incorporation into the nucleic acid, l i p i d and cold TCA-soluble fractions was increased. Glucose-l-C^4, glucoser2-G14 and glucose-6-C"1,4 were also used as substrates. The results agreed with the observations of other workers that the Entner-Doudoroff and pentose phosphate pathways predominate in the oxidation of glucose by P. aeruginosa. Almost a l l the C-l of glucose was eliminated as carbon dioxide and that which was incorpo-rated was largely found in compounds such as amino acids, protein and nucleic acid which can be synthesized with the participation of carbon fixation. The C-2 of glucose was predominantly incorporated into li p i d and the C-6 into nucleic acid which may be the result of the major role of the Entner-Doudoroff pathway in glucose catabolism. Carbon dioxide fixation appeared to be important in cellular synthe-sis and in the synthesis of °t -ketoglutaric acid which undoubtedly arises via the tricarboxylic acid cycle. It appeared likely that the enzymes for &t -ketoglutaric acid oxidation and transport of the compound into the cell were repressed by growth on a glucose medium. The oxidation of extracellular st -ketoglutaric acid by glucose-grown P. aeruginosa required protein synthesis and was prevented by the presence of chloramphenicol. i i i . TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW 3 I. Early studies on oxidative assimilation 3 II. Inhibition of oxidative assimilation 7 III. Products of oxidative assimilation 9 MATERIALS AND METHODS 16 I. Organism and media 16 II. Preparation of washed c e l l suspensions 16 III. Manometric measurements 17 IV. Assimilation experiments 17 1. Incubation with glucose 17 2. Fractionation of whole cells 18 3. Hydrolysis of c e l l fractions 19 V. Chromatographic and electrophoretic methods 20 1. Chromatography of supernatants and c e l l fractions 20 2. Detection reagents 20 3. Preparation and chromatography of keto acid derivatives 21 VI. Isotopic methods 21 VII. Analytical methods 22 1. Ammonia determinations 22 2. Glucose determinations 22 3. Chemical determinations of keto acids 23 4. Glutamic dehydrogenase assay for U. -ketoglutaric acid 23 5. Protein determinations 24 iv Page 6. Ribonucleic acid determinations 24 EXPERIMENTAL RESULTS AND DISCUSSION 25 I. Oxidation of glucose-C^ 4 by resting cells 25 1. Manometric observations 25 2. The distribution of C 1 4 27 3. Identification of radioactive compounds in the supernatants 29 II . Products of oxidative assimilation of glucose 37 1. Distribution of intracellular radioactivity i n c e l l fractions 37 2. Studies on the composition of the various c e l l fractions 39 III. Stimulation of oxidative assimilation by ammonia 44 17. Oxidative assimilation of specifically-labelled glucose-C 1 4 50 V. The influence of C 1 2 0 2 on oxidative assimilation of glucose-C 1 4 55 VI. The oxidation of ^ -ketoglutaric acid 57 VII. The influence of chloramphenicol on oxidative assimilation 61 VIII. The influence of uncoupling agents on oxidative assimilation 70. 1. Sodium azide 70 2. Dinitrophenol 78 GENERAL DISCUSSION 81 SUMMARY 85 BIBLIOGRAPHY 87 TABLE OF FIGURES Figure Page 1 Oxygen uptake by washed ce l l s i n the presence of glucose 26 2 Distribution of C 1 4 added to washed cells and calculated uptake of ammonia 28 3 Standard curve for the determination of <<-ketoglutaric acid with glutamic dehydrogenase 35 4 Composition of the reaction supernatant 36 5 Absorption spectra of c e l l fractions 40 6 Influence of added NHg on oxygen uptake 45 7 Distribution of C 1 4 i n the presence of added NHg and calc-ulated uptake of NHg 47 8 Composition of the reaction supernatant in the presence of added NHg 48 9 Distribution of C 1 4 added as uniformly- or variously-labelled glucose 53 10 Oxygen uptake by washed c e l l s in the presence of various substrates 59 11 Influence of chloramphenicol on oxygen uptake 62 12 Distribution of C 1 4 in the presence and absence of chloram-phenicol 64 13 Influence of chloramphenicol on oxygen uptake in the presence of added HH^ 67 14 Influence of chloramphenicol on distribution of C 1 4 i n the presence of added NEg 68 15 Influence of azide on oxygen uptake 71 16 Influence of azide on the distribution of C 1 4 72 75 17 Influence of azide on oxygen uptake in the presence of added 18 Influence of azide on the distribution of C 1 4 i n the presence of added NH^ 76 19 Influence of dinitrophenol on oxygen uptake 79 v i . ACKNOWLEDGEMENT I would like to express my sincere appreciation to Dr. J.J.R. Campbell for his interest and helpful criticism during the course of the experimental work and the writing of this thesis. 1. INTRODUCTION Blhen washed suspensions of microorganisms are incubated in the presence of an exogenous carbon source the oxidation of a portion of the substrate may provide energy for the assimilation of the remainder into cellular constituents. This assimilation has been termed "oxidative assimilation? and since i t can occur in the absence of a nitrogen source the assimilated carbon might be expected to be accumulated in a limited number of cell materials. More recently, radioactive substrates have been used to determine the products synthesized by washed cell suspen-sions of microorganisms from a single carbon source. In general, these studies have shown that a primary product of assimilation may be formed which may later be utilized by the cells for energy or for synthetic reactions. It had been observed in this laboratory that when washed cell suspensions of Pseudomonas aeruginosa are incubated with glucose the total oxygen uptake is approximately two-thirds of the theoretical value for complete oxidation of the substrate to carbon dioxide and water (65). This organism therefore appeared to possess a mechanism for the oxidative assimilation of glucose. Warren, Ells and Campbell (83) had shown that the endogenous respiration of P. aeruginosa resulted in the production of ammonia, and that this ammonia was rapidly reincorporated into, cellu-lar material when glucose was added. No increase in intracellular carbo-hydrate could be detected after the addition of glucose, indicating that nitrogenous compounds undoubtedly accounted for much of the substrate shunted into oxidative assimilation. In the present study oxidative assimilation by P. aeruginosa was 3. followed in greater detail by using C x*-labelled glucose. The products of assimilation were determined by fractionation of the cells at various time intervals. Certain products of the incomplete oxidation of glucose which were found to accumulate outside the cells at various stages of the experiment were identified. The influence of added ammonia and of inhibitors such as azide and chloramphenicol on the incorporation of 14 radioactivity from glucose-C was studied. A limited amount of inform-ation on the pathways of degradation of glucose by this organism was obtained by the use of variously-labelled glucose-C 1 4 and evidence for the role of carbon dioxide fixation in cellular synthesis was obtained. 3. LITERATURE REVIEW I. Early studies on oxidative assimilation In the early work on the biochemistry of microorganisms i t was generally assumed that assimilation was restricted to proliferating cells. It was thought possible, therefore, to study the eatabolic processes, uncomplicated by anabolism, i f use was made of so-called "resting", or washed, cell suspensions in a buffer solution containing only a nitrogen-free substrate in experiments of short duration. In such experiments an excess of substrate was used to ensure a relatively constant rate of respiration during the experimental period and in many cases, in which the substrate had the elementary composition of carbo-hydrate, determination of the respiratory quotient suggested that com-plete combustion of the substrate took place. Cook and Stephenson (27), on the other hand, measured the total oxygen consumed during the u t i l i -zation of relatively small amounts of substrate by washed suspensions of Escherichia coli. The amount of oxygen consumed with substrates such as glucose, acetate and lactate was only two-thirds or three-quarters of the amount necessary for complete oxidation. The cause of this incomplete conversion of the substrate to carbon dioxide and water could not be decided. The studies of Barker (5) in 1936 on the oxidation of a number of simple organic compounds by the colorless alga, Prototheca zopfii, suggested that this incomplete oxidation might result from the conversion of a substantial part of the substrate into cell material and he termed this phenomenon "oxidative assimilation*. When cells were incubated with limited amounts of different substrates the amount of oxygen consumed and 4 carbon dioxide produced were found to vary with the nature of the sub-strate but not according to the amounts required for complete combustion of the substrate. Qualitative tests for various products of incomplete oxidation in the medium were negative. Since the molecular quantities of the reactants and of one of the products, namely carbon dioxide, were known i t was possible to deduce balanced equations for the oxidation of the substrates. For example, the equation for oxidation of acetate could be regarded as: CHgCOOH + 0 2 > C02+ HgO + (CHgO) In each case the portion of the substrate which was not oxidized to carbon dioxide and water had the empirical composition, ( C H g O ) . Since this organism was known to synthesize and store glycogen, Barker conclu-ded that glycogen was the "primary product" of oxidative assimilation of organic substrates and that this compound might subsequently be used as the raw material for a l l secondary syntheses within the cells on prolonged incubation or under conditions where growth was possible. Studies by Giesberger (37) on various Spirillum species, by Clifton (21) on Pseudomonas calco-acetica and on E. coli, and by Doudoroff (30) on Pseudomonas saccharophila lent further support to the theories of oxidative assimilation as advanced by Barker. Their invest-igations were conducted by the Warburg manometric technique. By this means the disappearance of the original substrate could generally be detected by a more or less sharp decrease in the rate of oxygen uptake. The actual amounts of oxygen used, and of carbon dioxide produced, in this i n i t i a l conversion of the substrate were estimated and they proposed equations similar to those postulated by Barker in which the assimilated 5 material had the empirical formula (CHgO)^. In addition, the experiments of Winzler and Baumberger (88) on the heat production by non-proliferating yeast during alcoholic fermentation suggested that assimilation of a considerable part of the substrate was not limited to respiring systems, but might also occur during fermentation. Manometric observations by van Niel and Anderson (80) of the metabolism of sugars by yeast suspen-sions under anaerobic conditions furnished evidence of a more direct nature for the occurrence of fermentative assimilation. However, experi-ments with Streptococcus faecalis f a i l e d to demonstrate assimilation during anaerobic sugar decomposition by homofermentative l a c t i c acid bacteria. A source of error i n the manometric determinations of the extent of assimilation was the uncertainty as to whether the endogenous respiration of the organisms continued, or was inhibited, by the addition of an exogenous carbon source. Also the presence of products of incomplete oxidation in the medium was usually determined by qualitative tests for a limited number of typical end-products, or established intermediates, of the oxidation of the substrate. Such tests were generally negative but i n other experiments, i n which carbon balances were carried out, the medium frequently contained significant amounts of unidentifiable compounds. The efficiency of assimilation of a particular substrate was found to vary with the organism studied. For example, E. c o l i and P. oalco-acetica assimilated three moles of carbon from one mole of glucose while P. saccharophila and Saccharomyces cerevisiae incorporated four moles of carbon from one mole of glucose. The early experiments also demonstrated that the extent of assimilation of a substrate appeared 6. to be a function of i t s chemical constitution rather than of the free energy available (24,30). The same amount of carbon was assimilated from lactate as from pyruvate, or from succinate as from fumarate, the free energy of oxidation of the f i r s t being greater than that of the second i n each pair. Likewise, the free energy of combustion of suc-cinate i s greater than that of lactate or pyruvate yet only one carbon was assimilated by E. coli from one molecule of each substrate. There-fore, the a b i l i t y of an organism to produce and to u t i l i z e building blocks from the substrate appeared to be as important in the assimilatory process as was the free energy inherent in the oxidation of the substrate. Attempts were made to compare the assimilation from certain com-pounds during c e l l multiplication with assimilation shown by washed suspensions of the same organism. I t might be expected that there would be a greater u t i l i z a t i o n of assimilated material i n an actively-growing culture and that a portion of this material would be expended by the cells i n their pursuit of energy and also as non-utilizable waste from the various syntheses carried out by the c e l l s . Whelton and Doudoroff (84) observed that the average amount of assimilation in cultures of P. saccharophila was generally less than i n resting c e l l suspensions of this organism. With glucose the ratio of assimilation during growth to-assimilation by resting cells was 0.90; corresponding ratios were 0.83 for maltose, 0.80 for sucrose, 0.84 for trehalose, 0.72 for lactate, 0.60 for pyruvate and 0.62 for acetate. No values for residual carbon i n the culture or suspension medium were reported and since the extent of assimilation was calculated from manometric data the results are suggestive rather than conclusive. They do indicate 7. that under optimal conditions at least some substrates are assimilated almost to the same extent by growing as by resting c e l l s . Siegel and Clifton (72) found by manometric and carbon-balance studies that the efficiency of assimilation by E. c o l l was 55-60% during growth in a glucose medium, which was as high or even higher than that observed with washed suspensions. However, later studies by Roberts et a l (70) on assimilation of C 1 4-labelled glucose by E. c o l l during growth for one generation i n a glucose-basic salts medium indicated a u t i l i z a t i o n of only 50% of the glucose disappearing from the culture medium for cellular synthesis, while approximately 25% of the glucose u t i l i z e d was secreted into the medium, primarily as acetate. Whether this was due to the particular growth conditions or i s peculiar to the strain of E. c o l l studied remains to be determined. II; Inhibition of oxidative assimilation In 1937 Clifton (21) studied the p o s s i b i l i t y of bringing about a complete oxidation of the substrate by blocking the asslmilatory pro-cesses with suitable c e l l poisons. C r i t i c a l concentrations of sodium azide and 2,4-dinitrophenol appeared to prevent synthesis by P. calco- acetica and E. c o l l In the presence of acetate and forced the reaction in the direction of complete oxidation of the substrate to carbon dioxide and water. The rate of oxygen uptake was somewhat reduced at these concentrations of the inhibitors and higher concentrations reduced the rate of respiration to a negligible value. Clifton and Logan (24) extended the observations with E. c o l l and demonstrated that azide and dinitrophenol, i n suitable concentrations, caused the complete oxidation of acetate, lactate, pyruvate, glycerol, fumarate, succinate 8 . and glucose. It was also noted that the extent to which dinitrophenol decreased the rate of oxidation varied with the substrate. Doudoroff (30) found that P. saccharophila oxidized a number of sugars, as weli as pyruvate and lactate, to completion in the presence of dinitrophenol. During the course of oxidation of glucose i n the presence of this inhibitor, pyruvate accumulated i n the suspension medium and, being u t i l i z e d at a slower rate than glucose, increased the time required for complete oxidation. In the following years many observations were made on the increase in oxygen consumption in the presence of azide and dinitrophenol with various c e l l types, including bacteria, yeasts, embryonic tissue and plant and animal tissues. Considerable variations were noted with different test agents and particularly with dinitrophenol the observa-tions depended on the substrates used (22,67,69). These poisons were also found to inhibit the assimilation of ammonia by bacteria (8,9) and to inhibit the Pasteur effect in yeasts and other tissue preparations displaying this phenomenon (69,77,91). The mechanism of action of these compounds can now be explained by their a b i l i t y to "uncouple* phosphorylation, that i s , they abolish the synthesis of high energy phosphate (adenosine triphosphate) associated with electron transport without impairing the oxidation of the substrate (13). The "uncoupling! effect of azide and dinitrophenol has been shown i n bacterial extracts in addition to mitochondrial preparations, although higher concentrations may be required to demonstrate this effect (11,64). 9 III. Products of oxidative assimilation Since bacteria appear to assimilate organic carbon with unimpaired efficiency in the absence of a nitrogen source, under such conditions i t could be expected that the assimilated carbon would accumulate i n a limited group of c e l l materials or "primary products". As mentioned previously, the empirical formula for the material assimilated by Protetheca zopfli led Barker to conclude that a carbohydrate, probably glycogen, was the product of assimilation (5). The empirical formulas presented by other workers also suggested the synthesis of carbohydrate. Winzler i n 1940 (87) demonstrated an increase i n the reducing sugar content, after hydrolysis with sulfuric acid, of yeast suspensions during the oxidation of acetate; This could account for approximately 80$ of the carbon theoretically assimilated. However, other workers concluded that l i p i d was the primary product of synthesis by yeast when acetate was the substrate (59). During the oxidative assimilation of glucose by Saccharomyees cerevisiae Pickett and Clifton (69) found that the increase i n readily hydrolyzable carbohydrate was approximately equal to the increase i n the dry weight of the c e l l s and therefore con-cluded that l i t t l e or no fat had been synthesized. Fales (34) noted differences i n the type of carbohydrate formed during fermentative or oxidative assimilation of glucose by washed yeast c e l l s . A transient alkali-insoluble carbohydrate other than glycogen or trehalose appeared to be the primary product of assimilation. It was synthesized at a more rapid rate than the alkali-soluble reserve carbohydrates and was converted to other products during later stages of glueose u t i l i z a t i o n i n which the alkali-soluble carbohydrates continued to be synthesized. Midwinter and 10. Batt (63) studied the distribution of assimilated C 1 4 from propionate-3-C 1 4 into cellular components of Nocardia corallina. They found that 37% of the counts were recovered i n the l i p i d fraction, which was equi-valent to approximately 30% of the dry weight of the c e l l s , while 52% were present i n a carbohydrate fraction which constituted less than 8% of the cellular material. The very high activity of the carbohydrate fraction suggested that carbohydrate was the immediate cellular consti-tuent on the assimilation pathway. Jackson and Johnson (49) studied the u t i l i z a t i o n and assimilation of sucrose and acetate by Torulopsis  u t i l i s i n a nitrogen-free medium. In the presence of substrate amounts of sucrose with substrate or trace amounts of uniformly-labelled acetate the radioactivity was predominantly incorporated into l i p i d and carbo-hydrate. However, i f uniformly-labelled acetate was used alone almost 50% of the assimilated material was found to be protein. Bacteria may also assimilate a considerable portion of the substrate carbon into polysaccharide, which w i l l be present as reserve food within the c e l l s or as structural components, particularly i n their c e l l walls or capsules (6,86). Dagley and Dawes (28) studied the factors influen-cing the polysaccharide content of E. c o l i during growth on various substrates. Levine et a l (55) found that glycogen was very widespread among enteric bacteria grown on the surface of carbohydrate-containing agar medium. The glycogen content was influenced strongly by the content of the medium, the age of the culture, the temperature of incubation and the density of the inoculum. Glycogen levels as high as 48% of the dry weight were reported. The accumulation of an alkali-stable polyglucose, of a glycogen-like nature, by E. c o l i c e l l s during nitrogen-starvation 11. in the presence of glucose was studied by Holme and Palmstierna (46). 14 During the last phase of this accumulation C -labelled glucose was added and the radioactivity was found almost exclusively in the glyco-gen. If the cells were then transferred to a medium containing ammon-ium chloride but no carbon source the glycogen was broken down and utilized for protein synthesis. It was shown that those glucose residues which were the last to be incorporated during the net synthe-sis of glycogen were the fi r s t to be split off by the cells under conditions causing the breakdown of glycogen. Oxidative assimilation of glucose and other compounds by Sarcina  lutea was studied by Binnie, Dawes and Holms (10). Cells harvested from a peptone medium usually contained carbohydrate as 10$ of the dry weight. During incubation of washed suspensions with glucose the carbohydrate content increased to as much as 28$ of the dry weight. When uniformly-labelled glucose was used as the substrate approximately 55$ of the radioactivity appeared within the cells and a good corre-lation was found to exist between the radioactivity assimilated and the increase in polysaccharide content of the cells, fractionation of the cells and analyses of the fractions confirmed that the major product of assimilation was a single compound which on hydrolysis gave rise to glucose. Experiments carried out with variously-labelled glucose, in which the specific activity of the product was compared with the specific activity of the substrate, suggested that the glucose was assimilated as an intact molecule without appreciable dilution. After the withdrawal of external glucose, the assimilated material was utilized, the rate of disappearance being much less in the later stages* 12. The endogenous respiration bore no simple relationship to the assimi-lated polysaccharide and, while the polysaccharide undoubtedly made some contribution to endogenous metabolism, i t was clearly not the sole substrate u t i l i z e d . When acetate or pyruvate were the substrates no increase i n the carbohydrate content of the organism was detected. By using variously-labelled compounds i t was found that the carboxyl group of pyruvate was largely eliminated prior to assimilation, i n d i -cating that pyruvate i s assimilated as a two-carbon fragment such as acetate. The bulk of the radioactivity incorporated from acetate or pyruvate appeared i n the cold 5% trichloracetic acid pool of metabolic intermediates. Ho attempt was made to identify the material present i n this fraction although aeetate i t s e l f did not appear to be accumulated by the c e l l s . The work of Wiame and Doudoroff (85) on the oxidative assimilation 14 ' * of C -labelled substrates by P. saccharophila supported the view that two-carbon fragments, of the nature of "active acetate", were the funda-mental building blocks in assimilation by this organism. Thus the carboxyl carbon of lactate was almost completely oxidized, the remaining two-carbon fragment being primarily used for synthetic reactions. Both carboxyl carbons of succinate were converted to carbon dioxide, while the methylene carbons were largely assimilated. Both carbon atoms of acetate were assimilated, the methyl carbon being favored, especially i n the presence of a nitrogen source. The nature of the primary products of assimilation by P. saccharophila was not elucidated. Both the "glycogen" and the l i p i d fractions of the cells which had oxidized labelled acetate or glucose had relatively low specific a c t i v i t i e s as compared with the 13. activity of other cellular constituents. These workers were unable to identify the radioactive cell constituents and suggested tentatively that the assimilated carbon flowed into many different cell materials including protein. It has been known for many years that Bacillus species contain a polyester of /2rhydroxybutyric acid as a major cellular constituent.) i The work of Macrae and Wilkinson (60) in 1958, provided convincing support that poly- /3 -hydroxybutyrate functions as an intracellular reserve of carbon and energy in Bacillus megaterium and Bacillus cereus. They found that glucose, pyruvate or fi -hydroxybutyrate were suitable substrates for the synthesis of this polymer by washed cell suspensions. Acetate, although unable to induce synthesis when present as the sole carbon source, greatly enhanced the formation of poly-/3 -hydroxybutyrate in the presence of these substrates. In the absence of an external carbon source degradation of this compound occurred rapidly. This polymer is chloroform-soluble and ether-insoluble and can be isolated by treating the cells with alkaline hypochlorite and extracting with chloroform. In the usual fractionation procedures for separating cellular constituents poly-^5 -hydroxybutyrate would be present in the ^protein? fraction. The widespread occurrence of poly-/3 -hydroxybuty-rate in Gram negative bacteria, including several non-pigmented Pseudomonas species, was demonstrated by Forsyth, Hayward and Roberts in 1958 (35). These observations led Doudoroff and Stanier to re-examine the products of oxidative assimilation by P. saccharophila (31). when 14 freshly harvested cells of this organism were incubated with C -14. labelled glucose only 21% of the glucose which disappeared was found in the cells, and of this two-thirds was accounted for as poly-(6-hydrozybutyrate. The extent of assimilation was very much less than that customarily observed in manometric experiments with starved cells. When starved cells were used more than 50% of the carbon of glucose was assimilated and the main product was poly-/3 -hydroxybutyrate. This polymer was also the major compound formed during assimilation of acetate and butyrate. Other experiments showed that poly-^?-hydroxy-butyrate could serve as the substrate for endogenous respiration of P. saccharophila in the absence of an exogenous substrate. These workers also studied the photosynthetic assimilation from acetate and butyrate in Bhodospirillum rubrum. At least 60% of the total carbon assimilated from either substrate was found as poly-/3-hydroxybutyrate. Therefore, with the advent of the use of radioactive substrates the nature of the products of oxidative assimilation has been more accurately determined. In the limited number of examples which have been studied a primary product of assimilation appears to be synthesized when washed suspensions of microorganisms are incubated with a carbon source and this compound may later be utilized by the cells for energy or secondary syntheses. In yeast the primary product appears to be carbohydrate or lipid while in certain bacteria i t is a carbohydrate and in others poly-/4-hydroxybutyrate. A considerably different situation was recently reported for the oxidative assimilation of glucose by B. cereus. Gronlund and Campbell (40) had shown that Pseudomonas aeruginosa, Pseudomonas fluorescens, Achromobacter species, £. coli. Bacillus subtilis, Saccharomyees 15. i eerevisiae and Streptococcus faecalls consistently produced appreciable quantities of ammonia during endogenous respiration. When glucose was added to respiring cell suspensions, a l l organisms except S. faecalls reincorporated the accumulated ammonia. Clifton and Sobek (25) found that considerable amounts of ammonia were also formed during endogenous respiration of B. cereus. Clifton (23) found that when washed cell suspensions of this organism were incubated in phosphate buffer in the presence of exogenous C 1 4-labelled glucose 50$ of the substrate was assimilated by the cells. The radioactivity was taken up rapidly into the metabolic pool soluble in cold 5$ trichloracetic acid and from this pool the label passed into other cellular fractions, primarily materials soluble and insoluble in hot 5$ trichloracetic acid, which may consti-tute the endogenous reserves of the organism* Ho evidence was obtained for the synthesis of significant amounts of poly-^-hydroxybutyrate. Clifton therefore suggested that the oxidative assimilation of glucose by B. cereus may serve, at least in part, to replenish or replace the nitrogenous endogenous substrates of the organism* 16 MATERIALS AND METHODS I. Organism and media The organism used throughout these studies was the obligate aerobe, Pseudomonas aeruginosa strain ATCC 9027. Stock cultures were transferred at regular intervals on glycerol peptone agar and stored at 5°C. The routine medium for the growth of this organism was composed of NH4H3PO4, 0.3%; E2HPO4, 0.2%; glucose, 0.2%; iron, 0.5 p.p.m. MgS04«7H20 was added after s t e r i l i z i n g to a concentration of 0.1%. Prior to each experiment the organism was transferred for two consecutive days i n this medium. A 1% inoculum was then added to 100 ml. quantities of the medium i n Roux flasks and the cells were grown for 20 hours at 30°C. In the experiment i n which the cells were grown i n the presence of 14 C -labelled glucose, 30 ml. quantities of the medium were used i n Erlenmeyer flasks so that a similar surface/volume ratio was obtained. After the cells had grown for 14 hours at 30°C the radioactive glucose was added aseptically and incubation was continued for 6 hours. II. Preparation of washed c e l l suspensions The cells were harvested by centrifugation i n the cold and washed twice i n cold 0.05 M tris(hydroxymethyl)aminomethane (Tris) buffer pH 7.2. The washed cells were then resuspended to approximately ten times the growth concentration unless otherwise indicated. The dry weight of ce l l s was determined by drying 5 ml. of the suspension to constant weight at 100°C and correcting for the dry weight of buffer present. 17 III. Manometric measurements Manometrio measurements were carried out in a Warburg respircmeter at 30°C. Each vessel contained 1 ml. of the cell suspension (approxi-mately 6 mg. dry weight cells when cells were resuspended to ten times the growth concentration), 0.05 M Trie buffer pH 7.2, and substrate, inhibitors or other additions where required to a total volume of 3.0 ml. When necessary, solutions of the substrate or inhibitor were adjusted to pH 7.2. In the experiments on the influence of inhibitors the cells were in contact with the inhibitor for 30 minutes before the substrate was added. The rate of oxygen uptake was expressed as the QOg or pi, of oxygen uptake in 60 minutes per mg. dry weight of cells. Carbon dioxide production was estimated by the direct method of Umbreit, Burris and Stauffer (78) for determinations of the respiratory quotient (R.Q,.). IV. Assimilation experiments 1. Incubation with glucose The experiments on the oxidative assimilation of C l 4-labelled glucose were carried out in Warburg respirometer vessels in order that the oxygen consumption could be followed. A typical reaction mixture contained: 0.1 ml. glucose (50 juM/ml.); 1.0 ml. cell suspension (appro-ximately 6 mg. dry weight cells); 1.9 ml. Tris buffer, pH 7.2 (0.05 M); and 0.15 ml. KOH (20$) in the center well. Concentrations of other components, where added, will be given in the text. The glucose was added from the side arm after temperature equilibration at 30°C. In the experiment on the influence of a non-radioactive carbon dioxide atmosphere on the incorporation of C 1 4-labelled glucose, the potassium 18 hydroxide was omitted from the center well and several l i t e r s of a 10% carbon dioxide-90% a i r mixture were flushed through the gas phase above the reaction mixture. The reaction was stopped at the stated time intervals by pipetting the contents of the vessel into ice-cold Pyrex centrifuge tubes con-taining 1 ml. of buffer and the tubes were immediately centrifuged i n the cold. The supernatant was decanted and held in ice or frozen at -18°C. Residual supernatant adhering to the walls of the centrifuge tube was removed by absorption on a paper swab. The c e l l pellet was fractionated immediately. 2. Fractionation of whole c e l l s The fractionation procedure was a modification of the method of Roberts j3_t a l (70). The c e l l pellet i n the Pyrex centrifuge tube was resuspended i n 2 ml. of cold d i s t i l l e d water and 0.2 ml. of the sus-pension was removed for estimating the radioactivity of the unfrac-tionated c e l l s . (Since washing the c e l l pellet did not remove signi-ficant amounts of compounds found i n the reaction supernatant this step was not routinely carried out.) An equal volume of cold 10% trich l o r -acetic acid (TCA) was added to the remainder of the c e l l suspension to give a f i n a l concentration of 5% TCA (w/v) and the tube was held at 5°C for 30 minutes. The mixture was centrifuged, the supernatant being the cold TCA-soluble fraction. The residue from the cold TCA extraction was suspended i n 4 ml. of 75% ethanol (v/v) which had been adjusted to pH 2.5 with dilute sulfuric acid (25) and incubated at 45°C for 30 minutes. The supernatant removed by centrifugation i s referred to as the alcohol-soluble fraction. The pellet was suspended i n 2 ml. of 75% 19. ethanol (v/v) plus 2 ml. of ether and the mixture was centrifuged after incubation at 45°0 for 15 minutes. The supernatant was the alcohol- ether-soluble fraction. The residue was suspended i n 4 ml. of 5$ TCA and heated at 90°C for 30 minutes. After centrifugation the super-natant or hot TCA-soluble fraction was removed and the residual protein was dissolved i n 4 ml. of 0.5 N NaOH. After an aliquot of the alcohol-soluble fraction had been removed for radioactive measurement an equal volume of ether was added to the tube. Two ml. of d i s t i l l e d water was then added causing a separation into two phases. After shaking, the ether phase was removed and the extraction repeated a total of three times by adding more ether. The ether fractions were combined and the total volume determined. This alcohol-soluble, ether-soluble material and the alcohol-ether fraction previously obtained were considered to contain the l i p i d and phospho- l i p i d of the c e l l . The portion of the alcohol-soluble fraction which was not extracted by ether was termed the alcohol-soluble protein. 3. Hydrolysis of c e l l fractions The residual protein was washed with acid alcohol and with ether to remove traces of TCA. It was then hydrolyzed i n a sealed glass tube with 1.0 N HCI at 108°C for 6 hours. The hydrolyzate was dried and suspended in 1 ml. of d i s t i l l e d water. This solution was placed on a column of Dowex 50 H + and washed with 4 column volumes of d i s t i l l e d water. The amino acids were eluted with 1.5 N NH4OH. The eluate was boiled to remove the ammonia and concentrated i n the flash evaporator to obtain the solution for chromatography. The water wash was also concentrated and chromatographed. 20 The hot TCA fraction was extracted repeatedly with ether to remove the TCA and hydrolyzed in 1 N HC1 for 6 hours at 108°C, or in 0.5 N HC1 for 1 hour. The hydrolyzates were evaporated to dryness and suspended in distilled water for chromatography. V. Chromatographic and electrophoretic methods 1. Chromatography of supernatants and cell fractions Routine chromatography of the supernatants of the reaction mixtures and of the various cell fractions was carried out on Whatman No.l paper using the sec-butanol-formic acid-water (70:10:20 v/v) solvent of Roberts et al (70). Prior to chromatography of the cold and hot TCA-soluble fractions the TCA was removed by repeated extraction with ether and the fractions were adjusted to neutral pH. The solvent n-butanol-acetic acid-water (65:10:27 v/v) was used for the chromatography of amino acids and protein hydrolyzates on Whatman No.l paper. Organic acids were chromatographed on Whatman No.l paper using the sec-butanol-formic acid-water solvent, n-butanol-acetic acid-water (60:15:25 v/v) and ethanol-ammonia-water (80:5:15 v/v). Paper electrophoresis was carried out at 750 volts with Whatman No.3 mm. paper. The buffer was 0.05 M ammonium carbonate, pH 8.5 Following chromatography or electrophoresis for the time intervals stated in the text the paper was dried in an oven at 50°C and the radioactive areas were located before the application of reagents. 2. Detection reagents Amino acids were detected by dipping the chromatograms in acetone-acetic acid (90:10 v/v) with 0.5% ninhydrin. The paper was allowed to dry at room temperature and the color developed by heating at 70°C for 21. 10 minutes. Reducing sugars were located by dipping the sheets in acetone containing 0.5$ saturated AgNOg and drying, followed by immersion in 0.6 H NaOH in 70$ ethanol and then a 5$ sodium thio-sulfate solution. Organic acids were detected with an acid-base indicator spray (1) or the aniline-xylose reagent described by Smith (74). The latter method, which detects 10 ;ag. of most organic acids was generally used. 3, Preparation and chromatography of keto acid derivatives The preparation of the standard keto aeid-2,4-dinitrophenylhydra-zones was carried out by the method described by Smith (74). The hydrazones of unknown radioactive compounds were prepared by incubation for 2 hours at 37°C with 2,4-dinitrophenylhydrazine in 2 N HCI and extracted with ethyl acetate and chromatographed directly. Alternative-ly the unknown compounds were incubated with pyruvic or °t-ketoglutaric acid during the preparation of the respective standard dinitrophenyl-hydrazones. Chromatography was carried out using Whatman No. 4 paper with the solvent n-butanol-ethanol-ammonia (70:10*20 v/v) (35). The dinitrophenylhydrazones were detedted visually and by their absorption in the ultra-violet region. The spots were developed by dipping in 2$ NaOH in 90$ ethanol and the shade and intensity of the resulting color was noted. By these methods 1 to 2 ;ug. of the dinitrophenylhydrazones could be detected after chromatography. VI. Isotopic methods Uniformly-labelled glucose-©^4, glucose-l-C 1 4, glueose-2-C14 and 14 glucose-6-C were obtained from Merck and Go. Ltd. and were diluted with non-radioactive glucose solutions so that 5 pM. of glucose with a 22. specific activity of approximately 3.5 p. curies per 5 pM. was added to each reaction vessel. For the experiment in which the cells were grown 14 in the presence of glucose-U-C , 15 juM of glucose with a specific activity of 80 p. curies per 15 pM was added to the medium. The counts per minute added in each experiment were verified by plating and counting dilutions of the glucose solution. Aliq.uots of the reaction supernatants, cell suspensions and cell fractions were plated in duplicate on stainless steel planchets and dried under an infra-red lamp. The planchets were counted at infinite thinness using a Nuclear-Chicago scaler Model 181 A equipped with a gas-flow counter having a thin-end-window Geiger tube. Corrections were made for coincidence and background. Whenever possible at least 1000 counts were recorded so that the statistical deviation was less than 3.3%. Radioactive areas on chromatograms were determined by running one-inch strips through a Nuclear-Chicago Model C 100 B Actigraph II with the gas flow counter, a Model 1620 B Analytical Count Ratemeter and a Chart Recorder. VII. Analytical methods 1. Ammonia determinations The ammonia in the supernatant from the reaction mixture was determined by the Conway microdiffusion method (26). 2. Glucose determinations Glucose was determined by the ••glueostat* method of Jforthington Biochemicals, Freehold, N.J. 23 3. Chemical determination of keto acids A modification of the method of Friedemann (36) mas used to determine oC-ketoglutaric acid and pyruvic acid. The separate determi-nation of the keto acids i s based on differences i n the rate of reaction with 2,4-dinitrophenylhydrazine with mono- and dibasic keto acids and the fact that monocarboxylic acid dinitrophenylhydrazones are prefer-entially extracted from acid aqueous solutions by aromatic hydrocarbons; the dibasic acid dinitrophenylhydrazones by aliphatic and aromatic alcohols. In the pyruvic acid assay pyruvic acid standards, oC-keto-glutaric acid standards and the unknown were incubated with the 2,4-dinitrophenylhydrazine for 5 minutes and the dinitrophenylhydrazones were extracted with benzene. In the c<-ketoglutaric acid assay the above materials were incubated with the 2,4-dinitrophenylhydrazine for 25 minutes and the dinitrophenylhydrazones were extracted with benzyl alcohol. Centrifugation at low speeds was required to separate the phases when benzyl alcohol was the solvent. Mechanical shaking was substituted for nitrogen bubbling i n the extraction procedure. The optical densities of the f i n a l solutions were read at 435 mu and 390 mu which allowed the relative amounts of each acid present i n the unknown solutions to be determined. 4. Glutamic dehydrogenase assay for <*i-ketoglutaric acid The oxidation of reduced diphosphopyridine nucleotide (DPNH) by glutamic dehydrogenase, as measured in the spectraphotometer at 340 mu in the presence of excess ammonia and limiting ot -ketoglutaric acid, was used for the quantitative estimation of ot-ketoglutaric acid. The enzyme was supplied by the Nutritional Biochemical Co. and i t appeared 24, to lose activity rapidly. However, i f any precipitated material was removed by centrifugation and the enzyme solution was used during a short time interval, a satisfactory assay was achieved which appeared to be very sensitive and specific. The procedure used was as follows: 0.08 ml. of DPNH (1.5 pM/ml.), 0.7 ml. of NH4CI (600 pM/ml.), 0 to 0.06 ml. of <*-ketoglutaric acid (1 pM/ml.) or 0.1 ml. of unknown solution, and 0.05 M phosphate buffer, pH 7.6, to give a total volume of 0.96 ml. were added to cuvettes and the optical density was set at 0.500 at 340 mu on the Beckman D.TJ. spectraphotometer. At zero minutes 0.04 ml. of the enzyme solution was added to the cuvettes and after mixing the total decrease i n optieal density was determined. A l l the reactants used were carefully adjusted to pH 7.6 and the appropriate controls were carried out. If the substrate, enzyme, NH4CI or DPNH were omitted there was no significant change i n the optical density. 5. Protein determinations Protein was determined by the method of Lowry et a l (58) using double-strength reagents, the optical density being measured at 500 mu. Egg albumin was used for establishing the standard curve. 6. Ribonucleic acid determinations Ribonucleic acid was measured by the orcinol method (71). Adenosine-3'-monophosphate was used as the standard. 25. EXPERIMENTAL RESULTS AND DISCUSSION I. Oxidation, of glucose-C 1 4 by resting c e l l s 1. Manometric observations Typical curves for the uptake of oxygen during dissimilation of glucose by P. aeruginosa are shown i n Figure 1. These results were obtained with equivalent dry weights of cells and the values for endogenous oxygen consumption have been subtracted, as previous work (65) had shown that endogenous respiration continued at the same rate in the presence or absence of glucose. The i n i t i a l rapid rate of oxidation of glucose had a QOg of approximately 110 and a respiratory quotient (R.Q.) of 0.9. The break i n the curve, which was shown to coincide with the disappearance of glucose, occurred when the oxygen uptake was 45-50$ of the theoretical value for complete oxidation. This was followed by the second stage, characterized by an R.Q. of 1.0 to 1.05 and a low QOg that varied with the ratio of substrate to c e l l s . For example, the Q,02 during the second stage was 8 to 9 with 3 juM of glucose, 12 to 15 with 5 pM of glucose, and 22 with 10 uM of glucose. This stage continued u n t i l 65-70$ of the theoretical oxygen uptake was achieved. From the oxygen uptake values one might expect that approximately 50$ of the glucose had been assimilated during the i n i t i a l stage and that the oxygen uptake during the second stage was due to the oxida-tion of assimilated material. 26 0 30 60 90 120 T I M E ( M I N U T E S ) F I G U R E I . O X Y G E N U P T A K E B Y W A S H E D C E L L S I N T H E P R E S E N C E O F G L U C O S E 27. 2. The distribution of In an effort to determine the distribution of the carbon during 14 the oxidation of glucose, 5 juM of uniformly-labelled glucose-C was 14 used as the substrate. Figure 2 shows the percentage of the C added which was present i n the reaction supernatant and i n the c e l l s at various time intervals. The amount of C 1 4 evolved as carbon dioxide was calculated by subtracting the radioactivity of the c e l l s and supernatant from that added to the vessel. This value agreed f a i r l y well with the carbon dioxide evolution found by manometric experiments. The very low incorporation of C 1 4 into the c e l l s was surprising: for example, at 30 minutes only 10% of the radioactivity was present i n the cells while 50% assimilation was suggested by the manometric data, and at 120 minutes approximately 16% of the C 1 4 had been incorporated whereas the oxygen uptake values suggested 35% assimilation. Ammonia determinations were carried out on the supernatants of the endogenous controls. The results are presented i n Table 1. Since a l l evidence indicates that the endogenous respiration of this organism continues unabated i n the presence of an oxidizable substrate such as glucose (65) and since ammonia i s a major product of endogenous respir-ation (83) i t was assumed that ammonia formation by resting c e l l s would be the same i n the presence or absence of substrate. The amount of ammonia reincorporated into the c e l l s was calculated as being that amount released by endogenously respiring cells minus that which was present i n the reaction vessel containing added glucose; The calculated ammonia uptake i s shown graphically in Figure 2 and compared with the 14 incorporation of C into the c e l l s . The similarity i n the shape of 28. 100 8 0 " 6 0 % C 1 4 ADDED 4 0 2 0 % C % C 14 14 SUPERNATANT C E L L S O-X-— C % C 1 4 C 0 2 (CALC.) - - X pM N UPTAKE (CALC.) 5 /i M NH. - - 2 3 0 12 0 F IGURE 2 6 0 9 0 -T IME (MINUTES) D ISTRIBUTION OF C 1 4 ADDED TO WASHED C E L L S AND C A L C U L A T E D U P T A K E OF N H , 29 the curves suggested that ammonia was the factor which determined the rate of assimilation of carbon by the c e l l . Table 1 The production and reincorporation of ammonia NH^ present per 3 ml. reaction vessel Calculated Time Endogenous Glucose MH^  uptake min. pM }M pM. 0 0.27 0.27 0 15 0.50 0 0.50 30 0.73 0 0.73 60 1.20 0.14 1.06 120 1.72 0.42 1.30 The concentration of glucose i n the reaction supernatant was followed and i t was verified that a l l the glucose had disappeared by 30 minutes when the break i n the oxygen uptake occurred. Since at least 35% of the C was s t i l l present i n the supernatant at this time i t was apparent that some product, or products, of the incomplete oxidation of glucose accumulated outside the c e l l s . Investigations were carried out i n an attempt to identify these radioactive materials. 3. Identification of radioactive compounds in the supernatants The results of chromatography of the reaction supernatants with the solvent sec-butanol-formic acid-water are shown i n Table 2. The chromatograms were run for 20 hours and the radioactive areas were determined by running strips through the Actigraph II. The values are presented i n order of decreasing intensity of the radioactivity i n the spots and compared with the value for glucose. 30. Table 2 Chromatography of reaction supernatants i n see-butanol-formie acid-water Sample C 1 4-labelled materials % Glucose .20 Supernatant 15 min. .20, .68, .35, .80 30 min. .68, .35, .80, 0 60 min. .68, .35, .80, 0 120 min. o, .35, other small peaks The radioactivity i n the supernatant at 15 minutes appeared to be largely due to glucose while a compound with an Rf value of .68, which w i l l be referred to as compound A, was very prominent from 30 to 60 minutes. With the exception of glucose and the compound with an Rf value of .20 none of the spots reacted strongly with any of the location reagents tested. Samples of the compounds with the R^  values of .68 (compound A), .35 (compound £) and .80 (compound C) were prepared by running streaks of the supernatant at 30 minutes in sec-butanol-formie acid-water and elution from the dried chromatograms with d i s t i l l e d water. The eluates were concentrated i n the flash evaporator. The concentrated compound B was found to react with ninhydrin and i t was identified by chromatography and electrophoresis using known amino acids for comparison. Typical results are shown i n Table 3. It was concluded from these results that this compound was glutamic acid. 31. Table 3 Chromatography and electrophoresis of compound B and known amino acids Sample Compound B Glutamic acid Aspartic acid Proline Alanine Threonine Chromatography % .35 .35 .26 .41 .28 Ele c tropho re s i s at pH 8.5 for 1 hr. cm. to anode 7.3 7.5 8.4 - 0.1 0.2 Comments ninhydrin purple yellow purple The concentrated compound A gave a definite acid reaction when chromatograms were dipped i n an acid-base indicator, therefore various organic acids were used as standards ITable 4). Compound C corresponded very closely to succinic acid and was also present each time compound A was chromatographed even when compound A was eluted and chromatographed several times. It was suspected that compound A was oC-ketoglutaric acid from the Rf values and from the fact that succinic acid appeared to be derived from i t . However the possibility that i t was pyruvic or oxalaeetic acid or contained a mixture of the keto acids could not be ruled out. Since free keto acids are not particularly stable the presence of streaks or multiple spots for these compounds was not surprising. Table 4 Chromatography and electrophoresis of compounds A and C and organic acids Sample sec-butanol-formic acid-water n-butanol-acetic acid-water ethanol-ammonia-water electrophoresii at pH 8.3 for: Zh. hr. i f hr *f cm. to anode Compound A .68 (.79) .67 (.78) .51 (.46) 26 23-24 Compound C .79 .77 .46 29 25 c^-ketoglutaric acid .69 .71 .49 26 23 Pyruvic acid .67-.79 ,67-.77 .48-.66 24-26 19-22 Succinic acid .78 .77 .45 29 24 Oxaloacetic acid .69-.92 .63-.80 0 -.54 24-27 17-23 Malic acid .59 .63 .37 28 -Fumaric acid .85 ;83 .48 30 25 Aconitic acid .80 .75 .27 28 -Glyoxyllc acid .48,.58 .47,.70 .20,,32 - -C i t r i c acid .52 - - - -2-ketogluconic acid .16 _ ( ) indicates second radioactive peak with slightly less activity 33. Further identification of compound A was carried out by the preparation and chromatography of 2,4-dinitrophenylhydrazone deriva-tives. Two procedures were carried out for the identification of compound A. In the fi r s t the dinitrophenylhydrazone of A was prepared directly and in the second compound A was incubated with pyruvic or <?<.-ketoglutarie acid during the preparation of the dinitrophenylhydra-zones. The results in Table 5 indicate that oi-ketoglutaric acid is the principal keto acid in compound A by a comparison of the Bj values, 14 the color developed by alkali and the preparation of a C -labelled dinitrophenylhydrazone in the presence of oC-ketoglutaric acid but not in the presence of pyruvic acid. Table 5 v  Chromatography of keto acid 2,4-dinitrophenylhydrazones Sample 2,4-dinitrophenyl-hydrazine Oxaloacetic DNPH 2-ketogluconic DNPH Pyruvic DNPH ot -ketoglutaric DNPH A DNPH Pyruvic -f A DNPH ^-ketoglutaric +A DNPH Bj Badioactivity Color with alkali reagents 0 .68 .84 .17 .31 .48 .72 0 .30 0 .30 .45 .72 0 .30 0 .30 none none 0 .30 purple-brown yellow-tan reddish-brown reddish-brown orange reddish-brown yellow-tan purple-brown (overspotted) green purple-brown (overspotted) green reddish-brown yellow-tan purple-brown (overspotted) green DNPH = 2,4-dinitrophenylhydrazone. 34 Chemical assays for <?<.-ketoglutaric and pyruvic acid in the reaction supernatants were carried out by the modification of the method of Friedemann (36) described in Methods. The 30 minute reaction supernatant was calculated to contain approximately 1.62 pM. of p<. -keto-glutaric acid per 3 ml. and less than 0.15 ;uM pyruvic acid per 3 ml. This method has several drawbacks when used for routine estimations of radioactive materials: i t involves the use of several tubes, i t re-quires shaking and centrifugation of radioactive solutions, i t is quite time consuming and fairly large samples must be used when the keto acid concentration is low. An enzymatic assay with glutamic dehydrogenase was therefore developed and is described in Methods. The standard curve obtained for oc-ketoglutaric acid is shown in Figure 3. Routine determinations of oL-ketoglutaric acid were carried out by this method and the results agreed with those found by the chemical methpd. Typical results are shown in Figure 4 and correlated with the glucose concentration and the percentage of the added C 1 4 present in the supernatant at various time intervals. The concentra-tion of -ketoglutaric acid in the supernatant continued to rise as long as glucose was being oxidized and i t f e l l as soon as the glucose had disappeared. The slow secondary rate of oxygen uptake after the disappearance of glucose thus corresponded to the gradual oxidation of some of the <*L-rketoglutaric acid, while the remaining c<.-ketoglu-taric acid was incorporated into cellular material as ammonia was produced by the endogenous respiration. After the disappearance of oC-ketoglutaric acid at 120 minutes at least 10% of the C 1 4 added was always present in the supernatant. 35 . 0 2 . 0 4 . 06 M oc- K E T O G L U T A R I C ACID F IGURE 3. STANDARD CURVE FOR THE DETERMINATION OF oC-KETOGLUTARIC ACID WITH GLUTAMIC DEHYDROGENASE 3 6 . T I M E (M INUTES ) F I G U R E 4. COMPOS IT ION OF T H E REACT ION S U P E R N A T A N T 37. The majority of this radioactivity remained at the origin during chromatography in sec-butanol-formic acid-water and could not be eluted from the paper. It was suspected that this material was protein or other large molecular weight compounds. Glutamic acid was also present in the supernatant as were a number of unidentified compounds. The absorption spectra of the supernatant i n the Beckman D.TJ. spectraphotometer showed the presence of ultra-violet absorbing material as well. Since the radioactivity of the supernatant at 120 minutes i s two-thirds of that found i n the cells i t may be important to carry out further studies of the nature and origin of the compounds which are present. It i s not l i k e l y that these compounds arise from c e l l l y s i s as there i s very l i t t l e change i n the number of viable cells during the two-hour incubation period. II. Products of the oxidative assimilation of glucose 1. Distribution of intracellular radioactivity i n c e l l fractions The products of oxidative assimilation in the presence of 5 pM of 14 glucose-C were determined by fractionation of the whole cells using a modification of the method of Roberts _et a l described previously. The results are shown i n Table 6. The radioactivity was found to be present i n a l l the major fractions and a rough estimation of the specific activity of each fraction was obtained by comparing the per-centage of incorporated C 1 4 found i n each fraction during oxidation of glucose with the percentage of the total radioactivity i n each fraction of washed c e l l suspensions harvested after growth for 20 hours i n a complete medium containing uniformly-labelled glucose-C 1 4. The cold TCA fraction appeared to be important during the early stages of 38. Table 6 14 Incorporation of C from uniformly-labelled glucose into P.aeruginosa fo of total C i 1 4 added to vessel Time Cold TCA-soluble Lipid, Phospho-lipid Alcohol-soluble protein Hot TCA-soluble Resi-dual protein Total in fractions Unfrac-tionated cells min. * fo fo * fo fo 15 2.2 2.2 0.2 0.9 2.9 8.4 7.8 30 2.8 2.9 0.4 1.3 4.0 11.4 10.8 60 2.5 2.8 0.5 1.4 5.5 12.7 12.8 120 2.4 3.4 6.6 1.8 7.7 15.9 16.3 % of incorporated 14 G * found in each fraction 15 26 26 3 11 34 100 30 25 25 4 11 35 100 60 20 22 4 11 43 100 120 15 21 4 11 49 100 Growth eompo- 6 16 3 16 59 100 sition 39* i oxidative assimilation, as did the lipid fraction. Since the cold TCA fraction is assumed to contain metabolic intermediates its early label-ling was not surprising. The role of the lipid fraction was of con-siderable interest since the protein fraction contains the greatest percentage of the activity and the phospholipids of P. aeruginosa (73) and other bacteria (48) may be important in the uptake of amino acids by the cells or as a site of protein synthesis (38). The fact that most of the products of oxidative assimilation of glucose were apparent-ly nitrogenous agreed with the proposal that the ammonia available might be the factor which determined the assimilation of carbon into the cell. 2. Studies on the composition of the various cell fractions It was considered important to verify that the composition of each fraction of P. aeruginosa was as would be expected from the work of Roberts et al (70) using Escherichia coli. These investigations were carried out by determining the absorption spectra, the protein and ribonucleic acid (RNA) content and the chromatographic behaviour of the fractions or hydrolyzates of the fractions. The hot TCA and cold TCA fractions were extracted repeatedly with ether to remove the TCA before use, and very l i t t l e radioactivity passed into the ether phase. The absorption spectra of each fraction as determined using a Beckman D.TJ. spectraphotometer is shown in Figure 5. Only the hot TCA fraction absorbed strongly at 260 mu while the protein residue had a peak at 280 mu. Traces of TCA which were present in a l l the fractions caused considerable absorption at the lower wavelengths. 40 12 "t 0.D, 0.8 •+ 0 .4 -+ 260 280 W A V E L E N G T H F I G U R E 5. A 8 S 0 R P T I 0 N S P E C T R A OF C E L L F R A C T I O N S 41. The results of protein and RNA determinations of various fractions after 30 minutes incubation with glucose and of the fractions of washed suspensions of 20 hour cells are shown in Table 7. Table 7 Protein and ribonucleic acid content of cell fractions Fraction Incubated 30 min. with glucose Cold TCA-soluble Lipid alcohol-soluble protein Hot TCA-soluble Residual protein Protein ;ag./5.3mg. RNA /ig./5.3 mg. dry wt.cells dry wt.cells 60 80 380 2,400 80 100 640 60 20 hr. cells Cold TCA-soluble Lipid alcohol-soluble protein Hot TCA-soluble Residual protein 40 80 420 2,480 75 110 790 60 None of the fractions gave significant readings in the anthrone test for carbohydrate although a reasonable standard curve was obtained. It appeared that the hot TCA fraction contained considerable protein and this was also noticeable when chromatography of the various fractions in see-butanol-formic acid-water was carried out for 20 hours and the movement of the radioactive materials determined by running strips through the Actigraph II. Table 8 shows the Rf values of the radioactive 42. Table 8 Chromatography of c e l l fractions and of hydrolyzates of fractions Fraction Cold TCA-soluble Alcohol-soluble (a) alcohol-soluble ether-soluble (b) alcohol-soluble e ther-insoluble Hot TCA-soluble (a) hydrolyzate at 90° or 108° Residual protein (a) hydrolyzate KH4OH eluate 12 peaks H20 eluate Comments probably protein complex, some ninhydrin -f alcohol-soluble protein l i p i d , phospholipid n w alcohol-soluble protein probably protein mauve under u.v. (CMP) abs. under u.v. (UMP?) mauve under u.v. (guanine) abs. under u.v. (adenine) ninhydrin + u.v. absorption, etc. faint ninhydrin +• ninhydrin -f \- + 4- 4. ninhydrin+corr. to known amino acids Rf of G Relative material amt. C A* 0 + .20-.40 +• + + 0 -+ front +-(- + + front —(  -I- -*- H-0 -r + + .12 + .15 .30 •40-.80 -f-.43 + + .2-.6 -f + + .8 4-.12,.15,.30,.43 0 +- + -+ + 0-.5 4- 4-43. compounds of the fractions, of hydrolyzates of the hot TCA, and residual protein hydrolyzates which were chromatographed in n-butanol-acetic acid-water (63:10:27 v/v) rather than the see-butanol-formic acid-water solvent. It can be concluded from these results that the cold TCA fraction contains a number of compounds including amino aeids, other unidenti-fied small molecules and small amounts of protein. The chromatographic behaviour of the alcohol-soluble fraction is the same as that described by Roberts .et al and i t appears justifiable to term the alcohol-soluble, ether-soluble material lipid or phospholipid, and the alcohol-soluble, ether-insoluble material protein. The four spots detected in the hot TCA fraction by ultra-violet light are probably cytidylic acid, uri-dylic acid, adenine and guanine. These were the constituents of the hot TCA fraction described by Roberts jet al. The radioactivity from Rf .40 to .80 was absent in hot TCA fractions prepared from cells grown for 20 hours in the presence of glucose-C14 and the compounds responsible for the activity were not identified. It is obvious that the radioactivity of the hot TCA fraction is only partially due to nucleic acid material and that considerable protein or some large molecule which on hydrolysis gives rise to ninhydrin positive material is present. The ratio of the radioactivity found at the origin to that found in ultra-violet absorbing material was higher in hot TCA fractions from washed cell suspensions which are oxidizing glucose than in hot TCA fractions from washed cell suspensions of cells grown in the presence of radioactive glucose. The presence of protein in this fraction was not reported by Roberts _et al. However, Park and 44 Hancock (66) found ninhydrin positive material in this fraction which apparently arose from the cell wall mucopeptide of Staphylocoecus  aureus. Since Gram negative bacteria also have a mucopeptide (or protein) component in their eell walls i t is possible that this i s the origin of the "protein* in the hot TCA fraction of P. aeruginosa. The residual protein fraction seemed to consist almost entirely of protein which yielded known amino acids on hydrolysis. If poly-/3-hydroxybutyrate was formed i t would be present in this fraction. No evidence was found for this compound and forsyth, Hayward and Roberts (35) could not detect poly-y3 -hydroxybutyrate in the green-pigmented pseudomonads. In this laboratory specific assays for the presence of this polymer have also been negative (45). No evidence was obtained for the presence of a significant amount of a polysaccharide material in any of the fractions. The results obtained using this fractionation procedure were quite reproducible and a good correlation was found between duplicate experiments. III. Stimulation of oxidative assimilation By ammonia Since assimilation occurred primarily into nitrogenous compounds and appeared to depend on the ammonia available from endogenous respi-ration, the influence of added ammonia on the oxidative assimilation of glueose-C14 was studied. The washed cell suspensions were incubated in the presence of 5 ,uM of uniformly-labelled glucose and 5 /uM of ammonia as (NH^ JgSO^ . The oxygen uptake with glucose in the presence and absence of added ammonia and with the corresponding endogenous controls is.shown in figure 6. The presence of added ammonia had 45 I. G L U C O S E T I M E (M INUTES ) F IGURE 6. I N F L U E N C E OF ADDED N H , ON OXYGEN UPTAKE 46. l i t t l e influence on the endogenous respiration of the organism but decreased markedly the final oxygen uptake in the presence of glucose, suggesting that more assimilation had occurred. The R.%. during the in i t i a l stage of glucose oxidation was increased to 1.05 from approxi-mately 0.9 by the addition of ammonia. Figure 7 shows the distribution of carbon from glucose-0 1 4 between the cells, supernatant and calculated carbon dioxide. The rapid evolution of C^Og is in agreement with the increase in the R.Q. value for the i n i t i a l period in the presence of added ammonia. This increased rate may have resulted from an increased catabolism of glucose by the pentose phosphate pathway of oxidation. Holzer (47) has summarized the evidence that oxidation by the pentose phosphate pathway is limited by a deficiency of oxidized triphospho-pyridinenucleotide (TPN). Additional TPNH oxidation, for example during the reductive amination of oC-ketoglutaric acid to glutamic acid, may thus accelerate glucose metabolism by this pathway. There was a very marked stimulation of assimilation in the presence of added ammonia so that by 30 minutes 32$ of the added glucose was converted to cellular material. The incorporation of ammonia was compatible with this assimilation. The radioactivity in the supernatant decreased to a low value as the glucose disappeared and no ^-ketoglutaric acid appeared in the supernatant (Figure 8 ) . This organism contains a strong glutamic dehydrogenase and the coincident removal of ammonia and c<-ketoglutaric acid indicates that carbon was being assimilated by way of glutamic acid. 14 The pattern of incorporation of C into the various cell fractions is shown in Table 9. Maximum incorporation was observed by 30 to 60 47. 10 0 8 0 -t 6 0 % c ' 4 AD 0 ED 4 0 4 2 0 + A-o-% C -0 % c -0 % C 14 14 14 S U P E R N A T A N T C E L L S C 0 2 (CALC.) -X /i M N H 3 U P T A K E (CALC.) 4-4 .x- -— o - «t3 /*M N H. T I 0, 120 1 : — i 3 0 6 0 9 0 T IME (M INUTES) F IGURE 7. DISTRIBUTION O F C 1 4 IN T H E P R E S E N C E OF A D D E D N H 3 AND C A L C U L A T E D U P T A K E OF N H j 48. 10 0 H 8 0 % C 1 4 ADDED 6 0 -4 0 2 0 "• C 1 4 (%) JJLM G L U C O S E -O /tM oC-KETOGLUTARIC ACID 3 0 - 4 jx M P R E S E N T - - 2 r 0 120 F IGURE 8 6 0 9 0 T IME (MINUTES) COMPOSITION OF T H E R E A C T I O N SUPERNATANT IN T H E P R E S E N C E OF A D D E D N H , 49. Table 9 Incorporation of 0 -carbon from uniformly-labelled glucose into washed  suspensions of P.aeruginosa in the presence of 5 uM ammonia Time % of total C 1 4 added to vessel  Cold Lipid, Alcohol- Hot Eesi- Total Unfrac-TCA- Phospho- soluble TCA- dual in tionated min. soluble lipid protein soluble protein fractions cells fo £ $ % £ £ 15 3.5 4.4 0.4 1.6 8.6 18.5 17.1 30 4.5 5.5 1.2 2.9 18.4 32.5 31.1 60 3.7 5.3 1.6 2.9 19.8 33.3 33.2 120 2.7 5.0 0.6 2.8 17.5 28.6 27.6 £ of incorporated 0 found in each fraction 15 19 24 2 9 46 100 30 14 17 4 9 56 100 60 11 16 5 9 59 100 120 9 17 3 10 61 100 Growth compo-sition 6 16 3 16 59 100 50 minutes after which time the residual protein and compounds of the cold TCA fraction appear to he oxidized. As might be expected, in the presence of added ammonia the distribution of radioactivity in each cellular fraction corresponded much more closely to that found in cells grown in a complete medium containing glucose-6 1 4. An exception was the hot TCA fraction. The relatively low incorporation of glucose into nucleic acid material during oxidative assimilation by washed cell suspensions may possibly be due to a slow rate of turnover of this material or to lack of phosphate. IV. Oxidative assimilation of specifically-labelled glucose-C14 Carbohydrate metabolism in P., aeruginosa and the closely related organism P. fluorescens has been reviewed by De Ley (29) and Wood (90). In both species the Embden-Meyerhof pathway appears to be absent or relatively unimportant. This may be the result of the lack of the enzyme, phosphofructokinase, and is supported by the inability of resting cells to ferment glucose. On the other hand the complete system for the Entner-Doudoroff and pentose phosphate pathways are present (39,41,90). Since the kinase for glucose is present in glucose-grown P. aeruginosa (19,32,39,41) glucose can apparently enter these pathways directly. Experiments with dried cells and cell-free extracts of -P. aeruginosa strain ATCC 9027 have demonstrated that this organism possesses the enzymes for the oxidation of a l l compounds of the tricarboxylic acid cycle and the condensing enzyme for the entry of acetyl coenzyme A into the cycle (16,18). The terminal oxidation of glucose is thus likely to occur by the tricarboxylic acid cycle. The presence of the enzyme isocitritase was fi r s t observed in acetate-grown 51. cells of this organism (16,17) and. i t has now been established that the glyoxylate cycle, involving isocitritase and malate synthetase, is im-portant during growth on acetate. However, the enzymes of the glyoxy-late cycle, particularly isocitritase, are repressed when cells are grown on other substrates such as glucose (54). Attempts have been made to estimate the relative importance of the pentose phosphate pathway and the Entner-Doudoroff pathway in the oxidation of glucose by P. aeruginosa and P. fluoreseens by using specifically-labelled glucose-C14. Lewis et al (56) used glucose-I-CS14, glucose-2-C14 and glucose-6-C14 and followed the distribution and 14 quantity of C present in acetate and pyruvate. The results were cor-rected for endogenous dilution by using uniformly-labelled glucose-C14. Prom the results they concluded that the pentose phosphate pathway accounted for one-half or more of the glucose molecules catabolized by resting cells of P. fluoreseens and that the remaining substrate was dealt with according to the Entner-Doudoroff pathway. Similar results were obtained for P.aeruginosa. Stern, Wang and Gilmour (75) carried out radiorespirometric studies on the utilization of glucose by P. aeruginosa strain ATCC 9027 in a culture medium optimal for growth. From 34-43% of the radioactivity from glucose-2-C14, glucose-3:4-C14 and glucose-6-C14 was incorporated into the cells. Only 9% of the C 1 4 of glucose-l-C 1 4 was incorporated. From the rate of release of C 1 40g from the variously-labelled substrates, they estimated that the Entner-Doudoroff pathway accounted for 71% of the glucose oxidation and the pentose phosphate pathway accounted for 29%. However, such results are only approximations since they do not take into account the effect 52. of recycling v i a the pentose phosphate pathway and other factors (50). In the present study, oxidative assimilation of glucose-l-C 1 4, 14 14 14 glucose-2-C , glueose-6-C and uniformly-labelled glucose-;C were compared. The distribution of radioactivity between the c e l l s , supernatant, and the calculated C 1 40g evolution at various time inter-vals i s shown i n Figure 9. It was found that almost a l l the C-l was released as carbon dioxide, which i s in agreement with the major role of the Entner-Doudoroff and pentose phosphate pathways. No attempt was made to estimate the relative participation of the pathways of glucose dissimilation but observations were made on the nature of the radioactive products appearing in the cells during assimilation. The results of fractionation of the cells after 30 minutes incubation are presented i n Table 10. The radioactivity which was incorporated into the cells from glucose-l-C 1 4 was found predominantly i n compounds such as amino acids, protein and nucleic acids which are known to arise via carbon dioxide fixation. This suggests that carbon dioxide fixation may play an important role i n the synthetic reactions during oxidative assimilation of glucose by P. aeruginosa. The preferential incorp-oration of G-2 into l i p i d and C-6 into nucleic acid was found i n three different experiments. This may be the result of the major role of the Entner-Doudoroff pathway i f i t i s assumed that the pyruvic acid from C-l to C-3 w i l l be more li k e l y to be metabolized via acetate, whereas the glyceraldehyde-3-phosphate from C-4 to C-6 may react with carbon dioxide at the phosphoenolpyruvate level to form fumarate and hence aspartate, or go back via a reversal of the aldolase reaotion to glucose-6-phosphate which gives rise to ribose-5-phosphate. 100 o 1- 1 r— 1 0 3 0 6 0 9 0 120 T IME (M INUTES) F IGURE 9. D ISTRIBUTION OF C 1 4 A D D E D AS U N I F O R M L Y -OR V A R I O U S L Y - L A B E L L E D G L U C O S E 54. Table 10 Incorporation of radioactivity from variously-labelled and uniformly- labelled, glucose after 50 minutes incubation Substrate % of.C added to vessel Cold Lipid, Alcohol- Hot Resi- Total Unfrac-TCA- Phospho- soluble TCA dual in tionated soluble * l i p i d * protein soluble protein fractions fo f> % cells fo Glucose-U-C14 5.1 2.8 0.4 1.2 4.4 11.9 11.5 Glucoserl-C 1 4 0.6 0.2 0.1 0.5 1.3 2.5 2.5 Glucose-2-C 5.4 4.4 0,3 0.7 5.0 15.8 15.0 14 Glucose-6-C 3.2 2.5 0.4 2.0 4.5 12.6 15.2 14 % of incorporated C in each fraction Glucose-U-C14 26 24 5 10 57 100 Glucose-l-C 1 4 24 8 4 12 52 100 Glucose-2-C14 25 52 2 5 56 100 14 Glucose-6-C 25 20 5 16 56 100 55 Chromatography of the reaction supernatants indicated that the principal radioactive compound present at 30 minutes was oC -ketoglutaric acid regardless of the position of the C 1 4 in the substrate. This suggested that the od.-ketoglutaric acid was formed via the tricarboxylic acid cycle rather than by a direct pathway from glucose involving a five-one spliti The lower total activity present in c<-ketoglutaric acid 14 when glucose-l-C was the substrate could be the result of dilution 14 19 of C Og formed from C-l of glucose by 0 0% f r o m carbons 2 to 6 prior to carbon dioxide fixation for entry into the tricarboxylic acid cycle. Y. The influence of C120p on oxidative assimilation of glucose-C14 To obtain further information on the importance of carbon dioxide fixation in the oxidative assimilation of glucose, an experiment was carried out in which washed suspensions of P. aeruginosa were incubated with uniformly-labelled glucose-C14 with either air or a 10% C1S02-90% air mixture as the gas phase. The distribution of radioactivity between the cells and supernatant and the calculated C 1 40 P evolution is shown in Table 11. Table 11 The influence of the gas phase on the distribution of C 14 % C added to vessel Gas phase Time min. Cells Supernatant air 10% C02-90% air 30 30 15.6 11.4 36.8 30.4 47.6 58.2 air 10% C02-90% air 60 60 15.9 14.0 33.6 20.6 50.5 65.4 56. It is apparent that there was a very marked decrease (10 to 30$) in the radioactivity incorporated into the cells and the supernatant 1? when the gas phase contained C Og in a high concentration, whereas 14 the C Og evolution increased. This is probably due to the greater dilution of C l 40g prior to carbon dioxide fixation and is supported by the results of fractionation of the cells (Table 12). Incorpora-tion into the lipid fraction was not decreased significantly but incorporation into the protein and hot TCA fractions was definitely lowered. Table 12 The influence of C 1 20g on the incorporation of C 1 4 into cell fractions . 14 % C added to vessel Cold Lipid Hot Resi- Total Unfrac-TCA- ale.-sol. TCA- dual in tionated Gas phase Time soluble protein soluble protein fractions cells min. $ $ % $ $ $ air 30 3.6 3.9 1.6 6.5 15.6 15.1 10$ C0g-90$ air 30 2.4 3,2 1.1 4.7 11.4 10.8 air 60 3.3 3.7 1.4 7.5 15.9 15.6 10$ C02-90$ air 60 3.4 3.9 1.1 6.1 14.0 15.2 Hauser and Karnovsky (43) also concluded that carbon dioxide fixa-tion was important in the synthesis of cellular constituents by P. aeruginosa. In an experiment in which cells were grown in a synthe-tic medium with glycerol as the carbon source and was present in 57 the atmosphere they calculated 27% of the bacterial carbon was supplied by carbon dioxide. Wang and Ikeda (82) found that the biosynthesis of aspartic acid by glucose-grown P. fluoreseens relied heavily, i f not exclusively, on the fixation of carbon dioxide by the -f C 3 mechanism. In P. aeruginosa there appears to be a partial block in the t r i -carboxylic acid cycle at oC-ketoglutaric acid during the oxidation of glucose and thus in addition to the usual synthesis of amino acids and nucleic acids there is an accumulation of X-ketoglutaric acid. There-fore, the role of carbon dioxide fixation to form oxaloacetic acid, and permit entry of carbon units into the cycle, is clearly of great importance. VI. The oxidation of c^r-ketoglutaric acid The oxygen uptake by washed cell suspensions of P. aeruginosa in the presence of 5 ^ iM of <<-ketoglutaric acid is shown in Figure 10, The values for endogenous oxygen uptake were subtracted from those found in the presence of the substrate on the basis of the results obtained using various concentrations of cells with 3 )M of ^-keto-glutaric acid, as shown in Table 13. Table 13 Oxidation of 3 JUM ^-ketoglutaric acid by cell suspensions  of varying concentrations OR uptake 180 min. % theoretical 0 g uptake Dry wt. Substrate Endog. not Endog. , sells Endog. Substrate -endog. subtracted subtracted mg. ; i l . ;ul. 1° * 2.65 90 224 134 83 49 5,30 170 299 129 111 48 7.95 268 411 143 153 53 58 If the endogenous respiration had been suppressed in the presence of <*L-ketoglutaric acid, the same oxygen uptake in the presence of substrate should have been obtained in a l l cases. However, the total oxygen uptake increased proportionally with the increase in endogenous respiration, and i f the endogenous values were subtracted the percent-age of oxidation was found to be relatively constant in a l l cases. The oxygen uptake curve, during the oxidation of <*:-ketoglutaric acid, was characterized by an i n i t i a l lag followed by a more rapid linear rate which continued until approximately 50$ of the oxygen uptake for complete combustion was achieved. In the presence of 5 jxM. glucose and 5 /uM ^-ketoglutaric acid a diauxic oxygen uptake curve was observed as shown in Figure 10. The lag in the oxygen uptake when glucose-grown cells of P. aeruginosa are incubated with ^-ketoglutaric acid suggests that the cells do not contain a permease for this substrate. Campbell and Stokes (18) observed that intact cells of P. aeruginosa which had been grown in a glucose or acetate medium displayed a lag period prior to the linear rapid rate of oxidation of several tricarboxylic acid intermed-iates, whereas dried cell preparations could oxidize these compounds immediately. They attributed the i n i t i a l lag to the time necessary for the elaboration of a system for transporting the substrate across the cell membrane. Kogut and Podoski (53) and Barrett and Kallio (7) showed that cell-free extracts of P. fluorescens oxidized intermediates of the tricarboxylic acid cycle rapidly and linearly, whereas with whole cells there was a lag period before the linear rate of oxidation was reached. Clarke and Meadow (20) confirmed and extended the observations 59 5 j iM G L U C O S E + oC-KETOGLUTARATE 0 60 120 180 T IME (MINUTES) F IGURE 10. OXYGEN U P T A K E BY WASHED C E L L S IN T H E P R E S E N C E OF VARIOUS S U B S T R A T E S 60. with P. aeruginosa. Chloramphenicol inhibited the adaptation to sub-strates by whole cells of the organism but was without effect on the oxidation by cell-free extracts; This evidence supported the hypothe-? sis that the first step in the uptake of tricarboxylic acid cycle compounds consists in the action of inducible-permeases. In the present experiment the addition of 6 mg. of chloramphenicol to the reaction mixture caused the rate of oxygen uptake with -ketoglutaric acid to remain at a very low value, indicating that glucose-grown cells do not possess the complete enzyme system for the entry and oxidation of ^-ketoglutaric acid (Figure 10). The intracellular rate of -ketoglutaric acid oxidation by washed cell suspensions in the presence of glucose can not be calculated with-out an exact knowledge of the total «r-ketoglutaric acid formed during glucose dissimilation. Obviously i t must be lower than the rate of a l l oxidation steps between glucose and c<-ketoglutaric acid in order for the compound to accumulate in the supernatant, and once i t is out-Side the cells its oxidation depends on the synthesis of the permease. It can not be definitely concluded from the results presented in this thesis that the presence of glucose represses the formation of the enzymes for ot -ketoglutaric acid entry and oxidation. Preliminary experiments indicate that this may be the case. The rate of oxidation of ot-ketoglutaric acid by cell-free extracts of ot-ketoglutaric acid-grown P. .aeruginosa is greater than that of extracts from glucose-grown cells (14), and the QOg o f ^-ketoglutaric acid oxidation by whole cells grown on this substrate is as high as that of glucose-grown whole cells for glucose (15). Therefore, the presence of glucose during 61 growth of the organism undoubtedly decreases the intracellular level of the c*i-ketoglutaric acid oxidase system as well as repressing the •synthesis of the permease for this compound. ¥11. The influence of chloramphenicol on oxidative assimilation The influence of chloramphenicol on oxidative assimilation was studied in view of the observations that chloramphenicol inhibited the oxidation of extracellular ^-ketoglutaric acid, and that much of the material synthesized during oxidative assimilation of glucose appeared to be protein. Figure 11 shows the oxygen uptake of washed cell sus-pensions of P. aeruginosa during endogenous respiration or the oxida-tion of 5 }M of glucose in the presence and absence of 6 mg. of chloramphenicol. The endogenous oxygen uptake was slightly stimulated by chloramphenicol but ammonia production remained the same. The presence of chloramphenicol decreased the final oxygen uptake during the oxidation of glucose and appeared to inhibit the second stage known to be due to the oxidation of ^ -ketoglutaric acid. Table 14 shows that ammonia uptake in the presence of glucose was also decreased by the inhibitor. Table 14 The influence of chloramphenicol on ammonia reincorporation NH? uptake per 3 ml. reaction vessel  Time Control = Glucose Glucose 4- Chloramphenicol min. jiM. yuM 30 0.70 0,70 60 0.98 0.72 150 1.10 0.64 62. 5 0 0 4 0 0 -• / l l . 0 U P TAK E 3 0 0 •• 2 0 0 100 F I G U R E 3 0 6 0 9 0 T I M E (MINUTES) 120 I N F L U E N C E O F CHLORAMPHENICOL ON OXYGEN U P T A K E 63 If uniformly-labelled glucose-C * was used as the substrate the distribution of radioactivity shown in Figure 12 was found in the presence and absenee of chloramphenicol. The presence of chlorampheni-col caused a slight increase in the C 1 4 incorporated into the cells and released as C 1 40g at 30 minutes. After this time the radioactivity in the cells decreased and the radioactivity of the supernatant de-creased much more slowly than in the control. Chromatography of the supernatants of the reaction mixtures containing chloramphenicol showed that -ketoglutaric acid was s t i l l present in considerable quantity at 150 minutes. The high supernatant activity and low final oxygen uptake thus appeared to be due to inhibition of the synthesis of the permease for ^-ketoglutaric acid. The incorporation of radioactivity into cellular fractions in the presence of chloramphenicol is shown in Table 15. Results for the control were similar to those presented in Table 6. The presence of chloramphenicol increased the incorporation of radioactivity into the cold TCA-soluble, lipid and hot TCA-soluble fractions, whereas incorpo-ration into the residual protein was greatly decreased especially in the later stages. These results are in agreement with those of other workers who found that chloramphenicol specifically inhibits protein synthesis in bacteria (42,62,79,89) and with the observation that con-centrations of chloramphenicol which inhibit protein synthesis by P. aeruginosa stimulate the incorporation of phosphate when resting cells are incubated with glucose (76). The chloramphenicol-resistant 14 uptake of small amounts of C into the protein fraction may represent synthesis of cell wall peptides (12). 64. I O O i 8 0 % c 1 4 ADD E D 60 -• 4 O -• 20 -• w w o CELLS SUPERNATANT C 0 g (CALC.) G L U C O S E G L U C O S E + CHLORAMPHENICOL w V w w 30 120 150 F IGURE 12. 6 0 90 T IME (M INUTES ) DISTRIBUTION O F . C 1 4 IN T H E P R E S E N C E AND A B S E N C E OF C H L O R A M P H E N I C O L 65. Table 15 Incorporation of 0 from uniformly-labelled glucose in the  presence of chloramphenicol fo of total (/ added to vessel -Cold Lipid, Alcohol- Hot Resi- Total Uhfrac-TCA- Phospho- soluble TCA- dual in tionated Time soluble lipid protein soluble protein fractions cells min. fo fo % fo % % % 30 3.2 4.0 0.3 2.5 1.3 11.3 11.3 60 2.3 4.1 0.3 2.5 1.3 10.5 11.0 150 1.9 3.8 0.3 2.3 1.2 9.5 10.2 fo of incorporated C 1 4 in each fraction  30 28 35 3 22 12 100 60 22 39 3 24 12 100 150 20 40 3 24 13 100 66 Corresponding experiments were done with 5 ,uM of ammoniafcadded to the reaction mixtures. Oxygen uptake in the presence of glucose was increased by chloramphenicol (Figure 13) and the incorporation of ammonia was decreased especially at 60 and 150 minutes (Table 16). The endogenous respiration and ammonia production were not affected. Table 16 The influence of chloramphenicol on incorporation of ammonia in the presence of excess ammonia Net NHft uptake per 3 ml.reaction vessel  Time Control = Glucose Glucose 4- Chloramphenicol min. 30 3.14 2.36 60 3;20 1.35 150 2.70 0.96 Incorporation of G 1 4 into the cells in the presence of chloramphenicol was stimulated by the addition of ammonia but to a lesser extent than in the absence of the inhibitor, as shown in Figure 14. Maximum incor-poration was observed at 30 minutes and the material synthesized appeared to be unstable. The supernatant activity was slightly higher than the control but very l i t t l e ^-ketoglutaric acid was present at 30 minutes and none was present at 60 to 150 minutes. The presence of ammonia apparently prevents the accumulation of oC-ketoglutaric acid outside the cells so that inability to synthesize the permease for this compound does not cause a decrease in the final oxygen uptake for 5 ^uM of glucose. The distribution of radioactivity in the cellular fractions when chloramphenicol was present is shown in Table 17. The C 1 4 of the pro-tein fraction was specifically decreased and more radioactivity was present in the lipid and hot TCA fractions. 67. 5 0 0 4 0 0 -u I. 0 . U P T A K E 3 0 0 " ' 1. G L U C O S E + N H 3 + C H L O R A M P H E N I C O L 2. G L U C O S E + N H 3 3. E N O G E N O U S + N H + C H L O R A M P H E N I C O L 3 4. E N O G E N O U S + N H . 2 0 0 I O O - • 3 . 4 . 0 - * 3 O , [ — 6 0 9 0 T I M E ( M I N U T E S ) 120 F I G U R E 13. I N F L U E N C E O F C H L O R A M P H E N I C O L O N O X Y G E N U P T A K E IN T H E P R E S E N C E O F A D D E D N H , 68 10 0 C E L L S \ S U P E R N A T A N T C 0 2 (CALC.) 0 3 0 6 0 9 0 120 150 TIME ( M I N U T E S ) F I G U R E 14. I N F L U E N C E OF C H L O R A M P H E N I C O L ON DISTRIBUTION OF C 1 4 IN T H E P R E S E N C E OF ADDED N H j 69. Table 17 Incorporation of C 1 4 from uniformly-labelled glucose in the presence of chloramphenicol and added ammonia fo of total C 1 4 added to vessel Time Cold TCA-soluble Lipid, Phospho-lipid Alcohol-soluble protein Hot TCA-soluble Resi-dual protein Total in fractions Unfrac-tionated cells min. fo fo fo fo fo fo 30 7.4 6.5 0.5 5.3 2.0 21.7 22.4 60 3.3 7.1 0.4 4.4 1.8 17.0 17.1 150 2.4 6.9 0.5 4.3 1.8 15.9 16.8 fo O f incorporated C 1 4 in each fraction 30 34 30 2 25 9 100 60 19 42 2 26 11 100 150 15 44 3 27 11 100 70. VIII. The influence of uncoupling agents on oxidative assimilation 1. Sodium azide Horris, Campbell and Ney (65) reported that M/1000 sodium azide increased the oxygen uptake by P. aeruginosa-in the presence of glucose from 64$ to 90$ of the theoretical values. Higher concentrations of azide inhibited both the rate and the total amount of oxygen uptake. It was of interest, therefore, to determine the influence of this inhibitor 14 on the incorporation of C from radioactive glucose by washed suspen-sions of this organism. Various concentrations of azide were tested for their effect on oxygen consumption in the presence of 5 pM. of glueose. At concentrations from M/1000 to M/300 an increase of approx-imately 10$ was found in the final oxygen uptake. At the higher con-centrations this was accompanied by a pronounced decrease in the rate of oxygen consumption as shown in Figure 15. Concentrations of azide less than M/1000 gave results intermediate between the results for the control and for M/1000. Concentrations greater than M/300 almost completely inhibited oxidation. Endogenous respiration was not affected by M/1000 azide but both the oxygen uptake and the ammonia production were decreased in the presence of M/300 azide. As was expected, the calculated reincorporation of ammonia was lower in the presence of the inhibitor. Radioactive experiments were carried out using 5 pM of uniformly-labelled glucose-C14 and the results are shown in Figure 16. If a concentration of M/1000 azide was used the increase in the oxygen uptake at 60 minutes was found to be largely due to the disappearance 14 of C from the supernatant rather than to a decrease in assimilation. 71. 6 0 0 0, U P T A K E 4 0 0 " 2 0 0 - - . 1. G L U C O S E + M / 3 0 O A Z I D E 2. G L U C O S E + M/IOOO A Z I D E 3. G L U C O S E 4. ENDOGENOUS 5. ENDOGENOUS 4- M / I 0 0 0 AZIDE 6. E N D O G E N O U S + M /300 AZ I DE 150 F IGURE 15. 6 0 9 0 120 T IME (MINUTES) I N F L U E N C E O F AZ IDE ON O X Y G E N U P T A K E 72. 0 3 0 6 0 90 120 150 T I M E ( M I N U T E S ) F I G U R E 16. I N F L U E N C E OF A Z I D E ON THE DISTRIBUTION OF C 1 4 73. Chromatography confirmed that much less oL-ketoglutaric acid accumu-lated in the supernatant in the presence of M/1000 azide, none being detectable at 30 minutes and very small amounts being present at 60 minutes. There are several possible explanations of this phenomenon: the intracellular rate of oC-ketoglutaric oxidation may be increased, the participation of the pentose phosphate pathway relative to the Entner-Doudoroff pathway may be increased so that less ^-ketoglutaric acid is formed from glucose, or since the rate of glucose disappearance, and hence oC-ketoglutaric acid production, is decreased more ^-keto-glutaric acid could be oxidized intracellularly as i t was formed rather than being secreted from the cells. The increase in the per-centage of the theoretical oxygen consumption at 150 minutes in the presence of M/1000 azide could be accounted for by the decrease in the 14 C of the cells and supernatant. At a concentration of M/300 azide assimilation of C 1 4 into the cells was decreased even further; No <<-ketoglutaric acid was found in the reaction supernatant and the glucose was present until the break in the oxygen uptake curve. The distribution of the radioactivity incorporated into the cells in the presence of M/1000 and M/300 azide is shown in Table 18. The control gave results comparable to those found in Table 6 and i t is obvious that azide does not selectively affect the incorporation into any of the cell fractions. Its mechanism of action is undoubtedly the uncoupling of oxidative phosphorylation so that less energy is available for synthetic reactions. The corresponding results for the influence of azide when 5 pH of ammonia were added to the vessels are shown in Figures 17 and 18. 74. Table 18 Incorporation of C 1 4 from uniformly-labelled glucose in the presence of azide fo of total C 1 4 added to vessel Cold Lipid, Alcohol-- Hot Resi- Total Unfrae-Gone. TCA- Phospho- soluble TCA- dual in tionated Azide Time soluble lipid protein soluble protein fractions cells min. % * fo fo fo % fo M/1000 30 2.2 1.6 0.4 0.6 3.5 8.3 8.1 60 2.2 2.0 0.3 0.9 4.8 10.2 11.0 150 1.9 1.8 0.3 0.9 6.7 11.6 11.1 M/300 30 1.2 1.0 0.2 0.3 1.1 3.8 3.7 60 1.0 1.1 0.2 0.4 1.5 4.2 4.3 150 0.6 1.4 0.3 0.5 2.5 5.3 5.6 fo of incorporated C found in each fraction M/1000 30 27 19 4 7 43 100 60 22 20 3 8 47 100 150 17 15 3 8 57 100 M/300 30 32 26 4 8 30 100 60 24 26 5 9 36 100 150 13 26 5 9 47 100 75. M o2 U PTAKE 4 0 0 " 2 0 0 - " 1. G L U C O S E + N H 3 + - M / 3 0 0 AZIDE 2. G L U C O S E + N H 3 + tylOOO AZIDE 6 0 0 4 3 ' G L U C 0 S E + N H 3 4. ENDOGENOUS 4 NH; 5. ENDOGENOUS 4 N H 3 + M / l O O O A Z I D E 6 - ENDOGENOUS + N H 3 4 M / 3 0 0 AZIDE 3 0 6 0 9 0 120 T IME (M INUTES) 150 F IGURE 17. I N F L U E N C E OF AZIDE ON O X Y G E N U P T A K E IN T H E P R E S E N C E OF A D D E D N H 3 76 10 0 8 0 -% c 1 4 A D D E D 60 4 0 2 0 - -C E L L S 0 C O N T R O L SUPR. • M / I 0 0 0 AZIDE C O , (CALC.) A M/3 0 0 AZIDE 3 O I 5 0 6 0 9 0 12 0 T IME (M INUTES ) F I G U R E 18. I N F L U E N C E OF AZ IDE ON THE D ISTR IBUT ION OF C 1 4 IN T H E P R E S E N C E OF ADDED N H , 77 Table 19 Incorporation of C from uniformly-labelled glucose in the  presence of azide and added ammonia % of total Gx* added to vessel Cone. Azide Time min. Cold TCA-soluble % Lipid, Phospho-lipid $ Alcohol-soluble protein * Hot TCA-soluble fo Resi-dual protein Total in fractions * Unfrac-tionated cells % M/1000 30 2.6 2.3 0.3 0.7 5.8 11.7 11.7 60 3.5 2.6 0.6 1.2 9.1 17.0 17.6 150 2.4 2.5 0.5 1.2 9.3 15.9 16.3 M/300 30 1.1 0.8 0.1 0.3 1.1 3.4 3.4 60 1.3 1.3 0.2 0.3 1.8 4.9 5.3 150 0.9 1.9 0.3 0.6 3.5 7.2 7.3 ia of incorporated C 1 4 found in each fraction M/1000 30 26 19 4 7 44 100 60 22 20 3 8 47 100 150 17 15 3 8 57 100 M/300 30 31 24 4 8 33 100 60 26 26 4 6 38 100 150 13 26 4 8 49 100 78 As expected, the increase in oxygen consumption, when compared to control experiments with no added azide, was more pronounced in the presence of added ammonia since oxidative assimilation was increased in the control. At a concentration of M/1000 azide, the addition of ammonia caused an increase in assimilation but at a concentration of M/300 azide, assimilation was the same regardless of the ammonia available. Table 19 shows that the distribution of radioactivity in the various cell fractions at 150 minutes was approximately the same as control values which were similar to those in Table 9. 2. Dinitrophenol Previous work in this laboratory (65), with glucose as the sub-strate, failed to demonstrate any increase in oxygen consumption by P. aeruginosa when concentrations of 2,4-dinitrophenol ranging from M/4000 to M/1000 were added to the reaction mixtures. Manometric observations of the influence of various concentrations of dinitro-phenol on the oxygen uptake by this organism, during endogenous respiration and during the oxidation of 5 pM of glucose, were made in the course of the present work. Typical results are shown in Figure 19, The endogenous respiration was markedly increased by this inhibitor, as was observed by Doudoroff (30) for the endogenous respiration of P. saccharophila. It is impossible to decide on the basis of present experiments whether this increase in endogenous respiration also occurs in the presence of substrate. However, in calculating the oxygen uptake values shown in Figure 19, i t was assumed that the same endogenous respiration occurred in the presence or absence of substrate. At a concentration of M/1000 dinitrophenol 79. 6 0 0 6 0 120 TIME ( M I N U T E S ) I 8 0 F I G U R E 19. I N F L U E N C E OF D I N I T R O P H E N O L ON O X Y G E N U P T A K E 80. a slight stimulation of total oxygen uptake was routinely observed. Increasing the concentration of dinitrophenol had a very strong inhibitory effect on the rate of oxygen uptake and also appeared to decrease the final oxygen uptake. The inhibition of rate did not commence at zero time but at a later time, depending on the concen-tration of the inhibitor. The reason for this is not known but studies with radioactive substrate might aid in interpreting this data. 81 GENERAL DISCUSSION The manometric results for the oxidation of glucose by washed c e l l suspensions of P. aeruginosa suggested that approximately 50% of the substrate was assimilated during the i n i t i a l stage of oxidation and that the slow secondary rate of oxidation which followed might be due to the oxidation of assimilated material. This would be similar to the extent of oxidative assimilation of glucose by resting cells of other bacterial species mentioned in the Literature Review. However, using uniformly-labelled glueose-C 1 4 as the substrate i t was found that during the period of rapid glucose oxidation only a small per-centage of the C 1 4 was assimilated into the c e l l s , whereas a large quantity of o^-ketoglutaric acid accumulated in the supernatant. The slow secondary rate of oxygen uptake corresponded to the gradual oxidation of some of the oC-ketoglutaric acid while the remaining ^-ketoglutaric acid was incorporated as ammonia became available from the breakdown of endogenous reserves. The incorporated radio-act i v i t y was found in a l l major c e l l fractions and most of the pro-ducts of oxidative assimilation of glucose were nitrogenous. The addition of ammonia greatly increased the amount of C 1 4 assimilated and prevented accumulation of &c -ketoglutaric acid. The production of <=<-ketoglutaric acid was f i r s t reported by Lockwood and Stodola (57) i n 1946. They showed that when a strain of P. fluoreseens was grown in a medium containing glucose, and with a high carbon to nitrogen ratio, up to 18 moles of °<L -ketoglutaric acid per 100 moles glucose consumed accumulated in the growth medium. Koepsell and co-workers (51,52) obtained yields of up to 55 moles of «l-ketoglutaric acid per 100 moles glucose i n subsequent studies on 82. the production of keto acids by this organism. They considered i t unlikely that -ketoglutaric acid was formed directly from the Cg chain of the sugar molecule since pyruvic acid seemed to give rise to <<-ketoglutaric acid, and concluded i t probably was formed by way of the tricarboxylic acid cycle. This was further confirmed by Kogut and Podoski (53) who showed that washed suspensions of this organism oxidized succinate incompletely and with the accumulation of ^.-keto-glutaric and pyruvic acids. The accumulation of c^-ketoglutaric acid by growing cells or washed cell suspensions of at least 10 other genera of bacteria (2,3,4,61,68) and by molds (44,81) has been report-ed. In most of these instances the organisms had the ability to oxidize this compound and therefore its accumulation appeared to be the result of a low level of the oC-ketoglutaric acid oxidase system, rather than of the absence of the enzyme. In the present study chloramphenicol was found to inhibit the oxidation of extracellular ^-ketoglutaric acid added to washed cell suspensions of P. aeruginosa or formed during glucose oxidation. It is probable that growth on glucose represses both the intracellular level of enzymes for s><:-keto-glutaric acid oxidation and the specific permease for the transport of this compound across the cell membrane. The results with variously-labelled glucose suggested that ^-ketoglutaric acid was formed via the tricarboxylic acid cycle and this was supported by the decreased radioactivity of the supernatant when the gas phase contained 10$ C 1 20 2. In a soil and water organism such as P. aeruginosa, in which assimilation appears to rely almost exclusively on the available 83 ammonia, the presence of a partial metabolic block at the ^-ketoglu-taric acid stage of carbohydrate oxidation i s indeed advantageous, conserving as i t does the carbon compound which will act as the point of entry to protein and nucleic acid synthesis when ammonia becomes available, either by diffusion, leaching or from endogenous storage products. Oxidative assimilation of glucose by P. aeruginosa does not appear to involve the synthesis of a "primary product" of assimilation such as that proposed by Barker and found in yeast and those bacteria which form poly-/5-hydroxybutyric acid or a glucose polymer. Poly-,3-hydroxybutyrie acid has never been found in this organism (35,45) and Warren, Ells and Campbell (83) were unable to detect an increase in cellular carbohydrate during oxidative assimilation of glucose by P. aeruginosa using chemical assays. If a compound such as glycogen was a major reserve material in this organism i t would be expected that the enzymes for glucose oxidation would be constitutive. However, Hamilton and Dawes (41) found that this is not so. Cells grown on organic acids had low basal levels of glucose and gluconic dehydrogen-ase, hexokinase, gluconokinase, glucose-6-phosphate and 6-phospho-gluconate dehydrogenase, and the dehydrase and aldolase of the Entner-Doudoroff pathway. The levels of these enzymes were increased i f the cells were subsequently incubated with glucose. Since most of the material which was synthesized by P. aeruginosa from the radioactive glucose was nitrogenous, and since endogenous respiration supplies the ammonia for this synthesis, oxidative assimi-lation may serve, at least in part, to replenish the endogenous reserves 84 of the organism. Clifton (23), in 1962, reached a similar conclusion in a study of the oxidative assimilation of B. cereus. His results differ from those found for P. aeruginosa in that a much higher per-centage of the radioactive carbon from glucose was assimilated. This may be due to the fact that the rate of glucose oxidation as compared to endogenous respiration is lower in this organism, so that excess ammonia may possibly be present in the supernatant throughout the period of glucose oxidation by B. cereus. It should be emphasized that a l l studies reported in this work were carried out with freshly-harvested cell suspensions of P. aerugi-nosa which had been grown on a glucose-basic salt medium. It might be expected that the results would be different i f the cells were grown under different conditions or on a different medium, or i f the washed cell suspensions were starved prior to incubation with glucose. Tor example, the findings of Doudoroff and Stanier'(31) have been mentioned in which freshly-harvested cells of P. saccharophila assimilated only 21$ of the glucose while starved cells assimilated 50$. Starved cells, that is cells which have been aerated in a non-nutrient medium to reduce their endogenous respiration, have been used by most workers in studies of oxidative assimilation. They were not used in the present experi-ments on P. aeruginosa since early studies in the laboratory (65) indicated that i t was not possible to diminish the endogenous reserves of this organism by the aeration of washed cell suspensions in a buffer solution. 85. SUMMARY When washed cell suspensions of P. aeruginosa were incubated in 14 the presence of C -labelled glucose only small amounts of radioactivity were found to be incorporated into the cells and at -ketoglutaric acid accumulated in the supernatant. A portion of the -ketoglutaric acid was oxidized after the disappearance of glucose while the remaining e<-ketoglutaric acid was incorporated as ammonia became available from the breakdown of endogenous reserves. Most of the material synthesized by the cells during oxidative assimilation was nitrogenous, the ammonia being supplied by the endogenous respiration. The cold TCA-soluble fraction and the lipid fraction appeared to be important during the early stages of oxidative assimilation, while the largest percentage of the incorporated radioactivity was found in the protein fraction. Since chloramphenicol specifically inhibited incorporation of G 1 4 into the latter fraction i t appeared that actual protein synthesis was involved. In the presence of added ammonia assimilation was greatly increased and no oC-ketoglutaric acid was found in the supernatant fluid. Results obtained during the oxidation of variously-labelled glucose were in agreement with the major roles of the Entner-Doudoroff and pentose phosphate pathways in glucose dissimilation by P. aeruginosa. 14 When glucose-l-C was used as the substrate, almost a l l the radio-activity was lost as carbon dioxide and the compounds which were found to be labelled in the cells were those which could arise via carbon dioxide fixation. The importance of carbon dioxide fixation in cellular synthesis by this organism was also indicated by the decrease in C 1 4 i / 86. incorporated into the cells when a C 0 g atmosphere was used as the gas phase during oxidation of uniformly-labelled glucose. It appeared that growth of the organism in a glucose medium represses, at least partially, the enzymes for transport of oL -ketoglu-taric acid into the cells and for oxidation of this compound, thus allowing the transient accumulation of -ketoglutaric acid outside the cells during glucose oxidation. 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