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

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I METABOLIC ACTIVITIES OF PSEUDOMONAS AERUGINOSA WHEN GROWN ON A 2-KET0GLUC0NIC ACID MEDIUM by ROBERT WILLIAM HILL A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE i n the Department of Dairying We accept t h i s t h e s i s as conforming to the standard required from candidates for the degree of MASTER OF SjDIENCE IN AGRICULTURE Members of the Department of THE UNIVERSITY OF BRITISH COLUMBIA October 1952 - i -ABSTRACT The current concept of the pathway of the intermediate car-bohydrate metabolism of P. aeruginosa ATCC 9027 i s that glucose i s oxidized to carbon dioxide and water by way of glucose, glu-conic, 2-ketogluconic and pyruvic acids. The pathway of terminal r e s p i r a t i o n i s the conventional Kreb's t r i c a r b o x y l i c a c i d cycle. However, the mechanism by which 2-ketogluconic acid i s oxidized to pyruvic acid has not been elucidated. The f i r s t concern i n the present work was to es t a b l i s h the accumulation of 2-ketoglu-conic acid during glucose oxidation by P. aeruginosa ATCC 9027 and also to produce enough calcium 2-ketogluconate to serve as substrate i n studies of i t s oxidation. The i n i t i a l problem on the metabolism of 2-ketogluconic acid grown c e l l s of P. aeruginosa was to compare the enzymic pattern of these c e l l s with c e l l s grown on a glucose medium. The data obtained under these conditions,confirmed the previous conclusions that 2-ketogluconic acid i s i n the dire c t pathway of glucose oxidation. Additional evidence f o r the minor r o l e of the Embden-Meyer-hoff pathway i n t h i s organism was obtained when i t was shown that c e l l s grown on a 2-ketogluconic acid medium contained only traces of the enzyme aldolase. The amount of t h i s enzyme detected would be s u f f i c i e n t to permit the g l y c o l y t i c pathway to function as a synthesizing mechanism f o r pentoses or perhaps heptuloses but would not be s u f f i c i e n t to account for any major amount of glucose. - i i The major problem of establishing the presence of i n t e r -mediates occurring between 2-ketogluconic and pyruvic acids was approached from two points of view: f i r s t , by attempting to pro-duce a c e l l preparation which had a l i m i t e d a b i l i t y to oxidize 2-ketogluconic acid and which would accumulate intermediates i n a manner s i m i l a r to that of c e l l preparations which accumulate 2-ketogluconic acid during glucose oxidation, and second, by the blocking of the enzyme system of 2-ketogluconic acid grown c e l l s by an i n h i b i t o r at a point intermediate between 2-ketogluconic acid and pyruvic a c i d . Neither approach yielded p o s i t i v e r e s u l t s . Further studies were made on the basis of an early observa-t i o n that c e l l s which had been heated showed an impaired gluconic acid and 2-ketogluconic acid o x i d i z i n g a c t i v i t y . It was found subsequently that l i v e c e l l s heated to 55°C. f o r at least ten minutes would oxidize glucose but not gluconic acid or 2-keto-gluconic acid and that further heating nearly completely destroyed the a b i l i t y of the c e l l s to assimulate glucose. Lyophilized preparations of such heat treated c e l l s s t i l l retained the a b i l i t y to oxidize glucose or gluconic acid to 2-ketogluconic acid. This can be interpreted as i n d i c a t i n g that 2-ketogluconic acid i s on the direct oxidative pathway of glucose oxidation. ACKNOWLEDGMENT I wish to express my sincere thanks to Dr. J.J.R. Campbell f o r h i s d i r e c t i o n of t h i s work and f o r h i s invaluable assistance and i n t e r e s t . I also wish to thank the National Research Council f o r the grant to carry out t h i s i n v e s t i g a t i o n . R.W.H TABLE OF CONTENTS Abstract ' i Introduction 1 I. Production of calcium 2-ketogluconate. (a) Introduction 3 (b) Experimental i . Production 6 i i . I s o l a tion arid p u r i f i c a t i o n 10 I I . The comparison of the metabolic a c t i v i t i e s 13 of glucose grown and '2-ketogluconic acid grown c e l l s . I I I . Determination of the presence of the 14 enzyme aldolase. IV. Study of dried c e l l preparations. (a) Introduction 17 (b) Methods 18 (c) Results 22 V. Influence of metabolic i n h i b i t o r s on the 24 oxidation of glucose and 2-ketogluconic ' acid. VI. , E f f e c t of heat shocking on the enzyme 28 structure of c e l l s grown on 2-ketogluconic acid. VII. Addendum 1 30 VIII. Addendum 2 32 IX. Bibliography 35 INTRODUCTION In the past few years i t has been established that the oxidation of glucose by Pseudomonas aeruginosa ATCC 9027 pro-ceeds by way of glucose, gluconic, 2-ketogluconic and pyruvic acids (12, 39, 48 and 51). It has also been shown that the system of terminal r e s p i r a t i o n i s the t r i c a r b o x y l i c acid cycle (11, 13 and 14). There i s no evidence of phosphorylation between glucose and 2-ketogluconic acid and so glucose-6-phosphate i s not an intermediate (12 and 21). The proposal of t h i s pathway has been greeted by a great deal of skepticism (21) l a r g e l y because of the almost universal presence i n aerobic tiss u e and microorganisms of the Warburg-Dickens system (18) of glucose oxidation. When glucose i s oxidized v i a t h i s pathway i t i s through g l u c o s e s -phosphate, 6-phosphogluconolactone, a postulated 6-phospho-2-ketogluconolactone, ribulose -5-phosphate, ribose-5-phosphate, and eventually to acetic acid, C0 2 and water. The opponents of the glucose, gluconic, 2-ketogluconic acid scheme have maintained that the two l a t e r compounds are formed merely i n side reactions from members of the Warburg-Dickens pathway. However, using c e l l free extracts of P. aeruginosa 9027 and P. fluorescens, Wood and Schwerdt (55, 55a)' have been able to show that these organisms do have many of the enzymes of the Warburg-Dickens pathway, but that they lack hexokinase, and therefore cannot oxidize glucose by way of t h i s phosphorylated scheme. It would appear that the two schemes are operative i n the metabolism of these organisms. - 2 -Glucose i s oxidized by way of gluconic and 2-ketogluconic acids to t r i o s e s . A phosphorylated t r i o s e then condenses with pyruvic acid, to give 6-phosphogluconic acid which i n turn can be oxidized and decarboxylated to y i e l d ribose-5-phosphate (55a). The Warburg-Dickens enzymes thus serve as the pentose forming pathway of these organisms. The great hiatus i n our knowledge of the glucose - 2 -ketogluconic acid scheme at the present time i s the immediate fate of 2-ketogluconic acid. It i s possible that i t i s d i r e c t l y d i s -similulated to pyruvate, but i t i s equally possible that i t may be a stepwise degradation through 5 carbon and 4 carbon compounds. There i s also the p o s s i b i l i t y of phosphorylation of 2-ketogluconic acid, decarboxylation to a pentose and then a 3-2 s p l i t of the pentose. As a res u l t of t h i s s i t u a t i o n t h i s thesis i s concerned with attempts to i d e n t i f y the intermediates, i f any, between 2-keto-gluconic acid and pyruvic acid. - 3 -PART I. PRODUCTION OF CALCIUxM 2-KETOGLUCONATE a. Introduction Although the evidence available at the present time indicates that many bacteria oxidize glucose by way of 2-ketogluconic acid i t was not u n t i l 1935 that Bernhauer and Gorlick (3, 4, and 5) f i r s t demonstrated that t h i s acid was produced during b a c t e r i a l d i s -simulation of glucose. The organism which they studied was Acetobacter suboxydans. The enzyme which oxidizes the intermediate, 2-ketogluconic acid, Is destroyed by intensive aeration, and so t h i s acid accumulates under conditions which favour the oxidation of the 2-ketogluconic acid dissimulating enzyme. In the experi-ments of Bernhauer and Gorlick (3) the destruction of the 2-keto-gluconic acid oxidizing enzyme and therefore the enhancement of the accumulation of the acid was accomplished by incubating the culture at r e l a t i v e l y low temperatures f o r periods of 30 to 60 days. Similar work was done by Utkin (57) who reported the accumu-l a t i o n of 5-ketogluconic acid from a culture of Acetobacter. How-ever, h i s data would indicate that 2-ketogluconic acid rather than 5-ketogluconic acid was the end product. Pervozvanskii (41, 42) placed 2-ketogluconic acid production on a firmer basis when he demonstrated that "onic" or "ketonic n acids were a function of the s p e c i f i c b a c t e r i a l s t r a i n used. He suggested the i n d u s t r i a l p o s s i b i l i t i e s of the process by showing that C a + + , aeration and agi t a t i o n were necessary f o r rapid and near complete conversion of glucose to 2-ketogluconic acid. Ward (53) 1939, working on the gluconic acid fermentation - 4 -process i s o l a t e d a contaminating organism which accumulated 2-ketogluconic acid. Using the previously published p i l o t plant process (54) with which the Peoria group had done much work, Ward and h i s co-workers (30, 31, 34, 49, 54) were able to demon-strate that 2-ketogluconic acid accumulation was a two step pro-cess from glucose to gluconic acid and f i n a l l y to 2-ketogluconic a c i d . Lockwood, Ward, Stubbs, Roe and Tabenkin (30) 1940, sub-sequently obtained a patent on 2-ketogluconic acid production using a Pseudomonas species grown with intense aeration i n an alkaline medium with either glucose or calcium gluconate -as carbon course. Blaisten (8) 1947, an Argentine worker, produced 2-ketogluconic acid from glucose i n 80$ y i e l d using Lockwood's method. The 2-ketogluconic acid was used i n the production of a potential antioxidant, arabinoascorbic acid. Ikeda (26) 1948, and Asai and Ikada (1) 1950, obtained s i m i l a r r e s u l t s using shake cultures of P. fluorescens. Serratia marcescens, and Gluconobacter  species. They obtained 75-&0$ y i e l d s of 2-ketogluconic acid from glucose and from traces to 83$ y i e l d s of 2-ketogluconic acid from potassium gluconate. The occurrence of gluconic and 2-ketogluconic acids as normal intermediates i n glucose oxidation has been demonstrated by Norris and Campbell (39) 1949, using P. aeruginosa ATCC 9027 i n s t i l l culture. . De Ley and Cornut (17) 1951, used P: putida. obtained 90-97$ y i e l d s of 2-ketogluconic acid s t a r t i n g with calcium gluconate. In 1951 (43) a B r i t i s h patent was given f o r the production of 2-keto-hexonic acid from hexonic acid i n 90-95$ y i e l d using an organism - 5 -c a l l e d Cyanococcus chromospirans. It was thus f e l t that the demonstration under intense aerobic conditions of 2-ketogluconic acid accumulation using P. aeruginosa  ATCC 9027 would add to our knowledge of the metabolism of t h i s s t r a i n and would provide us with a supply of 2-ketogluconic acid f o r use i n further experimental work. b. Experimental i . Production The d e f i n i t e i d e n t i f i c a t i o n of 2-ketogluconic acid as a ba c t e r i a l end-product was f i r s t carried out by Bernhauer and Gorlick (3) 1935, Bernhauer and Knoblock (4, 5) 1940, and Knoblock and Tietze (27) 1941, with Acetobacter suboxydans using calcium gluconate or glucose plus calcium carbonate as s t a r t i n g materials. With t h e i r system they could show, depending on con-di t i o n s , an accumulation of calcium 2-ketogluconate or calcium 5-ketogluconate. This has been subsequently seriously questioned and attributed by other workers to the use of impure cultures as no known organism has since been shown to form both intermediates. Lockwood et a l (54,and 31) 1937, developed a p i l o t plant operation using P. fluorescens which accumulated 2-ketogluconic acid from glucose or gluconic acid. Lockwood's method has been used by others (1, 8, 17, 26 and 28) f o r the laboratory production of 2-ketogluconic acid because, at the present time, the market price i s p r o h i b i t i v e . The procedure used i n the present work i s a modification of the method of Lockwood. P. aeruginosa ATCC 9027 and a modified medium of Norris and Campbell (11) with glucose as a carbon source and urea as a nitrogen source was used. This synthetic medium f a c i l i t a t e d the p u r i f i c a t i o n with no apparent loss i n y i e l d over Lockwood's medium which contained crude corn steep l i q u o r . The prevention of foaming during the i n i t i a l stages of fermentation was p a r t i a l l y controlled by additions of saturated mineral o i l and - 7 -octadecanol. Corn o i l and 3$ Alkaterge C helped control the foaming during the l a t e r stages of fermentation. Foaming apparent-l y i s the l i m i t i n g factor i n that i t determines the amount of a i r flow and agitation possible i n a system i n which a i r flow through the fermentor i s induced by vacuum rather than by pressure d i f f e r e n t i a l as i s usual. Using t h i s method i t was possible to produce approximately 1000 grams of commercial grade calcium 2-ketogluconate and 300 grams of pure calcium 2-ketogluconate, containing no detectable contamination with glucose or gluconic acid. Paper chromatography using ethanol-ammonium hydroxide as ascending solvent and ortho-phenylendiame or ammoniacal s i l v e r n i t r a t e as developer f a i l e d to show any contamination. Manometric studies using glucose and gluconic acid oxidizing c e l l preparations showed traces of fermentable contaminating substances. The organism used was Pseudomonas.aeruginosa ATCC 9027 carried on stock agar slopes * at 0-5°C. afte r i n i t i a l 48 hour growth at 30 GC. These stocks were sub-cultured at monthly i n t e r v a l s . A stock culture slope was used as an.inoculum f o r 10 ml. of yeast glucose broth *. After 48 hours at 30°C. the 10 ml. of culture was transferred to 100 ml. of yeast glucose broth i n a 500 ml. Florence f l a s k . The f l a s k was incubated 72 hours on a rotary shaker or u n t i l heavy t u r b i d i t y appeared. This culture was used as inoculum f o r a small scale fermentor containing one l i t e r of production medium *.. The inoculum was introduced into the small fermentor by way of the o i l addition port or tube. The temperature i was controlled by p a r t i a l submersion of the fermentor i n a constant * Addendum 1 - 8 -temperature water bath. Aeration was accomplished by reducing the pressure within the fermentor with a trapped water jet vacuum pump. The incoming a i r was f i l t e r e d and s t e r i l i z e d by passage through a 4" x 1" tube of s t e r i l e cotton wool. The a i r was then l e d through two porous stone blocks at the bottom of the fermentor. Agitation was accomplished by aeration alone. The production medium* was composed of glucose, calcium carbonate, potassium acid phosphate, magnesium sulfate, iron sulfate and urea. The urea was s t e r i l i z e d separately by f i l t r a t i o n through u l t r a f i n e sintered glass f i l t e r and added through the o i l port to the s t e r i l i z e d medium i n the f e r -mentor just a f t e r inoculation. The glucose, calcium carbonate and other mineral s a l t s were s t e r i l i z e d f o r one hour at 1Z\ l b s . / i n . 2 . Prior to s t e r i l i z a t i o n a 1/8" to l/4 " layer of saturated mineral o i l and octadecanol,.which had been s t e r i l i z e d previously, was added to the main batch. The mineral o i l and octadecanol with enough water to f l o a t the o i l were s t e r i l i z e d f o r three hours at 12g l b s . / i n . 2 . During fermentation the foaming was p a r t i a l l y i: controlled by the addition of s i m i l a r l y s t e r i l i z e d corn o i l and 3% Alkaterge C. This o i l was added by means of a s t e r i l e pipette -to the small fermentor or by a s t e r i l e vented bottle to the large fermentor (Plate 2). The one l i t e r fermentor was incubated f o r 48-72 hours and then transferred as inoculum to the large fermentor containing 6000 ml. of production medium i n a 12 l i t e r f l a s k (Plate 1). The trans f e r was done by reducing the pressure on the larger vessel while they were connected v i a t h e i r respective o i l addition ports. * Addendum 1 - 8a -P R O D U C T I O N F E R M E . N T O B P L A T E N O . / V A C U U M L I N E . O U T C O T T O N Vtfooi A I R . \ N - r A v C E _ S A M P L I N G D E V / C E & O i i _ _ A O D I - T I O N C L A M P I R . O B B E . R T U B E . ^i_-vv'teAPpe.o C O V E R . V A C U U M L I M E . O u n - c r f-^-rHTii/CoTTOM W O O L M £ . D I A L _ E L V E . 1 _ COM3T&NT B A . T H Ca CO, - '8b -O I L ADD\TION F L A S K P L A T E NO. X C O T T O N W O O L ' G L A S S T U & I I N G I - R U B B E R . T U B I N G , £ 5 i u LEVEL. av-- WATER. s5CO ML. ElRi_t.NM&YElR. r v _ A . S K S t e r i l e urea was added immediately aft e r inoculation and a i r flow gradually increased u n t i l stable conditions were manifest. Foam-ing was the l i m i t i n g f a c t o r to. adequate aeration and agi t a t i o n e s p e c i a l l y toward the end of the run. The temperature control and other factors were sim i l a r to the small scale fermentor. The f e r -mentation was complete i n 3 to 5 days at which time the calcium carbonate, some of which s e t t l e s to the bottom, no longer went into solution. The end.point of the fermentation was checked by two d i f f e r e n t methods. The f i r s t method was to stop the run on the l e v e l l i n g o f f of the levorotatory readings of the polarimeter. The second method, used to check the f i r s t , was to make p i l o t quantitative i s o l a t i o n s of 2-ketogluconic acid u n t i l these also l e v e l l e d o f f , at which time the run was stopped. The percentage conversion varied from run to run so that a r b i t r a r y values could not be used to stop the runs. - 1 0 -b. Experimental i i . I s o l ation and P u r i f i c a t i o n ______________________________ y The y i e l d of crude product was between 4 0 0 and 7 0 0 grams per 1 1 2 0 grams of i n i t i a l glucose or a 3 6 to 6 2 % y i e l d . The essential steps i n the i s o l a t i o n and p u r i f i c a t i o n were f i l t r a t i o n , concentration, decolorization, and c r y s t a l l i z a t i o n . On completion of fermentation the broth was mixed with 1 to 2% supercel and f i l t e r e d through a supercel precoated f i l t e r cloth on a Buchner funnel or a s i m i l a r l y treated basket centrifuge. The r e s u l t i n g clear l i q u o r was treated with 6 0 to 1 0 0 gram batches of decolorizing charcoal u n t i l e s s e n t i a l l y c o l o r l e s s . The decoloriza-t i o n was carried, out by heating the l i q u o r and washings to 6 0 ° C , agitating, then cooling the mixture and f i l t e r i n g . The resultant clear l i q u o r was a very pale yellow green color. The decolorized l i q u o r was then vacuum d i s t i l l e d batchwise to one-third volume or to a t h i n syrup depending on the r e l a t i v e amounts of calcium 2-ketogluconate and glucose present. The d i s t i l l a t i o n was done from a controlled water bath at 4 5 - 5 5°C. -under vacuum. If necessary the concentrated broth was decolorized again and r e f i l t e r e d . The concentrated broth was cooled to 0 ° - 5°C. and l e f t at t h i s temperature overnight to c r y s t a l l i z e . The pale yellow-white c r y s t a l l i n e mass, was vacuum f i l t e r e d and then s l u r r i e d with hot 80% alcohol and r e f i l t e r e d . This procedure removed most of the contaminating glucose. The s o l u b i l i t y of p u r i f i e d calcium 2-ketogluconate i n water was 1 9 grams per 1 0 0 ml. at 3 0°C. and 1 7 grams per 1 0 0 ml. at 2 0 °C. It was only very s l i g h t l y soluble i n 95% alcohol. It i s because of - 11 -these s o l u b i l i t y c h a r a c t e r i s t i c s that alcohol washing, rather than r e c r y s t a l l i z a t i o n , i s emphasized. The s l i g h t l y moist c r y s t a l s are very sensitive to heating i n excess of 45°C. To remove the l a s t traces of glucose the cr y s t a l s obtained from the above procedure were dissolved at 80-90°C. i n a minimum of water, decolorized with charcoal; cooled, f i l t e r e d and cry-s t a l l i z e d out at 0-5°C. The f i l t e r e d c r y s t a l s were s l u r r i e d with. 70$ ethyl alcohol, f i l t e r e d and r e s l u r r i e d with 95$ ethyl alcohol, f i l t e r e d , and dried below 45°C The r e s u l t i n g c r y s t a l s were a f i n e white c r y s t a l l i n e mass which, on drying, yielded an e s s e n t i a l l y pure product. The purity of the product was checked chromatographically using ethanol 80 parts water,16 parts and ammonium hydroxide 4 parts on ascending and descending Whatman No. 1 f i l t e r paper. Ammoniacal s i l v e r n i t r a t e , ortho and meta phenylenediamine (29), brom cresol green and 2,6 dichlorobenzenone-indophenol (7) were used as spray developers. There was no evidence of gluconic acid or of glucose or of other contaminating reducing or a c i d i c products present. There was a trace of glucose and some color i n the crude product, however. Manometric determinations using 18 oxygen equivalents of c a l -cium 2-ketogluconate as substrate yielded 329 micro l i t e r s of oxygen uptake (as the average value f o r twenty runs). This was lower than the glucose controls by an average of 8 m i c r o l i t e r s . This indicates the presence of 2.4$ of •.. non-oxidiaable substances and may be due to incomplete water removal or contaminating s a l t s . Using a dried preparation of P. aeruginosa and 18 oxygen equivalents - 12 -of calcium 2-ketogluconate as substrate the average difference be-tween endogenous r e s p i r a t i o n and calcium 2-ketogluconate f o r nine runs was 1.7 m i c r o l i t e r s of oxygen. The control glucose f l a s k s contained 18 oxygen equivalents and took up 72 m i c r o l i t e r s of oxygen. Assuming the fermentable contamination was glucose there would be 2% glucose present. However, t h i s difference of 1.7 micro-l i t e r s i s well within the l i m i t s of experimental error with the equipment and procedure used. - 13 -PART I I . THE COMPARISON OF THE METABOLIC ACTIVITIES OF GLUCOSE GROWN AND 2-KETOGLUCONIC ACID GROWN CELLS Ce l l s of P. aeruginosa harvested from the glucose medium* re-quired a period of adaptation before attacking 2-ketogluconic acid at a maximum rate (39). Stanier (21 and 44) has argued that i n l i g h t of h i s theory of simultaneous adaptation, 2-ketogluconate can therefore not be considered an intermediate i n glucose oxidation. However, i f 2-ketogluconic acid i s an intermediate i n glucose oxida-t i o n then c e l l s grown on either glucose or 2-ketogluconic acid should exhibit the same spectrum of adaptive enzymes. In an e f f o r t to check t h i s assumption the enzymic a c t i v i t y of c e l l s harvested from media containing glucose* as sole carbon source and media con-taining 2-ketogluconic a c i d * as sole carbon source were compared. The compounds compared were glucose, gluconic, 2-ketogluconic, pyruvic, eCketoglutaric, fumaric, succinic, c i t r i c and acetic acids. Allowing f o r the normal variations between runs the two types of c e l l s showed no differences i n t h e i r enzymic make-up as i indicated by t h e i r a b i l i t i e s to attack the substrates tested (Plates 3 to 11). As a further comparison, glucose and 2-ketoglu-conic acid grown c e l l s which were grown on a rotat i n g shaker f o r IB hours at 30°C. were compared (Plates 12 to 14). The enzymic pattern i s the same as that f o r the c e l l s harvested from stationary medium and again no differences can be detected between the c e l l s grown on glucose and those grown on 2-ketogluconic acid. * Addendum 2 3001-GUOCOSE. STILL. GROWN P L A T E . N O . •+ A G L U C O S E . B . G L U C O N I C A C I D C 2 - K E T O G L O C O N I C A C I D Z- K_TO GLUCONIC A c i D STILL GROWN TIME (MINUTES) 300 GLUCOSE. STILL. G e o w i s 2- KCTOGLUCONIC ACID STILL GROWN P L A T E . N O . 5 A G L U C O S E . B G L U C O N I C A C I D C E - K E T O G L U C O N I C A C I D 200U CL o 100 A c 20 40 60 c TIME (MINUTES) ZO AO 60 - 13d -C/°0 3MVJLdO z 0 3 0 0 l GLUCOSE. STIUL GROWN P L A T E : N O . 7 A K . E . T O G L U T A R . I C A C I D B F U M A R J C A C I D c P Y R U V I C A C I D 2.- K E . T O G L U C O N I C A C I O S T I L L GROWN zoo 3 a o IOO a I I I- I I I I I 20 40 60 6 0 ICO O T I M E (MINUTES) • A • -» B • C zo AO j l i l SO I C O 3 0 0 GLUCOSE STIUL. GBOWM P L A T E . N O . B A <<- K E . T O O i _ U T - A R . I C . A C I D E> F ' U M A R . t C A C I O C P Y R - O V I C A C I D 2 0 0 l — Id M O 100 2 - K E L T O G L O C O N I C A C I O S f l L L . G f t O W N 2 0 4 0 L 6 0 O 2 0 T I M E (MINUTES) A K C 4 0 6 0 30O G L U C O S E . S T I L L GeowM PLATL NO. 7 A A C E - T I C A C I D E> S U C C I I S I C A C I D ZOO I bJ _: < <x o N o too 2,-KETOGL.UCONIC ACIO STILL GROWN B 20 40 €0 0 TIME. ( M I N U T E S ) 20 40 60 P L A T E . NO. 10 200 3 hi < 0-D IOO GLUCOSE. STILL GROWN A A C E T I C A C I D e> S U C C I N I C A C I D C C I T R I C A C I D « B £ - KETOGLOCONIC ACID S T I L L • GROWN 20 40 60 O TIME (MINUTES) 20 40 60 300 -GUUCOSE. STICL. GROWN 2 - Kero GUUCON IC ™ GROWN ACID STII_L PLATE NO. // A ACELTIC ACID B S O O C - I N I C A C I D c C I T R I C , A C I D e A • —0 A ZOO 3. UPTAKE J m g% # g, O 100 *~—"™ ™ - c * f B i i i. -JL 1 1 / — • I ———~~* C 1 1 0 20 40 Go 0 20 40 60 T I M E (MINUTES) .3001 P L . A X E L N O . / Z, G L U C O S E S H A K E L R . Geowrs A - G L U C O S E B - G L o e o r - f i C A c i o C - 2 - K E T O G L U C O M I C A C I D S t O O l _: _ a "too 2.- KETOGLOCONIC ACID SHAKCER. GROWN A c l i l t IO Z.O 30 -40 AO 60 VO OO 90 IOO O 'O ZO 30 AO SO <SO TO SO SO IOO T I M E (MINUTES) 300 P L A T E . NO. / 3 ZOO id _ O GLUCOSE SHAKER. GROWN A - ° C K E T O G L U T A R I C . A C I O B - F U M A R . I C A C I D c - P Y R U V I C A C I D J£- KETOGLOCONIC ACID SHAKER. GROWN J I I I • » • 1 c J I I L _ l L l _ L O KD 20 «30 -40 SO 60 70 ao 90 loo o l O Z.O -SO -40 50 60 70 So 90. IOO TIME CMINUTES) 300 GU-UCOSE. SHAKER GROWN E - k _ E _ T O G L U C O N I C A c i O S H A x K E L i e . PLATE. NO. A - A C E 1 T I C A C I D . B - S U C C I N I C A C I D C - C iT f5»o A C I D Z O O I bJ _ O (4 O lOOl IO - 3 0 -40 «50 60 7 0 So 9 0 IOO o »Q 2 d -30 -SO 6 0 7 0 80 S O l o o TIME (MINUTES) - 14 -PART I I I . DETERMINATION OF THE PRESENCE OF THE ENZYME ALDOLASE In c o n s i d e r i n g the r e s u l t s so f a r obtained i n t h i s l a b o r a t o r y (11, 12, 13, 14, 3#, 39, 48 and 51) i t would be c o n s i s t e n t t o ex-pect P. aeruginosa t o have a r e s t r i c t e d aldolase-enzyme since the phpsphorylated Embden-Meyerhoff intermediates could not be detected and do not appear to f u n c t i o n to a measurable extent i n the path-way of glucose o x i d a t i o n (12). To demonstrate i f t h i s concept i s corre c t the al d o l a s e determination of Dounce and Beyer (20) 194#, was f o l l o w e d . This t e s t was chosen because i t i s considered i n -d i c a t i v e of an Embden-Meyerhoff system i n that i t measures the t r i o s e s formed from fructose-lrr6-,diphosphate. This procedure was c a r r i e d out on s e v e r a l d i f f e r e n t c e l l p reparations of P. aeruginosa  ATCC 9027. Streptococcus f a e c a l i s B18 was used as a standard since i t was known to have an a c t i v e a l d o l a s e system (47). The S. f a e c a l i s c e l l s were used as an acetone p r e p a r a t i o n . The pre-parations of pseudomonas c e l l s were so weak i n the enzyme a l d o l a s e (Table 1) t h a t 10 mg. of c e l l s were used f o r comparison against 0.1 t o 1.0 mg. of S. f a e c a l i s . Ten mg. of c e l l s was the l i m i t beyond which the blank reading became high enough t o impair the s e n s i t i v i t y of the t e s t . The r e s u l t s obtained (Table 1) are con s i s t e n t w i t h those of Campbell and N o r r i s (12) i n which they found no members of the Embden-Meyerhoff pathway present. Working w i t h P. aeruginosa and P. f l u o r e s c e n s , Wood and Schwerdt (55) demonstrated the presence of glucose-6-phosphate o x i d i z i n g enzyme and a 6-phosphogluconic o x i d i z i n g enzyme. However, n e i t h e r organism had hexokinase and 15 -therefore had no means of converting glucose to glucose-6-phosphate. They also found traces of aldolase a c t i v i t y which might mean that the Warburg-Dickens scheme (18) exists i n our organism as a syn-t h e t i c system f o r production of necessary intermediates. Horecker and Smyrniotis (25a) have described a system which causes the accumulation of sedoheptulose from pentose phosphate. Aldolase was required presumably to s p l i t the pentose, t r i o s e and a 2 carbon fragment. The two dioses combine to form tetrose which i n turn combines with previously formed t r i o s e to y i e l d sedohep-tulose. This could conceivably account f o r the aldolase a c t i v i t y which we found although the intermediates required by t h i s reaction have not been formed. - 16 -TABLE I Results of Aldolase Determination Organism Preparation of Cells A c t i v i t y * S. f a e c a l i s Acetone preparation 1000 P. aeruginosa Acetone preparation 1+2 P. aeruginosa Acetone preparation 32 P. aeruginosa Alumina ground debris 66 P. aeruginosa Sloppy dried 33 P. aeruginosa Lyophilized 3 Based on 1 mg. of S. f a e c a l i s equals 1000 units. - 17 -PART IV. STUDY OF DRIED CELL PREPARATIONS a. Introduction It i s known that when c e l l s of P.. aeruginosa are dried under vacuum with phosphorous pentoxide as desiccant, they r e t a i n the a b i l i t y to quantitatively oxidize glucose or gluconic acid to 2-ketogluconic acid (48). It was hoped that by modifying t h i s treatment c e l l s of pseudomonas could be dried to a stable form which would r e t a i n the a b i l i t y to quantitatively convert 2-ketogluconic acid to further intermediates. The quantitative conversion i s possible because dried c e l l preparations have no syn-t h e t i c a b i l i t i e s and therefore no oxidative assimulation. Using sloppy dried c e l l s as a comparitive standard, a method previously used i n t h i s laboratory on glucose grown c e l l s (48), i t was found that the treatments t r i e d did not preserve the a b i l i t y of the organism to oxidize 2-ketogluconic acid. - 1. -b. Methods It was established manometrlcally that carboxymethylcellulose (CMC) could not be oxidized appreciably by our s t r a i n of pseudo-raonas and so t h i s compound was added i n 20 and 40 mg. amounts to enough l i v e c e l l s , harvested from a 2-ketogluconic acid medium*, to give ten 20 mg. batches of dried c e l l s a f t e r l y o p h i l i z a t i o n . This was done on the assumption that CMC would form a protective coating around the i n d i v i d u a l c e l l and prevent the oxidative des-t r u c t i o n of the 2-ketogluconic acid oxidizing enzyme. Cysteine (37) could not be used as a protective agent since these bacteria oxidize i t . For the same reason g e l a t i n could not be used although i t has been used by other workers (35, 10) f o r various organisms. It has been demonstrated (36) that substrate bound enzymes are more stable than those not bound to the substrate, therefore, 2-ketogluconic acid grown c e l l s * * were sloppy dried i n the pre-sence of 0.05% calcium 2-ketogluconate. In another experiment sodium thioglycolate at 0.3$ concentration was used i n an attempt to prevent the oxidation of the "SH" groups on the enzyme during drying by acting as an anti-oxidant. Sucrose at a concentration of 0.5$ was used as a possible means of protecting the l a b i l e enzyme system during drying (35) as was 0.9$ NaCl, yeast extract 0.05$, mineral medium of Norris and Campbell (11), M/30 phosphate buffer at pH 7.4, and d i s t i l l e d water. Carbowax has been used as an embedding material i n tissue s l i c e studies (25), and under these conditions i t appeared to help * Addendum 1 ** Addendum 2 - 19 -preserve the enzymes of the plant t i s s u e . It was therefore f i r s t tested manometrically as a possible protective coating f o r bacteria during drying. P. aeruginosa was found to attack t h i s compound with vigor and so could not be used. In the studies on l y o p h i l i z a t i o n (22) use was made of both bulk l y o p h i l i z a t i o n , i n which the resultant dried c e l l s were ground i n a i r , and stored i n bulk at 0-5°C., and of a procedure suited to smaller quantities i n which the 2-ketogluconic grown c e l l s * were dried i n ind i v i d u a l v i a l s . Each v i a l contained 20 mg. of dried c e l l s which i s enough f o r one Warburg cup.- On completion of drying, the v i a l s were sealed o f f under vacuum. Three methods were used to arrive at a low enough dew point or vapour pressure to maintain the c e l l s below the t e r t i a r y point of water during drying. In the bulk l y o p h i l i z i o n the water was trapped from the vacuum system by dry ice and acetone. The i n d i v i d u a l v i a l s were dried using Freon 23 i n a mechanical r e f r i g e r a t i o n system. The t h i r d method made use of the chemical desiccant, phosphorous pent-oxide i n a large desiccator under high vacuum. The P2O5 was layered between glass wool to increase the e f f e c t i v e area of the desiccant. Under these conditions the vapour pressure was reduced almost to that of l i q u i d a i r and the c e l l s which were i n the desiccator dried by sublimation. Sloppy dried c e l l s (4$) i s a method again u t i l i z i n g P2O5 as the desiccant but i n t h i s case a t h i n layer was used on the bottom of a desiccator and only a water jet vacuum was drawn on the desiccator. Under these conditions the t h i n paste of 2-ketogluconic * • Addendum 2 i - 20 -acid grown c e l l s * took i n excess of 48 hours to dry and the drying was from the l i q u i d state: rather than the frozen state as i n l y o p h i l i z a t i o n . Sloppy dried c e l l s under nitrogen were prepared from 2-keto-gluconic acid grown c e l l s * which were treated s i m i l a r l y to sloppy dried c e l l s except that the desiccator was flushed with nitrogen, evacuated and reflushed with nitrogen f i v e to six times before evacuating f o r the f i n a l time. The nitrogen was added i n an e f f o r t to reduce the p o s s i b i l i t y of oxidation of the l a b i l e 2-ketogluconic acid enzyme during drying. Acetone dried c e l l s (52) were prepared by pipetting a s l u r r y of washed, 2-ket6gluc6riic acid grown c e l l s * into 25 volumes of dry acetone at 0-5°C. The supernatant was decanted o f f af t e r the c e l l s had s e t t l e d . The c e l l s were repipetted into 10 volumes of dry.cold ether which was decanted o f f and the remaining c e l l s re-pipetted into 10 volumes of fresh cold dry ether. The resultant s l u r r y was f i l t e r e d on a buchner funnel and was then dried under vacuum to remove the ether. The c e l l preparation from t h i s pro-cedure was l i g h t and f r i a b l e and contained no appreciable water. Alumina ground c e l l s (32) c e l l s were prepared by adding three times the.weight of washed dried alumina grinding powder to the c e l l paste from one l i t e r of 2-ketogluconic acid medium*. The grinding was carried out for three minutes using maximum pressure i n a c h i l l e d mortar and pestle. The mixture was extracted three times with cold 40 ml. quantities of W/lOO phosphate buffer at pH 7.4. The alumina was centrifuged o f f at s u f f i c i e n t l y low speed * Addendum 2 1 - 21 -that the c e l l s of pseudomonas would not come down i n a reasonabl time. The c e l l debris was brought down at 6000 R.P.M. The supe natant and the c e l l debris were tested for t h e i r a c t i v i t y on 2-ketogluconic acid. - 22 -c. Results • Carboxymethylcellulose apparently offered no protection since the preparation had no a b i l i t y to oxidize 2-ketogluconate. None of the additions t r i e d assisted i n preserving any 2-ketogluconic acid oxidizing enzyme (Table I I ) . Variation i n pH of the buffer used i n Warburg studies on a l y o p h i l i z e d preparation f a i l e d to influence the i n a b i l i t y to oxidize 2-ketogluconic acid. S/rensen's phosphate buffer was used at pH 5, 6.6, 7.4 and 8.2 and also Veranol buffer at pH 7.4. Lyophilization, a f t e r the methods des-cribed, f a i l e d to preserve the enzyme. 2-ketogluconic acid c e l l s grown at a temperature of 15°C. f o r one week were prepared i n the normal way* and afte r l y o p h i l i z a t i o n f a i l e d to show any 2-keto-gluconic oxidizing a b i l i t y . This was done on the supposition that the observations of Gale (23) might be applicable i n t h i s case. This worker noted that amino acid decarboxylases were formed to a larger extent when the organisms were cultivated at a lower than i optimum temperature. Acetone dried c e l l s showed poor a c t i v i t y on glucose and none on 2-ketogluconic acid. Sloppy dried c e l l s and sloppy dried c e l l s prepared under nitrogen contained no detectable 2-ketogluconic oxidizing enzyme. Neither the supernatant nor the c e l l debris of alumina ground c e l l s showed any discernible a b i l i t y to oxidize 2-ketogluconic acid. Although many methods were t r i e d i n an attempt to f i n d a method of preserving t h i s enzyme none..were found. Howe.ver, t h i s does not mean that the problem has been ex-hausted or that a solution i s not possible. * Addendum 2 - 23 -TABLE II C e l l Preparations Method of Preparation Sloppy dried c e l l s Sloppy dried c e l l s under nitrogen Lyophilized c e l l s (aJ bulk - dry ice and acetone (b) bulk - P 20 5 (c) sealed i n vacuum individual v i a l s (d) i n presence of CMC Sloppy dried i n presence of: (a) 0.9$ NaCl (b) 0.05$ yeast extract (c) mineral media (d) phosphate buffer pH 7.4 (e) d i s t i l l e d water (f) 2-ketogluconic acid Acetone dried Alumina ground (a) debris (b) supernatant A c t i v i t y  Glucose 2-ketogluconic Normal None None Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal S l i g h t : A c t i v i t y Normal None None None None None None None (increased endogenous) None None None None None None None Lyophilized c e l l s grown at 15°C. Normal None - 24 -PART V. THE INFLUENCE OF METABOLIC INHIBITORS ON THE OXIDATION OF GLUCOSE AND 2-KETOGLUCONIC ACID A study of enzyme i n h i b i t o r s was instigated i n the hope of f i n d i n g one which would act on the enzyme system of P. aeruginosa at some point beyond the 2-ketogluconic acid stage. The following i n h i b i t o r s were tested: The l a t t e r two compounds i n h i b i t e d to some degree the oxida' t i o n of the substrates while the remainder showed no i n h i b i t i o n Neutralized i n h i b i t o r s were placed i n the Warburg cups with the buffer and c e l l s i n the main compartment and-incubated f o r 30 minutes at 30°C. i n the Warburg bath p r i o r to t i p p i n g i n the sub-strate. The influence of the i n h i b i t o r on the rate of endogenous re s p i r a t i o n was determined i n each experiment. The generalized r e s u l t s can be seen i n the accompanying table (Table I I I ) . The f i r s t run r e s u l t s with sodium selenite at a concentration of 0.00065 M indicated that t h i s concentration caused a decrease in rate of oxygen uptake but not i n amount. The concentration of selenite was increased to 0.0013 M and the i n h i b i t i o n of 2-keto-gluconic acid o x i d i z a t i o n was 50$ while the oxidation of glucose was unimpaired (Plate No. 15). From these data i t would appear that oxidation of 2-keto-gluconic acid was being interfered-with at some intermediate stage. sodium azide (16) sodium arsenate (50) 2,4 dinitrophenol (19) o c t y l alcohol (46) caffeine (46) a c r i f l a v i n e ( 6) methylene blue (45) iodoacetate (33) hydrazine sulfate (20) sodium f l u o r i d e (24) sodium arsenite (15) sodium selenite (40) over the range of concentrations used. - 24a -J 6 CL Z - 25 -I f t h i s was t r u e i t should have been p o s s i b l e t o detect the i n t e r -mediate compound accumulated at the point of i n h i b i t i o n . In order to increase the p o s s i b i l i t y of detecting, or even i s o l a t i n g , t h i s p o s t u l a t e d intermediate, a l a r g e Warburg f l a s k c o n t a i n i n g 30 ml. of r e a c t i o n mixture and the substrate was increased to 36 oxygen equ i v a l e n t s per 3 ml. or t w i c e the normal concentration o f 2-keto-g l u c o n i c a c i d . The r e s u l t s ( P l a t e No. 16) i n d i c a t e d a decrease i n r a t e o f oxygen uptake only and not the 50$ i n h i b i t i o n to be expected i f the enzyme had been poisoned. ; The experiment was repeated using selenium at a concentration of 0.0026 M. This r e s u l t e d i n a 61+% i n h i b i t i o n of 2-ketogluconic a c i d o x i d a t i o n . The contents of the l a r g e Warburg v e s s e l s c o n t a i n i n g endogenous p l u s selenium and j a l s o 2-ketogluconic a c i d p l u s selenium were each c e n t r i f u g e d t o remove the c e l l s and the supernatant concentrated to 5 ml. This was passed through an IRA 40 r e s i n column to remove Na, K, and Mg ions. These ions i n t e r f e r e w i t h the movement o f 2-ketogluconic a c i d on a chromatograph by slowing i t down. They also tended to mask some of the chromatographic developers. The d e i o n i z e d l i q u i d s , i n c l u d i n g washings, were again concentrated, to 0.5 ml. and run chromatographically on Whatman #1 paper w i t h an endogenous c o n t r o l p l u s selenium, and calcium 2-ketogluconate as a standard. The ascending system was used w i t h ethanol 86 p a r t s , water 16 p a r t s , and ammonium hydroxide 4 p a r t s , as a solvent mixture. The developers used were: ortho phenylendiamine {2% i n 80$ ethanol) meta phenylendiamine {2% i n 80$ ethanol) ammoniacal s i l v e r n i t r a t e brom e r e s o l green (1$ i n 95$ ethanol adjusted to pH 7.0) 2- KETOGuoooisio A C I D S T I _ . U _ G R O W I N P L A T E , NO. /6> ZOO U J 0-3 N O 3<i OXVGEJM EJCiOIVM-E-NTS O 0026 M SOD"OM SCUENITE. 09 T I M E (MINUTES) - 26 -o r c i n o l (9) phloroglucinol (9) r e s o r c i n o l (9) napthol (9) napthoresorcinol (9) ninhydrin Hanes' and Isherwood's phosphate reagent (2) 2,6 dichlorobenzenoneindophenol (7) The only reducing spots observed were those due to 2-keto-gluconic acid from the 2-ketogluconic plus selenium cup and calcium 2-ketogluconate standard. A l l other compounds detected were present i n the endogenous plus selenium as well as i n the 2-keto-gluconic acid plus selenium f l a s k s . It would thus appear that selenium did not act on the enzyme within the c e l l but combined with the 2-ketogluconic acid and prevented i t from entering the c e l l . When 2-ketogluconic acid did cross the c e l l membrane apparently i t was converted to G0 2 and water. The i n h i b i t i o n which was obtained with a concentration of 0.0001 M. sodium arsenite apparently resulted i n the accumulation of 2-ketogluconic acid since a nearly t h e o r e t i c a l oxygen uptake of 68 m i c r o l i t e r s of oxygen was used by the c e l l s i n the presence of arsenite and with glucose as substrate while only 4 m i c r o l i t e r s of oxygen was taken up by 2-ketogluconic acid under the same condi-tions (Plate No. 17). The t h e o r e t i c a l oxygen uptake i n the oxida-t i o n of 18 oxygen equivalents of glucose to 2-ketogluconic acid i s 67.2 m i c r o l i t e r s of oxygen. In a confirmatory experiment the oxygen uptake of glucose and of 2-ketogluconic acid was 80 micro-l i t e r s and 24 m i c r o l i t e r s respectively (Plate No. 17). It would appear that sodium arsenite i n h i b i t e d the 2-ketogluconic acid o x i d i z i n g enzyme with the same effect as excessive aeration. 2 -KE_-TO GLUCONIC A C I D STIUL. GROWIN C L L L S 0} - 27 -TABLE III Summary of Inhibiti o n Studies Concentration Within I n h i b i t i o n Compound the Warburg Cup Glucose 2-ketogluconate Sodium azide 0.015 M, 0.12M ...Nil N i l Sodium arsenate 0.013 M, 0.026 M, 0.054 M N i l N i l 2,4 dinitrophenol 1x10" 5m, 5 x 1 0 " 1 x 1 0 - % ! N i l N i l Octyl alcohol Saturated solution Complete Complete ' Sodium f l u o r i d e 0.016 M, 0.032 M, 0.16 M N i l N i l Caffeine 0..025 M N i l N i l A c r i f l a v i n e 0.035 M, 0.07 M, 0.35 M N i l N i l Methylene Blue 5xl0-A-M, 5X10-5m N i l N i l Iodoacetate 0.001 M, 0.005 M N i l N i l Hydrazine Sulfate 0.0037 M, 0.0148 M N i l N i l Sodium Arsenite* (See text) 70$ 100$ Sodium Selenite** (See text) 40-50$ 50-60$ * Plate 17 ** Plates 15 and 16 _ 28 -PART VI. EFFECT OF HEAT SHOCKING ON THE ENZYME STRUCTURE OF CELLS GROWN ON 2-KETOGLUCONIC ACID During the high speed centrifuging of a culture, unavoidable heating occurred which reduced the rate of oxidation of gluconic and 2-ketogluconic acids but not the rate of glucose oxidation. Since t h i s might be interpreted as ind i c a t i n g that glucose i s not oxidized by way of gluconic and 2-ketogluconic acids i t was de-cided to explore the observation further. The r e s u l t s with l i v e c e l l s which had been heated to 4,5°C. f o r one hour indicated a similar reduced rate of oxidation of gluconic and 2-ketogluconic acids (Plate 18, 19). However, 55°C. f o r 30 minutes almost com-pl e t e l y abolished the a b i l i t y to oxidize gluconic acid and 2-keto-gluconic acid whereas the a b i l i t y to attack glucose was unimpaired. When these heat treated c e l l s were dried i n vacuum they then possessed the a b i l i t y to oxidize gluconate to 2-ketogluconate acid (Plate 20). This treatment therefore merely destroyed the a b i l i t y of gluconate to cross the membrane of the heat treated c e l l but not the a b i l i t y of t h i s c e l l to oxidize gluconic acid. This phenomenon might p a r t i a l l y be explained by Dicken's l a t e s t scheme (1.8) i n which he postulates the presence of 6-phosphogluconolactone and 6-phospho-2-ketogluconolactone rather than 6-phosphogluconic acid and 6-phospho-2-ketogluconic acids as Intermediates of glu-cose oxidation. The presence of a heat l a b i l e enzyme to convert gluconic acid to gluconolactone would then be indicated except that t h i s would not account f o r the a b i l i t y of the l y o p h i l i z e d c e l l to oxidize gluconic acid. Heat treated 2-ketogluconic acid grown c e l l s either l y o p h i l i z e d - 2_a -U 1 * He. a t 300 TteELATELD SS°C Jor /O MIV. 2-KE.TOGLOCONJC ACID GROWN PLATE. No. /«? 200 bJ f a 3 100 ro - 29 -or sloppy dried are comparable to normal 2-ketogluconic acid grown c e l l s l y o p h i l i z e d or sloppy dried when tested on glucose or glu-conic acid (Plate 20). C e l l s heated f o r one-half hour at 55°C. manifest the character-i s t i c r e s u l t s of c e l l s heated f o r only 10 minutes at t h i s tempera-ture (Plate 18 and 19). The glucose curve was e s s e n t i a l l y s i m i l a r to that of untreated c e l l s . The c e l l s treated for one hour at 55°C. appear to have l o s t the a b i l i t y to assimulate glucose but not the a b i l i t y to oxidize i t . Nearly t h e o r e t i c a l oxygen uptake, 403 m i c r o l i t e r s , was achieved with glucose as a substrate. This technique may be useful as a means of preparing c e l l s f o r i n h i b i -t i o n studies since c e l l s so treated would tend to accumulate higher y e i l d s of intermediates than i s usual because normally i n l i v e c e l l s l / 2 to l / 3 of the available substrate i s assimulated and does not take part i n the oxidative process. HEAT TREATED 2 -KETOGLUCONIC ACID STILL. GROWN LYOPHILIZED CELLS O 30 60 SO 120 ~ 0 " 3 0 6 © 90 1*3 TIME (MINUTES) - 30 -PART VII. ADDENDUM I 1. Stock Culture Agar Medium Tryptone , 1 $ K 2HP0 4 0.3$ Glucose 0.1$ Glycerol 0.3$ Liver Extract 10$ by volume Agar 0.5$ Gelatin 2$ Adjust to pH 7.2. 2. Yeast - Glucose . Medium (49) 5$ glucose 0.5$ yeast extract Adjust to pH 7.2. 3. Production Medium Glucose 16 $ CaC03 2.7$ K2HP0^ 0.3$ MgS0^.7H20 0.1$ FeS0 4 .7H 20 0.05$ Urea 0.2$ ( s t e r i l i z e d separately) - 31 -Mineral Medium of Campbell and Norris (11) NH^H2P0^ 0.3$ K2H P0^ 0.3$ MgS0^.7H20 0.1$ ( s t e r i l i z e d separately) Glucose 0.1$ ( s t e r i l i z e d separately) Fe as F e C l 2 0.5 ppm Adjust to 7.4. PART VIII. ADDENDUM 2 METHODS USED WHICH ARE COMMON TO PHYSIOLOGICAL STUDIES Throughout t h i s study the methods used have been those out-l i n e d by Umbreit Burris and Stauffer (56). The organism used was Pseudomonas aeruginosa ATCC 9027 unless otherwise noted. In the preparation of c e l l s the mineral media* of Campbell and Norris (11) was used with 1$ glucose or sodium 2-ketogluconate as carbon source. The glucose was s t e r i l i z e d as a 10$ solution by autoclaving. The sodium 2-ketogluconate was s t e r i l i z e d as a 10$ solution through a sintered u l t r a f i n e glass f i l t e r . Magnesium sulfate was s t e r i l i z e d separately by autoclaving as a 10$ solution. The two phosphates and iron were s t e r i l i z e d a f t e r n e u t r a l i z a t i o n i n 9° ml. quantities at the indicated strength i n Roux b o t t l e s by autoclaving. The medium was compounded just p r i o r to inoculation. One ml. of a 18-20 hour old culture was used as inoculum. The sodium 2-ketogluconate was made up as a 50$ weight by volume solution from calcium 2-ketogluconate by stoichiometric replacement of C a + + with sodium sulfate and f i l t e r i n g o f f the calcium s u l f a t e . This solution was stored at 0-5°C. and r e d i s -solved p r i o r to use. The inoculum was sub-cultured monthly on stock culture agar slopes* incubated for 48 hours at 30°C..... and stored at 0-5°C. A stock culture slope was used as an inoculum f o r 10 ml. of * Addendum 1 - 33 -2-ketogluconic acid mineral medium af t e r incubation f o r 24 hours at 30°C. The tube of media was used to inoculate a s i m i l a r tube of 2-ketogluconic acid mineral medium. This was repeated three times to insure an a c t i v e l y growing culture. After three such tr a n s f e r s the culture was used as inoculum f o r 100 ml. Roux bottles of 2-ketogluconic acid mineral medium which were incubated at 30°C. fo r 18 to 20 hours and harvested.' The harvested c e l l s were re sus-pended i n 1/25 volume of 0.9$ saline and recentrifuged.. The re-sulting, c e l l paste was used immediately or was stored at 0-5°C. fo r periods up to three days. The washed c e l l s were made up i n M/30 S^rensen's phosphate buffer at pH 7.4 to a concentration such that a 1:50 d i l u t i o n gave a 60-70$ l i g h t absorption reading on a Fisher electrophotometer with a 525 ytt f i l t e r using d i s t i l l e d water as a blank. The Warburg cups were made up to a f i n a l volume of 3.15 ml. consisting of: 0.15 ml. of 20$ K0H i n the center well; 0.5 ml. of standardized c e l l suspension, 1.5 ml. of M/30 SpVensen's phosphate buffer at pH 7.4, and water to make up to 3 nil. i n the main com-partment. The volume of substrate or i n h i b i t o r was compensated for by an appropriate reduction i n volume of water. The concen-t r a t i o n of substrate was such that 0.2 ml. gave 18 oxygen equi-valents or 403.2 m i c r o l i t e r s of oxygen uptake on complete oxida-t i o n of the substrate to CO2 and water. The Warburg was at 120 o s c i l l a t i o n s per minute. The tempera-ture of the bath was 30 * 0.1°C. The c e l l s used i n the metabolic studies were grown on a medium - 34 -containing sodium 2-ketogluconate as the sole carbon source i n stationary Roux bottles. After addition of inoculum the volume of the medium was 100 ml. PART IX. BIBLIOGRAPHY 1. Asai, Toshinobu, and Ikeda, Yonosuiki, Manufacture of i s o -ascorbic acid. I. 2-ketogluconic acid forming s t r a i n s . Jour. Agr. Chem. S o c , Japan. 22, 50-1, 1948 (CA /£, 6191 i ) . 2. Bandurski, R.S., and Axelrod, B., The chromatographic i d e n t i -f i c a t i o n of some b i o l o g i c a l l y important phosphate esters, Jour. B i o l . Chem. 123, 405, 1951. 3. Bernhauer, K., and Gorlick, B., Oxidations with acetic acid bacteria. IV. Formation of 2-ketogluconic acid by B. gluconi-cum. Biochem. Z. 280, 367-74, 1935 (CA 30, 1085 3). 4. Bernhauer, K., and Knoblock, H., Oxidations with acetic acid bacteria. VI. Comparative study on the formation of reducing sugar carboxylic acids and the preparation of 2-ketogluconic acid. Biochem. Z. 308-15, 1940 (CA14, 4102'). 5. Bernhauer, K., and Knoblock, H., Decomposition of glucose by Acetobacter suboxidans. Naturwissenschaften, 26, 819, 193$ (CA 23, 4282V). 6. 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I k e d a , Y o n o s h u i k i , P s e u d o o x i d a t i v e b a c t e r i a . I . S e l e c t i o n o f new o x i d a t i v e b a c t e r i a . J o u r . A g r . Chem. S o c , J a p a n . 24t, 51-5, 1950, p u b . 1951 (CA 7139 g h ) . I I . Shake c u l t u r e method, i b i d . p . 56-9. V . G l u c o s e o x i d a s e and g l u -c o n i c a c i d o x i d a s e o f v a r i o u s o x i d a t i v e b a c t e r i a , i b i d . p . 147-50. 27. K n o b l o c k , H. and T i e t z e , H . , F o r m a t i o n o f r e d u c i n g s u g a r c a r -bo x y l i c a c i d s by a c e t i c a c i d b a c t e r i a V I I . Biochem. Z... 309, 399-414, 1941 (CA 4 6 4 0 3 ) . 28. K u l k a , D . , H a l l , A...N. and W a l k e r , T . K . , F o r m a t i o n o f 2 - k e t o -d - g l u c o n i c a c i d , 5 - k e t o - d - g l u c o n i c a c i d and t a r t a r i c a c i d by a c e t o b a c t e r s p e c i e s . N a t u r e , I67, 905, 1951. 29. L a n n i n g , M . C . and Cohen, C . 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Marmur, J . and S c h l e n k , F . , G l y c o l a l d e h y d e and g l y c o l a l d e -hyde p h o s p h a t e a s r e a c t i o n componants i n e n z y m a t i c p e n t o s e f o r m a t i o n . A r c h . Biochem. B i o p h y s i c s , jtl> 1 5 4 - 5 , 1951. 3 4 . M o y e r , A . J . , W e l l s , P . A . , S t u b b s , J . J . , H e r r i e k , H . T . and May, O . E . , G l u c o n i c a c i d p r o d u c t i o n , . Ind. E n g . Chem., 29, 777-31, 1937. - 38 -35. Sumner, J.B. and Myrback, K.;, The Enzymes, Vol. 1, No. 1, page 536, 1950. Academic Press, New York. 36. Sumner, J.B. and Myrback, K., The Enzymes, Vol. 1, No. 1, page 931, 1950. . Academic Press, New York. 37. Naylor, H.B. and Smith, P.A., Factors a f f e c t i n g the v i a b i l i t y of S erratia marcescens during dehydration and storage. Jour. Bact. 52, 565-73, 1946. 38. Norris, F.C., Campbell, J.J.R. and Ney, P.W., The intermediate metabolism of Pseudomonas aeruginosa. I. 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