THE METABOLISM OF ORGANIC ACIDS

BI FSEUDQMONAS AERUGINOSA A.T.C. 9027

JUDITH FRANCES MDORE WHITAKER

A THESIS SUBMITTED IN PARTIAL FULFILMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF ARTS

i a the Departments

of

Biology and Botany

and

Dairying

We accept this thesis as conforming to the

standard required for candidates for the

degree of M&STER OF ARTS.

Members tf£ the Department of Biology and Botany*

Hemo<3i7s—or the i/epartment of Dairying.

THE UNIVERSITY OF BRITISH COLUMBIA

Ap r i l , 1951.

ABSTRACT

The present study has been concerned with the oxidation of

the 4-carbon dicarboxylic acids: succinate, fumarate and malate, in the

striot aerobe, Pseudomonas aeruginosa A.T.C. 9027 e -Hesting oells harvested

from a mineral medium in 'which any one of these compounds was the sole

carbon source, metabolized al l three acids immediately and at a constant

and rapid rate© This observation has suggested the operation of the

enzymatic inter-relationship:

Succinate f umarate<r— malate

It is very likely that the oxidation of these substrates follows the

familiar tricarboxylic acid cycle, which has already been shown to occur

in P. aeruginosa 9027*

Questionable quantitative data for the oxidation of the

dicarboxylic acids by dried cell preparations has been obtained. These

preparations did not require either cytochrome c or pyocyanine as a carrier

system for succinate oxidation. The optimum pH range of succinic dehydrogenase

in succinate-grown and acetate-grown dried cells lay between pH 7.0 and 8.0,

an observation which coinoides with values found for other species©

The effect of the two metabolic inhibitors, malonate and uranyl

nitrate, on the respiration of both resting and dried cells of P. aeruginosa

has been investigated. Malonate v/as not inhibitory in moderately high

concentration at pH 7.0, but when the pH was lowered, maximum inhibition

was effected by low concentrations. Apparently, permeability functions as

a governing factor in malonate inhibition, a suggestion which was borne

out in experiments with dried cells, in which the ce l l membrane has been

altered during the drying process. These cells were strongly inhibited

at pH 7.0 by low, concentrations of malonate* An interesting though

complicating feature of the malonata experiments with resting cells'

has "been that P. aeruginosa possesses an adaptive enzyme system for

metabolizing the inhibitor. The inhibitory effect of uranyl nitrate

on the oxidation of Krebs intermediates or of glucose varied with the

substrate - maximum inhibition being obtained with isocitrate oxidation,

and none with malate oxidation, while tho inhibition of other oxidations

f o i l between these two extremes. The use of this non-specific inhibitor

in metabolic studies on whole cells has been discussed and, on the basis

of the present and other studios, its use has been precluded*

AC KNCJl'/LEDG-EMEJHT

Without the unfailing assistance and encouragement

of Dr» J« J. R» Campbell, the enthusiastic interest

of Dean B. A. Eagles, and the financial support of

the Research Council of Ontario, this thesis could

never have been written*

TABLE OF CONTENTS

INTRODUCTION .. . 1

PART I WARBURG STUDIES WITH RESTING CELLS

Introduction 10 Methods

Bacteriological ».... 11 Chemical ...... ««e«.o«.........«.«.«............ 12

Experimental 14 Discussion ........................................» 19

PART II THE METABOLISM OF DRIED CELLS

Introduction •••.••••••.••••••••••..•••«««..•«.•««.. 20 Methods 21 Experimental ....................................... 23 Discussion •••• •.«•••.••...•.•. ••••...« 32

PART III INHIBITOR STUDIES WITH MALONATE AND URANYL NITRATE

Introduction ....................................... 34 Methods 36 Experimental......

A. Malonate Studies ..........«••.............. 38 B. Inhibition by Uranyl Nitrate •• 52

Discussion ......................................... 61

SUMMARY 64

65

- 1 -

DTTRODUCTION

Since carbohydrate is the main source of energy for liv i n g c e l l s ,

the mechanism of i t s degradation has been extensively investigated. In

general, carbohydrate breakdown occurs i n two phases: the anaerobic phase

which ends i n pyruvio acid formation, and the aerobic phase which involves

the u t i l i z a t i o n of pyruvate via tha oxidative pathway. Some aspects of

this second phase have been studied i n the present thesis.

The f i r s t attempts to formulate a scheme of carbohydrate oxidation

are found i n the papers of Thunberg (149),and, of Knoop (69). These workers

suggested that pyruvate was f i r s t converted to acetate, two molecules

of iriiich condensed to give succinate (the Thunberg-WIeland condensation).

The succinate was then cyclically re-converted to acetate, via fumarate,

malate, oxalacetate and pyruvate. The chief weakness of this scheme lay

in the complete lack of evidence in support of the condensation.

A similar scheme was proposed by Toeniessen and Brinkman (151),

except that the condensation involved two molecules of pyruvate, instead of

acetate, an unknown polymerization product being formed, which was then

transformed to succinic acid.

The f i r s t real evidenoe that organic acids function i n respiration

was contained i n the work of Szent-Gyorgyi and his associates (7,8,49), who

found thatsuocinate (or i t s four-oarbon oxidation products)) could stimulate

catalytically the respiration of pigeon breast muscle brei, and that this

oatalytic effect was inhibited by malonate. Szent-^Gyorgyi suggested that

the main, function of succinate, fumarate, malate and oxalacetate i n muscle

"is not to serve as fuel, but to serve as a catalyst j as a catalytic hydrogen

carrier between foodstuff (e.g. pyruvate) ard cytochrome."(147)• The

Szent-Gyorgyi scheme has met with considerable criticism* f i r s t , because i t

rests on the assumption that the succinate systaa is the. only one in animal

tissues which can act directly with the cytochromes, and, second, beoause

the carrier function of oxalacetate~=-^malate merely results i n the accumul­

ation of reduced coenzyme I, with no effective transport of hydrogen taking

place.

Shortly after Szent-Gyorgyi had proposed this scheme, Krebs announced

the discovery that, i n addition to succinate, fumarate, malate and oxalacetate,

o6-ketog!utaric and c i t r i c acids also act catalytically on the respiration

of minced muscle, and that their effects are also inhibited by malonate.

On the basis of further investigations, Krebs ( 72) proposed that pyruvate

and oxalacetate might react together to form a 7-carbon compound, from which

citrate, "C-ketoglutarate, and the 4-carbon dicarboxylic acids would then be

re-formed. What had formerly been considered simply as a chain or series of

reactions now became in effect a cycle, the ' c i t r i c acid cycle'. The 7-carbon

condensation product (oxalocitraconio acid) has never been isolat ed, but

Krebs was able to demonstrate the formation of substantial amounts of c i t r i c

acid when pyruvate and oxalaoatata condensed in an anaerobic system (73, 75).

He therefore suggested that pyruvate condensed with oxalacetate to give

citrate, which was oxidized via cis-aconitate, isocitrate, oxalosuccinate,

oC -ketoglutarate and the 4-carbon dicarboxylic acids, with the resulting

oxalacetate becoming available to condense again with pyruvate araid to

repeat the cycle. On the basis of tracer studies, W0od et al.(l60) proposed

- 3 -

that ois-aconitate, and not citrate, i s the i n i t i a l condensation product.

They showed that isotopic carbon, introduced as carbon 'dioxide together

with pyruvate, led to the formation of <<-ketoglutarate which .contained

isotopic carbon only in the carboxyl group next to the ketone group; i f

°c-ketoglutarate was derived from a symmetrical molecule (citrate), the

fixed carbon would be equally distributed in the two carboxyl groups.

Therefore, they concluded that^-ketoglutarate arose from cis-aoonitate,

and that citrate was outside the cycle but in equilibrium with cis-aconitate

through the mediation of aconitase.

Recently, Ogston (103) on purely theoretical grounds, has suggested

that the asymmetrical, occurrence of an isotope in a product cannot be taken

as conclusive evidence against i t s having arisen from a symmetrical preoursor.

He maintains that an asymmetric enzyme (aconitase) can indeed distirg uish

between the identical groups (end carboxyls) of the compound (citrate) which

i t attacks. This conclusion i s based on the fact that enzyme and symmetrical

substrate combine at three points, and that two of the three combination

sites are catalytically different. Stern & Ochoa (l38) have now shown

that citrate is the i n i t i a l product of condensation between oxalacetate and

some "active" 2-carbon compound (derived from pyruvate, and not pyruvate

i t s e l f ) , the condensation being catalysed by condensing enzyme, Coenzyme A,

Hg*" or MET* and adenosine triphosphate. The "active" 2-carbon compound may­

be acetic acid or the acetyl radical or possibly acetyl phosphate (55).

By a series of hydrations, dehydrpgenations, and decarboxylations,

citrate i s dissimilated in turn to the following compounds; cis-aconitate,

isocitrata, oxalosuccinate,<<-ketoglutarate, succinate, fumarate, malate

and thence back to oxalacetate. The modern concept of the tricarboxylic

acid cycle is shown in Figure I. Thescheme from oxalacetate back to

succinate corresponds to the catalytic system of Szent-Gyorgyio For each

complete cycle, one molecule of acetate is completely oxidized to carbon

dioxide and wab er. A l l the steps in the cycle may be reversible under

aerobic conditions, but the only hydrogenations which appear to proceed

with any rapidity under these conditions are those connecting the

Szent-GyBrgyi system from oxalacetate to succinate.

The experimental work upon which the Krebs cyole is based was

performed largely with minced pigeon breast muscle (72,73,75). Ochoa

(99, 100,101,140), Wood (158), Barron (126), Vennesland, Peters and

others have demonstrated and discussed the occurrence of the various

reactions in other animal tissue, including kidney, heart and liver.

D.S. Green et a l . (50, 51) have obtained a preparation, the "cyolophorase

system"', which they have defined as a " c omplex of enzymes which catalyses

the oomplete oxidation of pyruvic acid by way of the Krebs oitrio acid

cycle." Microscopic examination of cyolophorase has indicated that i t

is essentially a suspension of intaot mitoohondria, and the enzyme activity

has been shown to be related to the structure of these maoro-granules (141).

- Evidenoe that pyruvate is oxidized through the tricarboxylic acid

cycle in plant tissues comes from three sources: (a) al l the acids which

are intermediates in the Krebs cycle are present inyplantsj (b) a number

of the enzymes catalyzing the reactions of the cycle have been found; (c) the few investigations of pyruvate oxidation in vivo (15,19,21,78) have

presented evidence conforming with the four criteria originally set forth

-2H (CHg.COOH) CH3.CO' (CH3.C0~P04)

COOH.CO.CHg.COOH Oxalacetate

- 2H

COOH.C(OH).CH2.COOH CHg.COOH

Citrate H20

C00H.C.CH2.C00H CH.COOH

+ H20

Cis-aconitate

V COOH.CH.CHg.COOH

CH(OH).COOH

- 2H

Isocitrate

COOH.CH.CHg.COOH CO.COOH

Oxalosucc inate

COOH.CHOH.CH2.COOH

A Malate

COOH.CH:CH.COOH A

Fumarate

-2H

COOH.CHg.CHg.COOH

Succinate -2H -CO2 +H20

CH2.CH2.COOH CO.COOH

od-ketoglutarate

Figure I. The tricarboxylic acid cycle

by Krebs in support of the cycle (72:) • The general picture of the Krebs

cycle in plant tissue has been reviewed by Bonner (20) and by Goddard &

Meeuse(48)»

Scattered information has appeared during the past five years

which indicates that the Krebs cycle is present in microorganisms, Ajl &

Workman (4,5) in studies on the replacement of oarbon dioxide by various

intermediates of the cycle^ suggested that at least parts of the scheme were

operative in Escherichia ooli and Aerobaoter aerogenes, Baskett & Hinshelwood

(16,17) on the baas of growth experiments, have proposed that the cycle oocurs

in Bacterium lactis aerogenes. However, Karlsson & Barker (62) were not

able to demonstrate the presence of this system in Azotobacter agilis, either

by the teohnique of simultaneous adaptation or by radioactive traoer exper­

iments, Umbreit (l53),in a review of this data, suggested that part of the

cycle is present, while Stern & Oohoa (139) using extracts of Barker's

organism obtained the condensation of oxalaoetate with "active " aoetate

to citrate. They were able to showithat E, ooli (139) and Streptococcus

faecalis (70) also possess this condensation enzyme. Indirect evidence for

the oxalacetate-"active" acetate condensation in B,coli was given by

Oginsky et a l . (102) Oand by Umbreit (154) in studies on the bactericidal

effects of streptomycin. Further evidenoe for the participation of acetate

in the cycle has been provided by the action of fluoroacetate in inhibiting

the conversion of aoetate to citrate in yeast and baoteria (59)• Hovelli &

Lipmann (96) have found acetyl phosphate to be more effective than acetate

plus adenosine triphosphate in the formation of oitrate by extracts of

E, coli. Using radioactive carbon as a traoer, Saz & Krampitz (128)

- 7 -

demonstrated the same condensation in a lysate of Micrococcus lysodeikticus.

Campbell & Stokes (29) have presented evidence for the tricarboxylic acid

cycle in cells of gseudoraonas aeruginosa, harvested from either a glucose

or an aoetate medium* Several years ago, I»ockwood & Stodola (84) found that

P, fluoresoens produced considerable quantities of<<-ketoglutarate, one of °^

the key intermediates in the cycle, when cells were grown on a glucose

medium under conditions of intense aeration*

In addition to the condensation enzyme, bacterial preparations

have been made of the following enzymes in the Krebs oycle: succinic

dehydrogenase from Corynebaoterium diphtheriae (106), Salmonella aertryoke

(77) and B,ooli (2,57); fumarase from Propionibacterium Shermanii (74);

malio dehydrogenase from a stenothermophilio bacterium (87); oxalaoetio

decarboxylase from M, lysodeiktious (7l), and Azotobaoter vinelandii (108);

pyruvic oxidase from Proteus vulgaris (145)•

Barron et a l , (13) have investigated the occurrence of the Krebs

cycle in yeast, and have given a comprehensive review of previous work* The

cycle may also be operative in the metabolism of molds suoh as Aspergillus

niger (30), Neurospora orassa (82) and Streptomyees species (31), Working

with protozoans, Speck et_ a l , found the Krebs cycle present in the malaria

parasite, Plasmodium gallinaoeum (136), while Harvey obtained no evidence

for its occurrence in Trypanosoma hippicum (56),

Some reoent investigations of acetate oxidation have suggested

that an alternative scheme to the tricarboxylic acid oycle may function in

oertain microorganisms. Entrance into this cycle, the dicarboxylic aoid

cycle (12,42) is made by the Thunberg-lfieland reaction in which two moles

of acetate (possibly phosphorylated acetate) condense to form succinate, or

by the Wood-Werkman reaction of carbon dioxide fixation by pyruvate*

- 8 -

Using radioactive carbon as a tracer, Slade & Werkman (132) and

Kalnitsky et_ al , (60) have obtained evidence for the condensab ion of two

2-carbon molecules to succinic acid, with cell-free preparations of

Aerobacter indologenes ,and B.ooli respectively. Further swpport for this

scheme of acetate oxidation has been given by Ajl (3), working with

A, aerogenes and B.ooli, and by Lenti, al so employing B.ooli (81)• Ten

years ago, Wood & Workman (159) suggested that the Thunberg-Wieiand

condensation might occur in the organism, Propionibaoteriurn pentosaoeum,

but Krebs & Eggleston (74) rejected suoh a possibility in the allied species,

P» Shermanii, On the basis of inhibitor and adaptation experiments,

Barron et a l . (12) have concluded that the dicarboxylic acid oycle, and not

the tricarboxylic acid cycle, is operative in Corynebacterium oreatinovorans.

Foster & Carson (42) in studies on the mechanism of fumaric acid formation

by Rhizopus nigricans observed the four steps fitting the pattern of a

dicarboxylic acid oycle, entrance to the cycle being made by the Thunberg-

Wieiand condensation^

The experiments of Thimann, Bonner, Albaum and others (6,148),

on the action of iodoacetate and organic acids on the growth and proto­

plasmic streaming in Avena coleoptiles and whole seedlings, have suggested

that "a respiratory process, which accounts for only a small fraction of

the total respiration, and which involves the 4-carbon acids, is in conb rol

of growth. This particular respiratory process is the one promoted by

auxin," (148). Similarly, Ryan et a l , (127) deduced that a respiratory

process involving the 4-carbon dicarboxylic acids "supplies energy for

growth"' in Heurospora.

r Investigations carried out in this laboratory have shown that

Pseudomonas aeruginosa A.T.C. 9027 oxidizes glucose by way of gluconio

and 2-ketogluconic acids (95,142) and that i t does not possess an Embden-

Meyerhof scheme of metabolism (27)• Both pyruvate and acetate have been

isolated as intermediates in glucose breakdown (28,157). Recently, Campbell

& Stokes (29) presented evidence for the presence cf a tricarboxylic acid

oycle* It is therefore probable that glucose is broken down by the

following pathway:-

Glucose v Gluconate v ^ 2-ketogluconate — — .pyruvate ^acetate

The intermediary steps between 2-ketogluconate and pyruvate may involve a

stepwise degradation of Cg to Cg, C4 and Cg, with alternate phosphorylative

oxidation and decarboxylation); as proposed originally, by Dickens ( 3 6 , 3 . 7 ) .

It is likely that the C3 compound is pyruvate, whioh undergoes oxidative

decarboxylation to "active" acetate, at which point the Krebs cycle is

entered.

The present study has centred about the oxidation of the 4-carbon

dioarboxylio acids, succinate, fumarate and malate, a l l well-established

constituents of the tricarboxylic acid cycle. By using whole resting oells

and dried cell preparations of P. aeruginosa, the interrelationship:

Succinate Fumarate . _ Malate

has been demonstrated. Strong evidence for the occurrence of the enzyme

succinic dehydrogenase, which catalyses the reversible step between succinate

and fumarate, has been obtained in experiments with the competitive inhibitor,

malonate.

- 10 -

PART I

WARBURG STUDIES WITH RESTING CELLS

Numerous studies i n bacterial chemistry have been made with

"resting"' cells* The bacteria are grown in pure culture, centrifuged

and washed, and suspended i n water or saline in a concentration greater

than could be achieved by growth* According to Quastel & Whetham (117),

these "resting" 1 baoteria are f u l l y endowed with metabolic potentialities*

yet unable to multiply because of the lack of essential growth nutrients*

Cells i n this non-proliferating state are well suited for use i n metabolic

studies, as their respiratory mechanisms may be evaluated in the absence

of interfering growth reactions (91).

The pathway of glucose oxidation in P* aeruginosa as now

recognized was arrived at on the basis of resting c e l l studies (91,92)*

Campbell & Stokes (29) used both glucose- and acetate-grown cells to

obtain evidence for a tricarboxylic acid cycle in the same organism*

In the present study, cells have been grown with succinate,

fumarate, malate or acetate as the sole carbon source, end the metabolic

patterns of the c e l l s investigated, with particular reference to the

oxidation of the dicarboxylic acids*

- 11 -

METHODS

Bacteriological: The culture of P. aeruginosa A.T.C. 9027 employed v/as a

typical strongly pigmenting strain. Stocks were maintained in a liver

extract agar of the following composition: 1.0$ tryptone, 0.25$ K2HP04,

0.15& glucose, 0.3$ CaC03, 10.0$ liver extract, 1.5$ agar, pH 7.2. After

growth was initiated at 30° C, cultures were refrigerated. Fresh transfers

of the stock culture were made every month.

Medium for growth of the organism was prepared as follows:

0.3$ (NH4)H2P04, 0.3$ K2HP04, and 0.5 p.p.m. iron (as FeS04.4H20) were

adjusted to pH 6.8, dispensed in 100 ml. quantities in Roux flasks, and

sterilized by autoclaving at 15 lb. pressure for 15 min. Before flasks

were inoculated, the carbon source that had been sterilized through

sintered glass as a 10$ solution was added aseptically to a final conc­

entration of 0.3$ to 0.5$, and MgS04.7H20 that had been sterilized by

autoclaving as a 10$ solution was added to a final concentration of 0.1$.

When a dicarboxylic aoid was used as carbon souroe, i t was neutralized to

pH 6.5 with N/l HaOH before being made up to volume.

A 1$ inoculum of a 24 hr. culture, grown in ammonium phosphate

medium with appropriate carbon souroe, was employed. After subculture

from the refrigerated stock into Sullivan's medium (146), the culture was

always transferred at least twice at 24 hr. intervals in ammonium phosphate

medium with appropriate carbon source, before use as inoculum.

The cells for Warburg experiments were harvested after 21 to 24

hours incubation at 30° C , washed with half the growth volume of 0.9$

saline and finally made up to the desired concentration. This concentratio n

of oells gave a light transmission of 65$ to 75$ when 0.2 ml. of the

- 12 -

suspension was mad© upr.to 10.0 ml., and read in the Fisher Electrophotometer.

It is equivalent to 0.30 mg. bacterial nitrogen per ml. (92).

A conventional Warburg apparatus (155) was used to follow the

oxygen uptake, al l experiments being run at 31° C , pH 7.2. Warburg

cups contained 1.5 ml. Sorensen's phosphate buffer (M/l5), pH 7.2, and

0.5 ml. cell suspension in the main compartment. The substrate to be

oxidiged was tipped in from the side-arm after equilibration. The centre

well contained 0.15 ml. of 20$ K0H for the absorption of carbon dioxide.

Total volume of fluid in the cups was 3.15 ml. A l l substrates were

neutralized where necessary and added at suoh a concentration that 0.2 ml.

substrate was equivalent to 18 uM oxygen. The endogenous respiration

was always measured (94). Oxygen uptake due to substrate dissimilation

was determined by subtracting the endogenous oxygen uptake from the

total uptake.

Chemicalt With the exception of sodium pyruvate and cis-aconitic acid,

a l l substrates were commercial preparations. The pyruvate was prepared

in this laboratory by the method of Robertson (122), while cis-aconitate

was prepared from the trans- isomer by the procedure of Malachowski &

Maslowski (86).

Warburg vessels were cleaned by soaking overnight in detergent

solution, then rinsed and treated with hot 10$ M O 3 for an hour. Finally

they were washed with water and dried at 80° C.

- 13 -

EXPERIMENTAL

- Easting oells- of P. aoruginosa harvested from a mineral medium

in which succinate, fumarate or malate was the sole carbon source,

metabolize these dicarboxylic acids immediately and at a constant and

rapid rate (Figures II, III and IV). Other intermediates in the Krebs

cycle are oxidized more slowly or only after a period of adaptation.

This suggests that cells grown on one dicarboxylio acid («.g» succinate)

hava developed the enzymes for metabolizing not only that particular acid

but also fumarate and malate. On the other hand, i f the calls are grown

on acetate (Figure V), or on gluoose or pyruvate (as carried out in tha

present and other investigations, but not shorn), they metabolize the

dicarboxylic acids only after a lag period. According to Karstrom's

terminology (63), cells grown on any one of the 4-carbon dicarboxylio

acids possess "constitutive" enzymes for oxidizing a l l these compounds,

while calls grown on some other carbon source must first develop "adaptive"

enzymes for attacking these substrates.

The sharp division of enzymes into "'constitutive" and "adaptive"

has been questioned during recent years (161, 164). It may well be that it

the lag phase, characteristic of an "adaptive curve, represents the time

necessary for the development of the enzyme systems which will transport

the substrate across the ce l l membrane. It is therefore possible that cells

whioh have been grown on any one of the 4-oarbon dicarboxylio acids may

use the same system for transporting a l l these substrates across the c e l l .

Similarly, cells grown on another carbon souroe, would be expected to

show the same adaptive pattern for each of the 4-carbon acids. Results

obtained (29) with acetate oells or with glucose cells do nob indicate

-.14 -

I S f

ot- XET0GLUTA1Z47E

TYKL

•SULCI MATE

'ALATE

<7b

VATE

MINUTES

Figure I I . O x i d a t i o n of s u c c i n a t e , fumarate, mala te , ° ^ - k e t o g l u t a r a t e , pyruvate and ace ta te by 22 h r . c e l l s ha rves ted from a succ ina t e ammonium phosphate medium. C i t r a t e and i s o c i t r a t e were o x i d i z e d a t a l o w , r a t e , w h i l e c i s - a c o n i t a t e was not me tabo l i zed . ^

Warburg cups con ta ined 0.5 m l . c e l l suspens ion , 1.5 m l . M/15 phosphate b u f f e r pH 7 . 2 , 0 .2 m l . s u b s t r a t e . F i n a l volume 3 .0 m l . T h e o r e t i c a l oxygen uptake f o r complete o x i d a t i o n of any subs t r a t e was 403 u l .

- 15 -

SUCCINATE

PUMARATE

•8

441

3 s

MINUTES Figure III. Oxidation of succinate, fumarate, malate, o^ketoglutarate, pyruvate

and acetate by 23 hr. cells harvested from a fumarate ammonium phosphate medium. Citrate and cis-aconitate were metabolized at a low rate.

Warburg cups contained 0.5 ml. c e l l suspension, 1.5 ml. M/15 phosphate buffer pH 7.2, 0.2 ml. substrate. Final volume was 3.0 ml.

f Theoretical oxygen uptake for complete oxidation of any substrate was 403 u l .

- 16 -

20 o MALATE

ACETATE

VCWTA&.TE

OMLACETATE

6b

MINUTES

Figure IV. Oxidation of malate, succinate, fumarate, oxalacetate, °<-ketoglutarate, pyruvate and acetate by 22 hr. cells harvested from a malate ammonium phosphate medium. Citrate and isocitrate were oxidized at a low rate, while cis-aconitate was not metabolized.

Warburg cups contained 0.5 ml. c e l l suspension, 1.5 ml; M/l5 phosphate buffer pH 7.2, 0.2 ml. substrate. Final volume was 3.0 ml. Theoretical oxygen uptake for complete oxidation of any substrate was 403.ul.

- 17 -

I*

Sao ACETATE

o

MINUTES.

Figure V. Oxidation of acetate, succinate, fumarate, malate and isocitrate by 21.hr. c e l l s harvested from an acetate ammonium phosphate medium. Citrate and cis-acoriitate were oxidized at a low rate.

Warburg cups contained 0.5 ml. c e l l suspension, 1.5 ml. phosphate buffer pH 7.2, 0.2 ml. substrate. Final volume was 3.0 ml. Theoretical oxygen uptate for complete oxidation of any substrate was 403.ul.

- 13 -

the operation of such a transport mechanism. The effect of uranyl

nitrate, vdiich inhibits the transport of substrate across the cell

membrane, has been studied in Part III, and evidence has been obtained

that the same transport mechanism is not used by a l l the 4-carbon

dicarboxylic acids.

- 19 -

' • DISCUSSION

It is suggested that the substrates succinate, fumarate and

malate are metabolized at a constant and rapid rate by cells of P.aeruginosa

A.T.C. 9027 whioh have been grown on any one of these compounds, because of

an enzymatic relationship among themj i.e. :

Succinate v 1 ~ Fumarate ^ Malate succinic fumarafce dehydrogenase

Such a relationship, "vfoioh w s first proposed by Quastel and

coworkers (114,115,116,117jl56) in studies with B. ooli and B.pyooyaneus,

is probably operative in other bacteria, including Azotobacter agilis (62)

and P.saocharophila.(l8)« The enzyme succinio dehydrogenase has been

obtained in cell-free extracts from E.ooli (2,57), S. aertryoke (77),

and C. diphtheriae{106). Conclusive proof of its presence in P.aeruginosa

A.T.C. 9027 is presented in Part III, while suggestive evidence has been

•given for its occurrence in a number of other bacteriaj Azotobacter agilis **• ' n i- r I I --. •

(62), B.laotis aerogenes (16), P. saccharophila (18), Moraxella lweffi

mutant S (85), B. typhosa (52), and certain stenothemophilic bacteria (47).

The enzyme fumarase has been prepared from Propionibacterium Shermanii (74).

In addition to succinate, fumarate and malate,(Ney (91) has shown

that oxalacetate is metabolized at a constant and rapid rate in succinate-

grown cells, and that this substrate is decarboxylated anaerobioally, no

doubt to pyruvate. It is very likely, therefore, that succinate oxidation

follows the familiar tricarboxylic acid cycle, the existence of which in

P. aeruginosa A.T.C. 9027 has already been proposed by Campbell & Stokes (29).

- 20

PART II

THE METABOLISM OF DRIED CELLS

It has been observed in Part I that whole resting oells of

P. aeruginosa metabolize sudcinate, fumarate and malate at approximately

the same rate, regardless of which of these three substrates has been

used for growth. This suggests that there is an enzymatically controlled

relationship between the three compounds. Other intermediates in the

Krebs cycle are metabolized only after a lag phase or period of adaptation,

whioh may represent the time necessary for the elaboration of a system

for transporting the compounds across the cell membrane.

By using dried cell preparations of P. aeruginosa, Campbell &

Stokes (29) were able to increase the range of substrates attacked. Ia

the absence of the complicating factor of permeability, a clear quantit­

ative picture was obtained. In the present study, dried cell preparations

of succinate- sad aoetate- grown cells were used in the hope of finding

a quantitative relationship amongst the 4-carbon dicarboxylic acids.

The possible carrier systems and optimum pH of the succinate-

oxidizing enzyme in these dried cells were also investigated.

METHODS

To obtain oells for driad preparations, 20 litres of ammonium

phosphate medium containing succinate or acetate as sole carbon source,

were dispensed in Roux flasks and inooulated with a 24 hour culture of

P. aeruginosa A.T.CV 9027. The cells were collected at the end of 24

hours with a Sharpies centrifuge at 48,000 r.p.m. They were washed once

with 0.9$ saline and re-oentrifuged in a Servall High Speed centrifuge

at 5,000 r.p.m. The resulting pink cell paste was suspended in 50 ml.

distilled water and spread in a thin layer on a large Petri plate. The

cells were dried slowly (3 to 5 days) in vacuo over phosphoric pentoxide

at room temparature. The resulting tanrglassy residue was ground to a

fine powder in a mortar, and stored in air in the freezing compartment of

a refrigerator. The cells were used after a storage period of one week

or more. Two preparations of succinate cells were made, weighing 4.8 and

7.7 grams respectively. One preparation of acetate cells was made, 20

litres of medium yielding 6.2 grams of cells.

For Warburg studies, the dried cells were weighed accurately and

suspended in distilled water so that; each ml. of suspension contained 20

mgm. of cells. An even milky suspension of cells, which could be pipetted

accurately, was obtained after the dried cells had stood for 10 to 15

minutes. Each Warburg vessel contained 1.0 ml. of this suspension. In

addition, a l l Warburg vessels contained 2 uM MgS04.7H20, 2 uM MnCIg

0.4 mMAlCl3, 0.4 mM CaCl 2, 107 ug. DPN, and 5 uM substrate, neutralized,

to pH 7. All runs were made at 31° C , and pH 7.2 or 7.4. In experiments

on possible oarrier systems, 1 and 4 mg. of cytochrome o, and 0.001$ and

0.01$ final concentration of pyocyanine were used. The cytochrome c was

- 22 -

the produot of the Sigma Chemical Company, while the pyocyanine had been

prepared in the laboratory by the method of Elema & Sanders (39).

The optimum pH for succinate oxidation was found by the standard

Thunberg method (155). In the tube were placed 1.0 ml. buffer (phthalate

for pH 4 to 5, phosphate for pH 5.5 to 8, and borate for pH 8 to 10), 1.0

ml. cell suspension (containing 20 mg. dried cells), and 0.3 ml. of 0.01M

malonate. .To the side-bulb were added 0.5 ml. ljlOOO methylene blue, and

0.5 ml. 0.05M sodium succinate. A standard was included which contained

a l l the components of the above system (cells inactivated by boiling for

20 minutes) but with methylene blue at l/lO normal concentration. This

tube represented 90$ reduction of the methylene blue and was used as the

end point of reduction. An endogenous control was also run. Thunberg

tubes were evacuated with a strong water suction pump for 3 minutes before

they were sealed. Following a 10 minute equilibration period at 31° C,

the substrate and methylene blue were tipped in. The end point of the

reaction was recorded as the time required for the colour intensity of

the experimental tube to be reduced to that of the boiled cell standard.

At the end of the reduction period, the seals were checked to confirm the

presence of a vacuum throughout the experiment.

- 23 -

EXPERIMENTAL

Dried oell preparations ,o"£ bacteria have been used for many

years. These preparations have a high endogenous respiration for 4 or

5 days, which drops off to a negligible value when the cells are stored

in the refrigerator. The enzymatic activity remains constant for

several months thereafter. These observations were made forty years

ago by Shattock & Dudgeon (130) and later by Callow (25)• They have been

confirmed by Sleeper et a l . (134) with P. fluoresoens. In this laboratory,

Stokes & Campbell (142) obtained quantitative data on glucose oxidation

by P. aeruginosa, while Campbell & Stokes (29) have presented evidence

for a Krebs cycle in the same organism, both studies being made with dried

oell preparations.

With a view to obtaining a quantitative picture of the oxidation

of the 4-carbon dicarboxylic acids in P.aeruginosa, cells were grown on

succinate and on acetate and dried in vacuo. In Warburg experiments

(Figures VI,VII,VIII), the oxygen consumption was low in comparison to that

obtained by Campbell & Stokes (29) with glucose- or aoetate - grown cells.

In Figure VI, although the values for oxygen consumption are

approximately half the theoretical, a definite relationship is seen, i.e.

24 ul oxygen are required for the step succinate to fumarate, 28 ul for the

step succinate to malate (theoretical in each case is 56 ul), while 56 ul

are used in the oxidation of succinate to oxalacetate (theoretical 112 ul).

The succinate preparation was also able to oxidize citrate, cis-aconitate,

isocitrate, and ^-ketoglutarate, while pyruvate and aoetate were not

metabolized (Figure VII). The enzymes for oxidizing pyruvate and acetate

were probably destroyed during the drying process. ' Similarly, Campbell &

Stokes (29) found that the acetate enzyme was inactivated i f drying took

Bo

MINUTES

Figure VI. Oxidation of succinate, fumarate, malate and oxalacetate by dried cells harvested from a succinate,ammonium phosphate medium.

Warburg cups contained 20 mg. dried cells, 0.5 ml. M/5 phosphate buffer pH 7.2, 2 uM MgSe^HgO, 2 uM MnClg, 0.4 mM A1C13, 0.4 mM CaClg, 107 ug. DPN, and 5 uM substrate. Final volume was 3.0 ml.

- 25 -

Figure VII, Oxidation of succinate, fumarate, isocitrate, 'X'-ketoglutarate, cis-aconitate and citrate by dried cells harvested from a succinate ammonium phosphate medium. Pyruvate and acetate were not metabolized.

Warburg cups contained 20 mg. dried c e l l s , 0.5 ml. M/5 phosphate buffer pH 7.2, 2 uM MgSO^HgO, 2 uM MnClg, 0.4 mM AICI3, 0.4 mM CaCl2, 107 ug DPN, and 5 ui£ substrate. Final volume was 3.0 ml.

- 26 -

longer than 48 hours, an observation whioh was also made by Barron et a l .

(12) with dried cells of C. oraatinovorans.

With aoetate-grown cells, the quantitative picture for the

stepwise oxidation of the 4-carbon dicarboxylic acids more closely approached

the theoretical (Figure VIII). Here the differences in oxygen uptake betweeen

succinate and fumarate and between succinate and malate are 45 and 433 ul

respectively (theoretical in both cases, 56 ul).

With succinate as substrate, dried cells take up approximately

one-third of the oxygen consumed by resting cells. Since the latter have

their oarrier systems and oof actors intact, i t is possible that the low

oxygen uptake charaoteristio of dried cells may arise from the absence of

these necessary constituents. The system for succinate oxidation, known

as the succinoxidase system, has been studied mainly with animal tissue

homogenates (65,106,133) where i t is generally supposed to consist of the

following steps:

Succinate .... Sucoinic dehydrogenase . ... cytochrome b —> cytochrome c

—•^•cytochrome a > cytochrome oxidase (ag) oxygen.

Of the cytochromes, only "c" has been isolated as a soluble pure protein (64),

evidence for the others being based on spectrophotometry observation. In

addition, succinic dehydrogenase and cytochrome oxidase are closely associated

and form an integral part of the large granules or mitochondria in certain

animal cells (58,129). Although the evidence for the existenoe of cytochrome

as a oarrier system in certain species of bacteria is controversial (137,163)

and no large granules are present as sites of succinic dehydrogenase and

cytochrome oxidase activity, i t is probable that P.aeruginosa possesses

the various constituents of the cytochrome system (45,137).

- 27 -

SUCCINATE

1b MINUTES

Figure V X I I . Oxidation o f succinate, fumarate and malate b y dried cells harvested from an acetate ammonium phosphate medium.

Warburg cups contained 20 mg. dried cells, 0.5 ml. M/5 phosphate buffer pH 7.2, 2 uM MgSO^HgO, 2 uM MnClg 0.4 ml A1C13 0.4 ml CaClg, 107 ug. DPN, and 5 uM substrate. Final volume was 3.0 ml.

- 28. -

There is also the possibility of pyocyanine acting as a carrier

sinoe i t is produced copiously by F.aeruginosa on a succinate ammonium

phosphate medium, can be reversibly oxidized and reduced, and has been

found capable of acting as a carrier system with various dehydrogenase

systems in vitro (38).

In addition, a number of metal ions are required as cofactors

for dehydrogenase systems. Ca and Al enhance the activity of animal

succinoxidase (112,113), while Mgt+ and Mn++ are important cofactors for

a large number of enzymes in the glycolytic; and Krebs cycles in both

animal and bacterial tissues (80). These four ions, therefore, were

included in a l l experimental systems.

The effeot of various concentrations of cytochrome c and of

pyocyanine on oxygen uptake are given in Tables I and II. No significant

increase in oxygen consumption is evident. In these particular experiments

there was no endogenous respiration. However, in another experiment where

the oells did oxidize their storage produots, no increase in oxygen uptake

occurred in the presence of added cytochrome o or pyocyanine*

Table I

The effeot of cytochrome o on the oxygen uptake of

dried succinate-grown oells. Substrate: 5VuM succinate.

pH 7.2 .

Oxygen uptake (ul) Time

No 1 mg. 4 mg. (minutes) cytochrome cytochrome cytochrome

60 48 54 43 100 73 75 79 140 91 101 86 180 133 137 131

- 29 -

Table II

The effeot of pyooyanine on the oxygen uptake of dried

suooinate-grown cells* Substrate; 5 uM succinate. pH 7.4 •

T j j n 9 Oxygen uptake (ul) No 0.001$ 0.01$

(minutes) pyocyanine pyooyanine pyocyanine

30 30 37 37 60 ' 70 69 76 90 90 87 97

120 113 105 121 150 131 124 142

The optimum pH of succinic;dehydrogenase has been determined by

the Thunberg method for both succinate and acetate dried cells of

F.aeruginosa,(Figures IX and X). With succinate cells, the optimum

lies between pH 7.0 and 7.5,, while with acetate cells i t occurs at

pH 8.0 .

- 3 0 -

Figure IX. Effect of pH on reduction of M.B. by dried succinate-grown cells, (2 preparations).

- 3 1 -

F i g u r e X. E f f e c t o f pH o n r e d u c t i o n o f M.B. b y d r i e d a c e t a t e - g r o w n c e l l s .

- 32 -

DISCUSSION

The non-stimulatory effect of cytochrome c on succinate oxidation

by P. aeruginosa may be the result of one or more factors* F i r s t , there is

the possibility that cytochrome o is not a limiting factor- i n this oxidation.

Or, i f we assume that the cytochromes are limiting i n this preparafc ion, the

non-stimulatory effect may result either from the inactivation of cytochrome

oxidase during the drying process, or from the requirement for some cytochrome

component other than "c":. Discussion is restricted to the latter two

p o s s i b i l i t i e s .

"Very l i t t l e work has bean done with bacterial cytochrome oxidase

since Kubowitz & Haas (76) f i r s t obtained evidence for i t s occurrence.-

However, Borei (22) found that the cytochrome oxidase of horse heart became

inactive with increasing age, due to aL terations i n the colloidal struoture.

Such an alteration may also occur i n dried cells of P. aeruginosa. Support

for this possibility i s contained in the work of Militzer et a l . (88) who

found cytochrome oxidase to be the limiting enzyme for the cytochrome chain

in thermophile No. 2184.

The evidenoe for the cytochrome system (a,b and c) i n P.aeruginosa

(45,137) is based solely on spectrophotometric observation. There i s ,

therefore, no guarantee that the bacterial components are structurally

identical with those i n animal tissue(45). Support for this view may be

found i n the fact that neither of the succinoxidase preparations made thus

far from bacterial oells (77,106) require cytochrome c as a carrier.

As noted in the preceding section, pyocyanine does not inorease

succinate oxidation by dried cells of P. aeruginosa. Actually the r . ' .v

evidence for pyocyanine as a carrier system i s fragmentary. Friedheim (43)

_ 33 -

observed an increased oxygen consumption when pyocyanine was added to

suspensions of B. pyooyaneus, an observation which was later confirmed by

Norris (93), Reed & Boyd (121) also comment on the respiratory significance

of the pigment in B. pyocyaneus cultures, while Dickens & Mcllwain (38)

found it capable of acting as a carrier with the hexosemonophosphate system

of yeast juice and red blood cells. However, oontrary results were obtained

by Barron et a l . with C. creatinovorans (12), and by Keilin & Hartree (65)

with a heart muscle preparation. <In these cases, pyocyanine was inhibitory.

The optimum pH for suooinio dehydrogenase in suocinate-grown dried

oells lies between pH 7.0 and 7.5, while with aoetate cells i t occurs at

pH 8.0. These values coincide with others in the literatures Ohlsson (104)

in the original study on bacterial succinic dehydrogenase found. pH 9.0 to

be optimum, while Quastel & Whetham (117), three years later, found a pH

of 7.4 to be the most suitable for the reduction of methylene blue by

resting oells of B.coli. Subsequently, Cook & Aloook (34) found pH 7.6

to 8.0 to be optimum for the aerobic oxidation of succinate by E. coli.

In more recent work, Kun & Abood (77) showed that oell-free preparations

of succinoxidase from S« aertrycke have their optimum activity at pH 7.4,

while Pappenheimer & Hendee (106) found pH 8.0 to be optimum for suocinio

dehydrogenase from C. diphtheriae.

The anaerobic reduction of methylene blue by resting oells of

P. aeruginosa was studied by Randies & Birkeland (120). Guggenheim (52,53)

investigated the same property in a number of pathogens (S. typhosa,.

CI. butylioum, CI. parabotulinumi, and CI. welohii.)

- 34 -

PART III

INHIBITOR STUDIES WITH MALONATE AND URANH, NITRATE

Certain specific information concerning the pathways of cellular

oxidation may be obtained by studying the effects of metabolic inhibitors*

In general, these compounds f a l l into two main groups, depending on the

manner in which they effect inhibition* The first and by far the larger

group act by inhibiting the activity of enzyme systems directly* They

may do so by combining with the enzyme system through one of the following:

the activating protein, the prosthetic group or the carrier system, or by

competing with or otherwise reduoing the effective concentration of substrate*

Malonate, which inhibits the enzyme suecinio dehydrogenase by competing with

the normal substrate for a position on the enzyme surface, has been chosen

from this group*

The second group of inhibitors act indirectly by altering one or

more of the varied meohanisms which regulate the rate and direction of

enzymatic reactions in the living oell. One of these regulating mechanisms

is the state of the oell surface, which is important not only because of

its property of selective permeability, but also because of its more direct

role in the "active" transfer of materials into the cell* That this "active"

transfer is oonnected with phosphorylation has been suggested by Barron (90)

and by Rothstein (123,124) in studies on the metabolism of glucose in yeast

and bacteria, using uranyl nitrate as an inhibitor* An investigation of

the effect of this inhibitor on the metabolism of organic acids and glucose

in P* aeruginosa was undertaken in the present study* In recent years,

ample evidence has appeared for a coupling between the oxidation of organic

acids or glucose and phosphorylation (10,11)• Uranyl nitrate, therefore,

appeared to be a useful tool -whose action might provide more concrete

evidence for this connection between aerobic metabolism and phosphorylation*

- 36 -

METHODS

The methods of maintaining stock cultures were similar to those

described in Part I. Growth medium for the preparation of resting cell

suspensions was similar to that used previously. Resting oell suspensions

of 21 to 24 hours cultures were prepared as in Part I. Dried cell prepar­

ations were the same as described in Part II.

Warburg' experiments were conducted as in Part I. In malonate

studies, Sorensen's phosphate buffer was used, while M/7 veronal buffer

was employed xvith uranyl nitrate. 0.3 ml. of inhibitor was added at ten

times the final concentration desired. Thirty to forty minutes contact

between inhibitor and cells was allowed before tipping in the substrate

from the side-arm. In malonate inhibition studies at pH's 5.0 to 5.6,

substrate and mal onate were both neutralized with N/l NaOH to the required

pH using a Beckman pH meter. Uranyl nitrate was also neutralized with

N/i NaOH before being added to the Warburg cups. Percentage inhibition

has been expressed in terms of the % 2 ( u ) values.

Malonate inhibition of dried oell preparations was studied by

both the Warburg method and by the Thunberg technique. In the latter

instance, 0.3 ml. of ten times the desired concentration of malonate was

left in contact with the cell suspension in the main tube for 30 minutes

before adding succinate and methylene blue from the side-bulb.

Chemically-clean glassware was used for determining the influence

of magnesium on growth. This was prepared by soaking overnight in 10$

nitric acid, washing ten times with tap water, three times with distilled

water, three times with re-distilled (glass) water, and finally autoclaving

for 15 minutes at 15 pounds pressure. The basal medium, consisting of

- 37 -

0.11$ KH40H, 0.11$ K2HP04, 0.5 p.p.m. Fe^* (as FeS04.4H20), and 0.33$

sodium succinate, was dispensed in 9 ml. amounts into 125 ml. Erlenmeyer

flasks, and autoclaved. MgS04.7H20 \ms made up in concentrations ranging

from 0.001$ to 10$i and autoolaved separately. 1 ml. of the various Mg++

solutions was added aseptically to the basal medium. Final concentrations

of various constituents were: 0.1$ HH OH, 0.1$ K2HP04, 0.5 p.p.m. Fe ,

0.3$ sodium succinate, and Mg + + ranging from 0.0001$ to 1$.

The inoculum was prepared from a' 21 hour culture of the organism

in basal medium •+• 0.05$ Hg + + . These cells w ere harvested by centrifug-

ation, and washed twice with water re-distilled from glass. They were

re-suspended to give a barely visible turbidity. One drop of this suspension

was employed as inoculum for 10 ml© of medium. Following incubation for

24 hours, the turbidity of the suspension was read in a Fisher Eleotrophoto-

meter using Filter 525 B.

- 38 -

EXPERIMENTAL

A. Malonate Studies

The inhibition of the anzyme succinic dehydrogenase by malonate

was first reported by Quastel & Whet ham (118). Subsequent work (34,49,

116,119) has confirmed their observation and demonstrated a high degree of

specificity for the action of this inhibitor. Szent-Gyorgyi and coworkers

(8,49) observed that malonate inhibited the respiration of pigeon breast

muscle and that succinate was formed oxidatively from fumarate in the presence

of malonate. Further investigations of these effects by Krebs and his assoc­

iates (72,73,75) have furnished some of the strongest evidence for the

oxidation of pyruvate by the tricarboxylic acid cycle.

Malonate is assumed to inhibit succinic dehydrogenase by competing

with the enzyme's normal metabolite, sucoinate, for the "hot spots'" or active

centres on the enzyme surface. These centres are thought to be oriented

specifically to pick up succinate as a preliminary to its dehydrogenation,

but may also adsorb closely related molecules such as malonate. This

phenomenon of competitive inhibition, first observed by Qjuastel (115),

results in a more or less complete blocking of the enzymatic reaction© The

effective concentrations of malonate are relative rather than absolite -

i.e. the ratio of suocinate to malonate is the important factor - since the

affinity of the inhibitor for the enzyme is considerably greater than that of

the substrate. Potter & DuBois ( i l l ) obtained 50$ inhibition with, a v

substrate/inhibitor ratio as low as 50/l, while in earlier work Krebs &

Eggleston (73) required a ratio of 9.5/1 •

Initial experiments were conducted with dried cell preparations.

A preliminary study of the effect of pH on malonate inhibition was made

according to the Thunberg teohnique. The tubes contained 20 mg. dried cells

(suspended in 1 ml.), 1.0 ml. phthalate, phosphate or borate buffer

(depending on the desired pH), and 0.3 ml* of either web er or 0*01 M

malonate* In the side-bulb were placed 0*5 ml* of 0.05M suooinate and

0*5 ml* of 1*1000 methylene blue* The ganeral procedure was carried out

as in Part I* Results are given in Tables III and IV.

Table III The effect of pH on malonate inhibition of two

different dried preparations of suocinate-grown cells

Preparation A Preparation B Reduction time Inhibition Reduction t ime Inhibition

(minutes) (per cent) (minutes) (per Succ. Succ. Me i l . Succ. Succ.-+-Mal.

4.1 165 165 0 160 160 0 4.6 108 87 0 115 110 0 5.5 57 129 55 40 67 40 5.9 56 90 39 6*5 40 91 56 35 65 46 7.0 23 55 58 16 43 63 7.5 14 50 72 18 50 64 8*0 17 60 72 20 55 64 9.0 20 40 50 20 44 55

10.0 35 60 42 35 75 53

Note: Endogenous controls showed no reduction at 180 minutes.

Table IV The effect of pH on malonate inhibition of succinic dehydrogenase activity in acetate-grown dried cells

Reduction time (min.) Inhibition pH Succ. Succ . •+• Malon* (per oent)

4.1 180 180 0 5.1 63 57 0 6.0 37 59 37 7.0 27 47 43 7.2 27 42 36 7.5 26 41 37 8.0 24 45 47 9.0 31 46 33

10.0 48 68 29 Note: Endogenous controls showed no reduction at 170 minutes.

- 40 -

It may be concluded that malonate exerts its inhibitory effect

in the pH range of 7.0 to 8.0. This is also the optimum pH range for

succinate dehydrogenation in dried cell preparations, as found in Part II«

Qoiastel & Yfooldridge (119) carried out their experiments at pH 7.4, using

normal resting cells and toluene-treated cells of B. ooli. Since a l l

previous studies with P. aeruginosa have been done at pH 7.2 and 7.4, i t

was decided to use pH 7.4 for further investigation of malonate inhibition.

Warburg studies of the effect of malonate on succinate oxidation

were carried put as in Part II, with both succinate- and acetate-grown dried

cells. The results are summarised in Table V.

Table V. The effect of malonate on the oxygen uptake by dried cells.

Warburg vessels, contained 20 mg, cells, 0.5 ml. M/5 phosphate buffer pH 7.4, 2 uM. MgS04.7H20, 2 uM MnCl2, 0.4 mM AICI3, 0.4 mM CaCl2, 5 o r 10 succinate in side-arm. Malonate was in contact with cells 30 minutes before -.tipping in substrate.

Sxpt. Dried Time Suocin- Malonate Oxygen uptake (ul) Inhibition No. Cells ate.

I Succinate 60 ' 5 uM " 120 « t i 1 8 Q ,1

II Succinate 66 5 uM: " 120 " " 180 "

III Acetate 60 5 uM « 120 11 " 60 " " 120 "

17 Aoetate 60 10 uM " 120 "

60 '» 120 '«

Succ. SuccMale (per c

0.001 M ,51 12 77 11 83 18 78 n 98 34 65

0.005 M 45 0 100 11 59 0 100 n 69 0 loo

0.001 M 62 0 100 tt 89 43 51

0.005 M .62 0 100 11 89 14 84

0.001 M 94 47 50 t i 135 87 36

0.005M 94 9 90 11 135 41 60

It is apparent that malonate in concentrations ranging from

0.001M to 0,005M is able to inhibit the oxidation of suooinate by dried

cell preparations of ff, aeruginosa, harvested from either succinate- or

acetate- ammonium phosphate medium, Table V, Since Krebs (72) considers

that in these concentrations malonate acts exclusively upon the enzyme

succinic dehydrogenase, we may conclude that both types of cells contain

this enzyme. The fact that at a concentration of 0.001 M, malonate

produces a greater inhibitory effect on sucoinate-grown oells than on

acetate-grown cells would tie in with earlier observations that cells which

have been grown on succinate contain a higher relative concentration of

succinic dehydrogenase than those grown on acetate.

In studies with 0.05M malonate at pH 7,4, Ney (91) was unable

to inhibit either the rate or the extent of oxidation of succinate or

glucose by glucose-arhmonium phosphate resting cells of P, aeruginosa.

These experiments were repeated in the present study, using both 0,01M

and 0.05M malonate at pH 7.2 and pH 7.4 • The cells were harvested from a

medium in which sodium succinate constituted the sole oarbon source.

The effect of 0.01M malonate on the oxidation of suocinate,

fumarate and glucose, is given in Table 71. This concentration of malonate

inhibited the rate of oxidation of both succinate and fumarate, but not that

of glucose.

The inhibitory effect of malonate, in concentrations 0.01M and 0.05M,

on the oxidation of suocinate and fumarate has been computed in the form of

^)o2(K) v a l , U 9 S i n ^&^9 VII. Experiment 1 is shown in Figure XI. It is

apparent that 0.01M and 0.05M malonate inhibit the rate of oxidation of

succinate, and to a lesser extent that of fumarate.

- 42 -

Table 71

Oxidations by suooinate-grown resting c e l l s , i a the presence of 0.01M malonate» pH 7>2 . •

Warburg substrate

Time (minutes)

Endog. •+• Malonate

Oxygen uptake (ul)

Substrate Total Endog, + Malonate* Inhibition Substrate (per oent)

Succinate 20 18 174 192 145 25 it 40 38 219 247 230 7 II 60 70 221 291 267 8

Fumarate 20 18i. 164 182 131 29 II 40 38 209 247 192 22 II 60 70 218 288 228 21

Glucose 20 18 29 47 55 0 n 40 38 50 88 . 112 0 it 60 . 70 73 143 190 0

Table 711

Oxidations by auooinate-grown resting c e l l s ,

i n the presence of O.OlM.and 0,05M malonate

Experiment Number pH

7.2 it

Warburg Substrate

Succinate it

Malonate (Molar)

0.01 0.05

9: D 2 ( u )

3220 2040 1190

Inhibition (per cent)

37 63

II

t i

it

t i

7.4: It

Fumarate it

Succinate it

Fumarate tt

0.01 0.05

0.01 0.05

0.01 0.05

2740 2740 1605

2222 1900 1105

2395 1940 1255

0 41

14 50

19 48

- 43 -

12 Q

3

FUMARATE +0OS MAJJOHAU-

\TE_ MALONATE

SUCCINATE HATE

CCINATEU-001H MALtbUATE

SUCCINATE +005~H MALONATE

_MINUTES 4o

Figure X L . Oxidation of succinate and fumarate by 21 hr. cells harvested from succinate-ammonium phosphate medium, i n the presence of 0.O1M and 0.05M malonate.

Warburg cups contained 0.5 ml. c e l l suspension^!.. 5. ml. M/15 phosphate buffer pH 7.2, 0.3 ml. malonate, o.2/substrate. Final volume was 3.0 ml. Theoretical oxygen uptake for complete oxidation of any substrate was 403 u l .

- 44 -

Similar results have been obtained by other workers. Bernstein

(18) observed a decrease in the rate of oxygen uptake by cells of P. sacchar-

ophila when employing 0.01M solutions of malonate. With 0.05M malonate, he

obtained a 71$ inhibition of amount of succinate oxidation and a 59$. inhibition

in the case of fumarate. In studies with a suooinoxidase preparation from

S. aertrycke endotoxin, Kun & Abood (77) obtained 100$ inhibition of succinate

oxidation in the presence of 0.01M malonate. Quastel and his associates

(116,118,119) worked with concentrations of 0.02M malonate and higher to

obtain inhibitory effects in their Thunberg studies with B. coli, B.prodigiosus,

and B. proteus. Quastel & Wheatley (116) reported a 44$ inhibition; of

succinate oxidation in the presence of 0.067M malonate, over a period of 2

hours. In the same study, succinate oxidation by fresh cells of B. coli

was inhibited 24$, while with cells whioh had been stored at 0° C. for

several days a 90$ inhibition was observed* The increase in inhibition

may have been due to an increase of cellular permeability. Most

experiments with animal tissue have been carried out with lower concentrations

of malonate than the above (1,111), although Stoppani (143) required 0.01M

malonate to give a 99$ Inhibition of his succinoxidase preparation from heart

muscle or liver. As to inhibition of glucose oxidation by malonate, Harvey

(56) noted that this compound exerted no effeot at 0.01M in experiments

with Trypanosoma hippicum.

A l l these investigations have been made using malonate concentrations

higher than those recommended by Krebs (72) . Das (35) suggested that at

higher concentrations malonate may interfere with enzymes other than suocinio

dehydrogenase. In this connection, Pardee & Potter (107) claim that malonat<a

inhibits the oxidations of the Krebs cycle by at least two mechanismss

low concentrations (0.004M) completely block suocinate oxidation, -while

higher concentrations (0»02M) effect inhibition of oxalacetate oxidation,

presumably by forming a' complex with free or bound magnesium, a necessary

oofactor for the oxidative decarboxylation of oxalacetate, Evans et al.(41)

also noted that 0,01M malonate completely inhibits the decarboxylation of

oxalacetate. Further evidence that oxalacetate oxidation is the point of

inhibition by malonate in higher concentrations was provided by Potter (110),

He found that malate oxidation is not inhibited when oxalacetate is removed

from the system by transamination, Lwoff & Cailleau (85) working with the

S mutant of Moraxella Lwoffi observed that oxalacetate oxidation was completely

inhibited by malonate at a ratio of malonio acid j oxalacetio aoid of 10,

An interesting though complicating feature of the present studies

on malonate inhibition has been that P, aeruginosa possesses an adaptive

enzyme system for metabolizing the inhibitor, Karlsson & Barker (62) and

Karlsson (61) have found that Azotobacter agilis also is able to adapt to

malonate, .Similarly, Baskett & Hinshelwood (16) observed that cells of

B,lactis aerogenes became adapted to malonate after training on glucose, the

normal growth medium. The original report on the bacterial oxidation of

malonate was made by Butterworth & Walker employing B, pyooyaneus (24)•

To provide conclusive proof for the existence of such an adaptive

enzyme system in P, aeruginosa, the following experiment was conducted.

Cells which had been grown on suocinate and on aoetate mineral media for

21 hours were divided into two parts. One was set aside as a control -

non-adapted cells; the other was incubated for 1 hour with malonate (in the

same concentration as the original growth substrates - via, 0»25$Q added to

the medium - adapted oells. In subsequent Warburg studies, the succinate

" - 46' -

enzyme of both groups of cells was saturated with its substrate in a

concentration of 5 uM, and with a high concentration of malonate (o»011l) . .

The resulting curves (Figures XII and XIII) indicate that there are

indeed two separate enzyme systems present - one for metabolizing succinate,

the other for oxidizing malonate. In the case of the non-adapted succinate-

grown cells, malonate had l i t t l e inhibitory effect, The endogenous curves

indicate that the organism is able to adapt to malonate. With the malonate-

adapted, succinate-grown oells, two distinot curves are apparent. The curve

for oxidation of suocinate (5 uM) by the" adapted cells may be an example of

the "uiauxie" effect of Monod (89), in which a double growth cycle consisting

of two exponential phases is operative. Each curve in this instance would

correspond to the exolusive utilization of one of the constituents in the

growth medium (i.e. either succinate or malonate) due to the inhibitory

effect of one of the compounds on the formation of the enzyme for attacking

the other. The phenomenon and its explanation would appear to be valid only

in the oase of compounds whioh are closely related in structure. Cells whioh

have been grown on acetate show adaptive curves fbrboth suocinate and malonate.

In previous experiments at pH 7 .2 and 7 , 4 , inhibition by malonate in

low concentrations was obtained with dried cell preparations, while high

concentrations -were necessary to inhibit whole resting cells* This suggests

that permeability is involved} i.e. dried oells, in which the cell membrane

has been destroyed or altered, are inhibited, while whole cells, with the

membrane intact, show no inhibition. Turner & Hanly (152) in experiments

with carrot slices found that potassium malonate at pH 4 , 0 caused "inhibition

of that fraction of respiration which is inhibited by cyanide," They conoluded

that although the actual inhibitor is the malonic ion, only undissociated

malonio acid serves as cell penetrant, so that a low pH is essential. Similar

- 47 -

to^H SUCCINATE•_ J

^u/f SUCCINATE /0-0fM MALONATE

MINUT£&

Figure XII. Oxidation of succinate and malonate by non-adapted succinate-grown cells (unbroken lines) and by cells which have been adapted to malonate for 1 hour (broken line s ) . Warburg cups contained 0.5 ml. c e l l suspension, 1.5 ml. M/15 phosphate

buffer pH 7.2, O.g. ml. substrate. Final volume was 3.0 ml.

- 48 -

SUCCINATE

SUCCINATE

SUCCINATE:

ACETATE

SUCCWATB •fO-OIH MALONATE

SUCC/NAT1 / 0.01 M MALONATE

MINUTES

Figure XIII. Oxidation of succinate and malonate by non-adapted acetate cells (unbroken lines) and by acetate cells which have been adapted to malonate for 1 hour (broken lines).

Warburg cups contained 0.5 ml. c e l l suspension, 1.5 ml. M/15 phosphate buffer, pH 7.2, 6.2 ml. substrate. Final volume was 3.0 ml.

results were obtained by Bonner & Wildman (21), Bonner (19), and Laties (78), .

with, spinaoh leaves, oat ooleoptiles and barley roots respectively, Gunsalus

(54) working with B, subtilis noted good inhibition at low pH's while none

ocourred at pH 7.0 • The same effect of pH was observed by Barron et a l ,

with C, creatinovorans (12) and with baker's yeast (13),

Results obtained with P, aeruginosa are given in Tables VIII, EC

and X, and in Figure XIV, A l l experiments were conducted at pH 5,0 to

5,6, since at lower values (i.e. pH 3,7 and pH 4,5) no oxidation of substrate

took place. Maximum inhibition of rate and extent of oxygen consumption on

succinate, with succinate-grown cells, was- found at pH 5,0 in the presence

of 0•005M malonate, Malonate affected only the rate of oxidation of other

Krebs intermediates studied, viz. fumarate, malate and V-katoglutarate.

There was no inhibitory effect on,pyruvate oxidation.

Table VIII '

The influence of pH and concentration on malonate inhibition of succinate oxidation,using succinate-grown cells.

Experiment Malonate QQ . Inhibition Final Og Inhibition Number pH (molar) 2 ( N ) of rate % uptake(ul) of amount %

(at 60«) I 5*6 — 895 200

" 0.001 747 16 170 15 M 0.005 494 45 110 45

(at 80') II 5.0 ~ 975 275

III 5.0 n

0.001 812 17 275 0 0.005 283 71 80 71

(at 60') 1845 235

0.002 1 6 4 0 11 225 4 0.003 1 245 33 180 23

- 50 -

Table IX

Inhibition, of succinate and fumarate oxidation by 0*00511 malonate using fumarate-grown oells at pH 5*1

Experimental Conditions °2(N)

Inhibition of 0 2 uptake .."Inhibition rate $ at 90' (ul) of amt. %

Fumarate 880 Fumarate + malonate 706

Succinate 840 Succinate 4-malonate 350

20

58

230 225;

220 130 41

Table X

Inhibition by 0.005M malonate of the oxidation of malate, -ketoglutarate and pyruvate, using oells grown on each of these substrates.

Experimental Conditions

Malate Malate + malonate

PH

5.1 n

-ketoglutarate 5.5 c<-ketoglutarate + "

malonate

Pyruvate' 5*5 Pyruvate 4 malonate "

°2(N)

1310 940

1170 985

1590 1470

Inhibition Og uptake Inhibition of rate % at 90' ul* of amt, %

28

16

215 220

210 195

260 262

Quastel & Wheatley (116) used 0*07M malonate to obtain 30$ inhibition

of fumarate oxidation by B* pyooyaneus over 2 hours, aconcentration which did

not affeot B, coli* These results were typical for organisms which oxidize

fumarate faster than succinate; those with slower fumarate oxidation were

inhibited to the extent of about 90$. Using the same concentration of malonate,

they observed a 6$ inhibition of the amount of oxygen uptake with malate over

- 51

MINUTES Figure XIV. Inhibition of succinate oxidation by various concentrations

of malonate on succinate-grown c e l l s . Warburg cups contained 0.5 ml. c e l l suspension, 1.5 ml. M/l5 phosphate buffer pH 5.0, 0.3 ml. malonate, 0.2 ml. substrate. Final volume was 3.0 ml. Theoretical oxygen uptake for complete oxidation of any substrate was 403 u l .

SUCCINATE

SUCCINATE +0001H MALONATE

SUCCINATE +0-00$ M

MALONATE

SUCCINATE + 0OOS M MALONATE

a period of 2 hours, an amount which is but slightly significant. Although

only one experiment on inhibition was carried out, pyruvate'oxidation by

P, aeruginosa was.found not to be inhibited significantly. This finding

agrees with Lenti's .(81) observation that malonate affects the succinate

but not the pyruvate oxidation of B, co l i . On the other hand, Barron et a l .

(13) found that malonate completely inhibited the oxidation of pyruvic acid ,

by yeast, a finding confirmed by Speck et_ a l , (136) with the malaria parasite.

Similar results were obtained by Szent-Gyorgyi aid his coworkers (8,49) and

by Krebs et a l , (73) using pigeon breast muscle brei, Pardee & Potter (107)

found that 0.004M malonate strongly inhibited pyruvate oxidation in homogen-

ates of rat heart, kidney and brain, but not in liver, Lehninger ( 7 9 ) has

noted that liver homogenates can oxidize pyruvate to acetoacetate in the

presence of malonate,

B, Inhibition by Uranyl Hitrate

Barron (14,90) and Rothstein (123,124) have shown that uranyl

nitrate in low concentrations inhibits metabolism' in whole cells of yeast

and bacteria by combining with the protein of the cell surface. This

uranium-protein complex can be reversed by a number of compounds, including

phosphate, whioh form more strongly associated complexes with the uranyl

ion than does protein (131).

Initial experiments were designed to obviate any complication by

phosphate either in the form of an insoluble precipitate in the culture

medium, or as inorganio phosphate attached to the cell surface. To avoid

precipitation of magnesium ammonium phosphate from the mineral medium, the

concentrations of KH4"*" and P04""" were reduced to 0,1$ while that of Mg*+

was lowered to 0,01$ or 0,005$, depending on the carbon source present.

- 53 -

At this concentration, optimal growth of the bacterium wis maintained, as

shown in Figure XV, which confirms the previous work of Burton et a l . (23).

It was found that by washing the cells three times with 0.9$ saline, the

concentration of phosphate attached to the cell surfaoe could be lowered

to a negligible value. A l l experiments were oonducted with veronal as

buffer© The oxygen consumption of P. aeruginosa at pH 7.2 is the same

whether veronal or phosphate is used as buffer.

x 10 M to 1 x 10_<i M has been investigated. Concentrations of 1 x 10 M

to 4 x 10"^ M reduced the endogenous respiration of P. aeruginosa, as shown

with gluoose oells in Table XI. The higher concentrations, 1 x 10 M and 2

1 x 10 M, caused agglutination of the cells. The inhibition of endogenous

respiration by lower concentrations is probably due to a combination of the

uranyl ion with the proteinaceous storage material of this organism (26),

while at higher concentrations, uranium may be aoting as a general protein

precipitant (131).

The effect of uranyl nitrate in concentrations ranging from 7.5

Table XI The effect of ^ 2 ( ^ 3 ) 2 on the endogenous respiration of cells harvested from a gluoose ammonium phosphate medium.

Duration of experiment was 70 minutes.

Experiment Number.

Experimental Conditions

Oxygen Uptake (ul)

Inhibition (per cent)

I Control 65 35 41 28 81 63 © 0

1 x lO" 4 M UJ 46 37 57

II

2.5 x lO" 4 M U 4 x 10"4 M U Control 1 x lO" 4 M U 1 x 10"3 M U 1 x 10-2 M U

22 100 100

- 54 -

Figure XV. The influence of magnesium on the growth of F. aeruginosa

- .55-

The effect of uranyl nitrate on the oxidation of the Krebs. inter­

mediates and of glucose by cells which have been grown on these compounds,

is shown in Tables<XII to XV, and Figure XVI. Uranyl nitrate in concentrat­

ions up to 4 x 10"*4 M did not effect a significant inhibition of glucose

oxidation by P. aeruginosa 9027* (Table XII). In contrast to this finding,

Barron (14,90), employing the same species found an inhibition of 25$ with

1 x l O - 4 ! U0 2+ + , and of 69$ with 4 x HT 4 M U0 2

+ + •

Table XII The effect of U02(N03)2 on the oxidation of gluoose by 22 hour cells harvested from a gluoose mineral medium.

Inhibition (per cent)

5 13 17

T h e inhibitory action of uranyl nitrate on the oxidation of the

dicarboxylio acids, succinate, fumarate and malate is shown in Table XIII*

Succinate and fumarate oxidations were decreased only -when a relatively

high concentration of U02 was used, while malate was completely unaffected •

Barron (14) found i t necessary to employ 1 x 10"^ M uranyl nitrate to

inhibit succinate oxidation of E. coli, but at this concentration P.aeruginosa

was agglutinated. The absence of inhibition with malate may be explained

by the fact that this and other hydroxypolycarboxylic acids are able to

reverse uranium inhibition of isolated enzyme systems (131). Barron et a l ,

(14) also observed no inhibition of malate oxidation in yeast, when they

used concentrations of U0 2+ completely toxic to glucose oxidation*

(Molar) * ( »

— 1050 1 x 10"*4 1000

2.5 x 10"4 '925 4 x 10"*4 875

Table XIII

The effect of U02(NQ3)2 on the oxidation of succinate, fumarate

and malate by 21 hour oells harvested from an ammonium phosphate

medium containing one of these compounds as sole carbon souroe.

Experiment Humber®

Experimental Conditions

U0 2(N0 3) 2

(molar) 4 ° (N) Inhibition (per cent)"

I Succinate it it n

1 x 10" 4

2.5 x l O " 4

4 x 10" 4

807 eo4 7853 616

0 3 24

II Fumarate tt it

7.5 x 10~ 5

1 x l O " 4

510 518 480

0 6

III Fumarate ti 4 x l O " 4

522 320 39

IV Malate n tt

1 x 10" 4

4 x 10" 4

1100 1200 1000

0 9

Higher concentrations of uranyl nitrate (2.5 x 10 M and 4 x 10" 4 M)

were required for significant inhibition cf the oxidation of pyruvate and

oc -ketoglutarate (Table XIV), and of acetate (Table XV). The most striking

data were obtained with isooitrate (Figure XVI), i n which even the lowest

concentration (7.5 x 10"^ M) caused greater than 50$ inhibition, and

higher concentrations ( l x 10*"4 M and 2.5 x 10" 4 M) were almost completely

inhibitory. Citrate oxidation, on the .other hand, was but slightly

affected by these conoentrations (Table XV).

- &7; -

Table XIV

The effect of U02(N03)2 on the oxidation of pyruvate and

<-ketoglutarate by 22 hour oells harvested from an ammonium

phosphate medium containing either of these compounds as C souroe

Experiment Number.

Experimental Conditions

UO2(N0?)2

(molar) Q02(N) Inhibition

(per cent)

I Pyruvate 11 tt n

1 x lO**4

2.5 x 10-4 4 x l O - 4

1485 1251 10E8 1034

16 31 30

II <*. -ket oglut ar at e 11 it 7.5 x 10"5

1 x 10"4

538 488 437

9 19

III «* -ketoglutarat e it 11

1 x 10"4

4 x lO""4

1375 1115 920

19 33

Table XV

The effect of 1102(^03)2 °n the oxidation of acetate and

citrate by 24 hour oells harvested from an ammonium phosphate

medium containing either of these compounds as sole C souroe.

Experimental Conditions

Aoetate u ti tt

Citrate tt ti n

U02(NO,)2

(molar)

1 x 10"* 2.5 x l O - 4

4 x 10**4

1 x 10~* 2.5 xlO" 4

4 x lO" 4

°2 Uptake (ul)

(at 120') • 280 248 193 170

(at 105') 158 160 142 112

Inhibition (per cent)

11 31 39

0 10 29

- 58 -

Figure XVI. The inhibitory effect of U0 2(N03) 2 on the oxidation of isocitrate by 22 hour cells harvested from an isocitrate ammonium phosphate medium. Warburg cups contained 0.5 ml. c e l l suspension, 1.5 ml. M/7 veronal buffer pH 7.2, 0.3 ml. U02(N0 3) 2, 0.2 ml. substrate.. Final volume was 3.0 ml.

- 59 -

Attempts were made to reverse the inhibition of uranyl nitrate,

by the addition of phosphate, which forms a strongly associated complex

with the toxio ion. The oxidizable substrate was added .to the main oomparfc-

ment of the Warburg cup (instead of to the side-arm), together with veronal

buffer, cells and water to volume, Uranyl nitrate was added to one side-arm,

and phosphate buffer to the other (0.3 ml. of each). The UOg++ was added

from the first side-arm when the cells had been metabolizing their substrate

at a constant rate for 30 minutes. Fifty minutes later, phosphate was

added from the second sidefearm. In experiments with glucose and with succin­

ate oells, no significant decrease in oxidation was observed when the

uranium'was added in this manner, and hence, no reversal of inhibition

was effected by phosphate. Apparently, i t is necessary to have the

uranium and oells in oontact for well over an hour to obtain measurable

inhibition. This finding is in contradiction to the observation^ of

Rothstein (123,124) and Barron (14,90), who obtained an instantaneous

uranium uptake by yeast cells. In addition, Barron (14) always used 38° C.

for bacterial studies, while a l l experiments with P. aeruginosa ATC 9027

have been oonducted at 31° C. At the latter temperature, a much slower

rate of reaction would be operative.

Uranyl nitrate as an inhibitor on dried cell preparations provided

additional evidence that the cell membrane has been altered duringthe drying

process. Concentrations of 4 x 10""4 M and 5 x 10""4 M which had effected a

25$ inhibition with whole resting cells, showed no toxic effect on dried

cells,-in fact, a slight stimulation was observed (Table XVI).

- 6 0 -

Table XVI

The effeot of UOgCNOjOg o n ^he oxidation of succinate by dried

oells harvested from a succinate ammonium phosphate medium.

Experiment UOgCNOg^ Oxygen Uptake % Inhibition

Number (molar) At 60» At 120* At 60* At 120'

17 65 £ x 10-4 21 75.

II — 30 65: S x 10"4 33 70 0 0

DISCUSSION

From studies of malonate inhibition in P. aeruginosa it may be

concluded that this organism possesses the enzyme succinic dehydrogenase*

Additional evidence for a close relationship amongst the compounds succinate,

fumarate and malate was also provided. While malonate strongly inhibits

both the rate and extent of oxidation of suocinate, only rate is affected

in the case of both fumarate and malate. The rate of «W:etoglutarate

oxidation was also inhibitadj an observation which indicates an enzymatio

relationship between this compound and succinates Non-inhibition of

pyruvate oxidation might suggest that this intermediate is not oxidized

via the same meohanism (the Krebs cycle) as those listed above. However,

such a conclusion seems unlikely in the light of other findings (157),

nor can i t be drawn on the basis of a single experiment.

Turning now to uranium, i t is believed to inhibit cellular

metabolism indirectly by combining with the cell surface,, thus causing

decreased permeability to the passage of oxidizable substrates. According

to Knaysi (66,67,68) and others (135), the cell membrane consists of a

lipoid-protein system, with associated ribonucleic acid. Apparently,

uranium complexes with the protein constituents of the membrane, through

certain "active" groups, which Barron (90) and Rothstein (123,124) suggest

may be phosphates, phosphate esters or polyphosphates. Moreover, this

uranium-protein complex can be reversed by the addition of inorganic phosphate

at low concentrations. These facts point to some relationship between

phosphorylation and the "active" transfer of substrates across the cell

membrane. Additional support for this suggestion was found by Gale (46)

in that the passage of. glutamic acid into the cell of Streptococcus ffigpalis

is dependent on an exergonic metabolic reaction involving phosphate.

- 62 -

It also seemed possible that the energy for transport might arise

from a coupling between phosphorylation at the "active" sites in the oell

surface, and the oxidation of substrate - once the reaction had been primed

by energy from some other coupled reaction (44)• That there is a coupling

between phosphorylation and the oxidation of glucose and of certain Krebs

intermediates has been shown by a number of investigators (9,11,32,97,98,

109,144), in studies made mainly with aaimal tissue. Evidence for the

oxidative formation of energy-rich phosphate bonds in bacterial metabolism

has been presented by Lipmann (83),with Lactobaoillus delbruckij by TissiSres

(150) with Aerobaoter aerogenes, and by Hersey & Agl (57) with cell-free

preparations of E,coli.

When the present study was undertaken, i t was hoped that some

association might be uncovered between uranium inhibition and the "active"

(possibly phosphorylative) transfer of substrate across the oell membrane.

However, i t is very difficult to interpret;, the results of the experiments,

since the concentration of uranyl nitrate required for significant inhibition

was not the same in each case. For example, isocitrate oxidation was strongly

inhibited by a l l concentrations used, while malate showed no inhibition what­

soever. Inhibition falling between these two extremes was observed with the

other substrates. Similar findings were reported by Rothstein & Meier (125)

in work on molybdate inhibition of surface phosphatases in yeast. It may be

that the protein with which the uranium complexes is not only "active"1 in

the transport of substrate, but is itself the enzyme for metabolizing the

substrate. Thus, different enzymes would show different sensitivities

to uranium. Such an hypothesis i s , however, far beyond the present bounds

of experimental verification.

- 63 -

The greater part of the work on metabolic inhibitors has dealt

with the inhibition of isolated systems. The application of inhibition

studies to the metabolism of whole oells would appear to be open to

question, especially when so l i t t l e i s known of a cell's actual metabolic

pathways, A reduction i n respiration may imply that an inhibitor i s

acting on one or more of the enzymes concerned with carbohydrate metabolism

(162). In addition, an inhibitor may cause an organism to use an alternative

metabolic pathway. Until more i s known of the direct and alternative

pathways i n bacterial metabolism, the use of a non-specific inhibitor such

as uranium is precluded.

- m -

SUMMARY <

Resting cells of P. aeruginosa harvested from a mineral medium,

in which succinate or fumarate or malate was the sole carbon source,

metabolized al l three compounds immediately and at a constant and rapid

rate, thus indicating an enzymatic inter-relationship.

The oxygen consumption of dried oells was considerably lower

than that observed with resting cells. These preparations did not

require cytochrome c or pyocyanine as a carrier system. Optimum pH for

suocinic dehydrogenase in succinate and acetate dried cells lay between

pH 7.0 and 8.0 •

Moderately high concentrations of malonate were neoessary to

inhibit succinate oxidation by resting cells at pH 7.0, but when the pH

was lowered, maximum inhibition v/as effected by low concentrations.

P. aeruginosa possesses an adaptive enzyme system for metabolizing the

inhibitor. Dried cell preparations were strongly inhibited by low

concentrations of malonate at pH.7.0, thus indicating that permeability

is an important factor in malonate inhibition with P. aeruginosa.

Low concentrations of uranyl nitrate caused varying degrees of

inhibition with resting cells in the presence of the Krebs intermediates

or glucose, the most striking effect being observed in the case of

isocitrate oxidation. No reversal of uranium inhibition by phosphate

was obtained.

- 65 -

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