Purine degradation in Pseudomonas aeruginosa and Pseudomonas testosteroni

111

Biochimica et Biophysica Acta, 499 (1977) 111--118 © Elsevier/North-Holland Biomedical Press

BBA 28295

PURINE DEGRADATION IN PSEUDOMONAS A E R UGINOSA A N D PSEUDONIONAS T E S T O S T E R O N I

G.P.A. BONGAERTS, I.L. SIN *, A.L.J. PETERS and G.D. VOGELS

Department of Microbiology, Faculty of Science, University of Nijmegen, Ni]megen (The Netherlands) (Received January 17th, 1977)

Summary 1. Adenine, hypoxanthine, xanthine and guanine are broken down in Pseu. domonas aeruginosa and Pseudomonas testosteroni to allantoin by the concerted action of the enzymes adenine deaminase, guanine deaminase, NAD ÷dependent xanthine dehydrogenase and uricase. 2. Uric acid is broken down by an unstable, membrane-bound uricase with an unusually low pH optimum. 3. In both strains adenine inhibits growth and xanthine dehydrogenase. A second type of inhibition is manifest only in Ps. testosteroni and concerns the regulation of the biosynthesis of amino acids of the aspartate family. Enzymic studies showed that in this strain aspartate kinase is inhibited by AMP.

Introduction

The reactions involved in the degradation of allantoin have been intensively studied in a variety of organisms [1--7], including several Pseudomonas species [1,2,4--6]. However, few studies were made on the initial steps in the catabolism of purines in bacterial systems [8]. The aim of the present study was to investigate the capacity of Pseudomonas aeruginosa and Pseudomonas testosteroni to grow on the various purine bases and to study their degradation to allantoin. Materials and Methods

Organisms and cultivation methods. Ps. aeruginosa V3003 and Ps. testosteroni ATCC 15667 were used. In this study the nomenclature of Stanier et al. [9] will be followed, which implies that Pseudomonas acidovorans ATCC * Present address: Agricultural Research Council Unit, Department of Zoology, Oxford, U.K.

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15667, strain 21 of Den Dooren de Jong [10], is called Ps. testosteroni. This strain was used in our previous studies on the purine metabolism of Pseudo. monas species [1,3,4,11]. Ps. testosteroni was grown aerobically at 30°C in media containing the following salts per liter: 6.8 g KH2PO4; 8.9 g Na2HPO4 • 2H:O; 0.2 g MgSO4 • 7H20; 0.1 mg MnC12 • 4H20; 0.2 mg ferric ammonium citrate; 2 mg CaCI~; 10 mg sodium molybdate. Ps. aeruginosa was grown aerobically at 30°C in a medium with a mineral composition as described by Kalt~ wasser [12]. The media were supplemented with either (a) Nutrient Broth (Oxoid; 30 g/l), (b) one of the following compounds (3 g/l), hypoxanthine, xanthine, guanine or uric acid, or (c) sodium fumarate (15 g/l) and KNO~ (5 g/l). The media containing Nutrient Broth, xanthine or guanine were sterilized b y autoclaving for 10 min at 121°C, while the other media were filtersterilized. Growth was followed spectrophotometrically at 600 nm. Preparation o f cell-free extracts. The collected cells were washed in 40 mM phosphate buffer (pH 7.2) or in 0.1 M Tris • HC1 buffer (pH 8.0), supplemented with 0.125 M KC1. The latter buffer was used in our studies on homoserine dehydrogenase (L-homoserine:NAD ÷ oxidoreductase, EC 1.1.1.3). The cells were resuspended in the same buffer, and disrupted in a MSE 100-W ultrasonic disintegrator by sonication for 90 s. The sonicated cell suspension was centrifuged at 1 0 0 0 0 0 × g for 20 rain. The supernatant is referred to as crude extract. Preparation o f toluenized cells. 0.3 ml of 10% (v/v) toluene in ethanol was added to 300 ml culture. The culture was shaken vigorously for 15 s and the cells were then pelletted, washed once with 40 mM phosphate buffer (pH 7.2) and resuspended in 5 ml of the same buffer. Enzyme assays. Xanthine dehydrogenase (xanthine:NAD ÷ oxidoreductase, EC 1.2.1.37) was determined as described b y Watt [13] by using a total incubation volume of 2 ml, and a NAD ÷ concentration of 0.6 raM. The products formed when extract was incubated with NAD ÷ and either hypoxanthine or xanthine were investigated by chromatography on cellulose-thin layers of 10-~1 samples taken from incubation mixtures at various times. Development was for 10 cm in 0.01% Na4EDTA in water, and purines were visualized under ultraviolet light. A c o m p o u n d with an RF equal to that of uric acid was produced in incubations containing hypoxanthine or xanthine. No other products or intermediates were detected. Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) and adenine deaminase (adenine aminohydrolase, EC 3.5.4.2) were assayed b y measuring the ammonia produced when extract was incubated with guanine or adenine, respectively, in 40 mM phosphate buffer (pH 7.2) at 30°C. The ammonia was determined b y the phenol-hypochlorite method [14], using a standard curve constructed in the presence of adenine or guanine as appropriate. Guanine was present in incubations at a saturating concentration, adenine at 3.0 raM. No ammonia was produced, over a 30 min incubation period, in the absence of these purines. The products formed b y the adenine- and guanine-degrading enzymes were investigated b y chromatography on Whatman No. 1 paper. The solvent system consisted of water saturated with amyl alcohol [15,16]. Hypoxanthine was produced from adenine, and xanthine from guanine.

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Uricase (urate:oxygen oxidoreductase, EC 1.7.3.3) was measured by following the disappearance of uric acid. 0.5 ml cell suspension was incubated in a total volume of 2.5 ml of 1 mM uric acid in 50 rnM Tris • HC1 (pH 7.6) at 30°C, in a 50 ml Erlenmeyer flask shaking at a speed of 100 rev./min in a reciprocal shaker bath. 0.20-ml samples were added to 3.8 ml of 0.1 M HC1, centrifuged for 10 rain at 12000 × g, and the absorbance of uric acid in the supernatant read at 283 nm (E283 -- 12.0 • 103 M -~ • cm-~). The product formed by uricase action was investigated by chromatography on Whatman No. 1 paper, using the solvent system of 9 parts water saturated with amyl alcohol and 1 part formic acid [15,16]. Aspartate kinase (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) was assayed essentially by the hydroxamate method described by Datta and Prabash [17]. Homoserine dehydrogenase was assayed essentially by the method described by Truffa-Bachi et al. [18]. Protein was determined by the method of Lowry et al. [19], using bovine serum albumin as a standard. Results

Growth on various purines Both Ps. testosteroni and Ps. aeruginosa were capable of growing on 0.3% uric acid, xanthine, hypoxanthine or guanine as sole carbon, nitrogen and energy source, but not on 0.3% adenine. The growth of Ps. testosteroni was accompanied by the production of large quantities of ammonia and a sharp increase in the pH of the growth medium, making it necessary to use a rather strongly buffered growth medium in order to obtain a reasonable yield of cells. During growth of Ps. aeruginosa no marked increase of pH was observed. Enzymes o f purine degradation Cell-free extracts prepared from Ps. testosteroni grown on a variety of media were assayed for the enzymes guanine deaminase, adenine deaminase and xanthine dehydrogenase (Table I). Xanthine oxidase activity was found only to be 3--6% of xanthine dehydrogenase activity, so the enzyme was subsequently assayed as a dehydrogenase. Cell-free extracts prepared from Ps. aeruginosa, grown on some purines, were assayed for the same enzymes. Uricase could not be detected in crude extracts of either strain, although it was present in suspensions of whole cells. After sonication in water, activity was obtained only in the membrane fraction, which was sedimented at 100000 × g. Similar results were obtained by Kaltwasser for the uricase of Alcaligenes eutrophus (Hydrogenomonas H16), Ps. aeruginosa and Micrococcus denitrificans [20]. Uricase activity was not released from the particles when they were treated with sodium dodecyl sulphate or guanidine, both in saturated solution at 0°C, or a 10% solution of Tween 80; full activity was retained in the particulate fraction. Treatment of suspensions of Ps. aeruginosa or Ps. testosteroni cells with toluene in ethanol, and subsequent washing of the toluenized cells yielded a system retaining 95% of the activity of untreated cells, giving a linear rate of

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TABLE LEVELS

I OF

PURINE-DEGRADING ON PURINES

ENZYMES

IN CELLS

O F PS. T E S T O S T E R O N I

AND

PS. A E R U .

GINOSA GROWN

E n z y m e assays w e r e p e r f o r m e d as d e s c r i b e d in t h e t e x t . Crude e x t r a c t w a s u s e d for t h e assay o f a d e n i n e d e a m i n a s e , guanine d e a m i n a s e and x a n t h i n e d e h y d r o g e n a s e 0 w h i l e uricase w a s a s s a y e d in t o l u e n i z e d c e l l s . V a l u e s + S . E . w e r e derived f r o m t w o t o five d e t e r m i n a t i o n s o n d i f f e r e n t cultures. O t h e r values w e r e d e t e r m i n e d in d u p l i c a t e o n o n e culture o n l y . S p e c i f i c a c t i v i t y is e x p r e s s e d as p-tool p r o d u c t f o r m e d per rain per mg protein. Carbon, n i t r o g e n and energy source for cell g r o w t h Uric acid

S p e c i f i c activity Guanine deaminase Ps. t e s t o s t e r o n i Ps. aeruginosa

--**

Hypoxanthine

Ps. t e s t o s t e r o n i

0.I0

Xanthine

Ps. t e s t o s t e r o n i Ps. aeruginosa

Guanine

Fumarate + nitrate

0.028

0.08

*

* +- 0 . 0 2

--

Ps. testosteroni Ps. aeruginosa

0.027 +- 0.002

Ps. t c s t o s t e r o n i Ps. a e r u g t n o s a

0.007

0.28

0.020

Adenine deaminase 0.034

0.012 --

0.021

0.30

+- 0 . 0 4

--

0.081 0.021

0.31 0.12

+ 0.04

0.37 --

0.071

0.II --

+ 0.002

Uricase

--

--+ 0 . 0 0 1

X a n t h i n e dehydrogenase

0.20

+- 0 . 0 2

0.065

+- 0 . 0 0 4

0.21 --

0.071

0.010

0.003

0.015

0.008

0.004

* V a l u e s f o r d e t e r m i n a t i o n o n t w o sepsxate c u l t u r e s w e r e i d e n t i c a l . ** N o t d e t e r m i n e d .

uric acid disappearance for 20 min, and showing an activity proportional to the volume of cell suspension added. Freezing and thawing of the toluenized cell suspension, as described for the assay of phosphofructokinase in Escherichia coli [21] had no effect on the uricase activity observed. Levels of uricase in cells of Ps. testosteroni and Ps. aeruginosa are shown in Table I. The uricase of both Pseudomonas strains has the unusually low pH optimum of 7.5, compared to the pH optima of around 9 shown by uricases from most other sources [8]. Inhibitory effects of adenine on growth Adenine inhibited the growth of both pseudomonads (Table II). It is concluded that adenine exerts an inhibitory effect at t w o levels. Adenine inhibits the xanthine dehydrogenase activity of Ps. testosteroni in a non-competitive way [11]. This effect was also observed for Ps. aeruginosa and is specific to the purine, since adenosine, adenosine 3':5'-monophosphate, AMP and ADP do n o t influence xanthine dehydrogenase activity of either strain. This inhibitory effect may explain the large generation times observed in media containing adenine and purines, of which the degradation depends on the activity of xanthine dehydrogenase. However, a second less drastic inhibitory effect was observed for Ps. testosteroni. The generation time of Ps. testosteroni growing in fumarate/nitrate medium is enlarged considerably in the presence of adenine (Fig. 1). Since fumarate and nitrate are used as carbon and nitrogen source in this medium, the inhibitory effect of adenine could be a regulatory one exerted on an essential biochemical pathway. To elucidate this we tried to abolish this

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T A B L E II GENERATION

T I M E S O F PS. T E S T O S T E R O N I A N D P 8 . A E R U G I N O S A IN V A R I O U S M E D I A Mean generation time (min)

Medium

Ps. testosteroni

Adenine (0.3%) Hypoxanthine ( 0 . 1 % ) + adenine (0.1%) Hypoxanthine (0.1%) Uric acid ( 0 . 1 % ) + adenine (0.1%) Uric acid ( 0 . 1 % ) Fumatate (0.15%) + nitrate (0.05%) + adenine (0.1%) Fumarate (0.15%) + nitrate ( 0 . 0 5 % )

Ps. aeruginosa

>600 >600

>600 >600

100 215

110 45

65 330

35 75

105

70

inhibitory effect by adding a variety of compounds. Addition of thiamine, or histidine and succinate, which exerted positive effects in the relieve of the adenine inhibition in Aerobacter aerogenes [22,23] did n o t cause any effect in our case. The combination of the four amino acids of the aspartate family, methionine, threonine, isoleucine, and lysine, caused a drastic lowering of the generation time. This effect is mainly caused by the presence of isoleucine, but threonine exerts also a beneficial effect (Table III). The stimulation of growth depends on the amount of these amino acids added to the growth medium. On account of these data, it was concluded that adenine or one of its derivatives may exert a regulatory effect on the amino acids of the aspartate family by controlling the activity of one o f the enzymes involved. Therefore, we tested the effect of adenine, adenosine, adenosine 3':5'-monophosphate, and AMP on the regulatory enzymes c o m m o n in the biosynthesis, viz. aspartate kinase and Generation time [rain)

300

200

100

0

I

5

I

10 Adenine[raN)

F i g , 1. I n h i b i t o r y e f f e c t o f a d e n i n e o n g r o w t h o f Ps. testosteroni. Cells w e r e c u l t i v a t e d o n a f u m ~ x a t e / nitrate m e d i u m t o w h i c h t h e i n d i c a t e d a m o u n t o f a d e n i n e w a s a d d e d .

116

T A B L E III INFLUENCE OF ADDITIONS ON THE GENERATION

T I M E O F PS. T E S T O S T E R O N I

T h e g r o w t h m e d i u m c o n t a i n e d 0 . 1 5 % s o d i u m f u m a t a t e a n d 0 . 0 5 % K N O 3. A d e n i n e c o n c e n t r a t i o n w a s 0 . 1 % . T h e i n h i b i t i n g e f f e c t o f a d e n i n e is d e f i n e d as t h e r a t i o o f t h e m e a n g e n e r a t i o n t i m e s m e a s u r e d i n t h e presence and in the absence of adenine. Addition

Final concentration (/~g/rnl)

None M e t , T h r , Ile a n d L y s Ile Thr Asp Leu, Glu, Tyr and Phe Set, Ag, Ala and Gly His, t h i a m i n e a n d u r a c i l His, t h i a m i n e a n d u r a c i l

Mean generation time (min)

-all 2 0 0 200 200 1000 all 2 0 0 all 2 0 0 all 5 0 all 2 0 0

With adenine

Without adenine

Inhibiting effect of adenine

270 105 105 180 200 200 210 235 250

II0 85 80 130 75 75 85 115 120

2.4 1.25 1.3 1.4 2.7 2.7 2.5 2.0 2.1

TABLE IV INFLUENCE OF AER UGINOSA

AMP ON ASPARTATE

AMP concentration (mM)

0 1.2 4 12

KINASE

ACTIVITY

O F PS. T E S T O S T E R O N I

A N D PS.

Percentage of activity Ps. t e s t o s t e r o n i

Ps. aeruginosa

100.0 45.0 34.2 33.3

100.0 100.0 109.0 109.5

homoserine dehydrogenase. Homoserine dehydrogenase was n o t inhibited, even when relatively high concentrations (6--10 mM) of adenine or its derivatives were used. Aspartate kinase activity, however, appeared to be influenced directly or indirectly by AMP, which influence depended on the concentration applied (Table IV), but the enzyme was insensitive for adenine and the other

adenine -

guanine

adenine

guanine

deaminase

deaminase

~. h y p o x a n t h i n e

1 1 1

xanthine

dehydrogenase

xanthine

dehydrogenase

, xanthine

uric acid

allantoin S C H E M E I.

uricase

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derivatives. Therefore, the inhibiting action of adenine on the growth of Ps. testosteroni may result from an inhibitory effect exerted by AMP or an AMP derivative on aspartate kinase causing a requirement for the amino acids of the aspartate family, especially isoleucine and threonine, in the growth medium. Since the enzyme of Ps. aeruginosa is not influenced by the presence of AMP the inhibiting effect of adenine is much lower for this organism. Discussion

The results presented show that both Ps. aeruginosa and Ps. testosteroni possess the enzymes necessary to catalyze the Scheme I reactions (facing page). The reactions involved in the breakdown of allantoin have been already demonstrated and intensively studied previously [1,2]. The detection of a reasonably stable, soluble xanthine dehydrogenase is in contrast to earlier reports on the unstable nature of xanthine oxidase from Ps. aeruginosa [24, 25], where sonication of cells was reported to result in its destruction. This observation m a y be explained if the enzyme, studied by Dikstein et al. [24] as an oxidase, was indeed functioning primarily as a dehydrogenase, using intracellular N A D ÷ as substrate. The N A D H so formed could presumably be readily oxidized in an intact cell, but not in a cell-free extract, thus explaining the observed loss of activity on preparation of a cell-freesupernatant. Although the most intensively studied xanthine oxidase, milk xanthine oxidase, prefers O2 as electron acceptor, there are numerous reports of enzymes from m a m m a l s [26, 27], birds [28], insects [12,29], and anaerobic bacteria [30,31], which appear to function physiologically as dehydrogenases rather than as oxidases, preferring N A D + or ferredoxins as electron acceptors. The enzyme from these two pseudomonads probably also functions physiologically as a dehyd2ogenase. The uricase could not be obtained in soluble form. After sonication in water it could be detected bound to membrane particles,from which it could not be released. After sonication in phosphate buffer activity was almost completely lost. Growth of Ps. aeruginosa and Ps. testosteroni on adenine proceeds very slowly. The sensitive enzyme appears to be xanthine dehydrogenase, which is inhibited non~ompetitively by adenine [11], but not by other adenine derivatives. This result m a y reflect a physiological control mechanism, by which adenine can be preserved, and used immediately for nucleotide synthesis under physiological conditions. In Ps. testosteroni an additional inhibitory effect of adenine on growth was observed. Attempts to relieve this effect, showed that only amino acids of the aspartate family were able to do so. A sensitive spot for an indirect effect of adenine was found to be located in the biosynthetic pathway of these amino acids. A M P produced from adenine by adenine phosphoribosyl transferase (AMP:pyrophosphate phosphoribosyltransferase, E C 2.4.2.7) or a derivative derived from it appears to inhibit the activity of aspartate kinase, which enzyme has a key function in the biosynthesis of threonine, isoleucine, methionine and lysine from aspartate. Inhibition by adenine could not be relieved by the addition of thiamine, or by histidine and succinate, as was found for A. aerogenes [22,23], in which strain the inhibitory effect was explained as a feedback inhibition of purine nucleotide synthesis [23], nor

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does the inhibitory effect resemble the bacteriostatic effect of adenine in E.

coli, where it was ascribed to the inhibition of the de novo synthesis of pyrimidine nucleotides [32]. Inhibition of growth by adenosine and deoxyadenosine was reported for Ps. acidouorans [33], but because of the scarce data a comparison of the results with ours is not yet possible. References 1 2 3 4 5 6 7 S 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 26 29 30 31 32 33

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