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249 FEMS MicrobiologyLetters 4 (1978) 249-253 © CopyrightFederation of European MicrobiologicalSocieties Published by Elsevier/North-HollandBiomedicalPress U T I L I Z A T I O N O F P U R I N E S AS N I T R O G E N SOURCE BY F A C U L T A T I V E P H O T O T R O P H I C BACTERIA W. ARETZ, H. KASPARI and J.-H. KLEMME Institut far Mikrobiologie der Universitdt, Meekenheimer Allee 168, 5300 Bonn 1, FederalRepublic of Germany Received 3 July 1978 1. Introduction The ability to grow with purines as nitrogen, carbon or energy sources is widespread among bacteria [1]. Recently, it was reported that the facultative phototrophic bacterium, Rhodopseudomonas palustris, was able to use azaguanine as nitrogen source [2]. Considering the fact that the bacterial pathways of aerobic and anaerobic purine degradation differ widely, it seemed of interest to study purine utilization by facultative phototrophic bacteria in more detail. In the present communication, we give a summary of growth experiments with adenine, guanine, xanthine and uric acid as N-sources conducted with 12 strains of the Rhodo~pirillaceae and show that the enzymes uricase (EC 1.7.3.3) and xanthine dehydrogenase (EC 1.2.1.37) do not participate in uric acid degradation by a newly isolated strain ofRps, capsulata. The properties of a partially purified xanthine dehydrogenase isolated from hypoxanthine-grown cells of this strain will be reported. 2. Material and Methods Purine-utilizing Rhodospirillaceae were grown in a medium of the following composition (quantities are specified per liter of deionized water): purine compound, 0.5 g; sodium-D,L-malate, 4 g (or D-fructose, 2.5 g); MgS04 • 7 I-I20, 0.2 g; CaCI2 • 2 H20, 0.05 g; potassium phosphate, pH 6.8, 10 mM; trace elements solution (see [3]), 1 ml; nicotinamide, 1 mg; thiamine-HC1, 1 mg; biotin, 0.01 mg; p-aminobenzoic acid, 0.2 rag; pH adjusted to 6.8. The culture media were sterilized by filtration. Photosynthetic cultures were grown in completely filled screw-cap bottles (50 or 500 ml volume) at 30°C and about 2500 lux; heterotrophic cultures were grown in the dark in erlenmeyer flasks Filled for 1/6 of their volume with culture medium. Incubation was at 30°C on a rotary shaker. The culture media contained either 0.1% (w/v) (NH4)2SO4 or 0.05% (w/v) of a putine compound (adenine, guanine, hypoxanthine, xanthine, uric acid) as N-source. Growth was followed by measuring the absorbance at 660 nm in a "Spectronic 20".colorimeter (Bausch and Lomb). Uric acid was analyzed enzymatically with the "Utica Quant" test system of Boehringer, Mannheim, following the instructions of the manufacturer. NI-I~ was determined colorimetrieally by using the test combination for urea of Boehringer, Mannheim, whereby the enzymatic step involving urease was omitted. / For thin-layer chromatography, commercial DCplates Ce1400-10 UV2s4 (Macherey, Nagel & Co., Dtiren) were used. The chromatograms were developed with a mixture of n-butanol, acetic acid and water (2 : 1 : 1), dried in the air and sprayed either with Pauly's reagent [4] for the detection of imidazol compounds, ninhydrin for the detection of amino acids, or Ehrlich's reagent (1 g p-dimethylaminobenzaldehyde in 95 ml of 96% ethanol plus 5 ml conc. HC1) for the detection of urea, indole derivatives and ureido compounds. Purines were detected on the plates under UV-light and identified according to their fluorescence and their Rrvalues. Cells were ruptured by ultrasonic oscillation. The homogenates were separated into soluble and particulate fractions by centrifugation at 140 000 g for 90 250 min (4°C). Protein in the extracts was determined according to the method of Lowry et al. [5]. Uricase (EC 1.7.3.3) activity of cell homogenates was determined manometrically at 37°C following the method of Leone [6]. The activity of xanthine dehydrogenase (EC 1.2.1.37) was assayed spectrophotometrically at 30°C in reaction mixtures containing 125 mM potassium phosphate, pH 7.5; 1 mM NAD and 1 mM hypoxanthine. To follow reduction of pyridine nucleotides, absorbance changes at 340 nm were recorded. Reduction of 2,6-dichlorophenolindophenol (DPIP) was followed at 600 nm, that of K3Fe(CN)6 at 430 nm, and that of cytochrome c at 550 nm. Analytical gel electrophoresis in polyacrylamide gels was performed with the disc-electrophoresis system of Desaga GmbH, Heidelberg, using a gel concentration of 7% and Tris-glycine buffer, pH 8.3 [7]. Protein bands were stained with Coomassie brilliant blue and xanthine dehydrogenase activity bands were localized by incubating the gels for 15 min at room temperature in 12 ml of a mixture containing 125 mM K-phosphate, pH 7.5; 1.5 mM hypoxanthine; 0.75 mM NAD; 0.1 mg phenazine methosulfate and 3 mg nitroblue tetrazolium chloride. NAD, NADH, NADP, cytochrome c and the assay kits for uric acid and urea were obtained from Boehringer, Mannheim. Allantoin, allantoic acid, 4-amino-5.imidazol-carboxamide, formiminoglycine and hypoxanthine were purchased from Sigma Chemic GmbH, Mianchen. Protamine sulfate and nitroblue tetrazolium chloride were obtained from Serva, Heidelberg, and all other chemicals from Merck, Darmstadt. 3. Results and Discussion Table 1 gives a survey of the strains used in this study, and Table 2 summarizes the results of growth experiments conducted with these strains. Obviously, the ability to use purine compounds as N-source is a common property of RhodospiriUaceae. It should be noted in this connection that, with the exception of Rps. capsulata AI, none of the strains was enriched and isolated in pufine containing media. With purines as N-source, the majority of the strains showed much better growth under aerobic, dark than under anaer- TABLE 1 Strains of Rhodospirillaceae used in this study Strain Species Kbl Rps. capsulata R8 R10 AI Rps. capsulata Rps. capsulata Rps. capsulata R1 R6 lal Rps. palustris Rps. palustris Rps. palustris 11/1 Rps. palustris le7 Rps. sphaeroides 29/1 Rps. gelatinosa FR1 R. rubrum Ha S1 R. rubrum R. rubrum Source DeutscheSammlung yon Mikroorganismen, GiSttingen (DSM) No. 155 New isolate New isolate New isolate from medium with uric acid as N-source New isolate New isolate Lehrstuhl fur Mikrobiologie der Universitfft Freiburg Lehrstuhlftir Mikrobiologie der Universit~it Freiburg Lehrstuhlfar Mikrobiologie der Universit~tt Freiburg Lehrstuhl far Mikrobiologie der Universit[t Freiburg Lehrstuhl far Mikrobiologie der Universit~tt Freiburg DSM No. 107 DSM No. 467 obic, light conditions. This is particularly evident in the case of adenine as N-source. Only the three R. rubrum strains were able to use this compound under both growth conditions. Rps. palustris seems to be unable to utilize adenine as N-source, possibly because of the lack of adenine deaminase (EC 3.5.4.2). In the other strains, however, either the transport of adenine into the cell or the biosynthesis and/or the activity of adenine deaminase must be under strict metabolic control. None of the strains was able to grow in purine containing media in the absence of a suitable carbon source. For further experiments on the mechanism of purine degradation, the newly isolated Rps. capsulata strain AI was chosen. In photosynthetic cultures with as N-source, the growth rate (at 2500 lux) was 0.19 h -1 (malate or fructose as C-source). In photosynthetic cultures with uric acid as N-source, the growth rate was considerably lower (0.13 h-l). It seems, therefore, that the enzymatic steps of uric acid degradation were growth-limiting. The next experiment was to show that the photoassimilation of the N-atoms of the purine molecule 251 TABLE 2 Utilization of purine compounds as N-sourcesby Rhodospidllaceaeunder different culture conditions (aerobically in the dark and anaerobically in the light) The organisms were grown in culture media with Na-D,L-malateas C-source and the various purines as N-sources.Photosynthetic cultures were grown in completely filled 13-mlscrew-captubes at 30°C and about 2500 lux. Aerobic cultures were grown in the da~k at 30°C in 250-ml erlenmeyer flasks containing 40 ml of culture medium. The flasks were incubated on a rotatory shaker at 150 rev./min. Growth was judged visually after 3 days. Species Rps. capsulata Rp~. sphaeroides Rps. gelatinosa Rps. palustris R. rubrum Number of strains tested 3 1 1 4 3 Growth with Adenine Guanine Uric acid Xanthine Aerob. dark Anaer. light Aerob. dark Anaer. light Aerob. dark Anaer. light Aerob. dark Anaer. light ++ + (+) + .+ ++ ++ + ++ + ++ + (+) + ++ ++ ++ (+) + + ++ ++ (+) ++ ++ ++ ++ + + + + + + + ++;~,~erygood growth; +, good growth; (+), weak growth; -, no growth. occurred via NI~. Cells from a photosynthetic culture with ~ as N-source (fructose as C-source) were harvested, washed two times with sterile 0.9% (w/v) Na~-solution and finally resuspended to an A~6o of 0.74n sterile 70 mM potassium phosphate containing 1.2~mM uric acid (pH 6.8). The suspension was gassed with N~, magnetically stirred, and incubated at 30°C and 2500 lux. At different times, samples were withdrawn from the suspension and analyzed for NI-~ and uri~acid. Because of the lack of a carbon source, the A6~0 of the cell suspension did not increase significancy during 3 days. The ratio between the NI~ libe~ted and the uric acid degraded ranged from 2.82 ~to 3.18 in the various samples. Interestingly, the degradation of uric acid under anaerobic conditions was dependent on light. To identify the nature of the missing N-com. pound, cell-free samples of the culture medium were concentrated by freeze drying and then subjected to thin-layer chromatography. After development of the plates with Ehrlich's reagent, a yellow spot with an Rfvalue of 0.43 became visible. It must be noted that this compound could not be detected in cell-free samples of growing cultures, regardless of whether urate or Nl~ was used as N-source. Comparison with the chromatographic behaviour of possible inter- mediates of the aerobic and anaerobic degradation pathway for uric acid [1] revealed that the unknown substance was not identical with 4-amino-5-imidazolecarb0xamide, formiminoglycine, glycine, serine, allantoin, allantoic acid, oxamic acid or urea. The nature of this substance remains to be elucidated. In order to get further insight into the mechanism of urate degradation in R p s . c a p s u l a t a AI, the key enzymes of the aerobic (uricase) and the anaerobic, clostridial (xanthine dehydrogenase) degradation pathway were determined. Interestingly, uricase activity could not be detected in homogenates neither from heterotrophically nor from photosynthetically grown cells. To investigate the possibility that xanthine dehydrogenase was involved in the primary steps of uric acid degradation, extracts of cells grown at the expense of different N-sources were analyzed for xanthine dehydrogenase activity. As has been reported for other bacteria [8], extracts from hypoxanthine-grown cells contained much higher xanthine dehydrogenase activities (0.04 units/rag protein) than extracts from xanthine-grown cells (0.014 units/rag protein). Importantly, extracts of cells grown at the expense of uric acid or NI~ as N-sources were completely devoid of xanthine dehydrogenase activity. The enzyme was stabilized in crude extracts by 252 TABLE 3 Activity of purified xanthine dehydrogenasefrom Rhodopseudomonas capsulata AI with different electron donors and acceptors Reaction rates were measured in mixtures with 125 mM K-phosphate, pH 7.5, and the indicated concentrations of substrates. Electron donor Hypoxanthine (1 mM) Xanthine (1 mM) Hypoxanthine (1 mM) Electron acceptor NAD (1 mM) NAD (1 mM) t NAD (1 mM) NADP (1 mM) DPIP (0.07 mM) KaFe(CN)6 (1 mM) Cytochrome c Spec. act. (units/mg protein) a 3.06 2.21 3.06 0.10 0.95 0.65 0 a One unit is the enzyme activity catalyzing the reduction of 1 #mole of substrate per min. 1 mM EDTA and could be partially purified from hypoxanthine-grown cells by using conventional methods (precipitation of nucleic acids by protamine sulfate, fractionation of the extract by (NH4)2SOa-precipitation, heat denaturation of inactive proteins and adsorption onto and elution from calcium phosphate gel). The enzyme was purified about 80-fold up to a specific activity of about 3 units/mg protei n. At this stage, the enzyme preparation still contaihed 4 different protein bands in polyacrylamide gel electrophoresis slabs. Judged by activity stains, the enzyme comprised about 40% of the total protein in the purest fraction obtained. The optimal pH for enzyme activity was 8.3. Table 3 shows the relative activities of the enzyme with different electron donors and electron acceptors. Hypoxanthine was the most effective electron donor and NAD the most effictive electron acceptor. The Km values (determined by the graphical method of Eisenthal and Cornish-Bowden [9]) were 53/aM for hypoxanthine and 61/aM for NAD, respectively. Although the purified enzyme catalyzed the reduction of uric acid with NADH as electron donor with a rate of about 3% of that of the inverse reaction, it can be excluded that xanthine dehydrogenase plays a role in the anaerobic-light degradation of uric acid by Rps. capsulata AI, since the enzyme could be found only in cultures l~rown with xanthine or hypoxanthine as N-source. According to our present knowledge, the anaerobic degradation of uric acid in clostridia [ 10] and Veillonella alcalescens [11 ] requires that this compound is initially reduced to xanthine by xanthine dehydrogenase. If phototrophic bacteria would make use of the same anaerobic degradation pathway, one would expect to find xanthine dehydrogenase in sufficiently high activities in Rps. capsulata AI cells grown photosynthetically with uric acid as N-source. Our experiments have shown, however, that this enzyme is induced only in cells grown with xanthine or hypoxanthine as N-source. Thus, although the purified enzyme catalyzed the reduction of uric acid with NADH as electron donor, it can be excluded that it plays a role in anaerobic uric acid degradation. The absence of uricase (EC 1.7.3.3) and xanthine dehydrogenase (EC 1.2.1.37) in uric acid-grown cells ofRps, capsulata AI suggests the existence of a novel, possibly light-dependent, enzymatic mechanism of urate degradation. Acknowledgements These investigations were supported by a grant from the Deutsche Forschungsgemeinschaft. References [I] Vogels,G.D. and van der Drift, C. (1976) Bacteriol. Rev. 40, 403-468. [2] Malofeeva,I.V. and Laush, D. (1976) Microbiology (USSR)45,441--442. 253 ~3] Pfennig, N. and Lippert, K.D. (1966) Arch. MikrobioL 55, 245-256. ~4] Hais, I.M. and Macek, K., Handbuch der Papierchromatographie (1963) Gustav Fischer Verlag, Jena. ~5] Lowry, O.H., Rosebrough, H.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. |6] Leone, E. (1955) in: Methods in Enzymolgy (Colowick, S.P. and Kaplan, N.O., Eds.), Vol. 2, pp. 485-489, Academic Press, New York. i[7 ] Maurer, H.R., Disc Electrophoresis and Related Tech- niques of Polyacrylamide Gel Electropho~esis ( 1971 ) Walter de Gmyter, Berlin. [8] Ohe, T. and Watanabe, Y. (1977) Ag~. Biol. Chem. (Tokyo) 41, 1161-1170. [9] Eisenthal, R. and Cornish-Bowden, A. (1974) Biochem. J. 139, 715-720. [10] Bradshaw, W.H. and Barker, H.A. (1960) J. BioL Chem. 235, 3620-3629. [11] Smith, S.T., Rajagopalan, K.V. and Handler, P. (1967) J. BioL Chem. 242, 4108-4117.