Download Utilization of purines as nitrogen source by facultative phototrophic

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.
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