FEMS Microbiology Letters 77 (1991) 175-180 175

© 1991 Federation of European Microbi.ological Societies 0378-1097/91/$03.50

ADONIS 037810979100079E

FEMSLE 04258

Pyrimidine catabolism in Pseudomonas aeruginosa

Seunghee K i m and Thomas P. West

Department of Chemistry, South Dakota State University, Brookings, SD, U.S.A.

Received 31 May 1990

Revision received 6 August 1990
Accepted 21 August 1990

Key words: Pseudomonas aeruginosa; Pyrimidine; Dihydropyrimidine dehydrogenase;

Dihydropyrimidinase; N-Carbamoyl-fl-alanine amidohydrolase; Reductive catabolism

1. SUMMARY 2. INTRODUCTION

Pyrimidine catabolism in Pseudomonas aeru-
ginosa was investigated. It was found that the
pyrimidine bases uracil and thymidine as well as
their respective reductive catabolic products could
be utilized as sole sources of nitrogen. Reductive
degradation of the pyrimidine bases was noted.
The reductive catabolic pathway enzymes dihy-
dropyrimidine dehydrogenase, dihydropyri-
midinase and N-carbamoyl-fi-alanine amido-
hydrolase were all detected in minimal medium
grown cells. Induction of pyrimidine catabolism
by uracil was observed in this pseudomonad.
Pyrimidine degradation in P. aeruginosa was not
subject to catabolite repression.
Correspondence to: T.P. West, Olson Biochemistry Laborato-
ries, South Dakota State University, Box 2170, Brookings, SD
57007, U.S.A.
There are two known pathways of pyrimidine
catabolism in bacteria [1]. These pathways involve
either reductive or oxidative catabolism of
pyrimidine bases. The reductive pathway, which
appears prevalent in bacteria, involves 3 en-
zymatic steps where uracil or thymine is ultimately
converted to fi-alanine or/3-aminoisobutyric acid,
respectively, as well as carbon dioxide and am-
monia [1]. In the oxidative pathway, uracil is
degraded to urea and malonic acid while thymine
is degraded to 5-methylbarbituric acid [1]. Few
studies have examined pyrimidine base catabolism
by the aerobic pseudomonads. Much of the previ-
ous work examining such catabolism concerned
Pseudomonas facilis; it was found that reductive
degradation of uracil was involved [2]. Moreover,
this pathway was shown to be inducible in P.
facilis [3].
With respect to Pseudomonas aeruginosa, thin-
layer chromatographic analysis revealed that uracil
or thymine, present in culture medium as a sole
176
source of nitrogen, was converted by the pseudo-
monad to /3-alanine and /3-aminoisobutyric acid,
respectively [4]. The isolation of P. aeruginosa
strains unable to utilize uracil as a nitrogen source
but capable of using/3-alanine as a nitrogen source
has been reported but the strains were not char-
acterized further [5]. In this study, the catabolism
of the pyrimidine bases uracil and thymine by P.
aeruginosa was investigated.
3. MATERIALS A N D M E T H O D S
P. aeruginosa PAO1 was the strain used in this
report [6]. It was grown in a previously described
minimal medium [7]. The nitrogen source uracil,
dihydrouracil, N-carbamoyl-/~-alanine, fl-alanine,
thymine, dihydrothymine or fl-aminoisobutyric
acid was included i n the medium at a concentra-
tion of 0.2%. The carbon source glucose or suc-
cinate was present in the medium at a concentra-
tion of 0.4%.
Liquid medium cultures were used to investi-
gate the ability of the strain to utilize the pyrimi-
dine bases and their catabolic products. Ap-
proximately 10 9 cells were inoculated into the
appropriate sole nitrogen source medium. The in-
oculated medium (5 ml) was aerated at 200 rpm
for 96 h at 37 o C. Growth was measured spectro-
photometrically at 600 nm. Bacterial cell con-
centration (colonies/M) was estimated from a
previously determined A600 vs cell concentration
calibration curve.
For enzyme assays, bacterial cultures (250 ml)
were grown at 37 ° C on a rotary shaker (200 rpm)
until late exponential phase and then were
harvested. When determining if the increase in
pyrimidine catabolic enzyme activities observed
after growth on uracil was dependent upon pro-
tein synthesis, strain PAO was initially grown in
500 ml minimal medium containing 0.4% am-
monium sulfate until mid-exponential phase. The
ceils were collected, washed and resuspended into
a glucose minimal medium containing uracil as its
sole nitrogen source. This culture was divided into
two separate cultures of equal volume. To only
one of these cultures was added 0.1 m g / m l chlor-
amphenicol. Both cultures were grown at 3 7 ° C
for 2 h and then harvested. Harvested cells were
washed With water. Each pellet was resuspended
in 4 ml of 20 m M Tris-HC1 buffer (pH 7.5)
containing 1 m M E D T A and 1 m M 2-
mercaptoethanol. After the cells were sonically
disrupted in ice for a total of 5 rain, the extract
was centrifuged at 10900 × g for 30 min at 4°C.
Following overnight dialysis against 1 liter of soni-
cation buffer, the cell-free extract was immediately
assayed.
Enzyme assays were performed at 37 o C. Dihy-
dropyrimidine dehydrogenase activity was mea-
sured using an assay mix (1 ml) that contained 0.1
M Tris-HC1 buffer (pH 7.5), 0.2 mM N A D H , 1
m M uracil and cell-free extract [8]. The oxidation
of N A D H to N A D was followed at 340 nm over a
10 rain period. Specific activity was expressed as
nmol dihydrouracil f o r m e d / m i n per mg protein.
Dihydropyrimidinase activity was assayed over a
30 min period using a mix (1 ml) that contained
0.1 M Tris-HC1 buffer (pH 7.5), 10 mM MgC12,
0.1 mM dihydrouracil and cell-free extract. En-
zyme activity was determined with a colorimetric
assay at 466 nm for N-carbamoyl-fi-alanine [9]
using an experimentally calculated absorption
coefficient. Specific activity was expressed as nmol
N-carbamoyl-fi-alanine p r o d u c e d / m i n / m g pro-
tein. N-Carbamoyl-fl-alanine amidohydrolase was
assayed over a 20 min period using a mix (1 ml)
that contained 0.1 M Tris-HC1 buffer (pH 7.5), 10
m M MgC-12, 0.1 m M N-carbamoyl-fl-alanine and
cell-free extract. The ureido group determination
method I of Prescott and Jones [101 was used to
measure the disappearance of N-carbamoyl-/3-
alanine at 466 nm. An experimentally derived
absorption coefficient was determined to calculate
activity. Specific activity was expressed as nmol
N-carbamoyl-/3-alanine utilized/min per mg pro-
tein.
Protein was measured by the Bradford method
[11] with lysozyme as a standard. Specific activity
of each enzyme was the average of 2 separate
determinations that were reproducible within
+10%.
 177

4. RESULTS AND DISCUSSION Table 1

As can be seen in Table 1, strain PAO1 is
capable of utilizing both pyrimidine bases and the
intermediates of their reductive catabolism as
nitrogen sources. Uracil is utilized far more read-
ily as a sole nitrogen source than thymine by P.
aeruginosa.
The three enzymes associated with the reduc-
tive catabolic pathway, namely dihydropyrimidine
dehydrogenase, dihydropyrimidinase and N-
carbamoyl-B-alanine amidohydrolase, were as-
sayed. They were detected in cell-free extracts of
strain PAO1 (Table 2). Similarly, it has been re-
ported that P. facilis degraded pyrimidine bases
utilizing the reductive pathway [2]. The three re-
ductive pathway enzyme activities were present in
extracts from cytosine-grown cells of P. facilis. In
addition, the reductive pathway of pyrimidine
catabolism may be present in Pseudomonas putida
since it contains dihydropyrimidinase activity [12].
In pseudomonads, it is known that catabolite
repression of enzyme synthesis occurs after growth
on succinate [13]. To learn if catabolite repression
was a factor in P. aeruginosa pyrimidine catabo-
lism, the catabolic enzyme activities were de-
termined in extracts prepared from glucose-grown
cells relative to succinate-grown cells (Table 2). It
appears that catabolite repression does not regu-
late this pathway. Considering that this catabolic
Growth of Pseudomonas aeruginosa on pyrimidines and their
reductive catabolic products
Cells were grown for 96 h at 3 7 ° C in a glucose minimal
m e d i u m with a 0.2% ( w / v ) concentration of each nitrogen
source. Results represent the average of 2 separate determina-
tions that were reproducible within _+10%. After 96 h at 37 ° C,
control cultures of minimal m e d i u m or a glucose minimal
lacking a nitrogen source contained 8.81x109 cells/mi or
4.75 × 10 v cells/ml, respectively.
Nitrogen source Bacterial cells/ml ( x 1 0 - 9)
Uracil 13
Dihydrouracil 4.7
N-Carbamoyl-fl-alanine 9.0
fl-Alanine t4
Thymine 3.3
Dihydrothymine : 3.9
fl-Aminoisobutyfic acid 13
pathway provides nitrogen to the cell, the lack of
catabolite repression would seem to be logical.
Strain PAO1 was also grown on both pyrimi-
dine and dihydropyrimidine bases as sole nitrogen
sources. Only uracil, when included as a sole
nitrogen source (generation time 190 min), ap-
peared to increase all three enzyme activities sig-
nificantly (Table 2). Growth of this strain on
thymine as a nitrogen source (generation time 430
r n i n ) increased the activities of dihydropyri-
midinase and N-carbamoyl-fl-alanine amido-

Table 2
Effect of growth medium upon pyrimidine catabolic enzyme activities in P. aeruginosa
Nitrogen and carbon sources were included in the growth medium as indicated in the text. Cell-free extracts, prepared from cells
harvested in exponential phase, were assayed for the pyrimidine catabolic enzyme activities as described in the MATERIALS AND
METHODS,

Nitrogen Carbon Enzyme specific activity

source source ( n m o l / m i n per mg protein)
Dihydropyrimidine Dihydro- N-Carbamoyl-fl-alanine
dehydrogenase pyrimidinase amidohydrolase
A m m o n i u m sulfate Succinate 0.34 0.72 0.61
A m m o n i u m sulfate Glucose 0.56 0.73 0.37
Uracil Glucose 1.7 2.0 3.9
Thymine Glucose 0.46 1.1 0.46
Dihydrouracil Glucose 0.36 0.51 0.15
Dihydrothymine Glucose 0.34 0.73 0.46
178
hydrolase slightly but decreased dehydrogenase
activity (Table 2). This result would seem to corre-
late well with the greater ability of this strain to
utilize uracil relative to thymine as a nitrogen
source. After P. aeruginosa growth on either dihy-
drouracil (generation time 150 min) or dihydro-
thymine (generation time 225 rain) as a sole
nitrogen source, the pathway enzyme activities, in
general, decreased (Table 2). At least with respect
to dihydropyrimidinase, this agrees with the find-
ing in P. putida that this enzyme failed to be
induced after growth on dihydrouracil [12].
It was concluded that a metabolite of uracil
may have a role in the regulation of pyrimidine
catabolism at the level of gene expression. To test
this hypothesis, an experiment was performed to
learn whether the increase in pyrimidine catabolic
pathway enzyme activities was dependent upon
protein synthesis. Initially, the cells were grown in
a medium containing ammonium sulfate as its sole
nitrogen source and collected in mid-exponential
phase. The cells were washed and resuspended in
medium containing uracil as its sole nitrogen
source. After dividing this culture evenly with
respect to volume, the prokaryotic protein synthe-
sis inhibitor chloramphenicol was added to only
one culture. After 2 h of rotary shaking, the cells
were analyzed for their catabolic enzyme activities
(Table 3). The presence of protein synthesis would
seem to be necessary to observe increased
Table 3
Regulation of pyrimidine catabolic enzyme synthesis in P.
aeruginosa
Cultures were grown in minimal medium, resuspended in a
uracil-containing medium and were either treated with (+ Cm)
or without ( - Cm) chloramphenicol as stated in the ~ATER~ALS
ANDMETHODS.The resultant cell-free extracts were assayed for
the pyrimidine catabolic enzyme activities.
Enzyme Specific activity
(nmol/min per mg protein)
+ Cm - Cm
Dihydropyrimidine
dehydrogenase 0.24 0.61
Dihydropyrimidinase 0.98 2.6
N-Carbamoyl-fl-alanine
amidohydrolase 1.3 3.2
pyrimidine catabolic enzyme activities. From the
data, it appeared that dehydrogenase synthesis
responded to growth upon uracil more slowly than
the syntheses of the other two enzymes. It was
found that this enzyme increased to a specific
activity of 1.45 nmol dihydrouracil formed/rain
per mg protein after 4 h of growth on uracil as a
sole nitrogen source in the absence of chlor-
amphenicol. It is also unknown why the amido-
hydrolase level increased in the presence of chlor-
amphenicol compared to its level in cells grown on
a glucose medium containing the nitrogen source
ammonium sulfate (Table 2). In any case, induc-
tion of pyrimidine catabolism by uracil in P.
aerugmosa would seem to be present. In P. facilis.
the three reductive pathway enzymes are known to
be induced in the presence of uracil or thymine
indicating that species differences do exist with
respect to regulation of this pathway [31. Further
studies of pseudomonad pyrimidine catabolism
will be necessary to learn if any species have
common regulatory mechanisms of pathway gene
expression.
ACKNOWLEDGEMENTS
Published with the approval of the Director of
the South Dakota Agricultural Experiment Station
as Publication N u m b e r 2519 of the Journal Series.
We wish to thank M.S. Shanley and L.N. Ornston
for supplying the strain used in this study, The
expert technical assistance of Beth Hamer was
greatly appreciated.
REFERENCES
[1] Vogels, G.D. and van der Drift, C. (1976) Bacteriol. Rev.
40, 403-468.
[2] Kramer, J. and Kaltwasser, H. (1969) Arch. Mikrobiol. 68,
227-235.
[3] Kramer, J. and Kaltwasser, H. (1969) Arch. Mikrobiol. 69,
138-148.
[4] Fink, R.M., Cline, R.E. and Koch, H.M.G. (1954) Fed.
Proc. 13, 207-208.
[5] Matsumoto, H., Nakazawa, T., Ohta, S. and Terawaki, Y.
(1981) Genet. Res. Camb. 38, 251-266.
[6] Holloway,B.W. (1955) J. Gen. Microbiol. 13, 572-580.
[7] West, T.P. (1989) Arch. Microbiol. 151,220-222.

[8] Hunninghake, D. and Grisolia, S. (1965) Methods En-
zymol. 12A, 50-59.
[9] West, T.P., Shanley, M.S. and O'Donovan, G.A. (1982)
Anal. Biochem. 122, 345-347.
[10] Prescott, L.M. and Jones, M.E. (1969) Anal. Biochem. 32,
408-419.
179
[11] Bradford, M.M. (1976) Anal. Biochem. 72, 248-254.
[12] Morin, A., Hummel, W. and Kula, M.-R. (1986) Appl.
Microbiol. Biotechnol. 25, 91-96.
[13] Ornston, L.N. (1969) J. Biol. Chem. 241, 3800-3810.