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


Growth of Pseudomonas aeruginosa on pyrimidines and their
As can be seen in Table 1, strain PAO1 is reductive catabolic products
capable of utilizing both pyrimidine bases and the Cells were grown for 96 h at 3 7 ° C in a glucose minimal
intermediates of their reductive catabolism as 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-
nitrogen sources. Uracil is utilized far more read-
tions that were reproducible within _+10%. After 96 h at 37 ° C,
ily as a sole nitrogen source than thymine by P. control cultures of minimal m e d i u m or a glucose minimal
aeruginosa. lacking a nitrogen source contained 8.81x109 cells/mi or
The three enzymes associated with the reduc- 4.75 × 10 v cells/ml, respectively.
tive catabolic pathway, namely dihydropyrimidine Nitrogen source Bacterial cells/ml ( x 1 0 - 9)
dehydrogenase, dihydropyrimidinase and N-
Uracil 13
carbamoyl-B-alanine amidohydrolase, were as- Dihydrouracil 4.7
sayed. They were detected in cell-free extracts of N-Carbamoyl-fl-alanine 9.0
strain PAO1 (Table 2). Similarly, it has been re- fl-Alanine t4
ported that P. facilis degraded pyrimidine bases Thymine 3.3
Dihydrothymine : 3.9
utilizing the reductive pathway [2]. The three re-
fl-Aminoisobutyfic acid 13
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 pathway provides nitrogen to the cell, the lack of
since it contains dihydropyrimidinase activity [12]. catabolite repression would seem to be logical.
In pseudomonads, it is known that catabolite Strain PAO1 was also grown on both pyrimi-
repression of enzyme synthesis occurs after growth dine and dihydropyrimidine bases as sole nitrogen
on succinate [13]. To learn if catabolite repression sources. Only uracil, when included as a sole
was a factor in P. aeruginosa pyrimidine catabo- nitrogen source (generation time 190 min), ap-
lism, the catabolic enzyme activities were de- peared to increase all three enzyme activities sig-
termined in extracts prepared from glucose-grown nificantly (Table 2). Growth of this strain on
cells relative to succinate-grown cells (Table 2). It thymine as a nitrogen source (generation time 430
appears that catabolite repression does not regu- r n i n ) increased the activities of dihydropyri-
late this pathway. Considering that this catabolic 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
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hydrolase slightly but decreased dehydrogenase pyrimidine catabolic enzyme activities. From the
activity (Table 2). This result would seem to corre- data, it appeared that dehydrogenase synthesis
late well with the greater ability of this strain to responded to growth upon uracil more slowly than
utilize uracil relative to thymine as a nitrogen the syntheses of the other two enzymes. It was
source. After P. aeruginosa growth on either dihy- found that this enzyme increased to a specific
drouracil (generation time 150 min) or dihydro- activity of 1.45 nmol dihydrouracil formed/rain
thymine (generation time 225 rain) as a sole per mg protein after 4 h of growth on uracil as a
nitrogen source, the pathway enzyme activities, in sole nitrogen source in the absence of chlor-
general, decreased (Table 2). At least with respect amphenicol. It is also unknown why the amido-
to dihydropyrimidinase, this agrees with the find- hydrolase level increased in the presence of chlor-
ing in P. putida that this enzyme failed to be amphenicol compared to its level in cells grown on
induced after growth on dihydrouracil [12]. a glucose medium containing the nitrogen source
It was concluded that a metabolite of uracil ammonium sulfate (Table 2). In any case, induc-
may have a role in the regulation of pyrimidine tion of pyrimidine catabolism by uracil in P.
catabolism at the level of gene expression. To test aerugmosa would seem to be present. In P. facilis.
this hypothesis, an experiment was performed to the three reductive pathway enzymes are known to
learn whether the increase in pyrimidine catabolic be induced in the presence of uracil or thymine
pathway enzyme activities was dependent upon indicating that species differences do exist with
protein synthesis. Initially, the cells were grown in respect to regulation of this pathway [31. Further
a medium containing ammonium sulfate as its sole studies of pseudomonad pyrimidine catabolism
nitrogen source and collected in mid-exponential will be necessary to learn if any species have
phase. The cells were washed and resuspended in common regulatory mechanisms of pathway gene
medium containing uracil as its sole nitrogen expression.
source. After dividing this culture evenly with
respect to volume, the prokaryotic protein synthe-
sis inhibitor chloramphenicol was added to only ACKNOWLEDGEMENTS
one culture. After 2 h of rotary shaking, the cells
were analyzed for their catabolic enzyme activities Published with the approval of the Director of
(Table 3). The presence of protein synthesis would the South Dakota Agricultural Experiment Station
seem to be necessary to observe increased 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
Table 3 expert technical assistance of Beth Hamer was
Regulation of pyrimidine catabolic enzyme synthesis in P. greatly appreciated.
aeruginosa
Cultures were grown in minimal medium, resuspended in a
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