Regulation of pyrimidine formation in Pseudomonas oryzihabitans

440

Journal of Basic Microbiology 2007, 47, 440–443

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Short Communication

Regulation of pyrimidine formation
in Pseudomonas oryzihabitans

Thomas P. West

Department of Biology and Microbiology, South Dakota State University, Brookings, SD 57007, USA

The regulation of pyrimidine formation in the opportunistic human pathogen Pseudomonas
oryzihabitans was investigated at the level of enzyme synthesis and at the level of activity for the
pyrimidine biosynthetic pathway enzyme aspartate transcarbamoylase. Although pyrimidine
supplementation of succinate-grown P. oryzihabitans cells produced little effect on the de novo
pyrimidine biosynthetic pathway enzyme activities, pyrimidine limitation experiments
undertaken using an orotidine 5

′-monophosphate decarboxylase mutant strain isolated from

P. oryzihabitans ATCC 43272 indicated that repression of enzyme synthesis by pyrimidines was
occurring. Following pyrimidine limitation of the succinate-grown decarboxylase mutant
strain cells, aspartate transcarbamoylase and dihydroorotase activities were found to increase
by about 3-fold while dihydroorotate dehydrogenase and orotate phosphoribosyltransferase
activities were also observed to increase relative to their activities in the mutant strain cells
grown on excess uracil. At the level of enzyme activity, aspartate transcarbamoylase in
P. oryzihabitans was strongly inhibited by pyrophosphate, ADP, ATP and GTP in the presence of
saturating substrate concentrations.

Keywords: Pyrimidine / Biosynthesis / Regulation / Aspartate transcarbamoylase / Pseudomonas

Received: February 02, 2007; returned for modification: February 19, 2007; accepted March 03, 2007

DOI 10.1002/jobm.200710333

Introduction

Despite the bacterium Pseudomonas  oryhabitans being
considered an opportunistic human pathogen (Freney
et al. 1988, Bendig et al. 1989, Marin et al. 2000), there is
a paucity of knowledge regarding its nucleic acid meta-
bolism. With the increasing clinical significance of
P. oryzihabitans in hospital infections, a better under-
standing of its nucleic acid metabolism would seem
to be necessary to learn how it might be controlled
biologically. With pyrimidine biosynthesis being an
important component of nucleic acid metabolism, in-
vestigating the regulation of pyrimidine biosynthesis in
P. oryzihabitans should provide new insights into how
its nucleic acid metabolism is controlled. The de  novo
pyrimidine biosynthetic pathway, consisting of five
enzymes, culminates with the production of UMP.
The five pathway enzymes include aspartate trans-

Correspondence: Dr. T.P. West, Department of Biology and Micro-
biology, South Dakota State University, Box 2104, Brookings, SD 57007,
USA
E-mail: Thomas.West@sdstate.edu

carbamoylase (EC 2.1.3.2), dihydroorotase (EC 3.5.2.3),
dihydroorotate dehydrogenase (EC

1.3.3.1), orotate

phosphoribosyltransferase (EC

2.4.2.10) and orotidine

′-monophosphate (OMP) decarboxylase (EC 4.1.1.23).

Each of the enzymes are encoded by the genes pyrB,
pyrC, pyrD, pyrE and pyrF, respectively (O’Donovan and
Neuhard 1970). Feedback inhibition of the initial en-
zyme aspartate transcarbamoylase has been demonstrat-
ed in pseudomonads (Adair and Jones 1972, Vickrey
et al. 2002). Previous studies have explored pyrimidine
biosynthesis in other known opportunistic pathogenic
species of Pseudomonas (Isaac and Holloway 1968, Chu
and West 1990, West 1997, Santiago and West 2002,
2003) but not in the emerging human pathogen

P. oryzihabitans. Taxonomically, it has been shown that
P. oryzihabitans is closely related to the human pathogen
Pseudomonas aeruginosa (Anzai et al. 1997). In this work,
the regulation of pyrimidine biosynthesis by pyrimidi-
nes at the level of enzyme synthesis in P. oryzihabitans
ATCC 43272 was investigated. Moreover, the regulation
of the enzyme aspartate transcarbamoylase was studied
to learn whether its activity was also controlled.

Journal of Basic Microbiology 2007, 47, 440–443

Regulation of pyrimidine biosynthesis

441

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Materials and methods

Pseudomonas oryzihabitans ATCC 42372 (Kodama et al.
1985) and the mutant strain PT117 were used in this
study. The OMP decarboxylase mutant strain of P. oryzi-
habitans ATCC 42372, designated PT 117, was isolated by
ethylmethane sulfonate mutagenesis and resistance to
5-fluoroorotic acid as reported previously (Santiago and
West 2002). The strains were grown in a modified
minimal medium as has been described previously
(West 1989). Succinate (0.4%) was used as the carbon
source. Batch cultures (25 ml) were inoculated in sterile
125 ml Erlenmeyer flasks using overnight cultures.
When a pyrimidine base was supplemented, a concen-
tration of 50 mg l

–1

was utilized. All cultures were

shaken (200 rpm) at 30 °C. Growth was followed spec-
trophotometrically at 600 nm.
To prepare the P. oryzihabitans cell extracts used
to assay the de novo pyrimidine biosynthetic pathway
enzyme activities, three separate 25

ml cultures

were used. The cells were collected by centrifugation
during late exponential phase and washed. During the
pyrimidine limitation experiments, strain PT 117 was
grown in succinate minimal medium containing
50 mg l

–1

uracil to the late exponential phase of growth.

After collecting the cells, they were washed and resus-
pended in succinate minimal medium. After 1 or 2 h
of pyrimidine limitation at 30

°C, the cells were

collected by centrifugation and washed. The wash-
ed cells were resuspended in 2.5 ml of 20 mM Tris-HCl
buffer (pH 8.0) containing 1 mM 2-mercaptoethanol.
Each cell suspension was subjected to ultrasonic

disruption for a total of 4 min (30 s bursts) in ice and
the cell extracts were centrifuged at 1,930

× g

for 15 min at 4 °C. Following dialysis of each extract
for 18

h against resuspension buffer (300

ml) at

4 °C,  the  de novo pathway enzyme activities were deter-
mined. Aspartate transcarbamoylase activity was as-
sayed at 30 °C using a reaction mix (1 ml) that con-
tained 0.1 M Tris-HCl buffer (pH 8.5), 10 mM L-aspartate
(pH 8.5), 1 mM dilithium carbamoylphosphate and cell
extract (West 1994). An effector concentration of 5 mM
was present in the reaction mix. The concentration
of carbamoylaspartate was measured according to
method I of Prescott and Jones (1969). The activities
of dihydroorotase, dihydroorotate dehydrogenase, oro-
tate phosphoribosyltransferase and OMP decarboxy-
lase were assayed at 30 °C as previously described
(West 1997). Protein was determined by the method of
Bradford (1976) using lysozyme as the standard. Specific
activity was expressed as nmol · min

–1

· (mg protein)

–1

30 °C.

Results and discussion

It was determined that the five de novo pyrimidine bio-
synthetic pathway enzyme activities were detectable in
the P. oryzihabitans ATCC 43272 cells (Table 1). The effect
of supplementing pyrimidine bases to the succinate-
grown wild type strain cells (generation time 240 min)
on its pathway enzyme activities was investigated. The
addition of orotic acid (generation time 234 min) or
uracil to the medium (generation time 246 min) re-
sulted in a slight increase in transcarbamoylase activity
relative to the activity in the unsupplemented cells
(Table 1). Orotic acid inclusion in the medium elevated
dihydroorotase activity compared to its activity in cells
grown with no supplement (Table 1). Dihydroorotase
activity was decreased slightly after uracil addition
relative to its activity in cells grown in the absence of a
pyrimidine base (Table 1). Orotic acid or uracil supple-
mentation of the medium increased dehydrogenase
activity by at least 1.5-fold compared to its activity in
the cells grown in unsupplemented medium (Table 1).
Addition of either pyrimidine base to the culture me-
dium resulted in a slight increase in orotate phosphori-
bosyltransferase activity (Table 1). In the orotic acid-
grown cells of the P. oryzihabitans wild-type strain, OMP
decarboxylase activity was depressed by more than two-
fold relative to its activity in unsupplemented cells
(Table 1). In contrast, the decarboxylase activity in the
uracil-grown cells of P. oryzihabitans was elevated slightly
compared to its activity in the unsupplemented medium.
An OMP decarboxylase mutant strain of P. oryzihabi-
tans, designated PT

117, was isolated by chemical

mutagenesis and resistance to 5-fluoroorotic acid
(Santiago and West 2002). Decarboxylase activity in the
mutant strain grown in uracil-containing medium
(generation time 259 min) was not detectable (Table 1).
The mutant strain was found to utilize uracil, cytosine,
uridine or cytidine as a pyrimidine source. With

the isolation of the mutant strain, it was possible to
undertake pyrimidine limitation experiments to

learn whether the pyrimidine biosynthetic pathway in
P. oryzihabitans is controlled at the level of enzyme syn-
thesis. A prior investigation has shown that the depres-
sion of the pyrimidine nucleotide pools caused by
pyrimidine limitation of auxotrophic strains can pro-
duce pyrimidine pathway enzyme derepression in bac-
teria (West et al. 1983). After pyrimidine limitation of
strain PT 117 cells for 1 h, aspartate transcarbamoylase
activity increased by 2.8-fold compared to its activity in
uracil-grown cells (Table 1). After 2 h of pyrimidine
limitation, transcarbamoylase activity rose by 3.2-fold
compared to the activity of the cells grown on saturat-

442 T.

West

Journal of Basic Microbiology 2007, 47, 440 – 443

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Table 1. Effect of pyrimidine supplementation and pyrimidine limitation on de novo pyrimidine biosynthetic enzyme activities in
P. oryzihabitans strains.

Enzyme

Specific activity of strain

ATCC

43272

117

addition

Orotic

acid Uracil

Excess

uracil

Limiting

uracil

(1 h)

Limiting uracil
(2 h)

Aspartate transcarbamoylase

50.0 (1.1)

67.5 (1.0)

61.8 (0.6)

20.4 (0.7)

57.3 (0.6)

65.5 (0.5)

Dihydroorotase

46.0 (0.4)

55.8 (0.6)

43.3 (0.3)

19.9 (0.8)

63.1 (0.8)

60.7 (0.1)

Dihydroorotate dehydrogenase 1.8 (0.0)

3.5 (0.1)

2.7 (0.0)

1.9 (0.0)

1.9 (0.1)

2.4 (0.1)

Orotate
phosphoribosyltransferase

27.8 (0.3)

34.6 (0.6)

35.6 (0.4)

23.6 (0.5)

31.9 (0.8)

30.0 (0.3)

OMP decarboxylase

12.4 (0.5)

5.1 (0.1)

14.3 (0.2)

<0.7 (0.1)

<1.0 (0.3)

<0.7 (0.1)

Expressed as nmol · min

–1

· (mg protein)

–1

. Results indicate the mean of 3 separate trials and the number in parentheses

represents the standard deviation of the mean.

The strain was grown in minimal medium alone or medium containing 50 mg l

–1

orotic acid or uracil at 30 °C.

The strain was grown at 30 °C in minimal medium with excess uracil (50 mg l

–1

) or starved for uracil for 1 or 2 h under

limiting uracil growth conditions.

OMP decarboxylase, orotidine 5

′-monophosphate decarboxylase.

ing uracil levels (Table 1). In a similar fashion, dihydro-
orotase activity in the mutant cells limited for
pyrimidines for 1 or 2 h elevated by more than 3-fold
relative to its activity in the uracil-grown cells (Table 1).
Pyrimidine limitation of strain PT 117 cells for 1 h had
no effect on dehydrogenase activity while 2 h of pyrimi-
dine limitation increased its activity slightly compared
to its activity in the cells supplemented with uracil
(Table 1). Pyrimidine limitation of the mutant cells for
1 or 2 h was found to slightly increase orotate phospho-
ribosyltransferase activity relative to the cells grown
with excess uracil (Table 1). By virtue of the pyrimidine
limitation experiments, it appeared that de novo biosyn-
thetic enzyme synthesis could be derepressed which sug-
gested that repression by a pyrimidine-related compound
at the transcriptional level was occurring.
With

P. oryzihabitans being an opportunistic human

pathogen, its transcriptional regulation of the pyrimi-
dine biosynthetic pathway was compared to the human
pathogen  Pseudomonas  aeruginosa because both species
have been shown to be taxonomically-related (Anzai
et  al. 1997). Similar to the finding observed for the de
novo pyrimidine biosynthetic enzyme activities in

P.  oryzihabitans (Table 1), the pathway enzyme activities
were not repressible by a uracil-related compound in
P.  aeruginosa (Isaac and Holloway 1968). While pyrimi-
dine limitation of pyr mutant strains of P. aeruginosa did
not derepress its de novo pyrimidine biosynthetic path-
way enzyme activities (Isaac and Holloway

1968),

derepression of the de novo pyrimidine biosynthetic
pathway enzyme activities was observed in the P. oryzi-
habitans strain PT 117 cells (Table 1). More than a three-
fold derepression of aspartate transcarbamoylase and

dihydroorotase activities was noted in the pyrimidine-
limited PT 117 cells compared to the uracil-grown mu-
tant cells (Table 1). In contrast to P. aeruginosa, it ap-
peared that de novo pyrimidine biosynthetic pathway
enzyme synthesis in P. oryzihabitans was subject to regu-
lation by pyrimidines.
With

the

de novo pyrimidine biosynthetic pathway

enzyme aspartate transcarbamoylase being highly regu-
lated in pseudomonads (Adair and Jones 1972, Vickrey
et al. 2002), the control of its in vitro activity was studied
in P. oryzihabitans (Table 2). The K

(standard deviation)

of aspartate transcarbamoylase for its substrate

L-aspartate or carbamoylphosphate was determined to
be 1.18 mM (0.08) or 0.45 mM (0.08), respectively, in
P. oryzihabitans ATCC 43272 cell extracts. The K

of the

Table 2. Effect of possible effectors on aspartate transcarba-
moylase activity in Pseudomonas oryzihabitans ATCC 43272.

Effector

Specific

activity

Relative

activity

Control 50.6

(0.2)

100

Pyrophosphate

1.0

(0.1)

UDP

4.7

(0.1)

CDP 30.5

(0.8)

ADP

0.8

(0.1)

GDP 21.6

(0.2)

UTP

3.1

(0.2)

CTP

8.5 (0.4)

ATP

0.3

(0.1)

GTP

0.6

(0.2)

The concentration of each effector was 5 mM.

Expressed as nmol carbamoylaspartate formed · min

–1

· (mg

protein)

–1

at 30 °C. Results indicate the mean of 3 separate

trials and the number in parentheses represents the
standard deviation of the mean.

Expressed in %.

Journal of Basic Microbiology 2007, 47, 440–443

Regulation of pyrimidine biosynthesis

443

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P. oryzihabitans transcarbamoylase for carbamoylphos-
phate was similar to what was observed for the

P. aeruginosa aspartate transcarbamoylase while its K

for L-aspartate was much lower (Vickrey et al. 2002).
The K

of the P. aeruginosa aspartate transcarbamoylase

for its substrate L-aspartate or carbamoylphosphate was
2.6 mM or 0.49 mM, respectively (Vickrey et al. 2002)
Putative transcarbamoylase effectors were investigat-
ed under saturating substrate concentrations where
10

mM L-aspartate and 1

mM carbamoylphosphate

were present in the assay mix (Table 2). Relative to the
effectors studied (Table 2), the most potent inhibitors of
the P. oryzihabitans aspartate transcarbamoylase activity
were ATP, GTP, ADP and pyrophosphate although all
the ribonucleotide triphosphates were highly inhibi-
tory. Similar to P. oryzihabitans, the transcarbamoylase
activity of the taxonomically-related species P. aerugi-
nosa, was also inhibited by the ribonucleotide triphos-
phates (Vickrey et al. 2002).
  In conclusion, pyrimidine biosynthesis in P. oryzihabi-
tans  was repressible by a pyrimidine-related compound
at the level of enzyme synthesis and its aspartate trans-
carbamoylase activity was highly regulated by pyro-
phosphate and ribonucleotides. Although control of
pyrimidine biosynthetic enzyme synthesis existed for
P.  oryzihabitans  but not for P.  aeruginosa, both transcar-
bamoylases from these closely-related pseudomonads
exhibited a similar pattern of effector inhibition. The
findings of this study should be helpful in understand-
ing how nucleic acid metabolism in the emerging, hu-
man pathogen P. oryzihabitans is regulated.

Acknowledgements

This research work was supported by funds from the
South Dakota Agricultural Experiment Station. The
technical assistance of Beth Reed Nemmers was appre-
ciated.

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((Funded by:● South Dakota Agricultural Ex-

periment Station))