Pseudomonas cepacia ATCC 17759. The presence of the de novo pyrimidine biosynthetic pathway enzyme activi- ties was confirmed in this strain. Following ...
Arch Microbiol (1990) 154: 407 - 409
Archives of
Microbiology
9 Springer-Verlag1990
Short communications
Regulation of pyrimidine biosynthesis in Pseudomonas cepacia Thomas P. West x,2 and Chien-peng Chu 2,. 1 Department of Biological Sciences, Institute of Genetics, University of Southern Mississippi, Hattiesburg, MS 39406, USA z Department of Chemistry, South Dakota State University, Brookings, SD 57007, USA Received December 12, 1989/Accepted April 13, 1990
Abstract. Pyrimidine biosynthesis was investigated in Pseudomonas cepacia ATCC 17759. The presence of the de novo pyrimidine biosynthetic pathway enzyme activities was confirmed in this strain. Following transposon mutagenesis of the wild-type cells, a mutant strain deficient for orotidine 5'-monophosphate decarboxylase activity (pyrF) was isolated. Uracil, cytosine or uridine supported the growth of this mutant. Uracil addition to minimal medium cultures of the wild-type strain diminished the levels of the de novo pyrimidine biosynthetic enzyme activities, while pyrimidine limitation of the mutant cells increased those de novo enzyme activities measured. It was concluded that regulation of pyrimidine biosynthesis at the level of enzyme synthesis in P. cepacia was present. Aspartate transcarbamoylase activity was found to be regulated in the wild-type cells. Its activity was shown to be controlled in vitro by inorganic pyrophosphate, adenosine 5'-triphosphate and uridine 5'-phosphate.
Key words: Pyrimidine biosynthesis - Pseudomonas cepacia - Transposon mutagenesis - Aspartate transcarbamoylase
rimidine biosynthesis in Pseudomonas aeruginosa and Pseudomonas putida (Isaac and Holloway 1968; Condon et al. 1976). Although significant regulation at the level ofpyrimidine gene expression could not be substantiated, these studies provided evidence that aspartate transcarbamoylase activity was strongly influenced by various effectors including pyrimidine and purine nucleotides (Isaac and Holloway 1968; Condon et al. 1976). It has also been established that aspartate transcarbamoylase purified from Pseudomonas fluorescens is controlled by nucleotides (Neumann and Jones 1964; Adair and Jones 1972). Prior studies have not investigated whether pyrimidine biosynthesis is regulated in Pseudomonas r even though it is a clinically significant microorganism among individuals with cystic fibrosis. In this work, possible regulation of de novo pyrimidine biosynthesis was examined in P. cepacia ATCC 17759. This included investigating if pyrimidine biosynthetic enzyme synthesis was regulated in vivo by pyrimidines. Control of in vitro aspartate transcarbamoylase activity in P. cepacia cells was also studied. Materials and methods
Pyrimidine biosynthesis has not been extensively investigated in pseudomonads. Aspartate transcarbamoylase (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 5'-monophosphate (OMP) decarboxylase (EC 4.1.1.23) are the enzymes unique to the de novo synthesis of the pyrimidine nucleotide UMP in bacteria (O'Donovan and Neuhard 1970). Prior studies have explored the regulation of py* Present address: Department of Microbiology and Immunology, Temple University, Philadelphia, PA 19140, USA Offprint requests to: T. P. West
Strains utilized were Pseudomonas cepacia ATCC 17759 (Stanier et al. 1966) and its derivative strain CW2001 (Tn5:pyrb). A minimal medium of Stanier (1947) was modified as previously described (West 1989). After autoclaving, glucose (22.2 mM) was added to the medium as a carbon source. For solid medium, 2% agar was added. All liquid cultures were provided with aeration (200 rpm) and incubated at 25~ The doubling times of the mutant strains in various media were derived spectrophotometrically. The turbidity at 600 nm of each culture was examined at selected time intervals. The isolation of strain CW2001 involved transposon mutagenesis of P. cepacia ATCC 17759 using the transfer of a "suicide" plasmid. The plasmid pJB4JI, which contains Tn5, was transferred from Escherichia coli to P. cepacia by bacterial conjugation (Beringer et al. 1978; Gantotti et al. 1981; Monticello et al. 1985). Transfer was accomplished by mixing exponential phase cultures of E. coli and P. cepacia (5 : 1 ratio). After the ceils were collected on 0.45 IxM membrane filters (25 ram), these filters were placed on minimal medium agar plates containing 50 mg/1 kanamycin and
408 Table 1. Effect of uracil upon the specific activities of the pyrimidine de novo biosynthetic pathway enzymes in ATCC 17759. Strains were grown in minimal medium at 25~ and when added, uracil was present at a concentration of 50 mg/1. Cells were harvested, sonically disrupted, centrifuged, dialyzed and the extracts assayed for enzyme activities. The results are the average of 2 separate determinations __ i0% Enzyme
Aspartate transcarbamoylase Dihydroorotase Dihydroorotate dehydrogenase Orotate phosphoribosyltransferase OMP decarboxytase
Growth conditions (additions) None
Uracil
32 45 7.6 50 12
24 32 6.1 46 9.8
uracil. After 2 4 - 48 h at 30 ~C, the cells were washed from the filters using 0.85% NaC1. Dilutions of these suspensions were made and then used to inoculate a minimal medium containing novobiocin (50 rag/t), uracil (50 mg/1), and kanamycin (50 rag/l) (Monticello et al. 1985). The bacteria were grown overnight in this medium at 30~C. Each culture of the mutagenized cells was diluted and spread onto minimal medium agar plates containing uracil (50 rag/l) and kanamycin (50 rag/l). After 48 h at 30~ the resultant colonies were screened on solid medium for both uracil auxotrophy and kanamycin resistance. Strain CW2001 was detected by this screening and was subsequently characterized. The mutant strain was tested for its ability to grow on uracil, cytosine, dihydrouracil, uridine, eytidine, UMP and CMP. Approximately 10 7 cells of the mutant strain were first spread onto minimal medium agar plates. To the center of each plate was placed a glass mierofiber filter disk (2.1 cm in diameter) saturated with a sterile solution of the test compound (1.5 mg/ml). Following 8 days at 25 ~C, all plates were examined and recorded as positive if there was confluent growth surrounding the disk. To investigate de novo pyrimidine biosynthetic pathway enzyme activities in P. cepacia, cell extracts were prepared from 25 ml cultures, Cells were collected by centrifugation during the late exponential phase of growth, washed and resuspended in 2 mI of 20 mM Tris-HC1 buffer (pH 8.0) containing 1 mM 2-mercaptoethanol (Condon et al. 1976). The suspension was subjected to ultrasonic disruption for a total of 3 min in ice. The disrupted cells were centrifuged 1930 x g for 15 min at 4 ~C. The resultant extract was dialyzed for 18 h against 50 vol. resuspension buffer at 4~ and was then assayed. For the pyrimidine starvation experiment, strain CW2001 was grown in minimal medium containing 50 rag/1 uracil and kanamycin to the late exponential phase of growth. The ceils were collected, washed and resuspended in minimal medium containing 50 rag/1 kanamycin while maintaining sterile conditions. After 2 h ofpyrimidine starvation, the cells were collected and extracts prepared as described above. All assays were performed at 25~ Aspartate transcarbamoylase was assayed using a reaction mix (1 ml) that contained 100 mM Tris-HC1 buffer (pH 8.5)~ 10raM L-aspartate pH 8.5, I mM dilithium carbamoylphosphate and cell-free extract (Adair and Jones 1972). Dihydroorotase was assayed using a modified assay mixture (l ml) that contained 100mM Tris-HC1 buffer (pH 8.5), 1 mM EDTA, 2 mM L-dihydroorotate and cell-free extract (Beckwith et al. 1962). When assaying aspartate transcarbamoylase or dihydroorotase activity, the concentration of carbamoyl aspartate was determined according to Method I of Prescott and Jones (1969). A modified assay mixture (1 ml) for dihydroorotate dehydrogenase, containing 100mM Tris-HC1 buffer (pH 8.5),
Table 2. Effect of pyrimidine limitation upon specific activities of the pyrimidine de novo biosynthetic pathway enzymes in strain CW2001. Under uracil excess growth conditions, strain CW2001 was grown at 25~ in minimal medium containing 50 mg/l uracil and 50 mg/l kanamycin. During uracil starvation growth conditions, strain CW2001 cultures containing minimal medium plus uracil (50 rag/l) and kanamycin (50 rag/l) was collected in late exponential phase, washed, resuspended in minimal medium containing kanamycin (50 rag/l) and shaken for a period of 2 h at 25 ~C. Cells were harvested, sonically disrupted, centrifuged, dialyzed and the extract assayed for enzyme activities. The results are the average of two separate determinations + 10%. ND: not determined Enzyme
Aspartate transcarbamoylase Dihydroorotase Dihydroorotate dehydrogenase Orotate phosphoribosyltransferase OMP decarboxylase
Growth conditions Uracil excess
Uracil starvation
17 27 6.6 40 < 0.56
84 105 15 80 ND
2 mM L-dihydroorotate and cell-free extract, was used (Kelln et al. 1975). The reaction was followed by observing the increase in absorbance at 290 nm using a molar absorption coefficient of 6500 M - lcm- ~. The final de novo pyrimidine pathway enzymes orotate phosphoribosyltransferase and OMP decarboxylase were assayed according to the protocols developed by Schwartz and Neuhard (1975). Protein was determined by the method of Bradford (1976) where lysozyme served as the standard protein. Specific activity was expressed as nmol substrate utilized or product formed/rain -mg protein at 25 ~C. All values represented the average of two separate determinations that differed by no more than 10%.
Results and discussion T h e existence a n d p o s s i b l e r e g u l a t i o n o f de n o v o p y r i m idine b i o s y n t h e s i s w a s i n v e s t i g a t e d in Pseudomonas cepacia. A s was o b s e r v e d f o r P. aeruginosa a n d P. putida (Isaac a n d H o l l o w a y 1968; C o n d o n et al. 1976), the de n o v o p y r i m i d i n e b i o s y n t h e t i c p a t h w a y e n z y m e activities in P. cepacia are i d e n t i c a l to t h o s e f o u n d in o t h e r b a c t e r i a (Table 1). I n this study, a n O M P d e c a r b o x y l a s e m u t a n t s t r a i n o f P. cepacia was i s o l a t e d b y t r a n s p o s o n m u t a g e n e s i s . T h e r e s u l t a n t strain isolated, C W 2 0 0 1 , was c h a r a c t e r i z e d for its a b i l i t y to g r o w u p o n v a r i o u s c o m p o u n d s . N e i t h e r d i h y d r o u r a c i l , cytidine, U M P n o r C M P were a b l e to s u p p o r t the p y r i m i d i n e r e q u i r e m e n t for s t r a i n g r o w t h . It is n o t c l e a r w h y c y t i d i n e d i d n o t s u p p o r t the g r o w t h o f this strain. In a d d i t i o n to uracil, this s t r a i n c o u l d utilize c y t o s i n e o r u r i d i n e to m e e t its a u x o t r o p h i c r e q u i r e m e n t for p y r i m i d i n e s . T h e respective d o u b l i n g time o f strain CW2001 at 25 ~ C o n each o f these p y r i m i d i n e s (50 rag/l) p r e s e n t in the glucose m i n i m a l m e d i u m was 67, 229 o r 159 rain. Previously, m u t a n t s t h a t l a c k e d either a s p a r t a t e transcarbamoylase, dihydroorotate dehydrogenase, orotate p h o s p h o r i b o s y l t r a n s f e r a s e o r O M P d e c a r b o x y l a s e a c t i v i t y were i s o l a t e d f r o m P. aeruginosa (Isaac a n d Hol-
409 loway 1968). These uracil-requiring mutants failed to grow upon intermediates of the pyrimidine pathway. Also, the isolation of uracil-requiring mutants from P. putida has been reported where only cytosine could substitute for uracil as an alternate pyrimidine source (Condon et al. 1976). In this work, possible regulation of de novo pyrimidine biosynthetic pathway enzyme synthesis by pyrimidines was explored for P. cepacia. It appeared that the de novo pyrimidine pathway activities in P. cepacia were slightly repressed by growth in a uracil-containing medium (Table 1). Repression by a uracil-related compound was most significant for aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase (Table 1). With the isolation of strain CW2001, it was possible to study the effect of pyrimidine limitation upon the de novo pyrimidine pathway enzyme activities (Table 2). The levels o f aspartate transcarbamoylase, dihydroorotase, dehydroorotate dehydrogenase and orotate phosphoribosyltransferase activities were not derepressed after 1 h of pyrimidine starvation. In contrast, pyrimidine limitation of the mutant cells for 2 h resulted in nearly a fivefold increase in aspartate transcarbamoylase activity as well as about a fourfold increase in dihydroorotase activity (Table2). Only about a twofold increase in dihydroorotate dehydrogenase or orotate phosphoribosyltransferase activity was observed (Table 2). The increase observed for each de novo pathway enzyme activity following pyrimidine limitation seemed to correlate with the degree that each was repressed by growth in a uracil-containing medium. Transcriptional regulation of pyrimidine biosynthesis would seem to be a factor in this microorganism. Regulation of de novo enzyme synthesis by pyrimidines has also been investigated in P. aeruginosa and P. putida. In P. aeruginosa, neither growth upon uracil nor conditions of limiting uracil caused any major change in the levels of aspartate transcarbamoylase, dihydroorotase, dehydroorotate dehydrogenase and orotate phosphoribosyltransferase (Isaac and Holloway 1968). Similarly, the five de novo enzymes unique to pyrimidine biosynthesis were not repressible by uracil in P. putida (Condon et al. 1976). In contrast, starvation o f P. putida uracil-requiring mutants caused the de novo pyrimidine pathway enzyme activities to nearly double indicating some form of regulation to be present. In any case, pyrimidine biosynthesis in P. cepacia would seem to be more highly regulated than in P. putida or P. aeruginosa at the level of gene expression. In most bacteria, aspartate transcarbamoylase is known to be an important regulatory enzyme in the de novo pyrimidine biosynthetic pathway (O'Donovan and Neuhard 1970). This enzyme has been investigated previously in various pseudomonads. In P. aeruginosa, this enzyme was found to be inhibited by ATP, UTP, or CTP (Isaac and Holloway 1968). In Pseudomonasfluorescens, PPi as well as pyrimidine and purine nucleotides inhibit transcarbamoylase activity (Neumann and Jones 1964; Adair and Jones 1972). Inhibition of the P. putida aspartate transcarbamoylase by PPi, ATP, U T P or CTP has been shown (Condon et al. 1976). In this investigation, in vitro regulation of aspartate transcarbamoylase
activity in the P. cepacia wild-type strain was found to occur. The wild-type strain transcarbamoylase specific activity of 33 nmol carbamoyl aspartate/min 9 protein (no effector added) was found to decrease to a specific activity of 4.6 or 22 in the presence of 5 m M PPi or ATP, respectively. Significant inhibition of its enzyme activity by U T P or CTP was not observed at the same concentration. The ribonucleotide U M P (1 raM) was shown to activate transcarbamoylase specific activity (48 nmol carbamoyl a s p a r t a t e / m i n . m g protein) approximately 1.5-fold. Its activation by a pyrimidine ribonucleotide may indicate that the P. cepacia enzyme differs from the previously studied pseudomonad transcarbamoylases. In conclusion, de novo pyrimidine biosynthesis in P. cepacia does appear to be regulated at the level of enzyme synthesis and at the level of enzyme activity for aspartate transcarbamoylase. This pseudomonad species would seem to mandate further study with respect to its regulation of pyrimidine biosynthesis.
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