Antonie van Leeuwenhoek 80: 163–167, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
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Pyrimidine base catabolism in Pseudomonas putida biotype B Thomas P. West Olson Biochemistry Laboratories, Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA (Tel.: +1-605-688-5469; Fax: +1-605-688-6295; E-mail:
[email protected]) Accepted 5 June 2001
Key words: dihydropyrimidine dehydrogenase, dihydropyrimidinase, amidohydrolase, Pseudomonas putida, pyrimidine catabolism, nitrogen source
Abstract Reductive catabolism of the pyrimidine bases uracil and thymine was found to occur in Pseudomonas putida biotype B. The pyrimidine reductive catabolic pathway enzymes dihydropyrimidine dehydrogenase, dihydropyrimidinase and N-carbamoyl-β-alanine amidohydrolase activities were detected in this pseudomonad. The initial reductive pathway enzyme dihydropyrimidine dehydrogenase utilized NADH or NADPH as its nicotinamide cofactor. The source of nitrogen in the culture medium influenced the reductive pathway enzyme activities and, in particular, dihydropyrimidinase activity was highly affected by nitrogen source. The reductive pathway enzyme activities in succinate-grown P. putida biotype B cells were induced when uracil served as the nitrogen source. The catabolism of the pyrimidine bases uracil and thymine as a source of cellular nitrogen in bacteria occurs using two distinct pathways (Vogels & van der Drift 1976). The oxidative catabolic pathway, which exists in few bacterial species, degrades uracil or thymine to urea plus malonic acid or 5methylbarbituric acid, respectively (Vogels & van der Drift 1976; Patel & West 1987). The reductive pathway of pyrimidine catabolism, which is active in numerous bacterial species, involves three enzymatic steps (Vogels & van der Drift 1976). The initial pathway enzyme, dihydropyrimidine dehydrogenase (EC 1.3.1.2), converts uracil and thymine to dihydrouracil and dihydrothymine, respectively. The subsequent enzyme dihydropyrimidinase (EC 3.5.2.2) synthesizes N-carbamoyl-β-alanine and N-carbamoylβ-aminoisobutyric acid from dihydrouracil and dihydrothymine, respectively. The last reductive pathway enzyme N-carbamoyl-β-alanine amidohydrolase (EC 3.5.1.6) produces β-alanine or β-aminoisobutyric acid from N-carbamoyl-β-alanine or N-carbamoyl-βaminoisobutyric acid, respectively. Numerous bacteria have been shown to utilize the reductive pathway of uracil and thymine catabolism and this includes several species of the genus Pseudomonas (Vogels & van der Drift 1976; West et al. 1985;
Kim & West 1991; Xu & West 1992; West 1997, 1998). The genus Pseudomonas has undergone extensive taxonomic evaluation following ribosomal RNA phylogenetic studies. As a consequence, species of Pseudomonas have been assigned to ribosomal RNA homology group I (De Vos & De Ley 1983). Within homology group I, it was proposed to assign the species into the Pseudomonas fluorescens, Pseudomonas stutzeri or Pseudomonas alcaligenes DNA homology groups (De Vos & De Ley 1983). Pyrimidine base catabolism has been investigated in the P. stutzeri and P. alcaligenes DNA homology groups (West 1991; Xu & West 1992). In contrast, much less work has examined pyrimidine base catabolism in the P. fluorescens DNA homology group (Kim & West 1991). The microorganism Pseudomonas putida biotype B ATCC 17536, which has been assigned to the P. fluorescens DNA homology group, has not been previously investigated. In this study, the ability of P. putida to degrade pyrimidine bases as nitrogen sources was examined. By developing additional biochemical criteria to help differentiate the species within the Pseudomonas DNA homology groups, a further refinement of the current taxonomic classification of the pseudomonad species may be possible.
164 Pseudomonas putida biotype B ATCC 17536 was the strain used in this study (Stanier et al. 1966). The minimal medium was prepared as previously stated (West 1989). The nitrogen sources were included in the medium at a concentration of 0.2% (w/v). The carbon source, succinate or glucose (0.4%, w/v), was added to the medium after autoclaving. To study the ability of the strain to utilize pyrimidine bases and their catabolic products as nitrogen sources, liquid medium cultures (5 ml) were inoculated with about 106 cells and aerated at 200 rpm for 96 h at 30◦ C. Growth was monitored spectrophotometrically at 600 nm and values were used to calculate generation times. Bacterial cell concentration (cells/ml) was estimated from a previously derived A600 versus cell concentration calibration curve. To prepare cell extracts for enzyme assays, shake flask cultures (250 ml) of P. putida cells were grown on a rotary shaker (200 rpm) at 30◦ C. The cultures were harvested during the late exponential phase. When examining whether the rise in pyrimidine catabolic enzyme activities was dependent on protein synthesis, the strain was grown in 500 ml of succinate minimal medium containing (NH4 )2 SO4 and the cells were collected in mid-exponential phase. After washing the cells, they were resuspended into a succinate minimal medium containing uracil as its sole nitrogen source. This suspension was divided into two cultures of equal volume (250 ml). Chloramphenicol (0.1 mg/ml) was added to only one of these cultures and both cultures were grown for 300 min at 30◦ C. The cells were collected by centrifugation at 10 400 × g at 4◦ C for 20 min, washed and suspended in 4 ml of 20mM Tris– HCl buffer (pH 7.5) containing 1 mM EDTA and 1 mM 2-mercaptoethanol. The suspended cells were sonically disrupted at maximum power at 4◦ C using 30-s bursts for a total of 5 min. The extract was centrifuged at 10 900 × g for 30 min at 4◦ C. The cell-free extract was dialyzed overnight at 4◦ C versus 11 of resuspension buffer. Dihydropyrimidine dehydrogenase and dihydropyrimidinase activities were assayed at 30◦C as previously described (West 1998). The activity of Ncarbamoyl-β-alanine amidohydrolase was assayed at 30◦C by monitoring the release of NH+ 4 during the reaction. The assay mixture (1 ml) consisted of 0.1 M Tris-HCl buffer (pH 7.5), 10 mM MgCl2 , 1 mM Ncarbamoyl-β-alanine and cell extract. Activity was determined over a period of 30 min and the reaction was stopped by the addition of 50% (w/v) trichloroacetic acid (0.1 ml). After removing the precipitate by
centrifugation, the supernatant was assayed for NH+ 4 using glutamate dehydrogenase (Tamaki & Mizutani 1987). Pyridine nucleotide transhydrogenase activity was assayed using a mixture (1 ml) containing 50 mM Tris-HCl buffer (pH 7.5), 0.1 mM NADPH, 0.1 mM thionicotinamide adenine dinucleotide (tNAD+ ) and cell extract by monitoring the formation of tNADH from tNAD+ at 400 nm using a molar absorptivity of 11.3×103 M−1 cm−1 (French et al. 1997). Protein was measured by the method of Bradford (1976) where lysozyme served as the standard protein. Enzyme specific activity was stated as nmol product formed per min (mU) per mg protein at 30◦ C. All values represented the mean of three independent determinations where three separate extracts were assayed. The catabolism of uracil or thymine to NH3 and β-alanine or β-aminoisobutyric acid, respectively, involves a three-step pathway (Vogels & van der Drift 1976). The reductive products of pyrimidine catabolism were found to support the growth of P. putida biotype B as nitrogen sources. After 96 h at 30◦C, the strain utilized uracil (13.3×107 cells/ml), dihydrouracil (10.1×107 cells/ml), β-alanine (8.2×107 cells/ml), dihydrothymine (11.6×107 cells/ml) and β-aminoisobutyric acid (7.5×107 cells/ml) as nitrogen sources more effectively than it used thymine (2.7×107 cells/ml) as a nitrogen source to sustain its growth after 96 h at 30◦C. Control cultures of the strain grown for 96 h at 30◦ C in succinate minimal medium 6.6×107 cells/ml or in succinate minimal medium lacking a nitrogen source 6×106 cells/ml. The limited utilization of thymine as a nitrogen source by P. putida biotype B cells may indicate that thymine uptake by the cells of this pseudomonad occurs with a low efficiency. To confirm the presence of the reductive pathway in P. putida biotype B cells, the catabolic enzymes of the reductive pathway were assayed. The initial enzyme of the reductive pathway, dihydropyrimidine dehydrogenase, has been shown to require a nicotinamide cofactor. To identify this cofactor, extracts prepared from P. putida cells grown on succinate/uracil (generation time 687 min) or succinate/thymine (generation time 900 min) and glucose/uracil (generation time 643 min) were studied. In extracts prepared from cells grown on succinate/uracil, dehydrogenase activity was observed only when NADH (0.2 mM) served as the nicotinamide cofactor using 1 mM uracil (0.23 mU/mg protein) or thymine (0.32 mU/mg protein) as the substrate. Cells grown on succinate/thymine only exhibited dehydrogenase activity when NADH (0.2 mM) served
165 as the nicotinamide cofactor using 1 mM uracil (0.13 mU/mg protein) or thymine (0.10 mU/mg protein) as the substrate. In the succinate-grown cells that utilized uracil or thymine as the nitrogen source, dehydrogenase activity with NADPH (0.2 mM) as a cofactor was not detectable. In contrast, extracts prepared from cells grown on glucose/uracil exhibited dehydrogenase activity when 0.2 mM NADPH (0.03 mU/mg protein) or 0.2 mM NADH (0.12 mU/mg protein) served as a cofactor (1 mM uracil or thymine as the substrate). It should be noted that dehydrogenase activity was 4-fold higher on NADH than on NADPH. Similar results were observed with P. stutzeri since its dihydropyrimidine dehydrogenase also used either nicotinamide cofactor although NADH was the preferred cofactor (Xu & West 1992). A possible explanation for the ability of the dihydropyrimidine dehydrogenase activity present in the extracts from the glucose-grown cells to utilize either nicotinamide cofactor is likely related to the presence of the enzyme pyridine nucleotide transhydrogenase. This enzyme is known to be active in pseudomonads (Kaplan 1955; French et al. 1997). Interestingly, the ability of the dehydrogenase to use either cofactor appears only in the extracts prepared from the glucose/uracil-grown cells (5.70 mU/mg protein) where transhydrogenase activity was about 50% lower than that observed in the succinate/uracil-grown cells (10.74 mU/mg protein). Transhydrogenase activity (13.74 mU/mg protein) was also elevated in the succinate-grown cells utilizing thymine as a nitrogen source. It may be that the transhydrogenase activity produces a subsaturating concentration of NADPH relative to the Km of the P. putida dehydrogenase for this substrate in the succinate-grown cells. This could account for no detectable dehydrogenase activity being found in the extracts prepared from the succinate-grown cells when NADPH served as the cofactor. In contrast, the concentration of NADPH available to the dehydrogenase reaction may have increased to a saturating level in the glucose-grown cells due to the reduction in transhydrogenase activity. This could explain the dehydrogenase activity becoming detectable in the glucose-grown cells when NADPH served as the cofactor. Although NADPH serves as the nicotinamide cofactor for dihydropyrimidine dehydrogenase in a variety of species (West 1997, 1998), it is not clear whether NADH serves as the cofactor for the P. putida dehydrogenase or whether its apparent specificity for NADH results from interference by pyridine nucleotide transhydrogenase.
The effect of nitrogen source on the levels of the pyrimidine reductive catabolic pathway enzyme activities in P. putida was next investigated. With respect to the initial catabolic enzyme dihydropyrimidine dehydrogenase, low activity was observed when the strain was grown on (NH4 )2 SO4 (generation time 112 min), cytosine (generation time 355 min), dihydrouracil (generation time 245 min) or dihydrothymine (generation time 372 min) as a nitrogen source and succinate as a carbon source (Table 1). Growth of the strain on uracil or thymine as a nitrogen source and succinate as a carbon source resulted in a 4.6-fold or 2.6-fold elevation of dehydrogenase activity relative to its activity in (NH4 )2 SO4 -grown cells (Table 1). If β-alanine (generation time 118 min) or β-aminoisobutyric acid (generation time 219 min) served as the nitrogen source, dehydrogenase activity in the succinate-grown cells was enhanced by 1.8-fold compared to the activity detected in cells utilizing (NH4 )2 SO4 as a nitrogen source (Table 1). Relative to (NH4 )2 SO4 min as a nitrogen source (generation time 115 min), glucosegrown cells contained a 2.4-fold higher level of dehydrogenase activity when uracil served as the nitrogen source (Table 1). Of the three pathway enzymes, the activity of the enzyme dihydropyrimidinase was influenced to the greatest degree by variation of the nitrogen source. In the succinate-grown cells, dihydropyrimidinase activity increased by more than 89-, 59- or 44-fold when thymine, uracil or dihydrothymine, respectively, replaced (NH4 )2 SO4 as a nitrogen source (Table 1). Using glucose as a carbon source, dihydropyrimidinase activity increased 159fold after growth on uracil relative to (NH4 )2 SO4 as a nitrogen source (Table 1). Dihydropyrimidinase activity in P. putida biotype B succinate-grown cells rose by more than 19-fold when dihydrouracil served as the nitrogen source compared to (NH4 )2 SO4 as a nitrogen source (Table 1). In contrast, dihydropyrimidinase activity in P. putida strain IFO 12996 glycerol-grown cells failed to be induced after the addition of dihydrouracil to the culture medium (Ogawa et al. 1994). This may indicate that strain-specific differences exist in P. putida relative to the inducibility of dihydropyrimidinase. The third enzyme of the pathway N-carbamoyl-β-alanine amidohydrolase was found to be affected the least by the source of nitrogen in the medium. A 2.4- and 1.2-fold increase relative to the activity observed for cells grown on (NH4 )2 SO4 was seen for cells grown on succinate/β-alanine and glucose/uracil, respectively (Table 1).
166 Table 1. Effect of growth conditions on pyrimidine reductive catabolic enzyme activities in P. putida biotype B Nitrogen source
Carbon source
Dihydropyrimidine dehydrogenase (mU/mg protein)
Dihydropyrimidinase (mU/mg protein)
N-Carbamoyl-β-alanine amidohydrolase (mU/mg protein)
(NH4 )2 SO4 (NH4 )2 SO4 Uracil Uracil Cytosine Thymine Dihydrouracil Dihydrothymine β-Alanine β-Aminoisobutyric acid
Succinate Glucose Succinate Glucose Succinate Succinate Succinate Succinate Succinate Succinate
0.05±0.01 0.05±0.01 0.23±0.01 0.12±0.00 0.05±0.00 0.13±0.00 0.04±0.01 0.04±0.00 0.09±0.01 0.09±0.00
0.23±0.02 0.09±0.00 13.61±0.41 14.34±0.49 2.88±0.09 20.56±0.67 4.48±0.04 10.28±0.20 0.23±0.01 0.13±0.00
0.48±0.02 0.80±0.06 1.13±0.09 0.98±0.04 0.66±0.01 0.71±0.01 0.91±0.03 0.81±0.02 1.15±0.01 0.76±0.01
The strain was grown in succinate or glucose minimal medium containing the indicated nitrogen source at 30◦ C. Each specific activity is the mean of three separate determinations ± standard deviation. Table 2. Regulation of pyrimidine reductive catabolic enzyme synthesis in P. putida biotype B Enzyme
Specific activity (mU/mg protein) +Cm −Cm
Dihydropyrimidine dehydrogenase Dihydropyrimidinase N-Carbamoyl-β-alanine amidohydrolase
0.09 ± 0.01 0.54 ± 0.02 0.57 ± 0.05
0.18 ± 0.01 5.69 ± 0.09 0.90 ± 0.05
Cells were grown at 30◦ C in succinate minimal medium, washed, and resuspended in succinate minimal medium in which uracil served as the sole nitrogen source. The medium was either treated with chloramphenicol (+Cm) or was untreated (−Cm) for 300 min. Each activity (mU = nmol/min) is the mean of three separate determinations ± standard deviation.
The induction of the reductive pathway enzyme activities after growth of succinate-grown P. putida cells on uracil as a nitrogen source was also investigated using the prokaryotic protein synthesis inhibitor chloramphenicol. In the absence of the inhibitor, dehydrogenase, dihydropyrimidinase and amidohydrolase activities were found to increase by 2.0-, 10.5- and 1.6-fold, respectively, when compared to their activities in the chloramphenicol-treated cells (Table 2). The rise in the catabolic enzyme activities seemed to indicate that the synthesis of the reductive pathway enzymes was being induced by uracil as a nitrogen source. In species representing the three DNA homology groups (including P. aeruginosa, P. pseudoalcaligenes, P. alcaligenes or P. stutzeri), the response of the reductive pathway activities appears to be strongly influenced by nitrogen source (Kim & West 1991; West, 1991; Xu & West 1992). It is clear that differences even exist between P. aeruginosa and P. putida, which
are classified within the same homology group, since their growth on a given pyrimidine-related compound as a nitrogen source did not have a uniform effect on the reductive pathway enzyme activities. Increases in the pyrimidine reductive pathway enzyme activities after growth of P. aeruginosa or P. stutzeri on specific nitrogen sources has been previously demonstrated to be inducible (Kim & West 1991; Xu & West 1992) similar to what was witnessed in this study for P. putida enzyme synthesis. Overall, the catabolism of the pyrimidine bases uracil and thymine by P. putida was dependent upon the nitrogen source present in the growth medium. Discernible biochemical differences in pyrimidine catabolism between the DNA homology groups within the genus Pseudomonas in how they respond to nitrogen source were noted and these differences may prove useful in future taxonomic evaluation studies of pseudomonad species.
167 Acknowledgements This study was funded by the South Dakota Agricultural Experiment Station. The expert technical assistance of Beth Strohfus was greatly appreciated.
References Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of dye-binding. Anal. Biochem. 72: 248–254 De Vos P & De Ley J (1983) Intra- and intergeneric similarities of Pseudomonas and Xanthomonas ribosomal ribonucleic acid cistrons. Int. J. Syst. Bacteriol. 13: 487–509 French CE, Boonstra B, Bufton KA & Bruce NC (1997) Cloning, sequence, and properties of the soluble pyridine nucleotide transhydrogenase from Pseudomonas fluorescens. J. Bacteriol. 179: 2761–2765 Kaplan NO (1955) Pyridine nucleotide transhydrogenase. Methods Enzymol. 2: 681–687 Kim S & West TP (1991) Pyrimidine catabolism in Pseudomonas aeruginosa. FEMS Microbiol. Lett. 77: 175–179 Ogawa J, Kaimura T, Yamada H & Shimizu S (1994) Evaluation of pyrimidine- and hydantoin- degrading enzyme activities in aerobic bacteria. FEMS Microbiol. Lett. 122: 55–60
Patel BN & West TP (1987) Oxidative catabolism of uracil by Enterobacter aerogenes. FEMS Microbiol. Lett. 40: 33–36 Stanier RY, Palleroni NO & Doudoroff M (1966) The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 42: 159– 271 Tamaki N & Mizutani N (1987) Purification and properties of βureidopropionase from the rat liver. Eur. J. Biochem. 169: 21–26 Vogels GD & van der Drift C (1976) Degradation of purines and pyrimidines by microorganisms. Bacteriol. Rev. 40: 403–468 West TP (1989) Isolation and characterization of thymidylate synthetase mutants of Xanthomonas maltophila. Arch. Microbiol. 151: 220–222 West TP (1991) Pyrimidine base and ribonucleoside utilization by the Pseudomonas alcaligenes group. Antonie van Leeuwenhoek 59: 263–268 West TP (1997) Reductive catabolism of uracil and thymine by Burkholderia cepacia. Arch. Microbiol. 168: 237–239 West TP (1998) Isolation and characterization of an Escherichia coli B mutant strain defective in uracil catabolism. Can. J. Microbiol. 44: 1106–1109 West TP, Traut TW, Shanley MS & O’Donovan GA (1985) A Salmonella typhimurium strain defective in uracil catabolism and β-alanine synthesis. J. Gen. Microbiol. 131: 1083–1090 Xu G & West TP (1992) Reductive catabolism of pyrimidine bases by Pseudomonas stutzeri. J. Gen. Microbiol. 138: 2459–2463
