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Regulation of pyrimidine nucleotide formation in Pseudomonas taetrolens ATCC 4683
ARTICLE IN PRESS Microbiological Research 159 (2004) 29–33
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Regulation of pyrimidine nucleotide formation in Pseudomonas taetrolens ATCC 4683 Thomas P. West Olson Biochemistry Laboratories, Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA Accepted 2 January 2004
KEYWORDS Pseudomonas taetrolens; Pyrimidine; Biosynthesis; Aspartate transcarbamoylase
Abstract The regulation of the de novo pyrimidine biosynthetic enzymes in the food spoilage agent Pseudomonas taetrolens ATCC 4683 was investigated. The de novo pyrimidine biosynthetic enzyme activities were determined in P. taetrolens ATCC 4683 cells and in cells from an auxotroph deficient for orotidine 50 -monophosphate decarboxylase activity. Pyrimidine supplementation to the culture medium affected the biosynthetic enzyme activities in ATCC 4683 cells. Transcriptional regulation of the biosynthetic pathway by pyrimidines was indicated after the auxotroph was subjected to pyrimidine limitation. At the level of enzyme activity, aspartate transcarbamoylase activity was strongly inhibited by pyrophosphate, ADP, ATP, UDP, UTP and GTP. Transcriptional regulation of pyrimidine synthesis in P. taetrolens was not as highly controlled as in the taxonomically-related species Pseudomonas fragi although both species contained transcarbamoylase activities subject to significant nucleotide inhibition. & 2004 Elsevier GmbH. All rights reserved.
Introduction Although Pseudomonas taetrolens is known to produce an undesirable mustiness odor in eggs and meats, the study of its pyrimidine biosynthesis has not been investigated despite the possibility that the findings could help in its biological control (Levine and Anderson, 1932; Tompkin and Shaparis, 1972; Daise et al., 1986). Moreover, the findings could prove helpful from a biochemical perspective to the ongoing taxonomic analysis of Pseudomonas species. Taxonomically, P. taetrolens has been
classified within the Pseudomonas chlororaphis group (Anzai et al., 2000). This group contains the species Pseudomonas fragi which is also a food spoilage agent (Anzai et al., 2000). Five enzymes comprise the de novo pyrimidine biosynthetic pathway that ultimately produces UMP (O’Donovan and Neuhard, 1970). The first enzyme unique to only pyrimidine biosynthesis is aspartate transcarbamoylase (EC 2.1.3.2). This enzyme synthesizes carbamoylaspartate from its substrates L-aspartate and carbamoylphosphate. The other four pathway enzymes include dihydroorotase (EC 3.5.2.3),
E-mail address: Thomas [email protected] (T.P. West). 0944-5013/$ - see front matter & 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2004.01.007
ARTICLE IN PRESS 30
dihydroorotate dehydrogenase (EC 1.3.3.1), orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 50 -monophosphate (OMP) decarboxylase (EC 4.1.1.23). The pyrimidine biosynthetic pathway appears to be regulated at the level of enzyme synthesis by pyrimidines in the P. chlororaphis group member P. fragi (West, 2002). In addition, aspartate transcarbamoylase activity in P. fragi was controlled by nucleotides (West, 2002). In this study, pyrimidine biosynthesis in P. taetrolens ATCC 4683 was investigated for regulation by pyrimidines at the level of enzyme synthesis while possible control of its aspartate transcarbamoylase activity was also explored.
Materials and methods Strain and growth conditions P. taetrolens ATCC 4683 and strain PT111 were the strains used in this study (Levine and Anderson, 1932; Stanier et al., 1966). The minimal medium was prepared as previously stated (West, 1989). The carbon source succinate (0.4%, w/v) was added to the medium after autoclaving. Batch cultures (25 ml) were inoculated in sterile 125 ml Erlenmeyer flasks using overnight cultures. When a pyrimidine base was added, its concentration was 50 mg/l. All cultures were shaken (200 rpm) at an incubation temperature of 301C. To derive generation times, growth was followed spectrophotometrically at 600 nm. For the pyrimidine limitation experiments, the pyrimidine auxotrophic strain was grown in succinate minimal medium containing 50 mg/l uracil to the late exponential phase of growth. The cells were collected, washed and resuspended in succinate minimal medium. After 2 or 4 h of pyrimidine limitation at 301C, the cells were collected and extracts prepared as stated above.
T.P. West
succinate minimal medium containing uracil (50 mg/l) and then were grown in this medium for 48 h at 301C. The cell starvation and D-cycloserine procedure was repeated. The surviving cells from this procedure were grown for 15 h at 301C in succinate minimal medium containing uracil (50 mg/ml). Dilutions of this culture were spread onto solid succinate minimal medium containing uracil (50 mg/l). Colonies present on the solid medium after 72 h of growth at 301C were screened for uracil auxotrophy and strain PT111 was identified. The ability of the mutant strain to grow upon carbamoylaspartate, dihydroorotic acid, orotic acid, uracil, dihydrouracil, cytosine, uridine, UMP, cytidine or CMP to meet its pyrimidine requirement was explored. This involved spreading approximately 107 cells of the mutant strain onto succinate minimal medium agar plates. To the center of each plate was placed a glass microfiber filter disk (2.1 cm in diameter) saturated with a sterile solution of the test compound (1.5 mg/ml). All plates were examined daily over a period of 8 days at 301C for confluent growth.
Preparation of cell extracts To prepare cell extracts for use during the enzyme assays, shake flask cultures (25 ml) of P. taetrolens cells were grown on a rotary shaker (200 rpm) at 301C. The cultures were harvested during the late exponential phase and were collected by centrifugation at 10,400g at 41C for 20 min. The cells were washed and resuspended in 2.5 ml of 20 mM Tris-HCl buffer (pH 8.0) containing 1 mM 2-mercaptoethanol. The suspension was subjected to ultrasonic disruption for a total of 4 min in ice. The disrupted cells were centrifuged at 1930g for 15 min at 41C. The resultant extract was dialyzed for 18 h against resuspension buffer (300 ml) at 41C and was then assayed.
Enzyme assays Mutant isolation The uracil auxotrophic strain of P. taetrolens ATCC 4683 was isolated using ethyl methanesulfonate mutagenesis (Watson and Holloway, 1976) and subsequent outgrowth in nutrient broth at 301C. The mutagenized cells were collected and resuspended in 0.85% NaCl. After starving the cells for 2.5 h at 301C, the cells were collected and resuspended in succinate minimal medium containing D-cycloserine (1 mg/ml) and shaken for 2 h at 301C (West, 1997). The cells were collected, washed twice with 0.85% NaCl, resuspended in
All assays were performed at 301C. Aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase, orotate phosphoribosyltransferase and OMP decarboxylase activities were assayed as previously described (West, 1997). When studying regulation of transcarbamoylase activity, an effector concentration of 5 mM was present in the assay mix. The Km values of aspartate transcarbamoylase for L-aspartate and carbamoylphosphate were calculated from Lineweaver-Burk plots. Protein was measured by the method of Bradford (1976) where lysozyme served as the
ARTICLE IN PRESS Regulation of pyrimidine nucleotide formation
31
standard protein. Enzyme specific activity was stated as nmol/min/mg protein at 301C. All values represented the mean of three independent determinations where three separate extracts were assayed.
Results and discussion The five de novo pyrimidine biosynthetic pathway enzymes were active in the succinate-grown cells (generation time 161 min) of P. taetrolens ATCC 4683 (Table 1). The effect of pyrimidine base supplementation to the growth medium of the wild type strain on its pyrimidine biosynthetic pathway enzyme activities was examined. Compared to the activity of succinate-grown cells, orotic acid supplementation to the medium (generation time 165 min) resulted in a slight decrease in transcarbamoylase activity while the addition of uracil to the medium (generation time 205 min) increased this enzyme activity by 1.2-fold (Table 1). Dihydroorotase activity rose by 1.4-fold when orotic acid was included in the medium relative to its activity in the medium alone (Table 1). In contrast, the presence of uracil in the medium depressed
dihydroorotase activity to 65% of its activity in cells grown in unsupplemented medium (Table 1). Similar to what was observed for dihydroorotase activity, the presence of orotic acid in the medium elevated dehydrogenase activity by 1.2-fold relative to its activity in cells grown in unsupplemented medium (Table 1). Dihydroorotate dehydrogenase activity was diminished by the inclusion of uracil in the minimal medium compared to its activity in cells grown without a pyrimidine base added (Table 1). Pyrimidine supplementation had little effect on orotate phosphoribosyltransferase activity in the P. taetrolens cells compared to its activity in cells grown on only the minimal medium (Table 1). Relative to OMP decarboxylase activity in the unsupplemented cells, this enzyme activity was increased by 1.2-fold in cells grown on medium containing orotic acid but uracil addition to the medium lowered its activity (Table 1). An OMP decarboxylase deficient strain of P. taetrolens was isolated by chemical mutagenesis and D-cycloserine counterselection (West, 1997). No detectable decarboxylase activity was observed in the succinate-grown strain PT111 cells (generation time 178 min) supplemented with uracil (Table 2). The mutant strain was able to grow on uracil, cytosine or uridine as a pyrimidine source.
Table 1. Effect of exogenous pyrimidines on pyrimidine biosynthetic pathway enzyme activities in P. taetrolens ATCC 4683 Enzyme
Aspartate transcarbamoylase Dihydroorotase Dihydroorotate dehydrogenase Orotate phosphoribosyltransferase OMP decarboxylase
Specific activity None
Orotic acid
Uracil
89.872.9 37.271.0 3.270.0 38.771.2 6.770.2
85.572.8 51.471.1 3.970.1 40.672.5 7.770.1
107.170.6 23.371.4 2.770.1 39.471.3 5.870.2
The strain was grown in succinate minimal medium containing the indicated pyrimidine base at 301C. Specific activity of each enzyme is expressed as nmol/min/mg protein at 301C. Results are the mean of three separate determinations7S.D.
Table 2. Effect of pyrimidine nucleotide limitation on pyrimidine biosynthetic pathway enzyme activities in P. taetrolens strain PT111 Enzyme
Aspartate transcarbamoylase Dihydroorotase Dihydroorotate dehydrogenase Orotate phosphoribosyltransferase OMP decarboxylase
Specific activity þ Uracil
Uracil (2 h)
Uracil (4 h)
116.272.6 44.471.1 4.070.2 44.671.3 o0.470.1
81.072.3 101.077.8 3.470.1 72.373.4 o0.970.3
72.672.2 85.371.4 6.770.2 61.871.1 o0.670.1
Strain PT111 was grown at 30oC in succinate minimal medium containing 50 mg/l uracil or was subjected to pyrimidine nucleotide limitation in succinate minimal medium for 2 or 4 h at 301C as described in the text. Specific activity of each enzyme is expressed as nmol/min/mg protein at 301C. Results are the mean of three separate determinations7S.D.
ARTICLE IN PRESS 32
Since P. taetrolens has been reported to contain the enzymes cytosine deaminase and nucleoside hydrolase (Sakai et al., 1968, 1976), the utilization of cytosine or uridine as a pyrimidine source to support the growth of the mutant strain is not surprising. In P. fragi, the OMP decarboxylase mutant strain grew on uracil, cytosine, uridine or cytidine when glucose served as the carbon source (West, 2002). With the mutant strain PT111 isolated, the limitation of the mutant cells for pyrimidine nucleotides was undertaken to detect possible regulation of pyrimidine biosynthetic pathway enzyme synthesis in P. taetrolens. It has been shown previously the deprivation of the pyrimidine nucleotide pools can result in the derepression of pyrimidine pathway enzyme synthesis in bacteria (West et al., 1983). In strain PT111 cells (generation time 108 min), pyrimidine limitation decreased aspartate transcarbamoylase activity by 30% or 37%, respectively, after 2 or 4 h of pyrimidine limitation relative to its activity in uracil-grown cells (Table 2). In contrast, dihydroorotase activity in strain PT111 cells limited for pyrimidines for 2 h rose by 2.3-fold compared to its activity in cells grown with excess uracil (Table 2). Following 4 h of pyrimidine-limiting conditions, dihydroorotase activity was 1.9-fold higher than its activity in uracilgrown cells (Table 2). Interestingly, pyrimidine limitation of strain PT111 cells for 2 h produced a decrease in dehydrogenase activity while this enzyme activity rose by 1.7-fold compared to its activity in cells grown with a saturating level of uracil (Table 2). The mutant cells were found to contain elevated levels of orotate phosphoribosyltransferase activity following pyrimidine limitation. When the mutant cells were deprived of uracil for 2 h, phosphoribosyltransferase increased by 1.6-fold relative to the activity in the uracilgrown cells (Table 2). Following 4 h of pyrimidine starvation, phosphoribosyltransferase activity remained 1.4-fold elevated compared to the activity detected in the cells grown under saturating uracil conditions. The pyrimidine limitation experiments showed that derepression of the synthesis of at least three of the de novo biosynthetic enzymes was occurring which indicates repression by a pyrimidine-related compound at the transcriptional level. The transcriptional regulation of the pyrimidine biosynthetic pathway in P. taetrolens ATCC 4683 can be compared to P. fragi ATCC 4973 since it is the only species of the P. chlororaphis group whose regulation of pyrimidine biosynthesis has also been investigated. The regulation of pyrimidine biosynthesis in P. fragi was examined using a medium containing glucose as its carbon source (West,
T.P. West
2002). The levels of the de novo pyrimidine biosynthetic enzymes in P. taetrolens and P. fragi were compared. The levels of aspartate transcarbamoylase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase were found to be higher in P. taetrolens than P. fragi (West, 2002). The dihydroorotase and OMP decarboxylase activities were higher in P. fragi than in P. taetrolens (West, 2002). In P. fragi, aspartate transcarbamoylase and dihydroorotase activities appeared to be repressed by a uracil-related compound while orotic acid addition to the culture medium appeared to slightly repress the transcarbamoylase, phosphoribosyltransferase and decarboxylase activities (West, 2002). Following pyrimidine limitation of the pyrF mutant strain of P. fragi, all de novo pyrimidine biosynthetic pathway enzyme activities were derepressed (West, 2002). Relative to the P. fragi mutant strain grown under saturating uracil conditions, transcarbamoylase, dihydroorotase, dehydrogenase and phosphoribosyltransferase activities were increased in the pyrF strain by 2.1fold, 3.4-fold, 3.7-fold and 2.9-fold, respectively, following 4 h of pyrimidine limitation (West, 2002). Clearly, a higher degree of derepression of the de novo pyrimidine biosynthetic pathway enzyme activities was observed in the P. fragi mutant cells (West, 2002) than was noted for the P. taetrolens strain PT111 cells (Table 2). It can be concluded that the regulation of the de novo pyrimidine biosynthetic pathway enzyme synthesis in the species assigned to the P. chlororaphis group is not identical and varies according to species. The regulation of the in vitro activity of the pathway enzyme aspartate transcarbamoylase was also studied (Table 3). Earlier studies have shown that this enzyme regulates pyrimidine synthesis at the level of enzyme activity in pseudomonads (Adair and Jones, 1972; Condon et al., 1976). The Km of aspartate transcarbamoylase for its substrate L-aspartate or carbamoylphosphate was calculated (standard deviation) to be 2.38 mM (0.23) or 0.12 mM (0.02), respectively, in cell extracts of P. taetrolens ATCC 4683. The Km of the P. taetrolens transcarbamoylase for carbamoylphosphate and L-aspartate differed greatly from the P. fragi aspartate transcarbamoylase since the Km for its substrate L-aspartate or carbamoylphosphate was 1.18 mM or 0.32 mM, respectively (West, 2002). Possible transcarbamoylase effectors were screened under saturating substrate concentrations where 10 mM L-aspartate and 1 mM carbamoylphosphate were present in the assay mix (Table 3). Of the effectors screened (Table 3), pyrophosphate, ADP, ATP, UDP, UTP and GTP were the most potent inhibitors of aspartate transcarbamoylase activity
ARTICLE IN PRESS Regulation of pyrimidine nucleotide formation
Table 3. Effect of possible effectors on aspartate transcarbamoylase activity in cell extracts of P. taetrolens ATCC 4683 Effector
Specific activity
Relative activity (%)
Control Pyrophosphate UDP CDP ADP GDP UTP CTP ATP GTP
94.672.9 3.270.1 11.470.1 32.771.0 8.470.3 30.870.3 3.070.0 29.870.3 1.870.2 1.070.1
100 3 12 35 9 33 3 32 2 1
The concentration of each effector in the assay mix was 5 mM. Specific activity is expressed as nmol carbamoylaspartate formed/min/mg protein at 301C. Each value is the mean of three separate trials7S.D.
in P. taetrolens. The regulation of the P. fragi transcarbamoylase by pyrophosphate, ADP, ATP, UTP and GTP is similar to what was observed for the P. taetrolens transcarbamoylase (West, 2002). In summary, pyrimidine biosynthesis in P. taetrolens was repressible by a pyrimidine-related compound at the level of enzyme synthesis and its aspartate transcarbamoylase activity was highly regulated by pyrophosphate and ribonucleotides. Although the regulation of aspartate transcarbamoylase activity was similar in P. taetrolens and P. fragi, their transcriptional regulation of pyrimidine biosynthetic enzyme synthesis appeared to differ. These results should prove helpful to future studies attempting to taxonomically assign the most highly related species of Pseudomonas together.
Acknowledgements This work was funded by the South Dakota Agricultural Experiment Station.
33
References Adair, L.B., Jones, M.E., 1972. Purification and characteristics of aspartate transcarbamoylase from Pseudomonas fluorescens. J. Biol. Chem. 247, 2308–2315. Anzai, Y., Kim, H., Park, J.-Y., Wakabayashi, H., Oyaizu, H., 2000. Phytogenetic affiliation of the pseudomonads based on 16S rRNA sequence. Int. J. Syst. Evol. Microbiol. 50, 1563–1589. Bradford, M.M., 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. Condon, S., Collins, J.K., O’Donovan, G.A., 1976. Regulation of arginine and pyrimidine biosynthesis in Pseudomonas putida. J. Gen. Microbiol. 92, 375–383. Daise, R.L., Zottola, E.A., Epley, R.J., 1986. Potato-like odor of retail beef cuts associated with species of Pseudomonas. J. Food Prot. 49, 272–273. Levine, M., Anderson, D.Q., 1932. Two species of bacteria causing mustiness in eggs. J. Bacteriol. 23, 337–347. O’Donovan, G.A., Neuhard, J., 1970. Pyrimidine metabolism in microorganisms. Bacteriol. Rev. 34, 278–343. Sakai, T., Watanabe, T., Chibata, I., 1968. Metabolism of pyrimidine nucleotides in bacteria. J. Ferment. Technol. 46, 202–213. Sakai, T., Yu, T.-S., Omata, S., 1976. Distribution of enzymes related to cytidine degradation in bacteria. Agric. Biol. Chem. 40, 1893–1895. Stanier, R.Y., Palleroni, N.O., Doudoroff, M., 1966. The aerobic pseudomonads: A taxonomic study. J. Gen. Microbiol. 42, 159–271. Tompkin, R.B., Shaparis, A.B., 1972. Potato aroma of lamp carcasses. Appl. Microbiol. 24, 1003–1004. Watson, J.M., Holloway, B.W., 1976. Suppressor mutations in Pseudomonas aeruginosa. J. Bacteriol. 125, 780–786. West, T.P., 1989. Isolation and characterization of thymidylate synthetase mutants of Xanthomonas maltophila. Arch. Microbiol. 151, 220–222. West, T.P., 1997. Pyrimidine biosynthesis in Pseudomonas stutzeri ATCC 17588. Anton. Leeuw. 72, 175–181. West, T.P., 2002. Control of pyrimidine synthesis in Pseudomonas fragi. Lett. Appl. Microbiol. 35, 380–384. West, T.P., Herlick, S.A., O’Donovan, G.A., 1983. Inverse relationship between thymidylate synthetase and cytidine triphosphate synthetase activities during pyrimidine limitation in Salmonella typhimurium. FEMS Microbiol. Lett. 18, 275–278.
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SHORT COMMUNICATION
Regulation of pyrimidine nucleotide formation in Pseudomonas taetrolens ATCC 4683 Thomas P. West Olson Biochemistry Laboratories, Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007, USA Accepted 2 January 2004
KEYWORDS Pseudomonas taetrolens; Pyrimidine; Biosynthesis; Aspartate transcarbamoylase
Abstract The regulation of the de novo pyrimidine biosynthetic enzymes in the food spoilage agent Pseudomonas taetrolens ATCC 4683 was investigated. The de novo pyrimidine biosynthetic enzyme activities were determined in P. taetrolens ATCC 4683 cells and in cells from an auxotroph deficient for orotidine 50 -monophosphate decarboxylase activity. Pyrimidine supplementation to the culture medium affected the biosynthetic enzyme activities in ATCC 4683 cells. Transcriptional regulation of the biosynthetic pathway by pyrimidines was indicated after the auxotroph was subjected to pyrimidine limitation. At the level of enzyme activity, aspartate transcarbamoylase activity was strongly inhibited by pyrophosphate, ADP, ATP, UDP, UTP and GTP. Transcriptional regulation of pyrimidine synthesis in P. taetrolens was not as highly controlled as in the taxonomically-related species Pseudomonas fragi although both species contained transcarbamoylase activities subject to significant nucleotide inhibition. & 2004 Elsevier GmbH. All rights reserved.
Introduction Although Pseudomonas taetrolens is known to produce an undesirable mustiness odor in eggs and meats, the study of its pyrimidine biosynthesis has not been investigated despite the possibility that the findings could help in its biological control (Levine and Anderson, 1932; Tompkin and Shaparis, 1972; Daise et al., 1986). Moreover, the findings could prove helpful from a biochemical perspective to the ongoing taxonomic analysis of Pseudomonas species. Taxonomically, P. taetrolens has been
classified within the Pseudomonas chlororaphis group (Anzai et al., 2000). This group contains the species Pseudomonas fragi which is also a food spoilage agent (Anzai et al., 2000). Five enzymes comprise the de novo pyrimidine biosynthetic pathway that ultimately produces UMP (O’Donovan and Neuhard, 1970). The first enzyme unique to only pyrimidine biosynthesis is aspartate transcarbamoylase (EC 2.1.3.2). This enzyme synthesizes carbamoylaspartate from its substrates L-aspartate and carbamoylphosphate. The other four pathway enzymes include dihydroorotase (EC 3.5.2.3),
E-mail address: Thomas [email protected] (T.P. West). 0944-5013/$ - see front matter & 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2004.01.007
ARTICLE IN PRESS 30
dihydroorotate dehydrogenase (EC 1.3.3.1), orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 50 -monophosphate (OMP) decarboxylase (EC 4.1.1.23). The pyrimidine biosynthetic pathway appears to be regulated at the level of enzyme synthesis by pyrimidines in the P. chlororaphis group member P. fragi (West, 2002). In addition, aspartate transcarbamoylase activity in P. fragi was controlled by nucleotides (West, 2002). In this study, pyrimidine biosynthesis in P. taetrolens ATCC 4683 was investigated for regulation by pyrimidines at the level of enzyme synthesis while possible control of its aspartate transcarbamoylase activity was also explored.
Materials and methods Strain and growth conditions P. taetrolens ATCC 4683 and strain PT111 were the strains used in this study (Levine and Anderson, 1932; Stanier et al., 1966). The minimal medium was prepared as previously stated (West, 1989). The carbon source succinate (0.4%, w/v) was added to the medium after autoclaving. Batch cultures (25 ml) were inoculated in sterile 125 ml Erlenmeyer flasks using overnight cultures. When a pyrimidine base was added, its concentration was 50 mg/l. All cultures were shaken (200 rpm) at an incubation temperature of 301C. To derive generation times, growth was followed spectrophotometrically at 600 nm. For the pyrimidine limitation experiments, the pyrimidine auxotrophic strain was grown in succinate minimal medium containing 50 mg/l uracil to the late exponential phase of growth. The cells were collected, washed and resuspended in succinate minimal medium. After 2 or 4 h of pyrimidine limitation at 301C, the cells were collected and extracts prepared as stated above.
T.P. West
succinate minimal medium containing uracil (50 mg/l) and then were grown in this medium for 48 h at 301C. The cell starvation and D-cycloserine procedure was repeated. The surviving cells from this procedure were grown for 15 h at 301C in succinate minimal medium containing uracil (50 mg/ml). Dilutions of this culture were spread onto solid succinate minimal medium containing uracil (50 mg/l). Colonies present on the solid medium after 72 h of growth at 301C were screened for uracil auxotrophy and strain PT111 was identified. The ability of the mutant strain to grow upon carbamoylaspartate, dihydroorotic acid, orotic acid, uracil, dihydrouracil, cytosine, uridine, UMP, cytidine or CMP to meet its pyrimidine requirement was explored. This involved spreading approximately 107 cells of the mutant strain onto succinate minimal medium agar plates. To the center of each plate was placed a glass microfiber filter disk (2.1 cm in diameter) saturated with a sterile solution of the test compound (1.5 mg/ml). All plates were examined daily over a period of 8 days at 301C for confluent growth.
Preparation of cell extracts To prepare cell extracts for use during the enzyme assays, shake flask cultures (25 ml) of P. taetrolens cells were grown on a rotary shaker (200 rpm) at 301C. The cultures were harvested during the late exponential phase and were collected by centrifugation at 10,400g at 41C for 20 min. The cells were washed and resuspended in 2.5 ml of 20 mM Tris-HCl buffer (pH 8.0) containing 1 mM 2-mercaptoethanol. The suspension was subjected to ultrasonic disruption for a total of 4 min in ice. The disrupted cells were centrifuged at 1930g for 15 min at 41C. The resultant extract was dialyzed for 18 h against resuspension buffer (300 ml) at 41C and was then assayed.
Enzyme assays Mutant isolation The uracil auxotrophic strain of P. taetrolens ATCC 4683 was isolated using ethyl methanesulfonate mutagenesis (Watson and Holloway, 1976) and subsequent outgrowth in nutrient broth at 301C. The mutagenized cells were collected and resuspended in 0.85% NaCl. After starving the cells for 2.5 h at 301C, the cells were collected and resuspended in succinate minimal medium containing D-cycloserine (1 mg/ml) and shaken for 2 h at 301C (West, 1997). The cells were collected, washed twice with 0.85% NaCl, resuspended in
All assays were performed at 301C. Aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase, orotate phosphoribosyltransferase and OMP decarboxylase activities were assayed as previously described (West, 1997). When studying regulation of transcarbamoylase activity, an effector concentration of 5 mM was present in the assay mix. The Km values of aspartate transcarbamoylase for L-aspartate and carbamoylphosphate were calculated from Lineweaver-Burk plots. Protein was measured by the method of Bradford (1976) where lysozyme served as the
ARTICLE IN PRESS Regulation of pyrimidine nucleotide formation
31
standard protein. Enzyme specific activity was stated as nmol/min/mg protein at 301C. All values represented the mean of three independent determinations where three separate extracts were assayed.
Results and discussion The five de novo pyrimidine biosynthetic pathway enzymes were active in the succinate-grown cells (generation time 161 min) of P. taetrolens ATCC 4683 (Table 1). The effect of pyrimidine base supplementation to the growth medium of the wild type strain on its pyrimidine biosynthetic pathway enzyme activities was examined. Compared to the activity of succinate-grown cells, orotic acid supplementation to the medium (generation time 165 min) resulted in a slight decrease in transcarbamoylase activity while the addition of uracil to the medium (generation time 205 min) increased this enzyme activity by 1.2-fold (Table 1). Dihydroorotase activity rose by 1.4-fold when orotic acid was included in the medium relative to its activity in the medium alone (Table 1). In contrast, the presence of uracil in the medium depressed
dihydroorotase activity to 65% of its activity in cells grown in unsupplemented medium (Table 1). Similar to what was observed for dihydroorotase activity, the presence of orotic acid in the medium elevated dehydrogenase activity by 1.2-fold relative to its activity in cells grown in unsupplemented medium (Table 1). Dihydroorotate dehydrogenase activity was diminished by the inclusion of uracil in the minimal medium compared to its activity in cells grown without a pyrimidine base added (Table 1). Pyrimidine supplementation had little effect on orotate phosphoribosyltransferase activity in the P. taetrolens cells compared to its activity in cells grown on only the minimal medium (Table 1). Relative to OMP decarboxylase activity in the unsupplemented cells, this enzyme activity was increased by 1.2-fold in cells grown on medium containing orotic acid but uracil addition to the medium lowered its activity (Table 1). An OMP decarboxylase deficient strain of P. taetrolens was isolated by chemical mutagenesis and D-cycloserine counterselection (West, 1997). No detectable decarboxylase activity was observed in the succinate-grown strain PT111 cells (generation time 178 min) supplemented with uracil (Table 2). The mutant strain was able to grow on uracil, cytosine or uridine as a pyrimidine source.
Table 1. Effect of exogenous pyrimidines on pyrimidine biosynthetic pathway enzyme activities in P. taetrolens ATCC 4683 Enzyme
Aspartate transcarbamoylase Dihydroorotase Dihydroorotate dehydrogenase Orotate phosphoribosyltransferase OMP decarboxylase
Specific activity None
Orotic acid
Uracil
89.872.9 37.271.0 3.270.0 38.771.2 6.770.2
85.572.8 51.471.1 3.970.1 40.672.5 7.770.1
107.170.6 23.371.4 2.770.1 39.471.3 5.870.2
The strain was grown in succinate minimal medium containing the indicated pyrimidine base at 301C. Specific activity of each enzyme is expressed as nmol/min/mg protein at 301C. Results are the mean of three separate determinations7S.D.
Table 2. Effect of pyrimidine nucleotide limitation on pyrimidine biosynthetic pathway enzyme activities in P. taetrolens strain PT111 Enzyme
Aspartate transcarbamoylase Dihydroorotase Dihydroorotate dehydrogenase Orotate phosphoribosyltransferase OMP decarboxylase
Specific activity þ Uracil
Uracil (2 h)
Uracil (4 h)
116.272.6 44.471.1 4.070.2 44.671.3 o0.470.1
81.072.3 101.077.8 3.470.1 72.373.4 o0.970.3
72.672.2 85.371.4 6.770.2 61.871.1 o0.670.1
Strain PT111 was grown at 30oC in succinate minimal medium containing 50 mg/l uracil or was subjected to pyrimidine nucleotide limitation in succinate minimal medium for 2 or 4 h at 301C as described in the text. Specific activity of each enzyme is expressed as nmol/min/mg protein at 301C. Results are the mean of three separate determinations7S.D.
ARTICLE IN PRESS 32
Since P. taetrolens has been reported to contain the enzymes cytosine deaminase and nucleoside hydrolase (Sakai et al., 1968, 1976), the utilization of cytosine or uridine as a pyrimidine source to support the growth of the mutant strain is not surprising. In P. fragi, the OMP decarboxylase mutant strain grew on uracil, cytosine, uridine or cytidine when glucose served as the carbon source (West, 2002). With the mutant strain PT111 isolated, the limitation of the mutant cells for pyrimidine nucleotides was undertaken to detect possible regulation of pyrimidine biosynthetic pathway enzyme synthesis in P. taetrolens. It has been shown previously the deprivation of the pyrimidine nucleotide pools can result in the derepression of pyrimidine pathway enzyme synthesis in bacteria (West et al., 1983). In strain PT111 cells (generation time 108 min), pyrimidine limitation decreased aspartate transcarbamoylase activity by 30% or 37%, respectively, after 2 or 4 h of pyrimidine limitation relative to its activity in uracil-grown cells (Table 2). In contrast, dihydroorotase activity in strain PT111 cells limited for pyrimidines for 2 h rose by 2.3-fold compared to its activity in cells grown with excess uracil (Table 2). Following 4 h of pyrimidine-limiting conditions, dihydroorotase activity was 1.9-fold higher than its activity in uracilgrown cells (Table 2). Interestingly, pyrimidine limitation of strain PT111 cells for 2 h produced a decrease in dehydrogenase activity while this enzyme activity rose by 1.7-fold compared to its activity in cells grown with a saturating level of uracil (Table 2). The mutant cells were found to contain elevated levels of orotate phosphoribosyltransferase activity following pyrimidine limitation. When the mutant cells were deprived of uracil for 2 h, phosphoribosyltransferase increased by 1.6-fold relative to the activity in the uracilgrown cells (Table 2). Following 4 h of pyrimidine starvation, phosphoribosyltransferase activity remained 1.4-fold elevated compared to the activity detected in the cells grown under saturating uracil conditions. The pyrimidine limitation experiments showed that derepression of the synthesis of at least three of the de novo biosynthetic enzymes was occurring which indicates repression by a pyrimidine-related compound at the transcriptional level. The transcriptional regulation of the pyrimidine biosynthetic pathway in P. taetrolens ATCC 4683 can be compared to P. fragi ATCC 4973 since it is the only species of the P. chlororaphis group whose regulation of pyrimidine biosynthesis has also been investigated. The regulation of pyrimidine biosynthesis in P. fragi was examined using a medium containing glucose as its carbon source (West,
T.P. West
2002). The levels of the de novo pyrimidine biosynthetic enzymes in P. taetrolens and P. fragi were compared. The levels of aspartate transcarbamoylase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase were found to be higher in P. taetrolens than P. fragi (West, 2002). The dihydroorotase and OMP decarboxylase activities were higher in P. fragi than in P. taetrolens (West, 2002). In P. fragi, aspartate transcarbamoylase and dihydroorotase activities appeared to be repressed by a uracil-related compound while orotic acid addition to the culture medium appeared to slightly repress the transcarbamoylase, phosphoribosyltransferase and decarboxylase activities (West, 2002). Following pyrimidine limitation of the pyrF mutant strain of P. fragi, all de novo pyrimidine biosynthetic pathway enzyme activities were derepressed (West, 2002). Relative to the P. fragi mutant strain grown under saturating uracil conditions, transcarbamoylase, dihydroorotase, dehydrogenase and phosphoribosyltransferase activities were increased in the pyrF strain by 2.1fold, 3.4-fold, 3.7-fold and 2.9-fold, respectively, following 4 h of pyrimidine limitation (West, 2002). Clearly, a higher degree of derepression of the de novo pyrimidine biosynthetic pathway enzyme activities was observed in the P. fragi mutant cells (West, 2002) than was noted for the P. taetrolens strain PT111 cells (Table 2). It can be concluded that the regulation of the de novo pyrimidine biosynthetic pathway enzyme synthesis in the species assigned to the P. chlororaphis group is not identical and varies according to species. The regulation of the in vitro activity of the pathway enzyme aspartate transcarbamoylase was also studied (Table 3). Earlier studies have shown that this enzyme regulates pyrimidine synthesis at the level of enzyme activity in pseudomonads (Adair and Jones, 1972; Condon et al., 1976). The Km of aspartate transcarbamoylase for its substrate L-aspartate or carbamoylphosphate was calculated (standard deviation) to be 2.38 mM (0.23) or 0.12 mM (0.02), respectively, in cell extracts of P. taetrolens ATCC 4683. The Km of the P. taetrolens transcarbamoylase for carbamoylphosphate and L-aspartate differed greatly from the P. fragi aspartate transcarbamoylase since the Km for its substrate L-aspartate or carbamoylphosphate was 1.18 mM or 0.32 mM, respectively (West, 2002). Possible transcarbamoylase effectors were screened under saturating substrate concentrations where 10 mM L-aspartate and 1 mM carbamoylphosphate were present in the assay mix (Table 3). Of the effectors screened (Table 3), pyrophosphate, ADP, ATP, UDP, UTP and GTP were the most potent inhibitors of aspartate transcarbamoylase activity
ARTICLE IN PRESS Regulation of pyrimidine nucleotide formation
Table 3. Effect of possible effectors on aspartate transcarbamoylase activity in cell extracts of P. taetrolens ATCC 4683 Effector
Specific activity
Relative activity (%)
Control Pyrophosphate UDP CDP ADP GDP UTP CTP ATP GTP
94.672.9 3.270.1 11.470.1 32.771.0 8.470.3 30.870.3 3.070.0 29.870.3 1.870.2 1.070.1
100 3 12 35 9 33 3 32 2 1
The concentration of each effector in the assay mix was 5 mM. Specific activity is expressed as nmol carbamoylaspartate formed/min/mg protein at 301C. Each value is the mean of three separate trials7S.D.
in P. taetrolens. The regulation of the P. fragi transcarbamoylase by pyrophosphate, ADP, ATP, UTP and GTP is similar to what was observed for the P. taetrolens transcarbamoylase (West, 2002). In summary, pyrimidine biosynthesis in P. taetrolens was repressible by a pyrimidine-related compound at the level of enzyme synthesis and its aspartate transcarbamoylase activity was highly regulated by pyrophosphate and ribonucleotides. Although the regulation of aspartate transcarbamoylase activity was similar in P. taetrolens and P. fragi, their transcriptional regulation of pyrimidine biosynthetic enzyme synthesis appeared to differ. These results should prove helpful to future studies attempting to taxonomically assign the most highly related species of Pseudomonas together.
Acknowledgements This work was funded by the South Dakota Agricultural Experiment Station.
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