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Keywords:

  • aspartate transcarbamoylase;
  • biosynthesis;
  • Pseudomonas;
  • pyrimidines;
  • regulation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Aims:  To investigate the regulation of de novo pyrimidine biosynthesis in the bacterium Pseudomonas resinovorans ATCC 14235.

Methods and Results:  The pyrimidine biosynthetic pathway enzymes were measured in cell extracts from P. resinovorans ATCC 14235 and from an auxotroph lacking orotate phosphoribosyltransferase activity. Pyrimidine biosynthetic pathway enzyme activities in ATCC 14235 were affected by the addition of pyrimidine bases to the culture medium. The de novo enzyme activities of the phosphoribosyltransferase mutant strain increased after pyrimidine starvation indicating possible repression of the pathway by a pyrimidine-related compound. Aspartate transcarbamoylase activity in ATCC 14235 was inhibited in vitro by ATP, UTP and pyrophosphate.

Conclusions:  Pyrimidine biosynthesis in P. resinovorans was regulated at the level of enzyme synthesis and at the level of activity for aspartate transcarbamoylase. Its regulation of enzyme synthesis seemed to be similar to what has been observed in the taxonomically related species Pseudomonas oleovorans.

Significance and Impact of the Study:  This study found that pyrimidine biosynthesis is regulated in P. resinovorans. This could prove helpful to future studies investigating polyhydroxyalkanoate production by the bacterium.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgements
  7. References

The ability of Pseudomonas resinovorans to produce medium-chain-length polyhydroxyalkanoates on various substrates has been demonstrated and the polyhydroxyalkanoates can be extracted from the cells with high purity (Anderson and Dawes 1990; Ramsay et al. 1992; Ashby et al. 1998; Hampson and Ashby 1999). Despite the increasing importance of this bacterium for commercial biopolyester production because of its isogenic polyhydroxyalkanoate synthase genes (Solaiman 2000, 2003; Solaiman et al. 2002), relatively little is known about its nucleic acid metabolism. In particular, the regulation of pyrimidine biosynthesis in this species of Pseudomonas has not been investigated. The de novo pyrimidine biosynthetic pathway is responsible for UMP formation and consists of five enzymes unique to pyrimidine biosynthesis (O'Donovan and Neuhard 1970). 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 five pathway enzymes. Taxonomically, P. resinovorans has been assigned to the Pseudomonas aeruginosa group that also includes the species P. aeruginosa, Pseudomonas pseudoalcaligenes and Pseudomonas oleovorans (Anzai et al. 2000). Regulation of pyrimidine biosynthesis in P. aeruginosa, P. pseudoalcaligenes and P. oleovorans has been examined in earlier studies (Isaac and Holloway 1968; West 1994; Haugaard and West 2002). At the level of pyrimidine gene expression, little regulation was observed for P. aeruginosa (Isaac and Holloway 1968). In contrast, it did appear that control of pyrimidine gene expression was occurring in P. pseudoalcaligenes and P. oleovorans (West 1994; Haugaard and West 2002). The in vitro regulation of aspartate transcarbamoylase activity has also been investigated in these species and it was found that pyrophosphate as well as pyrimidine and purine nucleotides served as its effectors (Isaac and Holloway 1968; West 1994; Haugaard and West 2002).

Pyrimidine biosynthesis in P. resinovorans ATCC 14235 has not been previously studied. Such an analysis could also prove fruitful from a taxonomic perspective as the control of pyrimidine biosynthesis in P. resinovorans could be compared with previously investigated Pseudomonas species. In this report, both in vivo regulation of pyrimidine biosynthetic enzyme synthesis by pyrimidines and in vitro regulation of aspartate transcarbamoylase activity in P. resinovorans were explored.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Micro-organisms and culture media

Pseudomonas resinovorans ATCC 14235 (Delaporte et al. 1961) and mutant strain PT115, isolated in this study, were used in this study. The strains were grown in a modified minimal medium as has been described previously (West 1989). Succinate medium (SM) and glucose medium (GM) contained 0·4% sodium succinate and 0·4% glucose as the carbon source respectively. Batch cultures (25 ml) were inoculated in sterile 125 ml Erlenmeyer flasks using overnight cultures. When supplemented, the concentration of orotic acid or uracil in the medium was 50 mg l−1. All cultures were shaken (200 rev min−1) at an incubation temperature of 30°C. Growth was followed spectrophotometrically at 600 nm and the measurements were used to derive generation times.

Mutant isolation

The isolation of the uracil-requiring mutant strain of P. resinovorans involved the use of 1% ethylmethane sulfonate mutagenesis for 90 min (Watson and Holloway 1976) and outgrowth in nutrient broth at 30°C. The mutagenized cells were collected and resuspended in 14·55 mmol l−1 NaCl. The isolation of mutant strain PT115 involved spreading the mutagenized cells on glucose minimal medium agar plates containing 5-fluoroorotic acid (50 mg l−1) and uracil (1 mg l−1) and incubating the plates at 30°C (Santiago and West 2002). The smallest colonies on the pyrimidine analogue-containing solid medium after 72–96 h were screened for uracil auxotrophy on glucose minimal medium agar plates alone or containing uracil (50 mg l−1). The auxotrophic strain PT115 was identified during this screening. The ability of each strain to grow on carbamoylaspartic acid, dihydroorotic acid, orotic acid, uracil, cytosine, dihydrouracil, uridine, cytidine, UMP and CMP to meet its pyrimidine requirement was investigated. This involved spreading about 106 cells of the mutant strain onto minimal medium agar plates. To the centre of each plate was placed a glass microfibre filter disk (2·1 cm in diameter) saturated with a sterile solution of the test compound (1·5 mg ml−1). All plates were examined daily over a period of 8 days at 30°C for confluent growth.

Preparation of cell extracts

To assay the de novo pyrimidine biosynthetic pathway enzyme activities in P. resinovorans, cell extracts were prepared from three separately grown 25 ml cultures. Cells were collected by centrifugation, washed and resuspended in 20 mmol l−1 Tris-HCl buffer pH 8·0 containing 1 mmol l−1 2-mercaptoethanol. The suspension was disrupted ultrasonically (Heat Systems-Ultrasonics Model W-380; Farmingdale, NY, USA) at maximal microprobe power for a total of 4 min (30 s bursts with 1 min resting between bursts) in ice. The disrupted cells were centrifuged at 1930 g for 15 min at 4°C. The resultant extract was dialysed for 18 h against resuspension buffer (300 ml) at 4°C and was then assayed.

For the pyrimidine limitation experiments, strain PT115 was grown in SM or GM containing uracil. The cells were collected in mid-exponential phase, washed and resuspended in their respective medium lacking uracil. After 2 or 4 h of pyrimidine limitation at 30°C, the cells were collected and extracts prepared as stated above.

Enzyme assays

All assays were performed at 30°C. Aspartate transcarbamoylase activity was measured using an assay mix (1 ml) that contained 0·1 mol l−1 Tris-HCl buffer pH 8·5, 10 mmol l−1l-aspartate (pH 8·5), 1 mmol l−1 dilithium carbamoylphosphate and cell-free extract (Haugaard and West 2002). When investigating the in vitro control of transcarbamoylase activity, the effector concentration present in the reaction mix was 5 mmol l−1. The Km values of aspartate transcarbamoylase for l-aspartic acid and carbamoylphosphate were derived from Lineweaver–Burk plots. Dihydroorotase was assayed using a modified reaction mixture (1 ml) that contained 0·1 mol l−1Tris-HCl buffer pH 8·5, 1 mmol l−1 EDTA, 1 mmol l−1 dihydroorotate and cell-free extract (Haugaard and West 2002). When assaying transcarbamoylase or dihydroorotase activity, the concentration of carbamoylaspartate was measured using method I of Prescott and Jones (1969). Dihydroorotate dehydrogenase, orotate phosphoribosyltransferase and OMP decarboxylase activity were assayed as previously described (Haugaard and West 2002). Protein was determined by the method of Bradford (1976) where lysozyme served as the standard. Specific activity was expressed as nmol substrate utilized or product formed min−1 (mg protein)−1 at 30°C where each value represents the mean of three independent determinations. The Student's t-test was used during the statistical analysis.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgements
  7. References

The levels of the five de novo pyrimidine biosynthetic activities were initially investigated in P. resinovorans ATCC 14235 (Table 1). As has been demonstrated in previous investigations studying pyrimidine biosynthesis in other taxonomically related pseudomonads (Isaac and Holloway 1968; West 1994; Haugaard and West 2002), five de novo pyrimidine biosynthetic pathway enzyme activities were present in the type strain of P. resinovorans grown on succinate (generation time 108 min) or glucose (generation time 153 min) as a carbon source (Table 1). When succinate served as the carbon source, orotic acid supplementation (generation time 96 min) only depressed aspartate transcarbamoylase activity while the activities of dihydroorotase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase were increased (Table 1). OMP decarboxylase activity was unaffected when the cells were grown on orotic acid (Table 1). The effect of uracil addition (generation time 90 min) on the biosynthetic enzyme activities in succinate-grown ATCC 14235 cells was investigated. Transcarbamoylase and dihydroorotase activities were both depressed with transcarbamoylase activity being reduced by nearly 50% (Table 1). Uracil supplementation also increased dehydrogenase activity by 1·5-fold while phosphoribosyltransferase and decarboxylase activities remained unaffected (Table 1). Using glucose as the carbon source, orotic acid addition (generation time 144 min) produced an increase in aspartate transcarbamoylase, dihydroorotase, orotate phosphoribosyltransferase or OMP decarboxylase activity (Table 1). In contrast, dihydroorotate dehydrogenase activity was highly repressed after growth on orotic acid (Table 1). The effect of uracil supplementation (generation time 139 min) in glucose minimal medium on de novo enzyme activities of P. resinovorans was explored and it was found that transcarbamoylase, dihydroorotase, dehydrogenase and decarboxylase activities were depressed (Table 1). Dehydrogenase activity was affected to the greatest degree as its activity dropped by 37% (Table 1). Only orotate phosphoribosyltransferase activity was unaffected by uracil addition (Table 1).

Table 1.  Effect of exogenous pyrimidines on pyrimidine biosynthetic enzyme activities in Pseudomonas resinovorans ATCC 14235 cells
EnzymeSpecific activity of SM cells [nmol min−1 (mg protein)−1]Specific activity of GM cells [nmol min−1 (mg protein)−1]
SMSM + orotic acid (50 mg l−1)SM + uracil (50 mg l−1)GMGM + orotic acid (50 mg l−1)GM + uracil (50 mg l−1)
  1. ATCC 14235 was grown at 30°C. Cells were harvested, disrupted ultrasonically, centrifuged, dialysed and the extract assayed for pyrimidine de novo enzyme activities. Each value represents the mean of three independent determinations (SD). Superscripts in the same row of SM or GM data that do not have a common letter are statistically different (P < 0·01) using t-test.

Aspartate transcarbamoylase57·2 (3·4)a43·8 (1·6)b30·6 (0·4)c64·8 (1·0)a80·0 (1·9)b38·6 (1·6)c
Dihydroorotase43·1 (1·7)a50·5 (0·5)b37·9 (0·2)c29·0 (4·2)a49·2 (1·6)b19·8 (0·5)c
Dihydroorotate dehydrogenase0·4 (0·0)a0·7 (0·0)b0·6 (0·0)c3·0 (0·5)a0·2 (0·1)b1·1 (0·0)c
Orotate phosphoribosyltransferase30·2 (2·4)a33·0 (0·4)a30·5 (1·9)a24·4 (2·8)a43·5 (1·2)b25·2 (0·5)a
OMP decarboxylase12·7 (0·2)a12·8 (0·4)a12·8 (0·2)a9·3 (0·4)a12·3 (0·2)b7·3 (0·1)c

As it was observed that uracil addition to the ATCC 14235 glucose medium cultures repressed the levels of four de novo enzyme activities, repression of the synthesis of the de novo pyrimidine pathway enzymes appeared possible. A similar pattern of repression by a uracil-related compound of de novo pyrimidine biosynthetic enzyme activities in P. oleovorans has been witnessed previously (Haugaard and West 2002). To identify repression of de novo pyrimidine pathway enzyme synthesis, pyrimidine starvation experiments can be utilized. Previous studies have shown that derepression of de novo pyrimidine pathway enzyme synthesis can be observed following the starvation of a pyrimidine auxotroph for uracil over several hours (West 1994; Haugaard and West 2002). To more closely study any regulation by pyrimidine-related compounds, it was first necessary to isolate a uracil auxotroph of P. resinovorans ATCC 14235. Strain PT115, a uracil-requiring strain, was isolated by a combination of ethylmethane sulfonate mutagenesis and resistance to the pyrimidine analogue 5-fluoroorotic acid. The ability of strain PT115 to utilize pyrimidine biosynthetic pathway intermediates and products, including carbamoylaspartate, dihydroorotate, orotic acid, uracil, dihydrouracil, cytosine, uridine, cytidine, UMP and CMP, after 8 days of growth at 30°C was explored. It was found that this strain was capable of growth on only uracil and cytosine when succinate or glucose served as the carbon source. Other studies have reported the isolation of pyrimidine-requiring mutants from pseudomonads that are taxonomically related to P. resinovorans. The isolation of P. aeruginosa mutant strains deficient for aspartate transcarbamoylase, dihydroorotate dehydrogenase, orotate phosphoribosyltransferase or OMP decarboxylase activity has been reported (Isaac and Holloway 1968). Intermediates of the de novo pyrimidine pathway could not support the growth of these mutants. An orotate phosphoribosyltransferase mutant strain of P. pseudoalcaligenes ATCC 17440 was isolated and it could utilize uracil or cytosine as a pyrimidine source (West 1994). Mutants of P. oleovorans ATCC 8062 lacking either aspartate transcarbamoylase or dihydroorotase activity were reported (Haugaard and West 2002). It was shown that these uracil-requiring strains could also utilize cytosine as a pyrimidine source (Haugaard and West 2002).

The activities of the five de novo pathway enzymes were assayed so that the pyrimidine biosynthetic pathway enzyme activity diminished by the mutation in strain PT115 could be determined (Table 2). Orotate phosphoribosyltransferase activity in succinate-grown (generation time 116 min) cells of strain PT115 was reduced by 16-fold, from 30·5 to 1·9 nmol min−1 (mg protein)−1, compared with its activity in the parent strain grown on succinate and excess uracil (Table 2). Phosphoribosyltransferase activity in glucose-grown (generation time 99 min) cells of strain PT115 was reduced by 17-fold, from 25·2 to 1·5 nmol min−1 (mg protein)−1, relative to its activity in the glucose-grown cells of the parent strain supplemented with uracil (Table 2). The effect of pyrimidine-limiting growth conditions on the levels of the de novo pyrimidine biosynthetic pathway enzymes was also examined relative to carbon source. When succinate-grown strain PT115 cells were starved for pyrimidines for 2 h, it was observed that aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase and OMP decarboxylase activities increased by 2·5-, 9-, 18- and 3·3-fold, respectively, relative to the activities in the strain grown with saturating uracil (Table 2). Phosphoribosyltransferase activity in the mutant strain also increased after 2 h of pyrimidine limitation (Table 2). After 4 h of pyrimidine-limiting growth conditions for the succinate-grown mutant strain cells, the pyrimidine biosynthetic activities were still elevated compared with the cells grown in excess uracil but they had dropped relative to the activities observed after 2 h of pyrimidine-limiting growth conditions for strain PT115 (Table 2). When glucose served as the carbon source, the pyrimidine biosynthetic enzyme activities increased after 2 h of pyrimidine-limiting growth conditions relative to their activities in uracil-grown cells of strain PT115 (Table 2). After 4 h of pyrimidine-limiting growth conditions, aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase and OMP decarboxylase activities increased by 3·7-, 6·8-, 5·6- and 4·4-fold, respectively, compared with their activities in strain PT115 cells grown in the presence of excess uracil (Table 2).

Table 2.  Influence of pyrimidine-limiting growth conditions on de novo pathway enzyme activities in mutant strain PT115
EnzymeSpecific activity of SM cells [nmol min−1 (mg protein)−1]Specific activity of GM cells [nmol min−1 (mg protein)−1]
SM + uracilSM − uracil (2 h)SM − uracil (4 h)GM + uracilGM − uracil (2 h)GM − uracil (4 h)
  1. Strain PT115 was grown at 30°C in SM or GM containing 50 mg l−1 uracil under excess uracil growth conditions. Under limiting uracil growth conditions, the strain was initially grown at 30°C in SM or GM containing 50 mg l−1 uracil. After the cells were collected, washed and resuspended in SM or GM, respectively, the culture was shaken for 2 or 4 h at 30°C. The cells were processed and the extracts assayed for pyrimidine de novo enzyme activities at 30°C. Each value represents the mean of three separate determinations (SD). Superscripts in the same row of SM or GM data that do not have a common letter are statistically different (P < 0·01) using t-test.

Aspartate transcarbamoylase43·8 (1·7)a107·9 (9·1)b81·7 (1·5)c36·8 (1·0)a53·3 (1·3)b136·0 (1·5)c
Dihydroorotase35·4 (1·1)a317·4 (4·3)b103·1 (2·0)c40·6 (0·6)a107·5 (1·3)b275·6 (2·0)c
Dihydroorotate dehydrogenase0·2 (0·0)a3·6 (0·1)b1·9 (0·1)c0·9 (0·0)a1·8 (0·2)b5·0 (0·2)c
Orotate phosphoribosyltransferase1·9 (0·6)a4·8 (0·4)b1·6 (0·1)a1·5 (0·2)a3·3 (0·8)b4·2 (1·2)b
OMP decarboxylase11·5 (0·3)a37·9 (1·7)b33·6 (0·9)c13·6 (0·3)a22·2 (0·3)b60·1 (0·5)c

From the above data, it does appear that in vivo regulation of de novo pyrimidine biosynthetic enzyme synthesis does exist in P. resinovorans ATCC 14235. De novo pyrimidine biosynthetic pathway enzyme synthesis has been studied previously in the taxonomically related species P. aeruginosa, P. pseudoalcaligenes and P. oleovorans to learn if gene expression of the pathway was regulated by pyrimidines. It was observed in P. aeruginosa that the synthesis of aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase or orotate phosphoribosyltransferase was not repressible by a uracil-related metabolite in glucose-grown cells (Isaac and Holloway 1968). In succinate-grown P. pseudoalcaligenes cells, the five de novo enzyme activities appeared to be repressible by a uracil-related metabolite. Using the pyrE mutant strain, it was possible to witness a threefold derepression of aspartate transcarbamoylase and dihydroorotate dehydrogenase activities as well as observe a 1·5-fold increase in dihydroorotase and OMP decarboxylase activities after pyrimidine limitation in succinate-grown cells (West 1994). In P. oleovorans ATCC 8062, uracil or orotic acid addition induced dihydroorotate synthesis in succinate-grown cells (Haugaard and West 2002). In this pseudomonad, dihydroorotase, dihydroorotate dehydrogenase, orotate phosphoribosyltransferase and OMP decarboxylase activities could be derepressed by 2·1-, 3·2-, 2·3- and 1·8-fold, respectively, following pyrimidine limitation for 4 h of an aspartate transcarbamoylase-deficient strain grown on succinate as the carbon source (Haugaard and West 2002). When the glucose-grown cells of the transcarbamoylase mutant strain were starved for 4 h, dihydroorotase, dihydroorotate dehydrogenase, orotate phosphoribosyltransferase and OMP decarboxylase activities could be derepressed by 3-, 2·7-, 4·4- and 6·8-fold respectively (Haugaard and West 2002). The degree to which the de novo pyrimidine pathway enzyme activities in P. resinovorans could be derepressed appeared most similar to what was observed for P. oleovorans.

As the de novo enzyme aspartate transcarbamoylase is regulated by pyrophosphate and nucleotides in other pseudomonads (Isaac and Holloway 1968; West 1994; Haugaard and West 2002), the regulation of the P. resinovorans ATCC 14235 transcarbamoylase activity was investigated as to the effect of these compounds (Table 3). A number of effectors screened proved inhibitory to transcarbamoylase activity (Table 3). It can be seen that ATP, UTP and pyrophosphate were highly inhibitory to the enzyme (Table 3). In cell-free extracts of P. resinovorans ATCC 14235, the Km (standard deviation) of aspartate transcarbamoylase for its substrate l-aspartate or carbamoylphosphate was calculated to be 1·63 (0·26) mmol l−1 or 0·22 (0·05) mmol l−1, respectively. With 10 mmol l−1l-aspartate and 1 mmol l−1 carbamoylphosphate being used in the assay of transcarbamoylase activity, it would appear that the above inhibitors were effective when substrate concentrations were saturating. Regulation of aspartate transcarbamoylase has been examined in pseudomonads taxonomically related to P. resinovorans. The nucleotides ATP, UTP and CTP inhibited the P. aeruginosa transcarbamoylase while these nucleotides as well as pyrophosphate inhibited the P. pseudoalcaligenes enzyme (Isaac and Holloway 1968; West 1994). The P. oleovorans aspartate transcarbamoylase was strongly inhibited by UDP, UTP, GTP, ATP and pyrophosphate (Haugaard and West 2002). The transcarbamoylases from P. oleovorans and P. resinovorans were similar in that CTP was less effective as an inhibitor than the other nucleotide triphosphates tested (Haugaard and West 2002).

Table 3.  Effect of possible effectors on in vitro aspartate transcarbamoylase activity in Pseudomonas resinovorans
EffectorSpecific activity [nmol min−1 (mg protein)−1]Percentage activity
  1. Each value is the mean of three independent determinations (SD).

Control63·7 (4·5)100
Pyrophosphate7·1 (0·4)11
ADP15·2 (0·7)24
ATP1·6 (0·2)3
GDP60·6 (0·9)95
GTP14·8 (0·3)23
UDP34·8 (0·4)55
UTP3·7 (0·5)6
CDP63·9 (2·0)100
CTP47·8 (2·2)23

Overall, it can be concluded that de novo pyrimidine synthesis in P. resinovorans is regulated at both the level of enzyme synthesis and the level of enzyme activity. The regulation of the de novo pyrimidine biosynthetic pathway in this pseudomonad appeared to most closely resemble how the pathway was controlled in the taxonomically related species P. oleovorans that is also capable of producing high levels of polyhydroxyalkanoates.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Financial support for this study was provided by the South Dakota Agricultural Experiment Station.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgements
  7. References
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