Pyrimidine Biosynthetic Enzyme Activities Research Articles

Pyrimidine nucleotide synthesis in the emerging pathogen Pseudomonas monteilii.

Regulation of pyrimidine biosynthesis by pyrimidines in the emerging, opportunistic human pathogen Pseudomonas monteilii ATCC 700476 was evident. When wild-type cells were grown on succinate in the presence of uracil or orotic acid, the activities of all 5 pyrimidine biosynthetic enzymes were depressed while the activities of 3 of the enzymes decreased in glucose-grown cells supplemented with uracil or orotic acid compared with unsupplemented cells. Pyrimidine limitation of succinate- or glucose-grown pyrimidine auxotrophic cells lacking orotate phosphoribosyltransferase activity resulted in more than a doubling of the pyrimidine biosynthetic enzyme activities relative to their activities in uracil-grown cells. Independent of carbon source, pyrimidine-limited cells of the pyrimidine auxotrophic cells deficient for dihydroorotase activity generally resulted in a slight elevation or depression of the pyrimidine biosynthetic enzyme activities compared with their activities in cells grown under saturating uracil conditions. Aspartate transcarbamoylase activity in P. monteilii was regulated at the enzyme activity level, since the enzyme was strongly inhibited by CTP, UMP, GMP, GDP, ADP, and UTP. In summary, the regulation of pyrimidine biosynthesis in P. monteilii could be used to control its growth or to differentiate it biochemically from other related species of Pseudomonas.

Regulation of the Pyrimidine Biosynthetic Pathway in Pseudomonas mucidolens

Control of pyrimidine biosynthesis was examined in Pseudomonas mucidolens ATCC 4685 and the five de novo pyrimidine biosynthetic enzyme activities unique to this pathway were influenced by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. When uracil was supplemented to glucose-grown ATCC 4685 cells, activities of four de novo enzymes were depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the glucose-grown mutant strain cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase activities to increase by more than 3-fold while OMP decarboxylase activity increased by 2.7-fold. The syntheses of the de novo enzymes appeared to be regulated by pyrimidines. At the level of enzyme activity, aspartate transcarbamoylase activity in P. mucidolens ATCC 4685 was subject to inhibition at saturating substrate concentrations. Transcarbamoylase activity was strongly inhibited by UTP, ADP, ATP, GTP and pyrophosphate.

Effect of carbon source on pyrimidine formation in Pseudomonas fluorescens ATCC 13525

The effect of carbon source on the regulation of the de novo pyrimidine biosynthetic enzymes in the type strain of Pseudomonas fluorescens was investigated. The de novo pyrimidine biosynthetic enzyme activities were measured in P. fluorescens ATCC 13525 cells and in cells from an auxotroph deficient for orotate phosphoribosyltransferase activity where glucose or succinate served as the carbon source. Pyrimidine supplementation to the culture medium affected the biosynthetic enzyme activities in ATCC 13525 cells. Pyrimidine Limitation of the mutant strain indicated transcriptional regulation of the biosynthetic pathway by pyrimidines that was influenced by carbon source. Transcriptional regulation of pyrimidine synthesis in P. fluorescens appeared to be more highly regulated than was observed for the taxonomically related species Pseudomonas aeruginosa or Pseudomonas putida.

Regulation of pyrimidine biosynthesis in Acidovorax delafieldii

The regulation of de novo pyrimidine biosynthesis in the nutritionally versatile bacterium Acidovorax delafieldii ATCC 17505T was influenced by carbon source and pyrimidine supplementation. Uracil supplementation of succinate-grown A. delafieldii ATCC 17505T cells produced a greater decrease in the de novo pyrimidine biosynthetic pathway enzyme activities than did orotic acid supplementation. The presence of orotic acid or uracil in the medium of the glucose-grown wild type cells generally increased the pyrimidine biosynthetic enzyme activities. After the pyrimidine limitation of an A. delafieldii orotate phosphoribosyltransferase mutant strain, the pyrimidine biosynthetic pathway enzyme activities in the glucose-grown mutant cells were more highly derepressed than in the succinate-grown mutant cells.

Regulation of pyrimidine nucleotide formation in Pseudomonas taetrolens ATCC 4683

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 5′-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.

Effect of carbon source on pyrimidine biosynthesis in Pseudomonas alcaligenes ATCC 14909

The effect of carbon source on the regulation of the de novo pyrimidine biosynthetic enzymes in Pseudomonas alcaligenes ATCC 14909 was investigated. The de novo pyrimidine biosynthetic enzymes were measured in extracts of P. alcaligenes ATCC 14909 cells and of cells from an auxotroph deficient for orotate phosphoribosyltransferase activity. Pyrimidine biosynthetic enzyme activities in ATCC 14909 were influenced by pyrimidine supplementation to the culture medium but not by the carbon source present. Pyrimidine limitation of the auxotroph elevated the de novo enzyme activities indicating that this pathway may be controlled at the transcriptional level by a pyrimidine-related compound. Its regulation seemed to be subject to less transcriptional control by a pyrimidine-related compound than what was observed in the closely related species Pseudomonas pseudoalcaligenes.

Control of pyrimidine synthesis in Pseudomonas fragi.

To study the regulation of the de novo pyrimidine biosynthetic enzymes in the food-spoilage agent Pseudomonas fragi ATCC 4973. The de novo pyrimidine biosynthetic enzymes were measured in extracts of Ps. fragi ATCC 4973 cells and of cells from auxotrophs deficient for dihydroorotase or OMP decarboxylase activity. Pyrimidine biosynthetic enzyme activities in ATCC 4973 were influenced by pyrimidine supplementation to the culture medium. The pyrimidine limitation of each auxotroph elevated the de novo enzyme activities, indicating that this pathway may be repressible by a pyrimidine-related compound. Aspartate transcarbamoylase activity in ATCC 4973 was inhibited in vitro by pyrophosphate and purine or pyrimidine nucleotides. Pyrimidine synthesis in Ps. fragi appeared to be controlled at the transcriptional level and at the level of activity for aspartate transcarbamoylase. Its transcriptional regulation seemed to be more highly controlled than what was observed in the closely related species Pseudomonas putida and Pseudomonas fluorescens. This study demonstrates that pyrimidine synthesis is regulated in Ps. fragi. This could prove useful to future studies examining its biological control and its taxonomic assignment.

Effect of the carbon source succinate on pyrimidine synthesis in Burkholderia cepacia

AbstractThe effect of the carbon source succinate on pyrimidine synthesis in the pathogen Burkholderia cepacia ATCC 25416 was investigated. The five de novo pathway enzyme activities of pyrimidine biosynthesis in B. cepacia were shown to be affected when ATCC 25416 cells were grown in the presence of uracil. Pyrimidine limitation of a uracil auxotrophic B. cepacia strain lacking orotidine 5′‐monophosphate decarboxylase activity increased aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase activities by at least 1.5‐fold which indicated that these pathway enzymes may be regulated by a uracil‐related compound. The levels of the de novo pyrimidine biosynthetic enzyme activities in B. cepacia ATCC 25416 cells appeared to be influenced to a greater degree in cells grown on succinate than on glucose as a carbon source.

Regulation of pyrimidine biosynthesis in Pseudomonas cepacia

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.