Pyrimidine metabolism in Acinetobacter calcoaceticus

The metabolism of thymine, thymidine, uracil, and uridine has been investigated in five different strains of Acinetobacter calcoaceticus. Attempts to isolate thymine and thymidine auxotrophic mutants were not successful. Consistent with this finding was the observation that uptake of radioactive thymine or thymidine could not be demonstrated. Search for enzymes capable of transforming thymine via thymidine to thymidine-5'-monophosphate in crude extracts was performed, and the following enzymes were absent judging from enzyme assays: thymidine phosphorylase (EC 2.4.2.4), trans-N-deoxyribosylase (EC 2.4.2.6), and thymidine kinase (EC 2.7.1.21). The enzymes responsible for the phosphorylation of thymidine-5'-monophosphate to thymidine-5'-triphosphate were present in crude extracts. Radioactive uracil was readily incorporated into both ribonucleic acid and deoxyribonucleic acid, the ratio being 6:1, and radioactivity was found only in pyrimidine bases. No uptake of uridine could be demonstrated. Uridine-5'-monophosphate pyrophosphorylase (EC 2.4.2.9) activity was detected in crude extracts, suggesting that uracil is converted directly to uridine-5'-monophosphate which is then phosphorylated to uridine-5'-triphosphate or transformed to other ribo- and deoxypyrimidine nucleotides.     

Depression of uracil uptake by ammonium in Neurospora crassa.

The mechanism of uracil uptake and one aspect of its regulation were studied in germinated conidia of Neurospora crassa. Uracil was found to be taken up by a transport mechanism that did not exhibit Michaelis-Menten kinetics. Rather, the kinetic patterns indicated two separate systems or a single transport mechanism with negative cooperativity. Cytosine and thymine inhibited uracil uptake, but uridine did not. The mutant strain uc-5-pyr-1, which failed to transport uracil, was used in reversion studies and to map the uc-5 locus. Spontaneous reversion rates at the uc-5 locus were found to be approximately 2 x 10(-8), indicating that the uc-5 lesion results from a single mutation. Loss of the uracil transport function through a single mutation favors the model of a single transport mechanism with negative cooperativity. Uracil uptake was significantly decreased in the presence of NH 4+, and evidence is presented for repression by NH4+ of a uracil transport system. Growth rates of pyrimidine-requiring and wild-type strains measured in the presence and absence of NH4+, with uracil as the pyrimidine supplement, showed that NH4+ decreased the growth rates of the pyrimidine-requiring strains significantly, while having no effect on wild-type growth rates.     

Metabolism of pyrimidine bases and nucleosides in Bacillus subtilis.

In Bacillus subtilis, uracil (Ura), uridine (Urd), and deoxyuridine (dUrd) are metabolized through pathways similar to those of enteric bacteria. Ura is probably converted to uridine 5'-monophosphate by uridine 5'-monophosphate pyrophosphorylase. More than 95% of dUrd added to cultures is converted to Ura and deoxyribose-1-phosphate. Although dUrd kinase activity is detectable in vitro, this enzyme does not seem to play an important role in the metabolism of dUrd. The metabolism of cytosine (Cyt), cytidine (Cyd), and deoxycytidine (dCyd) in B. subtilis appears to be different from that in enteric bacteria. Cytosine cannot be used by Ura-requiring mutants as pyrimidine source. dCyd is deaminated by dCyd-Cyd deaminase or phosphorylated to dCyd nucleotides by dCyd kinase. Cyd is deaminated by dCyd-Cyd deaminase of phosphorylated by Cyd kinase. This Cyd kinase activity has never been reported for B. subtilis.     

Pyrimidine Salvage Pathways In Toxoplasma Gondii

ABSTRACT. Pyrimidine salvage enzyme activities in cell-free extracts of Toxoplasma gondii were assayed in order to determine which of these enzyme activities are present in these parasites. Enzyme activities that were detected included phosphoribosyltransferase activity towards uracil (but not cytosine or thymine), nucleoside phosphorylase activity towards uridine, deoxyuridine and thymidine (but not cytidine or deoxycytidine), deaminase activity towards cytidine and deoxycytidine (but not cytosine, cytidine 5'-monophosphate or deoxycytidine 5'-monophosphate), and nucleoside 5'-monophosphate phosphohydrolase activity towards all nucleotides tested. No nucleoside kinase or phosphotransferase activity was detected, indicating that T. gondii lack the ability to directly phosphorylate nucleosides. Toxoplasma gondii appear to have a single non-specific uridine phosphorylase enzyme which can catalyze the reversible phosphorolysis of uridine, deoxyuridine and thymidine, and a single cytidine deaminase activity which can deaminate both cytidine and deoxycytidine. These results indicate that pyrimidine salvage in T. gondii probably occurs via the following reactions: cytidine and deoxycytidine are deaminated by cytidine deaminase to uridine and deoxyuridine, respectively; uridine and deoxyuridine are cleaved to uracil by uridine phosphorylase; and uracil is metabolized to uridine 5'-monophosphate by uracil phosphoribosyltransferase. Thus, uridine 5'-monophosphate is the end-product of both de novo pyrimidine biosynthesis and pyrimidine salvage in T. gondii.     

Enzymes of Pyrimidine Metabolism in Mycoplasma mycoides subsp. mycoides

The major pathways of ribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides have been proposed from studies on its use of radioactive purines and pyrimidines. To interpret more fully the observed pattern of pyrimidine usage, cell extracts of this organism have been assayed for several enzymes associated with the salvage synthesis of pyrimidine nucleotides. M. mycoides possessed uracil phosphoribosyltransferase, uridine phosphorylase, uridine (cytidine) kinase, uridine 5'-monophosphate kinase, and cytidine 5'-triphosphate synthetase. No activity for phosphorolysis of cytidine was detected, and no in vitro conditions were found to give measurable deamination of cytidine. Of the two potential pathways for incorporation of uridine, our data suggest that this precursor would largely undergo initial phosphorolysis to uracil and ribose-1-phosphate. Conversely, cytidine is phosphorylated directly to cytidine 5'-monophosphate in its major utilization, although conversion of cytidine to uracil, uridine, and uridine nucleotide has been observed in vivo, at least when uracil is provided in the growth medium. Measurements of intracellular nucleotide contents and their changes on additions of pyrimidine precursors have allowed suggestions as to the operation of regulatory mechanisms on pyrimidine nucleotide biosynthesis in M. mycoides in vivo. With uracil alone or uracil plus uridine as precursors of pyrimidine ribonucleotides, the regulation of uracil phosphoribosyltransferase and cytidine 5'-triphosphate synthetase is probably most important in determining the rate of pyrimidine nucleotide synthesis. When cytidine supplements uracil in the growth medium, control of cytidine kinase activity would also be important in this regard.     

Nitrogen metabolite repression of fluoropyrimidine resistance and pyrimidine uptake in Neurospora crassa

Summary Wild type Neurospora crassa was shown to be more resistant to 5-fluoro-uracil, 5-fluoro-uridine and 5-fluoro-2-deoxyuridine in the presence of ammonia than in its absence. This differential resistance may in part be accounted for by the observation that both uracil and uridine uptake in germinating conidia is under nitrogen metabolite regulation. The uptake of uracil and uridine is increased on poor nitrogen sources in wild type, is unaffected by nitrogen source in a nit-2 mutant strain while a gln-1 mutant strain showed intermediate behaviour.Wild type also showed increased resistance to all three fluoropyrimidines with increased temperature and both uracil and uridine uptake in the wild type increased with temperature.Supported by S.R.C. grant GR/A/64655F.P. Buxton was supported during the period of this work by an S.R.C. Research Studentship     

5-Fluoropyrimidine-resistant mutants of pneumococcus

Three classes of 5-fluorpyrimidine-resistant mutants of Diplococcus pneumoniae have been characterized. The mutant strain upp is resistant to high concentrations of the fluoropyrimidine bases fluorouracil (FU) and fluorocytosine (FC); strain upp has a defective uridine monophosphate pyrophosphorylase. The mutant strain udk is resistant to inhibition by fluorouridine (FUR) and exhibits defective uridine kinase activity. The mutant strain fun is resistant to inhibition by the nucleosides fluorodeoxyuridine, fluorodeoxycytidine, and FUR, but shows normal activity for all pyrimidine pathway enzymes tested. This strain may be defective in the activity of a transport system that governs the cellular uptake of pyrimidine ribo- and deoxyribonucleosides. Biochemical studies on wild-type and fluoropyrimidine-resistant pneumococci are discussed with respect to the transport and early metabolism of preformed pyrimidine precursors by this organism.     

Metabolism of pyrimidine bases and nucleosides in Neisseria meningitidis.

In Neisseria meningitidis, uridine, deoxyuridine, cytosine, cytidine, or deoxycytidine could not be used by uracil-requiring mutants as pyrimidine sources. Consistent with these findings, only 5-fluorouracil of the different fluoropyrimidine bases and nucleosides showed any inhibitory effect on the growth of four prototrophic strains of N. meningitidis. Likewise, only radioactive uracil was readily incorporated into nucleic acids, whereas uptake of radioactive uridine, cytosine, or cytidine could not be demonstrated. Uracil was converted to uridine 5'-monophosphate by uracil phosphoribosyltransferase, whereas enzyme activities for conversion of cytosine or any of the nucleosides were not detectable in meningococcal extracts.     

Regulation of purine utilization in bacteria. VI. Characterization of hypoxanthine and guanine uptake into isolated membrane vesicles from Salmonella typhimurium.

Uptake of hypoxanthine and guanine into isolated membrane vesicles of Salmonella typhimurium TR119 was stimulated by 5'-phosphoribosyl-1'-pyrophosphate (PRPP). For strain proAB47, a mutant that lacks guanine phosphoribosyltransferase, PRPP stimulated uptake of hypoxanthine into membrane vesicles. No PRPP-stimulated uptake of guanine was observed. For strain TR119, guanosine 5'-monophosphate and inosine 5'-monophosphate accumulated intravesicularly when guanine and hypoxanthine, respectively, were used with PRPP as transport substrates. For strain proAB47, IMP accumulated intravesicularly with hypoxanthine and PRPP as transport substrates. For strain TR119, hypoxanthine also accumulated when PRPP was absent. This free hypoxanthine uptake was completely inhibited by N-ethylmaleimide, but the PRPP-stimulated uptake of hypoxanthine was inhibited only 20% by N-ethylmaleimide. Hypoxanthine and guanine phosphoribosyltransferase activity paralleled uptake activity in both strains. But, when proAB47 vesicles were sonically treated to release the enzymes, a three- to sixfold activation of phosphoribosyltransferase molecules occurred. Since proAB47 vessicles lack the guanine phsophoribosyltransferase gene product and since hypoxanthine effectively competes out the phosphoribosylation of guanine by proAB47 vesicles, it was postulated that the hypoxanthine phosphoribosyltransferase gains specificity for both guanine and hypoxanthine when released from the membrane. A group translocation as the major mechanism for the uptake of guanine and hypoxanthine was proposed.     

Repression of Enzyme Synthesis of the Pyrimidine Pathway in Salmonella typhimurium

It has been reported by other workers that a uridine and probably also a cytidine nucleotide are required for maximal repression of aspartate transcarbamylase encoded by the gene pyrB in Salmonella typhimurium. We have identified the repressing metabolites for three more biosynthetic enzymes, namely, dihydroorotate dehydrogenase (encoded by pyrD), orotidine-5'-monophosphate pyrophosphorylase (encoded by pyrE), and orotidine-5'-monophosphate decarboxylase (encoded by pyrF), as well as examining the repression profiles of aspartate transcarbamylase in more detail. Using a specially constructed strain of S. typhimurium (JL1055) which lacks the enzymes for the interconversion of cytidine and uridine compounds, thus allowing the independent manipulation of endogenous cytidine and uridine nucleotides, we found that a cytidine compound is the primary effector of repression in all cases except for aspartate transcarbamylase where little repression is observed in excess cytidine. For aspartate transcarbamylase, we found that the primary repressing metabolite is a uridine compound.     

UMP pyrophosphorylase of Tetrahymena pyriformis. Partial purification and properties.

UMP pyrophosphorylase (EC 2.4.2.9, UMP:pyrophosphate phosphoribosyltransferase) was purified approximately 85-fold from exponentially growing cells of Tetrahymena pyriformis GL-7. It was found to have a molecular weight of 36,000, and was active over a broad pH range, with an optimum at 7.5. The enzyme exhibited a temperature optimum at 40 °C, above which irreversible inactivation began to occur. The apparent K_m values for uracil and phosphoribosyl pyrophosphate (PRPP) were 0.4 and 6.9 m, respectively. The pyrophosphorylase exhibited a pyrimidine base specificity for uracil, although 5-fluorouracil was utilized by the enzyme. Neither cytosine, orotic acid, nor 6-azauracil competed with uracil for the enzyme or inhibited the production of UMP from uracil and PRPP. Although most triphosphates had little effect on pyrophosphorylase activity, UTP and dUTP, each at a concentration of 1 mm, depressed UMP formation by 86 and 59%, respectively. Thus, UMP pyrophosphorylase may be sensitive to feedback inhibition by the product of the pathway it initiates. UMP pyrophosphorylase specific activity in extracts of Tetrahymena grown in a medium containing uracil as the sole pyrimidine source was threefold higher than that in extracts of cells grown on uridine or UMP.     

Mutations affecting uridine monophosphate pyrophosphorylase or the argR gene in Escherichia coli. Effects on carbamoyl phosphate and pyrimidine biosynthesis and on uracil uptake

Summary In the course of experiments directed towards the isolation of mutants of Escherichia coli K12 with altered regulation of the synthesis of carbamoylphosphate synthetase, two types of mutations were found to affect the cumulative repression of this enzyme by arginine and uracil. Alteraction of the arginine pathway regulatory gene, argR, was shown to reduce the repressibility of the enzyme by both end products while mutations affecting uridine monophosphate pyrophosphorylase (upp) besides affecting uracil uptake preclude enzyme repression by uracil or cytosine in the biosynthesis of carbamoylphosphate and the pyrimidines. The upp mutations were located on the chromosome near the gua operon. Mutations previously designated as uraP are shown to belong to this class.The relation that could exist between the loss of uridine monophosphate pyrophosphorylase and the impairment of uracil uptake is discussed.A new method for isolating argR mutants in arginine-less strains is described.     

A protonmotive force as the source of energy for galactoside transport in energy depletedEscherichia Coli

Summary Uracil transport inSaccharomyces cerevisiae is mediated by a specific permease which does not recognize other pyrimidines such as uridine, cytosine, thymine, 2-hydroxypyrimidine or 5-amino-uracil; hypoxanthine and 6-amino-uracil slightly inhibit the uptake of uracil in a strain lacking cytosine permease activity. Wild type cells concentrate extracellular uracil before its transformation into UMP and subsequent incorporation into nucleic acids. A strain lacking UMP pyrophosphorylase and uridine ribohydrolase (strainfur 1–8 rh, in which the endogenous production as well as the utilization of uracil are lacking) is able to concentrate¹⁴C-2 uracil from the medium. At the same time no other¹⁴C-2 labelled compound could be detected in this strain, thus suggesting that the uptake of uracil in yeast occurs by active transport which is not coupled to the UMP pyrophosphorylase. The optimal pH of uracil uptake in standard growth conditions was 4.3. It was deduced from experiments performed on strainfur 1–8 rh with³H-5 and¹⁴C-2 uracil that the intracellular pool of uracil is recycled once the steady-state has been reached. First order kinetics with similar rate constants were observed for uracil efflux in strainfur 1–8 rh (k min^–1=0.75±0.08) as well as in the strain lacking uracil permease,fur 1–8 rh fur 4–6 (k min^–1=0.60±0.08). The intracellular pool of¹⁴C-2 uracil can be chased in strainfur 1–8 rh by addition of³H uracil without inducing a large initial acceleration of the exit rate (the rate constant remained at 0.60). 2-4-dinitrophenol inhibits the uptake of uracil but also reduces the efflux of uracil in strainfur 1–8 rh fur 4–6. These data and the comparison with cytosine transport in the same organism support the hypothesis that, whereas uracil uptake is a permease mediated active transport, the efflux of uracil does not involve the uracil uptake permease. A coefficient of permeability of 7.4×10^–7 cm sec^–1 was calculated for uracil.     

Uracil phosphoribosyltransferase from Acholeplasma laidlawii: partial purification and kinetic properties

Uracil phosphoribosyltransferase was purified 34-fold from sonicated extracts of Acholeplasma laidlawii by ammonium sulfate precipitation, binding to DEAE-Sephadex, Sephadex G-200 chromatography, and hydroxylapatite chromatography. The molecular weight of the enzyme by gel filtration was approximately 80,000. The pH optimum for phosphoribosylation was around 7.5, and the optimum MgCl2 concentration was 5 mM. Initial velocity studies were conducted over a wide range of both uracil and 5-phosphoribosyl-1-pyrophosphate (P-Rib-PP) concentrations, and various equations for biomolecular reaction mechanisms were fitted to the data by nonlinear regression. When the equation for an ordered sequential mechanism was fitted to the data, the Kia thus obtained was not statistically different from zero. This is interpreted as evidence for a nonsequential ("ping-pong") reaction. Graphic analysis of the data by the Hanes-Woolf linear transform supported this conclusion. The enzyme has high affinity for uracil (KmUra = 4.2 microM; KmP-Rib-PP = 66 microM), which provides supporting evidence that this activity is responsible for the incorporation of uracil and uridine into nucleotides.     

Transport of adenine, hypoxanthine and uracil into Escherichia coli.

Uptake of adenine, hypoxanthine and uracil by an uncA strain of Escherichia coli is inhibited by uncouplers or when phosphate in the medium is replaced by less than 1 mM-arsenate, indicating a need for both a protonmotive force and phosphorylated metabolites. The rate of uptake of adenine or hypoxanthine was not markedly affected by a genetic deficiency of purine nucleoside phosphorylase. In two mutants with undetected adenine phosphoribosyltransferase, the rate of adenine uptake was about 30% of that in their parent strain, and evidence was obtained to confirm that adenine had then been utilized via purine nucleoside phosphorylase. In a strain deficient in both enzymes adenine uptake was about 1% of that shown by wild-type strains. Uptake of hypoxanthine was similarly limited in a strain lacking purine nucleoside phosphorylase, hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase. Deficiency of uracil phosphoribosyltransferase severely limits uracil uptake, but the defect can be circumvented by addition of inosine, which presumably provides ribose 1-phosphate for reversal of uridine phosphorylase. The results indicate that there are porter systems for adenine, hypoxanthine and uracil dependent on a protonmotive force and facilitated by intracellular metabolism of the free bases.     

The FUR1 gene of Saccharomyces cerevisiae: cloning, structure and expression of wild-type and mutant alleles

The FUR1 gene of Saccharomyces cerevisiae encodes uracil phosphoribosyltransferase (UPRTase) which catalyses the conversion of uracil into uridine 5'-monophosphate (UMP) in the pyrimidine salvage pathway. The FUR1 gene is included in a 2.1 kb genomic segment of DNA and is transcribed into a 1 kb poly(A)+mRNA. Sequencing has determined a 753 bp open reading frame capable of encoding a protein of 251 amino acids. The FUR1 genes for three recessive fur1 alleles, having different sensibilities to 5-fluorouridine (5-FUR) but identical levels of resistance to 5-fluorouracil (5-FU), were cloned and sequenced. Single bp changes located in different regions of the gene were found in each mutant. Two in vitro-constructed deletions of the FUR1 gene have been integrated at the chromosomal locus, giving strains with 5-FURR and 5-FURR mutant phenotype. Assays of UPRTase, uridine kinase, uridine ribohydrolase and uridine 5'-monophosphate nucleotidase enzymatic activities, in extracts of strains where the FUR1 gene is overexpressed or deleted, indicate that the FUR1 encoded protein possesses only UPRTase activity.     

Positive selection for uracil auxotrophs of the sulfur-dependent thermophilic archaebacterium Sulfolobus acidocaldarius by use of 5-fluoroorotic acid.

Uracil auxotrophs of Sulfolobus acidocaldarius were positively selected by using 5-fluoroorotic acid. The wild-type strain was unable to grow in medium containing 5-fluoroorotic acid, whereas the mutants grew normally. Positive selection could be done for the auxotrophs. Mutants deficient in orotidine-5'-monophosphate pyrophosphorylase activity were isolated.     

Pathways of Nucleotide Biosynthesis in Mycoplasma mycoides subsp. mycoides

By measuring the specific activity of nucleotides isolated from ribonucleic acid after the incorporation of (14)C-labeled precursors under various conditions of growth, we have defined the major pathways of ribonucleotide synthesis in Mycoplasma mycoides subsp. mycoides. M. mycoides did not possess pathways for the de novo synthesis of nucleotides but was capable of interconversion of nucleotides. Thus, uracil provided the requirement for both pyrimidine ribonucleotides. Thymine is also required, suggesting that the methylation step is unavailable. No use was made of cytosine. Uridine was rapidly degraded to uracil. Cytidine competed effectively with uracil to provide most of the cytidine nucleotide and also provided an appreciable proportion of uridine nucleotide. In keeping with these results, there was a slow deamination of cytidine to uridine with further degradation to uracil in cultures of M. mycoides. Guanine was capable of meeting the full requirement of the organism for purine nucleotide, presumably by conversion of guanosine 5'-monophosphate to adenosine 5'-monophosphate via the intermediate inosine 5'-monophosphate. When available with guanine, adenine effectively gave a complete provision of adenine nucleotide, whereas hypoxanthine gave a partial provision. Neither adenine nor hypoxanthine was able to act as a precursor for the synthesis of guanine nucleotide. Exogenous guanosine, inosine, and adenosine underwent rapid cleavage to the corresponding bases and so show a pattern of utilization similar to that of the latter.     

Properties of three distinct pyrimide transport systems in yeast. Evidence for distinct energy coupling.

In Saccharomyces cerevisiae the uptake of cytosine, uracil and uridine is mediated by three permeases. Using mutants blocked in the metabolic utilization of these three compounds we were able to study their specific uptake. Cytosine and uridine show simple saturation kinetics, whereas uracil uptake is a biphasic process. A comparison of the effects of several inhibitors of energy metabolism on these uptake systems was made. Striking differences were found. 2,4-Dinitrophenol (10(-3) M) and NaN3 (10(-2) M) inhibit the entry of the three compounds to similar extent, but chlorhexidine (10(-5) M) and Dio 9 (50 microgram/ml) which are ATPase inhibitors in vitro strongly impaired cytosine and uridine entry and remained without effect on uracil uptake. We provisionally conclude that these systems may be energized by different mechanisms. In the case of cytosine and uridine permease, a membrane ATPase is possibly involved in the process of energetic coupling whereas this does not seem to be so for uracil.     

Pyrimidine biosynthetic pathway of Pseudomonas fluorescens

Pyrimidine biosynthesis in Pseudomonas fluorescens strain A126 was investigated. In this study, de novo pyrimidine biosynthetic pathway mutant strains were isolated using both conventional mutagenesis and transposon mutagenesis. The resulting mutant strains were deficient for either aspartate transcarbamoylase, dihydroorotase or orotate phosphoribosyltransferase activity. Uracil, uridine or cytosine could support the growth of every mutant strain selected. In addition, the aspartate transcarbamoylase mutant strains could utilize orotic acid to sustain their growth while the orotidine-5'-monophosphate decarboxylase mutant strains grew slowly upon uridine 5'-monophosphate. The wild-type strain and the mutant strains were used to study possible regulation of de novo pyrimidine biosynthesis in P. fluorescens. Dihydroorotase specific activity more than doubled after the wild-type cells were grown in orotic acid relative to unsupplemented minimal-medium-grown cells. Starving the mutant strains of pyrimidines also influenced the levels of several de novo pyrimidine biosynthetic pathway enzyme activities.