Thymine metabolism in Escherichia coli

Kammen, Harold O.

doi:10.1016/0005-2787(67)90008-1

Cited by 82 publications

(9 citation statements)

References 19 publications

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“…4). In this case thymidine is completely degraded to thymine during the first 5-7 min (as seen from Table 1) and deoxyadenosine added after 9 min acts as a donor of deoxyribosyl groups favouring the uptake of the thymine which has emerged [l, 3,5,6].…”

Section: Resultsmentioning

confidence: 99%

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Thymidine Uptake in Bacteria: The Effect of Purine Nucleosides

Vyacheslavov¹,

Mosevitsky²

1977

European Journal of Biochemistry

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The kinetics of thymidine uptake by Escherichia coli and Bacillus subtilis cells in the presence of adenine and guanine nucleosides was investigated. The initial concentration of thymidine in the growth medium was 0.35 pg/ml while the initial concentration of purine nucleosides ranged from 25 to 250 pg/ml. Adenine nucleosides when present at a concentration more than 50 pg/ml strongly inhibit thymidine uptake by the bacteria. The duration of the inhibition depends on the initial concentration of adenine nucleoside in the growth medium. At an initial concentration of deoxyadenosine (or adenosine) of 250 pg/ml the time of inhibition of thymidine uptake was about 60 min. During this period thymidine is almost completely preserved from the action of bacterial thymidine phosphorylase.Guanine nucleosides (guanosine or deoxyguanosine) do not markedly inhibit thymidine uptake by bacteria even at a concentration of 250 pg/ml. It is shown that they do protect thymidine from the phosphorolytic action of the thymidine phosphorylase although much less effectively than adenine nucleosides.It is suggested that some areas in the bacterial membrane where thymidine phosphorylase is located are not available to guanine nucleosides.Exogenous thymidine is incorporated into prototrophic bacteria in a very inefficient way: the process stops after a few minutes [l-41. This is due to the interaction of thymidine phosphorylase with external thymidine and its conversion to thymine [2]. Boyce and Setlow [I] showed that simultaneous addition of deoxyadenosine to a growing Escherichia coli culture leads to a more efficient uptake of the supplied thymidine. The authors concluded that in the presence of deoxyadenosine the exogenous thymidine is preserved from the degradative action of thymidine phosphorylase and thus can be taken up by cells for a longer time. This interpretation of deoxyadenosine action was accepted in subsequent papers deoxyadenosine even increased the rate of thymidine incorporation.In this work we used a broad range of adenosine and deoxyadenosine concentrations to study their effect on thymidine uptake by cells of E. coli and 3. subtilis. Our results show that adenosine and deoxyadenosine do inhibit the thymidine uptake in both species. The inhibition is practically complete provided an adenine nucleoside is present in sufficiently high excess.Guanosine and deoxyguanosine do not compete with thymidine for transport into the cell [9,10] but according to the data presented both show a definite protective action for thymidine against its phosphorolysis by the thymidine phosphorylase. MATERIALS AND METHODS ChemicalsThymine, thymidine, adenosine, deoxyadenosine, guanosine, deoxyadenosine, and 2-deoxyribose 1 -phosphate (all of A grade) were purchased from Calbiochem (U.S.A.). Casein hydrolysate and Bacto

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Section: Resultsmentioning

confidence: 99%

“…It may be suggested that some sites in the cell wall and/or cytoplasmic membrane [5,18] where thymidine phosphorylase is localized (particulary the sites placed near or inside the channels for thymidine and adenine nucleosides transport) are not available to guanine nucleosides.…”

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confidence: 99%

Thymidine Uptake in Bacteria: The Effect of Purine Nucleosides

Vyacheslavov¹,

Mosevitsky²

1977

European Journal of Biochemistry

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“…ribonucleoside triphosphates seems wasteful for the cell. However, in view of the fact that the availability of deoxyribosyl groups (in the form of deoxyribose-1-P or deoxyribonucleosides [45,46]), is the limiting factor in the anabolism of exogenous thymine, this "waste" may turn out to be of great importance in counteracting a situation that might eventually lead to death due to lack of thymine.…”

Section: Discussionmentioning

confidence: 99%

Turnover of the Deoxyribonucleoside Triphosphates in Escherichia coli 15 T during Thymine Starvation

Neuhard

Thomassen²

1971

European Journal of Biochemistry

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Since hydroxyurea specifically inhibits the biosynthesis of deoxyribonucleotides in Escherichia coli, analysis of the deoxyribonucleoside triphosphate pools following hydroxyurea addition makes possible a determination of the turnover of these compounds in vivo. This method has been used in the present work to study the effect of thymine starvation of E. coli S 5 T on the turnover of dGTP, dATP, and dCTP. From the results obtained the following conclusions may be made.I n exponentially growing cultures DNA synthesis can account for the entire turnover of the deoxyribonucleoside triphosphates.Removal of thymine from the growth medium results in the appearance of catabolic reactions that degrade dATP and dCTP. During the first 20 min of thymine starvation the rates of degradation of dATP and dCTP are of the same magnitude as those accounted for by DNA polymerization during exponential growth. After 75 min, however, the decay rate of dCTP has increased 5-fold while the rate of dATP decay remains constant. During the entire period of thymine starvation the rate of decay of dGTP is very low, i.e. one sixth of that observed during exponential growth.The end products of the catabolic reactions involved are to a large extent excreted into the growth medium as diphenylamine-reacting material.By determining the rate of accumulation of the individual deoxyribonucleoside triphosphates following thymine deprivation, and adding these values to the decay rates, determined in the presence of hydroxyurea, it is possible to estimate the endogenous rate of synthesis of dGTP, dATP and dCTP under these conditions. The results show that removal of thymine from E . coli 15 T results in a significant reduction in the rate of dGTP synthesis, essentially no effect on the rate of dATP synthesis, and an increasing rate of dCTP synthesis reaching 6-fold values after 75 min of starvation.I n previous publications [ 1,2] we have shown that removal of thymine from the medium of thymine auxotrophic mutants of Escherichia coli results in large alterations in their deox yribonucleoside triphosphate pools. Immediately upon removal of thymine from Escherichia coli 15 T and its derivatives, dATP starts to accumulate reaching 3 to 4-fold values after 30 min. The dCTP pool remains constant during the first 30 min after which time it increases linearly reaching SO to 15-fold values 90 min after thymine starvation is initiated. I n contrast, the dGTP pool does not change during the entire period of thymine starvation. It was furthermore observed [2] that when thymine auxotrophic strains are grown on suboptimal levels of exogenous thymine, there is an Enzymes. Ribonucleoside &phosphate reductase or reduced thioredoxin : ribonucleoside-5'-diphosphate oxidoreductase (EC I&--.--) ; dCTP deaminase or deoxycytidine-5'-triphosphate aminohydrolase (EC 3.5.4.-) ; dUTP pyrophosphatase or deoxyuri&ne-5'-triphosphate nucleotidohydrolase (EC 3.6.1.-).inverse relationship between the size of the thymidine nucleotide pool of the cells and the extent of accumulation of...

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“…The high dUMP pools in exponentially growing thy-dramutants indicated an increased catabolism of the pyrimidine deoxyribonucleotides, which might supply deoxyribosyl groups for thymidine synthesis. If this was the case one might expect addition of deoxyribonucleosides, which are potential deoxyribosyl donors for thymidine synthesis [25], to repress this catabolism and t o normalize the pyrimidine deoxyribonucleotide pools. That this does in fact happen is shown in Fig.…”

Section: Specific Effects On Pyrimidinementioning

confidence: 99%

“…thymine + dRib-I-P + dThd + Pi, Wild type cells do not incorporate exogenous thymine, except when deoxyribosyl groups are supplied by addition of deoxyribonucleosides to the medium [1,25]. This suggests a very low level of potential deoxyribosyl donors in these cells, a level which must be raised in a thymine requiring strain to meet the increased demands for thymidine synthesis under conditions where the thymidylate synthetase pathways are blocked.…”

Section: Thymine Incorporation In Different Anda-mutantsmentioning

confidence: 99%

Deoxyribonucleoside Catabolism and Thymine Incorporation in Mutants of Escherichia coli Lacking Deoxyriboaldolase

Munch‐Petersen¹

1970

Eur J Biochem

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Mutants of E'scherichia coli lacking the enzyme deoxyriboaldolase cannot catabolize the deoxyribose moiety of deoxyribonucleosides. When deoxyribonucleosides are added to the growth medium a severe growth inhibition is observed under aerobic as well as anaerobic conditions. The toxic substance is presumably deoxyribose-5-phosphate, which is shown t o accumulate in the cells. Normal growth is restored when this compound is broken down or if a ribonucleoside is added simultaneously t o the medium.dra-Mutants arise spontaneously as secondary mutants from cultures of thymine-requiring mutants, isolated by means of the trimethoprim selection procedure.dra-Mutants may also be obtained by mutagenization, penicillin treatment, and selection on thymidine as carbon source. Mutants, isolated in this way, have an additional mutation in the deoxyribonucleoside catabolizing system, which has been identified as an altered thymidine phosphorylase.A number of different thy-and dra-strains have been screened for incorporation of exogenous thymine in relation to pyrimidine deoxyribonucleotide pools. Low pools of dTTP and high pools of dUMP are found in all thymine incorporating mutants. It is concluded that incorporation depends primarily on the cellular pools of thymine nucleotides, which by regulatory mechanisms determine the availability of deoxyribosyl donors for thymidine synthesis. Catabolism of deoxyribonucleosides in Esckrichia cola' occurs through the following reactions :Purine deoxyribonucleoside purine basePyrimidine deoxyribonucleoside pyrimidine base dRib-1-P + Or'+ pi purine nueleoside phosphorylasethymidine phosphorylaseIf deoxyribonucleosides are added to the growth medium, the enzymes catalyzing these reactions are induced several-fold [l-61, and the deoxyribose moiety of the added deoxyribonucleoside is rapidly metabolized. One of the intermediates in the metabolic sequence, dRib-5-P, is supposed to be the inducer If the cells lack deoxyriboaldolase, for which dRib-5-P is the substrate, addition of deoxyribonucleosides causes inhibition of growth. dRib-5-P has been suggested [6,8,9] as the actual inhibitor, and this is supported by the results presented in this paper where the inhibitory effect and its consequences for cell metabolism have been studied more closely.Thymine auxotrophy is another metabolic condition in which the metabolism of deoxyribonucleosides becomes important. Since auxotrophic cells have lost the methylating systems which normally lead to the formation of dTTP, thymine for DNA-synthesis must be supplied from the outside. The initial reaction in the sequence of processes that lead to thymine incorporation into DNA (see Fig.1 Fig.l), and the methylation and deamination of dCTP to dTTP as reported by Holldorf and coworkers [1l,i2] (Reaction 2 in Fig.1 I n thymine auxotrophs the loss of these pathways necessitates a channeling of the flow of deoxyribosyl groups required for synthesis of dTTP through deoxyribonucleosides and deoxyribose-1-phosphate for synthesis of thymidine from exogenous thymin...

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Thymine metabolism in Escherichia coli

Cited by 82 publications

References 19 publications

Thymidine Uptake in Bacteria: The Effect of Purine Nucleosides

Thymidine Uptake in Bacteria: The Effect of Purine Nucleosides

Turnover of the Deoxyribonucleoside Triphosphates in Escherichia coli 15 T during Thymine Starvation

Deoxyribonucleoside Catabolism and Thymine Incorporation in Mutants of Escherichia coli Lacking Deoxyriboaldolase

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