JOURNAL OF BACTZRIOLOGY, JUlY 1976, p. 1-6 Copyright © 1976 American Society for Microbiology
Vol. 127, No. 1 Printed in U.S.A.
Utilization of Exogenous Pyrimidines as a Source of Nitrogen by Cells of the Yeast Rhodotorula glutinis 0.
A. MILSTEIN
AND
M. L. BEKKER*
Microbiological Department of the Latvian State University, Riga, and Leningrad Nuclear Physics Institute Academy of Sciences of the USSR, Gtachina, Leningrad District, 188350 USSR* Received for publication 26 November 1975
Uptake and intracellular transformation of pyrimidines supplying cells of the yeast Rhodotorula glutinis with nitrogen have been studied. The amine nitrogen of cytosine was found to be the easiest to utilize. The presence in the medium of inorganic ammonia along with cytosine had a slight effect on cytosine deaminase (EC 3.5.4.1) activity. The uracil produced entered into the nutrient medium with no fission break of the pyrimidine ring. In the absence of any other source of nitrogen, the cells of the yeast R. glutinis utilized nitrogen of the pyrimidine ring of oxypyrimidines. Catabolism of uracil followed the reductive pattern, with release of carbon dioxide; this was accompanied by synthesis of the key enzyme of pyrimidine catabolism, dihydrouracil dehydrogenase (EC 1.3.1.1), whose activity rose 10-fold. With thymine as the sole source of nitrogen, the lagphase growth of the yeast cells was maximum. Catabolism of the pyrimidine ring of thymine was possibly preceded by its transformation into uracil. With no source of nitrogen easily utilized, the uridine 5'-monophosphate content in the
generally acid-soluble pool rose. Our discussion of the regulation of catabolism of exogenous pyrimidine bases by the yeast R. glutinis takes into account the fact that transformations of pyrimidine bases are determined by how easily the cells can use a particular base as a source of nitrogen. The features characterizing the uptake of ex- determined by how easily the cells can use a ogenous pyrimidines, their involvement in cell particular base as a source of nitrogen. metabolism, and their effect on the de novo MATERIALS AND METHODS synthesis of pyrimidine nucleotides in bacteria and fungi are described in a number of publicaGrowth cells. The yeast used was R. glutinis tions (5, 14, 19, 23). It is also known that many (Fresenius)ofHarrison. Organisms were cultured in species of the yeast are capable of utilizing Reader medium (26), with (NH4)2SO4 substituted by exogenous cytosine and oxypyrimidines as the 4 mM cytosine, uracil, or thymine. When two pyrimsole source of nitrogen (8, 9, 21, 29). In this idine bases were used simultaneously, their total connection, it is of interest to study the catabo- concentration did not exceed 4 mM. '4C-labeled lism pathways for pyrimidines and their regu- pyrimidines (in the second position of the heterowere added to give a radioactivity of 3 ,uCi/ml. lation in microorganisms. Whereas much infor- cycle) The inoculatum was grown in a petri dish on a mation is now available concerning the degra- dense nutrient of the same content for 48 h dation of exogenous pyrimidine in bacteria (4, at 28 C. A 1-mlmedium suspension of cells washed with 6, 7, 15, 20, 30), there have been only few and phosphate buffer was added to the culture medium incomplete studies on yeasts. These studies so that there were 5 x 106 to 5.5 x 106 cells/ml in deal with the enzyme activity of cytosine catab- the medium. Cells were cultured at 27 C under inolism (16, 18) and accumulation of transforma- tensive aeration. Growth was monitored by direct counting of the tion products of thymine, 5-methylcytosine, number of cells in a counting chamber. and uracil in the culture medium (25, 29). Separation and determination of nitrogen bases, In this paper we present data on catabolism and nucleosides. Free nucleotides and nucleotides, pathways for exogenous cytosine and the oxy- their derivatives were extracted from cells for 1 h pyrimidines uracil and thymine in cells of the with 0.6 N HC104 and for 0.5 h with 0.2 N HC104 at 0 no yeast Rhodotorula glutinis when there are to 4 C. Chlorates were precipitated in the form of sources of nitrogen except pyrimidines. Trans- potassium salt. Neutral extract was applied to a port, intracellular transformations, and regula- column with Dowex I x 8 (200 to 400 mesh) in the tion of these processes are shown to be affected formate form. Nonsorbed compounds were removed by the nitrogen supply ofthe cells; i.e., they are by washing the column with water; the others were 1
2
MILSTEIN AND BEKKER
eluted with solutions of formic acid and ammonium formate (13) at a rate of 0.15 to 0.2 ml/min. The volume of the fractions was 7 to 8 ml. Combined fractions of the corresponding peaks were lyophilized, dissolved in a small volume of water, and chromatographed on paper in the following solvents: (i) n-butanol-ethanol-HCOOH-water (30:36:5:20); (ii) n-butanol-acetate-water (12:5:5); (iii) isopropyl2 N HCI (65:35); (iv) ethanol-i M ammonium acetate, pH 7.5 (5:2); and (v) ethyl acetate water (3:1). Pyrimidine derivatives were identified according to the position of a peak analyzed on the elution curve, from the chromatographic mobility of a particular compound in the above solvents, and from ultraviolet absorption spectra of the chromatogram eluate in acid and alkaline media. Determination of enzyme activity. Dihydrouracil dehydrogenase (EC 1.3.1.1) was determined according to a method proposed by Reichard and Skold (27) in a 3-ml incubation mixture containing 200 ,umol of phosphate buffer, pH 7.3, 0.5 ,umol of reduced nicotinamide adenine dinucleotide (NADH), and 0.1 ml of supernatant fluid with the cell homogenate. The reaction started after addition to the incubation mixture of 25 ,.mol of uracil or thymine. Optical density of the mixture was taken at 340 nm and 25 C for 5 min against the control, in which there were all the compounds of the incubation mixture except uracil or thymine. The rate of decrease in the optical density at 340 nm caused by transformation of NADH into NAD, when pyrimidine is being transformed into dihydropyrimidine, corresponds to the enzyme activity being determined. A decrease in optical density of 0.001 unit/min was considered one activity unit. Cytosine deaminase (EC 3.5.4.1) was determined according to Sakai et al. (28) in 3 ml of incubation fluid containing 200 jAmol of phosphate buffer, pH 7.0, and 0.1 ml of cell homogenate supernatant. The reaction was started by adding 50 ,umol of cytosine to the reaction mixture. Optical density of the mixture was measured at 285 nm, 25 C, for 5 min. A decrease in optical density of 0.001 unit/min was considered one activity unit. Specific activity of the enzymes was expressed as units per 1 mg of protein. Chemicals. Nitrogen bases, nucleosides, and nucleotides were from Reanal, Hungary. NADH was from Calbiochem. [2-14C]uracil (specific activity, 5.1 mCi/mmol) and [2-'4C]thymine (specific activity, 25.6 mCi/mmol) were from Isotop, USSR.
J. BACTERIOL.
Utilization of exogenous pyrimidines. Shortly after the inoculation of yeast into a medium with a pyrimidine as the sole source of nitrogen, a decrease in the concentration of the compound under study occurred, the decrease in cytosine concentration being observed earlier than in that of oxypyrimidines (Fig. 2). Utilization of oxypyrimidines as sources of nitrogen assumes a break in the heterocyclic ring. Pyrimidine catabolism of the reductive
.81 E
8
12
16
go
JOURS FIG. 1. Growth of the yeast R. glutinis on media with different sources of nitrogen. Abscissa indicates the incubation time; ordinate indicates 1 g of cell number per ml. Cultivation conditions are described in the text. The cells were cultivated in media with 0.1% (NH4)2SO4 (a); 4 mM cytosine (A); 4 mM uracil (U); and 4 mM thymine (x).
RESULTS HOURS Effect of exogenous pyrimidines as the FIG. 2. Content ofpyrimidine bases in culture mesource of nitrogen on yeast growth. R. glu- dia as a function of growth of R. glutinis. Abscissa tinis could grow in a medium with cytosine, indicates the incubation time; ordinate indicates the uracil, or thymine as the sole source of nitro- concentration of a base in the cultural medium. The were cultivated in media with 4 mM thymine gen. When the yeast grew on a medium with cells (a); 4 mM uracil (U); and 4 mM cytosine (A) as sole oxypyrimidines as the sole source of nitrogen, source nitrogen. (x) Uracil concentration in the the lag phase was longer compared with that culture of medium in the presence ofcytosine as the sole observed in the case of cytosine-containing me- source of nitrogen. Concentration of pyrimidines in dium. The lag phase was maximum in a me- the medium was determined by paper chromatogradium with thymine (Fig. 1). phy (see text).
VOL. 127, 1976
type proceeded with formation, along with other products, of carbon dioxide whose carbon originated from the second position in the pyrimidine ring. With [2-14Clpyrimidine, a decrease in total radioactivity of the culture may thus serve as a quantitative measure of the reductive catabolism of the corresponding compound. Five hours after starting incubation of the yeast in a medium with uracil as the sole source of nitrogen, a decrease in total radioactivity was observed. Catabolism of thymine began somewhat later (Fig. 3). Thymine (2 mM) added to a medium with uracil immediately after or 6 h after inoculation of the yeast had no effect on the extent of uracil catabolism. Addition of uracil into a yeast culture growing on a medium with labeled thymine inhibited thymine catabolism. The presence in the culture medium of nonorganic ammonia also drastically decreased the extent of pyrimidine catabolism, which was not observed until 14 to 15 h after growth began. Twenty-four hours after inoculation of the yeast, the total radioactivity of the culture given thymine or uracil as the sole source of nitrogen decreased by 89 or 94%, rocinarfluals A iP1 1) 1). umpmu.sV'kY (Tahlo
3
USE OF EXOGENOUS PYRIMIDINES BY R. GLUTINIS
At later stages of growth of the cells on me-
TABLE 1. Changes in total radioactivity ofa culture of the yeast R. glutinis incubated in medium with labeled thymine and uracil for 24 h Radioactive compound"
Additional source of tDtalradeion nitrogen activity (%)b
[2-'4Clthymine
[2-'4C]thymine 12-'4C]thymine [2-'4C]thymine
12-'4C]thymine' 12-'4C]thymine 12-'4C]uracil 12-'4C]uracil [2-'4C]uracile [2-'4C]uracil
Uracil (2 mM) Uracil (4 mM) Cytosine (2 mM) Uracil (2 mM) (NH4)2SO4 (0.1%)
Thymine (2 mM) Thymine (2 mM) (NH4)2SO4 (0.1%)
89 76 0 59 8 21 94 89 85 15
" Specific activity of the culture was 3 ACi/ml. A nonlabeled compound was added to the labeled one so that the total concentration of this base was 2 mM. b The total radioactivity of the culture is the sum of the radioactivity of cells and cultural medium per milliliter of culture. It was determined as follows: 0.1 ml of culture was placed into the aluminum target and was dried at 105 C. Radioactivity was measured by a gas-flow counter. The radioactivity of the culture before the beginning of cultivation was taken as 100%. c In this sample, radioactive compound was added 6 h after beginning cultivation on the medium with "additional source of nitrogen."
dium with thymine as the
source of nitrogen,
both thymine and products of its transformation into uracil, oxymethyluracil and carboxyuracil (0.90, 0.27, and 0.12 ,umol per g [dry *=weight] of cells, respectively) were found in the _ '4o cells. The concentration of uridine 5'-monophosphate in acid-soluble compounds in cells 3\s ] 7S growing on a medium with thymine and uracil was much higher than that in cells growing on % to a medium with ammonium sulfate as the sole 5s 0s source of nitrogen (3.22, 2.24, and 0.51 ,umol per g [dry weight] of cells, respectively). Enzymes of pyrimidine catabolism. When the yeast was incubated on a medium with 4 6 * t0 a a cytosine as the sole source of nitrogen, cytosine deaminase (EC 3.5.4.1) was found. The enzyme HOUR6 could be seen as early as 3 to 4 h after the FIG. 3. Catabolism of [2-'4C]uracil and [2-bgnigo nuaio nti eim m in th medium e '4C]thymine as a function of growth of R. glutinis. monium sf whenb resen Abscissa indicates the incubation time; ordinate in- with h sulfate e esecton theactium dicates the total radioactivity of the culture. For the with cytosine had lIttle effect on the activity of determination of total radioactivity of the culture, see this enzyme (Fig. 4). Its maximum activity was footnotes of Table 1. The cells were cultivated in a observed at the beginning and midpoint of the medium with (1) 2 mM [2-'4C]thymine plus 2 mM logarithmic phase of growth. The product ofthe nonlabeled uracil (U); (2) 2 mM[2-'4C]thymine plus reaction, uracil, was released into the culture 2 mM nonlabeled uracil added 6 h after beginning medium, where it could be found at all times 5 ofcultivation (0); (3)2 mM[2-'4CJthymine (0); (4)2 h after the beginning of cultivation. As stated mM [2-14C]uracil plus 2 mM nonlabeled thymine above, the total radioactivity of cells and cul(A); and (5) 2 mM [2-'4C]uracil plus 2 mM nonla- ture fluid was drastically decrased when pyrimbeled thymine added 6 h after the beginning of idines were the sole source of nitrogen in the of the cultivation (0). Total radioactivity before the begin- med ine which soles ning of cultivation was 3 uCi/ml. To obtain the base medium, which indicates that the pyrimidine total concentration as indicated above, nonlabeled ring undergoes breakage that follows the reductive pattern, C02 being liberated. The first enbases were added to the labeled ones.
4
J. BACTERIOL.
MILSTEIN AND BEKKER
TABLE 2. Activity of dihydrouracil dehydrogenase in the yeast R. glutinis during the logarithmic phase of growth Source of nitrogen Source of nltrogen
4
8
12
16
20
HOURS FIG. 4. Cytosine deaminase activity in cells of R. glutinis. Abscissa indicates the incubation time; ordinate indicates the enzyme activity. The cells were cultivated in media with 0.1% (NH4)2SO4plus 4 mM cytosine (a) and 4 mM cytosine (A).
zyme to involve pyrimidines in such transformations is dihydrouracil dehydrogenase (EC 1.3.1.1.). With no exogenous pyrimidines, the activity of this enzyme was 1; it rose sixfold when uracil or thymine was present in the medium in addition to ammonium sulfate, and it rose tenfold without uracil (Table 2). Under these conditions, the enzyme activity rose exponenfially until 14 to 16 h of incubation (Fig. 5).
DISCUSSION The data presented above indicate that without easily utilized sources of nitrogen, the cells of the yeast R. glutinis can use exogenous pyrimidines as a source of nitrogen. Amine nitrogen of cytosine is the easiest to utilize, almost as easy as inorganic ammonia. Indicative of this is the fact that cytosine deaminase activity does not decrease markedly in the presence of ammonium sulfate. In addition, cytosine inhibits utilization of thymine and uracil as sole sources of nitrogen when they are present together in the nutrient medium. With no cytosine, catabolism of oxypyrimidines is considerably enhanced. The breakage of the pyrimidine ring occurs as a reductive process, which occurs after a decrease in the radioactivity of the culture (Table 1, Fig. 3) and a sharp increase in dihydrouracil dehydrogenase activity when the yeast grows on a medium with oxypyrimidines as the sole source of nitrogen (Table 2). Increase in the enzyme activity and extent of decrease in the radioactivity of the medium correlate well (compare Fig. 3 with Fig. 5). When cells grow on the medium containing
act of enSp zyme"
1 (NH4)2SO4 46.0 Thymine (4 mM) 6.2 Thymine (4 mM) + (NH4)2SO4 (0. 1%) 60.2 Uracil (4 mM) 6.7 Uracil (4 mM) + (NH4)2SO4 (0. 1%) 20.3 Thymine (2 mM) + cytosine (2 mM) 19.5 Uracil (2 mM) + cytosine (2 mM) a Expressed as optical density units per milligram of protein.
r. wo S
HOURS FIG. 5. Dihydrouracil dehydrogenase activity in cells of R. glutinis. Abscissa indicates the incubation time; ordinate indicates the enzyme activity. The cells were cultivated in media with 0.1% (NH4)2S04 (a); 0.1% (NH4)2S04 plus 4 mM thymine (-OZ-*-); 0.1% (NH4) 2S04 plus 4 mM uracil (-*-*-); 4 mM thymine (0); and 4 mM uracil (a).
uracil, the key enzyme of uracil catabolism, dihydrouracil dehydrogenase, is induced in the cells; the activity of this enzyme is very low in the cells cultured on the standard medium and remains at the same level during the whole period of incubation (Fig. 5). We failed to find in R. glutinis the direct pathway of reductive catabolism of thymine reported for liver cells (12, 23). Our findings indicate that a more probable explanation is that exogenous thymine incorporated by the cells is first transformed into uracil through demethylation, as was reported for liver cells (11,12) and Neurospora crassa (1-3, 10, 22, 24, 31, 32; M. T. Abbott and R. M. Fink, Fed. Proc. 21:377, 1962; R. M. Fink and K. Fink, Fed. Proc. 21:377,
USE OF EXOGENOUS PYRIMIDINES BY R. GLUTINIS
VOL. 127, 1976
1962). This pathway is probably a narrow place for the whole process of utilization of nitrogen of thymine. The following facts suggest that this may be the case: (i) the yeast cells begin to multiply on a medium with thymine later than on a medium with uracil (Fig. 1); (ii) catabolism of thymine begins markedly later than that of uracil (Fig. 3); (iii) uracil inhibits catabolism of thymine (Fig. 3); and (iv) at the later stages.of cell growth on the medium with thymine, both thymine and products of its transformation into uracil are found in the cells. Other authors also found these products in culture medium when cultivating R. glutinis (29). On the basis of the evidence presented, the following suggestion can be made concerning the use of exogenous pyrimidines as sole source of nitrogen. (i) Cytosine is incorporated into the cells and deaminated to form uracil and ammonia. (ii) Uracil is utilized as a source of nitrogen, and the ammonia is partly taken into the medium. With no other source of nitrogen, there is a breakage in the pyrimidine ring of uracil, and the transformation of thymine into uracil is inhibited. Thymine catabolism begins with transformation into uracil. When the cells are deficient in nitrogen, all cell functions of importance, including biosynthesis of nucleic acids, are inhibited. Similar results have been previously reported for the yeast Candida utilis (17). Under this condition, the content of pyrimidine nucleotides and uridine 5-'-monophosphate in the acid-soluble pool is increased. The products in this pool inhibit their own biosynthesis, directing transformation of uracil along the pathway of catabolism. In this way the cells make use of regulatory mechanisms to ensure their basic needs. To summarize, the experimental results presented suggest that when exogenous pyrimidines are the sole source of nitrogen for the yeast R. glutinis, their transport into the cell and their intracellular transformations are governed by the ease with which cells are capable of liberating nitrogen from these com-
pounds. LITERATURE CITED 1. Abbott, M. T., T. A. Dragila, and R. P. McCroskey. 1968. The formation of 5-formyluracil by cell-free preparation from Neurospora crassa. Biochim. Biophys. Acta 169:1-6. 2. Abbott, M. T., R. I. Kadner, and R. M. Fink. 1964. Conversion of thymine to 5-hydroxymethyluracil in a cell-free system. J. Biol. Chem. 239:156-159. 3. Abbott, M. T., E. K. Schandl, R. F. Lee, T. S. Parker, and R. I. Midgett. 1967. Cofactor requirements of
thymine 7-hydroxylase. Biochim. Biophys. Acta 132:325-528. 4. Ban, J., L. Vitalle, and E. Kos. 1972. Thymine and
5
uracil catabolism in E. coli. J. Gen. Microbiol. 73:267-272. 5. Beck, C. F., J. L. Ingraham, J. Neuhard, and E. Thomassen. 1972. Metabolism of pyrimidines and pyrimidine nucleosides by Salmonella typhimurium. J. Bacteriol. 110:219-228. 6. Campbell, L. L. 1957. Reductive degradation of pyrimidines. I. The isolation and characterization of a uracil fermenting bacterium, Clostridium uracilicum nov. spec. J. Bacteriol. 73:220-224. 7. Campbell, L. L. 1957. Reductive degradation of pyrimidines. II. Mechanism of uracil degradation by Clostridium uracilicum. J. Bacteriol. 73:225-229. 8. DiCarlo, F. I., A. S. Schultz, and A. M. Kent. 1952. On the mechanism of pyrimidine metabolism by yeasts. J. Biol. Chem. 199:333-343. 9. DiCarlo, F. I., A. S. Schultz, and D. K. McManus. 1951. The assimilation of nucleic acid derivatives and relative compounds by yeasts. J. Biol. Chem. 189:151-157. 10. Fink, R. M., and K. Fink. 1962. Utilization of radiocarbon from thymidine and other precursors of ribonucleic acid in Neurospora crassa. J. Biol. Chem. 237:2289-2290. 11. Fink, K., R. E. Cline, R. B. Henderson, and R. M. Fink. 1956. Metabolism of thymine (methyl-5C'4 or -2C'4) by rat liver in vitro. J. Biol. Chem. 221:425433. 12. Fink, R. M., C. McGaughey, R. E. Cline, and K. Fink. 1956. Metabolism of intermediate pyrimidine products in vitro. J. Biol. Chem. 218:1-7. 13. Gilbert, D. A., and E. W. Yemm. 1958. Soluble nucleotides and nucleoside amino acid compounds of yeasts. Nature (London) 182:1745-1746. 14. Grenson, M. 1969. The utilization of exogenous pyrimidines and the recycling of uridine-5-phosphate derivatives in Saccharomyces cerevisiae, as studied by means of mutants affected in pyrimidine uptake and metabolism. Eur. J. Biochem. 2:249-260. 15. Hayashi, O., and A. Kornberg. 1952. Metabolism of cytosine, thymine, uracil and barbituric acid by bacterial enzymes. J. Biol. Chem. 197:717-732. 16. Ipata, P. L., P. Marmocchi, G. Magni, R. Felicioli, and G. Polidoro. 1971. Baker's yeast cytosine deaminase. Some enzymic properties and allosteric inhibition by nucleosides and nucleotides. Biochemistry 10:4270-4276. 17. Jones, R. W., and D. G. Wild. 1973. Regulation of uptake of purines, pyrimidines and amino acid by Candida utilis. Biochem. J. 134:617-627. 18. Kream, J., and E. Chargaff. 1952. On the cytosine deaminase of yeast. J. Am. Chem. Soc. 74:5157-5160. 19. Lacroute, F. 1968. Regulation of pyrimidine biosynthesis in Saccharomyces cerevisiae. J. Bacteriol. 95: 824-832. 20. Lara, F. I. 1952. On the decomposition of pyrimidine by bacteria. I. Studies by means of the technique of simultaneous adaption. J. Bacteriol. 64:271-278. 21. LaRue, T. A., and I. F. T. Spencer. 1968. The utilization of purines and pyrimidines by yeasts. Can. J. Microbiol. 14:79-85. 22. McCroskey, R. P., W. R. Griswold, R. L. Sokoloff, E. D. Sevier, S. Lin, C. K. Liu, P. M. Shaffer, R. D. Palmatier, T. S. Parker, and M. T. Abbott. 1971. Studies pertaining to the purification and properties of thymine-7-hydrolase. Biochim. Biophys. Acta 227:264-275. 23. O'Donovan, G. A., and J. Neuhard. 1970. Pyrimide catabolism in microorganisms. Bacteriol. Rev. 34: 278-343. 24. Palmatier, R. D., R. P. McCroskey, and M. T. Abbott. 1970. The enzymatic conversion of uracil 5-carboxylic acid to uracil and carbon dioxide. J. Biol. Chem. 245:6706-6710.
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25. Piret, M. C., R. Cuokaert, and J. Christophe. 1964. Le catabolisme reductif de l'uracile cher Torulopiu utilis. Arch. Int. Physiol. Biochem. 72:257-265. 26. Reader, V. 1927. The relation of the growth of certain microorganisms to the composition of the medium. I. The synthetic culture medium. Biochem. J. 21: 901-912. 27. Reichard, P., and 0. Sk6ld. 1963. Pyrimidine synthesis and breakdown. Methods Enzymol. 6:177-197. 28. Salai, T., T. Watanabe, and I. Chibata. 1971. Metabolism of pyrimidine nucleotides in bacteria. II. Studies on the regulation system of the degradation of nucleotides in Pseudomonaa oleovorans. J. Ferment. Technol. 49.488-498.
J. BACTZRIOL. 29. Vilks, S. R., and M. J. Vitols. 1973. Assimilation and catabolism of 5-methylcytosine and thymine by Rhodotorula glutinis (Fres.) Harrison. Microbiologia 4:576-582. 30. Wang, T. P., and I. 0. Lampen. 1952. Metabolism of pyrimidines by soil bacterium. J. Biol. Chem. 194: 775-783. 31. Watanabe, M. S., R. P. McCroskey, and M. T. Abbott. 1970. The enzymatic conversion of 5-formyl-uracil to uracil 5-carboxylic acid. J. Biol. Chem. 245: 2023-2026. 32. Williams, L. G., and H. K. Mitchell. 1969. Mutants affecting thymidine metabolism in Neurospora crassa. J. Bacteriol. 100:383-389.
Vol. 127, No. 1 Printed in U.S.A.
Utilization of Exogenous Pyrimidines as a Source of Nitrogen by Cells of the Yeast Rhodotorula glutinis 0.
A. MILSTEIN
AND
M. L. BEKKER*
Microbiological Department of the Latvian State University, Riga, and Leningrad Nuclear Physics Institute Academy of Sciences of the USSR, Gtachina, Leningrad District, 188350 USSR* Received for publication 26 November 1975
Uptake and intracellular transformation of pyrimidines supplying cells of the yeast Rhodotorula glutinis with nitrogen have been studied. The amine nitrogen of cytosine was found to be the easiest to utilize. The presence in the medium of inorganic ammonia along with cytosine had a slight effect on cytosine deaminase (EC 3.5.4.1) activity. The uracil produced entered into the nutrient medium with no fission break of the pyrimidine ring. In the absence of any other source of nitrogen, the cells of the yeast R. glutinis utilized nitrogen of the pyrimidine ring of oxypyrimidines. Catabolism of uracil followed the reductive pattern, with release of carbon dioxide; this was accompanied by synthesis of the key enzyme of pyrimidine catabolism, dihydrouracil dehydrogenase (EC 1.3.1.1), whose activity rose 10-fold. With thymine as the sole source of nitrogen, the lagphase growth of the yeast cells was maximum. Catabolism of the pyrimidine ring of thymine was possibly preceded by its transformation into uracil. With no source of nitrogen easily utilized, the uridine 5'-monophosphate content in the
generally acid-soluble pool rose. Our discussion of the regulation of catabolism of exogenous pyrimidine bases by the yeast R. glutinis takes into account the fact that transformations of pyrimidine bases are determined by how easily the cells can use a particular base as a source of nitrogen. The features characterizing the uptake of ex- determined by how easily the cells can use a ogenous pyrimidines, their involvement in cell particular base as a source of nitrogen. metabolism, and their effect on the de novo MATERIALS AND METHODS synthesis of pyrimidine nucleotides in bacteria and fungi are described in a number of publicaGrowth cells. The yeast used was R. glutinis tions (5, 14, 19, 23). It is also known that many (Fresenius)ofHarrison. Organisms were cultured in species of the yeast are capable of utilizing Reader medium (26), with (NH4)2SO4 substituted by exogenous cytosine and oxypyrimidines as the 4 mM cytosine, uracil, or thymine. When two pyrimsole source of nitrogen (8, 9, 21, 29). In this idine bases were used simultaneously, their total connection, it is of interest to study the catabo- concentration did not exceed 4 mM. '4C-labeled lism pathways for pyrimidines and their regu- pyrimidines (in the second position of the heterowere added to give a radioactivity of 3 ,uCi/ml. lation in microorganisms. Whereas much infor- cycle) The inoculatum was grown in a petri dish on a mation is now available concerning the degra- dense nutrient of the same content for 48 h dation of exogenous pyrimidine in bacteria (4, at 28 C. A 1-mlmedium suspension of cells washed with 6, 7, 15, 20, 30), there have been only few and phosphate buffer was added to the culture medium incomplete studies on yeasts. These studies so that there were 5 x 106 to 5.5 x 106 cells/ml in deal with the enzyme activity of cytosine catab- the medium. Cells were cultured at 27 C under inolism (16, 18) and accumulation of transforma- tensive aeration. Growth was monitored by direct counting of the tion products of thymine, 5-methylcytosine, number of cells in a counting chamber. and uracil in the culture medium (25, 29). Separation and determination of nitrogen bases, In this paper we present data on catabolism and nucleosides. Free nucleotides and nucleotides, pathways for exogenous cytosine and the oxy- their derivatives were extracted from cells for 1 h pyrimidines uracil and thymine in cells of the with 0.6 N HC104 and for 0.5 h with 0.2 N HC104 at 0 no yeast Rhodotorula glutinis when there are to 4 C. Chlorates were precipitated in the form of sources of nitrogen except pyrimidines. Trans- potassium salt. Neutral extract was applied to a port, intracellular transformations, and regula- column with Dowex I x 8 (200 to 400 mesh) in the tion of these processes are shown to be affected formate form. Nonsorbed compounds were removed by the nitrogen supply ofthe cells; i.e., they are by washing the column with water; the others were 1
2
MILSTEIN AND BEKKER
eluted with solutions of formic acid and ammonium formate (13) at a rate of 0.15 to 0.2 ml/min. The volume of the fractions was 7 to 8 ml. Combined fractions of the corresponding peaks were lyophilized, dissolved in a small volume of water, and chromatographed on paper in the following solvents: (i) n-butanol-ethanol-HCOOH-water (30:36:5:20); (ii) n-butanol-acetate-water (12:5:5); (iii) isopropyl2 N HCI (65:35); (iv) ethanol-i M ammonium acetate, pH 7.5 (5:2); and (v) ethyl acetate water (3:1). Pyrimidine derivatives were identified according to the position of a peak analyzed on the elution curve, from the chromatographic mobility of a particular compound in the above solvents, and from ultraviolet absorption spectra of the chromatogram eluate in acid and alkaline media. Determination of enzyme activity. Dihydrouracil dehydrogenase (EC 1.3.1.1) was determined according to a method proposed by Reichard and Skold (27) in a 3-ml incubation mixture containing 200 ,umol of phosphate buffer, pH 7.3, 0.5 ,umol of reduced nicotinamide adenine dinucleotide (NADH), and 0.1 ml of supernatant fluid with the cell homogenate. The reaction started after addition to the incubation mixture of 25 ,.mol of uracil or thymine. Optical density of the mixture was taken at 340 nm and 25 C for 5 min against the control, in which there were all the compounds of the incubation mixture except uracil or thymine. The rate of decrease in the optical density at 340 nm caused by transformation of NADH into NAD, when pyrimidine is being transformed into dihydropyrimidine, corresponds to the enzyme activity being determined. A decrease in optical density of 0.001 unit/min was considered one activity unit. Cytosine deaminase (EC 3.5.4.1) was determined according to Sakai et al. (28) in 3 ml of incubation fluid containing 200 jAmol of phosphate buffer, pH 7.0, and 0.1 ml of cell homogenate supernatant. The reaction was started by adding 50 ,umol of cytosine to the reaction mixture. Optical density of the mixture was measured at 285 nm, 25 C, for 5 min. A decrease in optical density of 0.001 unit/min was considered one activity unit. Specific activity of the enzymes was expressed as units per 1 mg of protein. Chemicals. Nitrogen bases, nucleosides, and nucleotides were from Reanal, Hungary. NADH was from Calbiochem. [2-14C]uracil (specific activity, 5.1 mCi/mmol) and [2-'4C]thymine (specific activity, 25.6 mCi/mmol) were from Isotop, USSR.
J. BACTERIOL.
Utilization of exogenous pyrimidines. Shortly after the inoculation of yeast into a medium with a pyrimidine as the sole source of nitrogen, a decrease in the concentration of the compound under study occurred, the decrease in cytosine concentration being observed earlier than in that of oxypyrimidines (Fig. 2). Utilization of oxypyrimidines as sources of nitrogen assumes a break in the heterocyclic ring. Pyrimidine catabolism of the reductive
.81 E
8
12
16
go
JOURS FIG. 1. Growth of the yeast R. glutinis on media with different sources of nitrogen. Abscissa indicates the incubation time; ordinate indicates 1 g of cell number per ml. Cultivation conditions are described in the text. The cells were cultivated in media with 0.1% (NH4)2SO4 (a); 4 mM cytosine (A); 4 mM uracil (U); and 4 mM thymine (x).
RESULTS HOURS Effect of exogenous pyrimidines as the FIG. 2. Content ofpyrimidine bases in culture mesource of nitrogen on yeast growth. R. glu- dia as a function of growth of R. glutinis. Abscissa tinis could grow in a medium with cytosine, indicates the incubation time; ordinate indicates the uracil, or thymine as the sole source of nitro- concentration of a base in the cultural medium. The were cultivated in media with 4 mM thymine gen. When the yeast grew on a medium with cells (a); 4 mM uracil (U); and 4 mM cytosine (A) as sole oxypyrimidines as the sole source of nitrogen, source nitrogen. (x) Uracil concentration in the the lag phase was longer compared with that culture of medium in the presence ofcytosine as the sole observed in the case of cytosine-containing me- source of nitrogen. Concentration of pyrimidines in dium. The lag phase was maximum in a me- the medium was determined by paper chromatogradium with thymine (Fig. 1). phy (see text).
VOL. 127, 1976
type proceeded with formation, along with other products, of carbon dioxide whose carbon originated from the second position in the pyrimidine ring. With [2-14Clpyrimidine, a decrease in total radioactivity of the culture may thus serve as a quantitative measure of the reductive catabolism of the corresponding compound. Five hours after starting incubation of the yeast in a medium with uracil as the sole source of nitrogen, a decrease in total radioactivity was observed. Catabolism of thymine began somewhat later (Fig. 3). Thymine (2 mM) added to a medium with uracil immediately after or 6 h after inoculation of the yeast had no effect on the extent of uracil catabolism. Addition of uracil into a yeast culture growing on a medium with labeled thymine inhibited thymine catabolism. The presence in the culture medium of nonorganic ammonia also drastically decreased the extent of pyrimidine catabolism, which was not observed until 14 to 15 h after growth began. Twenty-four hours after inoculation of the yeast, the total radioactivity of the culture given thymine or uracil as the sole source of nitrogen decreased by 89 or 94%, rocinarfluals A iP1 1) 1). umpmu.sV'kY (Tahlo
3
USE OF EXOGENOUS PYRIMIDINES BY R. GLUTINIS
At later stages of growth of the cells on me-
TABLE 1. Changes in total radioactivity ofa culture of the yeast R. glutinis incubated in medium with labeled thymine and uracil for 24 h Radioactive compound"
Additional source of tDtalradeion nitrogen activity (%)b
[2-'4Clthymine
[2-'4C]thymine 12-'4C]thymine [2-'4C]thymine
12-'4C]thymine' 12-'4C]thymine 12-'4C]uracil 12-'4C]uracil [2-'4C]uracile [2-'4C]uracil
Uracil (2 mM) Uracil (4 mM) Cytosine (2 mM) Uracil (2 mM) (NH4)2SO4 (0.1%)
Thymine (2 mM) Thymine (2 mM) (NH4)2SO4 (0.1%)
89 76 0 59 8 21 94 89 85 15
" Specific activity of the culture was 3 ACi/ml. A nonlabeled compound was added to the labeled one so that the total concentration of this base was 2 mM. b The total radioactivity of the culture is the sum of the radioactivity of cells and cultural medium per milliliter of culture. It was determined as follows: 0.1 ml of culture was placed into the aluminum target and was dried at 105 C. Radioactivity was measured by a gas-flow counter. The radioactivity of the culture before the beginning of cultivation was taken as 100%. c In this sample, radioactive compound was added 6 h after beginning cultivation on the medium with "additional source of nitrogen."
dium with thymine as the
source of nitrogen,
both thymine and products of its transformation into uracil, oxymethyluracil and carboxyuracil (0.90, 0.27, and 0.12 ,umol per g [dry *=weight] of cells, respectively) were found in the _ '4o cells. The concentration of uridine 5'-monophosphate in acid-soluble compounds in cells 3\s ] 7S growing on a medium with thymine and uracil was much higher than that in cells growing on % to a medium with ammonium sulfate as the sole 5s 0s source of nitrogen (3.22, 2.24, and 0.51 ,umol per g [dry weight] of cells, respectively). Enzymes of pyrimidine catabolism. When the yeast was incubated on a medium with 4 6 * t0 a a cytosine as the sole source of nitrogen, cytosine deaminase (EC 3.5.4.1) was found. The enzyme HOUR6 could be seen as early as 3 to 4 h after the FIG. 3. Catabolism of [2-'4C]uracil and [2-bgnigo nuaio nti eim m in th medium e '4C]thymine as a function of growth of R. glutinis. monium sf whenb resen Abscissa indicates the incubation time; ordinate in- with h sulfate e esecton theactium dicates the total radioactivity of the culture. For the with cytosine had lIttle effect on the activity of determination of total radioactivity of the culture, see this enzyme (Fig. 4). Its maximum activity was footnotes of Table 1. The cells were cultivated in a observed at the beginning and midpoint of the medium with (1) 2 mM [2-'4C]thymine plus 2 mM logarithmic phase of growth. The product ofthe nonlabeled uracil (U); (2) 2 mM[2-'4C]thymine plus reaction, uracil, was released into the culture 2 mM nonlabeled uracil added 6 h after beginning medium, where it could be found at all times 5 ofcultivation (0); (3)2 mM[2-'4CJthymine (0); (4)2 h after the beginning of cultivation. As stated mM [2-14C]uracil plus 2 mM nonlabeled thymine above, the total radioactivity of cells and cul(A); and (5) 2 mM [2-'4C]uracil plus 2 mM nonla- ture fluid was drastically decrased when pyrimbeled thymine added 6 h after the beginning of idines were the sole source of nitrogen in the of the cultivation (0). Total radioactivity before the begin- med ine which soles ning of cultivation was 3 uCi/ml. To obtain the base medium, which indicates that the pyrimidine total concentration as indicated above, nonlabeled ring undergoes breakage that follows the reductive pattern, C02 being liberated. The first enbases were added to the labeled ones.
4
J. BACTERIOL.
MILSTEIN AND BEKKER
TABLE 2. Activity of dihydrouracil dehydrogenase in the yeast R. glutinis during the logarithmic phase of growth Source of nitrogen Source of nltrogen
4
8
12
16
20
HOURS FIG. 4. Cytosine deaminase activity in cells of R. glutinis. Abscissa indicates the incubation time; ordinate indicates the enzyme activity. The cells were cultivated in media with 0.1% (NH4)2SO4plus 4 mM cytosine (a) and 4 mM cytosine (A).
zyme to involve pyrimidines in such transformations is dihydrouracil dehydrogenase (EC 1.3.1.1.). With no exogenous pyrimidines, the activity of this enzyme was 1; it rose sixfold when uracil or thymine was present in the medium in addition to ammonium sulfate, and it rose tenfold without uracil (Table 2). Under these conditions, the enzyme activity rose exponenfially until 14 to 16 h of incubation (Fig. 5).
DISCUSSION The data presented above indicate that without easily utilized sources of nitrogen, the cells of the yeast R. glutinis can use exogenous pyrimidines as a source of nitrogen. Amine nitrogen of cytosine is the easiest to utilize, almost as easy as inorganic ammonia. Indicative of this is the fact that cytosine deaminase activity does not decrease markedly in the presence of ammonium sulfate. In addition, cytosine inhibits utilization of thymine and uracil as sole sources of nitrogen when they are present together in the nutrient medium. With no cytosine, catabolism of oxypyrimidines is considerably enhanced. The breakage of the pyrimidine ring occurs as a reductive process, which occurs after a decrease in the radioactivity of the culture (Table 1, Fig. 3) and a sharp increase in dihydrouracil dehydrogenase activity when the yeast grows on a medium with oxypyrimidines as the sole source of nitrogen (Table 2). Increase in the enzyme activity and extent of decrease in the radioactivity of the medium correlate well (compare Fig. 3 with Fig. 5). When cells grow on the medium containing
act of enSp zyme"
1 (NH4)2SO4 46.0 Thymine (4 mM) 6.2 Thymine (4 mM) + (NH4)2SO4 (0. 1%) 60.2 Uracil (4 mM) 6.7 Uracil (4 mM) + (NH4)2SO4 (0. 1%) 20.3 Thymine (2 mM) + cytosine (2 mM) 19.5 Uracil (2 mM) + cytosine (2 mM) a Expressed as optical density units per milligram of protein.
r. wo S
HOURS FIG. 5. Dihydrouracil dehydrogenase activity in cells of R. glutinis. Abscissa indicates the incubation time; ordinate indicates the enzyme activity. The cells were cultivated in media with 0.1% (NH4)2S04 (a); 0.1% (NH4)2S04 plus 4 mM thymine (-OZ-*-); 0.1% (NH4) 2S04 plus 4 mM uracil (-*-*-); 4 mM thymine (0); and 4 mM uracil (a).
uracil, the key enzyme of uracil catabolism, dihydrouracil dehydrogenase, is induced in the cells; the activity of this enzyme is very low in the cells cultured on the standard medium and remains at the same level during the whole period of incubation (Fig. 5). We failed to find in R. glutinis the direct pathway of reductive catabolism of thymine reported for liver cells (12, 23). Our findings indicate that a more probable explanation is that exogenous thymine incorporated by the cells is first transformed into uracil through demethylation, as was reported for liver cells (11,12) and Neurospora crassa (1-3, 10, 22, 24, 31, 32; M. T. Abbott and R. M. Fink, Fed. Proc. 21:377, 1962; R. M. Fink and K. Fink, Fed. Proc. 21:377,
USE OF EXOGENOUS PYRIMIDINES BY R. GLUTINIS
VOL. 127, 1976
1962). This pathway is probably a narrow place for the whole process of utilization of nitrogen of thymine. The following facts suggest that this may be the case: (i) the yeast cells begin to multiply on a medium with thymine later than on a medium with uracil (Fig. 1); (ii) catabolism of thymine begins markedly later than that of uracil (Fig. 3); (iii) uracil inhibits catabolism of thymine (Fig. 3); and (iv) at the later stages.of cell growth on the medium with thymine, both thymine and products of its transformation into uracil are found in the cells. Other authors also found these products in culture medium when cultivating R. glutinis (29). On the basis of the evidence presented, the following suggestion can be made concerning the use of exogenous pyrimidines as sole source of nitrogen. (i) Cytosine is incorporated into the cells and deaminated to form uracil and ammonia. (ii) Uracil is utilized as a source of nitrogen, and the ammonia is partly taken into the medium. With no other source of nitrogen, there is a breakage in the pyrimidine ring of uracil, and the transformation of thymine into uracil is inhibited. Thymine catabolism begins with transformation into uracil. When the cells are deficient in nitrogen, all cell functions of importance, including biosynthesis of nucleic acids, are inhibited. Similar results have been previously reported for the yeast Candida utilis (17). Under this condition, the content of pyrimidine nucleotides and uridine 5-'-monophosphate in the acid-soluble pool is increased. The products in this pool inhibit their own biosynthesis, directing transformation of uracil along the pathway of catabolism. In this way the cells make use of regulatory mechanisms to ensure their basic needs. To summarize, the experimental results presented suggest that when exogenous pyrimidines are the sole source of nitrogen for the yeast R. glutinis, their transport into the cell and their intracellular transformations are governed by the ease with which cells are capable of liberating nitrogen from these com-
pounds. LITERATURE CITED 1. Abbott, M. T., T. A. Dragila, and R. P. McCroskey. 1968. The formation of 5-formyluracil by cell-free preparation from Neurospora crassa. Biochim. Biophys. Acta 169:1-6. 2. Abbott, M. T., R. I. Kadner, and R. M. Fink. 1964. Conversion of thymine to 5-hydroxymethyluracil in a cell-free system. J. Biol. Chem. 239:156-159. 3. Abbott, M. T., E. K. Schandl, R. F. Lee, T. S. Parker, and R. I. Midgett. 1967. Cofactor requirements of
thymine 7-hydroxylase. Biochim. Biophys. Acta 132:325-528. 4. Ban, J., L. Vitalle, and E. Kos. 1972. Thymine and
5
uracil catabolism in E. coli. J. Gen. Microbiol. 73:267-272. 5. Beck, C. F., J. L. Ingraham, J. Neuhard, and E. Thomassen. 1972. Metabolism of pyrimidines and pyrimidine nucleosides by Salmonella typhimurium. J. Bacteriol. 110:219-228. 6. Campbell, L. L. 1957. Reductive degradation of pyrimidines. I. The isolation and characterization of a uracil fermenting bacterium, Clostridium uracilicum nov. spec. J. Bacteriol. 73:220-224. 7. Campbell, L. L. 1957. Reductive degradation of pyrimidines. II. Mechanism of uracil degradation by Clostridium uracilicum. J. Bacteriol. 73:225-229. 8. DiCarlo, F. I., A. S. Schultz, and A. M. Kent. 1952. On the mechanism of pyrimidine metabolism by yeasts. J. Biol. Chem. 199:333-343. 9. DiCarlo, F. I., A. S. Schultz, and D. K. McManus. 1951. The assimilation of nucleic acid derivatives and relative compounds by yeasts. J. Biol. Chem. 189:151-157. 10. Fink, R. M., and K. Fink. 1962. Utilization of radiocarbon from thymidine and other precursors of ribonucleic acid in Neurospora crassa. J. Biol. Chem. 237:2289-2290. 11. Fink, K., R. E. Cline, R. B. Henderson, and R. M. Fink. 1956. Metabolism of thymine (methyl-5C'4 or -2C'4) by rat liver in vitro. J. Biol. Chem. 221:425433. 12. Fink, R. M., C. McGaughey, R. E. Cline, and K. Fink. 1956. Metabolism of intermediate pyrimidine products in vitro. J. Biol. Chem. 218:1-7. 13. Gilbert, D. A., and E. W. Yemm. 1958. Soluble nucleotides and nucleoside amino acid compounds of yeasts. Nature (London) 182:1745-1746. 14. Grenson, M. 1969. The utilization of exogenous pyrimidines and the recycling of uridine-5-phosphate derivatives in Saccharomyces cerevisiae, as studied by means of mutants affected in pyrimidine uptake and metabolism. Eur. J. Biochem. 2:249-260. 15. Hayashi, O., and A. Kornberg. 1952. Metabolism of cytosine, thymine, uracil and barbituric acid by bacterial enzymes. J. Biol. Chem. 197:717-732. 16. Ipata, P. L., P. Marmocchi, G. Magni, R. Felicioli, and G. Polidoro. 1971. Baker's yeast cytosine deaminase. Some enzymic properties and allosteric inhibition by nucleosides and nucleotides. Biochemistry 10:4270-4276. 17. Jones, R. W., and D. G. Wild. 1973. Regulation of uptake of purines, pyrimidines and amino acid by Candida utilis. Biochem. J. 134:617-627. 18. Kream, J., and E. Chargaff. 1952. On the cytosine deaminase of yeast. J. Am. Chem. Soc. 74:5157-5160. 19. Lacroute, F. 1968. Regulation of pyrimidine biosynthesis in Saccharomyces cerevisiae. J. Bacteriol. 95: 824-832. 20. Lara, F. I. 1952. On the decomposition of pyrimidine by bacteria. I. Studies by means of the technique of simultaneous adaption. J. Bacteriol. 64:271-278. 21. LaRue, T. A., and I. F. T. Spencer. 1968. The utilization of purines and pyrimidines by yeasts. Can. J. Microbiol. 14:79-85. 22. McCroskey, R. P., W. R. Griswold, R. L. Sokoloff, E. D. Sevier, S. Lin, C. K. Liu, P. M. Shaffer, R. D. Palmatier, T. S. Parker, and M. T. Abbott. 1971. Studies pertaining to the purification and properties of thymine-7-hydrolase. Biochim. Biophys. Acta 227:264-275. 23. O'Donovan, G. A., and J. Neuhard. 1970. Pyrimide catabolism in microorganisms. Bacteriol. Rev. 34: 278-343. 24. Palmatier, R. D., R. P. McCroskey, and M. T. Abbott. 1970. The enzymatic conversion of uracil 5-carboxylic acid to uracil and carbon dioxide. J. Biol. Chem. 245:6706-6710.
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25. Piret, M. C., R. Cuokaert, and J. Christophe. 1964. Le catabolisme reductif de l'uracile cher Torulopiu utilis. Arch. Int. Physiol. Biochem. 72:257-265. 26. Reader, V. 1927. The relation of the growth of certain microorganisms to the composition of the medium. I. The synthetic culture medium. Biochem. J. 21: 901-912. 27. Reichard, P., and 0. Sk6ld. 1963. Pyrimidine synthesis and breakdown. Methods Enzymol. 6:177-197. 28. Salai, T., T. Watanabe, and I. Chibata. 1971. Metabolism of pyrimidine nucleotides in bacteria. II. Studies on the regulation system of the degradation of nucleotides in Pseudomonaa oleovorans. J. Ferment. Technol. 49.488-498.
J. BACTZRIOL. 29. Vilks, S. R., and M. J. Vitols. 1973. Assimilation and catabolism of 5-methylcytosine and thymine by Rhodotorula glutinis (Fres.) Harrison. Microbiologia 4:576-582. 30. Wang, T. P., and I. 0. Lampen. 1952. Metabolism of pyrimidines by soil bacterium. J. Biol. Chem. 194: 775-783. 31. Watanabe, M. S., R. P. McCroskey, and M. T. Abbott. 1970. The enzymatic conversion of 5-formyl-uracil to uracil 5-carboxylic acid. J. Biol. Chem. 245: 2023-2026. 32. Williams, L. G., and H. K. Mitchell. 1969. Mutants affecting thymidine metabolism in Neurospora crassa. J. Bacteriol. 100:383-389.
