Vol. 137, No. 3JOURNAL OF BACTERIOLOGY, Mar. 1979, p. 1145-11500021-9193/79/03-1145/06$02.00/0

Urea: Obligate Intermediate of Pyrimidine-Ring Catabolism inRhodosporidium toruloides

WILLIAM M. THWAITES,1* CRAIG H. DAVIS,' NEAL WALLIS-BIGGART,"'2LILLIAN M.WONDRACK,2 AND MITCHEL T. ABBOTT2

Biology Department,' and Chemistry Department and Molecular Biology Group,2 San Diego StateUniversity, San Diego, California 92182

Received for publication 7 September 1978

Urea has been shown to be an obligate intermediate in and the penultimateproduct of the catabolism of pyrimidine-ring nitrogen in Rhodosporidium toru-loides (Rhodotorula). One of a series of mutants selected for its inability to utilizeuracil as a sole source of nitrogen was unable to utilize urea also. The mutantaccumulated urea and failed to form "'CO2 during supplementation with[2-"C]uracil. Radioautograms from the resulting cell extracts and media failed toreveal expected intermediates. Cell-free extracts of the mutant were shown tolack urease activity. Revertants of the mutant were essentially wild type in alltested attributes. Elements of the reductive pathway for pyrimidine catabolismare present in Rhodosporidium (0. A. Milstein and M. L. Bekker, J. Bacteriol.127: 1-6, 1976), but is has not been determined whether this pathway is involvedwith production of urea.

Although a variety of pyrimidine catabolicpathways has been proposed in the course of thepast half century (e.g., 5, 7 and 26), only threepathways have been confirmed in subsequentinvestigations. One of these involves oxidationof the pyrimidine ring and the conversion ofuracil to urea with barbituric acid as the inter-mediate (14). This oxidative pathway has onlybeen demonstrated in a few genera of soil bac-teria including Corynebacterium, Mycobacte-rium, and Nocardia (14, 17), but its occurrencein mammals has not been excluded (3, 25).Another established pathway of pyrimidine

catabolism involves the oxidation of the methylgroup of thymine but does not degrade the py-rimidine ring (11, 13). This oxidative pathwayhas been detected in Neurospora (11), in Rho-dotorula (29, 33, 34), and in mammals (10).The third pathway involves the reduction of

the pyrimidine ring so that uracil is convertedto dihydrouracil, N-carbamyl-,8-alanine, and ,8-alanine. This reductive pathway has been as-cribed to a wide variety of organisms includingthe procaryotes Clostridium (4, 15) and Hydro-genomonas (16) and a broad spectrum of eu-caryotes including mammals (12), higher plants(28), Euglena (31), Neurospora (35), Saccharo-myces and Torulopis (23), and Rhodotorula (20,29). In the cases of Neurospora and Rhodoto-rula, a functional demonstration of the completepathway is lacking. In Neurospora the reductivepathway was inferred by the ability of dihy-drouracil and N-carbamyl-fi-alanine to serve as

pyrimidine supplements for auxotrophs (35). InRhodotorula the formation of 8-alanine has notbeen demonstrated (20 and 29). In mammals theformation of all the intermediates of the reduc-tive pathway has been demonstrated (12), buturea is observed routinely along with these in-termediates (8, 24, 26). These observations haveled Fink et al. (10) to suggest that mammals mayperform the bulk of pyrimidine catabolism viathe reductive pathway while also having a minorroute which is urea producing. They did notinvestigate this pathway but suggested thateither it might be of the oxidative type or per-haps involve an alternative branch of the reduc-tive pathway.

In this paper we report the finding that ureais an obligate intermediate in the catabolism ofpyrimidines in Rhodosporidium, the designationgiven to interfertile members of Rhodotorula,etc., by Banno (1). This finding is unanticipatedin previous papers concerning Rhodotorula inwhich elements of the reductive pathway havebeen shown (20, 29). Thus Rhodosporidium andmammals may have a similar array of enzymesinvolved with pyrimidine catabolism. Both haveurea-producing pathways and both have at leastportions of the reductive pathway. In Rhodo-sporidium it is now clear that the urea-produc-ing pathway is the only route capable of yieldingnitrogen. Thus this pathway has no functionalcompetitor as is apparently the case in mam-mals. It would seem that Rhodosporidium mightbe an ideal subject for the study ofthe eucaryotic

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pathway which produces urea as an intermedi-ate.

MATERIALS AND METHODSOrganisms. Wild types of Rhodosporidium toru-

loides ATCC 10788 (mating type a) and ATCC 10657(mating type a) were obtained from the AmericanType Culture Collection, Rockville, Md.

Chemicals. ['4C]urea (INC) was adjusted to a spe-cific activity of 0.1 Ci/mol. [2-`4C]uracil was from NewEngland Nuclear Corp. The specific activity of theuracil was adjusted in each experiment as indicated.All other fine chemicals were purchased from theSigma Chemical Co.Media. ATCC 10788 was found to have no complex

growth requirements. Thus 1 liter of minimal mediumcontained 20 ml of nitrogen-free salt solution, 2 g ofsucrose, and 0.5 g of ammonium sulfate. The nitrogen-free salt solution was similar to Vogel's medium N (6,30), except that KH2PO4 was reduced to 50 g, NH4NO3was omitted, MgSO4. 7H20 was increased to 25 g, and5 g ofNaCl was added to each liter. For tests of specificnitrogen sources, the ammonium sulfate was replacedwith a similar gram quantity of organic nitrogensource. All organic nitrogen sources were filter steri-lized. Medium YM (Difco) was used as a completemedium.Maintenance of stocks. Stocks were grown for 5

days at 24 to 28°C on YM slants and stored no longerthan 2 months at 4 to 8°C before transfer. Stocks havebeen stored on silica gel (6) for over 6 months withoutan apparent loss of viability.Growth tests. A water suspension from a slant was

added to 50 ml of liquid medium to give a startingconcentration of 104 cells per ml as determined in ahemocytometer. The 125-ml conical flasks were placedon a rotary shaker at 26°C for 5 days before counting.Compounds which failed to support the growth of wildtype were found to be non-inhibitory when added tominimal medium. Growth curves of wild type on min-imal medium show that, during the log phase, thedoubling time is about 2.5 h, and that at about 3 daysthe stationary phase is reached at a concentration of5 x 10" to 2 x 109 cells per ml.

Crossing. Mycelium was isolated from parentalyeasts and then allowed to form secondary yeast col-onies. When single cells were isolated from each of thesecondary colonies, most were found to be parentaltypes with a very low frequency of recombinantsregardless of the mutant strains crossed. We suspectthat most secondary yeasts originate as haploid blas-tospores rather than being the products of telosporegermination (1). Thus they are probably not productsof meOss.

Mutagenesis. A "Mineralight" lamp equipped withits shortwave filter gave a surface exposure of 180ergs/mm2 per s when placed 10 cm above a cell sus-pension. Cells in a water suspension approximately 3mm deep showed about 50% survival after a 1-minexposure.

Selection of UN-2. Irradiated cells from a slant ofwild-type ATCC 10788 were plated on YM until colo-nies were not more than 2 mm in diameter. Theseplates were used as masters to replicate (19) onto

plates containing uracil as the sole source of nitrogenand onto minimal plates. The replicated plates werethen compared on a replica plate comparator (27).The UN-2 mutant was found growing on the minimalplate but not on the uracil plate.The UN-2 mutant has the a mating type and the

same colony morphology and color as the ATCC 10788starting strain. The UN-2 mutant has been recoveredfrom a cross with ATCC 10657 in the a mating typeand is nutritionally undistinguishable from the originala mating type UN-2 mutant.

Revertant selection. Approximately 106 cells ofUN-2 was plated on each of a series of plates usinguracil as the nitrogen source. After 10 days of incuba-tion at 28°C, a few of the plates showed large growingcolonies among many unchanging microcolonies.These plates were discarded, and the remaining plateswere exposed to varying doses of UV light as in themutagenesis section. Six days after the irradiation theplates were scored for growing revertant colonies. Aconsistent response was noted in the number of re-vertant colonies seen. The density of growing coloniesper plate ranged from zero on the unirradiated controlplates to 16.5 on plates exposed for 2 min. MutantsUN-2R6 and UN-2R11 were arbitrarily selected forfurther study. Both of the selected revertants were ofthe expected mating type and had the same color andcolonial morphology as wild-type ATCC 10788 fromwhich the UN-2 mutant was selected.

Supplementation ofwhole cells with [2-14Clur-acil. Cells used in these studies were grown for 2 daysin liquid medium containing 1 g of uracil and 1 g ofammonium sulfate as nitrogen sources. The cells wereharvested by centrifugation (2,000 x g for 1 min) andsuspended in miniimal medium. In studies in which therate ofCO2 formation was determined, a 0.4-ml portionof the cell suspension (6.3 x 108 cells per ml) wasadded to a culture tube (10 by 75 mm) containing 0.1ml of 2.4 mM [2-"4C]uracil (4.1 Ci/mol). After the cellshad been incubated with shaking at 28°C for variousperiods, the amounts of CO2 produced were deter-mined as previously described (21).To analyze for the other products of uracil metab-

olism, 1 ml of the resuspended cells (10'0 cells per ml)was added to 4 ml of 0.6mM (2-14C]uracil (4.2 Ci/mol)and incubated at 28°C in a 25-mil flask agitated withan air-driven magnetic stirrer. After 1 h, 1 ml of theculture was harvested by centrifugation as before and50 p1 of the supernatant was subjected to paper chro-matography. The cells were extracted twice with 0.5-ml portions of absolute ethanol, and the two extractswere combined. A 100-Il volume of this extract waschromatographed along with nonlabeled referencecompounds. The detection of uracil, urea, dihydrour-acil, and N-carbamyl-fi-alanine was performed as de-scribed elsewhere (9). The solvents routinely usedwere designated as follows: FORM/t-Bu consisted oftert-butyl alcohol, methylethylketone, formic acid, wa-ter (40:30:15:15, vol/vol); NH3/t-Bu consisted of tert-butyl alcohol, methylethylketone, water, ammoniumhydroxide (40:30:20:10, vol/vol); EtAc/form consistedof upper phase from a mixture of ethyl acetate, water,formic acid (60:35:5, vol/vol); and t-Bu/fonn consistedof tert-butyl alcohol, methylethylketone, water, formicacid (44:44:11:0.26, vol/vol).

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Preparation of cell-free extracts. The culturesfor these studies were grown under forced aeration for3 days at 240C on yeast carbon base medium (22)supplemented with ammonium sulfate (5 g/liter) aspreviously described (33). The manner in which thesecells were harvested, powdered in a mortar containingliquid nitrogen, and further disrupted with glass beadsin the chamber of a Sorvall Omnimixer is also de-scribed in the previous reference (33). Sonic oscillationwas also used to further disrupt the cells powdered inliquid nitrogen. In the sonic disruption procedure, 15g of frozen pads of cells were powdered and thensuspended in 30 ml of 0.05 M Tris-hydrochloride (pH8.0), which contained 0.1 mM EDTA and 0.21 mMphenylmethyl sulfonylfluoride. The suspension was

exposed to the standard (1.90-cm) probe at about 74W/cm2 of a Biosonik III cell disruptor (Bronwill Sci-entific) for 15 min so that the temperature did notexceed 10°C. The homogenate was centrifuged at12,000 x g for 30 min to yield a supernatant solutionwhich is referred to as the crude extract. In preliminaryexperiments it was shown that the maximal urease-specific activity [both with respect to nanomoles perminute per gram (dry weight) and nanomoles perminute per miligram of protein] was obtained after 12min of sonic oscillation and that this activity did notdecrease appreciably when the sonic osciUation wascarried out for as long as 30 min. The homogenizingprocedure that utilized the Sorvall Omnimixer yieldedcomparable values for determinations of nanomolesper minute per milligram of protein but somewhatlesser values for nanomoles per minute per gram (dryweight). The data (see Tables 3 and 4) were obtainedusing crude extracts prepared with the sonic osciUationprocedure and are typical of those data obtained withseveral batches of cells grown at different times.Urease assay. To prepare the standard incubation

mixture, a 0.2-ml portion of the crude extract waspipetted into a culture tube (10 by 75 mm) whichcontained ["4C]urea (0.1 Ci/mol) in 0.025 ml of waterso that the final concentration of urea was 0.1 M. Thegenerated CO2 was trapped and counted as previouslydescribed (21). For measuring the specific activity ofan enzyme preparation, the protein concentration ofthe incubation mixture and the time of incubationwere varied to determine conditions under which therate of C02 formation was linear and proportional tothe enzyme concentration. This rate was determinedat a time in the incubation period when the amount ofsubstrate hydrolyzed did not exceed 10%. Proteinswere estimated with the Foline-Ciocalteu reagent (18).

RESULTS

Growth tests. Mutant UN-2 was selectedfrom wild-type ATCC 10788 after UV light treat-ment for its inability to utilize uracil as a solesource of nitrogen. It was recognized that anyblock between uracil and the release of ammo-nium should restrict the utilization of pyrimi-dine-ring nitrogen and that mutants deficient inpyrimidine transport would probably be of thetype found in Neurospora crassa (32) in thatthey would retain the wild-type ability to trans-

port nucleosides. The UN-2 mutant is deficientin the utilization of not only pyrimnidine bases,but deoxyribo- and ribonucleosides as well (Ta-ble 1). Thus the UN-2 mutant on the basis ofnutritional data appears to be a block betweenuracil and ammonium release. The failure ofUN-2 to utilize urea as a nitrogen source was anunexpected observation that suggested a loss ofurease activity.Catabolism of uracil. Since urea was not an

expected intermediate of uracil catabolism, itwas recognized that a loss of urease activitymight be coincidental to the block in uracilcatabolism. On the other hand, if urease werethe last step in the release of pyrimidine-ringnitrogen, then a mutant such as UN-2 shouldaccumulate urea during uracil supplementationand furthermore such a mutant should not re-lease the ureido carbon as C02 during suchsupplementation.

In several preliminary experiments in whichunlabeled uracil was added to minimal medium,samples of the media were subjected to one-dimensional paper chromatography at 24-h in-

TABLE 1. Growth of wild-type ATCC 10788, UN-2,UN-2R6, and UN-2RI1 on various sources of

nitrogenCell concn (106 cells/ml)a

Nitrogen source Wild-type UN-2b UN-2R6c UN-2R11cATCC10788"

None 0.65 0.11 0.11 0.22Anumonium sulfate 470 160 340 340

(minimal)Urea 1,000 0.20 400 400Dihydrouracil 380 0.15 200 240Uracil 560 0.15 600 440Uridine 208 0.83 190 200Deoxyuridine 300 0.19 220 160Uracil-5-carboxylic 42 0.20 150 82

acid5-Hydroxymethyl- 140 0.10 220 260

uracilThymine 600 0.07 280 224Thymidine 240 0.25 148 73Cytidine 148 5.5 340 280N-Carbamyl-,B-ala- 7.5 2.3

nineBarbituric acid 0.78 0.47Dihydroorotic acid 7.5 0.20Isobarbituric acid 2.6 0.50Dihydrothymine 0.41 0.65

a Cells were grown for 5 days at 26'C on a rotary shakerfrom an inoculum of 104 cells per ml and counted with ahemocytometer.

"Wild-type ATCC 10788 and mutant UN-2 were run onthree separate occasions. The median value is given. Com-pounds which did not support the growth of the wild typewere found to be noninhibitory when added to minimal me-dium.

c The revertants of UN-2 were only run once.

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tervaLs using the NH3/t-Bu solvent. With bothwild type and UN-2, a UV-absorbing spot cor-responding to uracil could be seen during theinitial stages of growth. After 3 days the mediumfrom UN-2 showed a distinct p-dimethylamino-benzaldehyde-positive spot which co-chromato-graphed with urea using all four solvents de-scribed in Materials and Methods. The mediumfrom wild type did not show such a spot. WhenUN-2 was grown on unsupplemented minimalmedium, the accumulation of urea did not occur.To determine if the accumulated urea was in

fact a product of uracil catabolism, the systemwas modified to use [2-'4C]uracil. Cells weregrown for 3 days in minimal medium supple-mented with unlabeled uracil and suspended inminimal medium to which labeled uracil wasadded. Figure 1 is of radioautographs exposedfor 1 month to two-dimensional chromatographsof cell extracts made after 1 h of supplementa-tion with labeled uracil. The figure shows apronounced accumulation of labeled urea by theUN-2 mutant extract. The medium from theUN-2 mutant also showed a heavy accumulationof radioactive urea. Detectable amounts of ureawere noted occasionally in the medium fromwild type after 3 h of supplementation withlabeled uracil, but the heavy accumulation of["4C]urea by UN-2 confirms the hypothesis thaturea is an intermediate of uracil catabolism.

In addition to labeled urea, uridine and UMPhave been tentatively identified in radioauto-graphs. Quite unexpectedly, however, neitherdihydrouracil, N-carbamyl-,/-alanine, nor bar-bituric acid have been detected even when ra-

dioautographs were exposed for 3 months. Onemight expect precursors of urea to be moreheavily labeled in the UN-2 mutant, yet exceptfor the accumulation of urea by UN-2 there isno other obvious difference between wild typeand mutant radioautographs. None of the uni-dentified compounds seen in the radioauto-graphs seems to be a precursor of urea by thiscriterion.

In experiments also using [2-'4C]uracil supple-mentation, but designed to measure '4CO2, theUN-2 mutant was shown to have little or noability to generate labeled CO2. This inabilitywas in marked contrast to that of wild-typeATCC 10788 (Table 2).Urease activity in cell-free extracts. The

data in Table 3 show that the UN-2 mutantcontains little if any urease activity in contrastto the wild-type strain. This observation is in

TABLE 2. CO2 release from cells supplemented with[2-'4C]uracil

C02 released (cpm)aStrain

ob 5 30 60 120 240

Wild-type 29 238 3,802 8,426 25,298 37,552ATCC10788

Mutant 0 3 17 25 69 82UN-2a Cultures (0.5 ml; 5 x 10" cells per ml) supplemented with

[2-'4C]uracil were enclosed with C02 absorbing paper andincubated for various times before the addition of trichloroa-cetic acid. Similar values were obtained when the cell numberwas doubled.

b Time of incubation in minutes.

0

a b c

FIG. 1. Cells were grown for 3 days at 27°C in minimal medium supplemented with uracil. Before theexperiment the cells were suspended in fresh minimal medium. At zero time [2-'4C]uracil was added so thatthe final concentrations were 0.5mM uracil and 2 x 109 cellsper ml. The specific activity ofthe uracil was 4.2Ci/mol. At 1 h, a 1-ml portion of the culture was harvested by centrifugation and extracted twice with 0.5-mlportions of absolute ethanol. A 100-pl volune of this extract was chromatographed with the NH4/t-Bu andFORM/t-Bu solvents as indicated. Radioautographs were exposed for 4 weeks. The locations of knowncompounds were determined with unlabeled reference compounds that were added to the extracts. (a) Map oflocations: A, dihydrouracil; B, urea; C, uracil; D, uridine; E, N-carbamyl-/3-alanine; F, barbituric acid(streaked in the FORM/t-Bu solvent); G, uridine monophosphate; H through L, unidentified labeled com-pounds seen in radioautographs. (b) Radioautograph from wild-type ATCC 10788. (c) Radioautograph fromUN-2 mutant.

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TABLE 3. Urease activity of several strainsSp actb

Straina . nmol/unmol/min per min perg (dry wt) mg of

protein

ATCC 10788 (wild type) 6,200 53.0UN-2 (mutant) 18.3 0.2UN-2R6 (revertant) 12,300 98.1

a Strains were grown in parallel and used to preparecrude extracts for the determination of urease activi-ties as described in the text.

b Each value represents the average of quadrupli-cate incubations.

complete agreement with all of those precedingit, including the observation that UN-2 was un-able to utilize urea as a nitrogen source (Table1). That the loss of activity in the UN-2 mutantis a reflection of active enzyme and not thepresence of an inhibitor was indicated by exper-iments in which extracts of the wild-type strainand the UN-2 mutant were combined in variousproportions and assayed (Table 4).Revertants of UN-2. As an independent

check that the lack of urease activity was notcoincidental to the loss ofpyrimidine-ring catab-olism, revertants of UN-2 were selected. Sincethe revertants were selected for their regainedability to utilize uracil as a nitrogen source, areacquisition of urease activity in such a mutantwould further substantiate the identity of theloss of urease with the loss of pyrimidine catab-olism.Two revertants, UN-2R6 and UN-2R11, were

chosen arbitrarily for further analysis. Thesewere checked for wild-type pyrimidine and ureautilization as measured by growth (Table 1) andfor urease activity (Table 3). The urease activityof the UN-2R11 strain was determined in onlyone study, but it had approximately the samespecific activities as did wild-type ATCC 10788.Supplementation with dihydrouracil. Al-

though dihydrouracil is able to serve as a solesource of nitrogen (Table 1), we were not able toconfinn that Rhodosporidium converts uracil todihydrouracil and N-carbamyl-fi-alanine as hasbeen reported (20, 29). In an experiment de-signed to follow the fate of dihydrouracil, cellswere grown for 6 days in minimal medium sup-plemented with 500 ,jg of dihydrouracil per ml.At intervals of 1 day, a 100-,l sample of mediumwas subjected to chromatography with the NH3/t-Bu solvent. Initially only a single spot identi-fied as dihydrouracil was seen, but after 4 daystwo new accumulations were discernible in thewild-type media. These were identified as uraciland N-carbamyl-fl-alanine. The UN-2 mutant

displayed all of these plus an additional spotwhich corresponded to urea.

DISCUSSIONIn this paper we have shown that uracil is

catabolized via a urea-producing pathway. Sincethe UN-2 mutant lacks urease and is unable toutilize pyrimidine-ring nitrogen, it also can beconcluded that the urea-producing pathway hasno functional competitor as it apparently doesin mammals (10).What is still unknown is the route by which

urea is removed from the pyrimidine ring. Ifbarbituric acid is shown to be the precursor ofurea, it would mark the first time this pathwayhad been demonstrated clearly in a eucaryote.Nevertheless, it is easy to imagine why barbi-turic acid might be held to very low levels insidethe cell. If the inhibition of pyrimidine synthesisand salvage by barbituric acid in rat brain ex-tracts (25) is a general phenomenon, the accu-mulation of this intermediate might impose apyrimidine requirement. If the reductive path-way is somehow involved, novel enzyme mech-anisms must be considered which would removethe urea moiety from dihydrouracil or N-carba-myl-fi-alanine. However, it is possible that thereductive sequence may have no relation to py-rimidine catabolism in Rhodosporidium. Al-though Neurospora crassa has elements of thispathway (35), it is not able to utilize cytosine,thymine, or uracil as sources of nitrogen (2).Finally our data cannot exclude the possibilitythat urea is released from the pyrimidine ring insome completely new fashion.

TABLE 4. Incubations of various combinations ofextracts from wild-type and UN-2 strains

Vol of extracts from Activity of urease in in-cubation mixtures

Expt strains (ml)' (nmol/min)bWild UN-2 Experi- Predictedctype mentalc

1 0.20 1,7302 0.15 0.05 1,280 1,3003 0.10 0.10 810 8674 0.05 0.15 410 4355 0.20 3

a Crude extracts were prepared from each strain andassayed for urease activity in the standard incubationmixture. It consisted of 0.2 ml of extract or combina-tion of extracts and 0.025 ml of an aqueous solution ofthe substrate.

b Each value represents the average of duplicateincubations.'The experimentally determined values of activity

are recorded under "Experimental." The values in the"Predicted" column were calculated from the "Exper-imental" values determined in experiments 1 and 5.

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ACKNOWLEDGMENTSThis work was supported by San Diego State University

Foundation grant number 263052 and National Science Foun-dation grant number PCM 76-81531 to W. M. Thwaites, andby U.S. Public Health Service grant number GM25055 to M.T. Abbott from the National Institute of General MedicalSciences.

The excellent technical assistance of Marlene DeMers isalso acknowledged.

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