JOURNAL OF BACTERIOLOGY, Mar. 1979, p. 1073-1080 Vol. 137, No. 30021-9193/79/03-1073/08$02.00/0

Enzymes of Pyrimidine Metabolism in Mycoplasma mycoidessubsp. mycoides

ALANA MITCHELL* AND LLOYD R. FINCHRussell Grimwade School of Biochemistry, University ofMelbourne, Parkville, Victoria 3052, Australia

Received for publication 3 January 1979

The major pathways of ribonucleotide biosynthesis in Mycoplasma mycoidessubsp. mycoides have been proposed from studies on its use of radioactive purinesand pyrimidines. To interpret more fully the observed pattern of pyrimidineusage, cell extracts of this organism have been assayed for several enzymesassociated with the salvage synthesis of pyrimidine nucleotides. M. mycoidespossessed uracil phosphoribosyltransferase, uridine phosphorylase, uridine (cyti-dine) kinase, uridine 5'-monophosphate kinase, and cytidine 5'-triphosphate syn-thetase. No activity for phosphorolysis of cytidine was detected, and no in vitroconditions were found to give measurable deamination of cytidine. Of the twopotential pathways for incorporation of uridine, our data suggest that this pre-cursor would largely undergo initial phosphorolysis to uracil and ribose-1-phos-phate. Conversely, cytidine is phosphorylated directly to cytidine 5'-monophos-phate in its major utilization, although conversion of cytidine to uracil, uridine,and uridine nucleotide has been observed in vivo, at least when uracil is providedin the growth medium. Measurements of intracellular nucleotide contents andtheir changes on additions of pyrimidine precursors have allowed suggestions asto the operation of regulatory mechanisms on pyrimidine nucleotide biosynthesisin M. mycoides in vivo. With uracil alone or uracil plus uridine as precursors ofpyrimidine ribonucleotides, the regulation of uracil phosphoribosyltransferaseand cytidine 5'-triphosphate synthetase is probably most important in determin-ing the rate of pyrimidine nucleotide synthesis. When cytidine supplements uracilin the growth medium, control of cytidine kinase activity would also be importantin this regard.

Mycoplasma mycoides subsp. mycoides re-quires for growth both uracil and guanine asprecursors of ribonucleotides (15). From an ex-amination ofthe incorporation of precursors intoRNA (9) and a consideration of the known path-ways of salvage nucleotide synthesis in otherbacteria (14), we have outlined possible path-ways of nucleotide metabolism in M. mycoides(9). Our results indicate that this organism lacksthe reactions of de novo synthesis of nucleotidesfrom simple precursors and displays a limitedability to interconvert nucleotides.Of the enzymes associated with the proposed

pathways, those relating to purine nucleotideshave been partially defined and were found todiffer from the corresponding Escherichia colienzymes in some aspects of their regulation (10,19). In relation to pyrimidine nucleotide metab-olism, uridine phosphorylase (EC 2.4.2.3) hasbeen detected in many strains of mycoplasmasduring the search for methods to assess myco-plasmal contamination of mammalian cell cul-tures (6).To extend this limited knowledge of the py-

rimidine nucleotide metabolism in mycoplas-mas, we have assayed cell extracts of M. my-coides for the various enzymes suggested fromour previous studies and have investigated as-pects of the kinetic properties and regulation ofthose enzymes likely to be significant in deter-mining the rate of in vivo nucleotide synthesis.To test the effectiveness of in vitro regulatorymechanisms in controlling the synthesis of ri-bonucleotides in vivo, we have measured theintracellular contents of some ribonucleotidesand the changes which occur in them in responseto the addition of supplementary nucleosides orbases as potential precursors of pyrimidine nu-cleotides.

MATERIALS AND METHODSOrganism and culture medium. M. mycoides

subsp. mycoides was kindly supplied by A. W. Rodwell,Commonwealth Scientific and Industrial Research Or-ganisation, Division of Animal Health. For estimationof intracellular nucleotide contents, cells were grownin a modified medium C2 of Rodwell (16) as previouslydescribed (9). For the preparation of extracts for assay

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1074 MITCHELL AND FINCH

of enzymes, cells were grown in PPLO broth (17).Chemicals and radiochemicals. Tris, 2,5-diphen-

yloxazole, and tetrasodium 5-phosphoribosyl-1-di-phosphate (P-Rib-PP) were all from the Sigma Chem-ical Co. Dithiothreitol, 2-phosphoenolpyruvate, pyru-vate kinase, and crystalline bovine serum albuminwere from Calbiochem. The radiochemical substrates[2-'4C]uracil (61 Ci/mol), [2-'4C]uridine (59 Ci/mol),[4-'4C]UMP (24 Ci/mol), and [4-'4C]UTP (48 Ci/mol)were all supplied by the Radiochemical Centre, Eng-land; [2-'4C]cytidine (43 Ci/mol) was a product of ICNChemical and Radioisotope Division.Preparation of extracts for enzyme assays. Ex-

tracts of M. mycoides were prepared by sonic oscilla-tion of cells from late-log-phase or stationary-phasecultures as previously described (10). Unless specified,extracts were dialyzed for 4 to 6 h against 100 mMTris-hydrochloride at 0°C. Protein content was esti-mated by the method of Lowry et al. (8) as previouslydescribed (10).Enzyme assays. (i) Uracil phosphoribosyl-

transferase. In the uracil phosphoribosyltransferase(UMP:pyrophosphate phosphoribosyltransferase, EC2.4.2.9) assay, unless otherwise specified, reaction mix-tures, incubated at 37°C, contained 5 mM MgSO4,50 ,uM ['4C]uracil, 500 ,uM P-Rib-PP, bovine serumalbumin (0.2 mg/ml), and extract protein (to approxi-mately 40 jig/ml) in 40 mM Tris-hydrochloride (pH8.0). When GTP was included, 1 mM 2-phosphoenol-pyruvate and pyruvate kinase were also added. Thereaction was initiated by the addition of ['4C]uracil toa mixture of the other components after preincubationof the mixture at 37°C. At various times, 5-jil sampleswere applied to the origins of polyethyleneimine-cel-lulose (PEI-C) thin layers for chromatographic isola-tion of the product for quantitation by liquid scintil-lation counting (11).

(ii) Uridine phosphorylase. In the uridine phos-phorylase (uridine:orthophosphate ribosyltransferase)assay, reaction mixtures, incubated at 37°C, werebuffered at pH 7.2 with 40mM Tris-hydrochloride andcontained 5 mM MgSO4, various concentrations of thesubstrates [14C]uridine and potassium phosphate, andextract protein (to approximately 160 ,g/ml). Thereaction was started by the addition of the ['4C]uridineto a mixture of the other components at 37°C. Samplesof the incubation mixture, taken at various times, werestreaked on the origins of PEI-C thin layers in theborate form (18) for chromatography and counting ofproducts as described earlier (9).

(iii) Uridine (cytidine) kinase. In the uridine(cytidine) kinase (ATP:uridine 5'-phosphotransferase,EC 2.7.1.48) assay, reaction mixtures, incubated at37°C, contained 5 mM Mg acetate, 1 mM '4C-labelednucleoside, 2 mM ATP, 40 mM Tris-hydrochloride(pH 7.8), and extract protein (to approximately 400jig/ml). Sampling, chromatography, and liquid scintil-lation counting were as described for uracil phospho-ribosyltransferase (11).

(iv) Cytidine deaminase. In the cytidine deami-nase (cytidine aminohydrolase, EC 3.5.4.5) assay, toassay for the formation of ['4C]uridine from ['4C]cyti-dine, cell extracts of M. mycoides were incubated at37°C in 40 mM Tris-hydrochloride (pH 7.2), contain-

ing 5 mM MgSO4. At the required times, 5- to 20-tilsamples of incubation mixture were spotted at theorigins of cellulose phosphate paper strips for chro-matography as described (9).

(v) UMP kinase. In the UMP kinase (ATP:UMPphosphotransferase) assay, reaction mixtures, incu-bated at 37°C, contained 5 mM ATP, 0.2 mM ["C]-UMP, 5 mM MgCl2, 40 mM Tris-hydrochloride (pH7.8), and extract protein to approximately 50jug/ml.Reaction was started by the addition of ['4C]UMP toa mixture of the other components at 37°C. At differ-ent times, 5-jul samples were applied to PEI-C thinlayers (11), 10 cm long. The origins had previouslybeen spotted with a marker solution (2 ,ul, containing3.3 mM UTP, UDP, and UMP). The chromatogramswere developed once with distilled water, dried, thenredeveloped in 1.0 M acetic acid containing 0.75 MLiCl. The first development moved any uracil or uri-dine to the front, whereas the redevelopment effec-tively separated UMP, UDP, and UTP which werelocated by the UV absorbance of the marker com-pounds to allow their excision for liquid scintillationcounting.

(vi) CTP synthetase. In the CTP synthetase(UTP:ammonia ligase [ADP], EC 6.3.4.2) assay, reac-tion mixtures, incubated at 37°C, contained 1 mMATP, 0.31 mM [14C]UTP, 0.5 mM GTP, 5.0 mMglutamine, and 10.0 mM Mg acetate in 37.5 mM Tris-hydrochloride (pH 7.2), and extract protein (to ap-proximately 400 jug/ml). The reaction was begun bythe addition of [14C]UTP to all other components at37°C. At various times, 5-jl samples were applied toPEI-C thin-layer strips (11), 20 cm in length. Theorigins had previously been spotted with a markersolution (2 Il, containing UTP, UDP, UMP, CTP,CDP, and CMP, each at a concentration of 1.6 mM).Chromatograms were developed for 9 cm in 1.0 Mformic acid containing 0.4 M LiCl, and for an addi-tional 9 cm in 1.0 M formic acid containing 1.0 M LiCl.This procedure effectively separated UTP and CTPfrom the corresponding nucleoside mono- and diphos-phates. The six nucleotides were located by the UVabsorbance of the marker compounds to allow theirexcision for liquid scintillation counting.Processing of kinetic results. For the estimation

of kinetic parameters as described earlier (10), thedata for kinetic experiments were fitted to the rateequations describing ordered sequential, equilibrium-ordered, and ping-pong reaction mechanisms (3) fortwo substrate reactions. For each of the enzymes stud-ied, best fit of the kinetic data was achieved with therate equation for the ordered sequential reactionmechanism.Measurement of intracellular ribonucleotide

contents. [32P]orthophosphate, to a specific activityof 60 to 70 Ci/mol, was added to cultures at a celldensity corresponding to approximately 25 jug of cel-lular protein per ml of culture. Uniform labeling wasensured by allowing at least two doubling times beforecommencement of sampling. Extraction of cells in ice-cold HCl04, and subsequent procedures for the esti-mation of nucleotides were performed as described byBagnara and Finch (1). To study the effects of addednucleosides and bases on the intracellular nucleotide

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PYRIMIDINE METABOLISM IN M. MYCOIDES 1075

content, the additions were made as a 1 mg/ml solu-tion to give a concentration of 20 pg/ml in the culture.

RESULTS

Uracil phosphoribosyltransferase. Toseek explanation of the ability of M. mycoidesto incorporate uracil, cell extracts were assayedfor uracil phosphoribosyltransferase. The reac-tion was found to be dependent on the presenceof Mg2+, and the rate was stimulated three- tosixfold by saturating concentrations of GTP,depending on the concentration of P-Rib-PPused (Fig. la). A double reciprocal plot of theGTP-dependent component of the rates shownin Fig. la gives straight lines (Fig. lb) intersect-ing the absicissa at -40 mM-1, suggesting thatthe dissociation constant (KD) for GTP from theenzyme is approximately 25 AM.The results ofinitial velocity studies at a series

of concentrations of uracil and P-Rib-PP and a

-2

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-5SEC.

0.0 0.1 0.2 0.3 0.AConcentration of GTP (mM)

ioo200

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E

-.50-5

E

(b)

o~~~~~~-50 0 50 100

[GTP' (mM )-'FIG. 1. Effect of GTP on the kinetics of uracil

phosphoribosyltransferase with respect to P-Rib-PP.Standard assay conditions were used, except that theconcentrations of P-Rib-PP were: 5 (0); 10(0); 25 (A); 100 (A); and 400 (0). (a)Direct plot of rate of reaction versus GTP concentra-tion. (b) Double reciprocalplot ofthe GTP-dependentcomponent of the data in a.

fixed concentration of 1 mM GTP gave Km val-ues of 2.6 ± 0.4 ,uM for uracil and 51.3 ± 6.7 ,uMfor P-Rib-PP and a Vm. of 120 ,umol min-m g-'of extract protein when fitted to the model foran ordered sequential mechanism.Table 1 shows the effects of several nucleo-

tides, added together with 1 mM GTP, on theactivity of uracil phosphoribosyltransferase. Noother purine or cytidine nucleotide significantlyaffected the rate of reaction. The inhibition byuridine nucleotides increases with inhibitor con-centration and in the order UTP, UDP, UMP(Table 2). Double reciprocal plots (Fig. 2) ofdata for the effect of P-Rib-PP concentration onthe rate of reaction at various concentrations ofUMP (Fig. 2) showed that the effect of UMPwas to change the slope, but not the intercept,ofthe linear curve, suggesting that the inhibitionby UMP was competitive with respect to P-Rib-PP. The data suggest Ki values for UMP in therange 19 to 22 ,uM with 0.1 mM GTP (Fig. 2a)and 30 to 50 ,uM with 1.0 mM GTP (Fig. 2b).Other data confirm Ki values in these ranges forUMP and suggest values of approximately 100

TABLE 1. Effect of nucleotides added together withI mM GTP on uracil phosphoribosyltransferase

Activity (% control) with added nucleo-tide (2 mM)

Nucleoside Mono-phos- Diphos- Triphos-

phate phate phate

Adenosine 97 104 101Inosine 94 94 103Guanosine 82 101 looaUridine 10 25 80Cytidine 108 101 108

a Final GTP concentration 2 mM-control.

TABLE 2. Response of uracilphosphoribosyltransferase to various concentrations

of uridine nucleotide added together with 1 mMGTP

Activity as % of control'Nucleo- Concn (mM) MM P-Rib 500MM P

tide 50uMPRb 50,MPPP Rib-PP

UMP 0.1 28 660.5 9 221.0 5 15

UDP 0.1 64 790.5 21 511.0 13 42

UTP 0.1 100 950.5 71 761.0 53 69

a Control assays contained 1 mM GTP, 50 or 500uM P-Rib-PP as indicated, and no added uridine nu-cleotide.

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0i-

cx

C

E-

o.E

0

10 20(PRPP-1 (arbitrary units)

0 10 20

IPRPPI 1 (arbitrary units)

FIG. 2. Double reciprocal plots of rate of reactionversus P-Rib-PP concentration for uracilphosphori-bosyltransferase for various concentrations of UMP.Except for varied P-Rib-PP concentration, standardassay conditions were used with 0.1 mM GTP (a) or

1.0mM GTP (b) and UMP at various concentrations:0 AM (0); 10 pM (0); 20 ,M (A); 50 uM (A); and 100

and 650 ,uM for UDP and UTP, respectively,with GTP at 1.0 mM.Without GTP, the rate of reaction followed a

sigmoidal curve when plotted against P-Rib-PPconcentrations over the range of 70 to 150 ,uM,and the enzyme appeared to be saturated withuracil at concentrations below 2.5 t,M. Also,without GTP, uracil phosphoribosyltransferasewas somewhat more susceptible to inhibition byuridine nucleotides and was slightly activated byGDP. Since it is likely that GTP is always pres-

ent in the cell, these latter two effects are prob-ably not of physiological importance.Uridine phosphorylase. The ability of M.

mycoides to use uridine as a precursor of RNA,probably after initial degradation to the freebase (9), is suggestive that this organism pos-sesses uridine phosphorylase. Cell extracts ofM.mycoides catalyzed a phosphate-dependent re-lease of uracil from uridine as expected for uri-dine phosphorylase. Similar to other organisms(4, 13), M. mycoides displayed no phosphorolyticactivity toward cytidine.The data for initial velocity studies at a series

of concentrations of phosphate and uridineyielded Km values of 1.99 ± 0.98 mM for phos-phate and 2.16 ± 0.68 mM for uridine, and aVmax of 860 pmol min-' g-' of protein when fittedto the equation for an ordered sequential reac-tion mechanism.Uridine (cytidine) kinase. The effective uti-

lization of cytidine by M. mycoides as a precur-sor of cytidine nucleotide in RNA (9) suggestedthe involvement of a cytidine kinase. This activ-ity and a uridine kinase activity were demon-strated in dialyzed cell extracts of M. mycoides.The specific activity of uridine (cytidine) kinasein these extracts was 12.5 ,umol min-' g-1 ofprotein with uridine as substrate and 6.5,molnin-n g-1 of protein with cytidine. The enzymerequired Mg2" for activity, although concentra-tions above 10 mM were somewhat inhibitory.ATP and to some extent GTP were substrates

of uridine kinase, but cytidine kinase used onlyATP. Inclusion of 1 mM cytidine in the assay ofuridine kinase caused 30% inhibition of this re-action. Studies on the effects of various nucleo-tides on uridine (cytidine) kinase are shown inTables 3 and 4. Both activities were inhibited byuridine and cytidine nucleotides, with cytidinekinase being more susceptible to inhibition byall effectors tested. These results contrast withthose for the E. coli enzyme, which was foundto be largely specific for GTP as phosphoryldonor and to be strongly inhibited by CTP, lessstrongly inhibited by CMP, and almost unaf-fected by uridine nucleotides (John Baird, M.Sc.thesis, University of Melbourne, Parkville, Vic-toria, Australia, 1973). Kinetic studies on uridine(cytidine) kinase were not undertaken, owing tothe instability of cytidine kinase and the diffi-culty in suppressing uridine phosphorylase inassays. Extensive dialysis was only partially suc-cessful in alleviating the latter problem becausephosphate in assay mixtures appeared to derivefrom ATP.Cytidine deaminase. Cytidine is incorpo-

rated into the UMP isolated from RNA of M.mycoides (9) and, upon addition of ['4C]cytidine

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TABLE 3. The effect ofpyrimidine nucleotides onuridine (cytidine) kinase

Added nucleotide Activity as % of control

(2 mM) Uridine kinase Cytidine kinase

None 100 100UTP 16 2UDP 23 7UMP 62 19CTP 18 1CMP 35 4

TABLE 4. Effect of CTP and CMP on cytidinekinase

Activity with added nucleotide (% con-Nucleotide concn trol)

(mM)CMP CTP

0.0 100 1000.1 73 670.5 37 151.0 17 12.0 10 1

to cultures, ["4C]uracil, derived via ['4C]uridine,accumulates extracellularly (9). These findingsimply that M. mycoides may possess cytidinedeaminase. However, no deamination of cytidinewas measurable on incubation of the nucleosidewith cell extracts with or without sulflhydrylreagents under conditions of varying pH or Mg2"concentrations. Cytidine deaminase is induciblein E. coli and Salmonella typhimurium (5, 12),but no activity was detectable in extracts of M.mycoides grown in medium C2 supplementedwith 2 mM cytidine.UMP kinase. For M. mycoides grown on

uracil as the sole precursor of pyrimidine ribo-nucleotides, the activity of UMP kinase may bea factor in controlling the supply of UTP andCTP to the cell. High levels of UMP kinase andsignificant levels of UDP kinase were measura-ble in cell extracts. The rate of formation ofUTP was greatly decreased by lowering theamount of extract protein in assays and, underthese conditions, the appearance of UDP andany UTP was taken as a measure of total UMPkinase activity. UMP kinase was activated byMg2" concentrations up to 5 mM, with slightinhibition occurring at higher concentrations.Analysis of initial velocity data at a series of

concentrations ofUMP and ATP gave Km valuesof 530 ± 90 uM for ATP, 60 ± 10 uM for UMP,and a V.. of approximately 200,umol min-' g-'of extract protein. Potential iAhibitory effects ofUDP and UTP were not investigated becauseof difficulties in ascertaining the effect of con-

taminant UMP on the specific activity of the['4C]UMP substrate.CTP synthetase. In keeping with the obser-

vations that M. mycoides can use uracil as thesource of both uridine and cytidine nucleotide,CTP synthetase was demonstrated in cell ex-tracts of the organism. Autoradiography of thechromatograms obtained in the assay of CTPsynthetase revealed the formation of UDP,UMP, CDP, and CMP, as well as CTP, from theradioactive UTP used as substrate. Owing tothese further complications to the assay of thealready complex CTP synthetase reaction, noattempt was made to study the kinetic proper-ties of this enzyme in crude cell extracts.The enzyme from M. mycoides resembled that

from E. coli in not requiring a sulfhydryl reagentfor activity (7). Although the addition of ATPand GTP was not absolutely essential to thedemonstration of activity with undialyzed cellextracts, their omission decreased activity bymore than 90%. Presumably, nucleotides presentat low levels in the extract may have accountedfor the background activity. Mg2" was essentialfor reaction. Preliminary studies (Table 5) showthat the enzyme was inhibited by CTP.The effects of addition of supplementary

precursors of pyrimidine nucleotides onthe intracellular contents of some ribonu-cleotides. Figure 3 shows the effects of additionof uridine to cells of M. mycoides grown in alow-phosphate modification (9) ofmedium C2 ofRodwell (16) with guanine as the sole source ofpurine nucleotide. The addition caused amarked and rapid increase in the level of UTPand a slower, significant increase in the contentof CTP. ATP was maintained at a somewhathigher steady-state level 5 min after the additionof uridine and GTP slowly decreased in concen-tration after an initial increase. When uridinewas added to cells grown on medium in whichadenine supplemented guanine to provide analternative source of purine nucleotide (resultnot shown), the effects on pyrimidine nucleo-tides were very similar to those shown in Fig. 3.None of the observed changes is attributable touridine providing a source of extra uracil, whichis already 0.18 mM in medium C2 (16), sincesupplementation with additional uracil failed toelicit any effect on the intracellular ribonucleo-

TABLE 5. Effect ofCTP on CTP synthetase

Concn of CTP (mM) Activity (umol min-' g-' ofprotein)

0.00 2.460.05 2.000.10 1.370.50 0.55

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tide contents of a culture grown in this medium(result not shown).The effects of cytidine on the ribonucleotide

levels of cells grown in medium with guaninealone or guanine plus adenine are shown in Fig.4. In each case, the addition of cytidine resultedin marked increases in the concentrations ofUDP and CTP. However, the culture containingguanine plus adenine (Fig. 4a) showed thegreater response, with CTP increasing steadilyto a maximum of fivefold by 15 min. This in-

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E:,10

a-xw

220-

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Zn~U L_

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A

-10 0 10 20 30Time (min)

FIG. 3. Effect of uridine on the intracellular ribo-nucleotides ofM. mycoides grown on guanine as thesole source ofpurine nucleotide. Uridine was addedat zero time to a concentration of 20 pg per ml ofculture, and samples were taken for estimation ofribonucleotides at the indicated times. Symbols: E,GTP; 0, ATP; A, CTP; A, UTP;E, GDP; 0, ADP.

Tim* (min) Time (min)

FIG. 4. Effect of cytidine on the intracellular ri-bonucleotides ofM. mycoides. The conditions were asdescribed for Fig. 2, with cytidine replacing uridine.(a) Cells grown on medium containing guanine andadenine. (b) Cells grown on medium containing gua-nine as the only source ofpurine nucleotide.

crease was largely retained over 80 min. Withthe culture containing guanine as the only purine(Fig. 4b), both CTP and UTP doubled to amaximum by 5 min. Thereafter, UTP fell rapidlyto be near its initial level by 10 min, whereasCTP remained slightly below the maximum. Inthe latter culture, but not the former, there wasan appreciable increase in the ratio of diphos-phate to triphosphate for the purine nucleotides.

DISCUSSIONThe present findings substantiate earlier pro-

posals, based on the incorporation of precursorsinto RNA (9), for the reactions of pyrimidinenucleotide biosynthesis in M. mycoides. Theorganism possessed uracil phosphoribosyltrans-ferase, UMP kinase and CTP synthetase for theprovision of all pyrimidine ribonucleotides fromuracil. The activation of uracil phosphoribosyl-transferase from M. mycoides by GTP is similarto the effects observed with this enzyme from E.coli K-12 (11), and from E. coli B and Klebsiellaaerogenes (unpublished data). The CTP synthe-tase also appears similar to that from E. coli (7)in being activated by GTP and inhibited byCTP. Uridine phosphorylase was very active incell extracts, in keeping with the extensive deg-radation of uridine in cultures of M. mycoides(8). The high level of this enzyme would suggestthat the direct phosphorylation of uridine toUMP via uridine kinase would only have a tran-sitory significance in cultures unless uridine issupplied continuously. The observations on nu-cleotide contents support this suggestion. Therapid increase in concentration ofUTP upon theaddition of uridine is likely to arise through theprovision of an alternative reaction for the syn-thesis of UMP via uridine kinase in addition tothat catalyzed by the phosphoribosyltransferase.Before the provision of this additional pathway,the synthesis of uridine nucleotides would beregulated at the point of UMP biosynthesis bythe activating effect of GTP and the inhibitoryeffect of uridine nucleotides on uracil phospho-ribosyltransferase, perhaps also influenced bythe P-Rib-PP concentration which lies in therange of 15 to 60 uM in the total cell water(unpublished data). The increase in GTP con-centration (Fig. 3) could further increase therate of UMP production through activation ofuracil phoshorbosyltransferase. To limit therate of UTP production back to the require-ments ofthe celL the rise in UTP would initiallyprovide feedback, on both uridine kinase anduracil phosphoribosyltransferase. Subsequently,the fall in concentration of GTP and decreasedavailability of uridine (9) would limit the capac-

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PYRIMIDINE METABOLISM IN M. MYCOIDES 1079

ity for UMP synthesis such that a rate of syn-

thesis appropriate to cellular requirements isonly achieved at a lower concentration of thefeedback inhibitor, UTP. The increased CTPconcentration is a predictable consequence ofelevated levels of ATP, GTP, and UTP. Thereturn toward the original CTP level would oc-

cur as the inhibition of CTP synthetase by CTPbecame effective.Our observations that M. mycoides is unable

to incorporate cytosine into RNA (9), whereas iteffectively uses cytidine, are consistent with thefailure of cell extracts to catalyze the phospho-rolysis of cytidine. The ability to use cytidine as

a ready source of cytidine nucleotide (9) findsexplanation in the availability of cytidine kinase.However the inability to demonstrate cytidinedeaminase activity in extracts gave no supportfor the plausible hypothesis that this enzyme

might account for our observation that cytidineserves as a precursor of a significant fraction ofuridine nucleotide in RNA (9). Although it isunlikely, cytidine may appear as uridine nucleo-tide following deamination at the nucleotidelevel. Alternatively, M. mycoides may resemblea pyrimidine-requiring mutant of S. typhimu-rium reported to lack detectable cytidine de-aminase, yet to retain the ability to convertcytidine to uridine (2). Possibly, a slow deami-nation to maintain a low concentration ofuridinemight allow this product to be preferentiallyused by a uridine kinase with a high affinity forit, rather than being degraded by a uridine phos-phorylase with a low affinity.With the addition of cytidine to cells growing

in medium with guanine or guanine plus ade-nine, a pathway for the direct synthesis of cyti-dine nucleotides, independent of UTP, becomesavailable via cytidine kinase. The very largedifference in the extent to which CTP concen-

trations increase in the two cultures may stemfrom the enhanced ability of cultures containingguanine as well as adenine to support the addi-tional phosphorylations required to maintain a

raised content of CTP and UTP. Feedback in-hibition by the increased concentration of CTPon its further synthesis from UTP may have theeffect of sparing UTP and so contributing to itsincreased concentration, as would activation ofuracil phosphoribosyltransferase by the initiallyenhanced GTP content. Also, any conversion ofcytidine to uridine would influence the intracel-lular content of uridine nucleotides by makingavailable a pathway for their synthesis via uri-dine kinase. The in vivo studies on the effects ofcytidine addition suggest that the concentrationof CTP necessary to inhibit CTP synthetasemust be considerably less than that required to

inhibit cytidine kinase, since the concentrationsof both CTP and UTP rise markedly. Such aneffect would account for the strong preferenceshown for cytidine as a source of cytidine nu-cleotide in RNA when both uracil and cytidineare available as precursors (9).

ACKNOWLEDGMENTSWe are indebted to A. W. Rodwell for helpful discussion

and for supplying the organism and some constituents of thegrowth medium.

The work was carried out during tenure of a Sir John andLady Higgins Research Award to A.M., with support from theAustralian Research Grants Committee.

LITERATURE CITED

1. Bagnara, A., and L R. Finch. 1972. Quantitative ex-traction and estimation of intracellular nucleoside tri-phosphates of Escherichia coli. Anal. Biochem. 45:24-34.

2. Beck, C. F., J. L. Ingraham, J. Neuhard, and E.Thomassen. 1972. Metabolism of pyrimidines and py-rimidine nucleosides by Salmonella typhimurium. J.Bacteriol. 110:219-228.

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