JOURNAL OF BACTERIOLOGY, Aug. 1968, p. 501-514Copyright @) 1968 American Society for Microbiology

Vol. 96, No. 2Prinited in U.S.A.

Characteristics of the deo Operon: Role in ThymineUtilization and Sensitivity to Deoxyribonucleosides'

MARGARET S. LOMAX' AND G. ROBERT GREENBERG

Department ofBiological Chemistry, The University ofMichigan, Ann Arbor, Michigan 48104

Received for publication 6 March 1968

Inability to grow on deoxyribonucleosides as the sole carbon source is charac-teristic of deo mutants of Escherichia coli. Growth of deoC mutants, which lackdeoxyribose 5-phosphate aldolase, is reversibly inhibited by deoxyribonucleosidesthrough inhibition of respiration. By contrast, deoB mutants are not sensitive todeoxyribonucleosides, and deoxyribose 5-phosphate aldolase and thymidine phos-phorylase are present at normal levels but are not inducible by thymidine. Organ-isms with the genotype deoB- thy- or deoC- thy- are able to grow on low levels ofthymine, whereas deoB+ thy- or deoC+ thy- strains require high levels of thyminefor growth. The deoB and deoC mutations are transducible with and map on thecounterclockwise side of the threonine marker. They are closely linked to deoA, a

gene determining thymidine phosphorylase. Merodiploids heterozygous for eitherthe deoB or deoC genes are resistant to deoxyribonucleosides and, in combinationwith the thy mutation, require high levels of thymine for growth. Cultures of thy+deoC> mutants are inhibited by thymidine until this compound has been com-

pletely degraded and excreted as deoxyribose and thymine, whereupon growthpromptly resumes at a normal rate. The inhibition of respiration in deoC strainsand the induction of thymidine phosphorylase and deoxyribose 5-phosphate aldo-lase in the wild-type organism are considered to result from the accumulation ofdeoxyribose 5-phosphate.

At least three enzymes appear to be required forthe catabolism of thymidine by Escherichia coli:thymidine (TdR) phosphorylase (thymidine:orthophosphate deoxyribosyltransferase, EC2.4.2.4); 1, 5-phosphodeoxyribomutase (PDM);and deoxyribose 5-phosphate (dR-5-P) aldolase(2-deoxy-D-ribose 5-phosphate acetaldehyde lyase,EC 4.1.2.4) (6, 18, 28, 29, 33).

TTdR dR phosphorylase thymine + dR-I-PPDM ) dR-5 dR-5-P aldolase

glyceraldehyde 3-phosphate + acetaldehyde

The three enzymes are all reported to be inducibleby deoxyribonucleosides (5, 7, 30; H. 0. Kam-men, Federation Proc. 26:809, 1967).

Thymine-requiring (thy) mutants isolated bymeans of the aminopterin selection procedure of

1A portion of this paper was presented at the67th Annual Meeting of the American Society forMicrobiology, New York, N.Y., 30 April to 4 May1967.

2 This work was carried out during the tenure of aPost-doctoral Fellowship from the American CancerSociety, 1964-1966.

Akada et al. (26) lack the enzyme thymidylatesynthetase which converts deoxyuridylate(dUMP) to thymidylate (dTMP), and thesemutants show a high thymine requirement forgrowth, i.e., 20 ,ug/ml or more. Recently, in-vestigators in several laboratories have shown thatmutants with a low thymine requirement, i.e., 1 to2 A.g/ml, can be selected directly from high thy-mine-requiring thy strains and that such lowthymine-requiring mutants are actually doublemutants. Alikhanian and co-workers (2) andOkada (25) have mapped the mutation to a lowthymine requirement near the thr locus. Breitmanand Bradford (5) have provided evidence that alow thymine-requiring thy- strain of E. coli 15(70V3) (4) lacks dR-5-P aldolase. They haveascribed the low thymine requirement of thisstrain to an accumulation of dR-1-P, which is acosubstrate in the conversion of thymine to TdR.These authors have suggested this compound isnormally the limiting factor in thymine utiliza-tion.

This paper reports studies of two genes, deoBand deoC, and provides evidence that they areinvolved in determining two of the enzymes of thepathway described above. The deo designation

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has been adopted to indicate a gene determiningan enzyme necessary for growth on deoxythymi-dine or deoxyuridine as the sole source of carbon(10). Phenotypically, organisms carrying muta-tions in either gene B or C and in the structuralgene (thy) for thymidylate synthetase, i.e., withthe genotypes deoTB thy- or deoC- thy-, exhibita low thymine requirement for growth, whereasdeoB+ thy- or deoC+ thy- strains require highthymine. The growth of strains carrying the deoC-mutation is inhibited by deoxyribonucleosides asa result of the inhibition of respiration, and TdRphosphorylase is inducible in these mutants.With deoTB strains, which are not sensitive todeoxyribonucleosides, neither TdR phosphorylasenor dR-5-P aldolase is inducible. Recently, amutant organism deficient in TdR phosphorylasewas isolated by Fangman and Novick (12), andthe mutation (deoA-) was then shown to becotransducible with the thr marker by Dale andGreenberg (10).The present investigation relates the properties

of deoB and deoC mutants to the genetic findingsofAlikhanian and co-workers and of Okada and tothe biochemical studies by Breitman and Bradfordon low thymine-requiring thy mutants. dR-S-Paldolase has been shown to be absent in deoCmutants but present in deoB mutants. Evidence ispresented that both the inhibitory effect of deoxy-ribonucleosides in deoC mutants and the induc-tion of TdR phosphorylase and dR-5-P aldolaseby deoxyribonucleosides in wild-type cultures re-sult from the accumulation of dR-5-P. In simul-taneous studies, Breitman and Bradford (7;personal communication) have found low thymine-requiring mutants of E. coli strain 15 (thy-) lack-ing phosphodeoxyribomutase which were notinducible. These workers have also concludedthat dR-5-P was the inducer.

MATERIALS AND METHODS

Biological materials. The bacterial strains employedin this study and their complete phenotypes arepresented in Table 1. The transducing phage Plbt(15) was kindly provided by E. Englesberg, andShigella 16, the indicator bacterium for this phage,was obtained from R. L. Somerville.

Media. The following media were used in thisstudy: nutrient broth (0.8% Difco Nutrient Brothand 0.5% NaCl); L broth, L agar, and soft agar(22); and the minimal medium of Vogel and Bonner(36) containing 0.2% glucose as the carbon source.Minimal medium was supplemented with 10 ,ug ofthymine per ml for low thymine requirers or 50pg of thymine per ml for high thymine requirers andL-amino acids (each at 20 ,g/ml) as required.

Culture methods. Bacterial stocks were maintainedon nutrient agar slants supplemented with 50 ,g ofthymine per ml for thy- strains. Overnight nutrientbroth cultures were centrifuged and washed oncewith an equal volume of saline before being used to

inoculate prewarmed minimal medium. Cultures wereincubated at 37 C on a New Brunswick gyratoryshaker in Erlenmeyer flasks having Klett colorimetertube side arms. Growth was monitored turbidimetri-cally with a no. 66 filter or by measuring the absorb-ance of samples at 590 mp with a Beckman DU mono-chromator fitted with a Gilford absorbance con-verter. A reading of 10 Klett units or 0.12 Aso repre-sented approximately 108 cells/ml in minimal medium.

Mating conditions. The procedure described byde Haan and Gross (11) was followed for conjugationexperiments.

Transfer of episome. Suitable samples of culturesof donor and recipient strains growing exponentiallyin nutrient broth were mixed to obtain a cell densityof 5 X 108 and a ratio of 10 F- to 1 F'. The frequencyof transfer of episomal markers was determined bydiluting and plating the culture on selective mediaafter incubation at 37 C for 60 min. To determine thekinetics of transfer of episomal markers, samples ofthe mating mixture were diluted into 10 ml of ice-cold diluting medium at 5-min intervals. Matingpairs were disrupted by vigorous agitation on aVortex mixer for 1 min. The mixture was plated onselective media as described above.

Transduction experiments. Transducing lysates ofphage Plbt were prepared by confluent lysis on L-agarplates (22). Lysates were stored in the cold overchloroform and were titered on L-agar plates withShigella 16 as the indicator bacterium. Transductionswere performed as described by Lennox (22). Re-combinants were usually streaked on homologousmedia to obtain single-colony isolates before testingfor unselected markers. To test for nutritional mark-ers, single colonies were picked with sterile woodensticks and transferred to homologous media and testplates in an array. Plates were incubated at 37 C andexamined for growth after 1 day. To test for sensi-tivity to deoxyribonucleosides, single colonies weresuspended in 0.5 ml of saline and streaked on selectiveagar plates containing thymidine (200 ,g/ml). Re-sistant colonies produced streaks which were visiblewithin 24 hr, whereas deoxyribonucleoside-sensitiveisolates were not visible for 2 to 3 days.

Disc diffusion assay for sensitivity to deoxyribonu-cleosides. This assay was a modification of the pro-cedure of Allen and Yanofsky (3) for quantitating thesensitivity of various strains to inhibitors. Ap-proximately 106 cells in 3.0 ml of minimal top-layeragar were poured onto minimal agar plates supple-mented with any required nutrients except thymine. A0.1-ml sample of the test solution was pipetted onto asterile filter-paper disc (13 mm in diameter) placed atthe center of the plate. Compounds to be tested fortheir effect on the inhibition by thymidine wereincorporated into the top-layer agar. After a 24- or48-hr incubation at 37 C, the distance from theedge of the disc to the line of growth was measured.

Oxygen-uptake studies. Bacterial cultures grown toa concentration of about 4 X 108 cells/ml in glucose-minimal salts medium were maintained in ice untiluse. Portions (1 ml) of the culture were transferredto the main compartment of Warburg flasks con-taining 0.1 ml of various nucleosides in the side armsand containing alkali in the center well. The uptake

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TABLE 1. Bacterial strainsa

Strain deoC deoB deoA thy arg pyrA his leu met pro thr trp ara gal lac str T6 Sex

Bb... + + + + + + + + + + + + + + +S S F-B3 (72)c..... + - + -+ + + + + + + + + + + SS FB/r-62d ..... - + + - + + + + + + + + + + + S S F-B307cd..... + - + - + + + + + + + + + + S S F-B84Ocd..... - + + -+ + + + + + + + + + + S S FHB45e .... . + + + + - + R S F-201. + - + - + + + + + + + + + + + S S F2779.......++ + +-+.R R F-296h... .. - + + + + + + + + + + + + + + S S F

297i..... + + + + + + + + + + + + + + S S F300i. + + + + + - + + + - + + S S F302k .............. + R+ + F-303k........-_++--+.R R F-3101.....-+-.....+ _ + + R R F311 ..+ + + + + - + + + + - + - + + S S F

318........ - ++ --+.R R F~-322"1....... - ++-+--+.R R P-3390....... - ++ +-+.-R R P-388P ....... + +-++ ++ ++ ++ ++ ++ S S F-F'araB24/araB24,7.. + ++ + + + + + + + + + _ + S S F'

The following symbols are used: +, wild type; -, inability to synthesize or utilize; S, sensitivity;R, resistance; deo, deoxyribonucleoside; thy, thymine; ara, arabinose; gal, galactose; lac, lactose;arg, arginine; pyrA, structural gene for carbamyl phosphate synthetase (27); his, histidine; leu, leucine;met, methionine; pro, proline; thr, threonine; trp, tryptophan; str, streptomycin; T6, bacteriophage T6.

b The prototrophic E. coli B strain from which all mutants were derived was obtained from J. Spizizenin 1957.

c Originally isolated by S. Brenner and obtained from N. Melechen, St. Louis University, in 1965.d These strains were kindly provided by F. Kaudewitz (21). B307c and B840c were isolated after treat-

ment of E. coli B with nitrous acid; B/r-62 was induced by decay of 32p incorporated into cells of E. coliB/r. The mutants were identified by replica plating; no other selection procedure was involved in theisolation of these mutants (F. Kaudewitz, personal communication).

e A B/r strain which was obtained from H. Boyer.f A deoTB thy- derivative isolated by plating a deoB+ thy- mutant (strain 104) on a suboptimal level

of tbymine. Strain 104 was isolated by means of a modification of the procedure of Okada et al. (26)which employs trimethoprim rather than aminopterin (34) as the selective agent. The procedure wasmodified further by selecting mutants directly on agar plates containing trimethoprim (5,gg/ml) andthymine (20 jug/ml) rather than in liquid culture (B. Dale and G. R. Greenberg, unpublished procedure).

9 T6R derivative of HB45 obtained by direct selection.h Constructed by means of the cross described in Table 6, in which B840c was the donor.i A thr- mutation induced by ultraviolet irradiation of E. coli B and isolated by the penicillin selection

procedure of Gorini and Kaufman (14).i A thr pyrA araA13 leu-1 derivative of strain B/r which was obtained from R. Helling, Department

of Botany, The University of Michigan.k Obtained by plating strain 277 (106 cells) on minimal medium containing all the required amino

acids plus thymine (5,ug/ml) and trimethoprim (5 ,g/ml).An arg+ derivative of 303 produced by transduction.A thy+ derivative of B840c produced by transduction.

n A pyrA- derivative of 310 produced by selecting a leu+ pyrA- recombinant from the following trans-duction: donor 311 (thr- pyrA- ara-) X recipient 310 (deoC- ara- leu-).

0 A thy+ derivative of 303 produced by transduction.P Obtained as a thr+ deoA- recombinant from the following cross: K12SH-28 (thr+ deoA-) (12) X 297

(thr- deoA+).q Obtained from D. Sheppard (32).

studies were carried out in an atmospheric medium. as uridine were added from a second arm after deter-The system was allowed to reach 37 C, and the mining the initial rate of oxygen uptake in the pres-nucleoside or water was tipped into the culture. In ence of the deoxyribonucleoside inhibitor. The detailssome cases (not presented), other compounds such are given in the legends of the figures.

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Preparation of extracts. Cultures were harvestedby centrifugation at 6,000 X g for 15 min in the cold.The cells were washed once with minimal mediumand recentrifuged. Extracts were prepared by grindingcells with alumina as described previously (31) andextracting with 0.05 M tris(hydroxymethyl)amino-methane-chloride, pH 7.4, containing 10-2 M 2-mercaptoethanol and 10-3 M ethylenediaminetetra-acetate.

Enzyme assays. TdR phosphorylase was assayed bythe method of Friedkin and Roberts (13). dR-5-Paldolase was measured by the procedure of Racker(29), with the addition only of alcohol dehydrogenase(Worthington Biochemicals Corp., Freehold, N. J.)and reduced nicotinamide adenine dinucleotide (SigmaChemical Co., St. Louis, Mo.) to the reaction mixturein order to follow acetaldehyde formation, or bymeasurement of dR-5-P formation (29).

Chemical analysis of cultures for deoxyribonucleicacid (DNA), ribonucleic acid (RNA), and protein. Theacid-insoluble material from 10 ml of culture wasprecipitated by the addition of 1 ml of cold 55%perchloric acid (PCA), separated by centrifugation,washed twice with cold 5% PCA, and resuspended in1.25 ml of 5% PCA. The nucleic acids were extractedby heating for 20 min at 90 C. After centrifugation,constant-size samples of supernatant fluid wereanalyzed for DNA by the Burton modification of thediphenylamine reaction (9) with deoxyadenosine asthe standard, and for RNA by the orcinol method(1) with adenosine as the standard. The residue wasdissolved in 5 ml of 0.02 M NaOH-0.02 M Na2CO3,and was analyzed for protein by the procedure ofLowry et al. (24).

Analysis of culture filtrates for thymine and deoxy-ribose. Portions of cultures were filtered throughHA45 filters (Millipore). The filtrate was analyzedfor deoxyribose by the diphenylamine reaction. TdRdoes not react in the diphenylamine test. Thyminewas determined from the absorbance of samples in0.1 NNaOHat300mju (19).

Chemicals. TdR (A grade) and dR-5-P were ob-tained from Calbiochem, Los Angeles, Calif. Allother deoxyribonucleosides, ribonucleosides, andbases used in this study were obtained from com-mercial sources, primarily Calbiochem. Trimethoprimwas the generous gift of G. H. Hitchings, Burroughs-Wellcome Laboratories. DL-Glyceraldehyde 3-phos-phate was prepared from the diethylacetal bariumderivative from Calbiochem. The D-glyceraldehyde3-phosphate concentration was measured by couplingto a-glycerophosphate dehydrogenase and triose-phosphate isomerase (Calbiochem).

RESULTS

Identification of deoB and deoC mutants. ThedeoB and deoC mutants were discovered whenseveral low thymine-requiring thy mutants weretested for their relative rates of growth on mini-nmal agar plates supplemented with thymine, TdR,or dTMP, by measuring the average size of thecolonies. Two of these strains displayed unex-pected growth characteristics on TdR-agar plates,

TABLE 2. Average size of colonies produced by thymutants on minimal agar supplemented with

thymine, thymidine, or thymidylate"

Thymine Thymidine Thymidylate

Strain (10 mg/ml) (20 pg/ml) (30 pg/ml)24 hr 48 hr 24 hr 48 hr 24 hr 48 hr

B3 (71)....... lb 3.3 0.8 3.8 1.1 4.1B307c....... 2.1 5.6 1.9 4.7 1.2 1.8B840c....... 1.7 4.5 0 0.9 1.0 1.0B/r-62.: .... 1.4 3.5 0 1.8 0.7 1.3

a The diameter of individual colonies was meas-ured under a low-power Bausch & Lomb stereo-scopic microscope fitted with an ocular rule. Thevalues given represent the average of 10 measure-ments and are in mm.

as shown in Table 2. All mutants formed smallbut distinguishable colonies on thymine-agarwithin 24 hr. Neither B840c nor B/r-62 formedcolonies on thymidine-agar within 24 hr. The ap-pearance of visible colonies after 48 hr indicatedthat the effect of thymidine was not bactericidal.All strains appeared to grow more slowly ondTMP at the level employed.

Further studies on the growth characteristics ofthese mutants in liquid medium supplementedwith either thymine (10 jig/ml) or TdR (20 ,ug/ml) showed that in TdR B/r-62 and B840c grewat a greatly reduced rate for 2 hr, after whichgrowth abruptly resumed at the normal rate. Thegrowth of B307c was normal in either medium.Therefore, low thymine-requiring strains, such asB307c, which grow normally on either thymine orTdR were designated deoB mutants, and strainssuch as B840c and B/r-62 which could grow onlow levels of thymine but were inhibited by TdRwere called deoC mutants. The complete pheno-types of these two classes of mutants are sum-marized in Table 3.

Expression of deoC mutation in thy+ and thybackgrounds. Both B840c and B/r-62 were con-verted to thy+ derivatives by transduction withphage Plbt which had previously been grown on awild-type organism. All thy+ transductants stillgrew slowly on TdR, indicating that this effectwas an inhibition unrelated to the nutritional re-quirement imposed upon the cell by the thy muta-tion. Furthermore, the deoC mutation was notlinked to the thy locus by transduction. These re-sults are summarized in Table 4, which lists thegrowth rate as mass doubling time of the originalstrains and thy+ transductants in medium supple-mented with thymine or high concentrations ofTdR. It can also be seen from these data thatTdR had no effect on the growth rate of wild-typeor deoTB thy- cells.

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TABLE 3. Phenotypes of deoB and deoC mutants

GenotypePhenotype

deoBF deoC+ thy- deoB- deoC+ thy- deoB+ deoC- thy- thy' deoB+ deoC-

Can utilize 1 to 2 peg of thymine No Yes Yes Not testableper ml for growth

Can utilize thymidine as a sole Yes No No Nocarbon source

Growth inhibited by deoxyribo- No No Yes Yesnucleosides

Thymidine phosphorylase activity Inducible Uninducible Inducible InducibleDeoxyribose 5-phosphate aldolase Inducible Uninducible Absent Absent

Inhibition of growth of a deoC mutant by TdR.The kinetics of growth of a thy+ deoC> strain incultures containing identical initial cell densitiesbut different amounts of thymidine was investi-gated. The length of the lag period was directlyproportional to the concentration of TdR in themedium (Fig. 1). If 50 ,ug of TdR per ml wasadded to a sample of the same culture at a lowerinitial cell density, the length of the lag periodwas increased proportionately to the decrease incell density. Thus, the length of the inhibitoryphase was a function of the concentration ofTdR per cell.

This proportionality and the fact that inhibitionwas reversible suggested that the effect might bedue either to the TdR itself or to a closely relatedmetabolite whose intracellular concentrationcould be maintained at an elevated level so longas a sufficiently high concentration of TdR re-mained in the medium. Therefore, the fate of theadded TdR and the relationship between TdRdegradation and inhibition were examined bymeasuring the appearance of thymine and deoxy-ribose in the medium. As shown in Fig. 2, thetime required for complete conversion of TdRto thymine and deoxyribose coincides with thelength of time the culture remained inhibited.The thymine and deoxyribose found in the culturefiltrate represented 100% and 75%, respectively,of the TdR originally present, a finding notgreatly different from the results with E. coli15 thy- (70V3) (5) in which equimolar concen-trations of thymine and deoxyribose were found.The deoxyribose could be either free deoxyriboseor a phosphorylated derivative, since the assayfor deoxyribose would not distinguish betweenthese compounds.

Inhibition by TdR of the synthesis of nucleicacids and protein in a deoC mutant. To determinewhich metabolic process was being inhibited byTdR and thus limiting the rate of growth, we in-vestigated the kinetics of the synthesis of nucleicacids and protein. The results shown in Fig. 3

TABLE 4. Summary ofgrowth rates ofdeoB and deoCmutants in minimal medium supplemented with

either thymine or thymidinea

Mass doubling time (min)

Genotype Strain ThydiMin- Thymine, 200-500imal 10 pg/mi -500l

deoB+ deoC+ thy+ B 43 49 44deoBT deoC+ thy- B307c - 49 47

302 - 48 48deoB+ deoC- thy- B840c - 49 127-267

B/r-62 - 67 159-192303 - 31b 100-116b

deoB+deoC thy+ 315 45 45 164296 43 140

a The mass doubling time for deoC mutantsgrowing in thymidine was determined from theinhibitory phase of the growth cycle. The valuefor deoC mutants in minimal medium or minimalmedium plus thymine and for deoC+ strains underall conditions were determined from the exponen-tial phase of the growth cycle. The range of valuesgiven for the mass doubling time of deoC mutantsrepresents the values obtained in several differentexperiments.

b The medium was supplemented with vitamin-free acid-hydrolyzed casein (0.2%) and L-trypto-phan (20 ,g/ml) in addition to thymine or thymi-dine.

indicate that, when TdR was added to an expo-nentially growing culture of thy+ deoC- cells,protein and RNA synthesis were quickly inhibited.DNA synthesis appeared to be inhibited onlyafter a delay of 15 or 20 min. Since these resultsmight derive from limitation of an energy supply,the effect ofthymidine on the respiration of deoC-cells was investigated.

Inhibition by TdR of oxygen uptake. The effectof TdR on the rate of oxygen uptake by suspen-sions of thy+ deoC cells in a glucose-salts mediumcan be seen in Fig. 4. In this experiment, the cul-

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F140/z2120/

o 100

IL 80/

Z60-0 40 Xw

p 20 X

0 10 2030 40 50 60 70 80 90 100TdR, pg/ml

FIG. 1. Influence of thymidine concentration on thelength of the period of inhibition of a deoC mutant.Thymidine was added to samples ofstrain 315 (deoC-thy+) growing exponentially in minimal medium. Growthwas followed turbidimetrically until each culture hadovercome the inhibition by thymidine and had reachedlate stationary phase. The length of the period ofinhibition was calculated from the resulting growthcurves by extrapolating the two linear portions of asemilog plot. The solid circles represent data fromcultures to which the thymidine had been added whenthe cell density was 20 Klett colorimeter units. Oneculture which received thymidine (50 ,g/ml) at aKlett reading of8 had a period ofinhibition of229 min.When this value was corrected for the difference inthe initial cell density of the culture, the value (0)also fell on the line.

tures were allowed to come to equilibrium in theWarburg flasks, and at time-zero the indicatedquantities ofTdR were tipped into the main com-partment of the flask and oxygen uptake wasmeasured. Because the cells had been exposed tolow temperatures, there was a short lag periodbefore a linear rate of oxygen uptake was ob-tained with the control vessel containing no TdR.Increasing the concentration of TdR in the reac-tion vessel greatly increased this lag period. TdRadded during oxygen-uptake measurementspromptly caused inhibition. Figure 5 shows thatTdR had no effect upon the rate of oxygen uptakeby the wild-type strain (B), whereas the thy+deoC- strain (296) was severely inhibited. Uridinewas able to overcome partially the inhibitory ef-fect of TdR. This effect also was observed whenuridine was added after the TdR inhibition haddeveloped. By means of the disc-diffusion tech-nique, it was determined that TdR showed thesame inhibition of growth of strain 296 whenglucose, acetate, or succinate was employedas the carbon source. That the decreased oxygen

1.0

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FIG. 2. Kinetics of the appearance of thymine anddeoxyribose in the culture medium of a deoC mutantinhibited by thymidine. Thymidine was added at theindicated time to an exponentially growing culture ofstrain 296 (thy+ deoC-) to yield a final concentrationof200,ug/ml (0.83 mole/ml).

uptake is a result of an inhibition of respiration,and not due to a difference in the mass of cells,is shown by the following. (i) An immediate de-crease occurs in oxygen uptake of about two tofive times below the control value in the presenceof TdR. In other experiments (not shown), TdRwas added while the oxygen uptake was beingmeasured, and an immediate decrease in uptakeoccurred. (ii) The growth rate in these smallvessels with the beginning concentration of 4 X108 cells/ml and with minimal agitation was quiteslow, with doubling times of perhaps 80 min.

Inhibition of deoC strains by other deoxyribo-nucleosides. By means of the disc diffusion assay,several bases, ribonucleosides, and deoxyribo-nucleosides were tested in place of TdR as pos-sible inhibitors of deoCc strains. The resultsshown in Table 5 indicate that all deoxyribo-nucleosides tested, except deoxycytidine, in-hibited this strain. On the basis of isotopedilution experiments, Lichtenstein et al. (23) havesuggested that deoxycytidine is metabolized dif-ferently than TdR. Ribonucleosides did not in-hibit thy+ deoC> strains. The free bases had noeffect on any strain tested, and none of the com-pounds inhibited the wild-type control. However,if 0.5 mg of any ribonucleoside was added to thetop-layer agar and TdR was placed on the disc,either no zone of inhibition was observed or thezone of inhibition was greatly decreased.

r-

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.5 GROWTH3o 4 THYMIDINE

PROTEIN.j07:

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.0340 80 120 160

TIME IN MINUTES

FIG. 3. Kinetics of growth, protein synthesis, DNAsynthesis, and RNA synthesis of a deoC mutant in-hibited by thymidine. Thymidine (50 jig/ml) was addedto one-half of a culture of strain 296 (thy+ deoC')growing exponentially in minimal medium at the timeindicated by the arrow. Samples ofthe inhibited cultureand the control culture containing no thymidine wereassayed for protein, DNA, and RNA. An As-A6a0reading of3.83 is equivalent to I limole ofdeoxyaden-osine in the diphenylamine reaction. An Awo of 14.5is equivalent to I jamole ofadenosine in the orcinol re-action.

Mapping of the deoB and deoC genes. In con-jugation studies involving a prototrophic Hfr anda thy- deoTB thr- female (strain 302), we ob-served that 91% of the thr+ recombinants hadreceived the deoB+ marker. The linkage of deoBto thr was confirmed by transductional crosses,which yielded a cotransduction frequency of48%. The deoC locus is also linked to thr. Theresults of transduction experiments in which twodifferent deoC mutants were used as donors indi-cated that the deoC marker was transferred withthe thr+ marker at a frequency of about 50%(Table 6). Therefore, the sensitivity to deoxyribo-nucleosides was not the result of several unlinkedmutations in the original strains.

Three-factor crosses to order the deoC genewith respect to thr and pyrA were also performedwith the three different deoC- alleles in our col-

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zw0

20 40 60 80 100 120 140 160MINUTES

FIG. 4. Inhibition of the oxygen uptake of a deoCmutant with increasing concentrations of thymidine.Cultures ofE. coil 296 (deoC-) were grown aerobicallyto a cell concentration of 4 X 108/ml; I-ml portionswere added to each flask. After equilibration at 37 C,0.1 ml of TdR or water was tipped in from the sidearm, and measurements were made.

10 20 30 40 50 60 70TIME, MINUTES

FIG. 5. Inhibition by thymidine of the oxygen up-take of deoC- strains and insensitivity of deoC+strains. Cultures of E. coli 296 (deoC-) and E. coliB (deoC+) were grown and treated as in the legendto Fig. 4. TdR (200 jig) and uridine (200 jig) wereadded to the indicated vessels at zero-time. The finalvolume was 1.2 ml.

lection. The results shown in Table 7 suggestthat the most probable order of markers is: deoCthr pyrA.The results of a three-factor cross to order the

deoA, deoC, and thr markers are presented inTable 8. The low frequency of recombination be-tween the deoA and deoC markers indicates thatthey are very closely linked. In addition the ab-sence of deoA- deoC- recombinants and thepresence of only a few deoA+ deoC+ recombi-nants is taken to indicate that the deoA- deoC-class requires four crossing-over events. There-

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fore, the most probable order of markers is:deoA deoC thr. Figure 6 shows the position ofthese genes in relationship to other markers onthe chromosome of E. coli.Dominance of the deoC+ and deoB+ alleles.

TABLE 5. Effect of deoxyribonucleosides and relatedcompounds on a deoC mutant (296)a

Copudtestedb Concn Zone ofCompound testedb Concn inhibition

mg/ml mm

Thymidine................. 10 17Deoxyuridine .............. 10 17.5Deoxycytidine ............. 10 1Deoxyadenosine ........... 5 14.5Deoxyguanosine ........... 10 17Bromodeoxyuridine ........ 10 12.5

a Inhibition was measured by the disc diffusionassay. The concentration given is of the test solu-tion applied to the disc. The plates were incubatedfor 24 hr.

b The following bases were not inhibitory at 1mg/ml: thymine, uracil, adenine, cytosine, andguanine. Adenosine (5 mg/ml) and the followingribonucleosides (10 mg/ml) were not inhibitory:thymine riboside, uridine, cytidine, and guano-sine (saturated solution). Deoxyribose (10 mg/ml)was not inhibitory. None of the compoundstested inhibited the wild-type control.

Merodiploids heterozygous for the deoC anddeoB markers were constructed by the introduc-tion of an episome (F' ara) which carries the wild-type alleles of both markers. This episome arosefrom Hfr B2, which has a point of origin nearthr and transfers markers in a counter-clockwisedirection (32). The merodiploid with the geno-type deoB+ deoC- thy- (strain 318)/F' araB24deoC+ was resistant to deoxyribonucleosides andrequired high levels of thymine for growth.The merodiploid having the genotype deoB-deoC+ thy- (strain 302)/F' araB24 deoB+deoC+ required high levels of thymine for growth.These results demonstrate that deoB+ and deoC+are dominant to their mutant alleles and thatthese genes must normally be responsible for theformation of physiologically active products.dR-S-P aldolase and TdR phosphorylase ac-

tivities in deoB and deoC mutants with and withoutinduction by TdR. Extracts of various strainsgrown in the presence or absence of levels ofTdR sufficient to induce TdR phosphorylase(100 ,tg/ml or greater) (30) were assayed fordR-5-P aldolase and for TdR phosphorylaseactivities (Table 9). The results indicate that TdRinduces the formation of both the aldolase andTdR phosphorylase in the wild-type strain(E. coli B). However, in deoB mutants, inductionof phosphorylase and aldolase did not occur or

TABLE 6. Cotransduction of deoC and thr markers"

Donor Genotype of donor Genotype of recipient recombinants tested No. deoC Cotransduction

B/r-62 thr+ deoC- thr- deoC+ 104 46 44B840c thr+ deoC- thr- deoC+ 104 55 53

a P1 lysate made on the indicated donor was used to infect the recipient strain 297. The deoC markerwas tested by spotting suspensions of single colonies on plates containing TdR (200 jug/ml).

TABLE 7. Ordering of deoC, thr, andpyrA by a three-factor transductional crossa

Cross Ib: deoC thr+ PyrA+ - deoC' thr pyrA deoC+ thr+ CrossA Ie:CrossespyrA+ - deo thrpyrAo

Classes of recombinants No. of recombinants if donor is: Required No. of Required

________________-crossing-over reobnns crossing-ov~erB/r-62 B840c regions d recombinants regionsd

pyrA+ thr+ deoC 16 12 1-4 28 2-4thr+ deoC+ 19 15 2-4 19 1-4thr- deoC- 0 0 1-2-3-4 41 3-4thr- deoC+ 19 27 3-4 2 1-2-3-4

a The pyrA+ recombinants were purified on selective media and tested for thr and deoC markers.b The recipient for this cross was strain 300.* The donor in this cross was strain 104 and the recipient strain 322.d Crossing-over regions (as shown in parentheses) if the order of markers is: (1) - deoC - (2) - thr -

(3) - pyrA - (4).

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TABLE 8. Ordering of deoA, deoC, and thr by athree-factor transductional cross,

Class of recombinants No. in Required crossing-class over regionsb

thr+ deoA+ deoC- 37 3-4thr+ deoA- deoC+ 45 1-4thr+ deoA+ deoC+ 2 2-4thr+ deoA- deoC- 0 1-2-3-4

a P1 lysate prepared on the donor strain 299(deoA- deoC+ thr+) was used to infect the re-cipient strain 339 (deoA+ deoC- thr-). The thr+recombinants (84) were selected and purified onselective agar. The deoA and deoC markers weretested in the following manner. The deoA+ deoC+recombinants were able to grow on thymidine assole carbon source. Neither deoA- nor deoC-mutants would grow on this medium. The deoA+deoC: recombinants were inhibited when streakedon minimal-glucose agar containing a high con-centration of TdR (200 jug/ml). Recombinantsthat did not grow on TdR as sole carbon sourcebut were not inhibited by TdR were assumed tobe deoA- recombinants; however, this class wouldinclude both deoA- deoC+ and deoA- deoWrecombinants. The deoA- deoC- recombinantsshould be inhibited by purine deoxyribosidessuch as AdR. The deoA- class was tested for AdRinhibition by the disc-diffusion assay; no AdR-sensitive recombinants were found. The deoAmarker was independently confirmed by deter-mining whether cultures of each recombinantcould degrade TdR to thymine and deoxyribose.

b Crossing-over regions (shown in parentheses)if the order is: (1) - deoA - (2) - deoC - (3) - thr -(4).

was only minimal. On the other hand, TdRphosphorylase was slightly elevated in deoC-cells and was greatly increased after exposure ofthe culture to TdR. In sharp contrast, TdR-induced or uninduced deoC- cells showed negli-gible or very low levels of dR-5-P aldolase. In someinstances, a slight and anomalous activity ap-peared to be present immediately after additionof the dR-S-P substrate to extracts of deoC- cellsbut stopped within minutes, whereas the activitiesobserved in E. coli B and in deoB cells continuedlinearly for periods of over 1 hr.

Strain 388 carrying a mutation in the deoAgene and lacking TdR phosphorylase (12) showedinduced levels of dR-5-P aldolase when grown ondeoxyadenosine. Strain 71 was obtained fromE. coli B3 (our original culture obtained fromN. Melechen in 1959) as a single-colony isolate onplates containing 2,ug of thymine/ml. Strain 71had constitutive levels of both TdR phosphorylaseand dR-5-P aldolase, about 20 times greater thanthe uninduced wild-type levels. These activities

deoA (deoB) deoC thr

str trp

FiG. 6. Genetic map (35) ofE. coli showing the posi-tion of the deo genes.

could be induced slightly further by growth inTdR. A culture of E. coli B3 (strain 72) obtainedfrom Dr. Melechen in 1965 had approximatelynormal levels of both dR-5-P aldolase and TdRphosphorylase activities, but these were increasedonly minimally by exposure to TdR. Neitherstrain 71 nor strain 72 is inhibited by TdR, andneither grows on TdR as the sole carbon source;both are therefore classified as deoBT organisms.The TdR phosphorylase to dR-5-P aldolase ratiosappear to be increased in strain 71, but in bothstrain 72 (ratios not shown) and strain 71 theratios were essentially the same in induced anduninduced cultures.

Because some extracts exhibited a relativelyrapid oxidation of reduced nicotinamide adeninedinucleotide, it was frequently not possible toassay dR-5-P aldolase by a one-step direct cou-pling of the acetaldehyde to the alcohol dehydro-genase reaction (or D-glyceraldehyde 3-phosphateto triose isomerase and glycerol-phosphate de-hydrogenase), especially when the aldolase levelwas low. Accordingly, in some instances aldolaseactivity was assayed by measuring the rate offormation of dR-5-P from acetaldehyde andglyceraldehyde 3-phosphate (29) at 25 C, by useof the Burton (9) modification of the diphenyl-amine reaction. By this procedure, very low levelsof aldolase activity could be detected withoutbackground interference. These results are shownin parentheses in Table 9.

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TABLE 9. Levels of dR-5-P aldolase and TdR phosphorylase in extracts of deoA, deoB, and deoC mutants"

dR-S-P aldolase TdR phosphorylaseGenotype Strain

(units/mg of protein)b (units/mg of protein)c Phosphorylase/aldolase

Uninduced Induced Uninduced Induced Uninduced Induced

Wild type

deoB- thy-

deoC- thy-

deoC thy+

deoA- thy+

BBB307cB307c20172(B3)71 (B3)B840cB/r-62296296388

6.7-7.2(2.8)e7.9

(13.8)13.3(6.3)

102Of

<1 .5Of(0)

87-127(43)e15.3(10.0)11.5(8.4)134

Of

Of(0)97

92-95

77

128114

1,470177

135

1,240-1,250

170

115172

2,1302,220720

1 ,180

6.7

13 .4d

9.7

9.6

14.5

11 .9d

11.1

10.0

15.9

a Uninduced values are from cultures grown in minimal medium (thy+ strains) or 10 /Ag/ml of thy-mine (thy- strains). Induced values are from cultures grown in 500 pgg/ml of thymidine, except forstrain 388 in which 500,ug/ml of AdR was used as the inducer.bProduct formed (m,umoles) per minute at 25 C.c Product formed (m,umoles) per minute at 37 C.d When more than one enzyme value is presented, extracts of at least two separate cultures were

assayed, and the ratios are based on average values.e The figures in parentheses represent the rates of formation of dR-5-P (9) from acetaldehyde and

D-glyceraldehyde 3-phosphate (29). The two values in parentheses opposite strains B307c and B arefrom different extracts than those assayed by the forward reaction.

f Anomalous rate first few minutes; see Results.

DISCUSSIONThese studies provide evidence that: (i) muta-

tions in either of two genes, deoB or deoC, willexplain the mutation of a thy- organism from ahigh to a low thymine requirement; (ii) the in-hibition of the growth of deoC mutants by deoxy-ribonucleosides appears to result from an inhibi-tion of respiration; (iii) the deoC gene determinesdR-5-P aldolase; and (iv) dR-5-P aldolase andTdR phosphorylase (determined by the deoAgene) are coordinately induced by thymidine andare controlled by genes in an operon responsiblefor the degradation of TdR. To account for thesefindings, we present a model which proposes thatdeoB determines phosphodeoxyribomutase andthat dR-5-P is both the inducer of this operon andthe inhibitor of deoC mutants. The phenotypes ofdeoB and deoC mutants are summarized in Table3.

Figure 7 describes the pathway for the degrada-tion of TdR (and other deoxyribonucleosides)and the gene-enzyme relationships suggested bythe present studies. The following evidence hasbeen presented to support these gene assign-ments.The deoC mutants clearly lack dR-5-P aldolase

activity (Table 9). In such mutants, TdR phos-phorylase is present at wild-type levels and is

inducible by TdR. These deoC mutations aretherefore not in a regulatory gene affectingall the enzymes of the pathway. This conclusionis also supported by the observation that thewild-type allele is dominant in merodiploids.Because deoB mutants cannot grow on TdR as a

sole source of carbon, they must be blocked insome step in the conversion of TdR to a sourceof energy. Since E. coli is unable to utilize eitherthymine or free deoxyribose as an energy source,the energy is considered to be derived from adeoxyribose phosphate moiety. Extracts of deoBmutants have been shown (Table 9) to containwild-type levels of TdR phosphorylase and dR-5-P aldolase, two of the three enzymes required forthe conversion of TdR to compounds which areintermediates in an energy-yielding pathway.Therefore, we conclude that the deoB mutantslack the third enzyme, phosphodeoxyribomutase,although this deduction has not yet been verifieddirectly by enzyme assays. The fact that thedeoB+ allele is also dominant to the deoB-allele in merodiploids supports the conclusionthat deoB mutants are deficient in some enzy-matic function and are not regulatory mutantsof the 00 type, although the dominance relation-ship per se does not eliminate a mutation of aregulatory gene of the positive control type (32).

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These gene-enzyme relationships also provide amodel to explain the phenotypes of deoB anddeoC mutants. Loss of either phosphodeoxyribo-mutase (deoB) or dR-5-P aldolase (deoC) wouldcause an accumulation of dR-i-P and an enhance-ment of the ability of a thy- cell to utilize lowconcentrations of thymine for DNA synthesis.However, mutants of these two genes would beexpected to differ with respect to other propertiesbecause different deoxyribose phosphates wouldaccumulate in each case. Only dR-i-P shouldaccumulate in deoB mutants, whereas bothdR-1-P and dR-5-P should accumulate in deoCmutants. Assuming that dR-5-P is the inhibitor(or the precursor of the inhibitor) of deoC mu-tants, it is to be expected that deoTB cells, whichcannot form dR-5-P, would be insensitive todeoyxribonucleosides.

This model also is consistent with the results ofstudies of the induction of thymidine phos-phorylase in deoC and deoB mutants. In the wild-type organism, this activity can be increased morethan 10-fold by the addition of high concentra-tions of TdR to the growth medium. The ob-servation that all deoxyribonucleosides induceTdR phosphorylase activity in E. coli K-12 hadled to the suggestion that dR-1-P could be theinducer (30). On the basis of our findings, sum-marized in the model shown in Fig. 7, the inducerof both TdR phosphorylase and dR-5-P aldolaseis considered to be dR-5-P. TdR phosphorylaseand dR-5-P aldolase activities could not be in-duced or showed only minimal induction in deoBmutants (Table 9), which according to our modelcannot form dR-5-P. On the other hand, TdRphosphorylase was induced in deoC mutants,which are able to form dR-5-P, and was normallyhigher in the absence of added inducer than thelevels found in uninduced wild-type cells. Thedifferent patterns of induction of TdR phos-phorylase observed in deoB; thy- and deoC- thy-strains help to explain the apparently contradic-tory reports of other workers concerning theinducibility or noninducibility of this enzyme inlow thymine-requiring thy- mutants (5, 6, 16, 30)and emphasizes the importance of distinguishingbetween these two classes of mutants when at-tempting to describe the properties of thy- strains.

Breitman and Bradford (5) also had suggestedthat the inducer could be dR-5-P. In a very recentreport, these workers (7) have found that certainlow thymine-requiring mutants of E. coli 15 thy-lacked phosphodeoxyribomutase activity and,therefore, would not be expected to form dR-5-P.TdR phosphorylase and dR-5-P aldolase couldnot be induced in these mutants, although in theparental strain these enzymes were apparentlycoordinately induced (5, 7). Their findings

GENE ORDER: deoA (deoB) deoC thr ovrA

PHOSPHORYLASE MUTASE ALDOLASE

TdR / dR-I-P \ Gly-3-P+ + _ dR-5-pF +Pi T Acetoldehyde

INDUCES INHIBITSPHOSPHORYLASE 02 UPTAKEAND ALDOLASE

FIG. 7. The deo gene-enzyme complex of E. coli.The deoB gene has not been ordered by genetic testsbut has arbitrarily been placed between deoA and deoCon the basis of the sequence of enzymatic reactions.Abbreviations used: phosphorylase, TdR phosphorylase;mutase, phosphodeoxyribomutase; aldolase, deoxy-ribose 5-phosphate aldolase; Pi, orthophosphate;dR-1-P, deoxyribose 1-phosphate; dR-5-P, deoxyribose5-phosphate; gly-3-P, glyceraldehyde 3-phosphate.

strengthen the model presented in this paper andour assignment of the mutase activity to thedeoB gene in E. coli B.

Recent studies by Hoffee (17) on deoxyribose-negative mutants of Salmonella typhimurium alsoare consistent with the hypothesis that dR-5-Pis the inducer of the thymidine pathway in E. coli.S. typhimurium can utilize free deoxyribose as thesole carbon source by virtue of an inducible path-way consisting of a permease, deoxyribosekinase, and a dR-5-P aldolase, all of which arecoordinately induced by deoxyribose. However,this organism contains a second form of dR-5-Paldolase which is induced by dR-5-P and co-ordinately regulated with TdR phosphorylase.Mutants of this second form of dR-5-P aldolasemap in the thr-ara region of the S. typhimuriumgenetic map and are therefore analogous to ourdeoC mutants (P. Hoffee, personal communica-tion).The close linkage of the deo genes (Tables 6-8)

and the clear biochemical relationship of theirproducts suggest that these genes may constitutean operon whose enzyme products are requiredfor the degradation of TdR and deoxyribose-phosphates. DeoA and deoC are clearly veryclosely linked (Table 8). Although the deoB genehas not been ordered in these studies, it showsthe same frequency of cotransduction with thras do deoA and deoC (about 50%). The mappingdata of other workers (2, 25) places all low-thymnine mutations to the left of thr. We havetherefore arbitrarily placed the deoB gene be-tween deoA and deoC.The concept of a deo operon is further sup-

ported by our observations that dR-5-P aldolaseand TdR phosphorylase are coordinately in-duced by TdR (Table 9). Other workers haveshown that TdR phosphorylase (5, 28, 30),

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phosphodeoxyribomutase (7; H. 0. Kammen,Federation Proc., p. 809, 1967), and dR-5-Paldolase (5, 7) were inducible by TdR. The re-quirement that an operon be regulated is also metby the finding that strain 71, classified as deoB,and derived from E. coli B3, is constitutive forboth TdR phosphorylase and dR-5-P aldolaseand thus may represent a regulatory mutation ofthe QO type. The exact nature or location of themutation in this organism remains to be deter-mined and is presently under investigation.The mutation to the low thymine requirement

has been described by Breitman and Bradford(4), Stacey and Simson (34), and Harrison (16),and has recently been mapped by Alikhanianet al. (2) and by Okada (25). The mutation hasbeen called Itr and thyR, respectively, by the lattertwo groups. Alikhanian and co-workers (2) alsodescribed a mutation to TdR sensitivity (td8),but suggested that the low thymine requirementof the same thy- strain might be due to the pres-ence of an additional mutation in the Itr gene.Our work confirms and extends the geneticstudies of these groups on mutations leading to alow thymine requirement (deoB or deoC) and tosensitivity to deoxyribonucleosides (deoC). Inaddition, the data presented here establish thatdeoC mutants show the low thymine phenotypeas well as deoxyribonucleoside sensitivity as aconsequence of the loss of dR-5-P aldolase ac-tivity. These observations are in accord with thefindings of Breitman and Bradford (5) that a lowthymine-requiring thy- strain of E. coli 15(70V3) lacked dR-5-P aldolase. These workersalso had observed that the growth of this strainwas inhibited by thymidine (5, 6) but did not re-late the inhibition to the loss of dR-5-P aldolase.In fact, they report that thymidine inhibited thegrowth of E. coli 15 thy- and E. coli 15, I, and II,all of which lacked phosphodeoxyribomutaseactivity (7). At the present time, we have noexplanation for the differences between the resultsof these authors and those described here.Whether these are species differences or otherkinds of mutations will have to await furtherstudy. The simultaneous genetic, enzymatic, andphysiological studies reported in the presentwork clearly establish that the inhibition, lowthymine requirement, and loss of dR-5-P aldolaseare phenotypic expressions of the same mutationin E. coli B. Eisenstark and co-workers (personalcommunication) have found that S. typhimuriumshows a mutation to deoxyribonucleoside sensi-tivity and that the same gene is responsible for alow thymine requirement in thy- cells. Theirmutants appear to correspond to our deoCmutants and to the mutants of Hoffee determin-

ing the second form of dR-5-P aldolase in S.typhimurium (17).The inhibitory effect of deoxyribonucleosides

on the oxygen uptake of deoC> cells (Fig. 4 and 5)is considered to be on the respiratory chain ratherthan in the carbon pathway, inasmuch as growthwas inhibited about equally well with glucose,succinate, or acetate as the carbon source. Sinceall macromolecular syntheses tested (DNA,RNA, and protein) were found to be inhibitedby TdR, it has been concluded that the inhibitionoccurs by a limitation in the energy source. Theauthors are aware of no evidence that inhibitionof protein, RNA, or DNA synthesis will decreaserespiration. Accordingly, the effect on respirationis considered to be the primary action of TdR.The site of action and the mechanism by whichthe apparent agent, dR-5-P, effects this inhibitionremain to be studied. The data in Fig. 5 and thetext indicate that ribonucleosides could overcomethe inhibitory effects of TdR. This effect of ribo-nucleosides may be explained by the recentdemonstration that these compounds inhibit TdRphosphorylase (8) and thus may act by preventingthe formation of dR-5-P.

Although the method of analysis of the deoxy-ribose excreted into the medium when TdR wasadded to deoC> cells did not distinguish betweendeoxyribose and its phosphorylated derivatives,in all probability free deoxyribose was excreted.Breitman and Bradford (4) have shown that freedeoxyribose was excreted by a low thymine-requiring derivative of E. coli strain 15 during thethymineless state. These observations suggestthat another enzyme, possibly a specific deoxyri-bose phosphate phosphatase, may operate in thedeoxyribonucleoside and deoxyribonucleotidedegradation pathway. It is not unreasonable thatsuch an enzyme would be part of the deo operonand would be inducible by dR-5-P. Normally, indeoC+ cells the dR-5-P aldolase is capable of de-grading dR-5-P derived from added TdR, and nodeoxyribose is excreted (5, 18). It is not knownwhether deoB- cells excrete deoxyribose.

It seems appropriate to speculate about thepossible role of dR-5-P in the control of respira-tion and perhaps of DNA synthesis. We considerthat the pathway: deoxyribonucleotide -* de-oxyribonucleoside ;=± free base + dR-1-P a#dR-5P -+ glyceraldehyde-3-P + acetaldehyderepresents the route of degradation of deoxyribo-nucleotides and deoxyribonucleosides. Normally,dR-1-P and dR-5-P probably do not accumulatein quantity because of the excess of mutase andaldolase activities. Any block in the conversion ofdUMP to dTMP or of the utilization of dTMP orof other deoxyribonucleotides for DNA synthesiswould be expected to cause an accumulation of

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deoxyribonucleotides and an increased formationof dR-5-P. Thus far, there is no evidence thatconditions created by a block of dR-5-P aldolaseor ofdTMP synthetase will inhibit growth throughan affect on respiration except by addition ofdeoxyribonucleosides to the media. It is possiblethat the affect is more subtle and might require anexamination of synchronized cells. If such a con-trol were to be operative, it could represent amechanism for synchronizing DNA synthesiswith energy need.

ACKNOWLEDGMENTS

This investigation was supported by Public HealthService grants AM 01669 from the National Instituteof Arthritis and Metabolic Diseases and Al 08133from the National Institute of Allergy and InfectiousDiseases. We thank Lee Gough for excellent technicalassistance. We are grateful to P. Hoffee, T. Breitman,and A. Eisenstark for sending us manuscripts of theirwork prior to publication.

Beverly Dale generously provided us with extractsof various deo and thy mutants, made available thedata for some of the TdR phosphorylase values, andcarried out the crosses in Table 8. We are gratefulto R. L. Somerville for advice and consultation.

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1. Albaum, H. G., and W. W. Umbreit. 1947.Differentiation between ribose-3-phosphate andribose-5-phosphate by means of the orcinolpentose reaction. J. Biol. Chem. 167:369-376.

2. Alikhanian, S. I., T. S. lljina, E. S. Kaliaeva,S. V. Kameneva, and V. V. Sukhodolec. 1966.A genetical study of thymineless mutants ofE. coli K-12. Genet. Res. 8:83-100.

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5. Breitman, T. R., and R. M. Bradford. 1967. Theabsence of deoxyriboaldolase activity in athymineless mutant of Escherichia coli strain 15:A possible explanation for the low thymine re-quirement ofsome thymineless strains. Biochim.Biophys. Acta 138:217-220.

6. Breitman, T. R., and R. M. Bradford. 1967.Metabolism of thymineless mutants of Escher-ichia coli. I. Absence of thymidylate synthetaseactivity and growth characteristics of two se-quential thymineless mutants. J. Bacteriol. 93:845-852.

7. Breitman, T. R., and R. M. Bradford. 1968.Inability of low thymine-requiring mutants ofEscherichia coli lacking phosphodeoxyribo-mutase to be induced for deoxythymidine phos-

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9. Burton, K. 1956. A study of the conditions andmechanism of the diphenylamine reaction forthe colorimetric estimation of deoxyribonucleicacid. Biochem. J. 62:315-323.

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13. Friedkin, M., and D. Roberts. 1954. The enzy-matic synthesis of nucleosides. 1. Thymidinephosphorylase in mammalian tissue. J. Biol.Chem. 207:245-256.

14. Gorini, L., and H. Kaufman. 1960. Selecting bac-terial mutants by the penicillin method. Science131:604-605.

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