JOURNAL OF BACTERIOLOGY, Mar. 1972, p. 1085-1096Copyright (0 1972 American Society for Microbiology

Vol. 109, No. 3Printed in U.S.A.

Isolation and Partial Characterization ofRegulatory Mutants of the Pyrimidine Pathway

in Salmonella typhimuriumGERARD A. O'DONOVAN AND JOHN C. GERHART

Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, andDepartment of Molecular Biology, University of California, Berkeley, California 94720

Received for publication 26 October 1971

Mutants of Salmonella typhimurium affected in the regulation of pyrimidinebiosynthesis were isolated by two methods. The first involved screening forbacteria able to feed a pyrimidine-requiring indicator strain, and the secondinvolved selection for bacteria simultaneously resistant to two pyrimidine ana-

logues, 5-fluorouracil and 5-fluorouridine, in a S. typhimurium strain unable todegrade 5-fluorouridine. Among the mutants isolated by these methods are

constitutive mutants, producing high levels of pyrimidine biosynthetic en-

zymes in the presence or absence of pyrimidines, and feedback modified mu-

tants, in which aspartate transcarbamylase is partially desensitized to its in-hibitor, cytidine triphosphate. No fully desensitized mutant has been found.The partially desensitized character cotransduces with the pyrB locus, that ofaspartate transcarbamylase. The constitutive character has been determined ina few cases to be localized in the region of leu and pro on the Salmonella map.

The pyrimidine pathway in the enteric bac-teria received early attention concerning itsregulation by feedback inhibition and repres-sion (3, 11, 29-31). Recent reviews have sum-marized the present status of physiologicalstudies on enzyme repression (21) and of en-zymological studies on feedback inhibition (9,21). Genetic analysis, which has so advancedthe understanding of regulation in severalamino acid biosynthetic pathways, has notbeen applied to the regulatory elements of thepyrimidine pathway since constitutive anddesensitized mutants have been lacking so far.At present, only structural genes specifyingthe enzymes of the pathway have been as-signed and localized for Escherichia coli (25)and Salmonella typhimurium (23 and Fig. 1).The patterns of genetic regulation in the

pyrimidine pathway are of potential interestsince the structural genes are individuallyscattered around the genome in E. coli (25, 26),and in S. typhimurium (28) and since enzymesynthesis for the different steps of the pathwayappears under the control of different re-pressing metabolites (1, 17, 18). Desensitizedmutants would probably only concern aspar-tate transcarbamylase and the control of itsactivity by the end product cytidine triphos-

phate (CTP). Since the protein chemistry ofthis enzyme has been well studied, desensi-tized forms of the enzyme could be analyzed inmore detail than is possible for allosteric pro-teins of other biosynthetic pathways. In partic-ular, mutant forms of the regulatory subunitha':e potential interest for understanding therole of this nonenzymatic protein in modifyingaspartate transcarbamylase activity.

In this report we describe a method for theselection of high-level constitutive mutants inS. typhimurium and some physiological char-acteristics of these mutants. Although noselective condition was found for desensitizedmutants of aspartate transcarbamylase, a largeset of partially desensitized mutants was ob-tained by screening for pyrimidine-overpro-ducing bacteria. The physiology of these mu-tants is described briefly.

MATERIALS AND METHODSBacterial strains. E. coli JC411 requires methio-

nine, arginine, histidine, and leucine and was sup-plied by A. J. Clark. S. typhimurium LT2 and bacte-riophage P22 were supplied by P. E. Hartman. TheS. typhimurium deletion mutant pyrA81 (28) whichrequires arginine and uracil for growth and the otherS. typhimurium pyrimidine-requiring deletion mu-

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(Cpsase) (ATCose)HCO- fpA C A CARBAMYL3HC PHOSPHATE ASPARTAT

2 ATP 2 ADP + Pi Aspartote Pi \( 2+ + pirC

Glutoffe Giutomote

CTP ADP DIHYDROOROTATE1" 1/20(CTPsase)cGuteG te/t

ATP co ppiPRPP2Glutomine HkM S 20<

UTP <r UDP -(I UMP-r OMP<&r

OROTATEgZDP k/nse) (lW A*w) (O4fd.cs) (OIVwJo)

FIG. 1. Pyrimidine biosynthetic pathway of Salmonella typhimurium and Escherichia coli. Genetic sym-bols for the enzymes are shown in italics. The enzymes shown in parentheses are abbreviated as follows: car-bamyl phosphate synthetase (CPSase); aspartate transcarbamylase (ATCase); dihydroorotase (DHOase); di-hydroorotate dehydrogenase (DHOdehase); orotidine-5'-phosphate pyrophosphorylase (OMPppase); orotidine-5'-phosphate decarboxylase (OMPdecase); uridine-5'-phosphate kinase (UMP kinase); uridine-5'-diphosphatekinase (UDP kinase); cytidine-5'-triphosphate synthetase (CTPSase).

tants were supplied by K. E. Sanderson. S. typhimu-rium DP-39 (18), a derivative of pyrA81 requiringarginine and uracil for growth (pyrA) as well aslacking the enzymes uridine phosphorylase (udp)and cytidine deaminase (cdd), was supplied by J. L.Ingraham. The strain DP-39 (18) was transduced toprototrophy by transferring the parental pyrA locusinto DP-39 with P22 phage and selecting for bacteriaable to grow in the absence of arginine and uracil. Thestrain retained the udp and cdd mutant loci and ishenceforth designated OG-39.Growth media. Bacteria were grown in a modi-

fied M-56 medium (10) containing the followingcomponents (in grams per liter of distilled water):Na2HPO4, 8.7; KH2PO4, 5.3; (NH4) 2S04, 2.0;MgSO4 .7H20, 0.10; Ca(NO3)2, 0.005; ZnSO4 .7H20,0.005; FeSO4.7H20, 0.005; and D-glucose, 2.0. ThepH was 7.0. Supplements, when required (in gramsper liter), were: L-amino acids, 0.02; pyrimidines,0.02. Solid medium was prepared by the addition of20 g of agar per liter.

Preparation of extracts. Bacteria were grown toa density of approximately 5 x 108 cells per ml, cen-trifuged, and resuspended at approximately 1010cells per ml in 40 mm potassium phosphate buffer,pH 7.0. Suspensions were frozen and stored for sonictreatment. Samples (0.5 to 1.0 ml) in 40 mm phos-phate buffer, pH 7.0, were contained in 5-ml cellu-lose nitrate centrifuge tubes (Spinco), sealed withparafilm, and sonically treated in a 9-kc Raytheonsonic oscillator at 0 C for about 3 min (3 x 60 sec).Samples were examined turbidometrically for visiblebreakage and immediately were sonically treatedagain if the cells did not appear disrupted.

Aspartate transcarbamylase. Aspartate trans-carbamylase was assayed by the method of Gerhartand Pardee (11). Cytidine (0.02 M) was sometimesused instead of CTP (0.2 mM) as the feedback inhib-itor of aspartate transcarbamylase in vitro.

Dihydroorotate dehydrogenase. The sensitivityof the assays of Beckwith et al. (3) and of Taylor andTaylor (27) was increased by the use of iodo-nitrotet-

razolium (INT) as the electron acceptor in the de-hydrogenase reaction. Stock solutions were preparedas follows: solution A, 0.1 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 8.7, con-taining 0.004 M KCN; solution B, INT chloride (3.2mg/ml) in water; solution C, 0.02 M L-dihydroorotatedissolved in 0.05 M Tris-hydrochloride, pH 8.1, andstored at 4 C. Assay tubes contained 0.5 ml of solu-tion A, 0.3 ml of solution B, 0.05 ml of solution C,and water and cell extract to yield a total volume of1.0 ml. The control tube, to indicate backgroundreduction of INT, contained all components exceptL-dihydroorotate. The reaction was initiated by theaddition of enzyme (generally in a volume of 0.05ml) and after mixing was allowed to proceed for 20min at 30 C. The reaction was stopped by the addi-tion of 0.1 ml of 1.0 N HCl. At this point, tubes couldbe stored several days at room temperature withoutfinal effect on the color yield. To determine the ex-tent of reaction, a 1.0-ml volume of n-butanol wasadded to each tube and the mixture was agitatedvigorously on a Vortex Jr. mixer to extract the re-duced INT into the organic phase. The emulsion ofthe aqueous and organic phases was broken by low-speed centrifugation in a clinical centrifuge. Theupper phase was withdrawn and read at 480 nm in acuvette of 1-cm path length.One unit of enzyme activity is defined as that

causing a change of 1.0 optical density unit at 480nm under the assay conditions described above. Theamount of reaction was proportional to enzyme con-centration up to two units of activity.

Zone centrifugation. The approximate molecularsize of the partially desensitized mutant aspartatetranscarbamylase was estimated by zone centrifuga-tion in a sucrose density gradient as follows. Asample of 0.4 ml of mutant enzyme extract (equiva-lent to 6.4 mg of protein) in 2 mM mercaptoethanol,pH 7.0; 40 mM potassium phosphate, pH 7.0; and 0.2mM ethylenediaminetetraacetic acid (EDTA), pH7.0, was layered onto a 5-ml linear gradient of 6 to25% sucrose. It was centrifuged in a Spinco model L

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centrifuge at 35,000 rev/min at -6 C in an SW39rotor for 16 hr. The sedimentation positions of wild-type aspartate transcarbamylase (12S) and catalyticsubunit (6S) of aspartate transcarbamylase were de-termined in separate samples centrifuged simultane-ously.

Drops were collected into 13 tubes from centri-figed samples. Fractions were then assayed to deter-mine aspartate transcarbamylase activity in thepresence of 15 mM aspartate, 3.6 mm carbamyl phos-phate, 40 mm potassium phosphate buffer. Fractionsfrom samples of partially desensitized mutants re-quired no dilution prior to assay.

Determination of acid-soluble nucleotide pools.Nucleoside triphosphates were determined by pre-viously published procedures (17, 19).

Chemicals. Chemicals were purchased from thefollowing sources: cytidine and CTP from SigmaChemical Co.; INT (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl-mono tetrazolium chloride) from Mann; N-methyl-N'-nitro-N-nitrosoguanidine (NTG) from K& K Laboratories. The 5-fluoro analogues were gen-erous gifts from W. E. Scott, Hoffman-La Roche,Inc., Nutley, N.J.

RESULTSIsolation of regulatory mutants of

Salmonella typhimurium. In screening forpyrimidine overproducers. Mutants defectivein the regulation of pyrimidine biosynthesiswere sought on the basis of their overproduc-tion and release of intermediates or productsof the pyrimidine pathway, or both. On solidmedium, colonies of such mutants were de-tected by their capacity to support the satellitegrowth of pyrimidine-requiring indicator bac-teria. Being relatively nonselective, such ascreening procedure was expected to yieldmany classes of regulatory mutants, amongthem constitutives and feedback-resistantmutants.

For the detection of pyrimidine-overpro-ducing bacteria on plates, an indicator strainwas obtained that accepted any of a largenumber of intermediates of the pyrimidinepathway to satisfy its auxotrophic require-ment. Strain construction began with S. typhi-murium pyrA81, a deletion mutant lackingcarbamyl phosphate synthetase activity andrequiring arginine and pyrimidine for growth.Like most pyrimidine auxotrophs of S. typhi-murium and E. coli, this strain does notreadily use pyrimidine intermediates such ascarbamyl aspartate, dihydroorotate, or orotatefor its requirement, presumably because ofimpermeability. A derivative of pyrA81 wastherefore selected in two steps: first, for its useof carbamyl aspartate, and second, for its useof orotate. The capacity to use orotate simulta-neously conferred upon the strain the capacityto use dihydroorotate. For use in screening,

this modified strain was starved for pyrimi-dines for 2 hr in liquid culture and then mixedin with the plate-pouring medium consisting ofminimal medium plus Casamino Acids and 2%molten agar at about 45 C to give a concentra-tion of 106 bacteria per ml. Plates of 20-mlmedium were poured within 5 min after thebacteria were introduced and were allowed tocool. Such indicator plates have been used aslong as a week after preparation when storedat 4 C. Ornithine, at 100 ,g/ml, was includedin the medium since it stimulated pyrimidineoverproduction in certain mutants. This effectis probably related to the role of ornithine asan activator and counter-inhibitor [againsturidine monophosphate (UMP)] of carbamylphosphate synthetase, causing an increasedflow of carbamyl phosphate into the pyrimi-dine pathway (1).An NTG-treated (2) culture of bacteria, after

phenotypic expression for 6 to 8 hr in minimalCasamino Acid medium, was spread on to thesurface of an agar plate containing indicatorbacteria. About 600 viable mutagenized bac-teria were spread on to each plate.

Colonies were examined after 2 to 3 days ofincubation of the plates at 37 C. Rare colonieswere found surrounded by a halo of secondarygrowth of the indicator bacteria in and on theagar medium. Such colonies were picked andpurified on complex media and retested forpyrimidine prototrophy (to eliminate the indi-cator strain) and pyrimidine overproduction.The frequency of overproducers was about 1 in104 colonies, thus necessitating 10 to 20 platesto obtain a single mutant. About 400 inde-pendent pyrimidine-overproducing mutantshave thus been isolated.

This type of screening indeed yielded mu-tants that overproduced pyrimidines as meas-ured in liquid cultures lacking the indicatorbacterium. Mutants were grown in glucoseminimal medium to 109 bacteria per ml anduracil was detected in the cell-free supernatantfluid of the mutants by its absorption spec-trum in alkaline solution after the method ofPaege and Schlenk (22). Orotate was estimatedby the method of Machida and Kuninaka (15).Some overproducers released as little as 1 ,gof uracil per ml, whereas others (e.g., HD58)produced as much as 100 ,g/ml.The primary product found in the medium

of the mutants was uracil, but traces of car-bamyl aspartate and up to 15 ,ug of orotate perml were observed in media of those strainswhich accumulated high concentrations ofuracil.Attempts were made to isolate a similar set

of overproducers in E. coli K-12, but with less

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success than for S. typhimurium. The wild-type E. coli parent (JC411) itself produced ahalo, reducing the certainty of mutant detec-tion. Recently, it has been observed that K-12strains, unlike S. typhimurium and E. coli Bstrains, generally overproduce about 30 qg oforotate per ml and release it into the medium(15, 21). This high background may explain thelow frequency of E. coli K-12 regulatory mu-tants isolated by this technique for pyrimidinebiosynthesis. E. coli B and S. typhimuriumstrains do not overproduce the intermediates(15). Studies to elucidate the mechanism(s)involved in this phenomenon are in progress(Machida and Kuninaka, manuscript in prep-aration).

Selection for doubly drug-resistant mu-tants. Mutants deficient in the regulation ofpyrimidine biosynthesis were expected to con-tain high internal pools of UMP and thereforehave increased resistance to toxic pyrimidine(nucleotide) analogues. Hence, drug resistancewas used as a condition for selecting pyrimi-dine overproducers. 5-Fluorouracil (5-FU) and5-fluorouridine (5-FUR) are toxic for S. typhi-murium, each being metabolized to 5-fluoro-UMP by different enzymes (21). Accordingly,selections for pyrimidine regulatory mutantswere done using mixtures of 5-FU and 5-FURsince two-step resistance would be rare. Thisobviated the problem encountered when eitherdrug alone is used because 5-FU-resistantmutants remain sensitive to 5-FUR and 5-FUR-resistant mutants may or may not be re-sistant to 5-FU. Indeed, in strains of S. typhi-murium that can degrade uridine (and hence5-FUR), most 5-FUR-resistant mutants so iso-lated are merely 5-FU resistant and remainsensitive to the nucleoside analogue (21). Forthese reasons, the direct selection required astrain of S. typhimurium which lacked uridinephosphorylase (udp) so that it would not de-grade 5-FUR to 5-FU. The strain OG-39 (seeabove) was used. 5-FU and 5-FUR, each at aconcentration of 0.5 to 1.0 ,ug/ml, were in-cluded in the minimal agar and about 107viable cells of an NTG-treated culture (2) of anLT-2 strain harboring the udp mutation werespread on each plate. The frequency of muta-tion to resistance was about 1 in 104, and allcolonies that grew proved to be high-level py-rimidine overproducers, accumulating uraciland uridine in the range of 10 to 100 ,g/ml inliquid culture. When higher concentrations ofthe two analogues were employed, resistantmutants did not arise.The high level overproducers obtained by

the screening method were also doubly re-sistant to the 5-FU and 5-FUR mixture, but,

since all of these strains contain an active uri-dine phosphorylase (udp+ can degrade 5-FURand uridine to 5-FU and uracil), the resistanceis primarily directed toward 5-FU.

Characterization of regulatory mutants.To identify pyrimidine-overproducing mutantsaffected in aspartate transcarbamylase, trans-duction was employed to select only thosemutants linked to pyrB, the gene that codesfor aspartate transcarbamylase. A pyrB dele-tion (pyrB124) was used as the recipient strainfor phage P22 lysates that had been grown onregulatory mutants obtained from thescreening procedure, and pyrimidine proto-trophic transductants were selected. Thoseprototrophic transductants that gave halos ofsecondary growth were considered excellentcandidates to have an aspartate transcarba-mylase modified in its regulatory properties.The pyrB gene and the gene for pyrimidineoverproduction were thus cotransduced inthese strains. Of all (407) the screened mu-tants, 30% of them were found to be linked topyrB. These strains were all low-level over-producers (1 to 10 ,ug/ml of uracil in liquidculture) and came only from the screeningtechnique.Table 1 summarizes the results of five such

mutants. As can be seen, the enzyme levels inthese mutants were quite low in the absence ofadded uracil, and in fact are comparable to the"fully repressed" levels found in the parentstrain, LT2, grown in uracil. Since these mu-tants excreted as much as 10 mg of uracil perml into the medium during their growth, thisis the expected result. All mutants testedshowed considerably less sensitivity to CTPinhibition than the parent strain (Table 2).Figure 2 compares the inhibition by increasingconcentrations of CTP on two of these mu-tants with the wild strain. It can be seen fromFig..2 that both mutants approach a maximumlevel of inhibition which is much less than thatfor the parental strain. Other properties suchas adenosine triphosphate (ATP) activationand substrate dependence have not been ex-amined. In no case, though, was a mutantfound with no sensitivity to CTP, as would beexpected if the regulatory or catalytic subunithad been modified to the extent of being in-capable of aggregation to form asparatatetranscarbamylase. Indeed, in zone centrifuga-tion experiments (see above) performed on ex-tracts from these 5 and 20 other mutants, as-paratate transcarbamylase sedimented at thevelocity of wild-type asparatate transcarbamy-lase, indicating the presence of regulatory andcatalytic subunits in a hexameric complex.Approximately 60% of the pyrimidine-over-

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TABLE 1. Specific activity of aspartate transcarbamylase and dihvdroorotate dehydrogenase, grown in thepresence and absence of uracil, as well as feedback inhibition of aspartate transcarbamylase in the wild strain,

LT2, and in five pyrimidine-overproducing mutants linked to pyrB by cotransduction

Ratio of aspartate Per centAspartate Dihydroorotate transcarbamylase inhibition

Strain Uracila transcarbamylase dehydrogenase specific activity of aspartatespecific activity" specific activity" without and with transcarbamylase

uracil by CTPd

LT2 + 0.06 0.11 87LT2 - 0.25 0.16 4.1 85HD1 + 0.07 0.14 50HD1 - 0.06 0.12 045HD2 + 0.05 0.15 47HD2 - 0.07 0.16 1.4 42HD23 + 0.05 0.09 12 35HD23 - 0.06 0.14 1 30HD27 + 0.05 0.14 12 38HD27 - 0.06 0.23 .35HD35 + 0.06 0.10 0.8 50HD35 - 0.05 0.22 48

a All cultures were grown in glucose minimal medium to which Casamino Acids (100 ,g/ml) were added.Uracil (50 Ag/ml) was used where stated (+).

Aspartate transcarbamylase activity was measured as previously described (11), using 5 x 108 cells pertube at 30 C for 30 min. One unit of specific activity for aspartate transcarbamylase is defined as thatcausing a change of 1.0 optical density unit at 560 nm per mg of protein at 30 C in 30 min in the colorimetricassay (11).

' Dihydroorotate dehydrogenase activity was measured by using a modification of previously describedmethods (3, 27) with 5 x 108 cells per tube at 30 C for 20 min. (See Materials and Methods.) One unit of spe-cific activity for dihydroorotate dehydrogenase is defined as that causing a change of 1.0 optical density unitat 480 nm per mg of protein at 30 C in 20 min.

d Inhibition was measured by using 5 mm aspartate, 3.6 mM carbamyl phosphate, and 0.2 mm cytidine tri-phosphate (CTP).

producing mutants proved to have elevatedlevels of aspartate transcarbamylase activitywhich were not decreased by exogenous uracil(20 ,g/ml). These mutants were classified asconstitutive, at least for aspartate transcarba-mylase synthesis. No mutant was less than 20-fold derepressed for aspartate transcarba-mylase and many were 100-fold derepressedover wild-type levels from cells grown in thepresence of uracil.Table 2 lists some of the properties of 12

constitutive mutants. Six of these were iso-lated by the screening technique and six bydirect selection, as indicated. The symbolpyrR for "pyrimidine regulatory," is used todesignate the genotype of these mutants. Forcomparison on levels of derepression, the wild-type LT2 and a deletion mutant for each genein the pyrimidine pathway are included. Thewild type and each of the deletions (auxo-trophs) were grown in excess uracil (repressed)and in limiting uracil (derepressed) before as-says were performed. Feedback inhibition ofaspartate transcarbamylase by CTP was exam-ined in all cases. The following key points maybe made from Table 2. (i) The specific activityof aspartate transcarbamylase was elevated

100- to 200-fold in the 12 mutants, comparedto the repressed level found in wild-type cells.(ii) The specific activity of dihydroorotatedehydrogenase was elevated three to sixfold inthe 12 mutants, compared to repressed wild-type level. This increase is strikingly smallerthan was found for aspartate transcarba-mylase. (iii) The level of aspartate transcarba-mylase in the constitutive mutants did notchange when uracil was added or removedfrom the growth medium; in contrast, the levelchanged 4-fold in wild-type bacteria, and from3- to 10-fold in the auxotrophs. (iv) The inhibi-tion of aspartate transcarbamylase by CTPwas not affected in the constitutive mutants.These mutants accumulated large amounts

of uracil in the medium, up to 100 ,ug/ml. Theaddition of other pyrimidines such as cytidineand uridine did not repress enzyme synthesiseven in mutants HD11, 12, and 14 which lackcytidine deaminase and use cytidine well as asource of CTP. Hence, these mutants behavedas high-level constitutive mutants. Beckwithet al. (3) also showed that aspartate transcar-bamylase and dihydroorotate dehydrogenaselevels do not vary proportionately in E. coliduring derepression.

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TABLE 2. Constitutive mutants for pyrimidine biosynthesis in Salmonella typhimurtum

Reaie Ratio of aspartate Per centRelative aspartate Relative transcarbamylase inhibition of

Straina Uracil' transcarbamylase dehydrogenase specific activity aspartate trans-activityc activityd without and with carbamylase

activity_ uracil by CTPeLT-2 + 1.0 1.0LT-2 - 4.1 1.5

Mutants isolated by screening techniqueHD4 + 93.3 6.0HD4 - 103.3 3.7HDl9 + 96.6 3.7HD19 - 85.0 4.5HD42 + 167.0 4.6HD42 - 167.3 3.5HD47 + 131.6 6.0HD47 - 100.0 5.6HD58 + 106.6 3.4HD58 - 105.0 3.6HD67 + 118.5 3.5HD67 - 83.3 3.6

Mutants isolated by double resistance to 5-fluorouridine and 5-fluorouracilHD1l + 73.3 2.8HD11 _ 73.3 3.3HD12 + 99.8 4.0HD12 - 87.0 4.4HD14 + 70.0 3.0HD14 - 73.3 2.9HD87 + 83.3 3.5HD87 - 101.6 2.6HD88 + 101.6 4.5HD88 - 96.6 4.7HD120 + 66.6 3.0HD120 - 70.0 3.0

Pyrimidine deletionsPyrA81 + 1.5 1.3PyrA81 Limit 4.5 2.6PyrB124 + 1.3PyrB124 Limit 3.3PyrC138 + 2.0 1.1PyrC138 Limit 16.6 3.0PyrD135 + 1.3PyrD135 Limit 13.3PyrE137 + 2.6 2.0PyrE137 Limit 20.0 5.0PyrF146 + 1.6 1.0PyrF146 Limit 9.6 2.2

A 1 894.1 90

899091838888888888908585

848488839090838284867981

1.1

0.9

1.0

0.8

1.0

1.4

1.0

0.9

1.0

1.2

0.9

1.0

3.0

8.3

10.0

7.5

5.8

7580

8085828082818085

a Extent of derepression is compared with the wild strain LT-2 and with six deletions (28) each lacking thesingle indicated enzyme.

° All cultures were grown in glucose minimal medium to which Casamino Acids (100 jg/ml) were added.Uracil (50 ,ug/ml) was used where stated (+). The pyrimidine auxotrophs were grown in 6 gg of uracil per mland then starved for 2 hr (limit) after uracil had been exhausted.

c Aspartate transcarbamylase activity was measured as previously described (11), using a sonically treatedextract equivalent to 5 x 10" cells per tube at 30 C for 30 min. When necessary, 1: 10 and 1: 100 dilutions weremade with constitutive mutants. The specific activity for aspartate transcarbamylase is as in Table 1. Com-puted by dividing aspartate transcarbamylase specific activity for all mutants grown in the presence and ab-sence of uracil by the wild type (LT-2) repressed aspartate transcarbamylase level, namely, 0.06 (Table 1).

d Dihydroorotate dehydrogenase activity was measured by using a modification of previously describedmethods (3, 27) with 5 x 108 cells per tube at 30 C for 20 min. (See Materials and Methods.) The specific ac-tivity for dihydroorotate dehydrogenase is as in Table 1. Computed as by dividing dihydroorotate trans-carbamylase specific activity for all mutants by the wild-type (LT-2) repressed dihydroorotate dehydro-genase level, namely, 0.11 (Table 1).

e Inhibition was measured by using 5 mM aspartate, 3.6 mM carbamyl phosphate and 0.2 mM cytidine triphos-phate (CTP).

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TABLE 3. Nucleoside triphosphate pools ofSalmonella typhimurium LT2a and six constitutivemutantsa of the pyrimidine biosynthetic pathway

StrainsPools"

LT2 HD58 HD67 HD42 HDll HD12 HD14

ATP 3.29 3.42 4.24 6.88 4.56 3.20 6.18CTP 0.81 1.12 1.14 1.73 1.36 1.22 1.68GTP 1.26 2.04 2.64 3.27 2.50 1.45 3.12UTP 1.13 1.02 1.06 1.49 1.31 1.24 1.57dATP 0.17 0.25 0.31 0.55 0.29 0.21 0.43dCTP 0.25 0.29 0.30 0.47 0.26 0.42 0.32dGTP 0.11 0.15 0.19 0.25 0.19 0.13 0.23dTTP 0.30 0.37 0.33 0.65 0.34 0.27 0.53

a Pools were determined in cells growing exponen-tially in glucose medium with 100 Ag of CasaminoAcids per ml and 32P-orthophosphate (3 iC1/Mmole).Strains HD58, HD67, and HD42 were isolated by the

l____________________________________lscreening technique, and strains HD11, HD12, and4 6 to - HD14 were selected directly by double drug resist-

CTP x leM ance (see text).CTAbbreviations: ATP, adenosine triphosphate;

IG. 2. Effect of increasing concentrations of CTP CTP, cytidine triphosphate; GTP, guanosine tri-the activity of wild-type LT2 aspartate transcar- phosphate; UTP, uridine triphosphate; dATP, deox-zylase and on two partially desensitized mutants, yadenosine triphosphate; dCTP, deoxycytidine tri-1 and HD2. Extractions and assays were per- phosphate; dGTP, deoxyguanosine triphosphate;ned as in Table 1. dTTP, deoxythymidine triphosphate.

C Values are exnressed as micromoles ner gramIn analyzing these mutants, measurements

of nucleotide pools were made to test whethertheir constitutivity was due to an inability toproduce adequate levels of endogenous uridinetriphosphate (UTP) and CTP, repressing me-

tabolites of the pyrimidine pathway (17, 18).Despite the gross overproduction of uridinemonophosphate (UMP) in these mutants whichaccumulate uracil in the medium, UTP andCTP synthesis could be limiting if there werea defective UMP kinase or CTP synthetase. Adefect at these late steps could not be over-

come by exogenous added pyrimidines. Asshown in Table 3, the UTP and CTP levels insix constitutive mutants were not lowered, andin fact were somewhat high, perhaps as a resultof constitutivity. Three of the mutants were ob-tained from screening, and three were fromdrug selection, the six being chosen at randomfrom the set of high-level constitutives de-scribed in Table 2. Thus, there is no evidencethat low endogenous pools of UTP and CTPare responsible for the constitutive phenotypeof these strains.The constitutive phenotype of HD47, HD58,

and HD67 was successfully transferred to wild-type LT2 strains with the selection of trans-ductants resistant to mixtures of 5-FU and 5-FUR. These doubly resistant transductantswere then assayed for aspartate transcarbamyl-ase activity and were found to be high-level

(dry weight).

constitutives. As a control, bacteria were pre-pared that had gained resistance to 5-FU and5-FUR in two separate selective steps andwhich lacked the enzymes uridine kinase (theudk locus) and UMP pyrophosphorylase (theupp locus). With these control strains, it wasnot possible to transduce double drug resist-ance. The two loci are known to be well sepa-rated (24). The pyrR character was not co-transduced with loci for enzymes of the pyrim-idine pathway, pyrA,B,C,D,E,F, G; with thelocus for cytidine deaminase (cdd); nor withthe udk or upp loci. Preliminary mapping byconjugation has shown the pyrR locus of thethree mutants HD47, HD58, and HD67 to belocated in the region between leu and pro on

the S. typhimurium map. None of the fol-lowing loci namely, leuB, panA, proB, andproC, known to be in this region (23), havebeen found to be cotransduced with pyrR.About 10% of the pyrimidine overproducers

were neither linked to pyrB by cotransductionlike the partially desensitized class nor were

they as constitutive (i.e., all had less than 20-fold derepressed levels of aspartate transcarba-mylase over the wild-type repressed level) asthe doubly resistant analogue class. Thoughthese strains responded to growth in exogenousuracil, they were less responsive than the wild

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strain. Aspartate transcarbamylase levels werealways above the basal repressed level. On theother hand, the constitutive mutants neverresponded to uracil. No linkage to any pyrimi-dine locus has been found for any of theseSalmonella mutants.

Only a few of these low-level overproducershave been examined in any detail. One strain,HD77, was shown to excrete 12 ,ug of orotate aswell as 20 ,ug of uracil per ml and the de novoenzymes of pyrimidine biosynthesis appearedto be derepressed in the strain. The organismwas temperature sensitive at 42 C, at whichtemperature a high concentration of uracil (100,tg/ml) was required for growth. Addition of200 ,ug of uracil per ml to the growth mediumat 35 C repressed partially aspartate transcar-bamylase formation, possibly indicating a defi-ciency in the formation of UTP and CTP.HD77 has normal feedback inhibition by CTP(Stockert and O'Donovan, manuscript in prep-aration).One final mutant class isolated by the

screening procedure should be mentioned.Among the class three types, we isolated someE. coli mutants which were low-level overpro-ducers and which have been characterized indetail. One such mutant isolated from E. coliJC411 is HD1038 which contains altered de-oxyribonucleoside triphosphate pools as com-pared to its parent when grown in minimalmedium. Aspartate transcarbamylase is dere-pressed about fourfold in HD1038, and feed-back inhibition is exactly as is found in JC411(19). Recently, the strain was shown to harbora mutation in deoxycytidine triphosphate(dCTP) deaminase, an enzyme that deami-nates dCTP to deoxyuridine triphosphate(dUTP) (20). No such mutation, designatedpaxA (20), has been found among the Salmo-nella overproducers (high or low level) in ourcollection, nor has any such mutant been iso-lated so far (J. Neuhard, personal communica-tion).

DISCUSSIONPyrimidine regulatory mutants. The py-

rimidine overproducing mutants collected inthe present study have been divided into threeclasses with the following characteristics: (i)Mutants in which aspartate transcarbamylaseis partially desensitized to its feedback inhibi-tor, CTP, comprised approximately 30% of thetotal mutants. In all cases, the mutated locuscotransduced with the pyrB locus, that of as-partate transcarbamylase. (ii) Mutants pro-duced aspartate transcarbamylase constitu-tively in levels at least 20-fold elevated above

wild-type repressed levels. Many of the mu-tants produced 100-fold elevated levels. Atleast one additional enzyme of the pathway,dihydroorotate dehydrogenase, was also dere-pressed. These constitutive mutants comprisedapproximately 60% of the total collected. Themutated locus in all cases did not cotransducewith any of six known loci for enzymes of thepyrimidine pathway. (iii) Miscellaneous mu-tants, which were not desensitized and whichsimultaneously had slightly elevated levels ofaspartate transcarbamylase (less than 20-foldwild-type repressed) and responded to uracil,comprised approximately 10% of the total. Themutated loci were not linked to any of sixknown loci of enzymes of the pyrimidinepathway.These classes will be discussed in more de-

tail below. Although over 100 mutants wereobtained in which aspartate transcarbamylasewas partially desensitized to its inhibitor CTP,no mutant was found to be fully desensitized.The absence of such mutants is surprising inlight of the in vitro information about aspar-tate transcarbamylase. Aspartate transcarba-mylase is known to be an oligomer which canbe easily dissociated into two kinds of proteinsubunits: one the catalytic subunit which bearsthe substrate binding sites and is enzymati-cally active, and the other the regulatory sub-unit which binds the nucleotide effectors, suchas CTP, and is essential for the control of en-zymatic activity. The catalytic subunit is sta-ble, fully active, and fully desensitized invitro. Its presence has been detected in vivo inbacteria derepressed under conditions of zincstarvation, where catalytic and regulatory sub-units cannot aggregate (Nelbach et al., Bio-chemistry, in press). From this information, itwas expected that pyrimidine-overproducingmutants would appear frequently in which thecatalytic subunit remains free and thereforedesensitized, due to the inability of the twokinds of subunits to aggregate. This inabilitycould conceivably derive from a defect in ei-ther the catalytic or regulatory subunit. How-ever, all mutants examined thus far do formaggregates of both subunits, since some inhibi-tion is evident in all cases, and the aggregateresembles the wild-type hexamer in sedimen-tation properties. The locus for partial desensi-tization of aspartate transcarbamylase cotrans-duces with pyrB, the locus for aspartate trans-carbamylase activity, that is, at least for thecatalytic subunit. The locus for desensitizationis either within the gene for the catalyticsubunit or within the gene for the regulatorysubunit, provided that gene is very near pyrB.The gene for the regulatory subunit has not

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been mapped, so these alternatives remainunresolved. On the chance that the genes forcatalytic and regulatory subunits are separateand that fully desensitized mutants would notcotransduce with pyrB, desensitization hasbeen tested as well in mutants not cotrans-ducing with pyrB (e.g., class three mutants),but no additional case of desensitization wasfound. Since the experimental means areavailable to dissociate and reconstitute mix-tures of mutant and wild-type aspartate trans-carbamylase in vitro (Gerhart and Holoubek,unpublished data), it will soon be possible toassign the defect to the catalytic or regulatorysubunit by nongenetic means. The absence of aclass of fully desensitized mutants is not un-derstood; in the light of past suggestions (6)and recent experiments (4, 13) on a mechan-istic relationship of feedback inhibition andrepression, the search for such mutants de-serves further attention.

Several formal explanations for partial de-sensitization are contained in the model ofMonod, Wyman, and Changeux (16) for allo-steric proteins, and discussed in detail by Ger-hart (9) for aspartate transcarbamylase withrespect to "nonexclusive" ligand binding. As-partate transcarbamylase from E. coli or S.typhimurium is never inhibited 100% by CTP.Indeed by increasing CTP concentration aprogressive reduction of activity is broughtabout which reaches a plateau, the level de-pending on the substrate concentration. At lowsubstrate concentration, up to 95% inhibitionis caused by CTP; at high substrate concentra-tion, less than 5% inhibition can be produced.Such partial competitive inhibition has beenexplained in terms of the nonexclusive bindingof CTP to aspartate transcarbamylase with theT state having less than twofold higher affinityfor CTP than the R state (5, 8). It was foundhere that some partially densensitized mu-tants are less inhibited maximally by CTPthan is wild-type aspartate transcarbamylase(about 40% inhibition under condition wherewild type was 85% inhibited with excess CTP),a finding that is expected if the R and T statesof the mutants have a still smaller differencein affinities for CTP than the states of thewild-type enzyme. If the two states had equalaffinities, CTP would not inhibit, even thoughthe enzyme bound CTP strongly and still un-derwent its allosteric transition. Thus, it isformally possible that mutation has affectedthe relative affinity of the T and R states forCTP. Other possibilities concern changes inthe relative, or absolute affinities of the twostates for substrates, or the relative stability ofthe two forms in the absence of ligands. It is

noteworthy that the wild-type enzyme is lessinhibited maximally by CTP when the pH ortemperature is lowered (O'Donovan and Ger-hart, unpublished data). These conditions arethought to affect the relative stability and sub-strate affinities of the two states; presumablymutation could have similar effects. As puri-fied preparations of the partially desensitizedmutants become available, some of the abovedistinctions can be made by kinetic analyses.Most of the mutants obtained by the double

drug resistance selection were high-level con-stitutive mutants for aspartate transcarba-mylase production, as were approximately halfof the mutants obtained by the screening pro-cedure. The highest level constitutives havebeen used for enzyme purification and are esti-mated to contain 2 to 3% of their protein asaspartate transcarbamylase (O'Donovan, Hol-oubek, and Gerhart, manuscript in prepara-tion). The production of another enzyme of thepathway, dihydroorotate dehydrogenase, wasdetermined in some of the mutants and, likeaspartate transcarbamylase, was found to beelevated constitutively, though at much lowerlevels than aspartate transcarbamylase (e.g.,100- to 200-fold increases of aspartate trans-carbamylase and only 4- to 6-fold increases ofdihydroorotate dehydrogenase). In the past,the pyrimidine pathway in E. coli has beenanalyzed with regard to the following proper-ties of importance for the characterization ofthese constitutive mutants.

(i) Under conditions of pyrimidine starva-tion in E. coli, the enzymes encoded by pyr-C,D,E, and F increase coordinately over asmall range, approximately 25-fold from fullyrepressed to fully derepressed levels (3). Onthe other hand, aspartate transcarbamylasedetermined by pyrB increases approximately400-fold under equivalent conditions (31).Furthermore, the pyrA product, carbamylphosphate synthetase, varies less than five-fold under these conditions. Enzyme produc-tion from pyrC,D,E, and F appears coordinate,whereas pyrA and B expression is not coordi-nate with these or with each other (3). ThepyrG gene product has not been studied inthis regard. Hence, the regulatory mechanismsgoverning enzyme production in this pathwayappear to differ from gene to gene.

(ii) Of the nine enzymes immediately in-volved with de novo pyrimidine ribonucleotidebiosynthesis in E. coli and S. typhimurium,seven have been localized genetically, andthese genes are all unlinked (3, 25, 28; Beckand Ingraham, Mol. Gen. Genet., in press).Hence, mutants of the classical operator typearising in this pathway would be constitutive

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for just one enzyme of the pathway.(iii) Different steps of the pathway are under

the control of different repressing metabolites,several steps being under the control of atleast two or three repressing metabolites each.Gene expression for pyrB appears under thecontrol of uridine and cytidine nucleotides (17,18), whereas pyrA expression is affected bycytidine and arginine supply, but not UTP (1,17). Arginine as well as UTP and CTP seemsto have some effect on pyrC,D,E,F. These en-zymes are now being studied in specially con-structed strains (Williams and O'Donovan,manuscript in preparation). The expression ofpyrG has not been studied. Hence, the possi-bility exists that several repressor proteins, ofdifferent ligand-binding specificity, act jointlyto control gene expression of the entire path-way. A mutation of a single repressor proteinwould be expected to yield a mutant constitu-tive for some, but not all, of the enzymes ofthe pathway.

Since the constitutive mutants obtained inthe present study were constitutive at least foraspartate transcarbamylase and dihydroorotatedehydrogenase production, it is plausible topropose that they represent an alteration of asingle repressor protein governing at leastpyrB and D. Constitutivity was transduced byP22 phage infection, indicating a single geneticlocus or closely grouped loci for the mutantcharacter. Preliminary experiments indicateconstitutivity also for the products of pyrC,E,and F as well (O'Donovan and Henderson,unpublished data). Aside from the possibilityof a defective repressor protein, apparent con-stitutivity would result if the intracellular pro-duction of UTP or CTP were impaired, e.g., bythe defectiveness of the kinase that elevatesUMP to the triphosphate level, or of the syn-thetase that converts UTP to CTP. To test thepossibility that repressing metabolites werelow in the mutants, six constitutive mutantswere selected at random for measurements ofintracellular nucleotide pools. UTP and CTPlevels were not lower than in the wild type,and in fact were slightly higher. Thus, the ex-planation of constitutivity in terms of deple-tion of repressor metabolites could not be sup-ported by our experiments. A more direct testof this possibility would be the assay and ge-netic identification of the pyrimidine nucleo-tide kinases, the genes for which have not beenlocated on the bacterial chromosome. One fur-ther possibility must be considered for ap-parent constitutivity, that of internal induc-tion. Lacroute (14) has found a clear inductionof pyrimidine enzymes by intermediates of the

pathway in Saccharomyces. If induction byintermediates also occurs in S. typhimurium,one cause for intermediates to accumulate inour mutants might be the partial defectivenessof a late enzyme of the pathway. However,such a mutant would presumably also besomewhat low in its nucleotide pools, a char-acteristic out of keeping with the findingsmentioned above. Furthermore, if the defectiveenzyme were late in the pathway, as for ex-ample the pyrE and F products, then the con-stitutive character should be cotransducedwith the pyrE or F locus; however, the consti-tutive character was not linked to known lociof the pathway. Furthermore, direct experi-ments to test induction by intermediates indoubly blocked pyrimidine auxotrophs, similarto the experiments of Lacroute, have given noevidence of induction (O'Donovan and Hen-derson, unpublished data) in S. typhimurium.

Approximately 10% of the pyrimidine-over-producing mutants did not fall into the classeswith desensitized or constitutive phenotypes.Furthermore, the mutant characters were notcotransduced with any of the loci pyrA throughpyrF. Some of the mutants have slightly ele-vated levels of aspartate transcarbamylase, butthese levels drop to repressed values in thepresence of high concentrations of uracil (100,ug/ml). Preliminary studies indicate that adefect exists in one of the enzymes beyond thepyrimidine nucleoside monophosphate [cyti-dine monophosphate (CMP) or uridine mono-phosphate (UMP) ] level in the formation ofUTP or CTP. These data derive from endoge-nous nucleoside mono- and triphosphate meas-urements and from assays for UMP(CMP)kinase. The enzyme that converts UTP toCTP, namely CTP synthetase, is normal in allthe mutants of this subclass (Stockert andO'Donovan, manuscript in preparation). Thereis another mutant type expected from experi-ments with pyrimidine overproducers of E.coli (19, 20), but not yet identified in S. typhi-murium, namely, those with a defective dCTPdeaminase. The mutant, characterized in E.coli, overproduces pyrimidines and containspools of dCTP 10- to 20-fold higher than inwild type (20).Problems in the selection of pyrimidine

regulatory mutants. High-level constitutivemutants were readily obtained in this study bya selection for bacteria simultaneously re-sistant to two antimetabolites, 5-FU and 5-FUR. In contrast, selections with 5-FU or 5-FUR singly did not yield constitutive bacteria,but instead mostly mutants unable to trans-port the drug or convert it to 5-FUMP, the

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intermediate necessary in both cases for thedrugs' toxic action (21). Mutants selectedagainst one drug remained sensitive to theother. Since 5-FU and 5-FUR are metabolizedto 5-FU monophosphate by different pathways(21), two mutational events are probably re-quired to confer simultaneous resistance to thedrug mixture. A much more frequent cause ofdouble resistance appears to be pyrimidineoverproduction due to constitutivity, whicharises in one step and combats the drug mix-ture by diluting the pools of toxic compoundswith high levels of UMP, uridine, and uracil.

It proved essential for the selection that theS. typhimurium strain lacked the enzyme uri-dine phosphorylase, eliminated by mutation.This enzyme disposes of 5-FUR by convertingit to 5-FU, thereby making the selective powerof a mixture of 5-FUR and 5-FU no greaterthan that of 5-FU alone. Indeed, attempts toselect constitutive mutants in bacteria pos-sessing uridine phosphorylase have failed,whereas attempts with uridine phosphorylase-negative mutants reliably yielded constitu-tives.

Selective conditions have not been found forthe isolation of desensitized mutants affectingaspartate transcarbamylase. Desensitized mu-tants are known for allosteric enzymes in sev-eral pathways of amino acid biosynthesis, byvirtue of the availability of analogues thatenter the bacterial cell, act as feedback inhibi-tors, and yet are not incorporated into macro-molecules (24). These analogues cause the cellto starve itself for the natural amino acid. Inthe case of aspartate transcarbamylase, invitro studies have shown the 5-halo and 5-methyl analogues of CTP to equal CTP asenzyme inhibitors. Even at the nucleosidelevel these analogues are equally inhibitory asCTP if present at 50-fold increased concentra-tion. Various cytidine analogues (e.g., 5-bro-mocytidine, cytosine arabinoside) have beentested as growth inhibitors of S. typhimurium,but without effect, even in strains lacking theenzyme cytidine deaminase, which rapidlydeaminates cytidine compounds to uridinecompounds (7, 17). Some of the cytidine ana-logues probably fail to be converted to the tri-phosphate level where they would act mosteffectively as false feedback inhibitors, on amole per mole basis. However, a more funda-mental problem probably obstructs such selec-tive schemes in the case of aspartate transcar-bamylase; no inhibitor, including CTP itself,reduces aspartate transcarbamylase activityto less than 5 to 10% in vitro, even at very highinhibitor concentrations (lla). In the bac-

terium, a reduction of aspartate transcarba-mylase activity to 5% is easily compensated by20-fold derepression of aspartate transcarba-mylase, since even 200-fold increases of ac-tivity are obtained under conditions of pyrimi-dine limitation. Thus, derepression can prob-ably offset excess inhibition. Selections arepresently underway using strains that are fullyderepressed for all the biosynthetic enzymes,but which have, nevertheless, low levels ofaspartate transcarbamylase (they have fullyderepressed levels of aspartate transcarba-mylase also but the enzyme is defective). It ishoped that with these strains excess inhibitioncannot be offset by any further derepressionand that feedback-resistant mutants can beisolated.

ACKNOWLEDGMENTS

We thank Tom Ostwald and H. Holoubek for their excel-lent technical assistance during the early phases of this workand T. Henderson for her recent excellent assistance. Thenucleoside triphosphate pool measurements were kindly per-formed by Gordon Edlin.

Most of this work was carried out during the tenure of aDernham Fellowship (J-104), California Division, AmericanCancer Society, in the Department of Molecular Biology,University of Califomia at Berkeley. This investigation wassupported by a U.S. Public Health Service research grantCA-07410 from the National Cancer Institute, and by grantsfrom the Robert A. Welch Foundation, Houston, Texas, andH-1670, Texas A&M University.

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bamyl phosphate synthesis in Salmonella typhimu-rium. J. Biol. Chem. 244:4033-4038.

2. Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965.Optimal conditions for mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine in Escherichia coli K12.Biochem. Biophys. Res. Commun. 18:788-795.

3. Beckwith, J. R., A. B. Pardee, R. Austrian, and F.Jacob. 1962. Coordination of the syn'thesis of the en-zymes in the pyrimidine pathway of E. coli. J. Mol.Biol. 5:618-634.

4. Blasi, F., S. M. Aloj, and R. F. Goldberger. 1971. Effectof histidine on the enzyme which catalyzes the firststep of histidine biosynthesis in Salmonella typhimu-rium. Biochemistry 10:1409-1417.

5. Changeux, J.-P., and M. M. Rubin. 1968. Allosteric in-teractions in aspartate transcarbamylase. III. Inter-pretation of experimental data in terms of the modelof Monod, Wyman, and Changeux. Biochemistry 7:553-561.

6. Cline, A. L., and R. M. Bock. 1966. Translational con-trol of gene expression. Cold Spring Harbor Symp.Quant. Biol. 31:321-333.

7. Furth, J. J., and S. S. Cohen. 1968. Inhibition of mam-malian DNA polymerase by the 5'-triphosphate of 1-O-D-arabinofuranosylcytosine and the 5'-triphosphateof 9-0-D-arabinofuranosyladenine. Cancer Res. 28:2061-2067.

8. CGrhart, J. C. 1964. Subunits for control and catalysis inaspartate transcarbamylase. Brookhaven Symp. Biol.17:222-231.

9. Gerhart, J. C. 1970. A discussion of the regulatory prop-erties of aspartate transcarbamylase from Escherichia

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11. Gerhart, J. C., and A. B. Pardee. 1962. The enzymologyof control by feedback inhibition. J. Biol. Chem. 237:891-896.

lla. Gerhart, J. C., and A. B. Pardee. 1964. Aspartate trans-carbamylase, an enzyme designed for feedback inhi-bition. Fed. Proc. 23:727-735.

12. Gerhart, J. C., and H. K. Schachman. 1965. Distinctsubunits for the regulation and catalytic activity ofaspartate transcarbamylase. Biochemistry 4:1054-1062.

13. Hatfield, G. W., and R. 0. Bums. 1970. Specific bindingof leucyl transfer RNA to an immature form of L-threonine deaminase: its implications in repression.Proc. Nat. Acad. Sci. U.S.A. 66:1027-1035.

14. Lacroute, F. 1968. Regulation of pyrimidine biosynthesisin Saccharomyces cereuisiae. J. Bacteriol. 95:824-832.

15. Machida, H., and A. Kuninaka. 1969. Studies on theaccumulation of orotic acid by Escherichia coli K-12.Agr. Biol. Chem. 33:868-875.

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17. Neuhard, J. 1968. Pyrimidine nucleotide metabolismand pathways of thymidine triphosphate biosynthesisin Salmonella typhimurium. J. Bacteriol. 96:1519-1527.

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20. O'Donovan, G. A., G. Edlin, J. A. Fuchs, J. Neuhard,and E. Thomassen. 1971. Deoxycytidine triphosphatedeaminase: characterization of an Escherichia colimutant lacking the enzyme. J. Bacteriol. 105:666-672.

21. O'Donovan, G. A., and J. Neuhard. 1970. Pyrimidinemetabolism in microorganism. Bacteriol. Rev. 34:278-343.

22. Paege, L. M., and F. Schlenk. 1952. Bacterial uracil ri-boside phosphorylase. Arch. Biochem. Biophys. 40:42-49.

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27. Taylor, W. H., and M. L. Taylor. 1964. Enzymes of thepyrimidine pathway of Escherichia coli. II. Intracel-lular localization and properties of dihydroorotic de-hydrogenase. J. Bacteriol. 88:105-110.

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