JOURNAL OF BACTERIOLOGY, OCt. 1976, p. 39-48Copyright © 1976 American Society for Microbiology

Vol. 128, No. 1Printed in U.S.A.

Structural and Regulatory Mutations Allowing Utilization ofCitrulline or Carbamoylaspartate as a Source ofCarbamoylphosphate in Escherichia coli K-12

CHRISTIANE LEGRAIN, VICTOR STALON, NICOLAS GLANSDORFF,* DANIEL GIGOT, ANDRIgPI1RARD, AND MARJOLAINE CRABEEL

Laboratoire de Microbiologie, Universite Libre de Bruxelles, Erfelijkheidsleer en Mikrobiologie, VrijeUniversiteit Brussel, and Institut de Recherches du Centre d'Enseignement et de Recherches des Industries

Alimentaires et Chimiques,* B-1070 Brussels, Belgium

Received for publication 18 May 1976

Escherichia coli mutants lacking carbamoylphosphate synthase require argi-nine and uracil for growth. It is, however, possible to obtain mutants in whichcarbamoylphosphate is obtained by phosphorolysis of citrulline or carbamoylas-partate. Citrulline utilizers are argG bradytrophs or strains in which thesynthesis of ornithine carbamoyltransferase (either of the F or I type) is specifi-cally derepressed by unstable chromosomal rearrangements or stable mutationsthat presumably affect the operators of those genes. Carbamoylaspartate utiliza-tion as a source of carbamoylphosphate appears to require more than onemutation; the best-understood strains are pyrD pyrH or pyrC pyrH mutants inwhich aspartate carbamoyltransferase activity is high and the pool of cytidinetriphosphate (feedback inhibitor of aspartate carbamoyl-transferase) is presum-ably low and in which channeling of carbamoylaspartate towards pyrimidinebiosynthesis is considerably reduced. Selection of enzyme overproducers basedon a metabolic dependency for a reversed enzymatic reaction can be regarded asa means for isolating regulatory mutants.

The de novo biosynthesis of arginine and thepyrimidines requires carbamoylphosphate (CP;see Fig. 1). The carbamoylation of aspartate iscatalyzed by aspartate carbamoyltransferase(ATCase; product of the pyrB locus) and consti-tutes the first step of the pyrimidine pathway;the carbamoylation of ornithine by ornithinecarbamoyltransferase (OTCase; step 6 of argi-nine biosynthesis) produces citrulline. In Esch-erichia coli K-12 the products of the closelyrelated genes argF and argl interact to give afamily of trimeric isozymic OTCases (11). Theequilibrium of both carbamoylations is highlyfavorable to the production of citrulline or car-bamoylaspartate and the release of inorganicphosphate. Equilibrium constants for the car-bamoylation of ornithine and aspartate at pH8.0 are 2.105 (V. Stalon, C. Legrain, and J. M.Wiame, submitted for publication) and 105 (3),respectively.

In all organisms investigated, ATCase ap-pears to fulfil an anabolic function exclusively.The reverse reaction can be measured by phos-pho- or arsenolysis of carbamoylaspartate (3);the rate observed is very low, and in E. coli (seebelow) it appears to be unable to meet the de-mands of a mutant deficient in CP production.

Phosphorolysis of citrulline by all anabolic OTC-ases (except by the Pseudomonas enzyme) ismeasurable (10; C. Legrain, V. Stalon, and J.M. Wiame, submitted for publication); the rateis low, but it nevertheless permits very slowgrowth of CP synthase (CPSase) mutants ofE.coli provided with citrulline as a source of CP(20; see below).Catabolic OTCases are known; their coupling

to carbamate kinase alleviates the constraintimposed by an equilibrium unfavorable to theproduction of CP.The metabolic significance of a particular

OTCase is a trait that appears amenable tomodification by genetic means: Stalon (22) hasshown that the catabolic OTCase ofPseudomo-nas can fulfil an anabolic function if, by one orpossibly several mutations, the allosteric con-straint that makes the enzyme normally unre-sponsive to low concentrations of CP is partlyreleased.

This in vivo approach of the extent to whichthe role of a particular enzyme is modifiableunder selective pressure allows us to probe theflexibility retained in genetic systems adaptedto particular situations; this may contribute toour understanding of metabolic evolution. We

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40 LEGRAIN ET AL.

rD

argD

Arginine Arginino- OrotateargH succinate Dihdro-pyrD -p\ ~~~~~orotate pyrt ,

OMPorgG\pyr

Citrulline Carbomoyl pyrFaspa rtote

UMPorg F,! pyrB PYrH

Glutomate Ornithine CAPFl

\ZgA orgEj carA UTParyC AcetyI- cprSor9D~~~~~~~~~~~~~~~~~~yargO ornithine

Glutomine CTPHCO5ATP

FIG. 1. Genetic loci relevant to this study and corresponding metabolic steps in arginine and pyrimidinebiosynthesis. For pyrH and pyrG mapping, see references 1 and 17.

therefore thought it of interest to determinewhich types of mutations could create condi-tions making E. coli able to use citrulline orcarbamoylaspartate for the production ofCP byan efficient "reversal" ofthe anabolic reactions.

MATERIALS AND METHODSStrains. The strains used in the study are de-

scribed in Table 1, the nomenclature of genetic lociis that used by Bachmann et al. (1); carA and -B(new symbols for pyrA, see reference 13) designatethe structural genes for CPSase.A carB8 derivative of 3000X111 was obtained by

using the following steps. An F- phenocopy of

3000X111 was crossed with HfrJEF8, Thi+ Met+ re-combinants were selected, and a thr carB8pro strainwas identified. A carB8 derivative of strain H1175was identified among Leu+ recombinants obtainedby transducing H1175 with phage 363 (5) grown onJEF8. Strain P678 carB8 was obtained as a Thi+Leu+ thr carB8 str recombinant from a mating be-tween strain P678 and JEF8. Strain P4X argR carB8was prepared as follows. A canavanine-resistant de-rivative of P4XSB25 was selected and characterizedas argR by enzyme assays. This strain was trans-duced by phage 363 grown on strain RC50, and aThr+ carA50 recombinant was identified. A recAderivative of the F- citrulline-utilizing (Citr-ut)strain mentioned in Results (section A, d) was pre-

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UREIDO COMPOUNDS AS CARBAMYLPHOSPHATE SOURCES 41

TABLE 1. List of strains used

Strain Genotype Source or refer-Strain Genotype ence

KL16-99 Hfr thi recA L. Brooks Low3000X111 Hfr thi la (pro lac argF) str+ F. JacobJEF8 Hfr metB thr carB8 str+ 13H1175 F- purA argI leu proA argF 6b

strLM5thy F- thi aspB argG thyA str This labora-

toryP678 F- thr leu thi str R. LavalleP4XSB25 Hfr thr metB str+ S. BourgeoisRC50 F- thi carA5O R. Lavall6; 13N3 3000X111 argF argI, lysogenic 12

for A argI-3N12 3000X111 argF argI, lysogenic 12

for A argI-12a A denotes deletion.b In reference 6 the strain was identified as F- K-12 Thr+

leu adeKproA cit (for citrulline) str. adeK is now recognizedas being identical to purA, not to purB.

pared from a thyA derivative of the latter (21) byconjugation with Hfr KL16-99; Thy+ recA recombi-nants were recognized as ultraviolet sensitive andby their inability to serve as recipients in transduc-tion experiments.

Growth of cultures and genetic analysis. The me-dia employed, the growth of cultures, and the proce-dures used in the genetic analysis have been de-scribed previously (4, 5, 13).Enzyme assays. The preparation of cell extracts

and the assays for OTCase (EC 2.1.3.3), ATCase (EC2.1.3.2, pyr-B product), CPSase (EC 2.7.2.9), anddihydroorotate dehydrogenase (EC 1.3.3.1, pyrDproduct) have been performed as previously de-scribed (13, 18). N-a-acetylornithinase (EC3.5.1.16), orotidine 5'-phosphate pyrophosphorylase(EC 2.4.2.10, pyrE product), and uridine 5'-mono-phosphate kinase (EC 2.7.4.-, pyrH product) wereassayed as described by Vogel and Bonner (23),Schwartz and Neuhard (19), and Ingraham andNeuhard (7), respectively.OTCase isoenzyme characterization and purifi-

cation. The characterization of OTCase isoenzymesby diethylaminoethyl-Sephadex chromatographyand the purification of the FFF and Ifl isoenzymeshave been performed as in our previous paper (11).

RESULTSRationale for mutant isolation. All mutants

were isolated from strains carrying the carB8deletion (13), which lack the heavier subunit ofCPSase. It is consequently totally dependent onarginine and uracil; neither ofthese substancesalone elicits the slightest growth response. Mu-tants able to use citrulline as a CP source maybe selected as colonies appearing on plates pro-vided with glucose, methionine, threonine, andL-citrulline (2,000 ,ug/ml) and devoid of uracil(GMT-citr plates). The presence of citrulline, aproduct of the reaction catalyzed by OTCase,

makes the normal anabolic function of thatenzyme dispensable and provides conditions forrepression ofenzyme synthesis in the arg path-way; little OTCase is thus made and, if singlecells are plated on GMT-citr plates, tiny colo-nies appear only after 3 days at 370C. Bettercitrulline utilizers were therefore selected.pyrB or car mutants do not grow on carbamoyl-aspartate as source of pyrimidines, but sponta-neous mutants able to do so are obtained easily(B. Perbal, Ph.D. thesis, Univ. of Paris VII,Paris, 1974; unpublished data). Such mutants ofthe carB8 strain do not grow on plates contain-ing carbamoylaspartate but lacking arginine.Attempts to select mutants capable of growingunder such conditions were made.

Citr-ut derivatives. (A) Mutants from argF+argI+ carB strain JEF8. The first Citr-utstrains were selected from Hfr strain JEF8,which carries both argF and argI in a func-tional state. They were obtained at a frequencyof about 1 out of 5 x 10-7 cells plated. After twopurifications on GMT-citr plates, they weregrown for about 20 generations in liquid mini-mal medium supplemented with citrulline(2,000 ug/ml) or arginine and uracil (100 ,g/ml); assays of OTCase and acetylornithinasewere performed on such cultures. All mutantsappeared to be of the same type, as judged fromthe following criteria (data in Table 2).

(1) OTCase specific activity was extremelyhigh in all cultures utilizing citrulline as theCP source. Acetylornithinase synthesis was re-pressed.

(2) A wide variation ofOTCase specific activ-ities was observed after growth on arginine anduracil. All mutants, however, are unstable:after growth on arginine and uracil they segre-gate cells growing slowly on citrulline. Datanot shown here demonstrated that there is anunambiguous relationship between the numberof cells able to develop into Citr-ut coloniesand the level of OTCase. It is thus quite likelythat the cells growing on citrulline obtain theirCP by phosphorolysis of this amino acid cata-lyzed by very high amounts of OTCase; thehighest activities observed are three to fourtimes that of an argR (genetically derepressed)mutant. If the specific activity of OTCase isfollowed during only four to five generationsafter the addition of arginine and uracil, tominimize the effects of segregation, OTCasesynthesis appears to be nonrepressible.

(3) The OTCase isoenzyme pattern was de-termined in four randomly chosen mutants. Forall four, the elution diagrams of extracts chro-matographed on diethylaminoethyl-Sephadexare practically identical (Fig. 2); they show thatonly the expression of the argF gene is affected,

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TABLE 2. Characteristics of Citr-ut mutants

Supplements added to minimal me-dium

GArginine and uracil L-CitrullineRelevant genotype of the Global muta- Mutant typeP No. (100 zg/ml) (2,000 isg/ml)carB8 parental strain quency testedquency ~~~~~~~~~~~~~~~Dou-Dou-

OTCase sp bling OTCase blingact' time sp act time

(min) (min)

argF+ argI+ (in JEF8) 5 x 10- Unstable argF constitutive 15 3,000 (4.8)c 55 3,100 140(JEF8C23)

argF- argI+ (in 3000X111- 1.5 x 10-9 Stable argI constitutive 1 168 (3.5) 55 85 130JEF8 hybrid) (NCI-0)

Stable argI constitutive 7 246 55 455 105(NCI-8)

argG bradytroph (NCI-23) 15 3.5 55 1,142 125

argF+ argI+ (in P678-JEF8 2 x 10-8 Stable argl constitutive 2 778 (7.0) 60 650 160hybrid) (P678C2)

Stable argF constitutive 1 380 50 292 160(P678C3)

a Each class is made of mutants whose OTCase specific activities do not differ by more than 25%.b Specific activities are given as micromoles per hour per milligram of protein and are given for one example of each

class. N-a-acetylornithinase data are given in the text.c Numbers in parentheses are enzyme values obtained for the parental strain.

the synthesis of the I isoenzyme remaining re-pressible. The FFF isoenzyme of the mutantswere eluted at the same buffer ionic strength asthe corresponding enzyme from the wild type.It shows the typical thermolability of the FFFmolecule (see accompanying paper, reference12); the kinetic parameters of the OTCase pres-ent in strain JEF8C23 are not significantly dif-ferent from those of all E. coli OTCases studiedup to now (12). This type ofmutant thus carriesan unstable mutation that specifically dere-presses the argF gene. One of the mutants(JEF8C23) has been analyzed further by ge-netic means.

(a) The mutation involved is strongly linkedto the original site ofthe argF gene. This can bedemonstrated by conjugation (data not shown)and transduction into a carB pro argF strain(Table 3). No co-transduction of the Citr-utcharacter could be observed with purA, amarker co-transducible with argI (6). TheproA-Citr-ut co-transduction index is about 1%(Table 3; and results of a replica-plating analy-sis), compared to the usual 20% observed be-tweenproA and argF when the wild type (JEF8or other K-12 strains) is used as donor (6). If anisogenic Car+ recipient is used, the proportionof Arg+ recombinants among the Pro+ can beestimated directly: it amounts to 10% (19/181).Both repressible and constitutive argF recom-binants are recovered among Pro+ transduc-tants; a possible interpretation is that JEF8C23

carries one or more extra copies ofargF, whichare not under control of the arg repressor.

(b) From Pro+ Citr-ut recombinants obtainedby conjugation or transduction, as fromJEF8C23 itself, one can obtain segregantsgrowing slowly on citrulline, among which 70%exhibit enzyme levels identical to that ofJEF8,the parental strain. This might also be ac-counted for by the presence of extra copies ofargF in JEF8C23.

(c) Since the F factor is inserted close to argFin Hfr JEF8C23, we examined the Citr-ut phe-notype to determine whether it is related to arearrangement involving the F factor itself.This appears unlikely since segregants ofJEF8C23 are F- (as checked by MS2 phagesensitivity) and since Pro+ Citr-ut recombi-nants recovered from a conjugation between aF- proA argF strain and Hfr JEF8 are F .

(d) The Citr-ut phenotype is stabilized by arecA mutation: a Pro+ Citr-ut F- recombinantof the above-mentioned conjugation (a) hasbeen purified and found to produce high levelsof OTCase and to be unstable. The strain wasthen made recA (see Materials and Methods)and examined again for OTCase. No segregantswere observed (among about 2,000 colonies)after 40 generations in the presence of arginineand uracil, a condition that brings about exten-sive segregation in the parental Rec+ Citr-utstrain.The latter result is also compatible with the

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UREIDO COMPOUNDS AS CARBAMYLPHOSPHATE SOURCES 43

In

a,E

ENCLi

2E

-

tn00-

Fraction numberFIG. 2. Distribution ofornithine carbamoyltransferase activity in diethylaminoethyl-Sephadex A-50 chro-

matography of dialyzed extracts ofE. coli K-12 JEF8C23 (lower, argF constitutive) and E. coli K-12 NCI8(upper, argI constitutive). The preparation was eluted with a linear potassium phosphate gradient, 50 to 250mM, at pH 7.5.

TABLE 3. Co-transduction of the Citr-ut character from JEF8C23 with proA

OTCase sp act (gmol/h/mg ofprotein) on minimal medium sup-

Recipient Donor Recombinants/ml plemented with:

Citrulline Arginine + ura-cil

purA argF argI JEF8C23 Pro+, 3,445 900 415carB8 proA Citr-ut, 450 11,200 (730

Pro+ Citr-ut, 28 820 485

Pur+ Citr-ut, 0

a Values obtained for two representatives of each class.

hypothesis that Citr-ut mutants harbor dupli-cations of the argF gene: various types of dupli-cations of chromosomal genes have indeed beenfound to be stabilized by a recA mutation (2).Since Citr-ut mutants exhibit normal repressi-bility of N-a-acetylornithinase (another en-zyme of the arg regulon), the lack ofrepressibil-ity of OTCase cannot be explained by exhaus-tive binding of the arg repressor on multiplecopies of the argF operator; rather, one has to

assume that one or more of these copies of theargF gene is not under control ofthe arg repres-sor.The results do not prove that JEF8C23 car-

ries a duplication of argF: one can imagineunstable insertions or transpositions thatwould be stabilized by recA and be compatiblewith most of the data available. As the struc-ture of the mutation is of interest, since it oc-curred in JEF8 but not in another K-12 strain

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44 LEGRAIN ET AL.

(see section c), further mapping studies withmarkers flanking argF and heteroduplex anal-ysis appear desirable.

Citr-ut mutants derived from strain JEF8exhibit a high level of FFF OTCase; since thesynthesis of the Ill isoenzyme is not affected inthose strains, one may wonder whether theargI gene product is less efficient in the cataly-sis of citrulline phosphorolysis than the argFenzyme. The accompanying paper (12) shows,however, that no significant difference can beobserved between the two kinds of enzymeswith respect to their kinetic parameters. There-fore, we tried to isolate Citr-ut mutants from astrain carrying a deletion of the F gene. Mu-tants were indeed obtained, but at about one-third of the frequency of Citr-ut mutants fromstrain JEF8. All mutants were found to be sta-ble even after a large number (about 20) ofgenerations. They are analyzed in the next sec-tion.

(B) Mutants from an argI+ AargF carB8strain. Two classes of mutants may be distin-guished on the basis of the results provided bythe OTCase assays.

(1) In the first group, exemplified by strainsNCI-0 and -8, OTCase specific activity was highand only weakly, or not at all, repressible (Ta-ble 2). NCI-0 appeared partly derepressed,whereas NCI-8 produced the OTCase level ofanargR argF- argI+ strain. When measured (inNCI-8), acetylornithinase was found to be re-pressed. These data immediately suggest thatstrain NCI-0 and NCI-8 contain operator muta-tions affecting argl. The following data supportthis interpretation.

Phage 363 lysates of strain NCI-0 and NCI-8were prepared and used to transduce two re-cipient strains: strain H1175 (Table 4), argI

argF purA leu proA, and the carB8 derivative(see Materials and Methods), argI argF carB8purA proA. Pur+ Arg+ recombinants obtainedwith strain H1175 were assayed for OTCaseactivity; in the case of the carB8 derivative ofH1175, Pur+ recombinants were first selectedin the presence of arginine and uracil and repli-cated onto citrulline plates to identify Citr-utrecombinants. Enzyme essays performed on anumber of recombinants (Table 4) show that inboth transductions the Citr-ut character and itsenzymatic manifestation, OTCase constitutiv-ity, are linked to argI. In addition, (i) the co-transduction index between purA and theCitr-ut character in strain H1175 carB8 (Pur+among Citr-ut, Citr-ut among Pur+) was about1%, the same as between purA and argI, and(ii) Citr-ut recombinants selected directly fromstrain H1175 carB8 are all argI constitutive.Preliminary results indicate that the argI

constitutive mutation of strain NCI-8 is domi-nant. Proof that the mutation involved has af-fected the operator of argI must await the con-struction of diploids harboring the mutationresponsible for constitutivity in the cis positionwith respect to a structural argI mutation.

Table 4 shows that in strains H1175 and H1175carB8 the mutations of strain NCI-0 determinea pattern of partial OTCase repressibility thatis more similar to that of strain NCI-8 than tothat of the original mutant itself. The reasonfor this is unknown. It should be noted, how-ever, that the data reported in Table 4 for trans-ductants of H1175 were obtained from culturesgrowing at the same rate (about 1-h doublingtime); this is not the situation for the resultsreported in Table 2.

(2) A second type of mutant exhibited highOTCase activity on citrulline, but OTCase syn-

TABLE 4. Transduction of the Citr-ut mutations ofNCI strains into argF argI recipients

Type and no. of recombi- OTCase sp acta in recombinants grown innants tested minimal medium supplemented with:

Donor Relevant genotype of recipi- Arginine Citrullineent. Not sArgim'ne + uracil Ctuln

Pur+ Arg Pur+ Citr-ut sp- (100 |Og/ ( (2,000 ug/plemented ml) ml)ug ml)

NCI-0 purA argF argI 4 540 240 250purA argF argI carB8 4 190 140

NCI-8 purA argF argI 5 1,100 330 300purA argF argI carB8 4 320 410

NCI-23 purA argF argI 4 35 3purA argF argI carB8 No recom-

binants

a Results are given as micromoles per hour per milligram of protein.

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UREIDO COMPOUNDS AS CARBAMYLPHOSPHATE SOURCES 45

thesis was repressible in the presence of argi-nine and uracil. It could be demonstrated thatmutants of this class are argG bradytrophs (seebelow); in those strains the derepression ofOTC-ase synthesis, resulting from a very slow con-version of the supplied citrulline into argi-nine, allowed a sufficient rate of citrullinephosphorolysis to support growth. Only one

mutant, NCI-23, has been submitted to all thetests reported.

(i) Pur+ Arg+ recombinants obtained betweenstrain H1175 and phage 363, grown on strainNCI-23, are normally repressible (Table 4),which was, at any rate, expected, since theexpression of argI is repressible in strain NCI-23 as well. (ii) no Pur+ Citr-ut recombinantscould be obtained with strain H1175 carB8,either by direct selection or by replica-platinganalysis of Pur+ recombinants. The mutationthus does not appear to be responsible for thesynthesis of a modified OTCase able to phos-phorolyze citrulline with a higher efficiencythan the wild-type enzyme. (iii) Thy+ Pro+ Car+Thr+ recombinants were selected from a matingbetween strains LM5 (F- thi argG aspB thyA)and NCI-23 (Hfr Apro lac argF carB8 thr) onminimal agar supplemented with glucose, thia-mine, aspartate, and arginine (see markers onFig. 1). Five of the phenotypically Arg+ recom-binants were checked for their growth behaviorand OTCase specific activity. All five recombi-nants exhibited long lags (3 to 5 h) when trans-ferred from a medium containing arginine inexcess to minimal medium, in contrast to theArg+ wild-type E. coli strain, for which a muchshorter or even no lag was usually observed.OTCase levels after growth without argininewere very high in the five recombinants. Thisbehavior is typical ofthe argG bradytrophs firstdescribed by Novick and Maas (15). The addi-tion of arginine to a culture of NCI-23 utilizingcitrulline as source of CP is expected to retardthe growth rate because of repression of OTC-ase synthesis. This was indeed observed (datanot shown); moreover, no growth occurredwhen single cells ofNCI-23 were plated on min-imal medium supplemented with citrulline(2,000 ,ug/ml) and arginine (100 ug/ml).

(c) Mutants derived from different argF+argl+ carB8 strains. Strain JEF8, as reportedin (A), gives a vast majority of unstable mu-tants in which the argF gene has escaped therepression normally exerted by arginine. argloperator mutants probably arose but were lostamong the majority type. For the same reason,we were not optimistic about recovering trueargF operator mutants from such a strain, noreven from an argF AargI derivative of JEF8.

Considering the interesting possibility that thefrequency of a particular type of chromosomalrearrangement could be dependent on deoxyri-bonucleic acid sequences, and/or on enzymespresent in some strains but absent in others, weselected Citr-ut mutants from the previouslyprepared strain P678 carB8 (see Materials andMethods) in which the chromosome is partiallyderived from strain P678 and partially fromstrain JEF8.

Citr-ut mutants were obtained; they were 1/25 as frequent as when Hfr JEF8 was used asthe parental strain. The three mutants ana-lyzed are stable; two could be argl operatormutants, as defined by the criteria reported in(B). The other one produced the FFF isoenzymeconstitutively, as determined by diethylamino-ethyl-Sephadex chromatography and thermola-bility tests (11, 12). A weak repression by argi-nine was still observed. The enzyme level wascomparable to that of an argR+ argF+ argI-strain (6). The strain is stable and is probablyan argF operator mutant.

(D) Behavior of argR strains. Since the de-repressed levels of OTCase observed in the mu-tants described in (A), (B), and (C) allow suffi-cient citrulline phosphorolysis to supportgrowth of car strains on citrulline, one maywonder why argR mutants have not been re-covered by our selection procedure. There is atleast one reason why argR mutants may not beable to utilize exogenous citrulline: the highlevel of argG and -H enzymes characteristic ofargR strains would channel much or even mostof the citrulline in the direction of argininebiosynthesis. Our interpretation is supportedby the fact that an argR carA50 derivative ofHfr P4X grows poorly on citrulline, althoughdefinitely better than JEF8, as compared to anyof the Citr-ut mutants described above.

Carbamoylaspartate-utilizing mutants. Aderivative of JEF8 carB8 able to use carbam-oylaspartate as source of pyrimidine wasselected on a minimal medium plate supple-mented with arginine (100 jig/ml) and carbam-oylaspartate (100 ug/ml). The mutant obtained(JEF8USU20) was then used for the selection ofstrains able to use carbamoylaspartate (4,000,g of DL form per ml) as the source ofCP, in theabsence of arginine.The selection was first attempted on plates

supplemented with carbamoylaspartate butwithout uracil. A few very slow-growing colo-nies were obtained and they were obtained onlywhen the cells were submitted to N-methyl-N'-nitro-N-nitrosoguanidine used as crystals onthe selective plates. Purification of these colo-nies on the same medium (N-methyl-N'-nitro-N-nitrosoguanidine omitted) gave visible

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46 LEGRAIN ET AL.

growth only after several days; the one strain(JEF8USU20-8G) that we finally analyzed is,in fact, a secondary mutant that appeared inthose streaks and shows significant and repro-ducible growth on carbamoylaspartate at con-

centrations above 500 ,ug/ml. In liquid mediumthe strain has a doubling time of 3 h in 4,000 ,gof DL-carbamoylaspartate per ml; ATCase, di-hydroorotate dehydrogenase, and orotidine 5'-phosphate pyrophosphorylase are substantiallyderepressed (Table 5) even in the presence ofuracil. This is easily explained by the findingthat the strain has a partial pyrH block, whichlimits the endogenous production of pyrimi-dine nucleotides from uridine 5'-monophos-phate. This type of mutant was obtained first inSalmonella (8), where it was originally thoughtto be a pyrR mutant, and later in E. coli (17).An additional mutation present in USU20-8Gdetermines a severe pyrC block, thus consider-ably slowing down the channeling of carba-moyl-aspartate towards pyrimidine biosynthe-S1S.The parental strain of USU20-8G is already

depressed for ATCase (Table 5) when grown on4,000 jIg of DL-carbamoylaspartate per ml (andarginine, of course). Therefore, the significanceof the two pyr blocks in USU20-8G for ATCaseactivity is not only that they allow derepressionof that enzyme, but probably also that theylimit the production of cytidine triphosphate, afeedback inhibitor of ATCase. We have re-

peated the selection on plates provided withcarbamoylaspartate (4,000 jig/ml) and uracil(100 ,g/ml). The presence of uracil maintainsthe cells under conditions of repression and al-lows the selection of mutants with a tight block

in an early step of the pyrimidine pathway.Again, N-methyl-N'-nitro-N-nitrosoguanidineproved to be necessary to obtain mutants. Oneof them has been analyzed extensively enoughto allow conclusions to be reached: it is a pyrDpyrH mutant (USU20-19). The pyrD block istight (a Car+ transductant shows no growth onminimal medium without uracil); the pyrHblock is, of course, not total, but rather severe.The pyrD block of mutant USU20-19, pre-

cluding channelling of carbamoylaspartatedown the pyrimidine pathway further than di-hydroorotate, and the ready reversibility of di-hydroorotate synthesis (16) from carbamoylas-partate may be regarded as conditions concur-ring to allow this strain to use carbamoylaspar-tate as a source of CP.

DISCUSSIONMutant derivatives of an E. coli strain de-

leted for the CPSase gene have been obtainedin which a simultaneous supply of arginine andpyrimidines is no longer an obligatory require-ment. The CP necessary for growth is obtainedby the reverse of the enzymatic reactionsthrough which it is normally channeled to-wards arginine or pyrimidine biosynthesis.The conditions necessary to make citrulline

an efficient source of CP, despite an equilib-rium constant extremely favorable for citrul-line synthesis, is a high level of an apparentlyunmodified OTCase; this occurs either by mu-tations that specifically relieve an OTCase genefrom repression by arginine or by partial blockof the enzyme subsequent to OTCase in argi-nine biosynthesis (bradytrophic argG muta-tion) resulting in a lower rate of citrulline con-

TABLE 5. Enzymes ofpyrimidine biosynthesis in carbamoylaspartate-utilizing mutantsa

Enzyme sp actMinimal me DMutant dium supple- Doubling DHOde- UMP ki-mented with

itime (min) ATCase DHOase" hase OMPppase UPase(pyrB) (Prc (pyrD) (Pr) (pyrH)JEF8USU20 A + CA 105 536 + 1.60 5.15

A + U 60 2.4 + 0.23 0.53 0.9

JEF8USU20-BG U + CA NM 447 ND 1.11 4.37A + U 180 378 ND 1.08 5.00 <0.05CA 180 469 1.10 4.50

JEF8USU20-19 A + U 174 860 + ND 4.35 NDU + CA 186 1,040 + ND 4.70

a Abbreviations: DHOase, Dihydroorotase; DHOdehase, dehydroorotate dehydrogenase; OMPppase, oro-tidine 5'-phosphate pyrophosphorylase; UMP, uridine 5'-monophosphate; NM, not measured; ND, notdetectable.

" A, Arginine; CA, carbamoylaspartate; U, uracil.c The activity of this enzyme was assayed only qualitatively by coupling with DHOdehase and estimating

orotate produced in the presence ofan extract of strain 30SOU6 providing thepyrD but not thepyrC product.

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UREIDO COMPOUNDS AS CARBAMYLPHOSPHATE SOURCES 47

version into arginine. The high level of OTC-ase, when combined with a functional ATC-ase, allows a high enough rate of citrullinephosphorolysis to supply the pyrimidine path-way with the CP required. This coupling of areversible OTCase with the reaction catalyzedby ATCase is formally similar to the situationobserved in the catabolism of citrulline in var-ious organisms, where citrulline phosphorol-ysis is coupled to the action of a carbamatekinase (see above).argR CPSase-less mutants, in which citrul-

line is readily channelled into argininosuccin-ate, are only weak citrulline utilizers.

Genetic derepression of OTCase is achievedeither by unstable chromosomal rearrange-ments or by stable mutations, probably of theoperator type. cis dominance tests have not yetbeen performed; it should be noted, however,that only one type of OTCase (of either the FFFor III type) is expressed constitutively in strainsharboring both the argF and I genes (see sec-tion C).A metabolically significant citrulline phos-

phorolysis is thus provided by mutations thatcause OTCase overproduction. A metabolicallysignificant carbamoylaspartate phosphorolysisis achieved by mechanisms bearing a certainsimilarity to those discussed above. ATCasederepression is brought about even in the pres-ence of uracil by a pyrH block, reducing therate of synthesis of uridine 5'-diphosphate fromuridine 5'-monophosphate, combined witheither a pyrC block or a pyrD block preventingcarbamoylaspartate from being channelledaway further than dihydroorotate, which, inturn, is readily converted to carbamoylaspar-tate by the reversible reaction catalyzed by di-hydroorotase. Operator mutants have not yetbeen obtained, despite the screening (not dis-cussed here) of a large number of carbamoylas-partate utilizers. Since they are needed for theunraveling of the poorly understood controlmechanisms regulating pyrimidine biosyn-thesis, they are being sought by localized muta-genesis.The present study offers precise implications,

in addition to the purely metabolic aspects, re-lated to the in vivo phosphorolysis of citrullineand carbamoylaspartate. It appears indeed thatthe selection of enzyme overproducers based ona metabolic dependence for a reversed biosyn-thetic reaction may be contemplated as ameans for isolating regulatory mutants. Manyenzymatic reactions, indeed, use as substratesmolecules that are required in more than oneessential pathway. A straightforward applica-tion of this methodology is given in the accom-panying paper by Messenguy, in which the se-

lection of operator mutants of the OTCase genein yeast is described (14). The isolation of OTC-ase-constitutive mutants in E. coli has beenperformed previously by Jacoby and Gorini (8),who selected for a growth-supporting level ofOTCase synthesis in a nonsense, streptomycin-(partially) suppressible argI mutant ofa partic-ular strain (B) in which arg genes are ex-pressed at a lower rate than in strain K-12. Thepresent method allows a more straightforwardisolation of such mutants, which are presentlyneeded at various stages of in vitro studies ofthe arg regulon.Another interesting point is the fact that the

chromosomal rearrangements that bring aboutconstitutive F-type OTCase synthesis occur inone strain but are not detected in another (seesection C). This observation provides a possibleapproach to analyzing the genetic determinants(particular deoxyribonucleic acid sequences,availability of particular enzymes) that controlthe production of certain classes of chromo-somal rearrangements.

ACKNOWLEDGMENTSThis work has been supported by the Belgian Funds for

Fundamental and Collective Research (FRFC-FKFO) andby the Belgian Funds for Scientific Research (FNRS).

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