JOURNAL OF BACTERIOLOGY, June 1982, p. 1130-11370021-9193/82/061130-08$02.00WO

Vol. 150, No. 3

Regulation of Phenylalanine Biosynthesis in Escherichia coliK-12: Control of Transcription of the pheA Operon

J. GOWRISHANKAR AND JAMES PHTARD*School ofMicrobiology, University ofMelbourne, Parkville, Victoria 3052 Australia

Received 27 October 1981/Accepted 21 January 1982

Bacteriophage X ppheA-lac was used to obtain strains of Escherichia coli K-12in which pheA and lacZ are each transcribed from a separate pheA promoter.Mutants in which both 3-galactosidase and chorismate mutase P-prephenatedehydratase (the pheA gene product) were derepressed were isolated, and a trans-acting gene (pheR) was identified. pheR was mapped at min 93 on the E. colichromosome; pheR mutants acquired the wild-type phenotype when either F117(which covers the 93-min region) or F116 (which covers min 59 to 65) wasintroduced into the cell. A rifampin resistance mutation, rpoB366, was found toderepress transcription of the pheA operon. pheR and rpoB366 affected twodifferent systems for the phenylalanine-mediated control of pheA. A mutation inmiaA (trpX), a gene known to be involved in attenuation in the tryptophan operon,was also shown to increase transcription of the pheA gene.

The biosynthesis of phenylalanine from inter-mediary products of glucose metabolism inEscherichia coli is achieved via (i) a commonpathway that subserves the synthesis of thearomatic amino acids and aromatic vitamins,followed by (ii) a terminal pathway for theconversion of chorismate (the final intermediatein the common pathway) to phenylalanine (Fig.1). The regulation of phenylalanine biosynthesisis predominantly effected by control ofthe activ-ity of the bifunctional enzyme chorismate mu-tase P-prephenate dehydratase (EC 5.4.99.5/4.2.1.51), which catalyzes the conversion ofchorismate to phenylpyruvate, the first step inthe terminal pathway. This enzyme, the productof the pheA gene, is subject both to feedbackinhibition (5) and to feedback repression ofsynthesis. The enzyme is maximally repressedeven when prototrophic strains are grown in theabsence of exogenous phenylalanine; it is dere-pressed only threefold when a multiple aromaticauxotroph is starved for phenylalanine in batchculture (10). A 10-fold derepression was, howev-er, demonstrated when such a strain was grownunder phenylalanine limitation in a chemostat(3).

E. coli mutants altered in the regulation ofphenylalanine biosynthesis have been isolatedwith the use of the amino acid analogs a- and p-fluorophenylalanine (FPA) (10). These mutantsare constitutively derepressed for chorismatemutase P-prephenate dehydratase; the muta-tions are cis-dominant and are closely linked tothe structural gene pheA. It was assumed thatthese mutations occurred in an operator locus

(pheO; hereinafter referred to as pheAo, follow-ing the nomenclature suggested by Bachmannand Low [1]), which controls the expression ofpheA. However, attempts to isolate strains withmutations in the postulated regulator gene pheRby this method of FPA selection were not suc-cessful.A pheR mutant of Salmonella typhimurium

has been obtained (7). This mutation is recessivein heterogenote strains carrying F116 of E. coliK-12, which permitted the direct inference thatthe gene pheR also exists in E. coli and is on theportion of the chromosome present on F116.

Recently, nucleotide sequence analysis of theleader region of pheA (24) suggested that thegene possesses an attenuation mechanism analo-gous to those described for other biosyntheticoperons in E. coli and S. typhimurium (for areview, see reference 20). In vitro transcriptionstudies of the pheA gene showed that 60%o oftranscripts initiated at the promoter terminate atthe proposed attenuator site in the leader (24).

In the accompanying paper (8), we describethe construction of the bacteriophage A ppheA-lac from a pheA::Mu dl (lac ApT) fusion strain.We describe below the use of A ppheA-laclysogens for the successful isolation and charac-terization of mutants in pheR and present genet-ic evidence for the presence of two distinct andindependent mechanisms for the transcriptionalregulation of the pheA operon.

MATERALS AND METHODSThe chemicals and growth media used, many of the

genetic techniques, and the methods for enzyme as-

1130

REGULATION OF pheA OPERON IN E. COLI 1131

p-P.TYR

,',-TRPPEP-.+ -..DAHP _-SA.- _--CA-PA-_-PPA-'iPHEE4P ~ *

ss ",Aromatic'% Vitamins

%---EnterochelinFIG. 1. Pathway of phenylalanine biosynthesis in

E. coli and its relationship to the synthesis of the otheraromatic amino acids and vitamins. PEP, Phospho-enolpyruvate; E4P, erythrose-4-phosphate; DAHP, 3-deoxy-D-arabinoheptulosonic acid-7-phosphate; SA,shikimic acid; CA, chorismic acid; PA, prephenicacid; PPA, phenylpyruvic acid. The conversion ofchorismate through prephenate to phenylpyruvate iscatalyzed by the bifunctional enzyme chorismate mu-tase P-prephenate dehydratase, a product of the pheAgene.

says are all described in the accompanying paper (8); AppheA-lac is also described therein. XNK370 (b221c1857 cI171::TnlO 0 UGA261) was from N. Kleckner.Phenyl-3-D-galactoside (PG) and o-FPA and p-FPAwere purchased from Sigma Chemical Co., St. Louis,Mo. Table 1 lists the strains of E. coli K-12 used.Transpoation oftWThlO. Random transpo-

sitions ofTnlO into the chromosome were obtained bythe method of Kleckner et al. (12).

Seletion for Tete clones ftom TnlO-crring strains.Quinaldic acid, ZnCI2, and chlortetracycline wereused in the selection for tetracycline-sensitive cellsfrom strains carrying TnWO, as described by Bochner etal. (2).PG seeton for mutants with icrased P-gaacto

ddase actvity. The method for selecting mutants withincreased ,-galactosidase activity was based on thatdescribed by Smith and Sadler (18).

Phenyblanine cross-feeding. The method used todetect cross-feeding was based on that described byGibson and Jones (6), except that 0.01% peptone wasomitted from the medium. The phenylalnnine auxo-troph AT2022 was used as the indicator strain in theseexperiments.

RESULTSPG selectIon for mutaswithin aed ,-

galactsidase activity. Two related strains,JP3156 and JP3290 (both Alac recA (A ppheA-lac)], were used to select for spontaneous mu-tants with increased ,B-galactosidase activity on0.01% PG plates, as described above. Mutantswere obtained at a frequency of between 10-4and 10-5 per cell plated. They were then purifiedand classified into cis- and trans-acting mutantson the basis of the following property. BothJP3156 and JP3290 carry an intact pheA gene inaddition to the pheA-lac fusion. Mutations in cisthat increase ,B-galactosidase activity do notderepress pheA, whereas the trans-acting muta-tions not only increase p-galactosidase activity,

but also increase the activity of chorismatemutase P-prephenate dehydratase. The dere-pression of chorismate mutase P-prephenate de-hydratase activity can be screened by phenylala-nine cross-feeding tests and then demonstratedmore definitively by assays of cell extracts forenzyme activity.The cis-acting mutations that were obtained

with the use of A ppheA-lac are not consideredfurther in this paper. Some of the trans-actingmutations are described below.

Isolation and mapping of pheR mutants. Oneclass of mutants obtained in the PG selectionhad a 10-fold derepression of 3-galactosidaseactivity and strongly cross-fed the phenylalanineauxotroph AT2022; this class also had a 20-foldincrease in the activity of perphenate dehydra-tase. The same mutant alleles, however,produced only 6- and 10-fold increases, respec-tively, in 3-galactosidase and prephenate dehy-dratase activities in other strains of differentpedigree. Two independently derived mutants(JP3297 and JP3299) were chosen for furtherstudy. The mutations causing this phenotypewere tentatively assigned to the gene pheR andgiven the allele numbers pheR372 and pheR374,respectively, for strains JP3297 and JP3299.Both mutations pheR372 and pheR374 were

recessive in F116 heterogenote strains (Table 2);this confirmed an earlier report by Gollub et al.(7), who found that a pheR mutation in S.typhimurium was recessive in heterogenotestrains carrying E. coli F116.

Further mapping ofpheR372 was achieved byusing transposon TnlO. A Tets derivative ofJP3297 was isolated by the method ofBochner etal. (2), and random chromosomal TnlO inser-tions were introduced by using XNK370, asdescribed above. Strains with TnlO insertionsnear pheR were then selected for by preparing aP1 kc lysate on the pool of cells, transducingJP3151, and then selecting for tetracyclineresistance and growth on 0.01% PG. One of thestrains so obtained, JP3302, was subsequentlyshown by P1 transductions to have TnlO linked95% to pheR.

Direct mapping of the pheR (TnlO) locusyielded results that were in conflict with theF116 data, which had implied that pheR was inthe 59- to 65-min region of the chromosome; inview of this anomaly, the precise map locationwas confirmed by a variety of genetic methods.Thus, the TnWO (and the pheR372 allele linked toit) in JP3302 was transferred into several Hfrstrains and mapped by interrupted matings withappropriate recipient strains. These experimentsestablished that there was only one Tetr locusand that this was situated at about 90 to 95 minon the map, close to and clockwise from metA(data not shown). The TnlO locus was shown to

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1132 GOWRISHANKAR AND PITTARD

TABLE 1. List of E. coli K-12 strains usedStrain I Genotype I Origin or reference

AT2022KLF16/KL110KLF17/KL132

JP3151

JP3156JP3290JP3297JP3298JP3299JP3302JP2998

JP2999JP3351JP3355JP3346

JP2604JP2982JP2983JP3281

JP3342JP3343JP3344JP3345JP3365

JP2898JP2899JP2987JP2988JP3314JP3349JP3350JP3371JP3372

his argE3 pro pheAlF116/argG6 metB his-I thyA23 leu-6 recAl rpsL104F117/pyrB31 thi-l thyA25 his-i pro-27 leu-6 thr-lrecAl rpsL9

F+ purE trp his argG ilv leu metA or metB rpsLAlacU169 (A ppheA-lac)

JP3151 recA56 srl-1300::TnlOF- derivative of JP3156JP3156 pheR372JP3290 pheR373JP3290 pheR374JP3151 pheR372 zjd-I::TnlOpheAo3Sl rpoB361 AlacUl69

tyrA4 rpoB361 AlacU169JP2998 pheR372 zjd-351::TnlOJP2999 pheR371 zjd-351::TnlOaroB351 tyrA4 rpsL pheR372 zjd-351::TnlO rpoB361AlacUl69 (A ppheA-lac)

aroG aroH aroP pheP367 his argE thi rpoB352 A- ArJP2604 argE+ rpoB+ thi+ TnlO (position not known)JP2982 rpoB366JP2604 AlacU169 (A ppheA-lac)

JP3281 argE+ rpoB366 pheR372 zjd-351::TnlOJP3281 argE+ rpoB366 pheR+ 4zd-351::Tn1OJP3281 argE+ rpoB352 pheR372 zjd-351::TnlOJP3281 argE+ rpoB352 pheR+ zjd-351::TnlOaroG aroH aroB351 pheR372 zjd-351::TnlO rpsLrpoB366 AlacUI69 (A ppheA-lac)

JP2604 his+ argE pheAo+ rpoB352JP2604 his+ argE pheAo351 rpoB352JP2604 his+ argE+ pheAo351 rpoB366JP2604 his+ argE+ pheAo+ rpoB366trpR thi AlacUI69 (XppheA-lac)JP3314 miaA (TnlO)JP3314 miaA+ (TnlO)JP3314 pheR372 zjd-351::TnlO rpoB352JP3314 pheR372 _jd-351::TnIO rpoB366

A. L. Taylor1414

From CSH57 (16), involving sev-eral intermediate strains

By P1 kc transductionFrom JP3156This workThis workThis workThis workFrom JP2869 (8) by PI kc trans-

duction8By P1 kc transductionBy P1 kc transductionFrom JP3141 (8) by Pl kc trans-

ductionsFrom JP2404 (19)By P1 kc transductionsThis workFrom JP2604, involving several

intermediate strainsBy P1 kc transductionsBy P1 kc transductionsBy P1 kc transductionsP1 kc transductionsFrom JP3342, involving several

intermediate strainsBy P1 kc transductionsBy P1 kc transductionsBy P1 kc transductionsBy P1 kc transductions8By P1 kc transductionBy P1 kc transductionsBy P1 kc transductionsBy P1 kc transductions

a The nomenclature for genetic symbols follows that described by Bachmann and Low (1) and fortranspositional insertions that described by Kleckner et al. (13). Allele numbers are indicated where they areknown. Fermentation markers are not described.

be cotransducible with ampA at a frequency of33.7% and with purA at frequencies of 5 to 10%o;ampA and purA were themselves cotransducibleat a frequency of 24%. These data suggested agene order of pheR (TnlO)-ampA-purA andplaced pheR (TnlO) at position 93.3 to 93.5 mnon the E. coli genetic map. The gene order wasconfirmed by three-factor crosses (data notshown). The relative disposition of TnlO withrespect to pheR, however, could not be ascer-tained from these mapping experiments. Therewas no linkage in transduction between pheR(TnlO) and metC, serA, thyA, lysA, or argA(markers in the 59- to 65-min region covered byF116 [14]). On the basis of these results, wedesignated the site of insertion of TnlO as zid-

351::TnlO, in accordance with the nomenclatureproposed by Kleckner et al. (13).The F-prime F117 covers the 93- to 98-min

region ofthe E. coli chromosome (14), and it wasintroduced into the strains JP3297 and JP3299 todetermine whether it also carried the pheR+gene. The presence of F117 in the cells de-creased the activities of ,B-galactosidase andprephenate dehydratase in both JP3297 andJP3299 to the fully repressed levels (Table 2).A number of genetic tests provided unequivo-

cal evidence for homology between F117 and thechromosomal pheR region. (i) When gene con-versions at pheR were selected for (with 0.01%PG) in a recA+ F117 heterogenote strain thathad the pheR372 4jd-351::TnlO on the chromo-

I---r

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REGULATION OF pheA OPERON IN E. COLI 1133

TABLE 2. Effect of F116 and F117 on the specific activities of prephenate dehydratase and 3-galactosidasein pheR strainsa

Sp actb

In haploid strains In hyAF6 hetero- In F117 heterogenotecStrain Relevant genotype genotec _____ ____

Prephenate c-Galacto- Prephenate ,B-Galacto- Prephenate B-Galacto-dehydratase sidase dehydratase sidase dehydratase sidase

JP3290 Parent pheR+ recA 4.8 145 3 74 3.2 75(A ppheA-lac)

JP3297 pheR372 90 1,580 4.1 100 4.2 93JP3299 pheR374 90 1,450 4 117 4 89JP3298 pheR373 32 575 5 90 4.5 114

a All strains were grown in repressing concentrations of aromatic end products.b Specific activities of prephenate dehydratase, anthranilate synthetase, and tryptophan synthetase are

expressed n milliunits per milligam of protein. P-Galactosidase activity is expressed in units as described byMiller (16).

c thyA mutants of the strains were selected as described by Miller (16). The F-primes 116 and 117 wereintroduced by conjugation, with KLF16/KL110 and KLF17/KL132, respectively, as donors.

some, it was shown that all of the coloniesobtained had also undergone gene conversionfor the closely linked Tetr locus. (ii) When F117was used to mobilize the chromosome of apheRzjd-351::TnlO strain into a pheR+ Tet' recipient(selecting for Leu+ recombinants after earlyinterruption), a percentage of the recombinantsobtained had become pheR Tetr. (iii) Lastly,when a strain carrying F117 was transduced toTetr with a P1 kc lysate prepared on JP3302, itwas shown that some of the transductants car-ried the TnlO on the F-prime itself.These same tests failed to provide any evi-

dence of homology between F116 and the chro-mosomal pheR region. Although this would ar-gue against the presence of any major region ofF117 on F116, it does not exclude the possibilityof a small aberrant translocation which hasadded the pheR locus to the F-prime F116.A second class of mutants obtained in the

original PG selection with JP3290 had four- andsevenfold elevations in the activity of f-galacto-sidase and prephenate dehydratase, respective-ly. In one of them (JP3298) the mutation wasagain shown to be recessive in both F116 andF117 merodiploid strains (Table 2); on this basis,this class is also believed to represent mutationsin pheR, with only a partial loss of repressoractivity.

Role ofpheR in the regulation ofpheA. Growthof an aroB pheR+ ppheA-lac lysogen underconditions of phenylalanine limitation in a che-mostat was shown earlier to produce a 10- to 12-fold increase in the activity of chorismate mu-tase P-prephenate dehydratase and a fourfoldincrease in ,B-galactosidase activity (8). To studythe physiological effect of pheR in the expres-sion of the pheA gene, an aroB derivative of apheR strain was constructed (JP3346) and grownin continuous culture in the chemostat under

similar conditions of phenylalanine limitation.Prephenate dehydratase activity increased butonly two- to threefold (Table 3). Unexpectedly,P-galactosidase activity did not change, but thismay represent an instability of the enzyme itselfunder the conditions of phenylalanine starvationin the chemostat; when the same strain wasgrown at a slower rate (D, 0.05 h-1) in thechemostat, the prephenate dehydratase activityremained elevated and the P-galactosidase activ-ity feli further to 200 U. Other data whichsupported this interpretation were the observa-tions that P-galactosidase activity in a pheR+pheA-lac fusion strain decreased during phenyl-alanine starvation in batch culture and that theactivity of P-galactosidase was derepressed lessin proportion to that of prephenate dehydratasewhen a pheR+ A ppheA-lac lysogen was grownunder phenylalanine limitation in the chemostat(8). The results therefore indicated that pheRmutants have an altered regulation of pheAexpression; they also suggested the presence of

TABLE 3. Specific activities of 0-galactosidase andprephenate dehydratase in strains grown under

phenylalanine limitation in the chemostataSp act'

Strain Relevant genotype i-Galacto- Prephe-sia nate dehy-dratase

JP3346 aroB pheR372 600 (600) 145 (65)(k ppheA-lac)

JP3365 aroB pheR372 rpoB366 600 (750) 140 (105)_ (A ppheA-lac) I _Ia Values in parentheses indicate the activities ob-

tained when the strains were assayed simultaneouslyafter growth in repressing concentrations of the aro-matic end products.bSee Table 2, footnote b.

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1134 GOWRISHANKAR AND PITTARD

one or more additional mechanisms involved inthe control of the pheA operon.To examine the interaction between pheR and

the operator of the pheA gene, pheR372 wasintroduced into a strain carrying the pheAo351mutation. pheAo3SI had earlier been isolated inthis laboratory (10) as a cis-dominant mutationthat was closely linked to and constitutivelyderepressed the pheA structural gene; it wastherefore postulated that it defined the operatorregion for a single-gene operon. Table 4 showsthe prephenate dehydratase activities of a nearlyisogenic set ofpheR pheAo strains; the enzymeactivity was elevated 10-fold in both thepheR372 and pheAo351 strains and was notfurther increased when the two mutations werebrought together in JP3351. This indicated thatmutations pheR372 and pheAo351 occurred indifferent genes involved in the same system ofcontrol affecting pheA expression.

Isolation and characterization of the rpoB366mutation. Strain JP2982 carries mutations inseveral genes concerned with aromatic aminoacid biosynthesis, rendering it extremely sensi-tive to low levels of p-FPA. In the course ofattempts to isolate and characterize FPA-resis-tant mutants in this strain, we unexpectedlydiscovered that a spontaneously isolated rifam-pin-resistant derivative of this strain, JP2983,was also resistant to 10-3 M p-FPA. The muta-tion was given the allele number rpoB366, andafter transfer by Pl kc transduction into wild-type strains it was seen to confer on these strainsan increased ability to excrete phenylalanine.Further studies were carried out to investigateits role, if any, in the regulation and expressionof the pheA gene. Another rpoB allele in ourlaboratory stocks that was tested, rpoB352, didnot confer FPA resistance in JP2982, as was truealso of the majority of spontaneously occurringrifampin resistance mutations. The rpoB352 al-lele was used as a control to rpoB366 in some ofthe experiments below.The phenotype of rpoB366 strains is apparent-

ly a consequence of increased activity of choris-mate mutase P-prephenate dehydratase be-

TABLE 4. Prephenate dehydratase activity inisogenic pheR pheAo strains grown in repressing

concentrations of aromatic end productsPrephenatedehydratase

Strain Relevant genotype sp act (mU/mg of

protein)

JP2999 pheR+ pheAo+ 10JP3355 pheR372 pheAo+ 95JP2998 pheR+ pheAo351 105JP3351 pheR372 pheAo3SI 104

cause, when compared with isogenic rpoB+strains, derivatives with the rpoB366 mutationshowed a two- to threefold elevation in prephen-ate dehydratase activity. When these derivativeswere also recA and lysogenic for A ppheA-lac, 3-galactosidase activity showed a similar twofoldincrease (data not shown). As noted previously(8), the recA mutation is required to stabilize theA ppheA-lac in these lysogens. The coordinatederepression of lacZ and pheA that is producedby the rpoB366 mutation in strains in whichthese two genes are independently expressedfrom pheAp indicates that rpoB366 acts at thelevel of transcription of the pheA operon.

In P1-mediated transductions, rpoB366 wasshown to be linked 40o to argE and 90 to thiA;three-factor crosses confirmed that the geneorder was argE-rpoB366-thiA (data not shown),in accord with the established location of rpoBon the E. coli linkage map. The effect ofthe rpoBallele was dominant over the wild type in resist-ance to rifampin in F112 heterogenote strains;prephenate dehydratase activity was also elevat-ed in these strains (data not shown).The combined effects ofpheR372 and rpoB366

were studied by the introduction ofpheR372 intoisogenic rpoB366 and rpoB352 strains (the latterused as a control), and the results of the pre-phenate dehydratase assays are shown in Table5. The mutations acted independently of oneanother in increasing the expression initiatedfrom pheAp. This conclusion was supported bythe further observation that rpoB366, as com-pared with rpoB3S2, doubled the activity ofprephenate dehydratase in a strain carrying thepheAo351 mutation (Table 5).When an aroB pheR rpoB366 strain lysogenic

for A ppheA-lac (JP3365) was grown under phe-nylalanine limitation in the chemostat, the pre-phenate dehydratase activity increased by about40%o (Table 3). The magnitude of the increasewas distinctly smaller than that seen with thearoB pheR strain JP3346. This indicated that therpoB366-mediated increase of pheA expressionrepresents a second mechanism of phenylala-nine-specific control of the operon; the mutationmay, however, only be providing partial releasefrom this second control mechanism. P-Galacto-sidase activity in JP3365 fell slightly upon star-vation in the chemostat, which again was pre-sumably a consequence of the instabilityexhibited by the enzyme under such conditions.Yanofsky and Horn (22) recently described

the isolation and characterization of severalrpoB mutations that alter the efficiency of atten-uation in the trp operon leader region. In light ofthat report, we too studied the effect of rpoB366on the expression of the trp operon. rpoB366 andrpoB3S2 (as control) were transduced into apheR trpR strain (JP3341) in which, as described

J. BACTERIOL.

REGULATION OF pheA OPERON IN E. COLI 1135

TABLE 5. Specific activity of prephenatedehydratase in isogenic rpoB pheAo strains grown in

repressing concentrations of the aromatic endproducts

Prephenatedehydratase

Strain Relevant genotype sp act (mU/mg of

protein)

JP3342 pheR372 rpoB366 112JP3343 pheR+ rpoB366 13JP3344 pheR372 rpoB3S2 52JP3345 pheR+ rpoB352 7

JP2987 pheAo351 rpoB366 130JP2988 pheAo+ rpoB366 13JP2899 pheAo351 rpoB3S2 72JP2898 pheAo+ rpoB352 7

in the accompanying paper (8), the lacZ gene isunder trp operon control and trpA is expressedfrom pheAp. The results of the enzyme assays(Table 6) showed that rpoB366 but not rpoB352increased the activity of anthranilate synthetaseand P-galactosidase (both expressed from trpEp)by amounts comparable to those obtained withthe miaA mutation (see below). We conclude,therefore, that rpoB366 diminishes attenuationcontrol in the trp operon.

Mutations in miaA. miaA (trpX) was originallyisolated by Yanofsky and Soil (21) as a mutationthat relieved transcription termination at theattenuator site of the trp operon. It has subse-quently been shown to affect the modification oftRNAIP, tRNAPhC, and tRNATY", the miaAgene product being in some way necessary forthe methyl-thio modification of isopentenyl ade-nine (msZi6-A) at one position in the anti-codonloop of each of these three tRNA species (4).miaA has also been reported to produce a two-fold increase in prephenate dehydratase activity(24).To study whether miaA also acts at the level

of transcription of the pheA operon, the muta-

tion was transduced, with the aid of the TnlOlinked to it (4), into JP3314, which carries thetrp-lacZ and pheAp-trpA fusions. The incomingmiaA mutation was screened by checking forincreased P-galactosidase activity, and then theprephenate dehydratase and tryptophan synthe-tase levels were determined in isogenic miaAand miaA+ strains. miaA increased chorismatemutase P-prephenate dehydratase activity (Ta-ble 6). That this effect was the result of anincreased rate of transcription of the pheA geneis shown by the fact that trpA expression frompheAp was also coordinately derepressed in thisstrain.

DISCUSSIONpheA-lac fusions used to obtain regulatory mu-

tants of the pheA operon. As described above, X

ppheA-lac was useful not only in selecting forregulatory mutants but also in differentiating themutations affecting genes acting in cis fromthose acting in trans. It was also of use inconfirming the futility of the FPA resistanceselection attempts for pheR mutants. Thus, of250 mutants resistant to 10-3 M o-FPA obtainedfrom a A ppheA-lac lysogen, approximately 20%were identified as pheA regulatory mutants on

the basis of phenylalanine cross-feeding tests;none, however, showed any increase in P-galac-tosidase activity, indicating that none of thesemutations acted in trans on the pheA operon. AspheR mutants themselves are significantly resis-tant to o-FPA and p-FPA, the basis for this biasagainst pheR during FPA resistance selectionremains unexplained.F116 effect on pheR. The direct mapping data

on pheR372 zjd-351: :TnlO unquestionably locat-ed the gene position at 93.3 to 93.5 min on thelinkage map. There are three possible explana-tions for the observation that the pheR muta-tions were recessive in F116 heterogenotes. (i)F116 carries an extragenic suppressor. This is an

TABLE 6. Enzyme specific activities in isogenic rpoB and isogenic miaA strainsaSp actc

Strain Genotypeb Anthranilate Tryptophan Prephenatesynthetase synthetase dehydratase ,-Galactosidase

JP3371 rpoB352 pheR372 4.0 21.2 7 750JP3372 rpoB366 pheR372 12.1 41.0 13 1,400JP3349 miaA 22.0 7 15 1,500JP3350 miaA+ 4.2 4.5 7 980

All strains were grown in repressing concentrations of the aromatic end products.b All strains were trpR and lysogenic for A ppheA-lac. The latter is integrated into the chromosome by

trp'CBA' homology, so that the genes for anthranilate synthetase and 3-galactosidase are expressed from trpEpand tryptophan synthetase and prephenate dehydratase activities represent expression from two separate pheApromoters (8).

c See Table 2, footnote b.

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1136 GOWRISHANKAR AND PIITARD

unlikely explanation, as the F116 effect wasnoted by us with all three of our independentpheR mutations (including one with partial lossof repressor activity) and also by Gollub et al.with their pheR mutation in S. typhimurium (7).(ii) The pheR phenotype is produced by theinteraction of mutations in two separate genes,and the presence of a functional gene productfrom either can make a strain pheR+. In thatcase it might be further postulated that F116carries one of these functional genes and that thecorresponding gene on the chromosome carriesa silent mutation in all of the strains we exam-ined. The complexity of this explanation, as wellas our current inability to conceive of a molecu-lar model for such gene-product interaction, alsomakes this possibility unlikely. (iii) The thirdpossibility, which appears to be the most likelyexplanation, is that F116 carries the pheR geneitself. It may be that in some strains ofE. coli K-12 other than those which we tested, the pheRgene is located in the 59- to 65-min region(homologous to the map location of pheR in S.typhimurium [7]) and that the parent Hfr fromwhich F116 was derived, AB312, was one suchstrain, or it may be that the gene pheR hasundergone an aberrant translocation from itsnormal site on the chromosome onto F116. Ourexperimental results failed to demonstrate anyhomology between F116 and the chromosomalpheR region, but the tests were genetic in natureand not sensitive enough to exclude the pres-ence of small regions of identity. The naturaloccurrence of inverted repeat sequences aboutthe argF gene segment, rendering that genetransposable, has been reported recently (9, 23),and it is possible that pheR too may be situatedon a transposable segment ofthe E. coli chromo-some. We are presently in the process of tryingto distinguish among these various possibilities.pheR and rpoB366 mutations. Our results from

the chemostat starvation studies indicated thatpheR and rpoB366 defined two independentmechanisms by which intracellular phenylala-nine concentration feeds back to repress theexpression of the gene for chorismate mutase P-prephenate dehydratase. Both mechanisms wereconcerned with transcriptional control of thepheA operon. pheR mutants showed a 10- to 20-fold derepression of chorismate mutase P-pre-phenate dehydratase, the exact magnitude ap-pearing to depend on strain background,whereas rpoB366 mutants showed a two- tothreefold increase in the activity of this enzyme.It is most likely that these are the only twomechanisms involved in the control ofpheA andthat the residual derepression seen during phe-nylalanine starvation of the pheR rpoB366 dou-ble mutant in the chemostat is merely a reflec-tion of the incomplete effect of the rpoB366

mutation. However, the existence of an addi-tional, albeit minor, mechanism of regulationcannot be ruled out.The mechanisms by which pheR and rpoB366

control the expression of pheA can reasonablybe conjectured at present, but their verificationwould necessarily depend on more detailed stud-ies at the molecular level. The fact that bothpheR and pheAo351 define a single mechanismof control would indicate that these representmutations in a gene coding for an aporepressormolecule and the operator of pheA, respective-ly; this then would be the classical operator/repressor mechanism of transcriptional control(11).There is substantial circumstantial evidence

for the presence of a mechanism of attenuationcontrol of the pheA operon. As mentioned earli-er, the most compelling evidence comes fromDNA sequencing studies of the pheA leader andfrom results of in vitro transcription of the pheAgene (24). The effect of miaA on the transcrip-tion of pheA lends support to this idea. Inanother study, McCray and Hermann (15)showed that Fe3" starvation produced a fivefoldincrease in prephenate dehydratase activity; andiron starvation was earlier (17) shown to affectthe methyl-thio modification of isopentenyl ade-nine (mszi6-A) in several tRNA species, includ-ing tRNAph'. This is the same modification thatis lost in miaA mutants (4).The effect of the rpoB366 mutation on pheA

fits very well with an attenuation model ofcontrol of the operon. Thus, rpoB366 would beanalogous to the Rif' termination relief muta-tions of the trp operon described by Yanofskyand Horn (22), and it probably alters the ,subunit of RNA polymerase to reduce the effi-ciency of termination at the attenuator site ofthepheA gene. The fact that rpoB366 increased theexpression of the trp operon lends further sup-port to a common mechanism underlying attenu-ation control in these two operons.

ACKNOWLEDGMENTSWe thank D. Eddy, S. Norton, and L. Vizard for technical

assistance.This work was supported by a grant from the Australian

Research Grants Committee. J.G. is the recipient of a Univer-sity of Melbourne postgraduate student award.

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VOL. 150, 1982