Vol. 138, No. 3JouRNAL OF BACTERIOLOGY, June 1979, p. 933-9430021-9193/79-0933/11$02.00/0

Aromatic Amino Acid Biosynthesis: Regulation of ShiimateKinase in Escherichia coli K-12

B. ELY AND J. PITTARD*

Department ofMicrobiology, University ofMelbourne, Parkville, 3052 Victoria, Australia

Received for publication 6 April 1979

Starvation of cells ofEscherichia coli K-12 for the aromatic amino acids resultsin an increased rate of synthesis of shikimate kinase activity. The two controllingamino acids are tyrosine and tryptophan, and starvation for both results inderepression. The product of the regulator gene tyrR also participates in thiscontrol, and shikimate kinase synthesis was derepressed in tyrR mutants. Chro-matography of cell extracts on diethylaminoethyl-Sephadex allowed partial sep-aration of two shikimate kinase enzymes and demonstrated that only one of theseis subject to specific repression control involving tyrR. By contrast, chromatog-raphy of cell extracts with G-75 or G-200 columns revealed a single-molecular-weight species of shikimate kinase activity with an apparent molecular weight of20,000. The levels of shikimate kinase in a series of partial diploid strains indicatedthat aroL, the structural gene for the tyrR-controlled shikimate kinase enzyme,is located on the E. coli chromosome between the structural genes proC andpurE. By means of localized mutagenesis, an aroL mutant of E. coli was isolated.The mutant was an aromatic prototroph and, by the criterion of column chro-matography, appeared to have only a single functional species of shikimate kinaseenzyme.

Commencing with the condensation of ery-throse-4-phosphate and phosphoenolpyruvateto form 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP), the common pathway ofaromatic biosynthesis proceeds in seven steps tothe branch point compound chorismic acid (Fig.1). The first step of the common pathway, whichis carried out by three isofunctional and inde-pendently regulated enzymes, is important incontrolling the flow of intermediates along thepathway. With the exception of shikimate ki-nase, the fifth enzyme, the other enzymes of thecommon pathway are synthesized constitutively(31; B. K. Ely and J. Pittard, Proc. Aust. Bio-chem. Soc. 8:56,1975).Attempts to isolate shikimate kinase mutants

of Escherichia coli by selecting aromatic auxo-trophs have been unsuccessful. Such mutantshave been isolated, however, in both Bacillussubtilis (25) and Neurospora crassa (12). Onehypothesis to explain this is that in E. coli thereare two or more shikimate kinase isoenzymes.The identification by Berlyn and Giles (5) oftwopeaks of shikimate kinase activity after densitygradient centrifugation of extracts of E. colisupports this hypothesis. In addition, two peaksof shikimate kinase activity have been resolvedwhen extracts of Salmonella typhimurium aresubjected to either density gradient centrifuga-tion (5) or to ion-exchange chromatography (23).

The importance of the regulation of shikimatekinase in the control of aromatic biosynthesishas been postulated for B. subtilis (16), wherethe single shikimate kinase enzyme is repressedand inhibited as part ofa regulatory trifunctionalenzyme aggregate (25, 26). Shikimate kinase isalso implicated in the control of aromatic bio-synthesis in N. crassa (33).Although specific regulation of shikimate ki-

nase has not been detected in S. typhimurium(13), preliminary results from this laboratoryhave indicated that shikimate kinase is specifi-cally regulated in E. coli (11). It is the purposeof this paper to provide further details on theregulation and multiplicity of the shikimate ki-nase enzymes of E. coli K-12 and to describe theisolation and characterization of a mutant strainlacking activity for one of the shikimate kinaseenzymes.(Some of this work has been briefly reported

elsewhere [B. K. Ely, and J. Pittard, Proc. Aust.Biochem. Soc. 8:56, 1975].)

MATERIALS AND METHODSOrganisms. Strains used in this work are all deriv-

atives of E. coli K-12. They are described in Table 1.Growth medium. The minimal medium used was

medium 56 as described by Monod et al. (22). This wassupplemented with 0.5% glucose and with thiamineand amino acids as required. When used as supple-

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934 ELY AND PITTARD

PEP

0OHC-C,

CIH-CH-C-

COOH

-5_ ~ CO0CH OOH OOHT _ Ci

HO T 1CH o III~~ E~IIEEI4 HO-C-OH -

-OH R-Ij-OH OH-OH H-OH OHo-0pQ3H2 D2OHSELP DAHPP

H OH "A , C O O

5A SAP EPSAP CA

FIG. 1. Outline of the reactions involved in thesynthesis of chorismate from erythrose-4-phosphateand phosphoenolpyruvate. Abbreviations: PEP,phosphoenolpyruvate; E4P, erythrose-4-phosphate;DHQ, 3-dehydroquinic acid; DHS, 3-dehydroshi-kimic acid; SA, shikimic acid; SAP, shikimic acid-3-phosphate; EPSP, 3-enol-pyruvyl shikimic acid 3-phosphate; CA, chorismic acid.

ments, amino acids were added at predetermined op-timal levels ranging from 5 x 10-4 to 2 x 10-3 M.

Buffers. The Veronal-hydrochloride and Tris-hy-drochloride buffers used were prepared by the meth-ods of Dawson and Elliott (8).

Chemicals. Sephadex G-75 and DEAE-Sephadexwere obtained from Pharmacia Fine Chemicals. D-Erythrose-4-phosphate dimethylacetal dicyclohexyl-ammonium salt was obtained from Fine Chemicals ofAustralia and was converted to free D-erythrose-4-phosphate by the method of Ballou et al. (4). Otherchemicals were generally of the highest quality avail-able commercially.Growth of cells and preparation of cell ex-

tracts. Cells were grown in minimal medium or inminimal medium supplemented with the aromaticamino acids and vitamins or in minimal medium sup-plemented with shikimic acid. These were added inthe following concentrations: L-phenylalanine, 10-3 M;DL-tryptophan, 5 x 10-4 M; L-tyrosine, 10-3 M; p-aminobenzoic acid, 4 x 106 M;p-hydroxybenzoic acid,4 x 10-6 M; 2,3-dihydroxybenzoic acid, 5 x 10-i M;and shikimic acid, 10-4 M.

TABLE 1. Description of E. coli K-12 strainsStrains Relevant markers Origin/reference

aroB351atrpA9605 his-29 ilv-1tyrR370 his-29 ilv-1his-29 ilv-IaroH367 aroG365aroH367 aroG365 tyrR352aroH367 aroF363aroH367 aroF363 tyrR363aroH367 aroG365 tyrR359recA13 tyrR390/F104 thr+ leu+ pro+purE42 recAl tyrR391/F254 lac+ purE+galK35 recAl tyrR392/F126 gar pyrD+his4 aroD5 recAl tyrR393/F148 his+ aroD+his-i recAl tyrR394/F129 his+tyrA2 recAl tyrR395/F142 tyrA+lac proC14purE355 tyrR366lac purE355 tyrR366/F254 lac+ purE+lac purE355 tyrR366/F144 purE+lac proC14 purE355 tyrR366/F13 procC+

lac+ purE+thyA23 recAl tyrR475/F116 thyA+lacpurE355 tyrR366/F13-4 lac+lacpurE355 tyrR366/F210purE+lac proC14 purE355 tyrR366lac aroL476purE355purE356 tyrR366

lac tyrR366lac aroL476 tyrR366

lac aroL476 tyrR366/F13 pro+ lac+ purE+aroL+

laclac aroL476his-29 ilv-1 tyrR366

297

trp+ tyrR370 transductant of JP2140trp+ transductant of JP2140

1717171717

tyrR derivative of CGSC4251btyrR derivative of CGSC4282tyrR derivative of CGSC4253tyrR derivative of CGSC4302tyrR derivative of CGSC4280tyrR derivative of CGSC4279tyrR366 transductant of AB1515F254 derivative of JP1065F144 derivative of JP1065F13C derivative of JP1065

tyrR derivative of CGSC4254F13-4C derivative of JP1065F210' derivative of JP1065trp+ transductant of JP1065NTG-inducedd derivative of JP1078 which

lacks shikimate kinase II (this paper)pur+ transductant of JP1078pur+ transductant of JP1078-1 selected at

370C.F13 derivative of JP1080

tyrR+ transductant of JP1079tyrR+ transductant of JP1080trp+ tyrR366 transductant of JP2140

a Genetic symbols used are described in reference (3).b CGSC strains were obtained from B. Bachmann.F42, F13, F13-4, and F210 are referred to in Hu et al. (15).

d NTG, N-methyl-N'-nitro-N-nitrosoguanidine.

AB2826JP2140JP2310JP2311AB3253AB3271AB3259JP544JP2034JP1059JP1060JP1061JP1062JP1063JP1064JP1065JP1067JP1069JP1071

JP1089JP1075JP1077JP1078JP1078-1

JP1079JP1080

JP1081

JP1082JP1083JP2143

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SHIKIMATE KINASE IN E. COLI 935

The cells were harvested in the mid-exponentialphase of growth, washed twice in chilled 0.9% NaCl,and suspended in 0.05 M Veronal buffer (pH 7.0)containing lo-3 M shikimic acid. Cell breakage wasachieved by treatment in a French press at 20,000 lb/inm. Crude cell extracts, obtained as the supernatantsafter centrifugation at 20,000 x g for 20 min, wereassayed immediately for enzyme activity.

Mutagenesis. Bacteria were mutagenized to 5 to10% survival with N-methyl-N'-nitro-N-nitrosoguani-dine by the method of Adelberg et al. (1).

Transduction. Transduction with phage Plkc wascarried out by the method described by Pittard andWallace (29).Mating conditions. Conditions under which mat-

ing experiments were carried out have been describedelsewhere (29).

Precipitation ofnucleic acid. When extracts wereto be used in chromatographic experiments, crude cellextracts were treated with fresh 2% protamine sulfate.This was added slowly to the constantly stirred, cooledcell extract (4C) at the ratio of 1 ml of protaminesulfate solution for 140 mg of protein in the crudeextract. After a further 30 min of stirring, the precipi-tate was removed by centrifugation at 18,000 x g for30 min. The supematant was then dialyzed for 12 hagainst 100 volumes of starting buffer (0.01 M Tris-hydrochloride, pH 7.5, containing 10-3 M dithiothrei-tol and 10-3 M shikimic acid).Column chromatography. (i) Ion-exchange

chromatography. The dialyzed cell extract was ad-justed to 0.2 M in NaCl and applied to a column (1.5by 28 cm) of A50 DEAE-Sephadex which had previ-ously been equilibrated in starting buffer containing0.2 M NaCl. The column was washed with approxi-mately 100 ml of starting buffer before a 200-ml lineargradient in NaCl (0.2 to 0.4 M NaCl in starting buffer)was applied. The flow rate was 10 ml/h, and 2.0-mlfractions were collected. All chromatographic proce-dures were carried out at 4C.

(ii) Gel filtration. The dialyzed cell extract wasadjusted to 0.1 M in NaCl and applied to a column ofSephadex G-75 (2.5 by 85 cm) previously equilibratedin starting buffer containing 0.1 M NaCl. The flowrate was 14 ml/h, and 2.0-ml fractions were collected.The apparent molecular weight was determined asdescribed by Andrews (2).Assay of shikimate kinase activity. When mea-

suring shikimate kinase activity in crude extracts, theassay used was based on that described by Pittard andWallace (29). The incubation mixture contained shi-kimic acid, 1 yimol; ATP, 4 ,umol; MgCl2, 5 gmol; NaF,10 ,umol; Veronal buffer (pH 9.0), 25 ,umol; cell extract,0.1 to 1.0 mg of protein in a total volume of 1.0 ml.

Kinase activity was determined by measuring thedisappearance of shikimic acid by the method of Gai-tonde and Gordon (10). Shikimate-3-phosphate wasestimated by the method of Morgan et al. (24).

Shikimate kinase activity was determined in frac-tions obtained by column chromatography by measur-ing the appearance of ADP and was based on theassay described by Morell and Sprinson (23). Theincubation mixture contained Veronal buffer (pH 9.0),50 ,umol; shikimic acid, 1 ,umol; ATP, 4 ,mol; MgC12, 5.tmol; NaF, 10 ,umol; and up to 0.5 ml of fraction in atotal volume of 1.0 ml. Incubation was at 37C for 30

min, after which 0.2 ml of 1 M Tris-hydrochloridebuffer (pH 7.8) was added and the mixture was boiledfor 2 min and finally chilled on ice. The amount ofADP was then determined. Because this assay is notspecific for shikimate kinase, the activity detected bythis method was verified to be due to shikimate kinaseby the specific but more time-consuming method de-scribed above.Assay of DAHP synthetase. The method de-

scribed by Camakaris and Pittard (7) was used toassay DAHP synthetase.Assay of chorismate mutase. The method de-

scribed by Im et al. (17) was used to assay chorismatemutase.

Protein estimation. Protein was estimated by themethod of Lowry et al. (20).

Specific activity. The specific activities of en-zymes are expressed as micromoles of product formedor substrate used per minute per milligram of protein(international units per milligram).

RESULTSDerepression of the synthesis of shiki-

mate kinase in cells starved for particulararomatic amino acids. Strain AB2826 is anaromatic auxotroph unable to carry out the sec-ond reaction of the common pathway. Conse-quently, strain AB2826 requires either the aro-matic end products or a suitable aromatic inter-mediate, such as shikimic acid, for growth. Al-though appropriately blocked aromatic auxo-trophs can grow in minimal medium supple-mented with only shikimate, they do so at a slowrate and are actually in a state of partial star-vation for the aromatic amino acids (6; J. Pit-tard, unpublished data). By adding an aromaticamino acid with the shikinic acid supplement,starvation for that particular amino acid is re-lieved, and the effect of this relief on enzymelevels can be measured.Table 2 shows the level of shikimate kinase in

cell extracts of strain AB2826 prepared after theTABLE 2. Shikimate kinase activity ofAB2826grown under conditions of varying aromatic

supplementationShikimate

Supplementa kinasesp act

Minimal medium + shikimate 0.045, 0.040(medium A)

Medium A + tyrosine 0.010Medium A + phenylalanine 0.035Medium A + tryptophan 0.015Medium A + phenylalanine + 0.020tryptophan

Medium A + phenylalanine + 0.015tyrosine

Medium A + tyrosine + 0.005tryptophan

Medium A + tyrosine + 0.005, 0.010tryptophan + phenylalaninea Concentration of supplements is given in the text.

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936 ELY AND PITTARD

growth of cells either in miniimal medium sup-plemented with shikimic acid (medium A) or inmedium A supplemented with one or more aro-matic amino acids. Cells grown in medium Ahave a level of shikimate kinase which is ap-proximately fourfold higher than the level foundin cells grown in medium A supplemented withthe three aromatic amino acids. The addition of-either tyrosine or tryptophan to medium A re-sults in the reduction of shikimate kinase levelsto those approaching the fully repressed value.On the other hand, the addition ofphenylalanineto the growth medium has only a marginal effecton the synthesis of shikimate kinase. In fact,where phenylalanine is present in the growthmedium with either tyrosine or tryptophan, en-zyme levels are slightly higher than those ob-served in the absence of phenylalanine.Effect ofmutations in the regulator genes

trpR and tyrR on the synthesis of shikimatekinase. Both tryptophan and tyrosine, in con-cert with the product of the regulator genes trpRand tyrR, respectively, are involved in therepression of other enzymes of aromatic biosyn-thesis in E. coli (7, 17, 21, 28, 31, 32, 34). To testwhether the product of either the trpR or tyrRgene is also involved in the repression of shiki-mate kinase, enzyme levels were measured inmutant strains in which one or other of theregulator gene products was nonfunctional.No changes were observed in the regulation of

shikimate kinase synthesis in the trpR mutant,strain JP2269 (data not shown). On the otherhand, in a number of tyrR mutants, the synthesisof shikimate kinase was derepressed (Table 3).A previous report from this laboratory (11)

had shown that the synthesis ofshikimate kinasewas derepressed in a tyrR mutant grown inminimal medium but was almost fully repressedwhen the mutnt was grown in the presence ofthe aromatic end products. To examine thisphenomenon more closely, shikimate kinase lev-

TABLE 3. Effect of different tyrR alleles on the levelof shikimate kinase activity

Shikimate kinase sp actStrain tyrR allele Group for cells grown in:

MM' MM and EPAB3253 + Wild 0.020 0.010

typeAB3259 + Wild 0.015 0.010

typeJP544 363 I 0.055 0.045AB3271 352 hIa 0.045 0.040JP2034 359 lIb 0.045 0.015

- MM, Minimal medium; MM + EP, minimal me-dium supplemented with the aromatic endproducts.

els were measured in a series of strains, eachcarrying a different tyrR allele. The tyrR alleleswhich have been classified in this laboratory fallinto one of three groups, depending on how theregulation ofDAHP synthetases (phe) and (tyr)is altered (18). In strains with group I allelessuch as tyrR363, the syntheses of both DAHPsynthetases (phe) and (tyr) are derepressed. Inall strains with group II alleles, the synthesis ofDAHP synthetase (phe) is over-repressed. Instrains with group IIa alleles such as tyrR352,the synthesis of DAHP synthetase (tyr) is de-repressed, even when cells are grown in thepresence of high concentrations of tyrosine (10-'M). On the other hand, in strains with group Ilballeles such as tyrR359, the synthesis of DAHPsynthetase (tyr) is derepressed only when cellsare grown in the absence of high concentrationsof tyrosine (18). The results in Table 3 show thediffering levels of shikimate kinase activity inbacterial strains possessing tyrR alleles repre-sentative of each group. It can be seen that whencells are grown in minimal medium the levels ofkinase activity are derepressed in all strains.When cells are grown in the presence of thearomatic end products, however, the level ofshikimate kinase is greatly reduced in the strainwith the class lIb tyrR allele, as has been ob-served for DAHP synthetase (tyr). In no casedoes the presence of a mutant tyrR allele resultin the phenomenon of over-repression of shiki-mate kinase synthesis as reported for DAHPsynthetase (phe).

Effect of amber suppressors. To confirmthe role of the tyrR gene product in the repres-sion of shikimate kinase, the level of shikimatekinase activity was examined in a single tyrRamber mutant (JP2202) and in four derivativesof this strain, each of which carries a differentsuppressor. The levels of shikimate kinase inthese strains (data not shown) indicate that thesuppressors supF and supC restore the tyrR+phenotype, and supU almost does so, whereassupD fails to restore the repressible state. Simi-lar conclusions to these have been drawn whenrepression of DAHP synthetase (tyr) was usedas an indicator of tyrR function (7).Column chromatography of shikimate

kinase activity. To obtain more informationabout the nature of the shikimate kinase activitywhich is derepressed in tyrR mutants, we usedcolumn chromatography of cell extracts onDEAE-Sephadex to examine kinase activity.Extracts were prepared of both tyrR' cells(JP2311) and tyrR celLs (JP2310) after theirgrowth in minimal medium supplemented withthe aromatic amino acids.

Figure 2 shows the elution profiles obtained

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SHIKIMATE KINASE IN E. COLI 937

Fraction No.FIG. 2. Elution profiles of shikimate kinase activity from DEAE-Sephadex columns. (a) Extract of tyrR'

strain JP2311; (b) extract of the tyrR strain JP2310; (c) extract ofAB2826 prepared from cells grown in thepresence of the three aromatic amino acids; (d) extract ofAB2826prepared from cells grown in the presenceof shikimic acid and phenylalanine. Symbols: 0, shikimate kinase activity; +, protein concentration.

after the ion-exchange chromatography of ex-tracts of tyrR + cells (Fig. 2a) and tyrR cells (Fig.2b). Fractions were assayed for shikimate kinase,DAHP synthetase, and chorismate mutase ac-tivities. Neither DAHP synthetase nor choris-mate mutase activities were recovered in therange of salt concentrations shown in Fig. 2.

In both the extracts of the tyrR+ and tyrRstrains, chromatography revealed two closelyassociated peaks of shikimate kinase activity,each of which was unstable (half life of less than10 h). The first peak of activity (I) eluted be-tween 0.27 and 0.30M NaCl, whereas the second(II) eluted between 0.30 and 0.34 M NaCl. Mostimportantly, it can be seen that the major peakof activity in the extract of the tyrR + strain (i.e.,shikimate kinase I) corresponded to the minorpeak of activity in the extract of the tyrR strain.It appears that only one peak of activity (shiki-mate kinase II) is effected by a mutation in tyrR.The shikimate kinase activity observed when

cell extracts of the aroB mutant strain AB2826were chromatographed on DEAE-Sephadex isalso shown in Fig. 2. In this case, strain AB2826had been grown either in minimal medium sup-plemented with shikimic acid and the three ar-omatic acids (Fig. 2c) or in minimal mediumsupplemented with shikimic acid and phenylal-anine only (Fig. 2d). It will be recalled that strainAB2826 is the strain on which the initial obser-vations of the repression of shikimate kinasewere made. Once again, chromatography re-solved two closely associated peaks of shikimate

kinase activity in each cell extract. Each peakeluted in the range of salt concentrations de-scribed above; on this basis, they correspondedto shikimate kinases I and II. Whereas shikimatekinase I was the predominant activity in thefully repressed cells (Fig. 2c), shikimate kinaseII was predominant in the cells grown underconditions of partial aromatic starvation (Fig.2d). By comparing these elution profiles, it ap-pears that either a mutation in the gene tyrR orstarvation for tyrosine and tryptophan dere-presses the synthesis of shikimate kinase II.Noninhibition of shikimate kinase activ-

ity. To examine eluted shikimate kinase activi-ties for sensitivity to inhibition, fractions con-taining shikimate kinase I or shikimate kinase IIactivity were assayed in the presence of variousaromatic products and intermediates. Aminoacids and biosynthetic intermediates such aschorismic acid and prephenic acid were testedat a final concentration of 1.0 mM, and aromaticvitamins were tested at a final concentration of0.1 mM. Neither the aromatic amino acids,either singly or all together, nor a mixture ofaromatic amino acids and vitamins at the con-centrations used caused inhibition of either shi-kimate kinase I or shikimate kinase II activity.Similarly, neither chorismic acid nor prephenicacid, two key biosynthetic intermediates in thearomatic pathway, had a significant effect onthese shikimate kinase activities (data notshown).Gel filtration (Sephadex G-75) chroma-

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938 ELY AND PITTARD

tography of cell extracts. In an attempt todistinguish shikimate kinase isoenzymes withdifferent molecular weights, cell extracts of thetyrR+ strain JP2311 or the amber tyrR mutantstrain HP2310 were used in gel filtration exper-iments. Cells were grown in minimal mediumsupplemented with the aromatic end products.Under these conditions, shikimate kinase Ishould constitute the major activity in the tyrR+cells, whereas we would expect shikimate kinaseII would constitute the major activity in tyrRcells. As a consequence of the instability ofeluted shikimate kinase activity, it was not pos-sible to use the activity eluted from ion-exchangecolumns in these latter experiments.

Figure 3 shows typical elution profiles ob-tained after chromatography of extracts of thetyrR+ strain (Fig. 3a) and the tyrR strain (Fig.3b). Only a single peak of shikimate kinase ac-tivity is eluted from each cell extract. In eachcase the peak of activity elutes at a positionwhich indicates an apparent molecular weight ofapproximately 20,000. Identical results were ob-tained with Sephadex G-200. Either both shiki-mate kinase I and II have similar molecularweights, or only one of the shikimate kinaseenzymes is being recovered.

In an additional attempt to differentiate be-tween different shikimate kinase isoenzymes inE. coli, extracts of the tyrR + and tyrR cells were

E

asCD00C\o

0

Fraction No.FIG. 3. Elution profiles of shikimate kinase activ-

ity from Sephadex G-75 columns. (a) Extract oftyrR*strain JP2311; (b) extract of tyrR strain JP2310.Symbols: 0, shikimate kinase activity; +, proteinconcentration.

examined for kinase activity over the pH range7.8 to 9.0. In each case, only similar broad pHoptimum curves were obtained (data notshown).Approximate map position of the struc-

tural gene for shikimate kinase II. In dere-pressed strains, kinase activity consists predom-inantly of shikimate kinase II. Tribe et al. (31)measured the rate of shikimate kinase synthesisin derepressed strains growing at two growthrates and used these data to predict a chromo-somal map location for this unidentified genethat we have called aroL. According to theircalculations, aroL should (because of the bidi-rectional nature of chromosomal replication) befound in a region of the E. coli chromosome neareither min 13 or 45.To test the prediction, a series of strains was

constructed, each carrying a different F-mero-genote which duplicated part of the region ofthe chromosome which had been predicted toinclude aroL. Should the presence ofa particularF-merogenote result in a cell being diploid foraroL, then extracts of the strain would be ex-pected to have approximately double the hap-loid, derepressed level of shikimate kinase dueto the gene dosage effect. Table 4 shows that, ofthe various different partial diploid strains ex-amined, only those which were diploid for genesin the region between min 9 and 15 (19) had

TABLE 4. Specific activity of shikinate kinase inextracts of strains with different F-prime factors'

Approx re-StrainF-merogen- tyrR al- gion for ShikimateStrain F~otre,genb trlelea- which the kinase spcell is dip- act

loid (min)

JP2143 366 0.042JP1059 F104 472 0-9 0.045JP1060 F254 471 9-15 0.098JP1061 F126 473 17-34 0.040JP1062 F149 366 32-39 0.044JP1063 F129 366 39-45 0.040JP1064 F142 366 45-50 0.046JP1089 F116 366 50-60 0.035JP1065 366 0.031JP1067 F254 366 -e 0.070JP1069 F144 366 - 0.073JP1071 F13 366 - 0.068JP1073 F42 366 - 0.040JP1075 F13-4 366 - 0.035JP1077 F210 366 - 0.034

a All strains were derepressed for the synthesis ofshikimate kinase II as a result of mutations in the genetyrR. The cells were grown in minimal medium sup-plemented with the aromatic end products.

b The approximate chromosomal region covered byeach F-prime is based on the data of Low (12).C-, Refer to Fig. 4.

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SHIKIMATE KINASE IN E. COLI 939

double the level of shikimate kinase activityfound in the haploid tyrR mutant JP2143.To determine the position of aroL more

closely, F254 and five other F-merogenotes, F42,F13, F13-4, F210, and F14-24, each of whichoverlaps the 9- to 15-min chromosomal region,were transferred into the tyrR mutant JP1065.Figure 4 shows the chromosomal region coveredby these F-merogenotes. It can be seen in Table4 that shikimate kinase levels were double thehaploid (JP1065) level only in those strainswhich carried F254, F144, or F13 (JP1067,JP1069, and JP1071). Each of the other malederivatives had a level of shikimate kinase activ-ity which was comparable to the haploid level.This probably means that aroL is located be-tween the genes proC and purE on the E. colichromosome (3).

Isolation of a mutant with decreased shi-kimate kinase activity. After the location ofaroL within a small region of the chromosome,localized mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine was used to isolate an aroLmutant. Since no specific selection was availablefor a mutant altered in only a single shikimatekinase enzyme, the procedure involved selectingstrains mutated in a gene near aroL and subse-quently assaying these for decreased shikimatekinase activity (see reference 14).The starting strain chosen was strain JP1078.

In addition to possessing a tyrR allele (to facili-tate screening for the loss of aroL-specified ac-tivity), strain JP1078 also has mutations inproCand purE, both of which are located in thevicinity of aroL. In one experiment, strainJP1078 was mutagenized, and selection wasmade for Pur+ survivors. Since the residual levelof shikimate kinase activity in an aroL mutantwas predicated to be very low, aromatic endproducts were included in the selective media toensure the recovery of any mutants lacking shi-kimate kinase II. Plates were incubated at 320C,this temperature having been shown in prelimi-

lao

F42

F13-4

F144~~~~~~~~~-region in whicharoQL is located

FIG. 4. Chromosomal regions carried by F-mero-genotes used in this work. This diagram is based onthe data ofHu et al. (15) and Low (19).

nary experiments to maximize the number ofPur+ clones. From these plates, 142 clones witha Pur+ phenotype were isolated and were ex-amined for shikimate kinase activity. StrainJP1079, a purE+ transductant of JP1078, wasused as a control. The results are not shown, butonly one strain, strain JP1078-1, had levels ofshikimate kinase activity which were signifi-cantly lower than the level found in strainJP1079.On closer examination, strain JP1078-1 was

found to have a requirement for adenine at 370C.To avoid this complication, strain JP1078-1 wastransduced with phage P1 which had been grownon the prototrophic strain W3110. Selection wasmade for Pur+ transductants at 370C. Of 10 suchtransductants that were purified and assayed, allhad low levels of shikimate kinase activity. Onestrain, strain JP1080, was retained, and Table 5shows the level of shikimate kinase and DAHPsynthetase activities in cell extracts of this strainand the parent JP1079. In strain JP1080 thelevel of shikimate kinase is reduced to approxi-mately 14% of that found in strain JP1079.

In both strains, however, the tyrR gene prod-uct remained nonfunctional, as was evidencedby the fully derepressed levels of both DAHPsynthetase (phe) and DAHP synthetase (tyr)(data not shown). Strain JP1080 behaved, there-fore, as would be expected for a strain which hadreceived an inactivating mutation in aroL.Location of the mutation causing the de-

crease in shikimate kinase activity inJP1080. To confirn that the aroL gene hadbeen mutated in JP1089, the F-merogenote F13(i.e., F-aroL+) was transferred into this strain.Table 5 shows that the presence of F13 restoredthe fully derepressed level of shikimate kinaseto the cell. We conclude that an inactivatingmutation has occurred in aroL and will refer tothis allele as aroL476.Because aroL mutants are aromatic proto-

TABLE 5. Specific activity of shikimate kinase incells grown in different media and mutated in the

genes tyrR, or aroL, or bothGrowth me- tyrR al- aroL al- Shikimate ki-

Strain dium lele lele nase sp actJP1082 MMa + + 0.017JP1082 MM + EP + + 0.010JP1083 MM + 476 0.004JP1079 MM 366 + 0.052JP1079 MM + EP 366 + 0.055JP1080 MM 366 476 0.005JP1080 MM + EP 366 476 0.004JP1081 MM + EP 366 4761+b 0.049

a MM, minimal medium; MM + EP, minimal me-dium Supplemented with the aromatic end products.

b JP1081 carries the F-merogenote F13.

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trophs (see below), no selection exists for thearoL marker. However, the possibility that aroLand purE were cotransducible was investigated.Phage Pl was grown on JP1O80 (tyrR366aroL476 purE+) and used to transduce strainJP1065 (tyrR366 aroL+ purE). After purifica-tion, 100 Pur+ transductions were examined forshikimate kinase activity. Each was found tohave derepressed, wild-type (i.e., aroL+) levelsof activity, indicating that aroL476 and purE+are probably less than 1% cotransducible.Regulation of shikimate kinase activity

in the aroL mutant. The isolation of a mutantin which shikimate kinase II was inactive al-lowed the regulation of shikimate kinase I to bemore closely examined. The effect of eitherchanges in aromatic supplementation or muta-tion in tyrR on shikimate kinase levels was in-vestigated in strains JP1080 (tyrR aroL), JP1083(tyrR' aroL), and the corresponding aroL+ de-rivatives. Table 5 shows that, in aroL mutantcells, the level of shikimate kinase activity wasthe same under all the conditions examined.Neither the presence of aromatic supplementa-tion in the growth medium nor the absence offunctional tyrR gene product affected the levelof kinase activity. In contrast, the shikimatekinase levels of aroL+ cells differed dependingon whether tyrR + cells were grown with or with-out aromatic supplements and whether the tyrRgene product was functional. In a repressed cell,aroL specifies approximately 50% of total shiki-mate kinase activity. However, in a tyrR mutantcell aroL specifies more than 90% of total activ-ity.

Despite the fact that E. coli synthesizes apermnease for shikimic acid (6), it cannot utilizeshikimic acid as a carbon/energy source. In con-trast, organisms such as Klebsiella pneumoniaecan do so (30). Catabolite repression effectsmany enzymes serving a catabolic function; todetermine whether shikimate kinase I, in addi-tion to its biosynthetic role, also retains a cata-bolic function and was subject to cataboliterepression, shikimate kinase levels were meas-ured in extracts of cells grown with either glyc-erol or glucose as a carbon source. No differencewas detected in the level of either shikimatekinase I or shikimate kinase II (data not shown).Growth characteristics of aroL mutant

cells. The failure in the past to isolate a shiki-mate kinase mutant of E. coli is consistent withthe postulate that simultaneous mutations inseparate genes are required to completely blockthis reaction. Mutation in only a single gene,e.g., aroL, would alter the phenotype onlyslightly and would not be expected to yield aro-matic auxotrophs. That this is the case is sup-

ported by the data of Table 6. In minimal me-dium with or without aromatic supplementationand in both tyrR+ and tyrR cells, the aroL476mutation appeared to have no significant effecton growth rates. In the case of strain JP1080(tyrR aroL) growing in minimal medium, therewas a slight increase in doubling time comparedwith an isogenic aroL+ derivative, but this mayreflect the fact that in tyrR mutant cells a dis-proportionate fraction of the chorismate pool isdirected into the tyrosine terminal pathway dueto derepression of chorismate mutase T-prephe-nate dehydrogenase enzyme. Possibly, the ab-sence of shikimate kinase II activity preventsthe supply of chorismic acid from fully meetingthe cell's increased demand. Table 6 also showsthe doubling times of cells in enriched minimalmedia. In this enriched medium (medium17AA), aroL mutant strains grow significantlyslower than aroL + strains. When aromatic endproducts are also provided (medium 2OAA), botharoL and aroL+ strains grow at similar rapidrates suggesting that shikimate kinase activitylimits growth of aroL mutants in medium 17AA.Column chromatography of shikimate

kinase activity present in cell extracts ofan aroL mutant. Chromatography on DEAE-Sephadex gave similar results whether cell ex-tracts were of JP1O80 (tyrR aroL) or of its tyrR'transductant JP1083. The elution profile ob-tained from chromatography of an extract ofJPlO80, together with that of the correspondingaroL + strain JP1079, is presented in Fig. 5. Theelution profile of the extract of JP1O80 showedonly a single peak of kinase activity which elutedin the salt concentration range of 0.25 to 0.31 MNaCl and therefore corresponded to shikimatekinase I.Gel filtration chromatography. When ex-

tracts of either JPlO80 or JP1083 were chromat-ographed on Sephadex G-75 gel, a single peak ofenzyme activity was eluted at a position which

TABLE 6. Doubling times of cells which aremutated in the genes tyrR, or aroL, or both

Doubling time (min) of

Strain tyrR aroL cells growing in:allele alleleMMa MM + EP 17AA 20AA

JP1079 366 + 73 71 45 48JP1O80 366 476 85 70 80 50JP1082 + + 75 74 48 40JP1083 + 476 70 73 72 45

a MM, Minimal medium; MM + EP, minimal me-dium supplemented with the aromatic end products;17AA, minil medium supplemented with the non-aromatic amino acids and vitamins; 20AA, medium17AA supplemented with aromatic end products.

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SHIKIMATE KINASE IN E. COLI 941

E

CD

CD0

0

E

0CM

0

CDz0

CD0

Fraction No. cFIG. 5. Elution profiles of shikimate kinase activ-

ity from DEAE-Sephadex columns. (a) Extract ofJP1079 (aroL+); (b) extract ofJP1l8O (aroL476). Sym-bols: *, shikimate kinase activity; +, protein concen-tration.

indicated a molecular weight of approximately20,000 (Fig. 5b). The profile obtained was thesame as indicated in Fig. 3, in which case extractsof aroL+ strains had been chromatographed(data not shown).

DISCUSSIONResults presented in this paper demonstrate

that the level of shikimate kinase in E. coli K-12can be repressed and derepressed. Both tyrosineand tryptophan, but not phenylalanine, are ap-parently involved in this control. A mutation inthe regulator gene tyrR abolishes the repressionof shikimate kinase, whereas a mutation in trpRhas no effect. It appears therefore that the tyrRgene product, apo-tyrR, is capable of interactionwith both tyrosine and tryptophan to repressthe synthesis of shikimate kinase. Apo-tyrR hasfor some time been known to interact with bothphenylalanine and tyrosine (or related mole-cules) to regulate the expression of several un-linked genes which specify enzymes involved inaromatic biosynthesis (7). More recently, Whippand Pittard (34) have reported on the aromatictransport systems ofE. coli and the involvementof tyrR in their regulation.

Since either tyrosine or tryptophan can causealmost complete repression of shikimate kinase,this particular control circuit is one of neithermultivalent repression (9) nor cumulative

repression (27). In the simplest model, both ofthe putative complexes, apo-tyrR-tyrosine andapo-tyrR-tryptophan, function as active repres-sor molecules in regulating the synthesis of shi-kimate kinase. A similar, although more compli-cated, system has been described for the com-mon aromatic transport system of E. coli inwhich apo-tyrR apparently interacts with any ofthe three aromatic amino acids as part of a singlerepression control (34).

Ion-exchange chromatography of cell extractsresolves two closely eluting peaks of shikimatekinase activity which we have referred to asshikimate kinase I and shikimate kinase II. In afully repressed cell, shikimate kinase I is thepredominant shikimate kinase activity. How-ever, a mutation in tyrR or the partial starvationof an aromatic auxotroph for tyrosine and tryp-tophan appears to alter only the level of shiki-mate kinase II which now becomes the cell'smajor shikimate kinase activity. Since a muta-tion in the regulator gene tyrR apparently failsto alter the level of shikimate kinase I, it isunlikely that the two peaks of shikimate kinaseactivity represent different aggregational statesof the one enzyme. This is confirmed by theisolation of a mutant of E. coli in which thetyrR-regulated (shikirnate kinase II) activity isabsent, whereas shikimate kinase I remains un-altered.When cell extracts of either tyrR+ or tyrR

cells are chromatographed on a molecular siev-ing gel, only a single peak of shikimate kinaseactivity is resolved. The apparent molecularweight of this activity is 20,000 and compareswith values of 17,000 reported for one of thepeaks of shikixnate kinase activity detected bydensity gradient centrifugation of extracts of E.coli (5) and of 30,000 for the more stable of thetwo activities resolved in extracts of Salmonellatyphimurium (23). From the data presentedhere, however, the possibility cannot be ruledout that shikimate kinases I and II have differentmolecular weights but stabilities which allow thedetection of only one of them in eluates from G-75 or G-200 columns.By the technique of localized mutagenesis, a

mutant strain of E. coli has been isolated inwhich the repressible, tyrR-regulated shikimatekinase isoenzyme (shikimate kinase II) is non-functional. The mutant strain, strain JP1080,and its tyrR' derivative, strain JP1083, werefound to be aromatic prototrophs that grew wellon minimal medium whether or not it was sup-plemented with aromatic amino acids and vita-mins. The major phenotypic difference betweenthe mutant and its parent was the loss in themutant of approximately 90% of the total shiki-

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mate kinase activity.The structural gene for shikimate kinase II

has been termed aroL. By assuming aroL to befunctional in all the F-primes used in mappingexperiments, we place the aroL locus betweenthe loci ofproC andpurE (i.e., min 9 and 12) onthe E. coli chromosome. In an experiment inwhich finer mapping of aroL was attempted, itwas shown that aroL is probably less than 1%cotransducible with purE+. This suggests thataroL is actually located closer to the gene acrAthan to purE.

Ion-exchange chromatography of extracts ofaroL mutants resolved only a single, sharplydefined peak of shikimate kinase activity. Thispeak eluted at a salt concentration which iden-tified it as the unregulated, shikimate kinase Ienzyme. When extracts of an aroL strain werechromatographed on a molecular sieving gel,only a single peak of activity, with apparentmolecular weight 20,000, was resolved.

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SHIKIMATE KINASE IN E. COLI 943

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