JOURNAL OF BACTERIOLOGY, Jan. 1970, p. 209-217Copyright © 1970 American Society for Microbiology

Vol. 101, No. 1Printed In U.S.A.

Mechanism of 3-Methylanthranilic Acid Derepressionof the Tryptophan Operon in Escherichia coli1

WILLIAM A. HELD AND OLIVER H. SMITH

Department oJ Biology, Marquette University, Milwaukee, Wisconsin 53233

Received for publication 11 October 1969

3-Methylanthranilic acid (3MA) inhibits growth and causes derepression of thetryptophan biosynthetic enzymes in wild-type strains of Escherichia coli. Previousreports attributed this effect to an inhibition of the conversion of 1-(o-carboxyphen-ylamino)-1-deoxyribulose 5-phosphate to indole-3-glycerol phosphate and a conse-quent reduction in the concentration of endogenous tryptophan. Our studies haveshown that 3MA-resistant mutants linked to the tryptophan operon have a feed-back-resistant anthranilate synthetase; mutants with an altered indole-3-glycerolphosphate synthetase were not found. 3MA or 7-methylindole can be metabolizedto 7-methyltryptophan, and 3MA, 7-methylindole, and 7-methyltryptophan leadto derepression of the tryptophan operon. Furthermore, 3MA-resistant mutantsare also resistant to 7-methylindole derepression. These results strongly suggestthat the primary cause of derepression by 3MA is through its conversion to 7-meth-yltryptophan, which can inhibit anthranilate synthetase, thereby decreasing theconcentration of endogenous tryptophan. Unlike 5- or 6-methyltryptophan, 7-meth-yltryptophan does not appear to function as an active corepressor.

Regulation of tryptophan biosynthesis inEscherichia coli occurs through the combinedeffects of repression of enzyme synthesis andfeedback inhibition of the branch-point enzyme.At least one regulator gene concerned withrepression has been mapped and is situated somedistance from the tryptophan operon nearthreonine on the E. coli chromosome (2). Unlikesome other biosynthetic operons (15, 16),tryptophanyl-transfer ribonucleic acid (tRNA)does not appear to be involved in repression(4). Regulation of tryptophan biosynthesis alsooccurs through feedback inhibition of *be producttryptophan, on the first enzyme in the biosyn-thetic sequence, anthranilate synthetase (1, 14).Derepression can occur whenever the level ofendogenous tryptophan becomes limiting (21).Analogues of tryptophan may be capable ofcausing derepression by limiting tryptophanproduction through feedback inhibition ofanthranilate synthetase.

3-Methylanthranilic acid (3MA), inhibitsgrowth and causes derepression of the trypto-phan biosynthetic enzymes in wild-type strains

I Taken in part from a thesis submitted to Marquette Univer-sity by William A. Held in partial fulfillment of the requirementsfor the Ph.D. degree. A preliminary report of part of this paperwas presented at the 69th Annual Meeting of the American Societyfor Microbiology, Miami Beach, Fla., 1969.

of E. coli. Previous reports attributed this effectto an inhibition of the conversion of 1-(o-carboxyphenylamino)-l-deoxyribulose 5-phos-phate to indole-3-glycerol phosphate and a con-sequent reduction in the concentration ofendogenous tryptophan (12). However, anthrani-late synthetase feedback-resistant mutants areknown to be resistant to the effects of this ana-logue (18). In this report, we present evidencethat 3MA is converted to 7-methyltryptophan(7MT), which can inhibit anthranilate synthe-tase, thereby decreasing the concentration of en-dogenous tryptophan and causing derepression.Unlike 5-methyltryptophan (5MT) or 6-methyl-tryptophan (6MT), 7-MT does not appear tofunction as an active corepressor.

MATERIALS AND METHODSOrganisms. Wild-type strains of E. coli K-12,

W1485, and W3110 were used in these studies. Mostof the mutants described are derived from W1485tna (a mutant lacking tryptophanase isolated in thislaboratory) after ultraviolet (UV) or nitrosoguani-dine treatment and plating on appropriate selectivemedia. Derepressed mutants (trpR) and mutantswith a feedback-resistant anthranilate synthetase(trpEFBR) were isolated by plating on minimal agarcontaining 5 X 10-4 M 5MT. 3-Methylanthranilate-resistant mutants (MAR) were isolated by plating onminimal agar containing 6.7 X 10-4 M 3MA. The

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large resistant colonies were picked, purified bysingle colony isolation, and further characterized byassaying whole cells for elevated levels of tryptophansynthetase (7). Of the 5MT-resistant mutants, trpRmutants produced elevated levels of tryptophansynthetase in the presence of repressing concentrationsof tryptophan (2.5 X 10-4 M). The trpEFBR mutantshad repressed enzyme levels and were identified bypreparing cell-free extracts and assaying anthranilatesynthetase in the presence of L-tryptophan. MARmutants did not produce elevated levels of tryptophansynthetase in the presence of 0.2 umole of 3MA/ml.These tentative conclusions were confirmed by pre-paring cell-free extracts of the mutants grown underappropriate conditions and assaying the tryptophanbiosynthetic enzymes.

Transduction procedures. The trpR, trpEIR, andMAR mutants were characterized genetically asbeing linked, or unlinked, to the tryptophan operonby transduction. Plkc lysates of the mutants wereprepared by the confluent lysis technique (11). Theselysates were used to transduce a cysB trp(A-E dele-tion) or other suitable recipient, selecting for cys+trp+ recombinants. Control lysates prepared fromwild-type strains and controls for sterility weredone routinely. The recombinants were tested todetermine whether they had concurrently receivedeither 5MT resistance or 3MA resistance. Resistanceto 5MT was determined by picking the colonies,diluting in saline, and spotting on 5MT plates.Growth within 24 hr was used as a criterion for 5MTresistance. To determine resistance to 3MA-inducedderepression, recombinants were assayed for trypto-phan synthetase by the whole-cell assay (7) aftergrowth in minimal medium containing 0.2,mole of3MA/ml. Cell-free extracts were prepared from afew of the recombinants, in each case, and the resultsof the whole-cell assay were verified by enzymeassays.

Isolation of MAR mutants linked to the trp region(trp MAR). A procedure similar to that used bySomerville and Yanofsky (18) to isolate 5MT-resistant mutants linked to the trp region was used toisolate trp-linked 3MA-resistant mutants. After UVtreatment, cells were inoculated into minimal mediumcontaining 0.2,mole of 3MA/ml and were grownovernight; 0.1 ml of this culture was used to inoculatea second tube containing 0.2,umole of 3MA/ml andagain grown overnight. Since 3MA inhibits growth,this procedure enriched for mutants which werenot inhibited by 3MA. A Plkc lysate of the secondculture was prepared and used to transduce a cysBtrp(A-E deletion), selecting for cys+ trp+ recombinantswhich were also resistant to 6.7 X 10-4 M 3MA.

Culture conditions. All strains were grown in theminimal medium of Vogel and Bonner (19) containing0.5% glucose and other supplements as desired. Thecultures were agitated on a rotary shaker at 37 C.Growth curves were obtained by growing 50-mlcultures in a 500-ml side arm flask and estimatinggrowth at various times by turbidity at 660 nm.One hundred Klett units correspond to a platecount of 7.2 X 108 cells/ml. The kinetics of enzymeappearance was measured in the following manner.

The prototrophic strain was grown overnight inminimal medium and harvested in the mid to latelog phase of growth. The cells were washed once withice-cold minimal medium and resuspended in pre-warmed minimal medium containing glucose and thedesired concentration of inhibitor (e.g., 7-methyl-indole). Samples of the cell culture were removed atappropriate times and poured over crushed ice. Cell-free extracts were then prepared and the enzymeactivity was determined.

Preparation of cell-free extracts. Cells were har-vested by centrifugation, washed once in saline, andresuspended in 0.1 M K2HPO4-HCl, pH 7.0, contain-ing 103 M ethylenediaminetetraacetate and 10-3 M 2-mercaptoethanol. The cells were broken by sonic os-cillation, and cell debris was removed by centrifuga-tion at 30,000 X g for 30 min.

Estimtion of 3-methylanthranilate and 7-methyl-indole in culture supernatant fluids. The amount of3MA in culture supernatant fluids was estimatedby acidifying 1.0 ml of the culture supernatant with0.1 ml of 1 N HCl and extracting in 5.0 ml of ethylacetate. The concentration of 3MA in the ethylacetate was determined spectrophotometrically at anabsorption maximum of 336 nm. In some cases, theconcentration of 3MA was determined fluorome-trically with 3MA as a standard. The concentrationof 7-methylindole was determined colorimetricallyby use of indole reagent, after basic extraction intotoluene (17). 7-Methylindole was used as a standard.Indoleglycerol was estimated spectrophotometricallyafter periodate oxidation to the aldehyde (17).Enzyme assays. The a subunit of tryptophan syn-

thetase (A protein) was assayed according to Smithand Yanofsky (17). Indole-3-glycerol phosphatesynthetase was assayed according to the method ofSmith and Yanofsky (17), except that incubationwas for 15 min at 37 C in 10.2 M tris(hydroxymethyl)-aminomethane (Tris), pH 7.8. Anthranilate synthe-tase activity was measured by following the increasein fluorescence at 390 nm (excitation 320 nm, un-corrected) with a Farrand spectrofluorometer. Thereaction mixture contained, in a total volume of 3.0ml, Tris, pH 7.5, 50,moles; MgCl2, 7.5 ,umoles;L-glutamine, 15 jumoles; chorismic acid, 0.5 pmole;enzyme extract; and distilled water. The reactionwas initiated by addition of enzyme to the reactionmixture prewarmed to 37 C. Chorismic acid wasprepared as the free acid according to the procedureof Gibson and Gibson (6). One enzyme unit isdefined as the production or consumption of 1 Amoleof product or substrate in 1 min at 37 C. Specificactivity is given as units of enzyme per milligram ofprotein. Protein was deternined by the method ofLowry et al. (13). In most cases, the enzyme levels areexpressed relative to those found in wild-type W1485tna, with a value of 1.0 assigned to the level of en-zymes found in W1485 tna grown in minimal me-dium.

Chemicals. 3-MA and 7-methylindole were ob-tained from Aldrich Chemical Co., Inc. Milwaukee,Wis. 'H-3MA was prepared by tritium exchange ofcrystallized 3MA (New England Nuclear Corp.,Boston, Mass.). The material obtained from New

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England Nuclear Corp. was heavily contaminatedwith radioactivity not associated with 3MA. Furtherexchange, charcoal treatment, and repeated crystal-lization did not remove all of the contaminatingradioactivity. However, descending paper chromatog-raphy in n-butyl alcohol-propanol-water (1:2:1)plus 1.0 ml of 1 N NH40H per 100 ml of solventwas capable of separating the contaminating radio-activity from the radioactive 3MA. After being chro-matographed twice, the resulting preparation con-tained no radioactivity which migrated at the solventfront, but a small amount of radioactivity migratedjust behind 3MA. This latter material constitutedfrom 5 to 10% ofthe total radioactivity and apparentlywas a breakdown product of3MA because the amountincreased with the age of the preparation. All studieswith 3H-3MA were with twice chromatographedmaterial. The specific radioactivity was 6.9 X 106counts per min per jAmole.7-MT was prepared from 7-methylindole by use of

whole cells of the tryptophan auxotroph trpC2. Themutant was grown overnight in limiting tryptophan(so that the tryptophan enzymes were derepressed).The cells were harvested by centrifugation, washed inminimal medium, and resuspended at a density ofapproximately 1010 cells/ml in an incubation mixturecontaining 1 ,Lmole of 7-methylindole/ml, 3.8 ,umolesof DL-serine/ml, 0.2,umole of pyridoxal phosphate/ml,minimal medium, and 0.2% glucose. The cells wereincubated for 2 hr at 37 C, by which time all of the7-methylindole had been converted to 7MT. Thesupernatant solution was boiled for 30 min and thencentrifuged at 20,000 X g for 30 min to remove

protein from lysed cells. The material was spottedalong the entire length of Whatman no. 1 paper andchromatographed in a solvent system consisting ofn-butyl alcohol-acetic acid-water (25:4:10). Stripscontaining the 7MT were cut out and eluted withdistilled water. The resulting 7MT migrates at anR, similar to that of 5MT or 6MT (RF, 0.56) butdifferent from that of L-tryptophan (RF , 0.50).L-Tryptophan could not be detected in the 7MTpreparation, although a small amount of materialwhich migrated at the same RF as serine was present.All other chemicals were obtained commercially.

RESULTS3-MA inhibited growth and, as shown in

Table 1, caused derepression in various wild-type and trpR (constitutive) strains of E. coli.At the growth-inhibiting concentrations of 3MAused, derepression was usually not coordinate,and the maximal level of enzyme formed wassomewhat below the derepressed level achievedby starving tryptophan auxotrophs for trypto-phan (about a 40-fold inciease in enzyme level).However, feedback-resistant anthranilate syn-thetase mutants (trpEFBR), 3MA-resistant mu-tants (MAR), or double mutants, both trpEFBRand trpR, which are resistant to this analogue,were not derepressed for either enzyme.

Previous reports attributed 3MA derepressionto an inhibition of indoleglycerol phosphatesynthetase (12). To test this hypothesis directly,

TABLE 1. Derepression of anthranilate synthetase and tryptophan synthetase A proteinby 3-methylanthranilate (3MA) and 7-methylindole (7MIn)

Relative specific activity'

Strain Anthranilate synthetase A protein

Minimal 3MAb 7MInb Minimal 3MA 7MInmedium medium

W1485 tna .............. 1.0 19.4 20.1 1.0 11.8 -cW1485.- 13.6 8.9 -W3110...... 13.1 - 7.0 -trpR39................. 4.4 37.8 16.5 4.4 24.6 8.8trpR4................. 3.4 28.6 3.1 18.2trpR trpEFBR44.......... 3.2 3.5 3.7 4.3 3.3trpR trpEFBR46.......... 2.0 4.1 3.8 5.7trpEFBR2O5 0............. 0.44 0.56 0.61 0.61 0.81trpEFBR142 ..............- 0.87 1.62 0.96MAR 13................ 0.37 0.87 2.25 0.42 0.72 2.2MAR1-9.-3.4.--.-.-MAR31. ..50 -MAR22.. 3.2 -

aThe enzyme levels are expressed relative to those found in W1485 tna grown in minimal media. Arelative specific activity of 1.0 corresponds to a specific activity of 0.0027 units/mg of anthranilate syn-thetase and 0.0152 units/mg of the a subunit of tryptophan synthetase (A protein).

b The concentration of 3MA or 7MIn in the growth media was 0.2 ,mole/ml.c Dashes indicate that the enzyme activity was not determined.

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the indoleglycerol phosphate synthetase in ex-tracts of mutants which were selected for re-sistance to 3MA (MAR), as well as those iso-lated as being resistant to 5MT (trpR39 and thedouble mutant trpR trpEFBR44), were tested forinhibition by 3MA. Table 2 shows that, althoughtrpEBFR and MAR mutants failed to derepresson 3MA, their indoleglycerol phosphate syn-thetase was inhibited by the analogue to thesame extent as trpR39, which does derepress.The genetic locus conferring feedback resist-

ance is located in the trpE gene, the structuralgene for anthranilate synthetase (18). That theseFBR mutants are also located within the trpoperon (presumably in the trpE region) is indi-cated in Table 3 by their linkage to the cysBgene. As has been shown previously, the trpRgene is unlinked to the trp operon. In the doublemutant trpR trpEFBR, the recombinants werenot constitutive, although they did have a feed-back-resistant anthranilate synthetase, indicatingthat the mutant was a double mutant.

Since trpEFBR mutants locate within the trpoperon, as would also be expected for mutantswhich become resistant to 3MA through analteration in indoleglycerol phosphate synthe-tase, a preliminary test of the linkage of MARmutants to trp was performed. Several mutantsselected for 3MA resistance were tested in thismanner and, as shown by the representativedata in Table 4, were found not to be trp-linked.We conclude that MAR 13 has a wild-type trpoperon and have evidence that the mechanismof its resistance to 3MA is only indirectly related

TABLE 2. Inhibition of indoleglycerol phosphatesynthetase by 3-methylanthranilate (3MA)

Strain" 3MA concn Inhibition

M %

trpR39 5 X 10- 60.01 X 10-3 76.2

trpR trpEFBR44 5 X 104 59.91 X 10- 71.6

MAR 13 trpA 5 X 10-4 68.41 X 1O-3 81.7

MAR 22 trpB 5 X 1O-4 72.71 X 10-3 84.5

a Strain trpR39 derepresses when grown in thepresence of 3MA, the other strains do not. MAR13 trpA and MAR 22 trpB are double mutantscontaining mutations in the trpA and trpB cistronsof the tryptophan operon. The double mutantswere used to increase the level of indoleglycerolphosphate synthetase in crude extracts.

TABLE 3. Linkage to cysB ofloci governtingresistance to S-methyltryptophan (5 MT)

Doncr ~~~~~No.of cys+ Percent-recombinants ageDonc r Recipient resistant cotrans-tMG ducedto5 with cysB

W1485 tna his cysB 0trpR39 his cysB 0trpA trpEFBR44 his cysB 68 53.1trpEFBR1-42 his cysB 63 49.2

a The number of cys+ recombinants tested was128.

TABLE 4. Nonlinkage to trp operon ofMAR 13

No. ofa No.wild-typea dere-Donor Recipient recomi- pressedbinants o Mbtested o M

W1485 MAR 13 trpA 27 0W1485 trpA2 7 7MAR 13 cysBt trpdel 34 34

MAR 13 trpA re- 7 0vertants

a Wild type are tryptophan prototrophs.b Wild-type recombinants were tested for dere-

pression in the presence of 3MA by the whole-cellassay as described in Materials and Methods.

to tryptophan metabolism (see succeedingpaper).To determine whether it was possible to iso-

late 3MA-resistant mutants linked to the trypto-phan operon, cells were selected for 3MA re-sistance and then transduced into a trp deletion(see Materials and Methods). Thus, the onlymutants which should grow on 3MA would bethose recombinants which received the trpgenes along with a mutation conferring 3MAresistance. Of 76 such mutants isolated, all werealso 5MT-resistant, and, of 11 tested, all had afeedback-resistant anthranilate synthetase, sug-gesting that anthranilate synthetase is moredirectly involved in 3MA derepression thanindoleglycerol phosphate synthetase. Attemptswere made to isolate 3MA-resistant mutants fromrevertants of the tryptophan auxotroph trpC2,which has an altered indoleglycerol phosphatesynthetase; however, none was found. Thus,there appear to be two classes of 3MA-resistantmutants, one of which is linked by transductionto the trp operon. These mutants are 5MT-resistant and have a feedback-resistant anthrani-late synthetase. The other class is unlinked bytransduction to the trp operon and may con-

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VOL. 101, 1970 MECHANISM OF 3-METHYLANTHRANILIC ACID DEREPRESSION

stitute a heterogeneous class of mutants. Thus,it appears unlikely that 3MA derepression iscaused solely by inhibition of indoleglycerolphosphate synthetase.

Previous hypotheses to explain the 3MA re-sistance of trpEFBR mutants postulated that theintracellular supply of tryptophan is high enoughto antagonize the action of the analogue (18).However, neither trpEFBR nor trpR mutantsappear to excrete significant quantities of trypto-phan, although double mutants which are bothtrpEFBR and trpR do excrete large amounts oftryptophan (Table 5).That the mechanism of 3MA-mediated growth

inhibition and derepression of the trp enzymesinvolves tryptophan limitation is indicated bythe ready reversal of these effects by exogenouslysupplied tryptophan. To examine whether thereversal was specific for tryptophan, a numberof intermediates in tryptophan biosynthesis weretested. Anthranilic acid, at low concentrations,appears to be as effective as indole in reversing3MA derepression (Table 6). If 3MA inhibits areaction in the tryptophan biosynthetic sequenceafter anthranilic acid (i.e., indoleglycerol phos-phate synthetase), one would expect anthranilicacid to be ineffective in reversing the inhibition,as it inhibits the same reaction (5).Although it has been previously reported that

3MA is not metabolized to any extent by E.coli (12), in several experiments it was noticedthat the fluorescence due to 3MA disappearedfrom the culture after growth of wild-type andtrpR strains of E. coli. Table 7 shows thatvarious wild-type and trpR strains, but nottrpEFBR or MAR strains, appear to metabolize3MA. Kinetic experiments with trpR39 indicatedrapid disappearance of 3MA from the cultureafter an initial lag period (Fig. 1). Concomitantwith the loss of 3MA from the culture super-

TABLE 5. L-Tryptophan accumulation in culturesupernatant fluids

Amt ofL-trypto han

Strain (nmoles)ml ofculture

supernatant fluida

W1485 tna 2.3

trpEFBR2S50....................... 5.9trpR39...........5 .2

trpR trpEFBR44 ...................... 39.0

The concentration of L-tryptophan was esti-mated fluorometrically in culture supernatantfluids, with L-tryptophan as a standard. Culturesupernatant fluids were boiled for 30 min andcentrifuged to remove any protein from lysedcells before assaying for tryptophan.

TABLE 6. Effect of some aromatic compounds on3MA derepression of anthranilate synthetase

in W1485 tna

Concn ofaromatic

Growth Additions corn- Relativeconditions pounds activitya

("moles/MI)

Minimal me- None 1.0dium Indole .10 0.55

Anthranilic .34 3.8acid

Minimal me- None - 17.0dium + 0.2 L-Trypto- .05 1.8Mmole of 3- phan 1.8MA/ml Indole .017 13.3

Indole .034 7.1Indole .068 2.5Indole .134 0.30Anthranilic .014 6.7

acidAnthranilic .029 2.3

acidAnthranilic .058 2.6

acidAnthranilic .116 3.3

acid

aThe anthranilate synthetase levels are re-pressed relative to that of W1485 tna grown inminimal medium.

TABLE 7. 3MA disappearance from culturesupernatant fluids

3MA remainingStrain after 18 hr

of growtha

IAmOleS/mlNone.33W1485tna. .11W1485.073Y mel..21trpR39...

HELD AND SMITH

Eg200 4.0

4I ~~~~~~~~3.0**

E Eo 100 K2.0 0c c0 aC C

mnG1.0

2 3 4 5 6

TIME (HRS)FiG. 1. Kinetics of 3MA disappearance by trpR39

Strain trpR39 was grown overnight in minimal medium.The cells were resuspended at a density of 2 X 108cells/mI in minimal medium containing 0.325 jAmole of3MA/ml and 0.S% glucose. 3MA concentration in theculture supernatant was estimated spectrophotometri-cally as described in Materials and Methods. InG=indoleglycerol.

TABLE 8. Conversion of3MA to 7-methylindole(7Mln) by trpR39 in the presence

of hydroxylaminea

3MA 7MI Indole formedime disappeared formed (no 3MA)

mi pmolcs/ml gmoles/mi pmolcs/ml

30 .034 .035 .00060 .053 .05590 .063 .069 .013120 .072 .076180 .085 .088 .013

The incubation mixture consisted of a minimalmedium containing 0.3 ,smole of 3MA/ml, 0.2%glucose, and 8.7 jumoles of hydroxylamine/mi. At30 and 90 min, an additional 8.7 jsmoles of hy-droxylamine/ml was added.

To establish whether 3MA was indeed me-tabolized, possibly all the way to the correspond-ing tryptophan derivative, 7-MT, several addi-tional experiments were performed. A trpR strainwas grown overnight in the presence of 3MA sothat the tryptophan enzymes were derepressed.The cells were washed and incubated with 3MAin the presence of hydroxylamine [hydroxylamineinhibits tryptophan synthetase and causes ac-cumulation of indole (20)]. If 3MA was beingmetabolized via the tryptophan pathway, itshould accumulate as the indole derivative,7-methylindole. Table 8 shows that the 3MAwas quantitatively converted to the correspond-

ing indole derivative, whereas cells incubatedwithout 3MA accumulated very little indole.The indole derivative was extracted from theculture supernatant fluid by basic extraction intoether, crystallized, and shown to have the samemelting range (79 to 81 C) as commerciallyavailable 7-methylindole.With the demonstration that 3MA was con-

verted to 7-methylindole, it became necessary todetermine whether 3MA is converted to 7MT bywhole cells of E. coli. W1485 tna was grownovernight on 0.267 ,mole of 3MA/ml so thatthe tryptophan enzymes were derepressed. Afterthe cells were harvested and washed, they weresuspended in minimal medium containing 0.2;umole of 'H-3MA/ml (see Materials and Meth-ods), 0.95 ,umole of DL-serine/ml, and 0.4%glucose, and were incubated for 2 hr at 37 C.The culture supernatant fluid was co-chromato-

f graphed with partially purified 7MT made frommethylindole (see Materials and Methods) inn-butylalcohol-acetic acid-water (25:4:10). Thepaper was sprayed with ninhydrin, and then wascut into 1.5-cm squares and counted. Figure 2shows that 'H-3MA is converted to 3H-7MT.Chromatography in n-butyl alcohol-propanol-water (1:2:1) plus 1.0 ml of 1 N NH40H per100 ml of solvent gave essentially the same re-sults, although the peaks were not as well sepa-rated.7-MT does no appear to be incorporated into

3MA

10-'

0L8

6

CY0

4

0 12 24 36

Distance from Origin (cm)FiG. 2. Conversion of 3H-3MA to 3H-7MT by

W1485 tna. Dashed line indicates radioactivity of'H-3MA, sample taken before 2-hr incubation. Solidline indicates radioactivity after 2-hr incubation. Theposition of3MA was determinedby its bluefluorescence;7MT, by ninhydrin staining.

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protein. Table 9 indicates that most of the radio-activity after 12 hr of growth of trpR39 on IH-3MA remained in the supernatant fluid. Less than0.018% of the original counts were precipitatedwith trichloroacetic acid, and more than 85%of the 'H-3MA had been metabolized.

Since 3MA can be converted to 7MT by E.coli, the question whether 3MA or 7MT causesderepression needed to be resolved. If 7MTcauses derepression, then 7-methylindole, whichcan be converted to 7MT, should also causederepression in wild-type strains. Table 1 showsthat 7-methylindole does effectively cause de-repression in wild-type and trpR strains of E.coli. Also, it is evident that trpEFBR and MARmutants are relatively unaffected by 7-methyl-indole. The kinetics of enzyme appearance whenlow concentrations of 7-methylindole (0.02Mmole/ml) were used indicate that derepressioncontinued for a period of time and then the cul-ture began to be repressed (Fig. 3). In thisexperiment, samples of the culture were tested atvarious times for the presence of 7-methylindole.After 1.5 hr, 7-methylindole was not detectablein the culture supernatant fluid, although de-repression continued for another 1.5 hr, indicat-ing that 7-methylindole probably does not directlycause derepression.

Since both 3MA and 7-methylindole are con-verted to 7MT, it appeared probable that 3MAand 7-methylindole cause derepression throughtheir conversion to 7MT. This could be testeddirectly by using 7MT prepared from 7-methyl-indole (see Materials and Methods). The fol-lowing experiment was designed to determine therelative effectiveness of 3MA, 7-methylindole, and7MT in causing derepression and growth in-hibition in a wild-type strain. W1485 tna wasgrown overnight in minimal medium; the cellswere harvested, washed in cold minimal medium,and resuspended in each of three flasks containingequal molar concentrations of 3MA, 7-methyl-indole, and 7MT. The initial concentration ofinhibitor was 0.01 ,umole/ml. At 1-hr intervals,growth was determined and an additional 0.01,umole of inhibitor/ml was added to maintain asource of inhibitor. 3MA only slightly inhibitedgrowth at this low concentration of inhibitor,although it did cause some derepression (Fig. 4).Both 7-methylindole and 7MT inhibited growthvery strongly and caused derepression to asimilar extent.As shown in Table 10, 7MT inhibited anthrani-

late synthetase and this inhibition appears to becompetitive with chorismic acid. Previousreports have shown that L-tryptophan inhibitionof anthranilate synthetase is competitive withchorismic acid (1). Although 7MT is not as

TABLE 9. Recovery of radioactivity from a cultureof trpR39 grown in the presence of 3H-3MAa

3MA TotalSample &.moles/ counts/minMl)

Supernatant fluid (0 hr) .224 6.68 X 106Supernatant fluid (12 hr). .033 5.52 X 106Crude extract (cold, tri-

chloroacetic acid -..._ 1.18 X 103Crude extract (hot tri-

chloracetic acid. 9.21 X 102

a Strain trpR was grown for 12 hr in 50 ml ofculture medium containing 0.224 1Amole of 3H-3MA/ml.

b The total counts per minute represents thetotal radioactivity in the supernatant fluid (50ml) or crude extract (3 ml).

>, 4

3

2

4-0

300

4-._2

2504-

%-

200

m0

150

1000 2 3 4

TIME (HRS)FIG. 3. Kinetics of derepression of W1485 tna on

0.02 jinole of 7-methylindole/ml. Enzyme activity isexpressed relative to the level of enzyme in W1485tna grown in minimal medium. ASase = anthrarilatesynthetase.

effective a feedback inhibitor of anthranilatesynthetase as L-tryptophan, it is more effectivethan 5MT or 6MT.

DISCUSSIONThe results of these experiments indicate: (i)

that 3MA is metabolized to 7MT in wild-typestrains of E. coli; (ii) that 3MA-resistant mutantslinked to the tryptophan operon have a feedback-resistant anthranilate synthetase and that mu-tants with an altered indoleglycerol phosphatesynthetase do not occur; and (iii) that 3MA,7-methylindole, and 7MT lead to derepression

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300

0)14n.t 250c

4-., 25004-0 1- 200

ft.o

0

100tI I I

0 2 4 6

TIME (HRS)FIG. 4. Derepression and growth inhibition by 3-methylanthranilic acid (3MA), 7-methylindole (7MIn), and

7-methyltryptophan (7MT) in W1485 tna. Cultures were harvested at the end of the growth period and cell-freeextracts were prepared. Enzyme activities are relative to the level of enzymes in W1485 tna grown in minimalmedium.

TABLE 10. Inhibition of anthranilate synthetaseaby tryptophan analogues

Inhibitor

L-Tryptophan

5-Methyl-tryptophan

6-Methyl-tryptophan

7-Methyl-tryptophan

Inhibitorconcn

M

1.7 X 10-63.4 X 10-63.4 X 10-1.7 X 104

3.4 X 10-61.7 X 10-4

1.7 X 10-4

1.7 X 1053.4 X 10-53.4 X 10-61.7 X 10-4

Chorismateconcn

M

1.7 X 10-61.7 X 10-61.7 X 10-'3.4 X 10-6

1.7 X 10-3.4 X 10-

3.4 X 10-

1.7 X 10-1.7 X 1061.7 X 10-43.4 X 105

a Partially purified anthranilate synthetase fromtrpA2/F'A2 was used as a source of the enzyme.

of the trp operon. These results suggest stronglythat the primary cause of derepression by 3MAis through its conversion to 7MT. Formation ofrelatively small amounts of 7MT inhibits the

initially low concentration of anthranilate syn-thetase present, reducing the concentration ofendogenous L-tryptophan. This leads to dere-pression of the tryptophan operon, the formationof more 7MT, and further inhibition of anthran-ilate synthetase, etc. 7MT, unlike 5MT or 6MT,apparently does not function as an active core-pressor so that inhibition of anthranilate syn-thetase can lead to derepression. Mutants with afeedback-resistant anthranilate synthetase are notinhibited by 7MT, do not derepress, and, there-fore, convert very little of the 3MA or 7-methyl-indole to 7MT.3MA inhibition of indoleglycerol phosphate

synthetase may contribute somewhat to 3MAderepression but is not the primary cause. An-thranilic acid causes a low-level derepression inwild-type strains, and 3MA would be expectedto cause a similar low-level derepression exceptfor its conversion to 7MT. Recent reports havealso indicated that anthranilic acid causes prod-uct inhibition of anthranilate synthetase fromSalmonella typhimurium (3). However, sinceanthranilic acid reverses 3MA derepression,product inhibition of anthranilate synthetase by3MA is probably not relevant to 3MA derepres-sion.

216 J. BACTERIOL.

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VOL. 101, 1970 MECHANISM OF 3-METHYLANTHRANILIC ACID DEREPRESSION

Anthranilate synthetase and phospho-ribosyl(PR) transferase activities normally exist as anenzyme complex in E. coli (8), and both activitiesare inhibited by L-tryptophan. Recent investiga-tions on the nature of this enzyme complex indi-cate that anthranilate synthetase and PR trans-ferase probably have separate tryptophan bind-ing sites, as tryptophan inhibition of PR trans-ferase is not related to feedback sensitivity ofanthranilate synthetase (9). Furthermore, tryp-tophan inhibition of PR transferase appears toreach a limiting value at about 60% inhibitionat 10-4 M L-tryptophan. Anthranilate synthetase,however, is more sensitive to tryptophan inhibi-tion and is completely inhibited (at low chorismicacid concentrations) at about 3 X 105 M. Thisdifferential sensitivity presumably allows metabo-lism of 3MA under conditions where anthranilatesynthetase may be strongly inhibited (i.e., in thepresence of 7MT).

Kinetic experiments indicate that 7-methyl-indole derepression ceases rather abruptly,followed by a period of relative repression andthen slight derepression again (see Fig. 3).Derepression may allow the cells to overcomethe inhibitory effect of 7MT and to make L-tryptophan even though 7MT is present. Synthe-sis of the tryptophan enzymes may "over-shoot"the critical level of enzymes required to over-come the inhibition by 7MT, resulting in tem-porary repression until the new "equilibriumstate" is established.Although 7MT does not appear to be incor-

porated into protein to any extent, studies werenot carried out to determine whether 7MTis charged to the tryptophanyl-tRNA. Previousinvestigators have shown that mutations affectingtryptophanyl-tRNA synthetase probably donot have a direct effect on regulation (4), al-though the analogue 5MT, long known-to causerepression of the trp operon, has been shown tobe incorporated at a low level into proteins ofE. coli. Presumably this incorporation proceedsthrough a charged tRNA intermediate (10).7-MT appears to be unique among tryptophan

analogues in causing derepression. Other trypto-phan analogues such as 4-, 5-, or 6-MT and 6-fluorotryptophan cause repression (4). Presum-ably, either 7MT does not bind or binds veryweakly to the tryptophan operon repressor, or7MT may bind to the repressor but the complexis relatively inactive in causing repression.

ACKNOWLEDGMENTSThis investigation was supported by National Science Founda-

tion grant GB-6807. One of us (W. A. H.) was supported by Pub-

lic Health Service Predoctoral Training grant STOI HD 00027-07.

LITERATURE CITED

1. Baker, T. I., and I. P. Crawford. 1966. Anthranilate synthe-tase. Partial purification and some kinetic studies on theenzyme from Escherichia coli. J. Biol. Chem. 241:5577-5584.

2. Cohen, G. N., and F. Jacob. 1959. Sur la repression de la syn-thesis des enzymes intervenant dans la formation du tryp-tophane chez Escherichia coli. Compt. Rend. 248:3490-3492.

3. Cordaro, J., H. Levy, and E. Balbinder. 1968. Product inhibi-tion of anthranilate synthetase in Salmonella typhimurium.Biochem. Biophys. Res. Commun. 33:183-189.

4. Doolittle, W. F., and C. Yanofsky. 1968. Mutants of Esch-erichia coli with an altered tryptophanyl-transfer ribonu-cleic acid synthetase. J. Bacteriol. 95:1283-1294.

5. Gibson, F., and C. Yanofsky. 1960. The partial purificationand properties of indole-3-glycerol phosphate synthetasefrom Escherichia coli. Biochim. Biophys. Acta 43:489-500.

6. Gibson, M. K., and F. Gibson. 1964. Preliminary studies onthe isolation and metabolism ofan intermediate in aromaticbiosynthesis: chorismic acid. Biochem. J. 90:248-256.

7. Ito, J., and I. P. Crawford. 1965. Regulation of the enzymesof the tryptophan pathway in Escherichia coli. Genetics52:1303-1316.

8. Ito, J., and C. Yanofsky. 1966. The nature of the anthranilicacid synthetase complex of Escherichia coli. J. Biol. Chem.241:4112-4114.

9. Ito, J., and C. Yanofsky. 1969. Anthranilate synthetase, anenzyme specified by the tryptophan operon of Escherichiacoli: comparative studies on the complex and the subunits.J. Bacteriol. 97:734-742.

10. Lark, K. G. 1969. Incorporaion of 5-methyltryptophan intothe protein of Escherichia coli 15T - (555-7). J. Bacteriol.97:980-982.

11. Lennox, E. S. 1955. Transduction of linked genetic charactersof the host by bacteriophage P1. Virology 1:190-206.

12. Lester, G., and C. Yanofsky. 1961. Influence of 3-methyl-anthranilic and anthranilic acids on the formation of tryp-tophan synthetase in Escherichia coli. J. Bacteriol. 81:81-90.

13. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Ran-dall. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275.

14. Moyed, H. S. 1960. False feedback inhibition: inhibition oftryptophan biosynthesis by 5-methyltryptophan. J. Biol.Chem. 235:1098-1102.

15. Neidhardt, F. C. 1966. Roles of amino acid activating enzymesin cellular physiology. Bacteriol. Rev. 30:701-719.

16. Roth, J., D. Silbert, G. Fink, M. Voll, D. Anton, P. Hartman,and B. Ames. 1966. Transfer-RNA and the control of thehistidine operon. Cold Spring Harbor Symp. Quant. Biol.31:383-392.

17. Smith, 0. H., and C. Yanofsky. 1962. Enzymes involved inthe biosynthesis of tryptophan, p. 748-806. In S. P. Colo-wick and N. 0. Kaplan (ed.), Methods in enzymology, vol.5. Academic Press Inc., New York.

18. Somerville, R. L., and C. Yanofsky. 1965. Studies on theregulation of tryptophan biosynthesis in Escherichia coil.J. Mol. Biol. 11:747-759.

19. Vogel, H. J., and D. M. Bonner. 1956. Acetyl-ornithinase ofEscherichia coli: partial purification and some properties.J. Biol. Chem. 218 97-106.

20. Yanofsky, C. 1955. On the conversion of anthranilic acid toindole. Science (Washington) 121:138-139.

21. Yanofsky, C. 1960. The tryptophan synthetase system. Bac-teriol. Rev. 24:221-245.

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