tryptophan synthetase levels in escherichia coli

TRYPTOPHAN SYNTHETASE LEVELS IN ESCHERICHIA COLI,SHIGELLA DYSENTERIAE, AND TRANSDUCTION HYBRIDS

RICHARD B. EISENSTEIN1 AND CHARLES YANOFSKYDepartment of Microbiology, Western Reserve University, Cleveland, Ohio,

and Department of Biological Sciences, Stanford University,Stanford, California

Received for publication August 30, 1961

ABSTRACT

EISENSTEIN, RICHARD B. (Western ReserveUniversity, Cleveland, Ohio) AND CHARLESYANOFSKY. Tryptophan synthetase levels inEscherichia coli, Shigella dysenteriae, and trans-duction hybrids. J. Bacteriol. 83:193-204. 1962-Shigella dysenteriae and Escherichia coli, strainsK-12 and B, were found to produce low levels oftryptophan synthetase, although some hybrids,formed by the introduction of the gene clusterconcerned with tryptophan synthesis from S.dysenteriae into E. coli, produced high levels ofthis enzyme system. A revertant obtained from atryptophan-requiring mutant also formed highlevels of tryptophan synthetase. The gene orgenes responsible for high enzyme production inthese strains was shown to be linked to the clusterof genes concerned with tryptophan synthesis.The cause of high enzyme production was investi-gated. Various lines of evidence, including stimu-lation of growth by tryptophan precursors, sensi-tivity to inhibition by 5-methyltryptophan,absence of accumulation of tryptophan, and re-pression of enzyme formation by anthranilic acidand tryptophan, suggested that high enzymeproduction in the strains examined results froma partial block in the tryptophan pathway andnot from resistance to repression by tryptophan.The conversion of shikimic acid-5-phosphate toanthranilic acid appears to be the partiallyblocked reaction in the strains studied.

It has been repeatedly demonstrated that theconcentration of the end product of a biochemicalpathway influences the formation of the enzymesin the pathway (Pardee, 1959). Thus, it has been

1 Taken, in part, from a dissertation submittedin partial fulfillment of the requirements forthe degree of Doctor of Medicine at WesternReserve University, Cleveland, Ohio.

shown that arginine concentration affects the for-mation of acetylornithinase (Vogel, 1957) andornithine transcarbamylase (Gorini and Maas,1958), two enzymes involved in the biosynthesisof this amino acid. Similarly, histidine influencesthe formation of four enzymes involved in thebiosynthesis of this amino acid (Ames and Garry,1959). Genetic effects on the level of the enzymeformed have also been observed, and, in some ofthese cases, it has been found that the formationof a specific enzyme or group of enzymes is nolonger affected by the end product of the pathwayin which the enzymes operate (Cohen and Jacob,1959; Gorini, 1960). The present study, whichgrew out of an attempt to determine whetherthere were qualitative differences between thetryptophan synthetases of Escherichia coli andShigella dysenteriae, is concerned with the level ofthis enzyme in these organisms and in varioushybrids.

MATERIALS AND METHODS

Bacterial stocks. The following auxotrophs ofthe K-12 strain of E. coli were employed: cys4,td4, A-78, T-41, T-3, T-16, and T-58. Of these,cys4 requires cysteine for growth and the othersrequire tryptophan. Strains B-82, B/1TN,B/1T7, and C-13T, tryptophan auxotrophs of theB strain of E. coli, were also used. Mutant T-3 isblocked in the synthesis of anthranilic acid andgrows on minimal medium supplemented withanthranilic acid, indole, or tryptophan. StrainsT-16 and T-58 are blocked in the conversion ofanthranilic acid to anthranilic ribulotide (Smithand Yanofsky, 1960) and respond to indole ortryptophan. Strain A-78 also grows on indole ortrvptophan but is blocked in the conversion ofindoleglycerol phosphate to tryptophan. Theother K-12 and the B tryptophan auxotrophs willgrow only on tryptophan. Strains B/1T7, B/1TN,and C-13T are deletion mutants in which a seg-

193

EISENSTEIN AND YANOFSKY

ment of the cluster of genes controlling trypto-phan formation was lost simultaneously with mu-tation to resistance to phage T-1 (Yanofsky andLennox, 1959). Strain B/1T7 lacks all the en-

zymes involved in tryptophan synthesis; strainB/1TN lacks only those concerned with the con-

version of indoleglvcerol phosphate to indole;and C-13T lacks those concerned with the con-

version of anthranilic acid to tryptophan. Otherstocks employed included the K-12 wild-typestrain and S. dysenteriae strain Sh/S (obtainedfrom S. E. Luria). In addition, an unusual re-

vertant (designated T-41R5), isolated fromstrain T-41, was examined. This strain formslarge amounts of tryptophan synthetase whengrown on an unsupplemented glucose-saltsmedium.

Preparation of extracts. All strains of E. coliwere grown on single strength minimal medium(Vogel and Bonner, 1956) supplemented with0.16 to 0.2% glucose and any required substance,e.g., tryptophan. S. dysenteriae was grown on thesame medium with supplements of 0.05%Casamino acids, and 0.4% nutrient broth, or

with 0.1% casein hydrolyzate plus low levels ofanthranilic acid, indole, or tryptophan. Cells were

grown 16 to 18 hr at 37 C with shaking and were

harvested by centrifugation. The sedimentedcells were washed once with cold 0.85% NaCl,suspended in 0.1 M potassium phosphate buffer(pH 7.8), and disrupted in a 9 kc Raytheon sonicoscillator or a Nossal disintegrator. The treatedmaterial was then centrifuged to remove celldebris, and the extracts obtained were stored at-15 C.Assay of tryptophan synthetase components. The

tryptophan synthetase of E. coli (and of S. dysen-teriae) is composed of two separate protein com-

ponents, termed A and B (Crawford andYanofsky, 1958). Together these two proteinscatalyze the following three reactions: (I) indole+ L-serine L-tryptophan, (II) indoleglycerolphosphate indole + triose phosphate, and(III) indoleglycerol PO4 + L-serine -* L-trypto-

phan + triose phosphate. Each protein compo-

nent may be quantitatively assayed in thepresence of a saturating amount of the secondcomponent, usually a threefold excess. Under theassay conditions employed, neither componenthas appreciable activity in any of the three reac-

tions in the absence of the other. The studiesreported here are concerned with reaction I, in

which pyridoxal phosphate is required as a co-enzyme. The unit of enzyme activity employedin this study is defined as the amount of enzyme(or of component A or B) that will convert 0.1,umole of indole to tryptophan in 30 min at 37 C.Assays were carried out both in 0.1 M potassiumphosphate buffer and in tris(hydroxymethyl)-aminomethane (tris) buffer, supplemented with0.03 ml of a saturated solution of NaCl per ml ofreaction mixture, since the activity sometimesdiffered under these two conditions. Tryptophansynthetase (or component A or B) specific ac-tivity is defined as the units of activity per milli-gram of extract protein. The protein content wasdetermined by the method of Lowry et al. (1951).

Whole cell assay of tryptophan synthetase. Cellswere grown overnight in 5 ml of single-strengthminimal medium with 0.12% glucose plus supple-ments as indicated. Turbidity was determinedwith a Klett colorimeter (660-m,u filter), portionswere centrifuged, and the cells were resuspendedin a mixture containing potassium phosphatebuffer (pH 7.8) 50 ,umoles; DL-serine, 40 ,umoles;pyridoxal phosphate, 0.3 Amole; and indole, 0.3,umole; in a total volume of 1 ml. Assay tubeswere incubated with shaking at 37 C for 30 min.

TABLE 1. Tryptophan synthetase content ofvarious hybrids

Transduction Extract specific

Isolate activity

testedDonor Recipien t Minimal Nutrient

medium medium

S. dysen- B/1T7 SR* T+-(1) 26 15teriae T+-(2) 164

T+- (3) 80T+- (4) 37 11T+-(5) 41 11T+- (6) 203 73T+- (7) 101T+- (8) 30

S. dysen- B/1T7 T+-(7) 78teriae T+- (8) 88

Controls

S. dysenteriae - 3.2, 3.5E. coli B 4.7 5.6E. coli K-12 1.9 2.2, 2.8

* SR (Streptomycin resistant); T+ (Tryptophanindependent).

194 [VOL. 83

TRYPTOPHAN SYNTHETASE LEVELS

The unreacted indole was extracted with tolueneand determined quantitatively as described else-where (Yanofsky, 1955). Three arbitrary classeswere distinguished: low enzyme producers, lessthan 0.1 ,mole of indole taken up per 3 X 109cells; intermediate producers, 0.1 to 0.2 ,umole ofindole taken up per 3 X 109 cells; and high pro-ducers, greater than 0.2 ,umole of indole taken upper 3 X 109 cells.Assay of the conversion of shikimic acid-5-phos-

phate to anthranilic acid. The synthesis of anthra-nilic acid by cell-free extracts was carried out asdescribed by Srinivasan (1959) and Moyed(1960). The reaction mixture contained tris buffer(pH 7.8), 20 ,umoles; MgCl2, 2.5 ,umoles; L-glutamine, 3 ,umoles; diphosphopyridine nucleo-tide (DPN), 0.25 ,umole; shikimic acid-5-phos-phate, 0.3 umole; and extract; in a final volumeof 0.5 ml. Incubation was at 37 C and 0.05-mlsamples were removed at 10-min intervals andpipetted into 2 ml of 0.1 M potassium phosphatebuffer (pH 6.0). The anthranilic acid present inthese samples was determined fluorometricallyusing an Aminco-Bowman spectrophotofluor-ometer. The barium salt of shikimic acid-5-phosphate was kindly supplied by Bernard Davisof Harvard University.

Transduction. Phage Plkc was used in all thetransduction experiments. The procedural detailsare described elsewhere (Lennox, 1955). Trans-duction hybrids bear the designations of both thedonor and recipient species, with the donor alwayswritten first. For example, Sh: K-12 indicates astrain derived from a transduction from S.dysenteriae into a K-12 auxotroph.

RESULTS

Initial observations with high enzyme producers.The initial observations which prompted thisstudy are shown in Table 1. Whereas E. colistrain B, E. coli K-12, and S. dysenteriae formrelatively little tryptophan synthetase whengrown on a glucose-salts medium or a richmedium, hybrids, produced by the introductionof the gene cluster concerned with tryptophansynthesis from S. dysenteriae into the deletionmutant, B/1T7, form large amounts of this en-zyme system. Previous studies had shown thatstrain B/1T7 lacked all the tryp genes and en-zymes concerned with the formation of anthra-nilic acid and its conversion to tryptophan(Yanofsky and Lennox, 1959; Yanofsky, 1957).

It appeared that the introduction of these genesfrom S. dysenteriae into E. coli strain B/1T7 wasresponsible for the production of high levels oftryptophan synthetase. It was also shown in im-munological tests that the strains with highenzyme activity formed correspondingly highlevels of the A and B proteins as antigens. Thus,the increased activity detected in these strains isnot due to alterations in the turnover number ofthe enzyme system but represents an increase inthe amount of specific enzyme proteins.To determine whether high enzyme production

was due to some abnormality associated withB/1T7 (the recipient) or to the genic materialintroduced from S. dysenteriae, hybrids producedwith other tryptophan auxotrophs were alsoexamined. Standard transductions (Table 2) wereperformed with phages grown on the donorsindicated, with the listed tryptophan auxotrophsas recipients. The plating medium did not containtryptophan; thus, tryptophan independence wasselected. Colonies were picked from the platesafter 3 to 4 days and streaked on minimal mediumfor single colony isolation. A stock was preparedfrom one colony from each plate. The results ofthe examination of these stocks are summarizedin Table 2. Both components, A and B, and totaltryptophan synthetase activity are high in thehybrids obtained in transductions from S. dysen-teriae into B/1T7. Furthermore, high enzyme-pro-ducing strains, although in the minority, werealso isolated from transductions involving strainsB-82 and td4 as recipients. Thus, high enzyme-producing transductants are obtained with otherE. coli auxotrophs as recipients. It is also evidentfrom the results presented in Table 2 that genicmaterial, permitting tryptophan synthetase for-mation, can be introduced from S. dysenteriaeinto K-12 and B tryptophan auxotrophs withoutconcomitant high enzyme production. Controltransductions with wild-type K-12 as the donororganism and td4 or B/1T7 as recipient yieldedstrains with enzyme levels equal to those pro-duced by the wild-type form of the recipients.When these observations were made, it was re-

called that a high tryptophan synthetase-pro-ducing strain had been isolated previously duringthe course of a reversion experiment with strainT-41. This strain, designated T-41R5, was in-cluded in this investigation of high tryptophansynthetase production. The tryptophan synthe-tase and A and B levels produced by this strain

1962] 195


TABLE 2. Tryptophan synthetase and A and B specific activities of various strains

Transduction Specific activity

Stock tested Tryptophan synthetase Component A Component B Ratio A/BDonor Recipient

Phosphate Tris buffer Tris buffer Tris buffer

T-41R5 12.0 14.8 29.6 22.9 1.3

E. coli K-12 1.4 1.4 2.8 1.8 1.7wild type (1.2-1.5*) (0.9-1.6) (2.2-3.3) (0.9-2.3) (1.1-2.5)

S. dysenteriae 2.5 1.9 4.3 2.9 1.5

E. coli B 3.1 3.2 5.2 4.5 1.3wild type (2.5-3.7) (1.8-4.5) (3.5-6.9) (2.3-6.6) (1.0-1.5)

S. dysen- B/1T7 SR T+- (3) 30 18 36 29 1.2teriae T+- (6) 150 121 306 257 1.2

T+- (8) 29 27 48 45 1.1

S. dysen- td4 SR 7 T+ isolates 1.2 1.1 3.3 1.4 2.3teriae (0.7-1.6) (0.9-1.4) (2.8-3.5) (1.1-1.8) (1.8-3.3)

T+- (8) 5.3 4.4 8.1 7.9 1.0

S. dysen- B-82 7 T+ isolates 2.6 2.6 6.1 4.7 1.2teriae (1.4-3.6) (1.3-3.6) (3.1-7.2) (2.3-6.3) (1.0-1.4)

T+- (3) 35 30 55 53 1.0

K-12 td4 SR 3 T+ isolates 1.8 2.0 3.4 3.0 1.1(1.6-2.0) (3.3-3.7) (2.9-3.1) (1.1-1.2)

K-12 B/1T7 SR 3 T+ isolates 3.5 4.3 7.6 6.6 1.2(2.1-4.5) (3.2-5.1) (7.1-8.0) (5.2-7.8) (1.1-1.4)

* Values in parentheses represent range of specific activities and A/B ratios detected in separate ex-periments. T+ indicates isolate which can grow in the absence of tryptophan.

are given in Table 2. Extracts of mutant T-41lack the B component of tryptophan synthetase.

Extract preparation and analyses of the typepresented in Table 2 involve many operations andas a result are time-consuming. To examinelarger numbers of transductants for tryptophansynthetase activity, a whole cell assay was de-veloped (Materials and Methods). This assaymethod gave good proportionality between num-bers of cells added and tryptophan synthetaseactivity detected (Fig. 1) for both high and lowtryptophan synthetase producers; therefore, itcould be used to assay large numbers of individualisolates for their tryptophan synthetase activity.It can also be seen from Fig. 1 that S. dysenteriae,E. coli B, and E. coli K-12 show less than one unitof tryptophan synthetase activity per 3 X 109

cells. In other tests the tryptophan synthetaseactivity of whole cells, toluene-treated cells, andextracts was compared, and no appreciable dif-ferences in tryptophan synthetase levels weredetected.During the course of standardizing the whole

cell assay, it was noticed that, although S. dysen-teriae formed fully active tryptophan synthetase,it was impaired at some step in the formation oftryptophan, since its growth was appreciablystimulated (three- to fivefold) by tryptophan,indole, or anthranilic acid. The other amino acidrequirements of the S. dysenteriae strain em-ployed were not determined, but could be satis-fied by tryptophan-free acid-hydrolyzed casein.

Genetic studies. The gene or genes responsiblefor the partial growth dependence of S. dysen-

196 [VOL. 83


3

w 2n

0

C

c

c01

0

I-

Shigella

,_K-12

2 3 4X 109cells assayed

FIG. 1. Whole cell assay: relationship betweennzumbers of cells added and tryptophan synthetaseactivity. Strains T-41R5 and Sh:K-12 (7) are highenzyme producers while the others form low levelsof tryptophan synthetase. A substrate concentrationwhich gives a maximum of 3 units of activity was

employed.

teriae on tryptophan was shown to be linked tothe genes controlling tryptophan synthetase for-mation (Table 3). Here 104 of 240 colonies testedfrom a transduction, with strain B-82 as recipient,responded to anthranilic acid. The test employedto detect anthranilic acid stimulation was tostreak loopfuls of a bacterial suspension ontoplates with or without an anthranilic acid supple-ment and to compare growth after 24 hr. Thismethod is somewhat crude and it is likely that theestimate of the number of anthranilic-stimulatedcolonies is low. Some colonies of each type were

tested for tryptophan synthetase activity, and itcan be seen (Table 3) that high-producing strainsappeared in both groups; however, all theanthranilic-stimulated isolates produced appre-ciable amounts of enzyme and were at least inter-mediate producers.

Strain T-41R5 was also examined in transduc-tion tests. When T-41 was employed as a re-

cipient, it became apparent that high enzymeproduction was associated with a gene distinctfrom the mutated region in T-41 (Table 3). Ap-parently strain T-41R5 differs from T-41 at twosites, one responsible for high enzyme productionand the other representing a functionally effectivesite corresponding to the defective site in strainT-41. The high enzyme-producing determinant isobviously closely linked to the genic region con-

trolling tryptophan synthetase formation, since

76% of the isolates which received genic ma-terial permitting tryptophan synthetase produc-tion were high producers. Thus, in both T-41R5and S. dysenteriae, genic material which can causehigh enzyme production is linked to the genescontrolling tryptophan synthetase formation.Attempts were made to localize further these

determinants in the bacterial genome by trans-duction into a series of mutants lacking differentsegments of the tryp gene cluster as a result ofdeletion. The rationale for this approach wasthat if a deletion mutant lacked genic materialcorresponding to the region responsible for highenzyme production, then only high-producingstrains could be obtained in transductions. On theother hand, if the corresponding region was notdeleted, some low-producing strains would be ob-tained among the tryptophan-independent stocksrecovered. The size of the deletions in the threestocks tested, in relation to the biosynthetic path-way of tryptophan formation, is shown in Fig. 2.The results of these transduction tests, summar-ized in Table 3, indicate that, with strain T-41R5as donor, low producers are recovered withB/1TN and C-13T as recipient but not withB/1T7. These findings indicate that the genicregion responsible for high enzyme production inT-41R5 is probably close to, but not in, the regiondeleted in C-13T and B/1TN.

Close linkage to the tryp region is also shownby the recovery of many high producers in thetransduction into strain td4 and into a strainbearing a closely linked marker, cys4. The loca-tions of these markers relative to the deletionsstudied are indicated in Fig. 2. Similar experi-ments carried out with S. dysenteriae or a highenzyme-producing Shigella: K-12 hybrid, stockSh:K-12 (7), as donors, were complicated by thefact that many of the recombinants obtainedwere stimulated by anthranilic acid. Such stocks,when grown on unsupplemented minimal me-dium, generally fell into at least the intermediateproducer class, and often produced just enoughenzyme to be placed in the high enzyme producerclass. With various auxotrophs as recipients andS. dysenteriae as donor (Table 3), both high- andlow-producing strains were recovered. Further-more, no low producers were recovered with theSh: K-12 hybrid as donor and the deletionmutants C-13T and B/1T7 as recipients. Thesefindings indicate that genic material closelylinked to the tryptophan synthetase genic region

1962] 197


TABLE 3. Whole cell assay results with isolates from various transductions

Number of isolates producingTransduction T+ isolates Stimulated by Isolates enzyme at following levels

Donor Recipientexamined anthranilic acid assayed

Donor Recipient HP* IPF LP*

T-41R5 T-41 50 38 0 12T-41 R5 td4 50 40 7 3T-41R5 Cys4 46 19 2 25T-41R5 B-82 47 23 6 18T-41R.5 B/1T7 82 82 0 0T-41R5 C-13T 45 42 0 3T-41R5 B/1TN, 15 9 0 6

S. dysenteriae B-82 240 104 112 NS* 7 15 9019 S* 18 1 0

S. dysenteriae B/1T7 89 89 27 S 16 11 0S. dysenteriae td4 94 19 14 NS 4 0 10

5S 4 1 0

Sh:K-12(7)t B/1T7 20 0 20 NS 20 0 0Sh:K-12(7) C-13T 18 0 18 NS 17 1 0

K-12 T-41 50 0 50 NS 0 4 46K-12 td4 25 0 25 NS 0 0 25K-12 B/1T7 9 0 9 NS 0 0 9

* Abbreviations used: NS (not stimulated by anthranilic acid); S (stimulated by anthranilic acid);HP = high producing; IP = intermediate producing; LP = low producing.

t A high enzyme-producing hybrid.

-- Anthronilic -O -* Indoleoglycerol Tryptophanacid phosphate

cystic

T,3 T16 T 41 td42

3

FIG. 2. Relative location of mutated sites in thedeleted in mutants B/1lTN, C-13T, and B/1T7.

is responsible for high enzyme production. Trans-ductions with K-12 as donor, also listed inTable 3, demonstrate that high producers are notrecovered with this low-producing donor strain.The few intermediate producers recovered were

barely out of the low producer class (1.2 units).Cause of high tryptophan synthetase production.

The previous findings indicated that the introduc-tion of certain genie material from strains T-41R5and S. dysenteriae into the proper stocks resultedin the production of high levels of tryptophansynthetase. Several explanations for this observa-tion were considered. First, it appeared possible

{ BilT7

strains examined. The thin lines represent the areas

that the high-producing strains, in contrast tonormal strains, were not subject to repression bytryptophan, and thus enzyme production pro-ceeded unregulated. According to this interpreta-tion both T-41R5 and S. dysenteriae would carrythe gene or genes concerned with regulatingtryptophan synthetase formation in a mutatednonfunctional form, or the S. dysenteriae repressorgene or genes could not function in E. coli. Muta-tions relieving repression in the tryptophan path-way have been described in E. coli (Cohen andJacob, 1959). A second possibility was that allthe high tryptophan synthetase-producing strains

198 [VOL. 83


I) aVJ -

8 - 1.6

U) ---- ,6004 - 0.8C /0x oCCr00.. 2/ 0.4.CC. 0

0 2 4 6 8 IO.pg anthranilic acid / ml

FIG. 3. The effect of anthranilic acid concentra-tion on tryptophan synthetase production. Thedashed line represents the growth response of mutantT-3 to the levels of anthranilic acid indicated. Thesolid lines represent tryptophan synthetase levels,X (T-3); 0 (T-41R5); 0 (Sh:K-12 (7), uppercurve); O (S. dysenteriae); * (K-12, lower curve)

had a partial block at some step in tryptophansynthesis, and this partial block limited thesupply of tryptophan. The resulting low endoge-nous concentration of this amino acid wouldthereby relieve repression. A situation analogousto this has been described by Gorini and Maas(1958) in studies of the ornithine transearbamyl-ase of E. coli. To be consistent with the data pre-sented here it would have to be further assumedthat other limitations, perhaps also in tryptophanformation, mask the effect of the partial block inS. dysenteriae.To distinguish between the possibilities men-

tioned, two general approaches were employed;the first was intended to determine whether tryp-tophan synthetase production by high producersis subject to repression by tryptophan, and thesecond, whether high-producing strains are, infact, partially blocked in tryptophan formation.To examine whether the high-producing strains

were resistant to repression, they were grown inthe presence or absence of high concentrations oftryptophan or its precursor, anthranilic acid.More extensive data were obtained with anthra-nilic acid, and typical results with this compoundare presented in Fig. 3. The high enzyme-produe-ing strains examined were T-41R5 and the hybridSh: K-12 (7). For comparison, a mutant blockedprior to anthranilic acid, strain T-3, was alsoexamined, as well as the parental organisms, E.coli K-12 and S. dysenteriae. It can be seen that atlow anthranilate concentrations T-3 produced

higher than normal tryptophan synthetase levels,but the level dropped to the wild-type controllevel as the anthranilic acid supplement ap-proached a concentration which supported maxi-mal growth. Similarly, the tryptophan synthetaselevel in T-41R5 and Sh: K-12 (7) dropped at ap-proximately the same anthranilate concentration.Similar inhibitions were observed with hightryptophan concentrations. Thus, tryptophansynthetase formation in high-producing strains isclearly subject to repression by tryptophan.

Several approaches were employed to deter-mine whether the high producers are partiallyblocked at some step in tryptophan synthesis.High-producing strains which were stimulated bvanthranilic acid in the streaking test obviouslyhave a partial block and were not examined fur-ther. In addition to this type, some hybrid strains

TABLE 4. Anthranilic acid stimtulation of growth*

Strain

Sh:K-12(7)(H.P.)

Sh: K-12 (10)(H.P.)

Sh: B(7)(H.P.)

Sh:K-12(11)(L.P.)

T-41R5 (H.P.)

K-12 (L.P.)

Sample

AtBABABABABAB

Inoculum dilution

1-10

Mint AAt

0 40 51 55 50 02 54 45 55 55 55 55 5

1-100

Min AA

0 00 50 05 50 00 50 05 50 05 50 05 5

1-103

Min AA

0 00 50 05 50 00 50 05 50 05 50 05 5

* Cultures were grown on minimal medium to apopulation of 2 to 5 X 108 cells/ml and diluted asindicated. An inoculum of 0.1 ml of each dilutionwas used for 5 ml of minimal medium with andwithout a 10 mg/ml anthranilic acid supplement.Growth was checked at different times and re-corded on a 0 to 5 scale. Turbidity measurementsin the Klett Colorimeter on K-12 and T-41R5cultures indicated that growth was identical onminimal medium and anthranilic acid-supple-mented medium.

t Min (single strength E. coli minimal medium);AA (the same medium supplemented with an-thranilic acid).

t A and B represent different sampling times,with B the later time.

19621 199


were recovered which were not stimulated byanthranilic acid but which were high producers(Table 3). Furthermore, T-41R5 and all high-producing stocks derived from it were not stimu-lated by anthranilic acid.A fairly sensitive method of detecting differ-

ences in growth rate was employed in examininghigh enzyme-producing strains. This involvedgrowing a log-phase culture in minimal medium,diluting the cells, and using equal inocula in freshminimal medium and in minimal medium supple-mented with anthranilic acid or tryptophan. Thecultures were then observed for visible turbidityand scored, using an arbitrary scale. The resultsobtained in one series of experiments are pre-sented in Table 4. The data obtained indicatethat three high-producing strains, Sh: K-12 (7),Sh:K-12 (10), and Sh:B (7), none of which wasdetectably stimulated by anthranilic acid in thestreaking test, reached visible turbidity first inthe anthranilic-supplemented medium. The con-trols and T-41R5 grew equally rapidly in bothmedia. Similar results were obtained with severalof these strains when tryptophan, rather thananthranilic acid, was employed as supplement.As an independent test of the conclusions reachedin the experiments in liquid medium, small num-bers of cells, ca. 5 to 20, were plated on minimalagar plates and on minimal agar plates supple-mented with anthranilic acid; and after 2 to 3days, colony size (diameter) was measured. Theresults were entirely consistent with those ob-tained in liquid medium. For example, colonies ofstrain Sh: K-12 (7) on minimal medium averaged2.3 i 0.3 mm and on minimal medium plusanthranilic acid averaged 3.2 1 0.3 mm.Only strain T-41R5, of the high-producing

types, failed to respond to tryptophan or trypto-phan precursors. This behavior need not excludethe existence of a partial block in tryptophansynthesis in this strain, since it is possible thatK-12 normally overproduces tryptophan andthat this slight overproduction is responsible forrepression of the enzymes involved in tryptophanbiosynthesis. T-41R5 could have a partial blockwhich reduces the rate of tryptophan synthesisto a level at which tryptophan is never present ata concentration sufficient to repress. It still mightbe capable of synthesizing tryptophan at a ratewhich permits normal growth.To examine this possibility, a test was sought

which would effectively measure the endogenous

levels of tryptophan and tryptophan precursorspresent during growth rather than growth de-pendence on tryptophan. The test ultimatelyselected was to examine sensitivity to an analogueof tryptophan, 5-methyltryptophan. It was rea-soned that, since inhibition by this analogue isspecifically reversed by tryptophan and trypto-phan precursors, the level of growth obtained inthe presence of the analogue would be an indica-tion of the internal concentrations of tryptophanand tryptophan-specific precursors. Sensitivity ofvarious high-producing and low-producing strainsto 5-methyltryptophan is shown in Table 5,where it can be seen that all the high producers,but none of the low producers, were inhibited bythe level of 5-methyltryptophan employed. It isalso evident that strain T-41R5 was more sensi-tive to the analogue than the wild type or a low-producing revertant, T41-R8, which was alsoisolated from T-41. However, T-41R5 was theonly high producer to form visible colonies after3 days. These results, although they do not giveany information on the mechanism of inhibitionby 5-methyltryptophan, indicate that the con-centration of tryptophan and tryptophan-specificprecursors is probably lower in the high-producingstrains, including T-41R5.As a verification of the method and the inter-

pretation, several presumed partial revertants

TABLE 5. 5-illethyltryptophan sensitivity ofhigh and low producers

Strain

K-12, wild typeT-41R5 (H.P.)T-41R8 (L.P.)Sh:K-12(7) (H.P.)Sh:K-12(18)

(H.P.)Sh: K-12 (11)

(L.P.)Sh:K-12 (15)

(L.P.)Sh:B(7) (H.P.)Sh:B(20) (L.P.)

Colony size in mm (averageof 5 or more colonies)

Minimal medium

2 days

2.83.22.72.02.0

2.8

2.9

3.14.2

3 days

4.24.6

3.43.3

3.9

4.0

* 5-MT (5-methyltryptophan).

Minimal mediumplus 0.05 jAg 5-

M:T*/nII

2 days 3 days

2.9 4.10 2.53.10 00 0

2.0

2.5

04.5

3.4

3.8

0

[VOL. 83200


TABLE 6. Conversion of shikimic acid-5-phosphateto anthranilic acid

S-5-P -+

Tryptophan synthe- Anth. A.tase components, AMoles Tryptophan*

Extract specific activity anth. A. inhibition ofS-5-P-

formed/hr/ Anth. Amg extract

A B protein

T-58t 31 17 9.2 45 to 60A-78 99 76 3.2T-41R5 22 19 0.6 43Sh:B (6) 151 147 0.06t 33Sh:K-12 (7) 71 72 0.12 50

* 4 X 10-5 M L-tryptophan. S-5-P (shikimicacid-5-phosphate).

t Grown in the presence of low levels of indole(tryptophan auxotrophs, T-58 and A-78). Grownon unsupplemented minimal medium (high enzymeproducers, T-41R5, Sh:B(6), and Sh:K-12(7)).

t Barely detectable.

were isolated from strain T-16, a mutant blockedbetween anthranilic acid and anthranilic ribulo-tide. (Genetic tests were not performed to dis-tinguish between partial reversion and suppres-sion.) These were obtained by plating T-16 cellson minimal medium and picking slow-growingtryptophan-independent colonies. These cells in-variably accumulated anthranilic acid. Theresults of whole cell assays on a number of thesepartial revertants showed that they were all highproducers. These revertants were then examinedfor sensitivity to 5-methyltryptophan, and allwere found to be inhibited by 5-methyltrypto-phan concentrations which do not inhibit thewild type.

Attempts to determine the site of the partial block.Culture filtrates of the various partially blockedhigh-producing strains (other than those derivedfrom T-16) were examined for anthranilic acid,anthranilic ribuloside, indoleglycerol, and indole.One of these compounds might be expected toaccumulate if the block in tryptophan synthesiswere at a reaction involving one of the compoundsas substrate. No trace of any of these compoundswas detected; therefore, it was concluded thateither the partial block was not severe enough tocause accumulation or was at a step prior toanthranilic acid.As a further test of this conclusion, extracts of

representatives of the different types of high-producing classes were examined semiquantita-

tively for the enzymes involved in the conversionof anthranilic acid to indoleglycerol phosphate.In all cases the enzyme activities were very high,at least 5 to 10 times that of the low-producingstrains. Thus, none of the enzymes betweenanthranilic acid and tryptophan appears to bepresent in a reduced amount, supporting the viewthat the partial block is prior to anthranilic acid.Culture filtrates were also examined for trypto-phan and none was detected. Neither was thisamino acid found in the supernatant solutions ob-tained following heating and centrifugation ofcrude extracts of high-producing strains.

Extracts of various high producers were alsoexamined in the conversion of shikimic acid-5-phosphate to anthranilic acid. Two tryptophanauxotrophs, strains A-78 and T-58, were em-ployed as controls. These strains form high levelsof tryptophan synthetase and other enzymes ofthe tryptophan pathway when grown on growth-limiting levels of indole or tryptophan. The hightryptophan synthetase-producing strains, T-41R5Sh:B (6), and Sh:K-12 (7), which presumablyhave partial blocks, are clearly distinguishablefrom the control stocks in that they showed littleanthranilic acid synthesis (Table 6). The absenceof appreciable activity could not be due to en-zyme imbalance or a rapid conversion of synthe-sized anthranilic acid to other intermediates,since the extracts employed did not metabolizeadded anthranilic acid in the presence of the sup-plements provided. Furthermore, the extracts donot contain an inhibitor, since mixtures withT-58 extracts were as active as T-58 extractsalone. The synthesis of anthranilic acid is a com-plex reaction and the activity values presentedshould be considered only a qualitative measureof enzyme activity. In fact, with the control ex-tracts, T-58 and A-78, there was a definite thresh-old effect, with little or no activity when less than2 to 3 mg of extract protein are present per ml.When the extract concentration was increasedthere was a sharp nonproportional increase inenzyme activity. This lack of proportionalitycould be eliminated by the addition of an extractof the deletion mutant B/1T7. Extracts of thismutant were devoid of activity but increased therate of anthranilic acid synthesis by T-58 andA-78 extracts 10-fold to 20-fold. The stimulatoryeffect of B/1T7 extracts is probably not due tothe presence of one pf the enzymes involved inthe synthesis of anthranilic acid, since extracts

19621 201


TABLE 7. Tryptophan synthetase levels in tryptophan-independent isolates derivedfrom Shigella dysenteriae

Transduction Number of isolates producingTransduction T+ isolates Stimulated by Isolates assayed enzymes at following levelexamined antbranilic acid

Donor Recipient HP IP LP

T-41R5 S. dysenteriae 20 0 20 20 0 0K-12 S. dysenteriae 39 11 28 NS* 0 0 28

11 S* 11 0 0

* See Table 3 for explanation of symbols.

of B/1T7 grown on repressing levels of trypto-phan were fully effective in stimulating the ac-tivity of T-58 extracts. Extracts of B/1T7 did notstimulate the synthesis of anthranilic acid byextracts of strains T-41R5, Sh:B (6), andSh: K-12 (7), suggesting that the activity of thelatter extracts is rate-limiting. In other tests itwas shown that tryptophan inhibited the synthe-sis of anthranilic acid by extracts of the variousstrains to the same extent (Table 6).

Studies with S. dysenteriae as recipient. One ofthe as yet unexplained observations is that, al-though introduction of S. dysenteriae genic ma-terial into E. coli stocks may result in high en-zyme production, S. dysenteriae itself does notform high levels of tryptophan synthetase. Onlylow levels of enzyme are formed by this organismdespite the fact that it appears to be partiallyblocked in tryptophan synthesis and grows onlypoorly in the absence of this amino acid or oneof its precursors. To obtain further informationrelevant to the behavior of this organism, severaltransduction experiments were performed withS. dysenteriae as recipient. In these experimentsthe limited ability of S. dysenteriae to grow with-out tryptophan was used as a selective character,and recombinants which grew well in the absenceof tryptophan were selected.The results obtained with wild-type K-12 and

T-41R5 as donors are presented in Table 7. Itcan be seen that with T-41 R5 as donor all therecombinants tested were high producers. Thusit would appear that the inability to produce highlevels of enzyme is not due to the other nutritionalrequirements of S. dysenteriae but is associatedwith the genic region concerned with tryptophansynthesis, since replacement of a portion or por-tions of this region yields high enzyme producers.The results obtained with wild-type K-12 asdonor show that replacement of a portion or por-tions of this region from a low enzyme producer,wild-type K-12, also gives high enzyme producers,

although less frequently. It should also be notedthat the recombinant high producers obtainedfrom the transduction K-12 -- S. dysenteriaeresponded to anthranilic acid in the streakingtest, indicating that they were still partiallyblocked in tryptophan synthesis. However, theywere capable of much faster growth in the ab-sence of tryptophan than the parental strain,S. dysenteriae.

DISCUSSION

The studies reported in this paper demonstratethat strain T-41R5 and some E. coli-S. dysen-teriae transduction hybrids form high levels of theA and B proteins of tryptophan synthetase. Twoalternative explanations were considered for theproduction of high levels of enzyme by thesestrains: a partial block in an earlier reaction, andresistance to repression by the end product of thepathway, tryptophan. The findings obtained inthis study have been interpreted as favoring thepartial block hypothesis. The growth of most ofthe high enzyme producers, with the notableexception of T-41R5, was stimulated by anthra-nilic acid or tryptophan. Stimulation of thegrowth of high producers is obviously an indica-tion of an impairment in tryptophan formation.In addition, all the high enzyme producers, in-cluding T-41R5 and strains derived from it, wereextraordinarily sensitive to inhibition by5-methyltryptophan, an analogue which inter-feres with tryptophan formation (Moyed, 1960).This extreme sensitivity would be expected if therate of tryptophan synthesis was low in the highenzyme producers. Consistent with this conclu-sion is the observation that tryptophan was notaccumulated in the culture medium of the highenzyme producers, although most of the assayedenzymes in the tryptophan pathway were presentin greater than normal amounts. Tryptophanmight be expected to be accumulated by organ-isms which are resistant to repression and form

202 [VOL. 83


high levels of enzymes; and, in fact, the onerepression-resistant mutant (tryptophan path-way) which has been isolated and studied (Cohenand Jacob, 1959) does accumulate this aminoacid. Finally, levels of anthranilic acid and tryp-tophan which just barely supported maximalgrowth of tryptophan auxotrophs were found torepress enzyme production in the high producers.Taken together, these observations strongly sug-gest that partial blocks in tryptophan synthesisare responsible for the production of high levelsof tryptophan synthetase in the strains examined.Enzymatic studies did lead to the detection of

a partial block in the conversion of shikimic acid-5-phosphate to anthranilic acid. The nature of thepartial block is not known; all that has been es-tablished is that there is very little enzyme ac-tivity in this reaction. One possible explanationof the partial block (suggested by Bernard Davisof Harvard University), that the enzyme(s) re-sponsible for the synthesis of anthranilic acid isparticularly sensitive to feedback inhibition bytryptophan, is unlikely in view of the findingthat the formation of anthranilic acid by extractsof strain T-41R5 is not unusually sensitive totryptophan. In view, however, of the fact thatfeedback-resistant mutants have been obtained(Moyed, 1960), it is not unlikely that mutantswith an increased feedback sensitivity will alsobe found. Studies with other microbial systemshave shown that partial blocks in the precedingreactions may lead to the formation of high levelsof specific enzymes (Gorini and Maas, 1958;Ames and Garry, 1959). In view of these obser-vations, it might be expected that any compoundwhich competes with one of the intermediates inthe tryptophan pathway could be employed tocontrol the internal supply of tryptophan in muchthe same way that a partial block would operate.Indeed, recent studies with many of the strainsemployed in the present investigation haveshown that high levels of enzyme are producedwhen 3-methyl anthranilic acid is added to theculture medium (Lester and Yanofsky, 1961).The transductions with strain T-41R5 as donor

demonstrate that the locus responsible for highenzyme production is distinct from the originalmutant site in strain T-41, but is closely linkedto this site. A group of 50 additional revertantsof strain T-41 was examined in other tests todetermine whether stocks of the T-41R5 type arefrequent among these revertants. High enzyme

producers were not detected. Furthermore, at-tempts to recover high enzyme producers bytransduction of T-41 to tryptophan independencewith phage grown on wild-type K-12 were alsounsuccessful. Thus, it would appear that the mu-tation responsible for high enzyme productionwas a rare event in strain T-41. The locus fromS. dysenteriae which is responsible for high en-zyme production by some of the hybrids was alsofound to be closely linked to the genes controllingtryptophan synthetase.The results of the transductions from wild-type

K-12 and T-41R5 into S. dysenteriae, and thefact that S. dysenteriae itself is a low enzyme pro-ducer, are difficult to explain. One could assumethat S. dysenteriae has not one but two blocks inanthranilic acid synthesis and any level of trypto-phan supplement supplied represses activity. Therecovery of high producers from the transductionswith wild-type K-12 as donor could be accountedfor by assuming that the more severe block in S.dysenteriae was eliminated but not a second,partial block. Thus, enough tryptophan could beproduced in the recovered strains to allow rapidgrowth in the absence of tryptophan, but notenough to effectively repress tryptophan synthe-tase production. When both blocks are eliminatedby transduction, tryptophan would be producedin amounts which allowed regulation of trypto-phan synthetase formation at the repressed wild-type level and low producers would be recovered.The T-41R5 transductions gave only high pro-ducers in the sample examined, probably by theintroduction of genic material carrying theT-41R5 partial block along with the gene or genespermitting rapid growth in the absence of tryp-tophan.

It is also of interest to compare the levels ofenzyme activity observed in the high producerswith those reported for a strain which is resistantto repression by tryptophan (Cohen and Jacob,1959). In the latter case tryptophan synthetaseproduction was increased only three- to fourfold,although in the present study, instances in whichenzyme levels were consistently increased by afactor of 50 were observed. Theoretically, a par-tial block could not lead to enzyme levels higherthan those obtained in an organism which iscompletely resistant to repression. Thus, otherfactors must play a role in the regulation ofenzyme formation, or mutations may occur whichimpart different degrees of repression resistance.

2031962]


The explanation for the variety of tryptophansynthetase levels observed in the S. dysenteriae-E. coli high enzyme-producing hybrids is notknown.

ACKNOWLEDGMENTS

This investigation was supported by grantsfrom the U. S. Public Health Service and theNational Science Foundation.The authors are greatly indebted to Virginia

Horn and Mary Hurd for technical assistance.

LITERATURE CITED

AMES, B. N., AND B. GARRY. 1959. Coordinaterepression of the synthesis of four histidinebiosynthetic enzymes by histidine. Proc.Natl. Acad. Sci. U. S. 45:1453-1461.

COHEN, G., AND F. JACOB. 1959. Sur la repressionde la synthese des enzymes intervenant dansla formation du tryptophan chez Escherichiacoli. Compt. rend. 248:3490-3492.

CRAWFORD, I, P., AND C. YANOFSKY. 1958. Onthe separation of the tryptophan synthetaseof Escherichia coli into two protein com-ponents. Proc. Natl. Acad. Sci. U. S., 44:1161-1170.

GORINI, L. 1960. Antagonism between substrateand repressor in controlling the formation ofa biosynthetic enzyme. Proc. Natl. Acad.Sci. U. S. 46:682-690.

GORINI, L., AND W. K. MAAS. 1958. Feed-backcontrol of the formation of biosyntheticenzymes, p. 469-479. In W. D. McElroy andB. Glass [ed.,, A symposium on the chemicalbasis of heredity. The Johns Hopkins Press,Baltimore.

LENNOX, E. S. 1955. Transduction of linked geneticcharacters of the host by bacteriophage P1.Virology 1:190-206.

LESTER, G., AND C. YANOFSKY. 1961. Influence of3-methylanthranilic and anthranilic acids onthe formation of tryptophan synthetase inEscherichia coli. J. Bacteriol. 81:81-90.

LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR,AND R. J. RANDALL. 1951. Protein measure-ment with the Folin phenol reagent. J. Biol.Chem. 193:265-275.

MOYED, H. S. 1960. False feedback inhibition:inhibition of tryptophan biosynthesis by5-methyltryptophan. J. Biol. Chem. 235:1098-1102.

PARDEE, A. B. 1959. The control of enzyme ac-tivity, p. 681-716. In P. D. Boyer, H. Lardy,and K. Myrback [ed.], The Enzymes. Aca-demic Press Inc., New York, vol. 1.

SMITH, 0. H., AND C. YANOFSKY. 1960. 1-(o-Carboxyphenylamino)-1-deoxyribulose 5-phos-phate, a new intermediate in the biosynthesisof tryptophan. J. Biol. Chem. 235:2051-2057.

SRINIVASAN, P. R. 1959. The enzymatic synthesisof anthranilic acid from shikimic-5-phosphateand L-glutamine. J. Am. Chem. Soc. 81:1772-3.

VOGEL, H. J. 1957. Repression and induction ascontrol mechanisms of enzyme biogenesis:The adaptive formation of acetylornithinase,p. 276-289. In W. D. McElroy and B. Glass,[ed-], A symposium on the chemical basisof heredity. The Johns Hopkins Press, Bal-timore.

VOGEL, H. J., AND D. M. BONNER. 1956. A con-venient growth medium for E. coli and someother microorganisms (Medium E). MicrobialGenetics Bull. 13:43-44.

YANOFSKY, C. 1955. Tryptophan synthetase fromNeurospora, p. 233-238. In S. P. Colowickand N. 0. Kaplan [eds.l, Methods in en-zymology, Academic Press Inc., New York,vol. 2.

YANOFSKY, C. 1957. Enzymatic studies with aseries of tryptophan auxotrophs of Escherichiacoli. J. Biol. Chem. 224:783-792.

YANOFSKY, C., AND E. S. LENNOX. 1959. Transduc-tion and recombination study of linkage re-lationships among the genes controllingtryptophan synthesis in Escherichia coli.Virology 8:425-447.

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tryptophan synthetase levels in escherichia coli

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