JOURNAL OF BACTERIOLOGY, Jan. 1976, p. 158-165Copyright 0 1976 American Society for Microbiology

Vol. 125, No. 1Printed in U.S.A.

Physiological Study of Ergot: Induction of Alkaloid Synthesisby Tryptophan at the Enzymatic Level

VERONICA M. KRUPINSKI, JAMES E. ROBBERS,* AND HEINZ G. FLOSS

Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences,Purdue University, West Lafayette, Indiana 47907

Received for publication 3 July 1975

The enhancement of ergot alkaloid production by tryptophan and its analoguesin both normal and high-phosphate cultures is more directly related to increaseddimethylallyltryptophan (DMAT) synthetase activity rather than to a lack ofregulation of the tryptophan biosynthetic enzymes. Thiotryptophan [$-(1-benzo-thien-3-yl)-alanine] is rather ineffective in the end product regulation oftryptophan biosynthesis, whereas tryptophan and 5-methyltryptophan are

potent effectors. The presence of increased levels of DMAT synthetase in ergotcultures supplemented with tryptophan or thiotryptophan, and to a lesser extentwith 5-methyltryptophan, suggests that the induction effect involves de novo

synthesis of the enzyme. Thiotryptophan and tryptophan but not 5-methyltryp-tophan can overcome the block of alkaloid synthesis by inorganic phosphate. Theresults with thiotryptophan indicate that the phosphate effect cannot beexplained merely on the basis of a block of tryptophan synthesis.

Tryptophan has a central role in the biosyn-thesis of the ergot alkaloids. It has been estab-lished unequivocally that tryptophan is a pre-cursor to the ergoline ring system of the alka-loids (26), and in 1964 Floss and Mothes (9)were the first to suggest that tryptophan mayalso act as an inducer of the alkaloid-synthesiz-ing enzymes. This proposal resulted from obser-vations that tryptophan stimulated alkaloidproduction if added early during the fermenta-tion period before the onset of alkaloid synthe-sis, but not when it was added after alkaloidformation had started, and that tryptophananalogues which were not alkaloid precursorsalso had a stimulatory effect. Evidence support-ing this suggestion was provided by Bu'Lockand Barr (4) when they found that proteinsynthesis was necessary to maintain alkaloidproduction. In addition, using tryptophan-sup-plemented cultures, they found that the seconddifferential of the alkaloid production curve,which would indicate the rate of the appearanceand disappearance of an enzyme(s) limiting therate of alkaloid synthesis, closely paralleled theexperimental curve for internal tryptophan con-centration, suggesting a direct relationship be-tween the rate of synthesis of this enzyme andthe amount of tryptophan within the mycelium.In this regard, we observed an increase in theendogenous, free-tryptophan pool prior to theonset of alkaloid production (17).

Several laboratories including our own pro-vided indirect evidence supporting an inductionof ergot alkaloid synthesis by tryptophan (4, 10,16, 17, 25). In addition, we showed that thewell-known inhibition of alkaloid synthesis byinorganic phosphate is, in some way, mediatedthrough tryptophan because it can be overcomeby the addition of tryptophan but not of 5-methyltryptophan (17). However, among thework done so far, there is little or no cleardemonstration of such an induction effect at theenzymatic level. Also, the tryptophan analoguesstudied usually are much less effective thantryptophan itself in stimulating alkaloid syn-thesis. In this paper we present observations in-dicating that thiotryptophan [,B-(1-benzothien-3-yl)-alanine], although not a substrate in thebiosynthesis, is equally as effective as trypto-phan in stimulating ergot alkaloid formation.Using thiotryptophan as the effector, we carriedout comparative studies on the levels of variousenzymes involved in alkaloid synthesis in in-duced and noninduced cultures.

MATERIALS AND METHODSOrganism and culturing procedures. The strain

of ergot used for this study was Claviceps species,strain SD58. It was originally isolated from sclerotiaobtained from the host Pennisetum typhoideum Rich-ard and is in the culture collection of the Departmentof Medicinal Chemistry and Pharmacognosy, PurdueUniversity.

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PHYSIOLOGICAL STUDY OF ERGOT 159

The organism was maintained on Czapek-Dox agarslants and grown in shake cultures at 24 C at 180 rpmon a New Brunswick model G54 rotary shaker. Culti-vation of the strain was performed by inoculatingflasks of liquid culture medium NL-406 with a portionof the mycelium from an agar slant. Culture mediumNL-406 consists of the following (per liter of distilledwater): mannitol, 50.0 g; sucrose, 50.0 g; succinicacid, 5.4 g; yeast extract (Difco), 3.0 g; KH,PO,, 0.1 g;MgSO4-7H,O, 0.3 g; FeSO4*7H,O, 0.01 g; ZnSO4-7H,O, 0.0044 g; and sufficient NH4OH to bring thepH of the solution to 5.4.The inoculum for induction experiments was pre-

pared by preculturing the organism in shake culturescontaining 100 ml of NL-406 medium with the yeastextract omitted. After 5 to 7 days of growth, 2-mlportions were used to inoculate fresh media.

After the organism had been transferred twice inpreculture, the entire 5- to 7-day-old mycelium from a100-ml shake culture was homogenized in a sterilizedWaring Blendor for 30 s and replaced by collecting themycelium on a sterile, sintered-glass funnel usingvacuum filtration. The mycelial pad was washedthree times with small volumes of sterile, deionizedwater and then transferred to 150 ml of sterile water.

Induction of alkaloid production. Two-millilitervolumes of resuspended mycelia were used to inocu-late experimental flasks, each containing 100 ml ofNL-406 culture medium (yeast extract omitted), a 4mM concentration of either DL-tryptophan, 5-methyl-DL-tryptophan, or DL-thiotryptophan and, in the sec-ond experiment, an additional 1.0 g of KH2PO4 perliter.

Cultures were harvested by suction filtration. Themycelium was washed and weighed, and a wedgesample of mycelium was also weighed separately. Thelarger portion of the mycelium was lyophilized andstored at 4 C for enzyme assays and free intracellulartryptophan quantitation. The wedge sample of myce-lium was dried at 55 C for 24 h.

Quantitation of alkaloids. The amount of alkaloidwas determined in micrograms per milligram ofmycelial dry weight.

Total mycelial dry weight was obtained from thefollowing relationship:total mycelial dry weight

= total mycelial wet weight xdry weight of mycelial wedgewet weight of mycelial wedge

Alkaloids were quantitated by making 2 ml ofculture filtrate alkaline with 0.2 ml of 10% NH40Hand extracting the alkaloids with 2 ml of CHCl,. Tothe residue from a known volume of this CHCl8extract was added 1 ml of 2% succinic acid and 2 ml ofvan Urk reagent (24) modified by the method ofAllport and Cocking (1); after 20 min, the opticaldensity at 580 nm was measured. The quantity ofalkaloid (micrograms per milliliter), calculated aselymoclavine, was determined by multiplying theabsorbancy at 580 nm by 38.7, which is the reciprocalof the slope of a standard curve based on elymocla-vine.

Investigation of DL-thiotryptophan as a precur-sor to the ergoline ring system of alkaloids. StrainSD58 was grown in NL-406 medium supplementedwith 4 mM DL-thiotryptophan. At the beginning of thegrowth period, 10 gCi of [2- "4C ]mevalonic acid wasadded through a membrane filter (Millipore Corp.) tothe culture. Using a partitioned CHCls extract of an11-day-old culture filtrate, thin-layer chromatogra-phy in CHCl,-methanol-NH,OH (80:20:0.2) was car-ried out on precoated Silica Gel G plates (Merck).Radioactive compounds were detected by using aradiochromatogram scanner, alkaloids were detectedby spraying with Dragendorff reagent (23), and indolealkaloids were detected by using Ehrlich reagent (1.0g of p-dimethylaminobenzaldehyde, 10 ml of water,and 20 ml of concentrated HCI). Additional separa-tion of the CHCl, extract of the 11-day-old culturewas effected by evaporating the extract to a dryresidue, which was taken up in a small volume ofmethanol and applied to a column prepared from 10 gof alumina (Brockmann). The alkaloids were elutedwith varying concentrations (0.5 to 10%) of methanolin CH,Cl,, and the fractions were chromatographedand treated as mentioned above.

Determination of free intracellular tryptophan.Tryptophan was assayed in the mycelium by themethod of Spiess and Chambers (20). Lyophilizedmycelial samples were finely ground in a mortar andpestle, and weighed portions (usually 100 mg) wereextracted with 5 ml of deionized water in a small testtube suspended in a boiling-water bath for 30 min.The mycelial powder was separated by centrifugation,and 6 ml of the supernatant fluid was adjusted to pH11 and extracted with 2 ml of CHCl, to removepenniclavine, which is sufficiently soluble in hotwater to interfere with the tryptophan quantitation.The extracted supernatant was assayed by the colori-metric method based on the reaction of tryptophanwith p-dimethylaminobenzaldehyde.Determination of DAHP synthetase activity.

The crude enzyme extract used for the assay of thetryptophan-sensitive isozyme of 3-deoxy-D-arabino-2-heptulosonic acid (DAHP) synthetase (EC 4.1.2.15)was prepared by the method of Schmauder andGroger (19). The powdered, lyophilized mycelium(500 mg) was mixed with 0.1 M phosphate buffer (pH7.2) containing 1 mM mercaptoethanol and groundwith dry ice in a mortar and pestle. After centrifuga-tion at 27,000 x g for 20 min, the supernatant waspassed through a Sephadex G-25-80 column.DAHP synthetase activity was determined by mea-

suring the amount of DAHP formed from erythrose4-phosphate and phosphoenolpyruvate, based on themethod of Sprinson and Srinivasan (21, 22). For theassay, a solution containing 0.5 Amol of phosphoenol-pyruvate, 1 pmol of L-tyrosine, 1 gmol of L-phenylala-nine, 0.5 pmol of D-erythrose 4-phosphate, 10 pmol ofpotassium phosphate buffer (pH 6.9), and 0.1 pumol ofmercaptoethanol was prepared to give a final volumeof 1 ml after the addition of enzyme. After preincuba-tion of the constituents at 37 C for 10 min, thereaction was initiated by the addition of enzyme, andafter 10 min of incubation at 37 C it was stopped by

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160 KRUPINSKI ET AL.

the addition of 0.4 ml of 10% trichloroacetic acid. Themixture was centrifuged and 0.25 ml of supernatantwas removed for the assay of DAHP. DAHP was

estimated by the method of Doy and Brown (5). Inthis assay, DAHP is oxidized to yield a compoundthat reacts with thiobarbituric acid to give a chromo-phore with an absorption maximum at 549 nm. Theconcentration of DAHP was calculated by using a

molar extinction coefficient of 41,000 (22).The enzymatic reaction was not linear even for a

short period of time; therefore, in accordance withprevious work on DAHP synthetase in Clavicepsspecies, strain SD58 (19), we used a 10-min incuba-tion period. Inherent in the interpretation of the datais the assumption that the differences in the amountof product formed reflect differences in the amount ofenzyme present rather than differences in the stabil-ity of enzyme.

Preparation of crude enzyme extract for anthra-nilate synthetase, tryptophan synthetase and di-methylallyltryptophan (DMAT) synthetaseassays. One gram of the powdered, lyophilized myce-lium was stirred at 0 to 4 C for 2 h with 15 ml of 0.5 Mpotassium phosphate buffer (pH 7.2) containing 20mM mercaptoethanol, 0.1 mM ethylenediaminetetra-acetate, and 20% glycerol. This suspension wascentrifuged at 27,000 x g for 20 min and the superna-tant was used for the assays.

Anthranilate synthetase assay. Anthranilate syn-

thetase was assayed by the method of Pabst et al.(15). The appearance of anthranilate was measuredspectrofluorometrically with excitation at 320 nm andemission at 400 nm. The assay was performed at 25 Cby adding to the cuvette 2 umol of MgCl,, 25 ,mol ofglutamine, 70 psmol of tris(hydroxymethyl)amino-methane-hydrochloride buffer (pH 7.8), and waterand/or enzyme to bring the volume to 0.9 ml. Thereaction was started by addition of 0.1 ml of 1 mMchorismate solution followed by measuring the initialrate (percent transmittance) for approximately 3 min.The percent transmittance of a standard solution ofanthranilic acid was read with each assay.

Tryptophan synthetase assay. Tryptophan syn-thetase (EC 4.2.1.20) activity was determined bymeasuring unreacted indole using Ehrlich reagent(27). The solution for incubation was prepared bymixing 8 ,umol of indole, 1.6 mmol of DL-serine, 20,gmol of glutathione, 1.6 Mmol of pyridoxal phosphate,1 mmol of potassium phosphate buffer (pH 7.8), and0.6 ml of saturated NaCl to bring the total volume to13 ml. For the incubation, 0.35 ml of water and/orcrude enzyme extract was mixed with 0.65 ml of themixture containing buffer, substrates, and cofactorsto give a total volume of 1 ml. Incubation was at 37 Cfor 2 h with boiled-enzyme controls to compensate forthe indole absorbed by protein but not converted totryptophan. Incubation was stopped by placing assay

tubes in ice and adding 3 ml of toluene. Indole was

extracted by shaking the tubes.For the indole assay, a 0.5-ml volume of the toluene

layer was mixed with 1.5 of Ehrlich reagent followedby 2 ml of 95% ethanol. After vigorous mixing followedby color development for 20 min, optical density was

measured at 540 nm. Optical density of an indolestandard (100 nmol/ml) was measured daily. Theamount of tryptophan formed was found by determin-ing the difference between the boiled control and theenzyme incubations, since the amount of tryptophanformed equals amount of indole used.DMAT synthetase. DMAT synthetase activity

was determined by the conversion of dimethylallyl-[1- 14C]pyrophosphate (DMAPP) to 4-DMAT. For theassay, 0.22 gmol of [1- "IC]DMAPP (with known radio-activity), 1 jUmol of L-tryptophan, a volume of crudeenzyme extract containing 1 mg of protein, andsufficient 0.01 M tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 8) containing 20 mM mer-captoethanol, 20 mM CaCl,, and 10% glycerol to givea total volume of 0.5 ml were mixed and incubated 1 hat 30 C. It has been determined that during thisperiod of incubation the enzyme activity is linearwith time (S.-L. Lee, H. G. Floss, and P. F. Heinstein,submitted for publication). The reaction was stoppedby the addition of 0.1 ml of 2 N HCl in 80% ethanol.After 15 min, 0.1 ml of this solution was spotted on astrip of Whatman no. 1 filter paper and dried. Thepaper was exposed to steam for approximately 15 minto remove volatile radioactive material. The radioac-tivity incorporated into 4-DMAT was counted in Braysolution. A blank without tryptophan was run todetermine background radioactivity due to polymeri-zation of DMAPP.

Protein estimations. Protein was estimated by themethod of Lowry et al. (14), with bovine serumalbumin as a standard.

Chemicals. DL-Tryptophan and 5-methyl-DL-tryp-tophan were obtained from Sigma Chemical Co.DL-Thiotryptophan [,B-(1-benzothien-3-yl)-alanine]was supplied by E. Campaigne, Indiana University,Bloomington, and was synthesized by the method ofAvakian et al. (2). DL-[2-'4C ]mevalonic acid (3.06mCi/mmol) as the DBED salt was purchased fromTracerlab, and (1-_"C]DMAPP was prepared as de-scribed by Heinstein et al. (11). Sodium D-erythrose4-phosphate was obtained from Calbiochem as thedimethylacetal of cyclohexylammonium-D-erythrose4-phosphate and was prepared for use in the DAHPassay by the method of Ballou et al. (3).

RESULTS AND DISCUSSIONTwo sets of experiments were carried out, one

in NL-406 lacking yeast extract and the other inthe same medium but with an elevated inor-ganic phosphate level to suppress alkaloid for-mation. In each case the effect of tryptophan,thiotryptophan, and 5-methyltryptophan on al-kaloid synthesis, free intracellular tryptophanconcentration, and on levels of four enzymes(three enzymes involved in tryptophan forma-tion and the first enzyme of the ergot alkaloidbiosynthetic pathway, DMAT synthetase [11])was investigated. Both sets of experiments gaveroughly comparable results.Our observations reaffirm previous work con-

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PHYSIOLOGICAL STUDY OF ERGOT 161

cerning the induction of alkaloid formation bytryptophan and its analogue 5-methyltrypto-phan (Fig. 1 and 2). In addition, they show, forthe first time, that the tryptophan analoguethiotryptophan can be equally as efficient as, ifnot more efficient than, the parent compound ininducing alkaloid formation. The greater en-hancement of alkaloid synthesis caused by thio-tryptophan than that caused by tryptophan(Fig. 1) becomes quite significant when oneconsiders that tryptophan can also serve as aprecursor to the alkaloids, whereas thiotrypto-phan cannot. If thiotryptophan could serve as asubstrate for the alkaloid-synthesizing en-zymes, the product of such a reaction would notcontain the indole ring system, which is re-quired for detection by the color reaction withp-dimethylaminobenzaldehyde used to visual-ize the alkaloids on chromatograms. We there-fore examined the possibility that thiotrypto-phan might serve as a substrate by feedingradioactively labeled mevalonic acid, a secondprecursor to the alkaloids, to thiotryptophan-supplemented cultures of the fungus. We coulddetect no major radioactive product that wouldbe the result of such a reaction; however, ourexperimental procedure does not rule out the

NL-406 CULTURE MEDIUM (YEAST EXTRACT OMITTED) PLUS

SC- O No Addition0 4mM DL-TryptophonA 4mM 5-mtthyl-DL-Tryptophon* 4mM DL-Thiotryptophon

2Lit

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NL-406 CULTURE MEDIUM (PLUS 1.0g./I. KH2P04,YEAST EXTRACT OMITTED) PLUS:o No Additiono 4mM DL-Tryptophon& 4mM 5-Msthyl-DL-Tryptophon* 4mM DL-Thiotryptophan

II

O 1 2 3 4 5 6 7 8 9 10 11 12 13 14AGE OF CULTURE (Days)

FIG. 2. Induction of alkaloid production by trypto-phan and its analogues in Claviceps species, strainSD58, grown in high-phosphate medium.

formation of trace compounds. Unequivocalsupport for our conclusion comes from observa-tions that DMAT synthetase does not acceptthiotryptophan as a substrate (Lee, Floss, andHeinstein, submitted for publication).One of the principle findings of this investiga-

tion is that the enhancement of alkaloid produc-tion by tryptophan and its analogues in bothnormal and high-phosphate cultures is moredirectly related to increased DMAT synthetaseactivity than to a lack of regulation of thetryptophan biosynthetic enzymes. Our dataillustrate that the biosynthesis of tryptophan instrain SD58 of the ergot fungus is under regula-tory control by the end product. It appears thatthiotryptophan is rather ineffective as a substi-tute for tryptophan in the end product regula-tion of tryptophan biosynthesis, whereas, asmight be expected, tryptophan itself is the mostpotent effector (Fig. 3 through 5). 5-Methyl-tryptophan approaches the ability of the parentcompound to control tryptophan biosynthesis.This agrees with the earlier data of Eberspiicheret al. (7), who observed that thiotryptophan didnot inhibit the tryptophan-sensitive isozyme ofDAHP synthetase in strain SD58 of ergot,whereas tryptophan and 5-methyltryptophancaused a 95 and 79% inhibition, respectively. In

AGE OF CULTURE (DAYS)

FIG. 1. Induction of alkaloid production in Clavi-ceps species, strain SD58, by tryptophan and itsanalogues.

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162 KRUPINSKI ET AL.

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AGE OF CULTURE (DAYS)

FIG. 3. Time course showing DAHP synthetase(tryptophan-sensitive isozyme) activity, based onmilligrams ofprotein, in induced cultures of Clavicepsspecies, strain SD58.

10_NL-406 CULTURE MEDRJM (YEAST EXTRACT OMTTED) PLLS:

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O 2 3 4 5 6 7 8 9 10 11 12 13 14AGE OF CULTURE (DAYS)

FIG. 5. Time course showing tryptophan synthe-tase activity, based on milligrams of protein, ininduced cultures of Claviceps species, strain SD58.

addition, it has been reported that anthranilatesynthetase is insensitive to inhibition by trypto-phan in a strain of Claviceps paspali (13)although not in Claviceps species, strain SD58(12). The enzyme in our experiments is clearlyunder control by tryptophan (Fig. 4); detailedstudies of the purified enzyme from Clavicepsspecies, strain SD58, showed normal feedbackinhibition by tryptophan (D. F. Mann, H. G.Floss, unpublished data). The data on trypto-phan synthetase obtained in this study (Fig. 5)differ considerably from that published earlier(17). This is due to a new procedure for theisolation of the enzyme, employing glycerol-containing buffers. As it turns out, only a verysmall fraction of the total amount of enzymepresent was extracted by the earlier procedure.Although the levels of tryptophan biosyn-

thetic enzymes do not correlate with alkaloidformation, there is a clear parallel betweenDMAT synthetase levels and alkaloid produc-tion. The increased enzyme levels in culturessupplemented with tryptophan or thiotrypto-phan and, to a lesser extent in those supple-mented with 5-methyltryptophan (Fig. 6), sug-gest that the induction effect involves de novosynthesis of the enzyme. Preliminary observa-tions from Gr6ger's laboratory (8) suggest thatthe same is true for another enzyme in thealkaloid biosynthetic pathway, chanoclavinecyclase. The fact that thiotryptophan producesas high levels of DMAT synthetase as trypto-phan both in normal (Fig. 6) and high-phos-

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2 3 4 5 6 7 8 9 10 11 12 13 14AGE OF CULTURE (DAYS)

FIG. 4. Time course showing anthranilate synthe-tase activity, based on milligrams of protein, ininduced cultures of Claviceps species, strain SD58.

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PHYSIOLOGICAL STUDY OF ERGOT 163

NL-406 CULTURE MEDIUM (YEAST EXTRACTO No Additiono 4mM DL-TrypoplA 4mM 5-melytl-DL-Trypplon* 4mM DL-Thiotryptophm

0 Z 3 4 5 6 7 8 9 10

AGE OF CULTURE (DAYS)

FIG. 6. Time course showing DM)activity, based on milligrams of protecultures of Claviceps species, strain Sl

OMITTED) PLUS phosphate medium, a high level of DMATsynthetase corresponds to a low cellular trypto-phan level (Fig. 7 and 9).

It is interesting that thiotryptophan, liketryptophan itself, can overcome the block ofalkaloid synthesis by inorganic phosphate (Fig.2). 5-Methyltryptophan, as in earlier experi-ments (17), has little effect in this respect. It isalso evident that in regular high-phosphatecultures the synthesis of the alkaloid-formingenzymes is initiated, possibly because a trigger-ing level of the inducer has not been reached.The results with thiotryptophan clearly showthat the phosphate effect cannot be explainedmerely on the basis of an inhibition of trypto-phan synthesis. A reasonable interpretationcould be based on the assumption that phos-phate causes a metabolic shift from higher tolower pentose phosphate shunt activity andfrom lower to higher glycolytic activity with aresultant decrease in the activity of the trypto-phan biosynthetic pathway (E. N. Udvardy, F.Fock, G. Wack, K. Zalay, and E. Zsoka, pre-

1'1 1'2 13 14 sented at the Conference on Medicinal Plants,Marianske Lazne, Czechoslovakia, April 1975).

AT synthetase Thiotryptophan and, to a lesser extent, 5-?in, in induced methyltryptophan would still trigger the forma-)58. tion of DMAT synthetase and the other en-

zymes of the alkaloid biosynthesis. Consump-phate cultures (Fig. 7), despite differences inthe cellular free-tryptophan levels (Fig. 8 and9), indicates that thiotryptophan must be aboutequally effective as tryptophan as an inducer.5-Methyltryptophan, on the other hand, seemsto be a considerably less effective inducer. Likethiotryptophan, this compound is not an alka-loid precursor (9). In addition, 5-methyltrypto-phan, while triggering the formation of alkaloid-synthesizing enzymes, limits tryptophan syn-thesis by false feedback inhibition and/orrepression, whereas thiotryptophan does not(Fig. 3 through 5). (Although the data are notshown, comparable results were also obtained inthe assay of the tryptophan biosynthetic en-zymes in high-phosphate cultures.) This ex-plains why 5-methyltryptophan is consistentlymuch less effective in stimulating ergot alkaloidsynthesis than tryptophan, whereas thiotrypto-phan is equally as effective or, in some experi-ments, even more effective than the parentcompound. The argument might be made thatthiotryptophan is not intrinsically an effector inthe induction process, but acts indirectly byinhibiting tryptophan degradation. However,this seems very unlikely because, in this case,DMAT synthetase levels should strictly corre-late with cellular tryptophan levels, which is nottrue. In the thiotryptophan experiment in high-

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o-4 rM IL-TryptophonA 4Md 5-Methyl-DL-Tryptophan* 4 mM DL-TNolryptophon

2 3 4 5 6 7 8 9 10 11 12 13 14AGE OF CULTURE (Days)

FIG. 7. Time course showing DMAT synthetaseactivity, based on milligrams of protein, in inducedcultures of Claviceps species, strain SD58, grown inhigh-phosphate medium.

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164 KRUPINSKI ET AL.

tion of tryptophan by alkaloid synthesis wouldturn on increased tryptophan formation in thethiotryptophan experiment, but not in the othercase, because 5-methyltryptophan would causean additional limitation of tryptophan synthe-sis. In agreement with this explanation, free-tryptophan levels are low in the thiotryptophanexperiment in high-phosphate medium (Fig. 9)as compared with that in the low-phosphatecultures (Fig. 8), yet DMAT synthetase levelsare comparable (Fig. 6).

In our opinion these results provide strongevidence that tryptophan, or a derivativethereof, is indeed an inducer of alkaloid synthe-sis in Claviceps species, strain SD58. The mech-anism by which this is accomplished most likelyinvolves the de novo synthesis of the enzymes inthe biosynthetic pathway leading from trypto-phan to the alkaloids as evidenced by theincreased specific activity in induced cultures ofDMAT synthetase, the first enzyme in thealkaloid biosynthetic pathway. Clear-cut exam-ples of an induction phenomenon in the produc-tion of secondary metabolites are rare in theliterature. The role of methionine in cephalo-

NL-406 CULTURE MEDIUM (PLUS 1.0g./I. KH2P04,YEAST EXTRACT OMITTED) PLUS:o No Additiono 4 mM DL-Tryptophon*& 4mM 5-Methyl-DL-Tryptophon* 4 mM DL-Thiotryptophan

NL-406 CULTURE MEDIUM (YEAST EXTRACT OMITTED) PLUS:O No Additiono 4mM DL-TryptophnA 4mM 5-methyl-DL-Tryptophm* 4mM DL-Thiotryptophan

I~~~ ~I0I

2 3 4 5 6 7 8 9 10 11 12 13 14AGE OF CULTURE (DAYS)

FIG. 8. Time course showing endogenous free-tryp-tophan levels in mycelia of induced cultures of Clavi-ceps species, strain SD58. In 5-methyl-DL-trypto-phan-induced cultures, the level of endogenous freetryptophan represents the sum offree tryptophan plus5-methyltryptophan.

AGE OF CULTURE (Days)

FIG. 9. Time course showing endogenous free-tryp-tophan levels in mycelia of induced cultures of Clavi-ceps species, strain SD58, grown in high-phosphatemedium. In 5-methyl-DL-tryptophan-induced cul-tures, the level of endogenous free tryptophan repre-sents the sum of free tryptophan plus 5-methyltrypto-phan.

sporin C formation (6) and in fosfomycin bio-synthesis (18), however, seems to be somewhatsimilar to that of tryptophan in ergoline biosyn-thesis. Finally, it might be noted that theresults of this study tend to argue against one ofthe concepts that is occasionally involved intrying to explain the formation of secondarymetabolites. Since little or no correlationseems to exist between alkaloid synthesis andthe ability to synthesize tryptophan and sincetryptophan biosynthesis in this organism iswell-regulated, it seems unlikely that the ergotalkaloids are "metabolic accidents" due to aregulatory defect in primary metabolism.

ACKNOWLEDGMENTSWe are greatly indebted to E. Campaigne, Department of

Chemistry, Indiana University, Bloomington, for a generoussupply of the thiotryptophan used in this study.

This work was supported by Public Healtlh Service re-search grants AM 11662 and CA 17482 from the NationalInstitute of Arthritis, Metabolism, and Digestive Diseases andthe National Cancer Institute, respectively.

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VOL. 125, 1976

LITERATURE CITED

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