Vol. 54, No. 10APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1988, p. 2504-25090099-2240/88/102504-06$02.00/0Copyright © 1988, American Society for Microbiology

Constitutive Expression of Enniatin Synthetase during FermentativeGrowth of Fusarium scirpiANDREAS BILLICH* AND RAINER ZOCHER

Institut fur Biochemie der Technischen Universitat Berlin, Franklinstrasse 29, D-1000 Berlin 10 (West),Federal Republic of Germany

Received 30 March 1988/Accepted 24 July 1988

The production of enniatins by Fusarium scirpi during fermentative growth in submerged cultures wasmeasured. The fungus produced the antibiotic during mycelial growth, but not during the stationary phase ofcultivation. By contrast, enniatin synthetase, the enzyme responsible for enniatin synthesis, was present duringgrowth, during the stationary phase, and even in spores. Similarly, the enniatin synthetase mRNA was presentat every stage of the cultivation of the fungus. Therefore, this multifunctional peptide synthetase is aconstitutive enzyme, the expression of which is not regulated by any specific mechanism. The findings stand incontrast to the common assumption that production of secondary metabolites underlies regulatory control,leading to separation of the trophophase and the idiophase.

Enniatins are a group of cyclic hexadepsipeptides pro-duced by various species of Fusarium. Many of thesefilamentous fungi are plant pathogens (8, 14), and the ennia-tins belong to a variety of low-molecular-weight phytotoxinsproduced by Fusarium species (11). Together with lycoma-rasmin, lycomarasminic acid, fusaric acid, fusarubin, andjavanicin, they induce wilt deseases in higher plants byinfluencing the water economy of the host (12). Besides theeffect of enniatins in the natural habitat of the fungi, theirantimicrobial (11) and immunomodulatory (N. Simon-La-voine and M. Forgeot, German patent 2851629, 1979) prop-erties are noteworthy. Enniatins consist of three units eachof an N-methylated branched-chain L-amino acid and aD-2-hydroxyisovaleric acid arranged in an alternating fash-ion (Fig. 1). The biosynthesis of these depsipeptides is wellunderstood. A multifunctional enzyme, consisting of one250-kilodalton polypeptide chain, synthesizes enniatins fromtheir primary precursors, i.e., valine, leucine or isoleucine,D-2-hydroxyisovaleric acid, ATP, and S-adenosylmethio-nine. This enzyme, designated enniatin synthetase, waspurified from Fusarium scirpi and studied in our laboratory(6, 7, 20-23). Since enniatin synthetase serves as a modelsystem for enzymes that synthesize peptidic phytotoxins, webecame interested in the regulation of its expression duringthe life cycle of the producer organism. Here we presentresults of our studies on the time course of enniatin produc-tion and enniatin synthetase and its mRNA in submergedcultures of F. scirpi.

MATERIALS AND METHODSCultivation of organisms. F. scirpi Lamb. et Fautr. ETH

1536/9 (previously designated F. oxysporum) and variantsthereof were maintained on FCM agar slants (3% molasses,1% cornsteep liquor, 1.5% agar) (22). Spore suspensions (107conidia per ml) were obtained by filtration of 4-day-oldsubmerged cultures maintained in acetate medium (2)through cotton wool.

All submerged cultures were run on a rotary shaker (115rpm, 27°C) in 500-ml Erlenmeyer flasks containing 100 ml ofmedium. The cultures were inoculated with 2 x 106 conidiaper flask.

* Corresponding author.

Either the FCM liquid medium (22) or the chemicallydefined media (FDM) of Madry et al. (17) was used; the lattercontained the following per liter of distilled water: 12.5 g ofglucose or 25 g of lactose, 4.25 g of NaNO3, 5 g of NaCl, 2.5g of MgSO4 7H20, 1.36 g of KH2PO4, 0.01 g ofFeSO4- 7H20, and 0.0029 g of ZnSO4 .7H20.

Nitrosoguanidine mutagenesis. Nitrosoguanidine mutagen-esis was done by the procedure described by Madry et al.(17).

Determination of enniatins. The content of total enniatinsin the fungal cultures was assayed spectrophotometrically bythe procedure described by Audhya and Russell (1).

Determination of cell dry weight. Defined volumes (10 to 25ml) of culture broth were suction-filtered through pre-weighed filter disks (MN 606; Schleicher & Schuell, Dassel,Federal Republic of Germany). The samples were dried for16 h at 105°C.

Preparation of crude extracts. Crude extracts of F. scirpiwere prepared as described by Zocher and Kleinkauf (22).Lyophilized mycelia or spores were ground to a fine powderin a mortar; for homogenization of spores, sea sand wasadded. The powder was suspended in 50 mM phosphatebuffer (pH 7.2) containing 4 mM dithiothreitol, and thesuspension was stirred for 1 h at 4°C. The extract wascentrifuged for 30 min at 16,000 x g and dialyzed against thesame buffer. The retentate was used for measurement ofenzymatic activity or for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis.Enzyme assay. Enniatin synthetase activity in crude ex-

tracts was determined as described by Zocher at al. (20) byusing L-[14C]valine as the radiolabel and measuring theformation of labeled enniatin B.

SDS-polyacrylamide gel electrophoresis. SDS-polyacryl-amide gel electrophoresis was done as described by Laemmli(15); gels contained 7.5% acrylamide and 0.2% bisacryl-amide. Fluorography was performed by using Amplify(Amersham, Braunschweig, Federal Republic of Germany),and the instructions of the manufacturer were followed. Gelsand fluorographs were scanned by using a thin-layear chro-matographic scanner (CS-930; Shimadzu).Western blots. Immunoblotting was performed as de-

scribed previously (7) by using monoclonal antibodies 21.1and 25.91, which are directed against enniatin synthetase.

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EXPRESSION OF ENNIATIN SYNTHETASE IN F. SCIRPI

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-CHCH(H3)-C

FIrg .d u ctures of ia Ei atin m, m C H(Cf 3LCIch 1cr ure nniatinrB,t EprHpa H; enniatin C,R =for-CH2CH(CH3)2.

Pulse-chase experiments. After 70 h of growth in sub-

merged cultures of FCM liquid medium, 50 ,uCi of L-

[35S]cysteine (600 Ci/mmol; Amersham) was added to 100 ml

of the fungal culture. After 60 m3of incubation, the myceliawere washed twice with FCM liquid medium and then

transferred to 100 ml of FCM liquid medium containing 1

mM L-cysteine. Every 2 h portions were removed, from

which crude extracts were prepared; samples were taken for

up to 14 h.

To measure the incorporation of radioactivity into totalprotein, 500Kl of ice cold 50% tricloroacetic acid was added

to 100ta of crude extract. After 30mg at 4°C, the sample

was filtered through filter disks (GFA; Schleicher &

Schuell). After the filters were washed with 7% trichloroace-

tic acid, they were dried and the radioactivity was measuredby liquid scintillation counting.

mRNA isolation. At different times after inoculation, my-

celia were harvested by suction filtration of the culture broth

through Kleenex tissue and washed with distilled water.Total RNA was isolated from 50 g of the wet mycelium by

the procedure of Flurkey and Kolattukudy (10). Poly(A)+mRNA was selected from the RNA preparation by chroma-

tography on oligo(dT)-cellulose as described by Aviv and

Leder (4), with the intermediate salt wash step omitted.

mRNA was quantitated by measuring the A260 and assuming1 A260 unit = 50 ,ug of RNA per ml. TheA26iJA280 ratio of the

isolated RNA was 1.6-1.7.

In vitro translation. In vitro translation was performed in a

nuclease-treated and amino acid-depleted rabbit reticulocyte

lysate (Amersham). The assay contained 3.4 ,l of lysate, 80

mM K+, 0.8mM Mg2w, 10 ,Ci of [35S]methionine (1,000 Ci!

mmol), 50oM each of the proteinaceous amino acids exceptmethionine, and 0.05 to 1 ,ug of mRNA in a total volume of

5 ,ul. After 1 h at 30°C a 1-pAw portion of the mixture was

subjected to precipitation with trichloroacetic acid on filter

disks, and acid-stable counts were detected by liquid scin-

tillation counting. The incorporation of [35S]methionine was

a linear function of the mRNA that was added to the

reticulocyte lysate under the conditions used. The remainder

of the assay mixture was analyzed by polyacrylamide gel

electrophoresis, and the content of enniatin synthetase syn-

thesized in vitro was determined by scanning the fluoro-

graphs of the gels (*e above). The amount of enniatin

synthetase formed was a linear function of the mRNA

concentration (data not shown).

RESULTS

Enniatin fermentation. When the enniatin-producing fun-gus F. scirpi was grown in submerged cultures, productionof the antibiotic occurred during mycelial growth; the ennia-tin content of the culture rose concomitantly with themycelial dry weight and remained at a constant level whenthe stationary phase was reached (Fig. 2). This pattern ofantibiotic production was observed both with the wild-typefungus, which produced a maximum of 25 mg of enniatin perliter, as well as with variants generated by nitrosoguanidinemutagenesis, which produced up to 50 times more of theantibiotic. In the studies described below, only the highproducer strain J5 was used; this was because of the highlevel of enniatin synthetase that is present in this strain.

Growth-associated enniatin production occurred in allmedia tested, i.e., in a complex cornsteep molasses medium(FCM liquid medium), in FCM liquid medium supplementedwith 1% glucose, and in defined medium (FDM) containingglucose or lactose as the sole carbon source. Growth ofcultures on FDM medium was retarded compared with thaton FCM liquid medium; the stationary growth phase andconstant enniatin titer were reached after 130 and 100 h ofgrowth, respectively (data not shown).The final enniatin titer could be raised by feeding the

precursor amino acid L-valine to the cultures on FCM liquidmedium during the exponential phase of growth, as has beendescribed for FDM cultures by Madry et al. (17) (data notshown). The enniatin content was also raised when L-valinewas added at the transition from the growth to the stationaryphase. The enniatin titer after 120 h on FCM liquid mediumwas about 1.5-fold higher when valine was included at aconcentration of 10 mM after 96 h. This indicates thatenniatin synthetase should still be active even in the station-ary phase.

Enniatin synthetase level. F. scirpi J5 was harvested atvarious times during growth on FCM liquid medium, andcrude extracts were prepared by homogenization of thelyophilized mycelium in phosphate buffer. The activity ofenniatin synthetase in these extracts was measured byincorporation of radiolabeled L-valine into enniatin B. Thetotal activity in the mycelium rose within about 20 h, startingfrom zero at 50 h and reached a maximum after about 70 h(Fig. 2); at this time, mycelial dry weight and the enniatintiter were about half of the maximal amounts. The totalactivity of enniatin synthetase per gram of mycelium peakedafter 60 h and decreased rapidly to 20% of its maximal valuewithin 35 h (Fig. 2).

In addition to the measurement of enzymatic activity,samples of the crude extracts were separated by SDS-polyacrylamide gel electrophoresis. Enniatin synthetase,which appeared as a 250-kilodalton band, was quantitated bydensitometric scanning of the Coomassie blue-stained gels.The plot of the enniatin synthetase content of the extractsversus the time of growth yielded a curve parallel to thatobtained by plotting the total enzymatic activity (Fig. 2).This means that the specific activity (in units per milligram ofprotein) of enniatin synthetase remained constant.

Enniatin synthetase was also detected by immunoblottingthe crude extracts by using monoclonal antibodies directedagainst the enzyme (7); with this sensitive technique enniatinsynthetase was even found in very young mycelia (30 to 50 hon FCM liquid medium). Therefore, we wondered whetherthe enzyme might be present in spores; indeed, the proteinand its activity were detectable in conidia that were freshlyharvested from cultures in FCM liquid medium. The ques-

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2506 BILLICH AND ZOCHER

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FIG. 2. Fermentation of F. scirpi J5 in submerged culture in FCM liquid medium. (A) Cell dry weight (0) and total enniatin content (0).(B) Activity of enniatin synthetase per liter of culture (x), activity of enniatin synthetase per cell (dry weight) (-), and content of the enniatinsynthetase protein per liter of culture (A). One pkat is the amount of enzyme catalyzing the formation of 1 pmol of enniatin BIS.

tion arose as to whether the depsipeptides produced by theenzyme were also present in the spores. A content of 2 mg ofenniatin per g (dry weight) of spores was determined.

Half-life of enniatin synthetase. The half-life of enniatinsynthetase in the mid-exponential phase of growth was

determined. After 70 h of growth on FCM liquid medium,pulse-chase experiments were performed, with L-[35S]cys-teine used as the radiolabel. At various times during thechase period, mycelia were harvested and crude extractswere separated by SDS-polyacrylamide gel electrophoresis.Gels were subjected to fluorography, and the amount ofenniatin synthetase was quantiated by densitometry (Fig. 3);the half-life of the enzyme calculated from these data was

found to be about 12 h.For comparison, the half-life of total soluble protein of the

cells was measured in similar experiments. The labeledproteins were precipitated with trichloroacetic acid on filterdisks, and radioactivity was determined by liquid scintilla-tion counting (data not shown). The half-life measured in thisway was about 12 h. Thus, the rates of degradation ofenniatin synthetase and total protein appeared to be similar.

Enniatin synthetase mRNA. Cellular RNA was isolatedfrom F. scirpi grown in FCM liquid medium. From the totalRNA, poly(A)+ mRNA was selected by chromatography on

oligo(dT)-cellulose. The mRNA was translated in a rabbitreticulocyte lysate. Results of the analysis of the totaltranslation products by SDS-polyacrylamide gel electropho-resis followed by fluorography are shown in Fig. 4. Thefungal proteins produced by mRNA-dependent in vitro syn-

thesis included enniatin synthetase, which appeared as a

prominent band at the 250-kilodalton position.

To estimate the enniatin synthetase mRNA level duringcultivation, total mRNA was isolated from mycelia that wereharvested at different periods of growth. The mRNA wastranslated in vitro, and the products were subjected topolyacrylamide gel electrophoresis followed by fluorog-raphy; the amount of enniatin synthetase formed was esti-mated by densitometric scanning of the fluorographs (Table1). In each case, the same amount of enniatin synthetase wasformed per microgram of added fungal mRNA; the amountwas equivalent to about 3% of the total protein synthesized.If one assumes that all mRNAs translated at the sameefficiency in our system, about 3% of total mRNA encodedfor enniatin synthetase. The total amount ofmRNA that wasisolated from the cultures and, consequently, the absoluteamount of enniatin synthetase mRNA varied considerably.The mRNA contents of exponentially growing mycelia weremuch higher than those of the cultures in the stationaryphase of growth (Table 2).

DISCUSSION

We showed that in submerged cultures of F. scirpi ondifferent media, production of enniatins occurs during my-celial growth but ceases at the beginning of the stationaryphase of fermentative growth. By contrast, a study byAudhya and Russell (3) on the production of enniatins instatic cultures of F. sambucinum led to somewhat differentresults. They observed that liquid surface cultures on semi-defined medium with glucose as th. carbon source passedthrough well-defined phases corresponding to the growthphase (trophophase) and the enniatin production phase (idio-

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EXPRESSION OF ENNIATIN SYNTHETASE IN F. SCIRPI

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t me (hours)FIG. 3. Determination of half-life of enniatin synthetase. Mid-exponential-phase cultures were incubated for 1 h with [35S]cysteine and

were then washed and transferred to FCM liquid medium containing 1 mM cysteine. After different times, mycelia were harvested andenniatin synthetase was measured in crude extracts (see text).

phase); when glucose was replaced by lactose, there was no

separation of the trophophase and the idiophase, but thegrowth rate and enniatin production were in balancethroughout the growth period, as was observed in our

experiments. To explain these contradictory findings,Audhya and Russell (3) have pointed out that glucose might

FIG. 4. Analysis of the translation products of F. scirpi mRNAin a rabbit reticulocyte lysate by SDS-polyacrylamide gel electro-phoresis followed by fluorography. Lane A, Control experiment (noaddition of fungal mRNA); lane B, addition of 0.5 ,ug of fungalmRNA; RNA was isolated from cells that were grown for 72 h on

FCM liquid medium. kDa, Kilodaltons.

repress or inhibit the synthesis of the depsipeptides. Fromresults of our experiments, there was no indication thatcatabolite repression is exerted by glucose. We agree withAudhya and Russell (3), however, in their speculation thatthe accumulation of acidic metabolities might bring aboutinhibition of the enzymes needed for enniatin synthesis.Production of acids led to an unfavorably low pH (as low as3.5) during the beginning of mycelial growth on their glu-cose-containing medium, and enniatin production was re-tarded. In our glucose-containing FDM medium, this drop inpH did not occur, and consequently, enniatin synthesis wasnot inhibited. Thus, the separation of production from thegrowth phase can be considered a laboratory artefact that isobserved when the fungus is grown on a rapidly utilizedcarbon source in a weakly buffered medium.The level of enniatin synthetase, the enzyme responsible

for enniatin synthesis, cannot be deduced simply from thetiter of its product in the culture, because the enzymaticactivity per gram of mycelium reaches its highest level about10 h before the enniatin content increases to its maximallevel. Furthermore, as much as 50% of the maximal synthe-

TABLE 1. In vitro translation of mRNA from F. scirpia

Content (arbitrary units) inTime (h) of vitro translation assay Relative amtgrowth Total Enniatin (%) of enzyme

protein synthetase

60 567,000 16,000 2.972 478,000 15,000 3.287 523,000 17,000 3.3110 532,000 14,000 2.8

A total of 0.5 ,ug of mRNA was used for in vitro translation (see text). Thereaction products were subjected to SDS-polyacrylamide gel electrophoresisfollowed by fluorography. Fluorographs were scanned with a densitometer,and the relative amounts of enniatin synthetase were calculated.

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2508 BILLICH AND ZOCHER

TABLE 2. Content of mRNA in cultures of F. scirpi

Time (h) of Mycelial dry weight Total mRNA contentgrowtha (g/liter) (,ug)/liter of culture

60 1.9 15265 2.5 18872 5.9 24774 6.1 23287 11.2 19096 12.0 120110 12.5 75

a The data were obtained from two independent fermentations. Thoseobtained at 60, 72, 87, and 110 h of growth were from fermentation 1; thoseobtained at 65, 74, and 96 h of growth were from fermentation 2.

sizing activity is found in the extracts of the cells afterenniatin production has stopped (100 h on FCM liquidmedium).

Since the time course of the levels of enzymatic activityand of enzyme protein coincide (i.e., the specific activity ofenniatin synthetase remains constant), the existence of somecovalent modification of the active enzyme to yield aninactive form at the end of the growth phase can be ex-cluded. Therefore, the reason for the cessation of antibioticproduction should lie in a shortage of the substrates neededfor enniatin synthetase; indeed, enniatin production could bestimulated by adding valine to the culture, even at the end ofthe growth phase.Many enzymes of secondary metabolism are repressed

during growth of the producer organism and are induced atthe end of the trophophase, e.g., penicillin acyltransferase(18) and candicidin synthetases (16). By contrast, enniatinsynthetase is present in conidia during mycelial growth andin the stationary phase of fermentative growth. Since thehalf-life of enniatin synthetase, like that of the total solubleprotein of the cells, is only about 12 h, the high content ofenniatin synthetase in the stationary phase can only beexplained by a continuous biosynthesis of the enzyme. Thisconclusion is substantiated by the observation that enniatinsynthetase mRNA is still present in the stationary phase;indeed, the relative amount of this message in the total poolof mRNA is constant. So there is no indication for a specificregulation of the enniatin synthetase gene. However, thetotal amount of mRNA present in the mycelium is lower inthe stationary phase than in the growth phase. The reductionof the enniatin synthetase level only reflects the decreasingamount of mRNA in the fungal cell. A drop in the rate ofprotein synthesis, which is preceded by a drop in the rate ofRNA and DNA biosynthesis, is generally observed at thetransition from the growth to the stationary phase of thecultivation of microorganisms (5); this drop is correlatedwith a decrease in the activities of the enzymes of primarymetabolism. Thus; enniatin synthetase, while producing apeptide that is conventionally regarded as a secondarymetabolite, behaves like a constitutive enzyme of primarymetabolism and in no way seems to be regulated by a specificmechanism either on a transcriptional or on a translationallevel.A major difference between the peptide antibiotics pro-

duced by Bacillus spp. and Streptomyces spp. and thestructurally related phytotoxins made by phytopathogenicfungi is that the latter are synthesized constitutively (J. D.Walton, Michigan State University, East Lansing, Mich.,personal communication). The reason for constitutive bio-synthesis of the fungal toxins, e.g., HC toxin (9), victorin(19) or AM toxin (13), should lie in the importance of their

role in the infection mechanism and in the progress of hostimpairment. Seen in the context of the biological role ofenniatins, it makes sense that enniatin synthetase as anenzyme that is responsible for phytotoxin production isexpressed continuously.

ACKNOWLEDGMENTS

We are indebted to S. Billich and U. Keller for valuable discus-sions. We thank P. Messner and M. Wernitz for skillful technicalassistance.

This study was supported by the Deutsche Forschungsgemeins-chaft, Sonderforschungsbereich 9, Teilprojekt C 3.

LITERATURE CITED1. Audhya, T. K., and D. W. Russell. 1973. Spectrophotometric

determination of enniatin A and valinomycin in fungal extractsby ion complexation. Anal. Lett. 6:265-274.

2. Audhya, T. K., and D. W. Russell. 1973. Production of enniatinA. Can. J. Microbiol. 19:1051-1054.

3. Audhya, T. K., and D. W. Russell. 1975. Enniatin production byFusarium sambucinum: Primary, secondary, and unitary me-tabolism. J. Gen. Microbiol. 86:327-331.

4. Aviv, H., and P. Leder. 1972. Purification of biologically activeglobin messenger RNA by chromatography on oligothymidylicacid-cellulose. Proc. Natl. Acad. Sci. USA 69:1408-1412.

5. Behal, V. 1986. Enzymes of secondary metabolism: regulationof their expression and activity, p. 265-282. In H. Kleinkauf,H. v. Doehren, H. Dornauer, and G. Nesemann (ed.), Regula-tion of secondary metabolite formation. VCH Publishers, Wein-heim, Federal Republic of Germany.

6. Billich, A., and R. Zocher. 1987. N-Methyltransferase functionof the multifunctional enzyme enniatin synthetase. Biochemis-try 26:8417-8423.

7. Billich, A., R. Zocher, H. Kleinkauf, D. G. Braun, D. Lavanchy,and H. Hockkeppel. 1987. Monoclonal antibodies to the mul-tienzyme enniatin synthetase. Biol. Chem. Hoppe-Seyler 386:521-529.

8. Booth, C. 1971. The genus Fusarium. Commonwealth Mycolog-ical Institute, Kew, England.

9. Comstock, J. C., and R. P. Scheffer. 1973. Role of host-selectivetoxin in colonization of corn leaves by Helminthosporiumcarbonum. Phytopathology 63:24-29.

10. Flurkey, W. H., and P. E. Kolattukudy. 1981. In vitro translationof cutinase mRNA: evidence for a precursor form of an extra-cellular fungal enzyme. Arch. Biochem. Biophys. 212:154-161.

11. Gaumann, E., L. Ettlinger, and St. Naef-Roth. 1950. Zur Gewin-nung von Enniatinen aus dem Myzel verschiedener Fusarien.Phytopathol. Z. 16:244-289.

12. Gaumann, E., St. Naef-Roth, and H. Kern. 1960. Zur phytoto-xischen Wirksamkeit der Enniatine. Phytopathol. Z. 40:45-51.

13. Kohmoto, K., I. D. Khan, Y. Renbutsu, T. Taniguchi, and S.Nishimura. 1976. Multiple host-specific toxins ofAlternaria maliand their effect on the permeability of host cells. Physiol. PlantPathol. 8:141-153.

14. Lacey, M. S. 1950. The antibiotic properties of fifty-two strainsof Fusarium. J. Gen. Microbiol. 4:122-131.

15. Laemmli, U. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

16. Liras, P., J. R. Villanueva, and J. F. Martin. 1977. Sequentialexpression of macromolecule biosynthesis and candicidin for-mation in Streptomyces griseus. J. Gen. Microbiol. 202:269-277.

17. Madry, N., R. Zocher, and H. Kleinkauf. 1983. Enniatin pro-duction by Fusarium oxysporum in chemically defined media.Eur. J. Appl. Microbiol. Biotechnol. 17:75-79.

18. Pruess, D. L., and M. J. Johnson. 1967. Penicillin acyltransfer-ase in Penicillium chrysogenum. J. Bacteriol. 94:1502-1525.

19. Yoder, 0. C., and R. P. Scheffer. 1969. Role of toxin in earlyinteractions of Helminthosporium victoriae with susceptible andresistant oat tissue. Phytopathology 59:1954-1959.

20. Zocher, R., U. Keller, and H. Kleinkauf. 1982. Enniatin synthe-

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tase, a novel type of multifunctional enzyme catalyzing depsi-peptide synthesis in Fusarium oxysporum. Biochemistry 21:44-48.

21. Zocher, R., U. Keller, and H. Kleinkauf. 1983. Mechanism ofdepsipeptide formation catalyzed by enniatin synthetase. Bio-chem. Biophys. Res. Commun. 110:292-299.

22. Zocher, R., and H. Kleinkauf. 1978. Biosynthesis of enniatin B:partial purification and charcterization of the synthesizing en-zyme and studies of the biosynthesis. Biochem. Biophys. Res.Commun. 81:1162-1167.

23. Zocher, R., J. Salnikow, and H. Kleinkauf. 1976. Biosynthesis ofenniatin B. FEBS Lett. 71:13-17.

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