the antibiotic cerulenin, noveltool for biochemistry ... · vol. 40, 1976 f r.!l ---i.1., _ 686...

BACTERiOLOGICAL REVIEWS, Sept. 1976, p. 681-697Copyright © 1976 American Society for Microbiology

Vol. 40, No. 3Printed in U.S.A.

The Antibiotic Cerulenin, a Novel Tool for Biochemistry as

an Inhibitor of Fatty Acid SynthesisSATOSHI OMURA

Kitasato University, Minato-ku, Tokyo, Japan

INTRODUCTION .............................................................. 681

PRODUCING STRAIN AND PRODUCTION OF CERULENIN .... ............. 681PHYSICOCHEMICAL PROPERTIES AND STRUCTURE .... .................. 682BIOLOGICAL PROPERTIES OF CERULENIN ................................ 683

ACTION MECHANISM OF CERULENIN ...................................... 684

EFFECT ON FATTY ACID SYNTHETASE .................................... 686

EFFECT ON THE ENZYMES INVOLVED IN STEROL SYNTHESIS .... ..... 689INHIBITION OF "POLYKETIDE" SYNTHESIS ............................... 689

APPLICATION AS A BIOCHEMICAL TOOL .................................. 691

LITERATURE CITED......................................................... 694

INTRODUCTIONOne ofthe most versatile uses of antibiotics is

as potent drugs for clinical application. In re-cent years, attention has also been paid to agri-cultural uses of antibiotics, such as for feedadditives for protecting plants and livestockagainst infectious diseases and for acceleratingtheir growth. They are also used as food addi-tives to retain freshness for an extended period.The usefulness of antibiotics is not limited

only to our daily needs, but also encompassesour research interests: they offer us remarkableexperimental devices for biochemistry - novelbiochemical tools, which have made a signifi-cant contribution to progress in this field (18).

Cerulenin, an antibiotic discovered by Hataet al. in 1960, was originally found as an anti-fungal antibiotic (30). Studies of its mode ofaction have revealed that it specifically inhibitsthe biosynthesis of fatty acids and sterols in-volving yeasts (55, 56). It should be particularlynoted that such specificity of cerulenin has beenused by investigators in various fields of bio-chemistry. In this connection, the present re-view deals with studies, which have hithertobeen reported, on the production, isolation,structure, and mode of action of cerulenin andits application as a biochemical tool. Unfor-tunately, the instability of the antibiotic in theanimal body prevents its use in therapy as anantimicrobial agent or as an antilipogenicagent.

PRODUCING STRAIN AND PRODUCTIONOF CERULENIN

The cerulenin-producing strain has someunique characteristics: it forms aerial hyphaeand conidiophore when applied on Czapek agaras shown in Fig. 1 and Fig. 2, respectively, and

it forms bluish-green aerial mycelium on maltagar. It has thus been classified as genus Ceph-alosporium and accordingly named Cephalo-sporium caerulens (49).The production of cerulenin was originally

performed under cultural conditions of 27 to32TC by using glucose and glycerol as carbonsources and peptone and corn steep liquor as ni-trogen sources (50). Unfortunately, however, itwas found that helvolic acid and steroidal anti-biotics (Fig. 3) were produced predominantlyover cerulenin under these conditions. Such dif-ficulties precluded the usefulness of thismethod for efficient production of cerulenin.Meanwhile, because much attention began tobe paid to its specific mode of action as aninhibitor of lipid biosynthesis, a method wasneeded to improve the yield of cerulenin. As amatter of course, the condition of fermentativeproduction of the antibiotic that was originallyreported was reexamined.As a beginning step to do so, the antimicro-

bial activities of cerulenin and helvolic acidwere reevaluated. Among the microorganismslisted, Candida albicans KF-1 and Corynebac-terium paurometablum KB-121 were chosen asthe most appropriate test microorganisms forthe selective bioassay of cerulenin and helvolicacid, respectively (36).Attempts were subsequently made to investi-

gate the medium for the selective production ofcerulenin. Although a previously employed pro-duction medium (50) that contained 6.0% glu-cose, 0.5% peptone, 0.3% NaCl, and 0.3%CaCO3 afforded mostly helvolic acid (350 ,tg/ml) and a small amount of cerulenin (50 ,tg/ml), further study revealed that a more suitablemedium for the efficient production ofceruleninshould contain 1% glucose, 3% glycerol, 0.5%peptone, and 0.2% NaCl. By using this produc-

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FIG. 1. Aerial hyphae of C. caerulens, cultured on Czapek agar at 270C for a week.

tion medium, 250 pug of cerulenin per ml wasobtained, with an almost negligible yield of hel-volic acid. Thus, this improved method offereda simple procedure to produce and isolate arelatively large amount of the antibiotic at onetime and in a short time.To prepare 3H-labeled cerulenin under these

producing conditions, further studies weremade with regard to the effect of the time of the[3H]acetate addition and the glucose concentra-tion in culture broth on the incorporation of[3H]acetate into cerulenin. The results showedthat the highest specific radioactivity (4.5 x10-3 cpm/mg) of 3H-labeled cerulenin was ob-tained by using the culture broth containing 2%glucose when [3H]acetate (1 ,uCi/ml) wasadded 14 h after the initiation of fermentation.'3C-labeled cerulenin was then prepared byadding the [L3C]acetate under these conditions.The nuclear magnetic resonance (NMR) spec-trum of the '3C-labeled cerulenin thus obtainedsuggested that its biosynthesis arises from sixmolecules of acetates. Therefore, the biosyn-thesis of cerulenin is closely related to that offatty acids, which are catalyzed by fatty acidsynthetase (4).

Helicoceras oryzae (K. Furuya and M. Shira-saka, Japan Kokai patent 45-21638), Sartoryasp. (T. Yamano, H. Yamamoto, and S. Itsumi,Japan Kokai patent 47-24156), Acrocylindriumsp. (Furuya and Shirasaka, patent 45-21638),etc., have been known as the producing strainsof cerulenin other than C. caerulens. On theother hand, it is known that helvolic acid,which tends to be produced simultaneouslywith cerulenin, is produced by Aspergillusfumigatus mutant helvola (9), Emericellopsisterricola (62), and Acrocylindrium oryzae (15).It is of interest to note in connection withchemotaxonomy that one compound arises fromvarious kinds of producing strains, as seen inthis case.

PHYSICOCHEMICAL PROPERTIES ANDSTRUCTURE

It should be noted that the isolation andstructural determination of antibacterial protolanostan-type antibiotics, helvolic acid (62),helvolinic acid (63), and 7-desacetoxyhelvolicacid (63), and the pentaprenyl terpenoid anti-biotic cepharonic acid (35), preceded those ofcerulenin despite the fact that all of these anti-

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CERULENIN, INHIBITOR OF FATTY ACID SYNTHESIS 683

FIG. 2. Conidiophore of C. caerulens, cultured on Czapek agar at 270C for a week.

biotics are produced by the same producingstrain (Fig. 3). This was mainly due to the factthat cerulenin is somewhat unstable in metha-nolic solutions and that it does not show a sin-gle spot on a thin-layer chromatography platewhen eluted with some solvent systems con-taining methanol. It was thus extremely diffi-cult to prove that it was a single compound.Notwithstanding, molecular weight and themolecular formula were finally determined forthe acid by mass and NMR spectroscopicmethods.Table 1 shows the physicochemical properties

of cerulenin (76). The infrared spectrum (Fig. 4)(3,425, 3,225, 1,663 cm-', KBr) and the evolu-tion of ammonia upon alkaline hydrolysis arecompatible with the presence of a primaryamide moiety in cerulenin (63). It is also sug-gested by its infrared spectrum that there aretrans-1,2-disubstituted olefin (969 cm-') and acarbonyl group (1,720 cm-'). In agreementwith the NMR, infrared, and mass spectra ofthe products obtained by the chemical trans-formations shown in Fig. 5 (63, 64), comparisonof the optical rotary dispersion curve of hexa-

hydrocerulenin with that of L-methyl malatereveals that the steric configuration of the two1,2-disubstituted olefins are both trans and thatthe absolute configuration of the cis-epoxide is(2S,(3R) (63, 64). By the use of NMR, the sol-vent effect at 100 MHz further establishes theposition of the two olefinic bonds. Thus it isunambiguously concluded that the structure ofcerulenin is (2S),(3R) 2,3-epoxy-4-oxo-7,10-do-decadienoyl amide (3).The fact that cerulenin is unstable in metha-

nolic solutions is attributed to the structuraltransformation of the epoxide moiety locatedbetween the two carbonyl groups on C1 and C4as evidenced by its CD spectrum (64).

BIOLOGICAL PROPERTIES OFCERULENIN

As indicated in Table 2 and Table 3, ceru-lenin possesses a remarkable growth-inhibitoryaction on various strains of mycobacteria,except Mycobacterium tuberculosis, Nocardia,and Streptomyces. A weak growth-inhibitoryactivity against other bacteria is also recog-nized. Furthermore, it inhibits significantly

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OAc

Helvolic acid (62)

CO2H

Helvolinic acid (63)

OH

0

Cephalonic acid (35)7-Desacetoxy helvolic acid (63)

FIG. 3. Products synthesized with cerulenin by C. caerulens.

TABLE 1. Physicochemical properties of cerulenin

Property Expt results

Molecular formula ... C12H1703N (mol wt 223.26)mp ... ... 93-940CIa]2.6. -12o (C 1.0, CHCl3)aSolubility ...... Soluble in CHCl3, CC14,

EtOAc, benzene; slightlysoluble in H20

Ultraviolet ...... End absorption in H20Infrared ..... . Fig. 2

a In a previous report (76), the optical rotation ofcerulenin was reported to be [a]25 +630 (C 2.0,MeOH); however, it was reexamined in CHCl3 be-cause of the instability of cerulenin in MeOH.

the growth of yeastlike fungi, such as Candida,Saccharomyces, Cryptococcus, Geotrichum, etc.,and Trichophyton, Epidermophyton, Micro-sporium, Piricularia oryzae, Ophiobolus miya-beanus, etc.

Cerulenin shows neither hemolytic action onvarious erythrocytes nor irritability on the con-jugative mucous membrane of rabbits. Its tox-icity to mice is, consequently, relatively low(51) (Table 4).

It has been found that cerulenin causes curl-ing of the microorganisms that belong to fila-mentous fungi, such as Trichophyton and Mi-

crosporium (51). Among other derivatives ofcerulenin in Fig. 5, the only one that exhibitsantimicrobial activity is tetrahydrocerulenin(16, 66). This in turn suggests that the presenceof the epoxide moiety is responsible for its sa-lient antimicrobial activity.

ACTION MECHANISM OF CERULENINIt has been reported that helvolic acid simul-

taneously produced with cerulenin inhibits theprotein synthesis of Escherichia coli (22; Ya-mano et al., patent 47-24156). Studies of theaction mechanism of cerulenin have begun withthe intriguing observation that the antibioticexhibits both antimicrobial and antifungal ac-tivity.At the first period, the influence of cerulenin

on the incorporation and the exogenous respira-tion of [32P]orthophosphoric acid, 14C-labeledamino acid, and [1-14C]glucosamine into eachbiopolymer by using the intact cells of Candidastellatoidea, which are markedly sensitive tocerulenin, was investigated (55). Nevertheless,no significant effects were observed. Subse-quently, a wide variety of biological substanceswere employed to investigate whether or notthe antifungal activity of cerulenin was re-versed by them. The antifungal activity of ceru-

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WAVENUMBER (cm-')FIG. 4. Infrared spectrum of cerulenin.

CH3CH=N-NH j NO2

TCH3CHO

NH3 NaQH

CH3 QOC(CH2)2COOCH3

, CH2N2Acids

03I Hs 1H

CH3 ,H H% CH CH C-C NH1% 0 % O4* .

HH CeCCCH2C 2len0 0

Cerul eni n

CrC12 H2/pt, AcOHI

t H H II I

CH3(CH2) 7-C-C_ ,C- C -NH2 -.CH3(CH2)7-C- CH2-CH- C-NH200 0 0 OH O

Tetrahydro-cerulenin Hexahydro-cerulenin~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ICH3CH=CH-CH2- CH=CH- CHCH2 C-CH2 CH-C-NH2

0 OH OulDnh

I Cro3, H2S04Acids

(dro-ceruienin1 CH2N2

CH3(CH2)7COOMeCH3(CH2)6COOMeCH3(CH2)5COOMe

FIG. 5. Chemical transformations of cerulenin (63).

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TABLE 2. Antibacterial spectrum of cerulenin

Test organism

Staphylococcus aureus FDA 209PS. albusS. citreusSarcina lutea, PCI 1001Diplococcus pneumoniae, type 1Streptococcus haemolyticus, Richard Cook NY-5S. haemolyticusS. haemolyticusEscherichia coli, NIHJKlebsiella pneumoniae PCI 602Proteus vulgaris OX 19P. morganiiSalmonella typhosaShigella dysenteriae ShigaS. flexneri E-20S. sonnei E-33Bacillus subtilis, PCI 219B. anthracisB. megateriumCorynebacterium diphtheriaeHaemophilus influenzaeMycobacterium ATCC 607M. avium FM. phleiM. smegmatisM. tuberculosis, H37R,M. tuberculosis, Aoyama B. FrankfurtM. tuberculosisNocardia asteroidesN. coeliacaStreptomyces griseus SN-15S. lavendulae 22A

MICa10010012.525255010010012.55012.512.55025505012.562.55050100

1.51.53.73.7

100100100

1.51.53.11.5

Mediumb

NNNNBBBBNNNNNNNNNNNBBGGGGyyyGGGG

a Minimal inhibitory concentration, micrograms per milliliter.b Media: N, Nutrient agar; B, 10% blood nutrient agar; G, 1% glycerol nutrient agar; Y, 1% Youman's

media. Incubation was at 37TC for 48 h. Strains of M. tuberculosis were cultured for 4 weeks at 37TC,Nocardia and Streptomyces were cultured for 7 days at 27TC, and other strains were cultured for 48 h at 37TC.

lenin is markedly reversed by ergocarciferol,laurate, and oleate, but is not reversed byother fatty acids, such as myristate and stear-ate. Moreover, it can be concluded that theamount of intracellular ergosterol in growingcells of C. stellatoidea incubated in the pres-ence of cerulenin appreciably decreases com-pared with that of the normal cells grown with-out cerulenin. It is thus suspected that ceru-lenin plays an important role as an inhibitor ofbiosynthesis of sterols and fatty acids. This isfurther supported by the experimental observa-tion that cerulenin inhibits the incorporation ofthe ['4C]acetate into sterols and fatty acids. Inthe presence of 0.1 ,ug of cerulenin per ml, theincorporation of the [1-_4C]acetate into fattyacid and sterol fraction is inhibited by almost50%, but its inhibitory effect is enhanced up to98.8 and 97.2%, respectively, at a concentra-tion of 2.5 pug of cerulenin per ml.Because cerulenin does not inhibit the incor-

poration of [2-"4C]mevalonate into the non-saponifiable fraction (NSF), it is suggested that

cerulenin works in an early stage of the path-way between acetate and mevalonate in steroland fatty acid biosyntheses. The action of avi-din, which inhibits acetyl-coenzyme A (CoA)carboxylase in yeast, is then compared withthat of cerulenin in a cell-free system ofSaccha-romyces cerevisiae (56). Expectedly, however,the action of cerulenin described earlier is dis-tinctive in that avidin does not inhibit the in-corporation of [1-_4C]acetate into NSF.As well as [1-_4C]acetate, cerulenin markedly

inhibits both the incorporation of [1-_4C]acetyl-CoA into fatty acid fraction and NSF and thatof [1,3-'4C]malonyl-CoA into NSF. This resultindicates that the antibiotic inhibits the stageof the postformation of malonyl-CoA and ace-tyl-CoA in fatty acid biosynthesis and the stepof preformation of mevalonate in sterol biosyn-thesis.

EFFECT ON FATTY ACID SYNTHETASEOn the basis of the finding that the antibiotic

cerulenin inhibits fatty acid and sterol

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TABLE 3. Antifungal spectrum of cerulenin

Test organism MI@a (g/ml)

Acrocylindrium oryzae 12.5Alternaria kikuchiana 50Aspergillus flavus 100A. fumigatus 50A. fumigatus, 4 strains" 12.5-50A. niger, 3 strains 100A. terreus 100A. ungiusb 100Botrytis cryptoneriae 12.5Candida albicans 1.5C. albicans, CA-7 0.8C. albicans, 10 strains" 0.8-3.7C. kruzei 6.25C. tropicalis 0.8C. stellatoidea 0.8Cephalosporium caerulens 100-Cladosporium wernicke 25Cryptococcus neogormans 1.5Epidermophyton ftoccosum 0.2Geotrichum sp. 3.7Gibberella fujikuroi 50G. saubinetii 50Glomerella cingulata 12.5Hormodendrum pedrosoi 50Kloeckera apiculata 50Microsporum gypseum, 2 strains" 12.5-25M. canis 6.3M. audouini 12.5Mucor mucedo 1.5Myxosporum rhois 6.25Ophiobolus miyabeanus 1.56Penicillium citrinum 50P. chrysogenum 12.5P. glaucum 12.5P. notatum 12.5Phialophora verrucasa 25Piricularia oryzae, 3 strains 6.25Pullularia pullulans 25Rhizopus nigricans 25Saccharomyces cerevisiae 0.8S. sake 0.8Sporotrichum schenckii 100Trichophyton asteroides 6.2T. asteroides, 2 strains" 3.7T. mentagrophytes 12.5T. interdigitale 12.5T. purpureum 6.2T. rubrum, 3 strains 6.2-3.1T. violaceum 12.5Trichoderma beigellii 12.5a MIC, Minimal inhibitory concentration.b From patient strain; fungi were cultured for 7

days at 270C.

syntheses in cell-free systems (56), the effects ofcerulenin on several fatty acid synthetases aris-ing from diverse sources have been investigated(83). Three major types of synthetases havebeen chosen; type I synthetases, represented bythe multienzyme complex (17) from rat liver,yeast, Euglena gracilis, Corynebacterium di-

TABLE 4. Toxicity of cerulenin

Administrative Acute toxicity Chronic toxicityroute (LD5s) (mgkg (mg/kg per

Intravenous 154 40 x 14 daysIntraperitoneal 211 40 x 30 daysSubcutaneous 245Oral 547a LDN, Mean lethal dose.

phtheriae, and Mycobacterium phlei, are deacti-vated by 50 to 100% with 4 to 20 ,ug of ceruleninper ml. The activity of type II synthetase,which consists of a nonaggregated individualenzyme and shows a requirement for acyl-car-rier protein (ACP) from E. coli and Euglena,are inhibited more than about 95% in the pres-ence of only 4 to 8 ,ug of cerulenin per ml,whereas the type II enzyme system from M.phlei shows only 10% inhibition with 100 ,tg ofcerulenin per ml. Type II systems from E. gra-cilis, which involves an ACP-dependent elon-gation of palmityl-CoA to stearate, show 77%inhibition with only 4 ,ug of cerulenin per ml.This extraordinary resistance of ACP-depend-ent synthetase from M. phlei against ceruleninstill remains unexplained.The activities of acyl-CoA transacylase,

malonyl-CoA transacylase, and 83-ketoacyl re-ductase, all of which come from the partialreaction of fatty acid synthetase (type II) fromM. phlei, are not affected by the antibiotic inconcentrations of even 100 gg/ml. On the otherhand, condensing enzyme (/3-ketoacyl thioestersynthetase) was highly sensitive to cerulenin asassayed by measuring 14CO2 fixation into malo-nyl-CoA in the presence of acetyl-CoA (83).Because the f3-ketoacyl-ACP synthetase has

not been dissociated in active form from thefatty acid synthetase of multienzyme complex,the effect of cerulenin on the isolated enzymecomponent could not be evaluated in that sys-tem. The effect of cerulenin and its analogue onhomogenous (8-ketoacyl-acyl carrier proteinsynthetase from E. coli has been investigated(16). The synthetase has been isolated as ahomogenous protein and studied in detail (1,25, 72, 75). The reaction catalyzed by this en-zyme proceeds in two partial reactions with anacyl-enzyme intermediate.

RCO-S-ACP + HS-E = RCO-S-E + ACP-SH (1)

RCO-S-E + HOOCCH2CO-S-ACP =

RCOCH2CO-S-ACP + CO2 + HS-E (2)

Sum: RCO-S-ACP + HOOCCH2CO-S-ACP =RCOCH2CO-S-ACP + C02 + ACP-SH (3)

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Thus, in reaction 1 the acyl group of an acyl-ACP is transferred to a sulfhydryl group of theenzyme to form an enzyme-thioester intermedi-ate (25). In reaction 2, the acyl group of theenzyme thioester is condensed with malonyl-ACP to form a (3-ketoacyl-thioester of ACP,CO2, and the free enzyme. The overall reaction,reaction 3, represents the condensation reac-

tion of fatty acid synthesis.The homogenous 8-ketoacyl-ACP synthetase

was inhibited 50% by cerulenin at concentra-tions of less than 5 ,uM. The tetrahydroceru-lenin also inhibited the enzyme, although sub-stantially higher concentrations of inhibitorwere required to reach similar inhibitory lev-els. The epoxide-free derivatives, dihydro- andhexahydrocerulenin, do not inhibit at even a

concentration of 5.0 mM when tested under thesimilar conditions. The ,3-ketoacyl-ACP syn-thetase also catalyzes a number of model reac-

tions that represent the component reactionsand, therefore, the effect of cerulenin on each ofthese reactions was investigated. The activityof the fatty acyl transacylase (reaction 1), mea-sured as the rate of [14C]hexanoyl ACP synthe-sis, and that of the decarboxylation in the ab-sence of fatty acyl-ACP are inhibited by ceru-lenin.The possibility that a covalent complex was

formed between acyl-ACP synthetase and ceru-

lenin was tested. When substrate quantities ofenzyme are treated with 3H-labeled ceruleninand subjected to trichloroacetic acid precipita-tion, the resultant precipitate contains boundcerulenin. The amount of cerulenin bound tothe enzyme is proportional to the extent of inhi-bition, and it is apparent that approximately 1mol of cerulenin is bound to 1 mol of enzymewhen inhibition of the enzyme approaches100%. It has been established that inhibition off3-ketoacyl-ACP synthetase by iodoacetamide isdue to the alkylation of one cysteine residue permole of enzyme (25). The demonstration thatinhibition by cerulenin is irreversible and thatit accompanied the binding of 1 mol each ofcerulenin and the enzyme suggests a highlyspecific interaction between the enzyme andthe inhibitor.

According to Appleby's report on the effect ofcerulenin on lipid synthesis in Crambe abyssin-ica tissues (12), the antibiotic is a potent inhibi-tor of fatty acid synthesis from [14C]acetate inleaves and developing seeds of plants. A 1 or

2 ,uM concentration of cerulenin inhibits theuptake of [14Clacetate into leaf lipids by 77 and82%, and into seed lipids by 80 and 83%. Theantibiotic inhibits equally the elongation of [1-140]oleate to erucic acid, which is the majorfatty acid of the seed. There is no significant

inhibition of fatty acid desaturation and acyla-tion of lipids in either tissue.Stumpf and his co-workers reported that the

de novo synthesis by fatty acid synthetase fromnaturing safflower seeds and avocado mesocarpwas fully inhibited by 10 pM cerulenin andthat the enzymatic elongation from C16 to C18was not affected by cerulenin (37). These find-ings led them to conclude that the enzymaticsystems involved in the two condensation reac-tions are distinctly different from each other.As previously demonstrated by Appleby, cer-

ulenin inhibits the elongation of [1-14C]oleicacid to erucic acid, whereas there is no effect ofthe antibiotic on the elongation from C16 to C18with avocado mesocarp. This result indicatesthat there should be various kinds of elongationsystems in plants.

EFFECT ON THE ENZYMES INVOLVEDIN STEROL SYNTHESIS

The target of cerulenin on fatty acid biosyn-thesis was made clear. The location of the ac-tion of sterol biosynthesis has been well ex-plored. Although it has been previously re-ported that cerulenin inhibits the incorporationof [14C]acetyl-CoA, but not that of [14C]meva-lonate, into NSF in a cell-free system of yeast,three more steps still remained to be investi-gated (58), namely, the steps catalyzed byacetoacetyl-CoA thiolase, 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) synthase, and HMG-CoA reductase (Fig. 6), which is known as therate-limiting enzyme of overall sterol synthesisin rat liver (74).

In a cell-free system of S. cerevisiae, ceru-lenin inhibits the incorporation of ['4C]acetyl-CoA into NSF by 67% at 9 x 10-6 M (2 jig/ml)(58). This inhibition is sufficient to account forthe inhibition of overall sterol synthesis invivo. On the contrary, the incorporation of[14C]HMG-CoA or [14C]mevalonate is not re-duced up to concentrations of 2.3 x 10-4 M. Thisfact suggests that cerulenin has no effect onHMG-CoA reductase activity and on the subse-quent steps of sterol synthesis. Therefore, thetwo catalytic steps, acetyl-CoA to acetoacetyl-CoA and then to HMG-CoA, must be affectedby cerulenin.The effect of cerulenin on the two enzymes,

acetoacetyl-CoA thiolase and HMG-CoA syn-thase, has been tested. Cerulenin has no effecton acetoacetyl-CoA thiolase activity at a con-centration of 1.8 x 10-3 M, but markedly in-hibits HMG-CoA synthase activity. When thetime of preincubation is reduced to 8 min from30 min, the inhibition is also reduced to 21%from 71% in the presence of cerulenin, whereasno inhibition is observed without preincuba-

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CERULENIN, INHIBITOR OF FATTY ACID SYNTHESIS

Acetate

(1), oCO2

Acetyl-CoA C

4) \

Acetoacetyl-CoA

Malc(5)

Acet

HMG-CoA Ceruleni

I(6)

Mevalonate

l

Sterols

+ Malonyl-CoA

)nyl-SEnz. (3)

tyl-S

Acetoacetyl-S-Enz.

Hydroxybutyryl-S-Enz.

l

Long-chain fatty acid

FIG. 6. Targets of cerulenin in biosyntheses of sterol and fatty acid (16, 56, 58, 83). (1) Acetyl-CoAsynthase; (2) acetyl-CoA carboxylase; (3) fatty acid synthetase; (4) acetoacetyl-CoA thiolase; (5) HMG-CoAsynthase; (6) HMG-CoA reductase.

tion. This therefore implies that the inhibitionappears to be noncompetitive.The fact that cerulenin specifically inhibits

the activity of HMG-CoA synthase suggeststhat the antibiotic possesses distinctly differentmodes of action from that of iodoacetamide (81),which deactivates both HMG-CoA synthaseand acetoacetyl-CoA thiolase.The experimental observation that the ceru-

lenin concentration required for 50% inhibitionof HMG-CoA synthase activity (1.1 x 10-3 M) ishigher than that required by inhibited incorpo-ration of ['4C]acetyl-CoA into NSF by the cell-free system has not been explained.

INHIBITION OF "POLYKETIDE"SYNTHESIS

Shortly after the discovery that ceruleninspecifically inhibited the biosynthesis of fattyacids, it was likewise expected that the anti-biotic would have an inhibitory effect on thebiosyntheses of macrolides (85), in which agly-cone is produced by condensation of acetate,malonate, or methyl malonate. At first, theaction of cerulenin on the biosynthesis of leuco-mycin-A3 (Fig. 7), one of the 16-memberedmacrolides (69), was investigated (67).The cerulenin added to the culture is metabo-

Me 17 isovalerylMeCHO I

mycarose

HO 9 5 mycaminoseMe O-t. ?

%) . ,,O~c m acet27Me 9Me r O < prop

buty

:ate

)lonate

yrateLeucomycin A -V

FIG. 7. Biosynthesis of leucomycin-A3 (69).

lized by the leucomycin-producing strain anddecreases as time goes on. However, the biosyn-thesis of leucomycin is fully inhibited until cer-ulenin is consumed completely. Some antibiot-ics are expected to be synthesized in vivothrough a hypothetical intermediate "polyke-tide." Tetracycline (Streptomyces aureofaciens)(52), cycloheximide (Streptomyces sp. OS-786)(84), and LA-1 (Streptomyces kitasatoensis 4-4-2) (68) (Fig. 8) have been used as examples ofsuch antibiotics and the effects of cerulenin ontheir production were studied (67). In eachcase it has been found that the production ofthe

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HTetracycline Cycloheximide

0CH - C-CH=CH-CH=CH=C=CH-CH=CH-COOH3

LA-1

Di hydro Streptomyci n

N.11 /CH-C - (CH2) 2-CH-COOH

° NHC=O

N\11 CH-C--(CH2) -CHN' "l I

CH -CH- C=O3,NH2

9CH3-CH- C-NH

NH2HO OH

NH2

Prumycin

Al azopepti nFIG. 8. Antibiotics tested and the effect of cerulenin on their production.

antibiotic, like that of leucomycin, is remarka-bly inhibited by cerulenin at a concentration of10 to 20 jig/ml, i.e., 1/10 to 1/5 of the minimalinhibitory concentration against the organismsproducing these antibiotics. On the other hand,even at a sufficiently high concentration thatreduces glucose consumption (100 ,ug/ml) ceru-lenin does not inhibit the synthesis of di-hydrostreptomycin (aminoglucoside antibiotic,Streptomyces sp. OS-3256) or prumycin (aminoacid amino sugar antibiotic, Streptomyces ka-gawaensis) (65), neither of which is expected tobe synthesized in vivo through the polyketide.The minimal inhibitory concentrations of ceru-lenin against the above organisms are 200 ag/ml for S. kitasatoensis 23-1 and S. kitasatoensis4-4-2 and more than 200 ug/ml for the otherorganisms.The mechanism of polyketide formation is

considered to be the polymerization of acetateand malonate, which resembles the condensa-tion of the two acids during the biosynthesis ofa long-chain fatty acid. It is thought that themechanism of polyketide formation lacks all orpart of three reactions, hydrogenation, dehy-dration, and a second hydrogenation, which oc-cur successively after condensation in fatty acidsynthesis. The enzymatic reaction involvingthe first condensation step is common to bothreactions. This implies that cerulenin inhibitsthe production of antibiotics synthesized in vivothrough the polyketide.The incorporation of labeled acetate and pro-

pionate into the aglycone moiety of the polyenemacrolides nystatin (8), candicidin (44), ampho-tericin B (43), and lucensomycin (46) has beendescribed (48). On the basis of this evidence ithas been proposed that the polyene macrolide

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CERULENIN, INHIBITOR OF FAT[Y ACID SYNTHESIS 691

antibiotics are synthesized via the polyketidepathway by a head to tail condensation of ace-tate and propionate, previously activated tomalonyl-CoA and methyl malonyl-CoA, respec-tively, in a manner similar to that found infatty acid biosynthesis. Using a phosphate-lim-ited, resting-cell system of Streptomyces gri-seus, Martin and McDaniel showed that ceru-lenin specifically inhibits the biosynthesis ofcandicidin (48). Cells in which candicidin syn-thesis is completely inhibited for 10 h by theaddition of 20 ,ug of cerulenin per ml remaincapable of candicidin synthesis. After removalof the cerulenin by washing and suspension ofthe cells in fresh medium, candicidin synthesisresumes at a lower rate (70%) than that of thecontrol.

Biosynthesis of flavonoids is an example forthe application of the acetate rule to the synthe-sis of various aromatic ring structures (7, 45).Recently, flavonone synthase, the enzyme cata-lyzing the formation of the flavonone narin-genin from p-coumaroyl-CoA and malonyl-CoA(Fig. 9), has been isolated and partially purifiedfrom fermentor-grown, light-induced cell sus-pension cultures of Petroselium hortense (40).The enzyme is strongly inhibited by ceruleninand further inhibited by the reaction productssuch as naringenin, CoA-SH, and several com-pounds reactive with sulfhydryl groups. Ap-proximately 80% inhibition of the reaction ca-

COO3 xI. OH

CH2-CO-S-CoA AOH

CoA-S>

E ~~~~0E OH

HO I

[oy~~~0

Fl avanone(Naringenin)

FIG. 9. Biosynthesis of the flavanone naringenin(40).

talyzed by flavonone synthase is achieved by0.5 ug (5 ug/ml) of cerulenin. The close similar-ity between the mechanisms of chain elonga-tion in fatty acid and flavonone synthesis issuggested by these results.

6-Methyl salicylic acid (Fig. 10) is one of thesimplest aromatic metabolites derived frompolyketide. Synthesis of 6-methyl salicylic acidusing the cell-free system from Penicillium ur-ticae is inhibited up to 60% by 100 ,ug of ceru-lenin. This is the same extent to which fattyacid biosynthesis is inhibited by cerulenin.On the basis of these results, along with the

effect of cerulenin on antibiotic production(67), it is possible, by examining the effect ofcerulenin on the production of a natural prod-uct, at least one of microbial origin, to identifywhether the product is synthesized through apolyketide intermediate. However, because theinhibitions of fatty acid and sterol synthesestake place simultaneously (60, 83), such anexperiment requires caution. Among otherthings, the product should not be synthesizedthrough mevalonic acid. Secondly, ceruleninmust not be added until the organism is fullygrown, as demonstrated by our experiment, toreduce the secondary effects of the inhibition oflipid synthesis as much as possible.

APPLICATION AS A BIOCHEMICALTOOL

To control the fatty acid or lipid compositionof bacterial membrane and to explore biochemi-cal and biogenetic interference of fatty acid (orlipids), the following methods have been used:(i) unsaturated fatty acid and saturated fattyacid auxotrophs (13, 77, 78); (ii) temperature-sensitive mutants that require saturated andunsaturated fatty acid for growth (28, 29), andthe mutants with temperature-sensitive phos-phatidyl serine decarboxylase (32); (iii) temper-ature control of fatty acid composition (11, 47);and (iv) the application of 3-decynoyl-N-acetyl-cysteamine, an inhibitor of fatty acid synthe-sis in E. coli (38, 39).

Cerulenin has proved useful for studying themetabolism and the function of fatty acids inthe cell membrane of bacteria (24) and yeast (5;J. Awaya, K. Otoguro, and S. Omura, unpub-lished data). Goldberg et al. demonstrated thatcerulenin, which inhibits the growth ofE. coli,specifically blocked lipid synthesis (24). I havereported that the cell growth of S. cerevisiaeATCC 12341, inhibited by cerulenin, is reversedby various exogenous fatty acids. Myristate(C14:0), pentadecanoate (C15:0), palmitate (C16:0),and oleate (C18g:) reverse effectively the inhibi-tion of growth by cerulenin. When these cellsare treated with pentadecanoate, over 90% of

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Me 0me

CH3COOH ; C-S-EnzYme 1COOH3 x HOOCCH2COOH J r OH

6-MSAFIG. 10. Biosynthesis of 6-methylsalicylic acid (6-MSA).

native even-carbon fatty acids are replaced byodd-carbon fatty acid. Those found in the re-versed cells treated with oleate are almost allunsaturated fatty acids. In both experiments,in the presence of myristate and pentadeca-noate along with cerulenin, these added fattyacids are metabolized to yield fatty acids withan extended carbon chain and unsaturatedfatty acids. This fact suggests that cerulenininhibits neither elongation nor dehydrogena-tion in S. cerevisiae. Accordingly, it has beensuggested from these experimental observa-tions that cerulenin could serve as a conven-ient alternative to mutants for studying mem-brane biogenesis (5, 24; Awaya et al., unpub-lished data).

Sporulation of microorganisms is an attrac-tive model for studies of cellular differentiation.Investigations have been made from biochemi-cal viewpoints, such as those on nucleic acid,protein, carbohydrate, and intermediate me-tabolism (20, 21, 27). However, there seem tobe fewer reports on lipid metabolism duringsporulation, especially in yeast, in spite of well-documented lipid accumulation in sporulatingcells (71). Esposito et al. (19) have found tworemarkable periods of lipid synthesis duringsporulation of S. cerevisiae, which have beenconfirmed by Illingworth et al. (34). Recently,Henry and Halvorson (33) have attempted aprecise analysis of this process to show thatmuch of the lipid synthesis is not specific tosporulation, and that, if the necessary diploidsfrom the auxotrophs for saturated (77) and un-saturated fatty acids (82) are constructed, thesestrains could be used to determine the lipidrequirement during sporulation.The inhibition of sporulation in S. cerevisiae

by cerulenin and its reversion by externallyadded fatty acid were studied (60). Both palmi-tate and oleate reverse the inhibition of sporu-lation by cerulenin. Pentadecanoate is the mosteffective among the saturated fatty acids eventhough fatty acids with additional carbons arenot detected in the resulting fatty acid of S.cerevisiae G 2-2.

It was unknown whether lipid synthesis dur-ing the earlier stage of sporulation occurredindependently of other sporulation-relatedmetabolic changes or as an early part of sequen-tial changes among the sporulation-related

changes. Ifthe former were the case, exogenousfatty acids should have rapidly reversed theinhibited sporulation in the cells pretreatedwith cerulenin. If the latter were true, exoge-nous fatty acids should have required severalhours to reverse the inhibited sporulation incells pretreated with the antibiotic and to re-verse the inhibited sporulation upon their addi-tion 12 h or more later to the sporulation me-dium. When cerulenin was added to the sporu-lation medium later than 12 h after the start ofincubation, the marked inhibition disappeared.When the fatty acid fraction extracted from thesporulated yeasts was added to the cells pre-treated with cerulenin for more than 6 h, sporu-lation became evident at 6 h after the additionof the fatty acid fraction. Therefore, sufficientsynthesis of fatty acids required for sporulationwas assumed to occur during the first 6 h inphase I of yeast sporulation.Nunn and Cronan have performed the exper-

iment using cerulenin to test in vivo if rel genecontrol of phospholipid synthesis is exertedsolely at the level of fatty acid synthesis (57).Phospholipid synthesis, as well as ribonucleicacid synthesis, is under control of the rel locusin E. coli (23, 53, 70, 80). Amino acid starvationof stringent (rel+) bacteria results in a two- tofourfold decrease in phospholipid synthesis (23,53, 70, 80). In contrast, starvation of a relaxed(rel+) strain results in decrease in phospholipidsynthesis.A requirement for an exogenous source of

fatty acid for growth of strain CP78 (rel+) andCP79 (rel+) has been engendered by the addi-tion of cerulenin. Addition of cerulenin in thepresence of an appropriate fatty acid supple-ment results in an 87% decrease in the incorpo-ration of ['4C]acetate into phospholipid (al-though growth continued). Under these condi-tions, amino acid starvation of strain CP78(rel+) results in a two- to fourfold inhibition ofthe rate of phospholipid synthesis. An identicalexperiment with strain CP79, the rel- deriva-tive, showed no inhibition of phospholipid syn-thesis when starved for amino acid. This dem-onstrates that the inhibition by phospholipidsynthesis observed in an amino acid-starvedstringent strain cannot be due solely to an inhi-bition of fatty acid synthesis.

Attempts have been made, by using ceru-

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lenin, to answer two questions as to how to stopoverall fatty acid synthesis in B. subtilis (87).(i) Is it possible to prevent the accumulation offree fatty acids under the conditions where denovo phospholipid biosynthesis is blocked inthese bacteria? (ii) Does the inhibition of lipidsynthesis with cerulenin influence the rate ofmacromolecular syntheses in B. subtilis?Although the bacteria ceased to grow upon

inhibiting de novo fatty acid biosynthesis bythe addition of cerulenin, they remained com-pletely viable. Addition of 12-methyl tetradeca-noate and palmitate to the culture medium ofcerulenin-treated cells retains growth of thebacteria, albeit at a reduced rate. Although thede novo synthesis of all lipid components of themembrane is blocked, citrate-Mg2+ transportactivity remains inducible and the induced cellsdo not lose this transport activity when treatedwith cerulenin. Shortly after the addition ofcerulenin, the rate of ribonucleic acid synthesisdrops rapidly and the rate of protein synthesisalso decreases, but more slowly than the for-mer. The rate of deoxyribonucleic acid synthe-sis remains almost unaffected.

Plasmid-mediated tetracycline resistance inS. aureus and enteric bacteria is usually in-ducible and appears to result from decreaseduptake of antibiotic. Because plasmid-mediatedtetracycline resistance probably results fromthe insertion of proteins into the membrane(42), it is of interest to know whether or notresistance can be expressed in the absence oflipid synthesis. Chopra has used cerulenin toresolve this problem (10). Addition of 125 ,ug ofcerulenin per ml to growing cultures of UB4012that contained plasmid pub... (specifying re-sistance to tetracycline) resulted in virtuallycomplete inhibition of fatty acid synthesis.Preincubation of UB4012 for 40 min with 5 tug oftetracycline per ml produced a marked fall inthe subsequent inhibition of protein synthesisby tetracycline, compared with organismsgrown initially in the absence of the drug. Ad-dition of 125 ,ug of cerulenin per ml duringpreexposure of UB012 to 5 ug of tetracycline perml had no effect on the subsequent degree ofinhibition of protein synthesis by tetracyclinecompared with organisms preincubated in thepresence of tetracylcine, but without cerulenin.On the basis of these results, it was concludedthat the induction of tetracycline resistance re-mained unchanged whether it appeared in theabsence of lipid synthesis or under normalgrowth conditions.The envelope of E. coli is composed of two

membranes: the inner-most cytoplasmic mem-brane and the rigid peptidoglycan layer. Bothmembranes contain protein and phospholipid,

which are synthesized either in the soluble cy-toplasm or at the cytoplasmic membrane itself.In addition, the outer membrane contains lipo-polysaccharide. Investigations of the assemblyof the cytoplasmic membrane in glycerol auxo-trophs have revealed that some proteins can besynthesized and incorporated into this mem-brane under conditions that do not permit thesynthesis of lipids (54, 86). Assembly is notnecessary for that of the cytoplasmic mem-brane. Thus, the protein, lipid, and lipopolysac-charide components must be translocated fromthe site of their synthesis inside the cell to thefinal location exterior to the cytoplasmic mem-brane and the peptidoglycan. It was of interestto know whether or not the insertion of a newlysynthesized protein into the outer membranerequires simultaneous lipid synthesis. Ceru-lenin was used to demonstrate that a minorprotein component of the outer membrane ofE.coli, which serves as the receptor for the phagelambda, can be synthesized and inserted intothe outer membrane during inhibition of lipidsynthesis (73).Marr and Ingraham (47) have reported that

the fraction of unsaturated fatty acid in theenvelope phospholipids of E. coli increasesmarkedly as the growth temperature is de-creased. This observation was confirmed by alarge number of investigators, but the mecha-nisms regulating this alteration were not fullyunderstood regardless of the considerableamount of work done in this field. There hadbeen two major interpretations to explain suchthermal regulation. One claimed that the en-zymes catalyzing phosphatidic acid synthesiswould be sensitive to temperature change (79)and the other suggested that a control at thelevel of fatty acid synthesis should also be re-quired.Cronan carried out experiments, a part of

which involved the utilization of cerulenin, toverify that both fatty acid synthesis and phos-phatidic acid synthesis accounted for one site ofthe thermal regulation of phospholipid fattyacid composition. When cerulenin-treated cul-tures are grown with a mixture of [3H]palmi-tate and cis-[14C]vaccenate at various tempera-tures in the presence of cerulenin, at least 85%of the fatty acid moieties in the phospholipidare derived from these exogenously suppliedfatty acids. These phospholipids are found tocontain different ratios of the two acids. Theseratios depend directly on the temperature ofgrowth.Because cerulenin possesses a number of re-

markable biological activities that have neverbeen found in other antibiotics, it will more andmore frequently be used as an unequivocal bio-

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chemical tool not only for the studies of fattyacid biosynthesis, but also for the biosynthesis,structure, and functions of membranes pro-duced by microorganisms.

Prospectively, the antibiotic will be used, asvarious other enzyme inhibitors have been, in avariety of ways, for biosynthetic studies of nat-ural organic compounds produced via polyke-tide and malonate and for enzymes involvedwith biosynthesis.

Despite the fact that cerulenin inhibits thebiosynthesis of sterol, its producing strain, C.caerulens, simultaneously affords sterol anti-biotics as described earlier. Why this biosyn-thetic system of sterol strongly resists ceru-lenin still remains a problem for future inves-tigation.

ACKNOWLEDGMENTSI wish to express my gratitude to H. Takeshima

and J. Awaya of the Kitasato Institute for a criticalreading of the manuscript and for valuable discus-sions.

ADDENDUMAfter submission of this manuscript it was re-

ported that Kamiryo et al., in a study of cerulenin,proved that acyl-CoA synthetase activity is requiredfor repression of yeast acetyl-CoA carboxylase byexogenous fatty acid (T. Kamiryo, T. S. Partha-sarathy, and S. Numa, Proc. Natl. Acad. Sci.U.S.A. 73:386-390, 1976).

LITERATURE CITED1. Alberts, A., W. R. M. Bell, and P. R. Vagelos.

1972. Acyl carrier protein. XV. Studies of f8-ketoacyl-acyl carrier protein synthase. J.Biol. Chem. 247:3190-3198.

2. Appleby, R. S. 1974. Inhibition by cerulenin oflipid synthesis in Crambe abyssinica tissues.Phytochemistry 13:2745-2748.

3. Arison, B. H., and S. Omura. 1974. Revisedstructure of cerulenin. J. Antibiot. 27:28-30.

4. Awaya, J., T. Kesado, and S. Omura. 1975.Preparation of 13C- and 3H-labeled ceruleninand biosynthesis with '3C-NMR. J. Antibiot.28:824-827.

5. Awaya, J., T. Ohno, H. Ohno, and S. Omura.1975. Substitution of cellular fatty acid inyeast cells by the antibiotic cerulenin andexogenous fatty acids. Biochim. Biophys.Acta 409:267-273.

6. Boldur, I., and D. Sompolinsky. 1974. Antigenspecific for bacteria resistance to tetracycline.Antimicrob. Agents Chemother. 6:117-120.

7. Brich, A. J., and F. W. Donovan. 1953. Studiesin relation to biosynthesis. I. Some possibleroutes to derivatives of orcinol and phloroglu-cinol. Aust. J. Chem. 6:360-368.

8. Brich, A. J., C. Holzapfel, R. W. Rickards, C.Djerassi, M. Suzuki, J. Westley, J. D.

Dutcher, and R. Thoma. 1964. Nystatin partVI. Chemistry and partial structure of theantibiotic. Tetrahedron Lett. 1491-1497.

9. Chain, E. H., W. Florey, M. A. Jennings, andT. I. Williams. 1943. Helvolic acid, and anti-biotic produced by Aspergillus fumigatus. Br.J. Exp. Pathol. 24:108-119.

10. Chopra, I. 1975. Induction of tetracycline resist-ance in Staphylococcus aureus in the absenceof lipid synthesis. J. Gen. Microbiol. 91:433-436.

11. Cronan, J. E., Jr., and P. R. Vagelos. 1972.Metabolism and function of the membranephospholipids of Escherichia coli. Biochim.Biophys. Acta 265:25-60.

12. Cronan, J. E., Jr. 1974. Regulation of the fattyacid composition of membrane phospholipidsof Escherichia coli. Proc. Natl. Acad. Sci.U.S.A. 71:3758-3762.

13. Cronan, J. E., Jr., and E. P. Gelmann. 1975.Physical properties of membrane lipids. Bio-logical relevance and regulation. Bacteriol.Rev. 39:232-256.

14. Cronan, J. E., Jr. 1975. Thermal regulation ofmembrane lipid composition of Escherichiacoli. Evidence for the direct control of fattyacid synthesis. J. Biol. Chem. 250:7074-7077.

15. Dachne, W. Von, H. Lorch, and W. 0. Godt-fredsen. 1968. Microbiological transforma-tions of fusidine-type antibiotics. A correla-tion between fusidic acid and helvolic acid.Tetrahedron Lett. 4843-4846.

16. D'Agnolo, G., I. S. Rosengeld, J. Awaya, S.Omura, and P. R. Vagelos. 1973. Inhibition offatty acid synthesis by the antibiotic ceru-lenin. Biochim. Biophys. Acta 326:155-166.

17. Delo, J., M. L. Ernst-Fonberg, and K. Bloch.1971. Fatty acid synthetase from Euglena gra-cilis. Arch. Biochem. Biophys. 143:384-391.

18. Demain, A. L. 1975. Why mode of action stud-ies? Chem. Technol. 287-289.

19. Esposito, M. S., R. E. Esposito, M. Arnaud,and H. 0. Halvorson. 1969. Acetate utiliza-tion and macromolecular synthesis duringsporulation of yeast. J. Bacteriol. 100:180-186.

20. Fowel, R. R., and M. E. Moorse. 1960. Factorscontrolling the sporulation of yeasts. I. Thepresporulation phase. J. Appl. Bacteriol. 23:53-68.

21. Fowell, R. R. 1967. Factors controlling thesporulation of yeasts. II. The sporulationphase. J. Appl. Bacteriol. 30:450-474.

22. Furuya, K., R. Enokita, and M. Shirasaka.1967. Studies on the antibiotics from fungi. I.Helicocerin, an antifungal antibiotic pro-duced by Helicoceras oryzae. Ann. SnakyoRes. Lab. 19:86-90.

23. Golden, N. G., and G. L. Powell. 1972. Strin-gent and relaxed control of phospholipidmetabolism in Escherichia coli. J. Biol.Chem. 247:6651-6658.

24. Goldberg, I., J. R. Walker, and K. Bloch. 1973.Inhibition of lipid synthesis in Escherichiacoli cells by the antibiotic cerulenin. Antimi-

BACTERIOL. REV.


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br.asm.org/

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nloaded from



crob. Agents Chemother. 3:549-554.25. Greenspan, M. D., A. W. Alberts, and P. R.

Vagelos. 1969. Acyl carrier protein. XIII. 38-Ketoacyl-acyl carrier protein synthetase fromEscherichia coli. J. Biol. Chem. 244:6477-6485.

26. Grisebach, H. 1959. Proceedings of the 4th in-ternational congress of biochemistry, Vienna,1958, vol. II, p. 56-70. Pergamon Press, Lon-don.

27. Harber, J. E., and H. 0. Halvorson. 1972. Regu-lation of sporulation in yeast. Curr. Top. Dev.Biol. 7:61-83.

28. Harder, E. M., I. R. Beacham, J. E. Cronan,Jr., K. Beacham, J. L. Honegaaer, and D. F.Silbert. 1974. Temperature-sensitive mutantsof Escherichia coli requiring saturated andunsaturated fatty acids for growth; isolationand properties. Proc. Natl. Acad. Sci. U.S.A.69:3105-3109.

29. Harder, E. M., R. C. Ladenson, S. D. Schim-mel, and D. F. Silbert. 1974. Mutants ofEscherichia coli with temperature-sensitivemalonyl CoA-acyl carrier protein transacyl-ase. J. Biol. Chem. 249:7468-7475.

30. Hata, T., Y. Sano, A. Matsumae, Y. Kamio, S.Nomura, and R. Sugawara. 1960. Study ofnew antifungal antibiotic. Jpn. J. Bacteriol.15:1075-1077.

31. Hata, T., A. Matsumae, S. Nomura, T. Kim,and K. Ryan. 1960. Studies on cerulenin, anew antifungal antibiotic. II. Biological char-acteristic and therapeutic effect of cerulenin.Abstr. 4th Annu. Meet. Jpn. Soc. Med. Mycol.Jpn. J. Med. Mycol. 1:382-383.

32. Hawrot, E., and E. P. Kennedy. 1975. Biogene-sis of membrane lipids: mutants of Esche-richia coli with temperature-sensitive phos-phatidyl serine decarboxylase. Proc. Natl.Acad. Sci. U.S.A. 72:1112-1116.

33. Henry, S. A., and H. 0. Halvorson. 1973. Lipidsynthesis during sporulation of Saccharomy-ces cerevisiae. J. Bacteriol. 114:1158-1163.

34. Illingworth, R. F., A. H. Rose, and A. Beckett.1973. Changes in lipid composition and finestructure ofSaccharomyces cerevisiae. J. Bac-teriol. 113:373-386.

35. Itai, A., S. Nozoe, K. Tsuda, S. Okuda, Y.Iitaka, and Y. Nakayama. 1967. The struc-ture of cephalonic acid a pentaprenyl terpen-oid. Tetrahedron Lett., p. 4111-4112.

36. Iwai, Y., J. Awaya, T. Kesado, H. Yamada, S.Omura, and T. Hata. 1973. Selective produc-tion of cerulenin by Cephalosporium caeru-lens KF-140. J. Ferment. Technol. 51:575-581.

37. Jaworski, J. G., E. E. Coldschmidt, and P. L.Stumpf. 1974. Fat metabolism in higherplants. Properties of the palmityl acyl carrierprotein: stearyl acyl carrier protein elonga-tion system in naturing safflower seed ex-tracts. Arch. Biochem. Biophys. 163:769-776.

38. Kass, L. R., and K. Bloch. 1967. On the enzy-matic synthesis of unsaturated fatty acid inEscherichia coli. Proc. Natl. Acad. Sci. U.S.A.58:1168-1173.

39. Kass, L. R. 1968. The antibacterial activity of 3-decynoyl-N-acetylcysteamine, inhibition invivo of 83-hydrodecanoyl thioester dehydrase.J. Biol. Chem. 243:3223-3228.

40. Kreuzaler, F., and K. Hahlbroch. 1975. Enzymicsynthesis on aromatic ring from acetate units.Partial purification and some properties offlavanone synthase from cell suspension cul-ture of Petroselinum hortense. Eur. J. Bio-chem. 56:205-213.

41. Kuroishi, H., Y. Takamura, T. Mizunaga, andT. Uemura. 1971. Factors influencing death ofbiotin-deficient yeast cells. J. Gen. Appl. Mi-crobiol. 17:29-42.

42. Levy, S. B., and L. McMurry. 1974. Detection ofan inducible membrane protein associatedwith R-factor mediated tetracycline resist-ance. Biochem. Biophys. Res. Commun.56:1060-1068.

43. Linke, H. A. B., W. Mechlinski, and C. P.Schaffner. 1972. Studies on candicidin biogen-esis. J. Antibiot. 27:155-160.

44. Liu, C. M., L. E. McDaniel, and C. P.Schaffner. 1972. Studies on candicidin bio-genesis. J. Antibiot. 25:161-171.

45. Lynen, F., and M. Tada. 1961. Die biochem-ischen Grundlagen der "Polyacetate-Regel."Angew. Chem. 73:513-519.

46. Manwaring, D. G., R. W. Richards, G. Gaudi-ano, and V. Nicolella. 1969. The biosynthesisof the macrolide antibiotic lucensomycin. J.Antibiot. 22:545-550.

47. Marr, A. G., and J. L. Ingraham. 1962. Effect oftemperature on the composition of fatty acidsin Escherichia coli. J. Bacteriol. 84:1260-1267.

48. Martin, J. F., and E. McDaniel. 1975. Specificinhibition of candicidin biosynthesis by thelipogenic inhibitor cerulenin. Biochim. Bio-phys. Acta 411:186-194.

49. Matsumae, A., Y. Kamio, and T. Hata. 1963.Studies on cerulenin. I. Studies on ceruleninproducing strain. J. Antibiot. Ser. A 16:236-238.

50. Matsumae, A., S. Nomura, and T. Hata. 1963.Studies on cerulenin. II. Biological assay andfermentation of cerulenin. J. Antibiot. Ser. A16:239-243.

51. Matsumae, A., S. Nomura, and T. Hata. 1964.Studies on cerulenin. IV. Biological charac-teristics of cerulenin. J. Antibiot. Ser. A 17:1-7.

52. McCormick, J. R. D. 1967. Biogenesis of anti-biotic substances, p. 73. Z. Vanek and Z. Hos-talek (ed.). Academic Press Inc., New York.

53. Merlie, J. P., and L. I. Pizer. 1973. Regulationof phospholipid synthesis in Escherichia coliby guanosine tetraphosphate. J. Bacteriol.116:355-366.

54. Mindich, L. 1971. Induction of Staphylococcusaureus lactose permease in the absence of gly-cerolipid synthesis. Proc. Natl. Acad. Sci.U. S. A. 68:420-424.

55. Nomura, S., T. Horiuchi, S. Omura, and T.Hata. 1972. The action mechanism of ceru-lenin. J. Biochem. (Tokyo) 71:783-786.

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56. Nomura, S., T. Horiuchi, T. Hata, and S.Omura. 1972. Inhibition of sterol and fattyacid biosynthesis by cerulenin in cell free sys-tems of yeast. J. Antibiot. 25:365-368.

57. Nunn, W. D., and J. E. Cronan Jr. 1974. Relgene control of lipid synthesis in Escherichiacoli. Evidence for eliminating fatty acid syn-thesis as the sole regulatory site. J. Biol.Chem. 249:3994-3996.

58. Ohno, T., J. Awaya, T. Kesado, and S. Omura.1974. Target of inhibition by the anti-lipo-genic antibiotic cerulenin of sterol synthesisin yeast. Biochem. Biophys. Res. Commun.57:1119-1124.

59. Ohno, H., T. Ohno, J. Awaya, and S. Omura.1975. Inhibition of 6-methylsalicylic acid syn-thesis by the antibiotic cerulenin. J. Biochem.78:1149-1152.

60. Ohno, T., J. Awaya, and S. Omura. 1976. Inhi-bition of sporulation by cerulenin and its re-version by exogenous fatty acids in Saccharo-myces cerevisiae. Antimicrob. Agents Chemo-ther. 8:42-48.

61. Okuda, S., S. Iwasaki, K. Tsuda, Y. Sano, T.Hata, S. Udagawa, Y. Nakayama, and H.Yamaguchi. 1964. The structure of helvolicacid. Chem. Pharm. Bull. 12:121-124.

62. Okuda, S., Y. Nakayama, and K. Tsuda. 1966.Studies on microbial products. I. Helvolic acidand related compounds. I. 7-Desacetoxy-hel-volic acid and helvolinic acid. Chem. Pharm.Bull. 14:436-441.

63. Omura, S., M. Katagiri, A. Nakagawa, Y.Sano, S. Nomura, and T. Hata. 1967. Studieson cerulenin. V. Structure of cerulenin. J.Antibiot. Ser. A 20:349-354.

64. Omura, S., A. Nakagawa, K. Sekikawa, M.Otani, and T. Hata. 1969. Studies on ceru-lenin. IV. Some spectroscopic features of ceru-lenin. J. Chem. Pharm. Bull. 17:2361-2363.

65. Omura, S., M. Katagiri, K. Atsumi, T. Hata,A. A. Jakubowski, E. Bleecker Springs, andM. Tishler. 1974. Structure of prumycin. J.Chem. Soc., p. 1627.

66. Omura, S., M. Katagiri, J. Awaya, T. Furu-kawa, I. Umezawa, N. Oi, M. Mizoguchi, S.Aoki, and M. Shindo. 1974. Relationship be-tween the structure of fatty acid amide deriv-atives and their antimicrobial activities. An-timicrob. Agents Chemother. 6:207-215.

67. Omura, S., and H. Takeshima. 1974. Inhibitionof the biosynthesis of leucomycin, a macrolideantibiotic, by cerulenin. J. Biochem. 75:193-195.

68. Omura, S., Y. Imai, H. Takeshima, and A. Nak-agawa. 1976. Structure of highly unsaturatedfatty acid from the leucomycin producingstrain. Abstr. 96th Annu. Meet. Pharm. Soc.Jpn.

69. Omura, S., A. Nakagawa, H. Takeshima, K.Atsumi, J. Miyazawa, F. Piriou, and G. Lu-kacs. 1975. Biosynthetic studies using '3C en-riched precursors on the 16-membered macro-lide antibiotic leucomycin A3. J. Am. Chem.Soc. 97:6600-6602.

BACTERIOL. REV.

70. Polakis, S. E., R. B. Guchhait, and M. D. Lane.1973. Stringent control of fatty acid synthesisin Escherichia coli. J. Biol. Chem. 248:7957-7966.

71. Pontefract, R. D., and J. J. Miler. 1962. Themetabolism of yeast sporulation. IV. Cytologi-cal and physiological changes in sporulatingcells. Can. J. Microbiol. 8:573-584.

72. Prescott, D. J., and P. R. Vagelos. 1970. Acylcarrier protein. XIV. Further studies of /3-ketoacyl-acyl carrier protein synthase fromEscherichia coli. J. Biol. Chem. 245:5485-5490.

73. Randall, L. L. 1975. Insertion of minor proteininto the outer membrane of Escherichia coliduring inhibition of lipid synthesis. J. Bacte-riol. 122:347-351.

74. Rodwell, V. W., D. J. McNamara, and J. D.Shapira. 1973. Regulation of hepatic 3-hy-droxy-3-methylglutaryl coenzyme A reduc-tase. Adv. Enzymol. 38:373-412.

75. Rosenfeld, I. S., G. D'Agnolo, and P. R. Va-gelos. 1973. Synthesis of unsaturated fattyacids and the lesion in feb B mutants. J. Biol.Chem. 248:2452-2460.

76. Sano, Y., S. Nomura, Y. Kamio, S. Omura, andT. Hata. 1967. Studies on cerulenin. III. Isola-tion and physico-chemical properties of ceru-lenin. J. Antibiot. Ser. A 20:344-348.

77. Scweizer, E., and H. Bolling. 1970. A Saccharo-myces cerevisiae mutant defective in satu-rated fatty acid biosynthesis. Proc. Natl.Acad. Sci. U.S.A. 67:660-666.

78. Silbert, D. F., and P. R. Vagelos. 1967. Fattyacid mutant of Escherichia coli lacking a ,B-hydroxy decanoyl thioester dehydrase. Proc.Natl. Acad. Sci. U.S.A. 58:1579-1586.

79. Sinensky, M. 1971. Temperature control of phos-pholipid biosynthesis in Escherichia coli. J.Bacteriol. 106:449-455.

80. Sokawa, Y., E. Nakao, and Y. Kaziro. 1970. Rcgene control in Escherichia coli is not re-stricted to RNA synthesis. Bidchim. Biophys.Acta 199:256-264.

81. Stewart, R. R., and H. J. Rudney. 1966. Thebiosynthesis of 8-hydroxy-f8-methylglutarylcoenzyme A in yeast. J. Biol. Chem. 241:1212-1221.

82. Tngle, M., A. Klar, S. Henry, and H. 0. Halvor-son. 1973. Ascospore formation in yeast, p.209-243. In 23rd Symposium of the Society forGeneral Microbiology.

83. Vance, D., I. Goldberg, 0. Mitsuhashi, K.Bloch, S. Omura, and S. Nomura. 1972. Inhi-bition of fatty acid synthestase by the anti-biotic cerulenin. Biochem. Biophys. Res.Commun. 48:649-656.

84. Vanek, Z., and M. Vondracek. 1966. Biogenesisof cycloheximide and of related compounds, p.982-991. Antimicrob. Agents Chemother.1965.

85. Vanek, Z., and J. Majer. 1967. Macrolide anti-biotics. In D. Gottlieb and P. D. Shaw (ed.),Antibiotics, vol. II, Biosynthesis. Springer-Verlag, New York.


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86. Willecke, K., and L. Mindich. 1971. Induction ofcitrate transport in Bacillus subtilis duringthe absence of phospholipid synthesis. J. Bac-teriol. 106:514-518.

87. Wille, W. E., E. Eisenstadt, and K. Willecke.

1975. Inhibition ofde novo fatty acid synthesisby the antibiotic cerulenin in Bacillus sub-tilis: effects on citrate-Mg2+ transport andsynthesis of macromolecules. Antimicrob.Agents Chemother. 8:231-237.

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