molecular genetic analysis and regulation of aflatoxin biosynthesis

8/10/2019 Molecular Genetic Analysis and Regulation of Aflatoxin Biosynthesis

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Appl Microbiol Biotechnol (2003) 61:8393DOI 10.1007/s00253-002-1199-x

M I N I - R E V I E W

D. Bhatnagar K. C. Ehrlich T. E. Cleveland

Molecular genetic analysis and regulation of aflatoxin biosynthesis

Received: 26 August 2002 / Revised: 4 November 2002 / Accepted: 8 November 2002 / Published online: 28 January 2003 Springer-Verlag 2003

Abstract Aflatoxins, produced by some Aspergillusspecies, are toxic and extremely carcinogenic fura-nocoumarins. Recent investigations of the molecular

mechanism of AFB biosynthesis showed that the genesrequired for biosynthesis are in a 70 kb gene cluster. Theyencode a DNA-binding protein functioning in aflatoxinpathway gene regulation, and other enzymes such ascytochrome P450-type monooxygenases, dehydrogenas-es, methyltransferases, and polyketide and fatty acidsynthases. Information gained from these studies has ledto a better understanding of aflatoxin biosynthesis bythese fungi. The characterization of genes involved inaflatoxin formation affords the opportunity to examine themechanism of molecular regulation of the aflatoxinbiosynthetic pathway, particularly during the interactionbetween aflatoxin-producing fungi and plants.

Introduction

Aflatoxins are a group of at least 16 structurally relatedpolyketide-derived furanocoumarins (Fig. 1). Thesemetabolites are primarily produced by some isolates ofAspergillus flavus and A. parasiticus on agriculturalcommodities, and less frequently by other Aspergillusspecies including A. pseudotamarii, A.bombycis, A.nomius, and an unnamed taxon from West Africa(Kurtzman et al. 1987; Cotty and Cardwell 1999; Ito etal. 2001; Peterson et al. 2001), and an isolate of A.ochraceoroseus (Frisvad 1985; Klich et al. 2000). Thereare four major aflatoxins: B1, B2, G1 and G2. Toxin-producing isolates ofA. flavus,A. ochraceoroseus and A.pseudotamariiproduce only the B-type, whereas the otherspecies produce both B and G-type aflatoxins.

Aflatoxins are potent carcinogens when ingested byanimals and humans (Eaton and Groopman 1994). Sincetheir discovery over 40 years ago, aflatoxins have been

shown to be immunosuppressive, mutagenic, teratogenicand hepatocarcinogenic in both experimental animals andhumans (Eaton and Groopman 1994). Contamination offoods and feeds remains a serious worldwide problem butis not a threat in most developed countries because ofcareful commodity screening (Bhatnagar et al. 2000;McAlpin et al. 2002). Aflatoxin contamination can arisefrom improper storage of commodities as well as beforeharvest in corn, peanuts, cottonseed and tree nuts. In thisarticle we have reviewed current research on the molec-ular regulation of aflatoxin biosynthesis, as well as thebiological and evolutionary significance of aflatoxinproduction. Previous reviews on this topic have covered

the literature up to 1998 (Minto and Townsend 1997;Payne and Brown 1998; Woloshuk and Prieto 1998;Bhatnagar et al. 2000; Cary et al. 2000a).

Biochemistry and molecular geneticsof aflatoxin biosynthesis

There are 21 enzymatic steps required for aflatoxinbiosynthesis and the genes for these enzymes have beencloned. Genes (aflR and aflJ) coding for proteins shownto be involved in transcriptional activation of most of thestructural genes are also part of the cluster (Table 1) (Caryet al. 2000a). Restriction mapping of cosmid and lambdaphage libraries of A. flavus and A. parasiticus DNAsshowed that all the genes are clustered within a 70-kbregion of the fungal genome (Fig. 2) (Trail et al. 1995b;Yu et al. 1995). Several otherAspergilli, e.g.,A. nidulans,make aflatoxin precursors such as sterigmatocystin (ST).The ST biosynthetic pathway is homologous to that in A.flavus and A. parasiticus, but the order of genes on thechromosomes is different (Fig. 2) (Brown et al. 1996).D. Bhatnagar ()) K. C. Ehrlich T. E. Cleveland

Southern Regional Research Center, ARS,USDA, New Orleans, LA 70124, USAe-mail: [email protected]: +1-504-2864419


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Aflatoxin formation

The synthesis of aflatoxin occurs through a series ofhighly organized oxidation-reduction reactions (Dutton1988; Bhatnagar et al. 1992; Minto and Townsend 1997).The currently accepted scheme is shown in Fig. 2.Aflatoxin biosynthesis begins with conversion of mal-onylCoA to a condensed polyketide noranthrone by theproducts of two fatty acid synthase genes (fas-1and fas-2)and a polyketide synthase gene (pksA) (Cary et al. 2000a).

No specific enzyme has yet been linked to the conversionof noranthrone to norsolorinic acid (NOR), the first stablemetabolite that can be isolated, but the conversion mayinvolve a monooxygenase (possibly cypA) and a dehy-drogenase (possibly norB) (Bhatnagar et al. 1992). Theconversion from NOR to averantin (AVN) involves adehydrogenase, encoded by the gene nor-1 (Chang et al.1992; Trail et al. 1994), but can also be catalyzed by thedehydrogenase encoded by norA (Cary and Bhatnagar1995). Some of the catalytic steps in the conversion ofAVR to versicolorin B (VERB) have not yet been

assigned to a specific gene in the cluster. Three genesare possible candidates for individual steps: cypX, moxY,and avfA, as indicated in Table 1. Two genes ver1(encoding a ketoreductase; Skory et al. 1992) and verA(encoding a cytochrome P-450 monooxygenase) arerequired for the conversion of versicolorin A (VERA) todemethylsterigmatocystin (DMST). The final step in theformation of aflatoxins is the conversion of O-methyl-

sterigmatocystin (OMST) or dihydro-O-methylsterigma-tocystin (DHOMST) to aflatoxins B1, B2, G1 and G2,requiring the presence of a NADPH-dependent mono-oxygenase, ordA (Prieto and Woloshuk 1997; Yu et al.1998). The formation of the G toxins involves anadditional step, possibly involving the enzyme encodedbyordB(Yu et al. 1998; Yabe et al. 1999). Another gene,aflT, encodes an ABC transporter protein that may benecessary for aflatoxin efflux from the cells.

Transcriptional regulation of aflatoxin biosynthesis

The production of aflatoxin by toxigenic Aspergilli isaffected by environmental and nutritional factors such astemperature, pH, carbon and nitrogen source, stressfactors, lipids, and certain metal salts (Cary et al.2000a). Some of these factors may affect expression ofgenes in the aflatoxin pathway. Of the 23 genes so farisolated that code for proteins involved in aflatoxinbiosynthesis, only one, aflR, appears to encode atranscription factor (Ehrlich et al. 1999b). Globally actingtranscription factors that respond to nutritional andenvironmental signals (Tag et al. 2000) may regulateexpression of some of these genes (Ehrlich and Cotty2002).

Isolation of the pathway-specific regulator gene, aflR

aflRwas first cloned from an A. flavus cosmid library byshowing that it could restore aflatoxin-producing abilityto a mutant blocked in all steps of aflatoxin biosynthesis(Payne et al. 1993). A homolog was subsequently isolatedfrom A. parasiticus transformed with a DNA fragmentcontaining aflR that caused the transformants to becomeorange-pigmented due to overproduction of aflatoxinbiosynthetic intermediates (Chang et al. 1993, 1995c).Overexpression ofaflRin A. flavusup-regulated aflatoxinpathway gene transcription and aflatoxin accumulation ina fashion similar to that found in A. parasiticus, butcolored colonies were not observed, a result suggestingthat subtle differences in biosynthesis occur in the twofungi (Flaherty and Payne 1997). These results suggestedthat an increase in the copy number of aflR somehowaltered normal regulation of aflatoxin biosynthesis. Me-tabolite feeding studies showed that a functional aflRallele is required for accumulation of NOR, the first stableintermediate in the aflatoxin biosynthetic pathway (Payneet al. 1993). When aflR was disrupted, the fungi wereincapable of aflatoxin metabolite production or transcrip-

Fig. 1AD Chemical structures of the aflatoxins. A The Btypeaflatoxins are characterized by a cyclopentane E-ring. Thesecompounds fluoresce blue under long-wavelength UV light. BThe G-type aflatoxins have a xanthone ring in place of thecyclopentane and fluoresce green. C Aflatoxins of the B2 and G2-type have a saturated bisfuranyl ring. D Aflatoxins B

2a and G

2ahave a hydrated bisfuranyl structure

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tion of nor-1, but otherwise grew normally. An aflRhomolog with only 31% identity to that ofA. parasiticus

or A. flavus was isolated from A nidulans, a fungus thatproduces ST rather than aflatoxin (Yu et al. 1996).Induced expression ofA. flavus aflR in A. nidulans, underconditions in which ST biosynthesis is normally sup-pressed, resulted in activation of genes in the STbiosynthetic pathway. These studies demonstrated thataflR function is conserved in widely different Aspergillusspp. Homologs of aflR have been identified from allaflatoxin and ST-producing Aspergilli so far examined(Chang et al. 1995a; Watson et al. 1999; Ehrlich et al.2002b)

Characterization of AflR and its rolein aflatoxin pathway gene expression

The deduced amino acid sequence of the 444 aa protein,AflR, contains a cysteine-rich motif, CTSCASSKVRC-TKEKPACARCIERGLAC, near its N-terminus (Wo-loshuk et al. 1994; Chang et al. 1995c). This domain ishomologous to Cys6-Zn2 domains of other fungal andyeast GAL4-type transcription factors involved in theregulation of many catabolic pathways (Burger et al.1991; Suarez et al. 1991; Kulmberg et al. 1992; Lamb et

al. 1996; Todd et al. 1998). Mutation of Cys6 to Trp,destroyed AflR function (Ehrlich et al. 1998). Preceding

the Cys6-Zn2domain is an arginine-rich domain, RRARK,that is homologous to nuclear localization regions in yeastand fungal GAL4-type proteins. As with mutation of thezinc cluster region, mutation of the amino acids in thenuclear localization region resulted in non-functionalAflR, presumably because the protein can no longer betranslocated to the nucleus. In some A. parasiticusisolates, a partial duplication of the gene cluster has beenfound (Chang and Yu 2002). The copy of aflR in thiscluster is predicted to encode a protein defective in itsnuclear localization domain (Cary et al. 2002). Compar-ison ofA. flavusandA. nidulansAflRs showed that, whileoverall amino acid identity is only 31%, the nuclear

localization signal domain and the Cys6-Zn2 domain are71% identical. Much higher identities were foundbetween the amino acid sequences of AflRs of otheraflatoxin-producing species of Aspergillus (>96%). Thezinc cluster region and the neighboring amino acidsimmediately downstream (linker region) have been shownto be necessary for sequence-specific DNA-binding inproteins of this type (Reece and Ptashne 1993). Non-conservative substitution of amino acids in the linkerregion also resulted in defective AflR (Ehrlich et al.1998).

Table 1 Genes in theAspergillus parasiticusaflatoxin gene clusterand their inferred protein sequence homology. AFB1 Aflatoxin B1,AFG1aflatoxin G1,AVNaverantin,AVR averufin,DMSTdemethyl-sterigmatocystin, HAVN5'-hydroxyaverantin, HVN hydroxyversi-

conal,NOR norsolorinic acid,OMST O-methylsterigmatocystin,STsterigmatocystin,VERAversicolorin A, VERBversicolorin B, VHAversiconal hemiacetal acetate, VHOH versiconal hemiacetal hy-droxide

Gene Protein homology Possible Function Stableintermediate

Reference

nor1 Dehydrogenasea Reduce NOR to AVNb AVN Zhou and Linz 1999norA Dehydrogenase Convert HAVN to AVR AVR Cary et al. 1996

norB Dehydrogenase Convert NOR anthrone to NOR NOR J. Yu, unpublishedcypA P450 monooxygenase Hydroxylate NOR-anthrone None J. Yu, unpublishedaflT ABC transporter Export aflatoxin from cell None P.-K. Chang, unpublishedpksA Polyketide synthase Convert hexanoylCoA to dodecaketide None Chang et al. 1995b;

Feng and Leonard 1995hexA Fatty acid synthase Convert malonylCoA to hexaketide None Watanabe et al. 1996hexB Fatty acid synthase Reduce hexaketide to hexanoylCoA None Watanabe et al. 1996afIR Cys6Zn2 DNAbindinga Regulate expression of genes in clusterb None Cary et al. 2000aaflJ Unknown AflR co-regulator None Meyers et al. 1998adhA Alcohol dehydrogenase Convert HAVN to AVRb AVR Chang et al. 2000bestA Esterasea Convert VHA to VHOHb VHOH Kusumoto and Hsieh 1996ver1 Dehydrogenase Convert VERA to DMSTb DMST Liang et al. 1996verA Monooxygenase Convert VHA to ST Unknown Matsushima et al. 1994verB Desaturase Convert VERB to VERAb VERA Yabe et al. 1991avnA P450 monooxygenase Convert AVN to HAVNb HAVN Yu et al. 1997avfA Dehydrogenase Convert AVR to VHAb VHA Yu et al. 2000aomtA O-Methyl transferasea Convert ST to OMSTb OMST Bhatnagar et al. 1988omtB O-Methyl transferase Convert DMST to STb ST Yu et al. 2000aordA Oxidoreductase Convert OMST to AFB1 AFB1 Prieto and Woloshuk 1997;

Yu et al. 1998vbs Dehydratasea Convert VHOH to VERBb VERB Silva et al. 1996cypX P450 monooxygenase Convert AVR to HVNb HVN Keller et al. 2000moxY Monooxygenase Convert VERA to DMST HVN Keller et al. 2000ordB Oxidoreductase Convert OMST to AFG1 AFG1 D.B., K.C.E. and T.E.C.

unpublished

a Protein function confirmedb Function confirmed by gene knockout in either Aspergillus parasiticus, Aspergillus flavus, or Aspergillus nidulans

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Several other domains appear to be highly conservedin AflRs from aflatoxin and ST-producing Aspergilli andmay constitute a portion of the transcription activationdomain. One of the conserved domains ranged fromaa 303 to aa 333 and contained the 66% identicalaa sequence, ILXCXCAXDXYXXXLVXLIVXKVLXW-YXAAA (where X = non-identical aa). This region, withpredominantly neutral amino acids, is predicted to have acoiled rather than a helical structure and may be involvedin interaction with basal transcription activating factors,such as the TATA-box-binding factor. An acidic amino

acid-rich domain (aa 349368) with the sequenceEERVLHHPSMVGEDCVDEED is homologous to acidicC-terminal regions in most GAL4-type transcriptionfactors and is believed to be characteristic of theactivation domain (Xiao et al. 1995). The C-terminal38 aa region (residues 408444) has runs of His,Arg, andacidic amino acids (HHPASPFSLLGFSGLEANLRHRL-RAVSSDIIDYLHRE) that again are probably importantfor transcription regulation (Xie et al. 2000). Pre-termi-nation ofaflRin Aspergillus sojaeat aa 384, in which thepredicted protein would be missing the C-terminal 37 aa,

Fig. 2AC Generally acceptedpathway for sterigmatocystin(ST) and aflatoxin biosynthesis.The genes and their corre-sponding enzymes are shown. AAflatoxin biosynthetic pathwaygene cluster in Aspergillus par-asiticus and A. flavus. B STbiosynthetic pathway genecluster in A. nidulans. The gene

names are labeled on the side ofthe cluster. Arrows indicate thedirection of gene transcription.Homologous genes in bothclusters have the same number.A putative hexose utilizationgene cluster is shown 3' to theaflatoxin pathway gene cluster.C Synthesis of aflatoxin. AFB1Aflatoxin B1, AFB2 aflatoxinB2, AFG1 aflatoxin G1, AFG2aflatoxin G2, AVNaverantin,AVNNaverufanin, AVR averu-fin, DHDMSTdihy-drodemethylstengmatocystin,DHOMSTdihydro-O-methyl-

sterigmatocystin, DHSTdihy-drosterigmatocystin,DMSTdemethylsterigmatocystin,HAVN5'-hydroxyaverantin, Metransferase methyltransferase,NOR norsolorinic acid, OMSTO-methylsterigmatocystin,VALversiconal,VERA versicolorinA, VERB versicolorin B, VHAversiconal hemiacetal acetate

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resulted in a protein with only 15% of the ability of wild-type AflR to activate expression (Matsushima et al. 2001;Takahashi et al. 2002). When the acidic amino acidsGlu423 and Asp439, were substituted with the basicamino acids Lys and His, the resulting AflR-carboxy-terminal GAL4-DNA binding domain fusion protein hada 10- to 15-fold lower ability to activate expression ofGAL1::lacZ in Saccharomyces cerevisiae than did the

intact AflR C-terminal fusion protein. Deletion of theacidic amino acids Asp365, Glu366, and Glu367 reducedthe activation potential by 33% and substitution ofArg427, Arg429, Arg431 with Leu caused a 50% dropin activation, whereas deletion of Glu444 had no effect. Amutation in which Asp436 (highlighted above) waschanged to His abolished activation entirely. Theseresults demonstrate that the carboxy terminal region iscritical to the transcription activation ability of AflR(Chang et al. 1999a). When the aflR region encodingaa 221444 (AFLRC) was fused to the A. parasiticus niiA(nitrite reductase) promoter, transformants overexpressedaflatoxin pathway precursors in a manner similar to that

found when extra copies of intact AflR were introducedinto the fungus. Since the AFLRC lacks the AflR DNA-binding domain, the increased metabolite productioncould result from titration of a putative AflR repressor(Chang et al. 1999b), thereby relieving inhibition. Asecond possibility is that the C-terminal domain of AflRcan stimulate expression with reduced efficiency evenwhen the protein is missing a DNA-binding domain(Chang et al. 1999a). A third possibility relates to theearlier observation that, in A. flavus, a 1.0 kb antisenseAflR (aflRas) transcript that overlaps the aflR promoterregion was detected. Although the role, if any, ofaflRasisstill unknown, it would be expected that increased levels

of AflR transcript would titrate out the antisense transcriptand thereby relieve possible inhibition (Woloshuk et al.1994).

Two other domains may be involved in aspects of thefine-tuning of regulation of AflR activity under differentenvironmental conditions. One unique site in AflR, whichis not present in other GAL4-type transcription factors sofar isolated from yeast and fungi, is a His-rich region(HAHRQAHTHAHAHSH, aa 103117). It is found in A.parasiticusand A. flavus AflRs, but is reduced in size inthe AflRs of other aflatoxin- or ST-producing Aspergilli(Ehrlich et al. 2002a). This region is predicted to be ametal-binding domain, but its function in AflR, so closeto the zinc binuclear motif (aa 2956), is still unknown.Another motif close to the carboxy terminus is not presentin A. parasiticusor A. flavusAflRs, but is found in AflRsfrom other aflatoxin-producing Aspergilli. This sequenceis Ser-rich and has a PEST value of +11.9, and should befunctionally comparable to PEST sites in other transcrip-tion factors such as mammalian Sp1. PEST sequenceshelp to target a protein for degradation by proteolysis(Rechsteiner 1988). Many regulatory proteins have PESTsequences that allow their fast turnover to reflect therequirements for a rapid response to external metabolicsignals. The turnover is probably mediated by a cascade

of phosphorylation that promotes ubiquitination andultimately proteolytic degradation. Such events mayreflect differences in the stability of aflatoxin productionby different types of aflatoxin- and ST-producing fungi.Phosphorylation may play a more direct role in AflRactivity, just as has been found for other Gal4-typetranscription factors (Parthun and Jaehning 1992). Phos-phorylation of key sites might prevent binding to either a

repressor protein or to the putative co-activator, AflJ (seebelow). AflR expressed in yeast has been shown to haveat least five phosphorylated sites based on the observationthat five differently migrating species detected on SDS-polyacrylamide gels collapsed to a single band afterphosphatase treatment (K.C. Ehrlich and J.W. Cary,unpublished results).

DNA-binding by AflR

AflR from bothA. nidulansandA. parasiticusbinds to thepalindromic sequence 5'-TCGN5CGA-3' (Fernandes et al.

1998; Ehrlich et al. 1999b). When possible sites contain-ing this motif at 168, and 81 were mutated in the A.nidulans stcU promoter, reporter expression was reduced6-fold, but when only one of these sites was mutated,expression was essentially unchanged. A putative far-upstream site at 762 appears to play little or no role inexpression. Based on mobility shift assays (EMSA), A.parasiticusAflR bound to the 5'-TCGN5CGA-3' motif inthe promoter regions of 11 of the aflatoxin biosynthesispathway genes known in A. parasiticus at the time of thestudy. Some of these genes, like stcUofA. nidulans, havemore than one AflR binding site. Unlike stcU, when eitherof the two AflR sites in the pksA promoter were

individually mutated, expression of the reporter constructwas reduced more than 100-fold indicating that, in thiscase, both AflR binding sites are critical to geneexpression. In the promoter of the gene encoding theP450 monooxygenase that oxidizes averantin to hydroxy-averantin, avnA, of the two possible AflR-binding sites,only mutation of the site closest to the transcription startsite affected expression (Cary et al. 2000b). In this casethe binding site is 5'-TCGN5CGG-3'. Footprinting anal-ysis showed that AflR protects a region 45 bp upstreamof the recognition motif, and that the preferred binding isto sequences with 5'-TCGG/CNNNC/GCGR-3'. Theamino acids most likely to make contact with the DNAin the zinc-binding domain ofA. nidulans AflR, (aa 3237, SRSKVK) are functionally identical to those in thezinc finger domain of A. parasiticus AflR (aa 3338,ASSKVR). In addition, the linker domains of the twotypes of AflR possess the same number and distribution ofLys and Arg residues, the residues critical for sequence-specific interaction. Therefore, it is not surprising that thetwo proteins recognize the same sequence even thoughtheir homology in this region is only 70%. By analogy tomost Gal4-type proteins that bind to a partially palin-dromic site, AflR probably binds to its recognition site asa dimer. However, no easily recognizable leucine repeat

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region is apparent in the sequence beyond the linkerregion, although the His-rich domain, which would beexpected to be a-helical, might be involved in dimeriza-tion as well as some other, poorly understood, functionwithin this protein.

Characterization of the aflR promoter

An intergenic region of 758 bp is located between thebidirectionally transcribedaflRandaflJgenes. To analyzepromoter function of the aflR gene, the entire 758-bpintergenic region as well as truncated forms of this regionwere used to drive expression of the Escherichia coli b-glucuronidase (GUS)-encoding gene uidA (Ehrlich et al.1999a). Removal of sequences in the promoter from 758to 280 had no apparent effect on promoter activity, butfurther truncation to 118 enhanced gene expressionnearly 5-fold. Therefore, there appears to be a negativeregulatory element in the region from 280 to 118.Further removal of bases 118 to 100 almost entirely

eliminated GUS gene expression. When the region from118 to 107 was deleted, there two-thirds of this activitywas lost. Therefore, sequences in the 18-bp region from100 to 118 appear to be critical for aflR promoteractivity. This region overlaps a 10-bp palindrome (120to 111) and a purine-rich region. EMSA using nuclearextracts from A. parasiticus and oligonucleotide ligandscovering the region from 81/173 revealed the presenceof a putative PACC-binding site (5'-GCCARG-3') (Espesoand Arst 2000). Binding to the aflR PacC site is consistentwith the function of this protein in repressing transcriptionof acid-expressed genes under alkaline conditions (Til-burn et al. 1995). Aflatoxin biosynthesis in A. flavus

occurs in acidic media, but is inhibited in alkaline media(Cotty 1988). It is possible that PacC binding to the 148/173 site has a negative effect on aflR expression.

The gene divergently transcribed from the aflRpromoter, aflJ, and the protein it encodes, AflJ, mayaffect AflR activity (Meyers et al. 1998; Chang et al.2000a; Ehrlich and Cotty 2002). AflJ has no knownsequence homologies with proteins identified in the yeastor fungal databases. Meyers et al. (1998) found thatdisruption of aflJ in A. flavus resulted in failure toproduce any aflatoxin pathway metabolites even thoughtranscripts for many of the aflatoxin pathway genes werestill made. This result suggests that AflJ does not affectAflR activity. Previously, Chang et al. (1995c) had foundthat aflR expression was enhanced in A. parasiticustransformants with aflR in which the aflJ region waspresent, compared to transformants in which this regionwas missing. In a different study Chang et al. (2000a)found that the nitrogen regulatory protein, AreA, bound toGATA sites in the aflR-aflJ intergenic region, suggestingthat nitrogen regulation of aflatoxin production could belinked to AreA control ofaflRand aflJexpression. Usinga yeast two-hybrid system, Chang et al. (1999a) were ableto show that AflJ binds to the carboxy-terminal region ofAflR. From the above results it is possible that AflJ is an

AflR coactivator but further work is necessary to provethis assumption.

Environmental and nutritional effects on aflatoxinproduction and the role of signaling pathways on aflatoxinpathway gene expression

Since sites in the aflR-aflJ intergenic region are recog-nized by transcription factors that are themselves regu-lated by environmental signals [pH regulates the activityof PacC (Tilburn et al. 1995) and nitrate regulates AreA],it is probable that, at least for nitrate, the effects onaflatoxin pathway gene transcription may, in part, bedirectly caused by changes in the expression of aflR oraflJresulting from activation by these factors. To supportthis observation we found that certain strains of aflatoxin-producingAspergillirespond differently to nitrate than doother strains, and that the differences could be correlatedwith differences in the number of possible GATA sites(ranging from five to nine) near theaflJstart site (Ehrlich

et al. 2002b). Other genes in the aflatoxin biosyntheticcluster have AreA and PacC binding sites at key positionsin their promoters that may affect their expression. Forexample, the1.7 kb intergenic region separating the nor-1andpksAgenes has two adjacent PacC sites nearly in themiddle that, from site-directed mutagenesis studies, affectexpression ofpksA, which encodes the pathway-specificpolyketide synthase necessary for the first steps information of the polyketide backbone (Ehrlich et al.2002a). In A. nidulans, the promoter region of the genestcU, which is necessary for conversion of VERA toDMST, contains a PacC-binding site immediately up-stream of its AflR-binding site and is probably involved in

expression of this gene.The mechanism of nitrate suppression is not clear. Theeffects of nitrate on aflatoxin formation indicate thatregulation of aflatoxin biosynthesis may be part of thenitrogen control circuit. Nitrate assimilation in fungi is atightly regulated process. The expression of nitratereductase and nitrite reductase genes requires both thelifting of nitrogen metabolite repression and specificinduction by nitrate (Marzluf 1997). Expression of genesinvolved in nitrate utilization is transcriptionally activatedby the global positive-acting regulatory factor, AreA.Marzluf (1997) suggested that nitrate increases thecytoplasmic NADPH/NADP ratio, which favors biosyn-thetic reductive reactions, and thus promotes utilization ofmalonyl coenzyme A and NADPH for fatty acid synthesisrather than for polyketide synthesis. Kachholz andDemain (1983) suggested that nitrate represses formationof active enzymes involved in the synthesis of alternariolmonomethyl ether in A. alternara. The transcription ofaflatoxin pathway genes is higher in nitrate medium intransformants containing an additional copy ofaflR thanin the untransformed fungus (Chang et al. 1995c; Flahertyand Payne 1997). This might be due to increased aflRcopy number, which elevates the basal levels of AFLR inthe transformants. More AFLR would then be available to

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bind to promoter sites, thereby increasing expression ofother aflatoxin pathway genes. Activation of transcriptioncould be modulated by the highly acidic domain ofAFLR. Preliminary data have shown that AFLR1 (arecombinant version of AFLR, but containing an intactzinc finger) also binds to sites in the promoter regions ofseveral aflatoxin biosynthetic genes and may therebyactivate their transcription (Ehrlich et al. 1999b).

The role of carbon utilization in the regulation ofexpression of genes involved in aflatoxin biosynthesis isnot, as yet, well understood. Unlike the biosynthesis ofmany other secondary metabolites, aflatoxin gene expres-sion is induced by the presence of simple carbohydrates,for example glucose, sucrose, or maltose, but not bypeptone, sorbose, or lactose (Payne and Brown 1998). Itshould be noted that all of the aflatoxin pathway genes sofar studied lack CreA sites in their promoters and,therefore, would not be expected to be subject to carboncatabolite repression mediated by the transcription factorCreA. However, an interesting possible role for CreA inaflR expression could be control of expression of the

antisenseaflRmRNA transcript, since two tandem CreA-binding sites, GCGGGGaGTGGGG, are present at thestart of this reported transcript. If carbon cataboliterepression prevents the expression of this transcript, nodecrease in the amount of AflR protein could occur.Another transcription factor that responds to simplesugars is Rgt1, a positively acting factor shown to benecessary for regulation of glucose transporter moleculeexpression (Ozcan et al. 1996). In S. cerevisiae, Rgt1functions as a transcriptional repressor in the absence ofglucose, but in the presence of high concentrations ofglucose it functions as a transcriptional activator. Apossible Rgt1 site is present in the promoter region ofA.

parasiticus aflJ(which, as discussed above, may encodean AflR cofactor) and may be involved in regulation of itsexpression.

Another indirect effect of glucose utilization onaflatoxin pathway gene expression could be the activationof a four gene sugar cluster downstream of the aflatoxingene cluster (Fig. 2) (Yu et al. 2000b). Activation ofgenes in this sugar cluster by an external hexose signalmay create a region of active chromatin that includes theneighboring aflatoxin gene cluster (Muro-Pasteur et al.1999). To support this observation, we and others foundthat when individual aflatoxin biosynthetic genes insert atsites other than the aflatoxin gene cluster following fungaltransformation, the expression of these genes is muchlower (500-fold) than it is when the genes insert into theaflatoxin cluster (Liang et al. 1997). Also, expression ofaflR in a partial duplicate copy of the aflatoxin pathwaycluster in some A. parasiticus isolates was not detectedeven though its promoter region was essentially intact(Chang and Yu 2002). Coordinated expression of thesetwo clusters could explain why the clustering of aflatoxingenes is necessary for aflatoxin production and why thecluster has been conserved in widely different taxaranging from A. nidulans to A. flavus (Walton 2000).

Another way that carbon source utilization could affectaflatoxin gene expression may be by inducing Gaprotein-dependent signaling in Aspergillus cells (Daniel et al.1998). High glucose levels should increase the level ofcAMP, which in turn activates cAMP-dependent proteinkinases. The level of these kinases is elevated inaflatoxin-producing cultures (Jayashree et al. 2000). Acorrelation between increased pool size of cAMP and

aflatoxin production had been observed previously (Khanand Venkitasubramanian 1986, 1987), and treatment ofcultures ofA. parasiticus with dibutyryl cAMP increasedaflatoxin biosynthesis (Bhatnagar et al. 1999). STproduction by A. nidulans appears to require inhibitionof FadA-dependent signaling (Hicks et al. 1997). FadA isthe alpha subunit of the A. nidulans heterotrimeric Gprotein. When FadA is bound to GTP, i.e., in its activeform, ST production (and sporulation) was repressed.However, in the presence of FlbA, a protein similar toRGS (regulators of G protein signaling)-type proteins, theintrinsic GTPase activity of FadA is stimulated, therebyleading to GTP hydrolysis, inactivation of FadA-depen-

dent signaling, and stimulation of ST production. Theactivity of RGS-type proteins is mediated by extracellularsignals that begin the signaling process by binding to acell-surface receptor. For the ST signaling cascade in A.nidulans, one possible product is synthesized by a GSI-type glutamine synthatase, FluG. G protein signalingmediates the levels of cAMP, which probably acts as thesecond messenger, affecting the activity of proteinkinases, which, in turn, may directly modulate the activityof AflR. Since AflR is probably active in its phosphor-ylated state, regulation of G protein signaling, cAMPlevels, and the activity of key protein kinases all shouldultimately affect aflatoxin and ST production by toxin-

producing Aspergilli.

Biological/evolutionary significanceof aflatoxin production

Aflatoxin does not appear to be essential to the growthand/or life-cycle of the fungus, and much speculationabout its role has been published (Bennett and Chris-tiansen 1983; Ciegler 1983; Lillehoj 1991; Jarvis andMiller 1996; Demain and Fang 2000). BuLock (1965)postulated that aflatoxin production was a mechanism forthe organism to release excess carbon when the fungus isgrowing in a carbon-rich environment, but little evidenceexists to support or defend this hypothesis and atoxigenicfungi can compete with toxigenic fungi on the samemedium (Cotty 1989).

Role of the cluster organization

Aflatoxin production may be a vestigal trait that hassurvived due to the clustered gene organization. Onehypothesis suggested that such an organization of genesmay allow coordinated regulation of the pathway (Walton

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2000). Another hypothesis is that the cluster organizationensures survival of the cluster by providing a mechanismfor horizontal gene transfer (Geiser et al. 1998, 2000).Evidence for such a transfer is still speculative. Manyspecies of Aspergilli contain some of the genes forsynthesis of aflatoxin and its precursors. BesidesA. flavusand A. parasiticus, A. nomius, A. pseudotamarii, A.bombycis, and A. ochraceoroseus (Klich et al. 2000;

Ehrlich et al. 2002b) make aflatoxins, while A. nidulans,A. ustus,A. variecolor,A. versicolorand possibly certainBipolaris spp. make ST (Cole and Cox 1987). Thearrangement of genes in theA. nidulans STgene cluster isdifferent from that in A. flavus or A. parasiticus (Fig. 2)and horizontal transfer of the different types of clusterbetween these species has not been found. The availabil-ity of a sexual stage, which facilitates recombination, inA. nidulans may account for the differences in geneorganization of the cluster in these species. Horizontalgene transfer may explain how genes similar to theaflatoxin pathway genes are present in species of fungiquite distant from Aspergilli, such as Dothistroma pini, a

pine forest fungus from New Zealand which makesVERA (Bradshaw et al. 2002). An argument againsthorizontal gene transfer of aflatoxin pathway genes is thatthe genes in the cluster quite closely follow the expectedevolutionary lineage of other non-clustered, essentialgenes in the same species isolated from widely divergentgeographical locations (Ehrlich et al. 2002b). A. flavusisolates separated by a vegetative compatibility systemthat precludes genetic mixing, also show no evidence forrecombination events that would be expected if theseevents were frequent rather than rare.

Aflatoxins as chemical signals

Aflatoxins could be chemical signals between species inan ecological niche (Lillehoj 1991) or serve to signalfungal development (Cotty 1988; Beppu 1992; Trail et al.1995a; Kale et al. 1996). Some of the genes involved inaflatoxin production appear to developmentally regulated(Mayorga and Timberlake 1992; Chang et al. 1995b) andan association between sclerotial formation and aflatoxinformation has been advanced (Bennett et al. 1978, 1979;Cotty 1988; Wang et al. 1996).

Role in defense

Aflatoxins toxicity may protect the fungus from com-petitors in the soil or during crop invasion (Demain andFang 2000). Aflatoxins are not particularly phytotoxic(McLean et al. 1995; Hasan 1999) and there is noevidence that they serve as virulence factors, sinceatoxigenic isolates are equally able to invade susceptiblecrop species (Cotty 1989). However, the atoxigenicisolates that are successful competitors retain the abilityto produce some of the aflatoxin biosynthesis enzymes(Cotty and Bhatnagar 1994). This result implies that some

of the biosynthetic proteins may be involved in othermetabolic processes that do contribute to fungal viru-lence.

Aflatoxins are toxic to insects and some aflatoxin-producing species have been associated with insect debris(Matsumura and Knight 1967; Reiss 1975; Wright et al.1982; Jarvis et al. 1984; Kurtzman et al. 1987; Llewellynet al. 1988; Drummond and Pinnock 1990). The lethal

concentration of aflatoxin for insects is quite high [ppmlevels; (Dowd 1992)] and such concentrations are rarelyfound in plants. These concentrations are found inindividual fungal colonies and could be toxic to feedinginsects. In spite of this, insects appear to be excellentvectors for mycotoxigenic fungi during plant invasion(Dowd 1992), suggesting that insects and aflatoxigenicfungi have adapted to one another. Fungi in the A. flavusgroup are thought to overwinter in the soil, perhaps assclerotia on crop debris, and overwintering structures(e.g., sclerotia) that contain aflatoxin may have a survivaladvantage compared to those from atoxigenic isolates.

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