Promoter elements involved in the expression of the Aspergillus parasiticus aflatoxin biosynthesis pathway gene avnA

Biochimica et Biophysica Acta 1491 (2000) 7^12 www.elsevier.com/locate/bba

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Promoter elements involved in the expression of the Aspergillus parasiticus a£atoxin biosynthesis pathway gene avnA Je¡rey W. Cary *, Beverly G. Montalbano, Kenneth C. Ehrlich Southern Regional Research Center, United States Department of Agriculture, PO Box 19687, 1100 R.E. Lee Blvd., New Orleans, LA 70179, USA Received 11 November 1999; received in revised form 3 February 2000; accepted 10 February 2000

Abstract One of the early genes in aflatoxin biosynthesis, avnA, encodes a pathway-specific cytochrome P-450 monooxygenase that catalyzes the hydroxylation of the polyketide anthraquinone, averantin. Based on L-glucuronidase (GUS) reporter and electrophoretic mobility shift assays, promoter sites upstream of 3118 bp in the 367-bp verB^avnA intergenic region are not required for avnA gene activity. Therefore, only the 3100 to 3110 site of the four putative binding sites for AFLR, the aflatoxin biosynthetic pathway transcription regulatory protein (consensus binding sequence : 5P-TCGN5 CGR-3P) was required for elevated avnA expression. ß 2000 Elsevier Science B.V. All rights reserved.

Biosynthesis of the a£atoxins, the potent toxic and carcinogenic metabolites of Aspergillus £avus and Aspergillus parasiticus, involves the coordinated expression of at least 21 genes which are clustered within a 75-kb region of one chromosome [1^3]. An early step in the biosynthesis involves conversion of the anthroquinone, averantin to 5P-hydroxyaverantin, a step catalyzed by a pathway-speci¢c cytochrome P-450 monooxygenase, in the P-450 family named CYP60A1 [4]. Knockout mutations of avnA led to accumulation of averantin and loss of a£atoxin accumulation, thereby demonstrating that the avnA gene product is uniquely responsible for the conversion. An intergenic region of 367 bp is located between the stop codon of verB and the start codon of avnA (Fig. 1). The former gene encodes a desaturase that is involved in conversion of versicolorin B to

* Corresponding author. Fax: +1-504-286-4419; E-mail: [email protected]

versicolorin A, one of the later steps in the a£atoxin biosynthetic pathway (Yu et al., unpublished results). This juxtaposition of an early and late gene in a£atoxin biosynthesis plus the fact that the avnA promoter region is relatively small prompted a detailed examination of the promoter elements involved in avnA gene activity. Transcription start point in A. parasiticus avnA: the avnA transcription start point (tsp) was mapped by primer extension analysis. Using an oligonucleotide primer with its 5P-end at +79 in the avnA coding region and mRNA from A. parasiticus grown on medium that induces genes involved in a£atoxin biosynthesis (glucose minimal salts medium, GMS [5]), an extension product of 111 bp was found, indicating that a single tsp is present 32 bp upstream of the ATG in avnA (Fig. 2). No primer extension product was obtained when mRNA was from fungi grown on peptone minimal salts medium, since avnA transcript was not present under these non-inducing conditions [6]. As is typical for fungal genes [7], the tsp is pre-

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Fig. 1. Sequence of the verB^avnA intergenic region. The genes verB and avnA are tandomly transcribed with the direction of transcription shown by the horizontal arrow. The vertical arrow marks the position of the transcription start point. Putative TATA box, AFLR and BRLA binding sites are indicated on the sequence in a di¡erent font. Bases converted to A's in site-directed mutagenesis studies (see text) are underlined. The sequence reported here can be found in the NCBI sequence database under accession number U62774.

ceded by a pyrimidine-rich block £ow 339 to 347. Two AT-rich regions (at 372 and 383) that resemble TATA-like elements may function as basal transcription elements for avnA. As expected, truncation of the promoter at 373 eliminated detectable promoter activity, while basal activity remained after truncation at 3100 (see Fig. 3 below). Functional analysis of the avnA promoter by GUS reporter assays: to analyze promoter function of the avnA gene, the entire intergenic region (avnA(p)) as

well as truncated forms of the region were used to drive expression of an Escherichia coli L-glucuronidase (GUS)-encoding gene uidA. Full-length and truncated versions of the avnA promoter (avnAp) were prepared by PCR of A. parasiticus genomic

C

Fig. 2. Mapping the avnA transcription start point (tsp) by primer extension analysis. A 24-bp oligonucleotide complementary to the avnA coding strand beginning at +79 was end-labeled in the presence of T4 polynucleotide kinase and 105 cpm of [Q-32 P]ATP and then annealed to 2 Wg A. parasiticus RH(pHX) mRNA from cultures either induced for a£atoxin pathway gene expression by growth on glucose minimal salts medium (mRNA-GMS) or grown in the non-inducing peptone minimal salts medium (mRNA-PMS). The primer was extended in the presence of SuperScript II RNase3 reverse transcriptase (BRL) at 52³C for 10 min. Products were separated on a 5% PAGE^7 M urea sequencing gel. The sequencing reactions from M13mp19 DNA primed with the 340 sequencing primer were run concurrently on the same gel for DNA size determination. The sequence surrounding the tsp is shown on the right side of the autoradiogram.

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Fig. 3. Functional activity of avnA promoter mutants. Activity is based on L-glucuronidase (GUS) expression in promoter-uidA (L-glucuronidase-encoding gene) construct DNAs transformed into A. parasiticus SU1(NiaD3 ) along with the plasmid pSL82 carrying a functional copy of A. parasiticus niaD as the selection marker. GUS activity was determined by £uorescence assay as previously described [14] on lysates from transformants grown for 48 h on either 2% yeast extract 6% sucrose (YES) or peptone minimal salts (PMS) agar plates. Relative activities were derived from the results of four separate assays using four clones each having a single copy of the promoter construct integrated at the niaD locus. The GUS activity for the unmutated 3308 clones was 0.31 þ 0.09 pmol/ min/mg protein. The `X's represent nucleotide substitutions in the putative AFLR binding sites as described in the text and in Fig. 1. A putative TATA site at 373 to 377 and a BRLA site at 3223 to 229 are indicated by ovals and triangles respectively. ND, not determined.

DNA using primers that introduced an upstream HindIII site and a NcoI site that overlapped the GUS gene translation initiation codon at the downstream end in the plasmid pBI221 (Clontech). A 1-kb region representing the avnA transcriptional terminator was ampli¢ed from genomic DNA using primers that introduced a 5P SacI site and a 3P EcoRI site, thereby allowing the avnA terminator to be placed downstream of the GUS coding region. Construct DNAs were transformed into A. parasiticus SU1(NiaD3 ) along with the plasmid pSL82 carrying a functional copy of A. parasiticus niaD as the selection marker [8]. Approximately 15% of the transformants showed GUS activity. Based on Southern blot analysis, most of these clones had a single copy of the plasmid integrated into the niaD chromosomal locus. Since GUS activity was much lower than that reported for other a£atoxin pathway genes in previous studies [9,10], it was possible that insertion of avnA(p)-GUS in the niaD locus caused a down-regulation (or lack of up-regulation) of expression. A similar e¡ect on expression of A. parasiticus trans-

formed with a ver1(p)-GUS construct was observed when insertion was at the niaD locus [9]. Removal of sequences in the promoter from 3367 to 3196 had no a¡ect on promoter activity, but further truncation to 3118 enhanced expression 3-fold (Fig. 3). Therefore, negative regulatory elements may be present in the region from 3196 to 3118. Further truncation to 3100 decreased promoter activity 6-fold, thereby removing an important positive regulatory element in this region. Functional activity of selected regions in the promoter was also studied by introducing point mutations in avnA(p) by overlap extension PCR as previously described [11] using the 3308 truncation plasmid as template. Constructs were prepared in which regions with homology to the AFLR binding motif, 5PTCGN5 CGR-3P [10,12], were altered (Fig. 1, underlined bases were changed from CG to AA). The sequences of the construct DNAs were con¢rmed by automated sequencing (results not shown). One of these regions (3147/3160) contained a GC-rich dyad symmetric inverted repeat 5-TCGGCCACTGCCGG-3P (palindrome under-

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Fig. 4. Electrophoretic mobility shift assays (EMSA) to identify sites that interact with sequence-speci¢c DNA-binding proteins. (A) Binding to AFLR1, a recombinant form of AFLR that contained 220 of the N-terminal 444 amino acids of the intact protein. (B) Binding to A. parasiticus nuclear extracts: G, extract from A. parasiticus RH(pHX) ([13]) grown on glucose minimal salts medium ; P, extract from A. parasiticus RH(pHX) grown on peptone minimal salts medium. The oligonucleotides used in this study are listed in Table 1. Oligonucleotides were labeled by primer extension in the presence of Klenow, [K-32 P]dCTP, and unlabeled dGTP, dATP, and dTTP. EMSA was performed at room temperature using 20^50 fmol radiolabeled oligonucleotide duplex and approximately 1.5 Wg crude protein. In all studies, the protein was ¢rst incubated with 5 pmol non-speci¢c 25-mer competitor oligonucleotide ([12]) and 1 Wg poly (dI-dC) for 10 min and then with the radiolabeled oligonucleotide for 15 min. A `+' indicates that 5 pmol of homologous competitor was added. A `-' indicates that no additional competitor was added. Reaction mixtures were run on 10% polyacrylamide, 1/2UTBE at 100 V for approximately 1 h. The dried gels were autoradiographed at 380³C using Kodak XAR5 ¢lm and an intensifying screen. B, bound (radiolabeled oligonucleotide bound speci¢cally to protein or proteins in the extract; F, free oligonucleotide; NS, non-speci¢c (radiolabeled oligonucleotide bound to protein in the extract that was not competed by excess homologous competitor (results not shown)).

lined) in which the triad repeat in the putative AFLR-binding site was separated by 8 bp rather than 5 bp. Mutagenesis of residues 3179 and 3180 or at 3158 and 3159 had no signi¢cant e¡ect on promoter activity, whereas mutagenesis of residues at 3108 and 3109 caused a loss of almost all activity. These results support the conclusion that only the 3100/3110 site is involved in gene activity. Localization of cis-acting elements in the avnA promoter by electrophoretic mobility shift assays (EMSA): EMSA with partially puri¢ed recombinant AFLR (AFLR1 [13]) showed that only an oligonucleotide overlapping the region from 3100 to

3110 (5P-TCGCAGCCCGG-3P) bound strongly to recombinant AFLR. As was previously shown [13], control bacterial lysates produced no band-shifts on EMSA gels. Possible weak binding was observed to the 3115/3136 oligonucleotide which overlapped a non-consensus AFLR-binding site, 5P-TCGCTCTTCGT-3P, but no speci¢c binding was found to the two other sites tested (Fig. 4A). Since removal of the 3115/3136 region by truncation did not decrease gene activity, but rather slightly increased activity, it is possible that this putative site is involved in negative rather than positive regulation of avnA transcription. Only the 392/3118 oligonucleotide

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Table 1 Oligonucleotides used for mobility shift assays of sites in the avnA promoter Positiona

Putative binding site for

392/3118

AFLR

3115/3136

AFLR

3140/3166

AFLR

3162/3190

AFLR

3213/3238

BRLA

Sequenceb

Binding in EMSA

5P-CGTTCTACTCGCAGCCCGGCTAACAAT-3P 3P-gcAAGATGAGCGTCGGGCCGATTGTTA-5P 5P-CCGATGGGCTTCTCGCTCTTCGTT-3P 3P-ggcTACCCGAAGAGCGAGAAGCAA-5P 5P-CTAGAATCGGCCACTGCCGGCGATGAA-3P 3P-gatcttAGCCGGTGACGGCCGCTACTT-5P 5P-AGAATTTCCTCGATATTCGGTATCCTAGA-3P 3P-tcttaaaGGAGCTATAAGCCATAGGATCT-5P 5P-AGAGAGACATCCCTCTGCCCAGCTGG-3P 3P-tctctcTGTAGGGAGACGGGTCGACC-5P

AFLR

PMS

Yes

No

Yes

Yes

No

Yes

No

No

No

No

a

Position relative to the translation start site of avnA. Uppercase letters are bases in the synthetic oligonucleotide ; lower case letters are bases derived from Klenow ¢ll-in. The bases in bold font are in regions with partial dyad symmetry or have homology either to an AFLR binding motif or to a BRLA binding motif (the 3213/3238 oligonucleotide). b

bound to protein (presumably AFLR) in a nuclear extract from A. parasiticus grown on glucose minimal salts medium, a medium that supports a£atoxin production (Fig. 4B). We had previously found that the same nuclear extract contained proteins that bound to oligonucleotides that overlap binding sites in the a£R promoter for the transcription factor involved in pH control, PACC, and in the norA promoter for AFLR [14]. In comparison, the other oligonucleotides tested (Table 1 and Fig. 4) overlapping bases from 3115 to 3136 (a C+T-rich region) and 3140 to 3166 (a G+C-rich palindrome) in the avnA promoter, bound to proteins in nuclear extracts from A. parasiticus grown in peptone minimal salts, a medium that does not induce a£atoxin production, but not to proteins from fungi grown on glucose minimal salts medium. These results provide further supportive evidence that certain sites in the intergenic region upstream of the 3100/3110 AFLR site may be involved in negative regulation of avnA expression and may partially explain why a£atoxin production does not take place when fungi are grown in peptone minimal salts medium ([14] and unpublished results). Similar GC-rich regions are present in some of the other a£atoxin pathway genes [15] and could play a similar role on gene expression. Although not detectable by EMSA, a much lower quantity of negative regulatory factors may also be present in nuclear extracts from fungi grown on glucose minimal salts medium, and could explain the decreased promoter

activity when these sites are present. A potential binding site for the transcription factor involved in regulation of developmental processes in Aspergillus, BRLA, was found in the avnA promoter at 3223 to 3229. Since the oligonucleotide overlapping this site is not recognized by proteins in either nuclear extract, it is unlikely to play a role in avnA expression.

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