Aspergillus nidulans Mutants Defective in stc Gene Cluster Regulation

Corresponding author: Nancy P. Keller, Department of Plant Pathology and Microbiology, Texas A&M University, 120 Peterson Bldg., College Station, TX 77843-2132., n-keller{at}tamu.edu (E-mail)

Communicating editor: M. E. ZOLAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The genes involved in the biosynthesis of sterigmatocystin (ST), a toxic secondary metabolite produced by Aspergillus nidulans and an aflatoxin (AF) precursor in other Aspergillus spp., are clustered on chromosome IV of A. nidulans. The sterigmatocystin gene cluster (stc gene cluster) is regulated by the pathway-specific transcription factor aflR. The function of aflR appears to be conserved between ST- and AF-producing aspergilli, as are most of the other genes in the cluster. We describe a novel screen for detecting mutants defective in stc gene cluster activity by use of a genetic block early in the ST biosynthetic pathway that results in the accumulation of the first stable intermediate, norsolorinic acid (NOR), an orange-colored compound visible with the unaided eye. We have mutagenized this NOR-accumulating strain and have isolated 176 Nor^- mutants, 83 of which appear to be wild type in growth and development. Sixty of these 83 mutations are linked to the stc gene cluster and are likely defects in aflR or known stc biosynthetic genes. Of the 23 mutations not linked to the stc gene cluster, 3 prevent accumulation of NOR due to the loss of aflR expression.

THE filamentous fungus Aspergillus nidulans produces a carcinogenic polyketide called sterigmatocystin (ST), the penultimate precursor in the aflatoxin (AF) biosynthetic pathway found in the related species A. parasiticus, A. flavus, and A. nomius (COLE and COX 1981 ). Both polyketides cause mammalian hepatocarcinomas (BRESSAC et al. 1991 ) and animal toxicities (ANDERSON et al. 1990 ), and both are thought to immunologically impair susceptible populations such as infants and the elderly (CARDWELL and MILLER 1996 ). A. parasiticus and A. flavus are common contaminants of a variety of agricultural products such as corn, peanuts, and cottonseed and the health risks associated with ingestion of AF have led to strict national and international guidelines regarding the acceptable levels of AF in food and feed.

The ST biosynthetic genes (stc genes) are clustered on chromosome IV of A. nidulans (BROWN et al. 1996B ). The cluster contains 24 coregulated genes (stcA through stcX), most of which encode ST biosynthetic enzymes (KELKAR et al. 1997 ; KELLER and HOHN 1997 ), and one pathway-specific regulatory gene, aflR, which encodes a 433-amino acid C6 zinc binuclear cluster DNA-binding protein (CHANG et al. 1993 ; PAYNE et al. 1993 ; YU et al. 1996 ; FERNANDES et al. 1998 ). Several lines of evidence indicate that AflR is a primary regulator of the other stc genes. aflR mRNA is detectable only after primary growth has ceased and stc gene expression follows shortly thereafter (YU et al. 1996 ). No stc gene transcripts can be detected in aflR deletion mutants (YU et al. 1996 ). AflR regulates stc gene expression by binding to the palindromic sequence 5'-TCG(N₅)CGA-3' found in the promoter regions of several stc genes (FERNANDES et al. 1998 ). As this same motif is found in AF cluster genes, and the A. flavus and A. nidulans aflR genes are functionally conserved (YU et al. 1996 ), it is likely that AflR regulation of AF cluster genes will be similar in other Aspergillus spp. Given that AflR has been shown to be a key regulator of ST production in Aspergillus species, novel strategies to control ST biosynthesis may be discovered from an understanding of the mechanisms that regulate the expression of aflR.

Although we have identified two members of a G protein signal transduction pathway that are required for normal aflR expression and activity and hence ST biosynthesis (HICKS et al. 1997 ), most of the genetic requirements for AflR function and stc cluster activation are unknown. In this article we present a screening method to rapidly identify pertinent ST mutants by taking advantage of the fact that the first stable intermediate in the ST/AF biosynthetic pathway, norsolorinic acid (NOR), is a visible orange pigment. NOR-accumulating mutants of A. parasiticus and A. flavus identified through mutagenesis techniques (LEE et al. 1971 ; PAPA 1982 ) have previously been used to visually identify AF-inducing and -inhibiting factors (KELLER et al. 1994 , KELLER et al. 1997 ) and AF deposition in living corn seed (KELLER et al. 1994 ). The production of NOR in A. nidulans requires at least four characterized stc genes; aflR, stcA, stcJ, and stcK. stcA encodes a polyketide synthase (YU and LEONARD 1995 ) and stcJ and stcK encode the and ß subunits, respectively, of a fatty acid synthase (BROWN et al. 1996A ). The fifth gene in the cluster, stcE, encodes a dehydrogenase that converts NOR to the next stable intermediate, averantin. By disrupting stcE, we created a NOR-accumulating strain which produces visible amounts of NOR when grown on oatmeal agar. Subsequent mutagenesis of this strain resulted in the visual identification of 176 mutants unable to accumulate NOR (Nor^- mutants). We describe the isolation and the genetic characterization of these mutants.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Fungal strains and growth conditions:
All fungal strains are listed in Table 1. Fungal strains were maintained on appropriately supplemented minimal medium (KAFER 1977 ) and stored as silica stocks. Standard genetic techniques were used for sexual crosses and diploid formation (PONTECORVO et al. 1953 ). Mutants were isolated on 1.0% oatmeal agar containing 0.25% Tween, 1.0% glucose, 0.5% yeast extract, and 1.6% agar plus appropriate supplements. For subsequent NOR assays, 1.0% oatmeal medium with 1.6% agar and appropriate supplements was used. All strains were incubated at 37°.

Nucleic acid manipulations:
stcE was disrupted by homologous integration of plasmid pJH3. A 3.98-kb EcoRV/HindIII fragment from pAHK25 (BROWN et al. 1996B ) was first subcloned into pK19 to create pJH1. pJH2 was created by inserting a polylinker containing a BamHI site into the PstI site within the coding region of stcE in pJH1. pJH3 was created by inserting a 1.8-kb BamHI fragment from pSalargB (BURSE et al. 1983 ) containing argB into the BamHI site in pJH2. pJH3 was used to transform A. nidulans strain RJH003 to arginine prototrophy. Subsequent transformants were visually screened for the ability to accumulate NOR and genomic DNA from such transformants was analyzed for the replacement of stcE with argB in the stcE locus. One transformant satisfying these requirements, TJH3.40, was used for the mutagenesis experiment. Additionally, TJH3.40 was sexually crossed to FGSC237 to introduce additional auxotrophies into the stcE background. One para-aminobenzoic acid auxotrophic progeny, RJH007 (Table 1) was used for diploid analysis. Disruption of stcE in RJH007 was confirmed by visualization of NOR and Southern analysis.

For mRNA extraction, wild-type and mutant strains were grown in 5 ml of liquid stationary complete medium (minimal medium with 2.0% dextrose, 0.2% peptone, 0.1% yeast extract, and 0.1% casein hydrolysate) in a 20-ml screw-cap vial. Cultures were set at a 45° angle to increase surface area and then incubated at 37° for 3 days after which fungal mycelium was harvested and frozen in liquid nitrogen. RNA was extracted with Trizol (GIBCO, Grand Island, NY) following the manufacturer's instructions. Total RNA (20 µg) was run on a 1.2% agarose gel containing 4% formaldehyde (v/v) and transferred to Hybond-N+ (Amersham, Arlington Heights, IL) nylon membrane following the manufacturer's instructions. DNA fragments from pAHK25 (a 1.29-kb XbaI/EcoRV fragment containing the aflR gene) and from pAHK27 (a 2.27-kb KpnI fragment containing the stcF, stcG, and stcH genes) were labeled with ³²P and hybridized to the nylon membranes at 60° for 16 hr. Membranes were washed for 30 min each in 2x SSC 0.1% SDS, 1x SSC 0.1% SDS, and 0.1x SSC 0.1% SDS at 60° and X-ray film was exposed overnight at -80°.

Mutagenesis:
Conidia of TJH3.40 were mutagenized with 4-nitro-1-quinoline oxide (NQO) as previously described by WIESER et al. 1994 . A total of 100,000 surviving colonies were screened on oatmeal agar (as described in Fungal strains and growth conditions) and 176 Nor^- mutants—hereafter referred to as MRB strains—were identified. The survival rate of the mutagenesis was between 1 and 10% (data not shown).

Genetic techniques:
Linkage of NQO-generated mutations to the stc cluster was determined by crossing each MRB strain to a stcE wild-type strain (FGSC89) and analyzing the meiotic progeny for accumulation of NOR. Linkage of the new mutation to the stc gene cluster would be indicated by an absence or low percentage of NOR-accumulating progeny due to a low frequency of recombination between the mutated gene and the stcE deletion. Mutations were determined to be unlinked to the stc gene cluster if 25% of the progeny were able to accumulate NOR. Approximately 200 meiotic progeny were analyzed from each cross.

The number of linkage groups or loci represented in the mutations unlinked to the stc cluster was determined by crossing each mutant in every pairwise combination. Each mutant was first crossed to FGSC410 to introduce a new genetic marker, in this case a mutation in the riboB gene, into the mutant background. Progeny from crosses between the original mutants and the recombinants were scored for NOR production. Two mutations were considered to be in the same or closely linked genes if none or a low percentage (<6%) of progeny accumulated NOR. If ~25% of the progeny accumulated NOR, the mutations were considered to be in different genes.

Diploids were generated between MRB strains and RJH007 to determine the dominant or recessive nature of each mutation. Diploids were tested on solid oatmeal medium for the accumulation of NOR and haploidization was induced by incorporation of 9 µl benomyl (2 mg/ml) into solid media.

NOR assays:
NOR production was assessed by growing all strains at 37° in liquid and solid media. All mutants were visually screened on solid oatmeal medium containing 1.0% oatmeal and appropriate supplements. The mutants not linked to the stc gene cluster were further assessed by growing them on solid oatmeal media at 30°, 37°, and 42° and on pH 4 and pH 8 media at 37°. This same subset of mutants was cultured on three different liquid media: (1) complete medium, (2) complete medium plus oatmeal, and (3) oatmeal. Mycelium was harvested after 3 days of stationary culture, frozen, lyophilized, crushed in a 1.7-ml centrifuge tube, and extracted with 750 µl chloroform for 10 min. An equal volume of water was added and the samples were vortexed and spun at 13,000 rpm for 10 min to separate the aqueous and organic phases. The organic phase was removed, dried, and resuspended in 50 µl chloroform. Twenty-five microliters of each extract was separated on a silica gel TLC plate in toluene:ethyl acetate:acetic acid (80:10:10) and visualized with long-wave UV light.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of Nor^- mutants:
We employed a visual screen for the identification of mutants defective in stc gene cluster activity. The mutants identified by this screen for NOR accumulation fell into two broad classes; 93 showed gross morphological defects and 83 exhibited growth and development resembling the original TJH3.40 strain. The 83 mutants whose growth and development resembled that of the original TJH3.40 strain were chosen for further analysis as we know that morphological mutants in A. nidulans are also defective in stc gene cluster activity (HICKS et al. 1997 ), and we wished to identify mutants in pathways more directly related to stc gene expression.

Linkage of mutations to the stc gene cluster:
To date it is known that four stc genes are required for NOR production, aflR, stcA, stcJ, and stcK. Mutations in any one of these four genes would lead to the loss of NOR production. Also, other genes have been found in the A. parasiticus and A. flavus aflatoxin clusters that are involved in aflatoxin production in these species and we believe it is possible that homologous genes that may be required for NOR production in A. nidulans exist in the region of the stc cluster. Therefore, MRB strains containing mutations linked to the cluster could have mutations in one of the four known stc cluster genes or in unidentified genes such as homologs of genes in the aflatoxin gene cluster. Linkage to the stc gene cluster was determined by crossing each MRB strain to a stcE wild-type strain (ST accumulating) and analyzing meiotic progeny for accumulation of NOR. MRB strains containing aflR, stcA, stcJ, and stcK mutations should produce a low percentage of NOR-accumulating progeny as such mutations would be closely linked to the stcE::argB locus. MRB strains containing mutations unlinked to the stc gene cluster would produce 25% NOR-accumulating progeny when crossed to a wild-type strain. Examination of 100–200 ascospore progeny of 60 of the 83 mutants showed between 0.99 and 6.2% recombination, suggesting that these 60 are linked to the stc gene cluster and represent candidate mutations in aflR, stcA, stcJ, stcK, or another linked locus.

Determination of linkage groups among the 23 unlinked mutants:
To determine the number of genes or loci represented in the 23 unlinked mutations, the mutants were crossed in every pairwise combination. This required first crossing each MRB strain to FGSC410 to introduce another genetic marker, a riboflavin mutation, into the mutant strains, thus generating RRB1 through RRB23. Each RRB strain, containing the stcE::argB2 disruption, the new mutation, and riboB2, was crossed to the 23 MRB mutants, and progeny were analyzed for the accumulation of NOR. As in the above test, NOR accumulation in 25% of the MRB x RRB progeny indicated that the mutations were in two separate linkage groups. If none of the progeny accumulated NOR, this suggested that the mutations were in the same or closely linked loci. From this analysis we determined that 5 mutants, MRB246, 278, 288, 308, and 379, contain mutations that are closely linked (and are therefore possibly allelic), whereas the remaining 18 mutants represented unique loci (Fig 1). In certain cases (indicated by an asterisk) viable ascospores were never generated after multiple attempts to cross two strains. We are unable to determine the genetic interaction in such cases.

View larger version (100K): In this window In a new window Download PPT slide   Figure 1. Linkage groups among the 23 unlinked mutations causing defects in NOR production. S indicates that the mutations in the two mutants are either in the same or in a closely linked gene. D indicates that the two mutations acted as two different, unlinked genes. An asterisk indicates that crosses between those two mutants never yielded viable ascospores.

mRNA and NOR analysis:
To determine whether the 23 unlinked mutations could define potential regulators/activators of aflR expression or AflR activity, we grew the mutants in three different media and analyzed culture extracts for NOR production and mRNA for aflR and stc gene transcripts. Cultures grown on liquid oatmeal in this manner did not produce enough mycelium for mRNA extraction, but organic extraction of NOR was possible. For 20 of the strains, transcript accumulation varied depending on growth medium. TLC analysis showed that all of these 20 mutants produced trace amounts of NOR that was visually undetectable in the oatmeal plate assay (Table 2). Only 3 mutants, MRB230, MRB234, and MRB300, consistently failed to produce NOR on 1.0% oatmeal plates or detectable aflR and stc transcripts (Fig 2). These 3 mutants represent the best candidates for nonconditional regulators of aflR.

View larger version (72K): In this window In a new window Download PPT slide   Figure 2. (A) RNA analysis of MRB mutants. DNA fragments used as probes are as described in MATERIALS AND METHODS. (B) Ethidium bromide-stained gel prior to transfer. Transcript sizes are as follows: aflR, 1.6 kb; stcF, 1.6 kb; stcG, 1.0 kb; and stcH, 0.6 kb.

In addition, all 23 mutants were tested for temperature and pH sensitivity. ST and AF biosynthesis has been shown to be pH regulated (KELLER et al. 1997 ) and AF biosynthesis is temperature regulated (SCHROEDER and HEIN 1967 ). The results from these experiments (Table 3) showed that several mutants were conditional for NOR production and/or had pleiotropic effects. For example, MRB326, MRB311, and MRB298 produced visible NOR at 29° but not 37° (the temperature used for the screen; Fig 3). This was in contrast to wild-type TJH3.40, which produced NOR at both 29° and 37° (Fig 3). Interestingly, NOR production is temperature sensitive in the wild type as it is not produced at 42° in TJH3.40 (Fig 3). Two mutants were pH sensitive; MRB234 could not grow at either pH extreme and MRB311 did not grow at pH 4 and at pH 8 had a fluffy phenotype and produced NOR.

View larger version (106K): In this window In a new window Download PPT slide   Figure 3. Temperature effects on NOR accumulation. A and B show strain TJH3.40 grown at 42° and 37°, respectively. C and D show strain MRB326 grown at 29° and 37°, respectively.

Dominant/recessive nature of mutations:
Diploids between MRB strains and another stcE disruption strain, RJH007, were created to determine the dominant or recessive nature of each mutation. For the 23 unlinked mutations, only one diploid (MRB265 x RJH007) did not produce NOR, indicating that MRB265 contains a dominant mutation. aflR and stc gene cluster transcripts were detectable in MRB265 (data not shown).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In an effort to reduce or eliminate AF contamination from food and feed supplies we are concentrating on understanding the genetic regulation of the Aspergillus gene cluster required for mycotoxin biosynthesis. A positively acting transcription factor present in the gene cluster, AflR (CHANG et al. 1993 ; PAYNE et al. 1993 ), is required for stc gene transcription (YU et al. 1996 ) and at least one AflR binding motif in the stc promoter (FERNANDES et al. 1998 ) is required for expression of the stc gene cluster. Other genes required for normal AF/ST production and aflR expression include flbA and fadA, genes involved in a G protein-signaling pathway required for Aspergillus developmental processes (HICKS et al. 1997 ). Obviously a more precise understanding of how aflR expression is regulated would greatly aid in designing strategies to reduce AF/ST production in agricultural commodities.

The design of the screen developed here ensured that we would obtain several different categories of Nor^- mutants, some of which would be defective in genes already found to be required for NOR production, including flbA, fadA, aflR, and three ST biosynthetic genes, stcA (encoding a polyketide synthase) and stcJ and stcK (encoding the and ß subunits, respectively, of a fatty acid synthase). Through genetic analysis, the mutations were determined to be linked or unlinked to the stc gene cluster. We are interested in characterizing those that are unlinked and thus represent the best possibilities for novel trans-acting regulators of ST/AF biosynthesis.

To assess how many loci are represented in the group of 23 unlinked mutants, meiotic progeny from each pairwise combination of mutants were analyzed for NOR production. This analysis revealed one linkage group that contained 5 mutants (MRB246, 278, 288, 308, and 379). MRB246, in contrast to the other four strains, was not able to grow at 42°, MRB308 did not express aflR in complete medium, and MRB278 grew poorly at pH 8. These differences could reflect distinct types of mutations in one gene or mutations in different, tightly linked genes. Interestingly, the remaining 18 mutants each fell into unique linkage groups, thus suggesting that our screen was not saturated. The high number of linkage groups may reflect the fact that a vast number of factors have been reported to influence AF production in A. flavus and A. parasiticus (ZAIKA and BUCHANAN 1987 ). Two of these factors are pH (KELLER et al. 1997 ) and temperature (SCHROEDER and HEIN 1967 ). An examination of the 23 mutants showed that several were pH (MRB230, MRB234, MRB278, MRB300, MRB311, MRB326, and MRB365) and temperature (MRB230, MRB234, MR246, MRB298, MRB311, and MRB326) sensitive with respect to growth and/or NOR production. Several mutants showed both pH and temperature sensitivity. We also found NOR production to be temperature sensitive in TJH3.40. Complementation of these 9 mutants may reveal some insight into pH and temperature control of ST/AF biosynthesis.

Finally, to further characterize the unlinked mutants in terms of the regulation of expression of the stc gene cluster, we looked for aflR and other stc gene cluster transcripts in these mutants. Only 3 of the 23 mutants lacked aflR and stc gene expression in all conditions tested. These 3 mutants fall into different linkage groups and appear to be specific regulators of aflR. The other 20 mutations likely affect some other aspect of ST biosynthesis, as aflR and other stc cluster genes were transcribed in one or more of the three media tested (Table 2). Perhaps these 20 mutations represent unlinked genes that are necessary for allocation of resources (e.g., acetyl CoAs) for polyketide formation or represent independent regulators of aflR. The mutations may block the availability of resources for the ST pathway or the localization of stc gene products to subcellular sites as is described for the penicillin biosynthetic pathway (MULLER et al. 1992 ). With the exception of the dominant mutation (MRB265), the genes denoted by the MRB mutations can be identified through classic complementation analysis with a wild-type cosmid library using the restoration of NOR production as the screen.

Aflatoxin contamination remains a problem worldwide and we believe that logical AF control strategies will be developed only through understanding the genetic regulation of the biosynthesis of this mycotoxin. To date we have found that all aspects of ST regulation in A. nidulans have been conserved with regard to AF regulation in the aflatoxigenic Aspergillus spp. including aflR function. Therefore it is likely that factors involved in the regulation of aflR should also be conserved in all species. If a novel approach to regulating ST production by regulating the expression of aflR exists, this might be applicable to controlling AF contamination in agricultural products.

	FOOTNOTES

¹ Present address: Cereon Genomics, LLP, Cambridge, MA 02139.

	ACKNOWLEDGMENTS

We thank Julie K. Hicks and Tiffany Sanford for construction of TJH3.40 and Elspeth Sharp for technical assistance. This work was supported by grant no. 96-35303-3415 from U.S. Department of Agriculture—Cooperative State Research Service to N.P.K. and T.H.A., by grant no. DGE-9354891 from National Science Foundation to N.P.K., T.H.A., and R.A.E.B., and by the Tom Slick Fellowship, Texas A&M College of Agriculture and Life Science, to R.A.E.B.

Manuscript received January 20, 1999; Accepted for publication June 7, 1999.

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				N. P. Keller, C. M. H. Watanabe, H. S. Kelkar, T. H. Adams, and C. A. Townsend Requirement of Monooxygenase-Mediated Steps for Sterigmatocystin Biosynthesis by Aspergillus nidulans Appl. Envir. Microbiol., January 1, 2000; 66(1): 359 - 362. [Abstract] [Full Text]