Sequence comparison of aflR from different Aspergillus species provides evidence for variability in regulation of aflatoxin production

Abstract

Aflatoxin contamination of foods and feeds is a world-wide agricultural problem. Aflatoxin production requires expression of the biosynthetic pathway regulatory gene, aflR, which encodes a Cys₆Zn₂-type DNA-binding protein. Homologs of aflR from Aspergillus nomius, bombycis, parasiticus, flavus, and pseudotamarii were compared to investigate the molecular basis for variation among aflatoxin-producing taxa in the regulation of aflatoxin production. Variability was found in putative promoter consensus elements and coding region motifs, including motifs involved in developmental regulation (AbaA, BrlA), regulation of nitrogen source utilization (AreA), and pH regulation (PacC), and in coding region PEST domains. Some of these elements may affect expression of aflJ, a gene divergently transcribed from aflR, that also is required for aflatoxin accumulation. Comparisons of phylogenetic trees obtained with either aligned aflR intergenic region sequence or coding region sequence and the observed divergence in regulatory features among the taxa provide evidence that regulatory signals for aflatoxin production evolved to respond to a variety of environmental stimuli under differential selective pressures. Phylogenetic analyses also suggest that isolates currently assigned to the A. flavus morphotype S_BG represent a distinct species and that A. nomius is a diverse paraphyletic assemblage likely to contain several species.

Introduction

Aspergillus section Flavi includes the species, A. parasiticus, A. flavus, A. nomius, A. bombycis, and A. pseudotamarii, which under certain conditions produce highly toxic and carcinogenic aflatoxins (Cotty and Cardwell, 1999; Egel et al., 1994; Ito et al., 2001; Peterson et al., 2001). In addition, more than 50 other species of filamentous fungi, including several species of Penicillium and a distantly related Chaetomium, have been reported to synthesize sterigmatocystin and other aflatoxin precursors (Barnes et al., 1994; Frisvad, 1985). Crops can become contaminated with aflatoxins when conditions favor growth of these fungi (Cotty et al., 1994). Annual costs resulting from crop losses and the need to limit food contamination have been estimated to be more than $100 million (Robens, 2001). Of the aflatoxin-producing species, A. flavus and A. parasiticus are the most common species implicated as causal agents of aflatoxin contamination (Cotty et al., 1994). Roles of agriculture in structuring communities of aflatoxin-producing fungi are unclear (Bayman and Cotty, 1993).

Aflatoxin-producing fungi are a mosaic of species that belong to divergent clades (Cotty et al., 1994). Within certain clades and along some lineages aflatoxin-producing ability is highly conserved, but in other clades aflatoxin production is either highly variable or lacking (Egel et al., 1994; Geiser et al., 2000). Aflatoxin-producing fungi reproduce in diverse ecological niches throughout warm climates (Cotty et al., 1994). Transcriptional regulation of aflatoxin biosynthesis might be contingent upon environmental signals particular to different niches. Nitrogen source, pH, and even antimicrobial agents are known to differentially influence aflatoxin biosynthesis among species, isolates, and strains (Cotty, 1988; Cotty and Cardwell, 1999). Variation in the molecular structure of aflatoxin regulatory genes and the molecular basis for divergent regulation of aflatoxin synthesis among strains has not been described.

The 23 genes involved in aflatoxin biosynthesis in A. flavus and A. parasiticus are part of a 75 kb cluster (Trail et al., 1995; Yu et al., 1995). Most of the genes are co-regulated by a single pathway-specific, DNA-binding protein, AflR (Ehrlich et al., 1999b). The gene encoding AflR resides in the cluster between early and late-acting genes involved in aflatoxin biosynthesis. AflR has a Cys₆Zn₂ DNA-binding domain and C-terminal transcription activation domain typical of GAL4-type fungal and yeast transcription factors. AflR binds to the partially palindromic consensus sequence 5^′-TCGN₅CGR-3^′ found in promoters of most of the aflatoxin biosynthesis genes (Chang et al., 1995; Ehrlich et al., 1999b).

A gene, aflJ, is divergently transcribed from aflR. This gene encodes a protein, AflJ, which also appears to be involved in aflatoxin gene regulation (Meyers et al., 1998). Although an exact function for this protein has not yet been identified, preliminary evidence suggests that it modulates AflR activity (P.-K. Chang, unpublished results). AflJ was found to be necessary for accumulation of some of the early precursor metabolites involved in the aflatoxin biosynthetic pathway (Meyers et al., 1998). In addition, transformants containing an extra copy of aflR but lacking an extra copy of aflJ have a reduced level of expression of aflR compared to transformants containing a second copy of both genes (Chang et al., 1995). In this report, we compared the nucleotide sequences of the aflJ/aflR intergenic region and aflR coding region in 28 isolates of five species of aflatoxin-producing fungi within Aspergillus section Flavi to deduce variations in regulatory region motifs and inferred protein structure, as well as phylogenetic relationships among these species.