Chromosomal location plays a role in regulation of aflatoxin gene expression in Aspergillus parasiticus.

The nor-1 gene in the filamentous fungus Aspergillus parasiticus encodes a ketoreductase involved in aflatoxin biosynthesis. To study environmental influences on nor-1 expression, we generated plasmid pAPGUSNNB containing a nor-1 promoter-beta-glucuronidase (GUS) (encoded by uidA) reporter fusion with niaD (encodes nitrate reductase) as a selectable marker. niaD transformants of A. parasiticus strain NR-1 (niaD) carried pAPGUSNNB integrated predominantly at the nor-1 or niaD locus. Expression of the native nor-1 and nor-1::GUS reporter was compared in transformants grown under aflatoxin-inducing conditions by Northern and Western analyses and by qualitative and quantitative GUS activity assays. The timing and level of nor-1 promoter function with pAPGUSNNB integrated at nor-1 was similar to that observed for the native nor-1 gene. In contrast, nor-1 promoter activity in pAPGUSNNB and a second nor-1::GUS reporter construct, pBNG3.0, was not detectable when integration occurred at niaD. Because niaD-dependent regulation could account for the absence of expression at niaD, a third chromosomal location was analyzed using pAPGUSNP, which contained nor-1::GUS plus pyrG (encodes OMP decarboxylase) as a selectable marker. GUS expression was detectable only when pAPGUSNP integrated at nor-1 and was not detectable at pyrG, even under growth conditions that required pyrG expression. nor-1::GUS is regulated similarly to the native nor-1 gene when it is integrated at its homologous site within the aflatoxin gene cluster but is not expressed at native nor-1 levels at two locations outside of the aflatoxin gene cluster. We conclude that the GUS reporter system can be used effectively to measure nor-1 promoter activity and that nor-1 is subject to position-dependent regulation in the A. parasiticus chromosome.     

Cloning of the Aspergillus parasiticus apa-2 gene associated with the regulation of aflatoxin biosynthesis.

An Aspergillus parasiticus gene, designated apa-2, was identified as a regulatory gene associated with aflatoxin biosynthesis. The apa-2 gene was cloned on the basis of overproduction of pathway intermediates following transformation of fungal strains with cosmid DNA containing the aflatoxin biosynthetic genes nor-1 and ver-1. Transformation of an O-methylsterigmatocystin-accumulating strain, A. parasiticus SRRC 2043, with a 5.5-kb HindIII-XbaI DNA fragment containing apa-2 resulted in overproduction of all aflatoxin pathway intermediates analyzed. Specific enzyme activities associated with the conversion of norsolorinic acid and sterigmatocystin were increased approximately twofold. The apa-2 gene was found to complement an A. flavus afl-2 mutant strain for aflatoxin production, suggesting that apa-2 is functionally homologous to afl-2. Comparison of the A. parasiticus apa-2 gene DNA sequence with that of the A. flavus afl-2 gene (G. A. Payne, G. J. Nystorm, D. Bhatnagar, T. E. Cleveland, and C. P. Woloshuk, Appl. Environ. Microbiol. 59:156-162, 1993) showed that they shared > 95% DNA homology. Physical mapping of cosmid subclones placed apa-2 approximately 8 kb from ver-1.     

Characterization of the function of the ver-1A and ver-1B genes, involved in aflatoxin biosynthesis in Aspergillus parasiticus.

The ver-1A gene was cloned and its nucleotide sequence was determined as part of a previous study on aflatoxin B1 (AFB1) biosynthesis in the filamentous fungus Aspergillus parasiticus SU-1. A second copy of this gene, ver-1B, was tentatively identified in this fungal strain. In this study, ver-1B was cloned by screening an A. parasiticus cosmid library with a ver-1A probe. The nucleotide sequence of ver-1B was determined. The predicted amino acid sequence of ver-1B had 95% identity with ver-1A. A translational stop codon, found in the ver-1B gene coding region, indicated that it encodes a truncated polypeptide. To confirm the function of the ver-1 genes in AFB1 synthesis, a plasmid (pDV-VA) was designed to disrupt ver-1A and/or ver-1B by transformation of the AFB1 producer A. parasiticus NR-1. One disruptant, VAD-102, which accumulated the pathway intermediate versicolorin A was obtained. Southern hybridization analysis of VAD-102 revealed that ver-1A but not ver-1B was disrupted. A functional ver-1A gene was transformed back into strain VAD-102. Transformants which received ver-1A produced AFB1, confirming that ver-1A is the only functional ver-1 gene in A. parasiticus SU-1 and that its gene product is involved in the conversion of versicolorin A to sterigmatocystin in AFB1 biosynthesis. A duplicated chromosomal region (approximately 12 kb) was identified upstream from ver-1A and ver-1B by Southern hybridization analysis. This duplicated region contained the aflR gene, which is proposed to be one regulator of AFB1, synthesis. A similar gene duplication was also identified in several other strains of A. parasiticus.     

Regulation of aflatoxin synthesis by FadA/cAMP/protein kinase A signaling in Aspergillus parasiticus

Analysis of fadA and pkaA mutants in the filamentous fungus Aspergillus nidulans demonstrated that FadA (Galpha) stimulates cyclic AMP (cAMP)-dependent protein kinase A (PKA) activity resulting, at least in part, in inhibition of conidiation and sterigmatocystin (ST) biosynthesis. In contrast, cAMP added to the growth medium stimulates aflatoxin (AF) synthesis in Aspergillus parasiticus. Our goal was to explain these conflicting reports and to provide mechanistic detail on the role of FadA, cAMP, and PKA in regulation of AF synthesis and conidiation in A. parasiticus. cAMP or dibutyryl-cAMP (DcAMP) were added to a solid growth medium and intracellular cyclic nucleotide levels, PKA activity, and nor-1 promoter activity were measured in A. parasiticus D8D3 (nor1::GUS reporter) and TJYP1-22 (fadAGA2R, activated allele). Similar to Tice and Buchanan [34], cAMP or DcAMP stimulated AF synthesis (and conidiation) associated with an AflR-dependent increase in nor-1 promoter activity. However, treatment resulted in a 100-fold increase in intracellular cAMP/DcAMP accompanied by a 40 to 80 fold decrease in total PKA activity. ThefadAG42R allele in TJYP1-22 decreased AF synthesis and conidiation, increased basal PKA activity 10 fold, and decreased total PKA activity 2 fold. In TJYP1-22, intracellular cAMP increased 2 fold without cAMP or DcAMP treatment; treatment did not stimulate conidiation or AF synthesis. Based on these data, we conclude that: (1) FadA/PKA regulate toxin synthesis and conidiation via similar mechanisms in Aspergillus spp.; and (2) intracellular cAMP levels, at least in part, mediate a PKA-dependent regulatory influence on conidiation and AF synthesis.     

黄曲霉毒素生物合成径调节基因aflR

黄曲霉毒素生物合成途径调节基因在黄曲霉毒素产生过程中发挥十分重要的作用,它为绝大多数黄曲霉毒素合成相关基因的表达所必需。黄曲霉毒素生物合成途径调节基因的启动子中,含有若干真菌转录因子同源物的假定结合位点。AflR蛋白是黄曲霉毒素生物合成途径中的主要正性转录因子,它调节大多数黄曲霉毒素合成相关基因,也包括其自身基因的表达。     

Structure and function of fas-1A, a gene encoding a putative fatty acid synthetase directly involved in aflatoxin biosynthesis in Aspergillus parasiticus.

A novel gene, fas-1A, directly involved in aflatoxin B1 (AFB1) biosynthesis, was cloned by genetic complementation of an Aspergillus parasiticus mutant strain, UVM8, blocked at two unique sites in the AFB1 biosynthetic pathway. Metabolite conversion studies localized the two genetic blocks to early steps in the AFB1 pathway (nor-1 and fas-1A) and confirmed that fas-1A is blocked prior to nor-1. Transformation of UVM8 with cosmids NorA and NorB restored function in nor-1 and fas-1A, resulting in synthesis of AFB1. An 8-kb SacI subclone of cosmid NorA complemented fas-1A only, resulting in accumulation of norsolorinic acid. Gene disruption of the fas-1A locus blocked norsolorinic acid accumulation in A. parasiticus B62 (nor-1), which normally accumulates this intermediate. These data confirmed that fas-1A is directly involved in AFB1 synthesis. The predicted amino acid sequence of fas-1A showed a high level of identity with extensive regions in the enoyl reductase and malonyl/palmityl transferase functional domains in the beta subunit of yeast fatty acid synthetase. Together, these data suggest that fas-1A encodes a novel fatty acid synthetase which synthesizes part of the polyketide backbone of AFB1. Additional data support an interaction between AFB1 synthesis and sclerotium development.     

Growth, sclerotia and aflatoxin production by Aspergillus parasiticus: influence of food preservatives

The influence of six food preservatives on control of aflatoxin production by Aspergillus parasiticus was tested in SMKY and defined media at three concentrations, viz., 0.1, 0.5 and 1.0%. Propionic acid completely inhibited the yield of mycelia and sclerotia, and aflatoxin production in culture medium, mycelia and sclerotia of A. parasiticus at all concentrations, whereas citric acid showed inhibition only at 0.5 and 1.0% concentrations. Sodium metabisulphite did not permit mycelial growth and aflatoxin biosynthesis in SMKY liquid medium but allowed production of sclerotia and aflatoxin on solid media, while the rest of the food preservatives had only marginal inhibitory effects.     

Effect of pyridazinone herbicides on growth and aflatoxin release by Aspergillus flavus and Aspergillus parasiticus.

The influence of pyridazinone herbicides on aflatoxin production by Aspergillus flavus and A. parasiticus was studied in liquid media. Mycelia production was not affected by 20, 40, or 60 micrograms of herbicide per ml; however, aflatoxin production by A. parasiticus was higher in media with herbicide, whereas A. flavus produced lower aflatoxin levels.     

Effect of pyridazinone herbicides on growth and aflatoxin release by Aspergillus flavus and Aspergillus parasiticus

The influence of pyridazinone herbicides on aflatoxin production by Aspergillus flavus and A. parasiticus was studied in liquid media. Mycelia production was not affected by 20, 40, or 60 micrograms of herbicide per ml; however, aflatoxin production by A. parasiticus was higher in media with herbicide, whereas A. flavus produced lower aflatoxin levels.