2.4.1 Genetic Regulation
Since the aflatoxin and ST biosynthetic pathway genes are found to be clustered on a single chromosome in both A. parasiticus and A. flavus and in A. nidulans, respectively, (Brown et al. 1996b; Woloshuk and Prieto 1998; Yu et al. 1995a), these genes are presumably expressed concurrently in the genome. In both the aflatoxin and ST gene clusters, there is a positive regulatory gene, aflR, for activating pathway gene transcription located in the middle of the gene clusters.
The aflR gene, coding for a sequence specific zinc binuclear DNA-binding protein, a Gal 4-type 47 kDa polypeptide, has been shown to be required for transcriptional activation of most, if not all, the structural genes (Chang et al. 1993; 1995b; 1999a,b; Ehrlich et al. 1998; Flaherty and Payne 1997; Payne et al. 1993; Yu et al. 1997; Woloshuk et al. 1994) by binding to the palindromic sequence 5'-TCGN5CGA-3' in the promoter region of the structural genes (Ehrlich et al. 1999a,b; Fernandes et al. 1998) in A parasiticus, A.flavus, and A. nidulans (Yu et al. 1996). In A. sojae, a nontoxigenic strain used in industrial fermentations, was found to contain a defective aflR gene in addition to potential other defects (Matsushima et al. 2001a,b; Takahashi et al. 2002). Thus, with the absence of the functional regulatory protein, no induction of aflatoxin can occur in this food grade Aspergillus. Additional factors involved in regulation of aflatoxin synthesis were evidenced by Flaherty and Payne (1997). Adjacent to the aflR gene in the aflatoxin gene cluster, a divergently transcribed gene, aflJ, was also found to be involved in the regulation of transcription (Meyers et al. 1998; Chang, personal communication). The exact mechanism by which aflJ modulates transcription of these pathway genes in concert with aflR is to be studied. A gene, aflT, encoding a membrane bound protein with homology to antibiotic efflux genes presumed to be for transporting aflatoxin out of the fungal cell, was discovered in A. parasiticus (Chang and Yu, unpublished, GenBank accession number: AF268071). The aflT gene might be involved in someway in aflatoxin secretion.
2.4.2 Nutritional Control (Carbon and Nitrogen Sources)
The best-known nutritional factors affecting aflatoxin biosynthesis are carbon and nitrogen sources (Adye and Mateles 1964; Bennettetal. 1979; Luchese and Harrigan 1993). It is clear that simple sugars such as glucose, sucrose, fructose, and maltose support aflatoxin formation, while peptone and lactose are not (Buchanan and Stahl 1984; Payne and Brown 1998). Woloshuk et al. (1997) reported the connection between alpha amylase activity and aflatoxin production in A.flavus. Yu et al. (2000c) identified a group of four genes that constitute a well-defined gene cluster related to sugar utilization in A. parasiticus next to the aflatoxin pathway gene cluster. The expression of the hxtA gene, encoding a hexose transporter protein, was found to be concurrent with the aflatoxin pathway cluster genes in aflatoxin-conducive medium. This is the first evidence that primary metabolism (sugar metabolism) and secondary metabolism (aflatoxin biosynthesis) are genetically linked on the chromosome. A close physical linkage between the two gene clusters could point to a relationship between the two clusters in reference to the processing of carbohydrates leading to the induction of aflatoxin biosynthesis. Lipid substrate was shown to be a good carbon source to support aflatoxin production (Fanelli and Fabbri 1989; Fanelli et al. 1983; 1995). A lipase gene, lipA, was cloned by Yu et al. (unpublished) in A. parasiticus and A. flavus. It was demonstrated that this lipase gene expression and subsequent aflatoxin production are induced by lipid substrate. Nonaflatoxin-conducive peptone medium supplemented with 0.5% soybean oil induces lipase gene expression and leads to aflatoxin formation (Yu et al. unpublished).
Nitrogen source plays an important role in aflatoxin production. Asparagine, aspartate, alanine, ammonium nitrate, ammonium nitrite, ammonium sulfate, glutamate, glutamine, and proline containing media support aflatoxin production; while sodium nitrate and sodium nitrite containing media do not (Davis et al. 1967; Eldridge 1964; Payne et al. 1983; Reddy et al. 1971; 1979). Studies by Kachholz and Demain (1983), Niehaus and Jiang (1989) suggested that nitrate represses AVF and aflatoxin formation. It was shown that while high temperature and nitrate support ST production in A. nidulans, the opposite was seen with A. parasiticus where these culture conditions repress synthesis of aflatoxin (Feng and Leonard 1998). Studies by Chang et al. (1995b) demonstrated that nitrate has suppressive effect on aflatoxin production and overexpression of aflR gene by additional copies of aflR could be required to overcome the negative regulatory effect on aflatoxin pathway gene transcription in the nitrogen control circuit. Chang et al. (1996) cloned a nitrogen utilization gene cluster consisting of two genes, niaD and niiA from A. parasiticus, A. oryzae, A. niger and A. nidulans and a nitrogen regulator, areA (Chang et al. 2000a) from A. parasiticus. In the intergenic region between aflR and aflJ several AreA binding motifs have been identified (Chang et al. 2000a). The AreA binding could prevent AflR binding. It seems that the aflatoxin formation and nitrogen metabolism are closely linked.
Aflatoxin production are also affected by many environmental factors (Bennett and Papa 1988; Demain 1972; Kale et al. 1994; Kale et al. 1996; Yabe et al. 1988b) such as physiological pH, temperature and water activity in the environment, volatile metabolites produced in host plant in response to fungal invasion, and developmental stages of the fungi.
Fungi have the ability to maintain internal pH and respond to the environmental changes. Studies by Cotty (1988) demonstrated the pH effect on aflatoxin formation and sclerotia development. Acidic pH condition favors aflatoxin biosynthesis in response to pH (Espeso et al. 1993; Keller et al. 1997). It is believed that pacC is a major transcriptional regulatory factor (Keller et al. 1997). In the promotor region of the regulatory gene, aflR, at least one pacC binding site has been identified (Ehrlich et al. 1999a). In the nonaflatoxin-inducing peptone medium, it was shown to be inhibitory to aflatoxin formation (Cotty 1988). The regulatory mechanism might be due to the binding of pacC to the PACC site in the aflR promoter region to repress the transcription of acid-expressed gene aflR and the aflatoxin formation (Espeso et al. 1993) since such medium keeps the medium (pH 7.4) at alkaline condition (Cotty 1988).
High water activity favors spore germination and mycelial growth. However, severe aflatoxin outbreak in corn was documented under hot weather and drought conditions (Brown et al. 1998). The mechanism of A. flavus infestation in corn under these conditions is not well understood. The possible scenarios may include a combination of these factors: (a) the plant defense mechanism is weakened under water stress conditions; (b) higher insect feeding and associated injuries to plant tissues, thus providing entry opportunities for fungal invasion; and (c) more fungal spores dispersed in the air under drier climate conditions.
Plant metabolites also play some role on aflatoxin formation. Zeringue (1991; 1996; 1993) reported plant metabolism and aflatoxin production. Wright et al. (2000) reported that at certain conditions, n-Decyl aldehyde reduces not only fungal growth of A. parasiticus but also aflatoxin production by over 95% compared with control. Octanal reduces fungal growth by 60%, however, increases aflatoxin production by 500%, while hexanal reduces fungal growth by 50%, but shows no effect on aflatoxin production. A relationship between fungal development (production of reproductive spore and survival structure sclerotia) and aflatoxin synthesis was studied (Garber and Cotty 1997). Wilson et al. (2001) reported that the 13(S)-hydroperoxide derivative of linoleic acid, the reaction product of lipoxygenase (encoded by L2 LOX gene from maize), involved in reducing aflatoxin production. The fungal morphology was significantly altered and toxin synthesis was inhibited during strain degeneration studies. The effect included the inhibition of expression of the toxin pathway genes, including aflR.
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