Genetic connection between fatty acid metabolism and sporulation in Aspergillus nidulans

In the Ascomycete fungus Aspergillus nidulans, the ratio of conidia (asexual spores) to ascospores (sexual spores) is affected by linoleic acid moieties including endogenous sporogenic factors called psi factors. Deletion of odeA (Delta odeA), encoding a Delta-12 desaturase that converts oleic acid to linoleic acid, resulted in a strain depleted of polyunsaturated fatty acids (18:2 and 18:3) but increased in oleic acid (18:1) and total percent fatty acid content. Linoleic acid-derived psi factors were absent in this strain but oleic acid-derived psi factors were increased relative to wild type. The Delta odeA strain was reduced in conidial production and mycelial growth; these effects were most noticeable when cultures were grown at 26 degrees C in the dark. Under these environmental conditions, the Delta odeA strain was delayed in ascospore production but produced more ascospores than wild type over time. This suggests a role for oleic acid-derived psi factors in affecting the asexual to sexual spore ratio in A. nidulans. Fatty acid composition and spore development were also affected by veA, a gene previously shown to control light driven conidial and ascospore development. Taken together our results indicate an interaction between veA and odeA alleles for fatty acid metabolism and spore development in A. nidulans.     

Aflatoxin production via cross-feeding of pathway intermediates during cofermentation of aflatoxin pathway-blocked Aspergillus parasiticus mutants.

Cofermentation of Aspergillus parasiticus strains (SRRC 163 and SRRC 2043) blocked at different steps in the aflatoxin B1 (AFB1) biosynthetic pathway in a synthetic liquid medium or on seeds (cottonseed, corn kernels, and peanuts) resulted in production of AFB1. Strain SRRC 2043 accumulated O-methylsterigmatocystin (OMST), a late precursor in AFB1 biosynthesis, whereas SRRC 163 accumulated averantin, an early precursor in the pathway. Strain SRRC 2043 secreted large amounts of OMST in culture relative to the amounts of several other pathway intermediates secreted into media (by other AFB1 pathway-blocked strains). AFB1 production occurred even when colonies of SRRC 163 and SRRC 2043 strains (producing no detectable AFB1) were grown together on an agar medium while physically separated from each other by a filter membrane (0.22-micron pore size). In addition, when mycelia of strain SRRC 163 were added to culture filtrates (containing no mycelia but containing secreted OMST) of strain SRRC 2043, AFB1 production occurred. The results suggested a chemical (rather than genetic) mechanism of complementation for AFB1 production between AFB1 pathway-blocked strains, since no mycelial contact was required between these strains for AFB1 production. The mechanism for chemical complementation involves secretion of OMST by SRRC 2043 and subsequent absorption and conversion of OMST to AFB1 by mycelia of strain SRRC 163.     

Aflatoxin production via cross-feeding of pathway intermediates during cofermentation of aflatoxin pathway-blocked Aspergillus parasiticus mutants.

Cofermentation of Aspergillus parasiticus strains (SRRC 163 and SRRC 2043) blocked at different steps in the aflatoxin B1 (AFB1) biosynthetic pathway in a synthetic liquid medium or on seeds (cottonseed, corn kernels, and peanuts) resulted in production of AFB1. Strain SRRC 2043 accumulated O-methylsterigmatocystin (OMST), a late precursor in AFB1 biosynthesis, whereas SRRC 163 accumulated averantin, an early precursor in the pathway. Strain SRRC 2043 secreted large amounts of OMST in culture relative to the amounts of several other pathway intermediates secreted into media (by other AFB1 pathway-blocked strains). AFB1 production occurred even when colonies of SRRC 163 and SRRC 2043 strains (producing no detectable AFB1) were grown together on an agar medium while physically separated from each other by a filter membrane (0.22-micron pore size). In addition, when mycelia of strain SRRC 163 were added to culture filtrates (containing no mycelia but containing secreted OMST) of strain SRRC 2043, AFB1 production occurred. The results suggested a chemical (rather than genetic) mechanism of complementation for AFB1 production between AFB1 pathway-blocked strains, since no mycelial contact was required between these strains for AFB1 production. The mechanism for chemical complementation involves secretion of OMST by SRRC 2043 and subsequent absorption and conversion of OMST to AFB1 by mycelia of strain SRRC 163.     

Molasses supplementation promotes conidiation but suppresses aflatoxin production by small sclerotial Aspergillus flavus

AIMS: To find a supplemental ingredient that can be added to routinely used growth media to increase conidial production and decrease aflatoxin biosynthesis in small sclerotial (S strain) isolates of Aspergillus flavus. METHODS AND RESULTS: Molasses was added to three commonly used culture media: coconut agar (CAM), potato dextrose agar (PDA), and vegetable juice agar (V8) and production of conidia, sclerotia, and aflatoxins by A. flavus isolate CA43 was determined. The effect of nitrogen sources in molasses medium (MM) on production of conidia, sclerotia and aflatoxins was examined. Water activity and medium pH were also measured. Conidia harvested from agar plates were counted using a haemocytometer. Sclerotia were weighed after drying at 45 degrees C for 5 days. Aflatoxins B(1) and B(2) were quantified by high-performance liquid chromatography. Addition of molasses to the media did not change water activity or the pH significantly. Supplementing CAM and PDA with molasses increased conidial production and decreased aflatoxins. Two-fold increased yield of conidia was found on MM, which, like V8, did not support aflatoxin production. Adding ammonium to MM significantly increased the production of sclerotia and aflatoxins, but slightly decreased conidial production. Adding urea to MM significantly increased the production of conidia, sclerotia and aflatoxins. CONCLUSIONS: Molasses stimulated conidial production and inhibited aflatoxin production. Its effect on sclerotial production was medium-dependent. Water activity and medium pH were not related to changes in conidial, sclerotial or aflatoxin production. Medium containing molasses alone or molasses plus V8 juice were ideal for conidial production by S strain A. flavus. SIGNIFICANCE AND IMPACT OF THE STUDY: Insight into molecular events associated with the utilization of molasses may help to elucidate the mechanism(s) that decreases aflatoxin biosynthesis. Targeting genetic parameters in S strain A. flavus isolates may reduce aflatoxin contamination of crops by reducing the survival and toxigenicity of these strains.     

Effects of laeA deletion on Aspergillus flavus conidial development and hydrophobicity may contribute to loss of aflatoxin production

LaeA of Aspergillus nidulans is a putative methyltransferase and a component of the velvet complex; it is thought to mainly affect expression of genes required for the production of secondary metabolites. We found that although Aspergillus flavus CA14 laeA deletion mutants showed no aflatoxin production, expression of some of the early genes involved in aflatoxin formation, but not the later genes, could still be detected. The mutants grown in minimal medium supplemented with simple sugars and on some complex media exhibited altered conidial development. On potato dextrose agar (PDA) medium the deletion mutants showed reduced conidial chain elongation, increased production of conidiophores, and decreased colony hydrophobicity when compared to the parental strain. The loss of hydrophobicity and the other developmental changes in the laeA deletion mutants could affect the ability of the fungus to produce aflatoxins.     

Conversion of a new metabolite to aflatoxin B2 by Aspergillus parasiticus

A new metabolite which could be converted to aflatoxin (AF) B2 was detected during cofermentation analysis of two nonaflatoxigenic strains (SRRC 2043 and SRRC 163) of Aspergillus parasiticus. SRRC 2043, which accumulates the xanthone O-methylsterigmatocystin (OMST), a late precursor in the AFB1 pathway, was observed to accumulate another chemically related compound (HOMST; molecular weight, 356); SRRC 163 is blocked early in the pathway and accumulates averantin. During cofermentation of the two strains, levels of OMST and HOMST were observed to be greatly reduced in the culture, with simultaneous production of AFB1, AFB2, and AFG1. Intact cells of SRRC 163 were able to convert pure OMST or its precursor, sterigmatocystin, to AFB1 and AFG1 without AFB2 accumulation; the same cells converted isolated HOMST to AFB2 with no AFB1 or AFG1 production. The results indicate that AFB2 is produced from a separate branch in the AF biosynthetic pathway than are AFB1 and AFG1; AFB2 arises from HOMST, and AFB1 and AFG1 arise from sterigmatocystin and OMST.     

Conversion of a new metabolite to aflatoxin B2 by Aspergillus parasiticus.

A new metabolite which could be converted to aflatoxin (AF) B2 was detected during cofermentation analysis of two nonaflatoxigenic strains (SRRC 2043 and SRRC 163) of Aspergillus parasiticus. SRRC 2043, which accumulates the xanthone O-methylsterigmatocystin (OMST), a late precursor in the AFB1 pathway, was observed to accumulate another chemically related compound (HOMST; molecular weight, 356); SRRC 163 is blocked early in the pathway and accumulates averantin. During cofermentation of the two strains, levels of OMST and HOMST were observed to be greatly reduced in the culture, with simultaneous production of AFB1, AFB2, and AFG1. Intact cells of SRRC 163 were able to convert pure OMST or its precursor, sterigmatocystin, to AFB1 and AFG1 without AFB2 accumulation; the same cells converted isolated HOMST to AFB2 with no AFB1 or AFG1 production. The results indicate that AFB2 is produced from a separate branch in the AF biosynthetic pathway than are AFB1 and AFG1; AFB2 arises from HOMST, and AFB1 and AFG1 arise from sterigmatocystin and OMST.     

adhA in Aspergillus parasiticus is involved in conversion of 5'-hydroxyaverantin to averufin

Two routes for the conversion of 5'-hydroxyaverantin (HAVN) to averufin (AVF) in the synthesis of aflatoxin have been proposed. One involves the dehydration of HAVN to the lactone averufanin (AVNN), which is then oxidized to AVF. Another requires dehydrogenation of HAVN to 5'-ketoaverantin, the open-chain form of AVF, which then cyclizes spontaneously to AVF. We isolated a gene, adhA, from the aflatoxin gene cluster of Aspergillus parasiticus SU-1. The deduced ADHA amino acid sequence contained two conserved motifs found in short-chain alcohol dehydrogenases-a glycine-rich loop (GXXXGXG) that is necessary for interaction with NAD(+)-NADP(+), and the motif YXXXK, which is found at the active site. A. parasiticus SU-1, which produces aflatoxins, has two copies of adhA (adhA1), whereas A. parasiticus SRRC 2043, a strain that accumulates O-methylsterigmatocystin (OMST), has only one copy. Disruption of adhA in SRRC 2043 resulted in a strain that accumulates predominantly HAVN. This result suggests that ADHA is involved in the dehydrogenation of HAVN to AVF. Those adhA disruptants that still made small amounts of OMST also accumulated other metabolites, including AVNN, after prolonged culture.     

Fate of the methyl group during the conversion of sterigmatocystin into O-methylsterigmatocystin and aflatoxin B1 by cell-free preparations of Aspergillus parasiticus

Cell-free extracts of fungal mycelia of two aflatoxin non-producing isolates of Aspergillus parasiticus (SRRC 163 and SRRC 2043) were utilized for the study of enzyme activities involved in the latter stages of aflatoxin biosynthesis. The post-microsomal fractions (105,000 x g supernatant) of both SRRC 163 and SRRC 2043 were able to convert sterigmatocystin (ST) into O-methylsterigmatocystin (OMST); whereas the microsomal (105,000 x g pellet) preparation of only SRRC 163 was able to convert OMST into aflatoxin B1 (AFB1). S-Adenosylmethionine (SAM) was the primary substrate for the ST to OMST (methyltransferase) enzymatic conversion; [3H]OMST of specific activity 0.93 Ci/mmol was obtained in a reaction containing the [3H]SAM substrate (specific activity 1 Ci/mmol). After the terminal enzymatic conversion of OMST into AFB1, none of the radiolabel of the methyl group from OMST was found in AFB1. It is postulated that the methylation of ST may be required for subsequent enzymatic oxidation of OMST to aflatoxin B1.     

Characterization of aflatoxin-producing fungi outside of Aspergillus section Flavi

Most aspergilli that produce aflatoxin are members of Aspergillus section Flavi, however isolates of several Aspergillus species not closely related to section Flavi also have been found to produce aflatoxin. Two of the species, Aspergillus ochraceoroseus and an undescribed Aspergillus species SRRC 1468, are morphologically similar to members of Aspergillus section Circumdati. The other species have Emericella teleomorphs (Em. astellata and an undescribed Emericella species SRRC 2520) and are morphologically distinctive in having ascospores with large flanges. All these aflatoxin-producing isolates were from tropical zones near oceans, and none of them grew on artificial media at 37 C. Aflatoxins and sterigmatocystin production were quantified by high-pressure liquid chromatography (HPLC) and confirmed by HPLC-mass spectrometry (LC-MS) detection. Phylogenetic analyses were conducted on these four species using A. parasiticus and Em. nidulans, (which produce aflatoxin and the aflatoxin precursor sterigmatocystin, respectively) for comparison. Two aflatoxin/sterigmatocystin biosynthesis genes and the beta tubulin gene were used in the analyses. Results showed that of the new aflatoxin-producers, Aspergillus SRRC 1468 forms a strongly supported clade with A. ochraceoroseus as does Emericella SRRC 2520 with Em. astellata SRRC 503 and 512.     

Sporogenic effect of polyunsaturated fatty acids on development of Aspergillus spp.

Aspergillus spp. are frequently occurring seed-colonizing fungi that complete their disease cycles through the development of asexual spores, which function as inocula, and through the formation of cleistothecia and sclerotia. We found that development of all three of these structures in Aspergillus nidulans, Aspergillus flavus, and Aspergillus parasiticus is affected by linoleic acid and light. The specific morphological effects of linoleic acid include induction of precocious and increased asexual spore development in A. flavus and A. parasiticus strains and altered sclerotium production in some A. flavus strains in which sclerotium production decreases in the light but increases in the dark. In A. nidulans, both asexual spore production and sexual spore production were altered by linoleic acid. Spore development was induced in all three species by hydroperoxylinoleic acids, which are linoleic acid derivatives that are produced during fungal colonization of seeds. The sporogenic effects of these linoleic compounds on A. nidulans are similar to the sporogenic effects of A. nidulans psi factor, an endogenous mixture of hydroxylinoleic acid moieties. Light treatments also significantly increased asexual spore production in all three species. The sporogenic effects of light, linoleic acid, and linoleic acid derivatives on A. nidulans required an intact veA gene. The sporogenic effects of light and linoleic acid on Aspergillus spp., as well as members of other fungal genera, suggest that these factors may be significant environmental signals for fungal development.     

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.     

Association of aflatoxin biosynthesis and sclerotialdevelopment in Aspergillus parasiticus

Secondary metabolism in fungi is frequently associated with asexual and sexual development. Aspergillus parasiticus produces aflatoxins known to contaminate a variety of agricultural commodities. This strictly mitotic fungus, besides producing conidia asexually, produces sclerotia, structures resistant to harsh conditions and for propagation. Sclerotia are considered to be derived from the sexual structure, cleistothecia, and may represent a vestige of ascospore production. Introduction of the aflatoxin pathway-specific regulatory gene, aflR, and aflJ, which encoded a putative co-activator, into an O-methylsterigmatocystin (OMST)-accumulating strain,A. parasiticus SRRC 2043, resulted in elevated levels of accumulation of major aflatoxin precursors, including norsolorinic acid (NOR), averantin (AVN), versicolorin A (VERA) and OMST. The total amount of these aflatoxin precursors, NOR, VERA, AVN and OMST, produced by the aflR plus aflJ transformants was two to three-fold that produced by the aflR transformants. This increase indicated a synergisticeffect of aflR and aflJ on the synthesis of aflatoxin precursors. Increased production of the aflatoxin precursors was associated with progressive decrease in sclerotial size, alteration in sclerotial shape and weakening in the sclerotial structure of the transformants. The results showed that sclerotial development and aflatoxin biosynthesis are closely related. We proposed that competition for a common substrate, such as acetate, by the aflatoxin biosynthetic pathway could adversely affect sclerotial development in A. parasiticus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Detection of toxigenic isolates of Aspergillus flavus and related species on coconut cream agar

A new readily-prepared medium, coconut cream agar, was developed for the detection of aflatoxin production by isolates of Aspergillus flavus and related species. Coconut cream agar, which comprised coconut cream (50%) and agar (1.5%), detected isolates of A. flavus more effectively than the synthetic media tested and was as effective as media containing desiccated coconut. Fluorescence colouring of colonies grown on coconut cream agar could be used to differentiate A. flavus from A. parasiticus and A. nomius. In addition, conidial colour of A. flavus and A. nomius was quite distinct from that of A. parasiticus.     

Substrate-induced lipase gene expression and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus

AIMS: To establish a relationship between lipase gene expression and aflatoxin production by cloning the lipA gene and studying its expression pattern in several aflatoxigenic and nontoxigenic isolates of Aspergillus flavus and A. parasiticus. METHODS AND RESULTS: We have cloned a gene, lipA, that encodes a lipase involved in the breakdown of lipids from aflatoxin-producing A. flavus, A. parasiticus and two nonaflatoxigenic A. flavus isolates, wool-1 and wool-2. The lipA gene was transcribed under diverse media conditions, however, no mature mRNA was detected unless the growth medium was supplemented with 0.5% soya bean or peanut oil or the fungus was grown in lipid-rich medium such as coconut medium. The expression of the lipase gene (mature mRNA) under substrate-induced conditions correlated well with aflatoxin production in aflatoxigenic species A. flavus (SRRC 1007) and A. parasiticus (SRRC 143). CONCLUSIONS: Substrate-induced lipase gene expression might be indirectly related to aflatoxin formation by providing the basic building block 'acetate' for aflatoxin synthesis. No direct relationship between lipid metabolism and aflatoxin production can be ascertained, however, lipase gene expression correlates well with aflatoxin formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Lipid substrate induces and promotes aflatoxin formation. It gives insight into genetic and biochemical aspects of aflatoxin formation.     

Identification of O-methylsterigmatocystin as an aflatoxin B1 and G1 precursor in Aspergillus parasiticus.

An isolate of Aspergillus parasiticus CP461 (SRRC 2043) produced no detectable aflatoxins, but accumulated O-methylsterigmatocystin (OMST). When sterigmatocystin (ST) was fed to this isolate in a low-sugar medium, there was an increase in the accumulation of OMST, without aflatoxin synthesis. When radiolabeled [14C]OMST was fed to resting mycelia of a non-aflatoxin-, non-ST-, and non-OMST-producing mutant of A. parasiticus AVN-1 (SRRC 163), 14C-labeled aflatoxins B1 and G1 were produced; 10 nmol of OMST produced 7.8 nmol of B1 and 1.0 nmol of G1, while 10 nmol of ST produced 6.4 nmol of B1 and 0.6 nmol of G1. A time course study of aflatoxin synthesis in ST feeding experiments with AVN-1 revealed that OMST is synthesized by the mold during the onset of aflatoxin synthesis. The total amount of aflatoxins recovered from OMST feeding experiments was higher than from experiments in which ST was fed to the resting mycelia. These results suggest that OMST is a true metabolite in the aflatoxin biosynthetic pathway between sterigmatocystin and aflatoxins B1 and G1 and is not a shunt metabolite, as thought previously.     

Biochemical analysis of oxidative stress in the production of aflatoxin and its precursor intermediates

The relevance of oxidative stress in the production of aflatoxin and its precursors was examined in different mutants of Aspergillus parasiticus, which produce aflatoxin or its precursor intermediates, and compared with results obtained from a non-toxigenic strain. In comparison to the non-toxigenic strain (SRRC 255), an aflatoxin producing strain (NRRL 2999) or mutants that accumulate aflatoxin precursors such as norsolorinic acid (by SRRC 162) or versicolorin (by NRRL 6196) or O-methyl sterigmatocystin (by SRRC 2043) had greater oxygen requirements and higher contents of reactive oxygen species. These changes were in the graded order of NRRL 2999 > SRRC 2043 > NRRL 6196 > SRRC 162 > SRRC 255, indicating incremental accumulation of reactive oxygen species, being least in the non-toxigenic strain and increasing progressively during the ternary steps of aflatoxin formation. Oxidative stress in these strains was evident by increased activities of xanthine oxidase and free radical scavenging enzymes (superoxide dismutase and glutathione peroxidase) as compared to the non-toxigenic strain (SRRC 255). Culturing the toxigenic strain in presence of 0.1–10 μM H₂O₂ in the medium resulted in enhanced aflatoxin production, which could be related to dose-dependent increase in [¹⁴C]-acetate incorporation into aflatoxin B₁ and increased acetyl CoA carboxylase activity. The combined results suggest that formation of secondary metabolites such as aflatoxin and its precursors by A. parasiticus may occur as a compensatory response to reactive oxygen species accumulation.     

Interrelation of growth media and water activity in sclerotia characteristics of Aspergillus section Flavi

AIMS: To examine sclerotium characteristics of two Aspergillus flavus and two A. parasiticus strains at different growth media and water stress. METHODS AND RESULTS: The effects of growth media and water activity (0.999, 0.971, 0.955 and 0.937) on characteristics of sclerotia production (number, size and volume) of four isolates of Aspergillus section Flavi were examined. There was total inhibition under the driest conditions (0.955 and 0.937). When an osmotic potential of 0.971 was generated in Czapek agar (CD) and maize meal extract agar with sucrose and sodium nitrate (MMEA S/N), an increase in sclerotial size and volume was observed. The amount of sclerotia produced by cultures at 0.999 a(w) value was higher on CD. CONCLUSIONS: The data show that the sclerotia characteristics of A. flavus and A. parasiticus have been influenced by water availability and growth media composition. SIGNIFICANCE AND IMPACT OF THE STUDY: The information obtained shows that if we know the nutritional and water stress requirements for sclerotia production, it could be possible to develop effective prevention strategies to inhibit the survival of these fungi in grain.