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Kenneth C Ehrlich

Publications and source records attributed to Kenneth C Ehrlich.

15 recordsLinked to original sources

Analysis of single nucleotide polymorphisms in three genes shows evidence for genetic isolation of certain Aspergillus flavus vegetative compatibility groups.

Genetic exchange by asexual filamentous fungi is presumed to be limited to isolates in the same vegetative compatibility group (VCG). To evaluate genetic isolation of Aspergillus flavus due to vegetative incompatibility, three gene regions were chosen that contained closely spaced nucleotides that were polymorphic among some of the six VCGs examined. A member of each VCG was collected from five regions across the southern United States. Isolates belonging to the same VCG had similar sets of single nucleotide polymorphisms regardless of isolate origin. The six VCGs formed four genetically distinct groups. Although recombination between certain pairs of VCGs could not be excluded, none was found for YV36, the VCG that includes the atoxigenic A. flavus isolate currently used to mitigate aflatoxin contamination in cotton in Arizona.

Aspergillus flavus↗

Aflatoxin-producing Aspergillus species from Thailand.

Aflatoxin-producing Aspergillus species were isolated from soil samples from ten different regions within Thailand. Aspergillus flavus was present in all of the soil samples. Unlike previous studies, we found no A. parasiticus or A. flavus capable of both B- and G-type aflatoxin production in any of the samples. A. pseudotamarii, which had not been previously reported from Thailand, was found in four soil samples. In two of the samples A. nomius was determined to be the most abundant aflatoxin-producing species. Based on sequence alignments for three DNA regions (Taka-amylase A (taa), the rRNA internal transcribed spacer (ITS), and the intergenic region for the aflatoxin biosynthesis genes aflJ and aflR) the A. nomius isolates separated into three well-supported clades. Isolates from one of the A. nomius clades had morphological properties similar to those found for S-type isolates capable of B and G aflatoxin production and could easily be mistaken for these isolates. Our results suggest that such unusual A. nomius isolates could be a previously unrecognized agent for aflatoxin contamination in Thailand.

Aflatoxins↗

Cladal relatedness among Aspergillus oryzae isolates and Aspergillus flavus S and L morphotype isolates.

Aspergillus flavus is the main etiological agent for aflatoxin contamination of crops. Its close relative, A. oryzae, does not produce aflatoxins and has been widely used to produce fermented foods. We compared the phylogeny of A. oryzae isolates and L- and S-type sclerotial isolates of A. flavus using single nucleotide polymorphisms in the omtA gene in the aflatoxin biosynthesis gene cluster and deletions in and distal to the norB-cypA intergenic region as phylogenetic signals. Aflatoxin-producing ability and sclerotial size also were weighted in the analysis. Like A. flavus, the A. oryzae isolates form a polyphyletic assemblage. A. oryzae isolates in one clade strikingly resemble an A. flavus subgroup of atoxigenic L-type isolates. All toxigenic S-type isolates closely resemble another subgroup of atoxigenic L-type isolates. Because atoxigenic S-type isolates are extremely rare, we hypothesize that loss of aflatoxin production in S-type isolates may occur concomitantly with a change to L-type sclerotia. All toxigenic L-type isolates, unlike A. oryzae, have a 1.0 kb deletion in the norB-cypA region. Although A. oryzae isolates, like S-type, have a 1.5 kb deletion in the norB-cypA region, none were cladally related to S-type A. flavus isolates. Our results show that A. flavus populations are genetically diverse. A. oryzae isolates may descend from certain atoxigenic L-type A. flavus isolates.

Aflatoxins↗

Aflatoxigenicity in Aspergillus: molecular genetics, phylogenetic relationships and evolutionary implications.

Aflatoxins (AFs) are toxic and carcinogenic secondary metabolites produced by isolates of Aspergillus section Flavi as well as a number of Aspergillus isolates that are classified outside of section Flavi. Characterization of the AF and sterigmatocystin (ST) gene clusters and analysis of factors governing regulation of their biosynthesis has resulted in these two mycotoxins being the most extensively studied of fungal secondary metabolites. This wealth of information has allowed the determination of the molecular basis for non-production of AF in natural isolates of A. flavus and domesticated strains of A. oryzae. This review provides an overview of the molecular analysis of the AF and ST gene clusters as well as new information on an AF gene cluster identified in the non-section Flavi isolate, Aspergillus ochraceoroseus. Additionally, molecular phylogenetic analysis using AF biosynthetic gene sequences as well as ribosomal DNA internal transcribed spacer (ITS) sequences between various section Flavi and non-section Flavi species has enabled determination of the probable evolutionary history of the AF and ST gene clusters. A model for the evolution of the AF and ST gene clusters as well as possible biological roles for AF are discussed.

Aflatoxins↗

The aflatoxin biosynthesis cluster gene, aflX, encodes an oxidoreductase involved in conversion of versicolorin A to demethylsterigmatocystin.

Biosynthesis of the toxic and carcinogenic aflatoxins by the fungus Aspergillus flavus is a complicated process involving more that 27 enzymes and regulatory factors encoded by a clustered group of genes. Previous studies found that three enzymes, encoded by verA, ver-1, and aflY, are required for conversion of versicolorin A (VA), to demethylsterigmatocystin. We now show that a fourth enzyme, encoded by the previously uncharacterized gene, aflX (ordB), is also required for this conversion. A homolog of this gene, stcQ, is present in the A. nidulans sterigmatocystin (ST) biosynthesis cluster. Disruption of aflX in Aspergillus flavus gave transformants that accumulated approximately 4-fold more VA and fourfold less aflatoxin than the untransformed strain. Southern and Northern blot analyses confirmed that aflX was the only gene disrupted in these transformants. Feeding ST or O-methylsterigmatocystin, but not VA or earlier precursor metabolites, restored normal levels of AF production. The protein encoded by aflX is predicted to have domains typical of an NADH-dependent oxidoreductase. It has 27% amino acid identity to a protein encoded by the aflatoxin cluster gene, aflO (avfA). Some of domains in the protein are similar to those of epoxide hydrolases.

Amino Acid Sequence↗

Divergent regulation of aflatoxin production at acidic pH by two Aspergillus strains.

Production of aflatoxins (AF) by Aspergillus flavus and A. parasiticus is known to occur only at acidic pH. Although typical A. flavus isolates produced more AF as the external pH became increasingly acidic, an atypical strain from West Africa produced less. The lower AF production was not well correlated with decreases in expression of the aflatoxin pathway regulatory gene, aflR, or of two other biosynthesis genes.

Aflatoxins↗

An isolate of Aspergillus flavus used to reduce aflatoxin contamination in cottonseed has a defective polyketide synthase gene.

Contamination of certain foods and feeds with the highly toxic and carcinogenic family of Aspergillus mycotoxins, the aflatoxins, can place a severe economic burden on farmers. As one strategy to reduce aflatoxin contamination, the non-aflatoxin-producing A. flavus isolate AF36 is currently being applied to agricultural fields to competitively exclude aflatoxin-producing Aspergillus species. We now show that the polyketide synthase gene (pksA) required for aflatoxin biosynthesis in AF36, and in other members of the same vegetative compatibility group, possesses a nucleotide polymorphism near the beginning of the coding sequence. This nucleotide change introduces a premature stop codon into the coding sequence, thereby preventing enzyme production and aflatoxin accumulation.

Aflatoxins↗

Aflatoxin biosynthesis cluster gene cypA is required for G aflatoxin formation.

Aspergillus flavus isolates produce only aflatoxins B1 and B2, while Aspergillus parasiticus and Aspergillus nomius produce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene, pksA, revealed that A. flavus isolates are missing portions of genes (cypA and norB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Gibberella zeae Tri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring of O-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.

Aflatoxins↗

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

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 SBG represent a distinct species and that A. nomius is a diverse paraphyletic assemblage likely to contain several species.

Aflatoxins↗

Molecular and functional characterization of a second copy of the aflatoxin regulatory gene, aflR-2, from Aspergillus parasiticus.

The genes required for the synthesis of aflatoxin (AF) in Aspergillus flavus and Aspergillus parasiticus have been shown to be clustered on a chromosome in these fungi. Transcription of most of these genes is dependent upon the activity of the aflR gene, also present on the gene cluster, which encodes a zinc binuclear cluster DNA-binding protein. While many strains of A. parasiticus have only one copy of aflR (aflR-1), many others contain a second copy of this gene (aflR-2) which resides on a duplicated region of the aflatoxin gene cluster. Targeted disruption of aflR-1 generated a number of non-aflatoxin producing transformants of A. parasiticus SU-1 which still harbored a wild-type aflR-2 gene. Southern and Northern hybridization analyses and ELISA assays demonstrated that aflR-1 had been successfully inactivated in strain AFS10. DNA sequence analysis showed that aflR-2 was capable of encoding a deduced 47 kDa protein. Northern and RT-PCR analysis of RNA from a toxin producing strain indicated that aflR-2 was transcribed at extremely low levels compared to aflR-1. RT-PCR analysis of RNA from AFS10 demonstrated that mRNAs of aflatoxin pathway genes were not processed to their mature forms. Functional analysis of aflr-2 protein in a yeast system showed that it was not activating transcription.

Aflatoxins↗

Promoter elements in the aflatoxin pathway polyketide synthase gene.

PksA catalyzes the formation of the polyketide backbone necessary for aflatoxin biosynthesis. Based on reporter assays and sequence comparisons of the nor1-pksA intergenic region in different aflatoxin-producing Aspergillus species, cis-acting elements for the aflatoxin pathway-specific regulatory protein, AflR, and the global-acting regulatory proteins BrlA and PacC are involved in pksA promoter activity.

Aflatoxins↗

Toxins of filamentous fungi.

Mycotoxins are low-molecular-weight secondary metabolites of fungi. The most significant mycotoxins are contaminants of agricultural commodities, foods and feeds. Fungi that produce these toxins do so both prior to harvest and during storage. Although contamination of commodities by toxigenic fungi occurs frequently in areas with a hot and humid climate (i.e. conditions favorable for fungal growth), they can also be found in temperate conditions. Production of mycotoxins is dependent upon the type of producing fungus and environmental conditions such as the substrate, water activity (moisture and relative humidity), duration of exposure to stress conditions and microbial, insect or other animal interactions. Although outbreaks of mycotoxicoses in humans have been documented, several of these have not been well characterized, neither has a direct correlation between the mycotoxin and resulting toxic effect been well established in vivo. Even though the specific modes of action of most of the toxins are not well established, acute and chronic effects in prokaryotic and eukaryotic systems, including humans have been reported. The toxicity of the mycotoxins varies considerably with the toxin, the animal species exposed to it, and the extent of exposure, age and nutritional status. Most of the toxic effects of mycotoxins are limited to specific organs, but several mycotoxins affect many organs. Induction of cancer by some mycotoxins is a major concern as a chronic effect of these toxins. It is nearly impossible to eliminate mycotoxins from the foods and feed in spite of the regulatory efforts at the national and international levels to remove the contaminated commodities. This is because mycotoxins are highly stable compounds, the producing fungi are ubiquitous, and food contamination can occur both before and after harvest. Nevertheless, good farm management practices and adequate storage facilities minimize the toxin contamination problems. Current research is designed to develop natural biocontrol competitive fungi and to enhance host resistance against fungal growth or toxin production. These efforts could prevent toxin formation entirely. Rigorous programs for reducing the risk of human and animal exposure to contaminated foods and feed also include economically feasible and safe detoxification processes and dietary modifications. Although risk assessment has been made for some mycotoxins, additional, systematic epidemological data for human exposure is needed for establishing toxicological parameters for mycotoxins and the safe dose for humans. It is unreasonable to expect complete elimination of the mycotoxin problem. But multiple approaches will be needed to minimize the economic impact of the toxins on the entire agriculture industry and their harmfulness to human and animal health.

Aflatoxins↗

Aflatoxin biosynthesis.

Aflatoxins are toxic and extremely carcinogenic natural secondary metabolites produced primarily by the fungi Aspergillus flavus and Aspergillus parasiticus. The biosynthesis of aflatoxins is a complex process involving multi-enzymatic reactions. Genetic studies of the molecular mechanism of aflatoxin B1 biosynthesis have identified an aflatoxin pathway gene cluster of 70 kilobase pairs in length consisting of at least 24 identified structural genes including a positive regulatory gene as transcription activator. The structural genes encode cytochrome P450 monooxygenases, dehydrogenases, oxidases, methyltransferases, a polyketide synthase and two unique fatty acid synthases. The aflatoxin biosynthesis and its genetic regulation are discussed in this review. The current knowledge of the relationship between fungal development and secondary metabolism is also summarized.

Journal Article↗