Browsing by Author "Gary A. Payne, Committee Chair"

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    Classical and Modern Genetic Approaches Reveal New Gene Associations with Aflatoxin Biosynthesis in Aspergillus parasiticus and A. flavus.
    (2006-04-11) Price, Michael Scott; Ross W. Whetten, Committee Member; Ralph A. Dean, Committee Member; Gary A. Payne, Committee Chair; Margaret E. Daub, Committee Member
    Production of aflatoxin (AF) in Aspergillus species is a highly regulated process involving transcriptional and post-transcriptional controls. Most of the regulation of AF production is focused through the pathway-specific transcriptional regulator aflR. While much is understood about the steps involved in biosynthesis, less is known about the regulatory circuits controlling AF production. A targeted cDNA microarray consisting of 768 genes was developed to investigate the effect of nitrogen source, carbon source, culture temperature and culture pH on AF production in A. parasiticus. Seventeen genes were identified as consistently differentially expressed with respect to AF, including three of the AF pathway structural genes. One of these genes, CA747470 was consistently downregulated with AF and was shown to repress AF production when overexpressed in A. flavus. Using an expanded cDNA microarray consisting of 5002 genes from an EST sequencing project (USDA-ARS, SRRC), we investigated the impact of aflR deletion on the transcriptome of A. parasiticus. In addition to the AF pathway genes, five additional genes were found to be regulated by aflR: niiA, hlyC, hypA, nadA, and hypB. These additional genes all possess putative consensus binding sites for AflR. The expression data from this study was also compared to the previous targeted array study by looking at expression of 324 genes shared by both microarrays. Expression profiles for the AF genes present on both arrays were consistent between experiments. CA747470 was shown to be highly expressed in all conditions. Overexpression of CA747470 resulted in increased radial growth and decreased AF production. Finally, a putative Rho-GDP dissociation inhibitor (Afrdi1) was deleted in A. flavus that was found to share a transcription profile with aflR with respect to AF. The Afrdi1 deletion strain exhibited repressed AF production, as well as a severe growth defect on minimal medium. The deletion mutant was phenotypically similar to the bem4 deletion strain of S. cerevisiae. The implication of this gene in AF regulation provides a direct link between vegetative growth and secondary metabolism in A. flavus. This work provides insight into the regulatory networks responsible for regulation of AF production in Aspergillus species, and indicates where future investigations are needed to understand the biology of this important mycotoxin.
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    Functional and comparative genomics of Aspergillus flavus to characterize secondary metabolism
    (2010-07-07) Georgianna, David Ryan; Dahlia M. Nielsen, Committee Member; James A. Alspaugh, Committee Member; David C. Muddiman, Committee Member; Gary A. Payne, Committee Chair
    Recently available genome sequences for Aspergillus flavus and A. oryzae were used to gain insight into the biosynthesis of secondary metabolites and to identify species-specific characters for these fungi. Transcriptome analyses, comparative genome hybridizations, and bottom-up proteomics were used to study two interrelated aspects of the ecology of A. flavus: 1) Regulation of secondary metabolism, with emphasis on the carcinogenic mycotoxin, aflatoxin, and 2) Discovery of key differences between A. flavus and the closely related domesticated species A. oryzae. Filamentous fungi such as A. flavus produce an abundance of diverse secondary metabolites, the most well-studied being aflatoxin. A defining feature of secondary metabolites is their production by clusters of genes. In this thesis I provide a comprehensive review of the current status of aflatoxin biosynthesis and regulation, including emerging genomic studies. I was the first to employ SILAC (stable isotope labeling by amino acids in cell culture), a technique to enable relative protein quantification by mass spectrometry, in a multicellular free-living prototroph. This technique allowed me to quantify 381 proteins during growth of A. flavus under conditions conducive (28°C) and non-conducive (37°C) for aflatoxin biosynthesis. From these studies I showed that enzymes needed for aflatoxin biosynthesis were lacking at 37°C. Additionally, I observed that protein concentration and transcript accumulation did not correlate well, with transcripts and proteins from genes within the aflatoxin cluster being a notable exception. I also showed through use of reporter constructs that the aflatoxin pathway specific transcription factor AflR is localized to the nucleus and active at 37°C even though aflatoxin is not produced and most genes in the pathway are not expressed. I have also studied the regulation of all predicted secondary metabolite gene clusters for A. flavus to better characterize their expression under environmental conditions. Of the predicted 55 secondary metabolite gene clusters in A. flavus, only three metabolites have been associated with a respective cluster. These are aflatoxin, cyclopiazonic acid, and aflatrem. Aside from aflatoxin, little knowledge is available about the regulation of other secondary metabolites in A. flavus. Transcriptional analysis of these secondary metabolite gene clusters over 28 experimental conditions showed the clusters to group into classes with similar profiles of expression. To further explore the correlations found by gene expression analysis, aflatoxin and CPA production were quantified under six cell culture environments known to be conducive or non-conducive for aflatoxin biosynthesis and in infected maize seeds. We found that CPA was not as tightly regulated as aflatoxin in response to cell culture environment however CPA and aflatoxin both accumulated similarly in developing maize seeds. I compared the genome of A. flavus with A. oryzae, a non-aflatoxigenic and domesticated species related to A. flavus. I hypothesized that insights gained from knowledge of the differences between these species would reveal new information on regulation of secondary metabolism, and possibly pathogenicity. I used comparative genome hybridization to characterize genomic content among three strains of each species. These results revealed that A. flavus and A. oryzae are strikingly similar with regard to DNA sequence. In addition to defining core sequence variations between these species I investigated the gene expression differences on substrates unique to each species’ ecological niche. Aspergillus oryzae has been used in fermentations through cultivation for thousand of years on wheat bran. Aspergillus flavus is an opportunistic pathogen of maize causing loss of crops through contamination with aflatoxin. From the expression analyses it was clear that despite similar genomes, A. flavus and A. oryzae use their genomes in vastly different ways. Among the most interesting of these differences was that A. flavus appears to be a much more capable producer of secondary metabolites.