Analysis of mechanisms regulating expression of the ver-1 gene, involved in aflatoxin biosynthesis

Abstract

Previous studies have shown that ver-1A encodes an enzyme which is directly involved in the conversion of versicolorin A to demethylsterigmatocystin during aflatoxin B 1 (AFB 1) biosynthesis in the filamentous fungus Aspergillus parasiticus. In this study, two different tools were utilized to study the regulation of ver-1A expression at the level of transcription and protein accumulation. First, a ver-1A cDNA was expressed in Escherichia coli with the vector pMAL-c2. The resulting maltose-binding protein-Ver-1A fusion protein was purified and used to generate polyclonal antibodies. Western blot analyses showed that these antibodies specifically recognized the Ver-1 protein (ϳ28 kDa) in cell extracts of Aspergillus parasiticus SU1. Second, a GUS (uidA; encodes ␤-glucuronidase) reporter system was developed by fusing the ver-1A promoter and transcription terminator to the GUS gene. Reporter constructs were transformed into A. parasiticus, resulting in a single copy of the ver-1A-GUS reporter integrated adjacent to the wild-type ver-1A gene (3 end) in the chromosome. Western blot analysis, Northern hybridization analysis, and a GUS activity assay were used to analyze transformants. The timing of appearance and pattern of accumulation of GUS transcript and GUS protein in transformants were consistent with the timing of appearance and pattern of accumulation of ver-1 transcript and Ver-1 protein. These data suggested that the GUS gene was under the same regulatory control as the wild-type ver-1 gene and confirmed that transcriptional regulation plays an important role in ver-1A expression. Integration of the ver-1A-GUS reporter construct at the niaD locus resulted in 500-fold-lower GUS activity, but the temporal pattern of accumulation of GUS activity was not affected. Therefore, chromosomal location can play a role in determining the level of gene expression in A. parasiticus and should be an important consideration when analyzing promoter function in this organism.

Completed sequence of aflatoxin pathway gene cluster in Aspergillus parasiticus1

“…However, the a£atoxin pathway gene, such as ordA, is expressed only within the a£atoxin gene cluster. Previous observations [30,31] and our experiment consistently demonstrated that chromosomal location is important for a£atoxin pathway gene expression in A. parasiticus. The mechanism for such transcriptional regulation is under investigation.…”

Overexpression of the Aspergillus nidulans histone 4 acetyltransferase EsaA increases activation of secondary metabolite production

“…Genes required for biosynthesis of SMs are located adjacent to one another within clusters in the genome, leading to the hypothesis that co-regulation of these gene clusters may be achieved through alterations in chromatin structure of the entire cluster (reviewed in Keller and Hohn, 1997;Hoffmeister and Keller, 2007). Initial evidence supporting this idea came from studies of the aflatoxin cluster in A. parasiticus, where translocation of two different biosynthetic genes outside of the cluster resulted in dramatically reduced gene expression (Liang et al, 1997;Chiou et al, 2002). In A. nidulans, removal of the COMPASS complex component cclA, responsible for histone 3 lysine 4 (H3K4) trimethylation, resulted in induction of the monodictyphenone cluster, producing emodin and several derivatives which were not previously believed to be produced by this species (Bok et al, 2009).…”

The initiation and pattern of spread of histone H4 acetylation parallel the order of transcriptional activation of genes in the aflatoxin cluster

“…In early studies we hypothesized that clustering of aflatoxin genes enabled such regulation to occur, although no conclusive data were reported (Trail et al ., 1995b). Further studies provided direct evidence that clustering affects aflatoxin gene regulation (Liang et al ., 1997). Integration of reporter plasmids bearing nor‐ 1 or ver‐ 1 promoters (genes encode ‘early’ and ‘middle’ enzymes, respectively) fused to uid A (encodes β‐ d ‐glucuronidase, GUS) (Jefferson, 1989) at locations outside the aflatoxin gene cluster ( nia D or pyr G) resulted in a loss of detectable promoter activity in comparison with integration into the nor ‐1 or ver‐ 1 loci (Chiou et al ., 2002).…”