Characterization of aflJ , a Gene Required for Conversion of Pathway Intermediates to Aflatoxin

Abstract

The genes encoding the aflatoxin biosynthetic pathway enzymes have been localized as a cluster to a 75-kb DNA fragment. The enzymatic functions of the products of most of the genes in the cluster are known, but there are a few genes that have not yet been characterized. We report here the characterization of one of these genes, a gene designated aflJ. This gene resides in the cluster adjacent to the pathway regulatory gene, aflR, and the two genes are divergently transcribed. Disruption of aflJ inAspergillus flavus results in a failure to produce aflatoxins and a failure to convert exogenously added pathway intermediates norsolorinic acid, sterigmatocystin, andO-methylsterigmatocystin to aflatoxin. The disrupted strain does, however, accumulate pksA, nor-1,ver-1, and omtA transcripts under conditions conducive to aflatoxin biosynthesis. Therefore, disruption ofaflJ does not affect transcription of these genes, andaflJ does not appear to have a regulatory function similar to that of aflR. Sequence analysis of aflJ and its putative peptide, AflJ, did not reveal any enzymatic domains or significant similarities to proteins of known function. The putative peptide does contain three regions predicted to be membrane-spanning domains and a microbodies C-terminal targeting signal.

A multiplex RT-PCR approach to detect aflatoxigenic strains of Aspergillus flavus

“…Among the 25 genes involved in aflatoxin biosynthesis (Yu et al 1995(Yu et al , 2004, we selected two genes, aflR and aflS, whose regulatory role in controlling the level of structural genes expression has been ascertained (Payne et al 1993;Woloshuk et al 1994;Flaherty and Payne 1997;Liu and Chu 1998;Meyers et al 1998;Takahashi et al 2002;Chang 2003;Ehrlich et al 2003;Chang 2004;Price et al 2006). In particular, aflR gene integrity has been considered a good candidate for diagnostic purposes in discriminating between aflatoxin producer and nonproducer strains of A. flavus .…”

“…In particular, aflR gene integrity has been considered a good candidate for diagnostic purposes in discriminating between aflatoxin producer and nonproducer strains of A. flavus . The role of aflS is less characterized than that of aflR (Liu and Chu 1998;Meyers et al 1998;Chang 2003Chang ,2004), but we did include it in our gene set in order to increase the predictability of the RT-PCR test. The other genes, aflD, aflO and aflQ, which encode for enzymes of the biosynthesis pathway, were among those that have been used by other authors; but we could not assess any preliminary advantage in their use when compared with that of other genes of the aflatoxin cluster.…”

Signalling pathways connecting mycotoxin production and sporulation

“…Deletion of aflJ reduces aflatoxin and sterigmatocystin production and biosynthetic gene transcription via an as yet unknown mechanism (R.A. Butchko & N.P. Keller, unpublished data;Meyers et al ., 1998). One study (Chang, 2003) provides evidence for a physical interaction between AflJ and AflR.…”

“…The sterigmatocystin/aflatoxin biosynthetic gene clusters include genes encoding two different transcriptional regulators: aflR (Chang et al ., 1993;Payne et al ., 1993;Woloshuk et al ., 1994;Yu et al ., 1996a) and aflJ (Chang, 2003;Meyers et al ., 1998). AflR encodes a Zn(II) 2 Cys 6 domain protein (Woloshuk et al ., 1994), which binds the palindromic sequence 5 ′ -TCG(N5)GCA-3 ′ found in promoters of aflatoxin and sterigmatocystin biosynthetic genes (Ehrlich et al ., 1999;Fernandes et al ., 1998).…”

expressed sequence tags for identification of genes with putative roles in aflatoxin contamination of crops

“…Genetic studies on aflatoxin biosynthesis in A. flavus and A. parasiticus led to the cloning of 25 clustered genes within a 70 kb DNA region responsible for the enzymatic conversions in the aflatoxin biosynthetic pathway [8,9]. Regulatory elements such as aflR [10,11] and aflS (aflJ) [12,13], nutritional and environmental factors [14,15], fungal developmental and sporulation [16][17][18][19] were also found to affect aflatoxin formation. In A. flavus there are eight chromosomes with an estimated genome size of about 33-36 Mbp that harbor an estimated 12,000 functional genes (reviewed in [7,20], Dr. Machida, personal communication).…”