Aspergillus nidulans verA is required for production of the mycotoxin sterigmatocystin.

Aspergillus nidulans produces the carcinogenic mycotoxin sterigmatocystin (ST), the next-to-last precursor in the aflatoxin (AF) biosynthetic pathway found in the closely related fungi Aspergillus flavus and Aspergillus parasiticus. We identified and characterized an A. nidulans gene, verA, that is required for converting the AF precursor versicolorin A to ST. verA is closely related to several polyketide biosynthetic genes involved in polyketide production in Streptomyces spp. and exhibits extended sequence similarity to A. parasiticus ver-1, a gene proposed to encode an enzyme involved in converting versicolorin A to ST. By performing a sequence analysis of the region 3' to verA, we identified two additional open reading frames, designated ORF1 and ORF2. ORF2 is closely related to a number of cytochrome P-450 monooxygenases, while ORF1 shares identity with the gamma subunit of translation elongation factor 1. Given that several steps in the ST-AF pathway may require monooxygenase activity and that AF biosynthetic genes are clustered in A. flavus and A. parasiticus, we suggest that verA may be part of a cluster of genes required for ST biosynthesis. We disrupted the verA coding region by inserting the A. nidulans argB gene into the center of the coding region and transformed an A. nidulans argB2 mutant to arginine prototrophy. Seven transformants that produced DNA patterns indicative of a verA disruption event were grown under ST-inducing conditions, and all of the transformants produced versicolorin A but negligible amounts of ST (200-fold to almost 1,000-fold less than the wild type), confirming the hypothesis that verA encodes an enzyme necessary for converting versicolorin A to ST.     

Requirement of monooxygenase-mediated steps for sterigmatocystin biosynthesis by Aspergillus nidulans

Sterigmatocystin (ST) and aflatoxin B(1) (AFB(1)) are two polyketide-derived Aspergillus mycotoxins synthesized by functionally identical sets of enzymes. ST, the compound produced by Aspergillus nidulans, is a late intermediate in the AFB(1) pathway of A. parasiticus and A. flavus. Previous biochemical studies predicted that five oxygenase steps are required for the formation of ST. A 60-kb ST gene cluster in A. nidulans contains five genes, stcB, stcF, stcL, stcS, and stcW, encoding putative monooxygenase activities. Prior research showed that stcL and stcS mutants accumulated versicolorins B and A, respectively. We now show that strains disrupted at stcF, encoding a P-450 monooxygenase similar to A. parasiticus avnA, accumulate averantin. Disruption of either StcB (a putative P-450 monooxygenase) or StcW (a putative flavin-requiring monooxygenase) led to the accumulation of averufin as determined by radiolabeled feeding and extraction studies.     

Aspergillus nidulans swoK encodes an RNA binding protein that is important for cell polarity

The Aspergillus nidulans swoK1 mutant is defective in polarity maintenance when grown at restrictive temperature (38 degrees C). Upon germination, the mutant extends a primary germ tube that swells to an enlarged, non-uniform cell with pronounced wall thickenings. The mutant is fully restored to wild-type growth when transformed with a plasmid containing the AN5802.2 ORF as designated in The Broad Institute A. nidulans sequence database. Genetic mapping places swoK in the same region of chromosome I, as that occupied by An5802.2 on the physical map. swoK is predicted to encode a protein that contains an N-terminal RRM (RNA Recognition Motif) and a highly repetitive C-terminus with numerous RD/DR and RS/SR dipeptides. We hypothesize that SwoK participates in one of the known functions of SR proteins (those that contain SR/RS repeats): mRNA maturation through the spliceosome and or transport of mRNAs out of the nucleus to sites of protein translation.     

The Aspergillus nidulans sfaD gene encodes a G protein beta subunit that is required for normal growth and repression of sporulation.

flbA encodes an Aspergillus nidulans RGS (regulator of G protein signaling) domain protein that antagonizes FadA (G(i)alpha-subunit of heterotrimeric G protein)-mediated growth signaling to allow asexual development. We previously defined and characterized five suppressors of flbA (sfa) loss-of-function mutations and showed that one suppressor (sfaB) resulted from a novel dominant-negative allele of fadA. In this report we show that a second suppressor gene (sfaD) is predicted to encode the beta subunit of a heterotrimeric G protein. Deletion of sfaD suppressed all defects resulting from complete loss-of-flbA function mutations, caused a hyperactive sporulation phenotype and severely reduced vegetative growth. However, the sfaD deletion could not suppress the growth activation caused by dominant-activating fadA alleles, indicating that constitutively active FadA can cause proliferative growth in the absence of Gbetagamma signaling. We propose that SfaD and FadA are both positive growth regulators with partially overlapping functions and that FlbA has an important role in controlling the activities of both proteins. Inactivation of signaling events stimulated by both components of the heterotrimeric G protein is essential for both sexual and asexual sporulation.     

Aspergillus nidulans swoF encodes an N-myristoyl transferase

Polar growth is a fundamental process in filamentous fungi and is necessary for disease initiation in many pathogenic systems. Previously, swoF was identified in Aspergillus nidulans as a single-locus, temperature-sensitive (ts) mutant aberrant in both polarity establishment and polarity maintenance. The swoF gene was cloned by complementation of the ts phenotype and sequenced. The derived protein sequence had high identity with N-myristoyl transferases (NMTs) found in fungi, plants, and animals. In addition, wild-type growth at restrictive temperature was partially restored by the addition of myristic acid to the growth medium. Sequencing revealed that the mutation in swoF changes the conserved aspartic acid 369 to a tyrosine. The predicted A. nidulans SwoF protein, SwoFp, was homology modeled based on crystal structures of NMTs from Saccharomyces cerevisiae and Candida albicans. The D369Y swoF mutation is on the opposite face of the protein, distal to the myristoyl coenzyme A and peptide substrate binding sites. In wild-type NMTs, D369 appears to stabilize a structural β-strand bend through two hydrogen bonds and an ionic interaction. These stabilizing bonds are abolished in the D369Y mutant. We hypothesize that a substrate of SwoFp must be myristoylated for proper polarity establishment and maintenance. The mutation prevents the proper function of SwoFp at restrictive temperature and thus blocks polar growth.     

myoA of Aspergillus nidulans encodes an essential myosin I required for secretion and polarized growth

We have identified and cloned a novel essential myosin I in Aspergillus nidulans called myoA. The 1,249-amino acid predicted polypeptide encoded by myoA is most similar to the amoeboid myosins I. Using affinity-purified antibodies against the unique myosin I carboxyl terminus, we have determined that MYOA is enriched at growing hyphal tips. Disruption of myoA by homologous recombination resulted in a diploid strain heterozygous for the myoA gene disruption. We can recover haploids with an intact myoA gene from these strains, but never haploids that are myoA disrupted. These data indicated that myoA encodes an essential myosin I, and this has allowed us to use a unique approach to studying myosin I function. We have developed conditionally null myoA strains in which myoA expression is regulated by the alcA alcohol dehydrogenase promoter. A conditionally lethal strain germinated on inducing medium grows as wild type, displaying polarized growth by apical extension. However, growth of the same myoA mutant strain on repressing medium results in enlarged cells incapable of hyphal extension, and these cells eventually die. Under repressing conditions, this strain also displays reduced levels of secreted acid phosphatase. The mutant phenotype indicates that myoA plays a critical role in polarized growth and secretion.     

stcS, a putative P-450 monooxygenase, is required for the conversion of versicolorin A to sterigmatocystin in Aspergillus nidulans.

Sterigmatocystin (ST) and aflatoxin are carcinogenic end point metabolites derived from the same biochemical pathway, which is found in several Aspergillus spp. Recently, an ST gene cluster, containing approximately 25 distinct genes that are each proposed to function specifically in ST biosynthesis, has been identified in Aspergillus nidulans. Each of these structural genes is named stc (sterigmatocystin) followed by a consecutive letter of the alphabet. We have previously described stcU (formerly verA) as encoding a keto-reductase required for the conversion of versicolorin A to ST. We now describe a second A. nidulans gene, stcS (formerly verB), that is located within 2 kb of stcU in the ST gene cluster. An stcS-disrupted strain of A. nidulans, TSS17, was unable to produce ST and converted ST/aflatoxin precursors to versicolorin A rather than ST, indicating that stcS functions at the same point in the pathway as stcU. Genomic sequence analysis of stcS shows that it encodes a cytochrome P-450 monooxygenase and constitutes a novel P-450 family, CYP59. Assuming that StcU activity mimics that of similar P-450s, it is likely that StcU catalyzes one of the proposed oxidation steps necessary to convert versicolorin A to ST. These results constitute the first genetic proof that the conversion of versicolorin A to ST requires more than one enzymatic activity.     

Calcium binding is required for calmodulin function in Aspergillus nidulans

To explore the structural basis for the essential role of calmodulin (CaM) in Aspergillus nidulans, we have compared the biochemical and in vivo properties of A. nidulans CaM (AnCaM) with those of heterologous CaMs. Neither Saccharomyces cerevisiae CaM (ScCaM) nor a Ca²⁺ binding mutant of A. nidulans CaM (1234) interacts appreciably with A. nidulans CaM binding proteins by an overlay assay or activates two essential CaMKs, CMKA and CMKB. In contrast, although vertebrate CaM (VCaM) binds a spectrum of proteins similar to that for AnCaM, it is unable to fully activate CMKA and CMKB, displaying a higher K_CaM and reduced V_max for both enzymes. In correlation with the biochemical analysis, neither ScCaM nor 1234 can support A. nidulans growth in the absence of the endogenous protein, whereas VCaM only partially complements the absence of wild-type CaM. Analysis of VCaM and AnCaM chimeras demonstrates that amino acid variations in both N- and C-terminal domains contribute to the inability of VCaM to activate CMKB, but differences in the N terminus are largely responsible for the reduced activity towards CMKA. In vivo, the chimeric molecules support growth equivalently, but only to levels intermediate between those of VCaM and AnCaM, suggesting that the reduced ability to activate the CaMKs is not solely responsible for the inability of VCaM to complement the absence of the wild-type protein. Thus, not only is Ca²⁺ binding required for CaM function in A. nidulans, but the essential in vivo functions of A. nidulans CaM are uniquely sensitive to the subtle amino acid variations present in vertebrate CaM.     

The Aspergillus nidulans uvsB gene encodes an ATM-related kinase required for multiple facets of the DNA damage response

In Aspergillus nidulans, uvsB and uvsD belong to the same epistasis group of DNA repair mutants. Recent observations suggest that these genes are likely to control cell cycle checkpoint responses to DNA damage and incomplete replication. Consistent with this notion, we show here that UVSB is a member of the conserved family of ATM-related kinases. Phenotypic characterization of uvsB mutants shows that they possess defects in additional aspects of the DNA damage response besides checkpoint control, including inhibition of septum formation, regulation of gene expression, and induced mutagenesis. The musN227 mutation partially suppresses the poor growth and DNA damage sensitivity of uvsB mutants. Although musN227 partially suppresses several uvsB defects, it does not restore checkpoint function to uvsB mutants. Notably, the failure of uvsB mutants to restrain septum formation in the presence of DNA damage is suppressed by the musN227 mutation. We propose that UVSB functions as the central regulator of the A. nidulans DNA damage response, whereas MUSN promotes recovery by modulating a subset of the response.     

An aflatoxin biosynthesis cluster gene encodes a novel oxidase required for conversion of versicolorin a to sterigmatocystin

Disruption of the aflatoxin biosynthesis cluster gene aflY (hypA) gave Aspergillus parasiticus transformants that accumulated versicolorin A. This gene is predicted to encode the Baeyer-Villiger oxidase necessary for formation of the xanthone ring of the aflatoxin precursor demethylsterigmatocystin.     

The Aspergillus nidulans rcoA gene is required for veA-dependent sexual development

The Aspergillus nidulans rcoADelta mutant exhibits growth and developmental defects. We show that the rcoADelta mutant lacks cleistothecia and is self-sterile. In crosses with wild-type strains, rcoADelta nuclei do not contribute to the cleistothecial walls. Furthermore, sexual development resulting from veA overexpression is rcoA dependent, indicating that rcoA lies downstream of veA in the sexual development pathway.     

Anthraquinones in the biosynthesis of sterigmatocystin by Aspergillus versicolor.

14C-labeled averufin, versiconal hemiacetal acetate, and versicolorin A were efficiently converted to sterigmatocystin by Aspergillus versicolor, thus providing experimental evidence that these anthraquinones are biosynthetic precursors of sterigmatocystin, a xanthone.