Nicotinate degradation has hitherto been elucidated only in bacteria. In the ascomycete Aspergillus nidulans, six loci, hxnS/AN9178 encoding the molybdenum cofactor-containing nicotinate hydroxylase, AN11197 encoding a Cys2/His2 zinc finger regulator HxnR, together with AN11196/hxnZ, AN11188/hxnY, AN11189/hxnP and AN9177/hxnT, are clustered and stringently co-induced by a nicotinate derivative and subject to nitrogen metabolite repression mediated by the GATA factor AreA. These genes are strictly co-regulated by HxnR. Within the hxnR gene, constitutive mutations map in two discrete regions. Aspergillus nidulans is capable of using nicotinate and its oxidation products 6-hydroxynicotinic acid and 2,5-dihydroxypyridine as sole nitrogen sources in an HxnR-dependent way. HxnS is highly similar to HxA, the canonical xanthine dehydrogenase (XDH), and has originated by gene duplication, preceding the origin of the Pezizomycotina. This cluster is conserved with some variations throughout the Aspergillaceae. Our results imply that a fungal pathway has arisen independently from bacterial ones. Significantly, the neo-functionalization of XDH into nicotinate hydroxylase has occurred independently from analogous events in bacteria. This work describes for the first time a gene cluster involved in nicotinate catabolism in a eukaryote and has relevance for the formation and evolution of co-regulated primary metabolic gene clusters and the microbial degradation of N-heterocyclic compounds.

Aspergillus nidulans has three high mobility group box (HMGB) proteins, HmbA, HmbB and HmbC that are chromatin-associated architectural proteins involved in DNA-related functions. By creating and studying deletion strains in both veA + and veA1 background, we have characterized the role of HmbA, HmbB and HmbC in sexual development. Expression of the mating-type MAT1-1 and MAT1-2 coding genes were found to be extremely down-regulated in all three mutants on day 4 of sexual development, which results in deficient ascospore production and/or ascospore viability in the mutants. In addition, we found that HmbA and HmbB play also a role in sensing of and response to environmental signals, while HmbC functionally interacts with VeA, a key regulator of the coordination of asexual and sexual development, as well as of secondary metabolism.

Aspergillus nidulans produces sterigmatocystin, a secondary metabolite mycotoxin, for the protection of its reproductive structures. Previous studies on grazing behavior of fungivore arthropods, regulation of sexual development, and secondary metabolite biosynthesis have revealed the association of sterigmatocystin biosynthesis with sexual reproduction, but the spatial distribution of sterigmatocystin producing hyphae within the colony has never been investigated. In this work, we aimed to locate the site of sterigmatocystin production within the colony by employing a yCFP reporter system. We demonstrated that the stcO promoter is active only in vegetative hyphae that surround groups of hülle cells and the activity decreases and eventually ceases as the distance between the hypha and the hülle cells increases. This phenomenon indicates that the vegetative mycelium might consist of morphologically uniform, but functionally different hyphae.

In Aspergillus nidulans a regulon including 11 hxn genes ( hxnS , T , R , P , Y , Z , X , W , V , M and N ) is inducible by a nicotinate metabolic derivative, repressible by ammonium and under stringent control of the nitrogen-state-sensitive GATA factor AreA and the specific transcription factor HxnR. This is the first report in a eukaryote of the genomic organization of a possibly complete pathway of nicotinate utilization. In A. nidulans the regulon is organized in three distinct clusters, this organization is variable in the Ascomycota . In some Pezizomycotina species all 11 genes map in a single cluster; in others they map in two clusters. This variable organization sheds light on cluster evolution. Instances of gene duplication followed by or simultaneous with integration in the cluster, partial or total cluster loss, and horizontal gene transfer of several genes (including an example of whole cluster re-acquisition in Aspergillus of section Flavi ) were detected, together with the incorporation in some clusters of genes not found in the A. nidulans co-regulated regulon, which underlie both the plasticity and the reticulate character of metabolic cluster evolution. This study provides a comprehensive phylogeny of six members of the cluster across representatives of all Ascomycota classes.

We describe an HxnR-dependent regulon composed of 11 hxn genes (hxnS, T, R, P, Y, Z, X, W, V, M and N). The regulon is inducible by a nicotinate metabolic derivative and repressible by ammonium and under stringent control of the GATA factor AreA. This is the first publication of a eukaryotic, complete nicotinate metabolic cluster including five novel genes. While in A. nidulans the regulon is organised in three distinct clusters, this organisation is variable in the Ascomycota. In some Pezizomycotina species all the 11 genes are organised in a single cluster, in other in two clusters. This variable organisation sheds light on cluster evolution. Instances of gene duplication, followed by or simultaneous with integration in the cluster; partial or total cluster loss; horizontal gene transfer of several genes, including an example of whole cluster re-acquisition in Aspergillus of section Flavi were detected, together with the incorporation in some clusters of genes not found in the A. nidulans co-regulated regulon, which underlie both the plasticity and the reticulate character of metabolic cluster evolution. This study provides the first comprehensive protein sequence comparison of six members of the cluster across representatives of all Ascomycota classes, including several hundreds of species.

Several strikingly different aerobic and anaerobic pathways of nicotinate breakdown are extant in bacteria. Here, through reverse genetics and analytical techniques we elucidated in Aspergillus nidulans, a complete eukaryotic nicotinate utilization pathway. The pathway extant in this fungus and other ascomycetes, is quite different from bacterial ones. All intermediate metabolites were identified. The cognate proteins, encoded by eleven genes (hxn) mapping in three clusters are co-regulated by a specific transcription factor. Several enzymatic steps have no prokaryotic equivalent and two metabolites, 3-hydroxypiperidine-2,6-dione and 5,6-dihydroxypiperidine-2-one, have not been identified previously in any organism, the latter being a novel chemical compound. Hydrolytic ring opening results in α-hydroxyglutaramate, a compound not detected in analogous prokaryotic pathways. Our earlier phylogenetic analysis of Hxn proteins together with this complete biochemical pathway illustrates convergent evolution of catabolic pathways between fungi and bacteria.

Several strikingly different aerobic and anaerobic pathways of nicotinate utilization had been described in bacteria. No similar work is extant in any eukaryote. Here we elucidate a complete eukaryotic nicotinate utilization pathway, by constructing single or multiple gene deleted strains and identifying metabolic intermediates by ultra-high performance liquid chromatography — high-resolution mass spectrometry. Enzymes catalyzing each step and all intermediate metabolites were identified. We previously established that the cognate eleven genes organized in three clusters constitute a regulon, strictly dependent on HxnR, a pathway-specific transcription factor. The first step, hydroxylation of nicotinic acid to 6-hydroxynicotinic acid is analogous to that occurring in bacterial pathways and is catalyzed by an independently evolved molybdenum-containing hydroxylase. The following enzymatic steps have no prokaryotic equivalents: 6-hydroxynicotinic acid is converted to 2,3,6-trihydroxypyridine through 2,5-dihydroxypiridine and the trihydroxylated pyridine ring is then saturated to 5,6-dihydroxypiperidine-2-one followed by the oxidation of the C6 hydroxyl group resulting in 3-hydroxypiperidine-2,6-dione. The latter two heterocyclic compounds are newly identified cellular metabolites, while 5,6-dihydroxypiperidine-2-one is a completely new chemical compound. Ring opening between C and N results in α-hydroxyglutaramate, an unprecedented compound in prokaryotic nicotinate catabolic routes. The pathway extant in A. nidulans, and in many other ascomycetes, is different from any other previously analyzed in bacteria. Our earlier phylogenetic analysis of Hxn proteins together with the complete novel biochemical pathway we now describe further illustrates the convergent evolution of catabolic pathways between fungi and bacteria.

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