The hallmark trait of fungal secondary-metabolite gene clusters is well established, consisting of contiguous enzymatic and often regulatory gene(s) devoted to the production of a metabolite of a specific chemical class. Unexpectedly, we have found a deviation from this motif in a subtelomeric region of Aspergillus fumigatus. This region, under the control of the master regulator of secondary metabolism, LaeA, contains, in its entirety, the genetic machinery for three natural products (fumitremorgin, fumagillin, and pseurotin), where genes for fumagillin and pseurotin are physically intertwined in a single supercluster. Deletions of 29 adjoining genes revealed that fumagillin and pseurotin are coregulated by the supercluster-embedded regulatory gene with biosynthetic genes belonging to one of the two metabolic pathways in a noncontiguous manner. Comparative genomics indicates the fumagillin/pseurotin supercluster is maintained in a rapidly evolving region of diverse fungal genomes. This blended design confounds predictions from established secondary-metabolite cluster search algorithms and provides an expanded view of natural product evolution.gene regulation | Zn(II) 2 Cys 6 transcription factor | FapR | biosynthesis | cluster evolution F ilamentous fungi are well known for their ability to produce a variety of natural products, so-called secondary metabolites that are not essential for growth under laboratory conditions (reviewed in refs. 1 and 2). However, the maintenance of the genetic information allowing fungi to produce secondary metabolites suggests that these small molecules provide essential benefits in environmental niches ranging from protection from fungivory (reviewed in ref.3) to chemical shields from UV radiation (4). Apart from providing evolutionary fitness to the producing organism in their natural habitat, many secondary metabolites are of major importance to humans because of their beneficial and deleterious effects as drugs and toxins, respectively.Fungal secondary metabolism has been characterized by physical linkage of the genes required for synthesis of specific metabolites and the distinct enzymatic machinery encoded by these genes (1, 2, 5). For instance, most fungal secondary metabolites belong to one of four chemical classes that are characterized based on the key or backbone enzymes that consist of polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), terpene cyclases (TCs), and prenyltransferases (PTs). Typically, cluster genes adjacent to these backbone genes code for accessory enzymes involved in either modification of the chemical scaffold, transcriptional control of cluster genes, transport of substrates and/or products, and resistance mechanisms. The most common regulatory genes of clusters encode fungal-specific C6 zinc binuclear cluster (Zn(II) 2 Cys 6 ) transcription factors (6), which, in general, exert positive transcriptional regulation of most of the genes within a single cluster (7). In addition to cluster-specific transcription factors, a higher order...

BackgroundOver the last ten years we have seen great efforts focused on revising amphibian systematics. Phylogenetic reconstructions derived from DNA sequence data have played a central role in these revisionary studies but have typically under-sampled the diverse frog family Microhylidae. Here, we present a detailed phylogenetic study focused on expanding previous hypotheses of relationships within this cosmopolitan family. Specifically, we placed an emphasis on assessing relationships among New World genera and those taxa with uncertain phylogenetic affinities (i.e., incertae sedis).ResultsOne mitochondrial and three nuclear genes (about 2.8 kb) were sequenced to assess phylogenetic relationships. We utilized an unprecedented sampling of 200 microhylid taxa representing 91% of currently recognized subfamilies and 95% of New World genera. Our analyses do not fully resolve relationships among subfamilies supporting previous studies that have suggested a rapid early diversification of this clade. We observed a close relationship between Synapturanus and Otophryne of the subfamily Otophryninae. Within the subfamily Gastrophryninae relationships between genera were well resolved.ConclusionOtophryninae is distantly related to all other New World microhylids that were recovered as a monophyletic group, Gastrophryninae. Within Gastrophryninae, five genera were recovered as non-monophyletic; we propose taxonomic re-arrangements to render all genera monophyletic. This hypothesis of relationships and updated classification for New World microhylids may serve as a guide to better understand the evolutionary history of this group that is apparently subject to convergent morphological evolution and chromosome reduction. Based on a divergence analysis calibrated with hypotheses from previous studies and fossil data, it appears that microhylid genera inhabiting the New World originated during a period of gradual cooling from the late Oligocene to mid Miocene.

Dimeric basic leucine zipper (bZIP) proteins are conserved transcriptional enhancers found in all eukaryotes. A recently reported and novel function for bZIPs is association of these proteins with secondary metabolite production in filamentous fungi. In particular a Yap-like bZIP termed RsmA (restorer of secondary metabolism A) was identified in Aspergillus nidulans that positively regulates the carcinogen sterigmatocystin. To assess for conserved function for RsmA, we examined a role of this protein in secondary metabolism in the pathogen A. fumigatus. RsmA was found to positively regulate gliotoxin where overexpression (OE) of rsmA led to 2–100 fold increases of twelve gli cluster metabolites in culture medium including the newly identified gli metabolite cyclo(L-Phe-L-Ser). Lungs from both wild type and OErsmA infected mice contained gliotoxin (2.3 fold higher in OErsmA treatment) as well as the gliotoxin precursor cyclo(L-Phe-L-Ser) (3.2 fold higher in OErsmA treatment). The data here presents a conserved role for RsmA in secondary metabolite cluster activation and suggests cyclo(L-Phe-L-Ser) may serve as an alternative marker for diagnosis of invasive aspergillosis.

LaeA is a conserved global regulator of secondary metabolism and development in filamentous fungi. Examination of Aspergillus fumigatus transcriptome data of laeA deletion mutants have been fruitful in identifying genes and molecules contributing to the laeA mutant phenotype. One of the genes significantly down regulated in A. fumigatus ΔlaeA is metR, encoding a bZIP DNA binding protein required for sulfur and methionine metabolism in fungi. LaeA and MetR deletion mutants exhibit several similarities including down regulation of sulfur assimilation and methionine metabolism genes and ability to grow on the toxic sulfur analog, sodium selenate. However, unlike ΔmetR, ΔlaeA strains are able to grow on sulfur, sulfite, and cysteine. To examine if any parameter of the ΔlaeA phenotype is due to decreased metR expression, an over-expression allele (OE::metR) was placed in a ΔlaeA background. The OE::metR allele could not significantly restore expression of MetR regulated genes in ΔlaeA but did restore sensitivity to sodium selenate. In A. nidulans a second bZIP protein, MetZ, also regulates sulfur and methionine metabolism genes. However, addition of an OE::metZ construct to the A. fumigatus ΔlaeA OE::metR strain still was unable to rescue the ΔlaeA phenotype to wildtype with regards gliotoxin synthesis and virulence in a zebrafish aspergillosis model.