Polyketide synthases (PKSs) and/or nonribosomal peptide synthetases (NRPSs) are central components of secondary metabolism in bacteria, plants, and fungi. In filamentous fungi, diverse PKSs and NRPSs participate in the biosynthesis of secondary metabolites such as pigments, antibiotics, siderophores, and mycotoxins. However, many secondary metabolites as well as the enzymes involved in their production are yet to be discovered. Both PKSs and NRPSs require activation by enzyme members of the 4-phosphopantetheinyl transferase (PPTase) family. Here, we report the isolation and characterization of Aspergillus nidulans strains carrying conditional (cfwA2) and null (⌬cfwA) mutant alleles of the cfwA gene, encoding an essential PPTase. We identify the polyketides shamixanthone, emericellin, and dehydroaustinol as well as the sterols ergosterol, peroxiergosterol, and cerevisterol in extracts from A. nidulans large-scale cultures. The PPTase CfwA/NpgA was required for the production of these polyketide compounds but dispensable for ergosterol and cerevisterol and for fatty acid biosynthesis. The asexual sporulation defects of cfwA, ⌬fluG, and ⌬tmpA mutants were not rescued by the cfwA-dependent compounds identified here. However, a cfwA2 mutation enhanced the sporulation defects of both ⌬tmpA and ⌬fluG single mutants, suggesting that unidentified CfwA-dependent PKSs and/or NRPSs are involved in the production of hitherto-unknown compounds required for sporulation. Our results expand the number of known and predicted secondary metabolites requiring CfwA/NpgA for their biosynthesis and, together with the phylogenetic analysis of fungal PPTases, suggest that a single PPTase is responsible for the activation of all PKSs and NRPSs in A. nidulans.Filamentous fungi represent a rich and diverse source of bioactive compounds derived from secondary metabolism. Indeed, many positive and negative effects that fungi have on human activity are mediated by secondary metabolites such as antibiotics and other pharmaceutical drugs, mycotoxins, or pathogen virulence factors (22, 30, 54-56, 60, 66). In contrast to primary metabolism, common to most living organisms, secondary metabolism is not essential for the immediate survival of the producing organism. Furthermore, different taxonomic groups produce different types of secondary metabolites.Despite their large chemical diversity, secondary metabolites can be grouped according to their primary metabolism precursors. Acetyl coenzyme A, shikimate, and amino acids are major secondary metabolite building units. Acetyl coenzyme A is used to produce terpenoids, steroids, carotenoids, and polyketides.

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SummaryOrgan growth during development is a highly regulated process with both temporal and spatial constraints. Epidermal stratification is essential for skin growth and development. Although the zebrafish has been well studied, it is not known when and how epidermal stratification occurs. This is because beyond the first five days of development our knowledge is currently limited. We found that epidermal stratification in zebrafish begins when the larvae reach a standard length (SL) of 6 mm at approximately 25 days of age. Over the next four days (from a SL of 6 to 9 mm), epidermis thickness increases almost four-fold. This represents a sudden increase in organ size, since for the previous 20 days of development, the epidermis has been only two layers thick. This pattern is different from that observed in mammals that undergo continuous stratification from E14.5–E18.5. To study how stem cell proliferation gives rise to the new epidermal layers, we used a combination of markers: one for cell proliferation (proliferating cell nuclear-antigen PCNA) and one for epidermal stem cells (P63 transcription factor). We identified, throughout the stratification process, two different waves of cell division. Initially, the most basal epidermal cells divided and generated a subset of suprabasal cells (possibly transient-amplifying cells); within the next several days, the basal cells stopped dividing, and the suprabasal cells began proliferation, giving rise to most of the cell types in the new layers. This part of the process is similar to what has been recently found during epidermal stratification in mammals.

An interesting question in developmental biology is why mutations in genes with functions essential for the majority of cells produce diseases affecting only specific tissues. For example, pigment dilution disorders are often the consequence of mutations in conserved vesicular traffic genes. In Hermansky-Pudlak, Griscelli, and Chediak-Higashi pigment dilution syndromes, vesicular traffic mutations affect several organs with one characteristic in common: to carry out their functions they depend to a great extent on lysosome-related organelles (LROs), such as the melanosomes in melanocytes. Conserved multimeric complexes, present in most cell types, target proteins to lysosomes or selected LROs using transport vesicles. By studying these diseases or the model organisms that are defective in these processes, we have learned that every cell type possesses a unique way to regulate its vesicular traffic machinery and to assemble its multimeric complexes. This is accomplished by subunits from these multimeric complexes acting in a cell-specific manner. Here, we review several fish pigment dilution mutants that represent models for human vesicular traffic diseases.

Aspergillus nidulans asexual sporulation (conidiation) is triggered by different environmental signals and involves the differentiation of specialized structures called conidiophores. The elimination of genes flbA-E, fluG, and tmpA results in a fluffy phenotype characterized by delayed conidiophore development and decreased expression of the conidiation essential gene brlA. While flbA-E encode regulatory proteins, fluG and tmpA encode enzymes involved in the biosynthesis of independent signals needed for normal conidiation. Here we identify afeA and tmpB as new genes encoding members the adenylate-forming enzyme superfamily, whose inactivation cause different fluffy phenotypes and decreased conidiation and brlA expression. AfeA is most similar to unknown function coumarate ligase-like (4CL-Lk) enzymes and consistent with this, a K544N active site modification eliminates AfeA function. TmpB, identified previously as a larger homolog of the oxidoreductase TmpA, contains a NRPS-type adenylation domain. A high degree of synteny in the afeA-tmpA and tmpB regions in the Aspergilli suggests that these genes are part of conserved gene clusters. afeA, tmpA, and tmpB double and triple mutant analysis as well as afeA overexpression experiments indicate that TmpA and AfeA act in the same conidiation pathway, with TmpB acting in a different pathway. Fluorescent protein tagging shows that functional versions of AfeA are localized in lipid bodies and the plasma membrane, while TmpA and TmpB are localized at the plasma membrane. We propose that AfeA participates in the biosynthesis of an acylated compound, either a p-cuomaryl type or a fatty acid compound, which might be oxidized by TmpA and/or TmpB, while TmpB adenylation domain would be involved in the activation of a hydrophobic amino acid, which in turn would be oxidized by the TmpB oxidoreductase domain. Both, AfeA-TmpA and TmpB signals are involved in self-communication and reproduction in A. nidulans.