Miguel A. Peñalva

Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.

Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus.

Aspergillus fumigatus is exceptional among microorganisms in being both a primary and opportunistic pathogen as well as a major allergen. Its conidia production is prolific, and so human respiratory tract exposure is almost constant. A. fumigatus is isolated from human habitats and vegetable compost heaps. In immunocompromised individuals, the incidence of invasive infection can be as high as 50% and the mortality rate is often about 50% (ref. 2). The interaction of A. fumigatus and other airborne fungi with the immune system is increasingly linked to severe asthma and sinusitis. Although the burden of invasive disease caused by A. fumigatus is substantial, the basic biology of the organism is mostly obscure. Here we show the complete 29.4-megabase genome sequence of the clinical isolate Af293, which consists of eight chromosomes containing 9,926 predicted genes. Microarray analysis revealed temperature-dependent expression of distinct sets of genes, as well as 700 A. fumigatus genes not present or significantly diverged in the closely related sexual species Neosartorya fischeri, many of which may have roles in the pathogenicity phenotype. The Af293 genome sequence provides an unparalleled resource for the future understanding of this remarkable fungus.

The Tip Growth Apparatus of Aspergillus nidulans

Hyphal tip growth in fungi is important because of the economic and medical importance of fungi, and because it may be a useful model for polarized growth in other organisms. We have investigated the central questions of the roles of cytoskeletal elements and of the precise sites of exocytosis and endocytosis at the growing hyphal tip by using the model fungus Aspergillus nidulans. Time-lapse imaging of fluorescent fusion proteins reveals a remarkably dynamic, but highly structured, tip growth apparatus. Live imaging of SYNA, a synaptobrevin homologue, and SECC, an exocyst component, reveals that vesicles accumulate in the Spitzenkörper (apical body) and fuse with the plasma membrane at the extreme apex of the hypha. SYNA is recycled from the plasma membrane by endocytosis at a collar of endocytic patches, 1-2 mum behind the apex of the hypha, that moves forward as the tip grows. Exocytosis and endocytosis are thus spatially coupled. Inhibitor studies, in combination with observations of fluorescent fusion proteins, reveal that actin functions in exocytosis and endocytosis at the tip and in holding the tip growth apparatus together. Microtubules are important for delivering vesicles to the tip area and for holding the tip growth apparatus in position.

Characterization of a Fungal Maleylacetoacetate Isomerase Gene and Identification of Its Human Homologue

We have previously used Aspergillus nidulans as a fungal model for human phenylalanine catabolism. This model was crucial for our characterization of the human gene involved in alcaptonuria. We use here an identical approach to characterize at the cDNA level the human gene for maleylacetoacetate isomerase (MAAI, EC 5.2.1.2), the only as yet unidentified structural gene of the phenylalanine catabolic pathway. We report here the first characterization of a gene encoding a MAAI enzyme from any organism, the A. nidulans maiA gene.maiA disruption prevents growth on phenylalanine (Phe) and phenylacetate and results in the absence of MAAI activity in vitro and Phe toxicity. The MaiA protein shows strong amino acid sequence identity to glutathione S-transferases and has MAAI activity when expressed in Escherichia coli. maiA is clustered with fahA and hmgA, the genes encoding the two other enzymes of the common part of the Phe/phenylacetate pathways. Based on the high amino acid sequence conservation existing between other homologous A. nidulans and human enzymes of this pathway, we used the MaiA sequence in data base searches to identify human expressed sequence tags encoding its putative homologues. Four such cDNAs were sequenced and shown to be encoded by the same gene. They encode a protein with 45% sequence identity to MaiA, which showed MAAI activity when expressed in E. coli. Human MAAI deficiency would presumably cause tyrosinemia that would be characterized by the absence of succinylacetone, the diagnostic compound resulting from fumarylacetoacetate hydrolase deficiency in humans and fungi. Culture supernatants of an A. nidulansstrain disrupted for maiA are succinylacetone-negative but specifically contain cis and/or trans isomers of 2,4-dioxohept-2-enoic acid. We suggest that this compound(s) might be diagnostic for human MAAI deficiency.

pH regulation is a major determinant in expression of a fungal penicillin biosynthetic gene.

Transcription of the ipnA gene encoding isopenicillin N synthetase, an enzyme of secondary metabolism, is under the control of the pH regulatory system in the fungus Aspergillus nidulans. External alkaline pH or mutations in pacC, the wide domain regulatory gene which mediates pH regulation, override carbon regulation of ipnA transcript levels, resulting in elevation of the levels of this message in sucrose broth. Strains carrying these mutations, which mimic growth at alkaline pH, produce higher levels of penicillins when grown in sucrose broth compared with the wild type strain grown under carbon derepressing conditions. ipnA transcription is regulated by carbon (C) source, but extreme mutations in creA (the regulatory gene mediating carbon catabolite repression) only slightly increase repressed transcript levels. Precise deletion of the only in vitro CreA binding site present in a region of the ipnA promoter involved in carbon regulation has no effect on ipnA expression. The levels of ipnA transcript in broths with acetate or glycerol as principal C sources are inconsistent with direct or indirect creA‐mediated transcriptional control of the gene. We conclude that a second, creA‐independent mechanism of carbon repression controls expression of this gene. All derepressing C sources tested result in alkalinization of the growth media. In contrast, all repressing C sources result in external acidification. Neither acidic external pH nor pal mutations, mimicking the effects of growth at acid pH, prevent carbon derepression, providing strong support for independent regulatory mechanisms, one mediating carbon regulation (via thus far unidentified genes) and another mediating pH regulation (through the pacC‐encoded transcriptional regulator). External pH measurements suggest that these two independent forms of regulation normally act in concert. We propose that external alkalinity represents a physiological signal which triggers penicillin biosynthesis.

YPXL/I Is a Protein Interaction Motif Recognized by Aspergillus PalA and Its Human Homologue, AIP1/Alix

ABSTRACT The zinc finger transcription factor PacC undergoes two-step proteolytic activation in response to alkaline ambient pH. PalA is a component of the fungal ambient pH signal transduction pathway. Its mammalian homologue AIP1/Alix interacts with the apoptosis-linked protein ALG-2. We show that both PalA and AIP1/Alix recognize a protein-protein binding motif that we denote YPXL/I, where Tyr, Pro, and Leu/Ile are crucial for its interactive properties. Two such motifs flanking the signaling protease cleavage site mediate direct binding of PalA to PacC, required for the first and only pH-regulated cleavage of this transcription factor. PalA can bind the “closed” (i.e., wild-type full-length) conformer of PacC, suggesting that PalA binding constitutes the first stage in the two-step proteolytic cascade, recruiting or facilitating access of the signaling protease, presumably PalB. In addition to recognizing YPXL/I motifs, both PalA and AIP1/Alix interact with the Aspergillus class E Vps protein Vps32 homologue, a member of a protein complex involved in the early steps of the multivesicular body pathway, suggesting that this interaction is an additional feature of proteins of the PalA/AIP1/Alix family.

Preferential localization of the endocytic internalization machinery to hyphal tips underlies polarization of the actin cytoskeleton in Aspergillus nidulans

AbpA, SlaB and AmpA, three demonstrated components of the endocytic internalization machinery, are strongly polarized in Aspergillus nidulans hyphae, forming a ring that embraces the hyphal tip, leaving an area of exclusion at the apex. AbpA, a prototypic endocytic internalization marker, localizes to highly motile and transient (average half life, 24 ± 5 s) peripheral punctate structures overlapping with actin patches, which also predominate in the tip. SlaB also localizes to peripheral patches, but these are markedly more abundant and cortical than those of AbpA. In contrast to its polarized distribution in hyphae, endocytic patches show random distribution during the isotropic growth phase preceding polarity establishment, but polarize as soon as a germtube primordium emerges from the swelled conidiospore. Thus, while endocytosis can occur along the hyphae, the apical predominance and the spatial organization of actin patches and of the above endocytic machinery proteins as a slightly subapical ring strongly suggests that tight spatial coupling of apical secretion and subapical compensatory endocytosis underlies hyphal growth. In agreement, the phenotype of a null slaB allele indicates that endocytosis is essential.

Characterization of a Penicillium chrysogenum gene encoding a PacC transcription factor and its binding sites in the divergent pcbAB–pcbC promoter of the penicillin biosynthetic cluster

Previous work established that pH regulation of gene expression in Aspergillus nidulans, a major determinant of penicillin biosynthesis, is mediated by the zinc‐finger transcription factor PacC, an activator of transcription of the isopenicillin N synthase gene. We characterize here the pacC gene from the efficient penicillin producer Penicillium chrysogenum, which functionally complements an A. nidulans pacC null mutation. It encodes a 641‐residue polypeptide showing 64% identity to A. nidulans PacC and containing three putative zinc fingers specifically recognizing a 5′‐GCCARG‐3′ hexanucleotide. Penicillium pacC transcript levels are higher under alkaline than under acidic growth conditions and elevate at late stages of growth. The gene contains three PacC‐binding sites in its 5′‐upstream region. Transcript levels of pcbC (encoding P. chrysogenum isopenicillin N synthase) are low on a repressing carbon source and elevated on a derepressing carbon source. With either carbon source, alkaline pH elevates pcbC transcript levels, correlating with the presence of seven PacC‐binding sites in the 1.1 kb pcbAB–pcbC intergenic region and strongly suggesting that pcbC is under direct pacC control. However, in contrast to the situation in A. nidulans, alkaline pH does not override the negative effects of a repressing carbon source, revealing differences in the regulation of the penicillin pathway between Penicillium and Aspergillus

Molecular Characterization of a Gene Encoding a Homogentisate Dioxygenase from Aspergillus nidulans and Identification of Its Human and Plant Homologues

We report here the first characterization of a gene encoding a homogentisate dioxygenase, the Aspergillus nidulans hmgA gene. The HmgA protein catalyzes an essential step in phenylalanine catabolism, and disruption of the gene results in accumulation of homogentisate in broths containing phenylalanine. hmgA putatively encodes a 448-residue polypeptide (M = 50,168) containing 21 histidine and 23 tyrosine residues. This polypeptide has been expressed in Escherichia coli as a fusion to glutathione S-transferase, and the affinity-purified protein has homogentisate dioxygenase activity. A. nidulans, an ascomycete amenable to classical and reverse genetic analysis, is a good metabolic model to study inborn errors in human Phe catabolism. One such disease, alkaptonuria, was the first human inborn error recognized (Garrod, A. E.(1902) Lancet 2, 1616-1620) and results from loss of homogentisate dioxygenase. Here we take advantage of the high degree of conservation between the amino acid sequences of the fungal and higher eukaryote enzymes of this pathway to identify expressed sequence tags encoding human and plant homologues of HmgA. This is a significant advance in characterizing the genetic defect(s) of alkaptonuria and illustrates the usefulness of our fungal model.

Activation of the Aspergillus PacC zinc finger transcription factor requires two proteolytic steps

The Aspergillus PacC transcription factor undergoes proteolytic activation in response to alkaline ambient pH. In acidic environments, the 674 residue translation product adopts a ‘closed’ conformation, protected from activation through intramolecular interactions involving the ≤150 residue C‐terminal domain. pH signalling converts PacC to an accessible conformation enabling processing cleavage within residues 252–254. We demonstrate that activation of PacC requires two sequential proteolytic steps. First, the ‘closed’ translation product is converted to an accessible, committed intermediate by proteolytic elimination of the C‐terminus. This ambient pH‐regulated cleavage is required for the final, pH‐independent processing reaction and is mediated by a distinct signalling protease (possibly PalB). The signalling protease cleaves PacC between residues 493 and 500, within a conserved 24 residue ‘signalling protease box’. Precise deletion or Leu498Ser substitution prevents formation of the committed and processed forms, demonstrating that signalling cleavage is essential for final processing. In contrast, signalling cleavage is not required for processing of the Leu340Ser protein, which lacks interactions preventing processing. In its two‐step mechanism, PacC processing can be compared with regulated intramembrane proteolysis.