Mark X. Caddick

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.

Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.

Regulation of gene expression by pH of the growth medium in Aspergillus nidulans

SummaryIn the fungus Aspergillus nidulans the levels of a number of enzymes whose location is at least in part extracellular (e.g. acid phosphatase, alkaline phosphatase, phosphodiesterase) and of certain permeases (e.g. that for γ-amino-n-butyrate) are controlled by the pH of the growth medium. For example, at acidic pH, levels of acid phosphatase are high and those of alkaline phosphatase are low whereas at alkaline pH the reverse is true. Mutations in five genes, palA, B, C, E and F, mimic the effects of growth at acid pH whereas mutations in pacC mimic the effects of growth at alkaline pH. palA, B, C, E and F mutations result in an intracellular pH (pHin) which is more alkaline than that of the wild type whereas pacC mutations result in a pHin more acidic than that of the wild type. This indicates that these mutations exert their primary effects on the regulation of gene expression by pH rather than on the pH homeostatic mechanism but that the expression of at least some component(s) of the pH homeostatic mechanism is subject to the pH regulatory system. It is suggested that pacC might be a wide domain regulatory gene whose product acts positively in some cases (e.g. acid phosphatase) and negatively in others (e.g. alkaline phosphatase). The products of palA, B, C, E and F are proposed to be involved in a metabolic pathway leading to synthesis of an effector molecule able to prevent the (positive and negative) action of the pacC product.These genes are, to our knowledge, the first examples of genes involved in the regulation of extracellular enzyme and permease synthesis by the pH of the growth medium to be described in any organism.

Mutational analysis reveals dispensability of the N-terminal region of the Aspergillus transcription factor mediating nitrogen metabolite repression.

Mutational analysis has enabled identification and localization of an upstream exon of the areA gene of Aspergillus nidulans mediating nitrogen metabolite repression. A mutation in the initiation codon and frameshift mutations, which revert by restoration of the reading frame, established the coding role of the exon and mutations affecting intron splicing in conjunction with DNA sequencing of reverse transcriptase polymerase chain reaction (RT—PCR) products localized the coding region intron. The resulting AREA translation product would have 876 residues. Deletion of the upstream exon such that translation of the remaining areA coding region would yield a protein containing only the 719 C‐terminal residues has only a subtle phenotype, very similar to those resulting from single amino acid replacements in upstream exon‐encoded regions of strong sequence similarity to the Neurospora crassa and Penicillium chrysogenum homologues. A number of areA mRNAs of different sizes are synthesised and appear to be functionally redundant. Synthesis of at least the smallest mRNA(s) is probably subject to autogenous activation. Suppression of frameshift mutations by compensating mutations preventing intron splicing suggests that insertion of a markedly hydrophobic sequence can impair AREA function. Finally, translational initiation for areA can occur within a region of at least 123 nucleotides.

Characterization of nitrogen metabolite signalling in Aspergillus via the regulated degradation of areA mRNA

AreA is the principal transcription factor involved in determining nitrogen utilization in Aspergillus nidulans. NH4+ and Gln are utilized preferentially but in their absence, AreA acts to facilitate the expression of genes involved in metabolizing alternative nitrogen sources. It is crucial to the function of AreA that its expression is tightly modulated by the quality and availability of nitrogen sources. One signalling mechanism involves regulated degradation of the areA transcript in response to NH4+ and Gln, which provides the first direct means of monitoring nitrogen signalling in this fungus. Here we assess the specificity of the transcript degradation response, determining that it responds qualitatively to a variety of additional nitrogen sources including Asn. Furthermore, the response to Gln and NH4+ requires the same discrete region of the areA 3′‐UTR but both NH4+ and Asn need to be metabolized to Gln before they are effective as a signal. However, NH4+ signalling is independent of AreA activity, unlike Gln and Asn signalling. A mutation in the structural gene for NADP‐linked glutamate dehydrogenase, gdhA, which disrupts metabolism of NH4+ to Glu, is additive with mutations in two distinct regions of areA that disrupt the previously identified signalling mechanisms. The triple mutant is both strongly derepressed and expresses very high levels of nitrate reductase activity. These data suggest nitrogen metabolism in A. nidulans is in part regulated in response to the intracellular levels of Gln via the regulated degradation of areA mRNA, but the intracellular Gln level is not the sole determinant of nitrogen metabolite repression.

Nitrogen regulation in fungi

Nitrogen regulation has been extensively studied in fungi revealing a complex array of interacting regulatory genes. The general characterisation of the systems inAspergillus nidulans andNeurospora crassa shall be briefly described, but much of this paper will concentrate specifically on the recent molecular characterisation ofareA, the principle regulatory gene fromA. nidulans which mediates nitrogen metabolite repression. Three areas shall be explored in detail, firstly the DNA binding domain, which has been characterised extensively by both molecular and genetic analysis. Secondly we shall report recent analysis which has revealed the presence of related DNA binding activities inA. nidulans. Finally we shall discuss the mechanism by which the nitrogen state of the cell is monitored by theareA product, in particular localisation of the domain within theareA product which mediates the regulatory response within the protein.

The GATA factor AreA regulates localization and in vivo binding site occupancy of the nitrate activator NirA

The GATA factor AreA is a wide‐domain regulator in Aspergillus nidulans with transcriptional activation and chromatin remodelling functions. AreA interacts with the nitrate‐specific Zn2‐C6 cluster protein NirA and both proteins cooperate to synergistically activate nitrate‐responsive genes. We have previously established that NirA in vivo DNA binding site occupancy is AreA dependent and in this report we provide a mechanistic explanation for our previous findings. We now show that AreA regulates NirA at two levels: (i) through the regulation of nitrate transporters, AreA affects indirectly the subcellular distribution of NirA which rapidly accumulates in the nucleus following induction; (ii) AreA directly stimulates NirA in vivo target DNA occupancy and does not act indirectly by chromatin remodelling. Simultaneous overexpression of NirA and the nitrate transporter CrnA bypasses the AreA requirement for NirA binding, permits utilization of nitrate and nitrite as sole N‐sources in an areA null strain and leads to an AreA‐independent nucleosome loss of positioning.

Structural genes for phosphatases in Aspergillus nidulans

Although the fungus Aspergillus nidulans has a multiplicity of phosphatases and of genes where mutations affect one or more phosphatases, we have succeeded in identifying structural genes for three phosphatases as well as one other gene which might encode a fourth. Using both conditional and non-conditional mutations, palD has been shown to be the structural gene for a phosphate-repressible alkaline phosphatase, palG to be the structural gene for a non-repressible alkaline phosphatase which apparently exists in two electrophoretically distinct forms (but whose rates of thermal inactivation are apparently very similar) and pacA to be the structural gene for both intracellular and secreted forms of a phosphate-repressible acid phosphatase. Colony staining techniques for the enzymes specified by palD and pacA have been described previously but we have now shown that the enzyme specified by palG can be detected by staining toluene-permeabilized colonies. Mutations in pacG lead to loss of non-repressible acid phosphatase as judged by colony staining and electrophoretic patterns but their effects on assays of activity in cell-free extracts are only marginal. Under phosphate-limited, but not phosphate-starved or phosphate-sufficient, conditions, pacG~ mutations also affect the regulation of other, phosphate-repressible phosphatases. None of these phosphatases, alone or in combination, plays an essential role.

C-terminal truncation of the transcriptional activator encoded by areA in Aspergillus nidulans results in both loss-of-function and gain-of-function phenotypes.

Mutations truncating as many as 143 C-terminal residues from the transcriptional activator encoded by the areA gene, mediating nitrogen metabolite repression in Aspergillus nidulans, do not significantly reduce the ability of the areA product to activate expression of most genes under areA control. Such mutations can even have a gain-of-function, derepressed phenotype, consistent with a critical role for this region in modulating the activity of the areA protein. However, expression of a few genes under areA control is substantially impaired by such C-terminal truncations, indicating that regions of an activator protein can play differing roles in the control of different structural genes. This underlines the advantages of being able to monitor effects of areA mutations on expression of large numbers of structural genes. Additionally, it is shown that truncation of as many as 153 C-terminal residues, virtually all amino acids C-terminal to the DNA-binding region, is compatible with retention of some areA function.

MOLECULAR CHARACTERISATION OF MEAB, A NOVEL GENE AFFECTING NITROGEN METABOLITE REPRESSION IN ASPERGILLUS NIDULANS

Mutations within the meaB gene elicit the inappropriate expression of several activities subject to nitrogen metabolite repression in Aspergillus nidulans and also have some unrelated phenotypic effects. We have cloned meaB and isolated a full length cDNA clone. Northern analysis has shown that meaB expression is not subject to nitrogen metabolite repression. meaB encodes a novel protein of 418 amino acids and contains a significantly high number of S/TPXX motifs, a motif common in transcriptional regulatory proteins. We have sequenced three mutations within meaB, two of which have an identical phenotype to that produced by gene disruption.