Andrew P. MacCabe

The 2008 update of the Aspergillus nidulans genome annotation: A community effort

The identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology.

Aspergillus niger mstA encodes a high affinity sugar:H + symporter which is regulated in response to extracellular pH.

A sugar-transporter-encoding gene, mstA, which is a member of the major facilitator superfamily, has been cloned from a genomic DNA library of the filamentous fungus Aspergillus niger. To enable the functional characterization of MSTA, a full-length cDNA was expressed in a Saccharomyces cerevisiae strain deficient in hexose uptake. Uptake experiments using 14C-labelled monosaccharides demonstrated that although able to transport D-fructose ( K(m), 4.5+/-1.0 mM), D-xylose ( K(m), 0.3+/-0.1 mM) and D-mannose ( K(m), 60+/-20 microM), MSTA has a preference for D-glucose (K(m), 25+/-10 microM). pH changes associated with sugar transport indicate that MSTA catalyses monosaccharide/H+ symport. Expression of mstA in response to carbon starvation and upon transfer to poor carbon sources is consistent with a role for MSTA as a high-affinity transporter for D-glucose, D-mannose and D-xylose. Northern analysis has shown that mstA is subject to CreA-mediated carbon catabolite repression and pH regulation mediated by PacC. A. niger strains in which the mstA gene had been disrupted are phenotypically identical with isogenic reference strains when grown on 0.1-60 mM D-glucose, D-mannose, D-fructose or D-xylose. This indicates that A. niger possesses other transporters capable of compensating for the absence of MSTA.

Carbon catabolite repression of the Aspergillus nidulans xlnA gene

Expression of the Aspergillus nidulans 22 kDa endoxylanase gene, xlnA, is controlled by at least three mechanisms: specific induction by xylan or xylose; carbon catabolite repression (CCR); and regulation by ambient pH. Deletion analysis of xlnA upstream sequences has identified two positively acting regions: one that mediates specific induction by xylose; and another that mediates the influence of ambient pH and contains two PacC consensus binding sites. The extreme derepressed mutation creAd30 results in considerable, although not total, loss of xlnA glucose repressibility, indicating a major role for CreA in its CCR. Three consensus CreA binding sites are present upstream of the structural gene. Point mutational analysis using reporter constructs has identified a single site, xlnA.C1, that is responsible for direct CreA repression in vivo. Using the creAd30 derepressed mutant background, our results indicate the existence of indirect repression by CreA.

Identification, isolation and sequence of the Aspergillus nidulans xlnC gene encoding the 34-kDA xylanase.

The xlnC gene encoding the 34-kDa xylanase (X34) of Aspergillus nidulans (An) has been cloned and sequenced, as has its corresponding cDNA. xlnC contains nine introns and shows considerable similarity to the xynA and xylP xylanase-encoding genes of A. kawachii (Ak) and Penicillium chrysogenum (Pc), respectively. Analysis of xylanase production in An multicopy transformants showed elevated levels of X34 and increased total xylanase activity, but no elevated production of other xylanases. Northern analysis demonstrated transcriptional induction by xylan and repression by glucose.

Consecutive gene deletions in Aspergillus nidulans: application of the Cre/loxP system

The ability to perform multiple gene deletions is an important tool for conducting functional genomics. We report the development of a sequential gene deletion protocol for the filamentous fungus Aspergillus nidulans using the Cre/loxP recombinase system of bacteriophage P1. A recyclable genetic marker has been constructed by incorporating loxP direct repeats either side of the Neurospora crassa pyr-4 gene (encodes orotidine 5′-monophosphate decarboxylase) which is able to complement the A. nidulans pyrG89 mutation. This construct can be directed to delete specific genomic regions by attaching flanking sequences corresponding to the desired target. The pyr-4 marker can subsequently be eliminated by Cre-catalysed recombination between the loxP sites. The recombinase gene (cre), which has been placed under the control of the A. nidulansxlnA (xylanase A) gene promoter thus providing a means to switch on (xylose induction) or off (glucose repression) recombinase expression, has been integrated into the genome of an A. nidulans mutant strain defective in orotidine 5′-monophosphate decarboxylase activity (pyrG89). We demonstrate the effectiveness of our deletion system by sequentially deleting two genes, yellow (yA) and white (wA), involved in the synthesis of conidial pigment.

Improving extracellular production of food-use enzymes from Aspergillus nidulans

Filamentous fungi, and particularly those of the genus Aspergillus, are major producers of enzymatic activities that have important applications in the food and beverage industries. Prior to the availability of transformation systems improvement of industrial production strains was largely restricted to the strategy of mutagenesis, screening and selection. Aspergillus nidulans is a genetically amenable filamentous fungus the ease of handling and analysis of which has led to its use as a model system for the investigation of eukaryotic gene regulation. Although not used industrially it is able to produce a wide variety of extracellular enzymatic activities. As a consequence of half a century of study a considerable resource of characterised mutants has been generated in conjunction with extensive genetic and molecular information on various gene regulatory systems in this micro-organism. Investigation of xylanase gene regulation in A. nidulans as a model for the production of food-use extracellular enzymes suggests strategies by which production of these enzymes in industrially useful species may be improved.

Identification of the mstE Gene Encoding a Glucose-inducible, Low Affinity Glucose Transporter in Aspergillus nidulans

The mstE gene encoding a low affinity glucose transporter active during the germination of Aspergillus nidulans conidia on glucose medium has been identified. mstE expression also occurs in hyphae, is induced in the presence of other repressing carbon sources besides glucose, and is dependent on the function of the transcriptional repressor CreA. The expression of MstE and its subcellular distribution have been studied using a MstE-sGFP fusion protein. Concordant with data on mstE expression, MstE-sGFP is synthesized in the presence of repressing carbon sources, and fluorescence at the periphery of conidia and hyphae is consistent with MstE location in the plasma membrane. Deletion of mstE has no morphological phenotype but results in the absence of low affinity glucose uptake kinetics, the latter being substituted by a high affinity system.

The wide-domain carbon catabolite repressor CreA indirectly controls expression of the Aspergillus nidulans xlnB gene, encoding the acidic endo-beta-(1,4)-xylanase X(24).

The Aspergillus nidulans xlnB gene, which encodes the acidic endo-beta-(1,4)-xylanase X(24), is expressed when xylose is present as the sole carbon source and repressed in the presence of glucose. That the mutation creA(d)30 results in considerably elevated levels of xlnB mRNA indicates a role for the wide-domain repressor CreA in the repression of xlnB promoter (xlnBp) activity. Functional analyses of xlnBp::goxC reporter constructs show that none of the four CreA consensus target sites identified in xlnBp are functional in vivo. The CreA repressor is thus likely to exert carbon catabolite repression via an indirect mechanism rather than to influence xlnB expression by acting directly on xlnB.

AcpA, a member of the GPR1/FUN34/YaaH membrane protein family, is essential for acetate permease activity in the hyphal fungus Aspergillus nidulans

In a previous study, alcS, a gene of the Aspergillus nidulans alc cluster, was shown to encode a protein that belongs to the GPR1/FUN34/YaaH membrane protein family. BLAST screening of the A. nidulans genome data identified additional genes encoding hypothetical proteins that could belong to this family. In this study we report the functional characterization of one of them, AN5226. Its expression is induced by ethanol and ethyl acetate (two inducers of the alc genes) and is mediated by the specific transcriptional activator of genes of the acetate-utilization pathway FacB. Growth of a null mutant (DeltaAN5226) is notably affected when acetate is used as sole carbon source at low concentration and in a high pH medium, i.e. when protonated acetate, the form that can enter the cell by passive diffusion, is present in low amounts. Consistently, expression of AN5226 is also induced by acetate, but only when the latter is present at low concentrations. (14)C-labelled acetate uptake experiments using germinating conidia demonstrate an essential role for AN5226 in mediated acetate transport. To our knowledge this report is the first to provide evidence for the identification of an acetate transporter in filamentous fungi. We have designated AN5226 as acpA (for acetate permease A).

Heterologous production in Saccharomyces cerevisiae of different Aspergillus nidulans xylanases of potential interest in oenology

Three Aspergillus nidulans endo-β-(1,4)-xylanases (X22, X24 and X34) have been produced in Saccharomyces cerevisiae laboratory strains. In fermenter cultivation, the resulting strains were able to secrete the heterologous xylanases when grown in minimal medium containing glucose as carbon source, yielding production levels between 0·5 and 1·0 U ml−1. From such a culture, the xylanases were purified in a single chromatographic step and then physico-chemically characterised. The yeast derived enzymes had the same enzymatic and biochemical properties as the fungal xylanases (ie molecular weights, pI values, pH and temperature optima, Km Michaelis constants and pH and temperature stabilities). The heterologously produced enzymes retained maximum activity in the presence of 10% (w/v) glucose or 10% (w/v) fructose, concentrations normally found in musts. However, ethanol at 12% (v/v) caused inhibitions of 20% for X22 and X24 and 48% for X34.