Aspergillus niger is an important industrial fungus expressing a broad spectrum of pectinolytic genes. The main constituent of pectin, polygalacturonic acid (PGA), is degraded into galacturonic acid (GA) by the combined activity of endo- and exo-polygalacturonases some of which are specifically induced by GA. The regulatory mechanisms that control the expression of genes encoding PGA-degrading enzymes are not well understood. Based on available genome-wide expression profiles from literature, we selected five genes that were specifically induced by GA. These genes include three exo-polygalacturonases (pgaX, pgxB and pgxC), a GA transporter (gatA), and an intracellular enzyme involved in GA metabolism (gaaB). These five genes contain a conserved motif (5'-TCCNCCAAT-3') in their promoter regions, which we named GARE (galacturonic acid-responsive element). Promoter deletion studies and site-directed mutagenesis of the conserved motif of the pgaX gene showed that the conserved element is required for GA-mediated induction. A set of promoter reporter strains was constructed by fusing the promoter region of the five above-mentioned genes to the amdS reporter gene. Expression of the amdS gene is quantitatively correlated with ability to utilise acetamide as an N-source, hence higher expression of amdS improves growth of the strain on acetamide and therefore can be used as an in vivo reporter for gene expression. Growth analysis of the reporter strains indicated that four genes (pgaX, pgxB, pgxC, and gatA) are specifically induced by GA. The in vivo promoter reporter strains were also used to monitor carbon catabolite repression control. Except for gaaB, all promoter-reporter genes analysed were repressed by glucose in a glucose concentration-dependent way. Interestingly, the strength of glucose repression was different for the tested promoters. CreA is important in mediating carbon catabolite repression as deletion of the creA gene in the reporter strains abolished carbon catabolite repression for most promoters. Interestingly, the pgxC promoter was still repressed by glucose even in the creA null background, suggesting a role for alternative repression mechanisms. Finally, we showed that low concentrations of GA are required to induce gene expression of pgaX, pgxB, and pgxC even under derepressing conditions. The results obtained are consistent with a model in which a GA-specific transcription factor is activated by GA or a GA-derivative, which binds to the conserved motif, possibly in combination with the HAP-complex, to drive GA-specific gene expression.
Keywords: Acetamidase; Carbon catabolite repression; CreA; Gene regulation; Pectin; Pectinolytic genes; Reporter genes.
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