Identification of the N-acetyl-D-glucosamine-inducible element in the promoter of the Trichoderma atroviride nag1 gene encoding N-acetyl-glucosaminidase.

We have investigated the regulation by N-acetyl-glucosamine of the nag1 gene of the mycoparasitic biocontrol fungus Trichoderma atroviride (= T. harzianum P1), which encodes a 73-kDa N-acetyl-beta-D-glucosaminidase. The use of translational fusions revealed that a 290-bp fragment of the 5' regulatory region of nag1 is sufficient to confer inducibility on the Aspergillus niger goxA gene. The region between positions -150 and -290, upstream of the nag1 coding region, was investigated using in vivo methylation protection analysis and electrophoretic mobility shift assays (EMSAs). Two neighbouring regions that interacted with regulatory proteins were identified, and bases essential for these interactions were determined in vitro. These data reveal protein binding to a CCCCT element at -240, a CCAGN(13)CTGG motif at -284, and a CCAAT-box which is present in the spacer of the latter motif. Evidence for the binding of a Hap2/3/5 complex to this CCAAT motif is presented. Protein binding to all three motifs was constitutive, and no differences were observed between induced and non-induced cultures. Mutation of either the CCAGN(13)CTGG or the AGGGG motif resulted in loss of inducibility of nag1 expression by N-acetyl-D-glucosamine in vivo.