Abstract
The cellulase system of the filamentous fungus Hypocrea jecorina (Trichoderma reesei) consists of several cellobiohydrolases, endoglucanases, and beta-glucosidases, encoded by separate genes, which are coordinately expressed in the presence of cellulose or the disaccharide sophorose. Using cell-free extracts from sophorose-induced and noninduced mycelia and various fragments of the cbh2 promoter of H. jecorina in electrophoretic mobility shift assay (EMSA) analysis and performing in vitro and in vivo footprinting analysis, we detected the nucleotide sequence 5'-ATTGGGTAATA-3' (consequently named cbh2-activating element (CAE)) to bind a protein complex with different migration in EMSA of induced and noninduced cell-free extracts. EMSA analysis, employing oligonucleotide fragments containing specifically mutated versions of CAE, revealed that protein binding requires the presence of an intact copy of either one of two adjacent motifs: a CCAAT (=ATTGG) box on the template strand and a GTAATA box on the coding strand, whereas a simultaneous mutation in both completely abolished binding. H. jecorina transformants, containing correspondingly mutated versions of the cbh2 promoter fused to the Escherichia coli hph gene as a reporter, expressed hph in a manner paralleling the efficacy of CAE-protein complex formation in EMSA, suggesting that the presence of either of both motifs is required for induction of cbh2 gene transcription. Antibody supershift experiments with anti-HapC antiserum as well as EMSA competition experiments with CCAAT binding promoter fragments of the Aspergillus nidulans amdS promoter suggest that the H. jecorina CCAAT box binding complex contains a homologue of HapC. The nature of the adjacent, GTAATA-binding protein(s) and its cooperation with the HapC homologue in cbh2 gene induction is discussed.
Highlights
Cellulose is the most abundant renewable carbon source on earth
We have previously identified the area between Ϫ361 and Ϫ170 in the 5Ј regulatory sequences of the cbh2 gene to be able in vitro to form a sophorosespecific DNA-protein complex [17]
Delimitation of the cbh2 Promoter Area, Which Binds a Protein Complex Unique for Sophorose Induction—When H. jecorina is pregrown on a carbon source not inducing cellulase formation and replaced to a medium containing the inducer sophorose, a transient accumulation of cbh1 and cbh2 mRNA is triggered (Fig. 1A)
Summary
Cellulose is the most abundant renewable carbon source on earth. Its recycling in nature occurs in a variety of habitats by the action of various pro- and eukaryotic microorganisms, which hydrolyse this homopolymer with extracellular enzyme systems [1]. To confirm the point mutations in the cbh2 promoter (generating a DraI restriction enzyme site) in transformants bearing the pSMZ2 and pSMZ4 plasmids, chromosomal DNA was digested with DraI/XhoII/ClaI, and hybridization was performed with a 2.6-kb XhoI/HindIII fragment of the vector pSMZ1 bearing the cbh2 promoter and terminator sequences and the hph structural gene labeled with [␣-32P]dCTP.
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