A 3.48-kb DNA region containing the gdhA gene, which codifies the NADP-dependent glutamate dehydrogenase enzyme from Botrytis cinerea, has been cloned and characterized. A fragment of 2351 nucleotides was sequenced and found to contain an ORF of 1350 bp that encodes a protein of 450 amino acids. The gene, containing two introns that showed polymorphic size between them, was located by pulsed-field gel electrophoresis in chromosome X in seven strains, which were isolated from several hosts and had different levels of pathogenesis. The protein was similar to the gdhA of various other organisms, with nine highly conserved motifs that included the known active site sequence. The cloned gene was proven to be functional since it complemented two different Aspergillus nidulans gdhA mutants, restoring high levels of NADP-dependent glutamate dehydrogenase activity to the transformants. gdhA was transcribed as a monocistronic transcript of 1.7 kb starting at an A or a T, located 40 or 47 bp, respectively, upstream from the initial ATG codon of the ORF. Transcription levels of the gdhA gene were high during the rapid growth phase. Very high expression levels of the gdhA gene were observed in media with asparagine as the nitrogen source, whereas glutamic acid repressed transcription of the gdhA gene. Similarly high levels of gdhA gene transcription were observed in media with acetate as the carbon source, while glycerol strongly repressed gdhA gene transcription. These results indicate that expression of the gdhA gene is subject to strong nitrogen and carbon regulation at the transcriptional level.
Copyright 2001 Academic Press.