Characterization of the gene encoding glutamate dehydrogenase ( gdhA) from the ruminal bacterium Ruminococcus...

The gene encoding glutamate dehydrogenase ( gdhA) in the ruminal bacterium Ruminococcus flavefaciens FD-1 was cloned. A degenerate primer based on the N-terminal amino acid sequence of the purified protein was used in conjunction with genome walking to obtain the complete ORF of 1,365 bp, capable of encoding a polypeptide of 455 amino acid residues. The translated ORF contained the amino acid motifs characteristic of the subfamily GDH S_50(I) small glutamate dehydrogenases, including the catalytic site, and matched the originally deduced N-terminal amino acid sequence. BLAST search yielded high scores with other GdhA sequences from a variety of organisms, the closest match being with the GdhA sequence of Corynebacterium glutamicum (63% amino acid identity). Classification of the GdhA enzyme from R. flavefaciens FD-1 as a GDH S_50(I) subfamily member was further supported by phylogenetic analysis. The transcript size determined by Northern blot analysis was in good agreement with the putative regulatory region of the gene and confirmed its monocistronic structure. R. flavefaciens GdhA activity appears to be regulated primarily at the level of transcription. Brief exposure to 20 mM NH(4)Cl prior to extraction did not alter the level of activity. Transcriptional regulation, studied with quantitative real-time RT-PCR, demonstrated a three-fold increase of the gdhA transcript concentration in ammonia-limited cells in comparison with an excess of ammonia in the medium. This is in agreement with the enzyme activity data obtained under ammonia- and carbon-limited growth conditions.