THE NADH-DEPENDENT GLUTAMATE-DEHYDROGENASE ENZYME OF BACTEROIDES-FRAGILIS BF1 IS INDUCED BY PEPTIDES IN THE GROWTH-MEDIUM

Bacteroides fragilis Bf1 possesses two enzymes having glutamate dehydr ogenase (GDH) activity. One is dual cofactor NAD(P)H-dependent, while the other has NADH-specific activity. The gene encoding the NADH-GDH ( gdhB) was cloned by complementation of the glutamate auxotrophic mutan t Escherichia coli MX3004 and the recombinant protein was characterize d with respect to the CDH activities present in the parental organism grown under different nitrogen conditions. The NAD(P)H-dependent GDH o f B. fragilis was confirmed to be most active under high ammonia condi tions, but the NADH-specific GDH levels were increased by high peptide concentrations in the growth medium and not regulated by the levels o f ammonia. Northern blotting analysis showed that gdhB regulation was at the transcription level, with a single transcript of similar to 1.6 kb being produced. GDH activity was demonstrated by zymography of the parental and recombinant enzymes. The recombinant GDH was NADH-specif ic and co-migrated with the equivalent enzyme band from B. fragilis ce ll extracts. The gdhB structural gene comprises 1335 bp and encodes a protein of 445 aa (49 kDa). Comparisons of the derived protein sequenc e with that of GDH from other bacteria indicated that significant sequ ence homology and conservation of functional domains exists with enzym es of Family I-type hexameric GDH proteins.