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 dehydrogenase (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 mutant Escherichia coli MX3004 and the recombinant protein was characterized with respect to the GDH activities present in the parental organism grown under different nitrogen conditions. The NAD(P)H-dependent GDH of B. fragilis was confirmed to be most active under high ammonia conditions, but the NADH-specific GDH levels were increased by high peptide concentrations in the growth medium and not regulated by the levels of ammonia. Northern blotting analysis showed that gdhB regulation was at the transcription level, with a single transcript of approximately 1.6 kb being produced. GDH activity was demonstrated by zymography of the parental and recombinant enzymes. The recombinant GDH was NADH-specific and co-migrated with the equivalent enzyme band from B. fragilis cell extracts. The gdhB structural gene comprises 1335 bp and encodes a protein of 445 aa (49 kDa). Comparisons of the derived protein sequence with that of GDH from other bacteria indicated that significant sequence homology and conservation of functional domains exists with enzymes of Family I-type hexameric GDH proteins.
Abrahams
Garth L
GL
Microbiology Department, University of Cape Town, Private Bag, Rondebosch 7701, South Africa.
Abratt
Valerie R
VR
Microbiology Department, University of Cape Town, Private Bag, Rondebosch 7701, South Africa.
D000595:Amino Acid Sequence; D000641:Ammonia / Q000494:pharmacology; D001441:Bacteroides fragilis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D004274:DNA, Recombinant / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors; D005969:Glutamate Dehydrogenase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology; D011994:Recombinant Proteins / Q000032:analysis
D004274:DNA, Recombinant; D010455:Peptides; D011994:Recombinant Proteins; D000641:Ammonia; D005969:Glutamate Dehydrogenase