The NAD(P)H-utilizing glutamate dehydrogenase of Bacteroides thetaiotaomicron belongs to enzyme family I, and its activity is affected by trans-acting gene(s) positioned downstream of gdhA.
Previous studies have suggested that regulation of the enzymes of ammonia assimilation in human colonic Bacteroides species is coordinated differently than in other eubacteria. The gene encoding an NAD(P)H-dependent glutamate dehydrogenase (gdhA) in Bacteroides thetaiotaomicron was cloned and expressed in Escherichia coli by mutant complementation from the recombinant plasmid pANS100. Examination of the predicted GdhA amino acid sequence revealed that this enzyme possesses motifs typical of the family I-type hexameric GDH proteins. Northern blot analysis with a gdhA-specific probe indicated that a single transcript with an electrophoretic mobility of approximately 1.6 kb was produced in both B. thetaiotaomicron and E. coli gdhA+ transformants. Although gdhA transcription was unaffected, no GdhA enzyme activity could be detected in E. coli transformants when smaller DNA fragments from pANS100, which contained the entire gdhA gene, were analyzed. Enzyme activity was restored if these E. coli strains were cotransformed with a second plasmid, which contained a 3-kb segment of DNA located downstream of the gdhA coding region. Frameshift mutagenesis within the DNA downstream of gdhA in pANS100 also resulted in the loss of GdhA enzyme activity. Collectively, these results are interpreted as evidence for the role of an additional gene product(s) in modulating the activity of GDH enzyme activity. Insertional mutagenesis experiments which led to disruption of the gdhA gene on the B. thetaiotaomicron chromosome indicated that gdhA mutants were not glutamate auxotrophs, but attempts to isolate similar mutants with insertion mutations in the region downstream of the gdhA gene were unsuccessful.
Baggio
L
L
Department of Animal Sciences, University of Nebraska, Lincoln 68583, USA.
Morrison
M
M
D000595:Amino Acid Sequence; D000643:Ammonium Chloride / Q000494:pharmacology; D001439:Bacteroides / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D001483:Base Sequence; D003001:Cloning, Molecular; D003106:Colon / Q000382:microbiology; D003470:Culture Media / Q000494:pharmacology; D004269:DNA, Bacterial; D018086:Glutamate Dehydrogenase (NADP+) / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009249:NADP; D017422:Sequence Analysis, DNA
D003470:Culture Media; D004269:DNA, Bacterial; D000643:Ammonium Chloride; D009249:NADP; D018086:Glutamate Dehydrogenase (NADP+)