The NAD(P)H-dependent glutamate dehydrogenase activities of Prevotella ruminicola B(1)4 can be attributed to one enzyme (GdhA), and gdhA expression is regulated in response to the nitrogen source available for growth.
Prevotella ruminicola B(1)4 possesses both NADPH- and NADH-linked glutamate dehydrogenase (GDH) activities, with the greatest specific activity being measured from ammonia-limited cultures. Relative to cells grown in the presence of 1 mM ammonium chloride, the NADPH-dependent activity was decreased approximately 10-fold when peptides were provided as a nitrogen source. Nondenaturing polyacrylamide gel electrophoresis (PAGE) was used to visualize the GDH protein(s) in cell extracts of P. ruminicola. For all growth conditions tested, only one GDH protein was detectable, and its relative abundance, as well as its reactivity with either NAD(P)+ or NAD(P)H, correlated well with the specific activities measured from whole-cell assays. Consistent with the findings from enzyme assays and PAGE activity gels, Northern (RNA) blot analysis revealed that expression of a gene encoding NAD(P)H-GDH activity was greatest in ammonia-grown cultures and that GDH activity is regulated in response to nitrogen source (ammonia versus peptides), probably at the level of transcription. A gene encoding the NAD(P)H-utilizing GDH activity (gdhA) was cloned, and its nucleotide sequence was determined and shown to contain an open reading frame of 1,332 bp which would encode a polypeptide of 48.8 kDa. The deduced amino acid sequence possesses three highly conserved motifs typical of family I GDHs, but several unique amino acid substitutions within these motifs were evident. These results are discussed within the context of ruminal nitrogen metabolism and the growth efficiency of succinate- and propionate-producing anaerobic bacteria.
Wen
Z
Z
Department of Animal Science, University of Nebraska-Lincoln 68583-0908, USA.
Morrison
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M
D000595:Amino Acid Sequence; D000643:Ammonium Chloride / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D005816:Genetic Complementation Test; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D018086:Glutamate Dehydrogenase (NADP+) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009584:Nitrogen / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D018720:Prevotella / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D000643:Ammonium Chloride; D011189:Potassium Chloride; D005969:Glutamate Dehydrogenase; D018086:Glutamate Dehydrogenase (NADP+); D009584:Nitrogen