Characterization of the gene encoding glutamate dehydrogenase (gdhA) from the ruminal bacterium Ruminococcus flavefaciens FD-1

D. A. Antonopoulos, Rustam Aminov, P. A. Duncan, B. A. White, R. I. Mackie

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Abstract

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 NH4Cl 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.
Original languageEnglish
JournalArchives of Microbiology
Volume179
Issue number3
Pages (from-to)184-190
Number of pages7
ISSN0302-8933
DOIs
Publication statusPublished - 2003
Externally publishedYes

Keywords

  • Amino Acid Sequence
  • Ammonia
  • Animals
  • Base Sequence
  • Cloning, Molecular
  • DNA Primers
  • Gene Expression Regulation, Bacterial
  • Gene Expression Regulation, Enzymologic
  • Genes, Bacterial
  • Glutamate Dehydrogenase
  • Gram-Positive Bacteria
  • Molecular Sequence Data
  • Phylogeny
  • Rumen
  • Ruminants
  • 7664-41-7 Ammonia
  • EC 1.4.1.2 Glutamate Dehydrogenase
  • ammonia
  • glutamate dehydrogenase
  • amino acid sequence
  • amino terminal sequence
  • article
  • controlled study
  • Corynebacterium glutamicum
  • enzyme active site
  • enzyme activity
  • enzyme structure
  • extraction
  • genetic analysis
  • genetic transcription
  • molecular cloning
  • multigene family
  • nonhuman
  • Northern blotting
  • nucleotide sequence
  • open reading frame
  • phylogeny
  • priority journal
  • protein purification
  • reverse transcription polymerase chain reaction
  • Ruminococcus
  • ruminococcus flavefaciens
  • sequence analysis
  • transcription regulation
  • Bacteria (microorganisms)
  • Corynebacterium
  • Posibacteria
  • Prokaryota
  • Ruminococcus flavefaciens
  • Glutamate dehydrogenase
  • Transcriptional regulation
  • MICROBIOLOGY
  • NITROGEN CONTROL
  • transcriptional regulation
  • Ruminococcus Glutamate dehydrogenase Transcriptional regulation
  • Actinomycetes and Related Organisms Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Irregular Nonsporing Gram-Positive Rods [08890] Corynebacterium glutamicum species
  • Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Gram-Positive Cocci [07700] Ruminococcus flavefaciens species rumen bacterium, strain-FD-1
  • Mammalia Vertebrata Chordata Animalia (Animals, Artiodactyls, Chordates, Mammals, Nonhuman Vertebrates, Nonhuman Mammals, Vertebrates) - Artiodactyla [85705] ruminant common
  • Microorganisms (Bacteria, Eubacteria, Microorganisms) - Bacteria [05000] bacteria common rumen bacteria common
  • Ruminococcus flavefaciens gdhA gene [Gram-Positive Cocci] characterization, functions
  • glutamate dehydrogenase 9001-46-1Q, 9029-11-2Q, 9029-12-3Q, 9001-46-1 EC 1.4.1.2 activities, analysis, functions, genes
  • nutrients limitation effects
  • 03502, Genetics - General
  • 03506, Genetics - Animal
  • 10062, Biochemistry studies - Nucleic acids, purines and pyrimidines
  • 10802, Enzymes - General and comparative studies: coenzymes
  • 13202, Nutrition - General studies, nutritional status and methods
  • 14004, Digestive system - Physiology and biochemistry
  • 31000, Physiology and biochemistry of bacteria
  • 31500, Genetics of bacteria and viruses
  • Digestive System
  • Enzymology
  • Molecular Genetics
  • Nutrition
  • bacterial growth characteristics
  • cistrons
  • gene functions
  • genomes
  • open reading frames
  • phylogenetic tree
  • transcription
  • Biochemistry and Molecular Biophysics
  • Ingestion and Assimilation
  • rumen digestive system
  • gene characterization genetic techniques, laboratory techniques
  • Northern blotting genetic techniques, laboratory techniques
  • phylogenetic analysis mathematical and computer techniques
  • reverse transcriptase-polymerase chain reaction genetic techniques, laboratory techniques

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