Identification of the glutamine synthetase adenylyltransferase of Azospirillum brasilense.
Glutamine synthetase, a key enzyme in nitrogen metabolism of both prokaryotes and eukaryotes, is strictly regulated. One means of regulation is the modulation of activity through adenylylation catalyzed by adenylyltransferases. Using PCR primers based on conserved sequences in glutamine synthetase adenylyltransferases, we amplified part of the glnE gene of Azospirillum brasilense Sp7. The complete glnE sequence of A. brasilense Sp245 was retrieved from the draft genome sequence of this organism (http://genomics.ornl.gov/research/azo/). Adenylyltransferase is a bifunctional enzyme consisting of an N-terminal domain responsible for deadenylylation activity and a C-terminal domain responsible for adenylylation activity. Both domains are partially homologous to each other. Residues important for catalytic activity were present in the deduced amino acid sequence of the A. brasilense Sp245 glnE sequence. A glnE mutant was constructed in A. brasilense Sp7 by inserting a kanamycin resistance cassette between the two active domains of the enzyme. The resulting mutant was unable to adenylylate the glutamine synthetase enzyme and was impaired in growth when shifted from nitrogen-poor to nitrogen-rich medium.
Van Dommelen
Anne
A
Centre of Microbial and Plant Genetics, K.U. Leuven, Heverlee, Belgium. Anne.Vandommelen@biw.kuleuven.be
Spaepen
Stijn
S
Vanderleyden
Jozef
J
D000595:Amino Acid Sequence; D015806:Azospirillum brasilense / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D055786:Gene Knockout Techniques; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009584:Nitrogen / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D009713:Nucleotidyltransferases; C020658:glutamine-synthetase adenylyltransferase; D009584:Nitrogen