ABSTRACT
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.
Subject(s)
Azospirillum brasilense/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Amino Acid Sequence , Azospirillum brasilense/genetics , Azospirillum brasilense/metabolism , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Gene Knockout Techniques , Molecular Sequence Data , Mutagenesis, Insertional , Nitrogen/metabolism , Sequence Alignment , Sequence Analysis, DNAABSTRACT
Sequencing the glnA genes of two chemically induced Azospirillum brasilense glutamine synthetase mutants revealed an Arg-->Cys mutation, corresponding to the glutamate binding site, in one mutant and an Asp-->Asn mutation, corresponding to the ammonium binding site, in the second mutant. The phenotypic changes in these mutants are discussed in relation to their genotypes.
Subject(s)
Azospirillum brasilense/genetics , Genes, Bacterial/genetics , Glutamate-Ammonia Ligase/genetics , Point Mutation , Amino Acid Substitution , Asparagine , Aspartic Acid , Azospirillum brasilense/enzymology , Binding Sites , Glutamate-Ammonia Ligase/metabolism , Quaternary Ammonium Compounds/metabolism , Recombinant Fusion Proteins/metabolismABSTRACT
The role of three key nitrogen regulatory genes, glnB (encoding the P(II) protein), glnZ (encoding the P(z) protein), and glnD (encoding the GlnD protein), in regulation of poly-3-hydroxybutyrate (PHB) biosynthesis by ammonia in Azospirillum brasilense Sp7 was investigated. It was observed that glnB glnZ and glnD mutants produce substantially higher amounts of PHB than the wild type produces during the active growth phase. glnB and glnZ mutants have PHB production phenotypes similar to that of the wild type. Our results indicate that the P(II)-P(z) system is apparently involved in nitrogen-dependent regulation of PHB biosynthesis in A. brasilense Sp7.
