Mutational analysis of GstI protein, a glutamine synthetase translational inhibitor of Rhizobium leguminosarum.
The small GstI protein (63 amino acids) of Rhizobium leguminosarum inhibits the expression of the glnII (glutamine synthetase II) gene, thus reducing the bacterial ability to assimilate ammonium. In order to identify the residues essential for its inhibitory activity, all the 53 non-alanine amino acid residues of GstI were individually mutated into alanine. Based on their capacity to inhibit glnII expression (in two genetic backgrounds) three groups of mutants were identified. The first group displayed an inhibitory activity similar to the wild-type; the second and the third ones showed partial and total loss of inhibitory activity, respectively. Several mutations of the latter group concerned residues conserved in two related sequences from Sinorhizobium meliloti and Agrobacterium tumefaciens. Additionally, we performed experiments to exclude a GstI-mediated mechanism of glutamine synthetase II inhibition/degradation. Finally, the protein was over expressed in Escherichia coli, purified and characterised.
Napolitani
Chiara
C
Istituto di Genetica e Biofisica 'A. Buzzati-Traverso', CNR, Via Marconi 10, 80125 Naples, Italy.
Mandrich
Luigi
L
Riccio
Anna
A
Lamberti
Alessandro
A
Manco
Giuseppe
G
Patriarca
Eduardo J
EJ
D016960:Agrobacterium tumefaciens / Q000235:genetics; D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D016384:Consensus Sequence; D004252:DNA Mutational Analysis / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011499:Protein Processing, Post-Translational; D012097:Repressor Proteins / Q000235:genetics* / Q000494:pharmacology; D016961:Rhizobium leguminosarum / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D016962:Sinorhizobium meliloti / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; C414318:GstI protein, Rhizobium leguminosarum; D012097:Repressor Proteins; D005974:Glutamate-Ammonia Ligase; D000409:Alanine