Existence of two ferredoxin-glutamate synthases in the cyanobacterium Synechocystis sp. PCC 6803. Isolation and insertional inactivation of gltB and gltS genes.
The first two genes of ferredoxin-dependent glutamate synthase (Fd-GOGAT) from a prokaryotic organism, the cyanobacterium Synechocystis sp. PCC 6803, were cloned in Escherichia coli. Partial sequencing of the cloned genomic DNA, of the 6.3 kb Hind III and 9.3 kb Cla I fragments, confirmed the existence of two different genes coding for glutamate synthases, named gltB and gltS. The gltB gene was completely sequenced and encodes for a polypeptide of 1550 amino acid residues (M(r) 168,964). Comparative analysis of the gltB deduced amino acid sequence against other glutamate synthases shows a higher identity with the alfalfa NADH-GOGAT (55.2%) than with the corresponding Fd-GOGAT from the higher plants maize and spinach (about 43%), the red alga Antithamnion sp. (42%) or with the NADPH-GOGAT of bacterial source, such as Escherichia coli (41%) and Azospirillum brasilense (45%). The detailed analysis of Synechocystis gltB deduced amino acid sequence shows strongly conserved regions that have been assigned to the 3Fe-4S cluster (CX5CHX3C), the FMN-binding domain and the glutamine-amide transferase domain. Insertional inactivation of gltB and gltS genes revealed that both genes code for ferredoxin-dependent glutamate synthases which were nonessential for Synechocystis growth, as shown by the ferredoxin-dependent glutamate synthase activity and western-blot analysis of the mutant strains.
Navarro
F
F
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Spain.
Chávez
S
S
Candau
P
P
Florencio
F J
FJ
D000594:Amino Acid Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics*; D004269:DNA, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D000594:Amino Acid Oxidoreductases; C042233:glutamate synthase (ferredoxin)