The NADP-glutamate dehydrogenase of the cyanobacterium Synechocystis 6803: cloning, transcriptional analysis and disruption of the gdhA gene.
The gdhA gene of Synechocystis PCC 6803, which encodes an NADP-dependent glutamate dehydrogenase (NADP-GDH), has been cloned by complementation of an Escherichia coli glutamate auxotroph. This gene was found to code for a polypeptide of 428 amino acid residues, whose sequence shows high identity with those of archaebacteria (42-47%), some Gram-positive bacteria (40-44%) and mammals (37%). The minimal fragment of Synechocystis DNA required for complementation (2kb) carries the gdhA gene preceded by an open reading frame (ORF2) encoding a polypeptide of 130 amino acids. ORF2 and gdhA are co-transcribed as a 1.9 kb mRNA, but shorter transcripts including only gdhA were also detected. Two promoter regions were identified upon transcriptional fusion to the cat reporter gene of a promoter probe plasmid. Transcription from the promoter upstream of ORF2 was found to be regulated depending on the growth phase of Synechocystis, in parallel to NADP-GDH activity. This promoter is expressed in Escherichia coli too, in contrast to the second promoter, located between ORF2 and gdhA, which was silent in E. coli and did not respond to the stage of growth in Synechocystis. Disruption of the cyanobacterial gdhA gene with a chloramphenicol resistance cassette yielded a mutant strain totally lacking NADP-GDH activity, demonstrating that this gene is not essential to Synechocystis 6803 under our laboratory conditions.
Chávez
S
S
Departamento de Bioquímica Vegetal y Biología Molecular, Universidad de Sevilla, Spain.
Reyes
J C
JC
Chauvat
F
F
Florencio
F J
FJ
Candau
P
P
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D005816:Genetic Complementation Test; D015698:Genomic Library; D018086:Glutamate Dehydrogenase (NADP+) / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D012333:RNA, Messenger; D011994:Recombinant Proteins; D018086:Glutamate Dehydrogenase (NADP+)