A protein is involved in accessibility of the inhibitor acetazolamide to the carbonic anhydrase(s) in the cyanobacterium Synechocystis PCC 6803.
A gene, zam (for resistance to acetazolamide), controlling resistance to the carbonic anhydrase inhibitor acetazolamide, is described. It has been cloned from a spontaneous mutant, AZAr-5b, isolated from the cyanobacterium Synechocystis PCC 6803, for its resistance to this drug (Bédu et al., Plant Physiol 93: 1312-1315, 1990). This mutant, besides its resistance to acetazolamide, displayed an absence of catalysed oxygen exchange activity on whole cells, suggestive of a deficiency in carbonic anhydrase activity. The gene was isolated by screening a genomic library of AZAr-5b, and selecting for the capacity to transfer the AZAr phenotype to wild-type cells. A system leading to forced homologous recombination in the host chromosome, using a platform vector, was devised in order to bypass direct selection difficulties. The putative encoded protein, 782 amino acids long, showed some homology with four eukaryotic and prokaryotic proteins involved in different cellular processes, one of them suppressing a phosphatase deficiency. The mutated allele of AZAr-5b showed an in-frame 12 nucleotide duplication, which should not interfere with translation, and might result from transposition of a mobile element. Integration into a wild-type genome of either the spontaneous mutated allele or one inactivated by insertional mutagenesis conferred the character of resistance, but not the deficiency in oxygen exchange, indicating that the two phenotypic aspects of AZAr-5b corresponded to two independent mutations. A working hypothesis explaining the phenotypes of the mutants is that the presence of the Zam protein would be necessary for the inhibitor to reach (one of) the two carbonic anhydrases present in this strain. This, however, would be a secondary action, the physiological role of the protein still being cryptic.
Beuf
L
L
Unité de Métabolisme Energétique, LCB-CNRS, Marseille, France.
Bédu
S
S
Cami
B
B
Joset
F
F
D000086:Acetazolamide / Q000494:pharmacology*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002256:Carbonic Anhydrases / Q000378:metabolism*; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000187:drug effects / Q000201:enzymology*; D004269:DNA, Bacterial; D004351:Drug Resistance; D004352:Drug Resistance, Microbial; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C093243:Zam protein, Synechocystis sp.; D002256:Carbonic Anhydrases; D000086:Acetazolamide