Cloning and inactivation of a gene essential to inorganic carbon transport of synechocystis PCC6803

Abstract

A clone (HP-1) which transforms the high CO2-requiring mutant (RKb) of Synechocystis PCC6803 defective in inorganic carbon transport to the wild-type (WT) phenotype was isolated from a WT genomic library. The clone contained a 5.4 kilobase-pair DNA insert. Complementation tests with subclones derived from HP-1 allowed the mutation in RKb to be located within 141 base-pair nucleotides. Sequencing of nucleotides in this region revealed an open reading frame encoding a hydrophobic protein consists of 80 amino acids. A defined mutant (M9) constructed by inactivating this putative inorganic carbon transport gene, designated ictA, was unable to transport CO2 and HCO3- into the intracellular inorganic carbon pool. Cloning and sequence analysis of the respective RKb gene revealed a base substitution which generates a stop codon in the middle of ictA.