Cloning and characterization of the genes required for inorganic carbon transport of Synechocystis PCC6803

Transformation of the high CO2-requiring mutants of Synechocystis PCC6803 defective in inorganic carbon (Ci) transport (RKa and RKb) by wild type (WT) DNA libraries restored their ability to grow under air levels of CO2. Two clones (PK-1 and HP-1), which complement RKa and RKb, respectively, were isolated from the libraries. PK-1 contained an 11.8-kilobase pair (kbp) DNA insert. The sequence of amino acid coded in the DNA in the region of the mutation showed an extensive homology to that of the ndh2 gene product of liverwort chloroplasts, which is suspected to be the subunit 2 of NADH dehydrogenase. Based on the result, we designated the gene mutated in RKa as ndh2. Inactivation of the ndh2 gene in the WT cells by inserting an aminoglycoside-3′-phosphotransferase gene generated a mutant (M57) that was unable to grow under low CO2 conditions. HP-1 contained a 5.4-kbp DNA insert. Sequencing of nucleotides in the region of the mutation revealed an open reading frame that codes a hydrophobic protein that consists of 80 amino acids. Insertional inactivation of this putative Ci transport gene, designated ictA, generated a high CO2-requiring mutant (M9). All these mutants (RKa, RKb, M9, and M57) showed very low activity of CO2 uptake into the intracellular Ci pools. The activity of HCO3− uptake was negligibly low in RKb, M9, M57 and high CO2-grown cells of RKa, and was about 10% the activity of wild type cells in low CO2-adapted cells of RKa. Key words: CO2-concentrating mechanism, inorganic carbon transport, Synechocystis PCC6803, mutant, NADH dehydrogenase, insertional inactivation.