Restoration of the wild-type locus in an RuBP carboxylase/oxygenase mutant of Synecbocystis PCC 6803 via targeted gene recombination

The interaction between homologous DNA sequences, distant from each other in the chromosome, was examined in the cyanobacterium Synechocystis PCC 6803. Most of the rbcL gene encoding the large subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) was duplicated in the genome by a targeted insertion of a 3′-truncated gene copy into the psbA-I locus. Both rbcL genes, in the psbA-I region and at the rbc locus, were non-functional; The former due to the 3′ truncation, and the latter due to a deletion in the 5′-region (creating a 5′ truncation) and a mutation associated with an insertion of the Rhodospirillum rubrum rbc gene, yielding a high-CO₂-requiring mutant ('cyanorubrum'). The 3′ and the 5′ truncated rbcL genes were linked to chloramphenicol and kanamycin resistance markers, respectively. Decreasing the kanamycin selective pressure concomitantly with exposure of the double resistance mutant to air, resulted in air-growing colonies. Analysis of their genomes, Rubisco proteins, and their ultrastructure revealed: 1) Reconstitution of a full-length cyanobacterial rbcL gene at the rbc locus; 2) simultaneous synthesis of the cyanobacterial (L₈S₈) and R. rubrum L₂) enzymes in meroploids containing both mutated and reconstituted rbcL genes; 3) reappearance of carboxysomes. Our results indicate extensive recombinatorial interactions between the homologous sequences at both loci leading to reconstitution of the cyanobacterial rbcL gene.