The 5'-flanking region of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase is crucial for growth of the cyanobacterium Synechococcus sp. strain PCC 7942 at the level of CO2 in air.

Transformation of the high-CO2-requiring mutants (hcr) O221 and E1 derived from the cyanobacterium Synechococcus sp. strain PCC 7942 by a wild-type DNA library restored their ability to grow at the level of CO2 in air. A plasmid (pE12) containing a 10-kilobase DNA insert was rescued from a O221 heterogenote and proved to transform both O221 and E1 to the wild-type phenotype. The capacity of the pE12 subclones to confer the wild-type phenotype to O221 transformants enabled the mapping of the mutation in O221 (designated hcrO221) within a 232-base-pair PstI-BstXI DNA restriction fragment. Sequence analysis revealed two open reading frames (ORFs) at positions -1745 to -1262 (ORFI) and -1218 to -393 (ORFII) upstream of the rbcL gene. A 3-kilobase PstI fragment of O221 was cloned, and hcrO221 was found to be a point mutation within the PstI-BstXI region -1309 nucleotides upstream of the rbcL gene. The significance of this flanking region for adaptation to air levels of CO2 was further demonstrated by the generation of new hcr mutants following insertional inactivation of wild-type DNA in the BstXI site. Electron microscopy revealed aberrant carboxysome structures in growing cells of the hcr mutants, a defect that was possibly related to the mutation, since transformation with pE12 derivatives restored the carboxysome structure to normal.