ANALYSIS OF A GENOMIC DNA REGION FROM THE CYANOBACTERIUM SYNECHOCOCCUS SP STRAIN PCC7942 INVOLVED IN CARBOXYSOME ASSEMBLY AND FUNCTION

We report on the sequencing and analysis of a 3,557-bp genomic DNA clo ne that is located between 4.8 and 1.2 kilobase pairs (kb) upstream of the rbcL gene and is capable of complementing a class of cyanobacteri um Synechococcus sp. strain PCC7942 mutants requiring a high level of CO2. The upstream 2,704 bp of this sequence is novel, the remaining 85 2 bp having been reported by other workers. Four new open reading fram es (ORFs) have been identified along with putative promoter elements. These ORFs, which could code for proteins of 7, 10.9, 11, and 58 kDa i n size, have been named ORF 64, ccmK, ccmL, and ccmM, respectively. Th e last three have been named ccm genes on the basis that insertional m utagenesis of each produces a phenotype requiring a high level of CO2 (i.e., each produces a lesion in the CO2 concentrating mechanism). The putative gene product for the large ccmM ORF has three internally rep eated regions and also has two possible DNA binding motifs. Two define d mutants in the 3,557-bp region, mutants PVU and P-N, have been more fully characterized. The PVU mutant has a drug marker inserted into th e ccmL gene, and it possesses abnormal rod-shaped carboxysomes. The P- N mutant is a 2.64-kb deletion of DNA from the same position in ccmL t o a region closer to rbcL. This mutant, which has previously been show n to lack carboxysomes and have soluble ribulosebiphosphate carboxylas e/oxygenase activity, has now been shown to have a predominantly solub le carboxysomal carbonic anhydrase activity. Both mutants were found t o possess carboxysomal carbonic anhydrase activities which are below w ild-type levels, and in the P-N mutant this activity appears to be uns table. The results are discussed in terms of the possible interactions of putative ccm gene products in the process of carboxysome assembly and function.