Isolation of ccmKLMN genes from the marine cyanobacterium, Synechococcus sp PCC7002 (Cyanobacteria), and evidence that CcmM is essential for carboxysome assembly

A high CO2 requiring mutant of the marine cyanobacterium Synechococcus PCC7 002 was generated using a random gene-tagging procedure. This mutant demons trated a reduced photosynthetic affinity for inorganic carbon (C-i) and acc umulated high internal levels of C-i that could not be used for photosynthe sis. Analysis of the mutant genomic DNA showed that the mutagenesis had dis rupted a cluster of genes involved in the cyanobacterial CO2 concentrating mechanism (CCM), the so-called cola genes, These characteristics are consis tent with a cyanobacterial mutant with defects in carboxysome assembly and/ or functioning. Further genomic analyses indicated that the genes of the Sy nechococcus PCC7002 operon, ccmKLMN, are structurally similar to those of t wo closely related cyanobacteria, Synechococcus PCC7942 and Synechocystis P CC6803, The Synechococcus PCC7002 ccmM gene, which encodes a polypeptide wi th a predicted size of 70 kDa, was the direct target of the mutagenesis eve nt. The CcmM protein has two distinct regions: an N-terminal region that sh ows similarity to an archaeon gamma carbonic anhydrase and a C-terminal reg ion that contains repeated domains demonstrating sequence similarity to the small subunit of Rubisco, Physiological analysis of a ccmM-defined mutant showed that these cells were essentially identical to the original mutant; they required high CO2 concentrations for growth, they had a low photosynth etic affinity for C-i, and they internalized C-i to high levels. Moreover, ultrastructural examination showed that both the original and the defined m utants lack carboxysomes. Thus, our results demonstrate that the ccmM gene of Synechococcus PCC7002 encodes a polypeptide that is essential for carbox ysome assembly and therefore for proper functioning of the cyanobacterial C CM.