Characterization of a mutant lacking carboxysomal carbonic anhydrase from the cyanobacterium Synechocystis PCC6803

A fully-segregated mutant (ccaA::kan(R)) defective in the ecaA gene, encoding a carboxysome-associated beta-carbonic anhydrase (CA), was generated in the cyanobacterium Synechocystis sp. PCC6803 by insertional mutagenesis. Immunoblot analysis indicated that the CcaA polypeptide was absent from the carboxysome-enriched fraction obtained from ccaA::kan(R), but was present in wild-type (WT) cells. The carboxysome-enriched fraction isolated from WT cells catalyzed O-18 exchange between (CO2)-C-13-O-18 and H2O, indicative of CA activity, while ccaA::kan(R) carboxysomes did not. Transmission and immunogold electron microscopy revealed that carboxysomes of WT and ccaA::kan(R) were of similar size, shape and cellular distribution, and contained most of the cellular complement of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The ccaA::kan(R) cells were substantially smaller than WT and were unable to grow autotrophically at air levels of CO2. However, cell division occurred at near-WT rates when ccaA::kan(R) was supplied with 5% CO2 (v/v) in air. The apparent photosynthetic affinity of the mutant for inorganic carbon (Ci) was 500-fold lower than that of WT cells although intracellular Ci accumulation was comparable to WT measurements. Mass spectrometric analysis revealed that the CA-like activity associated with the active CO2 transport system was retained by ccaA::kan(R) cells and was inhibited by H2S, indicating that CO2 transport was distinct from the CcaA-mediated dehydration of intracellular HCO3-. The data suggest that the ccaA mutant was unable to efficiently utilize the internal Ci pool for carbon fixation and that the high-CO2-requiring phenotype of ccaA::kan(R) was due primarily to an inability to generate enough CO2 in the carboxysomes to sustain normal rates of photosynthesis.