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

Abstract.

A fully-segregated mutant (ccaA::kan^R) defective in the ccaA gene, encoding a carboxysome-associated β-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 ¹⁸O exchange between ¹³C¹⁸O₂ 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.