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

Planta. 2002 Jan;214(3):456-67. doi: 10.1007/s004250100638.

Abstract

A fully-segregated mutant (ccaA::kanR) defective in the ccaA 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::kanR, 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::kanR carboxysomes did not. Transmission and immunogold electron microscopy revealed that carboxysomes of WT and ccaA::kanR 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::kanR 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::kanR 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::kanR 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::kanR was due primarily to an inability to generate enough CO2 in the carboxysomes to sustain normal rates of photosynthesis.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Biological Transport
  • Carbon / metabolism
  • Carbon Dioxide / metabolism*
  • Carbon Dioxide / pharmacology
  • Carbon Isotopes
  • Carbonic Anhydrases / genetics
  • Carbonic Anhydrases / metabolism*
  • Cells, Cultured
  • Cyanobacteria / enzymology*
  • Cyanobacteria / genetics
  • Cyanobacteria / ultrastructure
  • Hydrogen Sulfide / pharmacology
  • Immunohistochemistry
  • Microscopy, Electron
  • Molecular Sequence Data
  • Mutation
  • Oxygen / metabolism
  • Oxygen Isotopes
  • Photosynthesis / drug effects
  • Photosynthesis / physiology
  • Plastids / metabolism
  • Ribulose-Bisphosphate Carboxylase / metabolism
  • Time Factors

Substances

  • Carbon Isotopes
  • Oxygen Isotopes
  • Carbon Dioxide
  • Carbon
  • RbcL protein, plastid
  • Ribulose-Bisphosphate Carboxylase
  • Carbonic Anhydrases
  • Oxygen
  • Hydrogen Sulfide