Characterization of a mutant lacking carboxysomal carbonic anhydrase from the cyanobacterium Synechocystis PCC6803.
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.
So
Anthony K C
AK
Department of Botany, University of Toronto at Mississauga, Ontario, Canada.
John-McKay
Meryl
M
Espie
George S
GS
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002244:Carbon / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism* / Q000494:pharmacology; D002247:Carbon Isotopes; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D006862:Hydrogen Sulfide / Q000494:pharmacology; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D010103:Oxygen Isotopes; D010788:Photosynthesis / Q000187:drug effects / Q000502:physiology; D018087:Plastids / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism; D013997:Time Factors
D002247:Carbon Isotopes; D010103:Oxygen Isotopes; D002245:Carbon Dioxide; D002244:Carbon; C101625:RbcL protein, plastid; D012273:Ribulose-Bisphosphate Carboxylase; D002256:Carbonic Anhydrases; D010100:Oxygen; D006862:Hydrogen Sulfide