CATALYTIC EXCHANGE OF O-18 FROM (CO)-C-13-O-18-LABELED CO2 BY WILD-TYPE CELLS AND ECAA, ECAB, AND CCAA MUTANTS OF THE CYANOBACTERIA SYNECHOCOCCUS PCC7942 AND SYNECHOCYSTIS PCC6803

Expression constructs carrying the ecaA, ecaB, and ccaA genes from the cyanobacteria Synechococcus PCC7942 and Synechocystis PCC6803 were ge nerated and each transformed into Escherichia coli. Lysates from cells expressing recombinant protein were prepared and assayed using mass s pectrometry for their ability to catalyze the exchange of O-18 from (C O2)-C-13-O-18 to H2O. No carbonic anhydrase (CA) activity was detected from the cell lysates containing recombinant EcaA or EcaB proteins, w hereas the lysate harbouring the CcaA polypeptide clearly accelerated the rate of O-18 exchange. An ecaA deletion mutant in Synechococcus an d insertionally inactivated ecaB and ccaA mutants in Synechocystis wer e generated and similarly assessed for CA activity. All mutants displa yed a transient, ethoxyzolamide-sensitive, CA-like catalysis that was also exhibited by wild-type cells. The CcaA-deficient mutant showed a reduced capacity to exchange O-18 out of (CO)-C-13-O-18-labelled CO2 i n the light as well as an absolute requirement for high C-i for growth , reflecting the importance of the carboxysomal CA to the operation of the cyanobacterial CO2-concentrating mechanism. No detectable differe nces in O-18 exchange patterns, CO2 or HCO3- transport, or steady-stat e growth were observed between the ecaA and ecaB mutants and wild-type cells, indicating that neither EcaA nor EcaB play an essential cataly tic role in the functioning of the CO2-concentrating mechanism.