Expression and Function of Four Carbonic Anhydrase Homologs in the Deep-Sea Chemolithoautotroph Thiomicrospira crunogena

The hydrothermal vent chemolithoautotroph Thiomicrospira crunogena grows rapidly in the presence of low concentrations of dissolved inorganic carbon (DIC) (= CO2 + HCO3- + CO3-2). Its genome encodes alpha-carbonic anhydrase (alpha-CA), beta-CA, carboxysomal beta-like CA (CsoSCA), and a protein distantly related to gamma-CA. The purposes of this work were to characterize the gene products, determine whether they were differentially expressed, and identify those that are necessary for DIC uptake and fixation. When expressed in Escherichia coli, CA activity was detectable for alpha-CA, beta-CA, and CsoSCA but not for the gamma-CA-like protein. alpha-CA and CsoSCA but not beta-CA were inhibited by sulfonamide inhibitors. CsoSCA was also inhibited by dithiothreitol. When grown under DIC limitation in chemostats, T. crunogena transcribed csoSCA more frequently than when ammonia limited, while genes encoding alpha-CA and beta-CA were not differentially transcribed under these conditions. Cell extracts from T. crunogena grown under both DIC-and ammonia-limited conditions had CA activity that was strongly inhibited by sulfonamides, though extracts from nitrogen-limited cells had some CA activity that was resistant, perhaps due to a higher level of beta-CA activity. Based on predictions from the SignalP software program, subcellular location when expressed in E. coli, and carbonic anhydrase assays conducted on intact T. crunogena cells, alpha-CA is located in the periplasm. However, inhibition of alpha-CA by acetazolamide had only a minor impact on rates of DIC uptake or fixation. Conversely, inhibition of CsoSCA with ethoxyzolamide inhibited carbon fixation but not DIC uptake, consistent with this enzyme functioning to facilitate DIC interconversion and fixation within carboxysomes.