Exclusive localization of carbonic anhydrase in bacteriocytes of the deep-sea clam Calyptogena okutanii with thioautotrophic symbiotic bacteria

Summary Deep-sea Calyptogena clams harbor thioautotrophic intracellular symbiotic bacteria in their gill epithelial cells. The symbiont fixes CO 2 to synthesize organic compounds. Carbonic anhydrase (CA) of the host catalyzes the reaction CO 2 + H 2 O ↔ HCO 3 - + H + , and is assumed to facilitate inorganic carbon (Ci) uptake and transport to the symbiont. However, the localization of CA in gill tissue remains unknown. We therefore analyzed mRNA sequences, proteins and CA activity in Calyptogena okutanii using expression sequence tag, SDS-PAGE with LC-MS/MS. It was shown that acetazolamide-sensitive soluble CA was abundantly expressed in the gill tissue of C. okutanii, and the enzyme was purified by affinity chromatography. Mouse monoclonal antibodies against the CA of C. okutanii were used in Western blot analysis and immunofluorescence staining of the gill tissues of C. okutanii , showing that CA was exclusively localized in the symbiont-harboring cells (bacteriocytes) in gill epithelial cells. Western blot analysis and measurement of activity showed that CA was abundantly (26-72% of total soluble protein) detected in the gill tissues of not only Calyptogena clams but also deep-sea Bathymodiolus mussels that harbor thioautotrophic or methanotrophic symbiotic bacteria, but was not detected in a nonsymbiotic mussel, Mytilus sp. The present study showed that CA is abundant in the gill tissues of deep-sea symbiotic bivalves and specifically localizes in the cytoplasm of bacteriocytes of C. okutanii . These indicate that the Ci supply process to symbionts in the vacuole (symbiosome) in bacteriocytes is essential for symbiosis.