title: Exclusive localization of carbonic anhydrase in bacteriocytes of the deep-sea clam Calyptogena okutanii with thioautotrophic symbiotic bacteria.
abstract: Deep-sea Calyptogena clams harbor thioautotrophic intracellular symbiotic bacteria in their gill epithelial cells. The symbiont fixes CO2 to synthesize organic compounds. Carbonic anhydrase (CA) from the host catalyzes the reaction CO2 + H2O ↔ HCO3(-) + 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 and LC-MS/MS. We found 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, which showed 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 non-symbiotic 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. This indicates that the Ci supply process to symbionts in the vacuole (symbiosome) in bacteriocytes is essential for symbiosis.
authors.affiliation: Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Hongo
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Shigeru
authors.initials: S
authors.lastname: Shimamura
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Takaki
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Uematsu
authors.forename: Takashi
authors.initials: T
authors.lastname: Toyofuku
authors.forename: Hisako
authors.initials: H
authors.lastname: Hirayama
authors.forename: Ken
authors.initials: K
authors.lastname: Takai
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Nakazawa
authors.forename: Tadashi
authors.initials: T
authors.lastname: Maruyama
authors.forename: Takao
authors.initials: T
authors.lastname: Yoshida
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D049872:Bivalvia / Q000166:cytology / Q000201:enzymology* / Q000382:microbiology; D002256:Carbonic Anhydrases / Q000032:analysis* / Q000378:metabolism; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005880:Gills / Q000201:enzymology; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000737:chemistry; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D017423:Sequence Analysis, RNA; D013559:Symbiosis; D053719:Tandem Mass Spectrometry
chemical_list: D012333:RNA, Messenger; D002256:Carbonic Anhydrases
keywords: bacteriocyte; chemosynthetic ecosystem; gill; inorganic carbon uptake; intracellular; symbiosis