Exclusive localization of carbonic anhydrase in bacteriocytes of the deep-sea clamCalyptogena okutaniiwith thioautotrophic symbiotic bacteria

Abstract

, and is assumed to facilitate inorganic carbon (C i ) 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 C i supply process to symbionts in the vacuole (symbiosome) in bacteriocytes is essential for symbiosis. Supplementary material available online at

Comparative proteomics of related symbiotic mussel species reveals high variability of host–symbiont interactions

“…Possibly, CA in gill tissue may convert this methanotroph-derived CO 2 to HCO 3 − , thus immobilizing and concentrating it for efficient fixation by the thiotroph. A function of abundant host CA in providing chemoautotrophic symbionts with inorganic carbon has been suggested for several marine invertebrates, including various Bathymodiolus species, Calyptogena species, and Riftia pachyptila [33][34][35]. In B. thermophilus, which lacks a methanotrophic symbiont, CO 2 concentrations might be lower, which would require lower CA abundance, compared with B. azoricus.…”

Metabolic and physiological interdependencies in the Bathymodiolus azoricus symbiosis

“…The B. azoricus CA therefore likely represents a CO 2 -concentrating mechanism, which provides elevated levels of inorganic carbon for fixation by the thiotrophic symbiont ( Figure 4a ). High activity and protein expression of host CA was also described in gills of Bathymodiolus mussels hosting only methanotrophic symbionts, where CA may be involved in the elimination of CO 2 produced as an end product of methane oxidation ( Hongo et al , 2013 ). In addition to the host CA, we also found the CAs of the thiotroph and of the methanotroph expressed (BAT01672, BAZMOX_02303_3), albeit in very low abundances.…”

Electrical Collection of Membrane-intact and Dehydrogenase-positive Symbiotic Bacteria from the Deep-sea Bivalve <i>Calyptogena Okutanii</i>

“…The electrically retrieved bacteria obtained from the C. okutanii gill tissues were affiliated with a single species of symbiotic bacteria, consistent with the results of previous and continuing studies. 4,5,7 In negative control experiments, few or no symbiotic bacteria were attached to the large ITO electrode in the foot tissue homogenates and 70% EtOH-fixed gill tissue homogenates, respectively (Fig. 1C).…”