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

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.

Differences in gene expression of the ciliate Paramecium caudatum caused by endonuclear symbiosis with Holospora obtusa, revealed using differential display reverse transcribed PCR.

We identified six genes of Paramecium caudatum, which differentially expressed in Holospora obtusa-bearing and H. obtusa-free cells using differential display reverse transcribed PCR (DDRT-PCR). Northern blot analyses revealed that two of the genes, CA10-3 and CA20-2, were expressed extensively in the H. obtusa-free cell, while the other four, AS16-1, CS14, CS21 and CA17-1, were expressed more in the H. obtusa-bearing cell. Putative amino acid sequences of CA10-3, AS16-1 and CA17-1 showed high homologies with known genes related to intracellular signaling, transcription and aerobic metabolism. CS14 and CS21 also showed homologies with some genes whose products are still functionally unknown, but CA20-2 encoded a novel protein. We show in this study that H. obtusa alters multiple gene expression of the host after establishing endosymbiosis.