Using gene complementation to identify a SulP-family bicarbonate transporter in an N2-fixing cyanobacterial endosymbiont of an open ocean diatom

Abstract

ABSTRACTDiatom-Diazotrophic Associations (DDAs) contribute significantly to new and primary production in the world’s oceans, yet the understanding of how production is sustained is poorly resolved. These symbioses involve diatoms and N2-fixing, heterocyst-forming cyanobacteria of the genusRichelia, both partners being photosynthetic.Richelia euintracellularisresides in the cytoplasm ofHemiaulus hauckii, whereasRichelia intracellularisis periplasmic inRhizosolenia clevei. In the ocean, bicarbonate is taken up by phytoplankton to provide CO2for photosynthesis. The genomes of bothRicheliaendobionts (ReuHH01 and RintRC01, respectively) contain genes encoding SulP-family proteins, which are oxyanion transporters. To study the possible involvement of these transporters in bicarbonate uptake, we used complementation of aSynechocystissp. PCC 6803 mutant with its five CO2uptake systems inactivated, which is unable to grow in air levels of CO2. Three genes from RintRC01 and one gene and a DNA fragment containing four partial gene sequences from ReuHH01 were chemically synthesized, cloned under the control of a strong gene promoter and incorporated in the chromosome of theSynechocystismutant. One gene from RintRC01, RintRC_3892, complemented theSynechocystismutant to grow with air levels of CO2or with low bicarbonate concentrations. The complemented strain showed strong sodium-dependent, low affinity bicarbonate uptake, which, together with phylogenetic analyses, identified RintRC_3892 as a BicA protein. Additionally, RintRC_3892 transcripts were consistently detected in environmental samples from three ocean basins. No evidence for a bicarbonate transporter was found, however, for ReuHH01, suggesting different strategies for inorganic carbon uptake in the periplasmic and cytoplasmic endobionts.