The small protein SbtC is a functional component of the CO2 concentrating mechanism of cyanobacteria

Abstract

Oxygenic phototrophs fix CO2 via the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) that shows relatively low CO2 affinity and specificity. To circumvent low and fluctuating CO2 concentrations in aquatic systems, cyanobacteria and algae have evolved sophisticated inorganic carbon (Ci) concentrating mechanisms (CCMs). Bicarbonate transporters such as SbtA play a crucial role in the cyanobacterial CCM and hence are tightly regulated at multiple layers. The control of sbtA gene expression and corresponding transporter activity involves the PII-like protein SbtB, whose gene is frequently co-transcribed with sbtA. Here we report on the discovery of a so far non-annotated gene in the model Synechocystis sp. PCC 6803, which is located upstream of the sbtAB operon and encodes the small protein SbtC, composed of 80 amino acids. Presence of SbtC was confirmed by immunoblotting after fusing the sbtC-coding sequence to a Flag-tag. Similar to sbtAB, transcription of the sbtC locus is induced by low CO2 availability but controlled independently. Mutation of the sbtC locus in a wild-type background showed only a mild phenotype even under low CO2, but the diurnal growth was impaired as found before in the mutant {Delta}sbtB. Biochemical analysis provided evidence for a trimeric SbtABC complex in the membrane. Recombinant Synechocystis strains harboring only SbtA as single Ci uptake system with either deleted sbtB or sbtC genes showed that bicarbonate leakage from the cell was strongly elevated in both mutants. Our results provide evidence that SbtC contributes to the formation of the SbtAB complex, thereby regulating bicarbonate exchange at the cytoplasmic membrane. Well-conserved SbtC-like proteins encoded in the neighborhood of sbtAB exist in many cyanobacterial genomes pointing towards an important role in the cyanobacterial CCM.