Functional characterisation of bicarbonate transporters from the cyanobacterial SbtA2 family and subsequent expression in tobacco

Abstract Cyanobacteria rely on bicarbonate (HCO3-) as the primary inorganic carbon (Ci) source for photosynthesis in aquatic environments. To use of this Ci source, cyanobacteria employ CO2 concentrating mechanisms (CCMs) that elevate cytoplasmic HCO3- via plasma membrane transporters, enhancing carboxylation by carboxysomal Rubisco. The sodium-dependent SbtA1 transporter family is well-characterized in freshwater cyanobacteria, but the related SbtA2 family, prevalent in marine α-cyanobacteria, remains uncharacterised. Here, we report functional characterisation of SbtA2 homologues from marine Synechococcus spp., which exhibit high Ci uptake flux with apparent chloride dependence and intermediate HCO3- affinity (Km ≈ 150 µM), when assessed in E. coli. SbtA2 achieved internal Ci accumulation up to 24 mM within 30 seconds. Co-expression with the putative regulator SbtB2 reduced uptake activity, suggesting a regulatory role for this protein. These findings indicate that SbtA2 transporters contribute significantly to carbon acquisition in marine cyanobacteria. Given potential to enhance CO2 supply to Rubisco in C3 plants, we targeted SbtA2 to the tobacco chloroplast inner envelope membrane; however, this did not improve photosynthesis or growth. Our results highlight the functional diversity of cyanobacterial Ci transporters and suggest that additional components may be required for effective transfer of such systems into plant chloroplasts. Highlight SbtA2 bicarbonate transporters from some marine α-cyanobacteria are high flux transporters with an unusual chloride-dependence and show intermediate uptake affinity when expressed in E. coli. Initial attempts failed to demonstrate enhanced photosynthesis when directed to tobacco chloroplasts. Competing Interest Statement The authors have declared no competing interest. Footnotes Addition of Supplementary data files. Abbreviations - CA - carbonic anhydrase - CAfree - pecialized E. coli strain that lacks CAs - CCM - CO2-concentrating mechanism - CO2 - carbon dioxide - Ci - inorganic carbon - Cint - intercellular CO2 - HCO3- - bicarbonate - IEM - inner envelope membrane - IPTG - isopropyl β-D-1-thiogalactopyranoside LB Luria-Bertani - SbtA - sodium-dependent bicarbonate transporter