Heterologous expression in E. coli reveals the bicarbonate transporter BicA2 drives carbon uptake in marine Prochlorococcus spp

Abstract The widespread oceanic cyanobacterial Prochlorococcus genus is a major contributor to global carbon fixation, yet mechanisms enabling this lineage to elevate intracellular inorganic carbon as a substrate for photosynthesis remain unresolved. Cyanobacterial CO 2 concentrating mechanisms typically rely on membrane-bound bicarbonate (HCO - ) transporters SbtA1, SbtA2, BicA and BCT1, and CO 2 -to-HCO 3 - conversion uptake systems (CO 2 pumps; NDH-I 3 and NDH-I 4 ), to elevate a cellular HCO 3 - pool for use by Rubisco-containing carboxysomes. Evidence suggests Prochlorococcus harbours carboxysomes with a low-CO 2 -specificity Rubisco, implying a functional CCM dependent on active HCO 3 - uptake. However, canonical CO 2 pumps are absent, leaving distant HCO - transporter homologues, BicA2 and SbtA2, as prime candidates for HCO 3 - transport in this group. Yet these have not been functionally characterised. Here we demonstrate that BicA2 from P. marinus CCMP1375 mediates Na + -dependent HCO 3 - uptake in E. coli , while BicA2 from P. marinus CCMP1986 is inactive in its native form but acquired transport function through a single amino acid substitution during adaptive laboratory evolution. These findings confirm BicA2 as a low-affinity, Na + -dependent bicarbonate transporter with variable flux, revealing a previously uncharacterized CCM component in Prochlorococcus . This mechanistic insight reshapes our understanding of carbon acquisition strategies in the most abundant photosynthetic organism on Earth and highlights evolutionary plasticity in transporter function with implications for global biogeochemical cycles. Highlight This study provides the first mechanistic evidence of bicarbonate transport in Prochlorococcus , revealing functional and evolutionary flexibility in its CO 2 -concentrating mechanism components and reshaping our understanding of carbon acquisition in the most abundant marine photoautotroph.