The conserved protein CBA1 is required for vitamin B₁₂uptake in different algal lineages

Abstract Microalgae play an essential role in global net primary productivity and global biogeochemical cycling, but despite their phototrophic lifestyle, over half of algal species depend on a supply of the corrinoid vitamin B 12 (cobalamin) for growth. This essential organic micronutrient is produced only by a subset of prokaryotic organisms, which implies that for algal species to use this compound, they must first acquire it from external sources. Previous studies have identified protein components involved in vitamin B 12 uptake in bacterial species and humans. However, little is known about how it is taken up in algae. Here, we demonstrate the essential role of a protein, CBA1 (for cobalamin acquisition protein 1), in B 12 uptake in Phaeodactylum tricornutum , using CRISPR-Cas9 to generate targeted knockouts, and in Chlamydomonas reinhardtii , by insertional mutagenesis. In both cases, CBA1 knockout lines are no longer able to take up exogenous vitamin B 12 . Complementation of the C. reinhardtii mutants with the wildtype CBA1 gene restores B 12 uptake, and regulation of CBA1 expression via a riboswitch element can be used to control the phenotype. When visualised by confocal microscopy, a YFP-fusion with C. reinhardtii CBA1 shows association with membranes. A bioinformatics analysis found that CBA1-like sequences are present in all the major eukaryotic phyla. Its presence is correlated with B 12 -dependent enzymes in many, although not all, taxa, suggesting CBA1 has a conserved role. Our results thus provide insight into the molecular basis of algal B 12 acquisition, a process that likely underpins many interactions in aquatic microbial communities. One sentence summary Knockout mutants and physiological studies demonstrate that the CBA1 protein is essential for uptake of vitamin B 12 in both Chlamydomonas reinhardtii and the unrelated Phaeodactylum tricornutum .