Investigating the molecular basis of vitamin B12 uptake in Chlamydomonas reinhardtii

Approximately half of all algal species are dependent on an external supply of the corrinoid cobalamin (vitamin B12) for growth. Algal phyla differ in the proportion of species dependent on B12. Extremes of this are the Chlorophyta (30% B12 dependent) and the Dinophyta (91% B12 dependent). B12 is only synthesised by prokaryotic organisms, therefore, algae must rely on an external source of this nutrient. Little is known about how B12 is taken up in algae at the molecular level. One protein that has been characterised is Cobalamin Acquisition Protein 1 (CBA1) - identified in the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana - which was found to increase the B12 uptake rate when overexpressed. However, no proteins required for B12 uptake have been identified in green algae. In this study, the model green alga Chlamydomonas reinhardtii was used to investigate B12 uptake. Candidate genes related to B12 transport and binding were identified in C. reinhardtii from sequence similarity to genes known to bind or transport B12 in other organisms. Six genes were tested in vivo using knockdown and knockout strains. Lines with disruptions in Cre02.g081050, which encodes the C. reinhardtii CBA1 homologue (CrCBA1), were unable to take up B12, and complementation lines restored the ability to take up B12. To assess the subcellular location of CrCBA1, a line was made with a fluorescent tag at the C-terminus of CrCBA1. Confocal microscopy was used to image this line and showed its likely membrane association. To exert control over B12 uptake, a thiamine-responsive riboswitch was used to control CrCBA1 expression. In the absence of thiamine, the line could take up B12 to a similar extent as the wild type strain, but addition of thiamine impaired the ability of this line to take up B12. The presence of CBA1 was assessed in algae more generally, and sequences similar to CBA1 were identified in many algal species belonging to different phyla. This indicated that CBA1 is much more prevalent in nature than previously thought. The diversity of CBA1 sequences allowed highly conserved residues to be identified, which provides a promising route for future investigation. A forward genetic screen was undertaken to search for other proteins important in B12 uptake, using insertional mutagenesis. A reporter line was made with a paromomycin-resistance marker under control of a B12-repressible promoter, so that it showed repression of growth in paromomycin under high media vitamin B12 concentrations (+P+B12). As this line was thought to respond to intracellular B12 only, the screen was expected to detect mutants deficient in taking up B12 by selecting for those that grew in +P+B12. Insertional mutagenesis of the reporter strain was performed, and 8 insertional mutant lines were identified. One of these lines (1.G2) was found to be unable to take up B12. This was confirmed by incubating a fluorescent B12 analogue (B12-BODIPY) with algal cultures: 1.G2 did not show a B12-BODIPY signal that co-localised with the cell, whereas in lines able to take up B12, a B12-BODIPY signal with multiple circular foci within the cell was observed. The site of the transgene insertion was mapped using DNA sequencing to Cre12.g508644, however, knockout strains of this locus were able to take up B12, indicating that this gene was not responsible for B12 uptake. CRISPR/Cpf1 lines targeting Cre12.g508644 were made, however, no lines with successful genome editing of Cre12.g508644 were identified. Higher coverage DNA sequencing of 1.G2 was performed, which revealed that in addition to Cre12.g508644, there was also disruption of CrCBA1, by a putative transposon that was previously uncharacterised in C. reinhardtii. B12 transport was restored by transformation with CrCBA1, demonstrating the causality of this insertion. To identify genes that are differentially expressed in the presence of B12, RNA sequencing was performed. Seven genes were identified, 5 of which had not been associated with B12 before. RNA sequencing of 1.G2 found it had extensive transcriptomic differences compared to the background line. This provided new candidate genes to assess for association with CrCBA1 and B12 transport that could be investigated in future studies. Collectively, this work has contributed to a greater understanding of B12 acquisition in C. reinhardtii and algae more generally, and has provided algal lines, insight and techniques for future research in this area.

BBSRC funded DTP studentship