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

This review arose out of a course for graduate students in the life sciences at UCSF, “Peer Review in the Life Sciences,” which aims to introduce junior scientists to peer review in a critical yet constructive way. The students selected preprints to review, led discussions of them, drafted reviews, and revised them based on feedback from peers and instructors. The two student authors of this review chose to remain anonymous.

Summary:

This article sets out to discover the protein(s) involved in vitamin B₁₂ uptake in microalgae, which is poorly understood when compared to bacterial or mammalian uptake processes. Microalgae play essential roles in our ecosystem, thus understanding how they acquire the essential vitamin B₁₂  has ecological importance. Prior studies have demonstrated that a protein called CBA1 (cobalamin acquisition protein 1) is upregulated in times of B₁₂ scarcity and increases cellular uptake of B₁₂ when overexpressed; however, bioinformatics searches have not revealed homologous proteins in other species. This paper’s goals are to demonstrate the necessity of CBA1 in B₁₂ uptake for the diatom species Phaeodactylum tricornutum and to identify proteins required for B₁₂ uptake in another model algae, Chlamydomonas reinhardtii, ideally extending these findings to other, non-algae species.

The major successes of this paper include confirming the necessity of CBA1 in microalgal B₁₂ uptake in P. tricornutum through a CRISPR knockout of the gene, extending prior work and demonstrating the necessity of this gene for vitamin B₁₂ uptake in this species. Additionally, the authors rigorously demonstrated the existence of a similar gene required for B₁₂ uptake in C. reinhardtii and found CBA1 homologs in other species with a hidden Markov model (HMM) search. 

The authors’ rationale in designing their studies and drawing conclusions from their results was demonstrated clearly. However, as outsiders to the field, we did not have the required context to understand the decision to focus on P. tricornutum and C. reinhardtii in this work, so including a brief explanation of these species’ relevance to the field may help place these results within the context of the field.

The authors’ HMM search identified many CBA1 homologs that failed to be identified using conventional blast searches, implying that although these species share a  conserved mechanism of vitamin B₁₂ uptake, the sequences are quite diverged. Combined with their identification of C. reinhardtii genes that are homologous to human B₁₂ receptor-mediated endocytosis genes and structurally similar to a B₁₂-binding protein in Escherichia coli, this paper hints at a specific cellular mechanism for B₁₂ uptake and the role of CBA1 in this process. 

Major points:

1. It would be helpful to have additional justification for the organisms included in this study, as the similarities and differences between P. tricornutum and C. reinhardtii and how representative they are of algal diversity are unclear. More explanation on why these specific organisms were chosen, how they are model systems for larger families of algae, or why demonstrating the conserved role of CBA1 in these lineages is significant could help clarify the scope of the study and its impact on the field.

2. The authors selected for a rare event wherein a transposon disrupted the real CBA1 at the same time that they were conducting their insertional mutagenesis screen. The explanation of this series of events was somewhat confusing, partially because of our own pre-existing biases regarding what the results of an insertional mutagenesis screen should look like. It could be worthwhile to discuss the rarity of this occurrence when they first explain their results because their mention of the Cre12.g508644 locus adds an additional layer of complexity.

Minor points: 

1. The important role microalgae play in the ecosystem is clearly stated, but explicitly describing the overall significance of understanding B₁₂ uptake in microalgae would be helpful in contextualizing this research to a broader audience. 

2. The insertional mutagenesis workflow was somewhat confusing, so we recommend including a schematic illustrating the technique and selection markers used. 

3. In the pHyg3 plasmid, it is unclear why a β2-tubulin promoter was used to control the hygromycin resistance gene rather than the P_METE promoter. 

4. In Figure 3d samples IM4 and IM4::pAS_C2, why is the total amount of B₁₂ greater than the amount of B₁₂ added at the beginning of the assay? Similarly, in Figure 1c, the total amount of B₁₂ in the ΔCBA1-1, ΔCBA1-2, and ΔCBA1-3 conditions is lower than the amount added.

5. In the insertional mutagenesis experiment using the TPP-repressible riboswitch (Fig. 4), the complementation with pAS_C2 and pAS_C3 is effective in demonstrating that CrCBA1 is necessary for B₁₂ uptake. An additional complementation experiment with PtCBA1 would further demonstrate the functional conservation of CBA1 function across algal lineages.

6. In the B₁₂ deprivation experiment (Fig. 6), it is unclear what the starting concentration of B₁₂ was before it was removed. 

7. In Figure 6a, smaller increments on the x-axis would be helpful. A data point at 6 hours is mentioned, but it is difficult to identify because the axis jumps from 0 to 40 hours. 

8. Further explanation about the development of the authors’ hidden Markov model would be helpful in understanding its advantages over previous bioinformatics tools. For example, how would results from PSI-BLAST compare? Was the new model algorithm trained iteratively? 

9. In Figure 8a, assignments of Groups I-IX are unclear. Why do groups include varying amounts of total residues? 

10. Figure 8b is somewhat unclear. Though alignment of the CrCBA1 structure prediction to E. coli BtuF is discussed, an image of this alignment would be helpful for making comparisons between CrCBA1 and BtuF. For example, does the conserved cluster of CrCBA1 upper alpha helix residues align with a motif in BtuF? Additionally, we recommend looking at an AlphaFold prediction of the CrCBA1 structure. It would be an interesting case to compare to the Phyre2 prediction and study the sensitivity of sequence- versus structure-based homology searches. 

11. The authors speculate that CBA1 may be a B₁₂-binding protein, so a short statement about the significance of a B₁₂-binding protein in the mechanism of B₁₂ uptake in other organisms would be useful clarification. 

Competing interests

The author declares that they have no competing interests.