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Title:          Conserved cobalamin acquisition protein 1 is essential for vitamin B12 uptake in both Chlamydomonas and Phaeodactylum
Subject:        DOI: 10.1093/plphys/kiad564; Plant Physiology, 194, 2, 2023-10-21.; Abstract: Microalgae play an essential role in global net primary productivity and global biogeochemical cycling. Despite their phototrophic lifestyle, over half of algal species depend for growth on acquiring an external supply of the corrinoid vitamin B12 (cobalamin), a micronutrient produced only by a subset of prokaryotic organisms. Previous studies have identified protein components involved in vitamin B12 uptake in bacterial species and humans. However, little is known about its uptake in algae. Here, we demonstrate the essential role of a protein, cobalamin acquisition protein 1 (CBA1), in B12 uptake in Phaeodactylum tricornutum using CRISPR-Cas9 to generate targeted knockouts and in Chlamydomonas reinhardtii by insertional mutagenesis. In both cases, CBA1 knockout lines could not take up exogenous vitamin B12. Complementation of the C. reinhardtii mutants with the wild-type CBA1 gene restored B12 uptake, and regulation of CBA1 expression via a riboswitch element enabled control of the phenotype. When visualized by confocal microscopy, a YFP-fusion with C. reinhardtii CBA1 showed association with membranes. Bioinformatics analysis found that CBA1-like sequences are present in all major eukaryotic phyla. In algal taxa, the majority that encoded CBA1 also had genes for B12-dependent enzymes, suggesting CBA1 plays a conserved role. Our results thus provide insight into the molecular basis of algal B12 acquisition, a process that likely underpins many interactions in aquatic microbial communities.
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Author:         Andrew P. Sayer
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      Title: Conserved cobalamin acquisition protein 1 is essential for vitamin B12 uptake in both Chlamydomonas and Phaeodactylum
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        Title: Introduction
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       Item: 
        Title: Results
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         Item: 
          Title: P. tricornutum CBA1 knockout lines do not take up B12
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         Item: 
          Title: Insertional mutagenesis identified the C. reinhardtii homologue of CBA1
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         Item: 
          Title: CrCBA1 CLiP mutant is unable to take up B12 and is complemented by the WT CrCBA1 gene
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          Title: CrCBA1 shows an association with membranes and is highly upregulated under B12-deprivation
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         Item: 
          Title: Widespread distribution of CBA1 in algae
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        Title: Materials and methods
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          Title: Organisms and growth conditions
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          Title: Algal B12-uptake assay
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          Title: Generating P. tricornutum CBA1 knockout lines using CRISPR-Cas9
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          Title: Construct assembly and C. reinhardtii transformation
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          Title: Confocal laser scanning microscopy
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          Title: Reverse transcription quantitative PCR
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    Title: Conserved cobalamin acquisition protein 1 is essential for vitamin B12 uptake in both Chlamydomonas and Phaeodactylum
    Author: Andrew P. Sayer
    Subject: DOI: 10.1093/plphys/kiad564; Plant Physiology, 194, 2, 2023-10-21.; Abstract: Microalgae play an essential role in global net primary productivity and global biogeochemical cycling. Despite their phototrophic lifestyle, over half of algal species depend for growth on acquiring an external supply of the corrinoid vitamin B12 (cobalamin), a micronutrient produced only by a subset of prokaryotic organisms. Previous studies have identified protein components involved in vitamin B12 uptake in bacterial species and humans. However, little is known about its uptake in algae. Here, we demonstrate the essential role of a protein, cobalamin acquisition protein 1 (CBA1), in B12 uptake in Phaeodactylum tricornutum using CRISPR-Cas9 to generate targeted knockouts and in Chlamydomonas reinhardtii by insertional mutagenesis. In both cases, CBA1 knockout lines could not take up exogenous vitamin B12. Complementation of the C. reinhardtii mutants with the wild-type CBA1 gene restored B12 uptake, and regulation of CBA1 expression via a riboswitch element enabled control of the phenotype. When visualized by confocal microscopy, a YFP-fusion with C. reinhardtii CBA1 showed association with membranes. Bioinformatics analysis found that CBA1-like sequences are present in all major eukaryotic phyla. In algal taxa, the majority that encoded CBA1 also had genes for B12-dependent enzymes, suggesting CBA1 plays a conserved role. Our results thus provide insight into the molecular basis of algal B12 acquisition, a process that likely underpins many interactions in aquatic microbial communities.
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  Checksum: 8618ccce
   Type: CRC32
  Checksum: 1c43d69fa4fcccaa3f2548c5a0b5b861
   Type: MD5
  Checksum: d29a2642bf39ed44c78133cb6d77c19f51da104a
   Type: SHA-1
  Checksum: b07bce6a19ad87dd1aad6c73476f06934db30b0bc123c22c5ff697cddfe5626c
   Type: SHA-256