Extracellular vesicle-linked vitamin B12 acquisition via novel binding proteins in <i>Bacteroides thetaiotaomicron</i>

Vitamin B12 (cobalamin) and related cobamides are essential cofactors for many gut bacteria, yet their acquisition requires complex uptake systems due to limited availability. In the human gut commensal Bacteroides thetaiotaomicron, cobamide uptake is mediated by multiple operons encoding outer membrane proteins, transporters and uncharacterised lipoproteins, some of which are incorporated into bacterial extracellular vesicles (BEVs). Here, we advance the functional and structural understanding of this cobamide acquisition system by examining previously uncharacterised features. Bioinformatic and promoter-reporter analyses revealed four uptake operons, including novel genes we designate btuK, btuJ, btuL and btuX, with evidence for internal promoters and riboswitch regulation. Recombinant expression and binding assays identified ten cobamide-binding proteins, including three novel lipoproteins (BtuK1, BtuJ1 and BtuJ2). Biophysical measurements demonstrated affinities in the nano- to picomolar range, with BtuJ proteins displaying exceptionally tight binding. High-resolution crystal structures of BtuJ1 and BtuJ2 revealed an augmented β-jelly-roll fold, with conserved tyrosine residues forming a 'halo' around the corrin, suggesting a conserved binding mechanism within the IPR027828 protein family. Comparative proteomics of cells and BEVs under cobamide starvation showed selective enrichment of BtuJ and BtuL in BEVs. Functional assays demonstrated that BEV-mediated cobamide uptake depends specifically on BtuJ1 and BtuJ2, whereas BtuL promotes early-phase BEV release. These findings establish the BtuJ proteins as critical BEV-associated cobamide-binding components, provide structural insights into their tight binding, and suggest a model where BEVs act analogously to siderophores, capturing cobamides for delivery to cells. This work highlights the central role of BEVs in microbial nutrient competition.