BtuJ1, a Surface-exposed B12-binding Protein in Bacteroidota, Functions as an Extracellular Vitamin Reservoir That Enhances Fitness

The acquisition of vitamin B₁₂ and related cobamides is a key determinant for the fitness of Bacteroidota in the gut. Depending on the species, this uptake process relies on one to four transport systems centred on conserved core outer membrane (OM) complexes composed of the TonB-dependent transporter BtuB and the surface-exposed lipoprotein BtuG. Additionally, the surface-exposed lipoprotein BtuH, although not tightly associated with the BtuBG complex, contributes to cobamide uptake and provides a fitness advantage. Here, we report the functional and structural characterization of BtuJ1 from Bacteroides thetaiotaomicron (B. theta), an additional surface-exposed lipoprotein in B₁₂ uptake loci. BtuJ1 binds vitamin B₁₂ and cobinamide (an intermediate in B₁₂ biosynthesis) with low nM affinity, conferring a fitness advantage in B₁₂-limited environments. Regardless of B₁₂ availability, BtuJ1 is the most abundant of the B₁₂-transport components encoded by B. theta. Under B₁₂-replete conditions, BtuJ1 binds the vitamin, generating a readily available pool for transfer to the core BtuBG transport systems during periods of B₁₂ depletion as demonstrated by in vitro and in vivo B₁₂ transfer experiments. Together, these findings expand the known functionalities of the diverse accessory OM proteins employed by Bacteroidota and underscore the sophisticated strategies these human gut commensals use to secure vitamin B₁₂ in the competitive environment of the human gut.