Substrate preference of an ABC importer corresponds to selective growth on β-(1,6)-galactosides in Bifidobacterium animalis subsp. lactis

Bifidobacteria are exposed to substantial amounts of dietary beta-galactosides. Distinctive preferences for growth on different beta-galactosides are observed within Bifidobacterium members, but the basis of these preferences remains unclear. We previously described the first beta-(1,6)/(1,3)-galactosidase from Bifidobacterium animalis subsp. lactis Bl-04. This enzyme is relatively promiscuous, exhibiting only 5-fold higher efficiency on the preferred beta-(1,6)-galactobiose than the beta-(1,4) isomer. Here, we characterize the solute-binding protein (Bal6GBP) that governs the specificity of the ABC transporter encoded by the same beta-galactoside utilization locus. We observed that although Bal6GBP recognizes both beta-(1,6)- and beta-(1,4)-galactobiose, Bal6GBP has a 1630-fold higher selectivity for the former, reflected in dramatic differences in growth, with several hours lag on less preferred beta-(1,4)- and beta-(1,3)-galactobiose. Experiments performed in the presence of varying proportions of beta-(1,4)/beta-(1,6)-galactobioses indicated that the preferred substrate was preferentially depleted from the culture supernatant. This established that the poor growth on the nonpreferred beta-(1,4) was due to inefficient uptake. We solved the structure of Bal6GBP in complex with beta-(1,6)-galactobiose at 1.39 angstrom resolution, revealing the structural basis of this strict selectivity. Moreover, we observed a close evolutionary relationship with the human milk disaccharide lacto-N-biose-binding protein from Bifidobacterium longum, indicating that the recognition of the nonreducing galactosyl is essentially conserved, whereas the adjacent position is diversified to fit different glycosidic linkages and monosaccharide residues. These findings indicate that oligosaccharide uptake has a pivotal role in governing selectivity for distinct growth substrates and have uncovered evolutionary trajectories that shape the diversification of sugar uptake proteins within Bifidobacterium.