Eitinger T. Solitary BioY proteins mediate biotin transport into recombinant Escherichia coli

Energy-coupling factor (ECF) transporters form a large group of vitamin uptake systems in prokaryotes. They are composed of highly diverse, substrate-specific, transmembrane proteins (S units), a ubiquitous transmembrane protein (T unit), and homo-or hetero-oligomeric ABC ATPases. Biotin transporters represent a special case of ECF-type systems. The majority of the biotin-specific S units (BioY) is known or predicted to interact with T units and ABC ATPases. About one-third of BioY proteins, how-ever, are encoded in organisms lacking any recognizable T unit. This finding raises the question of whether these BioYs function as transporters in a solitary state, a feature ascribed to certain BioYs in the past. To address this question in living cells, an Esche-richia coli K-12 derivative deficient in biotin synthesis and devoid of its endogenous high-affinity biotin transporter was con-structed as a reference strain. This organism is particularly suited for this purpose because components of ECF transporters do not naturally occur in E. coli K-12. The double mutant was viable in media containing either high levels of biotin or a precursor of the downstream biosynthetic path. Importantly, it was nonviable on trace levels of biotin. Eight solitary bioY genes of proteo-bacterial origin were individually expressed in the reference strain. Each of the BioYs conferred biotin uptake activity on the re-combinants, which was inferred from uptake assays with [3H]biotin and growth of the cells on trace levels of biotin. The results underscore that solitary BioY transports biotin across the cytoplasmic membrane. Energy-coupling factor (ECF) transporters are a large group ofABC transporters for trace nutrients and intermediates of sal-vage pathways in prokaryotes. They consist of a transmembran