Simple SummarySecondary active transporters have major roles in transporting vital nutrients and other smaller molecules in and out of cells. Proton-coupled oligopeptide transporters comprise a family of secondary transporters with specificity towards very short peptides and a very large number of species contain these transporters. In bacteria, proton-coupled oligopeptide transporters play different physiological roles. However, they have also been shown to import antibiotics and other drugs; hence, these transporters are of particular interest in bacteria. Here we sought to characterize such a transporter from the bacteriumClostridium perfringens, the only and previously uncharacterized proton-coupled oligopeptide transporter from this bacterium. Our findings were surprising as conventional proton-coupled oligopeptide transport was low, but substrate exchange, an inducible transport mode observed among similar transporters, was more prominent. This finding was investigated in light of mutational studies based on the predicted 3D structure of the transporter.AbstractProton-coupled oligopeptide transporters (POTs) are a fundamental part of the cellular transport machinery that provides plants, bacteria, and mammals with nutrition in the form of short peptides. However, POTs are not restricted to peptide transport; mammalian POTs have especially been in focus due to their ability to transport several peptidomimetics in the small intestine. Herein, we studied a POT fromClostridium perfringens(CPEPOT), which unexpectedly exhibited atypical characteristics. First, very little uptake of a fluorescently labelled peptide β-Ala-Lys-AMCA, an otherwise good substrate of several other bacterial POTs, was observed. Secondly, in the presence of a competitor peptide, enhanced uptake of β-Ala-Lys-AMCA was observed due to trans-stimulation. This effect was also observed even in the absence of a proton electrochemical gradient, suggesting that β-Ala-Lys-AMCA uptake mediated by CPEPOT is likely through the substrate-concentration-driving exchange mechanism, unlike any other functionally characterized bacterial POTs.