The uptake of colicin U into sensitive cells is dependent on the Tol system. Like colicin A and the E-type colicins, colicin U contains the consensus pentapeptide DG(T/S)G(S/W) in the N-terminal 36 amino acids, which have been proposed to be involved in the interaction of colicin U with the Tol proteins. The role of the DGTGW motif in colicin U was studied by converting it into the TonB box motif DTMVV of colicin B. Uptake of the mutated colicin U (DTMVV) depended on TonB and ExbB and still remained TolA- and TolQ-dependent, but no longer required TolB. The Ton system could obviously replace the TolB function in the uptake of the mutated colicin U. The colicin U derivative is the first colicin whose uptake depends on the Ton and the Tol systems.