Uptake of a new colicin, colicin 10 (Col10), into cells of Escherichia coli required TonB, ExbBD (Ton system), but its cognate receptor, Tsx, functioned independently of Ton and TolQRAB (Tol system). Uptake of Col10 also required TolC which is unique for a Ton-coupled translocation through the outer membrane. A 2470 bp DNA fragment from the natural plasmid pCol10 encoding the Col10 activity (cta), immunity (cti) and lysis (ctl) genes was sequenced. The Cta, Cti and Ctl proteins, as deduced from the nucleotide sequences, consisted of 490 (M(r) 53,342), 96 (M(r) 11,586) and 43 (M(r) 4484) amino acid residues, respectively. Col10 (Cta) was highly homologous to colicin E1 in two regions which determined the common TolC requirement for uptake and the pore-forming activity. Col10 and E1 differed entirely in the regions which are predicted to determine the Ton dependence of Col10 and the Tol dependence of E1, and binding to the receptors Tsx and BtuB, respectively. The region responsible for the Ton-dependent uptake of Col10 was localized in the sequence ranging from residues 1 to 43 (Ton region), and the region responsible for the Tol-dependent uptake of colicin E1 extended from residues 1 to 34 (Tol region). Each Tol-dependent colicin contained a pentapeptide homologous to the sequence DGSGS in the Tol region of E1 which is proposed to be implicated in Tol-dependent uptake (TolA box). After the exchange of the Ton and the Tol regions between Col10 and E1, the Col10-E1 fusion protein was carried into cells via the Ton system and BtuB, whereas the E1-Col10 fusion protein was imported via the Tol system and Tsx. Although the immunity proteins of Col10 and E1 displayed a low homology, Cti conferred full immunity to E1, in contrast to the immunity protein of E1 which did not protect cells against Col10. It is proposed that Col10 belongs to the colicin E1, Ia, Ib group as opposed to the colicin A, B, N group of pore-forming colicins. Col10 consists of 4 domains of which two are very similar and two are very different to E1, supporting our previous proposal that colicins evolved by recombination of DNA fragments which encode uptake and activity domains.