NOVEL COLICIN-10 - ASSIGNMENT OF 4 DOMAINS TO TONB-DEPENDENT AND TOLC-DEPENDENT UPTAKE VIA THE TSX RECEPTOR AND TO PORE FORMATION

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 C ol10 also required TolC which is unique for a Ton-coupled translocatio n through the outer membrane. A 2470 bp DNA fragment from the natural plasmid pCol10 encoding the Col10 activity (cta), immunity (cti) and l ysis (ctl) genes was sequenced. The Cta, Cti and Ctl proteins, as dedu ced 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. Co l10 (Cta) was highly homologous to colicin E1 in two regions which det ermined the common TolC requirement for uptake and the pore-forming ac tivity. Col10 and E1 differed entirely in the regions which are predic ted to determine the Ton dependence of Col10 and the Tol dependence of E1, and binding to the receptors Tsx and BtuB, respectively. The regi on responsible for the Ton-dependent uptake of Col10 was localized in the sequence ranging from residues 1 to 43 (Ton region), and the regio n 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 sys tem and BtuB, whereas the E1-Col10 fusion protein was imported via the Tol system and Tsx. Although the immunity proteins of Col10 and E1 di splayed a low homology, Cti conferred full immunity to E1, in contrast to the immunity protein of E1 which did not protect cells against Col 10. It is proposed that Col10 belongs to the colicin E1, 1a, Ib group as opposed to the colicin A, B, N group of pore-forming colicins. Col1 0 consists of 4 domains of which two are very similar and two are very different to E1, supporting our previous proposal that colicins evolv ed by recombination of DNA fragments which encode uptake and activity domains.