THE N-TERMINAL DOMAIN OF COLICIN-E3 INTERACTS WITH TOLB WHICH IS INVOLVED IN THE COLICIN TRANSLOCATION STEP

Colicins use two envelope multiprotein systems to reach their cellular target in susceptible cells of Escherichia coil: the Tol system for g roup A colicins and the TonB system for group B colicins. The N-termin al domain of colicins is involved in the translocation step. To determ ine whether it interacts in vivo with proteins of the translocation sy stem, constructs were designed to produce and export to the cell perip lasm the N-terminal domains of colicin E3 (group A) and colicin B (gro up B). Producing cells became specifically tolerant to entire extracel lular colicins of the same group. The periplasmic N-terminal domains t herefore compete with entire colicins for proteins of the translocatio n system and thus interact in situ with these proteins on the inner si de of the outer membrane. In vivo cross-linking and co-immunoprecipita tion experiments in cells producing the colicin E3 N-terminal domain d emonstrated the existence of a 120kDa complex containing the colicin d omain and ToIB. After in vitro cross-linking experiments with these tw o purified proteins, a 120 kDa complex was also obtained. This suggest s that the complex obtained in vivo contains exclusively toIB and the colicin E3 domain. The N-terminal domain of a translocation-defective colicin E3 mutant was found to no longer interact with ToIB. Hence, th is interaction must play an important role in colicin E3 translocation .