Evidence that the immunity protein inactivates colicin 5 immediately prior to the formation of the transmembrane channel.

Determination and analysis of the nucleotide sequences of the activity, immunity, and lysis genes of colicin 5 assigned colicin 5 to the subclass of pore-forming colicins to which colicins 10, E1, Ia, Ib, and K belong. Mutational analysis of colicin 5 and exchange of DNA fragments between the most closely related colicins, colicins 5 and 10, and between their immunity proteins localized the regions that determine the reaction specificity between colicin 5 and its immunity protein to residues 405 to 424 of colicin 5, the region corresponding to the amphiphilic alpha-helix 6 of the similar colicins E1 and Ia. The specificity-conferring residues 55 to 58 and 68 to 75 of the immunity protein were localized in the cytoplasmic loop and the inner leaflet of the cytoplasmic membrane. The localization of the reactive regions of the immunity protein and the colicin close to the inner side of the cytoplasmic membrane suggests that the immunity protein inactivates colicin 5 shortly before the lethal colicin pores in the cytoplasmic membrane are opened.