QUANTIFICATION OF GROUP-A COLICIN IMPORT SITES

Pore-forming colicins are soluble bacteriocins which form voltage-gate d ion channels in the inner membrane of Escherichia coli. To reach the ir target, these colicins first bind to a receptor located on the oute r membrane and then are translocated through the envelope. Colicins ar e subdivided into two groups according to the envelope proteins involv ed in their translocation: group A colicins use the Tot proteins; grou p B colicins use the proteins TonB, ExbB, and ExbD. We have previously shown that a double-cysteine colicin A mutant which possesses a disul fide bond in its pore-forming domain is translocated through the envel ope but is unable to form a channel in the inner membrane (D. Duche, D . Baty, M. Chartier, and L. Letellier, J. Biol. Chem. 269:24820-24825, 1994). Measurements of colicin-induced K+ efflux reveal that preincub ation of the cells with the double-cysteine mutant prevents binding of colicins of group A but not of group B. Moreover, we show that the mu tant is still in contact with its receptor and import machinery when i t interacts with the inner membrane. From these competition experiment s, we conclude that each Escherichia coli cell contains approximately 400 and 1,000 colicin A receptors and translocation sites, respectivel y.