Interaction of 125I-labeled colicin E1 with Escherichia coli

Colicin E1 protein was labeled with (125)I to specific activities of up to 2 x 10(8) cpm/mg of protein and with no loss of the colicin biological activity. The labeled colicin bound to colicin E1-sensitive, tolerant, and immune E1-colicinogenic Escherichia coli. An E. coli mutant resistant to colicin E1 exhibited a much lower colicin-binding capacity. The average number of bound colicin molecules per sensitive cell increased as a function of the colicin concentration in the colicin cell interaction mixture and continued to increase even after loss of viability of the entire culture. Up to 2,400 colicin E1 molecules bound per cell, but saturation was not reached. Binding kinetics showed that maximum binding occurred within 2 to 5 min of colicin addition. Survival and binding assays indicated that one colicin killing unit corresponded to an average of about 100 colicin molecules bound per bacterial cell. This number, however, decreased to about 8 in more extensively washed cells. Trypsin digestion of the colicin-treated cells removed the majority of the cell-bound colicin, but in general provided little rescue from colicin killing. At low colicin concentrations, a linear relationship existed between survival and the number of trypsin-inaccessible colicin molecules. Under these circumstances and in agreement with single-hit kinetics, the relationship between the number of colicin killing units and the number of trypsin-inaccessible colicin molecules was close to 1. After trypsin digestion, cells that were nearly saturated with colicin retained about 200 trypsin-inaccessible colicin molecules per cell. The trypsin-inaccessible colicin might represent those colicin molecules that bound to the specific E colicin receptors of E. coli cells.