THE TOLA PROTEIN INTERACTS WITH COLICIN E1 DIFFERENTLY THAN WITH OTHER GROUP-A COLICINS

The 421-residue protein TolA is required for the translocation of grou p A colicins (colicins E1, E2, E3, A, K, and N) across the cell envelo pe of Escherichia coli. Mutations in TolA can render cells tolerant to these colicins and cause hypersensitivity to detergents and certain a ntibiotics, as well as a tendency to leak periplasmic proteins. TolA c ontains a long cc-helical domain which connects a membrane anchor to t he C-terminal domain, which is required for colicin sensitivity. The f unctional role of the or-helical domain was tested by deletion of resi dues 56 to 169 (TolA Delta 1), 166 to 287 (TolA Delta 2), or 54 to 287 (TolA Delta 3) of the alpha-helical domain of TolA, which removed the N-terminal half, the C-terminal half, or nearly the entire alpha-heli cal domain of TolA, respectively. TolA and TolA deletion mutants were expressed from a plasmid in an E. coli strain producing no chromosomal ly encoded TolA. Cellular sensitivity to the detergent deoxycholate wa s increased for each deletion mutant, implying that more than half of the TolA alpha-helical domain is necessary for cell envelope stability . Removal of either the N- or C-terminal half of the cc-helical domain resulted in a slight (ca. 5-fold) decrease in cytotoxicity of the Tol A-dependent colicins A, E1, E3, and N compared to cells producing wild -type TolA when these mutants were expressed alone or with TolQ, -R, a nd -B. In cells containing TolA Delta 3, the cytotoxicity of colicins A and E3 was decreased by a factor of >3,000, and K+ efflux induced by colicins A and N was not detectable. In contrast, for colicin E1 acti on on TolA Delta 3 cells, there was little decrease in the cytotoxic a ctivity (<5-fold) or the rate of K+ efflux, which was similar to that from wild-type cells. It was concluded that the mechanism(s) by which cellular uptake of colicin E1 is mediated by the TolA protein differs from that for colicins A, E3, and N. Possible explanations for the dis tinct interaction and unique translocation mechanism of colicin E1 are discussed.