The 421-residue
protein TolA is required for the translocation of group A
colicins (
colicins E1, E2, E3, A, K, and N) across the cell envelope of Escherichia coli. Mutations in TolA can render cells tolerant to these
colicins and cause
hypersensitivity to
detergents and certain
antibiotics, as well as a tendency to leak
periplasmic proteins. TolA contains a long alpha-helical domain which connects a membrane anchor to the C-terminal domain, which is required for
colicin sensitivity. The functional role of the alpha-helical domain was tested by deletion of residues 56 to 169 (TolA delta1), 166 to 287 (TolA delta2), or 54 to 287 (TolA delta3) of the alpha-helical domain of TolA, which removed the N-terminal half, the C-terminal half, or nearly the entire alpha-helical domain of TolA, respectively. TolA and TolA deletion mutants were expressed from a plasmid in an E. coli strain producing no chromosomally encoded TolA. Cellular sensitivity to the
detergent deoxycholate was 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 alpha-helical domain resulted in a slight (ca. 5-fold) decrease in cytotoxicity of the TolA-dependent
colicins A, E1, E3, and N compared to cells producing wild-type TolA when these mutants were expressed alone or with TolQ, -R, and -B. In cells containing TolA delta3, 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 action on TolA delta3 cells, there was little decrease in the cytotoxic activity (<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 distinct interaction and unique translocation mechanism of
colicin E1 are discussed.