Cloned diphtheria toxin within the periplasm of Escherichia coli causes lethal membrane damage at low pH

Acidic pH within endosomal vesicles of sensitive animal cells triggers a conformational change in diphtheria toxin (DT) that is believed to cause the B chain to insert into the vesicular membrane and the enzymic A chain to be released into the cytosol. In artificial lipid bilayers, DT forms ion-conductive channels under mildly acidic conditions (pH approximately 5). Here we report a related phenomenon in Escherichia coli strains that secrete certain cloned DT-related proteins into their periplasm: the cells are rapidly killed at pH 5 but remain unharmed at pH 7. Expression of full-length DT (an active-site mutant, to comply with the National Institutes of Health recombinant DNA guidelines) causes acid-sensitivity, whereas expression of the A chain alone does not. The killed cells are not lysed, but inner-membrane functions are impaired (membrane potential, active transport, and ion impermeability). We propose that acidification of DT within the periplasm induces its insertion into the inner membrane, lethally damaging the permeability barrier. This discovery provides a potentially important selection procedure for mutations affecting the membrane insertion function of DT. Similar approaches may be useful in studying other proteins that undergo condition-dependent interaction with membranes.