FIG. 2.

(a) HlyA activity secreted by E. coli SE5000 (pLG813, hlyCA), growing in LB-10 mM CaCl₂ at 30°C, expressing wild-type BD+ or mutant hlyD encoded by pLG815. ○/•, wild-type translocator (D⁺); /✚, V349-I; ⋄/⧫, T85-I; ▵/▴, V334-I; □/▪, L165-Q. Open symbols and dotted lines indicate growth (A₄₅₀, which was identical for all cultures); filled symbols and lines indicate hemolytic activity detected in culture supernatants (OD₅₄₃/OD₄₅₀ · ml⁻¹). (b) Immunodetection of HlyD in the isolated envelope fraction of cultures of SE5000 expressing different hlyD mutations. Cell envelopes were isolated as described in Materials and Methods. Protein samples (1.5 OD₄₅₀ equivalent cell loadings) were analyzed by SDS-PAGE (10% acrylamide), followed by immunodetection with anti-HlyD antibodies. Different HlyD null mutants (numbers above the tracks) were expressed from pLG815 BD⁻. Strains carrying plasmid vectors alone or the wild-type BD⁺ translocator were used as controls. All cultures also expressed hlyCA (pLG813). (c) The same results as shown in panel b, but with the conditional hlyD mutations.