Differential effects of ionic strength, divalent cations and pH on the pore-forming activity of Bacillus thuringiensis insecticidal toxins

The combined effects of ionic strength, divalent cations, pH and toxin concentration on the pore-forming activity of CrylAc and CrylCa were studied using membrane potential measurements in isolated midguts of Manduca sexta and a brush border membrane vesicle osmotic swelling assay. The effects of ionic strength and divalent cations were more pronounced at pH 10.5 than at pH 7.5. At the higher pH, lowering ionic strength in isolated midguts enhanced CrylAc activity but decreased considerably that of CrylCa. In vesicles, CrylAc had a stronger pore-forming ability than CrylCa at a relatively low ionic strength. Increasing ionic strength, however, decreased the rate of pore formation of CrylAc relative to that of CrylCa. The activity of CrylCa, which was small at the higher pH, was greatly increased by adding calcium or by increasing ionic strength. EDTA inhibited CrylAc activity at pH 10.5, but not at pH 7.5, indicating that trace amounts of divalent cations are necessary for CrylAc activity at the higher pH. These results, which clearly demonstrate a strong effect of ionic strength, divalent cations and pH on the pore-forming activity of CrylAc and CrylCa, stress the importance of electrostatic interactions in the mechanism of pore fomation by B. thuringiensis toxins.