Proteolytic Stability of Insecticidal Toxins Expressed in Recombinant Bacilli

Abstract

ABSTRACT The production of the vegetative mosquitocidal toxin Mtx1 from Bacillus sphaericus was redirected to the sporulation phase by replacement of its weak, native promoter with the strong sporulation promoter of the bin genes. Recombinant bacilli developed toxicity during early sporulation, but this declined rapidly in later stages, indicating the proteolytic instability of the toxin. Inhibition studies indicated the action of a serine proteinase, and similar degradation was also seen with the purified B. sphaericus enzyme sphericase. Following the identification of the initial cleavage site involved in this degradation, mutant Mtx1 proteins were expressed in an attempt to overcome destructive cleavage while remaining capable of proteolytic activation. However, the apparently broad specificity of sphericase seems to make this impossible. The stability of a further vegetative toxin, Mtx2, was also found to be low when it was exposed to sphericase or conditioned medium. Random mutation of the receptor binding loops of the Bacillus thuringiensis Cry1Aa toxin did, in contrast, allow production of significant levels of spore-associated protein in the form of parasporal crystals. The exploitation of vegetative toxins may, therefore, be greatly limited by their susceptibility to proteinases produced by the host bacteria, whereas the sequestration of sporulation-associated toxins into crystals may make them more amenable to use in strain improvement.