A natural hybrid of a Bacillus thuringiensis Cry2A toxin implicates Domain I in specificity determination

A PCR-RFLP method was used to identify cry2A toxin genes in a collection of 300 strains of Bacillus thuringiensis. From 81 genes identified, the vast majority appeared to be cry2Aa or cry2Ab, however three showed a different pattern and were subsequently cloned and sequenced. The gene cloned from strain HD395 was named cry2Ba2. Since the proteins encoded by the genes cloned from LS5115-3 and DS415 shared >95% sequence identity with existing toxins their genes were named cry2Aa17 and cry2Ab29 respectively by the toxin nomenclature committee. Despite this overall similarity these two toxins resembled natural hybrids, with Cry2Ab29 resembling Cry2Ab for the majority of the protein but then showing identity to Cry2Aa for the last 66 amino acids. For Cry2Aa17, Domains II and III most closely resembled Cry2Aa (99% identity) whilst Domain I was identical to that of Cry2Ab. The toxicity of the recombinant toxins was tested against Aedes aegypti and Spodoptera exigua, and it was found that the toxicity profile of Cry2Aa17 more closely matched the profile of Cry2Ab than that of Cry2Aa, thus implicating Domain I in specificity determination. This association of Domain I with toxicity was confirmed when hybrids were made between Cry2Aa and Cry2Ab.