Site-specific recombination and purifying selection influence Cry toxin formation and specialization.

In bacteria, multi-domain toxins represent a universal tool for determining the host range, being a cornerstone for public health and agriculture. Given high host specificity and potency, Cry toxins represent the most widely used bioinsecticides in agriculture. Their worldwide application grows annually; thus, the rational design of bioinsecticides requires a deeper understanding of the processes that orchestrate the origin and evolution of Cry toxins. The accepted views on how Cry toxins exchange domains postulate that homologous recombination sparks domain III swapping, altering host specificity and broadening strains' activities. This perspective, however, stems from laboratory experiments and has never been studied in nature. Here, we leveraged all available Cry sequences and uncovered the mechanism of the evolution of Cry toxins via site-specific recombination guided by XerCD recombinases that target insertions and conserved blocks between domain borders. The emergence of specialists from generalists is sparked by the purifying selection of specialized hybrids. Therefore, our model of domain shuffling caused by site-specific recombination of cry loci, followed by selection-driven specialization, illustrates how multi-domain toxins evolve and attune to specific hosts.