Regulatory diversity in Bacillus thuringiensis cry genes reveals flexible evolutionary strategies for in vivo toxin expression

Bacillus thuringiensis (Bt) is a highly effective insect pathogen, largely due to the expression of diverse insecticidal proteins upon sporulation. Among them, the three-domain Cry protein family represent the largest family, targeting a wide range of insect species and nematodes. While it is proposed that they have evolved from a common ancestral gene, the comparative analysis of their upstream regulatory regions revealed significant variability. To investigate this divergence, we employed Multiple Expectation maximization for Motif Elicitation (MEME) and Find Individual Motif Occurrences (FIMO) motive discovery tools, to identify conserved regulatory elements, including canonical -10 and -35 promoter motifs and Shine-Dalgarno (SD) sequences. Our analyses clearly revealed that upstream regulatory sequences are not conserved across the entire cry family. However, we identify subsets of genes with similar insect specificity which shared conserved motif architectures in their upstream regulatory sequences, suggesting a correlation between regulatory evolution and host range. Conversely, some proteins targeting the same insect order (e.g., Cry1 and Cry9Ca or Cry3 and Cry8) showed to be regulated by entirely different upstream sequences, indicating that Bt has evolved multiple regulatory strategies to achieve similar expression patterns. To test relevance of the upstream sequences, we cloned cry1Ab and cry4Ba genes under the control of heterologous upstream regions: P1P2 from lepidopteran-specific cry1Aa gene, and P4 from dipteran-specific cry4Ba gene. These constructions were expressed in non-toxic Cry- acrystalliferous Bt-backgrounds with distinct host specialization: Bt subsp thuringiensis 407 strain (lepidopteran adapted) and Bt subsp israelensis 4Q7 strain (dipteran adapted). Gene expression was assessed in vitro and in vivo after oral infection of lepidopteran and dipteran larvae with purified spores. Our findings indicate that Cry protein expression is influenced by both, the promoter identity, and Bt strain background, underscoring the evolutionary and functional significance of upstream regulatory sequences in the diversification and ecological success of Bt.