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

## TL;DR

This study explores how Bacillus thuringiensis regulates its insecticidal Cry proteins, finding that regulatory diversity allows flexible adaptation to different insect hosts.

## Contribution

The paper reveals that Bt uses multiple regulatory strategies for similar toxin expression, linking regulatory evolution to host specialization.

## Key findings

- Upstream regulatory sequences of cry genes are not conserved across the entire family but show partial conservation among genes targeting similar insect orders.
- Cry genes targeting the same insect order can have entirely different regulatory architectures, indicating independent evolutionary paths.
- Promoter identity and Bt strain background both influence Cry protein expression, highlighting the functional importance of regulatory diversity.

## Abstract

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.

Bacillus thuringiensis (Bt) is an effective insect pathogen that produces a wide variety of insecticidal proteins, including Cry toxins, which are highly specific toward certain lepidopteran, coleopteran, dipteran and nematodes. However, although Cry proteins share a common evolutionary origin, similar structural organization and mechanism of action, the evolution of their regulatory sequences revealed significant variability that is much less explored. Here, we systematically analyzed the upstream regions of cry genes, to understand how these regulatory elements evolved. We found that these regions are remarkably diverse, with partial conservation among toxins that target similar insect order, suggesting some adaptation to specific hosts during their evolution. Interestingly, we also identify cases where cry genes active against same insect order, such as cry3 and cry8 genes (both active against coleopteran), exhibit entirely distinct regulatory architecture, suggesting independent evolutionary paths, reflecting genetic flexibility, likely contributing to Bt´s ecological adaptation to different insect host. To explore their functional relevance, we engineered Bt strains to express the same Cry protein under different upstream sequences and evaluated toxin expression and activity in insect larvae. Our results suggest that Bt has evolved multiple transcriptional strategies to fine-tune cry gene expression, enabling successful adaptation to diverse insect environments.