Bacillus thuringiensis pathogenicity islands encode regulatory circuits controlling insecticidal Cry toxin expression during vegetative growth.

Bacillus thuringiensis (Bt) produces insecticidal toxins, including Cry and Vip3 proteins, that are widely used for biological pest control. Cry proteins are classically expressed during sporulation under the control of sporulation-specific σ factors, whereas Vip3 is produced during vegetative growth, suggesting distinct regulatory pathways. Notably, many cry and vip3A genes are clustered within pathogenicity islands (PAIs), such as BtPAI-1. However, whether these PAIs also encode regulatory mechanisms coordinating toxin expression remains unclear. Here, we identify VipR, a BtPAI-1-encoded transcriptional regulator, as an activator of insecticidal gene expression during the vegetative phase in Bt strains HD-1 and CT-43. In these strains, VipR promotes the transcription of BtPAI-1 associated insecticidal genes, including vip3A and selected cry genes, resulting in premature Cry protein accumulation and increased insecticidal activity. In addition, VipR contributes to the vegetative-phase expression of the non-BtPAI-1 cry9Aa genes in strain BGSC 4AE1. Phylogenetic analysis revealed that vipR is widely distributed in one-third of Bt strains, and is strongly associated with PAIs. Futhermore, heterologous expression of vipR in BGSC 4J5 and HD-73 was sufficient to activate vegetative-phase transcription of some cry independently of sporulation-specific σ factor cascade. These results support a role for VipR in coordinating vegetative-phase expression of insecticidal genes in the Bt strains examined and suggest that BtPAI-1 can encode both insecticidal determinants and regulatory functions that influence their expression. These findings provide new insights into the regulatory architecture of Bt pathogenicity islands and may facilitate the engineering of strains with enhanced insecticidal activity.