Paradigm shift for cry gene expression in Bacillus thuringiensis

In most Bacillus thuringiensis strains, the insecticidal cry genes are transcribed during sporulation by RNA-polymerases containing sigma factors E or K, leading to the formation of an insecticidal crystal within the mother cell along spore development. Some strains, such as the kurstaki HD1, a parent of commercial strains, also release insecticidal proteins Cry1I and Vip3A in the extracellular medium. vip3A gene expression is activated by the transcriptional regulator VipR at the onset of the stationary phase. Here, we expanded the VipR regulon to all the insecticidal-encoding genes of strain HD1 by identifying the VipR-binding box and conducting transcription assays. Unexpectedly, a VipR box was located in the promoter region of a putative N-acetylmuramoyl-L-alanine amidase gene upstream cry1Ac in strain kurstaki HD73, closely related to the HD1 but devoid of vipR. We demonstrated that introducing a functional vipR in this strain leads to the expression of the amidase-cry1Ac operon, resulting in an early and increased production of Cry1Ac. Investigating this phenotype, we showed that Cry1Ac was produced in a VipR-dependent manner in an HD73 {Delta}spo0A mutant. Similarly, an HD1{Delta}spo0A strain forms insecticidal crystals and produces all the insecticidal proteins encoded in its genome, including cry2Ab previously considered unexpressed in strain HD1. Finally, a genomic analysis revealed the presence of putative VipR-binding sequences upstream from insecticidal protein-encoding genes in several biopesticidal strains. Overall, our results break the dogma on the regulation of cry1A and cry2A genes and provide evidence of sporulation-independent Cry toxin production in biopesticidal Bt strains. ImportanceBacillus thuringiensis is a remarkably efficient entomopathogen due to its ability to produce various insecticidal proteins, such as Cry or Vip. This property has made it a highly effective biopesticide used worldwide. Our work breaks the dogma of cry1 and cry2 genes being regulated solely by sporulation-specific sigma factors and thus exclusively expressed during this process. Indeed, we demonstrated that the VipR regulator controls the transcription of all the insecticidal protein genes encoded by a strain closely related to that of commercial biopesticides and turns on their expression at the beginning of the stationary phase, leading to the production of insecticidal crystals independently of sporulation. By providing new knowledge on the regulation of insecticidal protein genes, these findings bring new insight for the genetic improvement of Bt strains used as commercial biopesticides.