Rap-Phr systems from pAW63 and pHT8-1 plasmids act together to regulate sporulation in the Bacillus thuringiensis kurstaki HD73 strain

Bacillus thuringiensis is a Gram-positive spore-forming bacterium pathogenic to various insect species. This property is due to the Cry toxins encoded by plasmid genes and mostly produced during sporulation. B. thuringiensis contains a remarkable amount of extrachromosomal DNA molecules and a great number of plasmid rap-phr genes. Rap-Phr quorum sensing systems regulate different bacterial processes, notably the commitment to sporulation in Bacillus species. Rap proteins are quorum sensors acting as phosphatases on Spo0F, intermediate of the sporulation phosphorelay, and are inhibited by Phr peptides that function as signaling molecules. In this study, we characterize the Rap63-Phr63 system encoded by the pAW63 plasmid from the B. thuringiensis var. kurstaki HD73 strain. Rap63 has a moderate activity on sporulation and is inhibited by the Phr63 peptide. The rap63-phr63 genes are co-transcribed and the phr63 gene is also transcribed from a σ^H-specific promoter. We show that Rap63-Phr63 regulates the sporulation together with the Rap8-Phr8 system harbored by the plasmid pHT8_1 of the HD73 strain. Interestingly, the deletion of both phr63 and phr8 genes in the same strain has a greater negative effect in sporulation than the sum of the loss of each phr gene. Despite the similarities in the Phr8 and Phr63 sequences, there is no crosstalk between the two systems. Our results suggest a synergism of these two Rap-Phr systems in the regulation of sporulation of B. thuringiensis at the end of the infectious cycle in insect, thus pointing out the role of the plasmids to the fitness of the bacteria.IMPORTANCEThe life cycle of Bacillus thuringiensis in insect larvae is regulated by quorum sensing systems of the RNPP family. After the toxemia caused by Cry insecticidal toxins, the sequential activation of these systems allows the bacteria to trigger a state of virulence (regulated by PlcR-PapR), then a necrotrophic lifestyle (regulated by NprR-NprX) and ultimately the sporulation is controlled by the Rap-Phr systems. Our study describes a new rap-phr operon carried by a B. thuringiensis plasmid and shows that the Rap protein has a moderate effect on sporulation. However, in combination with another plasmid rap-phr operon, both systems provide an effective control of sporulation when the bacteria develop in the cadavers of infected insect larvae. Overall, this study highlights the important adaptive role of the plasmid Rap-Phr systems in the developmental fate and the survival of B. thuringiensis within their ecological niche.