A novel Rap-Phr system inBacillus velezensisNAU-B3 regulates surfactin production and sporulation via interaction with ComA

Several quorum sensing systems occurring inBacillus subtilis, e.g. Rap-Phr systems, were reported to interact with major regulatory proteins, such as ComA, DegU, and Spo0A, in order to regulate competence, sporulation, and synthesis of secondary metabolites. In this study, we characterized a novel Rap-Phr system, RapA4-PhrA4, inBacillus velezensisNAU-B3. We found that therapA4andphrA4genes were co-transcribed in NAU-B3. WhenrapA4was expressed in the heterologous hostBacillus subtilisOKB105, surfactin production and sporulation were severely inhibited. However, when thephrA4was co-expressed, the RapA4 activity was inhibited. The transcription of the surfactin synthetasesrfAgene and sporulation-related genes were also regulated by the RapA4-PhrA4 system. In vitro results obtained from electrophoretic mobility shift assay (EMSA) proved that RapA4 inhibits ComA binding to the promoter of thesrfAoperon, and the PhrA4 pentapeptide acts as anti-activator of RapA4. We also found that the F24 residue plays a key role in RapA4 function. This study indicated that the novel RapA4-PhrA4 system regulates the surfactin synthesis and sporulation via interaction with ComA, thereby supporting the bacterium to compete and to survive in a hostile environment.