Investigation of the Autoregulator-Receptor System in the Pristinamycin Producer .

Pristinamycin biosynthesis in is governed by a complex hierarchical signaling cascade involving seven different transcriptional regulators (SpbR, PapR1, PapR2, PapR3, PapR4, PapR5, and PapR6). The signaling cascade is triggered by γ-butyrolactone (GBL)-like effector molecules, whereby the chemical structure of the effector, as well as its biosynthetic origin is unknown so far. Three of the pristinamycin transcriptional regulators (SpbR, PapR3, and PapR5) belong to the type of γ-butyrolactone receptor (GBLR). GBLRs are known to either act as "real" GBLRs, which bind GBLs as ligands or as "pseudo" GBLRs binding antibiotics or intermediates thereof as effector molecules. In this study, we performed electromobility shift assays (EMSAs) with SpbR, PapR3, and PapR5, respectively, in the presence of potential ligand samples. Thereby we could show that all three GBLRs bind synthetic 1,4-butyrolactone but not pristinamycin as ligand, suggesting that SpbR, PapR3, and PapR5 act as "real" GBLRs in . Furthermore, we identified a cytochrome P450 monooxygenase encoding gene as potential biosynthesis gene for the GBLR-interacting ligand. Inactivation of resulted in an increased pristinamycin production, which indicated that SnbU has a regulatory influence on pristinamycin production. EMSAs with culture extract samples from the mutant did not influence the target binding ability of SpbR, PapR3, and PapR5 anymore, in contrast to culture supernatant samples from the wild-type or the pristinamycin deficient mutant , which demonstrates that SnbU is involved in the synthesis of the GBLR-interacting ligand.