Isolation and Analysis of the Nisin Biosynthesis Complex NisBTC: further Insights into Their Cooperative Action

Lantibiotics are ribosomally synthesized and posttranslationally modified peptide antibiotics. Although the membrane-associated lantibiotic biosynthesis machinery has long been proposed to exist, the isolation of such a complex has not been reported yet, which limits the elucidation of the processive mechanism of lantibiotic biosynthesis. ABSTRACT Nisin is synthesized by a putative membrane-associated lantibiotic synthetase complex composed of the dehydratase NisB, the cyclase NisC, and the ABC transporter NisT in Lactococcus lactis. Earlier work has demonstrated that NisB and NisT are linked via NisC to form such a complex. Here, we conducted for the first time the isolation of the intact NisBTC complex and NisT-associated subcomplexes from the cytoplasmic membrane by affinity purification. A specific interaction of NisT, not only with NisC but also with NisB, was detected. The cellular presence of NisB and/or NisC in complex with precursor nisin (NisA) was determined, which shows a highly dynamic and transient assembly of the NisABC complex via an alternating binding mechanism during nisin dehydration and cyclization. Mutational analyses, with cysteine-to-alanine mutations in NisA, suggest a tendency for NisA to lose affinity to NisC concomitant with an increasing number of completed lanthionine rings. Split NisBs were able to catalyze glutamylation and elimination reactions in an alternating way as efficiently as full-length NisB, with no significant influence on the following cyclization and transport. Notably, the harvest of the leader peptide in complex with the independent elimination domain of NisB points to a second leader peptide binding motif that is located in the C-terminal region of NisB, giving rise to a model where the leader peptide binds to different sites in NisB for glutamylation and elimination. Overall, these combined studies provide new insights into the cooperative biosynthesis mechanism of nisin and thereby lay a foundation for further structural and functional characterization of the NisBTC complex. IMPORTANCE Lantibiotics are ribosomally synthesized and posttranslationally modified peptide antibiotics. Although the membrane-associated lantibiotic biosynthesis machinery has long been proposed to exist, the isolation of such a complex has not been reported yet, which limits the elucidation of the processive mechanism of lantibiotic biosynthesis. In this work, we present direct evidence for the existence of the nisin biosynthetic complex at the cytoplasmic membrane of L. lactis, producing fully modified precursor nisin. By analyses of the interactions within the intact NisBTC complex and the modification machinery NisABC, we were able to elucidate the cooperative action for the modification and transport of nisin. Inspired by the natural and documented degradation process of NisB, artificial split-NisBs were made and thoroughly characterized, demonstrating a crucial clue to the evolution of the LanB family. Importantly, our study also suggests that the leader peptide of NisA binds to two different recognition motifs, i.e., one for glutamylation and one for elimination.