Analysis of Cross-Functionality within LanBTC Synthetase Complexes from Different Bacterial Sources with Respect to...

Lanthipeptides belong to a family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) containing (methyl)lanthionine residues. Commonly, class I lanthipeptides are synthesized by a gene cluster encoding a precursor peptide (LanA), biosynthetic machinery (LanBTC), a protease (LanP), a two-component regulatory system (LanRK), and an immunity system (LanI and LanFEG). Although nisin and subtilin are highly similar class I lanthipeptides, the cross-regulation by LanRK and the cross-immunity by LanI and LanFEG between the nisin and subtilin systems have been proven to be very low. Here, the possibility of the cross-functionality of LanBTC to modify and transport nisin precursor (NisA) and subtilin precursor (SpaS) was evaluated in Bacillus subtilis and Lactococcus lactis. Interestingly, we found that a promiscuous NisBC-SpaT complex is able to synthesize and export nisin precursor, as efficiently as the native nisin biosynthetic machinery NisBTC, in L. lactis but not B. subtilis. The assembly of the NisBC-SpaT complex at a single microdomain, close to the old cell pole, was observed by fluorescence microscopy in L. lactis. In contrast, such a complex was not formed in B. subtilis. Furthermore, the isolation of the NisBC-SpaT complex and its subcomplexes from the cytoplasmic membrane of L. lactis by pulldown assays was successfully conducted. Our work demonstrates that the association of LanBC with LanT is critical for the efficient biosynthesis and secretion of the lanthipeptide precursor with complete modifications and suggests a cooperative mechanism between LanBC and LanT in the modification and transport processes. IMPORTANCE A multimeric synthetase LanBTC complex has been proposed for the in vivo production of class I lanthipeptides. However, it has been demonstrated that LanB, LanC, and LanT can perform their functionality in vivo and in vitro, independently of other Lan proteins. The role of protein-protein interactions, especially between the modification complex LanBC and the transport system LanT, in the biosynthesis process of lanthipeptides is still unclear. In this study, the importance of the presence of a well-installed LanBTC complex in the cell membrane for lanthipeptide biosynthesis and transport was reinforced. In L. lactis, the recruitment of SpaT from the peripheral cell membrane to the cell poles by the NisBC complex was observed, which may explain the mechanism by which the secretion of the premature peptide is prevented.