Subcellular Localization and Assembly Process of the Nisin Biosynthesis Machinery in Lactococcus lactis

Abstract

Nisin is the model peptide for LanBC-modified lantibiotics that are commonly modified and exported by a putative synthetase complex. Although the mechanism of maturation, transport, immunity, and regulation is relatively well understood, and structural information is available for some of the proteins involved (B. Li, J. P. J. Yu, J. S. Brunzelle, G. N. Moll, et al., Science 311:1464–1467, 2006, https://doi.org/10.1126/science.1121422 ; M. A. Ortega, Y. Hao, Q. Zhang, M. C. Walker, et al., Nature 517:509–512, 2015, https://doi.org/10.1038/nature13888 ; C. Hacker, N. A. Christ, E. Duchardt-Ferner, S. Korn, et al., J Biol Chem 290:28869–28886, 2015, https://doi.org/10.1074/jbc.M115.679969 ; Y. Y. Xu, X. Li, R. Q. Li, S. S. Li, et al., Acta Crystallogr D Biol Crystallogr 70:1499–1505, 2014, https://doi.org/10.1107/S1399004714004234 ), the subcellular localization and assembly process of the biosynthesis complex remain to be elucidated. In this study, we determined the spatial distribution of nisin synthesis-related enzymes and the transporter, revealing that the modification and secretion of the precursor nisin mainly occur at the old cell poles of L. lactis and that the transporter NisT is probably recruited later to this spot after the completion of the modification reactions by NisB and NisC. Fluorescently labeled nisin biosynthesis machinery was visualized directly by fluorescence microscopy. To our knowledge, this is the first study to provide direct evidence of the existence of such a complex in vivo . Importantly, the elucidation of the “order of assembly” of the complex will facilitate future endeavors in the investigation of the nisin secretion mechanism and even the isolation and structural characterization of the complete complex.