The length of a lantibiotic hinge region has profound influence on antimicrobial activity and host specificity

Lantibiotics are ribosomally synthesized (methyl)lanthionine containing peptides which can efficiently inhibit the growth of Gram-positive bacteria. As lantibiotics kill bacteria efficiently and resistance to them is difficult to be obtained, they have the potential to be used in many applications, e.g. in pharmaceutical industry or food industry. Nisin can inhibit the growth of Gram-positive bacteria by binding to lipid II and by making pores in their membrane. The C-terminal part of nisin is known to play an important role during translocation over the membrane and forming pore complexes. However, as the thickness of bacterial membranes varies between different species and environmental conditions, this property could have an influence on the pore forming activity of nisin. To investigate this, the so-called hinge region of nisin (residues NMK) was engineered to vary from one to six amino acid residues and specific activity against different indicators was compared. Antimicrobial activity in liquid culture assays showed that wild type nisin is most active, while truncation of the hinge region dramatically reduced the activity of the peptide. However, one or two amino acids extensions showed only slightly reduced activity against most indicator strains. Notably, some variants (+2, +1, -1, -2) exhibited higher antimicrobial activity than nisin in agar well diffusion assays against Lactococcus lactis MG1363, Listeria monocytogenes, Enterococcus faecalis VE14089, Bacillus sporothermodurans IC4 and Bacillus cereus 4153 at certain temperatures

Visualization and Analysis of the Dynamic Assembly of a Heterologous Lantibiotic Biosynthesis Complex in Bacillus subtilis

A membrane-associated lanthipeptide synthetase complex, consisting of the dehydratase NisB, the cyclase NisC, and the ABC transporter NisT, has been described for nisin biosynthesis in the coccoid bacterium Lactococcus lactis. Here, we used advanced fluorescence microscopy to visualize the functional nisin biosynthesis machinery in rod-shaped cells and analyzed its spatial distribution and dynamics employing a platform we developed for heterologous production of nisin in Bacillus subtilis. We observed that NisT, as well as NisB and NisC, were all distributed in a punctate pattern along the cell periphery, opposed to the situation in coccoid cells. NisBTC proteins were found to be highly colocalized, being visualized at the same spots by dual fluorescence microscopy. In conjunction with the successful isolation of the biosynthetic complex NisBTC from the cell membrane, this corroborated that the visual bright foci were the sites for nisin maturation and transportation. A strategy of differential timing of expression was employed to demonstrate the in vivo dynamic assembly of NisBTC, revealing the recruitment by NisT of NisBC to the membrane. Additionally, by use of mutated proteins, the nucleotide binding domain (NBD) of NisT was found to function as a membrane anchor for NisB and/or NisC. We also show that the nisin biosynthesis sites are static and likely associated with proteins residing in lipid rafts. Based on these data, we propose a model for a three-phase production of modified precursor nisin in rod-shaped bacteria, presenting the assembly dynamics of NisBTC and emphasizing the crucial role of NisBC, next to NisT, in the process of precursor nisin translocation. IMPORTANCE Nisin is a model antimicrobial peptide for LanBC-modified lantibiotics that are modified and transported by a membrane synthetase complex. Although the subcellular localization and the assembly process of such a complex in L. lactis have been described in our recent work (J. Chen, A. J. van Heel, and O. P. Kuipers, mBio 11:e02825-20, 2020, https://doi.org/10.1128/mBio.02825-20), it proved difficult to gain a more detailed insight into the exact LanBTC assembly in the L. lactis system. Rod-shaped cells, especially B. subtilis, are better suited to study the assembly dynamics of these protein complexes. In this work, we present evidence for the existence of the lanthipeptide biosynthetic complex by visualizing and isolating the machinery in vivo. The dynamic behavior of the modification machinery and the transporter within the cells was characterized in depth, revealing the dependence of first LanB and LanC on each other and subsequent recruitment of them by LanT during the machinery assembly. Importantly, the elucidation of the dynamic assembly of the complex will facilitate future studies of lanthipeptide transport mechanisms and the structural characterization of the complete complex

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

Nisin, a class I lantibiotic, is synthesized as a precursor peptide by a putative membrane-associated lanthionine synthetase complex consisting of the dehydratase NisB, the cyclase NisC, and the ABC transporter NisT. Here, we characterize the subcellular localization and the assembly process of the nisin biosynthesis machinery in Lactococcus lactis by mutational analyses and fluorescence microscopy. Precursor nisin, NisB, and NisC were found to be mainly localized at the cell poles, with a preference for the old poles. They were found to be colocalized at the same spots in these old pole regions, functioning as a nisin modification complex. In contrast, the transporter NisT was found to be distributed uniformly and circumferentially in the membrane. When nisin secretion was blocked by mutagenesis of NisT, the nisin biosynthesis machinery was also visualized directly at a polar position using fluorescence microscopy. The interactions between NisB and other components of the machinery were further studied in vivo, and therefore, the "order of assembly" of the complex was revealed, indicating that NisB directly or indirectly plays the role of a polar "recruiter" in the initial assembly process. Additionally, a potential domain that is located at the surface of the elimination domain of NisB was identified to be crucial for the polar localization of NisB. Based on these data, we propose a model wherein precursor nisin is first completely modified by the nisin biosynthesis machinery, preventing the premature secretion of partially modified peptides, and subsequently secreted by recruited NisT, preferentially at the old pole regions.IMPORTANCE 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

Employing the promiscuity of lantibiotic biosynthetic machineries to produce novel antimicrobials

As the number of new antibiotics that reach the market is decreasing and the demand for them is rising, alternative sources of novel antimicrobials are needed. Lantibiotics are potent peptide antimicrobials that are ribosomally synthesized and stabilized by post-translationally introduced lanthionine rings. Their ribosomal synthesis and enzymatic modifications provide excellent opportunities to design and engineer a large variety of novel antimicrobial compounds. The research conducted in this area demonstrates that the modularity present in both the peptidic rings as well as in the combination of promiscuous modification enzymes can be exploited to further increase the diversity of lantibiotics. Various approaches, where the modifying enzymes and corresponding leader peptides are decoupled from their natural core peptide and integrated in designed plug-and-play production systems, enable the production of modified peptides that are either derived from vast genomic data or designed using functional parts from a wide diversity of core peptides. These approaches constitute a powerful discovery platform to develop novel antimicrobials with high therapeutic potential

Specificity and Application of the Lantibiotic Protease NisP

Lantibiotics are ribosomally produced and posttranslationally modified peptides containing several lanthionine residues. They exhibit substantial antimicrobial activity against Gram-positive bacteria, including relevant pathogens. The production of the model lantibiotic nisin minimally requires the expression of the modification and export machinery. The last step during nisin maturation is the cleavage of the leader peptide. This liberates the active compound and is catalyzed by the cell wall-anchored protease NisP. Here, we report the production and purification of a soluble variant of NisP. This has enabled us to study its specificity and test its suitability for biotechnological applications. The ability of soluble NisP to cleave leaders from various substrates was tested with two sets of nisin variants. The first set was designed to investigate the influence of amino acid variations in the leader peptide or variations around the cleavage site. The second set was designed to study the influence of the lanthionine ring topology on the proteolytic efficiency. We show that the substrate promiscuity is higher than has previously been suggested. Our results demonstrate the importance of the arginine residue at the end of the leader peptide and the importance of lanthionine rings in the substrate for specific cleavage. Collectively, these data indicate that NisP is a suitable protease for the activation of diverse heterologously expressed lantibiotics, which is required to release active antimicrobial compounds

Genome-guided identification of novel head-to-tail cyclized antimicrobial peptides, exemplified by the discovery of pumilarin

The need for novel antibiotics in an era where antimicrobial resistance is on the rise, and the number of new approved antimicrobial drugs reaching the market is declining, is evident. The underused potential of post-translationally modified peptides for clinical use makes this class of peptides interesting candidates. In this study, we made use of the vast amounts of available genomic data and screened all publicly available prokaryotic genomes (~3000) to identify 394 novel head-to-tail cyclized antimicrobial peptides. To verify these in silico results, we isolated and characterized a novel antimicrobial peptide from Bacillus pumilus that we named pumilarin. Pumilarin was demonstrated to have a circular structure and showed antimicrobial activity against several indicator strains, including pathogens.MML was supported by ALW-NWO project SynMod (project number 855.01.162) and the FP7 project Synpeptide. AJvH was supported by NWO-STW programme GenBiotics (project number 10466)

Membrane composition and organization of Bacillus subtilis 168 and its genome-reduced derivative miniBacillus PG10

A form of lateral membrane compartmentalization in bacteria is represented by functional membrane microdomains (FMMs). FMMs are important for various cellular processes and offer application possibilities in microbial biotechnology. We designed a lipidomics method to directly measure relative abundances of lipids in detergent-resistant and detergent-sensitive membrane fractions of the model bacterium Bacillus subtilis 168 and the biotechnologically attractive miniBacillus PG10 strain. Our study supports previous work suggesting that cardiolipin and prenol lipids are enriched in FMMs of B. subtilis. Additionally, structural analysis of acyl chains of major phospholipids indicated that FMMs display increased order and thickness compared with the surrounding bilayer. Despite the 36% genome reduction, membrane and FMM integrity are largely preserved in miniBacillus PG10, as supported by analysis of membrane fluidity, flotillin distribution and gene expression data. The novel insights in FMM architecture reported here will contribute to further explore the biological significance of FMMs and the means by which FMMs can be exploited as heterologous production platforms. Moreover, our lipidomics method enables comparative FMM lipid profiling between different bacteria.ISSN:1751-7915ISSN:1751-790