Nisin system

The development of bacterial resistance against clinically relevant antibiotics is on the rise and represents a major scientific challenge. There is an ever growing demand for novel and improved antimicrobial agents. As potential antibiotics, bactericidal peptides that are secreted by many bacteria, mainly for self-defense purposes, have gained special interest. Among these are the so-called lantibiotics, which are ribosomally synthesized peptides that are posttranslationally modified and produced by a large number of Gram-positive bacteria. Nisin, probably the most well-known lantibiotic, is produced by the Gram-positive bacterium Lactococcus lactis and its antimicrobial activity is directed against microorganisms of similar type.

Nisin is produced by L. lactis as a prepeptide consisting of 57 amino acids and is the best-studied lantibiotic. After maturation, active nisin consists of 34 amino acids. Its antimicrobial activity against Gram-positive bacteria and was first described in 1928 by Roger Whittier who showed that a substance excreted by lactis cells inhibited growth of other species as well as the growth of the producer strain after the culture reached a certain cell density. Nisin is widely used as a food preservative since 50 years for example in milk products, canned vegetables, meat and fish and is marked with E number E234. Nisin is highly active against Gram-positive bacteria such as Bacillus cereus, Listeria monocytogenes, Enterococci, Staphylococci and Streptococci.

All proteins involved in nisin biosynthesis, modification, transport and immunity are encoded on a single operon. This operon consist of 11 genes and the function of the proteins is displayed in figure 1. The gene nisA encodes the prepeptide of the lantibiotic. The modification and secretion machinery is encoded by the genes nisBTCP. To protect the producer host against its own secreted antimicrobial peptide the immunity genes are nisI and nisFEG. Additionally nisin can auto-regulate its own biosynthesis via the two component system encoded on the genes nisK and nisR.

Nisin is ribosomally synthesized as a 57 amino acids long prepeptide. The prepeptide is subdivided in a N-terminal leader peptide comprising 23 amino acids and a 34 amino acid long propeptide. The whole maturation and biosynthesis process is shown in Figure 2. Prenisin is modified posttranslationally by a specific modification machinery, which is responsible for dehydration (NisB), cyclization (NisC), export (NisT) and cleavage of the leader peptide (NisP).

The dehydratase NisB is believed to be the initial interaction partner in modification. NisB dehydrates specific serine and threonine residues in prenisin, whereas the cyclase NisC catalyzes the (methyl)lanthionine formation. The fully modified prenisin is exported and the leader peptide is cleaved off by the extracellular protease NisP. Light scattering analysis demonstrated that purified NisB is a dimer in solution. Using size exclusion chromatography and surface plasmon resonance, the interaction of NisB and prenisin, including several of its modified derivatives, was studied. Unmodified prenisin binds to NisB with an affinity of 1.05 ± 0.25 mM, whereas the dehydrated and the fully modified derivatives bind with respective affinities of 0.31 ± 0.07 mM and 10.5 ± 1.7 mM. The much lower affinity for the fully modified prenisin related to a >20-fold higher off rate. For all three peptides the stoichiometry of binding was 1:1. Active nisin, which is the equivalent of fully modified prenisin lacking the leader peptide did not bind to NisB, nor did prenisin in which the highly conserved FNLD-box within the leader peptide was mutated to AAAA. Taken together our data indicate that the leader peptide is essential for initial recognition and binding of prenisin to NisB.

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A detailed in vitro study of the interaction between NisC and the nisin precursor peptides unraveled a specific interaction of NisC with the leader peptide independent of the maturation state. Furthermore, mutagenesis studies identified a specific binding sequence within the leader. Two amino acids (F_-18 and L_-16) within the highly conserved –FNLD- box of class I lantibiotics are essential for binding. They represent a potential general binding motif between leader peptides of lantibiotics with their specific cyclases. In summary, these in vitro data provide a new perception on the complexity of the lantibiotic modification machineries.

Moving one step further, we characterized the assembly of the NisBC complex in vitro, which is only formed in the presence of the substrate. The complex is composed of a NisB dimer, a monomer of NisC and one prenisin molecule. Interestingly, the presence of the last lanthionine ring prevented complex formation. This stoichiometry was verified by small-angle X-ray scattering  measurements, which revealed the first structural glimpse of a LanBC complex in solution. the data obtained in this study identified two factors influencing complex formation of the maturation enzymes NisB and NisC, respectively. First the core peptide, it can be dehydrated and also particular modified. In vitro the presence of the last (methyl)-lanthionine ring, ring E, abolished complex formation. Second, the N-terminal leader peptide plays an important role. The highly conserved -FNLD- box is an essential recognition factor for the modification enzymes NisB and NisC. Finally, the MALS-SEC analysis revealed the first quantitative data elucidating the stoichiometry of the nisin maturation complex. This complex revealed a molecular weight of approximately 291 kDa corresponding to a stoichiometry of 2:1:1 (NisB / NisC / prenisin peptide) in vitro.

The leader peptide serves as a signal sequence and ensures, that NisA remains inactive and secretion-competent within the cell. After translocation into the extracellular space the leader peptide is cleaved by the leader peptidase NisP resulting in active nisin. NisP is an extracellular subtilisin-like serine protease, which recognizes the cleavage site GASPR|IT located at the C-terminal end of the leader peptide. Here, we present the biochemical characterization of secreted and purified NisP (NisP_s) with its natural substrate, the fully modified NisA (mNisA). Furthermore, we determined the kinetic parameters of NisP_s in the presence of NisA in different modification states. Additionally, in vitro data revealed that NisP_s is able to cleave the leader peptide of mNisA efficiently. However, it is strictly dependent on the modification states of the core peptide. Thus, NisP_s has a sequence-based cleavage activity and the presence of at least one lanthionine-ring is crucial for optimal substrate recognition and subsequent cleavage.

Lagedroste, M., J. Reiners, S.H.J. Smits & L. Schmitt, (2019) Systematic characterization of position one variants within the lantibiotic nisin. Sci Rep 9: 935.

Reiners, J., A. Abts, R. Clemens, S.H. Smits & L. Schmitt, (2017) Stoichiometry and structure of a lantibiotic maturation complex. Sci Rep 7: 42163.

Lagedroste, M., S.H.J. Smits & L. Schmitt, (2017) Substrate Specificity of the Secreted Nisin Leader Peptidase NisP. Biochemistry 56: 4005-4014.

Abts, A., M. Montalban-Lopez, O.P. Kuipers, S.H. Smits & L. Schmitt, (2013) NisC binds the FxLx motif of the nisin leader peptide. Biochemistry 52: 5387-5395.

Mavaro, A., A. Abts, P.J. Bakkes, G.N. Moll, A.J. Driessen, S.H. Smits & L. Schmitt, (2011) Substrate recognition and specificity of the NisB protein, the lantibiotic dehydratase involved in nisin biosynthesis. The Journal of biological chemistry 286: 30552-30560.

Abts, A., A. Mavaro, J. Stindt, P.J. Bakkes, S. Metzger, A.J. Driessen, S.H. Smits & L. Schmitt, (2011) Easy and rapid purification of highly active nisin. Int J Pept 2011: 175145.