Identification of essential amino acid residues in the nisin dehydratase NisB.

Nisin is a posttranslationally-modified antimicrobial peptide that has the ability to induce its own biosynthesis. Serines and threonines in the modifiable core peptide part of precursor nisin are dehydrated to dehydroalanines and dehydrobutyrines by the dehydratase NisB, and subsequently cysteines are coupled to the dehydroamino acids by the cyclase NisC. In this study, we applied extensive site-directed mutagenesis, together with direct binding studies, to investigate the molecular mechanism of the dehydratase NisB. We use a natural nisin-producing strain as a host to probe mutant-NisB functionality. Importantly, we are able to differentiate between intracellular and secreted fully dehydrated precursor nisin, enabling investigation of the NisB properties needed for the release of dehydrated precursor nisin to its devoted secretion system NisT. We report that single amino acid substitutions of conserved residues, i.e., R83A, R83M, and R87A result in incomplete dehydration of precursor nisin and prevention of secretion. Single point NisB mutants Y80F and H961A, result in a complete lack of dehydration of precursor nisin, but do not abrogate precursor nisin binding. The data indicate that residues Y80 and H961 are directly involved in catalysis, fitting well with their position in the recently published 3D-structure of NisB. We confirm, by in vivo studies, results that were previously obtained from in vitro experiments and NisB structure elucidation and show that previous findings translate well to effects seen in the original production host.

The presence of modifiable residues in the core peptide part of precursor nisin is not crucial for precursor nisin interactions with NisB- and NisC.

Precursor nisin is a model posttranslationally modified precursor lantibiotic that can be structurally divided into a leader peptide sequence and a modifiable core peptide part. The nisin core peptide clearly plays an important role in the precursor nisin-nisin modification enzymes interactions, since it has previously been shown that the construct containing only the nisin leader sequence is not sufficient to pull-down the nisin modification enzymes NisB and NisC. Serines and threonines in the core peptide part are the residues that NisB specifically dehydrates, and cysteines are the residues that NisC stereospecifically couples to the dehydrated amino acids. Here, we demonstrate that increasing the number of negatively charged residues in the core peptide part of precursor nisin, which are absent in wild-type nisin, does not abolish binding of precursor nisin to the modification enzymes NisB and NisC, but dramatically decreases the antimicrobial potency of these nisin mutants. An unnatural precursor nisin variant lacking all serines and threonines in the core peptide part and an unnatural precursor nisin variant lacking all cysteines in the core peptide part still bind the nisin modification enzymes NisB and NisC, suggesting that these residues are not essential for direct interactions with the nisin modification enzymes NisB and NisC. These results are important for lantibiotic engineering studies.

Identification of distinct nisin leader peptide regions that determine interactions with the modification enzymes NisB and NisC.

Nisin is the most prominent and applied bacteriocin that serves as a model for class I lantibiotics. The nisin leader peptide importantly determines interactions between precursor nisin and its modification enzymes NisB and NisC that mature nisin posttranslationally. NisB dehydrates serines and threonines, while NisC catalyzes the subsequent coupling of the formed dehydroamino acids to form lanthionines. Currently, little is known about how the nisin leader interacts with NisB and even less is known about its interactions with NisC. To investigate the nisin leader peptide requirements for functional interaction with the modification enzymes NisB and NisC, we systematically replaced six regions, of 2-4 amino acids each, with all-alanine regions. By performing NisB and NisC co-purification studies with these mutant leader peptides, we demonstrate that the nisin leader regions STKD(-22-19), FNLD(-18-15) and PR(-2-1) importantly contribute to the interactions of precursor nisin with both NisB and NisC, whereas the nisin leader region LVSV(-14-11) additionally contributes to the interaction of precursor nisin with NisC.

When the leader gets loose: in vivo biosynthesis of a leaderless prenisin is stimulated by a trans-acting leader peptide.

The nisin leader is believed to be crucial for nisin biosynthesis. Here, by using a construct completely lacking the leader peptide, we show that an up to fivefold-dehydrated leaderless prenisin can be obtained, as judged by MALDI-TOF MS, and that some of these species are biologically active, thus suggesting that at least three lanthionine rings are present. Notably, by expressing the leader peptide in trans together with the leaderless prenisin, we were able to increase the dehydration/cyclization efficiency of both NisB and NisC, but still with limited efficiency until the fifth dehydratable residue (Thr13) was processed, thereby enabling three rings to form. This, for the first time, demonstrates that 1) the leader is not absolutely necessary for the dehydration reaction of class I lantibiotics to occur in vivo; 2) the leader acts in trans in vivo; 3) the leader increases the efficiency of modification. Based on previous work and our current study, a model for the interactions of NisB and NisC with prenisin is proposed, in which the leader induces a more active conformation and/or productive complex formation of the biosynthetic machinery, and, when covalently bound, is involved in increasing the efficiency of dehydration to the C-terminal end of the prenisin substrate molecule.

Determining sites of interaction between prenisin and its modification enzymes NisB and NisC.

Although nisin is a model lantibiotic, our knowledge of the specific interactions of prenisin with its modification enzymes remains fragmentary. Here, we demonstrate that the nisin modification enzymes NisB and NisC can be pulled down in vitro from Lactococcus lactis by an engineered His-tagged prenisin. This approach enables us to determine important intermolecular interactions of prenisin with its modification machinery within L. lactis. We demonstrate that (i) NisB has stronger interactions with precursor nisin than NisC has, (ii) deletion of the propeptide part keeping the nisin leader intact leads to a lack of binding, (iii) NisB point mutants of highly conserved residues W616, F342A, Y346F and P639A are still able to dehydrate prenisin, (iv) NisB Δ(77-79)Y80F mutant decreased the levels of NisB-prenisin interactions and resulted in unmodified prenisin, (v) substitution of an active site residue H331A in NisC leads to higher amounts of the co-purified complex, (vi) NisB is present in the form of a dimer, and (vii) the region FNLD (-18 to -15) of the leader is an important site for binding not only to NisB, but also to NisC.

Directionality and coordination of dehydration and ring formation during biosynthesis of the lantibiotic nisin.

The lantibiotic nisin is a potent antimicrobial substance, which contains unusual lanthionine rings and dehydrated amino acid residues and is produced by Lactococcus lactis. Recently, the nisin biosynthetic machinery has been applied to introduce lanthionine rings in peptides other than nisin with potential therapeutic use. Due to difficulties in the isolation of the proposed synthetase complex (NisBTC), mechanistic information concerning the enzymatic biosynthesis of nisin is scarce. Here, we present the molecular characterization of a number of nisin mutants that affect ring formation. We have investigated in a systematic manner how these mutations influence dehydration events, which are performed enzymatically by the dehydratase NisB. Specific mutations that hampered ring formation allowed for the dehydration of serine residues that directly follow the rings and are normally unmodified. The combined information leads to the conclusion that 1) nisin biosynthesis is an organized directional process that starts at the N terminus of the molecule and continues toward the C terminus, and 2) NisB and NisC are alternating enzymes, whose activities follow one after another in a repetitive way. Thus, the dehydration and cyclization processes are not separated in time and space. On the basis of these results and previous knowledge, a working model for the sequence of events in the maturation of nisin is proposed.

Stem cell based therapeutical approach of male infertility by teratocarcinoma derived germ cells.

Infertility affects 13-18% of couples and growing evidence from clinical and epidemiological studies suggests an increasing incidence of male reproductive problems. There is a male factor involved in up to half of all infertile couples. The pathogenesis of male infertility can be reflected by defective spermatogenesis due to failure in germ cell proliferation and differentiation. We report here in vitro generation of a germ cell line (SSC1) from the pluripotent teratocarcinoma cells by a novel promoter-based sequential selection strategy and show that the SSC1 cell line form mature seminiferous tubule structures, and support spermatogenesis after transplantation into recipient testes. To select differentiated germ cell population, we generated a fusion construct (Stra8-EGFP) harbouring the 1.4 kb promoter region of germ line specific gene Stra8 and coding region of enhanced green fluorescence protein. This region was sufficient to direct gene expression to the germinal stem cells in testis of transgenic mice. The purified cells expressed the known molecular markers of spermatogonia Rbm, cyclin A2, Tex18, Stra8 and Dazl and the beta1- and alpha6-integrins characteristic of the stem cell fraction. This cell line undergoes meiosis and can develop into sperm when transplanted into germ cell depleted testicular tubules. Sperm were viable and functional, as shown by fertilization after intra-cytoplasmic injection into mouse oocytes. This approach provides the basis that is essential for studying the development and differentiation of male germ line stem cell, as well as for developing new approaches to reproductive engineering and infertility treatment.