In vitro mutasynthesis of lantibiotic analogues containing nonproteinogenic amino acids.
ABSTRACT: Lantibiotics are ribosomally synthesized and post-translationally modified peptide antibiotics containing the characteristic thioether cross-links lanthionine and methyllanthionine. To date, no analogues of lantibiotics that contain nonproteinogenic amino acids have been reported. In this study, in vitro-reconstituted lacticin 481 synthetase was used in conjunction with synthetic peptide substrates containing nonproteinogenic amino acids to generate 11 analogues of lacticin 481. These analogues contained sarcosine and aminocyclopropanoic acid in place of Gly5, D-valine at position 6, 4-cyanoaminobutyric acid in place of Glu13, beta(3)-homoarginine at the position of Asn15, N-butylglycine and beta-Ala at Met16, naphthylalanine (Nal) at Trp19, 4-pyridynylalanine (Pal) at Phe21, and homophenylalanine (hPhe) at Phe23. Of these analogues, the Trp19Nal and Phe23hPhe mutants provided zones of inhibition larger than the parent compound in agar diffusion assays against the indicator strains Lactococcus lactis HP and Bacillus subtilis 6633. These two compounds also demonstrated improved MIC values against liquid cultures of L. lactis HP.
Project description:Lantibiotics are ribosomally synthesized and post-translationally modified peptide natural products that contain thioether cross-links formed by lanthionine and methyllanthionine residues. They exert potent antimicrobial activity against Gram-positive bacteria. We herein report production of analogues of two lantibiotics, lacticin 481 and nisin, that contain nonproteinogenic amino acids using two different strategies involving amber stop codon suppression technology. These methods complement recent alternative approaches to incorporate nonproteinogenic amino acids into lantibiotics.
Project description:Lantibiotics are peptide antimicrobial compounds that are characterized by the thioether-bridged amino acids lanthionine and methyllanthionine. For lacticin 481, these structures are installed in a two-step post-translational modification process by a bifunctional enzyme, lacticin 481 synthetase (LctM). LctM catalyzes the dehydration of Ser and Thr residues to generate dehydroalanine or dehydrobutyrine, respectively, and the subsequent intramolecular regio- and stereospecific Michael-type addition of cysteines onto the dehydroamino acids. In this study, semisynthetic substrates containing nonproteinogenic amino acids were prepared by expressed protein ligation and [3+2]-cycloaddition of azide and alkyne-functionalized peptides. LctM demonstrated broad substrate specificity toward substrates containing beta-amino acids, D-amino acids, and N-alkyl amino acids (peptoids) in certain regions of its peptide substrate. These findings showcase its promise for use in lantibiotic and peptide-engineering applications, whereby nonproteinogenic amino acids might impart improved stability or modulated biological activities. Furthermore, LctM permitted the incorporation of an alkyne-containing amino acid that can be utilized for the site-selective modification of mature lantibiotics and used in target identification.
Project description:Lantibiotics are ribosomally synthesized and post-translationally modified peptide natural products that contain the thioether structures lanthionine and methyllanthionine and exert potent antimicrobial activity against Gram-positive bacteria. At present, detailed modes-of-action are only known for a small subset of family members. Lacticin 481, a tricyclic lantibiotic, contains a lipid II binding motif present in related compounds such as mersacidin and nukacin ISK-1. Here, we show that lacticin 481 inhibits PBP1b-catalyzed peptidoglycan formation. Furthermore, we show that changes in potency of analogues of lacticin 481 containing non-proteinogenic amino acids correlate positively with the potency of inhibition of the transglycosylase activity of PBP1b. Thus, lipid II is the likely target of lacticin 481, and use of non-proteinogenic amino acids resulted in stronger inhibition of the target. Additionally, we demonstrate that lacticin 481 does not form pores in the membranes of susceptible bacteria, a common mode-of-action of other lantibiotics.
Project description:Lantibiotics are a family of antibacterial peptide natural products characterized by the post-translational installation of the thioether-containing amino acids lanthionine and methyllanthionine. Until recently, only a single naturally occurring stereochemical configuration for each of these cross-links was known. The discovery of lantibiotics with alternative lanthionine and methyllanthionine stereochemistry has prompted an investigation of its importance to biological activity. Here, solid-supported chemical synthesis enabled the total synthesis of the lantibiotic lacticin 481 and analogues containing cross-links with non-native stereochemical configurations. Biological evaluation revealed that these alterations abolished the antibacterial activity in all of the analogues, revealing the critical importance of the enzymatically installed stereochemistry for the biological activity of lacticin 481.
Project description:Lantibiotics are post-translationally modified peptide antimicrobial agents that are synthesized with an N-terminal leader sequence and a C-terminal propeptide. Their maturation involves enzymatic dehydration of Ser and Thr residues in the precursor peptide to generate unsaturated amino acids, which react intramolecularly with nearby cysteines to form cyclic thioethers termed lanthionines and methyllanthionines. The role of the leader peptide in lantibiotic biosynthesis has been subject to much speculation. In this study, mutations of conserved residues in the leader sequence of the precursor peptide for lacticin 481 (LctA) did not inhibit dehydration and cyclization by lacticin 481 synthetase (LctM) showing that not one specific residue is essential for these transformations. These amino acids may therefore be conserved in the leader sequence of class II lantibiotics to direct other biosynthetic events, such as proteolysis of the leader peptide or transport of the active compound outside the cell. However, introduction of Pro residues into the leader peptide strongly affected the efficiency of dehydration, consistent with recognition of the secondary structure of the leader peptide by the synthetase. Furthermore, the presence of a hydrophobic residue at the position of Leu-7 appears important for enzymatic processing. Based on the data in this work and previous studies, a model for the interaction of LctM with LctA is proposed. The current study also showcases the ability to prepare other lantibiotics in the class II lacticin 481 family, including nukacin ISK-1, mutacin II, and ruminococcin A using the lacticin 481 synthetase. Surprisingly, a conserved Glu located in a ring that appears conserved in many class II lantibiotics, including those not belonging to the lacticin 481 subgroup, is not essential for antimicrobial activity of lacticin 481.
Project description:Class I bacteriocins (lantibiotics) and class II bacteriocins are antimicrobial peptides secreted by gram-positive bacteria. Using two lantibiotics, lacticin 481 and nisin, and the class II bacteriocin coagulin, we showed that bacteriocins can be detected without any purification from whole producer bacteria grown on plates by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). When we compared the results of MALDI-TOF-MS performed with samples of whole cells and with samples of crude supernatants of liquid cultures, the former samples led to more efficient bacteriocin detection and required less handling. Nisin and lacticin 481 were both detected from a mixture of their producer strains, but such a mixture can yield additional signals. We used this method to determine the masses of two lacticin 481 variants, which confirmed at the peptide level the effect of mutations in the corresponding structural gene.
Project description:The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis strains. The genetic determinants of lacticin 481 production are organized as an operon encoded by a 70-kb plasmid. We previously reported the first three genes of this operon, lctA, lctM, and lctT, which are involved in the bacteriocin biosynthesis and export (A. Rincé, A. Dufour, S. Le Pogam, D. Thuault, C. M. Bourgeois, and J.-P. Le Pennec, Appl. Environ. Microbiol. 60:1652-1657, 1994). The operon contains three additional open reading frames: lctF, lctE, and lctG. The hydrophobicity profiles and sequence similarities strongly suggest that the three gene products associate to form an ABC transporter. When the three genes were coexpressed into a lacticin 481-sensitive L. lactis strain, the strain became resistant to the bacteriocin. This protection could not be obtained when any of the three genes was deleted, confirming that lctF, lctE, and lctG are all necessary to provide immunity to lacticin 481. The quantification of the levels of immunity showed that lctF, lctE, and lctG could account for at least 6% and up to 100% of the immunity of the wild-type lacticin 481 producer strain, depending on the gene expression regulation. The lacticin 481 biosynthesis and immunity systems are discussed and compared to other lantibiotic systems.
Project description:Class AII and AIII lantibiotics and mersacidin are antibacterial peptides containing unusual residues obtained by posttranslational modifications of prepeptides, presumably catalyzed by LanM. LctM, the LanM for lacticin 481, is essential for the production of this class AII lantibiotic. Using the yeast two-hybrid system, we showed direct contact between the prelacticin 481 and LctM, supporting the proposed LctM function. Sixteen domains are conserved between the 10 known LanM proteins, whereas three additional domains were found only in class AII LanM proteins and in MrsM, the LanM for mersacidin. All the truncated LctM proteins that we tested presented impaired LctA-binding activity.
Project description:Labeling of natural products with biophysical probes has greatly contributed to investigations of their modes of action and has provided tools for visualization of their targets. A general challenge is the availability of a suitable functional group for chemoselective modification. We demonstrate here that an N-terminal ketone is readily introduced into various lanthipeptides by the generation of a cryptic N-terminal dehydro amino acid by the cognate biosynthetic enzymes. Spontaneous hydrolysis of the N-terminal enamines results in ?-ketoamides that site-specifically react with an aminooxy-derivatized alkyne or fluorophore. The methodology was successfully applied to prochlorosins 1.7 and 2.8, as well as the lantibiotics lacticin 481, haloduracin ?, and haloduracin ?. The fluorescently modified lantibiotics were added to bacteria, and their cellular localization was visualized by confocal fluorescence microscopy. Lacticin 481 and haloduracin ? localized predominantly at sites of new and old cell division as well as in punctate patterns along the long axis of rod-shaped bacilli, similar to the localization of lipid II. On the other hand, haloduracin ? was localized nonspecifically in the absence of haloduracin ?, but formed specific patterns when coadministered with haloduracin ?. Using two-color labeling, colocalization of both components of the two-component lantibiotic haloduracin was demonstrated. These data with living cells supports a model in which the ? component recognizes lipid II and then recruits the ?-component.
Project description:A new lanthionine-containing bacteriocin, variacin, displaying a broad host range of inhibition against gram-positive food spoilage bacteria, has been identified from two strains of Micrococcus varians isolated from meat fermentations. The new bacteriocin was purified, and its amino-terminal end and total amino acid composition were determined. The structural gene was isolated and analyzed. Variacin is resistant to heat and pH conditions from 2 to 10. Its primary sequence shows significant homology to lacticin 481 to Lactococcus lactis, which is more pronounced for the probacteriocin than for the leader sequence. Variacin, like lacticin 481, contains lanthionine and beta-methyllanthionine residues, but its leader sequence clearly resembles nonlantibiotic leader sequences. In particular, the prepeptide contains glycine residues at positions -1 and -2 of the processing site.