Breaking Cycles: Saponification-Enhanced NMR Fingerprint Matching for the Identification and Stereochemical Evaluation of Cyclic Lipodepsipeptides from Natural Sources.

We previously described NMR based fingerprint matching with peptide backbone resonances as a fast and reliable structural dereplication approach for Pseudomonas cyclic lipodepsipeptides (CLiPs). In combination with total synthesis of a small library of configurational CLiP congeners this also allows unambiguous determination of stereochemistry, facilitating structure-activity relationship studies and enabling three-dimensional structure determination. However, the on-resin macrocycle formation in the synthetic workflow brings considerable burden and limits universal applicability. This drawback is here removed altogether by also transforming the native CLiP into a linearized analogue by controlled saponification of the ester bond. This eliminates the need for macrocycle formation, limiting the synthesis effort to linear peptide analogues. NMR fingerprints of such linear peptide analogues display a sufficiently distinctive chemical shift fingerprint to act as effective discriminators. The approach is developed using viscosin group CLiPs and subsequently demonstrated on putisolvin, leading to a structural revision, and tanniamide from Pseudomonas ekonensis COR58, a newly isolated lipododecapeptide that defines a new group characterized by a ten-residue large macrocycle, the largest to date in the Pseudomonas CLiP portfolio. These examples demonstrate the effectiveness of the saponification- enhanced approach that broadens applicability of NMR fingerprint matching for the determination of the stereochemistry of CLiPs.

Selenium reduction pathways in the colloidal synthesis of CdSe nanoplatelets.

Several established procedures are now available to prepare zinc blende CdSe nanoplatelets. While these protocols allow for detailed control over both thickness and lateral dimensions, the chemistry behind their formation is yet to be unraveled. In this work, we discuss the influence of the solvent on the synthesis of nanoplatelets. We confirmed that the presence of double bonds, as is the case for 1-octadecene, plays a key role in the evolution of nanoplatelets, through the isomerization of the alkene, as confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry. Consequently, 1-octadecene can be replaced as a solvent (or solvent mixture), however, only by one that also contains α protons to CC double bonds. We confirm this via synthesis of nanoplatelets in hexadecane spiked with a small amount of 1-octadecene, and in the aromatic solvent 1,2,3,4-tetrahydronaphthalene (tetralin). At the same time, the chemical reaction leading to the formation of nanoplatelets occurs to some extent in saturated solvents. A closer examination revealed that an alternative formation pathway is possible, through interaction of carboxylic acids, such as octanoic acid, with selenium. Next to shedding more light on the synthesis of CdSe nanoplatelets, fundamental understanding of the precursor chemistry paves the way to use optimized solvent admixtures as an additional handle to control the nanoplatelet synthesis, as well as to reduce potential self-polymerization hurdles observed with 1-octadecene.

Stereomeric Lipopeptides from a Single Non-Ribosomal Peptide Synthetase as an Additional Source of Structural and Functional Diversification in Pseudomonas Lipopeptide Biosynthesis.

In Pseudomonas lipopeptides, the D-configuration of amino acids is generated by dedicated, dual-function epimerization/condensation (E/C) domains. The increasing attention to stereochemistry in lipopeptide structure elucidation efforts has revealed multiple examples where epimerization does not occur, even though an E/C-type domain is present. While the origin of the idle epimerization in those E/C-domains remains elusive, epimerization activity has so far shown a binary profile: it is either 'on' (active) or 'off' (inactive). Here, we report the unprecedented observation of an E/C-domain that acts 'on and off', giving rise to the production of two diastereoisomeric lipopeptides by a single non-ribosomal peptide synthetase system. Using dereplication based on solid-phase peptide synthesis and NMR fingerprinting, we first show that the two cyclic lipopeptides produced by Pseudomonas entomophila COR5 correspond to entolysin A and B originally described for P. entomophila L48. Next, we prove that both are diastereoisomeric homologues differing only in the configuration of a single amino acid. This configurational variability is maintained in multiple Pseudomonas strains and typically occurs in a 3:2 ratio. Bioinformatic analysis reveals a possible correlation with the composition of the flanking sequence of the N-terminal secondary histidine motif characteristic for dual-function E/C-type domains. In permeabilization assays, using propidium iodide entolysin B has a higher antifungal activity compared to entolysin A against Botrytis cinerea and Pyricularia oryzae spores. The fact that configurational homologues are produced by the same NRPS system in a Pseudomonas strain adds a new level of structural and functional diversification to those already known from substrate flexibility during the recruitment of the amino acids and fatty acids and underscores the importance of complete stereochemical elucidation of non-ribosomal lipopeptide structures.

Charting the Lipopeptidome of Nonpathogenic Pseudomonas.

A major source of pseudomonad-specialized metabolites is the nonribosomal peptide synthetases (NRPSs) assembling siderophores and lipopeptides. Cyclic lipopeptides (CLPs) of the Mycin and Peptin families are frequently associated with, but not restricted to, phytopathogenic species. We conducted an in silico analysis of the NRPSs encoded by lipopeptide biosynthetic gene clusters in nonpathogenic Pseudomonas genomes, covering 13 chemically diversified families. This global assessment of lipopeptide production capacity revealed it to be confined to the Pseudomonas fluorescens lineage, with most strains synthesizing a single type of CLP. Whereas certain lipopeptide families are specific for a taxonomic subgroup, others are found in distant groups. NRPS activation domain-guided peptide predictions enabled reliable family assignments, including identification of novel members. Focusing on the two most abundant lipopeptide families (Viscosin and Amphisin), a portion of their uncharted diversity was mapped, including characterization of two novel Amphisin family members (nepenthesin and oakridgin). Using NMR fingerprint matching, known Viscosin-family lipopeptides were identified in 15 (type) species spread across different taxonomic groups. A bifurcate genomic organization predominates among Viscosin-family producers and typifies Xantholysin-, Entolysin-, and Poaeamide-family producers but most families feature a single NRPS gene cluster embedded between cognate regulator and transporter genes. The strong correlation observed between NRPS system phylogeny and rpoD-based taxonomic affiliation indicates that much of the structural diversity is linked to speciation, providing few indications of horizontal gene transfer. The grouping of most NRPS systems in four superfamilies based on activation domain homology suggests extensive module dynamics driven by domain deletions, duplications, and exchanges. IMPORTANCE Pseudomonas species are prominent producers of lipopeptides that support proliferation in a multitude of environments and foster varied lifestyles. By genome mining of biosynthetic gene clusters (BGCs) with lipopeptide-specific organization, we mapped the global Pseudomonas lipopeptidome and linked its staggering diversity to taxonomy of the producers, belonging to different groups within the major Pseudomonas fluorescens lineage. Activation domain phylogeny of newly mined lipopeptide synthetases combined with previously characterized enzymes enabled assignment of predicted BGC products to specific lipopeptide families. In addition, novel peptide sequences were detected, showing the value of substrate specificity analysis for prioritization of BGCs for further characterization. NMR fingerprint matching proved an excellent tool to unequivocally identify multiple lipopeptides bioinformatically assigned to the Viscosin family, by far the most abundant one in Pseudomonas and with stereochemistry of all its current members elucidated. In-depth analysis of activation domains provided insight into mechanisms driving lipopeptide structural diversification.

Altering in vivo membrane sterol composition affects the activity of the cyclic lipopeptides tolaasin and sessilin against Pythium.

Cyclic lipopeptides (CLiPs) are secondary metabolites produced by a variety of bacteria. These compounds show a broad range of antimicrobial activities; therefore, they are studied for their potential applications in agriculture and medicine. It is generally assumed that the primary target of the CLiPs is the cellular membrane, where they can permeabilize the lipid bilayer. Model membrane systems are commonly used to investigate the effect of lipid composition on the permeabilizing activity of CLiPs, but these systems do not represent the full complexity of true biological membranes. Here, we introduce a novel method that uses sterol-auxotrophic oomycetes to investigate how the activity of membrane-active compounds is influenced by alterations in membrane sterol composition. More specifically, we investigated how ergosterol, cholesterol, beta-sitosterol and stigmasterol affect the activity of the structurally related Pseudomonas-derived CLiPs tolaasin and sessilin against the oomycete Pythium myriotylum. Both compounds were effective against oomycetes, although tolaasin was considerably more active. Interestingly, tolaasin and sessilin effects were similarly reduced by the presence of sterols, with cholesterol showing the highest reduction of activity.

An Nuclear Magnetic Resonance Fingerprint Matching Approach for the Identification and Structural Re-Evaluation of Pseudomonas Lipopeptides.

Cyclic lipopeptides (CLiPs) are secondary metabolites secreted by a range of bacterial phyla. CLiPs from Pseudomonas in particular, display diverse structural variations in terms of the number of amino acid residues, macrocycle size, amino acid identity, and stereochemistry (e.g., d- versus l-amino acids). Reports detailing the discovery of novel or already characterized CLiPs from new sources appear regularly in literature. Increasingly, however, the lack of detailed characterization threatens to cause considerable confusion, especially if configurational heterogeneity is present for one or more amino acids. Using Pseudomonas CLiPs from the Bananamide, Orfamide, and Xantholysin groups as test cases, we demonstrate and validate that the combined 1H and 13C Nuclear Magnetic Resonance (NMR) chemical shifts of CLiPs constitute a spectral fingerprint that is sufficiently sensitive to differentiate between possible diastereomers of a particular sequence even when they only differ in a single d/l configuration. Rapid screening, involving simple matching of the NMR fingerprint of a newly isolated CLiP with that of a reference CLiP of known stereochemistry, can then be applied to resolve dead-ends in configurational characterization and avoid the much more cumbersome chemical characterization protocols. Even when the stereochemistry of a particular reference CLiP remains to be established, its spectral fingerprint allows to quickly verify whether a newly isolated CLiP is novel or already present in the reference collection. We show NMR fingerprinting leads to a simple approach for early on dereplication which should become more effective as more fingerprints are collected. To benefit research involving CLiPs, we have made a publicly available data repository accompanied by a 'knowledge base' at https://www.rhizoclip.be, where we present an overview of published NMR fingerprint data of characterized CLiPs, together with literature data on the originally determined structures. IMPORTANCE Pseudomonas CLiPs are ubiquitous specialized metabolites, impacting the producer's lifestyle and interactions with the (a)biotic environment. Consequently, they generate interest for agricultural and clinical applications. Establishing structure-activity relationships as a premise to their development is hindered because full structural characterization including stereochemical information requires labor-intensive analyses, without guarantee for success. Moreover, increasing use of superficial comparison with previously characterized CLiPs introduces or propagates erroneous attributions, clouding further scientific progress. We provide a generally applicable characterization methodology based on matching NMR spectral fingerprints of newly isolated CLiPs to natural and synthetic reference compounds with (un)known stereochemistry. In addition, NMR fingerprinting is shown to provide a suitable basis for structural dereplication. A publicly available reference compound repository promises to facilitate participation of the lipopeptide research community in structural assessment and dereplication of newly isolated CLiPs, which should also support further developments in genome mining for novel CLiPs.

The effect of membrane thickness on the membrane permeabilizing activity of the cyclic lipopeptide tolaasin II.

Tolaasin II is an amphiphilic, membrane-active, cyclic lipopeptide produced by Pseudomonas tolaasii and is responsible for brown blotch disease in mushroom. To better understand the mode of action and membrane selectivity of tolaasin II and related lipopeptides, its permeabilizing effect on liposomes of different membrane thickness was characterized. An equi-activity analysis served to distinguish between the effects of membrane partitioning and the intrinsic activity of the membrane-bound peptide. It was found that thicker membranes require higher local peptide concentrations to become leaky. More specifically, the mole ratio of membrane-bound peptide per lipid needed to induce 50% leakage of calcein within 1 h, Re 50, increased monotonically with membrane thickness from 0.0016 for the 14:1 to 0.0070 for the 20:1 lipid-chains. Moreover, fast but limited leakage kinetics in the low-lipid regime were observed implying a mode of action based on membrane asymmetry stress in this time and concentration window. While the assembly of the peptide to oligomeric pores of defined length along the bilayer z-axis can in principle explain inhibition by increasing membrane thickness, it cannot account for the observed limited leakage. Therefore, reduced intrinsic membrane-permeabilizing activity with increasing membrane thickness is attributed here to the increased mechanical strength and order of thicker membranes.