Daniela Reimer

A natural prodrug activation mechanism in nonribosomal peptide synthesis

We have identified a new mechanism for the cleavage and activation of nonribosomally made peptides and peptide-polyketide hybrids that are apparently operational in several different bacteria. This process includes the cleavage of a precursor molecule by a membrane-bound and D-asparagine-specific peptidase, as shown here in the biosynthesis of the antibiotic xenocoumacin from Xenorhabdus nematophila.

Determination of the Absolute Configuration of Peptide Natural Products by Using Stable Isotope Labeling and Mass Spectrometry

Structure elucidation of natural products including the absolute configuration is a complex task that involves different analytical methods like mass spectrometry, NMR spectroscopy, and chemical derivation, which are usually performed after the isolation of the compound of interest. Here, a combination of stable isotope labeling of Photorhabdus and Xenorhabdus strains and their transaminase mutants followed by detailed MS analysis enabled the structure elucidation of novel cyclopeptides named GameXPeptides including their absolute configuration in crude extracts without their actual isolation.

Genetic analysis of xenocoumacin antibiotic production in the mutualistic bacterium Xenorhabdus nematophila

Xenocoumacin 1 (Xcn1) and xenocoumacin 2 (Xcn2) are the major antimicrobial compounds produced by Xenorhabdus nematophila. To study the role of Xcn1 and Xcn2 in the life cycle of X. nematophila the 14 gene cluster (xcnA–N) required for their synthesis was identified. Overlap RT‐PCR analysis identified six major xcn transcripts. Individual inactivation of the non‐ribosomal peptide synthetase genes, xcnA and xcnK, and polyketide synthetase genes, xcnF, xcnH and xcnL, eliminated Xcn1 production. Xcn1 levels and expression of xcnA–L were increased in an ompR strain while Xcn2 levels and xcnMN expression were reduced. Xcn1 production was also increased in a strain lacking acetyl‐phosphate that can donate phosphate groups to OmpR. Together these findings suggest that OmpR‐phosphate negatively regulates xcnA–L gene expression while positively regulating xcnMN expression. HPLC‐MS analysis revealed that Xcn1 was produced first and was subsequently converted to Xcn2. Inactivation of xcnM and xcnN eliminated conversion of Xcn1 to Xcn2 resulting in elevated Xcn1 production. The viability of the xcnM strain was reduced 20‐fold relative to the wild‐type strain supporting the idea that conversion of Xcn1 to Xcn2 provides a mechanism to avoid self‐toxicity. Interestingly, inactivation of ompR enhanced cell viability during prolonged culturing.

A New Type of Pyrrolidine Biosynthesis Is Involved in the Late Steps of Xenocoumacin Production in Xenorhabdus nematophila

Bacteria of the genus Xenorhabdus live in symbiosis with nematodes of the genus Steinernema, and both form an entomopathogenic complex that is used commercially to kill several different insect larvae. Briefly, the nematode carries the bacteria in the gut of its free-living state called infective juvenile (IJ). IJs actively search the soil for insect larvae. After an insect is identified, the nematode infects the insect and regurgitates the bacteria, which kill the insect within 24 h post-infection. The insect cadaver is then digested by the bacteria and the nematodes, and after several cycles of nematode development, new IJs are formed, which carry the bacteria in the gut and leave the now empty insect carcass to find new prey. As there have been hints in the literature that small molecules (e.g. , secondary metabolites) produced by the bacterium are either involved in the pathogenesis against the insect or the symbiosis towards the nematode, we have started to search for ACHTUNGTRENNUNGsecondary metabolites produced by different Xenorhabdus ACHTUNGTRENNUNGspecies. During this work, we could identify xenocoumacins (XCNs) I and II (1 and 2, respectively, in Scheme 1), which were isolated several years ago from different strains of Xenorhabdus nematophila. Although both compounds show antibiotic activity, 1 is much more active and additionally shows good activity against different fungi. Currently, both compounds are thought to be involved in killing bacteria living inside the insect gut, where these bacteria would compete with XenoACHTUNGTRENNUNGrhabdus for food in the dead insect. We could identify 1 and 2 in several Xenorhabdus strains by their characteristic fragmentation pattern in HRESI-MS (see below). Moreover, a detailed analysis of two different XCN producer strains, namely X. nematophila AN6/1 and X. kozodoii DSM 17907, under different cultivation conditions led to the identification of four new XCN derivatives named XCN III–VI (3–6). Whereas only traces of 3 and 4 are observed throughout the cultivation process in strain AN6/1, 5 and 6 start to accumulate in significant amounts after 1 and 2 have been formed (after 8 h); this indicates a structural relationship (Figure 1). Interestingly, only traces of 5 and 6 were observed in cultures grown with the adsorber resin Amberlite XAD-16, which seems to protect 1 and 2 from their transformation or degradation, as has also been observed for other secondary metabolites.

Rhabdopeptides as insect-specific virulence factors from entomopathogenic bacteria.

Six novel linear peptides, named “rhabdopeptides”, have been identified in the entomopathogenic bacterium Xenorhabdus nematophila after the discovery of the corresponding rdp gene cluster by using a promoter trap strategy for the detection of insect‐inducible genes. The structures of these rhabdopeptides were deduced from labeling experiments combined with detailed MS analysis. Detailed analysis of an rdp mutant revealed that these compounds participate in virulence towards insects and are produced upon bacterial infection of a suitable insect host. Furthermore, two additional rhabdopeptide derivatives produced by Xenorhabdus cabanillasii were isolated, these showed activity against insect hemocytes thereby confirming the virulence of this novel class of compounds.

Xenortide Biosynthesis by Entomopathogenic Xenorhabdus nematophila.

The biosynthesis gene cluster of the xenortides and a new derivative, xenortide D, which is produced in only trace amounts, was identified in Xenorhabdus nematophila. The structure of xenortide D was elucidated using a combination of labeling experiments followed by MS analysis and was confirmed by synthesis. Bioactivity tests revealed a weak activity of tryptamine-carrying xenortides against Plasmodium falciparum and Trypanosoma brucei.

Thiol Probes To Detect Electrophilic Natural Products Based on Their Mechanism of Action

New methods are urgently needed to find novel natural products as structural leads for the development of new drugs against emerging diseases such as cancer and multiresistant bacterial infections. Here we introduce a reactivity-guided drug discovery approach for electrophilic natural products, a therapeutically relevant class of natural products that covalently modify their cellular targets, in crude extracts. Using carefully designed halogenated aromatic reagents, the process furnishes derivatives that are UV-active and highly conspicuous via mass spectrometry by virtue of an isotopically unique bromine or chlorine tag. In addition to the identification of high-value metabolites, the process facilitates the difficult task of structure elucidation by providing derivatives that are primed for X-ray crystallographic analysis. We show that a cysteine probe efficiently and chemoselectively labels enone-, β-lactam-, and β-lactone-based electrophilic natural products (parthenolide, andrographolide, wortmannin, penicillin G, salinosporamide), while a thiophenol probe preferentially labels epoxide-based electrophilic natural products (triptolide, epoxomicin, eponemycin, cyclomarin, salinamide). Using the optimized method, we were able to detect and isolate the epoxide-bearing natural product tirandalydigin from Salinispora and thereby link an orphan gene cluster to its gene product.

Biosynthesis of the antibiotic nematophin and its elongated derivatives in entomopathogenic bacteria

Nematophin, a known antibiotic natural product against Staphylococcus aureus for almost 20 years, is produced by all strains of Xenorhabdus nematophila. Despite its simple structure, its biosynthesis was unknown. Its biosynthetic pathway is reported using heterologous production in Escherichia coli. Additionally, the identification, structure elucidation, and biosynthesis of six extended nematophin derivatives from Xenorhabdus PB62.4 carrying an additional valine are reported. Preliminary bioactivity studies suggest a biological role of these compounds in the bacteria-nematode-insect symbiosis.

Thiol-Based Probe for Electrophilic Natural Products Reveals That Most of the Ammosamides Are Artifacts

To date, 16 members of the ammosamide family of natural products have been discovered, and except for ammosamide D each of these metabolites is characterized by an unusual chlorinated pyrrolo[4,3,2-de]quinoline skeleton. Several ammosamides have been shown to inhibit quinone reductase 2, a flavoenzyme responsible for quelling toxic oxidative species in cells or for killing cancer cells outright. Treatment of the extract from an ammosamide-producing culture (Streptomyces strain CNR-698) with a thiol-based reagent designed to label electrophilic natural products produced an ammosamide C-thiol adduct. This observation led us to hypothesize, and then demonstrate through experimentation, that all of the other ammosamides are derived from ammosamide C via nonenzymatic processes involving exposure to nucleophiles, air, and light. Like many established electrophilic natural products, reaction with the thiol probe suggests that ammosamide C is itself an electrophilic natural product. Although ammosamide C did not show substantial cytotoxicity against cancer cells, its activity against a marine Bacillus bacterial strain may reflect its ecological role.