Non-canonical Biosynthesis of the Brexane-Type Bishomosesquiterpene Chlororaphen through Two Consecutive Methylation Steps in Pseudomonas chlororaphis O6 and Variovorax boronicumulans PHE5-4.

A non-canonical biosynthetic pathway furnishing the first natural brexane-type bishomosesquiterpene (chlororaphen, C17 H28 ) was elucidated in the γ-proteobacterium Pseudomonas chlororaphis O6. A combination of genome mining, pathway cloning, in vitro enzyme assays, and NMR spectroscopy revealed a three-step pathway initiated by C10 methylation of farnesyl pyrophosphate (FPP, C15 ) along with cyclization and ring contraction to furnish monocyclic γ-presodorifen pyrophosphate (γ-PSPP, C16 ). Subsequent C-methylation of γ-PSPP by a second C-methyltransferase furnishes the monocyclic α-prechlororaphen pyrophosphate (α-PCPP, C17 ), serving as the substrate for the terpene synthase. The same biosynthetic pathway was characterized in the ß-proteobacterium Variovorax boronicumulans PHE5-4, demonstrating that non-canonical homosesquiterpene biosynthesis is more widespread in the bacterial domain than previously anticipated.

Chemical Ecology of Nematodes.

Nematodes represent the most abundant group of metazoans on earth. They utilize diverse chemicals to interact with con-specific and hetero-specific organisms, and are also impacted by compounds produced by other interacting organisms. In the first part of this review we discuss how nematode-derived glycolipids modulate their behavior and development, as well as the interactions with other organisms. Furthermore, we provide a short overview about other secondary metabolites produced by nematodes that affect different life traits of free-living nematodes. In the second part of this review we discuss how different bacteria-, nematode-, and plant-derived chemicals such as volatile organic compounds, root exudates, and plant defenses regulate the interaction between entomopathogenic nematodes, their symbiotic bacteria, insect prey, predators, and plants.

Dimerization of conserved ascaroside building blocks generates species-specific male attractants in Caenorhabditis nematodes.

Comparative ascaroside profiling of Caenorhabditis nematodes using HPLC-ESI-(-)-MS/MS precursor ion scanning revealed a class of highly species-specific ascaroside dimers. Their 2- and 4-isomeric, homo- and heterodimeric structures were identified using a combination of HPLC-ESI-(+)-HR-MS/MS spectrometry and high-resolution dqf-COSY NMR spectroscopy. Structure assignments were confirmed by total synthesis of representative examples. Functional characterization using holding assays indicated that males of Caenorhabditis remanei and Caenorhabditis nigoni are exclusively retained by their conspecific ascaroside dimers, demonstrating that dimerization of conserved monomeric building blocks represents a yet undescribed mechanism that generates species-specific signaling molecules in the Caenorhabditis genus.

Epimerization of an Ascaroside-Type Glycolipid Downstream of the Canonical ß-Oxidation Cycle in the Nematode Caenorhabditis nigoni.

A species-specific ascaroside-type glycolipid was identified in the nematode Caenorhabditis nigoni using HPLC-ESI-(-)-MS/MS precursor ion scanning, HR-MS/MS, and NMR techniques. Its structure containing an l-3,6-dideoxy-lyxo-hexose unit was established by total synthesis. The identification of this novel 4-epi-ascaroside (caenorhabdoside) in C. nigoni along with the previous identification of 2-epi-ascarosides (paratosides) in Pristionchus pacificus indicate that nematodes can generate highly specific signaling molecules by epimerization of the ascarylose building block downstream of the canonical ß-oxidation cycle.

Ascaroside Signaling in the Bacterivorous Nematode Caenorhabditis remanei Encodes the Growth Phase of Its Bacterial Food Source.

A novel class of species-specific modular ascarosides that integrate additional fatty acid building blocks was characterized in the nematode Caenorhabditis remanei using a combination of HPLC-ESI-(-)-MS/MS precursor ion scanning, microreactions, HR-MS/MS, MSn, and NMR techniques. The structure of the dominating component carrying a cyclopropyl fatty acid moiety was established by total synthesis. Biogenesis of this female-produced male attractant depends on cyclopropyl fatty acid synthase (cfa), which is expressed in bacteria upon entering their stationary phase.

Homologs of Caenorhabditis elegans Chemosensory Genes Have Roles in Behavior and Chemotaxis in the Root-Knot Nematode Meloidogyne incognita.

Nematode chemosensation is a vital component of their host-seeking behavior. The globally important phytonematode Meloidogyne incognita perceives and responds (via sensory organs such as amphids and phasmids) differentially to various chemical cues emanating from the rhizosphere during the course of host finding. However, compared with the free-living worm Caenorhabditis elegans, the molecular intricacies behind the plant nematode chemotaxis are a yet-unexploited territory. In the present study, four putative chemosensory genes of M. incognita, namely, Mi-odr-1, Mi-odr-3, Mi-tax-2, and Mi-tax-4 were molecularly characterized. Mi-odr-1 mRNA was found to be expressed in the cell bodies of amphidial neurons and phasmids of M. incognita. Mi-odr-1, Mi-odr-3, Mi-tax-2, and Mi-tax-4 transcripts were highly expressed in early life stages of M. incognita, consistent with a role of these genes in host recognition. Functional characterization of Mi-odr-1, Mi-odr-3, Mi-tax-2, and Mi-tax-4 via RNA interference revealed behavioral defects in M. incognita and perturbed attraction to host roots in Pluronic gel medium. Knockdown of Mi-odr-1, Mi-odr-3, Mi-tax-2, and Mi-tax-4 resulted in defective chemotaxis of M. incognita to various volatile compounds (alcohol, ketone, aromatic compound, ester, thiazole, pyrazine), nonvolatiles of plant origin (carbohydrate, phytohormone, organic acid, amino acid, phenolic), and host root exudates in an agar-Pluronic gel-based assay plate. In addition, ascaroside-mediated signaling was impeded by downregulation of chemosensory genes. This new information that behavioral response in M. incognita is modulated by specific olfactory genes can be extended to understand chemotaxis in other nematodes.

Guided cobamide biosynthesis for heterologous production of reductive dehalogenases.

Cobamides (Cbas) are essential cofactors of reductive dehalogenases (RDases) in organohalide-respiring bacteria (OHRB). Changes in the Cba structure can influence RDase function. Here, we report on the cofactor versatility or selectivity of Desulfitobacterium RDases produced either in the native organism or heterologously. The susceptibility of Desulfitobacterium hafniense strain DCB-2 to guided Cba biosynthesis (i.e. incorporation of exogenous Cba lower ligand base precursors) was analysed. Exogenous benzimidazoles, azabenzimidazoles and 4,5-dimethylimidazole were incorporated by the organism into Cbas. When the type of Cba changed, no effect on the turnover rate of the 3-chloro-4-hydroxy-phenylacetate-converting enzyme RdhA6 and the 3,5-dichlorophenol-dehalogenating enzyme RdhA3 was observed. The impact of the amendment of Cba lower ligand precursors on RDase function was also investigated in Shimwellia blattae, the Cba producer used for the heterologous production of Desulfitobacterium RDases. The recombinant tetrachloroethene RDase (PceAY51 ) appeared to be non-selective towards different Cbas. However, the functional production of the 1,2-dichloroethane-dihaloeliminating enzyme (DcaA) of Desulfitobacterium dichloroeliminans was completely prevented in cells producing 5,6-dimethylbenzimidazolyl-Cba, but substantially enhanced in cells that incorporated 5-methoxybenzimidazole into the Cba cofactor. The results of the study indicate the utilization of a range of different Cbas by Desulfitobacterium RDases with selected representatives apparently preferring distinct Cbas.

Exploring Modular Glycolipids Involved in Nematode Chemical Communication.

Chemical communication in nematodes has been known for over half a century, but the underlying molecular basis remained largely elusive. Recent advances in analytical techniques facilitated the characterization of a modular glycolipid library based on the dideoxysugar L-ascarylose, which modulates behavior and development in the model organism C. elegans. Ascaroside signaling is highly conserved in nematodes and represents a key factor in nematode chemical ecology. Ascaroside biosynthesis depends on the co-option of the peroxisomal ß-oxidation cycle and in addition integrates a large diversity of additional building blocks derived from various primary metabolic pathways to give rise to species-specific modular assemblies, thus, transcending the concept of strictly segregated primary versus secondary metabolism.

Comparative Ascaroside Profiling of Caenorhabditis Exometabolomes Reveals Species-Specific (ω) and (ω - 2)-Hydroxylation Downstream of Peroxisomal ß-Oxidation.

Chemical communication in nematodes such as the model organism Caenorhabditis elegans is modulated by a variety of glycosides based on the dideoxysugar l-ascarylose. Comparative ascaroside profiling of nematode exometabolome extracts using a GC-EIMS screen reveals that several basic components including ascr#1 (asc-C7), ascr#2 (asc-C6-MK), ascr#3 (asc-ΔC9), ascr#5 (asc-ωC3), and ascr#10 (asc-C9) are highly conserved among the Caenorhabditis. Three novel side chain hydroxylated ascaroside derivatives were exclusively detected in the distantly related C. nigoni and C. afra. Molecular structures of these species-specific putative signaling molecules were elucidated by NMR spectroscopy and confirmed by total synthesis and chemical correlations. Biological activities were evaluated using attraction assays. The identification of (ω)- and (ω - 2)-hydroxyacyl ascarosides demonstrates how GC-EIMS-based ascaroside profiling facilitates the detection of novel ascaroside components and exemplifies how species-specific hydroxylation of ascaroside aglycones downstream of peroxisomal ß-oxidation increases the structural diversity of this highly conserved class of nematode signaling molecules.

Halogenated C15 Acetogenin Analogues of Obtusallene III from a Laurenciella sp. Collected in Corsica.

NMR chemical profiling of a Laurenciella sp. using a computerized method developed in our laboratory resulted in the identification of five new compounds (1-5) and 17 known compounds, among which 3-(E)-laurenyne represented by far the most abundant metabolite. Compounds 1 to 5 were isolated and fully characterized by detailed spectroscopic analysis. The absolute configuration and structural features of compound 1 were determined by single-crystal X-ray diffraction analysis. Compounds 1 to 4 are 12-membered cyclic ether acetogenins that are present in solution as interconverting conformers exhibiting an (aR) configuration of the bromoallene unit together with an S configuration at C-4. Among these, compound 3 is the first obtusallene derivative with bromine substituents at both the C-7 and C-12 positions. Compound 5 is an acetogenin bearing a [5.5.1]bicyclotridecane ring system. A plausible biosynthetic route to 1-4 is proposed.

Ascaroside Profiling of Caenorhabditis elegans Using Gas Chromatography-Electron Ionization Mass Spectrometry.

Nematodes such as the model organism Caenorhabditis elegans produce various homologous series of l-ascarylose-derived glycolipids called ascarosides, which include several highly potent signals in intra and interspecies communication as well as cross-kingdom interactions. Given their low concentrations and large number of structurally similar components, mass spectrometric screens based on high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) are commonly employed for ascaroside detection and quantification. Here, we describe a complementary gas chromatography-electron ionization mass spectrometry (GC-EIMS) screen that utilizes an ascarylose-derived K1-fragment ion signal at m/z 130.1 [C6H14OSi]+● to highlight known as well as yet unidentified ascaroside components in TMS-derivatized crude nematode exometabolome extracts. GC-EIMS-based ascaroside profiling of wild-type and mutant C. elegans facilitates the analysis of all basic ascarosides using the same ionization technique while providing excellent resolution for the complete homologous series with side chains ranging from 3 to 33 carbons. Combined screening for m/z 130.1 along with side chain-specific J1 [M - 173]+ and J2 [M - 291]+ fragment ions, as well as additional characteristic marker ions from α-cleavage, enables convenient structure assignment of ca. 200 components from wild-type and peroxisomal ß-oxidation mutants including (ω - 1)-linked acyl, enoyl, ß-hydroxyacyl, and 2-ketoalkyl ascarosides along with their (ω)-linked or α-methyl isomers and ethanolamide derivatives, as well as 2-hydroxyalkyl ascarosides. Given the widespread availability of GC-MS and its increasing popularity in metabolomics, this method will promote the identification of ascarosides in C. elegans and other nematodes.

Selective MS screening reveals a sex pheromone in Caenorhabditis briggsae and species-specificity in indole ascaroside signalling.

The indole ascarosides (icas) represent a highly potent class of nematode-derived modular signalling components that integrate structural inputs from amino acid, carbohydrate, and fatty acid metabolism. Comparative analysis of the crude exo-metabolome of hermaphroditic Caenorhabditis briggsae using a highly sensitive mass spectrometric screen reveals an indole ascaroside blend dominated by two new components. The structures of isolated icas#2 and icas#6.2 were determined by NMR spectroscopy and confirmed by total synthesis and chemical correlation. Low atto- to femtomolar amounts of icas#2 and icas#6.2 act in synergism to attract males indicating a function as sex pheromone. Comparative analysis of 14 Caenorhabditis species further demonstrates that species-specific indole ascaroside biosynthesis is highly conserved in the Elegans group. Functional characterization of the dominating indole ascarosides icas#2, icas#3, and icas#9 reveals a high degree of species-specificity and considerable variability with respect to gender-specificity, thus, confirming that indole ascarosides modulate different biological functions within the Elegans group. Although the nematode response was usually most pronounced towards conspecific signals, Caenorhabditis brenneri, the only species of the Elegans group that does not produce any indole ascarosides, exhibits a robust response to icas#2 suggesting the potential for interspecies interactions.

The SMUL_1544 Gene Product Governs Norcobamide Biosynthesis in the Tetrachloroethene-Respiring Bacterium Sulfurospirillum multivorans.

UNLABELLED: The tetrachloroethene (PCE)-respiring bacterium Sulfurospirillum multivorans produces a unique cobamide, namely, norpseudo-B12, which, in comparison to other cobamides, e.g., cobalamin and pseudo-B12, lacks the methyl group in the linker moiety of the nucleotide loop. In this study, the protein SMUL_1544 was shown to be responsible for the formation of the unusual linker moiety, which is most probably derived from ethanolamine-phosphate (EA-P) as the precursor. The product of the SMUL_1544 gene successfully complemented a Salmonella enterica ΔcobD mutant. The cobD gene encodes an l-threonine-O-3-phosphate (l-Thr-P) decarboxylase responsible for the synthesis of (R)-1-aminopropan-2-ol O-2-phosphate (AP-P), required specifically for cobamide biosynthesis. When SMUL_1544 was produced in the heterologous host lacking CobD, norpseudo-B12 was formed, which pointed toward the formation of EA-P rather than AP-P. Guided cobamide biosynthesis experiments with minimal medium supplemented with l-Thr-P supported cobamide biosynthesis in S. enterica producing SMUL_1544 or S. multivorans Under these conditions, both microorganisms synthesized pseudo-B12 This observation indicated a flexibility in the SMUL_1544 substrate spectrum. From the formation of catalytically active PCE reductive dehalogenase (PceA) in S. multivorans cells producing pseudo-B12, a compatibility of the respiratory enzyme with the cofactor was deduced. This result might indicate a structural flexibility of PceA in cobamide binding. Feeding of l-[3-(13)C]serine to cultures of S. multivorans resulted in isotope labeling of the norpseudo-B12 linker moiety, which strongly supports the hypothesis of EA-P formation from l-serine-O-phosphate (l-Ser-P) in this organism. IMPORTANCE: The identification of the gene product SMUL_1544 as a putative l-Ser-P decarboxylase involved in norcobamide biosynthesis in S. multivorans adds a novel module to the assembly line of cobamides (complete corrinoids) in prokaryotes. Selected cobamide-containing enzymes (e.g., reductive dehalogenases) showed specificity for their cobamide cofactors. It has recently been proposed that the structure of the linker moiety of norpseudo-B12 and the mode of binding of the EA-P linker to the PceA enzyme reflect the high specificity of the enzyme for its cofactor. Data reported herein do not support this idea. In fact, norpseudo-B12 was functional in the cobamide-dependent methionine biosynthesis of S. enterica, raising questions about the role of norcobamides in nature.

Two Defensive Lines in Juvenile Leaf Beetles; Esters of 3-nitropropionic Acid in the Hemolymph and Aposematic Warning.

Juveniles of the leaf beetles in subtribe Chrysomelina have efficient defense strategies against predators. When disturbed, they transiently expose volatile deterrents in large droplets from nine pairs of defensive glands on their back. Here, we report on an additional line of defense consisting of the non-volatile isoxazolin-5-one glucoside and its 3-nitropropanoyl ester in the larval hemolymph. Because isoxazolin-5-one derivatives were not detectable in related leaf beetle taxa, they serve as a diagnostic marker for the Chrysomelina subtribe. Conjugation of isotopically labelled 3-nitropropionic acid to isoxazolin-5-one glucoside in vivo demonstrates its function as a carrier for the 3-nitropropanoyl esters. The previous identification of characteristic glucosides as precursors of the volatile deterrents underlines the general importance of glucosides for sequestration from food plants, and the subsequent transport in the hemolymph to the defense system. The combination of repellent volatiles with non-volatile toxic compounds in the hemolymph has the potential to create synergistic effects since the odorant stimulus may help predators learn to avoid some foods. The combination of the two defense lines has the advantage, that the hemolymph toxins provide reliable and durable protection, while the repellents may vary after a host plant change.

Mating dynamics in a nematode with three sexes and its evolutionary implications.

Nematodes have diverse reproductive strategies, which make them ideal subjects for comparative studies to address how mating systems evolve. Here we present the sex ratios and mating dynamics of the free-living nematode Rhabditis sp. SB347, in which males, females and hermaphrodites co-exist. The three sexes are produced by both selfing and outcrossing, and females tend to appear early in a mother's progeny. Males prefer mating with females over hermaphrodites, which our results suggest is related to the female-specific production of the sex pheromones ascr#1 and ascr#9. We discuss the parallels between this system and that of parasitic nematodes that exhibit alternation between uniparental and biparental reproduction.

Conserved nematode signalling molecules elicit plant defenses and pathogen resistance.

Plant-defense responses are triggered by perception of conserved microbe-associated molecular patterns (MAMPs), for example, flagellin or peptidoglycan. However, it remained unknown whether plants can detect conserved molecular patterns derived from plant-parasitic animals, including nematodes. Here we show that several genera of plant-parasitic nematodes produce small molecules called ascarosides, an evolutionarily conserved family of nematode pheromones. Picomolar to micromolar concentrations of ascr#18, the major ascaroside in plant-parasitic nematodes, induce hallmark defense responses including the expression of genes associated with MAMP-triggered immunity, activation of mitogen-activated protein kinases, as well as salicylic acid- and jasmonic acid-mediated defense signalling pathways. Ascr#18 perception increases resistance in Arabidopsis, tomato, potato and barley to viral, bacterial, oomycete, fungal and nematode infections. These results indicate that plants recognize ascarosides as a conserved molecular signature of nematodes. Using small-molecule signals such as ascarosides to activate plant immune responses has potential utility to improve economic and environmental sustainability of agriculture.

Spodoptera littoralis detoxifies neurotoxic 3-nitropropanoic acid by conjugation with amino acids.

Spodoptera littoralis is a phytophagous generalist. Its host range includes more than 40 plant species, some of which produce 3-nitropropanoic acid (3-NPA), an irreversible inhibitor of mitochondrial succinate dehydrogenase. Growth in larvae fed an artificial diet with a sublethal admixture of 3-NPA (4.2 µmol per g) was slowed significantly, but larvae experienced no increase in mortality. In contrast, larvae injected with 25.2 µmol/g (bodyweight) 3-NPA experienced acute toxicity and death. To study the detoxification mechanism of 3-NPA in S. littoralis, the insect frass was analyzed by HPLC-MS. Comparative analysis of 3-NPA-treated and -untreated control samples using HR-MS(2) revealed a group of differential signals that were identified as amino acid amides of 3-NPA with glycine, alanine, serine, and threonine. When sublethal amounts of stable isotope-labeled 3-NPA were injected into a larva's hemolymph, 3-NPA amino acid conjugates were identified as putative detoxification products. Bioassays with synthetic standards confirmed that the toxicity of the amides was negligible in comparison to the toxicity of free 3-NPA, demonstrating that amino acid conjugation in S. littoralis represents an efficient way to detoxify 3-NPA. Furthermore, biosynthetic studies using crude fractions of the gut tissue indicated that conjugation of 3-NPA with amino acids occurs in epithelial cells of the insect's gut. Taken together, these results suggest that the detoxification of 3-NPA in S. littoralis proceeds via conjugation to specific amino acids within the epithelial cells followed by export of the nontoxic amino acid conjugates to the hemolymph via as yet uncharacterized mechanisms, most likely involving the Malpighian tubules.

Combinatorial chemistry in nematodes: modular assembly of primary metabolism-derived building blocks.

The nematode Caenorhabditis elegans was the first animal to have its genome fully sequenced and has become an important model organism for biomedical research. However, like many other animal model systems, its metabolome remained largely uncharacterized, until recent investigations demonstrated the importance of small molecule-based signalling cascades for virtually every aspect of nematode biology. These studies have revealed that nematodes are amazingly skilled chemists: using simple building blocks from conserved primary metabolism and a strategy of modular assembly, C. elegans and other nematode species create complex molecular architectures to regulate their development and behaviour. These nematode-derived modular metabolites (NDMMs) are based on the dideoxysugars ascarylose or paratose, which serve as scaffolds for attachment of moieties from lipid, amino acid, carbohydrate, citrate, and nucleoside metabolism. Mutant screens and comparative metabolomics based on NMR spectroscopy and MS have so-far revealed several 100 different ascarylose ("ascarosides") and a few paratose ("paratosides") derivatives, many of which represent potent signalling molecules that can be active at femtomolar levels, regulating development, behaviour, body shape, and many other life history traits. NDMM biosynthesis appears to be carefully regulated as assembly of different modules proceeds with very high specificity. Preliminary biosynthetic studies have confirmed the primary metabolism origin of some NDMM building blocks, whereas the mechanisms that underlie their highly specific assembly are not understood. Considering their functions and biosynthetic origin, NDMMs represent a new class of natural products that cannot easily be classified as "primary" or "secondary". We believe that the identification of new variants of primary metabolism-derived structures that serve important signalling functions in C. elegans and other nematodes provides a strong incentive for a comprehensive re-analysis of metabolism in higher animals, including humans.