Charakterisierung von N-Acyl-glutaminkonjugaten aus dem Regurgitat von Lepidoptera Larven

Viele Pflanzen reagieren bei Herbivorenbefall, z. B. durch Lepidoptera-Larven, mit Abwehrreaktionen, unter anderem mit der Produktion von Duftstoffen. Da einfache mechanische Verwundung diese Verteidigungseaktion nicht einleitet, scheinen im Regurgitat (Vorderdarminhalt) befindliche chemische Komponenten Auslöser (Elicitoren) der pflanzlichen Abwehr zu sein. Bei der Charakterisierung der chemischen Zusammensetzung von Regurgitat wurden schwerpunktsmässig N-Acylglutaminkonjugate, die als Elicitoren angesehen werden, untersucht: die Stereochemie der 17-Hydroxygruppe von Volicitin [(17S)-N-(17-Hydroxylinolenoyl)-L-glutamin) wurde bestimmt und neue N-Acylglutaminkonjugate wie N-(15,16-Epoxylinoleoyl)-glutamin, N-(15,16-Dihydroxylinoleoyl)-glutamin und N-(17-Phosphonoxylinolenoyl)-glutamin sowie N-(17-Acyloxyacyl)-glutamine wurden mit Hilfe von LC-MS/MS, GC-MS und synthetisierten Referenzverbindungen identifiziert. Die Biosynthese von N-Acylglutaminkonjugaten wird in vitro von Darmbakterien der Lepidoptera-Larven katalysiert. Die Tensideigenschaften von N-Acylglutaminen sind vermutlich Ursache für ihre Elicitorwirkung auf manche Pflanzen. Das zur Beschreibung der Interaktionen von Pflanze, Herbivor und Predator verwendete tritrophische System muss daher erweitert werden, um auch die mikrobielle Komponente zu berücksichtigen

Anaerobic Degradation of the Plant Sugar Sulfoquinovose Concomitant With H2S Production: Escherichia coli K-12 and Desulfovibrio sp. Strain DF1 as Co-culture Model

Sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) is produced by plants and other phototrophs and its biodegradation is a relevant component of the biogeochemical carbon and sulfur cycles. SQ is known to be degraded by aerobic bacterial consortia in two tiers via C3-organosulfonates as transient intermediates to CO2, water and sulfate. In this study, we present a first laboratory model for anaerobic degradation of SQ by bacterial consortia in two tiers to acetate and hydrogen sulfide (H2S). For the first tier, SQ-degrading Escherichia coli K-12 was used. It catalyzes the fermentation of SQ to 2,3-dihydroxypropane-1-sulfonate (DHPS), succinate, acetate and formate, thus, a novel type of mixed-acid fermentation. It employs the characterized SQ Embden-Meyerhof-Parnas pathway, as confirmed by mutational and proteomic analyses. For the second tier, a DHPS-degrading Desulfovibrio sp. isolate from anaerobic sewage sludge was used, strain DF1. It catalyzes another novel fermentation, of the DHPS to acetate and H2S. Its DHPS desulfonation pathway was identified by differential proteomics and demonstrated by heterologously produced enzymes: DHPS is oxidized via 3-sulfolactaldehyde to 3-sulfolactate (SL) by two NAD+-dependent dehydrogenases (DhpA, SlaB); the SL is cleaved by an SL sulfite-lyase known from aerobic bacteria (SuyAB) to pyruvate and sulfite. The pyruvate is oxidized to acetate, while the sulfite is used as electron acceptor in respiration and reduced to H2S. In conclusion, anaerobic sulfidogenic SQ degradation was demonstrated as a novel link in the biogeochemical sulfur cycle. SQ is also a constituent of the green-vegetable diet of herbivores and omnivores and H2S production in the intestinal microbiome has many recognized and potential contributions to human health and disease. Hence, it is important to examine bacterial SQ degradation also in the human intestinal microbiome, in relation to H2S production, dietary conditions and human health

The Gene Cluster for Fluorometabolite Biosynthesis in Streptomyces cattleya: A Thioesterase Confers Resistance to Fluoroacetyl-Coenzyme A

SummaryA genomic library of Streptomyces cattleya was screened to isolate a gene cluster encoding enzymes responsible for the production of fluorine-containing metabolites. In addition to the previously described fluorinase FlA which catalyzes the formation of 5′-fluoro-5′-deoxyadenosine from S-adenosylmethionine and fluoride, 11 other putative open reading frames have been identified. Three of the proteins encoded by these genes have been characterized. FlB was determined to be the second enzyme in the pathway, catalyzing the phosphorolytic cleavage of 5′-fluoro-5′-deoxyadenosine to produce 5-fluoro-5-deoxy-D-ribose-1-phosphate. The enzyme FlI was found to be an S-adenosylhomocysteine hydrolase, which may act to relieve S-adenosylhomocysteine inhibition of the fluorinase. Finally, flK encodes a thioesterase which catalyzes the selective breakdown of fluoroacetyl-CoA but not acetyl-CoA, suggesting that it provides the producing strain with a mechanism for resistance to fluoroacetate

Revealing Genome-Based Biosynthetic Potential of Streptomyces sp. BR123 Isolated from Sunflower Rhizosphere with Broad Spectrum Antimicrobial Activity

Actinomycetes, most notably the genus Streptomyces, have great importance due to their role in the discovery of new natural products, especially for finding antimicrobial secondary metabolites that are useful in the medicinal science and biotechnology industries. In the current study, a genomebased evaluation of Streptomyces sp. isolate BR123 was analyzed to determine its biosynthetic potential, based on its in vitro antimicrobial activity against a broad range of microbial pathogens, including gram-positive and gram-negative bacteria and fungi. A draft genome sequence of 8.15 Mb of Streptomyces sp. isolate BR123 was attained, containing a GC content of 72.63% and 8103 protein coding genes. Many antimicrobial, antiparasitic, and anticancerous compounds were detected by the presence of multiple biosynthetic gene clusters, which was predicted by in silico analysis. A novel metabolite with a molecular mass of 1271.7773 in positive ion mode was detected through a high-performance liquid chromatography linked with mass spectrometry (HPLC-MS) analysis. In addition, another compound, meridamycin, was also identified through a HPLC-MS analysis. The current study reveals the biosynthetic potential of Streptomyces sp. isolate BR123, with respect to the synthesis of bioactive secondary metabolites through genomic and spectrometric analysis. Moreover, the comparative genome study compared the isolate BR123 with other Streptomyces strains, which may expand the knowledge concerning the mechanism involved in novel antimicrobial metabolite synthesis

Structural basis for the activity and substrate specificity of fluoroacetyl-CoA thioesterase FlK.

The thioesterase FlK from the fluoroacetate-producing Streptomyces cattleya catalyzes the hydrolysis of fluoroacetyl-coenzyme A. This provides an effective self-defense mechanism, preventing any fluoroacetyl-coenzyme A formed from being further metabolized to 4-hydroxy-trans-aconitate, a lethal inhibitor of the tricarboxylic acid cycle. Remarkably, FlK does not accept acetyl-coenzyme A as a substrate. Crystal structure analysis shows that FlK forms a dimer, in which each subunit adopts a hot dog fold as observed for type II thioesterases. Unlike other type II thioesterases, which invariably utilize either an aspartate or a glutamate as catalytic base, we show by site-directed mutagenesis and crystallography that FlK employs a catalytic triad composed of Thr(42), His(76), and a water molecule, analogous to the Ser/Cys-His-acid triad of type I thioesterases. Structural comparison of FlK complexed with various substrate analogues suggests that the interaction between the fluorine of the substrate and the side chain of Arg(120) located opposite to the catalytic triad is essential for correct coordination of the substrate at the active site and therefore accounts for the substrate specificity

Revealing genome-based biosynthetic potential of Streptomyces sp. BR123 isolated from sunflower rhizosphere with broad spectrum antimicrobial activity

Actinomycetes, most notably the genus Streptomyces, have great importance due to their role in the discovery of new natural products, especially for finding antimicrobial secondary metabolites that are useful in the medicinal science and biotechnology industries. In the current study, a genome-based evaluation of Streptomyces sp. isolate BR123 was analyzed to determine its biosynthetic potential, based on its in vitro antimicrobial activity against a broad range of microbial pathogens, including gram-positive and gram-negative bacteria and fungi. A draft genome sequence of 8.15 Mb of Streptomyces sp. isolate BR123 was attained, containing a GC content of 72.63% and 8103 protein coding genes. Many antimicrobial, antiparasitic, and anticancerous compounds were detected by the presence of multiple biosynthetic gene clusters, which was predicted by in silico analysis. A novel metabolite with a molecular mass of 1271.7773 in positive ion mode was detected through a high-performance liquid chromatography linked with mass spectrometry (HPLC-MS) analysis. In addition, another compound, meridamycin, was also identified through a HPLC-MS analysis. The current study reveals the biosynthetic potential of Streptomyces sp. isolate BR123, with respect to the synthesis of bioactive secondary metabolites through genomic and spectrometric analysis. Moreover, the comparative genome study compared the isolate BR123 with other Streptomyces strains, which may expand the knowledge concerning the mechanism involved in novel antimicrobial metabolite synthesi

N-(15,16-Dihydroxylinoleoyl)-glutamine and N-(15,16- epoxylinoleoyl)-glutamine isolated from oral secretions of lepidopteran larvae

N-(15,16-Dihydroxylinoleoyl)-glutamine (1) and N-(15,16- epoxylinoleoyl)-glutamine (2) and were identified in the regurgitant of lepidopteran larvae (Spodoptera exigua and Spodoptera frugiperda) by LC-MS. After methanolysis and derivatisation with MSTF

N-(17-acyloxy-acyl)-glutamines: Novel surfactants from oral secretions of lepidopteran larvae

N-(17-Acyloxy-acyl)-glutamine conjugates such as N-(17-linolenoyloxy-linolenoyl)-glutamine (6), N-(17-linolenoyloxy-linoleoyl)-glutamine (7), N-(17-linoleoyloxy-linolenoyl)-glutamine (8), and N-(17-linoleoyloxy-linoleoyl)-glutamine (9) were identified

Bacillus sp. G2112 Detoxifies Phenazine-1-carboxylic Acid by N5 Glucosylation

Microbial symbionts of plants constitute promising sources of biocontrol organisms to fight plant pathogens. Bacillus sp. G2112 and Pseudomonas sp. G124 isolated from cucumber (Cucumis sativus) leaves inhibited the plant pathogens Erwinia and Fusarium. When Bacillus sp. G2112 and Pseudomonas sp. G124 were co-cultivated, a red halo appeared around Bacillus sp. G2112 colonies. Metabolite profiling using liquid chromatography coupled to UV and mass spectrometry revealed that the antibiotic phenazine-1-carboxylic acid (PCA) released by Pseudomonas sp. G124 was transformed by Bacillus sp. G2112 to red pigments. In the presence of PCA (>40 µg/mL), Bacillus sp. G2112 could not grow. However, already-grown Bacillus sp. G2112 (OD600 > 1.0) survived PCA treatment, converting it to red pigments. These pigments were purified by reverse-phase chromatography, and identified by high-resolution mass spectrometry, NMR, and chemical degradation as unprecedented 5N-glucosylated phenazine derivatives: 7-imino-5N-(1′β-D-glucopyranosyl)-5,7-dihydrophenazine-1-carboxylic acid and 3-imino-5N-(1′β-D-glucopyranosyl)-3,5-dihydrophenazine-1-carboxylic acid. 3-imino-5N-(1′β-D-glucopyranosyl)-3,5-dihydrophenazine-1-carboxylic acid did not inhibit Bacillus sp. G2112, proving that the observed modification constitutes a resistance mechanism. The coexistence of microorganisms—especially under natural/field conditions—calls for such adaptations, such as PCA inactivation, but these can weaken the potential of the producing organism against pathogens and should be considered during the development of biocontrol strategies.publishe

Ammonia Released by Streptomyces aburaviensis Induces Droplet Formation in Streptomyces violaceoruber

Streptomyces violaceoruber grown in co-culture with Streptomyces aburaviensis produces an about 17-fold higher volume of droplets on its aerial mycelium than in single-culture. Physical separation of the Streptomyces strains by either a plastic barrier or by a dialysis membrane, which allowed communication only by the exchange of volatile compounds or diffusible compounds in the medium, respectively, still resulted in enhanced droplet formation. The application of molecular sieves to bioassays resulted in the attenuation of the droplet-inducing effect of S. aburaviensis indicating the absorption of the compound. 1H-NMR analysis of molecular-sieve extracts and the selective indophenol-blue reaction revealed that the volatile droplet-inducing compound is ammonia. The external supply of ammonia in biologically relevant concentrations of ≥8 mM enhanced droplet formation in S. violaceoruber in a similar way to S. aburaviensis. Ammonia appears to trigger droplet production in many Streptomyces strains because four out of six Streptomyces strains exposed to ammonia exhibited induced droplet production.publishe