Isolation and identification of natural products and biosynthetic pathways from Photorhabdus and Xenorhabdus

The entomopathogenic bacteria of the genera Photorhabdus and Xenorhabdus display perfect model organisms to gain insights into the sophisticated interplay between symbiosis and pathogenicity. Moreover, numerous publications in the last years have demonstrated that these bacteria represent a rich source of secondary metabolites, which is exemplified in this work with the description of the novel xenofuranone compounds. The recently available genome sequence of Photorhabdus luminescens TT01 pointed out that many biosynthetic gene clusters remain silent as the corresponding product cannot be detected. The heterologous expression of a nonribosomal peptide synthetase, which resulted in the successful production of indigoidine, depicts one way to gain access on these cryptic gene clusters. In addition the genome sequence also enabled the identification of biosynthesis genes of the already known compound families of stilbenes and anthraquinones. Thereby a type II polyketide synthase cluster was identified, which is responsible for anthraquinone biosynthesis, representing only the second known type II PKS derived compound from a Gram-negative bacterium. Furthermore the identification of genes involved in stilbene biosynthesis led to the discovery of a unique and novel pathway, strongly differing from plant derived stilbenes.Entomopathogene Bakterien der Gattungen Photorhabdus und Xenorhabdus eignen sich hervorragend als Modelorganismen um Einblicke in das komplizierte Wechselspiel zwischen Symbiose und Pathogenität zu erhalten. Darüber hinaus haben zahlreiche Publikationen der letzten Jahre gezeigt, dass diese Bakterien eine reiche Quelle an Sekundärstoffen darstellen. In der vorliegenden Arbeit wird dies anhand der neu beschriebenen Xenofuranone verdeutlicht. Die veröffentlichte Genomsequenz von Photorhabdus luminescens TT01 offenbarte, dass die meisten Biosynthese Gencluster "verwaist" sind, das heißt es ließ sich bisher kein dazugehöriges Produkt detektieren. Die erfolgreiche heterologe Expression einer nichtribosomalen Peptidsynthetase und der damit verbundenen Produktion von Indigoidin, zeigte eine Möglichkeit auf um Zugang zu solchen "verwaisten" Genclustern zu erhalten. Des Weiteren erlaubte die Genomsequenz nach Biosynthesegenen zu suchen, deren Produkte wie zum Beispiel die Stilbene oder die Anthrachinone bereits bekannt waren. Auf diese Weise konnte ein Typ II Polyketidsynthasecluster der für die Biosynthese der Anthrachinone verantwortlich ist identifiziert werden. Die Anthrachinone sind damit erst das zweite bekannte Beispiel eines Typ II PKS erzeugten Produktes aus einem Gram-negativen Bakterium. Zusätzlich gelang es die Gene, die an der Stilbenbiosynthese beteiligt sind zu identifizieren und damit einen neuen und einzigartigen Stoffwechselweg, welcher stark abweichend zur pflanzlichen Biosynthese funktioniert zu beschreiben

The expression of stlA in Photorhabdus luminescens is controlled by nutrient limitation

Photorhabdus is a genus of Gram-negative entomopathogenic bacteria that also maintain a mutualistic association with nematodes from the family Heterorhabditis. Photorhabdus has an extensive secondary metabolism that is required for the interaction between the bacteria and the nematode. A major component of this secondary metabolism is a stilbene molecule, called ST. The first step in ST biosynthesis is the non-oxidative deamination of phenylalanine resulting in the production of cinnamic acid. This reaction is catalyzed by phenylalanine-ammonium lyase, an enzyme encoded by the stlA gene. In this study we show, using a stlA-gfp transcriptional fusion, that the expression of stlA is regulated by nutrient limitation through a regulatory network that involves at least 3 regulators. We show that TyrR, a LysR-type transcriptional regulator that regulates gene expression in response to aromatic amino acids in E. coli, is absolutely required for stlA expression. We also show that stlA expression is modulated by σS and Lrp, regulators that are implicated in the regulation of the response to nutrient limitation in other bacteria. This work is the first that describes pathway-specific regulation of secondary metabolism in Photorhabdus and, therefore, our study provides an initial insight into the complex regulatory network that controls secondary metabolism, and therefore mutualism, in this model organism

mRNA Inventory of Extracellular Vesicles from Ustilago maydis

Extracellular vesicles (EVs) can transfer diverse RNA cargo for intercellular communication. EV-associated RNAs have been found in diverse fungi and were proposed to be relevant for pathogenesis in animal hosts. In plant-pathogen interactions, small RNAs are exchanged in a cross-kingdom RNAi warfare and EVs were considered to be a delivery mechanism. To extend the search for EV-associated molecules involved in plant-pathogen communication, we have characterised the repertoire of EV-associated mRNAs secreted by the maize smut pathogen, Ustilago maydis. For this initial survey, we examined EV-enriched fractions from axenic filamentous cultures that mimic infectious hyphae. EV-associated RNAs were resistant to degradation by RNases and the presence of intact mRNAs was evident. The set of mRNAs enriched inside EVs relative to the fungal cells are functionally distinct from those that are depleted from EVs. mRNAs encoding metabolic enzymes are particularly enriched. Intriguingly, mRNAs of some known effectors and other proteins linked to virulence were also found in EVs. Furthermore, several mRNAs enriched in EVs are also upregulated during infection, suggesting that EV-associated mRNAs may participate in plant-pathogen interactions

Pathogen invasion-dependent tissue reservoirs and plasmid-encoded antibiotic degradation boost plasmid spread in the gut

Many plasmids encode antibiotic resistance genes. Through conjugation, plasmids can be rapidly disseminated. Previous work identified gut luminal donor/recipient blooms and tissue-lodged plasmid-bearing persister cells of the enteric pathogen; Salmonella enterica; serovar Typhimurium (; S; .Tm) that survive antibiotic therapy in host tissues, as factors promoting plasmid dissemination among Enterobacteriaceae. However, the buildup of tissue reservoirs and their contribution to plasmid spread await experimental demonstration. Here, we asked if re-seeding-plasmid acquisition-invasion cycles by; S; .Tm could serve to diversify tissue-lodged plasmid reservoirs, and thereby promote plasmid spread. Starting with intraperitoneal mouse infections, we demonstrate that; S; .Tm cells re-seeding the gut lumen initiate clonal expansion. Extended spectrum beta-lactamase (ESBL) plasmid-encoded gut luminal antibiotic degradation by donors can foster recipient survival under beta-lactam antibiotic treatment, enhancing transconjugant formation upon re-seeding.; S; .Tm transconjugants can subsequently re-enter host tissues introducing the new plasmid into the tissue-lodged reservoir. Population dynamics analyses pinpoint recipient migration into the gut lumen as rate-limiting for plasmid transfer dynamics in our model. Priority effects may be a limiting factor for reservoir formation in host tissues. Overall, our proof-of-principle data indicates that luminal antibiotic degradation and shuttling between the gut lumen and tissue-resident reservoirs can promote the accumulation and spread of plasmids within a host over time

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Isolierung und Untersuchung von Naturstoffen und Biosynthesewegen aus Photorhabdus und Xenorhabdus

The entomopathogenic bacteria of the genera Photorhabdus and Xenorhabdus display perfect model organisms to gain insights into the sophisticated interplay between symbiosis and pathogenicity. Moreover, numerous publications in the last years have demonstrated that these bacteria represent a rich source of secondary metabolites, which is exemplified in this work with the description of the novel xenofuranone compounds. The recently available genome sequence of Photorhabdus luminescens TT01 pointed out that many biosynthetic gene clusters remain silent as the corresponding product cannot be detected. The heterologous expression of a nonribosomal peptide synthetase, which resulted in the successful production of indigoidine, depicts one way to gain access on these cryptic gene clusters. In addition the genome sequence also enabled the identification of biosynthesis genes of the already known compound families of stilbenes and anthraquinones. Thereby a type II polyketide synthase cluster was identified, which is responsible for anthraquinone biosynthesis, representing only the second known type II PKS derived compound from a Gram-negative bacterium. Furthermore the identification of genes involved in stilbene biosynthesis led to the discovery of a unique and novel pathway, strongly differing from plant derived stilbenes.Entomopathogene Bakterien der Gattungen Photorhabdus und Xenorhabdus eignen sich hervorragend als Modelorganismen um Einblicke in das komplizierte Wechselspiel zwischen Symbiose und Pathogenität zu erhalten. Darüber hinaus haben zahlreiche Publikationen der letzten Jahre gezeigt, dass diese Bakterien eine reiche Quelle an Sekundärstoffen darstellen. In der vorliegenden Arbeit wird dies anhand der neu beschriebenen Xenofuranone verdeutlicht. Die veröffentlichte Genomsequenz von Photorhabdus luminescens TT01 offenbarte, dass die meisten Biosynthese Gencluster "verwaist" sind, das heißt es ließ sich bisher kein dazugehöriges Produkt detektieren. Die erfolgreiche heterologe Expression einer nichtribosomalen Peptidsynthetase und der damit verbundenen Produktion von Indigoidin, zeigte eine Möglichkeit auf um Zugang zu solchen "verwaisten" Genclustern zu erhalten. Des Weiteren erlaubte die Genomsequenz nach Biosynthesegenen zu suchen, deren Produkte wie zum Beispiel die Stilbene oder die Anthrachinone bereits bekannt waren. Auf diese Weise konnte ein Typ II Polyketidsynthasecluster der für die Biosynthese der Anthrachinone verantwortlich ist identifiziert werden. Die Anthrachinone sind damit erst das zweite bekannte Beispiel eines Typ II PKS erzeugten Produktes aus einem Gram-negativen Bakterium. Zusätzlich gelang es die Gene, die an der Stilbenbiosynthese beteiligt sind zu identifizieren und damit einen neuen und einzigartigen Stoffwechselweg, welcher stark abweichend zur pflanzlichen Biosynthese funktioniert zu beschreiben

Fast direct detection of natural dyes in historic and prehistoric textiles by flowprobe™-ESI-HRMS

Dyestuff analyses were performed directly from the surface of different bluish and reddish colored historic textile samples by flowprobe™-electrospray ionization-high-resolution mass spectrometry (flowprobe™-ESI-HRMS). This real-time in situ microextraction method allowed rapid, reliable and minimal-destructive analysis without extra and time-consuming sample preparation and required only a minimum amount of valuable archaeological material. As demonstrated for indigo-type and anthraquinone dyes this technique is useful for the analysis of various types of textiles regardless of their fiber matrix, appearance or handicraft and is also suitable for investigating fragile archeological fibers. Thus, flowprobe™-ESI-HRMS is a promising analytical tool for characterizing organic colorants in objects of archaeological interest.ISSN:2046-206

Activation, structure, biosynthesis and bioactivity of glidobactin-like proteasome inhibitors from Photorhabdus laumondii

The glidobactin-like natural products (GLNPs) glidobactin A and cepafungin I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development. Their biosynthetic gene cluster (BGC) plu1881–1877 is present in entomopathogenic Photorhabdus laumondii but silent under standard laboratory conditions. Here we show the largest subset of GLNPs, which are produced and identified after activation of the silent BGC in the native host and following heterologous expression of the BGC in Escherichia coli. Their chemical diversity results from a relaxed substrate specificity and flexible product release in the assembly line of GLNPs. Crystal structure analysis of the yeast proteasome in complex with new GLNPs suggests that the degree of unsaturation and the length of the aliphatic tail are critical for their bioactivity. The results in this study provide the basis to engineer the BGC for the generation of new GLNPs and to optimize these natural products resulting in potential drugs for cancer therapy

A Desaturase‐Like Enzyme Catalyzes Oxazole Formation in Pseudomonas Indolyloxazole Alkaloids

Indolyloxazole alkaloids occur in diverse micro‐ and macroorganisms and exhibit a wide range of pharmacological activities. In spite of their ubiquitous occurrence and simple structures, the biosynthetic pathway remained unknown. Here, we used transposon mutagenesis in the labradorin producer Pseudomonas entomophila to identify a cryptic biosynthetic locus encoding an N ‐acyltransferase and a non‐heme diiron desaturase‐like enzyme. Heterologous expression in E. coli demonstrates that both enzymes are sufficient to produce indolyloxazoles. Probing their function in stable‐isotope feeding experiments, we provide evidence for an unusual desaturase mechanism that generates the oxazole by decarboxylative cyclization. © 2021 Wiley‐VCH GmbHISSN:1433-7851ISSN:1521-3773ISSN:0570-083

Heterologous pulcherrimin production in Saccharomyces cerevisiae confers inhibitory activity on Botrytis conidiation

Pulcherrimin is an iron (III) chelate of pulcherriminic acid that plays a role in antagonistic microbial interactions, iron metabolism, and stress responses. Some bacteria and yeasts produce pulcherriminic acid, but so far, pulcherrimin could not be produced in Saccharomyces cerevisiae. Here, multiple integrations of the Metschnikowia pulcherrima PUL1 and PUL2 genes in the S. cerevisiae genome resulted in red colonies, which indicated pulcherrimin formation. The coloration correlated positively and significantly with the number of PUL1 and PUL2 genes. The presence of pulcherriminic acid was confirmed by mass spectrometry. In vitro competition assays with the plant pathogenic fungus Botrytis caroliana revealed inhibitory activity on conidiation by an engineered, strong pulcherrimin-producing S. cerevisiae strain. We demonstrate that the PUL1 and PUL2 genes from M. pulcherrima, in multiple copies, are sufficient to transfer pulcherrimin production to S. cerevisiae and represent the starting point for engineering and optimizing this biosynthetic pathway in the future.ISSN:1567-1356ISSN:1567-136