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Bacterial endosymbionts protect beneficial soil fungus from nematode attack.

Abstract (Expand)

Fungi of the genus Mortierella occur ubiquitously in soils where they play pivotal roles in carbon cycling, xenobiont degradation, and promoting plant growth. These important fungi are, however, … threatened by micropredators such as fungivorous nematodes, and yet little is known about their protective tactics. We report that Mortierella verticillata NRRL 6337 harbors a bacterial endosymbiont that efficiently shields its host from nematode attacks with anthelmintic metabolites. Microscopic investigation and 16S ribosomal DNA analysis revealed that a previously overlooked bacterial symbiont belonging to the genus Mycoavidus dwells in M. verticillata hyphae. Metabolic profiling of the wild-type fungus and a symbiont-free strain obtained by antibiotic treatment as well as genome analyses revealed that highly cytotoxic macrolactones (CJ-12,950 and CJ-13,357, syn necroxime C and D), initially thought to be metabolites of the soil-inhabiting fungus, are actually biosynthesized by the endosymbiont. According to comparative genomics, the symbiont belongs to a new species (Candidatus Mycoavidus necroximicus) with 12% of its 2.2 Mb genome dedicated to natural product biosynthesis, including the modular polyketide-nonribosomal peptide synthetase for necroxime assembly. Using Caenorhabditis elegans and the fungivorous nematode Aphelenchus avenae as test strains, we show that necroximes exert highly potent anthelmintic activities. Effective host protection was demonstrated in cocultures of nematodes with symbiotic and chemically complemented aposymbiotic fungal strains. Image analysis and mathematical quantification of nematode movement enabled evaluation of the potency. Our work describes a relevant role for endofungal bacteria in protecting fungi against mycophagous nematodes.

Authors: H. Buttner, S. P. Niehs, K. Vandelannoote, Z. Cseresnyes, B. Dose, I. Richter, R. Gerst, M. T. Figge, T. P. Stinear, S. J. Pidot, C. Hertweck

Date Published: 14th Sep 2021

Publication Type: Journal

Multimodal Molecular Imaging and Identification of Bacterial Toxins Causing Mushroom Soft Rot and Cavity Disease

Abstract (Expand)

Soft rot disease of edible mushrooms leads to rapid degeneration of fungal tissue and thus severely affects farming productivity worldwide. The bacterial mushroom pathogen Burkholderia gladioli pv. … agaricicola has been identified as the cause. Yet, little is known about the molecular basis, the spatial distribution and the biological role of antifungal agents and toxins involved in this infectious disease. We combine genome mining, metabolic profiling, MALDI-Imaging and UV Raman spectroscopy, to detect, identify and visualize a complex of chemical mediators and toxins produced by the pathogen during the infection process, including toxoflavin, caryoynencin, and sinapigladioside. Furthermore, targeted gene knockouts and in vitro assays link antifungal agents to prevalent symptoms of soft rot, mushroom browning, and impaired mycelium growth. Comparisons of related pathogenic, mutualistic and environmental Burkholderia spp. indicate that the arsenal of antifungal agents may have paved the way for ancestral bacteria to colonize niches where frequent, antagonistic interactions with fungi occur. Our findings not only demonstrate the power of label-free, in vivo detection of polyyne virulence factors by Raman imaging, but may also inspire new approaches to disease control.

Authors: Benjamin Dose, Tawatchai Thongkongkaew, David Zopf, Hak Joong Kim, Evgeni V. Bratovanov, María García-Altares Pérez, Kirstin Scherlach, Jana Krabbe, Claudia Ross, Ron Hermenau, Sarah P. Niehs, Anja Silge, Julian Hniopek, Michael Schmitt, Jürgen Popp, Christian Hertweck

Date Published: 7th Jul 2021

Publication Type: Journal

Mining and unearthing hidden biosynthetic potential.

Abstract (Expand)

Genetically encoded small molecules (secondary metabolites) play eminent roles in ecological interactions, as pathogenicity factors and as drug leads. Yet, these chemical mediators often evade detection, … and the discovery of novel entities is hampered by low production and high rediscovery rates. These limitations may be addressed by genome mining for biosynthetic gene clusters, thereby unveiling cryptic metabolic potential. The development of sophisticated data mining methods and genetic and analytical tools has enabled the discovery of an impressive array of previously overlooked natural products. This review shows the newest developments in the field, highlighting compound discovery from unconventional sources and microbiomes.

Authors: K. Scherlach, C. Hertweck

Date Published: 23rd Jun 2021

Publication Type: Journal

The Termite Fungal Cultivar Termitomyces Combines Diverse Enzymes and Oxidative Reactions for Plant Biomass Conversion.

Abstract (Expand)

Macrotermitine termites have domesticated fungi in the genus Termitomyces as their primary food source using predigested plant biomass. To access the full nutritional value of lignin-enriched plant … biomass, the termite-fungus symbiosis requires the depolymerization of this complex phenolic polymer. While most previous work suggests that lignocellulose degradation is accomplished predominantly by the fungal cultivar, our current understanding of the underlying biomolecular mechanisms remains rudimentary. Here, we provide conclusive omics and activity-based evidence that Termitomyces employs not only a broad array of carbohydrate-active enzymes (CAZymes) but also a restricted set of oxidizing enzymes (manganese peroxidase, dye decolorization peroxidase, an unspecific peroxygenase, laccases, and aryl-alcohol oxidases) and Fenton chemistry for biomass degradation. We propose for the first time that Termitomyces induces hydroquinone-mediated Fenton chemistry (Fe(2+) + H2O2 + H(+) --> Fe(3+) + (*)OH + H2O) using a herein newly described 2-methoxy-1,4-dihydroxybenzene (2-MH2Q, compound 19)-based electron shuttle system to complement the enzymatic degradation pathways. This study provides a comprehensive depiction of how efficient biomass degradation by means of this ancient insect's agricultural symbiosis is accomplished. IMPORTANCE Fungus-growing termites have optimized the decomposition of recalcitrant plant biomass to access valuable nutrients by engaging in a tripartite symbiosis with complementary contributions from a fungal mutualist and a codiversified gut microbiome. This complex symbiotic interplay makes them one of the most successful and important decomposers for carbon cycling in Old World ecosystems. To date, most research has focused on the enzymatic contributions of microbial partners to carbohydrate decomposition. Here, we provide genomic, transcriptomic, and enzymatic evidence that Termitomyces also employs redox mechanisms, including diverse ligninolytic enzymes and a Fenton chemistry-based hydroquinone-catalyzed lignin degradation mechanism, to break down lignin-rich plant material. Insights into these efficient decomposition mechanisms reveal new sources of efficient ligninolytic agents applicable for energy generation from renewable sources.

Authors: F. Schalk, C. Gostincar, N. B. Kreuzenbeck, B. H. Conlon, E. Sommerwerk, P. Rabe, I. Burkhardt, T. Kruger, O. Kniemeyer, A. A. Brakhage, N. Gunde-Cimerman, Z. W. de Beer, J. S. Dickschat, M. Poulsen, C. Beemelmanns

Date Published: 15th Jun 2021

Publication Type: Journal

GNPS-guided discovery of xylacremolide C and D, evaluation of their putative biosynthetic origin and bioactivity studies of xylacremolide A and B.

Abstract (Expand)

Targeted HRMS(2)-GNPS-based metabolomic analysis of Pseudoxylaria sp. X187, a fungal antagonist of the fungus-growing termite symbiosis, resulted in the identification of two lipopeptidic congeners of … xylacremolides, named xylacremolide C and D, which are built from d-phenylalanine, l-proline and an acetyl-CoA starter unit elongated by four malonyl-CoA derived ketide units. The putative xya gene cluster was identified from a draft genome generated by Illumina and PacBio sequencing and RNAseq studies. Biological activities of xylacremolide A and B were evaluated and revealed weak histone deacetylase inhibitory (HDACi) and antifungal activities, as well as moderate protease inhibition activity across a panel of nine human, viral and bacterial proteases.

Authors: F. Schalk, J. Fricke, S. Um, B. H. Conlon, H. Maus, N. Jager, T. Heinzel, T. Schirmeister, M. Poulsen, C. Beemelmanns

Date Published: 24th May 2021

Publication Type: Journal

Mind the mushroom: natural product biosynthetic genes and enzymes of Basidiomycota.

Abstract (Expand)

Covering: up to September 2020 Mushroom-forming fungi of the division Basidiomycota have traditionally been recognised as prolific producers of structurally diverse and often bioactive secondary … metabolites, using the methods of chemistry for research. Over the past decade, -omics technologies were applied on these fungi, and sophisticated heterologous gene expression platforms emerged, which have boosted research into the genetic and biochemical basis of the biosyntheses. This review provides an overview on experimentally confirmed natural product biosyntheses of basidiomycete polyketides, amino acid-derived products, terpenoids, and volatiles. We also present challenges and solutions particular to natural product research with these fungi. 222 references are cited.

Authors: M. Gressler, N. A. Lohr, T. Schafer, S. Lawrinowitz, P. S. Seibold, D. Hoffmeister

Date Published: 28th Apr 2021

Publication Type: Journal

Frankobactin Metallophores Produced by Nitrogen-Fixing Frankia Actinobacteria Function in Toxic Metal Sequestration.

Abstract (Expand)

A series of new metallophores, referred to as frankobactins, were extracted from cultures of the symbiotic and nitrogen-fixing actinobacterium Frankia sp. CH37. Structure elucidation revealed a … 2-hydroxyphenyl-substituted oxazoline core and a chain composed of five proteinogenic and nonproteinogenic amino acids, suggesting nonribosomal peptide synthesis as the biosynthetic origin. By whole-genome sequencing, bioinformatic analysis, and comparison with other Frankia strains, the genetic locus responsible for the biosynthesis was detected. Spectrophotometric titration of frankobactin with Fe(III) and Cu(II) and mass spectrometry established the 1:1 (metal:frankobactin) coordination. Uptake experiments suggested that frankobactin A1 (1) did not serve to recruit iron, but to detoxify Cu(II). As frankobactin A1 prevents the cellular entry of Cu(II), it could play a crucial role in the symbiosis of Frankia sp. and its host in the reclamation of copper-contaminated soil.

Authors: J. F. Mohr, F. Baldeweg, M. Deicke, C. F. Morales-Reyes, D. Hoffmeister, T. Wichard

Date Published: 23rd Apr 2021

Publication Type: Journal

Biosynthesis of Sinapigladioside, an Antifungal Isothiocyanate from Burkholderia Symbionts.

Abstract (Expand)

Sinapigladioside is a rare isothiocyanate-bearing natural product from beetle-associated bacteria ( Burkholderia gladioli ) that may protect the beetle offspring against entomopathogenic fungi. The … biosynthetic origin of sinapigladioside has been elusive, and little is known about bacterial isothiocyanate biosynthesis in general. On the basis of stable-isotope labeling, bioinformatics, and mutagenesis we identified the sinapigladioside biosynthesis gene cluster in the symbiont and found that an isonitrile synthase plays a key role in the biosynthetic pathway. Genome mining and network analyses indicate that related gene clusters are distributed across various bacterial phyla including producers of both nitriles and isothiocyanates. Our findings support a model for bacterial isothiocyanate biosynthesis by sulfur transfer onto isonitrile precursors.

Authors: B. Dose, S. P. Niehs, K. Scherlach, S. Shahda, L. V. Florez, M. Kaltenpoth, C. Hertweck

Date Published: 19th Mar 2021

Publication Type: Journal

Iron Coordination Properties of Gramibactin as Model for the New Class of Diazeniumdiolate Based Siderophores.

Abstract (Expand)

Gramibactin (GBT) is an archetype for the new class of diazeniumdiolate siderophores, produced by Paraburkholderia graminis, a cereal-associated rhizosphere bacterium, for which a detailed solution … thermodynamic study exploring the iron coordination properties is reported. The acid-base behavior of gramibactin as well as its complexing ability toward Fe(3+) was studied over a wide range of pH values (2</=pH</=11). For the latter the ligand-competition method employing EDTA was used. Only two species are formed: [Fe(GBT)](-) (pH 2 to 9) and [Fe(GBT)(OH)2 ](3-) (pH>/=9). The formation of [Fe(GBT)](-) and its occurrence in real systems was confirmed by LC-HRESIMS analysis of the bacteria culture broth extract. The sequestering ability of gramibactin was also evaluated in terms of the parameters pFe and pL0.5 . Gramibactin exhibits a higher sequestering ability toward Fe(3+) than EDTA and of the same order of magnitude as hydroxamate-type microbial siderophores, but smaller than most of the catecholate-type siderophores and much higher than the most known phytosiderophores.

Authors: S. Gama, R. Hermenau, M. Frontauria, D. Milea, S. Sammartano, C. Hertweck, W. Plass

Date Published: 5th Feb 2021

Publication Type: Journal

Lipopeptide-mediated bacterial interaction enables cooperative predator defense

Abstract (Expand)

Bacteria are inherently social organisms whose actions should ideally be studied within an interactive ecological context. We show that the exchange and modification of natural products enables two … unrelated bacteria to defend themselves against a common predator. Amoebal predation is a major cause of death in soil bacteria and thus it exerts a trong selective pressure to evolve defensive strategies. A systematic analysis of binary combinations of coisolated bacteria revealed strains that were individually susceptible to predation but together killed their predator. This cooperative defense relies on a Pseudomonas species producing syringafactin, a lipopeptide, which induces the production of peptidases in a Paenibacillus strain. These peptidases then degrade the innocuous syringafactin into compounds, which kill the predator. A combination of bioprospecting, coculture experiments, genome modification, and transcriptomics unravel this novel natural product-based defense strategy.

Authors: Shuaibing Zhang, Ruchira Mukherji, Somak Chowdhury, Lisa Reimer, Pierre Stallforth

Date Published: 1st Feb 2021

Publication Type: Journal

N-Heterocyclization in Gliotoxin Biosynthesis is Catalyzed by a Distinct Cytochrome P450 Monooxygenase.

Abstract (Expand)

Gliotoxin and related epidithiodiketopiperazines (ETP) from diverse fungi feature highly functionalized hydroindole scaffolds with an array of medicinally and ecologically relevant activities. Mutation … analysis, heterologous reconstitution, and biotransformation experiments revealed that a cytochrome P450 monooxygenase (GliF) from the human-pathogenic fungus Aspergillus fumigatus plays a key role in the formation of the complex heterocycle. In vitro assays using a biosynthetic precursor from a blocked mutant showed that GliF is specific to ETPs and catalyzes an unprecedented heterocyclization reaction that cannot be emulated with current synthetic methods. In silico analyses indicate that this rare biotransformation takes place in related ETP biosynthetic pathways.

Authors: D. H. Scharf, P. Chankhamjon, K. Scherlach, J. Dworschak, T. Heinekamp, M. Roth, A. A. Brakhage, C. Hertweck

Date Published: 15th Jan 2021

Publication Type: Journal

Taking Different Roads: l-Tryptophan as the Origin of Psilocybe Natural Products.

Abstract (Expand)

Psychotropic fungi of the genus Psilocybe, colloquially referred to as "magic mushrooms", are best known for their l-tryptophan-derived major natural product, psilocybin. Yet, recent research … has revealed a more diverse secondary metabolism that originates from this amino acid. In this minireview, the focus is laid on l-tryptophan and the various Psilocybe natural products and their metabolic routes are highlighted. Psilocybin and its congeners, the heterogeneous blue-colored psilocyl oligomers, alongside beta-carbolines and N,N-dimethyl-l-tryptophan, are presented as well as current knowledge on their biosynthesis is provided. The multidisciplinary character of natural product research is demonstrated, and pharmacological, medicinal, ecological, biochemical, and evolutionary aspects are included.

Authors: C. Lenz, A. Sherwood, R. Kargbo, D. Hoffmeister

Date Published: 26th Nov 2020

Publication Type: Journal

Genomics-Driven discovery of natural products from symbiotic Burkholderia species

Abstract

Not specified

Editor:

Date Published: 20th Nov 2020

Publication Type: Doctoral Thesis

Polyhalogenation of Isoflavonoids by the Termite-Associated Actinomadura sp. RB99.

Abstract (Expand)

Based on high-resolution tandem mass spectrometry (HR-MS(2)) and global natural products social molecular networking (GNPS), we found that plant-derived daidzein and genistein derivatives are polyhalogenated … by termite-associated Actinomadura species RB99. MS-guided purification from extracts of bacteria grown under optimized conditions led to the isolation of eight polychlorinated isoflavones, including six unreported derivatives, and seven novel polybrominated derivatives, two of which showed antimicrobial activity.

Authors: S. Rak Lee, F. Schalk, J. W. Schwitalla, R. Benndorf, J. Vollmers, A. K. Kaster, Z. W. de Beer, M. Park, M. J. Ahn, W. H. Jung, C. Beemelmanns, K. H. Kim

Date Published: 23rd Oct 2020

Publication Type: Journal

Targeted Discovery of Tetrapeptides and Cyclic Polyketide-Peptide Hybrids from a Fungal Antagonist of Farming Termites.

Abstract (Expand)

Herein, we report the targeted isolation and characterization of four linear nonribosomally synthesized tetrapeptides (pseudoxylaramide A-D) and two cyclic nonribosomal peptide synthetase-polyketide … synthase-derived natural products (xylacremolide A and B) from the termite-associated stowaway fungus Pseudoxylaria sp. X187. The fungal strain was prioritized for further metabolic analysis based on its taxonomical position and morphological and bioassay data. Metabolic data were dereplicated based on high-resolution tandem mass spectrometry data and global molecular networking analysis. The structure of all six new natural products was elucidated based on a combination of 1D and 2D NMR analysis, Marfey's analysis and X-ray crystallography.

Authors: F. Schalk, S. Um, H. Guo, N. B. Kreuzenbeck, H. Gorls, Z. W. de Beer, C. Beemelmanns

Date Published: 15th Oct 2020

Publication Type: Journal

Rhizobactin B is the preferred siderophore by a novel Pseudomonas isolate to obtain iron from dissolved organic matter in peatlands.

Abstract (Expand)

Bacteria often release diverse iron-chelating compounds called siderophores to scavenge iron from the environment for many essential biological processes. In peatlands, where the biogeochemical cycle … of iron and dissolved organic matter (DOM) are coupled, bacterial iron acquisition can be challenging even at high total iron concentrations. We found that the bacterium Pseudomonas sp. FEN, isolated from an Fe-rich peatland in the Northern Bavarian Fichtelgebirge (Germany), released an unprecedented siderophore for its genus. High-resolution mass spectrometry (HR-MS) using metal isotope-coded profiling (MICP), MS/MS experiments, and nuclear magnetic resonance spectroscopy (NMR) identified the amino polycarboxylic acid rhizobactin and a novel derivative at even higher amounts, which was named rhizobactin B. Interestingly, pyoverdine-like siderophores, typical for this genus, were not detected. With peat water extract (PWE), studies revealed that rhizobactin B could acquire Fe complexed by DOM, potentially through a TonB-dependent transporter, implying a higher Fe binding constant of rhizobactin B than DOM. The further uptake of Fe-rhizobactin B by Pseudomonas sp. FEN suggested its role as a siderophore. Rhizobactin B can complex several other metals, including Al, Cu, Mo, and Zn. The study demonstrates that the utilization of rhizobactin B can increase the Fe availability for Pseudomonas sp. FEN through ligand exchange with Fe-DOM, which has implications for the biogeochemical cycling of Fe in this peatland.

Authors: S. Kugler, R. E. Cooper, J. Boessneck, K. Kusel, T. Wichard

Date Published: 7th Oct 2020

Publication Type: Journal

Streptomyces smaragdinus sp. nov., isolated from the gut of the fungus growing-termite Macrotermes natalensis.

Abstract (Expand)

The taxonomic position of a novel aerobic, Gram-positive actinobacteria, designated strain RB5(T), was determined using a polyphasic approach. The strain, isolated from the gut of the fungus-farming … termite Macrotermes natalensis, showed morphological, physiological and chemotaxonomic properties typical of the genus Streptomyces. Based on 16S rRNA gene sequence analysis, the closest phylogenetic neighbour of RB5(T) was Streptomyces polyrhachis DSM 42102(T) (98.87 %). DNA-DNA hybridization experiments between strain RB5(T) and S. polyrhachis DSM 42102(T) resulted in a value of 27.4 % (26.8 %). The cell wall of strain RB5(T) contained ll-diaminopimelic acid as the diagnostic amino acid. Mycolic acids and diagnostic sugars in whole-cell hydrolysates were not detected. The strain produced the following major phospholipids: diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol-mannoside and phosphatidylserine. The menaquinone profile showed hexa- and octahydrogenated menaquinones containing nine isoprene units [MK-9(H6) and MK-9(H8)]. The strain exhibited a fatty acid profile containing the following major fatty acids: 12-methyltridecanoic acid (iso-C14 : 0) 12-methyltetradecanoic acid (anteiso-C15 : 0), 13-methyltetradecanoic acid (iso-C15 : 0) and 14-methylpentadecanoic acid (iso-C16 : 0). Here, we propose a novel species of the genus Streptomyces - Streptomyces smaragdinus with the type strain RB5(T) (=VKM Ac-2839(T)=NRRL B65539(T)).

Authors: J. W. Schwitalla, R. Benndorf, K. Martin, J. Vollmers, A. K. Kaster, Z. W. de Beer, M. Poulsen, C. Beemelmanns

Date Published: 25th Sep 2020

Publication Type: Journal

Chemical Mediators at the Bacterial-Fungal Interface.

Abstract (Expand)

Interactions among microbes are key drivers of evolutionary progress and constantly shape ecological niches. Microorganisms rely on chemical communication to interact with each other and surrounding … organisms. They synthesize natural products as signaling molecules, antibiotics, or modulators of cellular processes that may be applied in agriculture and medicine. Whereas major insight has been gained into the principles of intraspecies interaction, much less is known about the molecular basis of interspecies interplay. In this review, we summarize recent progress in the understanding of chemically mediated bacterial-fungal interrelations. We discuss pairwise interactions among defined species and systems involving additional organisms as well as complex interactions among microbial communities encountered in the soil or defined as microbiota of higher organisms. Finally, we give examples of how the growing understanding of microbial interactions has contributed to drug discovery and hypothesize what may be future directions in studying and engineering microbiota for agricultural or medicinal purposes.

Authors: K. Scherlach, C. Hertweck

Date Published: 8th Sep 2020

Publication Type: Journal

Helper bacteria halt and disarm mushroom pathogens by linearizing structurally diverse cyclolipopeptides.

Abstract (Expand)

The bacterial pathogen Pseudomonas tolaasii severely damages white button mushrooms by secretion of the pore-forming toxin tolaasin, the main virulence factor of brown blotch disease. Yet, fungus-associated … helper bacteria of the genus Mycetocola (Mycetocola tolaasinivorans and Mycetocola lacteus) may protect their host by an unknown detoxification mechanism. By a combination of metabolic profiling, imaging mass spectrometry, structure elucidation, and bioassays, we found that the helper bacteria inactivate tolaasin by linearizing the lipocyclopeptide. Furthermore, we found that Mycetocola spp. impair the dissemination of the pathogen by cleavage of the lactone ring of pseudodesmin. The role of pseudodesmin as a major swarming factor was corroborated by identification and inactivation of the corresponding biosynthetic gene cluster. Activity-guided fractionation of the Mycetocola proteome, matrix-assisted laser desorption/ionization (MALDI) analyses, and heterologous enzyme production identified the lactonase responsible for toxin cleavage. We revealed an antivirulence strategy in the context of a tripartite interaction that has high ecological and agricultural relevance.

Authors: R. Hermenau, S. Kugel, A. J. Komor, C. Hertweck

Date Published: 31st Aug 2020

Publication Type: Not specified

Nocardia macrotermitis sp. nov. and Nocardia aurantia sp. nov., isolated from the gut of the fungus-growing termite Macrotermes natalensis.

Abstract (Expand)

The taxonomic positions of two novel aerobic, Gram-stain-positive Actinobacteria, designated RB20(T) and RB56(T), were determined using a polyphasic approach. Both were isolated from the fungus-farming … termite Macrotermes natalensis. Results of 16S rRNA gene sequence analysis revealed that both strains are members of the genus Nocardia with the closest phylogenetic neighbours Nocardia miyunensis JCM12860(T) (98.9 %) and Nocardia nova DSM44481(T) (98.5 %) for RB20(T) and Nocardia takedensis DSM 44801(T) (98.3 %), Nocardia pseudobrasiliensis DSM 44290(T) (98.3 %) and Nocardia rayongensis JCM 19832(T) (98.2 %) for RB56(T). Digital DNA-DNA hybridization (DDH) between RB20(T) and N. miyunensis JCM12860(T) and N. nova DSM 44481(T) resulted in similarity values of 33.9 and 22.0 %, respectively. DDH between RB56(T) and N. takedensis DSM44801(T) and N. pseudobrasiliensis DSM44290(T) showed similarity values of 20.7 and 22.3 %, respectively. In addition, wet-lab DDH between RB56(T) and N. rayongensis JCM19832(T) resulted in 10.2 % (14.5 %) similarity. Both strains showed morphological and chemotaxonomic features typical for the genus Nocardia, such as the presence of meso-diaminopimelic acid (A2pm) within the cell wall, arabinose and galactose as major sugar components within whole cell-wall hydrolysates, the presence of mycolic acids and major phospholipids (diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol), and the predominant menaquinone MK-8 (H4, omega-cyclo). The main fatty acids for both strains were hexadecanoic acid (C16 : 0), 10-methyloctadecanoic acid (10-methyl C18 : 0) and cis-9-octadecenoic acid (C18 : 1 omega9c). We propose two novel species within the genus Nocardia: Nocardia macrotermitis sp. nov. with the type strain RB20(T) (=VKM Ac-2841(T)=NRRL B65541(T)) and Nocardia aurantia sp. nov. with the type strain RB56(T) (=VKM Ac-2842(T)=NRRL B65542(T)).

Authors: R. Benndorf, J. W. Schwitalla, K. Martin, Z. W. de Beer, J. Vollmers, A. K. Kaster, M. Poulsen, C. Beemelmanns

Date Published: 21st Aug 2020

Publication Type: Journal

Functional Variation in Dipteran Gut Bacterial Communities in Relation to Their Diet, Life Cycle Stage and Habitat.

Abstract (Expand)

True flies and mosquitos (Diptera) live in habitats and consume diets that pose specific demands on their gut bacterial communities (GBCs). Due to diet specializations, dipterans may have highly diverse … and species-specific GBCs. Dipterans are also confronted with changes in habitat and food sources over their lifetime, especially during life history processes (molting, metamorphosis). This may prevent the development of a constant species- or diet-specific GBC. Some dipterans are vectors of several human pathogens (e.g., malaria), which interact with GBCs. In this review, we explore the dynamics that shape GBC composition in some Diptera species on the basis of published datasets of GBCs. We thereby focus on the effects of diet, habitats, and life cycle stages as sources of variation in GBC composition. The GBCs reported were more stage-specific than species- or diet-specific. Even though the presence of GBCs has a large impact on the performance of their hosts, the exact functions of GBCs and their interactions with other organisms are still largely unknown, mainly due to the low number of studies to date. Increasing our knowledge on dipteran GBCs will help to design pest management strategies for the reduction of insecticide resistance, as well as for human pathogen control.

Authors: R. Sontowski, N. M. van Dam

Date Published: 17th Aug 2020

Publication Type: Journal

Food-Poisoning Bacteria Employ a Citrate Synthase and a Type II NRPS To Synthesize Bolaamphiphilic Lipopeptide Antibiotics.

Abstract (Expand)

Mining the genome of the food-spoiling bacterium Burkholderia gladioli pv. cocovenenans revealed five nonribosomal peptide synthetase (NRPS) gene clusters, including an orphan gene locus (bol). Gene … inactivation and metabolic profiling linked the bol gene cluster to novel bolaamphiphilic lipopeptides with antimycobacterial activity. A combination of chemical analysis and bioinformatics elucidated the structures of bolagladin A and B, lipocyclopeptides featuring an unusual dehydro-beta-alanine enamide linker fused to an unprecedented tricarboxylic fatty acid tail. Through a series of targeted gene deletions, we proved the involvement of a designated citrate synthase (CS), priming ketosynthases III (KS III), a type II NRPS, including a novel desaturase for enamide formation, and a multimodular NRPS in generating the cyclopeptide. Network analyses revealed the evolutionary origin of the CS and identified cryptic CS/NRPS gene loci in various bacterial genomes.

Authors: B. Dose, C. Ross, S. P. Niehs, K. Scherlach, J. P. Bauer, C. Hertweck

Date Published: 11th Aug 2020

Publication Type: Not specified

Sulfonium Acids Loaded onto an Unusual Thiotemplate Assembly Line Construct the Cyclopropanol Warhead of a Burkholderia Virulence Factor.

Abstract (Expand)

Pathogenic bacteria of the Burkholderia pseudomallei group cause severe infectious diseases such as glanders and melioidosis. Malleicyprols were identified as important bacterial virulence factors, yet … the biosynthetic origin of their cyclopropanol warhead has remained enigmatic. By a combination of mutational analysis and metabolomics we found that sulfonium acids, dimethylsulfoniumpropionate (DMSP) and gonyol, known as osmolytes and as crucial components in the global organosulfur cycle, are key intermediates en route to the cyclopropanol unit. Functional genetics and in vitro analyses uncover a specialized pathway to DMSP involving a rare prokaryotic SET-domain methyltransferase for a cryptic methylation, and show that DMSP is loaded onto the NRPS-PKS hybrid assembly line by an adenylation domain dedicated to zwitterionic starter units. Then, the megasynthase transforms DMSP into gonyol, as demonstrated by heterologous pathway reconstitution in E. coli.

Authors: F. Trottmann, K. Ishida, J. Franke, A. Stanisic, M. Ishida-Ito, H. Kries, G. Pohnert, C. Hertweck

Date Published: 3rd Aug 2020

Publication Type: Journal

Scalable Hybrid Synthetic/Biocatalytic Route to Psilocybin.

Abstract (Expand)

Psilocybin, the principal indole alkaloid of Psilocybe mushrooms, is currently undergoing clinical trials as a medication against treatment-resistant depression and major depressive disorder. The … psilocybin supply for pharmaceutical purposes is met by synthetic chemistry. We replaced the problematic phosphorylation step during synthesis with the mushroom kinase PsiK. This enzyme was biochemically characterized and used to produce one gram of psilocybin from psilocin within 20 minutes. We also describe a pilot-scale protocol for recombinant PsiK that yielded 150 mg enzyme in active and soluble form. Our work consolidates the simplicity of tryptamine chemistry with the specificity and selectivity of enzymatic catalysis and helps provide access to an important drug at potentially reasonable cost.

Authors: J. Fricke, R. Kargbo, L. Regestein, C. Lenz, G. Peschel, M. A. Rosenbaum, A. Sherwood, D. Hoffmeister

Date Published: 2nd Jul 2020

Publication Type: Journal

Insect-Associated Bacteria Assemble the Antifungal Butenolide Gladiofungin by Non-Canonical Polyketide Chain Termination.

Abstract (Expand)

Genome mining of one of the protective symbionts ( Burkholderia gladioli ) of the invasive beetle Lagria villosa revealed a cryptic gene cluster coding for the biosynthesis of a novel antifungal … polyketide with a glutarimide pharmacophore. Targeted gene inactivation, metabolic profiling and bioassays led to the discovery of the gladiofungins as yet overlooked components of the antimicrobial armory of the beetle symbiont, specifically against the entomopathogenic fungus Purpureocillium lilacinum . By mutational analyses, isotope labeling and in silico analyses of the modular polyketide synthase, we found that the rare butenolide moiety of gladiofungins derives from an unprecedented polyketide chain termination reaction involving a glycerol-derived C3 building block. The key role of an A-factor synthase (AfsA)-like offloading domain was corroborated by CRISPR-Cas-mediated gene editing, which facilitated the precise excision within a PKS domain.

Authors: S. P. Niehs, J. Kumpfmuller, B. Dose, R. F. Little, K. Ishida, L. V. Florez, M. Kaltenpoth, C. Hertweck

Date Published: 26th Jun 2020

Publication Type: Not specified

Information theory tests critical predictions of plant defense theory for specialized metabolism.

Abstract (Expand)

Different plant defense theories have provided important theoretical guidance in explaining patterns in plant specialized metabolism, but their critical predictions remain to be tested. Here, we … systematically explored the metabolomes of Nicotiana attenuata, from single plants to populations, as well as of closely related species, using unbiased tandem mass spectrometry (MS/MS) analyses and processed the abundances of compound spectrum-based MS features within an information theory framework to test critical predictions of optimal defense (OD) and moving target (MT) theories. Information components of plant metabolomes were consistent with the OD theory but contradicted the main prediction of the MT theory for herbivory-induced dynamics of metabolome compositions. From micro- to macroevolutionary scales, jasmonate signaling was confirmed as the master determinant of OD, while ethylene signaling provided fine-tuning for herbivore-specific responses annotated via MS/MS molecular networks.

Authors: D. Li, R. Halitschke, I. T. Baldwin, E. Gaquerel

Date Published: 25th Jun 2020

Publication Type: Journal

Macroalgal-bacterial interactions: identification and role of thallusin in morphogenesis of the seaweed Ulva (Chlorophyta).

Abstract (Expand)

Macroalgal microbiomes have core functions related to biofilm formation, growth, and morphogenesis of seaweeds. In particular, the growth and development of the sea lettuce Ulva spp. (Chlorophyta) … depend on bacteria releasing morphogenetic compounds. Under axenic conditions, the macroalga Ulva mutabilis develops a callus-like phenotype with cell wall protrusions. However, co-culturing with Roseovarius sp. (MS2) and Maribacter sp. (MS6), which produce various stimulatory chemical mediators, completely recovers morphogenesis. This ecological reconstruction forms a tripartite community which can be further studied for its role in cross-kingdom interactions. Hence, our study sought to identify algal growth- and morphogenesis-promoting factors (AGMPFs) capable of phenocopying the activity of Maribacter spp. We performed bioassay-guided solid-phase extraction in water samples collected from U. mutabilis aquaculture systems. We uncovered novel ecophysiological functions of thallusin, a sesquiterpenoid morphogen, identified for the first time in algal aquaculture. Thallusin, released by Maribacter sp., induced rhizoid and cell wall formation at a concentration of 11 pmol l-1. We demonstrated that gametes acquired the iron complex of thallusin, thereby linking morphogenetic processes with intracellular iron homeostasis. Understanding macroalgae-bacteria interactions permits further elucidation of the evolution of multicellularity and cellular differentiation, and development of new applications in microbiome-mediated aquaculture systems.

Authors: T. Alsufyani, G. Califano, M. Deicke, J. Grueneberg, A. Weiss, A. H. Engelen, M. Kwantes, J. F. Mohr, J. F. Ulrich, T. Wichard

Date Published: 11th Jun 2020

Publication Type: Journal

S-Adenosyl-l-Methionine Salvage Impacts Psilocybin Formation in "Magic" Mushrooms.

Abstract (Expand)

Psychotropic Psilocybe mushrooms biosynthesize their principal natural product psilocybin in five steps, among them a phosphotransfer and two methyltransfer reactions, which consume one equivalent of … 5'-adenosine triphosphate (ATP) and two equivalents of S-adenosyl-l-methionine (SAM). This short but co-substrate-intensive pathway requires nucleoside cofactor salvage to maintain high psilocybin production rates. We characterized the adenosine kinase (AdoK) and S-adenosyl-l-homocysteine (SAH) hydrolase (SahH) of Psilocybe cubensis. Both enzymes are directly or indirectly involved in regenerating SAM. qRT-PCR expression analysis revealed an induced expression of the genes in the fungal primordia and carpophores. A one-pot in vitro reaction with the N-methyltransferase PsiM of the psilocybin pathway demonstrates a concerted action with SahH to facilitate biosynthesis by removal of accumulating SAH.

Authors: R. Demmler, J. Fricke, S. Dorner, M. Gressler, D. Hoffmeister

Date Published: 4th May 2020

Publication Type: Journal

Mining Symbionts of a Spider-Transmitted Fungus Illuminates Uncharted Biosynthetic Pathways to Cytotoxic Benzolactones

Abstract (Expand)

A spider-transmitted fungus (Rhizopus microspo- rus) that was isolated from necrotic human tissue was found to harbor endofungal bacteria (Burkholderia sp.). Metabolic profiling of the symbionts revealed … a complex of cytotoxic agents (necroximes). Their structures were characterized as oxime-substituted benzolactone enamides with a peptidic side chain. The potently cytotoxic necroximes are also formed in symbiosis with the fungal host and could have contributed to the necrosis. Genome sequencing and computational analyses revealed a novel modular PKS/NRPS assembly line equipped with several non-canonical domains. Based on gene-deletion mutants, we propose a biosynthetic model for bacterial benzolactones. We identified specific traits that serve as genetic handles to find related salicylate macrolide pathways (lobata- mide, oximidine, apicularen) in various other bacterial genera. Knowledge of the biosynthetic pathway enables biosynthetic engineering and genome-mining approaches.

Authors: Sarah Niehs, Benjamin Dose, Sophie Richter, Sacha J. Pidot, Timothy P. Stinear, Hans-Martin Dahse, Christian Hertweck

Date Published: 10th Feb 2020

Publication Type: Not specified

Both Biosynthesis and Transport Are Involved in Glucosinolate Accumulation During Root-Herbivory in Brassica rapa.

Abstract (Expand)

The optimal defense theory predicts that plants invest most energy in those tissues that have the highest value, but are most vulnerable to attacks. In Brassica species, root-herbivory leads to the … accumulation of glucosinolates (GSLs) in the taproot, the most valuable belowground plant organ. Accumulation of GSLs can result from local biosynthesis in response to herbivory. In addition, transport from distal tissues by specialized GSL transporter proteins can play a role as well. GSL biosynthesis and transport are both inducible, but the role these processes play in GSL accumulation during root-herbivory is not yet clear. To address this issue, we performed two time-series experiments to study the dynamics of transport and biosynthesis in local and distal tissues of Brassica rapa. We exposed roots of B. rapa to herbivory by the specialist root herbivore Delia radicum for 7 days. During this period, we sampled above- and belowground plant organs 12 h, 24 h, 3 days and 7 days after the start of herbivory. Next, we measured the quantity and composition of GSL profiles together with the expression of genes involved in GSL biosynthesis and transport. We found that both benzyl and indole GSLs accumulate in the taproot during root-herbivory, whereas we did not observe any changes in aliphatic GSL levels. The rise in indole GSL levels coincided with increased local expression of biosynthesis and transporter genes, which suggest that both biosynthesis and GSL transport play a role in the accumulation of GSLs during root herbivory. However, we did not observe a decrease in GSL levels in distal tissues. We therefore hypothesize that GSL transporters help to retain GSLs in the taproot during root-herbivory.

Authors: A. J. Touw, A. Verdecia Mogena, A. Maedicke, R. Sontowski, N. M. van Dam, T. Tsunoda

Date Published: 31st Jan 2020

Publication Type: Journal

Injury-Triggered Blueing Reactions of Psilocybe "Magic" Mushrooms.

Abstract (Expand)

Upon injury, psychotropic psilocybin-producing mushrooms instantly develop an intense blue color, the chemical basis and mode of formation of which has remained elusive. We report two enzymes from … Psilocybe cubensis that carry out a two-step cascade to prepare psilocybin for oxidative oligomerization that leads to blue products. The phosphatase PsiP removes the 4-O-phosphate group to yield psilocin, while PsiL oxidizes its 4-hydroxy group. The PsiL reaction was monitored by in situ (13) C NMR spectroscopy, which indicated that oxidative coupling of psilocyl residues occurs primarily via C-5. MS and IR spectroscopy indicated the formation of a heterogeneous mixture of preferentially psilocyl 3- to 13-mers and suggest multiple oligomerization routes, depending on oxidative power and substrate concentration. The results also imply that phosphate ester of psilocybin serves a reversible protective function.

Authors: C. Lenz, J. Wick, D. Braga, M. Garcia-Altares, G. Lackner, C. Hertweck, M. Gressler, D. Hoffmeister

Date Published: 20th Jan 2020

Publication Type: Journal

Simultaneous Production of Psilocybin and a Cocktail of beta-Carboline Monoamine Oxidase Inhibitors in "Magic" Mushrooms.

Abstract (Expand)

The psychotropic effects of Psilocybe "magic" mushrooms are caused by the l-tryptophan-derived alkaloid psilocybin. Despite their significance, the secondary metabolome of these fungi is … poorly understood in general. Our analysis of four Psilocybe species identified harmane, harmine, and a range of other l-tryptophan-derived beta-carbolines as their natural products, which was confirmed by 1D and 2D NMR spectroscopy. Stable-isotope labeling with (13) C11 -l-tryptophan verified the beta-carbolines as biosynthetic products of these fungi. In addition, MALDI-MS imaging showed that beta-carbolines accumulate toward the hyphal apices. As potent inhibitors of monoamine oxidases, beta-carbolines are neuroactive compounds and interfere with psilocybin degradation. Therefore, our findings represent an unprecedented scenario of natural product pathways that diverge from the same building block and produce dissimilar compounds, yet contribute directly or indirectly to the same pharmacological effects.

Authors: F. Blei, S. Dorner, J. Fricke, F. Baldeweg, F. Trottmann, A. Komor, F. Meyer, C. Hertweck, D. Hoffmeister

Date Published: 13th Jan 2020

Publication Type: Journal

Antifungal potential of secondary metabolites involved in the interaction between citrus pathogens.

Abstract (Expand)

Numerous postharvest diseases have been reported that cause substantial losses of citrus fruits worldwide. Penicillium digitatum is responsible for up to 90% of production losses, and represent a problem … for worldwide economy. In order to control phytopathogens, chemical fungicides have been extensively used. Yet, the use of some artificial fungicides cause concerns about environmental risks and fungal resistance. Therefore, studies focusing on new approaches, such as the use of natural products, are getting attention. Co-culture strategy can be applied to discover new bioactive compounds and to understand microbial ecology. Mass Spectrometry Imaging (MSI) was used to screen for potential antifungal metabolites involved in the interaction between Penicillium digitatum and Penicillium citrinum. MSI revealed a chemical warfare between the fungi: two tetrapeptides, deoxycitrinadin A, citrinadin A, chrysogenamide A and tryptoquialanines are produced in the fungi confrontation zone. Antimicrobial assays confirmed the antifungal activity of the investigated metabolites. Also, tryptoquialanines inhibited sporulation of P. citrinum. The fungal metabolites reported here were never described as antimicrobials until this date, demonstrating that co-cultures involving phytopathogens that compete for the same host is a positive strategy to discover new antifungal agents. However, the use of these natural products on the environment, as a safer strategy, needs further investigation. This paper aimed to contribute to the protection of agriculture, considering health and ecological risks.

Authors: J. H. Costa, C. I. Wassano, C. F. F. Angolini, K. Scherlach, C. Hertweck, T. Pacheco Fill

Date Published: 9th Dec 2019

Publication Type: Journal

Same Difference? Low and High Glucosinolate Brassica rapa Varieties Show Similar Responses Upon Feeding by Two Specialist Root Herbivores.

Abstract (Expand)

Glucosinolates (GSLs) evolved in Brassicaceae as chemical defenses against herbivores. The GSL content in plants is affected by both abiotic and biotic factors, but also depends on the genetic background … of the plant. Since the bitter taste of GSLs can be unfavorable for both livestock and human consumption, several plant varieties with low GSL seed or leaf content have been bred. Due to their lower GSL levels, such varieties can be more susceptible to herbivore pests. However, low GSL varieties may quickly increase GSL levels upon herbivore feeding by activating GSL biosynthesis, hydrolysis, or transporter genes. To analyze differences in herbivore-induced GSL responses in relation to constitutive GSL levels, we selected four Brassica rapa varieties, containing either low or high root GSL levels. Plants were infested either with Delia radicum or Delia floralis larvae. The larvae of both root flies are specialists on Brassica plants. Root samples were collected after 3, 5, and 7 days. We compared the effect of root herbivore damage on the expression of GSL biosynthesis (CYP79A1, CYP83B2), transporter (GTR1A2, GTR2A2), and GSL hydrolysis genes (PEN2, TGG2) in roots of low and high GSL varieties in conjugation with their GSL levels. We found that roots of high GSL varieties contained higher levels of aliphatic, indole, and benzyl GSLs than low GSL varieties. Infestation with D. radicum larvae led to upregulation of indole GSL synthesis genes in low and high GSL varieties. High GSL varieties showed no or later responses than low varieties to D. floralis herbivory. Low GSL varieties additionally upregulated the GSL transporter gene expression. Low GSL varieties did not show a stronger herbivore-induced response than high GSL varieties, which indicates that there is no trade-off between constitutive and induced GSLs.

Authors: R. Sontowski, N. J. Gorringe, S. Pencs, A. Schedl, A. J. Touw, N. M. van Dam

Date Published: 5th Dec 2019

Publication Type: Journal

Cyclopropanol Warhead in Malleicyprol Confers Virulence of Human- and Animal-Pathogenic Burkholderia Species.

Abstract (Expand)

Burkholderia species such as B. mallei and B. pseudomallei are bacterial pathogens causing fatal infections in humans and animals (glanders and melioidosis), yet knowledge on their virulence factors … is limited. While pathogenic effects have been linked to a highly conserved gene locus (bur/mal) in the B. mallei group, the metabolite associated to the encoded polyketide synthase, burkholderic acid (syn. malleilactone), could not explain the observed phenotypes. By metabolic profiling and molecular network analyses of the model organism B. thailandensis, the primary products of the cryptic pathway were identified as unusual cyclopropanol-substituted polyketides. First, sulfomalleicyprols were identified as inactive precursors of burkholderic acid. Furthermore, a highly reactive upstream metabolite, malleicyprol, was discovered and obtained in two stabilized forms. Cell-based assays and a nematode infection model showed that the rare natural product confers cytotoxicity and virulence.

Authors: F. Trottmann, J. Franke, I. Richter, K. Ishida, M. Cyrulies, H. M. Dahse, L. Regestein, C. Hertweck

Date Published: 1st Oct 2019

Publication Type: Journal

Genomics-Driven Discovery of NO-Donating Diazeniumdiolate Siderophores in Diverse Plant-Associated Bacteria.

Abstract (Expand)

Siderophores are key players in bacteria-host interactions, with the main function to provide soluble iron for their producers. Gramibactin from rhizosphere bacteria expands siderophore function and … diversity as it delivers iron to the host plant and features an unusual diazeniumdiolate moiety for iron chelation. By mutational analysis of the grb gene cluster, we identified genes (grbD and grbE) necessary for diazeniumdiolate formation. Genome mining using a GrbD-based network revealed a broad range of orthologous gene clusters in mainly plant-associated Burkholderia/Paraburkholderia species. Two new types of diazeniumdiolate siderophores, megapolibactins and plantaribactin were fully characterized. In vitro assays and in vivo monitoring experiments revealed that the iron chelators also liberate nitric oxide (NO) in plant roots. This finding is important since NO donors are considered as biofertilizers that maintain iron homeostasis and increase overall plant fitness.

Authors: R. Hermenau, J. L. Mehl, K. Ishida, B. Dose, S. J. Pidot, T. P. Stinear, C. Hertweck

Date Published: 9th Sep 2019

Publication Type: Journal

Genome Mining Reveals Endopyrroles from a Nonribosomal Peptide Assembly Line Triggered in Fungal-Bacterial Symbiosis

Abstract (Expand)

The bacterial endosymbiont (Burkholderia rhizoxinica) of the rice seedling blight fungus (Rhizopus microsporus) harbors a large number of cryptic biosynthesis gene clusters. Genome mining and sequence … similarity networks based on an encoded nonribosomal peptide assembly line and the associated pyrrole- forming enzymes in the symbiont indicated that the encoded metabolites are unique among a large number of tentative pyrrole natural products in diverse and unrelated bacterial phyla. By performing comparative metabolic pro fi ling using a mutant generated with an improved pheS Burkholderia counterselection marker, we found that the symbionts ’ biosynthetic pathway is mainly activated under salt stress and exclusively in symbiosis with the fungal host. The cryptic metabolites were fully characterized as novel pyrrole-substituted depsipeptides (endopyrroles). A broader survey showed that endopyrrole production is a hallmark of geographically distant endofungal bacteria, which produce the peptides solely under symbiotic conditions.

Authors: Sarah Niehs, Benjamin Dose, Kirstin Scherlach, Sacha J. Pidot, Timothy P. Stinear, Christian Hertweck

Date Published: 8th Jul 2019

Publication Type: Not specified

Disease-free monoculture farming by fungus-growing termites.

Abstract (Expand)

Fungus-growing termites engage in an obligate mutualistic relationship with Termitomyces fungi, which they maintain in monocultures on specialised fungus comb structures, without apparent problems with … infectious diseases. While other fungi have been reported in the symbiosis, detailed comb fungal community analyses have been lacking. Here we use culture-dependent and -independent methods to characterise fungus comb mycobiotas from three fungus-growing termite species (two genera). Internal Transcribed Spacer (ITS) gene analyses using 454 pyrosequencing and Illumina MiSeq showed that non-Termitomyces fungi were essentially absent in fungus combs, and that Termitomyces fungal crops are maintained in monocultures as heterokaryons with two or three abundant ITS variants in a single fungal strain. To explore whether the essential absence of other fungi within fungus combs is potentially due to the production of antifungal metabolites by Termitomyces or comb bacteria, we performed in vitro assays and found that both Termitomyces and chemical extracts of fungus comb material can inhibit potential fungal antagonists. Chemical analyses of fungus comb material point to a highly complex metabolome, including compounds with the potential to play roles in mediating these contaminant-free farming conditions in the termite symbiosis.

Authors: S. Otani, V. L. Challinor, N. B. Kreuzenbeck, S. Kildgaard, S. Krath Christensen, L. L. M. Larsen, D. K. Aanen, S. A. Rasmussen, C. Beemelmanns, M. Poulsen

Date Published: 19th Jun 2019

Publication Type: Journal

Melleolides impact fungal translation via elongation factor 2.

Abstract (Expand)

Melleolides from the honey mushroom Armillaria mellea represent a structurally diverse group of polyketide-sesquiterpene hybrids. Among various bioactivites, melleolides show antifungal effects against … Aspergillus and other fungi. This bioactivity depends on a Delta2,4-double bond present in dihydroarmillylorsellinate (DAO) or arnamial, for example. Yet, the mode of action of Delta2,4-unsaturated, antifungal melleolides has been unknown. Here, we report on the molecular target of DAO in the fungus Aspergillus nidulans. Using a combination of synthetic chemistry to create a DAO-labelled probe, protein pulldown assays, MALDI-TOF-based peptide analysis and western blotting, we identify the eukaryotic translation elongation factor 2 (eEF2) as a binding partner of DAO. We confirm the inhibition of protein biosynthesis in vivo with an engineered A. nidulans strain producing the red fluorescent protein mCherry. Our work suggests a binding site dissimilar from that of the protein biosynthesis inhibitor sordarin, and highlights translational elongation as a valid antifungal drug target.

Authors: M. Dorfer, D. Heine, S. Konig, S. Gore, O. Werz, C. Hertweck, M. Gressler, D. Hoffmeister

Date Published: 15th May 2019

Publication Type: Journal

Metallophore profiling of nitrogen-fixing Frankia spp. to understand metal management in the rhizosphere of actinorhizal plants.

Abstract (Expand)

Frankia spp. are widespread nitrogen-fixing soil bacteria, which often live in symbiosis with a broad range of hosts. Metal homeostasis plays a crucial role in the success of the symbiosis regarding … the acquisition of essential trace metals and detoxification of potentially toxic elements. We have hypothesised that Frankia releases many organic ligands with a broad spectrum of affinity for essential and toxic metals. We coined the term 'ligandosphere' to describe the entirety of excreted metal complexing agents and ligands derived from the dissolved organic matter. Using metal isotope-coded profiling (MICP); metallophores of physiological important and toxic trace metals were identified by the addition of stable metal isotope pairs such as 54Fe/58Fe, 63Cu/65Cu, 66Zn/68Zn or 95Mo/98Mo. Liquid chromatography coupled to a mass spectrometer revealed strong variations of the metallophore profile in between the 14 test-strains. In total, about 83 organic ligands were identified as binding to one of the tested metals. The predicted sum formula of the major Fe binding ligands and MS/MS experiments suggested that several metallophore candidates have a similar molecular backbone. Growth experiments with a hyper-producer of metallophores revealed a positive relationship between metallophore production and the concentration of Cu in the growth medium. The present study provides the first comprehensive overview of the complexity of Frankia's ligandosphere. It opens a path to a deeper understanding of mechanisms that regulate metal homeostasis in frankiae. Deciphering these mechanisms is important since the fitness of actinorhizal plants and their potential in ecological restoration relies heavily on their symbiosis with frankiae.

Authors: M. Deicke, J. F. Mohr, S. Roy, P. Herzsprung, J. P. Bellenger, T. Wichard

Date Published: 17th Apr 2019

Publication Type: Journal

Mechanistic characterization of three sesquiterpene synthases from the termite-associated fungus Termitomyces.

Abstract (Expand)

Three terpene synthases from the termite associated fungus Termitomyces were functionally characterized as (+)-intermedeol synthase, (-)-gamma-cadinene synthase and (+)-germacrene D-4-ol synthase, with … the germacrene D-4-ol synthase as the first reported enzyme that produces the (+)-enantiomer. The enzymatic mechanisms were thoroughly investigated by incubation with isotopically labeled precursors to follow the stereochemical courses of single reaction steps in catalysis. The role of putative active site residues was tested by site directed mutagenesis of a highly conserved tryptophan in all three enzymes and additional residues in (-)-gamma-cadinene synthase that were identified by homology model analysis.

Authors: I. Burkhardt, N. B. Kreuzenbeck, C. Beemelmanns, J. S. Dickschat

Date Published: 27th Mar 2019

Publication Type: Journal

Production Options for Psilocybin: Making of the Magic.

Abstract (Expand)

The fungal genus Psilocybe and other genera comprise numerous mushroom species that biosynthesize psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine). It represents the prodrug to its dephosphorylated … psychotropic analogue, psilocin. The colloquial term "magic mushrooms" for these fungi alludes to their hallucinogenic effects and to their use as recreational drugs. However, clinical trials have recognized psilocybin as a valuable candidate to be developed into a medication against depression and anxiety. We here highlight its recently elucidated biosynthesis, the concurrently developed concept of enzymatic in vitro and heterologous in vivo production, along with previous synthetic routes. The prospect of psilocybin as a promising therapeutic may entail an increased demand, which can be met by biotechnological production. Therefore, we also briefly touch on psilocybin's therapeutic relevance and pharmacology.

Authors: J. Fricke, C. Lenz, J. Wick, F. Blei, D. Hoffmeister

Date Published: 18th Jan 2019

Publication Type: Journal

Melleolides from Honey Mushroom Inhibit 5-Lipoxygenase via Cys159.

Abstract (Expand)

5-Lipoxygenase (5-LO) initiates the biosynthesis of pro-inflammatory leukotrienes from arachidonic acid, which requires the nuclear membrane-bound 5-LO-activating protein (FLAP) for substrate transfer. … Here, we identified human 5-LO as a molecular target of melleolides from honey mushroom (Armillaria mellea). Melleolides inhibit 5-LO via an alpha,beta-unsaturated aldehyde serving as Michael acceptor for surface cysteines at the substrate entrance that are revealed as molecular determinants for 5-LO activity. Experiments with 5-LO mutants, where select cysteines had been replaced by serine, indicated that the investigated melleolides suppress 5-LO product formation via two distinct modes of action: (1) by direct interference with 5-LO activity involving two or more of the cysteines 159, 300, 416, and 418, and (2) by preventing 5-LO/FLAP assemblies involving selectively Cys159 in 5-LO. Interestingly, replacement of Cys159 by serine prevented 5-LO/FLAP assemblies as well, implying Cys159 as determinant for 5-LO/FLAP complex formation at the nuclear membrane required for leukotriene biosynthesis.

Authors: S. Konig, E. Romp, V. Krauth, M. Ruhl, M. Dorfer, S. Liening, B. Hofmann, A. K. Hafner, D. Steinhilber, M. Karas, U. Garscha, D. Hoffmeister, O. Werz

Date Published: 17th Jan 2019

Publication Type: Journal

Algae induce siderophore biosynthesis in the freshwater bacterium Cupriavidus necator H16.

Abstract (Expand)

Cupriachelin is a photoreactive lipopeptide siderophore produced by the freshwater bacterium Cupriavidus necator H16. In the presence of sunlight, the iron-loaded siderophore undergoes photolytic … cleavage, thereby releasing solubilized iron into the environment. This iron is not only available to the siderophore producer, but also to the surrounding microbial community. In this study, the cupriachelin-based interaction between C. necator H16 and the freshwater diatom Navicula pelliculosa was investigated. A reporter strain of the bacterium was constructed to study differential expression levels of the cupriachelin biosynthesis gene cucJ in response to varying environmental conditions. Not only iron starvation, but also culture supernatants of N. pelliculosa were found to induce cupriachelin biosynthesis. The transcription factors involved in this differential gene expression were identified using DNA-protein pulldown assays. Besides the well-characterized ferric uptake regulator, a two-component system was found to tune the expression of cupriachelin biosynthesis genes in the presence of diatom supernatants.

Authors: C. Kurth, I. Wasmuth, T. Wichard, G. Pohnert, M. Nett

Date Published: 27th Nov 2018

Publication Type: Journal

Conspecificity of the model organism Ulva mutabilis and Ulva compressa (Ulvophyceae, Chlorophyta).

Abstract (Expand)

As one of the most abundant and ubiquitous representatives of marine and brackish coastal macrophytobenthos communities, the genus Ulva is not only an important primary producer but also of ecological … and morphogenetic interest to many scientists. Ulva mutabilis became an important model organism to study morphogenesis and mutualistic interactions of macroalgae and microorganisms. Here, we report that our collections of Ulva compressa Linnaeus (1753) from Germany are conspecific with the type strains of the model organism U. mutabilis Foyn (1958), which were originally collected at Olhao on the south coast of Portugal and have from that time on been maintained in culture as gametophytic and parthenogenetic lab strains. Different approaches were used to test conspecificity: (i) comparisons of vegetative and reproductive features of cultured material of U. mutabilis and German U. compressa demonstrated a shared morphological pattern; (ii) gametes of U. compressa and U. mutabilis successfully mated and developed into fertile sporophytic first-generation offspring; (iii) molecular phylogenetics and species delimitation analyses based on the Generalized Mixed Yule-Coalescent method showed that U. mutabilis isolates (sl-G[mt+]) and (wt-G[mt-]) and U. compressa belong to a unique Molecular Operational Taxonomic Unit. According to these findings, there is sufficient evidence that U. mutabilis and U. compressa should be regarded as conspecific.

Authors: S. Steinhagen, A. Barco, T. Wichard, F. Weinberger

Date Published: 28th Oct 2018

Publication Type: Journal

Iterative l-Tryptophan Methylation in Psilocybe Evolved by Subdomain Duplication.

Abstract (Expand)

Psilocybe mushrooms are best known for their l-tryptophan-derived psychotropic alkaloid psilocybin. Dimethylation of norbaeocystin, the precursor of psilocybin, by the enzyme PsiM is a critical step … during the biosynthesis of psilocybin. However, the "magic" mushroom Psilocybe serbica also mono- and dimethylates l-tryptophan, which is incompatible with the specificity of PsiM. Here, a second methyltransferase, TrpM, was identified and functionally characterized. Mono- and dimethylation activity on l-tryptophan was reconstituted in vitro, whereas tryptamine was rejected as a substrate. Therefore, we describe a second l-tryptophan-dependent pathway in Psilocybe that is not part of the biosynthesis of psilocybin. TrpM is unrelated to PsiM but originates from a retained ancient duplication event of a portion of the egtDB gene that encodes an ergothioneine biosynthesis enzyme. During mushroom evolution, this duplicated gene was widely lost but re-evolved sporadically and independently in various genera. We propose a new secondary metabolism evolvability mechanism, in which weakly selected genes are retained through preservation in a widely distributed, conserved pathway.

Authors: F. Blei, J. Fricke, J. Wick, J. C. Slot, D. Hoffmeister

Date Published: 18th Oct 2018

Publication Type: Journal

Insights into the Evolution of Multicellularity from the Sea Lettuce Genome.

Abstract (Expand)

We report here the 98.5 Mbp haploid genome (12,924 protein coding genes) of Ulva mutabilis, a ubiquitous and iconic representative of the Ulvophyceae or green seaweeds. Ulva's rapid and abundant … growth makes it a key contributor to coastal biogeochemical cycles; its role in marine sulfur cycles is particularly important because it produces high levels of dimethylsulfoniopropionate (DMSP), the main precursor of volatile dimethyl sulfide (DMS). Rapid growth makes Ulva attractive biomass feedstock but also increasingly a driver of nuisance "green tides." Ulvophytes are key to understanding the evolution of multicellularity in the green lineage, and Ulva morphogenesis is dependent on bacterial signals, making it an important species with which to study cross-kingdom communication. Our sequenced genome informs these aspects of ulvophyte cell biology, physiology, and ecology. Gene family expansions associated with multicellularity are distinct from those of freshwater algae. Candidate genes, including some that arose following horizontal gene transfer from chromalveolates, are present for the transport and metabolism of DMSP. The Ulva genome offers, therefore, new opportunities to understand coastal and marine ecosystems and the fundamental evolution of the green lineage.

Authors: O. De Clerck, S. M. Kao, K. A. Bogaert, J. Blomme, F. Foflonker, M. Kwantes, E. Vancaester, L. Vanderstraeten, E. Aydogdu, J. Boesger, G. Califano, B. Charrier, R. Clewes, A. Del Cortona, S. D'Hondt, N. Fernandez-Pozo, C. M. Gachon, M. Hanikenne, L. Lattermann, F. Leliaert, X. Liu, C. A. Maggs, Z. A. Popper, J. A. Raven, M. Van Bel, P. K. I. Wilhelmsson, D. Bhattacharya, J. C. Coates, S. A. Rensing, D. Van Der Straeten, A. Vardi, L. Sterck, K. Vandepoele, Y. Van de Peer, T. Wichard, J. H. Bothwell

Date Published: 24th Sep 2018

Publication Type: Journal

Genomics-driven discovery of a linear lipopeptide promoting host colonization by endofungal bacteria

Abstract (Expand)

The rice seedling blight fungus Rhizopus microsporus weakens or kills plants by means of a potent toxin produced by endobacteria (Burkholderia rhizoxinica) that live within the fungal hyphae. The success … of the highly attuned microbial interaction is partly based on the bacteria ’ s ability to roam and re-colonize the fungal host. Yet, apart from the toxin, chemical mediators of the symbiosis have remained elusive. By genome mining and comparison we identi fi ed a cryptic NRPS gene cluster that is conserved among all sequenced Rhizopus endosymbionts. Metabolic pro fi ling and targeted gene inactivation led to the discov- ery of a novel linear lipopeptide, holrhizin A, which was fully characterized. Through in vitro and in vivo assays we found that holrhizin acts (A) as a biosurfactant to reduce surface tension, (B) in fl uences the for- mation of mature bio fi lms and thus cell motility behavior that ultimately supports the bacterial cells to (C) colonize and invade the fungal host, consequently supporting the re-establishment of the exceptional Burkholderia-Rhizopus symbiosis. We not only unveil structure and function of an linear lipopeptide from endofungal bacteria but also provide a functional link between the symbiont’ s orphan NRPS genes and a chemical mediator that promotes bacterial invasion into the fungal host.

Authors: Sarah Niehs, Kirstin Scherlach, Christian Hertweck

Date Published: 31st Aug 2018

Publication Type: Not specified

Unexpected Bacterial Origin of the Antibiotic Icosalide Two-Tailed Depsipeptide Assembly in Multifarious Burkholderia Symbionts

Abstract (Expand)

Icosalide is an unusual two-tailed lipocyclopeptide antibiotic that was originally isolated from a fungal culture. Yet, its biosynthesis and ecological function have remained enigmatic. By genome mining …g and metabolic pro fi ling of a bacterial endosymbiont (Burkholderia gladioli) of the pest beetle Lagria villosa, we unveiled a bacterial origin of icosalide. Functional analysis of the biosynthetic gene locus revealed an unprecedented nonribosomal peptide synthetase (NRPS) that incorporates two β -hydroxy acids by means of two starter condensation domains in di ff erent modules. This unusual assembly line, which may inspire new synthetic biology approaches, is widespread among many symbiotic Burkholderia species from diverse habitats. Biological assays showed that icosalide is active against entomopathogenic bacteria, thus adding to the chemical armory protecting beetle offspring. By creating a null mutant, we found that icosalide is a swarming inhibitor, which may play a role in symbiotic interactions and bears the potential for therapeutic applications.

Authors: Benjamin Dose, Sarah Niehs, Kirstin Scherlach, Laura V. Florez, Martin Kaltenpoth, Christian Hertweck

Date Published: 30th Aug 2018

Publication Type: Not specified

Genomics-Driven Discovery of a Symbiont-Specific Cyclopeptide from Bacteria Residing in the Rice Seedling Blight Fungus

Abstract (Expand)

The rice seedling blight fungus Rhizopus microsporus harbors endosymbiotic bacteria (Burkholderia rhizoxinica) that produce the virulence factor rhizoxin and control host development. Genome mining … indicated a massive inventory of cryptic non- ribosomal peptide synthetase (NRPS) genes, which have not yet been linked to any natural products. The discovery and full characterization of a novel cyclopeptide from endofungal bac- teria is reported. In silico analysis of an orphan, symbiont-spe- cific NRPS predicted the structure of a nonribosomal peptide, which was targeted by LC-MS/MS profiling of wild-type and engineered null mutants. NMR spectroscopy and chemical deri- vatization elucidated the structure of the bacterial cyclopep- tide. Phylogenetic analyses revealed the relationship of starter C domains for rare N-acetyl-capped peptides. Heptarhizin is produced under symbiotic conditions in geographically con- strained strains from the Pacific clade; this indicates a potential ecological role of the peptide.

Authors: Sarah Niehs, Benjamin Dose, Kirstin Scherlach, Martin Roth, Christian Hertweck

Date Published: 16th Aug 2018

Publication Type: Not specified

Role of Chemical Mediators in Aquatic Interactions across the Prokaryote-Eukaryote Boundary.

Abstract (Expand)

There is worldwide growing interest in the occurrence and diversity of metabolites used as chemical mediators in cross-kingdom interactions within aquatic systems. Bacteria produce metabolites to protect … and influence the growth and life cycle of their eukaryotic hosts. In turn, the host provides a nutrient-enriched environment for the bacteria. Here, we discuss the role of waterborne chemical mediators that are responsible for such interactions in aquatic multi-partner systems, including algae or invertebrates and their associated bacteria. In particular, this review highlights recent advances in the chemical ecology of aquatic systems that support the overall ecological significance of signaling molecules across the prokaryote-eukaryote boundary (cross-kingdom interactions) for growth, development and morphogenesis of the host. We emphasize the value of establishing well-characterized model systems that provide the basis for the development of ecological principles that represent the natural lifestyle and dynamics of aquatic microbial communities and enable a better understanding of the consequences of environmental change and the most effective means of managing community interactions.

Authors: T. Wichard, C. Beemelmanns

Date Published: 15th Aug 2018

Publication Type: Journal

Gramibactin is a bacterial siderophore with a diazeniumdiolate ligand system.

Abstract (Expand)

Genome mining and chemical analyses revealed that rhizosphere bacteria (Paraburkholderia graminis) produce a new type of siderophore, gramibactin, a lipodepsipeptide that efficiently binds iron with a … logbeta value of 27.6. Complexation-induced proton NMR chemical shifts show that the unusual N-nitrosohydroxylamine (diazeniumdiolate) moieties participate in metal binding. Gramibactin biosynthesis genes are conserved in numerous plant-associated bacteria associated with rice, wheat, and maize, which may utilize iron from the complex.

Authors: R. Hermenau, K. Ishida, S. Gama, B. Hoffmann, M. Pfeifer-Leeg, W. Plass, J. F. Mohr, T. Wichard, H. P. Saluz, C. Hertweck

Date Published: 1st Aug 2018

Publication Type: Journal

An antifungal polyketide associated with horizontally acquired genes supports symbiont-mediated defense in Lagria villosa beetles.

Abstract (Expand)

Microbial symbionts are often a source of chemical novelty and can contribute to host defense against antagonists. However, the ecological relevance of chemical mediators remains unclear for most … systems. Lagria beetles live in symbiosis with multiple strains of Burkholderia bacteria that protect their offspring against pathogens. Here, we describe the antifungal polyketide lagriamide, and provide evidence supporting that it is produced by an uncultured symbiont, Burkholderia gladioli Lv-StB, which is dominant in field-collected Lagria villosa. Interestingly, lagriamide is structurally similar to bistramides, defensive compounds found in marine tunicates. We identify a gene cluster that is probably involved in lagriamide biosynthesis, provide evidence for horizontal acquisition of these genes, and show that the naturally occurring symbiont strains on the egg are protective in the soil environment. Our findings highlight the potential of microbial symbionts and horizontal gene transfer as influential sources of ecological innovation.

Authors: L. V. Florez, K. Scherlach, I. J. Miller, A. Rodrigues, J. C. Kwan, C. Hertweck, M. Kaltenpoth

Date Published: 26th Jun 2018

Publication Type: Journal

Chemical warfare between leafcutter ant symbionts and a co-evolved pathogen.

Abstract (Expand)

Acromyrmex leafcutter ants form a mutually beneficial symbiosis with the fungus Leucoagaricus gongylophorus and with Pseudonocardia bacteria. Both are vertically transmitted and actively maintained by … the ants. The fungus garden is manured with freshly cut leaves and provides the sole food for the ant larvae, while Pseudonocardia cultures are reared on the ant-cuticle and make antifungal metabolites to help protect the cultivar against disease. If left unchecked, specialized parasitic Escovopsis fungi can overrun the fungus garden and lead to colony collapse. We report that Escovopsis upregulates the production of two specialized metabolites when it infects the cultivar. These compounds inhibit Pseudonocardia and one, shearinine D, also reduces worker behavioral defenses and is ultimately lethal when it accumulates in ant tissues. Our results are consistent with an active evolutionary arms race between Pseudonocardia and Escovopsis, which modifies both bacterial and behavioral defenses such that colony collapse is unavoidable once Escovopsis infections escalate.

Authors: D. Heine, N. A. Holmes, S. F. Worsley, A. C. A. Santos, T. M. Innocent, K. Scherlach, E. H. Patrick, D. W. Yu, J. C. Murrell, P. C. Vieria, J. J. Boomsma, C. Hertweck, M. I. Hutchings, B. Wilkinson

Date Published: 7th Jun 2018

Publication Type: Journal

Two Types of Threonine-Tagged Lipopeptides Synergize in Host Colonization by Pathogenic Burkholderia Species.

Abstract (Expand)

Bacterial infections of agriculturally important mushrooms and plants pose a major threat to human food sources worldwide. However, structures of chemical mediators required by the pathogen for host … colonization and infection remain elusive in most cases. Here, we report two types of threonine-tagged lipopeptides conserved among mushroom and rice pathogenic Burkholderia species that facilitate bacterial infection of hosts. Genome mining, metabolic profiling of infected mushrooms, and heterologous expression of orphan gene clusters allowed the discovery of these unprecedented metabolites in the mushroom pathogen Burkholderia gladioli (haereogladin, burriogladin) and the plant pathogen Burkholderia glumae (haereoglumin and burrioglumin). Through targeted gene deletions, the molecular basis of lipopeptide biosynthesis by nonribosomal peptide synthetases was revealed. Surprisingly, both types of lipopeptides feature unusual threonine tags, which yield longer peptide backbones than one would expect based on the canonical colinearity of the NRPS assembly lines. Both peptides play an indirect role in host infection as biosurfactants that enable host colonization by mediating swarming and biofilm formation abilities. Moreover, MALDI imaging mass spectrometry was applied to investigate the biological role of the lipopeptides. Our results shed light on conserved mechanisms that mushroom and plant pathogenic bacteria utilize for host infection and expand current knowledge on bacterial virulence factors that may represent a new starting point for the targeted development of crop protection measures in the future.

Authors: T. Thongkongkaew, W. Ding, E. Bratovanov, E. Oueis, M. A. Garci A-Altares, N. Zaburannyi, K. Harmrolfs, Y. Zhang, K. Scherlach, R. Muller, C. Hertweck

Date Published: 18th May 2018

Publication Type: Journal

Biocatalytic Production of Psilocybin and Derivatives in Tryptophan Synthase-Enhanced Reactions.

Abstract (Expand)

Psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine) is the main alkaloid of the fungal genus Psilocybe, the so-called "magic mushrooms." The pharmaceutical interest in this psychotropic natural … product as a future medication to treat depression and anxiety is strongly re-emerging. Here, we present an enhanced enzymatic route of psilocybin production by adding TrpB, the tryptophan synthase of the mushroom Psilocybe cubensis, to the reaction. We capitalized on its substrate flexibility and show psilocybin formation from 4-hydroxyindole and l-serine, which are less cost-intensive substrates, compared to the previous method. Furthermore, we show enzymatic production of 7-phosphoryloxytryptamine (isonorbaeocystin), a non-natural congener of the Psilocybe alkaloid norbaeocystin (4-phosphoryloxytryptamine), and of serotonin (5-hydroxytryptamine) by means of the same in vitro approach.

Authors: F. Blei, F. Baldeweg, J. Fricke, D. Hoffmeister

Date Published: 11th May 2018

Publication Type: Journal

Thermodynamic study on 8-hydroxyquinoline-2-carboxylic acid as a chelating agent for iron found in the gut of Noctuid larvae

Abstract (Expand)

The recognition of quinolinic carboxylic acids as natural chelants and the recent observation of a high production of 8-hydroxyquinoline-2-carboxylic acid (8-HQA) in the gut of several Noctuid larvae … (e.g. Spodoptera littoralis) has inspired the study of the chelation properties of 8-HQA towards Fe2+ and Fe3+. Here, we report a detailed characterization of the thermodynamic solution behaviour of Fe2+/8-HQA and Fe3+/8-HQA systems as a function of the pH value. The acid–base properties of 8-HQA and its binding ability towards Fe2+ and Fe3+ have been investigated over a wide range of pH values (2.0 ≤ pH ≤ 11.0) by ISE-H+ (glass electrode) potentiometric titrations in KCl(aq) at I = 0.2 mol dm−3 and at T = 298.15 K. For both oxidation states, various FeLqHr species are formed, with q = 1, 2 (and 3), and −2 ≤ r ≤ 1. The presence of the main FeLqHr species was confirmed by HESI-HRMS. ESR measurements have also been performed to get some extra information on the Fe3+ coordination, indicating a distorted octahedral symmetry around the metal center. Quantum mechanical calculations have been carried out in order to characterize the structural features of selected metal complexes. The complexing ability of 8-HQA is generally much higher for Fe3+ than Fe2+. Nevertheless, the sequestering ability of 8-HQA towards these two oxidation states of this metal ion, obtained by the calculation of several pL0.5 values, resulted in it being highly dependent on the pH value: (i) at relatively low pH values, it is higher for Fe3+ (pL0.5 = 6.3 at pH = 3.0) than for Fe2+ (pL0.5 = 3.1 at pH = 3.0); (ii) it is almost the same at pH = 8.1 (Fe3+: pL0.5 = 8.3; Fe2+: pL0.5 = 8.1); (iii) it is higher for Fe2+ at high pH values (pL0.5 = 8.9 for Fe2+ and pL0.5 = 6.2 for Fe3+ at pH = 10.0). The determination of the stability constants of the Fe2+/8-HQA and Fe3+/8-HQA complexes was also complemented by data obtained by the ligand-competition approach, using EDTA as a competing ligand over a wide range of cation and ligand concentrations and ratios. This also allowed a more thorough investigation of both the Fe2+/EDTA and Fe3+/EDTA systems, providing an accurate stability constant dataset for the Fep(EDTA)qHr species under the above-mentioned experimental conditions, which are commonly used in biological studies.

Authors: Sofia Gama, Mariachiara Frontauria, Nico Ueberschaar, Giuseppe Brancato, Demetrio Milea, Silvio Sammartano, Winfried Plass

Date Published: 9th Apr 2018

Publication Type: Not specified

Verticillium dahliae-Arabidopsis Interaction Causes Changes in Gene Expression Profiles and Jasmonate Levels on Different Time Scales.

Abstract (Expand)

Verticillium dahliae is a soil-borne vascular pathogen that causes severe wilt symptoms in a wide range of plants. Co-culture of the fungus with Arabidopsis roots for 24 h induces many changes in the … gene expression profiles of both partners, even before defense-related phytohormone levels are induced in the plant. Both partners reprogram sugar and amino acid metabolism, activate genes for signal perception and transduction, and induce defense- and stress-responsive genes. Furthermore, analysis of Arabidopsis expression profiles suggests a redirection from growth to defense. After 3 weeks, severe disease symptoms can be detected for wild-type plants while mutants impaired in jasmonate synthesis and perception perform much better. Thus, plant jasmonates have an important influence on the interaction, which is already visible at the mRNA level before hormone changes occur. The plant and fungal genes that rapidly respond to the presence of the partner might be crucial for early recognition steps and the future development of the interaction. Thus they are potential targets for the control of V. dahliae-induced wilt diseases.

Authors: S. S. Scholz, W. Schmidt-Heck, R. Guthke, A. C. U. Furch, M. Reichelt, J. Gershenzon, R. Oelmuller

Date Published: 3rd Mar 2018

Publication Type: Journal

DeltaMS: a tool to track isotopologues in GC- and LC-MS data

Abstract (Expand)

Introduction: Stable isotopic labeling experiments are powerful tools to study metabolic pathways, to follow tracers and fluxes in biotic and abiotic transformations and to elucidate molecules involved …d in metal complexing. Objective: To introduce a software tool for the identification of isotopologues from mass spectrometry data. Methods: DeltaMS relies on XCMS peak detection and X13CMS isotopologue grouping and then analyses data for specific isotope ratios and the relative error of these ratios. It provides pipelines for recognition of isotope patterns in three experiment types commonly used in isotopic labeling studies: (1) Search for isotope signatures with a specific mass shift and intensity ratio in one sample set. (2) Analyze two sample sets for a specific mass shift and, optionally, the isotope ratio, whereby one sample set is isotope-labeled, and one is not. (3) Analyze isotope-guided perturbation experiments with a setup described in X13CMS. Results: To illustrate the versatility of DeltaMS, we analyze data sets from case-studies that commonly pose challenges in evaluation of natural isotopes or isotopic signatures in labeling experiment. In these examples, the untargeted detection of sulfur, bromine and artificial metal isotopic patterns is enabled by the automated search for specific isotopes or isotope signatures. Conclusion: DeltaMS provides a platform for the identification of (pre-defined) isotopologues in MS data from single samples or comparative metabolomics data sets.

Authors: Tim U. H. Baumeister, Nico Ueberschaar, Wolfgang Schmidt-Heck, J. Frieder Mohr, Michael Deicke, Thomas Wichard, Reinhard Guthke, Georg Pohnert

Date Published: 27th Feb 2018

Publication Type: Not specified

Analysis of basidiomycete pigments in situ by Raman spectroscopy.

Abstract (Expand)

Basidiomycetes, that is, mushroom-type fungi, are known to produce pigments in response to environmental impacts. As antioxidants with a high level of unsaturation, these compounds can neutralize highly … oxidative species. In the event of close contact with other microbes, the enzymatically controlled pigment production is triggered and pigment secretion is generated at the interaction zone. The identification and analysis of these pigments is important to understand the defense mechanism of fungi, which is essential to counteract an uncontrolled spread of harmful species. Usually, a detailed analysis of the pigments is time consuming as it depends on laborious sample preparation and isolation procedures. Furthermore, the applied protocols often influence the chemical integrity of the compound of interest. A possibility to noninvasively investigate the pigmentation is Raman microspectroscopy. The methodology has the potential to analyze the chemical composition of the sample spatially resolved at the interaction zone. After the acquisition of a representative spectroscopic library, the pigment production by basidiomycetes was monitored for during response to different fungi and bacteria. The presented results describe a very efficient noninvasive way of pigment analysis which can be applied with minimal sample preparation.

Authors: J. P. Tauber, C. Matthaus, C. Lenz, D. Hoffmeister, J. Popp

Date Published: 8th Feb 2018

Publication Type: Journal

Macroalgal-bacterial interactions: Role of dimethylsulfoniopropionate in microbial gardening by Ulva (Chlorophyta).

Abstract (Expand)

The marine macroalga Ulva mutabilis (Chlorophyta) develops into callus-like colonies consisting of undifferentiated cells and abnormal cell walls under axenic conditions. Ulva mutabilis is routinely … cultured with two bacteria, the Roseovarius sp. MS2 strain and the Maribacter sp. MS6 strain, which release morphogenetic compounds and ensure proper algal morphogenesis. Using this tripartite community as an emerging model system, we tested the hypothesis that the bacterial-algal interactions evolved as a result of mutually taking advantage of signals in the environment. Our study aimed to determine whether cross-kingdom crosstalk is mediated by the attraction of bacteria through algal chemotactic signals. Roseovarius sp. MS2 senses the known osmolyte dimethylsulfoniopropionate (DMSP) released by Ulva into the growth medium. Roseovarius sp. is attracted by DMSP and takes it up rapidly such that DMSP can only be determined in axenic growth media. As DMSP did not promote bacterial growth under the tested conditions, Roseovarius benefited solely from glycerol as the carbon source provided by Ulva. Roseovarius quickly catabolized DMSP into methanethiol (MeSH) and dimethylsulphide (DMS). We conclude that many bacteria can use DMSP as a reliable signal indicating a food source and promote the subsequent development and morphogenesis in Ulva.

Authors: R. W. Kessler, A. Weiss, S. Kuegler, C. Hermes, T. Wichard

Date Published: 2018

Publication Type: Journal

Dissimilar pigment regulation in Serpula lacrymans and Paxillus involutus during inter-kingdom interactions.

Abstract (Expand)

Production of basidiomycete atromentin-derived pigments like variegatic acid (pulvinic acid-type) and involutin (diarylcyclopentenone) from the brown-rotter Serpula lacrymans and the ectomycorrhiza-forming … Paxillus involutus, respectively, is induced by complex nutrition, and in the case of S. lacrymans, bacteria. Pigmentation in S. lacrymans was stimulated by 13 different bacteria and cell-wall-damaging enzymes (lytic enzymes and proteases), but not by lysozyme or mechanical damage. The use of protease inhibitors with Bacillus subtilis or heat-killed bacteria during co-culturing with S. lacrymans significantly reduced pigmentation indicating that enzymatic hyphal damage and/or released peptides, rather than mechanical injury, was the major cause of systemic pigment induction. Conversely, no significant pigmentation by bacteria was observed from P. involutus. We found additional putative transcriptional composite elements of atromentin synthetase genes in P. involutus and other ectomycorrhiza-forming species that were absent from S. lacrymans and other brown-rotters. Variegatic and its precursor xerocomic acid, but not involutin, in return inhibited swarming and colony biofilm spreading of Bacillus subtilis, but did not kill B. subtilis. We suggest that dissimilar pigment regulation by fungal lifestyle was a consequence of pigment bioactivity and additional promoter motifs. The focus on basidiomycete natural product gene induction and regulation will assist in future studies to determine global regulators, signalling pathways and associated transcription factors of basidiomycetes.

Authors: J. P. Tauber, R. Gallegos-Monterrosa, A. T. Kovacs, E. Shelest, D. Hoffmeister

Date Published: 6th Dec 2017

Publication Type: Journal

Plant-mediated RNAi silences midgut-expressed genes in congeneric lepidopteran insects in nature.

Abstract (Expand)

BACKGROUND: Plant-mediated RNAi (PMRi) silencing of insect genes has enormous potential for crop protection, but whether it works robustly under field conditions, particularly with lepidopteran pests, … remains controversial. Wild tobacco Nicotiana attenuata and cultivated tobacco (N. tabacum) (Solanaceae) is attacked by two closely related specialist herbivores Manduca sexta and M. quinquemaculata (Lepidoptera, Sphingidae). When M. sexta larvae attack transgenic N. attenuata plants expressing double-stranded RNA(dsRNA) targeting M. sexta's midgut-expressed genes, the nicotine-ingestion induced cytochrome P450 monooxygenase (invert repeat (ir)CYP6B46-plants) and the lyciumoside-IV-ingestion induced beta-glucosidase1 (irBG1-plants), these larval genes which are important for the larvae's response to ingested host toxins, are strongly silenced. RESULTS: Here we show that the PMRi procedure also silences the homologous genes in native M. quinquemaculata larvae feeding on irCYP6B46 and irBG1-transgenic N. attenuata plants in nature. The PMRi lines shared 98 and 96% sequence similarity with M. quinquemaculata homologous coding sequences, and CYP6B46 and BG1 transcripts were reduced by ca. 90 and 80%, without reducing the transcripts of the larvae's most similar, potential off-target genes. CONCLUSIONS: We conclude that the PMRi procedure can robustly and specifically silence genes in native congeneric insects that share sufficient sequence similarity and with the careful selection of targets, might protect crops from attack by congeneric-groups of insect pests.

Authors: S. Poreddy, J. Li, I. T. Baldwin

Date Published: 15th Nov 2017

Publication Type: Not specified

Calcium carbonates: induced biomineralization with controlled macromorphology

Abstract (Expand)

Biomineralization of (magnesium) calcite and vaterite by bacterial isolates has been known for quite some time. However, the extracellular precipitation has hardly ever been linked to different … morphologies of the minerals that are observed. Here, isolates from limestone-associated groundwater, rock and soil were shown to form calcite, magnesium calcite or vaterite. More than 92 % of isolates were indeed able to form carbonates, while abiotic controls failed to form minerals. The crystal morphologies varied, including rhombohedra, prisms and pyramid-like macromorphologies. Different conditions like varying temperature, pH or media components, but also cocultivation to test for collaborative effects of sympatric bacteria, were used to differentiate between mechanisms of calcium carbonate formation. Single crystallites were cemented with bacterial cells; these may have served as nucleation sites by providing a basic pH at short distance from the cells. A calculation of potential calcite formation of up to 2 g L−1 of solution made it possible to link the microbial activity to geological processes.

Authors: Aileen Meier, Anne Kastner, Dennis Harries, Maria Wierzbicka-Wieczorek, Juraj Majzlan, Georg Büchel, Erika Kothe

Date Published: 6th Nov 2017

Publication Type: Not specified

In Vivo Isotopic Labeling of Symbiotic Bacteria Involved in Cellulose Degradation and Nitrogen Recycling within the Gut of the Forest Cockchafer (Melolontha hippocastani).

Abstract (Expand)

The guts of insects harbor symbiotic bacterial communities. However, due to their complexity, it is challenging to relate a specific symbiotic phylotype to its corresponding function. In the present … study, we focused on the forest cockchafer (Melolontha hippocastani), a phytophagous insect with a dual life cycle, consisting of a root-feeding larval stage and a leaf-feeding adult stage. By combining in vivo stable isotope probing (SIP) with 13C cellulose and 15N urea as trophic links, with Illumina MiSeq (Illumina-SIP), we unraveled bacterial networks processing recalcitrant dietary components and recycling nitrogenous waste. The bacterial communities behind these processes change between larval and adult stages. In 13C cellulose-fed insects, the bacterial families Lachnospiraceae and Enterobacteriaceae were isotopically labeled in larvae and adults, respectively. In 15N urea-fed insects, the genera Burkholderia and Parabacteroides were isotopically labeled in larvae and adults, respectively. Additionally, the PICRUSt-predicted metagenome suggested a possible ability to degrade hemicellulose and to produce amino acids of, respectively, 13C cellulose- and 15N urea labeled bacteria. The incorporation of 15N from ingested urea back into the insect body was confirmed, in larvae and adults, by isotope ratio mass spectrometry (IRMS). Besides highlighting key bacterial symbionts of the gut of M. hippocastani, this study provides example on how Illumina-SIP with multiple trophic links can be used to target microorganisms embracing different roles within an environment.

Authors: P. Alonso-Pernas, S. Bartram, E. M. Arias-Cordero, A. L. Novoselov, L. Halty-deLeon, Y. Shao, W. Boland

Date Published: 28th Oct 2017

Publication Type: Not specified

Strategies and ecological roles of algicidal bacteria.

Abstract (Expand)

In both freshwater and marine ecosystems, phytoplankton are the most dominant primary producers, contributing substantially to aquatic food webs. Algicidal bacteria that can associate to microalgae … from the phytoplankton have the capability to control the proliferation and even to lyse them. These bacteria thus play an important role in shaping species composition in pelagic environments. In this review, we discuss and categorise strategies used by algicidal bacteria for the attack on microalgae. We highlight the complex regulation of algicidal activity and defence responses that govern alga-bacteria interactions. We also discuss how algicidal bacteria impact algal physiology and metabolism and survey the existing algicidal metabolites and enzymes. The review illustrates that the ecological role of algicidal bacteria is not yet fully understood and critically discusses the challenges in obtaining ecologically relevant data.

Authors: N. Meyer, A. Bigalke, A. Kaulfuss, G. Pohnert

Date Published: 30th Sep 2017

Publication Type: Not specified

High-density cultivation of terrestrial Nostoc strains leads to reprogramming of secondary metabolome.

Abstract (Expand)

Terrestrial symbiotic cyanobacteria of the genus Nostoc exhibit a large potential for the production of bioactive natural products of the nonribosomal peptide, polyketide and ribosomal peptide classes, … yet most of the biosynthetic gene clusters are silent under conventional cultivation conditions. In the present study, we have utilized a high-density cultivation approach recently developed for phototrophic bacteria to rapidly generate biomass of the filamentous bacteria up to a density of 400 g wet weight/L. Unexpectedly, integrated transcriptional and metabolomics studies uncovered a major reprogramming of the secondary metabolome of two Nostoc strains at high culture density and a governing effect of extracellular signals in this process. The holistic approach enabled capturing and structural elucidation of novel variants of anabaenopeptin including one congener with potent allelopathic activity against a strain isolated from the same habitat. The study provides a snapshot on the role of cell-type specific expression for the formation of natural products in cyanobacteria.Importance Terrestrial filamentous cyanobacteria are a largely untapped source of small molecular natural products. Exploitation of the phototrophic organisms is hampered by their slow growth and the requirement of photobioreactors. The current study not only demonstrates the suitability of a recently developed two-tier vessel cultivation approach for the rapid generation of biomass of Nostoc strains but also demonstrates a pronounced up regulation of high value natural products at ultra-high culture densities. The study provides new guidelines for high-throughput screening and exploitation of small molecule natural products and can facilitate the discovery new bioactive products from terrestrial cyanobacteria.

Authors: A. Guljamow, M. Kreische, K. Ishida, A. Liaimer, B. Altermark, L. Bahr, C. Hertweck, R. Ehwald, E. Dittmann

Date Published: 25th Sep 2017

Publication Type: Not specified

NaMYB8 regulates distinct, optimally distributed herbivore defense traits.

Abstract (Expand)

When herbivores attack, plants specifically reconfigure their metabolism. Herbivory on the wild tobacco Nicotiana attenuata strongly induces the R2R3 MYB transcriptional activator MYB8, which was … reported to specifically regulate the accumulation of phenolamides (PAs). We discovered that transcriptional regulation of trypsin protease inhibitors (TPIs) and a threonine deaminase (TD) also depend on MYB8 expression. Induced distributions of PAs, TD and TPIs all meet predictions of optimal defense theory: their leaf concentrations increase with the fitness value and the probability of attack of the tissue. Therefore, we suggest that these defensive compounds have evolved to be co-regulated by MYB8.

Authors: M. Schafer, C. Brutting, S. Xu, Z. Ling, A. Steppuhn, I. T. Baldwin, M. C. Schuman

Date Published: 27th Aug 2017

Publication Type: Not specified

Metabolomic compounds identified in Piriformospora indica-colonized Chinese cabbage roots delineate symbiotic functions of the interaction

Abstract (Expand)

Root colonization by endophytic fungus Piriformospora indica facilitating growth/development and stress tolerance has been demonstrated in various host plants. However, global metabolomic studies are … rare. By using high-throughput gas-chromatography-based mass spectrometry, 549 metabolites of 1,126 total compounds observed were identified in colonized and uncolonized Chinese cabbage roots, and hyphae of P. indica. The analyses demonstrate that the host metabolomic compounds and metabolite pathways are globally reprogrammed after symbiosis with P. indica. Especially, γ-amino butyrate (GABA), oxylipin-family compounds, poly-saturated fatty acids, and auxin and its intermediates were highly induced and de novo synthesized in colonized roots. Conversely, nicotinic acid (niacin) and dimethylallylpyrophosphate were strongly decreased. In vivo assays with exogenously applied compounds confirmed that GABA primes plant immunity toward pathogen attack and enhances high salinity and temperature tolerance. Moreover, generation of reactive oxygen/nitrogen species stimulated by nicotinic acid is repressed by P. indica, and causes the feasibility of symbiotic interaction. This global metabolomic analysis and the identification of symbiosis-specific metabolites may help to understand how P. indica confers benefits to the host plant.

Authors: Mo Da-Sang Hua, Rajendran Senthil Kumar, Lie-Fen Shyur, Yuan-Bin Cheng, Zhihong Tian, Ralf Oelmüller, Kai-Wun Yeh

Date Published: 24th Aug 2017

Publication Type: Not specified

Bacteria-induced morphogenesis of Ulva intestinalis and Ulva mutabilis (Chlorophyta): a contribution to the lottery theory.

Abstract (Expand)

The green marine macroalgae of the class Ulvophyceae (Ulvophytes) are common algae distributed worldwide particularly in intertidal areas, which play a key role in aquatic ecosystems. They are potentially … valuable resources for food, animal feed and fuel but can also cause massive nuisance blooms. Members of Ulvaceae, like many other seaweeds, harbour a rich diversity of epiphytic bacteria with functions related to host growth and morphological development. In the absence of appropriate bacterially derived signals, germ cells of the genus Ulva develop into 'atypical' colonies consisting of undifferentiated cells with abnormal cell walls. This paper examines the specificity of bacteria-induced morphogenesis in Ulva, by cross-testing bacteria isolated from several Ulva species on two Ulva species, the emerging model system Ulva mutabilis and the prominent biofouler species Ulva intestinalis. We show that pairs of bacterial strains isolated from species other than U. mutabilis and U. intestinalis can fully rescue axenic plantlets generated either from U. mutabilis or U. intestinalis gametes. This laboratory-based study demonstrates that different compositions of microbial communities with similar functional characteristics can enable complete algal morphogenesis and thus supports the 'competitive lottery' theory for how symbiotic bacteria drive algal development.

Authors: F. Ghaderiardakani, J. C. Coates, T. Wichard

Date Published: 16th Aug 2017

Publication Type: Not specified

Piriformospora indica Reprograms Gene Expression in Arabidopsis Phosphate Metabolism Mutants But Does Not Compensate for Phosphate Limitation.

Abstract (Expand)

Piriformospora indica is an endophytic fungus of Sebacinaceae which colonizes the roots of many plant species and confers benefits to the hosts. We demonstrate that approximately 75% of the genes, which … respond to P. indica in Arabidopsis roots, differ among seedlings grown on normal phosphate (Pi) or Pi limitation conditions, and among wild-type and the wrky6 mutant impaired in the regulation of the Pi metabolism. Mapman analyses suggest that the fungus activates different signaling, transport, metabolic and developmental programs in the roots of wild-type and wrky6 seedlings under normal and low Pi conditions. Under low Pi, P. indica promotes growth and Pi uptake of wild-type seedlings, and the stimulatory effects are identical for mutants impaired in the PHOSPHATE TRANSPORTERS1;1, -1;2 and -1;4. The data suggest that the fungus does not stimulate Pi uptake, but adapts the expression profiles to Pi limitation in Pi metabolism mutants.

Authors: M. Bakshi, I. Sherameti, D. Meichsner, J. Thurich, A. Varma, A. K. Johri, K. W. Yeh, R. Oelmuller

Date Published: 28th Jul 2017

Publication Type: Not specified

Androgen-mediated sex bias impairs efficiency of leukotriene biosynthesis inhibitors in males.

Abstract (Expand)

Proinflammatory leukotrienes (LTs) are produced by 5-lipoxygenase (5-LO) aided by 5-LO-activating protein (FLAP). LT biosynthesis inhibitors are currently under clinical investigation as treatments for … respiratory and cardiovascular diseases. Here, we have revealed a sex bias in the efficiency of clinically relevant LT biosynthesis inhibitors, showing that their effects are superior in females. We found that androgens cause these sex differences by impeding the LT-biosynthetic 5-LO/FLAP complex assembly. Lower doses of the FLAP inhibitor MK886 were required to reduce LTB4 levels in exudates of female versus male mice and rats. Following platelet-activating factor-induced shock, MK886 increased survival exclusively in female mice, and this effect was abolished by testosterone administration. FLAP inhibitors and the novel-type 5-LO inhibitors licofelone and sulindac sulfide exhibited higher potencies in human blood from females, and bioactive 5-LO/FLAP complexes were formed in female, but not male, human and murine leukocytes. Supplementation of female blood or leukocytes with 5alpha-dihydrotestosterone abolished the observed sex differences. Our data suggest that females may benefit from anti-LT therapy to a greater extent than males, prompting consideration of sex issues in LT modifier development.

Authors: S. Pace, C. Pergola, F. Dehm, A. Rossi, J. Gerstmeier, F. Troisi, H. Pein, A. M. Schaible, C. Weinigel, S. Rummler, H. Northoff, S. Laufer, T. J. Maier, O. Radmark, B. Samuelsson, A. Koeberle, L. Sautebin, O. Werz

Date Published: 25th Jul 2017

Publication Type: Not specified

Microbial communities in carbonate rocks-from soil via groundwater to rocks.

Abstract (Expand)

Microbial communities in soil, groundwater, and rock of two sites in limestone were investigated to determine community parameters differentiating habitats in two lithostratigraphic untis. Lower … Muschelkalk and Middle Muschelkalk associated soils, groundwater, and rock samples showed different, but overlapping microbial communities linked to carbon fluxes. The microbial diversities in soil were highest, groundwater revealed overlapping taxa but lower diversity, and rock samples were predominantly characterized by endospore forming bacteria and few archaea. Physiological profiles could establish a differentiation between habitats (soil, groundwater, rock). From community analyses and physiological profiles, different element cycles in limestone could be identified for the three habitats. While in soil, nitrogen cycling was identified as specific determinant, in rock methanogenesis linked carbonate rock to atmospheric methane cycles. These patterns specifically allowed for delineation of lithostratigraphic connections to physiological parameters.

Authors: A. Meier, M. K. Singh, A. Kastner, D. Merten, G. Buchel, E. Kothe

Date Published: 7th Jul 2017

Publication Type: Not specified

Ferrihydrite associated organic matter (OM) stimulates reduction by &lt;i&gt;Shewanella oneidensis&lt;/i&gt; MR-1 and a complex microbial consortia

Abstract (Expand)

The formation of Fe(III) oxides in natural environments occurs in the presence of natural organic matter (OM), resulting in the formation of OM-mineral complexes that form through adsorption or … coprecipitation processes. Thus, microbial Fe(III) reduction in natural environments most often occurs in the presence of OM-mineral complexes rather than pure Fe(III) minerals. In this study we investigated to which extent the content of adsorbed or coprecipitated OM on ferrihydrite influences the rate of Fe(III) reduction by Shewanella oneidensis MR-1, a model Fe(III)-reducing microorganism, in comparison to a microbial consortium extracted from the acidic, Fe-rich Schlöppnerbrunnen fen. We found that increased OM contents led to increased rates of microbial Fe(III) reduction by S. oneidensis MR-1 in contrast to earlier findings with the model organism Geobacter bremensis. Ferrihydrite-OM coprecipitates were reduced slightly faster than ferrihydrites with adsorbed OM. Surprisingly, the complex microbial consortia stimulated by a mixture of electrons donors (lactate, acetate, and glucose) mimics S. oneidensis under the same experimental Fe(III)-reducing conditions suggesting similar mechanisms of electron transfer whether or not the OM is adsorbed or coprecipitated to the mineral surfaces. We also followed potential shifts of the microbial community during the incubation via 16S rRNA gene sequence analyses to determine variations due to the presence of adsorbed or coprecipitated OM-ferrihydrite complexes in contrast to pure ferrihydrite. Community profile analyses showed no enrichment of typical model Fe(III)-reducing bacteria, such as Shewanella sp. or Geobacter sp., but an enrichment of fermenters (i.e. Enterobacteria) during pure ferrihydrite incubations which are known to use Fe(III) as an electron sink. Instead, OM-mineral complexes favored the enrichment of microbes including Desulfobacteria and Pelosinus sp., both of which can utilize lactate and acetate as an electron donor under Fe(III) reducing conditions. In summary, this study shows that increasing concentrations of OM in OM-mineral complexes determines microbial Fe(III) reduction rates and shapes the microbial community structure involved in the reductive dissolution of ferrihydrite. Similarities observed between the complex Fe(III)-reducing microbial consortia and the model Fe(III)-reducer S. oneidensis MR-1 suggest electron shuttling mechanisms dominate in OM-rich environments, including soils, sediments, and fens, where natural OM interacts with Fe(III) oxides during mineral formation.

Authors: Rebecca E. Cooper, Karin Eusterhues, Carl-Eric Wegner, Kai Uwe Totsche, Kirsten Küsel

Date Published: 6th Jul 2017

Publication Type: Not specified

A succinct access to omega-hydroxylated jasmonates via olefin metathesis.

Abstract (Expand)

In higher plants, jasmonates are lipid-derived signaling molecules that control many physiological processes, including responses to abiotic stress, defenses against insects and pathogens, and development. … Among jasmonates, omega-oxidized compounds form an important subfamily. The biological roles of these omega-modified derivatives are not fully understood, largely due to their limited availability. Herein, a brief (two-step), simple and efficient (>80% yield), versatile, gram-scalable, and environmentally friendly synthetic route to omega-oxidized jasmonates is described. The approach utilizes olefin cross-metathesis as the key step employing inexpensive, commercially available substrates and catalysts.

Authors: G. H. Jimenez-Aleman, S. Secinti, W. Boland

Date Published: 1st Jul 2017

Publication Type: Not specified

Sex differences in prostaglandin biosynthesis in neutrophils during acute inflammation.

Abstract (Expand)

The severity and course of inflammatory processes differ between women and men, but the biochemical mechanisms underlying these sex differences are elusive. Prostaglandins (PG) and leukotrienes (LT) … are lipid mediators linked to inflammation. We demonstrated superior LT biosynthesis in human neutrophils and monocytes, and in mouse macrophages from females, and we confirmed these sex differences in vivo where female mice produced more LTs during zymosan-induced peritonitis versus males. Here, we report sex differences in PG production in neutrophils during acute inflammation. In the late phase (4-8 hrs) of mouse zymosan-induced peritonitis and rat carrageenan-induced pleurisy, PG levels in males were higher versus females, seemingly due to higher PG production in infiltrated neutrophils. Accordingly, human neutrophils from males produced more PGE2 than cells from females. Increased PG biosynthesis in males was accompanied by elevated cyclooxygenase (COX)-2 expression connected to increased nuclear factor-kappa B activation, and was abolished when LT synthesis was pharmacologically blocked, suggesting that elevated PG production in males might be caused by increased COX-2 expression and by shunting phenomena due to suppressed LT formation. Conclusively, our data reveal that the biosynthesis of pro-inflammatory PGs and LTs is conversely regulated by sex with consequences for the inflammatory response.

Authors: S. Pace, A. Rossi, V. Krauth, F. Dehm, F. Troisi, R. Bilancia, C. Weinigel, S. Rummler, O. Werz, L. Sautebin

Date Published: 21st Jun 2017

Publication Type: Not specified

In situ monitoring of molecular changes during cell differentiation processes in marine macroalgae through mass spectrometric imaging.

Abstract (Expand)

Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) was employed to discriminate between cell differentiation processes in macroalgae. One of the key developmental processes … in the algal life cycle is the production of germ cells (gametes and zoids). The gametogenesis of the marine green macroalga Ulva mutabilis (Chlorophyta) was monitored by metabolomic snapshots of the surface, when blade cells differentiate synchronously into gametangia and giving rise to gametes. To establish MSI for macroalgae, dimethylsulfoniopropionate (DMSP), a known algal osmolyte, was determined. MSI of the surface of U. mutabilis followed by chemometric data analysis revealed dynamic metabolomic changes during cell differentiation. DMSP and a total of 55 specific molecular biomarkers, which could be assigned to important stages of the gametogenesis, were detected. Our research contributes to the understanding of molecular mechanisms underlying macroalgal cell differentiation. Graphical abstract Molecular changes during cell differentiation of the marine macroalga Ulva were visualized by matrix assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI).

Authors: R. W. Kessler, A. C. Crecelius, U. S. Schubert, T. Wichard

Date Published: 11th Jun 2017

Publication Type: Not specified

Transcription Factors in Fungi: TFome Dynamics, Three Major Families, and Dual-Specificity TFs.

Abstract (Expand)

Transcription factors (TFs) are essential regulators of gene expression in a cell; the entire repertoire of TFs (TFome) of a species reflects its regulatory potential and the evolutionary history of … the regulatory mechanisms. In this work, I give an overview of fungal TFs, analyze TFome dynamics, and discuss TF families and types of particular interest. Whole-genome annotation of TFs in more than 200 fungal species revealed ~80 families of TFs that are typically found in fungi. Almost half of the considered genomes belonged to basidiomycetes and zygomycetes, which have been underrepresented in earlier annotations due to dearth of sequenced genomes. The TFomes were analyzed in terms of expansion strategies genome- and lineage-wise. Generally, TFomes are known to correlate with genome size; but what happens to particular families when a TFome is expanding? By dissecting TFomes into single families and estimating the impact of each of them, I show that in fungi the TFome increment is largely limited to three families (C6 Zn clusters, C2H2-like Zn fingers, and homeodomain-like). To see whether this is a fungal peculiarity or a ubiquitous eukaryotic feature, I also analyzed metazoan TFomes, where I observed a similar trend (limited number of TFome-shaping families) but also some important differences connected mostly with the increased complexity in animals. The expansion strategies of TF families are lineage-specific; I demonstrate how the patterns of the TF families' distributions, designated as "TF signatures," can be used as a taxonomic feature, e.g., for allocation of uncertain phyla. In addition, both fungal and metazoan genomes contain an intriguing type of TFs. While usually TFs have a single DNA-binding domain, these TFs possess two (or more) different DNA-binding specificities. I demonstrate that dual-specific TFs comprising various combinations of all major TF families are a typical feature of fungal and animal genomes and have an interesting evolutionary history involving gene duplications and domain losses.

Author: E. Shelest

Date Published: 20th May 2017

Publication Type: Not specified

antiSMASH 4.0-improvements in chemistry prediction and gene cluster boundary identification.

Abstract (Expand)

Many antibiotics, chemotherapeutics, crop protection agents and food preservatives originate from molecules produced by bacteria, fungi or plants. In recent years, genome mining methodologies have been … widely adopted to identify and characterize the biosynthetic gene clusters encoding the production of such compounds. Since 2011, the 'antibiotics and secondary metabolite analysis shell-antiSMASH' has assisted researchers in efficiently performing this, both as a web server and a standalone tool. Here, we present the thoroughly updated antiSMASH version 4, which adds several novel features, including prediction of gene cluster boundaries using the ClusterFinder method or the newly integrated CASSIS algorithm, improved substrate specificity prediction for non-ribosomal peptide synthetase adenylation domains based on the new SANDPUMA algorithm, improved predictions for terpene and ribosomally synthesized and post-translationally modified peptides cluster products, reporting of sequence similarity to proteins encoded in experimentally characterized gene clusters on a per-protein basis and a domain-level alignment tool for comparative analysis of trans-AT polyketide synthase assembly line architectures. Additionally, several usability features have been updated and improved. Together, these improvements make antiSMASH up-to-date with the latest developments in natural product research and will further facilitate computational genome mining for the discovery of novel bioactive molecules.

Authors: K. Blin, T. Wolf, M. G. Chevrette, X. Lu, C. J. Schwalen, S. A. Kautsar, H. G. Suarez Duran, E. L. C. de Los Santos, H. U. Kim, M. Nave, J. S. Dickschat, D. A. Mitchell, E. Shelest, R. Breitling, E. Takano, S. Y. Lee, T. Weber, M. H. Medema

Date Published: 2nd May 2017

Publication Type: Not specified

Antibiotic-producing symbionts dynamically transition between plant pathogenicity and insect-defensive mutualism

Abstract (Expand)

Pathogenic and mutualistic bacteria associated with eukaryotic hosts often lack distinctive genomic features, suggesting regular transitions between these lifestyles. Here we present evidence supporting … a dynamic transition from plant pathogenicity to insect-defensive mutualism in symbiotic Burkholderia gladioli bacteria. In a group of herbivorous beetles, these symbionts protect the vulnerable egg stage against detrimental microbes. The production of a blend of antibiotics by B. gladioli, including toxoflavin, caryoynencin and two new antimicrobial compounds, the macrolide lagriene and the isothiocyanate sinapigladioside, likely mediate this defensive role. In addition to vertical transmission, these insect symbionts can be exchanged via the host plant and retain the ability to initiate systemic plant infection at the expense of the plant's fitness. Our findings provide a paradigm for the transition between pathogenic and mutualistic lifestyles and shed light on the evolution and chemical ecology of this defensive mutualism.

Authors: L. V. Florez, K. Scherlach, P. Gaube, C. Ross, E. Sitte, C. Hermes, A. Rodrigues, C. Hertweck, M. Kaltenpoth

Date Published: 30th Apr 2017

Publication Type: Not specified

Induced Chemical Defense of a Mushroom by a Double-Bond-Shifting Polyene Synthase.

Abstract (Expand)

The antilarval mushroom polyenes 18-methyl-19-oxoicosaoctaenoic acid and 20-methyl-21-oxodocosanonaenoic acid appear in response to injury of the mycelium of the stereaceous mushroom BY1. We identified … a polyketide synthase (PPS1) which belongs to a hitherto completely uncharacterized clade of polyketide synthases. Expression of the PPS1 gene is massively upregulated following mycelial damage. The synthesis of the above polyenes was reconstituted in the mold Aspergillus niger as a heterologous host. This demonstrates that PPS1 1) synchronously produces branched-chain polyketides of varied lengths, and 2) catalyzes the unprecedented shift of eight or nine double bonds. This study represents the first characterization of a reducing polyketide synthase from a mushroom. We also show that injury-induced de novo synthesis of polyketides is a fungal response strategy.

Authors: P. Brandt, M. Garcia-Altares, M. Nett, C. Hertweck, D. Hoffmeister

Date Published: 26th Apr 2017

Publication Type: Not specified

d-Fructose-Decorated Poly(ethylene imine) for Human Breast Cancer Cell Targeting

Abstract (Expand)

The high affinity of GLUT5 transporter for d-fructose in breast cancer cells has been discussed intensely. In this contribution, high molar mass linear poly(ethylene imine) (LPEI) is functionalized … with d-fructose moieties to combine the selectivity for the GLUT5 transporter with the delivery potential of PEI for genetic material. The four-step synthesis of a thiol-group bearing d-fructose enables the decoration of a cationic polymer backbone with d-fructose via thiol-ene photoaddition. The functionalization of LPEI is confirmed by 2D NMR techniques, elemental analysis, and size exclusion chromatography. Importantly, a d-fructose decoration of 16% renders the polymers water-soluble and eliminates the cytotoxicity of PEI in noncancer L929 cells, accompanied by a reduced unspecific cellular uptake of the genetic material. In contrast, the cytotoxicity as well as the cell specific uptake is increased for triple negative MDA-MB-231 breast cancer cells. Therefore, the introduction of d-fructose shows superior potential for cell targeting, which can be assumed to be GLUT5 dependent.

Authors: C. Englert, M. Prohl, J. A. Czaplewska, C. Fritzsche, E. Preussger, U. S. Schubert, A. Traeger, M. Gottschaldt

Date Published: 4th Apr 2017

Publication Type: Not specified

JA-Ile-macrolactones uncouple growth and defense in wild tobacco

Abstract (Expand)

Small molecules capable of uncoupling growth-defense in plants are currently not known. In this study, for the first time, semi-synthetic analogues of the phytohormone JA-Ile are employed to uncouple … growth and defense responses in wild tobacco. The JA-Ile analogues are easily synthesized from inexpensive substrates via olefin metathesis.

Authors: G. H. Jimenez-Aleman, R. A. R. Machado, I. T. Baldwin, W. Boland

Date Published: 7th Mar 2017

Publication Type: Not specified

Evaluation of Dual 5-Lipoxygenase/Microsomal Prostaglandin E2 Synthase-1 Inhibitory Effect of Natural and Synthetic Acronychia-Type Isoprenylated Acetophenones

Abstract (Expand)

Among the pathways responsible for the development of inflammatory responses, the cyclooxygenase and lipoxygenase pathways are among the most important ones. Two key enzymes, namely, 5-LO and mPGES-1, … are involved in the biosynthesis of leukotrienes and prostaglandins, respectively, which are considered attractive therapeutic targets, so their dual inhibition might be an effective strategy to control inflammatory deregulation. Several natural products have been identified as 5-LO inhibitors, with some also being dual 5-LO/mPGES-1 inhibitors. Here, some prenylated acetophenone dimers from Acronychia pedunculata have been identified for their dual inhibitory potency toward 5-LO and mPGES-1. To gain insight into the SAR of this family of natural products, the synthesis and biological evaluation of analogues are presented. The results show the ability of the natural and synthetic molecules to potently inhibit 5-LO and mPEGS-1 in vitro. The potency of the most active compound (10) has been evaluated in vivo in an acute inflammatory mouse model and displayed potent anti-inflammatory activity comparable in potency to the drug zileuton used as a positive control.

Authors: A. Svouraki, U. Garscha, E. Kouloura, S. Pace, C. Pergola, V. Krauth, A. Rossi, L. Sautebin, M. Halabalaki, O. Werz, N. Gaboriaud-Kolar, A. L. Skaltsounis

Date Published: 28th Feb 2017

Publication Type: Not specified

Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus.

Abstract (Expand)

BACKGROUND: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent … carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus. RESULTS: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli. CONCLUSIONS: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

Authors: R. P. de Vries, R. Riley, A. Wiebenga, G. Aguilar-Osorio, S. Amillis, C. A. Uchima, G. Anderluh, M. Asadollahi, M. Askin, K. Barry, E. Battaglia, O. Bayram, T. Benocci, S. A. Braus-Stromeyer, C. Caldana, D. Canovas, G. C. Cerqueira, F. Chen, W. Chen, C. Choi, A. Clum, R. A. Dos Santos, A. R. Damasio, G. Diallinas, T. Emri, E. Fekete, M. Flipphi, S. Freyberg, A. Gallo, C. Gournas, R. Habgood, M. Hainaut, M. L. Harispe, B. Henrissat, K. S. Hilden, R. Hope, A. Hossain, E. Karabika, L. Karaffa, Z. Karanyi, N. Krasevec, A. Kuo, H. Kusch, K. LaButti, E. L. Lagendijk, A. Lapidus, A. Levasseur, E. Lindquist, A. Lipzen, A. F. Logrieco, A. MacCabe, M. R. Makela, I. Malavazi, P. Melin, V. Meyer, N. Mielnichuk, M. Miskei, A. P. Molnar, G. Mule, C. Y. Ngan, M. Orejas, E. Orosz, J. P. Ouedraogo, K. M. Overkamp, H. S. Park, G. Perrone, F. Piumi, P. J. Punt, A. F. Ram, A. Ramon, S. Rauscher, E. Record, D. M. Riano-Pachon, V. Robert, J. Rohrig, R. Ruller, A. Salamov, N. S. Salih, R. A. Samson, E. Sandor, M. Sanguinetti, T. Schutze, K. Sepcic, E. Shelest, G. Sherlock, V. Sophianopoulou, F. M. Squina, H. Sun, A. Susca, R. B. Todd, A. Tsang, S. E. Unkles, N. van de Wiele, D. van Rossen-Uffink, J. V. Oliveira, T. C. Vesth, J. Visser, J. H. Yu, M. Zhou, M. R. Andersen, D. B. Archer, S. E. Baker, I. Benoit, A. A. Brakhage, G. H. Braus, R. Fischer, J. C. Frisvad, G. H. Goldman, J. Houbraken, B. Oakley, I. Pocsi, C. Scazzocchio, B. Seiboth, P. A. vanKuyk, J. Wortman, P. S. Dyer, I. V. Grigoriev

Date Published: 16th Feb 2017

Publication Type: Not specified

Selective upregulation of TNFalpha expression in classically-activated human monocyte-derived macrophages (M1) through pharmacological interference with V-ATPase.

Abstract (Expand)

Pharmacological interference with vacuolar-type H(+)-ATPase (V-ATPase), a proton-translocating enzyme involved in protein transport and pH regulation of cell organelles, is considered a potential … strategy for cancer therapy. Macrophages are critically involved in tumor progression and may occur as pro-tumoral M2 phenotype, whereas classically-activated M1 can inhibit tumor development for example by releasing tumor-suppressing molecules, including tumor necrosis factor (TNF)alpha. Here, we show that targeting V-ATPase by selective inhibitors such as archazolid upregulates the expression and secretion of TNFalpha in lipopolysaccharide (LPS)- or LPS/interferon (INF)gamma-activated M1-like macrophages derived from human blood monocytes. In contrast, archazolid failed to elevate TNFalpha production from uncommitted (M0) or interleukin (IL)-4-treated M2-like macrophages. Secretion of other relevant cytokines (i.e., IL-1beta, IL-6, IL-10) or chemokines (i.e. IL-8 and monocyte chemotactic protein-1) from M1 was not affected by archazolid. Though V-ATPase inhibitors elevated the lysosomal pH in M1 comparable to chloroquine or ammonium chloride, the latter agents suppressed TNFalpha secretion. Archazolid selectively increased TNFalpha mRNA levels, which was abolished by dexamethasone. Interestingly, archazolid enhanced the phosphorylation and nuclear translocation of the p65 subunit of NFkappaB and stimulated phosphorylation of SAPK/JNK. In a microfluidically-supported human tumor biochip model, archazolid-treated M1 significantly reduced tumor cell viability. Together, our data show that V-ATPase inhibition selectively upregulates TNFalpha production in classically-activated macrophages along with NFkappaB and SAPK/JNK activation. Such increased TNFalpha release caused by V-ATPase inhibitors may contribute to tumor suppression in addition to direct targeting cancer cells.

Authors: L. Thomas, Z. Rao, J. Gerstmeier, M. Raasch, C. Weinigel, S. Rummler, D. Menche, R. Muller, C. Pergola, A. Mosig, O. Werz

Date Published: 13th Feb 2017

Publication Type: Not specified

Cultivation-Free Raman Spectroscopic Investigations of Bacteria.

Abstract (Expand)

Raman spectroscopy is currently advertised as a hot and ambitious technology that has all of the features needed to characterize and identify bacteria. Raman spectroscopy is rapid, easy to use, … noninvasive, and it could complement established microbiological and biomolecular methods in the near future. To bring this vision closer to reality, ongoing research is being conducted on spectral fingerprinting. This can yield a wealth of information, from even single bacteria from various habitats which can be further improved by combining Raman spectroscopy with methods such as stable isotope probing to elucidate microbial interactions. In conjunction with extensive statistical analysis, Raman spectroscopy will allow identification of (non)pathogenic bacteria at different taxonomic levels.

Authors: B. Lorenz, C. Wichmann, S. Stockel, P. Rosch, J. Popp

Date Published: 12th Feb 2017

Publication Type: Not specified

Sticking together: inter-species aggregation of bacteria isolated from iron snow is controlled by chemical signaling

Abstract (Expand)

Marine and lake snow is a continuous shower of mixed organic and inorganic aggregates falling from the upper water where primary production is substantial. These pelagic aggregates provide a niche for … microbes that can exploit these physical structures and resources for growth, thus are local hot spots for microbial activity. However, processes underlying their formation remain unknown. Here, we investigated the role of chemical signaling between two co-occurring bacteria that each make up more than 10% of the community in iron-rich lakes aggregates (iron snow). The filamentous iron-oxidizing Acidithrix strain showed increased rates of Fe(II) oxidation when incubated with cell-free supernatant of the heterotrophic iron-reducing Acidiphilium strain. Amendment of Acidithrix supernatant to motile cells of Acidiphilium triggered formation of cell aggregates displaying similar morphology to those of iron snow. Comparative metabolomics enabled the identification of the aggregation-inducing signal, 2-phenethylamine, which also induced faster growth of Acidiphilium. We propose a model that shows rapid iron snow formation, and ultimately energy transfer from the photic zone to deeper water layers, is controlled via a chemically mediated interplay.

Authors: J. F. Mori, N. Ueberschaar, S. Lu, R. E. Cooper, G. Pohnert, K. Kusel

Date Published: 1st Feb 2017

Publication Type: Not specified

Modulation of actin dynamics as potential macrophage subtype-targeting anti-tumour strategy

Abstract (Expand)

Tumour-associated macrophages mainly comprise immunosuppressive M2 phenotypes that promote tumour progression besides anti-tumoural M1 subsets. Selective depletion or reprogramming of M2 may represent … an innovative anti-cancer strategy. The actin cytoskeleton is central for cellular homeostasis and is targeted for anti-cancer chemotherapy. Here, we show that targeting G-actin nucleation using chondramide A (ChA) predominantly depletes human M2 while promoting the tumour-suppressive M1 phenotype. ChA reduced the viability of M2, with minor effects on M1, but increased tumour necrosis factor (TNF)alpha release from M1. Interestingly, ChA caused rapid disruption of dynamic F-actin filaments and polymerization of G-actin, followed by reduction of cell size, binucleation and cell division, without cellular collapse. In M1, but not in M2, ChA caused marked activation of SAPK/JNK and NFkappaB, with slight or no effects on Akt, STAT-1/-3, ERK-1/2, and p38 MAPK, seemingly accounting for the better survival of M1 and TNFalpha secretion. In a microfluidically-supported human tumour biochip model, circulating ChA-treated M1 markedly reduced tumour cell viability through enhanced release of TNFalpha. Together, ChA may cause an anti-tumoural microenvironment by depletion of M2 and activation of M1, suggesting induction of G-actin nucleation as potential strategy to target tumour-associated macrophages in addition to neoplastic cells.

Authors: C. Pergola, K. Schubert, S. Pace, J. Ziereisen, F. Nikels, O. Scherer, S. Huttel, S. Zahler, A. M. Vollmar, C. Weinigel, S. Rummler, R. Muller, M. Raasch, A. Mosig, A. Koeberle, O. Werz

Date Published: 31st Jan 2017

Publication Type: Not specified

Symbiont-Derived Antimicrobials Contribute to the Control of the Lepidopteran Gut Microbiota

Abstract (Expand)

Insects develop efficient antimicrobial strategies to flourish in a bacterial world. It has long been proposed that native gut microbiota is an important component of host defense; however, the responsible … species have rarely been isolated to elucidate the mechanism of action. Here we show that the dominant symbiotic bacterium Enterococcus mundtii associated with the generalist herbivore Spodoptera littoralis actively secretes a stable class IIa bacteriocin (mundticin KS) against invading bacteria, but not against other gut residents, facilitating the normal development of host gut microbiota. A mundticin-defective strain lost inhibitory activity. Furthermore, purified mundticin cures infected larvae. Thus, the constitutively produced antimicrobials by native extracellular symbionts create a significant chemical barrier inside limiting invader expansion. This unique property also benefits E. mundtii itself by providing a competitive advantage, contributing to its dominance within complex microbial settings and its prevalence across Lepidoptera, and probably promotes the long-term cooperative symbiosis between both parties.

Authors: Y. Shao, B. Chen, C. Sun, K. Ishida, C. Hertweck, W. Boland

Date Published: 21st Jan 2017

Publication Type: Not specified

Time Course Exo-Metabolomic Profiling in the Green Marine Macroalga Ulva (Chlorophyta) for Identification of Growth Phase-Dependent Biomarkers

Abstract (Expand)

The marine green macroalga Ulva (Chlorophyta) lives in a mutualistic symbiosis with bacteria that influence growth, development, and morphogenesis. We surveyed changes in Ulva's chemosphere, which … was defined as a space where organisms interact with each other via compounds, such as infochemicals, nutrients, morphogens, and defense compounds. Thereby, Ulva mutabilis cooperates with bacteria, in particular, Roseovarius sp. strain MS2 and Maribacter sp. strain MS6 (formerly identified as Roseobacter sp. strain MS2 and Cytophaga sp. strain MS6). Without this accompanying microbial flora, U. mutabilis forms only callus-like colonies. However, upon addition of the two bacteria species, in effect forming a tripartite community, morphogenesis can be completely restored. Under this strictly standardized condition, bioactive and eco-physiologically-relevant marine natural products can be discovered. Solid phase extracted waterborne metabolites were analyzed using a metabolomics platform, facilitating gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) analysis, combined with the necessary acquisition of biological metadata. Multivariate statistics of the GC-MS and LC-MS data revealed strong differences between Ulva's growth phases, as well as between the axenic Ulva cultures and the tripartite community. Waterborne biomarkers, including glycerol, were identified as potential indicators for algal carbon source and bacterial-algal interactions. Furthermore, it was demonstrated that U. mutabilis releases glycerol that can be utilized for growth by Roseovarius sp. MS2.

Authors: T. Alsufyani, A. Weiss, T. Wichard

Date Published: 12th Jan 2017

Publication Type: Not specified

Cell structure and microtubule organisation during gametogenesis of Ulva mutabilis Føyn (Chlorophyta)

Abstract (Expand)

Cell structure and microtubule organisation during gametogenesis of the green alga Ulva mutabilis was studied using light microscopy, transmission electron microscopy (TEM) and tubulin immunofluorescence. … Microtubules in vegetative cells are organised in parallel bundles traversing the cortical cytoplasm. During gametogenesis, induced blade cells are transformed to gametangia, depending on the maturity of the algae and the removal of regulatory sporulation inhibitors. This differentiation is accompanied by formation of a conical cell projection (papilla) towards the exterior of the thallus. Microtubules form a clear, basket-like configuration converging towards the conical tip, but not reaching it. The conical microtubule structure stops below the tip, leaving a circular “opening”. Parallel to the above, the cell wall of the tip is differentiated, forming a “cap”. Nuclear divisions start at this stage, finally forming the nuclei of future gametes. Cytokinesis takes place by membrane furrowing and vesicle fusion, giving rise to 16 oval-shaped gametes. The conical microtubule organisation is gradually depolymerised, and a cortical, intensely fluorescing microtubule bundle is formed in each gamete. At this stage, the cap at the conical cell wall projection is removed and the exit pore opens. The biflagellate gametes remain initially motionless, connected by thin cytoplasmic bridges. Finally, they are released to the environment upon additional removal of a swarming inhibitor accumulated in the growth medium during gametogenesis. Keywords: cell differentiation; gametogenesis; green macroalgae; immunofluorescence; microtubule cytoskeleton

Authors: Thomas Wichard, Christos Katsaros, Anne Weiss, Ira Llangos

Date Published: 11th Jan 2017

Publication Type: Not specified

Biophysical characterization and antineoplastic activity of new bis(thiosemicarbazonato) Cu(II) complexes

Abstract (Expand)

Aiming to explore alternative mechanisms of cellular uptake and cytotoxicity, we have studied a new family of copper(II) complexes (CuL1-CuL4) with bis(thiosemicarbazone) (BTSC) ligands containing … pendant protonable cyclic amines (morpholine and piperidine). Herein, we report on the synthesis and characterization of these new complexes, as well as on their biological performance (cytotoxic activity, cellular uptake, protein and DNA binding), in comparison with the parental CuIIATSM (ATSM=diacetyl-bis(N4-methylthiosemicarbazonate) complex without pendant cyclic amines. The new compounds have been characterized by a range of analytical techniques including ESI-MS, IR spectroscopy, cyclic voltammetry, reverse-phase HPLC and X-ray spectroscopy. In vitro cytotoxicity studies revealed that the copper complexes are cytotoxic, unlike the corresponding ligands, with a similar potency to that of CuATSM. Unlike CuATSM, the new complexes were able to circumvent cisplatin cross-resistance. The presence of the protonable cyclic amines did not lead to an enhancement of the interaction of the complexes with human serum albumin or calf thymus DNA. However, CuL1-CuL4 showed a remarkably augmented cellular uptake compared with CuATSM, as proved by uptake, internalization and externalization studies that were performed using the radioactive congeners 64CuL1-64CuL4. The enhanced cellular uptake of CuL1-CuL4 indicates that this new family of CuIIBTSC complexes deserves to be further evaluated in the design of metallodrugs for cancer theranostics.

Authors: E. Palma, F. Mendes, G. R. Morais, I. Rodrigues, I. C. Santos, M. P. Campello, P. Raposinho, I. Correia, S. Gama, D. Belo, V. Alves, A. J. Abrunhosa, I. Santos, A. Paulo

Date Published: 3rd Dec 2016

Publication Type: Not specified

Catechol, a major component of smoke, influences primary root growth and root hair elongation through reactive oxygen species-mediated redox signaling

Abstract (Expand)

Nicotiana attenuata germinates from long-lived seedbanks in native soils after fires. Although smoke signals have been known to break seed dormancy, whether they also affect seedling establishment and … root development remains unclear. In order to test this, seedlings were treated with smoke solutions. Seedlings responded in a dose-dependent manner with significantly increased primary root lengths, due mainly to longitudinal cell elongation, increased numbers of lateral roots and impaired root hair development. Bioassay-driven fractionations and NMR were used to identify catechol as the main active compound for the smoke-induced root phenotype. The transcriptome analysis revealed that mainly genes related to auxin biosynthesis and redox homeostasis were altered after catechol treatment. However, histochemical analyses of reactive oxygen species (ROS) and the inability of auxin applications to rescue the phenotype clearly indicated that highly localized changes in the root's redox-status, rather than in levels of auxin, are the primary effector. Moreover, H2 O2 application rescued the phenotype in a dose-dependent manner. Chemical cues in smoke not only initiate seed germination, but also influence seedling root growth; understanding how these cues work provides new insights into the molecular mechanisms by which plants adapt to post-fire environments.

Authors: M. Wang, M. Schoettner, S. Xu, C. Paetz, J. Wilde, I. T. Baldwin, K. Groten

Date Published: 24th Nov 2016

Publication Type: Not specified

Myxochelin-Inspired 5-Lipoxygenase Inhibitors: Synthesis and Biological Evaluation

Abstract (Expand)

A total of 48 analogues of the natural product myxochelin A were prepared and evaluated for their inhibitory effects on human 5-lipoxygenase in both cell-free and cell-based assays. Structure-activity … relationship analysis revealed that the secondary alcohol function and only chiral center of myxochelin A is not required for biological activity. By expanding the diaminoalkane linker of the two aromatic residues it was possible to generate a myxochelin derivative with superior activity against 5-lipoxygenase in intact cells.

Authors: S. Schieferdecker, S. Konig, S. Pace, O. Werz, M. Nett

Date Published: 23rd Nov 2016

Publication Type: Not specified

Sex ratio of mirid populations shifts in response to hostplant co-infestation or altered cytokinin signaling

Abstract (Expand)

Herbivore species sharing a host plant often compete. In this study, we show that host plant-mediated interaction between two insect herbivores - a generalist and a specialist - results in a sex ratio … shift of the specialist's offspring. We studied demographic parameters of the specialist Tupiocoris notatus (Hemiptera: Miridae) when co-infesting the host plant Nicotiana attenuata (Solanaceae) with the generalist leafhopper Empoasca sp. (Hemiptera: Cicadellidae). We show that the usually female-biased sex ratio of T. notatus shifts toward a higher male proportion in the offspring on plants co-infested by Empoasca sp. This sex ratio change did not occur after oviposition, nor is it due differential mortality of female and male nymphs. Based on pyrosequencing and PCR of bacterial 16S rRNA amplicons, we concluded that sex ratio shifts were unlikely to be due to infection with Wolbachia or other known sex ratio-distorting endosymbionts. Finally, we used transgenic lines of N. attenuata to evaluate if the sex ratio shift could be mediated by changes in general or specialized host plant metabolites. We found that the sex ratio shift occurred on plants deficient in two cytokinin receptors (irCHK2/3). Thus, cytokinin-regulated traits can alter the offspring sex ratio of the specialist T. notatus.

Authors: N. Adam, T. Erler, M. Kallenbach, M. Kaltenpoth, G. Kunert, I. T. Baldwin, M. C. Schuman

Date Published: 20th Nov 2016

Publication Type: Not specified

Illuminating a plant's tissue-specific metabolic diversity using computational metabolomics and information theory.

Abstract (Expand)

Secondary metabolite diversity is considered an important fitness determinant for plants' biotic and abiotic interactions in nature. This diversity can be examined in two dimensions. The first one … considers metabolite diversity across plant species. A second way of looking at this diversity is by considering the tissue-specific localization of pathways underlying secondary metabolism within a plant. Although these cross-tissue metabolite variations are increasingly regarded as important readouts of tissue-level gene function and regulatory processes, they have rarely been comprehensively explored by nontargeted metabolomics. As such, important questions have remained superficially addressed. For instance, which tissues exhibit prevalent signatures of metabolic specialization? Reciprocally, which metabolites contribute most to this tissue specialization in contrast to those metabolites exhibiting housekeeping characteristics? Here, we explore tissue-level metabolic specialization in Nicotiana attenuata, an ecological model with rich secondary metabolism, by combining tissue-wide nontargeted mass spectral data acquisition, information theory analysis, and tandem MS (MS/MS) molecular networks. This analysis was conducted for two different methanolic extracts of 14 tissues and deconvoluted 895 nonredundant MS/MS spectra. Using information theory analysis, anthers were found to harbor the most specialized metabolome, and most unique metabolites of anthers and other tissues were annotated through MS/MS molecular networks. Tissue-metabolite association maps were used to predict tissue-specific gene functions. Predictions for the function of two UDP-glycosyltransferases in flavonoid metabolism were confirmed by virus-induced gene silencing. The present workflow allows biologists to amortize the vast amount of data produced by modern MS instrumentation in their quest to understand gene function.

Authors: Dapeng Li, S. Heiling, I. T. Baldwin, E. Gaquerel

Date Published: 9th Nov 2016

Publication Type: Not specified

Crossing the blood-brain barrier: Glutathione-conjugated poly(ethylene imine) for gene delivery

Abstract (Expand)

The targeted drug delivery to the central nervous system represents one of the major challenges in pharmaceutical formulations since it is strictly limited through the highly selective blood-brain … barrier (BBB). l-Glutathione (GSH), a tripeptide and well-known antioxidant, has been studied in the last years as potential candidate to facilitate the receptor-mediated transcytosis of nanocarriers. We thus tested whether GSH decoration of a positively charged polymer, poly(ethylene imine), with this vector enables the transport of genetic material and, simultaneously, the passage through the BBB. In this study, we report the synthesis of GSH conjugated cationic poly(ethylene imine)s via ecologically desirable thiol-ene photo-addition. The copolymers, containing 80% primary or secondary amine groups, respectively, were investigated concerning their bio- and hemocompatibility as well as their ability to cross a hCMEC/D3 endothelial cell layer mimicking the BBB within microfluidically perfused biochips. We demonstrate that BBB passage depends on the used amino-groups and on the GSH ratio. Thereby the copolymer containing secondary amines showed an enhanced performance. We thus conclude that GSH-coupling represents a feasible and promising approach for the functionalization of nanocarriers intended to cross the BBB for the delivery of drugs to the central nervous system.

Authors: , A. K. Trutzschler, M. Raasch, T. Bus, P. Borchers, A. S. Mosig, A. Traeger,

Date Published: 19th Oct 2016

Publication Type: Not specified

A fast and direct liquid chromatography-mass spectrometry method to detect and quantify polyunsaturated aldehydes and polar oxylipins in diatoms

Abstract (Expand)

Polyunsaturated aldehydes (PUAs) are a group of microalgal metabolites that have attracted a lot of attention due to their biological activity. Determination of PUAs has become an important routine … procedure in plankton and biofilm investigations, especially those that deal with chemically mediated interactions. Here we introduce a fast and direct derivatization free method that allows quantifying PUAs in the nanomolar range, sufficient to undertake the analysis from cultures and field samples. The sample preparation requires one simple filtration step and the initiation of PUA formation by cell disruption. After centrifugation the samples are ready for measurement without any further handling. Within one chromatographic run this method additionally allows us to monitor the formation of the polar oxylipins arising from the cleavage of precursor fatty acids. The robust method is based on analyte separation and detection using ultra high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (UHPLC-APCI MS) and enables high throughput investigations by employing an analysis time of only 5 min. Our protocol thus provides an alternative and extension to existing PUA determinations based on gas chromatography-mass spectrometry (GC-MS) with shorter run times and without any chemical derivatization. It also enables researchers with widely available LC-MS analytical platforms to monitor PUAs. Additionally, non-volatile oxylipins such as ω-oxo-acids and related compounds can be elucidated and monitored.

Authors: Constanze Kuhlisch, Michael Deicke, Nico Ueberschaar, Thomas Wichard, Georg Pohnert

Date Published: 15th Oct 2016

Publication Type: Not specified

Regulation and Role of Fungal Secondary Metabolites.

Abstract (Expand)

Fungi have the capability to produce a tremendous number of so-called secondary metabolites, which possess a multitude of functions, e.g., communication signals during coexistence with other microorganisms, … virulence factors during pathogenic interactions with plants and animals, and in medical applications. Therefore, research on this topic has intensified significantly during the past 10 years and thus knowledge of regulatory mechanisms and the understanding of the role of secondary metabolites have drastically increased. This review aims to depict the complexity of all the regulatory elements involved in controlling the expression of secondary metabolite gene clusters, ranging from epigenetic control and signal transduction pathways to global and specific transcriptional regulators. Furthermore, we give a short overview on the role of secondary metabolites, focusing on the interaction with other microorganisms in the environment as well as on pathogenic relationships.

Authors: J. Macheleidt, D. J. Mattern, J. Fischer, T. Netzker, J. Weber, V. Schroeckh, V. Valiante, A. A. Brakhage

Date Published: 13th Oct 2016

Publication Type: Not specified

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