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35 Publications visible to you, out of a total of 35
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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Pseudoxylallemycins A-F, Cyclic Tetrapeptides with Rare Allenyl Modifications Isolated from Pseudoxylaria sp. X802: A Competitor of Fungus-Growing Termite Cultivars.

Abstract (Expand)

Based on fungus-fungus pairing assays and HRMS-based dereplication strategy, six new cyclic tetrapeptides, pseudoxylallemycins A-F (1-6), were isolated from the termite-associated fungus Pseudoxylaria … sp. X802. Structures were characterized using NMR spectroscopy, HRMS, and Marfey's reaction. Pseudoxylallemycins B-D (2-4) possess a rare and chemically accessible allene moiety amenable for synthetic modifications, and derivatives A-D showed antimicrobial activity against Gram-negative human-pathogenic Pseudomonas aeruginosa and antiproliferative activity against human umbilical vein endothelial cells and K-562 cell lines.

Authors: H. Guo, N. B. Kreuzenbeck, S. Otani, M. Garcia-Altares, H. M. Dahse, C. Weigel, D. K. Aanen, C. Hertweck, M. Poulsen, C. Beemelmanns

Date Published: 25th Jun 2016

Publication Type: Not specified

A fivefold parallelized biosynthetic process secures chlorination of Armillaria mellea (honey mushroom) toxins

Abstract (Expand)

The basidiomycetous tree pathogen Armillaria mellea (honey mushroom) produces a large variety of structurally related antibiotically active and phytotoxic natural products, referred to as the melleolides. … During their biosynthesis, some members of the melleolide family of compounds undergo monochlorination of the aromatic moiety, whose biochemical and genetic basis was not known previously. This first study on basidiomycete halogenases presents the biochemical in vitro characterization of five flavin-dependent A. mellea enzymes (ArmH1-ArmH5) that were heterologously produced in Escherichia coli. We demonstrate that all five enzymes transfer a single chlorine atom to the melleolide backbone. A fivefold secured biosynthetic step during natural product assembly is unprecedented. Typically, flavin-dependent halogenases are categorized into enzymes acting on free compounds as opposed to those requiring a carrier protein-bound acceptor substrate. The enzymes characterized in this study clearly turned over free substrates. Phylogenetic clades of halogenases suggest that all fungal enzymes share a common ancestor and reflect a clear divergence between ascomycetes and basidiomycetes.

Authors: J. Wick, D. Heine, G. Lackner, M. Misiek, , H. Jagusch, ,

Date Published: 15th Dec 2015

Publication Type: Not specified

Plant pathogenic anaerobic bacteria use aromatic polyketides to access aerobic territory

Abstract (Expand)

Around 25% of vegetable food is lost worldwide because of infectious plant diseases, including microbe-induced decay of harvested crops. In wet seasons and under humid storage conditions, potato tubers … are readily infected and decomposed by anaerobic bacteria (Clostridium puniceum). We found that these anaerobic plant pathogens harbor a gene locus (type II polyketide synthase) to produce unusual polyketide metabolites (clostrubins) with dual functions. The clostrubins, which act as antibiotics against other microbial plant pathogens, enable the anaerobic bacteria to survive an oxygen-rich plant environment.

Authors: G. Shabuer, K. Ishida, S. J. Pidot, M. Roth, H. M. Dahse,

Date Published: 7th Nov 2015

Publication Type: Not specified

Three Redundant Synthetases Secure Redox-Active Pigment Production in the Basidiomycete Paxillus involutus

Abstract (Expand)

The symbiotic fungus Paxillus involutus serves a critical role in maintaining forest ecosystems, which are carbon sinks of global importance. P. involutus produces involutin and other 2,5-diarylcyclopentenone … pigments that presumably assist in the oxidative degradation of lignocellulose via Fenton chemistry. Their precise biosynthetic pathways, however, remain obscure. Using a combination of biochemical, genetic, and transcriptomic analyses, in addition to stable-isotope labeling with synthetic precursors, we show that atromentin is the key intermediate. Atromentin is made by tridomain synthetases of high similarity: InvA1, InvA2, and InvA5. An inactive atromentin synthetase, InvA3, gained activity after a domain swap that replaced its native thioesterase domain with that of InvA5. The found degree of multiplex biosynthetic capacity is unprecedented with fungi, and highlights the great importance of the metabolite for the producer.

Authors: J. Braesel, S. Gotze, F. Shah, D. Heine, , , A. Tunlid, P. Stallforth,

Date Published: 27th Oct 2015

Publication Type: Not specified

Antimicrobial discovery inspired by ecological interactions

Abstract

Not specified

Author: C. Hertweck

Date Published: No date defined

Publication Type: Not specified

A giant type I polyketide synthase participates in zygospore maturation in Chlamydomonas reinhardtii.

Abstract (Expand)

Polyketide synthases (PKSs) occur in many bacteria, fungi and plants. They are highly versatile enzymes involved in the biosynthesis of a large variety of compounds including antimicrobial agents, … polymers associated with bacterial cell walls and plant pigments. While harmful algae are known to produce polyketide toxins, sequences of the genomes of non-toxic algae, including those of many green-algal species, have surprisingly revealed the presence of genes encoding type I PKSs. The genome of the model alga Chlamydomonas reinhardtii (Chlorophyta) contains a single type I PKS gene, designated PKS1 (Cre10.g449750), which encodes a giant PKS with a predicted mass of 2.3 MDa. Here, we show that PKS1 is induced in two-day old zygotes and is required for their development into zygospores, the dormant stage of the zygote. Wild-type zygospores contain knob-like structures (~50 nm diameter) that form at the cell surface and develop a central cell wall layer; both of these structures are absent from homozygous pks1 mutants. Additionally, in contrast to wild-type zygotes, chlorophyll degradation is delayed in homozygous pks1 mutant zygotes, indicating a disruption of zygospore development. In agreement with a role of the PKS in the formation of the highly resistant zygospore wall, mutant zygotes have lost the formidable desiccation tolerance of wild-type zygotes. Together, our results represent functional analyses of a PKS mutant in a photosynthetic eukaryotic microorganism, revealing a central function for polyketides in the sexual cycle and survival under stressful environmental conditions. This article is protected by copyright. All rights reserved.

Authors: N. Heimerl, E. Hommel, M. Westermann, D. Meichsner, M. Lohr, C. Hertweck, A. R. Grossman, M. Mittag, S. Sasso

Date Published: No date defined

Publication Type: Not specified

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