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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 of the infection, 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., A combination of genome mining, metabolic profiling, and imaging techniques (MALDI and UV Raman spectroscopy) provided insight into the arsenal of antifungals produced by the mushroom pathogen Burkholderia gladioli pv. agaricicola. Targeted gene knockouts and bioassays link these antifungals to prevalent symptoms of mushroom soft rot, browning, and impaired mycelium growth.

The tight association of the pathogenic fungus Rhizopus microsporus and its toxin-producing, bacterial endosymbionts (Mycetohabitans spp.) is distributed worldwide and has significance for agriculture, food production, and human health. Intriguingly, the endofungal bacteria are essential for the propagation of the fungal host. Yet, little is known about chemical mediators fostering the symbiosis, and universal metabolites that support the mutualistic relationship have remained elusive. Here, we describe the discovery of a complex of specialized metabolites produced by endofungal bacteria under symbiotic conditions. Through full genome sequencing and comparative genomics of eight endofungal symbiont strains from geographically distant regions, we discovered a conserved gene locus (hab) for a nonribosomal peptide synthetase as a unifying trait. Bioinformatics analyses, targeted gene deletions, and chemical profiling uncovered unprecedented depsipeptides (habitasporins) whose structures were fully elucidated. Computational network analysis and labeling experiments granted insight into the biosynthesis of their nonproteinogenic building blocks (pipecolic acid and β-phenylalanine). Deletion of the hab gene locus was shown to impair the ability of the bacteria to enter their fungal host. Our study unveils a common principle of the endosymbiotic lifestyle of Mycetohabitans species and expands the repertoire of characterized chemical mediators of a globally occurring mutualistic association.

Sinapigladioside is a rare isothiocyanate‐bearing natural product from beetle‐associated bacteria (Burkholderia gladioli) that might protect 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 into isonitrile precursors., Alternative pathways: Genetic analyses and labeling studies provide the first insights into the biosynthesis of the rare isothiocyanate natural product sinapigladioside from beetle‐associated bacteria that might protect the beetle offspring against entomopathogenic fungi. Contrary to known isothiocyanate biosynthetic pathways, sinapigladioside assembly involves an isonitrile synthase.

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‐β‐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., When life gives you citrate… Genome mining of food‐spoiling bacteria revealed unprecedented lipocyclopeptides (bolagladins) with antimycobacterial activity. A specialized citrate synthase and nonribosomal peptide synthetases (NRPSs) play key roles in the formation and loading of their unusual tricarboxylic fatty acid tail, thus generating a bolaamphiphilic natural product (see scheme).

In invertebrates, the cuticle is the first and major protective barrier against predators and pathogen infections. While immune responses and behavioral defenses are also known to be important for insect protection, the potential of cuticle-associated microbial symbionts to aid in preventing pathogen entry during molting and throughout larval development remains unexplored. Here, we show that bacterial symbionts of the beetle Lagria villosa inhabit unusual dorsal invaginations of the insect cuticle, which remain open to the outer surface and persist throughout larval development. This specialized location enables the release of several symbiont cells and the associated protective compounds during molting. This facilitates ectosymbiont maintenance and extended defense during larval development against antagonistic fungi. One Burkholderia strain, which produces the antifungal compound lagriamide, dominates the community across all life stages, and removal of the community significantly impairs the survival probability of young larvae when exposed to different pathogenic fungi. We localize both the dominant bacterial strain and lagriamide on the surface of eggs, larvae, pupae, and on the inner surface of the molted cuticle (exuvia), supporting extended protection. These results highlight adaptations for effective defense of immature insects by cuticle-associated ectosymbionts, a potentially key advantage for a ground-dwelling insect when confronting pathogenic microbes.

Significance Soil is a complex and competitive environment, forcing its inhabitants to develop strategies against competitors, predators, and pathogens. Identifying and understanding the molecular mechanisms has translational value for medicine, ecology, and agriculture. In this study, we show that a member of important soil-dwelling fungi (Mortierella) forms a tight alliance with toxin-producing bacteria (Mycoavidus) that live within the fungal hyphae and protect their host from nematode attack. This discovery is relevant since Mortierella species correlate with healthy soils and are used as plant growth–promoting fungi in agriculture. Unraveling an ecological role for fungal endosymbionts in Mortierella, our results contribute to the understanding of a mainspring in fungal–endobacterial symbioses and open the possibility for the development of new biocontrol agents., 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.

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 profiling 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.

Antibiotics from few culturable microorganisms have saved millions of lives since the 20th century. But with resistance formation, these compounds become increasingly ineffective, while the majority of microbial and with that chemical compound diversity remains inaccessible for cultivation and exploration. Culturing recalcitrant bacteria is a stochastic process. But conventional methods are limited to low throughput. By increasing (i) throughput and (ii) sensitivity by miniaturization, we innovate microbiological cultivation to comply with biological stochasticity. Here, we introduce a droplet-based microscale cultivation system, which is directly coupled to a high-throughput screening for antimicrobial activity prior to strain isolation. We demonstrate that highly parallelized in-droplet cultivation starting from single cells results in the cultivation of yet uncultured species and a significantly higher bacterial diversity than standard agar plate cultivation. Strains able to inhibit intact reporter strains were isolated from the system. A variety of antimicrobial compounds were detected for a selected potent antibiotic producer., eLife digest Antibiotics are chemicals derived from microorganisms that can kill the bacteria that harm human health. In the 20th and 21st centuries antibiotics saved millions of lives, but new strains of dangerous bacteria that cannot be killed by antibiotics, known as antibiotic resistant strains, are becoming more frequent. Most antibiotics are produced by only a small group of microorganisms, but many more microorganisms exist in nature. So it is possible that microorganisms outside this small group can produce different antibiotics that are effective against antibiotic resistant strains. Unfortunately, finding the microorganisms that produce these alternative antibiotics is challenging because researchers do not know which bacteria are producing these molecules and how to grow these microorganisms in the laboratory. To solve this problem, Mahler et al. developed a new method for growing a new subset of microorganisms in the laboratory. This would allow researchers to study the new microorganisms under controlled conditions, and determine whether any of the substances they produce have antibiotic properties. Mahler et al. generated tiny droplets that could only contain a single cell of a microorganism, so each microbe could grow alone in its own protected environment. Using this approach, it was possible to grow completely different types of microorganisms than with traditional techniques, and keep them isolated from each other. This allowed each different species of microbe to be screened for antimicrobial activity, allowing the identification of chemicals that could potentially be developed into new antibiotics. This new method is automated and miniaturized, paving the way for growing many more cells in few hours, with very low material and space requirements. These results showcase a way of growing new types of microorganisms in the laboratory, making it easier and faster to study them and determine what chemicals they produce. Understanding a greater variety of microorganisms in detail can help identify new chemicals for industrial applications, including new ways of combating infections.

Genome mining of one of the protective symbionts (Burkholderia gladioli) of the invasive beetle Lagria villosa revealed a cryptic gene cluster that codes 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 previously‐overlooked components of the antimicrobial armory of the beetle symbiont, which are highly active against the entomopathogenic fungus Purpureocillium lilacinum. By mutational analyses, isotope labeling, and computational 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 precise excision within a PKS domain., Genome mining, bioactivity and metabolic profiling revealed a novel polyketide (gladiofungin) that augments the antifungal armory of pest beetles’ protective symbionts. Its non‐canonical multimodular assembly line employs an unprecedented polyketide chain termination reaction to form a butenolide ring, as demonstrated by isotope labeling and CRISPR‐Cas gene editing.

A spider‐transmitted fungus (Rhizopus microsporus) 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 (lobatamide, oximidine, apicularen) in various other bacterial genera. Knowledge of the biosynthetic pathway enables biosynthetic engineering and genome‐mining approaches., Spider sense: The fungus Rhizopus microsporus, isolated from necrotic tissue resulting from a spider bite, was found to contain bacteria (Burkholderia sp.) that produce a complex of highly cytotoxic compounds (necroximes). Analysis of the PKS/NRPS hybrid assembly line and genome mining revealed the molecular basis of the biosynthesis of V‐ATPase‐inhibiting antitumor agents from diverse biological origins.

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 identified a cryptic NRPS gene cluster that is conserved among all sequenced Rhizopus endosymbionts. Metabolic profiling and targeted gene inactivation led to the discovery 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) influences the formation of mature biofilms 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.

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 and metabolic profiling 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 different 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.