Discovery and Biosynthesis of Anthrochelin, a Growth-Promoting Metallophore of the Human Pathogen Luteibacter anthropi.

Various human pathogens have emerged from environmental strains by adapting to higher growth temperatures and the ability to produce virulence factors. A remarkable example of a pathoadapted bacterium is found in the genus Luteibacter, which typically comprises harmless soil microbes, yet Luteibacter anthropi was isolated from the blood of a diseased child. Up until now, nothing has been known about the specialized metabolism of this pathogen. By comparative genome analyses we found that L. anthropi has a markedly higher biosynthetic potential than other bacteria of this genus and uniquely bears an NRPS gene locus tentatively coding for the biosynthesis of a metallophore. By metabolic profiling, stable isotope labeling, and NMR investigation of a gallium complex, we identified a new family of salicylate-derived nonribosomal peptides named anthrochelins A-D. Surprisingly, anthrochelins feature a C-terminal homocysteine tag, which might be introduced during peptide termination. Mutational analyses provided insight into the anthrochelin assembly and revealed the unexpected involvement of a cytochrome P450 monooxygenase in oxazole formation. Notably, this heterocycle plays a key role in the binding of metals, especially copper(II). Bioassays showed that anthrochelin significantly promotes the growth of L. anthropi in the presence of low and high copper concentrations, which occur during infections.

Toxin-Producing Endosymbionts Shield Pathogenic Fungus against Micropredators.

The fungus Rhizopus microsporus harbors a bacterial endosymbiont (Mycetohabitans rhizoxinica) for the production of the antimitotic toxin rhizoxin. Although rhizoxin is the causative agent of rice seedling blight, the toxinogenic bacterial-fungal alliance is, not restricted to the plant disease. It has been detected in numerous environmental isolates from geographically distinct sites covering all five continents, thus raising questions regarding the ecological role of rhizoxin beyond rice seedling blight. Here, we show that rhizoxin serves the fungal host in fending off protozoan and metazoan predators. Fluorescence microscopy and coculture experiments with the fungivorous amoeba Protostelium aurantium revealed that ingestion of R. microsporus spores is toxic to P. aurantium. This amoebicidal effect is caused by the dominant bacterial rhizoxin congener rhizoxin S2, which is also lethal toward the model nematode Caenorhabditis elegans. By combining stereomicroscopy, automated image analysis, and quantification of nematode movement, we show that the fungivorous nematode Aphelenchus avenae actively feeds on R. microsporus that is lacking endosymbionts, whereas worms coincubated with symbiotic R. microsporus are significantly less lively. This study uncovers an unexpected ecological role of rhizoxin as shield against micropredators. This finding suggests that predators may function as an evolutionary driving force to maintain toxin-producing endosymbionts in nonpathogenic fungi. IMPORTANCE The soil community is a complex system characterized by predator-prey interactions. Fungi have developed effective strategies to defend themselves against predators. Understanding these strategies is of critical importance for ecology, medicine, and biotechnology. In this study, we shed light on the defense mechanisms of the phytopathogenic Rhizopus-Mycetohabitans symbiosis that has spread worldwide. We report an unexpected role of rhizoxin, a secondary metabolite produced by the bacterium M. rhizoxinica residing within the hyphae of R. microsporus. We show that this bacterial secondary metabolite is utilized by the fungal host to successfully fend off fungivorous protozoan and metazoan predators and thus identified a fundamentally new function of this infamous cytotoxic compound. This endosymbiont-dependent predator defense illustrates an unusual strategy employed by fungi that has broader implications, since it may serve as a model for understanding how animal predation acts as an evolutionary driving force to maintain endosymbionts in nonpathogenic fungi.

Endofungal bacteria boost anthelminthic host protection with the biosurfactant symbiosin.

Effective protection of soil fungi from predators is crucial for their survival in the niche. Thus, fungi have developed efficient defence strategies. We discovered that soil beneficial Mortierella fungi employ a potent cytotoxin (necroxime) against fungivorous nematodes. Interestingly, this anthelminthic agent is produced by bacterial endosymbionts (Candidatus Mycoavidus necroximicus) residing within the fungus. Analysis of the symbiont's genome indicated a rich biosynthetic potential, yet nothing has been known about additional metabolites and their potential synergistic functions. Here we report that two distinct Mortierella endosymbionts produce a novel cyclic lipodepsipeptide (symbiosin), that is clearly of bacterial origin, but has striking similarities to various fungal specialized metabolites. The structure and absolute configuration of symbiosin were fully elucidated. By comparative genomics of symbiosin-positive strains and in silico analyses of the deduced non-ribosomal synthetases, we assigned the (sym) biosynthetic gene cluster and proposed an assembly line model. Bioassays revealed that symbiosin is not only an antibiotic, in particular against mycobacteria, but also exhibits marked synergistic effects with necroxime in anti-nematode tests. By functional analyses and substitution experiments we found that symbiosin is a potent biosurfactant and that this particular property confers a boost in the anthelmintic action, similar to formulations of therapeutics in human medicine. Our findings illustrate that "combination therapies" against parasites already exist in ecological contexts, which may inspire the development of biocontrol agents and therapeutics.

Bacterial endosymbionts protect beneficial soil fungus from nematode attack.

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.

An Unexpected Split-Merge Pathway in the Assembly of the Symmetric Nonribosomal Peptide Antibiotic Closthioamide.

Closthioamide (CTA) is a symmetric nonribosomal peptide (NRP) comprised of two diaminopropane-linked polythioamidated monomers. CTA is biosynthesized by Ruminiclostridium cellulolyticum via an atypical NRP synthetase (NRPS)-independent biosynthetic pathway. Although the logic for monomer assembly was recently elucidated, the strategy for the biosynthesis and incorporation of the diamine linker remained a mystery. By means of genome editing, synthesis, and in vitro biochemical assays, we demonstrate that the final steps in CTA maturation proceed through a surprising split-merge pathway involving the dual use of a thiotemplated intermediate. This pathway includes the first examples of an aldo-keto reductase catalyzing the reductive release of a thiotemplated product, and of a transthioamidating transglutaminase. In addition to clarifying the remaining steps in CTA assembly, our data shed light on largely unexplored pathways for NRPS-independent peptide biosynthesis.

Genome Editing Reveals Novel Thiotemplated Assembly of Polythioamide Antibiotics in Anaerobic Bacteria.

Closthioamide (CTA) is a unique symmetric nonribosomal peptide with six thioamide moieties that is produced by the Gram-positive obligate anaerobe Ruminiclostridium cellulolyticum. CTA displays potent inhibitory activity against important clinical pathogens, making it a promising drug candidate. Yet, the biosynthesis of this DNA gyrase-targeting antibiotic has remained enigmatic. Using a combination of genome mining, genome editing (targeted group II intron, CRISPR/Cas9), and heterologous expression, we show that CTA biosynthesis involves specialized enzymes for starter unit biosynthesis, amide bond formation, thionation, and dimerization. Surprisingly, CTA biosynthesis involves a novel thiotemplated peptide assembly line that markedly differs from known nonribosomal peptide synthetases. These findings provide the first insights into the biosynthesis of thioamide-containing nonribosomal peptides and offer a starting point for the discovery of related natural products.