Actin-targeting macrolides comprise a large, structurally diverse group of cytotoxins isolated from remarkably dissimilar micro- and macroorganisms. In spite of their disparate origins and structures, many of these compounds bind actin at the same site and exhibit structural relationships reminiscent of modular, combinatorial drug libraries. Here we investigate biosynthesis and evolution of three compound groups: misakinolides, scytophycin-type compounds and luminaolides. For misakinolides from the sponge Theonella swinhoei WA, our data suggest production by an uncultivated 'Entotheonella' symbiont, further supporting the relevance of these bacteria as sources of bioactive polyketides and peptides in sponges. Insights into misakinolide biosynthesis permitted targeted genome mining for other members, providing a cyanobacterial luminaolide producer as the first cultivated source for this dimeric compound family. The data indicate that this polyketide family is bacteria-derived and that the unusual macrolide diversity is the result of combinatorial pathway modularity for some compounds and of convergent evolution for others.

The as-yet uncultured filamentous bacteria "Candidatus Entotheonella factor" and "Candidatus Entotheonella gemina" live associated with the marine sponge Theonella swinhoei Y, the source of numerous unusual bioactive natural products. Belonging to the proposed candidate phylum "Tectomicrobia," Candidatus Entotheonella members are only distantly related to any cultivated organism. The Ca. E. factor has been identified as the source of almost all polyketide and modified peptides families reported from the sponge host, and both Ca. Entotheonella phylotypes contain numerous additional genes for as-yet unknown metabolites. Here, we provide insights into the biology of these remarkable bacteria using genomic, (meta)proteomic, and chemical methods. The data suggest a metabolic model of Ca. Entotheonella as facultative anaerobic, organotrophic organisms with the ability to use methanol as an energy source. The symbionts appear to be auxotrophic for some vitamins, but have the potential to produce most amino acids as well as rare cofactors like coenzyme F 420 . The latter likely accounts for the strong autofluorescence of Ca. Entotheonella filaments. A large expansion of protein families involved in regulation and conversion of organic molecules indicates roles in host-bacterial interaction. In addition, a massive overrepresentation of members of the luciferase-like monooxygenase superfamily points toward an important role of these proteins in Ca. Entotheonella. Furthermore, we performed mass spectrometric imaging combined with fluorescence in situ hybridization to localize Ca. Entotheonella and some of the bioactive natural products in the sponge tissue. These metabolic insights into a new candidate phylum offer hints on the targeted cultivation of the chemically most prolific microorganisms known from microbial dark matter.

Summary Intracellular replication of the deadly pathogen Mycobacterium tuberculosis relies on the production of small organic molecules called siderophores to scavenge iron from host proteins 1 . M. tuberculosis produces two classes of siderophores, lipid-bound mycobactin and soluble carboxymycobactin 2 , 3 . Functional studies revealed that iron-loaded carboxymycobactin is imported into the cytoplasm by the ABC transporter IrtAB 4 , which features an additional cytoplasmic siderophore interaction domain (SID) 5 . However, IrtAB’s predicted ABC exporter fold seemingly contradicts its import function. Here, we show that membrane-reconstituted IrtAB is sufficient to import mycobactins, which are then reduced by the SID to facilitate iron release. Structure determination by X-ray crystallography and cryo-EM confirms IrtAB’s ABC exporter fold, but also reveals structural peculiarities at the transmembrane region of IrtAB resulting in a partially collapsed inward-facing substrate binding cavity. The SID is positioned in close proximity to the inner membrane leaflet, which allows the reduction of membrane-inserted mycobactin. Enzymatic ATPase activity and in vivo growth assays show that IrtAB prefers mycobactin over carboxymycobactin as its substrate. Our study provides insights into an unusual ABC exporter that evolved as highly specialized siderophore import machinery in mycobacteria.

The polytheonamides are among the most complex and biosynthetically distinctive natural products known to date. These potent peptide cytotoxins are derived from a ribosomal precursor processed by 49 mostly non-canonical posttranslational modifications. Since the producer is a "microbial dark matter" bacterium only distantly related to any cultivated organism, >70-step chemical syntheses have been developed to access these unique compounds. Here we mined prokaryotic diversity to establish a synthetic platform based on the new host Microvirgula aerodenitrificans that produces hypermodified peptides within two days. Using this system, we generated the aeronamides, new polytheonamide-type compounds with near-picomolar cytotoxicity. Aeronamides, as well as the polygeonamides produced from deep-rock biosphere DNA, contain the highest numbers of D-amino acids in known biomolecules. With increasing bacterial genomes being sequenced, similar host mining strategies might become feasible to access further elusive natural products from uncultivated life.