The Alphaproteobacterium Rhizobium radiobacter F4 (RrF4) was originally characterized as an endofungal bacterium in the beneficial endophytic Sebacinalean fungus Piriformospora indica. Although attempts to cure P. indica from RrF4 repeatedly failed, the bacterium can easily be grown in pure culture. Here, we report on RrF4's genome and the beneficial impact the free-living bacterium has on plants. In contrast to other endofungal bacteria, the genome size of RrF4 is not reduced. Instead, it shows a high degree of similarity to the plant pathogenic R. radiobacter (formerly: Agrobacterium tumefaciens) C58, except vibrant differences in both the tumor-inducing (pTi) and the accessor (pAt) plasmids, which can explain the loss of RrF4's pathogenicity. Similar to its fungal host, RrF4 colonizes plant roots without host preference and forms aggregates of attached cells and dense biofilms at the root surface of maturation zones. RrF4-colonized plants show increased biomass and enhanced resistance against bacterial leaf pathogens. Mutational analysis showed that, similar to P. indica, resistance mediated by RrF4 was dependent on the plant's jasmonate-based induced systemic resistance (ISR) pathway. Consistent with this, RrF4-and P. indica-induced pattern of defense gene expression were similar. In clear contrast to P. indica, but similar to plant growth-promoting rhizobacteria, RrF4 colonized not only the root outer cortex but also spread beyond the endodermis into the stele. On the basis of our findings, RrF4 is an efficient plant growth-promoting bacterium.

The chemical analysis of insect-associated Actinobacteria has attracted the interest of natural product chemists in the past years as bacterial-produced metabolites are sought to be crucial for sustaining and protecting the insect host. The objective of our study was to evaluate the phylogeny and bioprospecting of Actinobacteria associated with fungus-growing termites. We characterized 97 Actinobacteria from the gut, exoskeleton, and fungus garden (comb) of the fungus-growing termite Macrotermes natalensis and used two different bioassays to assess their general antimicrobial activity. We selected two strains for chemical analysis and investigated the culture broth of the axenic strains and fungus-actinobacterium co-cultures. From these studies, we identified the previously-reported PKS-derived barceloneic acid A and the PKS-derived rubterolones. Analysis of culture broth yielded a new dichlorinated diketopiperazine derivative and two new tetracyclic lanthipeptides, named rubrominins A and B. The discussed natural products highlight that insect-associated Actinobacteria are highly prolific natural product producers yielding important chemical scaffolds urgently needed for future drug development programs.

SummaryHere we review discoveries of secondary metabolites from microbes associated with insects. We mainly focus on natural products, where the ecological role has been at least partially elucidated, and/or the pharmaceutical properties evaluated, and on compounds with unique structural features. We demonstrate that the exploration of specific microbial–host interactions, in combination with multidisciplinary dereplication processes, has emerged as a successful strategy to identify novel chemical entities and to shed light on the ecology and evolution of defensive associations.

Author contributions and conflict of interest Daniel Braga performed research, analyzed data (molecular biology, malachite green assay, mass spectrometry) and contributed to writing the manuscript, Daniel Last performed research and analyzed data (structure elucidation, Fno assay, biogas plant studies), Mahmudul Hasan performed research (CofC/D enzyme assays), Huijuan Guo performed research and analyzed data (structure elucidation), Daniel Leichnitz performed research (chemical synthesis), Zerrin Uzum performed research (microscopy), Ingrid Richter performed research (microscopy), Felix Schalk performed research (cofE constructs), Christine Beemelmanns designed research, acquired funding, analyzed data (structure elucidation, synthesis) and edited the manuscript, Christian Hertweck designed research, acquired funding and edited the manuscript, Gerald Lackner designed the study, acquired funding and wrote the original manuscript. The authors declare no conflict of interest.

This review on natural products containing a tropolonoid motif highlights analytical methods applied for structural identification and biosynthetic pathway analysis, the ecological context and the pharmacological potential of this compound class.