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.

The discovery of six new, highly substituted tropolone alkaloids, rubterolones A-F, from Actinomadura sp. 5-2, isolated from the gut of the fungus-growing termite Macrotermes natalensis is reported. Rubterolones were identified by using fungus-bacteria challenge assays and a HRMS-based dereplication strategy, and characterised by NMR and HRMS analyses and by X-ray crystallography. Feeding experiments and subsequent chemical derivatisation led to a first library of rubterolone derivatives (A-L). Genome sequencing and comparative analyses revealed their putative biosynthetic pathway, which was supported by feeding experiments. This study highlights how gut microbes can present a prolific source of secondary metabolites.

In constructed wetlands, organic pollutants are mainly degraded via microbial processes. Helophytes, plants that are commonly used in these systems, provide oxygen and root exudates to the rhizosphere, stimulating microbial degradation. While the treatment performance of constructed wetlands can be remarkable, a mechanistic understanding of microbial degradation processes in the rhizosphere is still limited. We investigated microbial toluene removal in a constructed wetland model system combining 16S rRNA gene sequencing, metaproteomics and (13) C-toluene in situ protein-based stable isotope probing (protein-SIP). The rhizospheric bacterial community was dominated by Burkholderiales and Rhizobiales, each contributing about 20% to total taxon abundance. Protein-SIP data revealed that the members of Burkholderiaceae, the proteins of which showed about 73% of (13) C-incorporation, were the main degraders of toluene in the planted system, while the members of Comamonadaceae were involved to a lesser extent in degradation (about 64% (13) C-incorporation). Among the Burkholderiaceae, one of the key players of toluene degradation could be assigned to Ralstonia pickettii. We observed that the main pathway of toluene degradation occurred via two subsequent monooxygenations of the aromatic ring. Our study provides a suitable approach to assess the key processes and microbes that are involved in the degradation of organic pollutants in complex rhizospheric ecosystems.

Three new isoflavonoid glycosides, termisoflavones A-C (1-3), and eight isoflavonoids (4-11) were isolated from termite-associated Streptomyces sp. RB1 recovered from the cuticle of the South African termite, Macrotermes natalensis. The structures of new compounds were determined by spectroscopic methods including 1D and 2D NMR and HR-MS analysis, as well as comparison of their NMR data with those of related isoflavonoid glycoside derivatives. The absolute configurations of the sugar moieties were clarified by chemical reactions. None of the isolates (1-11) displayed antifungal or antimicrobial activities (MICs > 100 μg/mL), whereas compounds 6 and 11 ameliorated cisplatin-induced kidney cell damage to 80% of the control value at a cisplatin dose of 25 μM.

Actinobacteria, one of the largest bacterial phyla, are ubiquitous in many of Earth’s ecosystems and often act as defensive symbionts with animal hosts. Members of the phylum have repeatedly been isolated from basidiomycete-cultivating fungus-farming termites that maintain a monoculture fungus crop on macerated dead plant substrate. The proclivity for antimicrobial and enzyme production of Actinobacteria make them likely contributors to plant decomposition and defense in the symbiosis. To test this, we analyzed the prophylactic (biosynthetic gene cluster [BGC]) and metabolic (carbohydrate-active enzyme [CAZy]) potential in 16 (10 existing and six new genomes) termite-associated Actinobacteria and compared these to the soil-dwelling close relatives. Using antiSMASH, we identified 435 BGCs, of which 329 (65 unique) were similar to known compound gene clusters, while 106 were putatively novel, suggesting ample prospects for novel compound discovery. BGCs were identified among all major compound categories, including 26 encoding the production of known antimicrobial compounds, which ranged in activity (antibacterial being most prevalent) and modes of action that might suggest broad defensive potential. Peptide pattern recognition analysis revealed 823 (43 unique) CAZymes coding for enzymes that target key plant and fungal cell wall components (predominantly chitin, cellulose, and hemicellulose), confirming a substantial degradative potential of these bacteria. Comparison of termite-associated and soil-dwelling bacteria indicated no significant difference in either BGC or CAZy potential, suggesting that the farming termite hosts may have coopted these soil-dwelling bacteria due to their metabolic potential but that they have not been subject to genome change associated with symbiosis. IMPORTANCE Actinobacteria have repeatedly been isolated in fungus-farming termites, and our genome analyses provide insights into the potential roles they may serve in defense and for plant biomass breakdown. These insights, combined with their relatively higher abundances in fungus combs than in termite gut, suggest that they are more likely to play roles in fungus combs than in termite guts. Up to 25% of the BGCs we identify have no similarity to known clusters, indicating a large potential for novel chemistry to be discovered. Similarities in metabolic potential of soil-dwelling and termite-associated bacteria suggest that they have environmental origins, but their consistent presence with the termite system suggests their importance for the symbiosis.

In recent years, investigations into the biochemistry of insect-associated bacteria have increased. When combined with analytical dereplication processes, these studies provide a powerful strategy to identify structurally and/or biologically novel compounds. Non-ribosomally synthesized cyclic peptides have a broad bioactivity spectrum with high medicinal potential. Here, we report the discovery of three new cyclic tripeptides: natalenamides A–C (compounds 1–3). These compounds were identified from the culture broth of the fungus-growing termite-associated Actinomadura sp. RB99 using a liquid chromatography (LC)/ultraviolet (UV)/mass spectrometry (MS)-based dereplication method. Chemical structures of the new compounds (1–3) were established by analysis of comprehensive spectroscopic methods, including one-dimensional (1H and 13C) and two-dimensional (1H-1H-COSY, HSQC, HMBC) nuclear magnetic resonance spectroscopy (NMR), together with high-resolution electrospray ionization mass spectrometry (HR-ESIMS) data. The absolute configurations of the new compounds were elucidated using Marfey’s analysis. Through several bioactivity tests for the tripeptides, we found that compound 3 exhibited significant inhibitory effects on 3-isobutyl-1-methylxanthine (IBMX)-induced melanin production. The effect of compound 3 was similar to that of kojic acid, a compound extensively used as a cosmetic material with a skin-whitening effect.

The taxonomic positions of two novel aerobic, Gram-positive actinobacteria, designated strains RB29T and RB68T, were determined using a polyphasic approach. Based on 16S rRNA gene sequence analysis, the closest phylogenetic neighbours of RB29T were identified as Actinomadura rayongensis DSM 102126T (99.2 % similarity) and Actinomadura atramentaria DSM 43919T (98.7 %), and for strain RB68T was Actinomadura hibisca DSM 44148T (98.3 %). Digital DNA–DNA hybridization (dDDH) between RB29T and its closest phylogenetic neighbours, A. rayongensis DSM 102126T and A. atramentaria DSM 43919T, resulted in similarity values of 53.2 % (50.6–55.9 %) and 26.4 % (24.1–28.9 %), respectively. Additionally, the average nucleotide identity (ANI) was 93.2 % (94.0 %) for A. rayongensis DSM 102126T and 82.3 % (78.9 %) for A. atramentaria DSM 43919T. dDDH analysis between strain RB68T and A. hibisca DSM 44148T gave a similarity value of 24.5 % (22.2–27.0 %). Both strains, RB29T and RB68T, revealed morphological characteristics and chemotaxonomic features typical for the genus Actinomadura , such as the presence of meso-diaminopimelic acid in the cell wall, galactose and glucose as major sugar components within whole-cell hydrolysates and the absence of mycolic acids. The major phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside. Predominant menaquinones were MK-9(H6) and MK-9(H8) for RB29T and MK-9(H4) and MK-9(H6) for RB68T. The main fatty acids were identified as 10-methyloctadecanoic acid (10-methyl C18:0), 14-methylpentadecanoic acid (iso-C16:0), hexadecanoic acid (C16:0) and cis-9-octadecanoic acid (C18 : 1 ω9c). Here, we propose two novel species of the genus Actinomadura : Actinomadura rubteroloni sp. nov. with the type strain RB29T (=CCUG 72668T=NRRL B-65537T) and Actinomadura macrotermitis sp. nov. with the type strain RB68T (=CCUG 72669T=NRRL B-65538T).

Growth from spores activated a biosynthetic gene cluster in Actinomadura sp. RB29, resulting in the identification of two novel groups of halogenated polyketide natural products, named maduralactomycins and actinospirols. The unique tetracyclic and spirocyclic structures were assigned based on a combination of NMR analysis, chemoinformatic calculations, X-ray crystallography, and 13 C labeling studies. On the basis of HRMS 2 data, genome mining, and gene expression studies, we propose an underlying noncanonical angucycline biosynthesis and extensive post-polyketide synthase (PKS) oxidative modifications.