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Sphingoid bases are important bioactive lipids found in a variety of organisms, serving as the backbone of sphingolipids, which regulate essential physiological processes. Here we describe the total synthesis and structure revision of halisphingosine A, a sphingoid base initially isolated from marine sponges. To address inconsistencies in the NMR interpretation of this natural product, we developed a synthetic route involving a late-stage enantioselective Henry reaction that allows access to multiple stereoisomers of the proposed halisphingosine core structure. Our library of 32 fully characterized synthetic stereoisomers enabled us to rectify the structure of halisphingosine A as (2R,3R,8R,Z)-2-aminooctadec-9-ene-1,3,8-triol, and to pursue further structure-activity relation (SAR) studies regarding their antimicrobial and cytotoxic potential. In summary, our study offers a yet unreported compound library along with validated analytical datasets of marine sphingoid base derivatives, which significantly affects future ecometabolomic marine research and will facilitate the identification of inhibitors of sphingolipid metabolism or antagonists of sphingolipid base-sensing receptors., (© 2024 Wiley‐VCH GmbH.)

The use of compounds produced by hosts or symbionts for defence against antagonists has been identified in many organisms, including in fungus-farming termites (Macrotermitinae). The obligate mutualistic fungus Termitomyces plays a pivotal role in plant biomass decomposition and as the primary food source for these termites. Despite the isolation of various specialized metabolites from different Termitomyces species, our grasp of their natural product repertoire remains incomplete. To address this knowledge gap, we conducted a comprehensive analysis of 39 Termitomyces genomes, representing 21 species associated with members of five termite host genera. We identified 754 biosynthetic gene clusters (BGCs) coding for specialized metabolites and categorized 660 BGCs into 61 biosynthetic gene cluster families (GCFs) spanning five compound classes. Seven GCFs were shared by all 21 Termitomyces species and 21 GCFs were present in all genomes of subsets of species. Evolutionary constraint analyses on the 25 most abundant GCFs revealed distinctive evolutionary histories, signifying that millions of years of termite-fungus symbiosis have influenced diverse biosynthetic pathways. This study unveils a wealth of non-random and largely undiscovered chemical potential within Termitomyces and contributes to our understanding of the intricate evolutionary trajectories of biosynthetic gene clusters in the context of long-standing symbiosis., (© 2024. The Author(s).)

Uropygial gland secretions of birds consist of host and bacteria derived compounds and play a major sanitary and feather-protective role. Here we report on our microbiome studies of the New Guinean toxic bird Pachycephala schlegelii and the isolation of a member of the Amycolatopsis genus from the uropygial gland secretions. Bioactivity studies in combination with co-cultures, MALDI imaging and HR-MS/MS-based network analyses unveil the basis of its activity against keratinolytic bacteria and fungal skin pathogens. We trace the protective antimicrobial activity of Amycolatopsis sp. PS_44_ISF1 to the production of rifamycin congeners, ciromicin A and of two yet unreported compound families. We perform NMR and HR-MS/MS studies to determine the relative structures of six members belonging to a yet unreported lipopeptide family of pachycephalamides and of one representative of the demiguisins, a new hexapeptide family. We then use a combination of phylogenomic, transcriptomic and knock-out studies to identify the underlying biosynthetic gene clusters responsible for the production of pachycephalamides and demiguisins. Our metabolomics data allow us to map molecular ion features of the identified metabolites in extracts of P. schlegelii feathers, verifying their presence in the ecological setting where they exert their presumed active role for hosts. Our study shows that members of the Actinomycetota may play a role in avian feather protection., (© 2024. The Author(s).)

The human microbiome emerges as a promising reservoir for diagnostic markers and therapeutics. Since host-associated microbiomes at various body sites differ and diseases do not occur in isolation, a comprehensive analysis strategy highlighting the full potential of microbiomes should include diverse specimen types and various diseases. To ensure robust data quality and comparability across specimen types and diseases, we employ standardized protocols to generate sequencing data from 1931 prospectively collected specimens, including from saliva, plaque, skin, throat, eye, and stool, with an average sequencing depth of 5.3 gigabases. Collected from 515 patients, these samples yield an average of 3.7 metagenomes per patient. Our results suggest significant microbial variations across diseases and specimen types, including unexpected anatomical sites. We identify 583 unexplored species-level genome bins (SGBs) of which 189 are significantly disease-associated. Of note, the existence of microbial resistance genes in one specimen was indicative of the same resistance genes in other specimens of the same patient. Annotated and previously undescribed SGBs collectively harbor 28,315 potential biosynthetic gene clusters (BGCs), with 1050 significant correlations to diseases. Our combinatorial approach identifies distinct SGBs and BGCs, emphasizing the value of pan-body pan-disease microbiomics as a source for diagnostic and therapeutic strategies., (© 2024. The Author(s).)

Feature-based molecular networking (FBMN) is a popular analysis approach for liquid chromatography-tandem mass spectrometry-based non-targeted metabolomics data. While processing liquid chromatography-tandem mass spectrometry data through FBMN is fairly streamlined, downstream data handling and statistical interrogation are often a key bottleneck. Especially users new to statistical analysis struggle to effectively handle and analyze complex data matrices. Here we provide a comprehensive guide for the statistical analysis of FBMN results, focusing on the downstream analysis of the FBMN output table. We explain the data structure and principles of data cleanup and normalization, as well as uni- and multivariate statistical analysis of FBMN results. We provide explanations and code in two scripting languages (R and Python) as well as the QIIME2 framework for all protocol steps, from data clean-up to statistical analysis. All code is shared in the form of Jupyter Notebooks ( https://github.com/Functional-Metabolomics-Lab/FBMN-STATS ). Additionally, the protocol is accompanied by a web application with a graphical user interface ( https://fbmn-statsguide.gnps2.org/ ) to lower the barrier of entry for new users and for educational purposes. Finally, we also show users how to integrate their statistical results into the molecular network using the Cytoscape visualization tool. Throughout the protocol, we use a previously published environmental metabolomics dataset for demonstration purposes. Together, the protocol, code and web application provide a complete guide and toolbox for FBMN data integration, cleanup and advanced statistical analysis, enabling new users to uncover molecular insights from their non-targeted metabolomics data. Our protocol is tailored for the seamless analysis of FBMN results from Global Natural Products Social Molecular Networking and can be easily adapted to other mass spectrometry feature detection, annotation and networking tools., (© 2024. Springer Nature Limited.)

Fungi constitute the Earth's second most diverse kingdom, however only a small percentage of these have been thoroughly examined and categorized for their secondary metabolites, which still limits our understanding of the ecological chemical and pharmacological potential of fungi. In this study, we explored members of the co-evolved termite-associated fungal genus Xylaria and identified a family of highly oxygenated polyketide-terpene hybrid natural products using an MS/MS molecular networking-based dereplication approach. Overall, we isolated six no yet reported xylasporin derivatives, of which xylasporin A (1) features a rare cyclic-carbonate moiety. Extensive comparative spectrometric (HRMS 2 ) and spectroscopic (1D and 2D NMR) studies allowed to determine the relative configuration across the xylasporin family, which was supported by chemical shift calculations of more than 50 stereoisomers and DP4+ probability analyses. The absolute configuration of xylasporin A (1) was also proposed based on TDDFT-ECD calculations. Additionally, we were able to revise the relative and absolute configurations of co-secreted xylacremolide B produced by single x-ray crystallography. Comparative genomic and transcriptomic analysis allowed us to deduce the putative biosynthetic assembly line of xylasporins in the producer strain X802, and could guide future engineering efforts of the biosynthetic pathway., (© 2024. The Author(s).)

The cosmopolitan marine Roseobacter clade is of global biogeochemical importance. Members of this clade produce sulfur-containing amino lipids (SALs) involved in biofilm formation and marine surface colonization processes. Despite their physiological relevance and abundance, SALs have only been explored through genomic mining approaches and lipidomic studies based on mass spectrometry, which left the relative and absolute structures of SALs unresolved, hindering progress in biochemical and functional investigations. Herein, we report the structural revision of a new group of SALs, which we named cysteinolides, using a combination of analytical techniques, isolation and degradation experiments and total synthetic efforts. Contrary to the previously proposed homotaurine-based structures, cysteinolides are composed of an N,O-acylated cysteinolic acid-containing head group carrying various different (α-hydroxy)carboxylic acids. We also performed the first validated targeted-network based analysis, which allowed us to map the distribution and structural diversity of cysteinolides across bacterial lineages. Beyond offering structural insight, our research provides SAL standards and validated analytical data. This information holds significance for forthcoming investigations into bacterial sulfonolipid metabolism and biogeochemical nutrient cycling within marine environments., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Toxicity has evolved multiple times across the tree of life and serves important functions related to hunting, defence and parasite deterrence. Toxins are produced either in situ by the toxic organism itself or associated symbionts, or acquired through diet. The ability to exploit toxins from external sources requires adaptations that prevent toxic effects on the consumer (autoresistance). Here, we examine genomic adaptations that could facilitate autoresistance to the diet-acquired potent neurotoxic alkaloid batrachotoxin (BTX) in New Guinean toxic birds. Our work documents two new toxic bird species and shows that toxic birds carry multiple mutations in the SCN4A gene that are under positive selection. This gene encodes the most common vertebrate muscle Nav channel (Nav1.4). Molecular docking results indicate that some of the mutations that are present in the pore-forming segment of the Nav channel, where BTX binds, could reduce its binding affinity. These mutations should therefore prevent the continuous opening of the sodium channels that BTX binding elicits, thereby preventing muscle paralysis and ultimately death. Although these mutations are different from those present in Neotropical Phyllobates poison dart frogs, they occur in the same segments of the Nav1.4 channel. Consequently, in addition to uncovering a greater diversity of toxic bird species than previously known, our work provides an intriguing example of molecular-level convergent adaptations allowing frogs and birds to ingest and use the same neurotoxin. This suggests that genetically modified Nav1.4 channels represent a key adaptation to BTX tolerance and exploitation across vertebrates., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Background: Although Basidiomycota produce pharmaceutically and ecologically relevant natural products, knowledge of how they coordinate their primary and secondary metabolism is virtually non-existent. Upon transition from vegetative mycelium to carpophore formation, mushrooms of the genus Psilocybe use L-tryptophan to supply the biosynthesis of the psychedelic tryptamine alkaloid psilocybin with the scaffold, leading to a strongly increased demand for this particular amino acid as this alkaloid may account for up to 2% of the dry mass. Using Psilocybe mexicana as our model and relying on genetic, transcriptomic, and biochemical methods, this study investigated if L-tryptophan biosynthesis and degradation in P. mexicana correlate with natural product formation., Results: A comparative transcriptomic approach of gene expression in P. mexicana psilocybin non-producing vegetative mycelium versus producing carpophores identified the upregulation of L-tryptophan biosynthesis genes. The shikimate pathway genes trpE1, trpD, and trpB (encoding anthranilate synthase, anthranilate phosphoribosyltransferase, and L-tryptophan synthase, respectively) were upregulated in carpophores. In contrast, genes idoA and iasA, encoding indole-2,3-dioxygenase and indole-3-acetaldehyde synthase, i.e., gateway enzymes for L-tryptophan-consuming pathways, were massively downregulated. Subsequently, IasA was heterologously produced in Escherichia coli and biochemically characterized in vitro. This enzyme represents the first characterized microbial L-tryptophan-preferring acetaldehyde synthase. A comparison of transcriptomic data collected in this study with prior data of Psilocybe cubensis showed species-specific differences in how L-tryptophan metabolism genes are regulated, despite the close taxonomic relationship., Conclusions: The upregulated L-tryptophan biosynthesis genes and, oppositely, the concomitant downregulated genes encoding L-tryptophan-consuming enzymes reflect a well-adjusted cellular system to route this amino acid toward psilocybin production. Our study has pilot character beyond the genus Psilocybe and provides, for the first time, insight in the coordination of mushroom primary and secondary metabolism., (© 2024. The Author(s).)

Members of the bacterial phylum Planctomycetota have recently emerged as promising and for the most part untapped sources of novel bioactive compounds. The characterization of more than 100 novel species in the last decade stimulated recent bioprospection studies that start to unveil the chemical repertoire of the phylum. In this study, we performed systematic bioinformatic analyses based on the genomes of all 131 described members of the current phylum focusing on the identification of type III polyketide synthase (PKS) genes. Type III PKSs are versatile enzymes involved in the biosynthesis of a wide array of structurally diverse natural products with potent biological activities. We identified 96 putative type III PKS genes of which 58 are encoded in an operon with genes encoding a putative oxidoreductase and a methyltransferase. Sequence similarities on protein level and the genetic organization of the operon point towards a functional link to the structurally related hierridins recently discovered in picocyanobacteria. The heterologous expression of planctomycetal type III PKS genes from strains belonging to different families in an engineered Corynebacterium glutamicum strain led to the biosynthesis of pentadecyl- and heptadecylresorcinols. Phenotypic assays performed with the heterologous producer strains and a constructed type III PKS gene deletion mutant suggest that the natural function of the identified compounds differs from that confirmed in other bacterial alkylresorcinol producers. KEY POINTS: • Planctomycetal type III polyketide synthases synthesize long-chain alkylresorcinols. • Phylogenetic analyses suggest an ecological link to picocyanobacterial hierridins. • Engineered C. glutamicum is suitable for an expression of planctomycete-derived genes., (© 2024. The Author(s).)

After conducting an in silico analysis of the cryptic mdk cluster region and performing transcriptomic studies, an integrative Streptomyces BAC Vector containing the mdk gene sequence was constructed. The heterologous expression of the mdk cluster in Streptomyces albus J1074 resulted in the production of the angucyclic product, seongomycin, which allowed for the assesment of its antibacterial, antiproliferative, and antiviral activities. Heterologous production was further confirmed by targeted knock-out experiments involving key regulators of the biosynthetic pathways. We were further able to revise the core structure of maduralactomycin A, using a computational approach., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

β-Amino acid-containing macrolactams represent a structurally diverse group of bioactive natural products derived from polyketides; however we are currently lacking a comprehensive overview about their abundance across bacterial families and the underlying biosynthetic diversity. In this study, we employed a targeted β-amino acid-specific homology-based multi-query search to identify potential bacterial macrolactam producers. Here we demonstrate that approximately 10% of each of the identified actinobacterial genera harbor a biosynthetic gene cluster (BGC) encoding macrolactam production. Based on our comparative study, we propose that mutations occurring in specific regions of polyketide synthases (PKS) are the primary drivers behind the variation in macrolactam ring sizes. We successfully validated two producers of ciromicin A from the genus Amycolatopsis, revised the composition of the biosynthetic gene cluster region mte of macrotermycins, and confirmed the ciromicin biosynthetic pathway through heterologous expression. Additionally, network-based metabolomic analysis uncovered three previously unreported macrotermycin congeners from Amycolatopsis sp. M39. The combination of targeted mining and network-based analysis serves as a powerful tool for identifying macrolactam producers and our studies will catalyze the future discovery of yet unreported macrolactams., (© 2023. The Author(s).)

The choanoflagellate Salpingoeca rosetta transitions from unicellular to multicellular forms in the presence of bacterial signaling molecules, such as sulfonosphingolipids (RIFs). We set out to characterize the abundance of RIF-like molecules within five different Bacteroidetes strains belonging to different genera. While four strains exhibited similar sulfonosphingolipid profiles with sulfobacin A as the dominant feature, the composition in Z. uliginosa differed distinctively. Targeted isolation yielded four sulfonosphingolipids, including the previously reported flavocristamide A. While none of the sulfonosphingolipids induced rosette formation, a negative impact on choanoflagellate growth and cell density was observed. In contrast, supernatant extracts of Zobellia depleted in sulfonosphingolipid-like features provoked rosette formation in S. rosetta indicating for the presence of yet another morphogenic compound class., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Characterizing ancient clades of fungal symbionts is necessary for understanding the evolutionary process underlying symbiosis development. In this study, we investigated a distinct subgeneric taxon of Xylaria (Xylariaceae), named Pseudoxylaria, whose members have solely been isolated from the fungus garden of farming termites. Pseudoxylaria are inconspicuously present in active fungus gardens of termite colonies and only emerge in the form of vegetative stromata, when the fungus comb is no longer attended ("sit and wait" strategy). Insights into the genomic and metabolic consequences of their association, however, have remained sparse. Capitalizing on viable Pseudoxylaria cultures from different termite colonies, we obtained genomes of seven and transcriptomes of two Pseudoxylaria isolates. Using a whole-genome-based comparison with free-living members of the genus Xylaria, we document that the association has been accompanied by significant reductions in genome size, protein-coding gene content, and reduced functional capacities related to oxidative lignin degradation, oxidative stress responses and secondary metabolite production. Functional studies based on growth assays and fungus-fungus co-cultivations, coupled with isotope fractionation analysis, showed that Pseudoxylaria only moderately antagonizes growth of the termite food fungus Termitomyces, and instead extracts nutrients from the food fungus biomass for its own growth. We also uncovered that Pseudoxylaria is still capable of producing structurally unique metabolites, which was exemplified by the isolation of two novel metabolites, and that the natural product repertoire correlated with antimicrobial and insect antifeedant activity., (© 2023. The Author(s).)

Macrotermitinae termites have farmed fungi in the genus Termitomyces as a food source for millions of years. However, the biochemical mechanisms orchestrating this mutualistic relationship are largely unknown. To deduce fungal signals and ecological patterns that relate to the stability of this symbiosis, we explored the volatile organic compound (VOC) repertoire of Termitomyces from Macrotermes natalensis colonies. Results show that mushrooms emit a VOC pattern that differs from mycelium grown in fungal gardens and laboratory cultures. The abundance of sesquiterpenoids from mushrooms allowed targeted isolation of five drimane sesquiterpenes from plate cultivations. The total synthesis of one of these, drimenol, and related drimanes assisted in structural and comparative analysis of volatile organic compounds (VOCs) and antimicrobial activity testing. Enzyme candidates putatively involved in terpene biosynthesis were heterologously expressed and while these were not involved in the biosynthesis of the complete drimane skeleton, they catalyzed the formation of two structurally related monocyclic sesquiterpenes named nectrianolins., (© 2023. The Author(s).)

Serine palmitoyltransferase catalyzes the first step of the sphingolipid biosynthesis. Recently, sphingolipid homeostasis has been connected to several human diseases, making serine palmitoyltransferases an interesting therapeutic target. Known and efficient serine palmitoyltransferase-inhibitors are sphingofungins, a group of natural products isolated from fungi. To further characterize newly isolated sphingofungins, we designed an easy to use colorimetric serine palmitoyltransferase activity assay using FadD, which can be performed in 96-well plates. Because sphingofungins exert antifungal activitiy as well, we compared the in vitro assay results with an in vivo growth assay using Saccharomyces cerevisiae. The reported experiments showed differences among the assayed sphingofungins, highlighting an increase of activity based on the saturation levels of the polyketide tail. IMPORTANCE Targeting the cellular sphingolipid metabolism is often discussed as a potential approach to treat associated human diseases such as cancer and Alzheimer's disease. Alternatively, it is also a possible target for the development of antifungal compounds, which are direly needed. A central role is played by the serine palmitoyltransferase, which catalyzes the initial and rate limiting step of sphingolipid de novo synthesis and, as such, the development of inhibitory compounds for this enzyme is of interest. Our work here established an alternative approach for determining the activity of serine palmitoyltransferase adding another tool for the validation of its inhibition. We also determined the effect of different modifications to sphingofungins on their inhibitory activity against serine palmitoyltransferase, revealing important differences on said activity against enzymes of bacterial and fungal origin.

The choanoflagellate Salpingoeca rosetta is an important model system to study the evolution of multicellularity. In this study we developed a new, modular, and scalable synthesis of sulfonolipid IOR-1A (six steps, 27 % overall yield), which acts as bacterial inhibitor of rosette formation in S. rosetta. The synthesis features a decarboxylative cross-coupling reaction of a sulfonic acid-containing tartaric acid derivative with alkyl zinc reagents. Synthesis of 15 modified IOR-1A derivatives, including fluorescent and photoaffinity-based probes, allowed quantification of IOR-1A, localization studies within S. rosetta cells, and evaluation of structure-activity relations. In a proof of concept study, an inhibitory bifunctional probe was employed in proteomic profiling studies, which allowed to deduce binding partners in bacteria and S. rosetta. These results showcase the power of synthetic chemistry to decipher the biochemical basis of cell differentiation processes within S. rosetta., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Cultures of a termite-associated and a free-living member of the fungal genus Podaxis , revived from spores maintained in century-old herbarium collections, were analyzed for their insecticidal and antimicrobial effects. Their secondary metabolomes were explored to uncover possible adaptive mechanisms of termite association, and dereplication of LC-HRMS/MS data sets led to the isolation of podaxisterols A-D ( 1 - 4 ), modified ergosterol derivatives that result from a Diels-Alder reaction with endogenous nitrosyl cyanide. Chemical structures were determined based on HRMS/MS and NMR analyses as well as X-ray crystallography. The putative origin of the endogenous fungal nitrosyl cyanide and ergosterol derivatives is discussed based on results obtained from stable isotope experiments and in silico analysis. Our "omics"-driven analysis of this underexplored yet worldwide distributed fungal genus builds a foundation for studies on a potential metabolic adaptations to diverse lifestyles.

Covering: findings from early 1980s until early 2022Microbial-derived cues of marine biofilms induce settlement and metamorphosis of marine organisms, a process responsible for the emergence of diverse flora and fauna in marine habitats. Although this phenomenon is known for more than 80 years, the research field has only recently gained much momentum. Here, we summarize the currently existing biochemical and microbial knowledge about microbial signalling molecules, con-specific signals, and synthetic compounds that induce or prevent recruitment, settlement, and metamorphosis in invertebrate larvae. We discuss the possible modes of action and conclude with perspectives for future research directions in the field of marine chemical ecology.

Herein, we demonstrate the applicability of the 2,5-dimethylpyrrolo unit as a complementary N -protecting group in the highly diastereoselective synthesis of more than 20 different anti -amino alcohols (63-90% yields with up to 20 : 1 dr). Cleavage of the pyrrolo- N -protecting group was accomplished, e.g. in the presence of NH 2 OH under microwave conditions with yields exceeding 80%. The applicability of the protecting groups was further demonstrated by a short total synthesis of the sphinganine-like natural product clavaminol A. The introduction of the N -pyrrolo protecting group also offers the possibility to analyse product mixtures by NMR measurements due to the absence of conformational isomers, which are otherwise common for N -protecting groups.

In this study, we analyzed if Actinomadura sp. RB99 produces siderophores that that could be responsible for the antimicrobial activity observed in co-cultivation studies. Dereplication of high-resolution tandem mass spectrometry (HRMS/MS) and global natural product social molecular networking platform (GNPS) analysis of fungus-bacterium co-cultures resulted in the identification of five madurastatin derivatives (A1, A2, E1, F, and G1), of which were four new derivatives. Chemical structures were unambiguously confirmed by HR-ESI-MS, 1D and 2D NMR experiments, as well as MS/MS data and their absolute structures were elucidated based on Marfey's analysis, DP4+ probability calculation and total synthesis. Structure analysis revealed that madurastatin E1 (2) contained a rare 4-imidazolidinone cyclic moiety and madurastatin A1 (5) was characterized as a Ga 3+ -complex. The function of madurastatins as siderophores was evaluated using the fungal pathogen Cryptococcus neoformans as model organism. Based on homology models, we identified the putative NRPS-based gene cluster region of the siderophores in Actinomadura sp. RB99., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Aromatic prenylated metabolites have important biological roles and activities in all living organisms. Compared to their importance in all domains of life, we know relatively little about their substrate scopes and metabolic functions. Here, we describe a new UbiA-like prenyltransferase (Ptase) Ubi-297 encoded in a conserved operon of several bacterial taxa, including marine Flavobacteria and the genus Sacchromonospora . In silico analysis of Ubi-297 homologs indicated that members of this Ptase group are composed of several transmembrane α-helices and carry a conserved and distinct aspartic-rich Mg 2+ -binding domain. We heterologously produced UbiA-like Ptases from the bacterial genera Maribacter , Zobellia , and Algoriphagus in Escherichia coli . Investigation of their substrate scope uncovered the preferential farnesylation of quinoline derivatives, such as 8-hydroxyquinoline-2-carboxylic acid (8-HQA) and quinaldic acid. The results of this study provide new insights into the abundance and diversity of Ptases in marine Flavobacteria and beyond., (Copyright © 2022, Amiri Moghaddam et al.)

δ-Hydroxy-β-keto esters and δ,β-dihydroxy esters are characteristic structural motifs of statin-type natural products and drug candidates. Here, we describe the synthesis of functionalized δ-hydroxy-β-keto esters in good yields and excellent enantioselectivities using Chan's diene and modified Mukaiyama-aldol reaction conditions. Diastereoselective reduction of δ,β-dihydroxy esters afforded the respective syn- and anti-diols, and saponification yielded the corresponding acids. All products were evaluated for their anti-inflammatory properties, which uncovered a surprising structure-activity relationship., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Covering: September 1972 to December 2020Explorations of complex symbioses have often elucidated a plethora of previously undescribed chemical compounds that may serve ecological functions in signalling, communication or defence. A case in point is the subfamily of termites that cultivate a fungus as their primary food source and maintain complex bacterial communities, from which a series of novel compound discoveries have been made. Here, we summarise the origins and types of 375 compounds that have been discovered from the symbiosis over the past four decades and discuss the potential for synergistic actions between compounds within the complex chemical mixtures in which they exist. We go on to highlight how vastly underexplored the diversity and geographic distribution of the symbiosis is, which leaves ample potential for natural product discovery of compounds of both ecological and medical importance.

Macrotermitinae termites have domesticated fungi of the genus Termitomyces as food for their colony, analogously to human farmers growing crops. Termites propagate the fungus by continuously blending foraged and predigested plant material with fungal mycelium and spores (fungus comb) within designated subterranean chambers. To test the hypothesis that the obligate fungal symbiont emits specific volatiles (odor) to orchestrate its life cycle and symbiotic relations, we determined the typical volatile emission of fungus comb biomass and Termitomyces nodules, revealing α-pinene, camphene, and d-limonene as the most abundant terpenes. Genome mining of Termitomyces followed by gene expression studies and phylogenetic analysis of putative enzymes related to secondary metabolite production encoded by the genomes uncovered a conserved and specific biosynthetic repertoire across strains. Finally, we proved by heterologous expression and in vitro enzymatic assays that a highly expressed gene sequence encodes a rare bifunctional mono-/sesquiterpene cyclase able to produce the abundant comb volatiles camphene and d-limonene. IMPORTANCE The symbiosis between macrotermitinae termites and Termitomyces is obligate for both partners and is one of the most important contributors to biomass conversion in the Old World tropic's ecosystems. To date, research efforts have dominantly focused on acquiring a better understanding of the degradative capabilities of Termitomyces to sustain the obligate nutritional symbiosis, but our knowledge of the small-molecule repertoire of the fungal cultivar mediating interspecies and interkingdom interactions has remained fragmented. Our omics-driven chemical, genomic, and phylogenetic study provides new insights into the volatilome and biosynthetic capabilities of the evolutionarily conserved fungal genus Termitomyces , which allows matching metabolites to genes and enzymes and, thus, opens a new source of unique and rare enzymatic transformations.

Sphingofungins belong to a group of structurally related sphingolipid inhibitors produced by fungi, which specifically inhibit serine palmitoyl transferases, enzymes catalyzing the initial step during sphingolipid biosynthesis. Sphingolipids are integral parts of the eukaryotic cell membrane, and disturbances in their homeostasis have been linked to various human diseases. It has been suggested that external interventions, via sphingolipid inhibitors, may represent a promising approach for alternative therapies. Here, we identified and elucidated the biosynthetic gene cluster responsible for the biosynthesis of sphingofungins B, C, and D in Aspergillus fumigatus . Moreover, in vitro analyses have shown that sphingofungin biosynthesis starts with the condensation of a C18 polyketide with the uncommon substrate aminomalonate. Furthermore, the investigations on sphingofungin E and F produced by Paecilomyces variotii pointed out that different aminomalonate derivatives are used as substrates for those chemical variants. This research boosts knowledge on the general biosynthesis of sphingolipid inhibitors in fungi.

We have analyzed the abundance of bacterial sulfonosphingolipids, including rosette-inducing factors (RIFs), in seven bacterial prey strains by using high-resolution tandem mass spectrometry (HRMS 2 ) and molecular networking (MN) within the Global Natural Product Social Molecular Networking (GNPS) web platform. Six sulfonosphingolipids resembling RIFs were isolated and their structures were elucidated based on comparative MS and NMR studies. Here, we also report the first total synthesis of two RIF-2 diastereomers and one congener in 15 and eight synthetic steps, respectively. For the total synthesis of RIF-2 congeners, we employed a decarboxylative cross-coupling reaction to synthesize the necessary branched α-hydroxy fatty acids, and the Garner-aldehyde approach to generate the capnine base carrying three stereogenic centers. Bioactivity studies in the choanoflagellate Salpingoeca rosetta revealed that the rosette inducing activity of RIFs is inhibited dose dependently by the co-occurring sulfonosphingolipid sulfobacins D and F and that activity of RIFs is specific for isolates obtained from Algoriphagus., (© 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Sphingofungins are fungal natural products known to inhibit the biosynthesis of sphingolipids which play pivotal roles in various cell functions. Here, we report a short and flexible synthetic approach towards the sphingofungin family. Key step of the synthesis was a decarboxylative cross-coupling reaction of chiral sulfinyl imines with a functionalized tartaric acid derivative, which yielded the core motif of sphingofungins carrying four consecutive stereocenters and a terminal double bond. Subsequent metathesis reaction allowed for the introduction of different side chains of choice resulting in a total of eight sphingofungins, including for the first time sphingofungin C (eight steps from commercially available protected tartaric acid with an overall yield of 6 %) and sphingofungin A (ten steps). All newly synthesized derivatives were tested for their antifungal, cell-proliferative and antiparasitic activity unraveling their structure-activity relations., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Fungus-farming termites host gut microbial communities that contribute to the pre-digestion of plant biomass for manuring the fungal mutualist, and potentially to the production of defensive compounds that suppress antagonists. Termite colonies are characterized by complex division of labor and differences in diet between termite size (minor and major) and morphological (worker and soldier) castes, and this extends to the composition of their gut microbial communities. We hypothesized that gut metabolomes should mirror these differences and tested this through untargeted LC-MS/MS analyses of three South African species of fungus-farming termites. We found distinct metabolomes between species and across castes, especially between soldiers and workers. Primary metabolites dominate the metabolomes and the high number of overlapping features with the mutualistic fungus and plant material show distinct impacts of diet and the environment. The identification of a few bioactive compounds of likely microbial origin underlines the potential for compound discovery among the many unannotated features. Our untargeted approach provides a first glimpse into the complex gut metabolomes and our dereplication suggests the presence of bioactive compounds with potential defensive roles to be targeted in future studies.

Morphotype switches frequently occur in Actinobacteria and are often associated with disparate natural product production. Here, we report on differences in the secondary metabolomes of two morphotypes of a Streptomyces species, including the discovery of a novel antimicrobial glycosylated macrolide, which we named termidomycin A. While exhibiting an unusual 46-member polyene backbone, termidomycin A (1) shares structural features with the clinically important antifungal agents amphotericin B and nystatin A1. Genomic analyses revealed a biosynthetic gene cluster encoding for a putative giant type I polyketide synthase (PKS), whose domain structure allowed us to propose the relative configuration of the 46-member macrolide. The architecture of the biosynthetic gene cluster was different in both morphotypes, thus leading to diversification of the product spectrum. Given the high frequency of genomic rearrangements in Streptomycetes, the metabolic analysis of distinct morphotypes as exemplified in this study is a promising approach for the discovery of bioactive natural products and pathways of diversification.

Trillions of microbes representing all kingdoms of life are resident in, and on, humans holding essential roles for the host development and physiology. The last decade over a dozen online tools and servers, accessible via public domain, have been developed for the analysis of bacterial sequences; however, the analysis of fungi is still in its infancy. Here, we present a web server dedicated to the comprehensive analysis of the human mycobiome for (i) translating raw sequencing reads to data tables and high-standard figures, (ii) integrating statistical analysis and machine learning with a manually curated relational database and (iii) comparing the user's uploaded datasets with publicly available from the Sequence Read Archive. Using 1,266 publicly available Internal transcribed spacers (ITS) samples, we demonstrated the utility of DAnIEL web server on large scale datasets and show the differences in fungal communities between human skin and soil sites., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Loos, Zhang, Beemelmanns, Kurzai and Panagiotou.)

In marine environments, the bacterially induced metamorphosis of larvae is a widespread cross-kingdom communication phenomenon that is critical for the persistence of many marine invertebrates. However, the majority of inducing bacterial signals and underlying cellular mechanisms remain enigmatic. The marine hydroid Hydractinia echinata is a well-known model system for investigating bacterially stimulated larval metamorphosis, as larvae transform into the colonial adult stage within 24 h of signal detection. Although H. echinata has served as a cell biological model system for decades, the identity and influence of bacterial signals on the morphogenic transition remained largely unexplored. Using a bioassay-guided analysis, we first determined that specific bacterial (lyso)phospholipids, naturally present in bacterial membranes and vesicles, elicit metamorphosis in Hydractinia larvae in a dose-response manner. Lysophospholipids, as single compounds or in combination (50 μM), induced metamorphosis in up to 50% of all larvae within 48 h. Using fluorescence-labeled bacterial phospholipids, we demonstrated that phospholipids are incorporated into the larval membranes, where interactions with internal signaling cascades are proposed to occur. Second, we identified two structurally distinct exopolysaccharides of bacterial biofilms, the new Rha-Man polysaccharide from Pseudoalteromonas sp. strain P1-9 and curdlan from Alcaligenes faecalis, to induce metamorphosis in up to 75% of tested larvae. We also found that combinations of (lyso)phospholipids and curdlan induced transformation within 24 h, thereby exceeding the morphogenic activity observed for single compounds and bacterial biofilms. Our results demonstrate that two structurally distinct, bacterium-derived metabolites converge to induce high transformation rates of Hydractinia larvae and thus may help ensure optimal habitat selection. IMPORTANCE Bacterial biofilms profoundly influence the recruitment and settlement of marine invertebrates, critical steps for diverse marine processes such as the formation of coral reefs, the maintenance of marine fisheries, and the fouling of submerged surfaces. However, the complex composition of biofilms often makes the characterization of individual signals and regulatory mechanisms challenging. Developing tractable model systems to characterize these coevolved interactions is the key to understanding fundamental processes in evolutionary biology. Here, we characterized two types of bacterial signaling molecules, phospholipids and polysaccharides, that induce the morphogenic transition. We then analyzed their abundance and combinatorial activity. This study highlights the general importance of multiple bacterial signal converging activity in development-related cross-kingdom signaling and poses the question of whether complex lipids and polysaccharides are general metamorphic cues for cnidarian larvae.

Macrotermitine termites have domesticated fungi in the genus Termitomyces as their primary food source using predigested plant biomass. To access the full nutritional value of lignin-enriched plant biomass, the termite-fungus symbiosis requires the depolymerization of this complex phenolic polymer. While most previous work suggests that lignocellulose degradation is accomplished predominantly by the fungal cultivar, our current understanding of the underlying biomolecular mechanisms remains rudimentary. Here, we provide conclusive omics and activity-based evidence that Termitomyces employs not only a broad array of carbohydrate-active enzymes (CAZymes) but also a restricted set of oxidizing enzymes (manganese peroxidase, dye decolorization peroxidase, an unspecific peroxygenase, laccases, and aryl-alcohol oxidases) and Fenton chemistry for biomass degradation. We propose for the first time that Termitomyces induces hydroquinone-mediated Fenton chemistry (Fe 2+ + H 2 O 2 + H + → Fe 3+ + • OH + H 2 O) using a herein newly described 2-methoxy-1,4-dihydroxybenzene (2-MH 2 Q, compound 19)-based electron shuttle system to complement the enzymatic degradation pathways. This study provides a comprehensive depiction of how efficient biomass degradation by means of this ancient insect's agricultural symbiosis is accomplished. IMPORTANCE Fungus-growing termites have optimized the decomposition of recalcitrant plant biomass to access valuable nutrients by engaging in a tripartite symbiosis with complementary contributions from a fungal mutualist and a codiversified gut microbiome. This complex symbiotic interplay makes them one of the most successful and important decomposers for carbon cycling in Old World ecosystems. To date, most research has focused on the enzymatic contributions of microbial partners to carbohydrate decomposition. Here, we provide genomic, transcriptomic, and enzymatic evidence that Termitomyces also employs redox mechanisms, including diverse ligninolytic enzymes and a Fenton chemistry-based hydroquinone-catalyzed lignin degradation mechanism, to break down lignin-rich plant material. Insights into these efficient decomposition mechanisms reveal new sources of efficient ligninolytic agents applicable for energy generation from renewable sources.

Insights into the genomic consequences of symbiosis for basidiomycete fungi associated with social insects remain sparse. Capitalizing on viability of spores from centuries-old herbarium specimens of free-living, facultative, and specialist termite-associated Podaxis fungi, we obtained genomes of 10 specimens, including two type species described by Linnaeus >240 years ago. We document that the transition to termite association was accompanied by significant reductions in genome size and gene content, accelerated evolution in protein-coding genes, and reduced functional capacities for oxidative stress responses and lignin degradation. Functional testing confirmed that termite specialists perform worse under oxidative stress, while all lineages retained some capacity to cleave lignin. Mitochondrial genomes of termite associates were significantly larger; possibly driven by smaller population sizes or reduced competition, supported by apparent loss of certain biosynthetic gene clusters. Our findings point to relaxed selection that mirrors genome traits observed among obligate endosymbiotic bacteria of many insects., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Targeted HRMS 2 -GNPS-based metabolomic analysis of Pseudoxylaria sp. X187, a fungal antagonist of the fungus-growing termite symbiosis, resulted in the identification of two lipopeptidic congeners of xylacremolides, named xylacremolide C and D, which are built from d-phenylalanine, l-proline and an acetyl-CoA starter unit elongated by four malonyl-CoA derived ketide units. The putative xya gene cluster was identified from a draft genome generated by Illumina and PacBio sequencing and RNAseq studies. Biological activities of xylacremolide A and B were evaluated and revealed weak histone deacetylase inhibitory (HDACi) and antifungal activities, as well as moderate protease inhibition activity across a panel of nine human, viral and bacterial proteases., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

High-resolution tandem mass spectrometry (HR-MS 2 )-based metabolomic studies of Amycolatopsis saalfeldensis , isolated from the "Saalfelder Feengrotten" caves in Germany, led to the isolation of three ribosomally synthesized and post-translationally modified type II thiopeptides, saalfelduracin B-D ( 1 - 3 ) and the known saalfelduracin A ( 4 ). The structures of all four compounds were determined by comparative two-dimensional NMR analysis and high-resolution tandem mass spectrometry.

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., (Copyright © 2021 Murphy et al.)

Marine bacteria are excellent yet often underexplored sources of structurally unique bioactive natural products. In this review we cover the diversity of marine bacterial biomolecules and highlight recent studies on structurally novel natural products. We include different compound classes and discuss the latest progress related to their biosynthetic pathway analysis and engineering: examples range from fatty acids over terpenes to PKS, NRPS and hybrid PKS-NRPS biomolecules.

Antibiotics are commonly used in the Intensive Care Unit (ICU); however, several studies showed that the impact of antibiotics to prevent infection, multi-organ failure, and death in the ICU is less clear than their benefit on course of infection in the absence of organ dysfunction. We characterized here the compositional and metabolic changes of the gut microbiome induced by critical illness and antibiotics in a cohort of 75 individuals in conjunction with 2,180 gut microbiome samples representing 16 different diseases. We revealed an "infection-vulnerable" gut microbiome environment present only in critically ill treated with antibiotics (ICU + ). Feeding of Caenorhabditis elegans with Bifidobacterium animalis and Lactobacillus crispatus , species that expanded in ICU + patients, revealed a significant negative impact of these microbes on host viability and developmental homeostasis. These results suggest that antibiotic administration can dramatically impact essential functional activities in the gut related to immune responses more than critical illness itself, which might explain in part untoward effects of antibiotics in the critically ill.

The taxonomic position of a novel aerobic, Gram-positive actinobacteria, designated strain RB5 T , was determined using a polyphasic approach. The strain, isolated from the gut of the fungus-farming termite Macrotermes natalensis , showed morphological, physiological and chemotaxonomic properties typical of the genus Streptomyces . Based on 16S rRNA gene sequence analysis, the closest phylogenetic neighbour of RB5 T was Streptomyces polyrhachis DSM 42102 T (98.87 %). DNA-DNA hybridization experiments between strain RB5 T and S. polyrhachis DSM 42102 T resulted in a value of 27.4 % (26.8 %). The cell wall of strain RB5 T contained ll-diaminopimelic acid as the diagnostic amino acid. Mycolic acids and diagnostic sugars in whole-cell hydrolysates were not detected. The strain produced the following major phospholipids: diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol-mannoside and phosphatidylserine. The menaquinone profile showed hexa- and octahydrogenated menaquinones containing nine isoprene units [MK-9(H 6 ) and MK-9(H 8 )]. The strain exhibited a fatty acid profile containing the following major fatty acids: 12-methyltridecanoic acid (iso-C 14 : 0 ) 12-methyltetradecanoic acid (anteiso-C 15 : 0 ), 13-methyltetradecanoic acid (iso-C 15 : 0 ) and 14-methylpentadecanoic acid (iso-C 16 : 0 ). Here, we propose a novel species of the genus Streptomyces - Streptomyces smaragdinus with the type strain RB5 T (=VKM Ac-2839 T =NRRL B65539 T ).

Based on high-resolution tandem mass spectrometry (HR-MS 2 ) and global natural products social molecular networking (GNPS), we found that plant-derived daidzein and genistein derivatives are polyhalogenated by termite-associated Actinomadura species RB99. MS-guided purification from extracts of bacteria grown under optimized conditions led to the isolation of eight polychlorinated isoflavones, including six unreported derivatives, and seven novel polybrominated derivatives, two of which showed antimicrobial activity.

Herein, we report the targeted isolation and characterization of four linear nonribosomally synthesized tetrapeptides (pseudoxylaramide A-D) and two cyclic nonribosomal peptide synthetase-polyketide synthase-derived natural products (xylacremolide A and B) from the termite-associated stowaway fungus Pseudoxylaria sp. X187. The fungal strain was prioritized for further metabolic analysis based on its taxonomical position and morphological and bioassay data. Metabolic data were dereplicated based on high-resolution tandem mass spectrometry data and global molecular networking analysis. The structure of all six new natural products was elucidated based on a combination of 1D and 2D NMR analysis, Marfey's analysis and X-ray crystallography., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The taxonomic positions of two novel aerobic, Gram-stain-positive Actinobacteria, designated RB20 T and RB56 T , were determined using a polyphasic approach. Both were isolated from the fungus-farming termite Macrotermes natalensis . Results of 16S rRNA gene sequence analysis revealed that both strains are members of the genus Nocardia with the closest phylogenetic neighbours Nocardia miyunensis JCM12860 T (98.9 %) and Nocardia nova DSM44481 T (98.5 %) for RB20 T and Nocardia takedensis DSM 44801 T (98.3 %), Nocardia pseudobrasiliensis DSM 44290 T (98.3 %) and Nocardia rayongensis JCM 19832 T (98.2 %) for RB56 T . Digital DNA-DNA hybridization (DDH) between RB20 T and N. miyunensis JCM12860 T and N. nova DSM 44481 T resulted in similarity values of 33.9 and 22.0 %, respectively. DDH between RB56 T and N. takedensis DSM44801 T and N. pseudobrasiliensis DSM44290 T showed similarity values of 20.7 and 22.3 %, respectively. In addition, wet-lab DDH between RB56 T and N. rayongensis JCM19832 T resulted in 10.2 % (14.5 %) similarity. Both strains showed morphological and chemotaxonomic features typical for the genus Nocardia , such as the presence of meso -diaminopimelic acid (A 2 pm) within the cell wall, arabinose and galactose as major sugar components within whole cell-wall hydrolysates, the presence of mycolic acids and major phospholipids (diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol), and the predominant menaquinone MK-8 (H 4 , ω-cyclo). The main fatty acids for both strains were hexadecanoic acid (C 16 : 0 ), 10-methyloctadecanoic acid (10-methyl C 18 : 0 ) and cis -9-octadecenoic acid (C 18 : 1 ω9 c ). We propose two novel species within the genus Nocardia : Nocardia macrotermitis sp. nov. with the type strain RB20 T (=VKM Ac-2841 T =NRRL B65541 T ) and Nocardia aurantia sp. nov. with the type strain RB56 T (=VKM Ac-2842 T =NRRL B65542 T ).

The taxonomic positions of two novel aerobic, Gram-positive actinobacteria, designated strains RB29 T and RB68 T , were determined using a polyphasic approach. Based on 16S rRNA gene sequence analysis, the closest phylogenetic neighbours of RB29 T were identified as Actinomadura rayongensis DSM 102126 T (99.2 % similarity) and Actinomadura atramentaria DSM 43919 T (98.7 %), and for strain RB68 T was Actinomadura hibisca DSM 44148 T (98.3 %). Digital DNA-DNA hybridization (dDDH) between RB29 T and its closest phylogenetic neighbours, A. rayongensis DSM 102126 T and A. atramentaria DSM 43919 T , 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 102126 T and 82.3 % (78.9 %) for A. atramentaria DSM 43919 T . dDDH analysis between strain RB68 T and A. hibisca DSM 44148 T gave a similarity value of 24.5 % (22.2-27.0 %). Both strains, RB29 T and RB68 T , 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(H 6 ) and MK-9(H 8 ) for RB29 T and MK-9(H 4 ) and MK-9(H 6 ) for RB68 T . The main fatty acids were identified as 10-methyloctadecanoic acid (10-methyl C 18:0 ), 14-methylpentadecanoic acid (iso-C 16:0 ), hexadecanoic acid (C 16:0 ) and cis -9-octadecanoic acid (C 18 : 1 ω9 c ). Here, we propose two novel species of the genus Actinomadura : Actinomadura rubteroloni sp. nov. with the type strain RB29 T (=CCUG 72668 T =NRRL B-65537 T ) and Actinomadura macrotermitis sp. nov. with the type strain RB68 T (=CCUG 72669 T =NRRL B-65538 T ).