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In recent years, research in the field of Microbial Ecology has revealed the tremendous diversity and complexity of microbial communities across different ecosystems. Microbes play a major role in ecosystem functioning and contribute to the health and fitness of higher organisms. Scientists are now facing many technological and methodological challenges in analyzing these complex natural microbial communities. The advances in analytical and omics techniques have shown that microbial communities are largely shaped by chemical interaction networks mediated by specialized (water-soluble and volatile) metabolites. However, studies concerning microbial chemical interactions need to consider biotic and abiotic factors on multidimensional levels, which require the development of new tools and approaches mimicking natural microbial habitats. In this review, we describe environmental factors affecting the production and transport of specialized metabolites. We evaluate their ecological functions and discuss approaches to address future challenges in microbial chemical ecology (MCE). We aim to emphasize that future developments in the field of MCE will need to include holistic studies involving organisms at all levels and to consider mechanisms underlying the interactions between viruses, micro-, and macro-organisms in their natural environments.

The production of natural product compound libraries has been observed in nature for different organisms such as bacteria, fungi and plants; however, little is known about the mechanisms generating such chemically diverse libraries. Here we report mechanisms leading to the biosynthesis of the chemically diverse rhabdopeptide/xenortide peptides (RXPs). They are exclusively present in entomopathogenic bacteria of the genera Photorhabdus and Xenorhabdus that live in symbiosis with nematodes delivering them to insect prey, which is killed and utilized for nutrition by both nematodes and bacteria. Chemical diversity of the biologically active RXPs results from a combination of iterative and flexible use of monomodular nonribosomal peptide synthetases including substrate promiscuity, enzyme cross-talk and enzyme stoichiometry as shown by in vivo and in vitro experiments. Together, this highlights several of nature's methods for diversification, or evolution, of natural products and sheds light on the biosynthesis of the bioactive RXPs. Nature has evolved a variety of different mechanisms to generate chemical diversity; however, the reactions responsible for generating such diverse chemical libraries are often not clear. Now, the mechanisms employed by entomopathogenic bacteria for the biosynthesis of a large family of bioactive peptides have been identified. These include substrate promiscuity, enzyme cross-talk and enzyme stoichiometry.

Non-Ribosomal Peptides (NRPs) represent a biomedically important class of natural products that include a multitude of antibiotics and other clinically used drugs. NRPs are not directly encoded in the genome but are instead produced by metabolic pathways encoded by biosynthetic gene clusters (BGCs). Since the existing genome mining tools predict many putative NRPs synthesized by a given BGC, it remains unclear which of these putative NRPs are correct and how to identify post-assembly modifications of amino acids in these NRPs in a blind mode, without knowing which modifications exist in the sample. To address this challenge, here we report NRPminer, a modification-tolerant tool for NRP discovery from large (meta)genomic and mass spectrometry datasets. We show that NRPminer is able to identify many NRPs from different environments, including four previously unreported NRP families from soil-associated microbes and NRPs from human microbiota. Furthermore, in this work we demonstrate the anti-parasitic activities and the structure of two of these NRP families using direct bioactivity screening and nuclear magnetic resonance spectrometry, illustrating the power of NRPminer for discovering bioactive NRPs. Current genome mining methods predict many putative non-ribosomal peptides (NRPs) from their corresponding biosynthetic gene clusters, but it remains unclear which of those exist in nature and how to identify their post-assembly modifications. Here, the authors develop NRPminer, a modification-tolerant tool for the discovery of NRPs from large genomic and mass spectrometry datasets, and use it to find 180 NRPs from different environments.

Non-ribosomal peptide synthetases (NRPSs) are giant enzyme machines that activate amino acids in an assembly line fashion. As NRPSs are not restricted to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would enable microbial production of a diverse range of peptides; however, the structural requirements for reprogramming NRPSs to facilitate the production of new peptides are not clear. Here we describe a new fusion point inside the condensation domains of NRPSs that results in the development of the exchange unit condensation domain (XUC) concept, which enables the efficient production of peptides, even containing non-natural amino acids, in yields up to 280 mg l −1 . This allows the generation of more specific NRPSs, reducing the number of unwanted peptide derivatives, but also the generation of peptide libraries. The XUC might therefore be suitable for the future optimization of peptide production and the identification of bioactive peptide derivatives for pharmaceutical and other applications. Non-ribosomal peptide synthetases have now been modified and de novo non-ribosomal peptide synthetases constructed using new assembly points within condensation domains. This approach enabled the production of new-to-nature peptides, including some carrying synthetic amino acids, as well as the generation of peptide libraries.

Microorganisms contribute to the biology and physiology of eukaryotic hosts and affect other organisms through natural products. Xenorhabdus and Photorhabdus ( XP ) living in mutualistic symbiosis with entomopathogenic nematodes generate natural products to mediate bacteria–nematode–insect interactions. However, a lack of systematic analysis of the XP biosynthetic gene clusters (BGCs) has limited the understanding of how natural products affect interactions between the organisms. Here we combine pangenome and sequence similarity networks to analyse BGCs from 45 XP strains that cover all sequenced strains in our collection and represent almost all XP taxonomy. The identified 1,000 BGCs belong to 176 families. The most conserved families are denoted by 11 BGC classes. We homologously (over)express the ubiquitous and unique BGCs and identify compounds featuring unusual architectures. The bioactivity evaluation demonstrates that the prevalent compounds are eukaryotic proteasome inhibitors, virulence factors against insects, metallophores and insect immunosuppressants. These findings explain the functional basis of bacterial natural products in this tripartite relationship. Entomopathogenic nematodes carrying Xenorhabdus and Photorhabdus bacteria prey on insect larvae in the soil. Now, a comprehensive analysis of the bacterial genome has revealed ubiquitous and unique families of biosynthetic gene clusters. Evaluation of the bioactivity of the natural products expressed by the most prevalent cluster families explains the functional basis of bacterial natural products involved in bacteria–nematode–insect interactions.

Our study aimed to identify the novel acaricidal compound in Xenorhabdus szentirmaii and X. nematophila using the easyPACId approach (easy Promoter Activated Compound Identification). We determined the (1) effects of cell-free supernatant (CFS) obtained from mutant strains against T. urticae females , (2) CFS of the acaricidal bioactive strain of X. nematophila (pCEP_kan_XNC1_1711) against different biological stages of T. urticae, and females of predatory mites, Phytoseiulus persimilis and Neoseiulus californicus , (3) effects of the extracted acaricidal compound on different biological stages of T. urticae, and (4) cytotoxicity of the active substance. The results showed that xenocoumacin produced by X. nematophila was the bioactive acaricidal compound, whereas the acaricidal compound in X. szentirmaii was not determined. The CFS of X. nematophila (pCEP_kan_XNC1_1711) caused 100, 100, 97.3, and 98.1% mortality on larvae, protonymph, deutonymph and adult female of T. urticae at 7 dpa in petri dish experiments; and significantly reduced T. urticae population in pot experiments. However, the same CFS caused less than 36% mortality on the predatory mites at 7dpa. The mortality rates of extracted acaricidal compound (xenocoumacin) on the larva, protonymph, deutonymph and adult female of T. urticae were 100, 100, 97, 96% at 7 dpa. Cytotoxicity assay showed that IC 50 value of xenocoumacin extract was 17.71 μg/ml after 48 h. The data of this study showed that xenocoumacin could potentially be used as bio-acaricide in the control of T. urticae; however, its efficacy in field experiments and its phytotoxicity need to be assessed in future.

The pyrrolobenzodiazepine tilivalline (1) was originally identified in the human gut pathobiont Klebsiella oxytoca, the causative agent of antibiotic-associated hemorrhagic colitis. Here we show the identification of tilivalline and analogs thereof in the entomopathogenic bacterium Xenorhabdus eapokensis as well as the identification of its biosynthesis gene cluster encoding a bimodular non-ribosomal peptide synthetase. Heterologous expression of both genes in E. coli resulted in the production of 1 and from mutasynthesis and precursor directed biosynthesis 11 new tilivalline analogs were identified in X. eapokensis. These results allowed the prediction of the tilivalline biosynthesis being similar to that in K. oxytoca.

Natural products have been proven to be important starting points for the development of new drugs. Bacteria in the genera Photorhabdus and Xenorhabdus produce antimicrobial compounds as secondary metabolites to compete with other organisms. Our study is the first comprehensive study screening the anti-protozoal activity of supernatants containing secondary metabolites produced by 5 Photorhabdus and 22 Xenorhabdus species against human parasitic protozoa, Acanthamoeba castellanii, Entamoeba histolytica, Trichomonas vaginalis, Leishmania tropica and Trypanosoma cruzi, and the identification of novel bioactive antiprotozoal compounds using the easyPACId approach (easy Promoter Activated Compound Identification) method. Though not in all species, both bacterial genera produce antiprotozoal compounds effective on human pathogenic protozoa. The promoter exchange mutants revealed that antiprotozoal bioactive compounds produced by Xenorhabdus bacteria were fabclavines, xenocoumacins, xenorhabdins and PAX peptides. Among the bacteria assessed, only P. namnaoensis appears to have acquired amoebicidal property which is effective on E. histolytica trophozoites. These discovered antiprotozoal compounds might serve as starting points for the development of alternative and novel pharmaceutical agents against human parasitic protozoa in the future.

It is well recognized that bacteria communicate via small diffusible molecules, a process termed quorum sensing. The best understood quorum sensing systems are those that use acylated homoserine lactones (AHLs) for communication. The prototype of those systems consists of a LuxI-like AHL synthase and a cognate LuxR receptor that detects the signal. However, many proteobacteria possess LuxR receptors, yet lack any LuxI-type synthase, and thus these receptors are referred to as LuxR orphans or solos. In addition to the well-known AHLs, little is known about the signaling molecules that are sensed by LuxR solos. Here, we describe a novel cell–cell communication system in the insect and human pathogen Photorhabdus asymbiotica . We identified the LuxR homolog PauR to sense dialkylresorcinols (DARs) and cyclohexanediones (CHDs) instead of AHLs as signals. The DarABC synthesis pathway produces the molecules, and the entire system emerged as important for virulence. Moreover, we have analyzed more than 90 different Photorhabdus strains by HPLC/MS and showed that these DARs and CHDs are specific to the human pathogen P. asymbiotica . On the basis of genomic evidence, 116 other bacterial species are putative DAR producers, among them many human pathogens. Therefore, we discuss the possibility of DARs as novel and widespread bacterial signaling molecules and show that bacterial cell–cell communication goes far beyond AHL signaling in nature. Significance Bacteria can communicate with each other by small diffusible molecules, a process termed quorum sensing. Many bacteria use acylated homoserine lactones (AHLs) as signals, which are sensed by so-called LuxR-type receptors. With the photopyrones from the insect pathogenic bacterium Photorhabdus luminescens , we recently identified the first quorum sensing molecules different from AHLs that are sensed by a LuxR-type receptor. Here we describe the second novel quorum sensing molecule sensed by a LuxR-type receptor of Photorhabdus species, PauR of the human pathogen Photorhabdus asymbiotica . We demonstrate that P. asymbiotica communicates via dialkylresorcinols (DARs) and cyclohexanediones (CHDs). As the synthesis pathway is widespread, and often present in human pathogens, we discuss DARs and CHDs as novel and widespread signaling molecules.

Background Bacteria of the genus Photorhabdus and Xenorhabdus are motile, Gram-negative bacteria that live in symbiosis with entomopathogenic nematodes. Due to their complex life cycle, they produce a large number of specialized metabolites (natural products) encoded in biosynthetic gene clusters (BGC). Genetic tools for Photorhabdus and Xenorhabdus have been rare and applicable to only a few strains. In the past, several tools have been developed for the activation of BGCs and the deletion of individual genes. However, these often have limited efficiency or are time consuming. Among the limitations, it is essential to have versatile expression systems and genome editing tools that could facilitate the practical work. Results In the present study, we developed several expression vectors and a CRISPR-Cpf1 genome editing vector for genetic manipulations in Photorhabdus and Xenorhabdus using SEVA plasmids. The SEVA collection is based on modular vectors that allow exchangeability of different elements (e.g. origin of replication and antibiotic selection markers with the ability to insert desired sequences for different end applications). Initially, we tested different SEVA vectors containing the broad host range origins and three different resistance genes for kanamycin, gentamycin and chloramphenicol, respectively. We demonstrated that these vectors are replicative not only in well-known representatives, e.g. Photorhabdus laumondii TTO1, but also in other rarely described strains like Xenorhabdus sp. TS4. For our CRISPR/Cpf1-based system, we used the pSEVA231 backbone to delete not only small genes but also large parts of BGCs. Furthermore, we were able to activate and refactor BGCs to obtain high production titers of high value compounds such as safracin B, a semisynthetic precursor for the anti-cancer drug ET-743. Conclusions The results of this study provide new inducible expression vectors and a CRISPR/CPf1 encoding vector all based on the SEVA (Standard European Vector Architecture) collection, which can improve genetic manipulation and genome editing processes in Photorhabdus and Xenorhabdus .