Endophytic fungi: hidden treasure chest of antimicrobial metabolites interrelationship of endophytes and metabolites

Endophytic fungi comprise host-associated fungal communities which thrive within the tissues of host plants and produce a diverse range of secondary metabolites with various bioactive attributes. The metabolites such as phenols, polyketides, saponins, alkaloids help to mitigate biotic and abiotic stresses, fight against pathogen attacks and enhance the plant immune system. We present an overview of the association of endophytic fungal communities with a plant host and discuss molecular mechanisms induced during their symbiotic interaction. The overview focuses on the secondary metabolites (especially those of terpenoid nature) secreted by endophytic fungi and their respective function. The recent advancement in multi-omics approaches paved the way for identification of these metabolites and their characterization via comparative analysis of extensive omics datasets. This study also elaborates on the role of diverse endophytic fungi associated with key agricultural crops and hence important for sustainability of agriculture

Biosynthetic gene cluster identification in plasmids and characterization of plasmids from animal-associated microbiota

Individual bacteria in complex microbial communities can acquire and accumulate new traits. These traits are reflective of their environment, being niche-specific. A major player in trait sharing is horizontal gene transfer (HGT). Plasmids, extrachromosomal DNA molecules, have a role in HGT and can change the host’s phenotype. Considering the transformative role of plasmids in bacterial lifestyle, we investigated the prevalence, distribution and products of biosynthetic gene clusters (BGCs) present in plasmids. Sequences available on the National Center for Biotechnology Information (NCBI) database (n=101 416) were run through two bioinformatic pipelines for BGC detection that apply different approaches, deepBGC and antiSMASH (antibiotics and secondary metabolites analysis shell). The highest percentage of plasmids with BGCs was detected in Actinobacteria but, apart from Chlamidiae and Tenericutes, all phyla had BGCs in their plasmids, with predictions varying according to the software used. The BGCs identified comprised a range of classes, indicating that plasmid encoded BGCs could be leveraged for the discovery of new molecules. In order to apply that concept to real-life examples, plasmids were isolated from animal-associated microbial communities and characterized. Plasmids from Escherichia coli isolated from wild birds (n=36) were screened for phenotypes of interest in human and animal health. Seven isolates displayed plasmid-encoded antibiotic resistance. Taxonomic identification of the hosts of plasmids isolated from bovid-associated microbiomes (n=38) was determined via 16S rRNA gene, and placed the majority of the isolated in the phylum Firmicutes, apart from a single Klebsiella pneumoniae isolate. Twelve plasmids were sequenced. Three plasmids from different hosts (pRAM-12, pRAM-19-2 and pRAM-30-2) shared 100% nucleotide sequence and a gene cluster for the bacteriocin cloacin. Two of those hosts shared not one, but two plasmids, pRAM-19-1 and pRAM-30-1, despite being in different phyla. This highlights the intimacy of gene sharing and the importance of HGT. pRAM-28 and pRAM-21 shared a plasmid that harbors the BGC for the bacteriocin aureocin A70, the only four peptide bacteriocin known to date. Additional analysis revealed two putative novel lanthipeptide gene clusters in pRAM-2. These results suggest that the plasmidome is a neglected source of secondary metabolites with the potential for molecule discovery. Furthermore, it can be leveraged to study genetic exchange in a community and how plasmid-encoded featured can mediate interactions in a microbiome

RESPONSES OF PREDATORY MYXOBACTERIA TO PREY SIGNALING MOLECULES & FEATURES OF A PSEUDOMONAS PREY AVOIDING PREDATION

Gram-negative unicellular myxobacteria, along with their multicellular lifestyle andbiologically active specialized metabolites, are known for the predatory interactions with Gram-negative/ Gram-positive bacteria and fungi. Although myxobacterial predation range have been exploited extensively, little is known about the prey associated molecules contributing to myxobacterial predator-prey dynamics. By employing transcriptomics and untargeted metabolomics approaches, we demonstrate two structurally distinct classes of signaling molecules from Gram-negative bacterial prey elicit significant omics responses from myxobacteria, Myxococcus xanthus and Cystobacter ferrugineus. An overlapping and general response to acylhomoserine lactones, whereas a distinctive response to a quinolone signaling molecule is observed from both myxobacteria. Similarly, by employing transcriptomics and classical microbiological assays, we demonstrate higher production of molecules like pyoverdine, phenazine-1-carboxylic acid, and alginate and resistance to aminoglycosides and tetracycline antibiotics are unique to a predation survivor Pseudomonas putida phenotype. In a predator-prey co-culturing, the predatory stress from myxobacterium C. ferrugineus selects for this P. putida phenotype that eludes subsequent myxobacterial predation. Overall, our study confirms that prey associated chemical components significantly direct responses from predatory myxobacteria

Computational and molecular study of terpene synthase genes in Trichoderma

[ES] Trichoderma comprende un amplio número de especies, de gran interés en el manejo de enfermedades de las plantas de cultivo y la industria. El amplio rango de estilos de vida de estas especies está respaldado por su diversidad de Metabolitos Secundarios (MSs). En este trabajo, se ha utilizado una combinación de enfoques de minería genómica y genómica comparativa generando una extensa visión sobre el potencial de biosíntesis de MSs en Trichoderma

Bioprospecting of Arctic marine microorganisms. Exploring microbial secondary metabolite production using the one strain-many compounds approach: isolation and characterization of secondary metabolites

Natural products have been used by humans since ancient times as benefactors for improved health. Prior to modern medicine and chemistry, these compounds remained hidden in the plants, animals and other organisms used to heal inflammation, wounds, headache and stomachache among other conditions. Since the start of modern drug discovery with isolation of morphine in 1805, numerous natural products have been isolated from plants, animals, macroorganisms and microorganisms. Today, natural products, or their derivatives, are used as pharmaceuticals within a wide range of therapeutic areas, including cancer, pathogenic infections, inflammation and pain. Microbial natural products have played a particularly important role in the field of antibiotics. The discovery of penicillin from the Pencillium rubens fungus by Alexander Fleming in 1928 marked the beginning of the “Golden Age” of antibiotics that lasted until 1962, where most antibiotic classes in clinical use today were discovered. Several marketed drugs originate from marine microorganisms. Marine microorganisms are underexplored, thus representing a potential source for discovering novel bioactive compounds. In this project, Arctic marine microorganisms were fermented under different conditions based on the OSMAC approach and evaluated for their production of antibacterial and cytotoxic compounds. In paper I, a Pseudomonas sp. bacterium was cultivated in different growth media. The fermentation extracts were fractionated and tested for bioactivity, revealing different bioactivity profiles of the fractions from the different media. Dereplication of the active fractions by UHPLC-HR-MS and molecular networking led to identification of six rhamnolipid compounds, including one novel mono-rhamnolipid. All six compounds had antimicrobial activities, while three had cytotoxic activities. In paper II, a fractionated extract of the bacterium Lacinutrix sp. displayed antibacterial activity. Dereplication of the active fraction resulted in identification of two lyso-ornithine lipids, 1 and 2. The compounds were isolated and their structures were elucidated with UHPLC-HR-MS and NMR. Bioactivity screening showed that 1 had antibacterial activity, while 2 had cytotoxic activity. In paper III, the fungus Digitatispora marina was fermented under different cultivation conditions. Fermentation extracts were fractionated and bioactivity screening of the fractions revealed antibacterial and cytotoxic activities. UHPLC-HR-MS analysis of the fractions showed a compound with an isotope distribution pattern for an ion with a single chlorine atom. The compound was isolated, and structure elucidation with NMR identified it as chlovalicin B. Its bioactive properties were broadly evaluated, revealing it had weak cytotoxic activity but no antimicrobial activities

Fungi use highly diverse approaches for plant biomass conversion as revealed through bioinformatic analysis

Plant biomass is a magnificent renewable resource and therefore is of major importance for ecology and the global carbon cycle. In nature, fungi play central roles in the degradation of plant biomass, as they are highly efficient degraders of plant polysaccharides. Their ability to break down complex plant polysaccharides, such as cellulose and hemicellulose, into simple sugars is essential for the recycling of organic material in ecosystems. When it comes to the conversion of plant biomass, two crucial aspects of plant biomass conversion are of paramount importance: primary sugar metabolism and the extensive repertoire of Carbohydrate-Active enZymes (CAZymes) involved in the degradation of complex plant biomass substrates. Therefore, this thesis enhanced our comprehension of the diversity of primary carbon metabolism and the enzymatic capabilities of fungi. By investigating these aspects, we aimed to unravel the intricate mechanisms underlying fungal biomass conversion and shed light on the evolutionary adaptations and functional variations across fungi. To conclude, this thesis showcases using a combination of bioinformatic and omics approaches could help us to obtain a deeper understanding of the molecular mechanisms involved in plant biomass conversion by fungi, and the differences in these approaches across the fungal kingdom