ALBERT

Description: This study aimed at isolating microorganisms associated with the mesopelagic jellyfish Periphylla periphylla collected in Irminger Sea at a depth of 325 m in July 2020. Three different solid cultivation media; Hastings, Marine agar and Wickerham media were used for the isolation of the associated microorganisms. A total of 43 bacteria were isolated from the inner and outer surfaces of the umbrella of P. periphylla, but unfortunately, no fungal strain was isolated. Isolates were further identified by Sanger sequencing of the 16S rRNA gene, and based on phylogenetic distinctiveness (differences in closest relative species according to the nucleotide BLAST), 16 bacteria belonging to 8 different genera were selected and subjected to an OSMAC cultivation regime approach using liquid and solid marine broth and glucose– yeast–malt media. After 7 days of cultivation, cultures were extracted with ethyl acetate and assessed for antimicrobial activity against fish and human pathogens. Based on antimicrobial activity assessment, four most bioactive strains; Polaribacter sp. SU124, Shewanella sp. SU126, Psychrobacter sp. SU143 and Psychrobacter sp. SU137, were prioritized for a comparative and untargeted metabolomics analysis using feature-based molecular networking. These findings highlight the biotechnological potential of P. periphylla-associated microbiota.

Description: Identification of 16 selected bacteria associated to the umbrella of Periphylla periphylla collected in the Irminger Sea based on sequencing of the 16S ribosomal RNA gene and BLAST against the NCBI nucleotide database. The table displays the first 3 hits from two blastn searches, one against all database entries and the second against type strains only. Nucleotide identities (%) with database hits and NCBI accession numbers assigned to the isolated bacteria are also displayed. Colonies were cultured either in marine broth, Hastings medium or Wickerham medium for comparison.

Description: Putative annotation of metabolites produced by the Psychrobacter sp. SU143, Psychrobacter sp. SU137, Shewanella sp. SU126 and Polaribacter sp. SU124 in the liquid and solid regime of Marine Broth and Glucose-Yeast-Malt media. Annotation was based on the m/z [M+H]+ or other adducts, retention time, predicted molecular formula, fragmentation pattern and source of the hit. The source of the ion is indicated by the area under a peak (peak area count). Confidence level of annotation are given based on the reporting standards proposed by Sumner, et al. 2007, i.e. identified compound, is putative annotation without reference standards, is putative characterized compound class, and is unknown compound. The mass spectrometry data used for the molecular networking analysis were deposited in the MassIVE Public GNPS database under the accession number MSV000091067.

Description: Microbial co‐cultivation is employed for awakening silent biosynthetic gene clusters (BGCs) to enhance chemical diversity. However, the selection of appropriate partners for co‐cultivation remains a challenge. Furthermore, competitive interactions involving the suppression of BGCs or upregulation of known, functional metabolite(s) during co‐cultivation efforts is also common. Herein, we performed an alternative approach for targeted selection of the best co‐cultivation pair. Eight marine sediment‐derived fungi were classified as strong or weak, based on their anti‐phytopathogenic potency. The fungi were co‐cultured systematically and analyzed for their chemical profiles and anti-phytopathogenic activity. Based on enhanced bioactivity and a significantly different metabolite profile including the appearance of a co‐culture specific cluster, the co‐culture of Plenodomus influorescens (strong) and Pyrenochaeta nobilis (weak) was prioritized for chemical investigation. Large‐scale co‐cultivation resulted in isolation of five polyketide type compounds: two 12‐membered macrolides, dendrodolide E (1) and its new analog dendrodolide N (2), as well as two rare azaphilones spiciferinone (3) and its new analog 8a-hydroxy-spiciferinone (4). A well‐known bis‐naphtho‐γ‐pyrone type mycotoxin, cephalochromin (5), whose production was specifically enhanced in the co-culture, was also isolated. Chemical structures of compounds 1–5 were elucidated by NMR, HRMS and [] 20/D analyses. Compound 5 showed the strongest anti‐phytopathogenic activity against Xanthomonas campestris and Phytophthora infestans with IC50 values of 0.9 and 1.7 µg/mL, respectively.

Description: Sea foam harbors a diverse range of fungal spores with biological and ecological relevance in marine environments. Fungi are known as the producers of secondary metabolites that are used in health and agricultural sectors, however the potentials of sea foam-derived fungi have remained unexplored. In this study, organic extracts of six foam-derived fungal isolates belonging to the genera Penicillium, Cladosporium, Emericellopsis and Plectosphaerella were investigated for their antimicrobial activity against plant and human pathogens and anticancer activity. In parallel, an untargeted metabolomics study using UPLC-QToF–MS/MS-based molecular networking (MN) was performed to unlock their chemical inventory. Penicillium strains were identified as the most prolific producers of compounds with an average of 165 parent ions per strain. In total, 49 known mycotoxins and functional metabolites were annotated to specific and ubiquitous parent ions, revealing considerable chemical diversity. This allowed the identification of putative new derivatives, such as a new analog of the antimicrobial tetrapeptide, fungisporin. Regarding bioactivity, the Penicillium sp. isolate 31.68F1B showed a strong and broad-spectrum activity against seven plant and human pathogens, with the phytopathogen Magnaporthe oryzae and the human pathogen Candida albicans being the most susceptible (IC50 values 2.2 and 6.3 µg/mL, respectively). This is the first study mining the metabolome of the sea foam-derived fungi by MS/MS-based molecular networking, and assessing their biological activities against phytopathogens

Description: Plant pathogens remain a significant threat to crop yield and crop quality. Their control with chemical pesticides is faced with public resistance because of associated human and environmental toxicities. There is therefore a need for more novel and ‘friendlier’ agrochemicals, such as natural products, for the control of plant disease. Fungi have a long history in crop protection as a source of bioactive compounds. This is exemplified by strobilurins, terrestrial fungal secondary metabolites, which have a large market share of current agricultural fungicides. However, efforts to discover new natural products from terrestrial fungi are limited by re-isolation of known compounds. This is in part, because a subset of biosynthetic gene clusters (BGCs) remain silent and are not transcribed in laboratory monoculture conditions. This study sought to exploit the biosynthetic potential of fungi for discovery of new anti-phytopathogenic natural products. Towards this aim, chemical investigations were performed: (i) on fungal isolates from a less studied ecosystem, Windebyer Noor – including an unexploited habitat, sea foam, (ii) on fungal isolates from less studied genera, and (iii) after employing biosynthetic gene activating strategies, such as co-cultivation and variation of cultivation media. Molecular networking-based metabolomics was employed to facilitate dereplication of fungal metabolites, and to prioritize fungal extracts for isolation of new compounds with anti-phytopathogenic activity. A unique and direct co-cultivation approach involving 21 fungal strains (out of 123 fungal isolates obtained from the Windebyer Noor) against four phytopathogens (two bacteria: Pseudomonas syringae and Ralstonia solanacearum, and two fungi: Magnaporthe oryzae and Botrytis cinerea) on solid agar was employed. Organic extracts of the mono- and co-cultures were monitored by comparison of their anti-phytopathogenic activities and metabolome by untargeted metabolomics, using MS/MS-based molecular networking (MN). The phytopathogens established interspecies communications with marine fungi, resulting in the production of 9 new compound clusters exclusively produced in the co-cultures. Overall, 62 compounds belonging to terpenes, alkaloids, peptides, and polyketides were annotated, in addition to several new putative metabolites with potential anti-phytopathogenic activities. This new combined approach of direct co-cultivation, metabolomics and anti-phytopathogenic activity assessments, allowed the prioritization of two co-cultures, Cosmospora sp. against M. oryzae and Acremonium sp. against P. syringae, for future isolation of natural products. Another new co-cultivation approach was employed between sediment-derived isolates obtained from the Windebyer Noor. Strains were categorized as ‘strong’ or ‘weak’ based on their anti-phytopathogenic potencies. Intra- and inter-categorial co-cultivations were monitored by their anti-phytopathogenic activities and chemical profiles. The co-culture of Plenodomus influorescens (strong) and Pyrenochaeta nobilis (weak), representing less-studied genera, was selected for chemical investigation. From the dichloromethane subextract of this prioritized co-culture, five natural products were isolated; two 12-membered macrolides, dendrodolide E and its new analog dendrodolide N, two azaphilones spiciferinone and its new analog 8a-hydroxy-spiciferinone and a known bis-naphtho-γ-pyrone type mycotoxin, cephalochromin. Structures of these compounds were elucidated by 1D and 2D NMR, HRMS and [α]D analyses. Cephalochromin was over expressed in P. influorescens in response to the interspecies crosstalk with P. nobilis, and further exhibited strong anti-phytopathogenic activity against Xanthomonas campestris and Phytophthora infestans. Fungal isolates derived from sea foam, an unexploited habitat, obtained from the Windebyer Noor, were investigated for their anti-phytopathogenic potential. The metabolome of six representative strains was studied using metabolomics, incorporating molecular networking. In total, 49 metabolites were putatively annotated to known bioactive compounds with several derivatives representing new natural products. Penicillium sp. isolate 31.68F1B showed four strain-specific molecular families with strong inhibitory activity against the phytopathogen M. oryzae (IC50: 2.2 μg/mL), and was prioritized for future isolation of novel natural products. This study has successfully highlighted the enormous potential of marine fungi for the discovery of anti-phytopathogenic compounds. Furthermore, the discovery of novel natural products and a novel approach in strain selection for co-cultivation in this study presents optimism for control of pathogens in the agrochemical industry. It has discovered new natural products and a new approach in strain selection for co-cultivation.

Description: The South Shetland Trough, Antarctica, is an underexplored region for microbiological and biotechnological exploitation. Herein, we describe the isolation and characterization of the novel bacterium Lacinutrix shetlandiensis sp. nov. WUR7 from a deep-sea environment. We explored its chemical diversity via a metabologenomics approach, wherein the OSMAC strategy was strategically employed to upregulate cryptic genes for secondary metabolite production. Based on hybrid de novo whole genome sequencing and digital DNA–DNA hybridization, isolate WUR7 was identified as a novel species from the Gram-negative genus Lacinutrix. Its genome was mined for the presence of biosynthetic gene clusters with limited results. However, extensive investigation of its metabolism uncovered an unusual tryptophan decarboxylase with high sequence homology and conserved structure of the active site as compared to ZP_02040762, a highly specific tryptophan decarboxylase from Ruminococcus gnavus. Therefore, WUR7's metabolism was directed toward indole-based alkaloid biosynthesis by feeding it with L-tryptophan. As expected, its metabolome profile changed dramatically, by triggering the extracellular accumulation of a massive array of metabolites unexpressed in the absence of tryptophan. Untargeted LC-MS/MS coupled with molecular networking, followed along with chemoinformatic dereplication, allowed for the annotation of 10 indole alkaloids, belonging to β-carboline, bisindole, and monoindole classes, alongside several unknown alkaloids. These findings guided us to the isolation of a new natural bisindole alkaloid 8,9-dihydrocoscinamide B (1), as the first alkaloid from the genus Lacinutrix, whose structure was elucidated on the basis of extensive 1D and 2D NMR and HR-ESIMS experiments. This comprehensive strategy allowed us to unlock the previously unexploited metabolome of L. shetlandiensis sp. nov. WUR7.

Description: It is widely accepted that the commensal gut microbiota contributes to the health and well-being of its host. The solitary tunicate Ciona intestinalis emerges as a model organism for studying host–microbe interactions taking place in the gut, however, the potential of its gut-associated microbiota for marine biodiscovery remains unexploited. In this study, we set out to investigate the diversity, chemical space, and pharmacological potential of the gut-associated microbiota of C. intestinalis collected from the Baltic and North Seas. In a culture-based approach, we isolated 61 bacterial and 40 fungal strains affiliated to 33 different microbial genera, indicating a rich and diverse gut microbiota dominated by Gammaproteobacteria. In vitro screening of the crude microbial extracts indicated their antibacterial (64% of extracts), anticancer (22%), and/or antifungal (11%) potential. Nine microbial crude extracts were prioritized for in-depth metabolome mining by a bioactivity- and chemical diversity-based selection procedure. UPLC-MS/MS-based metabolomics combining automated (feature-based molecular networking and in silico dereplication) and manual approaches significantly improved the annotation rates. A high chemical diversity was detected where peptides and polyketides were the predominant classes. Many compounds remained unknown, including two putatively novel lipopeptides produced by a Trichoderma sp. strain. This is the first study assessing the chemical and pharmacological profile of the cultivable gut microbiota of C. intestinalis

Description: Highlights: • A chiral HPLC method was validated to determine usnic acid (UA) enantiomer ratios. • Molecular dynamics simulation revealed chiral chromatographic mechanisms. • MS imaging was used for spatial distribution of UA in lichen cross sections. • Fluorescence microscopy was used for spatial imaging of UA in lichen cross sections. Abstract: Usnic acid is an antibiotic metabolite produced by a wide variety of lichenized fungal lineages. The enantiomers of usnic acid have been shown to display contrasting bioactivities, and hence it is important to determine their spatial distribution, amounts and enantiomeric ratios in lichens to understand their roles in nature and grasp their pharmaceutical potential. The overall aim of the study was to characterise the spatial distribution of the predominant usnic acid enantiomer in lichens by combining spatial imaging and chiral chromatography. Specifically, separation and quantification of usnic acid enantiomers in four common lichens in Iceland was performed using a validated chiral chromatographic method. Molecular dynamics simulation was carried out to rationalize the chiral separation mechanism. Spatial distribution of usnic acid in the lichen thallus cross-sections were analysed using Desorption Electrospray Ionization-Imaging Mass Spectrometry (DESI-IMS) and fluorescence microscopy. DESI-IMS confirmed usnic acid as a cortical compound, and revealed that usnic acid can be more concentrated around the algal vicinity. Fluorescence microscopy complemented DESI-IMS by providing more detailed distribution information. By combining results from spatial imaging and chiral separation, we were able to visualize the distribution of the predominant usnic acid enantiomer in lichen cross-sections: (+)-usnic acid in Cladonia arbuscula and Ramalina siliquosa, and (−)-usnic acid in Alectoria ochroleuca and Flavocetraria nivalis. This study provides an analytical foundation for future environmental and functional studies of usnic acid enantiomers in lichens.

Description: The genome of the wheat-pathogenic fungus Zymoseptoria tritici represents extensive presence-absence variation in gene content. Here, we addressed variation in biosynthetic gene cluster (BGC) content and biochemical profiles among three isolates. We analyzed secondary metabolite properties based on genome, transcriptome, and metabolome data. The isolates represent highly distinct genome architecture but harbor similar repertoires of BGCs. Expression profiles for most BGCs show comparable patterns of regulation among the isolates, suggesting a conserved biochemical infection program. For all three isolates, we observed a strong upregulation of a putative abscisic acid (ABA) gene cluster during biotrophic host colonization, indicating that Z. tritici interferes with host defenses by the biosynthesis of this phytohormone. Further, during in vitro growth, the isolates show similar metabolomes congruent with the predicted BGC content. We assessed if secondary metabolite production is regulated by histone methylation using a mutant impaired in formation of facultative heterochromatin (H3K27me3). In contrast to other ascomycete fungi, chromatin modifications play a less prominent role in regulation of secondary metabolites. In summary, we show that Z. tritici has a conserved program of secondary metabolite production, contrasting with the immense variation in effector expression, and some of these metabolites might play a key role during host colonization. IMPORTANCE Zymoseptoria tritici is one of the most devastating pathogens of wheat. So far the molecular determinants of virulence and their regulation are poorly understood. Previous studies have focused on proteinaceous virulence factors and their extensive diversity. In this study, we focus on secondary metabolites produced by Z. tritici. Using a comparative framework, we characterize core and noncore metabolites produced by Z. tritici by combining genome, transcriptome, and metabolome data sets. Our findings indicate highly conserved biochemical profiles with contrasting genetic and phenotypic diversity of the field isolates investigated here. This discovery has relevance for future crop protection strategies.