Culture-Dependent Microbiome of the Ciona intestinalis Tunic: Isolation, Bioactivity Profiling and Untargeted Metabolomics

Ascidians and their associated microbiota are prolific producers of bioactive marine natural products. Recent culture-independent studies have revealed that the tunic of the solitary ascidian Cionaintestinalis (sea vase) is colonized by a diverse bacterial community, however, the biotechnological potential of this community has remained largely unexplored. In this study, we aimed at isolating the culturable microbiota associated with the tunic of C.intestinalis collected from the North and Baltic Seas, to investigate their antimicrobial and anticancer activities, and to gain first insights into their metabolite repertoire. The tunic of the sea vase was found to harbor a rich microbial community, from which 89 bacterial and 22 fungal strains were isolated. The diversity of the tunic-associated microbiota differed from that of the ambient seawater samples, but also between sampling sites. Fungi were isolated for the first time from the tunic of Ciona. The proportion of bioactive extracts was high, since 45% of the microbial extracts inhibited the growth of human pathogenic bacteria, fungi or cancer cell lines. In a subsequent bioactivity- and metabolite profiling-based approach, seven microbial extracts were prioritized for in-depth chemical investigations. Untargeted metabolomics analyses of the selected extracts by a UPLC-MS/MS-based molecular networking approach revealed a vast chemical diversity with compounds assigned to 22 natural product families, plus many metabolites that remained unidentified. This initial study indicates that bacteria and fungi associated with the tunic of C.intestinalis represent an untapped source of putatively new marine natural products with pharmacological relevance

Combined genotyping, microbial diversity and metabolite profiling studies on farmed Mytilus spp. from Kiel Fjord

The blue mussel Mytilus is a popular food source with high economical value. Species of the M. edulis complex (M. edulis, M. galloprovincialis and M. trossulus) hybridise whenever their geographic ranges overlap posing difficulties to species discrimination, which is important for blue mussel aquaculture. The aim of this study was to determine the genetic structure of farmed blue mussels in Kiel Fjord. Microbial and metabolic profile patterns were studied to investigate a possible dependency on the genotype of the bivalves. Genotyping confirmed the complex genetic structure of the Baltic Sea hybrid zone and revealed an unexpected dominance of M. trossulus alleles being in contrast to the predominance of M. edulis alleles described for wild Baltic blue mussels. Culture-dependent and -independent microbial community analyses indicated the presence of a diverse Mytilus-associated microbiota, while an LC-MS/MS-based metabolome study identified 76 major compounds dominated by pigments, alkaloids and polyketides in the whole tissue extracts. Analysis of mussel microbiota and metabolome did not indicate genotypic dependence, but demonstrated high intraspecific variability of farmed mussel individuals. We hypothesise that individual differences in microbial and metabolite patterns may be caused by high individual plasticity and might be enhanced by e.g. nutritional condition, age and gender

Comparative Microbiome and Metabolome Analyses of the Marine Tunicate Ciona intestinalis from Native and Invaded Habitats

Massive fouling by the invasive ascidian Ciona intestinalis in Prince Edward Island (PEI, Canada) has been causing devastating losses to the local blue mussel farms. In order to gain first insights into so far unexplored factors that may contribute to the invasiveness of C. intestinalis in PEI, we undertook comparative microbiome and metabolome studies on specific tissues from C. intestinalis populations collected in invaded (PEI) and native regions (Helgoland and Kiel, Germany). Microbial community analyses and untargeted metabolomics revealed clear location- and tissue-specific patterns showing that biogeography and the sampled tissue shape the microbiome and metabolome of C. intestinalis. Moreover, we observed higher microbial and chemical diversity in C. intestinalis from PEI than in the native populations. Bacterial OTUs specific to C. intestinalis from PEI included Cyanobacteria (e.g., Leptolyngbya sp.) and Rhodobacteraceae (e.g., Roseobacter sp.), while populations from native sampling sites showed higher abundances of e.g., Firmicutes (Helgoland) and Epsilonproteobacteria (Kiel). Altogether 121 abundant metabolites were putatively annotated in the global ascidian metabolome, of which 18 were only detected in the invasive PEI population (e.g., polyketides and terpenoids), while six (e.g., sphingolipids) or none were exclusive to the native specimens from Helgoland and Kiel, respectively. Some identified bacteria and metabolites reportedly possess bioactive properties (e.g., antifouling and antibiotic) that may contribute to the overall fitness of C. intestinalis. Hence, this first study provides a basis for future studies on factors underlying the global invasiveness of Ciona species

Mapping the Surface Microbiome and Metabolome of Brown Seaweed Fucus vesiculosus by Amplicon Sequencing, Integrated Metabolomics and Imaging Techniques

the brown alga Fucus vesiculosus is a keystone marine species, which is subject to heavy surface colonisation. this study was designed to analyse the surface epibiome of F. vesiculosus in conjunction with the composition and spatial distribution of its surface metabolome. the amplicon sequencing, seM and CARD-FIsH imaging studies showed Alphaproteobacteria to predominate the epibiotic bacteria. Fungi of the class Eurotiomycetes were visualised for the first time on an algal surface. An untargeted metabolomics approach using molecular networks, in silico prediction and manual dereplication showed the differential metabolome of the surface and the whole tissue extracts. In total, 50 compounds were putatively dereplicated by UPLC-MS/MS, 37 of which were previously reported from both seaweeds and microorganisms. Untargeted spatial metabolomics by DESI-Imaging MS identified the specific localisation and distribution of various primary and secondary metabolites on surface imprints and in algal cross sections. The UPLC-MS, DESI-IMS and NMR analyses failed to confirm the presence of any surface-associated metabolite, except for mannitol, which were previously reported from F. vesiculosus. This is the first study analysing the seaweed surface microbiome in conjunction with untargeted surface metabolomics and spatial metabolomics approaches

Iron limitation of microbial phosphorus acquisition in the tropical North Atlantic

In certain regions of the predominantly nitrogen limited ocean, microbes can become co-limited by phosphorus. Within such regions, a proportion of the dissolved organic phosphorus pool can be accessed by microbes employing a variety of alkaline phosphatase (APase) enzymes. In contrast to the PhoA family of APases that utilize zinc as a cofactor, the recent discovery of iron as a cofactor in the more widespread PhoX and PhoD implies the potential for a biochemically dependant interplay between oceanic zinc, iron and phosphorus cycles. Here we demonstrate enhanced natural community APase activity following iron amendment within the low zinc and moderately low iron Western North Atlantic. In contrast we find no evidence for trace metal limitation of APase activity beneath the Saharan dust plume in the Eastern Atlantic. Such intermittent iron limitation of microbial phosphorus acquisition provides an additional facet in the argument for iron controlling the coupling between oceanic nitrogen and phosphorus cycles

Ciona intestinalis in the spotlight of metabolomics and microbiomics : New insights into its invasiveness and the biotechnological potential of its associated microbiota

The tunicate Ciona intestinalis is one of the most notorious invasive ascidian species. In Prince Edward Island (PEI, Canada), C. intestinalis causes heavy fouling on farmed mussels leading to significant economic losses. Except for general beneficial eco-physiological characteristics of invasive ascidians, reasons underlying C. intestinalis’ invasiveness remain obscure. This study aimed to shed light on two additional factors potentially promoting its invasion success, i.e., bioactive secondary metabolites and associated microbiota, which reportedly contribute to the invasiveness of other marine species. Therefore, microbiomes and metabolomes of invasive (PEI) and native (Helgoland and Kiel, Germany) C. intestinalis populations were comparatively studied, a novelty in invasive ascidian research. Apart from being problematic invasive species, ascidians and their associated microbiota are a rich source for bioactive marine natural products (MNPs) relevant for human health. However, the biodiscovery potential of C. intestinalis-associated microorganisms remains largely unknown. Accordingly, this doctoral research project targeted to explore bioactivities and the chemical repertoire of culturable bacteria and fungi associated with C. intestinalis. Amplicon sequencing-based bacterial community analysis of gut, tunic, and seawater (control) samples revealed species-specificity and a diverse microbiota (39 phyla). The UPLC-MS/MS-based untargeted metabolomics approach revealed a diverse chemical inventory dominated by alkaloids and lipids. In addition to core bacteria and metabolites present in all samples, also tissue- and location-specific bacteria and metabolites were observed. Notably, highest microbial and chemical diversity were detected in the invasive C. intestinalis population (PEI). In combination, these results suggest a high adaptive capacity of C. intestinalis. In addition, several detected bacteria and secondary metabolites reportedly have antimicrobial, antifouling, and other relevant bioactivities, potentially promoting its overall health, fitness, and competitiveness. In conjunction with microbiome data, this first global metabolome study on C. intestinalis indicated microbial associates and chemical weapons as additional relevant factors promoting its invasion success. Therefore, this work contributes important basic knowledge for future projects scrutinizing the invasiveness of C. intestinalis. To investigate the potential of microorganisms associated with C. intestinalis in marine biodiscovery, isolates were obtained from tunics (T) and guts (G) due to their pivotal functions for the ascidian’s defense against, e.g., pathogens, and their reportedly different bacterial communities. In total, 89 (T) and 61 (G) bacteria as well as 22 (T) and 40 (G) fungi were isolated and identified from Helgoland and Kiel specimens. Many extracts showed antibacterial (T: 42%, G: 64%), antifungal (T: 10%, G: 11%), and/or anticancer (T: 6%, G: 22%) activities. A 2-step selection procedure considering bioactivity and metabolite profiles was applied to prioritize the most promising MNPs producers. This led to the selection of seven tunic- and nine gut-derived microbial extracts affiliated to the fungal group of ascomycetes (69%) and the bacterial taxa Actinobacteria (25%) and Bacillus sp. (6%). Through an UPLC-MS/MS-based dereplication workflow including molecular networking, in-silico approaches and manual database comparison, 170 compounds belonging to >40 different chemical families were putatively annotated, displaying a vast chemical diversity. Although this represents a significant increase in annotation rates compared to previous studies, still many compounds even from well-studied organisms (e.g., Penicillium and Streptomyces spp.) remained unknown. In summary, this study demonstrated a huge pharmaceutical potential of the culturable microbiota associated with C. intestinalis, including discovery of various putatively novel compounds. Application of novel selection and integrated dereplication procedures proved successful for strain prioritization and compound annotation. Furthermore, this strategy highlighted particularly fungi as so far uncharted and exceptionally promising resource for putatively novel anticancer and antimicrobial lead compounds of high interest

Diversity, Bioactivity Profiling and Untargeted Metabolomics of the Cultivable Gut Microbiota of Ciona intestinalis

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