Impact of phosphate concentration on the metabolome of biofilms of the marine bacterium Pseudoalteromonas lipolytica.

INTRODUCTION: Marine biofilms are the most widely distributed mode of life on Earth and drive biogeochemical cycling processes of most elements. Phosphorus (P) is essential for many biological processes such as energy transfer mechanisms, biological information storage and membrane integrity. OBJECTIVES: Our aim was to analyze the effect of a gradient of ecologically relevant phosphate concentrations on the biofilm-forming capacity and the metabolome of the marine bacterium Pseudoalteromonas lipolytica TC8. METHODS: In addition to the evaluation of the effect of different phosphate concentration on the biomass, structure and gross biochemical composition of biofilms of P. lipolytica TC8, untargeted metabolomics based on liquid chromatography-mass spectrometry (LC-MS) analysis was used to determine the main metabolites impacted by P-limiting conditions. Annotation of the most discriminating and statistically robust metabolites was performed through the concomitant use of molecular networking and MS/MS fragmentation pattern interpretation. RESULTS: At the lowest phosphate concentration, biomass, carbohydrate content and three-dimensional structures of biofilms tended to decrease. Furthermore, untargeted metabolomics allowed for the discrimination of the biofilm samples obtained at the five phosphate concentrations and the highlighting of a panel of metabolites mainly implied in such a discrimination. A large part of the metabolites of the resulting dataset were then putatively annotated. Ornithine lipids were found in increasing quantity when the phosphate concentration decreased, while the opposite trend was observed for oxidized phosphatidylethanolamines (PEs). CONCLUSION: This study demonstrated the suitability of LC-MS-based untargeted metabolomics for evaluating the effect of culture conditions on marine bacterial biofilms. More precisely, these results supported the high plasticity of the membrane of P. lipolytica TC8, while the role of the oxidized PEs remains to be clarified.

Synergistic effects of temperature and light affect the relationship between Taonia atomaria and its epibacterial community: a controlled conditions study.

In the context of global warming, this study aimed to assess the effect of temperature and irradiance on the macroalgal Taonia atomaria holobiont dynamics. We developed an experimental set-up using aquaria supplied by natural seawater with three temperatures combined with three irradiances. The holobiont response was monitored over 14 days using a multi-omics approach coupling algal surface metabolomics and metabarcoding. Both temperature and irradiance appeared to shape the microbiota and the surface metabolome, but with a distinct temporality. Epibacterial community first changed according to temperature, and later in relation to irradiance, while the opposite occurred for the surface metabolome. An increased temperature revealed a decreasing richness of the epiphytic community together with an increase of several bacterial taxa. Irradiance changes appeared to quickly impact surface metabolites production linked with the algal host photosynthesis (e.g. mannitol, fucoxanthin, dimethylsulfoniopropionate), which was hypothesized to explain modifications of the structure of the epiphytic community. Algal host may also directly adapt its surface metabolome to changing temperature with time (e.g. lipids content) and also in response to changing microbiota (e.g. chemical defences). Finally, this study brought new insights highlighting complex direct and indirect responses of seaweeds and their associated microbiota under changing environments.

Temporal covariation of epibacterial community and surface metabolome in the Mediterranean seaweed holobiont Taonia atomaria.

An integrative multi-omics approach allowed monthly variations for a year of the surface metabolome and the epibacterial community of the Mediterranean Phaeophyceae Taonia atomaria to be investigated. The LC-MS-based metabolomics and 16S rDNA metabarcoding data sets were integrated in a multivariate meta-omics analysis (multi-block PLS-DA from the MixOmic DIABLO analysis) showing a strong seasonal covariation (Mantel test: p < 0.01). A network based on positive and negative correlations between the two data sets revealed two clusters of variables, one relative to the 'spring period' and a second to the 'summer period'. The 'spring period' cluster was mainly characterized by dipeptides positively correlated with a single bacterial taxon of the Alteromonadaceae family (BD1-7 clade). Moreover, 'summer' dominant epibacterial taxa from the second cluster (including Erythrobacteraceae, Rhodospirillaceae, Oceanospirillaceae and Flammeovirgaceae) showed positive correlations with few metabolites known as macroalgal antifouling defences [e.g. dimethylsulphoniopropionate (DMSP) and proline] which exhibited a key role within the correlation network. Despite a core community that represents a significant part of the total epibacteria, changes in the microbiota structure associated with surface metabolome variations suggested that both environment and algal host shape the bacterial surface microbiota.

Metabarcoding and metabolomics offer complementarity in deciphering marine eukaryotic biofouling community shifts.

Metabarcoding and metabolomics were used to explore the taxonomic composition and functional diversity of eukaryotic biofouling communities on plates with antifouling paints at two French coastal sites: Lorient (North Eastern Atlantic Ocean; temperate and eutrophic) and Toulon (North-Western Mediterranean Sea; mesotrophic but highly contaminated). Four distinct coatings were tested at each site and season for one month. Metabarcoding showed biocidal coatings had less impact on eukaryotic assemblages compared to spatial and temporal effects. Ciliophora, Chlorophyceae or Cnidaria (mainly hydrozoans) were abundant at Lorient, whereas Arthropoda (especially crustaceans), Nematoda, and Ochrophyta dominated less diversified assemblages at Toulon. Seasonal shifts were observed at Lorient, but not Toulon. Metabolomics also showed clear site discrimination, but these were associated with a coating and not season dependent clustering. The meta-omics analysis enabled identifications of some associative patterns between metabolomic profiles and specific taxa, in particular those colonizing the plates with biocidal coatings at Lorient.

Metabolome and proteome changes between biofilm and planktonic phenotypes of the marine bacterium Pseudoalteromonas lipolytica TC8.

A number of bacteria adopt various lifestyles such as planktonic free-living or sessile biofilm stages. This enables their survival and development in a wide range of contrasting environments. With the aim of highlighting specific metabolic shifts between these phenotypes and to improve the overall understanding of marine bacterial adhesion, a dual metabolomics/proteomics approach was applied to planktonic and biofilm cultures of the marine bacterium Pseudoalteromonas lipolytica TC8. The liquid chromatography mass spectrometry (LC-MS) based metabolomics study indicated that membrane lipid composition was highly affected by the culture mode: phosphatidylethanolamine (PEs) derivatives were over-produced in sessile cultures while ornithine lipids (OLs) were more specifically synthesized in planktonic samples. In parallel, differences between proteomes revealed that peptidases, oxidases, transcription factors, membrane proteins and the enzymes involved in histidine biosynthesis were over-expressed in biofilms while proteins involved in heme production, nutrient assimilation, cell division and arginine/ornithine biosynthesis were specifically up-regulated in free-living cells.

Anti-Bacterial Adhesion Activity of Tropical Microalgae Extracts.

The evolution of regulations concerning biocidal products aimed towards an increased protection of the environment (e.g., EU Regulation No 528/2012) requires the development of new non-toxic anti-fouling (AF) systems. As the marine environment is an important source of inspiration, such AF systems inhibiting the adhesion of organisms without any toxicity could be based on molecules of natural origin. In this context, the antibiofilm potential of tropical microalgal extracts was investigated. The tropics are particularly interesting in terms of solar energy and temperatures which provide a wide marine diversity and a high production of microalgae. Twenty microalgal strains isolated from the Indian Ocean were studied. Their extracts were characterized in terms of global chemical composition by high resolution magic angle spinning (HR-MAS) and nuclear magnetic resonance (NMR) spectroscopy, toxicity against marine bacteria (viability and growth) and anti-adhesion effect. The different observations made by confocal laser scanning microscopy (CLSM) showed a significant activity of three extracts from Dinoflagellate strains against the settlement of selected marine bacteria without any toxicity at a concentration of 50 µg/mL. The Symbiodinium sp. (P-78) extract inhibited the adhesion of Bacillus sp. 4J6 (Atlantic Ocean), Shewanella sp. MVV1 (Indian Ocean) and Pseudoalteromonas lipolytica TC8 (Mediterranean Ocean) at 60, 76 and 52%, respectively. These results underlined the potential of using microalgal extracts to repel fouling organisms.

Discrimination of Four Marine Biofilm-Forming Bacteria by LC-MS Metabolomics and Influence of Culture Parameters.

Most marine bacteria can form biofilms, and they are the main components of biofilms observed on marine surfaces. Biofilms constitute a widespread life strategy, as growing in such structures offers many important biological benefits. The molecular compounds expressed in biofilms and, more generally, the metabolomes of marine bacteria remain poorly studied. In this context, a nontargeted LC-MS metabolomics approach of marine biofilm-forming bacterial strains was developed. Four marine bacteria, Persicivirga (Nonlabens) mediterranea TC4 and TC7, Pseudoalteromonas lipolytica TC8, and Shewanella sp. TC11, were used as model organisms. The main objective was to search for some strain-specific bacterial metabolites and to determine how culture parameters (culture medium, growth phase, and mode of culture) may affect the cellular metabolism of each strain and thus the global interstrain metabolic discrimination. LC-MS profiling and statistical partial least-squares discriminant analyses showed that the four strains could be differentiated at the species level whatever the medium, the growth phase, or the mode of culture (planktonic vs biofilm). A MS/MS molecular network was subsequently built and allowed the identification of putative bacterial biomarkers. TC8 was discriminated by a series of ornithine lipids, while the P. mediterranea strains produced hydroxylated ornithine and glycine lipids. Among the P. mediterranea strains, TC7 extracts were distinguished by the occurrence of diamine derivatives, such as putrescine amides.

Surface metabolites of the brown alga Taonia atomaria have the ability to regulate epibiosis.

This study aimed to improve understanding of the strategies developed by the Mediterranean seaweed Taonia atomaria to chemically control bacterial epibiosis. An experimental protocol was optimized to specifically extract algal surface-associated metabolites by a technique involving dipping in organic solvents whilst the integrity of algal cell membranes was assessed by fluorescent microscopy. This methodology was validated using mass spectrometry-based profiles of algal extracts and analysis of their principal components, which led to the selection of methanol as the extraction solvent with a maximum exposure time of 15 s. Six compounds (A-F) were identified in the resulting surface extracts. Two of these surface-associated compounds (B and C) showed selective anti-adhesion properties against reference bacterial strains isolated from artificial surfaces while remaining inactive against epibiotic bacteria of T. atomaria. Such specificity was not observed for commercial antifouling biocides and other molecules identified in the surface or whole-cell extracts of T. atomaria.

Characterization and anti-biofilm activity of extracellular polymeric substances produced by the marine biofilm-forming bacterium Pseudoalteromonas ulvae strain TC14.

This study investigated soluble (Sol-EPS), loosely bound (LB-EPS), and tightly bound extracellular polymeric substances (TB-EPS) harvested from biofilm and planktonic cultures of the marine bacterium Pseudoalteromonas ulvae TC14. The aim of the characterization (colorimetric methods, FTIR, GC-MS, NMR, HPGPC, and AFM analyses) was to identify new anti-biofilm compounds; activity was assessed using the BioFilm Ring Test®. A step-wise separation of EPS was designed, based on differences in water-solubility and acidity. An acidic fraction was isolated from TB-EPS, which strongly inhibited biofilm formation by marine bacterial strains in a concentration-dependent manner. The main constituents of this fraction were characterized as two glucan-like polysaccharides. An active poly(glutamyl-glutamate) fraction was also recovered from TB-EPS. The distribution of these key EPS components in Sol-EPS, LB-EPS, and TB-EPS was distinct and differed quantitatively in biofilm vs planktonic cultures. The anti-biofilm potential of the fractions emphasizes the putative antifouling role of EPS in the environment.

Protective effects of ethyl acetate fraction of Lawsonia inermis fruits extract against carbon tetrachloride-induced oxidative damage in rat liver.

This study aimed to investigate the antioxidant properties of different fractions obtained from the fruits of Lawsonia inermis, a widely used medicinal plant, against carbon tetrachloride (CCl4)-induced oxidative stress in rat liver. The results show that several fractions obtained from L. inermis fruits possessed important antioxidant activity. Among them, the ethyl acetate (EA) fraction showed the highest antioxidant activity. Then, EA fraction was selected for the purification of potential antioxidant compounds. The hepatoprotective effects of EA fraction and its most active constituent, gallic acid (GA), were evaluated against CCl4-induced hepatotoxicity in rats. CCl4 induced oxidative stress by a significant rise in serum marker enzymes. However, pretreatment of rats with EA fraction of fruits of L. inermis at a dose of 250 mg kg(-1)body weight and GA significantly lowered some serum biochemical parameters (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase) in treated rats. A significant reduction in hepatic thiobarbituric acid reactive substances and an increase in antioxidant enzymes namely superoxide dismutase, catalase, and glutathione peroxidase by treatment with plant extract and GA, against CCl4-treated rats, were observed. Histopathological examinations showed extensive liver injuries, characterized by extensive hepatocellular necrosis, vacuolization, and inflammatory cell infiltration. This potential antioxidant activity is comparable to those of the major purified antioxidant compound, GA. Based on these results, it was observed that fruits of L. inermis protect liver from oxidative stress induced by CCl4 and thus help in evaluation of traditional claim on this plant.

Modulation of violacein production and phenotypes associated with biofilm by exogenous quorum sensing N-acylhomoserine lactones in the marine bacterium Pseudoalteromonas ulvae TC14.

Various phenotypes ranging from biofilm formation to pigment production have been shown to be regulated by quorum sensing (QS) in many bacteria. However, studies of the regulation of pigments produced by marine bacteria in saline conditions and of biofilm-associated phenotypes are scarcer. This study focuses on the demonstration of the existence of a QS communication system involving N-acylhomoserine lactones (AHLs) in the Mediterranean Sea strain Pseudoalteromonas ulvae TC14. We have investigated whether TC14 produces the violacein pigment, and whether intrinsic or exogenous AHLs could influence its production and modulate biofilm-associated phenotypes. Here, we demonstrate that the purple pigment produced by TC14 is violacein. The study shows that in planktonic conditions, TC14 produces more pigment in the medium in which it grows less. Using different approaches, the results also show that TC14 does not produce intrinsic AHLs in our conditions. When exogenous AHLs are added in planktonic conditions, the production of violacein is upregulated by C6-, C12-, 3-oxo-C8 and 3-oxo-C12-HSLs (homoserine lactones), and downregulated by 3-oxo-C6-HSL. In sessile conditions, 3-oxo-C8-HSL upregulates the production of violacein. The study of the biofilm-associated phenotypes shows that oxo-derived-HSLs decrease adhesion, swimming and biofilm formation. While 3-oxo-C8 and 3-oxo-C12-HSLs decrease both swimming and adhesion, 3-oxo-C6-HSLs decrease not only violacein production in planktonic conditions but also swimming, adhesion and more subtly biofilm formation. Therefore, TC14 may possess a functional LuxR-type QS receptor capable of sensing extrinsic AHLs, which controls violacein production, motility, adhesion and biofilm formation.

Cystophloroketals A-E, Unusual Phloroglucinol-Meroterpenoid Hybrids from the Brown Alga Cystoseira tamariscifolia.

Cystophloroketals A-E (1-5), five new phloroglucinol-meroditerpenoid hybrids, have been isolated together with their putative biosynthetic precursor, the monocyclic meroditerpenoid 6, from the Mediterranean brown alga Cystoseira tamariscifolia. They represent the first examples of meroditerpenoids linked to a phloroglucinol through a 2,7-dioxabicyclo[3.2.1]octane moiety. The chemical structures, including absolute configurations, were elucidated on the basis of extensive spectroscopic analysis (HR-ESIMS, 1D and 2D NMR, and ECD) and TDDFT ECD calculations. Compounds 1-6 were tested for their antifouling activity against several marine colonizing species (bacteria, fungi, invertebrates, micro- and macroalgae). Compound 6 showed high potency for the inhibition of macrofoulers (invertebrates and macroalgae), while cystophloroketals B (2) and D (4) displayed strong inhibition of the germination of the two macroalgae tested and moderate antimicrobial activities (bacteria, microalgae, and fungi).

Antitumor and antimicrobial potential of bromoditerpenes isolated from the red alga, Sphaerococcus coronopifolius.

Cancer and infectious diseases continue to be a major public health problem, and new drugs are necessary. As marine organisms are well known to provide a wide range of original compounds, the aim of this study was to investigate the bioactivity of the main constituents of the cosmopolitan red alga, Sphaerococcus coronopifolius. The structure of several bromoditerpenes was determined by extensive spectroscopic analysis and comparison with literature data. Five molecules were isolated and characterized which include a new brominated diterpene belonging to the rare dactylomelane family and named sphaerodactylomelol (1), along with four already known sphaerane bromoditerpenes (2-5). Antitumor activity was assessed by cytotoxicity and anti-proliferative assays on an in vitro model of human hepatocellular carcinoma (HepG-2 cells). Antimicrobial activity was evaluated against four pathogenic microorganisms: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans. Compound 4 exhibited the highest antimicrobial activity against S. aureus (IC50 6.35 µM) and compound 5 the highest anti-proliferative activity on HepG-2 cells (IC50 42.9 µM). The new diterpene, sphaerodactylomelol (1), induced inhibition of cell proliferation (IC50 280 µM) and cytotoxicity (IC50 720 µM) on HepG-2 cells and showed antimicrobial activity against S. aureus (IC50 96.3 µM).

Impact of hydrodynamics on community structure and metabolic production of marine biofouling formed in a highly energetic estuary.

Biofouling is a specific lifestyle including both marine prokaryotic and eukaryotic communities. Hydrodynamics are poorly studied parameters affecting biofouling formation. This study aimed to investigate how water dynamics in the Etel Estuary (Northwest Atlantic coasts of France) influences the colonization of artificial substrates. Hydrodynamic conditions, mainly identified as shear stress, were characterized by measuring current velocity, turbulence intensity and energy using Acoustic Doppler Current Profiler (ADCP). One-month biofouling was analyzed by coupling metabarcoding (16S rRNA, 18S rRNA and COI genes), untargeted metabolomics (liquid chromatography coupled with high-resolution mass spectrometry, LC-HRMS) and characterization of the main biochemical components of the microbial exopolymeric matrix. A higher richness was observed for biofouling communities (prokaryotes and eukaryotes) exposed to the strongest currents. Ectopleura (Cnidaria) and its putative symbionts Endozoicomonas (Gammaproteobacteria) were dominant in the less dynamic conditions. Eukaryotes assemblages were specifically shaped by shear stress, leading to drastic changes in metabolite profiles. Under high hydrodynamic conditions, the exopolymeric matrix increased and was composed of 6 times more polysaccharides than proteins, these latter playing a crucial role in the adhesion and cohesion properties of biofilms. This original multidisciplinary approach demonstrated the importance of shear stress on both the structure of marine biofouling and the metabolic response of these complex communities.

Correlation between synthesis variation of 2-alkylquinolones and the antifungal activity of a Burkholderia cepacia strain collection.

Twenty epiphytic and rhizospheric bacterial strains harbouring strong antifungal activities were isolated from the Tunisian environment. This group of bacteria was identified as Burkholderia cepacia genomovar I using 16S rDNA and recA fragment gene sequence analyses for two selected strains and RFLP technique for the eighteen other ones. This identification did not show variability between isolates despite the significant differences in the antifungal activities of their culture supernatant and the organic crude extract against Aspergillus niger and other phytopathogenic fungi. Chromatographic and mass spectrometric analyses of these extracts allowed us to confirm the difference between strains of the group. Their metabolic production showed differences in term of contents and quantities of secreted molecules, particularly those which were identified to be involved in the antifungal activities. Two metabolites, named Bc-255 and Bc-257 secreted by the entire group at different amounts, have been purified and tested separately against A. niger. Bc-255 showed an activity twice as high as those shown by Bc-257. The structural characterization of these two compounds by mass spectrometry and nuclear magnetic resonance spectroscopy allowed their identification as two analogous 2-alkylquinolones with only one difference at the alkyl chain.

Allelopathic Potential of Mangroves from the Red River Estuary against the Rice Weed Echinochloa crus-galli and Variation in Their Leaf Metabolome.

Mangroves are the only forests located at the sea-land interface in tropical and subtropical regions. They are key elements of tropical coastal ecosystems, providing numerous ecosystem services. Among them is the production of specialized metabolites by mangroves and their potential use in agriculture to limit weed growth in cultures. We explored the in vitro allelopathic potential of eight mangrove species' aqueous leaf extracts (Avicennia marina, Kandelia obovata, Bruguiera gymnorhiza, Sonneratia apetala, Sonneratia caseolaris, Aegiceras corniculatum, Lumnitzera racemosa and Rhizophora stylosa) on the germination and growth of Echinochloa crus-galli, a weed species associated with rice, Oryza sativa. Leaf methanolic extracts of mangrove species were also studied via UHPLC-ESI/qToF to compare their metabolite fingerprints. Our results highlight that A. corniculatum and S. apetala negatively affected E. crus-galli development with a stimulating effect or no effect on O. sativa. Phytochemical investigations of A. corniculatum allowed us to putatively annotate three flavonoids and two saponins. For S. apetala, three flavonoids, a tannin and two unusual sulfated ellagic acid derivatives were found. Some of these compounds are described for the first time in these species. Overall, A. corniculatum and S. apetala leaves are proposed as promising natural alternatives against E. crus-galli and should be further assessed under field conditions.

Antifouling properties of simple indole and purine alkaloids from the Mediterranean gorgonian Paramuricea clavata.

Chemical investigation of the Mediterranean gorgonian Paramuricea clavata resulted in the isolation of two new alkaloids, 2-bromo-N-methyltryptamine (1) and 3-bromo-N-methyltyramine (2), together with nine known compounds (3-10 and linderazulene). The bromoindole derivative 3 is reported herein for the first time from a natural source. The chemical structures of these compounds were assigned by spectroscopic analyses and comparison with literature values. The antifouling activity and toxicity of compounds 1-10 were assessed using three marine biofilm bacteria and the Microtox assay. In contrast to commercial antifoulants, bufotenine (5) and 1,3,7-trimethylisoguanine (10) showed significant antiadhesion activity against one bacterial strain while being nontoxic.

Environmental Burkholderia cepacia strain Cs5 acting by two analogous alkyl-quinolones and a didecyl-phthalate against a broad spectrum of phytopathogens fungi.

An environmental Burkholderia cepacia strain named Cs5 was isolated and identified first using API biochemical identification system and then with 16S rDNA and recA sequence homology search. This bacterium exhibited a broad spectrum of fungicidal activities against Alternaria alternata, Aspergillus niger, Fusarium culmorum, F. graminearum, F. oxysporum and Rhizoctonia solani. In the liquid conditions, the MIC of A. niger and R. solani were reached with, respectively, 1.25-2% of the Cs5 liquid culture supernatant. However, in the solid conditions, the same inhibition was caused in the presence of 3% of the Cs5 supernatant. The exhibition of these two fungi at low concentrations of supernatant Cs5 caused various morphological changes of their mycelia which were observed by confocal microscopy. Three antifungal compounds, named Cs5-255, Cs5-257 and Cs5-446, were purified from the Cs5 culture. The structural analysis of these molecules showed that Cs5-255 and Cs5-257 are analogous and belonged to the alkyl-quinolone family, while Cs5-446 was a didecyl-phthalate, isolated for the first time from a bacterium.

Integration of LC/MS-based molecular networking and classical phytochemical approach allows in-depth annotation of the metabolome of non-model organisms - The case study of the brown seaweed Taonia atomaria.

Untargeted LC-MS based metabolomics is a useful approach in many research areas such as medicine, systems biology, environmental sciences or even ecology. In such an approach, annotation of metabolomes of non-model organisms remains a significant challenge. In this study, an analytical workflow combining a classical phytochemical approach, using the isolation and the full characterization of the chemical structure of natural products, together with the use of MS/MS-based molecular networking with various levels of restrictiveness was developed. This protocol was applied to the marine brown seaweed Taonia atomaria, a cosmopolitan algal species, and allowed to annotate more than 200 metabolites. First, the algal organic crude extracts were fractionated by flash-chromatography and the chemical structure of eight of the main chemical constituents of this alga were fully characterized by means of spectroscopic methods (1D and 2D NMR, HRMS). These compounds were further used as chemical standards. In a second step, the main fractions of the algal extracts were analyzed by UHPLC-MS/MS and the resulting data were uploaded to the Global Natural Products Social Molecular Networking platform (GNPS) to create several molecular networks (MNs). A first MN (MN-1) was built with restrictive parameters and allowed the creation of clusters composed by nodes with highly similar MS/MS spectra. Then, using database hits and chemical standards as "seed" nodes and/or similarity between MS/MS fragmentation pattern, the main clusters were easily annotated as common glycerolipids and phospholipids, much rare lipids -such as acylglycerylhydroxymethyl-N,N,N-trimethyl-ß-alanines or fulvellic acid derivatives- but also new glycerolipids bearing a terpene moiety. Lastly, the use of less and less constrained MNs allowed to further increase the number of annotated metabolites.

Integration of spatio-temporal variations of surface metabolomes and epibacterial communities highlights the importance of copper stress as a major factor shaping host-microbiota interactions within a Mediterranean seaweed holobiont.

BACKGROUND: Although considered as holobionts, macroalgae and their surface microbiota share intimate interactions that are still poorly understood. Little is known on the effect of environmental parameters on the close relationships between the host and its surface-associated microbiota, and even more in a context of coastal pollutions. Therefore, the main objective of this study was to decipher the impact of local environmental parameters, especially trace metal concentrations, on an algal holobiont dynamics using the Phaeophyta Taonia atomaria as a model. Through a multidisciplinary multi-omics approach combining metabarcoding and untargeted LC-MS-based metabolomics, the epibacterial communities and the surface metabolome of T. atomaria were monitored along a spatio-temporal gradient in the bay of Toulon (Northwestern Mediterranean coast) and its surrounding. Indeed, this geographical area displays a well-described trace metal gradient particularly relevant to investigate the effect of such pollutants on marine organisms. RESULTS: Epibacterial communities of T. atomaria exhibited a high specificity whatever the five environmentally contrasted collecting sites investigated on the NW Mediterranean coast. By integrating metabarcoding and metabolomics analyses, the holobiont dynamics varied as a whole. During the occurrence period of T. atomaria, epibacterial densities and α-diversity increased while the relative proportion of core communities decreased. Pioneer bacterial colonizers constituted a large part of the specific and core taxa, and their decrease might be linked to biofilm maturation through time. Then, the temporal increase of the Roseobacter was proposed to result from the higher temperature conditions, but also the increased production of dimethylsulfoniopropionate (DMSP) at the algal surface which could constitute of the source of carbon and sulfur for the catabolism pathways of these taxa. Finally, as a major result of this study, copper concentration constituted a key factor shaping the holobiont system. Thus, the higher expression of carotenoids suggested an oxidative stress which might result from an adaptation of the algal surface metabolome to high copper levels. In turn, this change in the surface metabolome composition could result in the selection of particular epibacterial taxa. CONCLUSION: We showed that associated epibacterial communities were highly specific to the algal host and that the holobiont dynamics varied as a whole. While temperature increase was confirmed to be one of the main parameters associated to Taonia dynamics, the originality of this study was highlighting copper-stress as a major driver of seaweed-epibacterial interactions. In a context of global change, this study brought new insights on the dynamics of a Mediterranean algal holobiont submitted to heavy anthropic pressures. Video abstract.