Inferring and comparing metabolism across heterogeneous sets of annotated genomes using AuCoMe.

Comparative analysis of genome-scale metabolic networks (GSMNs) may yield important information on the biology, evolution, and adaptation of species. However, it is impeded by the high heterogeneity of the quality and completeness of structural and functional genome annotations, which may bias the results of such comparisons. To address this issue, we developed AuCoMe, a pipeline to automatically reconstruct homogeneous GSMNs from a heterogeneous set of annotated genomes without discarding available manual annotations. We tested AuCoMe with three data sets, one bacterial, one fungal, and one algal, and showed that it successfully reduces technical biases while capturing the metabolic specificities of each organism. Our results also point out shared and divergent metabolic traits among evolutionarily distant algae, underlining the potential of AuCoMe to accelerate the broad exploration of metabolic evolution across the tree of life.

Candidate genes involved in biosynthesis and degradation of the main extracellular matrix polysaccharides of brown algae and their probable evolutionary history.

BACKGROUND: Brown algae belong to the Stramenopiles phylum and are phylogenetically distant from plants and other multicellular organisms. This independent evolutionary history has shaped brown algae with numerous metabolic characteristics specific to this group, including the synthesis of peculiar polysaccharides contained in their extracellular matrix (ECM). Alginates and fucose-containing sulphated polysaccharides (FCSPs), the latter including fucans, are the main components of ECMs. However, the metabolic pathways of these polysaccharides remain poorly described due to a lack of genomic data. RESULTS: An extensive genomic dataset has been recently released for brown algae and their close sister species, for which we previously performed an expert annotation of key genes involved in ECM-carbohydrate metabolisms. Here we provide a deeper analysis of this set of genes using comparative genomics, phylogenetics analyses, and protein modelling. Two key gene families involved in both the synthesis and degradation of alginate were suggested to have been acquired by the common ancestor of brown algae and their closest sister species Schizocladia ischiensis. Our analysis indicates that this assumption can be extended to additional metabolic steps, and thus to the whole alginate metabolic pathway. The pathway for the biosynthesis of fucans still remains biochemically unresolved and we also investigate putative fucosyltransferase genes that may harbour a fucan synthase activity in brown algae. CONCLUSIONS: Our analysis is the first extensive survey of carbohydrate-related enzymes in brown algae, and provides a valuable resource for future research into the glycome and ECM of brown algae. The expansion of specific families related to alginate metabolism may have represented an important prerequisite for the evolution of developmental complexity in brown algae. Our analysis questions the possible occurrence of FCSPs outside brown algae, notably within their closest sister taxon and in other Stramenopiles such as diatoms. Filling this knowledge gap in the future will help determine the origin and evolutionary history of fucan synthesis in eukaryotes.

Degradation dynamics and processes associated with the accumulation of Laminaria hyperborea (Phaeophyceae) kelp fragments: an in situ experimental approach.

A high proportion of the kelp Laminaria hyperborea production is exported from kelp forests following seasonal storms or natural annual old blade loss. Transport of drifting kelp fragments can lead to temporary accumulations in benthic subtidal habitats. We investigated the degradation processes of L. hyperborea in a low subtidal sandy bottom ecosystem by setting up a 6-month cage experiment to simulate accumulations of kelp fragments on the seafloor. We monitored temporal changes in biomass, nutritional quality (C:N ratio), respiration, quantum efficiency of photosystem II (Fv /Fm ), bacterial colonization, and chemical defense concentrations. Biomass decomposition started after 2 weeks and followed a classic negative exponential pattern, leading to 50% degradation after 8 weeks. The degradation process seemed to reach a critical step after 11 weeks, with an increase in respiration rate and phlorotannin concentration in the tissues. These results likely reflect an increase in bacterial activity and a weakening of the kelp cell wall. After 25 weeks of degradation, only 16% of the initial biomass persisted, but the remaining large fragments looked intact. Furthermore, photosystems were still responding to light stimuli, indicating that photosynthesis persisted over time. Reproductive tissues appeared on some fragments after 20 weeks of degradation, showing a capacity to maintain the reproductive function. Our results indicate that L. hyperborea fragments degrade slowly. As they maintain major physiological functions (photosynthesis, reproduction, etc.) and accumulate on adjacent ecosystems, they may play a long-term ecological role in coastal ecosystem dynamics.

Identification and Characterization of a New Type III Polyketide Synthase from a Marine Yeast, Naganishia uzbekistanensis.

A putative Type III Polyketide synthase (PKSIII) encoding gene was identified from a marine yeast, Naganishia uzbekistanensis strain Mo29 (UBOCC-A-208024) (formerly named as Cryptococcus sp.) isolated from deep-sea hydrothermal vents. This gene is part of a distinct phylogenetic branch compared to all known terrestrial fungal sequences. This new gene encodes a C-terminus extension of 74 amino acids compared to other known PKSIII proteins like Neurospora crassa. Full-length and reduced versions of this PKSIII were successfully cloned and overexpressed in a bacterial host, Escherichia coli BL21 (DE3). Both proteins showed the same activity, suggesting that additional amino acid residues at the C-terminus are probably not required for biochemical functions. We demonstrated by LC-ESI-MS/MS that these two recombinant PKSIII proteins could only produce tri- and tetraketide pyrones and alkylresorcinols using only long fatty acid chain from C8 to C16 acyl-CoAs as starter units, in presence of malonyl-CoA. In addition, we showed that some of these molecules exhibit cytotoxic activities against several cancer cell lines.

Peptimapper: proteogenomics workflow for the expert annotation of eukaryotic genomes.

BACKGROUND: Accurate structural annotation of genomes is still a challenge, despite the progress made over the past decade. The prediction of gene structure remains difficult, especially for eukaryotic species, and is often erroneous and incomplete. We used a proteogenomics strategy, taking advantage of the combination of proteomics datasets and bioinformatics tools, to identify novel protein coding-genes and splice isoforms, assign correct start sites, and validate predicted exons and genes. RESULTS: Our proteogenomics workflow, Peptimapper, was applied to the genome annotation of Ectocarpus sp., a key reference genome for both the brown algal lineage and stramenopiles. We generated proteomics data from various life cycle stages of Ectocarpus sp. strains and sub-cellular fractions using a shotgun approach. First, we directly generated peptide sequence tags (PSTs) from the proteomics data. Second, we mapped PSTs onto the translated genomic sequence. Closely located hits (i.e., PSTs locations on the genome) were then clustered to detect potential coding regions based on parameters optimized for the organism. Third, we evaluated each cluster and compared it to gene predictions from existing conventional genome annotation approaches. Finally, we integrated cluster locations into GFF files to use a genome viewer. We identified two potential novel genes, a ribosomal protein L22 and an aryl sulfotransferase and corrected the gene structure of a dihydrolipoamide acetyltransferase. We experimentally validated the results by RT-PCR and using transcriptomics data. CONCLUSIONS: Peptimapper is a complementary tool for the expert annotation of genomes. It is suitable for any organism and is distributed through a Docker image available on two public bioinformatics docker repositories: Docker Hub and BioShaDock. This workflow is also accessible through the Galaxy framework and for use by non-computer scientists at https://galaxy.protim.eu . Data are available via ProteomeXchange under identifier PXD010618.

Structure/function analysis of a type iii polyketide synthase in the brown alga Ectocarpus siliculosus reveals a biochemical pathway in phlorotannin monomer biosynthesis.

Brown algal phlorotannins are structural analogs of condensed tannins in terrestrial plants and, like plant phenols, they have numerous biological functions. Despite their importance in brown algae, phlorotannin biosynthetic pathways have been poorly characterized at the molecular level. We found that a predicted type III polyketide synthase in the genome of the brown alga Ectocarpus siliculosus, PKS1, catalyzes a major step in the biosynthetic pathway of phlorotannins (i.e., the synthesis of phloroglucinol monomers from malonyl-CoA). The crystal structure of PKS1 at 2.85-Å resolution provided a good quality electron density map showing a modified Cys residue, likely connected to a long chain acyl group. An additional pocket not found in other known type III PKSs contains a reaction product that might correspond to a phloroglucinol precursor. In vivo, we also found a positive correlation between the phloroglucinol content and the PKS III gene expression level in cells of a strain of Ectocarpus adapted to freshwater during its reacclimation to seawater. The evolution of the type III PKS gene family in Stramenopiles suggests a lateral gene transfer event from an actinobacterium.

The Ectocarpus genome and the independent evolution of multicellularity in brown algae.

Brown algae (Phaeophyceae) are complex photosynthetic organisms with a very different evolutionary history to green plants, to which they are only distantly related. These seaweeds are the dominant species in rocky coastal ecosystems and they exhibit many interesting adaptations to these, often harsh, environments. Brown algae are also one of only a small number of eukaryotic lineages that have evolved complex multicellularity (Fig. 1). We report the 214 million base pair (Mbp) genome sequence of the filamentous seaweed Ectocarpus siliculosus (Dillwyn) Lyngbye, a model organism for brown algae, closely related to the kelps (Fig. 1). Genome features such as the presence of an extended set of light-harvesting and pigment biosynthesis genes and new metabolic processes such as halide metabolism help explain the ability of this organism to cope with the highly variable tidal environment. The evolution of multicellularity in this lineage is correlated with the presence of a rich array of signal transduction genes. Of particular interest is the presence of a family of receptor kinases, as the independent evolution of related molecules has been linked with the emergence of multicellularity in both the animal and green plant lineages. The Ectocarpus genome sequence represents an important step towards developing this organism as a model species, providing the possibility to combine genomic and genetic approaches to explore these and other aspects of brown algal biology further.

Genome structure and metabolic features in the red seaweed Chondrus crispus shed light on evolution of the Archaeplastida.

Red seaweeds are key components of coastal ecosystems and are economically important as food and as a source of gelling agents, but their genes and genomes have received little attention. Here we report the sequencing of the 105-Mbp genome of the florideophyte Chondrus crispus (Irish moss) and the annotation of the 9,606 genes. The genome features an unusual structure characterized by gene-dense regions surrounded by repeat-rich regions dominated by transposable elements. Despite its fairly large size, this genome shows features typical of compact genomes, e.g., on average only 0.3 introns per gene, short introns, low median distance between genes, small gene families, and no indication of large-scale genome duplication. The genome also gives insights into the metabolism of marine red algae and adaptations to the marine environment, including genes related to halogen metabolism, oxylipins, and multicellularity (microRNA processing and transcription factors). Particularly interesting are features related to carbohydrate metabolism, which include a minimalistic gene set for starch biosynthesis, the presence of cellulose synthases acquired before the primary endosymbiosis showing the polyphyly of cellulose synthesis in Archaeplastida, and cellulases absent in terrestrial plants as well as the occurrence of a mannosylglycerate synthase potentially originating from a marine bacterium. To explain the observations on genome structure and gene content, we propose an evolutionary scenario involving an ancestral red alga that was driven by early ecological forces to lose genes, introns, and intergenetic DNA; this loss was followed by an expansion of genome size as a consequence of activity of transposable elements.

The genome-scale metabolic network of Ectocarpus siliculosus (EctoGEM): a resource to study brown algal physiology and beyond.

Brown algae (stramenopiles) are key players in intertidal ecosystems, and represent a source of biomass with several industrial applications. Ectocarpus siliculosus is a model to study the biology of these organisms. Its genome has been sequenced and a number of post-genomic tools have been implemented. Based on this knowledge, we report the reconstruction and analysis of a genome-scale metabolic network for E. siliculosus, EctoGEM (http://ectogem.irisa.fr). This atlas of metabolic pathways consists of 1866 reactions and 2020 metabolites, and its construction was performed by means of an integrative computational approach for identifying metabolic pathways, gap filling and manual refinement. The capability of the network to produce biomass was validated by flux balance analysis. EctoGEM enabled the reannotation of 56 genes within the E. siliculosus genome, and shed light on the evolution of metabolic processes. For example, E. siliculosus has the potential to produce phenylalanine and tyrosine from prephenate and arogenate, but does not possess a phenylalanine hydroxylase, as is found in other stramenopiles. It also possesses the complete eukaryote molybdenum co-factor biosynthesis pathway, as well as a second molybdopterin synthase that was most likely acquired via horizontal gene transfer from cyanobacteria by a common ancestor of stramenopiles. EctoGEM represents an evolving community resource to gain deeper understanding of the biology of brown algae and the diversification of physiological processes. The integrative computational method applied for its reconstruction will be valuable to set up similar approaches for other organisms distant from biological benchmark models.

The Vanadium Iodoperoxidase from the marine flavobacteriaceae species Zobellia galactanivorans reveals novel molecular and evolutionary features of halide specificity in the vanadium haloperoxidase enzyme family.

Vanadium haloperoxidases (VHPO) are key enzymes that oxidize halides and are involved in the biosynthesis of organo-halogens. Until now, only chloroperoxidases (VCPO) and bromoperoxidases (VBPO) have been characterized structurally, mainly from eukaryotic species. Three putative VHPO genes were predicted in the genome of the flavobacterium Zobellia galactanivorans, a marine bacterium associated with macroalgae. In a phylogenetic analysis, these putative bacterial VHPO were closely related to other VHPO from diverse bacterial phyla but clustered independently from eukaryotic algal VBPO and fungal VCPO. Two of these bacterial VHPO, heterogeneously produced in Escherichia coli, were found to be strictly specific for iodide oxidation. The crystal structure of one of these vanadium-dependent iodoperoxidases, Zg-VIPO1, was solved by multiwavelength anomalous diffraction at 1.8 Å, revealing a monomeric structure mainly folded into α-helices. This three-dimensional structure is relatively similar to those of VCPO of the fungus Curvularia inaequalis and of Streptomyces sp. and is superimposable onto the dimeric structure of algal VBPO. Surprisingly, the vanadate binding site of Zg-VIPO1 is strictly conserved with the fungal VCPO active site. Using site-directed mutagenesis, we showed that specific amino acids and the associated hydrogen bonding network around the vanadate center are essential for the catalytic properties and also the iodide specificity of Zg-VIPO1. Altogether, phylogeny and structure-function data support the finding that iodoperoxidase activities evolved independently in bacterial and algal lineages, and this sheds light on the evolution of the VHPO enzyme family.

Empirical evidence for metabolic drift in plant and algal lipid biosynthesis pathways.

Metabolic pathway drift has been formulated as a general principle to help in the interpretation of comparative analyses between biosynthesis pathways. Indeed, such analyses often indicate substantial differences, even in widespread pathways that are sometimes believed to be conserved. Here, our purpose is to check how much this interpretation fits to empirical data gathered in the field of plant and algal biosynthesis pathways. After examining several examples representative of the diversity of lipid biosynthesis pathways, we explain why it is important to compare closely related species to gain a better understanding of this phenomenon. Furthermore, this comparative approach brings us to the question of how much biotic interactions are responsible for shaping this metabolic plasticity. We end up introducing some model systems that may be promising for further exploration of this question.

Hyper-accumulation of vanadium in animals: Two sponges compete with urochordates.

Vanadium (V) concentrations in organisms are usually very low. To date, among animals, only some urochordate and annelid species contain very high levels of V in their tissues. A new case of hyper-accumulation of V in a distinct animal phylum (Porifera), namely, the two homoscleromorph sponge species Oscarella lobularis and O. tuberculata is reported. The measured concentrations (up to 30 g/kg dry weight) exceed those reported previously and are not found in all sponge classes. In both Oscarella species, V is mainly accumulated in the surface tissues, and in mesohylar cells, as V(IV), before being partly reduced to V(III) in the deeper tissues. Candidate genes from Bacteria and sponges have been identified as possibly being involved in the metabolism of V. This finding provides clues for the development of bioremediation strategies in marine ecosystems and/or bioinspired processes to recycle this critical metal.

X-ray structure and mechanism of ZgHAD, a l-2-haloacid dehalogenase from the marine Flavobacterium Zobellia galactanivorans.

Haloacid dehalogenases are potentially involved in bioremediation of contaminated environments and few have been biochemically characterized from marine organisms. The l-2-haloacid dehalogenase (l-2-HAD) from the marine Bacteroidetes Zobellia galactanivorans DsijT (ZgHAD) has been shown to catalyze the dehalogenation of C2 and C3 short-chain l-2-haloalkanoic acids. To better understand its catalytic properties, its enzymatic stability, active site, and 3D structure were analyzed. ZgHAD demonstrates high stability to solvents and a conserved catalytic activity when heated up to 60°C, its melting temperature being at 65°C. The X-ray structure of the recombinant enzyme was solved by molecular replacement. The enzyme folds as a homodimer and its active site is very similar to DehRhb, the other known l-2-HAD from a marine Rhodobacteraceae. Marked differences are present in the putative substrate entrance sites of the two enzymes. The H179 amino acid potentially involved in the activation of a catalytic water molecule was confirmed as catalytic amino acid through the production of two inactive site-directed mutants. The crystal packing of 13 dimers in the asymmetric unit of an active-site mutant, ZgHAD-H179N, reveals domain movements of the monomeric subunits relative to each other. The involvement of a catalytic His/Glu dyad and substrate binding amino acids was further confirmed by computational docking. All together our results give new insights into the catalytic mechanism of the group of marine l-2-HAD.

Heterologous Expression and Biochemical Characterization of a New Chloroperoxidase Isolated from the Deep-Sea Hydrothermal Vent Black Yeast Hortaea werneckii UBOCC-A-208029.

The initiation of this study relies on a targeted genome-mining approach to highlight the presence of a putative vanadium-dependent haloperoxidase-encoding gene in the deep-sea hydrothermal vent fungus Hortaea werneckii UBOCC-A-208029. To date, only three fungal vanadium-dependent haloperoxidases have been described, one from the terrestrial species Curvularia inaequalis, one from the fungal plant pathogen Botrytis cinerea, and one from a marine derived isolate identified as Alternaria didymospora. In this study, we describe a new vanadium chloroperoxidase from the black yeast H. werneckii, successfully cloned and overexpressed in a bacterial host, which possesses higher affinity for bromide (Km = 26 µM) than chloride (Km = 237 mM). The enzyme was biochemically characterized, and we have evaluated its potential for biocatalysis by determining its stability and tolerance in organic solvents. We also describe its potential three-dimensional structure by building a model using the AlphaFold 2 artificial intelligence tool. This model shows some conservation of the 3D structure of the active site compared to the vanadium chloroperoxidase from C. inaequalis but it also highlights some differences in the active site entrance and the volume of the active site pocket, underlining its originality.

Proteomic studies highlight outer-membrane proteins related to biofilm development in the marine bacterium Pseudoalteromonas sp. D41.

Bacterial biofilm development is conditioned by complex processes involving bacterial attachment to surfaces, growth, mobility, and exoproduct production. The marine bacterium Pseudoalteromonas sp. strain D41 is able to attach strongly onto a wide variety of substrates, which promotes subsequent biofilm development. Study of the outer-membrane and total soluble proteomes showed ten spots with significant intensity variations when this bacterium was grown in biofilm compared to planktonic cultures. MS/MS de novo sequencing analysis allowed the identification of four outer-membrane proteins of particular interest since they were strongly induced in biofilms. These proteins are homologous to a TonB-dependent receptor (TBDR), to the OmpW and OmpA porins, and to a type IV pilus biogenesis protein (PilF). Gene expression assays by quantitative RT-PCR showed that the four corresponding genes were upregulated during biofilm development on hydrophobic and hydrophilic surfaces. The Pseudomonas aeruginosa mutants unable to produce any of the OmpW, OmpA, and PilF homologues yielded biofilms with lower biovolumes and altered architectures, confirming the involvement of these proteins in the biofilm formation process. Our results indicate that Pseudoalteromonas sp. D41 shares biofilm formation mechanisms with human pathogenic bacteria, but also relies on TBDR, which might be more specific to the marine environment.

Molecular cloning, structure, and reactivity of the second bromoperoxidase from Ascophyllum nodosum.

The sequence of bromoperoxidase II from the brown alga Ascophyllum nodosum was determined from a full length cloned cDNA, obtained from a tandem mass spectrometry RT-PCR-approach. The clone encodes a protein composed of 641 amino-acids, which provides a mature 67.4 kDa-bromoperoxidase II-protein (620 amino-acids). Based on 43% sequence homology with the previously characterized bromoperoxidase I from A. nodosum, a tertiary structure was modeled for the bromoperoxidase II. The structural model was refined on the basis of results from gel filtration and vanadate-binding studies, showing that the bromoperoxidase II is a hexameric metalloprotein, which binds 0.5 equivalents of vanadate as cofactor per 67.4 kDa-subunit, for catalyzing oxidation of bromide by hydrogen peroxide in a bi-bi-ping-pong mechanism (k(cat) = 153 s(-1), 22 °C, pH 5.9). Bromide thereby is converted into a bromoelectrophile of reactivity similar to molecular bromine, based on competition kinetic data on phenol bromination and correlation analysis. Reactivity provided by the bromoperoxidase II mimics biosynthesis of methyl 4-bromopyrrole-2-carboxylate, a natural product isolated from the marine sponge Axinella tenuidigitata.

Up-regulation of lipoxygenase, phospholipase, and oxylipin-production in the induced chemical defense of the red alga Gracilaria chilensis against epiphytes.

The red alga Gracilaria chilensis is commercially farmed for the production of agar hydrocolloids, but some susceptible algae in farms suffer from intense epiphyte growth. We investigated the induced chemical defense response of G. chilensis against epiphytes and demonstrated that an extract of an epiphyte-challenged alga can trigger a defense response. The hormonally active metabolites were purified by RP-HPLC. Treatment with the extract or the purified fraction changed the chemical profile of the alga and increased resistance against epiphyte spores. Semi-quantitative RT-PCR and enzyme assays demonstrated that this metabolic response occurs after an increase in lipoxygenase and phospholipase A2 activity. Although this suggests the involvement of regulatory oxylipins, neither jasmonic acid nor the algal metabolite prostaglandin E2 triggers comparable defense responses.

Functional Characterization of a L-2-Haloacid Dehalogenase From Zobellia galactanivorans DsijT Suggests a Role in Haloacetic Acid Catabolism and a Wide Distribution in Marine Environments.

L-2-halocid dehalogenases (L-2-HADs) have been mainly characterized from terrestrial polluted environments. By contrast, knowledge is still scarce about their role in detoxification of predominant halocarbons in marine environments. Here, phylogenetic analyses showed a wide diversity of homologous L-2-HADs, especially among those belonging to marine bacteria. Previously characterized terrestrial L-2-HADs were part of a monophyletic group (named group A) including proteins of terrestrial and marine origin. Another branch (named group B) contained mostly marine L-2-HADs, with two distinct clades of Bacteroidetes homologs, closely linked to Proteobacteria ones. This study further focused on the characterization of the only L-2-HAD from the flavobacterium Zobellia galactanivorans DsijT (ZgHAD), belonging to one of these Group B clades. The recombinant ZgHAD was shown to dehalogenate bromo- and iodoacetic acids, and gene knockout in Z. galactanivorans revealed a direct role of ZgHAD in tolerance against both haloacetic acids. Analyses of metagenomic and metatranscriptomic datasets confirmed that L-2-HADs from group A were well-represented in terrestrial and marine bacteria, whereas ZgHAD homologs (group B L-2-HADs) were mainly present in marine bacteria, and particularly in host-associated species. Our results suggest that ZgHAD homologs could be key enzymes for marine Bacteroidetes, by conferring selective advantage for the recycling of toxic halogen compounds produced in particular marine habitats, and especially during interactions with macroalgae.

Semi-Quantitative Targeted Gas Chromatography-Mass Spectrometry Profiling Supports a Late Side-Chain Reductase Cycloartenol-to-Cholesterol Biosynthesis Pathway in Brown Algae.

Sterols are biologically important molecules that serve as membrane fluidity regulators and precursors of signaling molecules, either endogenous or involved in biotic interactions. There is currently no model of their biosynthesis pathways in brown algae. Here, we benefit from the availability of genome data and gas chromatography-mass spectrometry (GC-MS) sterol profiling using a database of internal standards to build such a model. We expand the set of identified sterols in 11 species of red, brown, and green macroalgae and integrate these new data with genomic data. Our analyses suggest that some metabolic reactions may be conserved despite the loss of canonical eukaryotic enzymes, like the sterol side-chain reductase (SSR). Our findings are consistent with the principle of metabolic pathway drift through enzymatic replacement and show that cholesterol synthesis from cycloartenol may be a widespread but variable pathway among chlorophyllian eukaryotes. Among the factors contributing to this variability, one could be the recruitment of cholesterol biosynthetic intermediates to make signaling molecules, such as the mozukulins. These compounds were found in some brown algae belonging to Ectocarpales, and we here provide a first mozukulin biosynthetic model. Our results demonstrate that integrative approaches can already be used to infer experimentally testable models, which will be useful to further investigate the biological roles of those newly identified algal pathways.

Copper stress proteomics highlights local adaptation of two strains of the model brown alga Ectocarpus siliculosus.

Ectocarpus siliculosus is a cosmopolitan brown alga with capacity to thrive in copper enriched environments. Analysis of copper toxicity was conducted in two strains of E. siliculosus isolated from (i) an uncontaminated coast in southern Peru (Es32) and (ii) a copper polluted rocky beach in northern Chile (Es524). Es32 was more sensitive than Es524, with toxicity detected at 50 microg/L Cu, whereas Es524 displayed negative effects only when exposed to 250 microg/L Cu. Differential soluble proteome profiling for each strain exposed to sub-lethal copper levels allowed to identify the induction of proteins related to processes such as energy production, glutathione metabolism as well as accumulation of HSPs. In addition, the inter-strain comparison of stress-related proteomes led to identify features related to copper tolerance in Es524, such as striking expression of a PSII Mn-stabilizing protein and a Fucoxanthine chlorophyll a-c binding protein. Es524 also expressed specific stress-related enzymes such as RNA helicases from the DEAD box families and a vanadium-dependent bromoperoxidase. These observations were supported by RT-qPCR for some of the identified genes and an enzyme activity assay for vanadium-dependent bromoperoxidase. Therefore, the occurrence of two different phenotypes within two distinct E. siliculosus strains studied at the physiological and proteomic levels strongly suggest that persistent copper stress may represent a selective force leading to the development of strains genetically adapted to copper contaminated sites.