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.

Aldehyde perception induces specific molecular responses in Laminaria digitata and affects algal consumption by a specialist grazer.

In the marine environment, distance signaling based on water-borne cues occurs during interactions between macroalgae and herbivores. In the brown alga Laminaria digitata from North-Atlantic Brittany, oligoalginates elicitation or grazing was shown to induce chemical and transcriptomic regulations, as well as emission of a wide range of volatile aldehydes, but their biological roles as potential defense or warning signals in response to herbivores remain unknown. In this context, bioassays using the limpet Patella pellucida and L. digitata were carried out for determining the effects of algal transient incubation with 4-hydroxyhexenal (4-HHE), 4-hydroxynonenal (4-HNE) and dodecadienal on algal consumption by grazers. Simultaneously, we have developed metabolomic and transcriptomic approaches to study algal molecular responses after treatments of L. digitata with these chemical compounds. The results indicated that, unlike the treatment of the plantlets with 4-HNE or dodecadienal, treatment with 4-HHE decreases algal consumption by herbivores at 100 ng.ml-1 . Moreover, we showed that algal metabolome was significantly modified according to the type of aldehydes, and more specifically the metabolite pathways linked to fatty acid degradation. RNAseq analysis further showed that 4-HHE at 100 ng.ml-1 can activate the regulation of genes related to oxylipin signaling pathways and specific responses, compared to oligoalginates elicitation. As kelp beds constitute complex ecosystems consisting of habitat and food source for marine herbivores, the algal perception of specific aldehydes leading to targeted molecular regulations could have an important biological role on kelps/grazers interactions.

Pathogen exposure leads to a transcriptional downregulation of core cellular functions that may dampen the immune response in a macroalga.

Diseases in marine eukaryotic organisms caused by opportunistic pathogens represent a serious threat to our oceans with potential downstream consequences for ecosystem functioning. Disease outbreaks affecting macroalgae are of particular concern due to their critical role as habitat-forming organisms. However, there is limited understanding of the molecular strategies used by macroalgae to respond to opportunistic pathogens. In this study, we used mRNA-sequencing analysis to investigate the early antipathogen response of the model macroalga Delisea pulchra (Rhodophyta) under the environmental conditions that are known to promote the onset of disease. Using de novo assembly methods, 27,586 unique transcripts belonging to D. pulchra were identified that were mostly affiliated with stress response and signal transduction processes. Differential gene expression analysis between a treatment with the known opportunistic pathogen, Aquimarina sp. AD1 (Bacteroidota), and a closely related benign strain (Aquimarina sp. AD10) revealed a downregulation of genes coding for predicted protein metabolism, stress response, energy generation and photosynthesis functions. The rapid repression of genes coding for core cellular processes is likely to interfere with the macroalgal antipathogen response, later leading to infection, tissue damage and bleaching symptoms. Overall, this study provides valuable insight into the genetic features of D. pulchra, highlighting potential antipathogen response mechanisms of macroalgae and contributing to an improved understanding of host-pathogen interactions in a changing environment.

Accumulation of detached kelp biomass in a subtidal temperate coastal ecosystem induces succession of epiphytic and sediment bacterial communities.

Kelps are dominant primary producers in temperate coastal ecosystems. Large amounts of kelp biomass can be exported to the seafloor during the algal growth cycle or following storms, creating new ecological niches for the associated microbiota. Here, we investigated the bacterial community associated with the kelp Laminaria hyperborea during its accumulation and degradation on the seafloor. Kelp tissue, seawater and sediment were sampled during a 6-month in situ experiment simulating kelp detritus accumulation. Evaluation of the epiphytic bacterial community abundance, structure, taxonomic composition and predicted functional profiles evidenced a biphasic succession. Initially, dominant genera (Hellea, Litorimonas, Granulosicoccus) showed a rapid and drastic decrease in sequence abundance, probably outcompeted by algal polysaccharide-degraders such as Bacteroidia members which responded within 4 weeks. Acidimicrobiia, especially members of the Sva0996 marine group, colonized the degrading kelp biomass after 11 weeks. These secondary colonizers could act as opportunistic scavenger bacteria assimilating substrates exposed by early degraders. In parallel, kelp accumulation modified bacterial communities in the underlying sediment, notably favouring anaerobic taxa potentially involved in the sulfur and nitrogen cycles. Overall, this study provides insights into the bacterial degradation of algal biomass in situ, an important link in coastal trophic chains.

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.

Hormonally active phytochemicals from macroalgae: A largely untapped source of ligands to deorphanize nuclear receptors in emerging marine animal models.

Hormonally active phytochemicals (HAPs) are signaling molecules produced by plants that alter hormonal signaling in animals, due to consumption or environmental exposure. To date, HAPs have been investigated mainly in terrestrial ecosystems. To gain a full understanding of the origin and evolution of plant-animal interactions, it is necessary also to study these interactions in the marine environment, where the major photosynthetic lineages are very distant from the terrestrial plants. Here we focus on chemicals from red and brown macroalgae and point out their potential role as modulators of the endocrine system of aquatic animals through nuclear hormone receptors. We show that, regarding steroids and oxylipins, there are already some candidates available for further functional investigations of ligand-receptor interactions. Furthermore, several carotenoids, produced by cyanobacteria provide candidates that could be investigated with respect to their presence in macroalgae. Finally, regarding halogenated compounds, it is not clear yet which molecules could bridge the gap to explain the transition from lipid sensing to thyroid hormone high affinity binding among nuclear receptors.

Habitat and taxon as driving forces of carbohydrate catabolism in marine heterotrophic bacteria: example of the model algae-associated bacterium Zobellia galactanivorans DsijT.

The marine flavobacterium Zobellia galactanivorans DsijT was isolated from a red alga and by now constitutes a model for studying algal polysaccharide bioconversions. We present an in-depth analysis of its complete genome and link it to physiological traits. Z. galactanivorans exhibited the highest gene numbers for glycoside hydrolases, polysaccharide lyases and carbohydrate esterases and the second highest sulfatase gene number in a comparison to 125 other marine heterotrophic bacteria (MHB) genomes. Its genome contains 50 polysaccharide utilization loci, 22 of which contain sulfatase genes. Catabolic profiling confirmed a pronounced capacity for using algal polysaccharides and degradation of most polysaccharides could be linked to dedicated genes. Physiological and biochemical tests revealed that Z. galactanivorans stores and recycles glycogen, despite loss of several classic glycogen-related genes. Similar gene losses were observed in most Flavobacteriia, suggesting presence of an atypical glycogen metabolism in this class. Z. galactanivorans features numerous adaptive traits for algae-associated life, such as consumption of seaweed exudates, iodine metabolism and methylotrophy, indicating that this bacterium is well equipped to form profitable, stable interactions with macroalgae. Finally, using statistical and clustering analyses of the MHB genomes we show that their carbohydrate catabolism correlates with both taxonomy and habitat.

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 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.

Kelps feature systemic defense responses: insights into the evolution of innate immunity in multicellular eukaryotes.

Brown algae are one of the few eukaryotic lineages that have evolved complex multicellularity, together with Opisthokonts (animals, fungi) and Plantae (land plants, green and red algae). In these three lineages, biotic stresses induce similar local defense reactions. Animals and land plants also feature a systemic immune response, protecting the whole organism after an attack on one of its parts. However, the occurrence of systemic defenses has never been investigated in brown algae. We elicited selected parts of the kelp Laminaria digitata and monitored distant, nonchallenged areas of the same individual for subsequent defense reactions. A systemic reaction was detected following elicitation on a distant area, including an oxidative response, an increase in haloperoxidase activities and a stronger resistance against herbivory. Based on experiments with pharmacological inhibitors, the liberation of free fatty acids is proposed to play a key role in systemic signaling, reminiscent of what is known in land plants. This study is the first report, outside the phyla of Opisthokonts and Plantae, of an intraorganism communication leading to defense reactions. These findings indicate that systemic immunity emerged independently at least three times, as a consequence of convergent evolution in multicellular eukaryotic lineages.

Identification of novel IP receptor agonists using historical ligand biased chemical arrays.

By considering published structural information we have designed high throughput biaryl lipophilic acid arrays leveraging facile chemistry to expedite their synthesis. We rapidly identified multiple hits which were of suitable IP agonist potency. These relatively simple and strategically undecorated molecules present an ideal opportunity for optimization towards our target candidate profile.

Different speciation for bromine in brown and red algae, revealed by in vivo X-ray absorption spectroscopic studies.

Members of various algal lineages are known to be strong producers of atmospherically relevant halogen emissions, that is a consequence of their capability to store and metabolize halogens. This study uses a noninvasive, synchrotron-based technique, X-ray absorption spectroscopy, for addressing in vivo bromine speciation in the brown algae Ectocarpus siliculosus, Ascophyllum nodosum, and Fucus serratus, the red algae Gracilaria dura, G. gracilis, Chondrus crispus, Osmundea pinnatifida, Asparagopsis armata, Polysiphonia elongata, and Corallina officinalis, the diatom Thalassiosira rotula, the dinoflagellate Lingulodinium polyedrum and a natural phytoplankton sample. The results highlight a diversity of fundamentally different bromine storage modes: while most of the stramenopile representatives and the dinoflagellate store mostly bromide, there is evidence for Br incorporated in nonaromatic hydrocarbons in Thalassiosira. Red algae operate various organic bromine stores - including a possible precursor (by the haloform reaction) for bromoform in Asparagopsis and aromatically bound Br in Polysiphonia and Corallina. Large fractions of the bromine in the red algae G. dura and C. crispus and the brown alga F. serratus are present as Br(-) defects in solid KCl, similar to what was reported earlier for Laminaria parts. These results are discussed according to different defensive strategies that are used within algal taxa to cope with biotic or abiotic stresses.

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.

Importance of quorum sensing crosstalk in the brown alga Saccharina latissima epimicrobiome.

Brown macroalgae are colonized by diverse microorganisms influencing the physiology of their host. However, cell-cell interactions within the surface microbiome (epimicrobiome) are largely unexplored, despite the significance of specific chemical mediators in maintaining host-microbiome homeostasis. In this study, by combining liquid chromatography coupled to mass spectrometry (LC-MS) analysis and bioassays, we demonstrated that the widely diverse fungal epimicrobiota of the brown alga Saccharina latissima can affect quorum sensing (QS), a type of cell-cell interaction, as well as bacterial biofilm formation. We also showed the ability of the bacterial epimicrobiota to form and inhibit biofilm growth, as well as to activate or inhibit QS pathways. Overall, we demonstrate that QS and anti-QS compounds produced by the epimicrobiota are key metabolites in these brown algal epimicrobiota communities and highlight the importance of exploring this epimicrobiome for the discovery of new bioactive compounds, including potentially anti-QS molecules with antifouling properties.

Exploring uranium bioaccumulation in the brown alga Ascophyllum nodosum: insights from multi-scale spectroscopy and imaging.

Legacy radioactive waste can be defined as the radioactive waste produced during the infancy of the civil nuclear industry's development in the mid-20th Century, a time when, unfortunately, waste storage and treatment were not well planned. The marine environment is one of the environmental compartments worth studying in this regard because of legacy waste in specific locations of the seabed. Comprising nearly 70% of the earth's service, the oceans are the largest and indeed the final destination for contaminated fresh waters. For this reason, long-term studies of the accumulation biochemical mechanisms of metallic radionuclides in the marine ecosystem are required. In this context the brown algal compartment may be ecologically relevant because of forming large and dense algal beds in coastal areas and potential important biomass for contamination. This report presents the first step in the investigation of uranium (U, an element used in the nuclear cycle) bioaccumulation in the brown alga Ascophyllum nodosum using a multi-scale spectroscopic and imaging approach. Contamination of A. nodosum specimens in closed aquaria at 13 °C was performed with a defined quantity of U(VI) (10-5 M). The living algal uptake was quantified by ICP-MS and a localization study in the various algal compartments was carried out by combining electronic microscopy imaging (SEM), X-ray Absorption spectroscopy (XAS) and micro X-ray Florescence (µ-XRF). Data indicate that the brown alga is able to concentrate U(VI) by an active bioaccumulation mechanism, reaching an equilibrium state after 200 h of daily contamination. A comparison between living organisms and dry biomass confirms a stress-response process in the former, with an average bioaccumulation factor (BAF) of 10 ± 2 for living specimens (90% lower compared to dry biomass, 142 ± 5). Also, these results open new perspectives for a potential use of A. nodosum dry biomass as uranium biosorbent. The different partial BAFs (bioaccumulation factors) range from 3 (for thallus) to 49 (for receptacles) leading to a compartmentalization of uranium within the seaweed. This reveals a higher accumulation capacity in the receptacles, the algal reproductive parts. SEM images highlight the different tissue distributions among the compartments with a superficial absorption in the thallus and lateral branches and several hotspots in the oospheres of the female individuals. A preliminary speciation XAS analysis identified a distinct U speciation in the gametes-containing receptacles as a pseudo-autunite phosphate phase. Similarly, XAS measurements on the lateral branches (XANES) were not conclusive with regards to the occurrence of an alginate-U complex in these tissues. Nonetheless, the hypothesis that alginate may play a role in the speciation of U in the algal thallus tissues is still under consideration.

In vivo speciation studies and antioxidant properties of bromine in Laminaria digitata reinforce the significance of iodine accumulation for kelps.

The metabolism of bromine in marine brown algae remains poorly understood. This contrasts with the recent finding that the accumulation of iodide in the brown alga Laminaria serves the provision of an inorganic antioxidant - the first case documented from a living system. The aim of this study was to use an interdisciplinary array of techniques to study the chemical speciation, transformation, and function of bromine in Laminaria and to investigate the link between bromine and iodine metabolism, in particular in the antioxidant context. First, bromine and iodine levels in different Laminaria tissues were compared by inductively coupled plasma MS. Using in vivo X-ray absorption spectroscopy, it was found that, similarly to iodine, bromine is predominantly present in this alga in the form of bromide, albeit at lower concentrations, and that it shows similar behaviour upon oxidative stress. However, from a thermodynamic and kinetic standpoint, supported by in vitro and reconstituted in vivo assays, bromide is less suitable than iodide as an antioxidant against most reactive oxygen species except superoxide, possibly explaining why kelps prefer to accumulate iodide. This constitutes the first-ever study exploring the potential antioxidant function of bromide in a living system and other potential physiological roles. Given the tissue-specific differences observed in the content and speciation of bromine, it is concluded that the bromide uptake mechanism is different from the vanadium iodoperoxidase-mediated uptake of iodide in L. digitata and that its function is likely to be complementary to the iodide antioxidant system for detoxifying superoxide.

Discovery and characterization of NVP-QAV680, a potent and selective CRTh2 receptor antagonist suitable for clinical testing in allergic diseases.

Optimization of a 7-azaindole-3-acetic acid CRTh2 receptor antagonist chemotype derived from high throughput screening furnished a highly selective compound NVP-QAV680 with low nM functional potency for inhibition of CRTh2 driven human eosinophil and Th2 lymphocyte activation in vitro. The molecule exhibited good oral bioavailability in the rat, combined with efficacy in rodent CRTh2-dependent mechanistic and allergic disease models and was suitable for clinical development.

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.