L'influence des bactéries sur l'adaptation aux environnements changeants chez Ectocarpus : une approche de biologie systémique

Ectocarpus subulatus depends on its associated bacteria for growth in fresh water, which stresses the significance of the “holobiont” during abiotic stress. The aim of my thesis is to elucidate the molecular mechanisms that underlie this phenomenon. Targeted co-culture experiments require cultivable organisms. Therefore, I have cultivated and characterized 388 Ectocarpus-associated bacteria, which belong to 33 different genera. None of the cultivated bacteria tested had a beneficial effect on algal growth in fresh water. For functional studies, I continued to work with mild antibiotic-treated holobionts that differed in their response to fresh water. The metatranscriptome and metabolome of these holobionts were analyzed during acclimation. In-depth analysis is ongoing, but first indications point towards a change in the microbiome regarding nitrogen assimilation and virulence. In parallel and complementary to the above, potentially beneficial algal-bacterial cross-talk was predicted in silico using metabolic network analysis on a subset of cultivated bacteria, and the predictions were experimentally verified using co-culture experiments. Together, these results contribute to a better understanding of how the Ectocarpus holobiont responds during abiotic stress and especially how bacteria are involved in this process.Ectocarpus dépend de bactéries associées pour croitre en eau douce, ce qui souligne l'importance de l'holobionte lors de stress abiotique. Le but de ma thèse est d'élucider les mécanismes moléculaires qui sous-tendent ce phénomène. Les expériences de co-culture ciblées nécessitent des organismes cultivables. Par conséquent, j'ai caractérisé 388 bactéries associées à Ectocarpus, réparties en 33 genres. Aucune des bactéries cultivées testées n'a eu d'effet bénéfique sur la croissance des algues dans l'eau douce. J'ai continué à travailler avec des holobionts, traités aux antibiotiques doux, qui différaient dans leur réponse à l'eau douce. Le métatranscriptome/métabolome de ces holobionts ont été analysés pendant l'acclimatation. L'analyse approfondie est en cours, mais les premières indications indiquent un changement dans le microbiome en ce qui concerne l'assimilation de l'azote et la virulence. Concomitamment et complémentaire à ce qui précède, les interactions algues/bactéries potentiellement bénéfiques ont été prédites in silico à l'aide d'une analyse de réseau métabolique et les prédictions ont été vérifiées expérimentalement à l'aide de co-cultures. Ensemble, ces résultats contribuent à mieux comprendre comment l'holobiont d'Ectocarpus réagit au stress abiotique et surtout comment les bactéries sont impliquées dans ce processus

Exploring the Cultivable Ectocarpus Microbiome

Coastal areas form the major habitat of brown macroalgae, photosynthetic multicellular eukaryotes that have great ecological value and industrial potential. Macroalgal growth, development, and physiology are influenced by the microbial community they accommodate. Studying the algal microbiome should thus increase our fundamental understanding of algal biology and may help to improve culturing efforts. Currently, a freshwater strain of the brown macroalga Ectocarpus subulatus is being developed as a model organism for brown macroalgal physiology and algal microbiome studies. It can grow in high and low salinities depending on which microbes it hosts. However, the molecular mechanisms involved in this process are still unclear. Cultivation of Ectocarpus-associated bacteria is the first step toward the development of a model system for in vitro functional studies of brown macroalgal–bacterial interactions during abiotic stress. The main aim of the present study is thus to provide an extensive collection of cultivable E. subulatus-associated bacteria. To meet the variety of metabolic demands of Ectocarpus-associated bacteria, several isolation techniques were applied, i.e., direct plating and dilution-to-extinction cultivation techniques, each with chemically defined and undefined bacterial growth media. Algal tissue and algal growth media were directly used as inoculum, or they were pretreated with antibiotics, by filtration, or by digestion of algal cell walls. In total, 388 isolates were identified falling into 33 genera (46 distinct strains), of which Halomonas (Gammaproteobacteria), Bosea (Alphaproteobacteria), and Limnobacter (Betaproteobacteria) were the most abundant. Comparisons with 16S rRNA gene metabarcoding data showed that culturability in this study was remarkably high (∼50%), although several cultivable strains were not detected or only present in extremely low abundance in the libraries. These undetected bacteria could be considered as part of the rare biosphere and they may form the basis for the temporal changes in the Ectocarpus microbiome

Insights into the potential for mutualistic and harmful host‐microbe interactions affecting brown alga freshwater acclimation

International audienceMicrobes can modify their hosts' stress tolerance, thus potentially enhancing their ecological range. An example of such interactions is Ectocarpus subulatus, one of the few freshwater-tolerant brown algae. This tolerance is partially due to its (un)cultivated microbiome. We investigated this phenomenon by modifying the microbiome of laboratory-grown E. subulatus using mild antibiotic treatments, which affected its ability to grow in low salinity. Low salinity acclimation of these algal-bacterial associations was then compared. Salinity significantly impacted bacterial and viral gene expression, albeit in different ways across algal-bacterial communities. In contrast, gene expression of the host and metabolite profiles were affected almost exclusively in the freshwater-intolerant algal-bacterial communities. We found no evidence of bacterial protein production that would directly improve algal stress tolerance. However, vitamin K synthesis is one possible bacterial service missing specifically in freshwater-intolerant cultures in low salinity. In this condition, we also observed a relative increase in bacterial transcriptomic activity and the induction of microbial genes involved in the biosynthesis of the autoinducer AI-1, a quorum-sensing regulator. This could have resulted in dysbiosis by causing a shift in bacterial behavior in the intolerant algal-bacterial community. Together, these results provide two promising hypotheses to be examined by future targeted experiments. Although they apply only to the specific study system, they offer an example of how bacteria may impact their host's stress response

Metabolic Complementarity Between a Brown Alga and Associated Cultivable Bacteria Provide Indications of Beneficial Interactions

Brown algae are key components of marine ecosystems and live in association with bacteria that are essential for their growth and development. Ectocarpus siliculosus is a genetic and genomic model for brown algae. Here we use this model to start disentangling the complex interactions that may occur between the algal host and its associated bacteria. We report the genome-sequencing of 10 alga-associated bacteria and the genome-based reconstruction of their metabolic networks. The predicted metabolic capacities were then used to identify metabolic complementarities between the algal host and the bacteria, highlighting a range of potentially beneficial metabolite exchanges between them. These putative exchanges allowed us to predict consortia consisting of a subset of these ten bacteria that would best complement the algal metabolism. Finally, co-culture experiments were set up with a subset of these consortia to monitor algal growth as well as the presence of key algal metabolites. Although we did not fully control but only modified bacterial communities in our experiments, our data demonstrated a significant increase in algal growth in cultures inoculated with the selected consortia. In several cases, we also detected, in algal extracts, the presence of key metabolites predicted to become producible via an exchange of metabolites between the alga and the microbiome. Thus, although further methodological developments will be necessary to better control and understand microbial interactions in Ectocarpus, our data suggest that metabolic complementarity is a good indicator of beneficial metabolite exchanges in the holobiont

The genome of Ectocarpus subulatus – a highly stress-tolerant brown alga

International audienceBrown algae are multicellular photosynthetic stramenopiles that colonize marine rocky shores worldwide. Ectocarpus sp. Ec32 has been established as a genomic model for brown algae. Here we present the genome and metabolic network of the closely related species, Ectocarpus subulatus Kützing, which is characterized by high abiotic stress tolerance. Since their separation, both strains show new traces of viral sequences and the activity of large retrotransposons, which may also be related to the expansion of a family of chlorophyll-binding proteins. Further features suspected to contribute to stress tolerance include an expanded family of heat shock proteins, the reduction of genes involved in the production of halogenated defence compounds, and the presence of fewer cell wall polysaccharide-modifying enzymes. Overall, E. subulatus has mainly lost members of gene families down-regulated in low salinities, and conserved those that were up-regulated in the same condition. However, 96% of genes that differed between the two examined Ectocarpus species, as well as all genes under positive selection, were found to encode proteins of unknown function. This underlines the uniqueness of brown algal stress tolerance mechanisms as well as the significance of establishing E. subulatus as a comparative model for future functional studies