Harveyi Autoinducer

Léa Girard
email: lea.girard@obs-banyuls.fr

Élodie Blanchet

Laurent Intertaglia

Julia Baudart
email: julia.baudart@obs-banyuls.fr

Didier Stien

Marcelino Suzuki

V Alginolyticus Chanos

V Anguillarum Anguilla

V Crassostreae Crassostreae

Faury

A Fischeri Sparus Aurata

Scophthalmus Maximus Austin

V Ichthyoenteri Paralichthys

Kim

V Logei Salmo Salar Groff

Lapatra

V Metschnikovii Tridacna

Sutton Garrick

V Mimicus

Scolopes Léa Girard

Elodie Blanchet

Raphaël Lami
email: raphael.lami@obs-banyuls.fr

M Bruto

F Leroux

DOC Dissolved Organic Carbon DPD 4,5-dihydroxy 2,3-pentanedione

Keywords: ACP Acyl Carrier Protein AFLP Amplified Fragment Length Polymorphism AHLs ARDRA Amplified Ribosomal DNA Restriction V. aestuarianus subsp. francensis Crassostrea gigas Sparus aurata, Epinephelus malabaricus, Rachycentrib canadum, Mylio macrocephalus, Penaeus monodon, Macrobrachium rosenbergii larvae, Ruditapes decussates Quorum sensing, N-acyl-homoserine lactone, Vibrio tasmaniensis LGP32, Fractionation, Biosensors, UHPLC-HRMS/MS Quorum sensing, N-acyl-homoserine lactone, Aliivibrio fischeri, Euprymna scolopes Quorum sensing, N-acyl-homoserine lactone, Vibrio mediterranei, Phenotypic plasticity, Epigenetic memory

Après trois ans de travail acharné, de questionnements, de réflexion scientifique intense (ou pas !), mais aussi après des litres et des litres de culture, après mes fameuses journées marathon d'échantillonnage accompagné de ma cinquantaine de kilos de matériel et à peu près tout autant d'eau de mer, il est temps de remercier toutes les personnes qui m'ont accompagnées, guidées, aidées, encouragées ou réconfortées… Mon aventure scientifique ne se résume absolument pas au document suivant car ce fut également une formidable aventure humaine. La qualité de mon travail est entièrement dépendante du cadre dans lequel j'ai pu évoluer ces trois dernières années. Et pour résumer ce fut une bonne grosse partie de rigolade tout du long ! Je remercie mes encadrants Julia et Raphael mais aussi mon directeur Marcelino pour m'avoir laissé une marge de manoeuvre assez large tout en m'accompagnant réellement tout au long de cette aventure, cela n'a pas été facile tous les jours mais nous avons réussi à faire du bon boulot. Un remerciement particulier s'impose pour toi Julia qui m'accompagne depuis mon stage de Master 1, comme je le dis souvent tu es devenue ma maman scientifique, nous avons beaucoup partagé au cours de ces années et tu resteras mon modèle d'honnêteté et de rigueur scientifique dans un monde ou ce n'est pas toujours le cas.

De manière générale, je veux remercier toutes les personnes du LBBM, de Pierre Fabre, de la plateforme Bio2Mar ou encore de Microbia pour m'avoir fourni chaque jour ma dose journalière de rires qui est indispensable au bon fonctionnement de mon cerveau. Vous formez un cocon bienveillant et confortable qu'il est difficile de quitter. Je ne veux surtout pas attiser les jalousies au sein du groupe mais je dois quand même remercier quelques personnes individuellement.

Laurent le champion du monde de l'enthousiasme, de la blague et de la bonne humeur.

Nicole et Cécile mes compagnes de paillasse en salle pathogène et mes aides précieuses tout au long de ces trois ans. Nicole notre duo de choc est inégalable lors de la réalisation des manips les plus folles et le tout toujours dans la rigolade.

Le groupe des ménagères écolo, Elisa, Elodie et Steph, qui au court de ma thèse, l'une après l'autre, ce sont transformées en incubateurs ambulants pour leurs petits clones. Merci à Elisa pour toutes tes répliques détonantes, savant mélange d'italien de français et parfois d'anglais, devenues mythiques avec fou rire à la clef. "Je suis en situation d'emmergence là." "Ah oui des fois si il n'y a pas la chimique ce n'est pas bon !" Ce qui me permet de basculer sur Stéphanie qui en tant que Française est censée maitriser la langue de Molière, a fortement participée à l'élaboration de quiproquos énormes avec nos chères collègues étrangères pour nous conduire dans des discussions complètement incompréhensibles. "Ah oui oui l'anguillette le bébé de l'anguille." Alors que nous parlions d'aiguillettes de canard. Et Elodie ma colocataire de bureau un grand merci pour l'énorme réconfort que tu m'as apporté, pour les pots de nougatines et les tisanes adaptées à toutes les situations (lendemain de cuite, stress, bien-être, beauté..), pour ton aide dans les manips comme dans l'amélioration de ma diplomatie et enfin merci pour les crises de rires à en pleurer.

Fanny et Justine merci pour les soirées de décompression et les virées détentes avec les potins de l'OOB dans le hammam.

Un grand merci au gang des brésiliennes/portugaises, Carmen, Joana, Jocivania, Alice, Laura pour votre bonne humeur, vos bon petits plats, les caipirinha, la musique et la danse.

Evelyne merci pour ces discussions enrichissantes, tes conseils et ta présence réconfortante tout au long de cette aventure mais aussi pour les petites soirées resto ou bière en terrasse.

Un grand merci à toutes les autres personnes avec qui j'ai pu interagir, Karine, Gilles, Jocelyne, les petites rigolotes du 3ème Laurence et Agnès, François pour des discussions toujours passionnantes, Didier pour ses précieux conseils en chimie ou encore Florence et Christophe à Perpignan pour leur aide énorme lors de mes campagnes d'échantillonnage. Et enfin le plus important, ma famille et mes amis, je souhaite vous remercier pour vos encouragements, pour m'avoir poussée à poursuivre mes études et m'avoir soutenue dans les moments difficiles. Sans vous je n'en serai pas là aujourd'hui car vous m'avez aidée à me construire brique par brique tout au long de ma vie. " Les rencontres dans la vie sont comme le vent. Certaines vous effleurent juste la peau, d'autres vous renversent." Florence Lepetitdidier-Rossolin

CHAPITRE 1 Introduction 1. The genus Vibrio

Metabolism

Vibrio is a genus of Gram-negative rods, belonging to the Gammaproteobacteria class and widespread in aquatic environment. They are commonly motile, with at least one flagella, non-spore formers, and on average measure 1 µm wide and 2 to 3 µm long. The majority of the bacteria in the genus are chemoheterotroph, halophilic, mesophilic, oxidase positive and facultative fermentative (Breed et al., 1957 ;F. L. Thompson, Iida, and Swings 2004). Since it produces a large diversity of extracellular enzymes such as lipases, proteases, hydrolases and chitinases, Vibrio play a role in nutrient cycling by metabolizing macromolecules and thus mediating the decomposion of dissolved and particulate organic matter (Al-saari et al., 2015;Cottrell and Kirchman, 2000). Through the action of chitinases, Vibrio are able to degrade chitin -the main component of the exoskeleton of many marine organisms such as crustaceans and dinoflagellates -in N-acetyl-D-glucosamine which represent the largest pool of amino sugars in aquatic food webs (Cottrell et al., 2000;[START_REF] Riemann | Widespread N-acetyl-D-glucosamine uptake among pelagic marine bacteria and its ecological implications[END_REF]. Vibrio are also important producers of bioactive secondary metabolites such as antibiotics, antifungal or anti-algal compounds [START_REF] Long | Antagonistic interactions among marine pelagic bacteria[END_REF][START_REF] Mansson | Production of bioactive secondary metabolites by marine Vibrionaceae[END_REF]. Under unfavourable environmental conditions, such as low seawater temperature, nutrient deficiency or unsuitable salinity, Vibrio are able to enter into what is called a «Viable But Non Culturable» (VBNC) state. This physiological condition is characterized by cell morphology modifications, a significant decrease in metabolic activity but a maintainance of vital functions and virulence factors (Colwell, 2009;[START_REF] Oliver | Recent findings on the viable but nonculturable state in pathogenic bacteria[END_REF]. This survival strategy and these metabolic abilities may explain the ubiquitous occurrence of Vibrio in diverse aquatic environments like marine coastal area, estuaries and sediments.

Classification

Vibrio belongs to the Vibrionaceae family, where a total of 142 species are classified in seven genus: Aliivibrio, Echinimonas, Enterovibrio, Grimontia, Photobacterium, Salinivibrio and Vibrio (Sawabe et al., 2013) and as per February 2017, 116 Vibrio species are reported in the Belgian Coordinated Collections of Microorganisms of the Ghent University (http://www.straininfo.net; Figure 1.1). Novel Vibrio species are constantly being described, with, to my knowledge, at least 9 new species described in 2016, V. ishigakensis, V. bivalvicida, V. algivorus, V. galatheae, V. barjaei, V. gallaecicus, V. sonorensis, V. japonicus and V. cidicii (Beaz-Hidalgo et al., 2009;[START_REF] Orata | Characterization of clinical and environmental isolates of Vibrio cidicii sp. nov., a close relative of Vibrio navarrensis[END_REF]Doi et al., 2017;Dubert et al., 2016bDubert et al., , 2016a;;Fukuda et al., 2016;[START_REF] Gao | Vibrio ishigakensis sp. nov., in Halioticoli clade isolated from seawater in Okinawa coral reef area, Japan[END_REF][START_REF] González-Castillo | Vibrio sonorensis sp. nov. isolated from a cultured oyster Crassostrea gigas[END_REF][START_REF] Gram | Vibrio galatheae sp. nov., a member of the family Vibrionaceae isolated from a mussel[END_REF]. Sawabe et al. 2013). However, as the identification of environmental isolates by MLSA is laborious and time consuming, and as there is not a consensus regarding a molecular marker for a fast identification of environmental Vibrio strains, a multitude of different approaches can be found in the literature. In this work, different methods have been used to identify Vibrio isolates and a detailed overlook on the subject can be found in section 2.1. Identification.

Pathogenic Vibrio species

Many Vibrio species are a threat for human health and diverse marine and estuarine ecosystems worldwide. Among human pathogens we distinguish between Vibrio species causing outbreaks of cholera (V. cholerae serogroups O1 and O139) and the others, V. cholerae serogroups non-O1 and non-O139, V. parahaemolyticus, V. vulnificus, V. alginolyticus, V. fluvialis, V. furnisii, V. metschnikovii, V. mimicus and V. cincinnatiensis. The vast majority of vibriosis (vibrio induced infections) are mainly attributed to four species, V. cholerae, V. parahaemolyticus, V. vulnificus, and V. alginolyticus (Lemee, 2004). They are responsible for gastroenteritis, otitis, eye and wound infection and in some cases (i.e. patients with underlying pathologies) septicaemia [START_REF] Ardiç | Case report: Otitis due to Vibrio alginolyticus[END_REF]Bullen, 1991;[START_REF] Mclaughlin | Outbreak of Vibrio parahaemolyticus gastroenteritis associated with Alaskan oysters[END_REF][START_REF] Morris | Non-O Group 1 Vibrio cholerae gastroenteritis in the united states: clinical, epidemiologic, and laboratory characteristics of sporadic cases[END_REF][START_REF] Oliver | Wound infections caused by Vibrio vulnificus and other marine bacteria[END_REF]. The ingestion of contaminated raw seafood or water and the direct penetration of wounds are the major transmission pathways for these pathogenic microorganisms.

A large number of Vibrio species are also commensal or pathogenic for marine vertebrates and invertebrates and responsible for important economic losses in aquaculture. A nonexhaustive list of Vibrio species pathogenic for the marine fauna can be found Table 1.1. Briefly, V. anguillarum and V. salmonicida are the most important fish pathogens, causing infection and septicaemia mainly in eel and salmon fish farms [START_REF] Austin | Vibrios as causal agents of zoonoses[END_REF]Egidius et al., 1986). Concerning molluscs, V. splendidus and V. aestuarianus have been associated with summer outbreaks affecting Crassosstreae gigas shellfish farming and V. tapetis is the responsible agent for the brown ring disease in clams.

Methods for the identification and quantification of Vibrio spp.

Identification

It is important to note that strain identification among Vibrio spp. has considerably evolved with the advent of new identification methods and thus strain identification is usually carried out by a classic polyphasic approach that combines genetic, chemotaxonomic and phenotypic typing. Hereafter I present the most common methods for the identification of Vibrio strains.

Phenotypical and biochemical identification

Gram negative staining, positive oxidase test, growth on Thiosulfate-citrate-bile saltssucrose agar (TCBS; i.e. selective medium for Vibrio spp.) and facultative anaerobic growth are the first traits for the discrimination of Vibrio. In addition arginine deiminase and lysine and ornithine decarboxylase activity have been widely used to screen the diversity of environmental strains of Vibrio, and all the methods described above have been reported as reliable species identification schemes (Alsina and Blanch, 1994;[START_REF] Lányi | Classical and rapid identification methods for medically important bacteria[END_REF][START_REF] Macian | The arginine dihydrolase (ADH) system in the identification of some marine Vibrio species[END_REF]. In addition, commercially available standardized kits like API 20E, API 20NE or Biolog (testing for the utilisation of 95 carbon) are widely used, and allow the identification of strains by comparison with known organisms database [START_REF] Ben-Haim | Vibrio coralliilyticus sp. nov., a temperature-dependent pathogen of the coral Pocillopora damicornis[END_REF][START_REF] Kent | Characteristics and identification of Pasteurella and Vibrio species pathogenic to fishes using API-20E (Analytab Products) multitube test strips[END_REF][START_REF] Klingler | Evaluation of the Biolog automated microbial identification system[END_REF][START_REF] Vandenberghe | Phenotypic diversity amongst Vibrio isolates from marine aquaculture systems[END_REF]. However, since Vibrio strains often respond to their environments by modifying metabolic and enzymatic activities, they exhibit a wide phenotypic and biochemical variability that impact results. Thus when used alone phenotypic and biochemical assays can be less effective for the identification of environmental strains of Vibrio (Abbott et al., 1998;Croci et al., 2007). This limitation have therefore contributed to the development of alternative molecular approaches.

Whole genome based methods

The genome of Vibrio species is organized in two circular chromosomes, the so called Chromosome I (between 3.0 to 4.2 Mb), larger than the Chromosome II (between 0.8 to 2.4

Mb), and one or more facultative plasmids [START_REF] Okada | Vibrios commonly possess two chromosomes[END_REF]. The information is generally distributed in these chromosomes as follows : Chromosome I carries most of the essential cellular functions while Chromosome II harbors species-specific functions that lead to niche specialization [START_REF] Grimes | What genomic sequence information has revealed about Vibrio ecology in the ocean -a review[END_REF][START_REF] Reen | The genomic code: inferring Vibrionaceae niche specialization[END_REF]. In addition plasmids can carry specific information such as virulence traits (Bruto et al., 2016).

Currently, the gold standard for the identification and the description of a new bacterial strains is the DNA-DNA hybridization (DDH) assay, based on the complementarity of denaturated genomic DNA and evaluating the sequence similarity between a pair of strains [START_REF] Gomez-Gil | Molecular identification of Vibrio harveyi-related isolates associated with diseased aquatic organisms[END_REF][START_REF] Stackebrandt | Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology[END_REF]. A reassociation rate above 70% to a type strain of previously described species is often used to identify an unknown strain. However, for Vibrio species this hybridization percentage can exhibit high variability (between 29 and 79%) and even show very low percentage for some species belonging to the Harveyi and Splendidus clade (29 to 36%) (Thompson et al., 2006). Fortunately, the development of high troughput sequencing and the wide availability of relatively inexpensive whole genome sequencing has led to an ever-increasing number of strains with sequenced genomes, and the development of comparative genomic tools. Methods calculating Average nucleotide identity (ANI) has then been developed and represent an alternative (albeit not officialy recognized) method to DDH for species delineation. ANI values >95% between two strain genomes are often used to classify two strains in the same species, [START_REF] Goris | DNA-DNA hybridization values and their relationship to whole-genome sequence similarities[END_REF][START_REF] Urbanczyk | Contrasting inter-and intraspecies recombination patterns in the "Harveyi clade" Vibrio collected over large spatial and temporal scales[END_REF] whereas an ANI value > 99.9% was previously reported as the cuttoff to consider two isolates as belonging to the same "outbreak" strain (Olm et al., 2017;Snitkin et al., 2012).

Alternative genome-based methods have been extensively used for the identification of Vibrio strains such as Amplified fragment length polymorphism (AFLP) (Beaz Hidalgo et al., 2008;Beaz-Hidalgo et al., 2009;[START_REF] Sokolova | An AFLP approach to identify genetic markers associated with resistance to Vibrio vulnificus and Perkinsus marinus in eastern oysters[END_REF]Thompson et al., 2001;[START_REF] Vandenberghe | Phenotypic diversity amongst Vibrio isolates from marine aquaculture systems[END_REF], Repetitive extragenic palindromic elements PCR (REP-PCR) [START_REF] Gomez-Gil | Molecular identification of Vibrio harveyi-related isolates associated with diseased aquatic organisms[END_REF][START_REF] Johnson | Genetic relatedness among tdh + and trh + Vibrio parahaemolyticus cultured from gulf of Mexico oysters (Crassostrea virginica) and surrounding water and sediment[END_REF][START_REF] Rao | Genetic heterogeneity of non-O1 and non-O139 Vibrio cholerae isolates from shrimp aquaculture system: a comparison of RS-, REP-and ERIC-PCR fingerprinting approaches[END_REF]Turner et al., 2013) and Random amplified polymorphic DNA (RAPD) (Alavandi et al., 2006;[START_REF] Rodríguez | Molecular fingerprinting of Vibrio tapetis strains using three PCR-based methods: ERIC-PCR, REP-PCR and RAPD[END_REF]. Briefly, these fingerprinting methods are based on DNA fragments amplification, with or without initial restriction cutting of genomic DNA, using primers of arbitrary nucleotide sequences (RAPD) or targeting specific zones like repeated interspersed sequences (REP-PCR) or matching restriction patterns (AFLP). Since AFLP is expensive and technically demanding and RAPD display poor reproducibility and moderate resolution power, in this thesis research, genotyping of environmental strains of Vibrio was conducted by REP-PCR which decreased costs while showing high reproducibility and resolution power as previously described (Cano-Gomez et al., 2009;[START_REF] Gomez-Gil | Molecular identification of Vibrio harveyi-related isolates associated with diseased aquatic organisms[END_REF].

Gene sequence based methods

Sequencing of housekeeping genes

During the past two decades, the identification of Vibrio species has also been extensively studied using a wide diversity of genetic markers, and an overview is presented Table 1.2. Among those genetic markers, the 16S rRNA gene is coding for the 16S rRNA which is structural and essential for assembly of ribosomal 30S subunit and is involved in genetic translation in bacteria. The 16S rRNA molecule (measuring about 1500 bp) is thus present in all bacteria and has a mosaic structure, alternating between highly conserved universal regions, variable regions among major phylogenetic taxa and hypervariable regions between more closely related (all the way to species) bacterial taxa. These features make it the most widely used genetic marker for identification and phylogenetic studies of bacteria and other organisms, in particular studies considering distantly related taxa. Moreover, the existence of very large datasets of 16S rRNA gene are available through public databases such as NCBI or RDP (Ribosomal Database Project) with over 75000 sequences belonging to Vibrio spp., supports the use of this marker [START_REF] Maidak | A new version of the RDP (Ribosomal Database Project)[END_REF]. Furthermore, two gene fingerprinting methods are based on PCR amplification of this gene, the Ribosomal Restriction Fragment Length Polymorphism (RFLP) and the Amplified Ribosomal DNA Restriction Analysis (ARDRA), they are fast and relatively inexpensive; and have been used for epidemiology studies (Hernández and Olmos, 2004;Popovic et al., 1993;[START_REF] Pujalte | Virulence and molecular typing of Vibrio harveyi strains isolated from cultured dentex, Gilthead Sea Bream and European Sea Bass[END_REF]. However, despite its widespreads use, a number of studies have shown that 16S rRNA gene sequences are insufficiently discriminant for the identification of Vibrio species especially those belonging to the Splendidus clade (Le Roux et al., 2004).

Thus, alternative phylogenetic markers have been proposed for the identification of Vibrio mostly choose based on the criteria of wide distribution, preferentially being present in a single copy within a genome and harboring an alternation between variable and conserved regions [START_REF] Zeigler | Gene sequences useful for predicting relatedness of whole genomes in bacteria[END_REF]. Housekeeping genes, coding for essential functions in bacteria (i.e.

Table 1.2) meet these criteria but overall resolution is still heterogeneous within clades. In this thesis, we used different approaches for the identification and the phylogenetic comparison among environmental isolates of Vibrio using the 16S rRNA gene, gyrB and hsp60 gene.

MultiLocus Sequence Analysis or Typing (MLSA/MLST)

In prokaryotic taxonomy MLSA, a.k.a. MLST is a reliable molecular tool to determine the phylogenetic relationships and the identification of bacterial species. In fact, it has been proposed by some authors as a replacement for DNA-DNA hybridization (DDH) for species delineation [START_REF] Glaeser | Multilocus sequence analysis (MLSA) in prokaryotic taxonomy[END_REF]. MLSA is an analysis based on sequence data from multiple housekeeping genes and has become easier to perform with the increased availability of whole genome sequences in public databases. In the literature, different variations of this technique can be found for the phylogenetic analysis of Vibrio, with the use of 3 to 9 housekeeping genes for a final concatenated sequence between 2000 and 6000 bp (Pérez-Cataluña et al., 2016;Sawabe et al., 2013;Thompson et al., 2009). The discrimination of strains within a clade or between closely relative species necessitate an adaptation of this method which is at the origin of these variations [START_REF] Steinum | Multilocus sequence analysis of close relatives Vibrio anguillarum and Vibrio ordalii[END_REF]Tarazona et al., 2014;Thompson et al., 2008). While MLSA has been a successful tool for the reconstruction of the evolutionary history of the Vibrionaceae family and for the description of new Vibrio species (Sawabe et al., 2013), this technique still remains relatively expensive and time consuming when very large numbers of environmental isolates are considered.

Quantification

As all the quantification methods have weaknesses as well strengths and I will discuss several of these techniques pointing the criteria of choice for the quantification of Vibrio.

Culture-based quantification

Vibrio are relatively easy to isolate from both clinical and environmental samples as they grow well at temperatures between 15 and 30 °C (with the exception of psychrophilic

Vibrio species such as V. salmonicida) on obviously any kind of media with a sufficient amount of NaCl (Tryptone Soy Agar, Luria-Bertani Agar/Broth or Marine Agar/Broth).Vibrio species with public health interest such as V. parahaemolyticus, V. vulnificus and V. cholerae have been targeted for the development of a wide variety of selective medium allowing species identification after short (24 h) incubation periods. Among those, the ChromAgar TM and the ChromID media are the most used, and they differentiate these species based on different enzymatic activities. For the quantification of Vibrio in the environment TCBS has been extensively used (Hsieh et al., 2008;[START_REF] Oberbeckmann | Occurrence of Vibrio parahaemolyticus and Vibrio alginolyticus in the German Bight over a seasonal cycle[END_REF][START_REF] Pfeffer | A comparison of thiosulphate-citrate-bile salts-sucrose (TCBS) agar and thiosulphate-chloride-iodide (TCI) agar for the isolation of Vibrio species from estuarine environments[END_REF]Tall et al., 2013;Turner et al., 2009). Typically, Vibrio grown on TCBS exhibit different morphotypes and colours depending on their abilities to use sucrose, while a large majority of non-Vibrio species produce hydrogen sulphide and grow as small black colonies [START_REF] Pfeffer | A comparison of thiosulphate-citrate-bile salts-sucrose (TCBS) agar and thiosulphate-chloride-iodide (TCI) agar for the isolation of Vibrio species from estuarine environments[END_REF]. To date, this has been the only selective medium for the isolation of Vibrio although some other Vibrionaceae species like Photobacterium and Shewanella, members of the famility Enterobactericeae and some marine Actinobacteria also grow with similar colony morphology and colours (APPENDICES / PAPER 5). Despite the fact that TCBS culturing approach is relatively low cost and technically straightforward, all culture based quantification techniques only allow the quantification of culturable Vibrio cells and not the total viable Vibrio (i.e. culturable plus VBNC cells). This bias has encouraged the development of cultivation-independent methods which requires nucleic acid extraction or direct cell hybridization (described in 2.2.2 and 2.2.3).

Fluorescence In Situ Hybridization (FISH)

FISH is a non-destructive method, i.e. there is no need for nucleic acids extraction from cells, and is based on the hybridization of fluorescent probes, in most cases targeting the 16S rRNA, allowing the enumeration of fluorescently labelled cells by epifluorescence microscopy or solid phase cytometry and in rare cases flow cytometry. Over the years, FISH has become a very important tool in counting Vibrio in the environment (Eilers et al., 2000b(Eilers et al., , 2000a;;[START_REF] Heidelberg | Bacteria of the γ-subclass proteobacteria associated with zooplankton in Chesapeake bay[END_REF]. A large panel of FISH based approaches have been developed

to study pathogenic Vibrio species lifestyle and interactions in the environment [START_REF] Kirschner | Interaction of Vibrio cholerae non-O1/non-O139 with copepods, cladocerans and competing bacteria in the large alkaline lake Neusiedler See, Austria[END_REF][START_REF] Rosenberg | Microbial diseases of corals and global warming[END_REF][START_REF] Rosenberg | The Vibrio shiloi / Oculina patagonica model system of coral bleaching[END_REF][START_REF] Sawabe | Multi-probefluorescence in situ hybridization for the rapid enumeration of viable Vibrio parahaemolyticus[END_REF]. An approach combining the Direct Viable Count assay, the FISH technique and solid phase cytometry (DVC-FISH/SPC) was developed to quantify total viable Vibrio cells (APPENDICES / PAPER 5).

Real-time Polymerase Chain Reaction (PCR)

Real-time PCR is a modification of traditional PCR that can be effectively used for quantification of microorganisms. DNA is directly extracted from environmental samples and is used as template for PCR where PCR products are detected either by a double stranded DNA dye (e.g. SYBR Green) or fluorogenic probes. This technique is highly sensitive and can detect down to a few cells per ml of water (Fukushima et al., 2003;[START_REF] Lyon | TaqMan PCR for detection of Vibrio cholerae O1, O139, Non-O1, and non-O139 in pure cultures, raw oysters, and synthetic seawater[END_REF]Tall et al., 2013). Despite the high sensitivity of real time PCR techniques, a general problem persists, there is no differentiation between live and dead cells as DNA from dead cells can also serve as template for real time PCR. Additionally, the quantification using the 16S rRNA gene as target can lead to an overestimation/underestimation of Vibrio spp. as they are harboring between 5 to 11 copies of this gene (Fegatella et al., 1998;[START_REF] Le Roux | Occurrence and distribution of the human pathogen Vibrio vulnificus in a subtropical Gulf of Mexico estuary[END_REF][START_REF] Wolfe | Bioluminescent symbionts of the Caribbean flashlight fish (Kryptophanaron alfredi) have a single rRNA operon[END_REF]. Finally variable DNA extraction efficiencies as well as PCR inhibition are still a problem and need to be improved.

Ecology of Vibrio

Vibrio are heterotrophic bacteria able to use a large range of nutrients [START_REF] Austin | A Comparison of Methods for the Typing of Fish-Pathogenic Vibrio spp[END_REF]Johnson, 2013;[START_REF] Raghul | Biodegradation of polyvinyl alcohol-low linear density polyethylene-blended plastic film by consortium of marine benthic vibrios[END_REF][START_REF] West | Numerical taxonomy of phenanthrene-degrading bacteria isolated from the Chesapeake Bay[END_REF], to form biofilms in diverse surfaces [START_REF] Pruzzo | Pathogenic Vibrio species in the marine and estuarine environment[END_REF][START_REF] Yildiz | Vibrio biofilms: so much the same yet so different[END_REF] and to switch to a VBNC state when environmental conditions are unfavorable [START_REF] Oliver | Recent findings on the viable but nonculturable state in pathogenic bacteria[END_REF]. These properties allow them to increase their fitness and to evolve in as free-living cells or as associated with marine organisms and abiotic surfaces (Johnson, 2013). Metagenomic studies from global sampling campaigns have evaluated that Vibrio 16S rRNA gene sequences represents around 1% of all bacterioplankton sequences (Biers et al., 2009;[START_REF] Wietz | Latitudinal patterns in the abundance of major marine bacterioplankton groups[END_REF]. In aquatic environments, numerous studies have shown that the dynamic of Vibrio spp. in various region around the world follow a prominent seasonal cycle attributed to both spatial and temporal fluctuations of environmental parameters (Takemura et al., 2014).

Abiotic parameters

Temperature

The sea surface temperature (SST) is the main factor controlling the abundance and the diversity of culturable Vibrio in aquatic environments (Eilers et al., 2000b;[START_REF] Heidelberg | Bacteria of the γ-subclass proteobacteria associated with zooplankton in Chesapeake bay[END_REF]Tall et al., 2013;Vezulli et al., 2009;Turner et al., 2009). In temperate areas, the seasonal dynamic of Vibrio results in high abundances in summer when highest temperatures occur and a decrease in counts while temperature is going down. A temperature of 15-16°C have been identified as the breakdown point of Vibrio abundances (Vezulli et al., 2009;Thompson et al., 2010;[START_REF] Gomez-Gil | Molecular identification of Vibrio harveyi-related isolates associated with diseased aquatic organisms[END_REF]. In tropical areas SST appears to have less influence in Vibrio dynamic because SST is elevated and quite stable over the year [START_REF] Asplund | Water column dynamics of Vibrio in relation to phytoplankton community composition and environmental conditions in a tropical coastal area[END_REF]. Conversely, in northern waters less abundance variation is observed (Eiler et al., 2006;[START_REF] Oberbeckmann | Seasonal dynamics and modeling of a Vibrio community in coastal waters of the North Sea[END_REF]. However, in these areas, it appears that other parameters, such as salinity, nutrients or phytoplankton, better explain the variation of Vibrio abundances. It is also important to point out the effect of SST on the structure of Vibrio populations (diversity and abundance) and on Vibrio species by considering the "cold" and "warm" adapted species which predominate in their corresponding habitats. In a global warming perspective some authors are predicting higher physiologic and metabolic activities of Vibrio and an increase of human and animal Vibrio-related diseases (Vezzulli et al., 2010(Vezzulli et al., , 2015)).

Salinity

Salinity appears as another factor explaining the variance in total Vibrio abundances (Froelich et al., 2013;[START_REF] Nigro | Temporal and spatial variability in culturable pathogenic Vibrio spp. in Lake Pontchartrain, Louisiana, following hurricanes Katrina and Rita[END_REF][START_REF] Oberbeckmann | Seasonal dynamics and modeling of a Vibrio community in coastal waters of the North Sea[END_REF]Turner et al., 2009;[START_REF] Wetz | Trends in total Vibrio spp. and Vibrio vulnificus concentrations in the eutrophic Neuse River Estuary, North Carolina, during storm events[END_REF]. However, different responses are obtained according to the type of environments, the temperature and the species considered. Even if Vibrio are halophilic bacteria, able to grow within a wide range of salinity, each species have its own growth optima. For exemple V. cholera is the only species able to grow in freshwaters and V.

vulnificus, favoured at salinity between 5-25 ppt, occurs preferentially in estuarine environments but shows differential responses depending on temperature at higher salinities (Thompson et al., 2006). Generally, the distribution of Vibrio is often more strongly linked either to temperature or to salinity depending on the climate of the studied area (Lipp et al., 2001;Takemura et al., 2014).

Others abiotic parameters

Compared to the temperature and salinity, other abiotic parameters are limited in explaning the dynamic of Vibrio. Indeed in models where temperature or salinity have been already incorporated they explain relatively little of the variance (Eiler et al., 2006;Vezulli et al., 2009;Turner et al., 2009). Few studies have demonstrated the impact of dissolved organic carbon (DOC), nonetheless, associated parameters such as Chlorophyll a, turbidity, chitine, dissolved oxygen, nitrogen and phosphate have been positively correlated to Vibrio abundances (Blackwell and Oliver, 2008;Eiler et al., 2006;Froelich et al., 2013;[START_REF] Heidelberg | Bacteria of the γ-subclass proteobacteria associated with zooplankton in Chesapeake bay[END_REF][START_REF] Oberbeckmann | Seasonal dynamics and modeling of a Vibrio community in coastal waters of the North Sea[END_REF]. These associations highlight the relationships between these heterotrophic bacteria, nutrients and indirectly primary production.

Biotic parameters

Zooplankton

Several studies highlight the fact that Vibrio spp. can dominate zooplankton surface and gut-attached communities (Preheim et al., 2011;[START_REF] Simidu | Bacterial flora of phyto-and zoo-plankton in the inshore water of Japan[END_REF]Sochard et al., 1979). The detection frequency of pathogen species such as V. cholerae, V. vulnificus and V.

parahaemolyticus is often positively correlated to the abundance of chitinous zooplankton [START_REF] Kirschner | Interaction of Vibrio cholerae non-O1/non-O139 with copepods, cladocerans and competing bacteria in the large alkaline lake Neusiedler See, Austria[END_REF]Turner et al., 2014). In fact, some authors describe zooplankton as a major vector of dissemination of cholera [START_REF] Matson | Regulatory networks controlling Vibrio cholerae virulence gene expression[END_REF]. The detailed mechanisms or metabolical impact of chitin utilization have been widely studied for pathogenic species such as V. cholerae [START_REF] Meibom | The Vibrio cholerae chitin utilization program[END_REF], V. parahaemolyticus [START_REF] Kaneko | Adsorption of Vibrio parahaemolyticus onto chitin and copepods[END_REF], V.

vulnificus [START_REF] Wortman | Chitinase determinants of Vibrio vulnificus: gene cloning and applications of a chitinase probe[END_REF] and V. furnissii (Bassler et al., 1991). Since the exoskeleton of zooplankton, as well as the crabs carapace and other shellfish, is often made of chitine this may explain the preferential colonization of these ecological niches.

Phytoplankton

Diverse studies have demonstrated the positive impact of Chlorophyll a pigment concentrations on the seasonality of different Vibrio species (Caburlotto et al., 2010;Froelich et al., 2013;[START_REF] Oberbeckmann | Seasonal dynamics and modeling of a Vibrio community in coastal waters of the North Sea[END_REF][START_REF] Randa | Effects of temperature and salinity on Vibrio vulnificus population dynamics as assessed by quantitative PCR[END_REF]. This pigment is essential for oxygenic photosynthesis and commonly used as an index of phytoplankton biomass.

Despite high concentrations of Vibrio spp. on phytoplanktonic surfaces compared to surrounding water, little is known about species specific associations [START_REF] Simidu | Bacterial flora of phyto-and zoo-plankton in the inshore water of Japan[END_REF]. Eiler et al., 2006, have highlighted that dinoflagellates were important predictors of Vibrio spp. abundances. [START_REF] Rehnstam-Holm | Association between phytoplankton and Vibrio spp. along the southwest coast of India: a mesocosm experiment[END_REF], have showed contrasting correlations with three diatoms, Chaetoceros and Skeletonema were negatively correlated while Coscinodiscus was positively correlated to Vibrio spp. abundances. Furthermore, a bloom of a single Vibrio sp. was correlated with Chaetoceros compressus in the English Channel [START_REF] Gilbert | Defining seasonal marine microbial community dynamics[END_REF] and several studies have demonstrated the survival advantage and persistence of V. cholerae with two phytoplankton strains, the cyanobacteria Anabaena and the Cladophoraceae, Rhizoclonium fontanum (See section 4.5;Ferdous, 2009;Islam et al., 1989Islam et al., , 1990Islam et al., , 2004;;Mizanur et al., 2002).

Microalgae represent a large reservoir of bioactive secondary metabolites and antimicrobial compounds which can promote extremely complex eukaryote/prokaryote relationships and can impact prokaryote/prokaryote interactions in natural environments (Abad et al., 2011;Kersey and Munger, 2009;[START_REF] Scholz | Screening for competition effects and allelochemicals in benthic marine diatoms and cyanobacteria isolated from an intertidal flat (southern North Sea)[END_REF]. Indeed, many phytoplankton species have been found to produce antimicrobial compounds such as the cyanobacterium Synechocystis sp., Fischerella ambigua, Anabaena constricta and the dinoflagellates Amphidinium sp., Prorocentrum lima, Dinophysis fortii and Gambierdiscus toxicus (Mo et al., 2009;[START_REF] Nagai | Antimicrobial activities of polyether compounds of dinoflagellate origins[END_REF]Plaza et al., 2010;[START_REF] Volk | Bromoanaindolone, a novel antimicrobial exometabolite from the cyanobacterium Anabaena constricta[END_REF][START_REF] Washida | Karatungiols A and B, two novel antimicrobial polyol compounds, from the symbiotic marine dinoflagellate Amphidinium sp[END_REF]. Some others are also able to disrupt the communication between bacteria by producing Quorum sensing (QS) receptors antagonists (Natrah et al., 2011;Teplitski, 2004).

Population as ecological unit

In the last decade, the idea of considering bacterial population as ecological, genetic and social units has gained ground, following the previously well-known Bass-Becking hypothesis "Everything is everywhere, but the environment selects". Indeed, diverse studies have assessed the fine-scale distribution of Vibrionaceae and highlighted highly predictable population-habitat linkage with the distribution of closely phylogenetic relatives according to ecological niches preferences (Acinas et al., 2004;Chimetto Tonon et al., 2015;Hunt et al., 2008;Polz et al., 2013). In practice, these populations are defined by water fractionation folowed by taxonomic characterization of Vibrio isolates based on the reliable taxonomic markers hsp60 or pyrH and ecological association are inferred by the model AdaptML (Hunt et al., 2008).

In this context, ecologically defined Vibrio populations have been screened for potential social activities such as antibiotics production and resistance or siderophores production (Cordero et al., 2012a;Cordero et al., 2012b).

The concept of ecological units and niche partitioning will be further detailed in CHAPTER IV. However, the results of these studies showed (i) evidence of reproducibility of population structure among time as defined in Hunt et al., 2008 andSzabo et al., 2013;(ii) the synergy of public goods production at the population level (Cordero et al., 2012a;Cordero et al., 2012b) ;

(iii) that populations can be regarded as species-like units in the environment and frequently coexist and re-assemble on small-scale habitats.

Quorum sensing in Vibrio

Quorum sensing (QS) is a density dependent mechanism enabling bacteria to coordinate their genetic expression, behavior and physiological responses via the emission of small diffusible molecules. Bacteria release these molecules into the extracellular environment by diffusion or active transport. While the cell density increase, there is an accumulation of signaling molecules and when a critical threshold level is reached the signaling pathway is activated leading to transcriptional regulation of functional genes as well as QS molecules synthases themselves, hence the name of autoinducers (AIs). There are two types of molecules allowing [START_REF] Selvin | Vibrio alginolyticus associated with white spot disease of Penaeus monodon[END_REF] intraspecies communication (between phylogenetically close bacteria) with the N-acylhomoserine lactones or autoinducer 1 (AHLs or AI-1), a common trait in Gram negative bacteria and CAI-1 or DPO which are specific to some Vibrio species [START_REF] Cao | Purification and structural identification of an autoinducer for the luminescence system of Vibrio harveyi[END_REF]Henke and Bassler, 2004a;Miller et al., 2002a) and ( 2) interspecies or universal communication with the autoinducer 2 (AI-2) widely distributed in microorganisms (Keller and Surette, 2006;[START_REF] Vendeville | Making "sense" of metabolism: autoinducer-2, LUXS and pathogenic bacteria[END_REF][START_REF] Xavier | LuxS quorum sensing: more than just a numbers game[END_REF] (Figure 1.3 and 1.4).

The Aliivibrio fischeri model

Jonh Hastings group showed in the nineteen-seventies that two Vibrio species, namely A.

fischeri and V. harveyi, produced light at high cellular densities but not in diluted suspension (Nealson et al., 1970). Light production could be induced in dilute solution by adding cell-free culture supernatant and the compound responsible for this induction was later identified as an AHL and called an autoinducer (Eberhard et al., 1981). These studies were the starting point of QS and for the first time bacteria were shown to be able to produce, release and detect molecules to sense their own population density and to respond to it by coordinated behaviours.

The regulation of bioluminescence by A. fischeri in his symbiotic relationship with the squid Euprymna scalopes remain the most studied QS system. The A. fischeri QS system consist of the AHL synthase LuxI and the transcriptional regulator LuxR. LuxI synthesize the N-3-oxohexanoyl-homoserine lactone (3-oxo-C6-HSL) and LuxR consist of two domains, the carboxy terminal that binds the promoter of target genes and the amino terminal that binds the AHL (Choi and Greenberg, 1991;Egland and Greenberg, 1999;[START_REF] Fuqua | Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators[END_REF].

At low cell density, LuxI synthesizes 3-oxo-C6-HSL and LuxR is unstable and inactive as its three-dimensional structure is such that the amino terminal domain is masked. As cell density increase, the AHL molecules accumulates and bind to LuxR which stabilizes, activates and dimerizes in order to bind to the promoter of the luxICDABE operon. LuxR induces luciferase production and the expression of LuxI gene which leads to an produce exponential increase of concentration of AHL (Figure 1.3). Metagenomic studies have revealed that A. fischeri luxI/luxR system is widespread among α, β and Ɣ-Proteobacteria by describing homologous QS system in over 40 species (Doberva et al., 2015;Hao et al., 2010).

Figure 1.3:

The A. fischeri LuxI/LuxR QS system.

A second system was latter discovered in A. fischeri, the ainS/ainR with high homology with the V. harveyi luxM/luxN system, responsible for the production of N-octanoyl-homoserine lactone (C8-HSL) (Gilson et al., 1995;Kuo et al., 1994). This second AHL-mediated pathway also affects the luxICDABE operon which indicates a complex intracellular mechanism and is the first evidence of a multichannel QS system in Vibrio species. The master regulator is a protein with a DNA binding domain that exerts the transcriptional regulation of QS molecules synthases and several other functional genes. There are two types of master regulators, an intracellular AHL receptor like LuxR, VanR, VfqR or an AHLindependent version like VanT, LitR, HapR, AphA or SmcR. Multichannel QS sytems have been greatly studied in V. harveyi and V. anguillarum (Lin et al., 2009;Milton, 2006;[START_REF] Ng | Bacterial quorum-sensing network architectures[END_REF].

In V. harveyi, it has been demonstrated that bacteria have specific response to each individual AI but also in the simultaneous presence of all three AIs (AI-1, AI-2 and HAI-1) (Waters and Bassler, 2006). It has also been reported that the master regulator LuxR controls gene expression at both high and low densities while AphA only functions in absence of AIs [START_REF] Rutherford | AphA and LuxR/HapR reciprocally control quorum sensing in vibrios[END_REF]. Furthermore, it has been demonstrated that in V. anguillarum the two AHL circuits were somewhat connected and that the AHL production of VanI was regulated by VanM (Milton et al., 2001). Since an increasing number of genomes are sequenced, we often observe the genes corresponding to the different QS channels in many Vibrio strains and it would not be surprising to observe the same or similar intracellular regulations and connections. Finally, QS is much more complex than a simple schema where at high density one molecule activates one function and it is moving forward to be reconsidered as a balanced phenomenon involving multiple molecules and multiple intracellular responses. The AHL molecule structure is composed of a hydrophilic homoserine lactone ring moiety and a hydrophobic acyl side chain (i.e. Figure 1.4A). The acyl side chain can vary in length from 4 to 21 (Doberva, 2016), in substitutions at the beta-position (Figure 1.4A R1) and in the level of saturation. The different features of the acyl side chain determine the AHL specificity towards his membrane or intracellular receptor. It also appears that the nature of the AHL acyl side chain determine its diffusion through the membrane or the requirement of a transport mechanism (Fuqua and Greenberg, 1998;Pearson et al., 1999).

AHL synthases are enzymes in the acyltransferase class, requiring two substrates, Sadenosyl-L-methionine (SAM) and an acyl carrier protein (ACP). They catalyse the amide bond between the fatty acid carried by the ACP and the primary amine group in the SAM.

The source of the homoserine lactone ring is the final step of lactonisation of the SAM-acyl which results in the release of the AHL molecule and 5-methylthioadenosine (MTA) (Figure 1.6) (Hanzelka et al., 1999;[START_REF] Moré | Enzymatic synthesis of a quorum-sensing autoinducer through use of defined substrates[END_REF]Parsek et al., 1999;[START_REF] Schaefer | Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein[END_REF][START_REF] Val | In vivo evidence that S-adenosylmethionine and fatty acid synthesis intermediates are the substrates for the LuxI family of autoinducer synthases[END_REF]. In Vibrio species, there is different variants of the A. fischeri QS systems, homologues of the LuxI synthase like VanI or VfqI and homologues of the AinS synthase like LuxM, VanM or opaM (Bassler et al., 1993;[START_REF] Cao | Purification and structural identification of an autoinducer for the luminescence system of Vibrio harveyi[END_REF]; (Milton et al., 1997(Milton et al., , 2001) ) (Wang et al., 2013). 1.3. Among Vibrio species, the LuxM family synthases are much more widespread than the LuxI. Indeed, an inventory of their diversity has been carried out and phylogenetic trees can be found in Figure 1.7 and 1.8. The phylogeny of different Vibrio species based on luxM and luxI genes was determined and the DNA sequence similarities range from 32.9% to 99.2% and from 42.4% to 100% respectivelly. Remarkably, the observed phylogeny (Figure 1.7 and 1.8) is close to that observed with MLSA or 16S rRNA gene phylogenies suggesting that these systems have been continually linked to their evolutionary history (Lerat, 2004). It also appears that luxI and luxM are conserved at the species level with similarities among a single species varying from 96 to 100%. Regarding the activity of AHL synthases in environmental settings, unsuccessful studies in our research group, as well in other groups, points to the difficulty to assess in situ expression of these genes (Tait et al., 2010). In fact, this is not surprising considering the very low homology of AHL synthases among the different species of Vibrio which hampers the design of universal primers. On the other hand, the great sequence similarity that offers the focus on a single species can lead further studies on QS in natural habitats.

Finally, the involvement of synthases unrelated to LuxI nor LuxM, like the hdtS family or novel and undescribed synthases is also possible as seen in the present thesis with the work on V. mediterranei in the Chapter 3 [START_REF] Laue | The biocontrol strain Pseudomonas fluorescens F113 produces the Rhizobium small bacteriocin, N-(3-hydroxy-7-cis-tetradecenoyl)homoserine lactone, via HdtS, a putative novel N-acylhomoserine lactone synthase[END_REF]Whitehead et al., 2001). The AI-2 has been more recently discovered and, it can be found in different chemical forms S-THMF-borate, R-THMF or DPD depending on the considered species and allows a communication between all bacteria (Gram-negative and Gram-positive; i.e. Figure 1.4B) (De Keersmaecker et al., 2006;[START_REF] Schauder | The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule: AI-2 biosynthesis[END_REF]. The furanosyl borate diester or S-THMF identified in V. harveyi is the most widespread molecule, followed by the R-THMF produced by Salmonella typhimurium and the DPD which is an intermediate of the biosynthesis pathway in many bacterial species and but is also known to bind the AI-2

receptor (Chen et al., 2002;[START_REF] Lowery | An unexpected switch in the modulation of AI-2-based Quorum sensing discovered through synthetic 4,5-dihydroxy-2,3pentanedione analogues[END_REF]Miller et al., 2004;[START_REF] Winzer | LuxS: its role in central metabolism and the in vitro synthesis of 4-hydroxy-5-methyl-3(2H)-furanone[END_REF]. Finally, the addition of a boron atom lead to the furanosyl borate diester and make it one of the few known biosynthesized boron containing molecule. The conservation of luxS is also explained by its enzymatic activity involved in the Activated Methyl Cycle (AMC, methionine metabolism, [START_REF] Vendeville | Making "sense" of metabolism: autoinducer-2, LUXS and pathogenic bacteria[END_REF] making the communication function of the AI-2 molecule controversial. In fact, physiological studies have proven that LuxS is involved not only in QS but also in protein synthesis and sulphur metabolism (Doherty et al., 2006;[START_REF] Vendeville | Making "sense" of metabolism: autoinducer-2, LUXS and pathogenic bacteria[END_REF][START_REF] Yu | Function of luxS gene in sulfurmetabolism of Streptococcus mutans[END_REF]. On the other hand, [START_REF] Taga | Lsr-mediated transport and processing of AI-2 in Salmonella typhimurium[END_REF] , 1997;[START_REF] Mcdougald | SmcR-dependent regulation of adaptive phenotypes in Vibrio vulnificus[END_REF].

LuxP is the membrane receptor and LuxQ his associated kinase which transfers the information to the secondary messengers by a phosphorylation cascade. For most of Vibrio species the secondary messengers and the master regulator are shared by the different QS pathways (Figure 1.5; see section 4. 2.4;Milton, 2006).

Cholerae autoinducer-1 (CAI-1) Quorum sensing

The CAI-1 molecule was initially discovered in V. cholerae hence its name Cholerae autoinducer-1. CAI-1 is an α-hydroxyketone namely, S-3-hydroxytridecan-4-one (Figure 1.4C; R: OH) (Miller et al., 2002b). Like AHL molecules, this type of molecules can be very diverse varying in length, substitutions and saturation. A similar molecule, the Harveyi Autoinducer-1 (HAI-1) has been discovered in V. harveyi (Henke and Bassler, 2004a).

Currently, the diversity of these compounds is undoubtedly underestimated, since the synthase, CqsA, belongs to PLP dependant acyl-coA transferase family, and these enzymes are able to carry out about 140 different enzymatic reactions [START_REF] Kelly | The Vibrio cholerae quorum-sensing autoinducer CAI-1: analysis of the biosynthetic enzyme CqsA[END_REF]. However, an inventory of the cqsA gene in Vibrio strains indicates that it is quite conserved and much more widespread than only in the species V. cholerae and V. harveyi (Figure 1.10). CAI-1 bind to his receptor CqsS, for Cholerae quorum sensing Sensor, and share the intracellular signalling pathway with the different QS pathways (Figure 1.5; Milton, 2006).

Other molecules

In addition to the main communication molecules presented above, some other compounds appear important for the expression of specific traits in Vibrio species. Recently, the pyrazine DPO (Figure 1.4 D) has been described in V. cholerae as well as its receptor

VqmA, synthase Tdh and master regulator VqmR [START_REF] Papenfort | A Vibrio cholerae autoinducer-receptor pair that controls biofilm formation[END_REF]. VqmR is involved in the control of collective behaviours like biofilm formation and toxin production.

Diketopiperazines (DKPs) are small dipeptides which have antifungal and antibacterial activities, involved in QS for their biding properties to the master regulator LuxR [START_REF] Martins | Diketopiperazines: biological activity and synthesis[END_REF]. In pathogenic Vibrio species DKPs have been shown to be regulators of virulence factors genes (Park et al., 2006).

However, their function as signalling molecules or QS modulators is controversial (Campbell et al., 2009). Finally, the indole-3-acetic acid (IAA), synthetized from tryptophan, seems quite widespread in Vibrio as indicated in the study of [START_REF] Gutierrez | Production of the phytohormone indole-3-acetic acid by estuarine species of the genus Vibrio[END_REF] This small molecule has been firstly isolated in plants and plant-bacteria associations, but it appears to be a signalling molecule used by marine bacteria for interactions with diatoms [START_REF] Amin | Interactions between Diatoms and Bacteria[END_REF] Lee and Lee, 2010).

QS associated phenotypes

It is now clear that communication is widely widespread and controls many physiological processes in Vibrio. QS allows bacteria to act socially as a cohesive unit in order to synchronize their behaviour like multicellular organisms. The group detects and respond to the signal molecule by a population-wide alteration of gene expression. These processes are predicted to be effective when undertaken by a group of bacteria but detrimental for single cells (Bassler and Losick, 2006;[START_REF] Waters | Quorum sensing: cell-to-cell communication in bacteria[END_REF]. However, recent studies have shown that this model might be oversimplistic and revealed that a nonhomogeneous response is observed for the same QS stimuli. In fact numerous other genetic elements and environmental conditions can control these phenotypes and it appears that heterogeneous populations are sometimes more efficient in taking actions [START_REF] Anetzberger | Heterogeneity in quorum sensing-regulated bioluminescence of Vibrio harveyi[END_REF]Miyashiro and Ruby, 2012;Pérez and Hagen, 2010).

In Vibrio, QS associated phenotypes have been widely studied in well described species along with QS pathways and the regulatory mechanisms. A non-exhaustive list of functions controlled by different QS pathways or molecules can be found in Table 1.4. In addition to the perfectly described mechanism of bioluminescence, QS is an important component of the pathogenicity of Vibrio species and controls many virulence factors as well as some proteases allowing a considerable advantage for host colonization. QS is also determinant for the access to different ecological niches by regulating biofilm formation, exopolysaccharide production, motility or membrane protein expression. The impact of QS has also been observed on the production of extracellular products involved in nutrient acquisition (siderophore production), defence mechanisms or survival strategies such as the VBNC state (Ayrapetyan et al., 2014), pigment production or metabolism (Goo et al., 2015).

For less studied Vibrio species much work is needed for the characterization of QS pathways and the identification of signal molecules, prior to the study of QS associated phenotypes. Positive regulation of metalloprotease, siderophore and exopolysaccharide production, osmotic stress response Bassler et al., 1993[START_REF] Bassler | Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway[END_REF]Henke and Bassler, 2004;Lilley and Bassler, 2000;[START_REF] Mok | Vibrio harveyi quorum sensing: a coincidence detector for two autoinducers controls gene expression[END_REF] Positive regulation of metalloprotease production VvpE and repression of cytolisyn VvhA production Kim et al., 2003;Shao and Hor, 2001 4.5 AHL diversity

The overall cellular metabolism and the genetic diversity of both synthases and receptors are the most described factors impacting the diversity AHL structures. AHLs synthesis has been maintained throughout the evolution suggesting direct benefits for individual fitness (Chong et al., 2013). However, it has been shown that AHL-sensing by luxR is evolutionarily pliable and not frozen in its response to different acyl side chain length AHLs (Hawkins et al., 2007). Regarding to the AHL synthases, the amino-acid composition of the fatty-acid biding domain is crucial for the acyl-side chain length but also for its substitutions (Watson et al., 2002). During evolution, modifications in the AHL synthase genes are thus likely to modify the specificity of the enzyme for its substrate. Moreover, AHL production is also under the control of the fatty acid metabolism, in fact, any modulation of the cell metabolic state such as environmental fluctuations are able to modify the nature and the concentration of the produced AHLs (Gould et al., 2006;Sullivan et al., 2002). Finally, it has also been observed that other QS pathways (AI-2 and CAI-1) can also have an impact on AHL production in V. fluvialis just like in V. anguillarum where LuxM/N can influence the AHL production of LuxI (Milton et al., 2001;Wang et al., 2013).

To date, V. anguillarum still remains as the most studied Vibrio species in terms of AHL structural diversity with studies thoroughly identifying the AHLs pattern produced in the 10 serotypes and under different culture conditions (Buch et al., 2003;Buchholtz et al., 2006).

Surprisingly, even in the very well-studied A. fischeri and V. harveyi strains there is a lack of studies investigating their AHL diversity. Overeall, C4-HSL, OH-C6-HSL and oxo-C10-HSL are the most detected molecules in Vibrio species and a non-exhaustive list of characterized AHLs can be found in Table 1.5. While different AHL production patterns have been reported for different Vibrio strains in a single species, some molecules are common to different isolates of a species suggesting that these AHLs are needed for important function at the species level (i.e. Table 1.5). These results suggest a certain intraspecific diversity of AHL production but the ecological significance and the factors involved in maintaining this diversity remains poorly known.

4.6 Interactions with other organisms 4.6.1 Interference with bacterial QS Vibrio species can have symbiotic relationship with higher organisms, where they can be pathogenic, commensal or in mutualistic association [START_REF] Austin | Vibrios as causal agents of zoonoses[END_REF][START_REF] Nishiguchi | Evolution of symbiosis in the Vibrionaceae: a combined approach using molecules and physiology[END_REF][START_REF] Ruby | Lessons from a cooperative bacterial-animal association: The Vibrio fischeri-Euprymna scolopes light organ symbiosis[END_REF]. As some of these higher organisms have developed defence mechanisms in order to compete for ecological niches or to protect themselves against bacterial colonisation. QS can be disrupted by diverse metabolites and enzymes. All the QS inactivation mechanisms are connected behind the term Quorum quenching (QQ) and include the disruption of QS signalling pathways by antagonistic/agonistic molecules or the inactivation of QS molecules by enzymatic degradation (Rolland et al., 2016). The global interest on QQ has started with the discovery of the red algae Delisea pulchra and its halogenated furanones able to inhibit the signalling pathways by displacing AHLs from their receptors and inactivating the AI-2 synthase LuxS [START_REF] Givskov | Eukaryotic interference with homoserine lactonemediated prokaryotic signalling[END_REF][START_REF] Manefield | Evidence that halogenated furanones from Delisea pulchra inhibit acylated homoserine lactone (AHL)-mediated gene expression by displacing the AHL signal from its receptor protein[END_REF][START_REF] Zang | A naturally occurring brominated furanone covalently modifies and inactivates LuxS[END_REF].

Many studies have investigated the QQ properties of aquatic organisms and red algae, sponge, cyanobacteria, microalgae/phytoplanktonic species, or bacteria are able to disrupt QS by many different processes and I list those in Table 1.6. Vibrio have been associated to cyanobacterial blooms [START_REF] Berg | High diversity of cultivable heterotrophic bacteria in association with cyanobacterial water blooms[END_REF], especially as their derived organic matter constitute a non-negligible reservoir of nutrient [START_REF] Eiler | Growth response of Vibrio cholerae and other Vibrio spp. to cyanobacterial dissolved organic matter and temperature in brackish water: Cyanobacterial DOM, temperature and Vibrio growth[END_REF]. On the other hand complex QS based relationships could occur between Vibrio and cyanobacteria. Gloeothece PCC 6909 produce C8-HSL which play a role in its metabolism and biofilm formation [START_REF] Sharif | Quorum sensing in cyanobacteria: Noctanoyl-homoserine lactone release and response, by the epilithic colonial cyanobacterium Gloeothece PCC6909[END_REF]. Phosphorous acquisition by Trichodesmium sp. is activated by AHLs produced by its epibiotic heterotrophic bacteria (C8-HSL, C10-HSL, C12-HSL and C14-HSL) [START_REF] Van Mooy | Quorum sensing control of phosphorus acquisition in Trichodesmium consortia[END_REF]. Growth and nitrogenase activity of Anabaena sp. PCC 7120 is inhibited by oxo-C10-HSL and oxo-C12-HSL and thus this strains produces an AHL acylase with highest affinities for AHLs above C10 chains (Romero et al., 2008[START_REF] Romero | Quorum sensing N-acylhomoserine lactone signals affect nitrogen fixation in the cyanobacterium Anabaena sp. PCC7120: AHLs inhibit nitrogen fixation in cyanobacteria[END_REF].

Insofar as most of the AHLs mentioned above can be produced by Vibrio species (i.e. Table 1.5) they could possibly control the metabolism of cyanobacteria species in the environment.

Finally, to understand properly QS it is necessary to admit that the mechanisms previously described mostly from a one strain pure culture cannot be that simple when the studied bacteria is part of a complex assemblage like in marine environment. Many factors such as abiotic environmental variables, beneficial or disadvantageous interactions with other oragnisms, or their lifestyle must be integrated in our vision of bacterial communication.

Objectives of this thesis

Quorum sensing is an important mechanism among Vibrio, involved in many vital functions such as niche colonization (motility, biofilm formation, exopolysaccharides secretion…), survival strategies (osmotic stress response, VBNC state) and virulence. The most studied signaling molecules are N-acyl-homoserine lactones (AHL) allowing intraspecific communication. However, the current knowledge on AHL-mediated QS mechanisms, pathways, and associated phenotypes is limited to a few pathogenic and/or bioluminescent Vibrio species while their AHL diversity remains underestimated. In fact, the comparison between the inventory of AHL synthase genes (Figure 1.7 and 1.8) and the previously described AHL diversity (Table 1.5) make it clear that AHL diversity still unknown or largely underestimated among Vibrio species.

Consequently, the first objective of this thesis, developed in Chapter 2, was to characterize AHL diversity among environmental strains of Vibrio. To determine the structure of AHLs a methodological approach combining biosensor-based screening of HPLC fractions prior to characterization by UHPLC-HRMS/MS was developed (Publication 1, Chapter 2).

Finally, our approach was applied on two model species, V. tasmaniensis LGP32 a model organism for host-pathogen interactions in bivalves (Publication 1) and 5 strains of A. fischeri the model for QS and bioluminescence studies (Publication 2). The results revealed an unexpected diversity of AHL molecules but also a quite surprising intra-species diversity of AHL production pattern. However, this observations have been made on strains isolated from diverse geographical locations at different time and it is conceivable that strains have evolved differently depending on the environmental conditions. In marine ecosystems, the physiology and the dynamic of Vibrio spp. in terms of abundance and diversity, is dependent on various abiotic factors but also on interactions with higher organisms. To better understand the QS mechanisms in Vibrio spp., and its roles in the ecology of these bacteria it is fundamental to investigate the environmental conditions and the ecological populations in which AIs production occurs. Furthermore, when studied at a microscale, they show a functional structuration in ecological units consisting of phylogenetically close strains sharing habitat and social traits.

Thus, my second objective was to understand how Vibrio communicate within environmental populations. In order to answer basic questions such as who communicate?

When? Which AHL are produced? A first ecological study has been conducted to investigate the temporal dynamic of AHL production among Vibrio species in a Mediterranean lagoon.

With this aim a two years sampling study in the Salses-Leucate lagoon was assessed, multiple environmental parameters were measured and phytoplankton communities were identified. A total of 840 strains of Vibrio were isolated in two fractions (water <50 µm and attached to phytoplankton >50µm) and screened for their ability to produce AHLs. We observed a temporal dynamic of AHL production patterns within a single strain of V. mediterranei and revealed by a statistical approach the potential involvement of abiotic and biotic parameters in these variations while no significant correlation was found between AHL production phenotypes and their respective size fractions (Chapter 3, Publication 3). However, it appears that Vibrio populations are exhibiting reproducible fine-scale structure according to their lifestyle in the bacterioplankton. In this context it was necessary to know if these different AHL production phenotypes could be explained by the association with different microhabitats in the water column. A fine-scale resource partitioning sampling strategy allowed to identify several of ecological populations co-occuring in a marine environment (closely related Vibrio strains associated to different lifestyles such free-living, particle associated, zooplankton associated or oyster associated) [START_REF] Bruto | Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition[END_REF].

In order to determine if a common language is spoken within ecological populations sharing habitat preferences, a second ecological study was conducted. A sub-selection of ~ 230 isolates of Vibrio, previously identified as belonging to 24 ecological populations, were screened for their abilities to produce AHL and AI-2 signal molecules. AI-2 mediated QS was widespread and conserved within populations while AHL production was strain dependant within a single population. Within an AHL producing population, affiliated to V. rotiferianus, we investigated the link between their genetic diversity and AHL production patterns. The results indicate that AHL production patterns were not conserved within ecological populations. (Chapter 4, Publication 4).

Altogether, the results provided by our two environmental studies, highlighted the fact that different strains of a single genotype or ecological unit of Vibrio, in identical culture conditions, harbouring identical AHL synthase genes, are showing a large diversity of intraspecies AHL production patterns.

Abad, M.J., Bedoya, L.M., Bermejo, P., 2011 synthesis and sensing of this signaling molecule (Engebrecht et al., 1983;Engebrecht and Silverman, 1984). Later, the AinS/AinR system was identified and a second auto-inducer, Noctanoyl-L-HSL (C8-HSL), was characterized. This inducer is involved, especially, in early colonization factors important for the settlement of A. fischeri in the light organ (Gilson et al., 1995;Kuo et al., 1994;Lupp and Ruby, 2005). Since these initial studies, many works have revealed a wide genetic and phenotypic diversity in symbiotic strains of A. fischeri. Indeed, heterogeneous phenotypes have been observed, including bioluminescence intensity, motility, growth rate, siderophore production, colony pigmentation or competence in colonizing the light organ (S and D strains). These results support the existence of intraspecific differences related to host population biology among A. fischeri strains within the same light organ [START_REF] Bongrand | A genomic comparison of 13 symbiotic Vibrio fischeri isolates from the perspective of their host source and colonization behavior[END_REF][START_REF] Wollenberg | Population structure of Vibrio fischeri within the light organs of Euprymna scolopes squid from two Oahu (Hawaii) populations[END_REF]. However, no recent studies have aimed to characterize the AHL diversity that could hide behind these phenotypic variations.

B B C C 4 9 8 B B C C 1 6 5 80 V ib ri o ta sm a n ie n s is (T ); L M G 2 0 0 1 2 ; A J3 1 6 1 9 2 V ib ri o le n t u s (T ) C E C T 5 1 1 0 T ; A J 2 7 8 8 8 1 B B C C 6 7 B B C C 52 6 B B C C 85 0 B BC C 85 1 BB C C2 19 7 L G P3
Furthermore, with the development of more sensitive methods in analytical chemistry, a large diversity of novel AHL molecules can be detected (Girard et al., 2017;Patel et al., 2016). The AHL molecule consists of a hydrophilic homoserine lactone ring moiety and a hydrophobic acyl side chain. The latter can lead to a wide diversity of molecules through variations in length, substitutions at the beta-position and level of saturation [START_REF] Fuqua | Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone Quorum sensing[END_REF]. These different features result from AHL synthase specificity for its substrates and determine the interactions with the corresponding receptor (Collins et al., 2004;Hawkins et al., 2007). Considering that genetic diversity, metabolism and environmental factors are likely to modulate AHL production (Gould et al., 2006;Sullivan et al., 2002;Watson et al., 2002), it seems essential to characterize the AHL diversity among A. fischeri strains presenting different genetic and phenotypic features.

The aims of this study were to determine whether strains exhibit similar or different chemical languages and to investigate the possible link between AHL production and the genetic diversity (whole genome, AHL synthases and receptors) of these A. fischeri strains.

Our work was based on isolates previously collected in squids from different geographical locations, which were then genotyped (VfRep PCR fingerprints and whole-genome sequencing) and phenotyped, especially for their capacity to colonize the light organ (S and D strains; [START_REF] Bongrand | A genomic comparison of 13 symbiotic Vibrio fischeri isolates from the perspective of their host source and colonization behavior[END_REF]Lupp and Ruby, 2005;[START_REF] Mandel | A single regulatory gene is sufficient to alter bacterial host range[END_REF][START_REF] Wollenberg | Population structure of Vibrio fischeri within the light organs of Euprymna scolopes squid from two Oahu (Hawaii) populations[END_REF]. This initial investigation consisted of the identification of the produced AHL by a previously described bioguided UHPLC-HRMS/MS methodology from a sub-selection of A. fischeri strains (ES213, KB2B1, KB4B5, MB13B1 and ES114) (Girard et al., 2017). In the present study, we also focused our investigation on the genetic basis of production and the mechanisms for sensing these signaling molecules.

RESULTS & DISCUSSION

Detection of AHL signal molecules

We investigated AHL diversity by a non-targeted UHPLC-HRMS/MS method previously described by Girard et al., 2017, which HSL (N-3-hydroxy-dodecanoyl-homoserine lactone). The analysis of AHL standards was also used to build retention time curves for Cx-HSL, OH-Cx-HSL and oxo-Cx-HSL according to their acyl-side chain length. The curves presented a R 2 >0.99 (Figure S1), and allowed for the prediction of AHL retention times (Table 1) under our chromatographic conditions. The identification of OH-C9-HSL (N-3-hydroxy-nonanoyl-homoserine lactone) was confirmed by the presence of at least 2 fragment ions cited above as well as a retention time corresponding to the prediction curves (Table 1, Table S2). Other AHL-type compounds were revealed by the presence of at least 2 characteristic fragment ions at indicative retention times; however, the structure of these compounds needs to be confirmed by nuclear magnetic resonance spectroscopy (NMR) analysis. The presence of these AHLs has unfortunately been impossible to confirm because an NMR analysis requires large amounts of pure compounds. Notably, these compounds are produced at very low concentrations and purification steps can lead to substantial difficulties in obtaining enough material for structural elucidation. The aim of our work was to compare the AHL production patterns between strains under identical culture conditions. We identified seven AHLs common to all the analyzed strains (C6-HSL, C7-HSL, C8-HSL, OH-C8-HSL, C9-HSL, OH-C10-HSL and OH-C9-HSL) and two different AHL production patterns, one including ES213, KB2B1, KB4B5 and ES114

(OH-C12-HSL and C10-HSL) and a second with MB13B1 (oxo-C6-HSL oxo-C8-HSL) (Figure 1). Recently, Purohit et al., 2013 described the presence of six different AHLs in the strain ES114 (C4-HSL, 3-oxo-C6 HSL, C6-HSL, 3-OH-C8-HSL, C8-HSL, C10-HSL), and here we have identified three additional AHLs. Our extraction protocol was performed at an OD600 ~ 1.5; therefore, it is not surprising that we did not recover C4-HSL and oxo-C6-HSL, as the limit of detection (LOD) of our UHPLC-HRMS/MS method for these molecules is far beyond the previously detected concentrations (LOD >500 nmol.L -1 for C4-HSL as compared with 70 nmol.L -1 and LOD 10.90 nmol.L -1 oxo-C6-HSL as compared with 0.1 nmol.L -1 ).

Initially, it was demonstrated that oxo-C6-HSL is produced in lower quantities than C8-HSL.

As we detected oxo-C6-HSL only in MB13B1, it appears that this strain produces larger quantities of this molecule than the other four strains in identical culture conditions (Girard et al., 2017;Lupp and Ruby, 2004;Purohit et al., 2013;[START_REF] Ruby | Quorum signaling and symbiosis in the marine luminous bacterium Vibrio fischeri[END_REF]. To our knowledge, this is the first report which inventories the AHLs produced by A. fischeri and compares AHLs across different symbiotic strains.

Link between AHL production and genetic diversity of A. fischeri strains

We examined the relationship between AHL production patterns and the genetic diversity of the associated Aliivibrio fischeri strains. First, it is worth noting that while the strains KB4B5, ES114, KB2B1, and ES213 are phylogenetically distinct based on wholegenome comparisons, these strains all presented the same AHL pattern [START_REF] Bongrand | A genomic comparison of 13 symbiotic Vibrio fischeri isolates from the perspective of their host source and colonization behavior[END_REF]. Interestingly, these data reveal the absence of a link between whole genome diversity and the AHL production patterns of these strains. Furthermore, the clustering of these strains according to their AHL production patterns did not correlate with the colonization capacities In a second step, we then examined the genetic diversity of synthases and receptors involved in AHL meditated QS across A. fischeri strains by focusing our work on five key proteins: AinS, AinR, LuxI, LuxR and LitR. These five genes were identified by BLAST in the strains ES213, KB2B1, MB13B1 and KB4B5 (genome sequenced by [START_REF] Bongrand | A genomic comparison of 13 symbiotic Vibrio fischeri isolates from the perspective of their host source and colonization behavior[END_REF] using the ES114 strain as a reference (NCBI Accession number: NC_006840). First, we observed a clear separation between MB13B1 and the other strains in AinS, LuxR and LitRbased phylogenies, while no correlation was observed with LuxI and AinR (Figure S2). Such observations imply that AHL production through the AHL synthase AinS as well as signal reception by LuxR and LitR are likely to have substrate specificity, which may vary depending on genetic diversity. As suggested by Collins et al., 2004, LuxR is evolutionary pliable and not fixed in its response to AHLs of different acyl-side-chain lengths. However, we showed that the separation of MB13B1 in a LuxR phylogeny is explained by a difference of 3 amino acids, including 2 located in the amino-terminal domain responsible for the sensitivity to a broad spectrum of AHLs (Table 2, Collins et al., 2004;[START_REF] Fuqua | Signalling: Listening in on bacteria: acyl-homoserine lactone signalling[END_REF]. Some works have identified the key amino-acids for LuxR specificity towards historical signaling molecules but data are missing for other AHLs as described in the present study (Collins et al., 2004(Collins et al., , 2006;;Hawkins et al., 2007). The AinS synthase is still poorly studied, but Gilson et al., 1995 have demonstrated that the full-length AinS is necessary to produce AHL. This implies that the four observed amino-acid changes in MB13B1 can possibly have an impact on AHL synthesis. The study of LitR sequences indicates a change of polarity on one amino acid in MB13B1; however, the lack of structural information prevents any conclusions from being drawn (Table 2).

AinS Q42 → K Amidic → Basic Q292 → K Amidic → Basic S293 → A Hydroxylic → Aliphatic T298 → A Hydroxylic → Aliphatic LuxR T101 → V Hydroxylic → Aliphatic D142 → N Acidic → Amidic R176 → H Basic → Basic LitR S187 → P Hydroxylic → Aliphatic
So far, it has been demonstrated that the two AHL pathways LuxI/R and AinS/R are connected by an intracellular signaling cascade and also that C8-HSL, produced by AinS, is able to bind LuxR and activate is own expression [START_REF] Fidopiastis | LitR, a new transcriptional activator in Vibrio fischeri, regulates luminescence and symbiotic light organ colonization[END_REF][START_REF] Lupp | The Vibrio fischeri quorum-sensing systems ain and lux sequentially induce luminescence gene expression and are important for persistence in the squid host[END_REF]Lupp and Ruby, 2004). LitR, a HapR homolog, is also a regulatory protein involved in QS, standing between the AinS/AinR signaling pathways and the regulation of LuxR gene expression [START_REF] Fidopiastis | LitR, a new transcriptional activator in Vibrio fischeri, regulates luminescence and symbiotic light organ colonization[END_REF]. In this context, it may be possible that AinS, LuxR and LitR are co-evolving in order to maintain the connection between the two circuits. This could explain the link between their genetic diversity and the AHL production patterns among A.

fischeri strains. Remarkably, LuxI and AinR were found to be extremely conserved, with 100% similarity between protein sequences among A. fischeri strains, suggesting that LuxI signal synthesis has been maintained through evolution and that in some way it is important for individual fitness to keep this signal production active (Chong et al., 2013). The climate is typical of the NorthWestern Mediterranean zone characterized by sporadic but heavy rainfall (500 mm.yr -1 ), intense dry-ness and sunshine. For 2015 and 2016 the maximum precipitation was between 208 and 406 mm.yr -1 with an average of 50 days of rain by year.

CONCLUSION

The lagoon is submitted to strong winds (>16 m.s -1 , 150 d.yr -1 ) which play a major role in the hydrodynamics of the basin and generate strong resuspension events mainly during autumn, winter and spring seasons.

Globally, the salinity is high (mean = 32 psu) and fluctuates both seasonally and spatially between 27 and 41 psu [START_REF] Wilke | Spatio-temporal dynamics of physicochemical and chemical factors in the water of a heavily transformed mediterranean coastal lagoon, the Etang de Salses-Leucate (France)[END_REF]. There is a general positive salinity gradient from the southwestern to the northwestern part of the lagoon [START_REF] Boutière | Effet d'une crise dystrophique dans l'étang de Salses-Leucate[END_REF]. Chlorophyll a (Chl

Monitoring programs

Few bulk measurements such as salinity, water temperature, oxygen saturation and turbidity in the Salses-Leucate lagoon are monthly monitored since 2014 by the program "Observatoire de l'étang" carried out by the "Syndicat Mixte Rivage" available online (http://rivage-salsesleucate.org/missions/suivis-et-travaux/observatoire-de-letang/).

Minimal temperatures are recorded in winter between December and February and can reach 5°C while maximal temperatures are observed in summer between June and August reaching 25°C. In addition, because of its shallow depth, the lagoon is subjected to high evaporation rate in summer when temperatures and insolation increase. In this climatic context, seawater surface temperature and salinity evolve in parallel, they are increasing in summer and decreasing in winter with freshwater inputs and strong cold wind events (Figure 3.2A).

The turbidity is on average very low in the lagoon, however high sediments resuspension are following severe wind events during winter and at the beginning of spring. The recorded oxygen saturation is always >100% either supersaturated, indicating that there is more oxygen production then consumption in the lagoon especially because high concentrations of photosynthetic organisms occurs in the lagoon (Figure 3.2B). A total of seven sampling points are used for the different programs described below, and the survey is carried out on water and/or in the shellfish flesh and intervalvular liquid (F.L.I.) (Figure 3.3).Three major monitoring programs are conducted in the lagoon:

-The REPHY, for phytoplankton populations and phycotoxin monitoring.

-The REMI, for microbiological monitoring following Escherichia coli as an index of faecal contamination. -The ROCCH, for chemical contamination monitoring following Cadmium (Cd), Lead (Pb), Mercury (Hg), Zinc (Zn), Fluoranthene (Polycyclic Aromatic Hydrocarbon, PAH) and Polychlorinated biphenyl (CB153; PCBs).

The lagoon is considered as an area with high risk of phycotoxin contamination, having an alert period of nine months out of twelve and with a greater risk in mussels than in oysters

INTRODUCTION

Marine bacterial communities inhabiting marine coastal areas are subjected to rapid and somewhat unpredictable changes in their environment [START_REF] Lennon | Microbial seed banks: the ecological and evolutionary implications of dormancy[END_REF]. In order to cope with these changes, bacteria implement flexible strategies supporting a certain degree of phenotypic plasticity [START_REF] Agrawal | Phenotypic plasticity in the interactions and evolution of species[END_REF][START_REF] Chevin | Adaptation, plasticity, and extinction in a changing environment: Towards a predictive theory[END_REF]West-Eberhard, 2003). Bacteria in the genus Vibrio are well-known for their capacity of rapid physiological adaptation in response to changing environmental conditions, making them highly dynamic over short-term and seasonal scales (Takemura et al., 2014). These opportunistic bacteria are sensitive to unfavorable conditions by switching from an active free living phenotype into a "dormant" viable but not culturable (VBNC) phenotype, or by colonizing viscous surfaces such as biofilms (Chimetto Tonon et al., 2015;[START_REF] Li | The importance of the viable but non-culturable state in human bacterial pathogens[END_REF]Vezzulli et al., 2015b;[START_REF] Whitesides | Resuscitation of Vibrio vulnificus from the viable but nonculturable state[END_REF]. When favorable conditions reappear their high reactivity allow them to colonize new substrate and to be part of the microbial community associated with zooplankton, phytoplankton, and marine vertebrates and invertebrates [START_REF] Ben-Haim | Vibrio coralliilyticus sp. nov., a temperature-dependent pathogen of the coral Pocillopora damicornis[END_REF][START_REF] Heidelberg | Bacteria of the γ-subclass proteobacteria associated with zooplankton in Chesapeake bay[END_REF][START_REF] Soto | Salinity and temperature effects on physiological responses of Vibrio fischeri from diverse ecological niches[END_REF][START_REF] Wendling | Adaptation to enemy shifts: rapid resistance evolution to local Vibrio spp. in invasive Pacific oysters[END_REF].

In the past decades, our vision of bacterial communities has significantly changed, as microbial cells are no longer considered to behave individually but rather to act socially (Goo et al., 2015;[START_REF] West | Social evolution theory for microorganisms[END_REF][START_REF] West | The social lives of microbes[END_REF]. It is now clear that microorganisms perform social behaviours, synchronizing the expression of functional genes in a high cell-density manner by sensing their environment. This mechanism is generally known as Quorum sensing (QS) [START_REF] Fuqua | Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators[END_REF]. The communication between close relative members of Proteobacteria is mediated by the Type 1 auto-inducer (AI-1) also called N-Acyl-Homoserine Lactones (AHL).

Today, most of the studies are highlighting the processes controlled by QS in Vibrio species, i.e. QS-associated phenotypes, such as bioluminescence and symbiosis [START_REF] Bassler | Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway[END_REF][START_REF] Schwartzman | A conserved chemical dialog of mutualism: lessons from squid and vibrio[END_REF], biofilm formation [START_REF] Hammer | Quorum sensing controls biofilm formation in Vibrio cholerae[END_REF], toxins production or expression of virulence factors [START_REF] Ha | Quorum sensing-dependent metalloprotease VvpE is important in the virulence of Vibrio vulnificus to invertebrates[END_REF]Natrah et al., 2011;Zhu et al., 2002). Finally in vitro studies have also demonstrated the involvement of QS for the resuscitation of Viable but Non-culturable (VBNC) Vibrio cells (Ayrapetyan et al., 2014;[START_REF] Bari | Quorum-sensing autoinducers resuscitate dormant Vibrio cholerae in environmental water samples[END_REF].

However, Platt and Fuqua, 2010 presented the QS process itself, i.e. the production of signal molecules, as depending on its ecological context and impacted by a multiple aspects of natural environments.

In support to these view, some studies on AHL biosynthetic pathways are suggesting that any environmental factor likely to affect the primary metabolism of a bacteria can modify his spoken "language" (Gould et al., 2006;[START_REF] Hoang | Beta-ketoacyl acyl carrier protein reductase (FabG) activity of the fatty acid biosynthetic pathway is a determining factor of 3-oxo-homoserine lactone acyl chain lengths[END_REF]. Furthermore, several studies have reported heterogeneous AHL production phenotypes (APP) among isolates of a single Vibrio species, which underlines different AHL production patterns according to their origin (Buchholtz et al., 2006;García-Aljaro et al., 2012;Purohit et al., 2013;Rasmussen et al., 2014). Interestingly, few reports enhance the idea that QS need to be studied by making the link between microbiology and ecology, meaning to integrate the QS in its ecological context (Bassler and Losick, 2006;Keller and Surette, 2006;Persat et al., 2015).

Nonetheless, the produced AHLs by Vibrio spp. in order to adapt to their environment, including competitive environments with other organisms, remain largely underexplored. In the present study we investigated the temporal dynamics of AHL producing Vibrio spp.

isolates over a two-year sampling of a French Mediterranean lagoon subjected to highly variable environmental conditions and evaluated the possible links between phenotypic plasticity of AHL production and environmental variables.

MATERIALS & METHODS

Sample collection

A Physico-chemical parameters, as well as pigments were analyzed using standard methods and the methodological details can be found in Table 1.

Table 1: Assessment of environmental parameters measurements

Phytoplankton and picoplankton counts

Phytoplankton counts was carried out with an Olympus IMT inverted microscope in 5, 10, 25 or 50 ml plate chambers using the Utermöhl method updated by Karlson and collaborators [START_REF] Karlson | Microscopic and molecular methods for quantitative phytoplankton analysis[END_REF]. Replicates of different volumes were counted according to the size and the abundance of the cells. Small species were mainly counted in diagonal transects at 300x magnification and for the large or less abundant species half or whole chambers were scanned at 100x and 40x magnification. A minimum of one hundred total cells were counted for each counting. Different settling times were applied according chamber volume in a temperature-controlled room [START_REF] Karlson | Microscopic and molecular methods for quantitative phytoplankton analysis[END_REF].

Samples were collected for picoplankton enumeration (1,5 ml The PCR conditions were slightly optimized for the KAPA2G Fast DNA Polymerase and were as following, a 5 min initial denaturation step at 95 °C, followed by 40 cycles at 95 °C for 15 sec, 60 °C for 30 s and 72 °C for 30 s, and a final elongation step of 2 min at 72 °C.

PCR products were sent to Macrogen Europe for sequencing using the dideoxy-termination (Sanger) reaction with the primer gyrB UP1S (5'GAAGTCATCATGACCGTTCT GCA3') [START_REF] Yamamoto | PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains[END_REF] and to obtain sequences around 800 bp length using.

Detection of QS-active isolates

The methodological details concerning the screening of AHL producers and the identification of AHL compounds by UHPLC-HRMS/MS were strictly identical to Girard et al., 2017. Briefly, each isolate was tested on their capacity to produce AHLs using two biosensors, Pseudomonas putida (pKR-C12) and Escherichia coli (pJBA-132) corresponding to, respectively, the F117 and MT102 phenotypes in order to detect long-and short-chain AHLs. The tests were performed as described previously by [START_REF] Wagner-Döbler | Discovery of complex mixtures of novel long-chain Quorum sensing signals in free-living and host-associated marine Alphaproteobacteria[END_REF].

Genetic and genomic characterization of V. mediterranei isolates

For the ERIC-PCR genotyping of V. mediterranei, genomic DNA from 253 isolates was obtained by cellular lysis (3 quick cycles of liquid nitrogen freeze/70°C thaw) and DNA concentrations were measured spectrophotometrically and diluted to at 100 ng/µL. The ERIC-PCR reactions were performed using previously described protocols employing the ERIC1 and ERIC2 primers [START_REF] Hulton | ERIC sequences: a novel family of repetitive elements in the genomes of Escherichia coli, Salmonella typhimurium and other enterobacteria[END_REF][START_REF] Khan | Characterization of United States outbreak isolates of Vibrio parahaemolyticus using enterobacterial repetitive intergenic consensus (ERIC) PCR and development of a rapid PCR method for detection of O3: K6 isolates[END_REF]. PCR amplifications were performed with the GoTaq G2 Flexi polymerase (Promega) and the fingerprint profiles were visualized using a Typhoon FLA 9000 (GE Life Sciences). Gel images analysis were performed using the GelJ software [START_REF] Heras | GelJ -a tool for analyzing DNA fingerprint gel images[END_REF]. For whole genome sequencing, genomic DNA of V. mediterranei isolates (17 LN0615E and 21 LS0615E) was obtained with a classical CTAB-β-mercatpoethanol extraction protocol and sent for Illumina MiSeq DNA sequencing by Mr DNA (Shallowater, TX, USA) as described by [START_REF] Doberva | Genome sequence of Maribius sp. strain MOLA 401, a marine Roseobacter with a Quorum-sensing cell-dependent physiology[END_REF] Genomes were annotated using the RAST server [START_REF] Aziz | The RAST Server: Rapid annotations using subsystems technology[END_REF].

Multivariate analyses

A PERMANOVA analysis was used to test for significant differences in Vibrio assemblages as well in V. mediterranei phenotypes between the two size fractions (<50µm and > 50µm)

and among time (Anderson, 2001a).

The relationships between the G1 V. mediterranei assemblage (abundance of each AHL production phenotypes over time, Square-root transformed and Bray-Curtis distance matrix) and all measured environmental variables (Normalization and Euclidean distance matrix)

were investigated using a Distance Based Linear Model (DISTLM) [START_REF] Mcardle | Fitting multivariate models to community data: A comment on distance-based redundancy analysis[END_REF]. In order to visualize if the temporal variations of G1 phenotypes were co-occurring with specific phytoplankton taxa abundances, we conducted a second step of analysis consisting of a Principal Component Ordination (PCO) of V. mediterranei assemblage using phytoplankton taxa counts (Square-root transformed and Bray-Curtis distance matrix) as predictor variables (non-parametric Spearman rank correlation). All the multivariate analyses were performed using PRIMER v.7 and its add-on package PERMANOVA + (Anderson, 2001a;Anderson, 2001b;[START_REF] Mcardle | Fitting multivariate models to community data: A comment on distance-based redundancy analysis[END_REF].

RESULTS

Phenotypic and Genetic temporal variation of V. mediterranei isolates

The partitioning of Vibrio isolates, between water column including particles less than 50 µm and particles greater than 50 µm was monitored during spring and summer 2015/2016 and a total of 840 Vibrio strains were isolated. We did not observe differences in Vibrio assemblages between both fractions over time (data not shown, PERMANOVA, p value = 0.904; the temporal variation of the total Vibrio spp. can be found in supplementary material,

Fig S2).

Isolate identification based on gyrB gene sequences from both size fractions showed a seasonal dynamic of V. mediterranei isolates (Figure 1) in particular for 2016, starting from 0-28% of total Vibrio isolates during spring and increasing gradually to up to 97% (Summer 2016). Interestingly, the phenotyping of these isolates, under identical culture and screening conditions, revealed a temporal variation in APP among time. They varied for example from producing only long chain AHLs (F117 + /MT102 -) to producing long and short chain AHLs (F117 + /MT102 + ) in 2015, or from a predominance of isolates with no production (F117 - /MT102 -) to an exclusive production of long chain AHLs (F117 + /MT102) in 2016 (Figure 1B). In order to determine if these phenotypic changes were related to a shift between different genotypes of V. mediterranei, we genotyped the 253 V. mediterranei strains by ERIC-PCR [START_REF] Rivera | Enterobacterial repetitive intergenic consensus sequences and the PCR to generate fingerprints of genomic DNAs from Vibrio cholerae O1, O139, and non-O1 strains[END_REF]. A total of 22 genotypes were identified, G1 to G22 (i.e. Figure 1A and To implement the genetic characterization of V. mediterranei G1 isolates and better understand the genetic basis of this phenotypic variation, we obtained a draft whole genome sequence for two strains (17LN 0615E -F117 + /MT102 -and 21LN 0615E -F117 + /MT102 + phenotypes). A comparison between these genomes yielded an ANI value of 99.96% and since as previously proposed, two strains can be considered identical if they have a ANI value >99.9% (Olm et al., 2017;Snitkin et al., 2012). We consider that we observed phenotypic plasticity within a single strain of V. mediterranei. To summarize, we observed in 2015 and 2016, the predominance of a single strain of V. mediterranei, up to 97% of the total cultivable Vibrio, showing different APP over time.

Characterization of AHL production phenotypes of G1 isolates

In order to confirm that the observed APP were exhibiting different AHL patterns, we analyzed the AHLs produced by two representative isolates of each phenotype (F117 + /MT102 -

) and (F117 + /MT102 + ) of V. mediterranei G1 strains by UHPLC-HRMS/MS (i.e. Table S6 and S7). A total of 13 AHLs were detected and 8 were common to the four isolates, namely OH-C10-HSL, oxo-C10-HSL, OH-C11-HSL, OH-C12-HSL, oxo-C12-HSL, C12-HSL, C13-HSL and oxo-C13-HSL (Fig. 2 and Fig. S4). Three short chain AHLs, C6-HSL, oxo-C6-HSL, and oxo-C8-HSL were putatively identified as being responsible for the F117 + MT102 + phenotype. Interestingly, we observed that two long chain AHLs, oxo-C11-HSL and C14-HSL, were produced by the (F117 + /MT102 -) isolates but were not detected in the (F117 + /MT102 + ) isolates. Summarizing, the AHL analysis confirmed that the different APP detected by the biosensors are producing different AHLs patterns, with (F117 + /MT102 -) producing long acyl-side chains and (F117 + /MT102 + ) producing long plus short acyl-side chains.

Genetic basis of AHL production

One possibility to explain differences between strains with highly similar genomes would be that these differences are carried by non-identical AHL synthase genes. We searched for AHL synthase genes in the two genomes of G1 strains but somewhat surprisingly no luxI or luxM orthologs were found. Therefore we searched for genes coding for acyltransferases that could be involved in AHL synthesis, and identified 16 genes, surrounded by genes unaffiliated to predictable alternative metabolic pathways (e.g. fatty acid synthesis) in the annotation. The comparison between the two strains (17 LN0615E and 21 LS0615E) revealed a 100% identity on the 16 genes sequences, suggesting that the differences observed in AHL production patterns are not explained by different presumptive AHL synthases (Table S8).

Factors affecting the dynamic of G1 -AHL production phenotypes

We conducted a distance based linear modeling approach (DistLM) to investigate the relationships between the dynamic of the APP among G1 strains and the environmental variables. A total of 7 variables explained 83.4% of the temporal variation of G1 APP, and among those, phosphate concentration was the variable with highest explanatory value (37.4 %) and ammonium concentration the lowest with 4.5% (Figure 3 and Sequential tests, Table 2). The analysis is detailed Table 2.

Axis 1 (db RDA1) represented essentially a gradient in abundance of the entire genotype varying from high abundances at the left-end to low abundances or no counts at the right-end (July 2015 and April/May 2016). Samples with high counts of V. mediterranei G1 were associated with higher conductivity (higher temperature plus salinity), total dissolved nitrogen, and low phaeophytin concentrations while an inversed trend was observed for samples with low counts or where V. mediterranei G1 was not retrieved. Axis 2 (db RDA2) reflected the distribution of the different APP. A peak of phosphates and nitrites were associated with an increase of non AHL producers in the higher end of the axis and as the F117 + /MT102 - phenotype is dominating dates with high counts of G1 isolates, the same parameters are linked to counts of the F117 + /MT102 -phenotype at the lower end of db RDA2 axis. S4 and the contribution of each variable in the construction of PCO axis (Pearson correlation) is detailed in Table S5.

DISCUSSION

It is well known that the ability of Vibrio, as well of other gram negative bacteria, to survive by making biofilms, infecting hosts, modifying their metabolism or going into a VBNC state is modulated by QS [START_REF] Bondí | Affecting Pseudomonas aeruginosa phenotypic plasticity by Quorum sensing dysregulation hampers pathogenicity in murine chronic lung infection[END_REF]Goo et al., 2015;Persat et al., 2015;Ayrapetyan et al., 2014). In addition to the fact that QS allows bacteria to sense their number and coordinate their actions, variability of auto-inducers production can offer an advantageous response to adapt their actions depending on fluctuating environments. However, in order to survive in changing environment, bacteria have evolved to set up various adaptive responses.

Phenotypic plasticity is one of them and it is the direct influence of the environment on the development of individual phenotypes, in other words, bacteria can adapt to environmental selective pressures to maintain their own fitness.

Phenotypic diversity of QS systems has already been described for different strains of a same Vibrio species (Buchholtz et al., 2006;García-Aljaro et al., 2012;[START_REF] Joelsson | Genetic and phenotypic diversity of Quorum-sensing systems in clinical and environmental isolates of Vibrio cholerae[END_REF]Purohit et al., 2013) and temporal changes in AHL production during the development of natural biofilm including Vibrio species as been shown [START_REF] Huang | Evidence for the dynamics of acyl homoserine lactone and AHL-producing bacteria during subtidal biofilm formation[END_REF], but the scope of these earlier studies did not show clear evidence of QS phenotypic plasticity.

Our results show a clear short-term temporal variation in APP among environmental isolates of a Vibrio species (V. mediterranei) that were grouped in a single genotype (G1) based on gyrB sequences, REP-PCR and genomic analysis. This change of AHL patterns, was temporally coherent and in some cases reached the near totality of Vibrio isolates in a sample.

As the Average Nucleotide Identity among the two sequenced genomes was very high (99.96%) but not identical, it is always possible that the residual 0.04% would be responsible for the differences of APP by some unknown mechanism. Nonetheless, sequence analysis of acyl transferase genes in these genomes found no differences either in the number of these enzymes or their sequences.

While it appears that we have different APP belonged to a single genotype, the temporal sampling of Vibrio isolates in parallel to physicochemical parameters measurements allowed us to evaluate the variation in the "environment" leading to the proposed AHL production phenotypic plasticity. However, the sampling was originally designed to evaluate total Vibrio spp. diversity (and not just G1V. mediterranei), and thus in the following discussion we point to major trends and raise ideas for future studies.

Phosphate concentration appear to be one of the most explanatory variables in our DistLM analysis being correlated with high abundances of non AHL producers on 21 June 2016, where the near totality of strains belonged to the (F117 -/MT102 -) phenotype and coinciding with a peak of phosphate likely associated with sediment resuspension. The whole genome analysis of the G1 isolates revealed few copies of the ppk gene and several studies have demonstrated that polyphosphate kinase (ppk) enhance the ability of bacteria to survive at environmental stressors. Effectively, this enzyme is repressed in high phosphorus concentration and have a negative impact on AHLs production [START_REF] Jahid | Polyphosphate stores enhance the ability of Vibrio cholerae to overcome environmental stresses in a low-phosphate environment[END_REF][START_REF] Rashid | Polyphosphate kinase is essential for biofilm development, quorum sensing, and virulence of Pseudomonas aeruginosa[END_REF][START_REF] Silby | Requirement of polyphosphate by Pseudomonas fluorescens Pf0-1 for competitive fitness and heat tolerance in laboratory media and sterile Soil[END_REF]. The bloom of non AHL producers, the 2016/06/21, occurred when there was the highest phosphate concentration in the two years time series. We hypothesized that in these context there is low expression of ppk gene and down regulation of AHL production [START_REF] Rashid | Polyphosphate kinase is essential for biofilm development, quorum sensing, and virulence of Pseudomonas aeruginosa[END_REF].

The Salses-Leucate lagoon was characterized by high Cyanobacteria concentrations, mainly Synechococcus species, ranging between 3.86 x 10 5 to 2.88 x 10 8 cells/L, making them an important part of the biological component. Over the study, Cyanobacteria counts were highly correlated to the (FF17 + /MT102 + ) phenotypes abundances (Pearson correlation 77.3%;

p value = 0.002; June/July 2015) but also to the bloom of (F117 + /MT102 -) phenotypes (July 2016, Figure 4). In the past, Vibrio have been associated to cyanobacterial blooms [START_REF] Berg | High diversity of cultivable heterotrophic bacteria in association with cyanobacterial water blooms[END_REF], especially via their derived organic matter [START_REF] Eiler | Growth response of Vibrio cholerae and other Vibrio spp. to cyanobacterial dissolved organic matter and temperature in brackish water: Cyanobacterial DOM, temperature and Vibrio growth[END_REF].

Mutual interactions were described between Synechococcus species and heterotrophic bacteria [START_REF] Hayashi | Growth of the cyanobacterium Synechococcus leopoliensis CCAP1405/1 on agar media in the presence of heterotrophic bacteria[END_REF] but co-culture experiment between Vibrio and Synechococcus revealed a defective effect on the physiology of the cyanobacteria especially on iron, phosphate and nitrogen pathways [START_REF] Tai | Whole-genome microarray analyses of Synechococcus -Vibrio interactions[END_REF]. In the other hand, since Synechococcus harbors the luxR gene involved in the AHL signal reception and is able to inhibit the QS of Vibrio species, complex QS based relationships can occur between them [START_REF] Honda | Draft genome sequence of marine cyanobacterium Synechococcus sp. strain NKBG042902, which harbors a homogeneous plasmid available for metabolic engineering[END_REF][START_REF] Marsan | Draft genome sequence of Synechococcus sp. strain CB0101, isolated from the chesapeake bay estuary[END_REF][START_REF] Santhakumari | Inhibitory effect of marine cyanobacterial extract on biofilm formation and virulence factor production of bacterial pathogens causing vibriosis in aquaculture[END_REF][START_REF] Shimura | Complete genome sequence of a coastal cyanobacterium, Synechococcus sp. strain NIES-970[END_REF][START_REF] Yoshino | Draft genome sequence of marine cyanobacterium Synechococcus sp. Strain NKBG15041c[END_REF].

The correlation between different phenotypes of AHL producers can be related to changes in the cyanobacterial assemblages, switching between "Helpers" to probably benefit from their associated dissolved organic matter and "Competitors" for nutrient acquisition such as phosphate, nitrogen and iron.

Assuming that the differences in APP are not caused by a difference in the genome content the facts that 1) a near totality of strains isolated is non-AHL producing in a time point and AHL-producing in a subsequent time point, and 2) that this phenotype is observed after two rounds of isolation in rich medium, raises the intriguing possibility that epigenetic regulation might be at play. This idea of an epigenetic memory is reinforced by the fact that our observations were not revealed by in situ measurement but by strain isolation and phenotyping after cultivation in rich media. We hypothesize that V. mediterranei strains could keep a memory of the social traits expressed in the environment. It has been shown that the regulation of phenotypic variation is not always linked to changes in DNA sequence and can be epigenetic in nature [START_REF] Smits | Phenotypic variation in bacteria: the role of feedback regulation[END_REF]. Epigenetic represent heritable traits inferring gene expression and modulating cellular and physiological phenotypes depending on environmental factors [START_REF] Casadesus | Epigenetic gene regulation in the bacterial world[END_REF]West-Eberhard, 2003). Indeed, recently [START_REF] Kurz | Epigenetic memory emerging from integrated transcription bursts[END_REF] have shown that epigenetic mechanisms can be involved in the regulation of AHL-based QS systems and thus we hypothesize that V. mediterranei strains could keep a memory of the social traits expressed in the environment.

However, a consequent work on the description of the QS pathways in V. mediterranei still need to be done to understand and to study properly this epigenetic mechanism. Our work also raise the important question of the in situ versus isolates QS activity measurements. Since the in situ measurement of the expression of AHL synthases seems difficult due to the low sequence homology of these genes among the genus Vibrio (Tait et al., 2010) and the presence of unknown AHL synthases it seems important to highlight the opportunity to study QS on environmental isolates at least for V. mediterranei.

CONCLUSION

QS is a well-studied social trait in Vibrio, however despite the current knowledge obtained by in vitro studies on a few species there is an inconsiderable lack regarding to complex ecological communities. This work provides the first investigation on the dynamic of AHL producers in changing environmental conditions and open a new path for outgoing studies aiming to understand the QS mechanism in natural habitats and to investigate the epigenetic regulation of QS phenotypic plasticity. Finally, global climate change may lead to an increase of physiologic and metabolic activities of Vibrio and Vibrio-related diseases (Vezzulli et al., 2010(Vezzulli et al., , 2015a)), thus understanding the factors and interactions affecting QS in natural environments and the underlying physiological mechanisms appears increasingly valuable. -0,4613 -0,0634 -0,3167 0,6165 Navicula and assimilated -0,4203 -0,1030 -0,4395 0,7537 Nitzschia sp.

-0,2447 -0,3751 -0,8087 0,2763 Pseudo-Nitzschia sp.

0,0122 -0,1159 -0,0715 0,2894 Rhizosolenia sp. 0,3770 0,0141 0,0226 -0,0003 Thalassionema nitzschïodes 0,3770 0,0141 0,0226 -0,0003 Alexandrium sp.

-0,0268 -0,2175 0,4391 -0,5969 Amylax sp.

-0,3331 -0,8688 0,1870 -0,0066 Ceratium sp.

-0,4481 -0,0184 -0,8920 -0,0565 Dinophysis sp. 0,5759 0,0215 0,0346 -0,0004 Gonyaulax sp.

-0,3734 -0,8242 0,4209 -0,0642 Prorocentrum micans 0,5253 0,2231 0,4144 -0,3464 Prorocentrum minimum 0,1879 0,0392 -0,2027 0,0875 Protoperidinium sp.

-0,0147 0,0849 0,2631 -0,1889 Pyrophacus sp.

-0,4194 0,5014 0,3709 0,6596 Scrippsiella sp.

-0,6256 -0,2238 -0,1128 0,0574 Cryptophyta 0,7498 -0,3218 -0,4345 0,1154 NanoEuk 0,6491 -0,4180 -0,4407 0,1613 P icoEuk 0,8787 -0,1557 -0,1498 0,1512 Cyanobacteria -0,5851 0,2801 0,3576 -0,1657 Bacteria -0,8356 -0,1805 -0,4241 0,1272

INTRODUCTION

Quorum sensing (QS) is a density dependent mechanism allowing bacteria to sense their number and act as cohesive social units in order to perform processes which appear effective only when undertaken by the group (Bassler and Losick, 2006). The perfect example is the early studied bioluminescence mechanism that trigger V. fischeri within the enclosed environment offered by the light organ of his symbiotic partner Euprymna scolopes (Nealson et al., 1970). Indeed, as the number of cells in the light organ increase small diffusible molecules named auto-inducers (AIs) accumulate and when the threshold is reached the transcription of multiple genes is activated including AI synthase which result in a positive feedback loop and luciferase production (Engebrecht et al., 1983;Engebrecht and Silverman, 1984). There is two major auto-inducers in Vibrio spp., the autoinducer type 1 or N-acylhomoserine lactone, AHL allowing both interspecies communication and the autoinducer type 2, AI-2 mediating intraspecies or universal communication. However, this well described mechanism, at the basis of all QS studies, is characterized by an homogeneous system in the light organ where high-cell density state is between 10 8 and 10 11 [START_REF] Miller | Quorum sensing in bacteria[END_REF]Nealson et al., 1970). Considering that the average cell density per milliliters of seawater is comprised between 10 4 and 10 7 it then appears difficult for a single species to reach the 'highcell density state' within complex bacterioplankton communities [START_REF] Maranger | Viral abundance in aquatic systems: a comparison between marine and fresh waters[END_REF].

Many works tried to face this problem and concluded that it seems unlikely to consider only cell density as responsible for QS mechanisms and that it is necessary to include the spatial distribution of cells and mass transfer properties [START_REF] Delle Side | Quorum sensing: Complexity in the bacterial world[END_REF]Hense et al., 2007;Platt and Fuqua, 2010;[START_REF] West | Quorum sensing and the confusion about diffusion[END_REF]. These conclusions appears to be even more significant when spatial distribution of microorganism is known to be heterogeneous and finescale environments such as biofilms or phytoplankton phycosphere selects specific populations and support higher cell densities (Acinas et al., 2004;[START_REF] Azam | Microbial structuring of marine ecosystems[END_REF]Persat et al., 2015;Rolland et al., 2016).

In temperate coastal area, Vibrio spp. are widely correlated to bulk environmental variables, mainly temperature and salinity which contribute to their high seasonal dynamic.

Vibrio spp. can evolve under different lifestyles in the environment such as free-living, particle associated, phyto-and zooplankton associated or occupy niches specific to pathogens [START_REF] Austin | Bacterial fish pathogens: disease of farmed and wild fish[END_REF][START_REF] Main | Community-level and speciesspecific associations between phytoplankton and particle-associated Vibrio species in Delaware's Inland Bays[END_REF]Sochard et al., 1979;Takemura et al., 2014;Turner et al., 2013).

In the last decade many works have contributed to the understanding of spatial and temporal patterns among bacterioplankton (Acinas et al., 2004;Hunt et al., 2008). Previous works have determined that in a same water sample Vibrio are well differentiated into genetic clusters corresponding to similar resource preferences and that these population-habitat linkage are highly predictable over the years (Preheim et al., 2011;Szabo et al., 2013). Furthermore, it have been demonstrated that these ecological populations of Vibrio are acting as socially cohesive units of antibiotic production and resistance and iron acquisition (Cordero et al., 2012a;Cordero et al., 2012b).

In the present study we tested the hypothesis that phylogenetically close strains from a same ecological population, sharing micro-habitats, are producing cohesive signal molecules.

With this purpose, we investigated AIs production (N-acyl-homoserine lactone (AHL) and AI-2) among a sub-selection of 227 Vibrio strains previously affiliated to 24 ecological populations and associated to different size particles and to oysters [START_REF] Bruto | Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition[END_REF]. A combined procedure of biosensors and UHPLC-HRMS/MS signal molecules detection with a whole genome analysis was applied to determine the link between genetic diversity and AIs production within ecological population (Girard et al., 2017).

MATERIALS & METHODS

Population structure and affiliations

This work is based on the study of [START_REF] Bruto | Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition[END_REF] where the detailed procedures from the sampling to the Vibrio strains isolation and identification are detailed. Briefly, strains were isolated from oysters and four fractions, >60 µm, 60-5 µm, 5-1 µm and 1-0.2 µm, the hsp60 was sequenced as marker gene and partitioned according to their genetic and ecological similarities using AdaptML (Hunt et al., 2008). This work resulted in the affiliation of 1635 isolates to 24 ecological populations and a sub-selection of around 10 non-clonal strains by population were picked for signal molecules detection (i.e. Table S1). The 227 Vibrio isolates used in this study were previously partitioned into 24 populations according to their phylogeny on the hsp60 marker gene and their ecological and seasonal preferences by using AdaptML (Bruto et al., 2016). 17 populations where consistent with named species, V. orientalis #1, V.

fortis #2, V. chagasii #3, V. rotiferianus #5, V. crassotreae #11, V. splendidus #13 to 24, while second analysis was performed to study MS/MS spectrum and identify four fragment ions at m/z 102.055, 84.045, 74.06 and 56.05 which are specific of the homoserine lactone (HSL) moiety (Patel et al., 2016).

Furthermore, 27 commercial AHL standards were analyzed and used to build prediction curves for Cx-HSL, OH-Cx-HSL and oxo-Cx-HSL which allowed the confirmation of AHLs identifications (Table S2, Figure S1). The Hierarchical Cluster Analysis of V. rotiferianus strains according to their AHL production (binary data, Jaccard distances and Ward method)

was obtained with the PermutMatrix program v. 1.9.3 (Caraux and Pinloche, 2005).

RESULTS & DISCUSSION

QS activity among ecological units of Vibrio

In order to determine if strains of a single ecological population are sharing QS abilities we screened the 227 isolates for their ability to produce AHL (F117, MT102 and CV026) and AI-2 (BB170). Our results indicate that half of the 24 ecological populations produce 4,5,6,7,8,9,11,and 12) while only one produce AHL (i.e. blue dots, V. rotiferianus #5;

Figure 1). This is consistent with previous studies that have already shown that AHL producers represent a minority of Vibrio isolates (Girard et al., 2017;Rasmussen et al., 2014) while AI-2 production is a widespread among Vibrionaceae (Yang et al., 2011).

Remarkably, QS activity was detected in a large majority of summer isolates which can be explained by the strong influence of season on population structure. In summer, there is a higher diversity of Vibrio populations that are QS active while spring is dominated by V.

splendidus isolates that are AIs non-producers. Secondly, 6 out of 9 AI-2 positive populations (#5, 6, 7, 8, 11 and 12), were detected as positively associated to oysters [START_REF] Bruto | Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition[END_REF].

Considering that oyster microbiome is often composed of a large number of different genus and that oyster offers an aggregated spatial distribution of cells it is not surprising to detect interspecies communication and a conserved AI-2 production among these populations [START_REF] King | Analysis of stomach and gut microbiomes of the Eastern oyster (Crassostrea virginica) from Coastal Louisiana, USA[END_REF][START_REF] Lemire | Populations, not clones, are the unit of vibrio pathogenesis in naturally infected oysters[END_REF][START_REF] Trabal Fernández | Changes in the composition and diversity of the bacterial microbiota associated with oysters (Crassostrea corteziensis , Crassostrea gigas and Crassostrea sikamea) during commercial production[END_REF]. This could mean that AI-2 signal production, by mediating cooperation between different species, is contributing to the construction of complex assemblages of bacteria among hosts microbiota. showing AI-2 activity reported as the fold induction of the reporter strain BB170 over the background. The 24 ecological population were predicted by AdaptML and indicated by bold numbers (Hunt et al., 2008).

AHL production among the V. rotiferianus #5

Among the population 5 affiliated to V. rotiferianus, 6/10 strains 7H5-2, 8T1-2, 8T3-1, 8T5-6, 8T6-4 and 8GS-12, produced AHLs and were selected for AHL characterization by UHPLC-HRMS/MS. The comparison with the different standards allowed the direct identification of 13 AHLs, C6-HSL, C7-HSL, C8-HSL, C9-HSL, oxo-C8-HSL, OH-C8-HSL, OH-C10-HSL, C16:1-HSL, oxo-C6-HSL, C14-HSL, oxo-C12-HSL, C14:1-HSL and C16-HSL (Table S2). The analysis of AHL standards was also used to build prediction curves for Cx-HSL, OH-Cx-HSL and oxo-Cx-HSL and allowed the identification of C5-HSL, OH-C11-HSL, OH-C9-HSL, oxo-C11-HSL and oxo-C13-HSL (Table S3 andS4).

A total of four AHLs, C6-HSL, C7-HSL, C8-HSL, C9-HSL, seems to be conserved among the 6 strains while the production of other signal molecules seems heterogeneous and strain dependent (Figure 2). Surprisingly, while identical AHL patterns had been expected for different strains of a single ecological population these results indicate heterogeneous AHL patterns and strain-dependent ability to produce AHLs.

Link between AHL production and genetic diversity of V. rotiferianus strains

The whole genome sequencing of 5 isolates of V. rotiferianus have been assessed, 7G5-1 a non AHL producer and 7H5-2, 8T6-4, 8T5-6 and 8T1-2 previously analyzed by UHPLC-HRMS/MS. The affiliation to V. rotiferianus was confirmed by ANI values were >95% between our five strains and the type strain V. rotiferianus LMG 21460. Using comparative genomics, we examined the links between genetic diversity and AHL production patterns within the V. rotiferianus population. We were able to identify the different genetic elements of a multichannel QS pathways in the 5 V. rotiferianus strains (Figure 4) and these links were examined on the basis of whole genomes and the synthase LuxM involved in AHL production. We observed no correlation between AHL production patterns (Figure 2) and the genetic diversity of V. rotiferianus, neither on the basis of whole genome phylogeny nor LuxM (Figure 3) phylogenies. Surprisingly, the non AHL producer 7G5-1 possess the AHL synthase and receptor with 100% identity on amino-acids sequences with two AHL producers 8T6-4 and 7H5-2. Furthermore, with ANI values above 99.9% between the genomes these 3 strains can be considered as identical.

Two hypothesis can be made (i) 7G5-1 can be cheater avoiding the cost of AHL production but still benefiting from the signal molecule but it appears that this strain possess a functional AHL synthase; and (ii) Others elements are regulating AHL production and as 7G5-1 genome is containing all the genetic elements of a multichannel QS pathway (AHL, AI-2 and CAI-1 pathways, i.e Figure 4) it seems possible that, as observed in other Vibrio species (Milton et al., 2001;Wang et al., 2013), other QS pathways regulate AHL production in this strain.

Finally, within a single population we did not observe a cohesive pattern of AHL production but multiple responses at a strain-dependant level ranging from no production to production of diverse AHL patterns with identical synthase.

LuxN signal reception

In a second step we examined the AHL receptor biding properties between the 5 strains by comparison with well described LuxN in V. harveyi (Waters and Bassler, 2006).

LuxN is a nine-transmembrane domain protein harboring a N-terminus portion on the periplasmic side and a C-terminal histidine -kinase portion responsible for the signal transduction [START_REF] Jung | The Quorum-sensing hybrid histidine kinase LuxN of Vibrio harveyi contains a periplasmically located N-terminus[END_REF]. [START_REF] Swem | Deducing receptor signaling parameters from in vivo analysis: LuxN/AI-1 Quorum sensing in Vibrio harveyi[END_REF] have identified the AHL-biding domain of LuxN located in the transmembrane helices 4, 5, 6, and 7 (between L140 and G240) and discovered the amino acids important for AHL sensitivity in V. harveyi BB120 (red stars, Figure 5). No correlation between LuxN phylogeny and AHL production patterns were identified as well as we did not identify important amino acids changes in the AHL biding domain among the five strains (Figure S1 and Table S5). However, it is important to note that despite a low luxN sequence homology between V. harveyi and V. rotiferianus proteins are showing ~95% similarity with high conservation of important amino-acids for AHL reception and sensitivity (Figure 5). These results indicate that LuxN signal reception was maintained through evolution at least among the Harveyi clade. efficiency identified in V.harveyi BB120 [START_REF] Swem | Deducing receptor signaling parameters from in vivo analysis: LuxN/AI-1 Quorum sensing in Vibrio harveyi[END_REF].

CONCLUSION

Our results suggest that cooperation via signal molecules could be important for the structuration of natural Vibrio populations in marine environments. We did not observe any evidence that QS cheaters are abundant among Vibrio ecological units as AI-2 production level was homogeneous among populations while non AHL production was not explained by the presence of nonfunctional or the lack of AHL synthase. However, in order to confirm these first observations it seems necessary to extend that type of study to larger data set and to different climate zones. Furthermore, we revealed an unexpected diversity of AHL and allowed to move forward in the identification of AHL mediated QS pathways in V.

rotiferianus isolates. Taking into account that we still observe different isolates of a single strain with different AHL production pattern within a same ecological population, i.e. isolated from the same micro-habitat, it appears fundamental to understand the regulation mechanism of AHL production. 107.086 (11.26), 109.101 (13.49), 121.101 (10.82), 143.073 (19.14), 147.117 (17.05), 149.132 (14.57), 240.232 (21.21), 269.190 (17.54), 285.298 (18.29), 286.098 (18.34)

→ Y Basic → Aromatic I264 → T Aliphatic → Hydroxylic LuxN A128 → T Aliphatic → Hydroxylic S300 → A Hydroxylic → Aliphatic H491 →N Basic → Amidic A503 → V Aliphatic → Aliphatic Q688 → K Amidic → Basic D691 → A Acidic → Aliphatic 8T5-6 LuxM S123 → C Hydroxylic → Sulfur-containing H211 → Y Basic → Aromatic I264 → T Aliphatic → Hydroxylic LuxN A128 → T Aliphatic → Hydroxylic K494 → Q Basic → Amidic Q698 → P Amidic → Aliphatic

AHL diversity

QS is increasingly recognised as an elaborate system of communication allowing bacteria to act as multicellular organisms and to coordinate group activities. AHL mediated QS still one of the most studied system, firstly discovered in the bioluminescent Vibrio species, V. fischeri and V. harveyi, but playing an important role in many Gram negative bacteria [START_REF] Bassler | Small Talk[END_REF][START_REF] Eberl | N-acyl homoserinelactone-mediated gene regulation in Gram-negative bacteria[END_REF]. AHL constitutes a predominant class of signalling compounds and by definition promote intraspecies communication meaning that only the species that produce AHL can detect and respond to it [START_REF] Federle | Interspecies communication in bacteria[END_REF]. Indeed, this signal molecule is emerging as an integral component of bacterial global gene regulatory networks, not only for marine species but also for plant-associated or human pathogens (Whitehead et al., 2001;[START_REF] Withers | Quorum sensing as an integral component of gene regulatory networks in Gram-negative bacteria[END_REF]. Over the years, many studies have investigated AHL diversity among a large number of Gram negative species [START_REF] Cha | Production of acylhomoserine lactone quorum-sensing signals by Gram-negative plant-associated bacteria[END_REF][START_REF] Fekete | Identification of bacterial N-acylhomoserine lactones (AHLs) with a combination of ultra-performance liquid chromatography (UPLC), ultra-high-resolution mass spectrometry, and in-situ biosensors[END_REF][START_REF] Huang | Utilization of acyl-homoserine lactone quorum signals for growth by a soil Pseudomonad and Pseudomonas aeruginosa PAO1[END_REF]Patel et al., 2016).

For Vibrio spp., since the studies revealing the production of AHLs in V. fischeri (oxo-C6-HSL and C8-HSL) and in V. harveyi (OH-C4-HSL) little has been done to further investigate AHL diversity in these species [START_REF] Cao | Purification and structural identification of an autoinducer for the luminescence system of Vibrio harveyi[END_REF]Kuo et al., 1994). An

inventory of the characterized AHL diversity among Vibrio species is listed in the Introduction of this thesis work and revealed that only 14 species have been investigated for AHL production. Remarkably, the majority of these data are based on three studies (Buchholtz et al., 2006;Purohit et al., 2013;Rasmussen et al., 2014), one dedicated to V.

anguillarum an important fish pathogen and the two others are based on diversified Vibrio strains in collections. For example, (Ward's method based on Jaccard distances, PermutMatrix). Red dots are indicating the strains analysed in this thesis work. Based on Table 1.5, Chapter 1: Introduction.

Nonetheless, it seems possible that the specificity of AHL mediated QS is not entirely driven by the diversity of the produced AHLs but also by the sensitivity of AHL receptors.

Few studies on V. fischeri have demonstrated that controlled mutations on key amino acids can induce different LuxR variants, exhibiting a broadened response to diverse AHLs, and that two or three changes in the amino-acid sequence of LuxR can modulate its sensitivity toward AHLs (Collins et al., 2004(Collins et al., , 2006;;Hawkins et al., 2007). Therefore, it is plausible that two Vibrio species producing the same AHL but have evolved in way that their receptors are different (different amino-acid sequences) which induces a difference in the sensitivity for the AHL.

In this context, it seems essential to take into account the concentrations of each emitted AHL and our analytical approach remains to be tuned for AHL quantification. For example, in order to test this hypothesis, assuming that we have the emitted concentrations of AHLs, it would be interesting to compare the response of the receptors AinR and LuxN of V.

fischeri and V. rotiferianus to the C8-HSL (common to both species) by testing the AHL at different emitted concentrations. Similarly to some AHL-biosensors, genetic constructions coupling AinR to the galactosidase activity have already been developed to assess AHL specificity of the AinR receptor [START_REF] Kimbrough | Substrate specificity and function of the pheromone receptor AinR in Vibrio fischeri ES114[END_REF]. Using such approach we could determine if the emitted concentration by strain A can induce a response in strain B. However, one complication to this approach would be that some Vibrio species possess two systems of AHL production/sensing: the LuxI/R and the AinS/R (or LuxM/N). Currently, only V. fischeri and V. anguillarum are known to possess both AHL-mediated systems but it also has been the most studied species. In this case it will be interesting to determine beforehand which synthase is responsible for which part of their AHL production patterns, and subsequently, the specificity of the two receptors LuxR and AinR towards all produced molecules. The characterization of AHL production patterns of knock-out mutants of LuxI alone and AinS alone could give an information on which synthase is producing which AHL. This experiment also could highlight if there is a connection between both AHL mediated QS systems, as observed in V. anguillarum, where the LuxM dependent AHLs control the AHL production of LuxI (Milton et al., 2001).

1.2 Factors affecting AHL production: How diverse AHL production patterns for a single species can be explained?

Our work on V. fischeri symbiotic strains revealed a link between the genetic diversity of the AHL synthase AinS and their AHL production patterns. Indeed, it has been demonstrated that the specificity of AHL synthases, homologs to LuxI and LuxM, towards their substrates can be modified. Variable residues at the C-terminal end of the synthase were identified as the basis of the acyl chain specificity which means that changes in the synthases amino-acid sequences can alter the specificity and then modify AHL production patterns (Watson et al., 2002). We also revealed a temporal variation of AHL production patterns for different isolates of a single strain of V. mediterranei greatly explained by environmental variables and suggested that phenotypic plasticity of AHL production can offer an advantageous response to adapt their actions in response to fluctuating environments.

This can be explained by either the presence (or differential expression) of different synthases or by the fact that fatty acid biosynthetic pathway can determine the acyl chain lengths of AHLs, or a combination of both. In fact, available acyl-ACP pools in bacteria can be modified by metabolic changes and then any factor (i.e. environmental factors, nutrients availability, and interactions with other organisms) likely to modulate their metabolism and physiological state may alter AHL production patterns (Gould et al., 2006;Sullivan et al., 2002).

Altogether, these results support the idea that metabolic and environmental changes, the genetic diversity, or regulatory processes could modify the spoken "language" of Vibrio.

On the other hand, the study of V. rotiferianus strains lead us to the opposite conclusion, neither genetic diversity nor ecological niches could explain the intra-specific diversity of AHL production patterns. Adding complexity to the understanding of AHL-meditated QS, multichannel QS systems are widespread among Vibrio species and some authors have already reported the influence of other signal molecules, such as AI-2 or CAI-1, on the modulation of AHL production (Milton et al., 2001;Wang et al., 2013). Since the multichannel QS pathways is present in V. rotiferianus it would not be surprising that such connexion and regulations might be occurring in this species. the same culture conditions for the same species, seems strain or isolate dependant. Further studies need to be conducted in order to understand this intra-species diversity of AHL production, where comparative genomic, epigenomic and transcriptomic studies could help the understanding of this variation.

Prevalence of QS in Vibrio spp.

A large number of species in the genus Vibrio have been reported to produce AHL [START_REF] Garcia-Aljaro | Detection of acylated homoserine lactones produced by Vibrio spp. and related species isolated from water and aquatic organisms[END_REF]Girard et al., 2017;Purohit et al., 2013;Tait et al., 2010) and even more widely, to harbor the genes coding for AHL synthases (Introduction section 4.3.1), including representatives of almost all the previously defined Clades (Sawabe et al., 2013). However, in some cases, like what I described in Chapters 2 and 4) as well in other studies (Rasmussen et al., 2014) a large majority of the tested Vibrio strains are not detected as AHL producers.

Since prokaryotic genomes tend to be streamlined, the question remains for the reasons of this discrepancy. From my point of view and based on current knowledge it is difficult to explain, but hereafter I propose some possibilities. These strains have been tested for AHL production when grown on rich culture medium representing a homogeneous and closed system. Taking into account the information presented in the previous sections regarding the complexity of response strategies, it would not be surprising that culture conditions may not provide a suitable environment for AHL production. Indeed, under shaking we obtain a homogeneous culture of clonal cells which reach high cell densities but the lack of external stimulus corresponding to a specific niche association and interactions with other organisms that will hamper AHL production. Furthermore, QS is known to allow bacteria to undertake collectives responses such as virulence factor secretion, biofilm formation, nutrient acquisition or survival strategies, and since in culture condition they might have "ideal" conditions they therefore might nor need to realise coordinated responses nor to produce AHL.

On the other hand, multiple strains from a single genotype isolated from a single sample producing AHL in such conditions, i.e. after isolation and growth in rich media, with coherent phenotypes over time such as V. mediterranei. This seem to suggest that bacteria might keep a type of "memory" of the environment in which they were isolated, perhaps via an epigenetic mechanism. V. mediterranei AHL production patterns were obtained by testing cultures of freshly isolated colonies while the Vibrio collections (Chapters 2 and 4) were tested, for some of the strains, after years of conservation and multiple cycles of growth and cryoconservation.

Experiments following AHL production after multiple consecutive cycles of growth in rich media and after several steps of freezing/thawing could allow to test of this hypothesis.

3.

In situ QS studies: Complexity of signal production and transmission in the environment Considering all that has been discussed above, the best strategy to understand QS in environmental Vibrio stains, would be to determine AHL production in situ. While direct detection of AHLs in seawater is technically feasible and could allow to determine which

AHLs are there at a given time, it won't give any clues of the bacteria that are responsible for their production. On the other hand, in situ expression of AHL synthases by real time quantitative PCR approaches could allow the confirmation of AHL synthase activity in the environment but conversely it won't provide any information on the produced AHLs.

Furthermore, for the study of AHL production in Vibrio spp. it is impossible to design universal primers due to the extremely low homology of AHL synthases at the genus level.

However, we were able here to identify an opening window, by the design of species specific primers, withal this solution requires to know beforehand the different major species evolving in the studied environment. One original objective of this thesis was to apply this approach to V. mediterranei during our monitoring in the Leucate lagoon, but unfortunately we were not able to find neither LuxI nor LuxN homologs in their genomes, and thus future studies will require an identification of the AHL synthases in this species. Finally, in my opinion, a combination of culture-based and in situ approaches will be necessary to the full understanding of AHL production by Vibrio in the environment.

In addition to this understanding it will be necessary to take in consideration that for QS to be effective once the AHLs are produced and emitted outside a cell A, the molecules need to reach the partner cell B at a sufficient concentration to alter its gene expression. The first investigations on QS, have demonstrated that cellular density is the main factor controlling AHLs concentrations (Nealson et al., 1970). However, this concept was based on mixed liquid batch cultures or suspensions where all spatial heterogeneity is minimized leading to an average concentration of AHL that is primarily a function of its production. I wonder whether mechanisms determining AHL concentration are actually the same considering the different

In addition to all the factors likely to modulate AHL production there is also parameters to consider when evaluating the degradation rate or half-life of AHLs. First of all, abiotic factors are really important for the stability of signal molecules in the environment. Basic pH and elevated temperatures are well known to alter the stability of AHLs [START_REF] Yates | N-acylhomoserine lactones undergo lactonolysis in a pH-, Temperature-, and acyl chain length-dependent manner during growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa[END_REF]. It has also been demonstrated that long chain and unsubstituted AHLs are more stable in seawater [START_REF] Hmelo | Kinetic constraints on acylated homoserine lactone-based quorum sensing in marine environments[END_REF][START_REF] Horswill | The effect of the chemical, biological, and physical environment on quorum sensing in structured microbial communities[END_REF]. A second element, the biotic degradation of AHLs need to be considered as a large difference of degradation rate (20 to 50% higher) was observed between artificial sea water and natural seawater [START_REF] Hmelo | Kinetic constraints on acylated homoserine lactone-based quorum sensing in marine environments[END_REF].

This results revealed the attack from quorum quenching enzymes which is not surprising because many marine organisms are continuously described as responsible for the inactivation of AHLs by enzymatic degradation [START_REF] Dobretsov | Malyngolide from the cyanobacterium Lyngbya majuscula interferes with quorum sensing circuitry[END_REF][START_REF] Leadbetter | Metabolism of acyl-homoserine lactone quorumsensing signals by Variovorax paradoxus[END_REF][START_REF] Lin | Acyl-homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum-quenching enzymes[END_REF]Romero et al., 2008;Syrpas et al., 2014). To conclude, to consider cell density as a sole driver of AHL concentrations seems unreasonable and thus, led to the introduction of new conceptual definition such as efficiency sensing which includes a combination of cell density, mass transfer and spatial distribution of the cells. I believe this framework allow to have a closer vision of the reality with the probable existence of specific 'hotspots' with monospecific bursts of bacteria employing QS mechanisms.

Concluding Remarks

The general theories on QS mechanisms have been firstly made on the symbiotic species V.

fischeri who lives in an enclosed system consisting of the light organ of its partner Euprymna Scolopes. In this system, similarly to batch cultures, high cellular densities result in a sufficient concentration of AIs to alter gene expression and activate the well-known bioluminescent phenotype. However, the ever growing interest of the scientific community on the understanding of QS mechanisms have lead us to have a more complex vision of bacterial cell to cell communication, shaped by many factors including the genetic diversity and expression, the influence of the environment in which bacteria thrive, as well the interactions with other organisms. Through the several studies conducted in this thesis I hope to have helped to improve this understanding.

Figure 1 . 1 :

 11 Figure 1.1: Systematic classification of the genera in the family Vibrionaceae.

Figure 1 . 2 :

 12 Figure 1.2 : MLSA concatenated split network tree based on 16S rRNA, gapA, gyrB, ftsZ, mreB, pyrH, recA, topA and rpoA gene sequences proposed by Sawabe et al. 2013.

Figure 1 . 4 :

 14 Figure 1.4: Structures of QS molecules in Vibrio.

RFigure 1 . 5 :

 15 Figure 1.5: Summary diagram of the multichannel QS system in Vibrio. AHL mr: AHL membrane receptor (AinR, LuxN or VanN); AHL ir: AHL intracellular receptor; AHL S: AHL synthase; AHLir + Master regulator: LuxR, VanR, VfqR; Master regulator: other master regulator like VanT, LitR, HapR or SmcR. The tricolor components are shared by the three QS channels. The three QS pathways will be individually detailed in the section 4.3.

4. 3

 3 Molecules, biosynthesis and signalling pathways 4.3.1 Autoinducer-1 or N-Acyl-Homoserine-Lactone (AHL) QS

Figure 1 . 6 :

 16 Figure 1.6: Biosynthesis pathway of N-acyl-homoserine lactone. Adapted from Keller and Surette, 2006.

Figure 1 . 7 :

 17 Figure 1.7: Maximum likelihood tree of the luxM gene sequence (~ 1250 pb) of 20 Vibrio species using the General time reversible model (GTR+G+I, Mega). Bootstrap values are based on 500 replicates.

Figure 1 . 8 :

 18 Figure 1.8: Maximum likelihood tree of the luxI gene sequence (~ 600 pb) of 6 Vibrio species using the Tamura 1992 model (T92+G, Mega). Bootstrap values are based on 500 replicates.

Figure 1 . 9 :

 19 Figure 1.9: Biosynthesis pathway of AI-2 molecules. Adapted from Keller and Surette, 2006.

Figure 1 . 10 :

 110 Figure 1.10: Maximum likelihood tree of the cqsA gene sequence (~ 950 pb) of 20 Vibrio species using the Tamura 1992 model (T92+G, Mega). Bootstrap values are based on 500 replicates.

  -HSL, OH-C10-HSL, oxo-C8-HSL, oxo-C10-HSL and oxo-C12-HSL Rasmussen et al., S0207, S1106, S1728, S1730, S1732 UHPLC-HRMS OH-C6-HSL, OH-C10-HSL, oxo-C10-HSL and oxo-C12-HSL S1729 UHPLC-HRMS OH-C6-HSL, OH-C10-HSL, oxo-C6-HSL, oxo-C8-HSL, oxo-C10-HSL and oxo--HSL, OH-C10-HSL, OH-C11-HSL, oxo-C8-HSL, oxo-C9-HSL, oxo-C10-HSL, oxo-C11-HSL, oxo-C12-HSL Rasmussen et al., XJ85003 HPLC-MS/MS C10-HSL, oxo-C10-HSL, oxo-C12-HSL Wang et al., 2013 OH-C6-HSL Rasmussen et al., LMG 19031 (T) HPLC-MS/MS OH-C4-HSL Purohit et al., 2013 V. variabilis T01 HPLC-HRMS C6-HSL, oxo-C10-HSL, C12-HSL Mohamad et al., 2016 V. vulnificus CECT 5763, Biotype 2 HPLC-HRMS C4-HSL Valiente et al., 2009

  io sc op h th al m i (T ) ; C E C T 46 3 8; A 08 9; U 46 57 9 V ib rio ic ht hy oe nt er i (T ); D S M 14 39 7T ; A J4 21 shii ATC C 191 09T ; X 747 25 V ibri o hep ata rius (T) ; LMG 203 62T ; AJ3 450 63 Vib rio po nti cus (T) ; typ e stra in: CE CT 586 9; AJ 63 010 3 Vi bri o fur nis sii (T) ; AT CC 35 01 6 T ; X7 63 36 BB CC 10 26 BB C C 10 55 V ib rio m e ts ch ni ko vi i C IP 69 .

INTRODUCTION

  Quorum sensing was initially observed in the Squid-Vibrio symbiosis betweenAliivibrio fischeri and the small Hawaiian bobtail squid Euprymna scolopes. The bioluminescence mechanism that A. fischeri triggers in the light organ only at high cell density led to the identification of N-acyl-homoserine lactones (AHL). These initial investigations resulted in the identification of the N-3-oxo-hexanoyl-homoserine lactone (oxo-C6-HSL) and the description of the well-known QS system LuxI/LuxR, which regulates

  enables detection based on MS/MS fragmentation patterns and is supported by the analysis of 27 AHL standards (Supplementary information, TableS1). Basically, 5 A. fischeri strains, ES213, KB2B1, KB4B5, MB13B1 and ES114, were cultivated in Luria-Bertani (LB) Salt media at 28°C for 24 h by following the cultivation conditions applied by[START_REF] Bongrand | A genomic comparison of 13 symbiotic Vibrio fischeri isolates from the perspective of their host source and colonization behavior[END_REF]. Cultures were extracted with ethyl acetate, and then extracts were fractionated into 22 fractions using separative HPLC (Thermo Scientific, Courtaboeuf, France) and analyzed by UHPLC-HRMS/MS. AHL identification was based first on an analysis to determine molecular weights and retention times and to identify chromatographic peaks generating the fragment ions corresponding to the lactone ring fragment at m/z 102.0555. A second analysis was performed to study the MS/MS spectrum and identify four fragment ions at m/z 102.055, 84.045, 74.061 and 56.050, which are specific to the homoserine lactone (HSL) moiety(Patel et al., 2016). A comparison to 27 different commercial standards enabled the direct identification of 10 AHLs: C6-HSL (Nhexanoyl-homoserine lactone), oxo-C6-HSL (N-3-oxo-hexanoyl-homoserine lactone), C7-HSL (N-heptanoyl-homoserine lactone), C8-HSL (N-octanoyl-homoserine lactone), OH-C8-HSL (N-3-hydroxy-octanoyl-homoserine lactone), oxo-C8-HSL (N-3-oxo-octanoylhomoserine lactone), C9-HSL (N-nonanoyl-homoserine lactone), C10-HSL (N-decanoyl-homoserine lactone), OH-C10-HSL (N-3-oxo-decanoyl-homoserine lactone) and OH-C12-

Figure 1 :

 1 Figure 1: AHL production patterns in the 5 symbiotic A. fischeri strains. Neighbour joining tree of AinS sequence (355 aa) of 13 symbiotic A. fischeri strains using the JTT model (MEGA,[START_REF] Kumar | MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets[END_REF]. Bootstrap values are based on 1000 replicates.

(

  D-types: ES213 and KB2B1 or S-types: ES114, KB4B5 and MB13B1) or the sampling location of host squids (Kaneohe Bay, KB: ES114, KB4B5 and KB2B1 or Maunalua Bay, MB: ES213 and MB13B1; Wollenberg and Ruby, 2009).

Finally

  , clustering patterns observed for AinS, LuxR and LitR based phylogenies tend to support that minor differences in their amino-acid sequences might lead to slightly different dialogues among Aliivibrio fischeri strains. Our results indicate a trend where LuxI AHL production and AinR reception are quite stable, while AinS, LuxR and LitR exhibit variable signal synthesis and reception. Taking into account the unexpected diversity of AHL signaling molecules among symbiotic A. fischeri strains, it appears fundamental to understand which synthase, LuxI or AinS, is responsible for the synthesis of these molecules and the possible impact on the different components of the AHL-mediated QS pathways. Our work will move forward by identifying amino acid changes possibly implicated in the specificity of these proteins toward AHL synthesis and reception.

Figure

  Figure S1: Retention times curves for Cx-HSL, oxo-Cx-HSL and OH-Cx-HSL based on

Figure S2 :

 S2 Figure S2: Neighbour joining tree of 13 symbiotic A. fischeri strains using the JTT model

  higher in the southern than in the northern basin (Ifremer 2006). Seven sewage treatment plants (representing an overall capacity of 200 000 inhabitants) are located all around the lagoon. The 3 main facilities (Leucate, Port-Leucate and Le Barcarès) are located on the east side of the lagoon.

Figure 3 . 1 :

 31 Figure 3.1: Salses-Leucate lagoon and location of the sampling sites. Leucate North (42° 52' 44.9" N, 3°01'29.0" E) and Leucate South (42° 50'54.6" N, 3°00'08.7" E) (Carlier et al., 2009).

Figure 3 . 2 :

 32 Figure 3.2: Temporal dynamic of bulk measurements, salinity, water temperature, oxygen saturation and turbidity in the Salses-Leucate lagoon, data from Observatoire de l'étang reports between 2015 and 2016.

(

  [START_REF] Neaud-Masson | Bilan des résultats du système de vigilance des phycotoxines lipophiles -Année[END_REF][START_REF] Neaud-Masson | Bilan des résultats du dispositif de vigilance pour les phycotoxines lipophiles -Année[END_REF]. For 2015 and 2014, the REPHY monitoring reveals variations in chlorophyll a concentration between 0 and 5 µg.L -1 and a number of cells between 10 3 and 10 5 cells.L -1 . The identification of the most abundant phytoplankton taxa show de predominance of Chaetoceros species over the years 2014 and 2015 but also the high seasonal abundances of other genus of Bacillariophyceae and Dinophyceae (Figure 3.4; Qualité du Milieu Marin Littoral. Bulletin de la surveillance 2014. Départements du Gard, de l'Hérault, de l'Aude et des Pyrénées Orientales, 2015, Qualité du Milieu Marin Littoral. Bulletin de la surveillance 2015. Départements du Gard, de l'Hérault, de l'Aude et des Pyrénées Orientales, 2016).

Figure 3 . 3 :

 33 Figure 3.3: Geographical location of IFREMER sampling sites in the Salses-Leucate lagoon.

Figure 3 . 4 :

 34 Figure 3.4: IFREMER 2015 bulletin of the REPHY monitoring program.

Figure 3 . 5 :

 35 Figure 3.5: Diversity of total Vibrio spp. isolates by clade over the three sampling dates in 2015. A: Leucate South and B: Leucate North.

Figure 3 . 6 :

 36 Figure 3.6: AHL production phenotypes among Vibrio spp. isolates over the three sampling dates in 2015. A: Leucate South and B: Leucate North

  two years study was carried out during Spring and Summer 2015 and 2016, April to July at one sampling site in the South basin of the Salses-Leucate lagoon, in the Mediterranean coast of France, (42° 50'54.6" N, 3°00'08.7" E; Fig S1). Surface water wascollected in sterile bottles and pre-filtered through a 50 µm mesh size net. Phytoplankton cells were collected using a 5 min horizontal tow at 2 knots of a 50 µm plankton net (Diameter 30 cm, Length 1 m, 500 mL cod-end), and all samples were processed within three hours. For each sampling date, triplicate phytoplankton aliquots of 15 mL were fixed with 4% commercial neutral Lugol's solution (98.98 % distilled water/0.68 % potassium iodide/0.34 % iodine), stored in the dark at 4°C and taxonomic identification and enumeration were performed within two months. Surface temperature (°C), salinity (psu), dissolved oxygen (DO, % saturation), conductivity (mS) and pH were recorded at each sampling date using a model HQ40d multiparametric profiler (Hach).

  in triplicate fixed on board with 1% Glutaraldehyde and then quick-frozen at -80°C). The picoplankton abundances, including bacterioplankton, Cyanobacteria, picophytoeukaryotes, nanophytoeukaryotes and Cryptophyta were measured with a FACSCanto II (Becton Dickinson) flow cytometer.

Fig S3 )

 S3 Fig S3) and remarkably 83% of the V. mediterranei isolates over the two years study belonged to Genotype 1 (G1).

Figure 1 :

 1 Figure 1: Temporal dynamic of V. mediterranei isolates relative to all Vibrio spp. isolates. A. G1-G21 are Genotypes based on ERIC-PCR; B. APP: AHL Production Phenotypes; where F117+ and F117-are isolates detectable or not by the biosensor Pseudomonas putida (pKR-C12) and MT102+ and MT102-are detectable or not by the biosensor Escherichia coli (pJBA-132).

Figure 2 : 2 :

 22 Figure 2 : Heat-Map of AHL diversity in V. mediterranei G1 isolates by UHPLC-HRMS/MS (Ward's classification based on Jaccard index; Caraux and Pinloche, 2005). 1: 17LN0615E and 2: 2LS0615E corresponding to the F117 + MT102 + phenotype; 3: 21LN0615E and 4: 9LS0615E corresponding to the F117 + MT102 -phenotype. (*) unanticipated AHLs corresponding to any analyzed AHL standards. Grey squares represent the absence of AHL.

Figure 3 :

 3 Figure 3: The dbRDA ordination representing the results of the DistLM analysis in 2D. The DistLM is used to examine the relationship between the distribution and temporal dynamics of V. mediterranei G1 APP and all measured environmental variables. Vectors represent predictor variables used for the construction of the constrained ordination (the dbRDA diagram), and the length of the vectors represents the explanatory percentage of each variable.

Table 2 :

 2 Test statistics for Distance-based Linear Model (DISTLM) analyses marginal and sequential tests based on 'Forward' procedure and AIC criteria of V. mediterranei G1 phenotypes abundance at the 11 sampling dates. Marginal tests show how much variation each variable explains when considered alone, ignoring other variables. Sequential tests explain the cumulative variation attributed to each variable fitted to the model in the order specified, taking previous variables into account. A principal coordinates analysis (PCO) was performed in order to visualize which phytoplankton species co-occurred with the different phenotypes (Figure 4). As observed in the db RDA1, Axis 1 (PCO1) reflected the gradient in G1 abundances among the sampling dates with the lowest counts of G1 in the right-end (July 2015 and April/May 2016) and where picophytoeukaryotes, Cryptophyta, Unidentified nanophytoeukaryotes, Dinophysis sp. and Prorocentrum micans were present in their highest abundances. In the left-end of PCO axis 1 were samples with highest counts (June 2015 and June/July 2016) and associated with the highest counts for Grammatophora sp., Scrippsiella sp. and bacterioplankton abundances.Axis 2 (PCO2) mostly separates dates based on the presence of V mediterranei G1 AHL producers and non-producer. While Gonyaulax sp., Amylax sp., seemed to be associated to dates with the highest counts of non-producers (2016/06/21), Cyanobacteria and Pyrophacus sp. abundances were linked to AHL producers.

Figure 4 :

 4 Figure 4: Principal co-ordinate plot (PCO) of V. mediterranei G1 phenotypes for the first and second principal co-ordinates using the Bray-Curtis similarity matrix. Phytoplankton and Bacteria counts were used as predictor variables, are shown here only the variable with Pearson correlation >0.45. The analysis is detailed in TableS4and the contribution of each

Fig S2 :

 S2 Fig S2: Temporal dynamic of Vibrio spp. isolates.

Figure 1 :

 1 Figure 1: Population structure prediction and QS activity of a sub-selection of 227 Vibrio isolates from (Bruto et al., 2017). Phylogenetic tree of 227 isolates based on partial hsp60 marker gene. The inner ring represent the season of isolation, the middle ring indicates AHL production (synthesis: filled circle; no synthesis: empty circle) and the outer ring is a heatmap

Figure 2 :

 2 Figure 2: Hierarchical cluster analysis of AHL production among V. rotiferianus strains (Ward's method based on Jaccard distances, PermutMatrix).

Figure 3 :

 3 Figure 3: Neighbour joining tree of LuxM (389 aa) in V. rotiferianus strains using the JTT model (MEGA 7). Bootstrap values are based on 1000 replicates.

Figure 4 :

 4 Figure 4: Prediction of V. rotiferianus QS pathways.

Figure 5 :

 5 Figure 5: LuxM sequence alignment (304 aa) corresponding to the N-terminus domain responsible for AHL biding. Red stars are indicating key amino acid for AHL biding

Figure S1 :

 S1 Figure S1: Neighbour joining tree of LuxN (851 aa) in V. rotiferianus strains using the JTT model (MEGA 7). Bootstrap values are based on 1000 replicates.

1. 1

 1 Signal specificity: How AHL signalling can be species specific? Detailed AHL chemical characterization was performed for a total of 16 Vibrio strains belonging to V. tasmaniensis (n=1), V. fischeri (n=5), V. mediterranei (n=4) and V. rotiferianus (n=6) and also compared to what has been described in the literature to yield a global overview of AHL production patterns among Vibrio species (Figure5.1). It is clear that the comparison between different studies is difficult because the characterization of AHL production patterns have not been assessed with the same analytical tools and since methods differ in their sensitivity and resolution, it is therefore very likely that some AHL have not been always recovered in strains able of producing them.However, despite the high diversity of AHL among Vibrio species, it is worth to note that many AHLs are shared between Vibrio species and that the clustering does not indicate a species or a Clade specific communication pattern (Figure5.1). In this context, it is unlikely that AHL mediated communication is species specific and that one AHL is only detected by the species who produced it. Insofar as the same AHL is produced by different Vibrio species or even different gram-negative species sharing the same habitat it seems unjustified to consider AHL-mediated QS as species specific.

Figure 5 . 1 :

 51 Figure 5.1: Hierarchical cluster analysis of AHL production patterns among Vibrio spp.

Figure 5 . 2 :

 52 Figure 5.2: Summary diagram proposal of determinant factors for AHL production in Vibrio spp.

Fig. S2 .

 S2 Fig. S2. Weekly variations of the bulk environmental variables at the FB (black circle) and SOLA (open

Table 1 . 2 :

 12 Genetic markers for the identification of Vibrio strains.

	Gene	Function	Number of Vibrio spp. sequences in NCBI	Limitations	References
					Dorsch, Lane, and
	16S rRNA Ribosomal RNA 30S subunit	~22500	Resolution limited to the clade/species	Stackebrandt 1992; Ruimy et al. 1994; Vandamme et
					al. 1996
				No differentiation
	recA	DNA repair and maintenance	~ 12000	between : V.
	rpoA	RNA polymerase alpha subunit	~ 1500	neptunius/V. coralliilyticus and V.	F. L. Thompson et al. 2005
	pyrH	UMP kinase	~ 5400	anguillarum/V. ordalii
	gyrB	DNA gyrase beta subunit	~ 8300	-	F Le Roux et al. 2004
	gapA	Glyceraldehyde-3-phosphate dehydrogenase A	~ 2900	-	Nishiguchi and Nair, 2003
	toxR	Toxin production	~ 3500 Including a large majority of pathogenic species	-	Kim et al., 1999
	hsp60	Heat shock protein hsp60	~ 5200 Including a large majority of sequences annotated Vibrio sp.		Kwok et al., 2002
	mdk adk pgi	Midkine (neurite growth-promoting factor 2) Adenylate kinase Glucose-6-phosphate isomerase	-~ 7900 ~ 3700 Including a large majority of pathogenic species	Heterogeneous level of resolution in the different clades	Hunt et al. 2008
	dnaJ	Chaperone dnaJ	~ 12000		Nhung et al. 2007
	fur	Iron acquisition	~ 2800	Intraspecies similiraty > 95%	Machado and Gram, 2015

Table 1 .3: AHL

 1 signalling pathways in Vibrio. Adapted fromMilton, 2006 and Wang et al., 2013.

		A. fischeri	V. harveyi	V. anguillarum	V. fluvialis
	QS system	LuxI/R	AinS/R	LuxM/N	VanI/R	VanM/N	VfqI/R
	Membrane receptor	-	AinR	LuxN	-	VanN	-
	Secondary messengers	-	LuxU LuxO	LuxU LuxO	-	VanU VanO	-
	Intracellular receptor	LuxR	-	-	VanR	-	VfqR
	Master

regulator (DNA binding protein)

		LuxR LitR	LuxR LitR	LuxR AphA	VanR	VanT	VfqR HapR
	AHL synthase	LuxI	AinS	LuxM	VanI	VanM	VfqI

Table 1 .4: Functions

 1 of QS among Vibrio species.

	Species	Pathways or molecules	Function	References
	V. anguillarum	mutant vanT (luxR homolog -C6-HSL/OH-C6-HSL, AI-2, CAI-1)	Positive regulation of Metalloprotease EmpA production, Serine, biofilm and pigment production	Milton et al., 1997; Croxatto et al., 2002
		AI-2, CAI-1	Down-regulation of Vibrio polysaccharide genes (vps) and virulence factors, activates	Papenfort et al., 2017; Zhu
	V. cholerae		Hap metalloprotease	et al., 2002; Zhu and
				Mekalanos, 2003
		DPO	Repression of biolfilm formation
	A. fischeri	oxo-C6-HSL C8-HSL	Positive regulation of bioluminescence Positive regulation of bioluminescence, colonization factors and motility	Lupp and Ruby, 2005, 2004
			Positive regulation of extracellular
		hapR (CAI-1 and AI2)	protease production and repression of
	V. fluvialis		hemolysin and AHL production	Wang et al., 2013
		vfqI/vfqR (AHL)	Postive regulation of hemolysin production and cytotoxic acitivity
		C4-HSL	Positive regulation of bioluminescence	Cao and Meighen, 1989
	V. harveyi	AHL, AI-2, CAI-1

Table 1 .5: AHL

 1 diversity among Vibrio species.

	Species	Strain	Detection method	AHLs	References
	V. aestuarianus VIB281		TLC	oxo-C8-HSL, C8-HSL	Yang et al., 2011
	NB10, Serotype O1	HPLC-HRMS	OH-C6-HSL, oxo-C10-HSL, oxo-C12-HSL
	Serotype O2a, O2b, O3a, O7	HPLC-HRMS	OH-C6-HSL, oxo-C10-HSL
	Serotype O3b	HPLC-HRMS	oxo-C10-HSL, oxo-C12-HSL
					Buchholtz et al., 2006
	V. anguillarum

Table 1 . 6 :

 16 Interference with bacterial QS.

	Organism	Molecule	Impact	Référence(s)
	Delisea pulchra (Red algae)	Furanones	Antagonistic activity on AHL receptors and Inhibition of AI-2 synthesis	Givskov et al., 1996; Manefield et al., 1999; Zang et al., 2009
	Luffariella variabilis (Sponge)	Sesquiterpenes	Antagonistic activity on AHL receptors	Skindersoe et al., 2008
	Chlamydomonas reinhardtii,
	Chlamydomonas mutablis, Chlorella vulgaris, Chlorella fusca	Unidentified	Agonistic activity on AHL and AI-2 receptors	Teplitski et al., 2004
	(Chlorophyta)
	Chlorella saccharophila, Chlorella
	vulgaris, Tetraselmis
	suecica, Tetraselmis striata,
	Tetraselmis tetrathele
	(Chlorophyta), Nannochloropsis sp,
	(Ochrophyta), Isochrysis sp.
	(Haptophyta)

Unidentified

Inhibition of AHL QS pathway Natrah et al., 2011 Lygbya sp. (Cyanobacteria) Malyngolide AHL Innactivation Dobretsov et al., 2010 Anabaena sp. PC 7120 (Cyanobacteria) AiiC AHL-acylase Hydrolysis of the peptide bond (highest affinity with AHL > C10) Romero et al., 2008 Nannochloropsis oceanica and 24 microalgal species (Chlorophyta) Indole (IAA)/Auxins Inhibition of AHL QS pathway Hidalgo-Romano et al., 2014; Lu and Xu, 2015; Stirk et al., 2013; Yang et al., 2017 Bacillus sp. (Bacteria) Lactonase Hydrolysis of the homoserine lactone Dong et al., 2002

Variovorax paradoxus, Arthrobacter sp., Ralstonia sp., Pseudomonas sp. (Bacteria)

				Huang et al., 2003;
		AHL-acylase	Hydrolysis of the peptide bond	Leadbetter and Greenberg,
				2000; Lin et al., 2003
	Nitzschia pellucida (Bacillariophyta)	Haloperoxidase	Hydrolysis of oxo-HSL	Syrpas et al., 2014
	Echerichia coli	Enzyme LsrK	Degradation of AI-2	Roy et al., 2010

4.6.2 Effects of QS molecules in the interactions with other organisms

Maximum likelihood tree of 16S rDNA gene sequence (559 bp) of the 87 isolates and 35 type strains of Vibrio using the Kimura 2 parameter (K2+G+I, Mega). Bootstrap values are based on 100 replicates. Black shapes are indicating AHL producing strains.

  Dalsgaard, I., Nielsen, K.F.,Buchholtz, C., Larsen, J.L., Gram, L., 2005. Hayes, R.L., Polson, S.W., Polson, S.C., Goreau, T.J., Martinez, R.J., Smith, G.W., 2004. Relationship of Vibrio species infection and elevated temperatures to yellow blotch/band disease in Caribbean corals. Appl.Environ. Microbiol. 70, 6855-6864. Silva, B.S. de O., Moreira, A.P.B., Valle, C., Alves, N., Cavalcanti, G., Garcia, G., Lopes, R.M., Francini-Filho, R.B., de Moura, R.L., Thompson, C.C., Thompson, F.L., 2015. Diversity and ecological structure of vibrios in benthic and pelagic habitats along a latitudinal gradient in the Southwest Atlantic Ocean. PeerJ 3, e741. Wood, D.N.,Yu, L., Kirchman, D.L., 2000. Selected chitinase genes in cultured and uncultured marine bacteria in the alpha-and gamma-subclasses of the proteobacteria. Appl.Environ. Microbiol. 66, 1195-1201. Gusti, A.R., Zhang, Q., Xu, J.-L., Zhang, L.-H., 2002. Identification of quorumquenching N-acyl homoserine lactonases from Bacillus species. Appl. Environ. Microbiol. 68, Vibrio barjaei sp. nov., a new species of the Mediterranei clade isolated in a shellfish hatchery. Syst. Appl. Microbiol. 39, 553-556. Doi:10.1016/j.syapm.2016.09.001 Pernthaler, J., Amann, R., 2000a. Succession of pelagic marine bacteria during enrichment: a close look at cultivation-induced shifts. Appl. Environ. Microbiol. 66, 4634-4640. Doi:10.1128/AEM.66.11.4634-4640.2000 Amaro, C., Biosca, E.G., Garay, E., 1995. Biochemical and toxigenic properties of Vibrio furnissii isolated from a European eel farm. Aquaculture 132, 81-90. Purohit, A.A., Leiros, H.-K.S., Johansen, J.A., Kellermann, S.J., Bjelland, A.M., Parsek, M.R., Val, D.L., Dunlap, P.V., Cronan, J.E., Greenberg, E.P., 1999. Acylhomoserine lactone synthase activity of the Vibrio fischeri AinS protein. J. Bacteriol. Fries, J.S., Noble, R.T., 2008. Dynamics and predictive modelling of Vibrio spp. in the Neuse River Estuary, North Carolina, USA. Environ. Microbiol. 10, 57-64. Wang, Z., Malanoski, A.P., O'Grady, E.A., Wimpee, C.F., Vuddhakul, V., Alves Jr, N., Thompson, F.L., Gomez-Gil, B., Vora, G.J., 2009. Comparative genomic analyses identify the Vibrio harveyi genome sequenced strains BAA-1116 and HY01 as Vibrio campbellii. Skorupski, K., Lenz, D.H., Taylor, R.K., Bassler, B.L., 2002b. Parallel quorum sensing systems converge to regulate virulence in Vibrio cholerae. Cell 110, 303-314. Xavier, K.B., Campagna, S.R., Taga, M.E., Semmelhack, M.F., Bassler, B.L., Chalker, V.J., Kirke, D., Hardman, A., Camara, M., Williams, P., 2001. The LuxM homologue VanM from Vibrio anguillarum directs the synthesis of N-(3-Lee, K.-E., Baek, C.-H., Kim, I.H., Kwon, J.-H., Lee, W.K., Lee, K.-H., Kim, B.-S., Choi, S.-H.,Kim, K.-S., 2006. Cyclo(Phe-Pro) Modulates the Expression of ompU in Vibrio spp. J.Bacteriol. 188, 2214-2221. Doi:10.1128/JB.188.6.2214-2221.2006 Van Delden, C., Iglewski, B.H., 1999. Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals. J. Bacteriol. 181, 1203-1210. Alm, E.J., Hanage, W.P., 2013. Horizontal gene transfer and the evolution of bacterial and archaeal population structure. Trends Genet. 29, 170-175. Chien, D.M., Polz, M.F., 2014. Associations and dynamics of Vibrionaceae in the environment, from the genus to the population level. Front. Microbiol. 5. Hervio-Heath, D., Teillon, A., Boisset-Helbert, C., Delesmont, R., Bodilis, J., Touron-Bodilis, A., 2013. Diversity of Vibrio spp. isolated at ambient environmental temperature in the Eastern English Channel as determined by pyrH sequencing. J. Appl. Fall, S., Pereira, M.R., Ramos-Valle, M., Curtis, S.K., Kothary, M.H., Chu, D.M.T., Monday, S.R., Kornegay, L., Donkar, T., Prince, D., Thunberg, R.L., Shangraw, K.A., Hanes, D.E., Khambaty, F.M., Lampel, K.A., Bier, J.W., Bayer, R.C., 2003. Chlamydomonas reinhardtii secretes compounds that mimic bacterial signals and interfere with quorum sensing regulation in bacteria. Plant Physiol. 134,

	Quorum sensing signal molecules (acylated homoserine lactones) in gram-negative fish pathogenic bacteria. Dis. Aquat. Organ. 65, 43-52. Doi:10.3354/dao065043 Bruto, M., James, A., Petton, B., Labreuche, Y., Chenivesse, S., Alunno-Bruscia, M., Polz, M.F., Le Roux, F., 2016. Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition. ISME J. Doi:10.1038/ismej.2016.162 Buch, C., Sigh, J., Nielsen, J., Larsen, J.L., Gram, L., 2003. Production of acylated homoserine lactones by different serotypes of Vibrio anguillarum both in culture and during infection of rainbow trout. Syst. Appl. Microbiol. 26, 338-349. Doi:10.1078/072320203322497365 conditions and serotype. Syst. Appl. Microbiol. 29, 433-445. 4, 1051-1059. Doi:10.1021/cb900165y Cano-Gomez, A., Bourne, D.G., Hall, M.R., Owens, L., Høj, L., 2009. Molecular identification, typing and tracking of Vibrio harveyi in aquaculture systems: Current methods and future prospects. Aquaculture 287, 1-10. Doi:10.1016/j.aquaculture.2008.10.058 Doi:10.1128/AEM.70.11.6855-6864.2004 Chen, X., Schauder, S., Potier, N., Van Dorsselaer, A., Pelczer, I., Bassler, B.L., Hughson, F.M., 2002. Structural identification of a bacterial quorum-sensing signal containing boron. Nature 415, 545-549. Doi:10.1038/415545a units of antibiotic production and resistance. Science 337, 1228-1231. Doi, H., Osawa, I., Adachi, H., Kawada, M., 2017. Vibrio japonicus sp. nov., a novel member of the Nereis clade in the genus Vibrio isolated from the coast of Japan. PLOS ONE 12, e0172164. Doi:10.1371/journal.pone.0172164 Eiler, A., Johansson, M., Bertilsson, S., 2006. Environmental Influences on Vibrio Populations in Northern Temperate and Boreal Coastal Waters (Baltic and Skagerrak Seas). Fuqua, C., Greenberg, E.P., 1998. Self perception in bacteria: quorum sensing with acylated homoserine lactones. Curr. Opin. Microbiol. 1, 183-189. strains of Vibrio harveyi using PCR and RAPD. Appl. Microbiol. Biotechnol. 63, 722-727. Doi:10.1007/s00253-003-1451-z 523-532. Doi:10.1111/j.1749-7345.2006.00066.x Jobling, M.G., Holmes, R.K., 1997. Characterization of hapR, a positive regulator of the Lin, Y.-H., Xu, J.-L., Hu, J., Wang, L.-H., Ong, S.L., Leadbetter, J.R., Zhang, L.-H., 2003. -acyl homoserine lactone production. Front. Life Sci. 9, Doi:10.1080/21553769.2015.1066716 and Vibrio mimicus by multilocus sequence analysis (MLSA). Int. J. Syst. Evol. Microbiol. Preheim, S.P., Boucher, Y., Wildschutte, H., David, L.A., Veneziano, D., Alm, E.J., Polz, 17-23. Thompson, C.C., Thompson, F.L., Vicente, A.C.P., 2008. Identification of Vibrio cholerae Environ. Microbiol. Rep. 2, 81-89. Doi:10.1111/j.1758-2229.2009.00100.x F., Chan, K.-G., 2016. Vibrio variabilis T01: A tropical marine bacterium exhibiting unique N standardized ribotype scheme for Vibrio cholerae O1. J. Clin. Microbiol. 31, 2474-2482. Chimetto Tonon, L.A., Mincer, T., Polz, M.F., 2012b. Ecological populations of bacteria act as socially cohesive Croci, L., Suffredini, E., Cozzi, L., Toti, L., Ottaviani, D., Pruzzo, C., Serratore, P., Fischetti, R., Goffredo, E., Loffredo, G., Mioni, R., Vibrio parahaemolyticus Working Group, 2007. Comparison of different biochemical and molecular methods for the identification of Vibrio parahaemolyticus. J. Appl. Microbiol. 102, 229-237. Doi:10.1111/j.1365-2672.2006.03046.x Croxatto, A., Chalker, V.J., Lauritz, J., Jass, J., Hardman, A., Williams, P., Cámara, M., Milton, D.L., 2002. VanT, a homologue of Vibrio harveyi LuxR, regulates serine, metalloprotease, pigment, and biofilm production in Vibrio anguillarum. J. Bacteriol. 184, 1617-1629. Bacteriol. 188, 2885-2897. Doi:10.1128/JB.188.8.2885-2897.2006 Dong, Y.-H., 1754-1759. Dorsch, M., Lane, D., Stackebrandt, E., 1992. Towards a Phylogeny of the Genus Vibrio Based on 16S rRNA Sequences. Int. J. Syst. Bacteriol. 42, 58-63. Doi:10.1099/00207713-42-1-58 Dubert, J., Balboa, S., Regueira, M., González-Castillo, A., Gómez-Gil, B., Romalde, J.L., 2016a. brackish water. FEMS Microbiol. Ecol. 60, 411-418. Doi:10.1111/j.1574-6941.2007.00303.x Eilers, H., Pernthaler, J., Glockner, F.O., Amann, R., 2000b. Culturability and in situ abundance of pelagic bacteria from the North Sea. Appl. Environ. Microbiol. 66, 3044-3051. Doi:10.1128/AEM.66.7.3044-3051.2000 Esteve, C., Doi:10.1016/0044-8486(94)00381-W 41, 5134-5146. Microbiol. 57, 55-60. Doi:10.1007/BF03175050 Hansen, H., Willassen, N.P., 2015. The autoinducer synthases LuxI and AinS are responsible for temperature-dependent AHL production in the fish pathogen Aliivibrio salmonicida. BMC Microbiol. 15, 69. Doi:10.1186/s12866-015-0402-z Hanzelka, B.L., 181, 5766-5770. bacterioplankton species. Appl. Environ. Microbiol. 68, 5488-5497. harveyi and Vibrio parahaemolyticus. J. Bacteriol. 186, 3794-3805. Doi:10.1128/JB.186.12.3794-3805.2004 Hernández, G., Olmos, J., 2004. Molecular identification of pathogenic and nonpathogenic 160, 2464-2473. Doi:10.1099/mic.0.081729-0 Hsieh, J.L., Doi:10.1111/j.1462-2920.2007.01429.x Huang, J.J., Han, J.-I., Zhang, L.-H., Leadbetter, J.R., 2003. Utilization of acyl-homoserine lactone quorum signals for growth by a soil Pseudomonad and Pseudomonas aeruginosa PAO1. Appl. Environ. Microbiol. 69, 5941-5949. Doi:10.1128/AEM.69.10.5941-5949.2003 Hunt, D.E., David, L.A., Gevers, D., Preheim, S.P., Alm, E.J., Polz, M.F., 2008. Resource Microbiol. Immunol. 39, 831-837. Jayasree, L., Janakiram, P., Madhavi, R., 2006. Characterization of Vibrio spp. associated with diseased shrimp from culture ponds of Andhra Pradesh (India). J. World Aquac. Soc. 37, genotypic diversity. Environ. Microbiol. 11, 1959-1970. Doi:10.1111/j.1462-2920.2009.01918.x Leadbetter, J.R., Greenberg, E.P., 2000. Metabolism of acyl-homoserine lactone quorum-sensing signals by Variovorax paradoxus. J. Bacteriol. 182, 6921-6926. Leaño, E.M., Lavilla-Pitogo, C.R., Paner, M.G., 1998. Bacterial flora in the hepatopancreas of pond-reared Penaeus monodon juveniles with luminous vibriosis. Aquaculture 164, 367-374. Doi:10.1016/S0044-8486(98)00201-4 Lee, J.-H., Lee, J., 2010. Indole as an intercellular signal in microbial communities. FEMS Microbiol. Rev. 34, 426-444. Doi:10.1111/j.1574-6976.2009.00204.x Trachinotus ovatus in cage mariculture. Aquaculture 261, 17-25. Miller, S.T., Hughson, F.M., 2004. Salmonella typhimurium recognizes a chemically distinct form of the bacterial quorum-sensing signal AI-2. Mol. Cell 15, 677-687. Doi:10.1016/j.molcel.2004.07.020 Milton, D.L., 2006. Quorum sensing in vibrios: Complexity for diversification. Int. J. Med. Microbiol. 296, 61-71. Doi:10.1016/j.ijmm.2006.01.044 Milton, D.L., combination of sheath components. Microbes Environ. 17, 18-25. Doi:10.1264/jsme2.2002.18 Mo, S., Krunic, A., Chlipala, G., Orjala, J., 2009. Antimicrobial ambiguine isonitriles from the cyanobacterium Fischerella ambigua. J. Nat. Prod. 72, 894-899. Doi:10.1021/np800751j Mohamad, N.I., Adrian, T.-G.-S., Tan, W.-S., Muhamad Yunos, N.Y., Tan, P.-W., Yin, W.-Park, D.-K., Parsek, M.R., Val, D.L., Hanzelka, B.L., Cronan, J.E., Greenberg, E.P., 1999. Acyl homoserine-lactone quorum-sensing signal generation. Proc. Natl. Acad. Sci. U. S. A. 96, 4360-4365. Pearson, J.P., Pérez, P.D., Hagen, S.J., 2010. Heterogeneous response to a Quorum-sensing signal in the luminescence of individual Vibrio fischeri. PLoS ONE 5, e15473. Doi:10.1016/j.tig.2012.12.006 Popovic, T., Bopp, C., Olsvik, O., Wachsmuth, K., 1993. Epidemiologic application of a Doi:10.3389/fmicb.2014.00038 Tall, A., Microbiol. 114, 1713-1724. Doi:10.1111/jam.12181 Tall, B.D., 137-146. Doi:10.1104/pp.103.029918 Figure S2 : B B CC615 B B CC626 B B CC605 B BCC 579 BB CC5 76 BB CC 62 BB CC 241 5 BB CC 6 57 6 0 48 T; X7 47 14 C C 1 77 49 ; X5 C C 14 s (T ); AT (T ); AT lyt icu e ns 41 6 Vi br io al g ino Vi br io na tri eg V ib rio
	58, 617-621. Doi:10.1099/ijs.0.65461-0

. Marine compounds and their antimicrobial activities. Sci.

Microb. Pathog. Commun. Curr. Res. Technol. Adv. 51, 1293-1306.

Abbott, S.L., Seli, L.S., Catino, M., Hartley, M.A., Janda, J.M., 1998. Misidentification of unusual Aeromonas species as members of the genus Vibrio: a continuing problem. J. Clin.

Microbiol. 36, 1103Microbiol. 36, -1104. .

Acinas, S.G., Klepac-Ceraj, V.,

Hunt, D.E., Pharino, C., Ceraj, I., Distel, D.L., Polz, M.F., 2004

. Fine-scale phylogenetic architecture of a complex bacterial community. Nature 430, 551-554. Doi:10.1038/nature02649 Alavandi, S.V., Manoranjita, V., Vijayan, K.K.,

Kalaimani, N., Santiago, T.C., 2006.

Phenotypic and molecular typing of Vibrio harveyi isolates and their pathogenicity to tiger shrimp larvae. Lett.

Appl. Microbiol. 43, 566-570. Doi:10.1111/j.1472-765X.2006.01986.

x Allam, B.,

Paillard, C., Ford, S.E., 2002

. Pathogenicity of Vibrio tapetis, the etiological agent of brown ring disease in clams. Dis.

Aquat. Organ. 48,[221][222][223][224][225][226][227][228][229][230][231]

/dao048221

Al-saari, N., Gao, F., A.K.M. Rohul, A., Sato, Kazumichi, Sato, Keisuke, Mino, S., Suda, W., Oshima, K., Hattori, M., Ohkuma, M., Meirelles, P.M.,

Thompson, F.L., Thompson, C., A. Filho, G.M., Gomez-Gil, B., Sawabe, Toko, Sawabe, Tomoo, 2015

. Advanced microbial taxonomy combined with genome-based-approaches reveals that Vibrio astriarenae sp. nov., an agarolytic marine bacterium, forms a new clade in Vibrionaceae. PLOS ONE 10, e0136279. Doi:10.1371/journal.pone.0136279 Alsina, M.,

Blanch, A.R., 1994

. A set of keys for biochemical identification of environmental Vibrio species. J.

Appl. Bacteriol. 76, 79-85. Doi:10.1111/j.1365-2672.1994.tb04419.

x Alvarez,

Austin, Alvarez, Reyes, 1998

. Vibrio harveyi: a pathogen of penaeid shrimps and fish in Venezuela. J. Fish

Dis. 21, 313-316. Doi:10.1046/j.1365-2761.1998.00101.

x Amin, S.A.,

Green, D.H., Kupper, F.C., Carrano, C.J., 2009. Vibrioferrin, an

unusual marine siderophore: Iron binding, photochemistry, and biological implications. Inorg. Chem. 48, 11451-11458. Doi:10.1021/ic9016883 Bassler, B.L., Wright, M., Showalter, R.E., Silverman, M.R., 1993. Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence. Mol. Microbiol. 9, 773-786. Bassler, B.L., Wright, M., Silverman, M.R., 1994. Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway. Mol. Microbiol. 13, 273-286. Bassler, B.L., Yu, C., Lee, Y.C., Roseman, S., 1991. Chitin utilization by marine bacteria. Degradation and catabolism of chitin oligosaccharides by Vibrio furnissii. J. Biol. Chem. 266, 24276-24286.

Beaz Hidalgo, R.,

Cleenwerck, I., Balboa, S., De Wachter, M., Thompson, F.L., Swings, J., De Vos, P., Romalde, J.L., 2008

. Diversity of vibrios associated with reared clams in Galicia (NW Spain). Syst.

Appl. Microbiol. 31, 215-222. Doi:10.1016/j.syapm.2008.04.001

Beaz-Hidalgo, R., Doce, A., Pascual, J., Toranzo, A.E., Romalde, J.L., 2009. Vibrio gallaecicus sp. nov. isolated from cultured clams in north-western Spain. Syst. Appl.

Microbiol. 32, 111-117. Doi:10.1016/j.syapm.2008.12.002

Ben-Haim, Y., 2003. Vibrio coralliilyticus sp. nov., a temperature-dependent pathogen of the coral Pocillopora damicornis. Int. J.

Syst. Evol. Microbiol. 53,[309][310][311][312][313][314][315]

.1099/ijs.0.02402-0 Berg, K.A.,

[START_REF] Berg | High diversity of cultivable heterotrophic bacteria in association with cyanobacterial water blooms[END_REF]

High diversity of cultivable heterotrophic bacteria in association with cyanobacterial water blooms.

ISME J. 3, 314-325. Doi:10.1038/ismej.2008.110

Biers, E.J., Sun, S., Howard, E.C., 2009. Prokaryotic genomes and diversity in surface ocean waters: Interrogating the global ocean sampling metagenome. Appl.

Environ. Microbiol. 75, 2221-2229. Doi:10

.1128/AEM.02118-08

Biosca, E.G., Amaro, C., Esteve, C., Alcaide, E., Garay, E., 1991. First record of Vibrio vulnificus biotype 2 from diseased European eel, Anguilla anguilla L. J. Fish

Dis. 14, 103- 109. Doi:10.1111/j.1365-2761.1991.tb00581.

x Blackwell, K.D.,

Oliver, J.D., 2008

. The ecology of Vibrio vulnificus, Vibrio cholerae, and Vibrio parahaemolyticus in North Carolina Estuaries. J. Microbiol. 46, 146-153. Doi:10.1007/s12275-007-0216-2 Bruhn, J.B., Buchholtz, C., Nielsen, K.F., Milton, D.L., Larsen, J.L., Gram, L., 2006. Profiling of acylated homoserine lactones of Vibrio anguillarum in vitro and in vivo: Influence of growth Doi:10.1016/j.syapm.2005.12.007 Bullen, J.J., 1991. Hemochromatosis, Iron, and Septicemia Caused by Vibrio vulnificus. Arch. Intern. Med. 151, 1606. Doi:10.1001/archinte.1991.00400080096018 Caburlotto, G., Haley, B.J., Lleò, M.M., Huq, A., Colwell, R.R., 2010. Serodiversity and ecological distribution of Vibrio parahaemolyticus in the Venetian Lagoon, Northeast Italy.

Environ.

Microbiol. Rep. 2, 151-157. Doi:10.1111/j.1758-2229.2009.00123.

x Cai, J.,

Li, J., Thompson, K.D., Li, C., Han, H., 2007.

Isolation and characterization of pathogenic Vibrio parahaemolyticus from diseased post-larvae of abalone Haliotis diversicolor supertexta. J. Basic

Microbiol. 47, 84-86. Doi:10.1002/jobm.200610192

Campbell, J.,

Lin, Q., Geske, G.D., Blackwell, H.E., 2009

. New and unexpected insights into the modulation of LuxR-type Quorum sensing by cyclic dipeptides. ACS Chem. Biol. Cao, J.G., Meighen, E.A., 1989. Purification and structural identification of an autoinducer for the luminescence system of Vibrio harveyi. J. Biol. Chem. 264, 21670-21676. Cervino, J.M., Doi:10.7717/peerj.741 Choi, S.H., Greenberg, E.P., 1991. The C-terminal region of the Vibrio fischeri LuxR protein contains an inducer-independent lux gene activating domain. Proc. Natl. Acad. Sci. U. S. A. 88, 11115-11119. Chong, G., Kimyon, Ö., Manefield, M., 2013. Quorum sensing signal synthesis may represent a selective advantage independent of its role in regulation of bioluminescence in Vibrio fischeri. PLoS ONE 8, e67443. Doi:10.1371/journal.pone.0067443 Colwell, R.R., 2009. Viable but non-culturable bacteria in the marine environment and the biotechnological tools to detect them. Biotechnology, volume IX: Fundamentals in Biotechnology. Cordero, O. X., Ventouras, L.-A., DeLong, E.F., Polz, M.F., 2012a. Public good dynamics drive evolution of iron acquisition strategies in natural bacterioplankton populations. Proc. Natl. Acad. Sci. U. S. A. 109, 20059-20064. Doi:10.1073/pnas.1213344109 Cordero, O. X., Wildschutte, H., Kirkup, B., Proehl, S., Ngo, L., Hussain, F., Le Roux, F., Doi:10.1126/science.1219385 Cottrell, M.T., Kirchman, D.L., 2000. Natural assemblages of marine proteobacteria and members of the Cytophaga-Flavobacter cluster consuming low-and high-molecular-weight dissolved organic matter. Appl. Environ. Microbiol. 66, 1692-1697. Cottrell, M.T., De Keersmaecker, S.C.J., Sonck, K., Vanderleyden, J., 2006. Let LuxS speak up in AI-2 signaling. Trends Microbiol. 14, 114-119. Doi:10.1016/j.tim.2006.01.003 Doberva, M., 2016. Le quorum sensing bactérien dans l'environnement marin: diversité moléculaire et génétique des auto-inducteurs. Paris 6. Doberva, M., Sanchez-Ferandin, S., Toulza, E., Lebaron, P., Lami, R., 2015. Diversity of quorum sensing autoinducer synthases in the Global Ocean Sampling metagenomic database. Aquat. Microb. Ecol. 74, 107-119. Doi:10.3354/ame01734 Dobretsov, S., Teplitski, M., Alagely, A., Gunasekera, S.P., Paul, V.J., 2010. Malyngolide from the cyanobacterium Lyngbya majuscula interferes with quorum sensing circuitry: QS inhibitors from marine cyanobacteria. Environ. Microbiol. Rep. 2, 739-744. Doi:10.1111/j.1758-2229.2010.00169.x Doherty, N., Holden, M.T.G., Qazi, S.N., Williams, P., Winzer, K., 2006. Functional analysis of luxS in Staphylococcus aureus reveals a role in metabolism but not Quorum sensing. J. Dubert, J., Romalde, J.L., Prado, S., Barja, J.L., 2016b. Vibrio bivalvicida sp. nov., a novel larval pathogen for bivalve molluscs reared in a hatchery. Syst. Appl. Microbiol. 39, 8-13. Doi:10.1016/j.syapm.2015.10.006 Eaves, L., Ketterer, P., 1994. Mortalities in red claw crayfish Cherax quadncannatus associated with systemic Vibrio mimicus infection. Dis. Aquat. Organ. 19, 233-237. Doi:10.3354/dao019233 Eberhard, A., Burlingame, A.L., Eberhard, C., Kenyon, G.L., Nealson, K.H., Oppenheimer, N.J., 1981. Structural identification of autoinducer of Photobacterium fischeri luciferase. Biochemistry (Mosc.) 20, 2444-2449.

Egidius, E., Wiik, R.,

Andersen, K., Hoff, K.A., Hjeltnes, B., 1986

. Vibrio salmonicida sp. nov., a new fish pathogen. Int. J. Syst. Evol. Microbiol. 36, 518-520. Egland, K.A., Greenberg, E.P., 1999. Quorum sensing in Vibrio fischeri: elements of the luxl promoter. Mol. Microbiol. 31, 1197-1204. Eiler, A., Gonzalez-Rey, C., Allen, S., Bertilsson, S., 2007. Growth response of Vibrio cholerae and other Vibrio spp. to cyanobacterial dissolved organic matter and temperature in Appl. Environ. Microbiol. 72, 6004-6011. Doi:10.1128/AEM.00917-06 Eilers, H., Faury, N., 2004. Vibrio crassostreae sp. nov., isolated from the haemolymph of oysters (Crassostrea gigas). Int. J. Syst. Evol. Microbiol. 54, 2137-2140. Doi:10.1099/ijs.0.63232-0 Fegatella, F., Lim, J., Kjelleberg, S., Cavicchioli, R., 1998. Implications of rRNA operon copy number and ribosome content in the marine oligotrophic ultramicrobacterium Sphingomonas sp. strain RB2256. Appl. Environ. Microbiol. 64, 4433-4438. Ferdous, 2009. Survival of Vibrio cholerae O139 in Association with Anabaena variabilis in four different microcosoms. Am. J. Appl. Sci. 6, 439-444. Doi:10.3844/ajassp.2009.439.444 Froelich, B., Bowen, J., Gonzalez, R., Snedeker, A., Noble, R., 2013. Mechanistic and statistical models of total Vibrio abundance in the Neuse River Estuary. Water Res. 47, 5783-5793. Doi:10.1016/j.watres.2013.06.050 Fukuda, H., Doi, H., Chinen, A., Usuda, Y., 2016. Vibrio algivorus sp. nov., an alginate-and agarose-assimilating bacterium isolated from the gut flora of a turban shell marine snail. Int. J. Syst. Evol. Microbiol. 66, 3164-3169. Doi:10.1099/ijsem.0.001165

Fukushima, H., Tsunomori, Y., Seki, R., 2003. Duplex real-time SYBR green PCR assays for detection of 17 species of food-or waterborne pathogens in stools. J. Clin. Microbiol. Fuqua, W.C., Winans, S.C., Greenberg, E.P., 1994. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J. Bacteriol. 176, 269-275. (Penaeus monodon) and experimental challenge study against post larvae and juveniles. Ann. Hao, Y., Winans, S.C., Glick, B.R., Charles, T.C., 2010. Identification and characterization of new LuxR/LuxI-type quorum sensing systems from metagenomic libraries. Environ. Microbiol. 12, 105-117. Doi:10.1111/j.1462-2920.2009.02049.x Hawkins, A.C., Arnold, F.H., Stuermer, R., Hauer, B., Leadbetter, J.R., 2007. Directed evolution of Vibrio fischeri LuxR for improved response to butanoyl-homoserine lactone. Appl. Environ. Microbiol. 73, 5775-5781. Doi:10.1128/AEM.00060-07 Heidelberg, J.F., Heidelberg, K.B., Colwell, R.R., 2002. Seasonality of Chesapeake bay Doi:10.1128/AEM.

68.11.5488-5497.2002

Henke, J.M.,

Bassler, B.L., 2004a

. Three parallel quorum-sensing systems regulate gene expression in Vibrio harveyi. J.

Bacteriol. 186, 6902-6914. Doi:10.1128/JB.186.20.6902- 6914.2004

Henke, J.M.,

Bassler, B.L., 2004b

. Quorum sensing regulates type III secretion in Vibrio Hidalgo-Romano, B., Gollihar, J., Brown, S.A., Whiteley, M., Valenzuela, E., Kaplan, H.B., Wood, T.K., McLean, R.J.C., 2014. Indole inhibition of N-acylated homoserine lactone-mediated quorum signalling is widespread in Gram-negative bacteria. Microbiol. Read. Engl. partitioning and sympatric differentiation among closely related bacterioplankton. Science 320, 1081-1085. Doi:10.1126/science.1157890 Islam, M.S., Drasar, B.S., Bradley, D.J., 1990. Long-term persistence of toxigenic Vibrio cholerae 01 in the mucilaginous sheath of a blue-green alga, Anabaena variabilis. J. Trop. Med. Hyg. 93, 133-139. Islam, M.S., Drasar, B.S., Bradley, D.J., 1989. Attachment of toxigenic Vibrio cholerae 01 to various freshwater plants and survival with a filamentous green alga, Rhizoclonium fontanum. J. Trop. Med. Hyg. 92, 396-401. Islam, M.S., Mahmuda, S., Morshed, M.G., Bakht, H.B.M., Khan, M.N.H., Sack, R.B., Sack, D.A., 2004. Role of cyanobacteria in the persistence of Vibrio cholerae O139 in saline microcosms. Can. J. Microbiol. 50, 127-131. Doi:10.1139/w03-114 Iwamoto, Y., Suzuki, Y., Kurita, A., Watanabe, Y., Shimizu, T., Ohgami, H., Yanagihara, Y., 1995. Vibrio trachuri sp. nov., a new species isolated from diseased Japanese horse mackerel. Vibrio cholerae HA/protease gene hap, and its identification as a functional homologue of the Vibrio harveyi luxR gene. Mol. Microbiol. 26, 1023-1034. Lee, K.K., Yu, S.R., Chen, F.R., Yang, T.I., Liu, P.C., 1996. Virulence of Vibrio alginolyticus isolated from diseased tiger prawn, Penaeus monodon. Curr. Microbiol. 32, 229-231. Lemee, J.-M.F., 2004. Les infections à vibrions non cholériques en France: cas identifiés de 2001 à 2003 par le Centre national de référence des vibrions et du choléra. Lerat, E., 2004. The Evolutionary history of Quorum-sensing systems in bacteria. Mol. Biol. Evol. 21, 903-913. Doi:10.1093/molbev/msh097 Li, G., Zhao, D., Huang, L., Sun, J., Gao, D., Wang, H., Tan, Y., Liang, L., 2006. Identification and phylogenetic analysis of Vibrio vulnificus isolated from diseased Doi:10.1016/j.aquaculture.2006.07.013 Lilley, B.N., Bassler, B.L., 2000. Regulation of quorum sensing in Vibrio harveyi by LuxO and sigma-54. Mol. Microbiol. 36, 940-954. Lin, B., Acyl-homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum-quenching enzymes. Mol. Microbiol. 47, 849-860. Miller, M.B., hydroxyhexanoyl) homoserine lactone and N-hexanoylhomoserine lactone. J. Bacteriol. 183, 3537-3547. Doi:10.1128/JB.183.12.3537-3547.2001 Milton, D.L., Hardman, A., Camara, M., Chhabra, S.R., Bycroft, B.W., Stewart, G.S., Williams, P., 1997. Quorum sensing in Vibrio anguillarum: characterization of the vanI/vanR locus and identification of the autoinducer N-(3-oxodecanoyl)-L-homoserine lactone. J. Bacteriol. 179, 3004-3012. Miyashiro, T., Ruby, E.G., 2012. Shedding light on bioluminescence regulation in Vibrio fischeri. Mol. Microbiol. 84, 795-806. Doi:10.1111/j.1365-2958.2012.08065.x Mizanur, R. md., Islam, M.S., Khan, S.I., Rahim, Z., 2002. The chemotactic response of Vibrio cholerae 0139 to the mucilaginous sheath of blue-green algae is mediated by a Doi:10.1371/journal.pone.0015473 Pérez-Cataluña, A., Lucena, T., Tarazona, E., Arahal, D.R., Macián, M.C., Pujalte, M.J., 2016. An MLSA approach for the taxonomic update of the Splendidus clade, a lineage containing several fish and shellfish pathogenic Vibrio spp. Syst. Appl. Microbiol. 39, 361-369. Doi:10.1016/j.syapm.2016.03.010 Pfeffer, C., Oliver, J.D., 2003. A comparison of thiosulphate-citrate-bile salts-sucrose (TCBS) agar and thiosulphate-chloride-iodide (TCI) agar for the isolation of Vibrio species from estuarine environments. Lett. Appl. Microbiol. 36, 150-151.

Plaza, M., Santoyo, S., Jaime, L., García-Blairsy Reina, G., Herrero, M., Señoráns, F.J., Ibáñez, E., 2010. Screening for bioactive compounds from algae. J.

Pharm. Biomed. Anal. 51, 450-455. Doi:10.1016/j.jpba.2009.03.016

Polz, M.F., M.F., 2011. Metapopulation structure of Vibrionaceae among coastal marine invertebrates.

Environ.

Microbiol. 13, 265-275. Doi:10.1111/j.1462-2920.2010.

02328.x Takemura, A.F., Characterization of Vibrio fluvialis-like strains implicated in limp lobster disease. Appl. Environ. Microbiol. 69, 7435-7446. Doi:10.1128/AEM.69.12.7435-7446.2003 Tan, P.-W., Tan, W.-S., Yunos, N., Mohamad, N., Adrian, T.-G.-S., Yin, W.-F., Chan, K.-G., 2014. Short chain N-acyl homoserine lactone production in tropical marine Vibrio sinaloensis strain T47. Sensors 14, 12958-12967. Doi:10.3390/s140712958 Tan, W.-S., Yunos, N., Tan, P.-W., Mohamad, N., Adrian, T.-G.-S., Yin, W.-F., Chan, K.-G., 2014. Characterisation of a marine bacterium Vibrio brasiliensis T33 producing N-acyl homoserine lactone Quorum sensing molecules. Sensors 14, 12104-12113. Doi:10.3390/s140712104

Tarazona, E., Lucena, T., Arahal, D.R., Macián, M.C., Ruvira, M.A.,

Pujalte, M.J., 2014.

Multilocus sequence analysis of putative Vibrio mediterranei strains and description of Vibrio thalassae sp. nov. Syst.

Appl. Microbiol. 37, 320-328. Doi:10.1016/j.syapm.2014.05.005

Teplitski, M., Chen, H., Rajamani, S., Gao, M., Merighi, M., Sayre, R.T., Robinson, J.B.,

Rolfe, B.G., Bauer, W.D., 2004.

Table 1 :

 1 Predicted

			Retention time (Rt, min)
	Acyl-side chain length (C atoms)	Cx-HSL oxo-Cx-HSL OH-Cx-HSL
	5	7,85	6,97	NP
	6	8,43	7,56	NP
	7	8,83	8,15	8,09
	8	9,27	8,69	8,55
	9	9,57	9,04	8,92
	10	9,90	9,43	9,25
	11	10,13	9,74	9,59
	12	10,46	10,04	9,87
	13	10,63	10,33	10,15
	14	10,93	10,56	10,42
	15	11,15	10,83	10,63
	16	11,34	11,06	10,85
	17	11,49	11,27	11,05
	18	11,66	11,47	11,24

retention times for Cx-HSL, oxo-Cx-HSL and OH-HSL. Based on retention times curves of AHL standards (Figure

S1

). NP: Non Predictable.

Table 2 :

 2 Amino acids changes in A. fischeri MB13B1.

	Protein	Amino acid change(s)	Properties change(s)

Table S1 : UHPLC-HRMS data of AHL Standards.

 S1 Rt: Retention time. Theoretical and Observed mass correspond to the pseudo-molecular ion [M + H] + .

	AHL Standard	Molecular Formula Theorical Mass Observed Mass	Rt (min)
	C4-HSL	C8H13NO3	172.0968	172.0968	5.26
	C6-HSL	C10H17NO3	200.1281	200.1281	8.43
	OXO-C6-HSL	C10H15NO3	214.1074	214.1072	7.56
	C7-HSL	C11H19NO3	214.1438	214.1440	8.83
	C8-HSL	C12H21NO3	228.1594	228.1594	9.27
	OXO-C8-HSL	C12H19NO4	242.1387	242.1381	8.69
	OH-C8-HSL	C12H21NO4	244.1543	244.154	8.55
	C9-HSL	C13H23NO3	242.1751	242.1748	9.57
	C10-HSL	C14H25NO3	256.1907	256.1907	9.90
	OXO-C10-HSL	C14H23NO4	270.1700	270.1699	9.43
	OH-C10-HSL	C14H25NO4	272.1856	272.1856	9.25
	C11-HSL	C15H27NO3	270.2064	270.2063	10.13
	C12-HSL	C16H29NO3	284.2220	284.2220	10.46
	OXO-C12-HSL	C16H27NO4	298.2013	298.2013	10.04
	OH-C12-HSL	C16H29NO4	300.2169	300.2169	9.87
	C13-HSL	C17H31NO3	298.2377	298.2377	10.63
	C14-HSL	C18H33NO3	312.2533	312.2533	10.93
	C14:1-HSL	C18H31NO3	310.2377	310.2370	10.51
	OXO-C14:1-HSL	C18H29NO4	324.2169	324.2170	10.23
	OXO-C14-HSL	C18H31NO4	326.2326	326.2322	10.56
	OH-C14-HSL	C18H33NO4	328.2482	328.2482	10.42
	C15-HSL	C19H35NO3	326.2690	326.2689	11.15
	C16-HSL	C20H37NO3	340.2846	340.2846	11.34
	C16:1-HSL	C20H35NO3	338.2690	338.2704	10.93
	OXO-C16:1-HSL	C20H33NO4	352.2482	352.2497	10.61
	C18-HSL	C22H42NO3	368.3159	368.3155	11.66
	C18:1-HSL	C22H39NO3	366.3003	366.3003	11.40

Table S2 : UHPLC-HRMS data and identification of OH-C9-HSL in A. fischeri strains.

 S2 Rt: Retention time. Theoretical and observed mass correspond to the pseudo-molecular ion [M + H] + .

	Strain	Rt (min)	Observed mass	Molecular Formula	Delta ppm	Fragmentation	Name	Molecular Formula Identification Molecular Weight	Theorical Mass
						70.041 (16.93), 74.061 (9.53),
						75.027 (16.64), 91.055 (14.94),
						93.037 (31.41), 102.055 (12.41),
						105.070 (10.50), 109.076 (66.51),
	ES213		258,1701		0,115	122.084 (18.27), 135.092 (53.91),
						137.060 (14.71), 151.075 (81.97),
						229.097 (23.28), 244.120 (21.08),
						257.128 (13.45), 257.175 (16.47),
						258.110 (21.03), 259.144 (100.00)
						70.041 (10.26), 74.061 (9.68),
						75.027 (12.38), 91.055 (7.87),
						93.037 (16.40), 102.055 (35.76),
						105.070 (6.38), 109.076 (31.04),
	MB13B1		258,1702		0,718	122.084 (10.24), 135.092 (52.27),
						137.060 (8.18), 151.075 (33.32),
						229.097 (23.46), 244.120 (23.04),
						257.128 (6.53), 257.175 (6.67),
						258.110 (9.27), 259.144 (100.00)
						70.041 (16.69), 74.061 (10.36),
						75.027 (12.97), 91.055 (9.90),
						93.037 (15.18), 102.055 (18.65),
						105.070 (9.32), 109.076 (54.58),
	KB2B1	8,9	258,1698	C13H24NO4	-0,185	122.084 (12.89), 135.092 (53.85),	OH-C9-HSL C13H23NO4	257,1627	258,1700
						137.060 (10.73), 151.075 (69.36),
						229.097 (24.46), 244.120 (18.15),
						257.128 (21.52), 257.176 (18.03),
						258.110 (17.91), 259.144 (100.00)
						70.041 (14.37), 74.061 (13.51),
						75.027 (18.06), 91.055 (12.21),
						93.037 (29.99), 102.055 (20.07),
						105.070 (12.15), 109.076 (56.22),
	KB4B5		258,1696		-0,395	122.084 (12.35), 135.092 (45.94),
						137.060 (10.86), 151.075 (72.83),
						229.097 (22.02), 244.120 (18.48),
						257.128 (23.68), 257.177 (14.71),
						258.109 (18.25), 259.144 (100.00)
						70.041 (9.43), 74.061 (10.47),
						75.027 (36.97), 91.055 (8.93),
						93.037 (100.00), 102.055
						(14.35), 105.070 (9.00), 109.076
	ES114		258,1700		0,214	(45.02), 122.084 (9.23), 135.092 (76.51), 137.060 (9.31), 151.075
						(51.00), 229.097 (29.82), 244.120
						(11.06), 257.128 (15.74), 257.176
						(13.72), 258.110 (22.61), 259.144
						(100.00)

Table S4 :

 S4 PCO analysis, variation ex plained by individual axis.

	Axis	Eigenvalue Individual% Cumulative%
	PCO1	10339,0	79,9	79,9
	PCO2	2291,7	17,7	97,6
	PCO3	310,1	2,4	100,0
	PCO4	6,6	0,1	100,0

Table S5 :

 S5 Pearson correlation between each variable and V. mediterranei G1 phenotypes ordination by axis.

	Variable	PCO1	PCO2	PCO3	PCO4
	Chaetoceros + Bacteriastrum sp.	0,3957	-0,0746	0,0727	-0,0074
	Coscinodiscus sp.	0,2091	-0,0596	0,1201	-0,2093
	Diploneis + Cocconeis sp.	0,1765	0,3734	0,2915	0,4676
	Grammatophora sp.

Table S6 :

 S6 Predicted retention times for Cx-HSL, oxo-Cx-HSL and OH-HSL. Based on retention times curves of AHL standards (FigureS1). NP: Non Predictable.

			Retention time (Rt, min)
	Acyl-side chain length (C atoms)	Cx-HSL oxo-Cx-HSL OH-Cx-HSL
	5	7,85	6,97	NP
	6	8,43	7,56	NP
	7	8,83	8,15	8,09
	8	9,27	8,69	8,55
	9	9,57	9,04	8,92
	10	9,90	9,43	9,25
	11	10,13	9,74	9,59
	12	10,46	10,04	9,87
	13	10,63	10,33	10,15
	14	10,93	10,56	10,42
	15	11,15	10,83	10,63
	16	11,34	11,06	10,85
	17	11,49	11,27	11,05
	18	11,66	11,47	11,24

Table S7 :

 S7 UHPLC-HRMS data and identification of unexpected AHLs in V. mediterranei strains. Rt: Retention Time. Theorical mass correspond to the pseudo-molecular ion [M+H] + .

Table S8 :

 S8 Potential candidate genes for AHL production in G1 V. mediterranei isolates 17 LN0615E and 21 LS0615E. The genomes will be submitted to NCBI before September 2017.

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	cttccttattcactcctcttcaagttaggccgaggtttgggcaaatttgggatgcgctatggcaagaaaagagcgaaagtcgctcggcgaaaccttga
	gctcgcttttcctgagatgaatgagcaagagcttgatcgttttgttgaagagaactttaaaaactccggtatggctctgattgaaacaggaattacttggtt
	ttggccaacatggcgcttcaagcgtattttggttgcaaaagatatcagtgcgcttgaaaagcatgaagctgaaggtaaaggtgtactgctgtgttgtgta
	catgcgttaaatctcgaaattaccgctcgagctttcgcagtattagggcttcctggttacggagcttatcgtcctcacagtaaccctgcgtatgagttcatt atgaaaggacagatgatgaacatccctctcaccatcactgaaatcatgcgtcacgccgaccgcgtcaatggcgataccgagatagtctcggtgacct caataccgtggaagaacacgtaatggtaataaactgatctatcgaactgacctgaagtcgatgatccgcgttatgcgtcaaggccatcgtttgttctatt acgacaaccctaggcaccgctataccttgtcggacgccttttcacgaactcgacaattggcgaatgcactcaaatcactgggattaaaaaaaggtca tacctgatcaggattatggtcacaataagtcagtgtttgttccactatttgcggtcaaagacgcctgcacaacaacaggaaccagtattctagcttacac gcgcgtcgctacgcttgcatggaatgactaccgacatcttgagttgtactacgccatctcttgttctgggggagtattacacaccgtcaatcctagactc cagtaagtgtgccattgttcctgggtcagggtttagaactagcgaaggcaaatatgaaatcatcgcggctgaatccatagaaggcgactaccctaag tttgaacaacaaattgagtacatcatcaatcatgcagaagacaccatagtgttctttgaccccatcttcacacctcttattgaaaaactgcaaagcaaact aaagatgagactgccgcagccgcttacatgaataagtatgttgagcaaattatcctacgtgcgccagagcaatggttgtggatgcacaaacggttca ctctaccgttaaccacttcatcgcactaacggatcacgctcacttacctgaatctcaaatcgaaaacctattggactacgaaagttttatcgcagaccga agcactgtatttacttggcccgagctagaagaagatgacgccagttctttatgctatacctcggggacaacaggccatcccaaaggcgtgctttactc aatcgctgccagataaaactcttacagactcaaggtatcaataa
	acaccggtcaacagtccttcactcattaggcagtgctctacctgatgcgtttgggctttccatcgatgagtgcgtaatgcctatcgtacctatgtttcatgt >fig|6666666.207930.peg.568 OK Lipid A biosynthesis lauroyl acyltransferase (EC 2.3.1.-)
	taacggatggggtctggcgtacagtgccccgatgactgggactaaactcgtgatgcccggtcccaaaatggcagatggagctacattgactgagct atgaatattgtaccaagaccgttattttcagtgcgtatgctagcccctaaatactggggaatctggtgtgcctttggatttctcgctttcatcgtcaacgtcc
	tatcaacaatgaaggcgtgaccatgacagcgggtgtgcctgttgtgtggctgatgttgttggagcatttaaaacaatcagagcttcaagtaccaagtct ttccttactcctttttgcgcggactaggcactggaaccggattattggcgatgaaaatactcaagaaacggtcccaagttgccgcccaaaatctagcac
	caagcgaatcgtcgtcggtggctctgcttgtcctgaagtattaataagggagttcgatgaattatttaatgtctcgatgcaccatgcttggggtatgacc tttgttttcctcattacacagctcaacaacgcgatttaatactgagaaacacctttaaacgcactggcatcgcactgtttgaaacgggtatggcttggtttt
	gaaatgagcccccttggtaccttcaataaactcaagccaaagctacgttcacttgctgatgaacaaaagatggggtaccagcttaaacaaggtcgcat ggcctgaatggcgactacgcagaatctcttccgttgtcgacagggaagtccttttagaacaagaggcggcggggcgaggaaccttggtagtgtgca
	tgtgtttggcgtagatatgcgaatagaagattggcaaggcaatcaggtgccgtgggatggtcaccaatttggttcggtcaaagtacgaggcccgtgg gtcatcacctaaatctagaaatgaccgcttcaatattcggtcagtttgctcaaggttttggcgtttatcgacctcattcaaacctggtttacgagttcgtcca
	attgcatcagggtattacaagcaaggagaaagtgttgatagcaacggctactttgataccggagacgtagccagcattgatcctgatggttacatgca acatagaggtcgcacccgaagtggccatcaaatgattgaccgaacagatgtaagaaccatgcttagagcgcttaaaaagggatgccgactctggta
	aatcaccgatcgttcaaaagatgttattaagtcaggaggcgagtggattagctcaattgaactggagaatatcgctgtagggcaccctgatatcaagg tttgccagaccatgattatggtcatcacaactcagtatttgcaccgttttttgcagtgcagcaagctgctgtaacagcaggccccagcgtcctcattgat
	aagctgcagtgatcggtgtttcgcacaacaagtggggtgaacgcccgctgcttattgcagtcactcatgatgtgactgtgacggaatcagagctcttg gccacaaactgtgccgtaatttcaggggttactgtcgtacaagataatcattatacgttgcatattggcgaggatttgagcgagcaaattccaaggcgt
	gctcacttcgatggtaaagttgcgaagttctgtatccccgacgccgttgagtttattaacgaacttcctcacaccgccactggcaaactcagtaaaaaa gaccctgaaactgcagccaaggtactaaacaaggaactagaaggaatgattgagcgcgatattcccggttggatgtggctacataaacggtttaaga
	gatctgcgagatcactttaaagactatgttctcggttga cacgtccagagggcgcgagcagcctgtactcctaa
	>fig|6666666.207930.peg.2303 FIG002208: Acetyltransferase (EC 2.3.1.-) >fig|6666666.207930.peg.2462 Predicted hydrolase/acyltransferase
	atgcttattcgaactgaagcgccggccgatatacttgtcattgaccgattactcaaatcggtatttgatacggatgcagaagccaatttagtgatggcac gtggcctatcttgaattgaaccccaaactaacggccgctaagacggtcgtttttattcatggttggatggataatgccgccagtttccactctttaatcga
	tgagagagaatggtcgtctaacgttatctttggtagcgtgctctgatgaaggtgaagtcattgggcatctgttgtttacacctgtcactatcgatggcgac gcaggctgctgcacatcaggtaccttggcgagttattgcgattgatttgccagggcatggtcattcaacacataagtctgcgcaccatttttacaactttc
	gacttgtcatggcaaggtttagcaccgctagcggttgccaaggagcatcaggggcaaggagtggcatctgcactcgttgaagaggcattcaccact acgactacatcgatgatcttcaccgcattttgctcaaacttgaagcagttgatgtatatctagtgggacattcacttggtgcattaatcgcgagttgctata
	ttacctgagttcggctatcccgcttgtgtggtgcttggagacccaagctattacggacgattccagttttctgctgcagctgactatgatcttcattgtcag gtgcggcctttcctgaaaaagtcgcaggattagttcaaatcgaagcgcatgtccctctttctgaatccccagagcagagcgcagagcgactaagaaa
	tgggaagtaccagaaggtgcattccaagttcgttcattgacggaagattcggtggccgaacagcttgcgggccgtaaagggttggtggattactgcc cggtattgaaagtcgtgagcattggcgagaaaaatcaccacgtgccattccatccaaacaagatgcaatcaaaatgaggatgcgagcaacaaagtt
	ctgagtttaacggtctgtaa gcgggaagaaacagtcctcccgattgttgaacgtgacctgcaattgatcgatggtcagtggttttggcgtcatgacagtaagcttaaatgtgagtcggt
	ctatcgtatgacgcaagagcacgctgaggccattatgtctgcgattactgttcctcatttggtgattttggggcagcgtggttatcactatttacagaacc >fig|6666666.207930.peg.356 Lipid A biosynthesis (KDO) 2-(lauroyl)-lipid IVA acyltransferase ctcaaatcttatctcttcttgtcgatgcacagatagaaaaggtcgaaggcgatcaccattgccatttagagtcgccaaaaaaggtatttgagctaatactt (EC 2.3.1.-) ggactagttaacaaaaattaa
	atgagtaatcaacaacacgtagataagaacctctacaatcctacattccaatgggcattcttacacccaaaacactggggaacttggctcggtattctct ttgctgttttactggcttttgtacccgctaaaccacgcgataaatttgcccgatacatctctggtattatcgtcggcaaaaatggtcgtgtagtcaggcgta >fig|6666666.
	ctcgcctcaatctacaaaagtgttttccagaaaagtccgagcaagagatccagcatattgtcgaagagacgtttgctaaagcaggtcaatacttgttgg
	ggtattcagagtttttgattcgctcaactaaacacaaccagcagcgtggcgttatgatcggtgaagaaaatctgatgcctttattagatgctggtgagaa
	tgtgattatcttagcaccacacgcatgggctgttgattacccggcggtgatgctcgctgcgcaaggttacaaagtcgcaacgatcatgaagccacaa
	aacaaccctattggtgactggctgatgcatgttcagcgtatgcaatatggtggtcgtatctttgctcgtagtgctggagtgaaaccattcgtacgctctat
	taaagatgggtatatcggatattggctgcctgatgaggatcatggtccacaaaactcagtatttgtgcctttctttgctactgaaaaagccacattaaaag
	gctttggcaaaatgtctcgattatcgcgagcgaaagtagtacccgtcctgcctgcctataacgacaaaaccagcaaatacgaagtgcatattctgcct
	gctatcgacaacttccctacgggtgatgaggagcaagatgcacgggcgatgaataaagcgattgaagatttgctacgagataaacctgagcagtat
	atgtggaacctataccttctcgaaacccaacgtgacggtattcgtatctacgacaatatctaa
	>fig|6666666.207930.peg.3952 Predicted acyltransferase
	atgggcgaagttgggcattgttttcagtttgcatgggcgttcattgatggcttacactgccgctattctatccttttgagacgagcattggctatggatacg
	ttaaaattcggtatgctagaccccattaaactgccgattgccaaaaaactgtataaagcacactacccagctggcaaaccaaaaagtgatgaacgtatt
	ctcactcttagccaaggccacagcctctgtgccctcgtacgatttcgcaccatcgagcagtatcgattgatgaccggcatgctggttcttccagacttc
	cgacaacaaggtgtcggtcatcatttcatggcacaactgcgacaaaatgagctgaccaacaacgatttttgttttgcgctagaacatttacagggatttt
	atgggcaacatggctttaaagtgattgcccaagaagagctaccaggtcctcttaagcaactgttttctcgttatattcacggcggaaaagcactggcag
	ctatgcgatacattgatccccgttaa
	>fig|6666666.207930.peg.357 Lipid A biosynthesis lauroyl acyltransferase (EC 2.3.1.-)

207930.peg.3055 Predicted hydrolase or acyltransferase

	>fig|6666666.207930.peg.2141 acetyltransferase, GNAT family
	atgtttgaagtgaaagagattgctcaagaacagttaccgttggatattttgttattggctgacccagaggaagccgcgattaacgtttatcgaggccgat
	gtcatgcttttgctgtgtttatgcaagataggttgattggggcaacgttaattgaagtggataaggttaatggtaaagctgaactgttcaatatcgcaacct
	acccagagtaccatggcaaaggatttggcggagttctacttgatgaaacattagcgaagttagcgagccgaggtattagaattgtcgaattaggaaca
	ggtacatttggttatcaactctcgttttatcaaaagcatggttttcgagtagagagagtgattaaagacttctttctcgataattatgatgaaccgatcttcga
	acacggtattcaacacaaagacatgttacgcctagtttggcgagcagaagagattgacgagtag
	>fig|6666666.207930.peg.2103 acetyltransferase, GNAT family
	ttgcgtaaatttgcaacgcttcctgattaccaaggccagggtattggctcacaggtcatcgagtttgtcttgaacatgcttgaaaggcaaaatattgagg
	tattttggtgcgatgcaagggagtctgcgctggatttctataagagatttggaatggttattgaaggagacatattttacaaaaaaggcgtggcatacaa
	aaaggcgtcgttagttttatcaacgctaagatatgagtag
	>fig|6666666.207930.peg.4904 Acetyltransferase, GNAT family
	atggcgacttaccgacgagacttcctattattgaatgagtttagatataccatgacttttgaaacactgcttgatacagctcgaattcaacttgatcacatta
	ccagtaatgatgcaggcttctttgttgagttgatgaacagtgaaggatggctcaaaaacataggtgatcgcaatgttcgtacgtcagatcaagctcaag
	agtatctaaccaacggtttcatcaaagcctatcaagaattgggctttgggtattatgttgtgcgcctcaaatcatgttcaacaccaatcggaatctgtggtt
	tcttaaagaaacccgacctgagttacccagatttcggctacgcgttcttgcccagctatcaaggtaaagggtatgcgcatgaggcttgtagcgaactg
	ctgacttttgccattgatgctttccaacttccagtgattgacgcggtatcactgccccaaaattcacccagcatacgattactcgaaaaactgggatttca
	gtacatcggcttacatcacactaatagcgacagtccactttctttatatcgcttgagcataaagtga
	>fig|6666666.207930.peg.2099 Acetyltransferase (EC 2.3.1.-)
	atgaataataccaaactaaggtttcaggatattaccttacgcaagacgtcagccgaagatctagaaccgctttatcaacttatgacggctgacgaaga
	gtggacgaaatacaacggcccttacttcggttatgttcggccaacactatccgaatttaagcagcaacagttccaaaagttcctagcaggcgatacaa
	gcttggttatagattatttggggcgagcaataggcagtgttagctactattgggaagatgaaaacacgcgttggcttgaagctggtgtggccatttacg
	actcatcactttggggtaggggcatcgggttgaaagcgctgattccatgggtggagcatctttttgcaacattggagatcgagcgagtaggctttacta
	cttggtctggaaaccctggaatgatgcgatgtgccgagaagcttggaatgaagcaagaggcaaggctgcgcaaagttcgctattacttaggtgagta
	ctttgattcgataaagtacggtgtcttgcgtgaagagtggcaagacctctacaagcgttggttgagtcgagaccgagtttacttgtttgattggggtgac
	actctaatggtcgatgaccctacacaaactggaaaaatgtgtgattggtcaaatgttgaagtagtttctggtgccgtggagttgttgtcagcgctttctaa
	gctgtatcccatttatgttgcgaacaatgcacaagattctaatgaaatcgatataaaacaggcatttgaacgtggtggtttagctcaatacatcacaggct
	acttctgttttaacaatcttggcgtttctaaaaataacccagaattctatcggctcatcgcaggcaagttaggtattccggcaaacaagttgacgatgacc
	ggcgatcaaagggacaaggacatagtacccgctaaatccgttggtttagtcaccaactggcttaattcgagtggtgtgtccgacccgtatgatgttgg
	cttcgcctcgttgcttgagatattagataaagaggtgaaggtcgagtaa
	atgagtgacaaaatttacttcaacacttctaataaattcagttttaaaaagcgcatgattaaccttacaacgcgcgctcatcataagttggcgcctaatcat >fig|6666666.
	gcgaagaacaccgcgaagaagctattgttgacgcctgttcgttctaaaccaaagaattctgagcctcagggattggtaaagggtgagattgcgacgc
	cagaaggaatgttaaagacatatcgtcttggtacaggtcctgtgtggattttaactcatggttggtctggtacggcaagccaattcttcccactaatgga
	gcacatcgcaagtcgaggatttactgcgcttgcgtatgaccatcctgctcatggtgagagtgaaggtgcgtttgggcatattcctgcctttgttggcggt
	ttagaaggtctgctggataatgaagacgatgttgctgggttagttgggcatagcatgggcacggcttcggcgattgaatgtaagcacgcgaaacttca
	tgataaacctttgctgcttatcgcgccggttctcgactatcttgataatttgtttggtagcgtcgcgcgttctggctattcgatgcgtttgtttgaagcggta
	gtcgatgatatccaaacccaatatcaataccctttgcaaaccatcgatccctataacaaattagcgcttcgcaatgcgccaactattattgttcatgatga
	aaaggatcgtttcactaaatattccgtgtcgaagaaagcagcgcaagagatggtcaatgtagagttggtcacgacacatggacaaggtcatggtcgc
	gttatgcaatgtcaggaggcaaaatcgagctttgataagctgattggtgtttaa
	>fig|6666666.207930.peg.2142 putative acetyltransferase [EC:2.3.1.-] [KO:K03830]
	atgcgattgtcggttagagcagcgacgcaagacgatgcaaatgagttattggaattatatcggcaaacaattcgcactgtaaatcgcaatgattacaat
	gagatacagattagagcgtgggctcgtgatgagttagagacatccgcttttgaagagtatattgctcgatgtaagccttttgtggtgacaaaccaaact
	cagatactgggttatagtgatttacaagccgatggattaatagaccatttctattgtcaccaccaagcacagaggcaaggggtagggagtgtgttgatg
	cagaccattatcaataaagcgcgagaagctgatatttgtaggctgtactcctatgtgagcattacagcaaagccgtttttccttcattttggctttaacgta
	gtgcgaccaaaccaagctgacattcgtggcgtcgtgttagataactatctgatggaaagagtattaaaagatgaataa

207930.peg.2116 Acetyltransferase (EC 2.3.1.-)

	>fig|6666666.207930.peg.5164 8-amino-7-oxononanoate synthase (EC 2.3.1.47) / CqsA
	atgcacttaacaaaaatcaagaaggctctcccagactttatttcacagaagcatgacttcttcatcgctgacagaattagttctaattcggaccagaggc
	cagttgtttgcggtggtcgcccatcaaagggtgacattgtgatgcaaacaaatgattatttgaatctcaatagtcatagcgaaatcaaggaagctcatat
	ccaaggaatcatggagaatgatgaaagcttgctgatgtctggcgtttacttactcggagccgaaaaagaaaccgacttcgaaactaagttagcaactt
	tgacggggtttgatagttgcttagttactcagtcaggttggacagcaaacgttaatttgcttcaagcaatatgcgataaagacaccaatgtatacgtagat
	ttctttgcccacgcctcactttgggaaggtgctcgtatcgcaggtgccaacgttcacatgtttatgcataacaacgtgcgtcatttaaagaagcaactga
	acaaacatggtagcggcattattcttgttgattctatctacagcacaagtggaactattgccccattagtcgatctggtctctcttgcttatgaatacgactg
	cgcactcgtcgttgatgaatcacattcattaggtactcatggaccaaatggtgcaggtctcgttgctgagctaggcctaaccgagcatgtcgactatat
	cactgctagtctagcgaagacctttgcgtacagagcaggtgctatacttgcaaacaacaagacaaattattgtgtcccaatggtcgcgtttcccgctatt
	tttagctccgcaatgttaccttatgaactaaaaaggctgagtaaaacgtttgatgtcatcgtcgctgccgatgagaagagaaagacattggcgaaaaat
	gcagattacctcaatcacaagttacgtgaaatagggtttagcattccgagtgagtcccagattattggcattgatactggtgatgttgaaaacactataa
	aagcgagagagtttttcgaagacaacggtatctttggctctgtttttgctccaccagctactccaaacggtagacatattctccgtttatccataaatgca
	gatcatacaattgaagagcttgcgagagtcgtaaatgtttgtcaaaaagcttgggacaatgaagagatagcgtttaagtaa
	atgcttactctagagacacctcgattgcagctgagacaaattggtcgtagtgacttcaagcttttccgactattacaccaagatccagagattatccgttt
	gtgttttgatgccccttcgatggacgaaattaaacaacgattcgaagagcgactaccggtttggcagccaggtagcgatcagtggttatgcatgataat
	tgtgttaaaaagtaatcaagctaaagtcggtgttacagggttcaaggtatcaaatcgtgttgcagaagtcggttacctcatgctcaccgaatatcaagg
	gatgggtattgctacagagtcgctaaaatgcgttctagattggggaatatcgcaactcgacgtctcaaagtttaatgctgtcgtaactcaaggtaatcat
	ggctccgaacgagtgctagagaaatgtggattccacattaaagcgatagagaaagaggcgtatgttatcggaggaaaaacgtacgacgatattattt
	atcagcgcaatgtgacgaagagaggagagtaa
	>fig|6666666.207930.peg.4938 Acetyltransferase (EC 2.3.1.-)
	atgaaaatcagaacagaaactcacgaagatattaaacagatccactcaataacatatcaagcatttgagaaccacccacatcacgaaccaggtgcga
	aaccaacagaacatctcattgttgacggacttcgtcgtgacaacgcacttactgtttctcttgtgtgcgaagccaataacgaggttatcggtcatattgc
	gttttcaccaatcctaatagatggtgaagcttcaacttgggttggattgggtcctgtatctgtcattccagagcatcaaggcaagggcataggcggagc
	gctgattcgagaaggggaagctcaaatcaaacaactcggttatcaaggcattgtgttacttggagacccaagttattacggacgctttggtttcgaatct
	catccaaacctaacgtttgcagatgtcccagcagaatacttcttggtgaaaggatttgagaatactatccctaaaggtgaagtaagctatcacaaggcg
	ttctttgaaacggcataa

Table S2 :

 S2 UHPLC-HRMS data of AHL Standards. Rt: Retention time. Theorical and observed mass correspond to the pseudo-molecular ion [M + H] + .

	AHL Standard	Molecular Formula Theorical Mass Observed Mass	Rt (min)
	C4-HSL	C 8 H 13 NO 3	172.0968	172.0968	5.26
	C6-HSL	C 10 H 17 NO 3	200.1281	200.1281	8.43
	OXO-C6-HSL	C 10 H 15 NO 3	214.1074	214.1072	7.56
	C7-HSL	C 11 H 19 NO 3	214.1438	214.1440	8.83
	C8-HSL	C 12 H 21 NO 3	228.1594	228.1594	9.27
	OXO-C8-HSL	C 12 H 19 NO 4	242.1387	242.1381	8.69
	OH-C8-HSL	C 12 H 21 NO 4	244.1543	244.154	8.55
	C9-HSL	C 13 H 23 NO 3	242.1751	242.1748	9.57
	C10-HSL	C 14 H 25 NO 3	256.1907	256.1907	9.90
	OXO-C10-HSL	C 14 H 23 NO 4	270.1700	270.1699	9.43
	OH-C10-HSL	C 14 H 25 NO 4	272.1856	272.1856	9.25
	C11-HSL	C 15 H 27 NO 3	270.2064	270.2063	10.13
	C12-HSL	C 16 H 29 NO 3	284.2220	284.2220	10.46
	OXO-C12-HSL	C 16 H 27 NO 4	298.2013	298.2013	10.04
	OH-C12-HSL	C 16 H 29 NO 4	300.2169	300.2169	9.87
	C13-HSL	C 17 H 31 NO 3	298.2377	298.2377	10.63
	C14-HSL	C 18 H 33 NO 3	312.2533	312.2533	10.93
	C14:1-HSL	C 18 H 31 NO 3	310.2377	310.2370	10.51
	OXO-C14:1-HSL	C 18 H 29 NO 4	324.2169	324.2170	10.23
	OXO-C14-HSL	C 18 H 31 NO 4	326.2326	326.2322	10.56
	OH-C14-HSL	C 18 H 33 NO 4	328.2482	328.2482	10.42
	C15-HSL	C 19 H 35 NO 3	326.2690	326.2689	11.15
	C16-HSL	C 20 H 37 NO 3	340.2846	340.2846	11.34
	C16:1-HSL	C 20 H 35 NO 3	338.2690	338.2704	10.93
	OXO-C16:1-HSL	C 20 H 33 NO 4	352.2482	352.2497	10.61
	C18-HSL	C 22 H 42 NO 3	368.3159	368.3155	11.66
	C18:1-HSL	C 22 H 39 NO 3	366.3003	366.3003	11.40

Table S3 :

 S3 Predicted retention times for Cx-HSL, oxo-Cx-HSL and OH-HSL. Based on retention times curves of AHL standards. NP: Non Predictable.

			Retention time (Rt, min)
	Acyl-side chain length (C atoms)	Cx-HSL oxo-Cx-HSL OH-Cx-HSL
	5	7,85	6,97	NP
	6	8,43	7,56	NP
	7	8,83	8,15	8,09
	8	9,27	8,69	8,55
	9	9,57	9,04	8,92
	10	9,90	9,43	9,25
	11	10,13	9,74	9,59
	12	10,46	10,04	9,87
	13	10,63	10,33	10,15
	14	10,93	10,56	10,42
	15	11,15	10,83	10,63
	16	11,34	11,06	10,85
	17	11,49	11,27	11,05
	18	11,66	11,47	11,24

Table S4 :

 S4 UHPLC-HRMS data and identification of unexpected AHLs in V. rotiferianus strains. Rt: Retention Time. Theorical mass correspond to the pseudo-molecular ion [M+H] + .

								Identification
	Strain	Rt (min)	Observed mass	Molecular Formula	Delta ppm	Fragmentation	Name	Molecular Formula	Molecular Weight	Theorical Mass
						56.050 (7.40), 57.071
						(100.00), 58.074 (5.11),
						74.024 (6.88), 74.061
		7,85	186,1126 C9H16NO3	0,090	(27.29), 84.045 (3.61),	C5-HSL	C9H15NO3	185,1052	186,1125
						85.065 (20.64), 102.055
						(30.89), 130.065 (0.54),
						146.0607 (0.93)
						74.061 (20.57), 81.070
	7H5-2					(24.68), 83.086 (17.88), 93.070 (15.67), 95.086
						(24.93), 102.055 (96.93),
						107.086 (16.39), 109.101
		9,52	286,2016 C15H28NO4	0,315	(20.49), 121.101 (13.07), 143.073 (16.38), 147.117	OH-C11-HSL	C15H27NO4	285,1940	286,2013
						(17.76), 149.132 (18.61),
						240.232 (100.00), 269.190
						(18.87), 285.298 (17.67),
						286.098 (21.13), 287.201
						(32.24)
						56.050 (6.79), 57.071
						(100.00), 58.074 (4.56),
						74.024 (6.77), 74.061
		7,85	186,1125 C9H16NO3	-0,010	(27.59), 84.045 (2.70),	C5-HSL	C9H15NO3	185,1052	186,1125
						85.065 (21.07), 102.055
						(29.44), 130.065 (1.41),
						146.060 (1.95)
						74.061 (20.47), 81.070
	8T6-4					(18.10), 83.086 (16.21), 93.070 (13.50), 95.086
						(16.67),
		9,52	286,201 C15H28NO4 -0,325

102.055 (100.00),

Table S5 :

 S5 Amino acids changes in V. rotiferianus 8T1-2 and 8T5-6.

	Strain	Protein Amino acid change(s)	Properties change(s)
			S123 → C	Hydroxylic → Sulfur-containing
		LuxM	H211
	8T1-2

  Rasmussen and colleagues have investigated AHL diversity among 32 Vibrio strains (13 species) isolated from diverse geographical locations as part of the global marine research cruise Galathea 3. However this study does not include closely related strains from a single location or same habitats. Thereby, AHL diversity is currently understudied for a large majority of Vibrio species, as well as for closely related species, and environmental factors driving AHL production in realistic ecological contexts is missing.To enhance our knowledge on AHL production patterns among Vibrio species a bioguided UHPLC-HRMS/MS methodology was developed. This thesis work focused on AHL diversity among (i) important species such as V. fischeri the model symbiotic bacteria associated to the squid Euprymna scolopes; and V. tasmaniensis LGP32 a model bacterium for the study of host-pathogens interactions in bivalves; (ii) dominant Vibrio species in the environment such as V. mediterranei; and (iii) closely-related Vibrio strains that share habitat preferences such as V. rotiferianus and V. fischeri. Our results revealed that: (i) Vibrio species can produce a large diversity of AHLs; (ii) different strains of single species isolated from a single ecological niche (e.g. V. fischeri and V. rotiferianus) show different AHL production patterns; (iii) this phenomenon was also observed at a finer phylogenetic resolutions between different isolates of a single strain (i.e. genotype, e.g. V. mediterranei and V. rotiferianus) and that the dynamic of different AHL production phenotypes was related to changes in environmental conditions (V. mediterranei).

Table S2: Raw data table of environmental variables.

ACKNOWLEDGEMENTS

First, I am very thankful to all the members of the jury for your time and for the evaluation of my work. I hope anyone of you will be offended by the fact that the following acknowledgements are in

ACKNOWLEDGEMENTS

We would like to gratefully acknowledge Professor Marti Anderson for her support with all the statistical analyses using PRIMER 7. We are thankful to Christophe Canal for the sampling campaigns at Leucate Lagoon, and for the physico-chemical analysis performed on water samples. We also thanks Jocelyne Caparros for the nutrients analysis and Nicole Batailler for her technical assistance in some steps of the experimental work. Finally, we thanks the Bio2Mar platform for providing access to the analytical chemistry and molecular biology equipments. We would like to thank Laurence Fonbonne from the "Syndicat RIVAGE" for her help and for giving us the access to the lagoon. This work was supported by a Ph.D. grant from the University Pierre et Marie Curie Paris VI and the Museum National d'Histoire Naturelle (MNHN), France.

ACKNOWLEDGMENTS

We thank the French Ministry of Education, for a PhD fellowship to Lea Girard through the Doctoral School 227 of the University Pierre et Marie Curie and the National Museum of Natural History, Paris. We thank the CNRS (grant EC2CO-ROSEOCOM) and the Sorbonne Universités (grant ENDOQUO) for funding this research. We are grateful to the BIO2MAR platform (http://bio2mar.obs-banyuls.fr) for access to instrumentation and particularly Karine Escoubeyrou for providing support technical aid. We thank Dr Didier Stein and Dr Alice Rodrigues for ther support in analytical chemistry and Nicole Batailler for her technical assistance in some steps of the experimental work.

Thompson, C.C.,

 Vicente, A., Souza, R.C., Vasconcelos, A., Vesth, T., Alves, N., Ussery, D.W., Iida, T., Thompson, F.L., 2009. Genomic taxonomy of vibrios. BMC Evol. Biol. 9, 258. Doi:10.1186/1471-2148-9-258Thompson, F.L., Austin, B., Swings, J.G., American Society for Microbiology (Eds.), 2006.

The biology of vibrios. ASM Press, Washington, D.C. Thompson, F.L., Gevers, D., Thompson, C.C., Dawyndt, P., Naser, S., Hoste, B., Munn, C.B., Swings, J., 2005 Thompson, F.L., Iida, T., Swings, J., 2004. Biodiversity of vibrios. Microbiol. Mol. Biol. Rev. 68, 403-431. Doi:10.1128/MMBR.68.3.403-431.2004 Thompson, F.L., Thompson, C.C., Vicente, A.C.P., Klose, K.E., AVIB Group, 2010.

Vibrio2009: the third international conference on the biology of vibrios. (2014).

Vibrio isolation and characterization

Water samples (0.1, 1, 10 mL) were filtered onto 0.45μm pore-size nitrocellulose filters (Millipore) and plated on the selective medium thiosulfate-citrate-bile-sucrose (TCBS) [START_REF] Pfeffer | A comparison of thiosulphate-citrate-bile salts-sucrose (TCBS) agar and thiosulphate-chloride-iodide (TCI) agar for the isolation of Vibrio species from estuarine environments[END_REF] for the enumeration of total Vibrio CFUs after 24 h incubation at 20°C. Vibrio strains were isolated from two fractions: less than 50 µm assumed to represent Vibrio in the water column and greater than 50 µm corresponding phytoplankton and zooplankton-associated Vibrio. The <50 µm fraction consisted in the concentration of 20 L of water prefiltered on 50 µm using a model HF80S hollow fiber filter (Hemoflow). The concentrated water was back-washed with 500 mL of a wash solutions (0.01% sodium hexametaphosphate, and 0.5% Tween 80; Sigma-Aldrich). The >50 µm fraction consisted of the plankton net tow concentrate split into five subsamples of 15 mL and homogenized by very gentle sonication for 5 min in order to unbind phytoplankton-attached bacteria. For both fractions, a ten-fold serial dilution was made in artificial seawater and 100 µL of each was spread and plated in triplicate on TCBS agar. Thirty colonies by fraction and by sampling date were randomly picked using a gridded Petri dish after 24 h of incubation at 20°C.

Isolate identification

A total of 840 Vibrio isolates were identified based on gyrB gene sequences. A PCR was performed using colony biomass directly. The amplification reactions consisted of 12.5 µL of Kappa2G Master Mix (KAPA2G Fast Hotstart ReadyMix PCR kit, KapaBiosystems), 6.25 µL of 10 mM MgCl2 and 1.25 µL of each of universal gyrB primers UPIE

(5'GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYRA 3') and UP2AR

(3'AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGYCAT 5') (Le Roux et al., 2004). Cryptophyta 7,56 x 10 5 1,65 x 10 6 4,73 x 10 5 8,61 x 10 5 1,05 x 10 6 1,87 x 5 9,37 x 10 2 2,81 x 10 3 NanoEuk 6,97 x 10 6 1,01 x 10 7 5,78 x 10 6 7,37 x 10 6 4,91 x 10 6 4,42 x 6 1,97 x 10 6 1,80 x 10 6 PicoEuk 1,66 x 10 7 2,35 x 10 7 1,53 x 10 7 4,95 x 10 6 6,40 x 10 6 2,22 x 6 9,58 x 10 5 1,84 x 10 5 Cyanobacteria 3,86 x 10 5 6,41 x 10 7 3,25 x 10 7 2,74 x 10 7 4,92 x 10 7 6,81 x 7 1,08 x 10 8 1,23 x 10 8 Bacteria 4,74 x 10 9 7,52 x 10 9 3,53 x 10 9 2,14 x 10 10 6,11 x 10 9 1,34 x 10 10 1,20 x 10 10 1,13 x 10 10 the 7 remaining populations where affiliated to Vibrio sp. as no significant similarity where found with type strains. Most of the populations were well resolved while high sequence similarity was observed within the clade V. splendidus (#13 to 22).

SUPPLEMENTARY INFORMATION

Phylogenetic analysis.

V. rotiferianus species tree was reconstructed using a concatenated nucleotide alignment of 4076 core unicopy families. Families were determined by performing all-vs-all blastp using the proteome of each strain and proteins were grouped in the same family if they aligned with at least 80% identity on at least 80% of their respective length. The nucleotide sequence of genes encoding these proteins were aligned with Muscle [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF] and filtered with Gblocks [START_REF] Castresana | Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis[END_REF]. Phylogenetic tree was reconstructed with PhyML [START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0[END_REF] with the following parameters: GTR model of evolution, estimation of invariant sites, NNI topological search and node support was assessed with SH-like test.

Detection of QS active strains

AHL and AI-2 biosensors assays followed previously described protocols.

Pseudomonas putida F117 (pRK-C12 ; Kmr; ppuI::npt) was used for the detection of longchain AHLs, Escherichia coli MT102 (pJBA132) and Chromobacterium violaceum for the detection of short chain AHLs [START_REF] Andersen | gfp-based N-acyl homoserine-lactone sensor systems for detection of bacterial communication[END_REF]Girard et al., 2017;[START_REF] Riedel | N-acylhomoserine-lactone-mediated communication between Pseudomonas aeruginosa and Burkholderia cepacia in mixed biofilms[END_REF][START_REF] Surette | Quorum sensing in Escherichia coli and Salmonella typhimurium[END_REF]. V. harveyi BB170 (sensor1 -, sensor2 + ) was used for AI-2 detection (Surette andBassler, 1998, 1999). Vibrio strains were grown in Marine Broth (MB) at 25°C

for 24h under shaking condition and culture supernatants were tested in triplicate for each assay.

AHL Identification by UHPLC-HRMS/MS

We investigated AHL diversity by a non-targeted UHPLC-HRMS/MS method previously described by Girard et al., 2017

CHAPITRE 5 Discussion & Perspectives

lifestyles of Vibrio in the environment. It is well known that the distribution of microorganisms as well as Vibrio spp. does not occur evenly and that specific populations appear to be associated to particular niches (Acinas et al., 2004;Chimetto Tonon et al., 2015;Hunt et al., 2008). In fact, members of Vibrio spp. can thrive in the environment as freeliving, attached to particles or higher organisms (Preheim et al., 2011), as primary biofilms colonizers [START_REF] Huang | Utilization of acyl-homoserine lactone quorum signals for growth by a soil Pseudomonad and Pseudomonas aeruginosa PAO1[END_REF], or able to find refuge in the sediment when conditions are unsuitable [START_REF] Kaneko | The annual cycle of Vibrio parahaemolyticus in Chesapeake bay[END_REF]) (Figure 5.3). Within this kind of highly heterogeneous localized niches where chemistry can vary drastically it is unlikely that the same rules are affecting AHL concentrations. It was then proposed that AHL concentration is depending on production rate, degradation rate or half-life of the signal molecules, diffusion properties and mass transfer properties [START_REF] Alberghini | Consequences of relative cellular positioning on quorum sensing and bacterial cell-to-cell communication[END_REF]Hense et al., 2007).