Eugénie Grigorian

Agnès Groisillier #

François Thomas

Catherine Leblanc

Ludovic Delage
email: ludovic.delage@sb-roscoff.fr.

Correspondence Ludovic

Functional characterization of a L-2-haloacid dehalogenase from Zobellia galactanivorans suggests a role in haloacetic acid catabolism and a wide distribution in marine environment

Je suis de ceux qui pensent que la science est d'une grande beauté. Un scientifique dans son laboratoire est non seulement un technicien : il est aussi un enfant placé devant des phénomènes naturels qui l'impressionnent comme des contes de fées » Marie Curie

Z. galactanivorans membrane fractions (5 deposits). In the corresponding gel charged with radioactive fractions, the bands #1, #2 and #3 showed the most intense signal and then cut in the non-radioactive gel.

The 125 I -radioactivity values correspond to the mean and standard deviation of five independent measurements for each. …………………………………………………………………………….……163 Figure 4-6: 3D model representation of zobellia_1580 OmpA-like protein based on CgOmpA (PDB accession: 5WTL). The overall structure is colored in turquoise and shown as ribbons. The Tyr425 is colored in red and shown as sticks. ……………………………………………………………………………...………165 Figure S4-1: 125 I -radioactivity measured on the mutant strain membrane fraction, migrated on a SDS-PAGE gel and placed on miniGita. …………………………………………………………………………….……171 Figure S4-2: 125 I -radioactivity measured on the wild-type strain membrane fraction, migrated on a SDS-PAGE gel and placed on miniGita. ……………………………………………………..………………………171 Figure 5-1: Hypothetical model of halogen-related metabolism within Z. galactanivorans (adapted from Fournier, 2014)…………………………………………………………………………………………………………..…176 Table 0-1: Abundance of halogen elements in the Earth's crust and the oceans [START_REF] Greenwood | Chemistry of The Elements[END_REF][START_REF] Guillory | CRC Handbook of Chemistry and Physics. 90 th Edition[END_REF]…………….…………………………………………………………………………..…..12 Table 0-2: Some examples of biochemically and structurally characterized halogenating enzymes…….………18 Table 0-3: Structural characterization of vanadium-dependent haloperoxidases (adapted from [START_REF] Leblanc | Vanadium haloperoxidases: From the discovery 30 years ago to X-ray crystallographic and V K-edge absorption spectroscopic studies[END_REF]. ………………………………………………………………………………………………………………….…27 Table 0-4: Biochemically and structurally characterized L-2-HADs enzymes from bacteria…………….….33-34 Table 3-1: Kinetic parameters of Zg-VIPO1, and Zg-VIPO2 (Fournier et al., 2014). …………………………128 Table 3-2: Kinetic parameters of Zg-VIPO1, VIPO2 and VIPO3 (Fournier et al., 2014) S4-1: Predictions of Zg-VIPOs subcellular protein localization using 7 different generalist tools developed to discriminate the sorting between 4 or 5 compartments (membranes are not dissociated for ProtComb whereas outer and inner membranes are separated in others). Resulting localization or probability scores are reported in the table. The best prediction of Zg-VIPOs are mentioned in bold in each line and in bold and underlined in the total predictions column. Potential secondary compartments are shown in bold in this latter column. Legend: not determined (nd). ………………………………………………………..……169 Table S4-2: Predictions of the presence/absence of signal peptides (SP) and the type of these signals in Zg-IPOs protein sequences using 12 different specialized tools. Results are reported with the score probability, the location of cleavage site when found and the length of the signal peptide in amino acids. The best predictions are in bold in each line and are summarized in the total predictions column. Legend: type I signal peptide (SPI), type II signal peptide (SPII), not determined (nd). …………………………………170

List of Tables

General introduction

1. Biogeochemical cycle of halogens

Definition of halogens

Fluorine (F), chlorine (Cl), bromine (Br) and iodine (I) belong to the group of halogens, located on the 17 th column of the periodic table. Astatine (At) is at the intersection between halogen and metalloid elements, highly unstable, and not considered as being part of the halogen group.

The word "halogen" means "salt-producing". These chemical elements produce salts by reacting with metals, for example: calcium fluoride (CaF2), sodium chloride (NaCl), silver bromide (AgBr) or potassium iodide (KI) [START_REF] Greenwood | Chemistry of The Elements[END_REF].

Halogens are natural chemical elements that are heterogeneously distributed between different Earth system reservoirs. Because of their reactivity, halogens do not occur in free elemental state, but are more often found in ionic forms (e.g., X -, where X is either Cl, Br or I). They can also be found as diatomic molecules (F2, Cl2, Br2, I2) and these gaseous forms can be highly reactive, oxidizing and toxic to organisms at high concentrations [START_REF] Greenwood | Chemistry of The Elements[END_REF]. Chlorine, bromine and iodine are indeed often used as disinfectants; their toxicity depends on their speciation and concentration levels. Halogens can also form bonds with carbon, producing organic halogenated compounds. Those are widely diverse and come from biogenic sources as well as anthropogenic.

Inorganic halogenated compounds

The inorganic halogenated compounds are divided between gaseous halogen molecules (F2, Cl2, Br2 and I2), ionic forms, and halogen salts, and their total concentrations vary by several order of magnitude, depending of halogen type and natural sources (Table 1). Globally, fluorine-and iodine-containing products are the least abundant found on Earth, even though fluorine is the richest halogen found in the Earth's crust [START_REF] O'hagan | Fluorine-containing natural products[END_REF][START_REF] Gribble | The diversity of naturally produced organohalogens[END_REF]. The least abundant halogen in the Earth's crust and in oceans is iodine, whereas chlorine is the most abundant in oceans (Table 1). [START_REF] Greenwood | Chemistry of The Elements[END_REF][START_REF] Guillory | CRC Handbook of Chemistry and Physics. 90 th Edition[END_REF].

Earth's crust

The most widespread chemical species of chlorine on the Earth's crust is the inorganic halide mineral, sodium chloride (NaCl). Similarly, NaCl is the most abundant inorganic halogen found in oceans, salt lakes, brines, and chloride ions represent half of the ocean total salinity [START_REF] Greenwood | Chemistry of The Elements[END_REF][START_REF] Atashgahi | Microbial synthesis and transformation of inorganic and organic chlorine compounds[END_REF]. The bromine inorganic species are less abundant than the chlorine and fluorine ones in crustal rocks, however they are more abundant in ocean waters (Table 01). Regarding iodine, it is very rare on Earth but it is mostly found in oceans as inorganic trace species (~0.5 µM) [START_REF] Wong | Seasonal variations in the speciation of dissolved iodine in the Chesapeake Bay[END_REF]. In the ocean, dissolved inorganic iodine exists as iodate (IO3 -) (< 0.1 to 0.45 µM) and as iodide (I -) (< 0.01 to 0.3 µM) depending on the pH and the temperature of waters. The iodide is concentrated in surface waters, whereas the iodate dominates in deeper waters [START_REF] Wong | Seasonal variations in the speciation of dissolved iodine in the Chesapeake Bay[END_REF][START_REF] Schwehr | Sensitive determination of iodine species, including organo-iodine, for freshwater and seawater samples using high performance liquid chromatography and spectrophotometric detection[END_REF].

Organic halogenated compounds

Halogens can form covalent bonds with carbon, C-F being the strongest and least chemically reactive. The bond strength weakens and the reactivity increases moving down the halogen column of the periodic table, meaning that iodinated organic compounds are the most instable among natural halogenated compounds [START_REF] Greenwood | Chemistry of The Elements[END_REF]. The organic halogenated compounds can derive from human activities, geochemical processes or biogenic sources. They can also be volatile and emitted in the atmosphere.

Over 5000 halogenated organic compounds (HOCs) have been identified until now, and they constitute one of the largest groups of natural chemical products (for review, [START_REF] Gribble | Biological activity of recently discovered halogenated marine natural products[END_REF].

These halogenated organic compounds are structurally and chemically very diverse. For example, polyvinyl chloride (PVC), a widely used polymer in plastic production, represents the largest single end-use of chlorine. Other industrial halogenated compounds (such as chlorinated hydrocarbons), can be used as solvents, biocides, flame retardants, fumigants, degreasers and chemical intermediates. Since the 1960s, it is publicly known that the dissemination of those compounds in the environment results in terrestrial, aquatic and atmospheric contaminations which strongly impact the functioning and the health of ecosystems (for review, [START_REF] Häggblom | Halogenated Organic Compounds -A Global Perspective[END_REF].

The HOCs are also naturally produced by a wide diversity of living organisms such as bacteria, plants, fungi, algae, lichens, insects and mammals [START_REF] Siuda | Naturally occurring halogenated organic compounds[END_REF][START_REF] Van Pée | Biosynthesis of Halogenated[END_REF][START_REF] Gribble | Naturally Occurring Organohalogen Compounds[END_REF][START_REF] Gribble | The diversity of naturally occurring organobromine compounds[END_REF]; as well as during abiotic natural processes like volcanic emissions, forest fire and other pyrogenic processes [START_REF] Jordan | Volcanogenic halocarbons[END_REF][START_REF] Gribble | The diversity of naturally produced organohalogens[END_REF]. Soil, sediments, groundwater, salt lakes and oceans have been found to be the major source of volatile halogenated organic compounds (VHOCs) emission [START_REF] Asplund | Naturally produced adsorbable organic halogens (AOX) in humic substances from soil and water[END_REF][START_REF] Keppler | Halocarbons produced by natural Oxidation processes during degradation of organic matter[END_REF][START_REF] Weissflog | Sediments of salt lakes as a new source of volatile highly chlorinated C1/C2 hydrocarbons[END_REF][START_REF] Ruecker | Predominance of biotic over abiotic formation of halogenated hydrocarbons in hypersaline sediments in Western Australia[END_REF]. Most chlorinated and fluorinated organic compounds come from terrestrial anthropogenic sources, whereas most iodinated and brominated organic compounds derived from marine biogenic sources [START_REF] Gribble | The diversity of naturally produced organohalogens[END_REF][START_REF] Ballschmiter | Pattern and sources of naturally produced organohalogens in the marine environment: Biogenic formation of organohalogens[END_REF]. In terrestrial environments, the forest soils are known as a main source of volatile organochlorine compounds including chloromethane, chloroform and other alkylchlorides [START_REF] Keppler | Halocarbons produced by natural Oxidation processes during degradation of organic matter[END_REF]. The ocean is a major source of VHOCs such as methyl halides (CH3X where X is either Cl, Br or I), and a large majority of these biogenic compounds are produced by a diversity of marine organisms [START_REF] Brownell | Production of methyl halides by Prochlorococcus and Synechococcus[END_REF][START_REF] Paul | Production and role of volatile halogenated compounds from marine algae[END_REF][START_REF] Sato | Methyl halide production by cultures of marine thraustochytrids, Aurantiochytrium sp., Botryochytrium radiatum, and Schizochytrium sp[END_REF]. It is essential to consider all those diverse biotic and abiotic sources in the global halogen biogeochemical cycle.

Biogeochemical cycle of halogens

Both land-and marine-emitted volatile organic and inorganic halogenated compounds participate to a complex biogeochemical cycle (Figure 0-1) leading to the formation of highly reactive halogen radicals in the atmosphere [START_REF] O'dowd | Marine aerosol formation from biogenic iodine emissions[END_REF]. Those are responsible of destroying tropospheric ozone (O3), a greenhouse gas that is very toxic to humans, animals and plants when present in the low atmosphere layers [START_REF] Lazrus | Stratospheric halogen measurements[END_REF][START_REF] Carpenter | Iodine in the marine boundary layer[END_REF]. The iodinated volatile compounds such as methyl iodide (CH3I) and diiodomethane (CH2I2) could contribute significantly to ozone depletion in the marine boundary layer [START_REF] Carpenter | Update on Ozone-Depleting Substances (ODSs ) and Other Gases of Interest to the Montreal Protocol[END_REF].

Inorganic iodine compounds like hypoiodous acid (HIO) and I2 are also an important source of atmospheric iodine and participate to tropospheric ozone destruction [START_REF] Carpenter | Atmospheric iodine levels influenced by sea surface emissions of inorganic iodine[END_REF].

After reacting with ozone, they can form iodine oxide species (OIO and IO) that result to new aerosols particles, which are precursors to cloud condensation nuclei [START_REF] O'dowd | Marine aerosol formation from biogenic iodine emissions[END_REF][START_REF] Saiz-Lopez | Boundary layer halogens in coastal Antarctica[END_REF][START_REF] Küpper | Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry[END_REF]. During rainfall, halogenated compounds reach soils where they can accumulate and/or be metabolized by living organisms. They can also return to oceans through runoff waters and underground rivers. The marine macroalgae contribute approximately to 70% of the total atmospheric bromoform (CHBr3) [START_REF] Carpenter | On temperate sources of bromoform and other reactive organic bromine gases[END_REF]. The annual global production of methyl iodide (CH3I) is estimated 1-4 x 10 11 g/year [START_REF] Moore | Methyl iodide distribution in the ocean and fluxes to the atmosphere[END_REF], but the macroalgal contribution is less than 0.2% [START_REF] Laturnus | Ultraviolet radiation affects emission of ozone-depleting substances by marine macroalgae: Results from a laboratory incubation study[END_REF]. The marine phytoplankton and the cyanobacteria are the primary contributors of CH3I emissions in the atmosphere with 1.2 to 38 x 10 9 g/year produced, followed by the bacterial production with 1.6 x 10 9 g/year [START_REF] Moore | Methyl iodide distribution in the ocean and fluxes to the atmosphere[END_REF][START_REF] Amachi | Microbial Contribution to Global Iodine Cycling: Volatilization, Accumulation, Reduction, Oxidation, and Sorption of Iodine[END_REF][START_REF] Brownell | Production of methyl halides by Prochlorococcus and Synechococcus[END_REF].

The marine macroalgae are also known to be strong accumulators of iodine in the case of brown algae, and especially Laminariales [START_REF] Küpper | Iodine uptake in Laminariales involves extracellular, haloperoxidase-mediated oxidation of iodide[END_REF] and, in a lesser extent, of bromine in the case of red algae [START_REF] Latham | Temperature stress-induced bleaching of the coralline alga Corallina officinalis: A role for the enzyme bromoperoxidase[END_REF]. The red and brown macroalgae are therefore important biogenic producers of halogenated compounds in the coastal marine areas [START_REF] Carpenter | On temperate sources of bromoform and other reactive organic bromine gases[END_REF][START_REF] Laturnus | Marine macroalgae in Polar Regions as natural sources for volatile organohalogens[END_REF]. For example, red algal species of Asparagopsis or Laurencia genera are the source of more than 100 organohalogens (for review, [START_REF] Gribble | Biological activity of recently discovered halogenated marine natural products[END_REF]. Moreover, Fucus vesiculosus and Ascophyllum nodosum, two brown algal species belonging to Fucales, have been found to be strong emitters of molecular iodine [START_REF] Huang | The seaweeds Fucus vesiculosus and Ascophyllum nodosum are significant contributors to coastal iodine emissions[END_REF]. The Laminaria genus is known to be producer of organic (methyl iodide, diiodomethane) but mainly inorganic iodinated compounds above seaweed beds [START_REF] Carpenter | Iodine in the marine boundary layer[END_REF][START_REF] Leblanc | Iodine transfers in the coastal marine environment: the key role of brown algae and of their vanadium-dependent haloperoxidases[END_REF]. For these kelps, inorganic compound emissions, essentially I2, actually dominate over the organic species by at least five orders of magnitude [START_REF] Küpper | Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry[END_REF].

Biological importance of halogen metabolism in marine organisms

Biotic stress responses and halogenated compounds

The marine organisms are exposed to biotic stresses from herbivores, bacteria, fungi, virus, during their life cycle. Naturally produced halogenated compounds are suggested to be involved in chemical defense, even though biological or ecological roles have been established for few of them. For example, the red alga Asparagopsis taxiformis released brominated compounds, mahorone and 5-bromomahorone, which were found to be highly toxic to the marine bacterium Vibrio fisheri [START_REF] Greff | Mahorones, highly brominated cyclopentenones from the red alga Asparagopsis taxiformis[END_REF]. The bromoform and dibromoacetic acid, both natural products produced by the red alga Asparagopsis armata, were shown to be efficient antibacterial compounds [START_REF] Paul | Chemical defence against bacteria in the red alga Asparagopsis armata: Linking structure with function[END_REF]. The halophenols and haloterpens are other secondary metabolites produced by red algae, and potential herbivore deterrents [START_REF] Hay | Marine plant-herbivore interactions: the ecology of chemical defense[END_REF].

Fewer brominated metabolites have been identified in brown algae, but some members of this group, such as Laminariales, also featured iodinated compounds (for review, La Barre et al., 2010). Laminaria digitata can accumulate iodine at an average of 30,000-fold the seawater concentration. The cyanobacteria Leptolyngbya crossbyana produces an organic chlorinated compound that inhibits quorum sensing against Vibrio harveyi [START_REF] Choi | Honaucins A-C, Potent Inhibitors of Inflammation and Bacterial Quorum Sensing Synthetic Derivatives.pdf[END_REF]. The brominated compounds, synthesized by the green alga Ulvella lens, inhibit the growth of sea urchins [START_REF] Agatsuma | Inhibitory effect of 2,4-dibromophenol and 2,4,6-tribromophenol on larval survival and metamorphosis of the sea urchin Strongylocentrotus nudus[END_REF]. Many of those marine halogenated compounds showing strong biological activities using laboratory bioassays have been investigated in the research field of natural product chemistry, to develop drugs against a wide range of bacterial and fungal pathogens but also for anti-tumoral or anti-inflammation applications (for review, [START_REF] Gribble | Biological activity of recently discovered halogenated marine natural products[END_REF].

Oxidative stress responses and iodine metabolism

As said above, molecular iodine emissions by kelps have been shown to be also related to environmental oxidant stress such as changes in temperature, salinity, nutrient depletion, UV radiation, especially during low tide and desiccation [START_REF] Laturnus | Ultraviolet radiation affects emission of ozone-depleting substances by marine macroalgae: Results from a laboratory incubation study[END_REF][START_REF] Mata | Carbon/nutrient balance in relation to biomass production and halogenated compound content in the red alga Asparagopsis taxiformis (bonnemaisoniaceae)[END_REF][START_REF] Leedham | Emission of atmospherically significant halocarbons by naturally occurring and farmed tropical macroalgae[END_REF]. Releases of iodide have also been observed in L. digitata, Fucus serratus and Kallymenia antarctica upon stress [START_REF] Gall | A survey of iodine content in Laminaria digitata[END_REF][START_REF] Truesdale | The biogeochemical effect of seaweeds upon close-to natural concentrations of dissolved iodate and iodide in seawater -Preliminary study with Laminaria digitata and Fucus serratus[END_REF][START_REF] Chance | Release and transformations of inorganic iodine by marine macroalgae[END_REF].

The release of halogenated compounds by the red alga Gymnogongrus antarcticus was highly affected after its exposure to different environmental conditions [START_REF] Laturnus | Low-molecular-weight organoiodine and organobromine compounds released by polar macroalgae -The influence of abiotic factors[END_REF].

Endogenous defense elicitors such as oligoguluronates, applied to the kelp L. digitata, induce an oxidative burst, i.e. a strong production of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), hypothesized to be part of antibacterial defense mechanisms [START_REF] Küpper | Oligoguluronates elicit an oxidative burst in the brown algal kelp Laminaria digitata[END_REF]. In addition, Laminariales seem to feature a unique capability to use accumulated iodine as antioxidant following biotic or abiotic stresses (for review, La Barre et al., 2010). The massive release of iodine, mainly I2, was proposed to have defense and antioxidant roles in brown algae during low tides [START_REF] Palmer | Iodine and halocarbon response of Laminaria digitata to oxidative stress and links to atmospheric new particle production[END_REF][START_REF] Küpper | Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry[END_REF][START_REF] Ball | Spectroscopic studies of molecular iodine emitted into the gas phase by seaweed[END_REF]. In L.

digitata, the iodide (I -) is accumulated in the cell wall of peripheral algal tissues (Verhaeghe et al. 2008b) and could act as an efficient antioxidant protection by detoxifying the excess of hydrogen peroxide and other ROS on kelp surfaces, producing oxidized iodine species, such as I2. Moreover, the defense elicitation seemed to specifically activate an iodine-related metabolism in the brown alga L. digitata, as observed by transcriptomic regulations of vanadium haloperoxidase enzymes (VHPO, cf. part 4) [START_REF] Cosse | Patterns of gene expression induced by oligoguluronates reveal conserved and environment-specific molecular defense responses in the brown alga Laminaria digitata[END_REF]. According to [START_REF] Strittmatter | Infection of the brown alga Ectocarpus siliculosus by the oomycete Eurychasma dicksonii induces oxidative stress and halogen metabolism[END_REF], when the brown alga Ectocarpus siliculosus is infected by Eurychasma dicksonii, the halogen metabolism is also upregulated in response to the pathogen attack. A bromoperoxidase was excreted after production of H2O2, and was the strongest upregulated protein during the infection. Similarly, the study of iodine-related metabolism in the marine microalga Tisochrysis lutea showed that a putative haloperoxidase might be involved in detoxifying ROS during an oxidative stress [START_REF] Javier | Characterization of Iodine-related molecular processes in the marine microalga Tisochrysis lutea (Haptophyta)[END_REF]. All these examples suggest a putative key role of halogen-related metabolism during protection against biotic and abiotic stresses in marine algae, with particular defense mechanisms in Laminariales, related to iodine metabolism.

Halogen-related metabolism in bacteria

A wide diversity of bacteria lives on the surfaces of macroalgae as part of their associated microbiome. The influence of the iodinated exudates of the kelp L. digitata on bacterial adhesion and biofilm formation was investigated by [START_REF] Salaün | Influence of Exudates of the Kelp Laminaria Digitata on Biofilm Formation of Associated and Exogenous Bacterial Epiphytes[END_REF] Even though all the bacteria used for the experiments were isolated directly from this species, the impacts of those exudates were different depending on bacterial strains and chemical nature of tested compounds. In some cases, the biofilm growth decreased (Pseudomonas sp. LD6), or on the contrary it was induced (Zobellia galactanivorans). Some bacteria strains appeared to possess adaptive traits to survive on those particular algal surfaces: for instance, Z. galactanivorans could use mannitol, a potential anti-fouler, as carbon source [START_REF] Salaün | Influence of Exudates of the Kelp Laminaria Digitata on Biofilm Formation of Associated and Exogenous Bacterial Epiphytes[END_REF].

It was previously assessed that methyl halide production by marine bacteria was of main importance as they were the second major producers [START_REF] Amachi | Microbial Contribution to Global Iodine Cycling: Volatilization, Accumulation, Reduction, Oxidation, and Sorption of Iodine[END_REF]. For instance, marine

Proteobacteria such as Erythrobacter and Pseudomonas were detected as sources of methyl

halides in the open ocean [START_REF] Fujimori | Methyl halide production by cultures of marine Proteobacteria Erythrobacter and Pseudomonas and isolated bacteria from brackish water[END_REF], and the emission rates were impacted by the water temperature [START_REF] Hirata | Effect of temperature on the production rates of methyl halides in cultures of marine Proteobacteria[END_REF]. Unfortunately, it is still not fully understood which bacteria and which enzymatic pathways are contributing to those emissions. Few studies have been dedicated to explore bacterial iodine metabolism. Some Proteobacteria, such as Pseudomonas sp. strain SCT, participate to the cycle of iodine by reducing IO3 -into I - [START_REF] Councell | Microbial reduction of iodate[END_REF][START_REF] Amachi | Dissimilatory Iodate Reduction by Marine Pseudomonas sp . Strain SCT[END_REF], while some α-Proteobacteria, such as Iodidimonas sp. Q-1, are able to oxidize I -into I2 (Amachi et al., 2005;[START_REF] Suzuki | Iodide oxidation by a novel multicopper oxidase from the Alphaproteobacterium strain Q-1[END_REF]. Furthermore, two strains of Roseovarius spp. have been described to emit methyl iodide (CH3I) and diiodomethane (CH2I2) thanks to GC-MS analysis [START_REF] Fuse | Production of free and organic iodine by Roseovarius spp[END_REF]. Both iodine-reducing and iodineoxidizing bacteria seem to be major contributors to the iodine cycle, but their exact global

implication is yet to be discovered (for review, [START_REF] Yeager | Microbial Transformation of Iodine: From Radioisotopes to Iodine Deficiency[END_REF].

Enzymatic mechanisms involved in halogenation and dehalogenation

The biological uses of halogens are not widespread in the tree of life, but some emblematic examples of this particular metabolism have been described in specific lineages. For instance, the mammalian thyroid system is known to efficiently concentrate iodine through specific iodide symporters and is the place of biosynthesis of iodinated thyroid hormones, involved in essential physiological regulations (for review, [START_REF] Rousset | Thyroid hormone synthesis and secretion. Endotext[END_REF]. During the biosynthesis of halogenated organic compounds (HOCs), the halogenation step corresponds to a chemical reaction binding halogen and carbon atoms and requires specific enzymatic mechanisms, which are diverse and have emerged independently in different lineages, such as those of animals, fungi, red and brown algae, and bacteria (for review, Agarwal et al., 2017). Another specialized enzymatic process is the dehalogenation which cleaves the carbon-halogen bond and leads to the recycling of halogens and/or the detoxification of HOCs. The enzyme-based microbial degradations of organohalogens have been reviewed in a large number of papers since the last 30 years. It has been established for instance that some terrestrial bacteria can use organohalogens for growth as a carbon source [START_REF] Fetzner | Bacterial dehalogenation[END_REF][START_REF] Goodwin | Bacterial oxidation of dibromomethane and methyl bromide in natural waters and enrichment cultures[END_REF], or as terminal electron acceptor for respiration [START_REF] Mccarthy | Identification of a covalent intermediate between glutathione and cysteine13 formed during catalysis by tetrachlorohydroquinone dehalogenase[END_REF][START_REF] Atashgahi | Microbial synthesis and transformation of inorganic and organic chlorine compounds[END_REF].

Organohalogens degradation by dehalogenases was also investigated for potential bioremediation applications (for review, Kurihara & Esaki 2008;[START_REF] Jugder | Reductive Dehalogenases Come of Age in Biological Destruction of Organohalides[END_REF]. Both halogenation and dehalogenation enzymatic mechanisms are presented in the following paragraphs.

Halogenating enzymes

Six groups of halogenating enzymes have been described based on their enzymatic mechanisms and cofactor specificity (for review, [START_REF] Xu | Independent evolution of six families of halogenating enzymes[END_REF]. Some examples are listed in The first group is represented by the flavin adenine dinucleotide (FAD)-dependent halogenases.

They are divided in two groups: those acting on free halide (usually chloride) and those acting on substrates tethered to a carrier protein (Ludewig et al., 2020). They were mostly isolated from several bacteria, but recently two putative fungal FAD-dependent halogenases have been reported to be involved in biosynthesis of halogenated metabolites [START_REF] Ferrara | Identification of a halogenase involved in the biosynthesis of ochratoxin A in Aspergillus carbonarius[END_REF][START_REF] Han | Genome-and MS-based mining of antibacterial chlorinated chromones and xanthones from the phytopathogenic fungus Bipolaris sorokiniana strain 11134[END_REF]. In addition, the first viral FAD-dependent halogenase has been described recently from a marine virus [START_REF] Gkotsi | A marine viral halogenase that iodinates diverse substrates[END_REF]. Despite having a wide diversity of substrates like indoles, pyrroles, phenols or aliphatic compounds [START_REF] Phintha | Dissecting the low catalytic capability of flavin-dependent halogenases[END_REF], the catalytic mechanism is highly conserved and involves two separated and distant binding sites for FAD cofactor and substrate. The catalytic reaction is dependent of the FAD cofactor that needs to be firstly reduced in FADH2 by a flavin-reductase, then undergoes a reaction with a water molecule to form a flavin-C(4a)-hydroperoxide intermediate [START_REF] Yeh | Flavin redox chemistry precedes substrate chlorination during the reaction of the flavindependent halogenase RebH[END_REF]. A chloride anion then reacts with FADH2 to form the corresponding hypochlorous acid (HOCl). This reactive HOCl species then moves through a tunnel from the FAD-binding site to the active site, where it interacts with a catalytic lysine residue to form a chloramine intermediate, followed by a regiospecific delivery to aromatic (tryptophan) residue (Figure 0-2A-C) (Ludewig et al., 2020).

The second group corresponds to the S-adenosyl-L-methionine (SAM)-dependent halogenases, which use a nucleophilic attack mechanism. These enzymes do not need to activate the halide by oxidation, but use SAM as a cofactor. Two classes of those enzymes are known: the 5'-halo-5'-deoxyadenosine synthases (5'-FDAS) and the halide methyl-transferases. The 5'-FDAS from Streptomyces cattleya is the only known fluorinating enzyme, which participates in the formation of fluoroacetate and fluorothreonine. It catalyzes the formation of C-F bond using a nucleophilic substitution on the C-5' carbon of the SAM co-substrate [START_REF] Dong | Crystal structure and mechanism of a bacterial fluorinating enzyme[END_REF][START_REF] Zhu | Mechanism of enzymatic fluorination in Streptomyces cattleya[END_REF]. The halide methyl-transferases catalyze a nucleophilic substitution reaction between SAM and halide ions to form methyl halides and S-adenosylhomocysteine (SAH)

(Figure 0-2D).
The third group comprises the non-heme iron-dependent halogenases, a class of highly homologous enzymes that halogenate an inactivated aliphatic carbon center, using non-heme iron (Fe 2+ ), O2 and α-ketoglutarate as cofactors. It is the only group of haloperoxidases that catalyzes reactions of radical halogenation (Ludewig et al., 2020). Those enzymes have been demonstrated to specifically catalyze the chlorination and bromination of organic compounds, and their resulting products are currently known to be amino acids and indole alkaloids derivatives (Figure 0-3A) [START_REF] Vaillancourt | Cryptic chlorination by a non-haem iron enzyme during cyclopropyl amino acid biosynthesis[END_REF][START_REF] Voss | Exploring the Biocatalytic Potential of Fe/α-Ketoglutarate-Dependent Halogenases[END_REF]. For instance, in Pseudomonas syringae, this enzyme is involved in the chlorination of threonine during the biosynthesis of syringomycin E, a phytotoxic molecule [START_REF] Vaillancourt | Cryptic chlorination by a non-haem iron enzyme during cyclopropyl amino acid biosynthesis[END_REF]. All these enzymes require substrates bound to a separate protein except for WelO5 from Hapalosiphon welwitschii, which acts on free substrates [START_REF] Hillwig | A new family of iron-dependent halogenases acts on freestanding substrates[END_REF].

The fourth group is composed of the heme iron-dependent haloperoxidases, firstly characterized in the terrestrial fungus Caldariomyces fumago [START_REF] Geigert | Epoxidation of alkenes by chloroperoxidase catalysis[END_REF]. Those enzymes oxidize halides with H2O2 (Figure 0-3B) to produce free hypohalogenous acid (HOX), highly reactive [START_REF] Kühnel | Crystal structures of chloroperoxidase with its bound substrates and complexed with formate, acetate, and nitrate[END_REF]. Consequently, they can halogenate a large panel of organic compounds and particularly aromatic, electron-rich substrates [START_REF] Hofrichter | Heme-thiolate haloperoxidases: versatile biocatalysts with biotechnological and environmental significance[END_REF]. They are divided into two superfamilies regrouping on one hand the bacterial, fungal and plant enzymes and on the other hand the animal peroxidases like mammalian myeloperoxidase, lactoperoxidase, eosinophil peroxidase, and thyroid peroxidase. A broad range of halogen specificity is encountered in these latter enzymes where myeloperoxidases can oxidize Cl -, Br - and I -, eosinophil peroxidases can catalyze bromination and iodination, and lactoperoxidases are specific to I -oxidation. Thyroid peroxidases are responsible of the thyroid hormone thyroxine biosynthesis (for review, Blasiak et al., 2009). The fifth group is represented by the cofactor-free haloperoxidases (HPOs), for which a catalytic mechanism of halide oxidation using hydrogen peroxide (H2O2) has been proposed to produce peroxoacid and hypohalogenous acid (HOX) intermediates [START_REF] Hofmann | Structural investigation of the cofactor-free chloroperoxidases[END_REF].

The HPOs have an active site pocket as well as a conserved substrate-binding site that may be involved in the halogenation of hydrophobic compounds (for review, [START_REF] Xu | Independent evolution of six families of halogenating enzymes[END_REF]. The cofactor-free haloperoxidases probably need to function with organic acids like benzoate or propionate as co-substrates. Eight structures of these enzymes, all belonging to bacteria, have been solved and released in the PDB database. Evolutionary relationship and similar overall topology have been found between HPOs and enzymes of the α/β hydrolase group, such as esterases and lipases (for review, [START_REF] Xu | Independent evolution of six families of halogenating enzymes[END_REF]. Among HPOs, the bromoperoxidase from Streptomyces aureofaciens [START_REF] Krenn | Purification and some characteristics of a non-haem bromoperoxidase from Streptomyces aureofaciens[END_REF] and the chloroperoxidase from Pseudomonas pyrrocinia [START_REF] Wolfframm | Chloroperoxidase-encoding gene from Pseudomonas pyrrocinia: sequence, expression in heterologous hosts, and purification of the enzyme[END_REF] appear to be involved in the biosynthesis of pyrrolnitrin and chlortetracycline antibiotics.

The last group refers to vanadium-dependent haloperoxidases (VHPOs). Like the previous group, they catalyze the oxidation of halides using H2O2, except that they feature vanadium as a cofactor. The biochemistry and characteristics of this group of halogenating enzymes are fully described later, in paragraph 4.

Dehalogenating enzymes

There are four types of dehalogenating enzymes described up to now based on their enzymatic mechanisms and substrate specificities (for review, Agarwal et al., 2017):

The first type belongs to the deiodinases from the thyroid hormone system of mammals. They are the only known dehalogenases from mammals. They play an essential role in human health as they control the thyroid hormone biosynthesis. Iodotyrosine deiodinases (IYD) are transmembrane proteins and they use flavin mononucleotide (FMN) as a cofactor. They scavenge iodide in the thyroid gland by catalyzing the Tyr-I bond of mono-and di-iodotyrosine (MIT and DIT), the byproducts of thyroid hormones T3 and T4. The scavenged iodide is then used to synthesize thyroid hormones again, creating an iodide cycle within the thyroid gland [START_REF] Thomas | Crystal structure of lodotyrosine deiodinase, a novel flavoprotein responsible for iodide salvage in thyroid glands[END_REF]. Iodothyronine deiodinases (IDs) are responsible for dehalogenating thyroxine (T4) into triiodothyronine (T3) hormone. These enzymes have the particularity to contain a selenocyteine in the active site (Figure 0-4B) (for review: [START_REF] Bianco | Deiodinases: Implications of the local control of thyroid hormone action[END_REF][START_REF] Gereben | Cellular and molecular basis of deiodinase-regulated thyroid hormone signaling[END_REF]. In addition, the mammalian deiodinases can display in vitro debrominase and dechlorinase activities on halogenated tyrosines (McTamney & Rokita, 2009).

(Figure 0-4A)
The last studies tend to demonstrate that IYDs are more widely spread in the nature than primarily thought as marine invertebrate and insect protein orthologues have been heterologously expressed and recently characterized at the biochemical and structural level [START_REF] Sun | Toward a Halophenol Dehalogenase from Iodotyrosine Deiodinase via Computational Design[END_REF]. Homologous IYD proteins are also found in bacteria without the Nterminal transmembrane segment. The recombinant IYD of Haliscomenobacter hydrossis exhibited a substrate specificity for halotyrosines [START_REF] Ingavat | Active Site Binding Is Not Sufficient for Reductive Deiodination by Iodotyrosine Deiodinase[END_REF], even though the nature of the biological substrates and metabolic roles of these bacterial IYDs have not yet been solved. The second type is represented by the reductive dehalogenases (RDHs) which can catalyze reactions with anthropogenic substrates making them enzymes of interest for bioremediation.

One subfamily has been isolated from anaerobic bacteria and these RDHs utilize a broad range of organohalogens as terminal electron acceptors in organohalide respiration chain, a particular energy-conserving metabolic process implicated in the global halogen cycle [START_REF] Hug | Diversity of reductive dehalogenase genes from environmental samples and enrichment cultures identified with degenerate primer PCR screens[END_REF].

In the other subfamily, glutathione S-transferase-like reductive dehalogenases are capable of dehalogenating toxic substrates by a catalytic attack of a cysteine thiolate on the halogen (Figure 0-5A). Examples of those enzymes involved in artificial chemical degradation are BphK from Burkholderia xenovorans strain LB400 [START_REF] Fortin | A glutathione S-transferase catalyzes the dehalogenation of inhibitory metabolites of polychlorinated biphenyls[END_REF] and PcpC from Flavobacterium sp. strain ATCC39723 [START_REF] Orser | Characterization of a Flavobacterium glutathione S-transferase gene involved in reductive dechlorination[END_REF].

The third type of dehalogenating enzymes catalyze oxidative dehalogenation, such as dioxygenases. The catalytic mechanism involves the replacement of a halogen with a hydroxyl group derived from a molecular oxygen or hydrogen peroxide (Figure 0-5B) (for review : Arora & Bae, 2014, and references therein). This type of dehalogenating enzymes is mainly involved in the degradation of chlorophenols and their derivatives. For example, dioxygenases are involved in the bacterial degradation pathway of 4-chlorophenol from Comamonas testosteroni JH5 [START_REF] Hollender | Degradation of 4-chlorophenol via the meta cleavage pathway by Comamonas testosteroni JH5[END_REF]. The last type is represented by hydrolytic dehalogenases (HDHs) that encompass widely diverse hydroxylase enzymes. They catalyze the substitution of a halogen by a hydroxyl group derived either from a water molecule or from a vicinal alcohol (Figure 5C) (for review, [START_REF] Jong | Structure and mechanism of bacterial dehalogenases: Different ways to cleave a carbon-halogen bond[END_REF]. The catalytic mechanisms of HDHs are named according to their substrate specificity. For example, the fluoroacetate dehalogenases are specific to fluorinated substrates, while the haloalcohol dehalogenases are specific to halohydrin substrates. The hydrolytic dehalogenation is commonly performed by haloalkane dehalogenases, 2-haloacid dehalogenases, 4-chlorobenzoyl-CoA dehalogenases and fluoroacetate dehalogenases (for reviews, [START_REF] Jong | Structure and mechanism of bacterial dehalogenases: Different ways to cleave a carbon-halogen bond[END_REF]Kurihara & Esaki, 2008). The most studied are the haloalkane dehalogenases that are widely distributed in the tree of life and are certainly predominant in marine environment (for review, [START_REF] Kunka | Haloalkane Dehalogenases From Marine Organisms[END_REF]. The other largely expanded group is the 2-haloacid dehalogenases (for review, Janssen et al., 2005). This latter is further described in paragraph 5.

GENERAL INTRODUCTION __________________________________________________________________________________________

-25 -

Vanadium-dependent haloperoxidases (VHPO)

Biochemical and structural characterization of VHPOs

The vanadium-dependent haloperoxidases (EC 1.11.1.10) catalyze the oxidation of halides (X -: I -, Br -or Cl -) in the presence of hydrogen peroxide. During the catalytic cycle, the vanadate cofactor coordinates one molecule of H2O2 to form a stable peroxovanadate intermediate. This intermediate will then react with halide. After this step, the oxidized halogen intermediate (X + which is a mixture of different halogen species such as OX -, X3 or X2) is very reactive and can halogenate a wide variety of nucleophilic organic substrates by a non-enzymatic reaction. In the absence of organic substrate, a second equivalent of H2O2 is oxidized to form an oxygen molecule and release the halide ion X -(Figure 0-6) (for review, [START_REF] Leblanc | Vanadium haloperoxidases: From the discovery 30 years ago to X-ray crystallographic and V K-edge absorption spectroscopic studies[END_REF]. The VHPOs are named according to the most electronegative halide they can oxidize. The vanadium-dependent chloroperoxidases (VCPO) can oxidize chloride, bromide and iodide; the vanadium-dependent bromoperoxidases (VBPO) can oxidize chloride and bromide; and the vanadium-dependent iodoperoxidases (VIPO) are specific to iodide oxidation (for reviews, [START_REF] Winter | Exploring the chemistry and biology of vanadiumdependent haloperoxidases[END_REF][START_REF] Leblanc | Vanadium haloperoxidases: From the discovery 30 years ago to X-ray crystallographic and V K-edge absorption spectroscopic studies[END_REF]and references through in).

2014)

In order to assess the VHPO enzymatic activities, a variety of sensible and reliable assays is available. The most common approach to determine VCPO and VBPO activities is the monitoring of monochlorodimedone (MCD) absorbance decrease at 290 nm [START_REF] De Boer | The reaction mechanism of the novel vanadiumbromoperoxidase. A steady-state kinetic analysis[END_REF]. It can detect halogenating activity quantitatively if the pH is below 6.5. Another qualitative method based on the halogenation of o-dianisidine can be used to determine chloro-, bromo-and iodo-peroxidase activities but only on native gels (Jordan & Vilter, 1991). The phenol red dye can also be used to determine VBPO and VCPO activities at 590 nm but only qualitatively and is also very useful as a visual screening assay [START_REF] De Boer | Vanadium containing bromoperoxidase: An example of an oxidoreductase with high operational stability in aqueous and organic media[END_REF]. Another quantitative spectrophotometric assay, used to determine both VBPO and VIPO kinetic parameters, has been developed by Verhaeghe et al., 2008a. It is based on the halogenation of thymol blue by following the absorbance increase at 620 nm. This method is also pH dependent and reliable between pH 7 and 8. Moreover, thymol blue is a dye that changes color during its halogenation and thus can be used for rapid screening assay of VIPO or VBPO activities, in solution or directly in gel. VIPO activity was previously assessed by the spectrophotometric detection of triiodide (I3 -) at 350 nm [START_REF] Björkstén | A Kinetic Study of the Horse-Radish Peroxidase-Catalyzed Oxidation of Iodide.pdf[END_REF]. I3 -is one of the intermediates formed during the catalytic reaction. However, this assay has been shown to present important quantitative biases and be less reliable than thymol blue assay for biochemical studies of VIPO (Verhaeghe et al., 2008a). All these enzymatic assays were applied to characterize VHPO catalytic activities of purified native or recombinant enzymes from seaweeds, bacteria, fungi and cyanobacteria [START_REF] Itoh | Characterization of nonheme type bromoperoxidase in Corallina pilulifera[END_REF][START_REF] Sheffield | Purification and characterization of the vanadium bromoperoxidase from the macroalga Corallina officinalis[END_REF][START_REF] Hemrika | Heterologous Expression of the the Vanadium-containing Chloroperoxidase from Curvularia inaequalis in Saccharomyces cerevisiae and Site-directed Mutagenesis of the Active Site Residues His496, Arg360, and Arg490[END_REF][START_REF] Colin | Vanadium-dependent iodoperoxidases in Laminaria digitata, a novel biochemical function diverging from brown algal bromoperoxidases[END_REF][START_REF] Wischang | Parameters for bromination of pyrroles in bromoperoxidase-catalyzed oxidations[END_REF][START_REF] Johnson | Characterization of a functional vanadium-dependent bromoperoxidase in the marine cyanobacterium Synechococcus sp. CC9311[END_REF][START_REF] Baharum | Molecular cloning, homology modeling and site-directed mutagenesis of vanadium-dependent bromoperoxidase (GcVBPO1) from Gracilaria changii (Rhodophyta)[END_REF]Fournier et al., 2014;[START_REF] Frank | Characterization of a Cyanobacterial Haloperoxidase and Evaluation of its Biocatalytic Halogenation Potential[END_REF][START_REF] Mckinnie | Characterization and Biochemical Assays of Streptomyces Vanadium-Dependent Chloroperoxidases[END_REF]. Up to this day, VHPOs from seaweeds have been shown to possess the highest catalytic activity compared to other organisms. According to [START_REF] Wischang | Molecular cloning, structure, and reactivity of the second bromoperoxidase from Ascophyllum nodosum[END_REF] and [START_REF] Colin | Vanadium-dependent iodoperoxidases in Laminaria digitata, a novel biochemical function diverging from brown algal bromoperoxidases[END_REF], VBPO-II from Ascophylum nodosum and VIPO from Laminaria digitata have respective kcat for iodide of 153 s -1 and 462 s -1 .

In terms of structural protein characterization, only 8 VHPOs from 7 different species have been studied, corresponding to 19 VHPO X-ray structures referenced in public databases up to now (listed in Table 0-3). These 3D structures correspond to either native, recombinant or derived enzyme forms (site-directed mutants, apo-proteins, etc) of VHPOs. Briefly, the tertiary structural foldings of VHPOs are highly conserved with helical bundles of alpha helices and identical arrangements of amino acid residues at the vanadium active sites, suggesting similar catalytic mechanisms. However, site-directed mutagenesis analyses of fungal VCPO, red algal VBPO and bacterial VIPO have shown that specific amino acids and hydrogen-bonding network around the vanadate center were both essential for catalytic properties and halide specificity (Fournier et al., 2014;for review, Leblanc et al. 2015). Other striking differences concern quaternary VHPO arrangements in solution between the different phyla, where monomers, covalently-bound homodimers, and homodimers self-rearranged into hexamers or dodecamers have been described (Table 0-3).

Occurrence, evolution and putative biological roles of VHPOs

In the marine organisms, it appears that the VBPOs are the most common VHPOs, especially within red and brown macroalgae (for review, [START_REF] Leblanc | Vanadium haloperoxidases: From the discovery 30 years ago to X-ray crystallographic and V K-edge absorption spectroscopic studies[END_REF]. VBPO activities also occur in a variety of diatoms species [START_REF] Moore | Bromoperoxidase and iodoperoxidase enzymes and production of halogenated methanes in marine diatom cultures[END_REF][START_REF] Hill | Release of reactive bromine and iodine from diatoms and its possible role in halogen transfer in polar and tropical oceans[END_REF], Cyanobacteria [START_REF] Johnson | Characterization of a functional vanadium-dependent bromoperoxidase in the marine cyanobacterium Synechococcus sp. CC9311[END_REF][START_REF] Frank | Characterization of a Cyanobacterial Haloperoxidase and Evaluation of its Biocatalytic Halogenation Potential[END_REF] and planktonic communities [START_REF] Archer | Fluorescent detection of bromoperoxidase activity in microalgae and planktonic microbial communities using aminophenyl fluorescein[END_REF]. Some marine bacteria such as Streptomycetes (for review, [START_REF] Mckinnie | Characterization and Biochemical Assays of Streptomyces Vanadium-Dependent Chloroperoxidases[END_REF] and terrestrial fungi [START_REF] Simons | Primary Structure and Characterization of the Vanadium Chloroperoxidase from the Fungus Curvularia inaequalis[END_REF][START_REF] Barnett | Isolation, characterization, and primary structure of the vanadium chloroperoxidase from the fungus Embellisia didymospora[END_REF] feature VCPOs. The first VIPOs were characterized in brown macroalgae, belonging to Laminariales, and it was relevant as they are the strongest known accumulators of iodine, particularly L. digitata [START_REF] Küpper | Iodine uptake in Laminariales involves extracellular, haloperoxidase-mediated oxidation of iodide[END_REF][START_REF] Colin | Vanadium-dependent iodoperoxidases in Laminaria digitata, a novel biochemical function diverging from brown algal bromoperoxidases[END_REF]. More recently, it was also shown that the marine flavobacterium Zobellia galactanivorans possesses two VIPOs, and a third VHPO, more divergent in amino acid sequence, that is yet uncharacterized at the biochemical and structural levels (Fournier et al., 2014;Barbeyron et al., 2016).

A previous phylogenetic analysis allowed the identification of numerous VHPO bacterial homologs in public protein databases and showed a large spreading of VHPO in bacterial lineages (Figure 0-7; Fournier et al. 2014). The phylogeny and conservation of 3D structures suggested a common origin for all VHPOs deriving from a marine bacterial ancestor, related to bacterial acid phosphatases. The algal VHPOs seem to be derived more precisely from VBPOs from Cyanobacteria (for review, [START_REF] Leblanc | Vanadium haloperoxidases: From the discovery 30 years ago to X-ray crystallographic and V K-edge absorption spectroscopic studies[END_REF][START_REF] Frank | Characterization of a Cyanobacterial Haloperoxidase and Evaluation of its Biocatalytic Halogenation Potential[END_REF]. Moreover, the emergence of VIPOs, i.e. the specificity for iodide oxidation, would result in a convergent evolution of the divergent phyla of Bacteroidetes and Laminariales (Fournier et al. 2014). In macroalgae, some VHPOs were shown to be up-regulated at the transcriptomic level and to be part of antioxidative responses following a stress (cf. paragraph 2.3). For instance, their detoxifying role was pointed out in the red algae Corallina officinalis and Gracilaria changii where the expression of VBPO enzyme increases during an abiotic stress [START_REF] Latham | Temperature stress-induced bleaching of the coralline alga Corallina officinalis: A role for the enzyme bromoperoxidase[END_REF][START_REF] Baharum | Molecular cloning, homology modeling and site-directed mutagenesis of vanadium-dependent bromoperoxidase (GcVBPO1) from Gracilaria changii (Rhodophyta)[END_REF]. The same results were observed for the brown algae L. digitata and E. siliculosus, respectively, upon defense elicitation [START_REF] Cosse | Patterns of gene expression induced by oligoguluronates reveal conserved and environment-specific molecular defense responses in the brown alga Laminaria digitata[END_REF] or during pathogen infection [START_REF] Strittmatter | Infection of the brown alga Ectocarpus siliculosus by the oomycete Eurychasma dicksonii induces oxidative stress and halogen metabolism[END_REF].

Other studies on Laminariales have suggested the involvement of VIPOs in the accumulation of iodide in the apoplast of those kelps [START_REF] Küpper | Iodine uptake in Laminariales involves extracellular, haloperoxidase-mediated oxidation of iodide[END_REF][START_REF] Küpper | Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry[END_REF]Verhaeghe et al., 2008b).

The study of iodine distribution in L. digitata by cryofixation and chemical imaging showed that it was mainly located in the apoplast of the meristoderm and the outer cortex tissues (Verhaeghe et al., 2008b). Following those results, it has been proposed a hypothesis about the direct involvement of an apoplastic VIPO into the highly selective H2O2-based oxidation of iodide from seawater, allowing its uptake and sequestration in L. digitata cell walls (Figure 012345678). The biological role of this accumulated iodide stock would be to protect the cortical cell layers against oxidative stress (Verhaeghe et al. 2008b), reinforcing its proposed role as antioxidant [START_REF] Küpper | Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry[END_REF]. However, the biological roles of algal VHPOs, which form very large multigenic families in some kelps [START_REF] Colin | The brown algal kelp Laminaria digitata features distinct bromoperoxidase and iodoperoxidase activities[END_REF][START_REF] Ye | Saccharina genomes provide novel insight into kelp biology[END_REF], are still not fully understood. Depending on their subcellular location, they could be involved in various physiological and metabolic processes. For instance, a recent study demonstrates the implication of VHPOs in phlorotannin incorporation into the cell wall of Fucales, as well as into the development of zygotes during embryogenesis [START_REF] Lemesheva | Vanadiumdependent haloperoxidase activity and phlorotannin incorporation into the cell wall during early embryogenesis of Fucus vesiculosus (Phaeophyceae)[END_REF].

The biological role of VHPOs has also been explored in fungi such as Curvularia inaequalis.

This plant pathogen fungus possesses VCPO and may use VCPO-produced hypochlorous acid to degrade lignocellulose in plant cell wall during hyphen penetration [START_REF] Winter | Exploring the chemistry and biology of vanadiumdependent haloperoxidases[END_REF].

This VCPO also possesses antimicrobial activity against Enterococcus faecalis biofilm [START_REF] Persoon | Effect of vanadium chloroperoxidase on Enterococcus faecalis biofilms[END_REF].

For biogeochemical cycling of halogens in marine environment (paragraph 1.4), VHPOs are likely to be some of key enzymes for producing biogenic halogenated compounds, emitted in marine environment. For instance, the VBPO activity has been linked to halocarbon emissions, especially methyl bromide (CH3Br), in Synechococcus thanks to an insertional inactivation of the gene [START_REF] Johnson | Halomethane production by vanadium-dependent bromoperoxidase in marine Synechococcus[END_REF], but their specific biological roles have not been studied yet. In marine Streptomyces sp. strains, VCPOs catalyze a chlorination reaction leading to the biosynthesis of antibiotics such as napyradiomycin [START_REF] Bernhardt | A stereoselective vanadium-dependent chloroperoxidase in bacterial antibiotic biosynthesis[END_REF] and merochlorin [START_REF] Kaysser | Merochlorins A-D, cyclic meroterpenoid antibiotics biosynthesized in divergent pathways with vanadium-dependent chloroperoxidases[END_REF]. In the marine flavobacteria Zobellia galactanivorans the presence of 3 VHPOs, including 2 VIPOs, could be related to the protection against algal chemical defenses or antioxidant responses (Fournier et al., 2014;Barbeyron et al., 2016). However, the biological roles of these VHPOs have yet to be confirmed experimentally.

Haloacid dehalogenases (HAD)

Classification of the HADs

There are several ways to classify the HADs according to their enzymatic or catalytic properties, and to their protein sequence or structure homologies.

First, they are ranged into three enzyme types based on their substrate specificities (for reviews Ang et al., 2018;[START_REF] Wang | Insights into the molecular mechanism of dehalogenation catalyzed by D-2-haloacid dehalogenase from crystal structures[END_REF] Finally, the HADs are also phylogenetically classified into two groups (Hill et al., 1999).

Similarly to catalytic regrouping, D-2-HAD and DL-2-HAD enzymes belong to the type I dehalogenases because of their high similarity in protein sequences. D-2-HADs and DL-2-HADs share about 30% similarities in their primary structures and α-helical hydrolase folding.

Recent advances about structure/function characterization of D-2-HADs have shown that HadD, from Pseudomonas putida AJ1, crystallized as a homotetramer with a substrate binding pocket located at the interface between two repeats. The amino acids surrounding the active pocket participated in the enzyme-substrate binding. Two residues, an aspartate (Asp205) and an asparagine (Asn131) have been demonstrated to directly activate the nucleophilic water molecule involved in the catalytic dehalogenation [START_REF] Wang | Insights into the molecular mechanism of dehalogenation catalyzed by D-2-haloacid dehalogenase from crystal structures[END_REF].

The L-2-HAD enzymes (EC 3.8.1.2) belong to the type II dehalogenases and are part of a large hydrolase superfamily, with diverse substrate specificities and are widely distributed among living organisms. This superfamily, firstly described by an iterative approach of sequence homology, contains three conserved domains [START_REF] Koonin | Computer analysis of bacterial haloacid dehalogenases defines a large superfamily of hydrolases with diverse specificity[END_REF]. The HAD superfamily includes phosphatases, phosphonatases, P-type ATPases, betaphosphoglucomutases, phosphomannomutases, epoxide hydrolases, or dehalogenases, which are involved in a variety of cellular processes, ranging from amino acid biosynthesis to detoxification (Burroughs et al., 2006). These hydrolases have all in common their ability to form a covalent enzyme-substrate intermediate via a conserved active site nucleophile aspartate, thus facilitating the cleavage of C-X (X = F, Cl, Br, I), P-C, or P-O bonds. Even if they gave their name to this superfamily, the L-2-HADs are the only "true" dehalogenases of the HAD superfamily enzymes. While the majority of enzymes in this superfamily requires a divalent metal ion cofactor for activity, the L-2-HADs are notable exceptions. Most superfamily members but not all have a cap domain, with variable folds and functions, in addition to a core domain always present [START_REF] Glasner | Evolution of enzyme superfamilies[END_REF].

Structural features and enzymatic properties of the L-2-HADs

The biochemical and structure/function characterizations of L-2-HADs have been conducted

during the past thirty years mainly on bacterial enzymes. Most structurally characterized L-2-HADs are dimers with two domains in each subunit: a characteristic alpha/beta core domain with a conserved alpha/beta hydrolase fold, similar to the "Rossmann-fold", and a small cap domain. The helical domains provide a tight homodimeric interface and a confined active site which limit substrate access, and therefore specificity, to small C2-C3 molecules (Van der Ploeg et al., 1991;Hisano et al., 1996;Ridder et al., 1997). The determination of the molecular bases for L-2-HADs aggregation into dimers has shown the importance of a 17 amino acids sequence [START_REF] Tsang | Identification of the dimerization domain of dehalogenase IVa of Burkholderia cepacia MBA4[END_REF]. The exchanges of the residues 73 to 89 in DehCI protein (monomeric L-2-HAD) by the residues 74 to 90 of DehIVa protein (dimeric L-2-HAD) led to the dimerization of recombinant mutated DehCI and reciprocally to the production of recombinant mutated DehIVa monomers. The roles of Phe58, Thr65, Leu78, and Phe92 were also important for the dimerization of DehIVa [START_REF] Tsang | Identification of the dimerization domain of dehalogenase IVa of Burkholderia cepacia MBA4[END_REF]. The diversity of possible substrates identified for some biochemically characterized L-2-HADs is displayed in Table 0-4. The substrates tested were usually brominated and chlorinated, and more rarely iodinated.

Not all of the enzyme activities listed in Table 0-4 have been tested with iodinated substrates.

The L-2-HADs are mostly specific towards short chained (C2-C3) mono-halogenated substrates, as shown for DehIVa [START_REF] Murdiyatmo | Molecular biology of the 2-haloacid halidohydrolase IVa from Pseudomonas cepacia MBA4[END_REF], DhlS5I (Köhler et al., 1998), dehDEH99 (Huang et al., 2011), DehCI and DehCII (Schneider et al., 1991;1993). Other L-2-HADs, such as DehRhb (Novak et al., 2013a), DhlB (Van der Ploeg et al., 1991) or DehL [START_REF] Cairns | Cloning, sequencing and expression in Escherichia coli of two Rhizobium sp. genes encoding haloalkanoate dehalogenases of opposite stereospecificity[END_REF], can also catalyze the dehalogenation of di-halogenated substrates, and for DhlB, dibromo-and dichloroacetic acids have been described as preferred substrates. Only four enzymes were shown to catalyze C4 substrates (L-2-halobutyric acid): L-DEX YL (Liu et al., 1994), HadL (Barth et al., 1992), L-2-HAD from P. ingrahamii (Novak et al., 2013b) and DehSft (Rye et al., 2009). The latter is even the unique known enzyme that can transform the L-2-bromohexanoic acid, with six carbons in its aliphatic chain. Up to now, only two L-2-HAD enzymes were shown to use fluorinated substrates: fluoroacetic acid can be dehalogenated by Bpro0530 and Rha0230 ( The structure determinations of recombinant L-2-HADs, complexed with their substrates, combined with site-directed mutagenesis analysis have allowed to identify the residues essential for the catalytic reaction processes (Kurihara et al., 1995;Pang & Tsang, 2001;Adamu et al., 2016). Nine amino acids (DTRSKYSND) around the active center are proposed to be involved in substrate binding and the formation of a hydrophobic halogen cradle (Kurihara et al., 1995;Ridder et al., 1997). The catalytic reaction starts by a nucleophilic attack on the alpha-carbon of the substrate by a conserved aspartic acid (Asp). This reaction results in cleaving the carbonhalogen bond, releasing one halide, and forming an enzyme-substrate ester bond. Then, an adjacent water molecule is activated by an Asp/Lys dyad to attack the intermediate's ester bond and to release the hydroxylated product (Schmidberger et al., 2007;Nakamura et al., 2009).

This last step is different for the only structurally characterized L-2-HAD of marine origin, as the water molecule is activated by a His/Glu dyad (Novak et al., 2013). This enzymatic reaction occurs with an inversion of chirality of the final product.

Occurrence and putative biological roles of L-2-HADs

Up to now, most of the identified and characterized L-2-HADs are present in organisms from terrestrial origin and, only few of them have been characterized from marine species. Some terrestrial bacteria are able to catabolize halogenated xenobiotic compounds to detoxify or use them as carbon sources. These halocarbons include chemicals derived from industrial activities and released in the environment. L-2-HAD enzymes like L-DEX YL and DhlB have been

shown to be part of metabolic pathways for the degradation of 1,2-dichloroethane (Janssen et al., 1994) and 2-chloroacrylate (Kurata et al., 2005), respectively.

In this context, some soil bacteria such as Burkholderia cepacia MBA4 or Xanthobacter autotrophicus GJ10 have been studied for their capacity to grow on and metabolize haloacid compounds. They possess the genetic tools to internalize and degrade a large range of anthropogenic HOCs that are often toxic [START_REF] Kwok | Proteomic analysis of Burkholderia cepacia MBA4 in the degradation of monochloroacetate[END_REF]Mena-Benitez et al., 2008;Horisaki et al., 2011). In B. cepacia MBA4, the L-2-HAD-encoding gene, dehIVa, and the downstream permease-encoding gene, dehIVp, form an inducible operon that mediates the transformation and uptake of halogenated acids (Yu et al., 2007). More generally, the activities of HADs have also been related to natural detoxification processes. For instance, the decomposition of forest soil organic matter leads to the production of chloroacetic acid, which will need to be detoxified (Matucha et al., 2003;Laturnus et al., 2005), and the conversion of chloroacetic acid to glycolate has been reported for X. autotrophicus GJ10 thanks to a L-2-HAD [START_REF] Torz | Biodegradation of monochloroacetic acid used as a sole carbon and energy source by Xanthobacter autotrophicus GJ10 strain in batch and continuous culture[END_REF].

The marine environment contains a large chemical diversity of natural halogenated compounds, some of them being highly specific and/or potentially toxic, as parts of marine algal chemical defenses (paragraph 2; Belkin, 1992;[START_REF] Paul | Chemical defence against bacteria in the red alga Asparagopsis armata: Linking structure with function[END_REF]. The halogenated compounds produced by kelps could be converted into haloacetic acids, and some red algae like Asparagopsis taxiformis contain haloacetic acids [START_REF] Woolard | Halogenated acetic and acrylic acids from the red alga Asparagopsis taxiformis[END_REF]Fenical, 1982). However, very little is known about marine dehalogenase enzymes and more generally about the reactions involved in the catabolic pathways of halogenated compounds and their biological and ecological roles in marine organisms.

Zobellia galactanivorans, a model marine bacterium for studying specific metabolic pathways

Zobellia galactanivorans Dsij T is a rod-shaped, Gram-negative, non-flagellated, marine

Flavobacterium first isolated in Roscoff (France) from the red alga Delesseria sanguinea [START_REF] Potin | Purification and characterization of a new kappa-carrageenase from a marine Cytophaga-like bacterium[END_REF]Barbeyron et al., 2001). This bacterium produces flexirubine, a pigment that colors its colonies in yellow/orange (Figure 10). Z. galactanivorans is chemoorganotrophic and strictly aerobic (i.e., it needs organic compounds as carbon source and oxygen as final electron acceptor). To this day, six other Zobellia strains have been characterized: Z. uliginosa, Z.

amurskyensis MAR_2009_138 (Barbeyron et al., 2001), Z. russellii, Z. laminariae, Z.

amuskyensis KMM 3526 T (Nedashkovskaya et al., 2004) and Zobellia sp. OII3 (Harms et al., 2017). All of them were isolated from coastal marine habitats and live on the surface of green, red and brown macroalgae [START_REF] Potin | Purification and characterization of a new kappa-carrageenase from a marine Cytophaga-like bacterium[END_REF]Nedashkovskaya et al., 2004). Since its discovery, Z. galactanivorans has been studied in the past 20 years for its ability to degrade polysaccharides constituting algal cell walls, thanks to specialized enzymes such as glycoside hydrolases, polysaccharide lyases and carbohydrate esterases (Barbeyron et al., 2016). Experimental research on Z. galactanivorans has benefited from genomic resources and genetic tools, developed for functional studies on algal polysaccharide degradation (for review, Lami et al., 2021, in press). For instance, the first operons of marine origins, specific to alginate and mannitol utilizations, have been described in this model bacterium [START_REF] Thomas | Characterization of the first alginolytic operons in a marine bacterium: From their emergence in marine Flavobacteria to their independent transfers to marine Proteobacteria and human gut Bacteroides[END_REF][START_REF] Groisillier | The mannitol utilization system of the marine bacterium Zobellia galactanivorans[END_REF]. Its entire genome was sequenced in 2010 and has opened the door to numerous research opportunities. Many of those are linked to algal polysaccharides consumption [START_REF] Thomas | Characterization of the first alginolytic operons in a marine bacterium: From their emergence in marine Flavobacteria to their independent transfers to marine Proteobacteria and human gut Bacteroides[END_REF]2013;2017;[START_REF] Groisillier | The mannitol utilization system of the marine bacterium Zobellia galactanivorans[END_REF]Ficko-Blean et al., 2017;[START_REF] Dudek | Regulation of alginate catabolism involves a GntR family repressor in the marine flavobacterium Zobellia galactanivorans DsijT[END_REF]. The developments of functional genomics (transcriptomics, heterologous protein overexpression, site-directed mutagenesis,…) and structure/function approaches (resolution of 3D structures) were major advances to unravel the biochemical and biological roles of enzymes [START_REF] Hehemann | Expression, purification and preliminary X-ray diffraction analysis of the catalytic module of a β -Agarase from the flavobacterium Zobellia galactanivorans[END_REF]2012;[START_REF] Labourel | The β-Glucanase ZgLamA from Zobellia galactanivorans Evolved a Bent Active Site Adapted for Efficient Degradation of Algal Laminarin[END_REF]2015;[START_REF] Ficko-Blean | Biochemical and structural investigation of two paralogous glycoside hydrolases from Zobellia galactanivorans: Novel insights into the evolution, dimerization plasticity and catalytic mechanism of the GH117 family[END_REF][START_REF] Naretto | The agar-specific hydrolase ZgAgaC from the marine bacterium Zobellia galactanivorans defines a new GH16 protein subfamily[END_REF]. More recently, this panel was completed with knock-out mutagenesis, which was a powerful tool to establish the in vivo function of alginate lyases (Zhu et al., 2017).

Living in close interactions with seaweeds, Z. galactanivorans is expected to display many genetic adaptations to interact with macroalgae. During the degradation of brown algal alginate cell walls, bacteria have to deal with algal oxidative responses and iodine-based chemical defenses [START_REF] Küpper | Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry[END_REF][START_REF] Cosse | Patterns of gene expression induced by oligoguluronates reveal conserved and environment-specific molecular defense responses in the brown alga Laminaria digitata[END_REF]. It has been shown that exudates of L. digitata elicited by oligoguluronates positively stimulated the biofilm growth of Z. galactanivorans [START_REF] Salaün | Influence of Exudates of the Kelp Laminaria Digitata on Biofilm Formation of Associated and Exogenous Bacterial Epiphytes[END_REF]. Moreover, the exposition of Z. galactanivorans cultures with diiodomethane (CH2I2) did not show strong effect on its biofilm formation [START_REF] Salaün | Interactions entre la macroalgue brune Laminaria digitata et ses épibiontes bactériens : études moléculaire et spectroscopiques : capacité d'adhésion et de formation de biofilm[END_REF].

Iodinated volatile compounds, such as I2 and CH2I2 were released by kelps in those conditions, and should indicate that Z. galactanivorans has developed enzymatic capacities to survive in the presence of toxic halogenated metabolites, even the iodinated ones.

Indeed, the genes encoding putative enzymes related to halogenation and dehalogenation metabolisms were annotated on Zobellia's genome (Barbeyron et al., 2016): three vanadiumdependent haloperoxidases, two of which already characterized as iodoperoxidases (VIPOs); one cofactor-free haloperoxidase (HPO), one iodotyrosine deiodinase (IYD), and one haloacid dehalogenase (HAD). While the first bacterial VIPOs were biochemically and structurally described in Z. galactanivorans (Fournier et al., 2014), no further analyses have been performed

on the other putative enzymes. In addition, besides the presence of putative iodination-related enzymes, Z. galactanivorans can accumulate iodine compared to other planktonic bacteria and surrounding medium, up to a hundred times more than the seawater concentration (Barbeyron et al., 2016).

Thesis project and outline

In the biogeochemical cycle of halogens, a wide diversity of actors are expected to be involved, from seaweeds to phytoplankton in oceans, to terrestrial plants, soil microbes and human industry on lands. The role of marine bacteria could be of importance in this cycle, especially in coastal regions, where they are living in close interactions with macroalgae, known to actively produce both brominated and iodinated compounds. However, very few information is available on enzymes connected to this metabolism, or even the biological and ecological importance of this metabolism for marine bacteria.

Z. galactanivorans represents a very interesting marine bacterial laboratory model to further explore its halogen metabolism, and more specifically that related to iodine. Based on previous work conducted in the Roscoff's team [START_REF] Fournier | Evolution des mécanismes d'accumulation et de transport de l'iode dans les organismes marins : étude de la structure/fonction des protéines du métabolisme iodé chez la bactérie Zobellia galactanivorans[END_REF] and current knowledge at the beginning of my thesis, a hypothetical model of the biochemical roles for the enzymes related to halogen metabolism in Z. galactanivorans was adapted and is presented in For that purpose, I developed different methodological approaches during my thesis, which are presented in four chapters, followed by a synthetic "conclusion and perspectives" part.

In chapter 1, I present the investigation of the enzymatic properties of the recombinant HAD enzyme of Z. galactanivorans (ZgHAD), in order to determine its biochemical characteristics and specific substrates. I produced a knock-out mutant, by deleting the corresponding gene and compared different cultures of the wild-type and mutant strains on selected halogenated compounds, in order to explore the biological role of ZgHAD. A second part of this chapter deals with the phylogenetic analysis of ZgHAD and the environmental distribution of homologous proteins.

In chapter 2, I focus on the structure resolution of ZgHAD. Site-directed mutagenesis and computational docking were performed to identify key amino acids involved in substrate fixation and in the water molecule activation during the catalytic reaction.

The third chapter is dedicated to the biochemical and structural studies of Z. galactanivorans VHPO3. Colorimetric assays were used to determine its halide specificity and crystallography tests were performed in the hope of solving its 3D structure. In view of this difficulty, structural models were built to explore the monomeric and dimeric folding of the protein.

Finally, the fourth chapter focuses on investigating the localization of Zg-VIPO1 enzyme within the bacterium, through Western-blot analysis, as well as its potential biological roles using the knock-out mutant approach. The ability to detoxify and/or accumulate iodine was explored in both cultures of wild-type and mutant strains, in particular using in vitro cultures with radioactive I 125 during a short research stay in the laboratoty of Prof. Seigo Amachi in Japan.

Chapter 1 ______________________________________________________________________________ -41 -

Chapter presentation

This chapter is dedicated to the first functional characterization of a L-2-haloacid dehalogenase (L-2-HAD) from a bacterium of the Bacteroidetes phylum, combining biochemical and gene knockout approaches. I showed that the unique L-2-HAD of Zobellia galactanivorans was involved in bromo-and iodoacetic acid catabolism, providing a potential selective advantage to this marine coastal Bacteroidetes.

Phylogeny and distribution analyses of homologous L-2-HADs in environmental sequence databases revealed an unexpected wide marine diversity, amongst which we identified two distinct clades of Bacteroidetes L-2-HADs. All these results provide insights towards a potential ecological role of Bacteroidetes L-2-HADs during biotic interactions in marine habitats.

INTRODUCTION

Halogenated metabolites are widely distributed on Earth. They are defined as organic compounds that covalently bind one or several halogen atom(s) such as fluorine, chlorine, bromine or iodine. Over 5,000 different natural organohalogens have been described, mainly from the marine environment [START_REF] Gribble | Biological activity of recently discovered halogenated marine natural products[END_REF]. They can be produced abiotically [START_REF] Dong | Photochemical origin of reactive radicals and halogenated organic substances in natural waters: A review[END_REF], but also derived from many different biogenic sources. For example, some chlorinated metabolites are found in land plants and fungi, while brominated metabolites have been mainly related to marine sources such as red and brown algae, or sponges (Amsler, 2008;[START_REF] Cabrita | Halogenated compounds from marine algae[END_REF][START_REF] Bialonska | Aplysinopsinsmarine indole alkaloids: chemistry, bioactivity and ecological significance[END_REF][START_REF] El-Demerdash | Chemical diversity and biological activities of marine sponges of the genus Suberea: a systematic review[END_REF]. Furthermore, during the last century and with industrial developments, man-made polyhalogenated organic products were synthesized and are now persistent organic environmental pollutants (Agarwal et al., 2017;Ang et al., 2018). To mitigate this environmental issue, nature-based solutions are a promising alternative for bioremediation.

During the last four decades, research efforts have identified microorganisms that can catabolize diverse mono or polyhalogenated compounds, and characterized a broad range of dehalogenases [START_REF] Hardman | Dehalogenases in soil bacteria[END_REF]Janssen et al., 1994;Copley S., 1998). These enzymes are highly diverse in terms of structure and biochemistry, but they all share the enzymatic capacity of cleaving carbon-halogen bonds. Depending on their catalytic mechanisms and substrates, they are classified in large superfamilies (Janssen et al., 2005;Ang et al., 2018). This haloacid dehalogenase (HAD) superfamily includes mostly enzymes that hydrolyze carbon-phosphorus or oxygen-phosphorus bonds, such as phosphoesterases, phosphonatases, P-type ATPases, or nucleotidases. Widely conserved among living Chapter 1 ______________________________________________________________________________ -47 -organisms, they are involved in a variety of cellular processes ranging from amino acid biosynthesis to detoxification (Burroughs et al., 2006). In plant pathogen bacteria, HADs might also be involved in biofilm formation during biotic interactions [START_REF] Li | Genome-wide mutagenesis of Xanthomonas axonopodis pv. citri reveals novel genetic determinants and regulation mechanisms of biofilm formation[END_REF].

The "true" dehalogenases of the HAD superfamily are categorized into two types. Type I contains both HADs that have an enantioselective dehalogenating activity on D-2haloacids (D-2-HADs), and those that have non-stereospecific mechanism accepting both D-or L-2-haloacids as substrates (DL-2-HADs). Enzymes from type II specifically act on L-2-haloacids (L-2-HADs) to produce the corresponding hydroxyalkanoic acids with an inverted chirality (Ang et al., 2018). The L-2-HADs are phylogenetically unrelated to D-2-HADs and DL-2-HADs exhibiting a distinct protein fold and catalytic mechanism (Hill et al., 1999).

Many genes encoding L-2-HADs have previously been identified in the genomes of bacteria from contaminated soils or freshwater environments (Copley S., 1997;Kurihara et al., 2008). Biochemical and structure/function characterizations of L-2-HAD enzymes have been conducted during the past thirty years to identify new enzymatic or biotechnological processes for detoxification of halogenated pollutants. The large majority of characterized L-2-HADs originate from terrestrial Proteobacteria such as DehIVa from Burkholderia cepacia MBA4 (Schmidberger et al., 2007), DhlB from Xanthobacter autotrophicus GJ10 ( Van der Ploeg et al., 1991), or L-DEX from Pseudomonas sp. strain YL (Nardi-Dei et al., 1994). The dhlB gene has been engineered into land plants by transformation, allowing effective treatment of soil and groundwater contaminated with halogenated solvents (Mena-Benitez et al., 2008).

Chapter 1 ______________________________________________________________________________ -48 -

The biological roles of these HADs in terrestrial and/or freshwater bacteria are mostly related to their adaptations to degrade xenobiotic compounds, derived from industrial activities and released in the environment (Copley, S., 1998;Hill et al., 1999;Janssen et al., 2005). However, sampling efforts for HAD-containing microorganisms mostly focused on polluted matrices, potentially biasing the overall representation of these enzymes.

Therefore, the phylogenetic diversity and biological roles of HADs might be underestimated in non-polluted environments (Janssen et al., 2005).

The marine environment contains a large range of natural halogenated compounds, some of them being highly specific and/or potentially toxic, as parts of chemical defenses of marine invertebrates or algae. For instance, while iodinated compounds are relatively rare on Earth, brown macroalgae are main producers of volatile iodocarbons that play a notable role in atmospheric chemistry [START_REF] Carpenter | Comparative genomics and CAZyme genome repertoires of marine Zobellia amurskyensis KMM 3526 T and Zobellia laminariae KMM 3676 T[END_REF]. Other natural sources of marine halogenated compounds could include volcanoes and organic matter efflux in estuaries [START_REF] Jordan | Volcanogenic halocarbons[END_REF][START_REF] Gribble | The diversity of naturally produced organohalogens[END_REF][START_REF] Gordon | Ecotoxicology of bromoacetic acid on estuarine phytoplankton[END_REF]. Very little is currently known about marine dehalogenase enzymes, and even less about the metabolic pathways involved in the catabolism of halogenated compounds by marine microorganisms. To date, only four marine L-2-HADs have been biochemically characterized: DehRhb from a Rhodobacteraceae bacterium associated to marine Polychaeta (Novak et al., 2013a); two enzymes from the sponge-associated Paracoccus sp. DEH99 and Pseudomonas stutzeri DEH130 (Huang et al., 2011;[START_REF] Zhang | Purification and characterization of a dehalogenase from Pseudomonas stutzeri DEH130 isolated from the marine sponge Hymeniacidon perlevis[END_REF]; and one from the psychrophilic strain Psychromonas ingrahamii DehPsi (Novak et al., 2013b). The best-studied marine L-2-HAD so far is DehRhb, whose structure and catalytic properties notably differ from soil enzymes (Novak et al., 2013a).

Chapter 1 ______________________________________________________________________________ -49 -In this context, this paper reports on the occurrence of L-2-HAD homologous proteins in microorganisms, i.e. marine bacteria and archaea, through large in silico genomic surveys; on the phylogeny of such proteins in the Bacteroidetes phylum, and on the biochemical characterization of the only haloacid dehalogenase identified on the genome of the marine flavobacterium Zobellia galactanivorans Dsij T . This bacterium has been developed as a laboratory model for marine Bacteroidetes, because of its capacities to degrade algal polysaccharides. Its genome also features several genes related to halogen metabolism (Fournier et al., 2014;Barbeyron et al., 2016). Thanks to a recent protocol development (Zhu et al., 2017), a genetic knockout approach was conducted, demonstrating the potential in vivo protective role of this L-2-HAD when Z. galactanivorans was exposed to toxic halogenated substrates. In addition, the distribution and expression of phylogenetically distinct L-2-HADs were analyzed using metagenomic and metatranscriptomic datasets, highlighting an underexplored marine biodiversity. Finally, the laboratory functional characterization of Z. galactanivorans L-2-HAD and environmental survey results on homologous proteins are discussed in light of the biological and ecological roles of these enzymes in marine Bacteroidetes in their natural coastal habitats.

RESULTS

Phylogenetic analysis of bacterial L-2-HADs

The phylogenetic analysis of microbial L-2-HAD proteins was based on a subset of 352 protein sequences, including the biochemically and structurally characterized enzymes (Table S1) and their closest counterparts, in addition to the closest relatives of ZgHAD, identified by BlastP search on protein databases (Table S2). L-2-HAD proteins mainly S2). By contrast, the second phylogenetic group (group B) contains, almost exclusively (138/150 sequences), L-2-HAD protein sequences belonging to microorganisms either from saline or from marine origins (Table S2). The group B is constituted of three well-supported clades and an isolated proteobacterial putative L-2-HAD from Halomonas (Fig. 1). A clade groups all other proteobacterial L-2-HAD proteins, including DehRhb. Interestingly, the Bacteroidetes L-2-HAD homologs are separated into two phylogenetic groups, named HAD1 and HAD2 clades. No Bacteroidetes L-2-HAD was yet biochemically characterized before this study. The two phylogenetic types of Bacteroidetes L-2-HADs share between 40 to 45% amino acid identity.

The phylogenetic group A (Fig. 1) features most of the biochemically characterized L-2-HADs (Table S1), and these enzymes share around 30% of identity at the protein sequence level. Furthermore, a motif of 9 amino acids (DTRSKYSND) essential for enzyme activity and substrate binding (Adamu et al., 2016) was strictly conserved between all the characterized L-2-HADs of group A (Fig. S1).

Chapter 1 ______________________________________________________________________________ -51 - The closest biochemically characterized protein homolog of ZgHAD is DehRhb from a marine Rhodobacteraceae, present in the group B, but they only share 31% of amino acid sequence identity (Table S1). Reciprocally, a motif of nine conserved amino acids Chapter 1 ______________________________________________________________________________ -52 -(DTF(S/T)KYAHD) present in the group B are equivalent to those of group A (Fig. S1).

Eight of those amino acids are strictly conserved in the sequences of DehRhb and ZgHAD.

Only a T124 present in DehRhb is replaced by a S131 in ZgHAD in this pattern (Fig. S1).

Study of the genomic context of ZgHAD and among Flavobacteriaceae

The phylogenetic analysis has shown that most Bacteroidetes featured only one L-2-HAD belonging to either the HAD1 or the HAD2 phylogenetic clade, but some of them also exhibited both L-2-HAD sequences (Table S2). Some intra-species variations occur: for example, Zobellia amurskyensis MAR_2009_138 (formerly Zobellia uliginosa; [START_REF] Carpenter | Comparative genomics and CAZyme genome repertoires of marine Zobellia amurskyensis KMM 3526 T and Zobellia laminariae KMM 3676 T[END_REF] possesses two different L-2-HAD-encoded genes whereas the Z. galactanivorans genome only encodes for ZgHAD, belonging to the HAD1 phylogenetic clade. The occurrence and genomic context of the different L-2-HAD-encoded genes were further analyzed in genomes of Zobellia species and closely related members of the Flavobacteriaceae family (Fig. 2). The HAD1-type gene (relative to the phylogenetic HAD1 clade) was present on the genome of all the 6 Zobellia strains among a conserved succession of 8 ORFs followed by 3 genes in the other orientation (Fig. S2). The analysis of the publicly available RNAseq data for Z. galactanivorans (Ficko-Blean et al., 2017) showed continuous transcript coverage over the three genes zgal_4181-zgal_4183 encoding MP/PP1/ZgHAD with a clear drop in coverage upstream zgal_4181 (Fig. S3), supporting the hypothesis that they might form an operon. The genetic context in the same genomic region for Maribacter and Zeaxanthibacter genomes showed the absence of the 3 homologous genes including the HAD1-type gene. The same situation was found in Cellulophaga genomes where a fourth gene (amidohydrolase) was also missing. Indeed, Chapter 1 ______________________________________________________________________________ -53 -the genomes of Leeuwenhoekiella (6 strains), Gramella (4 strains) and Zeaxanthibacter (one strain) have homologs of the ZgHAD gene, but without any genomic context conservation (Fig. 2 and Fig. S2). Only 3 of the 6 Zobellia strains feature a second L-2-HAD-encoding gene, belonging to the HAD2 phylogenetic clade, on their genomes (Fig. 2). The analysis of the genetic region showed that 3 genes are conserved around HAD2type gene, namely a transcriptional regulatory protein from the TetR family (TetR TF) upstream and an uncharacterized oxidoreductase (UOR) and an Asp/Glu aminotransferase (D/E AT) downstream (Fig. 2 and Fig. S2). The comparison with the genomes of other genera (Maribacter, Cellulophaga, Algibacter and Nonlabens) showed that they sometimes present a HAD2-type gene homolog but never a HAD1-type gene. It revealed also that the synteny of the TetR TF, HAD2-type and UOR encoding genes were always conserved. Furthermore, this study showed that some genera of Flavobacteriaceae like Tenacibaculum or Muricauda had no L-2-HAD-encoding gene.

Figure 2: Genetic context of the two types of haloacid dehalogenases identified in

Flavobacteriaceae. On the left, the phylogenetic tree of 10 genus clades was constructed using the comparative genomics tool for genome clustering of MaGe website. The yellow round and square symbols indicated the presence of homologs genes belonging, respectively, to HAD1 or HAD2 phylogenetic groups. The middle part represents the genetic context around HAD1-type genes found in Zobellia genomes. The synteny in this genomic region of other genera is indicated below (in black). In addition, the specific genomic context around HAD1-type genes is shown in Leeuwenhoekiella, Gramella and Zeaxanthinibacter genomes, respectively, in red, blue and green. At the right of the panel, the gene synteny is shown around HAD2-type gene. Conserved genes between species are shaded in black, partially conserved genes are shaded in grey and

Production, purification and enzymatic characterization of the recombinant ZgHAD

To characterize the biochemical activity of ZgHAD, the recombinant ZgHAD enzyme was produced in E. coli and purified to homogeneity (Fig. S4), yielding 300 mg.L -1 culture. SDS-PAGE confirmed the purity and correct size of the protein that appeared as a unique band at the expected 26.3 kDa molecular mass (Fig. S4). Based on size exclusion chromatography, the protein eluted as a homodimer in solution. A standard colorimetric assay solution based on phenol red was used to first determine the substrate specificity of recombinant ZgHAD. Thirteen substrates with different carbon chain lengths (C2 to C4) and halogen atom nature, position and number were tested (Fig. 3). Within 30 minutes, the recombinant ZgHAD showed activity only towards L-enantiomer substrates with short carbon chains (C2 and C3). The enzyme was active towards substrates, that were monoiodinated, monobrominated or monochlorinated on the α-carbon position. The recombinant ZgHAD enzyme was then able to react with iodoacetic acid (IAA), bromoacetic acid (BAA), chloroacetic acid (CAA), L-2-bromopropionic acid and L-2chloropropionic acid (Fig. 3). The fastest activity was observed with IAA and BAA, leading to an acidic yellow coloration within 5 min. The chloroacetic acid (CAA) and the L-2-bromopropionic acid were turned partially into their corresponding alcohols at the same time, giving an orange coloration. They were totally transformed between 10 and 30 min (Fig. 3). The conversion of the L-2-chloropropionic acid was achieved after more than 30 min. Longer incubations up to 16 hrs did not evidence activity on the 8 other substrates (data not shown), confirming that the D-2-haloacids, multi-chlorinated or brominated acetic acids, and the halobutyric acids cannot be processed by the recombinant ZgHAD.

To further compare the catalytic properties on both iodinated and brominated compounds, Chapter 1 ______________________________________________________________________________ -56 -IAA and BAA were used as substrates to calculate kinetic parameters of ZgHAD. The Vmax and Km value with IAA were 1.12 µM.sec -1 and 0.31 mM, respectively and with BAA they were 1.70 µM.sec -1 and 0.46 mM, respectively.

Effects of haloacetic acids on growth of different bacterial strains

The growth of Z. galactanivorans in the presence of BAA and IAA was compared to that of two Tenacibaculum bacterial species (T. discolor and T. gallaicum), which are phylogenetically closely-related to Z. galactanivorans (85.7% and 86.35% 16S rRNA identity respectively) but do not possess any L-2-HAD homolog gene. Bacterial cultures Chapter 1 ______________________________________________________________________________ -57 -were grown in liquid ZoBell medium with the highest concentrations of BAA (10 mM) or IAA (2 mM) tolerated by Z. galactanivorans. After 16 h, cell densities of both Tenacibaculum strains were < 5% in presence of BAA and IAA compared to unamended ZoBell medium. By contrast, the cell densities of Z. galactanivorans WT were still 20% with BAA and 30% with IAA compared to the unamended ZoBell condition. (Fig. 4A).

Therefore, the presence of a L-2-HAD-encoding gene in Z. galactanivorans compared to Tenacibaculum strains suggests an increased resistance to BAA and IAA.

In order to test this hypothesis, we investigated the effect of the zghad gene deletion on the Z. galactanivorans tolerance to haloacids. On solid medium, BAA or IAA induced clear growth inhibition zones for the wild-type strain (WT), with apparent diameters of 1.3 and 3.4 cm, respectively (Fig. 4B). No inhibition zone was observed with the buffer alone.

Compared to the WT, the inhibition zones observed for the zghad-deleted strain, Δhad, were larger, showing an increase of 85% for the BAA (apparent diameter 2.4 cm) and 35% for the IAA (4.6 cm). Consequently, the deletion effect appeared to be more pronounced with BAA than IAA. To confirm these results, we compared the growth rates of the WT and Δhad strains in liquid ZoBell medium supplemented with increasing concentrations of BAA or IAA, until reaching the upper toxic limit. For both halogenated compounds, all the culture conditions showed a reduced cell density for the Δhad mutant compared to the WT (Fig. 4C and4D). The greatest effect of the zghad deletion was obtained with 1 mM BAA where the growth of the WT strain was still 60% and that of the Δhad strain was only 10% compared to the control culture conditions without haloacid. Above 2 mM of BAA, the growth rate of the WT strain dropped at 25%, and reached 15% for 4 mM of BAA, whereas that of the Δhad strain remained under 10% of the corresponding control growth rate. The Chapter 1 ______________________________________________________________________________ -58 -growth rates of both WT and Δhad strains were close to 0% after incubation with 8 mM of BAA. More than 2 mM of IAA was lethal for both strains. However, the WT strain growth was higher than the Δhad mutant between 0.2 and 0.4 mM of IAA (Fig. 4D).

In order to further test the role of ZgHAD in haloacid tolerance, the recombinant E. coli expression strain (ZgHAD-BL21) was grown in parallel with a control E. coli strain transformed with an empty pFO4 plasmid (pFO4-BL21) (Fig. 4E). The control pFO4-BL21 strain did not grow with the upper BAA and IAA concentrations (4.9% and 6.4% of the relative growth compared to that of an unamended LB medium) while the ZgHAD-BL21 strain was able to reach 86% and 77%, respectively. The pFO4-BL21 strain was also strongly inhibited in the presence of 0.5 mM of BAA with 57% relative growth, compared to the ZgHAD-pFO4 strain, which showed no difference compared to the control (94% relative growth).

Distribution of the two L-2-HAD major phylogenetic groups in the environment

Our phylogenetic analysis suggested that the two different phylogenetic groups A and B, showing different conserved amino-acid signatures, could be differently distributed in the terrestrial and marine environments (Fig. 1). To test this hypothesis, we investigated (i) the overall metagenomic abundance distribution of L-2-HAD homologs in contrasted ecosystems using IMG/MER database (IMG/MER) and (ii) the specific marine distribution of the L-2-HADs through their gene number and gene expression using the Ocean Gene Atlas database (OGA). Among the 25 analyzed ecosystems corresponding to 2,500 metagenomes of IMG/MER (Table S3), a total of 4,265 hits were found. The group A L-2-HAD sequences were ten times more frequent (3,800 hits) than the group B L-2-HAD sequences (365 hits). This preponderance of group A L-2-HADs was verified for all the five major ecosystems of terrestrial, aquatic non-marine and marine habitats with 75 to 95% of the overall hit identification represented by the group A L-2-HAD sequences (Fig. 5A). Nevertheless, the environmental distribution differed in the 5 datasets (Fig. 5B-C).

The group A L-2-HAD sequences were almost equally distributed between the non-marine and marine fractions (43.9 and 56%, Fig. 5B). By contrast, the majority of group B hits originated from marine biotopes (72.6% altogether, Fig. 5C). As shown in Table S3, the group A L-2-HADs are encountered mostly in marine intertidal (764 hits) and coastal (554 hits) zones and in forest soils (560 hits). Moreover, they seemed to be more present in freeliving environments (1768 sequences representing 46.5% and 1195 sequences representing 31.4% for marine and terrestrial environments, respectively) than in host-associations (9.5% and 7.8% for marine and terrestrial hosts only). By contrast, the group B L-2-HADs were found in bacteria closely linked to marine hosts (28.8% of all the group B hits)

Chapter 1 ______________________________________________________________________________ -61 -compared to terrestrial hosts (6% of hits, Fig. 5C). Furthermore, they were particularly found in association with marine macroalgae (76 hits, representing 72.6% of the marine host-associated subset and 20.8% of all the group B hits). Other characteristics of this group B L-2-HADs revealed that these sequences are also principally present in the marine coastal and in the non-marine saline/alkaline environments (51 and 39 hits respectively, 31.9% and 90.7% of their subset, 14% and 10.7% of all the group B hits) (Table S3).

The distribution of the group A and group B L-2-HADs was assessed more precisely in marine environment using OGA (Table S4). Interestingly, a similar order of magnitude of group A sequences was obtained when the marine DehPsi and DehDEH99 proteins were used as queries (166 and 214 hits) while a highest number of hits (about 3 to 7 fold) was retrieved using the 15 terrestrial sequences. The three selected L-2-HAD protein sequences from the phylogenetic group B (ZgHAD, DehRhb and WP_038235908) allowed to retrieve 204 to 234 hits (Table S4), which all feature the conserved amino acid consensus DETF(S/T)KYAHD. The group A hits were detected in the metagenomes of 66 stations worldwide with an overall gene abundance of at least 10 -4 (Fig. 5D). Group A homologs were more abundant in temperate and tropical zones than in arctic and subarctic zones. By comparison, the group B hits were detected in the metagenomes of 64 stations also welldistributed around the globe but with lower abundances (Fig. 5E). We further evaluated the expression of these L-2-HAD homologs by searching hits in the OGA metatranscriptomes. Overall, the representation of group A sequences in metatranscriptomes was low compared to their corresponding detection in metagenomes.

The scatterplot of the normalized abundance in metatranscriptomes vs. metagenomes showed that all but one station fell below the 1:1 line, confirming a comparatively low Chapter 1 ______________________________________________________________________________ -62 -expression of the group A L-2-HAD-encoding genes in most oceanic regions (Fig. 5D).

Conversely, the metatranscriptomic abundance of group B L-2-HADs was more heterogeneous, and often higher than the corresponding metagenomic abundance for Atlantic and Pacific stations (Fig. 5E). The scatterplots demonstrated that the ratio of group B L-2-HAD-encoding genes abundance in metatranscriptomes vs. metagenomes was ≥ 1 for 75% of the 64 stations, suggesting a consistent gene expression. In particular, the ratios of 11 stations fell well above the 1:1 line (orange colored zone, Fig. 5E), with a The biochemical properties of this newly characterized marine L-2-HAD were compared to other previously characterized L-2-HADs, mostly in Proteobacteria. As also shown by the phylogenetic analysis, a poor conservation of amino acid sequences between L-2-HADs from group A and group B suggested differences in the conservation of the global protein folding, but also in the biochemical properties. ZgHAD exhibited highest activity towards C2 and C3 carbon-chain substrates with a preference for bromo and iodoacetate, similar to DehRhb (Novak et al., 2013a), DehIVa (Schmidberger et al., 2007), DehPsi (Novak et al., 2013b) and H-2 (Kawasaki et al., 1981) which use BAA as best substrate.

ZgHAD did not show any activity towards substrates with a longer carbon-chain or towards dihalogenated acid substrates, contrasting with other L-2-HADs like DehSft (Rye et al., 2009), DhlB (Ridder et al., 1997), DehPsi (Novak et al., 2013b) from the phylogenetic group A, suggesting some enzymatic specificities of the marine L-2 HADs from the phylogenetic group B. The Km value of ZgHAD for BAA was 0.46 mM, whereas it varied from 1.1 to 6.72 mM for other L-2-HADs. Notably its closest relative DehRhb showed a higher Km value of 6.72 mM (Novak et al., 2013a) revealing a lower affinity for BAA compared to ZgHAD. The Km of ZgHAD for IAA was lower (0.31 mM) compared to Chapter 1 ______________________________________________________________________________ -65 -BAA suggesting a higher affinity of ZgHAD for the iodinated substrate. The IAA has been previously shown to be a substrate for several L-2-HADs of the phylogenetic group A like DEH99 [START_REF] Zhang | Purification and characterization of 2-haloacid dehalogenase from marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis[END_REF], L-DEX (Liu et al., 1994), DhlS5I (Köhler et al., 1998), and H-2 (Kawasaki et al., 1981). Previous assays with the characterized L-2-HADs did not always include iodinated substrates. As iodide is a better leaving group than bromide or chloride, it is likely that most L-2-HAD enzymes, are also active on IAA and therefore on both brominated and iodinated short carbon chains, especially those from seawater origin, as the halocarbons are predominant in the marine environment. However it is the first time that IAA was reported to be a best substrate than other haloacids for a L-2-HAD. It would be interesting to confirm that by the calculation of enzymatic parameters with IAA for other L-2-HADs. In contrast, DEH130 of Pseudomonas stutzeri, isolated from the marine sponge Hymeniacidon perlevis, was unable to transform iodoacetic acid [START_REF] Zhang | Purification and characterization of a dehalogenase from Pseudomonas stutzeri DEH130 isolated from the marine sponge Hymeniacidon perlevis[END_REF].

Z. galactanivorans was able to grow in the presence of higher concentrations of BAA and IAA than its Δhad knockout mutant and, than both fish pathogens T. discolor and T.

gallaicum. The strong growth inhibition induced by BAA and IAA in strains lacking a L-2-HAD gene suggested it could confer a selective advantage to Z. galactanivorans in the presence of toxic haloacetic acids in its environment. This potential benefit was confirmed by the improved tolerance of the E. coli cells expressing ZgHAD to these haloacids. As ZgHAD enzyme was produced inside the E. coli cells (as it is also predicted for Z. galactanivorans), this result suggested that these bacteria could import BAA and IAA into their cytoplasm for detoxification. Soil bacteria such as Burkholderia cepacia MBA4 or Xanthobacter autotrophicus GJ10 have been studied for their capacity to grow on and Chapter 1 ______________________________________________________________________________ -66 -metabolize haloacid compounds. They possess the genetic tools to internalize and degrade a large range of organic halogenated compounds that are often toxic [START_REF] Kwok | Proteomic analysis of Burkholderia cepacia MBA4 in the degradation of monochloroacetate[END_REF]Mena-Benitez et al., 2008;Horisaki et al., 2011). In B. cepacia MBA4, the dehalogenase gene dehIVa and the downstream permease gene dehIVp were shown to form an inducible operon that mediates the transformation and uptake of halogenated acids (Yu et al., 2007).

No clear haloacid operon, permease gene or equivalent transport system were identified within Z. galactanivorans genome. Nevertheless, the detoxification of haloacetic acids was efficient in Z. galactanivorans suggesting that an unidentified transport system might exist.

The haloacetic acids are converted to glycolate by the L-2-HADs. The enzymes catalyzing the glycolate catabolism (Zgal_4160/Zgal_4169 and Zgal_4659) are predicted to be encoded by the genome of Z. galactanivorans as shown in its KEGG metabolic network and schematized in Fig. 6. They might link BAA and IAA degradations either to the glyoxylate cycle or to the vitamin B6 metabolism via glycolaldehyde (Fig. 6). The Δhad mutant of Z. galactanivorans resisted better to haloacetic acids than the Tenacibaculum strains lacking L-2-HAD. Other unidentified genes could therefore be involved in the resistance against haloacetic acids in Z. galactanivorans, as it was previously demonstrated for BAA resistance in E. coli [START_REF] Desai | Recruitment of genes and enzymes conferring resistance to the non-natural toxin bromoacetate[END_REF]. Altogether, our functional results and gene mining in Z. galactanivorans suggested that a complete degradation pathway of short carbon chain haloacids could be potentially present inside the cell, downstream of ZgHAD (Fig. 6). It would be interesting to further compare transcriptomic regulations in Z. galactanivorans and in its Δhad mutant in the presence of BAA or IAA to elucidate all the metabolic processes involved in haloacid intracellular degradation.

Chapter 1 ______________________________________________________________________________ -67 -All the available sequenced Zobellia isolates have one or two copies of L-2-HAD-encoding gene in their genome, a trait that might be a selective advantage linked to the presence of short haloacetic acids, in their natural environment. Indeed, coastal ecosystems could host a permanent natural production of toxic haloacids through various processes (Fig. 6). First, low-molecular-weight organic acids such as acetic acids are ubiquitous in seawater, produced either via respiration, microbial degradation of lipids or photochemical breakdown of dissolved organic matter (Liang et al., 2020). They might be converted to the corresponding halogenated acetic acids abiotically or biotically, as reported previously for other environments. For example, the decomposition of forest soil organic matter leads to the production of CAA (Matucha et al., 2003;Laturnus et al., 2005), potentially due to the action of fungal and bacterial chloroperoxidases [START_REF] Wever | Vanadium Chloroperoxidases: The Missing Link in the Formation of Chlorinated Compounds and Chloroform in the Terrestrial Environment?[END_REF]. In line with this, we hypothesize that the combination of vanadium-haloperoxidases and L-2-HADs could also contribute to the recycling of acetic acids dissolved in seawater through the production of BAA and IAA intermediates (Fig. 6). Second, brominated or iodinated compounds are produced by some other marine bacteria, and also by a range of marine macro-organisms living in close association with bacteria (e.g. corals, sponges, macroalgae) [START_REF] Gribble ; Gribble | Naturally Occurring Organohalogen Compounds -A Comprehensive Update[END_REF].

In particular, marine macroalgae are known to release organic brominated and iodinated toxic compounds in the seawater (Belkin, 1992;[START_REF] Paul | Chemical defence against bacteria in the red alga Asparagopsis armata: Linking structure with function[END_REF]. Some red algae like Asparagopsis taxiformis have been reported to produce haloacetic acids [START_REF] Woolard | Halogenated acetic and acrylic acids from the red alga Asparagopsis taxiformis[END_REF]Fenical, 1982). Moreover, haloalkanes like diiodoethane produced by kelps in biotic interactions could be sequentially transformed into haloacetic acids similarly to the dichloroethane degradation found in Xanthobacter autotrophicus GJ10 (Van der Ploeg et al., 1991).Since all known Zobellia species live with or close to macroalgae, including red Chapter 1 ______________________________________________________________________________ -68 -algae such as Delesseria sanguinea (Barbeyron et al., 2001) and brown algae such as Fucales and Laminariales (Nedashkovskaya et al., 2004a;[START_REF] Martin | The cultivable surface microbiota of the brown alga Ascophyllum nodosum is enriched in macroalgal-polysaccharide-degrading bacteria[END_REF], it is likely they regularly experience the release of haloacetic acids or haloalkanes (Fig. 6). The haloacetic acids present in the coastal seawater might also result from anthropogenic activities (e.g. agriculture, water-treatment stations). Chlorine residuals and humic acids of effluents can react with bromide-rich coastal seawaters (0.8 mM) to form BAA [START_REF] Gordon | Ecotoxicology of bromoacetic acid on estuarine phytoplankton[END_REF]. Similar reactions might occur for the IAA formation, as seawater contains 0.3 µM of iodide (La Barre et al., 2010). The catabolic properties towards both BAA and IAA might reflect the genetic adaptation of Zobellia to these halogen-rich marine ecological niches, and especially those formed by the coastal macroalgal beds (Fig. 6). The phylogenetic tree of L-2-HADs revealed two major distinct phylogenetic groups that were correlated to a specific amino acid signature corresponding to differences in catalytic sites. The phylogenetic group A contained a majority of terrestrial but also marine sequences and the phylogenetic group B was essentially composed of marine ones. Our study also highlighted the important frequency and diversity of marine L-2-HADs, especially of those belonging to the phylogenetic group A, which are still to be characterized as being important actors of halogenated compounds biotransformation and halogen biogeochemical cycle in open marine environments.

MATERIAL AND METHODS

Chemicals

Iodoacetic acid (I4386), bromoacetic acid (17000), chloroacetic acid (C19627), D-2bromopropionic acid (18165), L-2-bromopropionic acid (38551), D-2-chloropropionic acid (306800), L-2-chloropropionic acid (306797), trichloroacetic acid (91233), 3iodopropionic acid (I10457), 3-bromopropionic acid (101281), 2-chlorobutyric acid (24008), iodoacetamide (I1149), dibromoacetic acid (242357) were obtained from Merck.

Bacterial strains and plasmids

All the bacterial strains and plasmids used in this study are listed in Table S6.

Chapter 1 ______________________________________________________________________________ -72 -

Phylogenetic analysis

The selected set of L-2-HAD proteins was derived from NCBI BlastP queries against the RefSeq database. Sequences were loaded into the NGPhylogeny.fr "A la carte" pipeline and analyzed as follows. A total of 352 protein sequences were aligned using MAFFT under default parameters then cleaned with trimAl resulting in 165 informative positions over the 400 aligned positions (see Table S2 for complete information). A maximum likelihood phylogenetic analysis was carried out using default parameters of the PhyML-SMS tool allowing the best substitution model selection. Bootstrap analysis with 100 replicates was used to provide estimates for the phylogenetic tree topology and it resulted S3). The BlastP results of group A and group B sequences were expressed as hit number abundances in each dataset and subset, and as overall percentages.

Chapter 1 ______________________________________________________________________________ -73 -

Oceanic distribution and expression in OGA databases

The BlastP searches were carried out with the 20 representative L-2-HAD sequences against the Ocean Gene Atlas (OGA) database (http://taraoceans.mio.osupytheas.fr/ocean-gene-atlas), comprising metagenomic OM-RGCv2+G

and metatranscriptomic OM-RGCv2+T data collections from the Tara Oceans stations [START_REF] Villar | The Ocean Gene Atlas: exploring the biogeography of plankton genes online[END_REF]. A stringent e-value cutoff of 10 -30 was selected in order to cover a majority of each phylogenetic clade (Table S4). In a second step, we focused on the DehIVa and ZgHAD BlastP hits respectively as representative sequences of the phylogenetic group A and group B, to compare their geographic repartition, relative gene abundance (metaG) and relative gene expression (metaT) levels. All the data for BlastP searches can be downloaded and extracted at the website. Relative abundance values were calculated based on the sum of all the hits found at a precise site and showed on geographical maps. Relative abundance was further normalized by the number of hits in order to evaluate the average of gene abundance and gene expression for every OGA stations. The "global abundance / hits number" were calculated for each station and predicted as log10 correlation plots analyses of 66 and 64 spots respectively for DehIVa (group A) and ZgHAD (group B) homologs. Finally, environmental parameters were compared using the bubble plots tools available on the OGA website platform.

Genomic context decryption

The had gene context in Flavobacteriaceae was analyzed by the selective tool of synteny reconstruction on the Genoscope platform: Microscope (MaGe; https://mage.genoscope.cns.fr/microscope/home/). All the genomic regions containing a

Chapter 1 ______________________________________________________________________________ -74 -had gene (listed in Table S5) were then aligned to allow the identification of the conserved neighboring genes and the varying ones.

Gene cloning

The ZgHAD-encoding gene sequence (zgal_4183) was cloned from the genomic DNA of Zobellia galactanivorans Dsij T (Barbeyron et al., 2001), using primers Zgal_4183fw and Zgal_4183rv (Table S6). The PCR product was ligated into the pFO4 vector, using BamHI and EcoRI restriction sites and the T4 DNA ligase protocol (New England Biolabs). The recombinant vector was transformed firstly into Escherichia coli DH5α for sequence verification and secondly into E. coli BL21 (DE3) expression strain.

Overexpression and purification

The recombinant E. coli strain expressing ZgHAD was grown in LB medium containing 100 µg.ml -1 ampicillin at 37 °C to an optical density of ~1.0, then the temperature was lowered to 20 °C for 1 hour followed by a 20 h induction of protein production by the addition of 0.5 mM IPTG. After centrifugation (3,000 g, 30 min, 4 °C), the bacterial pellet was stored at -20 °C, then resuspended in 50 mM Tris (pH 7.5), 500 mM NaCl, bovine DNase I (500 U/µL), 0.1 mg/mL lysozyme, Complete™ Protease Inhibitor Cocktail (Merck) and 6 mM MgCl2 and lysed using a French press and the lysate was centrifuged at 23,000 g during 30 min and 4°C. The recombinant protein was purified by a two-step chromatography carried on an ÄKTA Avant purification system (GE Healthcare Life Sciences). The first step was performed on an immobilized nickel affinity HisTrap™ column (GE Healthcare Life Sciences) using an equilibration buffer composed of 50 mM

Substrate specificity and kinetic enzymatic parameters determination

The ZgHAD enzyme specificity was determined by adapting the colorimetric assay from Holloway et al., 1998. The detection was based on a pH decrease when the halogenated substrate was reduced to a hydroxyalkanoic acid. Upon dehalogenation, the release of halide ions X -decreased the pH inducing a color transition of the phenol red pH indicator (from pink to orange to yellow) that was monitored visually. The assay solution contained final concentrations of 0.3 mg.ml -1 recombinant ZgHAD enzyme, 1 mM HEPES, 1 mM EDTA, 20 mM sodium sulfate and 56 µM of phenol red (pH 8.2). The assay was adapted for 48-well plates to test 13 different substrates at 10 mM (iodoacetic acid, bromoacetic acid, chloroacetic acid, D-2-bromopropionic acid, L-2-bromopropionic acid, D-2chloropropionic acid, L-2-chloropropionic acid, trichloroacetic acid, 3-iodopropionic acid, 3-bromopropionic acid, 2-chlorobutyric acid, iodoacetamide, dibromoacetic acid) in 1 mL final volume. The plates were incubated at 20 °C during 30 min then photographed and left Chapter 1 ______________________________________________________________________________ -76 -The colorimetric assay was further used for measuring enzymatic kinetic parameters towards IAA or BAA as substrates. All reactions were performed in triplicates at 20 °C.

Each assay was carried out in a 180 µL reaction mixture. The final concentration of purified ZgHAD protein was 0.3 mg.ml -1 . The kinetics of the reaction was followed by measuring the decrease of absorbance at 560 nm for 10 minutes on a Spark® multimode microplate reader (Tecan Group Ltd. Switzerland). A standard curve was produced by mixing the assay solution with 1 M HCl to final concentrations of 0-2 mM in a total volume of 200 µL. The Lineweaver-Burke plots were used to calculate the Km and Vmax steady-state kinetic parameters.

Deletion of ZgHAD gene in Zobellia galactanivorans

A deletion mutant of the zghad gene was constructed following the previously described method from Zhu et al., 2017. A 2.2-kb fragment corresponding to the first 36 bp of the zghad gene with the region directly upstream was amplified using the primers OEG007 and OEG009 (Table S6). The fragment was digested with XbaI and SalI and ligated into the pYT313 vector cut with the same enzymes to generate the vector pEG1. Similarly, a 2.3-kb fragment corresponding to the last 35 bp of the zghad gene and the region directly downstream was amplified with the primers OEG008 and OEG010 (Table S6). The fragment was inserted between BamHI and XbaI sites of pEG1 to generate the suicide plasmid pEG3. Suicide plasmid was introduced into the wild type Z. galactanivorans Dsij T by conjugation with E. coli S17-1 λpir strain. Erythromycin resistance was used to select the cells having integrated the plasmid in their genome. Resulting colonies were grown overnight in Cytophaga medium (per liter: 1.0 g yeast extract, 1.0 g tryptone, 24.7 g/NaCl, Chapter 1 ______________________________________________________________________________ -77 -0.7 g KCl, 6.3 g MgSO4.7H2O, 4.6 g MgCl2.6H2O, 1.2 g CaCl2.2H2O, 0.2 g NaHCO3) without antibiotics at 30 °C to allow the loss of the plasmid. The cells were then plated onto Cytophaga-agar containing 5% sucrose. The sucrose-resistant colonies were checked for erythromycin sensitivity. The deletion of the zghad gene was confirmed by PCR using primer pairs OEG011-OEG012 (Table S6) and then verified by Sanger sequencing at the GENOMER platform (FR2424; Roscoff Biological Station).

Growth inhibition analyses of marine Flavobacteriaceae and phenotyping of the Z. galactanivorans Δhad mutant

The ability to grow and resist to increasing haloacid concentrations was tested using an antibiogram-like method. Z. galactanivorans wild type (WT) and its deleted had gene mutant (Δhad) were grown on ZoBell 2216-agar plates with a Whatman paper disc at the center. The paper was impregnated with 25 µl solution of either 500 mM BAA or IAA (shown to be in vitro substrates of ZgHAD) or Tris-glycine 100 mM pH 8.0 as negative control. The haloacid effect was determined by monitoring the size of the inhibition zone after 72 h at 20 °C. This experiment was realized two times independently.

Z. galactanivorans WT and both Tenacibaculum species (T. discolor DSM 18842 T and T.

gallaicum DSM 18841 T ) were grown in 3 mL ZoBell 2216 liquid medium with high concentrations of BAA and IAA (2 mM) buffered by 20 mM HEPES buffer pH 7.0 and during 16 h at 20 °C. Each medium was initially inoculated at 1/100 dilution with a twoday preculture. Z. galactanivorans WT strain and the mutant Δhad strain were also grown in ZoBell liquid medium in the same conditions with different concentration ranges of IAA or BAA dissolved in 200 mM HEPES buffer pH 7.0. The final concentrations were defined Chapter 1 ______________________________________________________________________________ -78 -from 0 to 1.6 mM and 0 to 8 mM respectively for IAA and BAA. Each concentration was tested in biological triplicates. The bacterial growth was measured by the mean of OD600 in the respect of technical duplicates. The growth rates were calculated from the slopes of the growth curves between 15 and 20 h, during the exponential phase. They were expressed as a percentage of the standard growth rate in ZoBell medium for each corresponding strain.

Phenotyping of recombinant E. coli tolerance to BAA and IAA

The E. coli BL21 expression strains containing the pFO4 vector alone (pFO4-BL21 strain) or recombined with the zghad gene (ZgHAD-BL21 strain) were grown in LB medium containing 100 µg.ml -1 ampicillin and IPTG overnight at 20 °C. Theses precultures were used to inoculate a fresh LB/Ampicillin medium supplemented with 0.5 mM IPTG at 1/100 dilution. Two concentrations distant of ten folds were tested in triplicates as follows, 0.5 and 5 mM for BAA and 0.16 and 1.6 mM for IAA. After 15 h of incubation at 20 °C, the final OD600 was measured and reported to that of an equivalent culture in standard conditions, without haloacids.

ACKNOWLEDGMENT

We would like to thank Tristan Barbeyron for giving us access to Tenacibaculum strains. This work benefited from the support of the French Government via the National Research Agency investment expenditure program IDEALG (ANR-10-BTBR-04) and via the Centre National de la Recherche Scientifique (CNRS). The PhD project of EG was also supported by Region Bretagne (ARED 2017, projet MHALIN).

INTRODUCTION

The haloacid dehalogenases (HAD) superfamily includes dehalogenating enzymes together with diverse enzymes that hydrolyze carbon-phosphorus bonds, such as epoxide hydrolases, phosphatases, phosphomutases, or nucleotidases. Widely conserved among living organisms, they are involved in a variety of cellular processes ranging from amino acid biosynthesis to detoxification (Burroughs et al., 2006). Since the industrial boom, halogenated xenobiotic pollutants are contaminating soils and aquatic environments. The accumulation of those toxic compounds led to the research of new tools for detoxification and bioremediation.

The "true" dehalogenases of the HAD superfamily are categorized into two types. Type I represents both HADs that have an enantioselective dehalogenating activity on D-2-haloacids (D-2-HADs), and those that have non-stereospecific mechanism accepting both D-or L-2haloacids as substrates (DL-2-HADs). Enzymes from type II specifically act on L-2-haloacids (L-2-HADs, E.C. 3.8.1.2) to produce the corresponding alcohols with an inverted chirality (Hill et al., 1999;Ang et al., 2018). Those enzymes degrade the substrate molecule by a nucleophilic attack on its halogenated Cα by an aspartic acid group that forms an ester bond and release the halide ion. The enzyme-substrate ester bond is then hydrolyzed by another nucleophilic attack by an activated water molecule. This reaction occurs with an inversion of chirality of the final product [START_REF] Liu | Reaction mechanism of L-2-haloacid dehalogenase of Pseudomonas sp. YL: Identification of Asp10 as the active site nucleophile by 18O incorporation experiments[END_REF][START_REF] Nardi-Dei | Bacterial DL-2-haloacid dehalogenase from Pseudomonas sp. strain 113: Gene cloning and structural comparison with D-and L-2-haloacid dehalogenases[END_REF].

There are currently five crystal structures of L-2-HADs available: L-DEX YL from Pseudomonas sp. (Hisano et al., 1996), DhlB from Xanthobacter autotrophicus GJ10 (Ridder et al., 1997), DehIVa from Burkholderia caribensis (Schmidberger et al., 2007;Pan et al., 2015), DehSft from Sulfolobus tokodaii (Rye et al., 2009) and DehRhb from Rhodobacteraceae (Novak et al., 2013). HAD proteins are dimers with two domains in each subunit: a characteristic alpha/beta core domain with a conserved alpha/beta hydrolase fold, similar to the "Rossmann-fold", and many of them have a small cap domain exhibiting varying folds and Chapter 2 ___________________________________________________________________________ -105 -functions. This cap domain is responsible for the diversification of chemistry within HAD superfamily [START_REF] Lahiri | Analysis of the Substrate Specificity Loop of the HAD Superfamily Cap Domain[END_REF]. Those enzymes transform the substrate(s) molecule(s) according to a conserved nucleophilic substitution involving a conserved aspartic acid group that forms an ester bond. In the case of L-2-HADs, that are the only true dehalogenases of the HAD superfamily, the enzyme-substrate ester bond is then hydrolyzed by another nucleophilic attack with an activated water molecule [START_REF] Liu | Reaction mechanism of L-2-haloacid dehalogenase of Pseudomonas sp. YL: Identification of Asp10 as the active site nucleophile by 18O incorporation experiments[END_REF][START_REF] Nardi-Dei | Bacterial DL-2-haloacid dehalogenase from Pseudomonas sp. strain 113: Gene cloning and structural comparison with D-and L-2-haloacid dehalogenases[END_REF]. In contrast to HAD in general, the cap domain is similar in all L-2-HADs and is composed of a four-helix bundle and the active site is flanked by a hydrophobic residue cavity between the core and cap domain (Ridder et al., 1997 ;Schmidberger et al., 2007 ;Rye et al., 2009).

The complete genome sequence of the marine flavobacterium Zobellia galactanivorans, revealed the presence of a type II HAD enzyme. Following cloning and purification, the biochemical characterization of the recombinant ZgHAD enzyme has been performed (Grigorian et al., in prep). Briefly, it is specific towards L-enantiomer substrates with short carbon chain (C2 and C3) and iodinated, brominated or chlorinated on the α-carbon position.

The fastest activity was observed with iodoacetic acid and bromoacetic acid, while the reaction with chloroacetic acid and L-2-bromopropionic acid was much slower. Thus, ZgHAD is a L-2-HAD.

In this paper, we describe the crystal structure resolution of ZgHAD. We present the highresolution three-dimensional structure of this mesophilic dehalogenase together with sitedirected mutagenesis, computational docking and comparison with other related enzymes previously described. The thermostability and solvent stability of this enzyme has also been assessed, in view of potential biotechnological applications.

Chapter 2 ___________________________________________________________________________ -106 -

MATERIALS AND METHODS

Gene cloning and site-directed mutagenesis

The ZgHAD gene sequence (Zobellia_4183) was cloned from the genomic DNA of Zobellia galactanivorans as described by Barbeyron et al., 2001; using primers Zgal_4183fw and Zgal_4183rv (Table S2) The PCR product was ligated into pFO4 vector using BamHI and EcoRI restriction sites and the T4 DNA ligase protocol (New England Biolabs). The recombinant vector was transformed firstly into Escherichia coli DH5α for sequence verification and secondly into E. coli BL21(DE3) expression strain.

ZgHAD mutants H179A and H179N were produced using QuickChange Lightning Sitedirected Mutagenesis (Agilent Technologies). Primers used are listed in Table S2 from supplementary data.

Protein overexpression and purification

As described in Chapter 1.

Thermal unfolding experiments

The proteins were diluted to a final concentration of 10 µM. For each condition, 10 µL of sample per capillary were prepared. The samples were loaded into UV capillaries and experiments were carried out using the Prometheus NT.48 (NanoTemper Technologies) that can detect changes in the fluorescence of tryptophan (Trp) residues in the proteins. The

Thermostability measurements

The thermostability of the protein was determined by incubation in the presence of monochloroacetic acid (MCA) at different temperatures between 10 and 90°C for 30 minutes.

The assay solution was added then incubated on ice for 1h. The solvent stability was investigated by incubating the enzyme in the presence of monochloroacetic acid and with ethanol, methanol, acetonitrile and dimethylsulfoxide (DMSO) at concentrations between 10% and 80% for 1h at room temperature. The assay solution was added then incubated on ice for 1h. The activity was determined by measuring the absorbance at 560 nm as previously described.

Crystallization, data collection, structure determination and refinement

The purified L-haloacid dehalogenase (ZgHAD) and its variants (ZgHAD H179A and ZgHAD H179N) were concentrated using a 10 kDa membrane Amicon Ultra-15 centrifugals Filters (Mercks Millipore) at 3600 g and 4°C until a final concentration of 15 mg/mL was reached.

Hanging drops were prepared by mixing 2μL of ZgHAD (15 mg/mL) and 1 µL of reservoir, and were equilibrated by vapor diffusion at 20°C. Diffraction-quality crystals appeared after approximately three days in a condition containing 0.33 M potassium thiocyanate and 31% (w/v) PEG3350 for ZgHAD and ZgHAD H1179A; and containing 25% (w/v) PEG3350, 0.1 M Tris-HCl pH 8.5 and 0.2 M NaCl for ZgHAD H179N. Crystals were soaked in their reservoir solutions supplemented with 10% (v/v) glycerol before flash-freezing in liquid nitrogen.

Diffraction data were collected at 100 K at microfocus beamline Proxima 2-A (Soleil, France).

The data were processed using XDS [START_REF] Kabsch | A significant role of Arg41 residue in the enzymatic reaction of haloacid dehalogenase L-DEX YL studied by QM/MM method[END_REF] and scaled with Aimless from the CCP4 program package [START_REF] Winn | Overview of the CCP4 suite and current developments[END_REF]. The structure of ZgHAD was solved by molecular replacement with the CCP4 suite program MolRep [START_REF] Vagin | Molecular replacement with MOLREP[END_REF] using the marine Rhodobacteraceae L-Haloacid Dehalogenase as the starting model (PDB code: 2YML).

Chapter 2 ___________________________________________________________________________ -108 -Iterative rounds of model building and refinement were carried out using Coot [START_REF] Emsley | Features and development of Coot[END_REF] and the Phenix.Refine module of PHENIX [START_REF] Adams | PHENIX: a comprehensive Python-based system for macromolecular structure solution[END_REF]. The validation of the crystal structures was performed with MolProbity [START_REF] Chen | MolProbity: all-atom structure validation for macromolecular crystallography[END_REF].

Computational docking

Computational docking of haloacetic acids (Cl-, Br-and I-) and of the 2-Bromopropionic acid to ZgHAD X-ray structure was performed using AutoDock Vina [START_REF] Trott | AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading[END_REF]. The initial coordinates of these molecules were generated from the SMILES string using PHENIX.eLBOW [START_REF] Liebschner | Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix[END_REF]. The ZgHAD protein was kept rigid during docking. A docking grid with dimensions 25 Å × 25 Å × 25 Å, encompassing the entire active site, was used. The calculation yielded 9 possible models, of which the one with the highest ranking was selected as the most likely. Then the complexes were energy minimized using the Yasara energy minimization server [START_REF] Krieger | Improving physical realism, stereochemistry, and side-chain accuracy in homology modeling: Four approaches that performed well in CASP8[END_REF].

Chapter 2 ___________________________________________________________________________ -109 -

RESULTS

Thermostability and solvent stability

A temperature gradient between 20°C and 95°C was applied in the aim to evaluate the stability zone of the protein fold with the Prometheus NT.48 (Nanotemper). ZgHAD is denaturated between 60 and 70°C with a melting temperature of 65°C (Fig. 1A). A similar experiment was achieved by Dynamic Light Scattering (DLS) measurement and exhibited a denaturing peak confirming the melting temperature of 65°C (data not shown). These correlated results suggest that ZgHAD is thermostable up to the maximal temperature of 65°C. The thermal stability of the enzymatic activity was investigated between 10°C and 90°C and measured after 30 minutes of incubation. The remaining activities were plotted as a percentage of initial activity (Fig. 1B).

ZgHAD activity was found to be stable up to 50°C and decreased strongly between 60°C and 70°C, from 80% remaining activity to a complete loss of activity. Again, both experiments, on protein denaturation and activity, correlate and show that ZgHAD turns completely inactive and unfolded at 70°C. The solvent stability of ZgHAD was assessed by incubating the enzyme with different concentrations of ethanol, methanol, acetonitrile and dimethyl sulfoxide (DMSO) for one hour before testing the residual activity (Fig. 1C). The effects of ethanol and methanol treatments are very similar. The activity of the enzyme is conserved up to 80% when 0% to 20% of these organic solvents were added. Then, the activity decreased to 20% in presence of 20% to 40% of alcohols. Above 40% of methanol or ethanol, the activity was almost zero. DMSO had the least inhibitory effect of all the solvent tested. Only 20% of activity loss was observed by the addition of up to 40% DMSO. The activity decreased to 60% with 50% of DMSO and higher concentrations drastically reduced the activity to 10% of the standard without any solvent. In acetonitrile the activity of ZgHAD was drastically reduced to 20% already in 10% of solvent.

It was completely inhibited between 20 and 30% of acetonitrile, and surprisingly it increased

Overall structure of ZgHAD

The crystal structure of ZgHAD was solved by the molecular replacement method at 1.6 Å, using the closest structural representative, which is DehRhb (PDB accession: 2YML) from a marine Rhodobacteraceae. The two proteins share 31% identity and 50% similarity in their amino acid sequences. ZgHAD crystallized with the space group P212121, and unit cell parameters are reported in Table 1. ZgHAD is a homodimer and its monomeric unit folds into two domains, comprising a core domain, formed by the residues 18-30 and 110-238, and a cap domain, formed by the residues 31-109 (Fig. 2). The core domain has a typical "Rossmann fold" which consists of six parallel β-strands surrounded by five α-helices and three 310 helices. All strands show β-strand α-helix β-strand connectivity, except for β-strands 5 and 6, which are connected by a β-turn. 310 helices are found before and after strand β3. The cap domain is composed of four α-helices and a 310 helix is inserted into the core domain between strand β1 and helix α5. The active site is located between the core and the cap domains, right after strand β1. The four helices of the cap domain shield the top of the active site cavity from the solvent (Fig. 2A). The ZgHAD protein crystallizes as a homodimer, with dimensions of 74 x 35 x 44 Å (Fig. 2B).

This oligomeric state is in agreement with the estimated size of the protein in solution as determined by gel filtration during the purification steps as referred in Grigorian et al., 2021.

Up to date, all other L-2-HADs (DehRhb, DehSft, DehIVa and DhlB) have been reported to occur as homodimers, as well. Similar to the dimers of other L-2-HADs, the two subunits are related by a two-fold symmetry axis running nearly parallel to the α2 helix. On dimer formation, 16% of each monomer's accessible surface is buried in comparison with 18.7% in DehRhb, 13.4% in DehSft, 13.5% in L-DEX-YL and 19% in DehlB. The subunit interface of ZgHAD is

Chapter 2 ___________________________________________________________________________ -113 -mainly formed by helices α2 and α3. In addition, a salt bridge between glutamate E53 of helix α2 of one subunit and arginine R188 of helix α8 of the other (and vice versa) reinforce the interaction between the two monomers.

Active site of ZgHAD

ZgHAD shares 31% of identical amino acids with DehRhb. Consequently there are 69% of differences in primary sequence between them. However, the active site of ZgHAD is strictly conserved with DehRhb except for one amino acid that changes from a serine (S120) in ZgHAD to a threonine (T124) in DehRhb (Fig. 3A). This corresponds to a minor difference, as both amino acids are exchangeable with a conservative function. Previous site-directed mutagenesis performed on other L-2-HADs has shown that nine amino acids are essential and conserved within those enzymes (Adamu et al., 2016). Those essential amino acids are highlighted in a protein alignment of structurally characterized dehalogenases (Fig. S1 and Table S1). There are three amino acids that are different in ZgHAD. The positively charged arginine that binds and stabilizes the halide ion in the active site of DhlB (R39) and L-DEX (R42) (Ridder et al., 1997 ;Kondo et al., 2014) is replaced by a non-polar phenylalanine (F43) in ZgHAD. In L-DEX and DehIVa, a serine residue (S175 and S176, respectively) is described to form a hydrogen bond with the catalytic aspartate (D10 and D11, respectively) to maintain a suitable orientation of its carboxyl group for the nucleophilic attack on the substrate (Hisano et al., 1996 ;Schmidberger et al., 2007). In ZgHAD and DehRhb, this serine is replaced by an alanine (A177) that cannot bind with the catalytic D14. Instead, a threonine (T18) and a lysine (K153) form a hydrogen bond with the carboxyl group of the catalytic aspartate (D14). In DehRhb, a histidine (H183) was proposed to participate to the activation of the catalytic water molecule instead of the conserved asparagine in other characterized L-2-HADs (Novak et al., 2013). Like

DehRhb, ZgHAD possesses a potential catalytic histidine at position 179 as part of the Chapter 2 ___________________________________________________________________________ -114 -hydrophobic pocket around the active site, which is an asparagine in the other L-2-HADs. This histidine in ZgHAD has been changed to alanine and asparagine by site directed mutagenesis to generate respectively ZgHAD_H179A and ZgHAD_H179N mutant enzymes. Both mutants were shown to be inactive using the colorimetric assay adapted from Holloway et al., 1998 (data not shown). To analyze potential structural rearrangements due to these mutations, we have also crystallized and solved the crystal structures of both ZgHAD_H179A and ZgHAD_H179N. No major structural differences were observed for the H179N and H179A mutants, except for the position of glutamate E17. In both cases, the main chain of E17 moved from 1 Å to 2 Å towards the active site and the side chain displayed an alternative conformer that moved the functional group by 6 Å closer to the catalytic residues, which may possibly interfere with substrate binding or water activation (Fig. 3B and3C). In the case of the mutant ZgHAD_H179N, the crystal structure revealed 13 dimers displaying a helical arrangement within the asymmetric unit, as shown in Figure 4A. Notably, the same space group and large unit cell parameters as for ZgHAD_H179N were also observed for certain wild-type ZgHAD crystals, indicating that this spatial arrangement (dependent on the pH of the crystallization condition) is also possible for the native protein. But due to diffraction at low resolution (i.e.

3.5 -3.2 Å), these crystals were not investigated further, since better diffracting crystals were obtained for the wild-type protein. For ZgHAD_H179N, when all independent dimers of the asymmetric unit are superimposed based on one monomer, these monomers match well with an rmsd between 0.288 and 0.445 (Fig. 4B). However, the other monomers, which are not included in the superimposition calculations, appear to display different relative positions with respect to the first monomer, with largest main chain distances of up to 2.7 Å (Fig. 4C) for the outer structural elements. This indicates that the dimer arrangement has some flexibility, allowing a rotational freedom at the interface of the two monomers.

Putative binding residues and docking analysis

Docking analyses with IAA, BAA, CAA and 2-BPA were undertaken. The binding energy of those substrates was comprised between -5.6 and -6.1 kcal/mol, meaning they can all be considered as preferable substrates for ZgHAD. The complexes with the best conformation were selected from nine docking positions for each ligand. V15, N16, S120, N121 and K153

were found to interact directly with each substrate through hydrogen bonding (Fig. 5). They each interact by forming a hydrogen bond to an oxygen atom of the carboxylate group present in the different substrates. There is no hydrogen bond observed between V15 and 2-BPA. N16

forms two hydrogen bonds with BAA and 2-BPA but only one with IAA and CAA.

According to Novak et al., 2013, a 'halogen cradle' is formed by the side chains of residues F47, I51, F66, N125 and W185. The corresponding amino acids in ZgHAD (F43, L47, F62, N121 and W181) are identically conserved and appear be similarly involved in stabilizing the halogen atom. In the complexes obtained by docking, the catalytic D14 is hydrogen bonded to T18 and K153, with D14-OD1 also at a distance of 3.0 Å from the C2 of substrate molecules (for IAA, BAA and CAA) and 4.6 Å from 2-BPA (Fig. 5).

DISCUSSION

L-2-HADs were shown to be robust enzymes as they have a high degree of thermostability and resistance in organic solvents. L-DEX from Pseudomonas sp. YL retained 100% of its activity when incubated at 60°C for 30 minutes (Liu et al., 1994) and DehRhb from Rhodobacteraceae retained 90% activity when incubated at 55°C (Novak et al., 2013). Similarly, we also observe a high thermal stability for ZgHAD as it conserves 100% of its activity when incubated up to 55°C. On the other hand, while ZgHAD was shown to be quite stable in ethanol, methanol, acetonitrile and DMSO at low concentrations, it is rapidly inactivated at high concentrations.

Similar results were presented for DehRhb (Novak et al., 2013) and DehSft (Rye et al., 2009).

ZgHAD and DehRhb appear to be more stable than DehSft, when incubated with the same organic solvents. The optimum pH was not determined since the activity assay is pH dependent, but according to previous studies, L-2-HADs proteins were generally reported to be alkaline (Van der Ploeg et al, 1991;Liu et al., 1994). In this respect, it is interesting to note the possible flexibility at the interface of the dimer at alkaline pH, as shown by the dimer positional variability of ZgHAD_H190N crystallized at pH 8.5 that could be important for the catalytic turnover.

The overall dimeric ZgHAD is similar to other L-2-HADs, but there are main differences regarding lengths of loops and interface interactions. The global structure is most similar to DehRhb (Novak et al., 2013), as indicated by the rmsd of 1.21 Å. In the same line, interactions involved in dimerization of ZgHAD are most similar to those of DehRhb and different from DehSft (Rye et al., 2009), L-DEX (Hisano et al., 1996) and DhlB (Ridder et al., 1997), as described by Novak et al., 2013. Nevertheless, some interesting differences between DehRhb and ZgHAD can be seen at the entrance of the active site.

ZgHAD shares low sequence identity (between 20 and 30 %) with the other characterized L-2-HADs. Interestingly, the marine ZgHAD confirms the differences of catalytic features

Chapter 2 ___________________________________________________________________________ -119 -described for DehRhb, in contrast to L-2-HAD originating from soil bacteria. While the aspartate D14 (ZgHAD numbering) performing the first step of the reaction is the main catalytic amino acid for all of these enzymes, the activation of a water molecule is done by a His/Glu dyad for both ZgHAD and DehRhb, whereas an Asp/Lys dyad is responsible of this step in the other structurally characterized enzymes (Novak et al., 2013 ;Schmidberger et al., 2007 ;Nakamura et al., 2009). A similar His/Glu dyad is well-known to operate in a same manner in haloalkane dehalogenases that are widespread in the marine environment [START_REF] Janssen | Evolving haloalkane dehalogenases[END_REF].

Among the nine residues depicted to be most important for catalytic activity (Table S1), the multiple alignment analysis shows that lysine and aspartic acid are fully conserved, while serine and tyrosine are less systematically conserved, but present in all bacterial and archaeal sequences (Fig. S1). Previous reports on site directed mutagenesis have already shown the importance of theses amino acids for protein activity (Kurihara et al., 1995;Pang & Tsang, 2001;Nakamura et al., 2009). Here we describe the high resolution structure of ZgHAD in the unbound state and computationally analyzed the structure by docking the substrate molecules into the active site. The catalytic amino acids are well-conserved with DehRhb and the overall structure showed a dimer with mixed α/β core domain and a four helix bundle cap domain. The catalytic site is located in a cavity between the core and cap domains. The core domain contributes to the catalytic residues and the cap domain stabilizes the substrate and determines substrate specificity [START_REF] Lahiri | Analysis of the Substrate Specificity Loop of the HAD Superfamily Cap Domain[END_REF]. Its 3D model structure is highly similar to the crystal structure of DehRhb from Rhodobacteraceae family (Novak et al., 2013). Only two important residues for substrate binding or catalysis are different between both proteins (L47 and S121 of ZgHAD vs. I51 and T124 of DehRhb). Since serine and threonine are neutral polar amino acids, the substitution between them is unlikely to introduce a strong significant change of the catalytic activity. The same conservative replacement could result for the exchange of hydrophobic amino acids isoleucine to leucine in ZgHAD. The recombinant ZgHAD was shown to have

Chapter 2

___________________________________________________________________________

-120 -preferable activity towards short-carbon-chain substrates and most specifically for C2 compared to C3 haloalkanoic acids. Iodoacetic acid and bromoacetic acid were found to be the best substrates and the catalytic turnover rates were similar for both substrates (Grigorian et al., in prep). When compared with DehRhb, we found that ZgHAD is more efficient to convert the bromoacetic acid. Consequently the replacement of these two amino acids mentioned earlier, which are the only underlined differences among those enzymes, might be associated with substrate affinity. Leucine 47 is located in the cap domain, which is responsible for substrate recognition and binding. However, this hypothesis requires further experimental examination such as site-directed mutagenesis. The high structural similarities, between ZgHAD and DehRhb, suggests that the equivalent His/Glu dyad might be responsible for water activation.

Table S1: Conserved essential amino acids for catalysis in L-DEX, DhlB, DehIVa, DehSft, DehRhb and ZgHAD

I. Introduction

During the annotation of Zobellia galactanivorans genome, 3 homologous VHPO-encoding genes were identified (Barbeyron et al., 2016). Two of them coded for Zg-VIPO1 and Zg-VIPO2, which were previously biochemically characterized as being VHPOs strictly specific to iodide oxidation and were used in structure/function analyses for studying the bases of iodide specificity (Fournier et al., 2014). Their kinetic parameters were measured, showing a high affinity to iodide (Table 3- et al., 2016).

Enzymes Km I-(mM) Kcat I-( sec -1
) Zg-VIPO1 0.22 ± 0.01 1.98 ± 0.05 Zg-VIPO2 0.22 ± 0.02 2.04 ± 0.08 Table 3-1: Kinetic parameters of Zg-VIPO1, and Zg-VIPO2 (Fournier et al., 2014).

The presence of several distinct VHPO activities was assessed earlier for macroalgae, based on protein purification, biochemical and structural characterizations: at least two VBPOs for Ascophyllum nodosum [START_REF] Wischang | Parameters for bromination of pyrroles in bromoperoxidase-catalyzed oxidations[END_REF][START_REF] Wischang | Molecular cloning, structure, and reactivity of the second bromoperoxidase from Ascophyllum nodosum[END_REF], two VBPOs for Corallina pilulifera [START_REF] Itoh | Characterization of nonheme type bromoperoxidase in Corallina pilulifera[END_REF][START_REF] Carter | Reactivity of recombinant and mutant vanadium bromoperoxidase from the red alga Corallina officinalis[END_REF], one VBPO and one VIPO for Laminaria digitata (Jordan et al., 1991;[START_REF] Colin | Vanadium-dependent iodoperoxidases in Laminaria digitata, a novel biochemical function diverging from brown algal bromoperoxidases[END_REF]. Moreover the marine bacterium Streptomyces sp. CNQ-525 possesses three characterized VCPOs [START_REF] Mckinnie | Characterization and Biochemical Assays of Streptomyces Vanadium-Dependent Chloroperoxidases[END_REF].

The third putative VHPO-encoding gene, zobellia_2250, only featured 28% of identity at the amino acid level with Zg-VIPO1 or Zg-VIPO2, while those latter shared 48% of identity. The main goal of this chapter is to characterize the biochemical function and the halide specificity of the third VHPO of Z. galactanivorans, as well as trying to obtain its 3D structure and compare it to Zg-VIPO1.

II.

Material and methods

II. 1. Zobellia_2250 gene cloning, overexpression and purification

Zobellia_2250 was cloned from the genomic DNA of Z. galactanivorans as described by Barbeyron et al., 2001. Specific oligonucleotides were designed according to carry SphI and PstI restriction sites. The forward and reverse primer sequences were 5′-ACCATCACGGATCCGCATGCCAGAAAAACCAAGAAGAACCAAGAGG-3′ and 5′ CTAATTAAGCTTGGCTGCAGCTATTGACCTAGGTGTTTGTTGTAG-3′ respectively.

The PCR product was ligated into a linearized pQE80L vector and then transformed into

Escherichia coli Stellar cells. Then, the extracted recombinant vector was firstly sequenced to ensure a correct DNA construction and secondarily transformed into E. coli BL21(DE3) expression cells, to produce the recombinant protein without the first 21 amino acids of the wild-type Zobellia_2250.

The recombinant bacteria were grown in LB medium in the presence of 100 µg/mL ampicillin at 37°C to an optical density of ~1.0, then the temperature was lowered to 20°C for 1 h followed by a 20 h induction of the protein production by the addition of 0.5 mM IPTG. The culture was then centrifuged at 3,000 g for 30 min at 4°C. The bacterial pellet was treated with a buffer containing 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 500 U/µl bovine DNase I, 0.1 mg/mL lysozyme, Complete™ Protease Inhibitor Cocktail (Merck) and 6 mM MgCl2. The bacterial cells were harvested using a French press and the lysate was centrifuged at 23,000 g for 30 min and 4°C. Then, the recombinant protein was purified by a two-step chromatography carried on an ÄKTA Avant purification system (GE Healthcare Life Sciences). The first step was performed on an immobilized nickel affinity HisTrap™ column (GE Healthcare Life Sciences) using an equilibration buffer composed of 50 mM Tris (pH 7.5), 500 mM NaCl and 50 mM imidazole and an equivalent elution buffer containing 500 mM imidazole instead of 50 mM.

The proteins were eluted with a constant gradient of imidazole concentration. The pooled fractions of the elution peak were concentrated on Centriprep devices to 3 mL and the protein sample was injected on a gel filtration HiLoad™ 16/600 Superdex™ 200 pg column (GE Healthcare Life Sciences). Two independent peaks of purified protein were obtained and pooled separately. Then, they were stored in a buffer containing 20 mM Tris/HCl pH 8.0 and 150 mM NaCl. The purity of the recombinant protein was analyzed using SDS-PAGE (Bio-Rad Mini-PROTEAN® precast gels and systems) and aliquots were frozen at -80°C. A second two-step chromatography purification was performed several months later with a thawed protein sample and a buffer containing 20 mM Tris/HCl pH 8.0 and 50 mM NaCl for the gel filtration. Dynamic

Light Scattering (DLS) measurement were performed on 20 µL of purified protein in a quartz cuvette using ZetaSizer Nano-S instrument (Malverne Instruments).

Protein concentration was determined by measuring absorbance at 280 nm and by using the Beer-Lambert law: A = ε * l * c. The molar attenuation coefficient (ε) was determined from the protein sequence on ProtParam server (https://web.expasy.org/protparam/) and was equal to 83690 M -1 .cm -1 .

II.2. Native gel and spectrophotometric haloperoxidase activity measurements

The VHPO activities were tested on native electrophoresis gels using the o-dianisidine as revealing agent. The running gel was prepared from 0.375 M Tris/HCl buffer pH 8.8 and 12% bis-acrylamide. The stacking gel consisted of 0.125 M Tris/HCl buffer pH 6.8 and 3.8% bisacrylamide. The polymerization was achieved chemically by the addition of 1 mM ammonium persulphate in the presence of 40 pM N,N,N',N'-tetramethyl-1,2-diaminoethane (TEMED) as catalyst accelerator. The samples were supplemented with a native buffer of 0.3 M Tris/HCl pH 6.8, 50% glycerol and 0.05% bromophenol blue. After migration, the gel was incubated in 0.1 mM of o-dianisidine solution, containing 100 mM potassium phosphate pH 7.4 and either 10 mM of potassium chloride, or bromide, or iodide, or no halide, for 2 h at room temperature with gentle shaking. The gel was rinsed with distilled water and placed in a solution of 0.37 mM hydrogen peroxide for revelation during few minutes to several hours.

The spectrophotometric assays used for the enzymatic activity measurements are based on the iodination of thymol blue (TB) and the production of a stable molecule, diiodothymolsulfonphthalein (TBI2), with spectral properties different from those of TB, as described by Verhaeghe et al., 2008b. Briefly, 10 µg of purified enzyme were added to the 250 µL reaction mixture, consisting of 0.1 M phosphate buffer pH 7.2, 100 µM TB, 80 µM to 2.5 mM KI, and 0.42 mM H2O2. The absorbance at 620 nm was recorded on a Safire2 spectrophotometer (Tecan Group Ltd., Switzerland) for 5 min. All reactions were performed in triplicate at 20°C in clear flat-bottomed 96-well microplates (Greiner UV-Star 96-well plates).The Lineweaver-Burke plots were used to calculate the Km and kcat steady-state kinetic parameters.

II.3. Protein stability and activity analysis

The Prometheus NT.48 (Nanotemper) measures the protein thermal unfolding by detecting smallest changes in the fluorescence emission of the tryptophans (ratio 330/350 nm). The Zg-VHPO3 protein was concentrated to 1 µM then centrifuged at 13,000 g for 5 minutes at 4°C.

The thermal unfolding experiments were carried out at a heating rate of 1°C/min. The F350/F330 measurements of the proteins, contained in glass capillaries, were performed by increasing temperature during 90 min.

The protein denaturing was also explored by heating 1 µg of proteins in a thermocycler (Bio-Rad) at different temperatures (from 10°C to 90°C) for 30 minutes. The samples were cooled on ice for 1 h then deposited on a native acrylamide gel. The running gel was prepared from 0.375 M Tris/HCl buffer pH 8.8 and 10% bis-acrylamide. The stacking gel consisted of 0.125 M Tris/HCl buffer pH 6.8 and 3.8% bis-acrylamide. The polymerization was achieved chemically by the addition of 1 mM ammonium persulphate 10% in the presence of 40 pM of the catalyst accelerator, N,N,N',N'-tetramethyl-1,2-diaminoethane (TEMED). After migration, the gel was briefly rinsed with milliQ water and incubated for 1 h in the elution buffer with 1 mM thymol blue and 15 µM potassium iodide. Then the precedent solution was discarded and replaced by 0.42 mM hydrogen peroxide for iodoperoxidase activity revelation.

II.4. Crystallogenesis

Prior to crystallization experiments by a vapor diffusion method, the purified proteins were concentrated at 11.6 mg/mL using a centrifugal filter 30K (Amicon Ultra) within a buffer containing 20 mM Tris-HCl pH 8.0 and 150 mM NaCl. The crystallization experiment was carried out as follows: a drop of the protein sample was mixed with one drop of a crystallization solution containing a precipitant reagent with or without a buffer, salts, organic solvents, polymers. This mixture was deposited next to a reservoir of the crystallization solution at a temperature of 18°C and the vapor diffusion between the drop and the reservoir led to the formation of crystals. This process was very slow and often the crystallization solution was either under-saturated (which prevented any crystal formation) or supersaturated (which generated an amorphous precipitate) (Figure 3-1).

Figure 3-1: Phase diagram of the conditions needed for crystallization by vapor diffusion. In the undersaturation area: the drop remained clear. The goal was to find the right ratio and the good protein/precipitant reagent concentrations in order to reach the nucleation area and promote crystal growth.

First, crystallization trials using the sitting-drop vapor-diffusion (Figure 3-2A) method were performed at room temperature using a Crystal Gryphon (Art Robbins Instruments). This pipetting robot allowed to automatically test 96 crystallization solutions at a time per plate. The tested commercial screens were PACT premier™, JCSG-plus™, Classics™, PEGs™, PEGs II™, SaltRx™, Natrix™ and pHClear™ (QIAGEN). The crystallization drops were composed of 0.1 µL of purified protein and 0.2 µL of crystallization solution that were deposited on a corning crystallization plate (Molecular Dimensions) and then sealed. After a few days, each well was observed with CrysCam™ Digital Microscope System (Art Robbins Instruments).

The best crystallization conditions were then manually reproduced and optimized on a 24-well XRL plate (Molecular Dimensions) for mass production with the hanging drop technic (Figure

3-2B).

The goal was to produce a single crystal, with sufficient size and quality in order to perform X-ray diffraction experiments at a Synchrotron beamline.

II.5. Structure modelling

The 3D structure modelling of Zg-VHPO3 was performed using Zg-VIPO1 structure (PDB:

4CIT, 28% of sequence identity) and NapH1 (PDB: 3W35, 31% of sequence identity) as models with SWISS-MODEL server (https://swissmodel.expasy.org/interactive). The figures were constructed by using PyMOL v2.0.6. Protein-protein interface analysis was performed on PDBePISA v1.52 server ( https://www.ebi.ac.uk/pdbe/pisa/).

III. Results and discussion

III.1. Purification of the third vanadium-dependent haloperoxidase from Z. galactanivorans

The third vhpo homologous gene (zobellia_2250) was heterologously expressed in E. coli and the recombinant protein was purified by a two-step chromatography using IMAC and gel filtration. One unique peak was obtained from the first chromatography (data not shown), but the enzyme was separated as a mixture of two major distinct peaks by gel filtration (Figure 3-3A). The first minor peak of gel filtration corresponded to high-size protein aggregations, the second peak was eluted as a protein of about 100 kDa, and the third peak as a protein of about 50 kDa (according to a calibration range with reference proteins) (Figure 3-3A). The theoretical molecular weight of the recombinant Zg-VHPO3 was calculated as being 53.5 kDa. Further electrophoresis analysis of each fraction led to the identification of single 50-kDa bands on denaturing polyacrylamide gel (Figure 3-3A), suggesting that the second and third peaks corresponded to dimeric and monomeric forms of the protein, respectively. The fractions of each peak were separately pooled to produce two dimer and monomer batches. Another step of gel filtration was performed several months later with 1 mL sample of the thawed dimeric batch.

It showed a major peak corresponding to the dimeric protein as well as a small peak corresponding to the monomeric protein (Figure 3-3B). Those results suggest that the separation between dimer and monomer batches is relatively stable. The smaller peak corresponding to the monomeric protein might be a minor contamination occurring during gel filtration separation. After this purification step, the dimer batch was concentrated up to 11.6 mg/mL and the monomer batch up to 5.3 mg/mL. The DLS analysis allowed to identify hydrodynamic diameters of 6.34 nm for the monomer batch and 7.4 nm for the dimer batch, which are also in agreement with monomer and dimer purifications (data not shown). The recombinant Zg-VIPO1 and Zg-VIPO2 were both purified as strictly monomeric proteins (Fournier et al., 2014). The recombinant Zg-VHPO3 purified here appeared to have two possible protein forms, dimeric or monomeric, in solution. In order to evaluate the impact of salt concentration on the Zg-VHPO3 protein folding, another two-step chromatography purification was performed by lowering the NaCl concentration of the elution buffer to 50 mM in the gel filtration chromatography (instead of 150 mM previously). As shown in Figure 34, there were still the major two peaks following the gel filtration, but the monomer peak was dominant in this case, according to UV absorbance and SDS-PAGE gel. It appears that low

NaCl concentration favors monomeric folding in solution. The Tris/NaCl elution buffer had a pH of 8, which was higher than the theoretical isoelectric point (pI = 6.16) of the recombinant Zg-VHPO3, and led to negatively charged proteins after purification. Protein-protein interactions usually increased in that case, which could explain the higher peak corresponding to the prevalence of dimeric folds in solution. The protein stability at different pH was also tested using DLS, but the purified recombinant Zg-VHPO3 was either

Chapter 3 __________________________________________________________________________________ -137 -aggregating at pH < 6 or precipitating at pH > 8 (data not shown). A clear effect of the pH on the dimeric or monomeric folding of Zg-VHPO3 cannot be concluded for now, as the nature of the buffer also impacted protein conformations: for example HEPES buffer at pH 8.0 induced protein precipitation, but not Tris buffer (data not shown).

III.2. Substrate specificity of Zg-VHPO3 and determination of steady-state kinetic parameters

The halogen specificity of the purified recombinant Zg-VHPO3 was determined by an odianisidine assay (Jordan & Vilter, 1991) on native protein electrophoresis gel. A red-brown color was only detected in the presence of iodide but not with bromide, chloride, or without halide showing a strict specificity of the enzyme towards iodide (Figure 345). A second experiment was performed using the thymol blue assay (Verhaeghe et al. 2008a). This colorimetric assay revealed changes of color, from yellow to blue, when oxidation occurred and only with iodide, for both enzyme batches (Figure 3456). The recombinant protein Zg-VHPO3 was then renamed Zg-VIPO3 as it was specific to iodide oxidation. With the same amount of protein, the resulting assay coloration was bright blue with the dimeric batch and green for the monomeric batch suggesting a faster and stronger reactivity of the dimeric enzyme compared to the monomeric one. Using this thymol blue assay, the steady-state kinetic parameters of the Zg-VIPO3 enzyme batches were determined. Both monomeric and dimeric purified batches were separately used, immediately after the purification steps (Table 3-2). As suggested by the in-well colorimetric assay (Figure 3456), the monomeric enzyme batch was less active than the dimeric one. Indeed, the kinetic parameters showed that even though the two batches had similar Km, i.e. similar iodide affinity, the kcat of monomeric enzyme batch was about 14 times lower than the dimeric one, confirming a more efficient activity of this latter form (Table 3-2). The most active batch, enriched in dimeric form, was kept for complete and further biochemical analyses. Compared to Zg-VIPO1 and Zg-VIPO2 kinetic parameters (Table 3-2), those of Zg-VIPO3 suggested that the iodide affinity was poor, with a higher Km value, and a catalytic turnover much lower, leading to a kcat/Km ratio for iodide more than 128-fold lower for Zg-VIPO3.

Enzymes

Km (Fournier et al., 2014) Chapter 3 __________________________________________________________________________________ -139 -Those results suggested that Zg-VIPO3 functions differently than its two homologues within Z. galactanivorans. Further investigation on its 3D structure might be of great interest to study a potentially different enzymatic mechanism.

III.3. Effect of temperature on the folding of Zg-VIPO3

The structure stability analysis by thermal shift of the freshly purified dimer and monomer batches revealed two different melting temperatures comprised between 39.2°C and 40.3°C for the first peak and between 53.2°C and 55.7°C for the second one (Figure 34567). These two peaks represented protein unfolding steps of the dimeric and monomeric forms, respectively, and suggested that the two batches had to be considered as mixtures of both forms in different equilibria. In parallel, to study the effect of temperature on the enzymatic activity, each batch was submitted to an increasing gradient of temperature for 30 min, and deposited on native gels to assess remaining activity using a thymol blue colorimetric assay. The results showed a better activity stability of the monomer form (still active after 50°C treatment) compared to the dimer form (fully active up to 30°C incubation) (Figure 345678). This suggested that Zg-VIPO3 as a dimer was first denatured and lost enzymatic activity with lower temperature than as a monomer. The monomeric form might be more stable in solution and better for in vitro experiments.

III.4. 3D structural studies of Zg-VIPO3

In order to determine the 3D structure of Zg-VIPO3, crystallographic tests were performed on the crystallography platform of the Station Biologique at Roscoff. First, many conditions were automatically tested with commercially available screening kits from QIAGEN. Both monomeric and dimeric purified protein batches were used.

After a few days, no crystal was formed with the monomeric batch, the drops stayed clear. On the other hand, half of the screens performed with the dimeric protein batch precipitated, especially in the SaltRx™ screen. It appeared that the dimeric form was very sensitive to high salt concentration, which was in contradiction with the fact that the monomeric form was dominant after purification with decreased NaCl concentration.

For the Zg-VIPO3 dimeric batch, only one microcrystal was obtained using the PEGs™ screen (0.1 M Tris-HCl pH 8.5 and 25% (w/v) PEG 4000). This condition was manually optimized with the hanging drop technic to obtain bigger crystals, but only an amorphous precipitate was obtained after few days. The nanocrystal was then frozen in liquid nitrogen and transported to Synchrotron SOLEIL to perform X-Ray diffraction, but it did not persist to the freezing process and melted. After unsuccessfully testing 768 crystallization conditions, no further investigation was performed according to these difficulties to obtain and keep stable crystals. Instead, it was decided to study a computational model of the recombinant Zg-VIPO3 protein.

Based on the protein sequence, two structural 3D models of Zg-VIPO3 were build using the two closest 3D VHPO available structures: the monomer of Zg-VIPO1 and the dimer of NapH1 (Figure 3456789). The latter was characterized as a VCPO from the marine bacterium strain, Streptomyces sp. CNQ-525 (McKinnie et al., 2018).The first model, based on Zg-VIPO1, showed a global structure folded into 11 α-helices, four 310-helices and no β-sheet. The vanadium-binding active site was located at the C-terminus and seemed to be easily accessible through a large tunnel on the surface (Figure 3-9A,C). The second model, based on NapH1, showed a global structure folded into 12 α-helices, three 310-helices and six β-sheets. Both active sites were found on opposing sides and seemed to be less accessible than in the first model (Figure 3 The dimeric structures were further compared by protein-protein interface analysis using PDBePISA tool (Table 3-3). The analyses performed on X-ray dimeric structures of NapH1 and An-VBPOI showed high Complex Formation Significance Score (CSS) scores of 1 and 0.92, respectively. 1 being the maximum score for complex formation, these results suggested that protein-protein interfaces are highly relevant for dimerization in both VHPOs. On the contrary, for Zg-VIPO3 model, the CSS score was null. This score is based on the total free energy of binding belonging to the interface in stable assemblies (Δ i G), and takes into account the number of hydrogen bonds, disulfide bonds and salt bridges. All those factors (Table 3-3) suggested that the interface did not play any role in dimer formation for Zg-VIPO3. Even though NapH1 was the closest homologue of Zg-VIPO3, this VCPO was structurally and biochemically quite different from Zg-VIPO1 (Fournier et al., 2014

IV. Conclusions

The main goal of this chapter was to biochemically and structurally characterize the third VHPO from Z. galactanivorans. After purification of the recombinant protein, overexpressed in E.

coli, colorimetric tests based on o-dianisidine and thymol blue halogenation showed that the enzyme was active and strictly specific to iodide oxidation and therefore, this new iodoperoxidase was renamed Zg-VIPO3. The recombinant proteins were purified as either monomers or dimers, stable in solution. By contrast, the two other VIPOs of Z. galactanivorans were shown to be strictly monomeric. The phenomena of multimerization in solution were already observed for other VHPOs of macroalgae, such as L. digitata [START_REF] Colin | The brown algal kelp Laminaria digitata features distinct bromoperoxidase and iodoperoxidase activities[END_REF], A.

nodosum (Wishang et al., 2012) or C. pilulifera [START_REF] Isupov | Crystal structure of dodecameric vanadium-dependent bromoperoxidase from the red algae Corallina officinalis[END_REF]. Changes in buffer salinity, pH and temperature during purification were explored to stabilize Zg-VIPO3 folding as monomer or as dimer only. The decrease of salinity might help to increase monomer folding in certain conditions of buffer and pH. The other tested parameters did not seem to influence the dimeric or monomeric folding state in solution, but some led to protein aggregation or precipitation.

The kinetic parameters showed that the catalytic turnover of the dimeric enzyme was up to 10 times faster than that of the monomeric Zg-VIPO3, but compared to the other characterized iodoperoxidases from Z. galactanivorans or Laminaria digitata, this VIPO was much less efficient to oxidize iodide. A potential reason for that would be a completely different biological role of Zg-VIPO3 compared to its homologues. In order to verify this hypothesis, a first step would be to solve the 3D structure of this enzyme and investigate the amino acids involved in its catalytic mechanism.

A lot of crystallogenesis experiments were attempted but did not lead to obtain adequate crystals for subsequent X-Ray data acquisitions. The experiments were performed at 18°C, and decreasing the temperature to 4°C may help to stabilize and homogenize protein folding during crystal generation and growth. In absence of X-ray data, the 3D model of Zg-VIPO3 was used to investigate dimer interface on PISA server. However this approach only suggested that the dimeric model was not naturally possible and the dimerization of Zg-VIPO3 would occur more likely by a different way. These modelling results were not conclusive, certainly because of significant structural divergences between Zg-VIPO3 and the other closest VHPO 3D

structures. Further investigations will be necessary to better understand the self-organization of Zg-VIPO3 in solution, to progress on its 3D structure resolution and to elucidate the molecular bases of dimerization. Introduction

Numerous bacterial strains have been isolated and characterized based on their capacity to interact with iodine or metabolize iodinated compounds. According to Amachi et al., 2005b, they are grouped into three categories: iodide-volatilizing bacteria belonging to Proteobacteria and Cytophaga-Flexibacter-Bacteroidetes (CFB) group [START_REF] Amachi | Radiotracer experiments on biological volatilization of organic iodine from coastal seawaters[END_REF], iodineaccumulating bacteria, such as the Flavobacteriaceae Arenibacter sp. strain C-21 [START_REF] Ito | Draft Genome Sequence of Arenibacter sp. Strain C-21, an Iodine-Accumulating Bacterium Isolated from Surface Marine Sediment[END_REF] and iodide-oxidizing bacteria, such as Iodidimonas sp. Q-1 [START_REF] Ehara | Draft genome sequence of strain Q-1, an iodide-oxidizing alphaproteobacterium isolated from natural gas brine water[END_REF] or Roseovarius sp. A-2 [START_REF] Yuliana | Draft Genome Sequence of Roseovarius sp. A-2, an Iodide-Oxidizing Bacterium Isolated from Natural Gas Brine Water, Chiba, Japan[END_REF]. A multicopper oxidase activity was linked to the iodide-oxidation activity for the two latter bacteria [START_REF] Yuliana | Draft Genome Sequence of Roseovarius sp. A-2, an Iodide-Oxidizing Bacterium Isolated from Natural Gas Brine Water, Chiba, Japan[END_REF][START_REF] Taguchi | Decolorization of recalcitrant dyes by a multicopper oxidase produced by Iodidimonas sp. Q-1 with iodide as a novel inorganic natural redox mediator[END_REF].

Zobellia galactanivorans was shown to accumulate iodine (Barbeyron et al, 2016), and to encode three vanadium-dependent iodoperoxidases (VIPOs), specific for iodide oxidation (Fournier et al., 2014; Chapter 3). The presence of three VIPOs and of other proteins potentially related to iodine metabolism such as a L-2 haloacid dehalogenase (Chapter 1) suggests that Z. galactanivorans has developed an active iodine metabolism, which may be related to antioxidant protection and its capacity to degrade algal polysaccharides and to cope with the high concentrations of iodide in brown algal cell walls (Barbeyron et al, 2016).

Up to now, however, the biological roles of such iodide-specialized enzymes have been poorly documented, especially in bacteria and in relation to iodine-rich environments. Taking advantage of biochemical and genetic tools developed in Zobellia in our laboratory, this chapter reports on the localization and expression of one of these VIPO proteins through immunolabelling and Western-blot analyses. Functional comparisons of the wild-type and the zg-vipo1 knock-out mutant strains were conducted to explore the role of the Zg-VIPO1 protein in iodine-rich medium and in relation to iodine uptake.

signal peptide or a lipoprotein signal was done using SignalP 5.0 [START_REF] Armenteros | SignalP 5.0 improves signal peptide predictions using deep neural networks[END_REF],

PrediSi [START_REF] Hiller | PrediSi: Prediction of signal peptides and their cleavage positions[END_REF], Phobius [START_REF] Käll | A combined transmembrane topology and signal peptide prediction method[END_REF], SOSUIsignal [START_REF] Gomi | High performance system for signal peptide prediction: SOSUIsignal[END_REF], SecretomeP [START_REF] Bendtsen | Non-classical protein secretion in bacteria[END_REF], Signal-3L [START_REF] Zhang | Signal-3L 3.0: Improving Signal Peptide Prediction through Combining Attention Deep Learning with Window-Based Scoring[END_REF], Signal-BLAST [START_REF] Frank | High-performance signal peptide prediction based on sequence alignment techniques[END_REF], LipoP [START_REF] Juncker | Prediction of lipoprotein signal peptides in Gram-negative bacteria[END_REF], DOLOP [START_REF] Babu | DOLOP -Database of bacterial lipoproteins[END_REF] and SPEPlip [START_REF] Fariselli | SPEPlip: The detection of signal peptide and lipoprotein cleavage sites[END_REF]. All of these programs are freely available on web server addresses.

II.4. Protein subcellular fractionation

After three days of bacterial cultures (corresponding to the end of the exponential growth phase), the pellet was collected by centrifugation at 9,000 g for 15 min, and rinsed twice with a wash buffer (19.45 g/L NaCl, 5.9 g/L MgCl2, 0.3 g/L CaCl2, 10 mM potassium phosphate pH 7.0). The pellet was then resuspended in (5mL/g of pellet) lysis buffer (3 mM EDTA, 100 mM Tris, 500 mM sucrose, pH 6.8) with 15 mg/mL of lysozyme and incubated for 20 min at 30°C

with stirring. An equal volume of distilled water was added and incubated at 30°C for an additional 20 min with stirring. After centrifugation at 20,000 g for 20 min at 4°C, the supernatant was collected as the periplasmic fraction. The resulting precipitate was resuspended in 50 mM Tris pH 7.5 buffer. The mixture was sonicated using a sonifier device to achieve lysis. After centrifugation for 1h at 20,000 g at 4°C, the supernatant corresponding to the cytoplasm and the precipitate corresponding to the membranes were separated. The latter fraction was resuspended with 1 mL of 50 mM Tris pH 7.5 buffer. The total protein concentration was determined by a colorimetric test with the Bio-Rad Protein Assay Dye Reagent according to standard recommendations. Aliquots of all the fractions were kept at -20°C before use.

II.5. Western blot analysis

An equal amount of protein fraction was deposited in each well of SDS-PAGE. Two gels were loaded and run in parallel to compare the immunorevelation signals with the protein quantities, revealed by a Coomassie blue coloration. The fractionated protein samples were supplemented with a 6-fold concentrated loading buffer (360 mM Tris HCl pH 6.8; 45% glycerol; 9% SDS; 9% DTT; 0.12% bromophenol blue) and they were previously denatured at 95°C for 5 min before being charged on a 12% SDS-PAGE (Bio-Rad system). Following the separation, the proteins were electro-transferred from the polyacrylamide gel to a nitrocellulose membrane (GE Healthcare) using Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was first incubated for 5 min with 5% milk in TBS-T buffer (20 mM Tris pH 7.6; NaCl 150 mM; 1% tween 20) to block free sites on the membrane. The immunodetection of target protein was realized either with the commercial anti-His tag directly to HRP for detection or with the anti-Zg-VIPO1. Both antibodies were diluted at 1/5000 in TBS-T buffer and incubated 1 h at room temperature with a gentle shaking. The membrane was then rinsed 3 times with TBS-T buffer for 5 min before adding the secondary antibody directed against rabbit IgG and couple with HRP at a final dilution of 1/5000 following the incubation with anti-Zg-IPO1. After 30 min of incubation, the membrane was washed again 3 times during 5 min with TBS-T buffer. The protein-antibody interaction was then revealed by ECL reagents (100 mM Tris pH 8.5; 44.3 mg/mL luminol; 14.8 mg/mL coumaric acid; 30% H2O2) and visualized by a Fusion FX camera using ChemiCapt software (Vilber Lourmat).

II.6. Deletion of the Zg-VIPO1-encoding gene

Single deletion mutant of zg-vipo1 (zobellia_1262) was constructed following the previously described method from Zhu et al., 2017. A 2.0-kb fragment corresponding to the first 30 bp of zobellia_1262 with the region directly upstream was amplified using the primers OEG001

(TTTTTTGGATCCAGATGAACAACACCAAGCTTATGGA) and OEG003

(TTTTTTCTGCAGCGATATTAGTGCGATAAGAATCTTCT). The fragment was digested with BamHI and PstI and ligated into pYT313 that had been digested with the same enzymes, to generate pEG1. Similarly, a 2.0-kb fragment corresponding to the last 30 bp of zg-vipo1 and the region directly downstream was amplified with the primers OEG002

(TTTTTTGCATGCGGTTGCCCAAAGAAAATTCCAAAGA) and OEG004

(TTTTTTCTGCAGCTATCCGATAAGAAAGTAGCCCAAA). The fragment was inserted between PstI and SpHI sites of pEG1 to generate the suicide plasmid pEG3. Suicide plasmid was introduced into the wild type Z. galactanivorans Dsij T by conjugation with E. coli S17-1 λ pir strain. Erythromycin resistance was used to select cells with the plasmid integrated in the genome. Resulting colonies were grown overnight in artificial ZoBell medium without antibiotics at 30°C to allow the loss of the plasmid. The cells were then plated onto artificial ZoBell-agar containing 5% sucrose. Sucrose-resistant colonies were checked for erythromycin sensitivity. The deletion of zg-vipo1 was confirmed by PCR using primer pairs: OEG005 (TATTTTGGCGTTAAACCCTATCATGG) and OEG006 (GGGTAAGTGTACTGGTGAAGTCC) and then verified by sequencing.

II.7. Iodine accumulation assays

The strains Z. galactanivorans Dsij T , Δipo1 and C. fucicola were cultured aerobically in 100 mL of artificial Zobell containing iodide (0.1 µM). After cultivation for 48 h, each culture was centrifuged at 4°C for 30 min at 10,000 g. Fresh pellets were then lyophilized, together with 50 mL of artificial ZoBell medium. The dried powder samples were analyzed for total iodine content using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) at the LABOCEA chemical analysis laboratory (Brest, France).

The C-21, Z. galactanivorans Dsij T , Δipo1 and C. fucicola strains were cultured aerobically in 100 mL of artificial ZoBell containing nonradioactive iodide (0.1 µM) and 125 I -(74 kBq mL -1 ).

After cultivation for 24 h, 700 µL of each culture was sampled and put on a silicone oil layer (500 µL; 35:65 mixture of SH556 and SH550; Toray Dow Corning Silicone) that had been placed over 100 µL of distilled water in a microcentrifuge tube. After centrifugation at 4°C for 3 min at 13,000 g, 50 µL of the supernatant sample was removed and placed in a scintillation vial. The distilled water layer containing the cell pellets were frozen at -40°C. The pellets in the microcentrifuge tubes were cut with dog nail clippers and were transferred to another scintillation vial. The activity of 125 I in each sample was measured with a NaI scintillation counter (Auto well gamma system ARC-370M, Aloka) for 10 min every 24h over 3 days. At the starting point (before bacterial cultivation), the medium was sampled to quantify the initial 125 I amount and then subsequent measurement values were subtracted to calculate the net uptake by the bacterial cells.

II.8. Mass spectrometry analysis of the iodine-enriched protein fractions

After 3-days cultures in the presence of 125 I, wild-type and mutant pellet cultures of Z.

galactanivorans were fractionated using the method described earlier. The radioactivity of 125 I in each fraction was measured with the NaI scintillation counter for 10 min. The protein fraction, with the most radioactive signal, was denatured at 100°C for 5 min and loaded on a 12% SDS-PAGE. After migration, the gel was placed on a miniGita scanning device (Elysia-Raytest) to determine which gel lanes and zones were the most radioactive. Then another SDS-PAGE gel was run with the corresponding protein fractions but coming from a non-radioactive culture. The corresponding Coomassie-blue bands were subsequently cut and sent for trypsin digestion and Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS) analysis of the peptides (Integrale Co, Ltd, Japan). The results of these proteomic data were analyzed by the freeware Scaffold Viewer 4.11.1, in particular for the iodination modifications.

II.9. Structure modelling

A 3D structure modelling of the Z. galactanivorans OmpA-like protein (Zobellia_1580) was performed using the OmpA protein from Capnocytophaga gingivalis (PDB accession: 5WTL, 60% sequence similarity) as a template using the SWISS-MODEL server (https://swissmodel.expasy.org/interactive). The figures were constructed using PyMOL 2.0.6.

III. Results and discussion

III.1. Subcellular localization prediction of Zg-VIPOs

In silico localization predictions were realized with the most accurate generalist program tools for Gram-negative bacteria allowing identification of protein assignment between several compartments [START_REF] Goudenège | CoBaltDB: Complete bacterial and archaeal orfeomes subcellular localization database and associated resources[END_REF][START_REF] Lertampaiporn | PSO-LocBact: A Consensus Method for Optimizing Multiple Classifier Results for Predicting the Subcellular Localization of Bacterial Proteins[END_REF]. The results of the seven different analyses showed that there was no prediction consensus for any of the 3 VIPO proteins (Table 4-1). Zg-VIPO1 would be likely assigned to periplasmic space but other secondary subcellular destinations were also predicted such as cytoplasm and outer membrane. Similarly, Zg-VIPO2 would be likely assigned in the cytoplasm, however, the periplasm and the outer membrane were also predicted as possibilities. Zg-VIPO3 had a higher probability of being localized in periplasm. Overall, there was relatively low agreement between programs. This difficulty to address a single compartment and to find a consensus between all the programs is illustrated by the results of PSORTb. This software is described as being the most effective generalist tool available [START_REF] Peng | Protein localization analysis of essential genes in prokaryotes[END_REF] Taking into account the difficulty in assigning the VIPOs into one subcellular compartment, a second approach was used to determine the presence and the nature of signal peptides using bioinformatic tools specialized for secretory pathway prediction, for lipoprotein sequence detection or for both (Table 4-2). The SignalP program is the most performant to date [START_REF] Armenteros | SignalP 5.0 improves signal peptide predictions using deep neural networks[END_REF] as it can discriminate between three types of bacterial signal peptides, corresponding to different secretory pathways, and also predict a peptide cleavage site. The summary of prediction results (last column, According to LipoP and SignalP, Zg-VIPO1 and Zg-VIPO2 were predicted to possess a signal peptide of type II (SPII) corresponding to a lipoprotein formation whereas Zg-VIPO3 could be exported by a signal peptide of type I (SPI). The length of the Zg-VIPO3 cleavable peptide might be of 21 amino acids with a cut between an alanine and a glutamine respectively in positions 21 and 22. In contrast, several sizes and cutting sites were proposed by the different programs for Zg-VIPO1 and Zg-VIPO2 signal peptides. The lipoproteins from Gram-negative bacteria are synthesized in the cytoplasm and then exported to the inner or to the outer membranes. The characteristic feature of all lipoproteins is a signal peptide sequence in the Nterminal end, followed by a cysteine residue [START_REF] Hayashi | Lipoproteins in Bacteria[END_REF]. Consequently, the serinecysteine peptide linkages, in positions 16-17 and 17-18 respectively, for Zg-VIPO1 and Zg-VIPO2, appeared to be the most reliable cleavage sites. Furthermore, the program Signal-BLAST, used to detect N-terminal peptides by homology with other known peptides of lipoproteins, indicated that Zg-VIPO1 had a signal peptide homologous to the penicillinbinding protein activator LpoB of Xenorhabdus nematophila (D3VIE0). This protein LpoB is located in the outer membrane where it activates bacterial cell wall polymerases [START_REF] Paradis-Bleau | Lipoprotein cofactors located in the outer membrane activate bacterial cell wall polymerases[END_REF]. Additionally the first twenty amino acids of Zg-VIPO2 were found to be similar to the lipoprotein Blc of Vibrio cholerae serotype O1 (Q08790), which has been demonstrated to be part of the outer membrane in E. coli [START_REF] Campanacci | The membrane bound bacterial lipocalin Blc is a functional dimer with binding preference for lysophospholipids[END_REF].

The amino acid in +2 position after the signal cleavage site was found to be essential to assign lipoproteins either to the inner or the outer membrane [START_REF] Seydel | Testing the "+2 rule" for lipoprotein sorting in the Escherichia coli cell envelope with a new genetic selection[END_REF]. The nature of this particular amino acid can change drastically the destination of the lipoprotein and also the orientation side of the protein in the corresponding membrane (cytoplasm or periplasm for the inner membrane and extracellular medium or periplasm for the outer membrane). The analysis of the +2 amino acids of Zg-VIPO1 and Zg-VIPO2 done by LipoP revealed, respectively, a lysine and a glycine at this position. Considering that the SPII pathway of Z. galactonivorans could strictly function as its equivalent in E. coli, it would direct Zg-VIPO1 in the outer membrane with an orientation to periplasmic space and Zg-VIPO2, equally into inner and outer

The fractionation of Z. galactanivorans subcellular compartments led to the separation of the periplasmic space, the cytoplasm and the total membranes. When no immunolabelling signal was revealed in periplasmic protein fractions, major 50 kDa bands were mainly detected in the membrane protein fraction and weakly in the cytoplasm (Figure 4-2). Since the inner and the outer membranes were not separated during protein fractionation and fell into the same fraction, it was impossible to conclude in which membrane Zg-VIPO1 was located. Nevertheless, those results were in accordance with the previous in silico analyses that might signify a possible outer membrane lipoprotein signal for Zg-VIPO1. The signal detected in the cytoplasm might be due to a contamination of the cytoplasm compartment by the membrane proteins as they are separated during the last step of fractionation by pipetting. We could also suppose that a part of the Zg-VIPO1 protein might stay in the cytoplasm where it is initially produced. As the predictor tools did not provide full evidence for a discriminant localization, it is possible that the cytoplasm might be the second place of residence of Zg-VIPO1. Further work on subcellular fractionation between all the bacterial compartments using sucrose gradients could help explore this in more detail.

III.2. Effects of an iodide increase on the wild-type and the Δipo1 knock-out mutant bacterial growth

In order to explore in vivo the biological roles of Zg-VIPO1 protein, a knock-out mutant of Z.

galactanivorans was produced by deleting the zobellia_1262 gene. The growth of cultures exposed to a gradient of increasing iodide concentrations, from 10 mM to 400 mM, was compared for both Z. galactanivorans wild-type (WT) and Δipo1 mutant strains. No major differences in growth curves were observed between the two strains, when comparing under control condition, or in the presence of each iodide concentration from 10 to 200 mM (Figure 4-3 A andB). During the one week of culture, the OD (optical density) of both strains was approximately similar at the concentrations of 0 mM and 10 mM iodide (orange and light blue curves, Figure 4-3). From the second day at 100 mM and from the third day at 200 mM iodide, the two strains seemed to have similarly increased growth compared to the control (ZoBell medium only). Up to 200 mM of iodide, there seem to be no effect on the growth of either strain. (Figure 4-3). In the cultures with 400 mM iodide, a growth stagnation was observed for both the WT and the Δipo1 mutant strains (green curves, Figure 4-3) during the 7 day kinetics.

The bacterial OD was lower during the first day and the first 72 h of cultures with 200 mM and 300 mM of iodide, respectively, suggesting a slowing-down of growth for both strains at these concentrations (yellow and dark blue curves, Figure 4-3). At the fourth day of culture with 300 mM iodide, cell densities of the WT strain appeared to reach those of the control condition, whereas the Δipo1 mutant cultures remained lower in comparison. This latter result suggested that the Δipo1 mutant strain could be more affected by a concentration of 300 mM iodide than the WT strain. Nevertheless, the growth level of the mutant strain was comparable to the WT strain under the same conditions and to the control after one week (light and dark blue curves, Figure 4-3). These results suggested a progressive adaptation of the strains to strong iodide concentrations or a possible transformation and iodovolatilisation to resist these unusual and unnatural quantities of iodine. Moreover, these experiments showed that a concentration of 300 mM iodide (1 million-fold higher than the concentration found in seawater) is the threshold beyond which cell division of Z. galactanivorans is no longer possible in a rich culture medium.

The fact that both WT and Δipo1 mutant strains had similar growth in the presence of 0 to 200 mM iodide demonstrated that the Zg-VIPO1 protein is potentially not involved in the tolerance of Z. galactanivorans to high amounts of iodide (up to 200 mM). However, this enzyme could participate in bacterial recovery when iodide concentrations of culture medium are closer to the tolerance limit (e.g. after 4 days with 300 mM iodide).

III.3. Comparison of iodine accumulation and uptake in wild-type and Δipo1

knock-out mutant strains

To monitor the iodine accumulation after 2 days of bacterial cultures and to test the potential involvement of Zg-VIPO1 in this process, ICP-MS analyses were performed on pellets of Z.

galactanivorans WT and Δipo1 mutant strains. In addition, another Bacteroidetes strain of Cellulophaga fucicola which does not possess any VHPO-encoding gene was used to compare the results. Z. galactanivorans pellets featured iodine contents of 6.85 ± 0.35 µg/g (Table 4-3),

whereas Δipo1 mutant and C. fucicola strains showed lower iodine concentration in their cell pellets (1.5± 0.00 µg/g and <1 µg/g respectively).

Sample name

Total iodine content (µg/g of pellet)

Artificial ZoBell medium below the detection threshold Zobellia galactanivorans WT 6.85 ± 0.35

Zobellia galactanivorans Δipo1 1.5 ± 0.00 In order to monitor iodine uptake in bacterial strains, the same culture conditions were performed for three days, in collaboration with Haruka Nakazawa, a Master student in the group of Prof. Seigo Amachi, who has a strong expertise in the use of radioactive iodide 125 I -as a tracer. The Arenibacter sp. C-21 strain was used as a positive control for the iodide uptake experiment (Amachi et al., 2005a). The separated bacterial pellets and surrounding media were

Cellulophaga fucicola <1

A B

Chapter 4 __________________________________________________________________________________ -161 -For the Z. galactanivorans WT strain, around 80% of total iodide was removed from the medium after 24h and transferred to cells, whereas almost no iodide was accumulated in the Δipo1 mutant cells, demonstrating that the deletion of zg-vipo1 gene provoked a decrease of internal iodine contents. Furthermore, the content of iodine in the cells of C. fucicola was null during the 72 h experiment and below the curve of the Δipo1 mutant. These results are in clear agreement with the precedent ICP-MS analyses and therefore strongly suggests that Zg-VIPO1 plays a key role in iodine concentration mechanism(s) in Z. galactanivorans.

In addition, based on the ICP-MS and radioactivity flux analyses, weak differences between the Z. galactanivorans Δipo1 mutant and C. fucicola could suggest an implication, albeit to a much lesser extent, of one of the other Zg-VIPOs, and more likely Zg-VIPO2 given its biochemical and structural similarities with Zg-VIPO1, in addition to its subcellular localization prediction.

III.4. Localization and LC-MS/MS analysis of iodine-enriched protein fractions

To further explore the iodine accumulation mechanisms in the different cell compartments, after 72 h, the 125 I --containing pellets of the Z. galactanivorans WT and Δipo1 mutant strains were separated and extracted into three protein samples corresponding to cytoplasm, periplasm, and total membrane-enriched fractions. Each protein fraction was then measured for radioactivity.

The highest radioactivity levels were detected in the membrane fractions for both strains, with significantly higher values for the Z. galactanivorans WT compared to the mutant (8.4-fold higher, Table 4-4). For the cytoplasmic-enriched fractions, there were approximately the same ratios of iodine content between the Z. galactanivorans WT and Δipo1 mutant samples (9.3fold higher, Table 4-4), but the 125 I -measurements were ~ 40-fold lower than for membrane fractions. As discussed above, this labelling in cytoplasm fractions might be due to 1) an artifact of a slight contamination of the cytoplasmic compartment by membrane debris occurring during fractionation, or 2) a real presence of radioactive iodine in the cytoplasmic compartment. This latter hypothesis remains to be demonstrated as in this case the iodide would need to be internalized by crossing the inner membrane by an energetic transport. The differences between the WT and Δipo1 mutant membrane and cytoplasmic-enriched fractions in iodide content and the simultaneous presence of Zg-VIPO1 in these fractions suggest that iodide could be associated directly or indirectly with this enzyme. In order to further investigate the nature of iodine-labelled proteins, the membrane-enriched protein fractions of both Z. galactanivorans and Δipo1 mutant strains were analyzed by Each band was cut on both gels and the radioactivity level was measured on those derived from radioactive gel (Figure 45). was annotated by an expert manual annotation of the genome of Z. galactanivorans, with a corresponded to an  fold of a peptidoglycan binding domain (Figure 4-6, right part). This latter domain could allow the anchoring of the outer membrane to the bacterial cell wall and ensure the cohesion of bacterial envelope supramolecular assembly [START_REF] Park | Mechanism of anchoring of OmpA protein to the cell wall peptidoglycan of the gram-negative bacterial outer membrane[END_REF].

Periplasm

According to their global structural conservation, the two OmpA-like proteins from Z. The level of Tyr425 iodination was very low therefore it was difficult to distinguish between a real, functional iodination of this protein or a putative artifact linked to a side effect of an iodinating enzyme like Zg-VIPO1. Four other OmpA-like proteins were also detected in the same dataset of LC-MS/MS spectra, but no peptide with a similar C-terminal iodinated tyrosine was obtained. As the number of spectra for these proteins was very low and the number of peptides identified in their C-terminal was minor, it was not excluded that some of them could also be iodinated. Nevertheless, as the OmpA-like protein ZoBellia_1580 and Zg-VIPO1 were isolated and identified in the same subcellular fraction, a close relationship might exist and remains to be demonstrated.

According to [START_REF] Feiters | Bromine is an endogenous component of a vanadium bromoperoxidase[END_REF], surface tyrosines of the Ascophyllum nodosum An-VBPO1 were found to be partially brominated and could also incorporate iodine to a lesser extent. Our precedent hypothesis was that Zg-VIPO1 could also present some tyrosine residues susceptible to be iodinated, particularly in a loop near the active site entry. Within that context, the Tyr263 was an interesting candidate, to explore a functional role of iodination, and its mutations with Ala/Phe/Ser affected the kinetic constants of the enzyme. One of the mutants even developed an activity towards bromide (Fournier et al., 2014).

As expected in this range of sizes (~50 kDa), peptides corresponding to both Zg-VIPO1 and Zg-VIPO2 proteins were detected from the trypsin digested band#1 extract in LC-MS/MS data, but no iodinated residue was identified. Unfortunately, no peptide including the Tyr263 residue was identified in this dataset, implying further tests are needed to validate this hypothesis of a potential Zg-VIPO1 iodination.

IV. Conclusions

The online prediction tools showed a low consensus for a clear and unique localization of each VIPO from Z. galactanivorans. It appeared probable that Zg-VIPO1 and Zg-VIPO2 could have a more similar but not identical localization, due to their predicted lipoprotein signal peptide, therefore anchored in a membrane (either outer, or inner). However, Zg-VIPO3 appeared to be more commonly predicted in the periplasm due to its potential SPI signal peptide. Even if these

VIPOs belonged to the same enzyme class and shared the same substrate specificity, they may not display the same in vivo function because of their possible different localizations.

Nonetheless, the in silico approaches have their limits and setting up in vitro experiments was necessary. The bacterial subcellular fractionation and the Zg-VIPO1 protein immunodetection allowed investigation of protein localization in the bacteria. Zg-VIPO1 protein was experimentally confirmed to be predominantly present in the membrane-enriched protein fractions (either the inner or the outer membrane), suggesting a biological role at the interface of the two membranes. However, no role in iodide detoxification was clearly unraveled, based on growth comparison between wild-type and Δipo1 mutant strains, although the capacity of Z.

galactanivorans to cope with high concentrations of iodide revealed that another VIPO, or other enzymatic pathways, might be involved in this protection mechanism. The gene knockout approach was particularly successful for exploring the potential role of VIPO in iodine uptake and concentration. Indeed, our results showed that Zg-VIPO1 was essential for a maximum iodide accumulation in the bacterium and that this accumulation seemed to be associated with the membranes. However, it was not proven that iodine was directly bound to any amino acid of this protein, only that the loss of Zg-VIPO1 enzyme led to a drastic reduction in iodine accumulation. This illustrated that the iodine trapping was achieved via mechanisms involving the oxidation of iodide via VIPO activity and probably a secondary iodination of molecules present in the same compartment. Zg-VIPO1 is certainly essential, however, it is not the only key player. The participation of other protein actors in this mechanism like the OmpA-like protein will need further experiments to be validated. The Δipo1 mutant concentrated iodine to a lesser extent than Z. galactanivorans WT suggesting that Zg-VIPO1 could not be fully replaced by another VIPO. Thus Zg-VIPO2 or Zg-VIPO3 might also be involved in iodine uptake but differences in precise localization, reactivity or expression level of these proteins could explain their low contribution to the process. The main goal of my thesis was to characterize at the biochemical, structural and functional levels some enzymes involved in the halogen-related metabolism in the bacterium Zobellia galactanivorans. The objective was to investigate the importance and the role of this specific metabolism in this model bacterium as marine bacteria may be significant actors in the global biogeochemical cycle of halogens, either as halogenated compounds producers, environmental detoxifiers or halogen accumulators. In this context, Z. galactanivorans possesses at least six enzymes identified as potentially interesting and my thesis was focused on two of them that might be involved in the VHOC production, iodide accumulation or detoxification.

First, I biochemically and structurally characterized the unique L-2-HAD protein, encoded on the genome of Z. galactanivorans. As expected from its belonging to this subfamily of HADs, this enzyme only transformed the short-chained halogenated compounds in S configuration, and was structurally similar to its closest homologues. Most structurally characterized homologues come from terrestrial bacteria, and are studied for their capacities to degrade toxic environmental halogenated compounds, and their potential in bioremediation. In order to determine if this enzyme gave a selective advantage to the bacterium, I decided to delete the zghad gene using a knock-out mutant approach. The implementation of this functional approach, still pioneer in Zobellia, was time-consuming, but worthwhile, as I obtained the first knock-out mutants at the end of 2019. Cultures of the wild-type and mutant strains with halogenated compounds illustrated a significant reduced growth for the mutant strain. This result brought forward that Z. galactanivorans might possess a selective advantage in the presence of toxic halogenated compounds, compared to other bacteria without a HAD enzyme.

However, the ZgHAD enzyme could only catabolize a few ranges of substrates and in vivo tests need to be performed to explore the bioremediation capacities of Z. galactanivorans. Sitedirected mutagenesis could be a complementary method to further investigate substrate specificity in the future and perhaps find a way to catabolize a greater variety of compounds.

Two closely-related VHPOs were biochemically and structurally characterized during a previous thesis work, as being VIPOs (Fournier et al., 2014). I studied the biochemistry of the recombinant enzyme obtained from the third VHPO encoded on the genome of Z.

galactanivorans, and established that it was also specific to iodide oxidation: this is the third characterized VIPO in this bacterium. Its structural characterization however could not be completed due to difficulties in stabilizing the protein folding in solution. When the two other VIPOs were shown to be monomers, Zg-VIPO3 was purified as a mixture of monomers and dimers. This observation reminds the biochemical behavior of algal VBPOs that were shown to be dimeric, but multimerizing in solution. Since the amino acid sequence of Zg-VIPO3 shares low similarities with its two closest crystallized homologues and is instead closer to a

Streptomycetes VCPO than the Z. galactanivorans VIPOs, the question of its biological role arises. Furthermore, steady-state kinetic analysis of this enzyme showed a very low activity towards iodide compared to Zg-VIPO1 and Zg-VIPO2. Consequently, the Zg-VIPO3 could have a different function in Z. galactanivorans. One hypothesis to be tested is a role in the biosynthesis of iodinated compounds, as found for the VCPO from Streptomyces sp. which is involved in the biosynthesis of halogenated antibiotics [START_REF] Mckinnie | Characterization and Biochemical Assays of Streptomyces Vanadium-Dependent Chloroperoxidases[END_REF]. In this case, producing a knockout mutant by deleting zg-vipo3 and perform metabolic profiling of iodinated compounds could help identify the biological role of Zg-VIPO3.

One way to determine the biological role of a protein is to study its localization within the organism. A first in silico approach was not efficient to assertively predict protein localization, for none of the VIPOs. At the contrary, western-blot analyses of protein fractions gave some clues about the localization of VIPOs in Z. galactanivorans and once again the gene knockout was successful to compare phenotypes of wild-type and mutant strains, building some conclusions about the in vivo role of Zg-VIPO1. This was the first functional evidence that Zg-VIPO1 is involved in the iodide accumulation, associated to the membrane fraction of the bacterium. However, it is still unknown if this process is comparable to the one taking place in kelps, as the two marine organisms feature very different extracytoplasmic chemical compositions. Now, it will be necessary to further investigate the precise localization of Zg-VIPO1 in membranes by a more discriminating technique, as sucrose gradient, to efficiently separate outer and inner membranes. The iodide uptake and concentration in Z. galactanivorans seemed similar in terms of rapidity and efficiency to that of Arenibacter sp. strain C-21.

Nonetheless, the enzymes involved in the uptake in this latter bacterium are unknown and may or may not be similar to those of Z. galactanivorans. Further comparisons of both bacterial genomes would be of interest.

Based on all results obtained during my thesis, the hypothetical model of halogen metabolism in Z. galactanivorans shown in figure 0-11, was updated in figure 5-1: When the iodide uptake by the mutant strain of Z. galactanivorans was compared to the uptake in C. fucicola, I noticed that the mutant strain was able to accumulate higher concentrations. It can be possible that one or the two other VIPOs is (are) also involved in this accumulation.

However, the low catalytic turnover of Zg-VIPO3, in addition to a probable different subcellular localization, seems to indicate another biological function, as already mentioned above. This strengthens the idea that Zg-VIPO2 may participate, in a lesser manner, in iodide accumulation. To validate this hypothesis, a single knockout deletion of zg-vipo2, then production of a double mutant of zg-vipo1 and zg-vipo2 are needed.

The presence of iodinated proteins in the membranes of Z. galactanivorans was investigated, and an OmpA-like protein connecting the outer membrane and the periplasmic peptidoglycan layer was identified. The OmpA protein family is well-studied in bacteria and has multiple functions, such as maintaining the structural integrity of the outer membrane (Smith et al., 2017[START_REF] Dai | Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bonddependent calcium-binding capability[END_REF]. Thus, this protein might be essential to incorporate iodide through the outer membrane. To further corroborate the hypothesis of a functional relationship between OmpA and Zg-VIPO1, deleting the OmpA-encoding gene in Z. galactanivorans and exploring if the bacterium is still able to accumulate iodine could be essential.

To analyze protein iodination in Z. galactanivorans, a more exhaustive LC-MS/MS analysis would be necessary, especially to target the C-term tyrosine of Zg-VIPOs. If more iodinated proteins are identified, it could validate their roles in iodide uptake.

Our results showed that iodine was associated with membrane protein fractions but the membranes have other components capable of reacting with oxidized iodine. Some electrophilic compounds with an aromatic core have been recently identified to be halogenated in some bacteria [START_REF] He | Chemical structure, biological roles, biosynthesis and regulation of the yellow xanthomonadin pigments in the phytopathogenic genus Xanthomonas[END_REF][START_REF] Martins | Chemistry, bioactivity and biosynthesis of cyanobacterial alkylresorcinols[END_REF][START_REF] Schöner | Aryl Polyenes, a Highly Abundant Class of Bacterial Natural Products, Are Functionally Related to Antioxidative Carotenoids[END_REF] and it would be interesting to evaluate the presence of such iodinated phenolic compounds or analogues in Z.

galactanivorans by metabolic profiling.

Important concentrations of I -captured in the kelp apoplasm might be released in the environment of Z. galactanivorans when the biomass degradation of these brown algae occurs.

The mechanisms of iodide concentration or detoxification could be important for bacteria in this case. However no involvement of Zg-VIPO1 has been correlated to the resistance of iodide high concentrations and the role of other enzymes like Zg-VIPO2 and Zg-VIPO3 in these processes would be interesting to study through gene deletion approaches.

Among the Z. galactanivorans halogenases, the putative cytoplasmic cofactor-free HPO is another interesting candidate to characterize: it should be first investigated further by biochemical exploration of its halogen specificity and substrates.

Thanks to the presence of three VIPOs and ZgHAD, with biochemical specificities towards iodine, it seems that the iodine-related metabolism is important in Z. galactanivorans, and by extension among other marine Bacteroidetes. The function of this specific metabolism in these bacteria is still unknown, as well as its importance during alga-bacteria interactions, or biofilm formation. Although we do not know which chemical compounds, excreted by algae, the bacteria are exposed to, the genome analysis has showed that this Bacteroidetes possesses genetic tools to keep up with oxidative stress. The surfaces of macroalgae provide a large interface with the bacterial microbiome and are also submitted to environment changes.

Résumé

Dans l'environnement marin, de nombreux organismes participent au cycle biogéochimique des halogènes en émettant des composés organiques volatils halogénés. Cependant les mécanismes enzymatiques responsables de la biosynthèse, de la captation et du stockage de ces métabolites particuliers sont souvent mal connus, notamment chez les bactéries marines. L'annotation du génome de Zobellia galactanivorans, une flavobactérie marine vivant à la surface des macroalgues, a permis d'identifier plusieurs gènes potentiellement impliqués dans ce métabolisme halogéné. Au cours de cette thèse, j'ai entrepris des analyses de biochimie et de biologie structurale, et développé des approches fonctionnelles à partir des gènes codant pour des iodoperoxydases à vanadium (VIPO), et pour une haloacide déhalogénase (HAD). J'ai déterminé les substrats préférentiels de l'enzyme ZgHAD et analysé sa stabilité enzymatique et ses paramètres cinétiques. J'ai également résolu la structure 3D de ZgHAD. La délétion du gène had chez un mutant a révélé que ce gène confèrerait un avantage sélectif à la bactérie en présence de BAA et IAA, des haloacides potentiellement toxiques et présent dans l'environnement marin. Par des analyses biochimiques, j'ai aussi caractérisé la troisième VIPO, spécifique à l'oxydation de l'iodure chez Z. galactanivorans (Zg-VIPO3). Grâce à la production d'un mutant knock-out pour zg-vipo1, j'ai montré l'implication directe de ce gène dans la captation d'iode par la bactérie, localisé par immunomarquage les activités VIPO dans des fractions membranaires et identifié, via des analyses LC-MS/MS, un autre acteur potentiel, une protéine membranaire, homologue de OmpA. Les résultats de ces travaux apportent une meilleure compréhension des rôles biologiques de ces enzymes et ont permis de proposer un modèle hypothétique du métabolisme halogéné de Z. galactanivorans, dans le contexte des interactions de cette bactérie marine avec son environnement.

Abstract

In the marine environment, numerous organisms participate in the biogeochemical cycle of halogens by emitting halogenated volatile organic compounds. However, the enzymatic mechanisms involved in the biosynthesis, uptake and storage of those particular compounds are often poorly known, especially in marine bacteria. The annotation of the genome of Zobellia galactanivorans, a marine flavobacterium living on the surface of macroalgae, allowed to identify several genes potentially involved in this halogen-related metabolism. During this PhD project, I have performed biochemical and structural analyses, and developed functional approaches on genes, coding for vanadium-dependent iodoperoxidases (VIPO) and for a haloacid dehalogenase (HAD). I have determined the specific substrates of the ZgHAD enzyme and analyzed its enzymatic stability, and steady-state parameters. The gene knockout approach revealed that the expression of had gene could provide a selective advantage to this bacterium in the presence of BAA and IAA, haloacids potentially toxic and present in the marine environment. I have also solved the 3D structure of ZgHAD. Through biochemical analyses, I have characterized the third VIPO, specific to iodide oxidation, in Z. galactanivorans (Zg-VIPO3). Thanks to the obtention of a zg-vipo1 gene knockout mutant, I have shown the direct involvement of this gene in iodine uptake in the bacteria, localized by immunolabelling the VIPO activities in membrane fractions and identified, through LC-MS/MS analyses, another potential actor, a membrane OmpA-like protein. The results of this work provide a better understanding of the biological roles of these enzymes and allow to propose a hypothetical model of the halogen metabolism of Z. galactanivorans, in a context of the interactions of this marine bacterium with its environment.

Figure 0 - 1 :

 01 Figure 0-1: Schematic overview of the global halogen biogeochemical cycle…………………………….……..14 Figure 0-2: Key steps of the catalytic mechanisms of flavin-dependent halogenases. (A) Formation of FADHOOH, which oxidizes X-to HOX. (B) Proposed formation of a lysine N-haloamine intermediate. (C) Electrophilic aromatic substitution of the tryptophan substrate. (D) Nucleophilic halogenases catalyze the attack of halide anions (X -) as nucleophiles on SAM. The fluorinase FlA and the chlorinase SalL produce halogenated adenosines, whereas halide methyl-transferases produce halomethanes. (Source: Senn, 2014)…………………………………………………………………………...……………..…….20 Figure 0-3: (A) Catalytic mechanisms of the non-heme iron halogenases (Source: Timmins & de Visser, 2018). (B) Key steps of the heme-dependent haloperoxidase mechanism (Source: Senn, 2014)………….……..21 Figure 0-4: Deiodination catalyzed by (A) iodotyrosine deiodinase (IYD) and (B) iodothyronine deiodinase (ID) (source: Fournier, 2014)………………………….…………………………………………………..……22 Figure 0-5: (A) reductive dehalogenation, (B) oxidative dehalogenation and, (C) hydrolytic dehalogenation (source: Sayyed & Patel, 2011)………………………………………………………..………….………24 Figure 0-6: Reactions of the VHPO/H2O2 system. X: Br, Cl, I. R-H: organic compound. (Fournier and Leblanc. 2014) …………………………………………………………………...…………………………………25 Figure 0-7: Phylogenetic analysis of VHPO proteins. A protein maximum likelihood (PML) tree was constructed using the multiple alignment of characterized and putative VHPO from 124 eukaryotic and selected bacterial species, and of five Bacterial Acid Phosphatases. When characterized at the biochemical level, the VHPO specificity towards halides is mentioned and refers to the legend code, i.e. iodo-(VIPO), bromo-(VBPO) or chloroperoxidase (VCPO) activity. The available 3D structures of VHPO are indicated by a star (from Fournier et al. 2014)…………………………………………………………………………………28 Figure 0-8: Hypothetic mechanisms of iodine uptake in L. digitata (from Verhaeghe et al. 2008b) …….………29 Figure 0-9: Mechanisms for hydrolytic dehalogenation of 2-haloacetic acids. (A) Reaction mechanism involving nucleophilic attack of an active site carboxylate group on the substrate leading to the formation of an ester intermediate. The reaction of L-2-haloacid dehalogenase (L-2-HAD) proceeds by this mechanism. (B) Reaction mechanism involving direct nucleophilic attack of water on the substrate. The reactions of D-2haloacid dehalogenase (D-2-HAD) and DL-2-haloacid dehalogenase (DL-2-HAD) proceed by this mechanism. L-2-Haloalkanoic acid is shown as the substrate here, but D-2-haloalkanoic acid can also serve as a substrate for this DL-2-HAD (Source: Kurihara & Esaki, 2008). …………………………………….31 Figure 0-10: (A) Picture of Z. galactanivorans colonies on agar plate. (B) Phase contrast microscopy image of a Z. galactanivorans liquid culture (scale bar 6 µm). ……………………………………………………….36 Figure 0-11: Hypothetical model of iodine metabolism within Z. galactanivorans and its interaction with the brown alga L. digitata (adapted from Fournier, 2014). ……………………………………………………38 Figure 3-1: Phase diagram of the conditions needed for crystallization by vapor diffusion. In the undersaturation area: the drop remained clear. The goal was to find the right ratio and the good protein/precipitant reagent concentrations in order to reach the nucleation area and promote crystal growth. ………………………132 Figure 3-2: Crystallization technics (A) sitting drop vapor diffusion and (B) hanging drop vapor diffusion (François Thomas, 2011) …………………………………………………………………………...……133 Figure 3-3: (A) Chromatogram and SDS-PAGE analyses of Zg-VHPO3 purification fractions by size exclusion chromatography. Elution buffer: 20 mM Tris/HCl, pH 8.0 and 150 mM NaCl. The polyacrylamide gel was stained with Coomassie Brilliant Blue R-250, revealing the presence of the recombinant protein in eluted fractions of the A280 peak (lanes 43 to 50 and 52 to 59). M = Precision Plus Protein™ Standards marker (Bio-Rad Laboratories) and molecular masses were indicated on the left of the lane. (B) Chromatogram analysis of the first peak from the previous size exclusion chromatography. Elution buffer: 20 mM Tris/HCl, pH 8.0 and 150 mM NaCl. Black dotted rectangle surrounded the 2 nd peak of the size exclusion chromatograph in A, corresponding to the dimeric form. …………………………………………..……135 Figure 3-4: Chromatogram and SDS-PAGE analyses of Zg-VHPO3 purification by size exclusion chromatography. Elution buffer: 20 mM Tris/HCl, pH 8.0 and 50 mM NaCl. The polyacrylamide gel was stained with Coomassie Brilliant Blue R-250, revealing the presence of the recombinant protein in eluted fractions of the A280 peak (lanes D10 to E4 and E8 to F2). M = Precision Plus Protein™ Standards marker (Bio-Rad Laboratories) and molecular masses were indicated on the left of the lane. ……………………136

Figure 0 - 1 :

 01 Figure 0-1: Schematic overview of the global halogen biogeochemical cycle (adapted from Fournier & Leblanc, 2014)

Figure 0 - 2 :

 02 Figure 0-2: Key steps of the catalytic mechanisms of flavin-dependent halogenases. (A) Formation of FADHOOH, which oxidizes X-to HOX. (B) Proposed formation of a lysine N-haloamine intermediate. (C) Electrophilic aromatic substitution of the tryptophan substrate. (D) Nucleophilic halogenases catalyze the attack

Figure 0

 0 Figure 0-3: (A) Catalytic mechanisms of the non-heme iron halogenases (Source: Timmins & de Visser, 2018). (B) Key steps of the heme-dependent haloperoxidase mechanism (Source: Senn, 2014).

Figure 0 - 4 :

 04 Figure 0-4: Deiodination catalyzed by (A) iodotyrosine deiodinase (IYD) and (B) iodothyronine deiodinase (ID) (source: Fournier, 2014)

Figure 0

 0 Figure 0-5: (A) reductive dehalogenation, (B) oxidative dehalogenation and, (C) hydrolytic dehalogenation (source: Sayyed & Patel, 2011).

Figure 0 - 6 :

 06 Figure 0-6: Reactions of the VHPO/H2O2 system. X: Br, Cl, I. R-H: organic compound. (Fournier and Leblanc.

Figure 0 - 7 :

 07 Figure 0-7: Phylogenetic analysis of VHPO proteins. A protein maximum likelihood (PML) tree was constructed using the multiple alignment of characterized and putative VHPO from 124 eukaryotic and selected bacterial species, and of five Bacterial Acid Phosphatases. When characterized at the biochemical level, the VHPO specificity towards halides is mentioned and refers to the legend code, i.e. iodo-(VIPO), bromo-(VBPO) or chloroperoxidase (VCPO) activity. The available 3D structures of VHPO are indicated by a star (from Fournier et al. 2014).

Figure 0 - 8 :

 08 Figure 0-8: Hypothetic mechanisms of iodine uptake in L. digitata (from Verhaeghe et al. 2008b)

  : -The L-2-haloacid dehalogenases (L-2-HADs), also named (S)-2-haloacid dehalogenases, only act on L-2-haloalkanoic acids to produce the corresponding D-2hydroxyalkanoic acids, -The D-2-haloacid dehalogenases (D-2-HADs), also named (R)-2-haloacid dehalogenases, only act on D-2-haloalkanoic acids to produce the corresponding L-2hydroxyalkanoic acids by a configuration-inverting mechanism, -The DL-2-haloacid dehalogenases (DL-2-HADs) can use both D-and L-2-haloalkanoic acids, producing a mixture of DL-2-hydroxyalkanoic acids. Secondly, these three types of HADs can be separated into two groups depending on their catalytic mechanism properties. In the case of L-2-HADs, it consists of a nucleophilic attack by a carboxylic amino acid followed by the hydrolysis of the esterified intermediate by a water molecule in a second step (Figure 0-9A). For the D-2-HADs and the DL-2-HADs, a direct nucleophilic attack mediated on the α-carbon by a hydrolytic water displaces the bound halogen of the substrate (Figure 0-9B).

Figure 0 - 9 :

 09 Figure 0-9: Mechanisms for hydrolytic dehalogenation of 2-haloacetic acids. (A) Reaction mechanism involving nucleophilic attack of an active site carboxylate group on the substrate leading to the formation of an ester intermediate. The reaction of L-2-haloacid dehalogenase (L-2-HAD) proceeds by this mechanism. (B) Reaction mechanism involving direct nucleophilic attack of water on the substrate. The reactions of D-2-haloacid dehalogenase (D-2-HAD) and DL-2-haloacid dehalogenase (DL-2-HAD) proceed by this mechanism. L-2-Haloalkanoic acid is shown as the substrate here, but D-2-haloalkanoic acid can also serve as a substrate for this DL-2-HAD (Source: Kurihara & Esaki, 2008).

Figure 0 -

 0 Figure 0-10: (A) Picture of Z. galactanivorans colonies on agar plate. (B) Phase contrast microscopy image of a Z. galactanivorans liquid culture (scale bar 6 µm).

Figure 11 .

 11 Besides the biochemical and structural characterization of the first two VIPOs(Fournier et al., 2014), it illustrates that most of the identified enzymes, through genomic annotation, still have putative biochemical and biological functions in Z. galactanivorans, and especially during interactions with L. digitata.

Figure 0 - 11 :

 011 Figure 0-11: Hypothetical model of iodine metabolism within Z. galactanivorans and its interaction with the brown alga L. digitata (adapted from Fournier, 2014).

  well-supported groups (Fig. 1), with an additional small clade of proteobacterial sequences related to the L-2-DhlB of Ancylobacter aquaticus UV5. The first group (group A) mostly comprises proteobacterial L-2-HAD sequences (122/182 sequences) that are divided into four clades, respectively containing the biochemically characterized L-2-HADs L-DEX, DhlB, DehIVa and DehPsi. Two other clades in group A contained archaeal proteins: one features terrestrial extremophile L-2-HADs together with the DehSft protein; the second clade contains a mix of Euryarchaea and Actinobacteria putative L-2-HADs. Most L-2-HAD homologs from group A have been identified in species of terrestrial or freshwater origins, but also few in marine species (Table

Figure 1 :

 1 Figure 1: Representative phylogenetic tree of the L-2-HAD proteins. This PhyML tree was constructed through an automated pipeline at the NGphylogeny.fr website including 352 archaeal and bacterial sequences (see Table S2). Only bootstrap values (100 replicates) > 70 % are provided. The tree is rooted by an outgroup of beta-phosphoglucomutase proteins, a distinct and closely related group of the HAD superfamily. Black colored circles indicate groups with at least an available 3D structure of L-2-HAD. Black branches represent putative L-2-HAD proteins from terrestrial or freshwater organisms, blue branches represent those from marine organisms and grey ones are those for mixed terrestrial/marine groups. The number of sequences in each group is indicated in brackets. DehSft, L-DEX, DhlB, DehIVa, DehPsi, DehRhb, ZgHAD, L-2-DhlB and BetaPGM proteins are indicated in their clade of belonging. Group A and group B define the two major phylogenetic groups, for which nine specifically conserved amino acids of the catalytic site are shown. The two distinct clades of Bacteroidetes L-2-HADs are named HAD1 and HAD2.

  Figure 2: Genetic context of the two types of haloacid dehalogenases identified inFlavobacteriaceae. On the left, the phylogenetic tree of 10 genus clades was constructed using the comparative genomics tool for genome clustering of MaGe website. The yellow round and square symbols indicated the presence of homologs genes belonging, respectively, to HAD1 or HAD2 phylogenetic groups. The middle part represents the genetic context around HAD1-type genes found in Zobellia genomes. The synteny in this genomic region of other genera is indicated below (in black). In addition, the specific genomic context around HAD1-type genes is shown in Leeuwenhoekiella, Gramella and Zeaxanthinibacter genomes, respectively, in red, blue and green. At the right of the panel, the gene synteny is shown around HAD2-type gene. Conserved genes between species are shaded in black, partially conserved genes are shaded in grey and absent genes are represented by dotted lines. PucB CT = PucB-like cytidyltransferase, AH = Amidohydrolase, MP = metallopeptidase, PP1 = putative protein 1, PP2 = putative protein 2, UDG = Uracyl-DNA-Glycolase, MutS2 = Recombination inhibitory protein, YuxK = Putative DCC family thioldisulfide oxidoreductase, CEP = Conserved exported protein of unknown function, SP = Secreted protein, HP = Hypothetical protein, CPUF = Conserved protein of unknown function, MoaC = Molybdenum cofactor biosynthesis protein, MCF = Molybdopterin converting factor, tG = trnaGly, GBR = Glyoxalase/bleomycin resistance, Tet TF = Tetracyclin transcription factor, UOR = Uncharacterized oxidoreductase, D/E AT = Asp/Glu amidotransferase. Arrow representations of genes indicate the orientation of the ORFs.

Figure 3 :

 3 Figure 3: Screening of ZgHAD specificity towards different halogenated substrates based on colorimetric assays. Pictures were taken after 0, 5, 10 and 30 minutes of enzymatic reaction. Substrates are : (1) iodoacetic acid ; (2) bromoacetic acid ; (3) chloroacetic acid ; (4) D-2bromopropionic acid ; (5) L-2-bromopropionic acid ; (6) D-2-chloropropionic acid ; (7) L-2chloropropionic acid ; (8) trichloroacetic acid ; (9) 3-iodopropionic acid ; (10) 3-bromopropionic acid ; (11) 2-chlorobutyric acid ; (12) iodoacetamide ; (13) dibromoacetic acid ; (-) no enzyme ; (+) with enzyme.

Figure 4 :

 4 Figure 4: Comparison of bacterial growth of different marine strains, Z. galactanivorans HAD knockout mutant and E. coli recombinant clones in presence of haloacetic acids (A) Growth rates of Tenacibaculum strains (T. discolor : TDIS and T. gallaicum : TGAL) compared to Z. galactanivorans wild type (ZGAL) in liquid cultures supplemented with 10 mM bromoacetic acid (BAA) and 2 mM iodoacetic acid (IAA). (B) Z. galactanivorans WT and Δhad growth on solid medium with BAA and IAA solutions deposited on a filter at the center of the plate. The diameter of inhibition around the disk was measured for each plate after 3 days of incubation at 20 °C. CTRL: negative control. (C, D) Growth rates of Z. galactanivorans wild-type and Δhad mutant in liquid cultures with different concentrations of BAA (C) and IAA (D). Z. galactanivorans (orange line, round dots) and Δhad mutant (green line, triangle dots). (E) Percentage of growth of ZgHADexpressing recombinant clones of E. coli (ZgHAD-BL21) compared to E. coli control strain (pFO4-BL21) in liquid cultures supplemented with either 0.5 or 5 mM BAA and 0.16 or 1.6 mM of IAA.

  metatranscriptome abundance exceeding 5 to 40 fold that in metagenome. The sample exhibiting the highest expression level for the group B (MetaT/MetaG ratio > 40) was located at the station 137 (sample TARA_B100001964, TARA_137_DCM) in the Pacific Ocean at about 500 km west off the coast of Mexico. It corresponds to water collected during late autumn at 40 m depth in the deep chlorophyll maximum layer. The temperature of the water was 20.5 °C and pigment detection indicated that chlorophyll a, fucoxanthin, lutein and zeaxanthin were present in the sample. The exploration of individual results for all group B L-2-HAD hits found in this particular sample suggests that mainly two homolog genes (corresponding to OM-RGC.v2.011753063 and OM-RGC.v2.014543634) are highly expressed. Both hits were affiliated to the Gammaproteobacterium Alteromonas australica.

Figure 5 :

 5 Figure 5: Analysis of environmental and oceanic distributions of L-2-HADs into IMG/MER metagenomes and OGA databases. (A) Global abundance representation of the group A homologs and group B homologs among L-2-HADs hit number in the full set of IMG/MER metagenomes. (B-C) Distribution of the group A (B) and the group B (C) sequence hits in 5 IMG/MER ecosystems sets. (D-E) Geographic distribution of group A (D) and group B (E) homolog relative abundances into metagenomic (metaG) and metatranscriptomic (metaT) OGA databases and scatterplots of the corresponding normalized gene abundance in metaT vs. metaG. Global abundance scales are represented by the grey circles at the bottom right of the maps.

Figure 6 :

 6 Figure 6: Hypothetical scheme of production and biotransformation of haloacetates by Z. galactanivorans, in macroalgae-dominated coastal habitats. ZgHAD and Zgal_4183 = L-2-Haloacid dehalogenase of Z. galactanivorans; BAA = bromoacetic acid; IAA = iodoacetic acid; Zgal_4160 = glyoxylate/hydroxypyruvate reductase; Zgal_4169 = glyoxylate reductase; Zgal_4659 = glycolaldehyde dehydrogenase; VHPO = vanadium-haloperoxidases. Dotted arrows

  Furthermore, the group B showed a separation of two distinct Bacteroidetes L-2-HAD clades, namely HAD1 (including ZgHAD) and HAD2. The comparative genomics in the Flavobacteriaceae family and transcriptomic analysis in Z. galactanivorans suggested that zghad featured a 3-gene operon with zgal_4181 and zgal_4182. Comparative genomics also demonstrated that the HAD1-type genes did not have the same genomic context in different Flavobacteriaceae. It brought to light that HAD1-type gene might be a mobile element in flavobacterial genomes or might have been acquired by multiple independent horizontal gene transfers. By contrast, the HAD2-type gene is often surrounded by the same two genes (TetR TF and UOR) suggesting a conserved genomic organization within Flavobacteriaceae. With the fact that its presence was frequent in Flavobacteriaceae genomes, this conserved pattern put forward the hypothesis that the HAD2-type gene was an ancestral gene, which still need to be characterized at the biochemical level. Additional functional and transcriptomic analyses would be necessary to further test these hypotheses.This phylogenetic overview was enlarged with environmental analyses through oceanic (OGA) and multi-ecosystemic (IMG/MER) data. Group A L-2-HADs are more widespread than group B, irrespective of the biotope. These group A L-2-HADs are almost equally Chapter 1 ______________________________________________________________________________ -70 -distributed between terrestrial and marine habitats. Moreover, the analyses of ocean genomic data revealed that these L-2-HADs were more frequent in the marine environment than initially thought, often exceeding group B sequences. The group B L-2-HADs appeared to be less frequent in nature but they are very selectively present in the marine environments, in particular in close association with macroalgae. Overall, the presence of toxic haloacids in marine environments likely drove the selection and evolution of L-2-HAD genes. Indeed, the strong prevalence of L-2-HADs in metagenomes from macroalgae or hypersaline waters suggests that these enzymes could be involved in the elimination of haloacetic acids favored in these particular environments. Considering a larger point of view, the important representation of L-2-HAD genes in oceanic stations could reflect strong biomass production spots like microalgal blooms. Metatranscriptomic analyses of OGA data revealed that group A L-2-HADs have a homogeneously low expression level around the globe while group B expression is likely induced in certain zones in response to yet-unknown stimuli. The high expression of group B L-2-HADs affiliated to Alteromonas at station TARA_137 where chlorophyll a, fucoxanthin, lutein and zeaxanthin were detected might suggest an association with microalgae and Flavobacteriaceae.CONCLUSIONBiochemical and functional characterizations of the only L-2-HAD encoded in the genome of Z. galactanivorans (ZgHAD) have shown its key role in the catabolism of IAA and BAA in this marine Bacteroidetes. ZgHAD could confer a selective advantage to live in haloacetic acid-producing niches, such as those formed by coastal macroalgae. An extensive phylogenetic analysis has revealed the occurrence of two sister groups of L-2-group A and group B. When the well-represented group A contains both terrestrial and marine sequences, the minor group B was mainly composed of marine L-2-HADs and contained two distinct Bacteroidetes clades. The search for group A and group B L-2-HADs homologs in environmental databases further confirmed world-wild, but distinct ecological distribution and expression levels of these two L-2-HAD groups, according to geographical origins, suggesting different ecological functions. For instance, the presence of homologs of ZgHAD could be the functional signature of bacterial haloacetic acid catabolism, especially during micro-and macroalgal interactions.

  in a newick file formatted with the program MEGA v10.1.1 to obtain the corresponding simplified dendrogram tree figure.Environmental IMG/MER data analysesL-2-HAD homologs were searched by BlastP in the environmental metagenomic //img.jgi.doe.gov/cgibin/mer/main.cgi?section=FindGenesBlast&page=geneSearchBlast), using DehIVa and ZgHAD sequences as the representative sequences of the phylogenetic group A and group B respectively. A stringent e-value cutoff of 10 -50 was selected to identify reliable hits of group A and group B with at least 38% amino acid identity into five datasets (Terrestrial environments, Terrestrial host-associated, Non-marine aquatic, Marine environments, Marine host-associated). Each dataset was constituted of 5 subsets comprising 100 selected metagenomes (Table

  7.5), 500 mM NaCl and 50 mM imidazole and an elution buffer containing 500 mM imidazole instead of 50 mM. Proteins were eluted with a constant gradient of imidazole concentration. The pooled fractions of the elution peak were concentrated on Centriprep devices and the protein sample was injected on a gel filtration HiLoad™ 16/600 Superdex™ 200 pg column (GE Healthcare Life Sciences). The purified protein was stored in a buffer containing 20 mM Tris (pH 8.0) and 150 mM NaCl. The purity of the recombinant protein was analyzed by SDS-PAGE (Bio-Rad Mini-PROTEAN® precast gels and systems).

  temperature gradient was set to an increase of 2°C/min in a range from 20 °C to 95 °C. Protein unfolding was measured by detecting the temperature-dependent change in tryptophan fluorescence at 330 and 350 nm emission wavelengths. The increase of the ratio of Trp fluorescence emission between 350 and 330 nm indicates the thermal unfolding transition midpoint of the protein.

  concentrations tested, with a stabilization allowing about 20% of residual activity in presence of 60 to 80% of acetonitrile solvent.

Figure 1 :

 1 Figure 1: (A) Thermal break point of ZgHAD as determined by the Prometheus NT.48. (B)Thermal stability of ZgHAD as determined by the incubation of the enzyme at varying temperatures for 30 minutes. (C) Solvent stability of ZgHAD as determined by the activity of the enzyme after exposure to different concentrations of ethanol, methanol, acetonitrile and DMSO for 1h.

  the cap domains of ZgHAD are similar to those of DehRhb (2YML), DehSft (2W43), DehIVa (2NO4) and L-DEX YL (1ZRM) with rmsd values between matching Cα positions of 1.21 Å over 212, 1.65 Å over 194, 1.77 Å over 213 and 1.30 Å over 203 residues, respectively.

Figure 2 :

 2 Figure 2: (A) Overall fold of ZgHAD monomer presented as a ribbon diagram colored by secondary structure elements. The α helices, β strands and loops are colored in green, red and orange respectively. (B) Ribbon diagram of ZgHAD dimer viewed with the two-fold horizontal axis. Each subunit is shown in a different color. The red arrows indicate the position of the catalytic site.

Figure 3 :

 3 Figure 3: Ribbon representation showing the active site residues in the structures of ZgHAD and DehRhb. The side chains of selected residues are shown as sticks (carbons are colored in green, pink, yellow or purple, oxygen red and nitrogen blue). (A) Superimposition of the structures of ZgHAD (green) and DehRhb (pink). (B) Superimposition of ZgHAD (green) and mutant H179N (yellow). (C) Superimposition of ZgHAD (green) and mutant H179A (purple). The red square shows the fixation site of the substrate. Orange doted lines highlight the distance of the movement of Glu17 between wild-type and mutant proteins.

Figure 4 :

 4 Figure 4: (A) Ribbon representation of the 13 ZgHAD_H179N dimers present in the asymmetric unit. The individual monomers of each dimer are colored with similar colors, highlighting the helical arrangement of the dimers. At one end, each circle of dotted lines surrounds one dimer. (B) Superimposition of all dimers based on the calculation of a single monomer of each (grey). The colored monomers highlight the variability of relative position to the gray monomers, which in contrast are almost identical. (C) Two of the dimers from (B), displaying the most distant relative orientations, are presented as ribbons and the amino acids E17 and N179, represented as sticks, highlight the position of the respective catalytic active sites.

Figure 5 :

 5 Figure 5: Illustration of the molecular docking calculations of various substrate molecules in the active site of ZgHAD. Selected residues are shown as sticks. (A) Docking of IAA (yellow) in ZgHAD (green) structures. (B) Docking of BAA (cyan) in ZgHAD (green) structure. (C) Docking of CAA (pink) in ZgHAD (green) structure. (D) Docking of 2BPA (purple) in ZgHAD (green) structure. Orange dotted line represent the distance between Asp 14 and the carbon 2 of the substrate. Black doted lines represent hydrogen bonds of the substrates with surrounding amino acids.

  1). The 3D structure of Zg-VIPO1 was obtained as a monomer, unlike algal VBPOs that are mostly homodimers, hexamers or dodecamers. The other bacterial VHPOs characterized up to now are the dimeric VCPO from Streptomyces sp. CNQ-525 (Liscombe et al., unpublished) and the dodecameric VBPO from Acaryochloris marina (Frank

Figure 3 - 2 :

 32 Figure 3-2: Crystallization technics (A) sitting drop vapor diffusion and (B) hanging drop vapor diffusion(François Thomas, 2011)

Figure 3 - 3 :

 33 Figure 3-3: (A) Chromatogram and SDS-PAGE analyses of Zg-VHPO3 purification fractions by size exclusion chromatography. Elution buffer: 20 mM Tris/HCl, pH 8.0 and 150 mM NaCl. The polyacrylamide gel was stained with Coomassie Brilliant Blue R-250, revealing the presence of the recombinant protein in eluted fractions of the A280 peak (lanes 43 to 50 and 52 to 59). M = Precision Plus Protein™ Standards marker (Bio-Rad Laboratories) and molecular masses were indicated on the left of the lane. (B) Chromatogram analysis of the first peak from the previous size exclusion chromatography. Elution buffer: 20 mM Tris/HCl, pH 8.0 and 150 mM NaCl. Black dotted rectangle surrounded the 2 nd peak of the size exclusion chromatograph in A, corresponding to the dimeric form.

Figure 3 - 4 :

 34 Figure 3-4: Chromatogram and SDS-PAGE analyses of Zg-VHPO3 purification by size exclusion chromatography. Elution buffer: 20 mM Tris/HCl, pH 8.0 and 50 mM NaCl. The polyacrylamide gel was stained with Coomassie Brilliant Blue R-250, revealing the presence of the recombinant protein in eluted fractions of the A280 peak (lanes D10 to E4 and E8 to F2). M = Precision Plus Protein™ Standards marker (Bio-Rad Laboratories) and molecular masses were indicated on the left of the lane.

Figure 3

 3 Figure 3-5: O-dianisidine assay on native polyacrylamide electrophoresis gel of the recombinant protein Zg-VHPO3, purified as monomer (M) and dimer (D) batches. The assay was performed in presence of potassium chloride, bromide or iodide.

Figure 3 - 6 :

 36 Figure 3-6: Thymol blue colorimetric assay of the recombinant protein Zg-VHPO3, purified as monomer and dimer batches. The assay was performed at room temperature for 1 h in clear flat-bottomed microplate wells, in presence of potassium iodide or potassium bromide. The negative control was performed without enzyme.

Figure 3 - 7 :

 37 Figure 3-7: Thermal denaturing analysis using Nanotemper NT48 of recombinant protein Zg-VIPO3, purified as (A) Dimer batch, or (B) Monomer batch. Each peak represented denaturing protein step.

Figure 3 - 8 :

 38 Figure 3-8: SDS-PAGE gel revealing the iodoperoxidase activities of recombinant protein in dimeric or monomeric batches, after heating treatment at 10°C to 90°C for 30 minutes. The native polyacrylamide electrophoresis gel was stained with thymol blue. M = Precision Plus Protein™ Standards marker (Bio-Rad Laboratories) and molecular masses were indicated on the left of the lane.

Figure 3 - 9 :

 39 Figure 3-9: Ribbon-type representation of Zg-VIPO3 3D model as monomer (A) based on Zg-VIPO1 (PDB accession number 4CIT) and as dimer (B) based on NapH1 (VCPO, PDB accession number 3W35). One monomer was colored in cyan and the other one in green. Red dotted circles showed the localization of the active sites. Corresponding surface representations of the monomer (C) and of the dimer (D) in side view with the vanadate molecule, represented in red.

Figure 4 - 2 :

 42 Figure 4-2: Anti-Zg-VIPO1 western blot analysis of the fractionated cellular compartments of Z. galactanivorans. 10 µg of protein samples were loaded in each well. Legend: periplasmic (P1, P2), cytoplasmic (C1, C2) and membrane (M1, M2) duplicates. Precision Plus Protein™ Dual Color Standard Marker (PM).

Figure 4 - 3 :

 43 Figure 4-3: Growth curves of Z. galactanivorans wild-type (A) and Δipo1 mutant (B) cultures with increasing concentrations of KI for 7 days. Values are mean ± s.e.m.

  measured each day for radioactivity. As shown in Figure4-4, for C. fucicola and Δipo1 mutant strains, the radioactive iodine contents remained stable in the medium, around 0.1-0.12 µM, between 24 h and 72 h (Figure4-4A). In comparison, based on the radioactive measurements, the iodide concentration decreased very rapidly in surrounding media of both Z. galactanivorans WT and Arenibacter sp. C-21 strains, up to 0.02 µM, and remained at this low level for the duration of the experiment. In parallel, after 24 h of culture, the radioactive iodide content significantly increased in the cell pellets of Arenibacter sp. C-21 and Z. galactanivorans WT strains (168 and 160 pmol per mg of dry cells respectively, Figure4-4B). At 48 h and 72 h of culture, the iodide content decreased slightly in the cell pellets of Z. galactanivorans WT and almost by half for the C-21 strain. For C. fucicola, the maximum iodide content in cell pellets reached only 0.48 ± 0.06 pmol per mg of dry cells after 48h of culture. The mutant strain Δipo1 cultures showed small increases of iodide content after 72h with 14 ± 0.33 pmol per mg of dry cells (Figure4-4B).

Figure 4 - 4 :

 44 Figure 4-4: Time course of iodide accumulation by Arenibacter sp. C-21 (black squares), Zobellia galactanivorans Dsij T WT (red squares), Zobellia galactanivorans Δipo1 (blue triangles) and Cellulophaga fucicola (grey diamond-shape) strains grown in artificial ZoBell medium containing iodide 125 I -. The results are expressed as the means +/-standard deviations of triplicate analyses. (A) Relative iodide content in the culture medium. (B) Measure of the radioactive iodide concentration in culture pellets during 3 days of culture.

  electrophoresis and the radioactivity was measured along the migration pattern. Only a background radioactivity was detected in the lane loaded with the protein fractions from the Δipo1 mutant strain (Figure S4-1). A uniquely high-level zone of 125 I -radioactivity was detected on the radioactive gel loaded with the WT membrane-enriched protein fractions (Figure S4-2), corresponding to the migration position of a band triplet, around 50 kDa, detected by Coomassie Blue in the equivalent non-radioactive gel run in parallel (Figure 4-5).

Figure 4 - 5 :

 45 Figure 4-5: Picture of the SDS-PAGE polyacrylamide gel, stained by Coomassie Blue and loaded with 20 µL of Z. galactanivorans membrane fractions (5 deposits).In the corresponding gel charged with radioactive fractions, the bands #1, #2 and #3 showed the most intense signal and then cut in the nonradioactive gel. The 125 I -radioactivity values correspond to the mean and standard deviation of five independent measurements for each.

  galactanivorans and C. gingivalis could feature conserved functions and cellular locations. In addition, the iodinated Tyr425 residue of Zobellia_1580 would be located in the peptidoglycan attachment domain, bathed in the periplasmic space.

Figure 4 - 6 :

 46 Figure 4-6: 3D model representation of zobellia_1580 OmpA-like protein based on CgOmpA (PDB accession: 5WTL). The overall structure is colored in turquoise and shown as ribbons. The Tyr425 is colored in red and shown as sticks.

A

  conserved chemical trapping by the cell wall structures in the apoplast of kelps as proposed by Verhaeghe et al. (2008b) could be different in Z. galactanivorans as these components are absent and the iodine concentration factor is very weak in comparison. Nevertheless, molecular structures present in the same compartment as Zg-VIPO1 could potentially play a role as iodine acceptors and explain the accumulation rate of iodine.

Figure S4- 1 :

 1 Figure S4-1: 125 I -radioactivity measured on the mutant strain membrane fraction, migrated on a SDS-PAGE gel and placed on miniGita.

Figure S4- 2 :

 2 Figure S4-2: 125 I -radioactivity measured on the wild-type strain membrane fraction, migrated on a SDS-PAGE gel and placed on miniGita.

Figure 5 - 1 :

 51 Figure 5-1: Hypothetical model of halogen-related metabolism within Z. galactanivorans (adapted from Fournier, 2014).

  Microbial communities form biofilms that differ depending on the seasons, their localization on the surface of the algae and the physiology of the algal hosts. During those interactions, many kinds of chemicals are secreted and can impact specific bacterial communities. Future experiments would consist of growing Z. galactanivorans WT and mutant strains with pieces of L. digitata, to analyze effects on growth/survival and genome regulation by transcriptomic approaches. To further explore compounds emitted by Z. galactanivorans in the presence of macroalgae, and their impact on biofilm formation with other bacteria, metabolomic approaches could be a complementary well-adapted tool.A last open question remains about the bacterial implication in the biogeochemical cycle of halogens. Since, Z. galactanivorans was shown to be a good bacterial model to study iodine metabolism, thanks to the numerous enzymes capable of iodination, it would be interesting to analyze the production of volatile halocarbons, and especially iodinated ones, by this bacteria in different physiological and environmental conditions. Moreover, the genome comparison of numerous microorganisms isolated from kelps, with the genome of Z. galactanivorans, would allow to question the occurrence and convergent evolution of iodine-specific enzymatic mechanisms in bacteria living on kelps surfaces and their potential importance in iodine biogenic cycling.

Table 4 - 4 :

 44 …………………...…138 Table 3-3: Protein-Protein interface analysis performed using PDBePISA online tool for the structure of Zg-VIPO3 dimeric model, VCPO of Streptomyces sp. CNQ-525 (NapH1, PDB accession number 3w35) and VBPOI of Ascophyllum nodosum (An-VBPOI, PDB accession number AQI9). ………………………...143 Table 4-1: Predictions of Zg-VIPOs subcellular protein localization using 7 different generalist tools developed to discriminate the sorting between 4 or 5 compartments (membranes are not dissociated for ProtComb whereas outer and inner membranes are separated in others). More detailed results are provided in Table S4-1………………………………………………………………………………………………………152 Table 4-2: Predictions of the presence/absence of signal peptides (SP) and the type of these signals in Zg-VIPOs protein sequences using 10 different specialized tools. The best predictions are summarized in the total predictions column. Legend: type I signal peptide (SPI), type II signal peptide (SPII). More detailed results are given in table S4-2. …………………………………………………………………………………...153 Table 4-3: Total iodine content of lyophilized bacterial pellets analyzed by ICP-MS for the Z. galactanivorans WT and ΔIPO1 mutant, and C. fucicola strains after 2-days cultured in artificial ZoBell medium. Values are mean ± std deviation. ……………………………………………………………………………..…159 Radioactive iodide detected in periplasm, cytoplasm and membranes fractions of Z. galactanivorans and ΔIPO1 mutant. Values are mean ± std deviation. ……………………………………………………162 Table

Table 0 - 1 :

 01 Abundance of halogen elements in the Earth's crust and the oceans

Table 0 - 2.

 0

	Name	Species	Activity/Cofactor	PDB accession	References
	Flavin-dependent halogenases	Pseudomonas fluorescens	Chloro/FADH2, O2	2AQJ	Dong et al., 2005
	Non-heme iron-dependent halogenases	Pseudomonas syringae Chloro/Fe2+, O2, α-KG 2FCT	Blasiak et al., 2006
		Pseudomonas syringae Bromo/Fe2+, O2, α-KG 2FCV	Blasiak et al., 2006
	Heme iron-dependent haloperoxidases	Caldariomyces fulmago	Chloro/Heme	1CPO	Sundaramoorthy et al., 1995
	SAM-dependent halogenases	Streptomyces cattleya	Fluoro/FDAS, SAM	1RQR	Dong et al., 2004
		Salinispora tropica	Chloro/SaIL, SAM	2Q6K	Eustaquio et al., 2008
	Cofactor-free haloperoxidases	Streptomyces aureofaciens	Bromo	1BRO	Hofmann et al., 1998
		Streptomyces aureofaciens	Chloro	1A7U	Hofmann et al., 1998
		Curvularia inaequalis	Chloro/vanadate	1VNC	Messerschmidt & Wever, 1996
	Vanadium-dependent haloperoxidase	Ascophyllum nodosum	Bromo/vanadate	1QI9	Weyand et al., 1999
		Zobellia galactanivorans	Iodo/vanadate	4CIT	Fournier et al., 2014

Table 0 -

 0

2: Some examples of biochemically and structurally characterized halogenating enzymes

Table 0 -3: Structural

 0 characterization of vanadium-dependent haloperoxidases (adapted from[START_REF] Leblanc | Vanadium haloperoxidases: From the discovery 30 years ago to X-ray crystallographic and V K-edge absorption spectroscopic studies[END_REF].

	Species	Activity PDB Accession # Structure	References
	Brown Alga
	Ascophyllum nodosum	VBPOI VBPOII	1QI9 5AA6	homodimer homohexamer	Weyand et al., 1999 Radlow et al., 2018
	Red Alga
	Corallina officinalis	VBPO	1QHB	dodecamer	Isupov et al., 2000
	Corallina pilulifera	VBPO	1UP8	dodecamer	Garcia-Rodriguez et al., 2005
	Fungi
	Curvularia inaequalis	VCPO	1VNC	monomer	Messerschmidt & Wever, 1996
	Bacteria
	Zobellia galactanivorans	VIPO1	4CIT	monomer	Fournier et al., 2014
	Streptomyces sp. CNQ-525 VCPO	3W35, 3W36	dimer	Liscombe et al., 2013 (unpublished)
	Acaryochloris marina	VBPO	5LPC	dodecamer	Frank et al., 2016
	VBPO: vanadium-dependent bromoperoxidase; VIPO: vanadium-dependent iodoperoxidase; VCPO:
	vanadium-dependent chloroperoxidase

Table 0 - 4 ,

 04 Chan et al., 2010).

	Protein ID	Species	Substrate of dehalogenation	Metabolic pathway	Origin	PDB on accessi	References
			bromoacetic acid
	DehRhb	Marine Rhodobacteraceae	chloroacetic acid L-2-bromopropionic acid L-2-chloropropionic acid	unknown	marine	2YML	Novak et al., 2013
			dichloroacetic acid
	DehSft	Sulfurisphaera tokodaii (formerly Sulfolobus tokodaii)	L-2-chloropropionic acid L-2-bromopropionic acid L-2-bromobutyric acid L-2-bromohexanoic acid	unknown	hot spring	2W43	Bachas-Daunert et al., 2009 Rye et al., 2009
							Murdiyatmo et
			bromoacetic acid				al., 1992
	DehIVa	Burkholderia cepacia MBA4	chloroacetic acid L-2-bromopropionic acid L-2-chloropropionic acid	2-haloalkanoic acid degradation	soil	2NO4	Pang & Tsang, 2001 Schmidberger et
							al., 2007
			L-2-chloropropionic acid
	L-DEX YL	Pseudomonas sp. strain YL	bromoacetic acid iodoacetic acid chloroacetic acid dichloropropionic acid	2-chloroacrylate degradation	soil	1JUD	Nardi-Dei et al., 1994 Liu et al., 1994
			L-2-chlorobutyric acid
	DhlB	Xanthobacter autotrophicus GJ10	dibromoacetic acid dichloroacetic acid chloroacetic acid bromoacetic acid L-2-chloropropionic acid	1,2-dichloroethane degradation	soil	1QQ5	Van der Ploeg et al., 1991 Ridder et al., 1997
	Bpro0530	Polaromonas sp. JS666	fluoroacetic acid bromoacetic acid chloroacetic acid	unknown	soil	3UM9	Chan et al.,
	Rsc1362	Ralstonia solanacearum GMI1000	chloroacetic acid bromoacetic acid	unknown	soil	3UMB Chan et al.,
	Rha0230	Rhodococcus jostii RHA1	fluoroacetic acid bromoacetic acid chloroacetic acid	unknown	soil	3UMG Chan et al.,
	DehDEH9 9	Paracoccus sp. DEH99	L-2-chloropropionic acid L-2-bromopropionic acid iodoacetic acid	2-haloalkanoic acid degradation	marine		Huang et al., 2011
			bromoacetic acid
			chloroacetic acid
		Psychromonas ingrahamii	dichloroacetic acid L-2-chloropropionic acid L-2-bromopropionic acid	unknown	marine		Novak et al., 2013
			L-2-chlorobutyric acid
			chloroacetic acid
	DhlS5I	Agrobacterium tumefaciens RS5	iodoacetic acid bromoacetic acid L-2-chloropropionic acid	unknown	soil		Köhler et al., 1998
			dichloroacetic acid
			bromoacetic acid
	H-2	Moraxella sp. strain B	L-2-chloropropionic acid iodoacetic acid chloroacetic acid	unknown	soil		Kawasaki et al.,
			dichloropropionic acid

Table 0 -

 0

4: Biochemically and structurally characterized L-2-HADs enzymes from bacteria

Table 1 :

 1 Data collection and refinement statistics for ZgHAD. Values in parentheses refer to the outer resolution shell.

	Chapter 2
	___________________________________________________________________________

  ).

		Zg-VIPO3 model	3W35 (NapH1)	1QI9 (An-VBPOI)
	Solvent-accessible area	7.0%	10.4%	23.5%
	Number of residues in the interface	10.3%	13.2%	24.1%
	Number of residues in the surface	93.4%	89%	91.2%
	Solvation free energy Δ i G (kcal/mol)	-7	-15.6	-47.6
	Hydrogen bonds	11	30	87
	Salt bridges	1	8	9
	Disulfide bond	0	0	2
	Complex Formation Significance Score	0	1	0.92

Table 3 - 3 :

 33 Protein-Protein interface analysis performed using PDBePISA online tool for the structure of Zg-VIPO3 dimeric model, VCPO of Streptomyces sp. CNQ-525 (NapH1, PDB accession number 3w35) and VBPOI of Ascophyllum nodosum (An-VBPOI, PDB accession number 1QI9).

  but it was unable to predict a clear localization for Zg-VIPO1 and Zg-VIPO3.

	Protein	PSORTb	CELLO2GO LocTree3	SOSUI-GramN	iLoc-Gneg	PSLpred	ProtCompB
	Zg-VIPO1	Not determined	Periplasmic Periplasmic Cytoplasmic	Outer membrane	Cytoplasmic Periplasmic
	Zg-VIPO2 Cytoplasmic Cytoplasmic Periplasmic Periplasmic	Outer membrane	Cytoplasmic	Membrane
	Zg-VIPO3	Not determined	Periplasmic Periplasmic Periplasmic Cytoplasmic	Periplasmic	Extracellular

Table 4 - 1 :

 41 Predictions of Zg-VIPOs subcellular protein localization using 7 different generalist tools developed to discriminate the sorting between 4 or 5 compartments (membranes are not dissociated for ProtComb whereas outer and inner membranes are separated in others). More detailed results are provided in TableS4-1.

Table 4

 4

	-2) suggested that the three Zg-VIPOs

Table 4 - 2 :

 42 Predictions of the presence/absence of signal peptides (SP) and the type of these signals in Zg-VIPOs protein sequences using 10 different specialized tools. The best predictions are summarized in the total predictions column. Legend: type I signal peptide (SPI), type II signal peptide (SPII). More detailed results are given in table S4-2.

Table 4 -

 4

3: Total iodine content of lyophilized bacterial pellets analyzed by ICP-MS for the Z. galactanivorans WT and Δipo1 mutant, and C. fucicola strains after 2-days cultured in artificial ZoBell medium. Values are mean ± std deviation.

Table 4 - 4 :

 44 Radioactive iodide detected in periplasm, cytoplasm and membranes fractions of Z. galactanivorans and Δipo1 mutant. Values are mean ± std deviation.

Table S4 - 2 :

 S42 Predictions of the presence/absence of signal peptides (SP) and the type of these signals in Zg-IPOs protein sequences using 12 different specialized tools. Results are reported with the score probability, the location of cleavage site when found and the length of the signal peptide in amino acids. The best predictions are in bold in each line and are summarized in the total predictions column. Legend: type I signal peptide (SPI), type II signal peptide (SPII), not determined (nd).

				Secretary Pathway tools		Lipoprotrein tools	Both
	Protein	Informations	PrediSi	Phobius	SOSUIsignal	SecretomeP	Signal-3L	Signal-BLAST	LipoP	DOLOP	SPEPLip	SignalP	total predictions
		Signal peptide no SP SP no SP no SP	SP	SP	LPSP no SP no SP LPSP	SP 7/12
		SP type							SPII			SPII	SPII
		Probability	0.4540 nd	nd 0.432871 0.984	100.0	6.3204	nd	nd	0.9935
							38/39
	Zg-IPO1	Cleavage site	nd	nd	nd	nd	(17/18)	S16/C17 S16/C17 nd	nd S16/C17 S16/C17
							(13/14)
							38
		SP lenght (aa)	nd	17	nd	nd	(17)	16	16	nd	nd	16	16
							(13)
		Signal peptide no SP SP no SP no SP	SP	SP	LPSP no SP no SP LPSP	SP 6/12
		SP type							SPII			SPII	SPII
		Probability	0.0927 nd	nd 0.146585 0.979	100.0 7.08603	nd	nd	0.9995
	Zg-IPO2						18/19
		Cleavage site	nd	nd	nd	nd	(19/20)	S17/C18 S17/C18 nd	nd S17/C18 S17/C18
							(38/39)
		SP lenght (aa)	nd	18	nd	nd	18	17	17	nd	nd	17	17
		Signal peptide	SP	SP no SP	SP	SP	SP	SP	no SP no SP	SP	SP 9/12
		SP type							SPI			SPI	SPI
	Zg-IPO3	Probability	0.7426 nd	nd 0.934241 0.996	100.0 10.5526	nd	nd	0.9054
		Cleavage site A21/Q22 nd	nd	nd	21/22 A20/A21 A21/Q22 nd	nd A21/Q22 A21/Q22
		SP lenght (aa)	21	21	nd	nd	21	20	21	nd	nd	21	21

Acknowledgments

Tout d'abord, je tiens à remercier mes encadrants Catherine Leblanc et Ludovic Delage qui m'ont donnée l'opportunité travailler sur ce sujet de thèse. Merci pour tous vos conseils, votre patience et votre aide au cours de ces 4 années. Merci de m'avoir fait confiance et de m'avoir soutenue quand j'en avais besoin. Sans vous je n'aurais pas pu y arriver.

Je remercie également la directrice de la Station Biologique Catherine Boyen de m'avoir accueillie, ainsi que tous les membres de l'équipe ABIE.

Abbreviations

Article in preparation

This chapter presents the structural characterization of recombinant ZgHAD, as well as the structure of two site-directed mutant of this protein: H179A and H179N. Computational docking has also been performed to study to substrate fixation in the active site of the enzyme. These data have been obtained thanks to the collaborative work of Mirjam Czjzek and Thomas Roret from the platform of crystallography in Roscoff.

Supporting information

Figure S1: Amino-acid sequence alignment of ZgHAD and his homologues DehRhb from Rhodobacteraceae, DehIVa from B. cepacia MBA4, L-DEX from Pseudomonas sp. YL, DehlB from X. autotrophicus and DehSft from S. tokodaii and using the programs MultAlin [START_REF] Corpet | Multiple sequence alignment with hierarchical clustering[END_REF] and ESPript [START_REF] Robert | Deciphering key features in protein structures with the new ENDscript server[END_REF]. Identical residues are shown in red boxes and residues with similar properties are in blue boxes. The green triangles highlight the residues that have been shown to be important for catalytic activity and substrate recognition (see Table S1). Materials and methods

II.1. Chemicals and antibodies

The monoclonal anti-polyHistidine-Peroxidase antibody (anti-His tag) produced in mice was obtained from Merck (reference: A7058). The rabbit polyclonal anti-Zg-VIPO1 (batch number:

1230005, New Zealand White rabbit) was produced by Covalab using the purified native recombinant protein. The total IgG fraction was purified using protein A beads column and stored at 4°C with a 0.02% (w/v) sodium azide preservative. The final concentration was 2.46 mg.ml -1 .

The secondary antibody coupled to Peroxidase was obtained from Bio-Rad. This anti-rabbit

IgG (H + L)-HRP Conjugate was produced in goats (reference: 170-5046).

Protein A gold conjugates (15 nm) for electronic microscopy were purchased from Cell Microscopy Center, Utrecht, Netherland.

II.2. Bacterial strains and culture conditions

All cultures of marine bacterial strains were carried out at 25°C with shaking at 180 rpm. Strains

Arenibacter sp. C-21 (NBRC culture collection, Chiba, Japan), Cellulophaga fucicola and Zobellia galactanivorans Dsij T (Roscoff culture collection, Roscoff, France) were grown in ZoBell (per liter: 1.0 g yeast extract, 5.0 g tryptone, sea water) or artificial ZoBell (per liter: 1.0 g yeast extract, 5.0 g tryptone, 24.7 g NaCl, 0.7 g KCl, 6.3 g MgSO4.7H2O, 4.6 g MgCl2.6H2O, 1.2 g CaCl2.2H2O, 0.2 g NaHCO3) media supplemented with or without 0.1 µM of KI.

II.3. In silico prediction of protein subcellular localization

The subcellular localization of Zg-VIPOs was predicted using the following generic tools:

PSORTb 3.0 [START_REF] Yu | PSORTb 3.0: Improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes[END_REF], CELLO2GO [START_REF] Yu | CELLO2GO: A web server for protein subCELlular lOcalization prediction with functional gene ontology annotation[END_REF], SOSUI-GramN [START_REF] Imai | SOSUI-GramN: high performance prediction for sub-cellular localization of proteins in Gram-negative bacteria[END_REF], LocTree3 [START_REF] Goldberg | LocTree3 prediction of localization[END_REF], iLoc-Gneg [START_REF] Xiao | A multi-label classifier for predicting the subcellular localization of gram-negative bacterial proteins with both single and multiple sites[END_REF]), PSLpred (Bahsin et al., 2005) and ProtCompB (http://www.softberry.com). The determination of the presence of a membranes, and also exposed to the periplasmic space (as deduced from the study of [START_REF] Seydel | Testing the "+2 rule" for lipoprotein sorting in the Escherichia coli cell envelope with a new genetic selection[END_REF].

Taken together, these in silico subcellular targeting results tend to predict Zg-VIPO1 and Zg-VIPO2 as anchored lipoproteins in the outer membrane, turned towards the periplasmic space.

Zg-VIPO2 could also be present at the inner membrane surface. In contrast, Zg-VIPO3 would be a periplasm-released protein, after cleavage of a SPI peptide signal.

The in vivo expression and localization of Zg-VIPO1 in Z. galactanivorans was investigated using a polyclonal antibody, shown to be specific for Zg-VIPO1 detection, as revealed by a 50 kDa band at the expected size of the recombinant protein (Figure 4-1). The anti-Zg-VIPO1 was also able to recognize some epitopes of Zg-VIPO2 as it cross-reacted at about 15% of intensity with this recombinant protein (Figure 4 putative N-terminal peptide signal assigning the protein to the external membrane or to the periplasm, which was in agreement with its detection in the total membrane protein fraction.

Zobellia_1580 corresponded to the most abundant protein detected in the band#1 gel sample, as it represented about 2.7% of all the peptide spectra. A total of 18 spectra corresponding to the iodinated zone of Zobellia_1580 were detected in the LS-MS/MS data, with two peptide motifs LSAIGYGEEK and LSAIGYGEEKPIASNNTR, but only one was found to be iodinated on the tyrosine (Tyr425). This suggests that Zobellia_1580 is probably partly iodinated with a minor percentage of at least 5.5%. OmpA proteins are well-studied outer membrane proteins in E. coli and possess many biological functions such as adhesion, host cell penetration and even participation in biofilm formation [START_REF] Smith | A molecular Swiss army knife: OmpA structure, function and expression[END_REF].

A 3D structure model of Zobellia_1580 was built with SWISS MODEL using the OmpA protein structure from Capnocytophaga gingivalis (CgOmpA; PDB accession: 5WTL) as a template (Figure 456). Only the second half of the protein was modeled (from glycine193 to lysine452) as the PDB structure of CgOmpA featured only 261/455 amino acids corresponding to the C-terminal domain. Zobellia_1580 featured a high degree of conservation with its counterpart: 63.6% of sequence identity and 57% of sequence coverage (51.5% of sequence identity for the overall sequence coverage). The N-terminal domain of the Zobellia_1580 protein was not modeled but a conserved domain possibly related to a putative MetA-pathway of phenol degradation was predicted in this region (Evalue of 1.17e-06 for a pfam 13557 domain in the region 47-191 of Zobellia_1580). Moreover, this N-terminal region was also similar to the TIGR04565 domain, OMP_myx_plus, known to form a transmembrane β-barrel structure embedded in the bacterial outer membrane. The central part of Zobellia_1580 (Figure 4-6, left part) could be considered as a linker region comprising five consecutive thrombospondin type 3 repeats (TT3Rs). This TT3R domain was shown to be an efficient calcium ion (Ca 2+ ) binding motif in CgOmpA [START_REF] Dai | Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bonddependent calcium-binding capability[END_REF]. The C-terminal modeled domain of Zobellia_1580

Conclusion and Perspectives

Fournier, J.B., Rebuffet, E., Delage, L., Grijol, R., Meslet-Cladière, L., Rzonca, J., et al. (2014) The vanadium iodoperoxidase from the marine Flavobacteriaceae species Zobellia galactanivorans reveals novel molecular and evolutionary features of halide specificity in the vanadium haloperoxidase enzyme family. Appl Environ Microbiol 80: 7561-7573.