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Ce travail a permis d’étudier la dynamique saisonnière des communautés microbiennes (cytométrie en flux et MiSeq ADNr16S) d'un étang sursalé thalassohalin. Malgré les variations de salinité (15,5 à 32 %), un core microbiome hautement stable (97,2± 2,1%) et dominé par Haloquadratum (40,3 à 57,4%) et Salinibacter (4,9 à 21,8%) a été décrit. De plus, des populations halotolérantes, capables de croitre rapidement, ont été détectées durant les épisodes de dilution des eaux. Dans un contexte physico-chimique contraignant où la biodégradation des hydrocarbures (HC) reste controversée, une première étude a été réalisée afin de comprendre le devenir d’un pétrole et son effet sur les communautés microbiennes actives de saumures proches de la saturation en sels (31%). Après biostimulation de ces communautés par l’ajout de matière organique labile et d'une température favorable (40°C), des phytolyptes actifs appartenant aux genres Haloarcula, Halobacterium et Halorubrum ont été détectés dans les microcosmes présentant de la biodégradation (12,8%) après 30 jours. Face aux limitations des processus d'autoépuration en contexte naturel (température plus faible), plusieurs approches de biostimulation testées (i.e. fertilisation minérale –NS ou organique –DS ; dilution) ont permis de forts taux d'atténuation des HC aliphatiques (97,8% et 54,5%) dans des saumures diluées (27,7 à 14%) et fertilisées (-DS et -NS). Dans ces mêmes microcosmes, des phylotypes actifs majoritaires appartenant aux genres Marinobacter et à la famille des Flavobacteriaceae (dont Psychroflexus) ont été détectés (MiSeq ADNr16S). L'opérabilité de ces traitements mériterait d'être testée à une plus large échelle. / This work performed on a thalassohaline hypersaline lake firstly considered seasonal dynamics of microbial communities (flow cytometry and MiSeq on 16S rRNA). Despite salinity fluctuations (15.5-32 %), a microbiome core highly stable at the genus level (97.2 ± 2.1 %) and dominated by Haloquadratum (40.3-57.4 %) and Salinibacter (4.9-21.8 %) was described. Interestingly, some halotolerant phylotypes exhibited rapid growths during dilutions episodes. In a controversial context concerning high salinity effects on hydrocarbons (HC) biodegradation, a study was conducted on close to salts-saturation brines (31 %) to gain insight into the fate of oil and it effects on active microbial communities after 15- and 30-days incubations. Significant oil biodegradation (12.8 %) was detected only after a 30-days incubation in LOM-amended microcosms while phylotypes belonging to Halobacteriaceae (Haloarcula, Halobacterium and Halorubrum) appeared as major active phylotypes. However, these low rates suggested that oil biodegradation should be lower under in situ conditions (lower temperature). Thus, among biostimulation approaches (mineral (-NS) or organic (-DS) amendments; dilution) used to improve self-cleaning processus, DS- or NS-amendments added to diluted brines (27.7 % to 14.0 %) allowed high attenuation rates of aliphatic HC with 97.8 % and 54.5 % respectively. Bacterial phylotypes belonging to Marinobacter and Flavobacteriaceae (e.g. Psychroflexus) were detected in NS- and DS-amended microcosms in which petroleum biodegradation occurred. This strategy will have, however, to be tested in other hypersaline systems (natural or industrial) in order to test its operational efficiency.

La saline de Sfax est un milieu hypersalin localisé dans la zone centrale de la côte est de Tunisie. Dans ce travail, nous avons isolé, identifié, et caractérisé des souches halophiles produisant des peptides antimicrobiens (halocines), afin de mieux comprendre leur rôle dans les interactions microbiennes au sein des milieux hypersalins. Deux étangs salins (TS18, 390 g.L-1 NaCl et M1, 200 g.L-1 NaCl) ont été choisis pour l’échantillonnage. Trente-cinq souches de procaryotes halophiles ont été isolées et caractérisées, dont 11 ont présenté une activité antimicrobienne. Parmi ces souches, 3 produisent des substances antimicrobiennes de nature protéique. A l’aide de PCR et RT-PCR nous avons montré que les souches Halobacterium salinarum ETD5 et ETD8 exprimaient le gène de l’halocine S8, un peptide de 3,6 kDa préalablement purifié d’une souche S8a non identifiée. Le peptide a été purifié à partir de cultures de la souche Hbt. salinarum ETD5. Après purification bioguidée, les fractions actives révèlent deux bandes de 8 et 14 kDa présentant une activité antimicrobienne. L’analyse par séquençage Nterminal et spectrométrie de masse a permis d’identifier ces deux halocines. La bande de 8 kDa correspond à une halocine S8 de 81 acides aminés qui subirait une maturation post-traductionnelle protéolytique différente de celle initialement décrite dans la littérature. Le clonage et le séquençage du gène codant le précurseur de l’halocine S8 démontrent que la séquence est identique chez les deux souches ETD5 et S8a. La bande de 14 kDa correspond à une nouvelle halocine, l’halocine S14. L’halocine S14 correspond à une forme tronquée en partie N-terminale de la Mn-superoxyde dismutase (SOD) d’Hbt. salinarum. Il pourrait s’agir d’évolution divergente d’un gène codant deux protéines distinctes, ou d’une maturation différente de la SOD. Ce travail permettra de mieux connaître les molécules intervenant dans les interactions microbiennes dans les milieux hypersalins, des milieux extrêmes susceptibles de révéler des structures et des modes d’action originaux. / The solar saltern of Sfax is a hypersaline located in the central area of the eastern coast of Tunisia. In this study, we isolated, identified, and characterized halophilic strains producing antimicrobial peptides (halocins), aiming to understand their role in microbial interactions in hypersaline environments. Two ponds (TS18, 390 g.L-1 NaCl and M1, 200 g.L-1 NaCl) were selected for sampling. Thirty-five halophilic strains have been isolated and characterized, among which 11 displayed antimicrobial activity. Three of them produced antimicrobial substances of proteinaceous nature. Using PCR and RT-PCR, we have demonstrated that Halobacterium salinarum ETD5 and ETD8 express the gene encoding halocin S8, a 3.6 kDa peptide previously isolated from a strain S8 unidentified. The peptide was purified from cultures of strain Hbt. salinarum ETD5. Following bioguided purification, the active fractions revealed two protein bands of 8 and 14 kDa exhibiting antimicrobial activity. N-terminal sequencing and mass spectrometry analyses allowed identification of these two halocins. The 8 kDa band corresponds to halocin S8, undergoing a different proteolytic post-translational processing from that originallydescribed. Cloning and sequencing of the gene encoding the precursor of halocin S8 showed that the sequence is identical for both strains ETD5 and S8a. The 14 kDa band is a new halocin termed halocin S14. Halocin S14 corresponds to an N-terminally truncated portion of the archaeal Mnsuperoxide dismutase (SOD). This could result from divergent evolution of a gene encoding two distinct proteins, or a different post-translational processing of SOD. Our study helps to better understand which molecules are involved in microbial interactions within hypersaline environments and and how they contribute to the competitions in such extreme environments, which are susceptible to give rise to original structures and modes of action.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / This study investigates ecological process and dynamic of the hypersaline tidal flats (HTF, locally knowns as âapicumâ). The HTF systems are characterized by flat landscape and hypersaline soil conditions that may occour in arid and semiarid regions of the world. Usually HTF can be found inside mangrove forest or as a transitional system between mangrove and dry land ecosystem. These areas are highly important for advances of mangrove forest in case of sea level increase. Besides, HTF have great economic value, as prioritarian areas to install aquaculture or saline ponds. Also it represents security zones for traditional populations, as artisanal fishermen. However, the ecological process which drives the HTFs systems remain poorly knew. The studies presented in this doctoral dissertation aim to fulfill some lacks of knowledge about ecological process that could drive the HTF dynamic. The chapter 1 is a bibliographical review about this system. On chapter 2 were evaluated possibles mechanisms used by plants in order to improve their survival in such stressed environment through the study of rhizosphere soils and comparison with bulk soils. On chapter 3, through an experimental study in the field, the possible effect of two crab species bioturbation on HTFs soils and it possible effects on soil properties were evaluated. The chapter 4 registered the evaluation of ecological interactions among biotic and abiotic fractions of the system using ecological modeling tools. In this sense, a structural ecological model was designed using microrelief elevation, soil properties, two plant species biomass and the covering areas of two crab species. Based on results obtained in these studies, it was possible to conclude that HTFs have a particular ecological dynamic and have close relation with mangrove forests due the nutrients, material and animal population flux shared by them. The plant community of HTFs areas is componed mainly by pioneersâ clonal halophytes species. These species seemed to have different strategies to deal with saline stress, an important limitant factor for their growing and distribution. The seasonal effects were noteworthy mainly in bulk soils. In this sense, it is possible that plant estabilize the environment in their benefit. The crabs were able to affect biogeochemical dynamic in HTF soils. However, the burrouing effects of crab species on soil properties were different and seemed reflect the species habit. Although bioturbation effects were important, the modifications on HTF soil properties were less evident than in mangrove forests. Environmental process related to salinity and nutrients availability are among the main factors to drive crabs distribution and plant growth in HTFs. These processes were dependent of microrelief variation. A combination of abiotic factors (microrelief elevation and soil properties) and positive relations among plant species seems to guide the ecological dynamics in HTFs systems. Conservation strategies of coastal environments areas must include the HTF protection due their importance for coastal environments on climate change scenario and economic importance, but mainly because their unique ecological characteristics. / Esse estudo teve como objetivo geral investigar processos ecolÃgicos e a dinÃmica do apicum. Esses sistemas se caracterizam por serem planÃcies hipersalinas que ocorrem em zonas costeiras de regiÃes Ãridas onde podem ser identificadas dentro de bosques de mangue ou entre o manguezal e ecossistemas de terra seca, como mata seca ou restinga. Os apicuns sÃo Ãreas estratÃgicas para recuo das florestas de mangue em caso de aumento do nÃvel do mar. AlÃm disso, possuem grande importÃncia econÃmica, visto que sÃo Ãreas preferenciais para instalaÃÃo de empreendimentos de aquicultura e salinas e por que representam zonas de seguranÃa para populaÃÃes tradicionais. Entretanto, muito pouco se sabe sobre os processos ecolÃgicos que regulam a dinÃmica do sistema. Os estudos compilados nessa tese visam de algum modo suprir lacunas sobre os processos ecolÃgicos que regem a dinÃmica desse sistema. O capÃtulo 1 traz uma ampla revisÃo bibliogrÃfica sobre esse sistema. No capÃtulo 2 foram avaliados possÃveis mecanismos utilizados pelas plantas para sobreviverem ao ambiente limitante atravÃs do estudo dos solos rizosfÃricos e comparaÃÃo com solos nÃo colonizados por plantas. No capÃtulo 3, atravÃs de um estudo experimental de campo foram avaliados os efeitos da bioturbaÃÃo de duas espÃcies de caranguejos nos solos de apicum e seus possÃveis efeitos sobre as condiÃÃes edÃficas. O capÃtulo 4 aprofunda-se na avaliaÃÃo das interaÃÃes ecolÃgicas que ocorrem entre o solo, microrelevo e a biota do sistema (plantas e animais). Nesse sentido, um modelo ecolÃgico estrutural foi construÃdo utilizando variaÃÃes na elevaÃÃo do relevo, propriedades do solo, biomassa das plantas e Ãrea de cobertura de caranguejos. Com base nos resultados, concluiu-se que o sistema apicum possui dinÃmica ecolÃgica muito prÃpria e possui uma relaÃÃo com os bosques de mangue graÃas ao compartilhamento de fluxos de matÃria, nutrientes e populaÃÃes de animais. A comunidade de plantas do apicum à composta, sobretudo por espÃcies clonais halÃfitas pioneiras e que parecem possuir estratÃgias diferenciadas para lidar com o estresse salino, fator limitante do seu crescimento e distribuiÃÃo. O efeito da sazonalidade à notÃvel, sobretudo nas Ãreas nÃo colonizadas por plantas, indicando que possivelmente as plantas estabilizam o sistema solo em seu beneficio. Diferentes espÃcies de caranguejos sÃo capazes de afetar a dinÃmica biogeoquÃmica do solo. A bioturbaÃÃo do solo pelas espÃcies estudadas provocou diferentes efeitos nas propriedades do solo, os quais parecem estar intimamente ligados as hÃbitos das espÃcies. Entretanto, as seus efeitos nos apicuns, embora importantes, sÃo menos intensos que em Ãreas de manguezais. Processos ambientais ligados à salinidade do solo e à disponibilidade de nutrientes estÃo entre os principais fatores que regem a distribuiÃÃo de caranguejos e crescimento das plantas nos apicuns. Esses processos sÃo intimamente dependentes das variaÃÃes do microrelevo. Uma combinaÃÃo entre fatores abiÃticos (elevaÃÃo do relevo e propiedades do solo) e relaÃÃes positivas entre espÃcies de plantas parecem se os princiapis agentes na dinÃmica ecolÃgica dos apicuns. EstratÃgias de conservaÃÃo de sistemas costeiros devem incluir a proteÃÃo dos apicuns visto seu importante papel ecolÃgico no cenÃrio de mudanÃas climÃticas e importÃncia para economia local, sobretudo por conta suas caracterÃsticas ecolÃgicas Ãnicas.

Les archées halophiles sont des procaryotes vivant dans des conditions d’extrême salinité. Ces micro-organismes colonisent les environnements hypersalins et synthétisent des peptides antimicrobiens appelés halocines, qui pourraient leur conférer un avantage sélectif sur leurs compétiteurs. Parmi les peptides antimicrobiens d’archées halophiles décrits, on distingue l’halocine C8, initialement purifiée à partir de la souche halophile Natrinema sp. AS7092. Ce travail de thèse a permis de montrer que la production d’halocine C8 était conservée chez plusieurs archées halophiles appartenant aux genres : Natrinema, Haloterrigena, Haloferax et Halobacterium. Une activité antimicrobienne associée des particules supérieures à 100 kDa non infectieuses suggère que l’halocine C8 pourrait être localisée dans des vésicules membranaires. Ce travail présente des résultats qui déboucheront sur une meilleure compréhension des compétitions microbiennes dans les environnements hypersalins et du rôle écologique des halocines. / Halophilic archaea are prokaryotes living in extremely high salinity conditions. Those microorganisms thrive in hypersaline environments and produce antimicrobial peptides named halocins, which may confer them a selective advantage over competitors. Among the known antimicrobial peptides produced by halophilic archaea, halocin C8 had been initially purified from the halophilic strain Natrinema sp. AS7092. This work demonstrates that halocin C8 production is conserved among several halophilic archaea belonging to genera Natrinema, Haloterrigena, Haloferax and Halobacterium. An antimicrobial activity has been associated with non-infectious particles larger than 100 kDa, suggesting that halocin C8 could be localized in membrane vesicles. Results obtained from this work will lead to a better understanding of microbial competitions arising in hypersaline environments and the ecological role of halocins

The current research project investigated the anoxygenic phototrophic and metal(loid) transforming bacteria of hypersaline and deep ocean hydrothermal environments. The East German Creek brine springs, an unusual flowing hypersaline system, was enumerated using classical techniques. Subterranean sulfide supported purple sulfur and nonsulfur bacteria, but at the highly oxygenated surface, aerobic anoxygenic phototrophs (AAP) were numerically dominant (up to 16-36% of cultivable bacteria). Strains (EG8, EG13, EG17, EG19) with unusual phylogenetic affiliation and novel photosynthetic and metal(loid) reducing traits were described taxonomically. Chromocurvus halotolerans gen. nov., sp. nov. was proposed as a second example of a gammaproteobacterial AAP. It exhibited bent rod-shaped cells, unusual among AAP. Facultatively anaerobic Charonomicrobium ambiphototrophicum gen. nov., sp. nov. was capable of both aerobic and anaerobic anoxygenic photosynthesis, and incapable of photoautotrophy, distinguishing it from both AAP and purple nonsulfur bacteria. Roseovarius vanadiphilum sp. nov. surprisingly produced 4.5 times more biomass and 2 times more bacteriochlorophyll (BChl) at extremely high NaVO3 concentration (7.5 g/l) than in metal-free medium. A second novel metabolic mode, anaerobic respiration on the toxic metalloid tellurate, was described for a relative of non-phototrophic Shewanella frigidimarina (ER-Te-48), from deep ocean hydrothermal vent Paralvinella worms at Explorer Ridge in the Pacific Ocean. Other strains respired on SeO32- (ER-Se-17L), VO3- (ER-V-6), and VO43- (AV-V-25). These organisms provided the first examples of anaerobic respiration on Te, Se and V at hydrothermal vents. High level resistance of AAP to metal(loid)s prompted investigation of the influence of TeO32- on photosynthetic pigment production in species including Erythromicrobium ramosum (from a terrestrial hydrothermal system) and Erythrobacter litoralis (from a hypersaline supralittoral system). Tellurite enhanced photosynthetic pigment production up to 3.4 times, consistent with an antioxidant carotenoid-based defense mechanism. However, in E. litoralis BChl precursors such as Mg protoporphyrin or its monomethyl ester also accumulated, indicating biosynthetic pathway interruption. In hydrothermal and hypersaline ecosystems, largely devoid of eukaryotic phototrophs but often enriched in metal(loid)s, AAP and metal(loid) reducers are key modulators of nutrient and toxin availability. The presented results on their ecology, physiology and biochemistry have important implications for theoretical understanding of extreme environments and hold potential for biotechnological applications.

The current research project investigated the anoxygenic phototrophic and metal(loid) transforming bacteria of hypersaline and deep ocean hydrothermal environments. The East German Creek brine springs, an unusual flowing hypersaline system, was enumerated using classical techniques. Subterranean sulfide supported purple sulfur and nonsulfur bacteria, but at the highly oxygenated surface, aerobic anoxygenic phototrophs (AAP) were numerically dominant (up to 16-36% of cultivable bacteria). Strains (EG8, EG13, EG17, EG19) with unusual phylogenetic affiliation and novel photosynthetic and metal(loid) reducing traits were described taxonomically. Chromocurvus halotolerans gen. nov., sp. nov. was proposed as a second example of a gammaproteobacterial AAP. It exhibited bent rod-shaped cells, unusual among AAP. Facultatively anaerobic Charonomicrobium ambiphototrophicum gen. nov., sp. nov. was capable of both aerobic and anaerobic anoxygenic photosynthesis, and incapable of photoautotrophy, distinguishing it from both AAP and purple nonsulfur bacteria. Roseovarius vanadiphilum sp. nov. surprisingly produced 4.5 times more biomass and 2 times more bacteriochlorophyll (BChl) at extremely high NaVO3 concentration (7.5 g/l) than in metal-free medium. A second novel metabolic mode, anaerobic respiration on the toxic metalloid tellurate, was described for a relative of non-phototrophic Shewanella frigidimarina (ER-Te-48), from deep ocean hydrothermal vent Paralvinella worms at Explorer Ridge in the Pacific Ocean. Other strains respired on SeO32- (ER-Se-17L), VO3- (ER-V-6), and VO43- (AV-V-25). These organisms provided the first examples of anaerobic respiration on Te, Se and V at hydrothermal vents. High level resistance of AAP to metal(loid)s prompted investigation of the influence of TeO32- on photosynthetic pigment production in species including Erythromicrobium ramosum (from a terrestrial hydrothermal system) and Erythrobacter litoralis (from a hypersaline supralittoral system). Tellurite enhanced photosynthetic pigment production up to 3.4 times, consistent with an antioxidant carotenoid-based defense mechanism. However, in E. litoralis BChl precursors such as Mg protoporphyrin or its monomethyl ester also accumulated, indicating biosynthetic pathway interruption. In hydrothermal and hypersaline ecosystems, largely devoid of eukaryotic phototrophs but often enriched in metal(loid)s, AAP and metal(loid) reducers are key modulators of nutrient and toxin availability. The presented results on their ecology, physiology and biochemistry have important implications for theoretical understanding of extreme environments and hold potential for biotechnological applications.