Biologie et Ecologie des Ecosystèmes Marins Profonds

A mesophilic, anaerobic, fermentative bacterium, strain BN3(T), was isolated from a producing well of a biodegraded oil reservoir in Canada. Cells were Gram-negative, non-motile rods that did not form spores. The temperature range for growth was 15-40 degrees C, with optimum growth at 37-40 degrees C. The strain grew with up 4 % NaCl, with optimum growth in the absence of NaCl. Tryptone was required for growth. Yeast extract and elemental sulfur stimulated growth. Growth was also enhanced during fermentation of glucose, arabinose, galactose, maltose, mannose, rhamnose, lactose, ribose, fructose, sucrose, cellobiose, lactate, mannitol and glycerol. Acetate, hydrogen and CO(2) were produced during glucose fermentation. Elemental sulfur and nitrate were used as electron acceptors and were reduced to sulfide and ammonium, respectively. The G + C content of the genomic DNA was 40.8 mol%. Phylogenetic analyses of the 16S rRNA gene sequence indicated that the strain was a member of the phylum 'Bacteroidetes', distantly related to the genera Bacteroides and Tannerella (similarity values of less than 90 %). The chemotaxonomic data (fatty acids, polar lipids and quinones composition) also indicated that strain BN3(T) could be clearly distinguished from its closest cultivated relatives. This novel organism possesses phenotypic, chemotaxonomic and phylogenetic traits that do not allow its classification as a member of any previously described genus; therefore, it is proposed that this isolate should be described as a member of a novel species of a new genus, Petrimonas gen. nov., of which Petrimonas sulfuriphila sp. nov. is the type species. The type strain is BN3(T) (= DSM 16547(T) = JCM 12565(T)).

Microbial communities in two production waters of a low-temperature and low-salinity petroleum reservoir in Canada were examined using cultural and molecular approaches. The predominant cultivated microorganisms were homoacetogens but sulfate-reducers, acetoclastic methanogens and denitrifiers also gave significant counts. The dominant members of the culturable population were affiliated with the Firmicutes, the "Deltaproteobacteria", the "Epsilonproteobacteria", the Spirochaetes and the Euryarchaeota. 16S rRNA gene clone libraries were also constructed from the total DNA collected from production waters. The bacterial library was entirely composed by a single phylotype related to Arcobacter. The archaeal phylotypes were generally very closely related to members of the orders Methanosarcinales and Methanomicrobiales. Consistent with earlier observations, our data suggest that methanogenesis is a dominant terminal process in the reservoir. Moreover, the cross-evaluation of culture-dependent and -independent techniques also indicates that, contrary to most studies, both acetoclastic and lithotrophic methanogens may be involved in this process. This first investigation of the microbial diversity in a non water-flooded low-temperature and low-salinity petroleum reservoir expands substantially our knowledge of the extent of microbial diversity and highlights the complexity of microbial communities involved in the oil field food chain.

Sub-sea-floor sediments may contain two-thirds of Earth's total prokaryotic biomass. However, this has its basis in data extrapolation from ~500-meter to 4-kilometer depths, whereas the deepest documented prokaryotes are from only 842 meters. Here, we provide evidence for low concentrations of living prokaryotic cells in the deepest (1626 meters below the sea floor), oldest (111 million years old), and potentially hottest (~100 degrees C) marine sediments investigated. These Newfoundland margin sediments also have DNA sequences related to thermophilic and/or hyperthermophilic Archaea. These form two unique clusters within Pyrococcus and Thermococcus genera, suggesting unknown, uncultured groups are present in deep, hot, marine sediments (~54 degrees to 100 degrees C). Sequences of anaerobic methane-oxidizing Archaea were also present, suggesting a deep biosphere partly supported by methane. These findings demonstrate that the sub-sea-floor biosphere extends to at least 1600 meters below the sea floor and probably deeper, given an upper temperature limit for prokaryotic life of at least 113 degrees C and increasing thermogenic energy supply with depth.

The potential for using marine microbes for biodiscovery is severely limited by the lack of laboratory cultures. It is a long-standing observation that standard microbiological techniques only isolate a very small proportion of the wide diversity of microbes that are known in natural environments from DNA sequences. A number of explanations are reviewed. The process of establishing laboratory cultures may destroy any cell-to-cell communication that occurs between organisms in the natural environment and that are vital for growth. Bacteria probably grow as consortia in the sea and reliance on other bacteria for essential nutrients and substrates is not possible with standard microbiological approaches. Such interactions should be considered when designing programmes for the isolation of marine microbes. The benefits of novel technologies for manipulating cells are reviewed, including single cell encapsulation in gel micro-droplets. Although novel technologies offer benefits for bringing previously uncultured microbes into laboratory culture, many useful bacteria can still be isolated using variations of plating techniques. Results are summarized for a study to culture bacteria from a long-term observatory station in the English Channel. Bacterial biodiversity in this assemblage has recently been characterized using high-throughput sequencing techniques. Although Alphaproteobacteria dominated the natural bacterial assemblage throughout the year, Gammaproteobacteria were the most frequent group isolated by plating techniques. The use of different gelling agents and the addition of ammonium to seawater-based agar did lead to the isolation of a higher proportion of Alphaproteobacteria. Variation in medium composition was also able to increase the recovery of other groups of particular interest for biodiscovery, such as Actinobacteria.

Identifying the function of uncultured microbes in their environments today remains one of the main challenges for microbial ecologists. In this article, we describe a new method allowing simultaneous analysis of microbial identity and function. This method is based on the visualization of oligonucleotide probe-conferred hybridization signal in single microbial cells and isotopic measurement using high-resolution ion microprobe (NanoSIMS). In order to characterize the potential of the method, an oligonucleotide containing iodized cytidine was hybridized on fixed cells of Escherichia coli cultured on media containing different levels of 13C or 15N. Iodine signals could clearly be localized on targeted cells and the isotopic enrichment could be monitored at the single-cell level. The applicability of this new technique to the study of in situ ecophysiology of uncultured microorganisms within complex microbial communities is illustrated.

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Recent studies indicate that ammonia is an important electron donor for the oxidation of fixed nitrogen, both in the marine water column and sediments. This process, known as anammox, has so far only been observed in a large range of temperature habitats. The present study investigated the role of anammox in hydrothermal settings. During three oceanographic expeditions to the Mid-Atlantic Ridge, hydrothermal samples were collected from five vent sites, at depths ranging from 750 to 3650 m from cold to hot habitats. Evidence for the occurrence of anammox in these particular habitats was demonstrated by concurrent surveys, including the amplification of 16S rRNA gene sequences related to known anammox bacteria, ladderanes lipids analysis and measurement of a (14)N(15)N dinitrogen production in isotope-pairing experiments at 60 and 85 degrees C. Together these results indicate that new deep-branching anammox bacteria may be active in these hot habitats.

Investigations of the diversity of culturable yeasts at deep-sea hydrothermal sites have suggested possible interactions with endemic fauna. Samples were collected during various oceanographic cruises at the Mid-Atlantic Ridge, South Pacific Basins and East Pacific Rise. Cultures of 32 isolates, mostly associated with animals, were collected. Phylogenetic analyses of 26S rRNA gene sequences revealed that the yeasts belonged to Ascomycota and Basidiomycota phyla, with the identification of several genera: Rhodotorula, Rhodosporidium, Candida, Debaryomyces and Cryptococcus. Those genera are usually isolated from deep-sea environments. To our knowledge, this is the first report of yeasts associated with deep-sea hydrothermal animals.

Plants alter chemical and physical properties of soil, and thereby influence rhizosphere microbial community structure. The structure of microbial communities may in turn affect plant performance. Yet, outside of simple systems with pairwise interacting partners, the plant genetic pathways that influence microbial community structure remain largely unknown, as are the performance feedbacks of microbial communities selected by the host plant genotype. We investigated the role of the plant circadian clock in shaping rhizosphere community structure and function. We performed 16S ribosomal RNA gene sequencing to characterize rhizosphere bacterial communities of Arabidopsis thaliana between day and night time points, and tested for differences in community structure between wild-type (Ws) vs clock mutant (toc1-21, ztl-30) genotypes. We then characterized microbial community function, by growing wild-type plants in soils with an overstory history of Ws, toc1-21 or ztl-30 and measuring plant performance. We observed that rhizosphere community structure varied between day and night time points, and clock misfunction significantly altered rhizosphere communities. Finally, wild-type plants germinated earlier and were larger when inoculated with soils having an overstory history of wild-type in comparison with clock mutant genotypes. Our findings suggest the circadian clock of the plant host influences rhizosphere community structure and function.

Protist plankton are major members of open-water marine food webs. Traditionally divided between phototrophic phytoplankton and phagotrophic zooplankton, recent research shows many actually combine phototrophy and phagotrophy in the one cell; these protists are the "mixoplankton." Under the mixoplankton paradigm, "phytoplankton" are incapable of phagotrophy (diatoms being exemplars), while "zooplankton" are incapable of phototrophy. This revision restructures marine food webs, from regional to global levels. Here, we present the first comprehensive database of marine mixoplankton, bringing together extant knowledge of the identity, allometry, physiology, and trophic interactivity of these organisms. This mixoplankton database (MDB) will aid researchers that confront difficulties in characterizing life traits of protist plankton, and it will benefit modelers needing to better appreciate ecology of these organisms with their complex functional and allometric predator-prey interactions. The MDB also identifies knowledge gaps, including the need to better understand, for different mixoplankton functional types, sources of nutrition (use of nitrate, prey types, and nutritional states), and to obtain vital rates (e.g. growth, photosynthesis, ingestion, factors affecting photo' vs. phago' -trophy). It is now possible to revisit and re-classify protistan "phytoplankton" and "zooplankton" in extant databases of plankton life forms so as to clarify their roles in marine ecosystems.

The shrimp Rimicaris exoculata dominates several hydrothermal vent ecosystems of the Mid-Atlantic Ridge and is thought to be a primary consumer harbouring a chemoautotrophic bacterial community in its gill chamber. The aim of the present study was to test current hypotheses concerning the epibiont's chemoautotrophy, and the mutualistic character of this association. In-vivo experiments were carried out in a pressurised aquarium with isotope-labelled inorganic carbon (NaH(13)CO(3) and NaH(14)CO(3)) in the presence of two different electron donors (Na(2)S(2)O(3) and Fe(2+)) and with radiolabelled organic compounds ((14)C-acetate and (3)H-lysine) chosen as potential bacterial substrates and/or metabolic by-products in experiments mimicking transfer of small biomolecules from epibionts to host. The bacterial epibionts were found to assimilate inorganic carbon by chemoautotrophy, but many of them (thick filaments of epsilonproteobacteria) appeared versatile and able to switch between electron donors, including organic compounds (heterotrophic acetate and lysine uptake). At least some of them (thin filamentous gammaproteobacteria) also seem capable of internal energy storage that could supply chemosynthetic metabolism for hours under conditions of electron donor deprivation. As direct nutritional transfer from bacteria to host was detected, the association appears as true mutualism. Import of soluble bacterial products occurs by permeation across the gill chamber integument, rather than via the digestive tract. This first demonstration of such capabilities in a decapod crustacean supports the previously discarded hypothesis of transtegumental absorption of dissolved organic matter or carbon as a common nutritional pathway.

Cold seeps, located along the Sonora Margin transform fault in the Guaymas Basin, were extensively explored during the 'BIG' cruise in June 2010. They present a seafloor mosaic pattern consisting of different faunal assemblages and microbial mats. To investigate this mostly unknown cold and hydrocarbon-rich environment, geochemical and microbiological surveys of the sediments underlying two microbial mats and a surrounding macrofaunal habitat were analyzed in detail. The geochemical measurements suggest biogenic methane production and local advective sulfate-rich fluxes in the sediments. The distributions of archaeal communities, particularly those involved in the methane cycle, were investigated at different depths (surface to 18 cm below the sea floor (cmbsf)) using complementary molecular approaches, such as Automated method of Ribosomal Intergenic Spacer Analysis (ARISA), 16S rRNA libraries, fluorescence in situ hybridization and quantitative polymerase chain reaction with new specific primer sets targeting methanogenic and anaerobic methanotrophic lineages. Molecular results indicate that metabolically active archaeal communities were dominated by known clades of anaerobic methane oxidizers (archaeal anaerobic methanotroph (ANME)-1, -2 and -3), including a novel 'ANME-2c Sonora' lineage. ANME-2c were found to be dominant, metabolically active and physically associated with syntrophic Bacteria in sulfate-rich shallow sediment layers. In contrast, ANME-1 were more prevalent in the deepest sediment samples and presented a versatile behavior in terms of syntrophic association, depending on the sulfate concentration. ANME-3 were concentrated in small aggregates without bacterial partners in a restricted sediment horizon below the first centimetres. These niche specificities and syntrophic behaviors, depending on biological surface assemblages and environmental availability of electron donors, acceptors and carbon substrates, suggest that ANME could support alternative metabolic pathways than syntrophic anaerobic oxidation of methane.

The subsurface realm is colonized by microbial communities to depths of >1000 meters below the seafloor (m.b.sf.), but little is known about overall diversity and microbial distribution patterns at the most profound depths. Here we show that not only Bacteria and Archaea but also Eukarya occur at record depths in the subseafloor of the Canterbury Basin. Shifts in microbial community composition along a core of nearly 2 km reflect vertical taxa zonation influenced by sediment depth. Representatives of some microbial taxa were also cultivated using methods mimicking in situ conditions. These results suggest that diverse microorganisms persist down to 1922 m.b.sf. in the seafloor of the Canterbury Basin and extend the previously known depth limits of microbial evidence (i) from 159 to 1740 m.b.sf. for Eukarya and (ii) from 518 to 1922 m.b.sf. for Bacteria.

The shrimp Rimicaris exoculata from hydrothermal vents on the Mid-Atlantic Ridge (MAR) harbours bacterial epibionts on specialized appendages and the inner surfaces of its gill chamber. Using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization (FISH), we examined the R. exoculata epibiosis from four vents sites along the known distribution range of the shrimp on the MAR. Our results show that R. exoculata lives in symbiosis with two types of filamentous epibionts. One belongs to the Epsilonproteobacteria, and was previously identified as the dominant symbiont of R. exoculata. The second is a novel gammaproteobacterial symbiont that belongs to a clade consisting exclusively of sequences from epibiotic bacteria of hydrothermal vent animals, with the filamentous sulfur oxidizer Leucothrix mucor as the closest free-living relative. Both the epsilon- and the gammaproteobacterial symbionts dominated the R. exoculata epibiosis at all four MAR vent sites despite striking differences between vent fluid chemistry and distances between sites of up to 8500 km, indicating that the symbiosis is highly stable and specific. Phylogenetic analyses of two mitochondrial host genes showed little to no differences between hosts from the four vent sites. In contrast, there was significant spatial structuring of both the gamma- and the epsilonproteobacterial symbiont populations based on their 16S rRNA gene sequences that was correlated with geographic distance along the MAR. We hypothesize that biogeography and host-symbiont selectivity play a role in structuring the epibiosis of R. exoculata.

Rimicaris exoculata dominates the megafauna of several Mid-Atlantic Ridge hydrothermal sites. Its gut is full of sulphides and iron-oxide particles and harbours microbial communities. Although a trophic symbiosis has been suggested, their role remains unclear. In vivo starvation experiments in pressurized vessels were performed on shrimps from Rainbow and Trans-Atlantic Geotraverse sites in order to expel the transient gut contents. Microbial communities associated with the gut of starved and reference shrimps were compared using 16S rRNA gene libraries and microscopic observations (light, transmission and scanning electron microscopy and FISH analyses). We show that the gut microbiota of shrimps from both sites included mainly Deferribacteres, Mollicutes, Epsilon- and Gammaproteobacteria. For the first time, we have observed filamentous bacteria, inserted between microvilli of gut epithelial cells. They remained after starvation periods in empty guts, suggesting the occurrence of a resident microbial community. The bacterial community composition was the same regardless of the site, except for Gammaproteobacteria retrieved only in Rainbow specimens. We observed a shift in the composition of the microbiota of long-starved specimens, from the dominance of Deferribacteres to the dominance of Gammaproteobacteria. These results reinforce the hypothesis of a symbiotic relationship between R. exoculata and its gut epibionts.

In September 2008, an expedition of the RV Urania was devoted to exploration of the genomic richness of deep hypersaline anoxic lakes (DHALs) located in the Western part of the Mediterranean Ridge. Approximately 40 nautical miles SE from Urania Lake, the presence of anoxic hypersaline lake, which we named Thetis, was confirmed by swath bathymetry profiling and through immediate sampling casts. The brine surface of the Thetis Lake is located at a depth of 3258 m with a thickness of ≈ 157 m. Brine composition was found to be thalassohaline, saturated by NaCl with a total salinity of 348‰, which is one of highest value reported for DHALs. Similarly to other Mediterranean DHALs, seawater-brine interface of Thetis represents a steep pycno- and chemocline with gradients of salinity, electron donors and acceptors and posseses a remarkable stratification of prokaryotic communities, observed to be more metabolically active in the upper interface where redox gradient was sharper. [(14) C]-bicarbonate fixation analysis revealed that microbial communities are sustained by sulfur-oxidizing chemolithoautotrophic primary producers that thrive within upper interface. Besides microaerophilic autotrophy, heterotrophic sulfate reduction, methanogenesis and anaerobic methane oxidation are likely the predominant processes driving the ecosystem of Thetis Lake.

Next-generation sequencing (NGS) opens up exciting possibilities for improving our knowledge of environmental microbial diversity, allowing rapid and cost-effective identification of both cultivated and uncultivated microorganisms. However, library preparation, sequencing, and analysis of the results can provide inaccurate representations of the studied community compositions. Therefore, all these steps need to be taken into account carefully. Here we evaluated the effects of DNA extraction methods, targeted 16S rRNA hypervariable regions, and sample origins on the diverse microbes detected by 454 pyrosequencing in marine cold seep and hydrothermal vent sediments. To assign the reads with enough taxonomic precision, we built a database with about 2,500 sequences from Archaea and Bacteria from deep-sea marine sediments, affiliated according to reference publications in the field. Thanks to statistical and diversity analyses as well as inference of operational taxonomic unit (OTU) networks, we show that (i) while DNA extraction methods do not seem to affect the results for some samples, they can lead to dramatic changes for others; and (ii) the choice of amplification and sequencing primers also considerably affects the microbial community detected in the samples. Thereby, very different proportions of pyrosequencing reads were obtained for some microbial lineages, such as the archaeal ANME-1, ANME-2c, and MBG-D and deltaproteobacterial subgroups. This work clearly indicates that the results from sequencing-based analyses, such as pyrosequencing, should be interpreted very carefully. Therefore, the combination of NGS with complementary approaches, such as fluorescence in situ hybridization (FISH)/catalyzed reporter deposition (CARD)-FISH or quantitative PCR (Q-PCR), would be desirable to gain a more comprehensive picture of environmental microbial communities.

An obligate piezophilic anaerobic hyperthermophilic archaeon, designated strain CH1(T), was isolated from a hydrothermal vent site named 'Ashadze', which is located on the Mid-Atlantic Ridge at a depth of 4100 m. Enrichment and isolation of the strain were carried out at 95 °C under a hydrostatic pressure of 42 MPa. Cells of strain CH1(T) were highly motile irregular cocci with a diameter of ~1-1.5 µm. Growth was recorded at 80-108 °C (optimum 98 °C) and at pressures of 20-120 MPa (optimum 52 MPa). No growth was observed under atmospheric pressures at 60-110 °C. Growth was observed at pH 6.0-9.5 (optimum 7.5-8.0) and in 2.5-5.5% (w/v) NaCl (optimum 3.5%). Strain CH1(T) was strictly anaerobic and grew on complex proteinaceous substrates, such as yeast extract, Peptone, and casein, as well as on sucrose, starch, chitin, pyruvate, acetate and glycerol without electron acceptors. The G+C content of the genomic DNA was 49.0±0.5 mol%. Analysis of 16S rRNA gene sequences revealed that strain CH1(T) belongs to the genus Pyrococcus. Based on its physiological properties and similarity levels between ribosomal proteins, strain CH1(T) represents a novel species, for which the name Pyrococcus yayanosii sp. nov. is proposed. The type strain is CH1(T) (=JCM 16557). This strain is also available by request from the Souchothèque de Bretagne (catalogue LMBE) culture collection (collection no. 3310).

Two novel, thermophilic piezophiles, capable of chemolithoautotrophic growth, are successfully cultivated and isolated from a black smoker chimney at the TAG field (Mid Atlantic Ridge: MAR) by using a piezophilic cultivation technique. Both strains (strains 106 and 108) represent dominant cultivated populations of the microbial communities in the chimney surface habitat. Strain 106 represents typically thin, long spiral cells under the piezophilic growth condition but short bent cells under the non-piezophilic condition. It is a strictly chemolithoautotrophic gammaproteobacterium using reduced sulfur compounds as the electron donors, and nitrate and O(2) as the electron acceptors. Based on the 16S rRNA gene sequence, strain 106 would represent a novel genus of the previously uncultivated group (Symbiont Group I; a potentially novel family) within the Gammaproteobacteria, and 'Thioprofundum lithotrophica' gen. nov., sp. nov. is proposed. Strain 108 is a short, oval rod at any of the growth pressures. It is a facultative chemoautotroph, capable of both chemolithoautotrophic growth with H(2) and S oxidations and organotrophic growth with complex organics or organic acids using nitrate and O(2) as the electron acceptors. The chemolithoautotrophic growth is strictly piezophilic and under the organotrophic growth condition, it grows at conventional pressures (0.1 MPa). Strain 108 is phylogenetically distinctive from any of the previously described genera of the family Rhodobacteraceae within the Alphaproteobacteria, and 'Piezobacter thermophilus' gen. nov., sp. nov. is proposed. The piezophilic cultivation technique can be a powerful tool to isolate and characterize the previously uncultivated phylotypes in the deep-sea hydrothermal vent environments.

The caridean shrimp Rimicaris exoculata dominates the fauna at several Mid-Atlantic Ridge hydrothermal vent sites. This shrimp has an enlarged gill chamber, harboring a dense ectosymbiotic community of chemoautotrophic bacteria associated with mineral oxide deposits. Until now, their acquisition is not fully understood. At three hydrothermal vent sites, we analyzed the epibionts diversity at different moult stages and also in the first stages of the shrimp life (eggs, hatched eggs (with larvae) and juveniles). Hatched eggs associated with young larvae were collected for the first time directly from gravid females at the Logachev vent site during the Serpentine cruise. An approach using 16S rRNA clone libraries, scanning and transmission electron microscopy, and fluorescent in situ hybridization was used. Molecular results and microscope observations indicated a switch in the composition of the bacterial community between early R. exoculata life cycle stage (egg libraries dominated by the Gammaproteobacteria) and later stages (juvenile/adult libraries dominated by the Epsilonproteobacteria). We hypothesized that the epibiotic phylotype composition could vary according to the life stage of the shrimp. Our results confirmed the occurrence of a symbiosis with Gammaproteobacteria and Epsilonproteobacteria, but more complex than previously assumed. We revealed the presence of active type-I methanotrophic bacteria colonizing the cephalothorax of shrimps from the Rainbow site. They were also present on the eggs from the Logachev site. This could be the first 'epibiotic' association between methanotrophic bacteria and hydrothermal vent crustacean. We discuss possible transmission pathways for epibionts linked to the shrimp life cycle.