Molecular and morphological characterization of four new ancyromonad genera and proposal for an updated taxonomy of the Ancyromonadida.

Ancyromonads are small biflagellated protists with a bean-shaped morphology. They are cosmopolitan in marine, freshwater, and soil environments, where they attach to surfaces while feeding on bacteria. These poorly known grazers stand out by their uncertain phylogenetic position in the tree of eukaryotes, forming a deep-branching "orphan" lineage that is considered key to a better understanding of the early evolution of eukaryotes. Despite their ecological and evolutionary interest, only limited knowledge exists about their true diversity. Here, we aimed to characterize ancyromonads better by integrating environmental surveys with behavioral observation and description of cell morphology, for which sample isolation and culturing are indispensable. We studied 18 ancyromonad strains, including 14 new isolates and seven new species. We described three new and genetically divergent genera: Caraotamonas, Nyramonas, and Olneymonas, together encompassing four species. The remaining three new species belong to the already-known genera Fabomonas and Ancyromonas. We also raised Striomonas, formerly a subgenus of Nutomonas, to full genus status, on morphological and phylogenetic grounds. We studied the morphology of diverse ancyromonads under light and electron microscopy and carried out molecular phylogenetic analyses, also including 18S rRNA gene sequences from several environmental surveys. Based on these analyses, we have updated the taxonomy of Ancyromonadida.

In-depth analyses of deep subsurface sediments using 454-pyrosequencing reveals a reservoir of buried fungal communities at record-breaking depths.

The deep subseafloor, extending from a few centimeters below the sediment surface to several hundred meters into sedimentary deposits, constitutes the deep biosphere and harbors an unexpected microbial diversity. Several studies have described the occurrence, turnover, activity and function of subseafloor prokaryotes; however, subsurface eukaryotic communities still remain largely underexplored. Ribosomal RNA surveys of superficial and near-surface marine sediments have revealed an unexpected diversity of active eukaryotic communities, but knowledge of the diversity of deep subseafloor microeukaryotes is still scarce. Here, we investigated the vertical distribution of DNA and RNA fungal signatures within subseafloor sediments of the Canterbury basin (New Zealand) by 454 pyrotag sequencing of fungal genetic markers. Different shifts between the fungal classes of Tremellomycetes, Sordariomycetes, Eurotiomycetes, Saccharomycetes, Wallemiomycetes, Dothideomycetes, Exobasidiomycetes and Microbotryomycetes were observed. These data provide direct evidence that fungal communities occur at record depths in deep sediments of the Canterbury basin and extend the depth limit of fungal presence and activity, respectively 1740 and 346 mbsf. As most of the fungal sequences retrieved have a cosmopolitan distribution, it indicates that fungi are able to adapt to the deep subseafloor conditions at record-depth and must play important ecological roles in biogeochemical cycles.

Phylogenetic and functional diversity of microbial communities associated with subsurface sediments of the Sonora Margin, Guaymas Basin.

Subsurface sediments of the Sonora Margin (Guaymas Basin), located in proximity of active cold seep sites were explored. The taxonomic and functional diversity of bacterial and archaeal communities were investigated from 1 to 10 meters below the seafloor. Microbial community structure and abundance and distribution of dominant populations were assessed using complementary molecular approaches (Ribosomal Intergenic Spacer Analysis, 16S rRNA libraries and quantitative PCR with an extensive primers set) and correlated to comprehensive geochemical data. Moreover the metabolic potentials and functional traits of the microbial community were also identified using the GeoChip functional gene microarray and metabolic rates. The active microbial community structure in the Sonora Margin sediments was related to deep subsurface ecosystems (Marine Benthic Groups B and D, Miscellaneous Crenarchaeotal Group, Chloroflexi and Candidate divisions) and remained relatively similar throughout the sediment section, despite defined biogeochemical gradients. However, relative abundances of bacterial and archaeal dominant lineages were significantly correlated with organic carbon quantity and origin. Consistently, metabolic pathways for the degradation and assimilation of this organic carbon as well as genetic potentials for the transformation of detrital organic matters, hydrocarbons and recalcitrant substrates were detected, suggesting that chemoorganotrophic microorganisms may dominate the microbial community of the Sonora Margin subsurface sediments.

Physiological features of Halomonas lionensis sp. nov., a novel bacterium isolated from a Mediterranean Sea sediment.

A novel halophilic bacterium, strain RHS90(T), was isolated from marine sediments from the Gulf of Lions, in the Mediterranean Sea. Its metabolic and physiological characteristics were examined under various cultural conditions, including exposure to stressful ones (oligotrophy, high pressure and high concentrations of metals). Based on phylogenetic analysis of the 16S rRNA gene, the strain was found to belong to the genus Halomonas in the class Gammaproteobacteria. Its closest relatives are Halomonas axialensis and Halomonas meridiana (98% similarity). DNA-DNA hybridizations indicated that the novel isolate is genotypically distinct from these species. The DNA G + C content of the strain is 54.4 mol%. The main fatty acids (C18:1ω7c, 2-OH iso-C15:0, C16:0 and/or C19:0 cyclo ω8c), main polar lipids (diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and an unidentified phosphoglycolipid) and major respiratory quinone (ubiquinone Q9) were determined. The novel isolate is heterotrophic, mesophilic, euryhaline (growth optimum ranging from 2 to 8% w/v NaCl) and is able to grow under stressful conditions. The strain accumulates poly-ß-hydroxyalkanoates granules and compatible solutes. Based on genotypic, chemotaxonomic and phenotypic distinctiveness, this isolate is likely to represent a novel species, for which the name Halomonas lionensis is proposed. The type strain of H. lionensis is RHS90(T) (DSM 25632(T) = CIP 110370(T) = UBOCC 3186(T)).

Microorganisms persist at record depths in the subseafloor of the Canterbury Basin.

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.

Phaeobacter leonis sp. nov., an alphaproteobacterium from Mediterranean Sea sediments.

A novel Gram-stain-negative, strictly aerobic, heterotrophic bacterium, designated 306(T), was isolated from near-surface (109 cm below the sea floor) sediments of the Gulf of Lions, in the Mediterranean Sea. Strain 306(T) grew at temperatures between 4 and 32 °C (optimum 17-22 °C), from pH 6.5 to 9.0 (optimum 8.0-9.0) and between 0.5 and 6.0% (w/v) NaCl (optimum 2.0%). Its DNA G+C content was 58.8 mol%. On the basis of 16S rRNA gene sequence similarity, the novel isolate belongs to the class Alphaproteobacteria and is related to the genus Phaeobacter. It shares 98.7% 16S rRNA sequence identity with Phaeobacter arcticus, its closest phylogenetic relative. It contained Q-10 as the only respiratory quinone, C(18:1)ω7c and C(16:0) as major fatty acids (>5%) and phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, diphosphatidylglycerol, two unidentified lipids and an aminolipid as polar lipids. The chemotaxonomic data are consistent with the affiliation of strain 306(T) to the genus Phaeobacter. Results of physiological experiments, biochemical tests and DNA-DNA hybridizations (with P. arcticus) indicate that strain 306(T) is genetically and phenotypically distinct from the five species of the genus Phaeobacter with validly published names. Strain 306(T) therefore represents a novel species, for which the name Phaeobacter leonis sp. nov. is proposed. The type strain is 306(T) ( =DSM 25627(T) =CIP 110369(T) =UBOCC 3187(T)).

DNA extractions from deep subseafloor sediments: novel cryogenic-mill-based procedure and comparison to existing protocols.

Extracting DNA from deep subsurface sediments is challenging given the complexity of sediments types, low biomasses, resting structures (spores, cysts) frequently encountered in deep sediments, and the potential presence of enzymatic inhibitors. Promising results for cell lysis efficiency were recently obtained by use of a cryogenic mill (Lipp et al., 2008). These findings encouraged us to devise a DNA extraction protocol using this tool. Thirteen procedures involving a combination of grinding in liquid nitrogen (for various durations and beating rates) with different chemical solutions (phenol, chloroform, SDS, sarkosyl, proteinase, GTC), or with use of DNA recovery kits (MagExtractor®) were compared. Effective DNA extraction was evaluated in terms of cell lysis efficiency, DNA extraction efficiency, DNA yield and determination of prokaryotic diversity. Results were compared to those obtained by standard protocols: the FastDNA®SPIN kit for soil and the Zhou protocol. For most sediment types grinding in a cryogenic mill at a low beating rate in combination with direct phenol-chloroform extraction resulted in much higher DNA yields than those obtained using classical procedures. In general (except for clay-rich sediments), this procedure provided high-quality crude extracts for direct downstream nested-PCR, from cell numbers as low as 1.1×10(6) cells/cm(3). This procedure is simple, rapid, low-cost, and could be used with minor modifications for large-scale DNA extractions for a variety of experimental goals.