Development of an ecotoxicological protocol for the deep-sea fauna using the hydrothermal vent shrimp Rimicaris exoculata.

In light of deep-sea mining industry development, particularly interested in massive-sulphide deposits enriched in metals with high commercial value, efforts are increasing to better understand potential environmental impacts to local fauna. The aim of this study was to assess the natural background levels of biomarkers in the hydrothermal vent shrimp Rimicaris exoculata and their responses to copper exposure at in situ pressure (30MPa) as well as the effects of depressurization and pressurization of the high-pressure aquarium IPOCAMP. R. exoculata were collected from the chimney walls of the hydrothermal vent site TAG (Mid Atlantic Ridge) at 3630m depth during the BICOSE cruise in 2014. Tissue metal accumulation was quantified in different tissues (gills, hepatopancreas and muscle) and a battery of biomarkers was measured: metal exposure (metallothioneins), oxidative stress (catalase, superoxide dismutase, glutathione-S-transferase and glutathione peroxidase) and oxidative damage (lipid peroxidation). Data show a higher concentration of Cu in the hepatopancreas and a slight increase in the gills after incubations (for both exposed groups). Significant induction of metallothioneins was observed in the gills of shrimps exposed to 4µM of Cu compared to the control group. Moreover, activities of enzymes were detected for the in situ group, showing a background protection against metal toxicity. Results suggest that the proposed method, including a physiologically critical step of pressurizing and depressurizing the test chamber to enable the seawater exchange during exposure to contaminants, is not affecting metal accumulation and biomarkers response and may prove a useful method to assess toxicity of contaminants in deep-sea species.

A novel polymer produced by a bacterium isolated from a deep-sea hydrothermal vent polychaete annelid.

AIMS: The objective of the present work was to describe an aerobic, mesophilic and heterotrophic marine bacterium, designated HYD657, able to produce an exopolysaccharide (EPS). It was isolated from a East Pacific Rise deep-sea hydrothermal vent polychaete annelid. METHODS AND RESULTS: This micro-organism, on the basis of the phenotypical features and genotypic investigations, can be clearly assigned to the Alteromonas macleodii species and the name A. macleodii subsp. fijiensis biovar deepsane is proposed. Optimal growth occurs between 30 and 35 degrees C, at pH between 6.5 and 7.5 and at ionic strengths between 20 and 40 g x l(-1) NaCl. The G + C content of DNA was 46.5%. This bacterium excreted, under laboratory conditions, an EPS consisting of glucose, galactose, rhamnose, fucose and mannose as neutral sugars along with glucuronic and galacturonic acids and a diacidic hexose identified as a 3-0-(1 carboxyethyl)-D-glucuronic acid. Its average molecular mass was 1.6 x 10(6) Da. CONCLUSIONS: The bacterium HYD657, for which the name A. macleodii subsp. fijiensis biovar deepsane is proposed, produces an unusual EPS in specific medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to its interesting biological activities, applications have been found in cosmetics. Its probable contribution to the filamentous microbial mat in the Alvinella pompejana microenvironment can be also mentioned.

Caloranaerobacter azorensis gen. nov., sp. nov., an anaerobic thermophilic bacterium isolated from a deep-sea hydrothermal vent.

A thermophilic, anaerobic, chemo-organotrophic bacterium, designated MV1087T, was isolated from a deep-sea hydrothermal chimney sample collected from the Mid-Atlantic Ridge. The cells were straight, motile and stained gram-negative. Growth was observed from 45 to 65 degrees C, with an optimum around 65 degrees C. No growth was observed at 40 or 70 degrees C. Growth was observed from pH 5.5 to 9.0 and the optimum pH was around 7. The salinity range for growth was 10-100 g sea salt l(-1) (corresponding to 6.5-65 g NaCl l(-1)) with an optimum at 30 g sea salt l(-1) (20 g NaCl l(-1)). Strain MV1087T was heterotrophic, able to ferment proteinaceous substrates, such as brain/heart infusion and gluten, and carbohydrates, such as glucose, xylan and starch. The DNA G+C content was 27 mol%. Phylogenetic analyses using 16S rDNA sequences indicated that strain MV1087T belonged to cluster XII of the Clostridium subphylum. Due to its phenotypic and genotypic characteristics, isolate MV1087T is proposed as a novel species of a new genus, Caloranaerobacter azorensis gen. nov., sp. nov. The type strain is MV1087T (= CNCM I-2543T = DSM 13643T).

Marinitoga camini gen. nov., sp. nov., a rod-shaped bacterium belonging to the order Thermotogales, isolated from a deep-sea hydrothermal vent.

A thermophilic, anaerobic, chemo-organotrophic sulfur-reducing bacterium, designated MV1075T, was isolated from a deep-sea hydrothermal chimney sample collected on the Mid-Atlantic Ridge. Cells were rod-shaped with a sheath-like outer structure, motile with polar flagella and stained Gram-negative. They appeared singly, in pairs or in short chains. The temperature range for growth was 25-65 degrees C, with an optimum at 55 degrees C. Growth was observed from pH 5 to pH 9, and the optimum pH was around 7. The salinity range for growth was 15-70 g sea salt l(-1) (corresponding to 10-45 g NaCl l(-1)), with an optimum at 30 g l(-1) (20 g NaCl l(-1)). The isolate was able to grow on a broad spectrum of carbohydrates or complex proteinaceous substrates. Sulfur was not necessary for growth. Growth was inhibited by H2, but, in presence of sulfur, this inhibition was removed and H2S was produced. The G+C content of the genomic DNA was 29 mol %. Phylogenetic analyses of the 16S rRNA gene located the strain within the order Thermotogales, in the domain Bacteria. On the basis of 16S rDNA sequence comparisons, in combination with morphological and physiological characteristics, it is proposed that the isolate should be described as a novel species of a new genus, Marinitoga gen. nov., of which Marinitoga camini sp. nov. is the type species. The type strain is MV1075T (= CNCM 1-2413T = DSM 13578T).

Microbial diversity in smoked salmon examined by a culture-independent molecular approach--a preliminary study.

Microbial biodiversity in sliced vacuum-packed cold smoked salmon was investigated using culture-independent molecular biology techniques. Sliced smoked salmon was stored for 25 days after being packed at 4 degrees C. DNA was extracted from sliced vacuum-packed cold smoked salmon. PCR DNA amplification were carried out using universal eubacterial primers corresponding to Escherichia coli 16S rRNA gene. 16S rRNA genes were amplified, cloned in E. coli and compared using Amplification Ribosomal DNA Restriction Analysis (ARDRA). 106 clones were studied and classified into 13 Operational Taxonomic Units (OTUs). Sequences obtained to describe those 13 OTUs were compared to GenBank data. They indicated the presence of Vibrio species. Enterobacteraceae and also marine psychrophilic clones related to Alteromonas macleodii, which were not encountered within cultures, but no Gram-positive species have been obtained. Those results indicate that bias in description of microbial diversity may be encountred in both molecular and cultural techniques.

Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans.

The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERS's collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30 degrees C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity.