Deep-sea Vent Chimney Research Articles

A Simple and Efficient RNA Extraction Method from Deep-Sea Hydrothermal Vent Chimney Structures.

RNA-based microbiological analyses, e.g., transcriptome and reverse transcription-quantitative PCR, require a relatively large amount of high quality RNA. RNA-based analyses on microbial communities in deep-sea hydrothermal environments often encounter methodological difficulties with RNA extraction due to the presence of unique minerals in and the low biomass of samples. In the present study, we assessed RNA extraction methods for deep-sea vent chimneys that had complex mineral compositions. Mineral-RNA adsorption experiments were conducted using mock chimney minerals and Escherichia coli total RNA solution, and showed that detectable RNA significantly decreased possibly due to adsorption onto minerals. This decrease in RNA was prevented by the addition of sodium tripolyphosphate (STPP), deoxynucleotide triphosphates (dNTPs), salmon sperm DNA, and NaOH. The addition of STPP was also effective for RNA extraction from the mixture of E. coli cells and mock chimney minerals when TRIzol reagent and the RNeasy column were used, but not when the RNeasy PowerSoil total RNA kit was used. A combination of STPP, TRIzol reagent, the RNeasy column, and sonication resulted in the highest RNA yield from a natural chimney. This indirect extraction procedure is simple, rapid, inexpensive, and may be used for large-scale RNA extraction.

Thermodesulfatator autotrophicus sp. nov., a thermophilic sulfate-reducing bacterium from the Indian Ocean.

A novel sulfate-reducing bacterium, strain S606T, was isolated from a sulfide sample collected at a depth of 2764 m from a deep-sea vent chimney wall in the Indian Ocean. Phylogenetic 16S rRNA gene sequence analyses placed strain S606T within the genus Thermodesulfatator, with highest sequence similarity of 98.2 % to Thermodesulfatator indicus DSM 15286T, followed by Thermodesulfatator atlanticus AT1325T (97.4 %). The average nucleotide identity (ANI) values between S606T and the two other type strains (T. indicus DSM 15286T and T. atlanticus AT1325T) were 79.2 % and 71.5 %, respectively. The digital DNA-DNA hybridization estimate values between S606T and these two type strains were 22.7±2.4 % and 18.1±2.3 %, respectively. Cells were Gram-stain-negative, anaerobic, motile rods (1-1.8×0.5-0.7 µm). The novel isolate grew at NaCl concentrations ranging from 1.5 to 4.5 % (optimum 2.5-3 %), from pH 5.5 to 8 (optimum 6.5-7.0) and at temperatures between 50 and 80 °C (optimum 65-70 °C). S606T grew chemolithoautotrophically in an H2/CO2 atmosphere (80 : 20, v/v; 200 kPa), used sulfate as a terminal electron acceptor, but not sulfur, sulfite nor thiosulfate. The predominant fatty acids were C16 : 0 (24.2 %), summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c, 26.3 %), C18 : 0 (22.2 %) and C18 : 1ω9c (9.2 %). The DNA G+C content of the chromosomal DNA was 43.1 mol%. The combined genotypic, chemotaxonomic and phenotypic traits show that S606T should be described as representing a novel species of the genus Thermodesulfatator, for which the name Thermodesulfatator autotrophicus sp. nov. is proposed. The type strain is S606T (=DSM 101864T=MCCC 1A01871T).

Metagenome reveals potential microbial degradation of hydrocarbon coupled with sulfate reduction in an oil-immersed chimney from Guaymas Basin

Deep-sea hydrothermal vent chimneys contain a high diversity of microorganisms, yet the metabolic activity and the ecological functions of the microbial communities remain largely unexplored. In this study, a metagenomic approach was applied to characterize the metabolic potential in a Guaymas hydrothermal vent chimney and to conduct comparative genomic analysis among a variety of environments with sequenced metagenomes. Complete clustering of functional gene categories with a comparative metagenomic approach showed that this Guaymas chimney metagenome was clustered most closely with a chimney metagenome from Juan de Fuca. All chimney samples were enriched with genes involved in recombination and repair, chemotaxis and flagellar assembly, highlighting their roles in coping with the fluctuating extreme deep-sea environments. A high proportion of transposases was observed in all the metagenomes from deep-sea chimneys, supporting the previous hypothesis that horizontal gene transfer may be common in the deep-sea vent chimney biosphere. In the Guaymas chimney metagenome, thermophilic sulfate reducing microorganisms including bacteria and archaea were found predominant, and genes coding for the degradation of refractory organic compounds such as cellulose, lipid, pullullan, as well as a few hydrocarbons including toluene, ethylbenzene and o-xylene were identified. Therefore, this oil-immersed chimney supported a thermophilic microbial community capable of oxidizing a range of hydrocarbons that served as electron donors for sulphate reduction under anaerobic conditions.

Comparative metagenomics of microbial communities inhabiting deep-sea hydrothermal vent chimneys with contrasting chemistries

Deep-sea hydrothermal vent chimneys harbor a high diversity of largely unknown microorganisms. Although the phylogenetic diversity of these microorganisms has been described previously, the adaptation and metabolic potential of the microbial communities is only beginning to be revealed. A pyrosequencing approach was used to directly obtain sequences from a fosmid library constructed from a black smoker chimney 4143-1 in the Mothra hydrothermal vent field at the Juan de Fuca Ridge. A total of 308,034 reads with an average sequence length of 227 bp were generated. Comparative genomic analyses of metagenomes from a variety of environments by two-way clustering of samples and functional gene categories demonstrated that the 4143-1 metagenome clustered most closely with that from a carbonate chimney from Lost City. Both are highly enriched in genes for mismatch repair and homologous recombination, suggesting that the microbial communities have evolved extensive DNA repair systems to cope with the extreme conditions that have potential deleterious effects on the genomes. As previously reported for the Lost City microbiome, the metagenome of chimney 4143-1 exhibited a high proportion of transposases, implying that horizontal gene transfer may be a common occurrence in the deep-sea vent chimney biosphere. In addition, genes for chemotaxis and flagellar assembly were highly enriched in the chimney metagenomes, reflecting the adaptation of the organisms to the highly dynamic conditions present within the chimney walls. Reconstruction of the metabolic pathways revealed that the microbial community in the wall of chimney 4143-1 was mainly fueled by sulfur oxidation, putatively coupled to nitrate reduction to perform inorganic carbon fixation through the Calvin-Benson-Bassham cycle. On the basis of the genomic organization of the key genes of the carbon fixation and sulfur oxidation pathways contained in the large genomic fragments, both obligate and facultative autotrophs appear to be present and contribute to biomass production.

Phylogenetic diversity of methanogenic, sulfate-reducing and methanotrophic prokaryotes from deep-sea hydrothermal vents and cold seeps

Microbial communities of methanogenic, sulfate-reducing and methanotrophic prokaryotes from deep-sea environments were investigated by molecular phylogenetic analysis of the deduced amino acid sequences of the genes encoding for the methyl coenzyme M reductase (mcrA), dissimilatory sulfite reductase (dsrAB) and particulate methane monoxygenase (pmoA), respectively. Clone libraries of PCR amplified genes were constructed using DNA extracted from deep-sea vent chimneys (Rainbow and Logatchev hydrothermal vent fields, Mid-Atlantic Ridge, Atlantic Ocean; 9°N East Pacific Rise, Pacific Ocean) and from vertically subsampled sediment cores from cold-seep areas (Blake Ridge, western Atlantic Ocean; Florida Escarpment, Gulf of Mexico). Recombinant clones were screened by RFLP and representative dsrAB, mcrA and pmoA genes were sequenced. The dsrAB sequences grouped primarily within the orders Desulfobacterales, Syntrophobacterales and the Gram-positive order Clostridales. Cold-seep mcrA sequences were distributed among the ANME-2c, -2d and -2e groups, which were previously shown to be associated with the anaerobic oxidation of methane. This study also reports the first mcrA sequences from a high-temperature, black smoker chimney (Logatchev) to group within the ANME-2e subgroup. The majority of the remaining hydrothermal vent mcrA sequences were primarily related to thermophilic members of the anaerobic, methanogenic order Methanococcales. A shift in the dominant ANME-2 group with depth in the sediment for both Florida Escarpment and Blake Ridge mcrA libraries was detected. ANME-2d related clones were detected in the top zones of both cores, with the frequency of ANME-2e related clones increasing with depth. All pmoA sequences retrieved from the cold-seep sites were found to be related to Type I methanotrophic members of the γ-proteobacteria, and were primarily distributed among three major clusters of sequences. No Type II pmoA sequences related to methanotrophic members of the α-proteobacteria were detected, suggesting that the methanotrophic communities in these cold-seep areas are dominated by Type I γ-proteobacteria.

Effect of variation of environmental conditions on the microbial communities of deep-sea vent chimneys, cultured in a bioreactor

Both cultivation and molecular techniques were used to investigate the microbial diversity and dynamic of a deep-sea vent chimney. The enrichment cultures performed in a gas-lift bioreactor were inoculated with a black smoker chimney sample collected on TAG site on the mid-Atlantic ridge. To mimic as close as possible environmental conditions, the cultures were performed in oligotrophic medium with nitrogen, hydrogen and carbon dioxide (N(2)/H(2)/CO(2)) gas sweeping. Also, the temperature was first settled at a temperature of 85 degrees C and colloidal sulphur was added. Then, the temperature was lowered to 60 degrees C and sulphur was omitted. Archaeal and bacterial diversity was studied in both culture and natural samples. Through 16S rRNA gene sequences analysis of the enrichment cultures microorganisms affiliated to Archeoglobales, Thermococcales were detected in both conditions while, Deferribacterales and Thermales were detected only at 65 degrees C in the absence of sulphur. Single-stranded conformational polymorphism and quantitative PCR permit to study the microbial community dynamic during the two enrichment cultures. The effect of environmental changes (modification of culture conditions), i.e. temperature, medium composition, electron donors and acceptors availability were shown to affect the microbial community in culture, as this would happen in their environment. The effect of environmental changes, i.e. temperature and medium composition was shown to affect the microbial community in culture, as this could happen in their environment. The modification of culture conditions, such as temperature, organic matter concentration, electron donors and acceptors availability allowed to enrich different population of prokaryotes inhabiting hydrothermal chimneys.

Continuous enrichment cultures: insights into prokaryotic diversity and metabolic interactions in deep-sea vent chimneys

The prokaryotic diversity of culturable thermophilic communities of deep-sea hydrothermal chimneys was analysed using a continuous enrichment culture performed in a gas-lift bioreactor, and compared to classical batch enrichment cultures in vials. Cultures were conducted at 60 degrees C and pH 6.5 using a complex medium containing carbohydrates, peptides and sulphur, and inoculated with a sample of a hydrothermal black chimney collected at the Rainbow field, Mid-Atlantic Ridge, at 2,275 m depth. To assess the relevance of both culture methods, bacterial and archaeal diversity was studied using cloning and sequencing, DGGE, and whole-cell hybridisation of 16S rRNA genes. Sequences of heterotrophic microorganisms belonging to the genera Marinitoga, Thermosipho, Caminicella (Bacteria) and Thermococcus (Archaea) were obtained from both batch and continuous enrichment cultures while sequences of the autotrophic bacterial genera Deferribacter and Thermodesulfatator were only detected in the continuous bioreactor culture. It is presumed that over time constant metabolite exchanges will have occurred in the continuous enrichment culture enabling the development of a more diverse prokaryotic community. In particular, CO(2) and H(2) produced by the heterotrophic population would support the growth of autotrophic populations. Therefore, continuous enrichment culture is a useful technique to grow over time environmentally representative microbial communities and obtain insights into prokaryotic species interactions that play a crucial role in deep hydrothermal environments.