The metabolic potential of the single cell genomes obtained from the Challenger Deep,Mariana Trench within the candidate superphylum Parcubacteria (OD1)

Candidate phyla (CP) are broad phylogenetic clusters of organisms that lack cultured representatives. Included in this fraction is the candidate Parcubacteria superphylum. Specific characteristics that have been ascribed to the Parcubacteria include reduced genome size, limited metabolic potential and exclusive reliance on fermentation for energy acquisition. The study of new environmental niches, such as the marine versus terrestrial subsurface, often expands the understanding of the genetic potential of taxonomic groups. For this reason, we analyzed 12 Parcubacteria single amplified genomes (SAGs) from sediment samples collected within the Challenger Deep of the Mariana Trench, obtained during the Deepsea Challenge (DSC) Expedition. Many of these SAGs are closely related to environmental sequences obtained from deep‐sea environments based on 16S rRNA gene similarity and BLAST matches to predicted proteins. DSC SAGs encode features not previously identified in Parcubacteria obtained from other habitats. These include adaptation to oxidative stress, polysaccharide modification and genes associated with respiratory nitrate reduction. The DSC SAGs are also distinguished by relative greater abundance of genes for nucleotide and amino acid biosynthesis, repair of alkylated DNA and the synthesis of mechanosensitive ion channels. These results present an expanded view of the Parcubacteria, among members residing in an ultra‐deep hadal environment.     

深海微生物高压适应与生物地球化学循环

深海是典型的高压环境,嗜压微生物是深海生态系统中的重要类群.随着深海采样技术的发展及高压微生物特殊培养设备的开发,已从深海环境中分离到一系列嗜压微生物,包括一些常压环境不能生长的严格嗜压菌.对这些嗜压菌的研究,不仅对微生物适应极端高压环境的机制有一定了解,而且发现了一些特殊的代谢产物.研究微生物高压嗜压机理,还有助于探索地球生命的温度压力极限及生命起源和演化等科学问题.从深海嗜压微生物多样性、深海微生物高压环境适应机理及深海微生物在生物地球化学循环中的作用等方面对嗜压微生物的研究进展进行综述.     

Capturing diversity of marine heterotrophic protists: one cell at a time

Recent applications of culture-independent, molecular methods have revealed unexpectedly high diversity in a variety of functional and phylogenetic groups of microorganisms in the ocean. However, none of the existing research tools are free from significant limitations, such as PCR and cloning biases, low phylogenetic resolution and others. Here, we employed novel, single-cell sequencing techniques to assess the composition of small (<10 μm diameter), heterotrophic protists from the Gulf of Maine. Single cells were isolated by flow cytometry, their genomes amplified, and 18S rRNA marker genes were amplified and sequenced. We compared the results to traditional environmental PCR cloning of sorted cells. The diversity of heterotrophic protists was significantly higher in the library of single amplified genomes (SAGs) than in environmental PCR clone libraries of the 18S rRNA gene, obtained from the same coastal sample. Libraries of SAGs, but not clones contained several recently discovered, uncultured groups, including picobiliphytes and novel marine stramenopiles. Clone, but not SAG, libraries contained several large clusters of identical and nearly identical sequences of Dinophyceae, Cercozoa and Stramenopiles. Similar results were obtained using two alternative primer sets, suggesting that PCR biases may not be the only explanation for the observed patterns. Instead, differences in the number of 18S rRNA gene copies among the various protist taxa probably had a significant role in determining the PCR clone composition. These results show that single-cell sequencing has the potential to more accurately assess protistan community composition than previously established methods. In addition, the creation of SAG libraries opens opportunities for the analysis of multiple genes or entire genomes of the uncultured protist groups.     

Whole genome sequencing of a snailfish from the Yap Trench (~7,000 m) clarifies the molecular mechanisms underlying adaptation to the deep sea

Hadal environments (depths below 6,000 m) are characterized by extremely high hydrostatic pressures, low temperatures, a scarce food supply, and little light. The evolutionary adaptations that allow vertebrates to survive in this extreme environment are poorly understood. Here, we constructed a high-quality reference genome for Yap hadal snailfish (YHS), which was captured at a depth of ~7,000 m in the Yap Trench. The final YHS genome assembly was 731.75 Mb, with a contig N50 of 0.75 Mb and a scaffold N50 of 1.26 Mb. We predicted 24,329 protein-coding genes in the YHS genome, and 24,265 of these genes were successfully functionally annotated. Phylogenetic analyses suggested that YHS diverged from a Mariana Trench snailfish approximately 0.92 million years ago. Many genes associated with DNA repair show evidence of positive selection and have expanded copy numbers in the YHS genome, possibly helping to maintain the integrity of DNA under increased hydrostatic pressure. The levels of trimethylamine N-oxide (TMAO), a potent protein stabilizer, are much higher in the muscles of YHS than in those of shallow-water fish. This difference is perhaps due to the five copies of the TMAO-generating enzyme flavin-containing monooxygenase-3 gene (fmo3) in the YHS genome and the abundance of trimethylamine (TMA)-generating bacteria in the YHS gut. Thus, the high TMAO content might help YHS adapt to high hydrostatic pressure by improving protein stability. Additionally, the evolutionary features of the YHS genes encoding sensory-related proteins are consistent with the scarce food supply and darkness in the hadal environments. These results clarify the molecular mechanisms underlying the adaptation of hadal organisms to the deep-sea environment and provide valuable genomic resources for in-depth investigations of hadal biology.     

Amino acid substitutions in malate dehydrogenases of piezophilic bacteria isolated from intestinal contents of deep-sea fishes retrieved from the abyssal zone

To examine the occurrence in other deep-sea bacteria of two amino acid substitutions (Ala-180 and His-229) in malate dehydrogenase (MDH) found previously in the deep-sea piezophilic Moritella sp. strain 2D2, we cloned and sequenced MDH genes of deep-sea piezophilic Moritella and Shewanella strains isolated from intestinal contents of deep-sea fishes, as well as other Moritella species from deep-sea water and sediments: M. marina, M. japonica, and M. yayanosii. The piezophilic Moritella strains had a Val residue or an Ala residue at position 180 and all the Moritella strains except for one had a His residue at position 229. However, four piezophilic-strain-specific substitutions at positions 103, 111, 229, and 283 were found to be completely conserved in the MDH of the intestinal Moritella strains of deep-sea fishes, indicating the substitutions may be habitat-specific. The piezophilic Shewanella strains had a Val residue and a Gln residue at positions 180 and 229, respectively. However, the MDHs of the Shewanella strains had five piezophilic-strain-specific substitutions at positions 61, 65, 107, 161, and 202. Therefore, the enzymatic strategies for responding to deep-sea high pressure environments of the MDHs between the genera Moritella and Shewanella are potentially different. Moreover, homology modeling shows these substitutions found in the MDHs of both genera except for position 229 in the subunit interface are located on the exposed region of the MDH molecules, indicating the substitutions may be related to the hydration state of the molecules.     

Phylogenetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes from deep-sea microorganisms

The phylogenetic diversity of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, E.C. 4.1.1.39) large-subunit genes of deep-sea microorganisms was analyzed. Bulk genomic DNA was isolated from seven samples, including samples from the Mid-Atlantic Ridge and various deep-sea habitats around Japan. The kinds of samples were hydrothermal vent water and chimney fragment; reducing sediments from a bathyal seep, a hadal seep, and a presumed seep; and symbiont-bearing tissues of the vent mussel, Bathymodiolus sp., and the seep vestimentiferan tubeworm, Lamellibrachia sp. The RuBisCO genes that encode both form I and form II large subunits (cbbL and cbbM) were amplified by PCR from the seven deep-sea sample DNA populations, cloned, and sequenced. From each sample, 50 cbbL clones and 50 cbbM clones, if amplified, were recovered and sequenced to group them into operational taxonomic units (OTUs). A total of 29 OTUs were recorded from the 300 total cbbL clones, and a total of 24 OTUs were recorded from the 250 total cbbM clones. All the current OTUs have the characteristic RuBisCO amino acid motif sequences that exist in other RuBisCOs. The recorded OTUs were related to different RuBisCO groups of proteobacteria, cyanobacteria, and eukarya. The diversity of the RuBisCO genes may be correlated with certain characteristics of the microbial habitats. The RuBisCO sequences from the symbiont-bearing tissues showed a phylogenetic relationship with those from the ambient bacteria. Also, the RuBisCO sequences of known species of thiobacilli and those from widely distributed marine habitats were closely related to each other. This suggests that the Thiobacillus-related RuBisCO may be distributed globally and contribute to the primary production in the deep sea.     

New selective amplifier genes containing c-Mpl for hematopoietic cell expansion

We previously developed "selective amplifier genes (SAGs)" which confer a growth advantage to transduced cells. The SAG is a chimeric gene encoding the G-CSF receptor (GCR) and the estrogen or tamoxifen (Tm) receptor and is able to expand transduced hematopoietic cells by treatment with estrogen or Tm. In the current study, we examined the in vitro efficacy of modified SAGs containing the thrombopoietin (TPO) receptor (c-Mpl) gene instead of GCR as a more potent signal generator. In addition, we constructed various mutant Mpl-type SAGs to abolish the responsiveness to endogenous TPO while retaining Tm-dependency. When Ba/F3 cells were retrovirally transduced with the Mpl-type SAGs, the cells showed Tm- and TPO-dependent growth even without IL-3. The Mpl-type SAGs induced more potent proliferation of Ba/F3 and cynomolgus CD34(+) cells than the GCR-type SAG. One mutant Mpl-type SAG (Delta GCRMplTmR) successfully lost the responsiveness to TPO without affecting the Tm-dependence.     

Genomic insights into the uncultivated marine Zetaproteobacteria at Loihi Seamount

The Zetaproteobacteria are a candidate class of marine iron-oxidizing bacteria that are typically found in high iron environments such as hydrothermal vent sites. As much remains unknown about these organisms due to difficulties in cultivation, single-cell genomics was used to learn more about this elusive group at Loihi Seamount. Comparative genomics of 23 phylogenetically diverse single amplified genomes (SAGs) and two isolates indicate niche specialization among the Zetaproteobacteria may be largely due to oxygen tolerance and nitrogen transformation capabilities. Only Form II ribulose 1,5-bisphosphate carboxylase (RubisCO) genes were found in the SAGs, suggesting that some of the uncultivated Zetaproteobacteria may be adapted to low oxygen and/or high carbon dioxide concentrations. There is also genomic evidence of oxygen-tolerant cytochrome c oxidases and oxidative stress-related genes, indicating that others may be exposed to higher oxygen conditions. The Zetaproteobacteria also have the genomic potential for acquiring nitrogen from numerous sources including ammonium, nitrate, organic compounds, and nitrogen gas. Two types of molybdopterin oxidoreductase genes were found in the SAGs, indicating that those found in the isolates, thought to be involved in iron oxidation, are not consistent among all the Zetaproteobacteria. However, a novel cluster of redox-related genes was found to be conserved in 10 SAGs as well as in the isolates warranting further investigation. These results were used to isolate a novel iron-oxidizing Zetaproteobacteria. Physiological studies and genomic analysis of this isolate were able to support many of the findings from SAG analyses demonstrating the value of these data for designing future enrichment strategies.     

Strain-level genomic variation in natural populations of Lebetimonas from an erupting deep-sea volcano

Chemolithoautotrophic Epsilonproteobacteria are ubiquitous in sulfidic, oxygen-poor habitats, including hydrothermal vents, marine oxygen minimum zones, marine sediments and sulfidic caves and have a significant role in cycling carbon, hydrogen, nitrogen and sulfur in these environments. The isolation of diverse strains of Epsilonproteobacteria and the sequencing of their genomes have revealed that this group has the metabolic potential to occupy a wide range of niches, particularly at dynamic deep-sea hydrothermal vents. We expand on this body of work by examining the population genomics of six strains of Lebetimonas, a vent-endemic, thermophilic, hydrogen-oxidizing Epsilonproteobacterium, from a single seamount in the Mariana Arc. Using Lebetimonas as a model for anaerobic, moderately thermophilic organisms in the warm, anoxic subseafloor environment, we show that genomic content is highly conserved and that recombination is limited between closely related strains. The Lebetimonas genomes are shaped by mobile genetic elements and gene loss as well as the acquisition of novel functional genes by horizontal gene transfer, which provide the potential for adaptation and microbial speciation in the deep sea. In addition, these Lebetimonas genomes contain two operons of nitrogenase genes with different evolutionary origins. Lebetimonas expressed nifH during growth with nitrogen gas as the sole nitrogen source, thus providing the first evidence of nitrogen fixation in any Epsilonproteobacteria from deep-sea hydrothermal vents. In this study, we provide a comparative overview of the genomic potential within the Nautiliaceae as well as among more distantly related hydrothermal vent Epsilonproteobacteria to broaden our understanding of microbial adaptation and diversity in the deep sea.     

Phylogenetic Diversity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Large-Subunit Genes from Deep-Sea Microorganisms

The phylogenetic diversity of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, E.C. 4.1.1.39) large-subunit genes of deep-sea microorganisms was analyzed. Bulk genomic DNA was isolated from seven samples, including samples from the Mid-Atlantic Ridge and various deep-sea habitats around Japan. The kinds of samples were hydrothermal vent water and chimney fragment; reducing sediments from a bathyal seep, a hadal seep, and a presumed seep; and symbiont-bearing tissues of the vent mussel, Bathymodiolus sp., and the seep vestimentiferan tubeworm, Lamellibrachia sp. The RuBisCO genes that encode both form I and form II large subunits (cbbL and cbbM) were amplified by PCR from the seven deep-sea sample DNA populations, cloned, and sequenced. From each sample, 50 cbbL clones and 50 cbbM clones, if amplified, were recovered and sequenced to group them into operational taxonomic units (OTUs). A total of 29 OTUs were recorded from the 300 total cbbL clones, and a total of 24 OTUs were recorded from the 250 total cbbM clones. All the current OTUs have the characteristic RuBisCO amino acid motif sequences that exist in other RuBisCOs. The recorded OTUs were related to different RuBisCO groups of proteobacteria, cyanobacteria, and eukarya. The diversity of the RuBisCO genes may be correlated with certain characteristics of the microbial habitats. The RuBisCO sequences from the symbiont-bearing tissues showed a phylogenetic relationship with those from the ambient bacteria. Also, the RuBisCO sequences of known species of thiobacilli and those from widely distributed marine habitats were closely related to each other. This suggests that the Thiobacillus-related RuBisCO may be distributed globally and contribute to the primary production in the deep sea.     

The unique 16S rRNA genes of piezophiles reflect both phylogeny and adaptation

In the ocean's most extreme depths, pressures of 70 to 110 megapascals prevent the growth of all but the most hyperpiezophilic (pressure-loving) organisms. The physiological adaptations required for growth under these conditions are considered to be substantial. Efforts to determine specific adaptations permitting growth at extreme pressures have thus far focused on relatively few gamma-proteobacteria, in part due to the technical difficulties of obtaining piezophilic bacteria in pure culture. Here, we present the molecular phylogenies of several new piezophiles of widely differing geographic origins. Included are results from an analysis of the first deep-trench bacterial isolates recovered from the southern hemisphere (9.9-km depth) and of the first gram-positive piezophilic strains. These new data allowed both phylogenetic and structural 16S rRNA comparisons among deep-ocean trench piezophiles and closely related strains not adapted to high pressure. Our results suggest that (i) the Circumpolar Deep Water acts as repository for hyperpiezophiles and drives their dissemination to deep trenches in the Pacific Ocean and (ii) the occurrence of elongated helices in the 16S rRNA genes increases with the extent of adaptation to growth at elevated pressure. These helix changes are believed to improve ribosome function under deep-sea conditions.     

Surface antigens of Toxoplasma gondii: variations on a theme

Toxoplasma gondii is an obligate intracellular protozoan parasite with an exceptionally broad host range. Recently, it has become apparent that the number of surface antigens (SAGs) it expresses may rival the number of genera it can infect. Most of these antigens belong to the developmentally regulated and distantly related SAG1 or SAG2 families. The genes encoding the surface antigens are distributed throughout the T. gondii genome, with remarkably little polymorphism observed at each locus. Results from a number of studies have suggested that the surface antigens play an important role in the biology of the parasite. For example, SAG3 null mutants generated by targeted disruption provide convincing evidence that this surface antigen, at least, functions during parasite attachment. Analyses of a SAG1 knockout in rodents, however, indicate that this surface antigen may play a crucial role in immune modulation or virulence attenuation. The current understanding of the SAG1 and SAG2 families will be discussed here.     

RESPONSE OF NORTH ATLANTIC RIGHT WHALES (EUBALAENA GLACIALIS) TO PLAYBACK OF CALLS RECORDED FROM SURFACE ACTIVE GROUPS IN BOTH THE NORTH AND SOUTH ATLANTIC

The surface active group (SAG) is the most obvious social interaction of the North Atlantic right whale ( Eubalaena glacialis ). SAGs are typically composed of an adult female with two or more males engaged in social behavior near the surface. Distinct calls, believed to be produced by the female, are associated with these groups. Calls recorded from three North Atlantic right whale SAGs and three South Atlantic right whale ( Eubalaena australis ) SAGs were played back to North Atlantic right whales to determine if these sounds are sufficient to attract males to the groups. Playbacks of gunshot sounds produced by North Atlantic right whales were used as a control stimulus. Thirty-six trials were carried out from 1999 to 2001 in the Bay of Fundy, Canada. Whales approached 27 of 31 SAG playbacks and 0 of 5 gunshot playbacks. Where sex was determined ( n = 28), all approaches to North Atlantic SAG recordings were by males. Individuals ( n = 22) of all age and sex classes approached South Atlantic SAG playbacks. These trials indicate that SAG calls from both populations are sufficient to attract right whales to SAGs and that males and females respond differently to stimuli from the North Atlantic. The difference in response to North and South Atlantic SAG stimuli was unexpected. Novelty, species differences in calls, and different seasonal or behavioral context for the recorded stimuli may be responsible for the differences in response.     

Exponential decline of deep-sea ecosystem functioning linked to benthic biodiversity loss

BACKGROUND: Recent investigations suggest that biodiversity loss might impair the functioning and sustainability of ecosystems. Although deep-sea ecosystems are the most extensive on Earth, represent the largest reservoir of biomass, and host a large proportion of undiscovered biodiversity, the data needed to evaluate the consequences of biodiversity loss on the ocean floor are completely lacking. RESULTS: Here, we present a global-scale study based on 116 deep-sea sites that relates benthic biodiversity to several independent indicators of ecosystem functioning and efficiency. We show that deep-sea ecosystem functioning is exponentially related to deep-sea biodiversity and that ecosystem efficiency is also exponentially linked to functional biodiversity. These results suggest that a higher biodiversity supports higher rates of ecosystem processes and an increased efficiency with which these processes are performed. The exponential relationships presented here, being consistent across a wide range of deep-sea ecosystems, suggest that mutually positive functional interactions (ecological facilitation) can be common in the largest biome of our biosphere. CONCLUSIONS: Our results suggest that a biodiversity loss in deep-sea ecosystems might be associated with exponential reductions of their functions. Because the deep sea plays a key role in ecological and biogeochemical processes at a global scale, this study provides scientific evidence that the conservation of deep-sea biodiversity is a priority for a sustainable functioning of the worlds' oceans.     

The Unique 16S rRNA Genes of Piezophiles Reflect both Phylogeny and Adaptation

In the ocean's most extreme depths, pressures of 70 to 110 megapascals prevent the growth of all but the most hyperpiezophilic (pressure-loving) organisms. The physiological adaptations required for growth under these conditions are considered to be substantial. Efforts to determine specific adaptations permitting growth at extreme pressures have thus far focused on relatively few γ-proteobacteria, in part due to the technical difficulties of obtaining piezophilic bacteria in pure culture. Here, we present the molecular phylogenies of several new piezophiles of widely differing geographic origins. Included are results from an analysis of the first deep-trench bacterial isolates recovered from the southern hemisphere (9.9-km depth) and of the first gram-positive piezophilic strains. These new data allowed both phylogenetic and structural 16S rRNA comparisons among deep-ocean trench piezophiles and closely related strains not adapted to high pressure. Our results suggest that (i) the Circumpolar Deep Water acts as repository for hyperpiezophiles and drives their dissemination to deep trenches in the Pacific Ocean and (ii) the occurrence of elongated helices in the 16S rRNA genes increases with the extent of adaptation to growth at elevated pressure. These helix changes are believed to improve ribosome function under deep-sea conditions.     

Psychropiezophilic microorganisms.

Bacteria living in the deep-sea have several unusual features that allow them to thrive in their extreme environment. Most isolated strains are not only piezophilic but also psychrophilic. In this short review, we summarize the current knowledge about psychrophiezophilic microorganisms with regard to their taxonomy and cellular fatty acids composition. Their biotechnological potential is also described.     

Leaf senescence in rice plants: cloning and characterization of senescence up-regulated genes.

To identify senescence-associated genes (SAGs) in rice leaves, senescence was induced by transferring rice seedlings into darkness. Senescence up-regulated cDNAs were obtained by PCR-based subtractive hybridization. Among 14 SAG clones characterized, 11 were found to be associated with both dark-induced and natural leaf senescence. Three clones were associated only with dark-induced leaf senescence. The possible physiological roles of these SAGs during rice leaf senescence are discussed.