Biodiversity's Big Wet Secret: The Global Distribution of Marine Biological Records Reveals Chronic Under-Exploration of the Deep Pelagic Ocean

Background

Understanding the distribution of marine biodiversity is a crucial first step towards the effective and sustainable management of marine ecosystems. Recent efforts to collate location records from marine surveys enable us to assemble a global picture of recorded marine biodiversity. They also effectively highlight gaps in our knowledge of particular marine regions. In particular, the deep pelagic ocean – the largest biome on Earth – is chronically under-represented in global databases of marine biodiversity.

Methodology/Principal Findings

We use data from the Ocean Biogeographic Information System to plot the position in the water column of ca 7 million records of marine species occurrences. Records from relatively shallow waters dominate this global picture of recorded marine biodiversity. In addition, standardising the number of records from regions of the ocean differing in depth reveals that regardless of ocean depth, most records come either from surface waters or the sea bed. Midwater biodiversity is drastically under-represented.

Conclusions/Significance

The deep pelagic ocean is the largest habitat by volume on Earth, yet it remains biodiversity''s big wet secret, as it is hugely under-represented in global databases of marine biological records. Given both its value in the provision of a range of ecosystem services, and its vulnerability to threats including overfishing and climate change, there is a pressing need to increase our knowledge of Earth''s largest ecosystem.     

The Impact of Whaling on the Ocean Carbon Cycle: Why Bigger Was Better

Background

Humans have reduced the abundance of many large marine vertebrates, including whales, large fish, and sharks, to only a small percentage of their pre-exploitation levels. Industrial fishing and whaling also tended to preferentially harvest the largest species and largest individuals within a population. We consider the consequences of removing these animals on the ocean''s ability to store carbon.

Methodology/Principal Findings

Because body size is critical to our arguments, our analysis focuses on populations of baleen whales. Using reconstructions of pre-whaling and modern abundances, we consider the impact of whaling on the amount of carbon stored in living whales and on the amount of carbon exported to the deep sea by sinking whale carcasses. Populations of large baleen whales now store 9.1×10⁶ tons less carbon than before whaling. Some of the lost storage has been offset by increases in smaller competitors; however, due to the relative metabolic efficiency of larger organisms, a shift toward smaller animals could decrease the total community biomass by 30% or more. Because of their large size and few predators, whales and other large marine vertebrates can efficiently export carbon from the surface waters to the deep sea. We estimate that rebuilding whale populations would remove 1.6×10⁵ tons of carbon each year through sinking whale carcasses.

Conclusions/Significance

Even though fish and whales are only a small portion of the ocean''s overall biomass, fishing and whaling have altered the ocean''s ability to store and sequester carbon. Although these changes are small relative to the total ocean carbon sink, rebuilding populations of fish and whales would be comparable to other carbon management schemes, including ocean iron fertilization.     

Distribution analysis of hydrogenases in surface waters of marine and freshwater environments

Background

Surface waters of aquatic environments have been shown to both evolve and consume hydrogen and the ocean is estimated to be the principal natural source. In some marine habitats, H₂ evolution and uptake are clearly due to biological activity, while contributions of abiotic sources must be considered in others. Until now the only known biological process involved in H₂ metabolism in marine environments is nitrogen fixation.

Principal Findings

We analyzed marine and freshwater environments for the presence and distribution of genes of all known hydrogenases, the enzymes involved in biological hydrogen turnover. The total genomes and the available marine metagenome datasets were searched for hydrogenase sequences. Furthermore, we isolated DNA from samples from the North Atlantic, Mediterranean Sea, North Sea, Baltic Sea, and two fresh water lakes and amplified and sequenced part of the gene encoding the bidirectional NAD(P)-linked hydrogenase. In 21% of all marine heterotrophic bacterial genomes from surface waters, one or several hydrogenase genes were found, with the membrane-bound H₂ uptake hydrogenase being the most widespread. A clear bias of hydrogenases to environments with terrestrial influence was found. This is exemplified by the cyanobacterial bidirectional NAD(P)-linked hydrogenase that was found in freshwater and coastal areas but not in the open ocean.

Significance

This study shows that hydrogenases are surprisingly abundant in marine environments. Due to its ecological distribution the primary function of the bidirectional NAD(P)-linked hydrogenase seems to be fermentative hydrogen evolution. Moreover, our data suggests that marine surface waters could be an interesting source of oxygen-resistant uptake hydrogenases. The respective genes occur in coastal as well as open ocean habitats and we presume that they are used as additional energy scavenging devices in otherwise nutrient limited environments. The membrane-bound H₂-evolving hydrogenases might be useful as marker for bacteria living inside of marine snow particles.     

Distribution and functions of TonB-dependent transporters in marine bacteria and environments: implications for dissolved organic matter utilization

Background

Bacteria play critical roles in marine nutrient cycles by incorporating and redistributing dissolved organic matter (DOM) and inorganic nutrients in the ocean. TonB-dependent transporter (TBDT) proteins allow Gram-negative bacteria to take up scarce resources from nutrient-limiting environments as well as siderophores, heme, vitamin B12, and recently identified carbohydrates. Thus, the characterization of TBDT distribution and functions is essential to better understand the contribution TBDT to DOM assimilation and its consequences on nutrient cycling in the environment.

Methodology/Principal Findings

This study presents the distribution of encoded known and putative TBDT proteins in the genomes of microorganisms and from the Global Ocean Survey data. Using a Lek clustering algorithm and substrate specificities, the TBDT sequences were mainly classified into the following three groups: (1) DOM transporters; (2) Siderophores/Vitamins transporters; and (3) Heme/Hemophores/Iron(heme)-binding protein transporters. Diverse TBDTs were found in the genomes of oligotroph Citromicrobium bathyomarinum JL354 and Citromicrobium sp JLT1363 and were highly expressed in the stationary phase of bacterial growth. The results show that the Gammaproteobacteria and the Cytophaga-Flavobacterium-Bacteroides (CFB) group bacteria accounted for the majority of the TBDT gene pool in marine surface waters.

Conclusions/Significance

The results of this study confirm the ecological importance of TBDTs in DOM assimilation for bacteria in marine environments owing to a wide range of substrate utilization potential in the ubiquitous Gammaproteobacteria and CFB group bacteria.     

Metagenomics reveals that detoxification systems are underrepresented in marine bacterial communities

Background

Environmental shotgun sequencing (metagenomics) provides a new way to study communities in microbial ecology. We here use sequence data from the Global Ocean Sampling (GOS) expedition to investigate toxicant selection pressures revealed by the presence of detoxification genes in marine bacteria. To capture a broad range of potential toxicants we selected detoxification protein families representing systems protecting microorganisms from a variety of stressors, such as metals, organic compounds, antibiotics and oxygen radicals.

Results

Using a bioinformatics procedure based on comparative analysis to finished bacterial genomes we found that the amount of detoxification genes present in marine microorganisms seems surprisingly small. The underrepresentation is particularly evident for toxicant transporters and proteins involved in detoxifying metals. Exceptions are enzymes involved in oxidative stress defense where peroxidase enzymes are more abundant in marine bacteria compared to bacteria in general. In contrast, catalases are almost completely absent from the open ocean environment, suggesting that peroxidases and peroxiredoxins constitute a core line of defense against reactive oxygen species (ROS) in the marine milieu.

Conclusions

We found no indication that detoxification systems would be generally more abundant close to the coast compared to the open ocean. On the contrary, for several of the protein families that displayed a significant geographical distribution, like peroxidase, penicillin binding transpeptidase and divalent ion transport protein, the open ocean samples showed the highest abundance. Along the same lines, the abundance of most detoxification proteins did not increase with estimated pollution. The low level of detoxification systems in marine bacteria indicate that the majority of marine bacteria have a low capacity to adapt to increased pollution. Our study exemplifies the use of metagenomics data in ecotoxicology, and in particular how anthropogenic consequences on life in the sea can be examined.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-749) contains supplementary material, which is available to authorized users.     

Depth-stratified functional and taxonomic niche specialization in the ‘core' and ‘flexible' Pacific Ocean Virome

Microbes drive myriad ecosystem processes, and their viruses modulate microbial-driven processes through mortality, horizontal gene transfer, and metabolic reprogramming by viral-encoded auxiliary metabolic genes (AMGs). However, our knowledge of viral roles in the oceans is primarily limited to surface waters. Here we assess the depth distribution of protein clusters (PCs) in the first large-scale quantitative viral metagenomic data set that spans much of the pelagic depth continuum (the Pacific Ocean Virome; POV). This established ‘core'' (180 PCs; one-third new to science) and ‘flexible'' (423K PCs) community gene sets, including niche-defining genes in the latter (385 and 170 PCs are exclusive and core to the photic and aphotic zones, respectively). Taxonomic annotation suggested that tailed phages are ubiquitous, but not abundant (<5% of PCs) and revealed depth-related taxonomic patterns. Functional annotation, coupled with extensive analyses to document non-viral DNA contamination, uncovered 32 new AMGs (9 core, 20 photic and 3 aphotic) that introduce ways in which viruses manipulate infected host metabolism, and parallel depth-stratified host adaptations (for example, photic zone genes for iron–sulphur cluster modulation for phage production, and aphotic zone genes for high-pressure deep-sea survival). Finally, significant vertical flux of photic zone viruses to the deep sea was detected, which is critical for interpreting depth-related patterns in nature. Beyond the ecological advances outlined here, this catalog of viral core, flexible and niche-defining genes provides a resource for future investigation into the organization, function and evolution of microbial molecular networks to mechanistically understand and model viral roles in the biosphere.     

Groups without Cultured Representatives Dominate Eukaryotic Picophytoplankton in the Oligotrophic South East Pacific Ocean

Background

Photosynthetic picoeukaryotes (PPE) with a cell size less than 3 µm play a critical role in oceanic primary production. In recent years, the composition of marine picoeukaryote communities has been intensively investigated by molecular approaches, but their photosynthetic fraction remains poorly characterized. This is largely because the classical approach that relies on constructing 18S rRNA gene clone libraries from filtered seawater samples using universal eukaryotic primers is heavily biased toward heterotrophs, especially alveolates and stramenopiles, despite the fact that autotrophic cells in general outnumber heterotrophic ones in the euphotic zone.

Methodology/Principal Findings

In order to better assess the composition of the eukaryotic picophytoplankton in the South East Pacific Ocean, encompassing the most oligotrophic oceanic regions on earth, we used a novel approach based on flow cytometry sorting followed by construction of 18S rRNA gene clone libraries. This strategy dramatically increased the recovery of sequences from putative autotrophic groups. The composition of the PPE community appeared highly variable both vertically down the water column and horizontally across the South East Pacific Ocean. In the central gyre, uncultivated lineages dominated: a recently discovered clade of Prasinophyceae (IX), clades of marine Chrysophyceae and Haptophyta, the latter division containing a potentially new class besides Prymnesiophyceae and Pavlophyceae. In contrast, on the edge of the gyre and in the coastal Chilean upwelling, groups with cultivated representatives (Prasinophyceae clade VII and Mamiellales) dominated.

Conclusions/Significance

Our data demonstrate that a very large fraction of the eukaryotic picophytoplankton still escapes cultivation. The use of flow cytometry sorting should prove very useful to better characterize specific plankton populations by molecular approaches such as gene cloning or metagenomics, and also to obtain into culture strains representative of these novel groups.     

Phylogeography of the Crown-of-Thorns Starfish in the Indian Ocean

Background

Understanding the limits and population dynamics of closely related sibling species in the marine realm is particularly relevant in organisms that require management. The crown-of-thorns starfish Acanthaster planci, recently shown to be a species complex of at least four closely related species, is a coral predator infamous for its outbreaks that have devastated reefs throughout much of its Indo-Pacific distribution.

Methodology/Principal Findings

In this first Indian Ocean-wide genetic study of a marine organism we investigated the genetic structure and inferred the paleohistory of the two Indian Ocean sister-species of Acanthaster planci using mitochondrial DNA sequence analyses. We suggest that the first of two main diversification events led to the formation of a Southern and Northern Indian Ocean sister-species in the late Pliocene-early Pleistocene. The second led to the formation of two internal clades within each species around the onset of the last interglacial. The subsequent demographic history of the two lineages strongly differed, the Southern Indian Ocean sister-species showing a signature of recent population expansion and hardly any regional structure, whereas the Northern Indian Ocean sister-species apparently maintained a constant size with highly differentiated regional groupings that were asymmetrically connected by gene flow.

Conclusions/Significance

Past and present surface circulation patterns in conjunction with ocean primary productivity were identified as the processes most likely to have shaped the genetic structure between and within the two Indian Ocean lineages. This knowledge will help to understand the biological or ecological differences of the two sibling species and therefore aid in developing strategies to manage population outbreaks of this coral predator in the Indian Ocean.     

Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins

Background

Generating autologous pluripotent stem cells for therapeutic applications will require the development of efficient DNA-free reprogramming techniques. Transfecting cells with in vitro-transcribed, protein-encoding RNA is a straightforward method of directly expressing high levels of reprogramming proteins without genetic modification. However, long-RNA transfection triggers a potent innate immune response characterized by growth inhibition and the production of inflammatory cytokines. As a result, repeated transfection with protein-encoding RNA causes cell death.

Methodology/Principal Findings

RNA viruses have evolved methods of disrupting innate immune signaling by destroying or inhibiting specific proteins to enable persistent infection. Starting from a list of known viral targets, we performed a combinatorial screen to identify siRNA cocktails that could desensitize cells to exogenous RNA. We show that combined knockdown of interferon-β (Ifnb1), Eif2ak2, and Stat2 rescues cells from the innate immune response triggered by frequent long-RNA transfection. Using this technique, we were able to transfect primary human fibroblasts every 24 hours with RNA encoding the reprogramming proteins Oct4, Sox2, Klf4, and Utf1. We provide evidence that the encoded protein is active, and we show that expression can be maintained for many days, through multiple rounds of cell division.

Conclusions/Significance

Our results demonstrate that suppressing innate immunity enables frequent transfection with protein-encoding RNA. This technique represents a versatile tool for investigating expression dynamics and protein interactions by enabling precise control over levels and timing of protein expression. Our finding also opens the door for the development of reprogramming and directed-differentiation methods based on long-RNA transfection.     

A phylometagenomic exploration of oceanic alphaproteobacteria reveals mitochondrial relatives unrelated to the SAR11 clade

Background

According to the endosymbiont hypothesis, the mitochondrial system for aerobic respiration was derived from an ancestral Alphaproteobacterium. Phylogenetic studies indicate that the mitochondrial ancestor is most closely related to the Rickettsiales. Recently, it was suggested that Candidatus Pelagibacter ubique, a member of the SAR11 clade that is highly abundant in the oceans, is a sister taxon to the mitochondrial-Rickettsiales clade. The availability of ocean metagenome data substantially increases the sampling of Alphaproteobacteria inhabiting the oxygen-containing waters of the oceans that likely resemble the originating environment of mitochondria.

Methodology/Principal Findings

We present a phylogenetic study of the origin of mitochondria that incorporates metagenome data from the Global Ocean Sampling (GOS) expedition. We identify mitochondrially related sequences in the GOS dataset that represent a rare group of Alphaproteobacteria, designated OMAC (Oceanic Mitochondria Affiliated Clade) as the closest free-living relatives to mitochondria in the oceans. In addition, our analyses reject the hypothesis that the mitochondrial system for aerobic respiration is affiliated with that of the SAR11 clade.

Conclusions/Significance

Our results allude to the existence of an alphaproteobacterial clade in the oxygen-rich surface waters of the oceans that represents the closest free-living relative to mitochondria identified thus far. In addition, our findings underscore the importance of expanding the taxonomic diversity in phylogenetic analyses beyond that represented by cultivated bacteria to study the origin of mitochondria.     

Influence of salinity on bacterioplankton communities from the Brazilian rain forest to the coastal Atlantic Ocean

Background

Planktonic bacteria are recognized as important drivers of biogeochemical processes in all aquatic ecosystems, however, the taxa that make up these communities are poorly known. The aim of this study was to investigate bacterial communities in aquatic ecosystems at Ilha Grande, Rio de Janeiro, Brazil, a preserved insular environment of the Atlantic rain forest and how they correlate with a salinity gradient going from terrestrial aquatic habitats to the coastal Atlantic Ocean.

Methodology/Principal Findings

We analyzed chemical and microbiological parameters of water samples and constructed 16S rRNA gene libraries of free living bacteria obtained at three marine (two coastal and one offshore) and three freshwater (water spring, river, and mangrove) environments. A total of 836 sequences were analyzed by MOTHUR, yielding 269 freshwater and 219 marine operational taxonomic units (OTUs) grouped at 97% stringency. Richness and diversity indexes indicated that freshwater environments were the most diverse, especially the water spring. The main bacterial group in freshwater environments was Betaproteobacteria (43.5%), whereas Cyanobacteria (30.5%), Alphaproteobacteria (25.5%), and Gammaproteobacteria (26.3%) dominated the marine ones. Venn diagram showed no overlap between marine and freshwater OTUs at 97% stringency. LIBSHUFF statistics and PCA analysis revealed marked differences between the freshwater and marine libraries suggesting the importance of salinity as a driver of community composition in this habitat. The phylogenetic analysis of marine and freshwater libraries showed that the differences in community composition are consistent.

Conclusions/Significance

Our data supports the notion that a divergent evolutionary scenario is driving community composition in the studied habitats. This work also improves the comprehension of microbial community dynamics in tropical waters and how they are structured in relation to physicochemical parameters. Furthermore, this paper reveals for the first time the pristine bacterioplankton communities in a tropical island at the South Atlantic Ocean.     

Tuna longline fishing around West and Central Pacific seamounts

Background

Seamounts have been identified as aggregating locations for pelagic biodiversity including tuna; however the topography and prevailing oceanography differ between seamounts and not all are important for tuna. Although a relatively common feature in oceanic ecosystems, little information is available that identifies those that are biologically important. Improved knowledge offers opportunities for unique management of these areas, which may advance the sustainable management of oceanic resources. In this study, we evaluate the existence of an association between seamounts and tuna longline fisheries at the ocean basin scale, identify significant seamounts for tuna in the western and central Pacific Ocean, and quantify the seamount contribution to the tuna longline catch.

Methodology/Principal Findings

We use data collected for the Western and Central Pacific Ocean for bigeye, yellowfin, and albacore tuna at the ocean basin scale. GLMs were applied to a coupled dataset of longline fisheries catch and effort, and seamount location information. The analyses show that seamounts may be associated with an annual longline combined catch of 35 thousand tonnes, with higher catch apparent for yellowfin, bigeye, and albacore tuna on 17%, 14%, and 14% of seamounts respectively. In contrast 14%, 18%, and 20% of seamounts had significantly lower catches for yellowfin, bigeye and albacore tuna respectively. Studying catch data in relation to seamount positions presents several challenges such as bias in location of seamounts, or lack of spatial resolution of fisheries data. Whilst we recognize these limitations the criteria used for detecting significant seamounts were conservative and the error in identification is likely to be low albeit unknown.

Conclusions/Significance

Seamounts throughout the study area were found to either enhance or reduce tuna catch. This indicates that management of seamounts is important Pacific-wide, but management approaches must take account of local conditions. Management of tuna and biodiversity resources in the region would benefit from considering such effects.     

Global patterns of bacterial beta-diversity in seafloor and seawater ecosystems

Background

Marine microbial communities have been essential contributors to global biomass, nutrient cycling, and biodiversity since the early history of Earth, but so far their community distribution patterns remain unknown in most marine ecosystems.

Methodology/Principal Findings

The synthesis of 9.6 million bacterial V6-rRNA amplicons for 509 samples that span the global ocean''s surface to the deep-sea floor shows that pelagic and benthic communities greatly differ, at all taxonomic levels, and share <10% bacterial types defined at 3% sequence similarity level. Surface and deep water, coastal and open ocean, and anoxic and oxic ecosystems host distinct communities that reflect productivity, land influences and other environmental constraints such as oxygen availability. The high variability of bacterial community composition specific to vent and coastal ecosystems reflects the heterogeneity and dynamic nature of these habitats. Both pelagic and benthic bacterial community distributions correlate with surface water productivity, reflecting the coupling between both realms by particle export. Also, differences in physical mixing may play a fundamental role in the distribution patterns of marine bacteria, as benthic communities showed a higher dissimilarity with increasing distance than pelagic communities.

Conclusions/Significance

This first synthesis of global bacterial distribution across different ecosystems of the World''s oceans shows remarkable horizontal and vertical large-scale patterns in bacterial communities. This opens interesting perspectives for the definition of biogeographical biomes for bacteria of ocean waters and the seabed.     

Potential virus-mediated nitrogen cycling in oxygen-depleted oceanic waters

Viruses play an important role in the ecology and biogeochemistry of marine ecosystems. Beyond mortality and gene transfer, viruses can reprogram microbial metabolism during infection by expressing auxiliary metabolic genes (AMGs) involved in photosynthesis, central carbon metabolism, and nutrient cycling. While previous studies have focused on AMG diversity in the sunlit and dark ocean, less is known about the role of viruses in shaping metabolic networks along redox gradients associated with marine oxygen minimum zones (OMZs). Here, we analyzed relatively quantitative viral metagenomic datasets that profiled the oxygen gradient across Eastern Tropical South Pacific (ETSP) OMZ waters, assessing whether OMZ viruses might impact nitrogen (N) cycling via AMGs. Identified viral genomes encoded six N-cycle AMGs associated with denitrification, nitrification, assimilatory nitrate reduction, and nitrite transport. The majority of these AMGs (80%) were identified in T4-like Myoviridae phages, predicted to infect Cyanobacteria and Proteobacteria, or in unclassified archaeal viruses predicted to infect Thaumarchaeota. Four AMGs were exclusive to anoxic waters and had distributions that paralleled homologous microbial genes. Together, these findings suggest viruses modulate N-cycling processes within the ETSP OMZ and may contribute to nitrogen loss throughout the global oceans thus providing a baseline for their inclusion in the ecosystem and geochemical models.Subject terms: Virus-host interactions, Biogeochemistry, Microbial biooceanography, Microbial ecology     

Prevalence of Papillomaviruses,Polyomaviruses, and Herpesviruses in Triple-Negative and Inflammatory Breast Tumors from Algeria Compared with Other Types of Breast Cancer Tumors

Background

The possible role of viruses in breast cancer etiology remains an unresolved question. We hypothesized that if some viruses are involved, it may be in a subgroup of breast cancers only. Epidemiological arguments drove our interest in breast cancer subgroups that are more frequent in Africa, namely inflammatory breast cancer (IBC) and triple-negative breast cancer. We tested whether viral prevalence was significantly higher in these subgroups.

Materials and Methods

One hundred fifty-five paraffin-embedded malignant breast tumors were randomly selected at the pathology laboratory of the University Hospital of Annaba (Algeria) to include one third of IBC and two thirds of non-IBC. They were tested for the presence of DNA from 61 viral agents (46 human papillomaviruses, 10 polyomaviruses, and 5 herpesviruses) using type-specific multiplex genotyping assays, which combine multiplex PCR and bead-based Luminex technology.

Results

Viral DNA was found in 22 (17.9%) of 123 tumors. The most prevalent viruses were EBV1 and HPV16. IBC tumors carried significantly more viruses (any type) than non-IBC tumors (30% vs. 13%, p<0.04). Similarly, triple-negative tumors displayed higher virus-positivity than non-triple-negative tumors (44% vs. 14%, p<0.009).

Conclusions

Our results suggest an association between the presence of viral DNA and aggressive breast cancer phenotypes (IBC, triple-negative). While preliminary, they underline the importance of focusing on subgroups when studying viral etiology in breast cancer. Further studies on viruses in breast cancer should be conducted in much larger samples to confirm these initial findings.     

Deciphering the heterogeneity in DNA methylation patterns during stem cell differentiation and reprogramming

Background

Human induced pluripotent stem cells (iPSCs) have a wide range of applications throughout the fields of basic research, disease modeling and drug screening. Epigenetic instable iPSCs with aberrant DNA methylation may divide and differentiate into cancer cells. Unfortunately, little effort has been taken to compare the epigenetic variation in iPSCs with that in differentiated cells. Here, we developed an analytical procedure to decipher the DNA methylation heterogeneity of mixed cells and further exploited it to quantitatively assess the DNA methylation variation in the methylomes of adipose-derived stem cells (ADS), mature adipocytes differentiated from ADS cells (ADS-adipose) and iPSCs reprogrammed from ADS cells (ADS-iPSCs).

Results

We observed that the degree of DNA methylation variation varies across distinct genomic regions with promoter and 5’UTR regions exhibiting low methylation variation and Satellite showing high methylation variation. Compared with differentiated cells, ADS-iPSCs possess globally decreased methylation variation, in particular in repetitive elements. Interestingly, DNA methylation variation decreases in promoter regions during differentiation but increases during reprogramming. Methylation variation in promoter regions is negatively correlated with gene expression. In addition, genes showing a bipolar methylation pattern, with both completely methylated and completely unmethylated reads, are related to the carbohydrate metabolic process, cellular development, cellular growth, proliferation, etc.

Conclusions

This study delivers a way to detect cell-subset specific methylation genes in a mixed cell population and provides a better understanding of methylation dynamics during stem cell differentiation and reprogramming.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-978) contains supplementary material, which is available to authorized users.     

Metabolic Flux Analysis of Mitochondrial Uncoupling in 3T3-L1 Adipocytes

Background

Increasing energy expenditure at the cellular level offers an attractive option to limit adiposity and improve whole body energy balance. In vivo and in vitro observations have correlated mitochondrial uncoupling protein-1 (UCP1) expression with reduced white adipose tissue triglyceride (TG) content. The metabolic basis for this correlation remains unclear.

Methodology/Principal Findings

This study tested the hypothesis that mitochondrial uncoupling requires the cell to compensate for the decreased oxidation phosphorylation efficiency by up-regulating lactate production, thus redirecting carbon flux away from TG synthesis. Metabolic flux analysis was used to characterize the effects of non-lethal, long-term mitochondrial uncoupling (up to 18 days) on the pathways of intermediary metabolism in differentiating 3T3-L1 adipocytes. Uncoupling was induced by forced expression of UCP1 and chemical (FCCP) treatment. Chemical uncoupling significantly decreased TG content by ca. 35%. A reduction in the ATP level suggested diminished oxidative phosphorylation efficiency in the uncoupled adipocytes. Flux analysis estimated significant up-regulation of glycolysis and down-regulation of fatty acid synthesis, with chemical uncoupling exerting quantitatively larger effects.

Conclusions/Significance

The results of this study support our hypothesis regarding uncoupling-induced redirection of carbon flux into glycolysis and lactate production, and suggest mitochondrial proton translocation as a potential target for controlling adipocyte lipid metabolism.     

Broad metabolic sensitivity profiling of a prototrophic yeast deletion collection

Background

Genome-wide sensitivity screens in yeast have been immensely popular following the construction of a collection of deletion mutants of non-essential genes. However, the auxotrophic markers in this collection preclude experiments on minimal growth medium, one of the most informative metabolic environments. Here we present quantitative growth analysis for mutants in all 4,772 non-essential genes from our prototrophic deletion collection across a large set of metabolic conditions.

Results

The complete collection was grown in environments consisting of one of four possible carbon sources paired with one of seven nitrogen sources, for a total of 28 different well-defined metabolic environments. The relative contributions to mutants'' fitness of each carbon and nitrogen source were determined using multivariate statistical methods. The mutant profiling recovered known and novel genes specific to the processing of nutrients and accurately predicted functional relationships, especially for metabolic functions. A benchmark of genome-scale metabolic network modeling is also given to demonstrate the level of agreement between current in silico predictions and hitherto unavailable experimental data.

Conclusions

These data address a fundamental deficiency in our understanding of the model eukaryote Saccharomyces cerevisiae and its response to the most basic of environments. While choice of carbon source has the greatest impact on cell growth, specific effects due to nitrogen source and interactions between the nutrients are frequent. We demonstrate utility in characterizing genes of unknown function and illustrate how these data can be integrated with other whole-genome screens to interpret similarities between seemingly diverse perturbation types.