Comparative metagenomics of bathypelagic plankton and bottom sediment from the Sea of Marmara

To extend comparative metagenomic analyses of the deep-sea, we produced metagenomic data by direct 454 pyrosequencing from bathypelagic plankton (1000 m depth) and bottom sediment of the Sea of Marmara, the gateway between the Eastern Mediterranean and the Black Seas. Data from small subunit ribosomal RNA (SSU rRNA) gene libraries and direct pyrosequencing of the same samples indicated that Gamma- and Alpha-proteobacteria, followed by Bacteroidetes, dominated the bacterial fraction in Marmara deep-sea plankton, whereas Planctomycetes, Delta- and Gamma-proteobacteria were the most abundant groups in high bacterial-diversity sediment. Group I Crenarchaeota/Thaumarchaeota dominated the archaeal plankton fraction, although group II and III Euryarchaeota were also present. Eukaryotes were highly diverse in SSU rRNA gene libraries, with group I (Duboscquellida) and II (Syndiniales) alveolates and Radiozoa dominating plankton, and Opisthokonta and Alveolates, sediment. However, eukaryotic sequences were scarce in pyrosequence data. Archaeal amo genes were abundant in plankton, suggesting that Marmara planktonic Thaumarchaeota are ammonia oxidizers. Genes involved in sulfate reduction, carbon monoxide oxidation, anammox and sulfatases were over-represented in sediment. Genome recruitment analyses showed that Alteromonas macleodii ‘surface ecotype'', Pelagibacter ubique and Nitrosopumilus maritimus were highly represented in 1000 m-deep plankton. A comparative analysis of Marmara metagenomes with ALOHA deep-sea and surface plankton, whale carcasses, Peru subsurface sediment and soil metagenomes clustered deep-sea Marmara plankton with deep-ALOHA plankton and whale carcasses, likely because of the suboxic conditions in the deep Marmara water column. The Marmara sediment clustered with the soil metagenome, highlighting the common ecological role of both types of microbial communities in the degradation of organic matter and the completion of biogeochemical cycles.     

Ultra-diffuse hydrothermal venting supports Fe-oxidizing bacteria and massive umber deposition at 5000?m off Hawaii

A novel hydrothermal field has been discovered at the base of Lōihi Seamount, Hawaii, at 5000 mbsl. Geochemical analyses demonstrate that ‘FeMO Deep'', while only 0.2 °C above ambient seawater temperature, derives from a distal, ultra-diffuse hydrothermal source. FeMO Deep is expressed as regional seafloor seepage of gelatinous iron- and silica-rich deposits, pooling between and over basalt pillows, in places over a meter thick. The system is capped by mm to cm thick hydrothermally derived iron-oxyhydroxide- and manganese-oxide-layered crusts. We use molecular analyses (16S rDNA-based) of extant communities combined with fluorescent in situ hybridizations to demonstrate that FeMO Deep deposits contain living iron-oxidizing Zetaproteobacteria related to the recently isolated strain Mariprofundus ferroxydans. Bioenergetic calculations, based on in-situ electrochemical measurements and cell counts, indicate that reactions between iron and oxygen are important in supporting chemosynthesis in the mats, which we infer forms a trophic base of the mat ecosystem. We suggest that the biogenic FeMO Deep hydrothermal deposit represents a modern analog for one class of geological iron deposits known as ‘umbers'' (for example, Troodos ophilolites, Cyprus) because of striking similarities in size, setting and internal structures.     

Usefulness of alkaline hydrogen peroxide oxidation to analyze eumelanin and pheomelanin in various tissue samples: application to chemical analysis of human hair melanins

Eumelanin and pheomelanin in tissue samples can be specifically measured as the markers pyrrole-2,3,5-tricarboxylic acid (PTCA) and 4-amino-3-hydroxyphenylalanine after acidic permanganate oxidation and hydroiodic acid hydrolysis, respectively. Those degradation methods, although widely applied, are not easily performed in most laboratories. To overcome this difficulty, we developed alkaline H(2)O(2) oxidation in 1 M K(2)CO(3) that produces, in addition to the eumelanin marker PTCA, thiazole-2,4,5-tricarboxylic acid (TTCA) and thiazole-4,5-dicarboxylic acid (TDCA) as markers for pheomelanin and pyrrole-2,3-dicarboxylic acid (PDCA) as a marker for 5,6-dihydroxyindole-derived eumelanin. Those four degradation products can be easily separated by HPLC and analyzed with ultraviolet detection. The alkaline H(2)O(2) oxidation method is simple, reproducible and applicable to all pigmented tissues. Its application to characterize eumelanin and pheomelanin in human hair shows that PTCA and TTCA serve as specific markers for eumelanin and pheomelanin, respectively, although some caution is needed regarding the artificial production of TTCA from eumelanic tissue proteins.     

Assessing protein oxidation by inorganic nanoparticles with enzyme‐linked immunosorbent assay (ELISA)

Growth in the nanotechnology industry is leading to increased production of engineered nanoparticles (NPs). This has given rise to concerns about the potential adverse and toxic effects to biological system and the environment. An important mechanism of NP toxicity is oxidative stress caused by the formation of reactive oxygen species (ROS) or via direct oxidation of biomolecules. In this study, a protein oxidation assay was developed as an indicator of biomolecule oxidation by NPs. The oxidation of the protein, bovine serum albumin (BSA) was evaluated with an enzyme‐linked immunosorbent assay (ELISA) to measure the protein carbonyl derivatives formed from protein oxidation. The results showed that some NPs such as Cu(0), CuO, Mn₂O₃, and Fe(0) caused oxidation of BSA; whereas, many of the other NPs tested were not reactive or very slowly reactive with BSA. The mechanisms involved in the oxidation of BSA protein by the reactive NPs could be attributed to the combined effects of ROS‐dependent and direct protein oxidation mechanisms. The ELISA assay is a promising method for the assessment of protein oxidation by NPs, which can provide insights on NP toxicity mechanisms. Biotechnol. Bioeng. 2013; 110: 694–701. © 2012 Wiley Periodicals, Inc.     

Thioesterase Superfamily Member 2/Acyl-CoA Thioesterase 13 (Them2/Acot13) Regulates Adaptive Thermogenesis in Mice

Members of the acyl-CoA thioesterase (Acot) gene family hydrolyze fatty acyl-CoAs, but their biological functions remain incompletely understood. Thioesterase superfamily member 2 (Them2; synonym Acot13) is enriched in oxidative tissues, associated with mitochondria, and relatively specific for long chain fatty acyl-CoA substrates. Using Them2^−/− mice, we have demonstrated key roles for Them2 in regulating hepatic glucose and lipid metabolism. However, reduced body weights and decreased adiposity in Them2^−/− mice observed despite increased food consumption were not well explained. To explore a role in thermogenesis, mice were exposed to ambient temperatures ranging from thermoneutrality (30 °C) to cold (4 °C). In response to short term (24-h) exposures to decreasing ambient temperatures, Them2^−/− mice exhibited increased adaptive responses in physical activity, food consumption, and energy expenditure when compared with Them2^+/+ mice. By contrast, genotype-dependent differences were not observed in mice that were equilibrated (96 h) at each ambient temperature. In brown adipose tissue, the absence of Them2 was associated with reduced lipid droplets, alterations in the ultrastructure of mitochondria, and increased expression of thermogenic genes. Indicative of a direct regulatory role for Them2 in heat production, cultured primary brown adipocytes from Them2^−/− mice exhibited increased norepinephrine-mediated triglyceride hydrolysis and increased rates of O₂ consumption, together with elevated expression of thermogenic genes. At least in part by regulating intracellular fatty acid channeling, Them2 functions in brown adipose tissue to suppress adaptive increases in energy expenditure.     

Human Indoleamine 2,3-Dioxygenase Is a Catalyst of Physiological Heme Peroxidase Reactions: IMPLICATIONS FOR THE INHIBITION OF DIOXYGENASE ACTIVITY BY HYDROGEN PEROXIDE*

The heme enzyme indoleamine 2,3-dioxygenase (IDO) is a key regulator of immune responses through catalyzing l-tryptophan (l-Trp) oxidation. Here, we show that hydrogen peroxide (H₂O₂) activates the peroxidase function of IDO to induce protein oxidation and inhibit dioxygenase activity. Exposure of IDO-expressing cells or recombinant human IDO (rIDO) to H₂O₂ inhibited dioxygenase activity in a manner abrogated by l-Trp. Dioxygenase inhibition correlated with IDO-catalyzed H₂O₂ consumption, compound I-mediated formation of protein-centered radicals, altered protein secondary structure, and opening of the distal heme pocket to promote nonproductive substrate binding; these changes were inhibited by l-Trp, the heme ligand cyanide, or free radical scavengers. Protection by l-Trp coincided with its oxidation into oxindolylalanine and kynurenine and the formation of a compound II-type ferryl-oxo heme. Physiological peroxidase substrates, ascorbate or tyrosine, enhanced rIDO-mediated H₂O₂ consumption and attenuated H₂O₂-induced protein oxidation and dioxygenase inhibition. In the presence of H₂O₂, rIDO catalytically consumed nitric oxide (NO) and utilized nitrite to promote 3-nitrotyrosine formation on IDO. The promotion of H₂O₂ consumption by peroxidase substrates, NO consumption, and IDO nitration was inhibited by l-Trp. This study identifies IDO as a heme peroxidase that, in the absence of substrates, self-inactivates dioxygenase activity via compound I-initiated protein oxidation. l-Trp protects against dioxygenase inactivation by reacting with compound I and retarding compound II reduction to suppress peroxidase turnover. Peroxidase-mediated dioxygenase inactivation, NO consumption, or protein nitration may modulate the biological actions of IDO expressed in inflammatory tissues where the levels of H₂O₂ and NO are elevated and l-Trp is low.     

Assays of plasma dehydrocholesteryl esters and oxysterols from Smith-Lemli-Opitz syndrome patients

Smith-Lemli-Opitz syndrome (SLOS) is caused by mutations in the gene encoding 3β-hydroxysterol-Δ⁷-reductase and as a result of this defect, 7-dehydrocholesterol (7-DHC) and 8-dehydrocholesterol (8-DHC) accumulate in the fluids and tissues of patients with this syndrome. Both 7- and 8-DHC are susceptible to peroxidation reactions, and several biologically active DHC oxysterols are found in cell and animal models of SLOS. Ex vivo oxidation of DHCs can be a confounding factor in the analysis of these sterols and their esters, and we developed HPLC/MS methods that permit the direct analysis of cholesterol, 7-DHC, 8-DHC, and their esters in human plasma, thus avoiding ex vivo oxidation. In addition, three oxysterols were classified as endogenously formed products by the use of an isotopically-labeled 7-DHC (d₇-7-DHC) added to the sample before workup, followed by MS analysis of products formed. Analysis of 17 SLOS plasma samples shows that 8-DHC linoleate correlates better with the SLOS severity score of the patients than other sterols or metabolites, including cholesterol and 7-DHC. Levels of 7-ketocholesterol also correlate with the SLOS severity score. 8-DHC esters should have utility as surrogate markers of severity in SLOS for prognostication and as endpoints in clinical trials.     

Regulation of lipid droplet size and phospholipid composition by stearoyl-CoA desaturase

Fatty acid desaturation regulates membrane function and fat storage in animals. To determine the contribution of stearoyl-CoA desaturase (SCD) activity on fat storage and development in the nematode Caenorhabditis elegans, we analyzed the lipid composition and lipid droplet size in the fat-6;fat-7 desaturase mutants independently and in combination with mutants disrupted in conserved lipid metabolic pathways. C. elegans with impaired SCD activity displayed both reduced fat stores and decreased lipid droplet size. Mutants in the daf-2 (insulin-like growth factor receptor), rsks-1 (homolog of p70S6kinase, an effector of the target of rapamycin signaling pathway), and daf-7 (transforming growth factor β) displayed high fat stores, the opposite of the low fat observed in the fat-6;fat-7 desaturase mutants. The metabolic mutants in combination with fat-6;fat-7 displayed low fat stores, with the exception of the daf-2;fat-6;fat-7 triple mutants, which had increased de novo fatty acid synthesis and wild-type levels of fat stores. Notably, SCD activity is required for the formation of large-sized lipid droplets in all mutant backgrounds, as well as for normal ratios of phosphatidylcholine (PC) to phosphatidylethanolamine (PE). These studies reveal previously uncharacterized roles for SCD in the regulation of lipid droplet size and membrane phospholipid composition.     

Geochemical Controls on Microbial Nitrate-Dependent U(IV) Oxidation

After reductive immobilization of uranium, the element may be oxidized and remobilized in the presence of nitrate by the activity of dissimilatory nitrate-reducing bacteria. We examined controls on microbially mediated nitrate-dependent U(IV) oxidation in landfill leachate-impacted subsurface sediments. Nitrate-dependent U(IV)-oxidizing bacteria were at least two orders of magnitude less numerous in these sediments than glucose- or Fe(II)-oxidizing nitrate-reducing bacteria and grew more slowly than the latter organisms, suggesting that U(IV) is ultimately oxidized by Fe(III) produced by nitrate-dependent Fe(II)-oxidizing bacteria or by oxidation of Fe(II) by nitrite that accumulates during organotrophic dissimilatory nitrate reduction. We examined the effect of nitrate and reductant concentration on nitrate-dependent U(IV) oxidation in sediment incubations and used the initial reductive capacity (RDC = [reducing equivalents] - [oxidizing equivalents]) of the incubations as a unified measurement of the nitrate or reductant concentration. When we lowered the RDC with progressively higher nitrate concentrations, we observed a corresponding increase in the extent of U(IV) oxidation, but did not observe this relationship between RDC and U(IV) oxidation rate, especially when RDC > 0, suggesting that nitrate concentration strongly controls the extent, but not the rate of nitrate-dependent U(IV) oxidation. On the other hand, when we raised the RDC in sediment incubations with progressively higher reductant (acetate, sulfide, soluble Fe(II), or FeS) concentrations, we observed progressively lower extents and rates of nitrate-dependent U(IV) oxidation. Acetate was a relatively poor inhibitor of nitrate-dependent U(IV) oxidation, while Fe(II) was the most effective inhibitor. Based on these results, we propose that it may be possible to predict the stability of U(IV) in a bioremediated aquifer based on the geochemical characteristics of that aquifer.     

Molecular Detection of Novel Anammox Bacterial Clusters in the Sediments of the Shallow Freshwater Lake Taihu

Previous studies have demonstrated the wide occurrence of anaerobic ammonium oxidizing (anammox) bacteria; however, there is very limited information on the distribution of these bacteria in freshwater habitats. In this study, the anammox bacterial communities were detected by molecular analysis targeting the 16S rRNA genes in the sediments of Lake Taihu, a large and shallow eutrophic freshwater lake in China. The recovery of specific 16S rRNA sequences with two stable monophyletic clusters indicated that anammox bacteria were present in Lake Taihu. A phylogenetic analysis indicated that these two groups represent two novel lineages within the first subgroup of anammox bacteria, independent of the treeing methods. High intra-lake variability in anammox bacterial diversity and community composition was observed, in particular, based on a 1% cut-off of 16S rRNA sequence variation. The spatial variability was largely related to the substrate availability, which was denoted by the correlations between the relative abundance of the two Taihu anammox bacterial groups and the concentrations of ammonium and nitrite. This indicates that the niche differentiation of anammox bacteria is linked to the environmental heterogeneity. These findings suggest that the freshwater lakes may accommodate different anammox bacterial communities and, thus, expand our knowledge on the diversity and distribution of anammox bacteria. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental files.