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Typically, healthy cardiac tissue utilizes more fat than any other organ. Cardiac hypertrophy induces a metabolic shift leading to a preferential consumption of glucose over fatty acids to support the high energetic demand. Calorie restriction is a dietary procedure that induces health benefits and lifespan extension in many organisms. Given the beneficial effects of calorie restriction, we hypothesized that calorie restriction prevents cardiac hypertrophy, lipid content changes, mitochondrial and redox dysregulation. Strikingly, calorie restriction reversed isoproterenol-induced cardiac hypertrophy. Isolated mitochondria from hypertrophic hearts produced significantly higher levels of succinate-driven H

Cardiolipin is the signature phospholipid of the mitochondrial inner membrane. It participates in shaping the inner membrane as well as in modulating the activity of many membrane-bound proteins. The acyl chain composition of cardiolipin is finely tuned post-biosynthesis depending on the surrounding phospholipids to produce mature or unsaturated cardiolipin. However, experimental evidence showing that immature and mature cardiolipin are functionally equivalents for mitochondria poses doubts on the relevance of cardiolipin remodeling. In this work, we studied the role of cardiolipin acyl chain composition in mitochondrial bioenergetics, including a detailed bioenergetic profile of yeast mitochondria. Cardiolipin acyl chains were modified by genetic and nutritional manipulation. We found that both the bioenergetic efficiency and osmotic stability of mitochondria are dependent on the unsaturation level of cardiolipin acyl chains. It is proposed that cardiolipin remodeling and, consequently, mature cardiolipins play an important role in mitochondrial inner membrane integrity and functionality.

The Carnitine Palmitoyltranferase I (CPT1) catalyzes the rate-limiting step of long-chain fatty acid (LCFA) mitochondrial β-oxidation. The enzyme promotes the conjugation of LCFA with l-carnitine, which allows LCFA to enter the mitochondria matrix. The structural features involved in CPT1 and LCFA-CoA interactions have not been fully elucidated, mainly due to the absence of CPT1 crystallographic data. Previous studies reported important residues (Lys556, Lys560, and Lys561) crucial to the CPT1 mechanism. Nonetheless, these studies have not explored the LCFA bindings. Using molecular modeling strategies, we aimed to understand the conformational changes in CPT1 structure induced by LCFA-CoA. For this purpose, a tridimensional CPT1A model was built by homology modeling using CRAT protein (PBD:1t7q, resolution 1.8 Å) as a template. We simulated the CPT1 structure in the presence and absence of LCFA-CoA by molecular dynamics (MD). By applying a principal component analysis (PCA), two states of apostructure CPT1 based on CoA-Loop (688-711) were observed. In contrast, just one state was evidenced along with smaller conformational subspaces in ligand-complexed simulations using LCFA-CoA. The CoA moiety of ligands interacts with charged residues, namely Lys560, Lys556, Arg563, and Arg645. The frequency of interactions observed for each of these residues is60% of simulation time, suggesting a dynamic profile of interactions in synergy with long-chain carbon interactions over α-I (478-492). Collectively, these features may be associated with the catalytic conformation of LCFA-CoA to CPT1a. Further calculations of free-energy for different fatty acids, such as alpha-linolenic (ALA), gamma-linolenic (GLA), and arachidonic (ARA) acids, yielded energy values ranging from -76.9 ± 15.9 to -68.5 ± 10.0 kcal mol

Radical mediated oxidation of polyunsaturated fatty acids (PUFA) is known to generate a series of polyoxygenated cyclic products (PUFA-On, n ≥ 3). Here, we describe the characterization of glutathione (GSH) conjugates bound to polyoxygenated docosahexaenoic (DHA-On, n = 3-9), arachidonic (ARA-On, n = 3-7), α-linolenic (ALA-O3), and linoleic (LA-O3) acid species. Similar conjugates were also characterized for N-acetylcysteine (NAC) and Cu,Zn-superoxide dismutase (SOD1). Extensive LC-MS/MS characterization using a synthetic α-linolenic hydroxy-endoperoxide (ALA-O3) derivative revealed at least two types of mechanisms leading to thiol adduction: a mechanism involving the nucleophilic attack by thiolate anion on 1,2-dioxolane to form a sulfenate ester-bonded conjugate and a mechanism involving cleavage of the dioxolane to form a α,β-unsaturated carbonyl followed by the Michael addition reaction. Finally, we detected a GSH conjugate with hydroxy-endoperoxide derived from linoleic acid (LA-O3) in mice liver. In summary, our study reveals the formation of a series of thiol conjugates that are bound to highly oxygenated PUFA species. GSH conjugates described in our study may potentially play relevant roles in redox and inflammatory processes, especially under high oxygen tension conditions.

Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of upper and lower motor neurons leading to muscle paralysis and death. While a link between dysregulated lipid metabolism and ALS has been proposed, lipidome alterations involved in disease progression are still understudied. Using a rodent model of ALS overexpressing mutant human Cu/Zn-superoxide dismutase gene (SOD1-G93A), we performed a comparative lipidomic analysis in motor cortex and spinal cord tissues of SOD1-G93A and WT rats at asymptomatic (~70 days) and symptomatic stages (~120 days). Interestingly, lipidome alterations in motor cortex were mostly related to age than ALS. In contrast, drastic changes were observed in spinal cord of SOD1-G93A 120d group, including decreased levels of cardiolipin and a 6-fold increase in several cholesteryl esters linked to polyunsaturated fatty acids. Consistent with previous studies, our findings suggest abnormal mitochondria in motor neurons and lipid droplets accumulation in aberrant astrocytes. Although the mechanism leading to cholesteryl esters accumulation remains to be established, we postulate a hypothetical model based on neuroprotection of polyunsaturated fatty acids into lipid droplets in response to increased oxidative stress. Implicated in the pathology of other neurodegenerative diseases, cholesteryl esters appear as attractive targets for further investigations.

Cardiac hypertrophy induces a metabolic shift, leading to a preferential consumption of glucose (over fatty acids) to support the high energetic demand. Typically, health cardiac tissue utilizes more fat than any other organ. Calorie restriction is a dietary procedure that induces health benefits and lifespan extension in many organisms. Given the beneficial effects of calorie restriction and the metabolic dysregulation seen during cardiac hypertrophy, we hypothesized that calorie restriction prevents cardiac hypertrophy, lipid, mitochondrial, and redox dysregulations. Strikingly, calorie restriction reversed isoproterenol-induced cardiac hypertrophy, lowered succinate driven mitochondrial H2O2 production, improved mitochondrial function (indicated as a higher Respiratory Control Ratio – RCR) and avoided mitochondrial superoxide dismutase (MnSOD) and glutathione peroxidase (GPX) repression. To gain insight into how calorie restriction could interfere with the metabolic changes induced by cardiac hypertrophy, we performed lipidomic profiling. Calorie restriction protected against the consumption of several triglycerides (TG) linked to unsaturated fatty acids, and the accumulation of TGs containing saturated fatty acids observed in hypertrophic samples. Cardiac hypertrophy induced an increase in ceramides, phosphoethanolamines and acylcarnitines (12:0, 14:0, 16:0 and 18:0) that were also reversed by calorie restriction. Altogether, our data demonstrate that hypertrophy changes the cardiac lipidome, causes mitochondrial disturbances and oxidative stress. All these changes are prevented by calorie restriction intervention in vivo. This study uncovers calorie restriction as a resource protect cardiac tissue and prevent cardiac hypertrophy-induced lipidomic remodeling.

The causative agent of citrus canker disease, Xanthomonas citri pv. citri, was found to produce copious amounts of outer membrane vesicles (OMVs), frequently forming long membranous tubes under different culture conditions. Lipidomic analysis revealed significant differences in lipid composition between purified vesicles in relation to whole cells. The results suggest an enrichment in saturated cardiolipins and a decrease in unsaturated lipids in the OMV samples, possibly granting them a more rigid structure while allowing their high degree of curvature caused by their small diameters. The vesicles’ proteome was found to be significantly enriched in TonB-dependent receptors related to the acquisition of different nutrients. These proteins are known to transport siderophores, which were evidenced to be present in purified X. citri OMVs, along with essential metals including iron, zinc, and manganese quantified by elemental analysis. The availability of vesicle-associated nutrients to be incorporated by cells was demonstrated by the use of OMVs as the sole carbon source for bacterial growth. At last, the vesicles also presented esterase and protease activities, which have been associated with virulence in phytopathogens. These evidences point that X. citri cells can use OMVs to share resources within microbial communities, which has potential implications for microbial interactions and plant colonization, affecting their survival and persistence on the host and in the environment.ImportanceThe shedding of outer membrane vesicles appears to be universal in Gram-negative bacteria and effectively constitutes a unique secretion pathway for diverse molecules and proteins. To study their possible functions in the citrus pathogen Xanthomonas citri, purified vesicles from this bacterium were studied by omics and functional approaches. Nutrient transporters were found associated to these structures, which were evidenced to contain siderophores and essential metals. The availability of these nutrients to be incorporated by cells was then demonstrated by showing that purified vesicles can be used as sole carbon sources for microbial growth. Additionally, the samples also presented esterase and protease activities which can contribute to the release of substrates from plant host tissues. These observations help to establish the developing idea of vesicles as shared bacterial resources which can participate in shaping host-associated microbial communities in contrast to other interactions such as bacterial competition.

Dietary sugar is an important determinant of the development and progression of nonalcoholic fatty liver disease (NAFLD). However, the molecular mechanisms underlying the deleterious effects of sugar intake on NAFLD under energy-balanced conditions are still poorly understood. Here, we provide a comprehensive analysis of the liver lipidome and mechanistic insights into the pathogenesis of NAFLD induced by the chronic consumption of high-sugar diet (HSD). Newly weaned male Wistar rats were fed either a standard chow diet or an isocaloric HSD for 18 weeks. Livers were harvested for histological, oxidative stress, gene expression, and lipidomic analyses. Intake of HSD increased oxidative stress and induced severe liver injury, microvesicular steatosis, and ballooning degeneration of hepatocytes. Using untargeted lipidomics, we identified and quantified 362 lipid species in the liver. Rats fed with HSD displayed increased hepatic levels of triacylglycerol enriched in saturated and monounsaturated fatty acids, lipids related to mitochondrial function/structure (phosphatidylglycerol, cardiolipin, and ubiquinone), and acylcarnitine (an intermediate lipid of fatty acid beta-oxidation). HSD-fed animals also presented increased levels of some species of membrane lipids and a decreased content of phospholipids containing omega-6 fatty acids. These changes in the lipidome were associated with the downregulation of genes involved in fatty acid oxidation in the liver. In conclusion, our data suggest that the chronic intake of a HSD, even under isocaloric conditions, induces lipid overload, and inefficient/impaired fatty acid oxidation in the liver. Such events lead to marked disturbance in hepatic lipid metabolism and the development of NAFLD.

Two commercial exogenous pulmonary surfactants, Curosurf and Survanta, are investigated. Their thermotropic behavior and associated structural changes for the samples in bulk are characterized and described. For Survanta, the obtained results of differential scanning calorimetry showed a thermogram with three peaks on heating and only a single peak on cooling. Curosurf on the other hand, presents calorimetric thermograms with only one peak in both the heating and cooling scans. This distinct thermotropic behavior between the two pulmonary surfactants, a consequence of their particular compositions, is associated with structural changes that were evaluated by simultaneous small- and wide-angle X-ray scattering experiments with in situ temperature variation. Interestingly, for temperatures below ∼35 °C for Curosurf and ∼53 °C for Survanta, the scattering data indicated the coexistence of two lamellar phases with different carbon chain organizations. For temperatures above these limits, the coexistence of phases disappears, giving rise to a fluid phase in both pulmonary surfactants, with multilamelar vesicles for Curosurf and unilamellar vesicles for Survanta. This process is quasi-reversible under cooling, and advanced data analysis for the scattering data indicated differences in the structural and elastic properties of the pulmonary surfactants. The detailed and systematic investigation shown in this work expands on the knowledge of the structure and thermodynamic behavior of Curosurf and Survanta, being relevant from both physiological and biophysical perspectives and also providing a basis for further studies on other types of pulmonary surfactants.

The oxidation of polyunsaturated fatty acids (PUFA) is a common stress response across biomes with potential to trigger impairment of cell growth and reproduction. The oxidative stress theory of coral bleaching induced by global warming has been widely accepted to explain coral reef decline, but its underlying physiological mechanism remains under debate. Here we used lipidomic and population density data to examine cell cultures of three coral reef symbionts after a heat shock (sudden rise of 12 °C for 4 hours). Heat tolerance in S. microadriaticum and C. goreaui was characterized by preservation of thylakoid-derived glycolipids. Conversely, heat sensitivity in B. minutum was linked to elevated concentrations of oxidized PUFA esterified to glycolipids, suggesting that culture growth had ceased due to severe oxidative damage. Our findings provide a basis to further understand the role played by oxidative stress in coral bleaching and reveal novel biomarkers for the monitoring of symbiont-coral health.

Mechanistic target of rapamycin complex (mTORC)1 activity is increased in adipose tissue of obese insulin-resistant mice, but its role in the regulation of tissue inflammation is unknown. Herein, we investigated the effects of adipocyte mTORC1 deficiency on adipose tissue inflammation and glucose homeostasis. For this, mice with adipocyte raptor deletion and controls fed a chow or a high-fat diet were evaluated for body mass, adiposity, glucose homeostasis, and adipose tissue inflammation. Despite reducing adiposity, adipocyte mTORC1 deficiency promoted hepatic steatosis, insulin resistance, and adipose tissue inflammation (increased infiltration of macrophages, neutrophils, and B lymphocytes; crown-like structure density; TNF-α, interleukin (IL)-6, and monocyte chemoattractant protein 1 expression; IL-1β protein content; lipid peroxidation; and de novo ceramide synthesis). The anti-oxidant, N-acetylcysteine, partially attenuated, whereas treatment with de novo ceramide synthesis inhibitor, myriocin, completely blocked adipose tissue inflammation and nucleotide oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3)-inflammasome activation, but not hepatic steatosis and insulin resistance induced by adipocyte raptor deletion. Rosiglitazone treatment, however, completely abrogated insulin resistance induced by adipocyte raptor deletion. In conclusion, adipocyte mTORC1 deficiency induces adipose tissue inflammation and NLRP3-inflammasome activation by promoting oxidative stress and de novo ceramide synthesis. Such adipose tissue inflammation, however, is not an underlying cause of the insulin resistance displayed by these mice.

Palmitoleic acid (POA, 16:1n-7) is a lipokine that has potential nutraceutical use to treat non-alcoholic fatty liver disease. We tested the effects of POA supplementation (daily oral gavage, 300 mg/Kg, 15 days) on murine liver inflammation induced by a high fat diet (HFD, 59% fat, 12 weeks). In HFD-fed mice, POA supplementation reduced serum insulin and improved insulin tolerance compared with oleic acid (OA, 300 mg/Kg). The livers of POA-treated mice exhibited less steatosis and inflammation than those of OA-treated mice with lower inflammatory cytokine levels and reduced toll-like receptor 4 protein content. The anti-inflammatory effects of POA in the liver were accompanied by a reduction in liver macrophages (LM, CD11c+; F4/80+; CD86+), an effect that could be triggered by peroxisome proliferator activated receptor (PPAR)-γ, a lipogenic transcription factor upregulated in livers of POA-treated mice. We also used HFD-fed mice with selective deletion of PPAR-γ in myeloid cells (PPAR-γ KOLyzCre+) to test whether the beneficial anti-inflammatory effects of POA are dependent on macrophages PPAR-γ. POA-mediated improvement of insulin tolerance was tightly dependent on myeloid PPAR-γ, while POA anti-inflammatory actions including the reduction in liver inflammatory cytokines were preserved in mice bearing myeloid cells deficient in PPAR-γ. This overlapped with increased CD206+ (M2a) cells and downregulation of CD86+ and CD11c+ liver macrophages. Moreover, POA supplementation increased hepatic AMPK activity and decreased expression of the fatty acid binding scavenger receptor, CD36. We conclude that POA controls liver inflammation triggered by fat accumulation through induction of M2a macrophages independently of myeloid cell PPAR-γ., Graphical abstract Unlabelled Image, Highlights • Palmitoleic acid (POA) supplementation reduced serum insulin and improved insulin tolerance; • Livers of POA-treated mice exhibited less steatosis and inflammation; • POA lowered the liver M1 macrophages population and the expression of inflammation-related immune-cell markers; • POA increased PPAR-γ, a transcription factor that regulates anti-inflammatory effects in macrophages; • However, POA reduced liver inflammation even in mice that lack PPAR-γ expression in myeloid cells; • POA controls liver inflammation through induction of M2a macrophages independently of PPAR-γ in myeloid cells.

Lack of effective treatments for aggressive breast cancer is still a major global health problem. We have previously reported that photodynamic therapy using methylene blue as photosensitizer (MB-PDT) massively kills metastatic human breast cancer, marginally affecting healthy cells. In this study, we aimed to unveil the molecular mechanisms behind MB-PDT effectiveness and specificity towards tumor cells. Through lipidomics and biochemical approaches, we demonstrated that MB-PDT efficiency and specificity rely on polyunsaturated fatty acid-enriched membranes and on the better capacity to deal with photo-oxidative damage displayed by non-tumorigenic cells. We found out that, in tumorigenic cells, lysosome membrane permeabilization is accompanied by ferroptosis and/or necroptosis. Our results also pointed at a cross-talk between lysosome-dependent cell death (LDCD) and necroptosis induction after photo-oxidation, and contributed to broaden the understanding of MB-PDT-induced mechanisms and specificity in breast cancer cells. Therefore, we demonstrated that efficient approaches could be designed on the basis of lipid composition and metabolic features for hard-to-treat cancers. The results further reinforce MB-PDT as a therapeutic strategy for highly aggressive human breast cancer cells.

Scope Glucose homeostasis and progression of nonalcoholic fatty liver disease (NAFLD) and hepatomegaly in severe lipoatrophic mice and their modulation by intake of a diet rich in omega 3 (n-3) fatty acids (HFO) are evaluated. Methods and results Severe lipoatrophic mice induced by PPAR-γ deletion exclusively in adipocytes (A-PPARγ KO) and littermate controls (A-PPARγ WT) are evaluated for glucose homeostasis and liver mass, proteomics, lipidomics, inflammation, and fibrosis. Lipoatrophic mice are heavier than controls, severely glucose intolerant, and hyperinsulinemic, and develop NAFLD characterized by increased liver glycogen, triacylglycerol, and diacylglycerol contents, mitotic index, apoptosis, inflammation, steatosis score, fibrosis, and fatty acid synthase (FAS) content and activity. Lipoatrophic mice also display liver enrichment with monounsaturated in detriment of polyunsaturated fatty acids including n-3 fatty acids, and increased content of cardiolipin, a tetracyl phospholipid exclusively found at the mitochondria inner membrane. Administration of a high-fat diet rich in n-3 fatty acids (HFO) to lipoatrophic mice enriches liver with n-3 fatty acids, reduces hepatic steatosis, FAS content and activity, apoptosis, inflammation, and improves glucose homeostasis. Conclusion Diet enrichment with n-3 fatty acids improves glucose homeostasis and reduces liver steatosis and inflammation without affecting hepatomegaly in severe lipoatrophic mice.

We sought to investigate how far the growth of Saccharomyces cerevisiae under full anaerobiosis is dependent on the widely used anaerobic growth factors (AGF) ergosterol and oleic acid. A continuous cultivation setup was employed and, even forcing ultrapure N2 gas through an O2 trap upstream of the bioreactor, neither cells from S. cerevisiae CEN.PK113–7D (a lab strain) nor from PE-2 (an industrial strain) washed out after an aerobic-to-anaerobic switch in the absence of AGF. S. cerevisiae PE-2 seemed to cope better than the laboratory strain with this extremely low O2 availability, since it presented higher biomass yield, lower specific rates of glucose consumption and CO2 formation, and higher survival at low pH. Lipid (fatty acid and sterol) composition dramatically altered when cells were grown anaerobically without AGF: saturated fatty acid, squalene and lanosterol contents increased, when compared to either cells grown aerobically or anaerobically with AGF. We concluded that these lipid alterations negatively affect cell viability during exposure to low pH or high ethanol titers.

Radiation induces the generation of platelet-activating factor receptor (PAF-R) ligands, including PAF and oxidized phospholipids. Alternatively, PAF is also synthesized by the biosynthetic enzymes lysophosphatidylcholine acyltransferases (LPCATs) which are expressed by tumor cells including melanoma. The activation of PAF-R by PAF and oxidized lipids triggers a survival response protecting tumor cells from radiation-induced cell death, suggesting the involvement of the PAF/PAF-R axis in radioresistance. Here, we investigated the role of LPCATs in the melanoma cell radiotherapy response. LPCAT is a family of four enzymes, LPCAT1-4, and modular nucleic acid nanoparticles (NANPs) allowed for the simultaneous silencing of all four LPCATs. We found that the in vitro simultaneous silencing of all four LPCAT transcripts by NANPs enhanced the therapeutic effects of radiation in melanoma cells by increasing cell death, reducing long-term cell survival, and activating apoptosis. Thus, we propose that NANPs are an effective strategy for improving radiotherapy efficacy in melanomas.

The nutrient sensors peroxisome proliferator-activated receptor γ (PPARγ) and mechanistic target of rapamycin complex 1 (mTORC1) closely interact in the regulation of adipocyte lipid storage. The precise mechanisms underlying this interaction and whether this extends to other metabolic processes and the endocrine function of adipocytes are still unknown. We investigated herein the involvement of mTORC1 as a mediator of the actions of the PPARγ ligand rosiglitazone in subcutaneous inguinal white adipose tissue (iWAT) mass, endocrine function, lipidome, transcriptome and branched-chain amino acid (BCAA) metabolism. Mice bearing regulatory associated protein of mTOR (Raptor) deletion and therefore mTORC1 deficiency exclusively in adipocytes and littermate controls were fed a high-fat diet supplemented or not with the PPARγ agonist rosiglitazone (30 mg/kg/day) for 8 weeks and evaluated for iWAT mass, lipidome, transcriptome (Rnaseq), respiration and BCAA metabolism. Adipocyte mTORC1 deficiency not only impaired iWAT adiponectin transcription, synthesis and secretion, PEPCK mRNA levels, triacylglycerol synthesis and BCAA oxidation and mRNA levels of related proteins but also completely blocked the upregulation in these processes induced by pharmacological PPARγ activation with rosiglitazone. Mechanistically, adipocyte mTORC1 deficiency impairs PPARγ transcriptional activity by reducing PPARγ protein content, as well as by downregulating C/EBPα, a co-partner and facilitator of PPARγ. In conclusion, mTORC1 and PPARγ are essential partners involved in the regulation of subcutaneous adipose tissue adiponectin production and secretion and BCAA oxidative metabolism.

Proton bioenergetics provides the energy for growth and survival of most organisms in the biosphere ranging from unicellular marine phytoplankton to humans. Chloroplasts harvest light and generate a proton electrochemical gradient (proton motive force) that drives the production of ATP needed for carbon dioxide fixation and plant growth. Mitochondria, bacteria and archaea generate proton motive force to energize growth and other physiologies. Energy transducing membranes are at the heart of proton bioenergetics and are responsible for catalyzing the conversion of energy held in high-energy electrons → electron transport chain → proton motive force → ATP. Whereas the electron transport chain is understood in great detail there are major gaps in understanding mechanisms of proton transfer or circulation during proton bioenergetics. This paper is built on the proposition that phospho- and glyco-glycerolipids form proton transport circuitry at the membrane's surface. By this proposition, an emergent membrane property, termed the hyducton, confines active/unbound protons or hydronium ions to a region of low volume close to the membrane surface. In turn, a von Grotthus mechanism rapidly moves proton substrate in accordance with nano-electrochemical poles on the membrane surface created by powerful proton pumps such as ATP synthase.

Analysis of cellular membrane lipids has been widely applied to describe the microbial community composition in natural systems. When combined with stable isotope probing (SIP) experiments, deuterium label (D 2 O) uptake into lipids enables assessment of microbial biomass production. We performed SIP on methane-rich, hydrothermally-heated sediments to examine the de novo production of individual archaeal intact polar lipids (IPLs) by mesophilic anaerobic methane oxidizing group-1 archaea (ANME-1). The greatest extent of label uptake was by phosphatidyl glycerol archaeol (PG-AR), reaching 50% of the medium’s label concentration in only 10 days. This indicates PG-AR as an important cell membrane lipid during the active growth phase of mesophilic ANME-1. Much less label uptake was into intact polar glycerol dibiphytanyl glycerol tetraethers (GDGTs), especially for those bound to diglycosidic head groups. The low production of these GDGTs contrasts with their predominance in ANME-1-dominated natural samples. We attribute the differential label uptake among individual IPLs to the ANME-1 tetraether biosynthetic pathway. This mechanism likely involves head-to-head condensation of two molecules of PG-AR and progressive substitution of PG by glycosidic head groups. The observation that ANME-1 invest in the synthesis of diethers during optimum growth suggests intact ARs and/or phosphate-bearing tetraethers as important biomarkers for actively growing populations in natural environments, while the diglycosidic GDGTs appear to signal stationary ANME-1 communities.

A low-sodium (LS) diet has been shown to reduce blood pressure (BP) and the incidence of cardiovascular diseases. However, severe dietary sodium restriction promotes insulin resistance (IR) and dyslipidemia in animal models and humans. Thus, further clarification of the long-term consequences of LS is needed. Here, we investigated the effects of chronic LS on gastrocnemius gene and protein expression and lipidomics and its association with IR and plasma lipids in LDL receptor knockout mice. Three-month-old male mice were fed a normal sodium diet (NS; 0.5% Na; n = 12–19) or LS (0.06% Na; n = 14–20) over 90 days. Body mass (BM), BP, plasma total cholesterol, triacylglycerol (TG), glucose, hematocrit, and IR were evaluated. LS increased BM (9%), plasma TG (51%), blood glucose (19%), and IR (46%) when compared with the NS. RT-qPCR analysis revealed that genes involved in lipid uptake and oxidation were increased by the LS: Fabp3 (106%), Prkaa1 (46%), and Cpt1 (74%). Genes and proteins (assessed by Western blotting) involved in insulin signaling were not changed by the LS. Similarly, lipid species classically involved in muscle IR, such as diacylglycerols and ceramides detected by ultra-high-performance liquid chromatography coupled to mass spectrometry, were also unchanged by LS. Species of phosphatidylcholines (68%), phosphatidylinositol (90%), and free fatty acids (59%) increased while cardiolipins (41%) and acylcarnitines (9%) decreased in gastrocnemius in response to LS and were associated with glucose disposal rate. Together these results suggest that chronic LS alters glycerophospholipid and fatty acids species in gastrocnemius that may contribute to glucose and lipid homeostasis derangements in mice.

Microglial activation by oleate and palmitate differentially modulates brain inflammatory status. However, the metabolic reprogramming supporting these reactive phenotypes remains unknown. Employing real-time metabolic measurements and lipidomic analysis, we show that both fatty acids promote microglial oxidative metabolism, while lipopolysaccharide (LPS) enhances glycolytic rates. Interestingly, oleate treatment was followed by enrichment in storage lipids bound to polyunsaturated fatty acids (PUFA), in parallel with protection against oxidative imbalance. Palmitate, in turn, induced a distinct lipid distribution defined by PUFA linked to membrane phospholipids, which are more susceptible to lipid peroxidation and inflammatory signaling cascades. This distribution was mirrored by LPS treatment, which led to a strong pro-inflammatory phenotype in microglia. Thus, although both oleate and palmitate preserve mitochondrial function, a contrasting lipid distribution supports differences in fatty acid-induced neuroinflammation. These data reinforce the concept that reactive microglial profiles are achieved by stimulus-evoked remodeling in cell metabolism.

Scope The mechanisms and involvement of uncoupling protein 1 (UCP1) in the protection from obesity and insulin resistance induced by intake of a high-fat diet rich in omega-3 (n-3) fatty acids are investigated. Methods and results C57BL/6J mice are fed either a low-fat (control group) or one of two isocaloric high-fat diets containing either lard (HFD) or fish oil (HFN3) as fat source and evaluated for body weight, adiposity, energy expenditure, glucose homeostasis, and inguinal white and interscapular brown adipose tissue (iWAT and iBAT, respectively) gene expression, lipidome, and mitochondrial bioenergetics. HFN3 intake protected from obesity, glucose and insulin intolerances, and hyperinsulinemia. This is associated with increased energy expenditure, iWAT UCP1 expression, and incorporation of n-3 eicosapentaenoic and docosahexaenoic fatty acids in iWAT and iBAT triacylglycerol. Importantly, HFN3 is equally effective in reducing body weight gain, adiposity, and glucose intolerance and increasing energy expenditure in wild-type and UCP1-deficient mice without recruiting other thermogenic processes in iWAT and iBAT, such as mitochondrial uncoupling and SERCA-mediated calcium and creatine-driven substrate cyclings. Conclusion Intake of a high-fat diet rich in omega-3 fatty acids protects both wild-type and UCP1-deficient mice from obesity and insulin resistance by increasing energy expenditure through unknown mechanisms.

Microbial life inhabiting subseafloor sediments plays an important role in Earth’s carbon cycle. However, the impact of geodynamic processes on the distributions and carbon-cycling activities of subseafloor life remains poorly constrained. We explore a submarine mud volcano of the Nankai accretionary complex by drilling down to 200 m below the summit. Stable isotopic compositions of water and carbon compounds, including clumped methane isotopologues, suggest that ~90% of methane is microbially produced at 16° to 30°C and 300 to 900 m below seafloor, corresponding to the basin bottom, where fluids in the accretionary prism are supplied via megasplay faults. Radiotracer experiments showed that relatively small microbial populations in deep mud volcano sediments (10 2 to 10 3 cells cm −3 ) include highly active hydrogenotrophic methanogens and acetogens. Our findings indicate that subduction-associated fluid migration has stimulated microbial activity in the mud reservoir and that mud volcanoes may contribute more substantially to the methane budget than previously estimated., Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe, 802

The majority of cells in nature probably exist in a stationary-phase-like state, due to nutrient limitation in most environments. Studies on bacteria and yeast reveal morphological and physiological changes throughout the stationary phase, which lead to an increased ability to survive prolonged nutrient limitation. However, there is little information on archaeal stationary phase responses. We investigated protein- and lipid-level changes inThermococcus kodakarensiswith extended time in the stationary phase. Adaptations to time in stationary phase included increased proportion of membrane lipids with a tetraether backbone, synthesis of proteins that ensure translational fidelity, specific regulation of ABC transporters (upregulation of some, downregulation of others), and upregulation of proteins involved in coenzyme production. Given that the biological mechanism of tetraether synthesis is unknown, we also considered whether any of the protein-level changes inT. kodakarensismight shed light on the production of tetraether lipids across the same period. A putative carbon-nitrogen hydrolase, a TldE (a protease inEscherichia coli) homologue, and a membrane bound hydrogenase complex subunit were candidates for possible involvement in tetraether-related reactions, while upregulation of adenosylcobalamin synthesis proteins might lend support to a possible radical mechanism as a trigger for tetraether synthesis.

Studies on microbial carbon cycling uniformly confirm that anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria represent the dominant and most active fraction of the sedimentary microbial community in methane-seep sediments. However, little is known about other frequently observed and abundant microbial taxa, their role in carbon cycling and association with the anaerobic oxidation of methane (AOM). Here, we provide a comprehensive characterization of stable carbon isotopes (δ 13 C) from several intact polar lipid (IPL) classes and metabolite pools in a downcore profile at a cold seep within the oxygen minimum zone off Pakistan. We aimed to evaluate microbial carbon metabolism using IPLs in relation to redox conditions, metabolites and 16S rRNA gene libraries. The 13 C-depleted signature of carbon pools and microbial metabolites in pore waters (e.g., dissolved inorganic carbon, lactate and acetate) demonstrated high accumulation of AOM-associated biomass and subsequent turnover thereof. ANMEs accounted for a small fraction of the archaeal 16S rRNA gene survey, whereas sequences of other uncultured benthic archaea dominated the clone libraries, particularly the Marine Benthic Group D. On the basis of lipid diversity and carbon isotope information, we suggest that structurally diverse phospho- and glycolipids, including the recently identified unsaturated tetraethers that are particularly abundant in this setting, are likely derived from archaea other than ANMEs. Through the evaluation of δ 13 C values of individual IPL, our results indicate heterotrophy as a possible metabolic pathway of archaea in these AOM-dominated sediments.

The C. elegans lipase-like 5 (lipl-5) gene is predicted to code for a lipase homologous to the human gastric acid lipase. Its expression was previously shown to be modulated by nutritional or immune cues, but nothing is known about its impact on the lipid landscape and ensuing functional consequences. In the present work, we used mutants lacking LIPL-5 protein and found that lipl-5 is important for normal lipidome composition as well as its remodeling in response to food deprivation. Particularly, lipids with signaling functions such as ceramides and mitochondrial lipids were affected by lipl-5 silencing. In comparison with wild type worms, animals lacking LIPL-5 were enriched in cardiolipins linked to polyunsaturated C20 fatty acids and coenzyme Q-9. Differences in mitochondrial lipid composition were accompanied by differences in mitochondrial activity as mitochondria from well-fed lipl-5 mutants were significantly more able to oxidize respiratory substrates when compared with mitochondria from well-fed wild type worms. Strikingly, starvation elicited important changes in mitochondrial activity in wild type worms, but not in lipl-5 worms. This indicates that this lipase is a determinant of mitochondrial functional remodeling in response to food withdrawal.

Sulfide 'chimneys' characteristic of seafloor hydrothermal venting are diverse microbial habitats. ¹³C/¹²C ratios of microbial lipids have rarely been used to assess carbon assimilation pathways on these structures, despite complementing gene- and culture-based approaches. Here, we integrate analyses of the diversity of intact polar lipids (IPL) and their side-chain δ¹³C values (δ¹³ C(lipid)) with 16S rRNA gene-based phylogeny to examine microbial carbon flow on active and inactive sulfide structures from the Manus Basin. Surficial crusts of active structures, dominated by Epsilonproteobacteria, yield bacterial δ¹³C(lipid) values higher than biomass δ¹³C (total organic carbon), implicating autotrophy via the reverse tricarboxylic acid cycle. Our data also suggest δ¹³C(lipid) values vary on individual active structures without accompanying microbial diversity changes. Temperature and/or dissolved substrate effects - likely relating to variable advective-diffusive fluxes to chimney exteriors - may be responsible for differing ¹³C fractionation during assimilation. In an inactive structure, δ¹³C(lipid) values lower than biomass δ¹³C and a distinctive IPL and 16S rRNA gene diversity suggest a shift to a more diverse community and an alternate carbon assimilation pathway after venting ceases. We discuss here the potential of IPL and δ¹³C(lipid) analyses to elucidate carbon flow in hydrothermal structures when combined with other molecular tools.

RATIONALE Studies of archaeal glycerol dibiphytanyl glycerol tetraethers (GDGTs) in the environment and cultures have exclusively focused on compounds with fully saturated alkyl moieties. Here we report a number of novel unsaturated GDGTs (uns-GDGTs) whose alkyl chains contain up to six double bonds and zero to two cyclopentyl moieties. METHODS The identification of these lipids was achieved via comparison of lipid distribution before and after hydrogenation, characteristic retention time patterns, and diagnostic ions using liquid chromatography/tandem mass spectrometry (LC/MS/MS), and ether cleavage products using gas chromatography/mass spectrometry (GC/MS). Isomerism resulting from different unsaturation patterns in the alkyl moieties was observed and specific positions of double bonds in the biphytene and biphytadiene moieties were tentatively assigned. RESULTS Uns-GDGTs were detected in sediment and microbial mat samples as both core lipids (CLs) and intact polar lipids (IPLs) associated with mono- or diglycosyl or phosphatidylglycerol headgroups. However, these lipids were overlooked in past investigations because conventional methods for archaeal lipid analysis are unsuitable for uns-GDGTs. Samples from distinct marine environments (Black Sea, Cariaco Basin, Discovery Basin, Eastern Mediterranean Sea, upwelling area off NW Africa, and seep sites off Crimea and Pakistan) were screened for uns-GDGTs using a new LC/MS protocol. The results show that uns-GDGTs contribute significantly to the archaeal lipid pool in anoxic methane-rich environments (Black Sea, Cariaco Basin, and both seep sites) but they were barely detected in the oxic or hypersaline settings. CONCLUSIONS The characteristic distribution of uns-GDGTs implies that they are attractive targets for future studies aiming at the chemotaxonomy of uncultivated archaea and regulation of uns-GDGT biosynthesis.

The flux of methane from the sea bed to the overlying water column is mitigated by the sulphate-dependent anaerobic oxidation of methane by marine microbes. Laboratory experiments point to the equilibration of stable carbon isotopes during the anaerobic oxidation of methane under sulphate-limited conditions.

A new clade of archaea has recently been proposed to constitute the seventh methanogenic order, the Methanomassiliicoccales , which is related to the Thermoplasmatales and the uncultivated archaeal clades deep-sea hydrothermal vent Euryarchaeota group 2 and marine group II Euryarchaeota but only distantly related to other methanogens. In this study, we investigated the membrane lipid composition of Methanomassiliicoccus luminyensis , the sole cultured representative of this seventh order. The lipid inventory of M. luminyensis comprises a unique assemblage of novel lipids as well as lipids otherwise typical for thermophilic, methanogenic, or halophilic archaea. For instance, glycerol sesterpanyl-phytanyl diether core lipids found mainly in halophilic archaea were detected, and so were compounds bearing either heptose or methoxylated glycosidic head groups, neither of which have been reported so far for other archaea. The absence of quinones or methanophenazines is consistent with a biochemistry of methanogenesis different from that of the methanophenazine-containing methylotrophic methanogens. The most distinctive characteristic of the membrane lipid composition of M. luminyensis , however, is the presence of tetraether lipids in which one glycerol backbone is replaced by either butane- or pentanetriol, i.e., lipids recently discovered in marine sediments. Butanetriol dibiphytanyl glycerol tetraether (BDGT) constitutes the most abundant core lipid type (>50% relative abundance) in M. luminyensis . We have thus identified a source for these unusual orphan lipids. The complementary analysis of diverse marine sediment samples showed that BDGTs are widespread in anoxic layers, suggesting an environmental significance of Methanomassiliicoccales and/or related BDGT producers beyond gastrointestinal tracts. IMPORTANCE Cellular membranes of members of all three domains of life, Archaea , Bacteria , and Eukarya , are largely formed by lipids in which glycerol serves as backbone for the hydrophobic alkyl chains. Recently, however, archaeal tetraether lipids with either butanetriol or pentanetriol as a backbone were identified in marine sediments and attributed to uncultured sediment-dwelling archaea. Here we show that the butanetriol-based dibiphytanyl tetraethers constitute the major lipids in Methanomassiliicoccus luminyensis , currently the only isolate of the novel seventh order of methanogens. Given the absence of these lipids in a large set of archaeal isolates, these compounds may be diagnostic for the Methanomassiliicoccales and/or closely related archaea.

Recent advances in lipidomic analysis in combination with various physiological experiments set the stage for deciphering the structure-function of haloarchaeal membrane lipids. Here we focused primarily on changes in lipid composition of Haloferax volcanii, but also performed a comparative analysis with four other haloarchaeal species (Halobacterium salinarum, Halorubrum lacusprofundi, Halorubrum sodomense and Haloplanus natans) all representing distinctive cell morphologies and behaviors (i.e., rod shape vs. pleomorphic behavior). Common to all five haloarchaea, our data reveal an extraordinary high level of menaquinone, reaching up to 72% of the total lipids. This ubiquity suggests that menaquinones may function beyond their ordinary role as electron and proton transporter, acting simultaneously as ion permeability barriers and as powerful shield against oxidative stress. In addition, we aimed at understanding the role of cations interacting with the characteristic negatively charged surface of haloarchaeal membranes. We propose for instance that by bridging the negative charges of adjacent anionic phospholipids, Mg2+ acts as surrogate for cardiolipin, a molecule that is known to control curvature stress of membranes. This study further provides a bioenergetic perspective as to how haloarchaea evolved following oxygenation of Earth's atmosphere. The success of the aerobic lifestyle of haloarchaea includes multiple membrane-based strategies that successfully balance the need for a robust bilayer structure with the need for high rates of electron transport - collectively representing the molecular basis to inhabit hypersaline water bodies around the planet.

Archaea are ubiquitous and abundant microorganisms on Earth that mediate key global biogeochemical cycles. The headgroup attached to the sn-1 position of the glycerol backbone and the ether-linked isoprenoid lipids are among the diagnostic traits that distinguish Archaea from Bacteria and Eukarya. Over the last 30 years, numerous archaeal lipids have been purified and described in pure cultures. Coupled high-performance liquid chromatography (HPLC) ion-trap mass spectrometry (ITMS) now enables the detection and rapid identification of intact polar lipids in relatively small and complex samples, revealing a wide range of archaeal lipids in natural environments. Although major structural groups have been identified, the lack of a systematic evaluation of MS/MS fragmentation patterns has hindered the characterization of several atypical components that are therefore considered as unknowns. Here, we examined mass spectra resulting from lipid analysis of natural microbial communities using HPLC/electrospray ionization (ESI)-ITMS(n), and depicted the systematics in MS(2) fragmentation of intact archaeal lipids. This report will be particularly useful for environmental scientists interested in a rapid and straightforward characterization of intact archaeal membrane lipids.

Glycolipids are prominent constituents in the membranes of cells from all domains of life. For example, diglycosyl-glycerol dibiphytanyl glycerol tetraethers (2Gly-GDGTs) are associated with methanotrophic ANME-1 archaea and heterotrophic benthic archaea, two archaeal groups of global biogeochemical importance. The hydrophobic biphytane moieties of 2Gly-GDGTs from these two uncultivated archaeal groups exhibit distinct carbon isotopic compositions. To explore whether the isotopic compositions of the sugar headgroups provide additional information on the metabolism of their producers, we developed a procedure to analyze the δ 13 C values of glycosidic headgroups. Successful determination was achieved by (1) monitoring the contamination from free sugars during lipid extraction and preparation, (2) optimizing the hydrolytic conditions for glycolipids, and (3) derivatizing the resulting sugars into aldononitrile acetate derivatives, which are stable enough to withstand a subsequent column purification step. First results of δ 13 C values of sugars cleaved from 2Gly-GDGTs in two marine sediment samples, one containing predominantly ANME-1 archaea and the other benthic archaea, were obtained and compared with the δ 13 C values of the corresponding biphytanes. In both samples the dominant sugar headgroups were enriched in 13 C relative to the corresponding major biphytane. This 13 C enrichment was significantly larger in the putative major glycolipids from ANME-1 archaea (∼15‰) than in those from benthic archaea (

The response of Arctic microbial communities to a variety of natural organic matter substrates, including peat, ice algae and ice-rafted debris was examined using bacterial regrowth experiments and compared to unamended controls. Bacterial growth and production were followed together with the phylogenetic composition using length-heterogeneity polymerase chain reaction (LH-PCR), and 16S rRNA gene cloning and sequencing. Intact phospholipids (IPLs) and fatty acids evaluated the relationship between lipids and bacterial community structure and the impact of varied organic substrates on microbial lipid synthesis. Differential responses to organic matter sources were observed, with ice algae supporting both higher bacterial growth and production than terrestrial-derived peat. In spite of disparate growth kinetics, the community composition remained similar in all amended incubations as was confirmed by automated ribosomal intergenic spacer analysis (ARISA). Gammaproteobacteria dominated the initial incubations, whereas in extended incubations with terrestrial peat Alphaproteobacteria dominated; in particular Sulfitobacter phylotypes closely related (>99%) to an Arctic sea-ice-associated member of the Roseobacter clade (ARK10278). Arctic bacterioplankton preferentially synthesized two phospholipids, phosphatidylethanolamine (PE) and phosphatidylglygerol (PG), with 18:0n, 18:1Δ11, 16:0n and 16:1Δ9 as the primary fatty acids. Overall, results show that organic matter source plays an important role in structuring bacterioplankton community composition, with similar IPL and fatty acid lipid distributions observed among phylogenetically distinct bacteria.

A ciclagem de matéria orgânica (MO) no ambiente marinho é um processo-chave para o ciclo global de carbono. Os sedimentos costeiros são de suma importância para a ciclagem de carbono pois atuam como receptores de grandes quantidades de MO alóctone (i.e. terrestre) e autóctone (i.e. marinho). A miríade dos componentes orgânicos e suas diferentes características dificultam o entendimento das fontes de MO em ambientes costeiros. Este trabalho visou entender a origem e a composição da MO (através de biomarcadores lipídicos) e a dinâmica da comunidade microbiana (método ATP) em sedimentos superficiais de diferentes ecossistemas marinhos da costa sudeste do Brasil: (i) margem continental de Cabo Frio; (ii) sistema lagunar de Saquarema; (iii) áreas costeiras e a plataforma continental de Ubatuba; (iv) e a plataforma adjacente ao estuário de Santos. Os resultados apontaram uma origem predominantemente autóctone para a MO nestes sistemas, com contribuição terrestre reduzida e limitada à áreas próximas à costa. Processos oceanográficos e forçantes ambientais são cruciais para a composição da MO sedimentar e são discutidas para cada um dos ecossistemas estudados. The cycling of the organic matter (OM) in the marine environment is a key process in the global carbon cycle. Coastal sediments are important to the global carbon cycle, since they receive large inputs from both marine and terrestrial OM. The myriad of organic compounds and their spectrum of reactivity complicate the understanding of OM sources in coastal environments. In this work, we aimed to access the origin and composition of the OM (through lipid biomarkers) and the microbial dynamics (ATP method) in surface sediments of diverse marine ecosystems from the SE Brazilian coast: (i) the continental margin off Cabo Frio; (ii) the lagoonal system of Saquarema; (iii) coastal and shelf areas from Ubatuba; and (iv) the continental shelf adjacent to Santos estuary. The results showed a dominance of autochthonous OM, with a minor fraction of the OM derived from terrestrial sources and restricted to areas close to the coast. Oceanographic processes and environmental forces are crucial to the composition of sedimentary OM and are discussed for each of those ecosystems.

The role of anaerobic oxidation of methane (AOM) in wetlands, the largest natural source of atmospheric methane, is poorly constrained. Here we report rates of microbially mediated AOM (average rate=20 nmol cm(-3) per day) in three freshwater wetlands that span multiple biogeographical provinces. The observed AOM rates rival those in marine environments. Most AOM activity may have been coupled to sulphate reduction, but other electron acceptors remain feasible. Lipid biomarkers typically associated with anaerobic methane-oxidizing archaea were more enriched in (13)C than those characteristic of marine systems, potentially due to distinct microbial metabolic pathways or dilution with heterotrophic isotope signals. On the basis of this extensive data set, AOM in freshwater wetlands may consume 200 Tg methane per year, reducing their potential methane emissions by over 50%. These findings challenge precepts surrounding wetland carbon cycling and demonstrate the environmental relevance of an anaerobic methane sink in ecosystems traditionally considered strong methane sources.

The main aim of the present study was to evaluate and compare temporal responses of the benthos in 2 continental shelf areas (Cabo Frio and Ubatuba) off the SE Brazilian coast. In Cabo Frio (23° S, 42° W), the western boundary coastal upwelling of the South Atlantic Central Water (SACW) enhances primary productivity, potentially increasing food supply to the benthic communi- ties via sinking of particulate organic carbon (POC). In contrast, POC fluxes in Ubatuba (23° S, 45° W) are expected to be comparatively lower because SACW remains subsurface. We analyzed the tempo- ral and spatial input of phytodetritus (concentration of chlorophyll a in sediments) and the benthic microbial biomass (estimated by ATP-based carbon content in sediments). Median surface chloro- phyll concentration was computed for all daily available SeaWiFS images (from 2001 and 2002) to fol- low chlorophyll a inputs. All parameters used to investigate benthic responses in the study areas showed consistently higher values in Cabo Frio than in Ubatuba. The results showed that benthic response to upwelling may last months, fueling the microbial communities in the Cabo Frio region.

This study investigates the benthic microbial responses to organic matter (OM) variations in quantity and sources in two shallow water bays (Fortaleza and Ubatuba Bays) on the SE coast of Brazil on six occasions during the year. The pelagic and benthic compartments of the bays were evaluated by: (i) nutrients and chlorophyll a (Chl a) in the water column; (ii) quantity and sources of OM in the sediment (Chl a, total organic carbon and total nitrogen and lipid biomarker composition); and (iii) microbial biomass in sediments as an indicator of active benthic response. Although there were changes in water-column nutrients during the year, Chl a was fairly constant, suggesting a regular supply of microalgae-derived OM to the sea bottom. Based on the composition of lipid biomarkers in sediments, OM sources were classified as mostly marine and with high contributions of labile (microalgae-derived) OM. Labile OM composition varied from diatoms in the summer to phytoflagellates in the winter and tended to accumulate in areas protected by physical disturbances in one of the bays. Microbial biomass followed this trend and was 160% higher in protected than in exposed areas. This study suggests that the coupling between labile OM and benthic microbial biomass occurs primarily in protected areas, irrespective of the time of the year. Since meio- and macrofaunal assemblages depend upon secondary microbial production within the sediments, this coupling may have an important role for the benthic food-web.

International audience; Subsurface microbial life contributes significantly to biogeochemical cycling, yet it remains largely uncharacterized, especially its archaeal members. This 'microbial dark matter' has been explored by recent studies that were, however, mostly based on DNA sequence information only. Here, we use diverse techniques including ultrastuctural analyses to link genomics to biology for the SM1 Euryarchaeon lineage, an uncultivated group of subsurface archaea. Phylogenomic analyses reveal this lineage to belong to a widespread group of archaea that we propose to classify as a new euryarchaeal order ('Candidatus Altiarchaeales'). The representative, double-membraned species 'Candidatus Altiarchaeum hamiconexum' has an autotrophic metabolism that uses a not-yet-reported Factor 420-free reductive acetyl-CoA pathway, confirmed by stable carbon isotopic measurements of archaeal lipids. Our results indicate that this lineage has evolved specific metabolic and structural features like nano-grappling hooks empowering this widely distributed archaeon to predominate anaerobic groundwater, where it may represent an important carbon dioxide sink.

The methane-rich, hydrothermally heated sediments of the Guaymas Basin are inhabited by thermophilic microorganisms, including anaerobic methane-oxidizing archaea (mainly ANME-1) and sulfate-reducing bacteria (e.g., HotSeep-1 cluster). We studied the microbial carbon flow in ANME-1/ HotSeep-1 enrichments in stable-isotope–probing experiments with and without methane. The relative incorporation of 13 C from either dissolved inorganic carbon or methane into lipids revealed that methane-oxidizing archaea assimilated primarily inorganic carbon. This assimilation is strongly accelerated in the presence of methane. Experiments with simultaneous amendments of both 13 C-labeled dissolved inorganic carbon and deuterated water provided further insights into production rates of individual lipids derived from members of the methane-oxidizing community as well as their carbon sources used for lipid biosynthesis. In the presence of methane, all prominent lipids carried a dual isotopic signal indicative of their origin from primarily autotrophic microbes. In the absence of methane, archaeal lipid production ceased and bacterial lipid production dropped by 90%; the lipids produced by the residual fraction of the metabolically active bacterial community predominantly carried a heterotrophic signal. Collectively our results strongly suggest that the studied ANME-1 archaea oxidize methane but assimilate inorganic carbon and should thus be classified as methane-oxidizing chemoorganoautotrophs.

Novel cardiolipins from Archaea were detected by screening the intact polar lipid (IPL) composition of microbial communities associated with methane seepage in deep-sea sediments from the Pakistan margin by high-performance liquid chromatography electrospray ionization mass spectrometry. A series of tentatively identified cardiolipin analogues (dimeric phospholipids or bisphosphatidylglycerol, BPG) represented 0.5% to 5% of total archaeal IPLs. These molecules are similar to the recently described cardiolipin analogues with four phytanyl chains from extreme halophilic archaea. It is worth noting that cardiolipin analogues from the seep archaeal communities are composed of four isoprenoidal chains, which may contain differences in chain length (20 and 25 carbon atoms) and degrees of unsaturation and the presence of a hydroxyl group. Two novel diether lipids, structurally related to the BPGs, are described and interpreted as degradation products of archaeal cardiolipin analogues. Since archaeal communities in seep sediments are dominated by anaerobic methanotrophs, our observations have implications for characterizing structural components of archaeal membranes, in which BPGs are presumed to contribute to modulation of cell permeability properties. Whether BPGs facilitate interspecies interaction in syntrophic methanotrophic consortia remains to be tested.

Organic matter quality, expressed as the proportion of chlorophyll a (Chl a) to degraded organic material (i.e. phaeopigments), is known to influence the structure of benthic associations and plays an important role in the functioning of the ecosystem. This study investigates the vertical distribution of microbial biomass, meiofauna and macrofauna with respect to organic matter variation in Ubatuba, Brazil, a southeastern, subtropical coastal area. On three occasions, samples were collected in exposed and sheltered stations, at high and low hydrodynamic conditions. We hypothesize that benthic assemblages will have high meio- and macrofaunal densities and high microbial biomass at the sediment surface at the sheltered site, and lower and vertically homogeneous microbial biomass and densities of meio- and macrofauna are expected at the exposed site. The accumulation of fresh organic matter at the sediment surface was observed at both stations over the three sampling dates, which contributed to the higher densities of meiofauna in the first layers of the sediment column. Macrofauna followed the same trend only at the exposed station, but changes in the number of species, biodiversity and feeding groups were registered for both stations. Microbial biomass increased at the sheltered station over the three sampling dates, whereas at the exposed station, microbial biomass was nearly constant. Physical exposure did not influence organic matter loading at the sites and therefore did not affect overall structure of benthic assemblages, which negates our original hypothesis. Most of the benthic system components reacted to organic matter quality and quantity, but relationships between different-sized organisms (i.e. competition and/or predation) may explain the unchanged microbial profiles at the exposed site and homogeneous vertical distribution of macrofauna at the sheltered site. In conclusion, the high quality of organic matter was a crucial factor in sustaining and regulating the benthic system, but coupled results showed that interactions between micro-, meio- and macrofauna can be highly complex.

The coastal upwelling off Cabo Frio (SE Brazilian coast, SEBC) represents an exception to the world’s oceans since the majority of the upwelling areas are located in eastern boundary current systems. Cabo Frio represents an interesting area for investigation due to its tight physical–biological interaction and the importance of the region as a major fishery area in the SEBC. We analyzed a suite of lipid biomarkers to apportion the main sources of organic matter in surface sediments of the continental shelf off Cabo Frio, comparing the area to non-upwelling regions off the SEBC (shelf break off Cabo Frio and continental shelf off Ubatuba). During spring and summer (the upwelling period), diatoms are probably the major sources of polyunsaturated fatty acids (PUFAs) and C28 sterols in surface sediments from Cabo Frio continental shelf. Sediments sampled in winter showed, in contrast, lower relative abundance of PUFAs and higher stanol/stenol ratio values. In deeper regions off Cabo Frio, elevated concentrations of alkenones, 24-methylcholest-5,22E-dien-3β-ol and 24-ethylcholest-5-en-3β-ol during the spring may be produced by prymnesiophytes or cryptophytes and cyanobacteria, respectively. In Ubatuba, the C27 and C28 sterols are likely derived from omnivorous salps and nanoflagellates. At non-upwelling areas, despite the increase in biomarker concentrations during spring and summer, lower concentrations of PUFAs, phytol and algal sterols than in shelf areas off Cabo Frio suggest the importance of the upwelling system to the rapid transfer of organic carbon to surface sediments. Our results suggest that spatial and temporal variability in organic matter production and deposition merits consideration for constraining the carbon budgets in the coastal region off Cabo Frio.

Metano (CH4) e dióxido de carbono (CO2) são gases de grande importância climática e energética, pois contribuem para o efeito de estufa, mas também para produção de energia, no caso do CH4. Do ponto de vista energético, reconhecer as diferentes fontes de geração de CH4 torna-se um fator crucial na avaliação das reservas mundiais de gás natural, uma vez que as estimativas atuais de reservas potenciais não levam em consideração as diferentes origens dos hidrocarbonetos presentes ou, na grande maioria, considera apenas a origem termogênica. A temperatura é um dos fatores mais importantes que afetam o crescimento microbiano e as reações biogeoquímicas ligadas à metanogênese. A Formação Irati (Permiano da Bacia do Paraná) ocorre na região sul da América do Sul e representa um dos folhelhos orgânicos mais importantes do mundo já que o conteúdo em carbono orgânico total (COT) atinge até 23% e cobre uma área de aproximadamente 700.000 km2. A história térmica deste importante reservatório de carbono é atípica por hospedar rochas ígneas do Cretáceo. Isto proporcionou a ocorrência de zonas termicamente imaturas até zonas com maturidade suficiente para a geração termogênica de CH4. Este estudo trata da influência da temperatura na geração de CH4 e CO2 biogênicos em folhelhos da Formação Irati. Para isto, amostras de folhelho foram utilizadas em experimentos de incubação realizados sob diferentes temperaturas (22°C, 50°C, 70°C e 80°C), com objetivo de avaliar a influência das condições térmicas sobre a geração biogênica de CH4 e CO2. Temperaturas mais elevadas promoveram maiores taxas de produção de CH4 e CO2. A produção biogênica de CH4 mostrou-se mais eficiente em condições de temperatura de 80°C, com um rendimento máximo de 2,45 ml/t.d em comparação com 0,49 ml/t.d a 22°C, 1,75 ml/t.d e 2,09 ml/t.d a 50°C e 70°C, respectivamente. A mesma tendência foi observada para o CO2. O potencial de produção máximo de CO2 foi observado sob condições térmicas de 80°C, atingindo 23467,37 ml/t.d. As diferenças de produção obtidas para as diferentes amostras analisadas parecem estar relacionadas com a composição química da matriz mineral, observando-se maior produção em amostras com maior quantidade de enxofre (S), quando submetidas a altas temperaturas (50°C, 70°C e 80°C). Além dos folhelhos da Formação Irati, níveis orgânicos das formações Água de Madeiros e Vale das Fontes, Jurássico da Bacia Lusitânica, foram também estudados, os quais são termicamente imaturos e apresentam querogênio tipicamente marinho, distinto do observado na Formação Irati. O aumento da geração biogênica de CH4 e CO2 com a elevação da temperatura também foi observado para amostras das formações Água de Madeiros e Vale das Fontes. Com o presente estudo, é quebrado o paradigma de que 80°C seria a temperatura máxima para a ocorrência de metanogênese em bacias sedimentares, ou reservatórios. Assim, a geração biogênica de CH4 é favorecida por temperaturas mais elevadas (até no mínimo 80°C), considerando-se que a microbiota dos experimentos decorridos durante o presente trabalho seria similar aquelas que ocorrem em bacias sedimentares, bem como o ecossistema. Este trabalho constitui o primeiro estudo que avaliou o efeito da temperatura na produção de gases de origem biogênica em folhelhos da Formação Irati (Bacia do Paraná) e das formações Água de Madeiros e Vale das Fontes (Bacia Lusitânica - Portugal). Methane (CH4) and carbon dioxide (CO2) have great environmental and economic importance due to their contribution to the greenhouse effect and climate change, but also as energy resource, in the case of CH4. From the energy point of view, recognizing the different sources of CH4 in sedimentary basins has a crucial factor in assessing the world\'s natural gas reserves, since the current estimates of hydrocarbons accumulations in sedimentary basins are based on thermogenic generation of hydrocarbons. However, few studies evaluate the importance of CH4 and CO2 generation as a product of organic matter biodegradation in sedimentary basins. This issue has great relevance to improve the estimates about the geological accumulations of CH4 and CO2. Temperature is one of the most important factors affecting microbial growth and biogeochemical processes responsible for CH4 and CO2 generation in subsurface environments. In this context, the Irati Formation (Permian of the Paraná Basin) in southern of South America represents one of the most organicrich shale around the world, reaching up to 23% of total organic carbon (TOC) and covering an area of approximately 700.000 km2. The thermal history of this important geological carbon pool is atypical because of the emplacement of igneous bodies during the Early Cretaceous. In this study, shale samples of the Irati Formation were used in incubation experiments performed under different temperatures (22°C, 50°C, 70°C and 80°C) to evaluate the influence of thermal conditions on biogenic generation of CH4 and CO2. Higher temperatures promoted higher production rates of CH4 and CO2. Biogenic CH4 production was more efficient when shale samples were incubated at 80°C, with a maximum yield of 2.45 ml/t.d compared to 0.49 ml/t.d at 22°C, 1.75 ml/t.d at 50°C and 2.09 ml/t.d at 70°C. The same trend was observed for CO2 generated as by-product of methanogenesis. The maximum production for CO2 was observed at 80°C, reaching 23467.37 ml/t.d. The differences in CH4 and CO2 production observed for different analyzed samples seem to be related to the composition of the mineral matrix, being observed higher production in samples with higher amount of sulfur. Additionaly, organic layers of the Água de Madeiros and Vale das Fontes Formations (Lusitanian Basin), which are thermally immature and present typically marine kerogen, were also submitted to incubation experiments to evaluate CH4 and CO2 generation. Higher biogenic generation of CH4 with the elevation of temperature was also observed for the Água de Madeiros and Vale das Fontes Formations. This suggests that biogenic CH4 generation is favored by higher temperatures, at least until 80°C, independent of the thermal maturity of the substrate, pointing that the temperature window (and depth zone) for the generation of biogenic CH4 in sedimentary basins is larger than suggested in previous studies.