Carbonic anhydrase activity in acetate grown Methanosarcina barkeri

Cell extracts (27000xg supernatant) of acetate grown Methanosarcina barkeri were found to have carbonic anhydrase activity (0.41 U/mg protein), which was lost upon heating or incubation with proteinase K. The activity was inhibited by Diamox (apparent K _i=0.5 mM), by azide (apparent K _i=1 mM), and by cyanide (apparent K _i=0.02 mM). These and other properties indicate that the archaebacterium contains the enzyme carbonic anhydrase (EC 4.2.1.1). Evidence is presented that the protein is probably located in the cytoplasm. Methanol or H₂/CO₂ grown cells of M. barkeri showed no or only very little carbonic anhydrase activity. After transfer of these cells to acetate medium the activity was induced suggesting a function of this enzyme in acetate fermentation to CO₂ and CH₄. Interestingly, Desulfobacter postgatei and Desulfotomaculum acetoxidans, which oxidize acetate to 2 CO₂ with sulfate as electron acceptor, were also found to exhibit carbonic anhydrase activity (0.2 U/mg protein).     

Environmental dynamics of dissolved black carbon in wetlands

Wetlands are ecosystems commonly characterized by elevated levels of dissolved organic carbon (DOC), and although they cover a surface area less than 2 % worldwide, they are an important carbon source representing an estimated 15 % of global annual DOC flux to the oceans. Because of their unique hydrological characteristics, fire can be an important ecological driver in pulsed wetland systems. Consequently, wetlands may be important sources not only of DOC but also of products derived from biomass burning, such as dissolved black carbon (DBC). However, the biogeochemistry of DBC in wetlands has not been studied in detail. The objective of this study is to determine the environmental dynamics of DBC in different fire-impacted wetlands. An intensive, 2-year spatial and temporal dynamics study of DBC in a coastal wetland, the Everglades (Florida) system, as well as one-time sampling surveys for the other two inland wetlands, Okavango Delta (Botswana) and the Pantanal (Brazil), were reported. Our data reveal that DBC dynamics are strongly coupled with the DOC dynamics regardless of location, season or recent fire history. The statistically significant linear regression between DOC and DBC was applied to estimate DBC fluxes to the coastal zone through two main riverine DOC export routes in the Everglades ecosystem. The presence of significant amounts of DBC in these three fire-impacted ecosystems suggests that sub-tropical wetlands could represent an important continental-ocean carrier of combustion products from biomass burning. The discrimination of DBC molecular structure (i.e. aromaticity) between coastal and terrestrial samples, and between samples collected in wet and dry season, suggests that spatially-significant variation in DBC source strength and/or degree of degradation may also influence DBC dynamics.     

A single-cell sequencing approach to the classification of large, vacuolated sulfur bacteria

The colorless, large sulfur bacteria are well known because of their intriguing appearance, size and abundance in sulfidic settings. Since their discovery in 1803 these bacteria have been classified according to their conspicuous morphology. However, in microbiology the use of morphological criteria alone to predict phylogenetic relatedness has frequently proven to be misleading. Recent sequencing of a number of 16S rRNA genes of large sulfur bacteria revealed frequent inconsistencies between the morphologically determined taxonomy of genera and the genetically derived classification. Nevertheless, newly described bacteria were classified based on their morphological properties, leading to polyphyletic taxa. We performed sequencing of 16S rRNA genes and internal transcribed spacer (ITS) regions, together with detailed morphological analysis of hand-picked individuals of novel non-filamentous as well as known filamentous large sulfur bacteria, including the hitherto only partially sequenced species Thiomargarita namibiensis, Thioploca araucae and Thioploca chileae. Based on 128 nearly full-length 16S rRNA-ITS sequences, we propose the retention of the family Beggiatoaceae for the genera closely related to Beggiatoa, as opposed to the recently suggested fusion of all colorless sulfur bacteria into one family, the Thiotrichaceae. Furthermore, we propose the addition of nine Candidatus species along with seven new Candidatus genera to the family Beggiatoaceae. The extended family Beggiatoaceae thus remains monophyletic and is phylogenetically clearly separated from other related families.     

The crystal structure of the apoenzyme of the iron-sulphur cluster-free hydrogenase

The iron-sulphur cluster-free hydrogenase (Hmd, EC 1.12.98.2) from methanogenic archaea is a novel type of hydrogenase that tightly binds an iron-containing cofactor. The iron is coordinated by two CO molecules, one sulphur and a pyridone derivative, which is linked via a phosphodiester bond to a guanosine base. We report here on the crystal structure of the Hmd apoenzyme from Methanocaldococcus jannaschii at 1.75 A and from Methanopyrus kandleri at 2.4 A resolution. Homodimeric Hmd reveals a unique architecture composed of one central and two identical peripheral globular units. The central unit is composed of the intertwined C-terminal segments of both subunits, forming a novel intersubunit fold. The two peripheral units consist of the N-terminal domain of each subunit. The Rossmann fold-like structure of the N-terminal domain contains a mononucleotide-binding site, which could harbour the GMP moiety of the cofactor. Another binding site for the iron-containing cofactor is most probably Cys176, which is located at the bottom of a deep intersubunit cleft and which has been shown to be essential for enzyme activity. Adjacent to the iron of the cofactor modelled as a ligand to Cys176, an extended U-shaped extra electron density, interpreted as a polyethyleneglycol fragment, suggests a binding site for the substrate methenyltetrahydromethanopterin.     

Endothelial Nitric Oxide Synthase Deficient Mice Are Protected from Lipopolysaccharide Induced Acute Lung Injury

Lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria induces acute lung injury (ALI) in mice. This injury is associated with lung edema, inflammation, diffuse alveolar damage, and severe respiratory insufficiency. We have previously reported that LPS-mediated nitric oxide synthase (NOS) uncoupling, through increases in asymmetric dimethylarginine (ADMA), plays an important role in the development of ALI through the generation of reactive oxygen and nitrogen species. Therefore, the focus of this study was to determine whether mice deficient in endothelial NOS (eNOS^-/-) are protected against ALI. In both wild-type and eNOS^-/- mice, ALI was induced by the intratracheal instillation of LPS (2 mg/kg). After 24 hours, we found that eNOS^-/-mice were protected against the LPS mediated increase in inflammatory cell infiltration, inflammatory cytokine production, and lung injury. In addition, LPS exposed eNOS^-/- mice had increased oxygen saturation and improved lung mechanics. The protection in eNOS^-/- mice was associated with an attenuated production of NO, NOS derived superoxide, and peroxynitrite. Furthermore, we found that eNOS^-/- mice had less RhoA activation that correlated with a reduction in RhoA nitration at Tyr³⁴. Finally, we found that the reduction in NOS uncoupling in eNOS^-/- mice was due to a preservation of dimethylarginine dimethylaminohydrolase (DDAH) activity that prevented the LPS-mediated increase in ADMA. Together our data suggest that eNOS derived reactive species play an important role in the development of LPS-mediated lung injury.     

Two-tier vessel for photoautotrophic high-density cultures

Two-tier vessels, developed for culturing of microalgae and cyanobacteria at high cell density on a shaken platform, were assembled from a flat lower chamber to be filled with a CO₂ buffer and an upper flat sterile chamber for the culture that was separated from the lower chamber by a porous polypropylene membrane. Diffusive gas exchange with the atmosphere was controlled by the O₂ outlet channel. Referred to surface area, rates of CO₂ transfer to a shaken weakly alkaline buffer solution across the membrane were higher than those reached on the conventional pathway through the free upper liquid surface. Membrane-mediated CO₂ supply enabled rapid growth of Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 up to ultrahigh cell density. The biomass (dry weight) concentration of Synechococcus cultures reached more than 30 g L^?1 on a buffered medium with adequate concentrations of mineral nutrients. An increase of 15 to 20 g L^?1 was observed during repeated two-day cycles. Separate pathways for CO₂ supply and oxygen outlet prevented significant loss of CO₂. Convective gas flow through the oxygen outlet channel enabled the estimation of the O₂ generation rate. The permeability of the channel for diffusive O₂/N₂ exchange limited the O₂ concentration to a moderate value. It is concluded that shaken flat cultures using CO₂ supply through a porous hydrophobic membrane and diffusive release of O₂ through a separate pathway are promising for research on microalgae and cyanobacteria.     

The Social Structure of Inachus phalangium,a Spider Crab Associated with the Sea Anemone

The behaviour of the spider crab Inachus phalangium (Fabricius, 1775), which lives in association with the sea anemone Anemonia sulcata (Pennant), was studied in the field. The crab was found in the littoral zone of the Mediterranean Sea near Banyuls sur Mer, France, in the whole depth range studied (0.5–25 m). The crabs had a long-lasting association with individual Anemonia sulcata, occasionally with Aiptasia mutabilis. Most crabs were found in association with the same anemone for several days, some crabs were found in association with the same anemone for longer than one month. In the areas studied, on average 65 Inachus phalangium were found on 100 anemones. Crabs released in the vicinity of anemones moved towards them and entered them. Inachus phalangium could walk between the tentacles of Anemonia sulcata and Aiptasia mutabilis without eliciting feeding reactions of the anemone. The crabs left the anemones for moulting. After moulting masking material was removed from the exuvia and used again. The animals returned into an anemone while still soft. Material used for masking, usually algae, could be picked off the body and eaten. Masking material may be a food reservoir in addition to providing camouflage. Anemones were left only during night-time. The crabs left their anemone to moult, to feed in the vicinity, fleeing from larger conspecifics, and to migrate to a different anemone. Outside the anemone's protection Inachus was eaten by several species of fish. Individuals appeared to avoid each other. 57% of all animals were found alone on an anemone. Large males and females were more frequently found alone than were small males and females. Fights were observed between members of the same and of the opposite sex. During fights, legs and claws could be torn off. Adult males migrated more often between anemones and moved over larger distances when migrating than did adult females. Adult males probably migrated in search of sexually mature females. Such a roving strategy is evolutionarily stable only when the higher costs (in terms of energy expenditure and mortality) are compensated for by a higher number of offspring than produced in the alternative, pair-bonding strategy.     

Evaluation of the 23S rRNA gene as target for qPCR based quantification of Frankia in soils

The 23S rRNA gene was evaluated as target for the development of Sybr Green-based quantitative PCR (qPCR) for the analysis of nitrogen-fixing members of the genus Frankia or subgroups of these in soil. A qPCR with a primer combination targeting all nitrogen-fixing frankiae (clusters 1, 2 and 3) resulted in numbers similar to those obtained with a previously developed qPCR using nifH gene sequences, both with respect to introduced and indigenous Frankia populations. Primer combinations more specifically targeting three subgroups of the Alnus host infection group (cluster 1) or members of the Elaeagnus host infection group (cluster 3) were specific for introduced strains of the target group, with numbers corresponding to those obtained by quantification of nitrogen-fixing frankiae with both the 23S rRNA and nifH genes as target. Method verification on indigenous Frankia populations in soils, i.e. in depth profiles from four sites at an Alnus glutinosa stand, revealed declining numbers in the depth profiles, with similar abundance of all nitrogen-fixing frankiae independent of 23S rRNA or nifH gene targets, and corresponding numbers of one group of frankiae of the Alnus host infection only, with no detections of frankiae representing the Elaeagnus, Casuarina, or a second subgroup of the Alnus host infection groups.     

Cholesteryl ester hydrolase activity is abolished in HSL macrophages but unchanged in macrophages lacking KIAA1363

Cholesteryl ester (CE) accumulation in macrophages represents a crucial event during foam cell formation, a hallmark of atherogenesis. Here we investigated the role of two previously described CE hydrolases, hormone-sensitive lipase (HSL) and KIAA1363, in macrophage CE hydrolysis. HSL and KIAA1363 exhibited marked differences in their abilities to hydrolyze CE, triacylglycerol (TG), diacylglycerol (DG), and 2-acetyl monoalkylglycerol ether (AcMAGE), a precursor for biosynthesis of platelet-activating factor (PAF). HSL efficiently cleaved all four substrates, whereas KIAA1363 hydrolyzed only AcMAGE. This contradicts previous studies suggesting that KIAA1363 is a neutral CE hydrolase. Macrophages of KIAA1363^−/− and wild-type mice exhibited identical neutral CE hydrolase activity, which was almost abolished in tissues and macrophages of HSL^−/− mice. Conversely, AcMAGE hydrolase activity was diminished in macrophages and some tissues of KIAA1363^−/− but unchanged in HSL^−/− mice. CE turnover was unaffected in macrophages lacking KIAA1363 and HSL, whereas cAMP-dependent cholesterol efflux was influenced by HSL but not by KIAA1363. Despite decreased CE hydrolase activities, HSL^−/− macrophages exhibited CE accumulation similar to wild-type (WT) macrophages. We conclude that additional enzymes must exist that cooperate with HSL to regulate CE levels in macrophages. KIAA1363 affects AcMAGE hydrolase activity but is of minor importance as a direct CE hydrolase in macrophages.