Nitrate-based niche differentiation by distinct sulfate-reducing bacteria involved in the anaerobic oxidation of methane

Diverse associations between methanotrophic archaea (ANME) and sulfate-reducing bacterial groups (SRB) often co-occur in marine methane seeps; however, the ecophysiology of these different symbiotic associations has not been examined. Here, we applied a combination of molecular, geochemical and Fluorescence in situ hybridization (FISH) coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS) analyses of in situ seep sediments and methane-amended sediment incubations from diverse locations (Eel River Basin, Hydrate Ridge and Costa Rican Margin seeps) to investigate the distribution and physiology of a newly identified subgroup of the Desulfobulbaceae (seepDBB) found in consortia with ANME-2c archaea, and compared these with the more commonly observed associations between the same ANME partner and the Desulfobacteraceae (DSS). FISH analyses revealed aggregates of seepDBB cells in association with ANME-2 from both environmental samples and laboratory incubations that are distinct in their structure relative to co-occurring ANME/DSS consortia. ANME/seepDBB aggregates were most abundant in shallow sediment depths below sulfide-oxidizing microbial mats. Depth profiles of ANME/seepDBB aggregate abundance revealed a positive correlation with elevated porewater nitrate relative to ANME/DSS aggregates in all seep sites examined. This relationship with nitrate was supported by sediment microcosm experiments, in which the abundance of ANME/seepDBB was greater in nitrate-amended incubations relative to the unamended control. FISH-NanoSIMS additionally revealed significantly higher ¹⁵N-nitrate incorporation levels in individual aggregates of ANME/seepDBB relative to ANME/DSS aggregates from the same incubation. These combined results suggest that nitrate is a geochemical effector of ANME/seepDBB aggregate distribution, and provides a unique niche for these consortia through their utilization of a greater range of nitrogen substrates than the ANME/DSS.     

ATP-dependent deoxyribonuclease in Bacillus subtilis and a mutant deficient in this activity

ATP-dependent DNAse activity was measured in rec⁺ and several rec strains of B. subtilis 168. One of the strains (marker recE₅) was found to lack this activity. The enzyme from the wild type was partially purified and some of its properties were determined. The pH optimum is 9.5. Activity is higher at 50° but inactivation occurs on standing at this temperature. The enzyme requires Mg²⁺ (10^?2M) or Mn²⁺ (2·10^?4M). ATP is an absolute requirement and the only other nucleoside triphosphate that can partially replace it is dATP. Lack of activity in the mutant does not seem to be due to the presence of an inhibitor. Results so far do not allow us to conclude as to whether or not the mutant produces an altered enzyme.     

Sulfite-oxido-reductase is involved in the oxidation of sulfite in Desulfocapsa sulfoexigens during disproportionation of thiosulfate and elemental sulfur

The enzymatic pathways of elemental sulfur and thiosulfate disproportionation were investigated using cell-free extract of Desulfocapsa sulfoexigens. Sulfite was observed to be an intermediate in the metabolism of both compounds. Two distinct pathways for the oxidation of sulfite have been identified. One pathway involves APS reductase and ATP sulfurylase and can be described as the reversion of the initial steps of the dissimilatory sulfate reduction pathway. The second pathway is the direct oxidation of sulfite to sulfate by sulfite oxidoreductase. This enzyme has not been reported from sulfate reducers before. Thiosulfate reductase, which cleaves thiosulfate into sulfite and sulfide, was only present in cell-free extract from thiosulfate disproportionating cultures. We propose that this enzyme catalyzes the first step in thiosulfate disproportionation. The initial step in sulfur disproportionation was not identified. Dissimilatory sulfite reductase was present in sulfur and thiosulfate disproportionating cultures. The metabolic function of this enzyme in relation to elemental sulfur or thiosulfate disproportionation was not identified. The presence of the uncouplers HQNO and CCCP in growing cultures had negative effects on both thiosulfate and sulfur disproportionation. CCCP totally inhibited sulfur disproportionation and reduced thiosulfate disproportionation by 80% compared to an unamended control. HQNO reduced thiosulfate disproportionation by 80% and sulfur disproportionation by 90%.     

一株嗜盐嗜碱硫氧化菌的筛选、鉴定及硫氧化特性

【背景】沼气和天然气等清洁能源中往往会含有一定量的硫化氢,硫化氢的存在不仅污染环境,而且对人类危害很大。【目的】以硫代硫酸钠为唯一硫源从巴丹吉林沙漠盐碱湖岸边沉积物中分离筛选得到一株硫氧化菌BDL05,并研究其硫氧化特性。【方法】通过形态观察、生理生化特征及16S rRNA基因序列分析对硫氧化菌BDL05进行鉴定。【结果】菌株BDL05为革兰氏阴性菌,弧状,其16S rRNA基因序列与Thiomicrospira microaerophila ASL 8-2的相似性达99.8%,将其命名为Thiomicrospira microaerophila BDL05。该菌氧化硫代硫酸盐的最适pH为9.3,最适总钠盐浓度为0.8mol/L,在以硫化钠为硫源的气升式反应器中单质硫的生成率为94.7%,生成速率为3.0 mmol/(L·h)。【结论】菌株Thiomicrospira microaerophila BDL05为嗜盐嗜碱硫氧化菌,其耐盐耐碱性较强,比生长速率快,硫化钠氧化能力较强,是一株在气体生物脱硫方面具有应用价值的菌株。     

The Structure of Sox17 Bound to DNA Reveals a Conserved Bending Topology but Selective Protein Interaction Platforms

Sox17 regulates endodermal lineage commitment and is thought to function antagonistically to the pluripotency determinant Sox2. To investigate the biochemical basis for the distinct functions of Sox2 and Sox17, we solved the crystal structure of the high mobility group domain of Sox17 bound to a DNA element derived from the Lama1 enhancer using crystals diffracting to 2.7 Å resolution. Sox17 targets the minor groove and bends the DNA by approximately 80°. The DNA architecture closely resembles the one seen for Sox2/DNA structures, suggesting that the degree of bending is conserved between both proteins and nucleotide substitutions have only marginal effects on the bending topology. Accordingly, affinities of Sox2 and Sox17 for the Lama1 element were found to be identical. However, when the Oct1 contact interface of Sox2 is compared with the corresponding region of Sox17, a significantly altered charge distribution is observed, suggesting differential co-factor recruitment that may explain their biological distinctiveness.     

Precursors of distinct size for chicken αA, αD and β globin mRNAs

The reconstitution of bovine cardiac troponin from its subunits has been investigated using hydrodynamic techniques. Gel filtration (Sephacryl S-300) and sedimentation velocity experiments indicate that troponin-C and troponin-I from a stable binary complex (1:1 mole ratio) with an apparent Stokes' radius of 36 Å (frictional ratio = 1.6). Troponin-C and troponin-T do not interact significantly while troponin-I and troponin-T undergo partial complex formation. The effect of subunit ratio on the reconstitution of whole troponin has been examined by SDS—polyacrylamide gel electrophoresis and gel filtration and the results suggest that native troponin contains the subunits in an equimolar ratio.     

Anaerobic oxidation of thiosulfate and elemental sulfur in Thiobacillus denitrificans

Thiobacillus denitrificans strain RT could be grown anaerobically in batch culture on thiosulfate but not on other reduced sulfur compounds like sulfide, elemental sulfur, thiocyanate, polythionates or sulfite. During growth on thiosulfate the assimilated cell sulfur was derived totally from the outer or sulfane sulfur. Thiosulfate oxidation started with a rhodanese type cleavage between sulfane and sulfone sulfur leading to elemental sulfur and sulfite. As long as thiosulfate was present elemental sulfur was transiently accumulated within the cells in a form that could be shown to be more reactive than elemental sulfur present in a hydrophilic sulfur sol, however, less reactive than sulfane sulfur of polythionates or organic and inorganic polysulfides. When thiosulfate had been completely consumed, intracellular elemental sulfur was rapidly oxidized to sulfate with a specific rate of 45 natom S°/min·mg protein. Extracellularly offered elemental sulfur was not oxidized under anaerobic conditions.     

两种鱼类Sox基因保守区的克隆和Sox9基因保守区内含子的遗传变异性

Sox基因家族是生物个体发育过程中关键的调控因子,尤其在睾丸、神经、肌肉和骨骼等组织分化和发育过程中发挥着重要的作用(Fosteretal.,1 994 ;PevnyandLovell Badge ,1 997;Schepersetal.,2 0 0 0 ;ClarksonandHarley ,2 0 0 2 ;Changetal.,2 0 0 2 ;Monteroetal.,2 0 0 2 ;Ga     

Roseobacter clade bacteria are abundant in coastal sediments and encode a novel combination of sulfur oxidation genes

Roseobacter clade bacteria (RCB) are abundant in marine bacterioplankton worldwide and central to pelagic sulfur cycling. Very little is known about their abundance and function in marine sediments. We investigated the abundance, diversity and sulfur oxidation potential of RCB in surface sediments of two tidal flats. Here, RCB accounted for up to 9.6% of all cells and exceeded abundances commonly known for pelagic RCB by 1000-fold as revealed by fluorescence in situ hybridization (FISH). Phylogenetic analysis of 16S rRNA and sulfate thiohydrolase (SoxB) genes indicated diverse, possibly sulfur-oxidizing RCB related to sequences known from bacterioplankton and marine biofilms. To investigate the sulfur oxidation potential of RCB in sediments in more detail, we analyzed a metagenomic fragment from a RCB. This fragment encoded the reverse dissimilatory sulfite reductase (rDSR) pathway, which was not yet found in RCB, a novel type of sulfite dehydrogenase (SoeABC) and the Sox multi-enzyme complex including the SoxCD subunits. This was unexpected as soxCD and dsr genes were presumed to be mutually exclusive in sulfur-oxidizing prokaryotes. This unique gene arrangement would allow a metabolic flexibility beyond known sulfur-oxidizing pathways. We confirmed the presence of dsrA by geneFISH in closely related RCB from an enrichment culture. Our results show that RCB are an integral part of the microbial community in marine sediments, where they possibly oxidize inorganic and organic sulfur compounds in oxic and suboxic sediment layers.     

A nested gene in Streptomyces bacteria encodes a protein involved in quaternary complex formation

The gene pgaM is involved in the biosynthesis of an angucycline-type polyketide antibiotic in Streptomyces sp. PGA64. It encodes a two-domain polypeptide consisting of an N-terminal flavoprotein oxygenase and a C-terminal short-chain alcohol dehydrogenase/reductase, which are fused together at the translational level as a result of end codon deletion. Here we show that translation also initiates at an internal start codon that enables independent expression of a separate reductase subunit, PgaMred. This confirms that the gene exhibits a rare viral-like arrangement of two overlapping reading frames that allows simultaneous expression of two alternative forms of the protein. Together, these two proteins associate to form a stable non-covalent complex, the native form of PgaM. The reductase subunit PgaMred is shown to provide enzyme stability and to affect the redox state of the oxygenase domain FAD. Finally, a model for the quaternary structure of the complex that explains the necessity for a nested gene system and the unusual behaviour of the protein subunits in vitro is presented.     

Stable sulfur isotope fractionation and discrimination between the sulfur atoms of thiosulfate during oxidation by Halothiobacillus neapolitanus

Growing cultures and nongrowing suspensions of Halothiobacillus neapolitanus selectively fractionated (32)S and (34)S during the oxidation of the sulfane- and sulfonate-sulfur atoms of thiosulfate. Sulfate was enriched in (32)S, with delta(34)S reaching -6.3 per thousand relative to the precursor sulfonate-sulfur of thiosulfate, which was progressively resynthesized from the thiosulfate-sulfane-sulfur during thiosulfate metabolism. Polythionates, principally trithionate, accumulated during thiosulfate oxidation and showed progressive increase in the relative (34)S content of their sulfonate groups, with delta(34)S values up to +20 per thousand, relative to the substrate sulfur. The origins of the sulfur in the sulfate and polythionate products of oxidation were tracked by the use thiosulfate labelled with (35)S in each of its sulfur atoms, enabling determination of the flow of the sulfur atoms into the oxidation products. The results confirm that highly significant fractionation of stable sulfur isotopes can be catalyzed by thiobacilli oxidizing thiosulfate, but that differences in the (34)S/(32)S ratios of the nonequivalent constituent sulfur atoms of the thiosulfate used as substrate mean that the oxidative fate of each atom needs separate determination. The data are very significant to the understanding of bacterial sulfur-compound oxidation and highly relevant to the origins of biogenic sulfate minerals.     

Interaction between SAP97 and PSD-95, two Maguk proteins involved in synaptic trafficking of AMPA receptors

Synapse-associated protein 97 (SAP97) and postsynaptic density 95 (PSD-95) are closely related membrane-associated guanylate kinase homologs (Maguks) implicated in the synaptic targeting and anchoring of alpha-amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid (AMPA)-selective glutamate receptors. Prompted by accumulating evidence for an oligomeric nature of Maguks, we examined the potential of SAP97 and PSD-95 to form heteromeric complexes. SAP97 and PSD-95 coimmunoprecipitated from rat brain detergent extracts and subsequent glutathione S-transferase pull-down and immunoprecipitation experiments showed that the interaction is mediated by binding of the N-terminal segment of SAP97 (SAP97(NTD)) to the Src homology 3 domain of PSD-95 (PSD-95(SH3)). In cultured hippocampal neurons, expression of green fluorescent protein-tagged PSD-95 triggered accumulation of SAP97 in synaptic spines, which was totally inhibited by coexpression of PSD-95(SH3). Furthermore, overexpression of green fluorescent protein-PSD-95 induced dendritic clustering of GluR-A subunit-containing AMPA receptors, which was strongly inhibited by cotransfection with SAP97(NTD) and PSD-95(SH3) constructs. Our results demonstrated a direct interaction between SAP97 and PSD-95 and suggested that this association may play a functional role in the trafficking and clustering of AMPA receptors.     

DNA distortion and specificity in a sequence-specific endonuclease

Five new structures of the Q138F HincII enzyme bound to a total of three different DNA sequences and three different metal ions (Ca²⁺, Mg²⁺, and Mn²⁺) are presented. While previous structures were produced from soaking Ca²⁺ into preformed Q138F HincII/DNA crystals, the new structures are derived from cocrystallization with Ca²⁺, Mg²⁺, or Mn²⁺. The Mn²⁺-bound structure provides the first view of a product complex of Q138F HincII with cleaved DNA. Binding studies and a crystal structure show how Ca²⁺ allows trapping of a Q138F HincII complex with noncognate DNA in a catalytically incompetent conformation. Many Q138F HincII/DNA structures show asymmetry, despite the binding of a symmetric substrate by a symmetric enzyme. The various complexes are fit into a model describing the different conformations of the DNA-bound enzyme and show how DNA conformational energetics determine DNA-cleavage rates by the Q138F HincII enzyme.     

Physical map of DNA from a new cauliflower mosaic virus strain

The restriction enzymes AluI, BamHI, BglII, EcoRI, HindIII, and SalI have been used to characterize and map a new cauliflower mosaic virus strain (Cabb-S). These fragments have been ordered by examining their overlapping regions after double enzymatic digestion. The single SalI cleavage site was chosen as the point of origin. We compare this strain with those already described.     

The relation between solution association and surface activity of the hydrophobin HFBI from Trichoderma reesei

Hydrophobins are small fungal surface active proteins that self-assemble at interfaces into films with nanoscale structures. The hydrophobin HFBI from Trichoderma reesei has been shown to associate in solution into tetramers but the role of this association on the function of HFBI has remained unclear. We produced two HFBI variants that showed a significant shift in solution association equilibrium towards the tetramer state. However, this enhanced solution association did not alter the surface properties of the variant HFBIs. The results show that there is not a strong relationship between HFBI solution association state and surface properties such as surface activity.     

Regulation of ammonium assimilation in Haloferax mediterranei: Interaction between glutamine synthetase and two GlnK proteins

GlnK proteins belong to the PII superfamily of signal transduction proteins and are involved in the regulation of nitrogen metabolism. These proteins are normally encoded in an operon together with the structural gene for the ammonium transporter AmtB. Haloferax mediterranei possesses two genes encoding for GlnK, specifically, glnK₁ and glnK₂. The present study marks the first investigation of PII proteins in haloarchaea, and provides evidence for the direct interaction between glutamine synthetase and both GlnK₁ and GlnK₂. Complex formation between glutamine synthetase and the two GlnK proteins is demonstrated with pure recombinant protein samples using in vitro activity assays, gel filtration chromatography and western blotting. This protein–protein interaction increases glutamine synthetase activity in the presence of 2-oxoglutarate. Separate experiments that were carried out with GlnK₁ and GlnK₂ produced equivalent results.     

Structural transitions of porin, a transmembrane protein

Conformational transitions of porin were monitored using 3 independent criteria: (i) oligomeric state as observed by SDS-polyacrylamide gel electrophoresis; (ii) spectroscopic titrations (ultraviolet and circular dichroism) and (iii) chemical modifications. Four pH-dependent transitions were observed with half-maximal changes occurring at pH values of 1.6, 3.5, 11.2 and 12.4. Two of these pH values differ significantly from intrinsic pK values of the constituent amino acids of this membrane protein. Since porin is very polar despite its location predominantly within the outer membranes, this may be due to ion pair formation in the hydrophobic environment of the membrane.