Deltaproteobacterium Strain KaireiS1, a Mesophilic, Hydrogen-Oxidizing and Sulfate-Reducing Bacterium From an Inactive Deep-Sea Hydrothermal Chimney.

A novel deltaproteobacterial, mesophilic, hydrogen-oxidizing, and sulfate-reducing bacterium (strain KaireiS1) was highly enriched from an inactive chimney located in the active zone of the Kairei hydrothermal vent field (Central Indian Ridge) in the Indian Ocean. Based on 16S rRNA gene analyses, strain KaireiS1 is the currently only cultured representative of a cluster of uncultured Deltaproteobacteria, positioned within the Desulfobulbaceae family, between the Desulfobulbus genus and the "Cable Bacteria." A facultative autotrophic lifestyle of KaireiS1 is indicated by its growth in the absence of organic compounds, measurements of CO2-fixation rates, and activity measurements of carbon monoxide dehydrogenase, the key enzyme of the reductive Acetyl-CoA pathway. Apart from hydrogen, strain KaireiS1 can also use propionate, lactate, and pentadecane as electron donors. However, the highest cell numbers were reached when grown autotrophically with molecular hydrogen. Hydrogen uptake activity was found in membrane and soluble fractions of cell-free extracts and reached up to 2,981±129 nmol H2*min-1*mg-1 of partially purified protein. Commonly, autotrophic sulfate-reducing bacteria from the Deltaproteobacteria class, thriving in hydrothermal vent habitats are described as thermophiles. Given its physiological characteristics and specific isolation source, strain KaireiS1 demonstrates a previously unnoticed potential for microbial sulfate reduction by autotrophs taking place at moderate temperatures in hydrothermal vent fields.

Metagenomics Meets Electrochemistry: Utilizing the Huge Catalytic Potential From the Uncultured Microbial Majority for Energy-Storage.

Hydrogen can in the future serve as an advantageous carrier of renewable energy if its production via water electrolysis and utilization in fuel cells are realized with high energy efficiency and non-precious electrocatalysts. In an unprecedented novel combination of structured electrodes with hydrogen converting enzymes from the uncultured and thus largely inaccessible microbial majority (>99%) we address this challenge. The geometrically defined electrodes with large specific surface area allow for low overpotentials and high energy efficiencies to be achieved. Enzymatic hydrogen evolution electrocatalysts are used as alternatives to noble metals. The enzymes are harnessed from the environmental microbial DNA (metagenomes) of hydrothermal vents exhibiting dynamic hydrogen and oxygen concentrations and are recovered via a recently developed novel activity-based screening tool. The screen enables us to target currently unrecognized hydrogenase enzymes from metagenomes via direct expression in a surrogate host microorganism. This circumvents the need for cultivation of the source organisms, the primary bottleneck when harnessing enzymes from microbes. One hydrogen converting metagenome-derived enzyme exhibited high activity and unusually high stability when dispersed on a TiO2-coated polyacrylonitrile fiber electrode. Our results highlight the tremendous potential of enzymes derived from uncultured microorganisms for applications in energy conversion and storage technologies.

Horizontal acquisition of hydrogen conversion ability and other habitat adaptations in the Hydrogenovibrio strains SP-41 and XCL-2.

BACKGROUND: Obligate sulfur oxidizing chemolithoauthotrophic strains of Hydrogenovibrio crunogenus have been isolated from multiple hydrothermal vent associated habitats. However, a hydrogenase gene cluster (encoding the hydrogen converting enzyme and its maturation/assembly machinery) detected on the first sequenced H. crunogenus strain (XCL-2) suggested that hydrogen conversion may also play a role in this organism. Yet, numerous experiments have underlined XCL-2's inability to consume hydrogen under the tested conditions. A recent study showed that the closely related strain SP-41 contains a homolog of the XCL-2 hydrogenase (a group 1b [NiFe]-hydrogenase), but that it can indeed use hydrogen. Hence, the question remained unresolved, why SP-41 is capable of using hydrogen, while XCL-2 is not. RESULTS: Here, we present the genome sequence of the SP-41 strain and compare it to that of the XCL-2 strain. We show that the chromosome of SP-41 codes for a further hydrogenase gene cluster, including two additional hydrogenases: the first appears to be a group 1d periplasmic membrane-anchored hydrogenase, and the second a group 2b sensory hydrogenase. The region where these genes are located was likely acquired horizontally and exhibits similarity to other Hydrogenovibrio species (H. thermophilus MA2-6 and H. marinus MH-110 T) and other hydrogen oxidizing Proteobacteria (Cupriavidus necator H16 and Ghiorsea bivora TAG-1 T). The genomes of XCL-2 and SP-41 show a strong conservation in gene order. However, several short genomic regions are not contained in the genome of the other strain. These exclusive regions are often associated with signs of DNA mobility, such as genes coding for transposases. They code for transport systems and/or extend the metabolic potential of the strains. CONCLUSIONS: Our results suggest that horizontal gene transfer plays an important role in shaping the genomes of these strains, as a likely mechanism for habitat adaptation, including, but not limited to the transfer of the hydrogen conversion ability.

Interaction of Cm(III) with human serum albumin studied by time-resolved laser fluorescence spectroscopy and NMR.

The complexation of Cm(III) with human serum albumin (HSA) was investigated using time-resolved laser fluorescence spectroscopy (TRLFS). The Cm(III) HSA species is dominating the speciation between pH 7.0 and 9.3. The first coordination sphere is composed by three to four H2O molecules and five to six coordinating ligands from the protein. For the complex formation at pH 8.0 a conditional stability constant of logK = 6.16 ±â€¯0.50 was determined. Furthermore, information on the Cm(III) HSA binding site were obtained. With increasing Cu(II) concentration the Cm(III) HSA complexation is suppressed whereas the addition of Zn(II) has no effect. This points to the complexation of Cm(III) at the N-terminal binding site (NTS) which is the primary Cu(II) binding site. NMR experiments with Cu(II), Eu(III) and Am(III) HSA show a decrease of the peak assigned to the His C2 proton of His 3, which is part of the NTS, with increasing metal ion concentration. This confirms the complexation of Eu(III) and Am(III) at the Cu(II) binding site NTS. The results presented in this study contribute to a better understanding of relevant biochemical reactions of incorporated actinides.

Microbially Mediated Hydrogen Cycling in Deep-Sea Hydrothermal Vents.

Deep-sea hydrothermal vents may provide one of the largest reservoirs on Earth for hydrogen-oxidizing microorganisms. Depending on the type of geological setting, hydrothermal environments can be considerably enriched in hydrogen (up to millimolar concentrations). As hot, reduced hydrothermal fluids ascend to the seafloor they mix with entrained cold, oxygenated seawater, forming thermal and chemical gradients along their fluid pathways. Consequently, in these thermally and chemically dynamic habitats biochemically distinct hydrogenases (adapted to various temperature regimes, oxygen and hydrogen concentrations) from physiologically and phylogenetically diverse Bacteria and Archaea can be expected. Hydrogen oxidation is one of the important inorganic energy sources in these habitats, capable of providing relatively large amounts of energy (237 kJ/mol H2) for driving ATP synthesis and autotrophic CO2 fixation. Therefore, hydrogen-oxidizing organisms play a key role in deep-sea hydrothermal vent ecosystems as they can be considerably involved in light-independent primary biomass production. So far, the specific role of hydrogen-utilizing microorganisms in deep-sea hydrothermal ecosystems has been investigated by isolating hydrogen-oxidizers, measuring hydrogen consumption (ex situ), studying hydrogenase gene distribution and more recently by analyzing metatranscriptomic and metaproteomic data. Here we summarize this available knowledge and discuss the advent of new techniques for the identification of novel hydrogen-uptake and -evolving enzymes from hydrothermal vent microorganisms.

Incorporation of transuranium elements: coordination of Cm(iii) to human serum transferrin.

The coordination environment of Cm(iii) bound at the Fe(iii) binding sites of transferrin was investigated using a combined experimental and theoretical approach. Complexation studies with two hTf/2N single point mutants, Y95F (Tyr → Phe) and H249A (His → Ala) were performed. The substitution of Tyr 95 by the non-complexing Phe prevents Cm(iii) from forming of a strong, multidentate complex with the mutant. In contrast, with the H249A mutant Cm(iii) complexation at the binding site still occurs although a slightly higher pH is required to form the complex. This elucidates that His plays a minor role and is not a key ligand like Tyr 95. MD/DFT calculations of Cm(iii) bound at the N-terminal binding site provide further structural information. All coordinating groups present in the Fe(iii) transferrin complex are also found for Cm(iii), i.e. Asp 63, Tyr 95, Tyr 188 and His 249. Additionally, two water molecules, one monodentate and one bidentate carbonate ion complete the coordination environment. This structure of the Cm(iii) hTf/2N complex is confirmed by vibronic sideband spectroscopy which allows an identification of the directly coordinating groups. The results underline an involvement of Asp 63, Tyr 95, Tyr 188 and His 249 as well as carbonate in Cm(iii) coordination at the transferrin Fe(iii) binding site.

Hydrothermal chimneys host habitat-specific microbial communities: analogues for studying the possible impact of mining seafloor massive sulfide deposits.

To assess the risk that mining of seafloor massive sulfides (SMS) from extinct hydrothermal vent environments has for changing the ecosystem irreversibly, we sampled SMS analogous habitats from the Kairei and the Pelagia vent fields along the Indian Ridge. In total 19.8 million 16S rRNA tags from 14 different sites were analyzed and the microbial communities were compared with each other and with publicly available data sets from other marine environments. The chimneys appear to provide habitats for microorganisms that are not found or only detectable in very low numbers in other marine habitats. The chimneys also host rare organisms and may function as a vital part of the ocean's seed bank. Many of the reads from active and inactive chimney samples were clustered into OTUs, with low or no resemblance to known species. Since we are unaware of the chemical reactions catalyzed by these unknown organisms, the impact of this diversity loss and bio-geo-coupling is hard to predict. Given that chimney structures can be considered SMS analogues, removal of sulfide deposits from the seafloor in the Kairei and Pelagia fields will most likely alter microbial compositions and affect element cycling in the benthic regions and probably beyond.

Novel hydrogenases from deep-sea hydrothermal vent metagenomes identified by a recently developed activity-based screen.

Hydrogen is one of the most common elements on Earth. The enzymes converting molecular hydrogen into protons and electrons are the hydrogenases. Hydrogenases are ubiquitously distributed in all three domains of life where they play a central role in cell metabolism. So far, the recovery of hydrogenases has been restricted to culture-dependent and sequence-based approaches. We have recently developed the only activity-based screen for seeking H2-uptake enzymes from metagenomes without having to rely on enrichment and isolation of hydrogen-oxidizing microorganisms or prior metagenomic sequencing. When screening 14,400 fosmid clones from three hydrothermal vent metagenomes using this solely activity-based approach, four clones with H2-uptake activity were identified with specific activities of up to 258 ± 19 nmol H2/min/mg protein of partially purified membrane fractions. The respective metagenomic fragments exhibited mostly very low or no similarities to sequences in the public databases. A search with hidden Markov models for different hydrogenase groups showed no hits for three of the four metagenomic inserts, indicating that they do not encode for classical hydrogenases. Our activity-based screen serves as a powerful tool for the discovery of (novel) hydrogenases which would not have been identified by the currently available techniques. This screen can be ideally combined with culture- and sequence-based approaches to investigate the tremendous hydrogen-converting potential in the environment.

Activity-Based Screening of Metagenomic Libraries for Hydrogenase Enzymes.

Here we outline how to identify hydrogenase enzymes from metagenomic libraries through an activity-based screening approach. A metagenomic fosmid library is constructed in E. coli and the fosmids are transferred into a hydrogenase deletion mutant of Shewanella oneidensis (ΔhyaB) via triparental mating. If a fosmid exhibits hydrogen uptake activity, S. oneidensis' phenotype is restored and hydrogenase activity is indicated by a color change of the medium from yellow to colorless. This new method enables screening of 48 metagenomic fosmid clones in parallel.