Characterization of microbiomic and geochemical compositions across the photosynthetic fringe.

Hot spring outflow channels provide geochemical gradients that are reflected in microbial community compositions. In many hot spring outflows, there is a distinct visual demarcation as the community transitions from predominantly chemotrophs to having visible pigments from phototrophs. It has been hypothesized that this transition to phototrophy, known as the photosynthetic fringe, is a result of the pH, temperature, and/or sulfide concentration gradients in the hot spring outflows. Here, we explicitly evaluated the predictive capability of geochemistry in determining the location of the photosynthetic fringe in hot spring outflows. A total of 46 samples were taken from 12 hot spring outflows in Yellowstone National Park that spanned pH values from 1.9 to 9.0 and temperatures from 28.9 to 92.2°C. Sampling locations were selected to be equidistant in geochemical space above and below the photosynthetic fringe based on linear discriminant analysis. Although pH, temperature, and total sulfide concentrations have all previously been cited as determining factors for microbial community composition, total sulfide did not correlate with microbial community composition with statistical significance in non-metric multidimensional scaling. In contrast, pH, temperature, ammonia, dissolved organic carbon, dissolved inorganic carbon, and dissolved oxygen did correlate with the microbial community composition with statistical significance. Additionally, there was observed statistical significance between beta diversity and the relative position to the photosynthetic fringe with sites above the photosynthetic fringe being significantly different from those at or below the photosynthetic fringe according to canonical correspondence analysis. However, in combination, the geochemical parameters considered in this study only accounted for 35% of the variation in microbial community composition determined by redundancy analysis. In co-occurrence network analyses, each clique correlated with either pH and/or temperature, whereas sulfide concentrations only correlated with individual nodes. These results indicate that there is a complex interplay between geochemical variables and the position of the photosynthetic fringe that cannot be fully explained by statistical correlations with the individual geochemical variables included in this study.

An examination of protist diversity in serpentinization-hosted ecosystems of the Samail Ophiolite of Oman.

In the Samail Ophiolite of Oman, the geological process of serpentinization produces reduced, hydrogen rich, hyperalkaline (pH > 11) fluids. These fluids are generated through water reacting with ultramafic rock from the upper mantle in the subsurface. On Earth's continents, serpentinized fluids can be expressed at the surface where they can mix with circumneutral surface water and subsequently generate a pH gradient (∼pH 8 to pH > 11) in addition to variations in other chemical parameters such as dissolved CO2, O2, and H2. Globally, archaeal and bacterial community diversity has been shown to reflect geochemical gradients established by the process of serpentinization. It is unknown if the same is true for microorganisms of the domain Eukarya (eukaryotes). In this study, using 18S rRNA gene amplicon sequencing, we explore the diversity of microbial eukaryotes called protists in sediments of serpentinized fluids in Oman. We demonstrate that protist community composition and diversity correlate significantly with variations in pH, with protist richness being significantly lower in sediments of hyperalkaline fluids. In addition to pH, the availability of CO2 to phototrophic protists, the composition of potential food sources (prokaryotes) for heterotrophic protists and the concentration of O2 for anaerobic protists are factors that likely shape overall protist community composition and diversity along the geochemical gradient. The taxonomy of the protist 18S rRNA gene sequences indicates the presence of protists that are involved in carbon cycling in serpentinized fluids of Oman. Therefore, as we evaluate the applicability of serpentinization for carbon sequestration, the presence and diversity of protists should be considered.

[FeFe]-hydrogenase abundance and diversity along a vertical redox gradient in Great Salt Lake, USA.

The use of [FeFe]-hydrogenase enzymes for the biotechnological production of H2 or other reduced products has been limited by their sensitivity to oxygen (O2). Here, we apply a PCR-directed approach to determine the distribution, abundance, and diversity of hydA gene fragments along co-varying salinity and O2 gradients in a vertical water column of Great Salt Lake (GSL), UT. The distribution of hydA was constrained to water column transects that had high salt and relatively low O2 concentrations. Recovered HydA deduced amino acid sequences were enriched in hydrophilic amino acids relative to HydA from less saline environments. In addition, they harbored interesting variations in the amino acid environment of the complex H-cluster metalloenzyme active site and putative gas transfer channels that may be important for both H2 transfer and O2 susceptibility. A phylogenetic framework was created to infer the accessory cluster composition and quaternary structure of recovered HydA protein sequences based on phylogenetic relationships and the gene contexts of known complete HydA sequences. Numerous recovered HydA are predicted to harbor multiple N- and C-terminal accessory iron-sulfur cluster binding domains and are likely to exist as multisubunit complexes. This study indicates an important role for [FeFe]-hydrogenases in the functioning of the GSL ecosystem and provides new target genes and variants for use in identifying O2 tolerant enzymes for biotechnological applications.

Competition for ammonia influences the structure of chemotrophic communities in geothermal springs.

Source waters sampled from Perpetual Spouter hot spring (pH 7.03, 86.4°C), Yellowstone National Park, WY, have low concentrations of total ammonia, nitrite, and nitrate, suggesting nitrogen (N) limitation and/or tight coupling of N cycling processes. Dominant small-subunit rRNA sequences in Perpetual Spouter source sediments are closely affiliated with the ammonia-oxidizing archaeon "Candidatus Nitrosocaldus yellowstonii" and the putatively nitrogen-fixing (diazotrophic) bacterium Thermocrinis albus, respectively, suggesting that these populations may interact at the level of the bioavailable N pool, specifically, ammonia. This hypothesis was evaluated by using a combination of geochemical, physiological, and transcriptomic analyses of sediment microcosms. Amendment of microcosms with allylthiourea, an inhibitor of ammonia oxidation, decreased rates of acetylene reduction (a proxy for N2 fixation) and nitrite production (a proxy for ammonia oxidation) and decreased transcript levels of structural genes involved in both nitrogen fixation (nifH) and ammonia oxidation (amoA). In contrast, amendment of microcosms with ammonia stimulated nitrite production and increased amoA transcript levels while it suppressed rates of acetylene reduction and decreased nifH transcript levels. Sequencing of amplified nifH and amoA transcripts from native sediments, as well as microcosms, at 2 and 4 h postamendment, indicates that the dominant and responsive populations involved in ammonia oxidation and N2 fixation are closely affiliated with Ca. Nitrosocaldus yellowstonii and T. albus, respectively. Collectively, these results suggest that ammonia-oxidizing archaea, such as Ca. Nitrosocaldus yellowstonii, have an apparent affinity for ammonia that is higher than that of the diazotrophs present in this ecosystem. Depletion of the bioavailable N pool through the activity of ammonia-oxidizing archaea likely represents a strong selective pressure for the inclusion of organisms capable of nitrogen fixation in geothermal communities. These observations help to explain the strong pattern in the codistribution of ammonia-oxidizing archaea and diazotrophs in circumneutral-to-alkaline geothermal springs.

Role of the Tyr-Cys cross-link to the active site properties of galactose oxidase.

The catalytically relevant, oxidized state of the active site [Cu(II)-Y·-C] of galactose oxidase (GO) is composed of antiferromagnetically coupled Cu(II) and a post-translationally generated Tyr-Cys radical cofactor [Y·-C]. The thioether bond of the Tyr-Cys cross-link has been shown experimentally to affect the stability, the reduction potential, and the catalytic efficiency of the GO active site. However, the origin of these structural and energetic effects on the GO active site has not yet been investigated in detail. Here we present copper and sulfur K-edge X-ray absorption data and a systematic computational approach for evaluating the role of the Tyr-Cys cross-link in GO. The sulfur contribution of the Tyr-Cys cross-link to the redox active orbital is estimated from sulfur K-edge X-ray absorption spectra of oxidized GO to be about 24 ± 3%, compared to the values from computational models of apo-GO (15%) and holo-GO (22%). The results for the apo-GO computational models are in good agreement with the previously reported value for apo-GO (20 ± 3% from EPR). Surprisingly, the Tyr-Cys cross-link has only a minimal effect on the inner sphere, coordination geometry of the Cu site in the holo-protein. Its effect on the electronic structure is more striking as it facilitates the delocalization of the redox active orbital onto the thioether sulfur derived from Cys, thereby reducing the spin coupling between the [Y·-C] radical and the Cu(II) center (752 cm(-1)) relative to the unsubstituted [Y·] radical and the Cu(II) center (2210 cm(-1)). Energetically, the Tyr-Cys cross-link lowers the reduction potential by about 75 mV (calculated) allowing a more facile oxidation of the holo active site versus the site without the cross-link. Overall, the Tyr-Cys cross-link confers unique ground state properties on the GO active site that tunes its function in a remarkably nuanced fashion.