The Oman Drilling Project established an “Active Alteration” multi-borehole observatory in dunite and harzburgite undergoing low-temperature serpentinization in the Samail ophiolite. The highly serpentinized rocks are in contact with strongly reducing fluids. Distinct hydrological regimes, governed by differences in rock porosity and fracture density, give rise to steep redox (Eh +200 to -750 mV) and pH (pH range 8.5 to 11.2) gradients within the 300 to 400 meter deep boreholes. The serpentinites and fluids host an active subsurface ecosystem. Microbial cell abundances vary at least 6 orders of magnitude, from ≤3.5*101 cells/g to 2.9*107 cells/gram. Low levels of biological sulfate reduction (2-1000 fmol/cm3/day) can be detected in rock cores, particularly in rocks in contact with reduced groundwaters with pH <10.5. Thermodesulfovibrio is the predominant sulfate reducer identified via metagenomic sequencing of adjacent groundwater communities. We infer that transport and reaction of microbially generated sulfide with the serpentine and brucite assemblages gives rise to optical darkening and sulfide overprinting, including the formation of tochilinite-vallerite group minerals, potentially serving as an indicator that this system is inhabited by microbial life. Olivine mesh-cores replaced with ferroan brucite and minor awaruite, abundant veins containing hydroandradite garnet and polyhedral serpentine, and late-stage carbonate veins are suggested as targets for future spatially-resolved life-detection investigations. The high-quality whole-round core samples that have been preserved can be further probed to define how life distributes itself and functions within a system where chemical disequilibria are sustained by low-temperature water/rock interaction, and how biosignatures of in-situ microbial activity are generated.

The potential for molecular hydrogen (H-OH- groundwaters bearing up to 4.05 μmol⋅L-1 H2 , 3.81 μmol⋅L-1 methane (CH4) and 946 μmol⋅L-1 sulfate (SO42-) revealed an ecosystem dominated by Bacteria affiliated with the class Thermodesulfovibrionia, a group of chemolithoheterotrophs supported by H2 oxidation coupled to SO42- reduction. In shallower, oxidized Mg2+-HCO3- groundwaters, aerobic and denitrifying heterotrophs were relatively more abundant. High δ13C and δD of CH4 (up to 23.9 ‰ VPDB and 45 ‰ VSMOW , respectively), indicated microbial CH4 oxidation, particularly in Ca2+-OH- waters with evidence of mixing with Mg2+-HCO3- waters. This study demonstrates the power of spatially resolving groundwaters to probe their distinct geochemical conditions and chemosynthetic communities. Such information will help improve predictions of where microbial activity in fractured rock ecosystems might occur, including beyond Earth.

Dissimilatory sulfite reductase is an ancient enzyme that has linked the global sulfur and carbon biogeochemical cycles since at least 3.47 Gya. While much has been learned about the phylogenetic distribution and diversity of DsrAB across environmental gradients, far less is known about the structural changes that occurred to maintain DsrAB function as the enzyme accompanied diversification of sulfate/sulfite reducing organisms (SRO) into new environments. Analyses of available crystal structures of DsrAB from Archaeoglobus fulgidus and Desulfovibrio vulgaris, representing early and late evolving lineages, respectively, show that certain features of DsrAB are structurally conserved, including active siro-heme binding motifs. Whether such structural features are conserved among DsrAB recovered from varied environments, including hot spring environments that host representatives of the earliest evolving SRO lineage (e.g., MV2-Eury), is not known. To begin to overcome these gaps in our understanding of the evolution of DsrAB, structural models from MV2.Eury were generated and evolutionary sequence co-variance analyses were conducted on a curated DsrAB database. Phylogenetically diverse DsrAB harbor many conserved functional residues including those that ligate active siro-heme(s). However, evolutionary co-variance analysis of monomeric DsrAB subunits revealed several False Positive Evolutionary Couplings (FPEC) that correspond to residues that have co-evolved despite being too spatially distant in the monomeric structure to allow for direct contact. One set of FPECs corresponds to residues that form a structural path between the two active siro-heme moieties across the interface between heterodimers, suggesting the potential for allostery or electron transfer within the enzyme complex. Other FPECs correspond to structural loops and gaps that may have been selected to stabilize enzyme function in different environments. These structural bioinformatics results suggest that DsrAB has maintained allosteric communication pathways between subunits as SRO diversified into new environments. The observations outlined here provide a framework for future biochemical and structural analyses of DsrAB to examine potential allosteric control of this enzyme.