Deep-branching acetogens in serpentinized subsurface fluids of Oman.

Little is known of acetogens in contemporary serpentinizing systems, despite widely supported theories that serpentinite-hosted environments supported the first life on Earth via acetogenesis. To address this knowledge gap, genome-resolved metagenomics was applied to subsurface fracture water communities from an area of active serpentinization in the Samail Ophiolite, Sultanate of Oman. Two deeply branching putative bacterial acetogen types were identified in the communities belonging to the Acetothermia (hereafter, types I and II) that exhibited distinct distributions among waters with lower and higher water-rock reaction (i.e., serpentinization influence), respectively. Metabolic reconstructions revealed contrasting core metabolic pathways of type I and II Acetothermia, including in acetogenic pathway components (e.g., bacterial- vs. archaeal-like carbon monoxide dehydrogenases [CODH], respectively), hydrogen use to drive acetogenesis, and chemiosmotic potential generation via respiratory (type I) or canonical acetogen ferredoxin-based complexes (type II). Notably, type II Acetothermia metabolic pathways allow for use of serpentinization-derived substrates and implicate them as key primary producers in contemporary hyperalkaline serpentinite environments. Phylogenomic analyses indicate that 1) archaeal-like CODH of the type II genomes and those of other serpentinite-associated Bacteria derive from a deeply rooted horizontal transfer or origin among archaeal methanogens and 2) Acetothermia are among the earliest evolving bacterial lineages. The discovery of dominant and early-branching acetogens in subsurface waters of the largest near-surface serpentinite formation provides insight into the physiological traits that likely facilitated rock-supported life to flourish on a primitive Earth and possibly on other rocky planets undergoing serpentinization.

Transformation of low-molecular-weight organic acids by microbial endoliths in subsurface mafic and ultramafic igneous rock.

A growing body of work indicates that continental subsurface rocks host a substantial portion of the Earth's biosphere. However, the activities of microbial cells inhabiting pore spaces and microfractures in subsurface rocks remain underexplored. Here, we develop and optimize microcosm assays to detect organic acid transformation activities of cells residing in mafic to ultramafic igneous rocks. Application of this assay to gabbro core from the Stillwater Mine, Montana, USA, revealed maximal methane production from acetate at temperatures approximating that of the mine. Controls show that these activities are not due to contamination introduced during drilling, exhumation, or laboratory processing of the core. The assay was then applied to rocks cored from the Samail Ophiolite, Oman, which is undergoing low-temperature serpentinization. Production of (i) carbon dioxide from acetate and formate and (ii) methane from formate were detected in a dunite/harzburgite rock core interfacing pH 9.6 waters, and estimates of microbial activities were up to three orders of magnitude higher in the rock core pore space than in corresponding waters. The detection of endolithic microbial activities in igneous rocks has implications for life detection on other planetary bodies where similar rock types prevail, such as Mars, Europa and Enceladus.

Molecular Evidence for an Active Microbial Methane Cycle in Subsurface Serpentinite-Hosted Groundwaters in the Samail Ophiolite, Oman.

Serpentinization can generate highly reduced fluids replete with hydrogen (H2) and methane (CH4), potent reductants capable of driving microbial methanogenesis and methanotrophy, respectively. However, CH4 in serpentinized waters is thought to be primarily abiogenic, raising key questions about the relative importance of methanogens and methanotrophs in the production and consumption of CH4 in these systems. Herein, we apply molecular approaches to examine the functional capability and activity of microbial CH4 cycling in serpentinization-impacted subsurface waters intersecting multiple rock and water types within the Samail Ophiolite of Oman. Abundant 16S rRNA genes and transcripts affiliated with the methanogenic genus Methanobacterium were recovered from the most alkaline (pH, >10), H2- and CH4-rich subsurface waters. Additionally, 16S rRNA genes and transcripts associated with the aerobic methanotrophic genus Methylococcus were detected in wells that spanned varied fluid geochemistry. Metagenomic sequencing yielded genes encoding homologs of proteins involved in the hydrogenotrophic pathway of microbial CH4 production and in microbial CH4 oxidation. Transcripts of several key genes encoding methanogenesis/methanotrophy enzymes were identified, predominantly in communities from the most hyperalkaline waters. These results indicate active methanogenic and methanotrophic populations in waters with hyperalkaline pH in the Samail Ophiolite, thereby supporting a role for biological CH4 cycling in aquifers that undergo low-temperature serpentinization.IMPORTANCE Serpentinization of ultramafic rock can generate conditions favorable for microbial methane (CH4) cycling, including the abiotic production of hydrogen (H2) and possibly CH4 Systems of low-temperature serpentinization are geobiological targets due to their potential to harbor microbial life and ubiquity throughout Earth's history. Biomass in fracture waters collected from the Samail Ophiolite of Oman, a system undergoing modern serpentinization, yielded DNA and RNA signatures indicative of active microbial methanogenesis and methanotrophy. Intriguingly, transcripts for proteins involved in methanogenesis were most abundant in the most highly reacted waters that have hyperalkaline pH and elevated concentrations of H2 and CH4 These findings suggest active biological methane cycling in serpentinite-hosted aquifers, even under extreme conditions of high pH and carbon limitation. These observations underscore the potential for microbial activity to influence the isotopic composition of CH4 in these systems, which is information that could help in identifying biosignatures of microbial activity on other planets.

Bioenergetic constraints on the origin of autotrophic metabolism.

Autotrophs form the base of all complex food webs and seemingly have done so since early in Earth history. Phylogenetic evidence suggests that early autotrophs were anaerobic, used CO2 as both an oxidant and carbon source, were dependent on H2 as an electron donor, and used iron-sulfur proteins (termed ferredoxins) as a primary electron carrier. However, the reduction potential of H2 is not typically low enough to efficiently reduce ferredoxin. Instead, in modern strictly anaerobic and H2-dependent autotrophs, ferredoxin reduction is accomplished using one of several recently evolved enzymatic mechanisms, including electron bifurcating and coupled ion translocating mechanisms. These observations raise the intriguing question of why anaerobic autotrophs adopted ferredoxins as central electron carriers only to have to evolve complex machinery to reduce them. Here, we report calculated reduction potentials for H2 as a function of observed environmental H2 concentration, pH and temperature. Results suggest that a combination of alkaline pH and high H2 concentration yield H2 reduction potentials low enough to efficiently reduce ferredoxins. Hyperalkaline, H2 rich environments have existed in discrete locations throughout Earth history where ultramafic minerals are undergoing hydration through the process of serpentinization. These results suggest that serpentinizing systems, which would have been common on early Earth, naturally produced conditions conducive to the emergence of H2-dependent autotrophic life. The primitive process of hydrogenotrophic methanogenesis is used to examine potential changes in methanogenesis and Fd reduction pathways as these organisms diversified away from serpentinizing environments. This article is part of a discussion meeting issue 'Serpentinite in the earth system'.

Dissolved silica affects the bulk iron redox state and recrystallization of minerals generated by photoferrotrophy in a simulated Archean ocean.

Chemical sedimentary deposits called Banded Iron Formations (BIFs) are one of the best surviving records of ancient marine (bio)geochemistry. Many BIF precursor sediments precipitated from ferruginous, silica-rich waters prior to the Great Oxidation Event at ~2.43 Ga. Reconstructing the mineralogy of BIF precursor phases is key to understanding the coevolution of seawater chemistry and early life. Many models of BIF deposition invoke the activity of Fe(II)-oxidizing photoautotrophic bacteria as a mechanism for precipitating mixed-valence Fe(II,III) and/or fully oxidized Fe(III) minerals in the absence of molecular oxygen. Although the identity of phases produced by ancient photoferrotrophs remains debated, laboratory experiments provide a means to explore what their mineral byproducts might have been. Few studies have thoroughly characterized precipitates produced by photoferrotrophs in settings representative of Archean oceans, including investigating how residual Fe(II)aq can affect the mineralogy of expected solid phases. The concentration of dissolved silica (Si) is also an important variable to consider, as silicate species may influence the identity and reactivity of Fe(III)-bearing phases. To address these uncertainties, we cultured Rhodopseudomonas palustris TIE-1 as a photoferrotroph in synthetic Archean seawater with an initial [Fe(II)aq ] of 1 mM and [Si] spanning 0-1.5 mM. Ferrihydrite was the dominant precipitate across all Si concentrations, even with substantial Fe(II) remaining in solution. Consistent with other studies of microbial iron oxidation, no Fe-silicates were observed across the silica gradient, although Si coprecipitated with ferrihydrite via surface adsorption. More crystalline phases such as lepidocrocite and goethite were only detected at low [Si] and are likely products of Fe(II)-catalyzed ferrihydrite transformation. Finally, we observed a substantial fraction of Fe(II) in precipitates, with the proportion of Fe(II) increasing as a function of [Si]. These experimental results suggest that photoferrotrophy in a Fe(II)-buffered ocean may have exported Fe(II,III)-oxide/silica admixtures to BIF sediments, providing a more chemically diverse substrate than previously hypothesized.

Single-cell measurement of microbial growth rate with Raman microspectroscopy.

Rates of microbial growth are fundamental to understanding environmental geochemistry and ecology. However, measuring the heterogeneity of microbial activity at the single-cell level, especially within complex populations and environmental matrices, remains a forefront challenge. Stable isotope probing (SIP) is a method for assessing microbial growth and involves measuring the incorporation of an isotopic label into microbial biomass. Here, we assess Raman microspectroscopy as a SIP technique, specifically focusing on the measurement of deuterium (2H), a tracer of microbial biomass production. We correlatively measured cells grown in varying concentrations of deuterated water with both Raman spectroscopy and nanoscale secondary ion mass spectrometry (nanoSIMS), generating isotopic calibrations of microbial 2H. Relative to Raman, we find that nanoSIMS measurements of 2H are subject to substantial dilution due to rapid exchange of H during sample washing. We apply our Raman-derived calibration to a numerical model of microbial growth, explicitly parameterizing the factors controlling growth rate quantification and demonstrating that Raman-SIP can sensitively measure the growth of microorganisms with doubling times ranging from hours to years. The measurement of single-cell growth with Raman spectroscopy, a rapid, nondestructive technique, represents an important step toward application of single-cell analysis into complex sample matrices or cellular assemblages.

Metapangenomic investigation provides insight into niche differentiation of methanogenic populations from the subsurface serpentinizing environment, Samail Ophiolite, Oman.

Serpentinization reactions produce highly reduced waters that have hyperalkaline pH and that can have high concentrations of H2 and CH4. Putatively autotrophic methanogenic archaea have been identified in the subsurface waters of the Samail Ophiolite, Sultanate of Oman, though the strategies to overcome hyperalkaline pH and dissolved inorganic carbon limitation remain to be fully understood. Here, we recovered metagenome assembled genomes (MAGs) and applied a metapangenomic approach to three different Methanobacterium populations to assess habitat-specific functional gene distribution. A Type I population was identified in the fluids with neutral pH, while a Type II and "Mixed" population were identified in the most hyperalkaline fluids (pH 11.63). The core genome of all Methanobacterium populations highlighted potential DNA scavenging techniques to overcome phosphate or nitrogen limitation induced by environmental conditions. With particular emphasis on the Mixed and Type II population found in the most hyperalkaline fluids, the accessory genomes unique to each population reflected adaptation mechanisms suggesting lifestyles that minimize niche overlap. In addition to previously reported metabolic capability to utilize formate as an electron donor and generate intracellular CO2, the Type II population possessed genes relevant to defense against antimicrobials and assimilating potential osmoprotectants to provide cellular stability. The accessory genome of the Mixed population was enriched in genes for multiple glycosyltransferases suggesting reduced energetic costs by adhering to mineral surfaces or to other microorganisms, and fostering a non-motile lifestyle. These results highlight the niche differentiation of distinct Methanobacterium populations to circumvent the challenges of serpentinization impacted fluids through coexistence strategies, supporting our ability to understand controls on methanogenic lifestyles and adaptations within the serpentinizing subsurface fluids of the Samail Ophiolite.

An untargeted exometabolomics approach to characterize dissolved organic matter in groundwater of the Samail Ophiolite.

The process of serpentinization supports life on Earth and gives rise to the habitability of other worlds in our Solar System. While numerous studies have provided clues to the survival strategies of microbial communities in serpentinizing environments on the modern Earth, characterizing microbial activity in such environments remains challenging due to low biomass and extreme conditions. Here, we used an untargeted metabolomics approach to characterize dissolved organic matter in groundwater in the Samail Ophiolite, the largest and best characterized example of actively serpentinizing uplifted ocean crust and mantle. We found that dissolved organic matter composition is strongly correlated with both fluid type and microbial community composition, and that the fluids that were most influenced by serpentinization contained the greatest number of unique compounds, none of which could be identified using the current metabolite databases. Using metabolomics in conjunction with metagenomic data, we detected numerous products and intermediates of microbial metabolic processes and identified potential biosignatures of microbial activity, including pigments, porphyrins, quinones, fatty acids, and metabolites involved in methanogenesis. Metabolomics techniques like the ones used in this study may be used to further our understanding of life in serpentinizing environments, and aid in the identification of biosignatures that can be used to search for life in serpentinizing systems on other worlds.

Intact polar lipidome and membrane adaptations of microbial communities inhabiting serpentinite-hosted fluids.

The generation of hydrogen and reduced carbon compounds during serpentinization provides sustained energy for microorganisms on Earth, and possibly on other extraterrestrial bodies (e.g., Mars, icy satellites). However, the geochemical conditions that arise from water-rock reaction also challenge the known limits of microbial physiology, such as hyperalkaline pH, limited electron acceptors and inorganic carbon. Because cell membranes act as a primary barrier between a cell and its environment, lipids are a vital component in microbial acclimation to challenging physicochemical conditions. To probe the diversity of cell membrane lipids produced in serpentinizing settings and identify membrane adaptations to this environment, we conducted the first comprehensive intact polar lipid (IPL) biomarker survey of microbial communities inhabiting the subsurface at a terrestrial site of serpentinization. We used an expansive, custom environmental lipid database that expands the application of targeted and untargeted lipodomics in the study of microbial and biogeochemical processes. IPLs extracted from serpentinite-hosted fluid communities were comprised of >90% isoprenoidal and non-isoprenoidal diether glycolipids likely produced by archaeal methanogens and sulfate-reducing bacteria. Phospholipids only constituted ~1% of the intact polar lipidome. In addition to abundant diether glycolipids, betaine and trimethylated-ornithine aminolipids and glycosphingolipids were also detected, indicating pervasive membrane modifications in response to phosphate limitation. The carbon oxidation state of IPL backbones was positively correlated with the reduction potential of fluids, which may signify an energy conservation strategy for lipid synthesis. Together, these data suggest microorganisms inhabiting serpentinites possess a unique combination of membrane adaptations that allow for their survival in polyextreme environments. The persistence of IPLs in fluids beyond the presence of their source organisms, as indicated by 16S rRNA genes and transcripts, is promising for the detection of extinct life in serpentinizing settings through lipid biomarker signatures. These data contribute new insights into the complexity of lipid structures generated in actively serpentinizing environments and provide valuable context to aid in the reconstruction of past microbial activity from fossil lipid records of terrestrial serpentinites and the search for biosignatures elsewhere in our solar system.

Parapatric speciation of Meiothermus in serpentinite-hosted aquifers in Oman.

The factors that control the distribution and evolution of microbial life in subsurface environments remain enigmatic due to challenges associated with sampling fluids from discrete depth intervals via boreholes while avoiding mixing of fluids. Here, using an inflatable packer system, fracture waters were isolated and collected from three discrete depth intervals spanning >130 m in a borehole intersecting an ultramafic rock formation undergoing serpentinization in the Samail Ophiolite, Sultanate of Oman. Near surface aquifer waters were moderately reducing and had alkaline pH while deeper aquifer waters were reduced and had hyperalkaline pH, indicating extensive influence by serpentinization. Metagenomic sequencing and analysis of DNA from filtered biomass collected from discrete depth intervals revealed an abundance of aerobes in near surface waters and a greater proportion of anaerobes at depth. Yet the abundance of the putatively obligate aerobe, Meiothermus, increased with depth, providing an opportunity to evaluate the influence of chemical and spatial variation on its distribution and speciation. Two clades of Meiothermus metagenome assembled genomes (MAGs) were identified that correspond to surface and deep populations termed Types I (S) and II (D), respectively; both clades comprised an apparently Oman-specific lineage indicating a common ancestor. Type II (D) clade MAGs encoded fewer genes and were undergoing slower genome replication as inferred from read mapping. Further, single nucleotide variants (SNVs) and mobile genetic elements identified among MAGs revealed detectable, albeit limited, evidence for gene flow/recombination between spatially segregated Type I (S) and Type II (D) populations. Together, these observations indicate that chemical variation generated by serpentinization, combined with physical barriers that reduce/limit dispersal and gene flow, allowed for the parapatric speciation of Meiothermus in the Samail Ophiolite or a geologic precursor. Further, Meiothermus genomic data suggest that deep and shallow aquifer fluids in the Samail Ophiolite may mix over shorter time scales than has been previously estimated from geochemical data.

Subsurface biogeochemical cycling of nitrogen in the actively serpentinizing Samail Ophiolite, Oman.

Nitrogen (N) is an essential element for life. N compounds such as ammonium ( NH 4 + ) may act as electron donors, while nitrate ( NO 3 - ) and nitrite ( NO 2 - ) may serve as electron acceptors to support energy metabolism. However, little is known regarding the availability and forms of N in subsurface ecosystems, particularly in serpentinite-hosted settings where hydrogen (H2) generated through water-rock reactions promotes habitable conditions for microbial life. Here, we analyzed N and oxygen (O) isotope composition to investigate the source, abundance, and cycling of N species within the Samail Ophiolite of Oman. The dominant dissolved N species was dependent on the fluid type, with Mg2+- HCO 3 - type fluids comprised mostly of NO 3 - , and Ca2+-OH- fluids comprised primarily of ammonia (NH3). We infer that fixed N is introduced to the serpentinite aquifer as NO 3 - . High concentrations of NO 3 - (>100 µM) with a relict meteoric oxygen isotopic composition (δ18O ~ 22‰, Δ17O ~ 6‰) were observed in shallow aquifer fluids, indicative of NO 3 - sourced from atmospheric deposition (rainwater NO 3 - : δ18O of 53.7‰, Δ17O of 16.8‰) mixed with NO 3 - produced in situ through nitrification (estimated endmember δ18O and Δ17O of ~0‰). Conversely, highly reacted hyperalkaline fluids had high concentrations of NH3 (>100 µM) with little NO 3 - detectable. We interpret that NH3 in hyperalkaline fluids is a product of NO 3 - reduction. The proportionality of the O and N isotope fractionation (18ε / 15ε) measured in Samail Ophiolite NO 3 - was close to unity (18ε / 15ε ~ 1), which is consistent with dissimilatory NO 3 - reduction with a membrane-bound reductase (NarG); however, abiotic reduction processes may also be occurring. The presence of genes commonly involved in N reduction processes (narG, napA, nrfA) in the metagenomes of biomass sourced from aquifer fluids supports potential biological involvement in the consumption of NO 3 - . Production of NH 4 + as the end-product of NO 3 - reduction via dissimilatory nitrate reduction to ammonium (DNRA) could retain N in the subsurface and fuel nitrification in the oxygenated near surface. Elevated bioavailable N in all sampled fluids indicates that N is not likely limiting as a nutrient in serpentinites of the Samail Ophiolite.

Presumptive Primary Cerebral T-Cell Lymphoma in a White-Tailed Deer (Odocoileus virginianus).

Primary central nervous system (CNS) lymphomas are rare in humans and even more uncommon in animals. We report the clinical, pathological and immunohistochemical features of a presumptive primary cerebral T-cell lymphoma (PCTCL) in an aged female white-tailed deer (Odocoileus virginianus) that had chronic progressive neurological disease characterized by ataxia, claudication and eventual circling. The animal was euthanized due to poor prognosis. Grossly, a 2.5 cm dark red, friable nodule effaced the cortical neuroparenchyma of the left anterior cingulate cortex (LACC). Microscopically, the meningeal vasculature and adjacent grey and white matter cortical neuroparenchyma of the LACC were infiltrated by a poorly demarcated, unencapsulated and densely cellular round cell neoplasm with a consistent angiocentric pattern. The neoplasm was associated with extensive regions of haemorrhage and liquefactive necrosis. Neoplastic cells immunolabelled for CD3 antigen and had high proliferative activity, as indicated by Ki-67 labelling. Based on the cytohistomorphological and immunohistochemical features and absence of metastasis, a diagnosis of PCTCL was determined. This case indicates that PCTCL should be considered in the differential diagnosis of neurological disease and intracranial, intra-axial CNS masses in deer.

Diversification of methanogens into hyperalkaline serpentinizing environments through adaptations to minimize oxidant limitation.

Metagenome assembled genomes (MAGs) and single amplified genomes (SAGs) affiliated with two distinct Methanobacterium lineages were recovered from subsurface fracture waters of the Samail Ophiolite, Sultanate of Oman. Lineage Type I was abundant in waters with circumneutral pH, whereas lineage Type II was abundant in hydrogen rich, hyperalkaline waters. Type I encoded proteins to couple hydrogen oxidation to CO2 reduction, typical of hydrogenotrophic methanogens. Surprisingly, Type II, which branched from the Type I lineage, lacked homologs of two key oxidative [NiFe]-hydrogenases. These functions were presumably replaced by formate dehydrogenases that oxidize formate to yield reductant and cytoplasmic CO2 via a pathway that was unique among characterized Methanobacteria, allowing cells to overcome CO2/oxidant limitation in high pH waters. This prediction was supported by microcosm-based radiotracer experiments that showed significant biological methane generation from formate, but not bicarbonate, in waters where the Type II lineage was detected in highest relative abundance. Phylogenetic analyses and variability in gene content suggested that recent and ongoing diversification of the Type II lineage was enabled by gene transfer, loss, and transposition. These data indicate that selection imposed by CO2/oxidant availability drove recent methanogen diversification into hyperalkaline waters that are heavily impacted by serpentinization.

Retrospective Survey of Amphibian Pathology Cases at Texas A&M University System (2016-2020).

Dramatic declines in amphibians worldwide highlight the need for a better understanding of diseases affecting these species. To delineate the health issues of amphibians submitted to Texas A&M University System (2016-2020), the databases were queried on the basis of defined selection criteria. A total of 502 anurans (157 frogs [44 species] and 345 toads [10 species]) and 30 caudatans (23 salamanders [6 species] and 7 newts [4 species]) were reviewed. A most likely cause of death or major pathological finding (CD-MPF) leading to euthanasia was identified in 295 (55%) Anura cases and 15 (50%) Caudata cases. Of the 532 records reviewed, anurans included 492 captive, seven free-ranging and three undetermined specimens. All caudatans were captive. The most common CD-MPF in anurans was infectious/inflammatory (228/295; 77%), involving mycobacteriosis (73/228; 32%), chlamydiosis (44/228; 19%) and mycosis (32/228; 14%). Neoplasia was less common (28/295; 9%). Infectious/inflammatory lesions (14/15; 93%) were the main CD-MPFs in caudatans. Infectious diseases are a significant threat to captive amphibians in Texas and these results may aid personnel involved in amphibian conservation programmes, veterinarians and diagnosticians.

Microbial Metabolic Redundancy Is a Key Mechanism in a Sulfur-Rich Glacial Ecosystem.

Biological sulfur cycling in polar, low-temperature ecosystems is an understudied phenomenon in part due to difficulty of access and the dynamic nature of glacial environments. One such environment where sulfur cycling is known to play an important role in microbial metabolisms is located at Borup Fiord Pass (BFP) in the Canadian High Arctic. Here, transient springs emerge from ice near the terminus of a glacier, creating a large area of proglacial aufeis (spring-derived ice) that is often covered in bright yellow/white sulfur, sulfate, and carbonate mineral precipitates accompanied by a strong odor of hydrogen sulfide. Metagenomic sequencing of samples from multiple sites and of various sample types across the BFP glacial system produced 31 metagenome-assembled genomes (MAGs) that were queried for sulfur, nitrogen, and carbon cycling/metabolism genes. An abundance of sulfur cycling genes was widespread across the isolated MAGs and sample metagenomes taxonomically associated with the bacterial classes Alphaproteobacteria and Gammaproteobacteria and Campylobacteria (formerly the Epsilonproteobacteria). This corroborates previous research from BFP implicating Campylobacteria as the primary class responsible for sulfur oxidation; however, data reported here suggested putative sulfur oxidation by organisms in both the alphaproteobacterial and gammaproteobacterial classes that was not predicted by previous work. These findings indicate that in low-temperature, sulfur-based environments, functional redundancy may be a key mechanism that microorganisms use to enable coexistence whenever energy is limited and/or focused by redox chemistry.IMPORTANCE A unique environment at Borup Fiord Pass is characterized by a sulfur-enriched glacial ecosystem in the low-temperature Canadian High Arctic. BFP represents one of the best terrestrial analog sites for studying icy, sulfur-rich worlds outside our own, such as Europa and Mars. The site also allows investigation of sulfur-based microbial metabolisms in cold environments here on Earth. Here, we report whole-genome sequencing data that suggest that sulfur cycling metabolisms at BFP are more widely used across bacterial taxa than predicted. From our analyses, the metabolic capability of sulfur oxidation among multiple community members appears likely due to functional redundancy present in their genomes. Functional redundancy, with respect to sulfur-oxidation at the BFP sulfur-ice environment, may indicate that this dynamic ecosystem hosts microorganisms that are able to use multiple sulfur electron donors alongside other metabolic pathways, including those for carbon and nitrogen.

Loihichelins A-F, a suite of amphiphilic siderophores produced by the marine bacterium Halomonas LOB-5.

A suite of amphiphilic siderophores, loihichelins A-F, were isolated from cultures of the marine bacterium Halomonas sp. LOB-5. This heterotrophic Mn(II)-oxidizing bacterium was recently isolated from the partially weathered surfaces of submarine glassy pillow basalts and associated hydrothermal flocs of iron oxides collected from the southern rift zone of Loihi Seamount east of Hawai'i. The loihichelins contain a hydrophilic headgroup consisting of an octapeptide comprised of D-threo-beta-hydroxyaspartic acid, D-serine, L-glutamine, L-serine, L-N(delta)-acetyl-N(delta)-hydroxyornithine, dehydroamino-2-butyric acid, D-serine, and cyclic N(delta)-hydroxy-D-ornithine, appended by one of a series of fatty acids ranging from decanoic acid to tetradecanoic acid. The structure of loihichelin C was determined by a combination of amino acid and fatty acid analyses, tandem mass spectrometry, and NMR spectroscopy. The structures of the other loihichelins were inferred from the amino acid and fatty acid analyses and tandem mass spectrometry. The role of these siderophores in sequestering Fe(III) released during basaltic rock weathering, as well as their potential role in the promotion of Mn(II) and Fe(II) oxidation, is of considerable interest.

Physiological adaptations to serpentinization in the Samail Ophiolite, Oman.

Hydration of ultramafic rock during the geologic process of serpentinization can generate reduced substrates that microorganisms may use to fuel their carbon and energy metabolisms. However, serpentinizing environments also place multiple constraints on microbial life by generating highly reduced hyperalkaline waters that are limited in dissolved inorganic carbon. To better understand how microbial life persists under these conditions, we performed geochemical measurements on waters from a serpentinizing environment and subjected planktonic microbial cells to metagenomic and physiological analyses. Metabolic potential inferred from metagenomes correlated with fluid type, and genes involved in anaerobic metabolisms were enriched in hyperalkaline waters. The abundance of planktonic cells and their rates of utilization of select single-carbon compounds were lower in hyperalkaline waters than alkaline waters. However, the ratios of substrate assimilation to dissimilation were higher in hyperalkaline waters than alkaline waters, which may represent adaptation to minimize energetic and physiologic stress imposed by highly reducing, carbon-limited conditions. Consistent with this hypothesis, estimated genome sizes and average oxidation states of carbon in inferred proteomes were lower in hyperalkaline waters than in alkaline waters. These data suggest that microorganisms inhabiting serpentinized waters exhibit a unique suite of physiological adaptations that allow for their persistence under these polyextremophilic conditions.

Phylogenetic Relationships and Functional Genes: Distribution of a Gene (mnxG) encoding a putative manganese-oxidizing enzyme in Bacillus species.

Several Bacillus and Paenibacillus species were isolated from Fe and Mn oxide minerals precipitating at a deep subsurface oxic-anoxic interface at Henderson Molybdenum Mine, Empire, CO. The isolates were investigated for their Mn(II)-oxidizing potential and interrogated for possession of the mnxG gene, a gene that codes for a putative Mn(II)-oxidizing enzyme in Bacillus species. Seven of eight Bacillus species were capable of Mn(II) oxidation; however, the mnxG gene was detected in only one isolate. Using sequences of known Bacillus species both with and without amplifiable mnxG genes and Henderson Mine isolates, the 16S rRNA and mnxG gene phylogenies were compared to determine if 16S rRNA sequences could be used to predict the presence or absence of an amplifiable mnxG gene within the genomes of the isolates. We discovered a strong correspondence between 16S rRNA sequence similarity and the presence/absence of an amplifiable mnxG gene in the isolates. The data revealed a complex phylogenetic distribution of the mnxG gene in which vertical inheritance and gene loss influence the distribution of the gene among the Bacillus species included in this study. Comparisons of 16S rRNA and functional gene phylogenies can be used as a tool to aid in unraveling the history and dispersal of the mnxG gene within the Bacillus clade.

Subsurface microbial diversity in deep-granitic-fracture water in Colorado.

A microbial community analysis using 16S rRNA gene sequencing was performed on borehole water and a granite rock core from Henderson Mine, a >1,000-meter-deep molybdenum mine near Empire, CO. Chemical analysis of borehole water at two separate depths (1,044 m and 1,004 m below the mine entrance) suggests that a sharp chemical gradient exists, likely from the mixing of two distinct subsurface fluids, one metal rich and one relatively dilute; this has created unique niches for microorganisms. The microbial community analyzed from filtered, oxic borehole water indicated an abundance of sequences from iron-oxidizing bacteria (Gallionella spp.) and was compared to the community from the same borehole after 2 weeks of being plugged with an expandable packer. Statistical analyses with UniFrac revealed a significant shift in community structure following the addition of the packer. Phospholipid fatty acid (PLFA) analysis suggested that Nitrosomonadales dominated the oxic borehole, while PLFAs indicative of anaerobic bacteria were most abundant in the samples from the plugged borehole. Microbial sequences were represented primarily by Firmicutes, Proteobacteria, and a lineage of sequences which did not group with any identified bacterial division; phylogenetic analyses confirmed the presence of a novel candidate division. This "Henderson candidate division" dominated the clone libraries from the dilute anoxic fluids. Sequences obtained from the granitic rock core (1,740 m below the surface) were represented by the divisions Proteobacteria (primarily the family Ralstoniaceae) and Firmicutes. Sequences grouping within Ralstoniaceae were also found in the clone libraries from metal-rich fluids yet were absent in more dilute fluids. Lineage-specific comparisons, combined with phylogenetic statistical analyses, show that geochemical variance has an important effect on microbial community structure in deep, subsurface systems.

Low-Temperature Sulfidic-Ice Microbial Communities, Borup Fiord Pass, Canadian High Arctic.

A sulfur-dominated supraglacial spring system found at Borup Fiord Pass (BFP), Ellesmere Island, Nunavut, Canada, is a unique sulfur-on-ice system expressed along the toe of a glacier. BFP has an intermittent flowing, subsurface-derived, glacial spring that creates a large white-yellow icing (aufeis) that extends down-valley. Over field campaigns in 2014, 2016, and 2017, numerous samples were collected and analyzed for both microbial community composition and aqueous geochemistry. Samples were collected from multiple site types: spring discharge fluid, aufeis (spring-derived ice), melt pools with sedimented cryoconite material, and mineral precipitate scrapings, to probe how microbial communities differed between site types in a dynamic freeze/thaw sulfur-rich system. Dissolved sulfate varied between 0.07 and 11.6 mM and was correlated with chloride concentrations, where the fluids were saltiest among spring fluids. The highest sulfate samples exhibited high dissolved sulfide values between 0.22 and 2.25 mM. 16S rRNA gene sequencing from melt pool and aufeis samples from the 2014 campaign were highly abundant in operational taxonomic units (OTUs) closely related to sulfur-oxidizing microorganisms (SOM; Sulfurimonas, Sulfurovum, and Sulfuricurvum). Subsequent sampling 2 weeks later had fewer SOMs and showed an increased abundance of the genus Flavobacterium. Desulfocapsa, an organism that specializes in the disproportionation of inorganic sulfur compounds was also found. Samples from 2016 and 2017 revealed that microorganisms present were highly similar in community composition to 2014 samples, primarily echoed by the continued presence of Flavobacterium sp. Results suggest that while there may be acute events where sulfur cycling organisms dominate, a basal community structure appears to dominate over time and site type. These results further enhance our knowledge of low-temperature sulfur systems on Earth, and help to guide the search for potential life on extraterrestrial worlds, such as Europa, where similar low-temperature sulfur-rich conditions may exist.