Theodore M. Flynn

THE THERMODYNAMIC LADDER IN GEOMICROBIOLOGY

A tenet of geomicrobiology is that anaerobic life in the subsurface arranges itself into zones, according to a thermodynamic ladder. Iron reducers, given access to ferric minerals, use their energetic advantage to preclude sulfate reduction. Sulfate reducers exclude methanogens in the same way, by this tenet, wherever the environment provides sulfate. Examining usable energy—the energy in excess of a cells internal stores—in subsurface environments, we find that in groundwater of near neutral pH the three functional groups see roughly equivalent amounts. Iron reducers hold a clear energetic advantage under acidic conditions, but may be unable to grow in alkaline environments. The calculations fail to identify a fixed thermodynamic hierarchy among the groups. In long-term bioreactor experiments, usable energy did not govern microbial activity. Iron reducers and sulfate reducers, instead of competing for energy, entered into a tightly balanced mutualistic relationship. Results of the study show thermodynamics does not invariably favor iron reducers relative to sulfate reducers, which in turn do not necessarily have an energetic advantage over methanogens. The distribution of microbial life in the subsurface is controlled by ecologic and physiologic factors, and cannot be understood in terms of thermodynamics alone.

Sulfur-mediated electron shuttling during bacterial iron reduction

How bacteria manage to breathe on rust In the absence of oxygen, anaerobic bacteria turn to other chemical compounds during respiration. This can be helpful in detoxifying heavy-metal pollution. Flynn et al. (see the Perspective by Friedrich and Finster) found that alkaline conditions prevent a detoxifying bug—Shewanella oneidensis—from using enzymes to reduce rust-like minerals. Instead, the bacteria reduce elemental sulfur compounds, generating hydrogen sulfide that reduces the iron indirectly. This interplay between anoxic biogeochemical cycles may explain why some anaerobic bacteria contain the genetic machinery necessary to reduce multiple compounds besides oxygen. Science, this issue p. 1039; see also p. 974 Bacterial respiration of ferric iron involves sulfur intermediates in alkaline conditions [Also see Perspective by Friedrich and Finster] Microbial reduction of ferric iron [Fe(III)] is an important biogeochemical process in anoxic aquifers. Depending on groundwater pH, dissimilatory metal-reducing bacteria can also respire alternative electron acceptors to survive, including elemental sulfur (S0). To understand the interplay of Fe/S cycling under alkaline conditions, we combined thermodynamic geochemical modeling with bioreactor experiments using Shewanella oneidensis MR-1. Under these conditions, S. oneidensis can enzymatically reduce S0 but not goethite (α-FeOOH). The HS– produced subsequently reduces goethite abiotically. Because of the prevalence of alkaline conditions in many aquifers, Fe(III) reduction may thus proceed via S0-mediated electron-shuttling pathways.

Natural polyreactive IgA antibodies coat the intestinal microbiota

Programmed recognition of microbiota Increasingly, we recognize that the gut is a specialized organ for maintaining microbial symbioses alongside nutritional functions. The gut produces large quantities of immunoglobulin A (IgA), which adheres to the surface of gut microbes. Bunker et al. discovered that antibodies produced by naïve small intestinal plasma cells are recirculated and enriched within Peyers patches, independently of exogenous antigen and T cell help. The resulting polyreactive IgAs are released into the gut lumen and bind to microbial surface glycans, thus innately recognizing the gut microbiota. Polyreactive IgAs appear to be a product of the coevolution of host and microbiota to maintain symbiotic homeostasis. Science, this issue p. eaan6619 Inherently polyreactive antibodies fuel homeostatic intestinal immunoglobulin A responses to the normal gut microbiota. INTRODUCTION Immunoglobulin A (IgA) is the most abundant mammalian antibody isotype, constituting more than 80% of all antibody-secreting plasma cells at steady state. IgA is particularly prevalent at barrier surfaces such as the intestinal mucosa, where it forms a first line of defense in conjunction with innate mediators, including mucus and antimicrobial peptides. IgA is thought to coat and contain the resident commensal microbiota and provide protection against enteric pathogens. IgA responses occur under normal homeostatic conditions and involve both T cell–dependent and T cell–independent pathways of differentiation in mucosa-associated lymphoid tissues such as Peyer’s patches. However, despite its abundance, the specificity of homeostatic IgA has long remained elusive. RATIONALE To elucidate the specificity and origins of homeostatic IgA, we performed unbiased, large-scale cloning and characterization of monoclonal antibodies (mAbs) from single murine IgA plasma cells and other B cell populations of different origins. All antibodies were expressed recombinantly with an IgG1 isotype to compare their reactivity independent of their monomeric or multimeric nature. RESULTS Panels of single cell–derived mAbs were cloned from various B cell and IgA plasma cell populations, and their microbiota-reactivity was characterized by using a combination of bacterial flow cytometry and 16S ribosomal RNA (rRNA) sequencing. Additionally, mAbs were assayed by enzyme-linked immunosorbent assay (ELISA) for polyreactivity—a peculiar property of certain antibodies that facilitates binding to a variety of structurally diverse antigens. Several insights emerged from this characterization: (i) Microbiota-reactive and polyreactive antibodies arose naturally in all naïve B cell populations but were significantly enriched among IgA-secreting plasma cells. (ii) Microbiota-reactive and polyreactive antibodies from naïve B cells and IgA plasma cells showed similar patterns of binding to a broad, but defined, subset of microbiota. This binding included many members of Proteobacteria but largely excluded those of Bacteroidetes and Firmicutes, the predominant phyla in the colon. Interestingly, broadly neutralizing antibodies against influenza virus, which had previously been shown to be frequently polyreactive, were also commonly microbiota-reactive and displayed binding patterns that resembled IgAs. These patterns of microbiota-reactivity thus appear to be a general property of polyreactive antibodies. (iii) The microbiota-reactive and polyreactive IgA repertoire emerged via a mechanism that was largely independent of T cell help or somatic hypermutation. Instead, naturally microbiota-reactive and polyreactive recirculating naïve B cells were selected to become IgA plasma cells in Peyer’s patches. Although some antibodies subsequently acquired somatic mutations, these did not substantially alter their reactivity. (iv) Differentiation of microbiota-reactive and polyreactive IgAs occurred independent of microbiota or exogenous dietary antigen. Analysis of germ-free mice and germ-free mice fed an antigen-free diet demonstrated that microbiota-reactive and polyreactive IgA plasma cells arose naturally, even in the absence of exogenous antigens. CONCLUSION We conclude that homeostatic intestinal IgAs are natural polyreactive antibodies with innate specificity to microbiota. These data suggest that IgA antibodies, though derived from the adaptive immune system, possess innate-like recognition properties that may facilitate adaptation to the vast and dynamic array of exogenous microbiota and dietary antigens encountered at mucosal surfaces. Large-scale analysis of mAbs reveals the specificity and origins of homeostatic intestinal IgA. Panels of single cell–derived mAbs were cloned from murine IgA plasma cells and other B cell populations and characterized for microbiota-reactivity by bacterial flow cytometry and 16S rRNA sequencing or for polyreactivity against structurally diverse antigens, including DNA, insulin, lipopolysaccharide (LPS), flagellin, albumin, cardiolipin, and keyhole-limpet hemocyanin (KLH) by ELISA. This approach revealed that intestinal IgAs are natural polyreactive antibodies with innate specificity to microbiota. FSC, forward scatter. Large quantities of immunoglobulin A (IgA) are constitutively secreted by intestinal plasma cells to coat and contain the commensal microbiota, yet the specificity of these antibodies remains elusive. Here we profiled the reactivities of single murine IgA plasma cells by cloning and characterizing large numbers of monoclonal antibodies. IgAs were not specific to individual bacterial taxa but rather polyreactive, with broad reactivity to a diverse, but defined, subset of microbiota. These antibodies arose at low frequencies among naïve B cells and were selected into the IgA repertoire upon recirculation in Peyer’s patches. This selection process occurred independent of microbiota or dietary antigens. Furthermore, although some IgAs acquired somatic mutations, these did not substantially influence their reactivity. These findings reveal an endogenous mechanism driving homeostatic production of polyreactive IgAs with innate specificity to microbiota.

Halomonas sulfidaeris-dominated microbial community inhabits a 1.8 km-deep subsurface Cambrian sandstone reservoir

A low-diversity microbial community, dominated by the γ-proteobacterium Halomonas sulfidaeris, was detected in samples of warm saline formation porewater collected from the Cambrian Mt. Simon Sandstone in the Illinois Basin of the North American Midcontinent (1.8 km/5872 ft burial depth, 50°C, pH 8, 181 bars pressure). These highly porous and permeable quartz arenite sandstones are directly analogous to reservoirs around the world targeted for large-scale hydrocarbon extraction, as well as subsurface gas and carbon storage. A new downhole low-contamination subsurface sampling probe was used to collect in situ formation water samples for microbial environmental metagenomic analyses. Multiple lines of evidence suggest that this H. sulfidaeris-dominated subsurface microbial community is indigenous and not derived from drilling mud microbial contamination. Data to support this includes V1-V3 pyrosequencing of formation water and drilling mud, as well as comparison with previously published microbial analyses of drilling muds in other sites. Metabolic pathway reconstruction, constrained by the geology, geochemistry and present-day environmental conditions of the Mt. Simon Sandstone, implies that H. sulfidaeris-dominated subsurface microbial community may utilize iron and nitrogen metabolisms and extensively recycle indigenous nutrients and substrates. The presence of aromatic compound metabolic pathways suggests this microbial community can readily adapt to and survive subsurface hydrocarbon migration.

The role of water in the structures of synthetic hallimondite, Pb2[(UO2)(AsO4)2](H2O)n and synthetic parsonsite, Pb2[(UO2)(PO4)2](H2O)n, 0 ≤ n ≤ 0.5

Abstract The crystal structures of synthetic hallimondite and synthetic parsonsite have been refined by fullmatrix least-squares techniques to agreement indices (hallimondite, parsonsite) wR2 of 5.5, and 7.6% for all data, and R1 of 2.7 and 3.4%, calculated for 3391 and 3181 unique observed reflections (|Fo| ≥ 4σF), respectively. Hallimondite is triclinic, space group P1̅, Z = 2, a = 7.1153(8), b = 10.4780(12), c = 6.8571(8) Å, α = 101.178(3)°, β = 95.711(3)°, γ = 86.651(3)°, V = 498.64(3) Å3, and is isostructural with parsonsite, triclinic, space group P1 . , Z = 2, a = 6.8432(5), b = 10.4105(7), c = 6.6718(4) Å, α = 101.418(1)°, β = 98.347(2)°, γ = 86.264(2)°, V = 460.64(5) Å3. In both structures, hexavalent uranium occurs as a uranyl pentagonal bipyramid. The uranyl polyhedra share an edge, forming dimers that are linked by edge- and vertex-sharing with arsenate or phosphate tetrahedra to form chains along [001]. Two symmetrically distinct Pb positions connect the chains. In hallimondite, a partially occupied oxygen atom is located in the cavity between the uranyl arsenate chains and Pb positions, and is attributed to an H2O group. The crystal of synthetic parsonsite investigated does not have appreciable electron density at this position, but its structural cavity is large enough to contain H2O. The presence of H2O in synthetic hallimondite, and its absence in synthetic parsonsite, are supported by the results of FTIR spectroscopy. In conjunction with thermogravimetric results from the literature, we suggest that the formula of parsonsite should be considered Pb2[(UO2)(PO4)2](H2O)n, and hallimondite, Pb2[(UO2)(AsO4)2](H2O)n, with 0 ≤ n ≤ 0.5 in each case.

Solute Concentrations Influence Microbial Methanogenesis in Coal-bearing Strata of the Cherokee Basin, USA.

Microorganisms have contributed significantly to subsurface energy resources by converting organic matter in hydrocarbon reservoirs into methane, the main component of natural gas. In this study, we consider environmental controls on microbial populations in coal-bearing strata of the Cherokee basin, an unconventional natural gas resource in southeast Kansas, USA. Pennsylvanian-age strata in the basin contain numerous thin (0.4–1.1 m) coalbeds with marginal thermal maturities (0.5–0.7% Ro) that are interbedded with shale and sandstone. We collected gas, water, and microbe samples from 16 commercial coalbed methane wells for geochemical and microbiological analysis. The water samples were Na–Cl type with total dissolved solids (TDS) content ranging from 34.9 to 91.3 g L−1. Gas dryness values [C1/(C2 + C3)] averaged 2640 and carbon and hydrogen isotope ratios of methane differed from those of carbon dioxide and water, respectively, by an average of 65 and 183‰. These values are thought to be consistent with gas that formed primarily by hydrogenotrophic methanogenesis. Results from cultivation assays and taxonomic analysis of 16S rRNA genes agree with the geochemical results. Cultivable methanogens were present in every sample tested, methanogen sequences dominate the archaeal community in each sample (avg 91%), and few archaeal sequences (avg 4.2%) were classified within Methanosarcinales, an order of methanogens known to contain methylotrophic methanogens. Although hydrogenotrophs appear dominant, geochemical and microbial analyses both indicate that the proportion of methane generated by acetoclastic methanogens increases with the solute content of formation water, a trend that is contrary to existing conceptual models. Consistent with this trend, beta diversity analyses show that archaeal diversity significantly correlates with formation water solute content. In contrast, bacterial diversity more strongly correlates with location than solute content, possibly as a result of spatial variation in the thermal maturity of the coalbeds.

Transhydrogenase and Growth Substrate Influence Lipid Hydrogen Isotope Ratios in Desulfovibrio alaskensis G20.

Microbial fatty acids preserve metabolic and environmental information in their hydrogen isotope ratios (2H/1H). This ratio is influenced by parameters that include the 2H/1H of water in the microbial growth environment, and biosynthetic fractionations between water and lipid. In some microbes, this biosynthetic fractionation has been shown to vary systematically with central energy metabolism, and controls on fatty acid 2H/1H may be linked to the intracellular production of NADPH. We examined the apparent fractionation between media water and the fatty acids produced by Desulfovibrio alaskensis G20. Growth was in batch culture with malate as an electron donor for sulfate respiration, and with pyruvate and fumarate as substrates for fermentation and for sulfate respiration. A larger fractionation was observed as a consequence of respiratory or fermentative growth on pyruvate than growth on fumarate or malate. This difference correlates with opposite apparent flows of electrons through the electron bifurcating/confurcating transhydrogenase NfnAB. When grown on malate or fumarate, mutant strains of D. alaskensis G20 containing transposon disruptions in a copy of nfnAB show different fractionations than the wild type strain. This phenotype is muted during fermentative growth on pyruvate, and it is absent when pyruvate is a substrate for sulfate reduction. All strains and conditions produced similar fatty acid profiles, and the 2H/1H of individual lipids changed in concert with the mass-weighted average. Unsaturated fatty acids were generally depleted in 2H relative to their saturated homologs, and anteiso-branched fatty acids were generally depleted in 2H relative to straight-chain fatty acids. Fractionation correlated with growth rate, a pattern that has also been observed in the fractionation of sulfur isotopes during dissimilatory sulfate reduction by sulfate-reducing bacteria.

Orenia metallireducens sp. nov. Strain Z6, a Novel Metal-Reducing Member of the Phylum Firmicutes from the Deep Subsurface

ABSTRACT A novel halophilic and metal-reducing bacterium, Orenia metallireducens strain Z6, was isolated from briny groundwater extracted from a 2.02 km-deep borehole in the Illinois Basin, IL. This organism shared 96% 16S rRNA gene similarity with Orenia marismortui but demonstrated physiological properties previously unknown for this genus. In addition to exhibiting a fermentative metabolism typical of the genus Orenia, strain Z6 reduces various metal oxides [Fe(III), Mn(IV), Co(III), and Cr(VI)], using H2 as the electron donor. Strain Z6 actively reduced ferrihydrite over broad ranges of pH (6 to 9.6), salinity (0.4 to 3.5 M NaCl), and temperature (20 to 60°C). At pH 6.5, strain Z6 also reduced more crystalline iron oxides, such as lepidocrocite (γ-FeOOH), goethite (α-FeOOH), and hematite (α-Fe2O3). Analysis of X-ray absorption fine structure (XAFS) following Fe(III) reduction by strain Z6 revealed spectra from ferrous secondary mineral phases consistent with the precipitation of vivianite [Fe3(PO4)2] and siderite (FeCO3). The draft genome assembled for strain Z6 is 3.47 Mb in size and contains 3,269 protein-coding genes. Unlike the well-understood iron-reducing Shewanella and Geobacter species, this organism lacks the c-type cytochromes for typical Fe(III) reduction. Strain Z6 represents the first bacterial species in the genus Orenia (order Halanaerobiales) reported to reduce ferric iron minerals and other metal oxides. This microbe expands both the phylogenetic and physiological scopes of iron-reducing microorganisms known to inhabit the deep subsurface and suggests new mechanisms for microbial iron reduction. These distinctions from other Orenia spp. support the designation of strain Z6 as a new species, Orenia metallireducens sp. nov. IMPORTANCE A novel iron-reducing species, Orenia metallireducens sp. nov., strain Z6, was isolated from groundwater collected from a geological formation located 2.02 km below land surface in the Illinois Basin, USA. Phylogenetic, physiologic, and genomic analyses of strain Z6 found it to have unique properties for iron reducers, including (i) active microbial iron-reducing capacity under broad ranges of temperatures (20 to 60°C), pHs (6 to 9.6), and salinities (0.4 to 3.5 M NaCl), (ii) lack of c-type cytochromes typically affiliated with iron reduction in Geobacter and Shewanella species, and (iii) being the only member of the Halanaerobiales capable of reducing crystalline goethite and hematite. This study expands the scope of phylogenetic affiliations, metabolic capacities, and catalytic mechanisms for iron-reducing microbes.

Thiosulfate oxidation by Thiomicrospira thermophila: metabolic flexibility in response to ambient geochemistry

Previous studies of the stoichiometry of thiosulfate oxidation by colorless sulfur bacteria have failed to demonstrate mass balance of sulfur, indicating that unidentified oxidized products must be present. Here the reaction stoichiometry and kinetics under variable pH conditions during the growth of Thiomicrospira thermophila strain EPR85, isolated from diffuse hydrothermal fluids at the East Pacific Rise, is presented. At pH 8.0, thiosulfate was stoichiometrically converted to sulfate. At lower pH, the products of thiosulfate oxidation were extracellular elemental sulfur and sulfate. We were able to replicate previous experiments and identify the missing sulfur as tetrathionate, consistent with previous reports of the activity of thiosulfate dehydrogenase. Tetrathionate was formed under slightly acidic conditions. Genomic DNA from T. thermophila strain EPR85 contains genes homologous to those in the Sox pathway (soxAXYZBCDL), as well as rhodanese and thiosulfate dehydrogenase. No other sulfur oxidizing bacteria containing sox(CD)2 genes have been reported to produce extracellular elemental sulfur. If the apparent modified Sox pathway we observed in T. thermophila is present in marine Thiobacillus and Thiomicrospira species, production of extracellular elemental sulfur may be biogeochemically important in marine sulfur cycling.

Parallelized, aerobic, single carbon-source enrichments from different natural environments contain divergent microbial communities

Microbial communities that inhabit environments such as soil can contain thousands of distinct taxa, yet little is known about how this diversity is maintained in response to environmental perturbations such as changes in the availability of carbon. By utilizing aerobic substrate arrays to examine the effect of carbon amendment on microbial communities taken from six distinct environments (soil from a temperate prairie and forest, tropical forest soil, subalpine forest soil, and surface water and soil from a palustrine emergent wetland), we examined how carbon amendment and inoculum source shape the composition of the community in each enrichment. Dilute subsamples from each environment were used to inoculate 96-well microtiter plates containing triplicate wells amended with one of 31 carbon sources from six different classes of organic compounds (phenols, polymers, carbohydrates, carboxylic acids, amines, amino acids). After incubating each well aerobically in the dark for 72 h, we analyzed the composition of the microbial communities on the substrate arrays as well as the initial inocula by sequencing 16S rRNA gene amplicons using the Illumina MiSeq platform. Comparisons of alpha and beta diversity in these systems showed that, while the composition of the communities that grow to inhabit the wells in each substrate array diverges sharply from that of the original community in the inoculum, these enrichment communities are still strongly affected by the inoculum source. We found most enrichments were dominated by one or several OTUs most closely related to aerobes or facultative anaerobes from the Proteobacteria (e.g., Pseudomonas, Burkholderia, and Ralstonia) or Bacteroidetes (e.g., Chryseobacterium). Comparisons within each substrate array based on the class of carbon source further show that the communities inhabiting wells amended with a carbohydrate differ significantly from those enriched with a phenolic compound. Selection therefore seems to play a role in shaping the communities in the substrate arrays, although some stochasticity is also seen whereby several replicate wells within a single substrate array display strongly divergent community compositions. Overall, the use of highly parallel substrate arrays offers a promising path forward to study the response of microbial communities to perturbations in a changing environment.