Daniel A. Stolper

Aerobic growth at nanomolar oxygen concentrations

Molecular oxygen (O2) is the second most abundant gas in the Earth’s atmosphere, but in many natural environments, its concentration is reduced to low or even undetectable levels. Although low-oxygen-adapted organisms define the ecology of low-oxygen environments, their capabilities are not fully known. These capabilities also provide a framework for reconstructing a critical period in the history of life, because low, but not negligible, atmospheric oxygen levels could have persisted before the “Great Oxidation” of the Earth’s surface about 2.3 to 2.4 billion years ago. Here, we show that Escherichia coli K-12, chosen for its well-understood biochemistry, rapid growth rate, and low-oxygen-affinity terminal oxidase, grows at oxygen levels of ≤ 3 nM, two to three orders of magnitude lower than previously observed for aerobes. Our study expands both the environmental range and temporal history of aerobic organisms.

Formation temperatures of thermogenic and biogenic methane

Making of methane deep underground Technologies such as hydraulic fracturing, or “fracking,” can now extract natural gas from underground reservoirs. Within the gas, the ratio of certain isotopes holds clues to its origins. Stolper et al. analyzed a wide range of natural gas, including samples from some of the most active fracking sites in the United States. Using a “clumped isotope” technique, the authors could estimate the high temperatures at which methane formed deep underground, as well as the lower temperatures at which ancient microbes produced methane. The approach can help to distinguish the degree of mixing of gas from both sources. Science, this issue p. 1500 Isotopic analysis of methane indicates the timing and location of hydrocarbon gas formation in natural settings. Methane is an important greenhouse gas and energy resource generated dominantly by methanogens at low temperatures and through the breakdown of organic molecules at high temperatures. However, methane-formation temperatures in nature are often poorly constrained. We measured formation temperatures of thermogenic and biogenic methane using a “clumped isotope” technique. Thermogenic gases yield formation temperatures between 157° and 221°C, within the nominal gas window, and biogenic gases yield formation temperatures consistent with their comparatively lower-temperature formational environments (<50°C). In systems where gases have migrated and other proxies for gas-generation temperature yield ambiguous results, methane clumped-isotope temperatures distinguish among and allow for independent tests of possible gas-formation models.

The kinetics of solid-state isotope-exchange reactions for clumped isotopes: A study of inorganic calcites and apatites from natural and experimental samples

Carbonate clumped-isotope geothermometry is a tool used to reconstruct formation or (re)equilibration temperatures of carbonate bearing minerals, including carbonate groups substituted into apatite. It is based on the preference for isotopologues with multiple heavy isotopes (for example, 13C16O218O2− groups) to be more abundant at equilibrium than would be expected if all isotopes were randomly distributed amongst all carbonate groups. Because this preference is only a function of temperature, excesses of multiply substituted species can be used to calculate formation temperatures without knowledge of the isotopic composition of water from which the mineral precipitated or other phases with which it may have equilibrated. However, the measured temperature could be modified after mineral growth if exchange of isotopes amongst carbonate groups within the mineral has occurred through internal isotope-exchange reactions. Because these exchange reactions occur through thermally activated processes, their rates depend on temperature and increase at higher temperatures. Thus internal isotope-exchange reactions could lead to effective re-equilibration at high temperatures, overprinting the original temperatures recorded during mineral growth. We measured clumped-isotope temperatures in carbonate bearing minerals (including apatites) from several carbonatites to constrain the kinetics of these internal isotope-exchange reactions. We observe two key trends for clumped-isotope temperatures in carbonatites: (i) clumped-isotope temperatures of apatites and carbonate-bearing minerals decrease with increasing intrusion depth and (ii) apatites record lower clumped-isotope temperatures than carbonate minerals from the same intrusion. We additionally conducted heating experiments at different temperatures to derive the temperature dependence for the rate constants that describe the alteration of clumped-isotope temperatures with time in calcites and apatites. We find that calcites exhibit complex kinetics as has been seen in previous studies. To quantify these results, we constructed a model that incorporates both diffusion of isotopes through the crystal lattice and isotope-exchange reactions between adjacent carbonate groups. We tested this model through comparison to previous measurements of optical calcites and brachiopods and to samples with known cooling histories and find that the model is able to reasonably capture kinetic data from previous experiments and the observed clumped-isotope temperatures of calcites assuming geologically reasonable cooling rates. A similar model for apatite over-predicts the observed clumped-isotope temperatures found in natural samples; we hypothesize this discrepancy is the result of annealing of radiation damage in our experiments, which lowers the diffusivity and rate of isotope exchange of carbonate groups compared to damaged natural samples. Finally, we constructed models to explore how heating can alter recorded clumped-isotope temperatures. Our model predicts that samples change in clumped-isotope temperatures in two stages. The first stage changes the recorded clumped isotope temperatures by <1 °C if held at 75 °C for 100 million years and by up to ∼40 °C if held at 120 °C, but the clumped-isotope temperatures does not reach ambient values through this low-temperature mechanism. A second, slower change becomes effective at temperatures above 150 °C and can take the measured clumped-isotope temperature up to the true ambient temperature. This result implies that old (hundreds of million years) samples that have only experienced mild (<100-125 °C) thermal histories could exhibit small but measurable (order 10 °C) changes in their clumped-isotope temperatures. We compared this heating model to clumped-isotope measurements from paleosol samples from the Siwalik Basin in Nepal, which were buried up to 5 km and then rapidly exhumed to the surface; these samples often do not give reasonable surface temperatures. The modeled temperatures agree with measured temperatures of these samples, suggesting that partial re-equilibration during shallow crustal burial is responsible for their elevated clumped-isotope temperatures.

Targeting a DNA binding motif of the EVI1 protein by a pyrrole-imidazole polyamide

The zinc finger protein EVI1 is causally associated with acute myeloid leukemogenesis, and inhibition of its function with a small molecule therapeutic may provide effective therapy for EVI1-expressing leukemias. In this paper we describe the development of a pyrrole-imidazole polyamide to specifically block EVI1 binding to DNA. We first identify essential domains for leukemogenesis through structure-function studies on both EVI1 and the t(3;21)(q26;q22)-derived RUNX1-MDS1-EVI1 (RME) protein, which revealed that DNA binding to the cognate motif GACAAGATA via the first of two zinc finger domains (ZF1, encompassing fingers 1-7) is essential transforming activity. To inhibit DNA binding via ZF1, we synthesized a pyrrole-imidazole polyamide 1, designed to bind to a subsite within the GACAAGATA motif and thereby block EVI1 binding. DNase I footprinting and electromobility shift assays revealed a specific and high affinity interaction between polyamide 1 and the GACAAGATA motif. In an in vivo CAT reporter assay using NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as an EVI1-responsive reporter, polyamide 1 completely blocked EVI1-responsive reporter activity. Growth of a leukemic cell line bearing overexpressed EVI1 was also inhibited by treatment with polyamide 1, while a control cell line lacking EVI1 was not. Finally, colony formation by RME was attenuated by polyamide 1 in a serial replating assay. These studies provide evidence that a cell permeable small molecule may effectively block the activity of a leukemogenic transcription factor and provide a valuable tool to dissect critical functions of EVI1 in leukemogenesis.

Surface processes recorded by rocks and soils on Meridiani Planum, Mars: Microscopic Imager observations during Opportunity's first three extended missions

The Microscopic Imager (MI) on the Mars Exploration Rover Opportunity has returned images of Mars with higher resolution than any previous camera system, allowing detailed petrographic and sedimentological studies of the rocks and soils at the Meridiani Planum landing site. Designed to simulate a geologists hand lens, the MI is mounted on Opportunitys instrument arm and can resolve objects 0.1 mm across or larger. This paper provides an overview of MI operations, data calibration, and analysis of MI data returned during the first 900 sols (Mars days) of the Opportunity landed mission. Analyses of Opportunity MI data have helped to resolve major questions about the origin of observed textures and features. These studies support eolian sediment transport, rather than impact surge processes, as the dominant depositional mechanism for Burns formation strata. MI stereo observations of a rock outcrop near the rim of Erebus Crater support the previous interpretation of similar sedimentary structures in Eagle Crater as being formed by surficial flow of liquid water. Well-sorted spherules dominate ripple surfaces on the Meridiani plains, and the size of spherules between ripples decreases by about 1 mm from north to south along Opportunitys traverse between Endurance and Erebus craters.

The utility of methane clumped isotopes to constrain the origins of methane in natural gas accumulations

Abstract Methane clumped-isotope compositions provide a new approach to understanding the formational conditions of methane from both biogenic and thermogenic sources. Under some conditions, these compositions can be used to reconstruct the formational temperatures of the gas, and this capability can be applied to common subsets of both biogenic and thermogenic systems. Additionally, there are examples in which clumped-isotope compositions do not reflect gas-formation temperatures but instead mixing effects and kinetic phenomena; such kinetic effects also occur in common and recognizable subtypes of biogenic and thermogenic gases. Here we review the use of methane clumped-isotope measurements for understanding the origin of methane in the subsurface. We review methane clumped-isotope measurements from numerous biogenic and thermogenic natural gas reservoirs. We then place these measurements in the context of common frameworks for identifying the formational conditions of methane including the use of methane δ13C and δD values and C1/C2–3 ratios. Finally, we propose a framework for how methane clumped isotopes can be used to identify the origin of methane accumulations.

Deciphering the diagenetic history of the El Abra Formation of eastern Mexico using reordered clumped isotope temperatures and U-Pb dating

Carbonates form ubiquitously throughout the history of deposition, burial, and uplift of basins. As such, they potentially record the environmental conditions at the time of formation. Carbonate clumped isotopes provide the temperature of precipitation but can be internally reordered if the host rock is exposed to elevated temperatures over geologic time scales. Here, we exploited this kinetic behavior by analyzing multiple generations of cements that capture the range of environments experienced by the El Abra Formation from eastern Mexico. From this, we developed a quantitative diagenetic history for these different phases of cementation. We observed a 70 °C range in clumped isotope temperatures from 64 °C to 134 °C for these cements, which is not compatible with their inferred precipitation environments. This suggests that bond reordering occurred during burial but did not fully reorder all cements to a common apparent temperature. We reconstructed original cement growth temperatures and the isotopic signature of the parent fluids to show that precipitation from a marine pore fluid began at 125 Ma, contemporaneous with deposition, and continued throughout burial to temperatures of at least 138 °C at 42 Ma. We show that precipitation of equant cements, which occluded 90% of the pore space, was coincident with Laramide-related burial to depths greater than 3800 m. A U-Pb age of diagenetic calcite of 77.1 ± 3.6 Ma provides independent support for our estimates of the absolute timing of precipitation of two distinct phases of the paragenesis. This is the first demonstration of the utility of integrating U-Pb age dating with reordered clumped isotope temperatures to provide quantitative constraints on the time-temperature history of cementation. Such information may ultimately lead to advances in our understanding of the formational environments and geological processes that drive diagenesis in carbonates for temperatures below the clumped isotope “blocking temperature.”

Methane on Mars and Habitability: Challenges and Responses.

Abstract Recent measurements of methane (CH4) by the Mars Science Laboratory (MSL) now confront us with robust data that demand interpretation. Thus far, the MSL data have revealed a baseline level of CH4 (∼0.4 parts per billion by volume [ppbv]), with seasonal variations, as well as greatly enhanced spikes of CH4 with peak abundances of ∼7 ppbv. What do these CH4 revelations with drastically different abundances and temporal signatures represent in terms of interior geochemical processes, or is martian CH4 a biosignature? Discerning how CH4 generation occurs on Mars may shed light on the potential habitability of Mars. There is no evidence of life on the surface of Mars today, but microbes might reside beneath the surface. In this case, the carbon flux represented by CH4 would serve as a link between a putative subterranean biosphere on Mars and what we can measure above the surface. Alternatively, CH4 records modern geochemical activity. Here we ask the fundamental question: how active is Mars, geochemically and/or biologically? In this article, we examine geological, geochemical, and biogeochemical processes related to our overarching question. The martian atmosphere and surface are an overwhelmingly oxidizing environment, and life requires pairing of electron donors and electron acceptors, that is, redox gradients, as an essential source of energy. Therefore, a fundamental and critical question regarding the possibility of life on Mars is, “Where can we find redox gradients as energy sources for life on Mars?” Hence, regardless of the pathway that generates CH4 on Mars, the presence of CH4, a reduced species in an oxidant-rich environment, suggests the possibility of redox gradients supporting life and habitability on Mars. Recent missions such as ExoMars Trace Gas Orbiter may provide mapping of the global distribution of CH4. To discriminate between abiotic and biotic sources of CH4 on Mars, future studies should use a series of diagnostic geochemical analyses, preferably performed below the ground or at the ground/atmosphere interface, including measurements of CH4 isotopes, methane/ethane ratios, H2 gas concentration, and species such as acetic acid. Advances in the fields of Mars exploration and instrumentation will be driven, augmented, and supported by an improved understanding of atmospheric chemistry and dynamics, deep subsurface biogeochemistry, astrobiology, planetary geology, and geophysics. Future Mars exploration programs will have to expand the integration of complementary areas of expertise to generate synergistic and innovative ideas to realize breakthroughs in advancing our understanding of the potential of life and habitable conditions having existed on Mars. In this spirit, we conducted a set of interdisciplinary workshops. From this series has emerged a vision of technological, theoretical, and methodological innovations to explore the martian subsurface and to enhance spatial tracking of key volatiles, such as CH4.