nai

Project Reports

• Control of Evolvability and Chromosomal Rearrangement by Stress

  Gross chromosomal rearrangements (GCRs) underlie much of evolution, changing the copy number of genes, allowing development of new functions by providing redundant genetic material, reassorting protein domains and reassorting regulatory elements. Some mechanisms of chromosomal rearrangement are understood, but most are not. Using a model system in Escherichia coli, we have shown that both point mutation and GCRs occur preferentially when the cells are stressed, and require several stress-responses to be activated. We seek to understand the regulation of GCR by discovering how stress regulates the process, and what is the decision that activates the GCR pathway rather than a parallel stress-induced point mutation pathway. These are important components of the mechanisms by which organisms evolve to adapt to new or changing environments.

  ROADMAP OBJECTIVES: 5.1

• Life Underground

  Our multidisciplinary team from USC, Caltech, JPL, DRI, and RPI is developing and employing field, laboratory, and modeling approaches aimed at detecting and characterizing microbial life in the subsurface—the intraterrestrials. We posit that if life exists, or ever existed, on Mars or other planetary body in our solar system, evidence thereof would most likely be found in the subsurface. This study takes advantage of unique opportunities to explore the subsurface ecosystems on Earth through boreholes, mine shafts, and deeply-sourced springs. Access to the subsurface, both continental and marine, and broad characterization of the rocks, fluids, and microbial inhabitants is central to this study. Our focused research themes require subsurface samples for laboratory and in situ experiments. Specifically, we seek to carry out in situ life detection and characterization experiments, employ numerous novel and traditional techniques to culture heretofore unknown intraterrestrial archaea and bacteria, and incorporate new and existing data into regional and global metabolic energy models.

  ROADMAP OBJECTIVES: 2.1 2.2 3.1 3.3 4.1 5.1 5.2 5.3 6.1 6.2 7.2

• Biosignatures in Ancient Rocks – Kasting Group

  The work by Ramirez concerned updating the absorption coefficients in our 1-D climate model. Harman’s work consisted of developing a 1-D code for modeling hydrodynamic escape of hydrogen from rocky planets.

  ROADMAP OBJECTIVES: 1.1 3.2 4.1 4.2 4.3 5.1 5.2 5.3 6.1 6.2 7.1 7.2

• Investigation 1: Habitability of Icy Worlds

  Habitability of Icy Worlds investigates the habitability of liquid water environments in icy worlds, with a focus on what processes may give rise to life, what processes may sustain life, and what processes may deliver that life to the surface. Habitability of Icy Worlds investigation has three major objectives. Objective 1, Seafloor Processes, explores conditions that might be conducive to originating and supporting life in icy world interiors. Objective 2, Ocean Processes, investigates the formation of prebiotic cell membranes under simulated deep-ocean conditions, and Objective 3, Ice Shell Processes, investigates astrobiological aspects of ice shell evolution.

  ROADMAP OBJECTIVES: 2.1 2.2 3.2 4.1 5.1 5.2 5.3 6.2 7.1 7.2

• Culturing Microbial Communities in Controlled Stress Micro-Environments

  In NAI Theme 4B, our goal in Year 1 has been to initiate our understanding of how cells structure their genomes in response to specific environmental stresses and to determine whether or not such mechanisms have been a major force in directing the evolution of cells in natural environments over evolutionary time. Natural environments are typically rather heterogeneous at small scales, as established by sampling from geothermal hot spring communities, and so it is important to understand the generic impact on the evolution and structure of microbial communities. Our first step towards probing this phenomenon has been to culture living bacterial populations within a small specially constructed microfluidic device (called the GeoBioCell), where strong physical, chemical and biological gradients can be imposed under carefully controlled conditions.

  ROADMAP OBJECTIVES: 3.2 3.4 4.1 4.2 5.1 5.2 5.3 6.1

• Investigation 2: Survivability on Icy Worlds

  Investigation 2 focuses on survivability. As part of our survivability investigation, we examine the similarities and differences between the abiotic chemistry of planetary ices irradiated with ultraviolet photons (UV), electrons, and ions, and the chemistry of biomolecules exposed to similar conditions. Can the chemical products resulting from these two scenarios be distinguished? Can viable microbes persist after exposure to such conditions? These are motivating questions for our investigation.

  ROADMAP OBJECTIVES: 1.1 2.2 3.2 5.1 5.3 6.1 6.2

• Experimental Evolution and Genomic Analysis of an E. Coli Containing a Resurrected Ancestral Gene

  In order to study the historical pathways and modern mechanisms of protein evolution in a complex cellular environment, we combined ancestral sequence reconstruction with experimental evolution. Our first goal was to identify how ancestral states of a protein effect cellular behavior by directly engineering an ancient gene inside a modern genome. We could then identify the evolutionary steps of this organism harboring the ancient gene by subjecting it to laboratory evolution, and directly monitoring the resulting changes within the integrated ancient gene as well as the rest of the host genome.

  ROADMAP OBJECTIVES: 3.4 4.1 5.1 5.2 6.1 6.2

• Project 1D: Potential for Microbial Iron Reduction in Chocolate Pots Hot Springs, Yellowstone National Park

  Iron biogeochemical cycling in circumneutral pH hot spring systems is an increasingly important astrobiological target, given recent discoveries on Mars by Curiosity. This study explored the potential for microbial reduction of ferric iron Fe(III) in the warm (ca. 40-60 C), circumneutral pH (ca. 6.0-6.5) Chocolate Pots (CP) hot springs in Yellowstone National Park. Endogenous microbial communities were able to reduce native CP Fe(III) oxides, as documented in most probable number (MPN) enumerations and ongoing enrichment culture studies. Microbial communities in the enrichments have been analyzed by high-throughput pyrosequencing of 16S rRNA gene amplicons. The sequencing revealed an abundance of the well-known Fe(III)-reducing bacterial species, Geobacter metallireducens, as well several other novel organisms with the potential to contribute to Fe(III) reduction. A shotgun metagenomic (paired-end Illumina sequencing) analysis of the enrichment cultures is in progress to explore the identity and function of G. metallireducens as well as other less well-characterized organisms in the cultures. Of particular interest are the likely presence of thermotolerance genes in the G. metallireducens metagenome, as well as outer membrane cytochrome genes that may be indicative of other Fe(III)-reducing organisms and provide evidence for pathways of electron flow in these cultures.

  ROADMAP OBJECTIVES: 2.1 5.1 6.1 7.1

• Project 1E: Metagenomic Analysis of Novel Chemolithoautotrophic Bacterial Cultures

  Metagenomic sequence information was obtained from two chemolithoautotrophic bacterial cultures: (1) an iron-oxidizing, nitrate-reducing culture that is capable of growth with either soluble or insoluble, mineral-bound (biotite, smectite) Fe(II) as the sole energy source; and (2) an aerobic iron/sulfur-oxidizing culture that grows with insoluble framboidal pyrite as the sole energy source. Both of these cultures carry-out novel neutral-pH lithotrophic microbial pathways, the discovery of which broadens our view of potential Fe/S based life on Earth (past and present) and other rocky planets. We hypothesize that genetic components of Fe/S oxidation identified in the metagenome of the cultures will bear resemblance to analogous components to be identified in other iron-oxidizing pure cultures being sequenced at JGI, together with existing published and unpublished information from other chemolithoautotrophic microorganisms. Identification of such genetic systems will enable comparative genomic analysis of mechanisms of extracellular phyllosilicate Fe/S redox metabolism, and facilitate development of techniques to detect the presence and expression of genes associated with chemolithotrophic Fe/S metabolism in various terrestrial environments.

  ROADMAP OBJECTIVES: 3.2 5.1 5.3 6.2

• Biosignatures in Relevant Microbial Ecosystems

  PSARC is investigating microbial life in some of Earth’s most mission-relevant modern ecosystems. These environments include the Dead Sea, the Chesapeake Bay impact structure, methane seeps, ice sheets, and redox-stratified Precambrian ocean analogs. We target environments that, when studied, provide fundamental information that can serve as the basis for future solar system exploration. Combining our expertise in molecular biology, geochemistry, microbiology, and metagenomics, and in collaboration with some of the planet’s most extreme explorers, we are deciphering the microbiology, fossilization processes, and recoverable biosignatures from these mission-relevant environments.

  PSARC Ph.D. (now postdoctoral researcher at Caltech) Katherine Dawson published a new paper documenting the anaerobic biodegradation of organic biosignature compounds pristane and phytane. PSARC Ph.D. Daniel Jones (now postdoctoral researcher at U. Minnesota) published a new paper that uses metagenomic data to show how sulfur oxidation in the deep subsurface environments may contribute to the formation of caves and the maintenence of deep subsurface microbial ecosystems. PSARC Ph.D. student Khadouja Harouaka published a new paper that represents some of the first available information about possible Ca isotope biosignatures. Lastly, the Macalady group published a paper showing how ecological models based on available energy resources can be used to predict the distribution of microbial populations in space and time.

  ROADMAP OBJECTIVES: 4.1 4.3 5.1 5.2 5.3 6.1 7.1 7.2

• Project 5: Geological-Biological Interactions

  We continue to study the intersection between geology and biology. We continue to explore how sub-seafloor interactions support deep ocean hydrothermal ecosystems. We study life’s adaption to extremes of pressure, cold, and salinity. We adapt and apply multiple isotopic sulfur geochemistry towards the understanding of microbial metabolism and as a means of detecting ancient metabolisms recorded in the rock record through characteristic sulfur isotopic signatures. We apply state-of-the-art methods to derive chemical and isotopic biosignatures of life in the Earth’s most ancient rocks.

  ROADMAP OBJECTIVES: 4.1 5.1 6.1 6.2 7.1

• Thermodynamics of Life

  Although thermodynamics dictates that all spontaneous processes must be purely dissipative and “destructive” (the notoriously ungenerous face of the “2nd law”), under particular circumstances a spontaneous process can be a compound of two mechanistically coupled sub-processes only one of which (necessarily the larger one), is dissipative while its coupled, lesser partner is literally “driven” to be creative and generative – that is, a process that can “do work”, “build stuff”, and “make things happen”. A system functioning in this way is technically an engine and all living systems are necessarily, examples of such thermodynamically compound and creative “engine” systems – while at the same time operating internally via a complex, interlinked clockwork of such engines.
  Moreover, living systems inherently belong to a special thermodynamic subclass of such engines, namely those that are “autocatalytic” (self-growing and self-stabilizing) in their operation. Arguably, in fact, it is the property of being autocatalytic thermodynamic engines which at root underlies the potency and magic of living systems and which at the same time constitutes life’s most assuredly universal, fundamental, and primitive property. However, as of yet, we understand the implications of these thermodynamic facts quite poorly – notwithstanding that they seem certain to materially impact questions regarding the origin of life, evolutionary dynamics, and community, trophic, and ecology-level organization.
  The present project undertakes to redress this situation to some extent by investigating the formal dynamical behavior of model systems made up of interacting, thermodynamically driven, autocatalytic engines.

  ROADMAP OBJECTIVES: 3.3 3.4 4.2 5.1 5.2

• Developing New Biosignatures

  The development and experimental testing of potential indicators of life is essential for providing a critical scientific basis for the exploration of life in the cosmos. In microbial cultures, potential new biosignatures can be found among isotopic ratios, elemental compositions, and chemical changes to the growth media. Additionally, life can be detected and investigated in natural systems by directing cutting-edge instrumentation towards the investigation of microbial cells, microbial fossils, and microbial geochemical products. Over the next five years, we will combine our geomicrobiological expertise and on-going field-based environmental investigations with a new generation of instruments capable of revealing diagnostic biosignatures. Our efforts will focus on creating innovative approaches for the analyses of cells and other organic material, finding ways in which metal abundances and isotope systems reflect life, and developing creative approaches for using environmental DNA to study present and past life.

  ROADMAP OBJECTIVES: 2.1 3.1 4.1 5.1 7.1

• Subsurface Exploration for Astrobiology: Oceanic Basaltic Basement Biosphere

  While extraterrestrial life is likely to exist within the subsurface of water-occupied objects such as Enceladus and Europa, the continued investigation of the subsurface biosphere on the earth provides important insight and implications for astrobiology. This research investigates a deep sub-seafloor basement biosphere. At the ocean floor, lying underneath an often times thick layer of sediment is hard basaltic rock, or basement. Seawater enters the basement and circulates within. It is now known that low temperature hydrothermal fluids (<100oC) circulate everywhere within the porous and permeable volcanic rocks of the upper ocean basement, providing temperature and chemical gradients that host extensive alteration of basement rocks and fluids and form plausible habitats for microbial life. While microbial activity has been observed in deeply buried sediments and exposed basement rock, few direct tests have been carried out in deep subseafloor basement rocks or fluids. A majority of the crustal hydrothermal flow and seawater-crustal fluid exchange, and the corresponding advective heat and mass output, occurs on the flanks of the mid-ocean ridge with basement ages of >1 million years old. This low-temperature ridge flank flow rivals the discharge of all rivers to the ocean and is about three orders of magnitude greater than the high temperature discharge at mid-ocean ridges. The resulting ridge flank chemical flux impacts ocean biogeochemical cycles and may sustain deep basement microbial communities. Access to uncontaminated fluids from subseafloor basement is problematic, especially where ridge flanks and ocean basins are buried under thick, impermeable layers of sediment (i.e., thick enough to act as a barrier to rapid exchange of fluids). We rely on custom designed instrumentation to collect large volume high integrity basement fluids, where the concentrations of microorganisms are often very low (e.g. about 1/10 of bottom seawater concentrations). By studying the chemical composition of crustal fluids, we have learned that several important energy sources, such as dissolved methane and hydrogen, are available. In addition, the isotopic signature of dissolved methane suggests that microbial production and consumption occurs in the basement environment. By filtering microbial biomass from the fluids and investigating their nucleic acids, we are investigating the evolutionary and functional characteristics of the diverse bacterial, archaeal, and viral communities that inhabit the deep subsurface of Earth. Our on-going research includes the investigation of temporal (at hourly-resolution) and spatial (at a few hundred meter scale) biogeochemical and biological variability in order to more effectively constrain our measured parameters. We are also characterizing the dissolved organic carbon pool in basement fluids to investigate the role that basement environment plays in the global carbon cycle.

  ROADMAP OBJECTIVES: 4.1 5.1 5.2 5.3 6.1 6.2

• Molecular Biosignatures: Hopanoid Sources in Modern Systems

  Molecular fossils preserved in sedimentary rocks provide a record of Earth’s early biosphere and its associated carbon cycle. Among the earliest and most abundant molecular fossils are the hopanoids. Derived primarily from bacteria, their diagenetic products, the hopanes, are detectable over timescales of billions of years and have been proposed to be among the most abundantly preserved molecules on Earth. However, an overall picture of their environmental, physiological, and taxonomic origins remains elusive. Are they primarily remnants of primary producers or of heterotrophic consumers? Do they primarily come from free-living marine communities, or from shallow mats, tidal zone communities, or even terrigenous runoff? Here we aim to obtain compound-specific carbon isotope data for hopanoids to infer their sources in modern systems, as proxies for understanding ancient environments.

  ROADMAP OBJECTIVES: 3.2 5.1 5.3 6.1

• The Long Wavelength Limit of Oxygenic Photosynthesis

  Oxygenic photosynthesis (OP) produces the strongest biosignatures at the planetary scale on Earth: atmospheric oxygen and the spectral reflectance of vegetation. Both are controlled by the properties of Chlorophyll a, its ability to perform the water-splitting to produce oxygen, and its spectral absorbance that is limited to red and shorter wavelength photons. We seek to answer what is the long wavelength limit at which OP might remain viable, and how. This would clarify whether and how to look for OP adapted to the light from red dwarfs or M stars, which emit little visible light but abundant far-red and near-infrared. Very recently discovered cyanobacteria have been found to harbor alternative chlorophylls adapted to spectral light environments very much like that of M stars. This projects uses field, lab, and modeling studies to study these far-red adapted cyanobacteria as analogues for extrasolar oxygenic photosynthesis pushing the long wavelength limit.

  ROADMAP OBJECTIVES: 3.2 4.2 5.1 5.3 6.2 7.2

• Molecular Biosignatures: Preservation in Mineral-Forming Ecosystems

  Molecular biosignatures are an important and informative means to reconstruct ancient ecosys-tems, especially, in the case of those dominated by microbes. Most microbes leave only fragmentary chemical records and fossilized hard-parts confined to those taxa with mineralized tests. Preservation can be significantly enhanced when these molecular biosignatures are encapsulated in minerals which are actively forming where the microbes are living and where they may be sequestered from the deleterious effects of oxygen and radiation. We studied several such organo-mineral associations comprising carbonate, silica and gypsum and document a diverse range of molecular biosignatures some of which would be preservable over long timescales. These results are relevant for the study of sediments on the ancient earth, but are also useful in predictive sense for the study of minerals on other planets.

  ROADMAP OBJECTIVES: 5.1 5.3 7.1

• Understanding Past Earth Environments

  For much of the history Earth, life on the planet existed in an environment very different than that of modern-day Earth. Thus, the ancient Earth represents a planet with a biosphere that is both dramatically different than the one in which we live, but that is also accessible to detailed study. As such, it serves as a model for what types of biospheres we may find on other planets. A particular focus of our work was on the “Early Earth” (formation through to about 500 million years ago), a timeframe poorly represented in the geological and fossil records but comprises the majority of Earth’s history. We have studied the composition, pressure and climate of the ancient atmosphere; the delivery of biologically available phosphorus; studied the sulfur, oxygen and nitrogen cycles; and explored atmospheric formation of molecules that were likely important to the origins of life on Earth.

  ROADMAP OBJECTIVES: 1.1 1.2 4.1 4.2 5.1 5.2 6.1

• Molecular Biosignatures: Reconstructing Events by Comparative Genomics

  Reconstructing ancient events in genome evolution provides a valuable narrative for planetary history. Phylogenetic analysis of protein families within microbial lineages can be used to detect horizontal gene transfers and the evolution of new metabolic pathways and physiologies, many of which are significant in reconstructing ancient ecologies and biogeochemical events. These gene transfers can also be used to constrain molecular clock models for early life evolution, applying principles of stratigraphy and date calibration. A better understanding of gene evolution, including partial horizontal gene transfer, is needed to improve these inferences and avoid systematic errors.

  ROADMAP OBJECTIVES: 3.2 3.4 4.1 4.2 4.3 5.1 5.2 6.1

• Project 3D: Microfossil Insights Into Proterozoic Microbial Ecology

  In a study of the chert-permineralized 1.8 Ga Duck Creek Dolomite, and underlying units, Western Australia, Schopf found that in sequences of 2.3 to 1.8 Ga age that indicate little environmental change, there has been no evolution of the form, function, or metabolic requirements of its biotic components. In a second study of sulfur-cycling bacteria from the 775 Ma chert-permineralized Bambui Group of Brazil, Schopf showed that pyritized microbes of this age were anaerobic sulfur-cyclers. This work, in addition to previous studies, forms the basis for ongoing studies of the biotic response to the Great Oxidation Event.

  ROADMAP OBJECTIVES: 4.1 5.1 5.2 6.1 7.2

• Stoichiometry of Life, Task 2a: Field Studies – Yellowstone National Park

  Yellowstone National Park harbors an array of hydrothermal ecosystems with widely varying geochemical characteristics and microbial communities. Our research aims to understand how the geochemistry of these hot springs shapes their constituent microbial communities including their composition and function. To accomplish this aim, we measure (1) physical and geochemical properties of hot spring fluids and sediments, (2) the rates of biogeochemical processes (i.e., methane oxidation, nitrogen fixation, microbial Fe cycling, photosynthesis, de-nitrification, etc.), and (3) markers for microbial community diversity (i.e., SSU rRNA, metabolic genes, lipids, proteins).

  ROADMAP OBJECTIVES: 5.1 5.2 5.3 6.1 6.2 7.2

• Stoichiometry of Life, Task 2b: Field Studies – Cuatro Cienegas

  Cuatro Cienegas is a unique biological preserve in México (state of Coahuila) in which there is striking microbial diversity, potentially related to extreme scarcity of phosphorus. We aim to understand this relationship via field sampling of biological and chemical characteristics and a series of enclosure and whole-pond fertilization experiments. We performed two studies to evaluate ecological impacts of nitrogen and/or phosphorus fertilization in a P-deficient and hyperdiverse shallow pond in the valley of Cuatro Cienegas, Mexico.

  ROADMAP OBJECTIVES: 5.1 5.2 5.3 6.1 6.2

• Stoichiometry of Life, Task 3b: Ancient Records – Genomic

  Task 3b team members are involved in deciphering genomic records of modern organisms as a way to understand how life on Earth evolved. At its core, this couples the integrated measurement and modeling of evolutionary mechanisms that drove the differences between extant genomes (and metagenomes), with experimental data on how environmental dynamics might have shaped these differences across geological timescales. This goal draws from team members’ expertise encompassing theoretical and computational biology, microbial evolution, and studying life in both extreme and dynamic environments across the planet.

  ROADMAP OBJECTIVES: 5.1 5.2 5.3