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Executive Summary

The Institute for Universal Biology (IUB) at the University of Illinois at Urbana-Champaign is focused on addressing one of the most fundamental questions in astrobiology, and indeed all of science: How does life begin and evolve?

To address such a grand question, we are studying in detail how life began and evolved on Earth, with the hope that we can extract general principles applicable to all life, wherever it may arise. These general principles, which do not make reference to specific chemical molecules or features of molecules, make up what is beginning to be called “universal biology”. Universal biology is a major conceptual ingredient of astrobiology, because a perspective that is rooted firmly in carbon-based life as we know it may be too narrow to encompass the potentially myriad ways that life may have arisen elsewhere in the universe. Thus, at the IUB, we focus on the processes of life, as much as possible, rather than the specific instantiation of these processes. Of course, it is impossible to understand the general properties of living systems without understanding deeply what goes on in the single example we currently have —- the Earth’s biosphere. Thus, our Institute’s work uses detailed analyses of carefully chosen specific model systems to help us probe most effectively the evolutionary mechanisms of life. For this reason, our thematic unity is captured by the title of our original grant proposal to NAI —- Towards Universal Biology: Constraints from Early and Continuing Evolutionary Dynamics of Life on Earth.

Our work is founded in the late Carl Woese’s pioneering comparison of molecular sequences to map out of evolutionary relationships of all cellular life on Earth, originally performed with George Fox with support from NASA. From his studies, we know now that life on Earth descended from a common ancestor or ancestral state, around 3.5 billion years ago. The IUB is attempting to see even further back in time, uncovering features of the first billion years of life that have until now been inaccessible to us. This can be accomplished by analyzing the behavior of judiciously chosen modern microbial systems and communities; by using detailed analyses of fully-sequenced genomes to explore for the first time cellular processes of the Archaea; and by focusing on specific organisms whose lifestyle and characteristics are exemplars for key evolutionary transitions. These experimental projects are combined with theoretical work which extends the scope of evolutionary theory —- currently very focused on genes —- to a period in time where genes as we know them had yet to emerge.

Our broad goals are to understand the major evolutionary transitions that occur in living matter, constrain the diversity of life, and govern the way in which energy and information are utilized by life. Through the NASA Astrobiology Institute system, we are exploring how molecules come to life, by investigating fundamental principles of biology from a multidisciplinary perspective, encompassing microbiology, geobiology, computational chemistry, genomics and even physics. In so doing, answers will begin to emerge to the question “Is there life beyond Earth?”

The specific objectives of our research are as follows:

1. Develop a mathematical understanding of the general physical principles underlying the emergence of life and the open-ended growth of complexity. Life to us means evolvable systems that spontaneously create ever-increasing levels of hierarchical organization with multiple levels of feedback. We wish to understand whether the phenomenon of life is a generic one, inexorably the outcome of the laws of physics, and what governs its complexity and diversity.

2. Constrain the nature of life before the Last Universal Common Ancestor (LUCA). The term “Last Universal Common Ancestor” implies that there were earlier organisms and modern life by chance descended from one of them. However, our prior work and the preliminary results in this proposal suggest that on generic and universal grounds, the modern era of vertical descent was preceded by a communal state for which there was no notion of “tree of life”. We wish to explore the properties of this state — the progenote — using detailed and sophisticated analyses of core translational machinery, building in some sense the genomic analogue of the Hubble telescope to see further back in time than has been possible by simple comparative genomics.

3. Explore how life made the transition from a communal progenote state to the present era of vertically-dominated evolution. To do this, we propose an unprecedentedly detailed study of the Archaeal domain and its transition to the Eukaryote lineage. The significance of this research is that we will obtain an understanding of why there are only three Domains of life, and not many more. We expect that our conclusions will be of general applicability to all life that balances the requirements of energy utilization, information processing, replication and evolvability.

4. Determine what factors govern the rate of evolution, and how organisms interact with their environment. How do cells observe their environment and regulate their response to stress? How do cells regulate their ability to evolve? We will answer these questions through detailed experimentation with microbial systems.

In year 1 of our project, we have made progress in 6 different project areas, described in this Annual Report. Highlights are listed briefly below.

Dynamics of self-programming systems. Living systems are unique in that they have the capacity to evolve. Evolving systems can reprogram themselves and so they are able to respond to perturbations by creating new functionality. This feature is something very different from physical systems, which obey a fixed or predetermined equation of motion. This project is a theoretical attempt to describe this state of affairs mathematically, and to construct computer programs that have the capacity to evolve and thus become more complex without this being “built in” by the original programmer. While being cognizant of existing work in this area, we have pursued two particular mathematical directions that are novel: [1] Populations of individuals whose interactions can be described by evolving cooperative games; and [2] Populations of individuals who interact through mathematical relationships encoded using recursive function theory.

For [1], we have succeeded in constructing evolutionary games, whose evolvability corresponds not to a reordering or redistribution of previously existing strategies (as is common place in the existing literature), but instead an innovation where novel strategies are added, which has the effect of increasing the dimension of the model. We are currently analyzing the mathematical properties of this class of dynamical systems.

For [2], we are trying to understand the grammar and syntax of evolving systems. The genetic code is an abstract aspect of a sophisticated and effective communication system, but the messages can be viewed in a real sense as having their own grammar and functionality. The languages of life have the interesting self-referential property of being able to rewrite or reprogram themselves, and this is what allows them to evolve (there may well be cultural analogues of course!). To explore this, we are developing the simplest model programming language that is a pure metaprogramming language for itself, i.e. each program in this language takes other programs of the same kind as inputs and produces new programs based on those inputs. How would communities of agents that can program themselves and each other behave? We are in the process of analyzing and simulating such systems, and have constructed a number of explicit examples that have the potential to exhibit open-ended evolution.

This work, part of Specific Objective 1, is an interdisciplinary collaboration between a mathematician (DeVille) and a physicist (Goldenfeld).

Thermodynamics of life. Living systems inherently belong to a special thermodynamic subclass of heat engines, that are “autocatalytic”: they are 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. These thermodynamic aspects of life are constraints that seem certain to materially impact questions regarding the origin of life, evolutionary dynamics, and community, trophic, and ecology-level organization. The present project is investigating the formal dynamical behavior of model systems made up of interacting, thermodynamically driven, autocatalytic engines, and relates these insights to a very practical proposal for a scenario for the origin of life in warm, alkaline hydrothermal vents. In collaboration with the NASA Astrobiology Institute at the Jet Propulsion Laboratory, Michael Russell and Elbert Branscomb have developed a scenario that is thermodynamically consistent with the redox disequilibria maintained in modern cells. Energy conversion processes in modern cells work by using externally supplied energy to pump protons from one side of a biological membrane to the other and then having them flow back to create ATP, which powers cell processes. Michael Russell’s theory, now elaborated by the recent work with Branscomb and others proposes that this roundabout process is a vestige of the conditions under which life evolved. In particular, the idea is that this is a natural physical consequence of a geochemical process called serpentinization that produced, for free, the system’s major components: cell-like compartments surrounded by membranes, the right proton concentration differences between the inside and outside of these mineral membranes, and primitive, “mineral-based” forms of the “turbine” motors needed to make a molecule like ATP.

This work, part of Specific Objective 1, is a collaboration between the IUB at Illinois and the Icy Worlds NAI at JPL, with personnel Branscomb (a biophysicist) working closely with Russell (a geologist).

Mining archaeal genomes for signatures of very early life. Carl Woese proposed that life started as semi-autonomous subcellular forms named progenotes. The progenotes lacked cell membranes and readily exchanged information, suggesting that aspects of information processing had already been developed. Woese further hypothesized that certain early life processes crossed a Darwinian threshold, where incorporation of new components of a processes was not tolerated. Goldenfeld and Woese later showed that prior to the Darwinian threshold, if it existed, the genetic code and the core machinery of life by extension could only have evolved so quickly (ie. within less than a billion years) if the progenote evolution was highly collective, corresponding to what is today called horizontal gene transfer. Horizontal gene transfer is the process of transferring genes between non-related organisms, as opposed to vertical transmission from parent to daughter cell. Today, we know that some genes can still be transferred horizontally, examples being those which confer antibiotic resistance to bacteria for example. But what about genes coding for central aspects of cellular function?

This project is determining whether translation, transcription, and replication have crossed the Darwinian threshold. To determine whether DNA replication has crossed the Darwinian Threshold, interchangeability of the DNA replication processivity factor known as the sliding clamp is being examined. It is only in the presence of the sliding clamp that DNA polymerases in extant organisms can gain the speed required to replicate their genomes. We have, expressed and purified a sliding clamp from each of the three domains of life (E. coli beta-subunit, M. acetivorans PCNA, and human PCNA). Sliding clamps are loaded in a clamp loader dependent manner; therefore, we have cloned, expressed and purified an archaeal clamp loader from M. acetivorans. Our next step is to determine whether an archaeal clamp loader can interact with each of the sliding clamps from the three domains of life and whether any of the interactions leads to loading of the sliding clamps onto DNA to orchestrate processive DNA synthesis.

This work, part of Specific Objectives 2 and 3, is a multidisciplinary collaboration primarily between Isaac Cann (a microbiologist) and Zaida Luthey-Schulten (a chemist).

The nature of the last Archaeal and Eukaryal ancestor. The Tree of Life, as developed by Carl Woese and collaborators, shows that there are two branch points for life. One is the root of all life, the so-called Last Universal Common Ancestor (LUCA) from which all life today has descended. The other is the root or branchpoint between the Eukarya and the Archaea. These two different roots should have rather different characteristics, because they involved two distinct evolutionary transitions. The LUCA transition is likely to correspond to the transition between horizontally-dominated and vertically-dominated evolution for core cellular machinery. But the Eukarya-Archaea transition corresponds to a different type of transition, one that involves far less fundamental change to the evolutionary process, since it occurred after the horizontal-vertical switch. This project is attempting to advance our understanding of this transition, using contemporary analogs of ancestral anaerobic eukaryotes (rumen ciliates), which are often associated with endosymbiotic archaea and bacteria in tightly associated communities. We study the evolution of this association using state-of-the-art metagenomic and ecological methods to gain a better understanding of the evolution of these types of associations and thus of eukaryotic evolutionary history.

As a first step to exploring this question, we have identified the architecture of the Archaeal genome in which some regions are highly exposed to gene flow and demonstrate communal dynamics where others do not. Variation in recombination rates across chromosomes has been shown to be a primary force shaping the architecture of genome divergence. In archaea, little is known about variation in recombination across the chromosome or how it shapes genome evolution. We identified significant variations in polymorphism occurring across the chromosomes of ten closely-related sympatric strains of the thermoacidophilic archaeon Sulfolobus islandicus, demonstrating how recombination defines the mosaic of variation in this asexually reproducing microorganism and providing insight into the evolutionary origins of genome architecture in this organism from the Archaeal domain.

A second step is to look at the evolution of early Eukarya. These likely evolved in low oxygen environments in long-term associations with anaerobic archaea and bacteria. Contemporary anaerobic microbial eukaryotes lacking mitochondria still persist, and a common reservoir for these organisms is the bovine rumen, a complex microbial ecosystem with intimately associated endo- and ectosymbiotic bacteria and archaea. We have explored the various forms of symbiosis present in the rumen through metagenomic comparative and population genomic analyses of a ciliate-methanogen consortium, identifying the symbiont genomes which are enriched in carbohydrate and amino acid metabolism subsystems. Our findings suggest that genomes of early anaerobic eukaryotes could have been shaped by horizontal gene transfer from similar intimately associated hydrogen-consuming archaea and bacteria.

This work, part of Specific Objectives 2 and 3, is led by Rachel Whitaker (a microbiologist at UIUC) and Scott Dawson (a microbiologist at UC Davis).

Control of evolvability and chromosomal rearrangement by stress. Genome plasticity or 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 universal mechanisms by which organisms evolve to adapt to new or changing environments, and are thus critical to astrobiology questions related to understanding the rate of emergence of life.

Research activity has used the lac assay to explore the way in which genome plasticity occurs through the suppression of the general and starvation stress-response regulator RpoS. The lac assay measures the frequency of cells that mutate to lactose utilization either by point mutation or by amplification of the leaky allele. Amplification is a proxy for GCR. So far, we have identified a number of genes, insertions and transposons that may inform how RpoS regulates GCR, and hence how stress regulates genomic change. In addition, we are probing the involvement of nucleoid associated proteins (NAPs) in stress-induced mutation. NAPs condense DNA, limiting access of DNA-acting proteins, namely proteins concerned in regulation, transcription, replication and repair. They are important in controlling the response of the genome to stressful environments. We find that 11 of 14 NAPs are required for both GCR and point mutation, showing that they do not control choice of mutation pathway.

This work, part of Specific Objective 4, is carried out by Susan Rosenberg and Philip Hastings, both microbiologists.

Culturing microbial communities in controlled stress micro-environments. 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.

Two major steps have been completed in Year 1 toward the accomplishment of our research goals. The first is that the Archaea Methanosaricina acetivorans, was chosen as a key lab organism on which to test microbial responses in our GeoBioCell microfluidic cells. The project team has worked extensively with this strain of Archaea, and its full genome has been sequenced to completion. Initial successful steps have been taken in the growth and culturing of this organism. . In addition, E. coli K12 was chosen as an added alternative to M. acetivorans, because it is known to grow easily and previous research shows it can be grown in GeoBioCells. Parallel batch experiments were done and growth curves were obtained by optical density (OD) measurement to determine the duration of lag phase, the growth rate, the generation time and the maximum concentration. These parameters are important for microbial inoculation and growth in the GeoBioCell. The second major step was the successful design and construction of a new Zeiss microscope, digital camera and anti-vibration table system on which the GeoBioCell is now being operated. The device itself was fabricated in the Micro and Nanotechnology (MNTL) at UIUC using standard photolithography techniques. We are currently testing the device using clean water and fluorescein to ensure that we can establish a gradient across the 1200 well array. Once this is established, the GeoBioCell will be inoculated with E. coli and adaptation to an antibiotic concentration gradient will be evaluated.

This project, part of Specific Objective 4, is a multidisciplinary collaboration involving Bruce Fouke (a geologist), Charlie Werth (a civil engineer), with involvement from Isaac Cann and Rod Mackie (both microbiologists by discipline).

Our Illinois-Baylor-UC Davis E/PO has been extremely active and has successfully fulfilled all of our mission goals for Year 1. This has included: (1) K-12 Formal Education: hiring of a Ph.D. graduate student named Susan Kelly, who was a former National Park Service range in Yellowstone National Park and is now establishing new middle school and high school Student Teacher Scientist Partnership (STSP) programs at 10 different public schools in Illinois and Montana; (2) Higher Education: successful filming, development and formal offering of a new Astrobiology University of Illinois College of Liberal Arts and Sciences On-Line course entitled GEOL 111 Emergence of Life (82 students were formally enrolled and successfully completed the course for formal credit); (3) Higher Education: offering of a new Illinois Campus Honors Program (CHP) course entitled CHP 395 Yellowstone Astrobiology, for which a capstone field course was conducted for 20 Illinois CHP students in Yellowstone where the students completed astrobiology-driven field experiments; and (4) General Web-Based Education: initial filming and development of a new Massively Open On-Line Course (MOOC) entitled Evolution Through the Lens of Astrobiology (to be offered in Spring 2014, with an expected enrollment of 200,000-300,000 students); and In addition, the writing and development of a new Yellowstone Astrobiology textbook is underway to be used with the new NAI MOOC, formal on-line and STSP courses. This book will be published by the University of Illinois Press and represents a collaborative effort between Co-PI Fouke and Tom Murphy who is an acclaimed wildlife photographer in Yellowstone (see his work at http://www.tmurphywild.com).