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Project Reports

• Project 1: The Origin of Homochirality

  Small biological molecules are frequently chiral, meaning that they can exist in both right-handed and left-handed forms. The two forms are identical except for the mirror symmetry that they break, and so would be expected to participate in chemical reactions in a way that does not depend on their chirality. When assembled into polymers, the resulting chains would therefore be expected to consist of a mixture of right and left-handed forms of the small molecules, a so-called racemic state. The surprise is that this is not true for the molecules of life. All chiral amino acids used by biology are left-handed and all chiral sugars are right-handed. That is, they are homochiral. This project is concerned with trying to find an explanation for this ubiquitous phenomenon, a universal aspect of all life on Earth. The specific question that is addressed is whether homochirality is a generic phenomenon of living systems, one that would be anticipated to arise if life were found elsewhere in the universe. Or is it instead some frozen accident related to the specific way that life arose on Earth? This question has been hotly debated in one form or other for over a hundred years, certainly since the time that Lord Kelvin coined the term “homochirality”. It is important for the Illinois NASA Astrobiology Institute for Universal Biology, because it is one of the two most evident universal phenomena of all life on Earth, the other being the universal genetic code. The phenomenon is important for another reason. The magnitude of the homochirality is 100%. It is not a slight imbalance in the abundance of right-handed vs. left-handed molecules. Thus, it is an unambiguous signal to measure, either from biological samples or remotely due to the effects of homochirality on the scattering of light waves. Specifically, homochiral solutions or suspensions will affect the polarization plane of electromagnetic waves, and so can readily be detected through optical means. The most exciting possibility in this regard is that if homochirality can be firmly established as a biological phenomenon, then its presence can be used as a biosignature of non-terrestrial life.

  ROADMAP OBJECTIVES: 1.2 3.2 3.4 4.1 4.2 7.1 7.2

• Project 2: Cells as Engines and the Serpentinization Hypothesis for the Origin of Life

  All life is, and must be, “powered” since all of its most essential and distinguishing processes have to be driven “up-hill” against their natural thermodynamic direction. By the 2nd law of thermodynamics, however, a process can only be made to proceed up-hill by being mechanistically linked, via a molecular device functioning as an engine, to another, more powerful, process that is moving in its natural, down-hill direction. On fundamental principles, we argue, such engine-mediated conversion activities must also have been operating at, and indeed have been the cause of, life’s emergence. But what then were life’s birthing engines, what sources of power drove them, what did they need to produce, and how did they arise in an entirely lifeless world? Promising potential answers to these and other questions related to the emergence of life are provided by the Alkaline Hydrothermal Vent/serpentinization (“AHV”) hypothesis, whose original propounder and lead proponent, Dr. Michael Russell of JPL, is a co-investigator on this project. The goal of the project is specifically to clarify the essential mechanistic modus operandi of all molecular engines that power life, and to see how the most fundamental and prerequisite of these could have arisen, and operated, in the structures and flows produced by the serpentinization process. Importantly, candidate answers to these questions can be put to definitive laboratory tests.

  ROADMAP OBJECTIVES: 1.1 1.2 3.1 3.2 3.3 3.4

• Project 3: Theory for the Darwinian Transition

  One of the key puzzles of astrobiology concerns the precision, uniqueness and rapidity of early evolution. In order for life to have evolved the main components of the modern cell as early 3.8 billion years ago, with a unique genetic code that is virtually optimal in terms of minimizing translational errors, the mode of evolution would have had to be different from the current vertical transmission of genes. We had shown in 2006 that the collective mechanism of horizontal gene transfer (HGT) is the only one capable of solving the puzzle of early evolution. The HGT means that the evolutionary process before LUCA can be thought of as a network of interactions rather than a tree, as would be the case in vertical gene transfer. The multiple connectivity of the network accelerates the evolution and allows rapid convergence to a unique, near-optimal genetic code. With all these advantages of HGT, why would it ever stop? Our project uses computer simulation of digital organisms in order to address these generic questions about the exit of life from the collective, progenote phase to the current era of vertically dominated evolution.

  This project is potentially important for understanding biosignatures of life. Even on Earth, we are familiar with the tree-like structure of individual organismal lineages. If life were a network, as we believe that it once was, the usual phylogenetic pattern of individuality and species would not apply. If we encounter life on other planets, we cannot be sure if it will be in the collective (progenote) phase or the vertical-dominated phase. Thus it is interesting to understand better the inexorability and timing of the Darwinian Transition.

  ROADMAP OBJECTIVES: 3.2 3.4 4.1 4.2 5.1 6.2

• Project 4: Experiment on Darwinian Transition

  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. We aim at 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. In Bacteria, the sliding clamp is the -subunit of Pol-III and in Archaea and Eukarya the functional homolog is proliferating cell nuclear antigen (PCNA). We have, therefore, expressed and purified a sliding clamp from each of the three domains of life (E. coli -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.

  ROADMAP OBJECTIVES: 3.2 3.4 4.2

• Project 5: Control of Evolvability and Chromosomal Rearrangement by Stress

  Using a bacterial system, we are investigating the regulation of genomic change by stress, both small changes in DNA sequence and larger chromosomal rearrangements. That mutation rates should be regulated by stress has wide significance for understanding mechanisms of evolution. Our major goal in this project is to discover how the general stress-response regulator switches from accurate modes of DNA repair to error-prone modes. We have established that mutation under stress only occurs if there are replicating blocking lesions in DNA, usually formed by reactive oxygen. We have shown apparent positive and negative regulation of the levels of oxygen radicals. We are now working to discover the origin of the radicals so that we can ask whether radicals form as an unavoidable byproduct of respiration, or whether this represents a dedicated mechanism to instigate mutation, and hence evolution.

  ROADMAP OBJECTIVES: 5.1

• Project 6: Life’s Diversity

  This project is on the theoretical modeling of life’s complexity and diversity, where we are modeling evolvability, diversity, and complexity in mathematical terms. Since these models are of high complexity, we are employing asymptotic and other approximate methods for their solution.

  ROADMAP OBJECTIVES: 4.2 6.2

• Project 7: Mining Archaeal Genomes for Signatures of Early Life: Comparison of Metabolic Genes in Methanogens

  Methanogens represent the largest diversity among the archaea and have the unique ability to generate methane from simple compounds such as carbon dioxide, acetate and methylamines which were common in the anaerobic environments of early Earth and perhaps Mars. Methane biosynthesis also requires the presence/uptake of important ions such as sulfates, sulfides, carbonates, phosphates, and various light metal ions. In this project, we are attempting to analyze the evolution of the methanogens’ central cellular functions of translation, transcription, replication, and metabolism. To accomplish this, we are constructing the metabolic and regulatory networks of Methanosarcina acetivorans, the most complex methanogen known, and using these models to establish a framework for studying the evolution of methanogens. Results will be tested through microfluidic studies using varying carbon and ion sources.

  ROADMAP OBJECTIVES: 1.1 2.1 3.1 3.2 3.3 3.4 4.1 4.2 5.1 5.2 5.3 6.1 6.2 7.1

• Project 8: The Evolution of the Eukaryote-Archaea Common Ancestor

  The goals of our lab with respect to the NAI project are to describe early evolutionary via genomic and cellular comparisons of diverse eukaryotes to diverse archaea. We are interested in comparing genomes from diverse free-living eukaryotes to investigate the origins and evolution of eukaryotic complexity. Evolutionary reconstructions of early eukaryotes are challenged by a lack of sufficient taxonomic sampling. Few genomes of free-living microbial eukaryotes are sequenced, despite their critical importance in ecology, evolution, and basic cellular biology. The real challenge to protistan genomics is actually quite mundane; it concerns the lack of available and cultivatable free-living protists (mainly heterotrophs) in the laboratory. Yet, a better understanding of the genomic content diverse eukaryotes facilitates the evolutionary analysis of archaeal genomes. To address these issues of poor taxonomic sampling of eukaryotic genomes, my lab has developed a molecular method to separate eukaryotic DNA from bacterial DNA. We have demonstrated conclusively that we can separate eukaryotic chromatin from a mixture of eukaryotic and bacterial genomic DNA. This method will be widely applicable to the study of protistan genomics. Currently, our lab is in the process of assembling and annotating ten eukaryotic genomes from my lab’s culture collection of over 100 amoeboid protists from diverse phylogenetic groups. Many of these amoeba represent novel phyla-level lineages of eukaryotes.

  One amoebal genome is form is Nuclearia sp., which is an amoeboid protist closely and a member of a primary “supergroup” of eukaryotes – the Optisthokonts. This supergroup includes all animals, fungi, and several types of unicellular or colonial protists including choanoflagllates. Thus, genomic analyses of Nuclearia will inform the evolution of complexity and multicelllularity in both Fungi and Animals.

  ROADMAP OBJECTIVES: 3.2 3.4 4.2 6.2

• Project 9: Metapopulation Structure

  Although often modeled as a single well mixed populations, microbes in terrestrial systems likely exist as metapopulations, isolated but connected by infrequent migration. This can change the evolution of complexity, increasing the effect of genetic drift and decreasing the effect of selection. It can increase diversity and the rate at which complexity evolves. We have argued that metapopulation structure may have existed in early life and been responsible for the rapid evolution of LUCA and diversification across the tree of life. We investigate microbial genome evolution in metapopulations in Yellowstone National Park. We find that indeed they represent evidence for both natural selection and genetic drift shaping these populations.

  ROADMAP OBJECTIVES: 3.2 3.4 4.2 6.2

• Project 10: Evolution Through the Lens of Codon Usage

  The sequences of protein encoding genes are subject to multiple levels of selection. First, amino acid changes that adversely alter protein function are unlikely to survive. In addition, the genetic code is degenerate; it includes alternative (synonymous) codons for most of the amino acids. The codon usages of genes reflect a balance between drift and selection for rapid and accurate translation of mRNAs into proteins, and in the case of horizontally transferred genes, the codon usages of their sources. Our studies of genes and their codon usages have led us to discover that: (i) most of the recently acquired genes come from such closely related organisms that their distinctive codon usages cannot be attributed to a phylogenetically distant source; (ii) the transfers commonly exceed recognized boundaries of microbial species; (iii) some genes do not drift to match the native codon usage of their current genome, but resemble the most recently acquired genes; (iv) many of the genes that are most up-regulated under starvation conditions also have this codon usage; and (v) a distinctive stress/starvation-associated codon usage is a recurring theme that is observed in diverse Bacteria and Archaea.

  These results have changed our understanding of the dynamics with which genetic novelties are shared in the biosphere, and revealed that there are selective forces on codon usage beyond those currently appreciated in the field.

  ROADMAP OBJECTIVES: 5.1 5.2 5.3 6.1

• Project 11: Culturing Microbial Communities in Controlled Stress Micro-Environments

  This project explores the adaptation and evolution of organisms under controlled environmental conditions, and compares the behavior across two Domains of Life in order to identify and quantify universal aspects of evolutionary response.

  ROADMAP OBJECTIVES: 6.1 6.2