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Project Reports

• 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

• The Nature of the Last Archaeal and Eukaryal Ancestor

  The evolutionary history of the eukaryotic cell is intimately linked evolution of atmospheric oxygen and with the endosymbiosis of bacterial symbionts to become the mitochondrial organelles. This project seeks to understand the evolutionary history of the eukaryotic cell 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.

  ROADMAP OBJECTIVES: 3.4 4.1 4.2 5.2 6.1

• 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.

  ROADMAP OBJECTIVES: 3.2 4.1 4.2 5.3 6.2

• 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

• 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. 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 b-subunit of Pol-III and in Archaea and Eukarya the functional homolog is proliferating cell nuclear anti-gen (PCNA). We have, therefore, 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.

  ROADMAP OBJECTIVES: 3.2 3.4 4.2

• 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