Metabolic Engineering Studies of Extreme Thermoacidophily

Extreme thermoacidophily (growth ~ 60[unreadable]C, pH s 3) is a remarkable growth physiology exhibited by certain members of the Crenarchaeota and Euryarchaeota, the major archaeal phyla. Interesting features of microorganisms in this group include: exploitation of large pH gradients across the cell membrane to drive proton pumping for maintaining a near neutral cytosol, resistance to normally toxic levels of base and heavy metals, capacity to grow autotrophically by fixing CO2, heterotrophically on peptides, or mixotrophically by using both modes, bioenergetics based on dissimilatory oxidation of reduced metal and sulfur species, unique membranes and S-Iayers, and growth stimulation by molecular H2[unreadable] By strategically integrating some or all of these physiological features into cellular metabolism, life in hot acid becomes possible. However, little is known about how this happens beyond biochemical characterization of a few proteins from these microorganisms and rudimentary pathway analysis. Yet, if the mechanisms behind these features were understood, insights into the biological strategies that define extreme thermoacidophily would result. Such insights have important scientific and technological importance. For example, it is clear that the Crenarcheaota (not all of which are extreme thermoacidophiles) comprise a significant fraction of the global biomass, and are relevant beyond extreme niches. These organisms play an expanding role in global carbon cycling, nitrogen cycling, symbiosis and eukaryotic interactions. The transcriptional and translational mechanisms observed in archaea are closely related to those found in eukaryotes and, thus, provide an excellent alternative perspective. Finally, clues to cellular survival under extreme conditions could lead to interesting medical strategies for surviving stress for higher eukaryotes. The objectives of this project are: 1) Determine the components of Iithotrophic pathways in the extreme thermoacidophile Metallosphaera sedula and how these processes relate to one another and integrate into overall cellular bioenergetics;2) Investigate the metallomics underlying M. sedula's intrinsic heavy and base metal resistance;3) Integrate the findings gained from aims 1 and 2 with studies on transcription factors, chromatin modification, and toxin-antitoxin loci to derive a conceptual model for the M. sedula transcription regulatory system.