LExEN: The Chemistry and Biology of Unusual Adaptive Solutes in Archaeoglobus Fulgidus and Natronococcus Occultus

点击翻译按钮获取中文摘要

Roberts9978250Two archaea, Archaeoglobus fulgidus and Natronococcus occultus, grow in extreme environments. Although both of these grow in moderate to high salt media (with N. occultus a true halophile), A. fulgidus is a hyperthermophile while N. occultus requires an external pH of 9.5 and is classified as a haloalkaliphile.It has been proposed that organisms that live and adapt to hypersaline environments can provide insightsinto life on early Mars. Halophilic archaea, in particular, may be important models for life on early Marswith sulfate-reducing archaea providing the most realistic models for extra-terrestrial environments. This project seeks to understand how both A. fulgidus and N. occultus respond to changes in external NaCl, including protein and solute (organic osmolyte) synthesis, K+ fluxes, and mechanisms for stabilization of intracellular macromolecules. The emphasis is on the biology of several unusual chemical compounds used as organic osmolytes, and their very specific role in the survival of these cells. Techniques used include in vivo NMR spectroscopy, atomic absorption, 35S-uptake and 2D SDS-PAGE, and computer simulations of water and the effect osmolytes have on water structure and dynamics. For A. fulgidus an important area to investigate is how growth temperature regulates di-inositol-1,1'-phosphate (DIP) synthesis and accumulation. This unusual organic solute is only accumulated and used to balance external osmotic pressure when the organism is grown above 80 degrees C. Cloning, overexpression, and characterization of enzymes involved in DIP synthesis will provide details on the biosynthetic machinery for this solute whose accumulation correlates with thermophily in many other archaea as well. Where possible overexpressed enzymes will be crystallized for structure determination. This goal will be particularly important for the novel A. fulgidus inositol-1-phosphate synthase that appears to be a class II aldolase, and for the proposed CTP:I-1-P cytidylyltransferase and DIP synthase activities (when they are identified and purified from the cells). Monitoring protein and message synthesis for these enzymes upon thermal stress should provide further insights into the temperature regulation of DIP accumulation. A related phosphodiester, di-a-glycerol phosphate, appears to be the major osmolyte at lower growth temperatures in A. fulgidus. Nothing is known about the biosynthesis of this solute from a-glycerol phosphate (although it is likely an activated glycerol phosphate is condensed with glycerol in analogy to the 'DIP synthase' reaction). NMR experiments are planned to elucidate the enzymology involved.