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

罗伯特9978250两种古生菌，黄褐古球菌和隐蔽纳氏球菌，生长在极端环境中。虽然这两种细菌都生长在中到高盐的介质中(潜伏藻是真正的嗜盐者)，但黄褐指藻是一种高度嗜热的细菌，而潜伏藻需要外部pH为9.5，属于嗜盐嗜碱菌。有人提出，生活在高盐环境中并适应高盐环境的有机体可以提供对早期火星生命的洞察。尤其是嗜盐古生菌，可能是火星早期生命的重要模型，而硫酸盐还原古生菌为外星环境提供了最现实的模型。本项目旨在了解黄褐指藻和隐匿藻如何响应外界盐分的变化，包括蛋白质和溶质(有机渗透物质)的合成，K+通量，以及细胞内大分子的稳定机制。重点是几种用作有机渗透分子的不寻常化合物的生物学，以及它们在这些细胞生存中的非常特殊的作用。使用的技术包括体内核磁共振光谱、原子吸收、35S吸收和2D SDS-PAGE，以及对水和渗透分子对水结构和动力学的影响的计算机模拟。对于黄曲霉来说，一个重要的研究领域是生长温度如何调节二肌醇-1，1‘-磷酸(DIP)的合成和积累。这种不寻常的有机溶质只有在生物体生长在80℃以上时才会积累并用于平衡外部渗透压力。参与DIP合成的酶的克隆、过度表达和表征将提供这种溶质生物合成机制的细节，这种溶质的积累也与许多其他古生物中的嗜热性相关。在可能的情况下，过度表达的酶将被结晶以进行结构测定。这一目标对于新的似乎是II类醛缩酶的黄曲霉肌醇-1-磷酸合成酶以及拟议的CTP：I-1-P胞苷转移酶和DIP合成酶活性(当它们被从细胞中鉴定和纯化时)尤其重要。监测这些酶在热胁迫下的蛋白质和信息合成，应该能为DIP积累的温度调节提供进一步的见解。在较低的生长温度下，一种相关的磷酸二酯，二a-甘油磷酸，似乎是黄曲霉的主要渗透调节物质。关于这种溶质是如何从α-甘油磷酸中生物合成的，我们一无所知(尽管它很可能是活化的甘油磷酸与甘油缩合，类似于‘DIP合成酶’反应)。计划进行核磁共振实验以阐明所涉及的酶学。

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.