Metabolic Activities and Gene Expression of Marine Psychrophiles in Cold Ice

使细菌在极低温下具有代谢活性的机制对极地微生物生态学、天体生物学、气候和低温保存具有相当重要的意义。这个研究项目有两个主要目标。首先，研究极地海洋嗜冷细菌在高于海水共晶温度（54℃）的冰冻条件下的代谢活动和基因表达，揭示与冬季海冰生态相关的冷适应机制。第二个目标是辨别亮氨酸并入蛋白质的嗜冷过程（显示发生在-196℃）是否相当于提供细胞存活的代谢活动，或者仅仅是在速冻样品中仍然可能发生的生化反应，而对存活没有任何影响。我们将通过以下方法研究共晶上下的细胞外和细胞内亲冷活性过程：(i)使用放射性示踪技术确定代谢活动的温度范围，如DNA合成、碳利用、呼吸和ATP生成，包括在液氦温度（-268.9℃）下的控制；（ii）使用全基因组和微阵列分析分析冰中的基因表达；（iii）使用原位显微镜技术检查外聚合物质（EPS）和冰微物理对观察到的活性的作用。这项研究的结果有望帮助确定细胞和遗传策略，这些策略允许细胞在极低的温度下在冰冷的基质中保持活性，或者在遇到更适宜生长的条件时再次恢复活性。这项研究是一项跨学科合作，涉及三个不同的机构，参与者包括海洋学、基因组学和地球物理科学。拟议的活动将支持女性研究人员开始职业生涯，并将作为几个本科生实验室项目的基础。

The mechanisms enabling bacteria to be metabolically active at very low temperatures are of considerable importance to polar microbial ecology, astrobiology, climate and cryopreservation. This research program has two main objectives. The first is to investigate metabolic activities and gene expression of polar marine psychrophilic bacteria when confronted with freezing conditions at temperatures above the eutectic of seawater (54C) to unveil cold adaptation mechanisms with relevance to wintertime sea-ice ecology. The second objective is to discern if psychrophilic processes of leucine incorporation into proteins, shown to occur to -196C, amount to metabolic activity providing for the survival of cells or are merely biochemical reactions still possible in flash-frozen samples without any effect on survival. We will examine extracellular and intracellular processes of psychrophilic activity above and below the eutectic by (i) determining the temperature range of metabolic activities such as DNA synthesis, carbon utilization, respiration and ATP generation using radioactive tracer technology, including a control at liquid helium temperature (-268.9C), (ii) analyzing gene expression in ice using whole genome and microarray analyses and iii) examining the role of exopolymeric substances (EPS) and ice micro-physics for the observed activity using an in-situ microscopy technique. Results of the proposed research can be expected to aid in the determination of cellular and genetic strategies that allow cells to maintain activity at extremely low temperatures within an icy matrix and/or to resume activity again when more growth-permissive conditions are encountered. The research is an interdisciplinary collaboration involving three different institutions with participants in Oceanography, Genomics, and Geophysical Sciences. The proposed activity will support the beginning professional career of a female researcher and will serve as the basis for several undergraduate student laboratory projects.