LExEn: Mercury Resistance in Archaea

这个项目正在调查汞和古生菌之间的关系。汞是一种从朱砂矿床中开采出来的剧毒重金属，但在其他方面很罕见。大多数细菌原核生物利用mer基因来获得抗汞性。mer基因高度保守，分布广泛，并经常在细菌中传播。令人惊讶的是，在古细菌原核生物中没有汞耐药性的报道。为了调查这个问题，研究人员对一个以巯基缺乏和氧化过剩为特征的天然富汞地热环境进行了研究。从位于莫哈韦沙漠的朱砂温泉池3-4 （pH值1.7,78℃，汞含量2 mg/l）中培养耐汞古菌。池3-4受到季节性降雨的强烈影响，同时池中溶质含量，特别是汞含量也发生了变化。本项目的工作集中在一种鉴定为solfataricus的分离物上。在这种分离物中维持古细菌的抗汞性需要选择压力。汞抗性和一个13kb内源质粒的共分离表明抗性是质粒编码的。汞的适应性反应被鉴定出来，并伴随着一个25 kDa蛋白的瞬时诱导。本LExEn项目的第一部分侧重于古细菌对汞的抗性和适应性。用于汞还原和挥发的酶测定将测试细菌mer型机制的存在。内源性质粒的鉴定将使鉴定可能的同源序列成为可能。在汞敏感受体的遗传转移研究中，将使用质粒缺失衍生物确定对汞抗性至关重要的区域。鉴定汞诱导的25 kDa蛋白有助于阐明其毒性机制。抗汞和适应之间的关系将集中在25 kDa蛋白的表达模式上。该项目的第二部分侧重于抗汞基因的起源和潜在转移。将完成池3-4中培养和未培养微生物的系统发育分析，并用于创建用于群落结构分析的特异性荧光标记探针。对群落组成的测量将确定汞含量季节性变化的后果。古菌抗汞基因探针将用于追踪与汞浓度峰值一致的Pool 3-4群落成员之间的汞抗性分布和转移。预期的结果应该区分古菌抗汞的可能来源，并建立对古菌汞毒性机制的理解。这一信息将扩展对极端氧化环境下生命的认识，并有助于古细菌嗜热菌的遗传操纵。

This project is investigating the relationship between mercury and archaea. Mercury is an extremely toxic heavy metal mined from cinnabar deposits but is otherwise rare. Most bacterial prokaryotes employ the mer genes to achieve mercury resistance. The mer genes are highly conserved, widely distributed, and frequently transmitted among bacteria. Surprisingly, there are no reports of mercury resistance among archaeal prokaryotes. To investigate this question studies have been undertaken on a naturally occurring mercury-rich geothermal environment distinguished by sulfhydryl deficiency and oxidative excess. Mercury resistant archaea were cultured from Pool 3-4, a cinnabar hot springs (pH 1.7, 78 degrees C, 2 mg/liter mercury) located in the Mojave Desert. Pool 3-4 is strongly influenced by seasonal rainfall with concomitant changes in pool solute levels, particularly mercury. Efforts in this project are focused on one isolate identified as Sulfolobus solfataricus. Maintenance of archaeal mercury resistance in this isolate requires selective pressure. Co-segregation of mercury resistance and a 13 kb endogenous plasmid indicates that resistance is plasmid encoded. A mercury adaptive response was identified which is accompanied by transient induction of a 25 kDa protein. The first part of this LExEn project focuses on archaeal mercury resistance and adaptation. Enzyme assays for mercury reduction and volatilization will test for presence of a bacterial mer-type mechanism. Characterization of the endogenous plasmid will enable identification of possible mer orthologous sequences. Regions critical for mercury resistance will be identified using plasmid deletion derivatives for genetic transfer studies with mercury sensitive recipients. Identification of the mercury induced 25 kDa protein will help clarify the mechanism of toxicity. The relationship between mercury resistance and adaptation will focus on expression patterns of the 25 kDa protein. The second part of the project focuses on the origin and potential transfer of mercury resistance genes. Phylogenetic analysis of cultured and uncultured microbes from Pool 3-4 will be completed and used to create specific fluor-labled probes for community structure analysis. Measurements of community composition will establish the consequences of seasonal variation in mercury levels. Archaeal mercury resistance gene probes will be used to track distribution and transfer of mercury resistance among members of the Pool 3-4 community coinciding with peak mercury concentrations. The anticipated results should distinguish possible origins of archaeal mercury resistance and establish an understanding of the mechanism of mercury toxicity in archaea. This information will extend knowledge about life in extremely oxidizing environments and contribute to the genetic manipulation of archaeal hyperthermophiles.