LExEn: Mercury Resistance in Archaea

该项目正在调查汞和古生菌之间的关系。汞是一种剧毒的重金属，从朱砂矿床中开采出来，但在其他方面很稀有。大多数细菌原核生物利用mer基因来实现抗汞能力。Mer基因高度保守，分布广泛，并经常在细菌之间传播。令人惊讶的是，没有古原核生物对汞产生抗药性的报道。为了研究这一问题，对自然形成的富含汞的地热环境进行了研究，这些环境的特点是巯基缺乏和氧化过剩。抗汞古菌是从位于莫哈韦沙漠的朱砂温泉(pH 1.7，78℃，2毫克/升汞)3-4池中培养出来的。水池3-4受到季节性降雨的强烈影响，并伴随着水池溶质水平的变化，特别是汞。该项目的工作重点是一种被确认为Sulfolobus solfararicus的菌株。在这种分离物中维持古生菌对汞的抗性需要选择性压力。汞抗性与一个13kb的内源质粒的共分离表明，抗性是由质粒编码的。鉴定出汞适应性反应伴随着一个25 kDa蛋白的瞬时诱导。该Lexen项目的第一部分侧重于古细菌对汞的抗性和适应。汞还原和挥发的酶检测将测试细菌聚合型机制的存在。内源质粒的特性将使鉴定可能的Mer同源序列成为可能。在汞敏感受体的遗传转移研究中，将使用质粒删除衍生品来识别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。汞抗性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汞抗性基因的起源和潜在的转移。将完成池3-4的培养和未培养微生物的系统发育分析，并用于创建特定的荧光标记探针，用于群落结构分析。对群落组成的测量将确定汞水平季节性变化的后果。古生代的汞抗性基因探针将被用来跟踪与汞浓度峰值一致的3-4池社区成员之间汞抗性的分布和转移。预期的结果应该区分古生菌汞抗性的可能来源，并建立对古生菌汞毒性机制的理解。这些信息将扩展关于极端氧化环境中生命的知识，并有助于对古生代嗜热细菌的遗传操作。

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.