Molecular studies of dissimilatory selenium reduction by subsurface microorganisms

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。智力优势：厌氧原核生物进行异化硒还原的能力是一种显着的生物适应性，它允许硒呼吸微生物在地球上填充生态位。年代的地下。在实验室实验中，多种厌氧菌已被证明使用硒酸盐作为终端电子受体生长。然而，基于现场的证据表明，硒氧阴离子还原支持地下微生物仍然难以捉摸。本研究旨在阐明厌氧硒呼吸的遗传决定因素，并利用这些分子指标量化沉积环境中硒还原菌的原位活性。本研究的目的是：1)鉴定不同硒还原菌菌株中硒酸盐还原酶的功能基因；2)硒酸还原酶基因的表达是硒还原活性的可靠标志；3)定量硒酸还原酶基因在硒氧阴离子还原活跃的缺氧沉积物中的原位转录水平。使用调查?收集新的硒呼吸分离物，他们将采用定向克隆技术和基因组-上下文分析来鉴定赋予硒酸还原酶活性的基因。利用这些遗传信息，将开发退化PCR引物来扩增硒酸盐还原酶的区域，这些区域在系统发育不同的硒还原细菌物种中是遗传保守区域。利用这些PCR引物，进行定量实时反转录（QRT） PCR实验，确定硒酸盐还原酶基因在硒酸盐作为唯一终端电子受体上生长时的转录反应。这些PCR引物在纯培养分离物上进行测试后，将在天然沉积物上进行QRT-PCR，以测量沉积物岩心中硒酸还原酶基因的mRNA水平，其中垂直地球化学剖面显示硒氧阴离子还原的发生。通过建立硒还原的遗传学和地球化学之间的联系，研究人员旨在为自然界中这种独特的生命模式的存在提供新的见解，并阐明沉积环境中控制硒循环的微生物过程。更广泛的影响：地球微生物学的本科生研究机会将通过与罗格斯大学荣誉项目的合作而启动。建立四年的本科生研究经验，以促进地质和生物科学界面的发现式学习。地质与环境地球科学专业的本科生将学习如何设计PCR引物并进行PCR反应来扩增硒酸还原酶基因。本科生研究项目的结果将通过校内和校外会议、本科生研究电子期刊和同行评议的科学期刊传播。该项目还将培养两名分子地球微生物学领域的研究生。地质科学研究生课程的研究生将学习克隆、排序和突变硒酸还原酶基因。从实验中获得的序列数据将被整合到罗格斯大学新地球微生物学课程的家庭作业中。本课程的学生将学习使用基于网络的搜索工具来分析和解释核苷酸序列数据。为了将研究活动整合到K-12科学教学中，研究人员建议使用罗格斯科学探索者？最先进的移动实验室？推进城市高民族学区地学教育。将开发一个90分钟的生物地球化学模块，向中学生传授物质如何在地圈和水圈之间移动的基本概念。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Intellectual Merit: The ability of anaerobic prokaryotes to perform dissimilatory selenium reduction is a remarkable biological adaptation that allows selenium respiring microorganisms to populate ecological niches in Earth?s subsurface. In laboratory experiments, a diverse range of anaerobes have been shown to grow using selenate as a terminal electron acceptor. However, field-based evidence for subsurface microorganisms that are supported by selenium oxyanion reduction remains elusive. In this study, it is proposed to elucidate the genetic determinants involved in anaerobic selenium respiration, and use these molecular proxies to quantify the in situ activity of Se-reducing bacteria in sedimentary environments. The objectives of this research are: 1) to identify the functional selenate reductase genes in diverse strains of Se-reducing bacteria; 2) to demonstrate that the expression of the selenate reductase gene is a reliable marker for Se-reducing activity; and 3) to quantify in situ transcript levels of the selenate reductase genes in anoxic sediments where selenium oxyanion reduction is actively occurring. Using investigators? collection of novel selenium respiring isolates, they will employ directing cloning techniques and genome-context analysis to identify the genes that confer selenate reductase activity. With this genetic information, degenerate PCR primers will be developed to amplify the regions of the selenate reductase that are genetically conserved regions among phylogenetically diverse species of Se-reducing bacteria. Utilizing these PCR primers, quantitative real-time reverse transcription (QRT) PCR experiments will be conducted to determine the transcriptional response of the selenate reductase gene during growth on selenate as the sole terminal electron acceptor. After these PCR primers have been tested on pure culture isolates, QRT-PCR will be performed on natural sediments to measure mRNA levels of selenate reductase genes in sediment cores where vertical geochemical profiles show the occurrence of selenium oxyanion reduction. By establishing a link between the genetics and geochemistry of selenium reduction, investigators aim to lend new insight into the existence of this unique mode of life in nature, and illuminate the microbial processes that govern the cycling of selenium in sedimentary environments. Broader Impacts: Undergraduate research opportunities in Geomicrobiology will be initiated through collaboration with the Rutgers Honors Program. Four-year undergraduate research experiences will be established to promote discovery-based learning at the interface of geological and biological sciences. Undergraduate students majoring in geology and environmental geosciences will learn how to design PCR primers and carry out PCR reactions to amplify selenate reductase genes. The results of undergraduate student research projects will be disseminated via on- and off-campus conferences, electronic journals for undergraduate research, and peer-review scientific journals. This project will also train two graduate students in the field molecular geomicrobiology. Graduate students from the Geological Sciences graduate program will learn to clone, sequence, and mutate selenate reductase genes. Sequence data obtained from the experiments will be integrated into homework assignments for the new Geomicrobiology course at Rutgers University. Students taking this course will learn to use web-based search tools to analyze and interpret nucleotide sequence data. To integrate research activities into the teaching of K-12 science, investigators propose to employ the Rutgers Science Explorer?a state-of-the-art mobile laboratory ? to advance geoscience education in high-minority urban school districts. A 90 minute module on biogeochemistry will be developed to teach middle school students the basic concepts of how matter moves between the geosphere and hydrosphere.