Molecular studies of dissimilatory selenium reduction by subsurface microorganisms

该奖项由2009年美国复苏和再投资法案(公法第111-5条)资助。智力价值：厌氧原核生物进行异化硒还原的能力是一种非凡的生物适应能力，使呼吸硒的微生物能够在地球上的生态位上繁殖？S地下。在实验室实验中，各种厌氧菌已经被证明使用硒作为终端电子受体来生长。然而，基于现场的证据表明地下微生物是由硒氧阴离子还原支持的，仍然难以捉摸。本研究旨在阐明厌氧性硒呼吸作用的遗传决定因素，并利用这些分子指标对沉积环境中硒还原细菌的原位活性进行定量研究。本研究的目的是：1)鉴定不同菌株的功能硒还原酶基因；2)证明硒还原酶基因的表达是硒还原活性的可靠标志；3)在硒氧阴离子还原活跃的缺氧沉积物中对硒还原酶基因的转录水平进行原位定量。利用调查人员？为了收集新的硒呼吸分离物，他们将使用定向克隆技术和基因组背景分析来识别赋予硒还原酶活性的基因。有了这些遗传信息，简并的PCR引物将被开发出来，以扩增硒还原酶的区域，这些区域在不同种类的降硒细菌中是遗传保守的区域。利用这些引物，进行实时定量逆转录(QRT)聚合酶链式反应(QRT)实验，以确定硒还原酶基因在作为唯一末端电子受体的硒酸根上生长过程中的转录反应。在纯培养分离株上测试了这些PCR引物后，将在天然沉积物上进行QRT-PCR，以检测垂直地球化学剖面显示硒氧阴离子还原发生的沉积物岩心中硒还原酶基因的mRNA水平。通过建立硒还原的遗传学和地球化学之间的联系，研究人员旨在对自然界中这种独特的生命模式的存在提供新的见解，并阐明控制沉积环境中硒循环的微生物过程。更广泛的影响：将通过与罗格斯大学荣誉计划的合作，启动本科生在地质微生物学方面的研究机会。将建立为期四年的本科生研究经验，以促进地质和生物科学相结合的基于发现的学习。地质学和环境地学专业的本科生将学习如何设计PCR引物，并进行PCR反应来扩增硒还原酶基因。本科生研究项目的结果将通过校内外会议、本科生研究电子期刊和同行评议科学期刊来传播。该项目还将培养两名分子地质微生物学领域的研究生。地质科学研究生专业的研究生将学习如何克隆、测序和突变硒还原酶基因。从实验中获得的序列数据将被整合到罗格斯大学新的地球微生物学课程的家庭作业中。学习这门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科学的教学中，研究人员提议使用罗格斯科学探索者？一个最先进的移动实验室？推进城市少数民族高中生学区地学教育。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运动的基本概念。

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.