Collaborative Proposal: The Genetics Underlying Prokaryote-Antimony Interactions, with Emphasis on Antimony Oxidation.

背景虽然文献中有大量关于锑（Sb）在自然沃茨、土壤和沉积物中赋存状态的报道，但对Sb的地球化学循环却知之甚少。特别是，有关环境微生物锑相互作用的信息仅限于研究记录锑在藻类中的积累，一些报告，记录在藻类文化中的锑形态的变化，和锑甲基化。几十年前发表了原核Sb氧化还原转化的唯一报告，但从未重复，也没有进一步的表征。理解原核生物在环境Sb氧化还原循环中可能发挥的作用的进展受到缺乏环境相关的纯微生物培养物能够Sb氧化和/或还原的限制。我们最近记录了Sb（III）氧化在遗传上易处理的根癌农杆菌土壤隔离，从而设置阶段的第二代研究，将重点放在确定的基因，编码的功能重要roxylate Sb（III）氧化。提议的研究。提出了一个强大的转座子诱变研究，其中将进行理论上的全基因组扫描，涉及数千个transconjugants筛选Sb耐受性的变化，特别是Sb（III）氧化能力的损失。在准备这一建议进行的初步实验已经确定了几个Tn 5-B22 transconjugants的改变，其耐受性的Sb（III），与一些表现出急性敏感性，甚至在低Sb（III）浓度，而其他人显然是现在能够增强的耐受性。此外，锑吸收特性和氧化的变化表明，在这些突变体中的一些。相对容易识别这些突变体表明一个更彻底的诱变筛选将产生许多额外的突变体，然后集体将其特征在于相对于突变的基因和编码的功能，然后由一个彻底的表征优先的突变体的子集，我们将把注意力集中在推定的调控蛋白和尚未确定的推定锑（III）氧化酶。其他感兴趣的突变体将涉及新的Sb转运蛋白。最后一个目标将是使用生物信息学的方法，以确定同源物的数据库为基础，设计PCR引物，用于探测的发生和表达，这些基因在已知含有显着升高水平的Sb。从拟议的实验结果预计将导致基本的发现，将有直接和变革性的影响，我们的理解如何以及为什么细菌相互作用，并氧化，锑（III）。因此，我们预计这项工作将作出一个民族的贡献，我们的锑地球微生物学的理解。更广泛的影响。锑是EPA的优先污染物，但我们对它在环境中的行为几乎一无所知。据推测，像其他类金属，锑在环境中的流动性是由其氧化还原形态的影响，但很少有关于微生物锑氧化还原活性，可能会影响锑的命运和运输的信息。从拟议的项目产生的数据应立即应用于生态研究，提高我们的理解的因素，可能会影响锑的流动性，从而提高我们的理解与锑污染的网站相关的健康风险。拟议的研究还将影响人力资源开发，为博士后科学家和本科生研究实习生提供地球生物学交叉培训机会，这是蒙大拿州立大学和圣路易斯奥比斯波加州理工学院两个实验室的紧密合作。代表性不足的群体将成为本科生招生的重点。最后，从拟议的研究产生的数据将在参与者适当的水平整合到由PI教授的本科和研究生课程，以及PI？的教育推广活动，涉及小学，高中，和科学奠定成人观众。

Background. While the literature contains numerous reports of antimony (Sb) occurrence in natural waters and in soils and sediments, little is known about Sb biogeochemical cycling. In particular, information relevant to environmental microbe-Sb interactions is limited to studies documenting Sb accumulation in algae, a few reports that document changes in Sb speciation in algal cultures, and Sb methylation. The lone report of prokaryotic Sb redox transformation was published decades ago, but never repeated nor followed by further characterization. Progress towards understanding the role that prokaryotes may play in environmental Sb redox cycling has been constrained by the lack of environmentally relevant pure microbial cultures capable of Sb oxidation and or reduction. We have recently documented Sb(III) oxidation in a genetically tractable Agrobacterium tumefaciens soil isolate, thus setting the stage for second generation studies that will focus on identifying the genes that encode for functions important to rokaryotic Sb(III) oxidation. Proposed Research. A robust transposon mutagenesis study is proposed, whereby a theoretical whole genome sweep will be conducted involving thousands of transconjugants to be screened for changes in Sb tolerance, and in particular loss of Sb(III) oxidation capacity. Preliminary experiments conducted in preparation of this proposal have identified several Tn5-B22 transconjugants that are altered with respect to their tolerance of Sb(III), with some demonstrating acute sensitivity at even low Sb(III) concentrations, whereas others apparently are now capable of enhanced tolerance. In addition, changes in Sb uptake properties and oxidation are indicated in some of these mutants. The relative ease of identifying these mutants suggest a more thorough mutagenesis screen will generate many additional mutants that then collectively will be characterized with respect to the mutated genes and encoded functions, followed by a thorough characterization of a prioritized subset of mutants where we will focus attention on putative regulatory proteins and the as-yet-to-be-identified putative Sb(III) oxidase. Other mutants of interest would involve novel Sb transporters. A final objective will then be to use a bioinformatics approach to identify homologues in the databases as the basis for designing PCR primers to be used to probe for the occurrence and expression of such genes in eothermal and mining environments known to contain significantly elevated levels of Sb. Intellectual Merit. Results from the proposed experiments are projected to lead to fundamental discoveries that will have immediate and transformative impacts on our understanding of how and why bacteria interact with, and oxidize, Sb(III). As such, we anticipate this work will make a oundational contribution to our understanding of Sb geomicrobiology. Broader Impacts. Antimony is an EPA priority pollutant, yet we know almost nothing about its behavior in the environment. Presumably, like other metalloids, Sb mobility in the environment is influenced by its redox speciation, yet there is very little information available concerning microbial Sb redox activity that could impact Sb fate and transport. Data generated from the proposed project should find immediate application to ecological studies, improving our understanding of the factors that could influence Sb mobility, and thus enhance our understanding of health risks associated with Sb-contaminated sites. The proposed research will also impact upon human resource development by providing geobiology cross-training opportunities for a postdoctoral-level scientist and undergraduate research interns in a tight collaborative effort of two laboratories located at Montana State University and Cal-Poly, San Luis Obispo. Underrepresented groups will be the focus of undergraduate student recruitment. Finally, data generated from the proposed research will be integrated at participant-appropriate levels into the undergraduate and graduate courses taught by the PIs, as well as the PI?s education-outreach activities that involve grade school, high school, and scientific-lay adult audiences.