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）在自然水体、土壤和沉积物中存在的报道，但对锑的生物地球化学循环知之甚少。特别是，与环境微生物-Sb相互作用相关的信息仅限于记录Sb在藻类中的积累的研究，一些报告记录了藻类培养中Sb物种形成的变化，以及Sb甲基化。原核生物Sb氧化还原转化的唯一报道是在几十年前发表的，但从未重复，也没有进一步的表征。了解原核生物在环境锑氧化还原循环中可能发挥的作用的进展受到缺乏与环境相关的能够氧化或还原锑的纯微生物培养物的限制。我们最近记录了在遗传上易于处理的农杆菌土壤分离物中Sb（III）的氧化，从而为第二代研究奠定了基础，该研究将集中于鉴定对核生物Sb（III）氧化重要功能编码的基因。提出了研究。一个强大的转座子诱变研究提出，其中理论全基因组扫描将进行涉及数千个转偶联筛选Sb耐受性的变化，特别是Sb（III）氧化能力的损失。为准备本提案而进行的初步实验已经确定了几种Tn5-B22转偶联物，它们对Sb（III）的耐受性发生了改变，其中一些在低Sb（III）浓度下表现出急性敏感性，而另一些现在显然能够增强耐受性。此外，在某些突变体中还发现了Sb吸收特性和氧化特性的变化。识别这些突变体相对容易，这表明更彻底的突变筛选将产生许多额外的突变体，然后将根据突变基因和编码功能对其进行总体表征，随后将对突变体的优先子集进行彻底表征，我们将重点关注假定的调节蛋白和尚未确定的假定的Sb（III）氧化酶。其他令人感兴趣的突变可能涉及新的Sb转运蛋白。最后的目标将是使用生物信息学方法在数据库中识别同源物，作为设计PCR引物的基础，用于探测这些基因在已知含有显着升高的Sb智力价值的热环境和采矿环境中的发生和表达。拟议实验的结果预计将导致根本性的发现，这些发现将对我们理解细菌如何以及为什么与Sb（III）相互作用和氧化产生直接和变革性的影响。因此，我们期望这项工作将为我们对Sb地球微生物学的理解做出基础贡献。更广泛的影响。锑是美国环保署优先考虑的污染物，但我们对它在环境中的行为几乎一无所知。据推测，与其他类金属一样，锑在环境中的迁移受到其氧化还原形态的影响，但关于微生物锑氧化还原活性可能影响锑命运和运输的信息很少。拟议项目产生的数据应立即应用于生态学研究，提高我们对可能影响Sb迁移的因素的理解，从而提高我们对与Sb污染场地相关的健康风险的理解。拟议的研究还将通过在位于蒙大拿州立大学和加州理工学院圣路易斯奥比斯波分校的两个实验室的紧密合作下，为博士后水平的科学家和本科生研究实习生提供地理生物学交叉培训机会，从而影响人力资源的发展。代表性不足的群体将成为本科招生的重点。最后，从拟议的研究中产生的数据将在参与者适当的水平上整合到PI教授的本科和研究生课程中，以及PI？包括小学、高中和科学专业的成人观众的教育推广活动。

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.