Molecular Mechanisms of Bacterial Metal Redox Transformations

描述（由申请人提供）：金属污染对人类健康的后果（即，毒性）取决于金属的生物利用度，而生物利用度又取决于金属的种类和形式。 可溶性金属物质，特别是游离金属离子，通常更具有生物利用度并且对细胞具有毒性，而不溶性金属形式的毒性较小。 细菌可能以各种方式与金属相互作用，导致金属生物利用度和毒性降低。 本项目将研究细菌氧化锰（II）和还原铬（VI）的机制。导致这些和其他金属从溶液中沉淀或去除的过程。这项研究的长期目标是，1）确定相关的基因和蛋白质，并表征这些过程的机制，包括那些通过暴露于有毒金属而开启的机制，2）开发金属生物利用度的生物标志物，以及3）操纵这些系统用于金属生物修复应用。 将采用分子生物学和生物化学方法来鉴定和表征参与Mn（II）氧化和Cr（VI）还原/Cr毒性的基因和蛋白质。 编码基因 Mn（II）氧化蛋白将被克隆，测序和分析关键氨基酸残基。 一种生物体/蛋白质将用作大规模天然或异源表达和纯化的模型，并主要使用质谱法进行详细表征（酶动力学、定位、辅因子和糖基化和金属结合位点）。 Mn、Cr、Pb和/或Cu对细胞Mn（II）氧化活性的影响将在生理和分子（例如，微阵列或蛋白质谱）水平，以开发Mn（II）氧化的环境控制模型。 铬的研究将集中在对铬的反应和不同形式的铬的吸收和毒性的基因调控。 将构建和表征在响应于Cr（VI）的上调的基因中缺陷的希瓦氏菌oneidensis MR-1突变体。 基因表达对Cr（VI）相对于其他金属（例如，Pb、As、Se）进行检测。 实时RT-PCR将被用来评估在实验室围隔（或现场）的铬（VI）的特定基因的表达和相关的表达模式，铬浓度和形态。 将使用TEM测试硫酸盐摄取缺陷的突变体在生理上耐受暴露于Cr（VI）的能力。 还将研究Cr（III）对基因表达的影响以及细菌铁载体与Cr（III）的络合作用。

DESCRIPTION (provided by applicant): The consequences to human health of metal pollution (i.e., toxicity) is dependent on metal bioavailability, which in turn is dependent on the species and form of the metal. Soluble metal species, particularly free metal ions, are generally more bioavailable and toxic to cells whereas the insoluble metal forms are less toxic. Bacteria may interact with metals in a variety of ways that lead to reduced metal bioavailability and toxicity. This project will examine the mechanisms of oxidation of manganese(II) and reduction of chromium(VI) by bacteria.processes that lead to the precipitation or removal of these and other metals from solution. The long-range goals of this research are, 1) to identify the genes and proteins involved and to characterize the mechanisms of these processes, including those that are turned on by exposure to toxic metals, 2) to develop biomarkers of metal bioavailability, and 3) manipulate these systems for metal bioremediation applications. Molecular biological and biochemical approaches will be employed to identify and characterize the genes and proteins involved in Mn(II) oxidation and Cr(VI) reduction/Cr toxicity. The genes encoding Mn(II)-oxidizing proteins will be cloned, sequenced and analyzed for key amino acid residues. One organism/protein will be used as a model for large-scale native or heterologous expression and purification and detailed characterization (enzyme kinetics, localization, cofactors and sites of glycosylation and metal-binding) largely using mass spectrometry. The effect of Mn, Cr, Pb, and/or Cu on the Mn(II)-oxidizing activity of cells will be examined at the physiological and molecular (e.g., microarrays or protein profiling) level in order to develop a model of environmental controls on Mn(II) oxidation. Chromium research will focus on genes regulated in response to Cr and the uptake and toxicity of different forms of Cr. Shewanella oneidensis MR-1 mutants deficient in genes up-regulated in response to Cr(VI) will be constructed and characterized. The specificity of gene expression to Cr(VI) relative to other metals (e.g., Pb, As, Se) will be tested. Real-time RT-PCR will be used to assess the expression of the Cr(VI) specific genes in laboratory mesocosms (or a field site) and correlate the expression patterns to Cr concentration and speciation. Mutants deficient in sulfate uptake will be tested for their ability to withstand exposure to Cr(VI) physiologically and using TEM. The effect of Cr(III) on gene expression and the complexation of Cr(III) by bacterial siderophores will also be investigated.