Molecular Mechanisms of Bacterial Metal Redox Transformations

说明(申请人提供)：金属污染(即毒性)对人类健康的影响取决于金属的生物可利用性，而生物利用度又取决于金属的种类和形态。可溶金属形态，特别是游离金属离子，通常具有更高的生物可利用性和对细胞的毒性，而不溶金属形态的毒性较小。细菌可能以各种方式与金属相互作用，导致金属的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)的机理，这些过程导致这些金属和其他金属从溶液中沉淀或去除。这项研究的长期目标是，1)识别涉及的基因和蛋白质，并表征这些过程的机制，包括那些因接触有毒金属而启动的过程；2)开发金属生物可利用性的生物标志物；3)操纵这些系统用于金属生物修复应用。分子生物学和生物化学方法将被用来鉴定和表征与锰(II)氧化和铬(VI)还原/铬毒性有关的基因和蛋白质。编码基因 锰(II)氧化蛋白将被克隆、测序并分析关键氨基酸残基。一个生物/蛋白质将被用作大规模原生或异源表达和纯化的模型，并在很大程度上利用质谱仪进行详细的表征(酶动力学、定位、辅因子和糖基化和金属结合部位)。将从生理和分子水平(例如，微阵列或蛋白质谱)来研究锰、铬、铅和/或铜对细胞中锰(II)氧化活性的影响，以开发一个环境控制锰(II)氧化的模型。铬的研究将集中在对铬反应的调控基因以及不同形态铬的吸收和毒性。本研究将构建缺铬突变株MR-1，并对突变株进行鉴定。将测试相对于其他金属(例如，铅、砷、硒)的铬(VI)基因表达的特异性。实时逆转录聚合酶链式反应将被用来评估铬(VI)特异性基因在实验室中(或现场)的表达，并将表达模式与铬浓度和形态相关联。缺乏硫酸盐吸收的突变体将被测试它们在生理上耐受铬(VI)暴露的能力和使用透射电子显微镜。还将研究铬(III)对基因表达的影响以及细菌铁载体对铬(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.