Molecular Mechanisms of Bacterial Metal Redox Transformations

细菌金属氧化还原转化的分子机制

• 批准号: 7598929
• 负责人: Bradley M. Tebo
• 金额: $ 24.84万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7598929
• 关键词: Adverse effects; Amino Acids; Animal Model; Arsenic; Bacillus (bacterium); Bacteria; Binding; Bioavailable; Biochemical; Biological; Biological Availability; Biological Markers; Bioremediations; Biosensor; Cadmium; Cells; Chemicals; Chromates; Chromium; Complex; Condition; Copper; DNA; Drug Metabolic Detoxication; Electrons; Elements; Engineering; Environment; Environmental Risk Factor; Enzyme Kinetics; Excision; Exposure to; Gene Expression; Gene Proteins; Genes; Genetic; Goals; Health; Heavy Metals; Homo; Homologous Protein; Human; Inorganic Sulfates; Ions; Iron; Laboratories; Lead; Link; Manganese; Marines; Mass Spectrum Analysis; Measures; Mediating; Mercury; Metabolic; Metabolic Pathway; Metal exposure; Metals; Modeling; Molecular; Mutagenesis; Nickel; Numbers; Organism; Oxidases; Oxidation-Reduction; Oxides; Pattern; Phase; Physiological; Plants; Pollution; Polymerase Chain Reaction; Precipitation; Process; Property; Protein Microchips; Proteins; Proteobacteria; Protocols documentation; Pseudomonas putida; Publishing; Range; Reaction; Regulation; Relative (related person); Research; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Running; Sampling; Sequence Analysis; Shewanella; Siderophores; Site; Soil; Solutions; Specificity; Staining method; Stains; Structure-Activity Relationship; Substrate Interaction; System; Testing; Time; Toxic effect; Unspecified or Sulfate Ion Sulfates; Variant; Zinc; base; catalyst; chromium hexavalent ion; cofactor; environmental agent; enzyme substrate; genome sequencing; glycosylation; in vivo; microbial; mutant; novel; oxidation; protein expression; protein purification; remediation; response; terminally differentiated effector memory (TEM) T cells; toxic metal; uptake

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.

描述（由申请人提供）：金属污染的人类健康的后果（即毒性）取决于金属生物利用度，这又取决于金属的物种和形式。 可溶性金属物种，尤其是游离金属离子，通常对细胞更有生物利用和毒性，而不溶性金属形式的毒性较小。 细菌可能以多种方式与金属相互作用，从而导致金属生物利用度和毒性降低。 该项目将检查细菌的氧化机制（II）（II）和铬（VI）的还原。这项研究的远距离目标是：1）确定所涉及的基因和蛋白质并表征这些过程的机制，包括通过暴露于有毒金属来打开的基因和蛋白质，2）开发金属生物利用度和金属生物利用度的生物标志物， 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化方法来识别和表征与Mn（II）氧化和CR（VI）还原/CR毒性相关的基因和蛋白质。 编码基因 Mn（II）氧化蛋白将被克隆，测序并分析关键氨基酸残基。 一种生物/蛋白质将用作大规模天然或异源表达和纯化和详细表征（酶动力学，定位，辅助因子和糖基化和金属结合位点）的模型。 Mn，Cr，Pb和/或Cu对细胞的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分析）水平上进行检查，以开发环境控制模型在Mn（II）氧化上。 铬研究将集中于对CR的响应以及不同形式的CR的摄取和毒性的基因。 Shewanella Oneidensis MR-1突变体在响应Cr（VI）上上调的基因缺乏的基因（VI）将被构建和表征。 基因表达对Cr（VI）的特异性相对于其他金属（例如Pb，AS，SE）的特异性将进行测试。 实时RT-PCR将用于评估实验室中孔（或现场位点）中CR（VI）特定基因的表达，并将表达模式与CR浓度和物种相关。 缺乏硫酸盐摄取的突变体将测试其在生理和使用TEM上承受CR（VI）的能力。 还将研究CR（III）对细菌铁载体基因表达和Cr（III）的络合的影响。