MOLECULAR MECHANISM OF BACTERIAL ARSENICAL TRANSPORT

细菌砷转运的分子机制

• 批准号: 2023371
• 负责人: LOUIS S TISA
• 金额: $ 10.03万
• 依托单位: UNIVERSITY OF NEW HAMPSHIRE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2023371
• 关键词: Staphylococcus aureus; arsenic; bacterial proteins; chimeric proteins; drug resistance; ion transport; membrane proteins; point mutation; protein purification; protein structure; site directed mutagenesis

DESCRIPTION: Bacterial resistance to arsenicals occurs by an extrusion and is encoded by the inducible arsenical resistance operon which is carried on conjugative R-factors or plasmids. The arsenical resistance system is a good model system for understanding antibiotic resistance including multidrug resistance. The overall goal of this investigation is the elucidation of the molecular mechanism of arsenical transport. This proposal will focus on a structural and functional characterization of the arsB gene product. The ArsB protein is an integral membrane protein predicted to be an anion channel. A topological model for the ArsB protein is an integral membrane protein predicted to be an anion channel. A topological model for the ArsB protein has been predicted from the nucleotide sequence. This model will be tested by a molecular genetic approach using lacZ and phoA gene fusions. In parallel, with the above study, a biochemical approach will also be used for the identification and purification of the ArsB protein. A fusion protein will be constructed between the arsB gene product and an N-terminal tag of six histidine residues. The resultant His-tagged ArsB protein will be solubilized and purified by affinity chromatography. Alternatively, the ArsB protein will be fused with the biotinylated domain of oxaloacetate decarboxylase from Klebsiella pneumoniae and purified by affinity chromatography. The resultant purified protein will be physiologically and biochemically characterized. Either in parallel with or subsequent to the above studies a collection of arsB point mutants will be generated in vitro by the use of oligonucleotide-directed random mutagenesis. Understanding the molecular mechanisms of anion transport will greatly increase our understanding of antibiotic resistances in human health. In addition to the potential health benefits, this study will make a significant contribution toward the infrastructure of biomedical research and development. Both graduate and undergraduate students will play an active role in this research project. Their research experience and training will help ensure a future generation of biomedical scientists for both the public and private sectors.

描述：细菌对砷的耐药性是通过挤压产生的， 由可诱导的砷抗性操纵子编码， 接合R-因子或质粒。 砷抗性系统是一种 了解抗生素耐药性的良好模型系统，包括 多药耐药 本次调查的总体目标是 阐明砷转运的分子机制。 本提案将侧重于以下方面的结构和功能特征： arsB基因产物。 ArsB蛋白是一种完整的膜蛋白 被预测为阴离子通道。 ArsB蛋白的拓扑模型 是一个完整的膜蛋白预测是一个阴离子通道。 一 ArsB蛋白的拓扑模型已经从 核苷酸序列。 这个模型将通过一个分子遗传学 方法使用lacZ和phoA基因融合。 与此同时，上述 研究中，生物化学方法也将用于识别和 纯化ArsB蛋白。 将构建融合蛋白 arsB基因产物和六个组氨酸的N末端标签之间 残基 所得His标记的ArsB蛋白将被溶解， 通过亲和层析纯化。 或者，ArsB蛋白将 与草酰乙酸脱羧酶的生物素化结构域融合， 肺炎克雷伯氏菌，并通过亲和层析纯化。 的 所得到的纯化蛋白质将在生理学和生物化学上 表征了 在上述研究的同时或之后， arsB点突变体的集合将通过使用 抗草甘膦定向随机诱变。 了解阴离子转运的分子机制将大大有助于 增加我们对人类健康中抗生素耐药性的了解。 在 除了潜在的健康益处外，这项研究还将使 对生物医学研究基础设施的重大贡献 发展先行者的要求 研究生和本科生都将扮演一个 在这个研究项目中发挥积极作用。 他们的研究经验和 培训将有助于确保下一代生物医学科学家， 公共和私营部门。