Conjugal Transfer of Bacteriodes Antibiotic Resistances

拟杆菌抗生素耐药性的夫妻传播

• 批准号: 7188639
• 负责人: Abigail A Salyers
• 金额: $ 32.65万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-30 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7188639
• 关键词: Amino Acid Sequence; Amino Acids; Antibiotic Resistance; Bacteria; Bacterial Chromosomes; Bacteroides; Binding; Binding Sites; Biochemical; Cells; Chromosomes; Class; Complex; DNA; DNA Binding; DNA Sequence; DNA-Binding Proteins; Development; Drug Industry; Elements; Ensure; Evaluation; Excision; Exposure to; Family; Food; Funding; Future; Gelshift Analysis; Gene Expression; Gene Family; Gene Transfer; Genes; Genetic Recombination; Genetic Transcription; Goals; Growth; Helix-Turn-Helix Motifs; Homologous Gene; Human; In Vitro; Integrase; Learning; Link; Measures; Mediating; Mutagenesis; Mutate; Mutation; Nature; Open Reading Frames; Operon; Phase; Phosphorylation; Process; Promoter Regions; Protein Databases; Proteins; RNA; RNA polymerase sigma 54; Range; Reaction; Regulation; Research; Research Personnel; Signal Transduction; Site; Structural Protein; System; Techniques; Testing; Tetracycline; Tetracycline Control; Tetracyclines; Topoisomerase; Virulence Factors; Work; base; fitness; genetic analysis; genetic regulatory protein; in vivo; interest; prevent; programs; protein function; research study; sensor

DESCRIPTION (provided by applicant): Conjugative transposons (CTns) are integrated self-transmissible elements. The first step in their transfer is excision from the bacterial chromosome to form a circular intermediate. A single-stranded copy of this intermediate is then transferred to a recipient cell where it integrates into the chromosome. CTns are proving to be major contributors to antibiotic resistance gene transfer. Yet little is know about their activities, particularly the process by which they excise from the chromosome. The focus of this proposal is on excision of a widely distributed CTn found in human colonic Bacteroides species, CTnDOT. CTnDOT has an unusually complex excision system that consists of an integrase (IntDOT).and 3 other proteins (Orf2c, Orf2d and Exc) that are essential for excision. Nothing is known about the mechanisms of Orf2c, Orf2d and Exc. We will purify these proteins and use a variety of biochemical techniques, ranging from gel shift analysis to footprinting, to determine how they bind DNA during the excision process and how they interact with each other and with IntDOT. Site-directed and random mutagenesis will be used to learn what amino acid residues are important for their functions. Orf2c, Orf2d and Exc also participate in the regulation of transfer genes. We will determine how these proteins contribute to control of transfer gene expression. Finally, we will characterize further three regulatory proteins, RteA, RteB and RteC, which are central regulators of CTnDOT functions including excision and transfer. Of special interest is the question of what RteA, the putative sensor protein, is sensing. The results of the proposed experiments will provide new information about the functions and regulatory machinery of a unique family of transmissible integrated elements. Information about excision might be used in the future to develop compounds that "cure" certain types of bacteria of genes on CTns that confer resistance to antibiotics or encode virulence factors. Also, information about how CTn functions are regulated is already beginning to be applied to the development of strategies that prevent their spread in nature and to guide evaluation of regulatory measures in the food and pharmaceutical industry.

描述（由申请人提供）：接合转座子（CTns）是整合的自传播元件。它们转移的第一步是从细菌染色体中切除，形成环状中间体。然后，该中间体的单链副本被转移到受体细胞，并在其中整合到染色体中。 CTns 被证明是抗生素抗性基因转移的主要贡献者。然而，人们对它们的活动知之甚少，特别是它们从染色体上切除的过程。该提案的重点是切除人类结肠拟杆菌属中广泛分布的 CTn，即 CTnDOT。 CTnDOT 具有异常复杂的切除系统，由整合酶 (IntDOT) 和切除所必需的其他 3 种蛋白质（Orf2c、Orf2d 和 Exc）组成。关于 Orf2c、Orf2d 和 Exc 的机制尚不清楚。我们将纯化这些蛋白质并使用各种生化技术，从凝胶位移分析到足迹分析，以确定它们在切除过程中如何结合 DNA 以及它们如何彼此相互作用以及与 IntDOT 相互作用。将使用定点和随机诱变来了解哪些氨基酸残基对其功能很重要。 Orf2c、Orf2d和Exc也参与转移基因的调控。我们将确定这些蛋白质如何有助于控制转移基因表达。最后，我们将进一步描述三种调节蛋白 RteA、RteB 和 RteC，它们是 CTnDOT 功能（包括切除和转移）的中央调节因子。特别令人感兴趣的是 RteA（假定的传感器蛋白）正在感知什么的问题。拟议实验的结果将提供有关独特的可传播整合元件家族的功能和调节机制的新信息。有关切除的信息将来可能会被用来开发化合物，“治愈”CTns上某些类型细菌的基因，这些细菌赋予抗生素抗性或编码毒力因子。此外，有关 CTn 功能如何监管的信息已开始应用于制定防止其在自然界传播的策略，并指导食品和制药行业监管措施的评估。