Conjugal Transfer of Bacteriodes Antibiotic Resistances

描述（由申请人提供）：共轭转座子（CTns）是集成的自传输元件。它们转移的第一步是从细菌染色体上切除，形成一个圆形的中间体。然后，这种中间产物的单链拷贝被转移到一个受体细胞，在那里它整合到染色体中。ctn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，尤其是它们从染色体上切除的过程。本提案的重点是切除广泛分布于人类结肠拟杆菌物种的CTn， CTnDOT。CTnDOT具有异常复杂的切除系统，由整合酶（IntDOT）组成。以及其他3种蛋白（Orf2c、Orf2d和Exc），它们是切除所必需的。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，从凝胶转移分析到足迹分析，以确定它们在切除过程中如何结合DNA，以及它们如何相互作用以及如何与IntDOT相互作用。位点定向和随机诱变将用于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如何参与控制转移基因的表达。最后，我们将进一步表征三种调节蛋白，RteA， RteB和RteC，它们是CTnDOT功能（包括切除和转移）的中心调节蛋白。特别令人感兴趣的问题是RteA，即假定的传感蛋白，正在感应什么。所提出的实验结果将提供关于一个独特的可传递整合元件家族的功能和调控机制的新信息。有关切除的信息可能在未来用于开发化合物，以“治愈”ctn上某些类型的细菌或基因，这些细菌或基因赋予抗生素抗性或编码毒力因子。此外，关于如何调节CTn功能的信息已经开始应用于制定防止其在自然界传播的战略，并指导对食品和制药工业中管制措施的评估。

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.