Conjugal Transfer of Bacteriodes Antibiotic Resistances

描述(申请人提供)：结合转座子(CTN)是整合的自我传递元件。它们转移的第一步是从细菌染色体上切除，形成环状中间体。然后，这种中间体的单链拷贝被转移到受体细胞，在那里它整合到染色体中。事实证明，CTN是抗生素耐药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。这项提案的重点是切除在人类结肠类杆菌中发现的一种广泛分布的CTN，CTnDOT。CTnDOT有一个异常复杂的切除系统，由整合酶(IntDOT)和其他3种对切除必不可少的蛋白质(Orf2c、Orf2d和exc)组成。Orf2c、Orf2d和exc的作用机制尚不清楚。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，以确定它们在切除过程中如何与DNA结合，以及它们如何相互作用以及如何与IntDOT相互作用。将使用定点突变和随机突变来了解哪些氨基酸残基对它们的功能是重要的。Orf2c、Orf2d和exc也参与了转移基因的调控。我们将确定这些蛋白质如何有助于控制转移基因的表达。最后，我们将进一步鉴定三个调节蛋白，RteA，RteB和RTEC，它们是CTnDOT功能的中心调节蛋白，包括切除和转移。特别令人感兴趣的是RteA，即假定的传感器蛋白，正在感知什么的问题。拟议的实验结果将提供关于一类独特的可传递整合元件的功能和调节机制的新信息。未来，有关切除的信息可能会被用来开发化合物，这种化合物可以“治愈”某些类型的细菌的CTN基因，这些基因赋予了抗生素耐药性或编码了毒力因子。此外，关于CTN功能如何受到监管的信息已经开始被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行业监管措施的评估。

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.