Amino acid prototrophy as a positive selective marker in Francisella

描述（由申请人提供）：本质上，土拉菌弗朗西斯菌引起人畜共患疾病土拉菌病。土拉菌因其高度传染性和通过雾化传播的潜在便利性而被列为a类精选病原体。世界范围内的恐怖活动导致人们越来越担心土拉菌可能被用作生物恐怖主义武器；作为回应，美国国家过敏和传染病研究所（National Institute of Allergy and Infectious Diseases）扩大了其生物防御研究项目，努力开发新的土拉菌疗法和疫苗。拟议研究的长期目标是为兔热病疫苗和疗法的开发确定新的靶点。然而，目前的遗传研究是有限的，因为缺乏可选择的遗传标记用于毒性土拉菌菌株。在本研究中，我们将开发基于氨基酸原生营养的非抗生素标记，作为土拉菌的阳性选择遗传标记。我们将使用遗传方法来鉴定补充土拉菌菌株Schu S4的异源基因，以便在缺乏必需氨基酸的特定培养基上生长。所得到的基因将用于产生几种新的土拉菌穿梭载体。拟议工作的结果将提供急需的遗传工具，这些工具将促进药物发现、疫苗开发、细胞生物学实验和毒力土拉菌菌株的微生物学实验。

DESCRIPTION (provided by applicant): In nature, Francisella tularensis causes the zoonotic disease tularemia. F. tularensis is a category A select agent because of its highly infectious nature and the potential ease of its dissemination by aerosolization. Worldwide terrorist activities have resulted in growing concerns about the potential use of F. tularensis as a bioterrorist weapon; in response, the National Institute of Allergy and Infectious Diseases has expanded its biodefense research program in an effort to develop new F. tularensis therapeutics and vaccines. The long term goal of the proposed research is to identify novel targets for the development of tularemia vaccines and therapeutics. However, current genetic research is limited due to a shortage of selectable genetic markers for use in virulent F. tularensis strains. In this proposal we will develop non-antibiotic markers based on amino acid prototrophy for use as positive selective genetic markers in F. tularensis. We will use a genetic approach to identify heterologous genes that complement F. tularensis strain Schu S4 for growth on defined media lacking essential amino acids. The resulting genes will be used to generate several new F. tularensis shuttle vectors. The results of the proposed work will provide much needed genetic tools which will facilitate drug discovery, vaccine development, cell biology experiments, and microbiological experiments with virulent F. tularensis strains.