Non-Antibiotic Selectable Markers for Bacillus Anthracis

描述（由申请人提供）：炭疽芽孢杆菌产生高致死率疾病。不幸的是，抗生素治疗选择仍然很少，治疗必须持续很长一段时间。这些观察结果强调了保持对当前抗生素治疗的敏感性的重要性。然而，能够对炭疽芽孢杆菌进行遗传操作以对其毒力特性进行科学研究的技术依赖于抗生素耐药性的引入。这是以抗生素耐药性标记的形式，允许选择所需的重组事件和维持质粒。因此，我们目前的科学探究方法可能会限制治疗选择，危及研究人员，甚至为恐怖分子提供现成的耐药病原体。本提案的目标有两个方面：开发用于炭疽芽孢杆菌的非抗生素选择性标记系统，以及开发使用这些标记的遗传系统，这将大大提高该生物的遗传操作效率。虽然这些工具是为我们正在进行的炭疽发病机制的研究而开发的，但它们将广泛适用于广泛的其他细菌病原体。提出了一个具体的目标，分为三个部分。首先，我们将定义对细菌素、重金属、化疗药物和除草剂具有抗性的标记物在炭疽芽孢杆菌中作为选择性标记物的能力。分析将包括研究标记物的活性和稳定性，以及评估潜在的不良特性，如诱导对抗生素的交叉抗性和对毒性的影响。其次，我们将开发新的反选择标记，作为删除基因的有力工具，因为当引入染色体时，它们可以选择罕见的重组事件，其中标记和链接的基因已被删除。由于目前尚不存在针对炭疽芽胞杆菌和大多数其他革兰氏阳性菌的反选择标记，我们建议开发两种创新的反选择标记系统。这些新的标记将极大地加速发现，因为它们将为炭疽芽孢杆菌的遗传操作带来新的效率。第三，我们将利用新发现的非抗生素选择性标记创建一系列载体，为炭疽芽孢杆菌和其他病原体的细菌遗传研究提供新的工具。

DESCRIPTION (provided by applicant): Bacillus anthracis produces highly lethal disease. Unfortunately, antibiotic treatment options remain few, and therapy must be continued for extended periods. These observations highlight the importance of preserving susceptibility to current antibiotic therapies. However, the very technologies that enable genetic manipulation of B. anthracis for scientific study of its virulence properties rely on introduction of antibiotic resistance. This is in the form of antibiotic resistance markers that permit selection of desired recombination events and maintenance of plasmids. Therefore, our current methods of scientific inquiry might limit therapeutic options, endanger researchers, and even provide ready made resistant pathogens for terrorists. The goals of this proposal are two fold: to develop non-antibiotic based selectable marker systems for use in Bacillus anthracis and to develop genetic systems using these markers that will dramatically increase the efficiency of genetic manipulation of this organism. Although being developed for our ongoing investigation of anthrax pathogenesis, these tools will be broadly applicable to a wide range of other bacterial pathogens. Proposed is one specific aim in three parts. In the first, we will define the ability of markers conferring resistance to bacteriocins, heavy metals, chemotherapeutics, and herbicides to function as selectable markers in Bacillus anthracis. Analysis will include study of the markers' activity and stability, as well as an assessment of potential undesirable properties such as induction of cross resistance to antibiotics and effects on virulence. In the second, we will develop new counterselectable markers that serve as a potent tool for deleting genes, since when introduced into the chromosome they enable selection of rare recombination events in which the marker and linked genes have been deleted. As counterselectable markers do not yet exist for B. anthracis and most other gram positive organisms, we propose development of two innovative counterselectable marker systems. These new markers will greatly accelerate discovery, as they will bring new efficiencies to genetic manipulation of B. anthracis. In the third, we will use newly characterized non-antibiotic selectable markers to create a series of vectors that will serve as new tools for bacterial genetic investigation of Bacillus anthracis and other pathogens. PUBLIC HEALTH RELEVANCE: The goals of this proposal are to develop new tools for analyzing gene function in Bacillus anthracis, the cause of anthrax. The new methods will provide an alternative to the introduction of antibiotic resistance into pathogens, previously needed for this type of research. These new tools will be safer for researchers and the public, accelerate discovery, and find broad use in investigation of a wide range of infectious organisms.