Non-Antibiotic Selectable Markers for Bacillus Anthracis

描述（由申请方提供）：炭疽杆菌产生高致死性疾病。不幸的是，抗生素治疗的选择仍然很少，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敏感性的重要性。然而，使B基因操作成为可能的技术。炭疽菌的毒力特性的科学研究依赖于抗生素耐药性的引入。这是以抗生素抗性标记的形式，其允许选择所需的重组事件和维持质粒。因此，我们目前的科学研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。该提议的目标有两个方面：开发用于炭疽芽孢杆菌的基于非抗生素的选择性标记系统，以及开发使用这些标记的遗传系统，其将显著提高该生物体的遗传操作的效率。尽管这些工具是为我们正在进行的炭疽病发病机制的研究而开发的，但它们将广泛适用于各种其他细菌病原体。提出了三个部分的具体目标。首先，我们将定义赋予细菌素、重金属、化疗药物和除草剂抗性的标记在炭疽芽孢杆菌中作为选择标记的能力。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耐药性和对毒力的影响。在第二，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，它们能够选择罕见的重组事件，其中标记和连锁基因已被删除。因为B的反选择标记尚不存在。炭疽和大多数其它革兰氏阳性生物，我们提出了两个创新的反选择标记系统的发展。这些新的标记将大大加速发现，因为它们将为B的遗传操作带来新的效率。炭疽病在第三部分中，我们将使用新表征的非抗生素选择标记来创建一系列载体，这些载体将作为炭疽芽孢杆菌和其他病原体的细菌遗传学研究的新工具。 公共卫生相关性：该提案的目标是开发新的工具，用于分析炭疽杆菌（炭疽病的病因）的基因功能。这些新方法将为将抗生素耐药性引入病原体提供一种替代方法，而这种方法以前需要进行此类研究。这些新工具将对研究人员和公众更安全，加速发现，并在广泛的传染性生物体调查中得到广泛应用。

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.