Non-Antibiotic Selectable Markers for Bacillus Anthracis

描述(申请人提供)：炭疽芽孢杆菌产生高致命性疾病。不幸的是，抗生素治疗选择仍然很少，治疗必须持续较长时间。这些观察结果突显了保持对当前抗生素疗法的敏感性的重要性。然而，能够对炭疽杆菌进行基因操作以科学研究其毒力特性的技术，依赖于引入抗生素耐药性。这是以抗生素耐药性标记的形式存在的，该标记允许选择所需的重组事件和保持质粒。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。这项提议的目标有两个：开发用于炭疽芽孢杆菌的非抗生素选择性标记系统，以及使用这些标记开发遗传系统，从而极大地提高对这种生物的遗传操作的效率。尽管这些工具是为我们正在进行的炭疽病发病机制研究而开发的，但它们将广泛适用于其他一系列细菌病原体。提出了三个部分的一个具体目标。首先，我们将定义对细菌素、重金属、化疗药物和除草剂产生抗药性的标记在炭疽杆菌中作为可选择标记的能力。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。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，因为当引入到染色体中时，它们能够选择罕见的重组事件，在这些事件中，标记和相连的基因已经被删除。由于炭疽杆菌和大多数革兰氏阳性菌尚不存在反选择标记，我们建议开发两个创新的反选择标记系统。这些新的标记将极大地加速发现，因为它们将为炭疽杆菌的基因操作带来新的效率。第三，我们将利用新鉴定的非抗生素可选标记创建一系列载体，作为炭疽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：这项提案的目标是开发新的工具来分析炭疽芽孢杆菌的基因功能，炭疽杆菌是炭疽病的原因。新方法将为将抗生素耐药性引入病原体提供另一种选择，此前这类研究需要这种方法。这些新工具将对研究人员和公众更安全，加速发现，并在广泛的感染性生物调查中得到广泛使用。

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.