Alternatives to antibiotic resistance selective markers for Francisella

描述（由申请人提供）： 拟议的研究旨在开发基于野生型弗朗西斯菌菌株所表现出的“天然”氨基酸营养缺陷型和糖敏感性的选择标记。我们发现了一个这样的F。novicida的组氨酸营养缺陷型，如果提供在弗朗西斯菌启动子下表达的hisD基因的产物，则组氨酸可以满足该缺陷型。因此，用组氨酸醇代替组氨酸在最小上生长提供了对hisD+标记的选择。我们将研究是否有可能找到一种类似的条件，在这种条件下催化甲硫氨酸生物合成最后一步的功能同样可以用于选择。此外，我们将检查野生型菌株的几种糖敏感性中的任何一种，例如对木糖的敏感性，是否可以开发用于选择。我们的总体计划是筛选敏感性并在F中开发标记物。novicida作为高毒力F.土拉热菌株该项目的最后一步将是使用一个或多个标记来产生克隆质粒和转座子，这些质粒和转座子在多个弗朗西斯菌属物种中发挥作用。(Some包括在最后一步中的实验涉及选择代理菌株;所有这些实验将作为通过WWAMI卓越研究中心独立支持的项目的一部分进行。

DESCRIPTION (provided by applicant): The proposed research seeks to develop selectable markers based on "natural" amino acid auxotrophies and sugar sensitivities exhibited by wild-type Francisella strains. We have identified one such F. novicida auxotrophy, for histidine, which can be satisfied by histidinol if the product of the hisD gene is supplied expressed under a Francisella promoter. Thus, growth on minimal with histidinol in place of histidine provides a selection for the hisD+ marker. We will examine whether it is possible to find an analogous condition in which functions catalyzing the last step of methionine biosynthesis can likewise be utilized for selection. In addition, we will examine whether any of several sugar sensitivities of the wild-type strain such as for xylose can be developed for selection. Our overall plan is to screen for the sensitivities and develop the markers in F. novicida as a surrogate for the highly virulent F. tularensis strain. The final step of the project will be to use one or more of the markers to generate cloning plasmids and transposons which function in multiple Francisella species. (Some of the experiments included in the final step involve select agent strains; all such experiments will be carried out as part of a project supported independently through the WWAMI Research Centers of Excellence.)