Alternative Genetic Selection in Bacillus anthracis and Francisella tularensis

描述（由申请方提供）：许多A类选择性病原菌天然对抗生素敏感，因此应避免引入抗生素耐药性选择性标记用于遗传研究。非抗生素选择性标记物的可用性有限，尚未评估其在这些细菌中的使用。待开发的标记应具有当基因以单拷贝存在于生物体中时具有表达的选择性表型、当在多拷贝质粒上表达时稳定、以及当插入多基因操纵子中时不对下游基因施加极性效应的特性。理想的性质还包括当敏感生物体在选择性条件下铺板时，所用的选择剂应提供低背景，并且敏感生物体与抗性生物体的MIC浓度之间的差异应较大。此外，能够容易且精确地切除选择性标记以产生缺乏标记盒的突变菌株将是有利的，使得抗微生物标记可以迭代地用于产生多重突变菌株。在这个项目中，我们将评估重金属抗性标记，包括对砷，镉，铬酸盐和汞的抗性以及对除草剂双丙氨膦的抗性，作为炭疽芽孢杆菌和土拉弗朗西斯菌遗传分析的潜在选择标记。将确定在两种宿主中选择所需的最小序列，并将进行突变研究以优化表达。将在两种宿主中验证这些标记物在多拷贝质粒和单拷贝插入上的使用。将创建并评估携带这些选择剂的转座子的构建。将完成侧翼为loxP位点的选择性标记盒的工程化，并将评价使用基于Cre重组酶的系统从这些选择剂细菌中的质粒或染色体靶标切除选择盒。

DESCRIPTION (provided by applicant): Many of the category A select agent bacteria are naturally sensitive to antibiotics and thus introduction of antibiotic resistance selective markers for genetic studies should be avoided. Non-antibiotic selective markers are limited in their availability and have not been evaluated for use in these bacteria. Markers to be developed should have the characteristics of having the selective phenotype expressed when the gene is present in single copy in the organism, being stable when expressed on multi-copy plasmids, and not exerting polar effects on downstream genes when inserted into a multi-gene operon. Desirable properties also include that the selective agent employed should give a low background when sensitive organisms are plated under selective conditions, and the difference between the MIC concentration of sensitive versus resistant organisms should be large. Furthermore, it would be advantageous to be able to easily and precisely excise the selective marker to create a mutant strain lacking the marker cassette so that the antimicrobial markers can be used iteratively to create multiply mutated strains. In this project, we will evaluate heavy metal resistance markers, including resistance to arsenite, cadmium, chromate, and mercury as well as resistance to the herbicide bialaphos as potential selective markers for genetic analysis of Bacillus anthracis and Francisella tularensis. The minimal sequence required for selection in the two hosts will be determined and mutational studies will be carried out to optimize expression. The use of these markers on multicopy plasmids and single copy insertions will be validated in the two hosts. Construction of a transposon bearing these selective agents will be created and evaluated. Engineering of the selective marker cassettes flanked by loxP sites will be accomplished and use of a Cre recombinase-based system to excise the selection cassette from plasmid or chromosomal targets in these select agent bacteria will be evaluated.