Alternative Genetic Selection in Bacillus anthracis and Francisella tularensis

描述(由申请人提供)：许多A类选择试剂细菌对抗生素自然敏感，因此应避免引入抗生素耐药性选择性标记进行遗传学研究。非抗生素选择性标记的可用性有限，尚未对其在这些细菌中的使用进行评估。待开发的标记应具有这样的特点：当基因在生物体中以单拷贝形式存在时，具有选择性的表型表达；当在多拷贝质粒上表达时，具有稳定性；当插入多基因操纵子时，不对下游基因产生极性效应。理想的特性还包括，当敏感生物在选择性条件下被电镀时，所使用的选择性试剂应该具有较低的背景，并且敏感生物和耐药生物的MIC浓度之间的差异应该很大。此外，能够容易和准确地切除选择标记以产生没有标记盒的突变菌株将是有利的，从而可以反复使用抗菌标记来产生多个突变菌株。在本项目中，我们将评估重金属抗性标记，包括对亚砷酸盐、镉、铬酸盐和汞的抗性，以及对除草剂Bialaphos的抗性，作为炭疽芽孢杆菌和土拉方杆菌遗传分析的潜在选择标记。将确定在两个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。这些标记在多拷贝质粒和单拷贝插入上的使用将在两个宿主中得到验证。含有这些选择性试剂的转座子的构建将被创建和评估。将完成以loxP位点为侧翼的选择标记盒的工程，并将评估使用基于Cre重组酶的系统从这些选择菌质粒或染色体靶标中切除选择盒。

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.