Summit Therapeutics plc, The Merrifield Centre, 12 Rosemary Lane, Cambridge, CB1 3LQ, UK.
Quadram Institute, Rosalind Franklin Road, Norwich Research Park, Norwich, NR4 7UQ, UK.
FEMS Microbiol Lett. 2020 Dec 14;367(22). doi: 10.1093/femsle/fnaa185.
The use of bacterial transposon mutant libraries in phenotypic screens is a well-established technique for determining which genes are essential or advantageous for growth in conditions of interest. Standard, inactivating, transposon libraries cannot give direct information about genes whose over-expression gives a selective advantage. We report the development of a system wherein outward-oriented promoters are included in mini-transposons, generation of transposon mutant libraries in Escherichia coli and Pseudomonas aeruginosa and their use to probe genes important for growth under selection with the antimicrobial fosfomycin, and a recently-developed leucyl-tRNA synthase inhibitor. In addition to the identification of known mechanisms of action and resistance, we identify the carbon-phosphorous lyase complex as a potential resistance liability for fosfomycin in E. coli and P. aeruginosa. The use of this technology can facilitate the development of novel mechanism-of-action antimicrobials that are urgently required to combat the increasing threat worldwide from antimicrobial-resistant pathogenic bacteria.
在表型筛选中使用细菌转座子突变文库是一种成熟的技术，可用于确定哪些基因对于在感兴趣的条件下生长是必需的或有利的。标准的失活转座子文库无法提供关于那些过表达赋予选择性优势的基因的直接信息。我们报告了一种系统的开发，其中在外向启动子被包含在微型转座子中，在大肠杆菌和铜绿假单胞菌中生成转座子突变文库，并将其用于探测在选择压力下生长的重要基因，包括抗菌药物磷霉素和最近开发的亮氨酰-tRNA 合成酶抑制剂。除了鉴定已知的作用机制和耐药性外，我们还确定碳-磷裂解酶复合物是大肠杆菌和铜绿假单胞菌中磷霉素耐药的潜在风险因素。该技术的应用可以促进新型作用机制抗菌药物的开发，这对于应对全球范围内日益严重的抗药性致病菌威胁是非常迫切的需求。
