A Novel High-Throughput Exploration of DNA Supercoiling toward the Therapeutic Manipulation of Bacteria

项目摘要 细菌的生存需要一个几乎恒定和特定的全球DNA超螺旋状态。 这是通过DNA松弛拓扑异构酶I的相反功能实现的， DNA压缩DNA促旋酶。我们提出了第一个无偏的高通量 探索控制这些酶的丰度和活性的基因， 在治疗上有着巨大的潜力。病原体如何变化 感染过程中的DNA超螺旋隐藏了克服感染的关键弱点。 目前DNA促旋酶抑制剂的毒性作用和耐药细菌的出现 这些代理人。我们将检查病原体肠道沙门氏菌血清型的突变体 鼠伤寒沙门氏菌在感染相关条件下通过部署改变DNA超螺旋 DNA超螺旋的荧光评价-或FEDS -一种新的高通量 在我们的实验室开发的方法，报告了全球DNA超螺旋状态， 活细菌。为了揭示改变DNA超螺旋的遗传基础，我们将鉴定 突变基因，并探索突变之间的遗传相互作用，以揭示 新的合成致命性的例子，从而揭示了新的遗传途径， 以及操纵DNA超螺旋的新方法。我们会找出DNA的碱基 通过确定DNA的量来确定所鉴定的突变体的超螺旋行为 促旋酶和拓扑异构酶I以及三磷酸腺苷的丰度， 腺苷二磷酸和特定的多胺-所有DNA促旋酶的关键调节剂 活动拟议中的研究计划开创了对一种疾病的生理学理解， 在广泛的细菌物种中可利用的基本细胞特性， 抗菌剂的新靶点。

PROJECT SUMMARY Bacterial survival requires a nearly constant and specific global DNA supercoiling status. This is achieved by the opposing functions of the DNA-relaxing topoisomerase I and DNA-compacting DNA gyrase. We propose the first unbiased high-throughput exploration of genes governing the abundance and activity of these enzymes, which hold great untapped promise for therapeutic manipulation. How pathogens change DNA supercoiling during infection conceals vulnerabilities critical to overcoming the toxic effects of current DNA gyrase inhibitors and emergence of bacteria resistant to these agents. We will examine mutants of the pathogen Salmonella enterica serovar Typhimurium for altered DNA supercoiling in infection-relevant conditions by deploying Fluorescence Evaluation of DNA Supercoiling – or FEDS – a new high-throughput method developed in our laboratory that reports the global DNA supercoiling status of living bacteria. To expose the genetic basis for altered DNA supercoiling, we will identify the mutated genes and explore genetic interactions among the mutations to reveal novel instances of synthetic lethality, thereby uncovering both novel genetic pathways and novel ways to manipulate DNA supercoiling. We will solve the bases for the DNA supercoiling behavior of the identified mutants by determining the amounts of DNA gyrase and topoisomerase I as well as the abundance of adenosine triphosphate, adenosine diphosphate, and specific polyamines – all critical regulators of DNA gyrase activity. The proposed research program pioneers the physiological understanding of an essential cellular property exploitable in a wide range of bacterial species to identify novel targets for antibacterial agents.