RNA-seq analyzes the effect of purA on transcriptome in Escherichia coli during ciprofloxacin treatment.

PurA encodes an adenylosuccinate synthetase that catalyzes the first step in the de novo synthesis of AMP. A deficiency in purA has been found to confer Escherichia coli tolerance to diverse types of antibiotics. To understand the molecular basis of the increased tolerance, we performed RNA-seq analysis to compare the transcriptional profiles of wild-type and ?purA mutant strains before and after ciprofloxacin treatment. The data showed that the purA deficiency suppresses transcript levels from NADH:quinone oxidoreductase genes prior to stress exposure, thereby predisposing E. coli to antibiotic tolerance by reducing respiration. During ciprofloxacin exposure, a purA deficiency suppressed a surge in expression of TCA cycle and ATP synthesis genes and the accumulation of intracellular ATP and ROS, thereby conferring bacterial tolerance to diverse antibiotic stresses. Overall design: Exponentially growing cultures of E. coli K-12 were treated with ciprofloxacin (5 MIC) for 0 min and 90 min. Cells were collected for total RNA extraction and subsequent RNA-seq. Differential expression of various conditions was analyzed.

PurA基因编码腺苷琥珀酸合成酶（adenylosuccinate synthetase），该酶催化腺苷一磷酸（AMP）从头合成的第一步反应。已有研究发现，purA基因缺陷可赋予大肠杆菌（Escherichia coli）对多种抗生素的耐受能力。为解析该耐受增强现象的分子机制，本研究开展了RNA测序（RNA-seq）分析，对比野生型与ΔpurA突变菌株在环丙沙星（ciprofloxacin）处理前后的转录组谱。
数据显示，在抗生素应激暴露前，purA基因缺陷会抑制NADH：泛醌氧化还原酶（NADH:quinone oxidoreductase）编码基因的转录水平，进而通过降低呼吸作用使大肠杆菌更易产生抗生素耐受。在环丙沙星处理过程中，purA基因缺陷可抑制三羧酸循环（TCA cycle）与ATP合成相关基因的表达激增，同时减少细胞内ATP与活性氧（ROS）的积累，从而使细菌对多种抗生素应激产生耐受。
整体实验设计：将处于指数生长期的大肠杆菌K-12菌株培养物用5倍最低抑菌浓度（MIC）的环丙沙星分别处理0分钟与90分钟，收集细胞用于总RNA提取及后续RNA-seq分析，并对不同处理条件下的基因差异表达情况进行了分析。