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

A deficiency in ptsI, encoding a cytoplasmic protein that serves as the gateway for the phosphoenolpyruvate:sugar phosphotransferase system, was found to confer tolerance to diverse types of disinfectants and antimicrobials. To understand the molecular basis of the increased tolerance, we performed RNA-seq analysis to compare the transcriptional profiles of the wild-type and the ΔptsI mutant strains before and after ciprofloxacin treatment. The data showed that cirpofloxacin treatment drastically increased the exprerssion of genes encoding enzymes in the TCA cycle and that the ptsI mutation suppressed the expression of most TCA genes and all ATP synthase genes, but elevated some of the genes involved in glycolysis and the pentose phosphate pathway (PPP) genes. Such transcriptomic alteration reduced stress-induced surge in respiration and accumutaion of toxic reaactive oxygen species, thereby conferring bacterial tolerance to diverse lethal stressors. Overall design: Mid-log-phase 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.

研究发现，ptsI基因编码一种作为磷酸烯醇丙酮酸糖磷酸转移酶系统（phosphoenolpyruvate:sugar phosphotransferase system）入口的细胞质蛋白，该基因的缺陷可使细菌对多种消毒剂与抗菌剂产生耐受性。为阐明该耐受性增强的分子基础，我们通过RNA测序（RNA-seq）分析，对比了野生型菌株与ΔptsI突变株在环丙沙星处理前后的转录谱。数据显示，环丙沙星处理可显著上调三羧酸循环（TCA cycle）相关酶编码基因的表达；而ptsI突变不仅抑制了大多数TCA循环基因与全部ATP合酶基因的表达，还上调了糖酵解与磷酸戊糖途径（pentose phosphate pathway, PPP）中的部分基因。此类转录组改变可降低应激诱导的呼吸爆发与毒性活性氧（reactive oxygen species, ROS）的积累，从而赋予细菌对多种致命应激源的耐受性。
整体实验设计：以大肠杆菌K-12（E. coli K-12）的中期对数生长期培养物为材料，使用5倍最低抑菌浓度（minimum inhibitory concentration, MIC）的环丙沙星分别处理0分钟与90分钟，收集细胞后提取总RNA并进行RNA-seq测序，随后分析不同处理条件下的基因差异表达情况。