RNA-Seq of Escherichia coli Nissle 1917 strains modified to produce different amounts of the bacteriocin microcin C

A popular strategy for enhancing the antibacterial properties of probiotic bacteria is to retrofit them with the ability to overproduce heterologous bacteriocins. This is often achieved from strong, non-native promoters. How the dysregulated overproduction of heterologous bacteriocins affects the fitness and antibacterial efficacy of the retrofitted probiotic bacteria is often overlooked. We conferred the prototypical probiotic Escherichia coli strain Nissle (EcN) the ability to produce different amounts of the bacteriocin microcin C (McC). Expression of the bacteriocin synthesis genes was driven from the native promoter (Pmcc-WT), or from promoters manipulated to be stronger (Pmcc-High) and weaker (Pmcc-Low) than the WT, in a plasmid-based system. Pmcc-Low and Pmcc-High retained their native regulation. A strain harbouring a non-functional promoter (Pmcc-Mut) produces no McC and was used as a control. Each strain was grown to early stationary phase, when production of McC starts, in Luria-Bertani broth at 37 degrees. The RNA was isolated and the effects of different levels of production of McC on the transcriptome of EcN was examined by RNA-Seq.

提升益生菌抗菌特性的常用策略之一，是对其进行基因工程改造，使其能够过量产生异源细菌素，该改造通常依托强效非天然启动子完成。而异源细菌素失调性过量产生对改造后益生菌的适应性与抗菌效能的影响，常被忽视。本研究使经典益生菌大肠杆菌Nissle菌株（Escherichia coli strain Nissle, EcN）具备合成不同剂量细菌素微菌素C（microcin C, McC）的能力。在质粒载体系统中，细菌素合成基因的表达分别由天然启动子（Pmcc-WT）、经改造得到的强于野生型的启动子（Pmcc-High）以及弱于野生型的启动子（Pmcc-Low）驱动；其中Pmcc-Low与Pmcc-High保留了天然调控特性。我们构建了携带无功能启动子（Pmcc-Mut）的菌株，该菌株无法合成McC，将其作为对照。将各菌株于37℃的卢里亚-伯特尼肉汤（Luria-Bertani broth，常简称LB培养基）中培养至早期稳定期——该阶段为McC的起始合成时期，随后提取RNA，通过RNA测序（RNA-Seq）分析不同水平的McC合成对EcN转录组的影响。