Examination of E. faecalis toxin-antitoxin (TA) toxin Fst function utilizing a pheromone-inducible expression vector with tight repression and large dynamic range

Tools for regulated gene expression in E. faecalis are extremely limited. In this study we describe the construction of an expression vector for E. faecalis, designated pCIE, utilizing the PQ pheromone-responsive promoter of plasmid pCF10. We demonstrate that this promoter is tightly repressed, responds to nanogram quantities of the peptide pheromone, and has a large dynamic range. To demonstrate its utility, the promoter was used to control expression of the toxic peptides of two par family toxin-antitoxin loci present in E. faecalis, parpAD1 of the pAD1 plasmid and parEF0409 located on the E. faecalis chromosome. Results demonstrated differences in the modes of regulation of toxin expression and in the effects of toxins of these two related systems. We anticipate that this vector will be useful for further investigation of par TA system function as well as the regulated expression of other genes in E. faecalis. Examination of cellular effects of toxin FstEF0409 at two different levels compared to unexposed cells and comparison of the effects of the two related toxins FstEF0409 and FstpAD1. Average of two biological replicates.

粪肠球菌（E. faecalis）的可调控基因表达工具极为有限。本研究构建了一种命名为pCIE的粪肠球菌表达载体，其依托质粒pCF10的PQ信息素响应启动子构建而成。研究证实该启动子具备严谨的阻遏特性，可响应纳克级别的肽类信息素，且拥有宽泛的动态调节范围。
为验证该载体的实用性，本研究利用该启动子调控粪肠球菌中两种par家族毒素-抗毒素（toxin-antitoxin, TA）基因座的毒性肽表达：分别为质粒pAD1携带的parpAD1，以及粪肠球菌染色体上的parEF0409。实验结果显示，这两种同源毒素-抗毒素系统的毒素表达调控模式以及毒素的细胞效应存在差异。
我们预期该表达载体可用于粪肠球菌中par TA系统功能的后续研究，以及其他基因的可调控表达实验。本研究分别在两个不同表达水平下检测了毒素FstEF0409对细胞的影响，并与未暴露的对照细胞进行比较；同时对比了两种同源毒素FstEF0409与FstpAD1的细胞效应。所有实验均设置两次生物学重复并取平均值。