假肠膜明串珠菌L64中mazEF类II型毒素-抗毒素系统的识别和鉴定

Identification and characterization of mazEF family type II toxin-antitoxin systems of Leuconostoc pseudomesenteroides L64

摘要:

目的 鉴定假肠膜明串珠菌L64基因组中编码的mazEF类II型毒素-抗毒素系统，并初步探讨mazEF系统协助宿主应对环境酸压力的分子机制。方法 在大肠杆菌中诱导表达MazF毒素，或同时表达其对应的抗毒素蛋白，以检测MazF是否抑制宿主的正常生长，并验证对应的抗毒素是否能中和MazF的毒性。基于lacZ报告系统及电泳迁移率变动分析(electrophoretic mobility shift assay, EMSA)等体内外试验确定mazEF系统的自调控机制。通过生物信息学预测及体内外试验确定受MazE调控的下游基因。结果 在假肠膜明串珠菌L64基因组编码的3对潜在mazEF毒素-抗毒素系统中，仅有mazEF1-Leup (OYT_01690-OYT_01685)编码真正的毒素-抗毒素系统。抗毒素MazE1-Leup (OYT_01685)通过与启动子中的回文序列(TAACAaaatgTGTTA)结合，抑制mazEF1-Leup启动子的转录；同时，MazE1-Leup通过与dlt-acpS-alr操纵子启动子中的相同回文序列(TAACAtattgaaatatatgTGTTA)结合，抑制dlt-acpS-alr的转录。结论 OYT_01690-OYT_01685编码一个真正的mazEF类毒素-抗毒素系统，该系统不仅参与自身转录的调控，还抑制dlt-acpS-alr操纵子的转录，从而协助假肠膜明串珠菌L64应对环境中的酸压力。

关键词:假肠膜明串珠菌;II型毒素-抗毒素系统;mazEF家族;回文序列;酸压力

Abstract:

Objective To identify mazEF family type II toxin-antitoxin systems of Leuconostoc pseudomesenteroides L64 and to elucidate the molecular roles of the mazEF systems in the host exposed to environmental acid stress.Methods Putative MazF toxins were induced alone or co-expressed with their cognate antitoxins in Escherichia coli. The toxic effect of MazF on bacterial growth and the antitoxic effects of cognate antitoxins were examined. The lacZ reporter system and electrophoretic mobility shift assay (EMSA) were used to decipher the auto-regulation mechanism of the mazEF system in vivo and in vitro. The putative target genes regulated by MazE were predicted and validated through in vivo and in vitro experiments.Results Among the three putative mazEF systems in L. pseudomesenteroides L64, mazEF1-Leup (OYT_01690-OYT_01685) encoded a functional type II toxin-antitoxin system. MazE1-Leup (OYT_01685) inhibited mazEF1-Leup transcription by binding to the palindromic sequence (TAACAaaatgTGTTA) in the promoter. In addition, MazE1-Leup inhibited transcription of the dlt-acpS-alr operon by binding to the similar palindromic sequence (TAACAtattgaaatatatgTGTTA) in the promoter of dlt-acpS-alr.Conclusion mazEF1-Leup (OYT_01690-OYT_01685) encodes a functional mazEF family type II toxin-antitoxin system. Beyond regulating its own operon, MazE1-Leup regulates the transcription of dlt-acpS-alr and finally assists L. pseudomesenteroides L64 in response to low acid stress.

Key words:Leuconostoc pseudomesenteroides;type II toxin-antitoxin system;mazEF family;palindromic sequence;acid stress