大肠杆菌mazG基因参与mazEF介导的严紧反应调控

The mazG gene involvement in the stringent response of Escherichia coli regulated by mazEF

目的探索mazG基因参与调控mazEF毒素-抗毒素系统(TAs)介导的细菌生长抑制与程序性死亡的分子机制,明确MazG蛋白真正的生理功能。方法以大肠杆菌MC4100为原型,通过relA基因的回复突变,获得relA野生型的菌株MC4200。通过同源重组的方法,构建大肠杆菌mazG、mazEF、mazEFG基因敲除菌株,测试mazG基因在不同基因背景菌株中过表达对细菌存活率的影响;通过rifampicin、H2O2及serine hydroxamate(SHT)等胁迫条件处理细菌,研究mazG及mazEFG操纵子缺失菌株的生长曲线与存活率;克隆大肠杆菌mazG基因,构建诱导型过表达质粒pET28a-mazG及突变体pET28a-mazG E38A,在BL21菌株中表达纯化MazG蛋白,通过酶学实验验证了MazG蛋白的去磷酸酶活性;测试突变体过表达对细菌存活率的影响。结果mazG过表达对大肠杆菌MC4200的生长无显著影响,但对mazEFG基因敲除株具有显著抑制作用;MazG的细胞毒性依赖于其NTP-PPase酶活性;mazEF的存在会显著抑制mazG的表型;mazEF/mazG/mazEFG敲除不影响大肠杆菌正常环境下的生长曲线;在胁迫条件下,mazG敲除菌株的存活率与mazEFG的敲除菌株基本一致,显著高于野生型。结论mazG基因参与mazEF诱导的细菌程序性死亡调控,并对细菌的生长抑制具有重要作用。本研究为TAs的研究提供了新的角度,有助于进一步理解TAs在细菌生长与死亡调节中的作用机制。

Objective To explore the molecular mechanism of mazG gene involved in regulating mazEF toxin-antitoxin sys⁃tem(TAs)mediated bacterial growth inhibition and programmed death,and to clarify the true physiological function of MazG protein.Methods The Escherichia coli(E.coli)strain MC4100 was used as a prototype and the relA gene was recovered to obtain the relA wildtype strain MC4200.E.coli mazG,mazEF,mazEFG and other series of gene knockout strains were constructed to test the effects of mazG gene overexpression in different genetic background strains on the survival rate of bacteria.Rifampicin,H2O2 and nalidixic acid and other stress conditions were used to treat the bacteria and study growth curve and survival rate of mazG gene and mazEFG operondeleted strains.The E.coli mazG gene and mutant were cloned into an inducible overexpression to construct pET28a-mazG and pET28amazG E38A.Then the protein was overexpressed in BL21 strain and purified using Ni-NTA resin.The dephosphatase activity of MazG protein was verified by enzyme experiments and the effect of mutant overexpression on bacterial survival was tested.Results The overexpression of mazG had no significant effect on the growth of E.coli MC4200,but had a significant inhibitory effect on the mazEFG gene knockout strain.The cytotoxicity of MazG depended on its NTP-PPase enzyme activity.The presence of mazEF significantly inhibited the phenotype of mazG;Knockout of the mazEF/mazG/mazEFG genes did not affect the growth curve of E.coli under normal envi⁃ronment.Under stress conditions,the survival rate of the mazG knockout strain was basically the same as that of the mazEFG knockout strain,which was significantly higher than that of the wild type.Conclusion The mazG gene is involved in the regulation of bacterial programmed cell death induced by mazEF and has an important role in bacterial growth inhibition.This study provides a new perspec⁃tive for the study of TAs and further understanding of its role in the regulation of bacterial growth and death.