摘要/Abstract
摘要： 目的 ·探究鼠伤寒沙门菌转录调控因子 PhoP的甲基化修饰与其活性的相关性，筛选 PhoP的甲基转移酶。方法 · Western blotting检测不同培养条件下鼠伤寒沙门菌中 PhoP的甲基化水平变化。通过生物信息学预测候选的甲基转移酶，体内过表达预测的甲基转移酶后检测 phoP及其下游基因的转录水平变化，找到可能的 PhoP甲基转移酶，并体外验证该甲基转移酶能否甲基化 PhoP。使用 real-time PCR检测高浓度 Mg2+、弱酸性等培养条件下， phoP与其甲基转移酶在转录水平的相对表达变化。结果 ·随着培养基中 Mg2+浓度升高， PhoP甲基化水平上升；而经过弱酸刺激后， PhoP甲基化水平下降。生物信息学预测并筛选到 9个甲基转移酶，其中体内过表达 STM14_0023后，phoP及其下游基因的转录水平下降且 PhoP蛋白水平也下降，但敲除 STM14_0023对上述分子的表达无影响。体外甲基化酶促反应显示 STM14_0023能够甲基化 PhoP，体内过表达 STM14_0023后也可提高 PhoP的甲基化水平，且在抑制 phoP表达的高浓度 Mg2+培养条件下 STM14_0023的转录水平升高。结论 · STM14_0023是 PhoP的甲基转移酶，甲基化修饰抑制 PhoP活性。
关键词: 鼠伤寒沙门菌, PhoP, 甲基化修饰, STM14_0023, 毒力
Abstract:
Objective · To investigate the correlation between methylation and the activity of transcriptional regulator PhoP in Salmonella enterica serovar Typhimurium (S. Typhimurium), and screen for the methyltransferase of PhoP. Methods · The methylation level of PhoP in S. Typhimurium under different culture conditions was determinedWestern blotting. The transcription levels of phoP and the genes regulatedphoP were examined to screen for the methyltransferase of PhoP after overexpressing methyltransferase candidates predictedbioinformatics. And then methyltransferase assay was verified in vitro. The transcription levels of phoP and its methyltransferase were determinedreal-time PCR in high concentration of Mg2+ or weak acid conditions. Results · As the concentration of Mg2+increased in the medium, the methylation level of PhoP increased, and its methylation level decreased after S. Typhimurium was stimulatedweak acid. In the screening of 9 methyltransferases predictedbioinformatics, over of STM14_0023 reduced the transcription level of phoP and its downstream genes and the protein level of PhoP in vivo, but knockout of STM14_0023 had no effect on the of phoP and its downstream genes. STM14_0023 was capable of methylating PhoP in vitro and could also increase the methylation level of PhoP after over of STM14_0023 in vivo. Under the condition of high concentration of Mg2+ when the of phoP was inhibited, the transcriptional level of STM14_0023 was reduced. Conclusion · STM14_0023 is the methyltransferase of PhoP, and methylation inhibits PhoP activity.
Key words: Salmonella enterica serovar Typhimurium, PhoP, methylation, STM14_0023, virulence
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