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首页> 外文期刊>Journal of bacteriology >Detrimental Effects of Hypoxia-Specific Expression of Uracil DNA Glycosylase (Ung) in Mycobacterium smegmatis
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Detrimental Effects of Hypoxia-Specific Expression of Uracil DNA Glycosylase (Ung) in Mycobacterium smegmatis

机译：耻垢分枝杆菌中尿嘧啶DNA糖基化酶（Ung）低氧特异性表达的有害影响

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Mycobacterium tuberculosis is known to reside latently in a significant fraction of the human population. Although the bacterium possesses an aerobic mode of metabolism, it adapts to persistence under hypoxic conditions such as those encountered in granulomas. While in mammalian systems hypoxia is a recognized DNA-damaging stress, aspects of DNA repair in mycobacteria under such conditions have not been studied. We subjected Mycobacterium smegmatis, a model organism, to the Wayne's protocol of hypoxia. Analysis of the mRNA of a key DNA repair enzyme, uracil DNA glycosylase (Ung), by real-time reverse transcriptase PCR (RT-PCR) revealed its downregulation during hypoxia. However, within an hour of recovery of the culture under normal oxygen levels, the Ung mRNA was restored. Analysis of Ung by immunoblotting and enzyme assays supported the RNA analysis results. To understand its physiological significance, we misexpressed Ung in M. smegmatis by using a hypoxia-responsive promoter of narK2 from M. tuberculosis. Although the misexpression of Ung during hypoxia decreased C-to-T mutations, it compromised bacterial survival upon recovery at normal oxygen levels. RT-PCR analysis of other base excision repair gene transcripts (UdgB and Fpg) suggested that these DNA repair functions also share with Ung the phenomenon of downregulation during hypoxia and recovery with return to normal oxygen conditions. We discuss the potential utility of this phenomenon in developing attenuated strains of mycobacteria.
机译：已知结核分枝杆菌潜伏在很大一部分人口中。尽管该细菌具有有氧代谢模式，但它能适应低氧条件下的持续性，例如肉芽肿中所遇到的条件。尽管在哺乳动物系统中，低氧是公认的破坏DNA的应激，但尚未研究在这种条件下分枝杆菌中DNA修复的方面。我们对模型生物 M 分枝杆菌 smegmatis 进行了Wayne的缺氧试验。通过实时逆转录酶PCR（RT-PCR）对关键的DNA修复酶尿嘧啶DNA糖基化酶（Ung）的mRNA进行分析，发现其在缺氧时下调。但是，在正常氧气水平下培养物恢复的一个小时内，Ung mRNA得以恢复。通过免疫印迹和酶分析法对Ung的分析支持了RNA分析结果。要了解其生理意义，我们在 M中错误表达了Ung。通过使用来自 M的 narK2 的低氧反应性启动子来实现包皮垢。结核病。尽管在缺氧状态下Ung的错误表达减少了C到T突变，但在正常氧水平下恢复时，它损害了细菌的存活。其他碱基切除修复基因转录本（UdgB和Fpg）的RT-PCR分析表明，这些DNA修复功能也与Ung共享缺氧和恢复正常氧气条件下恢复过程中下调的现象。我们讨论了这种现象在发展分支杆菌减毒株中的潜在效用。

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