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耻垢分枝杆菌转录因子Ms6174广泛调控功能的鉴定及其调控生物被膜形成的机理研究

Identification of the Global Transcription Regulator,Ms6174,and Its Regulatory Mechanisms of the Mycobacterium smegmatis Biofilm Formation
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摘要 在前期研究中,利用耻垢分枝杆菌(Mycobacterium smegmatis)转座插入突变体文库筛选并鉴定到的转录因子的编码基因ms6174突变后影响了其细菌菌落表面形态。利用凝胶阻滞迁移实验(electrophoretic mobility shift assay,EMSA)和β-半乳糖苷酶活性实验证实了Ms6174直接负调控其自身所在基因簇的表达。同时,利用EMSA实验、DNaseⅠ酶切实验以及蛋白质与染色质DNA荧光共定位等实验发现Ms6174具有拟核结合蛋白的特性,即Ms6174是耻垢分枝杆菌中新的拟核结合蛋白。进一步通过转录组测序分析发现,相较于耻垢分枝杆菌野生型菌株,ms6174敲除菌株中有682个基因的表达水平发生了显著变化,其中有387个基因上调表达,295个基因下调表达,142个脂质代谢相关基因的表达水平发生变化。最后,检测并比较了耻垢分枝杆菌ms6174敲除突变体菌株与野生型菌株的菌落形态与生物被膜形成的差异,发现敲除ms6174后耻垢分枝杆菌的菌落表面褶皱与气液表面生物被膜增多,因此,转录因子Ms6174负调控耻垢分枝杆菌生物被膜的形成。综上,本研究鉴定了分枝杆菌中一个新的拟核结合蛋白Ms6174,其通过调控耻垢分枝杆菌脂代谢相关基因的表达而影响细菌生物被膜的形成。本研究为发掘新的广泛调控因子拟核结合蛋白提供了基础,为解析分枝杆菌生物被膜形成的调控机理提供了依据。 The gene,ms6174,encoding a transcription factor which affected the formation of mycobacterial biofilm was identified in Mycobacterium smegmatis using transposition insertion mutant library screening as described previously.Subsequently,electrophoretic mobility shift assay(EMSA)andβ-galactosidase activity assays confirmed that Ms6147 negatively regulated the ms6147 operon.We found that Ms6174 has characteristics of a nucleoid-associated protein by EMSA,DNase I digestion,and fluorescence co-localization assays.Namely,Ms6174 is a novel nucleoid-associated protein in M.smegmatis.RNA-Seq data analysis showed that 682 genes expression were significantly changed in the ms6174 knockout strain compared with that of the wild-type strain,of which 387 genes were up-regulated and 295 genes were down-regulated.Among them,142 lipid metabolism related genes were changed.By analysis of the colony morphology and the formation of air-liquid interface pellicles,we found that wrinkles and air-liquid interface pellicles of the ms6174 knockout strain significantly increased compared to that of the wild-type strain.Therefore,it is found that Ms6174 negatively regulate mycobacterial biofilm formation,and it is inferred that the formation of mycobacterial biofilm is regulated by the expression of genes involved in lipid metabolism.The findings identified a new nucleoid-associated protein in mycobacteria and deepened our understanding of the regulatory mechanisms of mycobacterial biofilm formation.
作者 王瑞 王坤 郑慧霞 张景馨 金梅 李伟辉 Wang Rui;Wang Kun;Zheng Huixia;Zhang Jingxin;Jin Mei;Li Weihui(State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,Nanning,530004;College of Life Science and Technology,Guangxi University,Nanning,530004)
出处 《基因组学与应用生物学》 CAS CSCD 北大核心 2022年第11期2144-2155,共12页 Genomics and Applied Biology
基金 国家重点研发计划项目(2020YFA0907200) 国家自然科学基金项目(31870036) 亚热带农业生物资源保护与利用国家重点实验室-青年人才专项(SKLCUSA-a02)共同资助
关键词 耻垢分枝杆菌 转录调控 拟核结合蛋白 生物被膜 Mycobacterium smegmatis Transcriptional regulation Nucleoid-associated proteins Biofilms
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