DNA甲基化在铜绿假单胞菌中调控机制的研究

       DNA基化是细菌限制性修饰系统的重要组成部分，可以帮助原核生物抵御外源入侵的基因组，其调控基因表达参与胞内生理过程的机制也日渐引起关注。铜绿假单胞菌是一种重要的条件致病菌，然而关于这种细菌的DNA甲基化研究很少且缺乏系统性研究。本课题围绕铜绿假单胞菌临床分离株TBCF10839和LYSZa7展开。利用SMRT-seq测序及甲基化分析，预测和鉴定了DNA甲基转移酶和被修饰基序，并使用质谱、体外酶活测定等实验进行了验证。结合甲基转移酶突变菌株构建、转录组、表型测定等实验，对DNA甲基化影响基因表达调控的机制进行了研究。转录组学分析表明，TBCF10839中DNA 甲基转移酶 M.TBCFORFCP的缺失突变株ΔM.TBCFORFCP中一氧化氮还原酶（NOR）调节和编码基因（如nosR和norCBD）的表达显著下调。ΔM.TBCFORFCP在产NO的RAW 264.7巨噬细胞中胞内存活能力降低，在大蜡螟幼虫感染模型中毒力减弱，并且回补菌株均能恢复这些缺陷表型。生物信息学分析推测nosR 和norB上的M.TBCFORFCP识别基序的甲基化修饰可能通过影响DNR和RpoN等反式作用因子的结合来增强基因表达。此外，系统发育分析表明甲基转移酶M.TBCFORFCP酶的同源蛋白存在于多种铜绿假单胞菌以及其他细菌物种中。LYSZa7中DNA甲基转移酶可以广泛影响细菌转录水平的变化和表型的变化，包括水溶性荧光素铁载体产生、群集运动、生物被膜形成能力。因此，我们的工作为DNA甲基化和细菌毒力表型之间的关系提供了新的认识，为进一步探索DNA甲基转移酶调控铜绿假单胞菌致病性的分子机制奠定了基础。

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