Pseudomonas putida中czcRS双组分系统对锌离子内稳态调控机制的研究

Zinc is an essential nutrient that plays important roles in numerous physiological processes, as it serves as a cofactor of enzymes and is especially important in the maintenance of protein structure; but excess Zn can have significant toxicity and can act as a potent disrupter of biological system. Based on the gene knockout experiment and reporter gene fusion analysis, we found that three CzcRS two-component systems involved in the Zn homeostasis in Pseudomonas putida, but the transcriptional characteristics of these czcRSs were quite different. The transcription of czcRS1 was inhibited by Zn and Cd, while the transcription of czcRS2 and czcR3 was induced by Cd and Zn, respectively. Other divalent metal ions did not significantly influenced the czcRS transcription. In bacteria, gene transcription is mainly regulated by transcriptional factors. In this regard, this work attempts to screen and characterize the regulators that control the czcRS transcription using bacteria one-hybrid system, chromatin immunoprecipitation and electrophoretic mobility shift assay. The data obtained will valuable for understanding the detailed mechanisms of how P. putida keeps the equilibrium of zinc in cells.

锌离子是微生物生长所必需的过渡态金属离子，在维持酶的催化功能和蛋白质正常结构方面具有不可替代的作用；但当其超过一定浓度会严重影响细胞内的正常生理过程。申请者前期通过基因敲除、报告基因整合和逆转录定量PCR等研究，发现恶臭假单胞菌中三组CzcRS双组分系统与锌离子的输出密切相关，但三者的转录特性存在较大差别。其中czcRS1的转录受到锌离子与镉离子的抑制，而czcRS2与czcR3分别受镉离子与锌离子的诱导。其他二价金属离子没有显著改变czcRS的转录情况。由于原核生物主要依靠转录因子的作用调节靶基因的转录进程， 因此本项目拟综合运用细菌单杂交，染色质免疫共沉淀与凝胶迁移阻滞等技术在基因组范围内探索不同czcRS上游的转录调节蛋白，深入揭示P. putida维持细胞内Zn平衡的分子机制。

假单胞菌代谢类型多样，环境适应性强，分布广。基因组序列分析表明，假单胞菌属的细菌可以分为3个谱系、10个类群。不同类群的假单胞菌在应对环境锌离子胁迫方面表现出了较大的多样性。然而，比较转录组学研究发现，划分为不同类群的假单胞菌菌株均借助CzcRS双组分系统调节细胞内Zn2+的内稳态平衡。本研究以恶臭假单胞菌KT2440为研究对象，在转录组学的基础上，借助细菌单杂交和EMSA实验研究了主要Zn2+响应转录因子对czcRS2和czcR3的转录调节作用。结果表明，一种σ-54依赖型转录调控蛋白PP_5166可以与czcRS2和czcR3调控区内部的ACAW(2,3)CCG模体结合来增强二者的转录。Chip-seq分析显示，PP_5166还与外膜孔蛋白OprL，以及一个编码外膜多糖转运蛋白基因的上游区域发生了结合反应。因此，PP_5166基因不仅参与了假单胞菌中Zn2+内稳态平衡的调控，还与外膜结构的维持密切相关。

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