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。

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.