CzcR regulon with ChIPseq

Pseudomonas aeruginosa is a versatile opportunistic pathogen that thrives in hostile environments by tightly regulating zinc (Zn) homeostasis. The CzcRS two-component system is pivotal for Zn resistance, primarily by activating the CzcCBA efflux pump, yet its basal activity and full regulon remain poorly defined. Here, we mapped the CzcR regulon under zinc-sufficient (ZS) and zinc-excess (ZE) conditions in P. aeruginosa PAO1 using ChIP-seq. Under ZE, we identified 38 CzcR binding sites, potentially regulating 42 genes, many of which are linked to virulence, antibiotic resistance, and stress response. Under ZS, 12 binding sites were detected, revealing a distinct basal regulon associated with metabolic, transport, and transcriptional regulatory functions. Motif analysis uncovered a specific CzcR binding motif under ZE, absent in most basal targets, suggesting a conformation-dependent DNA recognition mechanism. Integration of ChIP-seq data with transcriptomics identified eight novel CzcR-regulated genes mostly implicated in virulence and colistin resistance. Our findings demonstrate that CzcR exhibits dual functionality: its basal, potentially phosphorylation-independent activity maintains physiological robustness, while its activated form orchestrates Zn detoxification and virulence modulation. This study expands our understanding of how P. aeruginosa integrates metal sensing with clinically relevant phenotypes, highlighting CzcR as a key regulator at the intersection of metal homeostasis and pathogenicity.

铜绿假单胞菌（Pseudomonas aeruginosa）是一种多能机会致病菌，可通过严格调控锌（Zn）稳态在恶劣环境中存活。CzcRS双组分系统（CzcRS two-component system）是其锌抗性的关键调控通路，主要通过激活CzcCBA外排泵（CzcCBA efflux pump）发挥功能，但其基础活性与完整调节子（regulon）的全貌仍未被充分阐明。本研究以铜绿假单胞菌PAO1为研究模型，利用染色质免疫共沉淀测序（ChIP-seq）绘制了锌充足（ZS）与锌过量（ZE）条件下的CzcR调节组图谱。在锌过量条件下，我们共鉴定出38个CzcR结合位点，潜在调控42个基因，其中多数与毒力、抗生素抗性及应激反应密切相关。在锌充足条件下，仅检测到12个结合位点，揭示了一套独特的基础调节子，其功能涉及代谢、转运及转录调控过程。基序分析发现，锌过量条件下存在特异性CzcR结合基序，而多数基础调控靶点并不具备该基序，提示CzcR存在构象依赖型DNA识别机制。将ChIP-seq数据与转录组学数据整合分析后，我们鉴定出8个全新的CzcR调控基因，这些基因主要参与毒力与粘菌素抗性调控。本研究结果表明，CzcR具备双重功能：其基础活性（可能为非磷酸化依赖）可维持菌体生理鲁棒性，而激活形式则协同调控锌解毒与毒力调控过程。本研究拓展了我们对铜绿假单胞菌如何整合金属感知与临床相关表型的认知，凸显了CzcR作为金属稳态与致病性交叉调控关键调控因子的重要地位。