The transcriptional regulator CueR positively regulates the expression of the cueRA operon to maintain copper homeostasis in Pseudomonas chlororaphis zm-1.

Copper serves as an indispensable trace element in living organisms, playing pivotal roles in fundamental physiological processes including enzyme assembly, antioxidant defense, and metabolic regulation. However, excess copper can induce oxidative damage. MerR-family regulator CueR acts as the core mediator of copper homeostasis in prokaryotes, but its mechanism of action in P. chlororaphis zm-1, a plant biocontrol bacterium that inhibits peanut stem rot, remains unclear. In this study, we investigated the regulatory function of CueR using molecular techniques including gene deletion, electrophoretic mobility shift assay, and quantitative real-time PCR. The results demonstrated that the transcriptional regulator gene cueR and the P1B-type ATPase gene cueA (involved in copper efflux) form the cueRA operon in strain zm-1, with a 4-bp overlap and co-transcriptional feature. CueR directly binds to the promoter region and positively activates cueRA expression upon copper ion induction, thereby driving CueA-mediated efflux of excess copper ions. The deletion of cueR or cueA significantly reduced copper tolerance, increased intracellular copper content, and impaired peanut rhizosphere colonization ability of the strain, while these phenotypes were restored in the complementation strains. This study deciphers the regulatory mechanism of the cueRA operon in P. chlororaphis and establishes a correlation analysis between this mechanism and the rhizosphere colonization capacity of the strain. The findings provide a molecular basis for understanding the diversity of copper homeostasis regulation in Pseudomonas and optimizing the biocontrol application of this bacterium.IMPORTANCEThe CueR-regulated copper efflux system is essential for bacterial survival under copper overload conditions. While this system has been extensively characterized in human pathogens, its function in plant-beneficial bacteria remains poorly understood. Here, we investigate the agricultural biocontrol strain P. chlororaphis zm-1 and delineate the regulatory mechanism of CueR through molecular approaches, leading to the identification of the cueRA operon. Furthermore, we establish a functional link between CueR and rhizosphere colonization, thereby providing a framework for understanding CueR-mediated regulation in plant-associated bacteria.