Regulatory Characterization of Two Cop Systems for Copper Resistance in Pseudomonas putida

Copper ions serve as essential cofactors for many enzymes but exhibit toxicity at elevated concentrations. In Gram-negative bacteria, the Cop system, typically encoded by <i>copABCD</i>, plays a crucial role in maintaining copper homeostasis and detoxification. The chromosome of <i>Pseudomonas putida</i> harbors two <i>copAB</i> clusters but lacks <i>copCD</i>, along with two <i>copR</i>-<i>copS</i> clusters that encode the cognate two-component system. Here, the roles of these Cop components in countering copper toxicity were studied. We found that <i>copAB2</i> was essential for full resistance to Cu²⁺ in <i>P. putida</i>, while <i>copAB1</i> made only a minor contribution, partially due to its low expression. The two-component systems CopRS1 and CopRS2 both played significant regulatory roles in copper resistance. Although they could compensate for the absence of each other to mediate copper resistance, they exhibited distinct regulatory effects. CopR1 bound to all four <i>cop</i> promoters and activated their transcription under copper stress. In contrast, though CopR2 bound to the same sites as CopR1 in each <i>cop</i> promoter, it significantly activated only <i>copAB2</i> and <i>copRS2</i> expression. Its competitive binding at the <i>copAB1</i> and <i>copRS1</i> promoters likely impeded CopR1-mediated activation of these genes. Overall, this study reveals the distinct contributions of the two Cop systems to copper resistance and their regulatory interplay in <i>P. putida</i>.