Cross-regulation and cross-talk of conserved and accessory two-component regulatory systems orchestrate Pseudomonas copper resistance

Bacteria use diverse strategies and molecular machinery to maintain copper homeostasis and to cope with its toxic effects. Some genetic elements providing copper resistance are acquired by horizontal gene transfer; however, little is known about how they are controlled and integrated into the central regulatory network. Here, we studied two copper-responsive systems in a clinical isolate of Pseudomonas paraeruginosa and deciphered the regulatory and cross-regulation mechanisms. To do so, we combined mutagenesis, transcriptional fusion analyses and copper sensitivity phenotypes. Our results showed that the accessory CusRS two-component system (TCS) responds to copper and activates both its own expression and that of the adjacent nine-gene operon to provide resistance to elevated levels of extracellular copper. The same locus was also found to be regulated by two core-genome-encoded TCSs - the copper-responsive CopRS and the zinc-responsive CzcRS. Although the target palindromic sequence - ATTCATnnATGTAAT - is the same for the three response regulators, transcriptional outcomes differ. Thus, depending on the operon/regulator pair, binding can result in different activation levels (from none to high), with the systems demonstrating considerable plasticity. Unexpectedly, although the classical CusRS and the noncanonical CopRS TCSs rely on distinct signaling mechanisms (kinase-based vs. phosphatase-based), we discovered cross-talk in the absence of the cognate sensory kinases. This cross-talk occurred between the proteins of these two otherwise independent systems. The entire locus is part of an Integrative and Conjugative Element, and was found in other Pseudomonas strains where its expression could provide copper resistance under appropriate conditions. The results presented here illustrate how acquired genetic elements can become part of endogenous regulatory networks, providing a physiological advantage. They also highlight the potential for broader effects of accessory regulatory proteins through interference with core regulatory proteins. Author SummaryTwo-component regulatory systems play a key role in bacterial life by detecting and integrating a wide range of signals, allowing bacteria to continuously monitor and adapt to a changing environment. They weave a complex network with a few highly interconnected phosphorelays and numerous cross-regulations. Our study reveals connections between zinc and copper homeostasis in a pathogenic bacterium, with cross-regulation observed for three independent, closely related transcriptional regulators, CzcR, CopR and CusR. Zinc and copper play a major role in host-pathogen interactions, and bacteria that synergize their responses to the two elements can harness a growth advantage and enhanced fitness in specific conditions. We also observed unexpected cross-talk between the core genome-encoded CopRS system and the horizontally acquired CusRS system, although the molecular control exerted by a histidine kinase on its cognate regulator differs. This plasticity observed within the two signaling systems ensures a normal regulatory response to the copper signal required to maintain copper homeostasis.