Pseudomonas aeruginosa is a ubiquitous Gram-negative environmental bacterium that can be isolated from a wide range of habitats and hosts. Its diversity and genetic complexity allow it to adapt to changing environments and exhibit high pathogenicity. It is a clinically important pathogen with the ability to resist antibiotics and heavy metals, attributed to its low membrane permeability and efflux mechanisms. Moreover, this organism serves as a noteworthy model for studying two- component systems' regulation, which are major players in exogenous signal transduction. Regulatory mechanisms enable P. aeruginosa to thrive in both host-associated and non-associated environments. Response regulators (RR), often activated by their sensor histidine kinase (HK), play a crucial role in gene expression regulation. Their function involves activating or repressing gene expression, orchestrating essential cellular processes. Therefore, the characterization of transcription factors (TFs) and their targets is vital in biological research.

The aim of this study is to characterise a two-component system, IrlRS, studied for the first time in Burkholderia pseudomallei due to its involvement in the regulation of heavy metal resistance and host invasion. However, information about it in P. aeruginosa is practically nonexistent. We determined that IrlR binds to the promoters of genes czcR and copR, encoding two regulators capable of activating the CzcCBA efflux pump, thus involved in heavy metal resistance. In addition to numerous connections with genes related to heavy metal import and export, this two-component system also possesses mechanisms to survive in case of excess zinc or cobalt and seems to be linked to zinc homeostasis in the bacterium Escherichia coli.

Finally, we report here that the transcriptional control of IrlRS relies on the metal-dependent phosphatase activity of IrlS. The phosphorylation of IrlR is likely independent of IrlS, and the gene activation by this system as well. However, in the absence of certain metals such as cobalt or zinc, IrlS dephosphorylates IrlR, rendering it inactive.