Regulation of copper homeostasis in Pseudomonas fluorescens SBW25

Abstract

Copper is an essential element for life, but too much copper is harmful: copper homeostasis must therefore be carefully regulated. When growing on plant surfaces, the plant growth-promoting bacterium Pseudomonas fluorescens SBW25 activates expression of a copper-transporting P1-type ATPase (CueA). Using a combination of transcriptional gene fusions and site-directed mutants, we show that copper-induced transcription of cueA is controlled by the MerR-type regulator, CueR; CueR is also required for activation of the copper chaperone protein encoded by cueZ (pflu0660). The promoters of cueA and cueZ are also responsive to the metal salts of gold, silver and mercury. In each case, CueR transduces the stimulus. Resistance to exogenously applied copper sulfate shows that cueA and cueR mutants are significantly less resistant than the wild type. This is consistent with the role of CueA as a copper efflux system and a general role for the CueR regulon in copper resistance. A search of the SBW25 genome for orthologues of genes predicted to play additional roles in copper homeostasis identified copCD of the known four-component copABCD system (unusually, copAB are absent from the SBW25 genome). Genetic studies showed that expression of copCD is controlled by copper and mediated by the CopRS two-component regulatory system. Mutants devoid of copCD or copS displayed an increased tolerance to copper and overexpression of copCD caused increased sensitivity. This is consistent with CopCD encoding a copper uptake system. Taken together, we suggest that the Cue and Cop systems are integral to copper homeostasis in P. fluorescens SBW25 with one (Cop) being active at low-copper environments and bringing copper into the cell, and the other (Cue) being active in high-copper environments and serving to export excess copper.