The Pseudomonas aeruginosa PAO1 two-component regulator, CarSR, regulates calcium homeostasis and calcium-induced virulence factor production through its regulatory targets, CarO and CarP.

ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life-threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca 2+ ) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca 2+ through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca 2+ level, followed by its removal to the basal submicromolar level. However, the molecular mechanisms responsible for regulating Ca 2+ -induced virulence factor production and Ca 2+ homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa to elevated [Ca 2+ ] in both planktonic cultures and biofilms. Among the genes induced by CaCl 2 in strain PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR ), while the closely related two-component regulators phoPQ and pmrAB were repressed by CaCl 2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR and performed transcriptome analysis of the mutant strain at low and high [Ca 2+ ]. Among the genes regulated by CarSR in response to CaCl 2 are the predicted periplasmic OB-fold protein, PA0320 (here called carO ), and the inner membrane-anchored five-bladed β-propeller protein, PA0327 (here called carP ). Mutations in both carO and carP affected Ca 2+ homeostasis, reducing the ability of P. aeruginosa to export excess Ca 2+ . In addition, a mutation in carP had a pleotropic effect in a Ca 2+ -dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca 2+ and responding through its regulatory targets that modulate Ca 2+ homeostasis, surface-associated motility, and the production of the virulence factor pyocyanin. IMPORTANCE During infectious disease, Pseudomonas aeruginosa encounters environments with high calcium (Ca 2+ ) concentrations, yet the cells maintain intracellular Ca 2+ at levels that are orders of magnitude less than that of the external environment. In addition, Ca 2+ signals P. aeruginosa to induce the production of several virulence factors. Compared to eukaryotes, little is known about how bacteria maintain Ca 2+ homeostasis or how Ca 2+ acts as a signal. In this study, we identified a two-component regulatory system in P. aeruginosa PAO1, termed CarRS, that is induced at elevated Ca 2+ levels. CarRS modulates Ca 2+ signaling and Ca 2+ homeostasis through its regulatory targets, CarO and CarP. The results demonstrate that P. aeruginosa uses a two-component regulatory system to sense external Ca 2+ and relays that information for Ca 2+ -dependent cellular processes.