Calcium homeostasis in Pseudomonas aeruginosa requires multiple transporters and modulates swarming motility

Pseudomonas aeruginosa is an opportunistic human pathogen causing severe acute and chronic infections. Earlier we have shown that calcium (Ca²⁺) induces P. aeruginosa biofilm formation and production of virulence factors. To enable further studies of the regulatory role of Ca²⁺, we characterized Ca²⁺ homeostasis in P. aeruginosa PAO1 cells. By using Ca²⁺-binding photoprotein aequorin, we determined that the concentration of free intracellular Ca²⁺ (Ca²⁺]_in) is 0.14 ± 0.05 μM. In response to external Ca²⁺, the Ca²⁺]_in quickly increased at least 13-fold followed by a multi-phase decline by up to 73%. Growth at elevated Ca²⁺ modulated this response. Treatment with inhibitors known to affect Ca²⁺ channels, monovalent cations gradient, or P-type and F-type ATPases impaired Ca²⁺]_in response, suggesting the importance of the corresponding mechanisms in Ca²⁺ homeostasis. To identify Ca²⁺ transporters maintaining this homeostasis, bioinformatic and LC–MS/MS-based membrane proteomic analyses were used. Ca²⁺]_in homeostasis was monitored for seven Ca²⁺-affected and eleven bioinformatically predicted transporters by using transposon insertion mutants. Disruption of P-type ATPases PA2435, PA3920, and ion exchanger PA2092 significantly impaired Ca²⁺ homeostasis. The lack of PA3920 and vanadate treatment abolished Ca²⁺-induced swarming, suggesting the role of the P-type ATPase in regulating P. aeruginosa response to Ca²⁺.