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Pseudomonas aeruginosa is an opportunistic human pathogen causing severe acute and chronic infections. Earlier we have shown that calcium (Ca(2+)) induces P. aeruginosa biofilm formation and production of virulence factors. To enable further studies of the regulatory role of Ca(2+), we characterized Ca(2+) homeostasis in P. aeruginosa PAO1 cells. By using Ca(2+)-binding photoprotein aequorin, we determined that the concentration of free intracellular Ca(2+) ([Ca(2+)]in) is 0.14±0.05μM. In response to external Ca(2+), the [Ca(2+)]in quickly increased at least 13-fold followed by a multi-phase decline by up to 73%. Growth at elevated Ca(2+) modulated this response. Treatment with inhibitors known to affect Ca(2+) channels, monovalent cations gradient, or P-type and F-type ATPases impaired [Ca(2+)]in response, suggesting the importance of the corresponding mechanisms in Ca(2+) homeostasis. To identify Ca(2+) transporters maintaining this homeostasis, bioinformatic and LC-MS/MS-based membrane proteomic analyses were used. [Ca(2+)]in homeostasis was monitored for seven Ca(2+)-affected and eleven bioinformatically predicted transporters by using transposon insertion mutants. Disruption of P-type ATPases PA2435, PA3920, and ion exchanger PA2092 significantly impaired Ca(2+) homeostasis. The lack of PA3920 and vanadate treatment abolished Ca(2+)-induced swarming, suggesting the role of the P-type ATPase in regulating P. aeruginosa response to Ca(2+).
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