The Pseudomonas aeruginosa PAO1 Two-Component Regulator CarSR Regulates Calcium Homeostasis and Calcium-Induced Virulence Factor Production through Its Regulatory Targets CarO and CarP
Authors: Guragain Manita , King Michelle M. , Williamson Kerry S. , Pérez-Osorio Ailyn C. , Akiyama Tatsuya , Khanam Sharmily , Patrauchan Marianna A. , Franklin Michael J. 
Published: January 2016

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.