Genetic tools to study c-di-GMP-dependent signaling in Pseudomonas aeruginosa.

Pseudomonas aeruginosa infections are often difficult or impossible to treat, mainly due to its ability to form antibiotic-resistant biofilms. Since c-di-GMP signaling strongly influences P. aeruginosa biofilm development and sensitivity to antibiotics, it is considered a promising target for the development of anti-biofilm drugs and it is under intensive investigation. However, studying c-di-GMP signaling in P. aeruginosa is challenging, mainly due to (1) the multiplicity of enzymes involved in c-di-GMP metabolism, (2) the difficulty to extract and measure c-di-GMP intracellular levels by chemical methods, and (3) the lack of genetic tools specifically dedicated to this purpose.Here, a bioluminescence-based reporter system convenient for studying cellular processes or compounds expected to cause an increase or a decrease in intracellular c-di-GMP levels produced by P. aeruginosa cultures is described. Bioluminescence is particularly appropriate in P. aeruginosa research, due to the high intensity of the signal and total lack of background noise. In addition, the use of genetic cassettes allowing the fine control of P. aeruginosa c-di-GMP intracellular levels via arabinose induction is described.Overall, the genetic tools described here could facilitate investigations tackling the c-di-GMP signaling process on different fields, from cellular physiology to drug-discovery research.