Dottorato in BIOCHIMICA

Titolo della tesi: RmcA controls c-di-GMP levels and biofilm formation in Pseudomonas species in response to L-Arginine

Bacteria can form organized community called biofilm allowing them to colonize the surfaces and to resist antimicrobial treatments and host defenses. The second messenger 3’-5’cyclic diguanylic acid (c-di-GMP) controls biofilm formation, maintenance, and dispersion in response to environmental signals, including nutrients and stressors. The intracellular levels of c-di-GMP are controlled by the rate of its synthesis and degradation, regulated by diguanylate cyclases (GGDEF signature) and phosphodiesterase (EAL and HD- GYP signatures), respectively. Among nutrients, Arginine represents one key metabolite in biofilm formation being at the crossroad of many metabolic processes. Moreover, in Pseudomonas aeruginosa, Arginine is the substrate for ATP production under low O2 levels and plays a key role in chronic infections, biofilm and antibiotic resistance. RmcA (Redox modulator of c-di-GMP) is a multidomain phosphodiesterase that links redox conditions to colony morphogenesis in P. aeruginosa, by modulating levels of c-di-GMP and wrinkling phenotype in response to phenazine availability. RmcA is a membrane protein composed by a periplasmic VFT domain, a transmembrane helix, four Per-Arnt-Sim (PAS) domains that are responsible for the periplasmic signal transduction to the catalytic moiety (GGDEF-EAL). RmcA is involved in biofilm maintenance pathway of P. aeruginosa, and the characterization of this protein is important to gain details of the molecular mechanism in terms of electron perceiving and nutrient sensing to finally control c-di-GMP consumption activity and treat chronic infection. Moreover, understanding mechanism involved in biofilm maintenance is relevant also in biotechnology field. In this thesis we have contributed to gain mechanistic details on the RmcA-based signal transduction by reaching three main goals: 1)The characterization of cytosolic portion of RmcA (cRmcA). I have found that RmcA regulates its phosphodiesterase activity by detecting the intracellular redox potential FAD/ FADH2 via the PASd domain. 2)The RmcA control via VFT domain in response to arginine. I have isolated the membrane protein and in vitro obtained the transduction of the periplasmic signal to the catalytic portion, resulting in the regulation of the phosphodiesterase activity in response to arginine. 3)The effect of RmcA in controlling P. putida metabolism in response to arginine. I have developed a novel approach for measuring the bacterial energy metabolism; the ΔrmcA mutant is affected by Arginine when it is the only carbon source.