TESTING AND CHARACTERIZATION OF GENES INVOLVED IN THE DEGRADATION AND THE PRODUCTION OF C-DI-GMP IN PSEUDOMONAS AERUGINOSE

Pseudomonas aeruginosa is an opportunistic pathogen which causes a diverse array of infections, most of them nosocomial and life threatening. Its resistance to a large range of antibiotics and ability to colonize and form biofilm on medical devices make it difficult to treat. Biofilm formation is regulated by cyclic diguanylate (c-di-GMP) which functions as a second messenger in bacteria and controls diverse cellular processes among them, including mobility. High levels of c-di-GMP induce high levels of biofilm formation and visa versa. c-di-GMP is synthesized by diguanylate cyclases (DGCs) from two molecules of GTP, degraded to 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG) by phosphodiesterases (PDEs) and broken down further by oligoribonuclease (Orn), a 3ʹ to 5ʹ exonuclease highly conserved among Actinobacteria. In this study, we demonstrate that orthologues of Orn from Bacillus subtilis reinstate the Orn phenotype to the wild type strain with an Orn knockout. In addition, we present the effects of PDE deletions on movement and biofilm formation of P. aeruginosa. In addition our work focused on SadB, a protein with an unknown function that contains two predicted active sites, one of them belonging to a family of proteins called Ybak-like possessing aminoacyl-tRNA editing activity, and the other one belonging to a family of proteins that contains the HDOD domain associated with phosphohydrolase activity. Studies show that the lack of SadB causes a decrease in levels of biofilm formation and an increase in mobility, as compared to the wild type strain. Here we also examine the role of SadB in biofilm formation and investigate whether its activity is related to the synthesis and degradation pathways of c-di-GMP.