Characterization of transporters involved in uptake of choline and other trimethylamine-containing compounds in the opportunistic pathogen Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa uses the trimethylamine-containing (TMAC) compounds: choline, betaine and carnitine as energy sources, osmoprotectants, and macromolecular precursors. The importance of these compounds in P. aeruginosa physiology is highlighted by the presence of multiple TMAC transporters encoded within its genome. The central goal of my study was to identify the roles of these transporters in uptake of TMACs at an osmolality comparable to that of blood and tissue fluids (280mOsm/kg/H2O).;The ABC transporter, CbcXWV was found to be essential for uptake of carnitine and betaine, and important for uptake of choline under physiological osmolality. The core Cbc transporter, composed of the permease CbcW and the ATPase CbcV, could interact with the SBPs: CbcX, BetX and CaiX which bind choline, betaine and carnitine respectively. While CbcX is encoded in the cbcXWV operon, caiX and betX are located at separate regions on the genome. CbcXWV is unusual among members of ABC transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that have distinct specificities for their substrates.;Under physiological osmolality, two Betaine-Choline-Carnitine family transporters, BetT1 and BetT3, were also found to be important for choline uptake. betT1 and betT3 expression was regulated by the repressor BetI and choline, whereas cbcXWV expression was induced by the activator GbdR and betaine. The betaine derived from choline taken up by BetT1 and BetT3 promoted subsequent GbdR-mediated cbcXWV induction. This work represents a systematic approach to unravel the mechanisms of choline uptake in P. aeruginosa, which has the most complex bacterial choline uptake systems characterized to date.;During growth in presence of choline, both lab and clinical Pseudomonas aeruginosa strains synthesize membrane phosphatidylcholine (PC). Mutants incapable of synthesizing PC were found to be indistinguishable from wild-type strains in antibiotic resistance profiles, biofilm formation and virulence mediated by Type III secretion system and hemolytic phospholipase C. A phenotypic microarray was further used to identify culture conditions wherein membrane PC could play a role of in P. aeruginosa. No clear conditions could be identified wherein wild-type and PC-deficient mutants exhibited phenotypic differences.