Complete Title of Thesis:
"Investigation of lipid chemotaxis by Pseudomonas aeruginosa"
Prepared under the direction of Michael L. Vasil, Ph.D.
Our focus was on investigating the signal as well as the recognition machinery that governs lipid chemotaxis in Pseudomonas aeruginosa. Some prokaryotes, including P. aeruginosa, have the ability to swim in aqueous environments using flagella and twitch across solid surfaces by extension and retraction of a type four pili. Microorganisms will utilize these mechanisms to direct their movement towards molecules of interest (an attractant) or away from one that is harmful in a behavior termed chemotaxis. The mechanisms associated with flagellar-mediated chemotaxis have been extensively studied, but the processes governing twitching-mediated chemotaxis have not yet been well characterized.
Previously, it was determined that P. aeruginosa twitches directionally up a gradient of the phospholipids phosphatidylethanolamine (PE) and phosphatidylcholine, and a specific extracellular phospholipase C, PlcB, is required for this complex behavior. In this study, we found that PE is metabolized into long chain fatty acids (LCFAs), and that the bacteria ultimately move up a gradient of unsaturated, but not saturated, LCFAs. This directional motility can be referred to as "metabolically-independent chemotaxis", rather than "energy taxis", because mutants that are unable to utilize LCFAs as a substrate for growth can still directionally twitch towards unsaturated LCFAs.
We also sought to identify the machinery required for lipid chemotaxis. Deletions were made within annotated yet uncharacterized genes encoding methyl-accepting chemotaxis proteins; unfortunately, this method did not reveal any individual genes that are required for this process. Therefore, an unbiased transposon screen was initiated to find mutants that are no longer able to directionally twitch towards PE. After screening 2000 mutants, two areas of the genome were selected for further investigation before focusing on the final operon candidate PA5455-PA5459. This previously uncharacterized operon is involved in A-band lipopolysachharide production. Further analysis of this operon shows that it influences social motility, a phenomenon required for chemotaxis. This finding introduces a new link between the bacterial cell surface and directed motility, and thus uncovers a new level of complexity to this phenotype.