Application of novel genetic methods for studies of unsaturated fatty acid synthesis in Pseudomonas aeruginosa.

摘要

In this dissertation, new genetic tools were developed that accelerate post-genomic studies. The tools developed here include a broad-host-range transposon Tn7-based site-specific gene integration system, a rapid and efficient bacterial transformation method, and a rapid gene replacement method. Mini-Tn7 vectors were developed for single-copy genetic complementation, gene and protein fusion analyses, reporter gene tagging and gene expression studies. A rapid transformation method developed in support of the mini-Tn7 system is generally applicable for delivery of genetic elements such as replicative and non-replicative plasmids, suicide delivery vectors, gene replacement vectors and chromosomal fragments containing selection markers. To facilitate high-throughput mutant construction, a combination of rapid transformation and Gateway recombinational cloning was implemented to greatly improve and accelerate a relatively tedious traditional gene replacement procedure. These tools were then utilized for studies on the mechanism and regulation of unsaturated fatty acid (UFA) biosynthesis in Pseudomonas aeruginosa.; In this study, we showed that UFA synthesis in P. aeruginosa occurs by two different pathways depending on the oxygen availability: (1) anaerobic enzymes encoded by the fabA and fabB of the type II fatty acid biosynthetic pathways; and (2) by aerobic desaturation of saturated fatty acids by DesA and DesB desaturases. DesA introduces a double bond into a fatty acyl group of membrane phospholipids, thus allowing cells to grow in the absence of FabA during aerobic growth. DesB desaturates exogenous saturated fatty acyl-CoAs with the aid of the DesC under the DesT repressor. Although two pathways exist for UFA production and the FabAB pathway is not required for aerobic growth, the FabA and FabB proteins are indispensable for anaerobic growth because of the oxygen-dependency of the enzymes of the aerobic pathway. This study demonstrated that transcriptional regulation of fabAB operon expression is complex, involving several promoters, a conserved DNA regulatory element and probably several unidentified regulatory factors. The inability to identify a specific DNA binding protein by genetic and biochemical methods suggests that fabAB operon expression may not simply be conducted by protein-DNA interactions, but rather by a more complex regulatory mechanism(s).