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|Title:||Escherichia coli long-chain fatty acid uptake: Molecular and biochemical investigation of proteins involved in fatty acid uptake across the cell envelope|
|Advisor:||Gerber, Gerhard E.|
|Abstract:||<p>E. coli can utilize long-chain fatty acids (LCFAs) as its sole carbon and energy source. This requires the coordinated expression of several proteins involved in the uptake and metabolism of LCFAs. The genes encoding these proteins are unlinked but regulated by the global repressor protein FadF, which makes up the fad regulon. We developed a novel method for monitoring LCFA permeation across the OM. Expression of FadL significantly increased labeling of the fusion protein. Treatment of E. coli with the outer membrane-permeabilizing reagent EDTA, also increased the labeling of the fusion protein to an even greater level. The role of Tsp in LCFA uptake has not been well defined. To determine if the periplasmic protease played a direct role in uptake, we investigated various ways in which Tsp might participate. Photoaffinity labeling with the photoreactive fatty acid suggested that Tsp has low affinity for fatty acids and is therefore unlikely to represent a periplasmic fatty acid binding protein. Preliminary evidence had suggested a link between Tsp protease and FadD cleavage, but our data demonstrated that Tsp is not required for FadD proteolysis. We then investigated the possibility that Tsp was mediating its effects on LCFA uptake through FadL. There were no apparent differences in the expression levels or LCFA affinity of endogenous FadL between tsp mutants and wild type E. coli . However, FadL overexpression experiments revealed that tsp mutants overexpressed the OM protein at reduced levels relative to wild type. Purification of FadD, the E. coli fatty acyl-CoA synthetase, is a difficult endeavor since low yields of unstable enzyme samples are typically obtained. Discrepancies in the description of native FadD complicate matters further. To overcome some of these technical difficulties, we developed overexpression systems for FadD and a FadD fusion protein. The FadD fusion protein was purified to high levels and free of degraded enzyme, which was a problem encountered when purifying FadD. We report that FadD enzyme is cleaved by the outer membrane protease OmpT, producing a 43-kDa carboxyl-terminal and a 19-kDa amino-terminal fragment, which remain tightly associated. OmpT-mediated proteolysis of FadD showed that the fatty acyl-CoA synthetase undergoes conformational changes in the presence of specific substrates including oleate, which inhibited cleavage. (Abstract shortened by UMI.)</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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