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dc.contributor.advisor Raetz, Christian R. H. en_US
dc.contributor.author Ingram, Brian O'Neal en_US
dc.date.accessioned 2010-05-10T19:57:15Z
dc.date.available 2012-05-01T04:30:05Z
dc.date.issued 2010 en_US
dc.identifier.uri http://hdl.handle.net/10161/2393
dc.description Dissertation en_US
dc.description.abstract <p>The lipid A component of lipopolysaccharide (LPS) in the nitrogen-fixing plant endosymbionts <italic>Rhizobium leguminosarum</italic> and <italic>Rhizobium etli</italic> is strikingly different when compared to that of enteric bacteria such as <italic>Escherichia coli</italic>. The <italic>Rhizobium</italic> species produce several unique enzymes that process the lipid A biosynthetic intermediate Kdo<sub>2</sub>-lipid IV<sub>A</sub>. These enzymes include a 1-phosphatase (LpxE), a 4´-phosphatase (LpxF), a 3-O-deacylase (PagL), and a lipid A oxidase (LpxQ). The biological functions and enzymological properties of many of the modification enzymes have remained unconfirmed and/or unknown. The purpose of these studies was to confirm the activities of these enzymes and to explore the functional significance of the resulting lipid A modifications.</p> <p>To confirm the proposed biological functions of the enzymes <italic>in vivo</italic>, homologs of the lipid A phosphatases, LpxE and LpxF, from <italic>Francisella novicida</italic> and the lipid A oxidase LpxQ, were expressed heterologously in combination in <italic>E. coli</italic>. The resulting novel lipid A hybrids were analyzed by thin-layer chromatography (TLC) and electrospray ionization-mass spectrometry (ESI-MS). </p> <p>The lipid A oxidase LpxQ, was characterized further biochemically. Two new purification procedures and a new <italic>in vitro</italic> assay were developed to analyze the properties of the enzyme. Purified LpxQ was shown to be dependent on oxygen and divalent cations for activity. Hydrogen peroxide was found to be a product of lipid A oxidation. A new fluorescence-based assay based on the detection of hydrogen peroxide was developed to monitor oxidation. LpxQ did not co-purifiy with any discernable cofactors, suggesting that it may employ a unique mechanism for the oxidation of lipid A.</p> <p>The biological roles of LpxE and LpxF in plant nodulation were analyzed. Deletion mutants of the two phosphatases were generated in <italic>R. etli</italic>. The mutant strains accumulated the expected structures, confirming the specificity of the enzymes. Single and double phosphatase mutants were able to fix nitrogen <italic>in planta</italic>. Antimicrobial susceptibility testing indicated that dephosphorylation of lipid A increases resistance to cationic antimicrobials.</p> <p>The biological role of the 3-O-deacylase, PagL, was also investigated. The <italic>pagL</italic> gene was identified using systematic homology searches. PagL was shown to be stimulated by calcium. A deletion mutant of the enzyme in <italic>R. etli</italic> was constructed and analyzed. The deletion mutant was found to be viable and unaltered in its ability to fix nitrogen. In conclusion, these studies have confirmed the roles of LpxE, LpxF, PagL, and LpxQ in Rhizobium lipid A biosynthesis and contributed new knowledge regarding the biochemical properties of LpxQ.</p> en_US
dc.format.extent 5718604 bytes
dc.format.mimetype application/pdf
dc.language.iso en_US
dc.subject Chemistry, Biochemistry en_US
dc.subject Biology, Microbiology en_US
dc.title Biochemical Characterization of Lipid A Modification Enzymes From Rhizobium leguminosarum and Rhizobium etli en_US
dc.type Dissertation en_US
dc.department Biochemistry en_US
duke.embargo.months 24 en_US

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