Outer Membrane Proteins Associated With Utilization of Chondroitin Sulfate by Bacteroides Thetaiotaomicron
Kotarski, Susan Frances
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Permalink
https://hdl.handle.net/2142/71167
Description
Title
Outer Membrane Proteins Associated With Utilization of Chondroitin Sulfate by Bacteroides Thetaiotaomicron
Author(s)
Kotarski, Susan Frances
Issue Date
1984
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Abstract
Bacteroides thetaiotaomicron employs a cell-associated inducible system to degrade chondroitin sulfate (CS), a mucopolysaccharide that may be a natural carbon source for this organism in the colon. The system that V. thetaiotaomicron uses to break down CS is not fully understood. Chondroitin lyase, an enzyme which cleaves CS into disaccharides, is not found on the cell surface and may have a periplasmic location. Presumably some components of the outer membrane (OM) facilitates entry of CS into the cell, but these have not been identified. The approach used to obtain an initial characterization of this OM system was to isolate the OM of B. thetaiotaomicron and then to identify proteins which are associated with CS utilization. Since the OM isolation procedures which have been used for other gram negative bacteria proved ineffective in purifying OM from B. thetaiotaomicron, a method was devised which purified the heretofore uncharacterized OM and inner membrane (IM). Membrane-associated respiratory enzymes and lipopolysaccharide-associated 3-hydroxy fatty acids were identified and used as quantitative markers of the IM and OM, respectively. Yields of IM and OM were about 20%. Each membrane type contained about 10% contamination by the other membrane type. The polypeptides of purified OM from cells grown on CS were compared with polypeptides of cells grown on other polysaccharides and on component sugars of CS. Comparative analysis included: polyacrylamide gel electrophoresis (PAGE) separation of proteins by molecular weight 2-dimensional PAGE separation by molecular weight and charge, 2-dimensional PAGE separation by heat of solubilization and differential staining of proteins containing carbohydrate and lipid moieties. There were five minor polypeptides and six more prominent polypeptides in the OM which appeared to be associated with growth on CS. Immunoblot analysis detected four of these polypeptides. Levels of these four polypeptides were coordinately regulated with the levels of chondroitin lyase. Potential functional roles of the OM in CS utilization were also explored.
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