Functional Characterization of Proteins Involved in Starch Utilization by Bacteroides Thetaiotaomicron

131 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2000.Bacteroides spp. are gram-negative obligate anaerobes, constitute 25--30% of the human colonic microflora, and are proposed to be the major polysaccharide fermenters in the colon. We believe that Bacteroides degrades these large substrates by binding them on the cell surface and hydrolyzing them to smaller fragments using cell-associated enzymes. This is in contrast to many other systems which use extracellular enzymes to hydrolyze the polysaccharide to digestible fragments. The Salyers lab has used starch utilization by B. thetaiotaomicron as a model to test this hypothesis. In this study, I have shown that B. thetaiotaomicron has an essential surface-exposed outer membrane-associated protein, SusG, which has starch-degrading activity. This protein may be hydrolyzing the starch to smaller fragments on the cell surface. The rest of this study focuses on the non-enzymatic OMPs, SusC-F, and discerning what role they played in the binding of starch to the cell surface. Previous studies had shown that binding mediated by outer membrane proteins was necessary for starch utilization. I was able to show that two proteins, SusC and SusD, were both needed for significant starch binding. It appears that another protein, SusE, enhanced binding, but that SusF played no detectable role in binding. As shown by protease accessibility studies, SusC, -E and -F are surface-exposed whereas SusD's exposure could not be determined. I also showed that the essential binding proteins, SusC and SusD, interact in the outer membrane. In addition, these proteins along with SusE, interact in vitro in order to bind starch. These results constitute the first direct evidence that these proteins form a multiprotein complex in the outer membrane that binds starch. The results of these studies have revealed the basic functions of these proteins as well as provided clues as to their assembly in the outer membrane. Hopefully, this study will help solidify the foundation to propel future study on the exact mechanism by which Bacteroides utilizes polysaccharides.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD