The normal human colon contains a dense bacterial population, which undoubtedly has many effects on the human body. One important activity of the colonic bacteria is the fermentation of dietary and host-derived polysaccharides. Fermentation products are absorbed by mucosal cells and used as a source of carbon and energy. The colonic fermentation is of interest to nutritionists because of its caloric significance. It is of interest to clinicians because imbalances in the composition of the microflora can lead to development of intestinal disease. Bacteroides is one of the numerically predominant genera of human colonic bacteria, and is known to be a versatile utilizer of polysaccharides. We are using the starch utilization system of Bacteroides thetaiotaomicron as a model system for learning more about how colonic bacteria utilize polysaccharides. Previous work has suggested a model for starch utilization. Starch is first bound to proteins on the bacterial surface then translocated across the outer membrane, possibly being clipped in the process. In the periplasm, starch is further degraded. The main goal of the proposed work is to test this model. We have identified 5 outer membrane proteins that appear to be involved in starch utilization. We will determine whether these proteins are surface-exposed and whether they form a complex. We will use a mixture of genetic and biochemical approaches to determine the roles of each of these proteins in starch utilization. We have also identified a periplasmic enzyme, whose role and interactions with the outer membrane proteins will be determined. Finally, we will characterize a new locus that appears to contain starch utilization genes and we will continue to search for other starch utilization genes. A secondary project has arisen as a result of the discovery by another group of a specific effect on mucosal cells that is associated with colonization by B. thetaiotaomicron. This is the first molecular change in mucosal cells to be attributed to a single bacterium, and is a major breakthrough in bacterium-host interactions. We will continue to provide mutants for this group to test. We are interested in testing the hypothesis that mucin degradation may be the trigger for the colonization-associated change in mucosal cells.
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