Genetic Analysis of Chondroitin Sulfate Utilization in Bacteroides Thetaiotaomicron (Cloning, Mutagenesis, Polysaccharide)

105 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1986.The goal of my project was to develop techniques for genetic analysis of Bacteroides thetaiotaomicron, a human colonic bacteria, which could be applied to determining the organization and regulation of the genes involved in the utilization of chondroitin sulfate, a complex polysaccharide which is probably a source of carbon and energy for the bacteria in the human colon. B. thetaiotaomicron produces two inducible chondroitin lyases (I and II) when it is grown on chondroitin sulfate. I have cloned in Escherichia coli the B. thetaiotaomicron gene which encodes for the production of chondroitin lyase II. This gene has been localized on the cloned fragment by (gamma)(delta) mutagenesis to a region of approximately 3.3 kb. To determine if the chondroitin lyase II gene is required for utilization of chondroitin sulfate, an insertional mutation was made within the chondroitin lyase II gene using a portion of the cloned gene in a suicide vector which I had constructed, pE3-1. The resulting strain was unable to produce chondroitin lyase II. However, it still was able to utilize chondroitin sulfate as its sole source of carbon. To determine if an increase in the copy number of the chondroitin lyase II gene would effect the level of chondroitin lyase II in B. thetaiotaomicron, the region required for the active production of chondroitin lyase II in E. coli was cloned into a multicopy shuttle vector, pE5-2, which I had constructed. The resulting plasmid, pEG800, was mobilized into B. thetaiotaomicron from E. coli. The presence of the chondroitin lyase II gene in multiple copies appeared to have no affect on the levels of chondroitin lyase in B. thetaiotaomicron. Moreover, when pEG800 was mobilized into Bacteroides uniformis, a closely related Bacteroides species which does not produce chondroitin lyase, no expression of the chondroitin lyase II gene was observed. Creation of a mutation upstream of the chondroitin lyase II gene in B. thetaiotaomicron showed that the chondroitin lyase II gene is not transcribed off the same promoter in Bacteroides spp. as that used in E. coli, and that the promoter is located at least two kb upstream of the chondroitin lyase II gene in B. thetaiotaomicron.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD