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 Title: Regulation of Bacteroides chondroitin sulfate utilization genes Author(s): Hwa, Vivian Doctoral Committee Chair(s): Salyers, Abigail A. Department / Program: Microbiology Discipline: Microbiology Degree Granting Institution: University of Illinois at Urbana-Champaign Degree: Ph.D. Genre: Dissertation Subject(s): Biology, Genetics Biology, Microbiology Abstract: Regulation of genes is probably important for survival of anaerobic bacteria that constitute the majority of bacteria in the human colon. Until recently, it was not possible to investigate regulation at the molecular level. I have completed the first such study using the chondroitin sulfate utilization (csu) system in Bacteroides thetaiotaomicron as the model system to study regulation.I have shown that expression of two genes encoding two of the degradative enzymes, csuC (chondro-4-sulfatase, 4-Sase) and csuB (chondroitin lyase II, CSaseII) is driven by a chondroitin sulfate-regulated promoter located upstream of both csuC and csuB. Using the promoterless E. coli uidA gene ($\beta$-glucuronidase, GUS) as a reporter gene, the region required for full functioning of the csuCB operon was localized, and was shown to be at least 500 bp in size. This regulatory region, supplied in trans on a multiple copy plasmid, decreased expression of the chromosomal csuB gene as well as the unlinked chromosomal csuA gene (CSaseI) by approximately 40%. In addition, the expression of another unlinked gene in the csu utilization pathway, csuE ($\beta$-glucuronidase, $\beta$-Gase) was derepressed. The DNA segment required for the derepression of csuE was larger than the region necessary for expression. Further evidence that the csuE is regulated differently from other csu genes, came from the finding that heparin (HP), a mucopolysaccharide that is structurally related to CS, induced expression of csuE but not the expression of other csu genes.Results of analysing four transposon generated mutants also indicated that different csu genes were regulated differently. The csu pathway could not be induced in mutant 46-4. However, expression of csuE, but not of the other enzymes, could still be derepressed in 46-4. Although 46-4 could still grow on HP, growth on HP did not lead to detectable $\beta$-Gase activity. This same phenotype was observed in mutant $\Delta$CS4. These results imply that the affect of HP on csuE expression was different from the derepression of csuE.Unlike mutant 46-4, mutants 46-1 and $\Delta$CS3 grew on HA but were unable to grow on HP. This strongly suggested that there was some connection between the CS and HP breakdown pathways. Analysis of 46-1 showed that the chondro-6-sulfatase (6-Sase, csuD) was missing. In addition, two-dimensional protein gel profiles revealed a total of four CS-associated proteins affected by the mutation: three soluble proteins, of which one was presumably the 6-Sase, were missing, and one membrane protein was now expressed constitutively. These results, in conjunction with the fact that 46-1 cannot grow on HP, suggested 46-1 could be a regulatory mutant. If so, the four CS-associated proteins affected by the transposon insertion in 46-1 are regulated differently from the other 27 CS-associated proteins detected in the two-dimensional gels. Mutant $\Delta$CS3 had the same phenotype as 46-1. Issue Date: 1991 Type: Text Language: English URI: http://hdl.handle.net/2142/22421 Rights Information: Copyright 1991 Hwa, Vivian Date Available in IDEALS: 2011-05-07 Identifier in Online Catalog: AAI9136622 OCLC Identifier: (UMI)AAI9136622
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