title: Analysis of two chondroitin sulfate utilization mutants of Bacteroides thetaiotaomicron that differ in their abilities to compete with the wild type in the gastrointestinal tracts of germfree mice.
abstract: Previously, we isolated two mutants of Bacteroides thetaiotaomicron that were unable to grow on the mucopolysaccharide chondroitin sulfate (CS). One of these mutants (46-1) was outcompeted by the wild type in the intestinal tracts of germfree mice, whereas the other mutant (46-4) competed equally with the wild type. In the present article, we report a detailed characterization of these two mutants. Assays of enzymes in the CS utilization pathway revealed that 46-1 did not express one of these enzymes, chondro-6-sulfatase. The absence of chondro-6-sulfatase activity in extracts from 46-1 allowed us to detect a previously unknown activity of another enzyme in the CS breakdown pathway, beta-glucuronidase. In addition to hydrolyzing its normal substrate (an unsulfated disaccharide), beta-glucuronidase also hydrolyzed the 6-sulfated disaccharide subunit of CS. Two-dimensional gel analysis of polypeptides produced by 46-1 showed that several proteins other than the 6-sulfatase were either missing or expressed aberrantly. Thus, 46-1 could be a regulatory mutant. Mutant 46-4 was unable to grow on CS, hyaluronic acid, or disaccharides of CS. Thus, expression of the CS pathway enzymes could not be induced. Nonetheless, the growth pattern of 46-4 and some other findings indicate that the structural genes for these enzymes were still intact. The most likely target of mutant 46-4 is a regulatory locus that is required for expression of CS utilization genes. A surprising characteristic of 46-1 was its inability to grow on heparin, a mucopolysaccharide which is structurally similar to CS but is utilized by a different pathway.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Microbiology, University of Illinois, Urbana 61801.
authors.forename: V
authors.initials: V
authors.lastname: Hwa
authors.forename: A A
authors.initials: AA
authors.lastname: Salyers
mesh_terms: D000818:Animals; D001439:Bacteroides / Q000235:genetics / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002809:Chondroitin Sulfates / Q000378:metabolism*; D004064:Digestive System / Q000382:microbiology*; D015180:Electrophoresis, Gel, Two-Dimensional; D005856:Germ-Free Life; D006493:Heparin / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation
chemical_list: D006493:Heparin; D002809:Chondroitin Sulfates