Mucin was isolated for incorporation in bacteriological media by reduction and proteolysis of mucous gel from porcine small intestine. Mucin prepared in this study contained (by weight) 37.2% protein, 58.7% carbohydrate (4.6% fucose, 9.4% mannose, 10.7% sialic acid, 13.5% galactose, and 20.5% hexosamine), and 3.7% ester sulfate. Fractionation of mucin on Sepharose CL-4B yielded one peak which eluted at the void volume. However, sodium dodecyl sulfate gel electrophoretic patterns contained 9 polypeptide bands of which 6 stained with periodic acid—Schiff reagent. Proline, serine, and threonine residues accounted for 26% (by weight) of the total protein in the preparation. Half-cysteine residues made up another 1%. Intrinsic viscosity of mucin prepared by reduction and proteolysis was 135 ml/g.
Mucin was incorporated into a minimal bacteriological medium as the sole-source of carbon and nitrogen. Enterotoxigenic and non-enterotoxigenic Escherichia coli grew equally well in mucin medium at levels comparable to growth in 3 mM glucose medium. Growth did not appear to be limited by availability of metabolizable substrates. Spent mucin medium supported growth in comparable numbers. E. coli P-155 produced heat-stable and heat-labile enterotoxins during growth in both mucin medium and fresh mucosal scrapings medium. E. coli utilized total hexose and protein in mucin medium at comparable levels (6 to 10%). Increases in reducing end groups (0.28 μ moles/ml) and free amino sugar end groups (0.04 μmoles/ml) during growth were detected. E. coli used approximately 15% of the total carbohydrate in mucin medium including 36% of the galactose, 15% of the fucose, and 27% of the mannose. Utilization of mucin by QL ggli produced minor changes in gel filtration patterns on Sepharose CL-4B.
Twelve strains of E. coli were examined for glycosidase activity during growth on mucin. All twelve produced a cell-bound and an extracellular α-fucosidase although the majority of activity was cell-bound. Although α-fucosidase was a constitutive enzyme of E. coli P-155, maximum activity was observed during exponential growth in mucin medium. Eleven strains produced cell-bound α-galactosidase. No extracellular activity of this enzyme was detected. Maximum levels of induced α-galactosidase activity were obtained in late exponential to early stationary growth of E. coli. E. coli ATCC 23723, a mutant of E. coli K12 lacking the galactoside permease gene, did not produce α-galactosidase activity during growth on mucin. No α-mannosidase activity was detected using nitrophenylmannoside as substrate.
Porcine small intestinal mucin was a positive chemoattractant for E. coli in capillary assays. Optimal chemotactic response by E. coli P-155 in capillary experiments was obtained at a mucin concentration of 1 mg dry wt/ml at a pH of 7.0. Spent mucin was still a positive chemoattractant for E. coli P-155 and 123 despite losing 15% of the total mucin carbohydrate. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/53860 |
| Date | January 1985 |
| Creators | Aimutis, William R. |
| Contributors | Food Science and Technology, Eigel, W.N., Keenan, Thomas W., Moore, W.E.C., Kornegay, Ervin T., Pierson, Merle D. |
| Publisher | Virginia Polytechnic Institute and State University |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation, Text |
| Format | x, 129 leaves, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | OCLC# 13193666 |
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