Document Type
Honors Project
First Advisor
Stephen F. Baron
Degree Award Date
Spring 5-2-2020
Keywords
Streptomyces, PHA depolymerase, N-acetylglucosamine (NAG), Colorimetric assay, Polyhydroxybutyrate (PHB), peptidoglycan hydrolase, autolysis, ammonium sulfate precipitation, native polyacrylamide gel electrophoresis, induction
Disciplines
Environmental Microbiology and Microbial Ecology
Abstract
Streptomyces sp. SFB5A is a filamentous, Gram-positive bacterium that makes a polyhydroxybutyrate (PHB) depolymerase to degrade PHB for energy usage. Its life cycle begins as spores germinate to form vegetative mycelia. Starvation prompts formation of aerial mycelia and ultimately spores after 5-7 days. During starvation, Streptomyces may use peptidoglycan hydrolases to degrade its own cell walls, releasing N-Acetylglucosamine (NAG). PHA depolymerase activity is detected during growth of strain SFB5A on NAG as the sole carbon source, suggesting that NAG induces PHA depolymerase synthesis. To test this hypothesis, strain SFB5A was grown for 4 days on PHB, and a colorimetric assay for NAG was performed on culture supernatants from samples taken every 12 h. NAG concentration peaked at about 24-36 h, when cells might be expected to autolyze. However, absorbance readings were only slightly above the hypothetical limit of detection, most likely due to very low NAG concentrations. Increasing the final concentration of borate buffer in the assay increased the assay sensitivity 2-fold, and suggested that culture supernatants could be concentrated at least 2-fold to increase detection. To detect peptidoglycan hydrolase secreted during autolysis, samples of culture supernatants were spotted onto agarose gels containing heat-killed cells. However, no clearing zones appeared after 6 h incubation. Instead, cells grew in the spotted areas after 24 h, probably because of residual cells in the supernatants. Concentrating supernatants by freeze drying might increase sensitivity and filtration of supernatants would remove residual cells.
Recommended Citation
Todd, Morgan. "Possible Role of N-Acetylglucosamine for Induction of Polyhydroxybutyrate Depolymerase in Streptomyces sp. SFB5A" Senior Honors Project, Bridgewater College, 2020.
Recommended Citation
Todd, Morgan. "Possible Role of N-Acetylglucosamine for Induction of Polyhydroxybutyrate Depolymerase in Streptomyces sp. SFB5A" Senior Honors Project, Bridgewater College, 2020.
Force Open Access
1