Molecular and Microbiological Studies on Regulation of the Polyhydroxybutyrate Depolymerase Gene of Streptomyces sp. 5A
Dr. Stephen Baron
Degree Award Date
Molecular Studies, Microbiological Studies, Polyhydroxybutyrate Depolymerase Gene, Streptomyces sp. 5A, PHB depolymerase synthesis, Phase II glycogen synthesis
Bacteriology | Biology | Microbiology | Molecular Biology
Streptomycetes are filamentous bacteria which sequentially form substrate mycelia, aerial mycelia and spores during growth. Polyhydroxybutyrate (PHB) depolymerase in Streptomyces sp. 5A is synthesized during growth on PHB but not glucose. Previous research suggests that PHB depolymerase synthesis may initially be triggered by developmental cues and/or nutrient limitation, and not necessarily by the presence of PHB. To determine whether this occurred at the transcriptional level, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify a 540-base pair segment of PHB depolymerase mRNA from glucose-grown and PHB-grown cells. RNA was extracted from cells using a commercial kit, treated with DNAse I, and negative controls were performed to detect genomic DNA (gDNA) contamination. Despite extensive efforts, gDNA contamination was still present in all samples, thus, mRNA could not reliably be quantified. Therefore, a microbiological approach was attempted. Since streptomycetes produce glycogen reserves during nutrient starvation and morphological transitions, glycogen can serve as a proxy indicator of both conditions. To further investigate regulation of PHB depolymerase synthesis, cell suspensions were incubated with PHB. Samples at time intervals were centrifuged, and supernatants assayed for PHB depolymerase activity. Cell pellets were assayed for glycogen content and cellular morphology. Both intracellular glycogen content and PHB depolymerase activity peaked between 24-48 hours, when pre-spore compartments became clearly visible. The glycogen probably represents Phase II deposits, which are subsequently degraded to support spore formation. Results suggest that PHB depolymerase synthesis is somehow linked to Phase II glycogen synthesis and early sporulation events.
Jones, Kara L., "Molecular and Microbiological Studies on Regulation of the Polyhydroxybutyrate Depolymerase Gene of Streptomyces sp. 5A" (2017). Honors Projects. 32.