Kara L. Jones

Document Type

Honors Project

First Advisor

Dr. Stephen Baron

Degree Award Date

Spring 2017

Keywords

Molecular Studies, Microbiological Studies, Polyhydroxybutyrate Depolymerase Gene, Streptomyces sp. 5A, PHB depolymerase synthesis, Phase II glycogen synthesis

Disciplines

Bacteriology | Biology | Microbiology | Molecular Biology

Abstract

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.

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