Edward R. Lyons

Degree Name

Master of Arts (MA)

Document Type

Thesis

School

Faculty of Arts and Sciences

Department

Biology

Date of Original Version

12-20-2021

Abstract

Polyhydroxyalkanoic acids (PHA) are bacterial carbon reserves with application as biodegradable biocompatible plastics. PHA synthase, encoded by phaC, creates the PHA polymer. Evidence presented by many researchers has emphasized the role of phasin proteins in PHA accumulation. Phasins have been implicated in PHA granule localization, PHA synthase activation, and possibly chaperone and nucleoid binding functions. The phaC gene from Vibrio species B-18 with and without an upstream gene showing homology to phasin genes (orf2) were placed into runaway replication vectors, which use temperature to control gene expression. The aim of this thesis is to explore earlier observations that lower temperatures and the presence of orf2 enhance PHA accumulation in enteric bacteria, specifically Escherichia coli and Klebsiella aerogenes. This study also sought to determine the correct transcriptional start site for PhaCB18, which has two potential start sites in close proximity. Cells containing the wildtype genes or one of three phaC mutations were subjected to SDS-PAGE analysis for gene expression and the ability to accumulate PHA via microscopy. The findings support the conclusion that the first ATG within phaC is the transcriptional start for PHA synthase. Furthermore, optimal PHA accumulation occurred only in the presence of orf2. However, this enhancing effect was only observed in cells grown at the lower temperatures that do not promote gene overexpression. Vibrio species B-18 was shown to have a lower growth optimum than either of the enteric hosts and this could explain why PHA accumulation occurred more readily in uninduced cells.

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