Abstract


Appuhamillage, Gayan A., Resistance of Escherichia coli towards toxic metalloidal oxyanions and gsha, gshb genes in their glutathione biosynthetic pathway, and an effort to produce an Escherichia coli strain with a higher glutathione content. Master of Science (Chemistry), August, 2013, Sam Houston State University, Huntsville, Texas, 84pp. (pdf version of this thesis)

Purpose

The purpose of this research was: (1) to determine the reducing power of two E. coli strains, AG1/pCA24NgshA and AG1/pCA24NgshB, for the toxic oxyanions SeO32-, SeO42-, and TeO32- in comparison to their wild type E. coli strain AG1 in terms of their relative ability to produce intracellular glutathione (GSH); and (2) to produce an E. coli strain having a better reducing power over the above strains, in terms of the production of intracellular GSH for the bioremediation of toxic metalloidal oxyanions.

Methods

In part (1), using bacterial precultures prepared in the absence and the presence of isopropyl-β-D-1-thiogalactopyranoside (IPTG), all of the following experimental methods were performed. Minimum inhibitory concentration measurements were carried out using 96-microwell plates by the OD600 method and the resazurin dye method. The specific growth rates were obtained by measuring the slope of the exponential phase of bacterial growth curves. These experiments were carried out to investigate the relative toxicity of each of the oxyanions towards each of the E. coli strains used. Intracellular GSH levels were measured using a colorimetric analysis with 5,5’-dithiobis(2-nitrobenzoic acid). Intracellular protein contents were measured with the standard Bradford assay method in order to estimate intracellular GSH levels present per mass of protein.

In part (2), polymerase chain reactions, restriction enzyme digestions, gene ligations, DNA purification, and gel electrophoresis techniques were used in an effort to achieve the goal.

Findings

In part (1), the relative toxicity of the oxyanions for the three E. coli strainsincreased in the order Na2SeO4 < Na2SeO3 < Na2TeO3. Specific growth rates confirmed that the presence of any of these toxicants was unfavorable for the normal growth of these E. coli strains.

The presence of higher GSH levels in AG1/pCA24NgshA and AG1/pCA24NgshB relative to AG1, caused the higher resistance of these two strains towards Na2SeO3 and Na2SeO4 and proved the involvement of gshA and gshB genes in the glutathione biosynthetic pathway. Higher IPTG levels caused a decrease of intracellular GSH.

In part (2), the E. coli strain BW 25113 could be used successfully as the source of the gshA gene for its amplification.

Keywords: Reducing power, Intracellular glutathione, IPTG, Minimum inhibitory, Gene ligations, Gel electrophoresis, Plasmid pSB1A2.

 

Thomas G. Chasteen
Thesis Director

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