Site-Directed Mutagenesis of the tutH Gene of Thauera Aromatica Strain T1 and Its Potential for Environmental Remediation of Toluene
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Toluene is a major component of gasoline and is a widely used industrial solvent. It contaminates surface and groundwater and poses a menacing threat to human health. Toluene is not a carcinogen; however, in mixtures it enhances the effect of carcinogens.
This research uses a molecular approach to study toluene biodegradation by the facultative, denitrifier Thauera aromatic strain T1 (T.aromatica strain T1). This strain is able to degrade toluene into non aromatic compounds under anaerobic conditions and use it as a sole carbon and energy source. Previous work identified the tutE tutFDGH gene cluster as essential for the first step of anaerobic toluene biodegradation.
The aim of this research is to determine the role of selected amino acids in the function of the TutH protein in toluene biodegradation by T.aromatica strain T1. This work determined if changes can be made to certain amino acids in the TutH protein without disrupting protein function. We identified amino acids of interest by computer analysis and used site-directed mutagenesis to generate the desired changes. Based on computer analysis, I formed a hypothesis that changing the amino acid leucine at position 266 into valine , changing serine at position 5 into theronine, or changing tyrosine at position 283 into phenyalanine will not disrupt the TutH protein function.
We performed site-directed mutagenesis on TutH and determined that two of these altered proteins (L266V and S5T) failed to complement a strain unable to produce TutH while altered protein containing Y283F succeeded in complementing a strain unable to produce TutH.
It is important to identify tolerated amino acids because researchers can use them to change the range of substrates. This research will further the understanding of the toluene metabolism in the environment and may help in the remediation of contaminated sites. Also, this research will contribute to the development of a more efficient toluene bio-degradation pathway. Ideally, this research could help in the conversion of toluene into a useful material that is safe for the environment and for public health.
Document number: ohiou1258525960
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1258525960
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