ANAEROBIC TOLUENE DEGRADATION: GENETIC ANALYSIS OF THE <i>TUTFDGH</i>OPERON OF <i>THAUERA AROMATICA</i>STRAIN T1
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Ohio University / OhioLINK
2007
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ndltd-OhioLink-oai-etd.ohiolink.edu-ohiou11945477262021-08-03T05:45:25Z ANAEROBIC TOLUENE DEGRADATION: GENETIC ANALYSIS OF THE <i>TUTFDGH</i>OPERON OF <i>THAUERA AROMATICA</i>STRAIN T1 Bhandare, Reena anaerobic toluene degradation T.aromatica strain T1 site-directed mutagenesis <p>Toluene is an aromatic hydrocarbon that is widely used in our everyday life. It is a major water-soluble constituent of petroleum and can pollute surface as well as ground waters. The toxic nature of toluene is responsible for causing severe health hazards. The study of toluene degrading bacteria has attracted attention because of their potential to clean up spills. <i>Thauera aromatica</i>strain T1 is one such bacterium capable of degrading toluene under anaerobic conditions.</p> <p>The <i>tutE tutFDGH</i>gene cluster is essential for the first step of anaerobic toluene degradation in <i>T. aromatica</i>strain T1. The <i>tutF</i>, <i>tutD</i>and <i>tutG</i>genes are proposed to code for the three subunits of the enzyme benzylsuccinate synthase, which is involved in the initial step of anaerobic toluene degradation pathway. The <i>tutE</i>gene is proposed to code for the enzyme benzylsuccinate synthase activase. The precise role of the <i>tutH</i>gene in toluene degradation is currently unknown, but it is proposed to have an ATP/GTP binding domain and is assumed to be involved in benzylsuccinate synthase complex formation. This is consistent with its proposed role as a chaperone of “ATPases Associated with a Variety of Cellular Activities” (AAA) class.</p> <p>Work presented here demonstrates that the gene <i>tutH</i>is essential for toluene metabolism. A plasmid carrying an in-frame <i>tutH</i>deletion was unable to produce wild-type TutH protein in a <i>tutG</i>chromosomal deletion background (chromosomal deletion in <i>tutG</i>does not result in production of TutH due to a polar effect on downstream genes). The resultant construct was unable to complement a polar <i>tutG</i>chromosomal mutation, indicating the importance of <i>tutH</i>in toluene degradation. Further, site-directed mutagenesis was used to identify amino acids in TutH that are essential for toluene metabolism. The TutH putative ATP/GTP binding domain was disrupted by changing glycine, lysine and serine at positions 52, 53 and 54 to alanine, arginine and alanine respectively. Additionally, other amino acids which are found to be highly conserved across closely related bacteria were also targeted for mutagenesis. Leucine and asparagine at positions 158 and 159 of TutH were changed to serine and alanine, glycine at position 161 was changed to alanine and arginine and phenylalanine at positions 177 and 178 were changed to alanine and serine, respectively. The resultant constructs were unable to complement a polar <i>tutG</i>chromosomal mutation, indicating that these amino acids are essential for toluene metabolism and might play important functional role. Additionally, using ProteoEnrich™ ATP Binders™ Kit (Novagen), it was demonstrated that the wild-type TutH protein can bind ATP, thereby supporting the possibility that it has a role in complex formation of benzylsuccinate synthase.</p> <p>Furthermore, attempts were made to identify amino acids in TutF and TutG proteins that are essential for toluene metabolism. Using site-directed mutagenesis cysteine and alanine at positions 9 and 10 of TutF were changed to tyrosine and serine respectively and cysteine at position 29 of TutG was changed to serine. The resultant mutant constructs were unable to complement relevant chromosomal deletions, indicating that the substituted amino acids are important for toluene metabolism.</p> 2007 English text Ohio University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1194547726 http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1194547726 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws. |
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NDLTD |
| language |
English |
| sources |
NDLTD |
| topic |
anaerobic toluene degradation T.aromatica strain T1 site-directed mutagenesis |
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anaerobic toluene degradation T.aromatica strain T1 site-directed mutagenesis Bhandare, Reena ANAEROBIC TOLUENE DEGRADATION: GENETIC ANALYSIS OF THE <i>TUTFDGH</i>OPERON OF <i>THAUERA AROMATICA</i>STRAIN T1 |
| author |
Bhandare, Reena |
| author_facet |
Bhandare, Reena |
| author_sort |
Bhandare, Reena |
| title |
ANAEROBIC TOLUENE DEGRADATION: GENETIC ANALYSIS OF THE <i>TUTFDGH</i>OPERON OF <i>THAUERA AROMATICA</i>STRAIN T1 |
| title_short |
ANAEROBIC TOLUENE DEGRADATION: GENETIC ANALYSIS OF THE <i>TUTFDGH</i>OPERON OF <i>THAUERA AROMATICA</i>STRAIN T1 |
| title_full |
ANAEROBIC TOLUENE DEGRADATION: GENETIC ANALYSIS OF THE <i>TUTFDGH</i>OPERON OF <i>THAUERA AROMATICA</i>STRAIN T1 |
| title_fullStr |
ANAEROBIC TOLUENE DEGRADATION: GENETIC ANALYSIS OF THE <i>TUTFDGH</i>OPERON OF <i>THAUERA AROMATICA</i>STRAIN T1 |
| title_full_unstemmed |
ANAEROBIC TOLUENE DEGRADATION: GENETIC ANALYSIS OF THE <i>TUTFDGH</i>OPERON OF <i>THAUERA AROMATICA</i>STRAIN T1 |
| title_sort |
anaerobic toluene degradation: genetic analysis of the <i>tutfdgh</i>operon of <i>thauera aromatica</i>strain t1 |
| publisher |
Ohio University / OhioLINK |
| publishDate |
2007 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1194547726 |
| work_keys_str_mv |
AT bhandarereena anaerobictoluenedegradationgeneticanalysisoftheitutfdghioperonofithaueraaromaticaistraint1 |
| _version_ |
1719424729808371712 |