{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/35988"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/35988","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"Effect of Autoregulated TxeR on the Expression of <I>Clostridium difficile</I> Toxins","abstract":"Clostridium difficile is a major nosocomial pathogen responsible for causing pseudomembranous colitis. It is estimated that 25% of antibiotic-associated diarrhea is due to C. difficile. These diseases result from intestinal tissue damage caused by two of the largest known bacterial toxins, A and B. Molecular studies of the C. difficile toxins have identified a 19.6 kb toxigenic element that contains both toxin genes flanked by three small open reading frames (ORFs). The focus of this study is to elucidate the function of the ORF, designated txeR, which is located at the beginning of the toxigenic element. The deduced amino acid sequence of txeR predicts a 22-kDa protein that contains a helix-turn-helix motif characteristic of DNA binding regulatory proteins. To determine if the protein TxeR regulates expression from the toxA, toxB, and txeR promoters, gene fusions were constructed that contained the various promoter regions and a reporter gene. The immunodominant region of toxin A located at the carboxy-terminus, termed the repeating units (ARU), was selected as the reporter gene. Expression studies were performed in Escherichia coli host strains. Levels of ARU expression were measured by enzyme-linked immunosorbent assay using an ARU-specific monoclonal antibody. Expression levels of ARU from the toxin B promoter region with TxeR supplied on the same plasmid (in cis) or on a different plasmid (in trans) were determined. In cis, ARU levels were 50-fold higher than strains without txeR. In trans, expression of ARU from the toxin B promoter region increased over 800-fold. When TxeR was supplied in trans to a toxin A promoter region-ARU fusion, expression levels of ARU increased over 500-fold. To test for autoregulation, TxeR was supplied in trans to the txeR promoter region fused to ARU. The effect was an increase of ARU expression up to 20-fold over background. These results suggest that TxeR is a trans-acting regulator that stimulates expression of the C. difficile toxins and is subjected to autoregulation.","abstract_html":"Clostridium difficile is a major nosocomial pathogen responsible for causing pseudomembranous colitis. It is estimated that 25% of antibiotic-associated diarrhea is due to C. difficile. These diseases result from intestinal tissue damage caused by two of the largest known bacterial toxins, A and B. Molecular studies of the C. difficile toxins have identified a 19.6 kb toxigenic element that contains both toxin genes flanked by three small open reading frames (ORFs). The focus of this study is to elucidate the function of the ORF, designated txeR, which is located at the beginning of the toxigenic element. The deduced amino acid sequence of txeR predicts a 22-kDa protein that contains a helix-turn-helix motif characteristic of DNA binding regulatory proteins. To determine if the protein TxeR regulates expression from the toxA, toxB, and txeR promoters, gene fusions were constructed that contained the various promoter regions and a reporter gene. The immunodominant region of toxin A located at the carboxy-terminus, termed the repeating units (ARU), was selected as the reporter gene. Expression studies were performed in Escherichia coli host strains. Levels of ARU expression were measured by enzyme-linked immunosorbent assay using an ARU-specific monoclonal antibody. Expression levels of ARU from the toxin B promoter region with TxeR supplied on the same plasmid (in cis) or on a different plasmid (in trans) were determined. In cis, ARU levels were 50-fold higher than strains without txeR. In trans, expression of ARU from the toxin B promoter region increased over 800-fold. When TxeR was supplied in trans to a toxin A promoter region-ARU fusion, expression levels of ARU increased over 500-fold. To test for autoregulation, TxeR was supplied in trans to the txeR promoter region fused to ARU. The effect was an increase of ARU expression up to 20-fold over background. These results suggest that TxeR is a trans-acting regulator that stimulates expression of the C. difficile toxins and is subjected to autoregulation.","abstract_has_math":false,"creators":["Barroso, Lisa Ann"],"institution":"Virginia Tech","degree_name":"Master of Science","degree_level":"masters","degree_discipline":"Biology (Microbiology)","degree_department":"Biology (Microbiology)","school":null,"contributors":[],"advisors":[],"committee_chairs":["Wilkins, Tracy D.","Falkinham, Joseph O. III"],"committee_members":["Bates, Robert C."],"year":1999,"date_issued":"1999-12-01","date_published":"1999-12-01","updated_at":"2026-07-22T22:18:42Z","subjects":["Clostridium difficile","autoregulation","expression system","regulatory protein"],"languages":[],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-120699-141047"],"render_values":[{"text":"etd-120699-141047","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/10919/35988","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeechair","label":"Committee Chair","values":["Wilkins, Tracy D.","Falkinham, Joseph O. 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It is estimated that 25% of antibiotic-associated diarrhea is due to C. difficile. These diseases result from intestinal tissue damage caused by two of the largest known bacterial toxins, A and B. Molecular studies of the C. difficile toxins have identified a 19.6 kb toxigenic element that contains both toxin genes flanked by three small open reading frames (ORFs). The focus of this study is to elucidate the function of the ORF, designated txeR, which is located at the beginning of the toxigenic element. The deduced amino acid sequence of txeR predicts a 22-kDa protein that contains a helix-turn-helix motif characteristic of DNA binding regulatory proteins. To determine if the protein TxeR regulates expression from the toxA, toxB, and txeR promoters, gene fusions were constructed that contained the various promoter regions and a reporter gene. The immunodominant region of toxin A located at the carboxy-terminus, termed the repeating units (ARU), was selected as the reporter gene. Expression studies were performed in Escherichia coli host strains. Levels of ARU expression were measured by enzyme-linked immunosorbent assay using an ARU-specific monoclonal antibody. Expression levels of ARU from the toxin B promoter region with TxeR supplied on the same plasmid (in cis) or on a different plasmid (in trans) were determined. In cis, ARU levels were 50-fold higher than strains without txeR. In trans, expression of ARU from the toxin B promoter region increased over 800-fold. When TxeR was supplied in trans to a toxin A promoter region-ARU fusion, expression levels of ARU increased over 500-fold. To test for autoregulation, TxeR was supplied in trans to the txeR promoter region fused to ARU. The effect was an increase of ARU expression up to 20-fold over background. These results suggest that TxeR is a trans-acting regulator that stimulates expression of the C. difficile toxins and is subjected to autoregulation."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Master of Science"]},{"key":"dc:title","label":"Title","values":["Effect of Autoregulated TxeR on the Expression of <I>Clostridium difficile</I> Toxins"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Wilkins, Tracy D.","Falkinham, Joseph O. III"],"dc:contributor.committeemember":["Bates, Robert C."],"dc:contributor.department":["Biology (Microbiology)"],"dc:creator":["Barroso, Lisa Ann"],"dc:date.accessioned":["2014-03-14T20:49:03Z"],"dc:date.available":["2014-03-14T20:49:03Z","2000-07-11"],"dc:date.issued":["1999-12-01"],"dc:description.abstract":["Clostridium difficile is a major nosocomial pathogen responsible for causing pseudomembranous colitis. It is estimated that 25% of antibiotic-associated diarrhea is due to C. difficile. These diseases result from intestinal tissue damage caused by two of the largest known bacterial toxins, A and B. Molecular studies of the C. difficile toxins have identified a 19.6 kb toxigenic element that contains both toxin genes flanked by three small open reading frames (ORFs). The focus of this study is to elucidate the function of the ORF, designated txeR, which is located at the beginning of the toxigenic element. The deduced amino acid sequence of txeR predicts a 22-kDa protein that contains a helix-turn-helix motif characteristic of DNA binding regulatory proteins. To determine if the protein TxeR regulates expression from the toxA, toxB, and txeR promoters, gene fusions were constructed that contained the various promoter regions and a reporter gene. The immunodominant region of toxin A located at the carboxy-terminus, termed the repeating units (ARU), was selected as the reporter gene. Expression studies were performed in Escherichia coli host strains. Levels of ARU expression were measured by enzyme-linked immunosorbent assay using an ARU-specific monoclonal antibody. Expression levels of ARU from the toxin B promoter region with TxeR supplied on the same plasmid (in cis) or on a different plasmid (in trans) were determined. In cis, ARU levels were 50-fold higher than strains without txeR. In trans, expression of ARU from the toxin B promoter region increased over 800-fold. When TxeR was supplied in trans to a toxin A promoter region-ARU fusion, expression levels of ARU increased over 500-fold. To test for autoregulation, TxeR was supplied in trans to the txeR promoter region fused to ARU. The effect was an increase of ARU expression up to 20-fold over background. These results suggest that TxeR is a trans-acting regulator that stimulates expression of the C. difficile toxins and is subjected to autoregulation."],"dc:description.degree":["Master of Science"],"dc:identifier.other":["etd-120699-141047"],"dc:identifier.uri":["http://hdl.handle.net/10919/35988"],"dc:publisher":["Virginia Tech"],"dc:rights":["In Copyright"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:subject":["Clostridium difficile","autoregulation","expression system","regulatory protein"],"dc:title":["Effect of Autoregulated TxeR on the Expression of <I>Clostridium difficile</I> Toxins"],"dc:type":["Thesis"],"thesis:degree_discipline":["Biology (Microbiology)"],"thesis:degree_level":["masters"],"thesis:degree_name":["Master of Science"],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-22T22:18:42Z"}