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Identification of stress-responsive transcription factors with protein-bound Escherichia coli genomic DNA libraries

Bacteria promoters along with operators are crucial elements in the control of gene expression in microbes in response to environmental stress changes. A genome-wide promoter DNA regulatory library is in demand to be developed for a microbe reporter method to monitor the existence of any given envir...

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Autores principales: Li, Xianqiang, Jiang, Xin, Xu, Meiying, Fang, Yun, Wang, Yan, Sun, Guoping, Guo, Jun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Berlin Heidelberg 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606416/
https://www.ncbi.nlm.nih.gov/pubmed/33140118
http://dx.doi.org/10.1186/s13568-020-01133-0
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author Li, Xianqiang
Jiang, Xin
Xu, Meiying
Fang, Yun
Wang, Yan
Sun, Guoping
Guo, Jun
author_facet Li, Xianqiang
Jiang, Xin
Xu, Meiying
Fang, Yun
Wang, Yan
Sun, Guoping
Guo, Jun
author_sort Li, Xianqiang
collection PubMed
description Bacteria promoters along with operators are crucial elements in the control of gene expression in microbes in response to environmental stress changes. A genome-wide promoter DNA regulatory library is in demand to be developed for a microbe reporter method to monitor the existence of any given environmental stress substance. In this study, we utilized Escherichia coli (E. coli) as a model system for the preparation of both cell lysates and genomic DNA fragments. Through enriching protein-bound DNA fragments to construct luciferase reporter libraries, we found that, of 280 clones collected and sequenced, 131 clones contained either the promoter-35 and -10 conservative sequences and/or an operator transcription factor binding sites (TFBS) region. To demonstrate the functionality of the identified clones, five of 131 clones containing LexA binding sequence have been demonstrated to be induced in response to mitomycin C treatment. To evaluate our libraries as a functional screening library, 80 randomly picked up clones were cultured and treated with and without MMC, where two clones were shown to have greater than twofold induction. In addition, two arsenite-responsive clones were identified from 90 clones, one having the well-known ArsR and another having the osmotically inducible lipoprotein (OsmE1). The newly discovered osmE1 has been quantitatively validated to be induced by arsenite treatment with real-time PCR in a dose response and time course manner. This enriching protein-bound DNA luciferase reporter libraries and functional screening facilitate the identification of stress-responsive transcriptional factors in microbes. We developed functional libraries containing E. coli genomic-wide protein-bound DNA as enhancers/operators to regulate downstream luciferase in response to stress.
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spelling pubmed-76064162020-11-04 Identification of stress-responsive transcription factors with protein-bound Escherichia coli genomic DNA libraries Li, Xianqiang Jiang, Xin Xu, Meiying Fang, Yun Wang, Yan Sun, Guoping Guo, Jun AMB Express Original Article Bacteria promoters along with operators are crucial elements in the control of gene expression in microbes in response to environmental stress changes. A genome-wide promoter DNA regulatory library is in demand to be developed for a microbe reporter method to monitor the existence of any given environmental stress substance. In this study, we utilized Escherichia coli (E. coli) as a model system for the preparation of both cell lysates and genomic DNA fragments. Through enriching protein-bound DNA fragments to construct luciferase reporter libraries, we found that, of 280 clones collected and sequenced, 131 clones contained either the promoter-35 and -10 conservative sequences and/or an operator transcription factor binding sites (TFBS) region. To demonstrate the functionality of the identified clones, five of 131 clones containing LexA binding sequence have been demonstrated to be induced in response to mitomycin C treatment. To evaluate our libraries as a functional screening library, 80 randomly picked up clones were cultured and treated with and without MMC, where two clones were shown to have greater than twofold induction. In addition, two arsenite-responsive clones were identified from 90 clones, one having the well-known ArsR and another having the osmotically inducible lipoprotein (OsmE1). The newly discovered osmE1 has been quantitatively validated to be induced by arsenite treatment with real-time PCR in a dose response and time course manner. This enriching protein-bound DNA luciferase reporter libraries and functional screening facilitate the identification of stress-responsive transcriptional factors in microbes. We developed functional libraries containing E. coli genomic-wide protein-bound DNA as enhancers/operators to regulate downstream luciferase in response to stress. Springer Berlin Heidelberg 2020-11-02 /pmc/articles/PMC7606416/ /pubmed/33140118 http://dx.doi.org/10.1186/s13568-020-01133-0 Text en © The Author(s) 2020 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Original Article
Li, Xianqiang
Jiang, Xin
Xu, Meiying
Fang, Yun
Wang, Yan
Sun, Guoping
Guo, Jun
Identification of stress-responsive transcription factors with protein-bound Escherichia coli genomic DNA libraries
title Identification of stress-responsive transcription factors with protein-bound Escherichia coli genomic DNA libraries
title_full Identification of stress-responsive transcription factors with protein-bound Escherichia coli genomic DNA libraries
title_fullStr Identification of stress-responsive transcription factors with protein-bound Escherichia coli genomic DNA libraries
title_full_unstemmed Identification of stress-responsive transcription factors with protein-bound Escherichia coli genomic DNA libraries
title_short Identification of stress-responsive transcription factors with protein-bound Escherichia coli genomic DNA libraries
title_sort identification of stress-responsive transcription factors with protein-bound escherichia coli genomic dna libraries
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606416/
https://www.ncbi.nlm.nih.gov/pubmed/33140118
http://dx.doi.org/10.1186/s13568-020-01133-0
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