Mutant HbpR transcription activator isolation for 2-chlorobiphenyl via green fluorescent protein-based flow cytometry and cell sorting
| العنوان: | Mutant HbpR transcription activator isolation for 2-chlorobiphenyl via green fluorescent protein-based flow cytometry and cell sorting |
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| المؤلفون: | Beggah, Siham, Vogne, Christelle, Zenaro, Elena, Van Der Meer, Jan Roelof |
| المصدر: | Microbial Biotechnology, vol. 1, no. 1, pp. 68-78 Microbial Biotechnology |
| سنة النشر: | 2008 |
| مصطلحات موضوعية: | transcription activator, Biphenyl Compounds, Green Fluorescent Proteins, Gene Expression Regulation, Bacterial, HbpR, green fluorescent protein-based flow cytometry, Flow Cytometry, Protein Structure, Tertiary, Bacterial Proteins, Mutation, Escherichia coli, Trans-Activators, Bacterial Proteins/chemistry, Bacterial Proteins/genetics, Biphenyl Compounds/metabolism, Escherichia coli/chemistry, Escherichia coli/cytology, Green Fluorescent Proteins/genetics, Green Fluorescent Proteins/metabolism, Protein Binding, Trans-Activators/chemistry, Trans-Activators/genetics, Research Articles |
| الوصف: | Summary Mutants were produced in the A‐domain of HbpR, a protein belonging to the XylR family of σ54‐dependent transcription activators, with the purpose of changing its effector recognition specificity from 2‐hydroxybiphenyl (2‐HBP, the cognate effector) to 2‐chlorobiphenyl (2‐CBP). Mutations were introduced in the hbpR gene part for the A‐domain via error‐prone polymerase chain reaction, and assembled on a gene circuitry plasmid in Escherichia coli, permitting HbpR‐dependent induction of the enhanced green fluorescent protein (egfp). Cells with mutant HbpR proteins responsive to 2‐CBP were enriched and separated in a flow cytometry‐assisted cell‐sorting procedure. Some 70 mutants were isolated and the A‐domain mutations mapped. One of these had acquired true 2‐CBP recognition but reacted hypersensitively to 2‐HBP (20‐fold more than the wild type), whereas others had reduced sensitivity to 2‐HBP but a gain of 2‐CBP recognition. Sequencing showed that most mutants carried double or triple mutations in the A‐domain gene part, and were not located in previously recognized conserved residues within the XylR family members. Further selection from a new mutant pool prepared of the hypersensitive mutant did not result in increased 2‐CBP or reduced 2‐HBP recognition. Our data thus demonstrate that a one‐step in vitro‘evolutionary’ adaptation of the HbpR protein can result in both enhancement and reduction of the native effector recognition. |
| وصف الملف: | application/pdf |
| اللغة: | English |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=pmid_dedup__::2f47a552516a53f2bcc096608ad1fb15 https://hdl.handle.net/11562/335319 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.pmid.dedup....2f47a552516a53f2bcc096608ad1fb15 |
| قاعدة البيانات: | OpenAIRE |
| الوصف غير متاح. |