Mutant HbpR transcription activator isolation for 2-chlorobiphenyl via green fluorescent protein-based flow cytometry and cell sorting.

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Serval ID
serval:BIB_808BE6B1F75F
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Mutant HbpR transcription activator isolation for 2-chlorobiphenyl via green fluorescent protein-based flow cytometry and cell sorting.
Journal
Microbial Biotechnology
Author(s)
Beggah S., Vogne C., Zenaro E., Van Der Meer J.R.
ISSN
1751-7915 (Electronic)
ISSN-L
1751-7915
Publication state
Published
Issued date
2008
Volume
1
Number
1
Pages
68-78
Language
english
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't
Publication Status: ppublish
Abstract
Mutants were produced in the A-domain of HbpR, a protein belonging to the XylR family of σ(54)-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.
Keywords
Bacterial Proteins/chemistry, Bacterial Proteins/genetics, Biphenyl Compounds/metabolism, Escherichia coli/chemistry, Escherichia coli/cytology, Flow Cytometry, Gene Expression Regulation, Bacterial, Green Fluorescent Proteins/genetics, Green Fluorescent Proteins/metabolism, Mutation, Protein Binding, Protein Structure, Tertiary, Trans-Activators/chemistry, Trans-Activators/genetics
Pubmed
Web of science
Create date
01/02/2008 13:04
Last modification date
20/08/2019 14:41
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