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Title: A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor

Abstract

A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires access to an expanded repertoire of TFs. Using homology modeling and ligand docking for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 107 variants of PobR, four were active when dosed with pNP, with two mutants showing a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production from hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show the fluorescence of the cells correlated with the catalytic efficiency of the PTE variant expressed in each cell. High selectivity between similar molecules (4HB versus pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo-structures of PobR scaffold with predetermined computational models are other significant results presented in this work.

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); National Institutes of Health (NIH) - National Institute of General Medical Sciences
OSTI Identifier:
1347611
DOE Contract Number:
AC02-06CH11357
Resource Type:
Journal Article
Resource Relation:
Journal Name: Nucleic Acids Research; Journal Volume: 44; Journal Issue: 17
Country of Publication:
United States
Language:
English

Citation Formats

Jha, Ramesh K., Kern, Teresa L., Kim, Youngchang, Tesar, Christine K., Jedrzejczak, Robert, Joachimiak, Andrzej, and Strauss, Charlie E E.. A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor. United States: N. p., 2016. Web. doi:10.1093/nar/gkw687.
Jha, Ramesh K., Kern, Teresa L., Kim, Youngchang, Tesar, Christine K., Jedrzejczak, Robert, Joachimiak, Andrzej, & Strauss, Charlie E E.. A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor. United States. doi:10.1093/nar/gkw687.
Jha, Ramesh K., Kern, Teresa L., Kim, Youngchang, Tesar, Christine K., Jedrzejczak, Robert, Joachimiak, Andrzej, and Strauss, Charlie E E.. Fri . "A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor". United States. doi:10.1093/nar/gkw687.
@article{osti_1347611,
title = {A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor},
author = {Jha, Ramesh K. and Kern, Teresa L. and Kim, Youngchang and Tesar, Christine K. and Jedrzejczak, Robert and Joachimiak, Andrzej and Strauss, Charlie E E.},
abstractNote = {A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires access to an expanded repertoire of TFs. Using homology modeling and ligand docking for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 107 variants of PobR, four were active when dosed with pNP, with two mutants showing a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production from hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show the fluorescence of the cells correlated with the catalytic efficiency of the PTE variant expressed in each cell. High selectivity between similar molecules (4HB versus pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo-structures of PobR scaffold with predetermined computational models are other significant results presented in this work.},
doi = {10.1093/nar/gkw687},
journal = {Nucleic Acids Research},
number = 17,
volume = 44,
place = {United States},
year = {Fri Sep 30 00:00:00 EDT 2016},
month = {Fri Sep 30 00:00:00 EDT 2016}
}