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 10(7) 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.

Figure 1.

Native and targeted effector molecules for PobR transcription factor (TF). (A) 4HB (native); (B) pNP (targeted). At around neutral pH, pNP is expected to be in equilibrium with a deprotonated phenoxide state.

Figure 2.

DoubleMut-IBD homology model and crystal structure. (A) Monomer of DoubleMut-IBD crystal structure (light gray) overlaid on DoubleMut homology model (black) and the template (PDB code: 2IA2, green). (B) Overlay of apo- (light gray) and holo- (magenta) crystal structures of DoubleMut, showing conformational changes in H216 and M241 side chains and rearrangement of loop L1 (residues 132–152). (C) DoubleMut-3HB crystal structure (magenta) overlaid on pre-determined ligand-docked homology models of DoubleMut with 4HB (light gray), 34DHB (black) and pNP (green). Carboxylate and nitro group of ligands showing polar interaction with backbone of S160, and side chains of T161 and N239 are highlighted with dotted lines. Figures were created using PyMOL (Version 0.99, Schrödinger, LLC).

Figure 3.

Engineered specificity switch in PobR TF. (A) Raw green fluorescent protein fluorescence of cells (405 nm excitation and 525 nm emission) expressing different variants of PobR and grown in the presence of pNP. (B) Switched response of PobR variants with 4HB. (C) Contrast ratio [(Induced fluorescence signal)/(Uninduced fluorescence signal)] of cells expressing PobR-wt, DoubleMut or pNPmut1. Error bars represent standard deviation from two sets of experiments performed on different days, sensor plasmid extracted from the cells after first set of experiments and retransformed in fresh competent cells to perform new experiments. (D) Comparison of the background signal (cells grown in the absence of pNP) of the sensor plasmid variants with a mutation in the possible protein dimer interface region (pNPmut1-Int), in the operator region (pNPmut1-O) or both combined (pNPmut1-1). (E) Contrast ratio of different variants of the sensor plasmid. Error bars represent standard deviation from experiments performed in triplicates.

Figure 4.

‘Smart’ microbial cells that hydrolyze paraoxon (PXN) and sense the product pNP. (A) Two plasmid system consisting of an Escherichia coli cell with a ‘sensor plasmid’ and an ‘enzyme plasmid’. Functioning of a smart cell is shown in steps I–VIII. IPTG induced expression of a phosphotriesterase (PTE) enzyme (steps I-III) hydrolyses exogenously supplied PXN substrate (steps IV–V), releasing pNP that activates the engineered TF, pNPmut1-1 (step VI) to express the reporter gene, gfp (step VII). Intracellular green fluorescent protein accumulation results in fluorescence of the cell (step VIII). The figure has been derived from our previous publication (26). (B) Substrate PXN and hydrolysis product pNP. (C) PTE variants (Native, F306E, F306H and F306K) expressed in ‘smart’ cells in the absence of substrate (top panel) and presence of 0.4 mM PXN (bottom panel, showing contrast ratio). Published catalytic efficiency of each PTE variant (30) is displayed in a box. Error bars represent standard deviation from two independent experiments performed on different days and divalent metal concentration between 0.2 and 0.5 mM. (D) Fluorescence histograms of ‘smart’ cells expressing native PTE and grown in the absence and presence of PXN (top panel). Fluorescence histograms of a mix of population with Native % as indicated and F306E, F306H and F306K in equal proportion, grown to an OD (600 nm) of 0.6 and then spiked with IPTG and PXN (bottom panel).