An Effective Strategy for a Whole-Cell Biosensor Based on Putative Effector Interaction Site of the Regulatory DmpR Protein

Abstract

Introduction and RationaleThe detection of bioavailable phenol is a very important issue in environmental and human hazard assessment. Despite modest developments recently, there is a stern need for development of novel biosensors with high sensitivity for priority phenol pollutants. DmpR (Dimethyl phenol regulatory protein), an NtrC-like regulatory protein for the phenol degradation of Pseudomonas sp. strain CF600, represents an attractive biosensor regimen. Thus, we sought to design a novel biosensor by modifying the phenol detection capacity of DmpR by using mutagenic PCR.MethodsBinding sites of ‘A’ domain of DmpR were predicted by LIGSITE, and molecular docking was performed by using GOLD to identify the regions where phenol may interact with DmpR. Total five point mutations, one single at position 42 (Phe-to-Leu), two double at 140 (Asp-to-Glu) and 143 (Gln-to-Leu), and two double at L113M (Leu-to- Met) and D116A (Asp-to- Ala) were created in DmpR by site-directed mutagenesis to construct the reporter plasmids pRLuc42R, pRLuc140p143R, and pRLuc113p116R, respectively. Luciferase assays were performed to measure the activity of luc gene in the presence of phenol and its derivatives, while RT-PCR was used to check the expression of luc gene in the presence of phenol.ResultsOnly pRLuc42R and pRLuc113p116R showed positive responses to phenolic effectors. The lowest detectable concentration of phenol was 0.5 µM (0.047 mg/L), 0.1 µM for 2, 4-dimethylphenol and 2-nitrophenol, 10 µM for 2, 4, 6-trichlorophenol and 2-chlorophenol, 100 µM for 2, 4-dichlorophenol, 0.01 µM for 4-nitrophenol, and 1 µM for o-cresol. These concentrations were measured by modified luciferase assay within 3 hrs compared to 6–7 hrs in previous studies. Importantly, increased expression of luciferase gene of pRLuc42R was observed by RT-PCR.ConclusionsThe present study offers an effective strategy to design a quick and sensitive biosensor for phenol by constructing recombinant bacteria having DmpR gene.

Catabolism of Phenol and Its Derivatives in Bacteria

“…Five different DmpR mutants active in E. coli cells enabled the detection of phenol, various chlorophenols, and nitrophenols, all of them commonly occurring priority pollutants. The DmpR regulator mutant designed by using a computational prediction was also applied in a highly sensitive phenol biosensor with the luciferase reporter gene (Gupta, Saxena, Saini, Mahmooduzzafar, Kumar, & Kumar, 2012). This whole-cell luminescence-based biosensor was successfully tested for detection of the phenolic compounds in mixtures (synthetic water) and its use was found to represent a simple, sensitive, and reproducible way to assess bioavailability and toxicity of these pollutants in environment.…”

A growth‐ and bioluminescence‐based bioreporter for the in vivo detection of novel biocatalysts

“…Also other advantages have led to an increase in the use of bioreporters. These include high specificity, high enantioselectivity, lower costs, reduced handling, measuring bioavailability instead of actual concentration, no requirement of artificial substrates and the possibilities of online monitoring and signal enhancement (van der Meer et al ., ; Gupta et al ., ; Mahr and Frunzke, ; van Rossum et al ., ).…”

Detection of Organic Compounds with Whole-Cell Bioluminescent Bioassays

“…This new reporter construct extended the range of detectable compounds to include 2-chlorophenol, 2, 4-dichlorophenol, 4-chloro-3-methylphenol, and 2- and 4-nitrophenol. Similarly, Gupta et al [65] performed a more defined DmpR mutation approach and linked gene expression to firefly luciferase within an E. coli host cell to create a bioluminescent bioreporter (pRLuc42R) capable of detecting phenol at a lower limit of 0.50 μM within a 3-h assay time frame, however, its ability to detect phenolic compounds in realworld samples has yet to be tested.…”