Development and Characterization of a Green Fluorescent Protein-Based Bacterial Biosensor for Bioavailable Toluene and Related Compounds

Abstract

A green fluorescent protein-based Pseudomonas fluorescens strain A506 biosensor was constructed and characterized for its potential to measure benzene, toluene, ethylbenzene, and related compounds in aqueous solutions. The biosensor is based on a plasmid carrying the toluene-benzene utilization (tbu) pathway transcriptional activator TbuT from Ralstonia pickettii PKO1 and a transcriptional fusion of its promoter PtbuA1 with a promoterless gfp gene on a broad-host-range promoter probe vector. TbuT was not limiting, since it was constitutively expressed by being fused to the neomycin phosphotransferase (nptII) promoter. The biosensor cells were readily induced, and fluorescence emission after induction periods of 3 h correlated well with toluene, benzene, ethylbenzene, and trichloroethylene concentrations. Our experiments using flow cytometry show that intermediate levels of gfp expression in response to toluene reflect uniform induction of cells. As the toluene concentration increases, the level of gfp expression per cell increases until saturation kinetics of the TbuTPtbuA1 system are observed. Each inducer had a unique minimum concentration that was necessary for induction, with K app values that ranged from 3.3 ؎ 1.8 M for toluene to 35.6 ؎ 16.6 M for trichloroethylene (means ؎ standard errors of the means), and maximal fluorescence response. The fluorescence response was specific for alkyl-substituted benzene derivatives and branched alkenes (di-and trichloroethylene, 2-methyl-2-butene). The biosensor responded in an additive fashion to the presence of multiple inducers and was unaffected by the presence of compounds that were not inducers, such as those present in gasoline. Flow cytometry revealed that, in response to toxic concentrations of gasoline, there was a small uninduced population and another larger fully induced population whose levels of fluorescence corresponded to the amount of effectors present in the sample. These results demonstrate the potential for green fluorescent protein-based bacterial biosensors to measure environmental contaminants.

Biosensors for environmental applications: Future development trends

“…Of particular concern for drinking water quality are water-soluble aromatic components (e.g., benzene, toluene, ethylbenzene, and xylenes) of petroleum products. Although many of these contaminants are readily biodegradable, they often persist in the environment [76]. A green fluorescent protein-based Pseudomonas fluorescens strain biosensor was constructed and characterized for its potential to measure benzene, toluene, ethylbenzene, and related compounds in aqueous solutions.…”

“…A green fluorescent protein-based Pseudomonas fluorescens strain biosensor was constructed and characterized for its potential to measure benzene, toluene, ethylbenzene, and related compounds in aqueous solutions. The biosensor is based on a plasmid carrying the toluene-benzene transcriptional activator [76]. Another microbial whole-cell biosensor, using E. coli with the promoter luciferase luxAB gene, was developed for the determination of water-dissolved linear alkanes by luminescence [77].…”

pA506, a Conjugative Plasmid of the Plant Epiphyte Pseudomonas fluorescens A506

“…In addition to fire blight control, the biocontrol agent also reduces the severity of frost damage caused by ice-nucleation-active bacteria (10). Beyond its commercial applications, A506 has been used as a biosensor of the chemical nature of microbial habitats (12)(13)(14)(15) and as a model organism for studies evaluating resource competition (16,17), survival and growth of bacteria on aerial plant surfaces (8,9,18,19), spatial patterns of bacterial cell aggregates on plants (20,21), and the viable but nonculturable condition that can occur in bacteria (22,23).…”

A Novel Fluorescent Protein-Based Biosensor for Gram-Negative Bacteria

“…These are mainly based on an in vitro pyrogen test involving the use of human cells such as leukocyte cell lines, isolated primary blood, and whole blood (4, 20). Recently, genetic engineering of an endotoxin-sensitive Limulus amoebocyte lysate protein, recombinant factor C expressed in a baculovirus system, produced an enzyme with remarkable sensitivity to endotoxin, at 0.001 endotoxin unit (EU)/ml (2).The green fluorescent protein (GFP) has been a popular choice for development of reporter-biosensors to detect various environmentally hazardous compounds (7,11,19).Through computer-aided simulation and rational design, we have recently developed a fluorescent biosensor for lipopolysaccharide and lipid A (the bioactive moiety of lipopolysaccharide) with enhanced green fluorescent protein (EGFP) as a scaffold protein (6). Previously, we have shown (5) that lipopolysaccharide or lipid A can interact strongly with short cationic amphipathic sequences of five alternating basic (B) and hydrophobic (H) residues (BHBHB).…”

“…The green fluorescent protein (GFP) has been a popular choice for development of reporter-biosensors to detect various environmentally hazardous compounds (7,11,19).…”