A cell array biosensor for environmental toxicity analysis

In this study, a cell-based array technology that uses recombinant bioluminescent bacteria to detect and classify environmental toxicity has been implemented to develop two biosensor arrays, i.e., a chip and a plate array. Twenty recombinant bioluminescent bacteria, having different promoters fused with the bacterial lux genes, were immobilized within LB-agar. About 2 microl of the cell-agar mixture was deposited into the wells of either a cell chip or a 384-well plate. The bioluminescence (BL) from the cell arrays was measured with the use of highly sensitive cooled CCD camera that measured the bioluminescent signal from the immobilized cells and then quantified the pixel density using image analysis software. The responses from the cell arrays were characterized using three chemicals that cause either superoxide damage (paraquat), DNA damage (mitomycin C) or protein/membrane damage (salicylic acid). The responses were found to be dependent upon the promoter fused upstream of the lux operon within each strain. Therefore, a sample's toxicity can be analyzed and classified through the changes in the BL expression from each well. Moreover, a time of only 2 h was needed for analysis, making either of these arrays a fast, portable and economical high-throughput biosensor system for detecting environmental toxicities.     

Evaluation of a high throughput toxicity biosensor and comparison with a Daphnia magna bioassay

A high throughput toxicity biosensor has been designed and constructed using recombinant Escherichia coli cells, containing stress specific promoters (recA, fabA, or katG) or constitutive promoters (lac) fused to luciferase genes originating from Vibrio fisheri. These genetically engineered cells were immobilized in 96 well plates. By optimizing cell immobilization conditions and the strains' response specificity to toxic chemicals, bioluminescent outputs decreased or increased dose-dependently upon adding test chemicals. However, to date the toxicity data obtained using this biosensor have not been compared with the results of other toxicity tests. Phenolics were chosen to evaluate the correlation between the LD50 and the EC50 (GC2) or EC120 (DPD2540) of Daphnia magna and E. coli, respectively. Toxicity data obtained from constitutive strains by bioluminescent level decrements were compared with the results from D. magna as a standard. LD50 values were used as parameters of D. magna toxicity and EC50 of EC120 values were used for the immobilized biosensor. In the DPD2540 test, phenolics, membrane damaging toxic chemicals, for testing immobilized stress specific bacterial strains trigger dose-dependant bioluminescence increase within specific concentration. Although the stress specific responsiveness from the strains could not be compared with D. magna's LD50 values, these responses offer additional information, such as upon the mode of toxic action in the sample, in addition to the cellular toxicity results as indicated by the EC50. This novel high throughput toxicity biosensor can be implemented to investigate the toxicity of any other soluble materials, and can be used as a standardization tool for the evaluation of toxicity.     

A bioluminescent sensor for high throughput toxicity classification

A high throughput toxicity monitoring and classification biosensor system has been successfully developed using four immobilized bioluminescent Escherichia coli strains, DPD2511, DPD2540, DPD2794 and TV1061, which have plasmids bearing a fusion of a specific promoter to the luxCDABE operon. The bioluminescence of DPD2511 increases in the presence of oxidative damage, DPD2540 by membrane damage, DPD2794 by DNA damage and TV1061 by protein damage. In the developed biosensor these strains are immobilized in a single 96 well plate using an LB-agar matrix, and are able to detect the toxicities of hydrogen peroxide, phenol and mitomycin C in water samples. As the concentration of each chemical was increased, the bioluminescence levels from the corresponding wells, containing either DPD2511, DPD2540, DPD2794 or TV1061, increased. This increase in bioluminescence followed a dose dependent response to the toxic chemicals within a specific concentration range. In particular, each test requires only 4 h to give clear bioluminescent response signature. Storage of the biosensor at 4 degrees C for 2 weeks caused no change in its dose-dependent response. The fast and easy detection of oxidative, membrane, protein and DNA damaging agents in aqueous environments is possible due to the high throughput capability of this biosensor.     

A whole cell bioluminescent biosensor for the detection of membrane-damaging toxicity

The recombinant bacteria strain DPD2540, containing afabA::luxCDABE fusion, was used to detect the toxicity of various chemicals in this study. Membrane damaging agents such as phenol, ethanol, and cerulenin induced a rapid bioluminescent response from this strain. Other toxic agents, such as DNA-damaging or oxidative-damaging chemicals, showed a delayed bioluminescent response in which the maximum peak appeared over 150min after induction. This strain was also tested for measurement of toxicity in field samples such as wastewater and river water effluents.     

Enhancement in the sensitivity of a gas biosensor by using an advanced immobilization of a recombinant bioluminescent bacterium

A genetically engineered bioluminescent bacterium (lac::luxCDABE) was immobilized to develop a whole cell biosensor for the detection of toxic gaseous chemicals. The toxicity of chemicals can be evaluated through the bioluminescent reaction as it reduces in intensity when the cells experience toxic or lethal conditions. This whole cell biosensor was fabricated, using an immobilization technique utilizing solid agar medium, for the measurement of toxicity through direct contact of the cells with the gas. To enhance the sensitivity of the biosenor, glass beads were used and the thickness of the agar layer was reduced. The bioluminescent response was measured using a fiber optic probe connected between the biosensor kit and a luminometer. As sample gaseous toxic chemicals, BTEX (Benzene, Toluene, Ethylbenzene, and Xylene) gases were selected and their vapors were produced by a gas generation system. The concentrations of the gaseous chemicals injected into the chamber were controlled by the time of exposure and were measured using a portable gas chromatograph (Allstech., USA). Additions of glass beads facilitated gas diffusion through the solid medium, making the biosensor more sensitive. In addition, a thinner matrix layer was more advantageous for the detection of gas toxicity.     

Monitoring and classification of PAH toxicity using an immobilized bioluminescent bacteria

An immobilized recombinant bioluminescent Escherichia coli strain, harboring a lac::luxCDABE fused plasmid, which shows lower bioluminescence levels when cellular metabolism is inhibited, was used to monitor the cellular toxicity of polycyclic aromatic hydrocarbons (PAHs). PAHs, classified as pericondensed (PCPAHs) or catacondensed (CCPAHs) according to their molecular structures, were differentiable according to the response of this biosensor. Only CCPAHs were found to cause cellular toxicity, resulting in a dose-dependent decrease in the bioluminescent output. The induction of cellular toxicity by CCPAHs and PCPAHs was compared with acute toxicity predictions obtained using the quantitative structure-activity relationship (QSAR) model. A good relationship was obtained between the toxicities determined with the bioluminescent response of the immobilized bacterium GC2 and the QSAR model. It was also found that the present study offers a new method of predicting the cellular toxicities of CCPAHs or PCPAHs using this biosensor.     

An oxidative stress-specific bacterial cell array chip for toxicity analysis

An oxidative stress-specific bacterial cell array chip was fabricated and implemented in the analysis of various different chemicals. The chip consisted of twelve toxicity responsive strains that respond specifically to different oxidative toxicities such as the generation of the superoxide radical, except for strain EBMalK, which was included as a negative control. Each bioluminescent strain carried a fusion of a stress gene promoter (sodA, pqi-5, soxR, fumC, soxS, inaA, hmp, malK, katG, zwf, fpr or pgi) to the bacterial lux reporter genes. A total of nine chemicals were selected to exhibit the capabilities of this array when analyzing different oxidative toxicities. Each of the chemicals were categorized according to their structure and their ability to form radicals in vivo: (I) paraquat, an active radical producer, (II) structural analogs of paraquat that produce radicals, (III) chemicals that are distinct from paraquat but still produce radicals and (IV) chemicals having similar structures as paraquat but do not produce radicals. The results found that each strain was responsive to one or more of the compounds tested but, as a definitive factor, the responses from the chip were dependent upon the production of radicals, i.e., the strains were unresponsive to compounds that were similar in structure to paraquat but lacked the ability to generate radicals. The specificity of the strains used in the chip was also demonstrated by their ability to discriminate between the superoxide radical and hydrogen peroxide. Therefore, this cell array chip could be implemented in characterizing and understanding the toxic impacts of newly synthesized chemicals and drugs in terms of toxicity classification and the nature of oxidative damage experienced by cells.     

Fabrication of a bio-MEMS based cell-chip for toxicity monitoring

A bio-MEMS based cell-chip that can detect a specific toxicity was fabricated by patterning and immobilizing bioluminescent bacteria in a microfluidic chip. Since the emitted light intensity of bioluminescent bacteria changed in response to the presence of chemicals, the bacteria were used as the toxicity indicator in this study. A pattern of immobilized cells was successfully generated by photolithography, utilizing a water-soluble and negatively photosensitive polymer, PVA-SbQ (polyvinyl alcohol-styrylpyridinium) as an immobilization material. Using the recombinant Escherichia coli (E. coli) strain, GC2, which is sensitive to general toxicity, the following were investigated for the immobilization: an acceptable dose of long-wavelength UV light, the biocompatibility of the polymer, and the effect of the chip-environment. We found that 10 min of UV light exposure, the toxicity of polymer (SPP-H-13-bio), and the other chip-environment did not inhibit cell metabolism significantly for making a micro-cell-chip. Detection of a specific toxicity was demonstrated by simply immobilizing the bioluminescent bacteria, DK1, which increased bioluminescence in the presence of oxidative damage in the cells. An injection of hydrogen peroxide of 0.88 mM induced 10-fold increase in bioluminescent intensity confirming the capability of the chip for toxicity monitoring.     

Chemical-specific continuous biomonitoring using a recombinant bioluminescent bacterium DNT5 (nagR-nagAa::luxCDABE)

The recombinant bioluminescent bacterium, DNT5, containing a nagR-nagAa::luxCDABE fusion, was tested in a multi-channel continuous monitoring system to evaluate its ability to detect benzoic acid derivatives. Seven chemicals, benzoic acid, salicylic acid, 2,5-dihydroxy benzoic acid, 3,5-dihydroxy benzoic acid, benzene, naphthalene and phenol, were used to characterize the responses of DNT5. This strain responded uniquely to each chemical, and these responses were then evaluated based upon the structures of each chemical. The greatest bioluminescent responses were to salicylic acid and benzoic acid, followed by 2,5-dihydroxy benzoic acid and 3,5-dihydroxy benzoic acid, but DNT5 was unresponsive when exposed to benzene, phenol and naphthalene, suggesting it has a strong preference for benzoic acid derivatives with few or no ring-substituted groups.     

A portable bioluminescence engineered cell-based biosensor for on-site applications

We have developed a portable biosensing device based on genetically engineered bioluminescent (BL) cells. Cells were immobilized on a 4 × 3 multiwell cartridge using a new biocompatible matrix that preserved their vitality. Using a fiber optic taper, the cartridge was placed in direct contact with a cooled CCD sensor to image and quantify the BL signals. Yeast and bacterial cells were engineered to express recognition elements, whose interaction with the analyte led to luciferase expression, via reporter gene technology. Three different biosensors were developed. The first detects androgenic compounds using yeast cells carrying a green-emitting P. pyralis luciferase regulated by the human androgen receptor and a red mutant of the same species as internal vitality control. The second biosensor detects two classes of compounds (androgens and estrogens) using yeast strains engineered to express green-or red-emitting mutant firefly luciferases in response to androgens or estrogens, respectively. The third biosensor detects lactose analogue isopropyl β-d-1-thiogalactopyranoside using two E. coli strains. One strain exploits the lac operon as recognition element for the expression of P. pyralis luciferase. The other strain serves as a vitality control expressing Gaussia princeps luciferase, which requires a different luciferin substrate. The immobilized cells were stable for up to 1 month. The analytes could be detected at nanomolar levels with good precision and accuracy when the specific signal was corrected using the internal vitality control. This portable device can be used for on-site multiplexed bioassays for different compound classes.     

Enhancing the sensitivity of a two-stage continuous toxicity monitoring system through the manipulation of the dilution rate.

Optimization of the dilution rates has been studied to provide an enhanced sensitivity to toxicity by several recombinant bioluminescent Escherichia coli strains, TV1061 (grpE::luxCDABE), DPD2794 (recA::luxCDABE) and DPD2540 (fabA::luxCDABE), in the two-stage continuous toxicity monitoring system. It was found that the sensitivity of both TV1061 and DPD2794 to a pulse injection of phenol and mitomycin C increased with a decrease in the dilution rate. The sensitivity, however, for all the strains to step injections of the toxic chemicals was found to increase with an increase in the dilution rate up to a certain dilution rate and then decreased, mainly due to the rapid washing out of the injected chemicals. The response kinetics of the strains were explained by evaluating the mode of action of the recombinant bioluminescent bacteria to toxicity with the dilution rate, the operating parameter of minibioreactors under consideration in this study.     

Toxicity monitoring of endocrine disrupting chemicals (EDCs) using freeze-dried recombinant bioluminescent bacteria

Five different freeze-dried recombinant bioluminescent bacteria were used for the detection of cellular stresses caused by endocrine disrupting chemicals. These strains were DPD2794 (recA::luxCDABE), which is sensitive to DNA damage, DPD2540 (fabA::luxCDABE), sensitive to cellular membrane damage, DPD2511 (katG::luxCDABE), sensitive to oxidative damage, and TV1061 (grpE::luxCDABE), sensitive to protein damage. GC2, which emits bioluminescence constitutively, was also used in this study. The toxicity of several chemicals was determined on the first four freeze-dried bacteria, while nonspecific cellular stresses were measured using GC2. Damage caused by known endocrine disrupting chemicals, such as nonyl phenol, bisphenol A, and styrene, was detected and classified according to toxicity mode, while others, such as phathalate and DDT, were not detected with the bacteria. These results suggest that endocrine disrupting chemicals are toxic in bacteria, and do not act via an estrogenic effect, and that toxicity monitoring and classification of some endocrine disrupting chemicals may be possible in the field using these freeze-dried recombinant bioluminescent bacteria.     

Saccharomyces cerevisiae BLYAS, a new bioluminescent bioreporter for detection of androgenic compounds

A Saccharomyces cerevisiae strain, capable of autonomous bioluminescence, was engineered to respond to androgenic chemicals. The strain, S. cerevisiae BLYAS, contains the human androgen receptor in the chromosome and was constructed by inserting a series of androgen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 that constitutively expressed luxA and luxB to create pUTK420. Cotransformation of this plasmid with a second plasmid (pUTK404), containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp), yielded a bioluminescent bioreporter responsive to androgenic chemicals. Using dihydrotestosterone (DHT) as a standard, the response time and the 50% effective concentration values were 3 to 4 h and (9.7 +/- 4.6) x 10(-9) M, respectively. The lower limit of detection in response to DHT was 2.5 x 10(-9) M, and in response to testosterone it was 2.5 x 10(-10) M. This strain is suitable for high-throughput screening of chemicals with potential for remote environmental monitoring systems because of the assay speed, sensitivity, and self-containment.     

In vitro antagonism of bioluminescent fungi by Trichoderma harzianum

Two species of bioluminescent fungi, Panellus stypticus and Omphalotus olearius were placed in contact with three different strains of interfungal pathogenic Trichoderma harzianum. Subsequent light emission by the luminous fungi and advance of the interfungal pathogens were compared. Relative differences among the pathogens were reflected in their rate of mycelial advance, the total area over which they produced spores upon the host fungi, and decreases in host bioluminescence. After ten days differences in the total surface areas of spore production varied from 1 to 53 per cent. Differences in the reduction of bioluminescence of the same material ranged over 2 orders of magnitude. Final reduction in luminescence ranged over 6 orders of magnitude. A marked reduction in bioluminescence was observed to precede the advance of spore production. The greatest reduction in luminescence was correlated with the presence of T. harzianum hyphae. Two strains of T. harzianum, NRRL 1698 and ATCC 58674, were effective against both bioluminescent fungi within the study period while a third strain, NRRL 13019, was only effective against Omphalotus olearius.     

Production of gibberellins and bikaverin by cells of Gibberella fujikuroi immobilized in carrageenan

The production of gibberellins and bikaverin by immobilized and free cells of Gibberella fujikuroi strains was followed. Both types of cells, free and immobilized, produced similar titers of the secondary metabolites during the normal growth cycle. The kinetics of nutrient use and product formation by the immobilized cells lagged behind that of the free cells and this was assumed to be the result of diffusional limitations imposed on the immobilized cells. A noticeable difference was that in the immobilized cells, all of the bikaverin was excreted into the medium for both strains of G. fujikuroi tested but in the free cell fermentation 44% was excreted for strain ACC 917 and only 10% for strain GF1a. Gibberellin and bikaverin could be produced in a semi-continuous fashion with both free and immobilized cells for a period of 16 d in a resuspension medium containing 0.12 mM or 0.60 mM ammonium chloride. No definite advantage, on a productivity basis, for using immobilized cells over free cells could be seen.     

Use of Saccharomyces cerevisiae BLYES expressing bacterial bioluminescence for rapid, sensitive detection of estrogenic compounds

An estrogen-inducible bacterial lux-based bioluminescent reporter was developed in Saccharomyces cerevisiae for applications in chemical sensing and environmental assessment of estrogen disruptor activity. The strain, designated S. cerevisiae BLYES, was constructed by inserting tandem estrogen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 (formerly pUA12B7) that constitutively express luxA and luxB to create pUTK407. Cotransformation of this plasmid with a second plasmid (pUTK404) containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp) yielded a bioluminescent bioreporter responsive to estrogen-disrupting compounds. For validation purposes, results with strain BLYES were compared to the colorimetric-based estrogenic assay that uses the yeast lacZ reporter strain (YES). Strains BLYES and YES were exposed to 17beta-estradiol over the concentration range of 1.2 x 10(-8) through 5.6 x 10(-12) M. Calculated 50% effective concentration values from the colorimetric and bioluminescence assays (n = 7) were similar at (4.4 +/- 1.1) x 10(-10) and (2.4 +/- 1.0) x 10(-10) M, respectively. The lower and upper limits of detection for each assay were also similar and were approximately 4.5 x 10(-11) to 2.8 x 10(-9) M. Bioluminescence was observed in as little as 1 h and reached its maximum in 6 h. In comparison, the YES assay required a minimum of 3 days for results. Strain BLYES fills the niche for rapid, high-throughput screening of estrogenic compounds and has the ability to be used for remote, near-real-time monitoring of estrogen-disrupting chemicals in the environment.     

Suppression of bacterial blight by a bacterial community isolated from the guttation fluids of anthuriums

Growth and survival of Xanthomonas campestris pv. dieffenbachiae in guttation fluids (xylem sap exuded from leaf margins) of anthuriums were suppressed by several bacterial strains indigenous to leaves of various anthurium cultivars. Inhibition of growth was not observed in filter-sterilized guttation fluids and was restored to original levels only by reintroducing specific mixtures of bacteria into filter-sterilized guttation fluids. The inhibitory effect was related to the species in the bacterial community rather than to the total numbers of bacteria in the guttation fluids. One very effective bacterial community consisted of five species isolated from inhibitory guttation fluids of two susceptible anthurium cultivars. The individual strains in this community had no effect on the pathogen, but the mixture was inhibitory to X. campestris pv. dieffenbachiae in guttation fluids. The populations of the individual strains remained near the initial inoculum levels for at least 14 days. The effect of the five inhibitory strains on reducing disease in susceptible anthurium plants was tested by using a bioluminescent strain of X. campestris pv. dieffenbachiae to monitor the progression of disease in leaves nondestructively. Invasion of the pathogen through hydathodes at leaf margins was reduced by applying the strain mixture to the leaves. When the strain mixture was applied directly to wounds created on the leaf margins, the pathogen failed to invade through the wounds. This bacterial community has potential for biological control of anthurium blight.