Identification of Yersinia enterocolitica using a random genomic DNA microarray chip.

AIMS: To fabricate a DNA chip containing random fragments of genomic DNA of Yersinia enterocolitica and to verify its diagnostic ability. METHODS AND RESULTS: A DNA microarray chip was fabricated using randomly fragmented DNA of Y. enterocolitica. Chips were hybridized with genomic DNA extracted from other Y. enterocolitica strains, other Yersinia spp. and bacteria in different genera. Genomic DNA extracted from Y. enterocolitica showed a significantly higher hybridization rate compared with DNA of other Yersinia spp. or bacterial genera, thereby distinguishing it from other bacteria. CONCLUSIONS: A DNA chip containing randomly fragmented genomic DNA from Y. enterocolitica can detect Y. enterocolitica and clearly distinguish it from other Yersinia spp. and bacteria in different genera. SIGNIFICANCE AND IMPACT OF THE STUDY: A microarray chip containing randomly fragmented genomic DNA of Y. enterocolitica was fabricated without sequence information, and its diagnostic ability to identify Y. enterocolitica was verified.

Enhanced degradation and toxicity reduction of dihexyl phthalate by Fusarium oxysporum f. sp. pisi cutinase.

AIMS: This research aims to investigate the efficiency of two lipolytic enzymes--fungal cutinase and yeast esterase--upon the biodegradation of dihexyl phthalate (DHP). METHOD AND RESULTS: During the enzymatic degradation of DHP dissolved in methanol, several degradation products were detected and their time-course changes were monitored using GC/MS. The DHP-degradation rate of cutinase was surprisingly high; i.e. almost 70% of the initial DHP (500 mg l(-1)) was decomposed within 4.5 h. Although the same amount of esterase was employed, more than 85% of the DHP remained after 3 days. Almost all the DHP was converted by cutinase into 1,3-isobenzofurandione (IBF), whereas hexyl methyl phthalate and IBF were abundantly produced by esterase. In addition, the toxicities of the DHP-degraded products by esterase were evaluated using various recombinant bioluminescent bacteria, which caused oxidative and protein damage, whereas the hydrolysis products from cutinase never caused any cellular damage in the methanol-containing reaction system. CONCLUSIONS: Cutinase starts to act as a DHP-degrader much earlier and faster than esterase, with high stability in ester-hydrolytic activity, therefore a plausible approach to the practical application of cutinase for DHP degradation in the DHP-contaminated environments may be possible. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the enhanced degradation and detoxification of DHP using Fusarium oxysporum f. sp. pisi cutinase.

Application of a multi-channel system for continuous monitoring and an early warning system.

A multi-channel continuous toxicity monitoring system developed in our laboratory, based on two-stage mini-bioreactors, was successfully implemented in the form of computer-based data acquisition. The multi-channel system consists of a series of a two-stage minibioreactor systems connected by a fiber optic probe to a luminometer, and uses genetically engineered bioluminescent bacteria for the detection of the potential toxicity from the soluble chemicals. This system can be stably and continuously operated due to the separation of the culture reactor from the test reactor and accomplish easy and long-term monitoring without system shut down by abrupt inflows of severe polluting chemicals. Four different recombinant bioluminescent bacteria were used in different channels so that the modes of the samples toxicities can be reasonably identified and evaluated based upon the response signature of each channel. The bioluminescent signatures were delivered from four channels by switching one at once, while the data is automatically logged to an IBM compatible computer. We also achieved the enhancement of the system through the manipulation of the dilution rate and the use of thermo-lux fusion strains. Finally, this system is now being implemented to a drinking water reservoir and river for remote sensing as an early warning system.

An Escherichia coli biosensor capable of detecting both genotoxic and oxidative damage.

A two-plasmid dual reporter Escherichia coli biosensor was developed using the genes for bacterial bioluminescence and a mutant of the green fluorescent protein, GFPuv4. To achieve this, the two plasmids, which were derivatives of pBR322 and pACYC184, had compatible origins of replication and different antibiotic selection markers: ampicillin and tetracycline. The parent strains DK1 and ACRG43, each carrying a single plasmid with one of the fusion genes (strain DK1 harboring a fusion of the katG promoter to the lux operon while in ACRG43, the recA promoter was fused with the GFP gene), were responsive to oxidative and DNA damage, respectively, resulting in higher bioluminescence or fluorescence under the relevant toxic conditions. The responses of the dual sensor strain, DUAL22, to various toxicants, e.g., mitomycin C, N-methyl-N-nitro--nitrosoguanidine, hydrogen peroxide and cadmium chloride, were characterized and compared with the responses of the parent strains to the same chemicals. Finally, several chemical mixtures that cause various stress responses were tested to demonstrate the ability of this biosensor to detect specific stress responses within a multiple toxicity environment.

Acclimation and repair of DNA damage in recombinant bioluminescent Escherichia coli.

AIMS: The aim of this study is to understand different adaptive responses in bacteria caused by three different mutagens, namely, an intercalating agent, an alkylating agent and a hydroxylating agent, and the repair systems according to the type of DNA damage, that is, DNA cross-linking and delayed DNA synthesis, alkylation and hydroxylation of DNA. A recombinant bioluminescent Escherichia coli, DPD2794 with the recA promoter fused to luxCDABE originating from Vibrio fischeri, was used in this study. METHODS AND RESULTS: The recombinant bioluminescent E. coli strain DPD2794, containing a recA promoter fused to luxCDABE from V. fischeri, was used to detect adaptive and repair responses to DNA damage caused by mitomycin C (MMC), and these responses were compared with those when the cells were induced with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and hydrogen peroxide (H2O2). The response ratio between the induced samples and that of the controls decreased suddenly when the induced culture was used in further inductions, indicating a possible adaptive response to DNA damage. DNA damage, or the proteins produced, because of MMC addition does not appear to be completely resolved until the seventh sub-culture after the initial induction, whereas simple damage, such as the base modification caused by MNNG and H2O2, appears to be repaired rapidly as evidenced by the quick recovery of sensitivity. CONCLUSIONS: These results suggest that it takes more time to completely repair DNA damage caused by MMC, as compared with a simple repair such as that required for the damage caused by MNNG and H2O2. Therefore, repair of the damage caused by these three mutagens is controlled by different regulons, even though they all induced the recA promoter. SIGNIFICANCE AND IMPACT OF THE STUDY: Using a bioluminescent E. coli harbouring a recA promoter-lux fusion, it was found that different adaptive responses and repair systems for DNA damage caused by several mutagens exists in E. coli.

Construction of a sodA::luxCDABE fusion Escherichia coli: comparison with a katG fusion strain through their responses to oxidative stresses.

A recombinant bioluminescent Escherichia coli strain, EBHJ, (sodA::luxCDABE), containing the promoter for the manganese superoxide dismutase ( sodA) gene fused to the Vibrio fischeri luxCDABE operon, was successfully constructed and characterized. Redox-cycling agents, such as paraquat and chromium, strongly induced a sodA- regulated response in dose-dependent manners, resulting in an increase of the bioluminescence. In a comparison with an existing oxidative stress responsive strain, DPD2511 (katG::luxCDABE), which is sensitive to H(2)O(2), the mechanism of chemicals that cause oxidative damage was elucidated via the key transcriptional factors involved in induction of the sodA and katG promoters, i.e. SoxRS and OxyR, respectively. It was found that responses from the katG- and sodA-based strains were significantly different dependent upon the chemicals being tested. Therefore, EBHJ, alone or in parallel with DPD2511, can be used to characterize and monitor chemicals that cause oxidative damage.

Application of recombinant fluorescent mammalian cells as a toxicity biosensor.

With respect to developing a more sensitive biosensor, a recombinant fluorescent Chinese Hamster Ovary cell line was used for the monitoring of various toxicants. Both cell lines, EFC-500 and KFC-A10, were able to detect toxicants sensitively. They were characterized with mitomycin C and gamma-ray as genotoxicants and bisphenol A, nonylphenol, ziram and methyl bromide as possible and known EDCs. When compared to each other, the response of KFC-A10 was generally more informative and sensitive. Compared to typical bacterial biosensor systems, these cell lines offered a sensitivity of 2- to 50-fold greater for the tested chemicals. Based on these results, the use of mammalian cells offers a sensitive biosensor system that is not only fast, cheap and reproducible but also capable of monitoring the endocrine-like characteristics of environmental toxicants.

Dynamic characterization of recombinant Chinese hamster ovary cells containing an inducible c-fos promoter GFP expression system as a biomarker.

An inducible reporter gene system for Chinese Hamster Ovary (CHO-DHFR(-)) cells has been developed and characterized with respect to its dynamic properties. The reporter gene system consists of the human c-fos promoter and variants of the green fluorescence protein (GFP), either EGFP with enhanced fluorescence or its destabilized form d2EGFP. The expression of wild-type EGFP or its destabilized form was studied in CHO-DHFR(-) cells in response to serum addition or deprivation. It was shown that serum-induced c-fos promoter mediated EGFP expression was considerably higher than expression from the human CMV promoter, a strong, constitutive promoter preferentially used for high-level expression in CHO cells. However, EGFP was less suitable for studying expression dynamics than d2EGFP due to the protein's long half-life in mammalian cells. The use of d2EGFP resulted in a significant improvement in the dynamic characteristics of the biomarker, particularly when the recombinant cells were selected for high-level GFP expression by subcloning or fluorescence activated cell/sorting (FACS). GFP expression in different subclones and cell populations sorted by FACS was characterized with respect to its dynamic responses in the presence or absence of serum in the culture medium. Significant differences in the GFP expression dynamics were observed for the isolated cell populations. The experimental results indicate that cells with high-level GFP expression also have a faster dynamic response and are thus, desirable for practical application of the reporter gene system e.g. in toxicity monitoring.

A multi-channel continuous toxicity monitoring system using recombinant bioluminescent bacteria for classification of toxicity.

A multi-channel system for continuous toxicity monitoring and classification of toxicity was developed based upon a previously developed two-stage minibioreactor system. The multi-channel system consists of a series of a two-stage minibioreactor systems connected by a fiber optic probe to a luminometer. Each channel was used for cultivating different recombinant bacterial strains, such as TV1061 (grpE::luxCDABE), DPD2794 (recA::luxCDABE), and DPD2540 (fabA::luxCDABE), which are induced by protein-, DNA-, and cell membrane damaging-agents, respectively. GC2 (lac::luxCDABE) is a bacterium expressing bioluminescence constitutively, which shows a reduction in its light level as cellular toxicity increases. Artificial wastewater samples were made by combining toxic chemicals, including Mitomycin C (a representative DNA damaging agent), phenol (a representative protein damaging agent), and cerulenin (a representative cell membrane damaging agent), and injecting this sample into each channel in order to simulate the detection of toxicity for mixed chemical samples. Each channel showed a specific bioluminescent response due to the toxic chemicals contained in the sample wastewater, while GC2 showed a general response to cellular toxicity. By using this multi-channel continuous toxicity monitoring system, classification of toxicity in field samples was found to be possible.

Soil biosensor for the detection of PAH toxicity using an immobilized recombinant bacterium and a biosurfactant.

A biosensor for detecting the toxicity of polycylic aromatic hydrocarbons (PAHs) contaminated soil has been successfully constructed using an immobilized recombinant bioluminescent bacterium, GC2 (lac::luxCDABE), which constitutively produces bioluminescence. The biosurfactant, rhamnolipids, was used to extract a model PAH, phenanthrene, and was found to enhance the bioavailability of phenanthrene via an increase in its rate of mass transfer from sorbed soil to the aqueous phase. The monitoring of phenanthrene toxicity was achieved through the measurement of the decrease in bioluminescence when a sample extracted with the biosurfactant was injected into the minibioreactor. The concentrations of phenanthrene in the aqueous phase were found to correlate well with the corresponding toxicity data obtained by using this toxicity biosensor. In addition, it was also found that the addition of glass beads to the agar media enhanced the stability of the immobilized cells. This biosensor system using a biosurfactant may be applied as an in-situ biosensor to detect the toxicity of hydrophobic contaminants in soils and for performance evaluation of PAH degradation in soils.

Physicochemical factors affecting the sensitivity of Ceriodaphnia dubia to copper.

The effects of physicochemical conditions, such as pH, water hardness, flow rates and natural organic substances on the sensitivity of Ceriodaphnia dubia to the toxic effects of copper were investigated using static bioassay cups and specially designed flow-through bioassay chambers. We found that C. dubia was very sensitive to pH changes and the total copper LC50 values of C. dubia neonates increased by 15-fold as the pH increased from pH 7 to 10. It was also observed that the LC50 values increased sharply upon increasing the water hardness value to 2.4 meq. In addition, increasing flow rates from zero to 50 mL hr(-1) also increased its sensitivity to copper, which was possibly due to hydrodynamic stress. The presence of natural organic substances (humic acid and dissolved organic matter) and suspended particles decreased the toxic effect of copper. This significant decrease in the toxicity of copper in the presence of natural organic materials can be explained by a reduction in the free ion concentration due to complexation. Furthermore, we observed that the kinetics of copper interactions with natural organic materials are a significant factor in the toxic effect of copper and that the acute LC50 values increased with increasing reaction time between solubilized copper and water-borne organics.

The continuous monitoring of field water samples with a novel multi-channel two-stage mini-bioreactor system.

Toxicity monitoring of field water samples was performed using a novel multi-channel two-stage mini-bioreactor system and genetically engineered bioluminescent bacteria for the continuous monitoring and classification of the toxicity present in the samples. The toxicity of various samples spiked with known endocrine disrupting chemicals and phenol was also investigated for system characterization. The field samples used in this study were obtained from two different sites on a monthly basis--from a drinking water treatment plant, referred to as site N, and from a stream near a dam which is currently being constructed, referred to as site T. These samples were either pumped or injected into the second mini-bioreactors to initiate the toxicity test. Most of the samples did not show any specific toxicity. However, one sample showed to have, based upon the detection results, and was deemed toxic. The samples spiked with phenol showed possible responses in the DPD2540 and TV1061 channels, indicating the occurrence of both membrane and protein damage due to phenol. In the tests using an endocrine disrupting chemical, bisphenol A, DNA damage was detected in the DPD2794 channel with a concentration of 2 ppm. Finally, a simple but novel early warning protocol that can be used in a drinking water reservoir and a suspected place where effluents of toxic materials enter the water sourse was suggested with a schematic diagram. In conclusion, this system showed good feasibility for use as a toxicity monitoring system in the field and as an early warning system, indicating if effluents are toxic.

Some observations in freeze-drying of recombinant bioluminescent Escherichia coli for toxicity monitoring.

A recombinant bioluminescent bacteria, containing a fabA::luxCDABE fusion gene, has been used to characterize freeze-drying methods, which may be conveniently used as a tool for the development of a portable biosensor. Through residual water, viability, biosensing activity and scanning electron microscopy analyses, the characteristics that four cryoprotectants, trehalose, sucrose, sorbitol, and mannitol, conferred on freeze-dried samples were elucidated, including the morphology, water content and activity of the cells. It was found that trehalose showed the best freeze-drying efficiency among the tested cryoprotectants and it might have a specific capacity limitation in protection of the cells during the freeze step. Humidity might result in damage to the cells, according to the viability, when exposed to air during storage, while the water remaining post freeze-drying showed good correlation with damage to the freeze-dried cells when under air-tight storage conditions. The results with other recombinant bioluminescent bacteria indicated that these findings might be general features of the freeze-drying processes.

Phenolic toxicity--detection and classification through the use of a recombinant bioluminescent Escherichia coli.

A genetically engineered Escherichia coli strain, DPD2540, containing a fabA::luxCDABE fusion that gives a bioluminescent output when membrane fatty acids are limited was used to determine the extent to which phenolics result in the limitation of membrane fatty acids. Tested phenolics were found to be classifiable into two groups according to the bioluminescent response they elicited and their pKa. A dose-dependent bioluminescent response, due to fatty acid limitation, was seen for phenolics with pKa values greater than seven, which exist mainly in the protonated form (HA), while no significant bioluminescent response was seen, compared with the control, for phenolics with pKa values lower than seven, which exist almost entirely as A-. A newly modified distribution model for phenolic compounds in the cellular membrane is proposed and used to predict the bioluminescent response induced by group I phenolics and the cellular toxicity for both groups. The [HA]*, obtained with this model, shows good correlation with the various bioluminescent responses produced by group I phenolics. It was also found that the distribution ratio between the medium and the cell membrane, K1, calculated as well using the proposed model, is a good representative parameter for the cellular toxicity of the phenolic compounds according to their substituted groups when compared with the conventional method of using the octanol-water partition coefficient, log Kow. As a new parameter, the critical concentration was also shown to be a good representative of the cellular toxicity for group I phenolics to the Escherichia coli cells.

Monitoring and classification of toxicity using recombinant bioluminescent bacteria.

Recombinant bioluminescent biosensing cells were used to detect and classify toxicity caused by various chemicals in water environments. Classification of the toxicity was realized based upon the chemicals' actions of toxicity by using DNA-, oxidative- and membrane-damage sensitive strains. Tested samples contained a single chemical or mixture of chemicals in media, wastewater, or river water. Finally, it is suggested that this method for classification of toxic chemicals in wastewater or other aqueous systems may be adopted for primary screening steps of the samples and can give useful information about the samples' characteristics.

Bacterial bioluminescent emission from recombinant Escherichia coli harboring a recA::luxCDABE fusion.

This paper describes the quantitative evaluation of a bioluminescence assay for DNA damaging agents with respect to the linearity, sensitivity, specificity and dependence on the cell culture status. A recombinant bacterium, DPD2794, harboring a plasmid with a recA promoter fused to the luxCDABE operon, showed a very sensitive response to DNA-damaging stress. DPD2794 was found to show no noticeable response to non-mutagenic agents, i.e. phenol, except for some false responses appearing soon after injection. DPD2794 also showed a highly sensitive response to Mitomycin C, which was found to be a growth-stage-dependent response, not a growth-rate-dependent response. In addition, the relationship between the bioluminescence emitted in vivo, luciferase activity measured in vitro, and the amount of Lux proteins expressed was determined. The intensity of the bioluminescence emitted was found to be proportional to the luciferase activity in vitro, while the bioluminescence also seems to be correlated with the level of Lux proteins expressed in these Escherichia coli cells, up to 230 min post induction.

Detection of radiation effects using recombinant bioluminescent Escherichia coli strains.

Effects of ionizing radiation (0.1-500 Gy) on recombinant Escherichia coli cells containing the stress promoters recA, grpE, or katG, fused to luxCDABE, were characterized by monitoring transcriptional responses reflected by the bioluminescent output. The minimum dose of gamma-irradiation detected by E. coli DPD2794 (recA::luxCDABE) was about 1.5 Gy, while the maximum response was obtained at 200 Gy. The amount of emitted bioluminescence increased proportionally with the gamma-ray doses which were found to elicit a DNA damage response in a range of 1-50 Gy. In addition, the cell growth rate was severely, but transiently, retarded by about 50 Gy. Quantification of the gamma-ray dose may be possible using the recA promoter fusion, since linear enhancement of the bioluminescence emission with increasing gamma-ray dose was observed. Other irradiated strains (50 Gy) responsive to either oxidative stress (DPD2511, katG::luxCDABE) or protein-damaging stress (TV1061, grpE::luxCDABE) did not display an increased bioluminescent output, while DPD2794 irradiated by the same dose of gamma-rays gave a significant bioluminescent output. This indicates that the recA promoter is the one most suitable for developing a biosensor for ionizing radiation.

A biosensor for the detection of gas toxicity using a recombinant bioluminescent bacterium.

A whole-cell biosensor was developed for the detection of gas toxicity using a recombinant bioluminescent Escherichia coli harboring a lac::luxCDABE fusion. Immobilization of the cells within LB agar has been done to maintain the activity of the microorganisms and to detect the toxicity of chemicals through the direct contact with gas. Benzene, known as a representative volatile organic compound, was chosen as a sample toxic gas to evaluate the performance of this biosensor based on the bioluminescent response. This biosensor showed a dose-dependent response, and was found to be reproducible. The immobilizing matrices of this biosensor were stored at 4 degrees C and were maintained for at least a month without any noticeable change in its activity. The optimal temperature for sensing was 37 degrees C. A small size of this sensor kit has been successfully fabricated, and found to be applicable as a disposable and portable biosensor to monitor the atmospheric environment of a workplace in which high concentrations of toxic gases could be discharged.

A two-stage minibioreactor system for continuous toxicity monitoring.

A two-stage minibioreactor system was successfully developed for continuous toxicity monitoring. This system consists of two minibioreactors in series. Recombinant Escherichia coli DPD2794 containing a RecA::luxCDABE fusion as a model strain was utilized to monitor environmental insults to DNA, with mitomycin C as a model toxicant. Pulse type exposures were used to evaluate the system's reproducibility and reliability. Step inputs of mitomycin C have been adopted to show the system's stability. The system's ability to monitor the possible upsets or accidental discharges of toxic chemicals was also evaluated with these step insults. All the data demonstrated that this two-stage minibioreactor system using recombinant bacteria containing stress promoters fused with lux genes is quite appropriate for continuous toxicity monitoring. Long-term operation and minimized media-usage have been investigated. Thus application to many different areas, including an early warning system of wastewater biotreatment plant upsets and the monitoring and tracking of accidental spills, discharges or failures in plant operation are plausible.