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.     

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.     

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.     

Gamma-radiation dose-rate effects on DNA damage and toxicity in bacterial cells

In order to investigate the relationship between radiation dose-rate and bacterial DNA damage as well as general cellular toxicity, two recombinant Escherichia coli strains, DPD2794 and GC2 were used. Following gamma-ray irradiation, these bioluminescent bacteria showed quantitative stress responses in terms of DNA damage and general toxicity depending on the dose rates of energy deposition, i.e. dose-rate of radiation. In addition, an inverse relationship was found, at lower dose rates between 0.5 and 1 Gy/h and a parabolic relationship at dose rates between 0.5 and 2.6 Gy/h.     

Response of bioluminescent bacteria to sixteen azo dyes

Recombinant bioluminescent bacteria were used to monitor and classify the toxicity of azo dyes. Two constitutive bioluminescent bacteria,Photobacterium phosphoreum andEscherichia coli, E. coli GC2 (lac::luxCDABE), were used to detect the cellular toxicity of the azo dyes. In addition, four stress-inducible bioluminescentE. coli, DPD2794 (recA::luxCDABE), a DNA damage sensitive strain; DPD2540 (fabA::luxCDABE), a membrane damage sensitive strain; DPD2511 (katG::luxCDABE), an oxidative damage sensitive strain; and TV1061 (grpE::luxCDABE), a protein damage sensitive strain, were used to provide information about the type of toxicity caused by crystal violet, the most toxic dye of the 16 azo dyes tested. These results suggest that azo dyes result in serious cellular toxicity in bacteria, and that toxicity monitoring and classification of some azo dyes, in the field, may be possible using these recombinant bioluminescent bacteria.     

A portable toxicity biosensor using freeze-dried recombinant bioluminescent bacteria

A portable biosensor has been developed to meet the demands of field toxicity analysis. This biosensor consists of three parts, a freeze-dried biosensing strain within a vial, a small light-proof test chamber, and an optic-fiber connected between the sample chamber and a luminometer. Various genetically engineered bioluminescent bacteria were freeze-dried to measure different types of toxicity based upon their modes of action. GC2 (lac::luxCDABE), a constitutively bioluminescent strain, was used to monitor the general toxicity of samples through a decrease in its bioluminescence, while specific toxicity was detected through the use of strains such as DPD2540 (fabA::luxCDABE), TV1061 (grpE::luxCDABE), DPD2794 (recA::luxCDABE), and DPD2511 (katG::luxCDABE). These inducible strains show an increase in bioluminescence under specific stressful conditions, i.e. membrane-, protein-, DNA-, and oxidative-stress, respectively. The toxicity of a sample could be detected by measuring the bioluminescence 30 min after addition to the freeze-dried strains. In an attempt to enhance the sensitivity of the freeze-dried cells, glucose and Tween 80 were tested as additives. It was found that the addition of glucose had a negative effect on the viability of the freeze-dried cells, while samples having Tween 80 showed an increase in their viability. On the other hand, the addition of either Tween 80 or glucose decreased the final bioluminescent response of DPD2540 in response to 4-chlorophenol. Using these strains, many different chemicals were tested and characterized. This portable biosensor, with a very simple protocol, can be used for field sample analysis and the monitoring of various water systems on-site.     

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.     

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.     

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.     

Enhancement of the multi-channel continuous monitoring system through the use of Xenorhabdus luminescens lux fusions

The enhancement of the multi-channel continuous toxicity monitoring system developed previously was studied. To achieve better and more stable results from the system, the use of thermo-lux fusion strains that express the luxCDABE genes from Xenorhabdus luminescens was evaluated. A total of six recombinant Escherichia coli strains with the promoters from three oxidative-stress responsive genes, i.e. the katG, sodA and pqi-5 genes, fused to either the lux genes from Vibrio fischeri or X. luminescens were characterized and their responses to different chemicals compared. It was found that the basal level bioluminescence (BL) from the thermo-lux fusion strains was always higher while that of the V. fischeri lux strains were always near or below the lower limit of detection of the system. For example, the katG::V. fischeri lux strain, DPD2511, gave no discernible response due to its low level expression while a fusion of the katG promoter with the X. luminescens lux operon was clearly responsive and capable of detecting hydrogen peroxide down to about 1 ppm. The use of the thermo-lux strains found them to be as sensitive as the V. fischeri lux strains while providing a brighter, more stable basal level bioluminescence, making the analysis and monitoring of water-borne toxicity more reliable.     

Characterization and optimization of two methods in the immobilization of 12 bioluminescent strains

Twelve recombinant bioluminescent bacteria have been immobilized within the wells of a 96-well plate using two different matrices--agar and sol-gel. All 12 strains were immobilized within individual wells of the plates and the sensitivity of the strains and the stability of the responses were determined for select chemicals. Each strain was exposed to seven well-characterized chemicals over a wide range of concentrations to demonstrate their individual selectivity for specific toxicants. Although the sensitivity of the immobilized cells was generally lower than cultures grown in liquid media, they were comparable. For example, strain DPD1710, which responds to DNA damage was able to detect mitomycin C, a genotoxin, at a minimum concentration of 0.6 ppb. When immobilized, the lower limit of detection was between 1 and 10 ppb. Finally, using compounds that are known to elicit a response from each of the strains, the stability of the bioluminescent responses were measured over an extended period of 4 weeks. Although the activity of several strains decreased over time, the majority of the strains used in both immobilized systems were still responsive.     

An<Emphasis Type="Italic"> Escherichia coli</Emphasis> 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-N-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.     

The Combined Effect of Hydrogen Peroxide and Ultraviolet Irradiation on Bacterial Spores

Ultra-violet (u.v.) light irradiation of spores of Bacillus subtilis in the presence of hydrogen peroxide produced a rapid kill which was up to 2000-fold greater than that produced by irradiation alone. A kill of 99–99% was produced by 30s u.v. irradiation of spores of 6 strains of Bacillus and Clostridium in the presence of hydrogen peroxide 1.0 g/100 ml but with the more resistant spores of 9 further strains, irradiation in the presence of hydrogen peroxide 2–5 g/100 ml followed by mild heating was required.     

On-line determination of glucose concentration throughout animal cell cultures based on chemiluminescent detection of hydrogen peroxide coupled with flow-injection analysis.

A flow-injection analysis (FIA) system for the on-line determination of glucose in animal cell cultures is described. The system is based on immobilized glucose oxidase (GOD). The hydrogen peroxide generated in the enzyme reaction is determined via a highly sensitive chemiluminescent reaction with luminol. Based on the measurement of the maximum emitted light intensity, the system was able to analyse hydrogen peroxide over the concentration range of 10(-7) to 10(-2) M. For glucose determination, the system has a linear range of 10(-5) to 5 x 10(-2) M glucose, with an r.s.d. of 3% at the 1 mM level (5 measurements). The influence of luminol and buffer concentrations, pH and temperature on the chemiluminescent reaction were investigated. The enzyme reactor used was stable for more than 4 weeks in continuous operation, and it was possible to analyse up to 20 samples per h. The system has been successfully applied to on-line monitoring of glucose concentration during an animal cell culture, designed for the production of human antithrombin III factor. Results obtained with the FIA system were compared with off-line results, obtained with a Yellow Springs Instrument Company Model 27 (YSI).     

Mycoplasma pneumoniae protects infected epithelial cells from hydrogen peroxide‐induced cell detachment

Epithelial cell shedding is a defence mechanism against infectious microbes that use these cells as an infection foothold and that eliminate microbes from infection foci by removing infected cells. Mycoplasma pneumoniae, a causative agent of respiratory infections, is known to adhere to and colonise the surface of ciliated airway epithelial cells; it produces a large amount of hydrogen peroxide, indicating its capability of regulating hydrogen peroxide‐induced infected cell detachment. In this study, we found that M. pneumoniae reduces exogenous hydrogen peroxide‐induced detachment of the infected cells from culture plates. This cell detachment occurred dependently of DNA damage‐initiated, poly (ADP‐ribose) polymerase 1 (PARP1)‐mediated cell death, or parthanatos. In cells infected with M. pneumoniae, exogenous hydrogen peroxide failed to induce DNA damage‐initiated poly (ADP‐ribose) (PAR) synthesis and concomitant increased cytoplasmic membrane rupture, both of which are biochemical hallmarks of parthanatos. The impairment of PAR synthesis was attributed to a reduction in the amount of cytosolic nicotinamide adenine dinucleotide (NAD), a substrate of PARP1, caused by M. pneumoniae. On the other hand, nonadherent mutant strains of M. pneumoniae showed a lower ability to reduce cell detachment than wild‐type strains, but the extent to which NAD was decreased in infected cells was comparable to that seen in the wild‐type strain. We found that NAD depletion could induce PARP1‐independent cell detachment pathways following stimulation with hydrogen peroxide and that M. pneumoniae could also regulate PARP1‐independent cell detachment in a cytoadhesion‐dependent manner. These results suggest that M. pneumoniae might regulate infected cell detachment induced by hydrogen peroxide that it produces itself, and such a mechanism may contribute to sustaining the bacterial infection.     

Utilization of H2O2 as the oxygen source by bacteria of the genera Pseudomonas and Rhodococcus

The growth of bacteria of the genera Pseudomonas and Rhodococcus in the presence of hydrogen peroxide as the sole source of oxygen was studied. The toxic effect of H2O2 in the concentration range of 100-200 microg/ml was shown to extend the lag phase by 2 to 3 days. Apart from the peroxide toxicity, the bacterial growth was inhibited by the toxic effect of dissolved oxygen in concentrations over 100 microg O2/ml; in the presence of a liquid hydrocarbon phase, this effect was alleviated. Under decreased partial pressure of oxygen in the presence of hydrocarbons (12-15 vol %), the culture growth was initiated at high initial concentrations of H2O2 (300 microg/ml). When hydrogen peroxide concentrations exceeded 320 microg/ml, no growth occurred, no matter how much hydrocarbon was added.