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.     

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.     

Detection of DNA damage by use of Escherichia coli carrying recA'::lux, uvrA'::lux, or alkA'::lux reporter plasmids.

Plasmids were constructed in which DNA damage-inducible promoters recA, uvrA, and alkA from Escherichia coli were fused to the Vibrio fischeri luxCDABE operon. Introduction of these plasmids into E. coli allowed the detection of a dose-dependent response to DNA-damaging agents, such as mitomycin and UV irradiation. Bioluminescence was measured in real time over extended periods. The fusion of the recA promoter to luxCDABE showed the most dramatic and sensitive responses. lexA dependence of the bioluminescent SOS response was demonstrated, confirming that this biosensor's reports were transmitted by the expected regulatory circuitry. Comparisons were made between luxCDABE and lacZ fusions to each promoter. It is suggested that the lux biosensors may have use in monitoring chemical, physical, and genotoxic agents as well as in further characterizing the mechanisms of DNA repair.     

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.     

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.     

Induction of umu gene expression by cross-links and other DNA lesions

The induction of umu gene expression by DNA cross-links was investigated in various strains of E. coli with different DNA-repair capacities. Expression was measured by quantifying enzymatic activity of beta-galactosidase produced under regulation of the umu promoter carried on a plasmid carrying the umuC-lacZ gene fusion. The treatment with MMC induced gene expression more efficiently in a wild-type strain when compared with an excision-repair-deficient strain (uvrA). In contrast, PUVA and cis-Pt treatment induced higher levels of the gene expression in the uvrA strain than in the wild-type strain, as did other DNA-damaging agents including 4NQO, MNNG and MMS. None of these chemicals induced umu expression in either lexA and recA strains. The mechanisms of the induction of umu expression by DNA cross-links in relation to DNA damage and repair are discussed.     

Distinct responses of a recA::luxCDABE Escherichia coli strain to direct and indirect DNA damaging agents.

The recombinant Escherichia coli strain DPD2794 containing a recA::luxCDABE fusion is used to detect genotoxicity of various chemicals. Genotoxic agents were previously categorized into two groups, Direct DNA Damaging (DDD) agents and Indirect DNA Damaging (IDD) agents; these two groups have been distinguished with this strain. Minimum detectable concentrations of the DDD agents were about one to five orders of magnitude lower than those of the IDD agents. The response patterns of this strain to DDD agents differed from those to IDD agents in terms of kinetics and the forms of the dose-dependent response.     

Repair of sister-chromatid exchange-inducing lesions in mutagen-treated cultures of human whole blood and purified fresh or frozen lymphocytes

Repair of mutagen-induced lesions that result in sister-chromatid exchanges was evaluated in 10 normal individuals. The mutagens used were mitomycin C (MMC), 4-nitroquinoline 1-oxide (4NQO), and N-methyl-N'nitro-N-nitrosoguanidine (MNNG). Cultures of whole blood, freshly purified lymphocytes, or purified lymphocytes cryopreserved for 6 months were analyzed after the mutagen treatments. All 3 mutagens induced reparable damage as evaluated by comparison of sister-chromatid exchanges between cultures that were given time to repair induced damage before 5-bromo-2'-deoxyuridine (BrdUrd) was added to the culture medium with those where BrdUrd was added immediately after the administration of the mutagens (MMC or 4NQO) or at culture initiation (MNNG). Repair of mutagen-induced DNA damage was detected in all 3 culture types; thus cryopreservation did not appear to alter the capacity of lymphocytes to repair mutagen-induced lesions. Quantitative differences in apparent repair capabilities were observed among individuals. Variability also existed among the different culture types within an individual, suggesting that caution should be exercised in interpreting these apparent differences.     

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.     

Prophage induction in Haemophilus influenzae and its relationship to mutation by chemical and physical agents

It is known that UV, X-rays, MMC and MMS are not mutagenic for H. influenzae, whereas HZ, EMS and MNNG are potent mutagens for this bacterium. All of these agents, however, are known to be both mutagenic and able to induce prophage in E. coli. We report here that all the agents except HZ induce prophage in H. influenzae, and EMS even induces in the recombination-defective recl mutant, which is non-inducible by UV, MMC, MNNG and MMS. MMS did not cause single-strand breaks or gaps in DNA synthesized after treatment of H. influenzae, but EMS and MNNG produced them. EMS caused more breaks in DNA synthesized before treatment than in that synthesized after treatment. On the other hand we did observe such breaks or gaps induced in E. coli in DNA synthesized posttreatment by EMS as well as by MMS and MNNG, at comparable survival levels.     

Improved bacterial SOS promoter∷lux fusions for genotoxicity detection

Escherichia coli strains containing plasmid-borne fusions of Vibrio fischeri lux to the recA promoter-operator region were previously shown to be potentially useful for detecting genotoxicants. In an attempt to improve past performance, the present study examines several modifications and variations of this design, singly or in various combinations: (1) modifying the host cell's toxicant efflux capacity via a tolC mutation; (2) incorporating the lux fusion onto the bacterial chromosome, rather then on a plasmid; (3) changing the reporter element to a different lux system (Photorhabdus luminescens), with a broader temperature range; (4) using Salmonella typhimurium instead of an E. coli host. A broad spectrum of responses to pure chemicals as well as to industrial wastewater samples was observed. Generally, fastest responses were exhibited by Sal94, a S. typhimurium strain harboring a plasmid-borne fusion of V. fischeri lux to the E. coli recA promoter. Highest sensitivity, however, was demonstrated by DPD3063, an E. coli strain in which the same fusion was integrated into the bacterial chromosome, and by DPD2797, a plasmid-bearing tolC mutant. Overall, the two latter strains appeared to perform better and seemed preferable over the others. The sensor strains retained their sensitivity following a 2-month incubation after alginate-embedding, but at the cost of a significantly delayed response.     

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.     

recA gene of Escherichia coli complements defects in DNA repair and mutagenesis in Streptomyces fradiae JS6 (mcr-6).

Streptomyces fradiae JS6 (mcr-6) is a mutant which is defective in repair of DNA damage induced by a variety of chemical mutagens and UV light. JS6 is also defective in error-prone (mutagenic) DNA repair (J. Stonesifer and R. H. Baltz, Proc. Natl. Acad. Sci. USA 82:1180-1183, 1985). The recA gene of Escherichia coli, cloned in a bifunctional vector that replicates in E. coli and Streptomyces spp., complemented the mutation in S. fradiae JS6, indicating that E. coli and S. fradiae express similar SOS responses and that the mcr+ gene product of S. fradiae is functionally analogous to the protein encoded by the recA gene of E. coli.     

The partial recA-lexA dependence of the adaptive response of Escherichia coli to exposure to methylmethane sulfonate

A study was made of the adaptive response to methylmethane sulfonate (MMS) in E. coli. (18 strains of B, WP2, and H/r30 groups, including three strains of bacteria with pKM101 plasmid). The adaptation of wild type cells and uvrA- and uvrB- mutants to non-lethal concentrations of MMS (10-30 mkg/ml during 90-120 min) leads to a significant increase in their resistance to lethal MMS concentrations (10-30 mM for 10-120 min): the dose modifying factor (DMF) being 1.5-1.8. In single recA or lexA mutants (or double recA uvr- and lexA uvr- mutants) the efficiency of adaptive response to MMS was significantly lower: the DMF being 1.1-1.2. In Bs-1 gamma R strain with intragenic suppressor of lexA gene the adaptive response efficiency was the same as in B/r (recA+lexA+) strain. There is no adaptive response to MMS in polA- strains. The adaptive response to MMS in E. coli is different from that to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methylnitrosourea (MNM), because in these two cases it is absolutely lexA-recA dependent. It is supposed that a partial recA-lexA dependence of the adaptive response to MMS in E. coli may be due to a specific MMS-induced lethal damage that induces an adaptive repair non-related to the system of recA-lexA-independent adaptive responses to MNNG and MNM. The presence of a plasmid of drug resistance pKM101 exerts no influence on the value, efficiency and recA-lexA-dependence of the adaptive response of E. coli to MMS.     

An integrated mini biosensor system for continuous water toxicity monitoring

An integrated water toxicity monitoring system that uses recombinant bioluminescent bacteria was successfully developed for the continuous monitoring and classification of toxicities present in water. This system consists of four channels arranged horizontally inside of a cylinder, with each channel having two small bioreactors that are vertically connected to each other to maintain a separation of the culture reactor from test reactor. This system is easily handled and installed, making its application in the field a potential reality. As well, it performed stably and continuously due to the vertical separation of the culture reactor from the test reactor and a long term operation was also performed because of its small working volume, i.e., only 1 ml for the 1st bioreactor and 2 ml for the 2nd. During an operation with four strains, i.e., EBHJ2, DP1, DK1 and DPD2794, which are responsive to superoxide damage (EBHJ2 and DP1), hydrogen peroxide (DK1), and DNA damage (DPD2794), the O.D. and bioluminescence of the bacterial cultures inside the system were constant when no chemical was injected. However, with the addition of paraquat, hydrogen peroxide or mitomycin C, the bioluminescent responses of the strains were found to be dose-dependent to different concentrations of these chemicals.