Optimization of Bacterial Whole Cell Bioreporters for Toxicity Assay

Publication Date (Web): September 11, 2009 .... using whole cell biosensors to detect chemicals (11-15) or toxicity (16-20). ...... Hydrol. 2001, 53 (...
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Environ. Sci. Technol. 2009, 43, 7931–7938

Optimization of Bacterial Whole Cell Bioreporters for Toxicity Assay of Environmental Samples †,‡,|

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YIZHI SONG, GUANGHE LI, STEVEN F. THORNTON,‡ IAN P. THOMPSON,§ STEVEN A. BANWART,‡ DAVID N. LERNER,‡ AND W E I E . H U A N G * ,‡ Department of Environmental Science and Engineering, Tsinghua University, Beijing, P.R. China, Department of Civil and Structural Engineering, Kroto Research Institute, University of Sheffield, Broad Lane, Sheffield S3 7HQ, U.K., Department of Engineering, University of Oxford, U.K., and State Key Joint Laboratory of Environment Simulation and Pollution Control, Beijing, P.R. China

Received May 6, 2009. Revised manuscript received August 4, 2009. Accepted August 19, 2009.

In a study to optimize bacterial whole cell biosensors (bioreporters) for the detection of environmental contaminants, we constructed a toxicity sensing strain Acinetobacter baylyi ADP1_recA_lux. The ADP1_recA_lux is a chromosomally based bioreporter which makes the sensing system stable and negates the need for antibiotics to maintain the trait. The ADP1_recA_lux is activated to express bioluminescence when it is exposed to DNA damaging toxicants. Since the ADP1_recA_lux constantly expresses a baseline level of bioluminescence, false negative results are avoided. The host strain, A. baylyi ADP1, is an ideal model strain typical of water and soil bacteria occurring in the natural environment, and it is more robust than E. coli in terms of viability, maintenance, and storage. The expression of reporter genes - luxCDABE cloned from Photorhabdus luminescens - is robust in a broad range of temperature (10-40 °C). The ADP1_recA_lux was used to detect a variety of toxic or potentially toxic compounds including mitomycin C (MMC), methyl methanesulfonate, ethidium bromide, H2O2, toluene, singlewall nanocarbon tubes (SWNCT), nano Au colloids (20 nm), pyrene, beno[a]pyrene, and UV light. These exposures revealed that the ADP1_recA_lux was able to detect both genotoxicity and cytoxicity, qualitatively and quantitatively. The optimal induction time of the ADP1_recA_lux bioreporter was 3 h, and the detection limits for MMC and benezo[a]pyrene were 1.5 nM and 0.4 nM, respectively. The ADP1_recA_lux was also used to detect toxicity of groundwater contaminated by a mixture of phenolic compounds, and the bioreporter toxicity detection was in a good agreement with chemical analysis. The optimized whole cell bioreporter ADP1_recA_lux could be * Corresponding author phone: +44 (0)114 2225796; fax: +44 (0)114 2225701; e-mail: [email protected]. † Tsinghua University. ‡ University of Sheffield. | State Key Joint Laboratory of Environment Simulation and Pollution Control. § University of Oxford. 10.1021/es901349r CCC: $40.75

Published on Web 09/11/2009

 2009 American Chemical Society

valuable in providing a simple, rapid, stable, quantitative, robust, and costly efficient approach for the detection of toxicity in environmental samples.

Introduction A broad range of contaminants (e.g., polycyclic aromatic hydrocarbons (PAHs)) has been designated by the European Union (EU) and U.S. Environmental Protection Agency (EPA) as priority pollutants because of their carcinogenic and mutagenic effects on humans and animals. However, the toxicity data for these specific contaminants in soil and groundwater are very limited. The assessment of environmental risks associated with aqueous and soil contamination requires a rapid and efficient method to evaluate their potential toxic impact on the environment and human health. Contaminants in the environment may exert two types of toxicity effects on human and animal cells. One type is cytoxicity, related to inhibition of enzyme activity, which usually results in an acute and immediate effect. The compounds responsible for cytoxicity can be enzyme inhibitors such as cyanide and heavy metal ions. Another type of toxicity is genotoxicity, which involves deleterious actions on cellular genetic materials (DNA or RNA) and is usually related to cancers and other genetic diseases. High concentrations of genotoxic compound can also result in cytoxic effects. Sometimes genotoxicity may not cause an immediate disease syndrome in humans or animals, but it has a longterm mutagenic effect on DNA integrity. Since chemical compounds are continually entering the environment, it is essential for safety assessments to effectively identify contaminants that could potentially damage DNA and lead to cancer and other genetic diseases. Chemical analysis-based toxicity assessment cannot detect the bioavailability of toxic compounds, as some compounds may bind to clay and other soil particles, making them unavailable to exert toxic effects. In addition, chemical analysis is not ideal for assessing environmental samples containing complex chemical mixtures which have additive, antagonistic, and synergistic toxic effects (1, 2). For example, a recent study in which the concentration of each compound in a mixture was below individual detectable toxicity effect showed the mixture was detrimental to fish (1). Traditionally, biological toxicity assessment includes animal tests and the Ames Salmonella typhimurium/microsome mutagenicity assay (Ames test) (3–7). Animal testing is less desirable as it is laborious and costly, and it requires the sacrifice of animals (2, 8, 9). Since all living organisms, including bacteria, animals, and humans, employ DNA to store genetic information, it is believed that a chemical capable of disrupting bacterial DNA would also have a similar effect on humans (3, 5, 6). The Ames test is basically a bacterial gene (his) reversion assay and has been widely accepted and used since the 1970s (3, 5, 6). However, the Ames test requires a large quantity of the test compound (1 g) and at least a 72 h incubation period (7, 10), which hampers its widespread use for either online or in situ monitoring. Since DNA damage can manifest itself in many different forms (e.g., point mutation, deletion, insertion, break, dimer formation, cross-linking, rearrangement), the Ames test must use a cocktail of his- Salmonella tester strains to encompass different types of DNA damage, each for a specific DNA target (4, 7). In recent years, a different approach has been developed using whole cell biosensors to detect chemicals (11–15) or toxicity (16–20). The whole cell biosensors have advantages over other detection methods because they 1) can detect VOL. 43, NO. 20, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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unpredictable additive toxic effects of complex mixtures; 2) can be used to simulate the response of human or animal cells to toxic compounds, as it is the response of the cells themselves that is monitored; 3) are highly sensitive, low cost, time-efficient, and easy to use (8); and 4) can be used for in situ or online measurement of contaminant toxicity, making them a desirable alternative to complex, costly, and labor-intensive chemical analysis or animal tests. To date, most toxicity bioreporters have used Escherichia coli, Salmonella sp., or Vibrio fischeri. There have been few, if any, reports regarding the development of bioreporters which can be used to detect both cytoxicity and genotoxicity simultaneously, while remaining stable and robust for industrial applications. In this study, we developed and applied a novel strategy for constructing a chromosomally based whole cell bioreporter ADP1_recA_lux using a soil bacterium Acinetobacter baylyi ADP1 as the host and applied this bioreporter to assess the bioavailability and toxicity of contaminants in groundwater. The ADP1_recA_lux bioreporter is chromosomally based, making it more stable than plasmid-based bioreporters and eliminating the need for antibiotics to maintain plasmids. We have used the ADP1_recA_lux bioreporter to test a variety of chemical compounds including polycyclic aromatic hydrocarbons (PAHs, e.g. pyrene and benzo[a]pyrene) and groundwater contaminated with a mixture of organic chemicals. The ADP1_recA_lux bioreporter demonstrated high sensitivity, specificity (to DNA damage), and robustness and is able to detect not only genotoxicity but also cytoxicity qualitatively and quantitatively.

Experimental Section Bacterial Strains, Plasmids, and Chemicals. The bacterial strains and plasmids used in this study are listed in Supporting Information Table S1. Unless otherwise stated, all chemicals were analytical grade reagents (Sigma UK). NADPH was from Promega, UK. The Luria-Bertani (LB) medium was used for cultivation of bacteria and induction studies. Where appropriate, antibiotics were used with a final concentration of 100 µg/mL ampicillin (LBA100) and 50 µg/mL kanamycin (LBK50) for Escherichia coli and of 150 µg/mL ampicillin (LBA150) and 10 µg/mL kanamycin (LBK10) for Acinetobacter baylyi ADP1 and ADP1_recA_lux. Primers were purchased from MWG Biotech and are listed in Supporting Information Table S2. Polymerase chain reaction (PCR) was carried out in 50 µL reactions containing 1 × reaction buffer, 0.2 mM of each deoxynucleoside triphosphate (Bioline), 0.2 µM of each primer, and 1-2 unit Taq DNA polymerase (Sigma). Molecular cloning of the ADP1_recA_lux is described in the Supporting Information. The ADP1_recA_lux Detection of DNA Damaging Compounds. Bioluminescence and Growth Measurement. To test the performance of bioreporter ADP1_recA_lux, the bioluminescence and OD600 were measured using a Synergy HT Multi-Detection Microplate Reader (Bio-Tek, UK). Measurement parameters were optimized. The parameters include inoculation ratio of overnight culture to fresh medium (1:10 and 1:25), incubation time before exposure to MMC (0, 3, 13-16 h), incubation and measurement temperature (28 and 37 °C), and shaking during incubation (150 rpm or no shaking). The optimization condition was determined as follows. One colony of A. baylyi ADP1_recA_lux was inoculated into 5-mL LBK10 liquid medium and incubated at 30 °C overnight (14-16 h) on a rotary shaker of 150 rpm. The cells of the overnight culture were harvested by centrifugation at 3000 rpm for 5 min and resuspended into 50-mL fresh LBK10 liquid medium. Two hundred µL of the cells and a 2 µL sample were transferred into each well of a 96-well microplate (96 well Optical flat bottom, Nunc) and mixed immediately for bioluminescence and OD600 measure7932

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ment. The 96-well microplate was incubated at 37 °C in a Synergy HT Microplate Reader (Bio-Tek, U.K.), and bioluminescence and OD600 were continuously recorded every 30 min for 16 h. All sample assessments were carried out with 3-12 replicates. Relative bioluminescence (absolute intensity of bioluminescence divided by OD600) was used to estimate the bioreporter induction level and genotoxicity. The induction factor was defined as relative bioluminescence of the sample divided by relative bioluminescence of a toxicant-free control. Kinetic Analysis of Bioluminescence Induced by MMC. MMC was added immediately into A. baylyi ADP1_recA_lux to achieve a final concentration of 0, 0.0015, 0.003, 0.015, 0.03, 0.15, 0.3, 1.5, 3, and 15 µM. The bioluminescence and OD600 of each sample were measured every 30 min at 37 °C. During sampling, the samples were shaken inside the Synergy HT Microplate Reader. The ADP1_recA_lux Induced by Other Compounds. Besides MMC, methyl methanesulfonate (MMS, 1800 µM), ethidium bromide (25 µM), H2O2 (100 µM), toluene (1000 µM), native SWNCT (200 µg/mL), and nano Au colloids (citrate coated, 20 nm, 0.68 µM) were directly added into the ADP1_recA_lux without any pretreatment. Ultraviolet (UV) radiation to bacteria was also investigated as follows: the cells were placed in a 96-well plate (Nunclon surface plate, Nunc) and then exposed to UV in a GeneFlash System (Syngene Bio Imagine, Model GVM20). The surface power of the UV light was 70 w/m2, and cell exposure time was 80 and 600 s. After UV irradiation the 96-well microplate with cells was loaded on the microplate reader for induction measurement. The ADP1_recA_lux Induced by Pyrene and Benzo[a]pyrene. Pyrene (PYR) and benzo[a]pyrene (BaP) were initially dissolved in dimethyl sulfoxide (DMSO) to produce 10 mg/ mL PYR/BaP stock solution. A series dilution of PYR (final concentration of 2.5 × 10-3, 2.5 × 10-2, 2.5 × 10-1, and 2.5 µM) and BaP (final concentration of 4.0 × 10-4, 2.0 × 10-3, 2.0 × 10-2, and 2.0 × 10-1 µM) was generated by serial dilution of the stock solution in 10% DMSO (v/v). The sample was tested with or without addition of the S9 enzyme (Sigma, UK). For the non-S9 treatment, the overnight culture of ADP1_recA_lux was diluted with fresh LB medium to 1:10, and 180 µL of suspended cells was then mixed with 2 µL of each diluted PYR or BaP sample plus 18 µL water in a 96-well plate. For the S9 treatment, 180 µL of cells was mixed with 1.25 µL of 20 × NADPH regeneration system solution A and 0.25 µL 100 × NADPH regeneration system solution B (Promega, UK), 2 µL of S9 (20 mg/mL), and 1.5 µL of water was mixed with 2 µL of each diluted PYR or BaP sample and 18 µL of water. The toxicant-free controls were prepared by adding all the above reagents but replacing PYR and BaP with water. The detection limit is determined when the relative bioluminescence induced by lowest concentrations of toxicants are greater than toxicant-free controls (p value 600 s) significantly inhibited cell growth (Figure S3) and reduced bioluminescence to less than the baseline level (Figure 3). This suggests that overexposure to high doses of genotoxic compounds could damage DNA so significantly that such damage decreases the cellular metabolic activity or kills cells. Based on U.S. EPA guidelines, toluene is not classified as to a human carcinogen (U.S. EPA, 1992). In agreement with this standard, we found that 1 mM toluene did not induce bioluminescence expression of ADP1_recA_lux but reduced bioluminescence lower than the baseline (Figure 3). The cell growth was not affected by 1 mM toluene (Figure S3), which suggests that toluene did not damage DNA but inhibited cellular activity. We found that 6.8 µM 20 nm Au colloids and native SWNCT did not show any genotoxic or cytoxic effects (Figure 3) and also had no detectable effect on the ADP1_recA_lux growth (Figure S3). The ADP1_recA_lux Quantitative Response to Pyrene and Benzo[a]pyrene with or without S9. BaP and PYR in the range of 0-200 µM and 0-250 µM, respectively, did not affect cell growth (Figure S4), although both compounds strongly induced bioluminescence of the bioreporters ADP1_recA_lux (Figure 4). It suggests that PYR and BaP are mutagenic (DNA damaging) compounds, but they did not show an acutely toxic effect. To illustrate the induction level of these two different compounds, the induction factor (IF) was used to estimate the toxicity effects. A. baylyi ADP1_recA_lux can be directly induced 7934

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FIGURE 4. The bioluminescence of ADP1_recA_lux can be induced by high-molecular weight PAHs (pyrene and benzo[a]pyrene). The addition of S9 improved detection. Benzo[a]pyrene (A) was more toxic than pyrene (B). by PYR and BaP without addition of S9 (Figure 4), but the inductions were higher in BaP with the presence of S9. This suggests that S9 acted as a P450 metabolic activation mixture, which has converted BaP to highly reactive diolepodies that can initiate the DNA-damaging reaction (48). Generally, there was a trend that the induction of A. baylyi ADP1_recA_lux became greater with increasing concentration of PYR and BaP (Figure 4). BaP was a more potent genotoxic compound than pyrene because the IF of BaP was 1.96 at a concentration as low as 2 × 10-3 µM (Figure 4A), while the IF of pyrene reached 1.91 at 2.5 µM (Figure 4B). This result is in a good agreement with other reports (23). Toxicity Assay for Contaminated Groundwater. Previous work has established the spatial distribution of biodegradation processes and microbiological activity within the aquifer at this field site (21, 22). The plume fringe has been shown to be the most microbiologically active zone in this and other contaminant plumes (24, 25), and microbial activity in the core of this plume is inhibited by the

proposed that this effect arises because the fringe of this plume harbors highly reactive genotoxic chemicals, associated with the increased phenol biodegradation activity here, whereas this did not occur in the plume core.

Discussion

FIGURE 5. The bioreporter ADP1_recA_lux was applied to detect toxicity of groundwater samples contaminated by phenolic compounds. (A) Profiles of total phenols and nitrate in groundwater obtained from a multilevel sampler installed at BH59 in the contaminant plume, showing background water quality, plume fringe, and plume core, with locations (arrows) of samples collected in this study. (B) The groundwater at the plume fringe showed a genotoxic effect, while the groundwater at the core of the plume indicated a cytoxic effect. contaminant matrix, presumably the high concentration of phenols or other compound(s) (26). The toxicity assessed by the bioreporter ADP1_recA_lux reflected the general level of contamination at the sampling points. The control groundwater sample, which was considered to be representative of the background groundwater outside of the contaminant plume, had the lowest toxicity (Figure 5). Interestingly, groundwater samples from the core of the plume (BH59: 14 to 27 m depth), where microbiological activity is relatively suppressed (21), have also reduced bioluminescence due to the high concentration of phenols (Figure 5 and Table S3 in the Supporting Information). In contrast, the samples (BH59: 10 and 30 m depth) from the plume fringe (Figure 5A), where microbiological activity is greatest (21), show genotoxic effects (Figure 5B). It is

Bacteria in the natural environment are constantly exposed to cosmic radiation and chemical hazards which provoke DNA damage. To maintain the integrity of bacterial genomes, bacteria have evolved an ‘SOS system’ that is used to rapidly detect and repair all types of DNA damage. The response of the SOS system is to activate a few genes associated with DNA repair; the response time can be as short as a few minutes. One of the key genes involving the SOS response to DNA damage is recA, which is universally present in bacteria, fungi, plants, and humans. RecA gene in A. baylyi ADP1 is induced and involved in DNA damage (29, 30), and it is also required for DNA repair (31). Contaminants in soil and water that can damage bacterial DNA would have a similar effect on human DNA, which links the whole cell bioreporters to human health (27, 28). A. baylyi ADP1 can naturally take up DNA from the environment and integrate the foreign DNA into its chromosome if the homologous DNA is present. This makes it an ideal model strain for gene manipulation and expression of heterologous genes (32), and we have used A. baylyi ADP1 to construct a variety of bioreporters (12–14). Many researchers have used whole cell biosensors, mostly E. coli or Salmonella sp., to detect general toxicity and genotoxicity (16, 20, 33–37). In an attempt to optimize the bioreporter system, we have fused luxCDBAE into recA of the chromosome of A. baylyi ADP1. There are several advantages of this new bioreporter. A comparison of the ADP1_recA_lux with Ames and other toxicity bioreporters is listed in Table 1. First, the ADP1_recA_lux bioreporter is a chromosomally based system. It has been proved that chromosomally based gene expression is more robust, reliable, and sensitive than plasmid based gene expression (16, 38). A group at MIT has demonstrated that the plasmid based gene expression inevitably suffers instability due to plasmids segregational and structural instability and allele segregation (38). The ADP1_recA_lux bioreporter should overcome the genetic instability, and it should remain extra stable because the recA gene has been knocked out. The ADP1_recA_lux bioreporter is also more convenient in practice since it negates the need for addition of any extra compounds, for example antibiotics to maintain plasmids. Second, the ADP1_recA_lux bioreporter could be either a ‘light-on’ or ‘light-off’ system depending on types or extents of toxicity. Toxicant activation bioreporter’s bioluminescence is referred to as a light-on, while diminishing bioluminescence is a light-off system (2). Most genotoxicity bioreporters are designed as light-on systems, in which toxicants damage DNA and hence activate reporter genes fused with SOS repairing genes. A key problem with lighton genetoxicity bioreporters, which restricts their practical application, is that it may generate false negative results where the decrease or lack of a bioreporter’s bioluminescence is due to high concentrations of toxicants or lethal compounds (9). A constitutive expression of a certain level (Figure 2 baseline) of recA in the ADP1_recA_lux can be exploited to avoid the false negative problem, because the decreased baseline indicates that the bioreporter was inhibited or inactivated (Figures 3 and 5). Third, the ADP1_recA_lux may be more suitable and robust for bioavailability and bioaccessibility testing of contaminants in soil and water. Acinetobacter sp. is a Gram-negative strain distributed in a broad range of natural environments, with typical populations of 105/g of soil and 105/mL of VOL. 43, NO. 20, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Comparison of the ADP1_recA_lux with Other Bioreporters genotoxicity test Ames

host strain Salmonella sp.

structure and stability reverse point mutations

Salmonella typhimurium.

MMC: 0.2

Umu fluxCDABE (Vibrio Umu test fischeri); plasmid based, less stable, need addition of antibiotics RecA fluxCDABE (Vibrio recA:luxCDABE Escherichia coli fischeri); plasmid based, less stable; need addition of antibiotics Salmonella RecNfluxCDABE (Vibrio Vitotox typhimurium. fischeri); plasmid based, less stable; need addition of antibiotics CdafluxCDABFE SOS lux test Escherichia coli (Photobacterium leiognathi); plasmid based, less stable; need addition of antibiotics Cdaf gfp (green fluorescent SOS gfp test Escherichia coli protein); plasmid based, less stable; need addition of antibiotics RecA fluxCDABE Acinetobacter ADP1_recA_lux (Photorhabdus luminescens); baylyi ADP1 chromosomally based, highly stable; no need of any extra compounds a

ref 20

H2O2: a

a applied to complex samples

MMC: 150

a

20

H2O2: 1.32 × 106

applied to complex samples

MMC: 14

a

35

MMC: 47

a

52

H2O2: 5.8 × 104

applied to complex samples

MMC: 4.3

a

20

applied to soils

17

at least 8 weeks at at 4 °C; directly applied to complex samples e.g. soils and groundwater

this study

H2O2: 2.0 × 104

H2O2: 8.5 × 104 MMC: 9 H2O2: 2.4 × 105 MMC: 1.5 H2O2 < 102

Note: ‘-‘ no data.

water (39, 40). The bioreporter ADP1_recA_lux can be a good representative species of soil and water bacteria, in terms of sensing bioavailability under environmental conditions. Finally, the bioluminescence reporter gene luxCDABE is more sensitive and rapid than other reporter genes, such as green fluorescent protein (GFP) and lac genes (2, 41, 42). The reporter genes luxCDABE in the bioreporter A. baylyi ADP1_recA_lux originated from Photorhabdus luminescens (43, 44), which are self-sufficient without the need to add any substrate to activate the bioluminescence. This genotype luxCDABE can be operated in a wider temperature range (10-40 °C) than luxCDABE from Fisheri vibro (12–14). More importantly, for practical applications, one of the main concerns is preserving and maintaining the active bacterial bioreporter (45). In our lab, we found A. baylyi ADP1_recA_lux can maintain viability and activity for at least 8 weeks, while E. coli barely survive for 2-3 weeks, when both were stored in liquid media or on agar plates at 4 °C. Therefore, the bioreporter ADP1_recA_lux integrates the benefits of both light-on and light-off systems into one detection system and provides a simple, stable, sensitive, robust, and rapid way to detect the toxicity of environmental samples. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous organic contaminants in environments such as sediments, soils, and groundwater, due to transportation, processing and distribution of fossil fuels, and combustion of organic material (46). One-, two-, and three-ring hydrocarbons are acutely toxic, while higher-molecular weight PAHs are genotoxic, causing DNA damage and potentially cancers (46). PAH contaminants such as benzo[a]pyrene pose a threat to human and animal health because of their mutagenic effect (46, 47). In most cases, higher molecular weight PAHs (e.g., benzo[a]pyrene) are usually not genotoxic unless they are oxidized to mutagenic compounds by oxygenase enzymes (48). Human or animal cytochrome P450s (CYP), which are usually monoxygenases, can convert PAHs to mutagens 7936

storage and application to complex samples

sensitivity (nM)

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(DNA-damaging compounds) (49, 50). In order to detect the toxicity of PAHs, it is required that the bioreporter bacteria can exert such oxidization, otherwise the SOS system cannot be activated. There is evidence suggesting that Acinetobacter sp. can oxidize a pyrene and other PAHs (46). A. baylyi ADP1 was originally isolated from soil and is a nutritionally versatile chemoheterotroph able to metabolize a large range of organic compounds (40, 51), and ADP1 harbors various enzymes for the oxidization of hydrocarbons. These enzymes may spontaneously or fortuitously convert pyrene or benzo[a]pyrene to genotoxic compounds. Figure 4 shows that bioluminescence of ADP1_recA_lux can be induced by pyrene and benzo[a]pyrene even in the absence of S9. It may suggest that the ADP1_recA_lux has converted pyrene and benzo[a]pyrene to mutagenic compounds and activated the recA gene. The bioreporter A. baylyi ADP1_recA_lux can easily detect pyrene and benzo[a]pyrene as low as 0.4 × 10-3 µM (∼0.0001 mg/L) and 2.0 × 10-3 µM (∼0.0005 mg/L), respectively, which covers the EPA wastewater standards for pyrene (0.067 mg/L) and benzo[a]pyrene (0.061 mg/L). The benzo[a]pyrene detection limit (0.0001 mg/L) is even lower than the EPA drinking water standard (0.0002 mg/L). This is the first report of the development and use of a chromosomally based soil bacterial bioreporter for the detection of pyrene and benzo[a]pyrene as well as contaminated groundwater. The bioreporter A. baylyi ADP1_recA_lux can therefore potentially contribute to the routine monitoring of the genotoxicity and cytoxicity of environmental samples.

Acknowledgments We thank NERC UK (project code NE/F011938/1) for funding this research. We also thank China National Natural Science Foundation (Project 40730738) for funding. We are grateful for a Chinese Council Scholarship to support S.Y. to visit the University of Sheffield for this study. We also thank Prof. Peter Dobson at Oxford University for providing SWNT and nano Au colloids.

Supporting Information Available Molecular cloning of the ADP1_recA_lux, Tables S1-S3, and Figures S1-S4. This material is available free of charge via the Internet at http://pubs.acs.org.

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