Noncovalent Binding of Polycyclic Aromatic Hydrocarbons with

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Noncovalent Binding of Polycyclic Aromatic Hydrocarbons with Genetic Bases Reducing the in Vitro Lateral Transfer of Antibiotic Resistant Genes Fuxing Kang,# Xiaojie Hu,# Juan Liu, and Yanzheng Gao* Institute of Organic Contaminant Control and Soil Remediation, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China S Supporting Information *

ABSTRACT: In current studies of noncovalent interactions of polycyclic aromatic hydrocarbons (PAHs) with genetic units, the impact of such interactions on gene transfer has not been explored. In this study, we examined the association of some widely occurring PAHs (phenanthrene, pyrene, benzo[g,h,i]perylene, and other congeners) with antibiotic resistant plasmids (pUC19). Small molecular PAHs (e.g., phenanthrene) bind effectively with plasmids to form a loosely clew-like plasmid−PAH complex (16.5−49.5 nm), resulting in reduced transformation of ampicillin resistance gene (Ampr). The in vitro transcription analysis demonstrated that reduced transformation of Ampr in plasmids results from the PAH-inhibited Ampr transcription to RNA. Fluorescence microtitration coupled with Fourier transform infrared spectroscopy (FTIR) and theoretical interaction models showed that adenine in plasmid has a stronger capacity to sequester small Phen and Pyre molecules via a π−π attraction. Changes in Gibbs free energy (ΔG) suggest that the CT−PAH model reliably depicts the plasmid−PAH interaction through a noncovalently physical sorption mechanism. Considering the wide occurrence of PAHs and antibiotic resistant genes (ARGs) in the environment, our findings suggest that small-sized PAHs can well affect the behavior of ARGs via above-described noncovalent interactions.

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INTRODUCTION The widespread of antibiotic resistant genes (ARGs) in the environment1−3 and their dissemination among various organisms4,5 pose a threat to public health. During the lateral genetic transfer (LGT) across organisms, free antibioticresistant plasmids (ARPs) may contract various substances and thus interact with many conventional pollutants,6,7 such as polycyclic aromatic hydrocarbons (PAHs).8,9 The interaction between plasmid and polycyclic aromatic hydrocarbons (PAHs) is dominated by known noncovalent interactions10 in the lack of biological enzymes. Nevertheless, it is not known whether this noncovalent PAH−plasmid interaction could affect the lateral transfer of ARGs, which awaits a proper investigation. PAHs, detected widely in natural water, sediment, and soils are usually chemically inert but can be readily taken up by plasmids through some physical interactions10−12 under a lack of active biological enzymes. For instance, the fluorescence studies demonstrated that small PAH molecules, such as phenenthrene (Phen) and pyrene (Pyre), can bind genetic bases, thus impeding the biological enzymolysis of DNA.8 Recent studies through computational chemistry revealed that noncovalent interactions of aromatics with free extracellular DNA can occur in vitro by molecular attraction via van der Waals’ forces (vdWs), hydrogen bonding, etc.10,13 These suggest that the strong affinity of certain PAHs to genetic bases may also alter the genetic or biological properties of © XXXX American Chemical Society

DNA. However, the biological significances of these noncovalent PAH−genetic interactions have not been sufficiently understood so far. Attractions between PAHs and genetic bases, such as by vdWs and H-bonding, are generally multifarious and almost imperceptible. Moreover, types of attractions or interactive sites cannot be discriminated precisely by conventional physical− analytical methods, such as by batch adsorption and potentiometric titration. Therefore, the main goal of the present study is to explore whether the genetic base−aromatic interaction affects the in vitro lateral transfer of antibiotic resistance genes through an understanding of the genetic base− PAH interactions. PAHs of different molecular sizes (including structural isomers) and plasmid pUC19 containing ampicillin resistance genes (Ampr) were used for investigation. The bacterial E. coli was used as host cells for plasmids. The fluorescence-microtitration technology in combination with the atomic force microscopy (AFM), scattering spectroscopy, and Fourier transformed infrared spectroscopy (FTIR) was used to assess the noncovalent interactions between genetic bases and PAHs. The efficiency of the Ampr transformation as influenced by the noncovalent plasmid−PAH interactions was determined. Received: May 8, 2015 Revised: July 27, 2015 Accepted: August 4, 2015

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DOI: 10.1021/acs.est.5b02293 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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of ampicillin sodium. The Petri dish was left upright for 30 min to ensure that the E. coli medium was fully imbibed by the solid culture medium. The dish was then inverted for 36 h at 37 °C. In addition, in order to rule out the effect of PAHs or methanol on E. coli growth, only PAHs or methanol (0−0.6 μmol/L) was added to E. coli medium containing ampicillin sodium (100 mg/L). The transfer efficiency was defined as the log ratio of the number of transformants (unit number) versus the mass of the added plasmid (0.25 μg). Transcription of Ampr Sequences in Vitro. The Ampr sequences were amplified with the addition of a T7 promoter (5′-TAATACGACTCACTATAGG-3′) by PCR using the pUC19 plasmid DNA as a template, with the designed DNA primers (5′-GGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTAT-3′ and 5′-GCGTAATACGACTCACTATAGGGAAAAAGGCCGCGTTGCTG-3′). The polymerase chain reaction (PCR) mixture (20 μL) contained 2 μL of DNA template (5 ng/μL), 10 μL of Premix Taq (TaKaRa, Premix Taq Version 2.0), and 0.5 μL of primers. The PCR was performed in a DNA Engine Thermal Cycler (TaKaRa, D8308), and the PCR program consisted of an initial denaturation at 94 °C for 5 min, 30 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, followed by a final extension step at 72 °C for 10 min. Obtained PCR products were examined by agarose gel electrophoresis. Afterward, trace PAHs were injected into the product system (about 1.1 ng, Ampr) to construct concentration gradients (PAHs, 0−0.5 μmol/L). Following 200 rpm shaking at 25 °C for 2 h, the products with PAHs were transferred into RNA with the Riboprobe in vitro transcription systems kit (from Promega Corporation). After being purified with the use of a RNA purification kit, the resulting RNA was quantified by real-time fluorescent PCR. Fluorescence Quenching Titration of PAHs by Plasmid. Three-dimensional excitation−emission matrix fluorescence spectroscopy combined with microtitration (EEMQM) was used to study the noncovalent plasmid−PAH interaction, as described previously.8 The plasmid stock solutions (10 mg/L) were gradually titrated into 20 mL of aqueous PAH solutions (0.056 μmol/L) using a chromatographic injector with an amount of 50 μL, followed by magnetic stirring for 20 min at 160 rpm, pH 7.0, and 25 °C. The fluorescent spectra and corresponding intensities were recorded at excitation (EX) wavelength of 250−310 nm and emission (EM) wavelength of 300−550 nm (the wavelength resolution being ≥2 nm) (F96PRO, LengGuang). The titration-reaction measurement was repeated until no significant change in fluorescence intensity was observed. The maximum concentrations of the added plasmid quencher in all PAH solutions (20 mL) were less than 6.0 × 10−12 mol/L. The examined maximal peaks (EX/EM, nm/nm) for six PAHs occur at 251/ 403 for Anthr, 271/364 for Phen, 341/389 for BenzAnthr, 332/ 375 for Pyre, 361/409 for BenzPyre, and 300/432 for BenzPery. The plasmid−PAH interaction could be well described in terms of the fluorescence intensity versus the quencher (plasmid) concentration using the Stern−Volmer equation16

The weak molecular attractions and binding energies obtained from molecular computation are used to discriminate the association mechanisms of a PAH with the genetic units. The Gibbs free energy change (ΔG) from molecular computation and the fluorescence microtitration data are used to explain the influence of the PAH−plasmid interaction energy on the reduced transformation of Ampr in plasmid.

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MATERIALS AND METHODS Materials. Test PAHs, anthracene (Anthr), phenanthrene (Phen), 1,2-benzanthracene (BenzAnthr), pyrene (Pyre), benzo[a]pyrene (BenzPyre), and benzo[ghi]perylene (BenzPery) (purity ≥98%) were purchased from Sigma-Aldrich. A short circular plasmid pUC19 (2686 base pairs) containing ampicillin resistance genes (Args), known well as the carrier of emerging genetic contaminants, was purchased from Takara Biotechnology Co., Ltd. (China). Escherichia coli (E. coli DH5α) preserved in our laboratory was used to prepare the competent cells, with the preparation method described earlier.14 Peptone, yeast extract, agar, and D-glucose in biotechnology grade, used as culture medium, were purchased from Sigma-Aldrich. Ampicillin sodium (>98%) was also purchased from Sigma-Aldrich. Physicochemical properties of PAHs, including the octanol−water partition coefficient (KOW), water solubility (SW), and molecular weight, are given in Table S1. Ultrapure water obtained via a Milli-Q gradient system (electric conductivity 18.2 MΩ × cm, Millipore, Bedford, MA, USA) was used to perform all experiments. Transformation of Ampr Exposed to PAHs. Experiments were performed in 5 mL brown glass vials equipped with Tefln-lined butyl rubber stoppers. Plasmid pUC19 (25 μg) was dissolved in a Tris-HCl solution (10 mmol/L, 500 μL, pH 7.0) in glass vials. The 5 μL of aqueous plasmid pUC19 was again added to Tris-HCl solution (10 mmol/L, 500 μL, pH 7.0) in glass vials to obtain the diluted plasmid (0.5 μg/mL). The test PAHs in methanol were added into the plasmid pUC19 at the level of 0−0.6 μmol/L, with the methanol−water ratio kept below 0.1%, and the PAH−plasmid mass ratio was 0−0.24. The samples were gently shaken manually for 5 s and then left to stand for 120 min to facilitate the association of the test PAH with plasmid pUC19. Aqueous samples were centrifuged in a 1.5 mL ultrafiltration centrifugal tube (molecular weight cut off 3000 Da, Millipore, USA) for 10 min at 4 °C and 6000g to save the plasmid (0.25 μg of plasmid), with the water-soluble PAH discarded. Here, the adsorbed PAHs, calculated as difference between total and water-soluble phase,15 were about ∼28 ± 0.3 μg/mg. The plasmid−PAH mixture was manipulated to transform to competent cells. Preparation of competent cells is described earlier.14 The plasmid pUC19 (0.25 μg) obtained by the above centrifugal process was dissolved in Tris-HCl solution to the original volume (500 μL) and then added to the competent cells (200 μL, OD560 nm = 0.4). The mixture of cells and plasmids was placed in an ice−water bath for 3 min, followed immediately by a heat-shocked stimulation at 42 °C for 90 s. Then, the samples were placed in an ice−water bath for 3 min. The 200 μL of bacterial solutions was inoculated to the SOC medium (750 mL, 2% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.05% (w/v) NaCl, 2.5 mmol/L KCl, 10 mmol/L MgCl2, and 20 mmol/L D-glucose), followed by incubation at 120 rpm for 60 min at 37 °C to express the Args. The 100 μL of bacterial liquid (about 300 cells) was uniformly spread on the surface of the LB solid culture media containing 100 mg/L

F0 = 1 + Kqτ0[Q ] = 1 + KSV[Q ] F

(1)

where F0 and F are the PAH fluorescence intensities without and with the quencher (plasmid), respectively. Kq is the bimolecular quenching rate constant; τ0 is the average lifetime of the fluorophore in the absence of quencher, and [Q] is the B

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Figure 1. AFM images (a−d) and scattering spectra (e) of plasmid DNA after reaction with PAHs. (a) plasmid only; (b) plasmid plus Phen; (c) plasmid plus Pyre; (d) plasmid plus BenzPery. (e) The scattering spectra were recorded by the synchronous fluorescence technique (excitation/ emission: 200−810 nm). The Raman, Mie, and Rayleigh scattering peaks were located at 325, 405, and 470 nm, respectively.

concentration of the quencher. KSV is the Stern−Volmer quenching constant. For a static quenching process, the following equation is used to determine the binding constant or association constant (KA) and the number of binding sites (n):17 log[(F0 − F )/F ] = log KA + n log[Q ]

combinations of four genetics (AG, AA, GG, AT, AC, and GC) using the Gabedit Software (Version 2.4.5)22 and computed their Gibbs free energies when combined with Phen and Pyre at 25 °C. It appears that the CT−Phen or CT−Pyre combination gives the lowest free enthalpy as compared to other combinations (the analytical process not shown). Finally, a simplified model for the genetic bases (5′-CT-3′) was employed to explore the noncovalent interaction between them. The geometry of PAHs was structured using the ChemBioOffice Software.23 All constructed geometries including the 5′-CT-3′ model and PAHs were initially optimized by using the Semi-Empirical Method PM7 (Mopac2012 program24), followed by both structural optimization and frequency analysis using the density functional theory (DFT) at a main def2-TZVP basis set, an auxiliary def2-TZVP/J basis set with dispersion-corrected DFT-D3 and GCP (DFT/ SVP).25−30 The wave functions obtained by computation were analyzed by the Multiwfn program,31 which would obtain the information about reduced density gradient (RDG), the bonding energy, and the Lorentz oscillator. The determined Lorentz oscillator was further used to draw the FTIR spectra for comparison with the experimental FTIR before and after the association of PAHs and plasmids. Solvent (water) effects were taken into consideration implicitly in the computation. Discriminating Binding Types. After obtaining the wave function for a PAH-5′-CT-3′ complex by the above model computation, a topological analysis and a graphic illustration of the distribution of the electron density were then conducted using the Multiwfn program.31 According to previous studies,10 the van der Waals (vdWs) attraction (including the π−π interaction), the H-bond attraction, and the nonbonded contact (such as the electron steric crowding toward aromatic nucleus) can be discriminated by the quantum-mechanical electron density (ρ(r)).32 The reduced electron density gradient (RDG), derived from the electron density and its first derivative, was a fundamental dimensionless quantity in DFT

(2)

Additionally, the synchronous fluorescence spectroscopy (EX/EM: 200−810 nm) was performed to measure the light scattering of aqueous plasmid sols before the titration with PAHs18,19 and to screen for the formation of plasmid−PAH floccules in sols after the titration with PAHs. AFM and FTIR Analyses. The atomic-force microscopic (AFM) analysis was applied to characterize the configuration of the plasmid20 before and after reaction with Phen, Pyre, and BenzPery. After mixing the plasmid with PAHs for 120 min at 25 °C, the aqueous samples were centrifuged in a 1.5 mL ultrafiltration centrifugal tube with a polyethersulfon membrane having a 3000 Da molecular weight cutoff (Millipore, USA) for 10 min at 4 °C and 6000g. The plasmid samples were resuspended, washed by Milli-Q water, and centrifuged three times to remove the free unadsorbed PAH in aqueous solution. The plasmid was redissolved by 1.5 mL of Milli-Q water, and then, it (200 μL) was titrated on the surface of mica at 37 °C until the samples became dry.21 Plasmid samples were imaged with a Multimode 8 AFM (Bruker, Germany). Additionally, a Fourier transform infrared spectroscopy (FTIR) analysis was performed to characterize the chemical structures of plasmid samples for finding the binding sites of a given plasmid−PAH combination. The FTIR spectra of freeze-dried plasmids mixed with KBr (mass ratio of 1:100) were acquired on a Nicolet NEXUS870 (Thermo Scientific, USA). Model Computation. On the basis of the number of determined binding sites for the plasmid−PAH interaction (n = 1) via the fluorescence microtitration combined with FTIR analysis, we structured six molecular models for possible C

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from the Raman scattering of plasmid−BenzPery seems to suggest a completely different conclusion due to its strongest Raman signal at 315 nm. This is likely attributed to the Raman scattering from unabsorbed BenzPery in aqueous solution, because it has a greater hydrophobic diameter than Phen and Pyre.36,37 Therefore, these results suggest that large PAHs are not active to bind the genetic bases of plasmids, presumably because it is difficult for them to penetrate into the inner genetic bases of plasmids even though BenzPery has a greater bioaffinity.38 The plasmid−PAH binding strength can be explored on the basis of the fluorescence quenching of PAHs following their sequestration by the plasmid. Figure 2a shows the Stern−

used to describe the deviation from a homogeneous electron distribution.10 By this analysis, the results were expressed by a colorized gradient isosurface, in which a weak interaction region is described by the equation:10 RDG(r ) =

|∇ρ(r )| 1 2 1/3 2(3π ) ρ(r )4/3

(3)

where the ρ(r), |∇ρ(r)|, and ∇ are the quantum-mechanical electron density, the gradient operator, and the modular arithmetic of the gradient operator for the quantum− mechanical electron density, respectively. From those results, the bonding types via noncovalent interactions were identified and drawn as the interaction isosurface of PAHs and genetic units. Binding Energy. After determination of the interaction sites, the binding energy was also computed using the simplified gene-base model (5′-CT-3′) at the above-mentioned DFT BLYP D3 GCP(DFT/SVP) def2-SVP def2-SVP/J level. It could be shown as follows: binding energy = ΔG = G5 ′‐CT‐3 ′···PAHs − (G5 ′‐CT‐3 ′ + G PAHs) (4)

where G5′‑CT‑3′···PAHs, G5′‑CT‑3′, and GPAHs are the Gibbs free energies (G) of the combined 5′-CT-3′-PAH species after adsorption, 5′-CT-3′, and PAHs, respectively.

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RESULTS AND DISCUSSION Noncovalent Interaction of Genetic Bases with Aromatics. The atomic force microscopy (AFM) images of plasmid pUC19 before and after reaction with PAHs are showed in Figure 1a−d. It provides a visual evidence for revealing the noncovalent interactions between plasmid and PAHs. The 1.2 μm × 1.2 μm planar region including the altitude scale of plasmid was scanned continuously for 20 min. In Figure 1a, the apparent width of the double-stranded DNA (plasmid pUC 19) only is ∼3 nm, which compares favorably with the expected diameter of 16.5−49.2 nm for plasmid−Phen (Figure 1b) and plasmid−Pyre complex (Figure 1c). In Figure 1b,c, the DNA strand of plasmid pUC19 after reaction with Phen and Pyre cannot be observed, but the loosely clew-like plasmid−Phen/Pyre complex can be clearly seen, suggesting that small-sized Phen (0.90 × 0.54 nm) and Pyre (0.89 × 0.66 nm) bind more actively with plasmid pUC19 than BenzPery (0.90 × 0.78 nm), because similar clews cannot be detected from the plasmid−BenzPery mixture (Figure 1d). In addition, it can be seen that there are more dots in Figure 1d, suggesting possibly that BenzPery has caused dissociation of some DNA strands to be in some random configuration. The scattered signals arising from singly sorbed molecules (or molecular complex that behave as single molecules) can be used to study the dielectric surfaces of the plasmid or plasmid− PAH complex in liquids at room temperature.33,34 The intensity of the scattered signal is proportional to the concentration of the plasmid sol.35 In Figure 1e, three distinct signals at 315, 415, and 470 nm are assigned to the Raman, Mie, and Rayleigh scattering peaks, respectively, according to the order they appear.36 With the exception of the Raman peak, both Mie and Rayleigh scattering intensities of plasmids with different PAH treatments appear in the order of plasmid−Pyre > plasmid− Phen > plasmid only ≥ plasmid−BenzPery. This reinforces that small Phen and Pyre exhibit a greater binding with plasmid pUC19 than BenzPery. Nonetheless, another piece of evidence

Figure 2. PAH−plasmid combination probed by plasmid-caused PAH fluorescence quenching. (a) Stern−Volmer plot; (b) Plot of log [(F0 − F)/F] vs log [Q]. This result was obtained by the microtitration experiments. The changes in fluorescence intensity of aromatic nucleus-related chromophores in PAHs were recorded during the plasmid titration. Each datum point shown in (a) and (b) is the average of three measurements.

Volmer plots (F0/F versus Q) for Phen, Pyre, and BenzPery at pH 7.0 and 25 °C (eq 1). By a linear data fitting, the calculated KSV values are 6.8 × 1018 L mol−1 for Phen, 2.5 × 1011 L/mol for Pyre, and 3.4 × 1010 L/mol for BenzPery. For the aromatic nucleus-related chromophores, the fluorescent lifetime (τ0) is known to be (1.5−12.8) × 10−8 s.39−41 Thus, from eq 1, the quenching rate constants (Kq) were calculated to be (0.5−4.5) × 1026, (0.2−1.7) × 1020, and (0.3−2.3) × 1019 L/mol/s for Phen−, Pyre−, and BenzPery−bioconjugates, respectively. Generally, the maximum Kq value for a diffusion-controlled quenching process of a biopolymer is about 2.0 × 1010 L/mol/ s.42 These higher quenching rate constants (Kq) suggest that a static quenching process occurs with the formation of a plasmid−PAH complex.43 A static quenching implies that the plasmid sequestrates PAHs through the formation of a genetic bases−PAH complex. D

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Environmental Science & Technology Figure 2b shows that the log [(F0 − F)/F] versus log [Q] plot follows a linear relationship (eq 2). From the slope of the fitted linear plot, the number of binding sites, n, was found to be 1.2 for Phen, 1.1 for Pyre, and 0.3 for BenzPery, respectively. These n values are approximately 1, except that of the plasmid− BenzPery association, suggesting that there is only a single binding site for small Phen/Pyre with genetic bases. The n value of Pyre ≈ BenzPyre ≈ BenzAnthr > BenzPery. Overall, the inhibitory rate decreases as the number of benzene ring E

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Figure 5. Comparison of the FTIR spectra of plasmid pUC19 before (a) and after reaction with Phen (b), Pyre (c), and BenzPery (d). Blue bar charts and red plots represent the Lorentz oscillators and infrared (IR) spectra from computation for a main def2-TZVP basis set, an auxiliary def2TZVP/J basis set with dispersion corrected DFT-D3 and gcp (DFT/SVP). The black plots are the experimental data. Red arrows represent the absorption bands related to adenine.

increases, i.e., three-ring PAHs > four-ring PAHs > five-ring PAHs. Binding Site. Changes in organic moieties and functional groups of plasmids based on the FTIR spectra of plasmids before and after reaction with PAHs are used to determine the plasmid−PAH noncovalent binding site. For the pristine plasmid (the black curve in Figure 5a), the vibrational bands of plasmid at 1691, 1649, 1605, and 1492 cm−1 are assigned to guanine (G), thymine (T), adenine (A), and cytosine (C) nitrogenous bases, respectively.8,44 The bands near 1369 and 1417 cm−1 are attributed to guanine and backbone vibrations related to asymmetric PO2−, respectively. The absorption bands near 1238 cm−1 indicate a B-form conformation of the plasmid.44 The FTIR spectra less than 900 cm−1 is related to the “fingerprint” zone caused by the functional groups, including phosphate and phenyl groups. After reaction with Phen (black curve in Figure 5b) and Pyre (black curve in Figure 5c), the absorption peak at 1605 cm−1 becomes weaker than that of the pristine plasmid (see the red arrow and the black curve in Figure 5a); this indicates that adenine, acting as a crucial site, is involved in the plasmid−PAH noncovalent interaction. The absorption band at 1649 cm−1 becomes also weaker after the plasmid is exposed to Phen (see the red arrow) but does not change after exposure to Pyre and BenzPery, which indicates that thymine can combine with the smallest Phen through the same dispersion force as that for the adenine−Phen complex (n = 1). Additionally, it should be noted that BenzPery does not cause an obvious change in absorption spectra of the plasmid, presumably because it is difficult to insert large BenzPery molecules between genetic bases.

The structured 5′-CT-3′ model for the genetic bases was used to assess the plasmid−PAH interaction. Figure 5a−d shows a comparison of the experimental FTIR spectra (the black plots) with the computed results of the 5′-CT-3′-PAH model (the red plots) on the basis of Lorentz oscillator (the blue plots). As seen, the model results exhibit a high precision except for the fingerprint region (with the wavenumber less than 900 cm−1). Additionally, for the fingerprint region (wavenumber 0) interactions (a negative value represents a strong bonding), whereas the density itself provides information regarding the bonding strength.10 Values near zero show a weak vdWs attraction. Gradient isosurfaces for a rich visualization show different vdWs interaction patterns between PAHs and genetic bases (Figure 6a,c,e). In Figure 6a, the bright green isosurface indicates the strong π−π attraction between adenine and Phen. The thymine−Phen combination, indicated also by the green F

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Figure 6. Gradient isosurfaces (a, c, and e) and the corresponding plots of the reduced density gradient versus the electron density multiplied by the sign of the second Hessian eigenvalues (b, d, and f). Results are shown for 5′-CT-3′-Phen (a and b), 5′-CT-3′-Pyre (c and d), and 5′-CT-3′BenzPery (e and f). The surfaces are colored on a blue−green−red scale according to values of sign(ƛ2)ρ, ranging from −0.1 to 0.1 au. Blue, green, and red indicate a strong attractive attraction, vdWs attraction, and strong nonbonded overlap, respectively. The N, H, C, O, and P in PAHs and 5′CT-3′ bases are labeled by blue, white, cyan, red, and brown, respectively. Solvent (water) effects were taken into consideration implicitly.

π−π interactions between adenine and Pyre dominate the plasmid−Pyre association. Results from visual gradient isosurfaces may also be used to explain the very weak BenzPery−plasmid binding (Figure 6e). A strong repulsion between the aromatic carbon from BenzPery and pentose carbon from the plasmid (see the pink arrow in Figure 6e) reflects that it is difficult to insert large PAH planes between genetic bases relative to the insertion of small Phen and Pyre. This view is also confirmed by the low-gradient spike at 0.027 au, which is indicative of a repulsion (Figure 6f). It appears that a strong molecular repulsion between BenzPery and pentose carbons in plasmids dominates the interactions, although a moderate (BenzPery−adenine) vdWs attraction can also be observed in Figure 6e. The repulsive force apparently offsets the vdWs attractions between BenzPery and adenine, resulting in a poor association of BenzPery with adenine in plasmids. Overall, the computational data reveal that Phen and Pyre bind efficiently with adenine in plasmids through the π−π attraction, retarding the transformation of Ampr. PAH−Genetic Base Binding Energy versus Ampr Transformation. A plot of ΔG computed according to 5′CT-3′-PAH models versus the association constant obtained by the fluorescence titration (logKA) is employed to further account for the relation between the genetic base−PAH

isosurfaces, show the same π−π interactions as for the Phen− adenine combination. This reconfirms that thymine combines with small Phen through the same dispersion force as adenine− Phen combination. Furthermore, Figure 6b shows the corresponding plots of RDG versus the electron density multiplied by the sign of the second Hessian eigenvalues. The low-gradient spikes found at 0.005−0.015 au show the weak repulsion of unsaturated electrons between CC/CN in adenine and CC in Phen. The spike at 0.055 au (the red arrow) appears to be the sterically crowded effect of unsaturated electrons inside Phen. It is worth noting that a set of spikes located at −0.005 to −0.015 au shows the vdWs interaction patterns between Phen and thymine. A greater negative value at −0.02 au, which is equivalent to a H-bonding strength,10 represents a strong π−π attraction between adenine and Phen (see the bright dark green isosurface in Figure 6a). Visual gradient isosurfaces of 5′-CT-3′-Pyre interaction only show a moderate vdWs attraction. In Figure 6c, the result seems to exhibit isosurfaces similar to the 5′-CT-3′-Phen interaction (Figure 6a). The low-gradient spikes corresponding to adenine−Pyre isosurfaces appear only at a negative value of less than −0.015 (Figure 6d), suggesting that there is a π−π isosurface between adenine and Pyre (also sees Figure 6c). These indicate that moderate vdWs attractions including weak G

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Environmental Science & Technology bonding energy and the reduced Ampr transformation. Figure 7a shows the calculated ΔG values for 6 PAHs (Anthr, Phen,

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Physicochemical properties for six PAHs (Table S1) and parameters of PAH−plasmid combinations obtained by the fluorescence microtitration (Table S2) (PDF)

AUTHOR INFORMATION

Corresponding Author

*Telephone/Fax: +86-25-8439-5019; e-mail: gaoyanzheng@ njau.edu.cn. Author Contributions #

F. Kang and X. Hu contributed equally to this paper.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation of China (Grants 41401543, 41171193), National Science Foundation for Postdoctoral Scientists of China (2014M561662), Natural Science Foundation of Jiangsu Province of China (BK20140725, BK20130030), and Priority Academic Program Development Foundation of Jiangsu Higher Education Institutions (PAPD). We appreciate the High Performance Computing Center at Nanjing University for offering the computational facilities.

■ Figure 7. Correlation of 5′-CT-3′-PAH binding energies at a major adenine−PAH site (computational ΔGCT‑PAHs) with association constant (KA, log-transformed) (a) and inhibitory rate for efficiency of transformation (b). Association constant (logKA) was obtained by the KA of Figure 2. The Gibbs free energy (G) of major noncovalent interaction (5′-CT-3′-PAHs) is calculated at a main def2-TZVP basis set, an auxiliary def2-TZVP/J basis set with dispersion corrected DFTD3 and gcp (DFT/SVP). The change in Gibbs free energy (ΔG) is calculated by the equation of ΔG = GPAH··CT··GA − (GPAHs + GCT··GA). Solvent (water) effects were taken into consideration implicitly. R2: correlation coefficient; p: possibility.

Pyre, BenzPyre, BenzAnthr, and BenzPery) before and after their interactions with the genetic 5′-CT-3′ fragment at a main def2-TZVP basis set level, an auxiliary def2-TZVP/J basis set with dispersion-corrected DFT-D3 and gcp (DFT/SVP). It shows a good linear dependence of ΔGCT‑PAHs (i.e., the binding energy) on the association constants (logKA) (with R2 = 0.94; probability, p < 0.01). Further, a good linear relation is found between ΔG and the rate of inhibition on the Ampr transformation (Figure 7b). The PAH inhibitory rate on Ampr transfer follows the order: BenzPery− < BenzPyre− < BenzAnthr− < Pyre− < Anthr− < Phen. This suggests that the association energy between a PAH and adenine site regulates the Ampr transformation in plasmids. In addition, the ΔG plot indicates that all active sites likely engage in a spontaneous physisorption (i.e., a noncovalent binding), because the ΔG is lower than 20 kJ/mol.45

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DOI: 10.1021/acs.est.5b02293 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.est.5b02293 Environ. Sci. Technol. XXXX, XXX, XXX−XXX