CdO Nanoparticle Toxicity on Growth, Morphology, and Cell Division

Nov 8, 2012 - Shweta , Durgesh K. Tripathi , Devendra Kumar Chauhan , Jose R. Peralta- ... T.V.M. Sreekanth , Muthuraman Pandurangan , G.R. Dillip , D...
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CdO Nanoparticle Toxicity on Growth, Morphology, and Cell Division in Escherichia coli Sk Tofajjen Hossain and Samir Kumar Mukherjee* Department of Microbiology, University of Kalyani, Kalyani 741235, India S Supporting Information *

ABSTRACT: This Article deals with the toxicological study of synthesized CdO nanoparticles (NPs) on Escherichia coli. Characterization of the CdO NPs was done by DLS, XRD, TEM, and AFM studies, and the average size of NPs was revealed as 22 ± 3 nm. The NPs showed bactericidal activity against E. coli. When NPs were added at midlog phase of growth, complete growth inhibitory concentration was found as 40 μg/mL. Bacterial cells changed morphological features to filamentous form with increasing CdO NPs exposure time, and thereafter resulted in filamentation-associated clumping. From AFM study, severe damage of the cell surface was found in CdO NPs-treated cells. CdO NPs were found to interfere with the expression level of two conserved cell division components, f tsZ and f tsQ, in E. coli at both transcriptional and translational levels. Interference of CdO NPs in proper septum formation without affecting the nucleoid segregation was also observed in confocal micrographs. The elevated intracellular oxidative stress due to CdO NPs exposure seems to be one of the reasons for the changes in cell morphology and expression of division proteins in E. coli.

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INTRODUCTION During the last few decades, continuous release of cadmium from different industries has created a threat to the environment due to its toxicity and long retention time in higher organisms and accumulation along food chains.1,2 Its toxic effects have been studied in several model organisms. Cd2+ is readily taken up by cells and thus can seriously damage the cell in several ways as a potent oxidative agent in cases of both prokaryotic and eukaryotic systems.3−5 Additional hazards are coming due to the release of nanosized Cd particles, especially CdO NPs, from the industries involved in manufacturing quantum dots for both medical diagnostic imaging and targeted therapeutics.6−8 Such particles have attracted a great deal of attention due to their potential interference in biological processes. Engineered nanoparticles (NPs) are now being released into the environment in many ways and are emerging as potential environmental contaminants.8−12 Upon environmental release, NPs could inhibit bacterial processes, as evidenced by laboratory studies.13,14 Thus, it warrants a better understanding of the consequences of released NPs in the environment. As bacteria perform many critical roles in ecosystem, any negative effect of NPs at hazardous concentration to the bacterial community affects the system in the long run. Laboratory study on the interactions between bacteria and different NPs could show the avenue to find their toxicological effect on microbiota. At the same time, bacteria as single cell organisms are good test models to study the NPs’ toxicity study. E. coli became one of the utmost popular models © 2012 American Chemical Society

used for studying the effects of metal stress, due to its generation time and rapid biological response to toxicants. The toxicity exerted by nanosized Cd particles has been found to be more profound in biological systems due to its higher surface area to volume ratio.15 The growth inhibitory concentration, when compared, is found to be less in eukaryotic systems than in prokaryotes;16,17 being evolutionarily primitive, prokaryotic organisms have evolved several strategies to combat the cadmium stress.18 Recent studies on formulated NPs demonstrate its interference in the bacterial system.19−22 Nanosilver has been shown for imparting antibacterial properties;19,23,24 nano-TiO2 and the oxides of other nanomaterials have also been reported for their antibacterial properties.25−27 It has been proposed that the induction of intracellular oxidative stress is a key event in the toxicity mechanisms of many nanomaterials.28 Once inside the cell, nanomaterials pose cytosolic oxidative stress by disturbing the balance between oxidant and antioxidant.29 In this work, the toxic effect of CdO NPs was studied using Escherichia coli. Emphasis was given to find the effect of CdO NPs on the cell viability, cell morphology, and the molecular mechanism of toxicity. Received: July 17, 2012 Revised: November 7, 2012 Published: November 8, 2012 16614

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following Lu et al.22 20 μL of cell extract was added into the reaction mixture containing 10 mM H2O2, 1.6 mM o-dianisidine, and 40 mM 3amino-1,2,4-triazole in potassium phosphate-EDTA solution (50 and 0.5 mM respectively, pH 7.0) at 25 °C. The change in absorbance was measured at 436 nm for 1 min. One unit of the peroxidase activity was defined as one unit change of absorbance at 436 nm/min. SOD activity was assayed following the xanthine oxidase/ cytochrome c method using a reaction mixture of 10 mM cytochrome c and 50 mM xanthine in potassium phosphate and EDTA solution (50 and 0.1 mM, respectively, pH 7.8) at 25 °C.32 The reduction of cytochrome c by superoxide anion was measured at 550 nm in a spectrophotometer. Reactions were initiated by adding xanthine oxidase in an amount that resulted in a change of absorbance of 0.025/min in the cytochrome c assay at pH 7.8. A 50% decrease in the rate of cytochrome c reduction was considered as one unit of the SOD activity. The protein content of each sample was determined by the folin−phenol method using bovine serum albumin (BSA) as standard.33 f tsZ and f tsQ Expression Study Following CdO NPs Exposure. RNA extraction, reverse transcription, and quantitative real-time PCR (qPCR) were performed to elucidate ftsZ and f tsQ expression in E. coli. Bacterial cells of midlog phase in LB were treated with 25 μg/mL CdO NPs for different time periods. Cells were harvested by centrifugation (3000g, 5 min, 4 °C), the cell pellets were resuspended in 1 mL of TRI reagent (Molecular Research Center, Cincinnati, OH), and cells were lysed by sonication. Total RNA was purified in TRI reagent as per manufacturer’s instructions and stored at −80 °C. Reverse transcription was done with random hexameric primers and ThermoScript reverse transcriptase (Invitrogen, Carlsbad, U.S.). mRNA was quantified by qPCR using gene-specific primers, molecular beacons, and AmpliTaq Gold polymerase (Applied Biosystems, U.S.) in a thermal cycler (Stratagene Mx4000, Agilent Technologies, U.S.). The PCR primer sequences and molecular beacons are given in Table S1, Supporting Information. Expression study was also done using CdCl2 (25 μg/mL)-treated E. coli cells as control. As a normalization factor, 16S rRNA copy number was used to enumerate bacterial transcripts.34 Molecular Biological Procedures for FtsZ and FtsQ Expression Study. Standard molecular biological procedures were followed for cloning and analysis of DNA, PCR, and transformation,35 and E. coli TOP10 cells were used for cloning. Enzymes used to manipulate DNA were from Roche Applied Science (Germany). All constructs were sequenced to verify their integrity. f tsZ was amplified from genomic DNA of E. coli using the sense primer 5′-AAAGGAT CCATGGAAATGTTTGAAC-3′- and the antisense primer 5′-TATAAAGCTTATCAGCTTGCTTACG CA-3′. The amplicon was cloned between asymmetric NcoI and HindIII sites in pET29b to construct plasmid pKM1. Sense primer for f tsQ (b0093) was designed ∼300bp upstream of b0091 (2744bp upstream of start codon of ftsQ). Reverse primer was at the end of gene without stop codon, but with His-tag. PCR product was cloned in promoter-less plasmid pMS236 between BglII and XbaI (underlined) to construct pKM2. The primers used in this study are 5′-AAAAGATCTCGTCGTTTGCCGCTCCGG TGCGTTAA-3′ (sense) and 5′-AAATCTAGATCAGTGGTGGTGGTGGTGGTGTTGTTGTTCT GCCTGTGCCTGATTTT-3′ (antisense). E. coli BL21 (DE3) harboring pKM1 was grown to an A600 of 0.5 at 37 °C. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a concentration of 0.25 mM, and growth was continued for 3 h. Polyclonal antibody against electroeluted recombinant FtsZ of E. coli K-12 MG1655 was raised in mouse by Imgenex, India. For the FtsQ expression study, E. coli cells were transformed35 with constructed pKM2 plasmid. Transformants were grown in LB supplemented with hygromycin (100 μg/mL), and at mid log phase 25 μg/mL CdO NPs were added. Thereafter, at certain intervals, cells were centrifuged (3000g, 5 min, 4 °C), and the cell pellets were lysed by sonication. Whole cell lysates were prepared by solubilizing with 2% Triton X-100 in 10 mM Tris-HCl (v/v, pH 7.5, 4 °C). For FtsZ expression study, E. coli cells were similarly treated with CdO NPs and at various time points, cell pellets, obtained by

MATERIALS AND METHODS

CdO Nanoparticle Preparation. CdO NPs have been synthesized in methanolic medium.30 Ethylene glycol (100 μL) with 200 mL of methanol was added dropwise to 100 mL of 0.1 M NaOH while it was continuously stirred. The resulting solution was continuously stirred for 1 h before 0.1 M CdCl2 (20 mL) solution was added to it. After 3 h of constant stirring, a milky white solution was obtained. The precipitate was washed in methanol and the solvent was then allowed to evaporate at 30 °C to obtain Cd(OH)2 NPs as a white powder state. Cd(OH)2 NPs were then placed in a furnace and heated to 250 °C for 5 h to obtain a brown CdO powder. Characterization of CdO Nanoparticles. The characterization of CdO NPs was conducted using dynamic light scattering (DLS), X-ray diffraction (XRD), atomic force microscopy (AFM), and transmission electron microscopy (TEM) before biological experiments. The size distribution was obtained by DLS (Malvern Zetasizer, UK). The CdO NPs were studied for their phase and average size employing XRD (D500 Siemens, Berlin, Germany) analysis. XRD study of CdO NPs was performed within a 2θ range of 20−80° using Cu Kα radiation. AFM study was also done to assess the structure of NPs.31 The dried film of NPs was scanned by AFM (Veeco, Innova, U.S.) in tapping mode, using the nanoprobe cantilever made of silicon nitride with a spring constant of 49 N/m. From the AFM image, the size of the NPs was measured using Veeco SPM Lab Analysis software. The size was further confirmed by TEM (FEI, Tecnai S-twin, U.S.). Toxic Effect of CdO NPs on Bacterial Growth. E. coli K-12 MG1655 was used as test bacteria and maintained in Luria−Bertani broth (LB). Bacterial cells were grown for 16 h in LB, and a 200 μL culture of each isolate was inoculated to 20 mL of LB and incubated at 37 °C. Growth inhibitory concentrations were determined by exposing the cells at midlog phase (A600 = 0.5) to different concentrations of CdO NPs and CdO inorganic powder (0−120 μg/mL) for 24 h. CdO NPs were added to the medium from a stock suspension of NPs (50 mg/mL) in sterilized Milli-Q water after vigorous mixing. After determining the complete growth inhibitory concentration of CdO NPs, added at midlog phase, sublethal concentration (25 μg/mL) was used for studying the CdO NPs toxicity in LB and compared to CdCl2treated, CdO inorganic powder-treated, and untreated cells as control. In each case, cell viability was measured by serial dilution of the culture after vortexing, followed by plating on solidified LB medium. The viable cell number was recorded by counting the number of bacterial colonies developed on the plate and expressed as CFU/mL. Microscopic Study of Bacterial Cells Exposed to CdO NPs. Microscopic observation of E. coli cells treated with 25 μg/mL CdO NPs was carried out under a phase contrast microscope (Leica DM 750, Germany) at different time intervals. Untreated, CdCl2-treated, and CdO inorganic powder (both 25 μg/mL concentration)-treated cells were considered as controls. AFM Study of the Cell Surface. E. coli cells of midlog phase exposed to NPs (25 μg/mL) as described above were fixed in 2.5% paraformaldehyde (w/v), 0.04% glutaraldehyde (w/v), and 30 mM phosphate buffer saline (PBS, pH 7.5). Fixed cells were washed twice and finally suspended in Milli-Q water to ∼106 cells/mL. A volume of 20 μL of bacterial suspension was placed on a 1.0 cm2 coverslip. The adhered cells were washed three times with Milli-Q water and dried in a vacuum. Height and phase data for the cells were captured using tapping mode AFM with standard Veeco phosphorus-doped siliconnitride cantilevers, having resonance frequencies of 245−284 kHz.31 An AFM study was also done using CdCl2 (25 μg/mL)-treated and untreated E. coli cells as control. The measurements were done using Veeco SPM Lab Analysis software. Enzyme Assay for Reactive Oxygen Species (ROS) Measurement. Peroxidase and super oxide dismutase (SOD) activities were assayed for qualitative determination of the state of intracellular ROS level in E. coli cells of midlog phase exposed to CdO NPs and CdCl2 (25 μg/mL). The cell pellets were harvested by centrifugation and lyzed by sonication in 20 mM PBS buffer (pH 7.5). The cell lysate were prepared by solubilizing the extract with 2% Triton X-100 in 10 mM Tris-HCl (v/v, pH 7.5, 4 °C). Peroxidase activity was measured 16615

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Figure 1. Characterization of synthesized CdO nanoparticles: (a) particle size distribution, (b) X-ray diffraction pattern, (c) TEM images, and (d) AFM image. centrifugation (3000g, 5 min, 4 °C), were lysed by sonication to obtain the cell lysate. Whole cell lysates were prepared with 2% Triton X-100 in 10 mM Tris-HCl (v/v, pH 7.5, 4 °C). Protein content was determined with folin−phenol reagent using bovine serum albumin as standard.33 Equal amounts of whole cell lysate were loaded and run on 10% SDS PAGE. The separated proteins were electroblotted onto polyvinylidene fluoride (PVDF) membrane, then blocked in blocking buffer [5% nonfat dry milk in 1X TBST (Tris-buffered saline containing 0.05% Tween 20, v/v, pH 7.5)], and probed with antihis antibody (G Bioscience, U.S.) for FtsQ or with anti FtsZ antisera for FtsZ at 4 °C for 16 h on a shaker. The membrane was then exposed to the second antibody, horseradish peroxidase (HRP)-conjugated antimouse antibody (G Bioscience, U.S.), and kept at 25 °C for 1 h followed by three washes with TBST. For control protein expression study, lysates of both CdO NPs-treated and CdCl2-treated cells were Western blotted using anti-RecA antibody as first antibody (Abcam, UK) and HRP-conjugated antirabbit antibody (G Bioscience, U.S.) as secondary antibody. The membrane was treated with HRP color development reagent solution containing Lumiglo (Cell Signaling Technology, U.S.) for 1 min, exposed to X-ray film, and then developed. For both FtsZ and FtsQ, whole cell lysates of untreated and CdCl2-treated (25 μg/mL) cells were considered as control and similarly processed. Confocal Microscopic Study of E. coli. Confocal microscopy was done to visualize cell septum by immunostaining using FtsZ antibody.37,38 E. coli cells of midlog phase were exposed to CdO NPs (25 μg/mL for 3 h) and fixed by incubation for 15 min at 25 °C and 30 min on ice in 2.5% paraformaldehyde (w/v), 0.04% glutaraldehyde (w/v), and 30 mM phosphate buffer saline (PBS, pH 7.5). The cells after washing with PBS were permeabilized by exposing to 1% Triton X-100 for 2 min, and then the cells were transferred to slides. The slides were washed with PBS and air-dried. The cell smear were then incubated with polyclonal FtsZ antibody developed in mouse, followed by staining with a Dylight 649 conjugated goat polyclonal secondary antibody to mouse IgG-H&L (Abcam, UK). The nucleoids were stained with 2 μg/mL DAPI (4′,6-diamino-2-phenylindole).39 The cells were observed under a fluorescence microscope (Zeiss LSM 510

Meta confocal microscope, Germany). For all microscopic studies, both untreated and CdCl2-treated E. coli cells were used as control.

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RESULTS Characterization of Nanoparticle. The CdO NPs synthesized in the laboratory were characterized by DLS, XRD, AFM, and TEM (Figure 1 and Figure S1, Supporting Information). DLS data showed relatively monodispersive nature of the CdO NPs with a geometric standard deviation (GSD) value of 1.19. Assuming a homogeneous strain across the CdO films, the average particle size (d) was estimated from the full-width at half-maximum (fwhm) of (111) peak corresponding to 2θ = 33.9° by using the Debye−Scherrer equation, d = 0.9λ/β cos θ, where, λ (1.5418 Å) is the wavelength of X-rays (Cu Kα), θ is the angle of reflection, and β is the widening of the Bragg XRD peak (following Warrens’s equation: β2 = β2sample − β2insrument, where β is the calculated fwhm, βsample is the fwhm of the peak of the sample, and βinstrument is the fwhm of the instrument error).40 The particle size of the CdO sample obtained from the fwhm of peak was found to be ∼22 nm. The (111), (200), (220), (311), and (222) reflections resemble the reference patterns for CdO (Joint Committee for Powder Diffraction Studies, File No. 050640). The sizes of the observed particles in TEM and AFM corroborate with the values obtained by XRD study. The average size of the NPs was found to be 22 ± 3 nm having mostly spherical nature (Table S2, Supporting Information). However, on the basis of the DLS measurement, the size distribution of NPs in aqueous solution was found to be larger than its actual size, which might be due to the hydrodynamic features in the aqueous phase. Toxic Effect on Bacterial Growth. CdO NPs were found to inhibit the growth of E. coli, and when they were added to the culture at midlog phase, the viable cell number declined 16616

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phase. Cell length of E. coli increased due to CdO NPs exposure (Table S3, Supporting Information). With the gradual progression of time of exposure to CdO NPs, cells became more filamentous in comparison to control ones (untreated, 25 μg/mL CdO inorganic powder-treated, or 25 μg/mL CdCl2treated). Cells showed filamentation associated clumping after 8 h treatment of CdO NPs (Figure 3). CdCl2-treated, CdO inorganic powder-treated, and untreated cells showed nearly similar length and morphology (Table S3, Supporting Information). CdO NPs exposure leading to morphological changes in E. coli cells raised a question as to whether it affects the cell structure and cell division or not; thus further experiments were conducted to elucidate the facts in this study. As we did not find significant differences in morphology and growth patterns between CdCl2-treated and CdO inorganic powder-treated cells, for further experimental study we considered CdCl2-treated cells as positive control. AFM Study of the Cell Surface of E. coli. In the AFM picture, topological changes of the CdO NPs-treated E. coli cells were observed and compared to control cells (Figure 4). As no significant changes were found among the two controls, only the pictures of CdCl2-treated cells are given for comparing the effect of Cd as nanoparticulate form. Toxic effects are usually accompanied by a change in bacterial surface morphology. CdCl2-treated cells have smooth surfaces in comparison to that of CdO-NPs-treated cells (Figures S3 and S4, Supporting Information). AFM images show that CdO-NPs treatment significantly changes the morphology of the bacterium. From higher magnification, it can be observed that pores on the surface of the CdO-NPs-treated bacterium are much deeper than those on the CdCl2-treated E. coli cell surface. Intracellular ROS Level. Intracellular ROS level might be the key factor in studying metal toxicity, which could be attributed by measuring perioxidase and SOD activities. Here, both peroxidase and SOD activities significantly decreased with exposure time to CdO NPs in comparison that in the CdCl2treated cells (Figure 5), suggesting increasing intracellular ROS level. Effect of CdO NPs on Cell Division Proteins of E. coli. Considering the roles of f tsZ and f tsQ in bacterial cell division, emphasis was given to understand the expression level of f tsZ

with increasing concentration of CdO NPs. At midlog phase, 25 μg/mL CdO NPs inhibit ∼50% of growth in E. coli, whereas 40 μg/mL completely ceased the growth. When cells were treated with CdO inorganic powder, ∼50% of growth was inhibited at 45 μg/mL concentration, and 120 μg/mL completely ceased the growth (Figure S2, Supporting Information). Sublethal concentration (25 μg/mL) was supplemented at midlog phase for studying the effect of CdO NPs on the survival in LB and compared to untreated, CdCl2-treated (25 μg/mL), and CdO inorganic powder-treated (25 μg/mL) cells as different controls. Cell viability declined significantly with the time of exposure (4−5 h) to CdO NPs; however, cells in different controls survived comparably well at the same culture conditions (Figure 2).

Figure 2. Survival of E. coli cells treated with 25 μg/mL CdO NPs in various time points and compared to untreated and 25 μg/mL CdCl2 or CdO inorganic powder-treated cells. Cells at midlog phase were exposed to CdO inorganic powder or CdCl2 or CdO NPs. Data are the mean of three replication ± SE.

Microscopic Observation of Cells. Microscopic observation of the CdO NPs (25 μg/mL)-treated cells was carried out under a phase contrast microscope (Leica DM 750, Germany) at various time points after addition of CdO NPs at midlog

Figure 3. Phase contrast micrographs of E. coli cells of midlog phase exposed to 25 μg/mL CdO NPs for 2 h (d), 4 h (e), and 8 h (f). As control, cells treated with 25 μg/mL CdCl2 for 4 h (a), cells treated with 25 μg/mL CdO inorganic powder for 4 h (b), and untreated cells of 4 h incubation (c). Bars are 10 μm. 16617

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Figure 4. CdO NPs-affected (25 μg/mL) surface morphology of E. coli cells. Selected magnified portion was shown with arrows sequentially. The red line-bordered part in the final magnified photograph shows the severe topological damage on the cell surface (a). Effect of CdCl2 (25 μg/mL) on surface morphology of E. coli cells. Selected magnified portion was showed with arrows sequentially. Smooth topology of the cell surface was visualized in the magnified photograph (b).

Figure 5. Effects of the CdO NPs and CdCl2 (25 μg/mL) on antioxidative enzyme activities in E. coli: peroxidase (a), superoxide dismutase (b) with exposure time. Data are the mean of three replications ± SE.

and f tsQ using E. coli K-12 MG1655 as model organism. The mRNA/16s rRNA ratio and relative mRNA expressions in both f tsZ and f tsQ decreased sharply with the time of CdO NPs exposure when compared to CdCl2-treated cells (Figure 6). For the protein expression study, FtsQ-His tag was also cloned under its native promoter in promoterless plasmid pMS2 (pKM2). pKM2 was transformed in E. coli K-12. The level of FtsQ was determined by immunoblotting of bacterial whole cell lysate using anti-His antibody. Similarly, FtsZ expression was determined by immunoblotting of bacterial whole cell lysate using anti FtsZ antisera. Cell lysates were normalized to the total protein concentration at the time of gel loading by protein quantification. The expression level of both FtsZ and FtsQ gradually decreased with time of CdO NPs exposure (Figure 7). As no significant changes were found among the two controls, only the data of CdCl2-treated cells are

given for comparing the effect of CdO NPs on FtsZ and FtsQ expression. For the control protein expression study, lysates of both CdO NPs-treated and CdCl2-treated cells were Western blotted using anti-RecA antibody, and no significant change was observed in the expression level of RecA with exposure time (Figure S5, Supporting Information). Visualization of Nucleoid and Septum Formation. Interference of CdO NPs on cell division was further confirmed by fluorescence microscopic study. The majority (∼90%) of untreated or CdCl2-treated (control) E. coli cells showed proper septum between the two nucleoids (Figure 8). On the contrary, CdO-NPs strongly inhibited proper septum formation in most of the cells (∼93%). As NPs-treated cells were unable to divide, the number of nucleoids was found to be higher than the control cells. However, the frequency of nucleoid/ micrometer of cell length was found to be unaltered in both 16618

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Figure 6. Expression levels (mRNA/16S rRNA) of ftsZ (a) and ftsQ (b) in E. coli treated with 25 μg/mL CdO NPs for different time periods and quantitated by qPCR, with relative mRNA expression at different time points (respective inset). Expression levels (mRNA/16S rRNA) of ftsZ (c) and ftsQ (d) in E. coli treated with 25 μg/mL CdCl2 were used as control.

in eukaryotes and in prokaryotes. Usually, the eukaryotic system is more sensitive than are the prokaryotic cells.7,16 Synthesized CdO NPs (22 ± 3 nm) showed bactericidal effect on E. coli. Antibacterial property of CdO NPs on E. coli was also reported earlier,16 but the authors did not report on the toxic effects on cell structure. Moreover, they reported nearly 70% inhibition of growth of E. coli in liquid medium treated with 1% CdO NPs, which is too high considering the general toxicity of cadmium to bacterial system. The cell length of E. coli increased due to CdO NPs exposure. With the progression of exposure time to CdO NPs, cells became more filamentous and showed filamentation associated clumping. There are few reports about the effect of metal−oxide NPs on such morphological changes in bacteria. Changes in bacterial cell size due to Fe304 and ZnO NPs exposure were reported;42−44 however, the reason of changing was not elucidated. It is evident that CdO NPs affect the cell division process, and thus cells took the filamentous form. This Article for the first time reports on the effect of CdO NPs on bacterial cell morphology, suggesting the toxic effect on bacterial cells and likely on cell division. Bacteria need involvement of various proteins for cell division and septum formation at the division site.45,46 Among those essential division proteins, FtsZ and FtsQ are reported to be highly conserved in bacteria.47,48 Both of the proteins play a pivotal role in septum formation by interacting with other cell division proteins.49−51 Gene expressions of both f tsZ and f tsQ decreased sharply with time of CdO NPs

Figure 7. Expression levels of FtsZ in E. coli treated with 25 μg/mL CdO NPs for different time periods and assessed by immunoblotting of whole cell lysate by anti FtsZ antisera (a) and compared to the expression level of that in 25 μg/mL CdCl2-treated cells (b). Expression levels of FtsQ in E. coli treated with 25 μg/mL CdO NPs for different time periods and assessed by immunoblotting of whole cell lysate by anti-His antibody (c) and compared to the expression level of that in 25 μg/mL CdCl2-treated cells (d).

CdO-NPs and control cells. As no significant changes were found among the two controls, only the pictures of CdCl2treated cells are given.

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DISCUSSION Reports on general toxicity of Cd in biological system are well documented;1,2 however, due to different physicochemical and biological properties of its nanosized counterparts, Cd NPs are recently getting more attention considering their toxicological impact.7,41 Cd NPs severely affect living organisms, and the magnitude of toxicity showed concentration dependency both 16619

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the E. coli cell morphology and expression of division proteins might be an indirect effect of the intracellular oxidative stress.

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CONCLUSION The present findings demonstrate the toxic effects of CdO NPs in bacteria, by affecting their morphology and cell division as a result of intracellular oxidative imbalance. Considering the ecotoxicological impact of CdO NPs, attention should be taken before releasing such particles into the environment by developing a scientifically defensible fact profile for the purpose of risk assessment.

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1: AFM photograph of synthesized CdO NPs and the height profile spectrum. Figure S2: Growth inhibitory effect of CdO NPs on E. coli. Cells at midlog phase were treated with CdO NPs and CdO inorganic powder at concentration ranging from 0 to 120 μg/mL. The viability of untreated cells was considered as control, and % viability of treated cells was calculated by comparing with the control sets. Figure S3: Sequentially magnified AFM photographs of CdO NPs-treated cells with the respective height profile spectrum of the surface topology (Z-data). Figure S4: Sequentially magnified AFM photographs of CdCl2-treated cells with the respective height profile spectrum of the surface topology (Z-data). Figure S5: Expression level of RecA in E. coli treated with 25 μg/mL CdO NPs for different time periods and assessed by immunoblotting of whole cell lysate by anti-RecA antibody (a) and compared to the expression level of that in 25 μg/mL CdCl2-treated cells (b). Table S1: Primer used for qPCR study. Table S2: A comparison of the particle size obtained for CdO NPs from XRD, TEM, and AFM studies. Table S3: Change in cell length due to CdO NPs, CdO inorganic powder, and CdCl2 exposure with time. This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 8. Interference of CdO NPs in proper septum formation without affecting the nucleoid segregation in E. coli. Bacterial cells of midlog phase treated with CdO NPs or CdCl2 (25 μg/mL). The FtsZ were stained with with FtsZ antisera developed in mouse, followed by staining with a Dylight 649 conjugated goat polyclonal secondary antibody to mouse IgG-H&L (red fluorescence). The nucleoids were stained with 2 μg/mL DAPI (blue fluorescence). The cells were observed under a confocal microscope. Shown are CdO NPs-treated cells: ftsZ (a), nucleoids (b), and merged (c). For control (CdCl2treated cells): ftsZ(d), nucleoids (e), and merged photograph (f) showing proper septum formation (indicated by arrow). Scale bar: 5 μm.

exposure. Downregulation of expression of different genes related to cell division including f tsZ in E. coli under Cd stress at nonlethal dose was also earlier reported.52 Declined expression of cell division gene ( f tsA) under Cd stress in Campylobacter jejuni was also reported.53 Downregulation of expression of both f tsZ and f tsQ in E. coli due to CdO NPs exposure is being reported here. Similarly, the expression level of both FtsQ and FtsZ proteins gradually decreased with time of CdO NPs exposure. Earlier documentation showed that changes in FtsZ and FtsQ expression level or its improper function under different stress conditions interfered with cell division, thus causing multinucleated filamentous structure of bacterial cell,39,54,55 which corroborates with this present study in E. coli K-12. The NPs-treated cells, when stained with DAPI, showed filamentous multinucleated bead structure, suggesting Cd-induced irregularities in cell septum formation. The majority of untreated or CdCl2-treated E. coli cells showed proper septum between the two nucleoids; in contrast, it was inhibited in CdO-NPs-treated cells. This result suggests that, due to CdO-NPs treatment, proper cell septum formation was inhibited without affecting nucleoid segregation. The development of oxidative stress as a common consequence resulting in cell damage due to NPs exposure and biological systems is well established.28,29,56,57 Metal oxide NPs, in particular, have been reported to increase oxidative stress, resulting in DNA damage and reduced viability of E. coli.58 In the present study, CdO NPs exposure lowered the SOD, and peroxidase activities that resulted increased ROS level, and thus developed an oxidative imbalance. The accumulation of ROS resulted in redundant free radicals that would oxidize biomolecules and as a consequence lead to metabolic or genetic dysfunction. Interference of CdO NPs on

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +91-3325828750 (ext. 332), +91-9433136617. Fax: +913325828282. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge technical support from the Department of Biophysics & Biochemistry, Department of Environmental Sciences, University of Kalyani, and the Department of Biotechnology, University of Calcutta. This work was financially supported by the Department of Science and Technology, Government of India, under PURSE program and the University of Kalyani, India.

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ABBREVIATIONS DLS, dynamic light scattering; XRD, X-ray diffraction; AFM, atomic force microscopy; TEM, transmission electron microscopy; NPs, nanoparticles; LB, Luria−Bertani broth; ROS, reactive oxygen species; SOD, super oxide dismutase; PBS, phosphate buffer saline; CFU, colony forming units; BSA, bovine serum albumin; IPTG, isopropyl β-D-1-thiogalactopyranoside; PVDF, polyvinylidene fluoride; TBST, Tris-buffered 16620

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saline with Tween 20; HRP, horseradish peroxidise; DAPI, 4′,6diamino-2-phenylindole; DIC, differential interference contrast

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