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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2012 Mar 12;109(13):4944–4949. doi: 10.1073/pnas.1107254109

Nanoalumina promotes the horizontal transfer of multiresistance genes mediated by plasmids across genera

Zhigang Qiu a,1, Yunmei Yu a,b,1, Zhaoli Chen a, Min Jin a, Dong Yang a, Zuguo Zhao a, Jingfeng Wang a, Zhiqiang Shen a, Xinwei Wang a, Di Qian c, Aihua Huang a, Buchang Zhang c, Jun-Wen Li a,2
PMCID: PMC3323979  PMID: 22411796

Abstract

Antibiotic resistance is a worldwide public health concern. Conjugative transfer between closely related strains or species of bacteria is an important method for the horizontal transfer of multidrug-resistance genes. The extent to which nanomaterials are able to cause an increase in antibiotic resistance by the regulation of the conjugative transfer of antibiotic-resistance genes in bacteria, especially across genera, is still unknown. Here we show that nanomaterials in water can significantly promote the horizontal conjugative transfer of multidrug-resistance genes mediated by the RP4, RK2, and pCF10 plasmids. Nanoalumina can promote the conjugative transfer of the RP4 plasmid from Escherichia coli to Salmonella spp. by up to 200-fold compared with untreated cells. We also explored the mechanisms behind this phenomenon and demonstrate that nanoalumina is able to induce oxidative stress, damage bacterial cell membranes, enhance the expression of mating pair formation genes and DNA transfer and replication genes, and depress the expression of global regulatory genes that regulate the conjugative transfer of RP4. These findings are important in assessing the risk of nanomaterials to the environment, particularly from water and wastewater treatment systems, and in the estimation of the effect of manufacture and use of nanomaterials on the environment.

Keywords: ecological safety, mating system, oxidative stress–response, conjugative transfer frequency, conjugation

The development of antibiotic resistance in bacteria is one of the most serious threats to global public health (1), as exemplified by the appearance of a new “superbug” in 2010 and by its dissemination among different bacterial genera in drinking water and seepage samples in New Delhi, India (2, 3). The spread of antibiotic-resistance genes is due to the selective pressures caused by increases in the use and misuse of antibiotics in medicine and animal feedstuffs (4). Also important is the presence of increasing amounts of these substances in the environment by horizontal transfer between bacteria rather than by the sequential modification of gene function by the accumulation of point mutations (5). Horizontal transfer often occurs between closely related strains or species of bacteria but occurs at a very low frequency across genera (6), although the medical importance of the latter is greater. Studies have demonstrated that water and wastewater, which are pools for antibiotic-resistant bacteria and -resistance genes, appear to contribute significantly to the acquisition of resistance against antibiotics by bacteria by horizontal DNA transfer (7, 8). Aquatic environments and water treatment processes are able to affect the efficiency of antibiotic-resistance gene transfer (9).

Nanomaterials have novel physical, chemical, and biological properties and have many potential applications for water treatment (10–13). These applications are increasing in number progressively and include adsorption, oxidation, and the minimization of contaminants in water and wastewater (14–18). We believe that the use of nanoalumina in water treatment processes will increase in the future because nanoalumina has a much greater surface area and a greater adsorption capacity for contaminants compared with alumina, which is currently being used as a good absorber for water treatment processes (19). However, it is unknown whether or how nanomaterials affect antibiotic-resistance gene transfer between bacteria, especially across genera. Some studies have indicated that nanomaterials can cause disruption to bacterial membranes, probably by the production of reactive oxygen species (20–22), and can deliver DNA or RNA molecules into animal or plant cells (23). Based on the above data, we hypothesized that nanomaterials that are present in water may promote the horizontal transfer of multidrug-resistance genes across bacterial genera by acting on cell membranes and/or regulating genes involved in plasmid transfer.

In this study, we systematically observed the effect of nanomaterials and aquatic factors on the horizontal transfer of antibiotic-multiresistance genes across genera. We demonstrate that the presence of nanoalumina in water results in a very significant increase in cross-genera conjugation of the mobilizable shuttle plasmid RP4 from Escherichia coli K12 to Salmonella Aberdeen Kauffmann 50312 and in other mating systems. We also explored the mechanisms that underlie this process with regard to morphological, biochemical, and molecular biological changes. The effect of nanoalumina in water on the promotion of horizontal transfer of antibiotic-multiresistance genes across genera is essential for guiding the manufacture and application of nanomaterials in the environment and in the evaluation of the possible consequences on ecology and bioremediation.

Results

Effect of Nanomaterials on the Conjugative Transfer of the RP4 Plasmid.

We observed the effect of several nanomaterials, such as nano-Al2O3, nano-TiO2, nano-SiO2, and nano-Fe2O3, on the conjugative transfer of the RP4 plasmid from E. coli to Salmonella to assess whether nanomaterials could promote transfer of the plasmid. The results (SI Appendix, Table 1) showed that all these nanomaterials could promote the conjugative transfer of the RP4 plasmid by 20- to 100-fold. Nanoalumina gave the most significant effect and was chosen as the indicator in subsequent experiments.

Effect of Single Factors on the Conjugative Transfer of the RP4 Plasmid.

First, we observed the effect of single factors (nanoalumina and bulk alumina concentration, bacteria concentration, mating temperature, mating time, and pH value) on the conjugative transfer of the RP4 plasmid in PBS. Bulk alumina at any concentration had no significant effect on conjugative transfer of RP4, but nanoalumina significantly promoted conjugative transfer. Conjugative transfer was the highest in the 5 mmol/L nanoalumina group, which was more than 100-fold higher than that of the control group (Fig. 1A). The conjugative transfer of RP4 increased with bacteria concentration (Fig. 1B). The conjugative transfer in each group increased with the prolongation of mating time, and the conjugative transfer of the 5 mmol/L nanoalumina group was markedly higher than that of the control group at each time point (Fig. 1C). To determine the impact of nanoalumina on RP4 transfer accurately, a simple mass action model described by Levin et al. (24) was used to explain the kinetics of RP4 conjugative transfer. The results (SI Appendix, Fig. 1) showed that the conjugational transfer rate constant of the 5 mmol/L nanoalumina group was approximately five orders of magnitude higher than that of the control group. The conjugation was lower at 4 °C and 15 °C than at 20 °C or higher, but showed no further increase above 20 °C (Fig. 1D). pH had no significant effect on plasmid transfer (SI Appendix, Fig. 2).

Fig. 1.

Fig. 1.

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Effects of a single factor on the conjugative transfer of RP4. (A) The effect of alumina concentration on the conjugative transfer of RP4 at pH 7.0, 25 ºC, 8-h mating time, and 109 cfu/mL bacteria. The concentration of nanoalumina had a significant effect on conjugative transfer (ANOVA, P < 0.05); significant differences between single concentration steps and the control (or bulk alumina group) were tested with the Student-Newman-Keuls (S-N-K) test, *P < 0.05 and **P < 0.01. (B) The effect of bacteria concentration on the conjugative transfer of RP4 at pH 7.0, 25 ºC, and 8-h mating time when nanoalumina concentration was 5 mmol/L. The bacteria concentration had a significant effect on conjugative transfer (ANOVA, P < 0.05); significant differences between the nanoalumina group and the control at single concentration steps were tested using the S-N-K test, *P < 0.05 and **P < 0.01. (C) The effect of mating times on the conjugative transfer of RP4 at pH 7.0, 25 ºC, and 109 cfu/mL bacteria when nanoalumina concentration was 5 mmol/L. Mating time had a significant effect on conjugative transfer (ANOVA, P < 0.05); significant differences between the nanoalumina group and the control at each time point were tested with the S-N-K test, *P < 0.05 and **P < 0.01. (D) The effect of mating temperature on the conjugative transfer of RP4 at pH 7.0, 8-h mating time, and 109 cfu/mL bacteria when nanoalumina concentration was 5 mmol/L. Mating temperature had a significant effect on conjugative transfer (ANOVA, P < 0.05); significant differences between the nanoalumina group and the control at each temperature point were tested with the S-N-K test, *P < 0.05 and **P < 0.01. The results of conjugation obtained with the nonconjugative plasmid pCB182 and tra-mutant RP4 were zero. This result confirmed that the uptake of naked DNA does not occur by transformation.

Effect of Main Factors on the Conjugative Transfer of the RP4 Plasmid.

We used orthogonal design [L64(421)] with the four variables to observe the effect on the conjugative transfer of RP4. Test conditions and results are shown in SI Appendix, Table 2. The statistical results show that every one of these four variables had a significant effect on conjugation, and that there was an interaction between the bacteria concentration and nanoalumina concentration. The ranking of these factors in order of importance of affecting the conjugative transfer was as follows: bacteria concentration > nanoalumina concentration > mating temperature > mating time > interaction between bacteria concentration and nanoalumina concentration. The effect on promotion of conjugative transfer of nanoalumina was greater than the effects of mating temperature and mating time.

The conjugation was highest under conditions of (i) nanoalumina concentration <5 mmol/L; (ii) 109 cfu/mL bacteria; and (iii) conjugation for 8 h and at 37 °C; it was more than 200-fold higher than when no nanoalumina was added.

Effect of Nanoalumina on the Horizontal Transfer of Conjugative Plasmids in Other Mating Systems.

Nanoalumina can not only promote the conjugative transfer of RP4 from E. coli to Salmonella but can also promote the conjugative transfer of RP4 in other mating systems (SI Appendix, Table 3). Nanoalumina (5 mmol/L) can promote the conjugative transfer of RP4 between bacteria of the same genus, more specifically from E. coli to E. coli, and increase the transfer by 200-fold. Nanoalumina also can significantly promote the conjugative transfer of RP4 from Gram-negative bacteria to Gram-positive bacteria, and increase the transfer by more than 50-fold. In other mating systems, from Enterococci to Enterococci, the conjugative transfer of RP4 could also be promoted by 5 mmol/L nanoalumina, which increased the transfer by 100-fold.

Nanoalumina also promoted the horizontal transfer of other conjugative plasmids, such as an unrelated conjugation system in the Gram-negative bacteria RK2 (SI Appendix, Fig. 3A) and the enterococcal pheromone-mediated conjugation system involving an endogenous enterococcal plasmid, pCF10 (SI Appendix, Fig. 3B). The results showed that nanoalumina could significantly promote RK2 and pCF10 conjugational transfer when the concentration of nanoalumina ranged from 0.05 to 50 mmol/L. The conjugative transfer frequencies of RK2 and pCF10 were also highest in the 5 mmol/L nanoalumina group, which induced an increase in transfer of more than 170- and 120-fold compared with the control group, respectively.

Effect of Nanoalumina on Cell Membranes and Its Distribution in Cells.

In the control and bulk alumina groups, the cell membranes of the bacteria were intact, cell borders were clear, and the cytoplasm was compact and distributed evenly (Fig. 2 A–C). The cytoplasm of the bacteria agglomerated, and parts of the cell membranes were undefined in the 5 mmol/L nanoalumina group (Fig. 2D). The cell membranes of the parent bacteria were both damaged severely in the 50 mmol/L group (Fig. 2E). Moreover, many spherical, highly dense particles were observed in these bacterial cells (Fig. 2 D and E). The elemental analysis results showed that there were high levels of aluminum in the regions around the highly dense particles in the bacteria (Fig. 2F). It indicated that nanoalumina not only damaged the structure of the bacterial membrane but also entered the cells. Atomic force microscopy (AFM) images of the bacteria (SI Appendix, Fig. 4) also showed that the cell membranes in the 5 and 50 mmol/L nanoalumina groups displayed wrinkles and fissures, and the cell membranes in the latter group were severely damaged, whereas the cell membranes in the control group and in all of the bulk alumina groups were smooth and intact. The AFM images also showed that nanoalumina could damage bacterial cell membranes.

Fig. 2.

Fig. 2.

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TEM detection of E. coli in ultrafine slices and elemental analysis. (A) In the control group, the cell membranes are distinct and the cytoplasm is compact. There were no highly dense particles in these cells. (B and C) The cell membranes of bacteria that were treated with different concentrations of bulk alumina (B, 5 mmol/L; C, 50 mmol/L) are distinguishable, and the cytoplasm is compact. There are no highly dense particles in these cells. (D and E) The cell membranes of bacteria that were treated with different concentrations of nanoalumina (D, 5 mmol/L; E, 50 mmol/L) were damaged, and the extent of damage increased with increasing concentration of nanoalumina. There were also many highly dense particles in the cells (indicated by arrows), and the number of highly dense particles increased with increasing concentration of nanoalumina. (Scale bars, 100 nm.) (F) The composition of chemical elements in the bacteria from D. Elemental aluminum (from nanoalumina) gave the highest counts; elemental copper originated from the copper net, whereas lead, arsenic, and other elements came from dye liquid or bacteria.

Effect of Nanoalumina on Bacterial Oxidative Stress–Response Systems.

Nanoalumina affected the oxidative stress–response systems of bacteria. The production of hydroxyl-free radicals (OH⋅) in bacteria increased with the increase in nanoalumina concentration. Moreover, the OH⋅ levels in the 5 and 50 mmol/L nanoalumina groups were significantly higher than those in the control and bulk alumina groups (Fig. 3A). In addition, the total antioxidative capacity (T-AOC), catalase (CAT) activity, superoxide dismutase (SOD) activity, and glutathione reductase (GR) activity of the bacteria in the treatment groups also increased with increases in nanoalumina levels and were significantly higher than those in the control and bulk alumina groups (Fig. 3 B–E). These results indicated that nanoalumina can affect the oxidative stress–response systems of bacteria.

Fig. 3.

Fig. 3.

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The effect of nanoalumina on bacterial antioxidant systems. The effects of alumina concentration on the activity of OH⋅ (A), T-AOC (B), SOD (C), CAT (D), and GR (E) at pH 7.0, 25 ºC, 8-h mating time, and 109 cfu/mL bacteria are shown. The concentration of nanoalumina had a significant effect on the activity of the five antioxidant system indicators (ANOVA, P < 0.05); no significant difference was found in the activities of the five antioxidant system indicators when conjugation was induced by bulk alumina. Significant differences between single concentration steps and the control (or the bulk alumina group) were tested using the S-N-K test, *P < 0.05 and **P < 0.01.

Effects of Nanoalumina on Bacterial Conjugation.

The conjugation results by transmission electron microscopy (TEM) are shown in Fig. 4. In the control and bulk alumina groups, although there were many close links between the bacteria, fewer compact electron-dense areas (i.e., in conjugation the outer cell membranes of the two cells appeared to be fused; partially magnified image in Fig. 4D) were observed between the outer cell membranes of the donor and recipient (Fig. 4 A–C). When 5 or 50 mmol/L nanoalumina was added to the mating experiments, we observed that conjugation occurred among numerous bacteria and that even a single bacterium may have conjugated with multiple other bacteria (Fig. 4 D and E). This finding indicated that nanoalumina significantly promotes bacterial conjugation.

Fig. 4.

Fig. 4.

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TEM micrographs of the conjugative junctions of mating cells. (A) The conjugative junctions of mating cells in the control group. The dotted arrows indicate that the bacteria did not conjugate or only came in contact with each other; the partially magnified images (Inset) marked with an “x” showed no conjugation. Very few compact electron belts were formed (solid arrow). (B and C) The conjugative junctions of mating cells in the bulk alumina groups (B, 5 mmol/L; C, 50 mmol/L). No conjugation was found. (D and E) The conjugative junctions of mating cells in the 5.0 mmol/L (D) and 50.0 mmol/L (E) nanoalumina groups. Bacteria were able to conjugate, shown by the presence of compact electron belts [the partially magnified images (Inset) marked with a “y” in D; arrows indicate the compact electron belts]. The arrows indicate that conjugation occurred both between two bacteria and between multiple bacteria. The conjugation of the 5 mmol/L nanoalumina group was more than that of the 50 mmol/L nanoalumina group, and the conjugation of the nanoalumina groups was much more than both that of the control and bulk alumina groups. (Scale bars, 100 nm.)

Effects of Nanoalumina on the Expression of Conjugation Genes and Global Regulatory Genes.

The RP4 plasmid conjugal transfer process requires a series of conjugation genes and corresponding involvement of regulatory genes. We found that the mRNA expression of the major global regulatory genes trbA and korB was repressed significantly with increases in nanoalumina concentrations compared with mRNA expression in the control groups (Fig. 5 D and E). As a result, the mating pair formation (Mpf) gene trbBp was activated, and trbBp mRNA expression increased significantly with increasing nanoalumina concentrations (Fig. 5A). The results showed that nanoalumina promoted the formation of mating pairs, which is the first step in the conjugative transfer process. The mRNA expression of the major global regulatory genes korA and korB was repressed significantly with increasing nanoalumina concentrations compared with mRNA expression in the control groups (Fig. 5 C and D). As a result, the DNA transfer and replication (Dtr) gene trfAp was activated, and trfAp mRNA expression increased significantly with increasing nanoalumina concentrations (Fig. 5B). Our study revealed that nanoalumina can also promote the second step of the conjugative transfer process.

Fig. 5.

Fig. 5.

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The mRNA expression levels of conjugation genes and global regulatory genes of RP4. The effects of alumina concentration on the expression levels of trbBp (A), trfAp (B), korA (C), korB (D), and trbA (E) at pH 7.0, 25 ºC, 8-h mating time, and 109 cfu/mL bacteria are shown. The concentration of nanoalumina had a significant effect on the expression levels of the conjugation genes and global regulatory genes of the RP4 plasmid (ANOVA, P < 0.05); no significant difference was found in the expression of the conjugation genes and global regulatory genes of RP4 when conjugation was induced by bulk alumina. Significant differences between single concentration steps and the control (or bulk alumina groups) were tested using the S-N-K test, *P < 0.05 and **P < 0.01.

Discussion

The spread of antibiotic resistance and the potential risks of nanotechnology are two worldwide concerns. Our results show that residual nanomaterials in water can promote the spread of antibiotic resistance. Here we demonstrate that nanomaterials can promote the horizontal transfer of multiresistance mediated by RP4, RK2, and pCF10 between bacteria. It is the shared nature of nanomaterials that enables them to promote the conjugative transfer of multiresistance plasmids. Out of a range of nanomaterials, nanoalumina had the most significant effect, and not only promoted the transfer of multiresistance plasmid RP4 between the same species of bacteria but also promoted the transfer of multiresistance plasmids across genera.

In this study, we focused on the effects of nanoalumina on RP4 plasmid transfer for two reasons: (i) Compared with other nanomaterials, nanoalumina had the greatest ability to enhance RP4 plasmid transfer (by more than 200-fold compared with the control); and (ii) alumina plays an important role in the regulation of the composition of soil water, sediment water, and other natural water systems (14–18). Nanoalumina, due to its huge surface area, has several applications in this field as an adsorbent and as a catalyst (19), and has now become one of the two market leaders of nanosized materials in the United States (25).

Our results showed that the natural conjugative transfer frequency of RP4 in water was very low, with 0.15 × 10−6 to 2.0 × 10−6 per recipient cell after an 8-h mating time, although the concentrations of both donor and recipient reached 108 to 109 cfu/mL (Fig. 1). These results were identical to those described by other groups (26, 27). The presence of nanoalumina in water significantly promoted the transfer of RP4. This transfer increased with increasing nanoalumina concentrations, and reached a peak value at 5 mmol/L nanoalumina, which was >200-fold that of the control. When the nanoalumina concentration was higher than 5 mmol/L, the transfer rate decreased, as higher nanoalumina concentrations were found to damage the bacterial membrane severely and kill most bacteria (SI Appendix, Table 4). Furthermore, the nanoparticles may have aggregated and thus become relatively less bioavailable at these concentrations compared with lower concentrations (28). More importantly, plasmid transfer can increase to a rate 200-fold higher than that found in the control under the optimum conditions. In contrast, no marked effect on the transfer of RP4 was observed with the addition of the same concentration of bulk alumina. Aquatic factors had no, or no significant, effect on conjugative transfer, in contrast to the findings described in some reports that temperature and pH may affect the conjugative transfer of drug-resistance traits (29, 30). These results should be noted, because the dosages of nanoalumina used in some previous research projects were much higher than the concentration used in our present study. For example, Kumar et al. (31) used 0.5 g/50 mL (100 g/L) of nanoalumina to defluorinate aqueous solutions. Afkhami et al. (32) used 0.05 g/50 mL (10 g/L) of nanoalumina to remove heavy metals from water. Although some water treatment operations, such as coagulation and filtering, are carried out to partially remove nanoalumina, evidence is emerging that coagulation alone is not a completely effective process to remove engineering nanoparticles from solutions, as this method only achieved variable removal of 20–80% of particles (33). We found that the concentration of nanoalumina particles in our study was 2.08 × 106 particles/mL, a level that can significantly promote the transfer of RP4 plasmids.

We also explored the potential mechanisms of nanoalumina in the promotion of conjugative transfer of RP4. Trieu-Cuot et al. (34) reported that the cell walls of Gram-positive bacteria constitute an important barrier for conjugal transfer of genetic information delivered from E. coli. We hypothesize that the cell membranes of Gram-negative bacteria also play an important barrier for conjugal transfer of RP4 delivered from E. coli. It is known that oxidative stress (the SOS response) can damage cell membranes and may promote the transfer of genes or nutrients (35–38). Our study showed that nanoalumina caused the SOS response of parent bacteria and also damaged the integrity of cell membranes, as was seen from the results of TEM and AFM.

Nanoalumina can enhance conjugation efficiency through the regulation of conjugative gene expression. We observed using TEM that nanoalumina significantly promoted bacterial conjugation. This phenomenon results from the promotion of RP4 conjugation gene expression by nanoalumina. trbBp expression was enhanced after treatment with nanoalumina, which led to the formation of more conjugants. In addition, nanoalumina promoted the expression of trfAp, which is a gene that plays an important role in the transfer and replication of RP4 (39, 40).

Nanoalumina may promote horizontal transfer by repressing the expression of global regulatory genes that are involved in RP4 conjugation. The RP4 plasmid is a typical IncP α-type plasmid, a broad-host-range conjugative plasmid. The conjugative transfer of this IncP plasmid depends on the product of the trfA and trbB genes. It is believed that korA and korB both exert roles in repression of trfA expression, and trbA and korB expression severely represses the trbB promoter (41, 42). We found in this study that korA, korB, and trbA mRNA expression was repressed significantly by nanoalumina. These results mean that trfA and trbB are activated and increase this expression, which provides the sequential steps that enhance the efficiency of transfer (43, 44).

In conclusion, this report describes the promotion of the horizontal transfer of antibiotic-resistance genes between bacteria by nanomaterials. Nanoalumina in water increased the horizontal transfer of multidrug-resistance genes across genera with an increase in the concentration of nanoalumina, density of parent bacteria, conjugation time, and temperature. In contrast, aquatic factors had no or little effect on the transfer of antibiotic-resistance genes. The mechanisms by which nanoalumina promotes the transfer of drug-resistance genes may involve the damage of bacterial membranes by oxidative stresses, an enhancement of the expression of conjugative genes, and the repression of global regulatory factor genes for RP4 plasmid conjugation. The findings described in this study suggest that the application of nanoalumina in water and waste treatment should be evaluated carefully so as not to cause public health and environmental and ecological hazards.

Materials and Methods

Bacterial Strains, Media, and Experimental Conditions.

A list of the bacterial strains and plasmids used in this study is given in SI Appendix, Table 5. The six mating pairs used in this study and the resistance used for all strains are described in SI Appendix, Text 1, and the culture conditions of donors and recipients are shown in SI Appendix, Text 2.

Preparation of Nanoalumina and Bulk Alumina Suspensions.

All of the nano- and bulk materials used in this study were purchased from Sigma-Aldrich, and the preparation methods of nanoalumina and bulk alumina suspensions are shown in SI Appendix, Text 3.

Conjugation Experiment Treated with Nanoalumina and Bulk Alumina.

Immediately after ultrasonication, nanoalumina or bulk alumina was added to the donor and recipient mixture and mixed by vortexing. The conjugation cultures were then placed in an incubator to mate. After a specific time, the cultures were vigorously mixed on a vortex mixer, and appropriate dilutions were plated on selection plates containing the appropriate antibiotics. After the overnight incubation of the selection plates at 37 ºC, the colonies were counted and the results are presented as cfu/mL and transfer frequency.

To determine the effect of the main factors on the conjugative transfer, an orthogonal design L64(421) in duplicate was used to evaluate the effects of bacteria concentration, nanoalumina concentration, mating time, mating temperature, and the interactions of these factors on conjugative transfer. The orthogonal experimental scheme is shown in SI Appendix, Table 1.

In parallel, all of the strains were respectively plated on selection plates containing the appropriate antibiotics to ensure that the bacteria growing on the selection plates were transconjugants and to exclude any spontaneous mutation of the strains. Another two control groups that used K12 bearing nonconjugative pCB182 or tra-mutant RP4 (SI Appendix, Table 5) as the donor were used to observe the transformation of free plasmids dissolved from dead bacteria in the presence of nanoalumina.

Sample preparation methods for TEM, antioxidant system detection, and real-time PCR are shown in SI Appendix, Text 4.

Transmission Electron Microscopy.

Samples for TEM detection were processed by standard procedures, and details are described in SI Appendix, Text 5.

Detection of Bacterial Antioxidant Systems.

Bacterial antioxidant systems were detected with appropriate kits from the market, and details are discussed in SI Appendix, Text 6.

Detection of mRNA Expression of Mpf Gene, Dtr Gene, and Global Regulatory Genes.

mRNA expression of all genes was quantified using real-time PCR, and details are discussed in SI Appendix, Text 7.

Statistical Analysis.

Data are expressed as mean ± SD. All data were analyzed with SPSS for Windows version 17.0. Details are shown in SI Appendix, Text 8.

Supplementary Material

Supporting Information
supp_109_13_4944__index.html (1KB, html)

Acknowledgments

We thank Prof. Julian E. Davies (University of British Columbia) for providing the RP4 plasmid, Dr. Gary Dunny (University of Minnesota) for providing the pCF10 plasmid, and Prof. Xingguo Wang (Hubei University, China) for providing the pCB182 plasmid. We would also like to thank Dr. Danfeng Yang, Jing Yin, and Zhuge Xi for providing several kinds of nanomaterials. We also thank Jingran Sun, Ji Pang, Jing Lang, Xiangfei Guo, Yabo Ouyang, Ankang Gu, Nannan Hou, Bin Zhang, Sha Liu, and Yunxiao Zhang for their assistance in the preparation of the manuscript. This work was supported by National Natural Science Foundation of China Grant 30870453.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1107254109/-/DCSupplemental.

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