1

Analysis of the Toxic Mode of Action by Silver Nano-1

Particles Using Stress-Specific Bioluminescent Bacteria 2

Ee Taek Hwang1,5, Jin Hyung Lee2,5, Yun Ju Chae1, Yeon Seok Kim1, Byoung Chan Kim3, Byoung-In 3

Sang4 and Man Bock Gu1* 4

5 1College of Life Sciences and Biotechnology, Korea University, Seoul, Rep. Korea 6

2Department of Environmental Science and Engineering, Gwangju Institute of Science and 7

Technology, Gwangju, Rep. Korea 8 3Diagnostics Group, Institut Pasteur Korea, Seoul, Rep. Korea 9

4Hazardous Substances Research Center, Korea Institute of Science and Technology, Seoul, Rep. 10

Korea 11 5Contributed equally to this work 12

13

Materials 14

The silver nanoparticles used in this study were purchased from the Nanopoly Company, Republic of 15

Korea, and have an average diameter of 10 nm. The silver nanoparticles were dissolved in Milli-Q water 16

at a final concentration of 10,000 mg L-1. The gold nanoparticles and AgNO3 were purchased from BB 17

International Company, UK and Sigma-Aldrich, USA, respectively. The gold nanoparticles also have an 18

average diameter of 10 nm. The silver nanoparticles were synthesized by a chemical reduction using reducing 19

agents such as hydrazine hydrate, formaldehyde and sodium formaldehydesulfoxylate, according the protocol of 20

the company [1,2,3]. 21

22

Silver nanoparticle characterization 23

The silver nanoparticles were characterized by Transmission Electron Microscope (TEM) imaging, salt 24

induced coagulation, X-ray diffraction (XRD) and Thermogravimetric/Differential Thermal Analysis 25

(TG/DTA). Agglomeration of the nanoparticles should be considered in toxicology study. In this study, 26

agglomeration was investigated by using TEM image and salt induced coagulation test (Supporting 27

Figures S1 and S2). Both indicate that the silver nanoparticles do not form aggregates with the 28

experimental conditions used. 29

2

We confirmed the size of the silver nanoparticles using transmission electron microscopy (TEM) 1

(Supporting Figure S1). This figure was included to show that the silver nanoparticles do not form 2

aggregates during the toxicity assay, even at high concentrations (50000 mg L-1). 3

4

5

6

7

8

9

10

Figure S1. TEM images of the silver nanoparticles. The silver nanoparticles were diluted 2-fold in LB media to 11

a final concentration of 50000 mg L-1. This picture was obtained after a two hour incubation in a rotary shaker at 12

37 °C, which are the same conditions as used for the toxicity assay experiments. Scale bar = 10 nm 13

14

We also tested the effects different salt concentrations had on the aggregation of the nanoparticles using 15

TEM (Supporting Figure S2). The media used was LB media and the salt concentration (NaCl) varied 16

from 0 to 20g L-1. Usually LB medium has 10 g L-1 NaCl. As shown in the figure, the silver 17

nanoparticles did not form aggregates with 10 g L-1 NaCl, which is the concentration used in this study 18

A. 19

20

B. 21

3

1

C. 2

3

D. 4

5

E. 6

4

1

Figure S2. TEM images of the silver nanoparticles in media containing different NaCl concentrations. The 2

silver nanoparticles were dissolved in a medium which has same composition as LB medium but with different 3

NaCl concentrations. The salt concentrations were A) 0 g L-1, B) 5 g L-1, C) 10 g L-1, D) 15 g L-1 and E) 20 g L-1. 4

Typically, LB medium has 10 g L-1 NaCl. The final concentration of silver nanoparticles in each sample was 50 g 5

L-1. Scale bar = 10 nm 6

7

Ө/ degree

Inte

nsity

8

Figure S3. XRD pattern for the silver nanoparticle used in this study. This figure indicates that the sample used 9

in this study is silver metal. This data was acquired and kindly supplied by the Nanopoly Company which 10

manufactured the silver nanoparticles as per our request. 11

12

5

-4

-3

-2

-1

0

1

2

3

4

Tem

pera

ture

diff

renc

e (o c/

mg)

0 200 400 600 800 1000

48.4

48.6

48.8

49.0

49.2

49.4

49.6

49.8

50.0

W

eigh

t (m

g)

Temperature (oC)Temperature/ °C

Wei

ght/

mg

Wei

ght d

iffe

renc

e/ °C

mg

-1

1

Figure S4. TG/DTA curves of the silver nanoparticle used in this study. The red line corresponds to the TG 2

curve and the blue the DT curve. This figure indicates that the silver sample is metal, not ion. This data was 3

acquired and kindly supplied by the Nanopoly Company as per our request. 4

5

Bacterial strains and toxicity assay 6

Seven recombinant Escherichia coli strains were used to evaluate the mechanisms by which the 7

samples are toxic. The E. coli hosts and plasmids used in this study are listed in Table S1. The 8

construction of each strain is described in the references cited. 9

10

Table S1. Description of the strains used in the toxicity assay. 11

Strains Plasmid Host Ref. Strains Plasmid Host Ref.

RFM443 N/A1 RFM4432 [4] DPD2794 pRecALux RFM443 [7]

DS1 pSodALux RFM443 [5] RecN pRecNLux RFM443 [8]

DK1 pKatGLux RFM443 [5] AlkA pAlkALux RFM443 [8]

DC1 pClpBLux RFM443 [6] NrdA pNrdALux RFM443 [8]

1 No recombinant plasmid 12

6

2 The genotype of RFM443 is (rpsL – (StrR), galK2, lac∆74). 1

2

Wild strain, RFM443, was used to measure the cell growth, which was determined at 600 nm using a 3

spectrophotometer (Supporting Figure S5). 4

A 5

0.01

0.1

1

10

0 50 100 150 200

Time/ min

Opt

ical

Den

sity

0 mg L-1

0.4 mg L-1

0.5 mg L-1

0.6 mg L-1

0.8 mg L-1

1.0 mg L-1

6 7

B 8

0.01

0.1

1

10

0 50 100 150 200

Time/ min

Opt

ical

Den

sity

0 mg L-1

0.4 mg L-1

0.5 mg L-1

0.6 mg L-1

0.8 mg L-1

1.0 mg L-1

9

Figure S5. Cell growth of wild type strain RFM443 in the presence of A) silver nanoparticles or B) gold 10

nanoparticles. 11

7

1

The toxicity assay was performed using flask tests. Initially, all the strains were streaked from glycerol 2

stocks onto agar plates containing 50 µg mL-1 ampicillin (Sigma, USA), except RFM443, which was 3

grown on LB agar. One colony, grown overnight, was inoculated into a culture tube containing 2 mL of 4

Luria-Bertani (LB) medium also containing 50 µg mL-1 ampicillin as needed and cultured for seed in a 5

rotary shaker at 30°C or 37°C according to the properties of the lux genes, i.e., 30ºC for the V. fischeri 6

lux strain (DPD2794) and 37ºC for RFM443 and the X. luminescens lux strains (all others) until 7

reaching an O.D. (600nm) of 0.8. From these seed cultures, 100 µL was transferred to 10 mL of sterile 8

LB, with ampicillin, in a 50 mL flask and grown to an optical density of 0.08 at a wavelength of 600 nm. 9

At this cell density, 20 µL of the silver nanoparticles were added to achieve the desired final 10

concentration. Each of the nanoparticle solutions was prepared by diluting the stock in LB so that 20 µL 11

would be added in each case. The O.D. and bioluminescence (BL) of the cultures were then measured 12

every 10 min for 2 hr using a spectrophotometer and a Turner 20e Luminometer, respectively. As a 13

control experiment, the same conditions were used, but without silver nanoparticles. The results were 14

then transferred to Microsoft Excel™, analyzed and are presented as the relative bioluminescence 15

(RBL), which is defined as the ratio of the sample’s BL to that of the control at the same time. For 16

growth inhibition tests, ampicillin was not used in the medium and only the optical density was 17

measured during culturing. 18

Enzyme detoxification experiments were the same as with the toxicity assay experiments except the 19

addition of the two enzymes (final concentration of 15 units mL-1 of catalase and superoxide dismutase). 20

21

A 22

0

5

10

15

20

0 0.1 0.2 0.3 0.4 0.5 0.6 0.8 1

Concentration of silver nanoparticle/ mg L-1

RB

L/ A

.U.

23

24

B 25

8

0

5

10

15

20

0 0.1 0.2 0.3 0.4 0.5 0.6 0.8 1

Concentration of silver nanoparticles / mg L-1

RB

L/ A

.U.

1

C 2

0

5

10

15

20

0 0.1 0.2 0.3 0.4 0.5 0.6 0.8 1

Concentration of silver nanoparticle /mg L-1

RB

L/ A

.U.

3

D 4

0

5

10

15

20

0 0.1 0.2 0.3 0.4 0.5 0.6 0.8 1

Concentration of silver nanoparticle / mg L-1

RB

L/ A

.U.

5

Figure S6. Responses from the DNA damaging responsive strains A) DPD2794 (recA::luxCDABE), B) 6

AlkA ( alkA::luxCDABE), C) RecN (recN::luxCDABE) and D) NrdA (nrdA::luxCDABE) to different 7

concentrations of silver nanoparticles. The results are presented as the maximum relative 8

bioluminescence seen for each test concentration. 9

9

1

A. 2

0

5

10

15

20

0 0.1 0.2 0.3 0.4 0.5 0.6 0.8 1

Concentration of silver microparticle mg L-1

RB

L/ A

.U.

3

B. 4

0

5

10

15

20

0 0.1 0.2 0.3 0.4 0.5 0.6 0.8 1

Concentration of silver microparticle/ mg L-1

RB

L/ A

.U.

5

C. 6

0

5

10

15

20

0 0.1 0.2 0.3 0.4 0.5 0.6 0.8 1

Concentration of silver microparticle/ mg L-1

RB

L/ A

.U.

7

D. 8

10

0

5

10

15

20

0 0.1 0.2 0.3 0.4 0.5 0.6 0.8 1

Concentration of silver microparticle/ mg L-1

RB

L/ A

.U.

1

Figure S7. Responses from strains A) DS1 (sodA::luxCDABE), B) DC1 (clpB::luxCDABE), C) DK1 2

(katG::luxCDABE) and D) DPD2794 (recA::luxCDABE) to different concentrations of micro-sized 3

silver particles. The results are presented as the maximum relative bioluminescence seen for each test 4

concentration. 5

6

Real-time quantitative RT-PCR assays 7

For real-time RT-PCR assays, three primers and probe sets (forward primer, reverse primer, reporter probe) for 8

the clpB, sodA and rrnH genes were purchased from Applied Biosystems (Poster City, CA, USA). Each primer 9

and probe set were designed to identify the sodA (forward primer: 5’-CAG CAG ACA AGA AAA CCG TAC 10

TG-3’, reverse primer: 5’-TTC CAG AAC AGG CTG TGG TTA G-3’, reporter probe: 5’-CCG CCA GCG TTG 11

TTG-3’), clpB (forward primer: 5’-GCC TGA AAG AAC GTT ACG AAT TGC-3’, reverse primer: 5’-GTC 12

GCC GCT GCA ACA AT-3’, reporter probe: 5’-CAC CAT GTG CAA ATT A-3’) and rrnH (forward primer: 13

5’-CGC TCA GGT GCG AAA GC-3’, reverse primer: 5’-GCA CAA CCT CCA AGT CGA CAT-3’, reporter 14

probe: 5’-TCC ACG CCG TAA ACG-3’)gene, with the rrnH gene being used as an endogenous control. 15

Reporter probes were labeled at the 5’ end with the fluorescent reporter dye 6-carboxy-fluorescein (FAM) and at 16

the 3’ end with a non-fluorescent quencher (NFQ). 17

For real-time RT-PCR assays, Escherichia coli strain RFM443 was grown in LB medium at 37 °C and 200 rpm. 18

When the optical density at 600 nm reached 0.08, silver nanoparticles were added into the flask to a 19

concentration of 0.4 mg L-1, since this was the maximum sub-lethal concentration, i.e., it does not inhibit the cell 20

growth. The cell suspension was harvested at 5, 10, 15, 20, 30 and 60 min after initiating the exposure by 21

centrifuging samples of the cultures at 13,000 rpm for 15 min. The total cellular RNA from the test and control 22

samples was extracted using the QIAGEN RNeasy mini columns (QIAGEN, USA) according to the 23

manufacturer’s instructions. The concentration and quality of the RNA extracted was determined using ND-1000 24

UV/Vis spectrophotometer (Nanodrop, Wilmington, USA). The ratios of UV 260/280 were between 1.8 and 2.1 25

11

for all the RNA samples. As a negative control, the total RNA from unexposed cells was extracted using the 1

same procedure in parallel. 2

For reverse transcription (RT), the RT-mixture was assembled using TagMan® Reverse Transcription Reagents 3

(Applied Biosystems, USA) on ice, with each sample containing 0.5 µg of total RNA, 1 X TaqMan® RT buffer, 4

5.5 mM magnesium chloride, 500 µM of each dNTP, 2.5 µM random hexamer, 0.4 U µL-1 RNase inhibitor, and 5

1.25 U µL-1 MultiScribeTM reverse transcriptase in a 100 µL reaction mixture. Thermocycling parameters for RT 6

included an incubation step at 25 ºC for 10 min, followed by a reverse transcription step at 48 ºC for 30 min and 7

a reverse transcriptase inactivation step at 95 ºC for 5 min in the GeneAmp® PCR system 9700 (Applied 8

Biosystems, USA). Real-time PCR reactions were then performed in triplicate in a MicroAmp Optical 96-well 9

plate (Applied Biosystems) using 2X TaqMan® Universial PCR Master mixture, 3 µl of total cDNA (four target 10

genes and rrnH) from the RT steps above, and 1.5 µL of the primers and probe sets in a 30 µL reaction volume. 11

Thermocycling parameters included an incubation step at 50 ºC for 2 min, followed by Taq DNA polymerase 12

activation at 95 ºC for 10 min and 40 PCR cycles of 95 ºC for 15 sec/60 ºC for 1 min. 13

Real-time RT-PCR assays were analyzed using the ABI PRISM 7000 sequence detection system (Applied 14

Biosystems). The gene expression levels of the target genes were normalized against the expression level of the 15

rrnH gene, which encodes for the 16S rRNA. The induction ratio was calculated using the function 2-∆∆CT 16

according to the ABI Prism 7000 SDS Software handbook where ∆∆CT = (CT, target gene - CT, rrnH)stressed - (CT, target gene 17

- CT, rrnH)control. [9] 18

19

A 20

0

2

4

6

8

10

12

5 10 20 30

Time / min

Gen

e ex

pres

sion

rat

io

21

12

1

B 2

0

2

4

6

8

10

12

10 20 30

Time / min

Gen

e ex

pres

sion

rat

io

3

4

Figure S8. Real Time RT-PCR gene expression results from wild type strain RFM443 for genes A) sodA and 5

B) clpB after an exposure to 0.4 mg L-1 silver nanoparticles. 6

7

Data analysis 8

All samples were performed in triplicate for error analysis. The standard deviations for the results are shown as 9

error bars within the graphs. 10

13

References 1

[1] K. Chou, C. Ren, Mater. Chem. Phys. 2000, 64, 241-246. 2

[2] P. K. Khanna, V. V. V. S. Subbarao, Mater. Lett. 2003, 57, 2242-2245. 3

[3] H. H. Nersisyan, J. H. Lee, H. T. Son, C. W. Won, D. Y. Maeng, Mater. Res. Bul. 2003, 38, 949-4

956. 5

[4] R. Menzel, Anal. Biochem. 1989, 181, 40-50. 6

[5] R. J. Mitchell, J. M. Ahn, M. B. Gu, J. Microbiol. Biotechn. 2005, 15, 48-54. 7

[6] R. J. Mitchell, M. B. Gu, Biosens. Bioelectron. 2006, 22, 192-199. 8

[7] J. H. Min, E. J. Kim, R. A. LaRossa, M. B. Gu, Mutation Res. 1999, 442, 61-68. 9

[8] J. M. Ahn, E. T. Hwang, C. H. Youn, D. L. Banu, B. C. Kim, M. B. Gu, In preparation 10

[9] K. J. Livak, T. D. Schmittgen, Methods 2001, 25, 402-408. 11

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