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Killing of Bacillus subtilis by exogenous hydroxyl radicals

Wu Chun-du*, Hou Ke-hui, He Hua-gang,

Zhu Shan-ying, Shi Lan, Yi Cheng-wu, Chu Jin-yu

School of Environment Jiangsu University

Zhenjiang, People’s Republic of China *E-mail: wucd@ujs.edu.cn

Wu Chun-du Yangzhou College of Environment and Resource

Yangzhou, People’s Republic of China

Abstract---In this study, we used Bacillus subtilis

168, a spore-producing gram-positive bacterium,

as a model to reveal the effects and mechanisms of

killing by exogenous hydroxyl radical (OH·). The

results showed that, exogenous OH· could kill B.

subtilis efficiently. Nearly 99% of them was killed

in 0.3 mg/L OH· solution, and 100% in 0.8 mg/L

OH· solution. The exogenous OH· could damage

biomolecules of B. subtilis. The quantity and the

integrities of DNA, RNA and protein decreased

with the increasing concentration of OH·. The

activities of superoxide dismutase (SOD), catalase

(CAT) and the content of H2O2,were analyzed after

treated with different concentration of exogenous

OH·. The results indicated that, the content of

H2O2 was decreased, the activities of SOD and CAT

were also changed.

Keywords: Bacillus subtilis, hydroxyl radicals,

killing, biomolecules, enzyme activity.

I. INTRODUCTION Bacillus subtilis is one kind of bud

spore-producing bacillus in extreme environment. Spores are the strongest resistance life body in the biosphere. So completely killing B. subtilis is a most important target while evaluate each kind of disinfection and antiseptic method in food industry and medical.

Hydroxyl radicals with a very high oxidation electrode potential (E0=2.80 V) is a

strong oxidant. It can react thoroughly and completely with almost all types of biomolecules: carbohydrates, nucleic acids, lipids, proteins and amino acids, organic, inorganic. The final products are carbon dioxide, water and some inorganic salts [1]. In recent years, hydroxyl radicals are used as a kind of advanced oxidation technique to resolve environmental problems. Hydroxyl radicals can be generated by Fenton reaction [2], UV/H2O2 [3] and strong electric-field ionization discharge [4].

II. MATERIALS AND METHODS A. Bacterial culture, growth and treatment

The gram-positive bacterium B. subtilis was used in this study and grown in LB medium at 37 ℃ for 6 hours. Bacterial culture was centrifuged at 5,000 rpm for 10 min. Cell pellet was washed and resuspended in sterile water with an OD600 of 1.0. Hydroxyl radicals were generated by strong ionization discharge [4], and the concentration was detected [5]. 10 ml of cell suspension was treated with different concentration of hydroxyl radicals for 10 min. Three repeats were set in the study [6]. B. Survival analysis of B. subtilis

100 μl of treated samples were diluted 10-fold in water and 50 μl of the dilutions were plated on LB-agar. After overnight incubation, colonies were counted to analyze the survivals of B. subtilis under different concentration of hydroxyl radicals.

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C. Extraction, concentration measurement of biomolecules

The treated samples were centrifuged at 6,000 rpm for 5 min and washed twice before biomolecules extraction. Extraction of DNA and RNA were performed as described by Maloy [7] and by Summers [8], respectively. RNA and DNA have an absorbance peak at 260 nm, so the concentration of DNA and RNA samples was calculated according to the value of OD260. Intracellular soluble protein was extracted by sonication at 200 w for 1 min after treatment with lysozyme (50 mg/ml) for 60 min at 4℃. The concentration of protein was determined using Bradford method [9]. D. Integrity assay of biomolecules

DNA and RNA samples were electrophoresis using 1% agar gel in 1×TAE buffer and stained with EB solution. E.Enzyme assay and H2O2 content determination

Superoxide dismutase (SOD) activity was determined by measuring its ability to inhibit the photochemical reduction of nitro blue tetrazolium chloride (NBT) [10]. The reaction mixture (2 ml) contained 50 mM phosphate buffer (PH7.8), 750 μM NBT, 130 mM Methionine, 20 μM riboflavin and 50 μl enzyme extract. Riboflavin was added last and the glass tube were shaken and illuminated with two 15-W fluorescent tubes for 15 min. Measure the absorbance of reaction mixture at 560 nm [11].

CAT activity was measured according the method of Beers and Sizer [12], with minor modification. The reaction texture (2 ml) consisted of 20 mM H2O2, 100 mM PBS (pH7.0), 50 μl enzyme extract. The reaction was started by addition of the extract, and monitored the decrease of H2O2 at 240 nm [11].

The content of H2O2 was determined according the method: mix well 1 ml enzyme extract, 0.1 ml of 5% Ti(SO4)2 and 0.2 ml of ammonium hydroxide, centrifuge at 8,000 rpm for 5 min, then remove the fluid suspension and wash the sediment with 1 ml acetone three times.

Dissolve the sediment in 1 ml of 2 M sulfuric acid solution and monitor the absorbance at 415 nm [13].

III. RESULTS A. Survival analysis of B. subtilis

To test effect exogenous hydroxyl radicals on Gram-positive bacteria, survivals of B.subtilis were analyzed. The result shown that, after treated by 0.1 mg/L and 0.2 mg/L OH· solution, the survival sharply drop to 30.5% and 3%, respectively. When the concentration of OH· increase to 0.3 mg/L and 0.4 mg/L, the survival was 0.6% and 0.38%. The concentration reached to 0.8 mg/L, there are still have one or two colony, maybe it is the spore germination.

B. Concentration measurement of biomolecules To evaluate the level of damage of major

biomolecules on B. subtilis, the concentrations of DNA, RNA and protein were measured. The results shown that amount of all tested reduced with the increasing of OH· and the degrees were different with each other. Overall, the degree of damage of intracellular soluble protein was severely during the three kinds of biomolecules and that of RNA was slightly.

Fig.1 Effects of OH· on the survival of B. subtilis

Fig.2 Effects of exogenous OH· on the DNA, RNA and Protein

C. Integrity assay of DNA and RNA

Molecule integrities were further assayed by agarose gel electrophoresis. The results showed no significant change of DNA bands was observed after treated by 0.1 to 0.3 mg/L OH· solution. After treated by 0.4 and 0.8 mg/L OH· solution, the size of DNA bands became shorter or disappeared (the left of Fig.3). After treated by 0.1 to 0.3 mg/L OH· solution, the bands of RNA maintained relatively complete. With the further increasing of exogenous hydroxyl radicals to 0.4 mg/L and 0.8 mg/L, obvious degradation of RNA was observed (the right of Fig. 3)

Fig. 3 Effects of exogenous OH· on DNA and RNA integrities.

Results of electrophoresis of DNA and RNA were showed in the

picture left and right, respectively. M1 was λ-Hind III digested

DNA marker, and M2 represented DL2000 marker. From lane 1

to 6, the concentrations of exogenous hydroxyl radicals used to

treat B. subtilis were 0, 0.1, 0.2, 0.3, 0.4, 0.8 mg/L, respectively.

D. Enzyme activity and H2O2 content

Superoxide dismutase (SOD) and Catalase (CAT) are important antioxidant enzymes in vivo. As the graph displayed that the activity of SOD

was maximum in 0.3 mg/L point, and sharp dropped with the OH· concentration increasing. The strange things was the activity of CAT no significant change. However, the content of H2O2 was reduced always.

Fig. 4 Enzyme activity and H2O2 content.

IV. DISCUSSION

As a strong oxidant, hydroxyl radicals can oxidize all kinds of organic matters including organisms and a variety of toxic, harmful, pollution in the environment. So, hydroxyl radicals were used to control water pollution, solid waste and sterilize Food et al, in the present research. In this study, we reported the effects of OH· on killing the B. stubtilis which is one of gram-positive bacteria producing spores. Efficient killing was obtained by low concentration of hydroxyl radicals. Only 0.2 mg/L OH· solution could damage almost all B. subtilis. We further analyzed the influence of OH· solution on the concentration and integrity of biomolecules, such as DNA, RNA and intracellular soluble protein. The results indicated the concentration of DNA, RNA and protein decreased with the increasing of OH· solution and the relative concentration of protein reduce fastest than that of nucleic acids (DNA and RNA). However, study the mechanisms of killing B. subtilis is not uncomplicated. In 0.1 mg/L exogenous hydroxyl radicals, the concentration and integrities of DNA, RNA and intracellular protein maintained relatively well, but the survival was exceedingly

M 1 1 2 3 4 5 6 1 2 3 4 5 6 M 2

low. So we researched the antioxidant system. SOD and CAT are the important antioxidant

enzymes. Superoxide anion (O2-) is toxic

byproducts of oxidative metabolism for the cell (Bowler et al., 1992). The dismutation of O2

- into H2O2 and oxygen by SOD is an important step in protecting the cell. However, H2O2 also being toxic must be eliminated by conversion to H2O in subsequent reactions by CAT. The results indicated that SOD and CAT activity increased with the concentration of OH· radicals increasing while H2O2 content is decreased. The max activity of SOD and CAT were at the 0.3 mg/L and 0.2 mg/L OH· radicals, respectively. Cell damage and protein degradation becomes more serious owing to the OH· solution increasing, the enzymes activity also decreased. In addition, after OH· treatment, the amount of B. subtilis dropped obviously, especially at 0.4 mg/L and 0.8 mg/L. The trend of H2O2 content was in line with the enzymes activities change on the whole.

ACKNOWLEDGEMENTS This research was supported by National

Natural Science Foundation of China (50778083), Innovation Foundation for Graduate Students of Jiangsu Province (CX09B_211Z), Qing Lan project of Jiangsu Province, and supported by Science and Technology Innovation Team of Jiangsu University.

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