SUPPLEMENTARY MATERIALAntibacterial mode of action of 1, 8-dihydroxy-anthraquinone from Porphyra haitanensis Against Staphylococcus aureusYuxi Wei 1*, Qi Liu2, Jia Yu1, Qiang Feng1,3, Ling Zhao2, Huiping Song1 and Wenxiu Wang1As one kind of anthraquinone dihydroxy derivatives, 1, 8-dihydroxy -anthraquinone (Dan) with strong antibacterial activity against Staphylococcus aureus was first isolated from Porphyra haitanensis. Here we report on the investigation of the antibacterial mode of action of Dan on the gram-positive bacterium Staphylococcus aureus. The results show that Dan strongly inhibited cell growth at logarithmic phase. In present study, the Dans antibacterial activity was analysed by using phosphorus standard solutionPSS, p-nitrophenyl phosphate (pNPP)o-nitrophenyl--D-galactopyanoside (ONPG), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results suggested that Dans antibacterial activity is due to its interaction with the cell wall and cell membrane, in which it increases cell envelope permeability and leads to the leakage of cytoplasm and the deconstruction of cell. The present study indicates that Dan as a natural product in seaweeds deserves further investigation for applications as an antibacterial bioactive substance in food safety control and drugs.Key wordsPorphyra haitanensis; 1, 8-dihydroxy-anthraquinone; antimicrobial action; Staphylococcus aureus1College of Life Sciences, Qingdao University, Qingdao 266071, PR China; 2Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, PR China; 3Center for Disease Control and Prevention of Jining City, Jining 272000, PR China*Corresponding author. E-mail address: yuxiw729@163.com Experimental

Materials

Porphyra haitanensis sample collected from the Jinjiang coastline (Fujian, China) in February 2013 was authenticated by Institute of Oceanology, Chinese Academy of Sciences. The voucher specimen (QD-SW-20130068) was deposited in the Biochemistry Lab of Qingdao University, Qingdao, China. 1, 8-dihydroxy- anthraquinone (Dan) was isolated from the sample by the reference method (Feng et al., 2013). Staphylococcus aureus (CAU0519) was supplied by Institute of Microbiology, Chinese Academy of Sciences. Bacterial cells were cultured and activated in fresh tryptone soybean broth (TSB) at 28 for 12 h.

Effect of Dan on the Growth Curve of S. aureus The experiment was carried out according to reference (Ai et al. 2007) with some modifications. BrieflyS. aureus culture was inoculated in nutrient broth medium with an inoculation amount of 106 CFU/mL and cultured at 37 with the air bath and homothermal vibrator at 150 rpm. Optical density (OD) at 550 nm was monitored with spectrophotometer every hour during a course of 13 hours as control group. The procedure for experimental group was the same as above except that bacterial suspension contained 80 g/mL Dan.

Effect of Dan on the phosphorus metabolism of S. aureus The experiment was carried out according to reference (Hancock & Rozek 2002, Qian et al. 2010, Zhai et al. 2006) with some improvements. S. aureus culture was inoculated in 5 mL of nutrient broth with an inoculation amount of 106 CFU/mL. Then 5 mL of glucose standard solution (1 mg/mL), 2 mL of KH2PO4 standard solution (0.17 mg/mL) were added. The mixture was cultured at 37 with the air bath and homothermal vibrator at 150 rpm. For each hour, the culture solution was centrifuged at 5000 rpm for 10 min. The supernatant (0.5 mL) was mixed with 4 mL CCl3COOH-FeSO4 solution (containing 10mg/mL thiourea, 100.0 mg/mL CCl3COOH and 0.6 mg/mL FeSO4) and layed up for 10 min. After each centrifugation, the OD value of the supernatant was determined by ammonium molybdate spectrophotometry at 630 nm as control group. The procedure for experimental group was the same as above except that bacterial suspension contained 80 g/mL of Dan.

Effect of Dan on the cell wall permeability of S. aureus determination of alkaline phosphatase activityMethods for bacterial culture and centrifugation of culture solution were the same as above. The experiment was performed as previously described (Wang et al. 2006). After centrifugation for each hour, the supernatant (2 mL) containing 80 g/mL Dan was mixed with 5 mL p-nitrophenyl phosphate (pNPPthe Sigma Chemical Co. St. Louis, MO, USA) substrate buffer. The mixture was heated at 40 for 5 min. A 2.0-mL portion of 0.5 mol/L Na2CO3 was added to stop reaction, and then absorbance reading was taken at 410 nm using a spectrophotometer. OD410 value was used to indicate the activity of alkaline phosphatase as experimental group. The procedure for control group was the same as above except that there was no Dan added.

Effect of Dan on the membrane permeability of S. aureus

Protein content determination of culture media Methods for bacterial culture and centrifugation of culture solution were the same as above. After centrifugation for each hour, the content of proteins in culture of both groups was assayed by Coomassie brilliant blue method at 595nm (Bradford 1976). -galactosidase activity assay of culture media. Methods for bacterial culture and centrifugation of culture solution were the same as above for both groups. The experiment was carried out according to references (Li et al., 2012; Ibrahim et al. 2000). After centrifugation for each hour, 4 mL o-nitrophenyl--D-galactopyanoside (ONPGthe Sigma Chemical Co. St. Louis, MO, USA) substrate buffer was mixed with 1.0 mL supernatant of culture solution obtained above (containing 80g/mL Dan) and the mixture was heated at 50 for 30 min. A 2.0-mL portion of 1.0 mol/L Na2CO3 was added to stop reaction, and then absorbance reading was taken at 420 nm using a spectrophotometer. OD420 value was used to indicate the activity of -galactosidase. The procedure for control group was the same as above except that there was no Dan added.

Sample Preparation of S. aureus and Observation by SEMThe experiment was carried out according to reference (Tang et al. 2010) with some improvements. S. aureus pure culture was inoculated with an inoculation amount of 106 CFU/mL, in nutrient broth medium (control group) or nutrient broth medium containing 80 g/mL Dan (experimental group) respectively. After being allotted into three tubes and cultured at 37 with the air bath and homothermal vibrator for 3, 5 and 7 h respectively, the culture solution was centrifuged at 5000 rpm for 10 min to collect bacteria cells. The pellets were prefixed in 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH7.3), postfixed in phosphate buffer (pH7.4), and dehydrated in graded ethanol solutions (30%, 50%, 70%, 80%, 90% and 100%). Pellets were then dried at critical point, coated with gold palladium by direct-current sputtering and examined with a HITACHI S-450 scanning electron microscope. Fig.S6 The morphology of S. aureus at incubation time of 0, 3, 5 and 7 h. Fig.S7 The morphology of S. aureus after incubation with Dan for 3, 5 and 7 h

Sample Preparation of S. aureus and Observation by TEM The experiment was carried out according to reference (Xing et al. 2009) with some improvements. The culturing, collecting, washing, fixing and dehydration procedures of S. aureus sample treated with Dan were the same as those used for preparing the sample observed by SEM. Pellets were embedded in Epon 812, fixed at 37, 45 and 65 and then cut using an ultramicrotome (UltracutE). Changes of ultrastructure of S. aureus treated by Dan were observed under transmission electron microscope (JEM-1200EX) after the thin sections were stained by uranyl acetate and lead nitrate. The same suspension was incubated in nutrient broth medium with distilled water instead of Dan as control group and the sample preparation for TEM observation was the same as that for experimental group. Fig.S8 The ultrastructure of S. aureus at incubation time of 0, 3, 5 and 7 h.

Fig.S9 The ultrastructure of S. aureus after incubation with Dan for 3, 5 and 7 h Statistical analysis All data were analysed by ANOVA method and expressed as mean standard deviation. ReferencesAi Q, Yu Q, Zhang H, Wu X, Wang X, Li X. (2007). Studies on Bacteriostatic action and antimicrobial mechanism of pyrolin against Staphylococcus Aureus. J Chin Inst Food Sci Technol 7(2):33-37Bradford MM. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizating the principle of protein-dye binding. Anal Biochem 1-2: 248-254Feng Q, Wei YX, Qi J, Wang CY, Wu TF. (2013). Studies on chemical constituents of Porphyra haitanensis. Mar Sci 37(5):15-18

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