Curr Microbiol (2014) 69:802–808 DOI 10.1007/s00284-014-0658-9
Seohaeicola nanhaiensis sp. nov., A Moderately Halophilic Bacterium Isolated from the Benthic Sediment of South China Sea Bai-Sheng Xie • Xiang-Lin Lv • Man Cai • Yue-Qin Tang • Yan-Nan Wang • Heng-Lin Cui Xue-Ying Liu • Yan Tan • Xiao-Lei Wu
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Received: 16 April 2014 / Accepted: 26 May 2014 / Published online: 16 July 2014 Ó Springer Science+Business Media New York 2014
Abstract An aerobic, Gram-staining negative, non-motile, and rod-shaped bacterial strain, SS011A0-7#2-2T, was isolated from the sediment of South China Sea with the depth of 1,500 m. Optimum growth occurred at pH 8.0, 30 °C, and 6 % (w/v) NaCl. Strain SS011A0-7#2-2T did not synthesize bacteriochlorophyll a or carotenoid, neither possess photosynthesis genes. Its genome DNA G?C content was 67.9 mol%. It contained Q-10 as the predominant ubiquinone and C18:1 x7c (52.3 %) as the major fatty acid. The major polar lipids were phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, unidentified phospholipid, and unidentified aminolipid. The 16S rRNA gene sequence analysis revealed that it was closely related to Seohaeicola saemankumensis SD-15T, Phaeobacter gallaeciensis BS 107T and Roseovarius pacificus 81-2T in Rhodobacteraceae, with the 16S
rRNA gene sequence similarities being 96.5, 95.7, and 95.6 %, respectively. However, the phylogeny of the 16S rRNA gene sequences revealed that strain SS011A0-7#2-2T was a member of the genus Seohaeicola. Strain SS011A07#2-2T was moderately halophilic which was different from Seohaeicola saemankumensis SD-15T, and it showed the enzyme activities and carbon source spectrum significantly different from Seohaeicola saemankumensis SD-15T. As its physiological and chemotaxinomic properties were different from those of Seohaeicola saemankumensis SD-15T, strain SS011A0-7#2-2T represents a novel species of the genus Seohaecola. The name Seohaeicola nanhaiensis sp. nov. is proposed, with strain SS011A0-7#2-2T (=LMG 27733T = CGMCC 1.12759T) as the type strain.
Introduction The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain SS011A0-7#2-2T is KF312716.
Electronic supplementary material The online version of this article (doi:10.1007/s00284-014-0658-9) contains supplementary material, which is available to authorized users. B.-S. Xie X.-L. Lv X.-Y. Liu Y. Tan X.-L. Wu (&) Department of Energy and Resources Engineering College of Engineering, Peking University, Beijing 100871, People’s Republic of China e-mail:
[email protected] M. Cai China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China Y.-Q. Tang College of Architecture and Environment, Sichuan University, Chengdu 610065, People’s Republic of China
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The genus Seohaecola was proposed by Yoon et al. [29]. At the time of writing, it includes only one species, Seohaeicola saemankumensis, with the type strain SD-15T Y.-N. Wang Institute of Biology, Henan Academy of Sciences, Zhengzhou 450008, People’s Republic of China H.-L. Cui School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, People’s Republic of China X.-L. Wu Institute of Engineering (Baotou) College of Engineering, Peking University, Baotou 014030, People’s Republic of China
B.-S. Xie et al.: Seohaeicola nanhaiensis sp. nov.
isolated from a tidal flat of Saemankum in the Yellow Sea, Korea. The predominant ubiquinone of the genus was Q10, meanwhile the major fatty acids were C18:1 x7c and 11-methyl C18:1 x7c. Seohaeicola belongs to the family Rhodobacteraceae, which were often found in freshwater, marine [2, 10, 11], and hypersaline environments [12, 18], including Phaeobacter gallaeciensis BS 107T, and Roseovarius pacificus 81-2T isolated from German Wadden Sea [21] and deep sea sediment [27], respectively. Phaeobacter and Roseovarius bacteria shared some common physiological and biochemical characteristics with Seohaeicola. For example, they have the same predominant ubiquinone and major fatty acids; they don’t produce photosynthetic pigments, which is different from the type genus Rhodobacter of Rhodobacteraceae. During the investigation into microbial community of a benthic sediment in South China Sea, we isolated many novel bacterial isolates [8, 9], including strain SS011A07#2-2T. Polyphasic analyses revealed that strain SS011A07#2-2T represents a novel species of the genus Seohaecola, for which the name Seohaecola nanhaiensis sp. nov. is proposed.
Materials and Methods Isolation and Cultivation Strain SS011A0-7#2-2T was isolated from a benthic sediment (salinity 33.4 %, temperature 3.1 °C) in South China Sea at a depth of 1,500 m, by the 10-fold dilution plating technique on natural seawater agar at 30 °C for 7 days. The natural seawater agar was made by melting 1.5 % (w/v) agar in surface seawater from Yellow Sea in Tsingtao, China, without amending any other nutrients. For maintenance and identification, strain SS011A0-7#2-2T was cultured in 2216E medium [22] (Difco; l-1 distilled water: Bacto peptone 5.00 g, Bacto yeast extract 1.00 g, Fe(III) citrate 0.10 g, NaCl 19.45 g, MgCl2 (anhydrous) 5.90 g, Na2SO4 3.24 g, CaCl2 1.80 g, KCl 0.55 g, NaHCO3 0.16 g, KBr 0.08 g, SrCl2 34.00 mg, H3BO3 22.00 mg, Na2SiO3 4.00 mg, NaF 2.40 mg, NH4NO3 1.60 mg, Na2HPO4 8.00 mg, 18 g agar for solid medium, pH 8.0) at 30 °C for 3 days. Seohaeicola saemankumensis SD-15T (KCTC 22175T), Phaeobacter gallaeciensis BS 107T (DSM 17395T) and Roseovarius pacificus 81-2T (CGMCC 1.7083T) were obtained from Korean Collection for Type Cultures (KCTC), Deutsche Sammlung fu¨r Mikroorganismen, und Zellkulturen (DSMZ) and China General Microbiological Culture Collection Center (CGMCC). These type strains were used as reference strains and cultivated simultaneously with strain SS011A0-7#2-2T.
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Morphological, Physiological, and Chemotaxonomic Tests The colonial morphology was observed after strain SS011A0-7#2-2T was grown on 2216E agar plate at 30 °C for 3 days. Cell morphology and flagellum type were viewed by the transmission electron microscopy (TEM) (JEDL, JEM-1400). The Gram staining was performed with standard protocol [24]. Growth under anaerobic condition was tested with 2216E agar plate according to standard protocol [6]. Growth at 4, 15, 20, 25, 30, 37, 40, 45, 50, and 55 °C was determined in 2216E medium, while NaCl and pH were kept at 2 % (w/v) and 8.0, respectively. The pH requirement for growth was determined in 2216E medium with pH values of 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0, while temperature and NaCl were kept at 30 °C and 2 % (w/v), respectively. NaCl requirement for growth was tested in 2216E medium with NaCl concentrations of 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9 % (w/v), while temperature and pH were kept at 30 °C and 8.0, respectively. All these tests were made in triplicates, and growth was determined with optical density (OD600). Activities of oxidase and catalase, hydrolysis of starch and Tween 80, H2S production as well as motility were determined according to previously described protocols [16, 28, 5, and 26, respectively]. After grown in dark for 3 days, cells of strain SS011A07#2-2T, Seohaeicola saemankumensis SD-15T, Phaeobacter gallaeciensis BS 107T, and Roseovarius pacificus 81-2T were harvested by centrifugation. They were then resuspended in acetone/methanol (7:2, v/v) solution at room temperature for 12 h to extract photosynthetic pigments, which were detected by the absorption spectrum at 300–900 nm using spectrophotometry (SHIMADZU UV1700) [3]. The pufM gene encoding the photosynthetic center was checked by PCR amplification with the primer set: pufM.557F (50 -CGC ACC TGG ACT GGA C) and pufM.750R (50 -CCC ATG GTC CAG CGC CAG AA) previously described protocol [1]. Carbon utilization spectrum was determined by Biolog GN II MicroPlate [17]. Tests of some enzyme activities and other physiological and biochemical properties were carried out by the API ZYM and API-20NE systems [4] according to manufacturer’s instructions, respectively. Simultaneously, the three reference type strains were also tested. Antibiotic resistance was tested on 2216E plate using the test strip with the antibiotics and their concentrations are listed in Supplementary Table S1. After strain SS011A0-7#2-2T and the three reference type strains were cultured in 2216E medium at 30 °C for 3 days, they were harvested and subjected to the analyses of fatty acid profiles and respiratory ubiquinones. Meanwhile, polar lipids were analyzed for strain SS011A0-7#2-2T,
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Phaeobacter gallaeciensis BS 107T, and Roseovarius pacificus 81-2T. Fatty acids were extracted and analyzed using the Sherlock system (Microbial ID Inc.) with the standard protocol [13]. Respiratory ubiquinones were extracted with methyl chloride/methanol (2:1, v/v) and analyzed by reversed-phase HPLC (Shim-pack, VP-ODS, Shimadzu) according to the protocol by Komagata & Suzuki [15]. Polar lipids were extracted using a chloroform/ methanol system and analyzed using one- & two-dimensional TLC, as described previously [14]. Merck silica gel
60 F254 aluminum-backed thin-layer plates were used in TLC analysis. Phylogenetic Analysis The DNA G?C content of strain SS011A0-7#2-2T was determined by extracting genomic DNA [19] and running thermal denaturation profile using DNA from E.coli K-12T as a control [20]. The 16S rRNA gene was amplified by universal bacterial primer pair: 8f (50 -AGA GTT TGA
Table 1 Different characteristics of strain SS011A0-7#2-2T and the three reference strains Characteristic
1
2
3
Cell shape
Rod
Rod, oval
Ovoid rod
Ovoid to rods
Cell size (lm)
0.7–0.9 9 1.4–1.6
0.7–1.8 9 1.0–10
0.6–0.8 9 1.4–1.9
0.5–0.6 9 0.9–1.7
Flagella Motility
– –
– –
T (range, optimum, °C)
4–30, 30
4-40, NT
25–35, 27-29
4–45, 25
pH (range, optimum)
7.0–8.0, 8.0
NT, 7.0–8.0
6.0–9.5, 7.5
6–9, 6.2–8.5
? ?
4
? ?
NaCl (range, optimum %)
0–8.0, 6.0
1.0–7.0, 2.0
0.01–1.5, 0.5-0.7
1–15, 2–12
H2S production
–
–
NT
–
Indole production
?
–
–
–
Oxidase
–
?
?
?
b-Glucosidase
–
–
?
?
Leucine Aminopeptidase
?
?
–
–
Enzyme type
Valine Aminopeptidase
–
–
?
?
Acid phosphatase
?
–
?
–
Phosphohydrolase
?
–
–
–
Glucose
?
–
–
–
Arabinose Mannose
? ?
– –
– –
– –
Carbon sources
Gluconate
?
–
–
–
Adipic acid
?
–
–
–
Malic acid
?
–
–
–
Phenylacetic acid
?
–
–
–
G?C content (mol%)
67.9
63.4
57.8
62.3
Polar lipids
AL, PC, PG, DPG, PE, PL
PC, PG, PE, L, PLs*
PC, PG, PE, AL
AL, PC, PG, DPG, PE, PL
Photosynthetic pigments
–
–
–
–
–
?
Nitrate reduction
T
– T
– T
Taxa: 1. Strain SS011A0-7#2-2 ; 2. Seohaeicola saemankumensis SD-15 ; 3. Phaeobacter gallaeciensis BS 107 ; 4. Roseovarius pacificus 81-2T. All strains are Gram-negative; negative for hydrolysis of starch, and Tween 80, in activities of arginine dihydrolase, urease, gelatinase, alkaline phosphatase, lipase (C14), cysteine aminopeptidase, trypsin, chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, b-fucosidase; lack of the abilities of glucose fermentation; positive for esterase (C4) and esterase lipase (C-8); cannot utilize mannitol, N-acetyl glucosamine, maltose, capric acid, citric acid as the sole carbon sources. All strains are positive for catalase activity. The predominant ubiquinones are Q10. Data were from this study, expected for those specially described ? positive, – negative, NT not tested or not reported, PE phosphatidylethanolamine, PG phosphatidylglycerol, DPG diphosphatidylglycerol, PC phosphatidylcholine, PL unidentified phospholipid, AL unidentified aminolipid *
Data from Yoon et al. (2009)
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TCC TGG CTC AG) and 1492r (50 -GGT TAC CTT GTT ACG ACT T) and sequenced. The multiple alignments of the 16S rRNA gene sequences were performed with the MEGA software version 5.05 [25]. The phylogenetic tree was reconstructed using the neighbor-joining method [23] and evaluated by bootstrap analysis (based on 1,000 resampling replicates). Maximum-parsimony [25] and maximum likelihood [7] algorithms were also used to test the stability of the tree topology.
Results and Discussion Cells of strain SS011A0-7#2-2T were Gram-negative, rodshaped, non-motile, and without flagellum. Its size was 0.7–0.9 9 1.4–1.6 lm. After 3 days incubation on 2216E agar plates, colonies were 0.5–0.8 mm in diameter, circular, smooth, convex, and light yellow in color. The growth ranges of NaCl concentration, pH value, and temperature were 0–8 % (w/v), 7.0–8.0, and 4–30 °C with optimum growth happened at 6 % (w/v), 8.0, and 30 °C, respectively. The strain did not contain photosynthesis pigments (bacteriochlorophyll a and carotenoid) and photosynthesis pufM genes. Strain SS011A0-7#2-2T was positive for activities of catalase, esterase (C4), esterase lipase (C8),
Table 2 Fatty acids of the novel isolate and the three reference type strains Fatty acid
1
2
3
4 ND
C10:0 3-OH
3.5
ND
1.7
C12:0
ND
ND
1.5
4.9
C12:1 3-OH
3.4
–
2.0
1.8
C12:0 3-OH
–
–
–
3.9
C14:0
ND
ND
ND
–
C14:0 3OH/C16:1 iso I
ND
ND
ND
ND
C15:0
ND
–
–
–
C16:1 x7c/C16:1x6c
1.1
ND
–
–
C16:0
12.3
8.0
14.3
7.3 4.0
C16:0 2-OH
4.2
–
4.4
C17:0
1.5
ND
ND
–
C17:0 iso
ND
ND
ND
ND
C17:0 10-methyl
ND
ND
ND
ND
C18:1 x7c C18:0
52.3 4.1
68.0 –
61.7 2.6
55.1 1.1
C18:1x7c 11-methyl
4.1
12.9
9.9
11.1
C19:0 cyclo x8c
17.6
5.4
ND
9.7
Taxa: 1. Strain SS011A0-7#2-2T; 2. Seohaeicola saemankumensis SD-15T; 3. Phaeobacter gallaeciensis BS 107T; 4. Roseovarius pacificus 81-2T –, fatty acids representing \1 %, ND not detected/not reported
leucine aminopeptidase, acid phosphatase, and phosphohydrolase. It was able to produce indole. The strain was negative to produce H2S, to hydrolyze starch and Tween 80, and to reduce nitrate and ferment glucose. In addition, the strain was negative for activities of arginine dihydrolase, urease, gelatinase, a/b-galactosidase, alkaline phosphatase, lipase (C14), cystine aminopeptidase, trypsin, chymotrypsin, b-glucuronidase, a/b-glucosidase, N-acetylb-glucosaminidase, a-mannosidase, and b-fucosidase. It could utilize glucose, mannose, arabinose, gluconate, adipic acid, malic acid and phenylacetic acid, D-arabitol, Dcellobiose, i-erythritol, D-fructose, L-fucose, D-galactose, gentiobiose, D-mannitol, sucrose, turanose, xylitol, pyruvic acid methyl ester, D-saccharic acid, and glycyl-L-aspartic acid as the sole carbon source, but could not utilize the other carbon sources in the API-20NE and Biolog GN II MicroPlate. Some of these properties were distinct from those of the three reference strains (Table 1). Strain SS011A0-7#2-2T was sensitive to choramphenicol, gentamicin, neomycin, penicillin, polymyxin, cefoxintin, rifampicin, ofloxacin, piperacillin, streptomycin, amoxicillin, ceftazidime, cefoperazone, cephalexin, and ampicillin at the given concentrations, but resistance to kanamycin, novobiocin, tetracycline, erythromycin, roxithromycin, oxacillin, vancomycin, norfloxacin, bacitracin, carbenicillin, and ciprofloxacin (Supplementary Table S1). All the other physiological properties are listed in Table 1 and in description of the species. The major cellular fatty acids of strain SS011A0-7#2-2T were C18:1 x7c (52.3 %), C19:0 cyclo x8c (17.6 %), and C16:0 (12.3 %) (Table 2). The presence of C18:1 x7c as the major fatty acid was consistent with Seohaeicola saemankumensis SD-15T and most of the species in family Rhodobacteraceae. However, the strain had a much higher amount of C19:0 cyclo x8c than Phaeobacter gallaeciensis BS 107T did (Table 2). In addition, strain SS011A0-7#2-2T had C10:0 3-OH, C12:1 3-OH, C16:1 x7c/C16:1x6c, C17:0, C18:0, which were absent in Seohaeicola saemankumensis
Fig. 1 Transmission electron micrograph of strain SS011A0-7#2-2T grown on 2216E agar plate at 30 °C for 3 days. Bar, 1 lm
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Fig. 2 Neighbor-joining phylogenetic tree based on 16S rRNA gene sequence relatedness showing the relationship between strain SS011A0-7#2-2T and other related strains. Bootstrap values ([50 %) are shown at nodes. Bar, 0.02 nucleotide substitution per nucleotides
T
Phaeobacter gallaeciensis BS107 (Y13244) 100
T
Phaeobacter inhibens T5 (AY177712) T
Phaeobacter daeponensis TF-218 (DQ981486)
81
T
Leisingera methylohalidivorans MB2 (AY005463)
95
T
Marinovum algicola ATCC 51440 (X78315) T
Seohaeicola saemankumensis SD-15 (EU221274) T
Seohaeicola nanhaiensis LMG 27733 (KF312716)
86
T
Roseovarius pacificus 2-81 (DQ120726) T
Donghicola eburneus SW-277 (DQ667965) T
Yangia pacifica DX5-10 (AJ877265) T
Roseivivax halodurans OCh 239 (D85829) T
78
Salipiger mucescens A3 (AY527274) T
Pseudoruegeria aquimaris SW-255 (DQ675021) T
Antarctobacter heliothermus EL-219 (Y11552)
93
T
Sagittula stellata E-37 (U58356) T
Thalassobius mediterraneus CECT 5383 (AJ878874) T
83 52
Roseobacter litoralis Och 149 (CP002623) T
Sulfitobacter pontiacus ChLG 10 (Y13155) T
Nereida ignava 2SM4 (AJ748748) T
Octadecabacter arcticus 238 (U73725) T
Jannaschia helgolandensis Hel 10 (AJ438157) T
Ketogulonicigenium vulgare DSM 4025 (AF136849) T
62
Oceanicola granulosus HTCC2516 (AY424896) T
Escherichia coli ATCC 11775 (X80725)
0.02
SD-15T. Strain SS011A0-7#2-2T contained Q10 as the main respiratory ubiquinone, which was same as the reference strains. It contained phosphatidylcholine (PC), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), unidentified phospholipid (PL), and unidentified aminolipid (AL) as the major polar lipids, which were similar as Roseovarius pacificus 81-2T, but different from the other two reference strains (Table 1). The DNA G?C content of strain SS011A0-7#2-2T was 67.9 mol%, higher than those of the three reference strains. Analysis of the nearly complete 16S rRNA gene sequence (1387 bp) revealed that strain SS011A0-7#2-2T was a member of family Rhodobacteraceae. It had a close phylogenetic relationship with Seohaeicola saemankumensis SD-15T, Phaeobacter gallaeciensis BS 107T, and Roseovarius pacificus 81-2T with 16S rRNA gene sequence similarities of 96.5, 95.7, and 95.6 %, respectively. In addition, it formed a stable cluster with Seohaeicola saemankumensis SD-15T (Figs. 1, 2, 3; Supplementary Fig. S1, S2). From the above analyses, strain SS011A0-7#2-2T is a member of the genus Seohaeicola, but different from Seohaeicola saemankumensis SD-15T in optimum NaCl tolerance, the enzyme activities, carbon source spectrum,
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and the DNA G?C content. Furthermore, strain SS011A07#2-2T had C10:0 3-OH, C12:1 3-OH, C16:1 x7c/C16:1 x6c, C17:0, C18:0 fatty acids, and unidentified AL, which were absent in Seohaeicola saemankumensis SD-15T. Therefore, strain SS011A0-7#2-2T represents to be a novel species of the genus Seohaecola, for which Seohaeicola nanhaiensis sp. nov is proposed. Description of Seohaeicola nanhaiensis sp. nov Seohaeicola nanhaiensis (nan.hai.en’sis. N.L. fem. adj. nanhaiensis pertaining to Nanhai (the South Sea in Chinese), China, where the type strain was isolated.) Cells are Gram-negative, rod-shaped, non-motile, without flagellum, and 0.7–0.9 9 1.4–1.6 lm in size. Colonies on 2216E agar plate are circular, smooth, convex, light yellow in color, and 0.5–0.8 mm in diameter after 3 days incubation at 30 °C. Under aerobic condition, strain SS011A0-7#2-2T can grow at NaCl concentration 0–8 % (w/v), pH 7.0–8.0, and temperature 4–30 °C; the optimum growth occurs at 6 % (w/ v), pH 8.0, and 30 °C. Anaerobic growth does not occur. Growth occurs in dark. Neither bacteriochlorophyll a and
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Fig. 3 Polar lipid patterns of strain SS011A0-7#2-2T after separation by two-dimensional TLC. F-first dimension of TLC; S second dimension of TLC, PE phosphatidylethanolamine, PG phosphatidylglycerol,
DPG diphosphatidylglycerol, PC phosphatidylcholine, PL unidentified phospholipid, AL unidentified aminolipid
carotenoid nor the photosynthesis genes are detected. Starch and Tween 80 are not hydrolysed and H2S is not produced either. The strain is positive for indole production, and esterase (C4), esterase lipase (C8), leucine aminopeptidase, acid phosphatase and phosphohydrolase activities. It is negative forarginine dihydrolase, urease, gelatinase, a/bgalactosidase, alkaline phosphatase, lipase (C14), cystine aminopeptidase, trypsin, chymotrypsin, b-glucuronidase, a/ b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, b-fucosidase activities. The strain is susceptible to choramphenicol, gentamicin, neomycin, penicillin, polymyxin, cefoxintin, rifampicin, ofloxacin, piperacillin,
streptomycin, amoxicillin, ceftazidime, cefoperazone, cephalexin and ampicillin, but resistant to kanamycin, novobiocin, tetracycline, erythromycin, roxithromycin, oxacillin, vancomycin, norfloxacin, bacitracin, carbenicillin and ciprofloxacin. It utilizes glucose, mannose, arabinose, gluconate, adipic acid, malic acid and phenylacetic acid, Darabitol, D-cellobiose, i-erythritol, D-fructose, L-fucose, Dgalactose, gentiobiose, maltose, D-mannitol, sucrose, turanose, xylitol, pyruvic acid methyl ester, D-saccharic acid and glycyl-L-aspartic acid as the sole carbon sources. The predominant ubiquinone is Q-10. The major fatty acids are C18:1 x7c (52.3 %), C19:0 cyclo x8c (17.6 %), and C16:0
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(12.3 %). Major polar lipids are PC, PG, DPG, PE, unidentified PL, and unidentified AL. The DNA G?C content of the type strain is 67.9 mol%. The type strain is SS011A0-7#2-2T (= LMG 27733T = CGMCC 1.12759T). Emended Description of Genus Seohaeicola Yoon et al. [29] The descriptions of the genus Seohaeicola are as given by Yoon et al. [29], with the following amendment. The major polar lipids also contain DPG. Genome DNA G?C contents range from 63.4 to 67.9 mol%. Acknowledgments This study was supported by National Natural Science Foundation of China (31225001) and the National High Technology Research and Development Program of China (2012AA02A703).
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