World J Microbiol Biotechnol DOI 10.1007/s11274-010-0396-5
ORIGINAL PAPER
Isolation, identification and screening of microorganisms for cytotoxic activities from deep sea sediments at different pacific stations Xiang Zeng • Xiang Xiao • Dehai Li Qianqun Gu • Fengping Wang
•
Received: 16 November 2009 / Accepted: 19 March 2010 Ó Springer Science+Business Media B.V. 2010
Abstract Bacteria and fungi were recovered from deep-sea sediments of the Middle Pacific, West Pacific, and East Pacific by using different isolation media. More than 150 strains were isolated, 90 of which were selected for initial identification and cytotoxic activity tests. The strains were identified by 16S rRNA gene sequencing and grouped into 19 different genera, including Acinetobacter, Arthrobacter, Bacillus, Burkholderia, Halomonas, Hyphomicrobium, Kocuria, Marinobacter, Microbacterium, Micrococcus, Nocardiopsis, Phialocephala, Planomicrobium, Planococcus, Leifsonia, Pseudoalteromonas, Pseudomonas, Psychrobacter, Shewanella, and Streptomyces. The cytotoxicities of the strains toward the mouse temperature-sensitive p34cdc2 mutant tsFT210, murine lymphoma P388 and human leukemia K562 cell lines were determined preliminarily by the sulforhodamine-B (SRB) or methyl thiazolyl
X. Zeng School of Life Sciences, Xiamen University, 361005 Xiamen, People’s Republic of China e-mail:
[email protected] X. Zeng X. Xiao F. Wang Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, SOA, Daxue Road No.184, 361005 Xiamen, People’s Republic of China X. Xiao F. Wang (&) School of Life Sciences and Biotechnology, State Key Laboratory of Marine Engineering, Shanghai JiaoTong University, 200240 Shanghai, People’s Republic of China e-mail:
[email protected] D. Li Q. Gu Key Laboratory of Marine Drugs, Chinese Ministry of Education, Institute of Marine Drugs and Food, Ocean University of China, 266003 Qingdao, People’s Republic of China
tetrazolium (MTT) bioassay method. The metabolites from three bacteria (Bacillus sp. EP39, Pseudomonas sp. WP133, and Pseudomonas sp. WP168) and one fungus (Phialocephala sp. FL30r) had strong cytotoxic activities (Inhibition rate [ 50%). Nine cytotoxic compounds belonging to phenazine, indole, polyether, and ester were isolated from them through bioassay-guided chromatography and identified by spectral methods. Keywords Deep sea sediments Culturable organisms Diversity Cytotoxic
Introduction The oceans constitute more than 70% of the earth’s total area. Of the total sea surface, only 7–8% is the coastal area and the rest are deep seas, 60% of which is covered by water more than 2,000 m in depth. The deep sea is a unique and extreme environment characterized by high pressure, low temperature (or high temperature at hydrothermal vents), lack of light, and variable salinity and oxygen concentration. It is the largest remaining unexplored area on the earth (Das et al. 2006). By using molecular-based, culture-independent microbiological methods, such as 16S rRNA gene library, PCR-DGGE (Polymerasechain reaction-denaturing gradient gel electrophoresis), PCR–RFLP (Polymerase chain reaction –restriction fragment length polymorphism), the microbial diversity in some hydrothermal vents, cold seeps, and deep-sea sediments have been investigated (Takai and Horikoshi 1999, Xu et al. 2004, Sogin et al. 2006). It has been revealed that the deep sea extreme environments contain highly diversified
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World J Microbiol Biotechnol Fig. 1 Map of the Pacific deep sea sites for collecting sediment samples. Dark circles indicate the sites
microbial communities. On the other hand, after Zobell (1941) studied on the cultural requirements of bacteria from sea water, a few investigators used the zobell’s 2216E media to isolate marine bacteria or give some modifications based on it to isolate acidophiles, alkaliphiles, and halophiles (Takami et al. 1999). Extracelluar enzyme-prodcing bacteria are also paid attention to screen, because of their important role for in situ biogeochemical processes and their potentials in biotechnology and industry applications (Dang et al. 2009). Raghukumar et al. (2004) and Damare et al. (2006) demonstrated the presence of fungi in deepsea sediments, but admitted that they were hard to detect because of inactive spores or low abundance by the limited isolation strategies. Briefly, many of isolates from several deep sea environments have been defined as novel microbial species (Bull et al. 2000). Microorganisms can sense, adapt, and respond to their environment quickly and can compete for defense and survival by producing unique secondary metabolites. The marine environment serves not only as a source of untapped taxonomic diversity of microorganisms but also as a source of novel microbial metabolites with unique chemical structures. Marine-derived microorganisms have attracted increasing attention in recent decades because of their great potential as producers of biologically active secondary metabolites such as hydrolase, antibiotics, and cytotoxins (Faulkner 2002, Chen et al. 2004, Zhang et al. 2005). Bioactive metabolites with novel structures have been isolated from the genera such as Actinomyces, Alteromonas, Bacillus, Pseudomonas, Streptomyces, and Vibrio from seawater, sediments, crustacean, sponges, mollusks, and fish in the marine environments (Kelecom 1999, 2002). The cytotoxic activities of some marinederived bacteria were tested, and it has been reported that the strains from seawater and sediments show less
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cytotoxic activity than those from seaweeds and invertebrates (Lin et al. 2005). Some cytotoxic compounds were also isolated from sediment-derived bacteria (mainly Streptomyces, Bacillus), and bacteria from sediments were considered a preferred source for cytoxicity screening (Gustafson et al. 1989, Trischman et al. 1998, Jeong et al. 2006, Gorajana et al. 2007). In the present study, different media were applied to isolate bacteria or fungi from several Pacific deep sea sediments. These strains were partially characterized and evaluated for cytotoxic activities. Our results indicated that the deep sea sediments provide large reservoirs of new bacterial species and novel bioactive compounds.
Materials and methods Sample collection Sediment samples were collected by a multi-core sampler at different sites from 2001 to 2003, respectively (Fig. 1). During the cruise of DaYang No. 1 in 2001, 3 samples were collected at the west Pacific WP site (E142°300 0800 , N8°000 1100 , 1,914 m in depth), east Pacific A site (W153°520 1900 , N7°330 4600 , 5,027 m) and middle Pacific MP site (W177°420 2000 , N10°350 0600 , 5,774 m). During the cruise of DaYang No.1 in 2002, 4 samples were collected at the sites of WP02-1 (E125°000 000 , N16°560 900 , 3,000 m), WP022 (E148°440 800 , N19°240 100 , 5,080 m), WP02-3 (E148°000 000 , N13°000 000 , 4,500 m) and WP02-4 (E141°430 700 , N09°460 800 , 2,900 m) in the west Pacific. During the cruise of DaYang No.1 in 2003, another 4 samples were obtained in the east Pacific at the sites of E2003-01 (W145°220 0900 , N9°100 2500 , 5,100 m), E2003-03 (W145°230 0400 , N7°370 2800 , 5,115 m), W2003-01 (W154°050 2800 , N10°500 3500 , 5,111 m), W2003-03
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(W154°040 5700 , N8°300 2000 , 5,059 m). The sediment cores were fractionated and transferred to sterile falcon tubes on a clean bench and kept at 4°C for shipping to the laboratory, then stored at -20°C. The pH and salinity of each sample were measured according to Munson et al. (1997). The total organic carbon content (TOC) of each sample was determined by the method of Gaudette et al. (1974). Isolation of deep sea bacteria from sediment samples After melting at 2–4°C, sediments were suspended and diluted with filtered ‘‘in situ’’ seawater or sterile artificial seawater containing 0.3% (wt/vol) NaCl, 0.07% (wt/vol) KCl, 0.53% (wt/vol) MgSO47H2O, 1.08% (wt/vol) MgCl26H2O and 0.1% (wt/vol) CaSO47H2O. Aliquots were taken out and spread onto different media for isolation (Table 1). Isolation media consisted of the following: (1) Marine 2216E agar plates (abbreviated as 2216E-N in Table 1) containing 0.5% (wt/vol) tryptone, 0.1% (wt/vol) yeast extract, 0.01% (wt/vol) FePO4, 3.4% (wt/vol) NaCl, 1.5% (wt/vol) agar, pH 7.6 ± 0.2; (2) Nitrate mineral salts (NMS) medium(Whittenbury et al. 1970) with 0.05–0.5% methanol at pH 6.8 for methylobacteria; (3) Medium A containing 0.05% (wt/vol) (NH4)2SO4, 0.05% (wt/vol) K2HPO4, 0.02% (wt/vol) CaCl2, 0.05% (wt/vol) NaNO3 and 1% (wt/vol) ammonium ferric citrate, pH 7.0; (4) Medium B containing 0.1% (wt/vol) FeSO47H2O, 0.2% (wt/vol) tryptone, 0.05% (wt/vol) yeast extract and 0.02% (wt/vol) MnSO4, pH 7.0–7.2; (5) M9 minimal medium with 1% only organic carbon source, such as carrageenan (Yantai Seaweed Industry Co., China), alginate (Qingdao Nanyang Seaweed Industry Co. Ltd., China), Tween80 (Sangon, China), olive oil (Leveking Bio-engineering, China) to isolate carrageenase-, alginate lyase-, esterase-, and lipase-producing bacteria; (6) Potato Dextrose Agar (PDA) for fungi species containing 20% (wt/vol) potatoes infusion, 2% (wt/vol)dextrose; 1.5% (wt/vol) agar; pH 7.2; (7) Modified marine 2216E(abbreviated as 2216E-M in
Table 1) agar plates of pH 3.0, 9.7 or NaCl concentration of 15% were used for detection of acidophiles, alkaliphiles, and halophiles as described previously by Takami et al. (1999). The plates were incubated at 4°C or 20°C for recovery of bacteria or fungi until colonies were visible. The strains were selected by different morphology of colonies. The piezotolerant species were enriched in the marine 2216E media under the pressure of 30 MPa (abbreviated as 2216E-P in Table 1) in the pin-closure pressure vessels by the dilution-to-extinction technique (Li et al. 2006). DNA isolation, PCR amplification and DNA sequencing The 16S rRNA gene fragments of the strains were amplified by colony PCR, which eliminated the need for DNA extraction. PCR amplification of the 16S rRNA gene fragments was according to the methods described previously. Primers EubF 933 (50 -GCACAAGCGGTGG AGCATGTGG-30 ) and EubR 1387 (50 -GCCCGGGAACG TATTCACCG-30 ), Eubac 27F (50 -AGAGTTTGATCC TGGCTCAG-30 ) and Eubac 1492R (50 -GGTTACCTTGTTACGACTT-30 ), were used to amplify the 500 bp or 1.5 kb fragment, of the bacterial 16S rRNA gene respectively. Fungal DNA was amplified using the highly conserved fungal rRNA gene primers ITS1 (50 -TCCGT AGGTGAACCTGCGG-30 ) and ITS4 (50 -TCCTCCG CTTATTGATATGC-30 ). The fragments were purified by ethanol precipitation. They were then digested for 8 h or more at 37°C with the restriction endonuclease MspI. The results were visualized by electrophoresis on a 5% (wt/vol) agarose gel containing ethidium bromide (0.5 mg L-1). Representative 16S rRNA gene fragments amplified by PCR with different RFLP patterns were selected, and the PCR products were ligated to pMD18-T vector (Takara, Japan) and transformed into Escherichia coli XL-Blue. Sequencing was performed with an ABI PEISM 377DNA
Table 1 Summary of the bacterial isolates from deep sea sediment samples with 9 different isolation media Phylum Medium
Proteobacteria
2216E
NMS ? Methanol MA ? Fe2? MB ? Mn2? Total Percentage(%)
M9?
N
M
P
TW/O A
K
20
14
3
21
6
3
4
0
0
71
78.89
High G ? C Gram-positive
0
0
0
2
0
0
5
2
6
15
16.67
Low G ? C Gram-positive
1
0
1
0
0
0
1
0
1
4
Total
21
14
4
23
6
3
10
2
7
90
100
Percentage (%)
23.33 15.56 4.44 25.56
2.22
7.78
100
100
6.67 3.33 11.11
4.44
2216E-N: normal marine 2216E agar; 2216E-M: modified marine 2216E agar with pH9.7 or 15% NaCl; 2216E-P: liquid 2216E with 30 MPa in pressure vessels; the minimal media M9 with organic matter (Tween/olive oil, alginate, carrageenan); the minimal media NMS with methanol; Medium A ? Fe2?; and Medium B ? Mn2?
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sequencer (Sangon, Shanghai, People’s Republic of China). The 16S rRNA gene sequences of the deep sea strains have already been deposited in the EMBL, GenBank and DDBJ nucleotide sequence databanks under accession numbers: AJ551089-AJ551094, AJ551096AJ551098, AJ551101, AJ551102, AJ551104, AJ551108, AJ551110-AJ551114, AJ551117, AJ551122-AJ551129, AJ551132, AJ551133, AJ551135, AJ551137, AJ551138, AJ551140, AJ551141, AJ560628, AJ715928, AM286803, AM396490-AM396492, AM396932-AM396935, AM403654AM403657, AM409192-AM409194, AM409196, AM410617, AM410618, AM410625, AM410627, AM410631, AM410898, AM410899, AM411063, AM411621, AM411992, AM411994, AM411996, AM412003, AM412004, AM412217, AM412548, AM412551-AM412555, AM418389-AM418391, AM421779, AM422562-AM522564, AM710607-AM710612, EF143420, EF143422, EF143424, EF143426, EF143428, EF143431, EF179615, EF186771, FM999988.
Crude extract preparation from deep sea strains Isolated bacteria were cultured for the production of secondary metabolites in 300 mL 2216E marine media contained in 500 mL Erlenmeyer flasks. Flasks were incubated on the rotatory shaker at 200 rpm and at 20°C. After 3– 5 days of cultivation, the broth was centrifuged at 5,0009g for 30 min to remove the cells, and the supernatant was extracted with ethyl acetate (100 mL 9 3) at a ratio of 1:1 (v/v) for three times. The isolated fungus strain was cultured at 28°C for 10 days on a rotary shaker (120 rpm) in 500 mL Erlenmeyer flasks containing 150 mL sea-water based culture media (2.0% (wt/vol) glucose, 20% (wt/vol) potato extract, 0.2% (wt/vol) yeast extract, 0.3% (wt/vol) peptone, 1% (wt/vol) NaCl, 0.08% (wt/vol) MgCl26H2O, and 0.1% (wt/vol) KCl). The whole broth was made and filtered through cheesecloth to separate the supernatant and mycelia. The former was extracted with ethyl acetate, while the latter was extracted with acetone. The acetone extraction was evaporated under reduced pressure to afford an aqueous solution and then extracted with ethyl acetate. The two ethyl acetate were combined. The ethyl acetate layer was concentrated under vacuum to give the crude extract, which was used for cytotoxic activities screening against the mouse temperature-sensitive p34cdc2 mutant tsFT210, murine lymphoma P388 and human leukemia K562 cell lines. The metabolites made by strains EP39, WP133, WP168 and FL30r with high cytotoxic activities ([50%) were chosen for further isolation of the bioactive compounds. The 100 L fermentation broths for EP39, WP133 and WP168, and 70 L for FL30r were extracted to give the crude extracts for further isolation of the bioactive compounds.
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Compound isolation, identification and cytotoxic activity evaluation The metabolites from different strains were screened for cytotoxic activities against tsFT210 cells, and further confirmed on K-562 and P388 cell lines using the sulforhodamine-B (SRB) method (Skehan et al. 1990). Cell suspensions (200 lL) were plated in 96-cell plates at a density of 5 9 104 cell mL-1. Two micro litters of the test compound solutions (in DMSO) at various concentrations (10-4, 10 -5, 10-6, 10-7, and 10-8 lM) were added to each well and the culture was incubated for 24 h at 37°C. Then, the cells were fixed with 12% trichloroacetic acid and the cell layer was stained with 0.4% SRB. The absorbance of SRB solution was measured at 515 nm. Dose response curves were generated and the concentration of each compound required to inhibit cell proliferation by 50% (IC50) was calculated from the linear portion of the log dose response curves. Cytotoxic activities were also determined by a colorimetric microassay based on the use of MTT method (Mosmann 1983). Cancer cell lines were put respectively into 96-cell plates, 90 lL per hole. After cultivation for 24 h, 10 lL extracts of the microorganisms were added into each hole. Five concentrations of each sample and three parallel holes for each concentration were designed. In addition, blank control holes were also prepared. After all the cells were cultured 48 h in 37°C and 5% CO2, 20 lL 5 mg/mL MTT solution was added into each hole. After another 4 h cultivation, 50 lL mixture of 10% SDS–5% isobutyl alcohol–0.01 mol/L HCl was introduced into the culture solution, the final optical density (OD) of it was determined by enzymatic spectroscopy instrument at 570 nm. The following formula was used to calculate the inhibition rate of cancer cells and the results in Table 1 were the averages of three parallel holes. Inhibitionrate ¼ ½ðthe OD of control the OD of sampleÞ=the OD of control 100% The crude extracts showing cytotoxic activities were chromatographed by repeated silica gel, LH-20, RP-18 and PHPLC to yield the pure compounds using bioassay guided isolation. The fractions of each chromatography were tested on the cancer cell lines and the active fractions were selected for further purification until obtained pure compounds. The structures of the compounds were further identified by using spectroscopic methods(1D and 2D NMR, together with UV, IR, MS or X-ray). The cytotoxic activities of pure compounds were evaluated on some of the cell lines K-562, P388, HL-60, BEL7402 or A-549 cell lines using SRB or MTT method. CDDP (cis-Diaminedichloroplatinum) was used as a positive control (IC50 values of 0.039 and 0.078 lM against P388 and K562, respectively).
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Results and discussion Isolation and characterization of sediment-derived bacteria from different Pacific sites We examined 11 sediment samples collected from different sites in the Pacific deep sea (West Pacific, abbreviated as WP; Middle Pacific, as MP; East Pacific, as EP) (Fig. 1). Analysis of environmental parameters of these sediment samples showed that they were from low temperatures (0– 4°C) and high pressures (1,914–5,115 m), and that they were oligotrophic samples with low total organic carbon (TOC) contents (ranging from 0.11 to 0.29%) and low organic matter (OM) (ranging from 0.18 to 0.51%). One hundred and fifty randomly selected colonies from different plates were purified for colony morphology and characterization. The strains selected were first screened by 16S rRNA gene PCR–RFLP, and 90 isolates were identified by 16S rRNA gene sequence analysis and compared with known sequences in GenBank. The evolutionary distances among the deep sea bacterial isolates in this study and the reference strains were computed, and a phylogenetic tree was constructed by the neighbor-joining method (Fig. 2). A diverse group of cultivable bacteria was retrieved, ranging from G- to G? bacteria, including Acinetobacter, Arthrobacter, Bacillus, Burkholderia, Halomonas, Hyphomicrobium, Kocuria, Leifsonia, Marinobacter, Microbacterium, Micrococcus, Nocardiopsis, Planomicrobium, Planococcus, Pseudoalteromonas, Pseudomonas, Psychrobacter, Shewanella, and Streptomyces. The isolated bacteria were checked for growth at different temperatures. Most of the strains could grow at 4°C but could not grow at temperatures above 37°C, indicating that most of them are cold-adapted bacteria. The strains were observed by microscopy, most were either rod- or coccus-shaped, while some exhibited a change in shape from rods to cocci during growth (photo not shown). Hydrolase activity was further analyzed according to Bowman (2001). Some isolates could produce hydrolytic enzymes, including amylase, esterase, galactosidase, gelatinase, lecithinase, lipase, and protease(data not shown). Different isolation media (organic matter, pH, salt concentrations, and pressure) were used, and they were selective for certain genera (Table 1). On marine 2216E medium, Halomonas (EP63-65, WP88) and Psychrobacter (WP229-231, WP240, WP243, MP244, EP245, and EP247) species were dominant, and another 2 piezotolerant Shewanella (WP264, WP265) were isolated. On M9 minimal medium supplemented with Tween or olive oil, Pseudomonas (WP127, WP129, WP130, WP133, WP145, WP155, WP159, and WP283) and Pseudoalteromonas (WP195, WP196, WP198, WP200, and WP201) species of the Gamma-Proteobacteria were predominantly isolated.
Using NMS plates with methanol, Microbacterium sp. (WP99), Micrococcus sp. (WP103), Planomicrobium sp. (WP116), Leifsonia sp. (WP117), Pseudomonas sp. (WP156, WP167, WP168), and Psychrobacter sp. (WP237, WP239) were isolated. Using medium A with Fe2?, 2 strains related to Streptomyces sp. (EP287, EP290) were isolated. Using medium B with Mn2?, Gram-positive strains of Arthrobacter sp. (EP289), Kocuria sp. (EP45, EP96), Nocardiopsis sp. (EP107, EP108), Planococcus sp. (EP118), and Streptomyces sp. (EP285, EP286, EP292-294) were isolated. Using minimal medium with carrageenan or alginate, most isolates (EP49, EP110, EP135, EP147, EP151, EP152, EP158, EP160, and EP259) were related to Pseudomonas sp., and 1 isolate (EP216) related to Psychrobacter sp. We also enriched the east Pacific samples under 30 MPa pressure, wherein 4 piezotolerant strains were isolated by the dilution-to-extinction technique; these strains were Planomicrobium sp. EP300, Serratia sp. EP301, Pseudomonas sp. EP302, and Shewanella sp EP303. High pressure (30-50 MPa) had no effect on either their cell morphology or growth. Nineteen alkaliphiles and halophiles were isolated using pH 9.7, 15% NaCl modified 2216E media, and all belonged to Halomonas species. We could not, however, isolate acidophiles from any of the deep sea sediment samples. Using PDA media, three fungi were isolated and for further cytotoxic tests. Two fungi (F1, F23) are both related to Penicillium sp. One fungus, namely, FL30r, related to Phialocephala sp., was isolated from the east Pacific site W2003-03. Among the isolated bacterial strains, three strains WP10, WP264, and WP265, have been identified as novel species: Arthrobacter ardleyensis sp. nov., Shewanella psychrophila sp. nov., and Shewanella piezotolerans sp. nov., respectively (Chen et al. 2005, Xiao et al. 2007). By constructing a 16S rRNA gene library and sequencing, we found that the Proteobacteria division, especially c-Proteobacteria, is predominant in the deep sea open ocean sediments, and Green nonsulfur bacteria and Cytophaga-Flexibacter-Bacteroides bacteria were also detected (Xu et al. 2004). Of the total isolates observed, more than half of the culturable strains were from the c-Proteobacteria subdivision, with the rest of b-Proteobacteria, G?, G- strains (Fig. 2). No a-, d-, e-Proteobacteria, green nonsulfur bacteria, Cytophaga/Flexibacter/ Bacteroides (CFB) phylum were isolated, partly because of lacking suitable isolating approaches in this study. When we used the marine 2216E medium, 2 genera, Halomonas and Psychrobacter, belonging to the c-Proteobacteria class, were the most frequently isolated species. By adding solo organic carbon (carrageenan, alginate), Pseudomonas strains were isolated as a result of their metabolic diversity. The addition of Tween80 or olive oil into minimal media made the Pseudomonas, Pseudoaltermonas species dominant. The oligotrophic medium containing Mn2? enriched the G?
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World J Microbiol Biotechnol 98 WP145,EP147,EP151-152,WP283 91 Pseudomonas fulva
EP110
43
95
Pseudomonas putida WP164165,WP167,WP168*
58 100
Pseudomonas stutzeri EP49 WP130,EP158
99
100 52 100 95
57
Pseudomonas synxantha WP129,WP127,WP155156,WP159160,,EP216,EP259
Pseudomonas rhodesiae WP133* Pseudomonas resinovorans EP98 100 Marinobacter salsuginis 91 WP83-88 70 Halomonas meridiana 74 Halomonas aquamarina
58
48
100
WP60,WP6869,WP71,EP6165 MP7678,EP79,EP80 WP3 100
25
Gamma-Proteobacteria
Acinetobacter junii WP240,WP243244,EP245,EP247,EP259
100
99
Psychrobacter marincola
92
WP233,WP235
100 99 69
EP303 WP264
100 48 100
WP74,WP229231,WP237,WP239 Psychrobacter pacificensis
Shewanella fidelia WP265 99
Shewanella benthica
100
100 100
WP93
Hyphomicrobium indicum
81
EP301 100
Serratia marcescens
WP196,WP198,EP205
74 99
94
Pseudoalteromonas WP195,WP200
100
Pseudoalteromonas elyakovii WP51
100
Burkholderia cepacia
Beta-Proteobacteria
100 EP300
Planomicrobium
100
EP118
100
99
Low G+C Gram-positives
Planococcus citreus EP39*
100
Bacillus pumilus
71 EP290,EP292-293 63 Streptomyces albidoflavus 100 EP285-287,EP294
Streptomyces sampsonii WP99
94
Microbacterium phyllosphaerae
100 100
WP70
100
100
Microbacterium oxydans WP117
100
Leifsonia rubeus 100
72
EP107
Nocardiopsis dassonvillei 100 EP45 99 Kocuria
76
High G+C Gram-positives
rosea
EP96 100
Kocuria carniphila
100
100
EP289
Arthrobacter oxydans 94
100
WP10
Arthrobacter bergeri 0.02
81 71
WP103
Micrococcus luteus
Fig. 2 Phylogenetic relationship between bacterial isolates on the basis of 16S rRNA gene sequences. The branching pattern was generated by the neighbor-joining method. The isolates were from
different Pacific cores (West Pacific, Middle Pacific, East Pacific, abbreviated as WP, MP, EP). The isolates indicated with an asterisk have high cytotoxic activities
species. These results imply that lower nutrient concentrations improve the initial isolation and recovery of diverse microorganisms (e.g., G? species), since they help avoid contamination and overgrowth by fast growing strains. Our observations are in agreement with the results of a previous study on marine-derived G? species (Gontang et al. 2007). On the other hand, isolation under ‘‘in situ’’ pressure
recovered 4 piezotolerant strains from the different genera that can survive under high pressure (50 MPa), namely, Planomicrobium, Serratia, Pseudomonas, and Shewanella. In this study, most of the isolates collected were piezotolerant but not obligately piezophilic. One of the main reasons might be the storage of the samples under the atmosphere pressure. Sampling and pure culture isolation of deep-sea
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piezophiles without loss of in situ pressure is required (Jannasch et al. 1983; Kim and Kato. 2010).
Table 2 Number of bacteria in different genus showing variable cytotoxic activities Genus
Inhibition rates
Screening for cytotoxic activity
\5%
These 90 isolates were tested for cytotoxic activities on a tsFT210 cell line by the SRB or MTT method. Eighteen extracts (20% of all extracts) showed almost no cytotoxic activity. Most of the isolates (39 strains, 43.33% of all extracts) showed weak cytotoxic activity (Inhibition rate = 5–25%). The other 30 extracts (33.33% of all extracts) showed moderate cytotoxic activity (Inhibition rate = 25–50%). The crude extracts of 3 strains belonging to the genera Bacillus and Pseudomonas (3.33% of all tested strains) showed high cytotoxic activities (Inhibition rate [ 50%). As shown in Table 2, active strains (with an inhibition rate [ 25%) were distributed among 10 genera: Bacillus (1/1 all in the genus), Halomonas (6/21), Leifsonia (1/1), Nocardiopsis (1/2), Planomicrobium (1/1), Planococcus (1/1), Pseudomonas (11/22), Pseudoalteromonas (1/7), Psychrobacter (2/14), and Streptomyces (6/8). The proportions of active strains belonging to the genera Pseudomonas (11/22) and Streptomyces (6/8) are far higher than those from any other genus. Of the 22 Pseudomonas strains, 2 extracts had high activities, 9 extracts showed medium activities with an inhibition rate [25%, while the remaining 11 extracts had little to no activity with an inhibition rate of \25%. Of the 8 Streptomyces isolates, 6 extracts showed activity higher than 25%, and 2 extracts had low activities. Among all these bacterial species, the extracts from EP39 (Bacillus sp.), WP133 (Pseudomonas sp.), and WP168 (Pseudomonas sp.) showed strong activities with inhibition rates of 78.67, 51.97, and 54.23%, respectively (Table 3). The growth curves related to the cytotoxic activities were further analyzed. For these bacteria (EP39, WP133, and WP168), the inhibition was obvious at 2–3 days (in their early stationary growth phase)
5–25%
Total 25–50%
[50%
Acinetobacter
0
1
0
0
1
Arthrobacter
0
1
0
0
1
Bacillus Burkholderia
0 1
0 0
0 0
1 0
1 1
Halomonas
3
12
6
0
21
Hyphomicrobium
1
0
0
0
1
Kocuria
1
0
0
0
1
Leifsonia
0
0
1
0
1
Marinobacter
0
1
0
0
1
Microbacterium
0
1
0
0
1
Micrococcus
0
1
0
0
1
Nocardiopsis
0
1
1
0
2
Planomicrobium
0
0
1
0
1
Planococcus
0
0
1
0
1
Pseudomonas
5
6
9
2
22
Pseudoalteromonas
2
4
1
0
7
Psychrobacter
4
8
2
0
14
Shewanella Streptomyces
1 0
1 2
0 6
0 0
2 8
18
39
30
3
90
Total
after inoculating 2216E medium at 20°C (data not shown). In addition, the extract from the fungus Phialocephala sp. FL30r showed strong cytotoxic activity, with an inhibition rate of 66.14% (Table 3). We found that most Pseudomonas, Streptomyces, and Firmicutes strains (such as Bacillus, Leifsonia, Planomicrobium, and Planococcus) exhibited a certain level of cytotoxic activity on cancer cells. The detection of high cytotoxicities associated with Bacillus and Pseudomonas strains was not unexpected as they have been demonstrated to be efficient producers of many biologically active
Table 3 Isolates with high cytotoxic activity and the structure of their active/novel compounds. Inhibition rate was valued against tsFT210 cells Isolates Closested species no (identity %)
16 s rRNA gene accession no.
EP39
AM396932 78.67
WP133 Pseudomonas gessardil (95%)
Structural elucidation of active/novel compounds
References
W2003-03 (5,059 m)
Phenazine Derivative; 9-Methyl-5,8, 10,13-tetraoxaheptadecane; Indole
Li et al. (2007c)
AM396933 51.97
WP02-3 (3,000 m)
Tributyl phosphate; Butyl isobutyl phthalate
This study
WP168 Pseudomonas stutzeri (99%)
AM396934 54.23
WP02-1 (4,500 m)
1H-indole-3-carbaldehyde
This study
FL30r
FM999988
W2003-03 (5,059 m)
Trisorbicillinoid; Bisorbicillinoid
Li et al. (2007a, b)
Bacillus pumilus (98%)
Phialocephala malorum (99%)
Inhibition Origin rate (%)
66.14
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were isolated (Li et al. 2007a). Compound A was only tested at 1 concentration (50 lM) because of the limited amount and showed cytotoxicity with an inhibition rate of 78.3% on P388 cells. Compound B (9-methyl-5, 8, 10, 13tetraoxaheptadecane) also demonstrated weak cytotoxic activity with an IC50 of 67 lM on P388 cells. Active compound C (indole) was cytotoxic with an IC50 of 74 lM on P388 cells (Table 3, Fig. 3). Eight compounds from Pseudomonas sp. WP133 and 12 compounds from Pseudomonas sp. WP168 were isolated and their cytotoxic activities were evaluated. Two compounds, tributyl phosphate (D) and butyl isobutyl phthalate (E) from Pseudomonas sp. WP133, showed cytotoxic activities on P388 cells with IC50 values of 63.4 lM and 97.6 lM, respectively. One active compound, 1H-indole-3-carbaldehyde (F), with an IC50 value of 88.9 lM on P388 cells was isolated from Pseudomonas sp. WP168 (Table 3; Fig. 3). Nitrogenated metabolites such as indole and phenazine are the most frequent chemical classes found in marine microorganisms (Kelecom 1999, 2002). Phenazine have been found in microorganisms such as Streptomyces and Pseudomonas, some of which have antimicrobial and cytotoxic activities (Laursen and Nielsen 2004). Compound A (phenazine derivative) from isolate Bacillus sp. EP39 was found to be a novel compound and could be a potential drug lead as an anticancer agent. Indole from bacteria and fungi, such as Alteromonas, Pseudomonas, Vibrio, and Penicillium, have been found to possess
compounds (Gontang et al. 2007, Jeong et al. 2008). Actinomycetes have traditionally represented one of the most important resources for the discovery of new active metabolites (Ayuso et al.2005). Some marine-derived actinomycetes such as Streptomyces have been found to be a good source for novel bioactive metabolites (Jeong et al. 2006, Liu et al. 2007, Martin et al. 2007). Furthermore, marine fungi have been recognized as one of the last, barely-tapped sources for new biologically active secondary metabolites (Vita-Marques et al. 2008). We isolated three fungal strains. Only one of them, which belong to Phialocephala as determined by 18S rRNA gene analysis, showed high cytotoxic activity. Novel bioactive compounds were isolated from this strain, our finding further indicates that more efforts should be devoted to searching for them from marine fungus. Isolation and structural elucidation of active compounds The metabolites produced by 3 bacterial isolates (Bacillus sp. EP39, Pseudomonas sp. WP133, and Pseudomonas sp. WP168) and 1 fungus (Phialocephala sp. FL30r) strongly inhibited the growth of cancer cells (P388 cells). The extracts were purified on silica gel, LH-20, RP18, and HPLC by the bioassay-guided method. From strain EP39 (Bacillus sp.), a novel phenazine derivative, compound A, along with 6 known compounds
A
B
N
C
O OH
O
N O
O
D
O
O
O
O
N H
E
O O P O
F
O
O
O O
OH N H
O
G
OH H
H O
HO
O
H
O
O
I
OH O H
H
O
HO HO
H
O
HO HO
O
OH OH
O
OH O H HO HO
H O
O HO HO
O O
HO HO
OH
Fig. 3 Active cytotoxic compounds isolated. A new phenazine derivative (A) and other two active compounds 9-methyl-5,8,10,13tetraoxaheptadecane (B) and indole (C) were isolated from Bacillus sp. EP39. Two active compounds, tributyl phosphate (D) and butyl
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isobutyl phthalate (E), were isolated from Pseudomonas sp. WP133. One active 1H-indole-3-carbaldehyde (F) was isolated from Pseudomonas sp. WP168. A novel trisorbicillinone A (G) and two new bisorbicillinoids (H, I) were isolated from Phialocephala sp. FL30R
World J Microbiol Biotechnol
antifungal and cytotoxic activities (Kelecom 1999). Compound C from EP39 and compound F from WP168 are both indoles with cytotoxic activities. In the malonate-derived metabolites, polyether is rarely found in marine microorganisms but often found in invertebrates (Kelecom 1999). Compound B classified to polyether was found from EP39, and that kind of chemical is rarely studied on cytotoxity. Compounds D and E from WP133, in the class of esters, have also been scarcely found with cytotoxities before. One novel trisorbicillinoid and 2 new bisorbicillinoids (compound G, H, I) were isolated from fungus Phialocephala sp. FL30r (Li et al. 2007b, c). Compound G showed cytotoxic activities with IC50 values of 9.10 lM, [100 lM, 30.21 lM on P388, A549 and K562 cells, respectively. Compound H was cytotoxic with IC50 values of 29.9, 103.4, and 56.3 lM on P388, A549, and K562 cells, respectively. Compound I was cytotoxic with IC50 values of 40.3, 97.6, and 68.2 lM on P388, A549, and K562 cells, respectively. The isolation and structural elucidation of these active compounds from their respective strains are shown in Table 3 and Fig. 3. Sorbicillinoids and bisorbicillinoids are metabolites that have been found in a variety of fungi and have attracted great attention for their interesting biological properties such as antifungal, antitumor, and antioxidative activities (Gerhard et al. 2005). Our study further indicates that marine bacteria and fungi from deep sea sediments could be an important source of cytotoxic metabolites. In conclusion, increasing attention has been focused on the potential applications of bacteria and fungi from deep sea environments. This study focused on isolating culturable microorganisms by different media from deep sea sediments at Pacific sites and demonstrated that they are rich sources of bioactive cytotoxic compounds. Acknowledgments We are grateful to the crews on HaiYang No.4 and DaYang No.1 for assisting in sample collection. This work was supported by the Chinese Ocean Mineral Resource R & D Association (DYXM-115-02-2-01) and Chinese National High-Tech R&D Program (2007AA091904).
References Ayuso A, Clark D, Gonza´lez I, Salazar O, Anderson A, Genilloud O (2005) A novel actinomycete strain de-replication approach based on the diversity of polyketide synthase and nonribosomal peptide synthetase biosynthetic pathways. Appl Microbiol Biotech 67:795–806 Bowman JP (2001) Methods for psychrophilic bacteria. In: Paul JH (ed) Methods in microbiology. Academic Press, New York, pp 591–614 Bull AT, Ward AC, Goodfellow M (2000) Search and discovery strategies for biotechnology: the paradigm shift. Microbiol Mol Biol Rev 64:573–606
Chen X, Zhang Y, Gao P (2004) Progress in deep-sea microbiology. J Marine Sci 28:61–66 Chen M, Xiao X, Wang P, Zeng X, Wang F (2005) Arthrobacter ardleyensis sp. nov., isolated from antarctic lake sediment and deep-sea sediment. Arch Microbiol 183:301–305 Damare S, Raghukumar C, Raghukumar S (2006) Fungi in deep-sea sediments of the Central Indian Basin. Deep-Sea Res- I 53:14–27 Dang H, Zhu H, Wang J, Li T (2009) Extracellular hydrolytic enzyme screening of culturable heterotrophic bacteria from deep-sea sediments of the Southern Okinawa Trough. World J Microbiol Biotechnol 25(1):71–79 Das S, Lyla PS, Khan SA (2006) Marine microbial diversity and ecology: importance and future perspectives. Curr Sci 90:1325– 1335 Faulkner DJ (2002) Marine natural products. J Nat Prod 19:1–48 Gaudette HE, Flight WR, Toner L, Folger DW (1974) An inexpensive titration method for determination of organic carbon in recent sediments. J Sediment Petrol 44:249–253 Gerhard B, Gerhard L, Tobias AMG, Tsuruta H, Jo¨rg M, Katja M, Stefan S, Karsten S, Ru¨diger S, Jutta W, Johannes FI, Sanja P, Olexandra B, Mu¨ller WEG (2005) The first sorbicillinoid alkaloids, the antileukemic sorbicillactones a and b, from a sponge-derived Penicillium chrysogenum strain. Tetrahedron Lett 61:7252–7265 Gontang EA, Fenical W, Jensen PR (2007) Phylogenetic diversity of Gram-positive bacteria cultured from marine sediments. Appl Environ Microbiol 73:3272–3282 Gorajana A, Venkatesan M, Vinjamuri S, Kurada BV, Peela S, Jangam P, Poluri E, Zeeck A (2007) Resistoflavine, cytotoxic compound from a marine actinomycete, Streptomyces chibaensis aubn1/7. Microbiol Res 162:322–327 Gustafson K, Roman M, Fenical W (1989) The macrolactins, a novel class of antiviral and cytotoxic macrolides from a deep-sea marine bacterium. J Am Chem Soc 111:7519–7524 Jannasch HW, Wirsen CO, Taylor CD (1983) Deep-sea bacteria: isolation in the absence of decompression. Science 216:1315– 1317 Jeong SY, Shin HJ, Kim TS, Lee HS, Park S, Kim HM (2006) Streptokordin, a new cytotoxic compound of the methylpyridine class from a marine-derived Streptomyces sp. Kordi-3238. J Antibiot 59:234–240 Jeong SY, Park SY, Kim YH, Kim M, Lee SJ (2008) Cytotoxicity and apoptosis induction of Bacillus vallismortis bit-33 metabolites on colon cancer carcinoma cells. J Appl Microbiol 104:796–807 Kelecom A (1999) Chemistry of marine natural products: yesterday, today, and tomorrow. An Acad Bras Cienc 71:249–263 Kelecom A (2002) Secondary metabolites from marine microorganisms. An Acad Bras Cienc 74:151–170 Kim SJ, Kato C (2010) Sampling, isolation, cultivation, and characterization of piezophilic microbes. In: Timmis KN (ed) Handbook of hydrocarbon and lipid microbiology. SpringerVerlag Press, Berlin Heidelberg, pp 3870–3880 Laursen JB, Nielsen J (2004) Phenazine natural products: biosynthesis, synthetic analogues, and biological activity. Chem Rev 104:1663–1685 Li S, Xiao X, Li J, Luo J, Wang F (2006) Identification of genes regulated by changing salinity in the deep-sea bacterium Shewanella sp. WP3 using RNA arbitrarily primed PCR. Extremophiles 10:97–104 Li D, Wang F, Xiao X, Zeng X, Gu Q, Zhu W (2007a) A new cytotoxic phenazine derivative from a deep sea bacterium Bacillus sp. Arch Pharm Res 30:552–555 Li D, Wang F, Cai S, Zeng X, Xiao X, Gu Q, Zhu W (2007b) Two new bisorbicillinoids isolated from a deep-sea fungus, Phialocephala sp. Fl30r. J Antibiot 60:317–320
123
World J Microbiol Biotechnol Li D, Wang F, Xiao X, Fang Y, Zhu T, Gu Q, Zhu W (2007c) Trisorbicillinone a, a novel sorbicillin trimer, from a deep sea fungus, Phialocephala sp. Fl30r. Tetrahedron Lett 48:5235–5238 Lin J, Yan X, Zheng L, Ma H, Chen H (2005) Cytotoxicity and apoptosis induction of some selected marine bacteria metabolites. J Appl Microbiol 99:1373–1382 Liu R, Zhu T, Li D, Gu J, Xia W, Fang Y, Zhu W, Gu Q (2007) Two indolocarbazole alkaloids with apoptosis activity from a marinederived actinomycete z (2) 039–2. Arch Pharm Res 30:270–274 Martin GDA, Tan LT, Jensen PR, Dimayuga RE, Fairchild CR, Raventos-Suarez C, Fenical W (2007) Marmycins a and b, cytotoxic pentacyclic c-glycosides from a marine sedimentderived actinomycete related to the genus Streptomyces. J Nat Prod 70:1406–1409 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Meth 65:55–63 Munson MA, Nedwell DB, Embley TM (1997) Phylogenetic diversity of archaea in sediment samples from a coastal salt marsh. Appl Environ Microblol 63:4729–4733 Raghukumar C, Raghukumar S, Sheelu G, Gupta SM, Nagender Nath B, Rao BR (2004) Buried in time: culturable fungi in a deep-sea sediment core from the Chagos Trench, Indian Ocean. Deep-Sea Res I 51:1759–1768 Skehan P, Storeng R, Scudiero D, Monks A, Mcmahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82:1107–1112 Sogin ML, Morrison HG, Huber JA, Welch DM, Huse SM, Neal PR, Arrieta JM, Herndl GJ (2006) Microbial diversity in the deep sea
123
and the underexplored ‘‘Rare biosphere’’. Proc Natl Acad Sci U S A 103:12115–12120 Takai K, Horikoshi K (1999) Genetic diversity of archaea in deep-sea hydrothermal vent environments. Genetics 152:1285–1297 Takami H, Kobata K, Nagahama T, Kobayashi H, Inoue A, Horikoshi K (1999) Biodiversity in deep-sea sites located near the south part of Japan. Extremophiles 3:97–102 Trischman JA, Jensen PR, Fenical W (1998) Guaymasol and epiguaymasol: aromatic triols from a deep-sea Bacillus isolate. Nat Prod Res 11:279–284 Vita-Marques AM, Lira SP, Berlinck RGS (2008) A multi-screening approach for marine-derived fungal metabolites and the isolation of cyclodepsipeptides from Beauveria felina. Quı´m Nova 31:1099–1103 Whittenbury R, Phillips KC, Wilkinson JF (1970) Enrichment, isolation and some properties of methane-utilizing bacteria. J Gen Microbiol 61:205–218 Xiao X, Wang P, Zeng X, Bartlett DH, Wang F (2007) Shewanella psychrophila sp. nov. and Shewanella piezotolerans sp. nov., isolated from west Pacific deep-sea sediment. Int J Syst Evol Microbiol 57:60–65 Xu M, Wang P, Wang F, Xiao X (2004) Microbial diversity in deepsea sediments collected from different stations of Pacific. Mar Biotechnol 6:S161–S167 Zhang L, An R, Wang J, Sun N, Zhang S, Hu J, Kuai J (2005) Exploring novel bioactive compounds from marine microbes. Curr Opin Microbiol 8:276–281 Zobell CE (1941) Studies on marine bacteria. I. The cultural requirements of heterotrophic aerobes. J Mar Res 4:42–75
