Diversity of Secondary Metabolites from Two Antarctic Microbes Rhodococcus sp. NJ-008 and Pseudomonas sp. NJ-011 ()
1. Introduction
Marine microbes from Antarctic area were well-known as bioactive compounds producers. In the past few years, some Chinese scientists have done fine chemical investigation from Antarctic fungui and led to the isolation of many interesting secondary metabolites, including new structures of two epipolythiodioxopiperazines and five diketopiperazines [1] , two novel polyketides [2] , six new peptaibols [3] , together with some known aromatic phenols [4] , anthraquinone derivatives and steroids [5] . During our first trip to Antarctic Ocean in 2010, some marine microbes associated with the Antarctic krill Euphausia superba had been obtained. Among these strains, Rhodococcus sp. NJ-008 was the only representative of Rhodococcus genus, while Pseudomonas sp. NJ-011 showed antimicrobial activity against Staphylococcus aureus. In the primary chemical investigation carried out for understanding the diversity of secondary metabolites from these Antarctic microbes, we found the methanol extracts of these two strains showed abundant & interesting ion peaks in HPLC-TOF MS tests. We herein report the diversity analysis on secondary metabolites of these two microbes, and the purification and structural elucidation of three compounds from Pseudomonas sp.NJ-011.
2. Experimental Section
2.1. General Procedures
All common solvents used were of anal. grade (Shanghai Chemical Plant). Solvents for HPLC and HPLC-TOF MS: Methanol and acetonitrile (Tedia, USA). Thin-layer chromatography (TLC): pre-coated silica gel GF254 plates (Qingdao Haiyang Chemical Co. Ltd.). Column chromatography (CC): HP-20 macroporos resin (Mitsubishi Chemical Industries Co., Ltd.) and Sephadex LH-20 (Pharmacia Biotech, Sweden). Semi-preparative HPLC was performed on a Shimadazu LC-20AT pump equipped with a Shimadazu SPD-M20A PDA detector and a YMC-Pack ODS-AQ column (250 ´ 10 mm, S-5 mm, 12 nm), flow rate: 2.5 mL×min−1. NMR spectra were recorded on a Bruker AM-400 spectrometer; d in ppm rel. to Me4Si, J in Hz. HR-ESI-MS were carried out on a Agilent 1290 HPLC-6224 TOF MS instrument; in m/z (rel. %).
2.2. Bacterial Source
Rhodococcus sp. NJ-008 and Pseudomonas sp. NJ-011 were isolated from the Antarctic krill Euphausia superba, collected in Jan 2010 in FAO 48.1 area, and were identified by Dr. Yang Qiao. The voucher specimens (No. DHS-NJ-008 and DHS-NJ-011) were deposited in East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences.
2.3. Cultivation and Extraction
Rhodococcus sp. NJ-008 in 2.0 L R2A liquid medium was shared with three 2000 mL Erlenmeyer flasks, and the flasks were incubated on a rotary shaker at 150 rpm for 7 d (28˚C). Pseudomonas sp. NJ-011 in 2.0 L GAUZE’s Medium NO.1 also in three 2000 mL flasks was incubated on the same rotary shaker as one batch culture.
Pseudomonas sp. NJ-011 in 8.0 L GAUZE’s Medium NO.1 in twelve 2000 mL flasks was incubated on the same rotary shaker at 150 rpm for 7 d (28˚C) as another batch culture.
The culture broth (2 L) of Rhodococcus sp. NJ-008 (or Pseudomonas sp. NJ-011) was centrifuged at 4000 rpm. The bacterial cells were frozen at −78˚C for 3 h, and then were extracted with 100 mL acetone and 100 mL methanol, successively. The organic solutions were concentrated under reduced pressure to afford a residue. The supernatant was evaporated to a salty dryness in vacuum. And these two parts were combined in 100 mL methanol, taking 1.0 mL supernatant for HPLC-TOF MS tests.
The culture broth (8 L) of Pseudomonas sp. NJ-011was extracted with ethyl acetate (1500 mL) for three times, and the mixture was treated with ultrasonic cleaner for 30 min each time. The organic solutions were combined and concentrated under reduced pressure to afford a residue (1.4 g).
2.4. HPLC-TOF MS test
HPLC column: YMC ODS-AA12S03-L546WT (4.6 ´ 75 mm, S-5 μm, 12 nm); injection volume: 10 mL; flow rate: 0.5 mL×min−1; mobile phase (CH3CN in H2O containing 0.1% HCOOH): 0 - 1.2 min 5%, 14 min 95%, 17 min 95%, 17.5 min 5%, 20 min 5%; TOF MS: positive ion mode ESI; gas temperature 340˚C, N2 8.0 L×min−1, nebulizer 40 psi. After test, the data of ion peaks with volume > 100,000 were extracted in TIC scan. Possible formula for each peak and the calculated exact mass for [M + H]+ were given by DNP database and ChemBio Draw Ultra version 12.0, respectively.
2.5. Isolation
Total extract (1.3 g) from 8 L culture broth of Pseudomonas sp. NJ-011 was absorbed by HP-20 macroporos resin (600 mL), and eluted with H2O (1500 mL) and methanol/H2O (10:90, 1500 mL) to remove some salts, sugars, amino acids and proteins, and then eluted with methanol/H2O (50:50 ® 80:20 ® 100:0, 2500 mL each)to afford three sections NJ-011-A (0.5 g), NJ-011-B (0.1 g) and NJ-011-C (0.4 g), successively. The section NJ-011-A was passed through a Sephadex LH-20 column, eluted with ethanol/H2O (70:30), to yield three fractions A1 to A3. Finally, A1 were purified by repeated semi-preparative HPLC with mobile phase of CH3CN/H2O-0.1%TFA (25:75 ® 70:30, 2.5 mL/min), and three compounds 1 (7.6 mg), 3 (2.3 mg), and 2 (3.5 mg) were obtained.
b-carboline (1): positive ion mode ESI m/z: 169.0758 [M + H]+; 1H-NMR (CD3OD, 400 MHz) d: 8.80 (1H, br s), 8.28 (1H, br s), 8.20 (1H, dt, J = 1.0, 8.0 Hz), 8.10 (1H, br d, J = 5.5 Hz), 7.57 (1H, m), 7.56 (1H, m), 7.27 (1H, ddd, J = 3.1, 5.1, 8.0 Hz); 13C-NMR (CD3OD, 100 MHz) d: 143.5, 138.8, 134.6, 130.9, 130.3, 123.3 (C ´ 2), 122.7, 121.4, 116.7, 113.3.
3-benzylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (2): positive ion mode ESI m/z: 245.1283 [M + H]+; 1H-NMR (CD3OD, 400 MHz) d: 7.28 (5H, m), 4.47 (1H, s), 4.08 (1H, m), 3.55 (1H, m), 3.39 (1H, m), 3.20 (2H, m), 2.11 (1H, m), 1.82 (2H, m), 1.34 (1H, m); 13C-NMR (CD3OD, 100 MHz) d: 169.9, 166.0, 135.3, 129.9 (2C), 128.3 (2C), 127.1, 58.4, 57.7, 44.8, 39.6, 28.4, 21.1.
3-isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (3): positive ion mode ESI m/z: 211.1438 [M + H]+; 1H-NMR (CD3OD, 400 MHz) d: 4.27 (1H, m), 4.14 (1H, m), 3.54 (2H, m), 2.63 (2H, m), 2.32 (1H, m), 2.02 (1H, m), 1.92 (1H, m), 1.67 (1H, m), 1.54 (1H, m), 0.99 (3H, d, J = 6.4 Hz), 0.98 (3H, d, J = 6.3 Hz); 13C-NMR (CD3OD, 100 MHz) d: 171.4, 167.5, 58.8, 53.2, 45.0, 37.9, 27.7, 24.3, 22.2, 21.9, 20.8.
3. Results and Discussions
3.1. Diversity Analysis on Secondary Metabolites
By high-resolution HPLC-TOF MS spectra (Table 1) of methanol extract solution from Rhodococcus sp. NJ- 008, 7 peaks were not given possible formula by DNP database, which may indicate the constituents with new structures. The possible formulae of 25 sample peaks were obtained among the total 32 observed peaks. Except for a plasticizer (Cpd19), the possible formulae indicated 13 - 15 compounds without nitrogen in their structures, and the other 9 - 11 were N-containing compounds. There were also 2S-containing metabolites, but no halogencontaining sample peaks were found by typical MS spectra. The relative contents (Vol%) of 14 sample peaks were higher than 2.0%, including 9 compounds not bearing N-atoms, 4 N-containing compounds, and another one without possible formula. So the chemical constituents of Rhodococcus sp. NJ-008 are mainly non-N-containing compounds, and the N-containing compounds showed a minor relative content.
The methanol extract solution of Pseudomonas sp. NJ-011 was also subjected to HPLC-TOF MS test. Among the total 53 observed peaks (Table 2), 10 peaks were not given possible formula by DNP database, which may indicate the constituents with new structures. The possible formulae of 43 sample peaks were obtained by highresolution HPLC-TOF MS spectra, including 29 N-containing compounds, and the other 14 compounds without nitrogen in their structures. There were also 1 metabolite which was both Sand N-containing compound, and another compound might bear P-atom, but no halogen-containing sample peaks were found by typical MS spectra. Most sample peaks showed very low relative contents with their Vol% much less than 1.0%. Besides a plasticizer (Cpd36), there were only 10 sample peaks with Vol% higher than 2.0%, including 9 N-containing compounds and another one without possible formula. So the chemical constituents of Pseudomonas sp. NJ-011 are mainly N-containing compounds, including some alkaloids and short polypeptides.
3.2. Structural Elucidation
Three compounds were obtained from the high polarity extract section of Pseudomonas sp. NJ-011, by different column chromatography and repeated preparative HPLC, and their structures were elucidated by comparison of
Table 1 . LC-TOF MS data of the NJ-008 methanol extract.
aNo possible formula given; bIon peak of plasticizer dibutyl phthalate [M + Na]+.
Table 2. LC-TOF MS data of the NJ-011 methanol extract.
aIon peak of plasticizer dibutyl phthalate [2M + Na]+.
HPLC-TOF MS, 1Hand 13C-NMR data with those reported. Their structures were identified as b-carboline (1) [6] , 3-benzylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (2, cyclo-(Pro-Phe)) [7] , and 3-isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (3, cyclo-(Pro-Leu)) [7] . These purified compounds were alkaloids or cyclic dipeptides of simple structures, and more microbial material of Pseudomonas sp. NJ-011 should be provided for exploration of the minor constituents with complicated structures.
4. Conclusion
Diversity analysis on secondary metabolites by HPLC-TOF MS tests exhibited that the chemical constituents of Pseudomonas sp. NJ-011 were mainly N-containing compounds including some alkaloids and short polypeptides, while those of Rhodococcus sp. NJ-008 were not N-containing ones. One alkaloid and two cyclic dipeptides were also isolated and identified from extract of Pseudomonas sp. NJ-011, which confirmed the chemical diversity analysis. There have been more than 200 compounds reported from the Pseudomonas genus, but only a small part of them were from marine derived Pseudomonas spp. Some known cyclic dipeptides [8] and two new a-pyrones [9] were reported recently, and more attention should be paid for chemical investigation on marine derived Pseudomonas spp.
Acknowledgements
We thank financial support from the National High Technology Research and Development Program (No. 2012AA092105), Chinese Polar Environment Comprehensive Investigation & Assessment Programmes (CHINARE2014-01-06), and the Natural Science Foundation of Shanghai (11ZR1450000).
NOTES
*Corresponding author.