Liquid Chromatography-Mass Spectrometry-Based Rapid Secondary-Metabolite Profiling of Marine Pseudoalteromonas sp. M2

The ocean is a rich resource of flora, fauna, and food. A wild-type bacterial strain showing confluent growth on marine agar with antibacterial activity was isolated from marine water, identified using 16S rDNA sequence analysis as Pseudoalteromonas sp., and designated as strain M2. This strain was found to produce various secondary metabolites including quinolone alkaloids. Using high-resolution mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis, we identified nine secondary metabolites of 4-hydroxy-2-alkylquinoline (pseudane-III, IV, V, VI, VII, VIII, IX, X, and XI). Additionally, this strain produced two novel, closely related compounds, 2-isopentylqunoline-4-one and 2-(2,3-dimetylbutyl)qunoline-4-(1H)-one, which have not been previously reported from marine bacteria. From the metabolites produced by Pseudoalteromonas sp. M2, 2-(2,3-dimethylbutyl)quinolin-4-one, pseudane-VI, and pseudane-VII inhibited melanin synthesis in Melan-A cells by 23.0%, 28.2%, and 42.7%, respectively, wherein pseudane-VII showed the highest inhibition at 8 µg/mL. The results of this study suggest that liquid chromatography (LC)-MS/MS-based metabolite screening effectively improves the efficiency of novel metabolite discovery. Additionally, these compounds are promising candidates for further bioactivity development.


Introduction
To date, the study of natural products has focused on the elaborate biosynthetic pathways in terrestrial plants and microorganisms. In the late 1960s, however, the search for novel metabolites took a new direction as the realm of exploration expanded to include marine plants and animals. This avenue of research was initiated primarily by academicians and facilitated by the development of colony showing the highest antimicrobial activity, designated strain M2, was selected for further study. The complete 16S rDNA sequence (1546 bp) of strain M2 was obtained. In the phylogenetic tree constructed using unrooted neighbor-joining algorithm, strain M2 fell within the clade comprising Pseudoalteromonas strains (Figure 1). It exhibited 16S rDNA gene sequence similarity values between 98.69% and 98.33% to Pseudoalteromonas prydzensis, P. atlantica, and P. espejiana, and between 95.33% to 98.31% to type strains of other Pseudoalteromonas species used in the phylogenetic analysis. This sequence has been submitted to GenBank and received accession number KJ407077. These results show that M2 was a Pseudoalteromonas strain, designated as Pseudoalteromonas sp. M2. comprising Pseudoalteromonas strains ( Figure 1). It exhibited 16S rDNA gene sequence similarity values between 98.69% and 98.33% to Pseudoalteromonas prydzensis, P. atlantica, and P. espejiana, and between 95.33% to 98.31% to type strains of other Pseudoalteromonas species used in the phylogenetic analysis. This sequence has been submitted to GenBank and received accession number KJ407077. These results show that M2 was a Pseudoalteromonas strain, designated as Pseudoalteromonas sp. M2.  Table 1 shows the 4-quinolones identified in the culture broth extract from Pseudoalteromonas sp. M2. Analysis of the ethyl acetate extract using ultra high performance liquid chromatography with high-resolution mass spectrometry (UHPLC-HRMS) revealed 11 quinolone compounds. The structure of the secondary metabolite from Pseudoalteromonas sp. M2 culture extract is shown in Figure 2. The high-resolution mass and MS/MS spectral characteristics of the 4-quinolones were compared to commercially obtained standards and published data.

Identification of Secondary Metabolites Using High-Resolution Mass Spectrometry
After extraction by ion chromatography (XIC), the LC-MS data were manually sorted to list such information as the retention time, m/z values for [M + H] + , and MS/MS fragmentation pattern, from base-peak chromatograms (Table 1). Each high-resolution MS spectrum and MS/MS spectrum peak were identified by AntiBase 2013 and in-house MS/MS spectral library, respectively. The differences in m/z values and retention time shifts were in accordance with the sequential decreases  Table 1 shows the 4-quinolones identified in the culture broth extract from Pseudoalteromonas sp. M2. Analysis of the ethyl acetate extract using ultra high performance liquid chromatography with high-resolution mass spectrometry (UHPLC-HRMS) revealed 11 quinolone compounds. The structure of the secondary metabolite from Pseudoalteromonas sp. M2 culture extract is shown in Figure 2.

Identification of Secondary Metabolites Using High-Resolution Mass Spectrometry
The high-resolution mass and MS/MS spectral characteristics of the 4-quinolones were compared to commercially obtained standards and published data.
After extraction by ion chromatography (XIC), the LC-MS data were manually sorted to list such information as the retention time, m/z values for [M + H] + , and MS/MS fragmentation pattern, from base-peak chromatograms (Table 1). Each high-resolution MS spectrum and MS/MS spectrum peak were identified by AntiBase 2013 and in-house MS/MS spectral library, respectively. The differences in m/z values and retention time shifts were in accordance with the sequential decreases or increases in the alkyl chain length on pseudane compound. All pseudane analogues had a main common fragment ion at m/z 159 that was derived from the loss of the alkyl chain. or increases in the alkyl chain length on pseudane compound. All pseudane analogues had a main common fragment ion at m/z 159 that was derived from the loss of the alkyl chain. * Commercial source. Searchable MS/MS spectra libraries based on the results of the liquid chromatography coupled with electrospray ionization (ESI) and tandem mass spectrometry (LC-MS/MS) with data-dependent acquisition using an ion trap mass spectrometer were compiled with regard to the identification and confirmation of the secondary metabolites from Pseudoalteromonas sp. M2. The main compound was identified as pseudane-V by AntiBase database search and confirmed by comparison analysis with a standard. The others peaks were detected before and after the major peaks ( Figure 3A) , which was tentatively identified as pseudane-III to XI based on the high-resolution mass and MS/MS production ions, respectively ( Figure 3C, Table 1). Thus, the LC-MS/MS analysis of the Pseudoalteromonas sp. M2 strains identified nine secondary metabolite peaks as known or putative structures, including pseudane-III (4.61 min), pseudane-IV (5.38 min), pseudane-V (6.14 min), pseudane-VI (6.89 min), pseudane-VII (7.60 min), pseudane-VIII (8.36 min), pseudane-IX (9.06 min), pseudane-X (9.72 min), and pseudane-XI (10.39 min), whereas another two peaks (6.07 and 6.75 min) were determined as unknown metabolites ( Figure 3B). The regular intervals of m/z values and retention time-shifts of the parent ions were Searchable MS/MS spectra libraries based on the results of the liquid chromatography coupled with electrospray ionization (ESI) and tandem mass spectrometry (LC-MS/MS) with data-dependent acquisition using an ion trap mass spectrometer were compiled with regard to the identification and confirmation of the secondary metabolites from Pseudoalteromonas sp. M2. The main compound was identified as pseudane-V by AntiBase database search and confirmed by comparison analysis with a standard. The others peaks were detected before and after the major peaks ( Figure 3A). The high-resolution mass spectrum showed an m/z 188. 1070 , which was tentatively identified as pseudane-III to XI based on the high-resolution mass and MS/MS production ions, respectively ( Figure 3C, Table 1).
Thus, the LC-MS/MS analysis of the Pseudoalteromonas sp. M2 strains identified nine secondary metabolite peaks as known or putative structures, including pseudane-III (4.61 min), pseudane-IV (5.38 min), pseudane-V (6.14 min), pseudane-VI (6.89 min), pseudane-VII (7.60 min), pseudane-VIII (8.36 min), pseudane-IX (9.06 min), pseudane-X (9.72 min), and pseudane-XI (10.39 min), whereas another two peaks (6.07 and 6.75 min) were determined as unknown metabolites ( Figure 3B). The regular intervals of m/z values and retention time-shifts of the parent ions were caused by sequential decreases or increases in the alkyl chain length. , which were consistent with the molecular formula C14H18ON (∆ppm −0.188) and C15H20ON (∆ppm 0.083), respectively. Interestingly, the two unknown metabolites have the same molecular formula and very similar MS/MS pattern with pseudane-IV and pseudane-V compounds, respectively. Moreover, the unknown compounds showed the same main dissociation fragment peaks, for example, m/z 146 and m/z 159, and these fragment peaks were derived from 4-hydroxy-2-alkylquinoline backbone by the loss of an alkyl chain. According to these data, the unknown peaks were clearly shown to be a new pseudane isomer.
Previously, Lépines's group reported 4-hydroxy-2-alkylquinolines series (pseudan-V~XIII) produced by a genetically engineered strain pqsL mutant derivative of PA14, indicating that this gene was involved in the biosynthesis of 4-hydroxy-2-alkylquinoline compounds in pathogenic Pseudomonas aeruginosa [17]. In this study, the novel Pseudoalteromonas sp. M2 strain producing a pseudane series with two novel compounds was screened using LC-MS based secondary metabolite screening methodology, from a total of 720 wild-type marine bacterial candidates. , which were consistent with the molecular formula C 14 H 18 ON (∆ppm´0.188) and C 15 H 20 ON (∆ppm 0.083), respectively. Interestingly, the two unknown metabolites have the same molecular formula and very similar MS/MS pattern with pseudane-IV and pseudane-V compounds, respectively. Moreover, the unknown compounds showed the same main dissociation fragment peaks, for example, m/z 146 and m/z 159, and these fragment peaks were derived from 4-hydroxy-2-alkylquinoline backbone by the loss of an alkyl chain. According to these data, the unknown peaks were clearly shown to be a new pseudane isomer.
Previously, Lépines's group reported 4-hydroxy-2-alkylquinolines series (pseudan-V~XIII) produced by a genetically engineered strain pqsL mutant derivative of PA14, indicating that this gene was involved in the biosynthesis of 4-hydroxy-2-alkylquinoline compounds in pathogenic Pseudomonas aeruginosa [17]. In this study, the novel Pseudoalteromonas sp. M2 strain producing a pseudane series with two novel compounds was screened using LC-MS based secondary metabolite screening methodology, from a total of 720 wild-type marine bacterial candidates.

Anti-Melanogenic Effect of Secondary Metabolites and New Compounds
To investigate the cytotoxic effects of the new compounds on melan-a cells, the cells were exposed to 2 to 8 µg/mL of pseudane-IV, 2-isopentylquinolin-4-one, pseudane-V, 2-(2,3-dimethylbutyl) quinolin-4-one, pseudane-VI, and pseudane-VII, pseudane-VIII, or pseudane-IX for three days, following which, cell viability was assessed using the CCK8 assay kit. None of the tested compounds exhibited toxicity at the concentration of 8 µg/mL, except for pseudane-VIII and IX ( Figure 4A). Anti-melanogenic effect was measured in terms of melanin content in the presence of 2 to 8 µg/mL of the test compounds. As shown in Figure 4B, all compounds showed an inhibitory effect on melanin synthesis in a dose-dependent manner. Among the eight compounds, 2-(2,3-dimethylbutyl)quinolin-4-one, pseudane-VI, and pseudane-VII showed 23.0%, 28.2%, and 42.7% inhibition of melanin synthesis in the melan-a cells, respectively. Especially, pseudane-VII showed the highest inhibitory activity (42.7%) at a concentration of 8 µg/mL. It has been reported that few active anti-melanogenic agents such as ginsenosides extracted from leaves of Panax ginseng [18] showed 35.5% inhibitory activity at 80 µM. In addition, cinnamic acid extracted from Cinnamomum cassia Blume and Panax ginseng exhibited 29% inhibitory effect on melanin synthesis at 500 µM [19]. Compared with ginsenoside and cinnamic acid, pseudane-VII showed strong inhibitory activity at concentrations of 8-33 µM (the concentration unit was converted from µg/mL to µM for comparison). However, the mechanism of the anti-melanogenic activity has not yet been investigated. Therefore, further in vitro and in vivo studies are necessary to determine the mechanism involved in the anti-melanogenic effect exerted by treatment with pseudane-VII.

Mar. Drugs 2016, 14, x 7 of 11
To investigate the cytotoxic effects of the new compounds on melan-a cells, the cells were exposed to 2 to 8 μg/mL of pseudane-IV, 2-isopentylquinolin-4-one, pseudane-V, 2-(2,3-dimethylbutyl) quinolin-4-one, pseudane-VI, and pseudane-VII, pseudane-VIII, or pseudane-IX for three days, following which, cell viability was assessed using the CCK8 assay kit. None of the tested compounds exhibited toxicity at the concentration of 8 μg/mL, except for pseudane-VIII and IX ( Figure 4A). Anti-melanogenic effect was measured in terms of melanin content in the presence of 2 to 8 μg/mL of the test compounds. As shown in Figure 4B, all compounds showed an inhibitory effect on melanin synthesis in a dose-dependent manner. Among the eight compounds, 2-(2,3-dimethylbutyl)quinolin-4-one, pseudane-VI, and pseudane-VII showed 23.0%, 28.2%, and 42.7% inhibition of melanin synthesis in the melan-a cells, respectively. Especially, pseudane-VII showed the highest inhibitory activity (42.7%) at a concentration of 8 μg/mL. It has been reported that few active anti-melanogenic agents such as ginsenosides extracted from leaves of Panax ginseng [18] showed 35.5% inhibitory activity at 80 μM. In addition, cinnamic acid extracted from Cinnamomum cassia Blume and Panax ginseng exhibited 29% inhibitory effect on melanin synthesis at 500 μM [19]. Compared with ginsenoside and cinnamic acid, pseudane-VII showed strong inhibitory activity at concentrations of 8-33 μM (the concentration unit was converted from μg/mL to μM for comparison). However, the mechanism of the anti-melanogenic activity has not yet been investigated. Therefore, further in vitro and in vivo studies are necessary to determine the mechanism involved in the anti-melanogenic effect exerted by treatment with pseudane-VII.

Isolation of Pseudane-Producing Bacterium
Golden sea squirt (Halocynthia aurantium) was collected from the East Sea, South Korea, and used as a source to isolate bacteria. Strain M2 was isolated by the standard dilution plating technique using marine agar 2216 (Becton Dickinson, Franklin Lakes, NJ, USA) with 3 g/L yeast extract (Difco) and 5 g/L protease peptone (Difco) at 22˝C and incubated under routine culture conditions. Microbial strains were isolated after incubation for two or three days. All bacterial isolates were examined for their antibacterial activity against Vibrio anguillarum on agar media. The isolate showing the largest halo, designated M2, was studied further.

Identification of Pseudoalteromonas sp.
Genomic DNA was extracted from Pseudoalteromonas sp. M2 for 16SrDNA analysis as previously described [20]. Polymerase chain reaction (PCR) was performed to amplify the 16S rDNA coding region using primers 5 1 -AGAGTTTGATCCTGGCTCAG-3 1 and 5 1 -ACGGTTACCTTGTTACGACTT-3 1 . The reaction mixture was subjected to initial denaturation at 95˝C for 10 min, followed by 30 cycles of denaturation at 95˝C for 1 min, annealing at 55˝C for 1 min, and extension at 72˝C for 1 min, with a final extension at 72˝C for 10 min, using a thermal cycler (TaKaRa, Shiga, Japan). The PCR product was subcloned into pGEM-T Easy vector, and transformed into Escherichia coli DH5α. DNA sequencing was performed using an Applied Biosystem Automated DNA Sequencer model 3130 with a dye-labeled terminator sequencing kit (Applied Biosystems, New York, NY, USA).
An unrooted neighbor-joining tree for the full sequence of the 16SrDNA was constructed based on the Kimura two-parameter model. Reference strains have been incorporated in the alignment and they were obtained from NCBI (http://www.ncbi.nlm.nih.gov). The sequences were aligned using Clustal X software [21], and the tree has been constructed using the MEGA 4 Software [22].

Secondary Metabolite-Profiling Using LC-MS
Strain M2 was inoculated into 5 mL marine broth 2216 (MB; Difco) and incubated for 48 h at 22˝C with shaking. The culture broth was transferred to a 15 mL tube and centrifuged at 11,000 g for 10 min. The supernatant was extracted with an equal volume of ethyl acetate. The dried ethyl acetate extract of M2 was dissolved in 50% methanol and 5 µL was analyzed to identify secondary metabolites by LC-MS technique.
All LC/MS analyses were carried out using an LTQ Orbitrap XL (Thermo Electron Co., Madison, WI, USA) coupled to an Accelar ultra-high pressure liquid chromatography system (Thermo, Waltham, MA, USA). Chromatographic separation of metabolites was conducted using a ACQUITY UPLC ® BEH C 18 column (2.1ˆ150 mm, 1.7 µm, Waters, Milford, MA, USA), operated at 40˝C and using mobile phases A (water) and B (acetonitrile with 0.1% formic acid) at flow rate of 0.4 mL/min. The initial gradient composition (95% A/5% B) was held for 0.5 min, increased to 80% B in 10 min, decreased to 0% A in 10.01 min, and held for 1.90 min. For recycling, the initial gradient composition was restored and allowed to equilibrate for 3 min. The LC-MS system consisted of heated electrospray ionization probe (HESI-II) as the ionization source. HESI was operated at 300˝C with spray voltage of 5.0 kV. The nebulizer sheath and auxiliary gas flow rates were set at 50 and 5 arb, respectively. MS analysis was performed with polarity switching, and the following parameters for MS/MS scan: m/z range of 100-1000; collision-induced dissociation energy of 45%; data-dependent scan mode. The Orbitrap analyzer was used for high-resolution mass spectra data acquisition with a mass resolving power of 30,000 FWHM (Full width at half maximum) at m/z 400. The data-dependent tandem mass spectrometry (MS/MS) experiments were controlled using menu-driven software provided with the Xcalibur system. All experiments were performed under automatic gain control conditions.

Extraction and Purification of Secondary Metabolites
To obtain secondary metabolites, the cell culture medium was centrifuged at 20,000 g for 30 min to remove precipitates. The supernatant was collected and treated with equal volume of ethyl acetate and shaken at 300 rpm for 15 min using a JEIO TECH RS-1 recipro shaker (Jeio Tech, Daejeon, Korea). Then the ethyl acetate layer (upper layer) was vacuum-dried using Speed-Vac (Labconco, Kansas, MA, USA) and the extract was diluted in 50% methanol (v/v in deionized water) to achieve concentrations of 540 mg/10 mL. Each extract was purified by high-pressure liquid chromatography (HPLC) on a Waters AutoPurification System (Waters, Milford, MA, USA) with a QDa detector and a Waters Xbridge prep C 18 Column (19ˆ250 mm, 5 µm) with a gradient of A (0.1% formic acid v/v in deionized water) and B (acetonitrile) at flow rate of 25 mL/min. The initial gradient composition (90% A/10% B) was held for 2.8 min, increased to 65% B in 43 min, and then decreased to 0% A in 45 min, where it was held for 5 min.

Cell Cultures
The Melan-A (murine Melan-A melanocyte) cell line, originally derived from C57BL/6 J (black, a/a) mice was received as a gift from Prof. Dorothy C. Bennett (St George's Hospital Medical School, London, UK). Melan-A cells are similar in characteristics to melanocytes in vivo and are widely used as a suitable substitute for normal primary mouse melanocytes in melanin metabolism tests. This cell line was cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), streptomycin-penicillin (100 µg/mL each), and 200 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, at 37˝C in 5% CO 2 . Cells were subcultured every three days up to a maximum of 40 passages. Confluent monolayers of melanocytes were harvested with a mixture of 0.05% trypsin and 0.53 mM EDTA (Gibco BRL, Grand Island, NY, USA).

Cell Viability Assay
Cell viability was determined via crystal violet staining. After four days of incubation with the test substances, the culture medium was removed and replaced with 0.1% crystal violet in 10% ethanol. The cells were then stained for 5 min at room temperature and rinsed with phosphate-buffered saline (PBS) three times. The crystal violet stain retained by adherent cells was extracted using 95% ethanol and absorbance was determined at a wavelength of 590 nm.

Measurement of Melanin Content
The cells were seeded in a 24-well plate (Corning, NY, USA) at a density of 1ˆ10 5 cells per well and allowed to attach overnight. They were then incubated in a fresh medium containing various concentrations of the test compounds for four days. After the cells had been washed with PBS, they were lysed with 250 µL 0.85 N KOH and transferred to a 96-well plate. The melanin content was estimated via absorbance measurements at a wavelength of 405 nm.

Conclusions
Pseudoalteromonas sp. M2 isolated from marine source was found to produce various secondary metabolites and novel compounds. Based on high-resolution MS and NMR spectroscopic analysis, two novel compounds, 2-isopentylquinolin-4-one and 2-(2,3-dimethylbutyl)quinolin-4-one are identified. The production of 9 quinolones (pseudane series III-XI), 2-isopentylqunoline-4-one, and 2-(2,3-dimetylbutyl)qunoline-4-one from a single wild-type marine bacterium has not been previously reported. We confirmed biological activity of the isolated compounds, including inhibition of melanin synthesis in Melan-A cells. This may be a useful approach to evaluate multi-functional biological activities to explore the potential therapeutic applications of this bacterium. Pseudane-VI, VII, and 2-(2,3-dimethylbutyl)quinolin-4-one may be promising candidates for the development of useful skin-lightening agents.
As shown in the present study, LC-MS/MS-based metabolite profiling of Pseudoalteromonas sp. M2 secondary metabolites is a useful technique to distinguish between known and unknown compounds, as well as to screen novel compounds without extensive culturing. Furthermore, the structure-based metabolite screening method, high-resolution LC-MS combined with MS/MS spectral library searches, minimizes both, time and resources utilized in redundant discovery efforts. The objective of this study was to develop a rapid, accurate, and more efficient technique compared with traditional biological activity-based screening methods for discovery of novel compounds from natural sources.