采用LC-MS法鉴定和定量植物相关桔黄假单胞菌和绿针铜绿假单胞菌中次级代谢产物

Izzah Shahid; Muhammad Rizwan; Samina Mehnaz

doi:10.21769/BioProtoc.2702

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Peer-reviewed

Identification and Quantification of Secondary Metabolites by LC-MS from Plant-associated Pseudomonas aurantiaca and Pseudomonas chlororaphis

采用LC-MS法鉴定和定量植物相关桔黄假单胞菌和绿针铜绿假单胞菌中次级代谢产物

Izzah Shahid

MR Muhammad Rizwan

Samina Mehnaz email

发布: 2018年01月20日第8卷第2期 DOI: 10.21769/BioProtoc.2702 浏览次数: 11644

评审: Neelanjan BoseGiovanna SalbitaniJohn F. C. Steele

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Abstract

Increased antibiotic resistance of plants and human pathogens and continuous use of chemical fertilizers has pushed microbiologists to explore new microbial sources as potential antagonists. In this study, eight strains of Pseudomonas aurantiaca and Pseudomonas chlororaphis, have been isolated from different plant sources and screened for their antagonistic and plant growth promoting potential (Shahid et al., 2017). All strains were compared with reference strain PB-St2 and their secondary metabolites were isolated by the use of solvent partitioning and subjected to LC/ESI/MS for confirmation of compounds. The ESI-mass spectra obtained were used to characterize the surfactants ionization behavior and [M + H]⁺ and [M + Na]⁺ ions were monitored for phenazines, derivatives of lahorenoic acid and cyclic lipopeptide (WLIP). LC-MS and HPLC methods were developed to see the elution of dominant metabolites in a single run to avoid the labor and separate methods of detection for all compounds. The method was found suitable and distinctively separated the compounds at different retention times in gradient flow. This method can be helpful to explore the metabolome of Pseudomonas sp. overall and in identification and quantification of strain specific metabolites.

Keywords: Phenazines (吩嗪)

White-line inducing principle (WLIP) (白线诱导原理（WLIP）)

Lahorenoic acid (Lahorenoic acid)

Pseudomonas aurantiaca (桔黄假单胞菌)

Pseudomonas chlororaphis (绿针铜绿假单胞菌)

LC-MS (LC-MS)

Background

Authors in this research group are working on bioformulations and have isolated a large number of bacteria, including pseudomonads with potential plant growth promoting abilities. Pseudomonads are known for their biocontrol ability through antibacterial and antifungal secondary metabolites. These secondary metabolites successfully counter several phytopathogens of economically important crops and Pseudomonas based biofertilizers developed so far, have been marketed as commercial products (Mandryk et al., 2007; Rovera et al., 2014). Pseudomonas aurantiaca and P. chlororaphis strains are well known biocontrol agents due to the production of phenazines and cyclic lipopeptides and their broad spectrum antagonistic activities. Although many compounds have been reported by Pseudomonas aurantiaca and P. chlororaphis to date, the list of unidentified compounds is long (Chin et al., 2001; Hu et al., 2014).

Major metabolites produced by our strains were, white line inducing principle (WLIP), 2-hydroxy phenazine (2-OH-PHZ), phenazine-1 carboxylic acid (PCA) and Lahorenoic acid A. Previously reported methods for the detection of these compounds usually account only for HPLC, and comparison of results with standards. Moreover, detailed information about their MS/MS spectra and LC-MS methods is not available. Many researchers described the short HPLC runs for the detection of phenazines that did not account for the metabolites that elute at later stages (El-sayed et al., 2008; Upadhyay and Srivastava, 2011).

Lahorenoic acid A was exclusively reported by our group (Mehnaz et al., 2013) and since then has not been reported by any other group. WLIP was also reported first time from Pseudomonas aurantiaca PB-St2 in the same paper, though it has been reported earlier from other species of Pseudomonas (Meng et al., 2014). Keeping these findings in mind, a method was established that accounts for all the basic details in detection of these compounds from Pseudomonas aurantiaca in particular and other pseudomonads in general. LC-MS method was established with long run time and gradient flow, to make the better separation of these compounds. For confirmation of results, MS/MS fragmentation patterns of all compounds were also observed. The same LC-MS method was used for HPLC detection of these compounds, however, a different column was used. Many research groups working on metabolomics do not have access to standards of every compound. It was difficult to get the standards by our group as well and therefore, purified fractions of these compounds manually collected through HPLC, were used as reference standards for relative quantification of detected compounds. This method is significant as it provides the details about four essential and main secondary metabolites of Pseudomonas sp. in a single LC-MS and HPLC run.

Materials and Reagents

Pipette tips (Axygen^TM 1,000 μl Universal Pipette Tips) (Corning, Axygen^®, catalog number: T-1000-B )
Sterile syringe filters of 0.2 µm (Corning, catalog number: 431225 )
Pasteur pipettes (e.g., Fisherbrand^TM disposable borosilicate glass Pasteur pipettes, Fisher Scientific, catalog number: 13-678-20A )
pH indicator strips (pH 0-14 Universal Indicator) (Merck, catalog number: 1095350001 )
Nine bacterial strains including eight isolates of Pseudomonas chlororaphis subsp. aurantiaca (GS-1, GS-3, GS-4, GS-6, GS-7, ARS-38, PBSt-2, FS-2) and one Pseudomonas chlororaphis subsp. chlororaphis (RP-4) isolate
Hydrochloric acid (HCl), 36.5-38% (Sigma-Aldrich, catalog number: 320331 )
Ethyl acetate, EMSURE^®ACS, ISO, Reag. Ph Eur. (Merck, catalog number: 1096232500 )
Sodium sulfate, anhydrous, ≥ 99% (Sigma-Aldrich, catalog number: 1614807 )
Methanol gradient grade for liquid chromatography, LiChrosolv^® Reag. Ph Eur (Merck, catalog number: 1060072500 )
Chloroform, EMSURE^® ACS, ISO, Reag. Ph Eur. (Merck, catalog number: 1024452500 )
Water, LC-MS grade, LiChrosolv^® (Merck, catalog number: 1153332500 )
Dimethyl sulfoxide (DMSO), ≥ 99% (Sigma-Aldrich, catalog number: 472301 )
Formic acid, 98-100%, EMSURE^® ACS, ISO, Reag. Ph Eur. (Merck, catalog number: 1002640510 )
Acetonitrile gradient grade for liquid chromatography, LiChrosolv^® Reag. Ph Eur (Merck, catalog number: 1000302500 )
Protease-peptone, microbiology grade (Sigma-Aldrich, catalog number: 82450 )
Glycerol, molecular biology grade, ≥ 99.5% (Sigma-Aldrich, catalog number: G9012 )
Potassium phosphate dibasic (K₂HPO₄), ACS reagent, ≥ 98% (Sigma-Aldrich, catalog number: P3786 )
Magnesium sulfate heptahydrate (MgSO₄·7H₂O), ACS reagent, ≥ 98% (Sigma-Aldrich, catalog number: 230391 )
Agar (for solid medium only), powder for Microbiology (Sigma-Aldrich, catalog number: 05040 )

Equipment

Microbiological static incubator (e.g., Memmert, model: IF55 standard incubator )
Microbiological incubator shaker (e.g., IKA, model: KS 4000 i control )
Pipettes (e.g., Sartorius, catalog numbers: 728020 , 728050 , 728060 and 728070 )
Large-volume centrifuge (e.g., HERMLE Labortechnik, model: Centrifuge ZK 496 )
Erlenmeyer flasks (e.g., PYREX^TM, 1,000 ml, 500 ml, Corning, PYREX^®, catalog number: 4980-1L )
Separatory funnels with stopcock and ring stand (e.g., 1,000 ml, 500 ml, VWR, catalog number: 89426-640 )
Autoclave
Glass beakers (e.g., PYREX^TM, 1,000 ml, 500 ml, 100 ml, 50 ml, DWK Life Sciences, KIMBLE, catalog number: 14000-2000 )
Glass stirrer
Rotary evaporator (e.g., Buchi Rotavapor^®R-100 (BÜCHI Labortechnik, model: Rotavapor^® R-100 ) with Re-circulating Chiller F-100/F-105 (BÜCHI Labortechnik, model: Recirculating Chiller F-100/F-105 ), Vacuum Pump V-100 (BÜCHI Labortechnik, model: Vacuum Pump V-100 ), water bath and Interface I-100 (BÜCHI Labortechnik, model: Interface I-100 ))
Fume hood (e.g., Labconco, model: 4’ Protector XStream Laboratory Hood, catalog number: 110410000 )
LC-MS (e.g., Thermo Finnegan LC-MS, ESI Ion Trap Mass Spectrometer, Thermo Electron, model: LCQ Advantage Max )
TLC plates (Silica Gel 60G F₂₅₄ 20 x 20 cm, e.g., Merck, catalog number: 100390 )
C18 column for MS (e.g., Thermo Hypersil Gold C18 column, Length 250 mm, 4.6 mm ID, 5 µm particle size, Thermo Fisher Scientific, catalog number: 25005-254630 , Lot No. 7648, S/N 0871381M)
HPLC system (e.g., WATERS, model: e2995 Separations Module , 2998 Photodiode Array (PDA) Detector)
HPLC column (e.g., Nucleosil C18 column, 4.6 x 250 mm, 5 µM; Macherey-Nagel, Germany)

Software

Xcalibur 2.0 software (Xcalibur^TM Software, control and process data from LC-MS instruments. It is Windows^TM based software that provides method setup, data acquisition, data processing and reporting. Data files are retrieved in Qual Browser and are processed for interpretation and analysis)

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Shahid, I., Rizwan, M. and Mehnaz, S. (2018). Identification and Quantification of Secondary Metabolites by LC-MS from Plant-associated Pseudomonas aurantiaca and Pseudomonas chlororaphis. Bio-protocol 8(2): e2702. DOI: 10.21769/BioProtoc.2702.