Isolation of Outer Membrane Vesicles from Phytopathogenic Xanthomonas campestris pv. campestris
从野油菜黄单胞菌野油菜致病变种分离外膜囊泡
发布: 2017年03月05日第7卷第5期 DOI: 10.21769/BioProtoc.2160 浏览次数: 13085
评审: Zhaohui LiuEmilie ViennoisMahmoud Kamal Ahmadi
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参见作者原研究论文
The authors used this protocol in:
May 2016
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Abstract
Gram-negative bacteria naturally release outer membrane vesicles (OMVs) to the surrounding environment. OMVs contribute to multiple processes, such as cell-cell communication, delivery of enzymes and toxins, resistance to environmental stresses and pathogenesis. Little is known about OMVs produced by plant-pathogenic bacteria, and their interactions with host plants. The protocol described below discusses the isolation process of OMVs from Xanthomonas campestris pv. campestris strain 33913, a bacterial pathogen of Crucifiers. Nevertheless, this protocol can be used and/or adapted for isolation of OMVs from other phytopathogenic bacteria to promote the study of OMVs in the context of plant-microbe interactions.
Keywords: OMVs (OMVs)
Outer membrane vesicles (外膜囊泡)
Extracellular vesicles (胞外囊泡)
Isolation (分离)
Purification (纯化)
Xanthomonas campestris pv. campestris (野油菜黄单胞菌)
Plant-microbe interactions (植物-微生物相互作用)
Background
Extracellular vesicle (EV) release is a process shared by many organisms from all domains of life. In Gram-negative bacteria, most EVs are a result of the outer membrane blebbing and eventually pinching off the bacterial cell wall, and are hence referred to as outer membrane vesicles (OMVs). The study of OMVs focuses on OMV biogenesis, cargo, functions and interactions with host organisms. To date, most of the study on OMVs focused on bacterial pathogens of humans and environmental bacteria, however very little research has been done on OMVs from phytopathogenic bacteria. The protocol described here was adapted from the protocol described by Chutkan et al. (2013) with slight modification, and presented here with the phytopathogen X. campestris pv. campestris. To our understanding this is the first, fully detailed, protocol for isolation of OMVs from phytopathogenic bacteria and we hope it could serve as a guiding protocol for other research groups interested in this topic.
Materials and Reagents
- Sterile single-use inoculating loop
- 200 μl pipette tips (such as: Corning, Axygen®, catalog number: # T-200-C )
- Parafilm (Bemis, catalog number: PM996 )
- Syringe-mounted 0.45-µM filter (EMD Millipore, catalog number: SLHV033RS )
- Polyethersulfone (PES) filter, 0.45-µM, Ø75-mM membrane diameter, with an attached 500-ml bottle. Operated by a vacuum pump (e.g., Guangzhou Jet Bio-Filtration, catalog number: FPE404500 )
- 1 ml and 10 ml sterile syringes (without needle)
1 ml syringes (such as: KDL Medical Product, catalog number [as written on the package]: 7290010993604)
10 ml syringes (such as: Medisposables, catalog number [as written on the package]: 9304-10/16933506093041 ) - Sterile 15-ml Falcon tube (such as: SPL Life Sciences, catalog number: 50015 )
- 12 ml polypropylene/polyallomer ultracentrifuge tubes (such as: Beckman Coulter, catalog number: 331372 ) – make sure that they are fit for the ultracentrifuge’s speed
- Syringe-mounted 0.22-µM filter (Axiva Sichem Biotech, catalog number: SFPV13 R )
- Sterile 1.5-ml tube (1.7-ml clear tube) (Corning, Axygen®, catalog number: # MCT-175-C )
- 1 ml pipette tips (such as: Corning, Axygen®, catalog number: # T-1000-C )
- Polystyrene (PS) aseptic Petri dishes, 90 x 15-mm (Miniplast Ein-Shemer, catalog number: 820-090-01-017 )
- Xanthomonas strain in glycerol stock stored at -80 °C – for the preparation of this protocol we used X. campestris pv. campestris (Xcc) 33913, which was obtained from American Type Culture Collection (ATCC)
- Difco nutrient agar (BD, catalog number: 21300 )
Note: It contains 3 g/L of beef extract, 5 g/L of peptone, and 15 g/L of agar, with a final pH of 6.8 ± 0.2 (as written on the powder’s container). - Cephalexin hydrate (Sigma-Aldrich, catalog number: C4895 ) – stock solution at concentration of 10 mg/ml
- Sterile dH2O
- 10x phosphate buffered saline (PBS) buffer (optional) (such as: Sigma-Aldrich, catalog number: P5493 )
- OptiPrep density gradient medium (Sigma-Aldrich, catalog number: D1556 )
- ‘Coomassie Plus – The Better Bradford Assay Kit’ (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23236 ) (optional)
- Lipid dye FM4-64FX (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: F34653 ) (optional)
- Materials and reagents required for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as indicated in the following protocol (optional): http://www.bio-protocol.org/e80 (‘[Bio101] Laemmli-SDS-PAGE’)
- Yeast extract powder (EMD Millipore, catalog number: 61931105001730 )
- Bacto tryptone (BD, Bacto, catalog number: 211705 )
- Sodium chloride (NaCl) (EMD Millipore, catalog number: 7647-14-5 )
- Sucrose crystallized (Duchefa Biochemie, catalog number: S0809 )
- Magnesium sulfate heptahydrate (MgSO4·7H2O) (EMD Millipore, catalog number: 105886 )
- NaOH or HCl (in order to adjust pH)
- Peptone (EMD Millipore, catalog number: 61930705001730 )
- L(+)-glutamic acid (Acros Organics, catalog number: 6106-04-3 )
- HEPES (Sigma-Aldrich, catalog number: H3375 )
- YEB medium (see Recipes)
- PSB medium (see Recipes)
- OptiPrep diluent buffer (see Recipes)
Note: (optional) – not necessary for the vesicles’ isolation process.
Equipment
- Biological hood
- Temperature controlled incubator shaker (28 °C), with a capability to reach 200 RPM (such as: Thermo Fisher Scientific, Thermo ScientificTM, model: MaxQTM 6000 )
- Two 2-L Erlenmeyer flasks
- Cuvette
- Polypropylene Copolymer (PPCO) 250-ml centrifuge bottles (such as: Thermo Fisher Scientific, Thermo ScientificTM, model: NalgeneTM PPCO Centrifuge Bottle ) – make sure that they are fit for the centrifuge’s speed
- High-speed centrifuge (e.g., Beckman Coulter, model: Avanti J-E , catalog number: 369003; or Thermo Fisher Scientific, model: SorvallTM RC-6 , catalog number: 74804*) and 6 x 250-ml rotor (such as: Beckman Coulter, model: JLA-16.250 , catalog number: 363930, dual-locking lid; or Thermo Fisher Scientific, SorvallTM, model: SLA-1500 , catalog number: 46615* [old model]) at a speed of approx. 32,000 x g
- Ultracentrifuge (e.g., Thermo Fisher Scientific, SorvallTM, model: Discovery 90SE , catalog number: 40130867*, Kendro Laboratory Products) and a 6 x 12-ml swinging bucket ultracentrifuge rotor such as: Thermo Fisher Scientific, SorvallTM, model: TH-641 , catalog number: 54295* (old model, Kendro Laboratory Products), or Thermo Fisher Scientific, Thermo ScientificTM, model: TH-641 , catalog number: 54295 (new model,) at a speed of approx. 175,000 x g
- Autoclave
- NanoSight device (Malvern) (optional)
- Transmission electron microscopy (optional)
- Equipment required for SDS-PAGE as indicated in the following protocol (optional): http://www.bio-protocol.org/e80 (‘[Bio101] Laemmli-SDS-PAGE’)
Notes
- *Catalog number of an old model.
- (optional) – not necessary for the vesicles’ isolation process.
- Important note: the centrifuge speeds mentioned above, were calculated with Science Gateway's centrifuge rotor speed calculator (http://www.sciencegateway.org/tools/rotor.htm) – if the centrifuge/ultracentrifuge did not give such option.
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Mordukhovich, G. and Bahar, O. (2017). Isolation of Outer Membrane Vesicles from Phytopathogenic Xanthomonas campestris pv. campestris. Bio-protocol 7(5): e2160. DOI: 10.21769/BioProtoc.2160.
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分类
微生物学 > 微生物细胞生物学 > 细胞器分离
微生物学 > 微生物-宿主相互作用 > 细菌
生物化学 > 蛋白质 > 分离和纯化
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