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Pyocyanin Extraction and Quantitative Analysis in Swarming Pseudomonas aeruginosa
群集铜绿假单胞菌中的绿脓菌素提取和定量分析   

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Abstract

This protocol describes the quantification of pyocyanin extracted from swarming colonies of Pseudomonas aeruginosa. Pyocyanin is a secondary metabolite and a major virulence factor, whose production is inducible and varies highly under different growth conditions. The protocol is based on the earlier developed chloroform/HCl extraction of pyocyanin from liquid cultures (Frank and Demoss, 1959). Swarming colonies together with the agar they occupy are split into two halves. Pyocyanin is extracted from one of them. Cells are collected from the other half and used to quantify total protein yield and normalize the estimated corresponding pyocyanin quantities.

Keywords: Pyocyanin(绿脓菌素), Swarming(群集), P. aeruginosa(铜绿假单胞菌), Chloroform extraction(氯仿萃取), Virulence factor(毒力因子), Motility(能动性)

Background

Pyocyanin is a blue redox-active phenazine pigment produced by a human pathogen, P. aeruginosa. Pyocyanin is present in large quantities in the sputum of cystic fibrosis patients infected by P. aeruginosa (Wilson et al., 1988) and plays a major role in the virulence of the pathogen (Lau et al., 2004). Pyocyanin production is regulated by quorum sensing, which involves a cell-density-dependent synthesis of signal molecules that modulate the expression of virulence genes (Fuqua et al., 2001). Swarming is a complex communal behavior developing in response to multiple environmental signals and enabling cell motility on a semi-solid surface and colonization of host tissues. Our earlier observations suggested that pyocyanin production in liquid cultures of P. aeruginosa differ significantly from that in swarming colonies, likely due to quorum sensing regulation. Therefore, we developed a protocol allowing extraction and quantification of pyocyanin secreted by swarming cells into the surrounding agar. The obtained quantities are normalized by total cellular protein extracted from the swarming cells. This protocol differs from commonly used quantification of pyocyanin extracted from liquid cultures (Frank and Demoss, 1959) cells scrapped from a solid agar surface (De Vleesschauwer et al., 2006), or agar itself, but without normalization per total cell protein (Gallagher and Manoil, 2001). Normalized quantification of pyocyanin from swarming colonies allows studying the regulation of pyocyanin production and secretion during swarming, which is an essential component of P. aeruginosa adaptation to a host environment.

Materials and Reagents

  1. Glass test tubes (VWR, catalog number: 47729-578 )
  2. Razor blade
  3. Plastic storage containers and spatulas
  4. Glass scintillation vials (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: B7999-6 )
  5. Glass Pasteur pipette
  6. 96-well flat bottom plates (Greiner Bio One, catalog number: 655-101 )
  7. 50 ml Falcon tubes (VWR, catalog number: 89039-658 )
  8. Whatman filter paper No.1 (GE Healthcare, catalog number: 1001-090 )
  9. 50 ml centrifuge vials
  10. 1.5 ml microcentrifuge tubes
  11. 0.2 μm filter
  12. 10 ml syringes
  13. PFTE lined caps for glass vials (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: B7815-24 )
  14. Pseudomonas aeruginosa PAO1
  15. Chloroform (Pharmco-AAPER, catalog number: 3090000DIS )
  16. HCl (Pharmco-AAPER, catalog number: 284000ACS )
  17. Sodium hydroxide (NaOH) (Thermo Fisher Scientific, Fischer Scientific, catalog number: 1310-73-2 )
  18. Bradford reagent (Alfa Aesar, catalog number: J61522-AP )
  19. Bovine serum albumin (BSA) (Akron Biotech, catalog number: AK8917-1000 )
  20. Monosodium glutamate (Sigma-Aldrich, catalog number: 1446600 )
  21. Glycerol (Thermo Fisher Scientific, Fisher Scientific, catalog number: G33 )
  22. Sodium phosphate monobasic dihydrate (NaH2PO4) (Sigma-Aldrich, catalog number: 71500 )
  23. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: 17835 )
  24. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 or VWR, catalog number: E529-500ML )
  25. Biotin (Gold Bio, catalog number: B-950-1 )
  26. Thiamine HCl (Gold Bio, catalog number: T-260-25 )
  27. Copper(II) sulfate pentahydrate (CuSO4·5H2O) (Sigma-Aldrich, catalog number: 209198 )
  28. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sigma-Aldrich, catalog number: Z0251 )
  29. Iron(II) sulfate heptahydrate (FeSO4·7H2O) (Sigma-Aldrich, catalog number: 215422 )
  30. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Avantor Performance Materials, J.T. Baker, catalog number: 2540-04 )
  31. Yeast extract (BD, Bacto, catalog number: 212750 )
  32. Tryptone (BD, Bacto, catalog number: 211705 )
  33. Agar (BD, Bacto, catalog number: 214010 )
  34. Glucose (Alfa Aesar, catalog number: A16828 )
  35. Casamino acids (Teknova, catalog number: C2000 )
  36. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: 230391 )
  37. Biofilm mineral medium (BMM) (see Recipes)
    1. 10x basal salt solution
    2. Vitamin solution
    3. Trace metals solution
  38. Luria Bertani (LB) agar plates (see Recipes)
  39. Swarming agar plates (see Recipes)
    1. Potassium phosphate buffer
    2. 1 mM FeSO4
    3. 20 mM MgSO4

Equipment

  1. Potato masher (see Notes)
  2. BioMateTM 3 spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 335904P )
    Note: This product has been discontinued. A similar model is BioMateTM 3S spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 840208400 ).
  3. MaxQ 4000 table top shake incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: SHKA4000 )
  4. SynergyTM Mx 2 Multi-Mode plate reader (BioTek Instruments) with Gen5TM 2.05 PC software (BioTek Instruments)
  5. Fume hood
  6. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: SorvallTM ST 40R ) with 50 ml bucket insert (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 75003674 )
  7. Water bath sonicator (Thomas Scientific, Branson®, catalog number: 1207K40 )
  8. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: SorvallTM RC 6 Plus Centrifuge ) with rotor type (Thermo Fisher Scientific, Thermo ScientificTM, model: F13-14x50 cy )
  9. Centrifuge (Eppendorf, model: 5424 )
  10. Table top standard orbital shaker (VWR, model: 3500 )
  11. 1 L graduated cylinder

Procedure

  1. Pyocyanin extraction
    1. Inoculate Pseudomonas aeruginosa strain PAO1 from frozen stock on an LB agar plate and incubate for 24 h at 37 °C.
    2. Pipette 5 ml of BMM into 3 sterile test tubes, inoculate isolated colonies of PAO1 grown on LB agar, and incubate for 16 h at 37 °C with shaking at 200 rpm.
    3. Measure OD600 (BioMate3 spectrophotometer) of the 3 biological replicates and normalize cell cultures to OD600 = 0.3 by diluting with fresh BMM.
    4. Inoculate 2 μl of normalized culture onto the center of a freshly made (3-4 h after pouring) swarming plate (described in Recipes section) and incubate for 24 h at 37 °C.
      Cut each swarming colony together with the agar it occupies in half using a razor blade. Transfer ½ of the swarming colony into a small plastic container using a plastic spatula (Figure 1) and add 5 ml of saline (0.85% NaCl).


      Figure 1. Transferring ½ of the swarming colony with the agar it occupies into a plastic container

    5. Using a potato masher, mash the agar into small pieces (Figure 2) and transfer the agar along with saline into a 40 ml glass scintillation vial (glass separation funnel can be used instead). Repeat for two other replicates.


      Figure 2. Smashing the collected swarming colony and agar into small pieces in preparation for extraction

    6. Rinse the container and the masher with another 5 ml of saline, collecting all the remaining pieces of agar, and combine with the first portion containing the smashed agar.
    7. Using a glass pipette, add 5 ml of chloroform to the glass vials containing smashed agar. Secure the glass vials to the table top orbital shaker and shake the vials at speed setting 3 for 15 min at room temperature. To avoid the toxic effect of chloroform, all the steps involving the solvent should be done in a fume hood.
    8. After shaking, stand the vials vertically to let the layers separate. This will take about 10 min.
    9. By using a glass Pasteur pipette, transfer the bottom chloroform layer into a clean scintillation vial.
      Note: If pyocyanin is present, this layer will be tinted blue.
    10. Repeat steps A7-A10 adding chloroform two more times to the glass vials containing smashed agar and combine all three chloroform fractions for each replicate in one vial.
    11. Add 5 ml of 0.2 N HCl and shake the vials using a table top orbital shaker at speed setting 3 for 15 min at room temperature.
    12. After shaking, stand the vials vertically to let the layers separate. This will take about 10 min.
    13. By using a glass Pasteur pipette, collect the top HCl layer and transfer 200 μl of it to a flat bottom 96-well plate.
      Note: If pyocyanin is present, this layer will be tinted red. Measure absorbance at 520 nm (SynergyTM Mx Biotek plate reader).
    14. Calculate the concentration of pyocyanin in μg/ml by using the extinction coefficient of 17.1 (Kurachi, 1958).

  2. Total cellular protein quantification
    Use the remaining half of the swarming colony from step A5 of pyocyanin extraction protocol above.
    1. Transfer the remaining ½ of the swarming colony into a small plastic container and pipette 5 ml of saline into it
    2. Using a potato masher, mash the agar into small pieces and transfer the agar along with saline into a 50 ml Falcon tube (Figure 2).
    3. Rinse the container and masher with 5 ml of saline to collect all the remaining pieces of agar and transfer to the Falcon tube used in the previous step, thus combining all the pieces of the mashed agar.
    4. Sonicate the mixture for 15 min in a Branson 2800 water-bath sonicator (with default setting), then vortex for 5 min. Repeat this step two more times.
    5. Gently pellet agar pieces by centrifuging for 2 min at 1,000 x g, carefully collect the supernatant without disturbing the agar, and transfer into a clean 50 ml Falcon tube.
    6. Add 10 ml of saline to agar pellet and repeat steps B4-B5. Add another 10 ml and repeat steps B4-B5. Combine all the collected supernatants and filter through a Whatman No. 1 filter to remove any leftover agar pieces. Apply vacuum if needed. Transfer the filtered supernatant into a clean 50 ml centrifuge vial.
    7. Centrifuge the filtrate at 17,000 x g for 10 min at 25 °C (Sorvall RC 6+ centrifuge with rotor type F13-14x50 cy). Collect the pellet, resuspend it in 1 ml of 1 N NaOH, and transfer into a 1.5 ml microfuge tube.
    8. Heat the samples at 90 °C for 10 min and centrifuge for 5 min at 15,000 x g (Eppendorf 5424 centrifuge) at room temperature.
    9. Transfer the supernatants to a new microfuge tube for Bradford analysis. Depending on the protein yield, samples may need to be diluted. In our experiments, we diluted samples 10 times with saline.
    10. Take 20 μl of each sample and mix with 200 μl of Bradford reagent in a 96-well flat bottom plate. Assay Bovine Serum Albumin protein standards in the same plate. Measure absorbance at 595 nm (SynergyTM Mx Biotech plate reader). Calculate protein concentration according to the BSA standard curve and use it to normalize the pyocyanin data.

Data analysis

Total amount of pyocyanin secreted by one half of the colony was determined (Figure 3B) and normalized by the total amount of cellular protein extracted from another half of the colony (Figures 3C and 3D). To calculate total protein, we used a calibration curve based on BSA standards plotted in Microsoft Excel. Only standard curves that yielded an R2 value above 0.95 were used. The measured and normalized production of pyocyanin correlates well with the visual observation of the mutant carP::Tn5 showing significantly reduced pyocyanin production in comparison to PAO1 swarming colonies. To test the accuracy and consistency of the measurements, the experiment was repeated three times, with three biological replicates each time. Standard deviation between biological replicates was below 11 %. We observed no additional deviation due to differences in the sizes of swarming colonies.


Figure 3. Quantification of pyocyanin production in P. aeruginosa swarming colonies. A. Photographs of swarming colonies formed by P. aeruginosa PAO1 and mutants with transposon disrupted (carP::Tn5) and in-trans complemented (carP::Tn5/carP) carP, encoding calcium-regulated beta-propeller protein (Guragain et al., 2016). Swarming medium [Recipes below] was complemented with 10 mM CaCl2. B. Amount of pyocyanin measured. C. Amount of protein quantified using the Bradford assay. D. Quantified pyocyanin normalized by total cellular protein.

Notes

  1. To enable accurate normalization of pyocyanin data, swarming colonies were cut in half, so one half can be used to measure pyocyanin, and the other to measure cell protein. We also tested an alternative approach: inoculating 2 separate plates and using one entire colony (or plate) for pyocyanin and the other colony (or plate) for protein quantification. However, colonies vary in diameters, and this prevented accurate normalization.
  2. The circular potato masher with 8 x 8 mm evenly spaced openings was used for convenient breaking agar into approximately even pieces.
  3. A non-inoculated swarming agar plate was used as a control to verify that the agar components do not interfere with pyocyanin and protein extraction and quantification.
  4. The number and the duration of sonication, vortexing, and extraction steps were optimized to allow maximum possible removal of swarming cells embedded into the agar, and efficient extraction of pyocyanin.
  5. 50 ml Falcon tubes were used for collecting cells for protein quantification, because the clear walls made it easy to visualize agar pellet. The latter was centrifuged at a low speed for a brief period to pellet only agar, but not cells. To pellet cells for the following lysis, samples were transferred into 50 ml centrifuge vials and centrifuged at 17,000 x g.
  6. Filtering the collected supernatant from step B6 removed all the agar pieces that were accidentally collected. If agar is not removed completely, it can interfere with cell lysis by NaOH.

Recipes

  1. Biofilm minimal media (BMM) (Sarkisova et al., 2005).
    Mix 100 ml of sterile 10x basal salt solution (Recipe 1a) to 900 ml of sterile nanopure water. Add 1 ml of vitamin solution (Recipe 1b), 200 μl of trace metals solution (Recipe 1c), and 20 μl of 1 M MgSO4.
    1. 10x basal salt solution
      In 1 L of nanopure water combine:
      15 g monosodium glutamate
      46 g glycerol
      0.18 g sodium phosphate monobasic dihydrate (NaH2PO4)
      0.78 g potassium phosphate dibasic (K2HPO4)
      84.7 g sodium chloride (NaCl)
      Adjust the pH to 7.0. Sterilize by autoclaving
    2. Vitamin solution
      Dissolve 1 mg of biotin in 10 ml of nanopure water. Aliquot 1 ml of biotin stock solution in fresh tube, add 50 mg thiamine HCl to it, and mix properly. Adjust the final volume to 100 ml with nanopure water. Filter sterilize using a 0.2 μm filter and store at 4 °C.
    3. Trace metals solution
      Dilute 10 ml concentrated HCl in 70 ml of nanopure water. Add the following ingredients:
      0.5 g CuSO4·5H2O
      0.5 g ZnSO4·7H2O
      0.5 g FeSO4·7H2O
      0.2 g MnCl2·4H2O
      Dissolve completely and adjust the final volume to 100 ml with nanopure water. Filter-sterilize using a 0.2 μm filter.
  2. Luria Bertani (LB) agar plates
    Combine the below ingredients in 1 L of nanopure H2O and autoclave
    5 g yeast extract
    5 g NaCl
    10 g tryptone
    15 g BD BactoTM agar
  3. Swarming agar plates (modified from [Overhage et al., 2007])
    For 1 L of media add (freshly made):
    3.6 g glucose
    5 g casamino acids
    100 ml potassium phosphate buffer (Recipe 3a)
    5 g BD BactoTM agar
    Autoclave for 20 min at 250 F and 20 PSI
    After autoclaving add:
    1 ml of 20 mM MgSO4 (final concentration = 0.02 mM) (Recipe 3b)
    10 ml of 1 mM FeSO4 (final concentration = 0.01 mM) (Recipe 3c)
    Using a 25 ml glass pipette, pipette 20 ml of medium into every plate. Then place the plates individually on a bench top to dry at room temperature for 3-4 h before inoculation.
    1. Potassium phosphate buffer
      Make 1 L of K2HPO4 (F.W:174.18). Add 107.99 g/1 L of nanopure H2O
      Make 1 L of KH2PO4 (F.W: 136.09). Add 84.37 g/1 L of nanopure H2O
      In a 1 L graduated cylinder, mix:
      615 ml 620 mM K2HPO4
      385 ml 620 mM KH2PO4
      Buffer pH should be 7
    2. 1 mM FeSO4
      To make 1 mM stock solutions add 0.0559 g of FeSO4 to 200 ml nanopure H2O. Filter sterilize and store at 4 °C
    3. 20 mM MgSO4
      To make 20 mM stock solutions add 0.9858 g of MgSO4 to 200 ml nanopure H2O. Filter sterilize and store at 4 °C

Acknowledgments

We thank Dr. Erica Lutter, Oklahoma State University, for sharing equipment and for helpful discussions. This work was supported by the Research Grant from OCAST (Award HR12-167).

References

  1. De Vleesschauwer, D., Cornelis, P. and Hofte, M. (2006). Redox-active pyocyanin secreted by Pseudomonas aeruginosa 7NSK2 triggers systemic resistance to Magnaporthe grisea but enhances Rhizoctonia solani susceptibility in rice. Mol Plant Microbe Interact 19(12): 1406-1419.
  2. Frank, L. H. and Demoss, R. D. (1959). On the biosynthesis of pyocyanine. J Bacteriol 77(6): 776-782.
  3. Fuqua, C., Parsek, M. R. and Greenberg, E. P. (2001). Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing. Annu Rev Genet 35: 439-468.
  4. Gallagher, L. A. and Manoil, C. (2001). Pseudomonas aeruginosa PAO1 kills Caenorhabditis elegans by cyanide poisoning. J Bacteriol 183(21): 6207-6214.
  5. Guragain, M., King, M. M., Williamson, K. S., Perez-Osorio, A. C., Akiyama, T., Khanam, S., Patrauchan, M. A. and Franklin, M. J. (2016). The Pseudomonas aeruginosa PAO1 two-component regulator CarSR regulates Calcium homeostasis and Calcium-induced virulence factor production through its regulatory targets CarO and CarP. J Bacteriol 198(6): 951-963.
  6. Kurachi, M. (1958). Studies on the biosynthesis of pyocyanine. (ii) : isolation and determination of pyocyanine. Bulletin of the Institute for Chemical Research, Kyoto University 36(6): 776-782.
  7. Lau, G. W., Hassett, D. J., Ran, H. and Kong, F. (2004). The role of pyocyanin in Pseudomonas aeruginosa infection. Trends Mol Med 10(12): 599-606.
  8. Overhage, J., Lewenza, S., Marr, A. K. and Hancock, R. E. (2007). Identification of genes involved in swarming motility using a Pseudomonas aeruginosa PAO1 mini-Tn5-lux mutant library. J Bacteriol 189(5): 2164-2169.
  9. Sarkisova, S., Patrauchan, M. A., Berglund, D., Nivens, D. E. and Franklin, M. J. (2005). Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms. J Bacteriol 187(13): 4327-4337.
  10. Wilson, R., Sykes, D. A., Watson, D., Rutman, A., Taylor, G. W. and Cole, P. J. (1988). Measurement of Pseudomonas aeruginosa phenazine pigments in sputum and assessment of their contribution to sputum sol toxicity for respiratory epithelium. Infect Immun 56(9): 2515-2517.

简介

该方案描述了从绿脓假单胞菌的群体群体提取的绿脓素的定量。绿脓素是次级代谢产物和主要毒力因子,其生产是可诱导的并且在不同生长条件下高度变化。该方案基于早先开发的氯仿/HCl从液体培养物中提取绿脓素(Frank和Demoss,1959)。聚集的菌落与它们所占据的琼脂一起分成两半。从它们之一提取绿脓菌素。从另一半收集细胞并用于定量总蛋白质产量并使估计的相应的绿脓素量标准化。
关键字:绿靛,Swarming,铜绿色,氯仿提取,毒力因子,运动性

[背景] 绿脓菌素是一种由人类病原体产生的蓝色氧化还原活性吩嗪色素。铜绿。绿脓菌素大量存在于感染有P的囊性纤维化患者的痰液中。 (Wilson等人,1988),并在病原体的毒力中起重要作用(Lau等人,2004)。绿脓菌素产生通过群体感应来调节,其涉及调节毒力基因的表达的信号分子的细胞密度依赖性合成(Fuqua等人,2001)。群集是响应于多种环境信号并使得细胞在半固体表面上的运动和宿主组织的建群而形成的复杂的共同行为。我们早先的观察表明,在液体培养物中的绿脓素生产。铜绿色与群体中的群体显着不同,可能是由于群体感应调节。因此,我们开发了一个协议,允许提取和定量的绿脓菌分泌的细胞成周围的琼脂分泌。所获得的量通过从群集细胞提取的总细胞蛋白质归一化。该方案不同于从固体琼脂表面(De Vleesschauwer等人,2006)废弃的液体培养物(Frank和Demoss,1959)提取的绿脓菌素或琼脂本身的常用定量,但是没有每个总细胞蛋白的归一化(Gallagher和Manoil,2001)。来自群体的绿脓菌的归一化定量允许研究在聚集期间的绿脓素生产和分泌的调节,其是p的必要组分。铜绿色适应于宿主环境。

关键字:绿脓菌素, 群集, 铜绿假单胞菌, 氯仿萃取, 毒力因子, 能动性

材料和试剂

  1. 玻璃试管(VWR,目录号:47729-578)
  2. 剃刀刀片
  3. 塑料存储容器和刮刀
  4. 玻璃闪烁瓶(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:B7999-6)
  5. 玻璃巴斯德吸液管
  6. 96孔平底板(Greiner Bio One,目录号:655-101)
  7. 50ml Falcon管(VWR,目录号:89039-658)
  8. 胰蛋白胨(BD,Bacto,目录号:211705)
  9. 琼脂(BD,Bacto,目录号:214010)
  10. 葡萄糖(Alfa Aesar,目录号:A16828)
  11. 酪氨酸(Teknova,目录号:C2000)
  12. 硫酸镁七水合物(MgSO 4·7H 2 O)(Sigma-Aldrich,目录号:230391)
  13. 生物膜矿物培养基(BMM)(参见配方)
    1. 10x基础盐溶液
    2. 维生素溶液
    3. 痕量金属溶液
  14. Luria Bertani(LB)琼脂平板(见Recipes)
  15. 聚集琼脂板(见配方)
    1. 磷酸钾缓冲液
    2. 1mM FeSO 4
    3. 20mM MgSO 4

设备

  1. 马铃薯捣碎器(见注释)
  2. BioMate TM分光光度计(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:335904P)
    注意:此产品已停产。类似的模型是BioMate TM suppectium分光光度计(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:840208400)。
  3. MaxQ 4000台式摇动培养箱(Thermo Fisher Scientific,Thermo Scientific TM ,型号:SHKA4000)
  4. Synergy TM sup/2 Mx 2多模式酶标仪(BioTek Instruments),具有Gen5 TM sup/2.02 PC软件(BioTek Instruments)
  5. 通风橱
  6. 离心机(Thermo Fisher Scientific,Thermo Scientific ,型号:Sorvall TM ST 40R),具有50ml桶插入物(Thermo Fisher Scientific,Thermo Scientific >,目录号:75003674)
  7. 水浴超声器(Thomas Scientific,Branson ,目录号:1207K40)
  8. 使用转子型(Thermo Fisher Scientific,Thermo Scientific TM )离心(Thermo Fisher Scientific,Thermo Scientific ,型号:Sorvall >,型号:F13-14x50 cy)
  9. 离心机(Eppendorf,型号:5424)
  10. 台式标准轨道摇床(VWR,型号:3500)
  11. 1升量筒

程序

  1. 绿脓菌素提取
    1. 在LB琼脂平板上接种来自冷冻储备物的铜绿假单胞菌菌株PAO1,并在37℃下孵育24小时。
    2. 移取5ml BMM到3个无菌试管中,接种生长在LB琼脂上的分离的PAO1菌落,并在37℃下以200rpm振荡孵育16小时。
    3. 测量3个生物重复的OD 600(BioMate 3分光光度计),并通过用新鲜BMM稀释将细胞培养物标准化至OD 600 = 0.3。
    4. 将2μl标准化培养物接种到新鲜制备的(3-4小时后倾倒)蜂群板的中心(在食谱部分描述),并在37°C孵育24小时。
      切割每个群集与琼脂,它占据一半使用剃刀刀片。使用塑料抹刀(图1)将一半的群落转移到一个小塑料容器中,加入5ml盐水(0.85%NaCl)。


      图1.将一半的群体与其占据的塑料容器中的琼脂转移

    5. 使用马铃薯捣碎机,将琼脂捣碎成小块(图2),并将琼脂与盐水一起转移到40毫升玻璃闪烁小瓶(玻璃分离漏斗可以代替)。对其他两个重复进行重复。


      图2.将收集的群体和琼脂粉碎成小块以备提取

    6. 用另一5ml盐水冲洗容器和捣碎器,收集所有剩余的琼脂块,并与含有捣碎的琼脂的第一部分合并。
    7. 使用玻璃吸管,向含有捣碎的琼脂的玻璃小瓶中加入5ml氯仿。将玻璃小瓶固定到桌面定轨振荡器上,并在室温下以速度设置3摇动小瓶15分钟。为避免氯仿的毒性作用,所有涉及溶剂的步骤都应在通风橱中进行
    8. 摇动后,垂直放置小瓶以使层分离。这将需要大约10分钟。
    9. 通过使用玻璃巴斯德吸管,将底部氯仿层转移到干净的闪烁瓶。
      注意:如果存在绿脓菌,该层将变成蓝色。
    10. 重复步骤A7-A10,向含有捣碎的琼脂的玻璃小瓶中再加入氯仿两次,并将每个重复的三个氯仿部分合并在一个小瓶中。
    11. 加入5ml 0.2N HCl,并使用台式定轨振荡器在速度设置3下在室温下摇动小瓶15分钟。
    12. 摇动后,垂直放置小瓶以使层分离。这将需要大约10分钟。
    13. 通过使用玻璃巴斯德吸管,收集顶部HCl层和转移200微升到平底96孔板。
      注意:如果存在绿脓菌素,该层将被着色为红色。测量520nm处的吸光度(SynergyTM Mx Biotek平板读数器)。
    14. 使用消光系数17.1(Kurachi,1958)计算以μg/ml计的绿脓素浓度。

  2. 总细胞蛋白定量
    使用上述绿脓素提取方案的步骤A5中剩下的一半群落菌落
    1. 转移剩余的一半的群集成一个小的塑料容器,并移取5毫升盐水进入它
    2. 使用马铃薯捣碎机,将琼脂捣碎成小块,并将琼脂与盐水转移到50ml Falcon管(图2)。
    3. 用5ml盐水冲洗容器和捣碎器以收集所有剩余的琼脂碎片,并转移到前面步骤中使用的Falcon管,从而合并所有碎片的捣碎的琼脂。
    4. 在Branson 2800水浴超声仪(默认设置)中超声处理混合物15分钟,然后涡旋5分钟。重复此步骤两次。
    5. 通过在1,000×g离心2分钟轻轻地沉淀琼脂碎片,小心地收集上清液而不打扰琼脂,并转移到干净的50ml Falcon管中。
    6. 向琼脂沉淀中加入10ml盐水,重复步骤B4-B5。加入另外10ml,并重复步骤B4-B5。合并所有收集的上清液并通过Whatman 1号过滤器过滤以除去任何剩余的琼脂片。如果需要,应用真空。将过滤的上清液转移到干净的50ml离心管瓶中。
    7. 在25℃下将滤液以17,000×g离心10分钟(具有转子类型F13-14x50cot的Sorvall RC 6+离心机)。收集沉淀,将其重悬在1ml 1N NaOH中,并转移到1.5ml微量离心管中。
    8. 将样品在90℃加热10分钟,并在室温下在15,000×g(Eppendorf 5424离心机)离心5分钟。
    9. 将上清液转移到新的微量离心管用于Bradford分析。根据蛋白质产量,样品可能需要稀释。在我们的实验中,我们用盐水稀释样品10次
    10. 取20μl每个样品,并与200μl的Bradford试剂在96孔平底板中混合。测定牛血清白蛋白标准品在同一板中。测量在595nm的吸光度(Synergy TM sup/Mx Biotech读板仪)。根据BSA标准曲线计算蛋白质浓度,并使用它来归一化绿脓菌素数据。

数据分析

测定由集落的一半分泌的绿脓菌素的总量(图3B),并通过从集落的另一半提取的细胞蛋白的总量标准化(图3C和3D)。为了计算总蛋白,我们使用基于在Microsoft excel中绘制的BSA标准的校准曲线。仅使用产生大于0.95的R 2值的标准曲线。绿脓菌素的测量和标准化产生与突变体pcP :: Tn5的视觉观察很好相关,显示与PAO1群集相比显着降低的绿脓菌素产生。为了测试测量的准确性和一致性,将实验重复三次,每次三次生物学重复。生物学重复之间的标准偏差低于11%。我们观察到由于群体大小的差异没有额外的偏差。


图3.在em中的绿脓素产量的定量。铜绿色群体。 A.由 P形成的群体的照片。 ( carP :: Tn5)和 in-trans 互补的突变体( carP :: Tn5/ carP )。聚集培养基[下面的食谱]补充有10mM CaCl 2。 B.测量的绿脓素的量。 C.使用Bradford测定法定量的蛋白质量。 D.通过总细胞蛋白标准化的量化的绿脓素

笔记

  1. 为了使精确的正常的绿脓菌数据正常化,将群集切成两半,因此一半可用于测量绿脓素,另一半用于测量细胞蛋白。我们还测试了一种替代方法:接种2个单独的平板,并使用一个完整菌落(或板)的绿脓素和其他菌落(或板)进行蛋白质定量。然而,菌落直径变化,这阻止了精确的标准化。
  2. 具有8×8mm均匀间隔开口的圆形马铃薯捣碎器用于方便地将琼脂破碎成大致均匀的碎片。
  3. 将未接种的聚集琼脂平板用作对照以验证琼脂组分不干扰绿脓菌素和蛋白质提取和定量。
  4. 优化超声处理,涡旋和提取步骤的次数和持续时间以允许最大可能地除去嵌入琼脂中的蜂群,并有效提取绿脓素。
  5. 使用50ml Falcon管来收集用于蛋白质定量的细胞,因为透明的壁使得容易观察琼脂沉淀。将后者在低速下离心短暂的时间,以仅沉淀琼脂,而不是细胞。为了将细胞沉淀用于以下裂解,将样品转移到50ml离心管中并以17,000×g离心。
  6. 过滤从步骤B6收集的上清液,除去所有的偶然收集的琼脂碎片。如果琼脂没有完全去除,它可以干扰NaOH的细胞裂解

食谱

  1. 生物膜基本培养基(BMM)(Sarkisova等人,2005)。
    混合100毫升无菌的10倍基础盐溶液(食谱1a)到900毫升无菌纳米纯水。加入1ml维生素溶液(配方1b),200μl痕量金属溶液(配方1c)和20μl1M MgSO 4。
    1. 10x基础盐溶液
      在1L纳米水中结合:
      15g谷氨酸单钠 46克甘油 0.18g磷酸二氢钠二水合物(NaH 2 PO 4)
      0.78g磷酸氢二钾(K 2 HPO 4)
      84.7g氯化钠(NaCl)
      调节pH至7.0。高压灭菌
      灭菌
    2. 维生素溶液
      将1mg生物素溶解在10ml的超纯水中。在新管中等分1ml生物素储备溶液,向其中加入50mg盐酸硫胺素,并适当混合。使用纳米纯水将最终体积调节至100 ml。使用0.2μM过滤器进行过滤灭菌并在4℃下储存。
    3. 微量金属污染
      稀释10毫升浓盐酸在70毫升超纯水中。添加以下成分:
      0.5g CuSO 4·5H 2 O 2 / 0.5g ZnSO 4·7H 2 O·h/v 0.5g FeSO 4·7H 2 O·h/v 0.2g MnCl 2·4H 2 O·m/2 完全溶解,使用纳米纯水将最终体积调节至100 ml。使用0.2μM过滤器过滤灭菌。
  2. Luria Bertani(LB)琼脂平板
    将以下成分在1L纳米H 2 O 2和高压釜
    中混合 5g酵母提取物
    5克NaCl
    10g胰蛋白胨
    15g BD Bacto TM 琼脂
  3. 聚集琼脂平板(修改自[Overhage等人,2007])
    对于1升培养基(新鲜制备):
    3.6克葡萄糖 5克酪蛋白氨基酸
    100ml磷酸钾缓冲液(配方3a) 5g BD Bacto TM 琼脂 在250 F和20 PSI下高压灭菌20分钟
    高压灭菌后,加入:
    1ml的20mM MgSO 4(终浓度= 0.02mM)(配方3b)
    10ml的1mM FeSO 4(终浓度= 0.01mM)(配方3c)
    使用25毫升玻璃移液管,吸取20毫升培养基到每个板。然后将板单独放在台面上,在室温下干燥3-4小时,然后接种。
    1. 磷酸钾缓冲液
      制备1L的K 2 HPO 4(F.W:174.18)。加入107.99g/1L纳米H 2 O 2 / 制备1L的KH 2 PO 4(F.W:136.09)。加入84.37g/l的纳米级H 2 O 2 / 在1L量筒中混合:
      615ml 620mM K 2 HPO 4
      385ml 620mM KH 2 PO 4 4/s 缓冲液pH值应为7
    2. 1mM FeSO 4
      为了制备1mM储备溶液,将0.0559g FeSO 4加至200ml纳米H 2 O 2。过滤灭菌并在4℃下保存
    3. 20mM MgSO 4 为了制备20mM储备溶液,将0.9858g MgSO 4加入到200ml纳米H 2 O 2中。过滤灭菌并储存在4°C

致谢

我们感谢俄克拉何马州立大学的Erica Lutter博士,分享设备和有益的讨论。这项工作是由OCAST的研究资助(奖HR12-167)支持。

参考文献

  1. De Vleesschauwer,D.,Cornelis,P.和Hofte,M。(2006)。  Mol Plant Microbe Interact 19(12):1406-1419。
  2. Frank,LH和Demoss,RD(1959)。  On吡啶核苷的生物合成。细菌 77(6):776-782。
  3. Fuqua,C.,Parsek,MR和Greenberg,EP(2001)。  Cell of gene expression by cell-to-cell communication:acyl-homoserine lactone quorum sensing。 Annu Rev Genet 35:439-468。
  4. Gallagher,LA和Manoil,C。(2001)。  PAO1通过氰化物中毒杀死秀丽隐杆线虫。 183(21):6207-6214。
  5. Guragain,M.,King,MM,Williamson,KS,Perez-Osorio,AC,Akiyama,T.,Khanam,S.,Patrauchan,MA和Franklin,MJ(2016)。< a class ="ke-insertfile "href ="http://www.ncbi.nlm.nih.gov/pubmed/26755627"target ="_ blank">绿脓杆菌 PAO1双组分调节剂CarSR调节钙稳态和钙 - 诱导的毒力因子产生通过其调节性靶向CarO和CarP。细菌199(6):951-963。
  6. Kurachi,M.(1958)。  对碧茱萸的生物合成的研究。 (ii):分离和测定卟啉。 <京都大学化学研究所通报 36(6):776-782。
  7. Lau,GW,Hassett,DJ,Ran,H.and Kong,F。(2004)。  绿脓杆菌感染中绿脓素的作用。 Trends Mol Med 10(12):599-606。 />
  8. Overhage,J.,Lewenza,S.,Marr,AK和Hancock,RE(2007)。  使用绿脓杆菌 PAO1 mini-Tn5-lux突变体文库鉴定参与群体运动性的基因。

  9. Sarkisova,S.,Patrauchan,MA,Berglund,D.,Nivens,DE和Franklin,MJ(2005)。  钙诱导的与粘液样细胞外基质相关的毒力因子绿脓假单胞菌生物膜细菌 187 (13):4327-4337。
  10. Wilson,R.,Sykes,DA,Watson,D.,Rutman,A.,Taylor,GW和Cole,PJ(1988)。< a class ="ke-insertfile"href ="http:痰中的铜绿假单胞菌吩嗪颜料的测量和它们对痰液溶胶对呼吸上皮的毒性的贡献的评价。 Infect Immun 56(9):2515-2517。
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引用:King, M. M., Guragain, M., Sarkisova, S. A. and Patrauchan, M. A. (2016). Pyocyanin Extraction and Quantitative Analysis in Swarming Pseudomonas aeruginosa. Bio-protocol 6(23): e2042. DOI: 10.21769/BioProtoc.2042.
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