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Quantification of Chlorophyll as a Proxy for Biofilm Formation in the Cyanobacterium Synechococcus elongatus
定量测定蓝藻细长聚球藻中作为生物膜形成代用指标的叶绿素

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Abstract

A self-suppression mechanism of biofilm development in the cyanobacterium Synechococcus elongatus PCC 7942 was recently reported. These studies required quantification of biofilms formed by mutants impaired in the biofilm-inhibitory process. Here we describe in detail the use of chlorophyll measurements as a proxy for biomass accumulation in sessile and planktonic cells of biofilm-forming strains. These measurements allow quantification of the total biomass as estimated by chlorophyll level and representation of the extent of biofilm formation by depicting the relative fraction of chlorophyll in planktonic cells.

Keywords: Biofilm(生物膜), Cyanobacteria(蓝藻), Synechococcus elongatus(细长聚球藻), Chlorophyll measurement(叶绿素测定), Sessile(无柄), Planktonic(浮游)

Background

Several recently published studies indicate an emerging interest in the mechanisms that underlie cell-aggregation and biofilm development in cyanobacteria (Fisher et al., 2013; Jittawuttipoka et al., 2013; Schatz et al., 2013; Enomoto et al., 2014; Schwarzkopf et al., 2014; Enomoto et al., 2015; Oliveira et al., 2015; Agostoni et al., 2016; Parnasa et al., 2016). We recently reported a self-biofilm-inhibitory mechanism that dictates planktonic growth of the model unicellular cyanobacterium Synechococcus elongatus PCC 7942 (Schatz et al., 2013; Nagar and Schwarz, 2015). Abrogation of the biofilm-inhibitory process by inactivation of particular genes results in robust biofilm development in this otherwise planktonic strain (Schatz et al., 2013; Nagar and Schwarz, 2015). These studies required quantification of the extent of biofilm development in various strains and under different conditions. Crystal violet is commonly used for quantification of biofilms in heterotrophic bacteria (O’Toole and Kolter, 1998). This staining procedure, however, quantifies only the sessile fraction of cells. Here we provide a detailed protocol for culture growth and quantification of cyanobacterial biofilms using chlorophyll measurement as a proxy for biomass accumulation in sessile as well as in planktonic cells. These measurements allow estimation of the total biomass accumulated and representation of the relative fraction of chlorophyll in sessile or in planktonic cells.

Materials and Reagents

  1. Custom-made Pyrex glass tubes for bacterial liquid cultures (200 x 32 mm, made from Pyrex tubing, Corning, catalog number: 8510-32-D)
    Note: Can order from http://www.degroot.co.il/.
  2. ‘Sponge plug’ (Plastic foam stoppers, 27 x 34 mm) (Jaece Industies, catalog number: L800-C )
  3. Pasteur pipettes (230 mm) (Romical, catalog number: 94-08401002 )
  4. 0.45 µm syringe filter (Sartorius, catalog number: 16555 )
  5. Filter Stericup-GP 250 ml Express Plus PES (0.22 μm) (EMD Millpore, catalog number: SCGPU05RE )
  6. Silicone tubing (6 x 9 mm, 4 x 6 mm) (Degania Silicone, catalog numbers: 2110600234 , 2110400434 , respectively)
  7. Sterile pipettes 1 and 25 ml
    1 ml pipettes (Corning, Costar®, catalog number: 4011 )
    25 ml pipettes (Corning, Costar®, catalog number: 4489)
  8. Sterilized pipette tips (Corning, Axygen®, catalog numbers: T-200-C , T-1000-C )
  9. Eppendorf tubes (1.5 ml) (Corning, Axygen®, catalog number: MCT-175-C )
  10. Synechococcus elongatus PCC7942
  11. Sodium nitrate (NaNO3) (Sigma-Aldrich, catalog number: S8170 )
  12. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Merck, catalog number: K26364082)
    Note: This product has been discontinued. Alternatively, Merck, catalog number: 105886 can be used.
  13. Calcium chloride dihydrate (CaCl2·2H2O) (ICN, catalog number: 10035-04-8)
    Note: This product has been discontinued. Alternatively, Bio-Lab, catalog number: 034205 can be used.
  14. Potassium phosphate dibasic (K2HPO4) (Honeywell International, Riedel-de-Haen, catalog number: 04248 )
  15. Ethylenediaminetetraacetic acid (Na2Mg·EDTA) (Bio-Lab, catalog number: 05142359)
    Note: This product has been discontinued. Alternatively, Biosolve, catalog number: 051423 can be used.
  16. Ferric ammonium citrate (C6H11FeNO7) (MP Biomedicals, catalog number: 02158040 )
  17. Citric acid (C6H8O7) (Frutarom, catalog number: 2355511000)
    Note: This product has been discontinued. Alternatively, Biosolve, catalog number: 030205 can be used.
  18. Boric acid (H3BO3) (Bio-Lab, catalog number: 02010591)
    Note: This product has been discontinued. Alternatively, Biosolve, catalog number: 020105 can be used.
  19. Manganese chloride hexahydrate (MnCl2·6H2O) (Duchefa Biochemie, catalog number: M0533 )
  20. Zinc sulfate heptahydrate (ZnSO4·7H2O) (CARLO ERBA Reagents, catalog number: 494907 )
  21. Sodium molybdate dihydrate (Na2MoO4·2H2O) (MP Biomedicals, catalog number: 194863 )
  22. Copper sulphate pentahydrate (CuSO4·5H2O) (Honeywell International, Riedel-de-Haen, catalog number: 12849 )
  23. Cobalt(II) nitrate hexahydrate (Co(NO3)2·6H2O) (Honeywell International, Riedel-de-Haen, catalog number: 12922)
    Note: This product has been discontinued. Alternatively, Sigma-Aldrich, catalog number: 239267 can be used.
  24. N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: H3375 )
  25. Sodium hydroxide (NaOH) (Frutarom, catalog number: 5553510 )
  26. Acetone ((CH3)2CO), A.C.S. reagent (Avantor Performance Materials, J.T. Baker®, catalog number: 9006-03 )
  27. Sodium bicarbonate (NaHCO3) (DAEJUNG CHEMICAL & METALS, catalog number: 7566-4100 )
  28. Sodium thiosulfate (Na2S2O3) (Sigma-Aldrich, catalog number: 217247 )
  29. Bacto agar (BD, BactoTM, catalog number: 214010 )
  30. LB agar (Lennox) (BD, DifcoTM, catalog number: 240110 )
  31. Antibiotics (added as appropriate according to the resistance of the particular strain)
    1. Spectinomycin dihydrochloride pentahydrate (Duchefa Biochemie, catalog number: S0188 )
    2. Kanamycine sulphate monohydrate (Duchefa Biochemie, catalog number: K0126 )
    3. Gentamycin sulphate (Duchefa Biochemie, catalog number: G0124 )
    4. Chloramphenicol (Duchefa Biochemie, catalog number: C0113 )
  32. Liquid BG11-medium (see Recipes)
    1. Stock I (100x concentrated)
    2. Stock II (100x concentrated)
    3. Stock III (100x concentrated)
    4. Stock V (1,000x concentrated)
  33. Solid BG11-medium (see Recipes)

Equipment

  1. Laminar flow hood
  2. Fume hood
  3. Autoclave
  4. Spectrophotometer (Agilent Technologies, model: Cary 100 , catalog number: 10069000) and respective cuvettes (Cell type P.L = 10 mm/EA) (Starna Cells, catalog number: 9-SOG-10)
  5. Benchtop centrifuge (MiniSpin, max. centrifugal force: 12,100 x g) (Eppendorf, model: MiniSpin® , catalog number: 5452000018)
  6. Refrigerator (4-7 °C)
  7. Growth rooms (30 ± 2 °C and 24 ± 2 °C for liquid cultures and cultures on solid medium, respectively)
    Note: Growing cultures on solid medium at 24 °C rather than 30 °C allows maintaining the cultures for longer periods.
  8. Light source
    For liquid cultures–incandescent light producing a flux of 20-30 μmol photons m-2  sec-1; Cultures on solid-BG11 are illuminated by fluorescent light (5 μmol photons m-2  sec-1)
  9. Pipet-aid
  10. Single-channel pipettes 200 and 1,000 μl
  11. Bubbling system
    1. Air compressor (Oil free air compressor) (Assouline Compressors, model: vs 204 50 )
    2. Cylinder carbon dioxide (CO2, compr. 99.5%) (Maxima, catalog number: GCDCU2.527)
    3. Flow meters (Tuttnauer company, 0-0.1 L/min, 1-10 L/min)
    4. High-flow air pressure regulator (0-2 PSI) (Marsh Bellofram, model: Type 70 , catalog number: 960-129-000)
    Note: To bubble cultures with 3% CO2 in air the following setup is used (Figure 1): CO2 gas is mixed with compressed air using flow meters to yield 3% CO2 in air. This mixture is humidified by bubbling into 1.5 L double distilled water (DDW) in a 2 L bottle to prevent water evaporation during bubbling of the cultures. The outlet from this bottle is passed through an empty 2 L bottle (serving to trap residual liquid) and through an air pressure regulator (200 mbar is appropriate for bubbling of ~300 culture tubes). Bubbling of CO2 enriched air from the latter into multiple cultures is obtained using home-made manifolds constructed from silicone tubing and appropriate T-bar connectors. The gas in each manifold channel is passed through a 0.45 µm filter (Figure 1). A short video is provided to demonstrate the flow rate in individual cultures tubes (Video 1).



    Figure 1. A scheme describing the setup used to bubble cultures with CO2-enriched air

    Video 1. Cyanobacterial cultures under bubbling

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player

Procedure

  1. Growing liquid cultures of Synechococcus elongatus PCC 7942 and biofilm-forming mutants thereof
    1. Round-bottom Pyrex tubes (20 cm in length, 3 cm in diameter) are used as growth vessels.
    2. A cotton-plugged Pasteur pipette (23 cm long) is inserted through a sponge-plug such that the tip of the Pasteur pipette is placed 1-2 cm above the bottom of the tube (Figure 2A, ‘growth set’).
    3. Growth sets are autoclaved in liquid cycle.
    4. Sterile liquid BG11-medium (25-50 ml) (see Recipes) is transferred into the Pyrex tube. The culture volume is adjusted to the biomass required for the designated experiment.
    5. Cells are scraped from solid-BG11 medium (see Recipes) with a 1 ml sterile plastic pipette and the biomass is shaken in the liquid medium (see Note 4).
      Important: To reproducibly observe biofilm formation, it is crucial to add the ferric ammonium citrate and citric acid components from a freshly made stock (see note in Recipe 1f). Autoclaved BG11 should be used within 4 days to inoculate cultures.
    6. Connect the growth set to the bubbling system (see description above) via 1 ml pipette tip (Figure 2A).
    7. The use of manifolds allows simultaneous bubbling of multiple cultures (Figure 2B, see Note under Equipment).


      Figure 2. Growing cyanobacterial cultures. A. Growth set connected to a single manifold channel; B. Cultures in the growth room.

  2. Quantification of biofilm
    1. Cells are cultured under continuous bubbling of 3% CO2 in air at 30 °C under continuous illumination with incandescent light (20-30 μmol photons m-2  sec-1).
    2. Starting cultures at the exponential phase are diluted (usually following 3 days of growth) to an optical density at 750 nm of 0.5.
    3. Experiments are initiated by an additional dilution of the cultures to an optical density at 750 nm of 0.5 (see Note 5).
    4. Biofilm development under this setup typically begins after 2-3 days.
    5. To verify that cultures are axenic ‘spot’ a 20 μl aliquot on solid LB agar.
    6. Percentage of chlorophyll in suspended cells serves to quantify biofilm formation as follows (Figure 3):
      1. Extraction of chlorophyll in planktonic cells is performed in 1.5 ml Eppendorf tubes. Remove an aliquot from the upper part of the suspended culture (see Note 6). In cases where the suspended fraction appears especially dense (planktonic strains or poor biofilm formers), a 0.2 ml sample is used for extraction. When robust biofilms are formed, 15 ml of planktonic cells are removed and concentrated 3 to 6-fold by centrifugation (5,000 x g, 10 min) prior to the chlorophyll extraction in 80% acetone (final concentration).
      2. Extraction of chlorophyll in the biofilm is performed in the growth tube. To separate the biofilm fraction, slowly remove the suspended cells. Biofilms tend to collapse during removal of the last few ml of liquid. Therefore, once clumps of biofilm detach and fall into the liquid, stop removing the culture. Measure the volume remaining in the tubes using a pipette (in our hands 2-5 ml are left) as well as the volume removed from the culture. The sum of these volumes (Vtotal) serves for calculation of the amount of chlorophyll in the suspended cells (see Data analysis). Extraction of chlorophyll is performed in 80% acetone at a final volume equal to Vtotal.
      3. Extraction of chlorophyll is carried out overnight in the refrigerator and chlorophyll is quantified based on absorbance as previously described (Arnon, 1949; Ritchie, 2006). Chlorophyll may be extracted with methanol instead of acetone. A step-by step protocol for measurement of chlorophyll a concentration in cyanobacteria is described by Zavřel et al. (2015).


        Figure 3. Quantification of biofilm based on chlorophyll measurement

Data analysis

The percentage of chlorophyll in suspension is calculated from the amount of chlorophyll in the planktonic and biofilm fractions. These values are calculated based on measured chlorophyll absorbances, sample volumes, and dilution factors, as described below. An example calculation is provided in Table 1.

Table 1. An example of calculations of chlorophyll (Chl) in planktonic and biofilm cells


  1. Calculate the amount of chlorophyll in suspended cells based on measured chlorophyll concentration and the volume of the suspended culture (Vtotal).

    Total Chl Planktonic (µg) = (OD663 - OD750) x 12.7 x (Dilution Factor) x Vtotal

  2. Calculate the amount of chlorophyll in the biofilm based on measured chlorophyll concentration and the volume of the acetone extraction, which should be Vtotal.
    1. If robust biofilm formation is observed, then the contribution from the remaining planktonic cells in the biofilm fraction is negligible and the total chlorophyll is calculated as follows:

      Total Chl Biofilm (µg) = (OD663 - OD750) x 12.7 x (Dilution Factor) x Vtotal

    2. To account for the contribution of remaining planktonic cells in the biofilm fraction, subtract the planktonic chlorophyll present in the fraction’s volume from the above value:

      Total Chl Biofilm Corrected (µg) = (Chl concentration in biofilm) x Vtotal - (Chl concentration in planktonic) x (Volume of Detached biofilm)

  3. Total chlorophyll in the culture is the sum of the amount of chlorophyll in planktonic cells and in the biofilm.

    Total Chl (µg) = Total Chl Biofilm + Total Chl Planktonic

    Or

    Total Chl (µg) = Total Chl Biofilm Corrected + Total Chl Planktonic

  4. The extent of biofilm formation is presented as percent of chlorophyll in planktonic cells.

    Chl in suspension (%) = (Total Chl Planktonic)/(Total Chl) x 100%

Notes

  1. All work with cyanobacterial cultures is carried out under sterile conditions using a laminar flow hood.
  2. The sponge plug is punched using an awl.
  3. For bubbling of small number of cultures, a mini aquarium pump (e.g., JBL ProAir a50, Art. No. 6054600) may be used instead of a compressor. As an alternative to mixing air and CO2 one may purchase 3% CO2 cylinders.
  4. A previous study revealed that a biofilm inhibitory substance is present in extracellular fluids from wild type culture as well as in old cultures of the biofilm forming mutant, T2SEΩ (Schatz et al., 2013). To avoid possible inhibitor accumulation in cultures of biofilm-forming mutants, cultures are initiated by inoculation of cells grown from solid agar. Make sure to dilute these starter cultures for ‘biofilm quantification assays’ while yet at exponential phase of growth.
  5. Starter cultures are inoculated from cultures grown on solid agar. Due to variations in the inoculum, the resulting liquid cultures vary in cell density. The dilution steps are important to ensure that experiments are initiated at the same density and a similar physiological state.
  6. First, take an aliquot from the upper part of the suspended culture and then remove the rest of the suspended fraction to avoid contamination of the extraction aliquot with biofilm cells.

Recipes

  1. Liquid BG11-medium (based on Stanier et al., 1971)
    1. Stock I, 100x concentrated (autoclave)
      150.00 g/L NaNO3
      6.50 g/L MgSO4·7H2O
      3.60 g/L CaCl2·2H2O
    2. Stock II, 100x concentrated (autoclave)
      3.05 g/L K2HPO4
      0.10 g/L Na2Mg·EDTA
    3. Stock III, 100x concentrated (prepare fresh, no need to sterilize)
      0.60 g/L C6H11FeNO7
      0.60 g/L C6H8O7
    4. Stock V, 1,000x concentrated (sterilize by filtration using Filter Stericup [0.22 μm])
      2.86 g/L H3BO3
      1.84 g/L MnCl2·4H2O
      0.22 g/L ZnSO4·7H2O
      0.39 g/L NaMoO4·2H2O
      0.08 g/L CuSO4·5H2O
      0.05 g/L Co(NO3)2·6H2O
    5. HEPES 119.15 g/L (25x concentrated) titrated to pH 8.0 with 10 N NaOH (399.97 g/L) (autoclave)
    6. Preparation of 1 L liquid-BG11:
      Start with ~500 ml DDW, add the required volume from the stock solutions and complete with DDW up to 1 L. Solution is sterilized by autoclaving (sterilization time: 40 min). Immediately remove from autoclave once the cycle is over (Long exposure to the high temperature may affect precipitation of minerals). Do not autoclave the medium more than once. Mix before aliquoting the medium to growth tubes
      Note: To reproducibly observe biofilm formation, it is crucial to use freshly made stock III each time liquid BG11 is prepared. Autoclaved BG11 is stored at room temperature. The medium should be used within 4 days to inoculate cultures.
  2. Solid BG11-medium
    1. Stock solutions as for liquid BG11
    2. Sodium bicarbonate 42 g/L (100x concentrated)
    3. Sodium thiosulfate 470 g/L (100x concentrated)
    4. Prepare two-fold concentrated liquid BG11 (BG11X2) by diluting appropriate volumes from stock solutions
    5. Prepare 3% Bacto agar in DDW
    6. Autoclave BG11X2 and 3% agar in separate containers
    7. Immediately following sterilization, combine equal volumes of BG11X2 and 3% agar to get 1.5% agar in BG11
    8. Cool to ~50-55 °C, dilute 100-fold NaHCO3 and Na2S2O3 stock solutions (at this step antibiotics may be added):
      Spectinomycin dihydrochloride pentahydrate (final concentration: 50 μg/ml)
      Kanamycine sulphate monohydrate (final concentration: 50 μg/ml)
      Gentamycin sulphate (final concentration: 7.5 μg/ml)
      Chloramphenicol (final concentration: 7.5 μg/ml)
    9. Pour ~50 ml into 90 mm Petri-dish

Acknowledgments

This protocol is modified from previous studies (Schatz et al., 2013; Parnasa et al., 2016). Growth medium is based on the protocol by (Stanier et al., 1971). Chlorophyll measurements are based on previous protocols (Arnon, 1949; Ritchie, 2006). Rakefet Schwarz and Susan Golden are supported by the program of the National Science Foundation and the US-Israel Binational Science Foundation (NSF-BSF 2012823). This study was also supported by a grant from the Israel Science Foundation (ISF 1406/14) to Rakefet Schwarz.

References

  1. Agostoni, M., Waters, C. M. and Montgomery, B. L. (2016). Regulation of biofilm formation and cellular buoyancy through modulating intracellular cyclic di-GMP levels in engineered cyanobacteria. Biotechnol Bioeng 113(2): 311-319.
  2. Arnon, D. I. (1949). Copper enzymes in isolated chloroplasts. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in beta vulgaris. Plant Physiol 24(1): 1-15.
  3. Enomoto, G., Ni Ni, W., Narikawa, R. and Ikeuchi, M. (2015). Three cyanobacteriochromes work together to form a light color-sensitive input system for c-di-GMP signaling of cell aggregation. Proc Natl Acad Sci U S A 112(26): 8082-8087.
  4. Enomoto, G., Nomura, R., Shimada, T., Ni Ni, W., Narikawa, R. and Ikeuchi, M. (2014). Cyanobacteriochrome SesA is a diguanylate cyclase that induces cell aggregation in Thermosynechococcus. J Biol Chem 289(36): 24801-24809.
  5. Fisher, M. L., Allen, R., Luo, Y. and Curtiss, R., 3rd (2013). Export of extracellular polysaccharides modulates adherence of the Cyanobacterium synechocystis. PLoS One 8(9): e74514.
  6. Jittawuttipoka, T., Planchon, M., Spalla, O., Benzerara, K., Guyot, F., Cassier-Chauvat, C. and Chauvat, F. (2013). Multidisciplinary evidences that Synechocystis PCC6803 exopolysaccharides operate in cell sedimentation and protection against salt and metal stresses. PLoS One 8(2): e55564.
  7. Nagar, E. and Schwarz, R. (2015). To be or not to be planktonic? Self-inhibition of biofilm development. Environ Microbiol 17(5): 1477-1486.
  8. Oliveira, P., Pinto, F., Pacheco, C. C., Mota, R. and Tamagnini, P. (2015). HesF, an exoprotein required for filament adhesion and aggregation in Anabaena sp. PCC 7120. Environ Microbiol 17(5): 1631-1648.
  9. O’Toole, G. A. and Kolter, R. (1998). Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol Microbiol 28(3): 449-461.
  10. Parnasa, R., Nagar, E., Sendersky, E., Reich, Z., Simkovsky, R., Golden, S. and Schwarz, R. (2016). Small secreted proteins enable biofilm development in the cyanobacterium Synechococcus elongatus. Sci Rep 6: 32209.
  11. Ritchie, R. J. (2006). Consistent sets of spectrophotometric chlorophyll equations for acetone, methanol and ethanol solvents. Photosynth Res 89(1): 27-41.
  12. Schatz, D., Nagar, E., Sendersky, E., Parnasa, R., Zilberman, S., Carmeli, S., Mastai, Y., Shimoni, E., Klein, E., Yeger, O., Reich, Z. and Schwarz, R. (2013). Self-suppression of biofilm formation in the cyanobacterium Synechococcus elongatus. Environ Microbiol 15(6): 1786-1794.
  13. Schwarzkopf, M., Yoo, Y. C., Huckelhoven, R., Park, Y. M. and Proels, R. K. (2014). Cyanobacterial phytochrome2 regulates the heterotrophic metabolism and has a function in the heat and high-light stress response. Plant Physiol 164(4): 2157-2166.
  14. Stanier, R. Y., Kunisawa, R., Mandel, M. and Cohen-Bazire, G. (1971). Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 35(2): 171-205.
  15. Zavřel, T., Sinetova, M.A., and Červený, J. (2015). Measurement of chlorophyll a and carotenoids concentration in Cyanobacteria. Bio-protocol 5: 1-5.

简介

最近报道了蓝细菌聚球蓝细菌PCC 7942中生物膜发育的自我抑制机制。 这些研究需要定量由生物膜抑制过程中受损的突变体形成的生物膜。 在这里,我们详细描述了叶绿素测量作为生物膜形成菌株无菌和浮游细胞中生物量积累的代用途。 这些测量可以通过叶绿素水平估计的总生物量进行定量,并通过描绘浮游细胞中叶绿素的相对分数来表示生物膜形成的程度。
【背景】几个最近发表的研究表明,蓝藻中细胞聚集和生物膜发育的基础的机制的新兴兴趣(Fisher等人,2013; Jittawuttipoka等人,2013; 2014; Enomoto等人,2014; Schwarzkopf等人,2014; Enomoto等人,,2015; Oliveira等人,2015; Agostoni等人,2016; Parnasa等人,2016)。我们最近报道了一种自生物膜抑制机制,其决定了单细胞蓝细菌聚球蓝细菌PCC 7942(Schatz等人,2013; Nagar和Schwarz,2015)的浮游生长)。通过灭活特定基因来消除生物膜抑制过程导致在这种其他浮游菌株(Schatz等人,2013; Nagar和Schwarz,2015)中的强大的生物膜发育。这些研究需要量化各种菌株和不同条件下生物膜发育的程度。结晶紫通常用于定量异养细菌中的生物膜(O'Toole和Kolter,1998)。然而,该染色程序仅量化细胞的固着部分。在这里,我们提供了使用叶绿素测量作为无菌和浮游细胞中生物量积累代用品的蓝藻生物膜的培养生长和定量的详细方案。这些测量可以估计总生物量的积累以及在无柄或浮游细胞中叶绿素相对分数的表示。

关键字:生物膜, 蓝藻, 细长聚球藻, 叶绿素测定, 无柄, 浮游

材料和试剂

  1. 定制的用于细菌液体培养的Pyrex玻璃管(200×32mm,由Pyrex管,Corning制造,目录号:8510-32-D)
    注意:可以从 http:// www.degroot.co.il /
  2. “海绵塞”(塑料泡沫塞,27 x 34毫米)(Jaece Industies,目录号:L800-C)
  3. 巴斯德移液器(230毫米)(Romical,目录号:94-08401002)
  4. 0.45μm注射器过滤器(Sartorius,目录号:16555)
  5. 过滤器Stericup-GP 250 ml Express Plus PES(0.22μm)(EMD Millpore,目录号:SCGPU05RE)
  6. 硅胶管(6×9mm,4×6mm)(Degania Silicone,目录号:2110600234,2110400434)
  7. 无菌移液器1和25 ml
    1 ml移液器(Corning,Costar ®,目录号:4011)
    25毫升移液器(康宁,Costar ®,目录号:4489)
  8. 灭菌移液器吸头(Corning,Axygen ®,目录号:T-200-C,T-1000-C)
  9. Eppendorf管(1.5ml)(Corning,Axygen ,目录号:MCT-175-C)
  10. 细胞聚球细胞PCC7942
  11. 硝酸钠(NaNO 3)(Sigma-Aldrich,目录号:S8170)
  12. 七水硫酸镁(MgSO 4·7H 2 O)(Merck,目录号:K26364082)
    注意:本产品已停产。或者,可以使用默克,目录号:105886。
  13. 氯化钙二水合物(CaCl 2·2H 2 O)(ICN,目录号:10035-04-8)
    注意:本产品已停产。或者,可以使用Bio-Lab,目录号:034205。
  14. 磷酸氢二钾(K 2 H 2 HPO 4)(Honeywell International,Riedel-de-Haen,目录号:04248)
  15. 乙二胺四乙酸(Na 2 Mg·EDTA)(Bio-Lab,目录号:05142359)
    注意:本产品已停产。或者,可以使用Biosolve,目录号:051423。
  16. 柠檬酸铁铵(C 6 H 11 11 FeNO 7)(MP Biomedicals,目录号:02158040)
  17. 柠檬酸(C 6 H 8 O 7)(Frutarom,目录号:2355511000)
    注意:本产品已停产。或者,可以使用Biosolve,目录号:030205。
  18. 硼酸(H 3 O 3 BO 3)(Bio-Lab,目录号:02010591)
    注意:本产品已停产。或者,可以使用Biosolve,目录号:020105。
  19. 六水合氯化锰(MnCl 2·6H 2 O)(Duchefa Biochemie,目录号:M0533)
  20. 硫酸锌七水合物(ZnSO 4·7H 2 O)(CARLO ERBA试剂,目录号:494907)
  21. 钼酸钠二水合物(Na 2 MoO 4·2H 2 O)(MP Biomedicals,目录号:194863)
  22. 五水硫酸铜(CuSO 4·5H 2 O)(Honeywell International,Riedel-de-Haen,目录号:12849)
  23. 硝酸钴(II)六水合物(Co(NO 3 3)2·6H 2 O)(Honeywell International,Riedel-de-Haen,catalog号码:12922)
    注意:本产品已停产。或者,可以使用Sigma-Aldrich,目录号:239267。
  24. N-2-羟乙基哌嗪-N'-2-乙磺酸(HEPES)(Sigma-Aldrich,目录号:H3375)
  25. 氢氧化钠(NaOH)(Frutarom,目录号:5553510)
  26. 丙酮((CH 3 3)2 CO),A.C.S.试剂(Avantor Performance Materials,J.T.Baker ,目录号:9006-03)
  27. 碳酸氢钠(NaHCO 3)(DAEJUNG CHEMICAL& METALS,目录号:7566-4100)
  28. 硫代硫酸钠(Na 2 S 2 O 3 O 3)(Sigma-Aldrich,目录号:217247)
  29. Bacto琼脂(BD,Bacto TM,目录号:214010)
  30. LB琼脂(Lennox)(BD,Difco TM,目录号:240110)
  31. 抗生素(根据特定菌株的耐药性适当添加)
    1. 倍他霉素二盐酸盐五水合物(Duchefa Biochemie,目录号:S0188)
    2. 硫酸卡那霉素一水合物(Duchefa Biochemie,目录号:K0126)
    3. 硫酸庆大霉素(Duchefa Biochemie,目录号:G0124)
    4. 氯霉素(Duchefa Biochemie,目录号:C0113)
  32. 液体BG11-介质(参见食谱)
    1. 库存我(100倍集中)
    2. 股票II(100倍集中)
    3. 股票III(100倍集中)
    4. 股票V(1,000倍集中)
  33. 固体BG11-培养基(参见食谱)

设备

  1. 层流罩
  2. 通风柜
  3. 高压灭菌器
  4. 分光光度计(Agilent Technologies,型号:Cary 100,目录号:10069000)和各自的比色杯(细胞类型P.L = 10mm / EA)(Starna Cells,目录号:9-SOG-10)
  5. 台式离心机(MiniSpin,最大离心力:12,100×g)(Eppendorf,型号:MiniSpin,目录号:5452000018)
  6. 冰箱(4-7°C)
  7. 生长室(液体培养物分别为30±2℃和24±2℃,分别在固体培养基上培养) 注意:在24°C而不是30°C的固体培养基上培养培养物可以使文化保持更长时间。
  8. 光源
    对于液体培养物 - 产生20-30μmol光子的光通量的白炽灯m sec -1 ;固体BG11上的培养物用荧光灯照射(5μmol光子m -2 sec -1
  9. 吸管
  10. 单通道移液器200和1,000μl
  11. 鼓泡系统
    1. 空气压缩机(无油空气压缩机)(Assouline压缩机,型号:vs 204 50)
    2. 气瓶二氧化碳(CO 2%,化合物99.5%)(Maxima,目录号:GCDCU2.527)
    3. 流量计(Tuttnauer公司,0-0.1L / min,1-10L / min)
    4. 高流量气压调节器(0-2 PSI)(Marsh Bellofram,型号70,型号:960-129-000)
    注意:要使用3%CO 2 在空气中鼓泡文化,使用以下设置(图1):CO 在空气中。通过在2L瓶中鼓泡入1.5L双蒸水(DDW)将该混合物加湿,以防止在培养物鼓泡期间水分蒸发。该瓶子的出口通过一个空的2升瓶(用于捕集残留液体),并通过空气压力调节器(200毫巴适用于〜300培养管的鼓泡)。使用由硅胶管和合适的T形棒连接器构成的自制歧管,将富集空气从后者进入多种文化的CO 。每个歧管通道中的气体通过0.45μm过滤器(图1)。提供了一个简短的视频来演示各个培养管中的流速(视频1)。



    图1.描述用CO气泡培养的设置的方案 2 <空>

    Video 1. Cyanobacterial cultures under bubbling

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player

程序

  1. 生长细胞聚球细胞PCC 7942及其生物膜形成突变体的液体培养物
    1. 圆底Pyrex管(长20厘米,直径3厘米)用作生长血管。
    2. 将一根棉塞巴斯德吸管(23厘米长)插入海绵塞中,使得巴斯德吸管的尖端位于管底部1-2厘米处(图2A,“生长组”)。
    3. 生长组在液体循环中进行高压灭菌
    4. 将无菌液体BG11-培养基(25-50ml)(参见食谱)转移到Pyrex管中。将培养体积调整为指定实验所需的生物量。
    5. 细胞用固体BG11培养基(参见食谱)用1毫升无菌塑料移液器刮擦,生物质在液体培养基中摇动(见附注4)。
      重要提示: 。高压灭菌BG11应在4天内用于接种培养物。
    6. 通过1毫升移液器吸头连接生长系统到冒泡系统(见上文)(图2A)
    7. 使用歧管允许同时鼓泡多种培养物(图2B,参见设备下的注释)。


      图2.生长的蓝藻培养物。 :一种。生长组合连接到单个歧管通道; B.成长室中的文化。

  2. 生物膜的定量
    1. 在连续照射下,在连续照射下,在白炽灯(20-30μmol光子),在30℃下在空气中连续鼓泡3%CO 2 / -1 )。
    2. 指数阶段的起始培养物经稀释(通常在3天生长后)至750nm处的光密度为0.5。
    3. 通过将培养物的另外稀释开始于750nm的光密度为0.5(参见附注5)。
    4. 此设置下的生物膜开发通常在2-3天后开始。
    5. 为了验证培养物是否为固体LB琼脂上的20μl等分试样的“斑点”。
    6. 悬浮细胞中叶绿素的百分比用于定量生物膜形成如下(图3):
      1. 在1.5ml Eppendorf管中进行浮游细胞中叶绿素的提取。从悬浮培养物的上部取出等分试样(见附注6)。在悬浮部分看起来特别密集(浮游菌株或不良生物膜形成物)的情况下,使用0.2ml样品进行提取。当形成坚固的生物膜时,在80%丙酮(最终浓度)中叶绿素提取之前,通过离心(5,000×g,10分钟)除去15ml浮游细胞并浓缩3至6倍。
      2. 生物膜中叶绿素的提取在生长管中进行。为了分离生物膜部分,缓慢除去悬浮细胞。生物膜倾向于在去除最后几毫升液体期间塌陷。因此,一旦生物膜结块分解并落入液体,就停止去除培养物。使用移液管(在我们手中留下2-5毫升)以及从培养物中取出的体积测量管中残留的体积。这些体积的总和(V total )用于计算悬浮细胞中叶绿素的量(参见数据分析)。在80%丙酮中进行叶绿素的提取,其最终体积等于V总计
      3. 叶绿素的提取在冰箱中进行过夜,叶绿素根据先前描述的吸光度进行定量(Arnon,1949; Ritchie,2006)。叶绿素可用甲醇代替丙酮萃取。 Zavřel等人(2015)描述了用于测量蓝细菌中叶绿素a浓度的逐步方案。


        图3.基于叶绿素测量的生物膜的定量

数据分析

悬浮液中叶绿素的百分比由浮游生物膜和生物膜组分中的叶绿素含量计算。这些值是根据测量的叶绿素吸光度,样品体积和稀释因子计算的,如下所述。表1中提供了一个示例计算。

表1.浮游生物膜细胞中叶绿素(Chl)的计算示例

  1. 根据测定的叶绿素浓度和悬浮培养物的体积计算悬浮细胞中叶绿素的含量(V )。
    总Chl浮游生物(μg)=(OD 663 - OD 750)×12.7×(稀释因子)×V

  2. 根据测定的叶绿素浓度和丙酮提取的体积计算生物膜中叶绿素的含量,该浓度应为V
    1. 如果观察到健壮的生物膜形成,则生物膜部分中剩余的浮游细胞的贡献可忽略不计,总叶绿素计算如下:

      总Chl生物膜(μg)=(OD 663 750)×12.7×(稀释因子)×V

    2. 为了解释生物膜部分中剩余的浮游细胞的贡献,从以上值减去部分体积中存在的浮游叶绿素:

      总Chl生物膜校正(μg)=(生物膜中的Ch1浓度)x V总计 - (浮游生物中的Chl浓度)x(分离生物膜的体积)

  3. 培养中总叶绿素是浮游细胞和生物膜中叶绿素含量的总和。

    总Chl(μg)=总Chl生物膜+总Chl浮游生物



    总Chl(μg)=总Chl生物膜修正+总Chl浮游生物

  4. 生物膜形成的程度以浮游细胞中叶绿素的百分比表示。

    悬浮液中的Chl(%)=(总Chl浮游生物)/(总Chl)×100%

笔记

  1. 所有使用蓝藻培养的工作都是在无菌条件下使用层流罩进行的。
  2. 海绵塞用锥子冲压。
  3. 为了冒泡少量的培养物,可以使用迷你水族馆泵(例如,例如JBL ProAir a50,Art.No.6054600)代替压缩机。作为混合空气和CO 2的替代方案可以购买3%CO 2气瓶。
  4. 以前的研究表明,生物膜抑制物质存在于野生型培养物以及生物膜形成突变体T2SEΩ(Schatz等人,2013)的旧培养物的细胞外液中。为了避免在生物膜形成突变体的培养物中可能的抑制剂积累,通过接种从固体琼脂生长的细胞来启动培养物。确保稀释这些起始培养物的“生物膜定量测定”,而在生长的指数阶段。
  5. 起始培养物从在固体琼脂上生长的培养物接种。由于接种物的变化,所得液体培养物的细胞密度变化。稀释步骤对于确保在相同密度和类似生理状态下开始实验是重要的。
  6. 首先,从悬浮培养物的上部取出等分试样,然后除去其余的悬浮液,以避免用生物膜细胞污染提取等分试样。

食谱

  1. 液体BG11-介质(基于Stanier等人,1971)
    1. 库存I,100x浓缩(高压釜)
      150.00g / L NaNO 3
      6.50g / L MgSO 4·7H 2 O
      3.60g / L CaCl 2·2H 2 O
    2. 库存二,100倍浓缩(高压釜)
      3.05g / L K 2 HPO 4
      0.10g / L Na 2 Mg·EDTA
    3. 库存III,100x浓缩(准备新鲜,不需要消毒)
      0.60g / L C 6 H 11 11 FeNO 7
      0.60g / L C 6 H 8 O 7
    4. 库存V,1,000倍浓缩(使用Filter Stericup过滤灭菌[0.22μm])
      2.86g / L H 3 3&lt; 3&lt; 3&gt;
      1.84g / L MnCl 2·4H 2 O
      0.22g / L ZnSO 4·7H 2 O
      0.39g / L NaMoO 4·2H 2 O 0.08g / L CuSO 4·5H 2 O
      0.05g / L Co(NO 3 3)2·6H 2 O
    5. 用10N NaOH(399.97g / L)(高压釜)滴定至119℃的HEPES 119.15g / L(浓缩25倍)。
    6. 1L液体BG11的制备:
      从约500ml DDW开始,从原液中加入所需体积,并加入DDW至1L。溶液用高压灭菌灭菌(灭菌时间:40分钟)。一旦循环结束,立即从高压釜中取出(长时间暴露于高温可能会影响矿物的沉淀)。不要多次高压灭菌介质。将培养基等分成生长管之前混合 注意:为了可重复地观察生物膜形成,每当制备液体BG11时,使用新鲜的原料III是至关重要的。高压釜BG11储存在室温下。培养基应在4天内用于接种培养物。
  2. 固体BG11-介质
    1. 液体BG11的库存解决方案
    2. 碳酸氢钠42g / L(100x浓缩)
    3. 硫代硫酸钠470 g / L(100x浓缩)
    4. 通过从储备溶液
      稀释适量的液体,制备双重浓缩液BG11(BG11X2)
    5. 在DDW中准备3%Bacto琼脂
    6. 将BG11X2和3%琼脂高压釜放置在不同的容器中
    7. 灭菌后立即加入等量的BG11X2和3%琼脂,以获得BG11中的1.5%琼脂。
    8. 冷却至〜50-55℃,稀释100倍NaHCO 3和Na 2 S 2 O 3 O 3 /储备溶液(在这一步可以添加抗生素):
      望角霉素二盐酸盐五水合物(终浓度:50μg/ ml)
      硫酸卡那霉素一水合物(终浓度:50μg/ ml)
      硫酸庆大霉素(终浓度:7.5μg/ ml)
      氯霉素(终浓度:7.5μg/ ml)
    9. 将90毫升培养皿倒入50毫升

致谢

该协议是从以前的研究(Schatz等人,2013; Parnasa等人,2016)修改的。生长培养基基于(Stanier等人,1971)的方案。叶绿素测量基于以前的方案(Arnon,1949; Ritchie,2006)。 Rakefet Schwarz和Susan Golden由美国国家科学基金会和美国 - 以色列两岸科学基金会(NSF-BSF 2012823)的计划提供支持。这项研究也得到以色列科学基金会(ISF 1406/14)给Rakefet Schwarz的资助。

参考

  1. Agostoni,M.,Waters,CM和Montgomery,BL(2016)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/26192200”target = “_blank”>通过调节工程蓝细菌中的细胞内环状二-GMP水平来调节生物膜形成和细胞浮力。 Biotechnol Bioeng 113(2):311-319。
  2. Arnon,D.I。(1949)。孤立叶绿体中的铜酶。&nbsp; 孤立叶绿体中的铜酶。寻常型的多酚氧化酶。植物生理学24(1):1-15。
  3. Enomoto,G.,Ni Ni,W.,Narikawa,R。和Ikeuchi,M。(2015)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih。 gov / pubmed / 26080423“target =”_ blank“>三种蓝细菌共同作用,形成用于细胞聚集的c-di-GMP信号传导的光色敏感输入系统。 Proc Natl Acad Sci USA em> 112(26):8082-8087。
  4. Enomoto,G.,Nomura,R.,Shimada,T.,Ni Ni,W.,Narikawa,R。和Ikeuchi,M。(2014)。&lt; a class =“ke-insertfile”href =“http: //www.ncbi.nlm.nih.gov/pubmed/25059661“target =”_ blank“>蓝细菌色素SesA是一种二氢叶酸环化酶,其诱导热球感球菌中的细胞聚集。 Biol Chem 289(36):24801-24809。
  5. Fisher,ML,Allen,R.,Luo,Y.和Curtiss,R.,3rd(2013)。&nbsp; 细胞外多糖的出口调节了蓝藻属细胞囊肿的粘附。 PLoS One 8(9):e74514。
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  7. Nagar,E.和Schwarz,R。(2015)。环境微生物 17(5):1477-1486。
  8. Oliveira,P.,Pinto,F.,Pacheco,CC,Mota,R.and Tamagnini,P。(2015)。 HesF,Anabaena中的细丝粘附和聚集所需的外蛋白。 PCC 7120. 环境微生物 17(5):1631-1648。
  9. O'Toole,GA和Kolter,R。(1998)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/9632250”target =“_ blank “>在荧光假单胞菌中起始生物膜形成WCS365通过多个收敛信号途径进行:遗传分析.Mol Microbiol 28(3):449-461 。
  10. Parnasa,R.,Nagar,E.,Sendersky,E.,Reich,Z.,Simkovsky,R.,Golden,S。和Schwarz,R。(2016)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/27558743”target =“_ blank”>小分泌蛋白质可使蓝细菌细菌聚球藻生物膜发育。 < em> Sci Rep 6:32209.
  11. Ritchie,RJ(2006)。&nbsp; 一致的分光光度法叶绿素丙酮,甲醇和乙醇溶剂的方程式。

    89(1):27-41。
  12. Schatz,D.,Nagar,E.,Sendersky,E.,Parnasa,R.,Zilberman,S.,Carmeli,S.,Mastai,Y.,Shimoni,E.,Klein,E.,Yeger, Reich,Z.和Schwarz,R。(2013)。&nbsp; 蓝藻细菌生物膜形成的自我抑制 环境微生物15(6):1786-1794。
  13. Schwarzkopf,M.,Yoo,YC,Huckelhoven,R.,Park,YM and Proels,RK(2014)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih蓝色细菌植物色素2调节异养代谢,并具有热和高光应激反应的功能。植物生理学164(4) :2157-2166。
  14. Stanier,RY,Kunisawa,R.,Mandel,M.和Cohen-Bazire,G.(1971)。单细胞蓝绿色藻类的纯化和性质(顺序Ch oc 35(2) :171-205。
  15. Zavřel,T.,Sinetova,MA和Červený,J。(2015)。&nbsp; 测定蓝细菌中的叶绿素a和类胡萝卜素浓度。生物方案5:1-5。
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引用:Sendersky, E., Simkovsky, R., Golden, S. S. and Schwarz, R. (2017). Quantification of Chlorophyll as a Proxy for Biofilm Formation in the Cyanobacterium Synechococcus elongatus. Bio-protocol 7(14): e2406. DOI: 10.21769/BioProtoc.2406.
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