Analysis of Metals in Whole Cells, Thylakoids and Photosynthetic Protein Complexes in Synechocystis sp. PCC6803

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New Phytologist
Jul 2017



Metals are essential in many biological processes, including oxygenic photosynthesis. Here we described a method to measure the metal pool in whole cells and thylakoids, including the bioactive pool in intact photosynthetic protein complexes in the model oxygenic cyanobacterium Synechocystis PCC6803. In the first part of the protocol, whole cells and thylakoid membranes are carefully prepared, in which the total metal concentrations are measured by inductively coupled plasma triple-quadrupole mass spectrometry (ICP-QQQ-MS). In the second part of the protocol, isolated thylakoids are solubilized to release the integral membrane proteins and the metal binding protein complexes. These intact photosynthetic protein complexes are subjected to size exclusion chromatography (SEC) and metal binding in the size separated complexes is analyzed by hyphenation with ICP-QQQ-MS.

Keywords: Cyanobacteria (蓝藻), Synechocystis (集胞藻), Manganese (锰), Iron (铁), Magnesium (镁), Thylakoid membranes (类囊体膜), Speciation analysis (形态分析), ICP-MS (ICP-MS)


The process of oxygenic photosynthesis requires metals due to their essential functions as cofactors and catalysts in the photosynthetic electron transport chain. The photosynthetic apparatus requires iron (Fe) in the form of either Fe-S clusters, heme-bridged Fe and non-heme Fe, copper (Cu) in the soluble mobile electron carrier protein plastocyanin, magnesium (Mg) in chlorophylls, calcium (Ca) and manganese (Mn) in the oxygen evolving complex of photosystem II (PSII). Tight control of metal allocation inside photosynthetic cells is essential for cell survival since an imbalanced metal accumulation induces mismetallation and inactivation of the various metallo-enzymes. An accurate analysis of the concentration and allocation of metals in photosynthetic cells is therefore important to investigate the role of key factors and proteins involved in metal homeostasis. While the method described in Brandenburg et al. (2017) accurately quantifies the periplasmic and intracellular pools of Mn in Synechocystis cells, the protocol presented here provides a wider overview, quantifying metals in whole Synechocystis cells, isolated thylakoids, and the bioactive metal pool in fractionated photosynthetic complexes. This protocol is partially based on the method described in Schmidt et al. (2015) for barley thylakoids.

Materials and Reagents

  1. Pipette tips
  2. 50 ml conical tubes (Greiner Bio One International, catalog number: 227261 )
  3. 1.5 and 2 ml Eppendorf tubes (Eppendorf, catalog numbers: 0030120086 and 0030120094 , respectively)
  4. Microwave Teflon tubes, 8 ml (VWR, catalog number: 525-0178 )
  5. Nylon membrane filters, 0.45 µm (Frisenette, catalog number: 13NY045-100 )
  6. Syringe, 1 ml (Frisenette, catalog number: 9161406 )
  7. NanoVipers fingertight fittings, 150 µm (Thermo Fisher Scientific, catalog number: 6041.5820 )
  8. Synechocystis sp. PCC6803 GT (glucose tolerant strain, from Dr. Himadri Pakrasi laboratory, Department of Biology, Washington University, St Louis, MO, USA)
  9. Multi-metal calibration standards for ICP-MS calibration (CPI International, catalog numbers: P/N4400-132565A and P/N4400-132565B )
  10. EDTA, BioUltra (Sigma-Aldrich, catalog number: E1644-100G )
  11. Tricine (Sigma-Aldrich, catalog number: T9784-25G )
  12. Lysozyme (Sigma-Aldrich, catalog number: 62971-50G-F )
  13. Sucrose, BioExtra (Sigma-Aldrich, catalog number: S7903-1KG )
  14. Sodium chloride (NaCl), BioExtra (Sigma-Aldrich, catalog number: S7653-250G )
  15. Magnesium chloride (MgCl2·7H2O), BioExtra (Sigma-Aldrich, catalog number: M2670-1KG )
  16. Na-ascorbate, BioExtra (Sigma-Aldrich, catalog number: 11140-250G )
  17. Sodium fluoride (NaF), BioExtra (Sigma-Aldrich, catalog number: S7920-100G )
  18. Liquid nitrogen
  19. PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, catalog number: 23225 )
  20. Glass beads (Sigma-Aldrich, catalog number: G4649 )
  21. Milli-Q water
  22. PlasmaPURE 67-69% HNO3 (SCP SCIENCE, catalog number: 250-039-175 )
  23. 30% H2O2 (prepare 15% H2O2 with Milli-Q water) (Sigma-Aldrich, catalog number: 31642 )
  24. n-Dodecyl-β-D-Maltopyranoside (β-DM) (Anatrace, catalog number: D310LA )
  25. Bis-Tris, BioExtra (Sigma-Aldrich, catalog number: B7535-100G )
  26. Glycerol, BioExtra (Sigma-Aldrich, catalog number: G6279 )
  27. Betaine, BioUltra (Sigma-Aldrich, catalog number: 61962-250G )
  28. Pefabloc SC (Roche Diagnostics, catalog number: 11429868001 )
  29. Apple leaves, standard reference material (Sigma-Aldrich, catalog number: NIST1515 )
  30. Glucose (Sigma-Aldrich, catalog number: G8270-1KG )
  31. Na2S2O3·5H2O (Merck, catalog number: 1.06516.0500 )
  32. FeNH4 citrate (MP Biomedicals, catalog number: 02158040 )
  33. Na2CO3 (Sigma-Aldrich, catalog number: S7795-500G )
  34. K2HPO4 (Sigma-Aldrich, catalog number: P8281 )
  35. NaNO3 (Sigma-Aldrich, catalog number: S8170-250 )
  36. MgSO4 (Sigma-Aldrich, catalog number: M2643-500G )
  37. CaCl2 (Sigma-Aldrich, catalog number: C5670-500G )
  38. Citric acid (Sigma-Aldrich, catalog number: 251275-100G )
  39. Na2-EDTA (Sigma-Aldrich, catalog number: E4884-500G )
  40. H3BO3 (Sigma-Aldrich, catalog number: B6768-500G )
  41. MnCl2 (Sigma-Aldrich, catalog number: 244589-50G )
  42. ZnSO4 (Sigma-Aldrich, catalog number: Z0251-100G )
  43. NaMoO4 (Sigma-Aldrich, catalog number: M1003-100G )
  44. CuSO4 (Sigma-Aldrich, catalog number: 209198-100G )
  45. Co(NO3)2 (Alfa Aesar, catalog number: 36418.22 )
  46. BG11-G (see Recipes)
  47. 100x BG-FPC (see Recipes)
  48. 1,000x trace element stock (see Recipes)
  49. FeNH4 citrate stock (see Recipes)
  50. Na2CO3 stock (see Recipes)
  51. K2HPO4 stock (see Recipes)
  52. EDTA washing buffer (see Recipes)
  53. Tricine buffer (see Recipes)
  54. Lysozyme solution (see Recipes)
  55. Homogenization buffer (see Recipes)
  56. Tricine-NaF buffer (see Recipes)
  57. Storage buffer (see Recipes)
  58. Solubilization buffer (see Recipes)
  59. Detergent solution (see Recipes)
  60. Mobile phase buffer (see Recipes)


  1. Pipettes
  2. Erlenmeyer flasks, 2 L (Fisher Scientific, catalog number: 11961566)
    Manufacturer: Pyrex, catalog number: 1130/30D .
  3. Orbital shaker (Eppendorf, New BrunswickTM, model: Innova® 2150, catalog number: M1194-0010 )
  4. Eppendorf centrifuge 5418 R, refrigerated (Eppendorf, model: 5418 R, catalog number: 5401000013 )
  5. Eppendorf centrifuge 5430 R, refrigerated (Eppendorf, model: 5430 R, catalog number 5428000210 )
  6. Beckman Coulter centrifuge (Beckman Coulter, model: Avanti® J-25 ) with type 19 rotor, fixed angle
  7. 250 ml bottles (Beckman Coulter, catalog number: 325620 )
  8. Microfluidizer Processor (Microfluidics, model: M-110L )
  9. Light microscope (ZEISS, model: Axiovert 135 TV )
  10. Bead mill TissueLyser II (QIAGEN, catalog number: 85300 )
  11. TissueLyser II adapter set (QIAGEN, catalog number: 69982 )
  12. Heraeus Fresco 21 Microcentrifuge, refrigerated (Thermo Fisher Scientific, catalog number: 75002425 )
  13. HPLC Microvials PP, 300 µl and screw cap PP, PFTE (VWR, catalog numbers: 548-0440 and 548-0787 )
  14. Size exclusion column, Biobasic SEC-1000 analytical column (300 x 7.8 mm) and guard column (30 x 7.8 mm), 5 µm particle size, 1000Å pore size (Thermo Fisher Scientific, catalog number: 73605-307846 and 73605-037821 )
  15. Pressurized Ultrawave System (Ultrawave system, Milestone Srl, Sorisole, Italy)
  16. HPLC system, bio-inert (i.e., the mobile phase and samples have no contact with metal parts) (Thermo Fisher Scientific, Dionex, model: UltiMate 3000 )
  17. DionexTM ICS-5000+ DP Dual Pumps (Thermo Fisher Scientific, model: ICS-5000+ DP )
  18. ICP-QQQ-MS (Agilent Technologies, model: 8800 Triple Quadrupole ICP-MS )
  19. Nebulizer, Ari Mist HP (Burgener Research, catalog number: AM HP 5500 )
  20. Gases for ICP: Liquid Argon, grade 5.0, and reaction gas: 20% Oxygen in Argon (AGA, catalog numbers: 107407 and 714039 )


  1. MassHunter 4.2 Workstation software 8800 ICP-QQQ data analysis (Agilent Technologies)


An overview of the entire protocol is given in Figure 1.

Figure 1. Schematic workflow illustrating the determination of the total metal concentrations in whole cells and thylakoids, and the more detailed analysis of metal allocation into photosynthetic complexes of Synechocystis. Capital letters (A-E) indicate the corresponding procedures in the protocol.

  1. Whole cell preparation
    1. Inoculate Synechocystis strains in 0.5 L of BG11-G growth medium (see Recipes), in 2 L Erlenmeyer flasks. Inoculate at least three independent cultures for each strain (biological replicates) to a final 0.05 OD750, following standard microbiological practices.
    2. Incubate the cultures at a light intensity of 50 µmol photons m-2 sec-1, and shake the flasks on an orbital shaker at 100 rpm until the cultures reach the late exponential phase (0.8-0.9 OD750). Applying these growth conditions the cultures should reach the desired density in about 5 days.
    3. Before collecting the cultures, measure the OD750 for each culture.
    4. For each culture, keep aside 50 ml culture for protein concentration determination in whole cells samples (Steps A8-A13).
    5. Pellet the remaining culture, centrifuging at 5,000 x g for 10 min (at room temperature), using the 250 ml bottles suitable for the type 19 rotor of the Beckman Coulter centrifuge. Repeat the centrifugation step to pellet the rest of the culture volume.
    6. Wash the cell pellet with 50 ml of EDTA washing buffer (see Recipes), pellet the cells at 5,000 x g for 10 min at room temperature, discard the supernatant and repeat this washing step two more times (Note 1). The supernatant from the first wash will appear slightly colored (with a yellow-orange shade), whereas the supernatant of the third wash should be clear (Figure 2).
    7. Wash the pellet with 50 ml of Tricine buffer (see Recipes), pellet the cells at 5,000 x g for 10 min, discard the supernatant and repeat this washing step two more times. Before the last centrifugation step, split the cell suspension into two parts, and pellet both cell fractions by centrifugation at 5,000 x g for 10 min at room temperature. One cell pellet constitutes the whole cell preparation ready to be analyzed (see Procedure C). The pellet for the whole cell preparation can be frozen in liquid nitrogen and stored in a freezer until analysis. The second cell pellet is directly further processed to obtain thylakoid membranes (Steps B1-B6).
      Note: The metal concentrations will be normalized on protein concentrations. Hence, protein concentration per cell number needs to be determined. For accurate determination of protein concentration in whole cell samples, complete cell rupture is essential.
    8. Treat the cells collected at Step A4 according to Steps A6-A7 and re-suspend the cell pellets to OD750 1 in Tricine buffer (see Recipes).
    9. Add 1 ml of lysozyme solution (see Recipes) to the cell suspension and gently shake it at room temperature for 30 min. In our hands, this step helps the subsequent rupture.
    10. Repeatedly rupture the cells in a microfluidizer processor, following manufacturer instruction. Seven to eight cycles are usually sufficient to obtain complete cell rupture.
    11. Examine the cells rupture under a light microscope to confirm complete rupture of the cells.
    12. Measure the protein concentration of the samples using the Pierce bicinchoninic acid (BCA) Protein Assay Kit (see Materials and Reagents) for colorimetric detection and quantitation of total protein. The determined protein concentration indicates the µg of proteins in 1 ml of cell lysate, and hence the µg of proteins in 1 ml of culture at 1 OD750.
    13. Calculate the protein concentration of the whole cell fraction collected in Step A7 by multiplying the protein concentration obtained in Step A12 with the OD750 value measured in Step A3 and by the initial culture volume of the collected cells, which in this case was 225 ml (500 ml of initial culture in Step A1 minus the 50 ml put aside in Step A4, and divided by 2 according to Step A7).

      Figure 2. Cell pellet washed three times with EDTA washing buffer. Cell pellets harvested as described in Step A5 are washed three times with the EDTA washing buffer (Step A6). The supernatant of the first wash has a distinct yellow-orange color (1st) which should fade during the second (2nd) and third (3rd) wash. A clear conical tube containing EDTA washing buffer (B) is shown for comparison.

  2. Thylakoids preparation (Note 2)
    Note: In the following steps, samples must be kept on ice, and it is recommended to perform all steps in dim green light to protect the photosynthetic complexes. All centrifugation steps are conducted at 4 °C.
    1. Re-suspend the cell pellets prepared in Steps A1-A7 in 1 ml of homogenization buffer (see Recipes) and transfer the cell suspension to a 2 ml Eppendorf tube holding 1 ml of glass beads.
    2. Rupture cells in the beads mill, using cooled racks (leave the racks 15 min at -20 °C). The beads mill disrupts the cells through high-speed shaking with glass beads. Cell disruption is performed during six cycles of beads beating, where each cycle involves 1 min of beads beating followed by 1 min of incubation on ice.
    3. Separate unbroken cells and glass beads by centrifugation for 2 min at 16,000 x g.
    4. Transfer the supernatant to a 1.5 ml Eppendorf tube and pellet thylakoid membranes for 1 h at 16,000 x g.
    5. Wash thylakoids two times in 500 µl of Tricine-NaF buffer (centrifuge for 20 min, 16,000 x g) and determine protein concentration using the BCA assay kit.
    6. Centrifuge thylakoids for 20 min, 16,000 x g and carefully discard the supernatant. The pellet is either digested for determination of total metal analysis (Procedure C) (Note 3) or re-suspended in storage buffer, frozen in liquid N2, and stored at -80 °C for up to 3 months until preparation for SEC-ICP-QQQ-MS analysis (Procedure D).

  3. Determination of total metal concentrations in whole cells and thylakoid membranes
    Note: Clean consumables and clean lab procedures must be used in all the following steps. It is recommended to include at least one certified reference (apple [Malus domestica] leaves, NIST 1515; National Institute of Standards and Technology) and one true blank in each digestion run.
    1. Thaw the pellets of whole cells and thylakoid membranes (prepared as described in Procedure A and B, respectively) and add 70% (v/v) HNO3 (see Note 4) directly to each sample in the Eppendorf tubes. Samples are pre-digested for two hours (leave the Eppendorf tubes open, loosely covered by a plastic bag to minimize contamination).
    2. Carefully transfer (by pipetting) the pre-digested samples to 8 ml Teflon digestion tubes.
    3. Add 15% (v/v) H2O2 (in a HNO3 to H2O2 ratio of 2.5 to 1, according to Step C1) (see Note 5).
    4. Digest the samples using a pressurized microwave system (e.g., Milestone, according to the detailed description reported in Hansen et al. [2013]).
    5. Transfer samples to a clean tube, rinsing the Teflon digestion tubes with Milli-Q water at least three times.
    6. Dilute samples to a final concentration of 3.5 % HNO3 (v/v) using Milli-Q water (see Note 6). Prepare the calibration standards for the external calibration of the metals of interest (covering a linear range of at least three orders of magnitude and 8 to 10 calibration points).
    7. Analyze the samples including calibration standards by ICP-MS.

  4. Thylakoid preparation for SEC-ICP-QQQ-MS measurements
    Note: In the following steps, samples must be kept on ice, and it is recommended to perform all steps in dim green light to protect the photosynthetic complexes. All centrifugation steps are conducted at 4 °C.
    1. Thaw the thylakoid membrane samples on ice and in darkness.
    2. Take a homogeneous sub-sample (in this example: corresponding to 300 µg of protein, see Step B5) (pipette gently up and down, avoid vortexing).
    3. Pellet the thylakoid membranes in a microcentrifuge by centrifugation at 7,000 x g for 2 min.
    4. Discard the supernatant and re-suspend (by gently pipetting up and down) the thylakoid membranes in 150 µl ice-cold solubilization buffer (freshly prepared, see Recipes) to a final protein concentration of 2 µg/µl.
    5. Add an equal volume of detergent solution (150 µl, see Recipes) to a final protein concentration of 1 µg/µl and solubilize the thylakoid membranes on ice and in darkness for 10 min.
    6. Solubilized proteins (supernatant) are separated from insoluble material (pellet) by centrifugation at 16,000 x g for 10 min.
    7. The solubilized proteins (supernatant) are immediately collected, and passed through a 0.45 µm nylon membrane filter using 1 ml syringes, into HPLC microvials.
    8. Keep samples on ice and in darkness until the subsequent speciation analysis (Procedure E).

  5. SEC-ICP-QQQ-MS measurements: Determination of metal binding in photosynthetic complexes (speciation analysis)
    Note: During speciation analysis, the HPLC autosampler and column compartment are kept cold at 6 °C. This part of the protocol is based on the method developed by Schmidt et al. (2015)
    1. Equilibrate the HPLC system and the size-exclusion column with the mobile phase buffer (see Recipes) for at least 1 h.
    2. Inject 50 µg of freshly solubilized thylakoid proteins onto the size-exclusion column, using the HPLC system.
    3. Perform isocratic protein fractionation and elution using mobile phase buffer at a flow rate of 1 ml/min for 35 min.
    4. For the online detection of metals bound in photosynthetic protein complexes, the column outlet is directly coupled to the ICP-QQQ-MS (Note 7).
    5. Time-resolved chromatograms with multi-element detection are recorded (Figure 3).

      Figure 3. Multi-metal chromatogram. Size-exclusion profiles recorded for the non-oxide ions 24Mg+ and 55Mn+ and the oxide ion 72FeO+. Individual Mn and Fe subfractions are assigned according to Gandini et al. (2017), the Mg profile is illustrated as a proxy for chlorophyll.

Data analysis

  1. Total metal concentrations
    The metal concentrations in whole cells and thylakoid membranes are determined from the external calibration curves for each metal, using the Masshunter software (the software enables automatic calculation of sample concentrations). The data are exported and the metal concentrations are normalized to protein concentrations for each sample. It is recommended to perform two-way analysis of variance (ANOVA) on metal concentrations from at least two independent experiments, each consisting of three independent preparations for each strain of interest, to take into account any variation between experiments. In this respect, we observed that the metal count of a strain differed more between two independent experiments than within independent cultures of the same experiment. This variability could be due to various reasons, such as slightly different log-phases at culture collection.
  2. Speciation analysis
    At least three independent samples for each strain are analyzed by SEC-ICP-MS. As for the total metal concentrations analysis, we strongly recommend analyzing at least two independent experiments. SEC-ICP-MS chromatograms can be individually prepared for each metal of interest, or by overlaying more metals to construct a single multi-metal chromatogram. Prepare the chromatogram for one representative sample. Data are presented as ion counts per second as a function of the retention time. The metal profiles may be background corrected or shown as raw data (Figure 3). Individual peaks are assigned according to Gandini et al. (2017).


  1. EDTA is used to remove the Mn storage pool located in the outer membrane and periplasmic space (Keren et al., 2002).
  2. The thylakoid preparation described here is actually composed by outer membrane, plasma membrane and thylakoids. However, thylakoids are, by far, the predominant membrane in the sample. The abundance of thylakoids in the preparation is particularly evident from the obtained green pellet and when the membrane preparation is loaded onto a sucrose step gradient (as described in Omata and Murata, 1985, see Figure 4).

    Figure 4. Membrane composition of the thylakoids preparation described in Procedure B. Thylakoids are the most abundant type of membrane, but other membranes are included in the preparation as well (plasma membrane and outer membrane). The sucrose step gradient is prepared according to Omata and Murata, 1985.

  3. If preferred, thylakoid pellets can be frozen in liquid N2 (to ensure the pellet is kept in the bottom of the Eppendorf tube) and stored in a freezer until digestion for total metal analysis.
  4. In the current protocol, 625 or 50 µl of 70% (v/v) HNO3 was added to the whole cell sample (25 ml OD7501 Synechocystis cells, approximately corresponding to 73 mg of proteins) and thylakoid pellets (corresponding to 0.73 mg protein), respectively. It is important to note, that the given protein amounts may be adjusted (and most likely further reduced). However, make sure that the whole cell and thylakoid pellets are completely covered with HNO3 during pre-digestion.
  5. Addition of H2O2 facilitates complete digestion at lower temperatures.
  6. To ensure accurate dilution, the dilution of each sample was performed using an analytical balance. The weight of each sample after dilution (the dilution factor) was noted for each sample for subsequent calculations and data analysis.
  7. The ICP-QQQ-MS is operated in MS/MS scan mode with oxygen as the reaction gas, enabling the sensitive simultaneous analysis of the oxide and non-oxide ions: 24Mg+, 55Mn+, and 72FeO+. The integration time is set to 0.1 sec per metal. It is recommended to tune the ICP-QQQ-MS for plasma and ion lenses conditions on a daily basis to ensure plasma robustness and maximum sensitivity.
  8. Synechocystis glucose tolerant strains (see Materials and Reagents) were cultivated in BG11 growth medium that contains glucose (BG11-G, see Recipes) in order to facilitate the study of photosynthetic mutants (Gandini et al. 2017). However, other Synechocystis strains in combination with the BG11 medium but without glucose (Rippka et al. 1979) can be used as well.


  1. BG11-G liquid growth medium, 1 L (see Note 8)
    5 ml 1 M Glucose
    4.7 g/L Na2S2O3·5H2O
    10 ml 100x BG-FPC
    1 ml FeNH4 citrate stock
    1 ml Na2CO3 stock
    1 ml K2HPO4 stock
    Bring to 1 L with ultrapure water and sterilize by autoclave
  2. 100x BG-FPC
    1.76 M NaNO3
    30.4 mM MgSO4
    24.5 mM CaCl2
    3.12 mM Citric acid
    0.279 mM Na2-EDTA
    1/10 (v/v) 1,000x trace element stock
  3. 1,000x trace element stock
    46.3 mM H3BO3
    9.15 mM MnCl2
    0.77 mM ZnSO4
    1.61 mM NaMoO4
    0.32 mM CuSO4
    0.17 mM Co(NO3)2
  4. FeNH4 citrate stock
    6 mg/ml FeNH4 citrate
    Sterilize by autoclave
  5. Na2CO3 stock
    198 mM Na2CO3
    Sterilize by autoclave
  6. K2HPO4 stock
    175 mM K2HPO4
    Sterilize by autoclave
  7. EDTA washing buffer
    5 mM EDTA
    20 mM Tricine, pH 7.9
  8. Tricine buffer
    20 mM Tricine, pH 7.9
  9. Lysozyme solution
    10 mg/ml Lysozyme
  10. Homogenization buffer
    0.4 M sucrose
    10 mM NaCl
    5 mM MgCl2
    20 mM Tricine, pH 7.9
    Freshly add: 10 mM Na-ascorbate, 10 mM NaF
  11. Tricine-NaF buffer
    5 mM Tricine, pH 7.9
    Freshly add: 10 mM NaF
  12. Storage buffer
    0.4 M sucrose
    10 mM NaCl
    5 mM MgCl2
    20 mM Tricine, pH 7.9
    20% glycerol
    Freshly add: 10 mM NaF
  13. Solubilization buffer
    25 mM Bis-Tris (pH 7/HCl)
    12.5% (v/v) glycerol
    2 M Betaine
    0.25 mg/ml pefabloc SC
  14. Detergent solution
    Note: The detergent solution is prepared from a 10% β-DM stock stored at -20 °C.
    2% (w/v) β-DM prepared in solubilization buffer
  15. Mobile phase buffer
    25 mM Bis-Tris (pH 7/TFA)
    0.03% β-DM


This research was supported by Independent Research Fund Denmark – Technology and Production Sciences (grant No. DFF-5054-00042 to S.B.S.). The authors declare no conflicts of interest or competing interests.


  1. Brandenburg, F., Schoffman, H., Keren, N. and Eisenhut, M. (2017). Determination of Mn concentration in Synechocystis sp. PCC6803 using ICP-MS. Bio-protocol 7(23): e2623.
  2. Gandini, C., Schmidt, S. B., Husted, S., Schneider, A. and Leister, D. (2017). The transporter SynPAM71 is located in the plasma membrane and thylakoids, and mediates manganese tolerance in Synechocystis PCC6803. New Phytol 215(1): 256-268.
  3. Hansen, T. H., de Bang, T. C., Laursen, K. H., Pedas, P., Husted, S. and Schjoerring, J. K. (2013). Multielement plant tissue analysis using ICP spectrometry. Methods Mol Biol 953: 121-141.
  4. Keren, N., Kidd, M. J., Penner-Hahn, J. E. and Pakrasi, H. B. (2002). A light-dependent mechanism for massive accumulation of manganese in the photosynthetic bacterium Synechocystis sp. PCC 6803. Biochemistry 41(50): 15085-15092.
  5. Omata, T. and Murata, N. (1985). Electron-transport reactions in cytoplasmic and thylakoid membranes prepared from the cyanobacteria (blue-green algae) Anacystis nidulans and Synechocystis PCC 6714. Biochim Biophys Acta 810(3): 354-361.
  6. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. and Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Microbiology 111(1): 1-61.
  7. Schmidt, S. B., Persson, D. P., Powikrowska, M., Frydenvang, J., Schjoerring, J. K., Jensen, P. E. and Husted, S. (2015). Metal binding in photosystem II super- and subcomplexes from barley thylakoids. Plant Physiol 168(4): 1490-1502.


金属在许多生物过程中是必不可少的,包括含氧光合作用。 在这里我们描述了测量全细胞和类囊体中金属库的方法,包括模型含氧蓝藻PCH6803中完整光合蛋白复合体中的生物活性库。 在方案的第一部分中,仔细制备全细胞和类囊体膜,其中通过电感耦合等离子体三重四极杆质谱(ICP-MS / MS)测量总金属浓度。 在该方案的第二部分,分离的类囊体被溶解以释放完整的膜蛋白和金属结合蛋白复合物。 将这些完整的光合蛋白复合物进行尺寸排阻色谱(SEC),并通过ICP-MS / MS进行连接分析尺寸分离的复合物中的金属结合。

【背景】氧光合作用的过程需要金属,因为它们在光合电子传递链中作为辅因子和催化剂的基本功能。光合装置需要Fe-S簇,血红素桥Fe和非血红素Fe形式的铁(Fe),可溶性移动电子载体蛋白质质体蓝素中的铜(Cu),叶绿素中的镁(Mg), Ca)和锰(Mn)在光合系统II(PSII)的放氧复合物中。严格控制光合细胞内的金属分配对于细胞存活至关重要,因为不平衡的金属累积诱导各种金属酶的错误金属化和失活。因此,准确分析光合细胞中金属的浓度和分配对于研究金属稳态中涉及的关键因子和蛋白质的作用是重要的。尽管Brandenburg等人(2017)中描述的方法精确地量化了集胞蓝细胞中Mn的周质和细胞内集合,但此处提供的方案提供了更广泛的概述,量化金属在整个集胞蓝细胞中,分离的类囊体和在分馏的光合复合体中的生物活性金属池。该协议部分基于Schmidt et al。(2015)中描述的大麦类囊体的方法。

关键字:蓝藻, 集胞藻, 锰, 铁, 镁, 类囊体膜, 形态分析, ICP-MS


  1. 移液器吸头

  2. 50 ml锥形管(Greiner Bio One International,目录号:227261)
  3. 1.5和2ml Eppendorf管(Eppendorf,目录号分别为0030120086和0030120094)
  4. 微波特氟龙管,8毫升(VWR,目录号:525-0178)
  5. 尼龙膜过滤器,0.45微米(Frisenette,目录号:13NY045-100)
  6. 注射器,1毫升(Frisenette,目录号:9161406)
  7. NanoVipers手拧配件,150μm(赛默飞世尔科技,产品目录号:6041.5820)
  8. Synechocystis sp。 PCC6803 GT(耐受葡萄糖的菌株,来自美国密苏里州圣路易斯华盛顿大学生物系Himadri Pakrasi实验室)
  9. 用于ICP-MS校准的多金属校准标准(CPI International,产品目录号:P / N4400-132565A和P / N4400-132565B)
  10. EDTA,BioUltra(Sigma-Aldrich,目录号:E1644-100G)
  11. Tricine(Sigma-Aldrich,目录号:T9784-25G)
  12. 溶菌酶(Sigma-Aldrich,目录号:62971-50G-F)
  13. 蔗糖,BioExtra(Sigma-Aldrich,目录号:S7903-1KG)
  14. 氯化钠(NaCl),BioExtra(Sigma-Aldrich,目录号:S7653-250G)
  15. 氯化镁(MgCl 2·7H 2 O),BioExtra(Sigma-Aldrich,目录号:M2670-1KG)
  16. Na抗坏血酸盐,BioExtra(Sigma-Aldrich,目录号:11140-250G)
  17. 氟化钠(NaF),BioExtra(Sigma-Aldrich,目录号:S7920-100G)
  18. 液氮
  19. Pierce BC BCA蛋白分析试剂盒(Thermo Fisher Scientific,目录号:23225)
  20. 玻璃珠(Sigma-Aldrich,目录号:G4649)
  21. Milli-Q水
  22. PlasmaPURE 67-69%HNO 3(SCP SCIENCE,目录号:250-039-175)
  23. 30%H 2 O 2(用Milli-Q水制备15%H 2 O 2)(Sigma -Aldrich,目录号:31642)
  24. 正十二烷基-β-D-麦芽吡喃糖苷(β-DM)(Anatrace,目录号:D310LA)
  25. Bis-Tris,BioExtra(Sigma-Aldrich,目录号:B7535-100G)
  26. 甘油,BioExtra(Sigma-Aldrich,目录号:G6279)
  27. 甜菜碱,BioUltra(Sigma-Aldrich,目录号:61962-250G)
  28. Pefabloc SC(Roche Diagnostics,目录号:11429868001)
  29. 苹果叶,标准参考物质(Sigma-Aldrich,目录号:NIST1515)
  30. 葡萄糖(Sigma-Aldrich,目录号:G8270-1KG)
  31. Na 2 S 2 O 3·5H 2 O(Merck,目录号:1.06516.0500) />
  32. FeNH 4柠檬酸盐(MP Biomedicals,目录号:02158040)
  33. Na 2 CO 3(Sigma-Aldrich,目录号:S7795-500G)
  34. K 2 HPO 4(Sigma-Aldrich,目录号:P8281)
  35. NaNO 3(Sigma-Aldrich,目录号:S8170-250)
  36. MgSO 4(Sigma-Aldrich,目录号:M2643-500G)
  37. CaCl 2(Sigma-Aldrich,目录号:C5670-500G)
  38. 柠檬酸(Sigma-Aldrich,目录号:251275-100G)
  39. Na 2 -EDTA(Sigma-Aldrich,目录号:E4884-500G)
  40. H 3 BO 3(Sigma-Aldrich,目录号:B6768-500G)
  41. MnCl 2(Sigma-Aldrich,目录号:244589-50G)
  42. ZnSO 4(Sigma-Aldrich,目录号:Z0251-100G)
  43. NaMoO 4(Sigma-Aldrich,目录号:M1003-100G)
  44. CuSO 4(Sigma-Aldrich,目录号:209198-100G)
  45. Co(NO 3)2(Alfa Aesar,目录号:36418.22)
  46. BG11-G(见食谱)
  47. 100x BG-FPC(见食谱)
  48. 1000x微量元素库存(请参阅食谱)
  49. FeNH 4柠檬酸盐储备(见食谱)
  50. Na 2 CO 3储备(见食谱)
  51. K 2 HPO 4股票(见食谱)
  52. EDTA洗涤缓冲液(见食谱)
  53. Tricine缓冲液(见食谱)
  54. 溶菌酶溶液(见食谱)
  55. 匀浆缓冲液(见食谱)
  56. Tricine-NaF缓冲液(见食谱)
  57. 存储缓冲区(请参阅食谱)
  58. 增溶缓冲液(见食谱)
  59. 洗涤剂解决方案(请参阅食谱)
  60. 流动相缓冲液(见食谱)


  1. 移液器
  2. 2升锥形瓶(Fisher Scientific,目录号:11961566)
    制造商:Pyrex,目录号:1130 / 30D。
  3. 定轨摇床(Eppendorf,New Brunswick TM,型号:Innova 2150,目录号:M1194-0010)
  4. Eppendorf离心机5418 R,冷藏(Eppendorf,型号:5418 R,目录号:5401000013)
  5. Eppendorf离心机5430 R,冷藏(Eppendorf,型号:5430 R,目录号5428000210)
  6. Beckman Coulter离心机(Beckman Coulter,型号:Avanti J-25),带19型转子,固定角度。
  7. 250毫升瓶(Beckman Coulter,产品目录号:325620)
  8. Microfluidizer处理器(Microfluidics,型号:M-110L)
  9. 光学显微镜(蔡司,型号:Axiovert 135电视)
  10. 珠磨机TissueLyser II(QIAGEN,目录号:85300)
  11. TissueLyser II适配器套件(QIAGEN,目录号:69982)
  12. Heraeus Fresco 21冷冻微量离心机(Thermo Fisher Scientific,目录号:75002425)
  13. HPLC微量管PP,300μl和螺帽PP,PFTE(VWR,目录号:548-0440和548-0787)
  14. 尺寸排阻柱,Biobasic SEC-1000分析柱(300×7.8mm)和保护柱(30×7.8mm),5μm粒径,1000孔径(Thermo Fisher Scientific,目录号:73605-307846和73605-037821)

  15. 加压Ultrawave系统(Ultrawave系统,Milestone Srl,Sorisole,意大利)
  16. HPLC系统,生物惰性(即流动相和样品不与金属部件接触)(Thermo Fisher Scientific,Dionex,型号:UltiMate 3000)
  17. Dionex TM ICS-5000 + DP双泵(Thermo Fisher Scientific,型号:ICS-5000 + DP)
  18. ICP-MS / MS(Agilent Technologies,型号:8800三重串联四极杆ICP-MS)
  19. 雾化器,Ari Mist HP(Burgener Research,目录号:AM HP 5500)
  20. 用于ICP的气体:5.0级液氩和反应气体:20%氩气中的氧气(AGA,目录号:107407和714039)


  1. MassHunter 4.2工作站软件8800 ICP-MS / MS数据分析(安捷伦科技)



图1.说明测定全细胞和类囊体中总金属浓度的示意性工作流程,以及更详细地分析金属分配入集胞藻的光合复合体 。大写字母(AE)表示协议中的相应程序。

  1. 全细胞制备
    1. 在2L锥形瓶中,在0.5L BG11-G生长培养基中接种集胞蓝细菌菌株(参见食谱)。按照标准微生物学实践,将每种菌株(生物学重复)的至少三个独立培养物接种至最终0.05OD 750。
    2. 在50μmol光子m-2秒-1的光强度下培养培养物,并在定轨摇床上摇动摇瓶,转速为100 rpm,直到培养物达到指数晚期相(0.8-0.9 OD 750分钟)。应用这些生长条件,培养物应在5天左右达到所需的密度。
    3. 在收集培养物之前,测量每种培养物的OD 750。
    4. 对于每种培养物,在整个细胞样品中保留50ml培养物用于蛋白质浓度测定(步骤A8-A13)。
    5. 沉淀剩余的培养物,使用适用于Beckman Coulter离心机的19型转子的250ml瓶子,在5000gxg离心10分钟(在室温下)。重复离心步骤,沉淀其余的培养体积。
    6. 用50ml EDTA洗涤缓冲液洗涤细胞沉淀(参见食谱),在室温下将细胞在5,000gxg下沉淀10分钟,丢弃上清液并重复该洗涤步骤两次(注1 )。第一次洗涤后的上清液会显得有些颜色(带有黄橙色的阴影),而第三次洗涤的上清液应该清澈(图2)。
    7. 用50ml Tricine缓冲液洗涤沉淀(参见食谱),将细胞以5,000μg×g沉淀10分钟,弃去上清液并重复该洗涤步骤两次。在最后一次离心步骤之前,将细胞悬浮液分成两部分,并通过在室温下以5,000μg×g离心10分钟来沉淀两种细胞组分。一个细胞沉淀物构成准备分析的整个细胞制备物(参见程序C)。用于整个细胞制剂的小球可以在液氮中冷冻并储存在冰箱中直至分析。直接对第二个细胞沉淀进一步处理以获得类囊体膜(步骤B1-B6)。
    8. 根据步骤A6-A7处理在步骤A4收集的细胞,并在Tricine缓冲液中重悬细胞沉淀至OD 750(参见食谱)。
    9. 加入1毫升溶菌酶溶液(见食谱)到细胞悬液中,并在室温下轻轻摇动30分钟。在我们的手中,这一步有助于随后的破裂。
    10. 按照制造商的说明反复使微流化器处理器中的细胞破裂。
    11. 在光学显微镜下检查细胞破裂以确认细胞完全破裂。
    12. 使用Pierce bicinchoninic acid(BCA)蛋白测定试剂盒(见材料和试剂)测定样品的蛋白质浓度,用于总蛋白的比色检测和定量。确定的蛋白质浓度指示1ml细胞裂解物中蛋白质的μg,并且因此表示在1OD 750℃下1ml培养物中的μg蛋白质。
    13. 通过将步骤A12中获得的蛋白质浓度乘以步骤A3中测量的OD 750值和收集的细胞的初始培养体积来计算步骤A7中收集的全部细胞级分的蛋白质浓度,其中在这种情况下是225ml(步骤A1中的500ml初始培养物减去步骤A4中放置的50ml,并根据步骤A7除以2)。


  2. 类囊体制剂(注2)
    1. 将步骤A1-A7中制备的细胞沉淀重新悬浮在1ml匀浆缓冲液中(见配方),并将细胞悬浮液转移到装有1ml玻璃珠的2ml Eppendorf管中。
    2. 珠磨机中的破裂细胞,使用冷却的架子(在-20℃保持架15分钟)。珠磨机通过用玻璃珠高速振荡破碎细胞。细胞破碎是在六个循环的珠子跳动过程中进行的,每个循环包括1分钟的珠子跳动,然后在冰上培养1分钟。
    3. 通过在16,000×gg离心2分钟分离未破碎的细胞和玻璃珠。
    4. 将上清液转移到1.5ml Eppendorf管中,并在16,000×gg下沉淀类囊体膜1小时。
    5. 在500μlTricine-NaF缓冲液(离心20分钟,16,000×g g)中洗涤两次类囊体并使用BCA测定试剂盒测定蛋白质浓度。
    6. 将类囊体离心20分钟,16,000×g,小心弃去上清液。将沉淀或者消化以测定总金属分析(程序C)(注3)或者重新悬浮在储存缓冲液中,在液体N 2中冷冻并且在-80℃下储存至多达3个月,直到准备SEC-ICP-MS / MS分析(程序D)。

  3. 全细胞和类囊体膜中总金属浓度的测定
    注:清洁消耗品和清洁实验室程序必须在以下所有步骤中使用。建议至少包括一个认证参考(苹果[Malus domestica]叶,NIST 1515;美国国家标准与技术研究院),每次消化过程中只有一个真正的空白。
    1. 解冻整个细胞和类囊体膜的颗粒(分别如步骤A和B中所述制备),并将70%(v / v)HNO 3(参见注释4)直接添加到Eppendorf管。样品预先消化2小时(将Eppendorf管打开,松散地用塑料袋盖住以减少污染)。
    2. 小心地将预消化的样品转移(通过吸取)到8ml特氟龙消化管中。
    3. 在HNO 3中加入15%(v / v)H 2 O 2 O 2(H 2 O 3)根据步骤C1),2.5比1的比率(参见注释5)。
    4. 使用加压微波系统(根据Hansen等人报道的详细描述,,Milestone, [2013])消化样品。
    5. 将样品转移到干净的管中,用Milli-Q水冲洗Teflon消化管至少三次。
    6. 使用Milli-Q水将样品稀释至终浓度为3.5%HNO 3(v / v)(参见注释6)。准备用于感兴趣金属的外部校准的校准标准(覆盖至少三个数量级和8到10个校准点的线性范围)。
    7. 通过ICP-MS分析样品包括校准标样。

  4. 用于SEC-ICP-MS / MS测量的类囊体制剂

    1. 在冰上和黑暗中解冻类囊体膜样品
    2. 取一个均匀的子样品(在本例中:相当于300μg蛋白质,参见步骤B5)(轻轻地上下移液管,避免涡旋)。
    3. 通过在7,000×gg离心2分钟使微量离心机中的类囊体膜颗粒化。
    4. 弃去上清液并重悬于150μl冰冷的溶解缓冲液(新鲜制备,参见食谱)中使类囊体膜重新悬浮(通过轻轻吹打上下)至最终蛋白质浓度为2μg/μl。
    5. 加入等体积的洗涤剂溶液(150μl,参见食谱)至1μg/μl的最终蛋白质浓度,并将类囊体膜在冰上和黑暗中溶解10分钟。
    6. 通过以16,000×gg离心10分钟将溶解的蛋白质(上清液)与不溶物质(沉淀物)分离。
    7. 立即收集溶解的蛋白质(上清液),并使用1ml注射器通过0.45μm尼龙膜过滤器进入HPLC微量瓶。
    8. 保持样品在冰上和黑暗中,直到后续的形态分析(程序E)。

  5. SEC-ICP-MS / MS测量:光合成络合物中金属结合的测定(形态分析)
    注意:在形态分析过程中,HPLC自动进样器和柱室在6°C保持冷却。该协议的这一部分基于Schmidt等人开发的方法。 (2015)
    1. 用流动相缓冲液平衡HPLC系统和大小排阻柱(参见食谱)至少1小时。
    2. 使用HPLC系统将50μg新溶解的类囊体蛋白注射到大小排阻柱上。

    3. 使用流动相缓冲液进行等度蛋白分离和洗脱,流速为1 ml / min,时间为35分钟
    4. 对于光合蛋白复合物中结合的金属的在线检测,色谱柱出口直接与ICP-MS / MS(注7)相连。
    5. 记录具有多元素检测的时间分辨色谱图(图3)。

    图3:多金属色谱图。对于非氧化物离子24Mg + 55和55nm记录的尺寸排阻分布曲线Mn + +和氧化物离子72 + FeO + +。根据Gandini等人(2017年)分配了单独的Mn和Fe亚组分,Mg分布图被证明是叶绿素的代用品。


  1. 总金属浓度
  2. 形态分析
    通过SEC-ICP-MS分析每个菌株至少三个独立的样品。至于总金属浓度分析,我们强烈建议分析至少两个独立实验。 SEC-ICP-MS色谱图可针对每种感兴趣的金属单独制备,或通过覆盖更多金属构建单一多金属色谱图。准备一个代表性样品的色谱图。数据以每秒离子计数作为保留时间的函数呈现。金属型材可以进行背景校正或作为原始数据显示(图3)。
    根据Gandini et。(2017)分配各个峰值。


  1. EDTA被用于去除位于外膜和周质间隙中的Mn存储池(Keren等,2002)。
  2. 这里描述的类囊体制剂实际上由外膜,质膜和类囊体组成。然而,到目前为止,类囊体是样品中主要的膜。制备过程中类囊体的丰度从得到的生球丸中特别明显,并且当膜制备物装载到蔗糖阶梯上时(如Omata和Murata,1985中所述,见图4)。


  3. 如果优选的话,可将类囊体小球冷冻在液体N 2中(以确保沉淀保留在Eppendorf管的底部)并储存在冰箱中直到消化完全金属分析。
  4. 在目前的方案中,将625或50μl的70%(v / v)HNO 3加入全细胞样品(25ml OD 7501集胞蓝细菌大约相当于73mg蛋白质)和类囊体小球(相当于0.73mg蛋白质)。重要的是要注意,给定的蛋白质含量可能会调整(最可能进一步降低)。但是,在预消化过程中,确保全细胞和类囊体颗粒完全被HNO 3覆盖。
  5. 添加H 2 O 2 2有利于在较低温度下完全消化。
  6. 为确保准确稀释,每个样品的稀释均使用分析天平进行。记录每个样品在稀释后的每个样品的重量(稀释因子),用于随后的计算和数据分析。
  7. ICP-MS / MS在MS / MS扫描模式下以氧气作为反应气体进行操作,可以灵敏地同时分析氧化物和非氧化物离子:+ 24 Mg + + 55 Mn + +和+ 72 FeO + +组成。积分时间设置为每个金属0.1秒。建议每天调整ICP-MS / MS的等离子和离子透镜条件,以确保等离子体稳定性和最大灵敏度。
  8. 在含有葡萄糖的BG11生长培养基(BG11-G,参见食谱)中培养集胞葡萄糖耐受性菌株(参见材料和试剂)以促进光合突变体的研究(Gandini等人。 2017)。然而,也可以使用其他集胞蓝细菌菌株与BG11培养基但不含葡萄糖的组合(Rippka等,1979)。


  1. BG11-G液体生长培养基,1L(见注8)
    5毫升1 M葡萄糖
    Na2.5g / L Na 2 S 2 O 3 0.5H 2 O
    10毫升100x BG-FPC
    1ml FeNH 4柠檬酸盐原料
    1毫升Na 2 CO 3储备
    1毫升K 2 HPO 4股票
  2. 100x BG-FPC
    1.76M NaNO 3
    30.4mM MgSO 4
    24.5mM CaCl 2 2/2 3.12 mM柠檬酸
    0.279mM Na 2 -EDTA
    1/10(v / v)1,000x微量元素库存
  3. 1000x微量元素库存
    46.3mM H 3 BO 3 3 9.15mM MnCl 2·/ 2 0.77mM ZnSO 4。
    1.61mM NaMoO 4 / sub>
    0.32mM CuSO 4
    0.17mM Co(NO 3)2:
  4. FeNH 4柠檬酸盐原料
    6毫克/毫升FeNH 4柠檬酸盐
  5. Na 2 CO 3储备
    198mM Na 2 CO 3 3 通过高压灭菌器消毒
  6. K <2> HPO <4>股票
    175mM K 2 HPO 4 4 通过高压灭菌器消毒
  7. EDTA洗涤缓冲液
    5 mM EDTA
    20 mM Tricine,pH 7.9
  8. Tricine缓冲液
    20 mM Tricine,pH 7.9
  9. 溶菌酶溶液
  10. 均化缓冲液
    0.4 M蔗糖
    10 mM NaCl
    5mM MgCl 2·/ 2 20 mM Tricine,pH 7.9
    新鲜加入:10mM Na-抗坏血酸盐,10mM NaF
  11. Tricine-NaF缓冲液
    5 mM Tricine,pH 7.9
    新鲜加入:10 mM NaF
  12. 存储缓冲区
    0.4 M蔗糖
    10 mM NaCl
    5mM MgCl 2·/ 2 20 mM Tricine,pH 7.9
    新鲜加入:10 mM NaF
  13. 增溶缓冲液
    25 mM Bis-Tris(pH 7 / HCl)
    12.5%(v / v)甘油
    2 M甜菜碱
    0.25 mg / ml pefabloc SC
  14. 洗涤剂解决方案
    在增溶缓冲液中制备2%(w / v)β-DM
  15. 流动相缓冲区
    25 mM Bis-Tris(pH 7 / TFA)


这项研究得到丹麦独立研究基金 - 技术和生产科学(批准号:DFF-5054-00042至S.B.S.)的支持。作者声明不存在利益冲突或利益冲突。


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引用:Gandini, C., Husted, S. and Schmidt, S. B. (2018). Analysis of Metals in Whole Cells, Thylakoids and Photosynthetic Protein Complexes in Synechocystis sp. PCC6803. Bio-protocol 8(12): e2889. DOI: 10.21769/BioProtoc.2889.