Determination of Intracellular Osmolytes in Cyanobacterial Cells

Materials and Reagents

1. 2 ml microcentrifuge tubes (Cypress
   
2. 10 ml Centrifuge tube, Snap-Cap (Kangjian)
   
3. 1 ml syringe (Jianshi)
   
4. Syringe membrane filters, 0.22 μm (Jinteng)
   
5. Synechocystis sp. PCC 6803
   
6. Nitrogen (Dehai)
   
7. CO₂ (Dehai)
   
8. MilliQ water (Millipore, Germany)
   
9. Potassium phosphate dibasic trihydrate (K₂HPO₄∙3H₂O) (Sinopharm Chemical Reagent, catalog number: 10017518 )
   
10. Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Sinopharm Chemical Reagent, catalog number: 10013018 )
    
11. Calcium chloride dihydrate (CaCl₂·2H₂O) (Sinopharm Chemical Reagent, catalog number: 20011160 )
    
12. Critic acid (Sinopharm Chemical Reagent, catalog number: 10007118 )
    
13. Ferric ammonium citrate (Sinopharm Chemical Reagent, catalog number: 30011428 )
    
14. EDTA·2Na (Sinopharm Chemical Reagent, catalog number: 10009717 )
    
15. Sodium carbonate (Na₂CO₃) (Sinopharm Chemical Reagent, catalog number: 10019260 )
    
16. Boric acid (H₃BO₃) (Sinopharm Chemical Reagent, catalog number: 10004818 )
    
17. Manganese(II) chloride tetrahydrate (MnCl₂·4H₂O) (Sinopharm Chemical Reagent, catalog number: 20026118 )
    
18. Zinc sulfate heptahydrate (ZnSO₄·7H₂O) (Sinopharm Chemical Reagent, catalog number: 10024018 )
    
19. Sodium molybdate dihydrate (Na₂MoO₄·2H₂O) (Sinopharm Chemical Reagent, catalog number: 10019818 )
    
20. Copper(II) sulfate pentahydrate (CuSO₄·5H₂O) (Sinopharm Chemical Reagent, catalog number: 10008218 )
    
21. Cobalt(II) chloride hexahydrate (CoCl₂·6H₂O) (Sinopharm Chemical Reagent, catalog number: 10007216 )
    
22. Sodium nitrate (NaNO₃) (Sinopharm Chemical Reagent, catalog number: 10019918 )
    
23. Sodium chloride (NaCl) (Sinopharm Chemical Reagent, catalog number: 10019318 )
    
24. Ethanol (Sinopharm Chemical Reagent, catalog number: 10009218 )
    
25. Glycerol standard (99%, Sinopharm Chemical Reagent, catalog number: 10010618 )
    
26. Glucosylglycerol standard (50%, Bitop, http://www.bitop.de/en/products/cosmetic-active-ingredients/glycoin)
    
27. Glucose standard (Sinopharm Chemical Reagent, catalog number: 10010518 )
    
28. Sucrose (Sinopharm Chemical Reagent, catalog number: 10021418 )
    
29. 50% Sodium chloride (NaOH) solution (Thermo Fisher Scientific)
    
30. BG11 medium (see Recipes)
    
31. Saturated NaCl solution prepared in BG11 media (see Recipes)
    
32. 80% ethanol (see Recipes)
    
33. Osmolytes standards (see Recipes)
    
34. 200 mM NaOH (see Recipes)
    

Equipment

1. 200, 1,000 ml Pipettes (Eppendorf, Germany)
   
2. Glass column photobioreactors (Sanhehuaxing, China) (Tan et al., 2011)
   
3. Centrifuge (Sigma-Zentrifugen, model: Sigma 1-14 )
   
4. Water bath (Yarong, model: B260 )
   
5. Organomation (Hengao, model: HGC-24A )
   
6. Ion chromatography (Thermo Fisher Scientific, Thermo Scientific^TM, model: Dionex^TM ICS-5000+ )
   
7. Dinex^TM CarboPac^TM analytical column (4 x 250 mm, Thermo Fisher Scientific, model: Dinex^TM CarboPac^TM PA10 )
   
8. -80 °C freezer (Thermo Fisher Scientific, Thermo Scientific^TM, model: Forma 705 )
   
9. Vortex-Genie 2 (Scientific Industries, model: Vortex-Genie 2 )
   
10. Autoclave (Hirayama, model: HV-50 )
    

Software

1. Chromeleon software (version 6.80, Thermo Fisher Scientific)
   
2. IBM SPSS Statistics (IBM, version 19)
   

Procedure

1. Cultivation of Synechocystis sp. PCC 6803
   Note: Monitor the growth of cyanobacterial cells by measuring the optical density at 730 nm (OD₇₃₀) with a spectrophotometer. Culture volume should be less than 150 ml in 200 ml columns.
   1. Inoculate Synechocystis cultures into liquid BG11 media in glass column photobioreactors at 30 °C under constant white fluorescent light with a light intensity of 100 μE/m²/sec. Adjust the initial OD₇₃₀ to 0.5.
      
   2. Bubble the cultures with CO₂-enriched air flow (3%).
      
   3. Add the saturated NaCl solution prepared in BG11 media into the late exponential phase culture (OD₇₃₀ ≈ 8-10) to reach a final NaCl concentration of 600 mM.
      
2. Harvesting cells: Centrifuge 2 ml aliquots of the above Synechocystis cultures at 12,000 x g for 5 min at room temperature. Cells (Figure 1A) can be stored at -80 °C if not proceed to the next step immediately.
   Note: Regularly, the highest intracellular glucosylglycerol concentration of Synechocystis would be reached within two days after salt shock. For sucrose, the highest concentration will be reached around 12 h after salt shock. If cells could not be extracted immediately, Synechocystis cells should be spun down by centrifugation and stored without supernatants at a -80 °C freezer.
   
3. Extraction of intracellular osmolytes from Synechocystis cells
   1. Re-suspend Synechocystis cells in 1 ml of 80% ethanol (v/v) (Figure 1B) and then incubate at 65 °C for 4 h.
      Note: When incubating at 65 °C, it is better to mix the samples gently by inverting tubes once for each hour.
      
   2. After centrifugation at 12,000 x g for 5 min at room temperature, transfer the supernatant to a clean 10 ml tube, and then dry at 55 °C under a stream of nitrogen.
      
   3. Dissolve the dry residues (Figure 1C) in 1 ml of Milli-Q water, and filter through membranes (0.22 μm).
      Note: For drying, it takes about 30 min. If the extracted samples (Figure 1D) could not be analyzed immediately, they should be stored in 4 °C for less than seven days (-20 °C for less than 30 days), and filtered again before analyzing by ion chromatography.
      
   
   
   Figure 1. Extraction of osmolytes from Synechocystis cells. A. Cells of Synechocystis for an osmolyte extraction experiment; B. Synechocystis cells re-suspended in 80% ethanol; C. The dry residues dried at 55 °C under a stream of nitrogen; D. The extracted osmolyte samples from Synechocystis cells.
   
   
4. Detection of intracellular osmolytes by ion chromatography
   1. Dilute the extracted samples to the suitable concentrations ranging from 1~20 mg/L.
      Note: Normally, the intracellular sucrose and glucosylglycerol concentrations in the wild type cells of Synechocystis sp. PCC 6803 are around 100 mg/L. Therefore, the samples should be diluted 5-10 fold.
      
   2. Subject 25 μl of samples to ICS-5000+ ion-exchange chromatography system equipped with an electrochemical detector and a Dinex^TM CarboPac^TM PA10 analytical column (4 x 250 mm, Thermo Fisher Scientific, Waltham, MA, USA). Elute the column with 200 mM NaOH at a flow rate of 1.0 ml/min.
      
   3. Mix the standards of glucosylglycerol, sucrose, and glycerol, dilute into 1, 5, 10, 15, 20 mg/L, and analyze by the same methods with the extracted samples.
      
5. Quantification of intracellular osmolytes
   1. For the standard mixtures with 15 mg/L of each osmolyte, retention times of glycerol, glucosylglycerol, glucose and sucrose using the method described above are 1.7, 2.1, 3.3 and 5.5 min, respectively (Figure 2A).
      
   2. Based on the mixed osmolyte standards, standard curves are made by using the peak area calculated by the Chromeleon software (Figure 2B).
      
   3. For quantification of intracellular osmolytes in cyanobacteria, areas of the target peak obtained by ion chromatography are calculated by the Chromeleon software, and then the osmolyte concentration is determined using the standard curve of osmolyte standards.
      
      
      Figure 2. Ion chromatography profile and standard curves of osmolyte standards. A. Chromatogram of the standard mixture. 15 mg/L of each kind of osmolyte standard were mixed together and analyzed by ion chromatography. Gly, Glycerol; GG, Glucosylglycerol; Glu, Glucose; Suc, Sucrose. B. Chromatogram of the sample isolated from Synechocystis cells. Standard curves of glycerol (C), glucosylglycerol (D), glucose (E) and sucrose (F) were established by plotting peak areas and concentrations of each kind of osmolyte standard.
      

Data analysis

1. For statistical analysis, IBM SPSS Statistics version 19 was used.
   
2. For comparing differences between two data sets, an independent samples t-test was performed.
   

Recipes

1. BG11 medium (Rippka et al., 1979)
   1. Prepare 8 kinds of 10x stocks as follows:
      Stock 1 (40 g/L K₂HPO₄·3H₂O)
      Stock 2 (75 g/L MgSO₄·7H₂O)
      Stock 3 (36 g/L CaCl₂·2H₂O)
      Stock 4 (6 g/L critic acid)
      Stock 5 (6 g/L ferric ammonium citrate)
      Stock 6 (1 g/L EDTA·2Na)
      Stock 7 (20 g/L Na₂CO₃)
      Stock A5 (2.86 g/L H₃BO₃, 1.81 g/L MnCl₂·4H₂O, 0.22 g/L ZnSO₄·7H₂O, 0.39 g/L NaMoO₄·2H₂O, 0.08 mg/L CuSO₄·5H₂O, 0.01 g/L CoCl₂·6H₂O)
      Autoclave Stocks 1 and 5 at 121 °C for 20 min. Store the autoclaved stocks and other stocks at 4 °C before use
      
   2. Dissolve 1.5 g NaNO₃ in 992 ml of ddH₂O, add 1 ml of Stocks 2, 3, 4, 6, 7 and A5 into the medium respectively
      
   3. Autoclave the medium at 121 °C for 20 min, and supplement with 1 ml of both Stocks 1 and 5 before inoculations
      
2. Saturated NaCl solution prepared in BG11 media
   Dissolve 292.5 g NaCl in BG11 media
   Adjust the final volume with BG11 media to 1 L
   Autoclave the NaCl solution at 121 °C for 20 min
   
3. 80% (v/v) ethanol
   For 100 ml of ethanol solution, supplement 80 ml of ethanol with Milli-Q water to reach the final volume of 100 ml
   
4. Osmolytes standards
   1. First, dissolve 6 mg of glycerol, sucrose, glucose and glucosylglycero in 100 ml of Milli-Q water
      
   2. Then, prepare different concentrations of osmolytes standards according to the following table
      
      
   3. 200 mM NaOH
      For 1 L of 200 mM NaOH solution, dilute 16 ml of 50% NaOH solution to the final volume of 1 L with Milli-Q water
      

   Acknowledgments

   The protocol was adopted from the publication ‘Effects of lowered and enhanced glycogen pools on salt-induced sucrose production in a sucrose-secreting strain of Synechococcus elongatus PCC 7942’ (Qiao et al., 2017). This work was supported by the National Science Fund for Distinguished Young Scholars of China (31525002 to X. Lu), Shandong Key basic Research project (ZR2017ZB0211), the Joint Sino-German Research Project (grant GZ 984 to X. Lu), the Shandong Taishan Scholarship (X. Lu), the National Science Foundation of China (31301018 to X. Tan), the Key Research Program of the Chinese Academy of Sciences (ZDRW-ZS-2016-3 to X. Tan) and Qingdao Innovative Leading Talent (15-10-3-15-(31)-zch). The authors have no conflicts of interest or competing interests to declare.

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