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Determination of Intracellular Osmolytes in Cyanobacterial Cells
蓝藻细胞内渗透调节物质的测定   

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Frontiers in Microbiology
Jun 2017

Abstract

Most of the cyanobacteria accumulate osmolytes including sucrose, glucosylglycerol, in their cells in response to salt stress. Here we describe a protocol of our laboratory for extraction and quantification of cyanobacterial intracellular sucrose and glucosylglycerol. We have confirmed this protocol was applicable to at least four kinds of cyanobacteria, filamentous cyanobacterium Anabaena sp. PCC 7120, unicellular cyanobacterium Synechocystis sp. PCC 6803, Synechococcus elongatus PCC 7942 and halotolerant unicellular cyanobacterium Synechococcus sp. PCC 7002.

Keywords: Osmolyte (渗透调节物质), Sucrose (蔗糖), Glucosylglycerol (甘油葡萄糖苷), Synechocystis (集胞藻), Cyanobacteria (蓝藻), Ion chromatography (离子色谱法)

Background

Osmolytes (or compatible solutes) are a group of low-molecular-weight organic solutes, and play important physiological roles on abiotic stress acclimation in microbes including cyanobacteria (Reed and Stewart, 1985; Klähn and Hagemann, 2011; Slama et al., 2015). For determination of intracellular osmolytes from cyanobacterial cells, several protocols have been established (Reed and Stewart, 1985; Hagemann et al., 1997; Motta et al., 2004; Du et al., 2013; Fa et al., 2015).

Among these methods, nuclear magnetic resonance (NMR) spectroscopy based method was the only one which could be directly applied on microbe cultures without any extraction procedure (Motta et al., 2004). However, this protocol has just been tested in cultures of Halomonas pantelleriensis and Sulfolobus solfataricus rather than in cyanobacterial cultures. For all other methods, 80% ethanol was used for extraction of osmolytes from cyanobacterial cells. After derivatization by some trimethyl-silyl reagents, the derivatives of osmolytes could be analyzed by gas chromatography (Reed and Stewart, 1985). Alternatively, the extracted osmolytes could be directly analyzed by high-performance liquid chromatography (HPLC) (Hagemann et al., 1997). Our lab has firstly reported our protocol for osmolyte determination by ion chromatography (IC) equipped with a carb-Pac®MA1 analytical column (Du et al., 2013). In this protocol (Du et al., 2013), the column was equilibrated with 600 mM NaOH with a flow rate of 0.4 ml/min, and the running time for one sample was 45 min. Later, the concentration of NaOH was increased to 800 mM, and the running time for each sample was shortened to 32 min (Song et al., 2016 and 2017). Recently, the PA10 analytical column was successfully used for osmolyte analysis by ion chromatography (Qiao et al., 2017), and the running time for one sample was further shortened to 10 min. It is worthy to note that our collaborator has established a novel method for osmolyte determination by combination of separation by capillary ion chromatography and detection by mass spectrometry (CIC-MS) (Fa et al., 2015). The NMR and CIC-MS based methods are suitable for determination of unknown osmolytes from cyanobacteria. Compared with these two methods as well as the GC and HPLC based methods, our IC based method has advantages on running time and accuracy which would be helpful for the high throughput osmolyte detection used in some cyanobacterial metabolic engineering reseaches.

Therefore, we detailedly present our recent IC-based protocol here for osmolyte determination 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 ScientificTM, model: DionexTM ICS-5000+ )
  7. DinexTM CarboPacTM analytical column (4 x 250 mm, Thermo Fisher Scientific, model: DinexTM CarboPacTM PA10 )
  8. -80 °C freezer (Thermo Fisher Scientific, Thermo ScientificTM, 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 DinexTM CarboPacTM 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.

    References

    1. Du, W., Liang, F., Duan, Y., Tan, X. and Lu, X. (2013). Exploring the photosynthetic production capacity of sucrose by cyanobacteria. Metab Eng 19: 17-25.
    2. Fa, Y., Liang, W., Cui, H., Duan, Y., Yang, M., Gao, J. and Liu, H. (2015). Capillary ion chromatography-mass spectrometry for simultaneous determination of glucosylglycerol and sucrose in intracellular extracts of cyanobacteria. J Chromatogr B Analyt Technol Biomed Life Sci 1001: 169-173.
    3. Hagemann, M., Richter, S. and Mikkat, S. (1997). The ggtA gene encodes a subunit of the transport system for the osmoprotective compound glucosylglycerol in Synechocystis sp. strain PCC 6803. J Bacteriol 179(3): 714-720.
    4. Klähn, S. and Hagemann, M. (2011). Compatible solute biosynthesis in cyanobacteria. Environ Microbiol 13(3): 551-562.
    5. Motta, A., Romano, I. and Gambacorta, A. (2004). Rapid and sensitive NMR method for osmolyte determination. J Microbiol Methods 58(2): 289-294.
    6. Qiao, C., Duan, Y., Zhang, M., Hagemann, M., Luo, Q. and Lu, X. (2017). Effects of lowered and enhanced glycogen pools on salt-induced sucrose production in a sucrose-secreting strain of Synechococcus elongatus PCC 7942. Appl Environ Microbiol.
    7. Reed, R. H. and Stewart, W. D. P. (1985). Osmotic adjustment and organic solute accumulation in unicellular cyanobacteria from freshwater and marine habitats. Mar Biol 88(1):1-9.
    8. 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. J Gen Microbiol 111(1):1-61.
    9. Slama, I., Abdelly, C., Bouchereau, A., Flowers, T. and Savoure, A. (2015). Diversity, distribution and roles of osmoprotective compounds accumulated in halophytes under abiotic stress. Ann Bot 115(3): 433-447.
    10. Song, K., Hagemann, M., Tan, X. and Lu, X. (2017). The response regulator Slr1588 regulates spsA but is not crucial for salt acclimation of Synechocystis sp. PCC 6803. Front Microbiol 8: 1176.
    11. Song, K., Tan, X., Liang, Y. and Lu, X. (2016). The potential of Synechococcus elongatus UTEX 2973 for sugar feedstock production. Appl Microbiol Biotechnol 100(18): 7865-7875.
    12. Tan, X., Yao, L., Gao, Q., Wang, W., Qi, F. and Lu, X. (2011). Photosynthesis driven conversion of carbon dioxide to fatty alcohols and hydrocarbons in cyanobacteria. Metab Eng 13(2): 169-176.

简介

大多数蓝细菌在其细胞中积累包括蔗糖,葡萄糖基甘油在内的渗透压以响应盐胁迫。 在这里,我们描述了我们实验室的蓝藻细胞内蔗糖和葡萄糖甘油的提取和定量方案。 我们已证实此方案适用于至少四种蓝藻,丝状蓝藻Anabaena sp。 PCC 7120,单细胞蓝藻集胞藻 sp。 PCC 6803,细长聚球蓝细菌PCC 7942和耐盐单细胞蓝细菌聚球蓝细菌。 PCC 7002。

【背景】Osmolytes(或相容溶质)是一组低分子量的有机溶质,对包括蓝细菌在内的微生物的非生物胁迫适应具有重要的生理作用(Reed和Stewart,1985;Klähn和Hagemann,2011; Slama等人。,2015)。为了确定来自蓝细菌细胞的细胞内渗透物,已经建立了几种方案(Reed和Stewart,1985; Hagemann等人,1997; Motta等人,2004; Du ,2013; Fa et al。,2015)。

在这些方法中,基于核磁共振(NMR)光谱的方法是唯一可以直接应用于微生物培养物而不需要任何提取程序的方法(Motta等人,2004年)。然而,这个方案刚刚在Halomonas pantelleriensis 和Sulfolobus solfataricus 培养物中进行过测试,而不是在蓝藻培养物中进行测试。对于所有其他方法,80%乙醇用于从蓝藻细胞中提取渗压剂。通过一些三甲基甲硅烷基试剂衍生后,可以通过气相色谱法分析渗压剂的衍生物(Reed和Stewart,1985)。或者,可以通过高效液相色谱法(HPLC)直接分析提取的渗透物(Hagemann等人,1997)。我们的实验室首先报道了我们用离子色谱仪(IC)测定渗压液的方法,该离子色谱仪配备了carb-Pac®MA1分析柱(Du等人,2013年)。在该方案中(Du等人,2013),柱用600mM NaOH以0.4ml / min的流速平衡,并且一个样品的运行时间为45分钟。之后,NaOH的浓度增加到800mM,每个样品的运行时间缩短到32分钟(Song等人,2016和2017)。最近,PA10分析柱已成功用于离子色谱分析渗析液(Qiao等人,2017),一个样品的运行时间进一步缩短为10分钟。值得注意的是,我们的合作者已经建立了一种通过毛细管离子色谱分离和质谱检测(CIC-MS)相结合的方法测定渗压剂的方法(Fa等人,2015年)。基于NMR和CIC-MS的方法适用于测定蓝藻中未知的渗透剂。与这两种方法以及基于GC和HPLC的方法相比,我们的基于IC的方法在运行时间和精度方面具有优势,这对于在一些蓝藻代谢工程研究中使用的高通量渗压剂检测是有帮助的。

因此,我们详细介绍了我们最近基于IC的协议,用于蓝藻细胞中渗透压测定。

关键字:渗透调节物质, 蔗糖, 甘油葡萄糖苷, 集胞藻, 蓝藻, 离子色谱法

材料和试剂

  1. 2毫升微量离心管(赛普拉斯
  2. 10 ml离心管,Snap-Cap(康健)
  3. 1毫升注射器(健氏)
  4. 注射器膜过滤器,0.22微米(金腾)
  5. Synechocystis sp。 PCC 6803
  6. 氮(德海)
  7. CO <2>(德海)
  8. MilliQ水(德国Millipore)
  9. 磷酸氢二钾三水合物(K 2 HPO 4·3H 2 O)(国药集团化学试剂,目录号:10017518)
  10. 硫酸镁七水合物(MgSO 4·7H 2 O)(国药集团化学试剂,目录号:10013018)
  11. 氯化钙二水合物(CaCl 2·2H 2 O)(国药集团化学试剂有限公司,产品目录号:20011160)
  12. Critic acid(国药集团化学试剂,目录号:10007118)
  13. 柠檬酸铁铵(国药集团化学试剂,目录号:30011428)
  14. EDTA·2Na(国药集团化学试剂,产品目录号:10009717)
  15. 碳酸钠(Na 2 CO 3)(国药集团化学试剂,目录号:10019260)
  16. 硼酸(H 3 BO 3)(Sinopharm Chemical Reagent,目录号:10004818)
  17. 氯化锰(II)四水合物(MnCl 2·4H 2 O)(国药集团化学试剂,目录号:20026118)
  18. 硫酸锌七水合物(ZnSO 4·7H 2 O)(国药集团化学试剂,目录号:10024018)
  19. 钼酸钠二水合物(Na 2 MoO 4·2H 2 O)(国药集团化学试剂,目录号:10019818)
  20. 五水合硫酸铜(II)(CuSO 4·5H 2 O)(国药集团化学试剂,产品目录号:10008218)
  21. 氯化钴(II)六水合物(CoCl 2·6H 2 O)(国药集团化学试剂,产品目录号:10007216)
  22. 硝酸钠(NaNO 3)(国药集团化学试剂,目录号:10019918)
  23. 氯化钠(NaCl)(国药集团化学试剂,目录号:10019318)
  24. 乙醇(国药集团化学试剂,产品目录号:10009218)
  25. 甘油标准(99%,国药集团化学试剂,目录号:10010618)
  26. 葡萄糖基甘油标准(50%,Bitop, http://www.bitop.de / en / products / cosmetic-active-ingredients / glycoin
  27. 葡萄糖标准品(国药集团化学试剂,目录号:10010518)
  28. 蔗糖(国药集团化学试剂,目录号:10021418)
  29. 50%氯化钠(NaOH)溶液(赛默飞世尔科技)
  30. BG11中(见食谱)
  31. 在BG11培养基中制备饱和NaCl溶液(见食谱)
  32. 80%乙醇(见食谱)
  33. Osmolytes标准(见食谱)
  34. 200 mM NaOH(见食谱)

设备

  1. 200,1000毫升移液器(Eppendorf,德国)
  2. 玻璃柱光生物反应器(中国Sanhehuaxing)(Tan等人,2011年)
  3. 离心机(Sigma-Zentrifugen,型号:Sigma 1-14)
  4. 水浴(亚龙,型号:B260)
  5. Organomation(Hengao,型号:HGC-24A)
  6. 离子色谱法(Thermo Fisher Scientific,Thermo Scientific TM,型号:Dionex TM ICS-5000 +)
  7. Dinex TM CarboPac TM分析柱(4×250mm,Thermo Fisher Scientific,型号:Dinex TM CarboPac TM TM) PA10)
  8. -80°C冰箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Forma 705)
  9. Vortex-Genie 2(Scientific Industries,型号:Vortex-Genie 2)

  10. 高压灭菌器(平山,型号:HV-50)

软件

  1. Chromeleon软件(版本6.80,赛默飞世尔科技)
  2. IBM SPSS Statistics(IBM,第19版)

程序

  1. Synechocystis sp。的培养PCC 6803
    注意:用分光光度计通过测量730nm处的光密度(OD <730> em>)来监测蓝细菌细胞的生长。 200 ml色谱柱的培养体积应小于150 ml。
    1. 在玻璃柱光生物反应器中,在30°C恒定的白色荧光下,以100μE/ m 2 /秒/秒的光强度将集胞藻培养物接种到液体BG11培养基中。将初始OD <730> 调整为0.5。
    2. 用CO 2浓化空气流(3%)使培养物鼓泡。
    3. 将BG11培养基中制备的饱和NaCl溶液加入晚指数期培养物(OD730≈8-10)以达到600mM的最终NaCl浓度。
  2. 收获细胞:在室温下,以12,000×gg离心2ml等份的上述集胞藻属培养物5分钟。如果不立即进入下一步,细胞(图1A)可以保存在-80°C。
    注意:经常在盐冲击后两天内达到集胞藻最高的细胞内葡萄糖甘油浓度。对于蔗糖,在盐冲击后约12小时达到最高浓度。如果不能立即提取细胞,则应通过离心将集胞藻细胞离心沉淀,并在-80°C冷冻箱中不含上清液。
  3. 从集胞蓝细胞中提取细胞内渗透物
    1. 重悬于1ml80%乙醇(v / v)(图1B)中的集胞蓝细胞,然后在65℃孵育4小时。
      注意:在65°C孵育时,最好每小时颠倒一次混合样品。
    2. 在室温下以12,000×g离心5分钟后,将上清液转移至干净的10ml管中,然后在55℃下在氮气流下干燥。
    3. 将干燥的残留物(图1C)溶解在1毫升Milli-Q水中,并通过膜(0.22微米)过滤。
      注:为了干燥,大约需要30分钟。如果提取的样品(图1D)不能立即分析,应在4℃下保存少于7天(-20℃,少于30天),并在离子色谱分析前再次过滤。 em>


    图1.从Synechocystis细胞中提取渗压剂A.用于渗压提取实验的集胞藻细胞 B.将集胞藻细胞重悬于80%乙醇中; C.干燥的残余物在55℃下在氮气流下干燥; D.从Synechocystis细胞提取的渗压液样品。

  4. 离子色谱法检测细胞内渗透物
    1. 将提取的样品稀释至1〜20 mg / L的合适浓度。
      注:正常情况下,集胞藻中野生型细胞内的蔗糖和葡萄糖基甘油浓度是不同的。 PCC 6803约为100 mg / L。因此,样品应稀释5-10倍。
    2. 将25μl样品加入配备有电化学检测器和Dinex TM CarboPac TM PA10分析柱的ICS-5000 +离子交换色谱系统(4×250mm,Thermo Fisher Scientific,Waltham,MA,USA)。
      用200 mM NaOH以1.0 ml / min的流速洗脱色谱柱
    3. 将葡萄糖甘油,蔗糖和甘油的标准混合,稀释成1,5,10,15,20 mg / L,并用提取的样品用相同的方法分析。
  5. 细胞内渗透物的定量
    1. 对于含有15mg / L各渗透质的标准混合物,使用上述方法的甘油,葡萄糖基甘油,葡萄糖和蔗糖的保留时间分别为1.7,2.1,3.3和5.5分钟(图2A)。
    2. 基于混合渗透压标准物,通过使用由Chromeleon软件计算的峰面积来制作标准曲线(图2B)。
    3. 为了定量分析蓝细菌细胞内的渗透物质,用Chromeleon软件计算离子色谱法得到的目标峰面积,然后用渗压液标准曲线确定渗透物浓度。


      图2.离子色谱图和渗压剂标准品的标准曲线A.标准混合物的色谱图。将15mg / L各种渗透压标准品混合在一起并通过离子色谱分析。 Gly,甘油; GG,葡萄糖基甘油; Glu,葡萄糖; Suc,蔗糖。 B.从集胞藻细胞中分离的样品的色谱图。通过绘制每种渗压剂标准品的峰面积和浓度来确定甘油(C),葡萄糖基甘油(D),葡萄糖(E)和蔗糖(F)的标准曲线。

数据分析

  1. 为进行统计分析,使用了IBM SPSS Statistics 19版。
  2. 为了比较两个数据集之间的差异,进行了独立样本t检验。

食谱

  1. BG11培养基(Rippka等人,1979)
    1. 准备8种10x股票如下:
      库存1(40g / L K 2 HPO 4·3H 2 O)
      库存2(75g / L MgSO 4·7H 2 O)
      库存3(36克/升CaCl 2·2H 2 O)
      库存4(6克/升批评酸)
      库存5(6克/升柠檬酸铁铵)
      库存6(1g / L EDTA·2Na)
      库存7(20g / L Na 2 CO 3)
      库存A5(2.86g / LH 3 BO 3,1.81g / L MnCl 2·4H 2 O, 0.22g / L ZnSO 4·7H 2 O,0.39g / L NaMoO 4·2H 2 O, 0.08mg / L CuSO 4·5H 2 O,0.01g / L CoCl 2·6H 2 O)
      高压灭菌器1和5在121°C下保持20分钟。
      使用前将储存在4°C的高压灭菌的股票和其他股票
    2. 将1.5g NaNO 3溶于992ml ddH 2 O中,分别向培养基中加入1ml储存液2,3,4,6,7和A5, >
    3. 将培养基在121°C高压灭菌20分钟,并在接种前补充1毫升股票1和5。
  2. 在BG11培养基中制备的饱和NaCl溶液

    在BG11培养基中溶解292.5克NaCl
    使用BG11介质调整最终音量至1 L 将121℃的NaCl溶液高压灭菌20分钟
  3. 80%(v / v)乙醇
    对于100毫升乙醇溶液,用Milli-Q水补充80毫升乙醇以达到最终体积100毫升
  4. Osmolytes标准
    1. 首先,将6毫克甘油,蔗糖,葡萄糖和葡糖基甘油溶解在100毫升Milli-Q水中
    2. 然后,根据下表制备不同浓度的渗压剂标准物
    3. 200 mM NaOH
      对于1 L的200 mM NaOH溶液,用Milli-Q水稀释16 ml 50%NaOH溶液至1 L的最终体积。

    致谢

    该方案从公开文本'Effects of reduced and enhanced glycogen pools on salt-induced sucrose production in a sucrose-secreting strain of the the homyces elongatus PCC 7942'(Qiao等人, 2017)。这项工作得到了中国杰出青年科学基金(31525002至X.鲁),山东省重点基础研究项目(ZR2017ZB0211),中德联合研究项目(授予GZ 984至X. Lu),中国科学院国家科学基金(31301018),中国科学院重点研究计划项目(ZDRW-ZS-2016-3至X.Tan),青岛创新领先企业山东泰山奖学金天赋(15-10-3-15-(31)-zch)。作者没有利益冲突或竞争利益声明。

    参考

    1. Du,W.,Liang,F.,Duan,Y.,Tan,X.和Lu,X.(2013)。 探索蓝藻对蔗糖的光合生产能力 Metab Eng em> 19:17-25。
    2. Fa,Y.,Liang,W.,Cui,H.,Duan,Y.,Yang,M.,Gao,J.and Liu,H。(2015)。 毛细管离子色谱 - 质谱法同时测定蓝细菌细胞内提取物中的葡萄糖基甘油和蔗糖。 a> J Chromatogr B Analyt Technol Biomed Life Sci 1001:169-173。
    3. Hagemann,M.,Richter,S.和Mikkat,S。(1997)。 ggtA基因编码在集胞蓝细菌中的渗透保护性化合物葡糖基甘油的转运系统的亚基, / em>的SP。菌株PCC 6803. J Bacteriol 179(3):714-720。
    4. Klähn,S.和Hagemann,M。(2011)。 蓝藻中兼容的溶质生物合成 Environ Microbiol 13( 3):551-562。
    5. Motta,A.,Romano,I.和Gambacorta,A。(2004)。 渗透压测定的快速和灵敏的NMR方法 J Microbiol方法 em> 58(2):289-294。
    6. Qiao,C.,Duan,Y.,Zhang,M.,Hagemann,M.,Luo,Q.和Lu,X。(2017)。 糖原分解降低和增强对蔗糖分泌株 Synechococcus elongatus PCC 7942. Appl Environ Microbiol 。
    7. Reed,R.H。和Stewart,W.D.P.(1985)。 淡水和海洋栖息地的单细胞蓝藻中的渗透调节和有机溶质积累 Mar Biol 88(1):1-9。
    8. Rippka,R.,Deruelles,J.,Waterbury,J.B.,Herdman,M.和Stanier,R.Y。(1979)。 蓝藻纯培养物的一般分配,菌株历史和特性。
    9. Slama,I.,Abdelly,C.,Bouchereau,A.,Flowers,T.和Savoure,A.(2015)。 非生物胁迫下盐生植物中积累的渗透保护化合物的多样性,分布和作用 Ann Bot 115(3):433-447。
    10. Song,K.,Hagemann,M.,Tan,X.和Lu,X.(2017)。 反应调节剂Slr1588调节 spsA ,但对盐的适应并不重要 Synechocystis sp。 PCC 6803. Front Microbiol 8:1176.
    11. Song,K.,Tan,X.,Liang,Y.和Lu,X.(2016)。 细长聚球蓝细菌UTEX 2973用于糖原料生产的潜力。应用微生物生物技术 100(18):7865-7875。
    12. Tan,X.,Yao,L.,Gao,Q.,Wang,W.,Qi,F。和Lu,X.(2011)。 光合作用驱动二氧化碳转化为蓝细菌中的脂肪醇和碳氢化合物 Metab Eng 13(2):169-176。
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引用:Tan, X., Song, K., Qiao, C. and Lu, X. (2018). Determination of Intracellular Osmolytes in Cyanobacterial Cells. Bio-protocol 8(8): e2812. DOI: 10.21769/BioProtoc.2812.
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