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Isolation of Heterocysts from Anabaena sp. PCC 7120
从鱼腥藻属 PCC 7120分离异型胞   

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Abstract

During combined nitrogen step-down, filaments of cyanobacterium Anabaena sp. PCC 7120 differentiate about 5-10% of vegetative photosynthetic cells into heterocysts, the specialized cells for N2 fixation (Walk, 1996). Heterocysts have a thick cell wall reducing permeation of O2 and consist of two additional layers composed of glycolipids and polysaccharides. The difference in structure and composition of the cell wall between heterocysts and vegetative cells allows separation and isolation of heterocyst. Heterocysts isolated by this protocol can be subjected to protein analysis and activity measurements, which do not require strict anaerobic conditions.

Keywords: Heterocyst(异形胞), Anabaena(鱼腥藻), Cyanobacteria(蓝藻), Nitrogenase(固氮酶), Nitrogen fixation(氮的固定)

Materials and Reagents

  1. Anabaena sp. PCC 7120
  2. BG110 growth medium-BG11 medium without addition of combined nitrogen (Rippka et al., 1979)
  3. 1 M 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid-KOH (TES) (pH 8.2)
  4. 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-NaOH (Hepes) (pH 7.2)
  5. 1 M NaCl
  6. 1 M Na-EDTA
  7. Sucrose (Merck KGaA, catalog number: K45136551417 )
  8. Lysozyme (N-acetylmuramide glycanhydrolase) (Roche Diagnostics, catalog number: 10837059001 )
  9. Extraction buffer (see Recipes)

Equipment

  1. 50 ml Falcon tubes
  2. 500 ml centrifuge vials (optional)
  3. Spectrophotometer (e.g. PerkinElmer, model: Lambda 25 )
  4. Sorvall GS-3 rotor centrifuge for 500 ml vials (optional)
  5. SL 16R centrifuge (6 x 50 ml) (e.g. Thermo Fisher Scientific)
  6. Vortex
  7. Sonicator (e.g. Labsonic U, B. Braun Melsungen AG)
  8. Orbital shaker (e.g. Infors HT, Labotron)
  9. Incubator (e.g. Certomat, B. Braun Melsungen AG)
  10. Bright-field microscope (40x magnification)
  11. Water bath or thermostat at 37 °C for Chlorophyll measurements

Procedure

  1. It is important to cultivate Anabaena sp. PCC 7120 in BG110 medium without combined nitrogen. In our laboratory, cultures are cultivated in BG110 supplemented with 10 mM TES-KOH (pH 8.2) at 30 °C under continuous light intensity of 50 μmol photons m-2 s-1 (PAR) and under agitation (about 120 RPM) to assure continuous gas exchange and kept at high CO2 (air enriched with 3% CO2).
  2. Harvest filaments during the logarithmic growth phase (OD750 = 1). To obtain 10-30 ml of purified heterocysts at the Chlorophyll (Chl) a concentration of 15-20 μg/ml use about 300-400 ml of cyanobacterial culture.
    Note: Chl a quantification, as described in Meeks and Castenholz (1971), is based on the extinction coefficient of 78,74 L/g/cm for Chl a in 90% methanol.



  3. Centrifuge filaments at 5,000 x g at room temperature for 5-10 min, discard the supernatant. If available, use a centrifuge with the rotor fitting 500 ml tubes or distribute the culture into 50 ml Falcon tubes and perform several rounds of centrifugations. In the end of centrifugation collect all cells in one 50 ml falcon tube and discard as much of growth medium as possible.
  4. Keep the pellet in falcon tube and add extraction buffer till about 30 ml, vortex thoroughly.



  5. Add 30 mg of fresh (!) lysozyme into the falcon tube, incubate for 1 h at 37 °C with continuous mixing on orbital shaker (200 RPM).
  6. Sonicate at 40 W for 1 min in water bath filled with ice-cold water to avoid excessive heating. Lysozyme treatment and sonication will digest vegetative cells, whereas heterocysts will remain intact. After this step all procedures should be performed at 4 °C and on ice.
  7. Centrifuge cell suspension at 1,000 x g for 5 min (4 °C), discard a supernatant (it can be used as a crude protein extract from vegetative cells).



  8. Resuspend the pellet in 10-15 ml of ice-cold extraction buffer, vortex thoroughly, centrifuge at 250 x g for 3 min (4 °C) and discard the supernatant. Repeat washing minimum 3 times or until the supernatant is not blue; the residual pellet containing heterocysts should be light-green.



  9. Check purity of heterocysts by microscope. Take small amount of pellet on a glass slide (optionally resuspend in small amount of extraction buffer) and visualize cells under a microscope (40x magnification). The pellet must be mostly formed by aggregates of intact roundish pale-green heterocysts, in some cells polar granules of cyanophycine might be visible. Alcian blue staining might also be used to confirm the absence of vegetative cells in preparation (Maldener et al., 2003).



    Note: Under the microscope vegetative cells and heterocysts can be easily distinguish by the shape of the cells and by the color. Due to the features of the camera, in this photo the color of cells is modified. Small arrows point to the heterocysts in filaments.
    Note: If the pellet still contains filaments consisting of several cells, then lysozyme treatment was not successful. In this case repeat lysozyme treatment with a fresh enzyme and continue with sonication.

  10. Freeze the pellet in liquid nitrogen and store at -80 °C to use later for protein isolation or resuspend in extraction buffer to reach the desirable concentration of Chl a for immediate activity measurements. After freezing heterocysts do not retain the activity.

Recipes

  1. Extraction buffer
    50 mM HEPES-NaOH (pH 7.2)
    0.4 M sucrose
    10 mM NaCl
    10 mM EDTA

Acknowledgments

The protocol was adopted from Ermakova et al. (2014), Fay (1980), Razquin et al. (1996), and Ow et al. (2009). The work was supported by the Academy of Finland (projects 271832) and Kone Foundation.

References

  1. Ermakova, M., Battchikova, N., Richaud, P., Leino, H., Kosourov, S., Isojarvi, J., Peltier, G., Flores, E., Cournac, L., Allahverdiyeva, Y. and Aro, E. M. (2014). Heterocyst-specific flavodiiron protein Flv3B enables oxic diazotrophic growth of the filamentous cyanobacterium Anabaena sp. PCC 7120. Proc Natl Acad Sci U S A 111(30): 11205-11210.
  2. Fay, P. (1980). Heterocyst isolation. Methods in Enzymology 69 No. Part C: 801-812.
  3. Ow, S. Y., Cardona, T., Taton, A., Magnuson, A., Lindblad, P., Stensjo, K. and Wright, P. C. (2008). Quantitative shotgun proteomics of enriched heterocysts from Nostoc sp. PCC 7120 using 8-plex isobaric peptide tags. J Proteome Res 7(4): 1615-1628.
  4. Razquin, P., Fillat, M. F., Schmitz, S., Stricker, O., Bohme, H., Gomez-Moreno, C. and Peleato, M. L. (1996). Expression of ferredoxin-NADP+ reductase in heterocysts from Anabaena sp. Biochem J 316 (Pt 1): 157-160.
  5. 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.
  6. Maldener, I., Hannus, S. and Kammerer, M. (2003). Description of five mutants of the cyanobacterium Anabaena sp strain PCC 7120 affected in heterocyst differentiation and identification of the transposon-tagged genes. FEMS Microbiol Lett 224(2): 205-213.
  7. Wolk, C. P. (1996). Heterocyst formation. Annu Rev Genet 30: 59-78.
  8. Meeks, J. C. and Castenholz, R. W. (1971). Growth and photosynthesis in an extreme thermophile, Synechococcus lividus (Cyanophyta). Arch Mikrobiol 78(1): 25-41.

简介

在组合氮降压期间,蓝细菌的鱼腥藻属 PCC 7120将约5-10%的营养光合细胞分化成异种细胞,这是用于N sub2固定的特化细胞(Walk,1996)。 杂合体具有厚的细胞壁,减少O 2的渗透,并且由两个由糖脂和多糖组成的附加层组成。 杂合细胞和营养细胞之间的细胞壁的结构和组成的差异允许异种细胞的分离和分离。 通过该方案分离的杂合体可以进行蛋白质分析和活性测量,其不需要严格的厌氧条件。

关键字:异形胞, 鱼腥藻, 蓝藻, 固氮酶, 氮的固定

材料和试剂

  1. 胰蛋白胨(BD,目录号:211705)
  2. 蛋白胨#3(BD,目录号:211693)
  3. 酵母提取物(BD,目录号:212720)
  4. NaCl(Acros Organics,目录号:207790250)
  5. 右旋糖(Thermo Fisher Scientific,目录号:D14-212)
  6. 胎牛血清(Atlanta Biologicals,目录号:S11150)
  7. Tris碱(Thermo Fisher Scientific,目录号:BP152)
  8. Na 2 HPO 4(Sigma-Aldrich,目录号:S5136)
  9. KCl(Sigma-Aldrich,目录号:P-9333)
  10. 10x PBS(Corning,目录号:46-013-CM)
  11. (Thermo Fisher Scientific,目录号:M35)
  12. CaCl 2(Sigma-Aldrich,目录号:C-4901)
  13. EDTA-Na 2 O 2·2H 2 O(Sigma-Aldrich,目录号:E4884)。
  14. D-生物素(Sigma-Aldrich,目录号:B4501)
  15. S-NHS-LC-生物素(Thermo Fisher Scientific,目录号:21335)
  16. Zwittergent 3-14(Fluka,目录号:40772)
  17. 蛋白酶抑制剂混合物(Roche Diagnostics,目录号:04693159001)
  18. 单体抗生物素蛋白磁珠,封闭缓冲液,再生缓冲液(Bioclone,目录号:MMI-101)
  19. 100%(w/v)TCA(Sigma-Aldrich,目录号:T0699)
  20. 丙酮(Thermo Fisher Scientific,目录号:A16F)
  21. 布鲁氏肉汤(见配方)
  22. 生物素化缓冲液(参见配方)
  23. TNKCM(参见食谱)
  24. 裂解缓冲液(见配方)
  25. TKE(参见食谱)
  26. TKEZ(参见食谱)
  27. 样品稀释缓冲液(参见配方)
  28. 阻止缓冲区(参见配方)
  29. 再生缓冲区(参见配方)
  30. 洗涤缓冲液(见配方)
  31. 洗脱缓冲液(参见配方)

设备

  1. CO 2振荡培养箱(ATR Biotech,型号:AJ125B)
  2. 台式离心机(Thermo Fisher Scientific,目录号:75004381)
  3. 超声波仪(Thermo Fisher Scientific,目录号:FB505)
  4. 台式离心机(Eppendorf,目录号:5424)
  5. 超速离心机(Beckman Coulter,目录号:A94469)
  6. Barnstead/Thermolyne Labquake Shaker Rotisserie(Labquake,目录号:C400110)
  7. DynaMag TM - Spin Magnet(Life Technologies,目录号:12320D)

程序

  1. 细胞生物素化
    1. 生长25ml的H。幽门螺杆菌液体培养物在含有10% 胎牛血清,直至晚对数期(OD <600 = 0.7)。
    2. 将来自25ml培养物的所有细菌以3,500×g在4℃下沉淀20分钟,并丢弃培养基。
    3. 将细菌沉淀重悬在10ml生物素化缓冲液中
    4. 在4℃下在3,500×g下沉淀细胞10分钟,并丢弃缓冲液。
    5. 将细菌沉淀重悬在10ml生物素化缓冲液中
    6. 加入S-NHS-LC-生物素至终浓度为200μM
    7. 在冰上孵育30分钟。
    8. 通过加入20ml TNKCM进行淬灭反应,在室温下孵育10分钟
    9. 在4℃下将细胞以3,500×g离心20分钟,并丢弃缓冲液。
    10. 将细菌沉淀重悬在10ml TNKCM中,在4℃下将细菌以3500×g离心10分钟,弃去缓冲液。重复两次以上的a  共三次洗涤。

  2. 细菌裂解和亚细胞分馏
    1. 在3ml裂解缓冲液+蛋白酶抑制剂混合物中重悬细菌沉淀
    2. 在冰上超声(4次,每次10秒,25%最大振幅)  裂解细菌,以7,000×g离心10分钟沉淀完整细菌,收集 裂解上清液
    3. 通过在4℃下以40,000×g离心裂解物30分钟来沉淀膜。
    4. 弃去上清液。
    5. 用TKE冲洗膜沉淀三次。
    6. 通过用干净的移液管尖端刮擦收集膜沉淀,并重悬于1ml TKEZ +蛋白酶抑制剂混合物中。
    7. 在4℃下溶解1小时
    8. 1 M Na-EDTA
    9. 蔗糖(Merck KGaA,目录号:K45136551417)
    10. 溶菌酶(N-乙酰甘露糖聚糖水解酶)(Roche Diagnostics,目录号:10837059001)
    11. 提取缓冲液(参见配方)

    设备

    1. 50ml Falcon管
    2. 500 ml离心管(可选)
    3. 分光光度计(例如,PerkinElmer,型号:Lambda 25)
    4. Sorvall GS-3转子离心机用于500 ml小瓶(可选)
    5. SL 16R离心机(6×50ml)(例如Thermo Fisher Scientific)
    6. 涡流
    7. 超声波仪(如 Labsonic U,B. Braun Melsungen AG)
    8. 轨道摇床(例如 Infors HT,Labotron)
    9. 孵化器(例如 Certomat,B.Braun Melsungen AG)
    10. 明视场显微镜(40x放大)
    11. 37℃的水浴或恒温器测定叶绿素。

    程序

    1. 培养鱼腥藻 sp。 PCC 7120在没有组合氮的BG11 0培养基中。 在我们的实验室中,将培养物在补充有10mM TES-KOH(pH 8.2)的BG11 0培养基中 (PAR)和搅拌(约120RPM)的连续光强度下,在50μmol光子的连续光强度下30℃,以确保连续气体交换并保持在高CO 2(富含3%CO 2的空气)。
    2. 在对数生长期(OD 750 = 1)中收获长丝。为了在叶绿素(Chl)获得10-30ml纯化的杂合体,15-20μg/ml的浓度使用约300-400ml的蓝藻培养物。
      注意:如Meeks和Castenholz(1971)所述,Chl的定量是基于90%甲醇中Chl a的消光系数78,74L/g/cm。



    3. 在室温下以5,000xg离心细丝5-10分钟,弃去上清液。如果可用,使用离心机与转子配件500毫升管或分配培养到50毫升Falcon管,并进行几轮离心。在离心结束时,将所有细胞收集在一个50ml的falcon管中,并尽可能多地丢弃生长培养基。
    4. 保持沉淀在猎鹰管,并添加提取缓冲液,直到约30毫升,彻底涡旋。



    5. 加入30mg新鲜(!)溶菌酶到falcon管中,在37℃下在定轨振荡器(200RPM)上连续混合孵育1小时。
    6. 在装有冰冷水的水浴中在40W超声处理1分钟,以避免过度加热。溶菌酶处理和超声处理将消化营养细胞,而异源细胞将保持完整。在该步骤之后,所有程序应在4℃和冰上进行。
    7. 在1,000×g离心细胞悬浮液5分钟(4℃),弃去上清液(其可以用作营养细胞的粗蛋白提取物)。



    8. 将沉淀重悬于10-15ml冰冷的提取缓冲液中,充分涡旋,250×g离心3分钟(4℃),弃去上清液。重复洗涤最少3次,或直到上清液不蓝色;含有异囊的残留球团应为浅绿色。



    9. 通过显微镜检查杂合子的纯度。取少量的小球在玻片上(任选地重悬在少量的提取缓冲液中),并在显微镜下观察细胞(40倍放大)。颗粒必须主要由完整的圆形浅绿色杂合体的聚集体形成,在一些细胞中,氰甙的极性颗粒可能是可见的。 Alcian蓝染色也可用于确认制备中营养细胞的缺失(Maldener等人,2003)。



      注意:在显微镜下,营养细胞和杂合细胞可以容易地通过细胞的形状和颜色来区分。由于相机的功能,在这张照片细胞的颜色被修改。小箭头指向细丝中的异囊。
      注意:如果颗粒仍然含有由几个细胞组成的细丝,则溶菌酶处理不成功。在这种情况下,用新鲜酶重复溶菌酶处理,并继续进行超声处理。

    10. 在液氮中冷冻沉淀并储存在-80℃下以便稍后用于蛋白质分离或重悬于提取缓冲液中以达到所需的Ch1a浓度,用于即时活性测量。冻结后异种细胞不保留活性。

    食谱

    1. 提取缓冲区
      50mM HEPES-NaOH(pH7.2) 0.4 M蔗糖 10mM NaCl 10 mM EDTA

    致谢

    该方案采用于Ermakova等人(2014),Fay(1980),Razquin等人(1996)和Ow等人(1996) (2009)。 这项工作得到了芬兰学院(项目271832)和Kone基金会的支持。

    参考文献

    1. Ermakova,M.,Battchikova,N.,Richaud,P.,Leino,H.,Kosourov,S.,Isojarvi,J.,Peltier,G.,Flores,E.,Cournac,L.,Allahverdiyeva, Aro,EM(2014)。 Heterocyst特异性黄病毒蛋白Flv3B能够使丝状蓝细菌的鱼腥藻属生长繁殖。 > sp。 PCC 7120. Proc Natl Acad Sci U S A 111(30):11205-11210。
    2. Fay,P。(1980)。 Heterocyst隔离。方法in Enzymology 69 No.Cart:801-812。
    3. Ow,S.Y.,Cardona,T.,Taton,A.,Magnuson,A.,Lindblad,P.,Stensjo,K.and Wright, 来自Nostoc sp的富集杂合子的定量鸟枪法蛋白质组学。 PCC 7120使用8重等量异位肽标签。蛋白质组学 7(4):1615-1628。
    4. Razquin,P.,Fillat,M.F.,Schmitz,S.,Stricker,O.,Bohme,H.,Gomez-Moreno,C.and Peleato,M.L。(1996)。 铁氧还蛋白-NADP +还原酶在来自鱼腥藻的异核中的表达生物化学316(Pt 1):157-160。
    5. Rippka,R.,Deruelles,J.,Waterbury,J.B.,Herdman,M。和Stanier,R.Y。(1979)。 蓝藻菌纯培养物的通用分配,菌株历史和特性 J Gen Microbiol 111(1):1-61。
    6. Maldener,I.,Hannus,S。和Kammerer,M。(2003)。 描述了蓝藻的五种突变体(Anabaena) sp菌株PCC 7120异核分化和转座子标记的基因的鉴定。 FEMS Microbiol Lett 224(2):205-213。
    7. Wolk,C.P。(1996)。 Heterocyst形成。 Annu Rev Genet 30:59-
    8. Meeks,J.C。和Castenholz,R.W。(1971)。 极端嗜热菌中的生长和光合作用,聚球藻(Cyanophyta)。 Arch Mikrobiol 78(1):25-41。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Ermakova, M. and Allahverdiyeva, Y. (2015). Isolation of Heterocysts from Anabaena sp. PCC 7120. Bio-protocol 5(8): e1456. DOI: 10.21769/BioProtoc.1456.
  2. Ermakova, M., Battchikova, N., Richaud, P.,Leino, H., Kosourov, S., Isojarvi, J., Peltier, G., Flores, E., Cournac, L.,Allahverdiyeva, Y. and Aro, E. M. (2014). Heterocyst-specificflavodiiron protein Flv3B enables oxic diazotrophic growth of the filamentouscyanobacterium Anabaena sp. PCC 7120. Proc Natl Acad Sci US A 111(30): 11205-11210.
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