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Rapid Nitrate Reduction Assay with Intact Microbial Cells or Spores
应用完整微生物细胞或孢子进行硝酸快速还原分析   

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Abstract

Many microorganisms have the capacity to use nitrate as a respiratory electron acceptor. Reduction of nitrate is catalyzed by a multi-subunit nitrate reductase that is often located associated with the cytoplasmic membrane and has its active site oriented toward the cytoplasm. This means that nitrate must be transported into the cell and often this occurs concomitantly with the export of the reduced nitrite product. Often nitrate and nitrite transport are coupled through the action of a nitrate: nitrite antiporter. Microbial cells, spores and mycelium harbour intracellular storage compounds such as trehalose or glycogen that, upon metabolism, function as endogenous electron donors for nitrate reduction. It is also possible to use glucose supplied exogenously as a substrate for nitrate reduction. The method described here allows the direct analysis of nitrate reduction by whole cell material without the requirement for artificial electron donors. This method is also applicable to the study of spores, particularly those of Streptomyces species (Fischer et al., 2013). The paper by Fischer et al. 2013 provides examples of datasets for the method presented below.

Keywords: Nitrate reductase(硝酸还原酶), Metabolism(代谢), Nitrite(亚硝酸盐), Respiration(呼吸), Dormancy(休眠)

Materials and Reagents

  1. Freshly harvested Streptomyces spores in water or mycelium in 50 mM MOPS buffer (pH 7)
  2. Tryptic Soy Broth (TSB) (Sigma-Aldrich, catalog number: 22092 )
  3. 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (Roth North America)
  4. Sulfanilic acid (Sigma-Aldrich, catalog number: 251917 )
  5. N-(1-naphthyl)-ethylenediamine dihydrochloride (Roth North America)
  6. Hydrochloric acid (Roth North America)
  7. Sodium hydroxide pellets (Roth North America)
  8. Soya flour (from local supermarket)
  9. D-mannitol (Sigma-Aldrich catalogue number 63560 )
  10. Agar-agar (Kobe I) (Roth North America)
  11. Sulfanilic acid solution (see Recipes)
  12. N-(1-naphthyl)-ethylenediamine solution (see Recipes)
  13. MOPS-buffer (see Recipes)
  14. SFM agar (see Recipes)

Equipment

  1. Cotton wool (standard issue from local pharmacy)
  2. 0.22 µm pore-size filters (PVDF) (Roth North America)
  3. 30 °C rotary shaker
  4. Baffled Erlenmeyer flasks (500 ml) (Glasgerätebau Ochs, Laborfachhandel e. K., catalog number: 100500 )
  5. Standard-sized plastic Petri dishes for bacterial growth and spore preparation
  6. Gas-tight glass Hungate tubes (16 ml) for anaerobic work with butyl rubber septa (Glasgerätebau Ochs, Laborfachhandel e. K., catalog number: 1020471 )
  7. Cooled table-top centrifuge (e.g. Eppendorf)
  8. Cylinder of pure nitrogen gas (e.g. Linde or local supplier)
  9. Needles 0.6 mm Gauge (B. Braun Melsungen AG)
  10. Water bath or heating block (Biometra)
  11. Spectrophotometer measuring absorption in the visible range

Procedure

  1. Incubation assay for Streptomyces spores
    1. Suspensions of freshly harvested spores, which were washed with water and filtered twice through cotton wool (Kieser et al., 2000), were adjusted to an OD450nm of 10 or 20 (1 ml of an OD450 = 1 is equivalent to 3.5 x 108 spores ml−1 or 2.37 x 108 cfu.).
    2. For the assay, 500 μl of the spore suspension was added to 500 μl of buffer (100 mM MOPS-NaOH, pH 7.0, 10 mM sodium nitrate) in gas-tight Hungate vials. The gas in the 15 ml headspace was exchanged against pure nitrogen and flushed with nitrogen for an additional 5 min through the closed septum.
    3. Hungate vials were incubated by slowly shaking at 30 °C for 5 h, or longer as necessary if the anticipated enzyme activity was low.
    4. At a defined time after starting the incubation the nitrate reduction reaction in these samples was terminated by heating the vials with the spores at 80 °C for 10 min. The heat-killed spores were then pelleted by centrifugation in a bench-top centrifuge (20,000 x g) for 5 min.
    5. The spore-free supernatant was used for nitrite determination either immediately or was stored at -20 °C until required.

  2. Incubation assay for Streptomyces mycelia grown in TSB liquid culture
    1. Suspensions of freshly grown, highly dispersed mycelium were washed twice with MOPS-buffer, and adjusted to the desired amount of mycelium using the methylene blue adsorption method (Fischer and Sawers, 2013; see notes below) (1,000 CAE are comparable with a dry weight of 1 mg Streptomyces mycelium growing in TSB.).
    2. For the assay, 500 μl of the mycelium suspension were added to 500 μl of buffer (100 mM MOPS-NaOH, pH 7.0, 10 mM sodium nitrate) in gas-tight Hungate vials. The gas in the 15 ml headspace was exchanged against pure nitrogen and flushed with nitrogen for an additional 5 min through the closed septum.
    3. The Hungate vials were incubated by slowly shaking at 30 °C for 30 min, or longer as necessary.
    4. For the determination of rates of nitrate reduction in mycelium, samples were removed at different times. The nitrate reduction reaction in these samples was terminated by heating the vials at 80 °C for 10 min.
    5. The mycelium-free supernatant, obtained by centrifuging the vials for 5 min at 5,000 x g in a bench-top centrifuge, was used for nitrite determination.

  3. Nitrite determination in the supernatant of bacterial cultures (Rider and Mellon, 1946)
    1. Aliquots (0.5 ml) of the supernatants were mixed by briefly pipetting up and down twice with one volume of freshly prepared sulfanilic acid solution and incubated at room temperature for at least 5 min. If the absorbance levels were too high, then supernatants were appropriately diluted (1:5 or 1:10) prior to repeating the nitrite determination assay.
    2. Subsequently, a half volume of N-(1-naphthyl)-ethylenediamine solution was added and the incubation was continued for exactly 10 min.
    3. The absorption was measured at 540 nm and the nitrite concentration was calculated using a calibration curve with nitrite.

Notes

  1. A modification of the method to allow a rapid and qualitative assessment for incubation/ growth of spores and mycelia on solid nutrient broth is described in Fischer et al. (2013).
  2. The precise details of the methylene blue adsorption method are available in Fischer and Sawers (2013). A bioprotocol detailing this method will be submitted in the near future.

Recipes

  1. Sulfanilic acid solution
    Sulfanilic acid (4% w/v) was solubilized in 25% (w/v) HCl at RT to deliver a saturated solution
  2. N-(1-naphthyl)-ethylenediamine solution
    Used as 0.2 % (w/v) solution in distilled water
    Can be stored at 4 °C for up to one week
  3. MOPS-buffer
    Adjusted to pH 7.2 with NaOH
    Sterilized by filtration through a 0.22 µm filter
  4. SFM agar
    20 g L−1 soya flour
    20 g L−1 D-mannitol
    Agar-agar (Kobe I)

Acknowledgments

This method was reliant on the assay to determine nitrite concentrations developed by Rider and Mellon (1946), published in Industrial and Engineering Chemistry Analytical Edition. This work was supported by the Deutsche Forschungsgemeinschaft (SA 494/4-1).

References

  1. Fischer, M. and Sawers, R. G. (2013). A universally applicable and rapid method for measuring the growth of Streptomyces and other filamentous microorganisms by methylene blue adsorption-desorption. Appl Environ Microbiol 79(14): 4499-4502.
  2. Fischer, M., Falke, D. and Sawers, R. G. (2013). A respiratory nitrate reductase active exclusively in resting spores of the obligate aerobe Streptomyces coelicolor A3(2). Mol Microbiol 89(6): 1259-1273.
  3. Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F. and Hopwood, D. A. (2000). Practical Streptomyces Genetics. Norwich: The John Innes Foundation.
  4. Rider, B. and Mellon, M. (1946). Colorimetric determination of nitrites. Industrial & Engineering Chemistry Analytical Edition 18(2): 96-99.

简介

许多微生物具有使用硝酸盐作为呼吸电子受体的能力。硝酸盐的还原由多亚基硝酸盐还原酶催化,所述多亚基硝酸盐还原酶通常位于与细胞质膜相关联并且其活性位点朝向细胞质。这意味着硝酸盐必须被运输到细胞中,并且通常这伴随着还原的亚硝酸盐产物的输出而发生。通常硝酸盐和亚硝酸盐转运通过硝酸盐:亚硝酸盐反向转运体的作用偶联。微生物细胞,孢子和菌丝体含有细胞内储存化合物,例如海藻糖或糖原,其在代谢后充当用于硝酸盐还原的内源性电子供体。还可以使用外源供应的葡萄糖作为硝酸盐还原的底物。这里描述的方法允许直接分析全细胞材料的硝酸盐还原,而不需要人工电子供体。该方法也适用于孢子的研究,特别是链霉菌属物种的研究(Fischer等人,2013)。 Fischer等人的论文2013年提供了下面介绍的方法的数据集示例。

关键字:硝酸还原酶, 代谢, 亚硝酸盐, 呼吸, 休眠

材料和试剂

  1. 在50mM MOPS缓冲液(pH7)中的水或菌丝体中新鲜收获的链霉菌孢子
  2. 胰蛋白酶大豆肉汤(TSB)(Sigma-Aldrich,目录号:22092)
  3. 3-(N-吗啉代)丙磺酸(MOPS)缓冲液(Roth North America)
  4. 对氨基苯磺酸(Sigma-Aldrich,目录号:251917)
  5. N-(1-萘基) - 乙二胺二盐酸盐(Roth North America)
  6. 盐酸(Roth北美洲)
  7. 氢氧化钠丸(Roth North America)
  8. 大豆粉(来自当地超市)
  9. D-甘露醇(Sigma-Aldrich目录号63560)
  10. 琼脂(Kobe I)(Roth北美)
  11. 对氨基苯磺酸溶液(见配方)
  12. N-(1-萘基) - 乙二胺溶液(参见配方)
  13. MOPS缓冲区(参见配方)
  14. SFM琼脂(见配方)

设备

  1. 棉毛(当地药店的标准问题)
  2. 0.22μm孔径过滤器(PVDF)(Roth North America)
  3. 30°C旋转振动器
  4. 将挡板锥形烧瓶(500ml)(GlasgerätebauOchs,Laborfachhandel e.K.,目录号:100500)
  5. 标准尺寸的塑料培养皿,用于细菌生长和孢子制备
  6. 气密玻璃用于用丁基橡胶隔片(GlasgerätebauOchs,Laborfachhandel e.K.,目录号:1020471)进行厌氧工作的Hungate管(16ml)
  7. 冷却的台式离心机(例如 Eppendorf)
  8. 纯氮气瓶(例如林德或本地供应商)
  9. 针0.6mm规格(B.Braun Melsungen AG)
  10. 水浴或加热块(Biometra)
  11. 分光光度计测量在可见光范围内的吸收率

程序

  1. 链霉菌孢子的孵育测定
    1. 新鲜收获的孢子悬浮液,用水洗涤 并通过棉绒过滤两次(Kieser等人,2000) 调节至OD 450nm的10或20(1ml的OD 450 = 1)是等效的 至3.5×10 8个孢子ml -1 或2.37×10 8 cfu。)。
    2. 对于测定,500   μl的孢子悬浮液加入500μl缓冲液(100mM)中 MOPS-NaOH,pH7.0,10mM硝酸钠)在气密的Hungate小瓶中。 的   将15ml顶部空间中的气体与纯氮气交换 用氮气冲洗通过封闭的隔膜另外5分钟
    3. 通过在30℃下缓慢摇动5小时孵育Hungate小瓶 如果预期的酶活性低,则需要更长时间
    4. 在开始孵育后的确定时间,硝酸盐还原 这些样品中的反应通过用小瓶加热来终止 孢子在80℃下10分钟。 然后将热灭活的孢子沉淀   在台式离心机(20,000×g)中离心5分钟
    5. 无孢子的上清液立即用于亚硝酸盐测定,或者储存在-20℃直至需要
  2. 对在TSB液体培养物中生长的链霉菌属菌丝体的孵育测定
    1. 洗涤新鲜生长的高度分散的菌丝体的悬浮液 用MOPS缓冲液洗涤两次,并调节至所需量的菌丝体 使用亚甲蓝吸附法(Fischer和Sawers,2013; 见下面的注释)(1,000 CAE与在TSB中生长的1mg链霉菌属菌丝的干重相当)。
    2. 对于测定,500μl 的菌丝体悬浮液加入到500μl缓冲液(100mM)中 MOPS-NaOH,pH7.0,10mM硝酸钠)在气密的Hungate小瓶中。 的   将15ml顶部空间中的气体与纯氮气交换 用氮气冲洗通过封闭的隔膜另外5分钟
    3. 通过在30℃缓慢摇动30分钟或根据需要更长时间来孵育Hungate小瓶
    4. 为了测定菌丝体中硝酸盐还原的速率, 在不同时间取出样品。 硝酸还原反应 在这些样品中通过在80℃下加热小瓶10秒而终止 min。
    5. 通过离心得到无菌的无菌上清液 使用在台式离心机中以5,000×g离心5分钟的小瓶   亚硝酸盐测定。

  3. 细菌培养物上清液中的亚硝酸盐测定(Rider和Mellon,1946)
    1. 通过简单吹吸混合上清液的等分试样(0.5ml)   并用一体积的新制备的对氨基苯磺酸降低两次 溶液并在室温下温育至少5分钟。 如果 吸光度水平太高,然后适当地加入上清液 在重复亚硝酸盐测定之前稀释(1:5或1:10) 测定
    2. 随后, 加入N-(1-萘基) - 乙二胺溶液,保温   继续正好10分钟。
    3. 在540nm测量吸收,并使用亚硝酸盐的校准曲线计算亚硝酸盐浓度

笔记

  1. 修改 方法,以允许快速和定性评估孵化/ 在固体营养肉汤上的孢子和菌丝体的生长在Fischer等人(2013)中描述。
  2. 亚甲蓝的确切细节 吸附方法可在Fischer和Sawers(2013)中找到。 生物协议详述 这个方法将在不久的将来提交。

食谱

  1. 对氨基苯磺酸溶液
    在室温下将对氨基苯磺酸(4%w/v)溶解在25%(w/v)HCl中以提供饱和溶液
  2. N-(1-萘基) - 乙二胺溶液
    用作蒸馏水中的0.2%(w/v)溶液 可以在4°C下存储长达一个星期
  3. MOPS缓冲区
    用NaOH调节至pH 7.2 通过0.22μm过滤器过滤灭菌
  4. SFM琼脂
    20g L -1 -1大豆粉 20g L -1 -D-甘露糖醇 琼脂(Kobe I)

致谢

这种方法依赖于由Rider和Mellon(1946)开发的测定亚硝酸盐浓度的测定,发表在Industrial and Engineering Chemistry Analytical Edition中。 这项工作得到了德意志交流协会(SA 494/4-1)的支持。

参考文献

  1. Fischer,M。和Sawers,R.G。(2013)。 用于衡量链霉菌属生长的普遍适用且快速的方法和其他丝状微生物通过亚甲基蓝吸附 - 解吸。 Appl Environ Microbiol 79(14):4499-4502。
  2. Fischer,M.,Falke,D。和Sawers,R.G。(2013)。 呼吸性硝酸还原酶仅在专性气单胞菌链球菌的休眠孢子中有效。 > A3(2)。 Mol Microbiol 89(6):1259-1273。
  3. Kieser,T.,Bibb,M.J.,Buttner,M.J.,Chater,K.F.and Hopwood,D.A。(2000)。 实用链霉菌遗传学。诺维奇:约翰·因尼斯基金会
  4. Rider,B。和Mellon,M。(1946)。 亚硝酸盐的比色测定。工业& Engineering Chemistry Analytical Edition 18(2):96-99。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Fischer, M., Falke, D. and Sawers, R. G. (2014). Rapid Nitrate Reduction Assay with Intact Microbial Cells or Spores. Bio-protocol 4(12): e1154. DOI: 10.21769/BioProtoc.1154.
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