Nitrite Reduction Assay for Whole Pseudomonas Cells

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The second step of the dissimilatory denitrification pathway in which nitrite (NO2-) is converted to nitric oxide (NO) is catalyzed by the enzyme nitrite reductase. Two distinct enzymes are found in nature that catalyze this reaction, and they contain different metal sites, either iron (Fe), in the form of heme, or copper (Cu) (Zumft, 1997). The Pseudomonas stutzeri (P. stutzeri) RCH2 strain used in this assay contains both an Fe and a Cu form of nitrite reductase. In this assay, total nitrite reductase activity can be measured in whole cells using fumarate or some other carbon source as an electron source by measuring the disappearance of nitrite over time (Thorgersen et al., 2015).

Keywords: Nitrite reductase(亚硝酸还原酶), Pseudomonas(铜绿假单胞菌), Griess(格里斯)

Materials and Reagents

  1. Borosilicate glass culture tubes, 16 x 125 mm (VWR International, catalog number: 47729-578 )
  2. Hungate tubes (16 x 125 mm) with butyl rubber stopper (Bellco Glass, catalog number: 2047-16125 )
  3. Pseudomonas cells
  4. M9, minimal salts, 5x (Sigma-Aldrich, catalog number: M6030 )
  5. Bacto yeast extract technical (BD Biosciences, catalog number: 288610 )
  6. Potassium phosphate (dibasic, powder) (VWR International, J.T. Baker®, catalog number: 3252-05 )
  7. Potassium phosphate (monobasic, crystal) (VWR International, J.T. Baker®, catalog number: 3246-05 )
  8. Sodium fumarate dibasic (Sigma-Aldrich, catalog number: F1506 )
  9. Sodium nitrate (VWR International, J.T. Baker®, catalog number: 3770-01 )
  10. Sodium nitrite (VWR International, J.T. Baker®, catalog number: 3780-01 )
  11. Griess reagent (Sigma-Aldrich, catalog number: G4410 )
  12. High purity 100% argon gas (Airgas, catalog number: ARHP300 )
  13. 50 mM potassium phosphate buffer (pH 7.0) (see Recipes)
  14. Assay buffer (see Recipes)


  1. Allegra 25R centrifuge (Beckman Coulter)
  2. Spectrophotometer (measuring absorption in the visible range)
  3. Innova 4230 refrigerated incubator shaker (New Brunswick Scientific)


  1. Cultures (5 ml) of P. stutzeri RCH2 were grown anaerobically with shaking (250 rpm) at 30 °C in Hungate tubes on M9 salts supplemented with 20 mM fumarate as a carbon source, 20 mM nitrate as an electron acceptor, and 0.5 g/L yeast extract. Cells were harvested during late log phase (OD650 0.7-0.9) by centrifugation at 4 °C (10 min at 5,000 x g) and washed once with pre-chilled (4 °C) 50 mM potassium phosphate buffer (pH 7.0) before being resuspended in the same buffer at approximately 3-7 x 109 cells/ml.
  2. In a culture tube, 500 μl of cell suspension was added to 4.5 ml of assay buffer. The assay was incubated at 30 °C with slow shaking (150 rpm) in an incubator. The volume of cells added can be changed to optimize reaction speed.
  3. Samples (100 μl) were taken every 5 min or longer as needed for 20 min to1 h, and were diluted into 900 μl distilled water. One ml of Griess reagent was added to the samples and they were incubated for 15 min at room temperature.
  4. The visible absorption at 540 nm was measured for each sample, and nitrite concentrations were calculated using a nitrite standard curve made in 50 mM potassium phosphate buffer (pH 7.0) (0-100 μM nitrite) (Figure 1). A unit of nitrite reductase activity catalyzed the reduction of 1 nmol of nitrite/min. A negative control using 50 mM potassium phosphate buffer (pH 7.0) without cells was also performed.

Representative data

Figure 1. Standard nitrite curve from 0-100 µM nitrite mixed 1:1 with Griess reagent. A. The red-pink color develops when the Griess reagent is added to the nitrite containing sample. B. A linear standard curve is shown with values ranging from 0-100 µM nitrite.


  1. If there is a concern about oxygen lability of the nitrite reductase enzyme, the method can optionally be performed anoxically with the use of sealed Hungate tubes where the headspace is exchanged with high purity 100% argon. The nitrite reductase activity of P. stutzeri RCH2 was stable in the presence of oxygen over the time period of the assay.
  2. Other carbon sources other than fumarate can be used to fit the carbon source preferences of the microorganism being studied including but not limited to lactate, pyruvate, and glucose. Nitrite reductase may not be as highly expressed if the microorganism can ferment the carbon source used rather than depending on nitrate or nitrite as electron acceptors. Additionally, cells were grown anaerobically in the presence of nitrate as an electron acceptor to increase nitrite reductase expression. Nitrite (2-10 mM) could also be used as an electron acceptor depending on the tolerance of the organism being assayed.


  1. 50 mM potassium phosphate buffer (pH 7.0)
    1. 1 M solutions of KH2PO4 (A) and K2HPO4 (B) were made separately.
    2. A mixture containing 39% (v/v) solution A and 61% (v/v) solution B was made.
    3. The pH of the mixture was adjusted to pH 7.0 with solution A or B before diluting.
    4. Dilute the stock solution 1:20 with distilled water (final concentration: 50 mM).
  2. Assay buffer
    50 mM potassium phosphate buffer (pH 7.0)
    40 mM fumarate
    1 mM nitrite (pH 7.0)


This material by ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory, is based upon work supported by the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research, under contract number DE-AC02-05CH11231.


  1. Thorgersen, M. P., Lancaster, W. A., Vaccaro, B. J., Poole, F. L., Rocha, A. M., Mehlhorn, T., Pettenato, A., Ray, J., Waters, R. J., Melnyk, R. A., Chakraborty, R., Hazen, T. C., Deutschbauer, A. M., Arkin, A. P. and Adams, M. W. (2015). Molybdenum availability is key to nitrate removal in contaminated groundwater environments. Appl Environ Microbiol 81(15): 4976-4983.
  2. Zumft, W. G. (1997). Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 61(4): 533-616.


其中亚硝酸盐(NO 2 - )被转化为一氧化氮(NO)的异化脱氮途径的第二步由亚硝酸盐还原酶催化。 在自然界中发现催化该反应的两种不同的酶,并且它们包含不同的金属位点,铁(Fe),血红素或铜(Cu)形式(Zumft,1997)。 用于该测定的 Pseudomonas stutzeri ( stutzeri )RCH2菌株含有Fe和Cu形式的亚硝酸还原酶。 在该测定中,可以通过测量亚硝酸盐随时间的消失,使用富马酸盐或一些其它碳源作为电子源在全细胞中测量总亚硝酸盐还原酶活性(Thorgersen等人,2015)。

关键字:亚硝酸还原酶, 铜绿假单胞菌, 格里斯


  1. 硼硅酸盐玻璃培养管,16×125mm(VWR International,目录号:47729-578)
  2. 具有丁基橡胶塞(Bellco Glass,目录号:2047-16125)的橡胶管(16×125mm)
  3. 假单胞单元格
  4. M9,极小盐,5x(Sigma-Aldrich,目录号:M6030)
  5. Bacto酵母提取物技术(BD Biosciences,目录号:288610)
  6. 磷酸钾(二碱价,粉末)(VWR International,J.T.Baker ,目录号:3252-05)
  7. 磷酸钾(一碱价,晶体)(VWR International,J.T.Baker ,目录号:3246-05)
  8. 富马酸二钠(Sigma-Aldrich,目录号:F1506)
  9. 硝酸钠(VWR International,J.T.Baker ,目录号:3770-01)
  10. 亚硝酸钠(VWR International,J.T.Baker ,目录号:3780-01)
  11. Griess试剂(Sigma-Aldrich,目录号:G4410)
  12. 高纯度100%氩气(Airgas,目录号:ARHP300)
  13. 50 mM磷酸钾缓冲液(pH 7.0)(参见配方)
  14. 测试缓冲区(参见配方)


  1. Allegra 25R离心机(Beckman Coulter)
  2. 分光光度计(测量可见光吸收)
  3. Innova 4230冷藏保温摇床(New Brunswick Scientific)


  1. 培养物(5ml)。 Stutzeri RCH2在30℃下在补充有20mM富马酸盐作为碳源,20mM硝酸盐作为电子受体的M9盐和0.5g/L酵母提取物的Hungate管中摇动(250rpm)厌氧生长, 。通过在4℃下离心(在5,000xg下10分钟),在对数后期(OD <650> 0.7-0.9)期间收获细胞,并用预冷冻的(4℃) ℃)50mM磷酸钾缓冲液(pH 7.0)中,然后以约3-7×10 9个细胞/ml重悬于相同缓冲液中。
  2. 在培养管中,将500μl细胞悬浮液加入4.5ml测定缓冲液中。将测定在30℃下在缓慢摇动(150rpm)下在孵育器中温育。可以改变加入的细胞体积以优化反应速度
  3. 每5分钟或更长时间取样(100μl)20分钟至1小时,并稀释到900μl蒸馏水中。将1ml Griess试剂加入样品中,并将它们在室温下孵育15分钟温度
  4. 测量每个样品在540nm的可见光吸收,并使用在50mM磷酸钾缓冲液(pH 7.0)(0-100μM亚硝酸盐)(图1)中制备的亚硝酸盐标准曲线计算亚硝酸盐浓度。 亚硝酸还原酶活性单位催化1nmol亚硝酸盐/分钟的还原。 还进行了使用不含细胞的50mM磷酸钾缓冲液(pH 7.0)的阴性对照。


图1.0-100μM亚硝酸盐与Griess试剂混合的1:1的标准亚硝酸盐曲线。 A.当将Griess试剂加入含亚硝酸盐的样品中时,产生红 - 粉色。 B.显示的线性标准曲线的值为0-100μM亚硝酸盐。


  1. 如果担心亚硝酸还原酶的氧不稳定性,则该方法可以任选地使用密封的Hungate管进行缺氧,其中顶空与高纯度100%氩交换。 P的亚硝酸还原酶活性。 stutzeri RCH2在氧的存在下在测定的时间段内是稳定的。
  2. 除富马酸盐外的其它碳源可用于适应所研究的微生物的碳源优选,包括但不限于乳酸盐,丙酮酸盐和葡萄糖。如果微生物可以发酵所用的碳源而不是依赖于硝酸盐或亚硝酸盐作为电子受体,亚硝酸还原酶可能不会高度表达。此外,细胞在作为电子受体的硝酸盐存在下厌氧生长以增加亚硝酸盐还原酶表达。根据所测定的生物体的耐受性,亚硝酸盐(2-10mM)也可以用作电子受体。


  1. 50mM磷酸钾缓冲液(pH7.0)
    1. 分别制备1M KH 2 PO 4(A)和K 2 HPO 4(B)的1M溶液。
    2. 制备含有39%(v/v)溶液A和61%(v/v)溶液B的混合物
    3. 在稀释前,用溶液A或B将混合物的pH调节至pH 7.0。
    4. 用蒸馏水稀释储备溶液1:20(最终浓度:50mM)。
  2. 测定缓冲区
    50mM磷酸钾缓冲液(pH7.0) 40mM富马酸盐 1mM亚硝酸盐(pH7.0)


本材料由ENIGMA(生态系统和网络与基因和分子组装集成)( http://enigma.lbl.gov ),劳伦斯伯克利国家实验室的科学焦点领域计划,是基于美国能源部,科学办公室,生物和环境研究办公室,合同号DE-AC02-05CH11231支持的工作。


  1. Thorersen,MP,Lancaster,WA,Vaccaro,BJ,Poole,FL,Rocha,AM,Mehlhorn,T.,Pettenato,A.,Ray,J.,Waters,RJ,Melnyk,RA,Chakraborty,R.,Hazen, TC,Deutschbauer,AM,Arkin,AP和Adams,MW(2015)。 钼的可用性是污染地下水环境中硝酸盐去除的关键。 Appl Environ Microbiol 81(15):4976-4983。
  2. Zumft,W.G。(1997)。 反硝化的细胞生物学和分子基础 Microbiol Mol Biol Rev < (4):533-616。

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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Thorgersen, M. P. and Adams, M. W. (2016). Nitrite Reduction Assay for Whole Pseudomonas Cells. Bio-protocol 6(10): e1818. DOI: 10.21769/BioProtoc.1818.

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