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Quantification of Flavin Production by Bacteria
细菌黄素产量的定量测定

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Abstract

This protocol provides a simple and fast method of quantification for intracellular flavin content, and for flavin secretion by bacteria. Intracellular flavins are extracted from bacterial pellets, and secreted flavins are examined in the cell growth medium. Flavins are separated and measured using HPLC with fluorescence detection, and quantified based on a comparison to standards.

Keywords: Riboflavin Biosynthetic Pathway(核黄素生物合成途径), FMN(FMN), FAD(时尚), Alpha-proteobacteria(α-变形), HPLC(高效液相色谱法)

Materials and Reagents

  1. Sinorhizobium meliloti (S. meliloti 1021, Galibert et al., 2001)
    Note: The protocol can be also applied to other bacteria.
  2. Bio-Rad Protein Assay Kit I (Sigma-Aldrich, catalog number: 500-0001 )
  3. Flavin standards:
    1. Riboflavin (Sigma-Aldrich, catalog number: 95170 )
    2. FMN (Sigma-Aldrich, catalog number: F2253 )
    3. FAD (Sigma-Aldrich, catalog number: F6625 )
    4. Lumichrome (Acros, catalog number: 146930010 )
    Note: FMN and FAD were further purified as described previously (Sandov et al., 2008).
  4. Ammonium formate
  5. Formic acid
  6. Methanol
  7. YMB medium (Somerville and Kahn, 1983) (see Recipes) (1 plate per sample)
  8. MMNH4 medium (Somerville and Kahn, 1983) (see Recipes) (6 ml per sample)
  9. Extraction buffer (see Recipes)
  10. HPLC mobile phase (see Recipes)
    Note: Except as otherwise noted, all other chemicals were obtained from Sigma-Aldrich.

Equipment

  1. Aluminum foil
  2. Eppendorf 1.7 ml tubes (Thermo Fisher Scientific, catalog number: 14-222-168 )
  3. 14 ml Falcon tubes (BD Biosciences, catalog number: 352059 )
  4. 0.22 µm syringe filter for HPLC sample preparation (Microsolv Technology, catalog number: 58022-N04-C )
  5. Scale (Ohaus Corporation, model: E10640 )
  6. Mini-centrifuge
  7. Shaker
  8. HPLC: Waters Alliance 2695 HPLC system linked to a 2475 fluorescence detector
  9. SunFire C18 reverse-phase column (4.6 x 150 mm, 3.5 µm)

Procedure

  1. Cell growth
    1. Grow S. meliloti on YMB plate for 48 h at 30 °C. The optimal temperature for S. meliloti growth is between 28 °C and 30 °C.
    2. Inoculate S. meliloti from the stock YMB plate to an OD600 of ~0.1 in 3 ml MMNH4 medium.
    3. Grow cells for 48 h at 30 °C, 250 rpm in 14 ml Falcon or glass tubes completely wrapped with foil to prevent light-induced flavin degradation. In case of testing flavin levels in light-grown culture or flavin degradation by light, the foil could be omitted.
    4. Dilute the cells 20-fold into 3 ml fresh MMNH4 medium and grow for 1 or 3 days at 30 °C, 250 rpm in 14 ml Falcon or glass tubes covered with aluminum foil. The length of cell growth could vary from several hours to 10+ days depending on the specific question of the research.
    5. Remove 1 ml of the growing culture for protein quantification.  These 1 ml samples can be frozen at -20 °C for later protein quantification. (The rest 2 ml of cell cultures will be used for flavin detection, steps B, C and D.)
    6. For protein assay, break the cells by sonication (Fisher Sonic Dismembrator 300 with the intermediate size attachment and a power setting of 60 %, three times for 3 min each at 4  °C).
    7. Remove cells debris by centrifugation (≥ 3,000 x g; room temperature; 20 min).
    8. Measure protein concentration in the cultures by using Bio-Rad Protein Assay Kit.
      Note: Carry out the rest of the procedure for flavin extraction and analysis under as limited lighting as possible since flavins are extremely light sensitive. Protect extracted and filtered flavins from light by storing in a light-tight container. For long-term storage, samples can be stored below 0 °C in a light-tight container.

  2. Separation of cells from conditioned media
    1. Harvest cells from 1 ml culture by centrifugation (≥ 10,000 x g; room temperature; 20 min) in pre-weighed Eppendorf tubes.  
    2. Transfer the supernatant to clean Eppendorf tubes. It will be used in step C and can be stored at -20 °C in foil wrapped Eppendorf tubes for later analysis.
    3. Wash the pellets with 1 ml MMNH4 buffer and centrifuge (≥ 10,000 x g; room temperature; 20 min).
    4. Pipet out all liquid and re-weigh the tube to measure the pellet mass. The pellet mass is expected to be at the range between 5 mg and 10 mg depending on the time of growth.
    5. The pellet will be used in step D can be stored at -20 °C in foil wrapped Eppendorf tubes for later analysis.

  3. To measure secreted flavins
    1. Filter the supernatant left after harvesting the bacteria (step B) using Microsolv 0.22 µm syringe filters.
    2. For riboflavin, FMN, and FAD detection, separate the samples by reverse-phase chromatography using a Waters Alliance 2695 HPLC system with a Waters SunFire C18 column (4.6 x 150 mm, 3.5 µm) maintained at 35 °C linked to a Waters 2475 fluorescence detector. Detect riboflavin, FMN, and FAD by using an excitation wavelength of 470 nm and an emission wavelength of 530 nm. Run mobile phase at a flow rate of 1 ml/min for 12 min per sample. Maintain the samples at 10 °C.
    3. For lumichrome detection, use excitation and emission wavelengths of 260 nm and 470 nm respectively. Use the mobile phase gradient program indicated in Table 1. The column and flow rate are the same as indicated above. Run mobile phase for 15 min. Column temperature 35 °C.
    4. Determine the flavin concentration by comparison to standards (0.05, 0.1, 0.5, 1.0 and 5.0 µM FAD, FMN and riboflavin).
    5. Normalize the flavin concentration against the protein concentration (as calculated above in step A8).

      Table 1. Solvent gradient program parameters for lumichrome detection
      Time (min)
      Water (%)
      Methanol (%)
      0
      77
      23
      8
      40
      60
      11
      40
      60
      12
      0
      100
      13
      0
      100
      14
      77
      23
      15
      77
      23
      *Flow rate 1 ml/min

  4. To measure intracellular flavin concentration
    1. Resuspend the cell pellets from step B in extraction buffer (1: 10 w/v), heat at 80 °C for 10 min, then centrifuge at 20,000 x g for 20 min at 4 °C.
    2. Filter the supernatants using a 0.22 µm syringe filter.
    3. Measure riboflavin, FMN, FAD, and lumichrome as described for the secreted flavins.

Representative data



Figure 1. Flavin secretion and accumulation by the S. meliloti strains. S. meliloti strains were grown in MMNH4 media. 1 ml of cell cultures was taken after 3 days of growth. The cells were spun down and the mass of the pellets was measured. Lumichrome (LMC), FMN, FAD and riboflavin (RF) concentration in the supernatants and the pellets were measured using HPLC. The flavin concentration was normalized against the protein concentration. Rm1021 - wild type strain; Rm1021ΔribA and Rm1021ΔribBA - Rm1021 mutants with decreased ability to secret flavins; Rm1021ΔribBAxpCPP30ribBA - Rm1021 mutant with restored ability to secret flavins. Data are averaged from at least three independent experiments.

Recipes

  1. YMB media for Rhizobium

    1 L
    Concentration
    Yeast Extract
    1 g

    Mannitol
    10 g
    54.9 mM
    Agar
    15 g


    Autoclave, cool to 55 °C, then add
    YMB Salt I
    10 ml
    YMB Salt II
    10 ml

    YMB Salt I
    1 L
    Concentration
    K2HPO4
    50 g
    287.06 mM
    NaCl
    10 g
    171.15 mM
    d-H2O
    960 ml


    YMB Salt II
    1 L
    Concentration
    MgSO4.7H2O
    20 g
    81.11 mM
    d-H2O
    1 L


  2. MMNH4 (minimal mannitol ammonia media for Rhizobium)

    per 1 L
    Concentration
    Mannitol
    10.0 g
    54.9 mM
    NH4Cl
    0.5 g
    9.34 mM
    Agar (for plates preparation)
    15.0 g

    d-H2O
    970 ml


    Autoclave, cool to 55 °C, then add:
    Biotin (0.2 mg/ml in 50% EtOH)
    1.0 ml
    Thiamine (2 mg/ml), filter sterilized
    1.0 ml
    Min Man Salts I
    10.0 ml
    Min Man Salts II
    10.0 ml

    Min Man Salts I

    per 1 L
    Concentration
    K2HPO4
    100 g
    574.12 mM
    KH2PO4
    100 g
    734.8 mM
    Na2SO4
    25 g
    174.8 mM
    d-H2O
    1 L

    Min Man Salt II

    per 1 L
    Concentration
    FeCl3.6H2O
    1.0 g
    3.7 mM
    Concentrated HCl
    adjust pH to ~7.0 (~1 drop)
    CaCl2.2H2O
    10.0 g
    68 mM
    MgCl2.6H2O
    25.0 g
    123 mM
    d-H2O
    1 L
    Autoclave

  3. Extraction buffer
    100 mM ammonium formate
    100 mM formic acid
    25% methanol
  4. HPLC mobile phase
    100 mM ammonium formate
    100 mM formic acid
    25% methanol
    For 1 liter of extraction buffer/ mobile phase, add the following components in the order below. Mix well after adding all the components.
    550 ml
    water
    100 ml   
    ammonium formate, 1 M, filtered through a 0.22 μm filter
    100 ml   
    formic acid, ≥98% pure, 1 M
    250 ml
    methanol, HPLC grade

Acknowledgments

This protocol was adapted from Yurgel et al., 2014. This work was supported by the Agricultural Research Center (WNP-00773) at Washington State University and grant DE-FG0396ER20225 Vol. 27, No. 5, 2014 / 445 from the Energy Biosciences Program at the United States Department of Energy, and by grant NSF-MCB 1052492 to S. Rajamani. This activity was funded, in part, with an Emerging Research Issues Internal Competitive grant from the Agricultural Research Center at Washington State University, College of Agricultural, Human, and Natural Resource Sciences and Biologically-Intensive Agriculture and Organic Farming (BIOAg) Internal Competitive grant from The Center for Sustaining Agriculture and Natural Resources (CSANR) at Washington State University to S. Yurgel. We thank M. Kahn for discussions and the Washington State University Laboratory for Biotechnology and Bioanalysis for sequencing support.

References

  1. Galibert, F., Finan, T. M., Long, S. R., Puhler, A., Abola, P., Ampe, F., Barloy-Hubler, F., Barnett, M. J., Becker, A., Boistard, P., Bothe, G., Boutry, M., Bowser, L., Buhrmester, J., Cadieu, E., Capela, D., Chain, P., Cowie, A., Davis, R. W., Dreano, S., Federspiel, N. A., Fisher, R. F., Gloux, S., Godrie, T., Goffeau, A., Golding, B., Gouzy, J., Gurjal, M., Hernandez-Lucas, I., Hong, A., Huizar, L., Hyman, R. W., Jones, T., Kahn, D., Kahn, M. L., Kalman, S., Keating, D. H., Kiss, E., Komp, C., Lelaure, V., Masuy, D., Palm, C., Peck, M. C., Pohl, T. M., Portetelle, D., Purnelle, B., Ramsperger, U., Surzycki, R., Thebault, P., Vandenbol, M., Vorholter, F. J., Weidner, S., Wells, D. H., Wong, K., Yeh, K. C. and Batut, J. (2001). The composite genome of the legume symbiont Sinorhizobium meliloti. Science 293(5530): 668-672.
  2. Sandoval, F. J., Zhang, Y. and Roje, S. (2008). Flavin nucleotide metabolism in plants: monofunctional enzymes synthesize fad in plastids. J Biol Chem 283(45): 30890-30900.
  3. Somerville, J. E. and Kahn, M. L. (1983). Cloning of the glutamine synthetase I gene from Rhizobium meliloti. J Bacteriol 156(1): 168-176.
  4. Yurgel, S. N., Rice, J., Domreis, E., Lynch, J., Sa, N., Qamar, Z., Rajamani, S., Gao, M., Roje, S. and Bauer, W. D. (2014). Sinorhizobium meliloti flavin secretion and bacteria-host interaction: role of the bifunctional RibBA protein. Mol Plant Microbe Interact 27(5): 437-445.

简介

这个协议提供了一种简单和快速的方法定量细胞内黄素含量,和黄素分泌的细菌。 从细菌团块提取细胞内黄素,并在细胞生长培养基中检查分泌的黄素。 分离黄酮并使用具有荧光检测的HPLC测量,并基于与标准品的比较进行定量。

关键字:核黄素生物合成途径, FMN, 时尚, α-变形, 高效液相色谱法

材料和试剂

  1. ( 1021,Galibert ,2001)
    注意:该协议也可应用于其他细菌。
  2. Bio-Rad蛋白测定试剂盒I(Sigma-Aldrich,目录号:500-0001)
  3. 黄酮标准:
    1. 核黄素(Sigma-Aldrich,目录号:95170)
    2. FMN(Sigma-Aldrich,目录号:F2253)
    3. FAD(Sigma-Aldrich,目录号:F6625)
    4. Lumichrome(Acros,目录号:146930010)
    注意:如前所述进一步纯化FMN和FAD(Sandov等人,2008)。
  4. 甲酸铵
  5. 甲酸
  6. 甲醇
  7. YMB培养基(Somerville和Kahn,1983)(参见Recipes)(每个样品1个平板)
  8. MMNH 4介质(Somerville和Kahn,1983)(参见Recipes)(每个样品6ml)
  9. 提取缓冲液(参见配方)
  10. HPLC流动相(参见配方)
    注意:除非另有说明,所有其他化学品均获自Sigma-Aldrich。

设备

  1. 铝箔
  2. Eppendorf 1.7ml管(Thermo Fisher Scientific,目录号:14-222-168)
  3. 14ml Falcon管(BD Biosciences,目录号:352059)
  4. 0.22μm用于HPLC样品制备的注射器过滤器(Microsolv Technology,目录号:58022-N04-C)
  5. Scale(Ohaus Corporation,型号:E10640)
  6. 微型离心机
  7. 振动器
  8. HPLC:Waters Alliance 2695 HPLC系统连接到2475荧光检测器
  9. SunFire C18反相柱(4.6×150mm,3.5μm)

程序

  1. 细胞生长
    1. 成长。 meliloti在YMB板上在30℃下培养48小时。 S的最佳温度。 meliloti生长在28℃和30℃之间。
    2. 接受 。在3ml MMNH 4培养基中从原液YMB平板将细胞密度调整至〜600的OD 600。
    3. 生长细胞在30°C下48小时,250 rpm在14毫升Falcon或玻璃管完全包装箔,以防止光诱导黄素降解。在测试光生长培养物中的黄素水平或通过光的黄素降解的情况下,可以省略箔。
    4. 将细胞稀释20倍至3ml新鲜MMNH 4培养基中,并在30℃,250rpm下在覆盖有铝箔的14ml Falcon或玻璃管中生长1或3天。细胞生长的长度可以从几小时到10+天,这取决于研究的具体问题。
    5. 取出1毫升的生长培养物进行蛋白质定量。这些1ml样品可以在-20℃冷冻用于后期蛋白质定量。 (其余2ml细胞培养物将用于黄素检测,步骤B,C和D.)
    6. 对于蛋白质测定,通过超声(Fisher Sonic Dismembrator 300,中间尺寸附件和功率设置为60%,每次3分钟,在4℃下)破碎细胞。
    7. 通过离心(≥3,000 x g ;室温; 20分钟)除去细胞碎片。
    8. 通过使用Bio-Rad蛋白质测定试剂盒测量培养物中的蛋白质浓度。
      ote:执行黄色素提取和分析程序的其余部分,因为黄素是非常光敏的。 通过存储在不透光的容器中保护提取和过滤的黄素从光。 对于长期储存,样品可以在0℃以下储存在不透光的容器中。

  2. 从条件培养基中分离细胞
    1. 通过在预称重的Eppendorf管中离心(≥10,000×g;室温; 20分钟)从1ml培养物收获细胞。  
    2. 转移上清液清洁Eppendorf管。 它将用于步骤C中,并且可以在-20℃下储存在箔包裹的Eppendorf管中,用于以后分析。
    3. 用1ml MMNH4缓冲液洗涤沉淀,离心(≥10,000×g;室温; 20分钟)。
    4. 移出所有液体,重新称量管以测量团块质量。 根据生长时间的不同,预期团块质量在5mg和10mg之间的范围内
    5. 颗粒将在步骤D中使用,可以在-20℃下储存在箔包裹的Eppendorf管中用于以后分析。

  3. 测量分泌黄素
    1. 使用Microsolv 0.22μm注射器过滤器过滤收获细菌后留下的上清液(步骤B)
    2. 对于核黄素,FMN和FAD检测,通过反相色谱法使用具有Waters SunFire C18柱(4.6×150mm,3.5μm)的Waters Alliance 2695 HPLC系统分离样品, 35℃连接到Waters 2475荧光检测器。通过使用470nm的激发波长和530nm的发射波长来检测核黄素,FMN和FAD。以1ml/min的流速运行流动相,每个样品12分钟。将样品保持在10°C。
    3. 对于荧光检测,分别使用260nm和470nm的激发和发射波长。使用表1所示的流动相梯度程序。柱和流速与上述相同。运行流动相15分钟。柱温35℃
    4. 通过与标准品(0.05,0.1,0.5,1.0和5.0μMFAD,FMN和核黄素)比较来确定黄素浓度。
    5. 根据蛋白质浓度标准化黄素浓度(如上步骤A8中计算的)
      表1. lumichrome检测的溶剂梯度程序参数
      时间(分)
      水(%)
      甲醇(%)
      0
      77
      23
      8
      40
      60
      11
      40
      60
      12
      0
      100
      13
      0
      100
      14
      77
      23
      15
      77
      23
      *流速1ml/min

  4. 测量细胞内黄素浓度
    1. 将来自步骤B的细胞沉淀物在提取缓冲液(1:10w/v)中重悬,在80℃加热10分钟,然后在4℃下以20,000×g离心20分钟。
    2. 使用0.22μm注射器过滤器过滤上清液。
    3. 测量核黄素,FMN,FAD和lumichrome,如分泌黄素所述。

代表数据



Fi gure 1。 黄病毒分泌和由 e meliloti meliloti 株 生长在MMNH 4介质中。 生长3天后取1ml细胞培养物。 将细胞离心并测量粒料的质量。 使用HPLC测量上清液和沉淀中的Lumichrome(LMC),FMN,FAD和核黄素(RF)浓度。 相对于蛋白质浓度标准化黄素浓度。 Rm1021 - 野生型菌株; Rm1021Δ ribA a ndRm1021Δ ribBA -Rm1021突变体具有降低的黄素调节能力; Rm1021ΔribBAxpCPP30 ribBA -Rm1021突变体,具有恢复的黄素的保藏能力。 从至少三次独立实验中平均数据。

食谱

  1. 用于根瘤菌的YMB培养基

    1 L
    集中
    酵母提取物
    1克

    甘露醇
    10克
    54.9 mM
    Agar
    15克


    高压灭菌,冷却至55°C,然后加入
    YMB盐I
    10 ml
    YMB盐II
    10 ml

    YMB盐I
    1 L
    集中
    K 2 HPO 4
    50克
    287.06 mM
    NaCl
    10克
    171.15 mM
    d H 2 O 2 960 ml


    YMB盐II
    1 L
    集中
    MgSO 4。 。 O
    20克
    81.11 mM
    d H 2 O 2 1 L


  2. MMNH4(用于Rhizo 的最小甘露醇氨培养基)

    每1 L
    集中
    甘露醇
    10.0克
    54.9 mM
    NH 4 Cl
    0.5克
    9.34 mM
    琼脂(用于平板制备)
    15.0克

    d H 2 O 2 970 ml


    高压灭菌,冷却至55°C,然后添加:
    生物素(0.2mg/ml,在50%EtOH中) 1.0 ml
    硫胺素(2mg/ml),过滤灭菌
    1.0 ml
    Min Man Salts I
    10.0 ml
    Min Man Salts II
    10.0 ml

    闵曼盐

    每1 L
    集中
    K 2 HPO 4
    100克
    574.12 mM
    KH 2 PO 4
    100克
    734.8 mM
    Na 2 4
    25克
    174.8 mM
    d H 2 O 2 1 L

    闵曼盐II

    每1 L
    集中
    FeCl 3 6H <2> O
    1.0 g
    3.7 mM
    浓HCl
    调节pH至〜7.0(〜1滴)
    CaCl 2 2H O
    10.0克
    68 mM
    MgCl 2 6H <2> O
    25.0克
    123 mM
    d H 2 O 2 1 L
    高压灭菌器

  3. 提取缓冲区
    100mM甲酸铵 100mM甲酸
    25%甲醇
  4. HPLC流动相
    100mM甲酸铵 100mM甲酸
    25%甲醇
    对于1升提取缓冲液/流动相,按以下顺序添加以下组分。 添加所有组分后混匀。
    550 ml

    100 ml   
    甲酸铵,1M,通过0.22μm过滤器过滤
    100 ml   
    甲酸,纯度≥98%,1M/B
    250 ml
    甲醇,HPLC级

致谢

该方案得自Yurgel等人,2014年。该工作得到华盛顿州立大学的农业研究中心(WNP-00773)的支持,并授予DE-FG0396ER20225 Vol。 27,No.5,2014/445,来自美国能源部的能源生物科学计划,并授予NSF-MCB 1052492给S.Rajamani。 这项活动资助了一部分来自农业部的新兴研究问题内部竞争性拨款 华盛顿州立大学农业,人类和自然资源科学与生物密集型农业和有机耕作学院(BIOAg)的研究中心。华盛顿州立大学维持农业和自然资源中心(CSANR)的内部竞争性资助。 Yurgel。我们感谢M. Kahn的讨论和华盛顿州立大学生物技术和生物分析实验室的测序支持。

参考文献

  1. Galibert,F.,Finan,TM,Long,SR,Puhler,A.,Abola,P.,Ampe,F.,Barloy-Hubler,F.,Barnett,MJ,Becker,A.,Boistard,P.,Bothe G.,Boutry,M.,Bowser,L.,Buhrmester,J.,Cadieu,E.,Capela,D.,Chain,P.,Cowie,A.,Davis,RW,Dreano,S.,Federspiel, NA,Fisher,RF,Gloux,S.,Godrie,T.,Goffeau,A.,Golding,B.,Gouzy,J.,Gurjal,M.,Hernandez-Lucas,I.,Hong,A.,Huizar, L.,Hyman,RW,Jones,T.,Kahn,D.,Kahn,ML,Kalman,S.,Keating,DH,Kiss,E.,Komp,C.,Lelaure,V.,Masuy, Palm,C.,Peck,MC,Pohl,TM,Portetelle,D.,Purnelle,B.,Ramsperger,U.,Surzycki,R.,Thebault,P.,Vandenbol,M.,Vorholter,FJ,Weidner,S 。,Wells,DH,Wong,K.,Yeh,KCand Batut,J。(2001)。 豆科植物共生体的复合基因组 hizobium ):668-672。
  2. Sandoval,F.J.,Zhang,Y.and Roje,S。(2008)。 植物中的黄酮核苷酸代谢:单功能酶在质体中合成fad。 Biol Chem 283(45):30890-30900。
  3. Somerville,J.E。和Kahn,M.L。(1983)。 克隆来自Rh的谷氨酰胺合成酶I基因 /em> 156(1):168-176。
  4. Yurgel,SN,Rice,J.,Domreis,E.,Lynch,J.,Sa,N.,Qamar,Z.,Rajamani,S.,Gao,M.,Roje,S。和Bauer, 。 罪 orhizobium melilot i 黄素分泌和细菌 - 宿主相互作用:双功能RibBA蛋白的作用。 Microbe Interact 27(5):437-445。
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引用:Yurgel, S. N., Lynch, J., Rice, J., Adhikari, N. and Roje, S. (2014). Quantification of Flavin Production by Bacteria. Bio-protocol 4(15): e1197. DOI: 10.21769/BioProtoc.1197.
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