搜索

Quantification of Hydrogen Sulfide and Cysteine Excreted by Bacterial Cells
定量测定细菌细胞分泌的硫化氢和半胱氨酸   

引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Journal of Bacteriology
Dec 2015

Abstract

Bacteria release cysteine to moderate the size of their intracellular pools. They can also evolve hydrogen sulfide, either through dissimilatory reduction of oxidized forms of sulfur or through the deliberate or inadvertent degradation of intracellular cysteine. These processes can have important consequences upon microbial communities, because excreted cysteine autoxidizes to generate hydrogen peroxide, and hydrogen sulfide is a potentially toxic species that can block aerobic respiration by inhibiting cytochrome oxidases. Lead acetate strips can be used to obtain semiquantitative data of sulfide evolution (Oguri et al., 2012). Here we describe methods that allow more-quantitative and discriminatory measures of cysteine and hydrogen sulfide release from bacterial cells. An illustrative example is provided in which Escherichia coli rapidly evolves both cysteine and sulfide upon exposure to exogenous cystine (Chonoles Imlay et al., 2015; Korshunov et al., 2016).

Keywords: Hydrogen sulfide (硫化氢), Thiols (硫醇), Cysteine (半胱氨酸), Escherichia coli (大肠埃希杆菌), Measurement (测定), Detection (检测)

Background

Reduced sulfur species are generated by microbes through several routes. Sulfate-reducing bacteria exploit the reductive process as an integral part of energy generation. Other bacteria release sulfide as a by-product of either the deliberate or adventitious degradation of sulfur species, including cysteine. We have observed that cysteine itself is excreted when intracellular levels are abnormally high, a situation that can occur through uncontrolled amino acid import or dysregulation of cysteine synthesis. These sulfur species are unusually reactive, as they bind metals with high avidity and also are among the few biomolecules that react chemically with molecular oxygen. The upshot is that reduced sulfur compounds can exert important effects upon cells. Hence, it can be important to track the dynamics of reduced sulfur compounds in a variety of contexts.

Thiol agents–notably, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB)–provide good spectroscopic probes of thiol concentrations. Unfortunately, they do not discriminate between organic thiols like cysteine and inorganic species like hydrogen sulfide. The evolution of the latter species has often been detected using lead acetate strips, which are suspended in the head space over sulfide-generating cultures. However, this method is slow and non-quantitative. For that reason, we have leveraged the volatility of hydrogen sulfide so that standard dyes can allow sulfide and organic thiols to be distinguished as they are generated in lab cultures. The methods are simple, quick, and sensitive.

Materials and Reagents

  1. 10 ml test tubes, as necessary (Fisher Scientific, catalog number: 14-961-27 )
  2. Polypropylene tubes, 2 ml, as necessary (Denville Scientific, catalog number: C2170 )*
  3. Polypropylene 2 ml tubes, as necessary (see Material and Reagents #2)**
  4. Parafilm* (Bemis, catalog number: PM996 )
  5. Pipette tips (1,000 μl; 200 μl) (Corning, catalog number: 4846 ; USA Scientific, catalog number: 1111-1006 )
  6. Cylinder with compressed air (AirGas Mid-America, breathing quality grade D)
  7. Cylinder with compressed nitrogen (AirGas Mid-America)**
  8. 50 ml flasks (Corning, PYREX®, catalog number: 4442-50 )
  9. Flask closures (Fisher Scientific, catalog number: 05-888 )
  10. Bacterial cell culture
  11. Ethylenediamine tetraacetic acid, disodium salt, dihydrate, EDTA (Fisher Scientific, catalog number: S311 )
  12. Cystine dihydrochloride (Sigma-Aldrich, catalog number: C2526 )
  13. 5,5-Dithiobis (2-nitrobenzoic acid), DTNB (Sigma-Aldrich, catalog number: D8130 )
  14. 4-Amino-N,N-dimethylaniline, DMPDA (Sigma-Aldrich, catalog number: 07750 )
  15. Ferric(III) chloride hexahydrate (Sigma-Aldrich, catalog number: F2877 )
  16. Potassium phosphate, mono- or dibasic (Fisher Scientific, catalog numbers: P284 , P288 )
  17. Ethanol, 100% (Decon Labs, catalog number: 2716 )
  18. Potassium hydroxide (Fisher Scientific, catalog number: P250 )
  19. Glucose (Fisher Scientific, catalog number: D16 )
  20. Ammonium sulfate (Fisher Scientific, catalog number: A702 )
  21. Sodium citrate (Fisher Scientific, catalog number: S279 )
  22. Hydrochloric acid (Sigma-Aldrich, catalog number: H1758 )
  23. Sodium sulfide nonahydrate (Sigma-Aldrich, catalog number: S4766 )
  24. Magnesium sulfate heptahydrate (Fisher Scientific, catalog number: M63 )
  25. Deionized water (University of Illinois deionizing system)
  26. Stock solutions (see Recipes)
Notes:
*These items are for DMPDA-based measurements.
**These items are for DTNB-based measurements.

Equipment

  1. For DMPDA-based measurements
    1. Pipettors, 1 and 0.2 ml (Mettler-Toledo, Rainin, catalog numbers: 17014382 , 17014384 )
    2. Microcentrifuge (Fisher Scientific, model: accuSpinTM Micro 17 , catalog number: 13-100-675)
    3. Spectrophotometer (Beckman Coulter, model: DU-640 )
    4. Shaking water bath (New Brunswick Scientific, model: G76D )
    5. Heater (Fisher Scientific, catalog number: 11-718 )

  2. For DTNB-based measurements
    1. Two 125 ml gas washing bottles with coarse fritted discs (Corning, PYREX®, catalog number: 31760-125C )
    2. Water bath (Shel-Lab, VWR, model: Model 1250 )

Procedure

  1. Measurement of excreted organic thiols
    1. Grow E. coli cultures (> 5 ml) to OD 0.1 in a medium that contains sulfate as the sole sulfur source, such as minimal A medium (Chonoles Imlay et al., 2015).
    2. Add 0.1 mM EDTA to the bacterial culture.
    3. Add 0.2 mM cystine to the culture. Turn the shaker on and let the culture mix for 10 sec.
    4. Remove 1 ml of bacterial culture every 2-5 min.
    5. Centrifuge the 1-ml aliquot (30-60 sec) using microcentrifuge (13,800 x g).
    6. Place 0.5 ml of supernatant into a test tube and bubble it vigorously with nitrogen for minimum 30 sec to remove hydrogen sulfide.
    7. Mix the bubbled supernatant with 0.5 ml of DTNB reagent by pipetting 3-4 times.
    8. Allow reaction for minimum 1 min and then measure the absorbance at 412 nm.
    9. Calculate the thiol concentration using extinction coefficient of 13 OD/mM of cysteine. Multiply the obtained value by 2 to correct for the dilution by the DTNB reagent.
    Note: Escherichia coli cells fed cystine excrete a significant amount of thiols (3-4 micromolar per minute at 0.1 OD). The excreted thiols are mostly cysteine (about 90%) and hydrogen sulfide (about 10%). Mass spectroscopic analysis has shown that the release of other biological thiols, i.e., glutathione, is negligible (Korshunov et al., 2016). Cysteine can be unstable in aerated medium because adventitious metals can catalyze its oxidation; therefore, EDTA is included to block this reaction. Nitrogen bubbling is necessary to remove hydrogen sulfide from the solution. Typical results are presented in Figure 1.


    Figure 1. Sample time course of cysteine excretion. The progressive accumulation of cysteine in the supernatant of wild-type (MG1655) E. coli cultures was tracked after cystine addition. Cells were at 0.1 OD in minimal glucose medium. Cystine was added at time point zero. The rate of cysteine accumulation in the medium is 3.1 μM/min.

  2. One-time sulfide measurements for sealed cultures
    1. Grow E. coli cultures to OD 0.1 in a medium that contains sulfate as the sole sulfur source, such as minimal A medium (Chonoles Imlay et al., 2015).
    2. Transfer 1 ml of bacterial culture into a 2 ml plastic tube in a heater at 37 °C.
    3. Add 0.1 mM cystine. Close the tube and seal it with Parafilm.
    4. Incubate for 10 min at 37 °C. Agitation is not necessary.
    5. Remove Parafilm, open the tube and quickly add 0.1 ml of 20 mM DMPDA followed immediately by 0.1 ml of 30 mM ferric chloride. Acidification will lyse cells; the DMPDA/ferric chloride will detect sulfide that had been released during the prior incubation period. Close the tube and seal it with Parafilm. To avoid the loss of hydrogen sulfide, do not pipet or mix the culture until the tube is closed and sealed.
    6. Shake the sealed tube and keep it in the dark at room temperature for 15 min. The control culture should not contain cystine.
    7. After the incubation, remove Parafilm and centrifuge the samples in a table centrifuge for 5 min at 13,800 x g.
    8. Carefully transfer 1 ml of the supernatant to clean plastic tubes.
    9. Measure the optical density at 650 nm.
    10. The extinction coefficient may vary depending on the medium and lies between 16-20 OD/mM of sulfide with typical mean of 17 OD/mM. In some media (for example, glycerol-containing minimal A medium) the absorbance value of the sample may drift over the time of incubation with DMPDA; therefore, the time span between the addition of DMPDA and the measurement of absorbance should be as consistent as possible and should not differ between samples by more than 2-3 min. A calibration curve using sodium sulfide in the 0.5-20 micromolar range should be obtained for each medium. This method was modified from Siegel (1965). Typical results are presented in Figure 2.


      Figure 2. Sample single-point sulfide measurements in bacterial cultures. A wild-type MG1655 cell culture (OD 0.1) in minimal A glucose medium was incubated for 10 min in the absence or presence of 0.2 mM of cystine, and the evolved hydrogen sulfide was then measured using DMPDA. Sterile medium was used as a blank; note the substantial background value. The suspension from cystine-fed wild-type cells accumulated 3.5 micromolar sulfide. The strain that was unable to import cystine into the cell (tcyP) did not produce any detectable sulfide.

  3. Time course for sulfide accumulation in open growing cultures
    1. Grow E. coli cultures to OD 0.1 in minimal A medium, with sulfate as the sole sulfur source.
    2. Place 12 ml of the culture into a 50 ml flask.
    3. Add 0.2 mM of cystine.
    4. Seal the flask with stopper. Shake the culture at 180 rpm.
    5. Take 1 ml samples every 5 min. Do not keep the flask open for more than 5 sec.
    6. Perform Steps B4-B8 from ‘one-time measurements for sealed cultures’ section.
    The time interval between the addition of DMPDA and actual measurement of a sample should be constant. Because a fraction of sulfide is lost during the withdrawal of the sample, the expected underestimation of the real sulfide production is about 10-20%. This value can be checked with standard samples of sodium sulfide.
    The DMPDA-based method for sulfide determination may not work properly if high cysteine concentrations (> 5 mM) are present in the medium. The weakness of this method is that, despite high reliability and sensitivity, the signal/background ratio is not optimal at low sulfide concentrations (see Figure 1). To address this situation, we developed a simple and sensitive DTNB-dependent system that employs an alkaline trap.

  4. DTNB-based procedure with an alkaline trap
    1. Grow E. coli culture to OD 0.1 in minimal A medium, with sulfate as the sole sulfur source.
    2. Connect the outlet of one gas-washing bottle with the fritted tube of the other (Figure 3).


      Figure 3. Arrangement for continuous trapping of sulfide released by cultures. A. Bottle 1 contains the active bacterial culture and may be located in a water bath. The inlet of the bottle 1 is connected to the cylinder with compressed air; the outlet of the bottle is connected to the inlet of the bottle 2 containing the alkaline solution. The outlet of the bottle 2 is not sealed. After addition of cystine to the bottle 1 the cap of this bottle is fixed by the rubber or metallic spring hooks to the bottle’s body. At intervals, samples from bottle 2 and removed, and sulfide is assayed. B. Photographic depiction of the sulfide trap.

    3. Fill the first bottle (bottle 1) with 75-100 ml of the bacterial culture (0.3-0.4 OD).
    4. Fill another bottle (bottle 2) with an equal volume of 0.1 M KPO4 buffer at pH 11.
    5. Set the air flow to 0.3-0.4 L/min. (Air flow is easily measured by quantifying the rate of water displacement when the line is directed into an inverted water-filled graduated cylinder.)
    6. Add 0.5 mM of cystine and seal the bottle. The flow of air passes through the bacterial culture and carries the released hydrogen sulfide to the alkaline solution in the next bottle. Hydrogen sulfide is deprotonated and thereby trapped in the alkaline solution of bottle 2.
    7. Periodically remove 0.5 ml of the alkaline solution and mix it with 0.5 ml of 0.5 mM DTNB solution in 0.5 M KPO4 (pH 7). Determine absorbance at 412 nm.
    8. Calculate hydrogen sulfide content using extinction coefficient of 26 OD/mM of hydrogen sulfide. Note that a correction should be made for the 1:1 dilution of the sample solution into the DTNB solution.
    The rate at which sulfide is taken away from bacterial culture is proportional to its concentration in the medium, so the rate of accumulation of sulfide in bottle 2 will increase over time until the rate of sulfide formation by cells in bottle 1 equals the rate of sulfide removal to bottle 2 (Figure 4). A small part of the sulfide from the gas mix will not be trapped in the alkaline solution of bottle 2 due to an incomplete gas:liquid exchange process. This portion can be determined by spiking a standard amount of sodium sulfide into bottle 1 and quantifying its recovery in bottle 2. The purpose of the frit is to create smaller bubbles that enhance exchange; the general technique can be replicated with pipets, but sulfide trapping may be less efficient.


    Figure 4. Sample time course of sulfide accumulation in an alkaline trap. Hydrogen sulfide formation was initiated by the addition of 0.5 mM cystine to an MG1655 culture (0.3 OD) in minimal A glucose medium. After addition of cystine the cell culture was sealed. Air flow rate was 0.35 l/min, the suspension volume was 75 ml, the trap volume was 75 ml. 0.1 KPO4 buffer taken as a blank. At intervals, aliquots were removed from the trap and thiol content was quantified by DTNB treatment. The initial lag represents the time needed for initial sulfide accumulation in the culture; the final slope represents the rate of sulfide evolution by the bacteria.

  5. Determination of the efficiency of the alkaline trap
    1. Fill bottle 1 with 75-100 ml of the medium containing 10 micromolar sodium sulfide.
    2. Fill bottle 2 with an equal volume of alkaline solution.
    3. Take 0.5 ml samples from each bottle and mix with 0.5 ml of the DTNB solution.
    4. Seal bottle 1 and adjust the air flow to 0.3-0.4 L/min.
    5. Bubble air for 10 min and repeat Step E3.
    6. Compare the change in OD from both bottles. The drop in OD from the pre- and post-gassing samples of bottle 1 represents the amount of sulfide that was removed by gassing. The rise in OD from the pre- and post-gassing samples of bottle 2 represents the amount of sulfide that was successfully trapped. Thus the efficiency of the trap is (ΔOD bottle 2)/(ΔOD bottle 1). The efficiency observed in our experiments was about 80-85%.
    Note: All our bacterial cultures were handled at 37 °C. For this reason, the washing tubes were kept in a pre-warmed water bath.

Data analysis

Cysteine-excretion and sulfide-excretion rates are calculated from the slope of the data (see Figures 1 and 3), using the absorbance values of standard samples to convert rate of absorbance change to rate of analyte concentration change. DMPDA determinations of sulfide in bacterial cultures rely on single measurements per sample; blanks from sulfide-free samples are subtracted before calculating the sulfide content of experimental samples. Technical replicates can be determined for each point and averaged, and best-fit curves are calculated. As discussed below, most variation arises from biological variance, which has different sources depending upon the phenomenon being studied; accordingly, workers are encouraged to measure biological replicates, but the nature and degree of the variation are not addressed here.
The technical variation among samples is modest. Three micromolar cysteine in culture supernatants was measured with a standard error of 2%. Three micromolar sulfide in culture supernatants was detected with SEM of 7%. Three micromolar sulfide in an alkaline trap was detected with SEM of 2%. Precision in the cysteine and sulfide-trap analyses is improved by obtaining multiple time points, if the experimenter is confident that the actual rates of excretion do not change over the course of the measurement.

Notes

Variability in data is dominated by variance among biological rather than technical replicates. There are a variety of scenarios that might attract the interest of biologists, and so we do not attempt to highlight the issues that might impinge upon fluctuations in cell behavior. In general, the excretion of hydrogen sulfide depends upon the efficiencies of cysteine/cystine import and the titers of desulfidases; each of the responsible proteins is regulated (Korshunov et al., 2016). Similarly, cysteine efflux rates also depend upon the induced levels of export systems (Chonoles Imlay et al., 2015). Therefore, experimenters who wish to track rates of sulfide or cysteine efflux are encouraged to tightly control cell-handling protocols, particularly with respect to medium, time, and temperature.

Recipes

  1. Stock solutions (prepared freshly, stored on ice)
    50 mM DTNB in 100% ethanol
    20 mM DMPDA in 6 M hydrochloric acid
    30 mM ferric chloride in 1 M hydrochloric acid
    50 mM cystine in 0.2 M hydrochloric acid
    50 mM EDTA, pH 8.0
  2. Buffers and medium stocks for the medium
    0.5 M phosphate buffer: 5 g KH2PO4, 11 g K2HPO4, 200 ml of deionized water; adjust pH to 7.0
    0.1 M phosphate buffer for the alkaline trap: 20 ml of 0.5 M phosphate buffer mixed with 80 ml of deionized water. Adjust pH to 11 using 6 M KOH
    0.5 M magnesium sulfate in deionized water, autoclave for 30 min
    20% glucose (w/v): 20 g glucose in 100 ml of deionized water, autoclave for 30 min
  3. Minimal A medium
    1.05 g K2HPO4
    0.45 g KH2PO4
    0.1 g (NH4)2SO4
    0.05 g sodium citrate dihydrate
    100 ml of deionized water
    Note: Autoclave the medium for 30 min, cool to room temperature, and then add 0.1 ml of 0.5 M magnesium sulfate and 1 ml of 20% glucose.

Acknowledgments

This work was supported by grant GM101012 from the National Institutes of Health. The DMPDA-based method was previously described in Siegel (1965), Anal Biochem 11: 126-132.
There are no conflicts of interest or competing interests to be declared.

References

  1. Chonoles Imlay, K. R., Korshunov, S. and Imlay, J. A. (2015). Physiological roles and adverse effects of the two cystine importers of Escherichia coli. J Bacteriol 197(23): 3629-3644.
  2. Korshunov, S., Imlay, K. R. and Imlay, J. A. (2016). The cytochrome bd oxidase of Escherichia coli prevents respiratory inhibition by endogenous and exogenous hydrogen sulfide. Mol Microbiol 101(1): 62-77.
  3. Oguri, T., Schneider, B. and Reitzer, L. (2012). Cysteine catabolism and cysteine desulfhydrase (CdsH/STM0458) in Salmonella enterica serovar typhimurium. J Bacteriol 194(16): 4366-4376.
  4. Siegel, L. M. (1965). A direct microdetermination for sulfide. Anal Biochem 11: 126-132.

简介

细菌释放半胱氨酸以调节细胞内池的大小。它们也可以通过硫的氧化形式的异化还原或通过细胞内半胱氨酸的故意或无意降解来释放硫化氢。这些过程会对微生物群落产生重要影响,因为排泄的半胱氨酸会自动氧化生成过氧化氢,而硫化氢是一种潜在的毒性物种,可通过抑制细胞色素氧化酶来阻断有氧呼吸。醋酸铅条可用于获得硫化物演化的半定量数据(Oguri et al。,2012)。在这里,我们描述的方法,允许更多的定量和歧视措施半胱氨酸和硫化氢释放细菌细胞。提供了一个说明性实例,其中当暴露于外源性胱氨酸时,大肠杆菌迅速产生半胱氨酸和硫化物(Chonoles Imlay等人,2015; Korshunov等人, ,2016)。

【背景】微生物通过几种途径产生了减少的硫物质。硫酸盐还原菌利用还原过程作为能量生成的组成部分。其他细菌释放硫化物,作为硫物质(包括半胱氨酸)的蓄意或偶然降解的副产物。我们观察到半胱氨酸本身是在细胞内水平异常高时排泄的,这种情况可能通过不受控制的氨基酸输入或半胱氨酸合成失调发生。这些硫物质具有非同寻常的反应性,因为它们以高亲和力与金属结合,也是与分子氧发生化学反应的少数生物分子之一。结果是减少的硫化合物可以对细胞产生重要影响。因此,跟踪各种情况下含硫化合物的动态变化是非常重要的。

硫醇试剂 - 特别是5,5-二硫代双(2-硝基苯甲酸)(DTNB) - 提供硫醇浓度良好的光谱探针。不幸的是,它们不区分有机硫醇如半胱氨酸和无机物质如硫化氢。后一物种的进化经常使用乙酸铅条检测,乙酸铅条悬浮在产生硫化物的培养物的顶部空间。但是,这种方法很慢且不定量。出于这个原因,我们利用了硫化氢的挥发性,因此标准染料可以让硫化物和有机硫醇在实验室培养中产生区分。这些方法简单,快速且灵敏。

关键字:硫化氢, 硫醇, 半胱氨酸, 大肠埃希杆菌, 测定, 检测

材料和试剂

  1. 根据需要10毫升试管(Fisher Scientific,目录号:14-961-27)
  2. 根据需要,聚丙烯管2毫升(Denville Scientific,目录号:C2170)*
  3. 根据需要加入2毫升聚丙烯管(请参阅材料和试剂#2)**
  4. Parafilm *(Bemis,目录号:PM996)
  5. 移液器吸头(1,000μl; 200μl)(Corning,目录号:4846; USA Scientific,目录号:1111-1006)
  6. 带压缩空气的气缸(AirGas Mid-America,呼吸质量等级D)
  7. 压缩氮气瓶(AirGas Mid-America)**
  8. 50毫升烧瓶(Corning,PYREX®,产品目录号:4442-50)
  9. 烧瓶盖(Fisher Scientific,目录号:05-888)
  10. 细菌细胞培养
  11. 乙二胺四乙酸,二钠盐,二水合物,EDTA(Fisher Scientific,目录号:S311)
  12. 胱氨酸二盐酸盐(Sigma-Aldrich,目录号:C2526)
  13. 5,5-二硫双(2-硝基苯甲酸),DTNB(Sigma-Aldrich,目录号:D8130)
  14. 4-氨基-N,N'-二甲基苯胺,DMPDA(Sigma-Aldrich,目录号:07750)
  15. 六水合氯化铁(III)(Sigma-Aldrich,目录号:F2877)
  16. 磷酸钾,一元或二元(Fisher Scientific,目录号:P284,P288)
  17. 乙醇,100%(Decon Labs,目录号:2716)
  18. 氢氧化钾(Fisher Scientific,目录号:P250)
  19. 葡萄糖(Fisher Scientific,目录号:D16)
  20. 硫酸铵(Fisher Scientific,目录号:A702)
  21. 柠檬酸钠(Fisher Scientific,目录号:S279)
  22. 盐酸(Sigma-Aldrich,目录号:H1758)
  23. 硫化钠九水合物(Sigma-Aldrich,目录号:S4766)
  24. 硫酸镁七水合物(Fisher Scientific,目录号:M63)
  25. 去离子水(伊利诺伊大学去离子系统)
  26. 库存解决方案(请参阅食谱)
    备注:
    *这些项目用于基于DMPDA的测量。
    **这些项目用于基于DTNB的测量。

设备

  1. 对于基于DMPDA的测量
    1. 移液管,1和0.2ml(Mettler-Toledo,Rainin,目录号:17014382,17014384)
    2. 微量离心机(Fisher Scientific,型号:accuSpinTM Micro 17,目录号:13-100-675)
    3. 分光光度计(Beckman Coulter,型号:DU-640)
    4. 摇晃水浴(New Brunswick Scientific,型号:G76D)
    5. 加热器(Fisher Scientific,目录号:11-718)

  2. 用于基于DTNB的测量
    1. 两个125毫升的气体清洗瓶,带有粗糙的多孔圆盘(Corning,PYREX ®,产品目录号:31760-125C)
    2. 水浴(Shel-Lab,VWR,型号:Model 1250)

程序

  1. 排泄的有机硫醇的测量
    1. 成长E。在含有硫酸盐作为唯一硫源如最小A培养基(Chonoles Imlay等人,2015)的培养基中,将大肠杆菌培养物(> 5ml)加入到OD 0.1中。 br />

    2. 在细菌培养基中加入0.1 mM EDTA
    3. 向培养物中加入0.2mM胱氨酸。打开摇床,让文化混合10秒。

    4. 每2-5分钟取出1毫升细菌培养液
    5. 使用微量离心机(13,800 x )离心1-ml等分试样(30-60秒)。
    6. 将0.5ml上清液放入试管中,用氮气剧烈鼓泡至少30秒以除去硫化氢。

    7. 用0.5 ml的DTNB试剂通过移液3-4次将发泡的上清液混合
    8. 允许反应至少1分钟,然后测量412nm处的吸光度。
    9. 用13 OD / mM半胱氨酸的消光系数计算硫醇浓度。将得到的值乘以2以纠正DTNB试剂的稀释。
    注意:喂食胱氨酸的大肠杆菌细胞分泌大量硫醇(在0.1OD下每分钟3-4微摩尔)。排泄出来的巯基主要是半胱氨酸(约90%)和硫化氢(约10%)。质谱分析显示其他生物硫醇即谷胱甘肽的释放可忽略不计(Korshunov等,2016)。半胱氨酸在充气介质中可能不稳定,因为偶发金属可以催化其氧化;因此,包含EDTA以阻断该反应。氮气鼓泡对于从溶液中除去硫化氢是必需的。典型的结果如图1所示。


    图1.半胱氨酸排泄的取样时间过程野生型(MG1655)E的上清液中半胱氨酸的累积累积。添加胱氨酸后追踪大肠杆菌培养物。细胞在最小葡萄糖培养基中为0.1OD。在时间点零时添加胱氨酸。
    培养基中半胱氨酸累积速率为3.1μM/ min
  2. 一次性硫化物测量密封文化
    1. 成长E。在含有硫酸盐作为唯一硫源的培养基如最小A培养基(Chonoles Imlay等,,2015)中培养大肠杆菌培养物至OD 0.1。
    2. 在37℃的加热器中将1ml细菌培养物转移到2ml塑料管中。
    3. 加入0.1 mM胱氨酸。关闭管道并用Parafilm密封。
    4. 37°C孵育10分钟。激动是没有必要的。
    5. 去除石蜡膜,打开试管并迅速加入0.1ml 20mM DMPDA,然后立即加入0.1ml 30mM氯化铁。酸化会裂解细胞; DMPDA /三氯化铁将检测在之前的潜伏期释放的硫化物。关闭管道并用Parafilm密封。为避免硫化氢的流失,请勿吸管或混合培养物,直至管道关闭并密封。
    6. 摇动密封管并在室温下避光保存15分钟。对照培养物不应含有胱氨酸。
    7. 孵育后,移除石蜡膜,并在13,800 x g的台式离心机中离心5分钟。
    8. 小心地将1毫升上清液转移到干净的塑料管中。
    9. 测量650 nm处的光密度。
    10. 消光系数可能因介质而异,介于16-20 OD / mM硫化物之间,典型平均值为17 OD / mM。在一些培养基(例如,含甘油的最小A培养基)中,样品的吸光度值可能随着与DMPDA一起温育而漂移;因此,添加DMPDA和吸光度测量之间的时间间隔应尽可能一致,样品间不应有差异超过2-3分钟。应使用0.5-20微摩尔范围内的硫化钠对每种培养基进行校准曲线。该方法由Siegel(1965)修改。典型的结果如图2所示。


      图2.细菌培养物中的样品单点硫化物测量将在最少的A葡萄糖培养基中的野生型MG1655细胞培养物(OD 0.1)在不存在或存在0.2mM胱氨酸,然后使用DMPDA测量放出的硫化氢。使用无菌培养基作为空白;注意大量的背景值。来自胱氨酸喂养的野生型细胞的悬液累积3.5微摩尔硫化物。无法将胱氨酸导入细胞( tcyP )的菌株不产生任何可检测到的硫化物。

  3. 开放生长期培养物中硫化物积累的时间过程
    1. 成长E。在最小A培养基中以0.1%硫酸盐作为唯一硫源,培养大肠杆菌培养物至OD 0.1。
    2. 将12ml培养物放入50ml烧瓶中。
    3. 加入0.2mM胱氨酸。
    4. 用塞子密封烧瓶。以180转/分摇动培养物。
    5. 每5分钟取1毫升样品。不要让烧瓶打开超过5秒钟。

    6. 从“密封培养的一次性测量”部分执行步骤B4-B8。
    添加DMPDA和实际测量样品之间的时间间隔应该是恒定的。由于在取样过程中会有部分硫化物丢失,预计实际硫化物产量低估约为10-20%。这个值可以用硫化钠的标准样品检查。
    如果培养基中存在高半胱氨酸浓度(> 5mM),则用于硫化物测定的基于DMPDA的方法可能无法正常工作。这种方法的缺点是,尽管可靠性和灵敏度很高,但在低硫化物浓度下信号/背景比率并不是最佳的(见图1)。为了解决这种情况,我们开发了一个简单而敏感的DTNB依赖系统,该系统采用碱性陷阱。

  4. 基于DTNB的程序与碱性陷阱
    1. 成长E。在最小的A培养基中以0.1%的硫酸盐作为唯一的硫源培养大肠杆菌培养物至OD 0.1。

    2. 将一个洗气瓶的出口与另一个洗瓶的出口连接起来(图3)。


      图3.连续捕获由培养物释放的硫化物的排列A.瓶1含有活性细菌培养物,可位于水浴中。瓶子1的入口用压缩空气连接到缸体上;瓶子的出口连接到含有碱性溶液的瓶子2的入口。瓶2的出口未被密封。在瓶子1中加入胱氨酸后,该瓶子的瓶盖通过橡胶或金属弹簧钩固定在瓶身上。每隔一段时间,从瓶子2取出样品并测定硫化物。 B.硫化物陷阱的照片描述。

    3. 用75-100ml的细菌培养物(0.3-0.4 OD)填充第一瓶(瓶1)。
    4. 用等体积的0.1M KPO缓冲液(pH 11)填充另一瓶(瓶2)。
    5. 设定空气流量为0.3-0.4 L / min。 (空气流量很容易通过量化管道被引入倒水充分量筒时的排水量来测量)。
    6. 加入0.5 mM胱氨酸并密封瓶子。空气流通过细菌培养物并将释放的硫化氢携带到下一瓶中的碱性溶液中。硫化氢被去质子化并因此被困在瓶子2的碱性溶液中。
    7. 定期除去0.5ml碱性溶液并将其与0.5ml在0.5M KPO 4(pH7)中的0.5mM DTNB溶液混合。测定412nm处的吸光度。
    8. 使用26 OD / mM硫化氢的消光系数计算硫化氢含量。请注意,应对样品溶液1:1稀释到DTNB溶液进行校正。
    从细菌培养物中除去硫化物的速率与其在培养基中的浓度成比例,因此瓶子2中硫化物的积累速率将随时间增加,直到瓶子1中的细胞形成硫化物的速率等于硫化物的速率去除瓶2(图4)。由于不完全的气体:液体交换过程,来自气体混合物的一小部分硫化物不会被困在瓶2的碱性溶液中。这部分可以通过将标准量的硫化钠加入瓶1并量化其在瓶2中的回收来确定。该熔块的目的是产生更小的气泡以增强交换;一般的技术可以使用移液器进行复制,但硫化物捕集可能效率较低。


    图4.碱性捕获器中硫化物积累的样品时间过程硫化氢的形成通过在最小的A葡萄糖培养基中添加0.5mM胱氨酸至MG1655培养物(0.3OD)开始。加入胱氨酸后,将细胞培养物密封。空气流量为0.35升/分钟,悬浮体积为75毫升,收集体积为75毫升。 0.1 KPO缓冲液4作为空白。每隔一段时间,将等分试样从捕集器中取出,通过DTNB处理定量硫醇含量。初始滞后表示培养物中初始硫化物积累所需的时间;最终斜率代表了细菌对硫化物进化的速度。

  5. 测定碱性阱的效率

    1. 用75-100毫升含有10微摩尔硫化钠的培养基填充瓶子1

    2. 用等体积的碱性溶液填充瓶子2

    3. 每个瓶子取0.5毫升样品,并与0.5毫升DTNB溶液混合。
    4. 密封瓶1并将空气流量调节至0.3-0.4 L / min。
    5. 鼓泡10分钟,然后重复步骤E3。
    6. 比较两个瓶子的OD变化。来自瓶子1的预充气和充气后样品的OD的下降表示通过充气去除的硫化物的量。来自瓶2的预充气和充气后样品的OD的上升表示成功捕获的硫化物的量。因此,陷阱的效率是(ΔOD瓶2)/(ΔOD瓶1)。在我们的实验中观察到的效率约为80-85%。
    注意:我们所有的细菌培养都是在37°C下处理的。出于这个原因,洗涤管保存在预热的水浴中。

数据分析

使用标准样品的吸光度值将吸光度变化率转换为分析物浓度变化率,从数据斜率计算半胱氨酸排泄率和硫化物排泄率(参见图1和3)。 DMPDA测定细菌培养物中的硫化物依赖于每个样品的单次测量;在计算实验样品的硫化物含量之前,减去不含硫化物样品中的空白。可以确定每个点的技术重复次数并取平均值,并计算出最佳拟合曲线。如下所述,大多数变化来自生物方差,根据所研究的现象,其具有不同的来源;因此,鼓励工作人员测量生物学复制品,但这里没有涉及变异的性质和程度。
样本之间的技术变化是适度的。用2%的标准误差测量培养上清液中的三微摩尔半胱氨酸。用7%的SEM检测培养上清液中3微摩尔硫化物。用2%的SEM检测碱性阱中的三微摩尔硫化物。如果实验者确信实际排泄率在测量过程中不发生变化,则通过获得多个时间点可以提高半胱氨酸和硫化物陷阱分析的精确度。

笔记

数据的变化主要取决于生物学方面的差异,而不是技术上的重复。有许多场景可能会吸引生物学家的兴趣,因此我们不试图强调可能影响细胞行为波动的问题。通常,硫化氢的排泄取决于半胱氨酸/胱氨酸输入的效率和脱硫酶的效价;每个负责的蛋白质都受到调节(Korshunov等人,2016年)。类似地,半胱氨酸流出速率也取决于诱导的出口系统水平(Chonoles Imlay等人,2015)。因此,鼓励希望跟踪硫化物或半胱氨酸流出速率的实验者严格控制细胞处理方案,特别是在培养基,时间和温度方面。

食谱

  1. 库存解决方案(新鲜制备,储存在冰上)
    在100%乙醇中的50mM DTNB
    在6M盐酸中的20mM DMPDA
    在1M盐酸中的30mM氯化铁
    在0.2M盐酸中的50mM胱氨酸
    50mM EDTA,pH8.0
  2. 缓冲液和媒介股票的媒介
    0.5M磷酸盐缓冲液:5g KH 2 PO 4 4,11g K 2 HPO 4 4,200ml的去离子水;调整pH值至7.0
    用于碱性阱的0.1M磷酸盐缓冲液:20ml与80ml去离子水混合的0.5M磷酸盐缓冲液。
    使用6 M KOH调节pH至11 去离子水中0.5M硫酸镁,高压灭菌30分钟 20%葡萄糖(w / v):在100ml去离子水中的20g葡萄糖,高压灭菌30分钟
  3. 最小中等
    1.05克K 2 HPO 4 4克/克 0.45克KH 2 PO 4 4 0.1克(NH 4)2 SO 4 4 /
    0.05克柠檬酸钠二水物

    100毫升去离子水 注意:将培养基高压灭菌30分钟,冷却至室温,然后加入0.1ml 0.5M硫酸镁和1ml 20%葡萄糖。

致谢

这项工作得到了美国国立卫生研究院GM101012资助。基于DMPDA的方法先前在Siegel(1965),Anal Biochem 11:126-132中描述。
没有利益冲突或竞争利益需要申报。

参考

  1. Chonoles Imlay,K. R.,Korshunov,S.和Imlay,J. A.(2015)。 两种大肠杆菌胱氨酸进口者的生理作用和不良反应 。 J Bacteriol 197(23):3629-3644。
  2. Korshunov,S.,Imlay,K.R。和Imlay,J.A。(2016)。 大肠杆菌细胞色素bd氧化酶阻止内源性和外源性呼吸抑制硫化氢。分子微生物学 101(1):62-77。
  3. Oguri,T.,Schneider,B。和Reitzer,L。(2012)。 肠道沙门氏菌中的半胱氨酸分解代谢和半胱氨酸脱氢酶(CdsH / STM0458) serovar鼠伤寒“ J Bacteriol 194(16):4366-4376。
  4. Siegel,L.M。(1965)。 硫化物的直接微量测定。 Anal Biochem 11:
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Korshunov, S. and Imlay, J. A. (2018). Quantification of Hydrogen Sulfide and Cysteine Excreted by Bacterial Cells. Bio-protocol 8(10): e2847. DOI: 10.21769/BioProtoc.2847.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。