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Determination of Oxygen Respiration Rates in Wetted Developmentally Arrested Spores of Streptomyces Species
链霉菌属物种潮湿抑制发育孢子的氧气呼吸率测定

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Abstract

Streptomyces species produce spores, which, while not as robust as endospores of Bacillus or Clostridium species, are capable of surviving for months or even years (Hopwood, 2006). During this time these spores remain viable, surviving by slowly degrading internal stores of carbon compounds, such as the carbohydrate trehalose. To enable metabolism to continue they must have access to an electron acceptor that allows the removal of the reducing equivalents that accumulate through metabolic activity. The most commonly used acceptor is oxygen. We describe the quantitative measurement of oxygen respiration rates by developmentally arrested spores of the streptomycete Streptomyces coelicolor (Fischer et al., 2013).

Keywords: Cytochrome oxidase(细胞色素氧化酶), Oxygen reduction(氧还原), Spores(孢子), Non-invasive optical oxygen-sensing(非侵入式光学氧传感), Streptomyces coelicolor(天蓝色链霉菌)

Materials and Reagents

  1. Freshly harvested Streptomyces spores in water
  2. Soya flour (from local supermarket)
  3. D-mannitol (Sigma-Aldrich, catalog number: 63560 )
  4. Agar-agar (Kobe I) (Roth North America)
  5. 3-(N-morpholino) propanesulfonic acid (MOPS) buffer (Roth North America)
  6. Chloramphenicol (Merck KGaA)
  7. Sodium hydroxide pellets (Roth North America)
  8. MOPS-buffer (see Recipes)
  9. SFM agar (see Recipes)

Equipment

  1. Cotton wool (standard issue from local Pharmacy) for spore preparation
  2. 0.22 µm pore-size filters (PVDF) (Sartorius AG)
  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. Needles (0.6 mm gauge) (B. Braun Melsungen AG)
  9. Water bath or heating block (Biometra)
  10. Spectrophotometer capable of measuring absorption in the visible range
  11. Optical ‘FirestingO2 oxygen meter’ (Pyro Science GmbH)
  12. Contactless optical oxygen ‘sensor spot’ (Pyro Science GmbH)
  13. ‘Bare fibre’ (Pyro Science GmbH)
  14. Adjustable mini magnetic stirrer (e.g. IKA) (10 mm x 0.3 mm magnetic stirrer bar) (e.g. Roth North America)

Software

  1. Firesting Logger Software (Pyro Science GmbH)

Procedure

  1. Streptomyces coelicolor was grown on SFM agar plates at 30 °C until colonies sporulated (Kieser et al., 2000). Spores were isolated by using cotton swabs and were washed with water and filtered twice through cotton wool (Kieser et al., 2000). Subsequently they 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. The oxygen reduction rate by spores was measured in 16 ml gas tight Hungate vials in a 30 °C room or chamber.
  3. The vials were filled with 5 ml of 50 mM MOPS buffer (pH 7.2), containing chloramphenicol (400 μg ml−1) and spore suspension with an OD450nm of 25 (Fischer et al., 2013).
  4. The spore suspension was stirred continuously with a magnetic stirrer at 800 rpm upto an oxygen saturation in the spore suspension. Oxygen saturation was determined empirically for each experiment performed and this process generally took approximately 2 min based on a stirring speed of 800 rpm. The vials were closed with a gas-tight rubber plug and simultaneously the pressure was equalized with a needle one time and the on-line measurement cycle was initiated, exactly as indicated in the protocol delivered by the manufacturer. An image of the experimental setup is available at http://www.pyro-science.com/optical_oxygen_meter_firesting_O2.html.
  5. The oxygen level in the remaining 11 ml headspace was monitored non-invasively and continuously over a period of 15 h using the oxygen-measuring system of Pyro Science GmbH and documented using the Firesting Logger Software. To do this, oxygen-dependent luminescence sensor spots were affixed to the glass wall of the tube within the headspace and the signals were measured through the vial’s glass wall using an optical oxygen meter (FirestingO2).
  6. The oxygen respiration rate was calculated as the moles of oxygen reduced per volume oxygen in the headspace with respect to the dry weight of spores (1 OD ml−1 = 0.12 mg dry weight). A representative example of data is shown in Fischer et al. (2013).

Recipes

  1. MOPS-buffer
    Adjusted to pH 7.2 with NaOH
    Sterilized by filtration through 0.22 µm filter
  2. SFM agar
    20 g L−1 soya flour
    20 g L−1 D-mannitol
    Agar-agar (Kobe I)

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (SA 494/4-1).

References

  1. 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.
  2. Hopwood, D. A. (2006). Soil to genomics: the Streptomyces chromosome. Annu Rev Genet 40: 1-23.
  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.

简介

< em>链霉菌属</em>物种产生孢子,其不如芽孢杆菌属或梭状芽孢杆菌属物种的内生孢子那样坚固,能够存活数月或甚至数年 Hopwood,2006)。 在此期间,这些孢子保持存活,通过缓慢降解碳化合物(例如碳水化合物海藻糖)的内部储存而存活。 为了使代谢能够继续,它们必须获得电子受体,其允许去除通过代谢活性积聚的还原当量。 最常用的受主是氧。 我们描述了通过发育阻止的链霉菌链霉菌(Streptomyces coelicolo)孢子的氧呼吸速率的定量测量(Fischer等人,2013)。

关键字:细胞色素氧化酶, 氧还原, 孢子, 非侵入式光学氧传感, 天蓝色链霉菌

材料和试剂

  1. 新鲜收获的链霉菌孢子在水中
  2. 大豆粉(来自当地超市)
  3. D-甘露醇(Sigma-Aldrich,目录号:63560)
  4. 琼脂(Kobe I)(Roth北美)
  5. 3-(N-吗啉代)丙磺酸(MOPS)缓冲液(Roth North America)
  6. 氯霉素(Merck KGaA)
  7. 氢氧化钠丸(Roth North America)
  8. MOPS缓冲区(参见配方)
  9. SFM琼脂(见配方)

设备

  1. 棉毛(当地药房的标准问题)孢子准备
  2. 0.22μm孔径过滤器(PVDF)(Sartorius AG)
  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. 针(0.6mm规格)(B.Braun Melsungen AG)
  9. 水浴或加热块(Biometra)
  10. 能够测量可见光范围内的吸收的分光光度计
  11. 光学"FirestingO 2"氧气计(Pyro Science GmbH)
  12. 非接触光学氧传感器点(Pyro Science GmbH)
  13. 'Bare fiber'(Pyro Science GmbH)
  14. 可调节的微型磁力搅拌器(例如IKA)(10mm×0.3mm磁力搅拌棒)(例如Roth North America)

软件

  1. Firesting Logger软件(Pyro Science GmbH)

程序

  1. 在30℃下在SFM琼脂平板上生长天蓝色链霉菌(Streptomyces coelicolor),直到菌落形成孢子(Kieser等人,2000)。 通过使用棉签分离孢子,并用水洗涤并通过棉绒过滤两次(Kieser等人,2000)。 随后,将它们调节至10或20的OD 450nm(1ml的OD 450 = 1等于3.5×10 8个孢子 ml -1 或2.37×10 8 cfu。)。
  2. 在30℃的室或室中,在16ml气密的Hungate小瓶中测量孢子的氧还原速率。
  3. 小瓶中装入5ml含有氯霉素(400μg/ml)的50mM MOPS缓冲液(pH7.2)和OD 450nm 25为25的孢子悬浮液Fischer等人,2013)。
  4. 用磁力搅拌器以800rpm将孢子悬浮液连续搅拌至孢子悬浮液中的氧饱和度。对于进行的每个实验,根据经验确定氧饱和度,并且基于800rpm的搅拌速度,该过程通常花费约2分钟。用气密性橡胶塞封闭小瓶,同时用针均衡一次压力,并开始在线测量循环,完全如制造商提供的方案中所示。实验设置的图片位于 http://www.pyro-science.com/optical_oxygen_meter_firesting_O < sub> 2 .html
  5. 使用Pyro Science GmbH的氧测量系统并且使用Firesting Logger软件记录,在15小时的时间内非侵入地并且连续地监测剩余的11ml顶部空间中的氧水平。为此,将氧依赖性发光传感器点固定到顶部空间内的管的玻璃壁,并使用光学氧计(Firesting2)通过小瓶的玻璃壁测量信号。 br />
  6. 氧气呼吸速率计算为相对于孢子干重(1OD ml -1 sup = 0.12mg干重)的顶部空间中每体积氧减少的氧的摩尔数。 在Fischer等人中示出了数据的代表性示例。 (2013)。

食谱

  1. MOPS缓冲区
    用NaOH调节至pH 7.2 通过0.22μm过滤器过滤灭菌
  2. 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.,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。
  2. Hopwood,D.A。(2006)。 土壤基因组学:链霉菌染色体。 > Annu Rev Genet 40:1-23。
  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
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引用:Fischer, M., Falke, D. and Sawers, R. G. (2014). Determination of Oxygen Respiration Rates in Wetted Developmentally Arrested Spores of Streptomyces Species. Bio-protocol 4(12): e1153. DOI: 10.21769/BioProtoc.1153.
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