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Aug 2020
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High-throughput Method for Detecting Siderophore Production by Rhizosphere Bacteria
根际细菌产生铁载体的高通量检测方法   

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Abstract

Siderophores, a key substance that microorganisms produce to obtain iron under iron-limited conditions, play an important role in regulating interactions between beneficial bacteria and pathogenic bacteria. A large number of bacteria were isolated from the rhizosphere, and we used the method presented here to assay the siderophore production by these rhizosphere bacteria. This method is a modified version of the universal chrome azurol S (CAS) assay that uses a 96-channel manual pipetting workstation. By combining the liquid CAS assay with the multi-channel pipette workstation, high-throughput and rapid detection of siderophore production can be achieved. In summary, this method can be used to gain a general understanding of siderophore production by rhizosphere bacteria.

Keywords: Rhizosphere bacteria (根际细菌), Siderophores (铁载体), CAS assay (铬天青S分析)

Background

Siderophores have an extremely strong affinity for ferric iron and are extremely important to bacterial survival. Almost all known bacterial species can produce siderophores (Miethke and Marahiel, 2007), which affect their interaction with microorganisms in close proximity. Siderophores produced by neighboring organisms can act as growth factors that promote the growth of unculturable microorganisms (Kaeberlein et al., 2002), and they also play a significant role in the biological control mechanism against certain phyto-pathogens. Over forty years ago, Kloepper et al. (1980) were the first to illustrate the importance of siderophore production as a mechanism of biological control of Erwinia carotovora by several plant growth-promoting Pseudomonas fluorescens strains such as A1, BK1, TL3B1, and B10. Siderophores play an important role in the composition and function of the rhizosphere microbiome and plant health.


The chrome azurol S (CAS) assay, the most common method for detecting siderophore production (Schwyn and Neilands, 1987), is based on a competition for Fe3+ between the ferric complex of the dye CAS and the siderophore. Increasing the knowledge of siderophore production by rhizosphere bacteria will contribute to a better understanding of the interactions among rhizosphere bacteria. High-throughput detection of siderophore production can be achieved by combining the CAS assay with a 96-channel manual pipetting workstation. In this method, siderophore production was assayed by a modified version of the universal chemical assay developed by Schwyn and Neilands (1987) in 2,150 representative bacterial members, which will help to better understand the abilities of rhizobacteria to produce siderophores.


Materials and Reagents

  1. 96-well plate (96-well clear; Costar, catalog number: 3599)

  2. Disposable Petri dish (90 mm; Jiangsu Kangjian Medical Products, catalog number: 161-0901)

  3. 96-well plate with 0.22-μm filter membranes (MultiScreenHTS GV Filter Plate, 0.22 µm, clear, sterile; Millipore®, catalog number: MSGVS2210)

  4. Pseudomonas aeruginosa PAO1 (Ghysels et al., 2005)

  5. Burkholderia cepacia H111 (Sathe et al., 2019)

  6. Pseudomonas aeruginosa PAO1ΔpvdDΔpchEF (Ghysels et al., 2005)

  7. Pseudomonas aeruginosa H111ΔorbJΔpchAB (Sathe et al., 2019)

  8. Tryptone (OXOID, catalog number: LP0042)

  9. Soy peptone (Chemical Reagent Co., Ltd. of Sinopharm Group, Shanghai Test, catalog number: 69047737)

  10. NaCl (Nanjing Chemical Reagent Co., Ltd. CAS: 7647-14-5)

  11. Agar (Fujian Jinyan Marine Biotechnology Co., Ltd., Chengfeng. CAS: 9002-18-0)

  12. K2PHO4 (Nanjing Chemical Reagent Co., Ltd. CAS: 7778-77-0)

  13. MgSO4·7H2O (Nanjing Chemical Reagent Co., Ltd. CAS: 10034-99-8)

  14. Glycerol (Chemical Reagent Co., Ltd. of Sinopharm Group, Shanghai Test, catalog number: 10010618)

  15. Casamino acids (DSLAB, catalog number: 18A0050)

  16. FeCl3 (Chemical Reagent Co., Ltd. of Sinopharm Group, Shanghai Test, catalog number: 10011918)

  17. Tris-HCl (1 M Tris-HCl, pH = 6.8, BL514A, 100 ml)

  18. Gelatin (Chemical Reagent Co., Ltd. of Sinopharm Group, Shanghai Test, catalog number: 10010328)

  19. Chrome azurol S (Chromeazurol S; Fluka, catalog number: 72687-25G)

  20. HTDMA (VETEC, catalog number: V900413-100G)

  21. Anhydrous piperazine (Piperazine, Reagentplus 99%, Sigma-Aldrich, CAS: 110-85-0)

  22. Glucose (Nanjing Chemical Reagent Co., Ltd. CAS: 50-99-7)

  23. Yeast extract (YEAST EXTRACT; OXOID, catalog number: LP0021)

  24. Beef extract (Chemical Reagent Co., Ltd. of Sinopharm Group, Wokai, catalog number: 69004461)

  25. Glycerin (Nanjing Chemical Reagent Co., Ltd. CAS: 56-81-5)

  26. 1/10 tryptone soya agar (1/10 TSA) (see Recipes)

  27. 1/10 tryptone soya broth (1/10 TSB) (see Recipes)

  28. MKB medium (see Recipes)

  29. Iron-rich medium (see Recipes)

  30. MS buffer solution (see Recipes)

  31. CAS assay solution (see Recipes)

Equipment

  1. 96-channel manual pipetting workstation (Suzhou SinoAnalysis Instrument Co., Ltd., SinoAnalysis, SC9000)

  2. Microplate centrifuge (Hunan Hirsch Instruments, Benchtop Low Speed Centrifuge, TD5A)

  3. Constant-temperature shaker (MIN QUAN, MQD-BIR)

  4. Microplate reader (SpectraMax M5, Sunnyvale, CA, USA)

  5. -80°C freezer (Haier, vertical ultra-low temperature storage box, DW-86L62, 2013 model)

  6. Centrifuge (Eppendorf AG, 22331 Hamburg, 5424EH062551)

  7. Pipette gun (Eppendorf Research plus)

  8. Autoclave (Tega SANYO Industry Co., Ltd, MLS-3780, Tottori, Japan)

  9. Constant-temperature and -humidity incubator (new seedlings, waterproof electric heating constant-temperature incubator, GNP-9080BS-III)

  10. Balance (Sartorius, model: BSA2202S)

  11. Vortex (SCIENTIFIC INDUSTRIES, USA)

Software

  1. R 3.1.2 program (https://github.com/shaohuagu/sidanalysis.git)

  2. Excel 2016

  3. SPSS 20.0

  4. Adobe Illustrator CS5

Procedure


Figure 1. Determination of siderophore production


  1. Rhizosphere soil sampling

    Rhizosphere soil samples were collected from tomato plants located in four different fields. The excess soil should be gently shaken off and discarded; the remaining soil attached to the roots is considered the rhizosphere soil (Hu et al., 2016) and should be collected for use.


  2. Isolation of rhizobacteria

    1. Mix 1 g rhizosphere soil with 9 ml MS buffer solution in a rotary shaker (170 rpm) at 30°C for 30 min.

    2. Dilute the soil suspension to a concentration of 10-5-10-6 g/ml with sterile water. Spread 100 μl diluted soil suspensions on 1/10 tryptone soya agar (TSA).

    3. After a 48-h incubation at 30°C in the dark, randomly pick 32 isolates per rhizosphere soil sample and restreak on TSA plates for colony purification.

    4. Culture all purified isolates in 100 μl tryptone soya broth (TSB) in 96-well microtiter plates at 30°C with shaking (rotary shaker at 170 rpm) for 18 h.

    5. Add 100 μl 30% (v/v) glycerin to the fermentation broth and mix well. Store the rhizobacteria at -80°C.


  3. Measuring siderophore production of rhizobacteria (Figure 1)

    1. Revive the isolates by transferring 5 μl respective freezer stocks into a clean 96-well plate containing 195 μl TSB per well. Culture the bacteria overnight at 30°C with shaking (rotary shaker set at 170 rpm).

    2. Transfer 10 μl overnight cultures into clean 96-well plates containing 190 μl MKB iron-limited medium and MKB iron-rich medium. Incubate for 48 h at 30°C with shaking (rotary shaker set at 170 rpm).

    3. Harvest the cell-free supernatant from the bacterial cultures by centrifugation (4,000 rpm, 5 min at 4°C) and filtration (using a 0.22-µm filter).

    4. Use the liquid version of the chrome azurol S (CAS) assay, in which 100 µl cell-free supernatant (three biological replicates for each of the 2,150 soil isolates) is added to 100 μl CAS assay solution in a 96-well plate using a 96-channel manual pipetting workstation. Add 100 μl MKB iron-limited meduim or MKB iron-rich medium to 100 μl CAS assay solution as a control group.

    5. Incubate the reaction mixture without agitation for 2 h at room temperature.

    6. The OD630 of the reaction mixture (A) and the control group (Ar) was measured using a plate reader (SpectraMax M5) at room temperature. Siderophores induce a color change in the CAS medium, which lowers the OD630 measurements.

    7. Siderophore production was quantitated using the following formula: 1 − A ÷ Ar.

    8. Organic acids in media components and other secreted compounds can also bind iron; therefore, it is essential to estimate the CAS signal background that is not due to siderophores. We assessed this signal background using defined siderophore-deficient mutants from two species (P. aeruginosa PAO1ΔpvdDΔpchEF and B. cenocepacia H111ΔorbJΔpchAB) (Ghysels et al., 2005; Sathe et al., 2019) and their corresponding wild types using the same protocol as described above. We then averaged the CAS background signals of the two siderophore-deficient mutants, which was used as a cut-off to distinguish siderophore producers from non-producers among our 2,150 rhizobacteria(Figure 2A).

Data analysis

  1. Isolation of rhizobacteria

    Almost 16% of the single colonies picked at random could not grow individually on TSA plates. Finally, 2,150 purified strains were isolated from 80 rhizosphere soil samples.


  2. Measuring siderophore production by rhizobacteria

    The calculation for the relative production of siderophores was obtained using this formula: siderophore unit = 1 − A ÷ Ar, which was used to estimate the levels of secreted siderophores in the supernatant of all 2,150 bacterial isolates. This assay serves as a proxy for siderophore production and revealed that up to 95% of the isolates produced siderophores, given that their CAS values exceeded those of siderophore-deficient control strains(Figure 2B). Estimating background CAS values is important since this assay also measures the binding activities of other organic iron-binding compounds. When the CAS assay was repeated under iron-rich conditions, we found that up to 99% of the siderophore producers upregulated siderophore production under iron-limited conditions as compared with under iron-rich conditions. Under iron limitation, siderophore production followed a bimodal distribution, with isolates producing either high or low quantities of siderophores(Figure 2B). These results suggest that siderophore production is a widespread trait across the taxa and sampling sites examined here under iron-limited conditions.



    Figure 2. Siderophore production of rhizobacteria under iron-limited and iron-rich conditions

Notes

  1. During the preparation of MKB medium, all supplies, including glass bottles, measuring cylinders, glass rods, and weighing spoons, must be iron-free. Glassware must be soaked in 6 M hydrochloric acid overnight and rinsed with distilled water several times to remove iron.

  2. Both MKB medium solutions need to be separately prepared and autoclaved at 115°C for 30 min.

Recipes

  1. 1/10 tryptone soya agar (1/10 TSA)

    1.5 g/L tryptone

    0.5 g/L soytone

    0.5 g/L sodium chloride

    15 g/L agar

    pH 7.0

  2. 1/10 tryptone soya broth (1/10 TSB)

    1.5 g/L tryptone

    0.5 g/L soytone

    0.5 g/L sodium chloride

    pH 7.0

  3. MKB medium

    2.5 g/L K2HPO4

    2.5 g/L MgSO4·7H2O

    15 ml/L glycerin

    5.0 g/L casamino acids

    pH 7.2

  4. Iron-rich medium

    Add FeCl3 to MKB medium to a final concentration of 50 μM

  5. MS buffer solution

    50 mM Tris-HCl, pH 7.5

    100 mM NaCl

    10 mM MgSO4

    0.01% gelatin

  6. CAS assay solution

    Add 1.5 ml 1 mM FeCl3 to 7.5 ml 1 mM Chrome azurol S and mix well. Add 50 ml 4 mmol/L HTDMA while stirring, followed by 30 ml 1 M piperazine solution. Finally, add deionized water to 100 ml

Acknowledgments

This research was financially supported by the National Natural Science Foundation of China (grant nos. 41922053 to Z.W., 41807045 to T.Y. and 31972504 to Y.X.) and the Natural Science Foundation of Jiangsu Province (grant nos. BK20180527 to T.Y. and BK20170085 to Z.W.). The Laboratory of Rhizosphere Micro-ecology (LorMe), College of Resources and Environmental Sciences, Nanjing Agricultural University, guaranteed the successful completion of this experiment.

Competing interests

The authors declare no competing interests.

Ethics

The experimental procedures involved in this article are in line with ethical and moral requirements, and have not caused adverse consequences to society or the environment.

References

  1. Ghysels, B., Ochsner, U., Mollman, U., Heinisch, L., Vasil, M., Cornelis, P. and Matthijs, S. (2005). The Pseudomonas aeruginosa pirA gene encodes a second receptor for ferrienterobactin and synthetic catecholate analogues. FEMS Microbiol Lett 246(2): 167-174.
  2. Hu, J., Wei, Z., Friman, V. P., Gu, S. H., Wang, X. F., Eisenhauer, N., Yang, T. J., Ma, J., Shen, Q. R., Xu, Y. C. and Jousset, A. (2016). Probiotic Diversity Enhances Rhizosphere Microbiome Function and Plant Disease Suppression. mBio 7(6).
  3. Kaeberlein, T., Lewis, K., Epstein, S. S. (2002). Isolating "uncultivable" microorganisms in pure culture in a simulated natural environment. Science 296(5570): 1127-1129.
  4. Kloepper, J. W., Leong, J., Teintze, M. and Schroth, M. N. J. C. M. (1980). Pseudomonas siderophores: A mechanism explaining disease-suppressive soils. 4(5): 317-320.
  5. Miethke, M. and Marahiel, M. A. (2007). Siderophore-based iron acquisition and pathogen control. Microbiol Mol Biol Rev 71(3): 413-451.
  6. Sathe, S., Mathew, A., Agnoli, K., Eberl, L. and Kummerli, R. (2019). Genetic architecture constrains exploitation of siderophore cooperation in the bacterium Burkholderia cenocepacia. Evol Lett 3(6): 610-622.
  7. Schwyn, B. and Neilands, J. B. (1987). Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160(1): 47-56.


简介

[摘要]铁载体, 一种 关键物质即微生物产生以获得铁受限条件下铁, 玩小鬼ortant角色 我Ñ调节相互作用之间有益菌和致病菌。甲升细菌ARGE数从根际分离,并且我们使用这里介绍的方法测定含铁细胞生产由这些根际细菌。此方法是通用的修改版本铬天青S(CAS)测定法,使用96通道手动移液工作站。通过将液体CAS分析与多通道移液器工作站结合使用,可以实现高通量和快速检测铁载体的产生。总之,该方法可以用来获得的一般理解铁载体生产的由根际细菌。

[背景]铁载体有三价铁极强的亲和力,是非常重要的 细菌升生存。几乎所有已知的细菌物种可以产生铁载体(Miethke和Marahiel,2007) ,从而影响IR相互作用与微生物在紧密接近。邻近生物产生的铁载体 可以作为促进不可培养 微生物生长的生长因子(Kaeberlein et al。,2002),并且它们在针对某些植物病原体的生物控制机制中也起着重要作用。四十多年前,Kloepper等人。(1980) 最早证明 铁载体的产生是通过数种促进植物生长的荧光假单胞菌菌株(例如A1,BK1,TL3B1和B10)对胡萝卜软腐欧文氏菌进行生物控制的机制的重要性。铁载体在根际微生物组的组成和功能以及植物健康中起着重要作用。

所述Ç hrome天青S(CAS)测定,m个用于检测铁载体生成的最常用方法(Schwyn和Neilands,1987年)是基于用于竞争的Fe 3+的染料CAS和的三价铁配合物之间的铁载体。增加对根际细菌生产铁载体的知识 会有所贡献 以一个更好地了解荷兰国际集团的互动小号根际细菌中。高-铁载体生产的通量检测可以通过CAS测定用96通道手动移液工作站组合来实现。在这种方法中,铁载体产量由开发通用化学法的修改版测定Schwyn和Neilands (1987)在2,150代表性细菌的成员,这将有助于以更好地理解 根细菌产生铁载体的能力。

关键字:根际细菌, 铁载体, 铬天青S分析

材料和试剂
1. 96孔板(96孔透明;Costar,目录号:3599)     
2.一次性陪替氏d ISH(90毫米;江苏康健医疗产品,目录号:161-0901)     
3. 96孔板用0.22 -微米滤膜小号(MultiScreenHTS GV滤板,0.22微米,透明的,无菌; Millipore公司® ,目录号:MSGVS2210)     
4.铜绿假单胞菌PAO1 (Ghysels等,2005)。     
5.洋葱伯克霍尔德菌H111 (Sathe et al。,2019)     
6.绿脓杆菌PAO1 Δ PVDD Δ pchEF (Ghysels等人,2005)     
7.绿脓杆菌H111 Δ orbJ Δ pchAB (萨特等人。,2019)     
8.胰蛋白((OXOID,目录号:LP0042)     
9.大豆p eptone(化学试剂有限公司的国药集团,上海测试,CATA登录号:69047737)     
10. NaCl(南京化学试剂有限公司CAS:7647-14-5) 
11.琼脂(福建金燕海洋生物技术有限公司,成峰。CAS:9002-18-0) 
12. K 2 PHO 4 (南京化学试剂有限公司CAS:7778-77-0) 
13. MgSO 4 · 7H 2 O(南京化学试剂有限公司CAS:10034-99-8) 
14.甘油(国药集团化学试剂有限公司,上海测试,目录号:10010618) 
15    酪蛋白一个的CID(DSLAB,CATA登录号:18A0050)
16. FeCl 3 (国药集团化学试剂有限公司,上海测试,目录号:10011918) 
17. Tris-HCl(1 M Tris-HCl,pH = 6.8,BL514A,100 ml) 
18岁    明胶(国药集团化学试剂有限公司,上海测试,目录号:10010328)
19.铬天青S(铬天青小号; Fluka公司,CATA登录号:72687-25G) 
20. HTDMA(VETEC,目录编号:V900413-100G) 
21.无水哌嗪(哌嗪,Reagentplus 99%,Sigma-Aldrich,CAS:110-85-0) 
22.葡萄糖(南京化学试剂有限公司CAS:50-99-7) 
23。    酵母提取物(酵母提取物; OXOID,CATA登录号:LP0021)
24.牛肉提取物(国药集团化学试剂有限公司,我开,目录号:69004461) 
25岁    甘油(南京化学试剂有限公司CAS:56-81-5)
26. 1/10胰蛋白try大豆琼脂(1/10 TSA)(请参阅食谱) 
27. 1/10胰蛋白try大豆汤(1/10 TSB)(请参阅食谱) 
28. MKB介质(请参阅食谱) 
29.富含铁的培养基(请参见食谱) 
30. MS缓冲溶液(请参阅食谱) 
31. CAS分析溶液(请参阅食谱) 
设备

96通道手动移液工作站(苏州SinoAnalysis仪器有限公司,SinoAnalysis ,SC9000)
微孔板离心机(湖南赫希仪器公司,台式低速离心机,TD5A)
常数-温度摇床(MIN泉,MQD-BIR)
酶标仪(SpectraMax M5,美国加利福尼亚州桑尼维尔)
-80°C冰柜(Haier,立式超低温储物盒,DW-86L62,2013年型号)
离心机(Eppendorf AG,22331汉堡,5424EH062551)
移液枪(Eppendorf Research plus)
高压釜(日本鸟取市MLS-3780 Tega SANYO Industry Co.,Ltd)
常数-温度和-湿度培养箱(新的幼苗,防水电热恒定-温度erature培养箱,GNP-9080BS-III)
天平(Sartorius,型号:BSA2202S)
Vortex(美国科学产业)

软件

R 3.1.2程序(https://github.com/shaohuagu/sidanalysis.git)
Excel 2016
SPSS 20.0
Adobe Illustrator CS5


程序



图URE 1.测定铁载体生产的

根际土壤采样
根际土壤样品从收集的番茄植株位于我Ñ在我们不同的领域 。多余的土壤应轻轻地甩掉并丢弃;残留在根部的土壤被认为是根际土壤(Hu等,2016),应收集起来使用。

隔离 根瘤菌
将1 g根际土壤与9 ml MS缓冲溶液混合 在旋转振动台上(170 rpm )在30°C下放置30分钟。
用无菌水将土壤悬浮液稀释至10 -5 -10 -6 g / ml的浓度。扩展 100个微升稀释土壤悬浮液 在1/10胰蛋白so大豆琼脂(TSA)上。
48后-在30℃小时的温育在黑暗中,从每个根际土壤样品中随机选择32个分离株, restreak上TSA平板菌落纯化。
培养物的LL纯化分离物在100微升胰蛋白胨大豆肉汤(TSB)我ñ 96孔微量滴定板在30℃下用18小时振荡(以170rpm旋转摇动器)。
加100微升 30%(v / v) 将甘油加入发酵液中并充分混合。小号撕开的根际细菌在- 80 ℃下。

              测量根瘤菌的铁载体产量(图1)
ř通过转移5只evive菌株微升各自冷冻股成一个干净的96孔板含有195微升TSB每孔。在摇动(旋转摇床设置为170 rpm)下于30°C过夜培养细菌。
转移10微升 过夜培养物到清洁含有190 96孔板微升MKB铁有限介质和MKB富含铁的平台。摇动(旋转摇床设定为170 rpm)在30°C下孵育48小时。
收获来自无细胞上清液的细菌培养物通过离心(4000转,在4℃下5分钟)和过滤(使用0.22 -微米的过滤器)。
使用铬的液体版本天青S(CAS)测定,在WH ICH 100微升无细胞上清液(三次生物学每个2150个土壤分离株的复制)加入到100微升 CAS测定溶液在96孔板用一个96通道手动移液工作站。100添加微升MKB铁有限meduim或MKB富含铁的培养基至100微升CAS测定溶液,作为对照组。
将反应混合物在不搅拌的情况下在室温下孵育2小时。
在OD 630的的反应混合物(A)和所述对照组(氩)中的溶液在室温下使用读板器(M5的SpectraMax)测量。 铁载体在CAS介质中引起颜色变化,从而 降低OD 630的测量值。
铁载体生产原为孔定量达ED使用下列公式:1 -甲÷氩气。
介质成分中的有机酸和 其他分泌的化合物也可以结合铁; 因此,必须估算出不是由铁载体引起的CAS信号背景。我们评估使用定义的铁载体缺陷型突变体的该信号从背景两个物种(铜绿假单胞菌PAO1 ΔpvdDΔpchEF和B.新洋葱伯克霍尔德杆菌H111 ΔorbJΔpchAB )(Ghysels等人,2005 ;萨特。等人,2019)和其相应的使用相同的野生型如上所述的协议。然后,我们平均两个铁载体缺陷型突变体的CAS背景信号,将其用作截止到我们的2150中区分非生产者铁载体生产者根际细菌(图2甲)。

数据分析

根瘤菌的分离
在TSA平板上,随机采摘的单菌落中几乎有16%不能单独生长。最后,2 ,150个纯化菌株从80根际土样中分离。

测量铁载体生产的 根瘤菌
用于计算的相对生产铁载体的小号-甲÷铁载体单元= 1:使用此公式获得的Ar ,其被用来估计在所有2150个细菌分离株的上清液中分泌的铁载体的水平。该测定用作铁载体生产的代理,并表明,高达产生铁载体的菌株95%,因为它们的CAS值超过第OSE铁载体缺陷型控制菌株 (图2 B )。估算背景CAS值很重要,因为此测定法还可以测量其他有机铁结合化合物的结合活性。当被富含铁的条件下重复中科院实验中,我们发现了铁限制的条件下,上调铁载体生产的铁载体生产的99%的较下的富含铁的条件。在铁的限制下,铁载体的产生遵循双峰分布,分离株产生大量或少量的铁载体(图2 B )。这些结果表明,铁载体的生产是在铁限制条件下在整个检查的分类单元和采样位点上的普遍性状。



图URE 2.铁限制和富含铁的铁载体下生产的根际细菌的条件

笔记

在MKB培养基的制备过程中,所有用品,包括玻璃瓶,量筒,玻璃棒,和称重匙,必须是无铁。玻璃器皿必须浸泡在6 M盐酸中过夜,并用蒸馏水冲洗几次以除去铁。
两个都 MKB介质解决方案需要单独准备和在115°C高压灭菌30分钟。

菜谱

1/10胰蛋白try大豆琼脂(1/10 TSA)
1.5 g L -1胰蛋白tone
0.5克L -1大豆蛋白
0.5克L -1氯化钠
15克L -1琼脂
pH值7.0
1/10胰蛋白so大豆汤(1/10 TSB)
1.5 g L -1胰蛋白tone
0.5克L -1大豆蛋白
0.5克L -1氯化钠
pH值7.0
MKB培养基
2.5克L -1 K 2 HPO 4
2.5 g L -1 MgSO 4 ·7H 2 O
15 ml L -1甘油
5.0 g L -1酪蛋白氨基酸
pH值7.2
富铁培养基
将FeCl 3添加到MKB培养基至终浓度为50μM
MS缓冲液
50 mM Tris - HCl,pH 7.5
100毫米氯化钠
10毫米硫酸镁4
0.01%明胶
CAS测定溶液
将1.5 ml 1 mM FeCl 3添加到7.5 ml 1 mM Chrome azurol S中,并充分混合。甲DD 50毫升4-毫摩尔/ L HTDMA而 搅拌,然后加入30毫升1 M哌嗪溶液。˚F inally ,加 去离子水至100毫升

致谢

这项研究得到了中国国家自然科学基金(ZW的授权号41922053,TY的41807045和YX的授权号31972504)和江苏省自然科学基金(TY的BK20180527和ZW的授权号BK20170085)的资助。实验室[R hizosphere中号ICRO生态学(LorMe南京农业大学),资源与环境科学学院,保证了该实验的顺利完成。

利益争夺

作者宣称没有利益冲突。

伦理

本文涉及的实验程序符合道德和道德要求,并且没有对社会或环境造成不利影响。

参考

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Kaeberlein ,T.,Lewis,K.,Epstein,SS(2002)。在模拟自然环境中的纯培养物中分离“无法培养的”微生物。科学296(5570):1127-1129。
Kloepper,JW,Leong,J.,Teintze,M。和Schroth,MNJCM(1980)。假单胞菌铁载体:解释抑制疾病的土壤的机制。4(5):317-320。
Miethke ,M.和Marahiel ,MA(2007)。基于铁载体的铁捕获和病原体控制。微生物分子生物学评论71(3):413-451。
Sathe,S.,Mathew,A.,Agnoli ,K.,Eberl ,L.和Kummerli ,R.(2019)。遗传结构限制了新细菌伯克霍尔德菌中铁载体合作的开发。Evol Lett 3(6):610-622。
Schwyn ,B。和Neilands ,JB(1987)。用于检测和测定铁载体的通用化学测定法。肛门生物化学160(1):47-56。
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引用:Gu, S., Wan, W., Shao, Z. and Wei, Z. (2021). High-throughput Method for Detecting Siderophore Production by Rhizosphere Bacteria. Bio-protocol 11(9): e4001. DOI: 10.21769/BioProtoc.4001.
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