Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

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

SG ShaoHua Gu
WW Wen Wan
ZS ZhengYing Shao
WZ Wei Zhong

发布: 2021年05月05日第11卷第9期 DOI: 10.21769/BioProtoc.4001 浏览次数: 6096

评审: Yufang LuAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Nature Microbiology, featuring study using the protocol.

Aug 2020

Advertisement

Technologies for RNA Detection
Special Issue on Protocols for Organoid Research
“科研可重复性与创新”系列网络研讨会
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

检测大肠杆菌中光激活的Cre介导的基因缺失效率

检测大肠杆菌中光激活的Cre介导的基因缺失效率

Yuta Koganezawa [...] Miki Umetani

2023年06月05日 2141 阅读

细菌和木材腐烂真菌共培养木块腐烂实验，分析木材降解过程中的协同/拮抗作用

细菌和木材腐烂真菌共培养木块腐烂实验，分析木材降解过程中的协同/拮抗作用

Julia Embacher [...] Martin Kirchmair

2023年10月05日 2077 阅读

利用微流控设备定量测量细菌在受控和静止化学梯度中的趋化性

利用微流控设备定量测量细菌在受控和静止化学梯度中的趋化性

Adam Gargasson [...] Harold Auradou

2025年02月20日 2003 阅读

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) search Siderophores (铁载体) search CAS assay (铬天青S分析) search

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

English
中文翻译

文章信息

版权信息

© 2021 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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.

ris ris Download Citation in RIS Format

分类

微生物学 > 微生物生理学 > 适应

生物科学 > 微生物学 > 微生物群落

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件