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Xanthoferrin Siderophore Estimation from the Cell-free Culture Supernatants of Different Xanthomonas Strains by HPLC

HPLC法估测不同黄单胞菌菌株培养的无细胞上清液中黄单胞菌铁载体

Sheo Shankar Pandey Sheo Shankar Pandey
Prashantee Singh Prashantee Singh
Biswajit Samal Biswajit Samal
Raj Kumar Verma Raj Kumar Verma
SC Subhadeep Chatterjee

发布: 2017年07月20日第7卷第14期 DOI: 10.21769/BioProtoc.2410 浏览次数: 9019

评审: Arsalan DaudiSrujana Samhita YadavalliBin Tian

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Abstract

Xanthomonads can scavenge iron from the extracellular environment by secreting the siderophores, which are synthesized by the proteins encoded by xss (Xanthomonas siderophore synthesis) gene cluster. The siderophore production varies among xanthomonads in response to a limited supply of iron where Xanthomonas campestris pv. campestris (Xcc) produces less siderophores than Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc). Siderophore production can be measured by HPLC and with the CAS (Chrome azurol S)-agar plate assay, however HPLC is a more accurate method over CAS-agar plate assay for siderophore quantification in Xanthomonads. Here we describe how to quantify siderophores from xanthomonads using HPLC.

Keywords: Xanthomonas siderophores (黄单胞菌属铁载体) search Xanthoferrin (黄单胞菌铁蛋白) search Bacterial siderophore estimation (细菌铁载体估测) search HPLC (HPLC) search Amberlite XAD-16 resin columns (Amberlite XAD-16树脂柱) search

Background

The Xanthomonas group of bacterial phytopathogens possess an xss (Xanthomonas siderophore synthesis) operon, which is required to produce xanthoferrin (an α-hydroxy carboxylate-type siderophore; similar to vibrioferrin) and to encode an outer membrane receptor involved in the siderophore-mediated iron uptake (Pandey and Sonti, 2010; Pandey et al., 2016a and 2016b). Siderophores are small iron-chelating compounds secreted by bacteria to utilize the insoluble form of iron (Neilands, 1995). For the past three decades, CAS (Chrome azurol S)-agar plate assay has been mostly employed to measure bacterial siderophores by monitoring the halo formation around bacterial colonies (Schwyn and Neilands, 1987; Pandey and Sonti, 2010). However, the above assay cannot reliably be used to quantify siderophores from bacteria which also secrete organic acids (e.g., oxalic acid, citric acid, and isocitric acids) along with siderophores, as the organic acids are capable of chelating iron from CAS dye to compromise the accuracy to quantify siderophore (Rai et al., 2015). Since the above mentioned assay only gives the idea about the bacterial siderophore production qualitatively, hence HPLC-mediated siderophore quantification is a better method than the CAS-agar plate assay for bacteria that produce both siderophores along with organic acids; such as Xanthomonas species (Rai et al., 2015; Pandey et al., 2016a and 2016b).

Materials and Reagents

  1. Pipette tips (Corning, Axygen®, catalog numbers: TF-300-R-S , TF-200-R-S , and TF-1000-R-S )
  2. Culture tubes (Borosil, catalog number: 9800U06 )
  3. Petri dishes (Tarson Products, catalog number: 460140-140MM )
  4. Stericup® filter units with 0.22 μm porosity (EMD Millipore, catalog number: SCGPU02RE )
  5. Centrifuge tubes: 500 ml, 50 ml, 15 ml, 2 ml, and 1.5 ml (Tarson Products, Kolkata, India)
  6. Cork borer, 8 ± 0.2 mm (HiMedia Laboratories, catalog number: LA737 )
  7. Syringe driven filter unit with 0.22 μm porosity (Millex®-GS) (EMD Millipore, catalog number: SLGS033SS )
  8. Xanthomonas campestris pv. campestris 8004, X. oryzae pv. oryzicola BXOR1, X. oryzae pv. oryzae (Indian isolate) and ∆xssA mutant in Xanthomonas siderophore synthesis gene A (Lab collections)
  9. Rifampicin
  10. 2,2’-dipyridyl (DP) (Sigma-Aldrich, catalog number: 14453 )
    Note: This product has been discontinued.
  11. Amberlite XAD16N, 20-60 mesh (Sigma-Aldrich, catalog number: XAD16-1KG )
  12. Methanol (Thermo Fisher Scientific, catalog number: Q32407 )
  13. Standard vibrioferrin (Fujita et al., 2011; see Notes 2 and 3)
  14. Trifluoroacetic acid (TFA) (Sigma-Aldrich, catalog number: T6508-25ML )
  15. Acetonitrile (CH3CN) (Sigma-Aldrich, catalog number: 34888-1L )
    Note: This product has been discontinued.
  16. Peptone (HiMedia Laboratories, catalog number: CR001 )
  17. Sucrose (HiMedia Laboratories, catalog number: GRM601-500G )
  18. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465 )
  19. Chrome Azural S (CAS) (Merck, catalog number: 1.02477.0025 )
  20. Ferric chloride (FeCl3) (Standard Reagents, catalog number: SRCF005C )
  21. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: 29507 )
  22. Hexadecyltrimethylammonium bromide (HDTMA) (Sigma-Aldrich, catalog number: H6269-250G )
  23. BactoTM agar (BD, BactoTM, catalog number: 214010 )
  24. BactoTM peptone (BD, BactoTM, catalog number: 211677 )
  25. Agar (HiMedia Laboratories, catalog number: RM026-500G )
  26. Peptone-sucrose (PS) medium (Tsuchia et al., 1982; see Recipes)
  27. CAS solution (Schwyn and Neilands, 1987; see Recipes)
  28. Peptone-sucrose-agar (PSA) medium (see Recipes)
  29. PSA-CAS-DP plate (see Recipes)

Equipment

  1. Conical flasks: 500 ml (Vensil Glass Works, catalog number: 1161 )
  2. Conical flasks: 2,000 ml (Borosil, catalog number: 4980030 )
  3. Micro-pipettes (Eppendorf, catalog number: EPPR4396 )
  4. Shaking incubator (Eppendorf, New BrunswickTM, model: Innova® 43 )
  5. SORVALL RC-5B PLUS Superspeed centrifuge (Thermo Fisher Scientific, Waltham, MA, USA)
  6. pH meter (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EC-PH510/11S )
  7. Agilent 1100 series HPLC system (Agilent Technologies, model: Agilent 1100 Series )
  8. Agilent C-18 (4.6 mm x 250 mm x 5 μm) column (Agilent Technologies, Santa Clara, CA, USA)
  9. Burette (BOROSIL, catalog number: 2123016 ), burette support stand and clamps
  10. Vacuum concentrator plus (Eppendorf, model: Concentrator Plus , catalog number: 5305000304)
  11. Millipore vacuum, pressure pump (EMD Millipore, catalog number: XI04 220 50 )
  12. Autoclave

Software

  1. ChemStation Software; Rev. A.10.02 [1757] (Agilent, Santa Clara, CA, USA)
  2. Microsoft Office Excel 2013 (Microsoft Corporation, Redmond, USA)

Procedure

English
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文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Pandey, S. S., Singh, P., Samal, B., Verma, R. K. and Chatterjee, S. (2017). Xanthoferrin Siderophore Estimation from the Cell-free Culture Supernatants of Different Xanthomonas Strains by HPLC. Bio-protocol 7(14): e2410. DOI: 10.21769/BioProtoc.2410.

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分类

微生物学 > 微生物-宿主相互作用 > 细菌

植物科学 > 植物免疫 > 宿主-细菌相互作用

生物化学 > 脂质 > 胞外脂质

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