High-throughput Method for Detecting Siderophore Production by Rhizosphere Bacteria

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 PAO1_{ΔpvdDΔpchEF} (Ghysels et al., 2005)

7. Pseudomonas aeruginosa H111_{ΔorbJΔpchAB} (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

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 PAO1_{ΔpvdDΔpchEF} and B. cenocepacia H111_{ΔorbJΔpchAB}) (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

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