High-throughput Siderophore Screening from Environmental Samples: Plant Tissues, Bulk Soils, and Rhizosphere Soils

Summary

We present a protocol for rapid screening of environmental samples for siderophore potential contributing to micronutrient bioavailability and turnover in terrestrial systems.

Abstract

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling in soils, to pathogen competition, plant growth promotion, and cross-kingdom signaling. Furthermore, siderophores are also of commercial interest in bioleaching and bioweathering of metal-bearing minerals and ores. A rapid, cost effective, and robust means of quantitatively assessing siderophore production in complex samples is key to identifying important aspects of the ecological ramifications of siderophore activity, including, novel siderophore producing microbes. The method presented here was developed to assess siderophore activity of in-tact microbiome communities, in environmental samples, such as soil or plant tissues. The samples were homogenized and diluted in a modified M9 medium (without Fe), and enrichment cultures were incubated for 3 days. Siderophore production was assessed in samples at 24, 48, and 72 hours (h) using a novel 96-well microplate CAS (Chrome azurol sulphonate)-Fe agar assay, an adaptation of the traditionally tedious and time-consuming colorimetric method of assessing siderophore activity, performed on individual cultivated microbial isolates. We applied our method to 4 different genotypes/Lines of wheat (Triticum aestivum L.), including Lewjain, Madsen, and PI561725, and PI561727 commonly grown in the inland Pacific Northwest. Siderophore production was clearly impacted by the genotype of wheat, and in the specific types of plant tissues observed. We successfully used our method to rapidly screen for the influence of plant genotype on siderophore production, a key function in terrestrial and aquatic ecosystems. We produced many technical replicates, yielding very reliable statistical differences in soils and within plant tissues. Importantly, the results show the proposed method can be used to rapidly examine siderophore production in complex samples with a high degree of reliability, in a manner that allows communities to be preserved for later work to identify taxa and functional genes.

Introduction

Siderophores are important biomolecules involved primarily in iron-chelation for bioavailability, but with a wide array of additional purposes in terrestrial and aquatic ecosystems ranging from microbial quorum sensing, signaling to microbial plant-hosts, plant growth promotion, cooperation and competition within complex microbial communities^1,2. Siderophores can be broadly classified according to their active sites and structural features, creating four basic types: carboxylate, hydroxamate, catecholate, and mixed types^3,4. Many microorganisms are capable of excreting more than one type of siderophore⁵ and in complex communities, a vast majority of organisms biosynthesize the membrane receptors to allow the uptake of an even wider variety of siderophores^1,6. Recent work indicates that siderophores are particularly important at the community level, and even in inter-kingdom communications and biogeochemical transfers^7,8,9,10,11.

Chrome azurol sulphonate (CAS) has been used for over 30 years as a chelating agent to bind iron (Fe) in such a way that addition of ligands (i.e., siderophores) can result in dissociation of the CAS-Fe complex, creating an easily identifiable color change in the medium¹². When the CAS is bound with Fe, the dye appears as a royal blue color, and as the CAS-Fe complex dissociates, the medium changes color according to the type of ligand used to scavenge the Fe¹³. The initial, liquid-based medium established by Schwyn and Neilands in 1987, has been modified in many ways to accommodate changing microbial targets¹⁴, growth habits and limitations¹⁵, as well as a variety of metals besides Fe, including aluminum, manganese, cobalt, cadmium nickel, lithium, zinc¹⁶, copper¹⁷, and even arsenic¹⁸.

Many human pathogens, as well as plant growth promoting microorganisms (PGPM) have been identified as siderophore-producing organisms^3,19,20, and important rhizosphere and endophytic PGPM often test positive for siderophore-production⁴. The traditional Fe-based liquid method has been adapted to microtiter testing of isolates in cultivation for siderophore production²¹. However, these techniques fail to recognize the importance of the microbial community as a whole (the microbiome), in cooperation and potential regulation of siderophore production in soils and plant systems²². For that reason, we have developed a high-throughput community-level assessment of siderophore production from a given environment, based on the traditional CAS assay, but with replication, ease of measurement, reliability, and repeatability in a microplate assay.

In this study, a cost-effective, high-throughput CAS-Fe assay for detecting siderophore production is presented to assess the enrichment of siderophore production from complex samples (i.e., soil and plant tissue homogenates). Bulk, loosely-bound, and tightly-bound rhizosphere soil (in terms of how the soil was bound to the root) were obtained along with grain, shoot, and root tissues from four distinct wheat (Triticum aestivum L.) genotypes: Lewjain, Madsen, PI561725, and PI561727. It was hypothesized that fundamental differences in the wheat genotypes could result in differences in recruitment and selection of siderophore producing communities. Of particular interest is the difference between microbial communities associated with the PI561725 isogenic line, which is aluminum tolerant because it possesses ALMT1 (Aluminum-activated Malate Transporter 1), compared with the aluminum sensitive PI561727 isogenic line, which possesses a non-aluminum responsive form of the gene, almt1^23,24,25,26. The chief objective of the study was to develop a straightforward, rapid method of quantitatively assessing siderophore production in siderophore enrichment cultures of complex sample types while preserving the cultures for future work.

Protocol

NOTE: Location of Field Site: Washington State University, Plant Pathology Farm (46°46’38.0”N 117°04’57.4”W). Seeds were sown using a mechanical planter on October 19, 2017. Each wheat genotype was planted in headrows, approximately 1 meter apart to avoid overlapping of root system. Plant and soil samples were collected on August 9, 2018, when plants were ready for harvest. Samples were gathered from three replicates of four wheat genotypes: PI561727, PI561725, Madsen, Lewjain.

1. Preparation of modified M9 medium

1. Use Na₂PO₄∙7H₂O (12.8 g/200 mL), KH₂PO₄ (0.3 g/200 mL), NaCl (0.5 g/200 mL), and NH₄Cl (1 g/20 mL) reagents to prepare the M9 salt solution.
2. Use 18 g of MgSO₄∙7H₂O in 100 mL of double deionized water (ddH₂O) to prepare 0.75 M MgSO₄∙7H₂O.
3. Make 1 M of CaCl₂∙2H₂O by adding 14.7 g of CaCl₂∙2H₂O with 100 mL of ddH₂O.
4. Prepare the buffer solution by dissolving 6.048 g of PIPES into 156 mL of ddH₂O with stirring. Adjust the pH to 6.8 with 5 M NaOH.
5. Prepare 20% glucose (dextrose monohydrate) by dissolving 20 g of glucose into 100 mL of ddH₂O.
6. Prepare solutions and individually autoclave all but the glucose. Filter sterilize the glucose solution for addition to the M9 media after autoclaving (0.22 µm).
7. Prepare the modified M9 medium by mixing 156 mL of PIPES Buffer solution, 40 mL of M9 salts solution, 20 μL of CaCl₂·2H₂O solution, 266 μL of MgSO₄·7H₂O, and 4 mL of 20% glucose solutions together in the biosafety cabinet, using sterile technique.
8. For preservation of the modified M9, seal the container, cover with aluminum foil to protect from UV, and place at 4 °C.

2. Preparation of CAS-Fe-Agar medium

1. Acid wash all glassware in 100 mM HCl/100 mM HNO₃ for a minimum of 2 h prior to utilization in the CAS assay.
2. Prepare an aluminum baking pan filled with laboratory grade sand and cover it with aluminum foil. Autoclave at 121 °C for 30 min and set aside.
3. Prepare HDTMA (hexadecyltrimethylammonium bromide) by adding 0.0365 g to 20 mL ddH₂O and place the solution at 37 °C to promote solubilization.
4. Prepare 10 mM HCl and generate 1 mM FeCl₃·6H₂O using 10 mM HCl as the solvent. Add 0.0302 g of CAS to 25 mL of ddH₂O while gently stirring with a sterile magnetic stir bar. Then add 5 mL of 1 mM FeCl₃·6H₂O (in 10 mM HCl) to the 25 mL of CAS solution while continuing to gently stir (the solution turns into dark reddish black color).
5. Slowly add the 20 mL of HDTMA solution while gently stirring, into the Fe-CAS solution (this yields a dark blue solution).
6. Prepare the buffer solution by dissolving 15.12 g of PIPES into 375 mL of ddH₂O, with gentle stirring. Adjust the pH to 6.8 with 5 M NaOH. Add water to bring the volume to 450 mL. Add 5 g of agarose to the solution. Autoclave the PIPES buffer solution and the CAS-Fe solution at 121 °C for 30 min.
7. Carefully add the entirety of the CAS-Fe solution to the entirety of the PIPES Buffer in the biosafety cabinet after each of them is autoclaved.
8. Place the mixed solution in a water bath at 50 °C.
   NOTE: Freshly prepare all the reagents in the CAS-Fe-Agar medium before each assay, as long-term storage at 50 °C results in precipitation of the CAS-Fe complex, and cooling results in solidified medium.
9. Place a sterile reagent boat in the sterile sand in the biosafety cabinet and heat to 50 °C. Transfer the CAS-Fe-Agar to the boat, then quickly aliquot 100 µL to each well in a clear, flat-bottom, sterile 96-well microplate.

3. Pyoverdine/EDTA standard preparation

1. Pyoverdine preparation
   1. Prepare 800 μM pyoverdine standard (mixture of succinic acid, 2-hydroxy glutaramide, and succinaminde forms of pyoverdine – see Table of Materials), in previously prepared modified M9 medium.
   2. Dilute this solution into 400, 200, 100, 50, 25, 12.5, and 6.25 μM solutions.
2. EDTA standard preparation
   1. Add 0.594 g of disodium ethylenediaminetetraacetic acid (EDTA: C₁₀H₁₄N₂Na₂O₈.2H₂O), to 500 mL of previously prepared modified M9 medium to prepare 3200 μM EDTA standard.
   2. Dilute this solution into 1600, 800, 400, 200, 100, 50, 25, 12.5, and 6.25 μM solutions.
3. Standard curve generation
   1. Add 100 μL of each concentration of pyoverdine and EDTA to separate wells of a 96-well microplate containing 100 μL of CAS-Fe Agar medium. Make duplicate technical replications of each concentration. Also, add blank wells with only M9 (no EDTA or pyoverdine).
   2. Using a microplate reader, measure absorbance (at 420 nm and 665 nm) after 1, 6, and 24 h incubation at 22 °C, and use absorbance measurements to generate standard curves.
      NOTE: For 420 nm measurements, subtract absorbance of blanks from the absorbance of pyoverdine or EDTA containing wells. For 665 nm, subtract absorbance of pyoverdine or EDTA containing wells from absorbance of blanks. Then, log₁₀(µM pyoverdine or EDTA) is regressed against the absorbance measurements. For ease of interpreting sample results, use absorbance as the x-axis and log₁₀(µM pyoverdine or EDTA) as the y-axis.

4. Collection of environmental samples: soil and plant tissues

1. Wash sampling equipment (shovels and scissors) with 0.22 µm filtered ddH₂O followed by 70% ethanol, and wipe with paper towels before sampling and in between samples to maintain sterile technique and reduce cross-contamination.
2. Excise the plant tissues (grains and shoots) from plants in the field, and place them in a labeled plastic storage bag, leaving enough shoot stubble for ease in uprooting the desired soil and root samples.
3. Excavate a small root ball approximately 15 cm deep and 23 cm wide and place it in a separate, labeled plastic bag for sample preparation in the laboratory environment. This step is similar to the methods of McPherson et al.²⁷.
4. Place all samples (grains, shoots, bulk soil, and root balls in separate bags) directly on ice and keep at 4 °C until samples are processed for the siderophore production assay.
5. Separate root associated soil samples into bulk, loosely-bound rhizosphere soil, and tightly-bound rhizosphere soil.
   1. Take the root balls out of the bags. Gently shake off soil from the root ball. Shaken off soil, along with the soil left in the bag comprises the “bulk” soil.
   2. Use a rubber mallet to further remove soil from the root ball. This is the loosely-bound rhizosphere soil.
   3. Generate tightly-bound rhizosphere soil by taking roots with the tightly-bound sample and putting them in a centrifuge tube. Add 30 mL of ddH₂O and vortex it for 2-3 minutes. Remove roots to get the tightly-bound rhizosphere soil slurry dilution.

5. Preparation of siderophore enrichment cultures and CAS-Fe siderophore production assay

NOTE: All glassware should be acid washed prior to beginning the assays.

1. Soil sample preparation (for each of the three soil sample types)
   1. Homogenize each soil sample within the sample bag, by mixing and turning the soil as much as possible without opening the bag.
      NOTE: This helps reduce natural soil spatial variability and aligns with normal soil sampling procedures. Other methods may be utilized to homogenize environmental samples, as appropriate, and depending on the experimental design.
   2. After each sample has been thoroughly mixed, aliquot and suspend 2.0 g of each soil in 20 mL of modified M9 medium, then dilute 10^-3 to a total volume of 20 mL in a sterile 50 mL centrifuge tube with a sterile foam plug to allow aeration.
   3. For tightly-bound rhizosphere samples, add 2 mL of the rhizosphere soil slurry to 20 mL of modified M9 medium, then dilute 10^-3 to a total volume of 20 mL in a sterile 50 mL centrifuge tube with a sterile foam plug to allow aeration.
2. Tissue sample (root, shoot and grain) preparation
   1. Surface sterilize the sample with 70% ethanol. Macerate 2.0 g of fresh tissue in 20 mL of modified M9 medium using a blender on high for 30 seconds. Transfer the sample to a sterile 50 mL centrifuge tube, then dilute 10^-3 to a total volume of 20 mL in a sterile 50 mL centrifuge tube with a sterile foam plug to allow aeration.
3. Enrichment of Siderophore Production Through Fe Limitation
   1. Incubate 50 mL centrifuge tubes at room temperature and shake at 160 rpm.

6. CAS-Fe agar assays for detection of siderophore production in environmental samples

1. At 24, 48, and 72 h after initiating the enrichment culture, remove 1 mL subsamples from the enrichment tubes using sterile technique and centrifuge at 10,000 x g for 1 min in 2 mL centrifuge tubes to pelletize the cells.
2. Collect the separated supernatant. Using sterile technique, add 100 μL of the supernatant to 100 μL solution of CAS-Fe-Agar in duplicate or triplicate in the microplate. Also add 100 µL of sterile M9 medium (as blanks). Then incubate the plate at 28 °C.
3. Suspend the remaining supernatant and pellet for each sample (not added to microtiter plate) into its own, sterile 2 mL centrifuge tube. Add 400 μL of sterile glycerol into each sample-supernatant tube and resuspend the pellet to create glycerol stocks. Freeze the stock at -80 °C for later analyses.
   NOTE: This step can be modified to generate glycerol stocks according to any preferred in-house protocol.
4. Measure absorbance at 6, 24, 48, and 72 h, at 420 nm wavelength.
5. Use standard curves generated from pyoverdine or EDTA to interpret sample absorbance measurements in terms of pyoverdine equivalents.
   NOTE: Pyoverdine was determined to be a superior standard compared with EDTA (vide infra), so EDTA was not used to interpret results in the current study.

Results

A pyoverdine mixture biosynthesized by Pseudomonas fluorescens was used as a standard to interpret and quantify absorbance (at 420 nm) of samples in terms of pyoverdine equivalents in µM. Figure 1 shows the relationship between absorbance (420 nm) and starting concentration of pyoverdine (Log₁₀ molarity in µM). EDTA did not provide an adequate standard because samples exhibited greater absorbance measurements than were attainable...

Discussion

The primary result of this work is the production of a new methodology that can be used to rapidly enrich for siderophore producing microbes while quantitatively measuring siderophore production/activity in the environmental sample. The methodology is quick, simple, and cost-effective, and the results show how it can be used to detect siderophore activity from complex and novel sample types (e.g., soil and plant tissue). The protocol also results in the production of glycerol stocks of the enrichment cultures, w...

Materials

Name	Company	Catalog Number	Comments
Agarose	Apex	LF451320014
Aluminum Baking Pan
Aluminum Foil
Ammonium chloride, granular	Fiesher Scientific	152315A
Autoclave and Sterilizer	Thermo Scientific
Calcium chloride dihydrate	Fiesher Scientific	171428
CAS (Chrome Azurol S)	Chem-Impex Int'l Inc)	000331-27168
Dextrose Monohydrate (glucose), crystalline powder	Fiesher Scientific	1521754
EDTA, disodium salt, dihydrate, Crystal	J.T.Baker	JI2476
Glycerol, Anhydrous	Baker Analyzed	C22634
HDTMA (Cetyltrimethylammomonium Bromide	Reagent World	FZ0941
Hydrochloride acid	ACROS Organic	B0756767
Infinite M200 PRO plate reader	TECAN
Iron (III) chloride hexahydrate, 99%	ACROS Organic	A0342179
Laboratory Fume Hood	Thermo Scientific
Laboratory Incubator	VWR Scientific
Magnesium Sulfate	Fiesher Scientific	27855
Niric Acid, (69-70)%	J.T.Baker	72287
PIPES buffer, 98.5%	ACROS Organic	A0338723
Potassium phosphate, dibaisc,powder	J.T.Baker	J48594
Pyoverdine	SIGMA-ALDRICH	078M4094V
Sand
SI-600R Shaker	Lab Companion
Sodium chloride, granular	Fiesher Scientific	136539
Sodium hydroxide, pellets	J.T.Baker	G48K53
Sodium phosphate, dibasic heptahydrate, 99%	ACROS Organic	A0371705