Siderophores are low molecular weight, metal-chelating biomolecules involved in iron cycling in the environment. This protocol allows for rapid high throughput assessment of siderophore activity in soil and plant samples. Previous methods for siderophore detection have eliminated the crucial environment of the microbial community.
Our technique allows detection within the relatively intact microbial community and the habitat in which it involved. Because iron availability is crucial to agriculture productivity, so in this protocol could be used to investigate the role of microbes in modulating the availability of iron to plants. Additionally, this method could be used to assess the impact of farm management practices on soil health and siderophore-producing communities as well as the development of such communities over time.
To begin, acid wash all glassware in 100 millimolar hydrochloric 100 millimolar nitric acid for a minimum of two hours prior to utilization in the CAS assay. Prepare an aluminum baking pan filled with laboratory-grade sand and cover it with aluminum foil. Autoclave at 121 degrees Celsius for 30 minutes and set aside.
Prepare HDTMA by adding 0365 grams to 20 milliliters of double deionized water, and place the solution in a water bath at 37 degrees Celsius to promote solubilization. Add 0302 grams of CAS to 25 milliliters of double deionized water while gently stirring with a sterile magnetic stir bar. Then add five milliliters of one molar iron chloride hexahydrate to the 25 milliliters of CAS solution while continuing to gently stir.
Now, slowly add the 20 milliliters of HDTMA solution into the iron CAS complex solution while gently stirring. Prepare the buffer solution by dissolving 15.12 grams of PIPES into 375 milliliters of double deionized water with gently stirring. Adjust the pH to 6.8 with five molar sodium hydroxide.
Then add water to bring the volume to 450 milliliters. Now add five grams of agarose to the solution. Autoclave the PIPES buffer solution and the iron CAS complex solution at 121 degrees Celsius for 30 minutes.
Carefully add the entirety of the iron CAS complex solution to the entirety of the PIPES buffer in the biosafety cabinet after each of them has been autoclaved. Place the mixed solution in a water bath at 50 degrees Celsius. Now place a sterile reagent boat in the sterile sand within the biosafety cabinet and heat to 50 degrees Celsius.
Transfer the iron CAS complex agar to the boat, then quickly aliquot 100 microliters to each well of a clear flat-bottomed sterile 96-well microplate. Prepare 800 micromolar pyoverdine standard in previously prepared modified M9 medium. Dilute the solution into 400, 200, 100, 50, 25, 12.5, and 6.25 micromolar solutions.
Add EDTA to 500 milliliters of previously prepared modified M9 medium to prepare the 3.2 millimolar EDTA standard. Dilute this solution into 1600, 800, 400, 200, 100, 50, 25, 12.5, and 6.25 micromolar solutions. To generate a standard curve, add 100 microliters of each concentration of pyoverdine and EDTA to separate wells of a 96-well microplate containing 100 microliters of iron CAS complex agar medium.
Make duplicate technical replications of each concentration. Also add blank wells with only M9.Using a microplate reader, measure absorbance after one, six, and 24 hours incubation at 22 degrees Celsius, and use absorbance measurements to generate standard curves. Wash the sampling equipment with 22 micron filter double deionized water followed by 70%ethanol and wipe with paper towels.
Perform the wash before sampling and in between samples to maintain sterile technique and reduce cross-contamination. After excising plant tissues and excavating a small root ball from plants in the field, place the root ball in a separate labeled plastic bag for sample separation in the laboratory environment. Place all samples directly on ice and keep at four degrees Celsius until samples are processed for the siderophore production assay.
Separate root associated soil samples into bulk, loosely bound rhizosphere soil and tightly bound rhizosphere soil. To do so, take the root balls out of the bags and gently shake off soil from the root ball. Shaken off soil, along with the soil left in the bag, comprise the bulk soil.
This is the loosely bound rhizosphere soil. Generate tightly bound rhizopshere soil by taking roots with the tightly bound sample and putting them in a centrifuge tube. Add 30 milliliters of double deionized water and vortex for two to three minutes.
Remove the roots to get the tightly bound rhizosphere soil slurry dilution. Homogenize each soil sample within the sample bag by mixing and turning the soil as much as possible without opening the bag. After each sample has been thoroughly mixed, aliquot and suspend two grams of each soil sample in 20 milliliters of modified M9 medium within a sterile 50 milliliter centrifuge tube.
Dilute the sample and then seal the tube with a sterile foam plug to allow aeration. For tightly bound rhizosphere samples, add two milliliters of the rhizosphere soil slurry to 20 milliliters of modified M9 medium within a sterile 50 milliliter centrifuge tube. Dilute the sample and then seal the tube with a sterile foam plug to allow aeration.
To prepare the tissue sample, surface sterilize the root, shoot, and grain with 70%ethanol. Macerate two grams of fresh tissue in 20 milliliters of modified M9 medium using a blender on high for 30 seconds. Then, transfer the sample to a sterile 50 milliliter centrifuge tube, dilute, and seal the tube with a sterile foam plug.
To enrich siderophore production through iron limitation incubate the 50 milliliter centrifuge tubes at room temperature and shake at 160 RPM. At 24, 48, and 72 hours after initiating the enrichment culture, remove one milliliter subsamples from the enrichment tubes using sterile technique. Centrifuge the subsamples at 10, 000 times G for one minute in two milliliter centrifuge tubes to pelletize the cells.
Using sterile technique, add 100 microliters of the supernatant to a 100 microliter solution of iron CAS complex agar in duplicate or triplicate within the microplate. Also add 100 microliters of sterile M9 medium as blanks. Then incubate the plate at 28 degrees Celsius.
Each plate should be sealed and covered with foil prior to placement in the incubator. Suspend the remaining supernatant and pellet for each sample into its own sterile two milliliter centrifuge tube. Add 400 microliters of sterile glycerol into each sample supernatant tube and re-suspend the pellet to create glycerol stocks.
Freeze the stock at minus 80 degrees Celsius for later analysis. Measure the absorbance at six, 24, 48, and 72 hours at a wavelength of 420 nanometers. A pyoverdine mixture biosynthesized by Pseudomonas fluorescens was used as a standard to interpret and quantify absorbance of samples in samples of pyoverdine equivalence in micromolar.
The relationship between absorbance at 420 nanometers and the starting concentration of pyoverdine is shown here. Siderophore activity of the 72 hour enrichment was assessed at 48 hours incubation to determine the influence of genotype and sample type on siderophore isolation. Siderophore activity in bulk soil samples was relatively low and did not exhibit differences between the wheat genotype from which the bulk soil was sampled.
However, enrichments of loosely bound soil isolated from the 725 genotype exhibited greater siderophore production as compared with the loosely bound soil from Madsen and 727 but not from Lewjain. Conversely, siderophore production in enrichments from tightly bound soil was not heavily influenced by genotype. Enrichment cultures of grain tissue yielded relatively low siderophore production regardless of genotype.
Enrichments of Lewjain shoot tissue had significantly lower siderophore production than the other genotypes, and 725 shoot tissue cultures resulted in more variable siderophore production. The greatest difference in siderophore activity was observed in root tissue enrichment cultures where 725 had more than 200%greater siderophore activity than all other genotypes. One of the most taxing aspects of executing the protocol is maintaining aseptic conditions while completing the many steps to generate the CAS iron agar microplate and testing samples for siderophore activity.
A wide array of chromatographic culture-based or DNA-based techniques can be applied either to the actual microtiter well or the glycerol preserved culture for further biochemical tests or genetic characterization and metabolic modeling. By using this protocol siderophore activity can later be linked with specific organisms responsible for that activity or subsequently targeted as a mechanism for larger ecological impacts. This protocol could easily be modified to assess siderophore production in enrichment cultures generated from samples gathered from other environmental compartments including air, water, and sediments.
