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, Xanthoferrin, Bacterial siderophore estimation, HPLC, Amberlite XAD-16 resin columns
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
Equipment
Software
Procedure
Data analysis
Load HPLC signals of sample (Procedure D; step D5) and blank (empty buffer elute from the XAD-16 resin column) to the offline mode of ChemStation software. Subtract the blank signal from sample signal and then auto-integrate to automatic identification of peaks. Record the RT (Retention Time) values and peak areas generated in the dialogue box. The pure xanthoferrin signals can be observed while monitoring the missing peak in the HPLC chromatogram of isolated siderophores from ∆xssA mutant of Xanthomonas spp. in comparison to their respective wild-type. From the active fractions of the xanthoferrin, the purest fraction can be isolated, vacuum-dried, weighed and utilized as ‘standard xanthoferrin’ for plotting the standard curve by taking different known amounts for HPLC runs. Draw the standard curve of xanthoferrin by plotting the ‘amounts vs. average peak areas’ obtained after respective HPLC runs (Figure 4). Determine the amount of unknown sample xanthoferrin siderophores using the standard curve. Validate the significance level in the difference, if any exists between the data obtained for two different sets, using a paired Student’s t-test. Note: For one set of experiments, take at least three independent biological replicates. Figure 4. Xanthoferrin standard curve; displaying the equation for depicting the amount of siderophores used in the HPLC run for getting respective peak areas
Notes
Recipes
Acknowledgments
We acknowledge Dr. Masaki J. Fujita (Hokkaido University, Japan) for providing the pure vibrioferrin for this study. We extend the acknowledgement to a previous study from our lab (Rai, 2015), from where we have modified the siderophore estimation protocol for Xcc. This work was supported by funding to SC from DBT-India, CSIR-HRDG-India, DST-SERB-India and CDFD core funding. SSP was recipient of JRF and SRF from CSIR-India. PS and BS were recipients of JRF and SRF from UGC-India. RKV was recipient of JRF and SRF from DBT-India.
References
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