Introduction

Our device relies on intracellular iron concentrations increasing as a result of higher siderophore concentrations. As a proof of concept, we measured the amount of iron in a enterobactin deficient strain, JC28, and its parental wild type background W3110 grown in LB, using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES). We found that intracellular iron was approximately 3 times higher in W3110.

Our reporter system utilises the Ferric Uptake Regulator Fur system to detect increases in intracellular iron levels caused by higher extracellular unbound siderophore concentrations. Therefore, we needed to demonstrate that the presence of siderophores increases intracellular iron levels, and to confirm that our JC28 strain does not produce enterobactin.

The Theory

Ideally, we would use the enterobactin deficient, JC28 strain and add known concentrations of siderophores into the medium, to see how it affects intracellular iron concentration. Initially, we cultured JC28 and W3110 in LB assuming enterobactin production was the only relevant factor differing between the two strains.

Figure 1. Schematic overview of Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES)¹

ICP-AES is a test for total iron concentration, and detects both free and protein associated iron. Ideally, we would use electron paramagnetic resonance (EPR) spectroscopy and measure free Fe(II) concentrations in living cells directly, which would be useful for the mathematical modelling of Fur activity. However, we did not have access to this technique. An increase in total iron would result in a higher free iron concentration as iron storage is triggered by Fur.

To measure the amount of intracellular iron, we used ICP-AES. This technique allows the measurement of trace metals at very low concentrations. The solution, in this case a suspension of lysed cells in acid, is passed through a nebuliser, to create an evenly dispersed mist (see Fig.1 for a schematic overview). The mist enters a plasma formed through the ionisation of argon gas by high power radio waves. At temperatures above 7000 K, Fe(II) and Fe(III) are both converted to free atoms in the plasma, so this technique is not specific to one oxidation state. In the plasma iron atoms become excited. As they de-excite specific frequencies of light are emitted due to quantum-mechanical effects. These frequencies are separated by a spectrometer and the intensity of emission is compared against known standards to calculate iron concentrations.

The data was normalised to the number of cells, based on the approximation that optical density is proportional to the number of cells, and that an OD600 of 1 corresponds to 8x10⁸ cells. The amount of intracellular iron was calculated using the equation below.

Equation 1.

Experimental protocol

1. 3 samples each of W3110 and JC28 were cultured overnight in LB
2. Samples were pelleted by centrifugation, and the supernatant was removed
3. Cells were washed 3 times in 1 ml PBS and recovered by centrifugation
4. Cells were resuspended in 2 ml PBS
5. 50 µl samples were taken for OD₆₀₀ measurements
6. 1 ml samples were taken and recovered by centrifugation
7. Pellets were suspended in 5 ml concentrated hydrochloric acid
8. Samples were lysed by 4 cycles of sonication and cold shock with an ice bath
9. Samples were heated to 90°C for 60 minutes
10. Samples were made up to 50 ml in deionised water using grade A glassware
11. 5 ml of each sample was immediately tested with a Spectro Ciros Vision ICP-ES machine

Results

Figure 2. Average number of iron atoms per cell of a siderophore-production difficient E. coli strain (JC28), and of its its parental wild type background (W3110), measured using ICP-AES. Measured with ICP-AES on samples prepared using the experimental protocol described above. P-values calculated using an unpaired 2-sample 2-tailed t-test; P<0.05, n = 3.

See appendix 1 for full data. Intracellular iron concentrations were calculated from the ICP readings and OD₆₀₀ measurements using equation 1 and the approximation that OD is proportional the number of cells with a constant of proportionality of 8x10⁸. The error bars on the graph represent the standard error of the mean.

The graph shows that the amount of intracellular iron in W3110 was significantly greater than in JC28 (Fig. 2). This provides indirect evidence that the JC28 strain lacks enterobactin production capabilities, and suggests that the presence of siderophores increases intracellular iron levels.

We initially explored a low cost iron concentration measurement method using Ferene-S, which produces a strong blue colour (peak absorption at 593 nm) in the presence of iron (II). We planned to measure iron concentrations using Ferene and then validate the accuracy by comparing to the ICP results. A discrepancy between the iron levels measured using Ferene and using ICP suggests that this method needs improving, but It may be useful to future teams looking to measure iron concentrations.

Theory

Figure 3. A structural representation of the Ferene-S salt.

Ferene (Fig. 3) binds to Fe(II) with high affinity, with a stoichiometry of 3 Ferene molecules per Fe(II) ion, to produce a water soluble, strongly coloured complex, with a peak absorbance at 593 nm.² We hoped to be able to use this to measure the amount of intracellular iron in E. coli.

Ferene can only bind to free iron(II) ions, but most of the iron in cells is stored as Ferritin-associated Fe(III). Therefore, we needed to design a method to release and reduce Fe(III) to Fe(II). We decided to use repeated sonication and cold shocking with an ice bath to lyse the cells. We used heat to denature the ferritin proteins, and ascorbic acid as a reducing agent, to solubilise the iron and convert it from Fe(III) to Fe(II) (see Fig.4). Spectroscopy can then be used to detect the intensity of absorption and hence calculate iron concentration through the Beer-Lambert law (below).

Equation 2.

Figure 4. Diagram of the iron release process used in the experimental protocol below. Cells are lysed, using sonication to release ferritin from the cells. Heat, in combination with ascorbic acid is used to denature ferritin and reduce all iron to Fe(II), so that Ferene can bind.

Results

To determine the molar extinction coefficient we produced a calibration curve of absorption at 593 nm of solutions made up with Fe(III), ascorbic acid, and Ferene with varying iron concentrations. Volumes and concentrations are shown in appendix 2. Ascorbic acid is in a large excess, and samples were left for approximately 1 hour, to give a constant absorption. Therefore, all the Fe(III) will have been reduced to Fe(II).

Figure 5. A calibration curve of the absorbance of a solution of iron(II) and Ferene at varying Fe(II) concentrations. The gradient of the linear region of the graph, with iron concentration being the sole limiting factor of absorbance, is shown.

At approximately 40 µM, the curve begins to level off due to Ferene not being in sufficient excess to fully bind all the iron (Fig. 5). The Ferene concentration is approximately 200 µM, therefore this shows that Ferene should be at least 5 times in excess of iron, to ensure full complexation. Since unbound Ferene exhibits low absorption at 593 nm, we used higher Ferene concentrations in our assay. By taking the gradient of the linear region of this graph, we can determine the molar extinction coefficient of the iron-Ferene complex.

Error analysis on the gradient of the linear region (Fig. 5) shows that the molar extinction coefficient of the iron-Ferene complex is 26100 +/- 300 mol^-1 L cm^-1.

Ferene iron assay protocol

1. Grow 5 x 10 ml cultures for each sample in the required medium overnight
2. Centrifuge all cultures and remove supernatant
3. Resuspend all 5 cultures in PBS and transfer to a single tube
4. Wash 3 times in 1 ml PBS and recovered by centrifugation.
5. Resuspend in 2 ml PBS
6. Take a 100 µl sample and dilute to take OD₆₀₀ measurement
7. Transfer 1 ml into a fresh tube, recovered by centrifugation.
8. Resuspend in 1 ml of 100 µM ascorbic acid solution
9. Lyse cells by sonicating and ice shocking 4 times
10. Heat sample to 90 °C for 1 hour
11. Transfer 700 µl to an Eppendorf tube
12. Add 300 uL of 10 µM Ferene solution, mix and incubate for 5 mins
13. If precipitate forms, centrifuge, and transfer supernatant into clean cuvette
14. Record absorbance at 593 nm

We initially tried performing the assay without the concentration steps (2-3), but we found that this gave absorption values approaching the lower limit of our spectrophotometer. We also tried using hydrochloric acid to improve the solubility and increase the rate of reduction of iron, but we found that low pHs decreased the absorption of iron-Ferene solutions. We performed this assay several times using LB (data in appendix 3) and tried using minimal media. We found that using minimal media, even with the concentration step, gave inconsistant results towards the lower limit of our spectrophotometer.

Figure 6. Samples of Ferene treated cell extract. The difference in absorbance is visible by eye. Water (blank), W3110 grown in LB (LW), JC28 grown in LB (LJ)

Figure 7. Number of iron atoms per cell of the JC28 and W3110 strains measured using the Ferene assay described above. Average number of iron atoms per cell of a siderophore-production difficient E.coli strain (JC28), and of its its parental wild type background (W3110), measured using our ferene-spectrophotometry assay

See appendix 3 for full data. Intracellular iron concentrations were calculated using absorbance at 593nm measurements with the molar extinction coefficient obtained from the calibration curve (Fig. 5), and the approximation that OD₆₀₀ is proportional to number of cells with a constant of proportionality of 8x10⁸. The error bars on the graph represent the standard error of the mean.

Although there was a large difference between the measured amount of iron in W3110 and JC28, the amount of iron detected was less than the amount measured using ICP-AES (Fig. 7). This suggests that using this technique not all the iron present is being detected. This could be due to the cells not being fully lysed or the iron not being fully released from proteins.

Due to time constraints this method was not investigated any further, but perhaps it could be improved by trying other lysis methods or allowing the reduction to take place overnight. We would recommend using Ferene as a cheaper alternative to ICP-AES to measure iron concentrations at the micro molar level. However, this option remains technically challenging and more work is required to construct the experimental framework.

References

1. Figure adapted from: Diadrasis, ICPAES/ICPMS, http://www.viaduct-diadrasis.net/methods/6
2. Hennessy DJ, Reid GR, Smith FE, Thompson SL. Ferene — a new spectrophotometric reagent for iron. Can J Chem. NRC Research Press Ottawa, Canada; 1984 Apr;62(4):721–4.

Appendices

APPENDIX 1.



APPENDIX 2.



APPENDIX 3.