Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation

The overall goal of this technique is to extract bacterial periplasmic contents by cell fractionation for biophysical characterization, including x-ray fluorescence, x-ray crystallography and radio metal uptake. This method can help us understand key biological relationships, including structure-function relationships of ABC transport systems and how they recognize their substrates, specifically, and transport them into the cell through the lipid bilayer. The main advantage of this technique is to separately evaluate the cell contents and to extract protein from its native environment with minimal cross-contamination.

We first had the idea for this method while trying to remove YfeA substrate, realizing, instead of using an artificial approach we could use the cell to produce apo protein. In addition to providing insight into substrate binding and transport across bacterial cell compartments, methods like this radiotracer assay apply to eukaryotic systems, which also have robust fractionation techniques. First, thaw a previously prepared bacterial subculture re-suspension at four degrees Celsius.

Then, pellet the thawed cells at 4, 000 times gravity for 20 minutes at 4 degrees Celsius. Next, re-suspend the cells in 50 milliliters of ice-cold high-salt buffer by aspirating with an automated pipette and a 25-milliliter tip. Incubate the suspension over ice for 20 minutes with occasional inversion for mixing.

After incubation, pellet the cells at 4, 500 times gravity for 20 minutes at 4 degrees Celsius. Re-suspend the cells in 15 milliliters of ice-cold low-salt buffer by aspirating with an automated pipette and a 25-milliliter tip. Then, incubate the suspension over ice for 20 minutes with occasional inversion for mixing.

Osmotic shock ruptures the gram-negative outer membrane, following a sudden drop in environmental salt concentration. Gently handling the cells is crucial to avoid instigating unwanted further rupture of the inner membrane, periplasm cytoplasm cross-contamination, and destabilization or aggregation of liberated periplasm proteins. Following this, pellet the spheroplasts at 4, 500 times gravity for 20 minutes at 4 degrees Celsius.

Then, recover the supernatant containing the periplasm. Re-suspend the pelleted spheroplasts in phosphate-buffered saline solution by aspirating with an automated pipette and a 25-milliliter tip. Then, lyse cells by three cycles of a French pressure cell press at 1, 500 psi.

Now, pellet the cellular debris at 50, 000 times gravity for 20 minutes at 4 degrees Celsius. Recover the supernatant containing the cytoplasm. Following fractionation and purification of the periplasmic fraction, mix 1.5 microliters of the purified protein with PEG 4000, sodium chloride, Bis-Tris buffer, and sodium azide in a sitting drop or hanging drop setup by aspirating with a pipette and a 10 microliter tip.

Incubate the crystallization drops at 293 Kelvin for two to four weeks. Following incubation, harvest the crystals and flash freeze them in a liquid nitrogen pool for shipment in the synchrotron. For x-ray fluorescence analysis, move the sample out of the beam and navigate to the Hutch tab in the software.

Use the Energy subheading to set the energy to 10 kiloelectron volts. Next, move the sample back into the beam and navigate to the Scan tab. Navigate to the Interactive tab.

Select Optimize fluorescence signal. Input four seconds under the Time subheading and select Take fluorescence spectrum. The spectrum is automatically plotted for viewing under the Plot tab.

Elements of interest can be selected to guide the analysis of the energy regions corresponding to their emission peaks. For metal energy absorption analysis move the sample out of the beam. Navigate to the Scan tab, then navigate to the Periodic Table tab and select the K-edge of elements of interest.

Next, navigate to the Hutch tab. Use the Energy subheading to set the energy to the value in the previous step in the kiloelectron volts. Move the sample into the beam.

Navigate to the Scan tab. Navigate to the Auto tab and input two seconds under the Time subheading. Now, select Start Scan and view the MAD scan using the Plot tab and select edge scan to determine the energy absorption edge.

Then, select done with the fluorescence. After preparing subculture with the radioactive tracer, aliquot one milliliter into individual 1.5 milliliter centrifuge tubes by aspirating with a pipette and a one-milliliter tip. Set the three replicate standards aside.

You will not need them again until the end of the experiment. Place the remaining tubes in a 37 degrees Celsius ThermoMixer and vortex at one times gravity. Following incubation, centrifuge the tubes at 15, 000 times gravity for 30 seconds using a benchtop centrifuge, preferably cooled to 4 degrees Celsius.

After discarding the supernatants, re-suspend the cells in one milliliter of ice-cold high-salt buffer by aspirating with a pipette and a one-milliliter tip and immediately place on ice for 20 minutes. After centrifugation, re-suspend the cells in ice-cold low-salt buffer by aspirating with a pipette and one-milliliter tip and incubate on ice for 20 minutes. After pelleting the cells at 15, 000 times gravity for 30 seconds, collect each of the supernatants into new 1.5 milliliter centrifuge tubes.

Measure the radioactivity in each fraction and standard. Use the amount of radioactivity in the standards to determine the total amount of radioactivity originally added to every sample. SDS-PAGE gel and gel filtration chromatograms were collected the evaluate the quality of protein purification from preparative periplasm fractionation.

A liner elution gradient for anion exchange chromatography significantly improves YfeA enrichment from making up 8%of the periplasm fraction to 49%of the anion exchange product. This composition is improved to 61%by gel filtration and the elution volumes of the apo protein and holo protein are identical. Purified apo and holo YfeA crystallize in the same crystallization conditions, though images of apo and holo YfeA crystal morphologies show differences in crystal growth between apo and holo YfeA states.

Given that zinc is the primary substrate bound to recombinant YfeA, x-ray fluorescence spectra can differentiate between a sample containing a strong zinc signal commensurate with holo YfeA that is indicative of cross-contamination or a weaker zinc signal suggestive of apo YfeA and minimal cross-contamination. A 60-minute snapshot of radioactive metal uptake fractionation comparing periplasmic and cytoplasmic fractions of E.coli cells expressing YfeA only, the full YfeABCDE transporter, and none of the YfeABCDE components reveals the consequences of expressing a single recombinant protein or protein complex on metal uptake. Once mastered the periplasmic compartment can be extracted from whole cells by fractionation.

The protein further purified by chromatography and the protein of interest in crystallization drops in approximately four hours and 30 minutes when the procedures are performed properly. While attempting the fractionation and purification procedure it is important to maintain the sample over ice during fractionation and at 4 degrees Celsius during purification. These conditions will improve the quality of fractionation and the stability of the purified protein.

Following this procedure other metals can be introduced, like copper or gallium, to measure competitive inhibition or transport rates of noncanonical substrates and to answer additional questions about substrate specificity and key functional amino acids. After watching this video you should have a good understanding of how to fractionate E.coli cells and perform biophysical analyses of the contents of a cell compartment, such as the periplasm, without contamination of the cytoplasm. Don't forget that working with radioactivity can be extremely hazardous and precautions, such as adequate waste management and using minimal radioactive reagent, should always be considered while performing this procedure.