Inorganic polyphosphate is a key element in bacterial stress response. But older methods for extracting and measuring polyP in bacteria are complex and labor-intensive. The main advantage of this technique is that it allows fast, sensitive, and inexpensive quantification of polyP levels in a variety of different bacterial species.
The procedure will be demonstrated by Arya Pokhrel, an undergraduate student from my laboratory. To begin, grow lactobacillus reuteri bacteria in malic enzyme induction medium without cystine at 37 degrees celsius overnight without shaking. Centrifuge one milliliters of this overnight culture in a 1.5 milliliter microcentrifuge tube to harvest enough cells to yield a total of 50 to 100 micrograms of cellular protein.
Remove the supernatant from the cell pellet completely, and then add 250 microliters GITC lysis buffer and resuspend by vortexing. Incubate at 95 degrees celsius for 10 minutes to lyse the cells. Store the lysate at 80 degrees celsius.
First, prepare BSA standards containing zero, 1, 2, and 4 milligrams per milliliter of BSA in GITC lysis buffer. Aliquot five microliters of thawed, well mixed cell lysates and five microliters of BSA standards to separate wells in a clear 96 well plate. Add 195 microliters of Bradford reagent to each well and measure absorbance at 595 nanometers in a plate reader.
Calculate the amount of protein in each well by comparison to the BSA standard curve as outlined in the manuscript. To determine the total amount of protein in each sample, multiply the resulting value by 05. To start polyphosphate extraction, add 250 microliters of 95%ethanol to each GITC lysed sample, and vortex to mix.
Pipette this mixture to a silica membrane spin column placed in a 1.5 milliliter centrifuge tube. Centrifuge at 16, 100 g for 30 seconds. Discard the flow through and add 750 microliters of the tris-hydrochloride, sodium chloride, EDTA, and ethanol mixture.
Centrifuge at 16, 100 gs for 30 seconds. Discard the flow through and centrifuge the spin column at 16, 100 gs for two minutes. Place the column in a clean 1.5 milliliter microfuge tube.
Add 150 microliters of 50 millimolar pH eight tris-hydrochloride, and incubate at room temperature for five minutes. Elute polyP by centrifuging at 8, 000 g for two minutes. Start by preparing standards containing zero, five, 50, or 200 micromolar potassium phosphate in 50 millimolar pH eight tris-hydrochloride.
Aliquot 100 microliters of each phosphate standard and 100 microliters of extracted polyP samples into separate wells of a clear 96 well plate. Prepare a master mix containing, per sample, 30 microliters of 5X ScPPX reaction buffer, 19 microliters water, and one microliter of purified ScPPX. Add 50 microliters of the master mix to each well of the 96 well plate.
And incubate for 15 minutes at 37 degrees celsius. On the day of detection, prepare a fresh working stock of detection solution by mixing at 9.12 milliliters of detection solution base with 88 milliliters of one molar ascorbic acid, and allow it to come to room temperature before use. Add 50 microliters of detection solution to each sample and standards in the 96 well plate.
To allow color development, incubate the plate at room temperature for approximately two minutes. Use a plate reader to measure absorbance at 882 nanometers and calculate the phosphate concentration of each sample by comparison to the potassium phosphate standard curve. Then convert the phosphate concentrations to nanomoles of polyP derived phosphate in each cell lysate and normalize cellular polyP content to total cellular protein, as described in the manuscript.
Wild-type E.coli, a gram-negative bacterium, grown in LB produced no polyP. But when grown in MOPS, produced about 192 nanomoles polyP per milligram of total protein. An E.coli delta ppk mutant, which lacks polyP kinase, produced no polyP in either medium.
A delta ppx mutant, which lacks exopolyphosphatase, produced approximately the same amount of polyP as the wild-type. And the delta phoB mutant, which is defective in phosphate transport, produced significantly less polyP than the wild-type. Wild-type L.reuteri, a gram-positive bacterium grown overnight in MEIC medium accumulated about 51 nanomoles polyP per milligram of total protein.
An L.reuteri ppk1 null mutant lacking polyP kinase contained less than half this amount. This presence of polyP in the ppk1 null mutant is probably due to L.reuteri containing a second polyP kinase. Mycobacterium smegmatis strain SMR five, grown in Hartmans-de Bont medium in the absence of ethanol accumulated about 141 nanomoles polyP per milligram of total protein.
While ethanol treatment resulted in a threefold increase. Following this procedure, acrylamide gel electrophoresis can be performed to assess differences in polyP chain length. While attempting this procedure, avoid common mistakes.
Remember to avoid getting bubbles in microtiter plate wells, and be careful to transfer your samples to the appropriate tube when switching from the column to the microfuge tube. Don't forget that working with GITC and strong acids can be hazardous, and proper protective equipment should always be worn while performing this procedure.
