Name: assaying for inorganic polyphosphate in bacteria
Uploaded: 2019-01-21T15:00:00
Description: Watch this Scientific Journal Video about Assaying for Inorganic Polyphosphate in Bacteria at JoVE.com

This project was supported by University of Alabama at Birmingham Department of Microbiology startup funds and NIH grant R35GM124590 (to MJG), and NIH grant R01AI121364 (to FW).

1. Purifying Yeast Exopolyphosphatase (ScPPX)
<ol>
	<li>Transform the E. coli protein overexpression strain BL21(DE3)31 with plasmid pScPPX26 by electroporation32 or chemical transformation33.</li>
	<li>Inoculate 1 L of lysogeny broth (LB) containing 100 &#181;g mL-1 ampicillin in a 2 L unbaffled flask with a single colony of BL21(DE3) containing pScPPX2 and incubate overnight at 37 &#176;C without shakin...

The protocol described here simplifies and accelerates quantification of polyP levels in diverse bacteria, with a typical set of 24 samples taking about 1.5 h to fully process. This permits rapid screening of samples and analysis of mutant libraries, and simplifies kinetic experiments measuring the accumulation of polyP over time. We have demonstrated that the protocol works effectively on representatives of three different phyla: proteobacteria, firmicutes, and actinobacteria, which are notorious for their resilient, di...

Inorganic polyphosphate (polyP) is a linear biopolymer of phosphoanhydride-linked phosphate units that is found in all domains of life1,2,3. In diverse bacteria, polyP is essential for stress response, motility, biofilm formation, cell cycle control, antibiotic resistance, and virulence4,5,6,7,8,9,10,11. Studies of polyP metabolism in bacteria therefore have the potential to yield fundamental insights into the ability of bacteria to cause disease and thrive in diverse environments. In many cases, however, the methods available for quantifying polyP in bacterial cells are a limiting factor in these studies.
There are several methods currently used to measure polyP levels in biological materials. These methods typically involve two distinct steps: extracting polyP and quantifying the polyP present in those extracts. The current gold standard method, developed for the yeast Saccharomyces cerevisiae by Bru and colleagues12, extracts polyP along with DNA and RNA using phenol and chloroform, followed by ethanol precipitation, treatment with deoxyribonuclease (DNase) and ribonuclease (RNase), and digestion of the resulting purified polyP with the S. cerevisiae polyP-degrading enzyme exopolyphosphatase (ScPPX)13 to yield free phosphate, which is then quantified using a malachite green-based colorimetric assay. This procedure is highly quantitative but labor-intensive, limiting the number of samples that can be processed in a single experiment, and is not optimized for bacterial samples. Others have reported extracting polyP from a variety of cells and tissues using silica beads (&#34;glassmilk&#34;) or silica membrane columns6,14,15,16,17,18. These methods do not efficiently extract short chain polyP (less than 60 phosphate units)12,14,15, although this is of less concern for bacteria, which are generally thought to synthesize primarily long-chain polyP3. Older methods of polyP extraction using strong acids19,20 are no longer widely used, since polyP is unstable under acidic conditions12.
There are also a variety of reported methods for quantifying polyP. Among the most common is 4&#8242;,6-diamidino-2-phenylindole (DAPI), a fluorescent dye more typically used to stain DNA. DAPI-polyP complexes have different fluorescence excitation and emission maxima than DAPI-DNA complexes21,22, but there is considerable interference from other cellular components, including RNA, nucleotides, and inositol phosphates12,15,16,23, reducing the specificity and sensitivity of polyP measurements made using this method. Alternatively, polyP and adenosine diphosphate (ADP) can be converted into adenosine triphosphate (ATP) using purified Escherichia coli polyP kinase (PPK) and the resulting ATP quantified using luciferase14,17,18. This allows the detection of very small amounts of polyP, but requires two enzymatic reaction steps and both luciferin and very pure ADP, which are expensive reagents. ScPPX specifically digests polyP into free phosphate6,12,13,24, which can be detected using simpler methods, but ScPPX is inhibited by DNA and RNA12, necessitating DNase and RNase treatment of polyP-containing extracts. Neither PPK nor ScPPX are commercially available, and PPK purification is relatively complex25,26.
PolyP in cell lysates or extracts can also be visualized on polyacrylamide gels by DAPI negative staining27,28,29,30, a method that does allow assessment of chain length, but is low-throughput and poorly quantitative.
We now report a fast, inexpensive, medium-throughput polyP assay that allows rapid quantification of polyP levels in diverse bacterial species. This method begins by lysing bacterial cells at 95 &#176;C in 4 M guanidine isothiocyanate (GITC)14 to inactivate cellular phosphatases, followed by a silica membrane column extraction optimized for rapid processing of multiple samples. The resulting polyP-containing extract is then digested with a large excess of ScPPX, eliminating the need for DNase and RNase treatment. We include a protocol for straightforward nickel affinity purification of mg quantities of ScPPX. Finally, polyP-derived free phosphate is quantified with a simple, sensitive, ascorbic acid-based colorimetric assay24 and normalized to total cellular protein. This method streamlines the measurement of polyP in bacterial cells, and we demonstrate its use with representative species of Gram-negative bacteria, Gram-positive bacteria, and mycobacteria.

<table>
	<tbody>
		<tr>
			<td>E. coli BL21(DE3)</td>
			<td>Millipore Sigma</td>
			<td>69450</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid pScPPX2</td>
			<td>Addgene</td>
			<td>112877</td>
			<td>available to academic and other non-profit institutions</td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Fisher Scientific</td>
			<td>BP1427-2</td>
			<td>E. coli growth medium</td>
		</tr>
		<tr>
			<td>ampicillin</td>
			<td>Fisher Scientific</td>
			<td>BP176025</td>
			<td></td>
		</tr>
		<tr>
			<td>isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)</td>
			<td>Gold Biotechnology</td>
			<td>I2481C</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Gold Biotechnology</td>
			<td>H-400-1</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium hydroxide (KOH)</td>
			<td>Fisher Scientific</td>
			<td>P250500</td>
			<td>for adjusting the pH of HEPES-buffered solutions</td>
		</tr>
		<tr>
			<td>sodium chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S27110</td>
			<td></td>
		</tr>
		<tr>
			<td>imidazole</td>
			<td>Fisher Scientific</td>
			<td>O3196500</td>
			<td></td>
		</tr>
		<tr>
			<td>lysozyme</td>
			<td>Fisher Scientific</td>
			<td>AAJ6070106</td>
			<td></td>
		</tr>
		<tr>
			<td>magnesium chloride (MgCl2)</td>
			<td>Fisher Scientific</td>
			<td>BP214-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Universal Nuclease</td>
			<td>Fisher Scientific</td>
			<td>PI88700</td>
			<td>Benzonase (Sigma-Aldrich cat. # E1014) is an acceptable substitute</td>
		</tr>
		<tr>
			<td>Model 120 Sonic Dismembrator</td>
			<td>Fisher Scientific</td>
			<td>FB-120</td>
			<td>other cell lysis methods (e.g. French Press) can also be effective</td>
		</tr>
		<tr>
			<td>5 mL HiTrap chelating HP column</td>
			<td>GE Life Sciences</td>
			<td>17040901</td>
			<td>any nickel-affinity chromatography column or resin could be substituted</td>
		</tr>
		<tr>
			<td>nickel(II) sulfate hexahydrate</td>
			<td>Fisher Scientific</td>
			<td>AC415611000</td>
			<td>for charging HiTrap column</td>
		</tr>
		<tr>
			<td>0.8 &#181;m pore size cellulose acetate syringe filters</td>
			<td>Fisher Scientific</td>
			<td>09-302-168</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford reagent</td>
			<td>Bio-Rad</td>
			<td>5000205</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris buffer</td>
			<td>Fisher Scientific</td>
			<td>BP1525</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrum&#160;Spectra/Por 4 RC Dialysis Membrane Tubing 12,000 to 14,000 Dalton MWCO</td>
			<td>Fisher Scientific</td>
			<td>08-667B</td>
			<td>other dialysis membranes with MWCO &#60; 30,000 Da should also work</td>
		</tr>
		<tr>
			<td>hydrochloric acid (HCl)</td>
			<td>Fisher Scientific</td>
			<td>A144-212</td>
			<td>for adjusting the pH of Tris-buffered solutions</td>
		</tr>
		<tr>
			<td>potassium chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>P217500</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP2294</td>
			<td></td>
		</tr>
		<tr>
			<td>10x MOPS medium mixture</td>
			<td>Teknova</td>
			<td>M2101</td>
			<td>E. coli growth medium</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>Fisher Scientific</td>
			<td>D161</td>
			<td></td>
		</tr>
		<tr>
			<td>monobasic potassium phosphate (KH2PO4)</td>
			<td>Fisher Scientific</td>
			<td>BP362-500</td>
			<td></td>
		</tr>
		<tr>
			<td>dibasic potassium phosphate (K2HPO4)</td>
			<td>Fisher Scientific</td>
			<td>BP363-500</td>
			<td></td>
		</tr>
		<tr>
			<td>dehydrated yeast extract</td>
			<td>Fisher Scientific</td>
			<td>DF0886-17-0</td>
			<td></td>
		</tr>
		<tr>
			<td>tryptone</td>
			<td>Fisher Scientific</td>
			<td>BP1421-500</td>
			<td></td>
		</tr>
		<tr>
			<td>magnesium sulfate heptahydrate</td>
			<td>Fisher Scientific</td>
			<td>M63-50</td>
			<td></td>
		</tr>
		<tr>
			<td>manganese sulfate monohydrate</td>
			<td>Fisher Scientific</td>
			<td>M113-500</td>
			<td></td>
		</tr>
		<tr>
			<td>guanidine isothiocyanate</td>
			<td>Fisher Scientific</td>
			<td>BP221-250</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin (protease-free)</td>
			<td>Fisher Scientific</td>
			<td>BP9703100</td>
			<td></td>
		</tr>
		<tr>
			<td>clear flat bottom 96-well plates</td>
			<td>Sigma-Aldrich</td>
			<td>M0812-100EA</td>
			<td>any clear 96-well plate will work</td>
		</tr>
		<tr>
			<td>Tecan M1000 Infinite plate reader</td>
			<td>Tecan, Inc.</td>
			<td>not applicable</td>
			<td>any plate reader capable of measuring absorbance at 595 and 882 nm will work</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Fisher Scientific</td>
			<td>04-355-451</td>
			<td></td>
		</tr>
		<tr>
			<td>silica membrane spin columns</td>
			<td>Epoch Life Science</td>
			<td>1910-050/250</td>
			<td></td>
		</tr>
		<tr>
			<td>ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Fisher Scientific</td>
			<td>BP120500</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microfuge tubes</td>
			<td>Fisher Scientific</td>
			<td>NC9580154</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium acetate</td>
			<td>Fisher Scientific</td>
			<td>A637-500</td>
			<td></td>
		</tr>
		<tr>
			<td>antimony potassium tartrate</td>
			<td>Fisher Scientific</td>
			<td>AAA1088922</td>
			<td></td>
		</tr>
		<tr>
			<td>4 N sulfuric acid (H2SO4)</td>
			<td>Fisher Scientific</td>
			<td>SA818-500</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium heptamolybdate</td>
			<td>Fisher Scientific</td>
			<td>AAA1376630</td>
			<td></td>
		</tr>
		<tr>
			<td>ascorbic acid</td>
			<td>Fisher Scientific</td>
			<td>AC401471000</td>
			<td></td>
		</tr>
	</tbody>
</table>

assaying for inorganic polyphosphate in bacteria

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. This procedure is straightforward, inexpensive, and allows reliable polyP quantification in diverse bacterial species. We present representative polyP quantification from the Gram-negative bacterium (Escherichia coli), the Gram-positive lactic acid bacterium (Lactobacillus reuteri), and the mycobacterial species (Mycobacterium smegmatis). We also include a simple protocol for nickel affinity purification of mg quantities of ScPPX, which is not currently commercially available.

The key steps of the protocol are diagrammed in simplified form in Figure 1.
To demonstrate the use of this protocol with Gram-negative bacteria, wild-type E. coli MG165539 was grown to mid-log phase in LB rich medium at 37 &#176;C with shaking (200 rpm), then rinsed and incubated for an additional 2 h in morpholinopropanesulfonate-buffered (MOPS) minimal medium<sup ...

Watch this Scientific Journal Video about Assaying for Inorganic Polyphosphate in Bacteria at JoVE.com

Assaying for Inorganic Polyphosphate in Bacteria

We describe a simple method for rapid quantification of inorganic polyphosphate in diverse bacteria, including Gram-negative, Gram-positive, and mycobacterial species.

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...

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