Assaying for Inorganic Polyphosphate in Bacteria

Summary

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

Abstract

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.

Introduction

Inorganic polyphosphate (polyP) is a linear biopolymer of phosphoanhydride-linked phosphate units that is found in all domains of life^1,2,3. In diverse bacteria, polyP is essential for stress response, motility, biofilm formation, cell cycle control, antibiotic resistance, and virulence^{4,5,6,7,8,9,10,11}. Studies....

Protocol

1. Purifying Yeast Exopolyphosphatase (ScPPX)

1. Transform the E. coli protein overexpression strain BL21(DE3)³¹ with plasmid pScPPX2⁶ by electroporation³² or chemical transformation³³.
2. Inoculate 1 L of lysogeny broth (LB) containing 100 µg mL^-1 ampicillin in a 2 L unbaffled flask with a single colony of BL21(DE3) containing pScPPX2 and incubate overnight at 37 °C without shaking, to an absorbance at 600 nm (A₆₀₀) of approximately 0.3, as measured in a spectrophotometer.
3. Start the culture shaking (180 rpm....

Results

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 °C with shaking (200 rpm), then rinsed and incubated for an additional 2 h in morpholinopropanesulfonate-buffered (MOPS) minimal medium

Discussion

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.......

Materials

List of materials used in this article

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