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J Vis Exp. 2019 Jan 21;(143). doi: 10.3791/58818.

Assaying for Inorganic Polyphosphate in Bacteria.

Author information

1
Department of Microbiology, University of Alabama at Birmingham.
2
Division of Infectious Diseases, University of Alabama at Birmingham.
3
Department of Microbiology, University of Alabama at Birmingham; mjgray@uab.edu.

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.

PMID:
30735204
DOI:
10.3791/58818

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