Background

Polyphosphate (polyP), a biopolymer composed of linear chains of up to 1,000 inorganic phosphate monomers, is found in cells of all three domains of life. Yet, bacteria are the only organisms for which the enzymes of polyP metabolism have been well studied. Bacterial polyP kinase (PPK), which converts ATP into polyP, catalyzes both forward and reverse reactions. While synthesis of polyP is clearly the favored reaction in the cell, by providing sufficient amounts of ADP in vitro, the enzyme can be used to generate ATP from polyP, making a luciferase-based ATP detection possible (Ault-Riché et al., 1998). Bacteria lacking PPK are defective in biofilm formation, motility, persistence, and various stress responses, and show significantly increased sensitivity towards hypohalous acids (i.e., bleach) stress or phosphate starvation (Figure 1) (Rao et al., 2009; Gray et al., 2014; Maisonneuve and Gerdes, 2014; Gray and Jakob, 2015; Groitl et al., 2017).


Figure 1. Synthesis of polyP and its role in Gram-negative Bacteria. The bacteria-specific polyphosphate kinase (PPK) reversibly catalyzes the conversion from ATP into polyP and ADP. Various functions for polyP have been described in Gram-negative bacteria, including its involvement in biofilm formation, colonization, motility, and formation of antibiotic-resistant persister cells. PolyP also contributes to the resistance of bacteria towards various stresses, including oxidative stress and starvation, and serves as metal chelator and Pi reservoir.

Given the many roles that polyP plays in Gram-negative bacteria, PPK became attractive as drug target to interfere with biofilm formation, make bacteria less persistent, and sensitize them towards physiological oxidants such as bleach (Dahl et al., 2017). Therefore, reliable and sensitive methods to determine the polyP levels in vivo are necessary. Several methods for the extraction and detection of polyP have been reported in Bio-protocol, including extraction of polyP with (i) perchloric acid, (ii) sodium hypochlorite, and (iii) phenol/chloroform and detection of polyP via visualization with urea-PAGE or colorimetric assays using malachite green or molybdenum blue (Gomez Garcia, 2014; Canadell et al., 2016; Ota and Kawano, 2017). In this protocol, we combined extraction of polyP via binding to glassmilk (Ault-Riché et al., 1998) with a very sensitive two-step enzyme-based detection system. First, the extracted polyP is converted into ATP by E. coli PPK in the presence of ultra-pure ADP. The ATP levels are then quantified using a luciferase-based detection system and corrected for cellular ATP. In comparison to the urea-PAGE or the colorimetric methods, the luciferase-based detection allows the quantification of much lower levels of polyP. This protocol has been successfully applied to quantify polyP levels from Pseudomonas aeruginosa.