An optimised protocol for the relative quantification of ATP and polyphosphates in the microalga Chlamydomonas reinhardtii and other microorganisms

Abstract Background Polyphosphates (polyP) and ATP are phosphate-containing metabolites present in prokaryotes and eukaryotes. PolyP are synthesised from ATP and have a wide variety of functions including phosphate and cation storage. In most microorganisms, a role of polyP in energetic metabolism has not been investigated. In the green microalga Chlamydomonas reinhardtii , polyP synthesis was suggested to buffer ATP concentration. To uncover polyP participation in energetic metabolism, relative quantification of both metabolites is needed. ATP and polyP half-lives differing greatly, harvesting generates biases in the relative quantification of these metabolites in current methods. We present here a protocol optimised to obtain maximal yield and integrate assay constraints linked to metabolite stability and purity. Methods Our optimised method quantifies ATP and polyP from the same cell extract, obtained by neutral phenol–chloroform extraction of cells directly pipetted from their culture. After a second chloroform extraction, ATP is quantified directly from the extract, while polyP measurement requires purification by ethanol precipitation. We tested statistical effects in our results with t-test, one-way analysis of variance or Kruskall-Wallis testing and appropriate post hoc testing. Results We show that the polyP/ATP ratio of the reference C. reinhardtii strain CC-4533 in exponential mixotrophic growth is around 70. The optimised protocol performs as well as specific protocols for either ATP or polyP measurements. Direct sampling from the culture works better than centrifugation to maintain physiological conditions and ATP content. Using spiking, we show that ATP is fully recovered. PolyP with 3 P units are poorly recovered (25%) while chains of 14 units and above are fully recovered. The method also works for other microorganisms, including yeast, diatoms and cyanobacteria. Finally, we show that the polyP chain length distribution extracted from CC-4533 is broad, reaching up to several thousand P with a mean around 200 P. Discussion Our protocol using neutral phenol–chloroform extraction on directly harvested samples allows quantification of true physiological ATP and polyP cell concentrations, and calculation of the polyP/ATP ratio from low-density samples with no other normalisation. It can be applied to other microorganisms or cells to investigate polyP physiology in a variety of physiological and stress conditions.