Rapid Fluorescence Assay for Polyphosphate in Yeast Extracts Using JC‐D7

• Published in: Yeast (Chichester, England) England), 2024-10, Vol.41 (10), p.593-604
• Main Authors: Deitert, Alexander, Fees, Jana, Mertens, Anna, Nguyen Van, Duc, Maares, Maria, Haase, Hajo, Blank, Lars Mathias, Keil, Claudia
• Format: Article
• Language: English
• Subjects: antibacterial properties; Bacteriostats; Cations; Fluorescence; fluorescent dyes; Fluorescent Dyes - chemistry; Fluorescent indicators; genes; Homeostasis; Hydrogen-Ion Concentration; industry; JC‐D7; Mercuric chloride; Metal ions; Microorganisms; phosphates; phosphorus; polymers; polyphosphate; Polyphosphates; Polyphosphates - chemistry; Polyphosphates - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Sensitivity analysis; Strains (organisms); temperature; toxicity; Yeast; yeasts; Zinc sulfate
• ISSN: 0749-503X; 1097-0061; 1097-0061
• DOI: 10.1002/yea.3979

ABSTRACT Polyphosphate (polyP) is an intriguing molecule that is found in almost any organism, covering a multitude of cellular functions. In industry, polyP is used due to its unique physiochemical properties, including pH buffering, water binding, and bacteriostatic activities. Despite the importance of polyP, its analytics is still challenging, with the gold standard being 31P NMR. Here, we present a simple staining method using the fluorescent dye JC‐D7 for the semi‐quantitative polyP evaluation in yeast extracts. Notably, fluorescence response was affected by polyP concentration and polymer chain length in the 0.5–500 µg/mL polyP concentration range. Hence, for polyP samples of unknown chain compositions, JC‐D7 cannot be used for absolute quantification. Fluorescence of JC‐D7 was unaffected by inorganic phosphate up to 50 mM. Trace elements (FeSO4 > CuSO4 > CoCl2 > ZnSO4) and toxic mineral salts (PbNO3 and HgCl2) diminished polyP–induced JC‐D7 fluorescence, affecting its applicability to samples containing polyP–metal complexes. The fluorescence was only marginally affected by other parameters, such as pH and temperature. After validation, this simple assay was used to elucidate the degree of polyP production by yeast strains carrying gene deletions in (poly)phosphate homeostasis. The results suggest that staining with JC‐D7 provides a robust and sensitive method for detecting polyP in yeast extracts and likely in extracts of other microbes. The simplicity of the assay enables high‐throughput screening of microbes to fully elucidate and potentially enhance biotechnological polyP production, ultimately contributing to a sustainable phosphorus utilization. Evaluation and application of the fluorescent dye JC‐D7 for polyphosphate detection in yeast extracts. Take‐Away The commercially available fluorescence sensor JC‐D7 was used to establish a fast, highly sensitive polyP detection assay tailored for yeast samples. First‐time evaluation of JC‐D7's robustness to various abiotic factors, including polyP counteracting metal cations and polyP extraction agents. Pre‐screening of yeast strains and rapid classification based on JC‐D7 polyP detection. Researchers and industry professionals can benefit from this powerful, easy‐to‐use method for detecting polyphosphates in yeasts and other organisms.