Alexander Deitert, Jana Fees, Anna Mertens, Duc Nguyen Van, Maria Maares, Hajo Haase, Lars Mathias Blank, Claudia Keil
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引用次数: 0
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.
期刊介绍:
Yeast publishes original articles and reviews on the most significant developments of research with unicellular fungi, including innovative methods of broad applicability. It is essential reading for those wishing to keep up to date with this rapidly moving field of yeast biology.
Topics covered include: biochemistry and molecular biology; biodiversity and taxonomy; biotechnology; cell and developmental biology; ecology and evolution; genetics and genomics; metabolism and physiology; pathobiology; synthetic and systems biology; tools and resources