Single-cell ICP-MS for assessing genetic modifications affecting intracellular phosphorus levels and polyphosphate...

Phosphorus is an essential element in bacterial metabolism, acting as a key regulator of primary and secondary metabolic processes in the model actinobacterium Streptomyces coelicolor. In this work, the capabilities of single cell-inductively coupled plasma-mass spectrometry (sc-ICP-MS) are challenged to address quantitative levels of phosphorus at the individual spore level as result of a genetic modification of the strain (sco1897::Tn5) previously established to be related to phosphate homeostasis and to pleiotropically affect secondary metabolism. After adequate sample cleaning protocols, the use of a triple quadrupole ICP-MS (permitting PO⁺ measurements) and phosphorus-free washing buffers polyatomic interferences and background signals can be effectively minimized, enabling the methodological challenge of an accurate phosphorus single-event detection for 1 μm bacteria. Different dwell times (100 μs-5 ms) were tested to assess their influence on data quality, showing that shorter dwell times significantly improved detection limits (up to 0.6 fg spore^-1 for 100 μs, an eightfold decrease with respect to the 5 ms one) and reduced variability. The results showed that the mutant strain exhibited a two-fold higher phosphorus content per spore compared to the wild type (12 fg P/spore and 4 fg P/spore respectively, with p < 0.001 in the case of 5 ms), confirming the role of the sco1897 region in phosphate regulation. Chromatographic separation of spore lysates followed by single-particle ICP-MS is applied, for the first time, to address the presence of phosphorus in particulate form consistent with polyphosphate granules (volutins) in Streptomyces coelicolor. These nanoparticles, below 100 nm in diameter, were confirmed by transmission electron microscopy and confocal imaging using DAPI staining, which displayed characteristic green fluorescence associated with polyphosphates. Overall, this study provides a methodological framework for the single cell and nanoparticle-level quantification of phosphorus in actinobacteria, linking genetic regulation with elemental composition and biogenic nanoparticle formation.