Discovering Polyphosphate and Polyhydroxyalkanoate-Accumulating Organisms Across Ecosystems: Phenotype-Targeted Genotyping via FACS-Sequencing

Intracellular biopolymers serve versatile functions, allowing microbes to adapt to fluctuating environmental conditions. The metabolic interdependence of dual biopolymers polyphosphate (polyP) and polyhydroxyalkanoates (PHA) is a defining feature of polyphosphate-accumulating organisms (PAOs), the key agents enabling enhanced biological phosphorus removal (EBPR). Beyond EBPR, PAOs accumulating both polyP and PHA (PHA-PAOs) in natural environments remain unexplored due to limited detection tools, despite hypothesized roles in nutrient cycling. This study presents a novel phenotype-targeted approach integrating triple-stained fluorescence-activated cell sorting with 16S rRNA gene sequencing (TriFlow-Seq) to detect, quantify, and phylogenetically characterize PHA-PAOs, validated by fluorescence imaging and single-cell Raman spectroscopy. TriFlow-Seq of EBPR samples confirmed known PAOs, including <i>Candidatus</i> Phosphoribacter, <i>Tetrasphaera</i>, and <i>Ca.</i> Accumulibacter, and revealed new candidate PHA-PAOs within the Rhodobacteraceae family. In maize rhizosphere, TriFlow-Seq led to the discovery of diverse PHA-PAOs, <i>Pseudomonas</i>, <i>Halomonas</i>, and <i>Nannocystis</i>. These genera participate in plant-growth-promoting functions including phosphate solubilization and phytohormone production, yet their simultaneous polyP and PHA accumulation has never been reported. Our findings revealed high PHA-PAO prevalence and distinct phylogenetic patterns associated with different maize genotypes, suggesting an overlooked role of biopolymer costorage in rhizosphere dynamics. This study establishes a pioneering approach to investigating PHA-PAO identities and roles across ecosystems.