The molecular mechanisms and evolution of polyphosphate accumulation in Candidatus Accumulibacter phosphatis

The evolution of complex traits is hypothesized to occur incrementally. Identifying the transitions that lead to extant complex traits may provide a better understanding of the genetic nature of the observed phenotype. A keystone functional group in wastewater treatment processes are polyphosphate accumulating organisms (PAOs), however the evolution of the PAO phenotype has yet to be explicitly investigated and the specific metabolic traits that discriminate non-PAO from PAO are currently unknown. Here, we perform the first comprehensive investigation on the evolution of the PAO phenotype using the model uncultured organism Candidatus Accumulibacter phosphatis (Accumulibacter) through time series metatranscriptomics, ancestral genome reconstruction, identification of horizontal gene transfer, and a kinetic/stoichiometric characterization of Accumulibacter Clade IIA. The time series metatranscriptomic analysis provided a transcriptional model of Accumulibacter under polyphosphate accumulating conditions identifying key regulatory mechanisms and links between glycogen and polyhydroxyalkanoate metabolism. Subsequently, the analysis of Accumulibacter's last common ancestor identified 135 laterally derived genes, including genes involved in glycogen, polyhydroxyalkanoate, pyruvate and NADH/NADPH metabolisms, as well as inorganic ion transport and regulatory mechanisms. In contrast, pathways such as the TCA cycle and polyphosphate metabolism displayed minimal horizontal gene transfer. We show that the transition from non-PAO to PAO coincided with horizontal gene transfer within Accumulibacter’s core metabolism; likely alleviating key kinetic and stoichiometric bottlenecks, such as anaerobically linking glycogen degradation to polyhydroxyalkanoate synthesis. These results demonstrate the potential of coupled metatranscriptomics and ancestral state reconstruction to identify key transitions leading to an extant complex phenotype in uncultivated organisms.