Members of the genus Tetrasphaera are considered to be putative polyphosphate accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) from wastewater. Although abundant in Danish full-scale wastewater EBPR plants, how similar their ecophysiology is to 'Candidatus Accumulibacter phosphatis' is unclear, although they may occupy different ecological niches in EBPR communities. The genomes of four Tetrasphaera isolates (T. australiensis, T. japonica, T. elongata and T. jenkinsii) were sequenced and annotated, and the data used to construct metabolic models. These models incorporate central aspects of carbon and phosphorus metabolism critical to understanding their behavior under the alternating anaerobic/aerobic conditions encountered in EBPR systems. Key features of these metabolic pathways were investigated in pure cultures, although poor growth limited their analyses to T. japonica and T. elongata. Based on the models, we propose that under anaerobic conditions the Tetrasphaera-related PAOs take up glucose and ferment this to succinate and other components. They also synthesize glycogen as a storage polymer, using energy generated from the degradation of stored polyphosphate and substrate fermentation. During the aerobic phase, the stored glycogen is catabolized to provide energy for growth and to replenish the intracellular polyphosphate reserves needed for subsequent anaerobic metabolism. They are also able to denitrify. This physiology is markedly different to that displayed by 'Candidatus Accumulibacter phosphatis', and reveals Tetrasphaera populations to be unusual and physiologically versatile PAOs carrying out denitrification, fermentation and polyphosphate accumulation.The ISME Journal advance online publication, 22 November 2012; doi:10.1038/ismej.2012.136.