ABSTRACTThe formation of stable foam in activated sludge plants is a global problem for which control is difficult. These foams are often stabilized by hydrophobic mycolic acid-synthesizingActinobacteria, among which areTsukamurellaspp. This paper describes the isolation from activated sludge of the novel double-stranded DNA phage TPA2. This polyvalentSiphoviridaefamily phage is lytic for mostTsukamurellaspecies. Whole-genome sequencing reveals that the TPA2 genome is circularly permuted (61,440 bp) and that 70% of its sequence is novel. We have identified 78 putative open reading frames, 95 pairs of inverted repeats, and 6 palindromes. The TPA2 genome has a modular gene structure that shares some similarity to those ofMycobacteriumphages. A number of the genes display a mosaic architecture, suggesting that the TPA2 genome has evolved at least in part from genetic recombination events. The genome sequence reveals many novel genes that should inform any future discussion onTsukamurellaphage evolution.