Three isolates of novel polyphosphate-accumulating Gram-positive cocci, obtained from activated sludge, belong to a new genus, Tetrasphaera gen. nov., and description of two new species, Tetrasphaera japonica sp, nov and Tetrasphaera australiensis sp nov.

Two isolates of Cram-positive cocci (Ben 109(T) and Ben 110) which could accumulate polyphosphate and were microscopically similar in appearance to so-called 'G-bacteria', appearing as tetrads, were isolated from samples of activated sludge biomass by micromanipulation and grown in axenic culture. On the basis of their phenotypic and chemotaxonomic characters and 16S On the basis of their phenotypic ana chemotaxonomic characters uno lp rDNA sequences, these isolates, together with strain T1-X7(T) isolated and described previously in Japan, belong to a new genus. These isolates are phylogenetically different from Tessaracoccus bendigoensis, Friedmanniella spumicola and Friedmanniella capsulate, Gram-positive cocci isolated previously in this laboratory. They are characterized by type A1 gamma peptidoglycan, with meso-diaminopimelic acid as the diagnostic diamino acid. The main cellular fatty acid of Ban 109T, Ben 110 and T1-X7(T) is '14-methylpentadecanoic acid (i-C-16:0). The major menaquinones of Ben 109(T) are MK-8(H-4), with MK-8(H-2) and MK-8 in trace amounts. In Ben 110 MK-8(H-4) and MK-G(H-4) are the major menaquinones, while T1-X7(T) has MK-8(H-4), MK-7(H-4) and MK-G(H-4) as its menaquinones. All three contain phosphatidylinositol. MK-G(H-4) as its menaquinones. nit three contain phosphatidylinostol. phosphatidylglycerol and diphosphatidylglycerol as their polar lipids. These properties, together with 16S rDNA sequence data, suggest that they all belong to a single new genus for which the name Tetrasphaera gen. nov. is proposed. However, the lipid, cellular fatty acid profiles and DNA-DNA similarity data suggest that Ben 109(T) and Ben 110 are sufficiently different from T1-X7(T) to represent a different species of the genus Tetrasphaera. Strain T1-X7(T) represents the type species Tetrasphaera japonica sp. nov. of this new genus, and strains Ben 109(T) and Ben 110 belong to the other species, Tetrasphaera australiensis sp. nov.