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 ac cumulate polyphosphate and were microscopically similar in appearance to so -called 'G-bacteria', appearing as tetrads, were isolated from samples of a ctivated sludge biomass by micromanipulation and grown in axenic culture. O n the basis of their phenotypic and chemotaxonomic characters and 16S On th e basis of their phenotypic ana chemotaxonomic characters uno lp rDNA seque nces, these isolates, together with strain T1-X7(T) isolated and described previously in Japan, belong to a new genus. These isolates are phylogenetic ally different from Tessaracoccus bendigoensis, Friedmanniella spumicola an d Friedmanniella capsulate, Gram-positive cocci isolated previously in this laboratory. They are characterized by type A1 gamma peptidoglycan, with me so-diaminopimelic acid as the diagnostic diamino acid. The main cellular fa tty 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 m ajor 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, togethe r 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 tha t Ben 109(T) and Ben 110 are sufficiently different from T1-X7(T) to repres ent a different species of the genus Tetrasphaera. Strain T1-X7(T) represen ts the type species Tetrasphaera japonica sp. nov. of this new genus, and s trains Ben 109(T) and Ben 110 belong to the other species, Tetrasphaera aus traliensis sp. nov.