Strain PYAN-1 (T = type strain), which was isolated from a pupal gut of the firefly beetle , and strains PIMN-1 and PIPN-2, which were isolated from guts of adult and fireflies, respectively, were demonstrated to be sterol-requiring mollicutes. Cells of the three strains were shown by electron and dark-field microscopy to be small, pleomorphic, nonhelical, nonmotile bodies surrounded by single membranes. No evidence of a cell wall was observed, and the organisms were not susceptible to 500 U of penicillin per ml. The three strains grew rapidly in SP-4 broth medium. Strains PIMN-1 and PIPN-2 grew in medium supplemented with bovine serum fraction, but strain PYAN-1 did not. All three strains grew on solid media when the cultures were incubated aerobically, but only strains PYAN-1 and PIPN-2 formed colonies when anaerobic conditions were employed. The three strains catabolized glucose but hydrolyzed neither arginine nor urea. All of the strains grew at temperatures of 18 to 32°C; strains PYAN-1 and PIMN-1 also grew at 10°C. The optimal temperature for growth for strains PYAN-1 and PIPN-2 was 30°C; strain PIMN-1 grew equally well at 30 or 32°C. None of the three strains grew at 37°C. The genome sizes of strains PYAN-1, PIMN-1, and PIPN-2 were about 527 (478 to 589), 570 (480 to 630), and 762 (635 to 871) megadaltons, respectively. The guanine-plus-cytosine contents of the DNAs of the strains, as determined by buoyant density, thermal denaturation, and high-performance liquid chromatography methods, were as follows: 27.4, 30.2, and 27.5 mol%, respectively, for strain PYAN-1; 28.8, 31.2, and 29.2 mol%, respectively, for strain PIMN-1; and 28.4, 30.4, and 31.2 mol%, respectively, for strain PIPN-2. All three strains were serologically unrelated to the type strains of previously established species (including ), to each other, and to 15 other unclassified sterol-requiring strains isolated from animals, plants, and insects. Along with , these three organisms represent an unusual cluster of strains belonging to the genus isolated from insects. Strain PYAN-1 (= ATCC 49194) is the type strain of sp. nov.; strain PIMN-1 (= ATCC 49195) is the type strain of sp. nov.; and strain PIPN-2 (= ATCC 49196) is the type strain of sp. nov.


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