Entomoplasma freundtii sp. nov., a new species from a green tiger beetle (Coleoptera: Cicindelidae).

A mollicute (strain BARC 318T) isolated from gut tissue of a green tiger beetle (Coleoptera: Cicindelidae) was found by dark-field microscopy to consist of non-helical, non-motile, pleomorphic coccoid forms of various sizes. In ultrastructural studies, individual cells varied in diameter from 300 to 1200 nm, were surrounded by a cytoplasmic membrane and showed no evidence of cell wall. The organisms were readily filterable through membrane filters with mean pore diameters of 450 and 300 nm, with unusually large numbers of organisms filterable through 200 nm pore membrane filters. Growth occurred over a temperature range of 15-32 degrees C with optimum growth at 30 degrees C. The organism fermented glucose and hydrolysed arginine but did not hydrolyse urea. Strain BARC 318T was insensitive to 500 U penicillin ml-1 and required serum or cholesterol for growth. It was serologically distinct from all currently described sterol-requiring, fermentative Mycoplasma species and from 12 non-sterol-requiring Mesoplasma species, 13 non-sterol-requiring Acholeplasma species and 5 previously described sterol-requiring Entomoplasma species. Strain BARC 318T was shown to have a G + C content of 34 mol% and a genome size of 870 kbp. The 16S rDNA sequence of strain BARC 318T was compared to 16S rDNA sequences of several other Entomoplasma species and to other representative species of the genera Spiroplasma and Mycoplasma, and to other members of the class Mollicutes. These comparisons indicated that strain BARC 318T had close phylogenetic relationships to other Entomoplasma species. On the basis of these findings and other similarities in morphology, growth and temperature requirements and genomic features, the organism was assigned to the genus Entomoplasma. Strain BARC 318T (ATCC 51999T) is designated the type strain of Entomoplasma freundtii sp. nov.

Effect of natural amphipathic peptides on viability, membrane potential, cell shape and motility of mollicutes.

The antibiotic activity of ten amphipathic peptides was investigated in six species of mollicutes belonging to the genera Acholeplasma, Mycoplasma and Spiroplasma. A. laidlawii was the most sensitive and M. mycoides subsp. mycoides SC the most resistant. Animal defence peptides (cecropins A and P1, and magainin 2) proved to be less potent than bee-venom mellitin and most of the peptides produced by bacteria (globomycin, gramicidin S, surfactin and valinomycin) or fungi (alamethicin). Gramicidin S was by far the most active peptide, with minimal inhibitory concentrations ranging from 2 to 50 nM. Alamethicin, gramicidin S, mellitin and surfactin had a cidal effect, whilst cecropins, globomycin, magainin 2, polymyxin B and valinomycin proved to be static. The peptides altered the membrane potential of spiroplasma cells with a potency independent of their linear or cyclic structure. However, globomycin depolarized the plasma membrane only weakly, whilst polymyxin B, in order to be active, required prior hyperpolarization of the membrane. The peptides also induced the loss of cell motility and helicity in spiroplasmas, suggesting that motility and cell shape in these bacteria are coupled to the transmembrane electrochemical gradient. Globomycin, an inhibitor of signal-peptidase II, prevented the growth of spiroplasmas, M. gallisepticum, and M. genitalium, but not that of A. laidlawii and M. mycoides subsp. mycoides SC, although the latter also synthesized membrane lipoproteins. Inhibition of spiralin processing by globomycin was demonstrated in S. citri and S. melliferum, with a more pronounced effect in the second species.