A phylogenomic and molecular markers based taxonomic framework for members of the order Entomoplasmatales: proposal for an emended order Mycoplasmatales containing the family Spiroplasmataceae and emended family Mycoplasmataceae comprised of six genera.

The "Spiroplasma cluster" is a taxonomically heterogeneous assemblage within the phylum Tenericutes encompassing different Entomoplasmatales species as well as the genus Mycoplasma, type genus of the order Mycoplasmatales. Within this cluster, the family Entomoplasmataceae contains two non-cohesive genera Entomoplasma and Mesoplasma with their members exhibiting extensive polyphyletic branching; additionally, the genus Mycoplasma is also embedded within this family. Genome sequences are now available for all 19 Entomoplasmataceae species with validly published names, as well as 6 of the 7 species from the genus Mycoplasma. With the aim of developing a reliable phylogenetic and taxonomic framework for the family Entomoplasmataceae, exhaustive phylogenetic and comparative genomic studies were carried out on these genome sequences. Phylogenetic trees were constructed based on concatenated sequences of 121 core proteins for this cluster, 67 conserved proteins shared with the phylum Firmicutes, 40 ribosomal proteins, three major subunits of RNA polymerase (RpoA, B and C) by different means and also for the 16S rRNA gene sequences. The interspecies relationships as well as different species groups observed in these trees were identical and robustly resolved. In all of these trees, members of the genera Mesoplasma and Entomoplasma formed three and two distinct clades, respectively, which were interspersed among the members of the other genus. The observed species groupings in the phylogenetic trees are independently strongly supported by our identification of 103 novel molecular markers or synapomorphies in the forms of conserved signature indels and conserved signature proteins, which are uniquely shared by the members of different observed species clades. To account for the different observed species clades, we are proposing a division of the genus Mesoplasma into an emended genus Mesoplasma and two new genera Tullyiplasma gen. nov. and Edwardiiplasma gen. nov. Likewise, to recognize the distinct species groupings of Entomoplasma, we are proposing its division into an emended genus Entomoplasma and a new genus Williamsoniiplasma gen. nov. Lastly, to rectify the long-existing taxonomic anomaly caused by the presence of genus Mycoplasma (order Mycoplasmatales) within the Entomoplasmatales, we are proposing an emendation of the family Mycoplasmataceae to include both Entomoplasmataceae plus Mycoplasma species and an emendation of the order Mycoplasmatales, which now comprises of the emended family Mycoplasmataceae and the family Spiroplasmataceae. The taxonomic reclassifications proposed here accurately reflect the species relationships within this group of Tenericutes and they should lead to a better understanding of their biological and pathogenic characteristics.

A test for measuring growth responses of mollicutes to serum and polyoxyethylene sorbitan.

A test is described that is useful for characterizing mollicutes in terms of the ability to maintain growth in medium containing 15 to 20% fetal bovine serum or in serum-free media with or without 0.04% Tween 80 (polyoxyethylene sorbitan). Representative Acholeplasma species maintained growth in serum-free medium, and about half of the strains tested grew well in Tween 80-supplemented medium. Representative Mycoplasma and Entomoplasma species did not maintain growth in either serum-free medium alone or when Tween 80 was added. Spiroplasma species and group representatives generally failed to sustain growth in serum-free medium with or without Tween 80, but at least four of the spiroplasmas tested maintained growth in serum-free medium. The representative Mesoplasma species grew in serum-free media only when Tween 80 was added, as did Mycoplasma lactucae. Although the test has obvious determinative uses for members of the class Mollicutes, it does not supplant the conventional methodology for assaying the cholesterol requirements of these organisms.