Phylogenetic Relationships among Members of the Class Mollicutes Deduced from rps3 Gene Sequences

A gene for a ribosomal protein, rps3, was amplified by PCR and sequenced from representatives of the class Mollicutes. Alignments of the deduced amino acid sequences allowed the construction of a phylogeny that is consistent with the phylogenetic trees created from 5s and 16s rRNA comparisons, including the position of the former Acholeplasma florum on the Mycoplusma branch, rather than with the classical Acholeplasmataceae. Additional confirmation of the phylogeny comes from the deduction that the UGA triplet encodes tryptophan in the rps3 gene from Mesoplasmuflorum, as it does in the mycoplasmas and spiroplasmas. The sequence data from Acholeplasma axanthum 743 and Acholeplusma sp. strain 5233 allow refinements to the phylogenetic tree within the Acholeplusmutuceae, providing evidence that the sterol requirement of Anueroplusma abactoclasticum (order Anaeroplasmatales) is a derived trait. It was also evident that the nonhelical plant-pathogenic members of the class Mollicutes, referred to as mycoplasmalike organisms or phytoplasmas, are more closely related to the true acholeplasmas (Acholeplusma laidlawii and strain 5233) than to other members of the Mollicutes. The placement of certain non-sterol-requiring, cell wallless bacteria from plants and insects into the family Acholeplasmataceae of the class Mollicutes was brought into question by 16s rRNA sequence data from a large number of these species (13,31,34). The 16s rRNA data indicated that two species that were classified as acholeplasmas, Acholeplasma frorum and the closely related Acholeplasma entomophilum, were phylogenetically closer to members of the Mycoplasmataceae and Spiroplasmataceae than to the other members of the Acholeplasmataceae. These data, together with a reassessment of nutritional requirements, led to the suggestion that they be renamed Mesoplasma florum and Mesoplasma entomophilum and, along with other closely related members of the Mollicutes, including all species of Spiroplasma, be placed in a new order, the Entomoplasmatales (31). The phylogenetic trees constructed from 5s and 16s rRNA data (27, 34) show that the Anaeroplasmataceae are more closely related to the classical Acholeplasmataceae than to the other mollicutes, even though species of Anaeroplasma require sterols, as do all species of Mycoplasma and Spiroplasma. The 16s rRNA sequence data from the plantpathogenic mycoplasmalike organisms (MLOs) indicated a phylogenetic position between the Acholeplasmatales and the Anaeroplasmatales (9, 10). Considering these relationships, the possibility arose that the difficulty in developing an in vitro culture medium for MLOs could be due to an oxygen-sensitive metabolism in MLOs or to incorrect guesses about their sterol requirements. To provide supplemental data to address the question of phylogeny of the MLOs, we have obtained sequence data from a gene for a ribosomal protein, rps3, for which comparable data were already available from Mycoplasma capricolum (19), Acholeplasma laidlawii (ll), and an MLO (11). In this report,