INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1993, p. 378-385 Vol. 43, No. 2 0020-7713/93/020378-08$02.00/0 Copyright 0 1993, International Union of Microbiological Societies NOTES Revised Taxonomy of the Class Mollicutes: Proposed Elevation of a Monophyletic Cluster of Arthropod-Associated Mollicutes to Ordinal Rank (Entomoplasmatalesord. nov.), with Provision for Familial Rank To Separate Species with Nonhelical Morphology (Entomoplasmataceaefam. nov.) from Helical Species (Spiroplasmataceae),and Emended Descriptions of the Order Mycoplasmatales, Family Mycoplasmataceae JOSEPH G. TULLY,’* JOSEPH M. BOVE,2 FREDERIC LAIGRET,2 AND ROBERT F. WHITCOMB3 Mycoplasma Section, Frederick Cancer Research and Development Center, National Institute of Allergy and Infectious Diseases, Frederick, Maryland 21 702’; Laboratoire de Biologie Cellulaire et Molhdaire, Institut National de la Recherche Agronomique and University of Bordeaux 11, 33883 Kllenave-d ’Ornon Ceda, France2; and Insect Biocontrol Laboratory, U.S. Department of Agriculture, Beltsville, Maryland 207053 On the basis of recent phylogenetic studies of 47 species within the class Mollicutes and recent molecular and physiologic findings that distinguish a new group of non-sterol-requiring insect and plant mollicutes from Acholeplasma species, we propose a revised taxonomy of the class Mollicutes. Order I (Mycoplasmutales)is retained as described earlier (S. Razin and E. A. Freundt, p. 740-742, in N. R. Krieg and J. G. Holt, ed,, Bergey ’s Manual of Systematic Bacteriology, vol, 1,1984), with provision for a single family (Mycoplasmataceae) and two genera (Mycoplasma and Ureaplasma) for classification of sterol-requiring mollicutes primarily associated with vertebrates. Emended descriptions of the order and family for the above taxa are hereby included. Proposed order I1 (Entomophmutales ord. nov,) contains subtaxa for either nonhelical (Entomoplas- mtuceae fam. nov.) or helical (Spirophmataceae)mollicutes; the latter family is hereby transferred from the order Mycoplasmatales. The new order and family permit designation and classification of a monophyletic cluster of mollicute strains associated primarily with arthropods. It is proposed that nonhelical, sterol- requiring insect and plant mollicutes previously designated Mycoplasma ellychniae (Tully et al., 1989), Mycoplasma melaleucae (Tully et al., 1990), Mycoplasma somnilux (Williamson et al., 1990), Mycophma luminosum (Williamson et al., 1990), and Mycophmu lucivorax (Williamson et al., 1990) be transferred to Entornoplasma gen. nov., family Entomoplasmataceae fam. nov., as Entomoplasma ellychniae comb. nov., Entomoplasma melaleucae comb. nov., Entomoplasma somnilux comb. nov, Entomoplasma luminosum comb. nov., and Entornoplasma lucivorax comb. nov. Furthermore, the genus Mesoplasma gen, nov. within the family Entomoplasmataceae fam. nov. is proposed for four non-sterol-requiring insect and plant mollicutes. This designation requires the transfer of Acholephma florum (McCoy et al., 1984), Acholeplasma entomophilum (Tully et al., 1988), Acholeplasma seirertii (Bonnet et al., 1991), and Mycoplasma ktucae (Rose et al., 1990) to Mesoplasma florum comb. nov., Mesoplasma entomophilum comb. nov., Mesoplasma seifertii comb. nov., and Mesoplasma luctucae comb. nov., respectively. Previously established orders Acholeplasmatales and Anaeroplasmatales within the class Mollicutes are designated orders I11 and IV, respectively, in the proposed revision. Classification and characteristics of lower taxa within these two orders remain as previously given. The proposal outlined here does not consider the large cluster of nonculturable, arthropod-associated, plant-pathogenic mollicutes (“mycoplasma-like organisms”) recently shown to be more closely related to members of the order Acholeplasmatales. The order Mycoplasmatales, first proposed in 1955 by species. It soon became apparent that a number of newly Freundt (ll), was later incorporated into the joint taxonomic described species in the genus Mycoplasma did not require proposals of Edward and Freundt in 1967 (9) to classify sterols or cholesterol for growth, and subsequent proposals sterol-requiring, wall-less prokaryotes in the class Molli- were made to separate these organisms into a new family CUtes (26). In these early endeavors, the single order encorn- (Acholeplasmataceae) and genus (Acholeplasma) within the Passed a small cluster of about 12 serologicallY distinct order Mycoplasmatales (10, 39). By 1983, fundamental al- terations in the perception of acholeplasma ecology had occurred, including observations that the organisms were * Corresponding author. commensals in a variety of animal hosts and could be 378 VOL. 43, 1993 NOTES 379 isolated from insects and plant surfaces (8, 20, 35, 39, 40). plasma/mycoides” group, a cluster of helical and nonhelical These and other major distinctions, particularly data show- sterol-requiring organisms phylogenetically distinct not only ing that Acholeplasma species had twice the genome size from members of the orders Acholeplasmatales and Anaero- (1,500 kbp) of Mycoplasrna species, were used in a 1984 plasmatales but also from the vast majority of members of proposal that elevated the family Acholeplasmataceae to the family Mycoplasmataceae. ordinal rank (order 11, Acholeplasmatales) within the class More recently, additional molecular distinctions between Mollicutes (12). Later, a cluster of strictly anaerobic anaero- the non-sterol-requiring insect- or plant-derived strains and plasmas from the rumens of bovine and ovine hosts were classic acholeplasmas have been documented. A. florum has raised to ordinal rank (order 111, Anaeroplasmatales) within been shown to possess a gene coding for one of the compo- the class (29). nents (enzyme 11) of a phosphoenolpyruvate-dependentsug- The taxonomic characterization and description of Spiro- ar-phosphotransferase system and to utilize UGA as a tryp- plasma citri in 1973 (32), the first helical, sterol-requiring tophan codon (21, 22). A functional phosphotransferase mollicute of plant and/or arthropod origin, caused certain system(s) appears to be absent in Acholeplasma spp. and in classification problems at higher taxonomic levels. The some nonfermentative Mycoplasma species but is present in morphology of the organism was clearly different from that Spiroplasma spp. and fermentative Mycoplasma species (3, of all other known mollicutes, and while the helical organ- 37,38). Likewise, the use of the UGA codon as a tryptophan isms required cholesterol, their 1,500-kbp genomes were signal is characteristic of Mycoplasma and Spiroplasma twice the size of those of other species of sterol-requiring species (27, 54). In contrast, Acholeplasma species utilize mollicutes. Eventually, S. citri and other species in the the UGA codon as a stop signal (36). Major differences in the genus Spiroplasma were elevated (34) to a family (Spiroplas- genome size among organisms in each of the two groups mataceae) within the order Mycoplasmatales. Although have also been disclosed recently. Data obtained by pulsed- many new spiroplasmas have been subsequently discovered field gel electrophoresis show that A. florum and other (43, 53), all have been shown to be associated with arthro- similar insect- or plant-derived strains have genome sizes in pods. the range of 860 to 1,100 kbp (570 to 730 MDa) (2, 25). In While higher classification of the spiroplasmas may have contrast, classic Acholeplasma species have genomes of been a minor dilemma, it soon became apparent that other 1,500 to 1,600 kbp (2, 24, 28). Thus, genome size, UGA newly isolated plant or insect mollicutes presented much codon usage, presence of a phosphotransferase system en- more serious problems in classification. The tropical flower zyme 11, and 16s rRNA sequences of the new insect and isolates described in 1979 by McCoy and associates (20) plant mollicutes clearly imply that they have greater affinities were the first to undergo extensive characterization. Three to members of the order Mycoplasmatales than to members serologically related strains (Ll, GF1, and PP2) appeared to of the order Acholeplasrnatales. possess most of the properties of acholeplasmas, including The sterol-Tween 80 test system described initially for A. rapid and extensive growth in broth medium at a tempera- florum (18, 40) and subsequently applied to other insect and ture optimum of about 30°C. Despite repeated testing, these plant mollicutes (1, 42, 44, 45, 52) provides a consistent strains could not be grown in medium devoid of cholesterol phenotypic marker for identification of strains that are or serum. However, it was eventually shown that growth did capable of growing in the presence of 0.04% Tween 80. A occur in serum- or cholesterol-free media containing 0.04% simplified method for testing sterol requirements, including Tween 80 (polyoqethylene sorbitan) (40). Since these flow- measuring growth promotion with Tween 80, has recently er-associated strains apparently grew in the absence of been proposed (31). serum or cholesterol, they were named Acholeplasma flo- On the basis of the above observations, we offer the rum (18). Two additional serologically distinct species, again following proposals for revision in the taxonomy of the class of insect or plant origin and with similar growth properties, Mollicutes (Table 1). were described more recently as Acholeplasma ento- Proposal for Entomoplasma gen. nov. for sterol-requiring mophilum (42) and Acholeplasma seiflertii