Spiroplasma Species Research Articles

Spiroplasma lineolae sp. nov., from the horsefly Tabanus lineola (Diptera: Tabanidae).

Spiroplasma strain TALS-2T from the viscera of the striped horsefly, Tabanus lineola, collected in Georgia was serologically distinct from other Spiroplasma species, groups, putative groups, and subgroups. Light and electron microscopy of cells of strain TALS-2T revealed helical motile cells surrounded only by a single cytoplasmic membrane. The organism grew in M1D and SP-4 liquid media. Growth also occurred in 1% serum fraction medium and in conventional horse serum medium. Growth in liquid media was serum dependent. The strain passed through 220-nm filter pores, but was retained in filters with 100-nm pores. The optimum temperature for growth was 30 degrees C. Multiplication occurred at temperatures from 20 to 37 degrees C, with a doubling time at the optimum temperature of 5.6 h in M1D broth. Strain TALS-2T catabolized glucose but hydrolyzed neither arginine nor urea. The guanine-plus-cytosine content of the DNA was 25 +/- 1 mol%. The genome size was 1,390 kbp. Six isolates serologically similar to strain TALS-2T were obtained from the same host in coastal Georgia. Three strains closely related to strain TALS-2T were isolated from the horsefly Poeciloderas quadripunctatus in Costa Rica. Strain TALS-2T (= ATCC 51749), a representative of group XXVII, is designated the type strain of a new species, Spiroplasma lineolae (Mollicutes: Entomoplasmatales).

Spiroplasma chrysopicola sp. nov., Spiroplasma gladiatoris sp. nov., Spiroplasma helicoides sp. nov., and Spiroplasma tabanidicola sp. nov., from Tabanid (Diptera: Tabanidae) Flies

Four spiroplasma strains, DF-1T, TG-1T, TABS-2T, and TAUS-1T, all of which were isolated from deerflies or horseflies (Diptera: Tabanidae), were serologically distinct from previously described spiroplasma species, groups, and subgroups. Strain DF-1Toriginated from a Maryland deerfly (Chrysops sp.); strain TG-1Twas isolated from a Maryland horsefly (Tabanus gladiator); strain TAUS-1Toriginated from a member of the Tabanus abdominalis-limbatinevris complex of horseflies collected in Maryland; and strain TABS-2Twas isolated from a horsefly (Tabanus abactor) collected in Oklahoma. Cells of all of the strains appeared to be helical and motile when they were examined by dark-field microscopy. Cells of strain DF-1Tgrowing in M1D medium were short helices with less than six turns; the helical cells of the other strains were long and usually had six or more turns. The short cells of strain DF-1Tpassed through 450- and 300-nm filter pores with no reduction in titer, but the longer cells of the other strains were partially retained by 450-nm-pore-size filters. Electron microscopic examination of all of the strains revealed wall-less cells surrounded only by a single cytoplasmic membrane. All of the strains grew well in SP-4 liquid media and in conventional mycoplasma or M1D media supplemented with horse or fetal bovine serum. Strains TABS-2T, TAUS-1T, and DF-1Trequired serum or sterol for growth, but strain TG-1Twas able to grow in the absence of serum or sterol. The optimum temperatures for growth of the four strains varied from 30 to 32°C, and growth occurred at 10 to 37°C. All of the strains catabolized glucose but did not hydrolyze urea. Only strain DF-1Thydrolyzed arginine. The guanine-plus-cytosine contents of the DNAs of the strains were: DF-1T, 29 ± 1 mol%; TG-1T, 26 ± 1 mol%; TABS-2T, 27 ± 1 mol%; and TAUS-1T, 26 ± mol%. The genome sizes of strains DF-1Tand TAUS-1Twere 1,270 and 1,375 kbp, respectively. Strain DF-1 (= ATCC 43209), the representative of spiroplasma subgroup VIII-2, is designated the type strain of a new species, Spiroplasma chrysopicola. We also propose that strain TG-1T(= ATCC 43525T), the designated representative of group XXIII, should be placed in a new species, Spiroplasma gladiatoris. In addition, group XXXII spiroplasma strain TABS-2 (= ATCC 51746) is designated the type strain of Spiroplasma helicoides sp. nov., and group XXXIII representative strain TAUS-1 (= ATCC 51747) is designated the type strain of another new species, Spiroplasma tabanidicola.

Spiroplasma alleghenense sp. nov., a New Species from the Scorpion Fly Panorpa helena (Mecoptera: Panorpidae)

Spiroplasma strain PLHS-1T from the gut of a common scorpion fly (Panorpa helena) collected in the West Virginia Allegheny Mountains was distinct from other spiroplasma species, groups, and subgroups as determined by reciprocal serological metabolism inhibition and deformation tests. However, when this strain was used as an antigen, it cross-reacted extensively with representatives of other groups. Light microscopy and/or electron microscopy of cells of strain PLHS-1T revealed helical motile cells surrounded by a single cytoplasmic membrane. The strain was resistant to penicillin, which confirmed that it had no cell wall. The organism grew well in M1D and SP-4 liquid media, in 1% serum fraction medium, and in conventional horse serum medium. The optimum temperature for growth was 30°C, at which the doubling time was 6.4 h. Multiplication occurred at temperatures from 10 to 32°C. Strain PLHS-1T catabolized glucose, hydrolyzed arginine but not urea, and required sterol for growth. The guanine-plus-cytosine content of the DNA was 31 ± 1 mol%, and the genome size was 1,465 kbp. Strain PLHS-1 (= ATCC 51752) is designated the type strain of a new species, Spiroplasma alleghenense.

Spiroplasma montanense sp. nov., from Hybomitra Horseflies at Northern Latitudes in North America

Spiroplasma strain HYOS-1Twas isolated from a tabanid fly, Hybomitra opaca. The organism was serologically distinct from other spiroplasma species, groups, and subgroups and was recently designated the representative of spiroplasma group XXXI. The cells of strain HYOS-1T, as determined by light microscopy, were long motile helices. Electron microscopic examination revealed wall-less cells delimited by a single membrane. The cells passed through 450- and 300-nm filter pores with a 10-fold reduction in titer, but failed to pass through 100-nm pores. Strain HYOS-1Tgrew very well in most conventional medium formulations for spiro-plasmas or other mollicutes. The organism grew at temperatures ranging from 5 to 41°C, and the optimum temperature was 32°C. The doubling time at the optimum temperature was 0.7 h, one of the shortest values obtained for members of the genus Spiroplasma. The strain catabolized glucose and hydrolyzed arginine but not urea. Growth of the organism was stimulated by cholesterol and serum, but the strain was nevertheless able to grow in the absence of sterols or serum. The guanine-plus-cytosine content of the DNA was about 28 ± 1 mol%, and the genome size was 1,225 kbp. On the basis of the experimental results reported here and previously reported data, group XXXI strain HYOS-1 (= ATCC 51745) is designated the type strain of a new species, Spiroplasma montanense.

Spiroplasma lampyridicola sp. nov., from the Firefly Beetle Photuris pennsylvanicus

Spiroplasma strain PUP-1Twas isolated from the gut fluids of a firefly beetle (Photuris pennsylvanicus) collected in Maryland. Cells of the strain were shown by dark-field microscopy to be helical, motile filaments. Ultrastructural examination by electron microscopy revealed filamentous cells bounded by a single cytoplasmic membrane and no evidence of a cell wall. The cells were not sensitive to 500 U of penicillin per ml and grew under aerobic conditions in M1D, SP-4, and M-2 broth formulations, as well as in conventional mycoplasma medium. The doubling times at 15, 20, 25, and 30°C were 83.1, 32.0, 14.9, and 9.8 h, respectively. Suboptimal growth occurred at 37°C, and no growth was apparent in cultures maintained at 10 or 40°C. The organism required cholesterol for growth and produced acid from glucose, fructose, and trehalose; arginine and urea were not hydrolyzed. The results of previous serological analyses of strain PUP-1Tindicated that the organism was not related to the then currently established Spiroplasma species or group representatives, and the organism was classified as the representative of group XIX. Additional testing of strain PUP-1Tagainst recently recognized Spiroplasma species or group representatives by both metabolism inhibition and deformation tests confirmed the unique serological status of the organism. The guanine-plus-cytosine content of the DNA was 26 ± 1 mol%, and the genome size was 1,375 kbp. These values clearly differentiate strain PUP-1Tfrom group XXI strain W115, with which it cross-reacted reciprocally at a low level in deformation and metabolism inhibition tests. We propose that strain PUP-1 (= ATCC 43206) should be recognized as the type strain of a new species, Spiroplasma lampyridicola.

Spiroplasma litorale sp. nov., from Tabanid Flies (Tabanidae: Diptera) in the Southeastern United States

Spiroplasma strain TN-1T (T = type strain), a strain serologically distinct from other spiroplasma species, groups, and subgroups, was isolated from the gut of a horsefly (Tabanus nigrovittatus) from a barrier island off the coast of North Carolina. Related strains were isolated from other Tabanus spp., T. atratus, T. americanus, T. gladiator, T. lineola, T. sulcifrons, and T. zythicolor, from coastal Georgia. Cells of strain TN-1T in culture were helical and motile with an average of 5 to 10 helical turns per cell. Electron microscopic studies determined that the cells of strain TN-1T were surrounded by a single cytoplasmic membrane, and there was no evidence of a cell wall. The spiroplasma grew well in MID and SP-4 liquid media. Serum fraction (1%) medium and conventional horse serum medium also supported growth of strain TN-1T. Fried-egg colonies were not produced; instead, the strain produced small diffuse colonies that were surrounded by satellite growth. The optimum temperature for growth was 32°C, but multiplication was observed at temperatures from 10 to 41°C. The doubling time at the optimum temperature (32°C) was 1.6 h. No growth was observed at 5 or 43°C. Spiroplasma strain TN-1T passed through 220-nm filter pores but failed to pass through 100-nm filter pores. Strain TN-1T catabolized glucose but hydrolyzed neither arginine nor urea. The guanine-plus-cytosine content of the DNA was about 25 ± 1 mol%, and the genome size was 1,370 kbp. Based on results from this study and previously published data, strain TN-1T (= ATCC 43211) (group XVIII) is designated the type strain of a new spiroplasma species, Spiroplasma litorale.

Spiroplasma diabroticae sp. nov., from the southern corn rootworm beetle, Diabrotica undecimpunctata (Coleoptera:Chrysomelidae).

Spiroplasma strain DU-1T (T = type strain), which was isolated from hemolymph of the corn rootworm Diabrotica undecimpunctata (Coleoptera:Chrysomelidae), was serologically distinct from other spiroplasma species, groups, and subgroups. Cells of strain DU-1T were shown by light microscopy to be helical motile filaments. Electron microscopy revealed cells bounded by a single cytoplasmic membrane, with no evidence of a cell wall. The organism was not sensitive to 500 U of penicillin per ml. Strain DU-1T grew well in SM-1, M1D, and SP-4 liquid media, in broth supplemented with 1% bovine serum fraction or conventional horse serum, and under both aerobic and anaerobic conditions. This organism did not appear to have a sterol requirement for growth, as has been reported for several other Spiroplasma species or strains. Optimal growth occurred at 32 degrees C, with a doubling time of 0.9 h; strain DU-1T multiplied at 10 to 41 degrees C but failed to grow at 5 or 43 degrees C. It produced acid from glucose but hydrolyzed neither arginine nor urea. The results of reciprocal serologic tests in which antigens or antisera to established Spiroplasma species, groups, subgroups, and putative groups were used indicated that strain DU-1T was serologically distinct. This organism has a DNA guanine-plus-cytosine content of 25 +/- 1 mol% and a genome size of 1,350 kbp. Strain DU-1T is a member of a cluster of fast-growing insect-associated spiroplasmas, as determined by sequence analysis of 16S rRNA. On the basis of the results of this study and previously published data, strain DU-1 (= ATCC 43210) is designated the type strain of a new species, Spiroplasma diabroticae.

Spiroplasma Species, Groups, and Subgroups from North American Tabanidae

Twenty-one triply cloned spiroplasma strains from the United States east of the Rocky Mountains, all isolated from tabanid (Diptera:Tabanidae) flies or serologically related to strains from tabanids, were compared reciprocally by spiroplasma deformation (DF) and metabolism inhibition (MI) serological tests. Many of the strains were also tested against 28 antisera representing known spiroplasma groups, subgroups, and putative groups isolated from nontabanid hosts. Relationships among strains were indicated by reciprocal cross-reactivity in both DF and MI tests. The strains were found to represent 11 recognized spiroplasma groups or subgroups. On the basis of serological, biochemical, and genomic data, strain BARC 1901 from Tabanus lineola appeared to represent a previously unrecognized candidate group. Strain BARC 2649, also from T. lineola, also appeared to represent a new group, but its morphology, arginine utilization, and some one-way serological crossing patterns suggested that it may be distantly related to group VIII spiroplasmas. Morphological, serological, and genomic data were used to place tabanid spiroplasma strains into three informal clusters. These are (i) groups IV (strain B31) and XXXI (strain HYOS-1); (ii) the three existing subgroups and a new candidate subgroup of group VIII represented by strain BARC 1357 plus ungrouped strain BARC 2649; and (iii) 14 strains, including EC-1 and TATS-1 (group XIV); strains TN-1 and TAAS-2 (group XVIII); strains TG-1, TASS-1, and BARC 4689 (group XXIII), strains TALS-2 (group XXVII), strain TABS-2 (group XXXII), and strains TAUS-1 and TABS-1 (group XXXIII) and ungrouped but closely related strains BARC 1901, BARC 2264 and BARC 2555. Analysis of tabanids from other geographic regions probably will substantially increase the number of known spiroplasma groups from this insect family.

Reduction of Benzyl Viologen Distinguishes Genera of the Class Mollicutes

We tested the ability of 62 growing strains belonging to the class Mollicutes to reduce the redox indicator and free-radical generator 1,1'-dibenzyl-4,4'-bipyridinium dichloride (benzyl viologen [BV]) to a blue-violet-purple color. BV was reduced by 12 Acholeplasma species but not by Acholeplasma multiforme PN525T (T = type strain). BV was also reduced by five of nine Mesoplasma species and by four of six Entomoplasma species. BV was not reduced by 19 Mycoplasma species, six Spiroplasma species, five unnamed Spiroplasma strains belonging to different serogroups, three Ureaplasma species, and one unnamed Ureaplasma strain. The BV-reducing ability was localized in the membrane of Acholeplasma laidlawii B-PG9 and was dependent on NADH. Reduction of BV could be expressed in mixed cultures, and this activity may be useful for recognizing the contaminating presence of an Acholeplasma species. The reductive BV response may have phylogenetic value. We believe that the test described in this paper readily distinguishes all Acholeplasma species and some Mesoplasma and Entomoplasma species from all Mycoplasma, Spiroplasma, and Ureaplasma species tested.

Spiroplasma corruscae sp. nov., from a firefly beetle (Coleoptera: Lampyridae) and tabanid flies (Diptera: Tabanidae).

Spiroplasma strain EC-1T (T = type strain), which was isolated from the gut of a lampyrid beetle (Ellychnia corrusca) in Maryland, was serologically distinct from other spiroplasma species and groups. Similar strains were obtained from other E. corrusca specimens, and, later, numerous isolates of similar or partially related strains were obtained from several species of tabanid files. Cells of strain EC-1T were helical, motile filaments that were bound by a single cytoplasmic membrane, and there was no evidence of a cell wall. The cells were filterable through 220-nm-pore-size membrane filters but not through 100-nm-pore-size membrane filters. The organism was absolutely resistant to penicillin (1,000 U/ml) and required sterol for growth. Strain EC-1T grew well in M1D and SP-4 liquid media and could be cultivated in the Edward formulation of conventional mycoplasma medium and in 1% serum fraction medium. Optimal growth occurred at 32 degrees C (doubling time, 1.5 h). Strain EC-1T multiplied at 10 to 41 degrees C, but not at 5 or 43 degrees C. This organism produced acid from glucose, but did not hydrolyze arginine or utilize urea. The guanine-plus-cytosine content of the DNA was determined to be 26.3 mol% by the melting temperature method and 27.0 mol% by the buoyant density method. As a result of our studies, strain EC-1 (= ATCC 43212) is designated the type strain of a new species, Spiroplasma corruscae.

Amended Data on Arginine Utilization by Spiroplasma Species

Hydrolysis of arginine is a classical diagnostic test for species in the mollicute order Entomoplasmatales. In this paper we report data for arginine utilization by spiroplasmas, as determined by standard methods. In addition, modified methods were developed for fastidious spiroplasmas, such as strain LD-1T (T = type strain), the Colorado potato beetle spiroplasma. Twenty-one spiroplasma strains representing 13 groups or subgroups and eight ungrouped spiroplasmas (seven of which represent putative groups) were studied. The arginine reactions of eight strains were the same as the reactions reported previously, but previously reported positive tests for spiroplasma subgroups I-5 and I-6 (Spiroplasma insolitum) could not be repeated, and the data for the latter taxa are corrected. Although other workers have reported that addition of carbohydrate to media may be necessary for the utilization of arginine, the presence of glucose tended to obscure arginine hydrolysis in our studies.

Spiroplasma syrphidicola sp. nov., from a syrphid fly (Diptera: Syrphidae).

Spiroplasma sp. strain EA-1(T) (T = type strain) (subgroup VIII-1), which was isolated from the syrphid fly Eristalis arbustorum, was serologically distinct from other spiroplasma species, groups, and subgroups, The cells of this strain, as revealed by dark-field light microscopy, were short, helical, and motile. An electron microscopic examination revealed wall-less cells delimited by a single membrane. The unusually short cells passed through 220-nm filter pores with no reduction in titer. The organisms grew well in SM-1, M1D, and SP-4 liquid media. Growth also occurred in conventional horse serum medium and 1% serum fraction medium. Strain EA-1(T) grew at temperatures between 10 and 41 degrees C, and optimum growth occurred at 32 degrees C. The doubling time at the optimal temperature was 1.0 h. The strain catabolized glucose and hydrolyzed arginine but did not hydrolyze urea. The guanine-plus-cytosine content of the DNA was 30 +/- 1 mol%. The genome size was about 1,230 kbp. Strain Ea-1 (= ATCC 33826), which represents subgroup VIII-1, is designated the type strain of a new species, Spiroplasma syrphidicola.

Mycoplasma sturni sp. nov., from the conjunctiva of a European starling (Sturnus vulgaris).

Strain UCMF(T) (T = type strain) was isolated from the conjunctiva of a European starling (Sturnus vulgaris) with conjunctivitis. Colonies grown on conventional mycoplasma agar possessed the typical fried-egg appearance observed with many mycoplasmal species. Electron micrographs of ultrathin sections of UCMF(T) revealed a pleomorphic cellular morphology; the cells ranged from spherical to elliptical or flask shaped. The cell size ranged from 0.3 to 0.5 microns. Strain UCMF(T) grows well in a variety of mycoplasma broth formulations at 25 degrees C, with rapid and heavy growth at 37 degrees C. No growth occurs at 42 degrees C. This organism ferments glucose but does not hydrolyze urea or arginine and has an absolute requirement for sterol from growth. Strain UCMF(T) does not hemagglutinate or hemadsorb chicken erythrocytes. The genome size is 870 kbp, and the guanine-plus-cytosine content is 31 mol%. Sequence analysis of the 16S rRNA gene demonstrated that this organism is unique and has not been described previously. Serological analysis confirmed that strain UCMF(T) is distinct from all previously identified Mycoplasma, Acholeplasma, Spiroplasma, Entomoplasma, and Mesoplasma species. This organism represents a new species, for which we propose the name Mycoplasma sturni. Strain UCMF (= ATCC 51945) is the type strain of M. sturni sp. nov.

Conformation, pore-forming activity, and antigenicity of synthetic peptide analogues of a spiralin putative amphipathic α helix

Current models depict spiralin as a bitopic transmembrane protein with the transbilayer domain being an amphipathic α helix. However, though secondary structure prediction methods suggest a helical conformation for the hypothetical transmembrane segment of spiralin, no potential transmembrane helices could be detected in this protein using the method of Von Heijne ( Von Heijne, G. (1992) J. Mol. Biol. 225, 487–494). Therefore, we have reconsidered the spiralin topological model by investigating the properties of the chemically synthesized peptides SM-BC3 (LNAVNTYATLAKAVLDAIQN-NH 2) and SC-R8A2 (LNAVNTYATLASAVLEAIKN-NH 2), corresponding to the hypothetical transmembrane segments of spiralins of two distinct spiroplasma species. The hydrophobic moment plot method suggests that these spiralin amino acid stretches are class G amphipathic α helices (i.e., helices localized on the surface of a globular protein domain). Circular dichroism spectra showed that both peptides have little ordered structure in aqueous solutions but adopt a mainly helical conformation in the presence of 25% trifluoroethanol or in detergent micelles (up to 74% α helix). Both peptides formed concentration- and voltage-dependent pores in planar lipid bilayers with a unitary conductance of 130 pS in 1 M KCl and with mean numbers of monomers per conducting aggregates of 6 for SC-R8A2 and 9 for SM-BC3. However, the two peptides displayed a haemolytic activity only at high concentrations (≫ 250 μM) and reacted with antibodies raised against membrane-bound spiralin. Together with previously published results, these data suggest that spiralin is a monotopic membrane protein anchored at the surface of the spiroplasma cell and that the 20-residue amphipathic segment is most probably a class G helix containing a B-cell epitope.

Spiroplasma velocicrescens sp. nov., from the vespid wasp Monobia quadridens.

Spiroplasma strain MQ-4T (T = type strain), which was isolated from the hemolymph of the vespid wasp Monobia quadridens, was serologically distinct from other spiroplasma species, groups, putative groups, and subgroups. Each strain MQ-4T cell was helical and motile and was surrounded by a single cytoplasmic membrane; there was no evidence of a cell wall. The strain grew well in 1% serum fraction medium, as well as in SM-1, M1D, and SP-4 liquid media, under both aerobic and anaerobic conditions. Strain MQ-4T grew at temperatures ranging from 10 to 41 degrees C but did not grow at 43 degrees C. The strain grew optimally at 37 degrees C with a doubling time of 0.6 h, the shortest doubling time recorded for any spiroplasma. Strain MQ-4T catabolized glucose and arginine but did not hydrolyze urea. The guanine-plus-cytosine content of the DNA was about 27.5 +/- 1 mol%. The genome size was 1,480 kbp (940 MDa). Strain MQ-4 (= ATCC 35262) is designated the type strain of a new species, Spiroplasma velocicrescens.

The topology of the promoter of RNA polymerase II- and III-transcribed genes is modified by the methylation of 5'-CG-3' dinucleotides.

In eukaryotic cells, RNA polymerase II- and III-transcribed promoters can be inactivated by sequence-specific methylation. For some promoter motifs, the introduction of 5-methyldeoxycytidine (5-mC) residues has been shown to alter specific promoter motif-protein interactions. To what extent does the presence of 5-mC in promoter or regulatory DNA sequences affect the structure of DNA itself. We have investigated changes in DNA bending in three naturally occurring DNA elements, the late E2A promoter of adenovirus type 2 (Ad2) DNA, one of our main model systems, the VAI (virus-associated) RNA gene of Ad2 DNA, and an Alu element associated with the human angiogenin gene. Alterations in electrophoretic mobility of differently permuted promoter segments in non-denaturing polyacrylamide gels have been used as assay system. In the late E2A promoter of Ad2 DNA, a major and possibly some minor DNA bending motifs exist which cause deviations in electrophoretic mobility in comparison to coelectrophoresed marker DNA fragments devoid of DNA bending motifs. DNA elements have been specifically in vitro methylated by the HpaII (5'-CCGG-3'), the FnuDII (5'-CGCG-3'), or the CpG DNA methyltransferase from Spiroplasma species (M-SssI; 5'-CG-3'). Methylation by one of these DNA methyltransferases influences the electrophoretic mobility of the three tested promoter elements very strikingly, though to different extents. It cannot be predicted whether sequence-specific promoter methylation increases or decreases electrophoretic mobility; these changes have to be experimentally determined. Methylation of the E. coli dcm (5'-CCA/TGG-3') sites in some of the DNA constructs does not make a contribution to mobility changes. It is concluded that sequence-specific methylations in promoter or regulatory DNA elements can alter the bending of DNA very markedly. This parameter may contribute significantly to the silencing of promoters, probably via altering spatial relationships among DNA-bound transcription factors.

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.

Spiroplasma cantharicola sp. nov., from Cantharid Beetles (Coleoptera: Cantharidae)

Spiroplasma strain CC-1T, isolated from the gut of the soldier beetle Cantharis carolinus, was serologically distinct from other spiroplasma species, groups, and subgroups. Cells of strain CC-1T were shown by light microscopy to be helical, motile filaments. Electron microscopy showed that the cells were bounded by a single cytoplasmic membrane, with no evidence of a cell wall. The organism was insensitive to penicillin. Strain CC-1T grew well in SM-1, MID, and SP-4 liquid media under aerobic or anaerobic conditions. The strain also grew in 1% serum fraction medium. Optimal growth occurred at 32°C, with a doubling time of 2.6 h, but the strain multiplied at temperatures of 10 to 37°C. Strain CC-1T produced acid from glucose but hydrolyzed neither arginine nor urea. The guanine-plus-cytosine (G+C) content of the DNA was 26 ± 1 mol%. Other uncloned isolates from C. carolinus exhibited similar or identical serological patterns. On the basis of the data presented here, strain CC-1T (= ATCC 43207), previously proposed as the representative strain of subgroup XVI-1, is designated the type strain of a new species, Spiroplasma cantharicola.

Spiroplasma monobiae sp. nov. from the Vespid Wasp Monobia quadridens (Hymenoptera: Vespidae)

Spiroplasma strain MQ-1T (T = type strain) from the hemolymph of the vespid wasp Monobia quadridens differed serologically from other spiroplasma species, groups, and subgroups. Cells of strain MQ-1T were helical and motile and possessed a single cytoplasmic membrane, with no evidence of a cell wall. The organism grew in conventional mycoplasma medium, in serum fraction, SM-1, MID, and SP-4 liquid media, and on SP-4 solid medium in either aerobic or anaerobic environments. The optimum temperature for growth was 32°C, but multiplication occurred over a wide temperature range (10 to 37°C). The doubling time at 32°C in MID medium was 1.9 h. Strain MQ-1T catabolized glucose but hydrolyzed neither arginine nor urea. Previous work showed that strain MQ-1T has a unique methylase, previously known only in eucaryotes. Also, strain MQ-1T induces production of tumor necrosis factor in bone marrow macrophages. The guanine-plus-cytosine content of the DNA was 28 ± 1 moI%. The genome size of strain MQ-1T was 940 kb (627 MDa); a similar strain, MQ-8, had a genome size of 985 kb (657 MDa). Strain MQ-1T and its allies have the smallest genomes known in the genus Spiroplasma. Strain MQ-1 (= ATCC 33825) is designated the type strain of a new species, Spiroplasma monobiae.

Spiroplasma clarkii sp. nov. from the Green June Beetle (Coleoptera: Scarabaeidae)

Spiroplasma strain CN-5T (T = type strain), isolated from the gut of the cetoniine scarabaeid beetle Cotinus nitida, was serologically distinct from other spiroplasma species, groups, and subgroups. Cells of strain CN-5T were shown by light microscopy to be helical, motile filaments. Cells in early passages exhibited strong translational motility that tended to be lost in later passages. Electron microscopy showed that the cells were bounded by a single cytoplasmic membrane with no evidence of a cell wall. The organism was not susceptible to penicillin. Strain CN-5T grew well in SM-1, M1D, and SP-4 liquid media and on solid SP-4 medium under aerobic or anaerobic conditions. The doubling time at 30°C, the optimum temperature, was 4.3 h. The strain also grew in 1% serum fraction medium. Strain CN-5T produced acid from glucose and catabolized arginine, but did not hydrolyze urea. The guanine-plus-cytosine content of the DNA was 29 ± 1 mol%. The genome size was 1,770 kb (1,186 MDa). Other uncloned isolates obtained from C. nitida or the cetoniine hermit flower beetle Osmoderma eremicola exhibited similar or identical serological patterns. Since no other hosts were discovered in extensive studies, strain CN-5T (previously designated group IX) appears to represent a cluster of relatively host-specific cetoniine beetle-associated strains. Strain CN-5 (= ATCC 33827) is designated the type strain of a new species, Spiroplasma clarkii.