J.M. Bové

Heterogeneity of Genome Sizes within the Genus Spiroplasma

Organisms belonging to the genus Spiroplasma are currently classified into 23 groups, 17 of which have been assigned species epithets. We determined the genome sizes of representatives of 20 groups by using pulsed-field gel electrophoresis. Each genome size was deduced from the mobility of linear nonrestricted DNA, as well as from the sum of the sizes of restriction fragments obtained after digestion with NotI, a restriction endonuclease with a limited number of restriction sites in spiroplasma DNA. The values which we obtained indicated that the genome sizes of members of the genus Spiroplasma range from 940 to 2,220 kbp.

Spiroplasma phoeniceum sp. nov., a new plant-pathogenic species from Syria.

Sixteen spiroplasma isolates, recovered over a 2-year period from symptomatic periwinkle plants (Catharanthus roseus) collected in eight different locations in Syria, were compared with other established Spiroplasma species or serogroups. Serological analysis of selected representatives of the new isolates revealed sharing of some antigenic components with several spiroplasmas currently classified within subgroups of group I of the genus. Strain P40Twas selected as the type strain and examined, meeting the criteria proposed by the International Committee on Systematic Bacteriology Subcommittee on the Taxonomy of Mollicutes. The organism was shown to belong to the class Mollicutes by its morphology, ultrastructure of its limiting membrane, colony characteristics, and filtration patterns. The helicity and motility of the cells indicated its placement within the family Spiroplasmataceae. Although some serological cross-reactions could be observed with representatives of group I subgroups, strain P40Tcould be readily distinguished from other plant or insect pathogenic spiroplasmas in subgroup I-1 (Spiroplasma citri), subgroup I-2 (S. melliferum), or subgroup I-3 (S. kunkelii) and from spiroplasmas assigned to subgroups I-4 through I-7 and groups II through XI. Cholesterol was required for growth. Glucose was fermented, and arginine was hydrolyzed. The base composition (guanine plus cytosine) of the deoxyribonucleic acid of strain P40Twas found to be 26 mol%. Deoxyribonucleic acid-deoxyribonucleic acid hybridization comparisons between strain P40Tand other subgroup I representatives revealed approximately 60% relatedness to S. citri and S. kunkelii and 50% relatedness to S. melliferum. Experimental transmission of two of the new isolates (P40Tand P354) occurred through inoculation of spiroplasma broth cultures into leafhoppers (Macrosteles fascifrons), multiplication of the organism in the insects, and subsequent transmission of the organism by insect feeding on aster or periwinkle plants. The organism was also successfully recovered from broth cultures of symptomatic tissues of experimentally infected periwinkle plants, thus fulfilling Kochs postulates. We propose that such strains be named Spiroplasma phoeniceum. Strain P40Thas been deposited in the American Type Culture Collection (= ATCC 43115T)

In vitro isolation of a transovarially transmitted bacterium from the leafhopper Euscelidius variegatus (Hemiptera: Cicadellidae)

Abstract Gram-negative, rod-shaped bacteria were present in the hemolymph of adults and nymphs from colonies of Euscelidius variegatus. the bacterium (designated BEV) was isolated from hemolymph of E. variegatus onto blood (sheep) agar or purple agar with or without added 10% horse or fetal bovine serum. BEV was microaerophilic, catalase- and oxidase-negative, and lacked flagella. Antisera to the cultured bacterium were used in fluorescent antibody staining of BEV-like isolates from E. variegatus or of macerated leafhoppers or smears from leafhoppers. Serological tests indicated that in vitro cultures of BEV were serologically and morphologically identical to the bacterium regularly seen in the hemolymph of E. variegatus from “infected” colonies. BEV was detected in and isolated from eggs and nymphs of E. variegatus reared without direct contact with their parents. BEV-free E. variegatus were injected with BEV cultures and the bacterium was reisolated from progeny of the injected insects. BEV appears to be a nonobligate symbiote of E. variegatus. It multiplied and was pathogenic to several other leafhopper species when inoculated by injection.

Division of group XVI spiroplasmas into subgroups

Some group XVI spiroplasmas, such as strains CC-1 (Spiroplasma cantharicola) and CB-1, are associated with cantharid beetles. Fifteen related but heterogeneous strains have been isolated from mosquitoes, other insects, and a flower in France and the United States. In the present study, these seventeen strains have been compared by deformation and metabolism inhibition serological tests, by one-dimensional protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and by determination of the guanine-plus-cytosine content of their DNA. Five of the 17 strains were further compared by DNA-DNA hybridization and by restriction enzyme (EcoRI and HindIII) analysis of their DNA. On the basis of the resulting data, we propose that group XVI be subdivided into three subgroups. Subgroup XVI-I is represented by strain CC-1 (ATCC 43207) from a cantharid beetle in the United States, and strain MQ-6 from a wasp; subgroup XVI-2 is represented by strain CB-1 (ATCC 43208) from a cantharid beetle and two strains from mosquitoes, all in the United States; and subgroup XVI-3 is represented by strain Ar-1357 (ATCC 51126) and contains 11 strains from mosquitoes and 1 strain from a flower, all from the Savoy region of France.

Spiroplasma leptinotarsae sp. nov., a mollicute uniquely adapted to its host, the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

Spiroplasma strain LD-1T (T = type strain), which was isolated from the gut of a Colorado potato beetle (Leptinotarsa decemlineata) larva collected in Maryland, was serologically distinct from other spiroplasmas. Similar isolates were obtained from other L. decemlineata specimens collected in various parts of North America, in Poland, and in other eastern European countries and from Leptinotarsa texana specimens collected in Texas. Cells of strain LD-1T, which in early passages were spiral, exhibited exceptionally rapid translational motility. This rapid motility and the spiral shape were lost after extended passage in culture. The organism required serum for growth. Originally isolated in coculture with insect cells in DCCM medium, strain LD-1T adapted to several media in the absence of cocultured cells. Use of anaerobic conditions allowed primary isolation in a variety of media. The organism did not grow in serum-free media containing 2% serum fraction. Optimal growth in M1D medium occurred at 30 to 37°C (doubling time, 7.2 h). On solid M1D medium containing 2.0% Noble agar (pH 6.25) at 30°C, strain LD-1T produced discrete colonies with numerous satellites. Strain LD-1T hydrolyzed arginine, but did not utilize urea; there was evidence of weak fermentation of glucose. The guanine-plus-cytosine content of the DNA was determined to be 25 ± 1 mol%, and the genome size was 1,085 kb. The results of extensive studies of the ecology of this spiroplasma suggest that it is host specific for Leptinotarsa beetles. Strain LD-1 (= ATCC 43213) is designated the type strain of a new species, Spiroplasma leptinotarsae.

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.

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, M1D, 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 M1D 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 mol%. 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 insolitum sp. nov., a New Species of Group I Spiroplasma with an Unusual DNA Base Composition

Spiroplasma strain M55, isolated from a fall flower in Maryland, showed patterns of partial serological cross-reactivity with the other seven group I spiroplasma subgroups but was not serologically related to other spiroplasma groups. Strain M55 had less than 70% DNA-DNA homology with group I subgroups previously assigned binomial names and was unique among the group I subgroups in possessing a higher guanine-plus-cytosine content in its DNA (28 ± 1 mol%, versus 26 ± 1 mol% for other group I subgroups). The genome size was 1,850 kb (1,233 MDa). Extensive data on the metabolism of strain M55 and the community ecology of this and similar strains have been reported. These circumstances fulfill criteria proposed by the Subcommittee on the Taxonomy of Mollicutes for elevation of mollicute subgroups to species status. Accordingly, strain M55 was characterized according to proposed minimal standards for species descriptions. Cells of strain M55 were shown by light microscopy to be helical, motile filaments. Electron microscopy showed that the cells possessed no cell wall and were bounded by a single membrane; they were insensitive to penicillin (1,000 U/ml). Strain M55 was culturable in M1D and SP-4 liquid media under aerobic (with or without enhanced carbon dioxide) or anaerobic environments. Optimal growth occurred at 30°C, with a doubling time of 7.2h. Growth occurred from 15 to 37°C but not at 10 or 42°C. Strain M55 did not utilize urea or hydrolyze arginine but did produce acid from glucose. As a consequence of these studies, strain M55 is designated the type strain (M55T; ATCC 33502T) of a new species, Spiroplasma insolitum.

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.