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Components of membranes isolated from Spiroplasma citri and corn stunt spiroplasma grown at 28 degrees C were analyzed. On a protein basis, lipid phosphorus was lower and cholesterol was higher in S. citri. Only minor differences between the two species were found in fatty acid composition, reduced nicotinamide adenine dinucleotide diaphorase, and adenosine triphosphatase.
In previous studies (A. Marais, J. M. Bove, and J. Renaudin, J. Bacteriol. 178:862-870, 1996), we have shown that the recA gene of Spiroplasma citri R8A2 was restricted to the first 390 nucleotides of the N-terminal part. PCR amplification and sequencing studies of five additional strains of S. citri have revealed that these strains had the same organization at the recA region as the R8A2 strain. In contrast to S. citri, Spiroplasma melliferum was found to contain a full-length recA gene. However, in all five S. melliferum strains tested, a TAA stop codon was found within the N-terminal region of the recA reading frame. Our results suggest that S. melliferum, as well as S. citri, is RecA deficient. In agreement with the recA mutant genotype of S. citri and S. melliferum, we have shown that these organisms are highly sensitive to UV irradiation.
The helical mycoplasma Spiroplasma citri was examined by electron microscopy with a newly developed transfer technique which preserves the helical morphology of the organism. The smallest viable cell was found to be a two-turn (elementary) helix. During the logarithmic phase of growth, organisms increased in length and divided by constriction, liberating two-turn elementary helices. The most frequently dividing parental helix was one with approximately four turns, yielding two elementary helices. Influence of pH and temperature on the morphology of the organism was also investigated. In unbuffered medium, growth of the organism produced a significant decrease in pH and a consequent formation of abnormal morphological forms and cell lysis. At 37 degrees C, cell division was inhibited, leading to a progressive disappearance of two-turn helices and an increase in the average length of other helices. Finally, helices were never seen to arise from round bodies at any stage of the growth cycle.
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The spiroplasmas are mollicutes characterized by motility and helical morphology. They were discovered through studies on corn stunt and citrus stubborn diseases. The stubborn agent was the first mollicute of plant origin to be obtained in culture and the first cultured mollicute to possess a helical morphology. The citrus pathogen has been known as Spiroplasma citri since 1973. The corn stunt agent was cultured in 1975 and fully characterized as Spiroplasma kunkelii by 1986. The third and only other phytopathogenic spiroplasma is Spiroplasma phoeniceum, cultured from naturally infected periwinkle plants in Syria and described in 1986. These three spiroplasmas are restricted to the phloem sievetubes of the infected plants and are transmitted from plant by various phloem feeding leafhopper vectors in which the spiroplasmas multiply. Following the pioneering work on S. citri and S. kunkelii, close to fifty other spiroplasma species or proposed species have been discovered. All spiroplasmas have been isolated from insects, ticks and plants. Insects are particularly rich sources of spiroplasmas. Some insect-derived spiroplasmas are entomopathogens. S. melliferum and S. apis are honey bee pathogens. They cross the insect-gut barrier and reach the hemolymph, where they multiply abundantly and kill the bee. Spiroplasma floricola is the agent of lethargy disease of Melolontha melolontha (cockchafer). Spiroplasma poulsonii infects the neotropical species of Drosophila, is transmitted transovarially and kills the male progeny of an infected female fly, hence the name sex ratio spiroplasma. Some insect-derived spiroplasmas are also found on plant (flower) surfaces. For instance, S. apis was cultured from the surfaces of flowers growing in the vicinity of affected beehives. This suggests that the plant surface spiroplasmas are deposited on these surfaces by contaminated insects. Many insect spiroplasmas are not pathogenic, are often restricted to the gut and may be regarded as mutualists or incidental commensals. Of the three known tick spiroplasmas, only Spiroplasma mirum obtained from rabbit ticks is pathogenic to the vertebrate animal (chick embryo, new-born rodents, adult rabbit), but only upon experimental inoculation of the spiroplasma. Strain SMCA induces high incidence of cataracts in new born rodents. With strain GT-48 no cataracts are observed, but fatal encephalitis occurs. Spiral membranous inclusions resembling spiroplasmas have been seen in brain biopsies taken from patients with Creutzfeldt-Jakob disease. However, failure to detect spiroplasmas by serology and culture points to the absence of spiroplasmal involvement in spongiform encephalopathies. Transposon Tn 4001 mutagenesis has been applied for the first time to Spiroplasma citri, and pathogenicity can now be studied at the genetic level. One Tn 4001 mutant does not multiply in the leafhoppers and is, therefore, not transmitted to the plant. Another mutant multiplies well in the plant and is transmitted to the plant, where it reaches high titers, but without inducing symptoms in the plant. In this non-phytopathogenic mutant, Tn 4001 is inserted in the spiroplasmal fructose operon, and the mutant is unable to use fructose. Finally, to study involvement of spiroplasmal motility in pathogenicity, a non-motile mutant has been obtained. Motility was restored by complementation with the wild type genes. This is the first time that successful complementation has been reported, not only in the spiroplasmas but in the mollicutes in general. Undoubtedly, studies on pathogenicity have entered a new era.
Significant changes have been made in the systematics of the genus Spiroplasma (class Mollicutes) since it was expanded by revision in 1987 to include 23 groups and eight sub-groups. Since that time, two additional spiroplasmas have been assigned group numbers and species names. More recently, specific epithets have been assigned to nine previously designated groups and three sub-groups. Also, taxonomic descriptions and species names have been published for six previously ungrouped spiroplasmas. These six new organisms are: Spiroplasma alleghenense (strain PLHS-1T) (group XXVI), Spiroplasma lineolae (strain TALS-2T) (group XXVII), Spiroplasma platyhelix (strain PALS-1T) (group XXVIII), Spiroplasma montanense (strain HYOS-1T) (group XXXI), Spiroplasma helicoides (strain TABS-2T) (group XXXII) and Spiroplasma tabanidicola (strain TAUS-1T) (group XXXIII). Also, group XVII, which became vacant when strain DF-1T (Spiroplasma chrysopicola) was transferred to group VIII, has been filled with strain Tab 4c. The discovery of these strains reflects continuing primary search in insect reservoirs, particularly horse flies and deer files (Diptera: Tabanidae). In the current revision, new group designations for 10 spiroplasma strains, including six recently named organisms, are proposed. Three unnamed but newly grouped spiroplasmas are strain TIUS-1 (group XXIX; ATCC 51751) from a typhiid wasp (Hymenoptera: Tiphiidae), strain BIUS-1 (group XXX; ATCC 51750) from floral surfaces of the tickseed sunflower (Bidens sp.) and strain BARC 1901 (group XXXIV; ATCC 700283). Strain BARC 2649 (ATCC 700284) from Tabanus lineola has been proposed as a new sub-group of group VIII. Strains TIUS-1 and BIUS-1 have unusual morphologies, appearing as helices at only certain stages in culture. In this revision, potentially important intergroup serological relationships observed between strain DW-1 (group II) from a neotropical Drosophila species and certain sub-group representatives of group I spiroplasmas are also reported.
Several spiroplasmas (helical, motile mollicutes) were previously shown to contain extrachromosomal DNA (E-DNA) elements in the form of viruses (double-stranded viruses or the replicative form of single-stranded viruses) or plasmids. These elements are now being investigated as potential vectors for use in spiroplasma transformation systems. Described herein is the first extensive survey of spiroplasma E-DNA in 23 spiroplasma groups (30 strains), a study facilitated by improvements in protocols for E-DNA extraction. E-DNA elements were found in spiroplasmas associated with leafhoppers/plants (spiroplasma subgroups I-1, I-3, and I-8), other insects (subgroups I-2, I-5, I-6, and I-7 and groups IV and XXII), and ticks (subgroup I-4 and groups V and VI). Elements, maintained by passage with their host spiroplasmas, were often lost after extended passage. Whether the current distribution of E-DNA elements is indicative of historical or proximate factors is not known. Many elements (about 75%) from group I spiroplasmas hybridized with Spiroplasma citri viruses SpV1 or SpV3. Of the elements associated with other spiroplasma groups, none hybridized with either virus. These include Spiroplasma apis strains B31 (18 kb) and L89 (18 and 20 kb), S. mirum strains SMCA (20 kb) and Anderson (16 and 20 kb), group VI strain Y32 (7, 9, 10, and 16 kb), and group XXII strain CT-1 (8 kb). Several of these elements will be characterized and examined for their suitability as spiroplasma cloning vectors.
A chemically defined medium, LD82, was formulated for in vitro cultivation of spiroplasmas. Medium LD82 supported good growth for four epiphytic and insect-pathogenic spiroplasmas, Spiroplasma floricola 23-6, Spiroplasma sp. strain SR3, Spiroplasma sp. strain brevi, and Spiroplasma sp. strain AS576, and of the phytopathogenic spiroplasmas Spiroplasma citri Maroc R8A2 and PC1. Titers of all six strains grown in defined medium LD82 reached 2.0 x 10 to 6.0 x 10 CFU/ml of culture. All spiroplasma strains tested formed colonies readily on agar medium LD82. None of the spiroplasmas formed typical fried-egg colonies. All formed diffuse colonies, but the forms of colonies differed somewhat among the spiroplasma strains. In preliminary studies of nutritional requirements, phospholipids slightly enhanced the growth of the epiphytic and insect-pathogenic strains in medium LD82 and were found essential for good growth of S. citri.
Representatives of several currently available spiroplasma serovars were used in feeding and injection experiments involving Drosophila pseudoobscura adult flies in order to evaluate the host-range of the spiroplasmas and to determine their pathogenicity for flies. Many of the isolates injected into flies either do not survive or grow poorly, and have no negative effects on longevity or fecundity. Two spiroplasma strains, honey-bee (serovar I-2) and cornstunt (serovar I-3), can grow to high titres in injected flies but are not pathogenic. In addition to the special pathology of male lethality caused by the sex-ratio organisms (spiroplasmas) which occur naturally in several neotropical species of Drosophila, two spiroplasma serovars resulted in the death of injected flies. Spiroplasmas isolated from syrphid flies (serovar VIII) and from Cotinus beetles (serovar IX) induced the appearance of symptoms of pathology that eventually culminated in death. All such flies which were examined for the presence of spiroplasmas in their haemolymph showed them to be present in very high concentrations. The numbers of progeny which these flies produced were greatly reduced, but did consist of equal numbers of flies of both sexes. Spiroplasmas from their infected female parents were not vertically (transovarially) transmitted. The results of the feeding experiments were all negative: none of the spiroplasmas persisted in the gut or appeared in the haemolymph. None of the flies which had been fed the spiroplasma suspension displayed any pathology.
The genus Spiroplasma (helical mollicutes: Bacteria: Firmicutes: Mollicutes: Entomoplasmatales: Spiroplasmataceae) is associated primarily with insects. The Mycoplasma mycoides cluster (sensu Weisburg et al. 1989 and Johansson and Pettersson 2002) is a group of mollicutes that includes the type species - Mycoplasma mycoides - of Mycoplasmatales, Mycoplasmataceae and Mycoplasma. This cluster, associated solely with ruminants, contains five other species and subspecies. Earlier phylogenetic reconstructions based on partial 16S rDNA sequences and a limited sample of Spiroplasma and Mycoplasma sequences suggested that the genus Mycoplasma was polyphyletic, as the M. mycoides cluster and the grouping that consisted of the hominis and pneumoniae groups of Mycoplasma species were widely separated phylogenetically and the M. mycoides cluster was allied with Spiroplasma. It is shown here that the M. mycoides cluster arose from Spiroplasma through an intermediate group of non-helical spiroplasmal descendants - the Entomoplasmataceae. As this conclusion has profound implications in the taxonomy of Mollicutes, a detailed phylogenetic study of Spiroplasma and its non-helical descendants was undertaken. These analyses, done with maximum-parsimony, provide cladistic status; a new nomenclature is introduced here, based on 'bottom-up' rather than 'top-down' clade classification. The order Entomoplasmatales consists of four major clades: (i) the Mycoides-Entomoplasmataceae clade, which contains M. mycoides and its allies and Entomoplasma and Mesoplasma species and is a sister lineage to (ii) the Apis clade of Spiroplasma. Spiroplasma and the Entomoplasmataceae are paraphyletic, but this status does not diminish their phylogenetic usefulness. Five species that were previously unclassified phylogenetically are basal to the Apis clade sensu strictu and to the Mycoides clade. One of these species, Spiroplasma sp. TIUS-1, has very poor helicity and a very small genome (840 kbp); this putative species can be envisioned as a 'missing link' in the evolution of the Mycoides-Entomoplasmataceae clade. The other two Spiroplasma clades are: (iii) the Citri-Chrysopicola-Mirum clade (serogroups I, II, V and VIII) and (iv) the ixodetis clade (serogroup VI). As Mesoplasma lactucae represents a basal divergence within the Mycoides-Entomoplasmataceae clade, and as Entomoplasma freundtii is basal to the Mycoides clade, M. mycoides and its allies must have arisen from an ancestor in the Entomoplasmataceae. The paraphyletic grouping that consists of the Hominis and Pneumoniae groups (sensu Johansson & Pettersson 2002) of Mycoplasma species contains the ancestral roots of Ureaplasma spp. and haemoplasmas. This clade is a sister lineage to the Entomoplasmatales clade. Serological classifications of spiroplasma are very highly supported by the trees presented. Genome size and G+C content of micro-organismal DNA were moderately conserved, but there have been frequent and polyphyletically distributed genome reductions. Sterol requirements were polyphyletic, as was the ability to grow in the presence of polyoxyethylene sorbitan-supplemented, but not serum-supplemented, media. As this character is not phylogenetically distributed, Mesoplasma and Entomoplasma should be combined into a single genus. The phylogenetic trees presented here confirm previous reports of polyphyly of the genus Mycoplasma. As both clades of Mycoplasma contain several species of great practical importance, a change of the genus name for species in either clade would have immense practical implications. In addition, a change of the genus name for M. mycoides would have to be approved by the Judicial Commission. For these reasons, the Linnaean and phylogenetic classifications of Mycoplasma must for now be discrepant.
Spiroplasmas are wall-less descendants of Gram-positive bacteria that maintain some of the smallest genomes known for self-replicating organisms. These helical, motile prokaryotes exploit numerous habitats, but are most often found in association with insects. Co-evolution with their insect hosts may account for the highly speciose nature of the genus Spiroplasma, with many spiroplasmas existing in obligate insect/plant transmission cycles. In addition to insect and plant hosts, spiroplasmas are found in association with ticks and crustaceans. Although most spiroplasma associations appear to be commensal, some cases of pathogenicity or mutualism have been described. Most notably, spiroplasmas have been identified as the causative agents of agricultural and aquacultural diseases and the sex ratio disorder in insects. Some spiroplasmas exhibit strict host and/or geographical ranges, but others are relative generalists. Species of the genus Spiroplasma have been traditionally classified into 34 groups based on cross-reactivity of surface antigens. Three of the serogroups contain closely related strain complexes that are further divided into subgroups. Phylogenetic reconstructions based on 16S rDNA sequence strongly support the closely related serogroups. To date, less than 40 Spiroplasma species have been fully characterized and given binomial names. Complete characterization of a new species involves numerous phenotypic and genotypic tests as outlined in the minimal standards document; this document is currently under revision to include phylogenetic data and a reevaluated set of required phenotypic and genotypic tests. The area of spiroplasma research is poised for major advances with new criteria for naming species in preparation, a dramatic increase in available molecular characters, the promise of full genome sequences, and advances in genetic tools for manipulation of these organisms.
Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative diseases of man and animals and are transmitted by a filterable pathogen whose identity is currently unresolved. Our data indicates that Spiroplasma, a wall-less bacterium, is involved in the pathogenesis of TSE. We searched for Spiroplasma ribosomal gene sequences in 10 scrapie-infected sheep brains and 10 normal sheep brains, 7 cervid samples infected with chronic wasting disease (CWD), and 7 normal cervid brains. DNA was extracted from these tissue samples and amplified by polymerase chain reaction (PCR) using primers specific for Spiroplasma-specific 16S rDNA. Specificity of the amplicon was determined by Southern blotting and DNA sequence analyses. Spiroplasma 16S rDNA was found in 8 of 10 scrapie-infected sheep brains and 6 of 7 CWD-infected tissue samples. All normal animal brain samples were negative. Spiroplasma 16S rDNA was also found in two human Creutzfeldt-Jakob diseased (CJD) brains but not in two age-matched normal human brains. DNA sequence analyses of the amplified PCR products from human and animal TSE cases revealed greater than 99% nucleotide sequence homology with Spiroplasma mirum. The presence of Spiroplasma DNA in TSE-infected tissues supports our hypothesis that Spiroplasma may be involved in the pathogenesis of these diseases.
Spiroplasma kunkelii distribution and infection mechanisms in the intestines and Malpighian tubules of Dalbulus maidis were investigated by transmission electron microscopy. Spiroplasmas were found between microvilli and in endocytic vesicles of the midgut epithelium. At the basal part, cytoplasmic vesicles contained multiple spiroplasmas with tube-like extensions and spiroplasmas accumulated between the laminae rara and densa of the basal lamina. Tip structures of flask-shaped spiroplasmas pierced the lamina densa that was discontinuous in close proximity to spiroplasmas. Spiroplasmas were found in hemolymph, crossed the basal lamina of Malpighian tubule epithelium and accumulated at high numbers in muscle cells that had cytopathogenic changes. S. kunkelii had perithrochous approximately 8nm diameter structures determined to be fimbriae protruding from the cell surface, and similar structures were adhering to the basal lamina of midgut epithelium and to external lamina of muscle cells. Further, spiroplasmas had pili-like appendages at one or both cell poles and appeared to conjugate. This is the first time that fimbriae and pili have been observed in a mollicutes.
We investigated the interactions between the endosymbionts Wolbachia pipientis strain wMel and Spiroplasma sp. strain NSRO coinfecting the host insect Drosophila melanogaster. By making use of antibiotic therapy, temperature stress, and hemolymph microinjection, we established the following strains in the same host genetic background: the SW strain, infected with both Spiroplasma and Wolbachia; the S strain, infected with Spiroplasma only; and the W strain, infected with Wolbachia only. The infection dynamics of the symbionts in these strains were monitored by quantitative PCR during host development. The infection densities of Spiroplasma exhibited no significant differences between the SW and S strains throughout the developmental course. In contrast, the infection densities of Wolbachia were significantly lower in the SW strain than in the W strain at the pupal and young adult stages. These results indicated that the interactions between the coinfecting symbionts were asymmetrical, i.e., Spiroplasma organisms negatively affected the population of Wolbachia organisms, while Wolbachia organisms did not influence the population of Spiroplasma organisms. In the host body, the symbionts exhibited their own tissue tropisms: among the tissues examined, Spiroplasma was the most abundant in the ovaries, while Wolbachia showed the highest density in Malpighian tubules. Strikingly, basically no Wolbachia organisms were detected in hemolymph, the principal location of Spiroplasma. These results suggest that different host tissues act as distinct microhabitats for the symbionts and that the lytic process in host metamorphosis might be involved in the asymmetrical interactions between the coinfecting symbionts.
Male-killing phenotypes are found in a variety of insects and are often associated with maternally inherited endosymbiotic bacteria. In several species of Drosophila, male-killing endosymbionts of the genus Spiroplasma have been found at low frequencies (0.1 to 3%). In this study, spiroplasma infection without causing male-killing was shown to be prevalent (23 to 66%) in Japanese populations of Drosophila hydei. Molecular phylogenetic analyses showed that D. hydei was infected with a single strain of spiroplasma, which was closely related to male-killing spiroplasmas from other Drosophila species. Artificial-transfer experiments suggested that the spiroplasma genotype rather than the host genotype was responsible for the absence of the male-killing phenotype. Infection densities of the spiroplasma in the natural host, D. hydei, and in the artificial host, Drosophila melanogaster, were significantly lower than those of the male-killing spiroplasma NSRO, which was in accordance with the hypothesis that a threshold infection density is needed for the spiroplasma-induced male-killing expression.
A simple and efficient osmotic lysis method was developed for enzyme studies in spiroplasmas. Log phase cells in R2 medium were harvested by centrifugation (19,600 x g for 30 min). Wash buffer supplemented with 0.23 M sucrose maintained the helicity of spiroplasma cells during washing. Osmotic lysis of spiroplasmas was achieved in H buffer that contained no sucrose. Sucrose at concentrations as low as 0.004 M dramatically increased the resistance of the spiroplasmas to osmotic lysis. NADH oxidase, lactate dehydrogenase, and malate dehydrogenase were detected in cell lysates of Spiroplasma floricola (23-6), Spiroplasma citri (R8A2), Spiroplasma apis (SR 3), and Spiroplasma melliferum (AS 576). Citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinyl coenzyme A synthetase, succinate dehydrogenase, and fumarase were not detected in cell lysates of S. floricola (23-6). NADH oxidase and malate dehydrogenase were found in the cytosol whereas lactate dehydrogenase was loosely associated with the cytomembrane.
Since its designation as a separate genus some 30 years ago, Spiroplasmas have been well documented in a wide range of hosts and as the causative agent of several plant and insect diseases. One major area of research is the continued identification and taxonomical characterization of new Spiroplasma sp. combined with a determination of phylogenetic relationships among the various Spiroplasma sp. and between the Spiroplasmas and other members of the Mollicutes and Eubacteria. Although most phylogenetic analyses have been dependent on 16S rDNA sequence data, progress in two Spiroplasma sp. genome sequencing projects will provide new genomic regions for comparative focus. The co-evolution of Spiroplasmas with their arthropod hosts has provided an additional research focus to study host specificity and attachment. The diversity of symbiotic relationships between Spiroplasmas and their hosts has led to the study of commensal, mutualistic, and pathogenic relationships. Pathogenesis in insect hosts or in plants, transferred by insect hosts, is a major research focus, which requires attachment and invasion into insect tissues beyond the initial infection site, and successful movement to other tissues. The diversity and adaptations that have occurred during the evolution of the Spiroplasmas with their hosts will be the primary focus of this article.
Three serologically distinct groups of spiroplasmas have been recovered from ticks. Spiroplasma mirum strains (from rabbit ticks, Haemaphysalis leporispalustris) and Y32 group (VI) spiroplasmas (from Ixodes pacificus) are the only spiroplasmas to have a clear association with these arthropods. Group (VI) spiroplasmas are distinguished by an unusual nonhelical morphology and their capacity to hemadsorb guinea pig erythrocytes. S. mirum strains are unique in their ability to induce cataracts or lethal brain infections in a number of young vertebrates and in their virulence for the chick embryo. The 277F spiroplasma, while initially recovered from a pool of rabbit ticks (H. leporispalustris), is related by certain serological and genetic properties to spiroplasmas in the S. citri complex (serogroup I). These relationships suggest that the 277F spiroplasma may not be a natural inhabitant of the rabbit tick.