Genome analysis-based union of the genus Mesoplasma with the genus Entomoplasma.

Early characterization of strains designated into the genera Entomoplasma and Mesoplasma was based upon biological and chemical characteristics. With the advent of 16S rRNA gene sequence analysis as an added taxonomic character, it became clear that the two genera did not form distinct and separate monophyletic clusters. A genome-level analysis of all 17 validly published species within the family Entomoplasmataceae has recently been performed. Phylogenetic analyses, comparisons of gene content, and the lack of genus-specific genes supported that species from the two genera are intermixed and should not be taxonomically separated. This level of analysis clearly reveals the necessity to revise the taxonomy of this family by merging the two genera into one, Entomoplasma. Additionally, it was definitively determined that the strain originally designated as Acholeplasma multilocale resides in this cluster and should be formally renamed as Entomoplasma multilocale. Merging Mesoplasma and Entomoplasma yields a paraphyletic genus, but is supported by cell morphology and ecology to be distinguished from the genera Spiroplasma and Mycoplasma.

Comparative genome analysis of Spiroplasma melliferum IPMB4A, a honeybee-associated bacterium.

BACKGROUND: The genus Spiroplasma contains a group of helical, motile, and wall-less bacteria in the class Mollicutes. Similar to other members of this class, such as the animal-pathogenic Mycoplasma and the plant-pathogenic 'Candidatus Phytoplasma', all characterized Spiroplasma species were found to be associated with eukaryotic hosts. While most of the Spiroplasma species appeared to be harmless commensals of insects, a small number of species have evolved pathogenicity toward various arthropods and plants. In this study, we isolated a novel strain of honeybee-associated S. melliferum and investigated its genetic composition and evolutionary history by whole-genome shotgun sequencing and comparative analysis with other Mollicutes genomes. RESULTS: The whole-genome shotgun sequencing of S. melliferum IPMB4A produced a draft assembly that was ~1.1 Mb in size and covered ~80% of the chromosome. Similar to other Spiroplasma genomes that have been studied to date, we found that this genome contains abundant repetitive sequences that originated from plectrovirus insertions. These phage fragments represented a major obstacle in obtaining a complete genome sequence of Spiroplasma with the current sequencing technology. Comparative analysis of S. melliferum IPMB4A with other Spiroplasma genomes revealed that these phages may have facilitated extensive genome rearrangements in these bacteria and contributed to horizontal gene transfers that led to species-specific adaptation to different eukaryotic hosts. In addition, comparison of gene content with other Mollicutes suggested that the common ancestor of the SEM (Spiroplasma, Entomoplasma, and Mycoplasma) clade may have had a relatively large genome and flexible metabolic capacity; the extremely reduced genomes of present day Mycoplasma and 'Candidatus Phytoplasma' species are likely to be the result of independent gene losses in these lineages. CONCLUSIONS: The findings in this study highlighted the significance of phage insertions and horizontal gene transfer in the evolution of bacterial genomes and acquisition of pathogenicity. Furthermore, the inclusion of Spiroplasma in comparative analysis has improved our understanding of genome evolution in Mollicutes. Future improvements in the taxon sampling of available genome sequences in this group are required to provide further insights into the evolution of these important pathogens of humans, animals, and plants.

Spiroplasma-like organisms closely associated with the gut in five leafhopper species (Hemiptera: Cicadellidae).

Spiroplasmas are bacteria in the Class Mollicutes that are frequently associated with insects and/or plants. Here, we describe the ultrastructure, localization, and occurrence of apparent commensal/symbiotic spiroplasma-like organisms (SLOs) in the midgut and hindgut of five leafhopper species from laboratory-reared colonies. Those found in Dalbulus elimatus, Endria inimica, and Macrosteles quadrilineatus were long and tubular shaped, whereas those in Dalbulus maidis and Graminella nigrifrons were shorter and mostly rod-shaped in their host organisms. These SLOs were found in great numbers in the gut lumen frequently associated with the gut microvilli, but unlike the plant-pathogenic mollicutes, they did not seem to invade the gut epithelium or other tissues in any of these five leafhopper species. Large accumulations of these gut-associated organisms were more commonly found by confocal laser scanning microscopy in males than in females and in crowded than in singly reared leafhoppers. Ultrastructural evidence suggests that these SLOs may be horizontally transmitted between leafhoppers by contamination of the mouth parts with leafhopper excretions.

Spiroplasma eriocheiris sp. nov., associated with mortality in the Chinese mitten crab, Eriocheir sinensis.

A motile bacterium, designated strain TDA-040725-5(T), was isolated from the haemolymph of a Chinese mitten crab, Eriocheir sinensis, with tremor disease. Based on 16S rRNA gene sequence analysis, the strain was phylogenetically distinct from other spiroplasmas but was closely related to Spiroplasma mirum ATCC 29335(T). Cells of strain TDA-040725-5(T) were variable in length and shape, helical and motile, as determined by phase-contrast light microscopy. Examination by electron microscopy revealed wall-less cells delimited by a single membrane. The strain grew in M1D or R-2 liquid media at 20-40 °C, with optimum growth at 30 °C. Doubling time at the optimal temperature was 24 h. The strain catabolized glucose and hydrolysed arginine but did not hydrolyse urea. The DNA G+C content was 29.7±1 mol%. The genome size was ~1.4-1.6 Mbp. Serological analysis, performed using the deformation test, did not reveal any reciprocal titres ≥320, indicating that strain TDA-040725-5(T) had minimal cross-reactivity to strains of recognized species of the genus Spiroplasma. Based on this evidence, strain TDA-040725-5(T) ( = CCTCC M 207170(T)  = DSM 21848(T)) represents a novel species of the genus Spiroplasma, for which the name Spiroplasma eriocheiris sp. nov. is proposed, belonging to the novel Spiroplasma serological group XLIII.

Spiroplasmas and phytoplasmas: microbes associated with plant hosts.

This review will focus on two distinct genera, Spiroplasma and 'Candidatus Phytoplasma,' within the class Mollicutes (which also includes the genus Mycoplasma, a concern for animal-based cell culture). As members of the Mollicutes, both are cell wall-less microbes which have a characteristic small size (1-2 microM in diameter) and small genome size (530 Kb-2220 Kb). These two genera contain microbes which have a dual host cycle in which they can replicate in their leafhopper or psyllid insect vectors as well as in the sieve tubes of their plant hosts. Major distinctions between the two genera are that most spiroplasmas are cultivable in nutrient rich media, possess a very characteristic helical morphology, and are motile, while the phytoplasmas remain recalcitrant to cultivation attempts to date and exhibit a pleiomorphic or filamentous shape. This review article will provide a historical over view of their discovery, a brief review of taxonomical characteristics, diversity, host interactions (with a focus on plant hosts), phylogeny, and current detection and elimination techniques.

An Australian environmental survey reveals moderate Spiroplasma biodiversity: characterization of four new serogroups and a continental variant.

An environmental survey of tabanid host spiroplasma carriage was undertaken at 10 collection sites in Australia during February 1999. A total of 164 tabanid flies, representing 27 species, were collected and sustainable spiroplasma isolations were made from 48 of the flies. The morphology of the cultured spiroplasmas, as observed in M1D medium under dark-field microscopy, was typical of either (i) Apis group spiroplasmas (relatively thick cells (approximately 150 nm) with six or more turns) or (ii) chrysopicola-syrphidicola-TAAS-1 clade spiroplasmas (narrower, often much shorter cells) serologically related to Spiroplasma serogroup VIII. Repetitive serological analyses, involving successive rounds of dilution cloning and serological reevaluation, identified one serotype referable to the Spiroplasma serogroup VIII strain complex and five putative members of the Apis clade. Apis clade placement for these five groups was verified using 16S rRNA phylogenetic analyses. Among the Apis clade members, one serotype representing 11 isolates was identified as a geographic variant of Spiroplasma turonicum. Spiroplasma turonicum (Tab4C) was originally isolated from a tabanid Haematopoda sp. in France. The other 34 isolates represented four new serogroups (= putative species). The following strains are proposed as representatives of the new serogroups: strain GSU5478 (group XXXIX), strain GSU5490 (group XL), strain GSU5508 (group XLI), and strain GSU5603 (group XLII). In summary, six serogroups were observed from isolations originating from seven distinct sample sites in Australia. Surprisingly, the serotype with the greatest geographical range (five sites from 16 degrees 48.9'S to 35 degrees 40.0'S) and the greatest host diversity (nine species over three genera) was the geographic variant of S. turonicum, which had only been reported previously in France.

Complete Genome Sequence of Spiroplasma phoeniceum Strain P40T, a Plant Pathogen Isolated from Diseased Plants of Madagascar Periwinkle [Catharanthus roseus (L.) G. Don].

The phytopathogen Spiroplasma phoeniceum was isolated from diseased plants of Madagascar periwinkle [Catharanthus roseus (L.) G. Don]. Here, we report the nucleotide sequence of the 1,791,576-bp circular chromosome and three plasmids of strain P40T This information serves as a resource for comparative analyses of spiroplasmal adaptations to diverse ecological niches.

Convergent Evolution among Ruminant-Pathogenic Mycoplasma Involved Extensive Gene Content Changes.

Convergent evolution, a process by which organisms evolved independently to have similar traits, provides opportunities to understand adaptation. The bacterial genus Mycoplasma contains multiple species that evolved independently to become ruminant pathogens, which represents an interesting study system for investigating the process. In this work, we determined the genome sequences of 11 Entomoplasma/Mesoplasma species. This new data set, together with the other available Mollicutes genomes, provided comprehensive taxon sampling for inferring the gene content evolution that led to the emergence of Mycoplasma Mycoides cluster. Our results indicated that the most recent common ancestor (MRCA) of the Mycoides-Entomoplasmataceae clade lost ∼15% of the core genes when it diverged from the Spiroplasma Apis clade. After this initial wave of genome reduction, relatively few gene gains or losses were inferred until the emergence of the Mycoides cluster. Compared with those Entomoplasmataceae lineages that maintained the association with insects, the MRCA of the Mycoides cluster experienced a second wave of gene losses, as well as acquiring >100 novel genes through horizontal gene transfer. These gene acquisitions involved many with the Mycoplasma Hominis/Pneumoniae lineages as the putative donors, suggesting that gene exchanges among these vertebrate symbionts with distinct phylogenetic affiliations may be important in the emergence of the Mycoides cluster. These findings demonstrated that the gene content of bacterial genomes could be exceedingly dynamic, even for those symbionts with highly reduced genomes. Moreover, the emergence of novel pathogens may involve extensive remodeling of gene content, rather than acquisition of few virulence genes.

Complete Genome Sequence of Spiroplasma sp. TU-14.

Spiroplasma sp. TU-14 was isolated from a contaminated sample of Entomoplasma lucivorax PIPN-2T obtained from the International Organization for Mycoplasmology collection. Here, we report the complete genome sequence of this bacterium to facilitate the investigation of its biology and the comparative genomics among Spiroplasma spp.

Complete Genome Sequence of Spiroplasma sp. NBRC 100390.

Spiroplasma sp. NBRC 100390 was initially described as a duplicate of S. atrichopogonis GNAT3597T (=ATCC BAA-520T) but later found to be different in the 16S rDNA sequences. Here, we report the complete genome sequence of this bacterium to establish its identity and to facilitate future investigation.

Complete Genome Sequence of Spiroplasma citri Strain R8-A2T, Causal Agent of Stubborn Disease in Citrus Species.

Spiroplasma citri causes stubborn disease in Citrus spp. and diseases in other plants. Here, we report the nucleotide sequence of the 1,599,709-bp circular chromosome and two plasmids of S. citri strain R8-A2T This information will facilitate analyses to understand spiroplasmal pathogenicity and evolutionary adaptations to lifestyles in plants and arthropod hosts.

Spiroplasmas: evolutionary relationships and biodiversity.

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.

Complete Genome Sequence of Spiroplasma turonicum Tab4cT, a Bacterium Isolated from Horse Flies (Haematopota sp.).

Spiroplasma turonicum Tab4c(T) was isolated from a horse fly (Haematopota sp.; probably Haematopota pluvialis) collected at Champchevrier, Indre-et-Loire, Touraine, France, in 1991. Here, we report the complete genome sequence of this bacterium to facilitate the investigation of its biology and the comparative genomics among Spiroplasma spp.

Found and Lost: The Fates of Horizontally Acquired Genes in Arthropod-Symbiotic Spiroplasma.

Horizontal gene transfer (HGT) is an important mechanism that contributed to biological diversity, particularly in bacteria. Through acquisition of novel genes, the recipient cell may change its ecological preference and the process could promote speciation. In this study, we determined the complete genome sequence of two Spiroplasma species for comparative analyses and inferred the putative gene gains and losses. Although most Spiroplasma species are symbionts of terrestrial insects, Spiroplasma eriocheiris has evolved to be a lethal pathogen of freshwater crustaceans. We found that approximately 7% of the genes in this genome may have originated from HGT and these genes expanded the metabolic capacity of this organism. Through comparison with the closely related Spiroplasma atrichopogonis, as well as other more divergent lineages, our results indicated that these HGT events could be traced back to the most recent common ancestor of these two species. However, most of these horizontally acquired genes have been pseudogenized in S. atrichopogonis, suggesting that they did not contribute to the fitness of this lineage that maintained the association with terrestrial insects. Thus, accumulation of small deletions that disrupted these foreign genes was not countered by natural selection. On the other hand, the long-term survival of these horizontally acquired genes in the S. eriocheiris genome hinted that they might play a role in the ecological shift of this species. Finally, the implications of these findings and the conflicts among gene content, 16S rRNA gene sequencing, and serological typing, are discussed in light of defining bacterial species.

Complete Genome Sequence of Spiroplasma turonicum Strain Tab4cT, a Parasite of a Horse Fly, Haematopota sp. (Diptera: Tabanidae).

Spiroplasma turonicum was isolated from a Haematopota sp. fly in France. We report the nucleotide sequence of the circular chromosome of strain Tab4c(T). The genome information will facilitate evolutionary studies of spiroplasmas, including symbionts of insects and ticks and pathogens of plants, insects, crustaceans, and humans.

Complete Genome Sequence of Spiroplasma kunkelii Strain CR2-3x, Causal Agent of Corn Stunt Disease in Zea mays L.

Spiroplasma kunkelii causes corn stunt disease of Zea mays L. in the Americas. Here, we report the nucleotide sequence of the 1,463,926-bp circular chromosome and four plasmids of strain CR2-3x. This information will facilitate studies of Spiroplasma pathogenicity and evolutionary adaptations to transkingdom parasitism in plants and insect vectors.

Spiroplasmas: evolution, adaptation and diversity.

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.

Integration of Ethics across the Curriculum: From First Year through Senior Seminar.

The Fisher College of Science and Mathematics (FCSM) at Towson University (TU) has integrated authentic research experiences throughout the curriculum from first year STEM courses through advanced upper-level classes and independent research. Our observation is that training in both responsible conduct of research (RCR) and bioethics throughout the curriculum was an effective strategy to advance the cognitive and psychosocial development of the students. As students enter TU they generally lack the experience and tools to assess their own competence, to apply ethical debates, to investigate scientific topics from an ethical perspective, or to integrate ethics into final conclusions. Student behavior and development follow cognitive models such as described in the theories put forth by Piaget, Kohlberg, and Erikson, both for initial learning and for how concepts are understood and adopted. Three examples of this ethics training integration are described, including a cohort-based course for first year students in the STEM Residential Learning Community, a cohort-based course for community college students that are involved in an NIH-funded Bridges to the Baccalaureate program, and a senior seminar in Bioethics in the Molecular Biology, Biochemistry and Bioinformatics Program. All three focus on different aspects of RCR and bioethics training, providing opportunities for students to learn about the principles of effective decision-making, critical and analytical thinking, problem solving, and communication with increasing degrees of complexity as they move through the curriculum.

Absence of Spiroplasma or other bacterial 16s rRNA genes in brain tissue of hamsters with scrapie.

Spiroplasma spp. have been proposed to be the etiological agents of the transmissible spongiform encephalopathies (TSEs). In a blind study, a panel of 20 DNA samples was prepared from the brains of uninfected hamsters or hamsters infected with the 263K strain of scrapie. The brains of the infected hamsters contained > or =10(10) infectious doses/g. The coded panel was searched for bacterial 16S rRNA gene sequences, using primers selective for spiroplasma sequences, primers selective for mollicutes in general, and universal bacterial primers. After 35 PCR cycles, no samples were positive for spiroplasma or any other bacterial DNA, while control Spiroplasma mirum genomic DNA, spiked at 1% of the concentration required to account for the scrapie infectivity present, was readily detected. After 70 PCR cycles, nearly all samples yielded amplified products which were homologous to various bacterial 16S rRNA gene sequences, including those of frequent environmental contaminants. These sequences were seen in uninfected as well as infected samples. Because the concentration of scrapie infectivity was at a known high level, it is very unlikely that a bacterial infection at the same concentration could have escaped detection. We conclude that the infectious agent responsible for TSE disease cannot be a spiroplasma or any other eubacterial species.

Spiroplasma leucomae sp. nov., isolated in Poland from white satin moth (Leucoma salicis L.) larvae.

Spiroplasma sp. strain SMA(T), isolated in Poland from white satin moth larvae, Leucoma salicis L. (Lepidoptera: Lymantriidae), was serologically distinct from other Spiroplasma species, groups or subgroups. Dark-field microscopy of the cells revealed the classical helical shape and subsequent transmission electron microscopy revealed cells surrounded by only a single cell membrane (lacking a cell wall). Growth of strain SMA(T) occurred in M1D medium at 30 degrees C. Strain SMA(T) catabolized both glucose and arginine, but did not hydrolyse urea. The G+C content of the DNA was 24+/-1 mol% as determined by melting temperature analysis. Serological analysis revealed a very weak cross-reactivity (positive reaction only up to a 1 : 80 dilution) with two Spiroplasma strains, 277F (Spiroplasma sp. group I-4) and LB-12 (Spiroplasma sp. group I-5). Strain SMA(T) (=ATCC BAA-521T=NBRC 100392T) is designated the type strain of a novel species, Spiroplasma leucomae sp. nov. (class Mollicutes: order Entomoplasmatales: family Spiroplasmataceae).