Detection of a novel intracellular microbiome hosted in arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) are important members of the plant microbiome. They are obligate biotrophs that colonize the roots of most land plants and enhance host nutrient acquisition. Many AMF themselves harbor endobacteria in their hyphae and spores. Two types of endobacteria are known in Glomeromycota: rod-shaped Gram-negative Candidatus Glomeribacter gigasporarum, CaGg, limited in distribution to members of the Gigasporaceae family, and coccoid Mollicutes-related endobacteria, Mre, widely distributed across different lineages of AMF. The goal of the present study is to investigate the patterns of distribution and coexistence of the two endosymbionts, CaGg and Mre, in spore samples of several strains of Gigaspora margarita. Based on previous observations, we hypothesized that some AMF could host populations of both endobacteria. To test this hypothesis, we performed an extensive investigation of both endosymbionts in G. margarita spores sampled from Cameroonian soils as well as in the Japanese G. margarita MAFF520054 isolate using different approaches (molecular phylotyping, electron microscopy, fluorescence in situ hybridization and quantitative real-time PCR). We found that a single AMF host can harbour both types of endobacteria, with Mre population being more abundant, variable and prone to recombination than the CaGg one. Both endosymbionts seem to retain their genetic and lifestyle peculiarities regardless of whether they colonize the host alone or together. These findings show for the first time that fungi support an intracellular bacterial microbiome, in which distinct types of endobacteria coexist in a single cell.

"Candidatus Bacilloplasma," a novel lineage of Mollicutes associated with the hindgut wall of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda).

Pointed, rod-shaped bacteria colonizing the cuticular surface of the hindgut of the terrestrial isopod crustacean Porcellio scaber (Crustacea: Isopoda) were investigated by comparative 16S rRNA gene sequence analysis and electron microscopy. The results of phylogenetic analysis, and the absence of a cell wall, affiliated these bacteria with the class Mollicutes, within which they represent a novel and deeply branched lineage, sharing less than 82.6% sequence similarity to known Mollicutes. The lineage has been positioned as a sister group to the clade comprising the Spiroplasma group, the Mycoplasma pneumoniae group, and the Mycoplasma hominis group. The specific signature sequence was identified and used as a probe in in situ hybridization, which confirmed that the retrieved sequences originate from the attached rod-shaped bacteria from the hindgut of P. scaber and made it possible to detect these bacteria in their natural environment. Scanning and transmission electron microscopy revealed a spherically shaped structure at the tapered end of the rod-shaped bacteria, enabling their specific and exclusive attachment to the tip of the cuticular spines on the inner surface of the gut. Specific adaptation to the gut environment, as well as phylogenetic positioning, indicate the long-term association and probable coevolution of the bacteria and the host. Taking into account their pointed, rod-shaped morphology and their phylogenetic position, the name "Candidatus Bacilloplasma" has been proposed for this new lineage of bacteria specifically associated with the gut surface of P. scaber.

Cytoskeleton of mollicutes.

Mollicutes are a class of bacteria that lack a peptidoglycan layer but have various cell shapes. They perform chromosome segregation and binary fission in a well-organized manner. Especially, species with polarized cell morphology duplicate their membrane protrusion at a position adjacent to the original one and move the new protrusion laterally to the opposite end pole before cell division. The featured various cell shapes of Mollicutes are supported by cytoskeletal structures composed of proteins. Recent progress in the study of cytoskeletons of walled bacteria and genome sequencing has revealed that the cytoskeletons of Mollicutes are not common with those of other bacteria. Mollicutes have special cytoskeletal proteins and structures that are sometimes not shared even by other mollicute species.

[Classification of mollicutes]. / Klasyfikatsiia molikutiv.

The paper deals, allowing for the newest achievements in the study of mollicute biology, with main principles and characteristics which would to be used in classification of these microorganisms. The present classification of mollicutes is based on the study of classical features of cells (morphology and ultrastructure of cells, morphology of colonies, requirements to nutrition components, biochemical and serological properties) and molecular biological properties (composition of cell proteins, properties of nucleic acids, and especially, ribosomal operons and ribosomal RNA), living conditions, ecological niches, etc. The author hopes that the work will help biologists, physicians, veterinaries, phytopathologists and other persons interested in the study of mollicutes to orient themselves in determining their systematic position.

Prokaryote infections in the New Zealand scallops Pecten novaezelandiae and Chlamys delicatula.

Four intracellular prokaryotes are reported from the scallops Pecten novaezelandiae Reeve, 1853 and Chlamys delicatula Hutton, 1873. Elongated (1025 x 110 nm), irregular (390 x 200 nm), or toroidal (410 x 200 nm) mollicute-like organisms (M-LOs) occurred free in the cytoplasm in the digestive diverticular epithelial cells of both scallop species. Those in P. novaezelandiae bore osmiophilic blebs that sometimes connected the organisms together, and some had a rod-like protrusion, both of which resemble the blebs and tip structures of pathogenic mycoplasmas. The M-LOs in C. delicatula had a slightly denser core than periphery. Round M-LOs, 335 x 170 nm, occurred free in the cytoplasm of agranular haemocytes in P. novaezelandiae, without apparent harm to the host cell. In P. novaezelandiae, 2 types of highly prevalent (95 to 100%) basophilic inclusions in the branchial epithelium contained Rickettsia-like organisms (R-LOs). Type 1 inclusions occurred in moderately hypertrophied, intensely basophilic cells, 8 to 10 microm in diameter, containing elongate intracellular R-LOs, 2000 x 500 nm. Type 2 inclusions were elongated and moderately basophilic in markedly hypertrophic branchial epithelial cells, 50 x 20 microm in diameter, containing intracellular organisms 500 x 200 nm in diameter. The possible roles of these organisms in pathogenesis is discussed.

Application of immunoelectron microscopy techniques in the diagnosis of phytoplasma diseases.

An immunoelectron microscopy technique was applied to label Chrysanthemum leuchanthemum phytoplasma in infected leaf tissues of Chrysanthemum leuchanthemum L. and Catharanthus roseus L. plants. Specific monoclonal antibodies at different dilutions and secondary antimouse antibody conjugated with colloidal gold particles of different sizes were used. The monoclonal antibodies demonstrated their specificity against the antigen; immunocytological methods permitted the precise localization and identification of phytoplasmas in thin sections from infected tissues.

Mollicutes-wall-less bacteria with internal cytoskeletons.

The structure and motility of the Mollicutes (Spiroplasma, Mycoplasma, and Acholeplasma) are briefly reviewed. The data are presented from the perspective of prokaryotic and eukaryotic motors, cytoskeletons, and cell motility. The Mollicutes are eubacteria derived from Clostridia by regressive evolution and genome reduction to produce the smallest and simplest free-living and self-replicating cells. Structurally, the Mollicutes are characterized by a complete lack of a cell wall and the presence of an internal cytoskeleton. Spiroplasma, which are helical cells with a flat, ribbon-like cytoskeleton, are amenable to structural and geometrical analysis. Motility and shape changes can be explained and modeled by the cytoskeleton acting as a linear motor.

Serological and molecular characterization of Mesoplasma seiffertii strains isolated from hematophagous dipterans in France.

Three strains of nonhelical mollicutes previously isolated in France from two different mosquitoes and one tabanid fly were designated strains Ar 2328 (isolated from Aedes detritus), Ar 2392 (isolated from Aedes caspius), and CP 13 (isolated from Chrysops pictus). All of these strains exhibited properties of the genus Mesoplasma, a recently described genus of non-sterol-requiring mollicutes isolated from plants and insects. The results of metabolism inhibition and growth inhibition tests revealed that these strains and Mesoplasma entomophilum TAC or Mesoplasma florum L1 were not serologically related, but all three dipteran strains reacted strongly with Mesoplasma seiffertii F7T (T = type strain) antibodies. Using metabolism inhibition and growth inhibition tests, we found that the dipteran strains were related to each other and to strain F7T but were not identical. We also found that they were able to multiply and persist in the central nervous systems of suckling mice inoculated intracerebrally, a property that makes their use as biological control agents for pest dipterans inadvisable. Scanning electron microscopy revealed marked differences in the morphologies of the colonies of the different strains on SP4 solid medium. The levels of DNA-DNA homology for strains Ar 2328, Ar 2392, CP 13, and F7T were more than 70%, indicating that these strains are closely related members of the same species, M. seiffertii. In addition, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that each strain produced about 40 protein bands. This technique also revealed differences between strains. Using the coefficient of Smeath-Jacquart, we constructed a dendrogram that allowed us to estimate of the levels of relatedness of these four strains. The results which we obtained were confirmed by two-dimensional protein electrophoresis results.

Acute bilateral optic disc neovascularization.

PURPOSE: A case of bilateral acute neovascularization of the optic disc and peripapillary epiretina of unknown etiology in a previously healthy 26-year-old woman is discussed. METHODS: The clinical course of the disease, investigation, and treatment are presented in detail. RESULTS: Histopathologic examination of epiretinal tissue removed from the second eye at vitrectomy provided evidence of inflammatory disease and suggested a possible etiology. CONCLUSION: The cause of this patient's disease remains uncertain. Known causes of this disease were excluded as far as possible. Histopathologic examination of the epiretinal membrane from the second eye demonstrated an unusually increased inflammatory response and multiple intracytoplasmic inclusion bodies in neutrophils resembling mollicute-like organisms.

Isolation and characterization of full-length chromosomes from non-culturable plant-pathogenic Mycoplasma-like organisms.

We describe the isolation and characterization of full-length chromosomes from non-culturable plant-pathogenic, mycoplasma-like organisms (MLOs). MLO chromosomes are circular and their sizes (640 to 1185 kbp) are heterogeneous. Divergence in the range of chromosome sizes is apparent between MLOs in the two major MLO disease groups, and chromosome size polymorphism occurs among some related agents. MLO chromosome sizes overlap those of culturable mycoplasmas; consequently, small genome size alone cannot explain MLO non-culturability. Hybridization with cloned MLO-specific chromosomal and 16S rRNA probes detected two separate chromosomes in some MLO 'type' strains. Large DNA molecules that appear to be MLO megaplasmids were also demonstrated. The ability to characterize full-length chromosomes from virtually any non-culturable prokaryote should greatly facilitate the molecular and genetic analysis of these difficult bacteria.