Comparative proteomic characteristic of mycoplasmas (Mollicutes)

Using one- and two-dimensional gel electrophoresis, various types of mass-spectrometry, and combinations of these two methods, saturating identification of mycoplasm Acholeplasma laidlawii proteome and study of proteins carrying posttranslational modifications were performed. We compared our data with earlier identified proteome of Mycoplasma gallisepticum. It was shown that M. gallisepticum and A. laidlawii expressed 61 and 58% of the proteins of the annotated open reading frames, respectively. All subunits of DNA-polymerase III were identified for A. laidlawii during our study, which indicates that our techniques enable detection of single copies of mollicutes proteins per cell. Metabolic pathways of respective mycoplasmas is compared in this research.     

Activity of dihydrofolate reductase in the mollicutesAcholeplasma laidlawii andMycoplasma gallisepticum and their susceptibility to antifolates

Mycoplasma gallisepticum andAcholeplasma laidlawii were found to possess dihydrofolate reductases exhibiting similar specific activities and kinetics, with values in the range of those reported for other microorganisms. The apparent Km values for dihydrofolate in enzymes ofM. gallisepticum andA. laidlawii are 7.95±0.13 and 7.50±0.11 M, and for NADPNH 8.46±0.25 and 9.32±0.18 M, respectively.M. gallisepticum is 3300-fold more resistant to methotrexate than isA. laidlawii; concentrations causing 50% inhibition were 200.00 and 0.06 M, respectively. This is in contrast to almost the same sensitivity to that drug exhibited by the dihydrofolate reductases of both microorganisms.M. gallisepticum is also 3600-fold more resistant to trimethoprim than isA. laidlawii, and the concentrations for 50% inhibition of growth were 1800.0 and 0.5 M, respectively. The high resistance was found to be due partially to a 130-fold lower affinity of the target enzyme for this antifolate, but another mechanism, presumably impaired transport, must also be involved. This is the first report of dihydrofolate reductase activity in Mollicutes.     

Isolation and Characterization of Mycoplasma and Acholeplasma from Apparently Healthy and Diseased (Infectious Sinusitis) Turkeys

Investigation of 136 turkeys (24 manifesting infra-orbital sinusitis, 112 apparently healthy) resulted in isolation of 79 strains of Mycoplasma and 4 of Acholeplasma. By the disc growth inhibition test with 16 reference antisera of avian serogroups, 55 strains were identified serologically and 28 remained unidentified. Thirteen strains of Mycoplasma gallisepticum, 1 of M. meleagridis, and 2 of Acholeplasma laidlawii were isolated from turkey sinusitis whereas serogroups C (2), D (19), F (8), M. meleagridis (4), M. anatis (4), A. laidlawii (2), and 28 unidentified strains were isolated from apparently healthy turkeys. Three patterns were recognized on the basis of glucose, maltose, and sucrose, fermentation. The most frequent, pattern I, included 13 M. gallisepticum strains whereas 5 M. meleagridis strains belonged to fermentation pattern III. Isolates were also studied for reduction of tetrazolium, methylene blue, potassium tellurite, resistance to methylene blue and sodium taurocholate, and production of arginine deiminase and “film and spots.” Inoculation of selected isolates into developing chick embryos revealed that 2 A. laidlawii strains were nonpathogenic and 13 M. gallisepticum, 1 serogroup D and 2 serogroup F strains were pathogenic, causing 50–100% mortality. In vitro antibiotic disc sensitivity tests indicated that rovamycin (solubilized spiramycin) may be recommended for turkey mycoplasmosis. Isolation of 2 A. laidlawii strains from turkey sinusitis and 4 M. anatis strains from apparently healthy turkeys appears interesting.     

Lipid and protein membrane components associated with cholesterol uptake by mycoplasmas

Membranes of Mycoplasma species take up 2–4 times more exogenous cholesterol than membranes of Acholeplasma species. To test whether the lower cholesterol uptake capacity of Acholeplasma is due to the high glycolipid content of their membranes, the phospholipids of Acholeplasma laidlawii and Mycoplasma capricolum membranes were hydrolyzed by phospholipase A². Digestion removed about 30% of the polar lipids of A. laidlawii, leaving the glycolipids and phospholglycolipids intact, and about 70% of the polar lipids of M. capricolum, the residue consisting mostly of sphingomyelin. Cholesterol uptake by the treated membranes from phosphatidylcholine/cholesterol vesicles decreased in rough proportion to the amount of polar lipid removed, indicating that the glycolipids in A. laidlawii membranes can participate in cholesterol uptake.Trypsin digestion of growing cells and isolated membranes of M. capricolum decreased cholesterol uptake by about one-half. Similar treatment of A. laidlawii cells and membranes had no effect on cholesterol uptake. These findings suggest the existence of protease-sensitive receptors on the cell surface of M. capricolum responsible for tighter contact with the cholesterol/phosphatidylcholine vesicles. It is proposed that the ability of Mycoplasma species to take up large quantities of exogenous cholesterol and phospholipids depends on the presence of protein receptors for cholesterol donors, receptors which are absent in Acholeplasma species.     

Effect of acriflavine on ultraviolet inactivation of Acholeplasma laidlawii

An increased sensitivity to inactivation was observed when ultraviolet light-irradiated Acholeplasma laidlawiiAn increase sensitivity to inactivation was observed when ultraviolet light-irradiated Acholeplasma laidlawii cells were plated on medium containing either acriflavine or chloramphenicol. Chloramphenicol reduced liquid holding recovery (dark repair) to about 10 percent of that in untreated irradiated cells. In acriflavine treated cells no dark repair could be observed and there was a progressive degradation of cell DNA during holding. While the primary effect of acriflavine may be to inhibit excision repair, since ultraviolet-irradiated Mycoplasma gallisepticum (cells which lack an excision repair mechanism) show a slight increase in inactivation when plated on medium containing acriflavine, the dye must also have some other effects on ultraviolet repair processes. Acriflavine treatment of A. laidlawii cells before ultraviolet irradiation has a protective effect, as seen by an increased cell survival.     

Immunological Analysis of Mycoplasma Membranes

The antigens responsible for the production of antibodies to Mycoplasma laidlawii and M. gallisepticum causing growth and metabolic inhibition of these organisms were localized in the cell membrane. Various membrane fractions were tested for serological activity. Membrane lipids were completely or almost completely inactive, whereas several preparations of defatted membrane proteins retained some serological activity, shown by their ability to stimulate metabolic inhibition antibody in rabbits and to adsorb metabolic inhibition antibody and form precipitation lines with an antiserum to the membrane. When the membranes were heated to 65 C for 1 hr, they virtually lost their ability to adsorb metabolic inhibition antibody, which suggests that the antigenic determinants are proteins. Serological activity was retained in reaggregated membranes obtained by dialysis against Mg²⁺ of membranes solubilized in sodium dodecyl sulfate. The amount of solubilized membrane protein and lipid incorporated into the reaggregated membranes could be regulated by varying the Mg²⁺ concentration. As the serological tests indicated that the various membrane antigens were selectively incorporated into the different reaggregated membranes, the use of controlled reaggregation of solubilized membranes is suggested as a new tool for the fractionation and antigenic analysis of membrane proteins.     

Biological Enrichment of Mycoplasma Agents by Cocultivation with Permissive Cell Cultures

In this study, we describe our results on the evaluation of the ability of different permissive mammalian cell lines to support the biological enrichment of mycoplasma species known to be bacterial contaminants of cell substrates. The study showed that this approach is able to significantly improve the efficiency of mycoplasma detection based on nucleic acid testing or biochemical technologies (e.g., MycoAlert mycoplasma detection). Of 10 different cell lines (Vero, MDBK, HEK-293, Hep-G2, CV-1, EBTr, WI-38, R9ab, MDCK, and High Five) used in the study, only MDCK cell culture was found to support the efficient growth of all the tested mycoplasmas (Mycoplasma arginini, M. bovis, M. fermentans, M. gallinaceum, M. gallisepticum, M. synoviae, M. hominis, M. hyorhinis, M. orale, M. salivarium, and Acholeplasma laidlawii) known to be most frequently associated with contamination of cell substrates and cell lines in research laboratories or manufacturing facilities. The infection of MDCK cells with serial dilutions of each mycoplasma species demonstrated that these common cell line contaminants can be detected reliably after 7-day enrichment in MDCK cell culture at contamination levels of 0.05 to 0.25 CFU/ml. The High Five insect cell line was also found to be able to support the efficient growth of most mycoplasma species tested, except for M. hyorhinis strain DBS1050. However, mycoplasma growth in insect cell culture was demonstrated to be temperature dependent, and the most efficient growth was observed when the incubation temperature was increased from 28°C to between 35 and 37°C. We believe that this type of mycoplasma enrichment is one of the most promising approaches for improving the purity and safety testing of cell substrates and other cell-derived biologics and pharmaceuticals.     

Proceedings: Freeze-drying of mycoplasma

The study shows the effect of protectants in the freeze-drying of Mycoplasma gallisepticum and M. synoviae, and the effect of the cultivation period required to prepare the bacterial suspension, on the survival of M. gallisepticum.Sucrose as a protectant is more effective in both Mycoplasmas. In detail, 12.5% sucrose produced the most satisfactory survival of M. gallisepticum by comparison with other protectants, and there was an optimal concentration. On the other hand, in M. synoviae higher survival than 90 % was obtained in 7.5%–17.5% sucrose.Survival of M. gallisepticum after freeze-dyring was affected by the growth stage of the organism at the time of preparation of the bacterial suspension.     

Development of a Site-Directed Integration Plasmid for Heterologous Gene Expression in Mycoplasma gallisepticum

Deciphering the molecular basis of the interactions between the parasite Mycoplasma gallisepticum and its avian hosts suffers from the lack of genetic tools available for the pathogen. In the absence of well established methods for targeted disruption of relevant M. gallisepticum genes, we started to develop suicide vectors and equipped them with a short fragment of M. gallisepticum origin or replication (oriC _MG). We failed to create a disruption vector, although by adding a further short fragment of the M. gallisepticum tufB upstream region we created a “Trojan horse” plasmid. This is fully integrated into the genomic DNA of M. gallisepticum, always at the same site, oriC _MG, and is able to carry and express any gene of interest in the genetic background of M. gallisepticum. Successful expression of a heterologous gene was shown with the lacZ gene of E. coli. When used for gene complementation or expression of hybrid genes in M. gallisepticum, a site-specific combined integration/expression vector constitutes an improvement on randomly integrating transposons, which might have unexpected effects on the expression of chromosomal genes.     

Identification of Mycoplasma Binding Proteins Utilizing A 100 Kilodalton Lung Fibroblast Receptor

Abstract

A 100 kilodalton glycoprotein receptor for Mycoplasma pneumoniae has been isolated from MRC-5 human lung fibroblasts. This receptor, as well as antireceptor serum, were both capable of inhibiting the attachment of ¹⁴C-labelled M. pneumoniae to MRC-5 fibroblasts. The receptor was also capable of inhibiting the attachment of C-labelled M. gallisepticum and M. genitalium, but not M. pulmonis, to MRC-5 fibroblasts. This indicates that a common sequence may exist in these binding proteins of M. pneumoniae, M. genitalium, and M. gallisepticum, This receptor and anti-receptor serum were utilized to probe M. pneumoniae, M. genitalium, and M. gallisepticum for their corresponding binding proteins. A 32 kilodalton protein in M. pneumoniae, a 90 kilodalton protein in M. genitalium and a 139 kilodalton protein in M. gallisepticum were recognized.     

Diverse Wild Bird Host Range of Mycoplasma gallisepticum in Eastern North America

Emerging infectious diseases often result from pathogens jumping to novel hosts. Identifying possibilities and constraints on host transfer is therefore an important facet of research in disease ecology. Host transfers can be studied for the bacterium Mycoplasma gallisepticum, predominantly a pathogen of poultry until its 1994 appearance and subsequent epidemic spread in a wild songbird, the house finch Haemorhous mexicanus and some other wild birds. We screened a broad range of potential host species for evidence of infection by M. gallisepticum in order to answer 3 questions: (1) is there a host phylogenetic constraint on the likelihood of host infection (house finches compared to other bird species); (2) does opportunity for close proximity (visiting bird feeders) increase the likelihood of a potential host being infected; and (3) is there seasonal variation in opportunity for host jumping (winter resident versus summer resident species). We tested for pathogen exposure both by using PCR to test for the presence of M. gallisepticum DNA and by rapid plate agglutination to test for the presence of antibodies. We examined 1,941 individual birds of 53 species from 19 avian families. In 27 species (15 families) there was evidence for exposure with M. gallisepticum although conjunctivitis was very rare in non-finches. There was no difference in detection rate between summer and winter residents, nor between feeder birds and species that do not come to feeders. Evidence of M. gallisepticum infection was found in all species for which at least 20 individuals had been sampled. Combining the present results with those of previous studies shows that a diverse range of wild bird species may carry or have been exposed to M. gallisepticum in the USA as well as in Europe and Asia.     

Preparation of reference strains for validation and comparison of mycoplasma testing methods

Aims: To optimize growth conditions for preparation of stocks of mycoplasma reference strains to obtain highly viable and disperse samples with low ratios of genomic copy (GC) number to that of colony forming units (CFU). These stocks are required for assessment of relative limits of detection (LOD) of alternative nucleic acid testing (NAT)‐based methods in comparison to the conventional microbiological methods. Methods and Results: A kinetics study was used to assess the changes in ratios between the numbers of GC and CFU at different growth phases of six different mycoplasma cultures Acholeplasma laidlawii, Mycoplasma gallisepticum, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma orale and Mycoplasma pneumoniae. All tested mycoplasmas demonstrated low GC/CFU ratios (≤10) within the log and early stationary growth phases. A significant increase in GC/CFU ratios was observed at the very late stationary and death phases, when the titre of cultures has declined. Similar patterns of GC/CFU profiles were observed for A. laidlawii and Myc. gallisepticum co‐cultured with suspension of Chinese hamster ovary (CHO) cells. Conclusions: Tested mycoplasma strains harvested at the exponential‐early stationary phases of growth demonstrated the lowest GC/CFU ratios and low propensity to form filamentous structures or aggregates under proposed conditions and can be used for the preparation of a mycoplasma reference panel for methods comparability study. Significance and Impact of the Study: This study shows that the preparation and use of viable mycoplasma reference strains with low CG/CFU ratios is the most reliable way to adequately evaluate the LOD of alternative NAT‐based mycoplasma testing methods.     

Analysis of membrane fractions from Mycoplasma gallisepticum

Membrane fractions have been isolated from Mycoplasma gallisepticum following a procedure derived from that described by Maniloff, J. and Quinlan, D.C. (J. Bacteriol. (1974) 120, 495–501). A light fraction F₁ was obtained which contained structures resembling the bleb-infrableb apparatus characteristic of M. gallisepticum. It was enriched in DNA and had an electrophoretic profile different from that of unfractionated membranes. Cholesterol-to-phospholipid ratios higher than two and elevated values of the ratio of saturated to unsaturated fatty acids were other characteristics of this fraction. The two other fractions isolated (F_II and F_IV) also differed from intact membranes by their cholesterol and phospholipid content as well as by their saturation ratios. The membrane fluidity of F_II and F_IV, estimated by fluorescence polarization, was similar to that of unfractionated membranes while a slight but significant difference was recorded for the light fraction. Possible relationships between the lateral heterogeneity of the M. gallisepticum membrane and the obtainment of fractions are discussed.     

Spatial Variation in an Avian Host Community: Implications for Disease Dynamics

Because many pathogens can infect multiple host species within a community, disease dynamics in a focal host species can be affected by the composition of the host community. We examine the extent to which spatial variation in species’ abundances in an avian host community may contribute to geographically varying prevalence of a recently emerged wildlife pathogen. Mycoplasma gallisepticum is a pathogen novel to songbirds that has caused substantial mortality in house finches (Carpodacus mexicanus) in eastern North America. Though the house finch is the primary host species for M. gallisepticum, the American goldfinch (Spinus tristis) and northern cardinal (Cardinalis cardinalis) are alternate hosts, and laboratory experiments have demonstrated M. gallisepticum transmission between house finches and goldfinches. Still unknown is the real world impact on disease dynamics of variation in abundances of the three hosts. We analyzed data from winter-long bird and disease surveys in the northeastern United States. We found that higher disease prevalence in house finches was associated with higher numbers of northern cardinals and American goldfinches, although only the effect of cardinal abundance was statistically significant. Nevertheless, our results indicate that spatial variation in bird communities has the potential to cause geographic variation in disease prevalence in house finches.     

Response of Black-Capped Chickadees to House Finch Mycoplasma gallisepticum

Tests for the presence of pathogen DNA or antibodies are routinely used to survey for current or past infections. In diseases that emerge following a host jump estimates of infection rate might be under- or overestimated. We here examine whether observed rates of infection are biased for a non-focal host species in a model system. The bacterium Mycoplasma gallisepticum is a widespread pathogen in house finches (Haemorhous mexicanus), a fringillid finch, but an unknown proportion of individuals of other songbird species are also infected. Our goal is to determine the extent to which detection of M. gallisepticum DNA or antibodies against the bacteria in a non-fringillid bird species is over- or underestimated using black-capped chickadees Poecile atricapillus, a species in which antibodies against M. gallisepticum are frequently detected in free-living individuals. After keeping black-capped chickadees in captivity for 12 weeks, during which period the birds remained negative for M. gallisepticum, four were inoculated with M. gallisepticum and four were sham inoculated in both eyes to serve as negative controls. Simultaneously we inoculated six house finches with the same isolate of M. gallisepticum as a positive control. All inoculated birds of both species developed infections detectable by qPCR in the conjunctiva. For the 6 weeks following inoculation we detected antibodies in all M. gallisepticum-inoculated house finches but in only three of the four M. gallisepticum-inoculated black-capped chickadees. All house finches developed severe eye lesions but none of the black-capped chickadees did. Modeling the Rapid Plate Agglutination test results of black-capped chickadees shows that the rate of false-positive tests would be not more than 3.2%, while the estimated rate of false negatives is 55%. We conclude that the proportion of wild-caught individuals in which we detect M. gallisepticum-specific antibodies using Rapid Plate Agglutination is, if anything, substantially underestimated.     

Reduction of Hydrogen Peroxide Accumulation and Toxicity by a Catalase from Mycoplasma iowae

Mycoplasma iowae is a well-established avian pathogen that can infect and damage many sites throughout the body. One potential mediator of cellular damage by mycoplasmas is the production of H₂O₂ via a glycerol catabolic pathway whose genes are widespread amongst many mycoplasma species. Previous sequencing of M. iowae serovar I strain 695 revealed the presence of not only genes for H₂O₂ production through glycerol catabolism but also the first documented mycoplasma gene for catalase, which degrades H₂O₂. To test the activity of M. iowae catalase in degrading H₂O₂, we studied catalase activity and H₂O₂ accumulation by both M. iowae serovar K strain DK-CPA, whose genome we sequenced, and strains of the H₂O₂-producing species Mycoplasma gallisepticum engineered to produce M. iowae catalase by transformation with the M. iowae putative catalase gene, katE. H₂O₂-mediated virulence by M. iowae serovar K and catalase-producing M. gallisepticum transformants were also analyzed using a Caenorhabditis elegans toxicity assay, which has never previously been used in conjunction with mycoplasmas. We found that M. iowae katE encodes an active catalase that, when expressed in M. gallisepticum, reduces both the amount of H₂O₂ produced and the amount of damage to C. elegans in the presence of glycerol. Therefore, the correlation between the presence of glycerol catabolism genes and the use of H₂O₂ as a virulence factor by mycoplasmas might not be absolute.     

Influence of FtsZ proteins from some mycoplasma species on the division process in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

FtsZ is a highly conserved protein that performs one of the key roles in cell division of most prokaryotes. At the present time it is believed that the main role of FtsZ in the cells of the microorganisms studied (Escherichia coli, Bacillus subtilis, etc.) is to control the process of cell wall synthesis at the site of future septum. In this regard, the presence of FtsZ in the cells of mycoplasmas – bacteria that lack the cell wall, looks intriguing. In the present work we investigated the influence of FtsZ proteins of three mycoplasmas – Mycoplasma hominis, Mycoplasma gallisepticum and Acholeplasma laidlawii – on the cytokinesis of E. coli. FtsZ proteins were able to interact with the E. coli division machinery, including inhibiting the cytokinesis process, while demonstrating various patterns of distribution in the cell. The data obtained support the hypothesis that FtsZ plays a significant role in the division of mycoplasma cells.     

Cholesterol-phosphatidylcholine dispersions as donors of cholesterol to Mycoplasma membranes

Growing cells of sterol-requiring Mycoplasma hominis and sterol non-requiring Acholeplasma laidlawii were used to test the ability of cholesterol-dipalmitoyl phosphatidylcholine dispersions to serve as cholesterol donors to these organisms. Dispersions with high cholesterol to phosphatidylcholine ratios were more effective than dispersions with low cholesterol to phosphatidylcholine ratios in donating cholesterol to the membranes of both mycoplasmas and in promoting growth of the sterol-requiring species. M. hominis took up almost three times as much cholesterol as did A. laidlawii. In addition, significant quantities of the phosphatidylcholine component of the dispersions were found to be associated with M. hominis membranes as against none in the A. laidlawii membrane preparations. In all cases, the percentage of cholesterol taken up by M. hominis from the dispersions exceeded that of phosphatidylcholine by a factor of 3–5. These results were interpreted to suggest that all the cholesterol taken up by A. laidlawii is transferred from the dispersion to the membranes by a process which involves only a transient contact between the organisms and the lipid dispersions, whereas a certain amount of the cholesterol taken up by M. hominis may also be derived from lipid dispersions adhering to or fusing with the cell membranes.     

Cytoskeletal Asymmetrical Dumbbell Structure of a Gliding Mycoplasma,Mycoplasma gallisepticum,Revealed by Negative-Staining Electron Microscopy

Several mycoplasma species feature a membrane protrusion at a cell pole, and unknown mechanisms provide gliding motility in the direction of the pole defined by the protrusion. Mycoplasma gallisepticum, an avian pathogen, is known to form a membrane protrusion composed of bleb and infrableb and to glide. Here, we analyzed the gliding motility of M. gallisepticum cells in detail. They glided in the direction of the bleb at an average speed of 0.4 μm/s and remained attached around the bleb to a glass surface, suggesting that the gliding mechanism is similar to that of a related species, Mycoplasma pneumoniae. Next, to elucidate the cytoskeletal structure of M. gallisepticum, we stripped the envelopes by treatment with Triton X-100 under various conditions and observed the remaining structure by negative-staining transmission electron microscopy. A unique cytoskeletal structure, about 300 nm long and 100 nm wide, was found in the bleb and infrableb. The structure, resembling an asymmetrical dumbbell, is composed of five major parts from the distal end: a cap, a small oval, a rod, a large oval, and a bowl. Sonication likely divided the asymmetrical dumbbell into a core and other structures. The cytoskeletal structures of M. gallisepticum were compared with those of M. pneumoniae in detail, and the possible protein components of these structures were considered.Mycoplasmas are commensal and occasionally pathogenic bacteria that lack a peptidoglycan layer (50). Several species feature a membrane protrusion at a pole; for Mycoplasma mobile, this protrusion is called the head, and for Mycoplasma pneumoniae, it is called the attachment organelle (25, 34-37, 52, 54, 58). These species bind to solid surfaces, such as glass and animal cell surfaces, and exhibit gliding motility in the direction of the protrusion (34-37). This motility is believed to be essential for the mycoplasmas'' pathogenicity (4, 22, 27, 36). Recently, the proteins directly involved in the gliding mechanisms of mycoplasmas were identified and were found to have no similarities to those of known motility systems, including bacterial flagellum, pilus, and slime motility systems (25, 34-37).Mycoplasma gallisepticum is an avian pathogen that causes serious damage to the production of eggs for human consumption (50). The cells are pear-shaped and have a membrane protrusion, consisting of the so-called bleb and infrableb (29), and gliding motility (8, 14, 22). Their putative cytoskeletal structures may maintain this characteristic morphology because M. gallisepticum, like other mycoplasma species, does not have a cell wall (50). In sectioning electron microscopy (EM) studies of M. gallisepticum, an intracellular electron-dense structure in the bleb and infrableb was observed, suggesting the existence of a cytoskeletal structure (7, 24, 29, 37, 58). Recently, the existence of such a structure has been confirmed by scanning EM of the structure remaining after Triton X-100 extraction (13), although the details are still unclear.A human pathogen, M. pneumoniae, has a rod-shaped cytoskeletal structure in the attachment organelle (9, 15, 16, 31, 37, 57). M. gallisepticum is related to M. pneumoniae (63, 64), as represented by 90.3% identity between the 16S rRNA sequences, and it has some open reading frames (ORFs) homologous to the component proteins of the cytoskeletal structures of M. pneumoniae (6, 17, 48). Therefore, the cytoskeletal structures of M. gallisepticum are expected to be similar to those of M. pneumoniae, as scanning EM images also suggest (13).The fastest-gliding species, M. mobile, is more distantly related to M. gallisepticum; it has novel cytoskeletal structures that have been analyzed through negative-staining transmission EM after extraction by Triton X-100 with image averaging (45). This method of transmission EM following Triton X-100 extraction clearly showed a cytoskeletal “jellyfish” structure. In this structure, a solid oval “bell,” about 235 nm wide and 155 nm long, is filled with a 12-nm hexagonal lattice. Connected to this bell structure are dozens of flexible “tentacles” that are covered with particles 20 nm in diameter at intervals of about 30 nm. The particles appear to have 180° rotational symmetry and a dimple at the center. The involvement of this cytoskeletal structure in the gliding mechanism was suggested by its cellular localization and by analyses of mutants lacking proteins essential for gliding.In the present study, we applied this method to M. gallisepticum and analyzed its unique cytoskeletal structure, and we then compared it with that of M. pneumoniae.     

Proteomic characterization of <Emphasis Type="Italic">Mycoplasma gallisepticum</Emphasis> nanoforming

The goal of this work was to create a model for the long persistence of Mycoplasma gallisepticum in depleted medium and under low growth temperature followed by proteomic study of the model. Nanoforms and revertants for M. gallisepticum were obtained. Proteomic maps were produced for different stages of the formation of nanoforms and revertants. It is shown that proteins responsible for essential cellular processes of glycolysis, translation elongation, and DnaK chaperone involved in the stabilization of newly synthesized proteins are crucial for the reversion of M. gallisepticum to a vegetative form. Based on the current data, it is assumed that changes in the metabolism of M. gallisepticum during nanoforming are not post-mortal, thus M. gallisepticum does not transform to uncultivable form, but remains in a reversible dormant state during prolonged unfavorable conditions.