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.     

The Genome of the Amoeba Symbiont “Candidatus Amoebophilus asiaticus” Reveals Common Mechanisms for Host Cell Interaction among Amoeba-Associated Bacteria

Protozoa play host for many intracellular bacteria and are important for the adaptation of pathogenic bacteria to eukaryotic cells. We analyzed the genome sequence of “Candidatus Amoebophilus asiaticus,” an obligate intracellular amoeba symbiont belonging to the Bacteroidetes. The genome has a size of 1.89 Mbp, encodes 1,557 proteins, and shows massive proliferation of IS elements (24% of all genes), although the genome seems to be evolutionarily relatively stable. The genome does not encode pathways for de novo biosynthesis of cofactors, nucleotides, and almost all amino acids. “Ca. Amoebophilus asiaticus” encodes a variety of proteins with predicted importance for host cell interaction; in particular, an arsenal of proteins with eukaryotic domains, including ankyrin-, TPR/SEL1-, and leucine-rich repeats, which is hitherto unmatched among prokaryotes, is remarkable. Unexpectedly, 26 proteins that can interfere with the host ubiquitin system were identified in the genome. These proteins include F- and U-box domain proteins and two ubiquitin-specific proteases of the CA clan C19 family, representing the first prokaryotic members of this protein family. Consequently, interference with the host ubiquitin system is an important host cell interaction mechanism of “Ca. Amoebophilus asiaticus”. More generally, we show that the eukaryotic domains identified in “Ca. Amoebophilus asiaticus” are also significantly enriched in the genomes of other amoeba-associated bacteria (including chlamydiae, Legionella pneumophila, Rickettsia bellii, Francisella tularensis, and Mycobacterium avium). This indicates that phylogenetically and ecologically diverse bacteria which thrive inside amoebae exploit common mechanisms for interaction with their hosts, and it provides further evidence for the role of amoebae as training grounds for bacterial pathogens of humans.Free-living amoebae, such as Acanthamoeba spp., are ubiquitous protozoa which can be found in such diverse habitats as soil, marine water, and freshwater and in many engineered environments (62, 100). They are important predators of prokaryotic and eukaryotic microorganisms, thereby having great influence on microbial community composition, soil mineralization, plant growth, and nutrient cycles (14, 100). Interestingly, many well-known pathogens of humans are able to infect, survive, and multiply within amoebae (39, 51). These protozoa can thus serve as reservoirs and vectors for the transmission of pathogenic bacteria to humans, as demonstrated for L. pneumophila and Mycobacterium avium (2, 115). It is also increasingly being recognized that protozoa are important for the adaptation of (pathogenic) bacteria to higher eukaryotic cells as a niche for growth (2, 24, 42, 78, 89).In addition to the many recognized transient associations between amoeba and pathogens, stable and obligate relationships between bacteria and amoebae also were described for members of the Alphaproteobacteria (11, 34, 48), the Betaproteobacteria (49), the Bacteroidetes (50), and the Chlamydiae (4, 12, 35, 52). These obligate amoeba symbionts show a worldwide distribution, since phylogenetically highly similar strains were found in amoeba isolates from geographically distant sources (51, 107). The phylogenetic diversity and the different lifestyles of these obligate intracellular bacteria—some are located directly in the host cell cytoplasm (11, 34, 48-50, 52), while others are enclosed in host-derived vacuoles (4, 35, 44)—suggest fundamentally different mechanisms of host cell interaction. However, with the exception of chlamydia-related amoeba symbionts (37, 46, 47), our knowledge of the biology of obligate intracellular symbionts of amoebae is still scarce.Comparative genomics has been extremely helpful for the analysis of intracellular bacteria. Numerous genome sequences from the Alpha- and Gammaproteobacteria and the Chlamydiae are available and have contributed significantly to our understanding of genome evolution, the biology of intracellular bacteria, and the interactions with their host cells (24, 26, 46, 79, 82). In this study, we determined and analyzed the complete genome sequence of “Candidatus Amoebophilus asiaticus” strain 5a2 in order to gain novel insights into its biology. “Ca. Amoebophilus asiaticus” is a Gram-negative, obligate intracellular amoeba symbiont belonging to the Bacteroidetes which has been discovered within an amoeba isolated from lake sediment (107). “Ca. Amoebophilus asiaticus” shows highest 16S rRNA similarity to “Candidatus Cardinium hertigii,” an obligate intracellular parasite of arthropods able to manipulate the reproduction of its hosts (131). According to 16S rRNA trees, both organisms are members of a monophyletic group within the phylogenetically diverse phylum Bacteroidetes, consisting only of symbionts and sequences which were directly retrieved from corals (113). Among members of the Bacteroidetes, the genome sequences of only three symbionts, which are only distantly related (75 to 80% 16S rRNA sequence similarity) to “Ca. Amoebophilus asiaticus,” have been determined to date: two strains of “Candidatus Sulcia muelleri,” a symbiont of sharpshooters, and “Azobacteroides pseudotrichonymphae,” a symbiont of an anaerobic termite gut ciliate (45, 72, 74, 127).The genome of “Ca. Amoebophilus asiaticus” is only moderately reduced in size compared to those of many other obligate intracellular bacteria (75, 123), but nevertheless, its biosynthetic capabilities are extremely limited. A large fraction of the genome consists of IS elements and an unparalleled high number of proteins with eukaryotic domains, such as ankyrin repeats, TPR/SEL1 repeats, leucine-rich repeats, and domains from the eukaryotic ubiquitin system, all of them most likely important for host cell interaction. Feature enrichment analysis across a nonredundant data set of all bacterial genomes showed that these domains are enriched in the genomes of bacteria (including several pathogens of humans) known to be able to infect amoebae, providing further evidence for an important role of amoebae in the evolution of mechanisms for host cell interaction in intracellular bacteria.     

Roles of Small,Acid-Soluble Spore Proteins and Core Water Content in Survival of Bacillus subtilis Spores Exposed to Environmental Solar UV Radiation

Spores of Bacillus subtilis contain a number of small, acid-soluble spore proteins (SASP) which comprise up to 20% of total spore core protein. The multiple α/β-type SASP have been shown to confer resistance to UV radiation, heat, peroxides, and other sporicidal treatments. In this study, SASP-defective mutants of B. subtilis and spores deficient in dacB, a mutation leading to an increased core water content, were used to study the relative contributions of SASP and increased core water content to spore resistance to germicidal 254-nm and simulated environmental UV exposure (280 to 400 nm, 290 to 400 nm, and 320 to 400 nm). Spores of strains carrying mutations in sspA, sspB, and both sspA and sspB (lacking the major SASP-α and/or SASP-β) were significantly more sensitive to 254-nm and all polychromatic UV exposures, whereas the UV resistance of spores of the sspE strain (lacking SASP-γ) was essentially identical to that of the wild type. Spores of the dacB-defective strain were as resistant to 254-nm UV-C radiation as wild-type spores. However, spores of the dacB strain were significantly more sensitive than wild-type spores to environmental UV treatments of >280 nm. Air-dried spores of the dacB mutant strain had a significantly higher water content than air-dried wild-type spores. Our results indicate that α/β-type SASP and decreased spore core water content play an essential role in spore resistance to environmentally relevant UV wavelengths whereas SASP-γ does not.Spores of Bacillus spp. are highly resistant to inactivation by different physical stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and high fluences of UV or ionizing radiation (reviewed in references 33, 34, and 48). Under stressful environmental conditions, cells of Bacillus spp. produce endospores that can stay dormant for extended periods. The reason for the high resistance of bacterial spores to environmental extremes lies in the structure of the spore. Spores possess thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents, such as the calcium chelate of dipicolinic acid and α/β-type small, acid-soluble spore proteins (α/β-type SASP), the last two of which protect spore DNA (6, 42, 46, 48, 52). DNA damage accumulated during spore dormancy is also efficiently repaired during spore germination (33, 47, 48). UV-induced DNA photoproducts are repaired by spore photoproduct lyase and nucleotide excision repair, DNA double-strand breaks (DSB) by nonhomologous end joining, and oxidative stress-induced apurinic/apyrimidinic (AP) sites by AP endonucleases and base excision repair (15, 26-29, 34, 43, 53, 57).Monochromatic 254-nm UV radiation has been used as an efficient and cost-effective means of disinfecting surfaces, building air, and drinking water supplies (31). Commonly used test organisms for inactivation studies are bacterial spores, usually spores of Bacillus subtilis, due to their high degree of resistance to various sporicidal treatments, reproducible inactivation response, and safety (1, 8, 19, 31, 48). Depending on the Bacillus species analyzed, spores are 10 to 50 times more resistant than growing cells to 254-nm UV radiation. In addition, most of the laboratory studies of spore inactivation and radiation biology have been performed using monochromatic 254-nm UV radiation (33, 34). Although 254-nm UV-C radiation is a convenient germicidal treatment and relevant to disinfection procedures, results obtained by using 254-nm UV-C are not truly representative of results obtained using UV wavelengths that endospores encounter in their natural environments (34, 42, 50, 51, 59). However, sunlight reaching the Earth''s surface is not monochromatic 254-nm radiation but a mixture of UV, visible, and infrared radiation, with the UV portion spanning approximately 290 to 400 nm (33, 34, 36). Thus, our knowledge of spore UV resistance has been constructed largely using a wavelength of UV radiation not normally reaching the Earth''s surface, even though ample evidence exists that both DNA photochemistry and microbial responses to UV are strongly wavelength dependent (2, 30, 33, 36).Of recent interest in our laboratories has been the exploration of factors that confer on B. subtilis spores resistance to environmentally relevant extreme conditions, particularly solar UV radiation and extreme desiccation (23, 28, 30, 34 36, 48, 52). It has been reported that α/β-type SASP but not SASP-γ play a major role in spore resistance to 254-nm UV-C radiation (20, 21) and to wet heat, dry heat, and oxidizing agents (48). In contrast, increased spore water content was reported to affect B. subtilis spore resistance to moist heat and hydrogen peroxide but not to 254-nm UV-C (12, 40, 48). However, the possible roles of SASP-α, -β, and -γ and core water content in spore resistance to environmentally relevant solar UV wavelengths have not been explored. Therefore, in this study, we have used B. subtilis strains carrying mutations in the sspA, sspB, sspE, sspA and sspB, or dacB gene to investigate the contributions of SASP and increased core water content to the resistance of B. subtilis spores to 254-nm UV-C and environmentally relevant polychromatic UV radiation encountered on Earth''s surface.     

The Longin Domain Regulates the Steady-State Dynamics of Sec22 in Plasmodium falciparum

The specificity of vesicle-mediated transport is largely regulated by the membrane-specific distribution of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. However, the signals and machineries involved in SNARE protein targeting to the respective intracellular locations are not fully understood. We have identified a Sec22 ortholog in Plasmodium falciparum (PfSec22) that contains an atypical insertion of the Plasmodium export element within the N-terminal longin domain. This Sec22 protein partially associates with membrane structures in the parasitized erythrocytes when expressed under the control of the endogenous promoter element. Our studies indicate that the atypical longin domain contains signals that are required for both endoplasmic reticulum (ER)/Golgi apparatus recycling of PfSec22 and partial export beyond the ER/Golgi apparatus interface. ER exit of PfSec22 is regulated by motifs within the α3 segment of the longin domain, whereas the recycling and export signals require residues within the N-terminal hydrophobic segment. Our data suggest that the longin domain of PfSec22 exhibits major differences from the yeast and mammalian orthologs, perhaps indicative of a novel mechanism for Sec22 trafficking in malaria parasites.Plasmodium falciparum exhibits a complex network of endomembrane organelles that are unique to this obligate intracellular parasite of human erythrocytes. They include parasite-induced tubules and vesicles in the infected host cell and specialized secretory structures collectively known as the apical complex. The asexual blood stages of the parasite develop within a parasitophorous vacuole (PV) and thus are separated from the external milieu by three lipid bilayers: the parasite plasma membrane (PPM), the PV membrane (PVM), and the erythrocyte plasma membrane. To survive inside these terminally differentiated human erythrocytes, P. falciparum remodels the host cell compartment by exporting numerous proteins into the erythrocyte cytoplasm (12, 15, 49, 50, 57). The mechanisms by which both soluble and membrane-bound proteins are transported, first into the PV lumen, followed by translocation across the PVM and transport within the erythrocyte cytosol, are not fully understood (9). A majority of the exported proteins contain bipartite signals that comprise a “recessed” N-terminal signal sequence and a Plasmodium export element/vacuolar translocation sequence (PEXEL/VTS) that is characterized by the consensus sequence RX(L/I)X(D/E/Q). These signals are predicted to facilitate the transport of proteins into the PV (using their recessed, or N-terminal, signal sequences) and translocation across the PVM (using their PEXEL/VTS motifs) (5, 23, 29, 34). However, a subset of the exported proteins lack either one or both signal elements and may require novel targeting motifs for transport beyond the PPM (20, 43). A majority of the proteins enter the parasite secretory system via the endoplasmic reticulum (ER), where they are incorporated into ER-derived vesicles and then transported through the “unstacked” Golgi bodies to their final destinations (45, 48, 55, 56). Membrane-bound vesicular elements have been detected in the infected host cell cytosol, suggesting the existence of an extraparasitic vesicle-mediated transport process in malaria parasites (22, 47, 52). How vesicle targeting is achieved in P. falciparum parasites remains elusive.Vesicle targeting and fusion in eukaryotic cells involves proteins of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family (25, 41, 42, 44). SNAREs are “tail-anchored” proteins that function by forming complexes that bridge vesicle and target membranes during fusion (6, 7, 24). Distinct sets of SNARE proteins localize to different intracellular transport pathways using processes that are not well understood. Increasing evidence suggests that the N-terminal regions of SNARE proteins contain signals required for their subcellular localization (4, 31, 53). These N-terminal regions include the three-helical Habc bundles of syntaxin SNAREs and the “profilin-like” folds of long VAMPs (vesicle-associated membrane proteins), also known as longin domains (7, 17, 33, 40, 46). The Sec22 gene products in mammals and yeast are longin domain-containing SNAREs that cycle between the ER and Golgi compartments (3, 19, 31, 32). We have identified a Sec22 ortholog in P. falciparum (PfSec22) that contains a PEXEL/VTS sequence insertion between the α2 and α3 segments of the longin domain preceded by a stretch of hydrophobic residues that spans a region between the β5 and α2 segments (2). In this study, we examined the distribution of PfSec22 in P. falciparum-infected erythrocytes and investigated the role of the atypical longin domain in its steady-state localization. Our data show that the P. falciparum ortholog of Sec22 partially associates with noncanonical destinations (tubovesicular network and intraerythrocytic vesicles) in the infected erythrocytes and that the N-terminal longin domain exhibits a dual function, mediating ER-to-Golgi apparatus trafficking, as well as retrieval from the Golgi apparatus.     

Immune Evasion Proteins of Murine Cytomegalovirus Preferentially Affect Cell Surface Display of Recently Generated Peptide Presentation Complexes

For recognition of infected cells by CD8 T cells, antigenic peptides are presented at the cell surface, bound to major histocompatibility complex class I (MHC-I) molecules. Downmodulation of cell surface MHC-I molecules is regarded as a hallmark function of cytomegalovirus-encoded immunoevasins. The molecular mechanisms by which immunoevasins interfere with the MHC-I pathway suggest, however, that this downmodulation may be secondary to an interruption of turnover replenishment and that hindrance of the vesicular transport of recently generated peptide-MHC (pMHC) complexes to the cell surface is the actual function of immunoevasins. Here we have used the model of murine cytomegalovirus (mCMV) infection to provide experimental evidence for this hypothesis. To quantitate pMHC complexes at the cell surface after infection in the presence and absence of immunoevasins, we generated the recombinant viruses mCMV-SIINFEKL and mCMV-Δm06m152-SIINFEKL, respectively, expressing the K^b-presented peptide SIINFEKL with early-phase kinetics in place of an immunodominant peptide of the viral carrier protein gp36.5/m164. The data revealed ∼10,000 K^b molecules presenting SIINFEKL in the absence of immunoevasins, which is an occupancy of ∼10% of all cell surface K^b molecules, whereas immunoevasins reduced this number to almost the detection limit. To selectively evaluate their effect on preexisting pMHC complexes, cells were exogenously loaded with SIINFEKL peptide shortly after infection with mCMV-SIINFEKA, in which endogenous presentation is prevented by an L174A mutation of the C-terminal MHC-I anchor residue. The data suggest that pMHC complexes present at the cell surface in advance of immunoevasin gene expression are downmodulated due to constitutive turnover in the absence of resupply.CD8 T cells recognize infected cells by interaction of their T-cell receptor (TCR) with a cell surface presentation complex composed of a cognate antigenic peptide bound to a presenting allelic form of a major histocompatibility complex class I (MHC-I) glycoprotein (77, 85, 97, 98). The number of such “peptide receptors” per cell has been estimated to be on the order of 10⁵ to 10⁶ for each MHC-I allomorph (for a review, see reference 82). Viral antigenic peptides are generated within infected cells by proteolytic processing of viral proteins, usually in the proteasome, and associate with nascent MHC-I proteins in the endoplasmic reticulum (ER) before the peptide-MHC (pMHC) complexes travel to the cell surface with the cellular vesicular flow (for reviews, see references 13, 87, 92, and 93). CD8 T cells have long been known to protect against cytomegalovirus (CMV) infection and disease in animal models (60, 72; reviewed in references 33 and 36) and in humans (9, 61, 67, 75, 76). As shown only recently in the murine CMV (mCMV) model of infection of immunocompromised mice by adoptive transfer of epitope-specific CD8 T cells, antiviral protection against CMV is indeed TCR mediated and epitope dependent. Specifically, memory cells purified by TCR-based epitope-specific cell sorting, as well as cells of a peptide-selected cytolytic T-lymphocyte line, protected against mCMV expressing the cognate antigenic peptide, the IE1 peptide 168-YPHFMPTNL-176 in this example, but failed to control infection with a recombinant mCMV expressing a peptide analogue in which the C-terminal MHC-I anchor residue leucine was replaced with alanine (3).Interference with the MHC-I pathway of antigen presentation has evolved as a viral immune evasion mechanism of CMVs and other viruses, mediated by virally encoded proteins that inhibit MHC-I trafficking to the cell surface (for reviews, see references 1, 24, 27, 29, 63, 70, 71, 84, and 95). These molecules are known as immunoevasins (50, 70, 89), as “viral proteins interfering with antigen presentation” (VIPRs) (95), or as negative “viral regulators of antigen presentation” (vRAPs) (34). Although the detailed molecular mechanisms differ between different CMV species in their respective hosts, the common biological outcome is the inhibition of antigen presentation. Accordingly, downmodulation of MHC-I cell surface expression is a hallmark of molecular immune evasion and actually led to the discovery of this class of molecules. Since CD8 T cells apparently protect against infection with wild-type CMV strains despite the expression of immunoevasins, the in vivo relevance of these molecules is an issue of current interest and investigation (for a review, see reference 14). As shown recently with the murine model, antigen presentation in infected host cells is not completely blocked for all epitopes, because pMHC complexes that are constitutively formed in sufficiently large amounts can exhaust the inhibitory capacity of the immunoevasins (40). Likewise, enhancing antigen processing conditionally with gamma interferon (IFN-γ) aids in peptide presentation in the presence of immunoevasins (18, 28). Thus, by raising the threshold of the amount of peptide required for presentation, immunoevasins determine whether a particular viral peptide can function as a protective epitope—an issue of relevance for rational vaccine design as well (94). Whereas deletion of immunoevasin genes gives only incremental improvement to the control of infection in immunocompetent mice (22, 51), expression of immunoevasins reduces the protective effect of adoptively transferred CD8 T cells in immunocompromised recipients (37, 40, 47, 48). In a bone marrow transplantation model, immunoevasins were recently found to contribute to enhanced and prolonged virus replication during hematopoietic reconstitution and, consequently, also to higher latent viral genome loads in the lungs and a higher incidence of virus recurrence (4). Notably, however, immunoevasins do not inhibit but, rather, enhance CD8 T-cell priming (5, 21, 22, 56), due to higher viral replication levels in draining lymph nodes associated with sustained antigen supply for the cross-priming of CD8 T cells by uninfected antigen-presenting cells (5).For mCMV, three molecules are proposed to function as vRAPs, only two of which are confirmed negative regulators that downmodulate cell surface MHC-I (34, 62, 89) and inhibit the presentation of antigenic peptides to CD8 T cells (34, 62). Immunoevasin gp40/m152 transiently interacts with MHC-I molecules and mediates their retention in a cis-Golgi compartment (96), whereas gp48/m06 stably binds to MHC-I molecules in the ER and mediates sorting of the complexes for lysosomal degradation by a mechanism that involves the cellular cargo sorting adaptor proteins AP1-A and AP3-A (73, 74). The third proposed immunoevasin of mCMV, gp34/m04 (46), also binds stably to MHC-I molecules. A function as a CD8 T-cell immunoevasin was predicted from some alleviation of immune evasion for certain epitopes and MHC-I molecules in cells infected with the deletion mutant mCMV-Δm04 (34, 42, 89), but gp34/m04 does not reduce the steady-state level of cell surface class I molecules and does not inhibit peptide presentation when expressed selectively after infection with mCMV-Δm06m152 (34, 62). The m04-MHC-I complexes are expressed on the cell surface (46) and appear to be involved in the modulation of natural killer cell activity (45).Here we give the first report on quantitating the efficacy of immunoevasins in terms of absolute numbers of pMHC complexes displayed at the cell surface. By comparing the fate of pMHC complexes already present at the cell surface in advance of immunoevasin gene expression with that of newly formed pMHC complexes, our data provide direct evidence to conclude that downmodulation of cell surface MHC-I molecules is secondary to an interruption of the flow of newly formed pMHC complexes to the cell surface.(Part of this work was presented at the 12th International CMV/Betaherpesvirus Workshop, 10 to 14 May 2009, Boston, MA.)     

Analysis of the Fine-Scale Population Structure of “Candidatus Accumulibacter phosphatis” in Enhanced Biological Phosphorus Removal Sludge,Using Fluorescence In Situ Hybridization and Flow Cytometric Sorting

To investigate the fine-scale diversity of the polyphosphate-accumulating organisms (PAO) “Candidatus Accumulibacter phosphatis” (henceforth referred to as “Ca. Accumulibacter”), two laboratory-scale sequencing batch reactors (SBRs) for enhanced biological phosphorus removal (EBPR) were operated with sodium acetate as the sole carbon source. During SBR operations, activated sludge always contained morphologically different “Ca. Accumulibacter” strains showing typical EBPR performances, as confirmed by the combined technique of fluorescence in situ hybridization (FISH) and microautoradiography (MAR). Fragments of “Ca. Accumulibacter” 16S rRNA genes were retrieved from the sludge. Phylogenetic analyses together with sequences from the GenBank database showed that “Ca. Accumulibacter” 16S rRNA genes of the EBPR sludge were clearly differentiated into four “Ca. Accumulibacter” clades, Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4. The specific FISH probes Acc444, Acc184, Acc72, and Acc119 targeting these clades and some helpers and competitors were designed by using the ARB program. Microbial characterization by FISH analysis using specific FISH probes also clearly indicated the presence of different “Ca. Accumulibacter” cell morphotypes. Especially, members of Acc-SG3, targeted by probe Acc72, were coccobacillus-shaped cells with a size of approximately 2 to 3 μm, while members of Acc-SG1, Acc-SG2, and Acc-SG4, targeted by Acc444, Acc184, and Acc119, respectively, were coccus-shaped cells approximately 1 μm in size. Subsequently, cells targeted by each FISH probe were sorted by use of a flow cytometer, and their polyphosphate kinase 1 (ppk1) gene homologs were amplified by using a ppk1-specific PCR primer set for “Ca. Accumulibacter.” The phylogenetic tree based on sequences of the ppk1 gene homologs was basically congruent with that of the 16S rRNA genes, but members of Acc-SG3 with a distinct morphology comprised two different ppk1 genes. These results suggest that “Ca. Accumulibacter” strains may be diverse physiologically and ecologically and represent distinct populations with genetically determined adaptations in EBPR systems.Enhanced biological phosphorus removal (EBPR) has been applied in many wastewater treatment plants to reduce the phosphorus that causes eutrophication in surface waters. EBPR employs polyphosphate-accumulating organisms (PAOs), which are enriched through alternating aerobic-anaerobic cycles (34). Since PAOs are essential for an understanding of EBPR, many candidates have been proposed as potential PAOs, such as Acinetobacter spp. (11), Tetrasphaera spp. (31), Microlunatus phosphovorus (36), Lampropedia spp. (40), and Gram-positive Actinobacteria (24). However, those organisms do not exhibit all of the characteristics of the EBPR biochemistry model. Recently developed culture-independent approaches such as PCR-clone libraries, fluorescence in situ hybridization (FISH), and microautoradiography (MAR) have highlighted an uncultured Rhodocyclus-related bacterium, “Candidatus Accumulibacter phosphatis” (henceforth referred to as “Ca. Accumulibacter”), as one of the most important PAO candidates (2, 5, 16, 22, 23, 27, 28, 47).Numerous studies have sought to investigate uncultured “Ca. Accumulibacter” and have shown the presence of genetically and physiologically different members with a global geographic distribution (3, 9, 22, 27, 39). For example, Kong et al. (22) identified two morphologically different “Ca. Accumulibacter” cells of small cocci and large coccobacilli labeled with PAOmix (PAO462, PAO651, and PAO846) in laboratory-scale EBPR reactors. Additional results showing phenotypic and morphological diversities of “Ca. Accumulibacter” cells also existed with respect to the different roles of denitrifying PAO (DPAO) in the EBPR process (3, 9, 23). Carvalho et al. (3) detected two different morphotypes of “Ca. Accumulibacter” with different nitrate reduction capabilities. The presence of other “Ca. Accumulibacter” strains with 15% genome sequence divergence from the dominant strains in metagenomic analyses is likely to explain these morphological and phenotypic differences (12). McMahon et al. (33) suggested the use of the polyphosphate kinase (ppk) gene, which is involved in the production of polyphosphate, for a finer elucidation of “Ca. Accumulibacter” diversity. He et al. (15) grouped “Ca. Accumulibacter” strains into five distinct clades, designated clades I, IIA, IIB, IIC, and IID, using ppk gene sequence information. Flowers and colleagues (9) previously reported that “Ca. Accumulibacter” cells of clade IA had nitrate reduction activity with phosphorus uptake but that “Ca. Accumulibacter” cells of clade IIA did not.FISH-fluorescence activated cell sorting (FACS) techniques have been used for the separation of specific microbial cells from complex microbial consortia and their metabolic gene analysis (14, 46). For example, Miyauchi et al. (35) sorted PAOmix probe-labeled “Ca. Accumulibacter” cells from EBPR sludge and analyzed their nitrite reductase gene (nirS) diversity. In the current study, we found that four different “Ca. Accumulibacter” clades (Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4) were present in the EBPR sludge of laboratory-scale reactors supplied with acetate as the sole carbon source. We analyzed their morphological characteristics and ppk gene sequence information using a suite of FISH and FACS approaches and linked fine-scale phylogenetic diversities of “Ca. Accumulibacter” strains with their morphological characteristics and metabolic genes. This study will be useful for further genetic and physiological studies of different “Ca. Accumulibacter” clades.     

Environmental Factors Shape Sediment Anammox Bacterial Communities in Hypernutrified Jiaozhou Bay,China

Bacterial anaerobic ammonium oxidation (anammox) is an important process in the marine nitrogen cycle. Because ongoing eutrophication of coastal bays contributes significantly to the formation of low-oxygen zones, monitoring of the anammox bacterial community offers a unique opportunity for assessment of anthropogenic perturbations in these environments. The current study used targeting of 16S rRNA and hzo genes to characterize the composition and structure of the anammox bacterial community in the sediments of the eutrophic Jiaozhou Bay, thereby unraveling their diversity, abundance, and distribution. Abundance and distribution of hzo genes revealed a greater taxonomic diversity in Jiaozhou Bay, including several novel clades of anammox bacteria. In contrast, the targeting of 16S rRNA genes verified the presence of only “Candidatus Scalindua,” albeit with a high microdiversity. The genus “Ca. Scalindua” comprised the apparent majority of active sediment anammox bacteria. Multivariate statistical analyses indicated a heterogeneous distribution of the anammox bacterial assemblages in Jiaozhou Bay. Of all environmental parameters investigated, sediment organic C/organic N (OrgC/OrgN), nitrite concentration, and sediment median grain size were found to impact the composition, structure, and distribution of the sediment anammox bacterial community. Analysis of Pearson correlations between environmental factors and abundance of 16S rRNA and hzo genes as determined by fluorescent real-time PCR suggests that the local nitrite concentration is the key regulator of the abundance of anammox bacteria in Jiaozhou Bay sediments.Anaerobic ammonium oxidation (anammox, NH₄⁺ + NO₂⁻ → N₂ + 2H₂O) was proposed as a missing N transformation pathway decades ago. It was found 20 years later to be mediated by bacteria in artificial environments, such as anaerobic wastewater processing systems (see reference 32 and references therein). Anammox in natural environments was found even more recently, mainly in O₂-limited environments such as marine sediments (28, 51, 54, 67, 69) and hypoxic or anoxic waters (10, 25, 39-42). Because anammox may remove as much as 30 to 70% of fixed N from the oceans (3, 9, 64), this process is potentially as important as denitrification for N loss and bioremediation (41, 42, 73). These findings have significantly changed our understanding of the budget of the marine and global N cycles as well as involved pathways and their evolution (24, 32, 35, 72). Studies indicate variable anammox contributions to local or regional N loss (41, 42, 73), probably due to distinct environmental conditions that may influence the composition, abundance, and distribution of the anammox bacteria. However, the interactions of anammox bacteria with their environment are still poorly understood.The chemolithoautotrophic anammox bacteria (64, 66) comprise the new Brocadiaceae family in the Planctomycetales, for which five Candidatus genera have been described (see references 32 and 37 and references therein): “Candidatus Kuenenia,” “Candidatus Brocadia,” “Candidatus Scalindua,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia.” Due to the difficulty of cultivation and isolation, anammox bacteria are not yet in pure culture. Molecular detection by using DNA probes or PCR primers targeting the anammox bacterial 16S rRNA genes has thus been the main approach for the detection of anammox bacteria and community analyses (58). However, these studies revealed unexpected target sequence diversity and led to the realization that due to biased coverage and specificity of most of the PCR primers (2, 8), the in situ diversity of anammox bacteria was likely missed. Thus, the use of additional marker genes for phylogenetic analysis was suggested in hopes of better capturing the diversity of this environmentally important group of bacteria. By analogy to molecular ecological studies of aerobic ammonia oxidizers, most recent studies have attempted to include anammox bacterium-specific functional genes. All anammox bacteria employ hydrazine oxidoreductase (HZO) (= [Hzo]₃) to oxidize hydrazine to N₂ as the main source for a useable reductant, which enables them to generate proton-motive force for energy production (32, 36, 65). Phylogenetic analyses of Hzo protein sequences revealed three sequence clusters, of which the cladistic structure of cluster 1 is in agreement with the anammox bacterial 16S rRNA gene phylogeny (57). The hzo genes have emerged as an alternative phylogenetic and functional marker for characterization of anammox bacterial communities (43, 44, 57), allowing the 16S rRNA gene-based investigation methods to be corroborated and improved.The contribution of anammox to the removal of fixed N is highly variable in estuarine and coastal sediments (50). For instance, anammox may be an important pathway for the removal of excess N (23) or nearly negligible (48, 54, 67, 68). This difference may be attributable to a difference in the structure and composition of anammox bacterial communities, in particular how the abundance of individual cohorts depends on particular environmental conditions. Anthropogenic disturbance with variable source and intensity of eutrophication and pollution may further complicate the anammox bacterium-environment relationship.Jiaozhou Bay is a large semienclosed water body of the temperate Yellow Sea in China. Eutrophication has become its most serious environmental problem, along with red tides (harmful algal blooms), species loss, and contamination with toxic chemicals and harmful microbes (14, 15, 21, 61, 71). Due to different sources of pollution and various levels of eutrophication across Jiaozhou Bay (mariculture, municipal and industrial wastewater, crude oil shipyard, etc.), a wide spectrum of environmental conditions may contribute to a widely varying community structure of anammox bacteria. This study used both 16S rRNA and hzo genes as targets to measure their abundance, diversity, and spatial distribution and assess the response of the resident anammox bacterial community to different environmental conditions. Environmental factors with potential for regulating the sediment anammox microbiota are discussed.     

New Device for High-Throughput Viability Screening of Flow Biofilms

Control of biofilms requires rapid methods to identify compounds effective against them and to isolate resistance-compromised mutants for identifying genes involved in enhanced biofilm resistance. While rapid screening methods for microtiter plate well (“static”) biofilms are available, there are no methods for such screening of continuous flow biofilms (“flow biofilms”). Since the latter biofilms more closely approximate natural biofilms, development of a high-throughput (HTP) method for screening them is desirable. We describe here a new method using a device comprised of microfluidic channels and a distributed pneumatic pump (BioFlux) that provides fluid flow to 96 individual biofilms. This device allows fine control of continuous or intermittent fluid flow over a broad range of flow rates, and the use of a standard well plate format provides compatibility with plate readers. We show that use of green fluorescent protein (GFP)-expressing bacteria, staining with propidium iodide, and measurement of fluorescence with a plate reader permit rapid and accurate determination of biofilm viability. The biofilm viability measured with the plate reader agreed with that determined using plate counts, as well as with the results of fluorescence microscope image analysis. Using BioFlux and the plate reader, we were able to rapidly screen the effects of several antimicrobials on the viability of Pseudomonas aeruginosa PAO1 flow biofilms.Bacterial biofilms are surface-attached communities that are encased in a polymeric matrix, which exhibit a high degree of resistance to antimicrobial agents and the host immune system (12, 16). This makes them medically important; diseases with a biofilm component are chronic and difficult to eradicate. Examples of such diseases are cystitis (1), endocarditis (31), cystic fibrosis (35), and middle-ear (17) and indwelling medical device-associated (20) infections. Biofilms also play important environmental roles in, for example, wastewater treatment (38), bioremediation (29, 30), biofouling (7), and biocorrosion (2). Better control of biofilms requires elucidation of the molecular basis of their superior resistance (by identifying resistance-compromised mutants) and identification of compounds with antibiofilm activity. While our understanding of these aspects of biofilms has increased (11, 15, 25-27, 36), further work, including development of accurate high-throughput (HTP) methods for screening biofilm viability, is needed.Two major biofilm models are studied in the laboratory, biofilms grown without a continuous flow of fresh medium and biofilms grown with a continuous flow of fresh medium; examples of these two models are microtiter well biofilms and flow cell biofilms, respectively. Methods have been developed for HTP screening of the viability of static biofilms (6, 28, 32, 33), but there are no methods for HTP screening of flow biofilms. The latter biofilms are typically grown in flow cells, which have to be examined individually to determine viability and thus cannot be used for rapid screening. An HTP screening method for flow biofilms is desirable, as these biofilms more closely approximate natural biofilms and can differ from static biofilms evidently due to hydrodynamic influences on cell signaling (22, 34). For example, the ability of rpoS-deficient Escherichia coli (lacking σ^S) to form flow biofilms is impaired, but its capacity to form biofilms under static conditions is enhanced (18).We describe here a new application of a recently developed device (8-10, 13), the “BioFlux” device consisting of microfluidic channels for biofilm growth. Other microfluidic devices have recently been used for biofilm formation (14, 19, 21, 23), but none of them has been used for HTP screening. The BioFlux device permits rapid measurement of the fluorescence of flow biofilms with a plate reader, which permits initial HTP screening of the viability of such biofilms.     

A Novel Dehalobacter Species Is Involved in Extensive 4,5,6,7-Tetrachlorophthalide Dechlorination

The purpose of this study was the enrichment and phylogenetic identification of bacteria that dechlorinate 4,5,6,7-tetrachlorophthalide (commercially designated “fthalide”), an effective fungicide for rice blast disease. Sequential transfer culture of a paddy soil with lactate and fthalide produced a soil-free enrichment culture (designated the “KFL culture”) that dechlorinated fthalide by using hydrogen, which is produced from lactate. Phylogenetic analysis based on 16S rRNA genes revealed the dominance of two novel phylotypes of the genus Dehalobacter (FTH1 and FTH2) in the KFL culture. FTH1 and FTH2 disappeared during culture transfer in medium without fthalide and increased in abundance with the dechlorination of fthalide, indicating their growth dependence on the dechlorination of fthalide. Dehalobacter restrictus TEA is their closest relative, with 97.5% and 97.3% 16S rRNA gene similarities to FTH1 and FTH2, respectively.4,5,6,7-Tetrachlorophthalide (commercially designated “fthalide”) is an effective fungicide for rice blast disease, which inhibits melanin biosynthesis and the formation of the mature appressorial cells of the rice blast pathogen on the host plant (5, 16). Fthalide has been reported to be reductively dechlorinated in soil (16) and compost (28), although its fates in paddy soil and the fthalide-dechlorinating bacteria are unknown. Besides fthalide, polychlorinated aromatic compounds are known to be reductively dechlorinated by the bacteria of several phyla. Six strains of Desulfitobacterium spp. of the phylum Firmicutes (2, 3, 6, 10, 23, 29) and Desulfomonile tiedjei DCB-1 of the phylum Proteobacteria (21) can dechlorinate polychlorinated phenols. Three strains of the phylum Chloroflexi can dechlorinate a variety of compounds, including polychlorinated phenols, benzenes, biphenyls, or dibenzo-p-dioxins: Dehalococcoides ethenogenes 195 (9, 19), Dehalococcoides sp. strain CBDB1 (1, 4), and strain DF-1 of Chloroflexi, collectively called the “o-17/DF-1 group” (18). Dehalococcoides spp. utilize hydrogen as an electron donor and acetate as a carbon source for growth coupled to the reductive dechlorination of chlorinated compounds (1, 12, 13, 19, 26). In contrast, Desulfitobacterium spp. can dechlorinate chlorinated compounds not only with hydrogen, but also organic acids, such as formate, pyruvate, lactate, or butyrate (3, 10, 23). Strain DF-1 can utilize hydrogen and formate for the dechlorination of polychlorinated biphenyls (PCBs) (18).In this study, bacteria that dechlorinate fthalide were enriched from a paddy soil with sequentially transferred cultures using a soil-free medium supplemented with single organic acids. Acetate, formate, lactate, and butyrate were used in this study because they are frequently used in the enrichment of dechlorinators and release hydrogen at different concentrations (8, 11, 14). Fthalide-dechlorinating bacteria in the enriched culture were phylogenetically identified based on the 16S rRNA gene with PCR-denaturing gradient gel electrophoresis (DGGE), a 16S rRNA gene clone library, and quantitative real-time PCR (qPCR).     

Abundance of Novel and Diverse tfdA-Like Genes,Encoding Putative Phenoxyalkanoic Acid Herbicide-Degrading Dioxygenases,in Soil

Phenoxyalkanoic acid (PAA) herbicides are widely used in agriculture. Biotic degradation of such herbicides occurs in soils and is initiated by α-ketoglutarate- and Fe²⁺-dependent dioxygenases encoded by tfdA-like genes (i.e., tfdA and tfdAα). Novel primers and quantitative kinetic PCR (qPCR) assays were developed to analyze the diversity and abundance of tfdA-like genes in soil. Five primer sets targeting tfdA-like genes were designed and evaluated. Primer sets 3 to 5 specifically amplified tfdA-like genes from soil, and a total of 437 sequences were retrieved. Coverages of gene libraries were 62 to 100%, up to 122 genotypes were detected, and up to 389 genotypes were predicted to occur in the gene libraries as indicated by the richness estimator Chao1. Phylogenetic analysis of in silico-translated tfdA-like genes indicated that soil tfdA-like genes were related to those of group 2 and 3 Bradyrhizobium spp., Sphingomonas spp., and uncultured soil bacteria. Soil-derived tfdA-like genes were assigned to 11 clusters, 4 of which were composed of novel sequences from this study, indicating that soil harbors novel and diverse tfdA-like genes. Correlation analysis of 16S rRNA and tfdA-like gene similarity indicated that any two bacteria with D > 20% of group 2 tfdA-like gene-derived protein sequences belong to different species. Thus, data indicate that the soil analyzed harbors at least 48 novel bacterial species containing group 2 tfdA-like genes. Novel qPCR assays were established to quantify such new tfdA-like genes. Copy numbers of tfdA-like genes were 1.0 × 10⁶ to 65 × 10⁶ per gram (dry weight) soil in four different soils, indicating that hitherto-unknown, diverse tfdA-like genes are abundant in soils.Phenoxyalkanoic acid (PAA) herbicides such as MCPA (4-chloro-2-methyl-phenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) are widely used to control broad-leaf weeds in agricultural as well as nonagricultural areas (19, 77). Degradation occurs primarily under oxic conditions in soil, and microorganisms play a key role in the degradation of such herbicides in soil (62, 64). Although relatively rapidly degraded in soil (32, 45), both MCPA and 2,4-D are potential groundwater contaminants (10, 56, 70), accentuating the importance of bacterial PAA herbicide-degrading bacteria in soils (e.g., references 3, 5, 6, 20, 41, 59, and 78).Degradation can occur cometabolically or be associated with energy conservation (15, 54). The first step in the degradation of 2,4-D and MCPA is initiated by the product of cadAB or tfdA-like genes (29, 30, 35, 67), which constitutes an α-ketoglutarate (α-KG)- and Fe²⁺-dependent dioxygenase. TfdA removes the acetate side chain of 2,4-D and MCPA to produce 2,4-dichlorophenol and 4-chloro-2-methylphenol, respectively, and glyoxylate while oxidizing α-ketoglutarate to CO₂ and succinate (16, 17).Organisms capable of PAA herbicide degradation are phylogenetically diverse and belong to the Alpha-, Beta-, and Gammproteobacteria and the Bacteroidetes/Chlorobi group (e.g., references 2, 14, 29-34, 39, 60, 68, and 71). These bacteria harbor tfdA-like genes (i.e., tfdA or tfdAα) and are categorized into three groups on an evolutionary and physiological basis (34). The first group consists of beta- and gammaproteobacteria and can be further divided into three distinct classes based on their tfdA genes (30, 46). Class I tfdA genes are closely related to those of Cupriavidus necator JMP134 (formerly Ralstonia eutropha). Class II tfdA genes consist of those of Burkholderia sp. strain RASC and a few strains that are 76% identical to class I tfdA genes. Class III tfdA genes are 77% identical to class I and 80% identical to class II tfdA genes and linked to MCPA degradation in soil (3). The second group consists of alphaproteobacteria, which are closely related to Bradyrhizobium spp. with tfdAα genes having 60% identity to tfdA of group 1 (18, 29, 34). The third group also harbors the tfdAα genes and consists of Sphingomonas spp. within the alphaproteobacteria (30).Diverse PAA herbicide degraders of all three groups were identified in soil by cultivation-dependent studies (32, 34, 41, 78). Besides CadAB, TfdA and certain TfdAα proteins catalyze the conversion of PAA herbicides (29, 30, 35). All groups of tfdA-like genes are potentially linked to the degradation of PAA herbicides, although alternative primary functions of group 2 and 3 TfdAs have been proposed (30, 35). However, recent cultivation-independent studies focused on 16S rRNA genes or solely on group 1 tfdA sequences in soil (e.g., references 3-5, 13, and 41). Whether group 2 and 3 tfdA-like genes are also quantitatively linked to the degradation of PAA herbicides in soils is unknown. Thus, tools to target a broad range of tfdA-like genes are needed to resolve such an issue. Primers used to assess the diversity of tfdA-like sequences used in previous studies were based on the alignment of approximately 50% or less of available sequences to date (3, 20, 29, 32, 39, 47, 58, 73). Primers specifically targeting all major groups of tfdA-like genes to assess and quantify a broad diversity of potential PAA degraders in soil are unavailable. Thus, the objectives of this study were (i) to develop primers specific for all three groups of tfdA-like genes, (ii) to establish quantitative kinetic PCR (qPCR) assays based on such primers for different soil samples, and (iii) to assess the diversity and abundance of tfdA-like genes in soil.     

Triskelion Structure of the Gli521 Protein,Involved in the Gliding Mechanism of Mycoplasma mobile

Mycoplasma mobile binds to solid surfaces and glides smoothly and continuously by a unique mechanism. A huge protein, Gli521 (521 kDa), is involved in the gliding machinery, and it is localized in the cell neck, the base of the membrane protrusion. This protein is thought to have the role of force transmission. In this study, the Gli521 protein was purified from M. mobile cells, and its molecular shape was studied. Gel filtration analysis showed that the isolated Gli521 protein forms mainly a monomer in Tween 80-containing buffer and oligomers in Triton X-100-containing buffer. Rotary shadowing electron microscopy showed that the Gli521 monomer consisted of three parts: an oval, a rod, and a hook. The oval was 15 nm long by 11 nm wide, and the filamentous part composed of the rod and the hook was 106 nm long and 3 nm in diameter. The Gli521 molecules form a trimer, producing a “triskelion” reminiscent of eukaryotic clathrin, through association at the hook end. Image averaging of the central part of the triskelion suggested that there are stable and rigid structures. The binding site of a previously isolated monoclonal antibody on Gli521 images showed that the hook end and oval correspond to the C- and N-terminal regions, respectively. Partial digestion of Gli521 showed that the molecule could be divided into three domains, which we assigned to the oval, rod, and hook of the molecular image. The Gli521 molecule''s role in the gliding mechanism is discussed.Mycoplasmas are commensal and occasionally parasitic bacteria with small genomes that lack a peptidoglycan layer (31). Several mycoplasma species form membrane protrusions, such as the headlike structure in Mycoplasma mobile and the attachment organelle in Mycoplasma pneumoniae (15, 19, 21, 22, 25, 33, 34, 36). On solid surfaces, these species exhibit gliding motility in the direction of the protrusion; this motility is believed to be involved in the pathogenicity of mycoplasmas (12, 13, 16, 20, 21). Interestingly, mycoplasmas have no surface flagella or pili, and their genomes contain no genes related to other known bacterial motility systems. In addition, no homologs of motor proteins that are common in eukaryotic motility have been found (11).M. mobile, which was isolated from the gills of a freshwater fish in the early 1980s, is a fast gliding mycoplasma (14). It glides smoothly and continuously on glass at an average speed of 2.0 to 4.5 μm/s, or three to seven times the length of the cell per second, exerting a force of up to 27 pN (8, 9, 24, 25, 32). Previously, we identified huge proteins involved in this gliding mechanism that are localized at the so-called cell neck, the base of the membrane protrusion (17, 26, 30, 35, 37, 39); we also visualized the putative machinery and the binding protein (1, 18, 23) and identified both the direct energy source used and the direct binding target (10, 27, 38). The force generated by the gliding machinery may be supported from inside the cell by a cytoskeletal “jellyfish” structure (28, 29). On the basis of these results, we proposed a working model, called the centipede or power stroke model, where cells are propelled by “legs” composed of Gli349 that repeatedly catch and release sialic acids fixed on the glass surface (5, 19, 21). These legs are driven by the force exerted by P42 through Gli521 molecules, which is supported by the jellyfish structure, based on energy from ATP hydrolysis.The Gli521 protein, which has an unusually high molecular mass (521 kDa), is suggested to have the role of force transmission, because a monoclonal antibody against this protein stops gliding, keeping the cells on a solid surface (35). About 450 molecules are estimated to be clustered in the gliding machinery with other component proteins, although their alignment has not been clarified (35, 37, 39). In this study, we isolated the Gli521 protein and studied its molecular shape using electron microscopy (EM) and biochemical analyses in order to understand the gliding mechanism.