Cell surface architecture of the cultivated DPANN archaeon Nanobdella aerobiophila.

The DPANN archaeal clade includes obligately ectosymbiotic species. Their cell surfaces potentially play an important role in the symbiotic interaction between the ectosymbionts and their hosts. However, little is known about the mechanism of ectosymbiosis. Here, we show cell surface structures of the cultivated DPANN archaeon Nanobdella aerobiophila strain MJ1T and its host Metallosphaera sedula strain MJ1HA, using a variety of electron microscopy techniques, i.e., negative-staining transmission electron microscopy, quick-freeze deep-etch TEM, and 3D electron tomography. The thickness, unit size, and lattice symmetry of the S-layer of strain MJ1T were different from those of the host archaeon strain MJ1HA. Genomic and transcriptomic analyses highlighted the most highly expressed MJ1T gene for a putative S-layer protein with multiple glycosylation sites and immunoglobulin-like folds, which has no sequence homology to known S-layer proteins. In addition, genes for putative pectin lyase- or lectin-like extracellular proteins, which are potentially involved in symbiotic interaction, were found in the MJ1T genome based on in silico 3D protein structure prediction. Live cell imaging at the optimum growth temperature of 65°C indicated that cell complexes of strains MJ1T and MJ1HA were motile, but sole MJ1T cells were not. Taken together, we propose a model of the symbiotic interaction and cell cycle of Nanobdella aerobiophila.IMPORTANCEDPANN archaea are widely distributed in a variety of natural and artificial environments and may play a considerable role in the microbial ecosystem. All of the cultivated DPANN archaea so far need host organisms for their growth, i.e., obligately ectosymbiotic. However, the mechanism of the ectosymbiosis by DPANN archaea is largely unknown. To this end, we performed a comprehensive analysis of the cultivated DPANN archaeon, Nanobdella aerobiophila, using electron microscopy, live cell imaging, transcriptomics, and genomics, including 3D protein structure prediction. Based on the results, we propose a reasonable model of the symbiotic interaction and cell cycle of Nanobdella aerobiophila, which will enhance our understanding of the enigmatic physiology and ecological significance of DPANN archaea.

Assembly properties of bacterial actin MreB involved in Spiroplasma swimming motility.

Bacterial actin MreB forms filaments composed of antiparallel double-stranded units. The wall-less helical bacterium Spiroplasma has five MreB homologs (MreB1-5), some of which are involved in an intracellular ribbon for driving the bacterium's swimming motility. Although the interaction between MreB units is important for understanding Spiroplasma swimming, the interaction modes of each ribbon component are unclear. Here, we examined the assembly properties of Spiroplasma eriocheiris MreB5 (SpeMreB5), one of the ribbon component proteins that forms sheets. Electron microscopy revealed that sheet formation was inhibited under acidic conditions and bundle structures were formed under acidic and neutral conditions with low ionic strength. We also used solution assays and identified four properties of SpeMreB5 bundles as follows: (I) bundle formation followed sheet formation; (II) electrostatic interactions were required for bundle formation; (III) the positively charged and unstructured C-terminal region contributed to promoting lateral interactions for bundle formation; and (IV) bundle formation required Mg2+ at neutral pH but was inhibited by divalent cations under acidic pH conditions. During these studies, we also characterized two aggregation modes of SpeMreB5 with distinct responses to ATP. These properties will shed light on SpeMreB5 assembly dynamics at the molecular level.

Promethearchaeum syntrophicum gen. nov., sp. nov., an anaerobic, obligately syntrophic archaeon, the first isolate of the lineage 'Asgard' archaea, and proposal of the new archaeal phylum Promethearchaeota phyl. nov. and kingdom Promethearchaeati regn. nov.

An anaerobic, mesophilic, syntrophic, archaeon strain MK-D1T, was isolated as a pure co-culture with Methanogenium sp. strain MK-MG from deep-sea methane seep sediment. This organism is, to our knowledge, the first cultured representative of 'Asgard' archaea, an archaeal group closely related to eukaryotes. Here, we describe the detailed physiology and phylogeny of MK-D1T and propose Promethearchaeum syntrophicum gen. nov., sp. nov. to accommodate this strain. Cells were non-motile, small cocci, approximately 300-750 nm in diameter and produced membrane vesicles, chains of blebs and membrane-based protrusions. MK-D1T grew at 4-30 °C with optimum growth at 20 °C. The strain grew chemoorganotrophically with amino acids, peptides and yeast extract with obligate dependence on syntrophy with H2-/formate-utilizing organisms. MK-D1T showed the fastest growth and highest maximum cell yield when grown with yeast extract as the substrate: approximately 3 months to full growth, reaching up to 6.7×106 16S rRNA gene copies ml-1. MK-D1T had a circular 4.32 Mb chromosome with a DNA G+C content of 31.1 mol%. The results of phylogenetic analyses of the 16S rRNA gene and conserved marker proteins indicated that the strain is affiliated with 'Asgard' archaea and more specifically DHVC1/DSAG/MBG-B and 'Lokiarchaeota'/'Lokiarchaeia'. On the basis of the results of 16S rRNA gene sequence analysis, the most closely related isolated relatives were Infirmifilum lucidum 3507LTT (76.09 %) and Methanothermobacter tenebrarum RMAST (77.45 %) and the closest relative in enrichment culture was Candidatus 'Lokiarchaeum ossiferum' (95.39 %). The type strain of the type species is MK-D1T (JCM 39240T and JAMSTEC no. 115508). We propose the associated family, order, class, phylum, and kingdom as Promethearchaeaceae fam. nov., Promethearchaeales ord. nov., Promethearchaeia class. nov., Promethearchaeota phyl. nov., and Promethearchaeati regn. nov., respectively. These are in accordance with ICNP Rules 8 and 22 for nomenclature, Rule 30(3)(b) for validation and maintenance of the type strain, and Rule 31a for description as a member of an unambiguous syntrophic association.

Architecture of Actinoplanes missouriensis sporangia and zoospores visualized using quick-freeze deep-etch electron microscopy.

The architecture of sporangia and zoospores of Actinoplanes missouriensis was analyzed at a high resolution using quick-freeze deep-etch replica electron microscopy. This analysis revealed that (i) sporangia were surrounded by at least 2 membranous layers with smooth surfaces, (ii) zoospores were enclosed by a fibrillar layer, and (iii) flagella were generated in a restricted area on the zoospore surface.

Structure and Function of Gli123 Involved in Mycoplasma mobile Gliding.

Mycoplasma mobile is a fish pathogen that glides on solid surfaces by means of its own gliding machinery composed of internal and surface structures. In the present study, we focused on the function and structure of Gli123, a surface protein that is essential for the localization of other surface proteins. The amino acid sequence of Gli123, which is 1,128 amino acids long, contains lipoprotein-specific repeats. We isolated the native Gli123 protein from M. mobile cells and a recombinant protein, rGli123, from Escherichia coli. The isolated rGli123 complemented a nonbinding and nongliding mutant of M. mobile that lacked Gli123. Circular dichroism and rotary-shadowing electron microscopy (EM) showed that rGli123 has a structure that is not significantly different from that of the native protein. Rotary-shadowing EM suggested that Gli123 adopts two distinct globular and rod-like structures, depending on the ionic strength of the solution. Negative-staining EM coupled with single-particle analysis revealed that Gli123 forms a globular structure featuring a small protrusion with dimensions of approximately 15.7, 14.7, and 14.1 nm for the "height," major axis and minor axis, respectively. Small-angle X-ray scattering analyses indicated a rod-like structure composed of several tandem globular domains with total dimensions of approximately 34 nm in length and 6 nm in width. Both molecular structures were suggested to be dimers, based on the predicted molecular size and structure. Gli123 may have evolved by multiplication of repeating lipoprotein units and acquired a role for Gli521 and Gli349 assembly. IMPORTANCE Mycoplasmas are pathogenic bacteria that are widespread in animals. They are characterized by small cell and genome sizes but are equipped with unique abilities for infection, such as surface variation and gliding. Here, we focused on a surface-localizing protein named Gli123 that is essential for Mycoplasma mobile gliding. This study suggested that Gli123 undergoes drastic conformational changes between its rod-like and globular structures. These changes may be caused by a repetitive structure common in the surface proteins that is responsible for the modulation of the cell surface structure and related to the assembly process for the surface gliding machinery. An evolutionary process for surface proteins essential for this mycoplasma gliding was also suggested in the present study.

Coordinated regulation of TORC2 signaling by MCC/eisosome-associated proteins, Pil1 and tetraspan membrane proteins during the stress response.

MCCs are linear invaginations of the yeast plasma membrane that form stable membrane microdomains. Although over 20 proteins are localized in the MCCs, it is not well understood how these proteins coordinately maintain normal MCC function. Pil1 is a core eisosome protein and is responsible for MCC-invaginated structures. In addition, six-tetraspan membrane proteins (6-Tsp) are localized in the MCCs and classified into two families, the Sur7 family and Nce102 family. To understand the coordinated function of these MCC proteins, single and multiple deletion mutants of Pil1 and 6-Tsp were generated and their MCC structure and growth under various stresses were investigated. Genetic interaction analysis revealed that the Sur7 family and Nce102 function in stress tolerance and normal eisosome assembly, respectively, by cooperating with Pil1. To further understand the role of MCCs/eisosomes in stress tolerance, we screened for suppressor mutants using the SDS-sensitive phenotype of pil1Δ 6-tspΔ cells. This revealed that SDS sensitivity is caused by hyperactivation of Tor kinase complex 2 (TORC2)-Ypk1 signaling. Interestingly, inhibition of sphingolipid metabolism, a well-known downstream pathway of TORC2-Ypk1 signaling, did not rescue the SDS-sensitivity of pil1Δ 6-tspΔ cells. These results suggest that Pil1 and 6-Tsp cooperatively regulate TORC2 signaling during the stress response.

Unraveling the Host-Selective Toxic Interaction of Cassiicolin with Lipid Membranes and Its Cytotoxicity.

Cassiicolin (Cas), a toxin produced by Corynespora cassiicola, is responsible for Corynespora leaf fall disease in susceptible rubber trees. Currently, the molecular mechanisms of the cytotoxicity of Cas and its host selectivity have not been fully elucidated. Here, we analyzed the binding of Cas1 and Cas2 to membranes consisting of different plant lipids and their membrane disruption activities. Using high-speed atomic force microscopy and confocal microscopy, we reveal that the binding and disruption activities of Cas1 and Cas2 on lipid membranes are strongly dependent on the specific plant lipids. The negative phospholipids, glycerolipids, and sterols are more sensitive to membrane damage caused by Cas1 and Cas2 than neutral phospholipids and betaine lipids. Mature Cas1 and Cas2 play an essential role in causing membrane disruption. Cytotoxicity tests on rubber leaves of Rubber Research Institute of Vietnam (RRIV) 1, RRIV 4, and Prang Besar (PB) 255 clones suggest that the toxins cause necrosis of rubber leaves, except for the strong resistance of PB 255 against Cas2. Cryogenic scanning electron microscopy analyses of necrotic leaf tissues treated with Cas1 confirm that cytoplasmic membranes are vulnerable to the toxin. Thus, the host selectivity of Cas toxin is attained by the lipid-dependent binding activity of Cas to the membrane, and the cytotoxicity of Cas arises from its ability to form biofilm-like structures and to disrupt specific membranes.

Structural dynamics of the chromo-shadow domain and chromodomain of HP1 bound to histone H3K9 methylated peptide, as measured by site-directed spin-labeling EPR spectroscopy.

The structural dynamics of the chromo-shadow domain (CSD) and chromodomain (CD) of human HP1 proteins essential for heterochromatin formation were investigated at the nanosecond and nanometer scales by site-directed spin labeling electron paramagnetic resonance and pulsed double resonance spectroscopy. Distance measurements showed that the spin-labeled CSD of human HP1α and HP1γ tightly dimerizes. Unlike CD-CD interaction observed in fission yeast HP1 in an inactivated state (Canzio et al., 2013), the two CDs of HP1α and HP1γ were spatially separated from each other, dynamically mobile, and ready for a Brownian search for H3K9-tri-methyl(me3) on histones. Complex formation of the CD with H3K9me3 slowed dynamics of the domain due to a decreased diffusion constant. CSD mobility was significantly (∼1.3-fold) lower in full-length HP1α than in HP1γ, suggesting that the immobilized conformation of human HP1α shows an auto-inactivated state. Differential properties of HP1α and HP1γ to form the inactive conformation could be relevant to its physiological role in the heterochromatin formation in a cell.

Tree of motility - A proposed history of motility systems in the tree of life.

Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement-producing protein architectures. Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility.

Use of a Ferritin L134P Mutant for the Facile Conjugation of Prussian Blue in the Apoferritin Cavity.

Since the bullfrog H-ferritin L134P mutant in which leucine 134 is replaced with proline was found to exhibit a flexible conformation in the C3 axis channel, homologous ferritins with the corresponding mutation have often been studied in terms of a mechanism of iron release from the mineral core within the protein cavity. Meanwhile, a ferritin mutant with the flexible channel is an attractive material in developing a method to encapsulate functional molecules larger than mononuclear ions into the protein cavity. This study describes the clathrate with a horse spleen L-ferritin L134P mutant containing Prussian blue (PB) without a frequently used technique, disassembly and reassembly of the protein subunits. The spherical shell of ferritin was confirmed in a TEM image of the clathrate. The produced clathrate (PB@L134P) was soluble in water and reproduced the spectroscopic and electrochemical properties of PB prepared using the conventional method. The catalytic activity for an oxidoreductive reaction with H2O2, one of the major applications of conventional PB, was also observed for the clathrate. The instability of PB in alkaline solutions, limiting its wide applications in aqueous media, was significantly improved in PB@L134P, showing the protective effect of the protein shell. The method developed here shows that horse spleen L-ferritin L134P is a useful scaffold to produce clathrates of three-dimensional complexes with ferritin.

Phylogenetic origin and sequence features of MreB from the wall-less swimming bacteria Spiroplasma.

Spiroplasma are wall-less bacteria which belong to the phylum Tenericutes that evolved from Firmicutes including Bacillus subtilis. Spiroplasma swim by a mechanism unrelated to widespread bacterial motilities, such as flagellar motility, and caused by helicity switching with kinks traveling along the helical cell body. The swimming force is likely generated by five classes of bacterial actin homolog MreBs (SMreBs 1-5) involved in the helical bone structure. We analyzed sequences of SMreBs to clarify their phylogeny and sequence features. The maximum likelihood method based on around 5000 MreB sequences showed that the phylogenetic tree was divided into several radiations. SMreBs formed a clade adjacent to the radiation of MreBH, an MreB isoform of Firmicutes. Sequence comparisons of SMreBs and Bacillus MreBs were also performed to clarify the features of SMreB. Catalytic glutamic acid and threonine were substituted to aspartic acid and lysine, respectively, in SMreB3. In SMreBs 2 and 4, amino acids involved in inter- and intra-protofilament interactions were significantly different from those in Bacillus MreBs. A membrane-binding region was not identified in most SMreBs 1 and 4 unlike many walled-bacterial MreBs. SMreB5 had a significantly longer C-terminal region than the other MreBs, which possibly forms protein-protein interactions. These features may support the functions responsible for the unique mechanism of Spiroplasma swimming.

Multilamellar and Multivesicular Outer Membrane Vesicles Produced by a Buttiauxella agrestis tolB Mutant.

Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important roles in various biological functions. Released vesicles are not uniform in shape, size, or characteristics, and little is known about this diversity of OMVs. Here, we show that deletion of tolB, which encodes a part of the Tol-Pal system, leads to the production of multiple types of vesicles and increases overall vesicle production in the high-vesicle-forming Buttiauxella agrestis type strain JCM 1090. The ΔtolB mutant produced small OMVs and multilamellar/multivesicular OMVs (M-OMVs) as well as vesicles with a striking similarity to the wild type. M-OMVs, previously undescribed, contained triple-lamellar membrane vesicles and multiple vesicle-incorporating vesicles. Ultracentrifugation enabled the separation and purification of each type of OMV released from the ΔtolB mutant, and visualization by quick-freeze deep-etch and replica electron microscopy indicated that M-OMVs are composed of several lamellar membranes. Visualization of intracellular compartments of ΔtolB mutant cells showed that vesicles were accumulated in the broad periplasm, which is probably due to the low linkage between the outer and inner membranes attributed to the Tol-Pal defect. The outer membrane was invaginating inward by wrapping a vesicle, and the precursor of M-OMVs existed in the cell. Thus, we demonstrated a novel type of bacterial OMV and showed that unconventional processes enable the B. agrestis ΔtolB mutant to form unique vesicles.IMPORTANCE Membrane vesicle (MV) formation has been recognized as a common mechanism in prokaryotes, and MVs play critical roles in intercellular interaction. However, a broad range of MV types and their multiple production processes make it difficult to gain a comprehensive understanding of MVs. In this work, using vesicle separation and electron microscopic analyses, we demonstrated that diverse types of outer membrane vesicles (OMVs) were released from an engineered strain, Buttiauxella agrestis JCM 1090T ΔtolB mutant. We also discovered a previously undiscovered type of vesicle, multilamellar/multivesicular outer membrane vesicles (M-OMVs), which were released by this mutant using unconventional processes. These findings have facilitated considerable progress in understanding MV diversity and expanding the utility of MVs in biotechnological applications.

Behaviors and Energy Source of Mycoplasma gallisepticum Gliding.

Mycoplasma gallisepticum, an avian-pathogenic bacterium, glides on host tissue surfaces by using a common motility system with Mycoplasma pneumoniae In the present study, we observed and analyzed the gliding behaviors of M. gallisepticum in detail by using optical microscopes. M. gallisepticum glided at a speed of 0.27 ± 0.09 µm/s with directional changes relative to the cell axis of 0.6 degree ± 44.6 degrees/5 s without the rolling of the cell body. To examine the effects of viscosity on gliding, we analyzed the gliding behaviors under viscous environments. The gliding speed was constant in various concentrations of methylcellulose but was affected by Ficoll. To investigate the relationship between binding and gliding, we analyzed the inhibitory effects of sialyllactose on binding and gliding. The binding and gliding speed sigmoidally decreased with sialyllactose concentration, indicating the cooperative binding of the cell. To determine the direct energy source of gliding, we used a membrane-permeabilized ghost model. We permeabilized M. gallisepticum cells with Triton X-100 or Triton X-100 containing ATP and analyzed the gliding of permeabilized cells. The cells permeabilized with Triton X-100 did not show gliding; in contrast, the cells permeabilized with Triton X-100 containing ATP showed gliding at a speed of 0.014 ± 0.007 µm/s. These results indicate that the direct energy source for the gliding motility of M. gallisepticum is ATP.IMPORTANCE Mycoplasmas, the smallest bacteria, are parasitic and occasionally commensal. Mycoplasma gallisepticum is related to human-pathogenic mycoplasmas-Mycoplasma pneumoniae and Mycoplasma genitalium-which cause so-called "walking pneumonia" and nongonococcal urethritis, respectively. These mycoplasmas trap sialylated oligosaccharides, which are common targets among influenza viruses, on host trachea or urinary tract surfaces and glide to enlarge the infected areas. Interestingly, this gliding motility is not related to other bacterial motilities or eukaryotic motilities. Here, we quantitatively analyze cell behaviors in gliding and clarify the direct energy source. The results provide clues for elucidating this unique motility mechanism.

Production and characterization of recombinant P1 adhesin essential for adhesion, gliding, and antigenic variation in the human pathogenic bacterium, Mycoplasma pneumoniae.

Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6 × His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications.

Detailed Analyses of Stall Force Generation in Mycoplasma mobile Gliding.

Mycoplasma mobile is a bacterium that uses a unique mechanism to glide on solid surfaces at a velocity of up to 4.5 µm/s. Its gliding machinery comprises hundreds of units that generate the force for gliding based on the energy derived from ATP; the units catch and pull sialylated oligosaccharides fixed to solid surfaces. In this study, we measured the stall force of wild-type and mutant strains of M. mobile carrying a bead manipulated using optical tweezers. The strains that had been enhanced for binding exhibited weaker stall forces than the wild-type strain, indicating that stall force is related to force generation rather than to binding. The stall force of the wild-type strain decreased linearly from 113 to 19 picoNewtons after the addition of 0-0.5 mM free sialyllactose (a sialylated oligosaccharide), with a decrease in the number of working units. After the addition of 0.5 mM sialyllactose, the cells carrying a bead loaded using optical tweezers exhibited stepwise movements with force increments. The force increments ranged from 1 to 2 picoNewtons. Considering the 70-nm step size, this small-unit force may be explained by the large gear ratio involved in the M. mobile gliding machinery.

Transformation of crustacean pathogenic bacterium Spiroplasma eriocheiris and expression of yellow fluorescent protein.

Spiroplasma eriocheiris, the cause of crab trembling disease, is a wall-less bacterium, related to Mycoplasmas, measuring 2.0-10.0 µm long. It features a helical cell shape and a unique swimming mechanism that does not use flagella; instead, it moves by switching the cell helicity at a kink traveling from the front to the tail. S. eriocheiris seems to use a novel chemotactic system that is based on the frequency of reversal swimming behaviors rather than the conventional two-component system, which is generally essential for bacterial chemotaxis. To identify the genes involved in these novel mechanisms, we developed a transformation system by using oriC plasmid harboring the tetracycline resistant gene, tetM, which is under the control of a strong promoter for an abundant protein, elongation factor-Tu. The transformation efficiency achieved was 1.6 × 10-5 colony forming unit (CFU) for 1 µg DNA, enabling the expression of the enhanced yellow fluorescent protein (EYFP).

Systematic Structural Analyses of Attachment Organelle in Mycoplasma pneumoniae.

Mycoplasma pneumoniae, a human pathogenic bacterium, glides on host cell surfaces by a unique and unknown mechanism. It forms an attachment organelle at a cell pole as a membrane protrusion composed of surface and internal structures, with a highly organized architecture. In the present study, we succeeded in isolating the internal structure of the organelle by sucrose-gradient centrifugation. The negative-staining electron microscopy clarified the details and dimensions of the internal structure, which is composed of terminal button, paired plates, and bowl complex from the end of cell front. Peptide mass fingerprinting of the structure suggested 25 novel components for the organelle, and 3 of them were suggested for their involvement in the structure through their subcellular localization determined by enhanced yellow fluorescent protein (EYFP) tagging. Thirteen component proteins including the previously reported ones were mapped on the organelle systematically for the first time, in nanometer order by EYFP tagging and immunoelectron microscopy. Two, three, and six specific proteins localized specifically to the terminal button, the paired plates, and the bowl, respectively and interestingly, HMW2 molecules were aligned parallel to form the plate. The integration of these results gave the whole image of the organelle and allowed us to discuss possible gliding mechanisms.

Unitary step of gliding machinery in Mycoplasma mobile.

Among the bacteria that glide on substrate surfaces, Mycoplasma mobile is one of the fastest, exhibiting smooth movement with a speed of 2.0-4.5 µm⋅s(-1) with a cycle of attachment to and detachment from sialylated oligosaccharides. To study the gliding mechanism at the molecular level, we applied an assay with a fluorescently labeled and membrane-permeabilized ghost model, and investigated the motility by high precision colocalization microscopy. Under conditions designed to reduce the number of motor interactions on a randomly oriented substrate, ghosts took unitary 70-nm steps in the direction of gliding. Although it remains possible that the stepping behavior is produced by multiple interactions, our data suggest that these steps are produced by a unitary gliding machine that need not move between sites arranged on a cytoskeletal lattice.

Gliding Direction of Mycoplasma mobile.

UNLABELLED: Mycoplasma mobile glides in the direction of its cell pole by a unique mechanism in which hundreds of legs, each protruding from its own gliding unit, catch, pull, and release sialylated oligosaccharides fixed on a solid surface. In this study, we found that 77% of cells glided to the left with a change in direction of 8.4° ± 17.6° µm(-1) displacement. The cell body did not roll around the cell axis, and elongated, thinner cells also glided while tracing a curved trajectory to the left. Under viscous conditions, the range of deviation of the gliding direction decreased. In the presence of 250 µM free sialyllactose, in which the binding of the legs (i.e., the catching of sialylated oligosaccharides) was reduced, 70% and 30% of cells glided to the left and the right, respectively, with changes in direction of ∼30° µm(-1). The gliding ghosts, in which a cell was permeabilized by Triton X-100 and reactivated by ATP, glided more straightly. These results can be explained by the following assumptions based on the suggested gliding machinery and mechanism: (i) the units of gliding machinery may be aligned helically around the cell, (ii) the legs extend via the process of thermal fluctuation and catch the sialylated oligosaccharides, and (iii) the legs generate a propulsion force that is tilted from the cell axis to the left in 70% and to the right in 30% of cells. IMPORTANCE: Mycoplasmas are bacteria that are generally parasitic to animals and plants. Some Mycoplasma species form a protrusion at a pole, bind to solid surfaces, and glide. Although these species appear to consistently glide in the direction of the protrusion, their exact gliding direction has not been examined. This study analyzed the gliding direction in detail under various conditions and, based on the results, suggested features of the machinery and the mechanism of gliding.

Structural Study of MPN387, an Essential Protein for Gliding Motility of a Human-Pathogenic Bacterium, Mycoplasma pneumoniae.

UNLABELLED: Mycoplasma pneumoniae is a human pathogen that glides on host cell surfaces with repeated catch and release of sialylated oligosaccharides. At a pole, this organism forms a protrusion called the attachment organelle, which is composed of surface structures, including P1 adhesin and the internal core structure. The core structure can be divided into three parts, the terminal button, paired plates, and bowl complex, aligned in that order from the front end of the protrusion. To elucidate the gliding mechanism, we focused on MPN387, a component protein of the bowl complex which is essential for gliding but dispensable for cytadherence. The predicted amino acid sequence showed that the protein features a coiled-coil region spanning residue 72 to residue 290 of the total of 358 amino acids in the protein. Recombinant MPN387 proteins were isolated with and without an enhanced yellow fluorescent protein (EYFP) fusion tag and analyzed by gel filtration chromatography, circular dichroism spectroscopy, analytical ultracentrifugation, partial proteolysis, and rotary-shadowing electron microscopy. The results showed that MPN387 is a dumbbell-shaped homodimer that is about 42.7 nm in length and 9.1 nm in diameter and includes a 24.5-nm-long central parallel coiled-coil part. The molecular image was superimposed onto the electron micrograph based on the localizing position mapped by fluorescent protein tagging. A proposed role of this protein in the gliding mechanism is discussed. IMPORTANCE: Human mycoplasma pneumonia is caused by a pathogenic bacterium, Mycoplasma pneumoniae This tiny, 2-µm-long bacterium is suggested to infect humans by gliding on the surface of the trachea through binding to sialylated oligosaccharides. The mechanism underlying mycoplasma "gliding motility" is not related to any other well-studied motility systems, such as bacterial flagella and eukaryotic motor proteins. Here, we isolated and analyzed the structure of a key protein which is directly involved in the gliding mechanism.