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Mycoplasma mobile is a parasitic bacterium that forms gliding machinery on the cell pole and glides on a solid surface in the direction of the cell pole. The gliding machinery consists of both internal and surface structures. The internal structure is divided into a bell at the front and chain structure extending from the bell. In this study, the internal structures prepared under several conditions were analyzed using negative-staining electron microscopy and electron tomography. The chains were constructed by linked motors containing two complexes similar to ATP synthase. A cylindrical spacer with a maximum diameter of 6 nm and a height of 13 nm, and anonymous linkers with a diameter of 0.9-8.3 nm and length of 14.7±6.9 nm were found between motors. The bell is bowl-shaped and features a honeycomb surface with a periodicity of 8.4 nm. The chains of the motor are connected to the rim of the bell through a wedge-shaped structure. These structures may play roles in the assembly and cooperation of gliding machinery units.

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Escherichia coli produces extracellular vesicles called outer membrane vesicles. In this study, we investigated the mechanism underlying the hypervesiculation of deletion mutant ΔrodZ of E. coli. RodZ forms supramolecular complexes with actin protein MreB and peptidoglycan (PG) synthase, and plays an important role in determining the cell shape. Because mreB is an essential gene, an expression-repressed strain (mreB R3) was constructed using CRISPRi, in which the expression of mreB decreased to 20% of that in the wild-type (WT) strain. In shaken-flask culture, the ΔrodZ strain produced >50 times more vesicles than the WT strain. The mreB-repressed strain mreB R3 showed eightfold higher vesicle production than the WT. ΔrodZ and mreB R3 cells were observed using quick-freeze replica electron microscopy. As reported in previous studies, ΔrodZ cells were spherical (WT cells are rod-shaped). Some ΔrodZ cells (around 7% in total) had aberrant surface structures, such as budding vesicles and dented surfaces, or curved patterns on the surface. Holes in the PG layer and an increased cell volume were observed for ΔrodZ and mreB R3 cells compared with the WT. In conditions of osmotic support using sucrose, the OD660 value of the ΔrodZ strain increased significantly, and vesicle production decreased drastically, compared with those in the absence of sucrose. This study first clarified that vesicle production by the E. coli ΔrodZ strain is promoted by surface budding and a burst of cells that became osmotically sensitive because of their incomplete PG structure.

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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.

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Bacterial spores, known for their complex and resilient structures, have been the focus of visualization using various methodologies. In this study, we applied quick-freeze and replica electron microscopy techniques, allowing observation of Bacillus subtilis spores in high-contrast and three-dimensional detail. This method facilitated visualization of the spore structure with enhanced resolution and provided new insights into the spores and their germination processes. We identified and described five distinct structures: (i) hair-like structures on the spore surface, (ii) spike formation on the surface of lysozyme-treated spores, (iii) the fractured appearance of the spore cortex during germination, (iv) potential connections between small vesicles and the core membrane and (v) the evolving surface structure of nascent vegetative cells during germination.

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Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and deliver microbial molecules to distant target cells in a host. OMVs secreted by probiotic probiotic strain Escherichia coli Nissle 1917 (EcN) have been reported to induce an immune response. In this study, we aimed to increase the OMV production of EcN. The double gene knockout of mlaE and nlpI was conducted in EcN because the ΔmlaEΔnlpI of experimental strain E. coli K12 showed the highest OMV production in our previous report. The ΔmlaEΔnlpI of EcN showed approximately 8 times higher OMV production compared with the parental (wild-type) strain. Quick-freeze, deep-etch replica electron microscopy revealed that plasmolysis occurred in the elongated ΔmlaEΔnlpI cells and the peptidoglycan (PG) had numerous holes. While these phenomena are similar to the findings for the ΔmlaEΔnlpI of K12, there were more PG holes in the ΔmlaEΔnlpI of EcN than the K12 strain, which were observed not only at the tip of the long axis but also in the whole PG structure. Further analysis clarified that the viability of ΔmlaEΔnlpI of EcN decreased compared with that of the wild-type. Although the amount of PG in ΔmlaEΔnlpI cells was about half of that in wild-type, the components of amino acids in PG did not change in ΔmlaEΔnlpI. Although the viability decreased compared to the wild-type, the ΔmlaEΔnlpI grew in normal culture conditions. The hypervesiculation strain constructed here is expected to be used as an enhanced probiotic strain.

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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.

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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.

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Membrane vesicles (MVs) are small spherical structures (20-400 nm) produced by most bacteria and have important biological functions including toxin delivery, signal transfer, biofilm formation, and immunomodulation of the host. Although MV formation is enhanced in biofilms of a wide range of bacterial species, the underlying mechanisms are not fully understood. An opportunistic pathogen, Pseudomonas aeruginosa, causes chronic infections that can be difficult to treat due to biofilm formation. Since MVs are abundant in biofilms, can transport virulence factors to the host, and have inflammation-inducing functions, the mechanisms of enhanced MV formation in biofilms needs to be elucidated to effectively treat infections. In this study, we evaluated the characteristics of MVs in P. aeruginosa PAO1 biofilms, and identified factors that contribute to enhanced MV formation. Vesiculation was significantly enhanced in the static culture; MVs were connected to filamentous substances in the biofilm, and separation between the outer and inner membranes and curvature of the membrane were observed in biofilm cells. By screening a transposon mutant library (8,023 mutants) for alterations in MV formation in biofilms, 66 mutants were identified as low-vesiculation strains (2/3 decrease relative to wild type), whereas no mutant was obtained that produced more MVs (twofold increase). Some transposons were inserted into genes related to biofilm formation, including flagellar motility (flg, fli, and mot) and extracellular polysaccharide synthesis (psl). ΔpelAΔpslA, which does not synthesize the extracellular polysaccharides Pel and Psl, showed reduced MV production in biofilms but not in planktonic conditions, suggesting that enhanced vesiculation is closely related to the synthesis of biofilm matrices in P. aeruginosa. Additionally, we found that blebbing occurred during bacterial attachment. Our findings indicate that biofilm-related factors are closely involved in enhanced MV formation in biofilms and that surface sensing facilitates vesiculation. Furthermore, this work expands the understanding of the infection strategy in P. aeruginosa biofilms.

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Peptidoglycan for elongation in Escherichia coli is synthesized by the Rod complex, which includes RodZ. Although various mutant strains of the Rod complex have been isolated, the relationship between the activity of the Rod complex and the overall physical and chemical structures of the peptidoglycan have not been reported. We constructed a RodZ mutant, termed RMR, and analyzed the growth rate, morphology, and other characteristics of cells producing the Rod complexes containing RMR. The growth and morphology of RMR cells were abnormal, and we isolated suppressor mutants from RMR cells. Most of the suppressor mutations were found in components of the Rod complex, suggesting that these suppressor mutations increase the integrity and/or the activity of the Rod complex. We purified peptidoglycan from wild-type, RMR, and suppressor mutant cells and observed their structures in detail. We found that the peptidoglycan purified from RMR cells had many large holes and different compositions of muropeptides from those of WT cells. The Rod complex may be a determinant not only for the whole shape of peptidoglycan but also for its highly dense structure to support the mechanical strength of the cell wall.

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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.

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Spiroplasma is a genus of pathogenic or commensal cell-wall-deficient helical bacterium. Spiroplasma -specific protein fibril and five classes of bacterial actins, MreB1-5, are involved in a helical ribbon structure responsible for helical-cell morphology and swimming motility. A gene for a hypothetical protein-SPE_1229, 7th protein-has been found in the locus coding mreB s. In this study, we characterized the 7th protein using in silico methods and found that it could be a lipoprotein whose gene is encoded downstream of mreB3 and conserved in a clade of Spiroplasma .

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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.

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Peptidoglycan (PG) is an essential component of the bacterial cell wall that protects the cell from turgor pressure and maintains its shape. In diderm (gram-negative) bacteria, such as Escherichia coli, the PG layer is flexible with a thickness of a 2-6 nm, and its visualization is difficult due to the presence of the outer membrane. The quick-freeze deep-etch replica method has been widely used for the visualization of flexible structures in cell interior, such as cell organelles and membrane components. In this technique, a platinum replica on the surface of a specimen fixed by freezing is observed using a transmission electron microscope. In this chapter, we describe the application of this method for visualizing the E. coli PG layer. We expect that these methods will be useful for the visualization of the PG layer in diverse bacterial species.

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Mycoplasma mobile forms a membrane protrusion at a pole as an organelle. M. mobile cells bind to solid surfaces and glide in the direction of the protrusion. In gliding motility, M. mobile cells catch, pull and release sialylated oligosaccharides on host cells. The observation of Mycoplasma species under light microscopy is useful for the analysis of adhesion ability and the motility mechanism.

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Isolating functional units from large insoluble protein complexes are a complex but valuable approach for quantitative and structural analysis. Mycoplasma mobile, a gliding bacterium, contains a large insoluble protein complex called gliding machinery. The machinery contains several chain structures formed by motors that are evolutionarily related to the F1-ATPase. Recently, we developed a method to purify functional motors and their chain structures using Triton X-100 and a high salt concentration buffer and resolved their structures using electron microscopy. In this chapter, we describe the processes of purification and structural analysis of functional motors for the gliding of M. mobile using negative-staining electron microscopy.

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Mycoplasma mobile is one of the fastest gliding bacteria, gliding with a speed of 4.5 µm s-1. This gliding motility is driven by a concerted movement of 450 supramolecular motor units composed of three proteins, Gli123, Gli349, and Gli521, in the gliding motility machinery. With general experimental setups, it is difficult to obtain the information on how each motor unit works. This chapter describes strategies to decrease the number of active motor units to extract stepwise cell movements driven by a minimum number of motor units. We also describe an unforeseen motility mode in which the leg motions convert the gliding motion into rotary motion, which enables us to characterize the motor torque and energy-conversion efficiency by adding some more assumptions.

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Optical tweezers enable us to measure the force generated by bacterial motility and motor proteins. Here, we describe a method, using optical tweezers and related techniques, to measure the force generated during Mycoplasma gliding. An avidin-conjugated polystyrene bead trapped by a focused laser beam is bound to the surface-biotinylated Mycoplasma cell, which pulls the bead from the trap center of the laser. The force generated by Mycoplasma is calculated from a displacement measured and a spring constant of the laser trap.

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Spiroplasma is a genus of wall-less helical bacteria with swimming motility unrelated to conventional types of bacterial motility machinery, such as flagella and pili. The swimming of Spiroplasma is suggested to be driven by five classes of MreB (MreB1-MreB5), which are members of the actin superfamily. In vitro studies of Spiroplasma MreBs have recently been conducted to evaluate their activities, such as ATPase, which is essential for the polymerization dynamics among classic actin superfamily proteins. In this chapter, we describe methods of purification and Pi release measurement of Spiroplasma MreBs using column chromatography and absorption spectroscopy with the molecular probe, 2-amino-6-mercapto-7-methylpurine riboside (MESG). Of note, the methods described here are applicable to other proteins that possess NTPase activity.

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Liquid-liquid phase separation (LLPS) is involved in various dynamic biological phenomena. In epithelial cells, dynamic regulation of junctional actin filaments tethered to the apical junctional complex (AJC) is critical for maintaining internal homeostasis against external perturbations; however, the role of LLPS in this process remains unknown. Here, after identifying a multifunctional actin nucleator, cordon bleu (Cobl), as an AJC-enriched microtubule-associated protein, we conducted comprehensive in vitro and in vivo analyses. We found that apical microtubules promoted LLPS of Cobl at the AJC, and Cobl actin assembly activity increased upon LLPS. Thus, microtubules spatiotemporally regulated junctional actin assembly for epithelial morphogenesis and paracellular barriers. Collectively, these findings established that LLPS of the actin nucleator Cobl mediated dynamic microtubule-actin cross-talk in junctions, which fine-tuned the epithelial barrier.

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