Structure and assembly of a bacterial gasdermin pore.

In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis1-3. Studies of human and mouse GSDM pores have revealed the functions and architectures of assemblies comprising 24 to 33 protomers4-9, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing more than 50 protomers. We determine a cryo-electron microscopy structure of a Vitiosangium bGSDM in an active 'slinky'-like oligomeric conformation and analyse bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning ß-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.

Visualization of Bacterial Microcompartment Facet Assembly Using High-Speed Atomic Force Microscopy.

Bacterial microcompartments (BMCs) are proteinaceous organelles widespread among bacterial phyla. They compartmentalize enzymes within a selectively permeable shell and play important roles in CO2 fixation, pathogenesis, and microbial ecology. Here, we combine X-ray crystallography and high-speed atomic force microscopy to characterize, at molecular resolution, the structure and dynamics of BMC shell facet assembly. Our results show that preformed hexamers assemble into uniformly oriented shell layers, a single hexamer thick. We also observe the dynamic process of shell facet assembly. Shell hexamers can dissociate from and incorporate into assembled sheets, indicating a flexible intermolecular interaction. Furthermore, we demonstrate that the self-assembly and dynamics of shell proteins are governed by specific contacts at the interfaces of shell proteins. Our study provides novel insights into the formation, interactions, and dynamics of BMC shell facets, which are essential for the design and engineering of self-assembled biological nanoreactors and scaffolds based on BMC architectures.

Cell alignment required in differentiation of Myxococcus xanthus.

During fruiting body morphogenesis of Myxococcus xanthus, cell movement is required for transmission of C-factor, a short range intercellular signaling protein necessary for sporulation and developmental gene expression. Nonmotile cells fail to sporulate and to express C-factor-dependent genes, but both defects were rescued by a simple manipulation of cell position that oriented the cells in aligned, parallel groups. A similar pattern of aligned cells normally results from coordinated recruitment of wildtype cells into multicellular aggregates, which later form mature fruiting bodies. It is proposed that directed cell movement establishes critical contacts between adjacent cells, which are required for efficient intercellular C-factor transmission.

Automated identification of Myxobacterial genera using Convolutional Neural Network.

The Myxococcales order consist of eleven families comprising30 genera, and are featured by the formation of the highest level of differential structure aggregations called fruiting bodies. These multicellular structures are essential for their resistance in ecosystems and is used in the primitive identification of these bacteria while their accurate taxonomic position is confirmed by the nucleotide sequence of 16SrRNA gene. Phenotypic classification of these structures is currently performed based on the stereomicroscopic observations that demand personal experience. The detailed phenotypic features of the genera with similar fruiting bodies are not readily distinctive by not particularly experienced researchers. The human examination of the fruiting bodies requires high skill and is error-prone. An image pattern analysis of schematic images of these structures conducted us to the construction of a database, which led to an extractable recognition of the unknown fruiting bodies. In this paper, Convolutional Neural Network (CNN) was considered as a baseline for recognition of fruiting bodies. In addition, to enhance the result the classifier, part of CNN is replaced with other classifiers. By employing the introduced model, all 30 genera of this order could be recognized based on stereomicroscopic images of the fruiting bodies at the genus level that not only does not urge us to amplify and sequence gene but also can be attained without preparation of microscopic slides of the vegetative cells or myxospores. The accuracy of 77.24% in recognition of genera and accuracy of 88.92% in recognition of suborders illustrate the applicability property of the proposed machine learning model.

Morphologies and phylogenetic classification of cellulolytic myxobacteria.

The evolutionary distances of the 16S rDNA sequences in cellulolytic myxobacteria are less than 3%, which units all the strains into a single genus, Sorangium. The size of myxospores and the shape of sporangioles, rather than fruiting body colors or swarm morphologies are consistent with the changes of the 16S rDNA sequences. It is suggested that there are at least two species in the genus Sorangium: one includes strains with small myxospores and spherical sporangioles, and the color of the fruiting bodies is normally orange or brown, though sometimes yellow or black. The second species has large myxospores, polyhedral sporangioles with many inter-cystic substrates, and normally deep brown to black color.

Enhygromyxa salina gen. nov., sp. nov., a slightly halophilic myxobacterium isolated from the coastal areas of Japan.

Six isolates of novel marine myxobacteria, designated strains SHK-1T, SMK-1-1, SMK-1-3, SMK-10, SKK-2, and SMP-6, were obtained from various coastal samples (mud, sands and algae) collected around Japan. All of the isolates had Gram-negative rod-shaped cells, motile by gliding and grew aerobically. They showed bacteriolytic action, fruiting body formation, and NaCl requirement for growth with an optimum concentration of 1.0-2.0% (w/v). In addition, divalent cationic components of seawater, such as Mg2+ or Ca2+, were also needed for growth. The major respiratory quinone was MK-7. The G+C content of genomic DNA ranged from 65.6 to 67.4 mol% (by HPLC). The isolates shared almost identical 16S rDNA sequences, and clustered with a recently described marine myxobacterium, Plesiocystis pacifica, as their closest relative on a phylogenetic tree (95.9-96.0% similarity). Physiological and chemotaxonomic differences between the new strains and strains of the genus Plesiocystis justify the proposal of a new genus. Therefore, we propose to classify the six isolates into a new taxon of marine myxobacteria with the name, Enhygromyxa salina gen. nov., sp. nov. The type strain is SHK-1(T) (JCM 11769(T) = DSM 15217(T) = AJ 110011(T)).

Assembly of robust bacterial microcompartment shells using building blocks from an organelle of unknown function.

Bacterial microcompartments (BMCs) sequester enzymes from the cytoplasmic environment by encapsulation inside a selectively permeable protein shell. Bioinformatic analyses indicate that many bacteria encode BMC clusters of unknown function and with diverse combinations of shell proteins. The genome of the halophilic myxobacterium Haliangium ochraceum encodes one of the most atypical sets of shell proteins in terms of composition and primary structure. We found that microcompartment shells could be purified in high yield when all seven H. ochraceum BMC shell genes were expressed from a synthetic operon in Escherichia coli. These shells differ substantially from previously isolated shell systems in that they are considerably smaller and more homogeneous, with measured diameters of 39±2nm. The size and nearly uniform geometry allowed the development of a structural model for the shells composed of 260 hexagonal units and 13 hexagons per icosahedral face. We found that new proteins could be recruited to the shells by fusion to a predicted targeting peptide sequence, setting the stage for the use of these remarkably homogeneous shells for applications such as three-dimensional scaffolding and the construction of synthetic BMCs. Our results demonstrate the value of selecting from the diversity of BMC shell building blocks found in genomic sequence data for the construction of novel compartments.

Taxonomic analysis of Sorangium strains based on HSP60 and 16S rRNA gene sequences and morphology.

The taxonomy of myxobacteria is based mainly on their morphological characteristics. The genus Sorangium belongs to the myxobacterial suborder Sorangiineae. Strains in the genus were classified either as one species, Sorangium cellulosum, by ignoring divergent morphological characteristics, or into several species; however, the latter classification is based on some dubious morphological characteristics and is inconsistent with the phylogeny constructed from 16S rRNA gene sequences. In this study, two HSP60 (groEL1 and groEL2) genes were amplified and sequenced from 22 Sorangium strains. The groEL1 and groEL2 gene sequences were highly conserved in Sorangium strains, suggesting that these two paralogous genes both play important roles in the life cycle. The phylogeny constructed by the groEL genes was rather consistent with the morphological characteristics of sporangioles. Including information from the phylogenetic analysis and morphological characteristics, it is suggested that the genus Sorangium includes two species.

Byssovorax cruenta gen. nov., sp. nov., nom. rev., a cellulose-degrading myxobacterium: rediscovery of 'Myxococcus cruentus' Thaxter 1897.

A rare, cellulose-decomposing myxobacterium is described, and a new genus name, Byssovorax, is proposed for it. The organism is almost certainly identical to the species 'Myxococcus cruentus' Thaxter 1897, and that species epithet is therefore revived for the novel bacterium: the type strain of Byssovorax cruenta gen. nov., sp. nov., nom. rev. is strain By c2(T) (=DSM 14553(T)=CIP 108850(T)). The G+C content of its DNA is 69.9 mol%. The 16S rRNA gene sequence shows that the species belongs to the family Polyangiaceae, suborder 'Sorangineae', of the Myxococcales. An emended description of the organism is given.

Evidence for motility-related fimbriae in the gliding microorganism Myxococcus xanthus.

An electron-microscopic examination of negatively stained preparations from cell lysates of Myxococcus xanthus and in situ samples of Myxococcus xanthus, Myxococcus virescens, and Myxococcus fulvus has demonstrated the presence of polar fimbriae, about 8.5 nm in diameter, on motile but not nonmotile cells.

Fruiting body morphogenesis in submerged cultures of Myxococcus xanthus.

Induced by starvation, the development of fruiting bodies by Myxococcus xanthus on glass and plastic surfaces under a layer of liquid was followed microscopically. Calcium ions and a neutral pH were required for development of a Myxococcus strain that grew dispersed in liquid culture. Initially asymmetric aggregates later became round, and sporulation followed aggregation.

Social gliding is correlated with the presence of pili in Myxococcus xanthus.

Myxococcus xanthus, an organism whose motility involves cell interactions, normally bears pili. Myxococcal pili are found only at cell poles, are less than 10 nm in diameter, and may be longer than a cell. Myxococcus has two basic patterns of cell movement, adventurous (A-motility) and social (S-motility). Pili are found to be completely correlated with the presence of S-motility. (The S-motility pattern has many groups of cells, almost no single cells, and is governed by a set of genes called system S.) On the other hand, A-motility is in dependent of piliation. (The A-motility pattern has many single, isolated cells and it is governed by a second set of genes called system A.) Electron microscopic examination of more than 40 genetically different strains shows that all A+S+ (wild-type) and A-S+ strains have pili, but A+S- and A-S- strains lack them. Mutations in four different loci belonging to system S were tested and were found to stop productions of pili: the loci sg1A, sg1B, sg1G, and tg1. When brought into contact with tg1+ cells, cells of a tg1- strain, which lack pili, become phenotypically S+, produce pili, and become S-motile. Both motility and the production of pili are transient when initiated in this way. Thus it appears that pili permit cells that are close to one another to move.

Morphogenesis of Stigmatella aurantiaca fruiting bodies.

Scanning electron micrographs of intermediate stages of fruiting body formation in the myxobacterium Stigmatella aurantiaca suggest that fruiting body formation can be divided into several stages distinguishable on the basis of the motile behavior of the cells. Aggregates formed at sites where cells glide as groups in circles or spirals. Thus, each aggregate was surrounded by a wide band of cells. Several streams of cells were pointed toward and connected to the wide band of cells at the base of the aggregate, suggesting directed cell movement toward the aggregate. The pattern of cells at the base of taller, more mature aggregates suggested that groups of cells enter the aggregate from the surrounding band of cells by changing the pitch of their movement, thus creating an ascending spiral. Stalk formation was characterized by a distinctly different pattern, which suggested that single cells emerge from the band of cells and move toward the aggregate, under it, and then vertically to create the stalk. At this stage, the aggregate appeared to be torn from the substrate as it was lifted off the surface. The cells in the completed stalks were well separated, and most had their long axes pointed in a vertical direction. A great deal of the stalk material appeared to be slime in which the cells were embedded and through which they were presumably moving in the live material. Some suggestions regarding factors that may direct the observed morphogenetic movements are discussed.

Fimbriation in gliding bacteria.

Of twenty-two strains of gliding prokaryotes examined, all but three were found to possess polar fimbriae. Fimbriae were not observed on two gliders, while Chloroflexus aurantiacus bore abundant peritrichous fimbriae. In some gliding bacteria, fimbriae were associated with 'holes' surrounded by an electron-transparent collar bearing 12 spike-like projections.

Ultrastructure of Polyangium cellulosum.

Polyangium cellulosum was examined with the transmission electron microscope and the scanning electron microscope. Freeze-fracturing and critical-point-drying techniques were employed with the latter instrument. Critical-point drying seemed to eliminate the distortion of cells and fruiting bodies. These instruments and techniques allowed for a detailed comparison of cell and fruiting-body ultrastructure. Lipid storage materials and mesosomes were found to be constant cell particulates in both vegetative cells and in the shortened myxospores.

Nuclear activity and cell division in the microcysts of Myxococcus fulvus demonstrated by electron micrography of sections.

Electron micrographs of sectioned fruiting bodies confirm that, in Myxococcus fulvus, approximately 10% of mature microcysts show appearances interpretable as typical bacterial nuclear activity and cell division. This suggests a simple mechanism for fruiting body development, and its existence bears upon the validity of the classical descriptions of microcyst maturation in myxobacteria.

Scanning electron microscopy of fruiting body formation by myxobacteria.

Scanning electron microscopy was used to follow fruiting body formation by pure cultures of Chondromyces crocatus M38 and Stigmatella aurantica. Vegetative cells were grown on SP agar and then transferred to Bonner salts agar for fructification. Fruiting in both species commences with the formation of aggregation centers which resemble a fried egg in appearance. In Chondromyces the elevated center or "yolk" region of the aggregation enlarges into a bulbous structure under which the stalk forms and lengthens. At maximum stalk height the bulb extends laterally as bud-like swellings appear. These are immature sporangia and are arranged in a distintive radial pattern around the top of the stalk. This symmetry is lost as more sporangia are formed. Stigmatella does not form a bulb; rather the yolk region of the aggregation center projects upward to form a column-like stalk which is nearly uniform in diameter throughout its length. At maximum stalk height, the terminus of the stalk develops an irregular pattern of bud-like swellings. These differentiate into sporangia. Stalks of 2-week-old mature fruiting bodies of both species appear to be cellular in composition. Stereomicrographs suggest orientation of these cells parallel to the long axis of the stalk. Stalks of 8-week-old fruiting bodies of Chondromyces were acellular and consisted of empty tubules, suggesting that the cells undergo degeneration with aging of the fruiting body.

Reexamination of the role of autolysis in the development of Myxococcus xanthus.

It has been widely reported that 80 to 90% of the cell population undergoes autolysis during sporulation in Myxococcus xanthus. A re-evaluation of the techniques used to measure autolysis in M. xanthus showed that the methods previously used to draw this conclusion are subject to artifacts, which result in a substantial underestimation of the number of cells present during development. We found that at least 80% of the cells that enter development survive throughout fruiting body formation. The cell loss that did occur appeared to be gradual over a period of at least 7 days. Our results suggest that autolysis is not an obligate stage in the development of M. xanthus. The data also showed that sporulating cells pass through a prespore stage in which they become osmotically and physically fragile and therefore difficult to harvest intact. The fragility was correlated with the change from a rod to a spherical shape. As the prespores differentiated into refractile spores, they lost fragility and became amenable to harvesting by standard protocols.