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.

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.

Cell surface modifications induced by calcium ion in the myxobacterium Stigmatella aurantiaca.

Calcium ion induces in the myxobacterium Stigmatella aurantiaca the ability to glide on solid surfaces and to become cohesive (D. F. Gilmore and D. White, J. Bacteriol. 161:113-117, 1985; B. J. Womack, D. F. Gilmore, and D. White, J. Bacteriol. 171:6093-6096, 1989). The addition of calcium ion to the growth medium resulted in the formation of extracellular fibrils, the appearance in the membrane fractions of a 30-kDa protein, and the accumulation in a low-speed centrifugal pellet of 10 polypeptides that cross-reacted with affinity-purified antibody to one of the polypeptides. One of the polypeptides, a 55-kDa protein, was present in the membrane fraction of control cells not incubated with calcium ion and was apparently translocated to the extracellular matrix during incubation in medium containing calcium ion. The 55-kDa protein was immunologically related to a 65-kDa protein located on the fibrils of another myxobacterium, Myxococcus xanthus.

Purification and properties of Myxococcus xanthus cell surface antigen 1604.

A cell surface antigen complex from Zwittergent-solubilized Myxococcus xanthus has been purified by immunoaffinity chromatography with monoclonal antibody (MAb) 1604 and by subsequent gel filtration. We propose that the cell surface antigen (CSA) 1604 complex participates in intercellular interactions. The apparent total molecular mass of the CSA 1604 complex is 200 kilodaltons (kDa), as determined by gel filtration and by electrophoresis and Western immunoblot probing with MAb 1604. The antigen epitope recognized by MAb 1604 is on a 51-kDa polypeptide. The CSA complex also contains 14% neutral carbohydrate and a 23-kDa polypeptide that lacks the 1604 epitope. The carbohydrate is most likely part of a lipopolysaccharide (LPS) associated with the CSA, because an MAb recognizing an O antigen epitope from the LPS of M. xanthus also reacted with CSA 1604 on Western immunoblots. Thus, the 200-kDa CSA complex consists of 97 +/- 6 kDa of protein and many associated LPS molecules. The LPS evidently produces the multiplicity of bands observed on Western immunoblots between 100 and 200 kDa. The association with LPS may contribute to the negative charge of the CSA 1604 complex, which has a pI of 4.3. The CSA was clustered on the surface of intact M. xanthus cells after labeling with MAb 1604 and immunogold. Furthermore, fractionation studies indicated that cells grown on a plastic surface had 50% of their total CSA 1604 in the cytosol, 39% in the membrane fraction, and 8% in the periplasm. Saturable binding studies with 125I-MAb 1604 indicated that there were 2,400 CSA 1604 sites per cell. The Kd for MAb 1604 binding to the cell was 9 nM.

Morphogenesis in Myxococcus xanthus and Myxococcus virescens Myxobacterales.

1. Myxococcus xanthus B and M. virescens V2 were compared with a view to establishing the control of their morphogenetic cycles. Both organisms are typical myxococci and on solid media with low concentrations of nutrient they form fruiting bodies, within which vegetative cells convert to myxospores. Ultrathin sections of vegetative M. virescens resembled those of M. xanthus and contained prominent heavily stained bodies, presumed to be polyphosphate granules. Shadowed preparations showed fimbriae associated with M. xanthus but not with M. virescens. 2. M. xanthus B converted to myxospores in liquid medium in response to certain alcohols. M. virescens V2 produced phase-refractile spheres, which were not viable and had an unusual ultrastructure. 3. The distributions of fruiting bodies on solid media containing 0.02% Casitone were recorded for the two species and were compared with a Poisson distribution. Cells responded to differences in cell density in a manner suggestive of a response to a chemotactic attractant. Cells growing vegetatively and also cells forming fruiting bodies produced 3',5'-cyclic adenosine monophosphate (cAMP) as measured by the incorporation of exogeneous [3H] adenosine into cAMP. 4. The significance of these findings for theories of fruiting body formation are discussed.