Polymerization of a single protein of the pathogen Yersinia enterocolitica into needles punctures eukaryotic cells.

A number of pathogenic, Gram-negative bacteria are able to secrete specific proteins across three membranes: the inner and outer bacterial membrane and the eukaryotic plasma membrane. In the pathogen Yersinia enterocolitica, the primary structure of the secreted proteins as well as of the components of the secretion machinery, both plasmid-encoded, is known. However, the mechanism of protein translocation is largely unknown. Here we show that Y. enterocolitica polymerizes a 6-kDa protein of the secretion machinery into needles that are able to puncture the eukaryotic plasma membrane. These needles form a conduit for the transport of specific proteins from the bacterial to the eukaryotic cytoplasm, where they exert their cytotoxic activity. In negatively stained electron micrographs, the isolated needles were 60-80 nm long and 6-7 nm wide and contained a hollow center of about 2 nm. Our data indicate that it is the polymerization of the 6-kDa protein into these needles that provides the force to perforate the eukaryotic plasma membrane.

Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins.

The non-fimbrial adhesins, YadA of enteropathogenic Yersinia species, and UspA1 and UspA2 of Moraxella catarrhalis, are established pathogenicity factors. In electron micrographs, both surface proteins appear as distinct 'lollipop'-shaped structures forming a novel type of surface projection on the outer membranes. These structures, amino acid sequence analysis of these molecules and yadA gene manipulation suggest a tripartite organization: an N-terminal oval head domain is followed by a putative coiled-coil rod and terminated by a C-terminal membrane anchor domain. In YadA, the head domain is involved in autoagglutination and binding to host cells and collagen. Analysis of the coiled-coil segment of YadA revealed unusual pentadecad repeats with a periodicity of 3.75, which differs significantly from the 3.5 periodicity found in the Moraxella UspAs and other canonical coiled coils. These findings predict that the surface projections are formed by oligomers containing right- (Yersinia) or left-handed (Moraxella) coiled coils. Strikingly, sequence comparison revealed that related proteins are found in many proteobacteria, both human pathogenic and environmental species, suggesting a common role in adaptation to specific ecological niches.

Gliding motility in cyanobacterial: observations and possible explanations.

Cyanobacteria are a morphologically diverse group of phototrophic prokaryotes that are capable of a peculiar type of motility characterized as gliding. Gliding motility requires contact with a solid surface and occurs in a direction parallel to the long axis of the cell or filament. Although the mechanistic basis for gliding motility in cyanobacteria has not been established, recent ultrastructural work has helped to identify characteristic structural features that may play a role in this type of locomotion. Among these features are the distinct cell surfaces formed by specifically arranged protein fibrils and organelle-like structures, which may be involved in the secretion of mucilage during locomotion. The possible role of these ultrastructural features, as well as consequences for understanding the molecular basis of gliding motility in cyanobacteria, are the topic of this review.

The junctional pore complex, a prokaryotic secretion organelle, is the molecular motor underlying gliding motility in cyanobacteria.

BACKGROUND: Whereas most bacteria move by means of flagella, some prokaryotes move by gliding. In cyanobacteria, gliding motility is a slow uniform motion which is invariably accompanied by a continuous secretion of slime. On the basis of these characteristics, a model has been proposed in which the gliding motility of cyanobacteria depends on the steady secretion of slime using specific pores, as well as the interaction of the slime with the filament surface and the underlying substrate. RESULTS: The structures of the pore apparatus of two different filamentous cyanobacteria have been characterized. In both species, pores are formed by a hitherto uncharacterized type of prokaryotic organelle that spans the entire multilayered cell wall and possesses structural properties expected for an organelle that is involved in the rapid secretion of extracellular carbohydrates. Light microscopic observations of the secretion process provided direct evidence that the pore complexes are the actual sites of slime secretion, that the secreted slime fibrils are elongated at about the same rate as the filament glides (up to 3 micrometer s-1), and that gliding movements are caused directly by the secretion of slime. CONCLUSIONS: It has been known for a long time that carbohydrate secretion has an important role in the gliding motility of various prokaryotes. Our results strongly suggest that slime secretion is not only a prerequisite for this peculiar type of motility in cyanobacteria, but also directly generates the necessary thrust for locomotion.

Structural and biochemical analysis of the sheath of Phormidium uncinatum.

The sheath of the filamentous, gliding cyanobacterium Phormidium uncinatum was studied by using light and electron microscopy. In thin sections and freeze fractures the sheath was found to be composed of helically arranged carbohydrate fibrils, 4 to 7 nm in diameter, which showed a substantial degree of crystallinity. As in all other examined motile cyanobacteria, the arrangement of the sheath fibrils correlates with the motion of the filaments during gliding motility; i.e., the fibrils formed a right-handed helix in clockwise-rotating species and a left-handed helix in counterclockwise-rotating species and were radially arranged in nonrotating cyanobacteria. Since sheaths could only be found in old immotile cultures, the arrangement seems to depend on the process of formation and attachment of sheath fibrils to the cell surface rather than on shear forces created by the locomotion of the filaments. As the sheath in P. uncinatum directly contacts the cell surface via the previously identified surface fibril forming glycoprotein oscillin (E. Hoiczyk and W. Baumeister, Mol. Microbiol. 26:699-708, 1997), it seems reasonable that similar surface glycoproteins act as platforms for the assembly and attachment of the sheaths in cyanobacteria. In P. uncinatum the sheath makes up approximately 21% of the total dry weight of old cultures and consists only of neutral sugars. Staining reactions and X-ray diffraction analysis suggested that the fibrillar component is a homoglucan that is very similar but not identical to cellulose which is cross-linked by the other detected monosaccharides. Both the chemical composition and the rigid highly ordered structure clearly distinguish the sheaths from the slime secreted by the filaments during gliding motility.

Oscillin, an extracellular, Ca2+-binding glycoprotein essential for the gliding motility of cyanobacteria.

Electron microscopic studies have demonstrated that various gliding filamentous cyanobacteria have trichome surfaces with a common structural organization. They contain an S-layer attached to the outer membrane and an array of parallel fibrils on top of the S-layer. In all species studied, the helical arrangement of these fibrils corresponds to the sense of rotation of the organism during the gliding movement. We have investigated the surface fibrils of Phormidium uncinatum using electron microscopic, spectroscopic and biochemical techniques. The fibrils consist of a single rod-shaped protein, which we refer to as oscillin. Oscillin is a 646 amino acid residue protein (Mr 65807; pI 3.63) and appears to be glycosylated. Sequence analysis reveals a two-domain structure: a 554 residue domain contains 46 repeats of a Ca2+-binding motif; it is followed by a 92 residue C-terminal domain, which might mediate its export. Filaments that do not express oscillin lose their ability to move. Homology studies suggest that similar proteins play comparable roles in other motile cyanobacteria. The structure of oscillin appears to favour a passive role in gliding.

Envelope structure of four gliding filamentous cyanobacteria.

The cell walls of four gliding filamentous Oscillatoriaceae species comprising three different genera were studied by freeze substitution, freeze fracturing, and negative staining. In all species, the multilayered gram-negative cell wall is covered with a complex external double layer. The first layer is a tetragonal crystalline S-layer anchored on the outer membrane. The second array is formed by parallel, helically arranged surface fibrils with diameters of 8 to 12 nm. These fibrils have a serrated appearance in cross sections. In all cases, the orientation of the surface fibrils correlates with the sense of revolution of the filaments during gliding, i.e., clockwise in both Phormidium strains and counterclockwise in Oscillatoria princeps and Lyngbya aeruginosa. The lack of longitudinal corrugations or contractions of the surface fibrils and the identical appearances of motile and nonmotile filaments suggest that this structure plays a passive screw thread role in gliding. It is hypothesized that the necessary propulsive force is generated by shear forces between the surface fibrils and the continuing flow of secreted extracellular slime. Furthermore, the so-called junctional pores seem to be the extrusion sites of the slime. In motile cells, these pores exhibit a different staining behavior than that seen in nonmotile ones. In the former, the channels of the pores are filled with electron-dense material, whereas in the latter, the channels appear comparatively empty, highly contrasting the peptidoglycan. Finally, the presence of regular surface structures in other gliding prokaryotes is considered an indication that comparable structures are general features of the cell walls of gliding microbes.