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Common and distinct structural features of Salmonella injectisome and flagellar basal body.

Kawamoto A, Morimoto YV, Miyata T, Minamino T, Hughes KT, Kato T, Namba K - Sci Rep (2013)

Bottom Line: The bacterial flagellum and injectisome export their component proteins for self-assembly.The injectisome lacks the flagellar basal body C-ring, but a wing-like disc and a globular density corresponding to the export gate platform and ATPase hexamer ring, respectively, are stably attached through thin connectors, revealing yet unidentified common architectures of the two systems.The ATPase ring is far from the disc, suggesting that both apparatuses are observed in an export-off state.

View Article: PubMed Central - PubMed

Affiliation: 1] Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan [2] Riken Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.

ABSTRACT
Bacterial pathogens use an injectisome to deliver virulence proteins into eukaryotic host cells. The bacterial flagellum and injectisome export their component proteins for self-assembly. These two systems show high structural similarities and are classified as the type III secretion system, but it remains elusive how similar they are in situ because the structures of these complexes isolated from cells and visualized by electron cryomicroscopy have shown only the export channel and housing for the export apparatus. Here we report in situ structures of Salmonella injectisome and flagellum by electron cryotomography. The injectisome lacks the flagellar basal body C-ring, but a wing-like disc and a globular density corresponding to the export gate platform and ATPase hexamer ring, respectively, are stably attached through thin connectors, revealing yet unidentified common architectures of the two systems. The ATPase ring is far from the disc, suggesting that both apparatuses are observed in an export-off state.

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Comparison of in situ structures of NC and FBB.The central sections of NC (a) and FBB (b) structures in situ after subtomogram average. (c), (d) The axial sections of isolated NC10 and FBB obtained by cryoEM image analysis are colored yellow and superimposed on their in situ structures, respectively. The cytoplasmic densities designated as the wing-like disc and the globular density in the text are indicated by red and blue arrows and are assigned to InvAC and InvC ATPase in NC and FlhAC and FliI ATPase in FBB, respectively. The white arrow indicates the connection between the wing-like disc and a putative structure of the sorting platform composed of SpaO, OrgA and OrgB in NC and that between the disc and the inner lobe of the C ring in FBB. The C ring conformation shows significant differences in the orientations of the cylindrical wall and the inner lobe between the isolated and in situ structures. OM: outer membrane, CM: cytoplasmic membrane. Scale bar, 10 nm.
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f4: Comparison of in situ structures of NC and FBB.The central sections of NC (a) and FBB (b) structures in situ after subtomogram average. (c), (d) The axial sections of isolated NC10 and FBB obtained by cryoEM image analysis are colored yellow and superimposed on their in situ structures, respectively. The cytoplasmic densities designated as the wing-like disc and the globular density in the text are indicated by red and blue arrows and are assigned to InvAC and InvC ATPase in NC and FlhAC and FliI ATPase in FBB, respectively. The white arrow indicates the connection between the wing-like disc and a putative structure of the sorting platform composed of SpaO, OrgA and OrgB in NC and that between the disc and the inner lobe of the C ring in FBB. The C ring conformation shows significant differences in the orientations of the cylindrical wall and the inner lobe between the isolated and in situ structures. OM: outer membrane, CM: cytoplasmic membrane. Scale bar, 10 nm.

Mentions: Although the structure of FBB isolated from Salmonella enterica has already been revealed by cryoEM image analysis6, we carried out cryoEM image analysis of FBB isolated from the same strain of Salmonellaenterica that we used for in situ structural analysis of NC and FBB, in order to compare the structures of in situ and isolated FBB as precisely as possible. We isolated and purified FBBs from a wild-type strain of Salmonella with modifications in the genetic background to increase the number of flagella per cell and to form the flagellar basal body without growing the filament and then carried out cryoEM single particle image analysis as described in Methods. The structure is shown in Figs. 1 inset, 4 and 5. We did not find any discernible differences in the structural features from the one previously published6.


Common and distinct structural features of Salmonella injectisome and flagellar basal body.

Kawamoto A, Morimoto YV, Miyata T, Minamino T, Hughes KT, Kato T, Namba K - Sci Rep (2013)

Comparison of in situ structures of NC and FBB.The central sections of NC (a) and FBB (b) structures in situ after subtomogram average. (c), (d) The axial sections of isolated NC10 and FBB obtained by cryoEM image analysis are colored yellow and superimposed on their in situ structures, respectively. The cytoplasmic densities designated as the wing-like disc and the globular density in the text are indicated by red and blue arrows and are assigned to InvAC and InvC ATPase in NC and FlhAC and FliI ATPase in FBB, respectively. The white arrow indicates the connection between the wing-like disc and a putative structure of the sorting platform composed of SpaO, OrgA and OrgB in NC and that between the disc and the inner lobe of the C ring in FBB. The C ring conformation shows significant differences in the orientations of the cylindrical wall and the inner lobe between the isolated and in situ structures. OM: outer membrane, CM: cytoplasmic membrane. Scale bar, 10 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842551&req=5

f4: Comparison of in situ structures of NC and FBB.The central sections of NC (a) and FBB (b) structures in situ after subtomogram average. (c), (d) The axial sections of isolated NC10 and FBB obtained by cryoEM image analysis are colored yellow and superimposed on their in situ structures, respectively. The cytoplasmic densities designated as the wing-like disc and the globular density in the text are indicated by red and blue arrows and are assigned to InvAC and InvC ATPase in NC and FlhAC and FliI ATPase in FBB, respectively. The white arrow indicates the connection between the wing-like disc and a putative structure of the sorting platform composed of SpaO, OrgA and OrgB in NC and that between the disc and the inner lobe of the C ring in FBB. The C ring conformation shows significant differences in the orientations of the cylindrical wall and the inner lobe between the isolated and in situ structures. OM: outer membrane, CM: cytoplasmic membrane. Scale bar, 10 nm.
Mentions: Although the structure of FBB isolated from Salmonella enterica has already been revealed by cryoEM image analysis6, we carried out cryoEM image analysis of FBB isolated from the same strain of Salmonellaenterica that we used for in situ structural analysis of NC and FBB, in order to compare the structures of in situ and isolated FBB as precisely as possible. We isolated and purified FBBs from a wild-type strain of Salmonella with modifications in the genetic background to increase the number of flagella per cell and to form the flagellar basal body without growing the filament and then carried out cryoEM single particle image analysis as described in Methods. The structure is shown in Figs. 1 inset, 4 and 5. We did not find any discernible differences in the structural features from the one previously published6.

Bottom Line: The bacterial flagellum and injectisome export their component proteins for self-assembly.The injectisome lacks the flagellar basal body C-ring, but a wing-like disc and a globular density corresponding to the export gate platform and ATPase hexamer ring, respectively, are stably attached through thin connectors, revealing yet unidentified common architectures of the two systems.The ATPase ring is far from the disc, suggesting that both apparatuses are observed in an export-off state.

View Article: PubMed Central - PubMed

Affiliation: 1] Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan [2] Riken Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.

ABSTRACT
Bacterial pathogens use an injectisome to deliver virulence proteins into eukaryotic host cells. The bacterial flagellum and injectisome export their component proteins for self-assembly. These two systems show high structural similarities and are classified as the type III secretion system, but it remains elusive how similar they are in situ because the structures of these complexes isolated from cells and visualized by electron cryomicroscopy have shown only the export channel and housing for the export apparatus. Here we report in situ structures of Salmonella injectisome and flagellum by electron cryotomography. The injectisome lacks the flagellar basal body C-ring, but a wing-like disc and a globular density corresponding to the export gate platform and ATPase hexamer ring, respectively, are stably attached through thin connectors, revealing yet unidentified common architectures of the two systems. The ATPase ring is far from the disc, suggesting that both apparatuses are observed in an export-off state.

Show MeSH