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Science 3 October 2008:
Vol. 322. no. 5898, pp. 92 - 96
DOI: 10.1126/science.1159572
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Reports

Molecular Architecture of the "Stressosome," a Signal Integration and Transduction Hub

Jon Marles-Wright1*, Tim Grant2*, Olivier Delumeau1{dagger}, Gijs van Duinen2{ddagger}, Susan J. Firbank1, Peter J. Lewis3, James W. Murray1§, Joseph A. Newman1, Maureen B. Quin1, Paul R. Race1, Alexis Rohou2, Willem Tichelaar2||, Marin van Heel2 and Richard J. Lewis1

1 Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle-upon-Tyne NE2 4HH, UK.
2 Faculty of Natural Sciences, Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK.
3 School of Environmental and Life Sciences, University of Newcastle, Collaghan, Newcastle, NSW 2308, Australia.


Figure 1 Fig. 1. Cryo-EM envelopes of stressosome structures. The cryo-EM envelopes of the three stressosome reconstructions shown in surface representation. (A) The final experimental EM-derived icosahedral molecular envelope of the RsbR146-274:RsbS core is shown in stereo as a gold semitransparent surface, with the map contoured at 3.0{sigma}, and the STAS domains of RsbR (blue) and RsbS (red) that constitute the scaffold of the stressosome are shown as ribbons. The resolution of this icosahedral reconstruction, by the Fourier shell correlation (FSC), 1/2-bit criterion, is 6.5 Å. (B) The RsbR:RsbS stressosome and (C) the RsbR:RsbS:RsbT stressosome ternary complex. To demonstrate clearly the detail in the structure, composite maps were generated where the core and peripheral regions were filtered to their FSC resolutions, contoured at 3{sigma} and 1.5{sigma}, respectively, and then recombined. In both panels, the N-terminal domains of RsbR are colored yellow and the stressosome core, comprising STAS domains from RsbS and RsbR, is colored blue. The density that corresponds to RsbT in (C) is colored purple. (D) "Bean" models showing the arrangements of subunits in the stressosome with an icosahedral mesh of 150 Å diameter. (Top) RsbR146-274:RsbS, with RsbR shown in blue and RsbS in red; (middle) RsbR:RsbS, with the N-terminal domains of RsbR shown in yellow; and (bottom) RsbR:RsbS:RsbT, with RsbT shown in purple. [View Larger Version of this Image (60K GIF file)]
 

Figure 2 Fig. 2. The quasi-atomic structure of the stressosome. (A) Two orthogonal views of the secondary-structure fitofasingle RsbS STAS domain in the icosahedral reconstruction of the RsbR146-274:RsbS. (Top) The electron density map contoured at 3.0{sigma} (to emphasize {alpha} helices); (bottom) the map shown as a semitransparent surface with RsbS fitted and shown as a cartoon continuously color-ramped from N terminus (blue) to C terminus (red). The experimental map is displayed as a semitransparent surface at 3.0{sigma} (to emphasize the {alpha} helices). (B) Orthogonal views of the interactions between RsbS (red) and RsbT (cyan) in the stressosome. (Left) The final RsbR:RsbS:RsbT EM envelope is shown as a semitransparent surface contoured at 1.5{sigma}. (Right) A view of a single molecule of RsbT poised above a single copy of RsbS to catalyze phosphorylation of Ser59 (magenta spheres) on receipt of stress. The residues known to be affected in the binding of RsbT to RsbS, or that are defective in signaling, are shown as spheres and colored green in RsbS and orange in RsbT (29). The peptide backbone of the ATP lid in RsbT is colored yellow. (C) Cartoon views of the sensory extensions of RsbR (blue) in the RsbR:RsbS complex that protrude from the core of the stressosome. The models for the N-terminal globin domain and C-terminal STAS domains are shown with an idealized helix illustrating the neck region (shown in cyan). Mutations that affect the activity of RsbR are shown as spheres and colored green, and phosphorylatable threonine residues are shown as red spheres (17, 19). The experimental map is shown as a semitransparent surface at 1.5{sigma}. [View Larger Version of this Image (48K GIF file)]
 

Figure 3 Fig. 3. Immunolocalization of stressosomes. (A) Pseudocolored image of stressosomes (green) and DNA (red) before induction of stress. A cartoon representation of the Bacillus (below) reveals the boundary of the cell. This cell contains two nucleoids. (B) The stressosomes immunolocalized with RsbR-specific antibodies from (A) are shown in monochrome. (C) A pseudocoloured image of a pair of cells 20 min after the induction of stress when {sigma}B activity is maximal, with stressosomes shown in green and DNA in red. The arrow indicates the position of the division septum between the two cells. (D) As in (C) but in the rsbR-null mutant strain, BSK5, with the green channel levels raised to show a faint background staining but no localization of stressosomes. (E) Monochrome version of (D). Scale bar, 2 µm. [View Larger Version of this Image (76K GIF file)]
 

Figure 4 Fig. 4. Response of B. subtilis to stress. Plots of differential β-galactosidase activity versus inducer concentration for ethanol (A), sodium chloride (B), and sodium azide (C). All data were collected and analyzed as described in the supporting online material. Data were fitted to either a three-parameter Hill equation or a single rectangular hyperbola. Error bars are SEMs calculated from three repeats of an identical experiment. [View Larger Version of this Image (12K GIF file)]
 

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