www.sciencemag.org/cgi/content/full/322/5898/92/DC1

Supporting Online Material for

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

Jon Marles-Wright, Tim Grant, Olivier Delumeau, Gijs van Duinen, Susan J. Firbank,

Peter J. Lewis, James W. Murray, Joseph A. Newman, Maureen B. Quin, Paul R. Race, Alexis Rohou, Willem Tichelaar, Marin van Heel,* Richard J. Lewis*

*To whom correspondence should be addressed. E-mail: r.lewis@ncl.ac.uk and

m.vanheel@imperial.ac.uk

Published 3 October 2008, Science 322, 92 (2008) DOI: 10.1126/science.1159572

This PDF file includes:

Materials and Methods SOM Text Figs. S1 to S8 Table S1 References

Other Supporting Online Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/322/5898/92/DC1)

Movie S1

Supporting Online Material to:

Molecular architecture of the ‘stressosome’, a signal integration and

transduction hub

by

Jon Marles-Wright, Tim Grant, Olivier Delumeau, Gijs van Duinen, Susan J. Firbank,

Peter J. Lewis, James W. Murray, Joseph A. Newman, Maureen B. Quin, Paul R.

Race, Alexis Rohou, Willem Tichelaar, Marin van Heel, Richard J. Lewis

Materials and Methods

RsbR:RsbS complexes

The cloning of full-length rsbR and rsbS from Bacillus subtilis strain SG38 to form a

bi-cistronic clone directing the simultaneous expression of both proteins has been

described previously (S1). Using the same procedures, an N-terminal truncation of

rsbR, corresponding to the first 145 residues, was cloned with rsbS using the NdeI and

BamHI restriction sites in pET11a to direct expression of the RsbR146-274:RsbS ‘core’

stressosome complex. Both complexes were expressed in E. coli BL21 (DE3) cells

grown at 37 °C in the presence of ampicillin at 100 μg/ml and induced at an A600 of

0.6 by the addition of IPTG to a final concentration of 1 mM. The cells were

harvested three hours after induction by centrifugation at 4,000 rpm. Both complexes

were purified using the same protocol. Cell pellets were resuspended in 30 ml of lysis

buffer (20 mM Tris.HCl, pH 8.5, 1 mM AEBSF, 1mM DTT) and lysed by sonication

at 4 °C. Soluble proteins were separated from cell debris by centrifugation (25,000 g,

30 min). The filtered supernatant was loaded onto a 25 ml Q-Sepharose column (GE

Healthcare) pre-equilibrated with buffer A (20 mM Tris.HCl pH 8.5, 1 mM DTT) and

bound proteins were eluted over a 200 ml linear gradient of buffer A plus 1 M NaCl.

The fractions containing the complexes were identified by 15 % SDS PAGE. The

complexes were then subjected to size exclusion chromatography using a Superdex

S200 16/60 column (GE Healthcare) pre-equilibrated in buffer B (20 mM Tris.HCl,

pH 8.5, 200 mM NaCl, 1 mM DTT). Those fractions corresponding to the void

volume of the gel filtration column, where the complexes elute, were then dialysed

into 20 mM Tris.HCl, pH 8.5, 1 M (NH4)2SO4, 1 mM DTT and purified further by

hydrophobic interaction chromatography on a 20 ml Phenyl Sepharose column (GE

Healthcare). Bound complexes were eluted with a 100 ml linear gradient of 20 mM

Tris.HCl, pH 8.5. The fractions corresponding to the RsbR:RsbS complexes were

purified to electrophoretic homogeneity by a further size exclusion step, as above, and

the void volume fractions were pooled and concentrated by centrifugation using a 30

kDa molecular weight cut-off centrifugal filter (Amicon).

RsbT

The cloning of RsbT from Bacillus subtilis into pGEX-6p-1 to create an N-terminal

GST fusion protein (to aid purification) has been described previously (S1). The

principal difference in procedures from those already published was the use of E. coli

Rosetta (DE3) cells (Novagen) as an expression vehicle, rather than BL21 (DE3), as

purification yields were improved in this strain.

RsbR:RsbS:RsbT complex

The purified RsbR:RsbS complex was mixed with an excess of RsbT and incubated at

4 °C for one hour before being subjected to gel filtration using a Superdex S200 16/60

column (GE Healthcare) equilibrated in buffer C (20 mM Tris.HCl pH 8.5, 200 mM

NaCl, 1 mM ADP, 1 mM DTT). Fractions corresponding to the void volume of the

column were analysed by 15 % SDS PAGE and only those fractions containing all

three proteins were pooled and concentrated by centrifugation as described above.

Tryptic digest of the RsbR:RsbS complex

The RsbR:RsbS complex at a concentration of ~1 mg/ml was incubated with trypsin

at 37 °C for one hour and then separated by SDS-PAGE. A band running at

approximately 17 kDa was excised from the gel and analysed by Edman sequencing

at the Pinnacle proteomics facility at Newcastle University. The products of

trypsinolysis were also subjected to gel filtration using a Superdex S200 16/60

column as before. Proteins in the fractions corresponding to the void volume, where

large complexes would elute, and those where recombinant N-RsbR normally elutes,

around 70 ml elution, were identified by SDS-PAGE.

Crystallisation and structure solution of MtRsbS

The Moorella thermoacetica RsbS protein (MtRsbS) was cloned, expressed and

purified as described by Quin et al (S2). Single wavelength anomalous dispersion

diffraction data were collected from a single MtRsbS crystal, grown in 20 % w/v PEG

3350, 0.2 M potassium thiocyanate and 0.1 M bis-tris-propane, pH 6.5. These crystals

were cryoprotected by direct transfer to 20 % v/v PEG 300, 20 % w/v PEG 3350, 0.2

M potassium thiocyanate and 0.1 M bis-tris propane, pH 6.5, before flash-cooling in

liquid nitrogen. Data were collected on beamline I04 of the Diamond Light Source at

a wavelength of 0.9699 Å; a total of 720 images were collected with 0.5 ° rotation

angle to a maximum resolution of 2.3 Å. Analysis of the diffraction data, integrated in

MOSFLM (S3) and scaled in SCALA (S4), revealed that the crystals belonged to

space group P212121, with unit cell dimensions of a = 51.96 Å, b = 60.55 Å and c =

88.49 Å, and with two molecules of MtRsbS per asymmetric unit (for data collection

statistics, see Table S1). The selenium heavy atom substructure was solved using the

HKL2MAP (S5) interface to SHELX (S6), and checked against an anomalous

difference Patterson map. The co-ordinates of the four selenium atoms found by

SHELXD were input to SOLVE (S7) and RESOLVE (S8) for phasing and phase

refinement using data to a maximum resolution of 3.5 Å. The phases were improved

so that the majority of both molecules in the dimer could be built by hand in COOT

(S9) to form an initial model. This initial model was used to commence refinement in

REFMAC5 (S10) and CNS (S11) using diffraction data to a maximum resolution of

2.3 Å, interspersed with rounds of manual rebuilding, until refinement converged. The

final refined model contains residues 6-120 in both chains and 43 ordered water

molecules (for refinement statistics, see Table S1).

Electron microscopy

Electron microscopy methods

Samples were prepared for electron cryo-microscopy data acquisition and applied to a

Quantifoil grid (S12). The grids were vitrified by plunging into liquid ethane (S13)

using an FEI “Vitrobot” (S14). The RsbR146-274:RsbS and RsbR:RsbS images were

collected in the FEI CM200-FEG microscope at a magnification of 50K, and the

RsbR:RsbS:RsbT images were collected in the FEI CM300-FEG microscope at a

magnification of 45K. All images were collected onto film following standard “low-

dose” techniques, under liquid-nitrogen conditions using a dose of ~15 e Å2.

Images were scanned using a Nikon Coolscan 9000 and coarsened by a factor of 2,

resulting in a pixel size of 2.54 Å for the RsbR146-274:RsbS and RsbR:RsbS datasets,

and a pixel size of 2.82 Å for the RsbR:RsbS:RsbT images. Individual particles were

selected manually using BOXER (S15). The RsbR146-274:RsbS data set contained

13,317 particles, the RsbR:RsbS complex contained 9,198 and the RsbR:RsbS:RsbT

data set contained 30,253 particles. All subsequent processing was carried out using

the IMAGIC (S16) package assuming D2 point-group symmetry, except in the case of

the RsbR146-274:RsbS reconstruction where icosahedral symmetry was assumed.

CTF estimation was performed using the IMAGIC TRANSFER program (S16) and

corrected via phase flipping. CTF-corrected particles were boxed, band-pass filtered

and normalised. Initial class averages were created following a reference free

alignment procedure (S17). Angles were assigned to the class averages using angular

reconstitution (S18), and 3D reconstructions were calculated using the exact-filter

back projection method. Angular reconstitution/multi-reference alignment refinement

steps were performed iteratively. The resolution of the final 3D reconstructions in D2

pointgroup symmetry was assessed by Fourier shell correlation between two random

halves of the dataset using the half-bit criterion (S19), and they are 7.1 Å for RsbR146-

274:RsbS, 8.0 Å for RsbR:RsbS and 8.3 Å for RsbR:RsbS:RsbT. The resolution of the

icosahedral RsbR146-274:RsbS reconstruction is 6.5 Å.

FSC comparison of the structures

The data used for the RsbR:RsbS and RsbR:RsbS:RsbT reconstructions were taken on

different microscopes and at different magnifications. The final maps were sampled at

2.54 Å and 2.8 Å, respectively. The RsbR:RsbS:RsbT map was therefore upscaled

using a scaling factor of 1.1 to approximately equalise the sampling. The core of each

reconstruction was then masked out using a softened spherical mask with a 35 pixel

radius. The two masked volumes were then compared using the FSC, yielding a

resolution of 8.9 Å using the ½-bit criterion (S19). This process was repeated for the

turrets corresponding to the N-terminal domains of RsbR, except that in this case a

softened shell mask of 20-pixel radius was used. This volume thus contained the N-

RsbR turrets and the areas corresponding to RsbT in the RsbR:RsbS:RsbT

reconstruction. Again, the masked reconstructions were compared via the FSC,

yielding a resolution of 16.8 Å using the ½-bit criterion (S19). Finally, we compared

two, non-symmetry related N-RsbR turrets, by masking them out of the RsbR:RsbS

reconstruction. Each was coarsely centred and cropped into a 48x48x48 pixel box.

The two were then aligned using a full brute-force cross-correlation alignment

approach. After alignment, the two were compared via the FSC, yielding a resolution

of 15.8 Å using the ½-bit criterion (S19).

Determination of the symmetry of the stressosome

The symmetry of the complexes was studied through the eigenimages of centred

particles. Analysis of the RsbR:RsbS complex indicated the structure had a mixed

symmetry Fig. S2B. The whole complex has an outer radius of 150 Å, which is

distinct from a core of 90 Å radius. Whereas the inner core shows two, three and five-

fold symmetry features, compatible with icosahedral symmetry, the outer part (radii

from 90 Å to 150 Å) exhibits clear five-fold and two-fold symmetries with other

features that are distinct from the core symmetry. The RsbR146-274:RsbS eigenimages

also show symmetry features that are consistent with the core of the full length

RsbR:RsbS and are indicative of icosahedral symmetry Fig. S2A. The eigenimages

for the ternary RsbR:RsbS:RsbT complex show very strong external features and the

same core features as the other two structures Fig. S2C.

Attempts to model the RsbR146-274:RsbS core with pseudo-icosahedral symmetry were

successful, with re-projections from the three-dimensional reconstruction Fig. S3

matching the class sum images used for the reconstruction. Attempts to model the full

length RsbR:RsbS complex as an icosahedron resulted in two models, neither of

which led to re-projections that matched fully the input class sums. The final structure

with D2 symmetry was modelled initially onto an icosahedron in vero loci using the

MagnetixTM model building system after making the assumption that each threefold

axis of the pseudo-icosahedron would consist of two dimers of RsbR and one of

RsbS, which is consistent with biochemical data indicating a ratio of two to one for

RsbR to RsbS (Fig. S4). The ratio was estimated by purifying co-expressed

RsbR:RsbS (and RsbR146-274:RsbS) complexes over 4 column steps and subjecting the

samples that were used for structure determination to SDS-PAGE, staining and then

densitometry. The ratio of RsbR:RsbS was found to be 1.93:1 (average of 4 gels) and

for RsbR146-274:RsbS 2.12:1. This allowed us to better comprehend the consequences

of the observed departures from icosahedral symmetry. This insight was then used as

the basis to assign angles to initial class sum images to start the reconstruction using

only D2 pointgroup symmetry. The re-projections from the final refined model using

this symmetry were clearly compatible with the input class sums Fig. S3. A random

distribution of RsbR and RsbS in the stressosome would not have revealed the

symmetry features we see in class sums or eigenimages of the data; both analyses are

independent of the final model. We assume that the RsbR146-274:RsbS complex

assembles in the same way as that of RsbR:RsbS, since both are co-expressed in a bi-

cistronic operon. Final plots for Fourier shell correlations are shown in Fig. S5.

As a further confirmation of the D2 symmetry, a model RsbR:RsbS system, based on

the assumptions above, had D2 symmetry imposed and was re-projected over 10,000

random angles. An eigenimage analysis was conducted on these projections, and the

results compared to the eigenimages obtained for the experimental RsbR:RsbS data

Fig. S6. Eigenimages from the model system clearly matched the experimental

RsbR:RsbS data providing verification of the D2 symmetry.

The cores of the RsbR:RsbS and RsbR:RsbS:RsbT structures are extremely similar,

taking into account the minor differences in resolution of the reconstructions. The N-

RsbR turrets appear different in Figs. 1B and 1C because this domain is somewhat

mobile in the complex, relative to the core, and thus its position and structure is less

well determined than the rigid core. Statistical analysis of the cores and separately the

turrets calculated between the RsbR:RsbS and RsbR:RsbS:RsbT reconstructions

reveal FSC values for the cores of 8.9 Å, and for the turrets 15.8 Å. It should also be

pointed out that the density for RsbT in the latter reconstruction was included in this

comparison, which will obviously contribute to this higher value, especially since the

presence of RsbT may have affected the position of the turrets, inducing a slight

rotation around the local dimer axis. The level of detail appears different for the core

and the turrets in the RsbR:RsbS and RsbR:RsbS:RsbT reconstructions due to the fact

that the turrets are mobile with respect to the core, leading to lower density values in

their part of the 3-D reconstruction and thus a different appearance in thresholded

surface renderings when compared to the rigid cores. The maps were contoured in

Figs. 1B and 1C at a level to generate the best contrast in the structure, in these cases

1.5 σ, which inevitably leads to the loss of the very strong features of the core (Fig.

1A, contoured at 3 σ) as the density level is decreased to reveal the turrets.

Stressosome interpretation with crystallographic models.

The crystallographic model of a single MtRsbS STAS domain was used to build

homology models of the STAS domains of RsbR and RsbS that are at the core of the

protein complex. The STAS domains of MtRsbS, and RsbR and RsbS from B.

subtilis, share 29 and 41 % sequence identity, respectively. A single STAS domain

was initially docked manually into the EM envelope of the core stressosome using

UROX (S20) and icosahedral symmetry operators were used to generate the full

stressosome core. The positions of the STAS domains were further refined using the

URO refinement module in UROX (S20). A real space correlation co-efficient of 87

% and real space R-factor of 46 % was obtained in comparing the final atomic model

to the experimental electron density map. The correct enantiomer of the model was

chosen on the basis of the fitting of α-helices of the STAS domains in the core of the

stressosome.

The N-terminal domain of RsbR (S1) was manually docked into each of the 'turret'

densities and positioned optimally using the modelling tools of CHIMERA (S21) and

refined in SITUS (S22). The initial RsbT-bound model of the stressosome was

obtained by least-squares superimposition of the STAS domain of SpoIIAA in the

SpoIIAA:SpoIIAB complex (S23) onto that of RsbS positioned in the stressosome.

The superimposition resulted in the positioning of SpoIIAB into the electron density

that corresponds to RsbT. The structure of SpoIIAB was then used to generate a

homology model for RsbT, which was then placed at the twenty RsbT-binding sites in

the stressosome and the initial model refined using SITUS (S22).

Immunofluorescence

Polyclonal rabbit anti-RsbR (S24) antibodies were purified using a protein A affinity

column (GE Healthcare) according to the manufacturer’s instructions. B. subtilis 168

trp+ (gift of E. Dervyn, Jouy-en-Josas) and, as a negative control, the rsbR null mutant

B. subtilis BSK5 (S25, gift of W. Haldenwang, Texas) were grown in buffered LB

(S26, S27), diluted from an unsaturated overnight culture, inoculated in fresh buffered

LB at A600 0.05 and grown to A600 of 0.3 at 37 °C with vigorous shaking (t0).

Following removal of the t0 sample, the environmental pathway of the σB-dependent

stress response was induced by the addition of 96 % (v/v) EtOH to the growing

culture to a final concentration of 4 % (v/v). Additional samples were taken at 20

minute intervals (t20, t40 etc) for the next 120 minutes. Sample fixation and

immunofluorescence were performed essentially as described previously (S28) using a

1:2000 dilution of the purified anti-RsbR. Cy3-conjugated goat anti-rabbit antibody

(GE Healthcare) was used at a 1:400 dilution. Finally, samples were counter-stained

with DAPI and mounted in anti-fading agent as described previously (S28).

Fluorescence microscopy was carried out as described previously, except images were

acquired using a Hamamatsu Orca AG cooled CCD and processed using MetaMorph

7.0.1 (Molecular Devices).

LacZ assay

Overnight cultures were diluted to an A600 of between 0.03-0.05 in fresh LB and

grown at 37 oC with shaking in the presence of varying concentrations of stress

inducer, ethanol (0 - 6.0 % in 0.5% increments), sodium chloride (0 – 1 M in 100 mM

increments) or sodium azide (0 – 100 μM in 10 μM increments). σΒ activity was

assayed by monitoring β-galactosidase activity from the chromosomally located σΒ-

dependent SPβ ctc::lacZ reporter (S29). Assays were performed using the method

described by Miller (S30) removing 0.75 ml samples from growing cultures every 5

minutes over a 35 minute period and also every 25 minutes over 450 minutes. The

data were analyzed as described by Costanzo and Ades (S31). β-galactosidase activity

was determined from the slope of the linear portion of a differential rate plot in which

β-galactosidase activity in a fixed volume of culture is plotted against cell density

(Fig. S7). This method corrects for increasing σΒ activity as a consequence of

increasing culture confluence. Gradients of linear differential rates were plotted as a

function of inducer concentration and fitted to an appropriate model (three parameter

Hill or single rectangular hyperbola). All experiments were performed in triplicate.

Although Fig. S7 and Fig. 4 originate from the longer time-scale experiments, the

plots of differential β-galactosidase activity vs. inducer concentration for ethanol,

sodium chloride and sodium azide for the shorter time scale produce near-identical

plots (data not shown).

Results and Discussion

Structure of the stressosome

The core of the stressosome is an icosahedron made up of the STAS domains from

RsbS and RsbR and the secondary structure elements of the proteins can clearly be

seen in the density maps generated for each structure (Fig. 1A-C). To confirm that the

N-terminal domain of RsbR corresponds to the peripheral turrets in the stressosome

structure, the RsbR:RsbS complex was subjected to tryptic digest and further purified

by size exclusion chromatography. Edman peptide sequencing revealed that the ~17

kDa digestion product that did not co-purify with RsbS in the void volume of the gel

filtration column was the N-terminal domain of RsbR. Therefore, the protrusions from

the core correspond to N-RsbR, and in the context of stressosomes found in B.

subtilis, these are the N-terminal signalling domains from the RsbR paralogues. This

conclusion correlates perfectly with the reconstruction of the structure of the complex

of RsbS with N-terminally-truncated RsbR (RsbR146-274:RsbS), which shows only the

icosahedral core of the complex (Fig. 1A, Fig. S2A) formed solely from STAS

domains.

The dimer interfaces of the RsbR and RsbS STAS domains in the stressosome differ

from those observed in other STAS domains; the STAS domain dimers that have been

observed in the PDB (2Q3L, 1VC1, 1H4X, 1H4Y) are all different from each other.

For instance, SpoIIAA has been crystallised in several different crystal forms, two of

which (1H4X and 1H4Y) contain two molecules per asymmetric unit, but the

‘dimers’, and the crystal packing, are quite different (S32) . Furthermore, SpoIIAA is

a monomer according to solution studies (S33) and in the structure of the

SpoIIAA:SpoIIAB complex, the SpoIIAA molecules do not contact one another

(S23). The dimer interfaces of the RsbR and the RsbS homology models contain

conserved leucine/proline and isoleucine/lysine residues at the N-terminus of the

STAS domain that pack against Glu109 and Gln110 in RsbS and Leu254 in RsbR in

the C-terminal helix in the dimer partner. The sequences of RsbR and its paralogues

are conserved in these regions, but there is no conservation in SpoIIAA and RsbV-like

sequences. Sequence alignments generated by probing the NCBI sequence database

with the protein sequences of B. subtilis RsbR, RsbS, SpoIIAA and RsbV confirm that

the key dimer-interface determining residues in the stressosome are not conserved in

those STAS domains that form neither dimers nor the larger stressosome complex Fig.

S8. The modelled three- and five-fold interfaces of RsbR and RsbS map to equivalent

surfaces on the RsbR and RsbS homology models Fig S8 and appear to be rich in

glutamic acid and lysine residues. Whilst conservation is less clear at these interfaces

than at the dimer interfaces there are still a number of more loosely conserved

residues in these regions Fig. S8. The relatively low sequence conservation at the

three- and five-fold interfaces may be relevant to the assembly of stressosomes in vivo

as the complexes must be dynamically constructed from all of the RsbR paralogues;

within the Bacillus cell, discrete stressosome species comprising only a single RsbR

paralogue do not exist, rather they contain a mixture of RsbR and other paralogues

and RsbS (S24, S34, S35, S36, S37). Thus the absence of strict sequence conservation

is consistent with the formation of multiple potential interfaces.

On the outer face of the structure, the site of phosphorylation of RsbS, Ser59, is

accessible to the active site of the RsbT kinase and the density attributed to RsbT in

the RsbR:RsbS:RsbT structure is seen above the RsbS STAS domains. There is one

density feature present per copy of RsbS, which is consistent with densitometry of

SDS-PAGE gels of purified RsbR:RsbS:RsbT complexes, which reveal a 1:1 ratio of

RsbS to RsbT. Superimposition of the SpoIIAA component of the SpoIIAA:SpoIIAB

complex (S23) onto the RsbS STAS domain in the stressosome places SpoIIAB in the

centre of the density that corresponds to RsbT. A homology model of RsbT based on

SpoIIAB has thus been constructed and placed at the appropriate positions in the

structure of the RsbR:RsbS:RsbT complex. The fact that this density is not as strong

as that seen for RsbR and RsbS could be explained by less than unity occupancy by

RsbT at the RsbT-binding site above RsbS, or by slight variations in the conformation

of RsbT, or by the fact that RsbT is mostly β-sheet, a secondary structure feature that

is harder to visualise at these resolutions.

Stressosome activation mechanism

We propose that activation of the complex in response to stress proceeds from a

conformational change in the signalling domains, which transmits the signal to the

STAS domains. The stress response is rapid and transient (S38). Peak σB activity is

achieved after ~20 minutes and is almost complete after about 40 minutes (S38). The

crystal structures of the sensor domain of the RsbR paralogue, YtvA (S39), show a

movement of the C-terminal helix - the ‘Jα’ helix according to the PAS/LOV domain

nomenclature (S40) - in response to changes in the state of the bound flavin co-factor.

YtvA is known to associate with RsbS and to mediate response of B. subtilis to UV

irradiation (S37, S41). How this signal elicits change in the STAS domains and leads

to the activation of RsbT is unknown; there are no gross structural differences seen in

the core of the RsbR:RsbS and RsbR:RsbS:RsbT structures at the resolution limit of

the models. Furthermore, there are limited movements seen in the crystal structures of

the YtvA sensory domain in the light and dark states (S39). However, the Jα helices

in the N-YtvA dimer are not structurally equivalent to each other in either the dark or

light state structures (2PR5 and 2PR6). The conformations of these two helices appear

fixed in the crystal by a series of important crystal contacts and these crystal contacts

may limit the magnitude of the conformational change that can occur without

destroying the crystal.

Several possibilities for stress signal transmission to RsbT logically present

themselves. First and most simply, the signal may be initiated in the sensing domain

and transmitted through the Jα helix to the STAS domains, which undergo structural

changes leading to their phosphorylation by RsbT, and the subsequent release of

RsbT. Second, the conformational change associated with the receipt of a stress signal

in the signalling domain leads to some change in RsbT, generating a greater rate of

nucleotide exchange and thus activation of its kinase function over the competing

phosphatase activity catalysed by RsbX. The conformational changes that occur on

the activation of RsbR and paralogues, such as YtvA, initially in their N-terminal

domains may result in the release of one RsbT molecule. The release of one copy of

RsbT may affect the adjacent RsbT molecule, causing a conformational change that

stimulates the phosphorylation of RsbS by RsbT and the release of RsbT. As each

RsbT disassociates from the stressosome after catalysing phosphoryl transfer to RsbS,

the affinity of neighbouring RsbTs for the stressosome is likely to be reduced and lead

to a rapid burst and co-operative release of multiple copies of RsbT as a function of a

single stress stimulus. The near simultaneous release of several copies of RsbT from

the stressosome is required to stimulate RsbU, which in vitro requires a 10-fold molar

excess of RsbT to RsbU for maximal phosphatase activity (S42). As the dimeric RsbU

only binds two monomers of RsbT, this requirement for a 10-fold molar excess more

likely reflects the weak affinity – 4 μM – of the RsbT:RsbU complex (S26), which is

ideally suited and balanced for a sensitive signalling switch that has to be turned on

and off to avoid inappropriate activation of σB. The concept that the stress response is

co-operative is supported by the data reported herein on the activation of a σB-

responsive ctc-lacZ reporter gene fusion. In response to both ethanol and salt, σB-

stimulating environmental insults (S35) that are likely to enter the cell by different

routes, the LacZ assay shows a co-operative response (Fig. 4). Iber et al (S43) have

previously shown that the SpoIIAA:SpoIIAB interaction is allosteric, which is key for

the control of σF and subsequent cell fate determination in Bacillus. SpoIIAA,

SpoIIAB and σF are homologous to RsbV, RsbW and σB and thus it could be

expected for allostery to be maintained in this system. Indeed, there is some evidence

for the co-operative binding of RsbV monomers to RsbW dimers (S27), but it is

highly unlikely that these binary interactions will have as such large an effect as we

observe in the cell during these experiments (Fig. 4). Clearly the cellular response to

these independent environmental stresses is complex and the precise point, or points,

at which allostery stems has yet to be clarified, and here a bio-mathematics approach

may be instructive in understanding this entire pathway at a systems level. Since co-

operativity is most commonly associated with oligomers, the stressosome is the

obvious point at which co-operativity will ensue in the response to environmental

insult.

Supplementary Figures

Fig. S1. Schematic of the σB pathway. Pre-stress, the anti-sigma factor RsbW sequesters σB and prevents it from directing RNA polymerase to σB-controlled promoters. In this state, RsbV is phosphorylated (RsbV-P) by the kinase activity of RsbW and hence RsbV is inactivated. Under stressful conditions, RsbV becomes dephosphorylated by one of two phosphatases and attacks the RsbW:σB complex and liberates σB to direct transcription of its regulon to provide the cell with stress-resistance. RsbV is thus the point at which the environmental and energy stress responses converge. Under energetic stress, the phosphatase RsbP is activated by RsbQ and dephosphorylates RsbV-P to allow it to form complexes with RsbW. The environmental stress response is somewhat more complicated with a large protein complex, termed the stressosome, acting to sequester the RsbU phosphatase-activator, RsbT, in the absence of stress. Under environmentally stressful conditions, RsbT phosphorylates the STAS domains of the stressosome proteins and disassociates, because of a reduced affinity for the phosphorylated proteins. RsbT switches its binding partner from the stressosome to the phosphatase RsbU. The RsbT:RsbU complex activates RsbV by its dephosphorylation. The phosphatase RsbX acts to remove phosphoryl groups from the stressosome and to mediate the duration of the stress response by ‘resetting’ the system. Ringed plus signs indicate positive regulators of σB activity, while ringed minus signs indicate those that are negative regulators.

Fig. S2. Eigenimages of stressosome cryo-EM datasets. Eigenimages showing symmetry elements associated with the RsbR146-274:RsbS stressosome core (A), the RsbR:RsbS stressosome showing the mixed symmetry that appears due to the N-terminal projections of RsbR in the RsbR:RsbS stressosome complex (B), and the RsbR:RsbS:RsbT structure (C) showing similar symmetry mismatch features to (B).

Fig. S3. Re-projections and class-sum images Class-sum images (top line in A, B, C) and re-projections (bottom line in A, B, C) for the RsbR146-274:RsbS reconstruction (A), the RsbR:RsbS reconstruction (B) and the RsbR:RsbS:RsbT reconstruction (C). In all three cases the re-projections match the class-sum images.

Fig. S4. Ratio of RsbR:RsbS in the stressosome (A) RsbR:RsbS and (B) RsbR146-274:RsbS complexes used in this study were co-expressed from a bicistronic operon and purified over 4 column steps. The samples that were used for structure determination were run on a SDS-PAGE gel, stained with Coomassie and subjected to densitometry. The ratios of RsbR:RsbS (A) and RsbR146-

274:RsbS (B) are indicated to the left hand side. The RsbR band in A appears heavy in comparison to RsbS, but this is because RsbR is approximately twice the mass of RsbS. In B, the bands corresponding to RsbR146-274 and RsbS are sufficiently resolved in order to determine their ratios.

Fig. S5. Fourier shell correlation (FSC) plots FSC for the icosahedral reconstruction of RsbR146-274:RsbS (A). The half-bit curve crosses the FSC curve at 6.5 Å. FSC for the D2 reconstruction of RsbR146-274:RsbS (B). The half-bit curve crosses the FSC curve at 7.1 Å. FSC for the D2 reconstruction of RsbR:RsbS (C). The half bit curve crosses the FSC curve at 8.0 Å. FSC for the D2 reconstruction of RsbR:RsbS:RsbT (D). The half-bit curve crosses the FSC curve at 8.3 Å.

Fig. S6. Experimental eigenimages confirm D2 symmetry (A) Eigenimages from the full length RsbR:RsbS reconstruction which appear to show a clear 10-fold symmetry (B) Eigenimages from re-projections of a model RsbR:RsbS with imposed D2 symmetry, which also exhibit a clear 10-fold symmetry, suggesting the 10-fold feature is a consequence of the centring and not a structural feature of the complex.

Fig S7. Example data from a β-galactosidase stress induction experiment using ethanol. (A) Growth curves for cells cultured in different concentrations of ethanol. (B) Corresponding β-galactosidase activities for samples shown in (A). (C) Plots of β-galactosidase activity vs. A600nm, with data fitted to a linear polynomial. Points are as follows, 0% ethanol, filled circles; 0.5% ethanol, filled squares; 1.0% ethanol, filled downward triangles; 1.5% ethanol, open squares; 2.0% ethanol, filled diamonds; 2.5% ethanol, open squares; 3.0% ethanol, open diamonds; 3.5% ethanol, open upwards triangles; 4.0% ethanol, filled upward triangles; 4.5% ethanol, outlined open upward triangles; 5.0% ethanol, dots; 5.5% ethanol, filled small circles and 6% ethanol, outlined open circles.

Fig. S8. Interfaces of RsbR and RsbS in the stressosome. Contact residues for the RsbS (left) and RsbR (right) STAS domains in the stressosome core. Surface contact residues for the STAS domain dimer interfaces are shown as yellow spheres, red at the five-fold interfaces and green at the three-fold axes. The conservation of interface residues in STAS domain proteins is illustrated in a sequence alignment of the B. subtilis STAS domain proteins SpoIIAA and RsbV, which do not form icosahedra, compared to RsbR and RsbS. Residues in contact sites are shown with identical colouring to the two panels above.

Video S1. Demonstration of the fit of the MtRsbS STAS domain into the D2 RsbR146-274:RsbS reconstruction. The video slabs through the density to demonstrate the quality of the fit. Secondary structure elements are clearly visible within the EM map and fit well with the crystal structure.

Table S1. MtRsbS data collection and refinement statistics Data collection Space group P212121 Unit cell dimensions (Å) a = 51.96, b = 60.55, c = 88.49 Resolution range (Å) 20.0 – 2.3 I/σ(I) 22.4 (6.2) Rmerge (%) 11.4 (47.8) Completeness (%) 99.9 (96.7) Anomalous completeness (%) 99.8 (93.5) No. of measurements 145 887 (4 295) No. of unique reflections 10 052 (342) Multiplicity 14.5 (12.6) Anomalous multiplicity 7.8 (8.1) Wilson B (Å2) 43.0 Refinement Rwork 0.228 (0.322) Rfree 0.280 (0.424) rmsd bond distance (Å) 0.012 rmsd bond angles (°) 1.458 No. of non-H atoms 1811 Mean B (Å2) 23.169 Ramachandran allowed region 100 % (Where values in parentheses refer to the highest resolution shell, 2.36 – 2.30 Å)

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