Associations between Bacillus subtilis {sigma}B regulators in cell extracts

Shrin Kuo, Shuyu Zhang, Robyn L. Woodbury{dagger} and W. G. Haldenwang

Department of Microbiology and Immunology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA

Correspondence
W. G. Haldenwang
Haldenwang{at}uthscsa.edu


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The general stress regulon of Bacillus subtilis is induced by the activation of the {sigma}B transcription factor. Activation of {sigma}B occurs as a consequence of the dephosphorylation of its positive regulator RsbV by one of two phosphatases that respond to either physical or nutritional stress. The physical stress phosphatase (RsbU) requires a second protein (RsbT) for activity. Stress is thought to initiate a process that triggers the release of RsbT from a large inhibitory complex composed of multiple copies of two protein species, RsbR (and/or its paralogues) and RsbS. The stress-derived signal driving RsbT release is unknown, but it fails to develop in B. subtilis lacking either ribosome protein L11 or the ribosome-associated protein Obg. RsbR, RsbS, RsbT, Obg and ribosomes elute in common high-molecular-mass fractions during gel-filtration chromatography of crude B. subtilis extracts. This paper reports the investigation of the basis of this coelution by the examining of associations between these proteins in extracts prepared from wild-type and mutant B. subtilis, and Escherichia coli engineered to express RsbR, RsbS and RsbT. Large RsbR/RsbS complexes, distinct from ribosomes, were detected in extracts of both B. subtilis and E. coli. In E. coli, high-molecular-mass forms of RsbS were less abundant when RsbR was absent, but in B. subtilis, only when both RsbR and its principal paralogues were missing from the extract was this form less abundant. This finding is consistent with the notion that the RsbR paralogues, present in B. subtilis but not E. coli, can substitute for RsbR in such complexes. RsbT was not bound to RsbR/RsbS in any extract that was examined, including one prepared from a B. subtilis strain with an RsbS variant (RsbS59SA) that is believed to continuously associate with RsbT. The high-molecular-mass forms of RsbT were found to be Triton-sensitive and independent of any other B. subtilis protein for their formation. These probably represent RsbT aggregates. The data suggest that the contribution of ribosomes/Obg to {sigma}B activation does not involve formation of a stable association between these proteins and the Rsb complex. In addition, the binding of RsbT to RsbS/RsbR appears to be more labile than the binding between the previously analysed Rsb proteins which form inhibitory complexes. This, and the apparent proclivity of RsbT to aggregate, suggests an inherent instability in RsbT which may play a role in its regulation.


{dagger}Present address: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The general stress regulon (GSR) of Bacillus subtilis is a collection of more than 200 genes whose expression is enhanced following exposure to physical (e.g. heat shock, osmotic shock, ethanol) or nutritional (e.g. azide treatment, glucose or phosphate limitation) stress (Hecker et al., 1996; Petersohn et al., 1999; Price et al., 2001; Voelker et al., 1994). Induction of the GSR is triggered by the activation of the {sigma}B transcription factor, an RNA-polymerase-binding protein that directs the resulting holoenzyme to GSR promoters (Benson & Haldenwang, 1992, 1993a, b; Boylan et al., 1992, 1993). {sigma}B is constitutively coexpressed with seven of its principal regulators (Regulator of Sigma B: rsbR, rsbS, rsbT, rsbU, rsbV, rsbW and rsbX) in an operon that is probably recognized by the cell's housekeeping {sigma} factor, {sigma}A (Kalman et al., 1990; Wise & Price, 1995). An internal {sigma}B-dependent promoter upregulates the expression of the operon's four downstream genes (rsbV, rsbW, sigB and rsbX) during periods of {sigma}B activity (Benson & Haldenwang, 1992; Boylan et al., 1992, 1993; Kalman et al., 1990).

RsbV and RsbW are the primary {sigma}B regulators. As illustrated in Fig. 1, RsbW is a {sigma}B-binding protein, able to sequester {sigma}B into an association that prevents it from joining RNA polymerase (Benson & Haldenwang, 1993b; Boylan et al., 1993). RsbV is an antagonist to the RsbW–{sigma}B complex (Benson & Haldenwang, 1993b; Dufour & Haldenwang, 1994). RsbW forms mutually exclusive complexes with either RsbV or {sigma}B (Delumeau et al., 2002; Dufour & Haldenwang, 1994). The ability of RsbV to compete for RsbW is dependent on RsbV's phosphorylation state (Dufour & Haldenwang, 1994). RsbW is both a binding protein and an RsbV-specific kinase. In unstressed B. subtilis, RsbW-dependent phosphorylation of RsbV inactivates RsbV as a potential {sigma}B release factor (Alper et al., 1996; Dufour & Haldenwang, 1994). The dephosphorylation and reactivation of RsbV-P are effected by two stress-activated phosphatases, RsbP and RsbU. Each of these enzymes responds to a particular class of stress: RsbP to nutritional stress and RsbU to physical stress (Kang et al., 1996; Vijay et al., 2000; Voelker et al., 1995a, b, 1996; Yang et al., 1996).



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Fig. 1. Model of {sigma}B activation. {sigma}B is held inactive in unstressed B. subtilis as a complex with an anti-{sigma}B protein, RsbW (W). {sigma}B is freed from RsbW when a release factor, RsbV (V), binds to RsbW. In unstressed B. subtilis, RsbV is inactive due to an RsbW-catalysed phosphorylation (V-P). Physical stress activates an RsbV-P phosphatase, RsbU (U), which reactivates RsbV. RsbT (T) is the RsbU activator. In unstressed B. subtilis, RsbT is believed to be bound to a negative regulator, RsbS (S), in a large complex composed of RsbR and a family of paralogous proteins (R*) that are thought to facilitate the RsbR/T interactions. Upon exposure to stress, RsbT phosphorylates and inactivates RsbS, freeing itself to activate the RsbU phosphatase. Obg, a ribosome-associated GTPase, and a ribosome-mediated process play essential, but unknown, roles in the activation of RsbT. RsbS-P is dephosphorylated and reactivated by a phosphatase, RsbX (X), that is encoded by one of the genes downstream of the sigB operon's {sigma}B-dependent promoter. RsbX levels become elevated when {sigma}B is active. This may facilitate the dephosphorylation of RsbS-P and the return of RsbT to an inactive complex with RsbS. Nutritional stress activates a separate pathway in which a novel RsbV-P phosphatase (P) and an associated protein (Q) dephosphorylate RsbV. The nutritional stress signal is unknown, but the triggering of this pathway coincides with a drop in ATP and is inhibited in RelA B. subtilis. The model is based on the references given in the text.

 
The nutritional stress phosphatase (RsbP) is cotranscribed with a predicted hydrolase (RsbQ) that is needed for RsbP's activity (Brody et al., 2001; Vijay et al., 2000). The metabolic inducer of RsbP/Q is unknown, but the conditions that trigger its activation are associated with a decrease in ATP levels, suggesting that changes in nucleotide pools may be involved in the activation process (Voelker et al., 1996; Zhang & Haldenwang, 2003).

The phosphatase that responds to physical stress (RsbU) requires an additional protein, RsbT, for activity (Yang et al., 1996). A current model envisions RsbT to be held inactive in unstressed B. subtilis in a complex with its binding partner RsbS. Exposure to physical stress enables RsbT to phosphorylate RsbS. This results in the release of RsbT and its activation of RsbU (Akbar et al., 1997; Chen et al., 2003; Voelker et al., 1995a, b; Yang et al., 1996). RsbS/RsbT interactions are believed to be modulated by RsbR and a family of related proteins (Akbar et al., 1997, 2001; Chen et al., 2003; Gaidenko et al., 1999). It has been recently demonstrated that RsbR, and by inference its paralogues, can self-associate into large-molecular-mass complexes (~106 Da) that can incorporate RsbS and RsbT (Chen et al., 2003). These complexes may represent the normal state of the RsbR, -S and -T proteins in unstressed B. subtilis. RsbR, like RsbS, can be phosphorylated by RsbT (Gaidenko et al., 1999). In vitro, RsbR is necessary for RsbS to bind and be phosphorylated by RsbT (Chen et al., 2003). The phosphorylation state of RsbR does not affect the binding of RsbT to RsbS in the RsbR/RsbS complex, but does influence the ability of RsbT to phosphorylate RsbS, with RsbR-P facilitating the phosphorylation reaction (Chen et al., 2003). Inhibition of RsbT is re-established through the activity of RsbX, an RsbS-P phosphatase that reactivates RsbS and allows it to again sequester RsbT (Voelker et al., 1995a, b; Yang et al., 1996).

Aside from RsbR, -S, and -T, there is evidence that a ribosome-associated process may contribute to stress activation of {sigma}B. {sigma}B fails to be induced by physical stress in B. subtilis strains that are either missing ribosome protein L11 or deficient in the ribosome-associated GTP-binding protein Obg (Scott & Haldenwang, 1999; Zhang et al., 2001).

We previously noted that RsbR, -S and -T, as well as Obg, cofractionate with ribosomes during gel-filtration chromatography of crude B. subtilis extracts (Scott et al., 2000). Obg was subsequently shown to bind to ribosome protein L13 in an affinity blot assay (Scott et al., 2000). Thus, Obg's cofractionation with ribosomes probably represents a direct interaction between these two entities; however, even though RsbT can interact with Obg in the yeast dihybrid assay (Scott & Haldenwang, 1999), the significance of RsbT, as well as RsbR and RsbS, in the ribosome-containing fractions is unknown. In the present work we explore the possible associations that exist between the Rsb proteins, Obg and ribosomes in extracts of wild-type and mutant B. subtilis, and Escherichia coli engineered to express RsbR, RsbS and RsbT. The data are consistent with Obg, but not RsbR, RsbS or RsbT, being ribosome-associated. The ribosome/Obg contributions to triggering stress induction of RsbT apparently do not involve interactions that persist in crude extracts. Additionally, and unlike the inactivating complexes that form between other {sigma}B regulators (e.g. RsbW–{sigma}B, RsbV–RsbW) which are readily identified in crude extracts, we find no evidence for persistent associations between the RsbR–RsbS complex and RsbT. Instead, RsbT appears to form Triton-sensitive aggregates in crude extracts, suggesting an inherent instability of free RsbT which may be related to its role as a stress-sensitive activator.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and plasmids.
All strains and their relevant genotypes are listed in Table 1. Except where indicated, the B. subtilis strains used in this study are derivatives of PY22.


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Table 1. Strains and plasmids

 
BSJ93, a strain carrying the rsbS59SA mutation, was made as follows. PCR-based oligonucleotide mutagenesis (Woodbury et al., 2004) was used to change codon 59 of rsbS in the plasmid pRT-2 (Smirnova et al., 1998) from TCA (Ser) to GCA (Ala), creating pJM63. The presence of rsbS59SA in pJM63 was confirmed by DNA sequencing. pJM63 encodes a KanR cassette flanked on one side by a 520 bp DNA segment encoding the region upstream of the sigB operon and on the other by PA rsbR rsbS59SA rsbT. Transformation of B. subtilis with linearized pJM63, followed by selection for KanR, allowed the isolation of clones in which the KanR cassette entered the B. subtilis chromosome by homologous recombination between the chromosome and the sequences bracketing KanR. To facilitate the identification of clones in which rsbS59SA was transferred with KanR to the recipient, pJM63 was transformed into a B. subtilis strain (XS352) (Smirnova et al., 1998) with a deletion extending from codon 25 of the 121 codon rsbS gene to codon 17 of the 133 codon rsbT gene. KanR clones containing RsbS and RsbT (detected by Western blot) should have acquired the plasmid-encoded rsbS59SA allele. BSJ93 was one such transformant. BSJ93 synthesizes RsbS and RsbT and is unable to activate {sigma}B following physical stress (the rsbS59SA phenotype; Kang et al., 1996).

BSK5 rsbR{Delta}2 was constructed as follows. A 502 bp EcoRI segment internal to rsbR in pRS11 (PA rsbRS; Smirnova et al., 1998) to create pJM49. This deletion terminates the synthesis of RsbR at codon 92 of the 274 codon rsbR gene. Using a strategy similar to that described above, BSK5 was identified following transformation of wild-type B. subtilis (BSA46) with linearized pJM49, yielding KanR clones which were screened by Western blot for the absence of RsbR but normal levels of the downstream rsbS gene product.

BSJ39 (RsbS) was created using plasmid p{Delta}S25. p{Delta}S25 consists of a PCR fragment that begins approximately 200 bp upstream of the sigB operon's transcriptional start site and ends at the fourth codon of RsbS, joined to a second fragment that extends from 10 codons before the rsbS carboxy terminus to the end of rsbT, cloned in pRT-2. KanR clones arising from transformation of linearized p{Delta}S25 into a wild-type B. subtilis strain (BSA46) were screened for the anticipated high {sigma}B activity (ctc : : lacZ) associated with the loss of RsbS on LB plates with X-Gal. Putative RsbS clones were then screened by Western blot for the presence of RsbR and T, and the absence of RsbS.

E. coli (TG-2) strains expressing various combinations of rsbR, -S and/or -T were constructed using pUC19-based plasmids. ECW1 carries pDRNT and expresses rsbR, -S and -T. ECW2 expresses rsbR and -S. It carries pDRS, a plasmid derived from pDRNT by cutting with NdeI and SphI to remove rsbT and recircularizing after treatment with mung bean nuclease to ‘blunt’ the ends. ECW4 expresses rsbT from plasmid pKAT-1. pKAT-1 is pUK19 bearing the sigB operon's PA promoter, amplified by PCR from PY22 as a 376 bp EcoRI/BamHI fragment (–315 to +60) and joined to a 450 bp BamHI/SphI fragment encoding rsbT (36 bp upstream of rsbT to 12 bp downstream of rsbT). ECW3 expresses rsbS and rsbT from pSK-X. To make pSK-X, rsbS and rsbT that had been amplified by PCR as a 715 bp HindIII/XbaI DNA fragment (23 bp upstream of rsbS to 23 bp downstream of rsbT) were cloned into pUC19. In plasmids pDNRT, pDRS and pKAT-1, the rsb genes are expressed from the sigB operon's PA promoter, which is recognized in E. coli, and in pSK-X they are expressed from Plac. Bacteria were routinely grown in LB at 37 °C.

Gel-filtration analysis.
Gel-filtration chromatography was performed as described previously (Scott et al., 2000). One litre of B. subtilis or E. coli culture (OD540, 0·4) was harvested on ice, washed with a low-salt buffer [10 mM Tris (pH 8·0), 50 µM EDTA, 1·5 mM MgCl2, 1 mM DTT, 0·03 % phenylmethyl sulfonyl fluoride (PMSF)] and resuspended in 5 ml of the same buffer. Following disruption in a French pressure cell, debris was removed by centrifugation (5 000 g for 10 min), and the resulting crude lysate loaded onto a 500 ml Sephacryl S-300 column equilibrated with the same buffer. Five millilitre fractions were collected, precipitated with 2 vols of ethanol and analysed by SDS-PAGE (13·5 % acrylamide) and Western blot. If a second round of gel filtration was to be used, the fractions containing the large-molecular-mass complexes were pooled and dialysed into buffer [10 mM Tris (pH 8·0), 50 mM KCl] containing either 10 mM magnesium acetate or no added Mg2+. The dialysed fractions were reapplied to the Sephacryl column, equilibrated with the buffer used for dialysis and fractionated/analysed as before.

Velocity-sedimentation analysis.
B. subtilis grown and harvested, as in the gel-filtration analysis, was resuspended in sedimentation buffer [20 mM Tris (pH 7·5), 10 mM Mg Cl2, 0·5 mM EDTA, 1 mM dithiotheritol, 0·1 M NH4Cl, 0·03 % PMSF], disrupted by passage through a French pressure cell, and centrifuged (8000 g, 15 min) to remove debris. Protease inhibitors were added to the following final concentrations: pepstatin, 1 µM; leupeptin, 1 µM; N-{alpha}-tosyl-L lysine chloromethyl ketone (TLCK), 100 µM; antitrypsin, 2 µg ml–1. One hundred microlitres of extract (OD280, 200) was layered onto 9 ml 10–30 % sucrose gradients prepared in the sedimentation buffer. Gradients containing identical samples were centrifuged (37 000 r.p.m. in a Sorvall TH641 swinging bucket rotor) for 1·5, 3 and 5 h. Fractions (0·5 ml) were collected from the gradients, precipitated with 2 vols of ethanol and analysed by SDS-PAGE and Western blot.

To analyse the high-molecular-mass complexes that were identified by gel filtration, 4 ml of pooled Sephacryl column fractions was precipitated with (NH4)2SO4 (0·56 g ml–1), resuspended in 0·5 ml of sedimentation buffer without PMSF, and dialysed overnight against 1 l of this buffer. Two hundred microlitres of dialysed extract was layered onto sucrose gradients and analysed as above.

General methods.
Transformations of B. subtilis and E. coli were performed by standard methods (Sambrook et al., 1989; Yasbin et al., 1973). Western blot analyses were done as previously described, using mouse monoclonal antibodies against the Rsb proteins and mouse polyclonal anti-Obg antibody (Dufour et al., 1996; Scott et al., 2000).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Fractionation of wild-type B. subtilis extracts
A number of the proteins that are required for stress-dependent regulation of {sigma}B elute in high-molecular-mass fractions during gel-filtration chromatography of crude B. subtilis extracts (Scott et al., 2000). Most of the RsbR, approximately half of the RsbS, and all of the detectable RsbT and Obg are present in fractions that also contain the bulk of the extracts' ribosomal proteins (Fig. 2a, lanes 2–10). To investigate whether this coelution represents a physical association between these proteins, gel-filtration fractions were pooled, and subjected to further analyses.



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Fig. 2. Gel-filtration chromatography of B. subtilis extracts. (a) B. subtilis (PY22) was harvested in exponential phase (OD540 0·4). Extracts were prepared as described in Methods and loaded onto a Sephacryl S-300 column. Samples of the protein-containing fractions were fractionated by SDS-PAGE. The protein profiles in each fraction were visualized by Coomassie-blue staining (upper panel) and Western blot (lower panel) using antibodies against RsbR, -S and -T and Obg as probes. Numbers at the top of the panel indicate fraction numbers with fraction 1 being the earliest-eluting fraction. The characteristic cluster of low-molecular-mass ribosomal proteins in the fast-eluting fractions is bracketed in the upper panel. The positions of Obg, RsbR, -S and -T are indicated in the lower panel, which also includes protein molecular-mass markers (Bio-Rad) in the first and last lanes. (b) Fractions 2–7 of the gel-filtration experiment illustrated in (a) were dialysed into buffer without Mg2+ and subjected to a second round of gel filtration using the same Sephacryl S-300 column that had been equilibrated with Mg2+-free buffer. The analyses of the indicated fractions are as in (a).

 
As a test of the dependence of the high-molecular-mass forms of Obg, RsbR, RsbS and RsbT on intact ribosomes, the ribosome component in the gel-filtration fractions was disrupted by dialysis against a buffer lacking Mg2+, a cation essential for ribosome integrity (Spirin, 1990). As a control, a sample was also dialysed against a similar buffer with 10mM MgCl2. The fractions dialysed against the Mg2+-containing buffer retained their original elution profile when chromatographed on a second gel-filtration column, i.e. Obg, ribosomes and Rsb proteins coeluted in the fast-exiting fractions (data not shown). In contrast, the sample that had been dialysed into magnesium-free buffer displayed an altered pattern (Fig. 2b), with the ribosomal proteins dispersed throughout the fractions. Consistent with Obg's presence in the higher-molecular-mass fractions being a consequence of ribosome association, the Obg protein shifted to a lower-molecular-mass position in the elution profile. However, despite the disassociation of ribosomes, RsbR and RsbS persisted in the original high-molecular-mass fractions. Thus, the presence of RsbR and RsbS in the high-molecular-mass fractions is not the result of a ribosome association. RsbT was not detected in the fractions from the second gel-filtration column, perhaps due to its dispersal among many fractions or its degradation.

In an alternative analysis, the high-molecular-mass fractions from the original gel-filtration column were concentrated by (NH4)2SO4 precipitation, dialysed into 10mM MgCl2 buffer and examined by velocity centrifugation. Samples of this material, as well as unfractionated whole-cell lysates, were layered in triplicate onto sucrose gradients and subjected to high-speed centrifugation. Representative gradients were removed at intervals throughout the centrifugation run and, following fractionation, analysed by SDS-PAGE and Western blot. As illustrated in Fig. 3, the velocity fractionation of both a whole-cell lysate (Fig. 3i) and the fast-eluting gel-filtration fractions (Fig. 3ii) resulted in the separation of the RsbR/RsbS proteins and the ribosomes during sedimentation. The RsbR/RsbS proteins sedimented more slowly than ribosomes, consistent with the sedimentation profile reported for the self-assembling RsbR/RsbS complex (Chen et al., 2003). RsbT's sedimentation through the gradient was distinct from that of either the ribosomes or the putative RsbR–RsbS complex. Additionally, RsbT's sedimentation pattern varied, depending upon whether the material analysed was pooled gel-filtration fractions or crude cell extract. The bulk of the RsbT in the whole-cell extract (Fig. 3i-c) remains in the uppermost portions of the gradient following centrifugation, apparently unassociated with fast-sedimenting components, while in the Sephacryl-fractionated sample, a second peak of RsbT is seen, trailing the ribosomal proteins but ahead of the putative RsbR/RsbS complex (Fig. 3ii).



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Fig. 3. Sedimentation analyses of B. subtilis extracts. Triplicate samples of a crude extract from wild-type B. subtilis were layered onto 10–30 % sucrose gradients and subjected to centrifugation at 37 000 r.p.m. in a Sorvall TH641 rotor for 1·5 (a), 3 (b) or 5 (c) hours. Fractions were collected from the bottom of the tube (fraction 1) and analysed as in Fig. 2. The top three gels in (i) depict Coomassie-blue staining, illustrating protein fractionation with sedimentation of the low-molecular-mass proteins of the ribosomes in the lead fractions. Below, the stained gels are samples from the same fractions analysed by Western blot using antibodies against the proteins indicated in the figure as probes. (ii) depicts triplicate samples of the fraction 2–7 material from the gel-filtration column in Fig. 2(a), concentrated by (NH4)2SO4 precipitation, centrifuged through sucrose gradients and analysed by Western blot for the sedimentation profile of RsbR, -S, -T and Obg. (iii) depicts triplicate samples of a crude extract from B. subtilis strain BSJ93 (rsbS59SA) centrifuged through sucrose gradients and analysed by Western blot for the positions of RsbR, -S and -T in the gradient profile.

 
Although Obg can specifically bind to ribosomal protein L13 (Scott et al., 2000), it too separated from the ribosomes during centrifugation. In other studies, we observed that GTP or nonhydrolysable GTP analogues (i.e. GIDP) can stabilize the association of Obg with ribosomes (S. Zhang, & W. Haldenwang, unpublished results). To ask whether the apparent Obg/ribosome complex that eluted from the Sephacryl column represents this GTP-stabilizable complex, the sedimentation analysis of the putative Obg/ribosome complex was repeated incorporating a non-hydrolysable GTP analogue (10 µM GIDP) in the sucrose gradient. In the presence of the GIDP, Obg sedimented with the ribosome components (Fig. 4c). Apparently, nucleotide-bound Obg is the form which most avidly binds ribosomes, or the ribosomes themselves are altered by the nucleotide to stabilize Obg binding.



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Fig. 4. Effects of GIDP on sedimentation of Obg with ribosomes. Pooled, early-eluting fractions (Fig. 2a, fractions 2–7) of a crude B. subtilis extract separated by Sephacryl chromatography concentrated and dialysed as in Fig. 3(ii) were subjected to centrifugation through 10–30 % sucrose gradients that were either unsupplemented (a, b) or contained 10 µM GIDP (c). The gradients were centrifuged for 5 h and analysed as in Fig. 3. (a) Coomassie-blue-stained gel; (b, c) Western blot analysis (anti-Obg, -RsbR, -RsbS and -RsbT antibody probes).

 
The data argue that Obg is ribosome-associated but RsbR, RsbS and RsbT are not part of an Obg/ribosome complex. Based on the velocity-sedimentation analysis, the presence of RsbT in the high-molecular-mass gel-filtration fractions appears to be independent of either RsbR or RsbS. The coincident elution of RsbR and RsbS in the high-molecular-mass gel-filtration fractions persisted in the centrifugation analysis, arguing for their presence in a common complex in these fractions, probably the large RsbR/RsbS multimer that is reported to self-assemble in vitro (Chen et al., 2003). RsbT is believed to be sequestered into the RsbR/RsbS complex in unstressed B. subtilis and released following stress-induced phosphorylation of RsbS. Although the nature of the high-molecular-mass form of RsbT is uncertain, it is formally possible that the RsbT remaining near the top of the velocity gradient could represent RsbT that was originally bound to RsbR/RsbS but was released from the complex as a consequence of the phosphorylation of RsbS during extract preparation or analysis. Such a circumstance was proposed to explain the failure of RsbT to cosediment with RsbR/RsbS in complexes prepared from purified components (Chen et al., 2003). In an attempt to circumvent this possibility, extracts were prepared from a B. subtilis strain with a mis-sense mutation in rsbS (rsbS59SA) which removes the target residue for phosphorylation by RsbT. B. subtilis strains carrying this rsbS allele are not stress-activable and, by the current model, should have RsbT permanently sequestered in the inactivating complex. As can be seen in Fig. 3iii, the presence of the mutant rsbS allele had no effects on RsbT's sedimentation profile. It remained in the slow-sedimenting fraction, free from the RsbR/RsbS complex. Thus, it is unlikely that the failure of RsbT to cofractionate with RsbR–RsbS is due to the phosphorylation of RsbS.

Gel-filtration profiles of Obg, RsbR, RsbS and RsbT in mutant B. subtilis
To better characterize the nature of the high-molecular-mass forms of RsbR, RsbS and RsbT that are found in B. subtilis extracts, cell lysates from B. subtilis mutants lacking RsbR, RsbS, RsbT, or RsbR and its three most homologous paralogues YkoB, YojH and YqhA (Akbar et al., 2001) were fractionated by gel filtration and analysed by Western blot (Fig. 5a–d). The abundance of RsbS in the high-molecular-mass fractions decreased only slightly, relative to its abundance in low-molecular-mass fractions, in the RsbR single mutant, but more dramatically in the mutant lacking multiple RsbR-like proteins. In contrast, the RsbT remained exclusively in the high-molecular-mass form regardless of the presence or absence of either RsbR or RsbS. These findings are consistent with the notion that RsbS, but not RsbT, is present in the high-molecular-mass fractions due to its association with RsbR, and that the RsbR paralogues can substitute for RsbR in such a complex. As would be expected from the current model, the absence of RsbT had no obvious effect on the abundance of the high-molecular-mass RsbR/RsbS complex and the loss of RsbS did not prevent the accumulation of the putative RsbR multimer. These results reinforce the notion that RsbT is not part of a stable complex with RsbR and RsbS in B. subtilis extracts. If the complex was initially present, it was unable to persist during the fractionation protocols that we employed.



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Fig. 5. Gel-filtration analyses of B. subtilis extracts lacking Rsb proteins. B. subtilis strains lacking one or more {sigma}B regulatory genes were grown, fractionated on Sephacryl S-300 and analysed as in Fig. 2(a). (a) BSK5 (RsbR); (b) PB629 (RsbR, YkoB, YojH, YqhA); (c) XS332 (RsbT); (d) BSJ39 (RsbS).

 
Formation of Rsb complexes in E. coli
To better judge the Bacillus-specific factors that contribute to the high-molecular-mass forms of RsbR, RsbS and RsbT, combinations of these proteins were expressed from plasmid-encoded genes in E. coli. Extracts prepared from E. coli carrying rsbR, rsbS and rsbT, fractionated by gel filtration and analysed by Western blot gave a profile (Fig. 6b) that was virtually indistinguishable from that seen in B. subtilis (Fig. 2b). As was also seen in B. subtilis (Fig. 5), the absence of RsbT had little effect on the formation of the high-molecular-mass form of RsbR/RsbS (Fig. 6c), but unlike the case in B. subtilis the high-molecular-mass form of RsbS was totally dependent on RsbR (Fig. 6d). Apparently the RsbR paralogues, present in B. subtilis but absent in E. coli, can substitute for RsbR and allow formation of a high-molecular-mass complex that can incorporate RsbS. As was also seen in B. subtilis, a high-molecular-mass form of RsbT occurs independently of RsbR and RsbS. The presence of this form of RsbT in E. coli extracts argues that it is independent of Bacillus-specific factors (Fig. 6e).



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Fig. 6. Gel-filtration chromatography of Rsb proteins in E. coli extracts. Crude extracts were prepared from E. coli strains carrying plasmids that allowed the expression of (a, b) rsbR, rsbS and rsbT (ECW1), (c) rsbR and rsbS (ECW2), (d) rsbS and rsbT (ECW3) or (e) rsbT (ECW4). Extract samples were fractionated on a Sephacryl S-300 column and analysed as in Fig. 2. (a) Coomassie-blue-stained gel; (b–e) Western blot analysis of the indicated fractions using antibody probes for the proteins depicted in each blot.

 
The failure of RsbT's large-molecular-mass form to be dependent on Bacillus-specific factors raises the possibility that it represents an inherent property of RsbT itself. During purification for biochemical studies and antibody production, RsbT, unlike the other Rsb proteins, showed a marked tendency to aggregate in the absence of detergents (A. Dufour & W. G. Haldenwang, unpublished results). If the RsbT found in the gel-filtration fractions represents a similar aggregation of free RsbT, its fractionation properties would be predicted to be affected by non-ionic detergents. We explored this possibility by running the gel-filtration fractionation of the E. coli extracts in the presence of 0·1 % Triton. The putative RsbR/RsbS complex was unaffected by the detergent treatment, while the RsbT shifted to a low-molecular-mass form that eluted in fractions that overlapped those containing the low-molecular-mass forms of RsbR and RsbS (Fig. 7a). The presence of all three of these proteins in some of the fractions is probably coincidental. Unlike the high-molecular-mass RsbR/RsbS complex, in which RsbR and RsbS abundance is seen to ‘peak’ in the same fractions, the peak amounts of each of these proteins are displaced one from another in the lower-molecular-mass elution profile. The independence of RsbT from RsbR and RsbS in the Triton-treated extract is reinforced by the observation that a Triton-treated extract from an E. coli strain that expresses rsbT alone has an elution profile similar to that of RsbT in a strain expressing all three proteins (Fig. 7b).



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Fig. 7. Gel-filtration chromatography of Triton-treated E. coli extracts. Triton X-100 was added to crude extracts prepared from E. coli strains ECW1 (rsbR, rsbS, rsbT) or ECW4 (rsbT) to a final concentration of 0·5 %. The extracts were then fractionated by gel filtration with buffer containing 0·1 % triton X-100. Fractions were collected. Successive fractions were pooled and analysed as in Fig. 2. (a, b) ECW1 Coomassie-blue-stained gel and Western blot, respectively; (c) ECW4 Western blot.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In previous work, a portion of the {sigma}B regulatory proteins RsbR and RsbS, and all of the RsbT, present in B. subtilis extracts were observed to coelute with the extract's ribosome/Obg population during gel-filtration chromatography (Scott et al., 2000). In light of the necessary, but undefined, roles that the ribosome and Obg play in the stress activation of {sigma}B, this coincident elution merited further study. The fractionation analyses reported in the present work demonstrate that the ribosome/Obg, RsbT and RsbR/RsbS complexes are independent components of the high-molecular-mass gel-filtration fractions. Velocity-gradient analyses allowed the separation of each of these as distinct entities. Additional support for the high-molecular-mass forms of RsbR and RsbS being unrelated to ribosome-association comes from the persistence of these forms under buffer conditions that caused the disassembly of ribosomes and the release of Obg. Although ribosomes and Obg contribute necessary inputs to the stress-activation process, these apparently do not involve long-lived associations that can be detected as complexes in crude extracts.

Recent biochemical experiments have shown that RsbR can form high-molecular-mass complexes which incorporate RsbS (Chen et al., 2003). It is thought that formation of such a complex is a prerequisite for RsbS to be able to sequester and inactivate RsbT. The high-molecular-mass RsbR/RsbS association that we observed in fractionated B. subtilis and in E. coli extracts probably represents this complex. As was seen in the biochemical experiments, RsbR is the essential element in its formation (i.e. the high-molecular-mass complex of RsbR occurs in the absence of RsbS, but the high-molecular-mass RsbS complex requires RsbR) and based on its formation when RsbR and RsbS are expressed in E. coli no additional Bacillus-specific proteins are needed. In Bacillus, in which a family of RsbR paralogues are thought to be able to substitute for RsbR (Akbar et al., 2001), the loss of RsbR itself has only a modest effect on the presence of a high-molecular-mass complex that incorporates RsbS. Only when RsbR and several RsbR paralogues are deleted does the abundance of the RsbS-containing complex show a noticeable decline.

A curiosity in the present study is the failure of RsbT to be part of the RsbR/RsbS complex. A favoured model for the stress-activation mechanism envisions RsbT as unavailable to activate the RsbU phosphatase due to its sequestration into the complex with RsbR/RsbS. This would be similar to the inactivation of {sigma}B by its sequestration in an RsbW/{sigma}B complex or the inactivation of RsbW by its binding to RsbV. Both of these complexes are readily discerned in fractionations of crude B. subtilis extracts (Dufour & Haldenwang, 1994), yet the association of RsbT with its putative inactivating complex is not evident. This is true even in extracts prepared from a B. subtilis strain with an altered RsbS (RsbS59SA) that should not release RsbT (Kang et al., 1996). Instead, two forms of RsbT were found in crude extracts: a low-molecular-mass form, observed when crude extracts were fractionated by velocity-gradient analysis, and a high-molecular-mass form, found following gel filtration, which, by virtue of its detergent sensitivity, probably represents aggregated RsbT.

In other stress-responsive systems, protein denaturation and chaperone-assisted protein-folding contribute to stress signalling and gene regulation (Narberhaus, 1999). It is plausible, although quite speculative, that the denaturation of RsbT, reflected in its apparent aggregation in crude extracts, could have regulatory significance. If RsbT is readily given to misfolding and aggregation, the RsbR/RsbS complex could not only serve as an inactivating RsbT-binding complex, but also play a positive role as an RsbT-chaperone complex, stabilizing and releasing a properly folded RsbT in response to a stress-generated trigger.

In an earlier analysis of the role of RsbS in {sigma}B regulation, it was reported that an rsbS allele (rsbS59SD), whose product mimics phosphorylated RsbS, was more effective in allowing RsbT to activate {sigma}B than was a deletion of rsbS (Kang et al., 1996). We have confirmed this result and, in addition, note that the rsbS59SD allele is codominant with its wild-type counterpart. We also found that the rsbS allele (rsbS59SA), whose product should sequester RsbT into stable inactive complexes (Kang et al., 1996; Yang et al., 1996), is recessive to the wild-type rsbS allele (A. Reeves, S. Zhang & W. Haldenwang, unpublished results). These phenotypes are unusual for an inhibitory system, where the capacity for negative control would be expected to be dominant. Although more complex models can be envisioned to explain these results, the dominance of wild-type rsbS over rsbS59SA, and rsbS59SD over the wild-type allele, could be simply explained if RsbS, when altered due to phosphorylation following stress or the rsbS59SD mutation, has a positive influence on RsbT activity. Detailed mutational analyses of rsbR, rsbS and rsbT could reveal the validity of this notion.


   ACKNOWLEDGEMENTS
 
We thank Janelle Scott and Natalya Smirnova for construction of several of the plasmids used in this work. This study was supported by US National Institutes of Health grant GM-48220. S. K. was supported, in part, by NIH training grant T32-AI-07271.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Akbar, S., Kang, C. M., Gaidenko, T. A. & Price, C. W. (1997). Modulator protein RsbR regulates environmental signalling in the general stress pathway of Bacillus subtilis. Mol Microbiol 24, 567–578.[Medline]

Akbar, S., Gaidenko, T. A., Kang, C. M., O'Reilly, M., Devine, K. M. & Price, C. W. (2001). New family of regulators in the environmental signaling pathway which activates the general stress transcription factor {sigma}B of Bacillus subtilis. J Bacteriol 183, 1329–1338.[Abstract/Free Full Text]

Alper, S., Dufour, A., Garsin, D. A., Duncan, L. & Losick, R. (1996). Role of adenosine nucleotides in the regulation of a stress-response transcription factor in Bacillus subtilis. J Mol Biol 260, 165–177.[CrossRef][Medline]

Benson, A. K. & Haldenwang, W. G. (1992). Characterization of a regulatory network that controls {sigma}B expression in Bacillus subtilis. J Bacteriol 174, 749–757.[Abstract]

Benson, A. K. & Haldenwang, W. G. (1993a). Regulation of {sigma}B levels and activity in Bacillus subtilis. J Bacteriol 175, 2347–2356.[Abstract]

Benson, A. K. & Haldenwang, W. G. (1993b). Bacillus subtilis {sigma}B is regulated by a binding protein (RsbW) that blocks its association with core RNA polymerase. Proc Natl Acad Sci U S A 90, 2330–2334.[Abstract]

Boylan, S. A., Rutherford, A., Thomas, S. M. & Price, C. W. (1992). Activation of Bacillus subtilis transcription factor {sigma}B by a regulatory pathway responsive to stationary-phase signals. J Bacteriol 174, 3695–3706.[Abstract]

Boylan, S. A., Redfield, A. R., Brody, M. S. & Price, C. W. (1993). Stress-induced activation of the {sigma}B transcription factor of Bacillus subtilis. J Bacteriol 175, 7931–7937.[Abstract]

Brody, M. S., Vijay, K. & Price, C. W. (2001). Catalytic function of an {alpha}/{beta} hydrolase is required for energy stress activation of the {sigma}B transcription factor or Bacillus subtilis. J Bacteriol 183, 6422–6428.[Abstract/Free Full Text]

Chen, C.-C., Lewis, R. J., Harris, R., Yudkin, M. D. & Delumeau, O. (2003). A supramolecular complex in the environmental stress signalling pathway of Bacillus subtilis. Mol Microbiol 49, 1657–1669.[CrossRef][Medline]

Delumeau, O., Lewis, R. J. & Yudkin, M. D. (2002). Protein-protein interactions that regulate the energy stress activation of {sigma}B in Bacillus subtilis. J Bacteriol 184, 5583–5589.[Abstract/Free Full Text]

Dufour, A. & Haldenwang, W. G. (1994). Interactions between a Bacillus subtilis anti-{sigma} factor (RsbW) and its antagonist (RsbV). J Bacteriol 176, 1813–1820.[Abstract]

Dufour, A., Voelker, U., Voelker, A. & Haldenwang, W. G. (1996). Relative levels and fractionation properties of Bacillus subtilis {sigma}B and its regulators during balanced growth and stress. J Bacteriol 178, 3701–3709.[Abstract]

Gaidenko, T. A., Yang, X., Lee, Y. M. & Price, C. W. (1999). Threonine phosphorylation of modulator protein RsbR governs its ability to regulate a serine kinase in the environmental stress signaling pathway of Bacillus subtilis. J Mol Biol 288, 29–39.[CrossRef][Medline]

Hecker, M., Schumann, W. & Voelker, U. (1996). Heat-shock and general stress response in Bacillus subtilis. Mol Microbiol 19, 417–428.[Medline]

Ju, J., Luo, T. & Haldenwang, W. G. (1998). Forespore expression and processing of the SigE transcription factor in wild-type and mutant Bacillus subtilis. J Bacteriol 180, 1673–1681.[Abstract/Free Full Text]

Kalman S. , Duncan, M. L., Thomas, S. M. & Price, C. W. (1990). Similar organization of the sigB and spoIIA operons encoding alternate sigma factors of Bacillus subtilis RNA polymerase. J Bacteriol 172, 5575–5585.[Medline]

Kang, C. M., Brody, M. S., Akbar, S., Yang, X. & Price, C. W. (1996). Homologous pairs of regulatory proteins control activity of Bacillus subtilis transcription factor {sigma}B in response to environmental stress. J Bacteriol 178, 3846–3853.[Abstract]

Narberhaus, T. (1999). Negative regulation of bacterial heat shock genes. Mol Microbiol 31, 1–8.[CrossRef][Medline]

Petersohn, A., Bernhardt, J., Gerth, U., Hoper, D., Koburger, T., Voelker, U. & Hecker, M. (1999). Identification of {sigma}B-dependent genes in Bacillus subtilis using a promoter consensus-directed search and oligonucleotide hybridization. J Bacteriol 181, 5718–5724.[Abstract/Free Full Text]

Price, C. W., Fawcett, P., Ceremonie, H., Su, N., Murphy, C. K. & Youngman, P. (2001). Genome-wide analysis of the general stress response in Bacillus subtilis. Mol Microbiol 41, 757–774.[CrossRef][Medline]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Scott, J. M. & Haldenwang, W. G. (1999). Obg, an essential GTP binding protein of Bacillus subtilis, is necessary for stress activation of transcription factor {sigma}B. J Bacteriol 181, 4653–4660.[Abstract/Free Full Text]

Scott, J. M., Ju, J., Mitchell, T. & Haldenwang, W. G. (2000). The Bacillus subtilis GTP binding protein Obg and regulators of the {sigma}B stress response transcription factor cofractionate with ribosomes. J Bacteriol 182, 2771–2777.[Abstract/Free Full Text]

Smirnova, N., Scott, J., Voelker, U. & Haldenwang, W. G. (1998). Isolation and characterization of Bacillus subtilis sigB operon mutations that suppress the loss of the negative regulator RsbX. J Bacteriol 180, 3671–3680.[Abstract/Free Full Text]

Spirin, A. S. (1990). Ribosome preparation and cell-free protein synthesis. In The Ribosome, pp. 56–70. Edited by W. E. Hill and others. Washington, DC: American Society for Microbiology.

Vijay, K., Brody, M. S., Fredlund, E. & Price, C. W. (2000). A PP2C phosphatase containing a PAS domain is required to convey signals of energy stress to the {sigma}B transcription factor of Bacillus subtilis. Mol Microbiol 35, 180–188.[CrossRef][Medline]

Voelker, U., Engelmann, S., Maul, B., Riethdorf, S., Voelker, A., Schmid, R., Mach, H. & Hecker, M. (1994). Analysis of the induction of general stress proteins of Bacillus subtilis. Microbiology 140, 741–752.[Medline]

Voelker, U., Dufour, A. & Haldenwang, W. G. (1995a). The Bacillus subtilis rsbU gene product is necessary for RsbX-dependent regulation of {sigma}B. J Bacteriol 177, 114–122.[Abstract]

Voelker, U., Voelker, A., Maul, B., Hecker, M., Dufour, A. & Haldenwang, W. G. (1995b). Separate mechanisms activate {sigma}B of Bacillus subtilis in response to environmental and metabolic stresses. J Bacteriol 177, 3771–3780.[Abstract]

Voelker, U., Voelker, A. & Haldenwang, W. G. (1996). Reactivation of the Bacillus subtilis anti-{sigma}B antagonist, RsbV, by stress- or starvation-induced phosphatase activities. J Bacteriol 178, 5456–5463.[Abstract]

Wise, A. A. & Price, C. W. (1995). Four additional genes in the sigB operon of Bacillus subtilis that control activity of the general stress factor {sigma}B in response to environmental signals. J Bacteriol 177, 123–133.[Abstract]

Woodbury, R. L., Luo, T., Grant, L. & Haldenwang, W. G. (2004). Mutational analysis of RsbT, an activator of the Bacillus subtilis stress response transcription factor, {sigma}B. J Bacteriol 186, 2789–2797.[Abstract/Free Full Text]

Yang, X., Kang, C. M., Brody, M. S. & Price, C. W. (1996). Opposing pairs of serine protein kinases and phosphatases transmit signals of environmental stress to activate a bacterial transcription factor. Genes Dev 10, 2265–2275.[Abstract]

Yasbin, R. E., Wilson, G. A. & Young, F. E. (1973). Transformation and transfection of lysogenic strains of Bacillus subtilis 168. J Bacteriol 113, 540–548.[Medline]

Zhang, S. & Haldenwang, W. G. (2003). RelA is a component of the nutritional stress activation pathway of the Bacillus subtilis transcription factor {sigma}B. J Bacteriol 185, 5714–5721.[Abstract/Free Full Text]

Zhang, S., Scott, J. M. & Haldenwang, W. G. (2001). Loss of ribosomal protein L11 blocks stress activation of the Bacillus subtilis transcription factor {sigma}B. J Bacteriol 183, 2316–2321.[Abstract/Free Full Text]

Received 18 June 2004; revised 23 July 2004; accepted 31 August 2004.



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