INTRODUCTION

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^B is a transcription factor that directs RNA polymerase (RNAP) to promoters of the Bacillus subtilis general stress regulon (14). Induction of the regulon occurs when ^B is activated by either a decline in ATP levels or the onset of any of a number of environmental stresses (e.g., heat shock, acid salt, and ethanol) (5, 7, 14, 18, 34, 38). ^B is present in the prestressed cell, but it is held inactive in a complex with an anti-^B protein, RsbW (6, 9). ^B is released from RsbW when a second protein (RsbV) binds to RsbW (9). The genes for RsbV, RsbW, ^B, and five additional ^B regulatory proteins (RsbR, -S, -T, -U, and -X) are cotranscribed as an eight-gene operon from a promoter (P_A) that is recognized by the B. subtilis housekeeping  factor, ^A (40). An internal ^B-dependent promoter (P_B) enhances expression of the downstream four genes during periods of ^B activity (i.e., P_A rsbR rsbS rsbT rsbU P_B rsbV rsbW sigB rsbX) (4, 5, 7). A likely mechanism for ^B control by the Rsb proteins is illustrated in Fig. 1. In unstressed cells, RsbV, the effector of ^B release, is inactive due to RsbW-catalyzed phosphorylation (9, 37). Under conditions of low ATP (e.g., entry into the stationary phase of growth) this phosphorylation reaction is thought to be inefficient, and ^B remains active (2, 37, 38). In addition to this ATP-responsive activation, diverse environmental stresses initiate a sequence of Rsb-dependent processes which reactivate RsbV (18, 37, 38, 40, 41). In stressed B. subtilis, RsbT, normally inactive and complexed to RsbS, phosphorylates RsbS and becomes free to activate the RsbV-P phosphatase, RsbU (12, 41). RsbU then dephosphorylates RsbV-P, allowing RsbV to displace ^B from RsbW. Negative regulation is reestablished when RsbX, a RsbS-P phosphatase, dephosphorylates RsbS-P (41). This enables RsbS to again bind and inactivate RsbT. Although much has been learned about how the Rsb proteins function to alternatively silence or activate ^B, the mechanism by which stress communicates with the Rsb proteins is unknown. Neither the signal that is generated by environmental stress nor the regulator that is its target has been identified. In E. coli, protein denaturation and chaperone activation play key roles in communicating environmental stress to stress response transcription factors (8, 13, 43). Although similar processes appear to control chaperone expression in B. subtilis (23, 24, 27), we and others have not found an obvious correlation between chaperone activity and ^B activity in B. subtilis (23, 29). In addition, the known Rsb proteins appear to be inadequate to respond to environmental stress and activate ^B when they are expressed in Escherichia coli (29). Taken together, these results argue that the signal communicating environmental stress to the Rsb proteins is likely to be novel and bacillus specific. Assuming that an unidentified B. subtilis protein communicates stress signals to the Rsb phosphatase/kinase cascade, we attempted to identify candidate proteins, based on their ability to physically interact with key Rsb proteins in the yeast Gal4 dihybrid system. By this approach, several B. subtilis genes were identified as Rsb interactors. Although many of the interactions appear to be fortuitous, a number of biologically relevant associations were found. These included several known interacting Rsb proteins, as well as Obg, an essential GTP-binding protein (21, 31, 32) which proved to be needed for stress activation of ^B. The pattern of ^B induction in various mutant B. subtilis strains indicated that Obg, or a process under its control, plays a role in activating RsbT, the most upstream effector of ^B's stress-induced pathway.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Model of B regulation. (Step 1) The anti-B protein RsbW (W) can form a mutually exclusive complex with either B or RsbV (V) (6, 9). When bound to RsbW, B is unavailable to RNA polymerase (E) (6). RsbV binding to RsbW allows the release of B and the formation of a B RNAP polymerase holoenzyme (E-B) (6). (Step 2) With ATP as a phosphate donor, RsbW can phosphorylate RsbV (2, 9). RsbV-P (V-P) is inactive as a B release factor (2, 9). During growth, relatively high ATP levels favor the phosphorylation and inactivation of RsbV, leaving B bound to RsbW (2, 38). If ATP levels fall, as when B. subtilis enters stationary phase, the phosphorylation of RsbV may be inefficient, leading to the persistence of active RsbV, the formation of RsbV-RsbW complexes, and the release of B (2, 38). (Step 3) The magnitude of the low-ATP activation of B is enhanced by dephosphorylation of a portion of the RsbV-P by an unknown mechanism (37). (Step 6) Environmental stress (e.g., heat shock, osmotic shock, and ethanol treatment) activates an RsbV-P phosphatase, RsbU (U), which creates active RsbV, regardless of ATP levels (37). (Step 4) RsbT (T), the activator of RsbU, is normally inactive due to an association with a negative regulator RsbS (S) (41). RsbR (R), an additional regulatory protein, binds to RsbS and RsbT (31) and is believed to play a structural role and to facilitate RsbT-RsbS interactions (1, 12). (Step 5) Upon exposure to stress, RsbT phosphorylates and inactivates RsbS and activates the RsbU phosphatase (12, 31). (Step 7) RsbS-P is dephosphorylated and reactivated by a phosphatase, RsbX (X) (31), which is encoded by one of the genes downstream of the sigB operon's B-dependent promoter (17). RsbX levels increase with increasing B activity (10). This may serve to limit the activation process and return RsbT to an inactive complex with RsbS.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and cultivation. All of the strains and plasmids used here are listed in Table 1. All BSJ and BSA strains are derivatives of PY22. Bacteria were grown in Luria-Bertani medium (LB [22]) at 37°C. The cells were exposed to ethanol or sodium azide during exponential growth at final concentrations of 4% or 2 mM, respectively. P_xyl and P_spac were induced by xylose (0.5%) or IPTG (isopropyl--D-thiogalactopyranoside; 1 or 0.1 mM), respectively. E. coli TG-2 was used as the host for cloning (26).

Library construction and yeast transformation. Libraries of B. subtilis DNA::GAL4 activator domain (AD) fusions were constructed from PY22 chromosomal DNA that had been partially digested with either SauIIIA or Tsp509I and ligated into either the BamHI sites in pACT-2, pGAD-GL, and pGAD-10 (Clontech Laboratories, Inc., Palo Alto, Calif.) or the EcoRI sites of pACT-2 and pGAD-GL, respectively. These clonings created potential translational fusions between the SauIIIA fragments and the Gal4 AD in all three reading frames and between the Tsp509I fragments and the Gal4-AD in two reading frames. The libraries were transformed, according to established protocols (Clontech Laboratories) into yeast strain Y190 containing resident plasmids that encoded binding domain (BD) fusions to RsbU, RsbT, and RsbX (pUV31, pUV76, or pUV145) (36). Plasmids that encoded interacting proteins were selected by GAL4-dependent histidine prototrophy and screened for GAL4-dependent -galactosidase activity (36). The B. subtilis DNA fragments of interest were amplified by PCR from positive colonies by using primers specific for the sequences immediately outside of the multiple cloning sites of the vectors. These DNAs were verified as the coding elements for proteins that interact with the rsb fusions by recloning into the AD vector and transformation into yeast cells carrying the appropriate rsb fusion. The DNA sequences that encoded interacting peptides were determined and their predicted protein sequences compared to the B. subtilis genome database (30a) to identify the genes from which they were derived.

Construction of plasmids for integration into target genes. pUSX19 was created by cloning a 1.7-kb BamHI-SphI DNA fragment containing the xylose promoter and repressor from pX-2 (20) into similar sites of pUS19 (4). The 5' ends (500 to 700 bp) of the genes that encoded protein fragments, which interacted with the Rsb proteins in the yeast two-hybrid system, were PCR amplified and cloned into the BamHI and SacI sites of pUSX19 by using sites that had been introduced during the amplification. This cloning placed the 5' ends of the genes downstream of P_xyl in pUSX19. When these plasmids were transformed into PY22, a Campbell-like integration of the plasmid into the B. subtilis chromosome disrupted the resident copy of the gene and positioned the sole intact copy of the target gene downstream of P_xyl. These strains were then grown with or without xylose to determine whether the isolated genes were essential for growth. A ctc::lacZ reporter system was introduced into these strains by transformation with BSA46 (3) chromosomal DNA and selection for a ctc::lacZ-linked antibiotic resistance (erm and cat). To assess the possible need for each Rsb interacting protein in ^B stress activation, strains containing the P_xyl fusions were grown in LB with or without xylose and exposed to stress (4% ethanol) during the exponential phase of growth (optical density at 540 nm of 0.2). The ^B activity was monitored by reporter gene (ctc::lacZ) expression. To determine the effects of enhanced levels of the interacting proteins on stress activation of ^B, the cultures were grown in the presence of 2% xylose, which induces P_xyl several 100-fold (20), and stressed as described above.

Construction of plasmids and strains for Obg and RsbT manipulations. pJM2, a P_spac::obg fusion plasmid, was constructed by cloning the obg gene, including its ribosomal binding site, which had been amplified by PCR into pBluescript (26), by using BamHI sites that had been placed at each end during the amplification. The orientation of the fragment in the plasmid was determined by restriction endonuclease digestion analysis. The obg segment was excised with HindIII and XbaI and cloned into the multiple cloning site of pDG148. pJM4 was constructed by PCR amplification of a 250-bp DNA fragment containing 150 bp of the 5' end of obg and 100 bp of upstream DNA. BamHI and BglII sites, introduced into the ends of the fragment during amplification, were used to clone the fragment into the BamHI site of pUSX19, creating a P_xyl::obg₁₅₀ fusion. pHV501T contains the HindIII/BamHI piece of rsbT from pDT11 (30). The fragment was cloned into pHV501 downstream of P_spac. The resulting plasmid was cut with ClaI and BamHI to remove lacZ, made blunt with the Klenow fragment of DNA polymerase, and religated. Transformation of PY22 with pHV501T places P_spac between rsbS and rsbT within the sigB operon (BSA400), due to a Campbell-like integration of the plasmid into rsbT.

General methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and -galactosidase activity assays were performed as previously described (10, 19). DNA manipulations were performed according to standard protocols (26). Transformation of naturally competent B. subtilis cells was carried out as described by Yasbin et al. (42). Yeast -galactosidase assays were performed as done previously (36). Predicted protein translations of the DNA fragments were determined by using DNAMAN software (Lynnon Biosoft Co.). Sequencing of DNA fragments identified in the yeast two-hybrid system screen was performed by the University of Texas Health Science Center at San Antonio Center for Advanced DNA Technologies.

	RESULTS

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Identification of Rsb interactors. We considered RsbT, RsbU, and RsbX as the most likely of the Rsb proteins to be targeted by stress-generated signals. To identify possible protein mediators of such signals, we screened B. subtilis chromosomal DNA for genes whose products could be shown to physically interact with RsbT, -U, or -X in the yeast dihybrid system. As described in Materials and Methods, yeast strains carrying Gal4-activatable his and lacZ genes, as well as plasmids encoding rsbT, rsbU, or rsbX fused to the Gal4 DNA BD, were transformed with plasmid libraries of B. subtilis chromosomal DNA. The plasmid libraries contain potential translational fusions between various B. subtilis genes and the Gal4 AD. Transformants in which the cloned B. subtilis DNA encoded a Gal4-AD protein which could associate with an Rsb/Gal4-BD and create a functional gene activator protein were selected as histidine prototrophs and screened for a Lac⁺ phenotype. After we verified that the His⁺ Lac⁺ activities of the positive clones were due to an interaction between the AD fusion proteins and the Rsb-BD proteins (Materials and Methods), the AD fusion plasmids were recovered from the yeast cells, transformed into E. coli, and screened by restriction endonuclease-Southern blot analyses to eliminate duplicate clones. A total of 31 unique B. subtilis DNA inserts were isolated from approximately 400 His⁺ Lac⁺ transformants. The insert DNA was sequenced by using DNA primers complementary to the vectors' translational fusion junctions. The identity of the B. subtilis element that had become fused to the Gal4-AD was determined by searching the B. subtilis genome sequence database (30a) for the sequences contained at the Gal4-AD fusion junctions. The results of this exercise are listed in Table 2. Eleven predicted intergenic regions were among the B. subtilis chromosome segments that displayed Gal4 activity when cloned into the AD vector. Presumably, these represent fortuitously generated AD fusion proteins that could pair with particular Rsb::Gal4-BD chimeras. In previous studies, where interactions between the various rsb genes were examined in the yeast dihybrid system, no RsbX interactions were detected with any of the other Rsb genes, but interactions were found between RsbT and RsbS/-R/-U and between RsbU and RsbV/-T (36). Our current screening of the B. subtilis genome detected two of these four associations: the interactions of RsbT with both RsbR and RsbS (Table 2).

Obg is necessary for stress activation of ^B. Obg is an essential B. subtilis protein (21, 31, 32). In order to study its possible role in ^B activation, we used a B. subtilis strain (BSJ-6) in which obg's expression was under the control of an IPTG-inducible promoter (32). Withholding IPTG from this strain leads to a depletion of Obg and a concomitant cessation of growth (32). Cultures of BSJ-6 were resuspended in LB with or without IPTG and monitored until growth had slowed in the culture lacking IPTG. Portions of the cultures were then stressed by treatment with ethanol. The ^B activity in both cultures was estimated by using a reporter gene fused to a ^B-dependent promoter (i.e., ctc::lacZ). B. subtilis with Obg (i.e., the IPTG-containing culture) immediately activated ^B after ethanol treatment; however, the strain in which the Obg levels had been depleted failed to activate ^B in response to ethanol stress (Fig. 2). Similar results were obtained with heat shock as the stress and Western blot analyses to judge the inducibility of the Rsb genes that are downstream of the sigB operon's ^B-dependent promoter (i.e., rsbV and -W, sigB, and rsbX) (data not shown).

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Effect of Obg depletion on ethanol induction of B. (Top panel) Growth of BSJ-6 (Pspac::obg) in LB with () or without () IPTG (100 µM). The arrows represent the points during growth when ethanol (4% final concentration) was added to the IPTG-induced (1) and the IPTG (2) cultures. The curves display the untreated cultures; the ethanol-treated cultures had their growth reduced by approximately 50% (not shown). (Bottom panel) ctc::lac expression in BSJ-6. Samples of the cultures represented in the top panel were analyzed for B-dependent -galactosidase activity. The arrows depict the times of ethanol addition in the IPTG+ (1) and IPTG (2) cultures. Closed symbols represent IPTG+ cultures with () or without () ethanol; open symbols depict the IPTG culture with () or without () ethanol.

View larger version (15K): [in this window] [in a new window]   FIG. 3.   ATP-responsive induction of B in Obg-depleted cells. BSJ-6 (Pspac::obg) was grown in the presence (A) or absence (B) of IPTG. The top portions of panels A and B illustrate the growth of the cultures without ( and ) or in the presence of 2 mM sodium azide () and ), which was added at the times indicated by the arrows. The lower portions of panels A and B display B-dependent -galactosidase activity of the cultures represented in the panels above them, with the arrows depicting the time of sodium azide addition. The triangles represent azide-treated cultures; the circles represent untreated cultures.

Obg effects on ^B are independent of Spo0A. Obg is essential for both growth and sporulation (21, 31, 32). During studies of the sporulation requirement for Obg, it was discovered that Obg is needed, either directly or indirectly, for the phosphorylation reaction that activates the transition-phase regulatory protein Spo0A (32). Given that both the activation of Spo0A and the stress induction of ^B require Obg, we asked whether the stress activation of ^B could be a Spo0A-mediated process. Wild-type and spo0A::cat strains of B. subtilis were exposed to ethanol stress and examined for ^B activation. ^B induction occurred regardless of the presence or absence of Spo0A (Fig. 4). Thus, the need for Obg in ^B activation is not likely to be due to an Obg effect on Spo0A.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Activation of B in Spo0A B. subtilis. B. subtilis BSA46 (wild type, circles) and BSJ-17 (spo0A::cat, triangles) were grown without stress in LB or exposed to ethanol (4%) during an early stage of exponential growth. Samples were taken from control (solid symbols) and ethanol-treated (open symbols) cultures at the times indicated and analyzed for B-dependent (ctc::lacZ) -galactosidase activity.

Obg interacts with RsbT and RsbW in the yeast dihybrid system. Knowing that high-molecular-weight complexes containing multiple Rsb proteins and unknown additional components are readily detectable in B. subtilis extracts (10) and that our initial screening of the B. subtilis library had not been exhaustive, we revisited the yeast dihybrid system to test whether Obg could interact with additional Rsb components. Specifically, we paired both the entire obg gene and the obg fragment which had interacted with RsbX with other rsb genes. rsbS was not included in the analyses due to its ability to activate Gal4-dependent promoters independently when fused to Gal4-BD.

Obg's essential role in ^B activation is independent of RsbX. Obg was initially identified as a potential ^B regulator on the basis of an interaction between its carboxy terminus and RsbX in the yeast dihybrid system. RsbX is a negative regulator of the stress-inducible pathway for ^B activation (3, 15, 17, 30, 33, 35). Thus, if Obg's essential role in ^B activation entails an effect on RsbX, we would expect this to involve a lifting of RsbX's negative control. If this is so, Obg should not be essential for the stress activation of ^B in the absence of RsbX.

View larger version (13K): [in this window] [in a new window]   FIG. 5.   Effects of Obg on activation of B in RsbX B. subtilis. BSJ-10 (Pspac::obg rsbX::spec rsbU194VA) was grown in LB with (A) or without (B) IPTG (0.1 mM). As in Fig. 2, cells were treated with ethanol (4%) at the times indicated by the arrows either during growth (A) or after the culture slowed due to Obg depletion (B). Symbols: triangles, ethanol-treated cultures; circles, untreated cultures.

RsbT can activate the ^B stress pathway in the absence of Obg. Stress is believed to set in motion a process (Fig. 1) whereby RsbT frees itself from its inhibitor and then binds and activates RsbU, the RsbV-P phosphatase (41). RsbV then interacts with RsbW to free ^B (2, 6, 9). There are several points at which an Obg-dependent function might be needed in this process. To determine whether Obg is required for the activation of RsbT or a downstream event, we took advantage of the observation that the induced expression of RsbT, in the absence of a corresponding synthesis of its inhibitor (RsbS), can drive activation of ^B in unstressed bacteria (18, 28). If the essential Obg-dependent function is limited to the activation of RsbT, then the induced synthesis of RsbT should activate ^B in Obg-depleted cells. Conversely, if Obg has a critical role in RsbT's ability to activate RsbU, RsbU's capacity to dephosphorylate of RsbV-P, or RsbV's ability to displace ^B from RsbW, then providing additional RsbT should not activate ^B in Obg-depleted cells.

View larger version (13K): [in this window] [in a new window]   FIG. 6.   Activation of B by RsbT overexpression in Obg-depleted B. subtilis. BSJ-13 (Pxyl::obg Pspac::rsbT) was grown in LB with or without xylose (0.5%) to maintain Obg levels (A) or to deplete the cell of Obg (B), respectively. At the times indicated by the arrows, IPTG (1 mM) was added to the exponentially growing cells (A), and the culture that had slowed due to Obg depletion (B) to induce Pspac upstream of rsbT. Samples were taken from IPTG-induced () and ) and control ( and ) cultures and were analyzed for B-dependent -galactosidase activity as in Fig. 2.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The general stress regulon of B. subtilis is induced when environmental stress communicates with a kinase/phosphatase pathway to trigger the activation of the ^B transcription factor (41). Neither the stress-generated signal that activates ^B nor the component within the pathway that initially responds to the signal are known. Previous studies indicated that the known ^B regulators are insufficient to detect and respond to stress and that the inducing signal is likely to be novel and Bacillus specific (29). This suggested that additional B. subtilis gene products are needed to communicate the presence of stress to the ^B activation cascade. In the current study, we attempted to identify such gene products by using the yeast dihybrid system to isolate B. subtilis proteins that could interact with important members of the ^B activation cascade and, as such, might be involved in this signaling. As is evident from our results, the dihybrid selection system readily detects peptides that can interact with Rsb fusion proteins to activate the yeast selection system. Although a few of these associations are clearly biologically relevant, i.e., known Rsb-Rsb interactions were detected, most of the isolates likely represent fortuitous interactions between domains on the fusion proteins. Fortunately, access to the B. subtilis genome database and our ability to readily alter the expression of the genes for these putative interactors allowed us to test whether the abundance or absence of their products could affect ^B stress activation and also allowed us to put aside unpromising candidates. By this approach, obg was detected in our current screening as a gene whose product influences ^B activity. If B. subtilis is depleted of Obg, stress activation of ^B, but not its ATP-responsive activation, is blocked. Overexpression of Obg from a high-copy-number plasmid had no effect on ^B activity (data not shown).

Obg is the second gene in the operon that encodes Spo0B, a critical protein in the phosphorelay which modulates the activity of the sporulation-transition state regulatory protein Spo0A (31). Obg is essential for both B. subtilis growth and sporulation (31, 32). It is a GTP binding protein (39) whose explicit function is unknown; however, there is evidence that Obg activity influences the initiation of chromosome replication (21) and the phosphorylation state of the postexponential-phase gene regulator, Spo0A (32). It is unclear whether Obg's activities in either of these processes is related to its effects on ^B activity. We note however, that the Obg-dependent activation of Spo0A is not needed for the ^B activation process, since stress activation of ^B occurs in the absence of Spo0A function.

Obg is a member of a unique family of small GTP binding proteins that have been identified in diverse organisms from bacteria and mammals (reviewed in reference 25). In bacteria, the Obg subfamily is speculated to monitor the state of intracellular GTP levels and to serve as a switch to promote growth when bound to GTP, but not when associated with GDP (25). The actual targets for this switch protein are unknown. Our yeast dihybrid data suggest that Obg may directly interact with a number of the ^B Rsb regulators; however, even if these interactions ultimately prove to be biologically relevant, the Rsb proteins cannot be Obg's only targets. Obg is essential for growth and sporulation, while neither the Rsb proteins nor ^B are essential for either process. Perhaps Obg, as a general sensor of intracellular GTP levels and a regulatory switch, has several targets within B. subtilis, allowing it to coordinate diverse functions in response to nucleotide changes.

Although it is clear that Obg is essential for stress activation of ^B, it remains possible that the apparent interaction of Obg with particular Rsb proteins is accidental and that the requirement for Obg in ^B stress activation is indirect. The putative RsbT-Obg interaction is, however, intriguing given that one function of the Obg class of proteins is intracellular signaling and that stress activation of RsbT fails to occur in its absence. Determining whether Obg directly communicates the existence of environmental stress to RsbT or is only indirectly involved in this process will likely require in vitro biochemical analyses.