May 14, 2007 - The general stress regulon of Bacillus subtilis is controlled by the activity state of B, a transcription factor that is switched on following exposure ...
JOURNAL OF BACTERIOLOGY, Sept. 2007, p. 6168–6175 0021-9193/07/$08.00⫹0 doi:10.1128/JB.00756-07 Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 189, No. 17
ClpP Modulates the Activity of the Bacillus subtilis Stress Response Transcription Factor, B䌤 Adam Reeves,1 Ulf Gerth,2 Uwe Vo ¨lker,3 and W. G. Haldenwang1* Department of Microbiology and Immunology, MC7758, University of Texas Health Science Center, San Antonio, Texas 78229-3900,1 and Institute for Microbiology2 and Interfacultary Institute for Genetics and Functional Genomics,3 Ernst Moritz Arndt University Interfaculty, Greifswald, Germany Received 14 May 2007/Accepted 18 June 2007
The general stress regulon of Bacillus subtilis is controlled by the activity state of B, a transcription factor that is switched on following exposure to either physical or nutritional stress. ClpP is the proteolytic component of an ATP-dependent protease that is essential for the proper regulation of multiple adaptive responses in B. subtilis. Among the proteins whose abundance increases in ClpPⴚ B. subtilis are several known to depend on B for their expression. In the current work we examine the relationship of ClpP to the activity of B. The data reveal that the loss of ClpP in otherwise wild-type B. subtilis results in a small increase in B activity during growth and a marked enhancement of B activity following its induction by either physical or nutritional stress. It appears to be the persistence of B’s activity rather than its induction that is principally affected by the loss of ClpP. B-dependent reporter gene activity rose in parallel in ClpPⴙ and ClpPⴚ B. subtilis strains but failed to display its normal transience in the ClpPⴚ strain. The putative ClpP targets are likely to be stress generated and novel. Enhanced B activity in ClpPⴚ B. subtilis was triggered by physical stress but not by the induced synthesis of the physical stress pathway’s positive regulator (RsbT). In addition, Western blot analyses failed to detect differences in the levels of the principal known B regulators in ClpPⴙ and ClpPⴚ B. subtilis strains. The data suggest a model in which ClpP facilitates the turnover of stress-generated factors, which persist in ClpP’s absence to stimulate ongoing B activity. The general stress regulon (GSR) of Bacillus subtilis encodes more than 150 genes whose products allow the bacterium to withstand physical insult or prolonged starvation (20, 29, 30). The GSR is controlled by the activity state of the B transcription factor (3, 5), an alternative RNA polymerase subunit that directs the enzyme to GSR promoters. In unstressed B. subtilis, B is unavailable to RNA polymerase due to its sequestration into an inhibitory complex with the anti-B protein RsbW (Fig. 1) (4). B is freed from RsbW when another protein (RsbV) binds to RsbW to allow B’s release (11, 12). RsbV’s phosphorylation state determines its ability to release B from RsbW. RsbW is both an RsbV/B binding partner and an RsbVspecific protein kinase (16). The RsbW-dependent phosphorylation of RsbV inactivates RsbV as a B release factor (16). Phosphorylated RsbV (RsbV-P) is reactivated when it is dephosphorylated by either of two stress-responsive phosphatases (RsbP and RsbU) (21, 37, 41). The RsbP phosphatase responds to nutritional stress (e.g., glucose or phosphate limitation), while the RsbU phosphatase is activated by physical stress (e.g., ethanol treatment or osmotic or heat shock) (1, 22, 38, 42, 44). The mechanisms by which stress is communicated to either phosphatase are unknown. Nutritional stress induction of RsbP coincides with a drop in cellular ATP; however, it is not known whether ATP levels themselves or other effectors trig-
ger the induction (46). The activation of RsbP also requires the product of a gene (rsbQ) that is cotranscribed with it (7). RsbP and RsbQ interact with each other in a yeast two-hybrid system (7); however, the contribution of RsbQ to RsbP’s activity remains undefined. The physical stress phosphatase (RsbU) also requires an additional protein (RsbT) for activity (43, 44). In unstressed B. subtilis, RsbT, in association with its primary negative regulator RsbS, is bound in large (⬎106 Da) multiprotein complexes termed “stressosomes” (9, 26). The principal structural element of the stressosome is believed to be formed as an assemblage of one or more members of a family of homologous proteins (RsbR, YkoB, YojH, and YqhA) (2, 9, 26). RsbS’s ability to inhibit RsbT requires the stressosome (1, 2). RsbS fails to block RsbT-dependent activation of RsbU in cells lacking the RsbR proteins. The roles of the individual RsbR paralogs in the stressosome are unknown; however, they are likely to be at least partially redundant (2, 26). The loss of any one RsbR protein does not significantly affect B activity. RsbS’s ability to inhibit RsbT is weakened only when multiple members of the RsbR family are lost (26). Physical stress activates B by triggering an RsbT-dependent phosphorylation of both the RsbR proteins and RsbS (25, 44). Phosphorylation of the RsbR proteins is believed to facilitate the phosphorylation of RsbS (9, 16, 26). Phosphorylation of RsbS is the pivotal event that allows the release of RsbT and the activation of RsbU. Restoration of B activity to prestress levels is effected by RsbX, a phosphatase that dephosphorylates and reactivates both RsbR and RsbS (8, 10, 44). The dephosphorylations allow RsbT to again be sequestered by the stressosome. RsbX levels increase following B activation;
* Corresponding author. Mailing address: Department of Microbiology and Immunology, MC7758, University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900. Phone: (210) 567-3957. Fax: (210) 567-6612. E-mail: haldenwang@uthscsa .edu. 䌤 Published ahead of print on 22 June 2007. 6168
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by the loss of ClpP during growth but more pronouncedly altered in ClpP⫺ strains following exposure to either physical or nutritional stress. It is the transience of B’s activation after exposure to stress, rather than its induction, that appears to be primarily affected by the loss of ClpP, with B activity persisting in the ClpP⫺ strains. The data implicate a ClpP-dependent process as part of the mechanism that limits the B response to stress. MATERIALS AND METHODS
FIG. 1. Model of B control. As depicted in the lower portion of the diagram, B is normally inactive, complexed with the anti-B protein, RsbW (W). B is freed from RsbW when a release factor, RsbV (V) binds RsbW in lieu of B. In the absence of stress, RsbV is unable to trigger B release due to an RsbW-catalyzed phosphorylation. RsbV is dephosphorylated by one of two stress-responsive phosphatases that uniquely respond to physical or nutritional stress. The nutritional stress phosphatase, RsbP (P), requires a coexpressed protein, RsbQ (Q), for activity. The RsbQ/P trigger is unknown, but its activation coincides with conditions that cause a drop in ATP. The physical stress phosphatase, RsbU (U), is activated by a second protein, RsbT (T), that is ordinarily bound to a negative regulator, RsbS (S), in a large (⬎106 Da) complex (stressosome) formed from a family of paralogous proteins (R). Following exposure to physical stress, an unknown mechanism allows the RsbR proteins and RsbS to become phosphorylated by RsbT. This frees RsbT to activate the RsbU phosphatase. RsbR-P and RsbS-P are dephosphorylated and reactivated by RsbX (X), a phosphatase whose levels increase following B activation. The model is based on the references given in the text.
however, RsbX abundance per se does not appear to be a significant factor in restricting B activity (40). B activity, triggered by either nutritional or physical stress is transient. Following exposure to a physical stress (i.e., ethanol treatment), B-dependent reporter gene activity peaks after 20 to 30 min and then declines (39). The decline in B activity could be due to a decrease in the activation process that drives the RsbT-dependent phosphorylation of RsbR and RsbS, an enhancement of the RsbX-dependent dephosphorylation of these proteins, or both. In a number of stress-induced systems, turnover of the triggering factors serves to limit the response and allow bacteria to resume balanced growth (reviewed in reference 18). It is plausible that similar proteolytic processes may contribute to the transience of the B response. Among the B. subtilis proteases, ClpP is a particularly interesting candidate for a possible modulator of B activity. ClpP is required for the proper functioning of a number of regulated processes in B. subtilis. These include competence development, motility, degradative enzyme synthesis, growth at high temperature, and sporulation (28). A possible interplay between ClpP and B is suggested by the observation that the levels of most of the B. subtilis proteins induced by stress or starvation, including several B-dependent gene products, are elevated in ClpP⫺ strains. In addition, the clpP promoter region includes a B-dependent promoter that augments the gene’s principal promoter which is dependent on A (17, 27). In the current work we examine the relationship of ClpP to the activity of B. We find that B activity is modestly elevated
Bacterial strains and plasmids. Strains and plasmids used in this study are shown in Table 1. All B. subtilis strains were derivatives of PY22. The rsbQ-P disruption (BZH47) was constructed by amplifying a 2.6-kbp DNA fragment from PY22 chromosomal DNA using oligonucleotide primers that hybridized 440 bases upstream of the rsbQ initiation codon and immediately downstream of the rsbP termination codon. Once cloned into pUC19, the resulting plasmid was linearized at a unique BglII site 252 bases into the 807-bp rsbQ gene and joined to an erm cassette cut with BamHI from pDG646 (19). The resulting plasmid (pWH87) was linearized and transformed into PY22 with selection for Ermr. The B-dependent reporter gene (Pctc::lacZ) was then introduced into an Ermr clone by transformation with chromosomal DNA from BSA46 (3) and selection for ctc::lacZ-linked Cmr. BZH47 is an Cmr Ermr clone, screened in liquid medium for the anticipated rsbQ::erm phenotype of B activation by physical but not nutritional stress. BSH80 is BSA46 made Cm::Tetr by transformation with the antibiotic resistance conversion vector pCm::Tc (36). BSH158 is BSH80 transformed to RsbU⫺ with chromosomal DNA from BSA70 (rsbU::kan) (3). ClpP⫺ strains were constructed by transformation with chromosomal DNA from QB4916 (clpP::spc) (28). ytvA was disrupted by targeting its coding sequence with an integrating plasmid (pARE189). pARE189 is pJM102 (21) into which a 396-bp DNA fragment (nucleotides 4 to 400 of the 783-bp ytvA coding sequence) amplified by PCR from PY22 chromosomal DNA had been cloned. pARE189 transformants, selected on the basis of the vector-encoded Cmr, were screened by PCR for integration of the plasmid within the chromosomal ytvA gene. pUC spc::kan was constructed by inserting the Spcr cassette of pDG1726 (19) as an EcoRI/BamHI fragment into these sites on pUC19. The resulting plasmid was cut at a unique ClaI site in the spc cassette and ligated to a Kanr cassette cut from
TABLE 1. Plasmids and strains used in this study Plasmid or strain Plasmids pUK19 pARE183 pWH87 pCM::TC pUC SpC::Kan pDG1726 pDG780 B. subtilis strains PY22 BSA46 BSA70 BSA419 QB4916 BSH80 BZH47 BSH115 BSH118 BSH121 BSH158 BSH159 BAR121 BAR126 XS16 BSH130 BSH131 BAR129
Relevant genotype or feature
Apr Apr Apr Apr Apr Apr Apr
Kanr Kanr ytv (4-400) rsbQ::erm Tetr Kanr Spcr Kanr
Wild type SP ctc::lacZ (Cmr Ermr) rsbU::kan PSPAC::rsbT SP ctc::lacZ clpP::spc SP ctc::lacZ (Tetr Erm) rsbQ::erm SP ctc::lacZ clpP::spc SP ctc::lacZ rsbQ::erm clpP::spec SP ctc::lacZ PSPAC::rsbT clpP::spec SP ctc::lacZ rsbU::kan SP ctc::lacZ rsbU::kan clpP::spc SP ctc::lacZ ytvA::pJM102 SP ctc::lacZ ytvA::pJM102 clpP::spc SP ctc::lacZ rsbT15IS rsbX::spc SP ctc::lacZ rsbT15IS rsbX::spc::kan SP ctc::lacZ rsbT15IS rsbX::kan clpP::spc SP ctc::lacZ ytvA::pJM102 rsbU::kan clpP::spc SP ctc::lacZ
Source, reference, or construction 31 This study This study 36 This study 19 19 3 3 3 33 28 pCM::TC3BSA46 This study QB49163BSH80 QB49163BZH47 QB49163BSA419 BSA703BSH80 QB49163BSH158 pARE1833BSH80 QB49163BAR121 pUC spc::KanXS16 QB49163BSH130 This study
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FIG. 2. Influence of the inactivation of ClpP on the expression of the SigB-dependent general stress regulon of B. subtilis. (Left) Representative autoradiogram of the cytosolic protein pattern of B. subtilis 168 grown in minimal medium at 37°C. (Right) Enlarged sections of gels prepared from crude protein extracts of cells of the B. subtilis wild-type strain 168 or its clpP mutant derivative pulse-labeled with L-[35S]methionine during exponential growth. Representative examples of the general stress proteins include Ctc, GsiB, SodA, YtxH, YsnF, and YvyD, labeled with arrowheads. In the 2D electrophoresis section of the clpP mutant, the location of ClpP is indicated by the square.
pDG780 with this same enzyme. The resulting plasmid transforms Spcr B. subtilis to Spcs Kanr. 2D-Gel electrophoresis. The B. subtilis wild-type strain 168 and its isogenic clpP derivative QB4916 (28) were grown in a defined minimal medium as described previously (37). During exponential growth, aliquots of the bacterial cultures were pulse-labeled with L-[35S]methionine (15 Ci/ml) for 5 min and harvested by centrifugation (10 min at 15,000 ⫻ g at 4°C). Cells were subjected to ultrasonic disruption using a Braun sonifier. Protein extracts containing equal amounts of radioactivity were separated according to established protocols by two-dimensional (2D) gel electrophoresis in the pH range of 4 to 7 (14). After exposure to phosphorimaging screens, images were recorded with a phosphorimager. General methods. B. subtilis was grown with shaking in Luria-Bertani (LB) medium (32). Physical stress was imposed by the addition of ethanol (4% final concentration) to logarithmically growing cultures. Nutritional stress activation was performed by allowing growth to halt in a low-phosphate synthetic medium (42). Samples taken for B-dependent -galactosidase activities were analyzed by the chloroform-permeabilized cell technique of Kenny and Moran (24). Western blotting assays were performed as previously described using mouse monoclonal antibodies against RsbR, RsbS, RsbT, RsbU, RsbV, RsbW, SigB, and RsbX (13). B. subtilis transformation was carried out by the method of Yasbin et al. (45).
RESULTS -dependent gene products are elevated in ClpPⴚ B. subtilis. In a study of ClpP’s role in protein degradation, Kock et al. noted that the levels of B itself and a number of proteins whose expression depend on B are elevated in strains of B. subtilis lacking ClpP (27). Given that ClpP appears to be an effector of bulk protein turnover in B. subtilis (27), this observation may reflect ClpP degradation of the gene products B
themselves or, alternatively, a ClpP-dependent modulation of B activity. In the latter case, the loss of ClpP might allow elevated B-dependent gene expression. We first sought to confirm the observation that B-dependent gene products are elevated in the absence of ClpP. To this end, wild-type and ClpP⫺ B. subtilis strains were grown in defined medium and labeled with L-[35S]methionine during the exponential growth phase. Extracts were prepared from these cultures and analyzed by 2D gel electrophoresis. Figure 2 shows phosphorimager recordings of representative gels. The left panel depicts a typical 2D gel image of extracts prepared from wild-type B. subtilis. The gel areas in the right panels illustrate gel regions of fractionated wild-type and ClpP⫺ extracts. Each gel area depicts the regions represented by the boxes in the left panel. The positions of the protein products of six B-dependent genes (ysnF, ctc, yvyD, sodA, ytxH, and gsiB) as well as clpP itself are indicated. As expected, ClpP is missing from the clpP::spc strain (Fig. 2, second panel from the bottom right). Consistent with the notion that B-dependent gene products are elevated in the absence of ClpP, all six of the representative B-dependent gene products are more abundant in the ClpP⫺ extract during exponential growth. ClpP restricts B activity following stress. To investigate whether the ClpP status of B. subtilis influences B-dependent transcription rather than turnover of B-dependent gene products, a clpP null mutation was introduced into B. subtilis strains with a B-dependent reporter gene (i.e., Pctc::lacZ). The recipient
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FIG. 3. B activity in ClpP⫹ and ClpP⫺ B. subtilis. B. subtilis strains carrying SP ctc::lacZ were grown in LB medium (A to C) or a PO4-limited medium (D). Cells shown in panels A to C were exposed to 4% ethanol (open symbols) at time zero or allowed to continue growth (filled symbols). The doubling times of the wild-type and ClpP⫺ cultures were 25 min and 43 min, respectively. Addition of ethanol increased the doubling time of each culture to approximately 60 min. The cultures shown in panel D were not exposed to ethanol but were allowed to enter stationary phase (indicated by the arrow). Samples were taken at the indicated times and assayed for -galactosidase as described in Materials and Methods. Symbols (open and filled as described above): panel A squares, BSH80 (wild type); panel A triangles, BSH115 (clpP::spc); panel B squares, BZH47 (rsbQ::erm) panel B triangles, BSH118 (rsbQ::erm clpP::spc); panels C and D squares, BSH158 (rsbU::kan); panels C and D triangles, and BSH159 (rsbU::kan clpP::spc).
strains contained either wild-type alleles of the B regulators or carried null mutations in either the physical (rsbU::kan) or nutritional (rsbQ::erm) stress pathway. When plated on medium containing X-Gal (5-bromo-4-chloro-3-indolyl-“⫺ transformants exhibited the small-colony morphology associated with the loss of ClpP, as well as a dark blue colony color indicative of heightened -galactosidase activity. Elevated B activity was evident in the ClpP⫺ strains regardless of whether the physical or nutritional pathway was disrupted. This implies that the loss of ClpP can enhance B activity through either the physical or nutritional stress pathway. To characterize the effects of ClpP on B activity, B. subtilis strains with wild-type or clpP null alleles were grown in liquid medium and exposed to physical (4% ethanol) or nutritional (PO4 limitation) stress. During growth, the ClpP⫺ strains displayed B-dependent -galactosidase activity that was slightly elevated compared to that of the ClpP⫹ parental strains (Fig. 3A to D). Distinct differences in B activity between the ClpP⫹ and ClpP⫺ variants of the ethanol-responsive strains (Fig. 3A and B) were evident following ethanol stress. Although B activity was similarly induced in both strains, the ClpP⫺ strain failed to exhibit the decline in B activity that normally follows stress induction. Typical of previous experiments with ClpP⫹ strains (35, 40), -galactosidase activity fell after 20 to 30 min in these strains. In contrast, B-dependent reporter gene activity persisted in the ClpP⫺ strain throughout the course of the experiment (Fig. 3A and B). This ongoing B activity is dependent on the pathway that normally activates B after exposure to physical stress. It occurs in a B. subtilis strain without an essential component of the nutritional pathway’s phosphatase, i.e., rsbQ::erm (Fig. 3B), but is absent from the strain lacking the physical stress pathway’s phosphatase
(rsbU::kan) (Fig. 3C). The profile of B-dependent reporter gene activity following ethanol treatment implies that ClpP is not involved in the activation of B by this physical stress but, instead, suggests that the absence of ClpP compromises the strain’s ability to reestablish prestress levels of B activity. To explore the possible effect of ClpP on the induction of B by nutritional stress, ClpP⫹ and ClpP⫺ strains of B. subtilis that lacked the physical stress pathway phosphatase (rsbU::kan) were grown and allowed to enter stationary phase in a PO4limited medium. Cessation of growth due to PO4 limitation is a strong inductant of B’s nutritional stress pathway (42). As illustrated in Fig. 3D, B-dependent reporter gene activity is induced in both the ClpP⫹ and ClpP⫺ cultures as the cultures slow (Fig. 3D, arrow) due to PO4 limitation. B activity in the ClpP⫹ strain slows soon after its induction but continues unabated in the ClpP⫺ strain. Thus, B activity, triggered by either the physical or nutritional stress pathways, persists in the absence of ClpP. Given that ClpP is involved in the turnover of a large number of diverse proteins, it is formally possible that the persistence of B-dependent reporter gene activity in the ClpP⫺ strains is a reflection of the half-life of the Ctc-LacZ fusion protein in the absence of ClpP rather than elevated B activity. To examine this possibility, ClpP⫹ and ClpP⫺ strains of B. subtilis were stressed with ethanol and then treated with chloramphenicol to block further protein synthesis at a time (30 min) after ethanol treatment when B-dependent -galactosidase activity of ClpP⫹ cultures begins to decline. If the loss of ClpP affects the stability of the Ctc-LacZ fusion protein, a difference in -galactosidase activity should become apparent between the chloramphenicol-treated ClpP⫹ and ClpP⫺ cultures. Alternatively, if the loss of ClpP has no effect on Ctc-
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FIG. 4. Effect of ClpP on reporter gene turnover. B. subtilis strains BSH80 (wild type; 䡺) and BSH115 (clpP::spc; ‚) were grown in LB medium to an optical density at 420 nm (OD420) of 0.2 and then treated with ethanol to 4% at time 1. After 30 min (arrow), chloramphenicol was added (100 g/ml) to half of each culture (filled symbols), and the incubation continued. Samples were taken at 15-min intervals and assayed for B-dependent -galactosidase activity. The data are presented as the OD420 of an assay mixture containing the cells from 1 ml of culture resuspended and permeabilized in 1 ml of assay buffer and incubated with substrate for 15 min at 25°C.
LacZ stability, both chloramphenicol-treated cultures should exhibit similar -galactosidase levels. To emphasize the absolute changes in -galactosidase levels within each culture, the Ctc-LacZ activity presented in Fig. 4 is depicted as a function of cell culture volume rather than specific activity (i.e., Miller units). As can be seen in Fig. 4, the -galactosidase activities of the ClpP⫹ strain remain constant over the course of the experiment (105 min) regardless of whether the culture had been treated with chloramphenicol. The decline in -galactosidase specific activity seen in the ClpP⫹ strains (Fig. 3) is therefore not a consequence of -galactosidase turnover but, rather, of a cessation of Ctc-LacZ synthesis and a reduction in specific -galactosidase activity as the culture density continues to increase. Consistent with the persistent elevation of specific -galactosidase activity in the ClpP⫺ strains (Fig. 3), loss of ClpP allows ongoing -galactosidase accumulation in the absence of chloramphenicol (Fig. 4). sigB operon protein levels in ClpPⴙ and ClpPⴚ strains. Changes in the levels of some of the B regulators can lead to B activation in the absence of stress (22, 33). It is therefore possible that the loss of the ClpP protease might influence stress-induced B activity by changing the levels of one or more of the known B regulators. To explore this possibility, ClpP⫹ and ClpP⫺ B. subtilis strains were grown and exposed to ethanol stress. Samples taken prior to ethanol addition and at 30 and 60 min after the stress application were examined by Western blotting for changes in the levels of B and the regulators known to be involved in the response to ethanol stress. B, its primary regulators (RsbV and RsbW), and the principal components of the physical stress pathway (RsbR, -S, -T, -U, and -X) are encoded in an eight-gene operon that is transcribed from a promoter likely recognized by the bacterium’s “housekeeping” form of RNA polymerase (i.e., A containing RNA polymerase) (43). An internal B-dependent promoter upregulates the expression of rsbV rsbW sigB rsbX when B becomes active (3, 6). Thus, the levels of these four gene products increase following stress. Figure 5 depicts a typical Western blotting result using monoclonal antibodies specific for each of
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FIG. 5. Western blot of sigB operon products in ClpP⫹ and ClpP⫺B. subtilis strains. BSH80 (Clp⫹) (lane groupings 1, 3, and 5) and BSH110 (Clp⫺) (lane groupings 2, 4, and 6) were grown to an optical density of 0.5 in LB medium and treated with ethanol (4%). Samples were taken immediately prior to ethanol addition (groups 1 and 2) or 30 min (groups 3 and 4) and 60 min (groups 5 and 6) after ethanol addition. Samples were processed as described in Materials and Methods and analyzed by Western blotting using monoclonal antibodies specific for each of the sigB operon gene products. Each lane grouping illustrates, in order, the antibody reactions to extract from 0.15, 0.45, and 1.35 ml of original culture volume.
the eight sigB operon gene products as probes. The anticipated stress-dependent elevation of RsbV, RsbW, B, and RsbX can be seen in the blot (Fig. 5, lower panels, lanes 3 to 6). B activity, as measured in reporter gene assays, (Fig. 3 and 4) diverges in ClpP⫹ and ClpP⫺ strains 30 min after exposure to physical stress. The Western blot reveals no obvious difference in the abundance of the sigB operon products in the ClpP⫹ and ClpP⫺ strains at 30 min or 60 min after stress induction. It therefore appears unlikely that the loss of ClpP affects B activity by altering the abundance of the known B regulators. ClpP’s loss influences the physical stress pathway upstream of RsbT release. Physical stress induction of B is triggered when exposure to stress causes the phosphorylation of RsbR and RsbS and the release of RsbT (25, 44). The physical stress pathway’s phosphatase can be artificially activated by heightened expression of RsbT relative to its principal negative regulator, RsbS (22, 23). If the loss of ClpP promotes heightened B activity by influencing events downstream of RsbT release, the absence of ClpP should elevate B activity over that seen in a ClpP⫹ strain when the system is triggered by induced RsbT synthesis. Conversely, if the loss of ClpP increases B’s activity by influencing events that facilitate the release of RsbT, the ClpP⫹ and ClpP⫺ strains should display similar activities. To examine these possibilities a clpP::spc allele was transformed into a B. subtilis strain that carries an IPTG-inducible promoter (PSPAC) within the sigB operon between rsbS and rsbT (BSA419). In such a strain, the addition of IPTG leads to an increase in RsbT levels relative to RsbS and activation of B. When RsbT is induced in the ClpP⫹ and ClpP⫺ strains, no significant differences in the levels of B activity are evident between the two strains (Fig. 6). This result is consistent with the participation of ClpP in events that are upstream of the release of RsbT. Presumably, this involves processes that control the phosphorylation states of RsbR and RsbS.
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FIG. 6. B activation by rsbT induction in ClpP⫹ and ClpP⫺ B. subtilis strains. B. subtilis strains BSA419 (PSPAC::rsbT) (squares) and BSH121 (PSPAC::rsbT clpP::spc) (triangles) were grown to an optical density of 0.25 in LB medium. IPTG (1 mM) was added to half of each culture (filled symbols) at time zero. Samples were taken at 20-min intervals and assayed as described in the legend of Fig. 4 for Bdependent -galactosidase activity. Open symbols, untreated cultures.
ClpP’s effect on B activity is not effected through RsbX or YtvA. There are two potential mechanisms by which the loss of ClpP could alter the phosphorylation state of RsbR and RsbS to allow the release of RsbT and the activation of B. Either ClpP could play a role in the activation or turnover of stressinduced factors that stimulate the phosphorylation of RsbR and RsbS, or, alternatively, a ClpP-dependent process could inhibit their dephosphorylation by an effect on RsbX. The later possibility seems especially plausible, given that the loss of either ClpP or RsbX results in a failure of the stress response to exhibit its normal transience. The loss of RsbX leads to growth-inhibiting levels of B activity, which gives rise to spontaneous suppressor mutations in the positive regulators of B that allow for more normal growth characteristics (35). One such mutation is an alteration of rsbT (rsbT15IS). RsbX⫺ strains that carry the rsbT15IS allele have relatively low B activity in the absence of stress but are still stress inducible (35). One phenotype associated with an rsbT15IS rsbX::spec strain, like that of the Clp⫺ strains, is persistent B activity following stress (35). The rsbT15IS rsbX::spec strain permits us to ask whether the loss of ClpP influences B activity via an effect on RsbX. If RsbX’s phosphatase activity is the target of ClpP’s loss, the ClpP mutation should not alter B activity in this strain. Alternatively, if it is the rate of RsbR/RsbS phosphorylation that is altered, the loss of ClpP may further enhance the strain’s B activity. ClpP⫹ and ClpP⫺ variants of an rsbT15IS rsbX::kan strain were grown, subjected to ethanol stress, and analyzed for B-dependent -galactosidase activity. As can be seen in Fig. 7, the level of B activity is substantially elevated in the ClpP⫺ strain, even in the absence of ethanol stress. This markedly elevated B activity seen during growth of the RsbX⫺ culture may reflect this strain’s response to the factors that are responsible for the modest increase in B activity seen in RsbX⫹ ClpP⫺ strains. In the current circumstance, the effect of ClpP’s loss may be amplified by the strain’s inability to dephosphorylate RsbR and RsbS. This could arise because the loss of ClpP serves as a stress-inducing event in itself or, alternatively, because the absence of ClpP might allow the factors which normally trigger these phosphorylations to accumulate. Although this result does not eliminate the possibility that the loss of ClpP could
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FIG. 7. Ethanol induction of B in RsbX⫺ ClpP⫹ and RsbX⫺ ClpP⫺ B. subtilis strains. B. subtilis strains, carrying rsbT15IS rsbX::kan mutations plus either a wild-type clpP allele (BSH130; squares) or clpP::spc (BSH131; triangles), were grown in LB medium to an optical density of 0.2; the cultures were split, and half of each culture was exposed to ethanol stress (4% ethanol) (time 1). Samples were taken at 15-min intervals and assayed for B-dependent -galactosidase activity. Open symbols, untreated cultures; filled symbols, ethanoltreated cultures.
also affect the activity of RsbX, it does show that ClpP exerts an RsbX-independent influence on B activity. Presumably, this involves heightened stimulation of RsbT-dependent phosphorylation of RsbR and RsbS. Recently an additional member of the RsbR family of proteins (YtvA) was demonstrated to have a positive effect on the activity of the B physical stress pathway (15). YtvA is believed to integrate signals of blue light signaling and disulfide stress to activate B. Overexpression of YtvA can activate B by a process that is dependent on the components of the physical stress pathway (15). Interestingly, expression of ytvA is positively regulated by the global regulatory protein Spx (15). This is significant because Spx abundance is elevated in ClpP⫺ strains of B. subtilis (47). It is therefore formally possible that YtvA could contribute to the heightened B activity that we observed in the ClpP⫺ strains. To test this possibility, we constructed ClpP⫺ strains that included disruptions in the ytvA gene (i.e., ytvA::pJM102) and examined the effect of this mutation on the activity of the physical and nutritional stress pathways. As illustrated in Fig. 8, activation of B by either physical stress (Fig. 8A) or nutritional stress (Fig. 8B) is largely unaffected by the loss of YtvA. This argues that the ClpP-dependent phenomenon which we have observed in these studies is independent of the YtvA protein and likely involves novel factors. DISCUSSION Targeted proteolysis has long been understood to be important to the proper functioning of regulatory circuits (reviewed in reference 18). In the current work we demonstrate that the B. subtilis ClpP protease participates in the regulation of the GSR’s transcription factor, B. B activity is modestly elevated in ClpP⫺ strains during growth and persists in these strains for an extended period, once induced by either nutritional or physical stress. The data raise the possibility that both physical and nutritional stresses trigger the formation of ClpP-sensitive factors that activate their respective pathways and linger in the absence of ClpP. In the case of the physical stress, these factors
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FIG. 8. Effect of ytvA on stress induction of B in ClpP⫺ B. subtilis. (A) B. subtilis strains BSH115 (clpP::spc) (triangles) and BAR126 (clpP::spc ytvA::pUK19) (squares) were grown in LB medium and assayed for B-dependent -galactosidase activity after ethanol treatment as described in the legend of Fig. 3. Open symbols, untreated cultures; filled symbols, treated cultures. (B) B. subtilis strains BSH159 (rsbU::kan clpP::spc) (〫) and BAR 129 (rsbU::kan clpP::spc ytvA:: pUK19) (‚) were grown and allowed to enter stationary phase in low-PO4 medium. Samples were taken at the times indicated and analyzed for B-dependent -galactosidase activity.
are likely to be novel, given that we could detect no obvious change in abundance of this pathway’s known components when ClpP⫹ and ClpP⫺ strains were compared. The existence of unknown Bacillus-specific factors in the physical stress pathway had been previously suggested by the failure of the known physical stress pathway components to activate B when reconstituted in E. coli (34). Recently, the first of what may prove to be a number of stressosome-activating proteins was identified. A B. subtilis blue light receptor protein (YtvA) was found to be able to associate with stressosome proteins and lead to B activation when overexpressed (15). YtvA is believed to function in activating B in response to potentially damaging blue light and not as a general element in stress reception (15). It is unknown whether YtvA is a ClpP substrate; however, given that the loss of YtvA is without effect on the persistence of B activity in ClpP⫺ B. subtilis, it is unlikely that YtvA is involved in the phenomena which we describe in the current work. Activation of B’s physical stress pathway is believed to occur via the enhanced phosphorylation of the RsbR and RsbS components of the stressosome (9, 16, 25, 26, 44). This higher level of phosphorylation could be the result of increased RsbT kinase activity, reduced RsbX phosphatase activity, or both. A previous observation that RsbX⫺ B. subtilis can still activate B following exposure to physical stress demonstrated that at least part of the induction process is independent of RsbX (40). Presumably, this is a result of an interaction between stressinduced factors and the stressosome itself. The finding that the
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loss of ClpP elevates B activity in the absence of RsbX (Fig. 7) is consistent with the notion that the factors affected by the loss of ClpP ultimately communicate with the stressosome to activate the system. It remains unclear whether ClpP could have an additional role in modulating RsbX activity. The transience of the B stress response is thought to involve a stress-dependent increase in the levels or the activity of the RsbX phosphatase (40, 44). This would presumably enhance the rate of RsbR/RsbS dephosphorylation and the reacquisition of RsbT into an inhibitory complex. The observation that B activity persists in the ClpP⫺ strain suggests that the mechanism which limits ongoing B activity may not be limited to changes in RsbX but could also include changes in the inducing signal itself. In such a model, stress would generate a ClpP-sensitive signal whose rapid turnover limits the persistence of the response. The loss of RsbX in otherwise wild-type B. subtilis leads to a high level of B activity in the absence of obvious stress (3, 6). A spontaneously high level of B activity in cells lacking the phosphatase that responsible for reactivating RsbR and RsbS implies either that the stressosome itself is inherently biased toward RsbR/RsbS phosphorylation or that, even in the absence of overt stress, induction signals are continuously received to trigger the phosphorylation reactions. Recent work (31) demonstrated that amino acid changes in the stressosome RsbR protein can enhance B activity in the absence of stress and that this phenomenon likely involved heightened phosphorylation of the mutant RsbR protein. The experiments did not, however, reveal whether the changes in RsbR had altered a “set point” in a potential stressosome bias toward autophosphorylation or created stressosomes that were more receptive to stress input signals. The finding that the loss of ClpP does not dramatically increase the activity of B in an RsbX⫹ strain during growth but does so in an rsbT15IS rsbX::spec strain argues for continuous stimulation of the stressosome rather than an inherent bias toward phosphorylation. If the background level of RsbR/RsbS phosphorylation is merely a consequence of the properties of the stressosome proteins themselves, it is not clear how the loss of ClpP could alter their inherent activities. A more likely possibility is that the stressosome is continually stimulated to phosphorylate RsbR and RsbS by background levels of stress-inducing signals which are amplified in the absence of ClpP. Activation of the nutritional stress pathway occurs coincident with conditions that lead to a drop in intracellular ATP levels (46). This induction could therefore be plausibly triggered by a metabolic cue (e.g., a drop in ATP) which directly alters the activity of the pathway’s phosphatase. The observation that a loss of ClpP leads to persistent B activity via the nutritional stress pathway argues that either normal cellular metabolism is significantly altered in ClpP’s absence to generate this cue or that the B trigger includes a ClpP-sensitive component. Persistent B activity upon entry into stationary phase, rather than a marked elevation of this activity during growth, is more readily reconciled with a failure to turn over an inducing factor than with a generalized change in metabolism. The ongoing B activity that occurs in ClpP⫺ B. subtilis may be a consequence of a failure of putative activators that are normally generated by stress to be properly turned over in the absence of ClpP or an additional stress-generated signal that
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appears uniquely in ClpP⫺ strains. In either case, this phenotype of the heightened B activity that results should be amenable to genetic analyses. Such studies offer the possibility of providing fresh insights into the B activation processes.
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ACKNOWLEDGMENTS We thank Annette Tschirner for excellent technical assistance. This work was supported by U.S. National Institutes of Health grant GM-48220 to W.G.H. and by the BMBF within the framework of the SYSMO program by grants 0313978A and 0313978B to U.V. and M. Hecker.
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