Exposure of bacteria to diverse growth-limiting stresses inducesthe synthesis of a common set of proteins which provide broadprotection against future, potentially lethal stresses . AmongBacillus subtilis and its relatives, this general stress responseis controlled by the ^B transcription factor . Signals of environmentaland energy stress activate ^B through a multicomponent networkthat functions via a partner switching mechanism, in which protein-proteininteractions are governed by serine and threonine phosphorylation.Here, we tested a central prediction of the current model forthe environmental signaling branch of this network . We usedisoelectric focusing and immunoblotting experiments to determinethe in vivo phosphorylation states of the RsbRA and RsbS regulators,which act in concert to negatively control the RsbU environmentalsignaling phosphatase . As predicted by the model, the ratioof the phosphorylated to unphosphorylated forms of both RsbRAand RsbS increased in response to salt or ethanol stress . However,these two regulators differed substantially with regard to theextent of their phosphorylation under both steady-state andstress conditions, with RsbRA always the more highly modified.Mutant analysis showed that the RsbT kinase, which is requiredfor environmental signaling, was also required for the in vivophosphorylation of RsbRA and RsbS . Moreover, the T171A alterationof RsbRA, which blocks environmental signaling, also blockedin vivo phosphorylation of RsbRA and impeded phosphorylation of RsbS . These in vivo results corroborate previous genetic analyses and link the phosphorylated forms of RsbRA and RsbSto the active transmission of environmental stress signals.

 
In Bacillus subtilis the general stress response triggers the synthesis of more than 150 proteins which confer resistanceto diverse lethal stresses [reviewed in references 14 and 23].Transcription of the general stress regulon is controlled by ^B, whose activity is regulated by a signal transduction networkin which key protein-protein interactions are determined byserine and threonine phosphorylation . This mechanism has beendubbed partner switching [4] and is found, wholly or in part, among evolutionarily diverse eubacteria [19, 22, 23] . Studyof this mechanism in B . subtilis should therefore help explainthe principles which govern a broad array of bacterial signalingpathways.

A model for the B . subtilis signaling network is shown in Fig. 1 . In this model, separate environmental and energy signalingpathways converge on the RsbV and RsbW regulators, which directlycontrol ^B by means of the partner switching mechanism [Fig.1A] . RsbW has two activities in unstressed cells . First, itacts as an anti- factor which binds ^B and prevents its associationwith RNA polymerase [3, 6] . Second, it acts as a serine kinasewhich specifically phosphorylates and inactivates the RsbV anti-anti- [3, 10] . Following an activating stress, the phosphate is removedfrom RsbV-P by either the RsbP energy phosphatase or the RsbUenvironmental phosphatase [26, 28, 30], leading RsbV to complex with RsbW and force the release of ^B . In this scheme, RsbW switchesits binding partner in response to stress.

 
The subject of this communication is a second partner switchfound in the environmental signaling branch [Fig . 1B] . In this switch, RsbS and RsbT are paralogs of RsbV and RsbW, respectively [16] . However, there are three significant differences betweenthe two switches . First, the target of the RsbS-RsbT switch is an enzyme, namely, the RsbU environmental signaling phosphatase [30] . Second, RsbT positively regulates RsbU activity, essentiallyserving as a regulatory subunit of the phosphatase [16, 17,30] . And third, RsbS alone is insufficient to sequester RsbTin an inactive complex . Also required is at least one of a familyof paralogous proteins, the RsbR coantagonists [1, 9, 18], whichpossess carboxyl-terminal domains that resemble the entire lengthof the smaller RsbS antagonist [Fig . 1C] . The first member ofthe RsbR family to be discovered was RsbRA, whose structuralgene lies in the sigB operon, immediately upstream from thatencoding RsbS [29] . The genes encoding the other family members—RsbRB,RsbRC, and RsbRD—are unlinked to the sigB operon and toeach other [1, 18, 20] . Notably, RsbRA, RsbRB, and RsbS copurifyfrom cell extracts in a large complex [18], and RsbRC and RsbRDhave recently been identified as constituents of the same complex[A . L . Weigel, T . J . Kim, S . Neissen, J . R . Yates, and C . W.Price, unpublished data] . The properties of this complex arethought to facilitate the sensing or transmission of environmentalstress signals [18].

According to the model shown in Fig . 1, in the absence of environmentalstress the RsbR coantagonists and the RsbS antagonist jointlybind RsbT, preventing its association with RsbU [9, 18] . Followingan environmental signal, such as acid, ethanol, heat, or saltstress, the RsbR family members and RsbS are specifically phosphorylatedby RsbT [1] . These phosphorylation events release RsbT to bindand activate RsbU by direct protein-protein interaction [9, 17, 30] . RsbU then communicates the stress signal to the downstreampartner switch by dephosphorylating RsbV-P, resulting in theactivation of ^B [28, 30] . This model further holds that theenvironmental signal is damped by the RsbX feedback phosphatase [24, 27, 30], whose expression is under ^B control [15] . Thebiochemical and genetic evidence supporting this model includesa consideration of the phenotypes caused by alteration of theresidues upon which RsbS and RsbRA are phosphorylated [2, 12, 16, 18, 30] . However, it has yet to be established whether thein vivo phosphorylation state of the RsbR family members andRsbS do in fact change as a result of environmental stress.

Here we test key predictions of the model and show that bothRsbRA and RsbS are indeed phosphorylated in vivo as part ofthe response to ethanol or salt stress and that this modificationrequires the RsbT kinase . However, we also find that the phosphorylationstates of RsbRA and RsbS differ substantially in both stressedand unstressed cells, with RsbRA the more highly phosphorylated.Based on these results, we propose a refinement of the modelfor environmental stress signaling.

 
Growth of bacterial strains. All B . subtilis strains were derivatives of the wild-type Marburgstrain [Table 1] . Cells were grown at 37°C in shake flaskscontaining buffered Luria broth lacking salt [7] . Early logarithmic cells were either used directly for unstressed controls [timezero] or stressed by the addition of salt or ethanol to a final concentration of 0.3 M or 4% [vol/vol], respectively . We usedtwo different methods to harvest cells for isoelectric focusing[IEF] analysis, depending upon the purpose of the experiment.

 
When preservation of the phosphorylation state of RsbS and RsbRAwas not an issue, as was the case for the experiments shownbelow in Fig. 2, we harvested 10 ml of culture by centrifugation at 4°C in a Sorvall SS34 rotor, run at 8,000 rpm for 5 min.The cell pellets were washed by resuspension in 1 ml of sonicationbuffer [50 mM Tris-HCl [pH 6.8], 150 mM NaCl, 1% Triton X-100,1 mM phenylmethylsulfonyl fluoride], after which they were transferredto a 1.5-ml Eppendorf tube . Cells were collected by centrifugationin a microcentrifuge [1 min at 4°C], resuspended in 0.5ml of sonication buffer, and then broken by sonication withfour 30-s treatments separated by 20 s on ice, using a SonicDismembrator [model 300; Fisher Scientific, Pittsburgh, Pa.]with a microtip on the 35% setting . Cell debris was removedby centrifugation in a microcentrifuge [10 min at 4°C],and the supernatants were analyzed by IEF.

 
When harvesting by centrifugation could significantly influencethe results, as in the experiments shown below in Fig . 3 and 4, we employed a rapid harvesting procedure . Here we filtered10 ml of cell culture through 25-mm-diameter MF-Millipore membranefilters with a pore size of 0.22 µm [Millipore, Billerica, Mass.] . The cells and the MF membranes were transferred to 1.5-ml Eppendorf tubes containing 1 ml of cold Z-buffer [100 mM sodium phosphate [pH 7.0], 10 mM KCl, 1 mM MgSO₄] supplemented with phosphatase inhibitors [50 mM NaF, 0.1 mM sodium orthovanadate, 60 mM ß-glycerophosphate, and 15 mM p-nitrophenylphosphate] . Following a brief mix to wash the cells from thefilters, the samples were collected by centrifugation in a microcentrifuge[30 s at 4°C], after which the membrane and supernatantwere removed . The cell pellet was then quickly frozen on dryice-ethanol and stored at –80°C until all sampleswere collected . This entire sampling procedure was accomplishedin less than 60 s . Prior to assay, cells were resuspended in0.5 ml of sonication buffer containing the phosphatase inhibitorsand broken by sonication, as described above for the standardharvesting procedure.

 
IEF. IEF of 20-µl cell extract samples was done in a Mini-ProteanII cell with a 15-well comb [Bio-Rad Laboratories, Hercules,Calif.], with the lower chamber containing 10 mM phosphoric acid as anolyte and the upper chamber containing 20 mM sodium hydroxide as catholyte . All samples were adjusted for equalamounts of protein and then run at 200 V for the first 0.5 hand at 300 V for another 2.5 h . However, to optimize isoformseparation, gel and sample preparations were different for RsbSand RsbRA.

For RsbS [calculated pI, 4.14], a 4% acrylamide gel was madeusing a stock 30% acrylamide-bis [29:1] solution [Bio-Rad Laboratories], 9.37 M urea, 1% Triton X-100, 22 mM 3-[[3-cholamidopropyl]-dimethylammonio]-1-propanesulfonate[CHAPS], 3.85% Pharmalyte, pH range 4.2 to 4.9 [Sigma, St . Louis,Mo.], 1.15% Pharmalyte pH 3.0 to 10.0 [Sigma], 0.027% ammoniumpersulfate, and 0.107% N,N,N',N'-tetramethylethylenediamine[TEMED] . After removing the residual liquid from the samplewells, the gel was assembled in the Mini-Protean II cell . Thereafter,the wells were filled with RsbS loading buffer [4 M urea, 1.2%Pharmalyte pH 4.2 to 4.9, 0.36% Pharmalyte pH 3.0 to 10.0] toreceive the samples . Protein concentration in each sample wasmeasured with the Bio-Rad protein assay reagent [Bio-Rad Laboratories],and equal amounts of protein were dried completely under vacuum.These dried samples were solubilized with 25 µl of RsbSsample buffer [5 M urea, 40 mM CHAPS, 0.77% Pharmalyte pH 4.2to 4.9, 0.23% Pharmalyte pH 3.0 to 10.0, 0.25 M dithiothreitol,and 0.075% sodium dodecyl sulfate [SDS]] . Twenty-microliteraliquots of these sample solutions were loaded into the bottomof the wells, using a long-tipped pipette to minimize mixingwith loading buffer . IEF was then conducted as described above.

For RsbRA [calculated pI, 4.73], a 5% acrylamide gel was made using a 30% acrylamide-bis solution [29:1], 8 M urea, 1% Triton X-100, 2% Pharmalyte pH 4.2 to 4.9, 0.6% Pharmalyte pH 3.0 to10.0, 0.021% ammonium persulfate, and 0.167% TEMED . After removingthe residual liquid from the wells, the gel was assembled andthe wells were filled with RsbRA sample buffer [2 M urea, 0.5%Pharmalyte pH 4.2 to 4.9, 0.15% Pharmalyte pH 3.0 to 10.0, 0.5%Triton X-100, 0.5% ß-mercaptoethanol, 0.01% bromphenolblue] . Sample protein concentration was measured, and the amountswere equalized . Each adjusted sample was then mixed with anequal volume of 4x RsbRA sample buffer [8 M urea, 2% PharmalytepH 4.2 to 4.9, 0.6% Pharmalyte pH 3.0 to 10.0, 2% Triton X-100,1% ß-mercaptoethanol, 0.04% bromphenol blue] and loadedinto the bottom of the wells.

Immunological methods. Anti-RsbRA and anti-RsbS antibodies [11] were kindly providedby William Haldenwang . Antibody specificity was confirmed byWestern blot analysis of wild-type and mutant cell extractsseparated on SDS-polyacrylamide gel electrophoresis . For analysisof the IEF gels, the separated proteins were transferred to polyvinylidene difluoride membranes [Bio-Rad Laboratories]. These membranes were blocked by immersion in 5% nonfat dried milk-TBS-T for 1 h [TBS-T is 10 mM Tris-HCl [pH 8.0], 150 mMNaCl, 0.5% Tween 20] . Membrane blots were exposed to primaryantibody in 5% nonfat dried milk-TBS-T for 1 h at 24°C,washed, and then incubated with peroxide-conjugated secondaryanti-mouse immunoglobulin G antibody [Santa Cruz BiotechnologyInc., Santa Cruz, Calif.] . The blots were washed, and boundantibody was visualized using the ECL Plus Western blottingdetection kit [Amersham Pharmacia Biotech, Piscataway, N.J.],according to the manufacturer's instructions, with the imagecaptured on Kodak BioMax light film [Eastman Kodak Company, Rochester, N.Y.] . For quantitative analysis, the exposed film was scanned and digitized using an Epson Perfection 636 [Epson America, Inc., Long Beach, Calif.], and the band intensitieswere determined using Quantity One version 4.1.1 software [Bio-Rad Laboratories].

PP assays. To determine if the RsbS and RsbRA isoforms were phosphorylated on serine, threonine, or tyrosine residues, we added 40 U of protein phosphatase [PP; New England BioLabs, Beverly, Mass.]per µl of cell extract, adjusted to 50 mM Tris-HCl [pH7.5], 0.1 mM EDTA, 5 mM dithiothreitol, 0.01% Brij 35, and 2mM MnCl₂ by the addition of 10x PP reaction buffer and 20 mMMnCl₂ . Extracts were incubated for 18 h at 30°C and thenanalyzed by IEF and immunoblotting . Controls included [i] cellextracts alone and [ii] cell extracts with PP reaction bufferbut no PP . During the course of these experiments, we discoveredthat one of the two RsbS isoforms was unstable in control ii,suggesting that it was the substrate of an endogenous activityin the cell extract [data not shown] . To clearly establish theeffect of PP, extracts for RsbS analysis were heated for 3 minat 100°C before adding PP and PP reaction buffer . By contrast,the RsbRA isoforms appeared stable in both control assays, andso these extracts were not subjected to heat treatment.

ß-Galactosidase accumulation assays. Strain PB198 carries a ^B-dependent ctc-lacZ reporter fusionat its amyE locus . Samples were collected at the indicated timesand assayed as described by Miller [21] . Cells were washed withZ-buffer and permeabilized using SDS and chloroform and thenincubated at 28°C in 1-ml reaction mixes containing o-nitrophenyl-ß-D-galactopyranoside. Reactions were stopped with 0.5 ml of 1 M Na₂CO₃ and centrifugedto remove cellular interference, and the A₄₂₀ was recorded.Protein levels were determined with the Bio-Rad protein assayreagent [Bio-Rad Laboratories] . ß-Galactosidase activity was defined as A₄₂₀ x 1,000 per min per mg of protein.

 
Separation and identification of RsbRA and RsbS isoforms. The RsbRA coantagonist and RsbS antagonist are known to be phosphorylated by the RsbT serine-threonine kinase in vitro [1, 12, 30] . Furthermore, phenotypes elicited by substitutions at the known or presumedsites of phosphorylation—threonines 171 and 205 of RsbRAor serine 59 of RsbS—support the hypothesis that modificationof these proteins influences the transmission of environmentalstress signals [2, 12, 16, 18, 30] . However, the phenotypesof some RsbRA substitutions are significantly altered by thepresence or absence of the other members of the RsbR coantagonistfamily [18] . In addition, any of these substitutions has thepotential to affect protein structure or function independentlyof phosphorylation.

We therefore sought to directly test the hypothesis by developing IEF protocols to separate the isoforms of RsbRA and RsbS inextracts of wild-type cells . These protocols are described inMaterials and Methods together with the two sampling methodswe used . For the work described in this section, cells wereharvested by a 5-min centrifugation at 4°C before beingwashed, chilled on ice, and frozen at –80°C . Thissampling procedure itself was expected to activate the generalstress response, yielding a mix of modified and unmodified regulatorsthat allowed us to test the method and identify the variousisoforms.

Turning first to RsbS, Fig . 2A shows that anti-RsbS antibodydetected two signals in wild-type extracts subjected to IEF, as would be expected for a regulator that is modified on a single residue [lane 1] . Neither of these signals was visible in extracts of mutant cells lacking RsbS [lane 2] . Two lines of evidence led us to conclude that the more acidic of these isoforms was phosphorylated RsbS.

First, PP is known to remove the phosphate group from serine,threonine, or tyrosine residues [31] . Significantly, incubation of wild-type extracts with PP decreased the signal of the moreacidic isoform of RsbS and increased that of the more basicisoform [Fig . 2A, lanes 3 to 5] . During the course of this experiment,we discovered that this conversion also occurred in the absenceof PP, presumably due to an endogenous enzyme which became activein the presence of PP buffer and MnCl₂ . Therefore, in orderto directly test the effect of the PP, we heated the cell extractsfor 3 min at 100°C before proceeding with the incubationsshown in Fig . 2A.

Second, we analyzed mutant extracts that contained versionsof RsbS in which serine 59 [S59] had been changed to eitheran alanine residue, which produces a form of RsbS that cannotbe phosphorylated in vitro [30], or to a negatively charged aspartate residue, which is thought to mimic a phosphoserine[16, 30] . Consistent with the results of the PP assay, the extractcontaining the S59A variant had only one signal [Fig . 2B, lane2], and this was near the position of the more basic isoformfound in wild-type extracts [lanes 1 and 4] . By contrast, theextract containing the S59D variant had no signal at this basicposition but did manifest a more acidic signal [lane 3] . Wenoted, however, the lack of close correspondence in the positionsof the more acidic isoform found in wild-type cells and the form found in mutant cells bearing the S59D variant . This is consistent with the acidity difference of phosphoserine andan aspartate residue and indicates that the aspartate substitutionis not an exact mimic for phosphoserine in RsbS.

For simplicity, we will refer to the more basic RsbS signalfound in wild-type cells as the unmodified form [RsbS] and themore acidic signal as the modified form [RsbS-P] . These sameterms can additionally apply to RsbRA, which also appears tobe present in two forms in wild-type cells.

Turning now to RsbRA, we had anticipated that signal interpretation would be complicated as a result of the two potential phosphorylation sites on this regulator . However, our IEF procedure readily separated three RsbRA isoforms, and their identities could bededuced from appropriate controls . As shown in Fig . 2C, the anti-RsbRA antibody first detected two signals in wild-typeextracts [lane 1], neither of which was visible in extractsof mutant cells lacking RsbRA [lane 2] . As was the case forRsbS, incubation with PP decreased the signal of the more acidicRsbRA isoform and increased that of the more basic isoform [lanes3 to 5] . In contrast to RsbS, here the more acidic RsbRA isoformwas relatively stable in cell extracts, and so no heat treatmentwas necessary prior to the incubations shown in Fig . 2C . BecauseRsbRA can be phosphorylated on two threonine residues [12], these results did not allow us to infer whether the more acidic signal reflected phosphorylation on threonine 171 [T171], on threonine 205 [T205], or both.

We addressed this issue by analyzing the mutant extracts shownin Fig . 2D, employing strains in which either or both threonine residues were changed to alanine or aspartate . The extracts bearing the T171 variants each had only a single signal: thesignal in the T171A variant of RsbRA [Fig . 2D, lane 2] was near the location of the more basic isoform found in wild-type cells [lanes 1 and 8], and the signal in the T171D variant [lane 3]was near the location of the more acidic isoform . By contrast,the extracts containing the T205 variants each had two signals:the T205A variant [lane 4] had signals which focused to similarpositions as those in wild-type extracts, whereas the T205Dvariant [lane 5] had one signal at about the same position asthe more acidic isoform in wild-type cells and a second, newsignal that was even more acidic . We interpret these resultsto indicate that in wild-type cells RsbRA is stably phosphorylatedon T171 but not on T205 . In this view, and given these growthand harvest conditions, the only circumstance under which themost acidic isoform appeared was when the T205 residue was alteredto the charged aspartate.

This interpretation was supported by the analysis of strains bearing double alterations at both T171 and T205 . The extract containing the T171A-T205A variant [lane 6] had only a singlesignal resembling the basic isoform in wild type, whereas theextract containing the T171D-T205D variant [lane 7] had a singlesignal near the position of the most acidic isoform found inthe T205D variant [lane 5] . As was the case for the aspartatesubstitution of RsbS, we note here that the aspartate substitutionsof RsbRA did not focus to the same positions as the presumedphosphorylated forms . This is consistent with the acidity differencebetween an aspartate residue and the phosphothreonine.

For simplicity, we will refer to the more basic RsbRA signalfound in wild-type cells as the unmodified form [RsbRA] andto the more acidic signal as the modified form [RsbRA-P] . Ourconclusion that RsbRA is normally phosphorylated on one andnot both threonine residues is further supported by our analysisof salt- and ethanol-stressed cells, which is reported in thefollowing section.

Shift of isoform balance during salt and ethanol stress. A rapid sampling method was needed to reproducibly detect thechanges in RsbS and RsbRA modification resulting from environmentalstress . For the experiments described here, we harvested thecells by filtration, washed them with a buffer containing amixture of phosphatase inhibitors, and then quickly froze themin dry ice-ethanol . The entire process was completed in 60 sor less . Using this approach, we found that the relative proportionsof RsbS-P and RsbRA-P increased following salt or ethanol stress.However, the two regulators manifested striking differencesboth in the balance of isoforms found in unstressed cells andin the kinetics of their phosphorylation after stress.

Figure 3A shows the data for a representative salt stress experiment,Fig . 3B graphically indicates the change in the phosphorylatedfraction of RsbS and RsbRA during the course of this experiment,and Fig . 3C summarizes the results of three independent saltstress experiments . We obtained similar results for the ethanolstress experiments shown in Fig. 3D to F . Notably, only theunmodified form of RsbS was found in unstressed cells . Immediatelyfollowing salt or ethanol stress, we detected a small amountof the modified form, with its peak level recorded at the 1-mintime point . In salt-stressed cells the modified form representedabout 10% of total RsbS [Fig. 3C], whereas during the strongerethanol stress the modified form represented about 20% of thetotal [Fig . 3F] . For each stress, the amount of this modifiedform then decreased, reverting to a new steady-state level thatwas somewhat higher than that found in prestress cells . Thisdecrease was presumably due to the action of the RsbX feedbackphosphatase [24, 27, 30] . Because the peak level of RsbS-P wasdetected at the first time point, the amount produced may have been underestimated . Nonetheless, the observed increase in RsbS-P correlated with the onset of the environmental stress response, measured indirectly by means of ß-galactosidase accumulationfrom a reporter fusion [Fig . 3B and E].

In sharp contrast to the case with RsbS, unstressed cells manifested a balance between the modified and unmodified forms of RsbRA, with about 60 to 70% of the total found in the modified form[Fig. 3] . Following salt or ethanol stress, the ratio of modified to unmodified RsbRA further increased, but more slowly than observed for RsbS, with the peak of the modified form recordedat the 5-min time point . Paralleling the observations for RsbS,this increase in the amount of RsbRA-P correlated with the onsetof the environmental stress response [Fig . 3B and E] . Based on the IEF patterns observed for the substituted forms of RsbRA [Fig . 2D], our interpretation of the results shown in Fig. 3is that in unstressed cells RsbRA is already largely phosphorylatedon T171, and environmental stress further increases the levelof this isoform . Neither of the stress conditions shown hereproduced a detectable signal representative of the isoform phosphorylatedon both T171 and T205 . Experiments described in the followingsection underscore the importance of the T171 residue for thein vivo phosphorylation of RsbRA.

Isoform balance is dependent on the RsbT kinase and the T171 residue of RsbRA. RsbT is known to specifically phosphorylate both RsbS and RsbRAin vitro, and direct biochemical analysis has identified T171 and T205 as the sites on which RsbRA is modified [12, 30] . Complementingand extending these in vitro results, genetic analysis has shownthat loss of RsbT kinase activity eliminates the environmentalstress response [16, 17] and that the T171A alteration of RsbRA substantially blocks environmental signaling [18] . In order to test the observed correlation between the environmental stress response and the increased phosphorylation of RsbS and RsbRA [Fig . 3], we next compared the isoforms present in wild-type and mutant cell extracts.

For these experiments, shown in Fig . 4, we examined ethanol-stressedcells over the 0-to-5-min interval found to embody the largestchange in isoform balance [Fig . 3] . In wild-type cells we observedtwo forms of RsbS and RsbRA [Fig. 4, wt lanes], and these manifestedessentially the same pre- and poststress balance noted in Fig.3 . By contrast, for the mutant lacking the RsbT kinase, onlythe unmodified forms of RsbS and RsbRA were detected [Fig . 4, rsbT lanes] . Yet another pattern was found for the mutant bearingthe T171A alteration of RsbRA . Here, the RsbS-P fraction wasreduced by a factor of three, calculated by scanning the relevantimages [Fig. 4A, compare wt and rsbRAT171A lanes] . This result was consistent with the known role of RsbRA in stimulating the in vitro phosphorylation of RsbS by RsbT [9, 12] and suggeststhat a similar in vivo activity is negatively affected by theT171A substitution . This substitution had a more profound effecton RsbRA phosphorylation, with the RsbRA-P fraction reduced below the limits of detection in both stressed and unstressed cells [Fig . 4B, compare wt and rsbRAT171A lanes] . From the resultsshown in Fig . 4, we conclude that phosphorylation of RsbRA andRsbS is dependent on RsbT in vivo and that phosphorylation ofRsbRA is dependent on the integrity of its T171 residue . Moreover,given the deleterious effects that loss of RsbT function orthe RsbRAT171A substitution have on environmental signalingin vivo [16-18], these results strengthen the correlation betweenRsbS and RsbRA phosphorylation and the transmission of environmentalstress signals.

 
We have tested a key prediction of the model shown in Fig . 1, which holds that the transmission of environmental stress signals is correlated with the phosphorylation of the RsbS antagonist and RsbRA coantagonist proteins . This correlation had been inferred from the phenotypes elicited by substitutions at the RsbS andRsbRA residues that are the known [or presumed] substrates ofthe RsbT serine-threonine kinase in vitro [2, 12, 16, 18, 30]. Here, we analyzed cell extracts by IEF and confirmed that the proportion of modified RsbS and RsbRA did indeed increase following salt and ethanol stress [Fig . 3] . In further confirmation of the model, we also showed that the appearance of the modified forms of both RsbS and RsbRA was dependent on the RsbT kinase[Fig. 4].

Our data suggest that RsbS is unmodified in unstressed cellsand that a surprisingly small amount of RsbS is rapidly phosphorylated during the stress response . Based on the limited range of stresses used in this study—a relatively weak salt stress and arelatively strong ethanol stress—it also appears thatboth the peak levels and the poststress, steady-state levelsof RsbS-P correlate with the strength of the stress . This relativelylow phosphorylation may reflect the fact that in cell extractsRsbS in found in a complex with RsbRA, RsbRB, and other proteins[9, 18], and some RsbS molecules in this complex could be inaccessibleto the RsbT kinase . In contrast, RsbRA appears to be substantiallyphosphorylated in unstressed cells and slowly becomes more fullyphosphorylated during the response . These trends are in accordwith previous genetic data, from which it was inferred that phosphorylation of RsbS is sufficient to trigger the environmental stress response [16] and that phosphorylation of RsbRA is aprerequisite for this response [18], as we shall discuss.

While this overall picture of the in vivo phosphorylation stateof RsbS and RsbRA is likely correct, we must also consider possible qualifications . Although our assay used a rapid harvesting procedure and yielded dependably reproducible results, it cannot be assured that the data shown in Fig . 3 precisely mirror the in vivo statusof the tested regulators . This is particularly the case with RsbS-P, which achieves its highest measured level by the first time point, taken 1 min after the stress . Moreover, RsbS-P islabile to the action of the RsbX feedback phosphatase [9, 27,30] . RsbX expression is induced by stress in a ^B-dependent manner[15], and RsbX activity may also increase in response to stress[24] . Therefore, considering the rapid kinetics of both itsphosphorylation and dephosphorylation, the amount of RsbS-Pmeasured in Fig . 3 may be an underestimate . Nonetheless, ourin vivo observations regarding the phosphorylation state ofRsbS corroborate previous genetic analyses, from which it wasdeduced that the phosphorylated form of RsbS is closely associatedwith the transmission of environmental stress signals [16].In these genetic experiments the RsbS S59A variant, which cannotbe phosphorylated, completely prevented environmental signaling.In contrast, the RsbS S59D variant, which is thought to mimicthe phosphorylated state, promoted continuous environmentalsignaling.

There is also some uncertainty surrounding our results regarding RsbRA-P . Although we interpret the data shown in Fig . 3 to indicatethat RsbRA is already 60 to 70% phosphorylated in unstressed cells, it remains possible that RsbRA is so readily phosphorylated by the RsbT kinase that even the rapid harvesting procedurewe employed could elicit the results shown . Moreover, whilethe experiment in Fig . 4 indicates that the integrity of the T171 residue is important for the appearance of RsbRA-P, we cannot exclude the possibility that phosphorylation at T205is also involved in environmental stress signaling . For example,a small amount of the T205-P isoform might be produced but goundetected in our assay, or T205-P might be extremely labile.However, the interpretation that RsbRA is primarily phosphorylatedon T171 agrees with our genetic analyses reported elsewhere,from which we infer that this phosphorylation event is requiredto permit the efficient transmission of environmental stresssignals [18].

This genetic analysis was conducted in strains from which the redundant RsbRA paralogs RsbRB, RsbRC, and RsbRD had been removedin order to clearly establish the effects of alterations atT171 and T205 of RsbRA [18] . Notably, these studies found that the RsbRA T171A variant largely blocked environmental stress signaling, whereas the T171D variant had a normal stress response. These phenotypes are in accord with our interpretation of thedata shown in Fig . 3 and 4—that in unstressed cells RsbRAis already substantially phosphorylated on T171, and the extentof this phosphorylation further increases after stress . If, as we surmise, the phenotype of the T171A alteration primarily reflects its effect on phosphorylation, these data imply that phosphorylation of T171 is normally a prerequisite for the environmental stress response, but it does not by itself trigger the response.

In contrast, the phenotypes caused by T205 alterations, together with our IEF studies, suggest that reversible phosphorylationof T205 is not part of the environmental stress response . Instrains in which RsbRA was the only coantagonist present, theT205A alteration caused continuous environmental signaling,whereas the T205D alteration had a normal stress response [18].One explanation for these results is that phosphorylation ofT205 is required for RsbRA to function as a coantagonist andthat the inability to phosphorylate T205 leads to loss of coantagonistfunction and, consequently, to continuous environmental signaling.This explanation would require that T205 is normally phosphorylatedin unstressed cells, which is contrary to our interpretationof the data shown in Fig. 2 to 4 . Because the T205A alteration has no effect on the accumulation of RsbRA protein in vivo [18], we suggest that its signaling phenotype only shows that this residue is important for RsbRA function, perhaps reflectingthe importance of a threonine [or aspartate] side chain to theRsbRA structure . In this view, the phosphorylation of T205 observedin vitro [12] does not normally occur in the in vivo environment.

Based on the sum of these results, we propose a new model of environmental signaling, shown in Fig . 5 . The significant featureof this model is that a substantial fraction of the RsbRA coantagonistis phosphorylated in unstressed cells grown under the conditionsused here . Following environmental stress, a small fractionof the RsbS antagonist is rapidly phosphorylated, leading to the release of RsbT and the activation of the general stress response . According to this model, the phosphorylation statesof RsbS and RsbRA [and presumably the other members of the RsbRcoantagonist family] are determined by the balance between theactivities of the RsbT kinase and the RsbX feedback phosphatase.RsbS and RsbRA are known to be phosphorylated by the RsbT kinasein vitro [1, 9, 12, 30], and we have shown here that the appearanceof the modified forms of RsbS and RsbRA is dependent on RsbTin vivo . Similarly, RsbS is known to be dephosphorylated bythe RsbX phosphatase in vitro [30], and preliminary evidencepoints to an RsbX-dependent dephosphorylation of RsbS and RsbRAin vivo [T . J . Kim, T . A . Gaidenko, and C . W . Price, unpublisheddata] . We therefore presume that the differential modificationof RsbS and RsbRA noted in unstressed cells reflects a greateractivity of the RsbT kinase toward RsbRA as a substrate, orperhaps a lesser activity of the RsbX phosphatase, comparedto their activities toward RsbS.

 
The present study supports the new model shown in Fig . 5, primarilyby coupling the in vivo phosphorylation of the RsbS antagonistand RsbRA coantagonist to the active transmission of environmentalstress signals . Adjacent genes encoding RsbS and RsbRA orthologsare common within the genomes of the Bacillales, including Listeriamonocytogenes [13] . And, as shown in Table 2, adjacent genesencoding RsbS and RsbRA orthologs, complete with conserved serineor threonine residues, are found in organisms representing evolutionarilydistinct lineages, including the clostridia, actinomycetes,cyanobacteria, gliding bacteria, filamentous anoxygenic phototrophicbacteria, and the beta and gamma subdivisions of Proteobacteria.This widespread distribution suggests that phosphorylation eventssimilar to those we have described here play an analogous rolein diverse signaling pathways.

 
We thank William Haldenwang for his generous gift of the anti-RsbRA and anti-RsbS antibodies and Kazuhiro Shiozaki for providingthe rapid harvesting protocol.

This research was supported by Public Health Service grant GM42077 from the National Institute of General Medical Sciences.

 
* Corresponding author . Mailing address: Department of Food Science and Technology, University of California, Davis, CA 95616 . Phone: [530] 752-1596 . Fax: [530] 752-4759 . E-mail: cwprice@ucdavis.edu .

Present address: Ajinomoto-Genetika Research Institute, 1 1-st Dorozhnyi Proezd, Moscow 113545, Russia.

1. Akbar, S., T . A . Gaidenko, C . M . Kang, M . O'Reilly, K . Devine, and C . W . Price. 2001 . New family of regulators in the environmental signaling pathway which activates the general stress factor ^B of Bacillus subtilis . J . Bacteriol . 183:1329-1338 .
2. Akbar, S., C . M . Kang, T . Gaidenko, and C . W . Price. 1997 . Modulator protein RsbR regulates environmental signaling in the general stress pathway of Bacillus subtilis . Mol . Microbiol . 24:567-578.
3. Alper, S., A . Dufour, D . A . Garsin, L . Duncan, and R . Losick. 1996 . Role of adenosine nucleotides in the regulation of a stress response transcription factor in Bacillus subtilis . J . Mol . Biol . 260:165-177.
4. Alper, S., L . Duncan, and R . Losick. 1994 . An adenosine nucleotide switch controlling the activity of a cell type-specific transcription factor in B . subtilis. Cell 77:195-205.
5. Altschul, S . F., T . L . Madden, A . A . Schaffer, J . Zhang, Z . Zhang, W . Miller, and D . J . Lipman. 1997 . Gapped BLAST and PSI-BLAST: a new generation of protein database search programs . Nucleic Acids Res . 25:3389-3402 .
6. Benson, A . K., and W . G . Haldenwang. 1993 . Bacillus subtilis ^B is regulated by a binding protein [RsbW] that blocks its association with core RNA polymerase . Proc . Natl . Acad . Sci . USA 90:2330-2334.
7. Boylan, S . A., A . R . Redfield, M . S . Brody, and C . W . Price. 1993 . Stress-induced activation of the ^B transcription factor of Bacillus subtilis . J . Bacteriol . 175:7931-7937.
8. Boylan, S . A., A . Rutherford, S . M . Thomas, and C . W . Price. 1992 . Activation of Bacillus subtilis transcription factor ^B by a regulatory pathway responsive to stationary-phase signals . J . Bacteriol. 174:3695-3706.
9. Chen, C.-C., R . J . Lewis, R . Harris, M . D . Yudkin, and O . Delumeau. 2003 . A supramolecular signaling complex in the environmental signaling pathway of Bacillus subtilis . Mol . Microbiol . 49:1157-1669.
10. Dufour, A., and W . G . Haldenwang. 1994 . Interactions between a Bacillus subtilis anti-sigma factor [RsbW] and its antagonist [RsbV] . J . Bacteriol . 176:1813-1820.
11. Dufour, A., U . Voelker, A . Voelker, and W . G . Haldenwang. 1996 . Relative levels and fractionation properties of Bacillus subtilis ^B and its regulators during balanced growth and stress . J . Bacteriol . 178:3701-3709.
12. Gaidenko, T . A., X . Yang, Y . M . Lee, and C . W . Price. 1999 . Threonine phosphorylation of modulator protein RsbR governs its ability to regulate a serine kinase in the environmental stress signaling pathway in Bacillus subtilis . J . Mol . Biol . 284:569-578.
13. Glaser, P., L . Frangeul, C . Buchreiser, C., Rusniok, A . Amend, et al. 2001 . Comparative genomics of Listeria species . Science 294:849-852 .
14. Hecker, M., and U . Völker. 2001 . General stress response of Bacillus subtilis and other bacteria . Adv . Microb . Physiol . 44:35-91.
15. Kalman, S., M . L . Duncan, S . M . Thomas, and C . W . Price. 1990 . Similar organization of the sigB and spoIIA operons encoding alternate sigma factors of Bacillus subtilis RNA polymerase . J . Bacteriol . 172:5575-5585.
16. Kang, C . M., M . S . Brody, S . Akbar, X . Yang, and C . W . Price. 1996 . Homologous pairs of regulatory proteins control activity of Bacillus subtilis transcription factor ^B in response to environmental stress . J . Bacteriol . 178:3846-3853.
17. Kang, C . M., K . Vijay, and C . W . Price. 1998 . Serine kinase activity of a Bacillus subtilis switch protein is required to transduce environmental stress signals but not to activate its target PP2C phosphatase . Mol . Microbiol . 30:189-196.
18. Kim, T . J., T . A . Gaidenko, and C . W . Price. 2004 A multicomponent protein complex mediates environmental stress signaling in Bacillus subtilis . J . Mol . Biol . 341:135-150.
19. Koonin, E . V., L . Aravind, and M . Y . Galperin. 2000 . A comparative-genomic view of the microbial stress response, p . 417-444 . In G . Storz and R . Hennge-Aronis [ed.], Bacterial stress responses . American Society for Microbiology, Washington, D.C.
20. Kunst, F., N . Ogasawara, I . Moszer, A . M . Albertini, G . Alloni, et al. 1997 . The complete genome sequence of the gram-positive bacterium Bacillus subtilis . Nature 390:249-256.
21. Miller, J . H. 1972 . Experiments in molecular genetics . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
22. Mittenhuber, G. 2002 . A phylogenomic study of the general stress response sigma factor ^B of Bacillus subtilis and its regulatory proteins . J . Mol . Microbiol . Biotechnol . 4:427-452.
23. Price, C . W. 2002 . General stress response, p . 369-384. In A . L . Sonenshein, R . Losick, and J . A . Hoch [ed.], Bacillus subtilis and its closest relatives: from genes to cells . American Society for Microbiology, Washington, D.C.
24. Scott, J . M., T . Mitchell, and W . G . Haldenwang. 2000 . Stress triggers a process that limits activation of the Bacillus subtilis stress transcription factor ^B . J . Bacteriol . 182:1452-1456 .
25. Tatusov, R . L., D . A . Natale, I . V . Garkavtsev, T . A . Tatusova, U . T . Shankavaram, B . S . Rao, B . Kiryutin, M . Y . Galperin, N . D . Fedorova, and E . V . Koonin. 2001 . The COG database: new developments in phylogenetic classification of proteins from complete genomes . Nucleic Acids Res . 29:22-28 .
26. Vijay, K., M . S . Brody, E . Fredlund, and C . W . Price. 2000 . A PP2C phosphatase containing a PAS domain is required to convey signals of energy stress to the ^B transcription factor of Bacillus subtilis . Mol . Microbiol . 35:180-188.
27. Voelker, U., T . Luo, N . Smirnova, and W . G . Haldenwang. 1997 . Stress activation of Bacillus subtilis ^B can occur in the absence of the ^B negative regulator RsbX . J . Bacteriol . 179:1980-1984.
28. Voelker, U., A . Voelker, and W . G . Haldenwang. 1996 . Reactivation of the Bacillus subtilis anti-^B antagonist, RsbV, by stress- or starvation-induced phosphatase activities . J . Bacteriol . 178:5456-5463.
29. Wise, A., and C . W . Price. 1995 . Four additional genes in the sigB operon of Bacillus subtilis that control activity of the general stress factor ^B in response to environmental signals . J . Bacteriol . 177:123-133.
30. Yang, X., C . M . Kang, M . S . Brody, and C . W . Price. 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.
31. Zhuo, S., J . C . Clemens, D . J . Hakes, D . Barford, and J . E . Dixon. 1993 . Expression, purification, and biochemical properties of a recombinant protein phosphatase . J . Biol . Chem . 268:17754-17761 .

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