General stress proteins protect Bacillus subtilis cells against a variety of environmental insults . This adaptive response is particularly important for nongrowing cells, to which it confersa multiple, nonspecific, and preemptive stress resistance . Inductionof the general stress response relies on the alternative transcription factor, SigB, whose activity is controlled by a partner switching mechanism that also involves the anti-sigma factor, RsbW, andthe antagonist protein, RsbV . Recently, the SigB regulon hasbeen shown to be continuously induced and functionally importantin cells actively growing at low temperature . With the exceptionof this chill induction, all SigB-activating stimuli identifiedso far trigger a transient expression of the SigB regulon thatdepends on RsbV . Through a proteome analysis and Northern blotand gene fusion experiments, we now show that the SigB regulonis continuously induced in cells growing actively at 51°C,close to the upper growth limit of B . subtilis . This heat inductionof SigB-dependent genes requires the environmental stress-responsivephosphatase RsbU, but not the metabolic stress-responsive phosphataseRsbP . RsbU dependence of SigB activation by heat is overcomein mutants that lack RsbV . In addition, loss of RsbV alone orin combination with RsbU triggers a hyperactivation of the generalstress regulon exclusively at high temperatures detrimentalfor cell growth . These new facets of heat induction of the SigBregulon indicate that the current view of the complex geneticand biochemical regulation of SigB activity is still incompleteand that SigB perceives signals independent of the RsbV-mediatedsignal transduction pathways under heat stress conditions.

 
In its natural habitats, Bacillus subtilis often faces hostile conditions that limit or prevent cell growth . Prevalence and proliferation of the bacterial cells require the constant monitoring of the environmental conditions and the mounting of appropriate genetic and cellular defense reactions [33, 51]. B . subtilisis well known for its ability to form highly resistant endosporesas a measure of last resort, thereby allowing survival of partof the bacterial population for extended time periods even underextreme conditions [44] . The synthesis of stress proteins constitutesanother route for cellular adaptation to unfavorable conditions.While stress-specific proteins defend the cell against particularenvironmental insults such as osmotic [12] or oxidative [26] stress, there is increasing evidence that general stress proteins provide cellular protection against a variety of environmental insults [24, 39] . In B . subtilis, synthesis of these generalstress proteins is controlled by the alternative transcriptionfactor SigB [8, 11, 22, 47] . The SigB-dependent general stressregulon comprises approximately 150 members [3, 25, 38, 40],and its induction affords cells with a multiple, nonspecific,and preemptive stress resistance [4, 20, 21, 48].

The activity of the sigma factor SigB is subjected to a tight biochemical regulation to [i] suppress the expression of theSigB regulon under nonstress conditions and [ii] allow rapidinduction of the whole regulon following the imposition of diversestresses [24, 39] . During exponential growth at 37°C, where increased levels of general stress proteins are not required,the activity of SigB is inhibited by binding to its primaryregulatory protein, the anti-sigma factor RsbW [Fig . 1], thereby preventing SigB's association with core RNA polymerase [5]. Release and thus activation of SigB from this inhibitory RsbW/SigB complex require the activity of the antagonist protein RsbV, which is capable of forming an alternative complex with RsbW[18] . This partner switching of RsbW critically depends on the phosphorylation state of RsbV [1, 18] . During growth, RsbV israpidly phosphorylated and thereby inactivated by the kinaseactivity of the anti-sigma factor RsbW . After exposure to stress,RsbV is dephosphorylated by one of two specific PP2C-type phosphatases[RsbU and RsbP] [46, 53], allowing binding of RsbV to RsbW andthereby disruption of the inhibitory RsbW/SigB complex [Fig.1].

 
SigB-activating stimuli can be allocated to two different groups based on the utilization of either the RsbU or the RsbP phosphatase. Sudden environmental stresses such as osmotic or thermal upshiftand exposure to ethanol activate the RsbU phosphatase [Fig. 1] via the action of additional regulatory proteins [e.g., RsbR,RsbS, RsbT, and RsbX] [53] . Metabolic stresses such as limitationof glucose, phosphate, or oxygen in turn are thought to activatethe RsbP phosphatase [46] . Each of these signals triggers atransient dephosphorylation of RsbVP [46, 49, 53] and the concomitanttransient induction of the SigB-dependent general stress regulon[11, 50] . This already complex picture of the control of SigBactivity was further complicated by the recent finding thatcontinued growth of B . subtilis in the cold triggers a long-lastinginduction of the SigB regulon independently of the antagonistprotein RsbV and the phosphatase RsbU or RsbP [Fig . 1] [13].

One of the environmental cues that transiently induces the SigB regulon is a heat shock from 37 to 48°C [8, 11, 47] . Preadaptationthrough a moderate heat shock [48°C] provides wild-typecells with resistance against a strong heat challenge [54°C],a resistance that is largely lost in a sigB mutant [48] . However,sigB mutants still display a considerable degree of inducibleheat resistance that depends on the induction of other classesof heat shock genes [43] . The heat shock stimulon of B . subtilis[25] comprises, besides the SigB regulon, at least four othergroups of heat shock genes: [i] the HrcA regulon encoding theGroESL and DnaK-GrpE-DnaJ chaperones [42]; [ii] the CtsR regulon coding for the proteases and/or chaperones of the Clp family [ClpP, ClpC, and ClpE] [17, 31]; [iii] the CssRS-controlledhtrA and htrB genes [16]; and [iv] a diverse group of heat shockgenes including htpG, lonA, ftsH, and clpX, for which no genetic control mechanism has been elucidated yet [43] . In addition to the changes in gene expression that occur subsequent to a heat shock [25, 38], uptake of compatible solutes such as glycinebetaine can also provide thermoprotection to B . subtilis cellspropagated close to their upper growth limit of 52°C [29].

In contrast to all other SigB-activating stimuli observed sofar, growth of B . subtilis in the cold [15°C] triggereda delayed but long-lasting induction of the entire SigB regulonthat does not depend on the antagonist protein RsbV [13] . These observations raise the question whether a similar atypical induction of the SigB-dependent general stress response also occurs whencells are propagated close to the maximal growth temperatureof B . subtilis. In this report, we show that the SigB regulonis induced in cells that are exponentially growing at 51°C.Like the previously observed chill induction [13], heat induction of the SigB regulon does not depend on the key regulatory protein RsbV . In contrast, a hyperinduction of the entire general stress regulon is observed in mutants lacking RsbV.

 
Bacterial strains, media, and growth conditions. The experiments conducted in this study were performed withthe B . subtilis strain 168 [33] or isogenic mutant derivatives of this wild-type strain [Table 1] . The chromosomal gsiB-bgaBreporter gene fusion was transferred into the B . subtilis strain168 background or its derivatives by transformation [15] . Theparental gene fusion strain BSM52 and the resulting derivativesare listed in Table 1 . Bacteria were routinely grown under vigorous agitation [220 rpm] in Spizizen's Minimal Medium [SMM] with0.5% [wt/vol] glucose as the carbon source, L-tryptophan [20 mg/liter], and a solution of trace elements [23] . Preculturesof B . subtilis strains were inoculated from exponentially growingovernight cultures propagated in SMM to a final optical densityat 578 nm [OD₅₇₈] of 0.1 . These precultures were allowed togrow to an OD₅₇₈ of 0.5, diluted to an OD₅₇₈ of 0.1, and subsequentlytransferred to the higher growth temperatures indicated in theindividual experiments . Ethanol stress was imposed onto thecells during exponential growth by the addition of ethanol toa final concentration of 4% [vol/vol] . For drug resistance selectionin B . subtilis, the following antibiotics were used [final concentrations are given in parentheses]: chloramphenicol [5 µg/ml], spectinomycin [200 µg/ml], kanamycin [20 µg/ml],and erythromycin [1 µg/ml].

 
RNA isolation and Northern blot analysis. Total RNA was isolated from B . subtilis cells growing exponentially[OD₅₇₈, 0.5 to 1.0] at 37 or 51°C by the acid-phenol method[2, 34] with the modifications introduced by Völker et al . [47] . Aliquots [4 µg] of the total RNA were used for the Northern blot analysis of the expression profile of gsiB. Digoxigenin-labeled antisense RNA probes were generated by in vitro transcription using a StripEZ kit [Ambion, Inc., Woodward, Tex.] and a gene-specific PCR product as the template . In thePCR with chromosomal DNA prepared from the B . subtilis strain168, one of the DNA primers carried the sequence of the T7 promoter [gsiB-for, 5'-GAGACCCGGGTTTTGTTTGTTTAAAAGAATTG-3', gsiB-rev, 5'-TAATACGACTCACTATAGGGAGGTCGTTGTTGCGGGCGT-3'] . The PCR fragmentwas subsequently used for in vitro RNA synthesis with commercially available T7 RNA polymerase [Ambion, Inc.], yielding a hybridization probe internal to the gsiB gene of 469 nt . Denaturing RNA electrophoresis on agarose gels, RNA transfer by diffusion onto a nylon membrane [NY13N; Schleicher & Schuell, Dassel, Germany], hybridization to gene-specific probes, and signal detection were performedas described by Holtmann et al . [28].

2-DE. Crude protein extracts for the separation by two-dimensionalprotein gel electrophoresis [2-DE] were prepared from 200-mlcultures grown in 2-liter flasks to an OD₅₇₈ of 0.6 . High-resolution2-DE with immobilized pH gradients [pH 4 to 7] in the firstdimension was performed as previously described [27] . Analyticalgels were stained with silver nitrate according to the methodof Blum et al . [10] . After scanning, analysis and quantificationof the 2-DE gel images were performed with the Delta 2D softwarepackage [Decodon GmbH, Greifswald, Germany] . Subsequent to thematching of the gel sets, the background intensity of all spotswas subtracted and relative spot volumes [background correctedfraction of total spot intensity on the gel contained in theindividual spot] of cultures grown at 37°C were comparedwith those of cultures grown at 51°C . Proteins were consideredto be repressed or induced when the relative spot volume differencesbetween cultures grown at 37°C and those grown at 51°C were observed to be at least fourfold . Two separate gels of each condition were analyzed, and only changes in the proteinpattern passing the cutoff criterion on both parallels wereconsidered significant . Proteins were identified by peptidemass fingerprinting as previously described [27] or by comparisonwith the master gel of B . subtilis 168 [http://microbio1.biologie.uni-greifswald.de/sub2d.htm] [14].

Western blot analysis. The Western blot analysis was carried out as described previously[7, 50] . The monoclonal antibodies raised against RsbS, RsbV,RsbW, SigB, and RsbX have been described [7, 18, 19].

Determination of BgaB activity. In this set of experiments, a ß-galactosidase gene[bgaB] from Bacillus stearothermophilus whose product [BgaB]is stable at high temperature was used [41] . For the determination of ß-galactosidase activity of gsiB-bgaB fusion strains [Table 1], cultures were propagated as described above . At appropriatetime points, 1-ml aliquots were harvested by centrifugationin a Heraeus tabletop centrifuge at 4°C . Cells were resuspendedin 800 µl of modified Z-buffer [41] and permeabilizedby addition of 10 µl of toluol; ß-galactosidase enzyme activity was then assayed at 52°C as previously described [41] . BgaB activity is given in units according to Miller [37].

 
High-temperature induction of the SigB-dependent general stress regulon. Brigulla et al . [13] recently reported that continued growth of B . subtilis in the cold [15°C] triggers a long-lasting induction of the SigB-controlled general stress regulon . This study challenged the belief that induction of the general stress regulon is only a transient response to various environmental stresses [24, 39], and it raised the question whether long-terminduction also occurred as a response to heat stress . A suddentemperature upshift from 37 to 48°C is known to transientlyinduce the SigB regulon [8, 11, 47], but how the SigB regulon responds to cultivation of B . subtilis close to its maximal growth temperature of 52°C has not yet been addressed [29]. High-resolution 2-DE is a convenient tool to monitor changesin the protein synthesis profile of the cells on a global scale[9] . We therefore carried out a comparative proteome analysisof cultures of B . subtilis 168 propagated at 37 or 51°C,and these experiments revealed extensive temperature-dependentdifferences in the cytosolic protein profile with more than128 repressed and 72 induced proteins at 51°C [data notshown] . These changes included increased levels of well-knownSigB-dependent general stress proteins such as GsiB, Dps, Ctc,SigB, KatE, YfkM, and YkzA at 51°C [Fig. 2] . Hence, continuedgrowth at 51°C prompts the induction of the SigB regulon,and, as expected, this phenomenon was not observed in a sigBmutant [Fig . 2].

 
As noted above, induction of proteins at high growth temperaturewas not confined to members of the SigB regulon but also included increased production of other typical heat shock proteins suchas DnaK, GrpE, GroEL, GroES, and HtpG [Fig . 2] . Consistent with their previously published induction pattern [43], these proteinsremained induced in a sigB mutant [Fig. 2B] . Notably, the amountof the heat shock proteins ClpP and ClpC, which are under thedual control of the CtsR repressor and SigB [17, 31], increased only in the high-temperature-grown cells of the wild-type andnot in those of the sigB mutant [Fig . 2].

High-temperature regulation of the sigB operon. SigB, its main regulatory proteins RsbW and RsbV, the phosphataseRsbU, and four additional regulatory proteins [RsbR, RsbS, RsbT,and RsbX] are encoded within the sigB operon [rsbR-rsbS-rsbT-rsbU-rsbV-rsbW-sigB-rsbX]. Basal levels of the sigB operon products are mainly provided by transcription initiating at a vegetative promoter positioned upstream of the rsbR gene [30, 52] . A second SigB-dependentpromoter located between rsbU and rsbV is induced by all SigB-activatingenvironmental and metabolic stimuli described thus far [13, 24, 39], thereby accounting for an amplification of the SigB-mediatedgeneral stress response [8, 11, 13] . The proteome experiments described above [Fig . 2] demonstrate that the SigB regulon isinduced in cells cultured at 51°C . Therefore, one would predict that the RsbV, RsbW, SigB, and RsbX proteins are synthesized in greater amounts in cells growing exponentially at 51°Cthan in cells cultivated at 37°C . To test this predictionexperimentally, we used a set of monoclonal antibodies directedagainst these proteins in Western blot analyses of crude proteinextracts prepared from B . subtilis 168 cultures grown eitherat 37 or at 51°C . As a control, we used a monoclonal antibodydirected against RsbS, which is also encoded by the sigB operonbut whose gene is located upstream of the SigB-dependent promoter.Thus, the cellular levels of RsbS do not respond to SigB activityand should not increase during heat stress . As documented inFig . 3, growth of the cells at 51°C triggered an increasein the levels of RsbV, RsbW, and SigB similar to that observedafter a 60-min exposure to 4% [vol/vol] ethanol, a well-knownstrong inducer of the SigB regulon [11, 50] . This increase was almost completely abolished in a sigB mutant . Residual low-level SigB-independent increases in the levels of RsbV and RsbW by heat shock have been observed before [50] . Neither ethanol norheat stress significantly influenced the level of the control protein RsbS [Fig . 3] . Thus, this Western blot analysis supportsthe initial observation of the proteome study of a high-temperature-mediatedincrease in the level of SigB, the master regulator of the generalstress response, and its main regulatory proteins, RsbW andRsbV.

 
High-temperature regulation of the sigB operon products in mutants lacking SigB regulatory proteins. Heat shock has been shown to induce the SigB regulon via theenvironmental branch of the signal transduction pathway thatis dependent on a functional RsbU protein [50] . Disruption ofthe rsbP gene did not prevent heat induction of SigB and itsmain regulators [RsbV and RsbW], whereas insertional inactivationof rsbU alone or in combination with an rsbP mutation almostcompletely abolished the accumulation of SigB, RsbV, and RsbWin cells growing actively at 51°C [Fig . 3] . With the exceptionof the chill induction of the SigB regulon [13], all SigB-activatingstimuli depend on an intact RsbV protein to elicit the inductionof the general stress regulon [24, 39] . We now observed thatRsbV was not required for heat induction of either RsbW, SigB,or RsbX [Fig . 3] . Surprisingly, heat induction of these proteinsoccurred in an rsbUV double mutant, and their increased levelswere even manifested in an rsbPUV triple mutant [Fig . 3] . Thus, RsbU seems to be essential for heat induction of sigB operon products in the presence of RsbV but is not required in cells carrying the rsbV312 frameshift allele [thus, lacking RsbV].

Hyperinduction of the SigB-dependent gsiB gene in an rsbV312 mutant. The Western blot analysis already provided hints for a hyperinductionof sigB operon products in strains lacking RsbV [Fig . 3] . Toinvestigate this phenomenon further, we carried out a Northernblot analysis of the gsiB gene, whose transcription is exclusivelydriven by a SigB-dependent promoter and is therefore a suitablereporter for SigB activity in the cell [35] . The gsiB gene was induced in cells cultured at 51°C, and this induction was prevented in a sigB mutant [Fig . 4] . A much stronger accumulationof the gsiB transcript was detected in rsbV312 mutant cellsgrown at 51°C than in the wild type [Fig . 4] . This strongtranscription of the gsiB gene, however, was strictly confinedto the high growth temperature, because there was no gsiB transcriptdetectable when the cells were grown at 37°C . The Northernblot analysis of gsiB thus provides strong evidence that RsbVis not necessary for high-temperature induction of SigB-dependentgenes . On the contrary, its loss leads to hyperactivation ofSigB . Hyperinduction of the whole SigB regulon in an rsbPUVtriple mutant at 51°C was also observed in the proteomeanalysis [Fig . 2B].

 
High-temperature induction of the gsiB gene is restricted to a narrow temperature range. A strain carrying a chromosomal gsiB-bgaB reporter gene fusionwas precultured at 37°C . Subsequent propagation of the cellswas continued in a range of temperatures between 37 and 51°C.This experiment was carried out to determine the growth temperatureabove which SigB is permanently released from its negative regulationby the anti-sigma factor RsbW . Expression of the gsiB-bgaB fusionremained repressed in cells that were cultivated between 37and 47°C [Fig . 5] . At 49°C, transcription of the reportergene fusion was induced in the wild-type strain, and the levelof induction was further increased when the cells were grownat 51°C [Fig . 5] . Fully consistent with the Northern blotanalysis of the gsiB transcript in temperature-challenged cells[Fig . 4], a hyperinduction of the gsiB-bgaB transcription in strains that lack RsbV alone or that are simultaneously defectivein the PP2C-type phosphatases RsbU and RsbP was observed [Fig. 5].

 
Time-resolved high-temperature induction of the gsiB-bgaB reporter gene fusion. The data reported in Fig . 5 argue for a permanent expressionof SigB-dependent genes in B . subtilis cells growing at 51°C,close to their maximal growth temperature of 52°C [29].By analyzing the ß-galactosidase activity of the gsiB-bgaBfusion during the first 6 h after the temperature shift from37 to 51°C, we investigated the kinetics of high-temperature-mediatedinduction of SigB-dependent genes . In accordance with previousdata, a sudden temperature upshift triggered an immediate activationof the gsiB-bgaB reporter gene in a wild-type strain that peakedat 20 min after the shift from 37 to 51°C [Fig . 6] . Thereafter, ß-galactosidase activity slowly declined but remainedsignificantly elevated above the preinduction basal level [37°C]throughout the experiment [Fig . 6] . Thermoinduction of the gsiB-bgaB fusion was entirely prevented by the presence of a sigB mutation.

 
A pattern of ß-galactosidase activity like that ofthe wild type was also observed in a gsiB-bgaB fusion straincarrying a mutation in the gene [rsbP] encoding the metabolicstress responsive phosphatase RsbP [Fig . 6] . This observation indicates that this branch of the SigB signal transduction cascade [Fig . 1] is not involved in thermosensing . In contrast, theintroduction of an rsbU mutation into the reporter strain completelyabolished the induction of the gsiB-bgaB gene fusion, thus demonstratingthat the PP2C-phosphatase RsbU is critically involved in sensinghigh temperature . Inactivation of the antagonist protein RsbVby the frameshift mutation rsbV312 caused a short delay in theinduction of the gsiB-bgaB fusion, but then the ß-galactosidaseactivity of this fusion continued to rise throughout the experiment,reaching sevenfold-higher levels than in the wild-type strainapproximately 6 h after the temperature upshift [Fig . 6] . Furthermore, delayed hyperinduction of the SigB-dependent reporter gene construct also occurred in an rsbPUV triple mutant, lacking the antagonist protein RsbV as well as both RsbVP phosphatases [Fig . 6].

Growth at high temperature. Growth experiments of B . subtilis under chill stress conditions[15°C] in a chemically defined minimal medium have shownthat a sigB mutant has a distinct growth disadvantage in comparisonto the wild-type strain [13] . Furthermore, such a sigB mutantis highly sensitive to severe heat shock [54°C] [48] . Evenafter preadaptation by a mild heat shock [48°C], a sigBmutant displayed extreme sensitivity to the strong heat challenge[54°C], whereas the same pretreatment conferred completeresistance to the wild-type strain [48] . We therefore investigatedwhether a growth phenotype is associated with a loss of SigBactivity in cells cultured at 51°C . We found that this wasnot the case [Fig . 7] . However, the growth experiments revealeda strong reduction in growth rate, which was associated withthe inactivation of rsbV [Fig . 7] that triggered hyperactivationof SigB [Fig . 3, 4, and 5] . This reduced growth rate [at 51°C]was also observed at high temperature in a strain combining mutations in rsbV, rsbU, and rsbP [Fig . 7].

 
The SigB-dependent general stress regulon of the soil bacteriumB . subtilis is engaged when cells experience a wide range of environmental or metabolic stresses [24, 39] . The physiologicalrole of general stress proteins for cellular protection is emphasizedby the finding that the disruption of the structural gene forits master regulator [SigB] causes sensitivity of the cell toa variety of stress factors, such as severe heat and salt shocks,ethanol treatment, low or high pH, and free oxygen radicals [4, 20, 21, 48].

Thus far, increased synthesis of the general stress proteinswas believed to be particularly important under harsh stressconditions under which the bacterial cells are no longer ableto grow [24, 39] . A new facet of the physiological functionof the SigB regulon was recently discovered by the finding thatthis regulon is induced to high levels in cells that activelygrow under chill stress conditions [15°C] [13] or that are subjected to a sudden temperature downshift from 37 to 20°C[36] . A heat shock at temperatures from 37 to 48°C has longbeen known to be one of the environmental cues that induce theSigB regulon [8, 11, 47] . The data reported here now demonstratea continuous high-level expression of SigB-dependent generalstress proteins in cells that actively grow at 51°C [Fig. 2, 5, and 6], close to the maximal growth temperature [52°C]of B . subtilis [29] . Growth at very high and low temperaturesis likely to reflect more closely the situation in natural habitatsof B . subtilis than the sudden harsh temperature up- or downshiftsused in the laboratory to induce the SigB response . A sigB mutantcannot cope effectively with low-temperature [15°C] growthconditions [13], but such a mutant is not at a significant growthdisadvantage when it is cultured at 51°C [Fig . 7] . Lackof a specific growth phenotype of a sigB mutant strain at hightemperature probably reflects the presence of redundant heatstress adaptation mechanisms in B . subtilis [43] . Indeed, we observed strong induction of heat shock proteins [GroEL, GroES,DnaK, and GrpE] controlled by the HrcA repressor and of theheat shock protein HtpG in cells that were exponentially growingat 51°C [Fig. 2] . It is well known that increased levelsof chaperones are needed when cells have to cope with high growth temperatures [43, 54].

It is firmly established that environmental stimuli such assalt shock, ethanol treatment, and heat shock transiently inducethe SigB response [8, 11, 47, 50] . In contrast, continued growthclose to the upper [Fig . 2, 5, and 6] or lower [13] temperaturelimits of B . subtilis leads to a sustained high-level expressionof the general stress response to provide cells with a sufficientlevel of general stress proteins . At these temperature boundaries,permanent high-level expression of the SigB regulon occurs onlyin narrow temperature ranges [Fig . 5] [13].

Heat and salt shock and ethanol treatment activate SigB viathe environmental branch of the signal transduction cascadethat relies on the activity of the PP2C-type phosphatase RsbU[Fig . 1] [49, 50, 53] . Whereas the requirement for RsbV in thesensing of salt shock and ethanol stress is firmly established,the role of RsbV in heat shock activation of SigB has been amatter of debate . Initially, SigB accumulation following heatshock had been observed to persist in cells lacking RsbV [8,11] . Due to the autoregulation of the sigB gene, this observationwas interpreted as indicating an RsbV-independent SigB activation. Subsequently, the majority of the heat shock induction of theSigB regulon was shown to proceed via RsbU and RsbV and residualSigB accumulation in rsbV or rsbU mutants seemed to be largely independent of SigB activity [50] . In a wild-type background,such a dependence on RsbU for thermoactivation of SigB was alsoobserved for cells that were continuously cultured at 51°C [Fig . 6] . In contrast, the metabolic stress-sensing PP2C-typephosphatase RsbP [Fig . 1] was not required for thermoinductionof SigB-dependent genes [Fig. 6] . The RsbU dependence of thermoactivationof SigB both after a heat shock and during continued growthat 51°C suggests that in a wild-type background both processesare mediated by the same signal transduction pathway relyingon a functional RsbV protein [Fig . 1].

However, a distinctively different regulation seems to occurin mutants that lack RsbV due to the frameshift mutation rsbV312. Thermoactivation of SigB during continuous growth at 51°Cwas also observed with strains carrying this rsbV312 mutation[Fig. 3 and 6], indicating that the requirement of RsbV canbe bypassed [Fig . 1] . To our great surprise, introduction ofthe rsbV312 allele into an rsbU mutant background overridesthe RsbU dependence and restores thermoactivation of SigB [Fig.3 and 6].

An RsbV-independent induction of general stress genes was also observed in chill-stressed B . subtilis cells [13] . However,under heat stress, one observes a hyperinduction of the SigB regulon in an rsbV312 mutant [Fig . 4 and 6] that is not seenin chill-stressed rsbV312 mutant cells [13] . This hyperinductionof SigB-dependent general stress genes is conserved both inan rsbUV double mutant and in an rsbPUV triple mutant [Fig. 2, 3, and 6] and is detrimental to the B . subtilis cell, sinceit results in a reduced growth rate at 51°C [Fig . 7].

Even if one cannot completely exclude that secondary effectssuch as a slightly modified RsbW/SigB ratio might contributeto the observed high-temperature activation of SigB in strainslacking RsbV, our data indicate that the current model for thegenetic and biochemical control of SigB activity [24, 39] afterexposure to heat seems to be incomplete [Fig . 1].

Heat stress is also a strong inducer of the SigB regulon inother gram-positive bacteria such as Staphylococcus aureus [32] and Bacillus cereus [45] . Neither microorganism encodes orthologuesof RsbT, RsbR, RsbS, and RsbX that are critically involved insignaling of environmental stress in B . subtilis [30, 46, 52,53], indicating that the perception of heat stress in theseorganisms might also involve signal transduction componentsthat have not yet been detected.

 
We are grateful to W . G . Haldenwang for providing the monoclonal antibodies directed against SigB and its regulatory proteinsand F . Spiegelhalter and G . Kuhnke for the construction of plasmids.We thank V . Koogle for her help in editing the manuscript.

Financial support for this study was provided by the Deutsche Forschungsgemeinschaft through the SFB-395 and the Graduiertenkolleg "Proteinfunktion auf atomarer Ebene," the Bundesministeriumfür Bildung und Forschung through the "Genomnetzwerk Göttingen,"the Max-Planck-Society, and the Fonds der Chemischen Industrie.

 
* Corresponding author . Mailing address: Laboratory for Microbiology, Department of Biology, Philipps-University Marburg, Karl-von-Frisch-Str., D-35032 Marburg, Federal Republic of Germany . Phone: 49-6421-2821529 . Fax: 49-6421-2828979 . E-mail: bremer@staff.uni-marburg.de.

Present address: 3M Deutschland GmbH, 41453 Neuss, Germany.

1. 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.
2. Ambulos, N . P., Jr., E . J . Duvall, and P . S . Lovett. 1987 . Method for blot-hybridization analysis of mRNA molecules from Bacillus subtilis. Gene 51:281-286.
3. Antelmann, H., J . Bernhardt, R . Schmid, H . Mach, U . Völker, and M . Hecker. 1997 . First steps from a two-dimensional protein index towards a response-regulation map for Bacillus subtilis. Electrophoresis 18:1451-1463.
4. Antelmann, H., S . Engelmann, R . Schmid, and M . Hecker. 1996 . General and oxidative stress responses in Bacillus subtilis—cloning, expression, and mutation of the alkyl hydroperoxide reductase operon . J . Bacteriol . 178:6571-6578.
5. 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.
6. Benson, A . K., and W . G . Haldenwang. 1992 . Characterization of a regulatory network that controls ^B expression in Bacillus subtilis. J . Bacteriol . 174:749-757.
7. Benson, A . K., and W . G . Haldenwang. 1993 . Regulation of ^B levels and activity in Bacillus subtilis. J . Bacteriol . 175:2347-2356.
8. Benson, A . K., and W . G . Haldenwang. 1993 . The ^B dependent promoter of the Bacillus subtilis sigB operon is induced by heat shock . J . Bacteriol . 175:1929-1935.
9. Bernhardt, J., J . Weibezahn, C . Scharf, and M . Hecker. 2003 . Bacillus subtilis during feast and famine: visualization of the overall regulation of protein synthesis during glucose starvation by proteome analysis . Genome Res . 13:224-237 .
10. Blum, H., H . Beier, and H . Gross. 1987 . Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels . Electrophoresis 8:93-99.
11. 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.
12. Bremer, E. 2002 . Adaptation to changing osmolarity, p . 385-391 . In A . L . Sonenshein, J . A . Hoch, and R . Losick [ed.], Bacillus subtilis and its closest relatives . From genes to cells . ASM Press, Washington, D.C.
13. Brigulla, M., T . Hoffmann, A . Krisp, A . Völker, E . Bremer, and U . Völker. 2003 . Chill induction of the SigB-dependent general stress response in Bacillus subtilis and its contribution to low-temperature adaptation . J . Bacteriol . 185:4305-4314 .
14. Büttner, K., J . Bernhardt, C . Scharf, R . Schmid, U . Mader, C . Eymann, H . Antelmann, A . Völker, U . Völker, and M . Hecker. 2001 . A comprehensive two-dimensional map of cytosolic proteins of Bacillus subtilis. Electrophoresis 22:2908-2935.
15. Cutting, S . M., and P . B . Vander Horn. 1990 . Genetic analysis, p . 27-74 . In C . R . Harwood and S . M . Cutting [ed.], Molecular biological methods for Bacillus. John Wiley & Sons, Chichester, United Kingdom.
16. Darmon, E., D . Noone, A . Masson, S . Bron, O . P . Kuipers, K . M . Devine, and J . M . van Dijl. 2002 . A novel class of heat and secretion stress-responsive genes is controlled by the autoregulated CssRS two-component system of Bacillus subtilis. J . Bacteriol . 184:5661-5671 .
17. Derre, I., G . Rapoport, and T . Msadek. 1999 . CtsR, a novel regulator of stress and heat shock response, controls clp and molecular chaperone gene expression in Gram-positive bacteria . Mol . Microbiol. 31:117-132.
18. 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.
19. Dufour, A., U . Völker, A . Völker, 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.
20. Engelmann, S., and M . Hecker. 1996 . Impaired oxidative stress resistance of Bacillus subtilis sigB mutants and the role of katA and katE. FEMS Microbiol . Lett . 145:63-69.
21. Gaidenko, T . A., and C . W . Price. 1998 . General stress transcription factor ^B and sporulation transcription factor ^H each contribute to survival of Bacillus subtilis under extreme growth conditions . J . Bacteriol . 180:3730-3733 .
22. Haldenwang, W . G., and R . Losick. 1980 . Novel RNA polymerase sigma factor from Bacillus subtilis. Proc . Natl . Acad . Sci . USA 77:7000-7004.
23. Harwood, C . R., and A . R . Archibald. 1990 . Growth, maintenance and general techniques, p . 1-26 . In C . R . Harwood and S . M . Cutting [ed.], Molecular biological methods for Bacillus. John Wiley & Sons, Chichester, United Kingdom.
24. Hecker, M., and U . Völker. 2001 . General stress response of Bacillus subtilis and other bacteria . Adv . Microb . Physiol . 44:35-91.
25. Helmann, J . D., M . F . Wu, P . A . Kobel, F . J . Gamo, M . Wilson, M . M . Morshedi, M . Navre, and C . Paddon. 2001 . Global transcriptional response of Bacillus subtilis to heat shock . J . Bacteriol . 183:7318-7328 .
26. Herbig, A . F., and J . D . Helmann. 2002 . Metal ion uptake and oxidative stress, p . 405-414 . In A . L . Sonenshein, J . A . Hoch, and R . Losick [ed.], Bacillus subtilis and its closest relatives . From genes to cells . ASM Press, Washington, D.C.
27. Hoffmann, T., A . Schütz, M . Brosius, A . Völker, U . Völker, and E . Bremer. 2002 . High-salinity-induced iron limitation in Bacillus subtilis. J . Bacteriol . 184:718-727 .
28. Holtmann, G., E . P . Bakker, N . Uozumi, and E . Bremer. 2003 . KtrAB and KtrCD: two K⁺ uptake systems in Bacillus subtilis and their role in the adaptation to hypertonicity . J . Bacteriol. 185:1289-1298 .
29. Holtmann, G., and E . Bremer. 2004 . Thermoprotection of Bacillus subtilis by exogenously provided glycine betaine and structurally related compatible solutes: involvement of the Opu transporters . J . Bacteriol . 186:1683-1693 .
30. 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.
31. Krüger, E., and M . Hecker. 1998 . The first gene of the Bacillus subtilis clpC operon, ctsR, encodes a negative regulator of its own operon and other class III heat shock genes . J . Bacteriol . 180:6681-6688 .
32. Kullik, I., and P . Giachino. 1997 . The alternative sigma factor ^B in Staphylococcus aureus: regulation of the sigB operon in response to growth phase and heat shock . Arch . Microbiol . 167:151-159.
33. Kunst, F., N . Ogasawara, I . Moszer, A . M . Albertini, G . Alloni, V . Azevedo, M . G . Bertero, P . Bessieres, A . Bolotin, S . Borchert, R . Borriss, L . Boursier, A . Brans, M . Braun, S . C . Brignell, S . Bron, S . Brouillet, C . V . Bruschi, B . Caldwell, V . Capuano, N . M . Carter, S . K . Choi, J . J . Codani, I . F . Connerton, and A . Danchin. 1997 . The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390:249-256.
34. Majumdar, D., Y . J . Avissar, and J . H . Wyche. 1991 . Simultaneous and rapid isolation of bacterial and eukaryotic DNA and RNA: a new approach for isolating DNA . BioTechniques 11:94-101.
35. Maul, B., U . Völker, S . Riethdorf, S . Engelmann, and M . Hecker. 1995. ^B-dependent regulation of gsiB in response to multiple stimuli in Bacillus subtilis. Mol . Gen . Genet . 248:114-120.
36. Mendez, M . B., L . M . Orsaria, V . Philippe, M . E . Pedrido, and R . R . Grau. 2004 . Novel roles of the master transcription factors Spo0A and ^B for survival and sporulation of Bacillus subtilis at low growth temperature . J . Bacteriol . 186:989-1000 .
37. Miller, J . H. 1972 . Experiments in molecular genetics . Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
38. Petersohn, A., M . Brigulla, S . Haas, J . D . Hoheisel, U . Völker, and M . Hecker. 2001 . Global analysis of the general stress response of Bacillus subtilis. J . Bacteriol . 183:5617-5631 .
39. Price, C . W. 2000 . Protective function and regulation of the general stress response in Bacillus subtilis and related gram-positive bacteria, p . 179-197 . In G . Storz and R . Hengge-Aronis [ed.], Bacterial stress responses . ASM Press, Washington, D.C.
40. Price, C . W., P . Fawcett, H . Ceremonie, N . Su, C . K . Murphy, and P . Youngman. 2001 . Genome-wide analysis of the general stress response in Bacillus subtilis. Mol . Microbiol . 41:757-774.
41. Schrögel, O., and R . Allmansberger. 1997 . Optimisation of the BgaB reporter system: determination of transcriptional regulation of stress responsive genes in Bacillus subtilis. FEMS Microbiol . Lett . 153:237-243.
42. Schulz, A., and W . Schumann. 1996 . hrcA, the first gene of the Bacillus subtilis dnaK operon, encodes a negative regulator of class I heat shock genes . J . Bacteriol . 178:1088-1093.
43. Schumann, W., M . Hecker, and T . Msadek. 2002 . Regulation and function of heat-inducible genes in Bacillus subtilis, p . 359-368. In A . L . Sonenshein, J . A . Hoch, and R . Losick [ed.], Bacillus subtilis and its closest relatives . From genes to cells . ASM Press, Washington, D.C.
44. Sonenshein, A . L. 2000 . Bacterial sporulation: a response to environmental signals, p . 199-215 . In G . Storz and R . Hengge-Aronis [ed.], Bacterial stress responses . ASM Press, Washington, D.C.
45. van Schaik, W., M . H . Tempelaars, J . A . Wouters, W . M . de Vos, and T . Abee. 2004 . The alternative sigma factor ^B of Bacillus cereus: response to stress and role in heat adaptation . J . Bacteriol . 186:316-325 .
46. 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.
47. Völker, U., S . Engelmann, B . Maul, S . Riethdorf, A . Völker, R . Schmid, H . Mach, and M . Hecker. 1994 . Analysis of the induction of general stress proteins of Bacillus subtilis. Microbiology 140:741-752.
48. Völker, U., B . Maul, and M . Hecker. 1999 . Expression of the ^B-dependent general stress regulon confers multiple stress resistance in Bacillus subtilis. J . Bacteriol . 181:3942-3948 .
49. Völker, U., A . Völker, 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.
50. Völker, U., A . Völker, B . Maul, M . Hecker, A . Dufour, and W . G . Haldenwang. 1995 . Separate mechanisms activate ^B of Bacillus subtilis in response to environmental and metabolic stresses . J . Bacteriol . 177:3771-3780.
51. Wipat, A., and C . R . Harwood. 1999 . The Bacillus subtilis genome sequence: the molecular blueprint of a soil bacterium . FEMS Microbiol . Ecol . 28:1-9.
52. Wise, A . 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.
53. Yang, X . F., 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.
54. Yura, T., M . Kanemori, and M . T . Morita. 2000 . The heat shock response: regulation and function, p . 3-18 . In G . Storz and R . Hengge-Aronis [ed.], Bacterial stress responses . ASM Press, Washington, D.C.

Free Online Full-text Article

What Is Bioengineering?, What Is Rhizobia?, What Is Dna?, What Is Bioassay?, What Is Nitrification?, s, Bacteria, s, Bacteriology, e, Microbe, s, Microbes, s, Bacterium, o, Yeasts, a, Microbial, i, Escherichia coli, a, Anaerobic bacteria, c, Microorganisms, c, S. cerevisiae, o, Prokaryotes, s, Streptomycin, n, Bacteriophage, a, Growth media, c, Lactobacillus, e, Escherichia coli, n, S. cerevisiae, s, Pseudomonas aeruginosa, a, Streptococcal, i, Escherichia coli, e, Staphylococcus, c, Escherichia coli, i, S. cerevisiae, n, Schizosaccharomyces, a, Enterobacters




 

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

Last modified: May 25, 2005