Proc. Natl. Acad. Sci. USAVol. 93, pp. 2488-2492, March 1996Genetics

The response regulator SprE controls the stability ofRpoS(sigma factor/stationary phase/ClpX/CIpP/proteolysis)

LESLIE A. PRATT AND THOMAS J. SILHAVY*Department of Molecular Biology, Princeton University, Princeton, NJ 08544

Communicated by Carol A. Gross, University of California, San Francisco, CA, November 30, 1995 (received for review September 29, 1995)

ABSTRACT In Escherichia coli, the sigma factor, RpoS, isa central regulator in stationary-phase cells. We have iden-tified a gene, sprE (stationary-phase regulator), as essentialfor the negative regulation ofrpoS expression. SprE negativelyregulates the rpoS gene product at the level of protein stability,perhaps in response to nutrient availability. The ability ofSprE to destabilize RpoS is dependent on the ClpX/ClpPprotease. Based on homology, SprE is a member of theresponse regulator family of proteins. SprE is the first re-sponse regulator identified that is implicated in the control ofprotein stability. Moreover, SprE is the first reported proteinthat appears to regulate rpoS in response to a specific envi-ronmental parameter.

rpoS encodes the stationary-phase sigma factor, os or RpoS (1,2), and is required for proper expression of many growth-phaseand osmotically regulated genes (3-10). The level of RpoS isbelieved to be regulated in response to a variety of signals,including nutrient availability (1, 11-13), cell density (14, 15),and osmolarity (16). The regulation of rpoS expression isaccordingly complex, with regulation occurring at the levels oftranscription (13, 15, 17), translation, and protein stability (11,12, 16, 18, 19). In addition, a number of small molecules havebeen implicated in regulation of rpoS expression, includingppGpp (20), cAMP (12, 17), homoserine lactone (14), andUDP-glucose (21). However, the mechanisms and proteinsinvolved in transmitting signals from the environment to rpoSremain a mystery. We have implemented a genetic screen toidentify components involved in regulating rpoS expressionand/or activity. We report the identification of a gene, sprE,which regulates the stability of the RpoS protein.

MATERIALS AND METHODSBacteria and Bacteriophage. All bacterial strains are deriv-

atives of MC4100 (F- araD139 A(argF-lac) U169 rpsL150 relA1flbB5301 deoCl ptsF25 rbsR) (22) and contain the allelesindicated in the figure legends. Bacteria were grown at 30°Cand standard microbiological techniques were used for strainconstruction (22, 23).

Screen for Mutations in rpoS Pathway. Strain LP89 [4(ompF-lacZ+)16-13 ompR107 recA::kan] was subjected to insertion mu-tagenesis using ANK1324 as described (24). Chloramphenicol-resistant isolates were selected on lactose MacConkey agarcontaining 10 mg of chloramphenicol per liter.

Biochemical Analysis. f3-Galactosidase assays were per-formed as described (25). Activities are expressed as (units/A600) X 103, where units = ,umol of orthonitrophenol formedper min. In Figs. 4 and 5, curves were drawn by inspection.Immunoblot analysis was performed as described (26). Pulse-labelings and immunoprecipitations were performed as de-scribed (27). Data were quantitated using the Phosphorlmager

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and the ImageQuant program, and half-lives were determinedusing CRICKET GRAPH III V.1.0 alias.PCR Amplification and DNA Sequence Analysis. sprE19::cam

is -97% linked to osmZ205::TnlO. Oligonucleotide primers wereused to amplify the DNA between osmZ and sprEl9::cam. TheDNA was amplified directly from the bacterial cells using PCR(28). The resulting PCR product was used as a template forDNAsequence analysis as described (28) with a few minor modifica-tions (29). The insertion joint was found to be 27 bp upstream ofthe translational start site of ORF37 (30). The transposon se-

quence appeared to have been rearranged; the details of thisaberrant insertion/rearrangement are unknown.

RESULTSBackground and Rationale: RpoS Is a Stationary-Phase-

Specific Negative Regulator of ompF. The screen for compo-nents affecting rpoS expression (described in the next section)was based on the observation that RpoS affects transcriptionof ompF, which encodes the outer membrane porin protein,OmpF. Osmoregulation of ompF is controlled by a two-component regulatory system comprised of the membranesensor/kinase, EnvZ, and the transcriptional regulator,OmpR. Certain mutations in ompR (e.g., ompR107) constitu-tively repress ompF transcription (31). We sought mutationsthat hinder or overcome the OmpR107-mediated repression ofompF by selecting Lac+ pseudorevertants of strain LP31[4(ompF'-lacZ+)16-13 ompR107]. The majority of mutationsisolated were located in rpoS. Indeed, a defined rpoS null allele(rpoS::kan) confers a Lac+ phenotype in the parent strainLP31. Similarly, rpoS::kan increases the Lac activity in a straincontaining ompR+ and 4(ompF'-lacZ+)16-13. However, thedifference in Lac activity is not as dramatic in a strain wild typefor ompR as in a strain harboring ompRlO7.RpoS affects ompF transcription in a stationary-phase-

specific fashion. During logarithmic growth, rpoS::kan doesnot cause an increase in the Lac activity of cells harboring4(ompF'-lacZ+)16-13 and ompRlO7. In contrast, rpoS::kancauses a 5-fold increase in Lac activity during stationary phase.This effect of RpoS on ompF transcription can be easilyvisualized on lactose MacConkey agar in strains containing4(ompF'-lacZ')16-13 and ompR107, indicating that most ofthe cells in such colonies are in stationary phase. A strain with4(ompF'-lacZ')16-13, ompR107, and rpoS+ appears light pink(Lac-/+) on lactose MacConkey agar, while an isogenicrpoS::kan derivatives is red (Lac+). These observations formthe basis of a simple screen to search for mutants in whichRpoS synthesis/activity is inhibited.Mutant Isolation. We reasoned that factors essential for

induction of rpoS would also be required for rpoS-mediatednegative regulation of ompF. Thus, the loss of an rpoS-positiveregulatory factor should result in an increase in ompF tran-scription similar to that observed in strains lacking a functionalrpoS gene. Moreover, the absence of both the regulatory factor

Abbreviation: ORF, open reading frame.*To whom reprint requests should be addressed.

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and rpoS should not have a cumulative effect on ompFtranscription.Based on the above rationale, the screen involved two steps.

First, insertion mutations conferring increased ompF tran-scription were isolated. ompF transcription was monitored ina strain containing an ompF'-lacZ+ operon fusion andompR107. Strain LP89 [4(ompF'-lacZ')16-13 ompR107recA::kan] was subjected to insertion mutagenesis. Mutantswith increased 4(ompF'-lacZ')16-13 transcription, as assayedby their red color on lactose MacConkey agar, were chosen.

Next, strains were constructed that contain ompR107,4(ompF'-lacZ')16-13, and either (i) each newly isolated in-sertion alone [4(ompF'-lacZ')16-13 ompR107X::cam], or (ii)each newly isolated insertion in combination with rpoS::kan[4(ompF'-lacZ')16-13 ompR107 X::cam rpoS::kan]. This al-lowed a distinction between those insertions that affect com-

ponents involved in the rpoS pathway from those insertionswhose effect on ompF transcription is independent of rpoS.f3-Galactosidase assays were performed on these strains withthe following prediction: Those insertions that function inde-pendently of rpoS should have a cumulative effect on ompFtranscription when combined with rpoS::kan, while thoseinsertions that affect components within the rpoS pathwayshould not.Of 61 insertions isolated, six map to the previously charac-

terized rpoS locus and 51 displayed levels of f3-galactosidaseactivities which suggested that they affect ompF transcriptionvia an rpoS-independent pathway (data not shown). Four ofthe insertions (5, 19, 34, and 46) appear to affect componentswithin the rpoS pathway. With each of these four insertions, thedouble-mutant strain (X::cam, rpoS::kan) exhibited a level of3-galactosidase activity similar to that observed in the strain

containing rpoS::kan alone (data not shown). Three of thesefour were found to be tightly linked to each other and mappedto approximately 5' on the Escherichia coli chromosome (thislocus will be described elsewhere). The final insertion(sprE19::cam; nomenclature explained below) caused a 4-foldincrease in ompF transcription and mapped to 27'.

sprE19::cam Causes Pleiotropic Defects in Stationary-PhaseDevelopment. Since rpoS affects ompF transcription in a station-ary-phase-specific fashion, components of the rpoS pathwayshould likewise have a stationary-phase-specific effect on ompF.As indicated by 3-galactosidase assays on strains harboring4(ompF'-lacZ+)16-13 and ompR107, sprE19::cam does not cause

an increase in ompF transcription during the logarithmic phase ofgrowth, while it causes a 4-fold increase in ompF transcriptionduring stationary phase. In addition, sprE19::cam hinders thestationary-phase induction of glycogen synthesis and the station-ary-phase induction of the catalase katE (data not shown).Finally, cells harboring sprE19::cam are defective in acquiring a

more spherical shape upon entering stationary phase (data notshown). Since these changes are all dependent on rpoS (7, 17, 32),sprE19::cam appears to render cells pleiotropically defective inrpoS-dependent stationary-phase development. These resultssuggest that sprE19::cam may exert its effects by altering theexpression/activity of rpoS. If the phenotypes conferred bysprE19::cam are actually due to decreased RpoS levels/activity,multiple copies of the rpoS gene might be able to play a

compensatory role. Indeed, we found that provision of rpoS on a

multicopy plasmid, pDEB2, partially suppresses the effects ofsprE19::cam on ompF, catalase induction, glycogen synthesis, andcell morphology (data not shown).

sprE19::cam Strains Have Decreased Levels of RpoS Protein.One possible interpretation of the above observation is thatsprE19::cam causes cells to accumulate less RpoS protein thantheir wild-type counterparts. To test this idea, wild-type andsprEl9::cam cells were grown in Luria broth and harvested atvarious stages during growth, and immunoblot analysis was

performed to determine the relative levels of RpoS. sprEl9::caminterferes with the growth-phase regulation of rpoS-that is,

RpoS never accumulates to the level observed in the wild-typecontrol during stationary phase (Fig. 1; compare lanes 1-4 withlanes 9-12). These results reveal that sprE19::cam confers itsstationary-phase defects by interfering with RpoS accumulation.

sprE19::cam Alters Expression of sprE, a Member of theResponse Regulator Family. Using standard techniques,sprE19::cam was found to be located 27 bp upstream of thetranslational start site of a previously identified, yet unchar-acterized, open reading frame (formerly designated ORF37 byref. 30). In light of this gene's involvement in the regulation ofrpoS, we have named it sprE (stationary-phase regulator). ThesprE ORF is predicted to encode a 337-amino acid protein andshares similarity with the response regulator family of proteins(30). Response regulators are part of signal transductionpathways in bacteria and allow adaptation to fluctuations inthe environment (reviewed in refs. 33 and 34). The effectsconferred by the sprE alleles described here are due to theresponse regulator SprE. The gene located downstream ofsprEis galU, and all strains are GalR and Gall.

Since the insertion does not actually interrupt the sprE ORF,the insertion could result in either increased or decreasedexpression of sprE. To distinguish between these possibilities,an insertion, sprE::IS1, located within the sprE ORF wasobtained (30). The IS1 element is located in the middle of thesprE gene (after the 197th amino acid), thus removing theC-terminal, presumed output domain of SprE (30). In anotherwise isogenic background, sprE::IS1 results in an in-creased level of RpoS during all phases of growth (Fig. 1;compare lanes 1-4 with lanes 5-8). Note that this phenotypeis the antithesis of that conferred by sprE19::cam (lanes 9-12).Indeed, the following observations indicate that sprE19::camaffects RpoS levels by increasing the expression of sprE: (i)sprE::IS1 is complemented by a plasmid containing sprE andflanking chromosomal DNA (pUM-E; ref. 30), whilesprE19::cam is not complemented by this plasmid (i.e.,sprE::IS1 is recessive while sprE19::cam is dominant); (ii) cellspossessing both sprE::IS1 and sprE19::cam are phenotypicallyindistinguishable from cells possessing sprE::IS1 alone (i.e., theeffects of sprE19::cam are blocked by the sprE null mutation);and (iii) the presence of sprE in multiple copies (pUM-E)mimics the effects of sprE19::cam. These experiments wereperformed by analyzing how the relevant genetic backgroundsaffect catalase activity and the Lac activity specified by eitheran 4(ompF'-lacZ1)16-13 operon fusion or the 4(rpoS742'-'lacZ) protein fusion described below (data not shown).sprE19::cam Hinders Posttranscriptional Regulation of

rpoS. Protein and operon fusions of lacZ to rpoS (16) were used

wild type sprE::ISI sprEl9::cam rpoS::kan

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FIG. 1. Immunoblot analysis of RpoS protein levels. Cells weregrown overnight in LB medium, subcultured 1:80, and grown until theyreached the cell densities indicated above the gel (OD600). Whole cellextracts were prepared, separated by SDS/PAGE (equal OD unitswere loaded in each lane) and subjected to immunoblot analysis withanti-RpoS antibodies. Lanes 1-4 contain extracts of strain LP423(MC4100 trpC::TnlO). Lanes 5-8 have extracts of strain LP422(MC4100 trpC::TnlO sprE::IS1). Lanes 9-12 have extracts of strainLP421 (MC4100 trpC::TnlO sprE19::cam). Lane 13 contains theextract of strain LP418 (MC4100 rpoS::kan).

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sprE+ sprE::IS110" 1' 3' 5' 12' 20,110"1 1' 3' 5' 12t 20'

RpoS742'-'lacZ

GroEL

FIG. 2. Pulse-chase analysis of 4(RpoS742'-'lacZ). Cells weregrown in glucose minimal medium until they reached OD600 = 0.3. Thecultures were pulse-labeled with [35S]methionine and chased withunlabeled methionine for the indicated times; 4(RpoS742'-'lacZ) andGroEL were immunoprecipitated. GroEL serves as an internal loadingcontrol. Genotypes of the strains are shown at the top.

to monitor rpoS expression in both wild-type and mutantstrains. These rpoS-lacZ fusions are present on a A prophageat Xatt; consequently, the strains possess a functional rpoSgene. ,B-Galactosidase assays performed on stationary-phasecells indicated that sprE19::cam does not alter the stationary-phase transcriptional induction of rpoS [assayed with the0(rpoS742'-lacZ1) operon fusion]. In contrast, sprE19::camcauses a 3-fold decrease in the Lac activity specified by the4(rpoS742'-'lacZ) protein fusion. These results suggest thatthe decreased level of RpoS protein in cells harboringsprE19::cam is attributable to aberrant posttranscriptionalregulation of rpoS.

sprE::IS1 Increases RpoS Stability. Results presented aboveindicate that SprE regulates rpoS at a posttranscriptional level,and thus SprE could influence either rpoS translation or RpoSstability. To determine more precisely how SprE influencesrpoS expression, the stability of the 4(RpoS742'-'lacZ) proteinfusion was analyzed in wild-type and sprE::IS1 cells. As re-ported by Schweder et al. (19), RpoS protein levels arereflected by the synthesis/degradation of such a fusion. Asillustrated in Fig. 2, sprE::ISI stabilizes the 4(RpoS742'-'lacZ)protein fusion, increasing the half-life from 2.7 to 21.4 min.These results indicate that SprE regulates rpoS expression atthe level of protein stability.SprE-Mediated Destabilization of RpoS Requires the

ClpX/ClpP Protease. Like SprE, ClpX/ClpP contributes todestabilization of RpoS, especially during logarithmic phase(19). Two lines of evidence indicate that sprE and clpX/clpP

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function in the same genetic pathway. First, 4(rpoS742'-'lacZ)strains harboring sprE::IS1, clpXA::kan (35), or both sprE::IS1and clpXA::kan, all display comparable levels of ,B-galactosi-dase activity. Specifically, during exponential growth in Luriabroth, the f3-galactosidase activity specified by 4(RpoS742'-'lacZ) is increased 2.4-fold by sprE::IS1, 2.5-fold byclpXA::kan,2.2-fold by clpPA::cam, 2.4-fold by sprE::ISI clpXA::kan, and2.8-fold by sprE::ISI clpPA::cam. Thus, the absence of bothfactors does not have a cumulative effect on 4(rpoS742'-'lacZ). Second, sprE19::cam does not affect the P-galactosi-dase specified by 0(rpoS742'-'lacZ) in strains harboringclpXA::kan or clpPA::cam. During stationary phase in Luriabroth, sprE19::cam causes a 3-fold decrease in the ,3-galacto-sidase activity specified by 4(RpoS742'-'lacZ), and eitherclpXA::kan or clpPA::cam is epistatic to this effect. Thus, theability of sprE19::cam to decrease the level of RpoS is entirelydependent on the ClpX/ClpP protease.

sprE::ISJ Alters Cell Density and Osmotic Regulation ofrpoS Expression. Because rpoS is regulated by numerousparameters, we analyzed how sprE::ISl affects rpoS regulationin response to various environmental conditions. First, wild-type and sprE::ISJ cells were grown in Luria broth, and rpoSexpression at various stages of growth was monitored with the4(rpoS742'-'lacZ) protein fusion (Fig. 3). Under these condi-tions, a number of parameters are altered as cells enterstationary phase. As illustrated in Fig. 3, sprE::IS1 decreasesthe stationary/logarithmic induction ratio from "20 to -5.Therefore, under these conditions, sprE::IS1 alters, but doesnot abolish, the regulation of rpoS. Thus, we are unable toconclude whether or not SprE plays a role in growth-phaseregulation of rpoS.Analogous results were observed with regard to osmotic

regulation of rpoS, as assayed with the 0(rpoS742'-'1acZ)protein fusion. Fig. 4 reveals that cells harboring sprE::ISldisplay different high and low osmolarity patterns of f3-galac-tosidase activity compared to their wild-type counterparts.High osmolarity cells containing sprE::ISI display an -4-foldhigher level of ,B-galactosidase activity during all phases ofgrowth. However, the 5- to 10-fold induction that occurs as celldensity increases is not significantly altered in the sprE::ISJstrain (compare Fig. 4 B and A). In low osmolarity, sprE::IS1cells possess a higher level of ,B-galactosidase activity, espe-cially at low cell densities. Consequently, '50-fold inductionnormally observed as wild-type cells enter stationary phase inlow osmolarity is reduced in cells bearing sprE::IS1 (compareFig. 4 B with A). sprE::ISI alters, but does not completelyblock, the growth-phase and osmotic regulation of rpoS ex-pression.

In sum, these results do not exclude a role for SprE in eithergrowth-phase or osmotic regulation of rpoS. However, the

4(rpoS742'-'1acZ)

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FIG. 3. Growth-phase effects on expression of theprotein fusion 4(rpoS742'-'lacZ) in various geneticbackgrounds. Cells were grown overnight in LB me-dium, subcultured 1:80, and grown until they reachedthe cell densities indicated on the abscissa; ,B-galacto-sidase assays were performed as described. Genotypesof the strains are as follows: R091 [MC41004(rpoS742'-'lacZ)]; LP800 [MC4100 04(rpoS742'-'lacZ) trpC::TnlO]; LP801 [MC4100 s0(rpoS742'-'lacZ)trpC::TnlO sprE::IS1]; LP802 [MC4100 4(rpoS742'-'lacZ) trpC::TnJO sprEl9::cam].

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protein fusion 4(rpoS742'-'lacZ) in wild-type and sprE::ISI strainbackgrounds. Cells were grown overnight in rich A medium with or

without 15% sucrose (23), subcultured 1:80 into the same medium, andgrown until they reached the cell densities indicated on the abscissa;,B-galactosidase assays were performed as described. (A) R091[MC4100 0(rpoS742'-'lacZ). (B) LP809 [MC4100 0(rpoS742'-'lacZ)sprE::IS ].

results demand that whether or not SprE contributes to thegrowth-phase or osmotic regulation of rpoS there must be atleast one other factor that also contributes to this regulation.Moreover, this factor(s) must function in an SprE-independentfashion.

sprE::IS1 Abolishes Carbon-Starvation Induction of rpoSExpression. Intriguingly, sprE::IS1 completely abolishes theinduction upon carbon starvation of the f3-galactosidase ac-tivity specified by the 4(rpoS742'-'lacZ) protein fusion (Fig.5). For this analysis, cells were grown in 0.1% glucose minimalmedium, subcultured 1:100 into 0.01% glucose, and thengrown for the times indicated in Fig. 5. For both wild-type andsprE::IS1 cells, the high level of 3-galactosidase activity at theearly time points is due to the high levels of RpoS742'-'lacZpresent in the dense overnight cultures grown in 0.1% glucoseminimal medium. As wild-type cells become deprived ofglucose, f3-galactosidase activity is induced even at very lowcell densities. Strains bearing sprE::IS1 possess increased a6-ga-lactosidase activity during logarithmic growth (Fig. 5B). No-tably, no further increase in P-galactosidase activity occurs

upon carbon starvation (Fig. SB). This effect is not specific toglucose; similar results were obtained when cells were starvedfor glycerol (data not shown). As cells enter stationary phasein Luria broth (Fig. 3), numerous changes occur (includingchanges in composition of the rich medium and increases incell density). Under the conditions described here (Fig. 5), thecells were starved in minimal medium at low cell density. Inthis way, we were able to alter a defined parameter, carbonsource availability. Thus, these results indicate that in response

4(rpoS742'-'lacZ) sprE::IS1

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FIG. 5. Effects of glucose starvation on expression of the proteinfusion, 4(rpoS742'-'lacZ) in wild-type and sprE::ISJ genetic back-grounds. Cells were grown overnight in 0.1% glucose minimal mediumand subcultured 1:80 into 0.01% glucose minimal medium. Cultureswere grown until the times indicated on the abscissa, OD600 were

determined, and 13-galactosidase assays were performed as described.El, OD600; *, corresponding ,B-galactosidase activities. (A) R091[MC4100 .(rpoS742'-'lacZ)]. (B) LP809 [MC4100 4(rpoS742'-'lacZ)sprE::ISJ].

to nutrient availability, rpoS expression is regulated in an

SprE-dependent fashion at the level of protein stability.

DISCUSSIONAlthough a number of small molecules have been shown toinfluence rpoS expression, the precise roles of these molecules,and how they fit into the regulatory network controlling rpoS,remain unclear. Moreover, most of the proteins reported to beinvolved in regulating rpoS expression function by influencingthe synthesis or degradation of these small molecules (12, 14,17, 20). One exception is the nucleoid protein, HNS. hns::neomutants possess increased levels of RpoS protein (36). This ispuzzling in light of their decreased levels of ppGpp (20) but islikely attributable to both the increased level of rpoS transla-tion and increased RpoS stability (36). However, hns mutantsare notoriously pleiotropoic and, consequently, the regulatoryimplications of such results are difficult to interpret.We utilized a genetic screen to identify two loci involved in

regulation of rpoS. One of these loci, sprE, functions tonegatively regulate rpoS expression at the level of proteinstability. sprE is a previously identified gene (30) with a

heretofore unknown function(s). It is predicted to encode a

protein of 337 amino acids, and it shares striking homology inits N-terminal domain with the response regulator family of

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2492 Genetics: Pratt and Silhavy

proteins (30). For example, the N-terminal 100-amino aciddomain of SprE is 33% identical and 55% similar to theN-terminal domain of the response regulator NtrC. In con-trast, the C-terminal region of SprE does not possess signifi-cant similarity to any of the response regulators.The response regulators fall into a number of subclasses

based on similarities in the C-terminal output domains. Theseproteins typically function as transcriptional activators and/orrepressors, with each subclass presumably interacting withDNA via a common motif located in the C-terminal domain ofthe protein (reviewed in ref. 33). SprE is the first example ofa response regulator that is implicated in regulation at the levelof protein stability. Indeed, the C-terminal, presumed outputdomain of SprE is not homologous to any of the responseregulators reported to data (30); it is not a member of any ofthe subclass of response regulators. SprE's unique outputdomain is noteworthy in light of its unusual role in regulatingprotein stability.

Identification of SprE is an important step in beginning tobuild an understanding of the regulatory network controllingrpoS. This is the first protein identified to regulate rpoS at aparticular level in response to a particular environmentalparameter. We propose that SprE monitors the availability ofnutrients and communicates this information to rpoS to con-trol the stability of RpoS. Since ClpX/ClpP is a protease andsince clpX/clpP and sprE are in the same genetic pathway, itseems likely that SprE regulates RpoS stability by controllingthe ClpX/ClpP-mediated degradation of RpoS. One possibil-ity is that SprE functions by regulating the levels of orproteolytic activity of ClpX/ClpP. However, as assayed byeffects on the ClpX/ClpP target, AO, neither a sprE nullmutation nor the presence ofsprE in multiple copies (pUM-E)appears to influence ClpX/ClpP activity (YanNing Zhou andSusan Gottesman, personal communication). Therefore, itseems likely that SprE functions by influencing the suscepti-bility of RpoS to ClpX/ClpP-mediated degradation.

We are grateful to Susan DiRenzo, Paul Danese, Christine Cosma,Janet Huie, Weihong Hsing, and Craig Parker for critical reading ofthe manuscript. We gratefully acknowledge Regine Hengge-Aronis forcommunicating unpublished results and for providing us with therpoS-lacZ fusions used in this paper. In addition, the observation thatsprE(rssB) influences RpoS stability was initially made in the labora-tory of Regine Hengge-Aronis, and she suggested that we look atstability as well (37). We also thank Susan Gottesman for communi-cating unpublished results and for the clpXA::kan and clpPA::camalleles. We thank Roberto Kolter, Regine Hengge-Aronis, HerbSchellhorn, and members of T.J.S.'s laboratory for helpful discussions.We are also grateful to: Debbie Siegele and Roberto Kolter for pDEB2and the rpoS::kan allele; Michael Bosl and Helga Kersten for providingthe sprE::IS1 allele; Richard Burgess for the monoclonal antibodies toRpoS; and Roberto Kolter and Herb Schellhorn for rpoS-lacZ fusions.L.A.P. was supported by New Jersey Commission on Cancer ResearchGrant 94-2003-CCR-00. T.J.S. was supported by National Institutes ofHealth Research Grant GM35791.

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