Multiple Pathways of Spx (YjbD) Proteolysis in Bacillus subtilis

ATP-dependent proteases degrade denatured or misfolded proteinsand are recruited for the controlled removal of proteins thatblock activation of regulatory pathways. Among the ATP-dependentproteases, those of the Clp family are particularly importantfor the growth and development of Bacillus subtilis. Proteolyticsubunit ClpP, together with regulatory ATPase subunit ClpC orClpX, is required for the normal response to stress, for developmentof genetic competence, and for sporulation. The spx (formallyyjbD) gene was previously identified as a site of mutationsthat suppress defects in competence conferred by clpP and clpX.The level of Spx in wild-type cells grown in competence mediumis low, and that in clpP mutants is high. This suggests thatthe Spx protein is a substrate for ClpP-containing proteasesand that accumulation of Spx might be partly responsible forthe observed pleiotropic phenotype resulting from the clpP mutation.In this study we examined, both in vivo and in vitro, whichClpP protease is responsible for degradation of Spx. Westernblot analysis showed that Spx accumulated in clpX mutant tothe same level as that observed in the clpP mutant. In contrast,a very low concentration of Spx was detected in a clpC mutant.An in vitro proteolysis experiment using purified proteins demonstratedthat Spx was degraded by ClpCP but only in the presence of oneof the ClpC adapter proteins, MecA or YpbH. However, ClpXP,either in the presence or in the absence of MecA and YpbH, wasunable to degrade Spx. Transcription of spx, as measured byexpression of spx-lacZ, was slightly increased by the clpX mutation.To exclude a possible effect of clpX and clpP on spx transcription,the spx gene was placed under the control of the IPTG (isopropyl-ß-D-thiogalactopyranoside)-induciblePspac promoter. In this strain, Spx accumulated when ClpX orClpP was absent, suggesting that ClpX and ClpP are requiredfor degradation of Spx. Taken together, these results suggestthat Spx is degraded by both ClpCP and ClpXP. The putative proteolysisby ClpXP might require another adapter protein. Spx probablyis degraded by ClpCP under as yet unidentified conditions. Thisstudy suggests that the level of Spx is tightly controlled bytwo different ClpP proteases.

INTRODUCTION

Top

Introduction

ATP-dependent proteases play an important role in many cellularprocesses in both prokaryotes and eukaryotes (reviewed in reference47). They function in the cell's response to stress by eliminatingdamaged or misfolded proteins and degrading truncated productsof aborted translation. They are also involved in degradationof specific, short-lived regulatory proteins, many of whichfunction in developmental processes. Members of one class ofATP-dependent proteases, the Clp proteases, are composed oftwo subunits, a regulatory ATPase component and the peptidase,ClpP (reviewed in reference 37). While ClpP alone exhibits peptidaseactivity, its association with the ATPase imparts substratespecificity and is required for the degradation of polypeptideslonger than six amino acids (41). The Clp ATPase hexamers bindto and unfold substrates and then translocate them to the tworings of ClpP heptamers (16, 40). Clp ATPases, in the presenceor absence of ClpP, also function as molecular chaperones (reviewedin reference 12). For example, ClpX chaperone activity is necessaryfor the dissociation of the MuA complex following strand transferduring Mu transposition (23). In Escherichia coli, ClpAP degradesRepA (46), bacteriophage P1 protein (22), and the MazE protein(1), which is involved in programmed cell death. ClpXP degradesMuA transposase (23), ${lambda}$ O (49), and the stationary-phase ${sigma}$ factorRpoS (39). Proteins from the translation of truncated mRNA aretargeted for the addition of 11-residue peptides to the carboxytermini, a process which is mediated by SsrA RNA (also called10Sa RNA or tm RNA) (42). The SsrA-tagged proteins are degradedby ClpAP and ClpXP (13).

In Bacillus subtilis, Clp proteases include those bearing theClpC, ClpE, and ClpX ATPase subunits (reviewed in reference38). Unlike E. coli clpP and clpX mutants, which grow normally(39), the B. subtilis clp mutants are highly pleiotropic andexhibit poor growth phenotypes on certain media such as minimalmedium and Luria-Bertani (LB) medium even under nonstress conditions(10, 26, 31). Immunocytochemical experiments showed that ClpCPand ClpXP are directly involved in degradation of misfoldedor denatured proteins after either heat shock or treatment withpuromycin (20). These treatments also induce transcription ofclpC, clpP, and clpX (10, 11, 19, 26, 27) and lead to increasedClpC and ClpP protein levels (20). ClpC, ClpP, and ClpX areessential for growth at high temperature following heat shockinduction (10, 19, 26, 27). Other common features observed inthe clpC, clpP, and clpX mutants include a salt-sensitive andnonmotile phenotype (10, 19, 26). No obvious phenotype whichis associated with the clpE mutation has been found so far (5).Recently, ClpXP was shown to be responsible for degradationof SsrA-tagged proteins in B. subtilis cells (48).

In addition to their growth phenotype and sensitivity to stress,mutants lacking ClpC, ClpP, or ClpX show defects in developmentalpathways including sporulation and competence (10, 26, 31, 32).A clpP null mutation confers the most severe sporulation defect,followed, in order of decreasing severity, by the clpC and clpXmutations. The sporulation phenotype of the clpC mutation appearedmore obvious at high temperature (32), while a mutation in clpE,encoding another ATPase subunit, produces no sporulation phenotype(5). Since ClpP is thought only to function with Clp ATPases,it is not clear why mutations in clpX, -C, and -E have littleor no sporulation phenotype. Perhaps the ATPases perform redundantfunctions during the sporulation process. That ClpP is necessaryfor entry into sporulation is supported by the finding thatSpo0A-dependent expression of spoIIA and spoIIG operons, whichencode ${sigma}$ ^F and ${sigma}$ ^E, respectively, was adversely affected by a clpPmutation (26). This result suggests that the phosphorelay leadingto the phosphorylation of Spo0A is defective in the clpP mutant.In fact, a spo0E mutation partially suppresses clpP; that is,Spo0A-dependent gene expression is observed in a spo0E clpPmutant background but sporulation was not restored (33). TheclpC mutation did not affect Spo0A-dependent expression of spoIIAand spoIIE; however, the mutation caused an impairment of ${sigma}$ ^F-dependentgene expression (35). Apparently ClpCP is required after phosphorylationof Spo0A and before activation of ${sigma}$ ^F. A recent study demonstratedthat ClpCP increased ${sigma}$ ^F activity by directly degrading SpoIIABand anti- ${sigma}$ ^F factor and by indirectly promoting polar septum formation,which is a prerequisite for the activation of ${sigma}$ ^F (35). ClpCPis also involved in the degradation of ${sigma}$ ^H, an RNA polymerasesigma subunit required for sporulation initiation, at high temperatureand might be responsible for the degradation of ${sigma}$ ^H after it accomplishesits task in early sporulation (32). Finally ClpX, but not ClpP,is required for ${sigma}$ ^H-RNA polymerase activity in vitro and in vivo(25). Thus the role of the ClpXP protease in sporulation, ifany, is elusive.

Although ClpC, ClpP, and ClpX are involved in competence development,the effect of clpC is opposite to that of clpP and clpX. BothclpP and clpX mutants are defective in competence (26, 31),while the clpC mutant shows a mec (medium-independent expressionof competence) phenotype (27), indicating a negative role forClpC. ClpC forms a complex with MecA (18) and ComK (45) in noncompetentcells; this complex sequesters transcription factor ComK inan inactive state (44). Thus, ClpC negatively regulates comK-dependentexpression of comK and late competence genes. In addition, ComKis degraded by ClpCP, and the proteolysis requires adapter proteinMecA (43). The small peptide ComS (7, 14), whose synthesis isactivated by a quorum-sensing pathway, accumulates in stationaryphase and releases ComK from the inhibitory complex (44). LiberatedComK is no longer a target of ClpCP; instead MecA and ComS aredegraded by ClpCP (43). Two additional roles for ClpP in competencehave been found. ClpP, together with ClpX, is required for srf(comS) expression (31). Amino acid substitutions in the carboxy-terminaldomain of the ${alpha}$ subunit of RNA polymerase suppress either clpXor clpP with respect to comS expression (31). Although the exactrole of ClpXP in comS expression remains to be elucidated, adirect involvement of ClpXP in comS transcription was suggested.Furthermore ClpP is also needed to degrade Spx (YjbD), the accumulationof which negatively affects competence and sporulation (28).The clpP spx double mutant completely and partially restoredcompetence and sporulation, respectively. In this study, weexamined how the level of Spx is regulated by ClpP proteasesand found that both ClpCP and ClpXP are involved in degradingSpx, probably under different conditions.

MATERIALS AND METHODS

Top

Materials and Methods

Bacterial strains and media. All B. subtilis strains listed in Table 1 are derivatives ofJH642. Construction of LAB2876 (clpX::spc) (25), LAB2972 (clpP::erm)(34), and ORB3834 (spx::neo) (28) was described previously.ORB3976 was constructed by transforming JH642 with chromosomalDNA prepared from QPB418 (clpC::tet) (35).

View this table:
[in this window]
[in a new window]
TABLE 1. B. subtilis strains and plasmids used in this study

A strain which carries the spx coding sequence downstream ofthe IPTG (isopropyl-ß-D-thiogalactopyranoside)-induciblePspac promoter was constructed as follows. Primers oMN01-173(5'-CGAGGAAGCTTAGATGTTCATCCTACTA-3') and oMN01-174 (5'-TACCAGCAGGTCGACAAATAAAAGAAGG-3')were used to amplify the spx gene by using JH642 chromosomalDNA as the template. The PCR product was digested with HindIIIand SalI and ligated with pDR66 digested with the same enzymes.After the spx sequence was verified, the resultant plasmid,pMMN497, was used to transform JH642 to generate ORB4075. ORB4075was generated by integration of pMMN497 at the amyE locus bydouble-crossover recombination and screening for the Amy^- (amylase-negative)phenotype. ORB4078 was constructed by transforming OEB4075 withchromosomal DNA prepared from LAB3834 (spx::neo). The insertionof the neo marker into spx located at the original locus andnot into Pspac-spx located at amyE was confirmed by testingthe genetic linkage between the chloramphenicol resistance (Cm^r;derived from pMMN497) and neomycin resistance (Neo^r) markers.ORB4079, ORB4080, and ORB4081 were constructed by transformingORB4078 with LAB2876 (clpX::spc), LAB2972 (clpP::erm), and ORB3976(clpC::tet) DNA, respectively.

B. subtilis cells were grown in rich media, 2x yeast extract-tryptone(YT) (29) and LB medium, Difco sporulation medium (DSM) (29),and one-step competence medium (CM) (8). Antibiotics were addedto the following concentrations: chloramphenicol, 5 µg/ml;erythromycin plus lincomycin, 1 and 25 µg/ml, respectively;tetracycline, 12.5 µg/ml; spectinomycin, 75 µg/ml;neomycin, 5 µg/ml; ampicillin, 25 µg/ml.

Purification of proteins. For production of proteins used in this study, the IMPACT system(New England BioLabs) (4), which utilizes the inducible self-cleavingintein tag, was used. Overproduction and purification of Spx(28) and ClpX (25) were previously described.

The clpP gene was amplified by PCR using primers oclpPex1 (5'-GGAGGAGCCATATGAATTTAAT-3')and oclpPex2 (5'-ATTAGGAAGAGCCCTTTTTGTCTTCTGTGTGA-3') and chromosomalDNA prepared from JH642 as the template. The PCR product digestedwith NdeI and SapI was cloned into pTYB1 (New England BioLabs),which had undergone digestion with the same enzymes. The resultantplasmid was digested with SapI, treated with T4 DNA polymerasein the presence of deoxynucleoside triphosphate to fill in thesingle-stranded end, and self-ligated to generate pClpP.

The clpC gene was amplified by PCR using primers oClpCex1 (5'-GGATGAATCCATATGATGTTTGGAAGATTT-3')and oClpCex2 (5'-TCCCCCGGGATTCGTTTTAGCAGTCGTTTT-3') and JH642chromosomal DNA as the template. The amplified fragment, afterbeing digested with NdeI and SmaI, was cloned into pTYB2 (NewEngland BioLabs) digested with the same enzymes to generatepClpC.

The mecA gene was amplified by PCR using JH642 chromosomal DNAand two primers, oMN01-156 (5'-GGAAGGTTGGCATATGGAAATTG-3') andoMN01-157 (5'-GACTTAGCTCTTCCGCATGATGCAAAGTGTTTT-3'). The amplifiedfragment, after being digested with NdeI and SapI, was insertedinto pTYB1 (digested with the same enzymes) to construct plasmidpSN3.

The ypbH gene was amplified by PCR using JH642 chromosomal DNAand two primers, oMN01-164 (5'-GAAGATCATATGCGGCTTGAGCGT-3')and oMN01-165 (5'-GCCGCCGCTCTTCCGCATGAAAAATGAGTTTGTA-3'). Theamplified fragment was inserted into pTYB1 by using the sameenzymes as those used for cloning the mecA gene to generatepSN5.

The sequences of all the genes cloned above were verified byDNA sequencing. The clpC gene in pClpC has a mutation in codon795; however, the amino acid (Asp) was not affected by the change,GAT to GAC. E. coli ER2566 (New England BioLabs) carrying pClpP,pClpC, or pSN3 was used to produce ClpP, ClpC, and MecA, respectively.E. coli BL21(DE3) carrying pLysS (Stratagene) and pSN5 was usedfor the production of YpbH. The proteins were purified by usinga chitin column as recommended by the manufacturer. PurifiedMecA and YpbH produced by this construct have no extra aminoacids. ClpP and ClpC have extra amino acids at their carboxyends (Gly-Ser-Ser-Tyr for ClpP and Pro-Gly for ClpC).

Purification of the Spx protein in the presence of CuSO_4. E. coli ER2566 carrying pMMN470 was cultured in 2x YT at 37°C.When the optical density at 600 nm reached 0.4, CuSO₄ was addedto the culture at the final concentration of 500 µM. Theprocedure for further purification was described previously(28).

In vitro degradation assay. An in vitro degradation assay was carried out as described previously(43) with slight modification. ClpP (4 µM) and Spx (4µM) were incubated at 37°C for 0 or 30 min in thepresence or absence of MecA (2.5 µM), YpbH (2.5 µM),ClpX (2.5 µM), and ClpC (2.5 µM) in a final volumeof 50 µl of reaction buffer (25 mM MOPS [morpholinepropanesulfonicacid]-KOH [pH 7.0], 100 mM KCl, 5 mM MgCl₂, 0.5 mM dithiothreitol,4 mM ATP, 2 mM phosphoenol pyruvate, 0.93 µM pyruvatekinase [Sigma]). Phosphoenol pyruvate and pyruvate kinase wereused to regenerate ATP. After the incubation, 10-µl portionsof the samples were transferred to 5 µl of stop solution.The proteins were analyzed on a 15% polyacrylamide-sodium dodecylsulfate (SDS) gel, followed by staining with Coomassie blue.

Western blot analysis. Cells were cultured in CM and harvested at T₂ (2 h after theonset of the stationary phase). Cells were broken by passagethrough a French press, and whole-cell extracts were used inWestern analysis or cell debris was first removed by centrifugation(17,000 x g for 15 min) to generate a cleared extract. The proteinconcentration of each sample was measured with a Bio-Rad proteinassay kit. Thirty micrograms of each protein was applied toa 15% polyacrylamide-SDS gel. After electrophoresis, the proteinswere transferred to a nitrocellulose membrane. Immunodetectionwas performed with the anti-Spx antibody (28) followed by incubationwith the secondary antibody conjugated to alkaline phosphatase.

Construction of a transcriptional spx-lacZ fusion. B. subtilis strain ORB3673, which has plasmid pPS34 insertedinto the yjbFG region, was constructed in a previous study (28).Chromosomal DNA prepared from ORB3673 was digested with ApaIand self-ligated. The ligation mixture was used to transformE. coli DH5 ${alpha}$ with selection for ampicillin resistance (Amp^r).Plasmid pMMN464, isolated from the Amp^r transformant, carries9.3 kbp of B. subtilis chromosomal DNA containing the 3' endsof oppB, oppCDE, yjbBCDE, and mecA and the 5' end of yjbF. pMMN464was cut with BglII, and the resulting 4.6-kbp fragment was recoveredfrom a low-melting-point agarose gel. The fragment was thendigested with BclI and HaeIII sequentially, which produces a1.4-kbp fragment containing the 3' end of yjbB, the entire yjbCgene, and the 5' end of spx (yjbD). The fragment was insertedinto promoter-probe vector pTKlac (17), which was digested withSmaI and BamHI to generate pSN16. JH642 was transformed withpSN16, and a Cm^r transformant was selected as ORB4059. ORB4065was isolated by transforming ORB4059 with chromosomal DNA preparedfrom LAB2876 (clpX::spc) with selection for Cm^r and Spc^r.

Assay of ß-galactosidase activity. Cells were grown in CM, DSM, or 2x YT, and samples were withdrawnat 30-min or 1-h time intervals during growth. ß-Galactosidaseactivity in each sample was determined as described previously(29) and is presented as Miller units.

RESULTS

Top

Results

Spx accumulates in clpP and clpX mutants but not in a clpC mutant. We have previously shown that Spx concentration was low in wild-typecells grown in CM but markedly higher in a clpP mutant (28).This suggests that Spx is a substrate of ClpP protease(s). InB. subtilis ATPase subunits ClpC, ClpX, and ClpE are known orthought to function with catalytic subunit ClpP (reviewed inreference 38). To identify which regulatory subunit is involvedin degradation of Spx, we first examined the amount of Spx ina clpX mutant. The result showed that Spx accumulated in theclpX mutant to a level similar to that observed in a clpP strain(Fig. 1A). In contrast, Spx concentration in a clpC mutant wasas low as that in the wild type (Fig. 1B). As was the case forthe clpC mutant, no increase in Spx concentration was observedin either the mecA or ypbH mutant. This result suggested thatdegradation of Spx is dependent primarily on ClpXP.

View larger version (18K):
[in this window]
[in a new window]
FIG. 1. Effect of mutations on Spx level as measured by Western blot analysis using anti-Spx antiserum. Samples were taken at T₂ (2 h after the onset of the stationary phase) from cells grown in CM. (A) Lane 1, molecular mass markers; lane 2, JH642 (wild type); lane 3, ORB3834 (spx::neo); lane 4, LAB2972 (clpP::erm); lane 5, LAB2876 (clpX::spc). (B) Lane 1, molecular mass markers; lane 2, JH642; lane 3, LAB2972; lane 4, ORB3976 (clpC::tet).

Spx is degraded in vitro by ClpCP but not ClpXP. Although the result described above suggests that Spx is a substrateof ClpXP, alternative interpretations, such as the possibilitythat a regulator of spx expression is a target of ClpXP protease,could not be excluded. To investigate whether Spx is degradedby ClpXP, in vitro proteolysis experiments using purified proteinswere carried out. We have previously found that Spx binds tocopper (30), and Fig. 2 shows the data from experiments usingcopper-bound Spx, prepared as described in Materials and Methods.However, a similar pattern of proteolysis was observed by usingthe copper-free Spx protein (data not shown). We also examinedthe effect of ClpCP protease on Spx degradation, since we observedthat Spx could form a complex with the ClpCP adapter, MecA,and could contribute to ComK inhibition (30). Degradation ofSpx was observed in the presence of ClpP, ClpC, and MecA (Fig.2, lanes 3 and 4). MecA, to a lesser extent, was also degradedby ClpCP, as previously reported (43). In the absence of MecA(lanes 1 and 2) or ATP (data not shown), Spx was stable afterincubation for 30 min. This indicates that MecA acts as theadapter for ClpCP-catalyzed proteolysis of Spx, as well as forMecA-dependent ComK and ComS proteolysis by ClpCP (43). Underthese same conditions, we observe that ComK is degraded by MecA-dependent,ClpCP-catalyzed proteolysis (data not shown), as was shown previously.YpbH is similar in amino acid sequence to MecA and is a possibleMecA paralog. Therefore we examined whether YpbH can act asthe adapter protein for ClpC. The result showed that Spx wasdegraded by ClpCP in the presence of YpbH (lanes 5 and 6), indicatingthat YpbH acts as the adapter for Spx proteolysis by ClpCP.We could not discern whether YpbH itself was degraded by ClpCPbecause YpbH and ClpP comigrated during SDS-polyacrylamide gelelectrophoresis. Surprisingly, Spx was not degraded in vitroin the presence of ClpX, ClpP, and ATP (lanes 7 and 8). Additionof MecA and YpbH did not promote ClpXP-dependent proteolysis(data not shown).

View larger version (37K):
[in this window]
[in a new window]
FIG. 2. Degradation of Spx in vitro. Spx and ClpP were incubated at 37°C in the presence of ATP and an ATP-generating system with (+) or without (-) ClpC, ClpX, MecA, and YpbH as described in Materials and Methods. Samples were taken at the times indicated and were analyzed by SDS-polyacrylamide gel electrophoresis, followed by staining with Coomassie blue. Lane M, molecular mass markers. PK, pyruvate kinase, used as an ATP-regenerating system.

Spx accumulates less in clpX and clpP cells grown in DSM than in cells grown in CM or 2x YT. Although Spx was degraded by ClpCP in vitro, Spx did not accumulatein the clpC mutant grown in CM. However, the clpC mutation mightaffect the concentration of Spx under different conditions.First we examined whether Spx accumulates in clpC cells culturedin DSM. The result showed that the level of Spx in the clpCstrain was as low as that in the wild type (data not shown).In addition, Spx accumulates in the clpX and clpP mutants, butat a level much lower than those in the same strains grown inCM (Fig. 3A) or 2x YT (data not shown). The band with a molecularweight lower than that of Spx, which may be a degradation productof Spx, was sometimes detected (as shown in lanes 2 and 3).

View larger version (25K):
[in this window]
[in a new window]
FIG. 3. Western blot analysis using anti-Spx antiserum. (A) Effect of culture media on Spx level as measured by Western blot analysis. Samples were taken at T₂ from cells grown in DSM and in CM. Lane 1, molecular mass markers; lane 2, LAB2876 (clpX::spc) grown in DSM; lane 3, LAB2972 (clpP::erm) grown in DSM; lane 4, LAB2876 grown in CM; lane 5, LAB2972 grown in CM. (B) Cells carrying Pspac-spx were grown in CM in the presence of 1 mM IPTG. Samples were taken at T₂. Lane 1, molecular mass markers; lane 2, ORB4078 (wild type); lane 3, ORB4081 (clpC::tet); lane 4, ORB4079 (clpX::spc); lane 5, ORB4080 (clpP::erm). (C) The wild type and the clpX mutant carrying Pspac-spx were grown in DSM or CM in the presence of 1 mM IPTG. Samples were taken at T₂. Lane 1, molecular mass markers; lane 2, ORB4078 in DSM; lane 3, ORB4078 in CM; lane 4, ORB4079 in DSM; lane 5, ORB4079 in CM.

The clpX mutation has no significant effect on spx transcription. That Spx is abundant in the clpX mutant whereas ClpXP was unableto degrade Spx in vitro might be explained if clpX negativelyregulates production of Spx at the level of spx transcription.To test this possibility, a transcriptional spx-lacZ fusionwas constructed as described in Materials and Methods and itsexpression in wild-type and clpX cells was examined. ß-Galactosidaseactivity from spx-lacZ was expressed constitutively in wild-typecells during exponential and stationary phases of growth inCM. Transcription of spx in the clpX mutant was slightly affectedby the clpX mutation (Fig. 4A). However, the increase in thespx transcription in the clpX mutant was two- to threefold atthe most, which cannot fully explain the large difference inSpx level between the wild type and the clpX mutant.

View larger version (17K):
[in this window]
[in a new window]
FIG. 4. Expression of spx-lacZ. (A) Cells were grown in CM. Time zero, onset of the stationary phase. ${circ}$ , ORB4059 (wild type); •, ORB4065 (clpX::spc). (B) ORB4059 (wild type) was grown in CM ( ${circ}$ ), DSM (•), or 2x YT ( ${square}$ ). ß-gal. act., ß-galactosidase activity.

We also examined whether the expression of spx is medium dependent.Levels of expression of spx when cells were grown in CM andDSM were similar, and the expression is higher in stationary-phasecultures grown in 2x YT (Fig. 4B). This result suggests thatthe smaller amount of Spx in the clpX and clpP mutant cellsgrown in DSM than in those grown in CM is attributable to posttranscriptionalregulation.

ClpXP is responsible for low levels of Spx in Pspac-spx cells. The results described above suggested that higher concentrationsof Spx in the clpX and clpP mutants are probably due to lossof proteolysis. To confirm this, spx was placed under the controlof IPTG-inducible promoter Pspac (50) and the effect of theclpX, clpP, and clpC mutations on the amount of Spx was examined.In these strains, Pspac-spx was integrated at the amyE locusand the original spx gene was mutated by insertion of a Neo^rgene cassette. When Pspac-spx strains with or without the clpmutations were grown in CM in the presence of 1 mM IPTG, Spxproteins were hardly detected in the wild type and the clpCmutant (Fig. 3B). In contrast, the amounts of Spx in the clpXand clpP mutant strains were dramatically increased. From theseresults we concluded that ClpXP is required for Spx degradation.The Spx level in the Pspac-spx clpX mutant was higher when cellswere grown in CM than when they were grown in DSM (Fig. 3C).However, the Spx concentration was significantly elevated evenin the DSM cultures in the absence of clpX. This result suggeststhat both ClpP and another protease(s) are responsible for thedegradation of Spx in B. subtilis cells grown in DSM.

DISCUSSION

Top

Discussion

Spx is highly conserved in gram-positive bacteria and was identifiedas the site of clpP suppressor mutations in Lactococcus lactis(9) and of clpX (and clpP) mutations in B. subtilis (28). Theaccumulated Spx in B. subtilis is harmful to cell growth oncertain media and to developmental pathways such as geneticcompetence and sporulation. This study indicated that the concentrationof Spx is controlled by multiple pathways of ClpP-dependentproteolysis.

Western blot analysis in a previous paper (28) and in this studyindicated that Spx markedly accumulated in the clpP and clpXmutants grown in CM or 2x YT. Using the Pspac-spx construct,we demonstrated that the amount of Spx is regulated either byClpXP-dependent proteolysis or, less likely, by an effect onspx translation that is indirectly influenced by ClpXP. Thepoor growth of the clpX and clpP mutants on minimal media (suchas CM) and rich media (such as LB medium and 2x YT) is causedby the increased amount of Spx under these growth conditions.In contrast, the clpX and clpP mutants grow relatively wellon DSM, where Spx accumulates to considerably lower levels.The growth of clpP spx and clpX spx mutants on CM and LB mediumis similar to that of the wild type. Furthermore the clpX andclpP mutants carrying the Pspac-spx construct grew well on CMand LB medium in the absence of IPTG; however, growth was severelyreduced in the presence of IPTG (data not shown). The growthof the Pspac-spx clpC strain was not affected by IPTG. Takentogether, these results are consistent with the hypothesis thataccumulation of Spx is unfavorable to normal cell growth. Italso indicates that the level of Spx in the clpP strain is nothigh enough to affect growth in DSM but is sufficient to inhibitsporulation because the spx mutation in the clpP backgroundonly partially restored sporulation (28). Our results also suggestedthat both ClpP and another protease(s) are involved in the degradationof Spx in DSM cultures.

Although Western blot analysis clearly showed that both clpXand clpP are responsible for maintaining low Spx concentrations,Spx is not degraded by ClpXP in vitro. In fact, at present,there is no experimental evidence demonstrating that B. subtilisClpXP is proteolytically active in vitro, although in vivo studieshave suggested various proteins as substrates. Comparison ofproteins by two-dimensional gel electrophoresis showed thatseveral proteins, such as GroEL, PpiB, PyrK, SucD, YhfP, YqkF,YugJ, and YvyD, were more abundant in clpP and clpX mutantsthan in the wild-type cells, suggesting that some of them mightbe the substrates for ClpXP (10). CtsR, the negative regulatorof class III heat-inducible genes such as clpP, clpE, and clpC,is degraded by ClpXP under nonstress conditions (6). Finallyevidence that SsrA-tagged proteins in B. subtilis are degradedby ClpXP was reported (48). We assume that the degradation ofSpx by ClpXP requires another protein, which may work as anadapter or a stimulator. In E. coli, ribosome-associated proteinSspB, by binding to SsrA-tagged proteins, enhances proteolysisby ClpXP but not by ClpAP (24). In the degradation of the stationary-phasesigma subunit of E. coli, RpoS, RssB acts as an adapter forClpXP-dependent RpoS proteolysis (3, 51). We are currently searchingfor a protein that may be involved in ClpXP-dependent proteolysisof Spx in B. subtilis.

The in vivo studies clearly indicated that ClpXP, not ClpCP,is responsible for maintaining low intracellular levels of Spx.In contrast, in vitro proteolysis experiments using purifiedproteins revealed that ClpCP, but not ClpXP, degraded Spx. Thedegradation of Spx by ClpCP requires adapter protein MecA orYpbH, a putative paralog of MecA. Although several ClpCP targetproteins were suggested by in vivo studies (see the introduction),only two cases have been analyzed in vitro. ComK and ComS arethe best-studied targets of ClpCP proteolysis in B. subtilis(43). The degradation of ComK and ComS by ClpCP also requiresMecA. The other substrate of ClpCP is CtsR. As mentioned above,CtsR is degraded by ClpXP under nonstress conditions; however,it becomes a target of ClpCP upon heat shock (21). In vitroproteolysis of CtsR by ClpCP was observed in the absence ofMecA. It has yet to be determined how MecA presents ComK, ComS,and Spx to ClpCP and how CtsR is directly recognized by ClpCP.There is a similar example from studies of ClpXP proteolysisin E. coli. The E. coli stationary sigma factor, ${sigma}$ ^s (39), and ${lambda}$ O (49) are substrates for ClpXP. Whereas the proteolysis of ${sigma}$ ^s requires response regulator RssB both in vivo and in vitro,the ClpXP-dependent proteolysis of ${lambda}$ O does not require RssB(51). Interestingly, RssB can also act like an anti- ${sigma}$ ^s factorwhen the cellular RssB/ ${sigma}$ ^s ratio is elevated and proteolysis isreduced (3). This is analogous to the ternary complex formedby MecA, ClpC, and ComK, which sequesters ComK. We have recentlyshown that Spx interacts with MecA, thus forming the quaternarycomplex and enhancing ComK binding to ClpC-MecA (30).

The contradictory in vitro and in vivo results of studies aimedat determining the role of ClpCP in Spx proteolysis are puzzling.One possibility is that the result of the in vitro proteolysisis an artifact. However, we think that this is unlikely becausethe proteolysis requires either MecA or YpbH. Another plausibleexplanation is that an unidentified protein is negatively involvedin ClpCP-dependent proteolysis of Spx in vivo. The protein couldbe an inhibitor that specifically binds to Spx and protectsit from proteolysis by ClpCP or could be the putative adapterfor ClpXP that binds to Spx. This interaction could render Spxresistant to proteolysis by ClpCP.

Although the increased amount of Spx is detrimental to cells,Spx is likely needed under certain conditions. The spx generesides downstream of yjbC, and these two genes probably constitutean operon (2). The transcription of the yjbC and spx genes wasinduced by heat, salt, and ethyl alcohol stress (36). A yjbCmutant and, to a lesser extent, an spx mutant exhibited a salt-sensitivephenotype (36). It is possible that Spx is produced under conditionsof extreme stress as one way to suppress developmental processesand allow the cell to devote its energy to dealing with thecellular damage caused by harsh conditions. It is apparent fromour studies and those of others that Spx is involved, positivelyand negatively, in various cellular functions in gram-positivebacteria.

ACKNOWLEDGMENTS

We thank Alan Grossman and Rich Losick for their kind gift ofa plasmid and a strain.

This work was supported by grant GM45898 from the National Institutesof Health.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, OGI School of Science & Engineering, Oregon Health & Science University, 20,000 NW Walker Rd., Beaverton, OR 97006-8921. Phone: (503) 748-7335. Fax: (503) 748-1464. E-mail: pzuber{at}bmb.ogi.edu.

REFERENCES

Top