involvement of rese phosphatase activity in down-regulation of

JOURNAL OF BACTERIOLOGY,0021-9193/01/$04.0010 DOI: 10.1128/JB.183.6.1938–1944.2001

Mar. 2001, p. 1938–1944 Vol. 183, No. 6

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Involvement of ResE Phosphatase Activity in Down-Regulationof ResD-Controlled Genes in Bacillus subtilis during

Aerobic GrowthMICHIKO M. NAKANO* AND YI ZHU

Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology,Beaverton, Oregon 97006

Received 15 September 2000/Accepted 20 December 2000

The ResD-ResE signal transduction system is required for aerobic and anaerobic respiration in Bacillussubtilis. The histidine sensor kinase ResE, by functioning as a kinase and a phosphatase for the cognateresponse regulator ResD, controls the level of phosphorylated ResD. A high level of phosphorylated ResD ispostulated to cause a dramatic increase in transcription of ResDE-controlled genes under anaerobic condi-tions. A mutant ResE, which retains autophosphorylation and ResD phosphorylation activities but is defectivein ResD dephosphorylation, allowed partially derepressed aerobic expression of the ResDE-controlled genes.The result indicates that phosphatase activity of ResE is regulated by oxygen availability and anaerobicinduction of the ResDE regulon is partly due to a reduction of the ResE phosphatase activity during anaer-obiosis. That elimination of phosphatase activity does not result in complete aerobic derepression suggests thatthe ResE kinase activity is also subject to control in response to oxygen limitation.

In two-component signal transduction systems (for reviews,see references 9 and 29) histidine kinases modulate the activityof response regulators via phosphorylation. Response regula-tors have autophosphatase activity, and half-lives for variousphosphorylated response regulators range from seconds tohours (36). The decay of phosphorylated response regulators isoften stimulated by the cognate sensor kinases that possessphosphatase activity (for a review, see reference 30). Since thelevel of phosphorylation of response regulators is determinedby the sum of kinase, phosphotransferase, and phosphataseactivities, how each of these activities is regulated is a key issuefor understanding the mechanism of signal transduction.

Bacillus subtilis, a gram-positive soil bacterium, can alternateits respiratory systems depending on the growth conditions.When nitrate is present in the absence of oxygen, cells undergonitrate respiration using nitrate reductase as terminal oxidase(for a review, see references 18 and 23). Anaerobic nitraterespiration, as well as aerobic respiration using cytochromeoxidases, is dependent on the ResDE signal transduction sys-tem (24, 31). The sensor kinase ResE and the response regu-lator ResD are required for transcription of resABCDE(resABC encodes proteins similar to those involved in cyto-chrome c biogenesis) (31), ctaA (required for cytochrome caa3

oxidase biosynthesis) (31), ctaB (cytochrome caa3 oxidase as-sembly factor) (13), qcrABC (encoding subunits of menaquin-ol:cytochrome c oxidoreductase) (31), fnr (encoding anaerobictranscriptional regulator) (24), nasDEF (nitrite reductase)(17), hmp (flavohemoglobin) (12), lctE (lactate dehydroge-nase) (4), and sbo-alb (subtilosin biosynthesis) (20). AllResDE-controlled genes so far tested are highly induced byoxygen limitation (21). Recent studies showed that purified

ResD directly interacts with promoter regions of some of thesegenes (22, 37).

We have previously shown that pgk-1, a mutation in pgk(phosphoglycerate kinase gene), suppresses resE but not resDmutations with respect to anaerobic growth in the presence ofnitrate and to ResDE-dependent gene expression (21). Thepgk-1 mutant displays very low but measurable phosphoglycer-ate kinase activity compared to the wild-type strain. Accumu-lation of a glycolytic intermediate, probably 1,3-diphosphoglyc-erate, was suggested to be responsible for the observedsuppressor effect of pgk-1. However, it remains to be examinedwhether 1,3-diphosphoglycerate can donate phosphate directlyto ResD or if a non-cognate kinase is involved in the ResE-independent ResD phosphorylation. During the study wefound that aerobic expression of the ResDE-controlled geneswas dramatically derepressed in the resE pgk-1 double mutant;however, the expression in the resE1 pgk-1 strain was similar tothat of the wild type, showing much lower expression underaerobic than anaerobic conditions. One possible explanationfor these results is that ResE has both kinase and phosphataseactivities under aerobic conditions but lacks phosphatase ac-tivity under anaerobic conditions. In this view, when ResD isphosphorylated by a pathway independent of ResE, and ResDphosphate (ResD;P) is not dephosphorylated by ResE phos-phatase, as in case of the resE pgk-1 mutant, a higher level ofResD;P would be attained, leading to robust activation ofResD-controlled genes. We also proposed that higher expres-sion of ResDE-controlled genes in the wild-type strain underanaerobic conditions is likely the result of higher ResD;P dueto reduced phosphatase activity of ResE (21). This hypothesiswas tested in this study by creating a mutant ResE that pos-sesses kinase activity but lacks phosphatase activity.

MATERIALS AND METHODS

B. subtilis strains and plasmids. B. subtilis strains and plasmids used in thisstudy are listed in Table 1.

* Corresponding author. Mailing address: Department of Biochem-istry and Molecular Biology, Oregon Graduate Institute of Science andTechnology, 20000 N.W. Walker Road, Beaverton, OR 97006. Phone:(503) 748-4078. Fax: (503) 748-1464. E-mail: [email protected].

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Construction of plasmids carrying truncated wild-type resE or resE378 gene.For production of wild-type and mutant ResE proteins, the IMPACT system(New England BioLabs) was used which utilizes the inducible self-cleaving inteintag (3). Plasmid pMMN424, which carries a truncated resE gene in pTYB4, wasconstructed previously (22). This plasmid was used to produce and purify asoluble form of the wild-type ResE protein lacking the N-terminal 195 aminoacids (total amino acids are 589) including two transmembrane regions and theperiplasmic region. The truncated protein was shown to have active kinaseactivity (22, 37). The last amino acid, Arg, is also replaced by Gly and Pro inwild-type and mutant ResE constructs.

Plasmid pMMN425, which was used to purify a truncated form of the mutantResE (T378R) protein, was constructed as follows. The upstream fragment ofresE was amplified by PCR using JH642 chromosomal DNA and two oligonu-cleotides, oMN98-47 (59-CATTCTTTTTATCAACCATGGTCACGTACCCT-39) and oMN98-49 (59GTCATGAGCTGAGACGACCGATCTCCAT-39). Thedownstream fragment of resE was amplified using two oligonucleotides,oMN98-48 (59-CAGACTCGATTTTACCCGGGTTTTGTCGGAATAT-39) andoMN98-50 (59-ATGGAGATCGGTCGTCTCAGCTCATGAC-39). oMN98-49and oMN98-50 are complementary and designed to generate the resE378 muta-tion. The PCR products were denatured at 94°C for 1 min and were successivelyincubated at 65°C for 2 min and 37°C for 1 min. The annealed mixture wastreated with T4 DNA polymerase in the presence of four deoxynucleosidetriphosphates at 37°C for 30 min. The aliquot of the reaction mixture was usedas a template for PCR using oMN98-47 and oMN98-48 to generate the truncatedresE378 gene. The PCR product was digested with NcoI and SmaI and clonedinto pTYB4, which was cleaved with the same enzymes to generate pMMN425.The inserted DNA was sequenced to verify the desired mutation, as well as theabsence of any extra mutation.

The truncated form of wild-type ResE was also produced as a protein fused tosix-histidine residues (His6). This His6-ResE protein was used to purify ResD;Pfrom His6-ResE by affinity chromatography. Plasmid pMMN424 was digestedwith SmaI and NcoI (blunt ended with T4 DNA polymerase), and the releasedresE fragment was cloned into pUC19 digested with SmaI. The resultant plasmidpMMN444 was digested with BamHI and KpnI to release resE, which wassubcloned into pPROEX-1 (GIBCO-BRL) that had been digested with the sameenzymes to generate pMMN446.

Purification of ResD and ResE proteins. ResD, wild-type, and mutant ResEproteins were overproduced in Escherichia coli ER2566 (New England Biolabs)as described previously (22, 37). His6-ResE was overproduced in E. coli BL21carrying pMMN446 and purified using Ni-nitrilotriacetic acid (NTA) resin col-umn chromatography as recommended by the manufacturer. The His6-ResEprotein has 44 extra amino acid residues including 6 histidines at the N terminusand 18 extra amino acids at the C terminus of ResE.

Autophosphorylation of ResE. The truncated wild-type and mutant ResEproteins (60 pmoles) were incubated in 60 ml of TEDG buffer (50 mM Tris-HCl,pH 8.0; 0.5 mM EDTA; 2 mM dithiothreitol [DTT]; 10% glycerol) containing 50mM KCl, 5 mM MgCl2, and 10 mM [g-32P]ATP (1 Ci/mmol). After incubationfor the indicated periods at room temperature, 10 ml of the reaction mixture wasremoved and added to 3 ml of a sodium dodecyl sulfate (SDS) sample buffer (250mM Tris-HCl, pH 6.5; 8% SDS; 8% 2-mercaptoethanol; 40% glycerol; 0.05%bromophenol blue). The proteins were separated by SDS–12% polyacrylamidegel electrophoresis and analyzed using a PhosphorImager (Molecular Dynam-ics).

Phosphorylation of ResD by ResE. The wild-type and mutant ResE proteins(960 pmol) were autophosphorylated with [g-32P]ATP for 30 min at room tem-perature as described above. The reaction mixture was applied to a SephadexG-75 column equilibrated with the same buffer. The fractions containing theradioactive ResE, which were free of ATP, were collected. An aliquot of thefractions was incubated with ResD (300 pmol) in 92 ml of TEDG phosphoryla-tion buffer, and ATP was added to 200 mM after 5 min.

Dephosphorylation of ResD;P. The His6-ResE protein (0.8 to 1.5 nmol) wasautophosphorylated with [g-32P]ATP as described above, except that DTT in thebuffer was replaced by 5 mM 2-mercaptoethanol because DTT reduces the Niions of the Ni-NTA resin used for immobilization of His6-ResE. After 30 min atroom temperature, Ni-NTA agarose was mixed into the reaction mixture andincubated by gently shaking for 15 min. The Ni-NTA resin was collected bycentrifugation and washed with the same buffer to remove unbound His6-ResEand unincorporated ATP until the radioactive signal in the wash buffer becameconstant. An equal amount of ResD was added to the resin and the mixture wasincubated for 10 min at room temperature. The reaction mixture was centri-fuged, and the supernatant containing ResD;P was collected, which was thenapplied to a Sephadex G-25 column. For the examination of autophosphatase

TABLE 1. B. subtilis strains and plasmids

Strain or plasmid Relevant feature(s) Source or reference

StrainsJH642 trpC2 pheA1 J. A. HochZB307A SPbc2D2::Tn917::pSK1 39LAB2000 trpC2 pheA1 SPbc2D2::Tn917::pML26 12LAB2234 trpC2 pheA1 DresE::spc 16LAB2252 trpC2 pheA1 SPbc2D2::Tn917::pMMN288 24LAB2537 trpC2 pheA1 amyE::pES17 (resA-lacZ) 21LAB2854 trpC2 pheA1 SPbc2D2::Tn917::pMMN392 17LAB3162 trpC2 pheA1 SPbc2D2::Tn917::sbo-lacZ 38ORB3303 resE::neo This studyORB3304 trpC2 pheA1 resE::neo This studyORB3331 trpC2 pheA1 amyE::pES17 resE::neo This studyORB3343 trpC2 pheA1 resE378 amyE::pES17 This studyORB3362 trpC2 resE378 This studyORB3370 trpC2 resE378 SPbc2D2::Tn917::pML26 This studyORB3371 trpC2 resE378 SPbc2D2::Tn917::pMMN392 This studyORB3372 trpC2 resE378 SPbc2D2::Tn917::pMMN288 This studyORB3373 trpC2 resE378 SPbc2D2::Tn917::sbo-lacZ This study

PlasmidspPROEX-1 ColE1 origin with His6, Ampr GIBCO-BRLpTYB4 ColE1 origin with intein tag, Ampr New England BiolabspUC18 ColE1 origin, Ampr 35pUC19 ColE1 origin, Ampr 35pMMN424 pTYB4 with resE, Ampr 22pMMN425 pTYB4 with resE378, Ampr This studypMMN444 pUC19 with resE, Ampr This studypMMN446 pPROEX-1 with resE, Ampr This studypYZ16 pUC18 with resE, Ampr This studypYZ24 pYZ16 with neo insertion in resE, Ampr Neor This study

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activity, ResD;P was incubated in the buffer with or without ATP (500 mM) atroom temperature. The purified ResD;P was also incubated with wild-type andmutant ResE (300 to 500 pmol) in the presence or absence of 500 mM ATP orADP for 10 min.

Construction of B. subtilis strains carrying the resE378 mutation. The mutantresE allele was introduced into B. subtilis as follows. Two fragments carrying the59-part and 39-part of resE were amplified by using JH642 chromosomal DNAand oligonucleotides oMN98-47 and oMN99-57 (59-CTGAAGCATGGGGATCCGTGTTCTCAG-39), as well as oMN98-48 and oMN99-56 (59-CTGAGAACACGGATCCCCATGCTTCAG-39). Two complementary oligonucleotides,oMN99-56 and oMN99-57, were designed to create a BamHI site in the resEgene. The PCR products, after annealing and being treated with T4 DNApolymerase as described above, were used as template for the second PCRreaction using oMN98-47 and oMN98-48. The PCR product digested with SmaIand NcoI (the end was filled in) was cloned into pUC18 digested with SmaI andHincII to generate pYZ16. A neomycin-resistant (Neor) cassette isolated frompDZ792 (8) digested with BamHI and BglII was inserted into the BamHI site ofpYZ16 to generate pYZ24. B. subtilis strains JH642 (trpC2 pheA1) and ZB307A(trpC21 pheA11) were transformed with pYZ24 that was linearized by ScaIcleavage, and Neor transformants were selected as ORB3304 and ORB3303,respectively. The transformants were generated by a double-crossover recombi-nation, as was confirmed by PCR analysis. LAB2537 carrying resA-lacZ wastransformed with ORB3304 chromosomal DNA and a chloramphenicol-resistant(Cmr) Neor transformant was chosen as ORB3331. ORB3304 was transformedwith ORB3331 chromosomal DNA and pMMN425 with selection for Cmr. ANeos Cmr transformant was chosen as ORB3343. Because ORB3331, likeORB3304, has the Neor cassette in resE, the neomycin sensitivity of ORB3343 isindicative of the replacement of resE::neo by the mutant allele of resE inpMMN425. This was further confirmed by sequencing the PCR product obtainedby using ORB3343 chromosomal DNA as a template and oMN98-47 andoMN98-48 as primers. The mutant resE strain without the lacZ fusion wasconstructed by transforming ORB3343 with ORB3303 chromosomal DNA. Afterselection with trp1, a Cms Neos transformant was chosen as ORB3362. ORB3362was used for transduction with phage lysates carrying hmp-lacZ (12), nasD-lacZ(17), fnr-lacZ (24), and sbo-lacZ (38) to construct strains ORB3370, ORB3371,ORB3372, and ORB3373, respectively.

Measurement of b-galactosidase activity. B. subtilis cells were grown in liquid2xYT medium (19) with 1% glucose and 0.2% KNO3 or in DS medium (19) with1% glucose and 0.2% KNO3 (starting optical density at 600 nm was 0.02). Cellswere cultured aerobically or anaerobically as previously described (24), andsamples were taken every 1 h to measure b-galactosidase activity as describedearlier (15). The maximal activity, which was attained at late exponential growth,was listed in Table 2.

Western blot analysis. Cells were grown as above until late exponential growthfor anaerobic cultures or T2 (2 h after the onset of the stationary phase) in thecase of aerobic cultures. After disruption by French press, cell debris was re-moved by centrifugation (17,000 3 g) for 15 min. The protein concentration ineach sample was determined by using the Bio-Rad assay kit. A total of 20 mg ofeach protein sample was loaded onto SDS–12% polyacrylamide gels. The pro-teins were detected by Western blot using a chromogenic alkaline phosphatasesubstrate, anti-ResE antibody (raised against purified ResE by Josman, LLC,Napa, Calif.), and secondary goat anti-rabbit alkaline phosphatase conjugate.

RESULTS AND DISCUSSION

Construction of a mutant ResE (T378R). The question ofhow the ResDE signal transduction pathway specifically acti-vates either aerobic or anaerobic respiration was addressed inthis study. Because ResD and ResE are needed both for aer-obic and anaerobic respiration and yet these genes are highlyinduced under anaerobic conditions in a ResDE-dependentmanner, several possibilities could be envisioned. One possi-bility is the presence of an unknown regulator that would onlybe active under anaerobic conditions. This putative regulatormay be controlled positively by the ResDE system or it may bea coactivator of ResD when oxygen is limited. Recent studiesindicated that ResD binds to the promoter regions of ctaA,resA, hmp, nasD, and fnr, suggesting that ResD activates tran-scription of these genes by interacting with their regulatory

regions (22, 37), arguing against the existence of a coactivatoror a ResD-controlled transcription activator. These results,together with studies on a resE suppressor mutant as describedabove (21), support the conclusion that the level of phosphor-ylation of ResD is the major factor for determining the induc-tion of these genes.

One possibility for why ResD is phosphorylated to higherlevels during anaerobic growth than during aerobic growth isreduced phosphatase activity of ResE under anaerobic condi-tions. This hypothesis was tested in this study by constructingkinase1 phosphatase2 ResE and examining the expression ofResDE-controlled genes in the strain producing the mutantResE. If the hypothesis is correct, one could expect dere-pressed aerobic ResDE-dependent gene expression in thestrain carrying such a mutant ResE. ResE belongs to the EnvZsubfamily of sensor kinases (6), and kinase1 phosphatase2

EnvZ mutants have been isolated (listed in reference 10). Onesuch mutant (envZ11) has an amino acid change (Thr247 toArg) in the vicinity of the conserved His243 residue (the site forautophosphorylation) (2). Interestingly, an E. coli sensor ki-nase CpxA with a change of the equivalent residue Thr to Prodisplays gain-of-function phenotype. The CpxA101 mutantalso lacks phosphatase activity for CpxR;P (25). Because thecorresponding residue (Thr378) is conserved in ResE, wechanged the Thr residue to Arg by site-directed mutagenesisusing PCR as described in Materials and Methods. To inves-tigate the biochemical activities of wild-type and mutant ResE,the soluble truncated ResE proteins were purified.

Autophosphorylation activities of wild-type and mutantResE. ResE, as with other sensor kinases, can undergo auto-phosphorylation in the presence of ATP (22, 37). Purifiedwild-type and mutant (T378R) ResE proteins were examinedfor autophosphorylation activity. The time course of incorpo-ration of the phosphoryl group from [g-32P]ATP into ResE is

TABLE 2. Aerobic and anaerobic expression of ResDE-controlledgenes

Fusion

b-Galactosidase activity (Miller unit)a in:

DSM 2xYT

Aerobic Anaerobic Aerobic Anaerobic

hmp-lacZWild type 1.7 6,480 3.5 6,550Mutant 18 8,170 47 5,390

nasD-lacZWild type 4.9 540 2.5 546Mutant 45 539 16 491

fnr-lacZWild type 5.5 78 6.6 199Mutant 47 133 42 224

resA-lacZWild type 22 377 16 430Mutant 227 593 199 605

sbo-lacZWild type 19 2,720 3.8 2,450Mutant 440 5,370 66 3,140

a Maximal activities during growth are shown. Standard deviations are lessthan 20% for each value.

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shown in Fig. 1. Both proteins have autophosphorylation ac-tivity, although the wild-type ResE was phosphorylated at ahigher rate than the mutant. EnvZ11 (2) and CpxA101 (25),carrying the corresponding mutation, exhibited increased ordiminished autophosphorylation activity, respectively. This re-sult indicates that the T378R mutation moderately affects theautophosphorylation activity of ResE.

Phosphorylation and dephosphorylation of ResD by ResEproteins. To examine transphosphorylation that is indepen-dent of the autophosphorylation reaction, wild-type and mu-tant ResE;P proteins free of ATP were purified by gel filtra-tion as described in Materials and Methods. The incubation ofResE;P with ResD resulted in phosphorylation of ResD (Fig.2). Phosphorylation of ResD either by wild-type or mutant

ResE occurred quickly and reached a maximum level within0.5 min. EnvZ catalyzes the dephosphorylation of OmpR;P inthe presence of ATP, ADP, or nonhydrolyzable analogs ofATP (1, 11). The addition of ATP to the reaction mixture alsostimulated the dephosphorylation of ResD;P in the reactioncontaining wild-type ResE, as shown by the absence of radio-labeled ResD and ResE after incubation for 5 min at roomtemperature (Fig. 2A and C). In contrast, the mutant ResE didnot stimulate ResD;P dephosphorylation by the addition ofATP (Fig. 2B and D). This result indicates that ResE functionsas a phosphatase for ResD;P, which is defective in the case ofthe T378R mutant.

It has been shown that some two-component regulatory pro-teins dephosphorylate response regulators in two differentways: through the phosphatase activity of sensor kinases, whichreleases Pi; and through reverse transphosphorylation, whichinvolves transfer of the phosphoryl group from response reg-ulators to sensor kinases. Reverse transphosphorylation hasbeen reported in several two-component regulatory systems,including NRII-NRI (33), CheA-CheY (28), a kinase2 phos-phatase1 EnvZ mutant (5), ArcB-ArcA (7), and PhoR-PhoP inB. subtilis (27). In an attempt to determine by which processResD is dephosphorylated, ResD;P was purified fromResE;P by using the His6-ResE construct and Ni-chelatechromatography (Materials and Methods).

Response regulators have autophosphatase activities, and akey residue in determining the magnitude of the activity isamino acid position 56 (in Spo0F), which is adjacent to the siteof phosphorylation of Asp54 (36). Response regulators con-taining an amino acid residue with a long side chain at theposition equivalent to 56 in Spo0F displayed a low autodephos-phorylation rate, and those carrying a residue with carboxy-amide or carboxylate side chain at that position had high de-phosphorylation rates (36). The corresponding amino acid inResD is Met, the same residue present in PhoB, OmpR, andVanR, which are known to exhibit inefficient autophosphataseactivity (36). Consistent with this observation, our resultshowed that autophosphatase activity of ResD is relativelyweak, and the half-life of ResD;P was calculated to be ca. 4 h(Fig. 3A and B). Addition of ATP did not show any significanteffect on autophosphatase activity (data not shown). This longhalf-life of ResD;P may explain why the phosphatase activityof ResE is regulated by oxygen tension. When oxygen concen-tration is increased, the cells need to rapidly decrease the levelof ResD;P by activating ResE phosphatase. A similar possi-bility was suggested in the case of the FixLJ system, whereFixJ;P has a relatively long half-life (4 h) (14).

In the presence of ATP or ADP, wild-type ResE stimulatesthe dephosphorylation of ResD (Fig. 3C). In contrast, in thepresence of the mutant ResE and ATP, reverse transphospho-rylation was observed. Reverse transphosphorylation was alsodetected in wild-type ResE if EDTA was present. This is insharp contrast to the phosphatase activity of the sensor kinases,which requires Mg21 and was inhibited by the presence ofEDTA. The reverse transphosphorylation from PhoP;P toPhoR does not require Mg21 (27) as in the case of the reactionfrom ResD;P to ResE.

Effect of the resE378 mutation on transcription of ResDE-controlled genes. The results using purified ResE proteins in-dicate that, unlike the wild-type ResE, the mutant ResE lacks

FIG. 1. Time course of autophosphorylation of ResE. Wild-type(A) and mutant (B) ResE proteins (60 pmol) were incubated at roomtemperature in 60 ml of TEDG buffer (50 mM Tris-HCl, pH 8.0; 0.5mM EDTA; 2 mM DTT; 10% glycerol) containing 50 mM KCl, 5 mMMgCl2 and 10 mM [g-32P]ATP (1 Ci/mmol). At the indicated times,10-ml samples were taken and analyzed by SDS–12% polyacrylamidegel electrophoresis and autoradiography. (C) Densitometry scanningof the gels.



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phosphatase activity, which could result in higher levels ofResD;P. Therefore, we examined whether the resE378 muta-tion affects ResDE-controlled gene expression in vivo. TheresE gene was replaced by the mutant allele as described inMaterials and Methods. The concentration of wild-type andmutant ResE proteins in aerobic and anaerobic cultures wasexamined by Western analysis using anti-ResE antibody (Fig.4). Higher levels of ResE proteins were detected in the wild-type ResE strain grown in 2xYT medium under anaerobicconditions (360%) than under aerobic conditions (100%). Incontrast, the level of mutant ResE was similar both in aerobic(450%) and anaerobic cultures (450%) and was as high as thelevel of the wild-type ResE protein under anaerobic condi-tions. The levels of ResE protein in the cells grown in DSmedium were as follows: aerobic wild-type cultures, 100%;anaerobic wild-type cultures, 410%; aerobic mutant cultures,200%; and anaerobic mutant cultures, 260% (data not shown).This indicates that the mutant ResE is indeed produced invivo. The higher mutant ResE concentration compared to thatof the wild type during aerobic growth probably reflects theautoregulation of the resE gene because it is transcribed pri-marily from the resA operon promoter which is dependent onResDE (31).

Various lacZ fusions of ResDE-controlled promoters wereintroduced in the wild-type and mutant strains. Expression ofthe fusions in both the strains grown aerobically and anaero-bically in 2xYT medium or in DS medium was examined (Ta-

ble 2). Aerobic expression of all genes was partly derepressedin the mutant cells grown in 2xYT or DS medium. AerobicnasD or fnr expression was six- to ninefold higher in the mutantthan in the wild-type ResE strain. In the case of hmp and resAexpression, aerobic expression was derepressed by 10- to 13-fold in the mutant strain. The mutation has a more drasticeffect on aerobic sbo expression, which resulted in a 17- to23-fold increase. In contrast, the anaerobic expression of thesegenes was either not affected at all or only slightly increased(up to twofold) by the mutation.

This result indicates that the resE mutant defective in phos-phatase activity leads to the partial derepression of theResDE-controlled genes under aerobic growth conditions.However, aerobic expression in the mutant strain is still lowerthan anaerobic expression, unlike the situation in the resEpgk-1 mutant, which showed complete derepression (21). Onepossible explanation of the difference is that ResD is phos-phorylated independently of ResE kinase in the resE pgk-1mutant, while ResE phosphatase activity is absent, resulting inhigher ResD;P levels than those in the resE378 mutant strain,which has reduced autokinase activity as shown in Fig. 1. Analternative, but not exclusive possibility, is that the kinase ac-tivity of ResE, like the phosphatase activity, is also regulated byoxygen limitation. In aerobic cultures of the resE378 strainwhich lacks phosphatase activity, the level of ResD;P is highenough to support a 6- to 20-fold induction compared to cul-tures of wild-type cells; however, the phosphorylation level

FIG. 2. Autophosphorylation and dephosphorylation of ResD. 32P-phosphorylated ResE (A) and ResE (T378R) (B) (960 pmol) were purifiedand then incubated with purified ResD (300 pmol) in 92 ml of TEDG phosphorylation buffer. After incubation for 0.5, 1, 2, and 5 min at roomtemperature, 10 ml of the reaction was transferred to the SDS buffer. At 5 min, ATP was added to 200 mM, and the reaction was continued for0.5, 1, 2, and 5 min. (C and D) Densitometry scanning of the gels in panels A and B, respectively. Symbols: E, ResE;P; F, ResD;P.



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could still be lower compared to anaerobic cultures, the cells ofwhich not only lack the phosphatase activity but might alsohave elevated kinase activity. Autophosphorylation activity ofthe sensor kinase FixL of Rhizobium meliloti is stimulated bylow oxygen tension, and the phosphatase activity of FixL;P(but not that of FixL) is depressed under anaerobic conditions(14). The ResDE regulon could be reciprocally regulated viakinase and phosphatase activity of ResE according to changesin the oxygen level, such as the expression of nitrogen fixationgenes in R. meliloti.

ResE is a membrane-associated protein with a type P linkerregion (periplasmic signal transducing), which is defined by thepresence of two amphipathic a-helices (AS1 and AS2) (34).The mechanism of signal transduction in this class of sensorswas proposed to involve a conformational change of theperiplasmic region brought about by binding to a signal ligand(34). The change in conformation is relayed through the cyto-

plasmic membrane and causes realignment of the two heliceswithin the linker region, which, in turn, alters the function ofthe C-terminal cytoplasmic domain. It remains to be deter-mined if the periplasmic region of ResE functions as the sig-nal-sensing domain and what the signal for ResE is that affectskinase and/or phosphatase activity. Interestingly, a PAS do-main, which is known to be an important signaling module forsensing changes in light, redox potential, and oxygen (32), wasidentified in a region adjacent to AS2 (SMART:http://smart.embl-heidelberg.de/ [26]). The involvement of the PAS do-main in the redox sensing of ResE remains to be examined.Future studies are also needed to determine whether the ki-nase and the phosphatase activities are affected by the samesignal or whether each activity is modulated by distinct signals.

ACKNOWLEDGMENTS

We are grateful to Peter Zuber for valuable discussions and criticalreading of the manuscript. We also thank F. Marion Hulett and LindaKenney for helpful advice.

This work was supported by NSF grant MCB9996014.

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FIG. 4. Western analysis of ResE. B. subtilis cells were grown aer-obically or anaerobically in 2xYT with 1% glucose and 0.2% KNO3. Atotal of 20 mg of each protein sample was separated by SDS–12%polyacrylamide gel electrophoresis. After electrophoresis, the proteinswere electrotransferred to a nitrocellulose filter and probed with anti-ResE antibody. Lanes: M, marker (79.0 kDa); 1, JH642 (wild type)grown aerobically; 2, JH642 grown anaerobically; 3, ORB3362(resE378) grown aerobically; 4, ORB3362 grown anaerobically; 5,LAB2234 (DresE) grown aerobically; 6, LAB2234 grown anaerobically.A cross-reacting band is detected both in resE1 and resE strains.

FIG. 3. (A and B) Time course of ResD;P dephosphorylation. (A)Purified ResD;P was incubated in TEDG buffer containing 50 mMKCl and 5 mM MgCl2. Samples were transferred at the indicated timesto the SDS buffer and subjected to electrophoresis followed by auto-radiography. Panel B shows densitometry scanning results of the gelshown in panel A. (C) Dephosphorylation of ResD;P by ResE. Pu-rified ResD;P was incubated at room temperature for 10 min in thesame buffer in the absence (2) or in the presence of ResE (wt) orResE (T378R) (m) proteins. ATP and ADP were added to 500 mM,and EDTA was added to 10 mM when indicated.



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involvement of rese phosphatase activity in down-regulation of

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