Journal of Bacteriology, March 2004, p . 1493-1502, Vol . 186, No . 5

Residues Required for Bacillus subtilis PhoP DNA Binding or RNA Polymerase Interaction: Alanine Scanning of PhoP Effector Domain Transactivation Loop and Helix 3

Yinghua Chen,¹ Wael R . Abdel-Fattah,¹ and F . Marion Hulett^1*

Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607¹

Received 22 September 2003/ Accepted 18 November 2003

 
Bacillus subtilis PhoP is a member of the OmpR family of response regulators that activates or represses genes of the Pho regulon upon phosphorylation by PhoR in response to phosphate deficiency. Because PhoP binds DNA and is a dimer in solution independentof its phosphorylation state, phosphorylation of PhoP may optimizeDNA binding or the interaction with RNA polymerase . We describealanine scanning mutagenesis of the PhoP loop and helix 3region of PhoPC [Val190 to E214] and functional analysis of the mutated proteins . Eight residues important for DNA binding were clustered between Val202 and Arg210 . Using in vivo andin vitro functional analyses, we identified three classes ofmutated proteins . Class I proteins [PhoP_I206A, PhoP_R210A, PhoP_L209A, and PhoP_H208A] were phosphorylation proficient and could dimerizebut could not bind DNA or activate transcription in vivo or in vitro . Class II proteins [PhoP_H205A and PhoP_V204A] were phosphorylationproficient and could dimerize but could not bind DNA prior tophosphorylation . Members of this class had higher transcriptionactivation in vitro than in vivo . The class III mutants, PhoP_V202Aand PhoP_D203A, had a reduced rate of phosphotransfer and coulddimerize but could not bind DNA or activate transcription invivo or in vitro . Seven alanine substitutions in PhoP [PhoP_V190A,PhoP_W191A, PhoP_Y193A, PhoP_F195A, PhoP_G197A, PhoP_T199A, and PhoP_R200A]that specifically affected transcription activation were broadlydistributed throughout the transactivation loop extending fromVal190 to as far toward the C terminus as Arg200 . PhoP_W191Aand PhoP_R200A could not activate transcription, while the otherfive mutant proteins showed decreased transcription activationin vivo or in vitro or both . The mutagenesis studies may indicatethat PhoP has a long transactivation loop and a short helix3, more similar to OmpR than to PhoB of Escherichia coli.

 
In Bacillus subtilis the response to phosphate deprivation is controlled by activation of genes previously silent and repressionof other genes whose products are antithetical to cell survivalin phosphate-limiting conditions . At least two global systemsare responsible for these changes, sigma B [3] and PhoP-PhoR. PhoP and PhoR comprise a prototypical two-component system in that the transmembrane sensor histidine kinase, PhoR, is autophosphorylated on a conserved histidine residue in response to a signal . The phosphoryl group is then transferred to a conserved aspartateresidue in the N-terminal domain of the response regulator,PhoP, thus activating PhoP for transcriptional regulation ofPho regulon genes . PhoPP is known to directly regulate 31genesin seven operons [14] and is directly or indirectly requiredfor eight additional genes [23, 24] . Among the genes that areactivated or repressed by PhoPP are the genes that encode ahigh-affinity phosphate ABC transporter, phosphatases [PhoA,PhoB, and PhoD], and biosynthetic proteins for essential anioniccell wall polymers [teichoic acid or teichuronic acid].

PhoP is a member of the winged helix OmpR subfamily of response regulators based on sequence similarities of the C-terminaleffector domains . The 240-amino-acid [aa] PhoP molecule is composedof two functional domains, an N-terminal receiver domain consistingof 119 aa [PhoPN] and a 102-aa C-terminal effector domain [PhoPC]involved in DNA binding and transactivation via RNA polymerase[RNAP] . A long linker region [19 aa] connects the regulatorydomain [PhoPN] to the output domain [PhoPC] . The receiver domainof PhoP has been structurally characterized [5] . The structure analysis revealed overall folds similar to those of two otherOmpR family proteins, PhoB from Escherichia coli [7] and DrrDfrom Thermotoga maritima [8], but showed that there are remarkabledifferences in the ß4-4 loop and 4 helix regions comparedto either PhoB or DrrD despite the high levels of sequence similarityexhibited by the three proteins in these regions . The PhoPNstructure analysis also showed that there is a novel asymmetricassociation between PhoPN protomers that supports the establishedDNA binding properties of PhoP . The dimer interface betweentwo PhoP monomers involves nonidentical surfaces such that eachmonomer in a dimer has a second surface available for further oligomerization . DNA footprinting studies have shown that there is cooperative binding of PhoP dimers at PhoP-activated promotersand have revealed that both phosphorylated and nonphosphorylateddimers can bind to the four 6-bp direct repeats, TT[A/C/T]A[C/T]A,spaced 4 to 6 bp apart that constitute the core binding regionfor PhoP at PhoP-activated promoters [12] . PhoP footprinting data for repressed promoters [6, 18] suggest that oligomerizationof PhoP along the DNA extends well into the coding region . Supportfor the physiological relevance of the asymmetric dimerizationinterface of PhoP was obtained when mutations designed to disruptthis interface resulted in PhoP mutant proteins that could bephosphorylated by PhoR but could not dimerize or bind DNA invitro and had no activity in vivo [9].

Our previous footprinting studies showed that there was cooperative binding between PhoP dimers at PhoP-activated promoters and that both nonphosphorylated and phosphorylated PhoP could bindto the four 6-bp repeats that form the core binding region forPhoP, suggesting that nonphosphorylated and phosphorylated PhoPdimers have structural relationships in common that allow similarbinding of their effector domains to target DNA . The fact thatphosphorylation is required for transcriptional activation orrepression suggests that it may enhance cooperativity for DNAbinding or interactions with components of the transcriptionmachinery . Studies described here were initiated to determinewhich residues of the effector domain, PhoPC, are required forPhoP DNA binding and which residues are required for transcriptionalregulation . For certain OmpR homologues the purified wingedhelix domain is sufficient for DNA binding to target DNA asa dimer [7, 15, 20] . In fact, the DNA binding domain of PhoB, the presumed E . coli orothologue of B . subtilis PhoP, has a higher affinity for target DNA than the nonphosphorylated intact PhoB protein has [13] . In contrast, the DNA binding domain ofPhoP was isolated and was shown to be a monomer in solution that required more than fivefold more protein for DNA binding to target DNA than nonphosphorylated intact PhoP required . In addition, expression of PhoPC in B . subtilis could not induce Pho regulon genes, at least in part due to the instability ofthis domain in vivo [Chen and Hulett, unpublished data] . Forthese reasons mutant proteins resulting from alanine scanningmutagenesis of the PhoP loop and the helix 3 region of PhoPCdomain were constructed with the intact PhoP protein for bothin vivo and in vitro functional analyses.

 
Strains and plasmids. E . coli DH5 [lab stock] and XL1-Blue [Stratagene] were usedas the hosts for plasmid construction [Table 1] . E . coli BL21[DE3][pLysS][Novagen] was used as the host for overexpressing the PhoP proteins.B . subtilis JH642 and MH6110, a derivative of JH642, were usedfor in vivo Pho induction experiments . When required, antibioticswere added at the following concentrations: for B . subtilisstrains, 5 µg of chloramphenicol per ml, 10 µg of spectinomycin per ml, or 10 µg of tetracycline per ml;and for E . coli strains, 100 µg of ampicillin per ml or50 µg of kanamycin per ml . 5-Bromo-4-chloro-3-indolyl-ß-D-galactopyranoside [X-Gal] was used at a concentration of 30 µg/ml, and isopropyl-ß-D-thiogalactopyranoside [IPTG] was used at a concentration of 1 mM.

 
To construct MH6110 [phoPR::Tet^r amyE::phoA-lacZ Sp^r], the chromosomalDNA from MH5923 [amyE::phoA-lacZ Sp^r] was used to transform MH5913 [phoPR::Tet^r] . Transformants were selected on platescontaining tryptose blood agar base [Difco] supplemented with0.5% glucose [TBABG] containing tetracycline and spectinomycin.The representative clones were confirmed by phenotypic screening.

To generate a B . subtilis strain with an IPTG-inducible phoPR operon, pWL29 was digested with ClaI, treated with the Klenow enzyme, and relegated, and the resulting plasmid was designated pCH101 . This plasmid was digested with BpuI102I and SphI, which removed a 178-bp fragment of the phoP gene 3' region, and then ligated with the 2.9-kbp [2,875-bp] BpuI102I/SphI fragment obtainedfrom pHT4phoPR that contained the 178 bp 3' of phoP, the completephoR gene, and 750 bp 5' of the polA gene, yielding pCH102.pCH102 was transformed into MH6110 and integrated into the chromosomeby homologous recombination . Transformants were selected forCm^r and screened for Sp^r and Tet^r . The position of homologousrecombination, 3' or 5' of the Tet^r gene in chromosome, wasdetermined by PCR to be in the phoR polA region downstream ofthe Tet^r gene in MH6110 . The resulting B . subtilis strain, MH6111, contains the complete phoPR operon under control of the P_spac promoter.

Plasmids similar to pCH102 but with a mutation at codons affecting PhoP amino acid residues 190 to 214 were constructed as follows.The 676-bp fragment containing the phoP gene 3' region and the phoR gene 5' region was released from pCH102 by digestion with ClaI and SacI and cloned into pBluescript KS[+] at the same sites in order to generate deltaPRKS+ . Most of the PhoP codons from the V190 codon to the E214 codon in plasmid pdeltaPRKS+were individually mutated to the alanine codon, GCG, by usinga QuickChange site-directed mutagenesis kit [Stratagene] andthe necessary primer pairs; the only exception was the A196codon, GCC, which was mutated to the V196 codon, GAT . Aftersequence confirmation, the 676-bp fragment with the requiredmutation was released from plasmid pdeltaPRKS+ by digestionwith ClaI and SacI and was used to replace the same region inpCH102, yielding the P_spac-controlled mutated phoPR operon with a ribosome binding site and a partial 5' polA gene . Plasmids pCH103 to pCH127 were transformed into MH6110, and representative transformants were used in in vivo experiments . The mutant strains were confirmed by PCR and DNA sequencing.

To construct a plasmid for overexpressing mutant PhoP proteins, the phoP gene was released from pCH01 [9] by digestion withNdeI and BamHI and cloned into pSKB3 at the same sites to generatepCH128 . The PhoP codons from V190 to E214 were individuallymutated as described above, yielding plasmids pCH129 to pCH153.For the PhoP codon substitution R113 to E113, plasmid pCH05 [9] was digested with NdeI and BamHI and cloned into pSKB3 atthe same sites to generate pCH154 . The required mutation wasconfirmed by DNA sequencing . These plasmids were transformedinto E . coli BL21[DE3][pLysS], and the representative transformantswere used to overexpress PhoP proteins.

phoA-lacZ plate assay. Low-phosphate complex medium [LPM] or low-phosphate complexmedium with 10 mM phosphate added [HPM] contained Noble agar[1.5%], X-Gal [30 µg/ml], and IPTG [1 mM] [26] . The freshtransformants generated as described above [typically 100 transformantsper construct] were selected and grown on TBABG agar plateswith antibiotics for 16 h at 37°C . The clones were thengrown on LPM or HPM agar containing X-Gal and IPTG for 16 hat 37°C.

Growth conditions and APase activity assay. Alkaline phosphatase [APase] activity was measured in cellsthat had been grown in low-phosphate defined medium [LPDM] asdescribed previously [9] with IPTG present at a concentrationof 1 mM throughout growth.

Western immunoblotting. Samples [50 ml] were taken from an LPDM culture at 11 h . Thecells were separated from the medium by centrifugation at 10,000x g for 10 min . The pellet fraction was suspended in 5 ml ofTris-EDTA containing 0.8 M sucrose, and then lysozyme was addedto the cell suspension at a final concentration of 5 mg/ml.After the mixture was incubated at 37°C for 10 min, thecells were collected by centrifugation at 5,000 x g for 10 minand washed twice with Tris-EDTA containing 0.8 M sucrose . The pellet fraction was then suspended in 5 ml of Tris-EDTA containing 0.3 M NaCl, 1 mM phenylmethylsulfonyl fluoride, and 1 mM MgCl₂ and subjected to sonication immediately . After centrifugation at 100,000 x g for 1 h, the supernatant fraction was used asthe soluble protein fraction . Equal volumes of an MH5913 [phoPR] cell extract containing different dilutions of purified wild-type PhoP [PhoP_WT] were used to generate a standard curve . Sodium dodecyl sulfate [SDS]-polyacrylamide gel electrophoresis [PAGE] separation and immunodetection were performed as described previously [9].

Overexpression and purification of PhoP proteins. E . coli BL21[DE3][pLysS] harboring a PhoP protein-overexpressingplasmid under control of a T7/lac promoter was incubated overnightat 37°C in Luria-Bertani medium containing ampicillin andwas used to inoculate 1 liter of the same medium at a ratioof 1:100 . The cells were grown at 30°C until the opticaldensity at 600 nm of the culture reached about 0.4 . IPTG wasthen added to the culture at a final concentration of 1 mM,and the culture was grown for another 3 h . The cells were harvestedby centrifugation at 4°C and washed with buffer A [1 M NaCl,5 mM MgCl₂, 10 mM dithiothreitol, 50 mM Tris-HCl [pH 7.8]].The cell pellets were then suspended in 50 ml of prechilledbuffer A on ice containing 1 mM phenylmethylsulfonyl fluorideand were immediately subjected to sonication . Disruption of the cells was confirmed by phase-contrast microscopy . After centrifugation at 40,000 x g for 1 h at 4°C, the supernatantfraction was filtered through a 0.45-µm-pore-size membrane[Amicon] . After 1/50 volume of 0.5 M imidazole in buffer A wasadded, the clear cell lysate was mixed with 2 ml of Ni-nitrilotriaceticacid [Qiagen] affinity resin preequilibrated with buffer A.After gentle shaking at 4°C for 30 min, the mixture wasloaded onto an Econo column [inside diameter, 2.5 cm; height,10 cm; Bio-Rad] . The column was washed with buffer A until theprotein concentrations in the elute were not detectable by theBio-Rad protein assay . The protein bound to the column was eluted by using 300 mM imidazole in buffer A . The protein fractions were dialyzed stepwise at 4°C against buffer A containing20% glycerol with decreasing concentrations of NaCl [1, 0.8,0.6, 0.4, 0.2, and 0.1 M] . The PhoP proteins were more than95% pure, as judged by SDS-PAGE.

Phosphotransfer assays. Glutathione S-transferase [GST]-*PhoR [30], prepared in phosphorylation buffer [50 mM KCl, 5 mM MgCl₂, 50 mM HEPES [pH 8.0]], was used to phosphorylate each PhoP . Boiled glutathione beads [400 mg] were washed with phosphorylation buffer and incubated with 200µg of GST-*PhoR on a rocker at room temperature for 10min . The unbound component was washed off the beads with 20volumes of phosphorylation buffer, and the extra buffer wasremoved by microcentrifugation for 10 s . Phosphorylation buffer[0.4 ml] with 20 µl of [-³²P]ATP [10 mCi/ml] was addedto the beads, and autophosphorylation of GST-*PhoR was performedat room temperature for 20 min . The beads were thoroughly washedwith phosphorylation buffer until there was no ATP in the flowthrough.The beads bound with GST-*PhoRP were suspended in 0.4 ml ofphosphorylation buffer containing 50 U of thrombin [Pharmacia]and incubated for 15 min at room temperature . The released *PhoRP was recovered by microcentrifugation through a Micro Bio-Spin chromatography column [Bio-Rad] . For phosphotrasfer reactions,*PhoRP was mixed with each PhoP protein at a molar ratio of1:15 in phosphorylation buffer . After incubation at room temperaturefor 10 or 60 s or 10 min, an equal-volume sample was taken andmixed with 4x SDS loading buffer containing 0.1 M EDTA [pH 8.0],and the sample was applied to an SDS—10% PAGE gel.

For native PAGE experiments, PhoP proteins were phosphorylated with GST-*PhoRP as described previously [9].

Determination of protein concentration. The protein concentration was determined by the Bradford methodby using a Bio-Rad protein assay kit as instructed by the manufacturer.

SDS-PAGE and native PAGE. SDS-PAGE was performed as described by Laemmli [17] . A 10% polyacrylamide separating gel was used for detection of His10-PhoP . An 8% native PAGE gel was prepared as described previously [9].

Quantitation of radioactivity. Radioactivity of proteins on SDS-PAGE or native PAGE gels wasdetected with Fuji medical X-ray film [Fuji] and/or a PhosphorImager[Molecular Dynamics] . When required, the image was quantitatedwith Imagequant, version 5.1.

Gel shift assays. Gel shift assays were done as described previously [19] . Theprobe was a 159-bp fragment containing the phoB promoter releasedfrom pRC696 [28] by digestion with BamHI and EcoRI and labeledwith the Klenow enzyme in the presence of 4 µl of [-³²P]dATP [6,000 Ci/mmol; 10 mCi/ml].

In vitro transcription assay. All components used for the transcription reaction were adjustedto the required concentrations and equilibrated in nuclease-freetranscription buffer [NFTB] [10 mM Tris-HCl [pH 8.0], 50 mMKCl, 5 mM MgCl₂, 1 mM CaCl₂, 0.1 mM EDTA, 5% glycerol], unlessindicated otherwise . To construct a template for the in vitrotranscription assay, the phoB promoter region [located at positions-221 to 105 relative to the translation start at position 1]was amplified by PCR by using primers FMH745 and FMH746 andstrain JH642 chromosomal DNA as the template . The PCR productwas extracted from a 1.2% agarose gel by using a gel extractionkit [Qiagen] and was further purified by phenol extraction andethanol precipitation . Finally, the phoB promoter was dissolvedin NFTB and stored at -20°C . B . subtilis ^A and core RNAPwere prepared as previously described [27] . To prepare E^A, coreRNAP was incubated with ^A at a molar ratio of 1:30 in NFTB onice for 30 min . For each reaction, addition of 5 µl oftemplate DNA [40 ng/µl] was followed by addition of 1µl of ATP [1 mM], 1 µl of *PhoR [4 µM], and 1 µl of each PhoP protein [wild type or mutant; concentration,6 µM], and the mixture was incubated at room temperaturefor 20 min . Then 2 µl of E^A [0.5 µM core RNAP] wasadded to the reaction mixture, followed by 5 µl of a nucleosidetriphosphate mixture [500 µM ATP, 500 µM GTP, 500 µM CTP, 50 µM UTP, 0.33 µM [-³²P]UTP [3,000Ci/mmol; 10 µCi/µl], and 4 U of RNasin per µlin NFTB], and the mixture was incubated at room temperaturefor another 30 min . The reactions were stopped by adding 7.5µl of stop buffer [7 M urea, 100 mM EDTA, 0.05% xylenecyanol, 0.05% bromophenol blue, 5% glycerol] and heating thepreparations at 75°C for 5 min . After electrophoresis ona sequencing gel, the transcripts were detected by PhosphorImageranalysis.

 
Experimental design and rationale. In this study, we set out to identify the amino acid residuesof the PhoP C-terminal effector domain required for DNA bindingor transcription activation, as well as to identify residuesthat may be involved in other functions, including protein stabilityor interdomain interactions in PhoP . To do this, we used theQuickChange method of site-directed mutagenesis to obtain single-alaninesubstitutions of 24 nonalanine residues of PhoP and one valinesubstitution of alanine, which constituted the putative transactivationloop and the DNA recognition helix . We introduced the mutationsinto the phoPR operon residing in plasmid pDH88 under controlof the P_spac promoter, and the plasmids were transformed intothe chromosome of a phoPR deletion strain via Campbell insertion[Fig . 1] . In the resulting strains, expression of the phoPRoperon was exclusively controlled by an inducer, IPTG.

 
Qualitative in vivo assessment of the effect of each PhoP mutation on PhoP function. The 36-fold increase in total APase activity upon phosphatestarvation represented the sum of the effects on three APases,PhoA, PhoB, and PhoD, which account for approximately 65, 30, and <5% of the total activity, respectively . The promoter-lacZ fusion of the major APase, PhoA, was inserted at the amyE locus in our constructs and used as a reporter of PhoPP transcriptionalactivity . Fresh transformants of each PhoP variant were pickedand transferred to a TBABG plate and incubated for 16 h at 37°C.Then the single clones were grown on HPM or LPM with IPTG andX-Gal to assess the relative promoter activities . The colony colors were scored after incubation of the plates at 37°Cfor 16 h . Parent strain MH6110 [phoPR] and strain MH6111 withan IPTG-inducible wild-type phoPR operon were included as phoA-lacZcontrols . Figure 2A shows that under phosphate-sufficient conditions [HPM], neither the wild-type strain nor the PhoP mutant strains activated phoA promoter expression . Under phosphate starvation conditions [LPM], the expression in strains having substitutionsat PhoP residues 192, 194, 196, 198, 201, 207, 212, and 213was similar to the phoA-lacZ expression [colony color score]in the strain with the wild-type phoPR operon [MH6111], suggestingthat substitutions at these residues had no effect on PhoP function. However, strains having substitutions at residues 191, 193,195, 199, 200, 202, 203, 206, 208, 209, and 210 showed no phoA-lacZ activity, indicating that each of these substitutions was deleterious to the PhoP function . Strains having the remaining PhoP substitutions exhibited decreased phoA-lacZ activity, indicating that these residues may affect the PhoP function.

 
Quantitative in vivo assessment of the effect of each PhoP mutation on Pho induction. To assess the Pho induction phenotype of each phoP mutationfurther, total APase specific activity was measured for each strain under P_i starvation conditions . Previous data showed that in strain MH6101 expressing the phoPR operon constitutively from the P_spac promoter, the APase specific activity increasedsharply upon phosphate starvation and reached a maximal valuethat was maintained as cells entered the stationary phase under phosphate starvation conditions [9] . We took advantage of theplateau in total APase specific activity to compare the total APase specific activities exhibited by the strains . Samples were taken from cultures grown in LPDM at 12 to 14 h, when thetotal APase specific activity remained constant . In agreementwith the plate assay data, strains having mutations that changedPhoP residues 192, 194, 196, 198, 201, 207, and 212 had levelsof APase specific activity similar to those of strains withthe wild-type phoPR operon [Fig . 2B], while strains having substitution mutations that affected residues 191, 193, 195, 199, 200, 202,203, 204, 205, 206, 208, 209, and 210 each exhibited a verylow level of APase specific activity, which was comparable tothe level of the parental strain [MH6110 phoPR::Tet^r phoA-lacZ].The remaining mutant strains had reduced total APase specificactivities, and the levels were less than 50% of the level ofthe wild-type phoPR operon strain [MH6111 P_spac phoPR] . Discrepanciesin the qualitative and quantitative expression assays were observedfor strains containing K212, V204, and H205 PhoP variants.

Similar PhoP concentrations [wild-type or mutant proteins] were detected in the soluble fraction of strains with phoPR expression controlled by IPTG. Because the PhoP protein concentration in cells may affect phoA-lacZand/or total APase expression during Pho induction, anti-PhoPCantiserum was used to immunodetect PhoP protein in the cells.Cells were grown in LPDM containing IPTG until the culture enteredstationary growth due to limiting phosphate concentrations andinduction of APase had plateaued . Cells were harvested, andthe proteins were extracted as described in Materials and Methods.Soluble proteins [12 µg] from each phoP mutant strainand the strain containing the wild type phoPR operon under P_spaccontrol [MH6111] were separated on an SDS-PAGE gel, blotted,and immunodetected with anti-PhoPC antiserum [Fig . 3A] . Theposition on the PhoP standard curve of PhoP in 12 µg ofsoluble proteins from MH6111 is indicated in Fig. 3B . The concentrationsof PhoP proteins induced with IPTG in strains having phoP mutationswere similar [within 5%] to the concentration of wild-type PhoPin MH6111 . The same results were obtained when lysed cell sampleswere analyzed similarly [data not shown] . Thus, the variabilityin the phoA-lacZ activities and/or the total APase activities[Fig . 2] was used to identify which residue changes affectedPhoP function.

 
In vitro phosphotransfer reactions were not altered in mostPhoP mutated proteins, and three of these proteins exhibiteda reduced phosphotransfer rate . To understand how PhoP functionwas affected by each residue change, PhoP mutant proteins wereoverexpressed in E . coli and purified by Ni-nitrilotriaceticacid affinity chromatography so that they were >95% pure,as judged by SDS-PAGE [data not shown] . The ability to receivea phosphoryl group from the cognate histidine kinase PhoR wasanalyzed by using the functional N-terminal truncated form ofPhoR, *PhoR [30] . *PhoRP, free of ATP, was used for phosphotransferto each mutated PhoP . Samples were taken after 10 and 60 s and10 min for analysis of phosphotransfer . In general, phosphoryltransfer from *PhoRP to most of the mutant Pho proteins wasmaximal within 10 s and remained stable, and there was onlya slight loss of P over time, which is typical of the wild-typePhoP protein [Fig. 4] . However, two Pho mutants, mutants 202and 203, exhibited reduced phosphotransfer rates from *PhoRP; there was only 20% of the wild-type PhoP labeling after 10 s, but the level increased to nearly 80% of the wild-type labelingafter 10 min . These two mutants identified residues locatedin the C-terminal domain that might cause a defect in phosphotransfervia domain-domain interaction . Howerver, most phoP variants behaved like the wild-type PhoP in phosphotransfer reactions, suggesting that the mutant proteins retained the overall structureof the wild-type PhoP.

 
PhoP changes that affect DNA binding. To identify residues that might be directly involved in theprotein-DNA interaction, PhoP-promoter DNA complexes were detectedby a gel mobility assay . The phoB promoter was chosen as thetarget because it is the simplest of the well-characterizedPho regulon promoters, with PhoP binding solely to the corebinding region between approximately positions-20 and -60 upstreamof the Pho-regulated promoter . Previous data had shown thatPhoP or PhoPP can bind to this promoter, although lower concentrationsof PhoPP [almost 25% the concentration of PhoP] were neededto bind [19] . Using PhoP_WT, we confirmed that 5 µM PhoPwas needed to form the protein-DNA complex, while 1.5 µMPhoPP was sufficient [data not shown] . PhoP_WT- or mutant PhoP-DNA complexes were detected by native gel electrophoresis underthe same conditions [Fig . 5] . Variants with mutations that created residue substitutions at positions 202, 203, 204, 205, 206, 208, 209, and 210 did not form complexes at the concentrationsused, while other PhoP variants were similar to PhoP_WT . These residues may be involved in direct PhoP-DNA interactions.

 
The fact that phosphorylation of PhoP in vitro increased theDNA binding efficiency in vitro [19] and the fact that in vivo a PhoR deletion strain did not express APase activity indicated that Pho induction required phosphorylation of the response regulator, PhoP . When the phosphorylated form of PhoP was usedto assess DNA binding, as expected, most complexes formed ata concentration that was approximately 25% of the required PhoP concentration [Fig . 6] . Surprisingly, phosphorylated forms of PhoP with residue substitutions at positions 204 and 205 formed complexes at PhoP_WT concentrations, while nonphosphorylated forms of PhoP with residue substitutions at position 204 or205 failed to bind DNA [Fig . 5] . Taken together, the results of the gel shift assay suggest that residues 202, 203, 204, 205, 206, 208, 209, and 210 might be involved in protein-DNA interactions . Phosphorylation of PhoP proteins with substituted residues at positions 204 or 205 may affect protein-DNA formationvia a mechanism that differs from the mechanism for other PhoPproteins.

 
It has been reported that PhoP mutant proteins that cannot dimerize are defective in binding to target DNA [9] . Native gel electrophoresisof nonphosphorylated or phosphorylated mutant proteins, thenative PhoP protein [dimer], and PhoP_R113E [monomer] was usedto assess the possibility that dimerization was affected bythe residue changes between I201 and K212 [Fig. 7] . The phosphorylatedor nonphoshorylated PhoP variants migrated at the rate of wild-typePhoP, not the higher rate of PhoP_R113E, suggesting that dimerizationwas not affected and therefore not the reason for a DNA bindingdeficiency.

 
In vitro transcription assays with E^A resulted in identificationof PhoP residue changes that affect transcriptional activityand potential RNAP interaction residues . To test the transcriptionalactivity of each PhoP mutant protein, runoff in vitro transcriptionassays were conducted with the phoB promoter as the template.Previous primer extension data showed that the phoB promoterhas two transcriptional start sites, P_v and P_s [10], which haverecently been shown to be dependent on ^A and ^E, respectively[W . R . Abdel-Fattah, Y . Chen, and F . M . Hulett, unpublisheddata] . Only the E^A promoter is PhoP regulated . To simplify thereaction, only transcription from the P_v promoter was tested. B . subtilis core RNAP was purified and mixed with B . subtilis ^A factor that had been overexpressed in and purified from E.coli to reconstitute active ^A holo-RNAP . As expected, PhoP DNAbinding-deficient substitutions at positions 202, 203, 206,209, and 210 did not activate transcription at all, while asubstitution at position 208 resulted in a decreased level oftranscript [perhaps 25% that of PhoP_WT] . In contrast, althoughthe PhoP proteins with residue substitutions at positions 190,191, 193, 195, 197, 199, and 200 bound DNA efficiently, theyactivated transcription at moderately to severely reduced levels.PhoP proteins with substituted residues at position 191 or 200yielded no detectable transcripts at all . Considering the invivo [Fig . 2] and in vitro [Fig . 8] data together, residues191 and 200 are probably two residues that directly interactwith RNAP, while residues 190, 193, 195, 197, and 199 may contributeto this interaction.

 
Functional analysis of PhoP mutant proteins derived from alanine scanning mutagenesis of the putative PhoPC transactivation loop[ loop] and DNA binding helix [ helix 3] showed that mutationsinvolving certain amino acid residues between PhoP_V190 and PhoP_E214were essential or important for PhoP function in vivo . In vitrofunctional analysis revealed that all mutated proteins defectivefor DNA binding had residue changes between PhoP_I202 and PhoP_E210, while mutations involving certain residues between PhoP_V190 and PhoP_I200 produced proteins that had reduced PhoP function in vivo and were unable or had reduced ability to activate transcription in vitro [Fig . 9].

 
Distribution of PhoP residues involved in DNA binding or transactivation appears more similar to the distribution determined for OmpR than to the distribution determined for PhoB of E . coli. The results of our analysis of mutations throughout the PhoPDNA binding-transactivation domain can be compared to the resultsof a mutational analysis and amino acid alignment of the domainsof two structurally characterized E . coli response regulators,OmpR [21] and PhoB [7] [Fig . 8] . The level of sequence identitywithin the DNA binding region of the three proteins is quite high, 47%, whereas the transactivation loop has no conserved residues in the three proteins . The eight mutations betweenV190 and E214 in PhoP that affect DNA binding are clusteredbetween V202 and R210 in helix 3, whereas the mutations affectingDNA binding of PhoB [20] or OmpR [1, 16, 29] are more broadlyspread throughout the corresponding region, especially in PhoB,in which mutations in the loop were shown to specifically affectDNA binding [20] and in which structural analysis [7] showedthat transactivation loop residues Val183, Glu191, and R193bind DNA . In contrast to mutations that affect PhoP DNA binding,mutations in PhoP that affect the transcription activation functionof PhoP were broadly distributed throughout the transactivationloop, extending as far C terminal as R200, a conserved residuein the three proteins that is part of an extended helix 3 inPhoB but is in the loop of OmpR.

PhoP DNA binding mutants fall into three classes. One class of PhoP DNA binding mutants, which included PhoP_I206A, PhoP_R208A, PhoP_L209A, and PhoP_H210A, had a phosphotransfer ratesimilar to that of the native PhoP and could dimerize, but neitherthe phosphorylated nor nonphosphorylated PhoP mutants couldbind DNA at concentrations appropriate for PhoP_WT . In general,the members of this class could not activate transcription ofthe phoB promoter in vitro or of total APases or phoA in vivo;the only exception was PhoP_H208A, which produced a weak transcriptin vitro but exhibited no transcription in vivo . Higher concentrationsof PhoP and/or E^A in vitro than in vivo may account for thedetectable transcriptional activity of PhoP_H208A observed invitro . The phenotype of the mutant proteins in this class isconsistent with residues that may directly interact with DNA.PhoB [E . coli] residues that correspond to PhoP_R210 bind DNAvia nonspecific interactions, while the residue correspondingto PhoP_H208 binds via both specific and nonspecific interactions[7] . In addition, especially for PhoP_I206A, PhoP_R210A, and PhoP_L209A, the phenotype is also consistent with disruption of conserved residues that stabilize secondary structural elements involvedin DNA recognition, a role determined for the analogous residuesof the E . coli ortholog, PhoB [7].

A second class of PhoP DNA binding mutants, including PhoP_V204A and PhoP_H205A, also had a phosphotransfer rate equal to that of the native PhoP and could dimerize, and the nonphosphorylated PhoP mutant proteins could not bind DNA at concentrations appropriate for nonphosphorylated PhoP_WT; however, the phosphorylated PhoP_V204A and PhoP_H205A proteins could . Transcription of phoA and thetotal APase activity in strains with these mutant phoP alleleswere quite low compared to those of the wild-type strain, but in vitro phoB transcription by using PhoP_H205APor PhoP_V204AP was more than 50% of the transcription observed when PhoP_WT was used . These data suggest that unlike the native PhoP [19], phosphorylation of PhoP_V204A and PhoP_H205A is required to changethe conformation of the mutant protein to allow DNA binding[compare Fig . 5 and 6] required for transcriptional activity,perhaps in a way similar to either that proposed for NarL [4]and CheB [11], in which functional regions of the effector domainsare blocked by the regulatory domains in the nonphosphorylatedstate, or that proposed for PhoB [7] of E . coli, in which it has been proposed that the position of the DNA binding region in the nonphosphorylated state prevents binding of the response regulator to the tandem repeats of the Pho box . Both structuraland genetic studies [2] implicated helix 5 of the receiver domainin the interdomain interactions in PhoB negative regulation.The phosphorylation-dependent phenotype for DNA binding of PhoP_H205Aor PhoP_V204A implies that a mutation in the C-terminal DNA bindingregion of PhoP may alter the interaction of the domain withthe PhoP N-terminal domain . A mutation in OmpR, OmpR_{V203 M},at the position analogous toV204A in PhoP, reduced DNA binding[29], and the protein exhibited functional properties that affectedboth DNA recognition and small-molecule phosphorylation of OmpR,leading to the conclusion that a single mutation in the OmpRDNA binding domain affects the N-terminal domain [31] . The E.coli PhoB residue corresponding to PhoP_V204 showed specificDNA interactions, while residues corresponding to PhoP_H205 had both specific and nonspecific DNA binding interactions, as wellas a structural role via hydrogen bonding to a conserved Gluresidue of helix 1 of the DNA binding domain [7].

A third class of PhoP DNA binding mutants, including PhoP_V202A and PhoP_D203A, have a reduced rate of phosphotransfer, can dimerize,and do not bind DNA or activate transcription in vitro or invivo . The reduced rate of phosphotransfer may again indicate that mutations in this DNA binding class affect the PhoPN structure in addition to the C-terminal domain structure . Residues of PhoB [7] or OmpR [22] corresponding to PhoP_V202 are residuesof the central hydrophobic core that maintain the tertiary structureof that domain . Residues of PhoB and OmpR analogous to PhoP_D203also link secondary structural elements by hydrogen bondingto the invariant tyrosine in the C-terminal hairpin that correspondsto PhoP_Y231 . All mutant PhoP proteins in this class were hypersensitivecompared to native PhoP in limited proteolysis experiments,which is consistent with the importance of these residues instabilization of the native PhoP conformation [data not shown].

Certain mutant PhoP proteins involving PhoP residues V190 to I201 are affected in in vivo and in vitro transcription. None of the PhoP_V190 to PhoP_I201 mutant proteins had phosphorylationor DNA binding defects, although a number had transcriptiondefects . The PhoP_W191A and PhoP_R200A mutant proteins could not activate in vivo or in vitro transcription, while PhoP_Y193A and PhoP_F195A abolished in vivo transcription but supported very reduced in vitro transcription compared to that of PhoP_WT. PhoP_V190A, PhoP_G197A, and PhoP_T199A showed reduced transcriptionin vivo or in vitro or both compared to the transcription ofPhoP_WT . These data indicate that PhoP residues V190, W191, Y193,F195, G197, T199, and R200 are candidates for RNAP interaction.PhoP_W191A is conserved in E . coli PhoB, in which it has beenshown to interact with the ⁷⁰ subunit of RNAP [20] . PhoP_R200Ais also conserved in PhoB, in which it is implicated in DNAbinding but not in RNAP interaction.

PhoP variants that are DNA binding proficient but are defectivein the RNAP interaction have Ala substitutions that are broadly distributed throughout 11 residues, from V190 to R200 [Fig. 9] . Since residues in the response regulator that contribute to the RNAP interaction generally belong to the transactivation loop, secondary structure considerations suggest that PhoP hasa longer transactivation loop and is more similar to OmpR thanto PhoB of E . coli . Mutations in PhoP that affect DNA binding[eight of nine contiguous residues] are in the highly conservedsequence that is part of helix 3 in either PhoB or OmpR . WhileOmpR mutations that affect DNA binding are confined to helix3, PhoB residues in both the loop and helix 3 have been shownby mutagenesis and/or PhoB DNA structure analysis to be importantfor binding . It is of interest to determine the relevance ofthese observations with respect to PhoPC structure and the PhoP-RNAPinteraction.

 
This work was supported by National Institutes of Health grant GM-33471 to F.M.H.

We thank J.-P . Samama for helpful discussions and suggestions.

 
* Corresponding author . Mailing address: Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, 900 S . Ashland Avenue [M/C 567], Chicago, IL 60607 . Phone: [312] 996-5460 . Fax: [312] 413-2691 . E-mail: Hulett@uic.edu .

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