When Bacillus subtilis is subjected to phosphate starvation, the Pho regulon is activated by the PhoP-PhoR two-componentsignal transduction system to elicit specific responses to thisnutrient limitation . The response regulator, PhoP, and its cognatehistidine sensor kinase, PhoR, are encoded by the phoPR operonthat is transcribed as a 2.7-kb bicistronic mRNA . The phoPRoperon is transcribed from two ^A-dependent promoters, P₁ andP₂ . Under conditions where the Pho regulon was not induced [i.e.,phosphate-replete conditions or phoR-null mutant], a low levelof phoPR transcription was detected only from promoter P₁ . During phosphate starvation-induced transition from exponential to stationary phase, the expression of the phoPR operon was up-regulated in a phosphorylated PhoP [PhoPP]-dependent manner; in additionto P₁, the P₂ promoter becomes active . In vitro gel shift assaysand DNase I footprinting experiments showed that both PhoP andPhoPP could bind to the control region of the phoPR operon.The data indicate that while low-level constitutive expressionof phoPR is required under phosphate-replete conditions forsignal perception and transduction, autoinduction is requiredto provide sufficient PhoPP to induce other members of the Phoregulon . The extent to which promoters P₁ and P₂ are activatedappears to be influenced by the presence of other sigma factors,possibly the result of sigma factor competition . For example,phoPR is hyperinduced in a sigB mutant and, later in stationaryphase, in sigH, sigF, and sigE mutants . The data point to a complex regulatory network in which other stress responses and post-exponential-phase processes influence the expression ofphoPR and, thereby, the magnitude of the Pho regulon response.

 
Bacillus subtilis responds to phosphate starvation by inducing or repressing genes of the phosphate stimulon, comprising: [i]the phosphate starvation-specific Pho regulon, [ii] the ^B-dependent general stress [^B-GS] regulon, and [iii] PhoP-PhoR/^B-independent phosphate starvation-inducible genes [2, 12, 15, 22] . The ^B-GS regulon has 200 members [29, 34], while the Pho regulon presentlyhas 31 members . Of the latter, five operons [phoPR, phoB-ydhF,pstSCA-pstBA-pstBB, phoD-tatAD, and tuaABCDEFGH] and seven monocistronicgenes [glpQ, phoA, tatCD, ykoL, yhaX, yhbH, and yttP] are inducedin response to phosphate starvation, and two operons [tagABand tagDEF] are repressed . The alkaline phosphatases [APases]PhoA and PhoB [4, 16], the phosphodiesterase-APase PhoD [6], and the glycerophosphodiesterase GlpQ [2] generate new sourcesof inorganic phosphate [P_i] from organic sources, such as deacylatedphospholipids, nucleic acids, and teichoic acid [3] . PhoD issecreted by the twin-arginine transporter [Tat] pathway, some components of which are encoded by members of the Pho regulon[19] . Pst, a high-affinity phosphate ABC transporter [35], facilitatesthe uptake of P_i at low P_i concentrations . Concomitant repressionof the teichoic acid operons [tagAB and tagDEF] and inductionof teichuronic acid operon [tuaA to tuaH] conserves phosphateby bringing about the controlled replacement of the phosphate-containingcell wall polymer teichoic acid with the non-phosphate-containingteichuronic acid [21, 24, 28] . The functions of five putative Pho regulon genes [ydhF, ykoL, yhaX, yhbH, and yttP] are presentlyunknown [2, 32, 36].

During phosphate starvation, genes of the Pho regulon are regulated by the PhoP-PhoR two-component signal transduction system [39, 40] . The PhoP response regulator is activated by its cognatesensor kinase, PhoR . Phosphorylated PhoP [PhoPP] induces theexpression of the phoPR operon about threefold from a low constitutivelevel of expression [17, 30, 32] and is required for the induction or repression of other members of the Pho regulon [15].

Phosphate starvation also induces the ^B-mediated general stressresponse, and the Pho and ^B-GS regulons interact to modulatethe levels to which each is activated . In the absence of theregulator of one of these regulons, the expression of the otherregulon is activated to a higher level [2, 32] . For maximalinduction of the Pho regulon, the respiration signal transductionsystem, ResD-ResE, is required [15] . If, despite these responses,phosphate starvation persists, a third response regulator, Spo0A,initiates sporulation and terminates the phosphate responseby repressing phoPR transcription via AbrB and ResD-ResE [15,17].

The induction or repression of Pho regulon genes is mediatedby the binding of PhoPP to Pho box sequences: direct repeatsof TT[A/T/C]ACA with a 5 ± 2-bp spacer [7] . For efficientbinding, four TT[A/T/C]ACA-like sequences with an 11-bp periodicityare required . In the case of genes induced by PhoPP, the PhoP-bindingsites are located on the coding strand of the promoter regionand on the noncoding strand of the promoter regions of PhoPP-repressed genes [25].

In the work described here we have used a combination of Northern hybridization, primer extension analyses, gel shift assays,and DNase I footprinting to analyze the transcriptional regulationof the phoPR operon . We compared the binding of PhoP and PhoPP to the promoter region of phoPR with that of two other putative members of the Pho regulon, namely ykoL and yhaX . In addition,the transcription of phoPR was studied in phoR, sigB, and abrBmutants as well as in a number of mutants deficient in variousstages of sporulation . The data confirm the role of PhoP inthe regulation of phoPR and identify two sigma A-like promoters[P₁ and P₂] with associated Pho boxes . Moreover, the extentto which P₁ and P₂ are activated appears to be influenced bythe presence of other sigma factors, possibly due to competitionbetween sigma factors for binding to core RNA polymerase.

 
Bacterial strains, plasmids, and media. B . subtilis strains and plasmids are listed in Table 1 . Strains were grown in Luria Bertani [LB] medium, low-phosphate medium[LPM; 0.42 mM P_i], or high-phosphate medium [5.0 mM P_i] [31]. E . coli XL1-Blue [Stratagene Europe, Amsterdam, The Netherlands] was used as the host for plasmid constructions, and E . coli BL21[D3] [Novagen, Madison, Wis.] was used for the productionof PhoP-His₆ and PhoR231-His₆ . When required, the concentrationsof antibiotics were the following: for E . coli, 100 µgof ampicillin [Ap] per ml and 25 µg of kanamycin [Km]per ml; for B . subtilis, 6 µg of chloramphenicol per ml,0.3 µg of erythromycin per ml, 25 µg of lincomycinper ml, 10 µg of Km per ml, and 12.5 µg of tetracyclineper ml . Isopropyl-ß-D-thiogalactopyranoside [IPTG]was used at 1 mM.

 
DNA manipulations and general methods. Plasmid and chromosomal DNA extraction, restriction endonucleasedigestion, agarose gel electrophoresis, transformation of E.coli cells, PCR, and bioinformatical analyses were carried outas described previously [30, 33] . Enzymes, molecular size markers,and deoxynucleotides were purchased from Roche Diagnostics,Ltd . [Lewes, United Kingdom], and from Amersham Pharmacia Biotech,Ltd . [Little Chalfont, United Kingdom].

Construction of plasmids. Plasmids pNHP and pNHR [Table 1] were constructed to preparedigoxigenin-labeled RNA probes for phoP and phoR, respectively.Primers NHP-FOR and NHP-REV [Table 2] were used for PCR amplification of a 621-bp fragment of phoP, and primers NHR-FOR and NHR-REV were used to amplify a 1,530-bp fragment of phoR . The PCR fragments were cloned into HindIII- and BamHI-digested pBluescript II KS[+] and were transformed into E . coli XL1-Blue . The resulting plasmids, pNHP and pNHR, were confirmed by sequencing the inserted DNA and adjacent vector sequences.

 
Plasmid pPE [Table 1] was constructed to generate a sequencing ladder for primer extension analysis . A 451-bp fragment from the 5' end of phoP was amplified by using primers PE-FOR and PE-REV [Table 2] and was cloned into BamHI- and EcoRI-digested pBluescript II KS[+] . The structure of pPE was confirmed by sequencing the inserted DNA and adjacent vector sequences.

For the production and purification of PhoP-His₆ and PhoR231-His₆ proteins a 734-bp fragment encoding PhoP and a 1,067-bp fragment encoding the cytoplasmic region of PhoR [from amino acid 231to the C terminus] were amplified by using primer pairs PP-FOR/PP-REV and R231-FOR/R231-REV, respectively [Table 2] . The BspHI-XhoI-digested PCR fragment containing phoP and the BsaI-XhoI-digested PCRfragment containing phoR231 were ligated into NcoI-XhoI-digested pET2816 [Table 1], and the mixtures were used to transform E.coli XL1-Blue . The structures of resulting plasmids pET-PhoPand pET-PhoR231 [Table 1], respectively, were confirmed by sequencingthe inserted DNA and adjacent vector sequences . The PhoP andPhoR231 proteins encoded by these plasmids contained His₆ tagsat their C termini [i.e., PhoP-His₆ and PhoR231-His₆].

RNA extraction, Northern hybridization, and primer extension. Total RNA of the B . subtilis strains was extracted with phenol [27] . Digoxigenin-labeled RNA probes specific for phoP and phoRwere synthesized in vitro with T7 RNA polymerase, using HindIII-linearizedpNHP and pNHR [Table 1], respectively, and the DIG NorthernStarter kit [Roche Diagnostics GmbH, Mannheim, Germany].

For primer extension analysis, primers PEPH1 and PEPH2 [Table 2], complementary to the region encoding the N terminus of phoP,were 5' end labeled with [-³²P]ATP [Amersham Pharmacia Biotech,Ltd.] by using T4 polynucleotide kinase [Promega] . Total RNA[4 µg], ³²P-labeled primer, and SuperScript II RNase H^- reverse transcriptase [Invitrogen Ltd., Paisley, United Kingdom]were used for the primer extension reaction . The primers usedfor reverse transcription were also used to prime dideoxy sequencingreactions from the corresponding phoP region of plasmid pPE[Table 1] as described previously [33].

Production and purification of PhoP-His₆ and PhoR231-His₆. Proteins PhoP-His₆ and PhoR231-His₆ were produced from E . coliBL21[D3] carrying pET-PhoP or pET-PhoR231, respectively, asdescribed previously [5].

Phosphorylation of PhoP by PhoR231. Purified PhoR231 [8 µM] was incubated in phosphorylationbuffer [0.1 M Tris-HCl [pH 8], 0.2 M KCl, 4 mM MgCl₂, 8 mM CaCl₂,0.5 mM dithiothreitol, 0.1 mM EDTA, 3.7% glycerol] in the presenceof PhoP [24 µM] for 10 min at room temperature [RT], followedby incubation with 2.5 µM [40 µCi] [-³²P]ATP for20 min at RT . The reaction was stopped by the addition of 1/5 volume of sodium dodecyl sulfate [SDS] blue loading buffer.The samples were analyzed by SDS-polyacrylamide gel electrophoresis [SDS-PAGE] . The dried gels were exposed to Hyperfilm ECL X-rayfilm [Amersham Pharmacia Biotech] . The radioactive gel imageswere scanned with a PhosphorImager [Storm 860; Molecular Dynamics]and were quantified by using Quantity One software [version4.3; Bio-Rad Laboratories, Hercules, Calif.].

Gel shift assay. For the preparation of DNA probes, fragments of the phoPR, yhaX,and ykoL promoter regions were amplified with primer pairs PhoP-FOR/PhoP-REV, YhaX-FOR/YhaX-REV, and YkoL-FOR/YkoL-REV . In each case the 5'ends of the forward [FOR] primers were labeled with [-³²P]ATP by using T4 polynucleotide kinase [Promega] . In the gel shift reactions, 4 µM PhoR231 and 0.1 µg of poly[dI-dC][Sigma] per µl were incubated with 0, 24, 47, 71, and95 µM PhoP in the presence or absence of 5 mM ATP for15 min at RT [23] . After addition of the DNA probe [1,000 cpmper µl] the mixture was incubated for a further 30 min.The samples were analyzed on a 6% native polyacrylamide gelby using Tris-glycine-EDTA buffer [38].

DNase I footprinting. The coding DNA strand was labeled as described for the gel shiftassay . The noncoding strand was labeled with [³²P] by usingreverse PCR primers . In the DNA binding reactions, a solutionof 4 µM PhoR231, 0.05 µg of bovine serum albuminper µl, and 0.1 µg of poly[dI-dC] per µl was incubated with 0, 19, 38, 57, and 76 µM PhoP in the presenceor absence of 5 mM ATP at RT for 15 min in binding buffer [20mM sodium phosphate buffer [pH 8], 50 mM NaCl, 2 mM MgCl₂, 1mM dithiothreitol, 10% glycerol] . After addition of the DNAprobe [500 cpm per µl] the mixture was incubated for afurther 30 min . DNase I [0.1 U in 10 mM MgCl₂-5 mM CaCl₂] wasadded to the reaction mixture, and digestion was conducted for1 min . The reactions were stopped with DNase I stop solution[0.4 M Na-acetate, 50 µg of calf thymus DNA [Sigma] perml, and 2.5 mM EDTA] . The samples were analyzed on a 6% polyacrylamidegel containing 7 M urea . A Maxam and Gilbert sequencing ladder[cleavage reactions at purine residues [A+G]] [38] was loadedon the same gel.

Protein assay. Protein concentration was determined by using the Bio-Rad proteinassay kit.

SDS-PAGE. SDS-PAGE was carried out as described previously [38]: a 16%separating gel in Tris-glycine buffer was used for the detectionof PhoP-His₆ and PhoR231-His₆ proteins, with low-molecular-size-rangeprestained protein standards as size markers [Bio-Rad] . Thegels were stained with GelCode blue stain reagent [Pierce].

Enzyme and P_i assays. APase production [32] and P_i concentration [10] were determined as described previously.

 
Transcription of the phoPR operon. lacZ transcriptional reporter studies indicated that the phoPR operon exhibits constitutive low-level expression during exponential growth [i.e., P_i-replete conditions] and is induced in response to phosphate starvation [32]: 4 h after the transition fromexponential to stationary phase, the expression of phoPR wasthreefold higher than that in the exponential phase [17, 30].To confirm these data and to determine the size of phoPR transcript[s],Northern blot analyses were performed on RNA extracted fromB . subtilis 168 at various times during growth and phosphatestarvation-induced stationary phase [Fig . 1] . The phoPR operonis located on a 2,742-bp region of the chromosome [Fig . 1B]between two terminators, T_mdh [bp 2977773 to 2977730 [20]; G = -19.8 kcal/mol] and T_phoPR [bp 2975014 to 2974989 [20]; G = -13.8 kcal/mol] . Using probes specific for phoP and phoR, a single prominent band was detected with an estimated sizeof 2.7 kb [Fig . 1C] . Compared with data for exponential phase, the amounts of this transcript were approximately threefold higher during transition phase and in stationary phase . Minorbands at 2.1 and 1 kb, seen only with the phoR probe, are likelyto represent degradation products that are missing regions homologousto the phoP probe . The putative processing sites at the 3' ends of the 1-kb and the 2.1-kb products coincide with inverted repeat sequences located 132 bp [AAGAAGCAAA and TTCTTCGCTT] and 1,221bp [TGCTCGATCT and ACGAGCTAAA] downstream of the translationstart site of phoR.

 
Primer extension analysis identified two major transcriptionalstart sites for the phoPR operon [Fig . 2] . The upstream transcriptionalstart site [TS₁] was mapped to a single nucleotide [A] located70 bp upstream of the phoP translational start site, while thesecond transcriptional start site [TS₂] mapped to two nucleotides[T and A] 49 and 48 bp upstream of the phoP translational startsite . TS₁ was preceded by the sequence 5' TTGTCG-N₁₄-CGCTAAAAT3' [P₁] [see Fig . 5], which is similar to the consensus sequence for a ^A promoter [13] . TS₂ was preceded by the sequence 5' TAAAAT-N₁₄-TGTTAAGAT3' [P₂] [see Fig. 5], which has a -10 region similar to theconsensus sequence for a ^A promoter . However, the -35 regionof P₂, which overlaps the -10 region of P₁, showed no homologyto the ^A consensus sequence . Because both promoters were inducedin response to phosphate starvation [Fig . 2], the region upstream of the operon was analyzed for the presence of putative PhoP binding sites . Three TT[A/T/C]ACA-like sequences were locatedfrom 7 to 33 bp upstream of the -35 region of the P₁ promoter, while the P₂ promoter was associated with four TT[A/T/C]ACA-like sequences [see Fig . 5] . In the latter case the location of thePho box-like sequences from 28 bp upstream to 3 bp downstream of TS₂ is unusual for a PhoP-regulated promoter.

 
Binding of PhoP and PhoPP to the region of the phoPR promoter. Gel shift assays and DNase I footprinting experiments were usedto analyze the binding of PhoP to the promoter region upstreamof phoPR . Because preparations of PhoP and PhoR were requiredfor these experiments, PhoP-His₆ and PhoR231-His₆ variants ofthese proteins were overexpressed and purified from E . coliBL21[D3] carrying plasmids pET-PhoP and pET-PhoR231, respectively[Table 1] . Both protein preparations exhibited greater than 95% homogeneity, as determined by SDS-PAGE with the GelCode blue stain reagent [Fig . 3A] . The functional activities of the purified proteins were determined with an in vitro phosphorylation assay . This confirmed that PhoR231-His₆ was autophosphorylated in the presence of [-³²P]ATP [Fig . 3B, lane 1] and was ableto phosphorylate PhoP-His₆ [Fig . 3B, lanes 2 and 3].

 
Gel shift assays were performed to determine whether PhoP isable to bind to the promoter region of phoPR [Fig . 4A] . PhoP and PhoPP decreased the mobility of the ³²P-labeled phoPR promoter probe in a concentration-dependent manner . No significant differences were observed in the retardation of the probe when PhoP was phosphorylated, indicating that PhoP and PhoPP bind to the phoPRpromoter region in vitro with similar efficiencies . Comparedto ykoL [Fig . 4B], relatively low amounts of the phoP probewere retarded by PhoP and PhoPP, reflecting their relative invitro levels of expression [200 nmol o-nitrophenyl [ONP]/min/opticaldensity unit for ykoL-lacZ and 18 nmol ONP/min/optical densityunit for phoP-lacZ at T₄] and induction ratios [250-fold forykoL and 3-fold for phoPR at T₄] [32].

 
DNase I footprinting was performed on both the coding and noncoding strands [Fig . 5] to locate the PhoP binding site on the phoPRpromoter region . PhoP and PhoPP protected the regions between-49 to -62 bp and -97 to -116 bp on the coding strand and between-53 to -70 bp and -115 to -145 bp on the noncoding strand . Theseregions correspond to the -10 sequence of promoter P₂ and the-35 sequence of promoter P₁ . In addition, relatively more probewas bound by PhoPP than by PhoP.

Binding of PhoP and PhoPP to the promoters of ykoL and yhaX. The binding of PhoP and PhoPP to the promoter region of phoPRwas compared with that of two other putative members of thePho regulon, namely ykoL and yhaX [32, 36] . Gel shift assays and DNase I footprinting experiments were performed to determine if PhoP binds to the ykoL and yhaX promoter regions . PhoP andPhoPP retarded the mobility of the ykoL promoter probe [Fig. 4B] in a concentration-dependent manner and to a greater extentthan was observed for the phoPR promoter probe [Fig. 4A] . DNaseI footprinting of the ykoL promoter region showed that regionsprotected by PhoP and PhoPP were located between +8 to -79 and-92 to -117 on the coding strand and between +30 to -65 and-92 to -111 on the noncoding strand [Fig. 6] . Both PhoP andPhoPP bound more efficiently to a region located at -35 to -63on the coding strand and at -43 and -65 on the noncoding strand.In this region of the ykoL promoter, just upstream of the -35 sequence, the level of protection was significantly higher thanwas observed with the phoPR promoter [Fig . 5] . The levels ofprotection afforded by the phosphorylated and nonphosphorylated forms of PhoP were similar . In contrast, the mobility of the yhaX probe was not influenced by PhoP and PhoPP in the gel shiftassay [Fig . 4C], and these proteins showed no protective effectin the DNase I footprinting experiments [data not shown], indicatingthat there are no PhoP binding sites in the region of the yhaXpromoter.

 
The effect of null mutations in the phoR and sigB genes on the transcription of the phoPR operon. Transcriptional studies with the lacZ reporter gene have shownthat the induction of phoPR in response to phosphate starvationis enhanced threefold in a sigB-null mutant [32] . In vitro transcriptionanalyses were performed to determine the promoter[s] involvedin this phenomenon . B . subtilis strains 168 [wild type], 168-PR[phoR], ML6 [sigB], and 168-PR-SB [phoR/sigB] were grown inLPM, and RNA was extracted throughout the growth cycle for Northernhybridization and primer extension analyses . Using a phoR-specificmRNA probe, Northern hybridization analysis showed that thelevel of induction of phoPR was twofold higher at T₀ and T₁in the absence of SigB [Fig . 7A] . Primer extension analysis[Fig . 7B] supported this result and showed that the P₁ and P₂promoters were both up-regulated at T₀ and T₅ . The data also confirmed that promoter P₁ was primarily responsible for the transcription observed during exponential growth [i.e., P_i-replete conditions] and in the absence of PhoR [i.e., phoR-null and phoR/sigB-null mutants] . Evidence from the Northern hybridizationexperiments that the phoPR promoters were slightly induced atT₂ and T₅ in the phoR-null and phoR/sigB-null mutants [Fig.7A] were not confirmed by the primer extension data [Fig . 7B]. It is likely that the observed weak transcription was due to the presence in the phoR-null mutant of the tetracycline resistance gene [17].

 
The effect of mutations in abrB, spo0A, sigH, sigF, and sigE on the transcription of the phoPR operon. Transcription of phoPR is controlled by a regulatory network that includes, in addition to the PhoP and PhoR proteins, atleast two other signal transduction systems, namely ResD-ResEand Spo0A [15] . When activation of the Pho regulon fails toovercome the phosphate deficiency, phosphorylated Spo0A [Spo0AP] initiates sporulation and indirectly represses phoPR transcription via AbrB and ResD-ResE [17] . We therefore studied the effectson phoPR expression of mutations in genes encoding the transitionstate regulator AbrB and regulators controlling various stagesof sporulation [Spo0A, SigH, SigF, and SigE] . Northern hybridizationanalyses revealed that at the phosphate starvation-induced transitionstage [T₀], phoPR transcription was induced about fourfold inthe abrB-null mutant and two- to threefold in the wild typeand sporulation-deficient mutants [Fig . 8A] . In late stationary phase [T₅] the transcription of phoPR decreased slightly inthe wild type and decreased markedly in the abrB-null mutant[Fig . 8A] . However, the activities of both promoters P₁ andP₂ were induced in the sporulation-specific mutants [Fig . 8].

 
In B . subtilis the phoPR operon encodes the response regulator and histidine sensor kinase responsible for activating or repressing genes of the Pho regulon in response to phosphate starvation. This bicistronic operon encodes a single major transcript of2.7 kb [Fig . 1C] . Primer extension analysis revealed the presence of two transcriptional start sites, TS₁ and TS₂ [Fig . 2], whichcorrespond to promoters P₁ and P₂ . The -35 and -10 sequencesof P₁ promoter and the -10 sequence of P₂ promoter were similar to the consensus sequence, TTGACA-N₁₄-TGNTATAAT, for ^A promotersof B . subtilis [13] . Our data indicate that the P₁ and P₂ promoterswere active in sigB [Fig . 7B], sigH, sigF, and sigE [Fig . 8B]mutants, indicating that they are recognized by ^A and not bythese alternative sigma factors.

There is evidence that factors compete for a limiting poolof core RNA polymerase [9, 14, 26, 37], and data presented hereand in previous work [32] are consistent with sigma factor competitionaffecting the expression of genes in the Pho regulon . For example,the Pho and ^B-GS regulons are induced in response to phosphatestarvation, and the cognate sigma factors ^A and ^B appear tocompete for the core enzyme, because ^A-dependent transcriptionof phoPR was enhanced twofold in a sigB mutant [Fig . 7A] . Laterin stationary phase ^H, ^F, and ^E may compete with ^A for the coreenzyme, because the transcriptional activities of the two ^A-dependent promoters of phoPR were significantly higher in the spo0A, sigH,sigF, and sigE mutants [stage 0, I, and II sporulation mutations]at T₅ than in the wild type [Fig. 8] and sigB mutants [Fig.7A] . However, we have not been able to provide direct evidencefor sigma factor competition, and it is important to recognizethat the inactivation of alternative sigma factors is likelyto have pleiotropic effects on gene regulation that could alsoaccount for the data.

Transcription of the phoPR operon was higher in the spo0A-null mutant than in the wild type [Fig . 8A] . This could be due to[i] direct repression by Spo0AP at putative 0A-boxes [TGNCGAA][41] located at the -35 sequences of P₁ promoter [98 to 105bp and 106 to 112 bp upstream of the translational start siteof phoP], [ii] competition between Spo0AP- and PhoPP-activated regulons for E^A holoenzyme, or [iii] indirect repression bySpo0AP via both AbrB and ResDE [17, 42].

The P₁ and P₂ promoters were induced at T₀ [Fig . 2], when thephosphate concentration in the medium decreased to below 0.1mM [Fig . 1A] . Gel shift assays and DNase I footprinting showedthat both PhoP and PhoPP were able to bind in the region ofphoPR promoters in vitro [Fig . 4A and 5] . However, in vivo only PhoPP was able to induce the transcription of P₁ and P₂ under phosphate starvation, because constitutive low-level transcription from the P₁ promoter was observed in the phoR-null mutant [Fig.7B].

The binding of PhoP and PhoPP to the phoPR promoter region wascompared to that with the ykoL and yhaX promoter regions . Transcriptionalreporter studies [32] showed that the ^A-dependent promoter ofykoL [36] was induced 250-fold in response to phosphate starvation,while the ^E-dependent promoter of yhaX [8] was induced 21-fold. The binding of PhoP and PhoPP to the ykoL promoter was verymuch more efficient than that to the weakly expressed phoPRpromoters [Fig . 4A and B and 5 and 6] . It was previously reportedthat yhaX was induced in response to phosphate starvation ina PhoP- and PhoR-dependent manner [32] . However, the presentstudies failed to demonstrate in vitro binding of PhoP and PhoPP in the region of the yhaX promoter [Fig . 4C] . This indicateseither that yhaX is activated by PhoP indirectly via anotherregulatory pathway or that binding of PhoPP to the yhaX promoterregion requires an additional factor[s].

 
We thank J . Errington [University of Oxford] for the gift ofstrains SWV215, BHI, 650, and 901 and M . A . Strauch for strainSWV119.

This work was funded by the European Commission [QLG2-CT-1999-01455] and the UK Biotechnology and Biological Sciences Research Council [13/PRES/12179].

 
* Corresponding author . Mailing address: School of Cell and Molecular Biosciences, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, United Kingdom . Phone: 44 191 222 7708 . Fax: 44 191 222 7736 . E-mail: colin.harwood@ncl.ac.uk .

Present address: DSM Nutritional Products, Biotechnology R&D,CH-4070 Basel, Switzerland.

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